Olives and Olive Oil in Health and Disease Prevention [2 ed.] 9780128195284

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Olives and Olive Oil in Health and Disease Prevention

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Olives and Olive Oil in Health and Disease Prevention Second Edition

Edited by

Victor R. Preedy Department of Nutrition and Dietetics, Genomics Centre, King’s College, London, United Kingdom; Department of Clinical Biochemistry, Genomics Centre, King’s College, London, United Kingdom

Ronald Ross Watson Mel and Enid Zuckerman College of Public Health and School of Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, AZ, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-819528-4 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisitions Editor: Megan R. Ball Editorial Project Manager: Lena Sparks Production Project Manager: Kumar Anbazhagan Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

Contents

List of contributors Acknowledgments

Part 1 General Aspects of Olives and Olive Oil

xvii xxv

1

Section 1.1 The plant, production, olives and olive oil and their detailed characterization

3

1. Table olives: types and trade preparations

5

3. Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement

22 23 24

27

Samanta Zelasco, Fabrizio Carbone, Luca Lombardo and Amelia Salimonti

Antonio-Higinio Sa´nchez, Jose´ Luis Ruiz-Barba, Antonio Lo´pez-Lo´pez and Alfredo Montan˜o 1.1 Introduction 5 1.2 Types of olives according to ripeness 5 1.3 Table olives according to trade preparations 7 1.4 Major processing methods 8 1.5 Composition of final products 12 1.6 Summary points 13 References 13

2. Naturally processed table olives, their preservation and uses 15 Manuel Brenes and Stanley George Kailis Abbreviations 2.1 Introduction 2.2 Factors to be considered in producing natural table olives 2.3 Natural table olive processing 2.4 Secondary processing of natural table olives 2.5 Preservation and storage methods for naturally processed table olives

2.6 Nutritional and health-related aspects of table olives 2.7 Concluding remarks on natural table olives References

15 15 15 16 21 21

Abbreviations 3.1 Origin, diffusion, and genetic resources 3.2 Phenotypic variability and breeding programs for the olive oil quality improvement 3.3 Olive genomics as tool for olive oil quality improvement 3.4 Conclusion and perspectives Mini-dictionary of terms References

4. The chemical composition of Italian virgin olive oils

27 28 30 32 43 43 44

51

Pierfrancesco Deiana, Maria Rosaria Filigheddu, Sandro Dettori, Nicola Culeddu, Antonio Dore, Maria Giovanna Molinu and Mario Santona Abbreviations 4.1 Introduction 4.2 Fatty acids 4.3 Sterols and triterpenic alcohols 4.4 Squalene 4.5 Phenolic compounds 4.6 Tocopherols 4.7 Comparisons of olive oils with other edible oils 4.8 Implications for human health and disease prevention References

51 51 53 56 57 57 58 59 59 60

v

vi

Contents

Section 1.2 Components of olives and olive plant product and uses 63 5. Bioactive ingredients in olive leaves

65

Abbreviations 8.1 Introduction 8.2 Spectroscopy for quality control 8.3 Conclusion Acknowledgment Mini-dictionary of terms References

91 91 93 95 95 95 96

N. Nenadis, V.T. Papoti and M.Z. Tsimidou Abbreviations 5.1 Introduction 5.2 Sampling 5.3 Postharvest treatment 5.4 Extraction procedures 5.5 Bioactivity of olive leaf extracts 5.6 Cardioprotective activity 5.7 Anticancer properties 5.8 Respiratory diseases 5.9 Diabetes 5.10 Conclusive remarks References Further reading

6. Detection of adulterations of extra-virgin olive oil by means of infrared thermography

65 65 65 67 68 71 71 73 74 74 74 74 78

79

Jose´ S. Torrecilla, John C. Cancilla, Sandra Pradana-Lopez and Ana M. Perez-Calabuig Abbreviations 6.1 Introduction 6.2 Infrared thermography 6.3 Detection of adulterated extra-virgin olive oil using infrared thermography 6.4 Conclusion Acknowledgment Mini-dictionary of terms References

79 79 80 81 82 83 83 83

7. Influence of the distribution chain on the quality of extra virgin olive oils 85 Jose´ S. Torrecilla and John C. Cancilla Abbreviations 7.1 Introduction 7.2 Quality of extra virgin olive oil 7.3 Conclusion Acknowledgment Mini-dictionary of terms References

8. Spectroscopy to evaluate the quality control of extra-virgin olive oils Jose´ S. Torrecilla, John C. Cancilla, Ana M. Perez-Calabuig and Sandra Pradana-Lopez

85 85 85 88 88 88 89

91

9. Chemical composition of fermented green olives

99

Alfredo Montan˜o and Antonio-Higinio Sa´nchez Abbreviations 9.1 Introduction 9.2 Major components of raw olives 9.3 Spanish-style green olives 9.4 Untreated green olives in brine 9.5 Summary points Mini-dictionary of terms References

99 99 99 100 105 106 107 108

10. Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil 111 Paloma Rodrı´guez-Lo´pez, Jesu´s Lozano-Sa´nchez, Isabel Borras-Linares, Tatiana Emanuelli, Javier A. Menendez and Antonio Segura-Carretero Abbreviations 10.1 Introduction: phenolic molecules in virgin olive oil 10.2 Why are the phenolic compounds in virgin olive oil so important? 10.3 Implications for human health and disease prevention 10.4 Phenolic contribution to the oxidative stability of virgin olive oil 10.5 Sensory properties affected by phenolics in virgin olive oil 10.6 Comparisons of olive oils with other edible oils Mini-dictionary of terms References

111 111 115 116 117 118 119 120 120

11. Polyphenol oxidase and oleuropein in olives and their changes during olive ripening 123 Francisca Ortega-Garcı´a, Santos Blanco, M. A´ngeles Peinado and Juan Perago´n Abbreviations 11.1 Introduction 11.2 Kinetic and molecular properties of PPO in the fruit and the leaf of olive trees of the Picual variety

123 123 124

Contents

11.3 Changes during ripening 11.4 Oleuropein concentration in fruit and leaf of olive during ripening 11.5 Effects of the variety of cultivar 11.6 Conclusion 11.7 Summary points References

125 127 127 128 128 128

Section 1.3 Stability, microbes, contaminants and adverse components and processes 131 12. Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains 133 Jose´ Marı´a Landete, He´ctor Rodrı´guez, Jose´ Antonio Curiel, Blanca de las Rivas, Fe´lix Lo´pez de Felipe and Rosario Mun˜oz List of abbreviations 12.1 Introduction 12.2 Phenolic compounds and Lactobacillus plantarum 12.3 Metabolism of phenolic compounds by Lactobacillus plantarum 12.4 Treatment of olive by-products by Lactobacillus plantarum Mini-dictionary of terms References

13. Microbial colonization of naturally fermented olives

133 133 133 135 141 142 143

Part 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil Section 2.1 General nutritional and health aspects

165

Vasanti Suvarna and Dhvani Sharma Abbreviations 14.1 Introduction 14.2 Composition of olive oil 14.3 Effect of olive oil on cardiovascular disease risk factors 14.4 Case studies 14.5 Conclusion References

165 165 167 168 169 170 171

15. Olive in traditional Persian medicine: an overview 175 Mohammad Mahdi Parvizi, Maryam Saki, Farhad Handjani and Mojtaba Heydari Abbreviations 15.1 Traditional Persian medicine 15.2 Olive in traditional Persian medicine 15.3 Implications of olive for human health and disease prevention in traditional Persian medicine 15.4 Implications of olive in medicine based on traditional Persian medicine 15.5 Implication of olive in dentistry and oral cavity based on traditional Persian medicine 15.6 Conclusion Mini-dictionary of terms References

175 175 175

176 177

181 182 182 182

145

E.Z. Panagou, C.C. Tassou and G.-J.E. Nychas Abbreviations 13.1 Introduction 13.2 Microbiota of olives References

14. Overview of olive oil in vascular dysfunction

vii

145 145 145 156

161

163

16. The bioavailability of olive oil phenolic compounds and their bioactive effects in humans

193

Rafael de la Torre, Montserrat Fito´ and Marı´a-Isabel Covas Abbreviations 16.1 Background 16.2 Bioavailability of olive oil phenolic compounds 16.3 Bioactive effects of olive oil phenolic compounds in humans 16.4 In vivo basic mechanisms assessed in human studies for explaining the bioactivity of olive oil rich in phenolic compounds 16.5 Conclusion References

193 193 193 196

200 201 201

viii

Contents

17. Mediterranean diet and role of olive oil

205

Mana Shahbaz, Emilio Sacanella, Iasim Tahiri and Rosa Casas Abbreviations 17.1 Introduction 17.2 What is the Mediterranean diet? 17.3 Extra-virgin olive oil 17.4 Conclusion Acknowledgments References

205 205 206 206 211 211 211

18. Probiotics from fermented olives

215

H. Abouloifa, Y. Rokni, N. Ghabbour, S. Karboune, M. Brasca, G. D’hallewin, R. Ben Salah, N. Ktari, E. Saalaoui and A. Asehraou Abbreviations 215 18.1 Introduction 215 18.2 Probiotic microorganisms isolated from fermented olives 215 18.3 Selection of probiotics from fermented olives 218 18.4 Safety properties of probiotics in human 219 18.5 Health-beneficial effects of probiotics from fermented olives 219 18.6 Technological properties of probiotics from fermented olives 220 18.7 Application of probiotics in olive fermentation 221 18.8 Application of probiotics in biopreservation of fermented olives 223 18.9 Application of probiotics from fermented olive in other foods fermentations 225 18.10 Conclusion 225 Mini-dictionary of terms 226 Summary points 226 References 226

19. Olive oil
contained phenolic compounds protect cells against H2O2-induced damage and modulate redox signaling by chelating intracellular labile iron 231 Alexandra Barbouti, Panagiotis Kanavaros, Panagiotis Kitsoulis, Vlasios Goulas and Dimitrios Galaris Abbreviations 19.1 Introduction 19.2 The concept of oxidative stress 19.3 Do free radical scavengers protect cells in conditions of oxidative stress?

231 231 232 232

19.4 Intracellular “labile iron” as mediator of oxidative stress
induced effects 19.5 Olive oil
contained compounds prevent H2O2-induced DNA damage by chelating intracellular labile iron 19.6 The role of iron in redox signaling 19.7 Olive oil
contained compounds modulate redox signaling through chelation of labile iron 19.8 Concluding remarks 19.9 Summary points References

233

233 234

235 235 235 236

20. Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors 239 Morales-Martı´nez Adriana, Montes Sergio, Sa´nchez-Mendoza Alicia, Quetzalli D. Angeles-Lo´pez, Jime´nez-Go´mez Joel, Martinez-Gopar Pablo Eliasib and Pe´rez-Severiano Francisca Abbreviations 20.1 Introduction 20.2 Fish oil 20.3 Olive oil 20.4 Implications for human health and disease prevention 20.5 Experimental models to study neurodegenerative diseases 20.6 Huntington’s disease and oils as therapeutic agents 20.7 Protective mechanism by polyunsaturated fatty acids in Huntington’s disease model 20.8 Conclusion Mini-dictionary of terms References

21. Olive oil and postprandial energy metabolism: implications for weight control

239 239 240 240 241 242 243 246 246 246 246

251

Mario J Soares, MBBS, MSc, PhD and Kaveri Pathak, APD, PhD Abbreviations 21.1 Introduction 21.2 Body weight regulation and nutrient partitioning 21.3 Can the type of fatty acid affect the rate of fat oxidation? 21.4 Postprandial fat oxidation in humans 21.5 Is there a preferential effect of olive oil in abdominal obesity? 21.6 Olive oil, satiety, and food intake

251 251 251 251 252 252 252

Contents

21.7 Mediterranean-style diets 21.8 Conclusion 21.9 Summary Acknowledgment Mini-dictionary of terms References

22. Effect of olive oil on metabolic syndrome

254 257 257 257 257 257

261

Asavari Joshi and Anand Zanwar Abbreviations 22.1 Introduction 22.2 Olive oil and metabolic syndrome 22.3 Implications for human health with special reference to metabolic health 22.4 Summary Mini-dictionary of terms References

Section 2.2 Cardiovascular

261 261 262 268 268 269 270

273

23. Olive and olive oil: a one stop herbal solution for the prophylaxis and management of cardiovascular disorders 275 Shanoo Suroowan, Bibi Sharmeen Jugreet, Nabeelah Bibi Sadeer and Mohamad Fawzi Mahomoodally Abbreviations 23.1 Introduction 23.2 Ethnobotanical uses of Olea europaea L. 23.3 Conclusion Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications in human health and disease prevention References

275 275 276 285 285 286 287 288

24. Extra-virgin olive oils storage: Effect on constituents of biological significance 291 Vita Di Stefano Abbreviations 24.1 Introduction 24.2 Nutritional quality alteration of extra-virgin olive oil 24.3 Storage of olive oil 24.4 Conclusion Highlights References

291 291 292 293 295 295 295

Section 2.3 Oxidative stress

ix

299

25. Antioxidants in olive oil phenolics: a focus on myoblasts

301

Paraskevi Kouka, Aristidis S. Veskoukis and Demetrios Kouretas Abbreviations 25.1 Introduction 25.2 Natural antioxidants: focus on olive oil constituents and their biological properties 25.3 Myoblasts and satellite cells: an overview 25.4 Reactive species, the antioxidant defense system and redox homeostasis 25.5 Oxidative
reductive stress and acute exercise 25.6 Olive extracts (mixtures) of bioactive compounds and their effects on myoblasts 25.7 In vivo effects of olive oil rich in biophenols in muscle redox regulation 25.8 Polyphenols and athletic performance 25.9 Conclusion Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

26. Antioxidant activity in olive oils

301 301 301 302 302 303 304 305 305 306 306 307 307 308

313

Gamze Guclu, Hasim Kelebek and Serkan Selli Abbreviations 26.1 Introduction 26.2 Natural antioxidants found in olive oil 26.3 Implications for human health and disease prevention 26.4 Conclusion References

Section 2.4 Cancer and immunology 27. Olives and olive oil compounds active against pathogenic microorganisms

313 313 314 319 321 321

327

329

Manuel Brenes, Eduardo Medina, Pedro Garcı´a, Concepcio´n Romero and Antonio de Castro Abbreviations 27.1 Introduction

329 329

x

Contents

27.2 Main antimicrobial compounds in olive oil 27.3 Main antimicrobial compounds in table olives Acknowledgment References

330 333 334 334

28. Olive oil in the prevention of breast and colon carcinogenesis 337 Aliza Hannah Stark and Zecharia Madar Abbreviations 28.1 Introduction 28.2 Breast cancer and olive oil 28.3 Colorectal cancer and olive oil 28.4 Conclusion: implications for human health and disease prevention Mini-dictionary of terms References

337 337 338 340 342 343 343

29. The effects of olive oil and other dietary fats on redox status on breast cancer 347 Marı´a Jesu´s Ramı´rez-Expo´sito, Marı´a Pilar CarreraGonza´lez and Jose´ Manuel Martı´nez-Martos Abbreviations 29.1 Introduction 29.2 Dietary fat and carcinogenesis parameters 29.3 Dietary fat and histopathology of breast tumors 29.4 Dietary fat and redox status 29.5 Dietary fat and hormonal status in breast cancer 29.6 Conclusion Mini-dictionary of terms Implications for human health and disease prevention References

347 347 348 348 350 354 355 355 355 356

30. Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy 359 Eva Batanero and Mayte Villalba Abbreviations 359 30.1 Introduction 359 30.2 Ole e 1 as a marker for sensitization to Oleaceae pollens 362

30.3 Ole e 2 and Ole e 10, two allergens associated with asthma 30.4 Ole e 3 and Ole e 8: Ca21-binding allergens 30.5 Ole e 7, a nonspecific lipid-transfer protein, and its clinical significance 30.6 Ole e 9 and pollen
latex
fruit syndrome 30.7 Other allergens from olive pollen: Ole e 4, Ole e 5, and Ole e 6 30.8 New approaches for new allergens: Ole e 11, Ole e 12, Ole e 14, and Ole e 15 30.9 The role of N-glycans in olive pollen allergy 30.10 Pollensomes: natural vehicles for pollen allergens 30.11 Recombinant olive pollen allergens as diagnostic and therapeutic tools 30.12 New concepts for specific immunotherapy using Ole e 1 as a model 30.13 Olive fruit: a new source of olive allergens Mini-dictionary terms References

363 364 364 365 365

366 367 367 368

369 371 371 372

31. Cancer preventive role of olives and olive oil via modulation of apoptosis and nuclear factor-kappa B activation 377 Vaishali Aggarwal, Gaurav Kumar, Diwakar Aggarwal, Mu¨kerrem Betu¨l Yerer, Ahmet Cumaoglu, Manoj Kumar, Katrin Sak, Sonam ˘ Mittal, Hardeep Singh Tuli and Gautam Sethi Abbreviations 31.1 Introduction 31.2 Chemistry and sources 31.3 Cancer prevention mechanisms 31.4 Conclusion and future perspectives References

32. Immune system and olive oil

377 377 378 378 384 384

389

Seyede Sanaz Seyedebrahimi Abbreviations 32.1 Introduction 32.2 Effects of olive oil components on immune responses 32.3 Olive oil and immune-mediated inflammatory diseases 32.4 Conclusion References

389 389 389 391 394 394

Contents

Section 2.5 Other effects, uses and diseases

399

33. Effect of olive oil on the skin

401

Diana Badiu and Rajkumar Rajendram Abbreviations 33.1 Introduction 33.2 Skin: a natural barrier. Structure and physiology 33.3 Clinical features and pathophysiology of aging conditions: wrinkles, pruritis, and xerosis 33.4 General beneficial properties and constituents of olive oil 33.5 The effects of olive oil on the skin 33.6 Conclusion Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention Summary points References

401 401 402

402 404 404 408 409 409 409 410 410

34. Extra-virgin olive oil, cognition and brain health 415 Elisabetta Lauretti, Luigi Iuliano and Domenico Pratico` Abbreviations 34.1 Introduction 34.2 Cognition, memory, and brain aging 34.3 Evidence of beneficial effects of extra-virgin olive oil on brain health and cognition in human 34.4 Evidence of beneficial effects of extra-virgin olive oil on cognition and neuroinflammation in aging rodents 34.5 Evidence of beneficial effects of extra-virgin olive oil on Alzheimer’s disease
associated memory and cognitive impairment 34.6 Extra-virgin olive oil and synaptic proteins 34.7 Extra-virgin olive oil and long-term potentiation 34.8 Conclusion Mini-dictionary of terms Comparisons of olive oil with other edible oils Implications for human health and disease prevention References

415 415 416

416

xi

35. The foundation for the use of olive oil in skin care and botanical cosmeceuticals 425 Edmund M. Weisberg and Leslie S. Baumann Abbreviations 35.1 Introduction 35.2 Chemistry 35.3 Dietary protection 35.4 Photoprotection 35.5 Topical applications for dermatologic conditions 35.6 Olive oil in combination 35.7 Cosmeceuticals 35.8 Conclusion 35.9 Summary points References

36. Olive oil and male fertility

425 425 426 428 429 430 431 432 432 432 433

435

Germa´n Domı´nguez-Vı´as, Ana Bele´n Segarra, Manuel Ramı´rez-Sa´nchez and Isabel Prieto Abbreviations 36.1 Diet and male fertility 36.2 Dietary lipid and male fertility 36.3 Male fertility and oxidative stress 36.4 Mediterranean diet, olive oil, and male fertility 36.5 The local renin
angiotensin system in the testis, dietary olive oil, and male fertility 36.6 Implications for human health and disease prevention 36.7 Comparisons of olive oils with other edible oils Mini-dictionary of terms References

435 435 436 437 437 438 439 439 440 440

418

419

37. Revealing the molecular mechanism of Olea europaea L. in treatment of cataract 445 Farid A. Badria and Abdullah A. Elgazar

419 420 420 420 420 421 421

Abbreviations 37.1 Introduction 37.2 Olive leaves, chemistry, biology, and therapeutics 37.3 Cataract: pathogenesis and current treatment 37.4 Plausible molecular mechanism of Olea europaea in treatment of cataract 37.5 Conclusion and future perspective References

445 445 446 449 450 454 454

xii

Contents

38. Olive leaf, DNA damage and chelation therapy

457

ˇ ˇ Andrea Cabarkapa-Pirkovi´ c, Lada Zivkovi´ c, Dragana Dekanski, Dijana Topalovi´c and Biljana Spremo-Potparevi´c Abbreviations 38.1 Olive leaf 38.2 Antioxidant effects of olive leaf, scavenging, and chelation 38.3 Effects of the olive leaf on the DNA damage 38.4 Chelation therapy and olive leaf Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

39. Olive polyphenols and chronic alcohol protection

457 457 458 460 462 464 464 465 466

471

Carla Petrella, Giampiero Ferraguti, Luigi Tarani, George N. Chaldakov, Mauro Ceccanti, Antonio Greco, Massimo Ralli and Marco Fiore Abbreviations 39.1 Alcohol consumption: effects and mechanisms 39.2 Polyphenols: a brief overview 39.3 Conclusion Acknowledgments Disclaimer Conflicts of interest References

40. Olive oil diet and amyloidosis: focus on Alzheimer’s disease

471 471 472 474 475 475 475 475

41. Benefits and challenges of olive biophenols: a perspective

483 484 484 485 485 486

489

Hassan Rasouli, Mehdi Hosseini Mazinani and Kamahldin Haghbeen Abbreviations 41.1 Introduction 41.2 An overview of plant polyphenols 41.3 Olive status in Iran and worldwide statistics 41.4 Pharmacological functionalities of olive biophenols 41.5 Recycling olive by-products for cosmetic industries 41.6 Limitations of polyphenols for clinical applications 41.7 Conclusion Mini-dictionary of terms Acknowledgments Conflict of interest statement Funding References

42. Treatment and valorization of olive mill wastewater

489 489 490 492 497 499 500 500 500 501 501 501 501

505

Parvin Mohammadnejad, Kamahldin Haghbeen and Hassan Rasouli

479

Elisabetta Lauretti Abbreviations 40.1 Introduction 40.2 Amyloid-β biology and function 40.3 Amyloid-β pathophysiology 40.4 Impact of extravirgin olive oil on amyloid-β pathology 40.5 Extravirgin olive oil inhibits amyloid-β peptide production and aggregation 40.6 Extravirgin olive oil induction of amyloid-β proteolytic cleavage and blood
brain barrier clearance

40.7 Extravirgin olive oil induction of autophagy activation and amyloid-β proteolytic clearance 40.8 Conclusion Mini-dictionary of terms Comparisons of extravirgin olive oils with other edible oils Implications for human health and disease prevention References

479 479 480 481 482 482 483

Abbreviations 42.1 Introduction 42.2 Olive oil production processes 42.3 Source of olive mill wastewater, its physical properties and chemical composition 42.4 Developments in treatment and valorization of olive mill wastewater 42.5 Exploitation of olive mill wastewater potentials as valuable source of nutraceutical 42.6 Safety concerns 42.7 Concluding remarks Acknowledgment References

505 505 505 507 509 514 516 516 516 516

Part 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

521

Section 3.1 Tyrosol and hydroxytyrosol

523

43. Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil 525 M. Emı´lia Juan and Joana M. Planas Abbreviations 43.1 Introduction 43.2 Maslinic acid, a pentacyclic triterpene from Olea europaea L 43.3 Cancer chemopreventive activity of maslinic acid in colon cancer cells in vitro 43.4 Cancer chemopreventive activity of maslinic acid in animal models in vivo 43.5 Implications for human health and disease prevention Mini-dictionary of terms Acknowledgments References

44. Hydroxytyrosol, olive oil, and use in aging

525 525 526 527 530 533 534 534 534

537

Mercedes Cano, Mario Mun˜oz, Antonio Ayala, Rafael Medina and Sandro Argu¨elles Abbreviations 44.1 Introduction 44.2 Cellular and molecular mechanism of aging 44.3 Beneficial effects of hydroxytyrosol and olive oil on molecular and cellular mechanisms of aging 44.4 Conclusion Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

537 537 538 540 542 542 543 544 544

45. Hydroxytyrosol and hydroxytyrosyl fatty esters: occurrence and antiinflammatory properties 547 Pierluigi Plastina Abbreviations

547

Contents

xiii

45.1 Introduction 45.2 Occurrence 45.3 Anti-inflammatory properties 45.4 In vitro studies 45.5 In vivo studies Mini-dictionary of terms Comparisons of olive oils with other edible oils References

547 548 549 549 550 551 551 552

46. Influence of olive oil on pancreatic, biliary, and gastric secretion: role of gastrointestinal peptides 557 Maria Dolores Yago, Maria Alba Martinez-Burgos, Namaa Audi, Mariano Man˜as and Emilio MartinezVictoria Abbreviations 46.1 Introduction 46.2 Olive oil and digestive secretion in dogs 46.3 Olive oil and digestive secretion in humans 46.4 Adaptation of digestive function and gastrointestinal peptides to dietary fat type: final considerations 46.5 Summary points Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

557 557 557 559 563 565 566 566 566 567

47. Effects of virgin olive oil on fatty acid composition of pancreatic cell membranes: modulation of acinar cell function and signaling, and cell injury 569 Maria Alba Martinez-Burgos, Maria Dolores Yago, Belen Lopez-Millan, Jose Antonio Pariente, Emilio Martinez-Victoria and Mariano Man˜as Abbreviations 47.1 Introduction 47.2 Dietary lipids and pancreatic secretion 47.3 Pancreatic secretion in anesthetized rats 47.4 Experiments in isolated pancreatic acini 47.5 AR42J studies 47.6 AR42J cell model of acute pancreatitis 47.7 Summary points Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

569 569 570 570 571 572 574 577 578 578 579 579

xiv

Contents

48. Hydroxytyrosol: features and impact on pancreatitis

581

Belen Lopez-Millan, Maria Alba Martinez-Burgos, Mariano Man˜as, Emilio Martinez-Victoria and Maria Dolores Yago Abbreviations 48.1 Introduction 48.2 Acute pancreatitis: key aspects 48.3 Lifestyle, Mediterranean diet, hydroxytyrosol, and acute pancreatitis 48.4 Summary points Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

49. The effects of extra-virgin olive oil minority compounds hydroxytyrosol and oleuropein on glioma

581 581 581 583 587 587 588 589 589

Section 3.2 Oleuropein 50. The usage of oleuropein on myocardium

593

593 593 599 599 599 600 600

603 605

Maria Tsoumani, Ioulia Tseti and Ioanna Andreadou Abbreviations 50.1 Introduction 50.2 The effect of oleuropein on cardiomyocytes 50.3 Oleuropein’s cardioprotective effect against myocardial ischemia
reperfusion injury 50.4 Molecular understanding of the protective role of oleuropein 50.5 The role of oleuropein in other cardiovascular disorders

611 611 611 612 612

51. Oleuropein and skin cancer

615

Siti Fathiah Masre Abbreviations 51.1 Introduction 51.2 Skin cancer 51.3 Beneficial properties of oleuropein 51.4 Conclusion References

615 615 615 617 621 621

52. Oleuropein, olive, and insulin resistance

Marı´a Jesu´s Ramı´rez-Expo´sito, Marı´a Pilar Carrera-Gonza´lez and Jose´ Manuel Martı´nez-Martos Abbreviations 49.1 Introduction 49.2 Conclusion Mini-dictionary of terms Implications for human health and disease prevention Acknowledgments References

50.6 Conclusion Mini-dictionary of terms Comparisons of olive oils with other edible oils Implications for human health and disease prevention References

605 605 606 607 610 610

625

Tomoko Ishikawa and Yoko Fujiwara Abbreviations 52.1 Introduction 52.2 The mechanism of insulin-induced hypoglycemia 52.3 Implications for human health and disease prevention 52.4 Oleuropein and olive on insulin resistance 52.5 Effects of metabolites of oleuropein on insulin resistance 52.6 Comparisons of olive oils with other edible oils 52.7 Conclusion Mini-dictionary of terms References

Section 3.3 Oleic acid

625 625 625 627 628 631 632 632 633 633

637

53. Oleic acid—the main component of olive oil on postprandial metabolic processes

639

Sergio Lopez, Beatriz Bermudez, Sergio Montserrat-de la Paz, Yolanda M. Pacheco, Almudena Ortega-Gomez, Lourdes M. Varela, Ana Lemus-Conejo, Maria C. Millan-Linares, Maria A. Rosillo, Rocio Abia and Francisco J.G. Muriana List of abbreviations 53.1 Introduction

639 639

Contents

53.2 Oleic acid on postprandial thrombogenesis 53.3 Oleic acid on postprandial fibrinolysis 53.4 Oleic acid on postprandial β-cell function and insulin sensitivity 53.5 Possible mechanisms by which oleic acid is acting on postprandial glucose homeostasis 53.6 Oleic acid on postprandial inflammation 53.7 Conclusion 53.8 Summary points 53.9 Acknowledgments References

641 641 642

644 645 646 647 647 647

54. Oleic acid and olive oil polyphenols downregulate fatty acid and cholesterol synthesis in brain and liver cells 651 Antonio Gnoni, Serena Longo, Fabrizio Damiano, Gabriele Vincenzo Gnoni and Anna Maria Giudetti Abbreviations 54.1 Introduction 54.2 Conclusion Mini-dictionary of terms Comparison of olive oils with other edible oils Implications for human health and disease prevention References

Section 3.4 Oleocanthal

651 651 655 655 656 656 656

659

55. Olive oil oleocanthal and estrogen receptor expression 661 Nehad M. Ayoub Abbreviations 55.1 Introduction 55.2 Oleocanthal 55.3 Estrogens and estrogen receptors 55.4 Impact of oleocanthal on estrogen receptor

661 661 661 663 665

55.5 Conclusion References

56. Neuroprotective effects of oleocanthal in neurological disorders

xv

667 667

671

Yazan S. Batarseh, Sweilem B. Al Rihani, Euitaek Yang and Amal Kaddoumi Abbreviations 56.1 Introduction 56.2 Oleocanthal induces brain amyloid-β clearance 56.3 Oleocanthal enhances blood
brain barrier integrity and function 56.4 Oleocanthal reduces neuroinflammation and oxidative stress 56.5 Conclusion References

671 671 672 673 674 676 677

57. S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor and its application to synergize targeted therapies and prevent breast cancer recurrence 681 Khalid A. El Sayed Abbreviation 57.1 Introduction 57.2 Conclusion References

58. Phenolic compounds in olive oil mill wastewater

681 682 689 689

693

Jose´ S. Torrecilla and John C. Cancilla Abbreviations 58.1 Introduction 58.2 Phenolic compounds in olive oil mill wastewater 58.3 Olive oil mill wastewater management 58.4 Conclusion Mini-dictionary of terms References Index

693 693 694 695 698 698 698 701

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List of contributors Rocio Abia, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain

Antonio Ayala, Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Seville, Spain

Abouloifa, Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco

Nehad M. Ayoub, Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology (JUST), Irbid, Jordan

H.

Morales-Martı´nez Adriana, Laboratory of Molecular Neuropharmacology and Nanotechnology, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico

Diana Badiu, Faculty of Medicine, Ovidius University of Constanta, Constanta, Romania Farid A. Badria, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt

Diwakar Aggarwal, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Ambala, India

Alexandra Barbouti, Department Histology-Embryology, University Medical School, Ioannina, Greece

Vaishali Aggarwal, Department of Histopathology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India

Eva Batanero, Deparment of Biochemistry and Molecular Biology, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain

Sa´nchez-Mendoza Alicia, Department of Pharmacology, National Institute of Cardiology Ignacio Cha´vez, Mexico City, Mexico

Yazan S. Batarseh, Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan

Ioanna Andreadou, Laboratoty of Pharmacology, School of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece

Leslie S. Baumann, Baumann Research and Cosmetic Institute, Miami, FL, United States

Quetzalli D. Angeles-Lo´pez, Laboratory of Molecular Neuropharmacology and Nanotechnology, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico; Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced studies of National Polithechnic Institute, Mexico City, Mexico Sandro Argu¨elles, Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain A.

Asehraou, Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco

Namaa Audi, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain

of Anatomyof Ioannina

R. Ben Salah, Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax, Sfax, Tunisia Beatriz Bermudez, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Santos Blanco, Cell Biology Section, Department of Experimental Biology, University of Jae´n, Jae´n, Spain Isabel Borras-Linares, Functional Food Research and Development Centre (CIDAF), Health Science Technological Park, Granada, Spain M. Brasca, Institute of Sciences of Food Production, National Research Council of Italy, Milano, Italy Manuel Brenes, Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain; Instituto de la Grasa (IG-CSIC), Campus University Pablo de Olavide, Seville, Spain xvii

xviii

List of contributors

Ana M. Perez-Calabuig, Department of Chemical and Materials Engineering, Complutense University of Madrid, Madrid, Spain; Departamento de Ingenierı´a Quı´mica y de Materiales, Universidad Complutense de Madrid, Madrid, Spain

Rafael de la Torre, Integrative Pharmacology and Systems Neurosciences, IMIM-Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, Spain; Biomedical Research Network Center on Obesity and Nutrition, Madrid, Spain

John C. Cancilla, Scintillon Institute, San Diego, CA, United States

Blanca de las Rivas, Institute of Food Science, Technology and Nutrition (ICTAN), CSIC, Madrid, Spain

Mercedes Cano, Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain

Pierfrancesco Deiana, Deparment of Agriculture, University of Sassari, Sassari, Italy

Fabrizio Carbone, Council for Agricultural Research and Economics, Research Centre for Olive, Fruit and Citrus Crops, Rende, Italy

Dragana Dekanski, Biomedical Research, Institute, Galenika a.d., Belgrade, Serbia

Marı´a Pilar Carrera-Gonza´lez, Experimental and Clinical Physiopathology Research Group CTS1039, Department of Health Sciences, School of Experimental and Health Sciences, University of Jae´n, Campus Universitario Las Lagunillas, Jae´n, Spain; Department of Nursing, Pharmacology and Physiotherapy, Faculty of Medicine and Nursing, University of Cordoba, IMIBIC, Co´rdoba, Spain Rosa Casas, Department of Internal Medicine, Clinic Hospital, August Pi and Sunyer Biomedical Research Institute (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER 06/03: Pathophysiology of Obesity and Nutrition, Institute of Health Carlos III, Madrid, Spain Mauro Ceccanti, Centro Riferimento Alcologico Regione Lazio, ASL Roma 1, Rome, Italy George N. Chaldakov, Department of Anatomy and Cell Biology, Medical University, Varna, Bulgaria Marı´a-Isabel Covas, NUPROAS (NUPROAS HB), Nacka˜, Sweden

Handelsbolag

Nicola Culeddu, National Council of Research (CNR), Institute of Biological Chemistry (ICB), Sassari, Italy Ahmet Cumao˘glu, Department of Biochemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey Jose´ Antonio Curiel, Functional Food Research and Development Center, Health Science Technological Park, Granada, Spain Fabrizio Damiano, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy Antonio de Castro, Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain Fe´lix Lo´pez de Felipe, Instituto de Ciencia y Tecnologı´a de Alimentos y Nutricio´n (ICTAN), CSIC, Madrid, Spain

R&D

Sandro Dettori, Deparment of Agriculture, University of Sassari, Sassari, Italy Vita Di Stefano, Department of Biological, Chemical, and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, Palermo, Italy German Domı´nguez-Vı´as, Unit of Physiology, Department of Health Sciences, University of Jae´n, Jae´n, Spain; Department of Physiology, Faculty of Health Sciences, Ceuta, University of Granada, Granada, Spain Antonio Dore, National Council of Research (CNR), Institute of Sciences of Food Production (ISPA), Sassari, Italy G. D’hallewin, Institute of Sciences of Food Production, National Research Council of Italy, Sassari, Italy Khalid A. El Sayed, School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, United States Abdullah A. Elgazar, Department of Pharmacognosy, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt Tatiana Emanuelli, Department of Food Technology and Science, Center of Rural Sciences, Federal University of Santa Maria, Santa Maria, Brazil Giampiero Ferraguti, Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Maria Rosaria Filigheddu, Deparment of Agriculture, University of Sassari, Sassari, Italy Marco Fiore, Institute of Biochemistry and Cell Biology, IBBC-CNR, Rome, Italy; Department of Sense Organs, Sapienza University of Rome, Rome, Italy Montserrat Fito´, Cardiovascular Risk and Nutrition Research Groups, IMIM-Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, Spain; Biomedical Research Network Center on Obesity and Nutrition, Madrid, Spain

List of contributors

Pe´rez-Severiano Francisca, Laboratory of Molecular Neuropharmacology and Nanotechnology, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico Yoko Fujiwara, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan Dimitrios Galaris, Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina, Greece Pedro Garcı´a, Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain N.

Ghabbour, Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco

Anna Maria Giudetti, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy Antonio Gnoni, Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari “Aldo Moro”, Bari, Italy Gabriele Vincenzo Gnoni, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy Vlasios Goulas, Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, Lemesos, Cyprus Antonio Greco, Department of Sense Organs, Sapienza University of Rome, Rome, Italy Gamze Guclu, Department of Food Engineering, Faculty of Agriculture, Cukurova University, Adana, Turkey Kamahldin Haghbeen, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Farhad Handjani, Department of Dermatology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Mojtaba Heydari, Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Mehdi Hosseini Mazinani, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Tomoko Ishikawa, Institute for Human Life Innovation, Ochanomizu University, Tokyo, Japan

xix

Luigi Iuliano, Department of Medico-Surgical Sciences and Biotechnologies., Sapienza University of Rome, Latina, Italy Jime´nez-Go´mez Joel, Laboratory of Molecular Neuropharmacology and Nanotechnology, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico, Asavari Joshi, Centre for Innovation in Nutrition Health Disease, Interactive Research School for Health Affairs, Bharati Vidyapeeth (Deemed to be University), Pune, India M. Emı´lia Juan, Departament of Biochemistry and Physiology, Facultat de Farma`cia i Cie`ncies de l’Alimentacio´ and Institut de Recerca en Nutricio´ i Seguretat Alimenta`ria (INSA-UB), Universitat de Barcelona (UB), Barcelona, Spain Bibi Sharmeen Jugreet, Department of Health Sciences, Faculty of Science, University of Mauritius, Re´duit, Mauritius Amal Kaddoumi, Department of Drug Discovery and Development, Harrison School of Pharmacy, Pharmacy Research Building, Auburn University, Auburn, AL, United States Stanley George Kailis, Australian Mediterranean Olive Research Institute, Perth, WA, Australia Panagiotis Kanavaros, Department Histology-Embryology, University Medical School, Ioannina, Greece

of Anatomyof Ioannina

S. Karboune, Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill University, Ste Anne de Bellevue, QC, Canada Hasim Kelebek, Department of Food Engineering, Faculty of Engineering, Adana Alparslan Turkes Science and Technology University, Adana, Turkey Panagiotis Kitsoulis, Department of AnatomyHistology-Embryology, University of Ioannina Medical School, Ioannina, Greece Paraskevi Kouka, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece Demetrios Kouretas, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece N. Ktari, Laboratory of Enzyme Engineering and Microbiology, University of Sfax, National School of Engineering of Sfax (ENIS), Sfax, Tunisia Gaurav Kumar, Department of Biochemistry, Delhi University, South Campus, New Delhi, India

xx

List of contributors

Manoj Kumar, Department of Chemistry, Maharishi Markandeshwar University, Sadopur, Ambala, India Jose´ Marı´a Landete, Food Technology Department, INIA-SGIT, Madrid, Spain Elisabetta Lauretti, Alzheimer’s Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, United States Ana Lemus-Conejo, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Luca Lombardo, Center Agriculture Food Environment (C3A), University of Trento, Trento, Italy; Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy Serena Longo, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy Sandra Pradana-Lopez, Department of Chemical and Materials Engineering, Complutense University of Madrid, Madrid, Spain; Departamento de Ingenierı´a Quı´mica y de Materiales, Universidad Complutense de Madrid, Madrid, Spain Sergio Lopez, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Belen Lopez-Millan, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain

Emilio Martinez-Victoria, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain Maria Alba Martinez-Burgos, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix,” University of Granada, Granada, Spain Jose´ Manuel Martı´nez-Martos, Experimental and Clinical Physiopathology Research Group CTS1039, Department of Health Sciences, School of Experimental and Health Sciences, University of Jae´n, Campus Universitario Las Lagunillas, Jae´n, Spain Siti Fathiah Masre, Centre for Toxicology and Health Risk Studies, Faculty of Health Sciences, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia Eduardo Medina, Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain Rafael Medina, Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain Javier A. Menendez, ProCURE (Program Against Cancer Therapeutic Resistance), Catalan Institute of Oncology, Hospital Dr. Josep Trueta de Girona, Girona, Spain Maria C. Millan-Linares, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Sonam Mittal, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

Antonio Lo´pez-Lo´pez, Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Seville, Spain

Parvin Mohammadnejad, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

Jesu´s Lozano-Sa´nchez, Department of Food Science and Nutrition, University of Granada, Granada, Spain; Functional Food Research and Development Centre (CIDAF), Health Science Technological Park, Granada, Spain

Maria Giovanna Molinu, National Council of Research (CNR), Institute of Sciences of Food Production (ISPA), Sassari, Italy

Zecharia Madar, Robert H. Smith Faculty of Agriculture, Food and Environment, School of Nutritional Sciences, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel Mohamad Fawzi Mahomoodally, Department of Health Sciences, Faculty of Science, University of Mauritius, Re´duit, Mauritius Mariano Man˜as, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix,” University of Granada, Granada, Spain

Alfredo Montan˜o, Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Seville, Spain Sergio Montserrat-de la Paz, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Mario Mun˜oz, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Seville, Spain Rosario Mun˜oz, Institute of Food Science, Technology and Nutrition (ICTAN), CSIC, Madrid, Spain

List of contributors

Francisco J.G. Muriana, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain N. Nenadis, Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece; NatPro-AUTH, Center for Interdisciplinary Research and Innovation (CIRI-AUTH), Thessaloniki, Greece

xxi

Joana M. Planas, Departament of Biochemistry and Physiology, Facultat de Farma`cia i Cie`ncies de l’Alimentacio´ and Institut de Recerca en Nutricio´ i Seguretat Alimenta`ria (INSA-UB), Universitat de Barcelona (UB), Barcelona, Spain Pierluigi Plastina, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy

G.-J.E. Nychas, Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, School of Food and Nutritional Sciences, Agricultural University of Athens, Athens, Greece

Domenico Pratico`, Alzheimer’s Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, United States

Francisca Ortega-Garcı´a, Biochemistry and Molecular Biology Section, Department of Experimental Biology, University of Jae´n, Jae´n, Spain

Rajkumar Rajendram, Department of Medicine, King Abdulaziz Medical City, King Abdullah International Medical Research Center, Ministry of National Guard—Health Affairs, Riyadh, Saudi Arabia; College of Medicine, King Saud bin Abdulaziz University of Health Sciences, Riyadh, Saudi Arabia; Nutritional Sciences Research Division, School of Life Sciences, King’s College London, London, United Kingdom

Almudena Ortega-Gomez, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Yolanda M. Pacheco, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain E.Z. Panagou, Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, School of Food and Nutritional Sciences, Agricultural University of Athens, Athens, Greece V.T. Papoti, Perrotis College, American Farm School, Thermi, Greece Jose Antonio Pariente, Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain Mohammad Mahdi Parvizi, Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Isabel Prieto, Unit of Physiology, Department of Health Sciences, University of Jae´n, Jae´n, Spain

Massimo Ralli, Department of Sense Organs, Sapienza University of Rome, Rome, Italy Marı´a Jesu´s Ramı´rez-Expo´sito, Experimental and Clinical Physiopathology Research Group CTS1039, Department of Health Sciences, School of Experimental and Health Sciences, University of Jae´n, Campus Universitario Las Lagunillas, Jae´n, Spain Manuel Ramı´rez-Sa´nchez, Unit of Physiology, Department of Health Sciences, University of Jae´n, Jae´n, Spain Hassan Rasouli, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Sweilem B. Al Rihani, Department of Drug Discovery and Development, Harrison School of Pharmacy, Pharmacy Research Building, Auburn University, Auburn, AL, United States

Kaveri Pathak, APD, PhD, Nutrition and Dietetics, School of Public Health, Curtin University, Bentley Campus, Perth, WA, Australia ´ ngeles Peinado, Cell Biology Section, Department M. A of Experimental Biology, University of Jae´n, Jae´n, Spain

He´ctor Rodrı´guez, Inflammation and Macrophage Plasticity Lab, CICbioGUNE, Derio, Spain

Juan Perago´n, Biochemistry and Molecular Biology Section, Department of Experimental Biology, University of Jae´n, Jae´n, Spain

Paloma Rodrı´guez-Lo´pez, Functional Food Research and Development Centre (CIDAF), Health Science Technological Park, Granada, Spain

Carla Petrella, Institute of Biochemistry and Cell Biology, IBBC-CNR, Rome, Italy ˇ Andrea Cabarkapa-Pirkovic, Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

Y. Rokni, Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco Concepcio´n Romero, Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain

xxii

List of contributors

Maria A. Rosillo, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Jose´ Luis Ruiz-Barba, Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Seville, Spain E. Saalaoui, Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco Emilio Sacanella, Department of Internal Medicine, Clinic Hospital, August Pi and Sunyer Biomedical Research Institute (IDIBAPS), University of Barcelona, Barcelona, Spain; CIBER 06/03: Pathophysiology of Obesity and Nutrition, Institute of Health Carlos III, Madrid, Spain

Dhvani Sharma, Department of Pharmaceutical Chemistry and Quality Assurance, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Mumbai, India Mario J Soares, MBBS, MSc, PhD, Nutrition and Dietetics, School of Public Health, Curtin University, Bentley Campus, Perth, WA, Australia Biljana Spremo-Potparevi´c, Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Aliza Hannah Stark, Robert H. Smith Faculty of Agriculture, Food and Environment, School of Nutritional Sciences, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel

Nabeelah Bibi Sadeer, Department of Health Sciences, Faculty of Science, University of Mauritius, Re´duit, Mauritius

Shanoo Suroowan, Department of Health Sciences, Faculty of Science, University of Mauritius, Re´duit, Mauritius

Katrin Sak, NGO Praeventio, Tartu, Estonia

Vasanti Suvarna, Department of Pharmaceutical Chemistry and Quality Assurance, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Mumbai, India

Maryam Saki, Health System Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Amelia Salimonti, Council for Agricultural Research and Economics, Research Centre for Olive, Fruit and Citrus Crops, Rende, Italy Antonio-Higinio Sa´nchez, Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Seville, Spain Mario Santona, Deparment of Agriculture, University of Sassari, Sassari, Italy Ana Bele´n Segarra, Unit of Physiology, Department of Health Sciences, University of Jae´n, Jae´n, Spain Antonio Segura-Carretero, Functional Food Research and Development Centre (CIDAF), Health Science Technological Park, Granada, Spain; Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain Serkan Selli, Department of Food Engineering, Faculty of Agriculture, Cukurova University, Adana, Turkey Montes Sergio, Department of Neurochemistry, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico Gautam Sethi, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Iasim Tahiri, Department of Internal Medicine, Clinic Hospital, August Pi and Sunyer Biomedical Research Institute (IDIBAPS), University of Barcelona, Barcelona, Spain Luigi Tarani, Department of Pediatrics, Medical Faculty, Sapienza University of Rome, Rome, Italy C.C. Tassou, Hellenic Agricultural Organization— DEMETER, Institute of Technology of Agricultural Products, Lykovrysi, Greece Dijana Topalovi´c, Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Jose´ S. Torrecilla, Department of Chemical and Materials Engineering, Complutense University of Madrid, Madrid, Spain; Departamento de Ingenierı´a Quı´mica y de Materiales, Universidad Complutense de Madrid, Madrid, Spain Ioulia Tseti, Uni-Pharma S.A., Athens, Greece M.Z. Tsimidou, Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece; NatPro-AUTH, Center for Interdisciplinary Research and Innovation (CIRI-AUTH), Thessaloniki, Greece

Seyede Sanaz Seyedebrahimi, Medical University of Kurdistan, Sanandaj, Islamic Republic of Iran

Maria Tsoumani, Laboratoty of Pharmacology, School of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece

Mana Shahbaz, Department of Internal Medicine, Clinic Hospital, August Pi and Sunyer Biomedical Research Institute (IDIBAPS), University of Barcelona, Barcelona, Spain

Hardeep Singh Tuli, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Ambala, India

List of contributors

Lourdes M. Varela, Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain Aristidis S. Veskoukis, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece Mayte Villalba, Deparment of Biochemistry and Molecular Biology, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain Edmund M. Weisberg, The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, United States Maria Dolores Yago, Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain

xxiii

Euitaek Yang, Department of Drug Discovery and Development, Harrison School of Pharmacy, Pharmacy Research Building, Auburn University, Auburn, AL, United States Mu¨kerrem Betu¨l Yerer, Department of Pharmacology, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey Anand Zanwar, Centre for Innovation in Nutrition Health Disease, Interactive Research School for Health Affairs, Bharati Vidyapeeth (Deemed to be University), Pune, India Samanta Zelasco, Council for Agricultural Research and Economics, Research Centre for Olive, Fruit and Citrus Crops, Rende, Italy ˇ Lada Zivkovi´ c, Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

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Acknowledgments The work of Dr. Watson’s editorial assistant, Bethany L. Stevens, in communicating with authors, editors, and working on the manuscripts was critical to the successful completion of the book. The author appreciates both Ms. Stevens and Lena Sparks, Editorial Project Manager at

Elsevier. Support for Ms. Stevens’ and Dr. Watson’ editing was graciously provided by Southwest Scientific Editing & Consulting, LLC. Direction and guidance from Elsevier’s staff Pat Gonzalez was critical.

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Part 1

General Aspects of Olives and Olive Oil

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Section 1.1

The plant, production, olives and olive oil and their detailed characterization

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Chapter 1

Table olives: types and trade preparations Antonio-Higinio Sa´nchez, Jose´ Luis Ruiz-Barba, Antonio Lo´pez-Lo´pez and Alfredo Montan˜o Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Sevilla, Spain

1.1 Introduction Table olives are the products prepared from the sound fruits of cultivated olive trees (Olea europaea L.). Consumption of table olives dates back to antiquity. The first author who described several methods to prepare edible olives according to their variety and degree of ripeness was Columela in CE 42.1 Table olive production was initially restricted to the producing regions, mainly around the Mediterranean Sea. Nowadays, however, olive elaboration has expanded to both North and South America, and even Australia. The world production of table olives is around 2.6
2.7 million tonnes/year. The main producers are the European Union (EU), Egypt, Turkey, Algeria, Syria, Morocco, Argentina, The United States, Peru, and Iran. Inside the EU, Spain is the main producer followed by Greece and Italy. Table 1.1 presents detailed data on production, export and import, and consumption of the main countries involved in table olive trade.2 Each olive grower country has its own and typical olive varieties. Of all the olive varieties that exist, only those having appropriate characteristics (Table 1.2) are used for table olive processing, and even fewer varieties are used for industrial preparation and international trade.3 The suitability of olives for table consumption is a function of size, shape, flesh-to-stone ratio, flesh finesse, taste, firmness, and ease of stone detachment. Olives weighing between 3 and 5 g are considered to be medium sized; over 5 g they are large. Olives that are more or less spherical facilitate processing operations and have a better market, although some elongated fruits do also find favor. The stone should come away easily from the flesh and a flesh-to-stone ratio of 5:1 is acceptable; the higher this ratio, the better the commercial value of the olives. The skin of the fruit should be fine, yet elastic and resistant to blows and to the action of alkalis and brine. At the international trade level the most important table olive

varieties are Manzanilla de Sevilla, Gordal sevillana, Hojiblanca, Kalamata, and Conservolea, and to a lesser extent Bella di Cerignola, Ascolana Tenera, and Picholine. There is a characteristic that is common to almost all olive varieties, which is their extremely bitter taste when tasted fresh. The glucoside oleuropein is responsible for it, and the different processing methods aim to remove this compound in order to obtain fruits with more palatable attributes. It could be said that there are too many processing methods at places where olives are consumed. In an attempt to normalize the different products the International Olive Council (IOC) has a Trade Standard Applying to Table Olives where the types, trade preparations, quality factors, and other properties are described.4 The objective of this chapter is to describe in detail the different kinds or classifications applicable to table olives, explaining the distinctive traits for each case.

1.2 Types of olives according to ripeness Table olives are classified in one of the following types according to the degree of ripeness of the fresh fruits: 1. Green olives: Fruits are harvested during the ripening period, prior to coloring and when they have reached normal size. Once processed, green olive color may vary from green to straw yellow. 2. Turning-color olives: Fruits are harvested before complete ripeness is attained, at the stage when color is changing from green to violet. After processing, this type of olives may vary from rose to wine rose or brown. 3. Black olives: Fruits are harvested when fully ripe or slightly before full ripeness is reached. Once processed, black olives may range from reddish black to violet black, deep violet, greenish black, and deep chestnut.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00019-5 © 2021 Elsevier Inc. All rights reserved.

5

6

PART | 1 General Aspects of Olives and Olive Oil

TABLE 1.1 World table olive production, exportation, importation, and consumption along the last eight seasons.a2 Country

Production average (%)

Exports average (%)

Imports average (%)

Consumption average (%)

Albania

31.6 (1.2)

2.7 (0.4)

3.4 (0.5)

31.3 (1.2)

Algeria

223.3 (8.5)

7.4 (1.2)

227.4 (8.9)

Argentina

104.4 (4.0)

0.3 (0.0)

35.1 (1.4)

Australia

3.7 (0.1)

17.3 (2.7)

21.1 (0.8)

Brazil

105.1 (16.7)

105.1 (4.1)

Canada

29.0 (4.6)

29.0 (1.1)

65.1 (10.0)

Croatia

0.4 (0.0)

0.2 (0.0)

0.6 (0.1)

0.9 (0.0)

Chile

22.4 (0.8)

2.8 (0.4)

12.0 (1.9)

31.3 (1.2)

Egypt

427.9 (16.2)

86.8 (13.4) 287.2 (44.2)

0.3 (0.0) b

97.1 (15.4)

342.2 (13.4) b

581.8 (22.7)

European Union

830.9 (31.5)

Iran

57.8 (2.2)

1.0 (0.2)

56.8 (2.2)

Iraq

8.0 (0.3)

15.2 (2.4)

23.1 (0.9)

Israel

16.8 (0.6)

5.4 (0.9)

21.8 (0.8)

4.1 (0.6)

4.1 (0.2)

0.3 (0.0)

Japan Jordan

30.7 (1.2)

5.6 (0.9)

1.5 (0.2)

26.1 (1.0)

Lebanon

21.3 (0.8)

1.8 (0.3)

2.9 (0.5)

22.6 (0.9)

Libya

3.0 (0.1)

10.1 (1.6)

13.1 (0.5)

Mexico

13.9 (0.5)

2.9 (0.4)

9.1 (1.4)

20.4 (0.8)

Morocco

112.5 (4.3)

80.8 (12.4)

0.1 (0.0)

31.5 (1.2)

Palestine

10.4 (0.4)

0.6 (0.1)

Peru

70.9 (2.7)

26.0 (4.0)

0.1 (0.0)

42.8 (1.7)

47.8 (7.6)

47.8 (1.9)

31.4 (5.0)

35.9 (1.4)

Russia Saudi Arabia

4.5 (0.2)

Syria

136.0 (5.1)

12.8 (2.0)

Switzerland

124.3 (4.9) 6.5 (1.0)

Tunisia

24.1 (0.9)

1.7 (0.3)

Turkey

400.9 (15.2)

66.8 (10.3)

Uruguay The United States

71.4 (2.7)

Other producing countries

15.0 (0.6)

5.4 (0.8)

Other nonproducing countries Total world

9.6 (0.4)

2641.8 (100)

649.2 (100)

6.5 (0.3) 22.1 (0.9) 340.7 (13.3)

1.8 (0.3)

1.8 (0.1)

142.7 (22.6)

209.6 (8.2)

10.0 (1.6)

25.0 (1.0)

68.9 (10.9)

68.9 (2.7)

630.7 (100)

2559.3 (100)

This table shows data on production, export and import, and consumption for the main countries involved in the table olive trade. a Average data (1000 tonnes) corresponding to harvest seasons 2010/11
2017/18, this last season’s data are provisional. b Without intracommunity trade.

Table olives: types and trade preparations Chapter | 1

7

TABLE 1.2 World olive varieties suitable for table or both table and oil extraction.3 Country

Table

Table and oil

Albania

Kalinjot

Algeria

Azeradj, Blanquette de Guelma, Sigoise

Argentina

Arauco

Chile

Azapa

Cyprus

Ladoelia

Croatia

Oblica

Egypt

Aggezi Shami, Hamed, Toffahi

France

Lucques

Greece

Chalkidiki

Amigdalolia, Kalamo´n, Konservolia, Mastoidis, Megaritiki

Israel

Kadesh, Merhavia

Barnea

Italy

Ascolana Tenera, Giarraffa, Nocellara del Belice, Oliva di Cerignola, Sant
Agostino, Santa Caterina

Carolea, Cassanese, Cellina di Nardo`, Cucco, Itrana, Majatica di Ferrandina, Nocellara Etnea, Pizz
e Carroga

Aglandau, Grossane, Picholine Languedoc, Salonenque, Tanche

Jordan

Rasi
i

Lebanon

Soury

Montenegro

Zutica

Morocco

Meslala

Haouzia, Menara, Picholine Marocaine

Palestine

Nabali Baladi

Portugal

Carrasquenha, Cordovil de Castelo Branco, Cordovil de Serpa, Galega Vulgar, Mac¸anilha Algarvia, Redondal

Spain

Aloren˜a, Gordal de Granada y Sevillana, Loaime, Manzanilla de Sevilla, Mollar de Cieza, Morona

Hojiblanca, Manzanilla Caceren˜a, Manzanilla Prieta, Morisca, Rapasayo, Villalonga

Syria

Abou-Satl, Kaissy

Doebli, Sorani

Tunisia

Meski

Che´toui, Gerboui, Oueslati

Turkey

Domat, Izmir Sofralik, Uslu

C ¸ ekiste, C ¸ elebi, Erkence, Gemlik, Memecik, Memeli

The United States

Mission

This table shows the major olive varieties grown worldwide suitable for table olive processing as well as those suitable for both table olive and oil extraction. The most important table olive varieties are Manzanilla de Sevilla, Gordal Sevillana, Hojiblanca, Kalamata, and Conservolea, and to a lesser extent Bella de Cerignola, Ascolana Tenera, and Picholine.

1.3 Table olives according to trade preparations As mentioned before, olive preparations are diverse and include not only traditional methods of processing but also those derived from them and improved by new technologies. The bitterness of olives may be removed by alkaline treatment, by immersion in water or brine to dilute the bitter compound oleuropein, or by biological processes. The product so obtained may be preserved in brine, dry salt, modified atmosphere, heat treatments, preservatives, acidifying agents, or, more commonly, a

mixture of several of these methods. Usually, the complete name of the preparation includes information on the type of raw materials, the procedure used for eliminating the bitterness, and the method for preserving the product. Olives may undergo the following trade preparations4: 1. Treated olives: Green, turning-color, or black olives that have undergone an alkaline treatment and are then placed under brine in which they undergo complete or partial fermentation. They might be preserved by the addition of acidifying agents. The bitterness of treated olives is removed by alkaline treatment.

8

2.

3.

4.

5.

PART | 1 General Aspects of Olives and Olive Oil

a. Treated green olives in brine. Examples are Spanish-style green olives, Picholine style, and Castelvetrano style. b. Treated olives turning-color olives in brine. c. Treated black olives. Natural olives: Green, turning-color, or black olives that are placed directly in brine in which they undergo complete or partial fermentation, being preserved or not by the addition of acidifying agents. The bitterness of natural olives is mainly removed by dilution. a. Natural green olives. b. Natural olives turning color. c. Natural black olives. Dehydrated and/or shriveled olives: Green, turningcolor, or black olives that have undergone or not a mild alkaline treatment, preserved in brine or partially dehydrated in dry salt, and/or by heating or by any other technological process. a. Dehydrated and/or shriveled green olives. b. Dehydrated and/or shriveled olives turning color. c. Dehydrated and/or shriveled black olives. Black olives in dry salt are the best example of this preparation. Olives darkened by oxidation: Green or turning-color olives preserved in brine, fermented or not, darkened by oxidation in an alkaline medium, and preserved in hermetically sealed containers subjected to heat sterilization. They get a uniform black color. a. Black olives. Other denominations for these olives are canned ripe olives or Californian-style olives. Specialties: Olives may be prepared by means distinct from the above described or in addition to them. Such specialties retain the name “olive” as long as the fruit used complies with the general definitions laid down before.

same day. There are two principal ways of processing treated green olives—one mediated by microbial fermentation (Spanish style) and the other without fermentation (Picholine and Castelvetrano styles).

1.4.1.1 Spanish-style green olives Also known as Sevillian-style green olives, this is one of the two main table olive products in the world. A flowchart with the steps of the process is presented in Fig. 1.1. The olives, once carefully harvested and transported to the factory, are treated in a dilute lye solution (sodium hydroxide) with concentrations ranging from 2.0% to 3.5% (w/v NaOH in water), depending on the variety and ripeness of the olives, temperature, and water quality.5,6 This alkaline treatment has several effects on the fruits such as the hydrolysis or elimination of the oleuropein, an increase of the fruit skin permeability, and other changes that aid subsequent fermentation.7,8 Treatment takes place in containers of various sizes in which olives are completely covered by the lye solution. The olives remain in this solution until the lye has penetrated two-thirds of the way through the flesh. At this stage, lye is removed and replaced by water. Lengthy or numerous washing steps properly eliminate alkali, but caution has to be taken at this step as it also drags along soluble sugars that will be needed for fermentation later on.9 After washing, olives are placed into suitable containers and covered with brine.10
12 Nowadays, olives are fermented into Spanish-style green olives Harvesting Transport Sorting and size grading (optional) Lye (NaOH) treatment

1.4 Major processing methods

Washing

Despite the numerous processing methods around the world, only some of them are economically important from a global standpoint. At the same time, some local methods have gained high value. The most important are explained in the following subsections.

Brining Fermentation Preservation

1.4.1 Treated green olives

Sorting

Green olives are harvested during the ripening cycle when they have reached normal size, but prior to color change. Table olives are machine-harvested in some cases but, owing to the large proportion of bruised fruit, the catching frames have to be handled with care and the fruit may even have to be immersed in a dilute alkaline solution while still in the orchard.5 Freshly harvested, the olives are taken to the factory for processing, if possible on the

Grading Pitting

Whole

Stuffing (optional)

Packing FIGURE 1.1 Flow diagram of Spanish-style green olive production.

Table olives: types and trade preparations Chapter | 1

large 10,000- to 15,000-kg glass fiber containers. Brine concentrations are typically in the range 9%
10% NaCl (w/v) to begin with but rapidly drop to 5%
6% at equilibrium owing to the high content of interchangeable water in the olives. The brine triggers the release of the fruit cell juices, forming a culture medium suitable for fermentation. In this broth culture, a complex and variable microbiota grows up whose evolution along fermentation time has been divided in up to three distinct stages.13
15 During the first stage (3
10 days), indigenous alkali and salt-tolerant microorganisms are responsible for the start of the fermentation, including Gram-negative bacteria as well as halophilic and alkaliphilic lactic acid bacteria (LAB).16,17 The origin of these microorganisms is diverse and may include the raw materials (olives, water, and salt) as well as diverse environmental elements at the factory’s facilities (pipes, pumps, tanks, and fermenters). This microbiota is able to reduce the high initial pH (10
11) to values in the range 6
7, more appropriate for the subsequent growth of common LAB during the second fermentation stage.18,19 This second stage is characterized by the exponential growth of homolactic LAB. These bacteria metabolize sugars into, mainly, lactic acid with a subsequent decrease in the pH value. The species Lactobacillus pentosus is nowadays recognized as the main responsible for this role,16,20 although in the past the species Lactobacillus plantarum was designated by several authors, most probably because of the lack of reliable molecular criteria to distinguish among these taxonomically very close LAB species.14,21 The second stage typically lasts 10
15 days, when pH values are around 4.5 units, being most of the sugars already metabolized.22 During the final, third stage, all remaining substrates are used up while LAB population steadily declines. A good indication that a proper fermentation has taken place is a pH value below 4.0 and free acidity in the range of 0.7%
1.2%. These conditions are essential since microorganisms of the Enterobacteriaceae family, eventual spoiling Clostridia, and other problematic microorganisms are killed or fail to grow at those pH values. On the other hand, different yeast species appear all along the fermentation.19,23 Fermentative yeasts do not cause deterioration, but oxidative yeasts, able to form films on the brine surface, may consume the organic acids produced by LAB and, therefore, raise the pH values while compromising long-term preservation of the fermented product.24 Starter cultures based on selected L. pentosus strains have been used in the past decades to ensure rapid and homogeneous lactic acid fermentation.20 Normal processes can be altered by the presence of undesirable microorganisms that may impair the lactic acid fermentation and confer poor sensorial properties to the olives or even compromise their preservation.25 Gaspocket formation in the olive skins (alambrado) may be

9

caused by Enterobacteriaceae during the first stage of the fermentation, while clostridia may cause butyric or putrid spoilage as well. In all cases, fermentation is controlled by ensuring the right pH and salt level. Obviously, it is also crucial to keep containers and the rest of the equipment in good sanitary conditions, and to use good quality water. When properly fermented, olives can be kept for a long time. However, the spoilage known as “zapaterı´a” may arise during the preservation of the fermented product. Zapaterı´a produces an unpleasant taste and odor, often coinciding with the rise of temperatures during the spring or early summer.26 The bacteria responsible for this spoilage are known for a long time and belong to the genera Clostridium and Propionibacterium.27,28 Again, the combination of brine concentration adjusted above 8.5% and pH values below 4.0 units helps to guarantee the correct preservation conditions. Finally, when olives are about to be marketed, the fruits are sorted and graded for the first or second time (Fig. 1.1). The original brine is replaced by a fresh one and the olives are packed in barrels, cans, or glass jars. Sometimes they are stoned (pitted), sliced, or stuffed with anchovies, pimento, etc. Pasteurization is not strictly necessary except for stuffed olives.29,30 The green Spanish-style table olives have sensory characteristics that make them highly appreciated by the consumers. In 2008 the IOC adopted a method for the sensory analysis of table olives, and since 2011 there has been a revised version of this method of sensory evaluation (COI/OT/MONo 1/Rev.2).31 The attributes to be evaluated for table olives were (1) negative attributes or defects (abnormal fermentations as putrid, butyric, and zapaterı´a; musty, rancid, cooking effect, soapy, metallic, earthy, and winey-vinegary defects); (2) descriptive gustatory sensations (salty, bitter, and acid); and (3) kinaesthetic sensations and texture (hardness, fibrousness, and crunchiness). Defects are unpleasant sensations caused by substances responsible for off-odors, which are formed during the fermentation and the preservation of olives, and are perceived directly or retronasally. For classification purposes, only the median of the defect predominantly perceived (DPP) was considered. According to the DPP intensity, the olives were classified into four categories (extra or fancy; first, 1st, choice or select; second, 2nd or standard; and olives that should not be sold as table olives). Recently, a lexicon to characterize the sensory profile of the green Spanish-style table olives has been developed and applied to the description of samples from diverse cultivars and production areas, using a trained panel and quantitative descriptive analysis (QDA). The results pointed to sensible differences among the sensory profiles of the samples due to cultivar and origin.32

10

PART | 1 General Aspects of Olives and Olive Oil

Picholine

Castelvetrano

Natural black olives

Harvesting

Harvesting

Harvesting

Transport

Transport

Lye (NaOH) treatment

Grading

Washes (3 in 1 day)

Lye (NaOH) treatment

Washes (2 in 1 day)

Salt addition

Brining (2 or 3)

Fresh conservation

Cold storage (5°C–7°C)

Washing

Packing

Packing

(A)

(B)

Transport Sorting and size grading Brining Fermentation (Aerating) Preservation

FIGURE 1.2 Flow diagrams of Picholine-style (A) and Castelvetranostyle (B) green olive production.

Sorting

1.4.1.2 Picholine-style green olives Fig. 1.2A shows the process diagram for this product, an example of NaOH-treated but not fermented fruits. Olives belonging to the Picholine Languedoc and Lucques varieties in the south of France are prepared in this manner, as are the other varieties in Morocco (Picholine Marocaine) and Algeria. The bitterness of the olives is removed by treating them with lye (2.0%
2.5% w/v NaOH) into which they are left for 8
12 h until the lye has penetrated three quarters of the way through the flesh. They are rinsed several times for 1 or 2 days and then placed in 5%
6% (w/v) brine for 2 days. A second brine is used at 7%, and the acidity is corrected by adding citric acid (pH 4.5). After 8
10 days the olives are ready to be eaten and retain their bright green color. Sometimes the consignment of the finished product has to be postponed, and it is necessary to store the olives, what is usually easy as long as temperature does not rise. In that case the olives can be left in 8% brine until the spring, when NaCl concentration should be raised to 10%. In large-scale facilities, they are kept in 3% brine in cold stores where the temperature is maintained between 5 C and 7 C. Before shipping the olives are washed repeatedly, stored, and packed in suitable containers in brine at 5%
6%.5,33

1.4.1.3 Castelvetrano-style green olives A flowchart of this product is presented in Fig. 1.2B. As with the Picholine style, it is an olive preparation where fermentation does not constitute a key step. This is a production method used in Italy, almost exclusively in the Castelvetrano region, using the olive variety Nocellara del Belice, and being mainly consumed in central and

Grading Packing (vinegar-Kalamata) FIGURE 1.3 Flow diagram of natural black olive production. Operations in brackets are optional.

southern Italy. Once olives arrive at the processing plant, they are graded, since only fruits with more than 19 mm in diameter are used. Selected olives are introduced into plastic vessels and covered with 1.8%
2.5% (w/v) NaOH solution, depending on the fruit ripeness and size. These vessels have 220 L total capacity and are filled with around 140 kg of fruits. After 1 h in the lye, 5
8 kg NaCl are added to each container and olives are kept in this alkaline brine for 10
15 days. A mild washing step is carried out before selling, which does not totally eliminate the soda, whose taste is appreciated by the consumers of these olives.5,34

1.4.2 Natural olives The designation “natural olives” applies to those fruits placed directly in brine, without any lye treatment to remove their bitterness. Although natural olives can be prepared from green, turning-color, or black fruits, the latter are more common. An example of the different production stages is outlined in Fig. 1.3. Natural black olives in brine are typical of the eastern Mediterranean and northern African countries: in Greece, they are made from the Conservolea variety, which grades at around 200 fruits/kg, and in Turkey, they are made with the Gemlik variety.33 To prepare natural black olives

Table olives: types and trade preparations Chapter | 1

the fruits are picked by hand when black ripe, but before the olives are overripen or shriveled by frost. They have to be transported as quickly as possible to the processing plant where they are stored, washed, and immersed in 8%
10% (w/v) NaCl brine. Large-scale plants use big 10- to 20-t tanks, while small-scale processors continue to use wooden vats. At the start of the fermentation the tanks are tightly sealed because the olives must not be exposed to air. The brine stimulates the microbial activity for fermentation and reduces the bitterness caused by oleuropein. Fermentation of these olives takes a long time because the diffusion of soluble components through the epidermis, in fruits not treated with alkali, is slow. A diverse microbiota grows in these brines, although yeasts are the microorganisms always present throughout the process. Enterobacteriaceae can be found during the first 7 or 15 days, but they disappear as the brine characteristics do not support their growth. The presence of LAB depends on the salt concentration and polyphenol content of the variety used. While traditional brining is carried out under anaerobic conditions, an aerobic method can be applied by bubbling air through a column disposed in the center of the fermenter. This system changes the ratio between fermentative and oxidative yeasts, and a final product with better quality is attained.6,14,35 When the bitterness of the fruits has been sufficiently weakened, the product can be marketed. As the color of the fruits fades during the process, it is corrected by aerating the olives for 2 or 3 days, sometimes after the addition of 0.1% (w/v) ferrous gluconate or lactate to turn them a deeper black appearance. Finally, the olives are selected and packed in barrels or inside varnished cans, which are filled with 8% fresh brine. They are popular on the markets because of their slightly bitter taste and aroma. Natural black olives can also be packed in vinegar (25% v/v) and may even be heat processed. A few grams of olive oil are then added to each can to form a surface layer. The Kalamata variety is prepared in this way, where the elongated medium-sized olives are slit to absorb the flavor of the marinade and then canned.31

1.4.3 Black olives in dry salt Although of Greek origin, dehydrated black olives are encountering a great consumer acceptance in many producing areas. These are prepared using overripe olives. They are vigorously washed and placed in baskets with alternating layers of dry salt equivalent to 15% of the weight of the olives.5 The final product is not bitter, but salty, and it looks like a raisin, being mostly for local consumption. The flowchart for the production of this olive preparation is outlined in Fig. 1.4.

11

Black olives in dry salt Harvesting Transport Washing Dry salt layers Curing Packing FIGURE 1.4 Flow diagram of black olives in dry salt production.

Olives darkened by oxidation Harvesting Transport Previous handlings Washing (Preservation in brine, fermentation) Lye treatment and air oxidation Washing (alkali neutralization) Brining Sorting and grading (Pitting, slicing, etc.) Packing Sterilization FIGURE 1.5 Flow diagram of olives darkened by oxidation. Operations in brackets are optional.

1.4.4 Olives darkened by oxidation These olives are also known as Californian-style black olives, ripe or semiripe olives, or simply black olives.33,36 Production flowchart of this preparation is outlined in Fig. 1.5. Fruits are harvested when their color is starting to change, before full maturity.13 Once in the production plant, olives are selected and may be directly processed or, more commonly, they are preserved before oxidation. Preservation is usually in brine and a fermentative process, comparable to that of natural black olives, takes place. Nevertheless, this preservation can also be done in acidified water, usually by adding acetic acid.37 With this method, discharge of sodium chloride in wastewater streams is remarkably reduced. From a microbiological

12

PART | 1 General Aspects of Olives and Olive Oil

TABLE 1.3 Physicochemical characteristics of the packing brine or of the juice after osmotic balance.4 Preparation

Minimum sodium chloride content (%)

Maximum pH limit

Minimum acidity expressed as % lactic acid

SCC, MAT

PR, R

P, S

SCC, MAT

PR, R

P, S

SCC, MAT

PR, R

P, S

Treated olives

5

4

GMP

4.0

4.0

4.3

0.5

0.4

GMP

Natural olives

6

6

GMP

4.3

4.3

4.3

0.3

0.3

GMP

Dehydrated and/or shriveled olives

10

10

GMP

GMP

GMP

GMP

GMP

GMP

GMP

Olives darkened by oxidation

GMP

GMP

GMP

GMP

GMP

GMP

GMP

GMP

GMP

This table shows the limits for the physicochemical parameters established in the “Trade Standard Applying to Table Olives” of the International Olive Council according to processing type and preservation method. GMP, Good manufacturing practice; MAT, modified atmosphere; P, pasteurization; PR, addition of preservatives; R, refrigeration; S, sterilization; SCC, specific chemical characteristics.

point of view the acid addition results in pH values that are not compatible with the growth of Enterobacteriaceae while allowing LAB growth in some instances. In any case, yeasts continue to be the most important microorganisms in these solutions. As this preservation step is not necessary, a complete fermentation is not required either. The characteristic operation in this preparation is the oxidation of the fruits. Processing methods for American varieties have been described in detail.14,38 In general, fruits are successively treated with sodium hydroxide solutions for varying periods of time to achieve a progressive penetration of the lye into the flesh. After each alkaline treatment the olives are immersed into water and oxidized by injecting air under pressure. This procedure is aimed at oxidizing phenolic compounds in the fruits, allowing the complete blackening of the fruit skin and a uniform coloration of the flesh. The phenolic compounds hydroxytyrosol (3,4-dihydroxyphenyl acetic acid) and caffeic acid, through their polymerization, were the promoters of the fruit darkening.39 Darker olives and higher oxidation rates were obtained at higher pH values.14,40 The number of lye treatments is usually between 3 and 5, although some processors apply two or even just one treatment. Penetration into the fruit is controlled so that the sodium hydroxide of the first treatment merely passes through the skin. Subsequent treatments are chosen so that they penetrate deeper into the pulp. The final lye treatment must reach the pit. The concentration of sodium hydroxide in the lye solution depends on the ripeness of the fruit, its variety, the environmental temperature, and the desired penetration speed. It varies between 1% and 2% NaOH (w/v). The highest concentration is usually used for the first treatment. The blackened olives are washed several times with water to remove most of the sodium hydroxide and lower the pH in the flesh to around 8.0 units. Generally, 0.1% (w/v) of

iron gluconate or lactate is added to the last wash to stabilize the color. The final canned product has sensorial properties very different from fermented fruits obtained by other processes. The pH values are between 5.8 and 7.9, and the NaCl content is between 1% and 3%. Due to these chemical characteristics, which do not guarantee safety, olives darkened by oxidation have to be sterilized to prevent any possibility of foodborne pathogen growth. The study of the sensory profile of black ripe table olives, from Spanish cultivars, has been carried out using an adaptation of the lexicon developed by QDA.41 The effects of cultivar and growing area on the sensory profiles were relevant.42

1.5 Composition of final products The numerous procedures to prepare table olives imply an ample range of characteristics in the different final products. However, they have to comply with the limits displayed in Table 1.3 to be marketed.4 The limits vary depending on the preparation system, and depending on the way preservation is guaranteed. Sodium chloride concentration, pH, and titratable acidity as lactic acid are the parameters to monitor. They can be analyzed in the brine or from the fruit juice but, in any case, once the osmotic balance between olives and packing brine has been attained. Olives darkened by oxidation have no requirement in relation with the cited parameters since they have to be sterilized to guarantee safety. Dehydrated olives must contain a minimum of 10% NaCl unless they are thermally treated. Natural or NaOHtreated olives vary in their requirements according to their preservation method. Apart from brine (water and food-grade salt) and olives, other possible ingredients are vinegar, olive oil, sugars, spices or aromatic herbs or natural extracts, and

Table olives: types and trade preparations Chapter | 1

authorized additives. Furthermore, any single or combination of edible material used as an accompaniment or stuffing is also allowed. Typical examples are pimento, capers, onions, and many others.

1.6 Summary points G

G

G

G

G

Fruits from olive trees may be prepared following many different methods usually related to the various producing areas. The different methods may, or may not, include a fermentation stage, and most of them are aimed at eliminating the natural bitterness of the fresh fruit. Spanish-style green olives are the only ones which include fermentation by LAB as a necessary step. Olives not treated with alkali usually support a fermentation by yeasts, while maintained in brine. Keeping quality may be guaranteed by physicochemical characteristics or thermal treatment.

References 1. Columela LJM (42). In: “De re rustica”. Santander: Sociedad Nestle´; 1979. 2.- IOC (International Olive Council). ,https://www.internationaloliveoil.org/what-we-do/economic-affairs-promotion-unit/#figures. Accessed 15.01.2020. 3. IOC. Cata´logo Mundial de Variedades de Olivo. Madrid: IOC; 2000. 4. IOC. Trade Standard Applying to Table Olives. Document COI/OT/ NC no. 1. Madrid: IOC; 2004. 5. Sa´nchez Go´mez AH, Garcı´a Garcı´a P. Elaboracio´n de aceitunas de mesa. In: Barranco D, Ferna´ndez-Escobar R, Rallo L, eds. El cultivo del olivo. 7th ed. Madrid: Ediciones Mundi-Prensa; 2017:869
941. 6. Sa´nchez AH, Garcia P, Rejano L. Elaboration of table olives. Grasas Aceites. 2006;57:86
94. 7. de la Borbolla y Alcala´ JMR. Sobre la preparacio´n de la aceituna estilo sevillano. El tratamiento con lejı´a. Grasas Aceites. 1981;32:181
189. 8. Brenes M, de Castro A. Transformation of oleuropein and its hydrolysis products during Spanish-style green olive processing. J Sci Food Agric. 1998;77:353
358. 9. de la Borbolla y Alcala´ JMR, Rejano Navarro L. Sobre la preparacio´n de la aceituna estilo sevillano. El lavado de los frutos tratados con lejı´a. Grasas Aceites. 1978;29:281
291. 10. Rejano L, Gonza´lez-Cancho F, de la Borbolla J-MR. Estudio sobre el aderezo de aceitunas verdes. XXIV Nuevos ensayos sobre el control de la fermentacio´n. Grasas Aceites. 1977;28:255
265. 11. de la Borbolla J-MR, Rejano Navarro L. Sobre la preparacio´n de la aceituna estilo sevillano. La fermentacio´n I. Grasas Aceites. 1979;30:175
185. 12. de la Borbolla J-MR, Rejano Navarro L. Sobre la preparacio´n de la aceituna estilo sevillano. La fermentacio´n II. Grasas Aceites. 1981;32:103
113. 13. Ferna´ndez-Dı´ez MJ, Castro R, Ferna´ndez AG, et al. Biotecnologı´a de la Aceituna de Mesa. Madrid: CSIC; 1985.

13

14. Garrido A, Garcı´a P, Brenes M. Olive fermentations. In: Rehm HJ, Redd G, eds. Biotechnology. Weinheim: VCH; 1995:593
627. 15. Garrido A, Ferna´ndez-Dı´ez MJ, Adams RM. Table Olives. Production and Processing. London: Chapman and Hall; 1997. 16. de Castro A, Montan˜o A, Casado FJ, Sa´nchez AH, Rejano L. Utilization of Enterococcus casseliflavus and Lactobacillus pentosus as starter cultures for Spanish-style green olive fermentation. Food Microbiol. 2002;19:637
644. 17. Lucena-Padro´s H, Ruiz-Barba J. Diversity and enumeration of halophilic and alkaliphilic bacteria in Spanish-style green table-olive fermentations. Food Microbiol. 2016;53:53
62. 18. Sa´nchez AH, Rejano L, Montan˜o A, de Castro A. Utilization at high pH of starter cultures of lactobacilli for Spanish-style green olive fermentation. Int J Food Microbiol. 2001;67:115
122. 19. Lucena-Padro´s H, Caballero-Guerrero B, Maldonado-Barraga´n A, Ruiz-Barba JL. Microbial diversity and dynamics of Spanish-style green table-olive fermentations in large manufacturing companies through culture-dependent techniques. Food Microbiol. 2014;42:154
165. 20. Ruiz-Barba JL, Jime´nez-Dı´az R. A novel Lactobacillus pentosuspaired starter culture for Spanish-style green olive fermentation. Food Microbiol. 2012;30:253
259. 21. Ruiz-Barba JL, Cathcart DP, Warner PJ, Jime´nez-Dı´az R. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture in Spanish-style green olive fermentations. Appl Environ Microbiol. 1994;60:2059
2064. 22. Montan˜o A, Sa´nchez AH, de Castro A. Controlled fermentation of Spanish-type green olives. J Food Sci. 1993;4:842
844. 23. Bautista-Gallego J, Rodrı´guez-Gomez F, Barrio E, Querol A, Garrido-Ferna´ndez A, Arroyo-Lo´pez FN. Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications. Int J Food Microbiol. 2011;147:89
96. 24. Arroyo-Lo´pez FN, Romero-Gil V, Bautista-Gallego J, et al. Yeasts in table olive processing: desirable or spoilage microorganisms? Int J Food Microbiol. 2012;160:42
49. 25. Lanza B. Abnormal fermentations in table-olive processing: microbial origin and sensory evaluation. Front Microbiol. 2013;4:1
7. 26. Montan˜o A, de Castro A, Rejano L, Brenes M. 4Hydroxycyclohexanecarboxylic acid as a substrate for cyclohexanecarboxylic acid production during the “Zapatera” spoilage of Spanish-style green table olives. J Food Prot. 1996;59:657
662. 27. Kawatomari T, Vaughn RH. Species of Clostridium associated with zapatera spoilage of olives. Food Res. 1956;21:481
490. 28. Plastourgos S, Vaughn RH. Species of Propionibacterium associated with zapatera spoilage of olives. Appl Microbiol. 1957;5:267
271. 29. Gonza´lez-Pellisso´ F, Rejano L, Gonza´lez-Cancho F. La pasterizacio´n de aceitunas estilo sevillano I. Grasas Aceites. 1982;33:201
207. 30. Gonza´lez-Pellisso´ F, Rejano L. La pasterizacio´n de aceitunas estilo sevillano II. Grasas Aceites. 1984;35:235
239. 31. IOC. Method for the Sensory Analysis of Table Olives. Document COI/OT/MO/Doc No 1/Rev. 2. Madrid: IOC; 2011. 32. Lo´pez-Lo´pez A, Sa´nchez-Go´mez AH, Montan˜o A, Corte´s-Delgado A, Garrido-Ferna´ndez A. Sensory profile of green Spanish-style table olives according to cultivar and origin. Food Res Int. 2018;108:347
356. 33.- IOC (International Olive Council). ,https://www.internationaloliveoil.org/olive-world/table-olives. Accessed 15.01.2020.

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34. Salvo F, Cappello A, Giacalone L. L
Olivicoltura nella Valle del Belice. Italy: Istituto Nazionale di Economia Agraria. Ministero delle Risorse Agricole, Alimentari e Forestali; 1995. 35. Garcı´a P, Dura´n MC, Garrido A. Fermentacio´n aero´bica de aceitunas maduras en salmuera. Grasas Aceites. 1985;36:14
20. 36. USDA United States Department of Agriculture. United States Standards for Grades of Canned Ripe Olives. Washington, DC: USDA United States Department of Agriculture; 1983. 37. De Castro A, Garcı´a P, Romero C, Brenes M, Garrido A. Industrial implementation of black ripe olive storage under acid conditions. J Food Eng. 2007;80:1206
1212. 38. Cruess WV. Pickling and canning of ripe olives. Commercial Fruits and Vegetable Products. 4th ed. New York: McGraw Hill; 1958.

39. Brenes Balbuena M, Garcı´a Garcı´a P, Garrido Ferna´ndez A. Phenolic compounds related to the black color formed during the processing of ripe olives. J Agric Food Chem. 1992;40:1192
1196. 40. Garcia P, Brenes M, Vattan T, Garrido A. Kinetic study at different pH values of the oxidation processes to produce ripe olives. J Sci Food Agric. 1992;60:327
331. 41. Lee SM, Kitsawad K, Sigal A, Flynn D, Guinard J. Sensory properties and consumer acceptance of imported and domestic sliced black ripe olives. J Food Sci. 2012;77:439
448. 42. Lo´pez-Lo´pez A, Sa´nchez-Go´mez AH, Montan˜o A, Corte´s-Delgado A, Garrido-Ferna´ndez A. Sensory characterisation of black ripe table olives from Spanish Manzanilla and Hojiblanca cultivars. Food Res Int. 2019;116:114
125.

Chapter 2

Naturally processed table olives, their preservation and uses Manuel Brenes1 and Stanley George Kailis2 1

Instituto de la Grasa (IG-CSIC), Campus University Pablo de Olavide, Seville, Spain, 2Australian Mediterranean Olive Research Institute, Perth,

WA, Australia

Abbreviations cv/s. GAP GHP GMP HACCP HHP IOC NaCl w/v w/w

cultivar/s good agricultural practices good hygienic practices good manufacturing processes hazard analysis and critical control point high hydrostatic pressure International Olive Council sodium chloride weight in volume weight in weight

2.1 Introduction Natural table olives are recognized worldwide as a unique foodstuff. These olives are produced from raw drupe fruit of the cultivated olive Olea europaea L. without the use of alkali, for example, sodium, potassium hydroxide, or wood ash. Raw olive fruits, mostly inedible, due to bitter polyphenols such as oleuropein and ligustroside in the flesh (pericarp), must be debittered to make them palatable. Natural table olives are gaining more and more credence in the marketplace because of present-day consumer desire and preference for natural and organic foods. Demand for naturally processed olives is also increasing for the following reasons: their richness in health-promoting bioactive substances; increased consumption of the Mediterranean diet; concerns by consumers for natural products perceived to be healthier and safe; and low environmental contamination compared to alkali-treated olives. Important influences on the production of quality natural table olives include the following: cultivar, preharvest factors, maturation state, harvesting method, postharvest factors, processing method, and preservation and storage

methods. This will involve selecting the appropriate cultivar, producing quality raw olives under good agricultural practices (GAP), and using good manufacturing processes (GMP) and good hygienic practices (GHP) to produce the final product. GMP involves quality assurance processes that include hazard analysis and critical control point (HACCP) principles. GHP prevents the introduction of spoilage and harmful microorganisms from external sources. The aim of natural table olive processing is to produce safe, palatable, and nutritious foodstuff.1 As indicated earlier, raw olive fruit is naturally bitter because of polyphenolic compounds present in the flesh, especially oleuropein, demethyloleuropein and ligustroside, and hydroxytyrosol glucosides.2 For natural olives, this bitterness must be partially or completely eliminated through leaching, weak acid hydrolysis, enzymatic or microbial hydrolysis, or oxidation of oleuropein resulting in less bitter polyphenols, such as hydroxytyrosol, so that final products are acceptable to consumers. Today, there are several challenges for the processing and commercialization of natural olives to get a homogeneous color of the final product (black, green/yellow, or turning color); achieve pitted and sliced olives so that olives retain their good texture; accelerate the debittering process, valorize the product, considering its nutritional characteristics, optimize the harvesting stage as well as to minimize spoilage drawbacks and many others.

2.2 Factors to be considered in producing natural table olives High-quality natural table olives can only be prepared from raw olives with outstanding pomological attributes with zero defects, that is, no damage during harvesting or

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00054-7 © 2021 Elsevier Inc. All rights reserved.

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PART | 1 General Aspects of Olives and Olive Oil

disease, frost, or harvest/postharvest storage. This can be achieved by paying attention to cultivar selection, growing environment, including pest control, maturation state, harvesting technology, postharvest handling, and storage before processing, that is, GAP. Degree of bitterness is another factor influenced by maturation state and cultivar, where generally it is greater in green-ripe olives, that is, green . turning color . black reflecting polyphenol levels and can influence the processing period. There are probably more than 2000 olive cultivars (genotypes) worldwide,3 and fruit from most olive cultivars can be processed into natural table olives; however, there are commercially viable, popular, and unique table olive cultivars as summarized in Table 2.1. Olive size and flesh:stone ratio are important considerations for table olive processing, consumption, and presentation. Broadly speaking, olives can be categorized as small (,3 g), medium (3
5 g), large (5
10 g), and extra-large also known as Queen ( . 10 g). Larger olives can take longer to process especially those with a high flesh:stone ratio. Furthermore, many consumers prefer larger olives (Gordal and Bella di Cerignola) rather than smaller ones, although small olives such as from Arbequina and Taggiasca cvs. are very popular. For natural olives, cultivars with fine skin and smooth flesh characteristics are preferable, for example, Kalamata and Gordal Sevillana cvs. For table olive production, three maturation states or ripeness levels are recognized—green-ripe, turning color, and naturally black-ripe olives. Color development of raw olive fruit goes from lime green (unripe), due to chlorophyll pigments, changing to straw yellow (green-ripe) due to chlorophyll and the presence of β-carotene becomes more evident. As they ripen further, the level of these pigments falls significantly, then skin starts to pigment with polyphenolic compounds such as anthocyanins cyanidin 3-glucoside and cyanidin 3-rutinoside, appearing at veraison (turning color or semiripe), followed by total skin and flesh pigmentation (naturally black-ripe). Olives at the green-ripe stage are used to produce natural green table olives by water curing; fermentation in brine, cracked olives, and occasionally for partially dehydrated olives. Turning color olives may have patchy or completely pigmented skin, rose to purple due to anthocyanins, but not the flesh. They can be processed by soaking in potable water or weak salt brine; spontaneous fermentation in brine; or as cracked olives with or without herbs and spices. When preparing naturally black-ripe olives by fermentation, to ensure firmness, olives are best harvested when the flesh is pigmented with anthocyanins, purple/brown/black, three quarters of the way through to the stone. These are used for producing Greek-style naturally black-ripe table olives, by spontaneous fermentation in brine, and Kalamata-style olives. Fully black-ripe

olives and overripe black olives are also used to prepare dehydrated olives. Ideally raw olives for table olive processing should be picked by hand to prevent skin and flesh damage. Fruits from some cultivars such as Ascolana Tenera, Gordal, and Manzanilla need to be handled carefully as the fruits bruise easily during harvesting and postharvest handling whereas olives from Barouni and Hojiblanca cvs. are more robust. Hand-harvesting is expensive compared to mechanical harvesting but has the significant advantage that fruit can be selectively removed, that is, size and color, reducing the need for sorting. This also favors small-scale table olive enterprises eliminating the need of expensive mechanical and optical sorting equipment. After harvesting, to prevent heat deterioration, raw olives should be transported in slatted ventilated crates to the processing facility. They are best processed within 1
2 days of harvesting; otherwise they should be stored at ambient temperature away from direct light, in a controlled atmosphere, at a controlled temperature. Respiratory activity has been detected in harvested olives several days after harvesting, causing changes in the fruit such as consumption of sugars—mainly mannitol, loss of moisture hence reduced weight, and decreased firmness through loss of texture.5

2.3 Natural table olive processing General considerations when processing natural table olives include removing leaves, twigs, and extraneous matter; color sorting and size grading to maintain the desired maturation state, removing damaged olives, and oversmall fruit; washing the raw olives by immersion or by spraying with potable water to remove orchard dust and agrochemical residues on the skin as well as environmentally acquired undesirable microorganisms, especially those that may cause food poisoning or spoilage. It is essential that high-quality potable water and food grade inputs and equipment are used during of natural table olive production. As under most circumstances, raw olive fruit need to be debittered before they can be consumed, the primary aim, that is, primary processing of natural table olives processing, is to reduce the level of bitterness of raw olives. A number of common natural table olive processing methods are available (Table 2.2).

2.3.1 Natural table olive processing by the archaic method (water/weak salt brine curing) Traditional or archaic table olive processing methods involve placing raw olives, green-ripe, turning color, or naturally black-ripe in potable water or weak salt brine

TABLE 2.1 Selected olive cultivars used in natural table olive processing.4 Cultivar

Country

Fruit sizea

Stone typeb

F:S ratioa

Oil contentb

Polyphenol levelsb

Aglandau

France

Medium

Clingstone

Medium

Med/high

Medium

Arauco

Argentina

Queen

Freestone

High

Medium

Medium

Arbequina

Spain

Small

Clingstone

Low

Med/high

Low

Ascolana Tenera

Italy

Queen

Freestone

Very high

Low/med

Medium

Ayvalik

Turkey

Large

Clingstone

Medium

High

Low/med

Azapa

Chile

Queen

Clingstone

High

Low

High

Barnea

Israel

Medium

Freestone

High

Med/high

Medium

Bella di Cerignola

Italy

Queen

Clingstone

Low/ med

Low

Low

Carolea

Italy

Large

Clingstone

Medium

Med/high

Low/med

Chalkidikis

Greece

Queen

Freestone

High

Low/med

High

Coratina

Italy

Med/large

Clingstone

Med/ high

Very high

Very high

Domat

Turkey

High

Clingstone

Med/ high

Medium

Low/med

Frantoio

Italy

Medium

Clingstone

Medium

Med/high

Med/high

Galega Vulgar

Portugal

Medium

Freestone

Low

Medium

Low

Gemlik

Turkey

Large

Freestone

Low/ med

High

Low/med

Gordal Sevillana

Spain

Queen

Clingstone

Medium

Low

Low

Hojiblanca

Spain

Large

Clingstone

Medium

High

Medium

Itrana

Italy

Large

Freestone

Medium

Medium

Medium

Kalamon

Greece

Large

Freestone

High

High

Low

Konservolia (Volos, Amfissis)

Greece

Large

Freestone

High

Med/high

Medium

Koroneiki

Greece

Small

Freestone

Low/ med

High

Very high

Ladoelia

Cyprus

Medium

Clingstone

Low

Low/med

Medium

Leccino

Italy

Medium

Freestone

Medium

Med/high

Medium

Manzanilla de Sevilla

Spain

Medium

Freestone

Med/ high

Medium

High

Memecik

Turkey

Large

Clingstone

High

Med/high

Medium

Meski

Tunisia

Med/large

Freestone

High

Low

Low

Mission (Californian)

United States

Medium

Freestone

Medium

Medium

Med/high

Nocellara del Belice

Italy

Large

Freestone

High

Medium

Low

Oblitza (Oblica)

Former Yugoslavia

Variable Small to large

Freestone

Med/ high

Med/high

Medium

Picholine Languedoc

France

Medium

Freestone

Med/ high

Low/med

High

Picholine Marocaine

Algeria

Medium

Freestone

Medium

Low/med

High

Sigoise

Algeria

Medium

Freestone

Med/ high

Low

High

Tanche

France

Medium

Freestone

Low/ med

High

Low

Toffahi

Egypt

Large

Clingstone

High

Low

Low

a

Fruit size—category size depends on growing conditions. Parameters will vary with maturation state and growing conditions.

b

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PART | 1 General Aspects of Olives and Olive Oil

TABLE 2.2 Common natural table olive processing methods. Naturally processed table olives Anaerobic fermentation

Kalamata-style traditional (short) or modern (long)

Salt dried

Heat dried

Green-ripe Turning color Naturally black ripe olives (also aerobic)

Naturally black olives

Overripe black olives

Green Turning color or naturally black olives

Spray rinse olives with potable water

Spray rinse olives with potable water

Spray rinse olives with potable water

Spray rinse olives with potable water

Size grade

Size grade

Size grade (optional)

Size grade (optional)

Whole olives (slit, crushed, or bruised as required)

Whole olives (slit—common procedure)

Blanch olives with hot water

Place in brine to ferment

Multiple daily rinsing with potable water (short method) or place in brine to ferment (long method)

Add dry salt to whole olives Complete or partial dehydration of olives Rinse in water then olive oil (optional)

Allow complete or partial fermentation Anaerobic - green, TC or black. Aerobic - black

Cease daily rinsing when olives are palatable (short method) or allow complete or partial anaerobic or aerobic fermentation (long method)

Soak olives in brine for a short period or add dry salt (optional) Oven dry at 50 C Complete or partial dehydration of olives

Aerate black olives to darken (optional) Remove defective olives Size grade

Remove defective olives Size grade

Pack under chemical conditions and preservatives (especially for green olives), or hot pack or pasteurize

Pack in vinegar brine with olive oil

Pack in containers without added brine (vacuum or modified atmosphere)

Pack in containers without added brine (vacuum or modified atmosphere)

Hot pack or pasteurize

Pasteurize (regular or tunnel)

Pasteurize (regular or tunnel)

followed by multiple daily changes until the olives have lost their bitterness. The term archaic reflects an ancient primitive processing method. During soaking, debittering of the raw olives occurs because water-soluble polyphenols, including oleuropein and others, diffuse into the soaking medium, hence reducing their levels in the olive flesh. There are drawbacks during processing olives by this method, such as olive fruit tends to darken as a consequence of polyphenol oxidation; excessive number of washing cycles may give rise to tissue softening and loss of texture due to changes polysaccharides and fiber; increased risk of microbiological spoilage because flesh pH is more than 4.5; loss of beneficial water-soluble beneficial compounds, such as antioxidants (polyphenols), polyols, vitamins, and minerals; and utilization of large volumes of soaking medium. As the olives debittered by this method lack aroma and flavor, they are mostly

packed in acid brine, aromatized with herbs, spices, or other condiments, including olive oil and vinegar. Suggested cultivars are Verdale, Konservolia, Kalamon, Gemlik, Carolea, Manzanilla as well as those related to Gordal Sevillana. Crushed, cracked, or bruised olives, preferably clingstone cultivars (Table 2.1), processed by this method are very popular for producing home-made cracked or bruised olives. It is also carried out at industrial scale with seasoned olives of the cvs. Ogliarola Salentina (Italy), Aloren˜a (Spain), Mac¸anilha Algarvia (Portugal).6
8 For example, the Alcaparra olives, originating in Portugal, are prepared with split green-ripe olives of cultivars such as Cobranc¸oso, Madural, Negrinha de Freixo, Santalhana, and Transmontana. Further information on cultivar suitability and olive style are presented in Table 2.3.

Naturally processed table olives, their preservation and uses Chapter | 2

19

TABLE 2.3 Table olive styles and suggested olive cultivars.4 Table olive style

Suggested cultivars

Greek-style black Naturally Black-ripe By fermentation or archaic method

Arauco, Ayvalik, Azapa, Azeradj, Barnea, Bosana, Buga, Carolea, Cassanese, Chalkidikis, C¸elebi, C¸ekiste, Che´toui, Cornicabra, Cucco, Empeltre, Erkence, Galega Vulgar, Gemlik, Gerboui, Giarraffa, Grossane, Hojiblanca, Itrana, Istrska belica, Kalinjot, Kalamon, Konservolia, Ladoelia, Lechı´n de Granada, Lechı´n de Sevilla, Memeli, Manzanilla de Sevilla, Manzanilla Caceren˜a, Mara, Mastoides, Memecik, Meski, Mission, Menara, Oblica, Picholine Languedoc, Picholine Marocaine, Picual, Rasi’i, Sigoise, Soury, Tanche (Olive de Nyons), Uslu, Verdale, Villalonga, and Zizula

Kalamata-style Naturally Black-ripe Short (soaking) and long method (fermentation)

Kalamon, Barouni, Leccino, Memecik, and Californian Mission

Natural green olives Green-ripe By fermentation or (Sicilian-style) or by archaic method

Aggezi shami, Alfafara, Amygdalolia, Ascolana Tenera, Arauco, Ayvalik (split), Azapa, Azeradj, Barnea, Barouni, Blanquette, Buga, Carolea, Carrasquenha, C¸ekiste, C ¸ elebi, Coratina, Cordovil de Serpa, Cornicabra, Cucco, Domat, Erkence, Frantoio, Gemlik, Gerboui, Giarraffa, Gordal Sevillana, Grimski-172, Hamed, Haouzia, Hojiblanca, Istrska belica, Itrana, Karolia Lesvou, Kadesh, Kaissy, Karydolia, Konservolia, Koroneiki, Ladoelia, Leccino, Lucques, Manzanilha Algarvia, Manzanilla de Sevilla, Manzanilla Caceren˜a, Mara, Memecik, Memeli, Meski, Mission (Californian), Morisca, Nikitski II, Nocellara del Belice, Nocellara Etnea, Menara, Nabali Baladi, Oblitza, Oliva di Cerignola, Picholine Languedoc, Picual, Pizz’e Carroga, Rasi’i, Redondal, Saloneque (split), Santa Caterina, Sant’Agostino, Sigoise, Souri, Toffahi, UC13A6 (Californian Queen), Vasilikada, Verdale, Villalonga, and Zizula

Turning color and cracked olives By fermentation or archaic method

Arbequina, Aglandau, Barnea, Edremit, Frantoio, Gaidourelia, Gordal Sevillana, Hojiblanca, Itrana, Kalamon, Kolybada, Manzanilla de Sevilla, Manzanilha Algarvia, Memecik, Picual, and Verdale

Naturally dried on tree

Cerasuol, Meski, Tanche, and Thrubolea

Heat dried Mainly naturally black-ripe

Arauco, Ascolano/a, Dolce Agogia, Majatica di Ferrandina, Pizz’e Carroga, Memecik, Sevillana (Olives of Criolla), Tonda di Cagliari and UC13A6 (Californian Queen)

Salt dried Mainly naturally black-ripe

Alats, Edremit, Kalamon, Thasitiki (Thrumba), Thrubolea Lesbos, Thrubolea Chios, Thrubolea Samos, Thrubolea, of Cyclades Islands, and Thrubolea of Ampadias Rethimnon

2.3.2 Natural table olive processing by fermentation Fermentation is an age-old method for debittering and preserving olives where lactobacilli and yeasts play major roles in the process. Placing raw olives at any maturation state in brine, usually 8%
10% w/v NaCl, initiates spontaneous fermentation due to endogenous lactic bacteria and yeasts closely associated with olive skin. Olives by fermentation are produced in most table olive
producing countries. Naturally fermented black-ripe olives are specifically called Greek-style black olives whereas fermented green-ripe olives are sometimes called Sicilian-style olives, but not to be confused with another Sicilian-style bright green olive, the alkali-treated Castelvetrano style. Fermented turning color olives are often used for cracked or bruised olives. During fermentation, debittering of raw olives is achieved through a number of mechanisms: diffusion of water-soluble

oleuropein from flesh into brine—hence lowering its level in the flesh; by weak hydrolysis of oleuropein and related compounds in the acid environment of the fermentation brine; and by β-glucosidase and esterase enzymes in olive flesh and from fermentative microorganisms.9,10 Fermentation is undertaken at around 25 C (20 C
30 C). Low-temperature starter cultures are of interest as at low temperatures, as experienced in some regions, fermentation is slow or nonexistent. High temperatures can promote growth of anomalous spoilage and/ or harmful microorganisms. Without temperature control, sodium chloride and pH levels are the principal influences during the fermentation period. Depending on salt concentration in brine, fermentation is carried out by lactic acid bacteria and/or yeasts. Although yeasts are always present at low salt levels (3%
6% w/v NaCl), here fermentation is mainly by lactic acid bacteria whereas at high salt levels ( . 8% w/v NaCl), it is predominantly by yeasts. Initial adjustment of the fermentation brine pH,

20

PART | 1 General Aspects of Olives and Olive Oil

with lactic or acetic acid to around pH 4.5, can also reduce the risk of food poisoning and microbial spoilage. Fermentation of naturally black-ripe table olives can also be undertaken under aerobic conditions. Air is injected through a central column in the fermenter to remove carbon dioxide produced by olive fruit and microorganisms.11 With the availability of oxygen (air), facultative anaerobes utilize aerobic respiration rather than anaerobic respiration. The end products by this method are much darker than that from anaerobic fermentation as well as differ in organoleptic characteristics. Natural green Sicilian table olives can be prepared from olives of Nocellara del Belice, Nocellara Etnea, Manzanilla, Brandofino, Castriciana, or Passalunara cvs. Natural black Itrana-style (Itrana cv.) olives protected designation of origin (PDO), traditionally known as Oliva nera di Gaeta, are an Italian natural table olive product from the Province of Latina. Raw green-ripe or turning color olives of Itrana cv. processed by fermentation known as Oliva Bianca di Itri are more tart and bitter than Gaeta olives and their kinesthetic characteristics such as hardness, fibrousness, and crunchiness are more noticeable.6 Kalamata-style olives are processed from naturally black-ripe olives of Kalamon cv. by the long method using natural fermentation. Once debittered, the olives are further embellished as for those prepared by the short method. Two styles of Spanish preparations of natural olives, Aceituna Aloren˜a de Ma´laga PDO and Aceituna de Mallorca PDO, have recognized appellations.12 There is current interest, especially at the industrial level, to add bacterial and/or yeast cultures to support more reliable and reproducible fermentation of natural table olives.13,14 One could argue that adding starter cultures is not in the spirit of natural processing table olive processing. However, IOC directives allow the use of starter cultures for table olives, including natural olives.15 In addition, the use of starter cultures is allowed for the production of organic food by European regulations.16 Once processed, the naturally black-ripe olives may be exposed to air to darken to a brown-black color. They are hot packed in acid brine, so that the packing brine or olive juice after osmotic balance has a final salt level of 6% w/v or more, a maximum pH of 4.3, and minimum acid of 0.3% as lactic acid.15 To reduce salt load, they can be packed in acid brine with 2%
3% w/v NaCl, with a maximum pH of 4.3 and minimum acid of 0.3% w/v as lactic acid then pasteurized with or without sodium sorbate to give a final sorbic acid level at equilibrium of 0.1% in the flesh. Quality table olives produced by fermentation should be firm, crisp, slightly bitter, and have retained some of olive flavor characteristics. Furthermore, aromatic products of bacterial and yeast fermentation add to the aroma and flavor of the olives. Generally, naturally black-ripe

olives, such as Greek-style, are less firm than those prepared from green-ripe olives due to the softening of the fruit during maturation. Nevertheless, Greek-style olives of the Konservolia cv., which are often soft and juicy, are very popular with consumers.

2.3.3 Natural table olive processing by partially dehydration Customarily, in the Mediterranean, raw olives have been debittered through partial dehydration: by allowing olive fruit to lose moisture while still on the olive tree, by using heat—sun or oven, or by layering olives in coarse salt. Drying lowers the water content of the flesh and decreases the water activity of the olives, hence aiding in their preservation. Partially dehydrated olives are usually wrinkled and dark brown to black in color. Colloquially, Greek salt-dried olives have been called Thrubolea, Thrumba-style olives, Throumbes, Alatsolies, staphyloelies, shriveled olives, raisin olives, date olives, and Kalahari olives.4 Once debittered, partially dehydrated olives can be rinsed in vinegar then extra-virgin olive oil before consumption. Olives of this category when exposed to air are prone to oxidation leading to rancidity, an unacceptable defect.

2.3.3.1 Partial dehydration of olives while on the tree Overripe black olives of some cultivars Thrubolea (Greece), Hurma (Turkey), Dhokar (Tunisia), Passulunastyle cv. Cerasuola (Sicily), Kalamon cv. (Greece), Tanche cv. (France), and Leccino (Italy) can be eaten directly off the tree because of low levels of bitterness, due to reduced amounts of oleuropein and related compounds in the flesh. Thrumba-style olives are prepared from Thrubolea cv. growing on the Greek Aegean islands, Crete, and some localities of Attica. Originally debittering of these olives was thought to be due to fungal activity, but more than likely oleuropein and similar glycosides degrade through enzyme activity during ripening and overripening. A small quantity of coarse salt is added to enhance taste as well as improve preservation. Of interest is that this type of olive still has residual amounts of oleuropein.17 A similar product to Thrumba-style olives is Hurma olives produced in Turkey. They are the sweet end product of raw olives from trees of the Erkence cv. grown in a particular area of the Karaburun Peninsula. These olives are dark brown in color that naturally shrivel and debitter while on the tree. The current view is that debittering may be due to enzymatic oxidation of oleuropein.18

Naturally processed table olives, their preservation and uses Chapter | 2

2.3.3.2 Partial dehydration of olives by heating Raw olives at all maturation states can be partially dehydrated when exposed to sunlight (heat) and natural air flow (evaporation). They can also be dehydrated in lowtemperature ovens, for example, 40 C
50 C with little loss of quality. Ferrandina-style olives are a well-recognized olive in Italy produced by heat drying raw olives of Majatica di Ferrandina cv. The olives are blanched by dipping them in very hot water (95 C) for a few minutes, to disrupt their skin, followed by storage in salt brine (7%
8% w/v) for a few days, that adds salt and helps debittering, then oven dried at 50 C for 1
2 days until their moisture content is around 10% w/w and water activity is around 0.7.19 A similar product to Ferrandina olives is Oinotria where naturally black-ripe olives of Cassanese cv. are used.20 There are also at least two methods for preparing Sardinian Scabbucci olives. In common is the use of ripe olives of cvs. Tonda di Cagliari or Pizz’e Carroga. With the first method, slit olives are placed in brine for a few days, washed with potable water, blanched with vinegarwater, and then sun dried. After frying with garlic and parsley, they are placed in olive oil. With the second variant, slit olives are placed in a container, covered with coarse salt, and mixed occasionally and let stand for a few days to sweeten. Boiling water is poured over the olives and stand for a few more days. The liquid is then drained and the olives covered with olive oil. Then garlic, parsley, and wine-vinegar are added. Peruvian Botija olives are prepared from green or black raw olives of an ancient cultivar possibly Sevillana cv. where they are allowed to undergo lacto-fermentation. Once debittered, they are partially dehydrated by application of heat. The final product is soft and moist with a tart and salty taste. In Azerbaijan, naturally black-ripe olives of cvs. Gara zytun and Shigin zytun cvs. are chilled, then dehydrated at 30 C
40 C until moisture levels of the olives reach 25%
30%. Heat-dried olives of the Domat and Gemlik cultivars are also very popular in Turkey.21

2.3.3.3 Partial dehydration of olives using dry salt Green-ripe, turning color, or naturally black-ripe olives from numerous cultivars can be used to prepare partially dehydrated olives. These include Megaritiki, Konservolia, Kalamata, Manzanilla, Barouni, Leccino, and Edremit. Naturally black-ripe olives, the most commonly processed, are left to fully ripen or overripen on the tree. The olives are harvested when some of the crop falls to the ground and those on the tree start to shrivel. Harvested olives are washed with pressurized water then either stored for a few days or processed immediately. Processing involves interlayering olives with course food

21

grade salt 150
200 g/kg raw olives.22 Processing time is 1
2 months, and the olives debitter due to enzymatic oxidation of oleuropein.23 When green-ripe olives of Manzanilla cv. are used, the final products are dark brown, wrinkled, and crisp. The only established limit for salt-dried olives is a minimum 8%
10% w/v salt in the olive juice.15,24

2.3.3.4 Partial dehydration of olives using microwaves A novel method for producing low salt-dried olives is to dry them using microwave technology. Drying times range from around 15 to 30 min with shorter drying times at lower microwave power. Polyphenol levels decrease by first order kinetics as microwave energy is increased without loss of quality.25,26 A short pre
salting period, as for Ferrandina-style olives, could give a more acceptable product for some consumers.

2.4 Secondary processing of natural table olives Once processed, the natural table olives, whole, cracked, or slit, can be marinated with herbs and spices. Fresh herbs and spices can be used in marinades when table olives are for immediate consumption. Otherwise quality food grade dried herbs, spices, olive oil, and vinegars can be used for packed aromatized olives. Larger olives can be destoned and sold as such or stuffed with pimento, nuts, anchovy, vegetables, or other suitable fillings. These additives provide extra phytonutrients, including polyphenols. However, making such additions can reduce the quality and shelf life of the final product. Processed olives are highly acceptable when used in food collations, pizzas, and olive breads and also used to prepare olive pastes and tapenades. Classic tapenades consisting of ground olives, capers, anchovy, garlic, olive oil, and lemon juice are eaten as a spread on toast or crackers. Olive paste, that is, ground olive flesh of naturally processed olives, is used as a cooking condiment.

2.5 Preservation and storage methods for naturally processed table olives All table olive products must comply with accepted food standards. They should be preserved in a manner that the presence of pathogens and toxins are minimized, and the products are safe to consume. Identified HACCP control points for natural table olive production include use of potable water; management of physical hazards—stones, glass fragments, and foreign materials; management of additives and food grade processing inputs; management of

22

PART | 1 General Aspects of Olives and Olive Oil

microbiological hazards—spoilage organisms (e.g., coliform bacteria, Clostridium butyricum); food poisoning risks (e.g., Salmonella, Escherichia coli, Cryptosporidium); and toxinproducing microorganisms (e.g., Clostridium botulinum, Staphylococcus aureus).

2.5.1 Bulk preservation of natural table olives Bulk quantities of natural olives can be preserved through a balance of pH, salt, and acid levels. Specifically, the olives are held in acid brine with a minimum 6% w/v sodium chloride, maximum pH of 4.3, and minimum acid level as lactic acid 0.3% w/v after osmotic balance.15 The holding solution can either be a fermentation brine, a new brine, or a combination of the two adjusted to the parameters mentioned earlier. Reusing fermentation brines is an environmentally responsible action. These bulk holding brines do not support the growth of pathogens,27,28 but some residual lactic acid bacteria and yeasts from fermentation may be present.15

2.5.2 Natural table olives in consumer packs Natural table olives are also presented and sold in consumer packs made of glass, plastic, cans, multilaminated pouches, or polyethylene bags packed in brine or modified atmospheres. The olives are removed from bulk containers, rinsed in potable water with or without prior washing in disinfectant, and then packed in filtered holding brine or a freshly prepared brine. Brine from fermenters is cloudy as it contains particulate matter, including residual bacteria and yeasts. Particles are removed initially through a coarse filter (5 μm) then a polishing filter (1 μm). At this point, as long as the processed olives are packed in acid/brine with the chemical characteristics indicated earlier, they are safely preserved. When an olive pack is opened, a layer of oxidative yeast can emerge at the air
brine interface after several days, reducing the acidity of the packing solution inducing malodourous spoilage. To prevent yeast growth the antifungal agent sorbic acid is added to the packing solution. A low level (0.025% w/v) of sorbic acid is helpful for around 1 week, whereas levels of 0.05%
0.1% w/v, at equilibrium, prevent the problem for several months. Sorbate does not alter the physiochemical characteristics of the packing solution or olive flavor. However, slight browning of green olives may occur in transparent containers exposed to light.

2.5.3 Preservation of natural table olives with heat treatment To overcome microbial problems with natural table olives especially at low salt levels, for example, 2%
3% w/v,

heat treatments such as pasteurization or hot bottling are used. Such treatments reduce the majority of vegetative organisms, minimize the risk of food borne diseases, and increase the shelf life of the product. Heat treatments may negatively affect color and texture of some types of natural table olives, but these changes may have to be accepted to ensure microbial safety and stability.

2.5.3.1 Preservation of natural table olives by pasteurization In the past, olives in consumer size packs were preserved only by their physicochemical characteristics that required low pH 4.3 or less, acidity of 0.3% w/v as lactic acid, and high salt levels of 6% w/v or more. However, at low salt levels, unacceptable brine pH values of 3.2
3.3 would be required to ensure no microbial growth. As consumers and health alerts are now focusing on lower salt levels, low salt olives need to be pasteurized. The reference microorganism for pasteurization is propionic bacteria as this bacterium has the greatest heat resistance of all potential microbiota. In principle, packed olives are placed in a water bath or retort, and when the olives have reached 62.4 C, they are maintained at that temperature for 15 min, that is, 15 pasteurization units. From a practical perspective, pasteurization is undertaken in a water bath at 80 C
90 C. A steam retort or tunnel pasteurizer can also be used.

2.5.3.2 Nonthermal pasteurization of natural table olives Nonthermal preservation methods have been investigated with table olives, including high hydrostatic pressure (HHP) cold pasteurization that has the potential to reduce the risk of spoilage and food poisoning while maintaining the organoleptic attributes of table olives.7 Furthermore, HHP has been demonstrated to be more effective than thermal pasteurization in decreasing yeast, mold, and mycotoxin levels as well as lengthening the shelf life of table olives.29 Green fermented table olives cv. Chalkidiki, subjected to HHP technology (400
500 MPa) for 15 or 30 min, revealed that only 500 MPa for 30 min was effective in lowering the olive microbiota below their detection limit after 5 months storage at 20 C without loss of quality except for minor degradation of color.30 However, this technology is not yet available for table olive packing, and the cost is very high in comparison with heat pasteurization.

2.6 Nutritional and health-related aspects of table olives Table olives and olive oil are classic components of the Mediterranean diet where they are consumed daily. Research has demonstrated that the Mediterranean diet reduces the risk

Naturally processed table olives, their preservation and uses Chapter | 2

of heart disease, some cancers, and degenerative diseases such as Parkinson’s and Alzheimer’s. Here, unsaturated fats—monounsaturated and polyunsaturated—antioxidants, and bioactive phytochemicals characteristic of olive oil and table olives are believed to play a significant role. Evidence from in vitro studies and animal models has provided some insights. Of interest is a recent randomized study with healthy volunteers, where they consumed natural green table olives that demonstrated antioxidant effects together with reduced fat mass and an increase in muscle mass.31 Even though there are many different types of table olive products, all natural table olives contain significant amounts of healthy fat, essential amino acids, vitamins, especially vitamin E, and sometimes ascorbic acid, minerals (phosphorous, iron, calcium, magnesium, potassium, sulfur), microelements (copper, zinc, manganese), fiber as well as phytonutrients such as polyphenols, phytosterols, squalene, and triterpenes. Where natural olives are salted, the level of sodium in the edible portion can be up 40 times greater by weight than the other minerals. The nutritional value of table olives can vary with the cultivar, maturation state, and processing method.32
34 Therefore, only generalized nutritional information can be given. For some types of natural olives, their energy value ranges from around 455 to 810 kJ/100 g of flesh with, dehydrated table olives . natural black olives . natural green olives.6 For protein levels, these range from trace amounts to 4.4% of flesh with dehydrated table olives . processed by natural fermentation.35 Polyphenols are considered to have antiinflammatory, antiallergic, antiatherogenic, antithrombotic, antibiotic, and antimutagenic effects as well modulate the human immune system. Fermented black olives rank highly as an important source of dietary polyphenols especially hydroxytyrosol. Based on European Food Safety Authority recommendations of 5 mg/day of hydroxytyrosol, 10 olives will make a significant contribution to the hydroxytyrosol pool. It is of interest that consumption of natural olives by ovariectomized rats prevented bone loss in these animals.36 Furthermore, consumption of natural olives has been correlated with plasma antioxidant status in humans, and a daily intake of natural green olives, 12 olives/day for 30 days, showed antiinflammatory and antioxidant effects.31,37 Vitamin E derivatives, especially α-tocopherol, are believed to play a role in the prevention of cardiovascular disease, atherosclerosis, and cancers. However, table olives have much less of these compounds than corresponding olive oils. Carotenoids pigments present in table olives are mainly β-carotene, lutein, and zeaxanthin. As they protect cells against oxidative damage, collectively carotenoids decrease the risk of cardiovascular disease, numerous cancers, agerelated macular degeneration, and photosensitivity associated with UV exposure. Consuming phytosterols can help reduce blood cholesterol by inhibiting its absorption. Although

23

phytosterols levels are low in table olives, they supplement other dietary phytosterols, including those from olive oils and commercially fortified foods, especially margarines. Table olives represent the highest dietary source of triterpenoids, including triterpenic acids maslinic acid and oleanolic acid, at much higher levels than in corresponding olive oils. Natural processing does not impact the levels of triterpenoids ( . 2 g/kg of flesh) in the finished product as these water-insoluble compounds cannot leach out of the flesh.38,39 A number of preclinical studies have revealed evidence that terpenoids, similar to those found in table olives, or synthetic derivatives possess chemopreventive actions against various cancers, including skin, prostate, breast, and colon. Moreover, natural table olives are also a good source of dietary fiber (2
5 g/100 g of flesh) and contribute significantly to daily dietary fiber requirements. Naturally processed olives contain more fiber than those treated chemically.40 With respect to the salt content in olives, over consumption of salt (sodium chloride) is detrimental to health with respect to hypertension and cardiovascular diseases. Several strategies can be followed to reduce salt intake, including restricting the daily intake of olives; reducing the salt in packaged table olive products; or using alternatives to salt such as calcium, magnesium, and/or potassium chlorides. Based on 1000 mg/100 g olive flesh, with 10 medium size olives/day, sodium consumption would be around 300
400 mg/day. Processing and packing table olives in acidulated low salt brine are possible as long as a thermal step such as pasteurization or sterilization is used. Most natural table olives by fermentation unless pasteurized can already carry live organisms. Hence, natural table olives by fermentation, especially those that are not heat treated, are considered as a potential matrix for conveying probiotics through the human gastrointestinal tract.41 During fermentation such organisms, that is, lactobacilli and yeasts, are closely associated with olive skin. However, the challenge will be to find strains of bacteria and/or yeasts with measurable probiotic activity. Furthermore, packing and storing can affect the viability of probiotic microorganisms. Green olives of Chalkidiki cv. fermented with potential probiotic strains of lactobacilli and native microbiota, packed under modified atmospheres stored at 4 C and 20 C, revealed high percentages after 6 months at 4 C with lower survival rates after 1 year.42

2.7 Concluding remarks on natural table olives When promoting the Mediterranean diet, the contribution of nutritional and health benefits of table olive is often

24

PART | 1 General Aspects of Olives and Olive Oil

neglected or ignored where the virtues of extra-virgin olive are extolled. Overall natural table olives offer more. Major aspects are higher levels of antioxidants with wide range nutrients that do not transfer into olive oil during its extraction from raw olives. Of concern are the high salt levels in some natural olives that can be harmful to health. This can be overcome by using existing partial dehydration processes, for example, natural, sun, and heat drying; existing and novel preservation processes such as hot or cold pasteurization, respectively; less sodium chloride in brines in conjunction with pasteurization; or by the addition of alternative salts. As more and more consumers around the world are interested in reducing their red meat consumption for health, philosophical, and/or environmental reasons, table olives are valuable vegetarian component to a daily diet.

References 1 2 3 4

5 6 7

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Kailis S, Harris D. Producing Table Olives. Collingwood: Landlinks Press; 2007. Medina E, Brenes M, Romero C, et al. Main antimicrobial compounds in table olives. J Agric Food Chem. 2007;55:9817
9823. Bartolini G, Prevost G, Messeri C, et al. Olive Germplasm: Cultivars and Worldwide Collections. Rome: FAO; 1998. Kailis S, Kiritsakis A. Table olives: processing, nutritional, and health implications. In: Kiritsakis A, Shahidi F, eds. Olives and Olive Oil as Functional Foods: Bioactivity, Chemistry and Processing. West Sussex: Wiley; 2017:295
324. Garcı´a P, Brenes M, Romero C, et al. Respiration and physicochemical changes in harvested olive fruits. J Hort Sci. 1995;70:925
933. Lanza B. In: Muzzalupo I, ed. Nutritional and Sensory Quality of Table Olives. IntechOpen.com; 2012. Chapter 16. Abriouel H, Benomar N, Ga´nvez A, et al. Preservation of Manzanilla Aloren˜a cracked green table olives by high hydrostatic pressure treatments singly or in combination with natural antimicrobials. LWT—Food Sci Technol. 2014;56:427
431. Mateus T, Santo D, Sau´ de C, et al. The effect of NaCl reduction in the microbiological quality of cracked green table olives of the Mac¸anilha Algarvia cultivar. Int J Food Microbiol. 2016;218:57
65. Ramı´rez E, Brenes M, Garcı´a P, et al. Oleuropein hydrolysis in natural green olives: importance of the endogenous enzymes. Food Chem. 2016;206:204
209. Marsilio V, Lanza B, Pozzi N. Progress in table olives debittering: degradation in vitro of oleuropein and its derivatives by Lactobacillus plantarum. J Am Oil Chem Soc. 1996;73:593
597. Garcı´a P, Dura´n MC, Garrido A. Aerobic fermentation of natural black olives in brine. Grasas Aceites. 1985;36:14
20. Romero C, Garcı´a P, Medina E, et al. The PDO and PGI table olives in Spain. Eur J Lipid Sci Technol. 2019;121:1800136. Panagou EA, Tassou C, Katsaboxakis CZ. Induced lactic acid fermentation of untreated green olives of the Conservolea cultivar by Lactobacillus pentosus. J Sci Food Agric. 2003;83:667
674. Servili M, Settanni L, Veneziani SE, et al. The use of Lactobacillus pentosus 1MO to shorten the debittering process time of black

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table olives (cv. Itrana and Leccino): a pilot-scale application. J Agric Food Chem. 2006;54:3869
3875. International Olive Council (IOC). Unified Quality Standard Applicable for Table Olives in International Trade. Madrid: IOC; 2004. European Commission. Council Regulation (EC) no 889/2008 on organic production and labelling of organic products with regard to organic production, labelling and control. Off J Eur Union. 2008; L250:1
84. Zoidou E, Melliou E, Gikas E. Identification of Throuba Thassos, a traditional Greek table olive variety, as a nutritional rich source of oleuropein. J Agric Food Chem. 2010;58:46
50. Susamci E, Romero C, Tuncay C, et al. An explanation for the natural debittering of Hurma olives during ripening on the tree. Grasas Aceites. 2017;68:e182. Marsilio V, Lanza B, Campestre C, et al. Oven-dried table olives: textural properties as related to pectic composition. J Sci Food Agric. 2000;80:1271
1276. Borzillo A, Iannotta N, Uccella N. Oinotria table olives: quality evaluation during ripening and processing by biomolecular components. Eur Food Res Technol. 2000;212:113
121. Ongen G, Sargin S, Tetik D, et al. Hot air drying of green table olives. Food Technol Biotechnol. 2005;43:181
187. Panagou EZ, Tassou C, Katsaboxakis KZ. Microbiological, physicochemical and organoleptic changes in dry-salted olives of Thassos variety stored under different modified atmospheres at 4 and 20 C. Int J Food Sci Technol. 2002;37:635
641. Ramı´rez E, Garcı´a-Garcı´a P, De Castro A, et al. Debittering of black dry-salted olives. Eur J Lipid Sci Technol. 2013;115:1319
1324. Codex Alimentarius. Codex standard for table olives. CODEX STAN 66-1981. 2013. Mahdhaoui B, Mechlouch RF, Mahjoubi A, et al. Microwave drying kinetics of olive fruit (Olea europaea L.). Int Food Res J. 2014;21:67
72. Icer F, Baysal I, Tastan O, et al. Microwave drying of black olive slices: effects on total phenolic contents and colour. GIDA J Food. 2014;39:323
330. Grounta A, Nychas GJN, Panagou EZ. Survival of food-borne pathogens on natural black table olives after post-processing contamination. Int J Food Microbiol. 2013;161:197
202. Medina E, Brenes M, Romero C, et al. Survival of foodborne pathogenic bacteria in table olive brines. Food Control. 2013;34:719
724. Tokusoglu O, Alpas H, Bozoglu F. High hydrostatic pressure effects on mold flora, citrinin mycotoxin, hydroxytyrosol, oleuropein phenolics and antioxidant activity of black table olives. Innov Food Sci Emerg Tecnnol. 2010;11:250
258. Argyri AA, Panagou EZ, Nychas GJE, et al. Nonthermal pasteurization of fermented green table olives by means of high hydrostatic pressure processing. Biomed Res Int. 2014;2014:515623. Accardi G, Aiello A, Gargano V, et al. Nutraceutical effects of table green olives: a pilot study with Nocellara del Belice olives. Immun Ageing. 2016;13:11. Alagna F, Mariotti R, Panara F, et al. Olive phenolic compounds: metabolic and transcriptional profiling during fruit development. BMC Plant Biol. 2012;12:162. Romero C, Brenes M, Yousfi K, et al. Effect of cultivar and processing method on the contents of polyphenols in table olives. J Agric Food Chem. 2004;52:479
484.

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¨ nal K, Nergiz C. The effect of table olive preparing methods and 34 U storage on the composition and nutritive value of olives. Grasas Aceites. 2003;54:71
76. 35 Lo´pez A, Garrido A, Montan˜o A. Proteins and amino acids in table olives: relationship to processing and commercial presentation. Ital J Food Sci. 2007;19:217
228. 36 Puel C, Mardon J, Kati-Coulibali S, et al. Black Lucques olives prevented bone loss caused by ovariectomy and talc granulomatosis in rats. Br J Nutr. 2007;97:1012
1020. 37 Kountouri AM, Mylona A, Kaliora AC, et al. Bioavailability of the phenolic compounds of the fruits (drupes) of Olea europaea (olives): impact on plasma antioxidant status in humans. Phytomedicine. 2007;14:659
667. 38 Romero C, Garcı´a A, Medina E, et al. Triterpenic acids in table olives. Food Chem. 2010;118:670
674.

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39 Alexandraki V, Georgalaki M, Papadimitriou K, et al. Determination of triterpenic acids in natural and alkaline-treated Greek table olives throughout the fermentation process. LWT— Food Sci Technol. 2014;58:609
613. 40 Lo´pez A, Jime´nez A, Garcı´a P, et al. Multivariate analysis for the evaluation of fiber, sugars and organic acids in commercial presentations of table olives. J Agric Food Chem. 2007;55:10803
10811. 41 Lavermicocca P, Valerio F, Lonigro SL, et al. Study of adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food. Appl Environ Microbiol. 2005;71:4233
4240. 42 Argyri AA, Nisiotou AA, Pramateftaki P, et al. Preservation of green table olives fermented with lactic acid bacteria with probiotic potential under modified atmosphere packaging. LWT—Food Sci Technol. 2015;62:783
790.

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Chapter 3

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Samanta Zelasco1, Fabrizio Carbone1, Luca Lombardo2,3 and Amelia Salimonti1 1

Council for Agricultural Research and Economics, Research Centre for Olive, Fruit and Citrus Crops, Rende, Italy, 2Center Agriculture Food

Environment (C3A), University of Trento, Trento, Italy, 3Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy

Abbreviations 3,4-DHPEAEDA 4CL ACP AFLP AM ANS ATPase C4H CH3 CHI CHS cM CREA-OFA DArT DFR ESTs EVOO F30 50 H F3H F30 H FADS2 FLS G3E Gb GBS GGPS GO GWAS hsa IFAPA IOC

3,4-DHPEA-elenolic acid dialdehyde 4-coumarate-CoA ligase acyl carrier protein amplified fragment length polymorphism association mapping anthocyanidin synthase adenosine triphosphatase cinnamate 4-hydroxylase coumarate 3-hydroxylase chalcone isomerase chalcone synthase centimorgan Agricultural Research and Economics, Research Centre for Olive, Fruit and Citrus Crops diversity arrays technology dihydroflavonol 4-reductase expressed sequence tags extra-virgin olive oil flavonol 30 50 -hydrogenase flavanone 3-hydroxylase flavonol 30 -hydrogenase fatty acid desaturases 2 flavonol synthase genotype 3 environment gigabase genotyping by sequencing geranylgeranyl pyrophosphate synthase gene ontology genome-wide association study homo sapiens Centre of the Agricultural, Fishery, Food and Organic Farming Research and Training Institute International Olive Oil Council

ISSR kb LD LG LTR LUS MAS MEP miRNAs mRNAs MUFAs MYB MYC N NGS OeCDPMES OeDH OeDXR OeDXS OeFPPS OeG10H OeGES OeHMBPPR OeHMBPPS OeLUPS OeMECPS OeSQS oeu PAL PPO

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00017-1 © 2021 Elsevier Inc. All rights reserved.

intersimple sequence repeat kilobase linkage disequilibrium linkage group long terminal repeat lupeol synthase marker-assisted selection 2-C-methyl-D-erythritol 4-phosphate micro-RNAs messanger RNAs monounsaturated fatty acids myeloblastosis myelocytomatosis nucleotide next-generation sequencing Olea europaea 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase Olea europaea arogenate dehydrogenase Olea europaea 1-deoxy-D-xylulose 5-phosphate reductoisomerase Olea europaea 1-deoxy-D-xylulose-5-phosphate synthase Olea europaea farnesyl diphosphate synthase Olea europaea geraniol-10-hydroxylase Olea europaea geraniol synthase Olea europaea 4-hydroxy-3-methylbut-2-enyl diphosphate reductase Olea europaea hydroxy-2-methyl-2-(E)-butenyl 4diphosphate synthase Olea europaea lupeol synthase Olea europaea 2-C-methyl-D-erythritol 2,4-cyclo-PP synthase Olea europaea squalene synthase Olea europaea phenylalanine ammonia lyase polyphenoloxidase

27

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PART | 1 General Aspects of Olives and Olive Oil

PRX PUFAs QTL RAPD RFLP SCAR SCPL SFAs siRNAs SNPs snRNA SSRs SUT1 TAGs TEs TFs UFGT UTR β-GLU

peroxidases polyunsaturated fatty acids quantitative trait loci random amplification of polymorphic DNA restriction fragment length polymorphism sequence-characterized amplified regions serine carboxypeptidase-like saturated fatty acids short interfering RNAs single nucleotide polymorphisms small nuclear RNA simple sequence repeats sucrose transporter 1 triacylglycerols transposable elements transcription factors UDP-glucose: anthocyanin: flavonoid glucosyltransferase untranslated region β-glucosidase

3.1 Origin, diffusion, and genetic resources Olive (Olea europaea L. subsp. europaea var. europaea) is the oldest tree crop in the Mediterranean basin, and its diffusion has been strongly influenced by geographic barriers, historical events, and climatic fluctuations that are still ongoing. Olive is a species characterized by phenotypic plasticity and wide genetic variability allowing it to survive under different and complex agroecological conditions.1 In fact, the most recent taxonomic review indicates that the Olea genus is considered to include 33 species and 9 subspecies, 6 of them (forming the Olea subsection) showing a diffusion under different climate conditions such as the Mediterranean basin (subsp. europaea), Macaronesia (subsp. cerasiformis and guanchica), Morocco (subsp. maroccana), Saharan mountains (subsp. laperrinei), and from South Africa to South Asia (subsp. cuspidata).2
4 The subspecies europaea includes two botanical varieties: cultivated olive (var. europaea) and its wild relative, usually named oleaster (var. sylvestris).4 A strict relationship has been recognized between cultivated and wild olives, and the latter one is currently considered the main wild ancestor of the cultivated olive based on similar morphology and ecological requirements and ploidy level.4,5 Although the olive tree is considered the icon of the Mediterranean area, its phylogeny has long been discussed. While its native origin in the Mediterranean basin has been definitively demonstrated, it still remains open where domestication took place.5,6 The double nature of the olive tree as a wild element of the Mediterranean vegetation and cultivated plant has caused confusion in researchers who have attempted to study its domestication. In fact, very often we are faced

with feral forms that derive from a hybridization between the cultivated and the wild olive trees.7 Molecular studies alongside fossil studies have highlighted how the ancestral of the cultivated olive tree was probably present in the Mediterranean area about 5 or 6 million years ago: three distantly related plastid DNA lineages have been identified, the divergence of which dates back probably in the late Pliocene or early Pleistocene with the establishment of the Mediterranean climate.7,8 To date, populations of wild olive trees are present in the eastern and western areas of the Mediterranean basin but with a clear genetic differentiation that shows the effect of distances and geographical isolation. Studies performed with nuclear markers substantially confirmed this differentiated gene pool, but the initial genetic configuration has been considerably altered by the gene flow due to the introgression of cultivated olive tree toward the wild olive, especially in the central and western parts of the Mediterranean area. These studies have also shown that the cultivated olive tree mainly derives from the eastern gene pool of the oleaster confirming most of the hypotheses believing the olive domestication occurred in the eastern Mediterranean basin.7 This hypothesis was furthermore corroborated by recent results obtained by GrosBalthazard et al.9 where more than 500,000 SNPs deriving from an RNA sequencing of 66 cultivated and wild olive trees were used for a deep genotyping in order to reveal the olive tree origin and genomic changes associated with its domestication. Although olive tree is clonally propagated, what is an irrefutable evidence is that the current pattern of genetic diversity of the olive tree is strongly influenced by introgression between olive tree cultivated and the wild one, a process that is still in progress and that makes the olive genetic resources almost intact and in constant evolution.10,11 Besides, some authors12,13 studying the polymorphism within single-copy genes showed affinity of cultivated olive with subsp. cuspidata and cerasiformis, confirming an introgression also among subspecies. Actually, this pattern complicates the management of genetic resources in olive trees, which is further aggravated by the presence of numerous cases of synonymy.14
16 More than 2600 varieties have been described,17,18 but still the whole framework is not considered exhaustive. From an analysis of the geographical origins of all the varieties present in the international OLEA database,19 Italy results to be the richest country with at least 730 putative unique varieties (Fig. 3.1) with a high genetic variability at local level,20
22 and the Council for Agricultural Research and Economics-Research Centre for Olive, Fruit and Citrus Crops (CREA-OFA) in Rende (Fig. 3.2) holds the richest collection of Italian germplasm. Analysis of the genetic structure of Italian varieties revealed a high number of admixed genotypes indicating how the Italian germplasm has accumulated high levels of

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FIGURE 3.1 The worldwide olive germplasm described in OLEA database. Data from Zelasco and Martire, unpublished data.

FIGURE 3.2 The CREA-OFA olive germplasm official collection. Data from Zelasco, unpublished picture.

variability over the centuries justified by the geographical position of the Italian peninsula, being in the center of the Mediterranean basin and considered a hybrid area. In addition, Italy is considered a diversification center for olive tree.22
24 The role of official olive tree collections

is crucial in the management of genetic resources and for breeding programs. The International Olive Council (IOC) in the True Healthy Olive Cultivars joint project with University of Cordoba has recently established the IOC network of germplasm banks where a survey of

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PART | 1 General Aspects of Olives and Olive Oil

international collections from 24 countries was conducted, and an attempting to standardize molecular characterization and phenotyping methodologies is currently in progress in order to solve the confusion existing for cultivars denomination, authenticate all the varieties, and promote certification protocols for international commercial purposes.11

3.2 Phenotypic variability and breeding programs for the olive oil quality improvement Olive oil is chemically composed of about 98%
99% of triacylglycerols (TAGs), the saponifiable fraction, mainly represented by monounsaturated fatty acids (MUFAs). For this reason the health effects of olive oil have been traditionally attributed to its high MUFA content, in particular, oleic acid; nevertheless, the minor components found in the unsaponifiable (nonpolar) and the soluble (polar) fraction are now recognized to confer important healthy characteristics.25 A great variability in the fatty acid composition of olive oils has been recorded. Olive oil contains for the most part (on average 75%) MUFAs, in the form of the omega-9 oleic acid (C18:1) described to range between 55% and 83%, and, to a minor extent, the omega-7 palmitoleic acid (C16:1) ranging from 0.3% to 3.5%, with traces of gadoleic/9-eicosenoic (C20:1 ω-11, ,0.4%) and heptadecenoic (C17:1, ,0.3%) acid. Furthermore, it has a modest content of polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). Among PUFAs, linoleic acid (C18:2 ω-6) has been reported to vary from 3.5% to 21% and alpha-linolenic (C18:3 ω-3) from 0% to 1.5%. Among SFAs, palmitic acid (C16:0) ranges between 7.5% and 20%, stearic acid (C18:0) between 0.5% and 5%, with only a marginal content of arachidic (C20:0, ,0.6%), heptadecanoic (C17:0, ,0.3%), behenic (C22:0; ,0.2%), lignoceric (C24:0; ,0.2%), and myristic (C14:0; ,0.03%) acids.26 The differences found are largely attributable to the genotype of the individual varieties. Accordingly, in a 7-year trial, the content of fatty acids in the oils obtained from 96 varieties cultivated in the CREA-OFA olive germplasm collection field ranged between 8.58% and 22.14% for palmitic acid, 1.21% and 3.37% for stearic acid, 49.32% and 83.37% for oleic acid, 3.64% and 25.57% for linoleic acid, and 0.40% and 1.36% for linolenic acid.27 The minor fraction is composed of substances responsible for the organoleptic qualities (color, odor, flavor, taste, and aftertaste), primarily present in the mature pulp and in the pits, which are dissolved in the oil via natural or technological processes, and the concentrations of which depend on the maturation stage and olive variety, pedo-climatic conditions, and management practices.28,29 These compounds, in increasing order of polarity, include hydrocarbons

(especially squalene and β-carotene), tocopherols, fatty alcohols, triterpenic alcohols, 4-methylesterols, sterols, triterpenic dialcohols, polar-colored pigments (chlorophylls and pheophitins), and polar phenols. The hydrocarbon squalene is the main component of the extra-virgin olive oil (EVOO) unsaponifiable fraction at a concentration ranging from 0.7 to 12 g/kg.30 Despite extraction technology and refining process may cause considerable reductions,31,32 squalene concentration is cultivar dependent, whereas its content in virgin olive oils from 28 olive varieties from the World Olive Germplasm Collection of Center of the Agricultural, Fishery, Food and Organic Farming Research and Training Institute in Cordoba ranged from 110 to 839 mg/100 g.33 Total phytosterol content in olive oil varies between 800 and 2600 ppm oil.34 The most abundant sterols are β-sitosterol (75%
90%), D5-avenasterol (5%
36%), and campesterol (3%); their level decreases during the storage of oil due to the increase of peroxides. Several factors influencing sterol composition and content in olive oil are the cultivar, crop year, and degree of fruit ripeness, storage time of fruits before oil extraction and the method of oil extraction.35 The main triterpenes present in EVOO are two hydroxyl pentacyclic triterpene acids (oleanolic and maslinic) and two dialcohols (uvaol and erythrodiol), the concentrations of which oscillate between 8.90 and 112.36 mg/kg.36 These compounds are mainly found in the epicarp of drupes, therefore, pomace olive oil—extracted from olive pomace after the first press with the use of solvents or other chemical processes—generally contains 10-fold higher concentration than EVOO.37 Olive oils from different cultivars, produced under the same conditions (extraction system, ripeness stage, and pedoclimatic and agronomic conditions), exhibited different amounts of total volatiles, ranging from 9 to 83 mg/kg.38 The phenolic component, mainly represented by phenolic alcohols (hydroxytyrosol and tyrosol), secoiridoids (oleuropein derivatives, oleacein, and oleocanthal), and lignans (e.g., pinoresinol), has attracted great attention in recent times because of the interesting biological properties of these molecules.39 The synergistic effect of these components makes olive oil a true functional food with unique characteristics, as indirectly confirmed by the less positive effects recorded with the consumption of monounsaturated fats from mixed plant and animal sources.40 In EVOO at least 36 structurally distinct phenolic compounds can be found.41,42 The great variability found in the phenolic composition of olive oils is strictly related to the olive harvesting time, oil extraction techniques, and quantification methodologies. Total phenolic content of an EVOO may range from 50 to B1000 mg/kg.43,44 Oleuropein is generally the most prominent phenolic compound in green olive fruits where it can reach 14% of dry matter,45 lowering during fruit maturation being slowly

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converted to elenolic acid glucoside and demethyloleuropein. In some varieties (e.g., Leccino), oleuropein can even fall to 0 when the olives are completely black.46 The decrease in the concentration of oleuropein and the increase in the concentration of hydroxytyrosol are furthermore a result of olive processing during oil production.47 In this regard the average content of oleuropein in the oil samples obtained from six Italian cultivars grown in different geographical areas of Calabria region ranged from 1 ppb to 11 ppm.48 Hydroxytyrosol in monovarietal oils has been reported to range between 0.36 and 41 mg/kg.49 The concentrations of oleocanthal in EVOOs have been reported to range from 8.3 (cv Taggiasca),50 to 87.9 (cv Cornicarba),51 to 92.8 mg/kg (cv Coratina),52 while oleacein content has been reported to vary between 11.5 mg/kg in the oil obtained from the cv Koroneiki53 and 253.9 mg/kg EVOOs from the Coratina variety.54 In addition, olive oil is generally characterized by high tocopherol content, ranging from 16355 to 141056 mg/kg in Spanish oils, from 2957 up to 80058 mg/kg in Italian oils, from 120 to 478 mg/kg in Tunisian oils,59 from 9860 to 56261 mg/kg in Greek oils, from 138 to 298 mg/kg in Portuguese oils,62 and from 97 to 403 mg/kg in Turkish oils.63 The main differences in olive oil composition, with particular regard to oleic/linoleic acid (C18:1/C18:2) ratio, triglyceride profile, and phenolic content,64
67 are attributable to the genetic peculiarities of the varieties of olive cultivated worldwide. In particular, Tous et al.68 and Uceda et al.,69 quantified that more than 70% of the variation in the fatty acid profile (apart from linolenic acid), phenolic composition, bitter index (K225), and oil stability was due to genetic effects. Furthermore, studies on olive oils from new olive obtained by crossbreeding selections showed a clear genotypic effect on the total variance of fatty acids70,71 and phenolic compounds.72,73 This makes the genetic improvement of olive for a better nutraceutical quality of the oil one of the principal goals of modern breeders. The review of the collections and the identification of true-to-type cultivars is a crucial step also for breeding programs and adopt strategies to mitigate climate changes, since they will allow to investigate the genotype by environment interaction useful both for the choice of more adapted varieties and/or parents1 and conduct association studies. Given the current poor knowledge on the genotype 3 environment (G 3 E) interaction and inheritance of olive characters due to the high intrinsic heterozygosity of the species, the choice of breeding programs should be based on highly adapted local germplasm cross-breeding programs and varieties that tend to be more self-fertile keeping a high-quality standard of the olive oil. Currently, a common effort is oriented toward the discovery, characterization, and creation of collections deriving from local spontaneous (wild and feral) and

31

monumental olive trees.74
78 The inclusion of this plant heritage within the genetic resources of olive trees contributes to expanding its biodiversity encouraging new breeding programs.33,34,79
82 For instance, Leo´n et al.74 conducted a diversity analysis on fatty acid composition (palmitic, palmitoleic, oleic, and linoleic acids) and minor components (tocopherols, sterols, and squalene) on wild genotypes, cultivars, parents, and progenies highlighted wide genetic diversity within and among groups. Most of the wild genotypes showed the highest mean content for C16:0 and C16:1 in contrast with much more lower values found in wild olives from Algeria83 and Tunisia.84 Furthermore, extreme values have been reported for subsp. cuspidata specimens from Kenya, with lower C18:1 content (up to 44.3%) and subsequently higher C18:2 content (up to 33.3%). Leo´n et al.74 found also the lowest average value for squalene increase the total tocopherol content by improving the antioxidant properties of the olive oil.78 The lowest average value for sterols was instead found in the progenies. In several cases the values of some oil components found in the wild olives exceeded or were below the threshold indicated for the commercial parameters established by IOC,85 defining the EVOO such as in the case of C16:0 content excluding them from olive oil production for commercial purposes. However, the inclusion of wild olive trees in breeding programs could help to increase the total tocopherol content by improving the antioxidant properties of the olive oil.74 Moreover, the enhancement of phenotypic screening of the olive germplasm allows to improve genomics applications such as association studies; increasing frequency and distribution of the sample by including a greater number of individuals with extreme values contributes to strengthening the correlation analysis between genotypic and phenotypic data. The olive growing can also benefit from cultivars of greater adaptability also in view of the ongoing climate change. A step forward was made thanks to a recent study by Mousavi et al.1 where G 3 E interaction of important characters was investigated. In this study, 14 varieties common to 6 different environments were studied for the variability of the following characteristics: fresh weight of the drupe, fruit moisture, oil content in the dry substance of the fruit, oleic acid, linoleic acid, palmitic acid, and oleic/ palmitic 1 linoleic acid ratio. Fruit moisture and oleic acid content were the most stable under the six environments considered confirming the high heritability of oleic acid as reported earlier, but with different degree of the trait plasticity among the cultivars. For instance, Picual and Koroneiki varieties widely cultivated especially in Spain and Greece, respectively, showed their maximum fitness for oleic acid (high and stable values) over all environments, while Moraiolo cultivar, a local Italian cultivar, expressed a major variability for almost all traits showing a reduced global adaptability.

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PART | 1 General Aspects of Olives and Olive Oil

3.3 Olive genomics as tool for olive oil quality improvement 3.3.1 Genome sequencing Conventional breeding in olive trees also presents another problem associated with the long generation time of the offspring resulting from controlled crosses. In olive the juvenile period is longer than in other fruit tree species, taking on average 12
13 years after germination.86,87 Forcing protocols have been performed leading to provide more than 80% of adult plants 4 years after planting in the field.88 However, they have not yet been widely tested. In the past decade the rapid evolution and widespread use of next-generation sequencing (NGS) techniques contributed to advancing in olive tree genomics knowledge although the biology complexity of the species and of its genome have significantly slowed the progress of research in this area in comparison with other crops, such as rice, tomato, or grapevine. In this contest, advanced genomic approaches are even more necessary for this species, which will contribute to a strong acceleration to olive breeding in the coming years. Olive tree shows a medium-sized (1.4
1.5 Gb), highly heterozygous, and repeat-rich nuclear genome, making its assembly a major challenge. Currently, three genomic sequences were released from wild89 and cultivated olives, the cultivars Farga and Picual.90,91 While for cultivated olive only the genome assembly was reported, a more information was produced in the wild olive where the author performed chromosome anchoring and a wide functional annotation too. Compared to the cultivated olive tree genome, the wild olive showed a larger genome size, although with a smaller number of genes. The assemblies were composed of about 1.4 Gb genomic sequences with an average scaffold N50 size of about 300 kb (230 and 440 kb, respectively), which is slightly larger than those from other Oleaceae plants, such as European ash (104 kb),92 but is much smaller than other plants with complex genomes, such as tea (920 kb).93,94 Moreover, compared to other model species, such as rice,95 the scaffold N50 sizes of the current assemblies are still somewhat unsatisfactory, due largely to the limitation imposed by the short-read length generated from NGS technology. In the future, additional efforts will be needed to further improve the quality and completeness of the olive genome assemblies, particularly with the upcoming advances in sequencing technologies (e.g., third-generation sequencing) and new algorithm-derived assemblers. In the wild and cultivated olive genomes, 50,684 and 56,349 protein-coding genes, respectively, were found. About 51% wild olive genome assembly was found to be composed of repetitive DNA, less than was found in the genome draft of Farga cultivar (63%).90 Among the most dominant transposable elements (TEs), long terminal repeat (LTR)

retrotransposons were found, independently representing 40.3% and 38.8% wild and cultivated olive genomes, respectively.89,96 Unver et al.89 conducted a near-complete representation and localization of genes, repeat elements, and small nuclear RNA (snRNA), as well as functional and metabolic annotations and an evolutionary analysis of olive oil biosynthesis genes. Several genes related to the lipids and secondary metabolites biosynthesis resulted significantly amplified and or contracted in the olive tree genome. It was observed that a large proportion of genes required for olive oil biosynthesis in oleaster has been maintained as duplicated genes, and most of them were shared with those from another oil-bearing crop, sesame (Sesamum indicum). However, the number of fatty acid metabolism genes was found to be significantly higher in sesame (n 5 164) than in oleaster (n 5 20), and this mechanism could contribute to explain how the sesame oil is a richer source of linoleic acid than olive oil. Besides, the concomitant contraction of gene families encoding degrading/catabolic enzymes (such as dehydrogenases and hydrolases) may also be responsible for the differential fatty acid accumulation in different oilbearing crops. Regard to secondary metabolism, interestingly, oleaster showed the highest number of coumarate 3-hydroxylase-encoding (CH3) genes, corresponding to 32% annotated genes of the phenylpropanoid pathway, in comparison with other sequenced plants. Moreover, other genes involved in the biosynthesis of secondary metabolites such as those of shikimate, carotenoid, terpene, and brassinosteroid signaling were identified in oleaster genome.

3.3.2 Molecular characterization, quantitative trait loci analysis, and association mapping studies A very consistent molecular characterization work has been carried out in the olive tree in traditionally cultivated area in the Mediterranean basin and several countries worldwide.11 More recently, a big effort was addressed toward solving the redundancies present in the collections97
100 using simple sequence repeats (SSRs) as molecular markers. These markers are characterized by a high polymorphism, a codominant inheritance, and a discriminating capacity superior to other molecular markers2 that make them particularly suitable for varietal characterization, development of genetic maps, and paternity analysis. However, several classes of markers have been and are still widely used in olive such as random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and inter-SSR (ISSR) markers.2 The main limitation of the SSRs is due to the fact that until recently, almost all the isolated SSRs were

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

characterized by repeated dinucleotide units that can cause stuttering phenomena and a consequent difficulty in allele sizing.14 New markers SSR from expressed sequence tags (ESTs) have recently been isolated.101
103 The SSRs represent currently a widespread and economic system for inter- and intravarietal molecular characterization also by virtue of the fact that a large part of the olive germplasm has been genotyped with these markers. Databases19,104 with reference allelic profiles and a set of markers selected for this purpose14 are available, allowing to compare SSR profiles from several authors, procedure that is particularly useful to identify true-to-type varieties.105 However, with the advent of NGS technology associated with the availability of a reference genome, the olive molecular characterization system is evolving toward SNP-producing systems such as genotyping by sequencing (GBS), genome resequencing, and transcriptomesequencing approaches. These markers have the advantage to be found throughout all the genomes, stable (i.e., are less mutable) and readily assayed using highthroughput genotyping protocols and automated data analysis.23 The GBS technology has also been adopted for the construction of genetic maps. Until a few years ago in olive trees, the most adopted genetic approach for the identification of regions or genes involved in the control of complex traits was the quantitative trait loci (QTL) analysis that provides for the establishment of a segregating biparental population where the recombination frequency among the markers used to genotype the population and between markers and genomic loci that influence the traits of interest are estimated.2 The olive tree is a highly heterozygous, self-incompatible species with long generation times, and therefore in order to adopt this approach, it is necessary an F1 map population where segregation is observed at the heterozygous loci for each parental taken separately. This approach, called pseudo-testcross, leads to the construction of two separate maps for each parent. The first genetic map was obtained in 2003106 through the use of 279 RAPDs, 304 AFLPs, some restriction fragment length polymorphism (RFLP) and SSR markers, and a progeny of 95 individuals deriving from the “Leccino” 3 “Dolce Agogia” cross. Two genetic maps were obtained covering 2765 cM and 22 major LGs in “Leccino” and 2445 cM and 27 major LGs in “Dolce Agogia,” respectively. The stearoyl-acyl carrier protein (ACP) desaturase encoding gene that converts stearic acid to oleic acid, isolated by Haralampidis et al.,107 was associated within group 4 of “Leccino.” However, the genome lengths were estimated to be 6465 and 6608 cM for “Leccino” and “Dolce Agogia,” respectively, demonstrating a low level of map coverage. Despite the progeny segregated for some characters, such as tree habit or ripening time, no QTLs were located on these maps.108 Subsequently, other genetic maps were

33

produced using a different numbers and types of markers such as AFLPs, ISSRs, RAPDs, sequence-characterized amplified regions (SCAR), SSRs, and diversity arrays technology (DArT),109
112 sometimes integrated with SSRs markers deriving from ESTs,113
115 but the saturation level shown was always medium low.2,116 Genetic maps with higher resolution capacity were obtained using GBS technology116,117 that led to the production of highdensity genetic maps passing from 400
600 markers previously used to over 5000 markers. When a map is saturated, the number of LGs corresponds to the haploid number of chromosomes which in the case of the olive tree is 23, and the addition of new markers should always result in an association within some LG. To date, the most advanced genetic map was obtained by Unver et al.89 using GBS technology, characterized by 23 LGs on which most of the 50,000 protein-coding genes that have been predicted could be anchored. GBS technique adopts methylation-sensitive restriction enzymes to reduce genome complexity, which allows recognition of low copy number genomic regions, including TEs and repeated regions that have not proliferated extensively.118 Its main limitation is precisely the random distribution of the sites recognized by restriction enzymes on the genome that are generally not found within coding sequences or having a functional meaning.119 The QTL analysis in olive trees is not too much suitable especially for complex traits related to maturity, although the recently tested forcing protocols could considerably shorten the juvenile phase.88 In particular, if the parameters related to the quality of the oil have to be assessed, it is not always easy to extract a sufficient quantity of oil from individual offspring if not after a certain number of years. All this could be overcome through the in vitro germination of the offspring embryos and their subsequent micropropagation.120 However, to date, some traits relating to characters related to the production and quality of the oil have been partially dissected. Atienza et al.121 used a genetic map previously developed by Dominguez-Garcia et al.112 for QTL mapping, analyzing 14 traits related to fruit characteristics (fruit size, pulp/stone ratio, and oil content) for 2 years. The authors detected 22 and 5 putative QTLs in “Arbequina” and “Picual” parents, respectively. The result of greater evidence in this work is given by the QTLs for the oil content, which were detected in both years in the same position in the LG of “Arbequina” maintaining similar differences in the allelic effect in the 2 years. These results indicated the existence of an important QTL for the oil content, explaining between 20% and 30% of the variation of the trait. Furthermore, the colocalization of the QTLs for the oil content, the pulp/kernel ratio, and the weight of the pulp, all negatively associated with other QTLs for the water content of the fruit, suggested that this region may be of great interest for the selection of

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PART | 1 General Aspects of Olives and Olive Oil

new oil and table cultivars. Five additional QTLs were detected in the map of “Picual.” Four of them clustered in LG 17 indicating the presence of an important QTL for fruit weight in this position, explaining around 12.7%
15.2% of the variability. The existence of alleles to increase the fruit size indicates the possibility to select genotypes better than “Picual” by combining the alleles from both parents. Recently, Herna´ndez et al.122 conducted a QTL analysis for fatty acid composition on the same mapping population described earlier121 but under only a single season. Two and eight QTLs were detected in “Picual” and “Arbequina” maps, respectively. A single QTL for the oleic acid was identified on LG 20 in Arbequina accounting for 41% of the phenotypic variance with a genotypic effect of 28.8, while a QTL for linoleic acid was colocated explaining 69.7% of the phenotypic variance with an allele effect of 7.9. Since the negative correlation between oleic and linoleic acid exists, this QTLs pattern indicates that allele increasing the linoleic acid content is inherited from “Arbequina.” At this resolution level of the genetic map, it was not possible to discern if a single segregating locus or clusters of linked QTLs independently affected the biosynthesis of oleic and linoleic acids. The authors individuated four QTLs for linolenic acid as well, two in “Picual” map on the LG 5 and 15, and two in “Arbequina” map on LG 14 and 19. The contribute to increase linolenic acid was from the “Arbequina” alleles, which explained about 15% of the phenotypic variation. A QTL for palmitoleic acid was found on the LG 13 in “Arbequina” map explaining 22.5% of phenotypic variance. Although the same genetic map was used, Herna´ndez et al.122 did not find any QTL for the acidic composition colocalized with those related to the oil content found by Atienza et al.121 This indicates how the two sections could be independent, and therefore the selection for the oil content would not affect the acidic composition and therefore the quality of the oil, allowing breeding programs aimed at improving both traits. A limit of the QTL analysis could be represented by the number of individuals in the population which, if low, can lead to an overestimation of the effect of the QTLs and to an underestimation of their number, since these parameters are strictly dependent on the size of the sample. An alternative to QTL analysis is represented by association mapping (AM) or linkage disequilibrium (LD) mapping. This approach increases the mapping resolution beyond the current capabilities of standard map populations, since this technique has the potential to identify a single polymorphism within a gene that is responsible for the differences of the phenotype. LD plays a fundamental role in association analysis and can be defined as the nonrandom association of alleles to different loci, loci that are not physically localized within a chromosome, but within an entire population. This association derives from the shared history of mutational and recombination events. All events that

drive a population away from the Hardy
Weinberg equilibrium, such as admixture, linkage, selection, contribute to influence LD levels.123 The LD analysis is the first step to undertake an association study because depending on how far it extends, the number and density of markers to be used will be defined. LD is strongly conditioned by the size of the population that can lead to the loss of rare allelic combinations due to genetic drift and this leads to an increase in LD levels. Generally, LD decays faster in outcrossing species because of the reproductive system, which promotes heterozygosity, and therefore recombination is more effective than highly homozygous self-pollinating individuals. The admixture also strongly influences the LD extent, as the phenomenon involves intermating and therefore favors the faster decay of LD. On the other hand, the presence of population stratification and an unbalanced distribution of alleles within these groups can result in spurious associations. Fortunately, the statistical methodologies developed allow us to estimate the effects of the genetic structure of the population on the extent of LD, making this technique widely accessible. Association analysis, therefore, correlates sequence polymorphisms with the variation of the phenotypic trait and is an extremely powerful tool for dissecting quantitative traits because the individuals that are sampled do not have to be closely related, which harnesses all of the meiotic and recombination events between those individuals to improve resolution. In fact, biparental populations are not used but natural or cultivated populations that must, however, include the greatest genetic variability of the species.123,124 In olive trees, this technique is considered to be the most suitable strategy given the biology of the species. Furthermore, the recurrent admixture between gene pools occurred during its varietal diversification process12 proven by the high degree of genetic diversity investigated between accessions124 encourages studies in this direction. Until a few years ago, genome sequence information was scarce; therefore the main association work concerned association studies by candidate gene. Researchers focused mainly on the biosynthetic fatty acid pathway for which regulation is known and where most of the genes were isolated in olive trees. Cultrera et al.12 scanned the level of variation within the ACP1, ACP2, sucrose transporter 1 (SUT1), and lupeole synthase (LUS) genes. The ACPs products are essential cofactors involved in the plastid biosynthesis of fatty acids and desaturation, as well as in other metabolic pathways. Both the genes have been characterized on a functional level showing that ACP1 increased the level of oleic and linolenic acids in tobacco, while ACP2 was mainly expressed during fruit development, suggesting its involvement in the biosynthesis of fatty acids. The SUT1 gene codes for a protein that has a central role in the transport of sucrose, while the LUS gene codes for an oxidosqualene cyclase involved in the production of a triterpene compound (Lupeol) capable of greatly influencing the health-giving properties of olive oil. The four genes were sequenced, and a

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

polymorphism analysis was conducted by comparing the gene sequences in 90 olive varieties. A correlation between SNPs and two phenotypic traits (fresh fruit weight and palmitoleic acid content) was also attempted, but no significant associations were detected. Another work has been conducted on 12 Tunisian varieties by Ben Ayed et al.,125 which identified two SNPs, FAD2.1, and FAD2.3 related to the content of oleic, linoleic, and palmitic acids on the fatty acid desaturases 2 (FADS2) gene. The homozygous (TT) genotype of FAD2.1 correlated positively with the content of oleic acid and negatively correlated with that of linoleic acid, while the homozygous (GG) genotype of FAD2.3 was negatively correlated with the content of oleic acid. Unfortunately, the study was conducted on a small population size and results obtained on only two varieties, “Chemlali de Sfax” and “Meski.” A stronger approach was adopted by Salimonti et al.126 who correlated polymorphisms of 50 untranslated region (50 UTR) intron of the FAD2-2 gene encoding a microsomal oleate desaturase127 with the variation of content of oleic and linoleic acids in 97 Italian olive varieties. The fatty acid composition was analyzed under 3 years, and the association study was conducted each year. This study highlighted how the compositional variability in the different olive varieties could be influenced by the presence of two SNPs (SNP23 and SNP26) in high LD, allocated in the 50 UTR-FAD2-2 intron, and how the heterozygous (CT) genotype for both the SNPs could have a superdominance effect in increasing the oleic acid content. However, as expected, being a polygenic character, the two SNPs explained low level of phenotypic variance ranging from 9% to 12% for SNP23 and 10% to 13% for SNP26 under 3 different years. The genetic structure analysis conducted on the 97 cultivars highlighted how all the cultivars from the Italian region, Abruzzo, showed a low oleic acid content clustering in the same group. A validation of these results through an analysis of gene expression is currently in progress but could help one to clarify the genetic
molecular basis of the natural variation of the acidic composition within the species, provide a useful marker for a marker-assisted selection (MAS) approach and produce editable alleles for genome editing approaches. Kaya et al.128 conducted a genome-wide association study (GWAS) as approach, using SNPs, AFLPs, and SSRs markers on 96 olive genotypes from diverse origins in relation to 5 important agronomic traits relevant to fruit and endocarp characteristics in olive finding 11 significant associations with traits related to endocarp and fruit. A few markers in association with traits such as stone weight and fruit weight were previously mapped by Khadari et al.,110 and a number of the associations identified in this study were located at loci where QTLs for the considered traits had already been reported113 showing as QTL and AM approaches can usefully integrate for the selection of regions of the genome involved in the control of agronomically important traits. However, the relatively high-cost and limited marker density

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of these methods led to the use of GBS approaches that provide low-cost, high-density genotype information.129 Only one GWAS has been conducted till today with GBS technology, which has provided a surprising number of high-quality SNPs (24977 SNPs) where even if a difference of the previous studies were used, both reference genomes currently available and 183 accessions of olive trees, greatly expanding the genetic variability that can be explored in olive trees.124 In almost all studies carried out so far by both candidate gene and GWAS, a low LD score has been identified,124,126,128 as expected, since olive is an outcrossing species. However, Cultrera et al.12 found a strong degree of association between the sites despite the high level of variation identified within the genes, even with an opposite trend, shared between coding regions and introns. The LD did not decay for almost the entire length of the fragment in the ACP genes and increased in the SUT1 gene forming “haplotype blocks.” A similar result was obtained during the study of the sequence variation of five single nuclear copy genes in native and invasive olive accessions where only one recombined haplotype was found.13 However, as reported earlier, the extent of the LD can vary in relation to the size and type of population used (natural vs nonnatural) and in olive trees, studies especially with the GWAS approach are still too scarce to define the extent of the LD. The critical point of this approach is the need to possibly have a more complete sequenced genome of olive trees cultivated to enhance the depth of genotyping and especially divided into chromosomes in order to identify the blocks of associated mutations (haplotypes).

3.3.3 Transcriptomics for olive oil quality In the last years an intensive use of ESTs, microarrays, large-scale gene expression (transcriptome) profiling, and associated informatics technologies has made possible to select a certain number of genes with a key role in several biological processes in olive. Table 3.1 shows large-scale gene expression studies published in the last 20 years for this species. Remarkably among these studies, those focusing on fruit development and ripening allowed to identify specific expression patterns of key genes involved in metabolic processes that determine the valuable chemical and sensory characteristics in olive oil. In fact, the specific biochemical programs resulting in fruit development and ripening phenomena carry to important changes as textural modification via alteration of cell turgor and cell wall structure and/or metabolism, generally enhanced susceptibility to opportunistic pathogens as well as modification of sugars, acids, and volatile profiles and alteration of chlorophyll, carotenoid, and/or flavonoid accumulation that affect nutritional quality, flavor, and aroma.

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PART | 1 General Aspects of Olives and Olive Oil

TABLE 3.1 Reports large-scale gene expression studies published in the last 20 years for olive (Olea europaea L.). Authors

Genotypes

Plant materials

Methodological approach

Aims of the work

Bruno et al., 2019

Olea europaea L. ssp. europaea, cv Carolea

Fruits harvested from three plant populations grown at different altitudes

Illumina HiSeq 2000 sequencer

To achieve information on the genetic networks controlling drupe ripening and their modulation in response to environmental conditions

Carbone et al., 2019

O. europaea L. ssp. europaea, cv Leucocarpa and cv Cassanese

Fruits harvested at two different ripening phases

Illumina HiSeq 2500 sequencer

Identification of specific miRNA involved in anthocyanin and flavonoid metabolism

Mougiou et al., 2018

O. europaea L. ssp. europaea, cv Koroneiki

Fruits harvested, for 4 consecutive years, in four stages of development

Illumina HiSeq 2000 sequencer

Identification of transcripts involved in the biosynthesis of secologanins and hydroxytyrosol; relative expression of the transcripts involved in the two biosynthetic pathways during fruit development

JimenezRuiz et al., 2018

O. europaea L. ssp. europaea, cv Arbequina

Seeds after two imbibition times, germinating embryos and seedlings

Illumina HiSeq 2000 sequencer

To elucidate the regulation of the transcriptomic response during the juvenile development period

LeyvaPerez et al., 2018

O. europaea L. ssp. europaea, cv Frantoio, tolerant to Verticillium dahliae and cv Picual, sensitive

Roots of plants inoculated with V. dahliae and sampled during the initial stages of infection

Illumina HiSeq 1000 sequencer

Identification of differences in the transcriptomic profile related to the susceptibility of a tolerant cultivar and one susceptible to infection with V. dahliae

JimenezRuiz et al., 2017

O. europaea L. ssp. europaea, cv Picual

Leaves and roots of plants, inoculated and not, with V. dahliae

Illumina HiSeq 1000 sequencer

Transcriptome analysis of plant
pathogen interaction (olive
V. dahliae)

Grasso et al., 2017

O. europaea L. ssp. europaea, cv Ortice, sensitive to Bactrocera oleae and cv Ruveia, tolerant

Drupes with fly sting, drupes with fly larvae, drupes with pupae, healthy drupes

454 GS FLX 1 Titanium sequencer

Study of the molecular response of drupes in two cultivars having different levels of tolerance to olive fly

Zafra et al., 2017

O. europaea L. ssp. europaea, cv Picual

Pistil, pollen, young leaves

SSH approach

To identify key genes involved in pollen and pistil functions, in order to elucidate the processes which happen during the pollen germination and the fertilization phase

Iaria et al., 2016

O. europaea L. ssp. europaea, cv Leucocarpa and Cassanese

Epicarpic and mesocarpic tissues of drupes

Illumina HiSeq 2000

To identify key genes involved in the fruit development and in the differential pattern of anthocyanins and flavonoids

Alagna et al., 2016

O. europaea L. ssp. europaea, cv Leccino and Dolce Agogia (selfincompatible) and Frantoio (self-compatible)

Flowers in different stages of development, from the differentiation of flower buds to the dehiscence of the anthers

454 GS FLX 1 Titanium sequencer

Transcriptome analysis of the flower with reference to the genetic basis that controls its development, sexual differentiation, the pollination process

Bazakos et al., 2015

O. europaea L. ssp. europaea, cv Kalamon

Leaves and roots from plants after salinity treatment.

454 GS FLX sequencer

Identification key genes for olive salt
response (Continued )

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

37

TABLE 3.1 (Continued) Authors

Genotypes

Plant materials

Methodological approach

Aims of the work

Guerra et al., 2015

O. europaea L. ssp. europaea, cv Leccino

Leaves

Illumina Truseq

Characterization of the molecular response to cold of leaf tissue

Carmona et al., 2015

O. europaea L. ssp. europaea, cv Picual

Ripe pollen and pistils at different stages of development, leaves and roots as a control.

454 GS FLX 1 Titanium sequencer, Sanger method

To study the transcriptome of the reproductive system

LeyvaPerez et al., 2015

O. europaea L. ssp. europaea, cv Picual

Cold-stressed leaves

Illumina HiSeq 1000 sequencer

To study the transcriptomic changes induced in leaves by above-freezing cold stress

MunozMerida et al., 2013

O. europaea L. ssp. europaea, cv Picual, Arbequina, Lechin de Sevilla, Picual 3 Arbequina

Mesocarp of the fruit and seeds in three stages of maturation, young stems and leaves, young, adult and dormant buds, young and adult roots

454 GS FLX 1 Titanium Sequencer, 454 GS FLX Sequencer, Sanger method

Assembly and functional annotation of 12 cDNA libraries from various tissues and different stages of development

Parra et al., 2013

O. europaea L. ssp. europaea, cv Picual

Fruit pericarp and AZ tissues

454 GS FLX sequencer

To study the transcriptomic profiling of AZ tissues at the last stage of fruit ripening

Yanik et al., 2013

O. europaea L. ssp. europaea, cv Ayvalik

Fruits (ripe, unripe); leaves (on-year and off-year)

Illumina HiSeq 2000 sequencer

To identify miRNA regulated by the plant alternate bearing

GilAmado and GomezJimenez, 2012

O. europaea L. ssp. europaea, cv Picual

Zone of abscission of drupes harvested in two periods (antecedent and corresponding to complete maturation)

454 GS FLX sequencer

Transcriptomic analysis of the abscission zone during cell separation for the study of the abscission control of the ripe fruit in the activation phase of the abscission zone

Alagna et al., 2012

O. europaea L. ssp. europaea, cv Coratina and cv Dolce d’Andria

Fruits harvested at three different development phases

EST dataset analysis cDNA-AFLP analysis RT-qPCR

To study the phenolic compounds profiles and identify the key genes of phenolic metabolism

Corrado et al., 2012

O. europaea L. ssp. europaea, cv Moraiolo and cv Leccino

B. oleae infested fruits

SSH approach RT-qPCR

To study the molecular pattern and related signaling pathways induced in drupe
fly interaction

Donaire et al., 2011

O. europaea L. ssp. europaea, cv Picual, cv Arbequina and cv Lechin de Sevilla

Juvenile and adult shoots

454 GS FLX sequencer

To identify and characterize miRNAs from different developmental stages and tissues

Alagna et al., 2009

O. europaea L. ssp. europaea, cv Coratina, with a very high phenolic content, and cv Tendellone, oleuropeinlacking

Fruits harvested at two different development stages

454 GS FLX sequencer

To enrich the sequence data available and identify key genes involved in fruit quality traits

Galla et al., 2009

O. europaea L. ssp. europaea, cv Leccino

Fruits sampled at three different development stages

SSH approach

Identification of differentially expressed genes in fruit during development period

AFLP, Amplified fragment length polymorphism; AZ, abscission zone; EST, expressed sequence tag; miRNA, micro-RNA; SSH, suppression subtractive hybridization.

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PART | 1 General Aspects of Olives and Olive Oil

Photosynthesis represents an important source of sugars for the mesocarp development and the olive oil synthesis. Plant cells require sugars for the synthesis of lipids, and acetyl-CoA is the precursor of the lengthening of the carbon chain in all fatty acids. The accumulation of transcripts related to the biosynthesis of structural proteins in the early fruit development highlighted by RNA sequencing approaches could be associated with the fast cell division that occurs in that stage, as well as the incremented expression of genes putatively associated with the biosynthesis of fatty acids and TAGs, observed during ripening, matching the accumulation of these metabolites in the late ripening stages.130,131 As expected, genes related to photosynthesis and carbohydrate metabolism had a contrasting expression trend during ripening, with up- and downregulation of their transcripts, respectively.130,132 Although the expression of some components of carbon fixation and starch and sucrose metabolism was modulated during the early stages of fruit development, numerous genes coding for enzymes involved in fatty acid biosynthesis, in glycolysis, in pyruvate metabolism, and in citrate cycle were most expressed during oil accumulation. It was observed this process began with the pit hardening phase and reached the highest level before the start of ripening. In particular, an increase in the expression of genes encoding fatty acid enzymes, such as acetyl-CoA carboxylase and enoyl-ACP reductase, actively involved in the synthesis of malonylCoA and in the elongation of the fatty acid chains was noted.132 FAD genes are involved in the biosynthetic pathway of unsaturated fatty acids and their expression pattern is modulated by several environmental factors. The olive FAD genes are among the most studied in the biosynthetic pathway of TAGs in all plant species.126 The oleic acid, which represents the main product of the synthesis of plastidial fatty acids, is widely activated in oleoyl-CoA and exported to the cytosol, where it is incorporated in the glycerolipids and can be further desaturated to linoleic acid and therefore to α-linolenic with consecutive action of the desaturases Δ12 and Δ15.133 This process can also take place within the plastid, and therefore there are two sets of enzymes that differ in their localization, lipid substrate, and electron donor system. The microsomial oleate desaturase was called FAD2, while the linoleate desaturase FAD3. On a plastidial level, the first one was called FAD6 and the second one FAD7/8. The genes encoding these enzymes have been extensively studied in the olive at molecular and biochemical level leading to the identification of numerous genes, in particular for the FAD2 gene that can be considered as belonging to a gene family.27,89,127,133,134 Herna´ndez et al.127 identified two genes (FAD2-1 and FAD2-2) differentially expressed in the endocarp and mesocarp, respectively, coding for two microsomal enzyme isoforms. The FAD2-2

gene expression was higher in the mesocarp in respect of the FAD2-1 and FAD6 genes expression, and the pattern of expression positively correlated with the content of linoleic acid during the fruit development and ripening phases. In both varieties studied (“Arbequina” and “Picual”), the expression level of the FAD2-2 gene increased at the beginning of ripening and differentiated in relation to the cultivar, keeping the higher expression in “Arbequina,” cultivar characterized by a higher content of linoleic acid than “Picual.” Unver et al.89 conducted an evolutionary analysis of oil biosynthesis comparing TAG biosynthesis genes between sesame and oleaster (var. sylvestris). It was observed, that within the FAD2 gene family, four genes were shared with sesame (FAD2-1, FAD2-2, FAD2-4, and FAD2-5 genes) while FAD2-3 gene evolved as a unique gene in oleaster. The expression analysis in different tissues collected from ripe and unripe fruits of the ortholog genes showed a downregulation in fruit tissues especially during the lipid-accumulation ripening stage while the FAD2-3 gene resulted actively expressed. These authors named FAD2-3 to the previously characterized FAD2-2 gene.127,134 Growing olive plants at different altitudes provided insight about the expression of some FAD genes, showing opposite trends during development. In fact, it was observed that FAD2-2 and FAD6 transcripts were upregulated during the veraison phase at high altitude, while an opposite pattern was observed at low altitudes where the FAD expression was generally lower and only FAD7 transcript levels appeared upregulated during the veraison phase, in according with a hypothesized greater stability of fatty acids at high altitudes. This differential expression pattern of FADs would confirm that transcriptomic profiling of these genes is modulated by external factors, mainly the temperature.135
137 Then, although a prolonged cold exposition of plants can cause frost damages, the maturation of olive fruit in cool environments may provide olive oil with peculiar qualitative characteristics. Matteucci et al.135 compared the expression of some fatty acid genes in two olive genotypes with opposite cold tolerance in order to investigate the relationship among fruit development, cold response, and expression of FAD genes. This study elucidated genotype-dependent differences related to the transcription of FAD genes (FAD2-1, FAD2-2, FAD6, and FAD7). Regardless of the genotype, the expression levels of FAD2-1 and FAD7 were always the highest in the early fruit development stages, while the expression level of FAD2-2 increased after exposure to cold during the oleogenic period. The unsaturated fatty acid content, after exposure to low temperature, increased in the drupes of the sensitive genotype, but not in those of the tolerant genotype, despite a greater transcription of FAD genes, suggesting that the adaptation of drupes to the cold requires posttranscriptional regulation of FAD

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

genes.135 Herna´ndez et al.134 showed that the transcripts of the FAD2-1 and FAD2-2 microsomal genes and those of the FAD6 and FAD7 plastidial genes appeared in all varieties studied (“Arbequina” and “Picual”) with the onset of the accumulation of lipids in the fruits and were present until complete veraison, when oil accumulation has been completed.135 Since an increase in FAD2-2 and FAD7 transcripts was observed in both genotypes, following exposure to cold, the genotype-specific polyunsaturation pattern must be the effect of a different posttranscriptional control. It was hypothesized that a greater production and/or stability of FAD2-2 and FAD7 transcripts occurs in the drupes of the sensitive genotype. Although the mesocarp of the olive drupes accumulates a wide range of secondary metabolites, the secoiridoids are the mainly represented compounds. Due to their insolubility in organic solvents, only a small portion is recovered in the oil, but for their sensorial and healthy properties, they represent the most important microconstituents of virgin olive oil, they are responsible for the sensorial notes of bitter and spicy, and, as antioxidants, they are also responsible for the oxidative stability of the oil. The accumulation of secoiridoids is a fine controlled process, and their expression and composition vary greatly among genotypes, tissues, development stages, and environmental conditions. A decreasing of total phenol content during fruit development in 12 different varieties (Coratina, Rosciola, Frantoio, Canino, Moraiolo, Leccino, Tendellone, Bianchella, Dritta, Dolce d’Andria, Nocellara del Belice, Nocellara etnea), grown under the same environmental conditions, was observed by Alagna et al.138 The authors, using a combined approach to monitor metabolic and transcriptional profiling detected a differential gene expression pattern in two genotypes with contrasting phenols content which, significantly correlated with the specific metabolites, was found at different developmental stages of drupe, indicating a regulation mechanism at transcriptional level of some major players putatively involved in biosynthesis of secondary compounds, as also reported by different authors.130,137,139 In fact, the levels of many transcripts putatively involved in the biosynthesis of secoiridoids were strong downregulated during the fruit development, in accordance with the reduction of oleuropein levels observed as well as many genes involved in the secoiridoid pathway are more expressed in the genotype with more high content of phenolic substances. Genotype-specific transcripts were prevalently identified in genotype with high phenolic content, supporting the hypothesis that its fruits may synthesize a broader range of secondary metabolites.130 Another study based on whole transcriptome sequencing showed a clear correlation between expression levels of genes and metabolites directly involved in biosynthesis of hydroxytyrosol and oleuropein, during fruit development.139 A hydroxytyrosol increase was instead observed

39

in black drupes that could be attributed to the destruction of complex phenolic molecules, rather than a transcriptional regulation mechanism. This breakdown is carried out by hydrolytic enzymes, esterases, and β-glucosidases (β-GLUs), which convert complex compounds, such as oleuropein, into simple phenols, such as hydroxytyrosol and elenolic acid, in order to recycle all the available molecules of esters of the oleuropein.140 A correlation between the 2-C-methyl-D-erythritol 4-phosphate (MEP) plastidial pathway and the transcriptional profiles of secoiridoids was also observed, supporting the hypothesis that this pathway, contributes primarily to the synthesis of secoiridoids. For six transcripts [1-deoxy-D-xylulose-5-phosphate synthase (OeDXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (OeDXR), 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (OeCDPMES), 2-C-methyl-D-erythritol 2,4-cyclo-PP synthase (OeMECPS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (OeHMBPPR), and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (OeHMBPPS)] putatively involved in the MEP pathway, an important downregulation during the fruit development was observed. The expression of putatively transcripts involved in the synthesis of the terpene portion of the secoiridoids also decreased considerably during the veraison period, in agreement with the oleuropein concentration reduction observed during fruit ripening.138 Some studies reported that water availability negatively influences phenol content in olive drupes and oil, in particular about the secoiridoids content.52,141
143 It has been suggested that the oleuropein catabolism regulated by glucosidase activity is particularly sensitive to water availability during the early stages of fruit development suggesting a positive effect of the water deficit mediated by β-GLU activity.143 3,4-DHPEA-elenolic acid dialdehyde (3,4-DHPEA-EDA) is hypothesized to be a product of the oleuropein catabolism linked to β-GLU activity. In fact, an inverse relationship between oleuropein content and its derivative 3,4-DHPEA-EDA during fruit development was observed.143,144 The expression of the polyphenoloxidase (PPO) genes was also influenced by the water status of the plant as well as by the fruit development stages. The levels of transcripts were higher in the plants irrigated during late stages of fruit ripening; however, in nonstressed drupes the PPO enzymes seem to have a minor role in the metabolism of secoiridoids. Peroxidases (PRX) are other enzymes that contribute to phenols oxidation, but their activity is limited by the availability of H2O2, which increases in stress conditions. The greatest PRX enzyme activity was identified during the early stages of fruit development, decreased in subsequent stages and increased again at ripening stage. During the early stages of fruit development, the PRX expression pattern and enzyme activity were related to the phenol

40

PART | 1 General Aspects of Olives and Olive Oil

content in immature fruits and were not influenced by water availability. A further comparative transcriptome-wide investigation on olive plants grown at different altitude level highlighted the correlation between expression of specific genes related to the MEP and mevalonate pathways and different climatic conditions.137 Interestingly, although in the drupes of plants grown at 10 and 200 masl the quantity of oleuropein decreased during the ripening, according to previous studies,130 an opposite trend was observed in the olives collected at 700 masl. Transcriptomic analysis revealed a higher number of differentially expressed genes in fruits collected at different altitudes.137 At 700 masl the opposite regulation of transcript genes related to biosynthesis (up-) and catabolism (down-) suggested a potential reduction or delay of degradation of oleuropein at higher masl, according to its greater accumulation during the ripening at the same altitude level; therefore a higher altitude level, the gene expression, resulted modulated so that greater stability of oleuropein was favored. The induction of oleuropein stability, detected in turning color drupes of plants grown at high masl, represents an important feature related to the better quality of the oil.137 Several studies highlighted a differential regulation of flavonoid levels among different genotypes and during fruit development.130,132,137,138,145 Flavonoids represent a large family of low molecular weight polyphenolic secondary metabolites that are widespread throughout the plant kingdom. Of these metabolites the anthocyanins, the most common pigments in plants, responsible for the color of all the main organs, possess numerous biological roles, such as protection from ultraviolet radiations and against numerous pathogens, antioxidant capacities and may provide health benefits as dietary complements. Aiming to identify the expressed genes related to biosynthesis of anthocyanins, a de novo transcriptome reconstruction of olive fruits was performed together with a full expression analysis in “Leucocarpa.” This cultivar is characterized by a switch-off in the epidermis color when fully ripe, and it represents an excellent experimental material to define the transcriptomic profile during the drupe ripening and identify the transcripts involved in the metabolism of flavonoids and anthocyanins.145 The transcript pattern of the flavonoid and anthocyanin pathways included a set of 11 identified transcripts: phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol 30 -hydrogenase (F30 H), flavonol 30 50 -hydrogenase (F30 50 H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose: anthocyanin: flavonoid glucosyltransferase (UFGT). Furthermore, different transcription factors (TFs) MYB, MYC, and WD, related to the

complex transcriptional regulation of the anthocyanin structural genes, were identified. By focusing on the pathways that control the biosynthesis of pigments and the natural reduction of photosynthetic pigments during the veraison phase, “Leucocarpa” was characterized by a significant downregulation of the CHS, DFR, and ANS transcripts, during the transition phase in comparison with the control gene expression pattern.145 All the results obtained agreed with a downregulation of flavonoid and anthocyanin metabolism in “Leucocarpa,” while an opposite trend was observed in the control cultivar. On the other hand, only for a few genes related to anthocyanin biosynthesis an expression peak, during fruit veraison, were observed.132 In particular, the PAL expression significantly increased in relation to the anthocyanin accumulation during veraison, as in the other study that linked a greater PAL transcript accumulation to the higher concentration of phenolic compounds at the early stages of fruit development.146 Besides, 4CL showed a greater expression only in the initial phase of fruit development.138 Moreover, a greater or prolonged activation of the anthocyanin pathway in olive trees grown at low altitudes compared to trees grown at high altitudes was reported, resulting in drupes with a higher index of ripeness. Although a different type of regulation could occur at the posttranscriptional level, the transcriptional pattern showed that during the veraison phase the genetic pathways related to both lipids and phenols were differentially modulated in drupes from different masls, according to the quantitative differences found for the lipid and phenolic composition.137 The deepening of transcript level regulation of ripening-mediated genes by suppression subtractive hybridization allowed to elucidate the function of other genes related to the biosynthesis of phenol compounds.132 Interestingly, a transient downregulation of transcript levels of the dihydrokaempferol 4-reductase, involved in oxidoreduction of flavonols to anthocyanidins, was observed at the veraison phase. Furthermore, two isoforms of a peroxidase specifically involved in four different phases of the phenylpropanoid synthesis were identified, as well as some enzymes involved in the biosynthesis of alkaloids, in the degradation of limonene and pinene and in the metabolism of caffeine.132 As also reported by Galla et al.,132 an upregulation of CHS and F3H key genes for the anthocyanin biosynthetic pathway was observed during the veraison stage. Moreover, the UFGT gene expression trend was interesting, this gene involved in the anthocyanin biosynthesis, at veraison stage resulted upregulated, but it was already transcribed in the young fruits. Two other key genes involved in the anthocyanin biosynthetic pathway regulation were ANS and DFR, the expression of which increased when the drupe coloring change starts.146 The flavor of fruits is

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

generally determined by hundreds of constituents, most of them generated during the ripening phase. Terpenoids have been identified at varying levels in the flavor profiles of most fruits. For this reason, researches focused on fruit development and ripening have to be able to identify genes involved in the biosynthesis of these metabolites differentially expressed among different genotypes and during fruit development.130,137,138 For example, transcript levels of farnesyl diphosphate synthase (FPPS) and squalene synthase (SQS) genes encoding two key enzymes for squalene synthesis showed a peak at veraison phase, while the lupeol synthase (LUPS) gene encoding for lupeol synthase, which catalyzes the formation of the triterpene lupeol, showed a different expression profile at green phase between genotypes with high- and low-fruit phenolic content, respectively, in agreement with the different squalene and triterpene accumulation observed during the fruit development.138 The levels of geranylgeranyl pyrophosphate synthase (GGPS) transcripts, involved in the geranylgeranyl pyrophosphate synthesis, with an important role in the synthesis of diterpenes and carotenoids, were very high at green phase compared to the other developmental phases in agreement with a great accumulation of carotenoids during the first phase of fruit development. It is interesting to note that some key genes of the biosynthesis of monoterpenoids and phenolic compounds, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), geraniol synthase (GES), geraniol-10-hydroxylase (G10H), and arogenate dehydrogenase (DH), were exclusively expressed at the first phase of fruit development, in conjunction with high levels of secoiridoids. A strong correlation between metabolic and transcriptional data was also identified for the biosynthesis of aromatic compounds, such as (1)-R-limonene, one of the most abundant monocyclic monoterpenes in nature, and 1,8-cineole, known as eucalyptol, a monoterpene oxide. The genes encoding for R-limonene synthase 1 and 1,8-cineole synthase showed an important differential expression during fruit development in the late stages when the greatest accumulations of limonene and eucalyptol were found.130,138 Bactrocera oleae, commonly known as olive fly, is the most important parasitic insect for this crop. The adult females of the fly pierce the drupe and release the eggs under the exocarp. The larvae consume the pulp, causing loss of tissue, premature fall of the fruit, and reduction of the yield in oil; there is also an increase in the oil acidity and the number of peroxides, the development of unpleasant aromas of worm, mold, and topsoil significantly reduces the chemical and sensorial quality of the oil. The main compounds negatively affected by B. oleae infestation are verbascoside, tyrosol, and hydroxytyrosol.147 Olive genotypes show a different degree of susceptibility to fly infestation, but the factors that determine the

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different susceptibility are still controversial. The interaction drupe
olive fly is little still studied at molecular level. The differential transcriptomic profiling, between drupes infested by fly larvae and healthy drupes, has shown a significant intraspecific variation between genotypes with opposite tolerance.148 About 2500 genes were found to be differentially regulated in the infested drupes of the tolerant genotype. The gene ontology (GO) annotation of the differentially expressed genes implied that the inducible resistance to fly involves a number of biological functions, cellular processes, and metabolic pathways, including those known to play a role in defense, in the response to oxidative stress, in organization of the cellular structure, in hormonal signaling, and in primary and secondary metabolism. More precisely, the unigenes were distributed in 14 GO terms for biological processes, 9 for cellular components, and 10 for molecular functions. The most numerous metabolic pathways, mapped in the KEGG database, were “biosynthesis of secondary metabolites,” “microbial metabolism in different environments,” “spliceosome,” “biosynthesis of plant hormones,” “ribosome,” “transport of RNA,” and “protein processing in endoplasmic reticulum.” The response to B. oleae by susceptible genotype concerned a limited set of genes and the majority (77%) were less expressed. Following the fly attack, more than 1000 genes were differentially expressed in drupes of tolerant genotype, compared to susceptible genotype; very few transcripts were shared between tolerant and susceptible genotypes, indicating a highly specific genotype response to fly infestation. Most of the genes induced by fly attach were more expressed in the tolerant genotype respect to the susceptible genotype, indicating a genotype-specific response. In the tolerant genotype the functional profile of down- and upregulated transcripts indicated that the most abundant categories of GO terms were related to chemical reactions and both biosynthetic pathways and protein modification processes; in particular, among genes induced by fly attack, those encoding proteins involved in disease resistance response and signaling, including receptors and TFs known as defense regulators of plants, were found. In particular, the genes coding for three β-GLUs were detected. In the fruits the β-GLU catalyzes the hydrolysis of oleuropein in a toxic structure, similar to glutaraldehyde, which acts as a defense mechanism against insects.149 Four serine carboxypeptidaselike (SCPL) genes, coding for proteins connected to the jasmonic acid pathway, were upregulated. Other upregulated genes, probably related to the jasmonic acid pathway, included two phospholipases.148 Following the attack, transcripts involved in the jasmonate signal transduction (lipoxygenase and the lipid transfer) and in phenylpropanoid metabolism pathway (trans-cinnamate 4-hydroxylase and caffeoyl-o-methyltransferase) were also detected.150 Finally, the response of tolerant genotype to B. oleae also includes genes

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PART | 1 General Aspects of Olives and Olive Oil

involved in the signaling of phytohormones, such as auxins, brassinosteroids, and ethylenes. The fly attack induces other stress-related cell messengers, reactive oxygen species, and calcium, as evidenced by the upregulation of five genes coding for calcium-dependent kinase proteins, for a calcium transporter adenosine triphosphatase (ATPase) at membrane level, four glutaredoxins, and one oxoglutarate. From KEGG analysis the metabolism of purines, associated with the drupe ripening, ranked first for the sequence number. However, regarding the number of enzymatic activities, the “biosynthesis of antibiotics,” including reactions associated with the biosynthesis of terpenoids, the pathway of the shikimate, and the biosynthesis of secondary metabolites, is first placed, followed by the enzymatic activities related to the starch and sucrose metabolism.148

3.3.4 Small nuclear RNA In plants, it is well known that the snRNAs, including short interfering RNAs (siRNAs) and micro-RNAs (miRNAs), when expressed, match with the UTR of target messanger RNAs (mRNAs) determining gene silencing through the transcript cleavage or the translational inhibition.151 In the same study described earlier,89 the authors have also elucidated the function of an siRNA sequence. This specific snRNA originated from a TE-rich genomic region and could bind specifically to the 50 UTR of duplicated copies of the FAD2 gene transcripts, repressing expression in drupe tissues. The authors hypothesized that this suppression mechanism as a result of gene expansion probably leads to a higher content of oleic acid in oleaster. An only FAD2 transcript, FAD2-3, due to the presence of an additional sequence of 12 nt at the siRNAbinding site, could not be influenced by the negative regulation of the siRNA even if the authors observed that an active expression of FAD2-3 gene in fruits leaded to the conversion of relatively low amount of oleic acid into linoleic acid. So far, some miRNAs have been experimentally analyzed and bioinformatically predicted in olive.152
155 While in three of them, deep sequencing has been used to report a catalog of snRNAs,152,153,155 in another study an in silico prediction of snRNAs, previously reported as miRNAs, was performed.154 These studies revealed that the miRNAs are primary regulators, targeting TFs involved in several morpho-physiologic processes, as fruit development, ripening, fruit alternate bearing, and secondary metabolism regulation. Fruit development and ripening are a result of a complex coordination of several biochemical pathways leading to variations in nutrient accumulation, texture, aroma, and color. Among them, there are pathways related to production of several secondary metabolites such as carotenoids and various phenol compounds, as anthocyanins, with key role in the

fruit postharvest performance and essential in a healthy diet for their antitumor, antineurodegenerative, antioxidative, antiaging, and antiinflammatory properties. A study aiming to identify miRNA related to alternate bearing, performed on ripe and unripe fruits and on leaves from high “on-year” and low “off-year” yield plants, allowed to detect 135 conserved miRNA belonging to 22 families, with 38 putative novel miRNA for the olive tree datasets.153 Of them, several miRNAs with different expression levels between the fruit libraries were observed, indicating a differential functional role of the involved genes in the development and ripening regulation. In particular, a key role in the transport of nutrients during the development and ripening was hypothesized for miR395, downregulated in the ripe fruit compared to the unripe ones, which targets transport and response-to-stimulus genes. Moreover, the miR166 and miR171 expression was significantly upregulated in the unripe fruit. These miRNAs target genes are involved in organ morphogenesis and developmental processes suggesting a potential ripening inhibition mediated by suppression of these genes. In our previous study,155 in order to elucidate miRNA putative roles in the biosynthesis and accumulation of secondary metabolisms and particularly of phenylpropanoids, we analyzed the drupes, at two different ripening stages (100
130 days after flowering), of two cultivars (“Leucocarpa” and “Cassanese”) showing opposite fruit pigmentation and significant differences in anthocyanin transcript profiles.145 A total of 218 transcripts were predicted as targets of 130 known and 492 putative novel miRNAs; some of them were involved in negative regulation of anthocyanin metabolic processes. As described in previous studies, significant upregulation of miR166 was observed in the unripe drupes. Interestingly, in “Leucocarpa,” which is an olive variety characterized by a switch-off in skin color at full ripeness, as described earlier, miR166 expression was found persistently high during the fruit ripening. The concomitant downregulation of the levels of its target transcript in both ripening stages suggest an inhibition of some typical ripening processes mediated by miRNA induction. Moreover, miR159 and miR168, involved in organismal development,153 showed an opposite expression pattern between the two cultivars. Their transcripts were higher in “Cassanese” ripe fruit than unripe fruit while in “Leucocarpa,” they were unchanged during ripening transition and similar to those of “Cassanese” unripe fruit. Also, the levels of their target genes were downregulated at the last ripening stages, suggesting a putative role in the biosynthesis of flavonoid metabolites. Then, miRNAs could play a role in olive fruit development tightly related to the production of several secondary metabolites, such as anthocyanins known for their benefits to the human health. In particular, it was observed that miRNA168a

Olive tree genetics, genomics, and transcriptomics for the olive oil quality improvement Chapter | 3

and 159a, provided through diet, were able to pass through human gastrointestinal track decreasing the LDL levels in the plasma.156,157 Nevertheless, in a recent study, through in silico prediction, O. europaea snRNAs, homologs of hsa-miR34a, were identified.154 The miRNA34 family (hsa-miR34) is an important class of human oncosuppressor miRNAs: his expression is frequently repressed in tumors than in normal tissues.158,159 The ability of oeu-miRNA34 to regulate tumorigenesis in human cells has been demonstrated. In fact, the transfection of synthetic oeu-miRNA34 reduced the protein expression of its targets, increased apoptosis, and decreased proliferation in different tumor cells. Moreover, the introduction of oeu-miRNA34 in hsa-miR34a-deficient tumor cells restores its function, whereas cells with normal expression of endogenous hsa-miR34a remained unaffected. The natural oeu-miRNA34 extracted from olive fruits induced the same effects as synthetic miRNA.154 In conclusion, miRNAs could represent potential molecules to promote the production of functional food and the development of novel natural nontoxic drugs also supporting antineoplastic strategies.

3.4 Conclusion and perspectives Olive is the most ancient tree crop originated in the Mediterranean basin and currently widespread in several countries worldwide. Although its domestication process is still under discussion, all the studies conducted so far on the diffusion of the olive tree and genotyping studies define it as a species characterized by a high level of genetic diversity and that the evolution toward new varieties is firmly influenced by the introgression between the cultivated form and the wild olive. Genetic diversity has also been demonstrated by the wide phenotypic variability studied under several environmental conditions although with different varieties but also in the context of germplasm collections under uniform environmental conditions, highlighting the genotypic component controlling fatty acid composition and phenolic profile. Most of the olive oil components play a role in determining its quality and its nutraceutical properties, and the level of knowledge on their typology and variability within the species is rather exhaustive. However, the olive tree is a species with a very complex biology that led to a delay in the acquisition of genomic information, a serious difficulty in managing breeding programs and a reduced knowledge of its physiology compared to those of all other tree crops. The recent sequencing of the genome, although not yet fully revealed, together with the numerous transcriptomic approaches are opening the doors to the understanding of genetic
molecular mechanisms that largely have a rate with the quality of the oil, a trait considered a priority in the genetic improvement of the species. In fact, only in

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the last 4 years, the genetic
molecular mechanisms able to explain the difference in fatty acid composition between the olive tree and an oleaginous species (sesame) were revealed. Similarly, regions of the genome and structural variants that could play a role in the modulation of the acidic composition within the species are being highlighted through QTL and AM approaches. In the case of phenolic compounds, those are specific within the Oleaceae family and for other secondary metabolites, numerous genes have been identified and the regulation points are highlighted through transcriptomic studies so far. Many other minor compounds the functional properties of which have been demonstrated are currently very little studied probably also due to their low variation range which implies greater difficulty in the application of these advanced approaches. Emerging studies about miRNAs have highlighted how these molecules could promote the production of functional food and the development of novel natural nontoxic drugs also supporting antineoplastic strategies An important role in clarifying the mechanisms that underlie the variability of expression of the traits that make up the quality of olive oil will be given by the resequencing of the genome and by the completion of the genome annotation. Genomes resequencing allows to the identification of all structural variation of the species potentially responsible for the variation in the expression of complex characters. The genome annotation identifies new genes by bioinformatic tools and assumes a functional characterization of genes as following step. Once these strategies are also applied to the olive tree, the knowledge of the species biology will probably be completely revealed.

Mini-dictionary of terms G

G

G

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NGS. Next-generation sequencing is a high-throughput method used to determine a portion of the nucleotide sequence of genomes and transcriptomes. It is based on DNA sequencing technologies that are capable of processing multiple DNA sequences in parallel. Gb. Gigabase, DNA sequence length in numbers of base pairs (bp), 1,000,000,000 bp. kb. Kilobase, DNA sequence length in numbers of base pairs (bp), 1,000,000 bp. TEs. Transposable elements are DNA sequences that can change their position within a genome, producing mutations and altering genetic identity and genome size. LTR. Long terminal repeat retrotransposons are the most abundant transposons in plants. They are a particularly interesting type of class I transposable element related to retroviruses and play important roles in alternative splicing, recombination, gene regulation, and defense mechanisms.

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PART | 1 General Aspects of Olives and Olive Oil

snRNA. Small nuclear RNA is one of the small RNA with an average size of 150 nt. It is a class of highly abundant RNA, localized in the nucleus with important functions in intron splicing and other RNA processing. SSRs. Simple sequence repeat or microsatellite is a tract of repetitive DNA in which certain DNA motifs, ranging in length from 1 to 6 or more bp, are repeated, typically 5
50 times. RAPD. Random amplification of polymorphic DNA markers are DNA fragments from polymerase chain reaction (PCR) amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence. AFLP. Amplified fragment length polymorphism is an analysis based on PCR used for mainly DNA fingerprinting. It is based on digestion of genomic DNA and binding of the restriction semisequences present on the fragments to specific adapters followed by a two selective amplification of some of these fragments. ISSR. Intersimple sequence repeat marker system detects polymorphisms in intermicrosatellite DNA regions without any prior sequence knowledge. ISSR markers are generated from single-primer PCR amplifications in which the primers are based on dinucleotide or trinucleotide repeat motifs. ESTs. Expressed sequence tags are fragments of messanger RNAs (mRNA) sequences derived through single sequencing reactions performed on randomly selected clones from cDNA libraries. SNPs. Single nucleotide polymorphisms are defined as loci with alleles that differ at a single base, with the rarer allele having a frequency of at least 1% in a random set of individuals in a population. QTL. A quantitative trait locus is a region of DNA associated with a particular quantitative character. QTL is closely associated with a gene that determines the phenotypic character in question or participates in its determination. RFLP. Restriction fragment length polymorphism is the most widely used hybridization-based molecular marker. The technique is based on restriction enzymes that reveal a pattern difference between DNA fragment sizes in individual organisms. cM. The centimorgan is the unit of measurement used to measure the percentage of recombinant genetic distance between two loci, or the gene map unit, “m.u.” (mapping unit). SCAR. Sequence-characterized amplified region is a random amplification of polymorphic DNA fragment sequenced. DArT. Diversity arrays technology is the name of a technology used in molecular genetics to develop sequence markers for genotyping and other techniques

G

G

G

G

G

G

for genetic analysis. DArT is based on microarray hybridizations that detect the presence versus absence of individual fragments in genomic representations. UTR. Untranslated region refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 50 side, it is called the 50 UTR (or leader sequence), or if it is found on the 30 side, it is called the 30 UTR (or trailer sequence). MAS. Marker-assisted selection or marker-aided selection is an indirect selection process where a trait of interest is selected based on a marker (morphological, biochemical or DNA/RNA variation) linked to a trait of interest. GWAS. Genome-wide association study also known as whole genome association study (WGA study, or WGAS) is an observational study of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait. TF. A transcription factor or sequence-specific DNAbinding factor is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. WD. proteins are made up of highly conserved repeating units usually ending with Trp-Asp (WD). The WD proteins contain varying numbers of WD repeats and act as protein
protein and protein
nucleic acid interaction domain. GO. Gene ontology is a major bioinformatics project to unify the representation of gene and gene product attributes across all species. More specifically, the project aims to (1) maintain and develop its controlled vocabulary of gene and gene product attributes; (2) annotate genes and gene products, and assimilate and disseminate annotation data; and (3) provide tools for easy access to all aspects of the data provided by the project, and to enable functional interpretation of experimental data using the GO, for example, via enrichment analysis.

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592. 99. Trujillo I, Ojeda MA, Urdiroz NM, et al. Identification of the Worldwide Olive Germplasm Bank of Cordoba (Spain) using SSR and morphological markers. Tree Genet Genomes. 2014;10: 141
155. 100. Muzzalupo I, Vendramin GG, Chiappetta A. Genetic biodiversity of Italian olives (Olea europaea) germplasm analyzed by SSR markers. Sci World J. 2014;2014:296590. 101. De la Rosa R, Belaj A, Mun˜oz-Me´rida A, et al. Development of EST-derived SSR markers with long-core repeat in olive and their use for paternity testing. J Am Soc Hortic Sci. 2013;138(4): 290
296. 102. Mariotti R, Cultrera NGM, Mousavi S, et al. Development, evaluation, and validation of new EST-SSR markers in olive (Olea europaea L.). Tree Genet Genomes. 2016;12:120. 103. Belaj A, De la Rosa R, Lorite IJ, et al. Usefulness of a new large set of high throughput EST-SNP markers as a tool for olive germplasm collection management. Front Plant Sci. 2018;9:1320. 104. Ben Ayed R, Ben Hassen H, Ennouri K, Marzoug BR, Rebai A. OGDD (Olive Genetic Diversity Database): a microsatellite markers’ genotypes database of worldwide olive trees for cultivar identification and virgin olive oil traceability. Database (Oxford). 2016;2016. bav090. 105. Mohamed B, Zelasco S, Ben Ali S, et al. Exploring olive trees genetic variability in the South East of Tunisia. Genet Mol Res. 2017;16:4. 106. De la Rosa R, Angiolillo A, Guerrero C, et al. A first linkage map of olive (Olea europaea L.) cultivars using RAPD, AFLP, RFLP and SSR markers. Theor Appl Genet. 2003;106(7):1273
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107. Haralampidis K, Milioni D, Sa`nchez J, Baltrusch M, Heinz E, Hatzopoulos P. Temporal and transient expression of stearoyl ACP carrier protein desaturase gene during olive fruit development. J Exp Bot. 1998;49(327):1661
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74. 109. Wu SB, Collins G, Sedgley M. A molecular linkage map of olive (Olea europaea L.) based on RAPD, microsatellite, and SCAR markers. Genome. 2004;47(1):26
35. 110. Khadari B, El Aabidine AZ, Grout C, et al. A genetic linkage map of olive based on amplified fragment length polymorphism, inter simple sequence repeat and simple sequence repeat markers. J Am Soc Hortic Sci. 2010;135(6):548
555. 111. El Aabidine AZ, Charafi J, Grout C, et al. Construction of a genetic linkage map for the olive based on AFLP and SSR markers. Crop Sci. 2010;50(6):2291
2302. 112. Dominguez-Garcia MC, Belaj A, De la Rosa R, et al. Development of DArT markers in olive (Olea europaea L.) and usefulness in variability studies and genome mapping. Sci Hortic (Amsterdam). 2011;136:50
60. 113. Ben Sadok I, Celton JM, Essalouh L, et al. QTL mapping of flowering and fruiting traits in olive. PLoS One. 2013;8:e62831. 114. Ben Sadok I, Moutier N, Garcia G, et al. Genetic determinism of the vegetative and reproductive traits in an F1 olive tree progeny evidence of tree ontogeny effect. Tree Genet Genomes. 2013;9(1):205
221. 115. Essalouh L, El Aabidine AZ, Contreras S, et al. Genomic and EST microsatellite loci development and use in olive: molecular tools for genetic mapping and association studies. Acta Hortic. 2014;1057(1057):543
550. 116. Ipek A, Yilmaz K, Sikici P, et al. SNP discovery by GBS in olive and the construction of a high-density genetic linkage map. Biochem Genet. 2016;54(3):313
325. 117. Marchese A, Marra FP, Caruso T, et al. The first high-density sequence characterized SNP-based linkage map of olive (Olea europaea L. subsp. europaea) developed using genotyping by sequencing. Austr J Crop Sci. 2016;10(6):857
863. 118. Elshire RJ, Glaubitz JC, Sun Q, et al. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One. 2011;6:e19379. 119. Barchi L, Acquadro A, Alonso D, et al. Single primer enrichment technology (SPET) for high-throughput genotyping in tomato and eggplant germplasm. Front Plant Sci. 2019;10:2005. 120. Rugini E, Cristofori V, Silvestri C. Genetic improvement of olive (Olea europaea L.) by conventional and in vitro biotechnology methods. Biotechnol Adv. 2016;34:687
696. 121. Atienza SG, De la Rosa R, Leo´n L, Martin A, Belaj A. Identification of QTL for agronomic traits of importance for olive breeding. Mol Breed. 2014;34(2):725
737. 122. Herna´ndez ML, Belaj A, Sicardo MD, et al. Mapping quantitative trait loci controlling fatty acid composition in olive. Euphytica. 2017;213:7. 123. Flint-Garcia SA, Thornsberry JM, Buckler IV ES. Structure of linkage disequilibrium in plants. Annu Rev Plant Biol. 2003;54:357
374. 124. Kaya HB, Akdemir D, Lozano R, et al. Genome wide association study of 5 agronomic traits in olive (Olea europaea L.). Sci Rep. 2019;9:18764.

125. Ben Ayed R, Ennouri K, Ben Hlima H, et al. Identification and characterization of single nucleotide polymorphism markers in FADS2 gene associated with olive oil fatty acids composition. Lipids Health Dis. 2017;16:138. 126. Salimonti A, Carbone F, Romano E, et al. Association study of the 50 UTR intron of the FAD2-2 gene with oleic and linoleic acid content in Olea europaea L. Front. Plant Sci. 2020;11:66. 127. Herna´ndez ML, Mancha M, Martı´nez-Rivas JM. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive. Phytochemistry. 2005;66(12):1417
1426. 128. Kaya HB, Cetin O, Kaya HS, Sahin M, Sefer F, Tanyolac B. Association mapping in Turkish olive cultivars revealed significant markers related to some important agronomic traits. Biochem Genet. 2016;54:506
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187. 134. Herna´ndez ML, Padilla MN, Mancha M, Martı´nez-Rivas JM. Expression analysis identifies FAD2-2 as the olive oleate desaturase gene mainly responsible for the linoleic acid content in virgin olive oil. J Agric Food Chem. 2009;57(14):6199
6206. 135. Matteucci M, D’Angeli S, Errico S, Lamanna R, Perrotta G, Altamura MM. Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness. J Exp Bot. 2011;62(10):3403
3420. 136. D’Angeli S, Altamura MM. Unsaturated lipids change in olive tree drupe and seed during fruit development and in response to cold-stress and acclimation. Int J Mol Sci. 2016;17:1889. 137. Bruno L, Picardi E, Pacenza M, et al. Changes in gene expression and metabolic profile of drupes of Olea europaea L. cv Carolea in relation to maturation stage and cultivation area. BMC Plant Biol. 2019;19:428. 138. Alagna F, Mariotti R, Panara F, et al. Olive phenolic compounds: metabolic and transcriptional profiling during fruit development. BMC Plant Biol. 2012;12:162. 139. Mougiou N, Trikka F, Trantas E, et al. Expression of hydroxytyrosol and oleuropein biosynthetic genes are correlated with metabolite accumulation during fruit development in olive, Olea europaea, cv. Koroneiki. Plant Physiol Biochem. 2018;128:41
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142. Caruso G, Gucci R, Urbani S, et al. Effect of different irrigation volumes during fruit development on quality of virgin olive oil of cv. Frantoio. Agric Water Manag. 2014;134:94
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1191.

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Chapter 4

The chemical composition of Italian virgin olive oils Pierfrancesco Deiana1, Maria Rosaria Filigheddu1, Sandro Dettori1, Nicola Culeddu2, Antonio Dore3, Maria Giovanna Molinu3 and Mario Santona1 1

Deparment of Agriculture, University of Sassari, Sassari, Italy, 2National Council of Research (CNR), Institute of Biological Chemistry (ICB),

Sassari, Italy, 3National Council of Research (CNR), Institute of Sciences of Food Production (ISPA), Sassari, Italy

Abbreviations α-T FA IOC LDL MUFA PC PDO PGI PUFA ROS SQ VOO

α-tocopherol fatty acids International Olive Council low-density lipoprotein monounsaturated fatty acids phenolic compounds protected designation of origin protected geographical indication polyunsaturated fatty acids reactive oxygen species squalene virgin olive oil

4.1 Introduction The Italian Peninsula is located in southern Europe into the Central Mediterranean Sea as a bridge between the continents of Europe and Africa. From the Alps firmly situated in Center Europe (44 N) to Lampedusa island (35 N), Italy is near 1300 km long and 600 km wide with a total area (including five large islands) of approximately 300,000 km2. The Apennine chain, extending from northwest to southeast, divides the western side overlooking the Tyrrhenian Sea from the eastern side bathed by the Adriatic Sea (Fig. 4.1). Starting from the Paleolithic period, the complex orography and succession of different peoples and cultures explain the presence of heterogeneous landscapes. The growing of olive cultivation, together with that of vine and wheat, represents the common feature that connects the many cultural landscapes of the peninsula.1 The olive tree cultivation and the use of the oil extracted from its fruits, first for body care and lighting,

then also as a food, started in Italy by the 8th century BCE from the Magna Graecia (southern Italy). From here, olive growing spread throughout the peninsula. At the end of the 19th century, Italy included 900,000 ha of olive groves, more than a third located in Apulia.2 Until the 1990s of the 20th century, the presence of 1.4 million hectares of olive groves made Italy the first producer in the world, contributing to 34% of the total production. Today, overtaken by Spain (and Tunisia for growing surfaces), Italian olive oil, although still appreciated for the quality of virgin olive oils (VOOs, i.e., oils mechanically or physically extracted from olive fruits3), represents only 15% of world production, being at the same time the first consumer and the second exporter.4 The 4-year period 2015
18 recorded an average yearly olive oil production of 317,717 tons, against basic domestic needs of 535,000 tons. In the same period, Italy imported 588,000 tons of olive oil and exported 375,000 tons.5 The production of Italian olive oil is extremely fluctuating (Fig. 4.2). Causes may be attributed both to the annual weather conditions (winter or spring frosts, summer droughts, and pest damages) and to the advanced age of olive groves that respond weakly to agronomic practices. Olive cultivation extends across 1,170,157 ha, managed by 825,201 holdings, with an average size of 1.42 ha.6 Growing areas are mainly hilly (61%), 28% spread in plain, and only 11% in the mountains. Apulia, Calabria, and Sicily host 53% of the olive farms and 62% of the Italian olive growing surface (Fig. 4.1). The recognized quality of the Italian VOOs can be partially motivated by the large number of mills (about 4000) that allow the timely olives processing.6 The high quality, but also the fragmentary of the Italian VOO

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00033-X © 2021 Elsevier Inc. All rights reserved.

51

52

PART | 1 General Aspects of Olives and Olive Oil

FIGURE 4.1 Italian olive groves surfaces according to CORINE Land Cover 2018 database and main varieties distribution by production area.

production, is confirmed by the presence of 42 protected designation of origin (PDO) brands and 4 protected geographical indication (PGI): almost 40% of the European VOO Geographical Indications. The production of certified oil, however, does not exceed 2%
3% of the total quantity, reaching 6% in terms of trade value.6 Another basic peculiarity of Italian olive growing is given by its high biodiversity. The millennial process of local selection and the introduction of allochthonous varieties have improved the diversity of the species that, today, includes 538 varieties, corresponding to 42% of the world’s biological diversity.7 The main contribution to national production comes from Coratina (Apulian variety), oils of which are characterized by a very high oleic acid and phenols content.8 Two other important Apulian varieties are Cellina di Nardo` and Ogliarola Salentina, which, together with Carolea (Calabria), Frantoio, Leccino (central Italy), and Coratina, cover around the 30% of the total cultivated trees. Other important varieties of central Italy are Ascolana Tenera (Marche),

Carboncella (Lazio, Marche), Itrana (Lazio), and Moraiolo (Tuscany, Umbria). In northwestern Italy, the “sweet” Taggiasca predominates along the coasts of Liguria. The northern limit of olive cultivation is the restricted area around Lake Garda (45 400 N), an important niche of PDO high-quality production. Here Casaliva, the principal variety, supplies light and fragrant oils.9 The two main Islands of Italy, Sicily and Sardinia, provide a relevant contribute, both for olive production and for genetic biodiversity with, respectively, 25 and 28 varieties.10
13 Some of the principal Sicilian varieties are Nocellara del Belice and Biancolilla, whereas Bosana and Semidana in Sardinia. The Italian VOO production is extremely varied in terms of chemical composition and organoleptic properties. The numerous varieties offer oils rich in polyphenols with fruity sensations and, often, a decisive spicy palate. Grass, artichoke, fresh almond, and tomato are the most frequent sensorial attributes.9,14 In addition, aromatic herbs, apple, banana, and berries flavors make some

The chemical composition of Italian virgin olive oils Chapter | 4

53

FIGURE 4.2 Italian olive oil production (years 2006
18, 2019 estimated)5 and respective contributions from south, center, insular, and north macro-areas.

Italian monovarietal VOOs unique. Those attributes can be felt in VOO all along the peninsula, and the specific balance between them characterizes each cultivar. The richness of the varietal heritage and the heterogeneity of cultivation environment are subject of great attention by researchers as they are strictly linked to VOO composition and nutraceutical properties.11,15
19 Moreover, the study of the interaction between variety and environment allows us to identify VOOs according to the variety or growing area, also at local scale.18,20 In turn, these two main factors affect others responsible for VOO quality. For instance, olives ripeness degree as well as soil available water content and related plant physiology are varietal-dependent factors and play an important role in determining VOO quality.8,21
23 The processing steps such as olives crushing type, malaxation parameters, oil extraction system, and storage conditions also contribute to profile the VOO composition.24
28 In addition, microbial component, both yeasts and bacteria, thanks to their enzymatic activity carried out during the transformation and storage phases plays an active role in determining VOO composition.29,30 Among the genetic, agronomic, technological, microbiological, and environmental influencing factors on the composition and quality of oils, the variety is recognized as predominant. For this reason, in this chapter, we will discuss the great variability of Italian VOO composition by monovarietal data. The aim is to offer a framework for the most important Italian cultivars about the VOO content of fatty acids, polyphenols, tocopherols, squalene, and sterols as chemical compounds with important effects for human health and disease prevention.

4.2 Fatty acids At first, VOO health benefits have been attributed to its distinctive fatty acids (FAs) profile, principally represented by the monounsaturated fatty acids (MUFA), in particular oleic acid (C18:1, 56%
82%), and in minor part by palmitoleic (C16:1, 0.1%
5.1%), eicosenoic (C20:1, ,0.4%), and heptadecenoic (C17:1, ,0.3%) acids. The reduction of lowdensity lipoprotein (LDL) cholesterol, the lower blood pressure, the downregulation of some genes are involved in cancer cells proliferation, and the protection to cell membranes oxidative stress has been linked to the intake of VOO with high oleic acid concentrations.31 Polyunsaturated fatty acids (PUFAs) of VOO are represented by linoleic acid (C18:2, ω-6) and α-linolenic acid (C18:3, ω-3), which may range between 3% and 19%, and 0.11% and 1.0%, respectively.8,11,14 These essential FAs are necessary for human well-being as their metabolites are of great importance for brain, liver, kidney, retina, and gonads functioning.31 Despite that, the excess of ω-6 with respect to ω-3 may have prooxidative effects; thus it is important to balance the intake of these two essential FAs. With this regard, VOOs provide an optimal ω-6/ω-3 ratio, ranging between 5:1 and 10:1. In addition, the presence of high MUFA concentration, with respect to PUFA, guarantees the VOO oxidative stability and large shelf life.8 Finally, a lower fraction of the VOO FA profile is represented by saturated fatty acid: palmitic acid (C16:0, 7.8%
17.3%), stearic acid (C18:0, 0.2%
3.2 %), arachidic acid (C20:0, ,0.7%), and margaric acid (C17:0, ,0.3%).

TABLE 4.1 Range values (minimum
maximum) reported by literature, of virgin olive oils principal fatty acids (%), total sterols (mg/kg), squalene (mg/kg), total polar phenols (mg/kg), and tocopherols (mg/kg) from 42 Italian varieties. Region of origin

Variety

Palmitic acid

Oleic acid

Linoleic acid

Abruzzo

Dritta Gentile di Chieti Intosso

14.0
15.1 15.1
16.5

69.4
73.3 66.1
66.4

12.3
14.2

Cellina di Nardo` Coratina Nociara Ogliarola Barese Ogliarola Salentina Peranzana

Apulia

Calabria

Campania

Emilia Romagna

Lazio

Total sterols

Squalene

Phenolic content

Tocopherols

References

7.5
9.7 11.8
12.8

4840 6180

450 289

159 164

[43,44]

66.1
75.7

6.8
13.2

7070

393

235

[43,44]

13.9
16.1

69.0
73.3

8.3
9.1

126
449

351

[20,45,46]

9.7
13.7 12.4
15.3 13.1
15.4

75.2
81.4 71.5
75.2 69.9
74.5

5.4
8.9 6.1
9.5 7.0
10.6

285
1039 167
350 134
434

115
301 87
223

[8,9,11,15
17,38,43,45
51]

14.1
15.6

68.9
72.4

7.1
10.2

134
746

323
639

[20,43,45,46,52]

12.4
14.7

69
73.9

9.2
11.0

190
379

286
295

[9,15,17,27,43,45,46]

Carolea Grossa di Gerace Ottobratica Sinopolese

13.3
16.0 12.2
15.9

69.6
74.9 60.7
72.7

6.0
8.7 10.3
17.6

1413
1796 1162
1906

148
716 102
349

197
356 177
401

[15,21,50,51,53
55]

12.7
16.8 11.9
16.0

66.2
74.1 70.3
77.7

7.1
11.4 4.3
7.2

1041
1808 1527
1840

199
1521 192
488

88
335 70
351

[21,38,41,47,53,55]

Ortice Pisciottana Ravece Rotondella

10.4
16.3 13.8
13.9 12.2
16.2 14.3
14.4

59.7
69.6 68.8
71.4 57.2
73.4 69.9
71.9

10.8
17.1 8.8
8.9 9.2
16.0 8.2
8.5

100 189 275
431 224

Correggiolo Ghiacciolo Nostrana di Brisighella

14.9 13.3 16.0

72.3 74.5
77.2 71
77.9

8.6 6.1
7.7 4.8
7.7

136
746 212
418 137
434

Carboncella Itrana

14.1 11.6
15.0

74.7
76.2 74.6
79.5

5.6
7.5 2.9
9.3

696
1498 1788

993
1483

4659

6550
6950 5705

[43,44]

[38,45
48,51] [17,45,46,51]

[21,41,53,54]

[21,38,41,47,53] [56
58] [56,57]

172

[9,15,56,57,59] [56,57] [60,61] [60,62] [60,62]

300
533 250
426

266
300 94
345

[14,63
65]

490
700

165
188

[66,67]

[9,11,15,38,47,48]

Liguria

Taggiasca

Lombardy

Casaliva

12.4

76.8

6.7

200
371

62
281

[9,22,66]

Marche

Ascolana Mignola Piantone di Mogliano Raggia

11.6
13.4 14.4
14.6 11.1
12.1

75.6
79.8 71.2
72.2 76.6
78.8

4.2
6.1 8.7
9.9 5.6
6.9

264
501 523
714 479
493

225

[9,14,63,68]

11.5
13.6

74.0
77.6

7.3
8.6

343
762

[9,14,63] [9,14,15,63]

[9,14,16,63]

Sardinia

Sicily

Tuscany

Bosana Nera di Oliena Semidana Tonda di Cagliari Tonda di Villacidro

12.1
14.4 12.6
13.9

68.6
74.3 71.2
74.3

9.3
12.9 7.9
11.8

1018
1068 1292

5887,9
7051 6824

161
817 155
509

136
354 199
231

[8,9,11,15,24,40,68,69]

13.0
15.3 14.7
16.5

65.7
73.5 62.0
68.5

9.6
16.1 13.7
19.1

1001
1015 1529

5837
8257 9221

135
564 142
507

110
298 127
247

[8,11,24,69]

13.2
13.3

71.8
74.3

9.6
9.9

1014

5336

197
451

163
289

[11,69]

Biancolilla Cerasuola Nocellara del Belice Tonda Iblea

10.6
14.8 7.8
12.4 10.9
14.5

69.7
78.0 73.6
82.4 69.1
76.4

5.7
12.3 7.9
8.7 6.3
11.4

1354

2136
8950 1040
4010 2499
9670

132
680 670
1340 317

178
223

[9,13,16,42,50,70,71,73]

9.7
10.4

67.4
80.2

8.6
14.8

6371
7474

560

193
217

[13,28,68,74]

Frantoio

10.1
15.9

70.6
81.8

4.9
10.0

3620
4300

95
958

29
249

[9,11,15,16,20,22,23,34
37,39,45,46,48,50,

632
1372

1612

[11,69]

[8,11]

[9,13,42,56,57,70
72] [13,42,71,72]

51,66,67,75
77]

Veneto

Leccino Moraiolo Pendolino

12.5
17.3 12.5
13.6 12.7
15.8

63.9
79.5 74.8
76.2 72.7
76.9

4.4
14.0 7.1
7.4 6.1
7.0

Grignano

11.6

78.6

5.5

1551
1637

3500
5200

1363 8260

100
1520 180
530 151
254

90
506 221
278 83
246

[9,11,15,20,36,38,39,43
48,51,66,67,72,76
78]

144
300

159

[44,46,66]

[9,15,35,39,46,67,68] [38,47,48,66,67]

56

PART | 1 General Aspects of Olives and Olive Oil

The FA composition of Italian VOO described in Table 4.1 shows a high variability, both between and within varieties. Such variability can be attributed principally to genetic factors (namely, variety) that regulate the response to environmental conditions,18,19,32,33 agronomical practices,23,34
37 and fruit maturation degree.8,21,38
42 According to Li et al.19 and Mailer et al.,32 when high temperatures and drought stress conditions occur during oil accumulation period, VOOs with reduced oleic acid and raised palmitic, linoleic, and α-linolenic acids relative contents are obtained. In some cases the limits imposed by the International Olive Council (IOC) can be exceeded.3 However, similar changes in FA profiles have also been obtained from particularly rainy growing areas.33 Those results may suggest that the environmental variable that mostly influences FA biosynthesis rate and composition is temperature, whereas rainfall regimes represent an indirect variable as it affects temperatures, daily thermal excursion, and solar radiation. However, due to the absence of clear evidence on these relationships, we could not end up in a safe conclusion. According to those authors, looking at the FA profiles of the Italian varieties involved, Coratina and Frantoio seem to have lower sensitivity to environmental changes than Leccino, Pendolino, Taggiasca, or Correggiolo, but higher if compared to other international varieties (e.g., Picual and Koroneiki).19,32,33 Looking at Italian productions, Leccino VOOs achieve the highest oleic acid (79.5%) and lowest palmitic and linoleic acid concentrations (12.5% and 4.4%) at hilly inland areas of Umbria,36,39 whereas opposite situations have been observed at coastal areas of Abruzzo, Apulia, and Calabria.38,43,45 On the other hand, as observed also by Li et al.19 and Mailer et al.,32 Frantoio VOOs produced along the Italian Peninsula followed the same trend of Leccino, but reporting lower FA variations.8,36,37,45 Similarly to Frantoio, Itrana, typical cultivar from Lazio, seems to be a relatively low sensitive variety. Indeed, VOOs produced from Lazio15 were in line with those obtained from Calabria38 region, even showing very low concentrations of linoleic acid (2.9%
3.6%). Oppositely, when grown in Sardinia, the same variety showed the aforementioned effects of arid climates.11,19 Biancolilla VOOs produced in Campania have considerably higher oleic acid (76.7%
78%)56,57 relative content than those produced in Sicily (69.7%
72.2%).42,70 Changes in FA composition can be found also at regional or local scale, where the variegate orography contributes to the creation of specific microclimates. With this regard, from Bosana, Carolea, Frantoio, Nostrana di Brisighella, Ortice, Semidana, and Tonda di Cagliari VOO productions, it is possible to observe further examples of varietal-specific sensitivity.40,53,58,62,69,75

Studies on Frantoio and Moraiolo VOO’s composition demonstrated that FAs are mostly affected by seasonal weather conditions rather than irrigation management.23,34,35,37 Furthermore, according to Rosati et al.36 organic soil management poorly influences FA composition, only little decreases of palmitoleic, stearic, and linoleic acids were observed, but not for all cultivars. In order to explain the high FA variability (Table 4.1), we should take into account also variations occurring during fruit maturation process, strictly connected to genetic and environmental factors. The genetic factor depends on the bearing (positive, negative, or negligible) and amounts of oleic, linoleic, or palmitic acid changes during fruit maturation period.8,11,38
41 Moreover, the same variety may assume different FA bearing changes according to the production area, where different bioclimatic conditions influence the timing and rate of maturation process. These are the cases of Coratina VOO grown in different Italian regions8,38 and Bosana VOO from different areas of Sardinia.8,40 If considered the influence of all the described factors, a first thought might be that central and northern areas of Italy can produce VOO characterized by slightly higher MUFA and lower PUFA content with respect to southern regions. However, looking at the single varieties is possible to observe many varieties with high oleic acid concentration, both in northern and southern Italy. For example, even grown under arid or semiarid conditions,79 Cerasuola, Coratina, Itrana, and Tonda Iblea contain the highest oleic acid concentrations registered for Italian VOO productions.8,13,38 Moreover, we can mention Ascolana Tenera and Piantone di Mogliano from Marche (79.9% and 78.8%, respectively),14,63 Casaliva and Grignano from Lake Garda area (76.8% and 78.6%, respectively),9,44 and Sinopolese (77.7%) from Calabria.38 Such evidences highlight the strong link of the numerous Italian varieties and the microclimates of their territory of origin. This means that Italian olive germplasm owns a great potential adaptability to climate changes, which are already affecting this sector.62

4.3 Sterols and triterpenic alcohols Sterols play an important role in mammalian membrane cells regulating its fluidity. Because of their activity in lowering intestinal cholesterol absorption and then cholesterol serum levels, sterols, stanols, and esterified respective forms have been widely used as addictive in functional foods.80 Sterols are widely present in plants. They act as precursor of some steroids involved in developmental processes regulating membrane fluidity, growth, and cell proliferation. VOO is a good source of sterols (usually 1000
2000 mg/kg) broadly lower than corn,

The chemical composition of Italian virgin olive oils Chapter | 4

rapeseed, and sunflower oils.80 Also cereals and some fresh vegetables are good sources of phytosterols. β-Sitosterol (74.2%
89.7%), Δ5-avenasterol (2.6%
18.5%), and campesterol (2.0%
4.6%) are the most representative sterols in VOO.11,54 Other sterols have been commonly reported at lower concentrations (often ,1.5%), among those sitostanol, stigmasterol, and clerosterol. Together with them are detected also the triterpene dialcohols (erythrodiol and uvaol). In the context of product adulteration control, analysis of sterols and triterpene dialcohol is a valid tool, and composition is regulated by IOC.3 However, due to some specific varietal features, together with environmental growing area conditions, it is not rare to observe outside IOC limit cases.11,32,70,81 Mailer et al.32 reported a general increasing trend of campesterol content and a decreasing trend of total sterols at VOO higher latitudes of Australian growing areas. Widely reported is the effect of fruit-ripening degree, also in this case strongly varietal-dependent.11,21,41,81 Some authors reported total sterols and β-sitosterol increase followed by an increase of Δ5-avenasterol.11,81 Among these two molecules, a strong negative relationship has been observed. In the cases of Ogliarola Garganica VOO, total sterols from ripe fruits dropped below the IOC limit of 1000 mg/kg. Conversely, between the main Calabrian varieties, Carolea, Ottobratica, Sinopolese, and Grossa di Gerace, all with high potential sterol content (respectively 1796, 1808, 1840, and 1906 mg/kg),47,53 were observed a different behavior.21,41 Among other Italian varieties, VOO from Frantoio, Leccino, Nociara, and Tonda di Cagliari can be considered a good sterolic source (Table 4.1).

4.4 Squalene Squalene (SQ) is a natural lipid, a triterpenoid hydrocarbon composed of 30 carbon atoms. SQ is widely present in nature. It could be found in animals, particularly in mammals, plants, fungal, and bacterial cells. Olive oil is the major vegetal edible source. It is present in lower amounts in other vegetable oils such as pumpkin, amaranth seed, soybean, sunflower, palm, and rice bran.82 SQ is present in skin surface lipids and, acting as antioxidant, contributes to protect skin from UV rays. An active role in the prevention of atherosclerosis diseases, colon and breast cancer, has been recognized. Thanks to its nutritional benefits and chemical properties, nowadays squalene is widely adopted in diseases management, pharmaceutical and cosmetic applications.82 In olive fruits, squalene acts as a precursor of sterols and triterpenoids. It is the principal molecule among the minor fractions of VOO composition. Content values may vary between 1000 and 12,000 mg/kg.82 Variations of squalene content (see Table 4.1) could be explained by

57

the influence of genetic, agronomic, agroclimatic, and technological factors. The most important one is the cultivar.11,44,74,76 It also affects SQ variations during fruit maturation, generally showing a decreasing trend.11,71 Little or negligible effects of extraction parameters, such as malaxing conditions or decanter type, have been reported.44,64 Similarly, no obvious changes can be observed during storage.64 This suggests a marginal role of SQ in the VOO oxidation process. On the other hand, environmental conditions related to the growing areas are relatively stronger, SQ concentration seems to be positively correlated to altitude and more humid conditions.33,58 Despite the rising interest in SQ health properties and potential applications, there are still few data available in recent literature about its levels in Italian VOOs. Among them, high concentrations, above 6000 mg/kg, were reported along the whole peninsula by Carboncella64 (Lazio), Gentile di Chieti and Intosso44 (Abruzzo), Bosana11 (Sardinia), Tonda Iblea74 (Sicily), whereas Grignano44 (Veneto), Biancolilla and Nocellara del Belice74 (Sicily), together with Semidana and Tonda di Cagliari11 (Sardinia), can be considered varieties with very high potential SQ content ( . 8000 mg/kg).

4.5 Phenolic compounds The VOO phenolic compounds (PC) include about 30 molecules from different chemical classes: phenolic alcohols, such as hydroxytyrosol (3,4-DHPEA) and tyrosol (pHPEA), phenolic acids (e.g., caffeic, vanillic, and p-coumaric), flavones (luteolin and apigenin), lignans (1-acetoxypinoresinol and pinoresinol), and secoiridoids. The latter group represents the largest fraction. The principal ones are the aglycon forms of oleuropein (3,4-DHPEAEA) and ligstroside (p-HPEA-EA), the dialdehydic forms of their decarboxymethylated derivatives, known as oleacein (3,4-DHPEA-EDA) and oleocanthal (p-HPEAEDA).83 The secoiridoids act as natural antioxidants protecting VOO against autoxidation processes during storage and are responsible for the bitter and pungent attributes. A considerable quantity of evidence indicates that VOO PC can exert biological activities due to their antioxidant, ibuprofen such as antiinflammatory and chemopreventive properties. A protective role against cardiovascular, metabolic diseases, and human cancer has been attributed to hydroxytyrosol, oleocanthal, and oleacein.31,84 Based on scientific evidences on health studies, in 2012 the European Food Safety Authority authorized the functional health claim on VOO polyphenols as they “contribute to the protection of blood lipids from oxidative stress.”85 This benefit became effective within a minimum concentration of 5 mg of hydroxytyrosol and its

58

PART | 1 General Aspects of Olives and Olive Oil

derivatives (i.e., oleuropein complex and tyrosol) in 20 g of VOO. From an analytic point of view, this health claim is still inaccurate because the European regulation does not indicate any specific analytical method.86 The difficult accessibility of secoiridoids standards, and to the possible generation of phenolic artifacts under specific analytical conditions, explains the great number of analytical techniques and methods proposed.83,86 This makes hard a direct comparison of data regarding individual PC. On the other hand, because of its simple and efficient procedure (although a low specificity due to the interference of non-PC), the total phenolic content of VOO is commonly measured by the well-known Folin
Ciocalteu colorimetric assay. The values of total phenolic content coming from some of the most relevant Italian varieties were summarized in Table 4.1. Literature data show a great variability, both between and within cultivars. Indeed, despite each variety reacts specifically to other sources of variability, abiotic stress conditions, fruit-ripening degree, or extraction technology became often predominant factors in determining the VOO phenolic amount and profile. Production areas differ in specific meteo-climatic conditions due to orography (altitude, slope, and exposition), latitude, distance from the sea, and soil type. With this regard, many studies reported some unambiguous varietal-specific changes on VOO phenolic profile according to production areas.20,58,60,66,72,75 The accumulation of PC in olive pulp came from the secondary metabolism, activated, as a defense, by biotic or abiotic stresses, specifically drought and high temperatures.87 Indeed the biosynthesis of PC, principally oleuropein, is promoted by water stress conditions at the earliest stages of fruit development.37,87 Conversely, if stress conditions occur at advanced fruit developmental stage, PC changes are negligible. Summer rainfall is much more effective than irrigation treatment in determining phenolic content.19,23,34,35 Moreover, higher temperatures and sunlight exposure are positively correlated to VOO phenolic concentration.34 Other agronomical practices, such as soil management, can modify phenolic amount.22,36,77 In particular, nitrogen fertilization seems to affect negatively phenolic metabolism of Leccino and Frantoio cultivars.36,77 It is widely known that PC in drupes gradually decrease and, accordingly, the same happens in VOOs. The enzymatic hydrolysis of oleuropein is the principal cause.87 Indeed, in VOO, the secoiridoids are those that suffer the greatest decrease, in particular oleuropein derivatives.8,81 Conversely, flavonoids and phenolic alcohols may assume an increasing trend.8,61 Lignans and phenolic acids change minimally.8,81 The amount and rate of phenolic loss, as well as the changing trends of individual molecules, is first varietal dependent. For instance,

Semidana, Tonda di Cagliari, Correggiolo, and Leccino are characterized by a rapid phenolic decrease, while others (e.g., Bosana, Coratina, Sinopolese, and Itrana) by a slow one.8,48,61,78 Oil extraction process strongly affects VOO phenols. For instance, the crushing process typology (hammer, blades, destoning), working speed, and temperatures determine the activation of endogenous enzymes (e.g., lipoxygenase, peroxidase, polyphenoloxidase, and β-glucosidase) and thus the potential accumulation of PC in VOOs.61,67 According to Morrone et al.,61 hammer crusher rather than blades can improve, until an increase of 54%, secoiridoids extraction in Correggiolo VOOs. Malaxation process promotes aggregation of oil drops and enzymatic activity, responsible for VOO phenolic and volatile profiles. Therefore as affecting oil yield and VOO quality, working time, temperature, and oxygen parameters have been deeply studied and optimized following varietal-specific requirements.25,26,48,81 Innovative technologies (e.g., ultrasounds and thermal conditioning) aimed at reducing malaxation time and oxidation of phenols reported strong increases of phenolic fraction, in some cases, almost doubled (e.g., Peranzana).26,81 Moreover, the introduction of innovative decanters, such as two-phase and real-time setup technologies, allowed to avoid additional water in the process increasing phenolic content.49,65 Finally, numerous filtration and storage systems have been proposed to enhance VOO shelf life. The use of argon or nitrogen seems to be the most effective technique, both to filter and to remove headspace O2.24,27,28,46,50,59 As we largely discussed, phenolic composition is affected by numerous factors and technological parameters. This explains the wide range of values reported by literature (Table 4.1). For this reason, it became hard to classify cultivars as “poor” or “rich” source of phenols. From the same cultivar can be produced VOO with total phenolic content lower than 100 mg/kg but also with concentrations up to 700 mg/kg and peaks above 1300 mg/ kg. With this regard, we can mention several examples along the whole peninsula: Bosana,8,24 Carolea,51,55 Cerasuola,72 Coratina,8,51 Correggiolo,60,61 Frantoio,37,75 Leccino,72,78 Mignola,14,63 Ogliarola Salentina,46,52 Raggia,14,63 and Ottobratica.48,55

4.6 Tocopherols Tocopherols are derivatives of the 2-methyl-6-chromanol linked to an isoprenoid chain of 16 carbon atoms. Tocopherols and tocotrienols are grouped under the collective name of vitamin E. Both have four natural isomers indicated with the Greek letters α, β, γ, and δ differing in methyl substituent’s number and position on the aromatic ring. Tocopherols act as chain-breaking antioxidants

The chemical composition of Italian virgin olive oils Chapter | 4

reacting with free radicals and quenching them. Vegetable seed oils (e.g., rapeseed, corn, sunflower, linseed, cottonseed oils) are the main source. They are also present in cereals and fruits, and in lesser quantities in foods of animal origin such as eggs, liver, and dairy products.88 Tocopherols are the second main class of antioxidants in VOO after polyphenols. Levels may vary between 70 and 600 mg/kg (Table 4.1). The main vitamin E isoform in VOOs is α-tocopherol (α-T) (95%
99%), the one with the greatest biological activity. Thanks to its antioxidant properties, α-T strongly contributes to VOO oxidative stability.66 Tocopherols contribute to the functional and nutraceutical VOO properties. As inhibitors of the reactive oxygen species (ROS) at cellular level, tocopherols contribute to reduce the risk of cancer, cellular aging, and cardiovascular diseases development.88 As well as the other VOO components, tocopherol levels are the result of the complex interaction between genetic factor, environmental conditions, and ripening stage. Similarly to squalene, warm temperatures and water availability during fruit development promote tocopherols concentration.32,33,66 Moreover, in the fruit maturation process, tocopherols gradually slow down, and decreasing rate follows varietal-specific trends.8,22,41,48 Agronomical practices,35,77 extraction technology,24,49,67 and storage24,27 marginally affect tocopherols concentration. As well as PC, tocopherols decrease during storage, confirming their active role in VOO protection from autooxidation.24 Most of the Italian cultivars are good sources of tocopherols, levels in VOO range between 200 and 300 mg/kg (Table 4.1). Only a few varieties, probably due to the small number of data currently available, did not achieve 200 mg/kg: Taggiasca,67 Ravece,68 Gentile di Chieti,44 Grignano,66 Dritta,44 whereas Leccino11 and Ogliarola Salentina52 stand out for very high α-T levels, up to 600 mg/kg. The other tocopherol isoforms, β and γ, are present in significantly lower quantities (from traces up to 15 mg/ kg).66,73 Unfortunately, information from literature is not complete, and in most cases only α-T levels are reported. The incomplete data suggest that the variability associated with the β and γ forms is greater than that related to α form.

4.7 Comparisons of olive oils with other edible oils The VOO has a unique composition if compared to other vegetable oils. It is obtained by mechanical extraction starting from the fruit of the olive tree. Olive fruits are rich in PC, up to 80 mg/g.87 Secoiridoids, exclusives

59

molecules of the Oleaceae family, are the most important phenolic class both from a quantitative and qualitative point of view.31,83,84 Numerous studies show that no other vegetable oil has the same compositional quality and stability as VOO.68,82,89,90 This derives not only by the levels of FAs, sterols, triterpenes, squalene, phenols, pigments, and tocopherols, but mostly from their synergistic action.91 For these reasons, VOO commercial prices are higher than other vegetable oils. Therefore it is exposed to numerous attempts of fraud, adulteration, and sophistications.3 With this regard, one of the priorities is to protect the image of the product and to be able of ensuring quality and authenticity.

4.8 Implications for human health and disease prevention Numerous studies linked the VOO consumption to several health benefits: lower risk of cardiovascular diseases, breast cancer, diabetes type 2, and the development of atherosclerosis and neurodegenerative diseases.31,84 Such nutraceutical properties come from the synergic activity of the several bioactive molecules of its “minor fraction” together with the unique FA composition, rich in MUFA and principally oleic acid.91 Conversely, other studies, comparing diets based on different vegetable oils, found out that olive oils (with no distinction between refined or virgin or high-quality products) were less effective in lowering total cholesterol, LDL, and triglycerides levels in blood than other vegetable oils richer in PUFA (n-3 and n-6) and phytosterols.92 Authors stress on the presence of some contrasting evidences on VOO health properties, probably due to the interaction between the other diets components or to different intake amounts and subjects involved.84,92 Anyway, findings agree with a fundamental aspect: only highquality VOOs, mostly those characterized by the presence of molecules with high antioxidant activity (hydroxytyrosol, tyrosol, and secoiridoids) can provide the aforementioned benefits.93 As we largely discussed, the Italian territory is a wide source of olive biodiversity. Furthermore, from each region is possible to achieve the best quality throughout the local varieties, optimally adapted to specific different bioclimates,9,11,13,66 or ongoing climate changes.79 Nowadays, the innovative and effective oil extraction technologies together with the knowledge of specific varietal requirements, in terms of stress conditions, nutrition, and harvest period, are spreading out. This makes possible, exploiting each varietal potential, the production of VOOs characterized by high levels of phenolics, largely above the minimum health claim levels,85 oleic acid, sterols, squalene, and tocopherols.

60

PART | 1 General Aspects of Olives and Olive Oil

References 1 2 3 4

5 6

7

8

9

10

11

12

13

14

15

16

17

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18 Culeddu N, Chessa M, Bandino G, et al. Classification of monovarietal Sardinian extra virgin olive oils by 1H NMR metabolomic approach. Eur J Lipid Sci Technol. 2017;119:1700035.

19 Li X, Flynn JD, Wang SC. The effects of variety, growing region, and drought stress on fatty acid and sterol compositions of California olive oil. J Am Oil Chem Soc. 2019;96:215
230. 20 Ragusa A, Centonze C, Grasso ME, et al. Composition and statistical analysis of biophenols in Apulian Italian EVOOs. Foods. 2017;6:90. 21 Piscopo A, Zappia A, De Bruno A, Poiana M. Effect of the harvesting time on the quality of olive oils produced in Calabria. Eur J Lipid Sci Technol. 2018;12:1700304. 22 Trombetta D, Smeriglio A, Marcoccia D, et al. Analytical evaluation and antioxidant properties of some secondary metabolites in northern Italian mono-and multi-varietal extra virgin olive oils (EVOOs) from early and late harvested olives. Int J Mol Sci. 2017;18:797. 23 Caruso G, Gucci R, Urbani S, et al. Effect of different irrigation volumes during fruit development on quality of virgin olive oil of cv. Frantoio. Agric Water Manage. 2014;134:94
103. 24 Fadda C, Del Caro A, Sanguinetti AM, et al. Changes during storage of quality parameters and in vitro antioxidant activity of extra virgin monovarietal oils obtained with two extraction technologies. Food Chem. 2012;134:1542
1548. 25 Selvaggini R, Esposto S, Taticchi A, et al. Optimization of the temperature and oxygen concentration conditions in the malaxation during the oil mechanical extraction process of four Italian olive cultivars. J Agric Food Chem. 2014;62:3813
3822. 26 Veneziani G, Esposto S, Taticchi A, et al. Cooling treatment of olive paste during the oil processing: impact on the yield and extra virgin olive oil quality. Food Chem. 2017;221:107
113. 27 Lozano-Sa´nchez J, Cerretani L, Bendini A, Gallina-Toschi T, Segura-Carretero A, Ferna´ndez-Gutie´rrez A. New filtration systems for extra-virgin olive oil: effect on antioxidant compounds, oxidative stability, and physicochemical and sensory properties. J Agric Food Chem. 2012;60:3754
3762. 28 Smeriglio A, Toscano G, Denaro M, De Francesco C, Agozzino S, Trombetta D. Nitrogen headspace improves the extra virgin olive oil shelf-life, preserving its functional properties. Antioxidants. 2019;8:331. 29 Santona M, Sanna ML, Multineddu C, et al. Microbial biodiversity of Sardinian oleic ecosystems. Food Microbiol. 2018;70:65
75. 30 Fancello F, Multineddu C, Santona M, et al. Bacterial biodiversity of extra virgin olive oils and their potential biotechnological exploitation. Microorganisms. 2020;8:97. 31 Lombardo L, Grasso F, Lanciano F, Loria S, Monetti E. Broadspectrum health protection of extra virgin olive oil compounds. In: Atta-Uhr-Rahman, ed. Studies in Natural Products Chemistry. Amsterdam: Elsevier; 2018:41
77. 32 Mailer RJ, Ayton J, Graham K. The influence of growing region, cultivar and harvest timing on the diversity of Australian olive oil. J Am Oil Chem Soc. 2010;87:877
884. 33 Yu L, Wang Y, Wu G, Jin J, Jin Q, Wang X. Quality and composition of virgin olive oils from indigenous and European cultivars grown in China. J Am Oil Chem Soc. 2019. 34 Caruso G, Gucci R, Sifola MI, et al. Irrigation and fruit canopy position modify oil quality of olive trees (cv. Frantoio). J Sci Food Agric. 2017;97:3530
3539. 35 Bucelli P, Costantini EAC, Barbetti R, Fanchini E. Soil water availability in rainfed cultivation affects more than cultivar some nutraceutical components and the sensory profile of virgin olive oil. J Agric Food Chem. 2011;59:8304
8313.

The chemical composition of Italian virgin olive oils Chapter | 4

36 Rosati A, Cafiero C, Paoletti A, et al. Effect of agronomical practices on carpology, fruit and oil composition, and oil sensory properties, in olive (Olea europaea L.). Food Chem. 2014;159:236
243. 37 Gucci R, Caruso G, Gennai C, Esposto S, Urbani S, Servili M. Fruit growth, yield and oil quality induced by deficit irrigation at different stages of olive fruit development. Agric Water Manage. 2019;212:88
98. 38 Poiana M, Mincione A. Fatty acids evolution and composition of olive oils extracted from different olive cultivars grown in Calabrian area. Grasas Aceites. 2004;55:282
290. 39 Portarena S, Farinelli D, Lauteri M, Famiani F, Esti M, Brugnoli E. Stable isotope and fatty acid compositions of monovarietal olive oils: implications of ripening stage and climate effects as determinants in traceability studies. Food Control. 2015;57:129
135. 40 Morrone L, Neri L, Cantini C, Alfei B, Rotondi A. Study of the combined effects of ripeness and production area on Bosana oil’s quality. Food Chem. 2018;245:1098
1104. 41 Mafrica R, Piscopo A, De Bruno A, et al. Integrated study of qualitative olive and oil production from three important varieties grown in Calabria (southern Italy). Eur J Lipid Sci Technol. 2019;121:1900147. 42 Chiavaro E, Vittadini E, Rodriguez-Estrada MT, et al. Monovarietal extra virgin olive oils: correlation between thermal properties and chemical composition. J Agric Food Chem. 2007;55:10779
10786. 43 Chiavaro E, Estrada MTR, Bendini A, Cerretani L. Correlation between thermal properties and chemical composition of Italian virgin olive oils. Eur J Lipid Sci Technol. 2010;112:580
592. 44 Ambra R, Natella F, Lucchetti S, Forte V, Pastore G. α-Tocopherol, β-carotene, lutein, squalene and secoiridoids in seven monocultivar Italian extra-virgin olive oils. Int J Food Sci Nutr. 2017;68:538
545. 45 Laddomada B, Colella G, Tufariello M, et al. Application of a simplified calorimetric assay for the evaluation of extra virgin olive oil quality. Food Res Int. 2013;54:2062
2068. 46 Baiano A, Gambacorta G, Terracone C, Previtali MA, Lamacchia C, La Notte E. Changes in phenolic content and antioxidant activity of Italian extra-virgin olive oils during storage. J Food Sci. 2009;74:C177
C183. 47 Giuffre` AM, Louadj L. Influence of crop season and cultivar on sterol composition of monovarietal olive oils in Reggio Calabria (Italy). Czech J Food Sci. 2013;31:256
263. 48 Giuffre` AM. The evolution of free acidity and oxidation related parameters in olive oil during olive ripening from cultivars grown in the region of Calabria, South Italy. Emir J Food Agric. 2018;30:539
548. 49 Squeo G, Tamborrino A, Pasqualone A, et al. Assessment of the influence of the decanter set-up during continuous processing of olives at different pigmentation index. Food Bioprocess Technol. 2017;10:592
602. 50 Di Serio MG, Giansante L, Di Loreto G, Di Giacinto L. Shelf life of extra-virgin olive oils: first efforts toward a prediction model. J Food Process Preserv. 2018;42:e13663. 51 Gambacorta G, Faccia M, Trani A, Lamacchia C, Gomes T. Phenolic composition and antioxidant activity of Southern Italian monovarietal virgin olive oils. Eur J Lipid Sci Technol. 2012;114:958
967. 52 Di Serio MG, Di Loreto G, Giansante L, et al. Influence of the nocturnal harvesting of olives from Salento (Italy) on the quality of the extra virgin olive oil. Grasas Aceites. 2014;65:044.

61

53 Piscopo A, De Bruno A, Zappia A, Ventre C, Poiana M. Characterization of monovarietal olive oils obtained from mills of Calabria region (southern Italy). Food Chem. 2016;213:313
318. 54 Di Serio MG, Di Giacinto L, Di Loreto G, et al. Chemical and sensory characteristics of Italian virgin olive oils from Grossa di Gerace cv. Eur J Lipid Sci Technol. 2016;118:288
298. 55 Piscopo A, De Bruno A, Zappia A, et al. Effect of olive storage temperature on the quality of Carolea and Ottobratica oils. Emir J Food Agric. 2018;30:563
572. 56 Di Vaio C, Nocerino S, Paduano A, Sacchi R. Characterization and evaluation of olive germplasm in southern Italy. J Sci Food Agric. 2013;93:2458
2462. 57 Grasso F, Paduano A, Corrado G, Ambrosino ML, Rao R, Sacchi R. DNA diversity in olive (Olea europaea L.) and its relationships with fatty acid, biophenol and sensory profiles of extra virgin olive oils. Food Res Int. 2016;86:121
130. 58 Di Vaio C, Nocerino S, Paduano A, Sacchi R. Influence of some environmental factors on drupe maturation and olive oil composition. J Sci Food Agric. 2013;93:1134
1139. 59 Sacchi R, Caporaso N, Paduano A, Genovese A. Industrial-scale filtration affects volatile compounds in extra virgin olive oil cv. Ravece. Eur J Lipid Sci Technol. 2015;117:2007
2014. 60 Rotondi A, Lapucci C, Morrone L, Neri L. Autochthonous cultivars of Emilia Romagna region and their clones: comparison of the chemical and sensory properties of olive oils. Food Chem. 2017;224:78
85. 61 Morrone L, Pupillo S, Neri L, Bertazza G, Magli M, Rotondi A. Influence of olive ripening degree and crusher typology on chemical and sensory characteristics of Correggiolo virgin olive oil. J Sci Food Agric. 2017;97:1443
1450. 62 Barbieri S, Bendini A, Gallina Toschi T. Recent amendment to product specification of Brisighella PDO (Emilia-Romagna, Italy): focus on phenolic compounds and sensory aspects. Eur J Lipid Sci Technol. 2019;121:1800328. 63 Cecchi T, Passamonti P, Alfei B, Cecchi P. Monovarietal extra virgin olive oils from the Marche region, Italy: analytical and sensory characterization. Int J Food Prop. 2011;14:483
495. 64 Sinesio F, Moneta E, Raffo A, et al. Effect of extraction conditions and storage time on the sensory profile of monovarietal extra virgin olive oil (cv Carboncella) and chemical drivers of sensory changes. LWT—Food Sci Technol. 2015;63:281
288. 65 Pastore G, D’Aloise A, Lucchetti S, et al. Effect of oxygen reduction during malaxation on the quality of extra virgin olive oil (cv. Carboncella) extracted through “two-phase” and “three-phase” centrifugal decanters. LWT—Food Sci Technol. 2014;59:163
172. 66 Tura D, Gigliotti C, Pedo` S, Failla O, Bassi D, Serraiocco A. Influence of cultivar and site of cultivation on levels of lipophilic and hydrophilic antioxidants in virgin olive oils (Olea europaea L.) and correlations with oxidative stability. Sci Hortic. 2007;112:108
119. 67 Lavelli V, Bondesan L. Secoiridoids, tocopherols, and antioxidant activity of monovarietal extra virgin olive oils extracted from destoned fruits. J Agric Food Chem. 2005;53:1102
1107. 68 Chen H, Angiuli M, Ferrari C, Tombari E, Salvetti G, Bramanti E. Tocopherol speciation as first screening for the assessment of extra virgin olive oil quality by reversed-phase high-performance liquid chromatography/fluorescence detector. Food Chem. 2011;125:1423
1429.

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69 Campus M, Sedda P, Delpiano D, et al. Variability in composition, sensory profiles and volatile compounds of Sardinian monovarietal virgin olive oils grown in different areas. Riv Ital Sostanze Grasse. 2013;90:237
248. 70 Naccari C, Rando R, Savo A, et al. Study on the composition and quality of several Sicilian EVOOs (harvesting year 2015). Riv Ital Sostanze Grasse. 2017;94:231
237. 71 Baccouri O, Cerretani L, Bendini A, et al. Preliminary chemical characterization of Tunisian monovarietal virgin olive oils and comparison with Sicilian ones. Eur J Lipid Sci Technol. 2007;109:1208
1217. 72 del Monaco G, Officioso A, D’Angelo S, et al. Characterization of extra virgin olive oils produced with typical Italian varieties by their phenolic profile. Food Chem. 2015;184:220
228. 73 Ranalli F, Ranalli A, Contento S, Casanovas M, Antonucci M, Simone GD. Bioactives and nutraceutical phytochemicals naturally occurring in virgin olive oil. The case study of the Nocellara del Belice Italian olive cultivar. Nat Prod Res. 2013;27:1686
1690. 74 Salvo A, La Torre GL, Rotondo A, et al. Determination of squalene in organic extra virgin olive oils (EVOOs) by UPLC/PDA using a single-step SPE sample preparation. Food Anal Methods. 2017;10:1377
1385. 75 Leporini M, Loizzo MR, Tenuta MC, et al. Calabrian extra-virgin olive oil from Frantoio cultivar: chemical composition and health properties. Emirates J Food Agric. 2018;30:631
637. 76 Pacetti D, Scortichini S, Boarelli MC, Fiorini D. Simple and rapid method to analyse squalene in olive oils and extra virgin olive oils. Food Control. 2019;102:240
244. 77 Ninfali P, Bacchiocca M, Biagiotti E, et al. A 3-year study on quality, nutritional and organoleptic evaluation of organic and conventional extra-virgin olive oils. J Am Oil Chem Soc. 2008;85:151
158. 78 Ciafardini G, Zullo BA. Improvement of commercial olive oil quality through an evaluation of the polyphenol content of the oily fraction of the olive fruit during its period of maturation. J Food Process Technol. 2014;5:12. 79 Pesaresi S, Biondi E, Casavecchia S. Bioclimates of Italy. J Maps. 2017;13:955
960. 80 Piironen V, Lindsay DG, Miettinen TA, Toivo J, Lampi AM. Plant sterols: biosynthesis, biological function and their importance to human nutrition. J Sci Food Agric. 2000;80:939
966. 81 Taticchi A, Selvaggini R, Esposto S, Sordini B, Veneziani G, Servili M. Physicochemical characterization of virgin olive oil obtained using an ultrasound-assisted extraction at an industrial scale: influence of olive maturity index and malaxation time. Food Chem. 2019;289:7
15.

82 Lou-Bonafonte JM, Martı´nez-Beamonte R, Sanclemente T, et al. Current insights into the biological action of squalene. Mol Nut Food Res. 2018;62:1800136. 83 Celano R, Piccinelli AL, Pugliese A, et al. Insights into the analysis of phenolic secoiridoids in extra virgin olive oil. J Agric Food Chem. 2018;66:6053
6063. 84 Foscolou A, Critselis E, Panagiotakos D. Olive oil consumption and human health: a narrative review. Maturitas. 2018;118:60
66. 85 European Community. Council Regulation No. 432/2012 of 16 May 2012 Establishing a list of permitted health claims made on foods, other than those referring to the reduction of disease risk, to children’s development, health. Off J Eur Union 2012;L136:1
40. 86 Tsimidou MZ, Sotiroglou M, Mastralexi A, Nenadis N, Garcı´aGonza´lez DL, Gallina Toschi T. In house validated UHPLC protocol for the determination of the total hydroxytyrosol and tyrosol content in virgin olive oil fit for the purpose of the health claim introduced by the EC Regulation 432/2012 for “Olive Oil Polyphenols”. Molecules. 2019;24:1044. 87 Cirilli M, Caruso G, Gennai C, et al. The role of polyphenoloxidase, peroxidase, and β-glucosidase in phenolics accumulation in Olea europaea L. fruits under different water regimes. Front Plant Sci. 2017;8:717. 88 Shahidi F, De Camargo AC. Tocopherols and tocotrienols in common and emerging dietary sources: occurrence, applications, and health benefits. Int J Mol Sci. 2016;17:1745. 89 Li C, Yao Y, Zhao G, et al. Comparison and analysis of fatty acids, sterols, and tocopherols in eight vegetable oils. J Agric Food Chem. 2011;59:12493
12498. 90 Javidipour I, Erinc¸ H, Ba¸stu¨rk A, Tekin A. Oxidative changes in hazelnut, olive, soybean, and sunflower oils during microwave heating. Int J Food Prop. 2017;20:1582
1592. 91 Schwingshackl L, Hoffmann G. Monounsaturated fatty acids, olive oil and health status: a systematic review and meta-analysis of cohort studies. Lipids Health Dis. 2014;13:154. 92 Ghobadi S, Hassanzadeh-Rostami Z, Mohammadian F, et al. Comparison of blood lipid-lowering effects of olive oil and other plant oils: a systematic review and meta-analysis of 27 randomized placebo-controlled clinical trials. Crit Rev Food Sci. 2019;59:2110
2124. 93 Schwingshackl L, Krause M, Schmucker C, Hoffmann G, Ru¨cker G, Meerpohl JJ. Impact of different types of olive oil on cardiovascular risk factors: a systematic review and network meta-analysis. Nutr Metab Cardiovas. 2019;29:1030
1039.

Section 1.2

Components of olives and olive plant product and uses

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Chapter 5

Bioactive ingredients in olive leaves N. Nenadis1,2, V.T. Papoti3 and M.Z. Tsimidou1,2 1

Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2NatPro-AUTH,

Center for Interdisciplinary Research and Innovation (CIRI-AUTH), Thessaloniki, Greece, 3Perrotis College, American Farm School, Thermi, Greece

Abbreviations CVDs DES MAE NCDs OLf PLE RSM TF TP UAE

cardiovascular diseases deep eutectic solvent microwave-assisted extraction noncommunicable diseases olive leaf pressurized liquid extraction response surface methodology total flavonoid total phenol ultrasound-assisted extraction

5.1 Introduction The evergreen olive tree (Olea europaea L., Oleaceae family) is treasured over the centuries for its fruits used for oil or table
olive production. In the last decades, its leaves [olive leaf (OLf)] attracted the interest of researchers beyond their traditional use as tea and are currently established as a rich source of bioactive secondary metabolites.1,2 Except for the unique bioactive secoiridoid oleuropein—its major constituent—other phenolic/nonphenolic bioactive compounds have been identified and quantified.3,4 Six are the most representative compounds in terms of abundance (Fig. 5.1).1,3
5 Complexity of biotic/abiotic factors has been mainly addressed for the phenolic compounds of OLf.3,6,7 Less is known for the triterpenoids.4,8,9 The chapter focuses on postharvest treatment and extractions means that can be currently applied to produce extracts in a green and safe manner for food, pharmaceutical, and medical uses. Regarding OLf bioactivity, the chapter focuses on applications that are related to the four most important noncommunicable diseases (NCDs), namely, cardiovascular diseases (CVDs), cancers, respiratory diseases, and diabetes based on the latest World Health Organization facts.1 These diseases are related to unhealthy diets and account for the 71% of all

deaths globally. Emphasis is given only on in vivo studies. Findings from in vitro ones, even when biological substrates are employed, remain a challenge for the extrapolation of in vivo health benefits.1 The major compounds or groups of bioactive compounds present in OLfs are presented in Fig. 5.1. Oleuropein, frequently found as the major OLf compound, is reported to range from traces to 143.2 mg/g dry leaf,3,5,6 luteolin-7-O-glucoside in the range 0.155
6.0 mg/g dry leaf,5 apigenin-7-glucoside from 0.12 to 2.33 mg/g dry leaf,5 and verbascoside from 0.2 to 18.6 mg/g dry leaf.3,5 Oleanolic (OA), which prevails in terpenoids, has been reported at concentrations # 34.5 mg/g dry leaf and maslinic (MA) in lower levels (#7.3 mg/g dry leaf).4,8,9 The analysis of the OLf bioactives is commonly achieved with chromatographic techniques. Liquid chromatography coupled to diode array detector can be used for both phenols and terpenic acids as all compounds bear double bonds.3,8,9 Electrospray ionization mode is appropriate for the ionization of both categories to achieve detection, although some authors propose for terpenoids atmospheric pressure chemical ionization as more suitable due to their low polarity.3,12 Terpenoids can also be determined using gas chromatography after derivatization.4 The latter is not feasible for the glucosides due to the large size of the derivatives, which exceeds the mass cutoff of the mass spectrometer detector. The composition of OLfs secondary metabolites depends on biotic/abiotic factors as expected for every plant material. Sampling practices, postharvest treatments, and extraction means affect then the final content in the major bioactive compounds mentioned earlier.

5.2 Sampling Leaves of different ages can be found concurrently all over tree canopy throughout the year.13,14 For an adult

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00056-0 © 2021 Elsevier Inc. All rights reserved.

65

66

PART | 1 General Aspects of Olives and Olive Oil

Structure/nomenclature/common name

O O O O

S E S

D

H

D

FIGURE 5.1 Major bioactive compounds reported in literature for OLfs.3
5 OLfs, Olive leafs.

O O

L

Bioactive ingredients in olive leaves Chapter | 5

tree, leaf life span is up to 3 years long; however, the majority of leaves fall down during the second year, especially when are found in shadow. OLfs can be distinguished from current and old season ones. The former are usually further subdivided into new and mature leaves. New leaves are defined as those growing toward the extreme tip of current-year shoots. Mature ones are those found between the middle and inner end of the same shoots. Apart from leaf age, cultivar, period of leaf development, tree age and vitality, as well as surrounding milieu affect leaf morphological characteristics. Leaf shape and size varies. Its upper side is light to dark green, while the opposite one is ash gray to green. Typical cultivar attributes (length, width, and shape) refer to mature leaves. Mean leaf axial dimensions (length and width) vary between 5
11 and 1
3 cm, respectively. It has to be stressed that sampling time, except for the developmental stage of the tree, is important as it affects canopy density and shading of leaves that make samples of exactly same characteristics (cultivar, orchard, environment, agricultural practices), simultaneously collected from different trees of the same orchard to present statistically significant different levels in individual phenolics (e.g., verbascoside ranged from 62 to 1278 μg as tyrosol/g dry leaf), as well as total content in bioactive classes [e.g., total phenols (TPs), 34
64 mg as oleuropein/g dry leaf and total flavonoids (TFs), 1885
3596 μg as morin/g dry leaf].6,7 Such differences reported by Papoti and Tsimidou,6 who studied the “within cultivar” effect, were considered more significant than the rest examined factors, that is, cultivar, leaf age, and sampling time. The latter, in addition to the fact that many parameters are interrelated, reveal that optimal values in bioactive characteristics cannot be easily predictable6 and comparison of results has to be done with caution from representative leaf populations. Biotic factors such as cultivar, leaf age, fungi and bacteria, as well as alternate bearing affect leaf content and composition in inorganic elements, fats, sugars, proteins, phenolics, uronic acids, and the Krebs cycle acids.3,15
18 Abiotic parameters such as water deficiency, salinity, frost, ultraviolet radiation, nutrients, and climatic conditions in the growing zone may notably affect qualitatively and quantitatively the composition.3 Information on the effect of the abovementioned factors is mainly available for oleuropein. Talhaoui et al.3 reported increase during the ripening of the olive drupe as a consequence to parallel increase in polyphenol protein oxidase content and activity in the leaves. The same applies in case of fungi and bacteria attack. This is verified by higher levels found in pathogen resistant cultivars. The levels of the respective secoiridoid seem to be higher in young leaves than old ones and the same has been observed in high-load seasons. Regarding the effect of abiotic factors, oleuropein tends to increase in OLfs in

67

response to water deficit in order to counteract the oxidation caused by the induced stress. It is also involved in the protection against salinity stress via serving as a “glucose reservoir for osmoregulation or high energyconsuming processes required for plant adaptation to salinity.” Furthermore, it accumulates in cold periods also as part of an antioxidant plant protection mechanism. Full sunlight reception favors oleuropein increase in comparison to OLfs from trees grown in the shade. Positive is the influence of nutrients as shown by spraying of leaves with a mixture of urea nitrogen, copper, manganese, and zinc. A decrease has been observed for orchards cultivated at sea level. Less is known for other bioactive phenols.3 An increase in the concentration of flavonoids during spring has been observed as a consequence of leaf renewal. Flavonoids such as luteolin-7-O-glucoside may contribute toward protection from ultraviolet radiation. This compound as well as apigenin-7-O-glucoside show increasing tendency upon boron deficiency. Little information is available for terpenoids. OA, as observed in the leaves of Picual and Cornezuelo cultivars, showed a minimum value between October and November reaching in December the levels found in July.9 According to another study,4 its concentration is low in young leaves, whereas in mature ones, its levels may become B2.5-fold higher. MA, following in abundance, presented the highest levels in the young leaves and after a decrease during leaf development, the concentration at maturity stage became almost equal to that at the beginning of the ontogenetic cycle. According to recent findings,8 concentrations of both terpenoids were not influenced by water shortage.

5.3 Postharvest treatment Postharvest treatment of this plant tissue has to follow general principles and precautions suggested for natural products.19 Immediate handling (e.g., freeze-dried in liquid nitrogen) or, alternatively, transportation to the laboratory in a portable refrigerator is a convenient means to avoid alterations in phenolic composition till analysis.20 Cryopreservation as a storing postharvest practice of OLfs is barely studied. The few available records indicate that freezing lowers the bioactive potential of the material21
23 and is, thus, not considered a good practice. Freezing entails thawing, which within 2
5 min may lead in sharp reduction (B50%) of main OLf bioactive content.1
3 Material should be washed with deionized water to remove dust while still green. After being thoroughly drained off, if not freshly used, leaves should be dried as soon as possible to eliminate biotransformations. Blanching prior drying,24,25 gamma irradiation pro or after drying,26 steam blanching, and ultraviolet-C

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PART | 1 General Aspects of Olives and Olive Oil

irradiation25 has been proposed to enhance the recovery of total and individual biophenols and improve quality and stability of the final formulation. Blanching before drying aims to disrupt cell walls and facilitate liberation of phenolic compounds, as well as to contribute to the inhibition of hydrolytic and oxidative enzymes release, avoiding thus loss of bioactive compounds. Drying conditions may change initial leaf phenolic content and composition27; for this reason a complete description of drying steps has to be given in any publication. Drying in conventional or ventilated ovens, under microwave irradiation or most commonly by freeze-drying is accomplished over different lengths of time and heat conditions. Dried material is preferably kept in sealed containers (glass) and stored in a dry and dark place. Plastic bags are not recommended. In conclusion, an ideal practice is not feasible, because individual bioactives are differently affected by the drying parameters in relevance to the compositional characteristics of the material. Ahmad-Qasem et al.28 reported that dehydrating leaves at 120 C lead to extracts with higher content in oleuropein, verbascoside, and generally in TP content when compared to freeze-dried and hot air-dried ones (70 C). Another study29 reported that fresh leaves had higher oleuropein content when dried at room temperature, compared to that dried at 50 C. Ovendrying of OLfs at 105 C for 3 h increased oleuropein content up to 38-fold as compared to fresh material.30 The highest concentrations of oleuropein, verbascoside, and luteolin-7-O-glucoside were obtained when extracts were prepared from leaves dehydrated at 120 C, compared to those of fresh or freeze-dried leaves.28 Drying method affected also verbascoside concentration that slightly increased in the case of the leaves dehydrated at 120 C and diminished under freeze-drying conditions. These two types of drying led to B17% degradation of luteolin-7-Oglucoside throughout storage. On the contrary, in extracts obtained from fresh leaves, its levels were quite stable over a month storage period according to the same authors. Benefits of microwave-drying are stressed31 for simplicity, short processing time, and low cost. Results of microwave-drying under optimum determined conditions (2.085 g sample at 459.257 W for 6 min drying) presented the best performance followed by freeze-drying, vacuumdrying, oven-drying, and ambient air-drying in terms of various indices, including oleuropein and TF contents. Similar were the findings of Elhussein and coworkers,32 who also employed microwave-, oven-, and vacuumdrying at several temperatures and found that the former was the fastest having the highest effective diffusivity and the lowest activation energy with an increase in oleuropein and TF contents. Few are the studies describing drying process using mathematical models24,31
40; however, information on how optimal conditions affect qualitatively and

quantitative the target bioactives of the material are scarce. Drying kinetics is influenced by the variability of morphometric and surface porosity of OLfs. Knowledge of how total content and composition in individual bioactive compounds, as well as the bioactivity of the material are affected by drying conditions is limited. Conduction of such studies to benefit from the technical advances in this field is needed.

5.4 Extraction procedures In most studies a range of conventional and advanced means and techniques have been applied and were recently reviewed.41
43 However, few are the publications with focus on the development and validation of extraction conditions. The majority adopts procedures that are repeatable but not necessarily satisfactory for quantitative recovery of bioactives. Publications at preparative scale ( . 1 kg) are rather limited. In most of the works quantities, ,5 g are treated. Literature search indicated that special attention has been paid to phenolic polar compounds. Water, methanol, ethanol, as well as aqueous alcohol mixtures are the usual solvents for phenol extraction from dry or fresh leaf material. Moreover, acetone, ethyl acetate, or a sequence of all of the above solvents has been also reported for the same compounds. Less polar bioactives (tocopherols, carotenoids, β-sitosterol, and squalene) have been identified in n-hexane extracts, whereas terpenoids [e.g., OA, ursolic acid (UA), MA, uvaol, and erythrodiol] in ethanol, ethyl acetate, or methylene chloride extracts. Supercritical fluid extraction has been also used for both polar and nonpolar compounds. Extraction techniques are critical parameters in the process depending upon objectives. Except for the preparation of infusions and decoctions, imitating traditional practices, maceration, agitation, shaking, blending, sonication, hydrodistillation, and Soxhlet extraction have been reported in combination with one or more solvents. Weaknesses such as low efficiency, degradation of thermolabile bioactive ingredients, long extraction periods, and high solvent consumption characterize many of them. Therefore in works aiming at future industrial applications, experimentation involved ultrasound-assisted extraction, superheated liquids, and microwave-assisted extraction.42 Interesting applications have been reported for phenolics and terpenoids.44,45 OA (87% purity) was obtained at a satisfactory yield (B2%) from OLfs.46 The parameters related to the extraction process affecting recovery of phytochemicals from plant materials are many, namely, solvent type, solid-to-solvent ratio, extraction method, temperature, pressure, time, and pH, so that application of experimental design provides a reliable strategy to optimize processes both at laboratory or industrial scale. One of the most frequently employed

TABLE 5.1 Application of response surface methodology for optimization of extraction conditions of olive leaf bioactives. Technique

Factors

Responses

Refs.

UAE

Ethanol concentration (0%
100% v/v) Solid/solvent (25
50 mg/mL) Time (20
60 min)

Extract yield, TP, antioxidant activity

48

PLE

Ethanol concentration (0%
100% v/v) Temperature (40 C
190 C) Cycles (1
3)

Extract yield, oleuropein, antioxidant activity

49

Solid
liquid extraction

Temperature (70 C
90 C) Time (50
70 min) Solid/water (1/10
1/100 g/mL)

TP, antioxidant activity

48

UAE

pH (3
11) Time (20
60 min) Temperature (30 C
60 C) Solid/solvent (500 mg/ 10
20 mL)

TP

50

Magnetic stirring

Glycerol concentration (7.5%
12.5% w/v) Time (10 C
320 C)

TP

51

UAE

Temperature (27 C
37 C) Ethanol concentration (10%
70%) Time (30
60 min)

TP, TF

52

DIC

Solvent extraction conditions: Ethanol concentration in water (0%
100%) Temperature (25 C
55 C ) Liquid/solid (10
40) DIC treatment conditions under optimal solvent extraction conditions: Saturated steam pressure (0.1
0.7 MPa) Cycles (1
3) Thermal treatment time (10
70 s)

TP

53

Reduced-pressure boiling extraction coupled with ultrasonication

Temperature (40 C
60 C) Ultrasonic power (480
720 W) EtOH concentration (65%
85%) Solid/solvent (1/20
1/40 g/mL)

Oleuropein

54

Steam explosion

Temperature (180 C
220 C) Process time (2
10 min) Milling time (0
15 s)

TP, sum of oleuropein, hydroxytyrosol, flavonoids (luteolin, apigenin, together with their corresponding 7-O-glucosides)

55

Stirring in water bath

Glycerol concentration (0%
60% w/v) 2-Hydroxypropyl-β-cyclodextrin concentration (1%
13% w/v) Temperature (40 C
80 C)

TP, antioxidant activity

56

Agitation

Time (1
59 min) pH (4
10) Agitation speed (80
480 rpm) Liquid/solid (20%
80% v/w) Temperature (30 C
60 C)

TP, TF

57

PLE

Cycles (1, 2) Temperature (60 C
100 C) Static times (5
15 min)

TP, TF, hydroxycinnamic acids, flavonols

58

(Continued )

TABLE 5.1 (Continued) Technique

Factors

Responses

Refs.

Solvent-free MAE

Microwave irradiation power (250
350 W) Time (2
3 min) Sample amount (5
10 g)

Oleuropein, TP

59

Homogenizing

EtOH concentration (10%
70%) Time (30
60 s) Solvent quantity (20
40 mL) Stirring speed (4000
10000 rpm)

Oleuropein, TP

1

UAE

Temperature (35 C
65 C) Time (5
15 min) EtOH concentration (25%
75%)

TP, TF, antioxidant activity

61

UAE Microfluidic extraction process

Frequency (20
80 kHz) Temperature (25 C
75 C) Power (40
100 W) pH (2
10) Temperature (20 C
80 C) Volumetric flow rate ratio of two phases (0.5
3.0) Two phases contact time (15
25 s)

Oleuropein

62

MAE using DES

Temperature (40 C
80 C) Irradiation time (10
40 min) Water (0%
70%)

Oleuropein, TP, sum of individual phenolics

63

UAE

Operation modes/cycles (pulsed/2
5, continuous/10) Liquid/solid (15
25 mL/g) Time (1
5 min)

Oleuropein, verbascoside, luteolin-4-O-glucoside

1

UAE

Time (20
60 min) Solvent/material (10
40) Temperature (40 C
80 C)

TP, TF, hydroxytyrosol, antioxidant activity

65

Stirring

Stirring speed (300
900 rpm) DES concentration (55%
85% w/w) Liquid/solid (20
100 mL/g) β-Cyclodextrin concentration (0.70%
2.10% w/v)

TP

66

PLE Maceration

Temperature (70 C
190 C) Leaf moisture content (4.7%
22.6%) Ethanol concentration (60%
80%) Temperature (40 C
85 C) Leaf moisture content (8.09%
48.7%) Solvent/solid (6
18) Ethanol concentration (40%
100%)

TP, TF, oleuropein, luteolin-7-O-glucoside, antioxidant activity

67

UAE

Temperature (15.9 C
44.1 C) Amplitude (23.8%
66.2%)

TP, TF, oleuropein, luteolin-7-O-glucoside, antioxidant activity

1

UAE MAE

Liquid/solid (4
20) Time (10
40 min) Liquid/solid (4
20) Time (1
5 min) Power (100
300 W)

TP, TF

69

(Continued )

Bioactive ingredients in olive leaves Chapter | 5

71

TABLE 5.1 (Continued) Technique

Factors

Responses

Refs.

Extraction using infrared apparatus or water bath

Ethanol concentration (40%
80%) Time (60
180 min) Temperature (38 C
77 C)

TP

70

UAE

Ethanol concentration (5%
85%) Temperature (15 C
75 C) Time (0
100 min) Solvent/solid (0.50
42.50 mL/g)

TP, oleuropein

71

DES, Deep eutectic solvents; DIC, instant controlled pressure drop; MAE, microwave-assisted extraction; PLE, pressurized liquid extraction; TF, total flavonoid; TP, total phenol; UAE, ultrasound-assisted extraction.

experimental designs for optimization of such procedures is the response surface methodology (RSM). It is considered a valuable statistical tool, as it allows evaluation of the main and interaction effects of multiple factors on one or more response variables, as well as determination of the optimum combination of all studied factors.47 Optimization is achieved by conduction of fewer experiments that save materials, consumables, labor, and time. RSM has been recently used for optimizing the recovery extraction process of phenolic bioactives from OLfs. Table 5.1 presents key points from related studies employing safe for consumption and use solvents since 2010.

5.5 Bioactivity of olive leaf extracts The NCDs are complex as evidenced by the various risk factors identified to be responsible for their promotion. Some of these are interrelated to more than one NCD; therefore an OLf extract/individual constituent, as described in the following paragraphs, exhibits multiple activities. Evidence has been obtained through testing diverse OLf extracts prepared with solvents safe for human use (water, ethanol, mixtures of water with ethanol or glycerol, or ethyl acetate fractions obtained from aqueous
methanol extracts).72
82 The extraction procedure, where mentioned, is simple like maceration or infusion or decoction preparation.72
75,83 In certain studies, standardized extracts from various companies active in the field of natural products were used and, thus, background information is not provided.76,79,81,82,84
87 Only for EFLA943, used in different studies,76,79,81,82 it is known that 80% ethanol was used for extraction and then a patented filtration (EFLA Hyperpure) took place before drying. These industrial extracts are rather standardized with regard to oleuropein (B15%
26%) or seldom in

hydroxytyrosol and/or TP content.76,79,88 In few cases the concentration of other phenols found at lower levels (verbascoside, luteolin glucosides, etc.) is also provided. Other bioactives such as triterpenoids, possibly present in a considerable amount due to their solubility in alcohols/ ethyl acetate,4,89 are usually ignored.84 The lack of appropriate characterization is a caveat for correct result interpretation.86 Extract administration protocols vary among different studies. In animal-based ones, water dispersion, intravenously dosing, or gavage supplementation has been reported.72
79,84,88 In human-based ones, tablets, aqueous dispersions, or solutions with glycerol
water, 1:1, v/v, and safflower oil-based capsules have been orally administered.81,82,85,86 As known,90 absorption and bioavailability of bioactives are affected by means of supplementation.

5.6 Cardioprotective activity The CVDs account for most NCD deaths on annual basis, (B55%/B17.9 million people).1 OLf extracts, as demonstrated by various in vivo studies (animal-/human-based), may assist protection from CVDs in different ways.72
82,84
88 The most common health effect is reduction of hypertension. Table 5.2 presents human studies on the issue.80
82,85
87 Findings of early studies83,91 were refined further in various animal and human studies. Such a positive effect has been associated with the reversal of the vascular changes provoked by NG-nitro-L-arginine-methyl ester when used as hypertension inducer or via angiotensinconverting enzyme inhibitor activity.76,82 The latter was deduced when an OLf extract presented similar activity to that of the medication captopril known to act in this way.82 Endocavitary recording in force-feed anesthetized dogs73 with glycerol ethanolic OLf extracts affected the

72

PART | 1 General Aspects of Olives and Olive Oil

TABLE 5.2 Human studies providing evidence on the cardiovascular protective effect of supplemented olive leaf extracts. Study

Extract type

Extract composition

Oral dose

Effect

Refs.

Two groups of patients (one receiving antihypertensive therapy)

Aqueous




4 times/day (400 ng) 3 months, after a 15-day use of a placebo

Hypotensive Slight decrease of glycemia and calcemia No effect on plasma lipids

80

Monozygotic twins with untreated suboptimal blood pressure, exceeding 120 mm Hg systolic or 80 mm Hg diastolic at rest

80% ethanol (Benolea EFLA943)a

Oleuropein content 20.8% (m/m)

Film tablet containing 500 mg of extract and Acaciae Gummi (carrier) and silica colloidalis anhydrica (one or two tablets) at breakfast for 8 weeks

Hypotensive, cholesterol lowering (dosedependent)

81

Double-blind, randomized, parallel, and active-controlled with stage-1 hypertension patients

80% ethanol (Benolea EFLA943)a

Oleuropein content 19.9% (m/m)

Film-coated caplet with 500 mg extract twice a day flat dose for 8 weeks

Hypotensive (comparable to captopril) improved lipid profile

82

Randomized, doubleblinded, placebocontrolled, and crossover trial with overweight middleaged men at risk of developing metabolic syndrome


a




Capsules containing 51.1 mg oleuropein, 9.7 mg hydroxytyrosol suspended in safflower oil for 12 weeks

Improved insulin sensitivity, pancreatic Bcell secretory capacity no effects on inflammation markers, lipid profile, ambulatory blood pressure, body composition, carotid intima
media thickness, or liver function

85

Randomized, doubleblind, placebocontrolled, crossover, and acute intervention trial—healthy volunteers







Capsules containing 400 mg extract in 672.5 mg safflower oil 4/day (providing 51.12 mg oleuropein, 9.67 mg hydroxytyrosol and ,1 mg of other phenols); monitoring up to 8 h

Positive modulation of vascular function and inflammation

86

Randomized, controlled, doubleblind, and crossover intervention trial with prehypertensive males


a

Concentrated liquid product (6.81 mg oleuropein/mL, 0.32 mg hydroxytyrosol/mL, 0.73 mg/mL oleoside, other phenols ,0.17 mg/mL, dissolved in 1:1 vegetable glycerol: water

10 mL, twice/day, with food 6 weeks/4-week washout

Hypotensive lipid lowering

87

a

Provided by an industry.

Bioactive ingredients in olive leaves Chapter | 5

sinusal cycle preventing consequently the acute arrhythmia. Aiming at having insight into the hypotensive effect of OLf extract84 using spontaneously hypertensive rats and measuring various parameters reduction in pressure has been associated with the improvement of the endothelial dysfunction due to amelioration in the vascular proinflammation and prooxidative status. Using the same animal model,79 the dose-dependent hypotensive effect was associated with improved systemic, regional hemodynamics, peripheral and regional vascular resistance. In the highest dose employed (50 mg/kg), a reduction in rat brain and kidneys blood inflow was observed. On the other hand, in a human study,85 no effect on blood pressure and other CVD-related inflammatory markers was found. Authors commended that findings were in line with the European Food Safety Authority conclusion on insufficient evidence to corroborate any health claim on an effect on blood pressure, lipid profile, or inflammation. Beyond hypertension, animal or human studies based on high-fat and/or -carbohydrate consumption known to incite CVDs have been also carried out.74,75,77,78,88 Dietary habits have been related to hypercholesterolemia, oxidative stress, or diabetes. The observed benefits have been linked to the decrease in serum total cholesterol, triacylglycerols, LDL cholesterol, the delay of lipid oxidation in various target organs, including heart and aorta, and an enhancement in antioxidant enzymes activity.77 Applying immunohistochemistry and real-time polymerase chain reaction in an animal study showed that a commercial OLf extract caused a downregulation of protein and/or mRNA expressions of inflammation factors, monocyte chemoattractant protein, vascular cell adhesion molecule, nuclear factor-kappa B, and tumor necrosis factoralpha.88 Improvement/normalization of disease symptoms, including collagen deposition in heart, cardiac stiffness, aortic ring reactivity, oxidative stress, and interleukin-8 production, has also been observed in parallel with the alleviation of hypertension.78 Interestingly, OLf extract supplementation is cheaper and nontoxic contrary to the most antihypertensive medications, which are costly and induce side effects at usual doses,92 making them inappropriate to treat patients with mild forms of hypertension. The aforementioned beneficial properties have been mostly related to oleuropein, although hydrolyzed OLf extracts containing mainly hydroxytyrosol (acid treatment) or oleuropein aglycone (enzymatic treatment) were of similar potency with the mother extract.77 Andreadou et al.93 examining OLf-isolated oleuropein administration [10 or 20 mg/(kg 3 day)] to rabbits subjected to ischemia for 3
6 weeks on infarct size, oxidative damage, and the metabolic profile found—using 1H-NMR—a dosedependent glycolysis metabolite profile. In both doses, reduction in the infarct size, improvement in the antioxidant defense, and a decrease in the circulating lipids were

73

observed. Supplementation of oleuropein (10 mg/kg) in spontaneous hypertensive rats improved but did not alter systemic or regional hemodynamics.79 Consequently, the authors suggested the contribution of other constituents to justify the changes in cardiac function, carotid, and renal hemodynamics upon OLf extract (EFLA943) supplementation. Protection from CVDs is also facilitated by OA. This was highlighted by Somova et al.,89 who examined pure OA and UA, as well as ethyl acetate isolates from Greek and African OLfs rich in triterpenoids on Dahl saltsensitive insulin-resistant rats (genetic model of hypertension). All isolates, provided for 6 weeks at 60 mg/kg b.w., prevented the development of severe hypertension and atherosclerosis, whereas the insulin-resistance was improved. Fonolla et al.94 affirmed also the presence of triterpenic acids in the commercial OLf extract (Olivia) administered after meal in hypercholesterolemic subjects that resulted in improved CVD risk markers, and oxidative and hepatic parameters. Recently,95 it was reported that plant-derived OA ameliorated the risk factors of CVDs and proposed its use as an alternative treatment to prevent the onset of these types of diseases during prediabetes stage even lacking dietary and lifestyle changes. An important property of oleuropein is that it may alleviate the structural, functional, and histopathological cardiac effects of acute or chronic doxorubicin chemotherapy, which retards/prevents tumor growth by blocking topoisomerase 2 enzyme.96
98 Proposed mechanisms for the protection are the suppression of oxidative/nitrosative stress, the interference with signaling molecules, and cardiomyocyte metabolism.

5.7 Anticancer properties Cancers account for the B28% of deaths derived annually from NCDs (B9 million people)1 and OLf extracts have attracted the interest of researchers to counteract its various types. Most studies have examined the activity against cell lines as recently reviewed.1 Although findings are promising, the authors stress that “the protective effect of olive polyphenols for cancer in humans remains anecdotal and clinical trials are required to substantiate these claims idea.” Despite the lack of clinical trials, there are few in vivo studies supporting the OLf extracts and constituents anticancer properties.99
107 Except for oleuropein and hydroxytyrosol, significant research has been dedicated to triterpenoids, which act in a multitude of modes (blocking of nuclear factor-kappa-B activation, induction of apoptosis, inhibition of signal transducer, activation of transcription and angiogenesis, protection from of oxidative stress, the enhancement of endogenous antioxidants, the improvement of the detoxification potential, and the disruption cell survival pathways).108
113

74

PART | 1 General Aspects of Olives and Olive Oil

5.8 Respiratory diseases

References

The respiratory diseases account for a substantial number of deaths (B12% of deaths derived annually from NCDs/ 3.9 million people) due to the inadequate treatment of multiresistant Gram-negative agents and Staphylococcus aureus oxacillin resistant. Still, scarce is the information on OLf extract effectiveness. Jozala et al.114 commenting on antimicrobial approaches against bacterial pathogens, which cause lower respiratory system infections, highlighted OLf extract as a possible candidate to treat pneumonia, although relying on an in vitro study.115 Few other works mainly focused on oleuropein standardized extracts were reported.1
3

¨ zcan MM, Mattha¨us B. A review: benefit and bioactive properties 1. O of olive (Olea europaea L.) leaves. Eur Food Res Technol. 2017;243(1):89
99. 2. El SN, Karakaya S. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev. 2009;67 (11):632
638. 3. Talhaoui N, Taamalli A, Go´mez-Caravaca AM, Ferna´ndezGutie´rrez A, Segura-Carretero A. Phenolic compounds in olive leaves: analytical determination, biotic and abiotic influence, and health benefits. Food Res Int. 2015;77:92
108. ´ , Rada M, Delgado T, Gutie´rrez-Ada´nez P, Castellano 4. Guinda A JM. Pentacyclic triterpenoids from olive fruit and leaf. J Agric Food Chem. 2010;58(17):9685
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140. 6. Papoti VT, Tsimidou MZ. Impact of sampling parameters on the radical scavenging potential of olive (Olea europaea L.) leaves. J Agric Food Chem. 2009;57(9):3470
3477. 7. Goulas V, Papoti VT, Exarchou V, Tsimidou MZ, Gerothanassis IP. Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts. J Agric Food Chem. 2010;58(6):3303
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5.9 Diabetes Diabetes is the less lethal among NCDs on annual basis (B5% annual deaths derived from NCDs/1.6 million people1). A recent review on randomized controlled trials related to diabetes induced in rats119 summarized—after the metaanalysis of available findings with the aid of SYRCLE’s risk of bias tool for animal studies—that the treatment raised insulin levels and reduced blood glucose levels. Some more studies were found74,85,120
122 on this issue. OLfs contain compounds acting as antagonists to TGR5 (member of G-protein receptor) which is activated by bile acids and act as a mediator of their endocrine functions, having, thus, influence in metabolic disorders.123 The antidiabetic effect of OLf extracts has been principally ascribed to oleuropein via affecting glucose-induced insulin release and increasing peripheral uptake of glucose.124 Oleuropein is a TGR5 antagonist.123 Ahamad et al.125 reviewed the multiple modes of oleuropein action to counteract the metabolic syndrome. Evidence is also provided for the hypoglycemic effect of OA and luteolin.120,123

5.10 Conclusive remarks OLfs are a good source of an array of bioactive compounds with synergistic effects. Preharvest, postharvest, and extraction practices and bioactivities have been extensively studied for polar phenolic compounds and the respective extracts. Less is known for the terpenoids. This plant material is the most promising renewable source of bioactives. Funding: Authors carried out this work in the frame of project “Upgrading the Plant Capital (PlantUp)” (MIS 5002803, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014
20) and cofinanced by Greece and the European Union (European Regional Development Fund).

Bioactive ingredients in olive leaves Chapter | 5

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1127. ˇ 58. Putnik P, Barba FJ, Spani´ c I, Zori´c Z, Dragovi´c-Uzelac V, Bursa´c Kovaˇcevi´c D. Green extraction approach for the recovery of polyphenols from Croatian olive leaves (Olea europaea). Food Bioprod Process. 2017;106:19
28. 59. Sahin ¸ S, Bilgin M. Selective adsorption of oleuropein from olive (Olea europaea) leaf extract using macroporous resin. Chem Eng Commun. 2017;204(12):1391
1400. 60. Sahin S, Samli R, Birteks Z, Tan AS, Barba FJ, Chemat F, et al. Solvent-free microwave-assisted extraction of polyphenols from olive tree leaves: antioxidant and antimicrobial properties. Molecules. 2017;22(7):1056. 61. Shirzad H, Niknam V, Taheri M, Ebrahimzadeh H. Ultrasoundassisted extraction process of phenolic antioxidants from Olive leaves: a nutraceutical study using RSM and LC
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2371. 62. Yasemi M, Heydarinasab A, Rahimi M, Ardjmand M. Microchannels effective method for the extraction of oleuropein compared with conventional methods. J Chem. 2017;2017. 63. Alan˜o´n ME, Ivanovi´c M, Go´mez-Caravaca AM, Arra´ez-Roma´n D, Segura-Carretero A. Choline chloride derivative-based deep eutectic liquids as novel green alternative solvents for extraction of phenolic compounds from olive leaf. Arab J Chem. 2020;13 (1):1685
1701. ˇ ˇ c M. Optimization of ultrasonic64. Giacometti J, Zauhar G, Zuvi´ assisted extraction of major phenolic compounds from olive leaves (Olea europaea L.) using response surface methodology. Foods. 2018;7(9):149. 65. Yao Q, Shen Y, Bu L, Yang P, Xu Z, Guo X. Ultrasound-assisted aqueous extraction of total flavonoids and hydroxytyrosol from olive leaves optimized by response surface methodology. Prep Biochem Biotechnol. 2019;49(9):837
845. 66. Chakroun D, Grigorakis S, Loupassaki S, Makris DP. Enhancedperformance extraction of olive (Olea europaea) leaf polyphenols using L-lactic acid/ammonium acetate deep eutectic solvent combined with β-cyclodextrin: screening, optimisation, temperature effects and stability. Biomass Convers Biorefin. 2019;1
12. 67. Lama-Mun˜oz A, del Mar Contreras M, Espı´nola F, Moya M, de Torres A, Romero I, et al. Extraction of oleuropein and luteolin-7-

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37. Abi-Khattar AM, Rajha HN, Abdel-Massih RM, Maroun RG, Louka N, Debs E. Intensification of polyphenol extraction from olive leaves using Ired-Irrads, an environmentally-friendly innovative technology. Antioxidants. 2019;8(7):227. ¨ , Kenar A, Akay MA. Green alcoholic Vural N, Algan Cavuldak O solvent and UAE extraction of oleuropein from the Olea europaea L. leaves: experimental design, optimization, and comparison with pharmacopoeia method. Sep Sci Technol. 2020;55:1813
1828. Circosta C, Occhiuto F, Gregorio A, Toigo S, De Pasquale A. The cardiovascular activity of the shoots and leaves of Olea europaea L. and oleuropein. Plant Med Phytother. 1990;24(4):264
277. Occhiuto F, Circosta C, Gregorio A, Busa G. Olea europaea L. and oleuropein: effects on excito-conduction and on monophasic action potential in anaesthetized dogs. Phytother Res. 1990;4(4):140
143. Bennani-Kabchi N, Fdhil H, Cherrah Y, Kehel L, El Bouayadi F, Amarti A, et al. Effects of Olea europaea var. oleaster leaves in hypercholesterolemic insulin-resistant sand rats. Therapie. 1999;54 (6):717
723. Bennani-Kabchi N, Fdhil H, Cherrah Y, El Bouayadi F, Kehel L, Marquie G. Therapeutic effect of Olea europaea var. oleaster leaves on lipidic and carbohydrate metabolism in obese and prediabetic sand rats (Psammomys obesus). Ann Pharm Fr. 2000;58(4):271
277. Khayyal MT, Elghazaly MA, Abdallah DM, Nassar NN, Okpanyi SN, Blood M-HK. pressure lowering effect of an olive leaf extract (Olea europaea) in L-NAME induced hypertension in rats. Arzneimittelforsch. 2002;52(11):797
802. Jemai H, Bouaziz M, Fki I, El Feki A, Sayadi S. Hypolipidimic and antioxidant activities of oleuropein and its hydrolysis derivative-rich extracts from Chemlali olive leaves. Chem Biol Interact. 2008;176(2
3):88
98. Poudyal H, Campbell F, Brown L. Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J Nutr. 2010;140(5):946
953. Ivanov M, Vajic UJ, Mihailovic-Stanojevic N, Miloradovic Z, Jovovic D, Grujic-Milanovic J, et al. Highly potent antioxidant Olea europaea L. leaf extract affects carotid and renal haemodynamics in experimental hypertension: the role of oleuropein. EXCLI J. 2018;17:29
44. Cherif N, Rahal M, Haouala B, Hizaoui F, Dargouth M, Gueddiche Z, et al. A clinical trial of a titrated Olea extract in the treatment of essential arterial hypertension. J Pharm Belg. 1996;51(2):69
71. Perrinjaquet-Moccetti T, Busjahn A, Schmidlin C, Schmidt A, Bradl B, Aydogan C. Food supplementation with an olive (Olea europaea L.) leaf extract reduces blood pressure in borderline hypertensive monozygotic twins. Phytother Res. 2008;22(9):1239
1242. Susalit E, Agus N, Effendi I, Tjandrawinata RR, Nofiarny D, Perrinjaquet-Moccetti T, et al. Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: comparison with captopril. Phytomedicine. 2011;18(4):251
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Bioactive ingredients in olive leaves Chapter | 5

83. Luibl E. Leaves of the olive tree in hypertension. Med Monatsschr Pharm. 1958;12:181
182. 84. Romero M, Toral M, Go´mez-Guzma´n M, Jime´nez R, Galindo P, Sa´nchez M, et al. Antihypertensive effects of oleuropein-enriched olive leaf extract in spontaneously hypertensive rats. Food Funct. 2016;7(1):584
593. 85. de Bock M, Derraik JGB, Brennan CM, Biggs JB, Morgan PE, Hodgkinson SC, et al. Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: a randomized, placebo-controlled, crossover trial. PLoS One. 2013;8 (3):e57622. 86. Lockyer S, Corona G, Yaqoob P, Spencer JPE, Rowland I. Secoiridoids delivered as olive leaf extract induce acute improvements in human vascular function and reduction of an inflammatory cytokine: a randomised, double-blind, placebo-controlled, cross-over trial. Br J Nutr. 2015;114(1):75
83. 87. Lockyer S, Rowland I, Spencer JPE, Yaqoob P, Stonehouse W. Impact of phenolic-rich olive leaf extract on blood pressure, plasma lipids and inflammatory markers: a randomised controlled trial. Eur J Nutr. 2017;56(4):1421
1432. 88. Wang L, Geng C, Jiang L, Gong D, Liu D, Yoshimura H, et al. The antiatherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr. 2008;47:235
243. 89. Somova LI, Shode FO, Ramnanan P, Nadar A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies Africana leaves. J Ethnopharmacol. 2003;84(2
3):299
305. 90. Vissers MN, Zock PL, Roodenburg AJ, Leenen R, Katan MB. Olive oil phenols are absorbed in humans. J Nutr. 2002;132 (3):409
417. 91. Kosak R, Stern P. Examination of the hypotensive activity of folium Olivae. Acta Pharm Jugosl. 1956;6:121
132. 92. Sweetman SC. Martindale: The Complete Drug Reference. 33rd ed. London: Pharmaceutical Press; 2002. 93. Andreadou I, Iliodromitis EK, Mikros E, Constantinou M, Agalias A, Magiatis P, et al. The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr. 2006;136(8):2213
2219. 94. Fonolla J, D
ıaz-Ropero P, de la Fuente E, Quintela JC. MS358 one-month consumption of an olive leaf extract enhances cardiovascular status in hypercholesterolemic subjects. Atheroscler Suppl. 2010;11(2):182. 95. Gamede M, Mabuza L, Ngubane P, Khathi A. Plant-derived oleanolic acid ameliorates markers associated with non-alcoholic fatty liver disease in a diet-induced pre-diabetes rat model. Diabetes Metab Syndr Obes. 2019;12:1953
1962. 96. Andreadou I, Sigala F, Iliodromitis EK, Papaefthimiou M, Sigalas C, Aligiannis N, et al. Acute doxorubicin cardiotoxicity is successfully treated with the phytochemical oleuropein through suppression of oxidative and nitrosative stress. J Mol Cell Cardiol. 2007;42(3):549
558. 97. Andreadou I, Papaefthimiou M, Zira A, Constantinou M, Sigala F, Skaltsounis AL, et al. Metabonomic identification of novel biomarkers in doxorubicin cardiotoxicity and protective effect of the natural antioxidant oleuropein. NMR Biomed. 2009;22 (6):585
592. 98. Andreadou I, Mikros E, Ioannidis K, Sigala F, Naka K, Kostidis S, et al. Oleuropein prevents doxorubicin-induced cardiomyopathy

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interfering with signaling molecules and cardiomyocyte metabolism. J Mol Cell Cardiol. 2014;69:4
16. Milanizadeh S, Bigdeli MR, Rasoulian B, Amani D. The effects of olive leaf extract on antioxidant enzymes activity and tumor growth in breast cancer. Thrita. 2014;3(1):e12914. Kimura Y, Sumiyoshi M. Olive leaf extract and its main component oleuropein prevent chronic ultraviolet B radiation-induced skin damage and carcinogenesis in hairless mice. J Nutr. 2009;139(11):2079
2086. Sumiyoshi M, Kimura Y. Effects of olive leaf extract and its main component oleuroepin on acute ultraviolet B irradiation-induced skin changes in C57BL/6J mice. Phytother Res. 2010;24 (7):995
1003. Hamdi HK, Castellon R. Oleuropein, a non-toxic olive iridoid, is an anti-tumor agent and cytoskeleton disruptor. Biochem Biophys Res Co. 2005;334(3):769
778. Granados-Principal S, Quiles JL, Ramirez-Tortosa C, CamachoCorencia P, Sanchez-Rovira P, Vera-Ramirez L, et al. Hydroxytyrosol inhibits growth and cell proliferation and promotes high expression of sfrp4 in rat mammary tumours. Mol Nutr Food Res. 2011;55(suppl 1):S117
S126. Carrera-Gonza´lez MP, Ramı´rez-Expo´sito MJ, Mayas MD, Martı´nez-Martos JM. Protective role of oleuropein and its metabolite hydroxytyrosol on cancer. Trends Food Sci Technol. 2013;31 (2):92
99. Sepporta MV, Fuccelli R, Rosignoli P, Ricci G, Servili M, Morozzi G, et al. Oleuropein inhibits tumour growth and metastases dissemination in ovariectomised nude mice with MCF-7 human breast tumour xenografts. J Funct Foods. 2014;8 (1):269
273. Shamshoum H, Vlavcheski F, Tsiani E. Anticancer effects of oleuropein. BioFactors. 2017;43(4):517
528. Imran M, Nadeem M, Gilani SA, Khan S, Sajid MW, Amir RM. Antitumor perspectives of oleuropein and its metabolite hydroxytyrosol: recent updates. J Food Sci. 2018;83(7):1781
1791. Petronellia A, Pannitterib G, Testaa U. Triterpenoids as new promising anticancer drugs. Anti-Cancer Drug. 2009;20(10):880
892. Kuttan G, Pratheeshkumar P, Manu KA, Kuttan R. Inhibition of tumor progression by naturally occurring terpenoids. Pharm Biol. 2011;49(10):995
1007. Patlolla JMR, Rao CV. Triterpenoids for cancer prevention and treatment: current status and future prospects. Curr Pharm Biotechnol. 2012;13(1):147
155. Sa´nchez-Quesada C, Lo´pez-Biedma A, Warleta F, Campos M, Beltra´n G, Gaforio JJ. Bioactive properties of the main triterpenes found in olives, virgin olive oil, and leaves of Olea europaea. J Agric Food Chem. 2013;61(50):12173
12182. Parikh NR, Mandal A, Bhatia D, Siveen KS, Sethi G, Bishayee A. Oleanane triterpenoids in the prevention and therapy of breast cancer: current evidence and future perspectives. Phytochem Rev. 2014;13(4):793
810. Pie¸t M, Paduch R. Ursolic and oleanolic acids as potential anticancer agents acting in the gastrointestinal tract. Mini-Rev Org Chem. 2019;16(1):78
91. Jozala AF, Grotto D, Novaes LCL, de Carvalho Santos-Ebinuma V, Gerenutti M, Del Fiol FS. Antimicrobial approaches against bacterial pathogens which cause lower respiratory system infections. In: Kon K, Rai M, eds. The Microbiology of Respiratory System Infections. London: Academic Press; 2016:211
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115. Pereira AP, Ferreira ICFR, Marcelino F, Valenta˜o P, Andrade PB, Seabra R, et al. Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobranc¸osa) leaves. Molecules. 2007;12(5):1153
1162. 116. Somerville V, Moore R, Braakhuis A. The effect of olive leaf extract on upper respiratory illness in high school athletes: a randomised control trial. Nutrients. 2019;11(2):358. 117. Khalili HR, Adeli Behrooz HR, Rashidi Nooshabadi MR, Geravandi S, Mohammadi MJ, Foruozandeh H. Evaluation of potential anti-fibrotic effect of oleuropein on bleomycin-induced pulmonary fibrosis in rat. Toxin Rev. 2018;1
9. 118. Kim YH, Choi YJ, Kang MK, Lee EJ, Kim DY, Oh H, et al. Oleuropein curtails pulmonary inflammation and tissue destruction in models of experimental asthma and emphysema. J Agric Food Chem. 2018;66(29):7643
7654. 119. Abunab H, Dator WL, Hawamdeh S. Effect of olive leaf extract on glucose levels in diabetes-induced rats: a systematic review and meta-analysis. J Diabetes. 2017;9(10):947
957. 120. Komaki E, Yamaguchi S, Maru I, Kinoshita M, Kakehi K, Ohta Y, et al. Identification of anti-α-amylase components from olive leaf extracts. Food Sci Technol Res. 2003;9(1):35
39. 121. El-Moneim MR Afify A, El-Beltagi HS, Fayed SA, El-Ansary AE. Enhancing effect of olive leaves extract on lipid profile and enzymes activity in streptozotocin induced diabetic rats. Fresenius Environ Bull. 2018;27(3):1875
1883.

122. Al-Attar AM, Alsalmi FA. Effect of Olea europaea leaves extract on streptozotocin induced diabetes in male albino rats. Saudi J Biol Sci. 2019;26(1):118
128. 123. Sato H, Genet C, Strehle A, Thomas C, Lobstein A, Wagner A, et al. Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea. Biochem Biophys Res Commun. 2007;362 (4):793
798. 124. Gonzalez M, Zarzuelo A, Gamez MJ, Utrilla MP, Jimenez J, Osuna I. Hypoglycemic activity of olive leaf. Planta Med. 1992;58(6):513
515. 125. Ahamad J, Toufeeq I, Khan MA, Ameen MSM, Anwer ET, Uthirapathy S, et al. Oleuropein: a natural antioxidant molecule in the treatment of metabolic syndrome. Phytother Res. 2019;33 (12):3112
3128.

Further reading 1 Bahloul N, Boudhrioua N, Kechaou N. Moisture desorptionadsorption isotherms and isosteric heats of sorption of Tunisian olive leaves (Olea europaea L.). Ind Crop Prod. 2008;28(2):162
176. 2 Sahin ¸ S, Samli ¸ R. Optimization of olive leaf extract obtained by ultrasound-assisted extraction with response surface methodology. Ultrason Sonochem. 2013;20(1):595
602.

Chapter 6

Detection of adulterations of extra-virgin olive oil by means of infrared thermography Jose´ S. Torrecilla1, John C. Cancilla2, Sandra Pradana-Lopez1 and Ana M. Perez-Calabuig1 1

Departamento de Ingenierı´a Quı´mica y de Materiales, Universidad Complutense de Madrid, Madrid, Spain, 2Scintillon Institute, San Diego,

CA, United States

Abbreviations CNN EVOO IT OPO ROO TAGs

convolutional neural network extra-virgin olive oil infrared thermography olive pomace oil refined olive oil triacylglycerols

6.1 Introduction In the food industry, one of the factors that have the greatest impact on consumers and overall society is adulteration. These adulterations can be carried out accidentally, by not following the established production and storage safety procedures, or simply on purpose. But regardless of the circumstances, all these reasons are considered fraudulent activities.1 When these actions are carried out to obtain a greater economic return, the foods on which they focus are fundamentally those that present a greater cost for the consumers, as could be the case of extra-virgin olive oils (EVOOs), or those sold in large quantities such as cereals.1,2 In any case, whether it is an intentional or accidental activity, and irrespective of the objectives pursued, it is necessary to have tools that are as simple as possible in order to detect adulterations before they are consumed, not only to protect society but also to be able to eradicate these illegal practices. EVOO is one of the few oils that is directly the juice of a fruit, namely, the olive. This is why all the varieties of EVOO have healthy and organoleptically pleasing characteristics. In order to maintain these characteristics from the milling process to the consumption of the oil

itself, it is necessary not only to protect it from adulteration but also to develop tools to control the quality of this juice.1 Even so, one of the foods that are subject to a greater number of adulterations worldwide focuses on the olive sector and, more specifically, on the production of EVOO, followed by the dairy and beekeeping sectors.3 EVOO is usually adulterated with other types of oils of lower quality and price [sunflower oil, corn oil, refined olive oil (ROO), olive pomace oil (OPO), etc.].4,5 Undoubtedly, adulterations made with oils more similar to EVOO, such as ROO or refined OPO, are usually more difficult to detect since they come from the same fruit.6 In either case the most common methods for detecting these fraudulent activities are based on complex analytical tools that, although offer very good analytical results, are not practical for recognizing adulterated oils in a portable, fast, and simple way.1 In order to solve these problems, portable and cost-effective tools are being proposed that present interesting adulteration detection rates as well as quality alterations.6 In 2019 the research group AlgoReach published research that echoed the application of totally noninvasive fluorescence-based tools for detecting fraudulent mixtures between EVOO, with a protected designation of origin, and other oils with errors in estimating the concentration of adulterant below 10%. The equipment had been designed using 3D printers and had been placed in a case to reduce costs and facilitate its transport to the production center or any other location where a quality measurement was required.7 Likewise, at the end of 2018, this same research group developed a tool for predicting the quality of the EVOO by means of optical photos of the olives themselves, representing a step forward in the olive sector.8 Also, during this

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00020-1 © 2021 Elsevier Inc. All rights reserved.

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same year, Mohd Khairi et al. presented a summary of noninvasive techniques: X-rays, thermal imaging, near-infrared spectroscopy, hyperspectral imaging, ultrasound, and terahertz. All of them for the detection of objects within the food itself could have been introduced at any stage of the production or distribution phases.9 Later, the research group AlgoReach presented a tool that by means of thermographic images could detect adulterations of different types of EVOO in a reliable way. This development led to the application of a patent and a subsequent publication,6 resulting in cost-effective and reliable method of detecting adulterations within EVOO, aiding the olive sector. This tool will be presented in more detail next.6

6.1.1 Comparisons between olive oils and other edible oils Currently, one of the greatest scourges in the field of food is the existence of fraudulent activities that reduce the quality of food, in some cases leading to very unfortunate situations. Among these activities, adulterations have been carried out since ancient times in search of economic benefit, forgetting about the health of the consumers. These problems are reflected in official summaries of the European Union as well as local administrations. They determine that EVOO is the food with more risk of suffering bad practices, fraudulent activities, and adulterations. Usually, these adulterations are based on mixing EVOO with seed oils that, in addition to being cheap, do not add flavor nor color to the oil, making it difficult to detect. Nevertheless, it is important not to forget that at the consumer level, it is necessary to know the composition of the oil that is being acquired, which is achieved via proper labeling, to ensure quality and safety. One way of detecting these differences between pure and adulterated samples is to focus on the chemical composition of the oil. Moreover, if these differences in composition are easy to detect, it makes the task of detecting these fraudulent activities easier. In particular, there are molecules that will serve as a reference for differentiating EVOO oils and seed oils, such as the concentration of triacylglycerol (TAG), which are different in each case.10,11 Likewise, the presence and quantity of TAG is relevant through the study of its thermodynamic behavior. This is different depending on the concentration and the matrix in which it is immersed in the different types of oil. Likewise, this thermodynamic behavior is easy to evaluate by means of simple thermographic cameras that are capable of measuring the evolution of the temperature in each of the oils analyzed. As these cameras do not use any incident radiation on the sample, they make it easier to maintain the integrity of the oil. In addition to these differences, when the adulterations are carried out in relatively low concentrations, it is

necessary to have chemometric tools to be able to detect those adulterated samples.6

6.1.2 Implications for human health and disease prevention The main organoleptic and health characteristics of EVOO are well known by many of the final consumers.12 Moreover, in the countries of the Mediterranean basin such as Spain and Italy, the use of these oils is requested in restaurants, hotels, and so on. Regarding this product, there are wide-ranging olive varietals that lead to many unique EVOOs with their own organoleptic profiles and tastes. But besides this, all of them, to a greater or lesser extent, have outstanding health characteristics.13 The profiles of monounsaturated fatty acids and phenolic fractions offer preventive effects against diseases such as different types of cancer (breast, colon, skin, among others) or harmful and relatively unknown neurodegenerative diseases.13 EVOO also plays an important role in the prevention of very common diseases with a very high incidence in society such as coronary heart disease.14,15 According to the World Health Organization, these diseases are among the top 10 causes of death in the world. This is why a healthy diet that includes foods such as EVOO, fruits, or vegetables, with preventive properties against these diseases, is relevant for the human society.13 These properties generate confidence in consumers regarding EVOO, and thus these characteristics must be protected, not only during production, where the highest possible quality standards are expected, but also by those responsible for distribution chains, to preserve these properties as much as possible until reaching the final consumer. That is why it is necessary to make these sectors aware of the need to have tools capable of monitoring quality in order to verify that what the customers are purchasing is in fact what they will consume. This confidence will increase not only the quality, but also the consumption, and with it the development of the olive sector will be facilitated, and the overall health of the consumers will benefit as well.6

6.2 Infrared thermography It is well known that temperature is one of the consequences of energy exchange and also that the rate and way at which this energy is exchanged depend on the physicochemical properties of the compounds of the material under analysis. Likewise, temperature is a variable that can be determined relatively easily by measuring the expansion of a material within a constant volume, based on a simple calibration. In addition to the expansion that is typical of a fluid or solid, there are other properties that can help determine temperature. For instance, equipment can be employed to measure

Detection of adulterations of extra-virgin olive oil by means of infrared thermography Chapter | 6

the temperature via electrical resistances or potential differences. During any industrial process, temperature monitoring is an important aspect. Generally, some instruments are used such as thermocouples and thermistors. The disadvantage of these instruments is that they can only determine the temperature at specific points, and, in addition, a stabilization of the measurement is necessary.16 In this area and based on the determination of the temperature at specific points, the differential scanning calorimeter (DSC) appears. This technique makes it possible to determine the quality, origin, and nature of the oil according to its freezing and melting points.10,17 In the food area, AlgoReach has used DSC combined with nonlinear models for the quantification of adulterating agents in EVOO, such as sunflower and corn oils, as well as ROO and refined olive ointment oils.18 However, the issue still remains as the temperature is from specific locations. Other ways of measuring temperature could be used to solve these difficulties, including measuring temperature through the infrared radiation emitted by the sample. At the beginning of the 19th century, William Herschel focused on measuring the temperature of the colors that came from breaking down sunlight into its various wavelengths. In these experiments, he found that a thermometer placed in blue radiation showed a lower temperature than a similar thermometer placed in the red one. The most surprising thing was that the temperature was even higher when a thermometer was placed close to red but in an area where no visible color was noticed (Fig. 6.1). With this information a relationship began to be established between the amount of infrared radiation emitted by an item and its temperature. Due to the wave characteristics of this radiation, the emission of each sample is formed by the infrared radiation emitted, reflected, and sometimes transmitted. As can be seen in Fig. 6.1, the infrared radiation is located in the less energetic zone of the visible electromagnetic spectrum. The wavelength of the infrared emission is between 780 and 1 3 106 nm.19 Within this region, three areas can be distinguished depending on the distance to the visible region in terms of wavelength: near-infrared (780
2500 nm), mid-infrared (2500
50,000 nm), and far-infrared (50,000
1 3 106 nm). In this line the infrared thermography (IT) technique is based on the analysis of the infrared radiation emitted by objects due to their temperature. Since each part of a

81

body can emit a different intensity of radiation depending on its temperature, it is easy to establish a color map where a color is set for each emitted electromagnetic radiation, which can be understood as a temperature map of the object. These maps or images are represented in photographs and are commonly called thermograms.20 These properties facilitate the development of a large number of applications for the medical sector, design, industry, etc. In spite of this number of applications, thermography, although it has been applied in various areas of the food sector such as the determination of food quality,9,16,21 no thermographic applications have been found that analyze whether the food, in this case the EVOO, is adulterated or not. The use of thermal imaging has gained popularity in agriculture and the food industry. The main advantages of this technique are that it is noninvasive, portable, and fast.16 One of the great difficulties that has historically kept thermographic studies at a lower level of development has been mainly the technological development of the equipment and, above all, the development of mathematical algorithms capable of dealing with the large amount of information generated by this technique. This is why the appearance of intelligent algorithms such as convolutional neural networks (CNNs) are capable of analyzing the images to achieve the necessary and complex objectives. This type of mathematical algorithms is especially suitable for treating images, and that is why they are able to classify thermograms of the EVOOs with different characteristics as we will see later.6 Moreover, the developed applications concerning thermal images have been feasible thanks to the abovementioned algorithms, which fall into the field of machine learning, and, specifically, deep learning.6,22
24

6.3 Detection of adulterated extra-virgin olive oil using infrared thermography The AlgoReach research group has integrated a thermographic camera (Optris PI 450 with a spectral range of 7500
14,000 nm, an accuracy of 6 2%, and an image collection frequency of 80 Hz) with intelligent mathematical tools based on CNNs. This thermographic equipment works in the spectral range of the mid- and far-infrared (vide supra). With this system a new and reliable method to detect adulterations is presented by comparing the FIGURE 6.1 Electromagnetic radiation and temperature.

Gamma rays

X-rays

Ultraviolet rays

400

500

600

Wavelength (nm)

700

Infrared rays

Shortwave Radar FM TV AM

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thermal evolution of pure and adulterated EVOO during its cooling process. The adulterated samples have been made in the laboratory itself, simply by mixing a controlled amount of olive oil (ROO or OPO) or sunflower oil as an adulterant, with a pure EVOO.25 In order to analyze the thermodynamic process, 6 mL of the EVOO under study are introduced into a transparent plastic cell measuring 1 3 1 3 7 cm3, which is heated to temperatures close to 45 C for a short time and then left to cool to 25 C under controlled, practically adiabatic conditions. Its walls are insulated against optic and thermal reflections. This enclosure has a front opening that allows the infrared radiation of the sample to escape the chamber and be registered by the thermographic camera. The oil sample is 25 cm from the camera lens, and the radiation is transformed into temperatures that are collected in a computer. This transformation is evaluated by the emissivity coefficient of the juice. This coefficient is used to quantify the ability of a given material to emit energy as infrared radiation. In this case the emissivity coefficient of all EVOO samples has been considered equal to 0.95.26,27 This cooling process from 45 C to 25 C, which is linked to and characteristic of the purity of the EVOO, is recorded on video by means of the thermographic camera mentioned earlier. Then, each of the videos is separated into frames (one for every 0.15 s), obtaining 22,782 thermographic images, distributed in 5515, 5716, 5673, and 5878 samples of pure EVOO, ROO, OPO, and sunflower oil, respectively.6 When the adulteration is considered (vide infra), the most important information is located between 39 C and 36 C. It should be noted that adulteration or abnormal activities to which EVOO is subjected during distribution or during prolonged storage will alter the lipid profile of the oil and thus alter the cooling profile over time. Specifically, it is considered that the thermal differences between the mixtures are fundamentally linked to the TAG composition of the oils. In other words, the TAGs have a significant influence on the thermal behavior observed in the EVOO samples.10,11 These lipid biomolecules are part of all oils of vegetable or animal origin. Considering this, it can be understood that the addition of oils of different origins would alter the TAG composition of EVOO samples resulting in characteristic thermal distributions related to the type and amount of adulterant present.6 For this purpose, CNN-based algorithms have been tested for the analysis and classification of different thermographic images of pure and adulterated EVOO samples containing amounts # 8% in weight of ROO, OPO, or sunflower oil. From an algorithmic point of view, eight mathematical models based on CNNs were used to classify EVOO samples and to estimate adulterant concentrations. Models were divided into single adulterant ones and global ones. The latter types have been developed,

including all samples regardless of the type of adulterant to achieve more versatile global tools for the detection of adulterated EVOO. In general, the statistical performance of the optimized CNNs in terms of classification as pure and adulterated has been focused on the development of an internal validation that prevents the overadjustment of the mathematical model. In this validation process the classification accuracy varies between 97% and 100%. As a final part of the work, we have tried to pinpoint the main sources of error of the developed tools. As expected, those errors in the classification were mainly focused on those samples that presented a lower concentration of the adulterants. And depending on the adulterant, those that presented a greater error were those adulterants more similar to the EVOO under study.6 Specifically, the major errors were clustered on samples containing ROO or OPO. Better results were obtained when it came to detecting adulterations made with sunflower oil. In the case of adulterations carried out with ROO, the errors coincide with the end of the cooling phase, within the temperature range of 37 C
36 C, which means that at these low temperatures the differences between the images of the different cases are less apparent. In the case of adulterations carried out with OPO, 2% of the samples were incorrectly classified as pure when they were not. These classification results are further valuable since the CNNs have been trained with images of samples that contain a relatively low concentration of adulteration, much lower than the typical adulteration range found in fraudulent commercial EVOOs. Despite this, the accuracy of these models is still very high and relevant to the safety of the EVOO sector, its consumers, and quality control in general.6

6.4 Conclusion This chapter has shown a way to detect adulteration and control the quality of EVOO, one of the foods that suffer most from this type of fraud. This quality control method is simple, easy to use, and economical for both consumers and producers. This tool consists of a thermographic camera and a mathematical tool that treats the collected thermal images. The mathematical part focuses on the study of the thermal progression of pure and adulterated EVOO samples through deep learning. Specifically, the use of algorithms based on CNNs was proposed. These mathematical models serve as systems to identify and quantify three different adulterations (ROO, OPO, and sunflower oil) between 0% and 8% in weight. The final optimized algorithms reach accuracies ranging from 97% to 100% for different sets of tests, considering both the estimation of the concentration of the adulterant and the type of adulterant. This study proposes an innovative system that merges a technique such as IT and a modern alternative of

Detection of adulterations of extra-virgin olive oil by means of infrared thermography Chapter | 6

mathematical modeling such as CNN. The system devised could serve as a powerful analytical system and the algorithmic option to control any stage of the production or distribution phases of EVOO, as well as potentially other foods or products as is the case of the application of these techniques to the quality control of honey. In addition, this tool is portable and versatile, very sensitive, and easy to operate while producing real-time results, which are very valuable features that could lead to a useful method of quality control for EVOO. As for the computing power required for the design of the models, it can be said that a powerful tool is necessary for the optimization of the intelligent models to be used, but that, once designed, the requirements for its use in real time is practically negligible, making the tool not only fast but also versatile and cost-effective.

Acknowledgment This work has been partially financed by the FEI program of the Universidad Complutense de Madrid under the references of the project FEI-EU-17-03 and FEI 18/10.

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Refined olive pomace oil. The olive oil that comes from a process of refining olive pomace oil Chemometric. Chemistry discipline that focuses on the application of mathematical and/or statistical methods to chemical data Electromagnetic radiation. Combination of oscillating electric and magnetic fields, which propagate through space transporting energy from one place to another without the need for a transmission medium Emissivity. Proportion of thermal radiation emitted by the surface of a material or object due to its temperature Infrared radiation. It is a type of electromagnetic radiation, longer than visible light but shorter than microwaves with a wavelength between 800 and 1 3 106 nm Quality control. The set of mechanisms, procedures, and technologies used to detect the presence of errors and/or nonconformities with respect to the purpose to be fulfilled with respect to the final customer Refined olive oil. The olive oil that comes from a process of refining lampante olive Spectroscopy. The study of the interaction between electromagnetic radiation and matter. This interaction can lead to an absorption or emission of incident energy Thermography. Technique that allows us to determine temperatures at a distance and without the need for physical contact with the object in study. Thermography allows the capture of infrared radiation of the electromagnetic spectrum

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Wavelength. Distance traveled by a periodic disturbance that is propagated by a medium in a single cycle. Also known as spatial period, representing the inverse of the frequency

References 1. Torrecilla JS. In: Torrecilla JS, ed. Olive: Its Processing and Waste Management. New York: Nova Science Publishers; 2010. 2. Izquierdo M, Lastra-Mejı´as M, Gonza´lez-Flores E, Pradana-Lo´pez S, Cancilla JC, Torrecilla JS. Visible imaging to convolutionally discern and authenticate varieties of rice and their derived flours. Food Control. 2020;110:106971. Available from: https://doi.org/ 10.1016/j.foodcont.2019.106971. 3. Moore JC, Spink J, Lipp M. Development and application of a database of food ingredient fraud and economically motivated adulteration from 1980 to 2010. J Food Sci. 2012;77(4). Available from: https://doi.org/10.1111/j.1750-3841.2012.02657.x. 4. Downey G, McIntyre P, Davies AN. Detecting and quantifying sunflower oil adulteration in extra virgin olive oils from the Eastern Mediterranean by visible and near-infrared spectroscopy. J Agric Food Chem. 2002;50(20):5520
5525. Available from: https://doi.org/10.1021/jf0257188. 5. Torrecilla JS, Cancilla JC, Matute G, Dı´az-Rodrı´guez P, Flores AI. Self-organizing maps based on chaotic parameters to detect adulterations of extra virgin olive oil with inferior edible oils. J Food Eng. 2013;118(4):400
405. Available from: https://doi.org/ 10.1016/j.jfoodeng.2013.04.029. 6. Izquierdo M, Lastra-Mejı´as M, Gonza´lez-Flores E, Cancilla JC, Aroca-Santos R, Torrecilla JS. Deep thermal imaging to compute the adulteration state of extra virgin olive oil. Comput Electron Agric. 2020;171:105290. Available from: https://doi.org/10.1016/j. compag.2020.105290. 7. Lastra-Mejı´as M, Aroca-Santos R, Izquierdo M, Cancilla JC, Mena ML, Torrecilla JS. Chaotic parameters from fluorescence spectra to resolve fraudulent mixtures of fresh and expired protected designation of origin extra virgin olive oils. Talanta. 2019;195:1
7. Available from: https://doi.org/10.1016/j.talanta.2018.10.102. 8. Pariente ES, Cancilla JC, Wierzchos K, Torrecilla JS. On-site images taken and processed to classify olives according to quality
the foundation of a high-grade olive oil. Postharvest Biol Technol. 2018;140:60
66. Available from: https://doi.org/10.1016/ j.postharvbio.2018.02.012. 9. Mohd Khairi MT, Ibrahim S, Md Yunus MA, Faramarzi M. Noninvasive techniques for detection of foreign bodies in food: a review. J Food Process Eng. 2018;41(6). Available from: https:// doi.org/10.1111/jfpe.12808. 10. Chiavaro E, Vittadini E, Rodriguez-Estrada MT, Cerretani L, Bendini A. Differential scanning calorimeter application to the detection of refined hazelnut oil in extra virgin olive oil. Food Chem. 2008;110(1):248
256. Available from: https://doi.org/ 10.1016/j.foodchem.2008.01.044. 11. Chiavaro E, Vittadini E, Rodriguez-Estrada MT, Cerretani L, Capelli L, Bendini A. Differential scanning calorimetry detection of high oleic sunflower oil as an adulterant in extra-virgin olive oil. J Food Lipids. 2009;16:227
244. 12. Bendini A, Cerretani L, Carrasco-Pancorbo A, et al. Phenolic molecules in virgin olive oils: a survey of their sensory properties, health

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effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules. 2007;12(8):1679
1719. Available from: https://doi.org/10.3390/12081679. Owen RW, Giacosa A, Hull WE, et al. Spacecrafts navigation signal research based on GNSS constellation. Lancet Oncol. 2000;1:107
112. Available from: https://doi.org/10.1007/978-3642-37404-3-4. Bubonja-sonje M, Giacometti J, Abram M. Antioxidant and antilisterial activity of olive oil, cocoa and rosemary extract polyphenols. Food Chem. 2011;127(4):1821
1827. Available from: https://doi. org/10.1016/j.foodchem.2011.02.071. Cicerale S, Lucas LJ, Keast RSJ. Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil. Curr Opin Biotechnol. 2012;23(2):129
135. Available from: https://doi. org/10.1016/j.copbio.2011.09.006. Vadivambal R, Jayas DS. Applications of thermal imaging in agriculture and food industry—a review. Food Bioprocess Technol. 2011;4 (2):186
199. Available from: https://doi.org/10.1007/s11947-010-0333-5. Ferrari C, Angiuli M, Tombari E, Righetti MC, Matteoli E, Salvetti G. Promoting calorimetry for olive oil authentication. Thermochim Acta. 2007;459(1
2):58
63. Available from: https://doi.org/ 10.1016/j.tca.2007.04.002. Torrecilla JS, Garcı´a J, Garcı´a S, Rodrı´guez F. Quantification of adulterant agents in extra virgin olive oil by models based on its thermophysical properties. J Food Eng. 2011;103(2). Available from: https://doi.org/10.1016/j.jfoodeng.2010.10.017. Meola C, Carlomagno GM. Recent advances in the use of infrared thermography. Meas Sci Technol. 2004;15(9). Available from: https://doi.org/10.1088/0957-0233/15/9/R01. Lowe S. A primer on infra-red thermography. Thermalcities. 2008;16
19. ,http://thermalcities.com/HOWITWORKS.htm..

21. Gowen AA, Tiwari BK, Cullen PJ, McDonnell K, O’Donnell CP. Applications of thermal imaging in food quality and safety assessment. Trends Food Sci Technol. 2010;21(4):190
200. Available from: https://doi.org/10.1016/j.tifs.2009.12.002. 22. Izquierdo M, Lastra-Mejı´as M, Gonza´lez-Flores E, Cancilla JC, Pe´rez M, Torrecilla JS. Convolutional decoding of thermographic images to locate and quantify honey adulterations. Talanta. 2020;209:120500. Available from: https://doi.org/10.1016/j. talanta.2019.120500. 23. Palancar MC, Arago´n JM, Migue´ns JA, Torrecilla JS. Application of a model reference adaptive control system to pH control. Effects of lag and delay time. Ind Eng Chem Res. 1996;35(11). Available from: https://doi.org/10.1021/ie960130 1 . 24. Torrecilla JS, Arago´n JM, Palancar MC. Modeling the drying of a high-moisture solid with an artificial neural network. Ind Eng Chem Res. 2005;. Available from: https://doi.org/10.1021/ ie0490435. 25. Mildner-Szkudlarz S, Jele´n HH. The potential of different techniques for volatile compounds analysis coupled with PCA for the detection of the adulteration of olive oil with hazelnut oil. Food Chem. 2008;110(3):751
761. Available from: https://doi.org/ 10.1016/j.foodchem.2008.02.053. 26. Gan-Mor S, Regev R, Levi A, Eshel D. Adapted thermal imaging for the development of postharvest precision steam-disinfection technology for carrots. Postharvest Biol Technol. 2011;59(3):265
271. Available from: https://doi.org/10.1016/j.postharvbio.2010.10.003. 27. Gonc¸alves BJ, de Oliveira Giarola TM, Pereira DF, de Barros Vilas Boas EV, de Resende JV. Using infrared thermography to evaluate the injuries of cold-stored guava. J Food Sci Technol. 2016;53(2):1063
1070. Available from: https://doi.org/10.1007/ s13197-015-2141-4.

Chapter 7

Influence of the distribution chain on the quality of extra virgin olive oils Jose´ S. Torrecilla1 and John C. Cancilla2 1

Department of Chemical and Materials Engineering, Complutense University of Madrid, Madrid, Spain, 2Scintillon Institute, San Diego, CA, United States

Abbreviations EVOO IOOC K232 K270 Mt

extra virgin olive oil International Olive Oil Council absorption of ultraviolet light at a wavelength of 232 nm absorption of ultraviolet light at a wavelength of 270 nm million tons

7.1 Introduction Edible vegetable oils represent a cornerstone in the diets of many cultures worldwide. More specifically, extra virgin olive oil (EVOO), which is directly olive juice, is a key piece in the Mediterranean diet.1 Among the most attractive characteristics of EVOO, we can highlight its variable and multicomponent organoleptic profile as well as its beneficial properties for health such as positive effects against digestive and cardiovascular diseases, several types of cancer, and more.2
5 In addition to these healthy characteristics, this oil represents a source of wealth for the European Community as the main producers are European.6,7 In the last campaign registered by the International Olive Oil Council (IOOC) (2017/18), 3.4 million tons (Mt) of EVOO were produced and, of these, 3.2 were produced in Europe. The IOOC forecasts a slight decrease in production on the European continent from 3.4 to 2.9 Mt of EVOO in the current campaign (2019/20), while the production of EVOO in countries outside of Europe will increase from 0.189 to 0.201 Mt. Considering total international imports of EVOO, according to the IOOC, a slight increase in the quantities imported is expected from 0.943 to 0.994 Mt in the 2017/18 to 2019/20 campaigns (data based on estimates), respectively. As with the vast majority of products, their market becomes more attractive to the extent that the producer ensures and manages quality properly, not only while being

produced, but also during its distribution chain. In the case of EVOO, several phenomena and environments are known to affect the quality of the product produced, helping understand and pinpoint crucial steps to protect its quality in the production process.8 Specifically, EVOO should be produced only through physical procedures leading to oils that have a low free acidity (,0.8%), a set of detectable organoleptic characteristics (perceptible fruitiness), and an absence of any sensory defect (e.g., musty, metallic, and rancid). It is important to emphasize that EVOO is a delicate food that is affected by a great number of variables and will never be better than at the time of production. Among others, the oil is affected by being exposed to light, humidity, temperature, and even just time.8,9 In the event that the conditions or environment surrounding the oil are not appropriate, a product that was originally an EVOO may downgrade to virgin olive oil, or even lampante olive oil, by the time it reaches the consumer. This is why a proper distribution chain and shelf life must be established, with controlled conditions, to ensure that the hard work invested to produce high-quality EVOO is not in vain.10

7.2 Quality of extra virgin olive oil This section will present not only the general measurements and analyses carried out on EVOOs within official quality control laboratories, but also those analytical prototypes that have been developed or are in the process of being produced for real-time quality control during the distribution chain.

7.2.1 Comparisons of olive oils with other edible oils The vegetable oil market is a key player in many economies. According to estimates by the German company Oil

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00015-8 © 2021 Elsevier Inc. All rights reserved.

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World, the production of vegetable oils in general (palm oil, soya, rape, sunflower, palm kernel, peanut, cotton, coconut, and olive) in the 2018/19 season will have increased by 2.3% compared to the 2017/18 season and by 27.2% compared to the 2011/12 season. Overall, vegetable oil production amounted to approximately 160 Mt in 2011/12, while slightly above 203 Mt in 2017/18. The mostly produced and consumed oils worldwide in recent seasons are, in decreasing order, palm oil, soybean oil, rapeseed oil, and sunflower oil, placing olive oil in ninth place, below maize oil and above coconut oil. The increase in world vegetable oil production in recent seasons was driven by the growth of palm, soybean, palm kernel, and maize oils. Global domestic consumption in 2011/12 was 156.9 Mt, while for the current cycle, it is expected to reach 202.6 Mt. Bearing in mind the quantity of vegetable oil produced and consumed, it is clear that in all cases the remains of the oils are not very high, Fig. 7.1. Although the general vegetable oil market is a key player in many economies, the market for EVOO is highly relevant not only for the economy of the countries of the Mediterranean Basin, but also for the economies of countries, such as United States or Australia, that are gradually becoming more recognized as producers of this juice.6,7 This is why high-quality levels must be maintained not only in the production stage but also during the entire distribution chain,8 so that consumption of this food, which ultimately provides the dividends for the sector’s subsistence, continues to grow.

7.2.2 Implications of olive oils for human health and disease prevention EVOO has a series of widely known health properties. However, some of them would need to be verified by

performing a greater number of validated in vivo experiments in order to guarantee them.11 Nevertheless, so far most of the health properties that are known are far from any doubt. These characteristics can be classified into three large groups according to the properties they present. In the first group, we can highlight those antimicrobial properties derived fundamentally from the polyphenols present in the oil. They mainly help against diseases of the digestive and respiratory systems.12,13 A second benefit relies on antioxidant properties that have a positive impact on diseases such as cancer, degenerative diseases, and promoting a healthy circulatory system.12,13 Finally, in the third group, the antiinflammatory properties of EVOO should be noted. These characteristics positively reflect on cardiovascular diseases, neurodegenerative diseases, arthritis, and even cancer.13,14 These healthy traits are directly correlated with the composition of the EVOO, which is highly dependent on the conditions to which the oil is exposed during the distribution chain. This is why the whole distribution chain must be adequate so that the composition of EVOO is maintained as much as possible, enabling the final consumer to access these desirable characteristics.

7.2.3 Laboratory quality control of extra virgin olive oil Without a doubt, official quality control procedures implemented by countries are vital in order to protect the quality of EVOOs, and they are done once the oil has been produced.8,15,16 Although these analyses are relatively long, they are conclusive in relation to different fraudulent activities that can be committed. The problem of carrying out lengthy chemical analyses, especially in the olive sector, is that olive growing takes approximately 4
5 months/year. During this time, many tons of EVOO must be evaluated. These conditions force official

FIGURE 7.1 Production (A) and consumption (B) of vegetable oils during three campaigns [2011/12 in black; 2017/18 in light gray; 2018/19 (estimations) in dark gray]. Oil World.

Influence of the distribution chain on the quality of extra virgin olive oils Chapter | 7

laboratories to carry out a great deal of work in short periods of time. This is why one of the lines of research is to facilitate finding low-quality oils by using analytical tools that can reduce the necessary amount of EVOO that must be officially assessed to validate its quality. The European Community has an established protocol or regulation that focuses on EVOO. The official regulations in this sector cover organoleptic, chemical, and physical characteristics of the EVOO, which enable the detection of any fraudulent activity that may appear while producing the food. That is why, in order to respond to the latest fraudulent activities, these regulations are updated over time to maintain the high quality of the food regardless of those who want to take illicit economic advantage of the regulations. An example is the Commission’s Implementing Regulation (EU) 2019/1604 of September 27, 2019, amending Regulation (EEC) 2568/91 on the characteristics of olive oil and olivepomace oil and on the relevant methods of analysis. A wide variety of analytical methods are available for carrying out quality control of EVOO outside or within its distribution chain. Among them, we can highlight analytical techniques, including vibrational (FTIR, MIR, NIR, and Raman spectroscopy), spectroscopic, chromatographic, and voltammetric.17
19 Quality control prototypes based on UV
vis spectroscopic techniques of repetitive analysis on EVOO samples are also being designed.20 Furthermore, other quality control pathways are based on the analysis of the volatile profile of the food,21 or through measurements with sophisticated equipment that enable evaluating the quality and even locate adulterations.22 These methods could be carried out both at origin and at destination, with samples being taken and analyses carried out in qualified centers for analysis. Esposto et al. determined a relationship between various quality parameters of the oil, including peroxide content, K232, and K270 with the storage time of EVOO.17 Likewise, once the EVOO is produced, and before it is marketed, the oil must be labeled so that the end customers know exactly the quality and main official reglementary

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specifications of the EVOO they are buying (quality, best before date, main composition, etc.). This label is a contract that the producer establishes with the client to certify that the quality of the product is ensured. As in the previous case, this regulation is also updated over time. In the European Community, all this is reflected and regulated in the Delegated Regulation (EU) 2018/1096 of the Commission of May 22, 2018, that modifies the Executing Regulation (EU) 29/2012 with regard to the requirements applicable to certain indications on the labeling of olive oil.

7.2.4 Real-time quality control of extra virgin olive oil Although these methods are still being carried out on a more formal basis, it should be known that it would be more appropriate for the producer, distributor, and consumer to carry out analytical measurements during real time so as to identify potential and/or specific points at which the quality of the EVOO could decline in order to locate the reasons and set possible improvements in the distribution chain.10,16 These methodologies would be all the more valuable for long-distance transport or lengthy storage periods.8 As a representative example, it is worth mentioning the designs put into operation by the AlgoReach research group (Universidad Complutense de Madrid). This group has integrated intelligent mathematical and spectroscopic tools based on visible and fluorescence spectroscopy, as well as thermographic techniques that monitor the evolution of the quality of EVOOs both during the transport and the storage of the product, Fig. 7.2.10,16,23
25 There is a relationship between the spectroscopic absorption of EVOO samples and their quality (US Standards for Olive Oil and Olive Pomace Oil Grades, 2010).10,19,23 Therefore it is expected that the analysis of absorbance over time will also provide information on the evolution of the quality of the EVOOs. Specifically, the degradation of EVOOs can be easily evaluated using mainly the variation of

FIGURE 7.2 Diagram of the distribution chain from production to the consumer.

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the concentrations of compounds present in the oil such as chlorophyll pigments (chlorophyll-a and chlorophyll-b) and carotenoids (lutein and β-carotene),26,27 compounds whose concentration can be inferred from spectroscopic measurements in the visible range. This has been the basis of some technological developments for the identification and quantification of adulterants.9,10,16,23,28 As in any biological matrix, the possibility of finding signal overlaps, mainly in spectroscopic analysis, is very common. Therefore signals must be treated by tools capable of managing and deciphering these overlaps in order to give the most reliable answers possible regarding quality estimation. In the case of EVOO, given the complexity and degree of overlapping absorption of the compounds that make up the oil, the use of chemometric tools based on nonlinear algorithms is indicated.9,28,29 The most common tools with this objective are based on neural networks, chaotic algorithms, and some algorithms of linear nature such as principal component analysis.19,30 With this integration between the analytical equipment and the mathematical tools, it is possible to control the quality of the EVOO and also have a record of how the exposures to light, time, and temperatures during storage affect EVOO since it is produced until it is acquired and consumed by the final customer. All this information would provide valuable feedback to evaluate and monitor EVOO throughout its distribution chain. This allows us to determine the phases where the food has been mistreated, enabling the improvement of production and mainly the distribution chain, helping protect the producers work and satisfying the client.10,16,23 These tools have no official status, so they cannot provide a direct administrative response to fraudulent activities. However, given the ease and speed with which they can be used, they facilitate the performance of a large number of analyses in a short period of time.8,16 These analyses could ensure that those doubtful samples in terms of quality can be removed from the distribution chain, thus ensuring the high-quality standards that must accompany the product at all times. The application of these tools is particularly suitable for olive harvesting periods where a large number of EVOO tons are produced over 4
5 months. In this context, with these tools, the oils would be classified into two groups: the samples with (1) undoubtfully proper quality or (2) with dubious or negative quality that contradicts labeling. In this line, only the analysis via official quality control would be carried out on the latter group. This would enable spending time only on the samples that are suspected of improper quality.

7.3 Conclusion Healthy dieting is a vital topic for every person and nation as well as a fundamental economic aspect. Ensuring that

food such as EVOO maintains the highest quality possible until it reaches the consumer is therefore crucial. To do so, the entire production and distribution chains must be monitored, so that the final customer not only consumes the product but also enjoys it safely and continues to trust it. This way, the commercialization and economic performance will be positive, linked with the satisfaction of the client. It should be noted that these needs are covered and protected by the various states and communities of states, in different ways, such as defining designations of origin, labeling, and chemical analysis of samples, among others. These chemical analyses can be carried out by official methods, which give a regulatory character to the techniques used, being able to definitively diagnose an activity as fraudulent from another that is not. On the other hand, there are also quicker and easier to use methods that can detect activities not subject to regulation in a shorter time. They can be used as a method of detecting a fraudulent sample quickly and separating it from the distribution chain so that it does not alter the quality of the product. Subsequently, this sample must be contrasted by official methods in order to ensure that the activity is truly fraudulent and to act in accordance with regulations.

Acknowledgment This work has been partially financed by the FEI program of the Universidad Complutense de Madrid under the references of the project FEI-EU-17-03 and FEI 18/10.

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Adulteration. Alteration or reduction of the quality and purity of a good by adding something that is foreign or due to improper handling. Artificial neural networks. Computational model consisting of a set of computing centers, called artificial neurons, connected to each other to transmit signals and adapt the models to the systems under study. Food security. Adequate availability and accessibility of food, while meeting quality and safe standards. Lampante olive oil. Defective olive oil that cannot be marketed until a refining process is carried out to enable consumption. Milling of grains or fruits. For virgin olive oil, the act of breaking the olive to form the paste from which the fat content present in the olives is extracted. Organoleptic characteristics. Physical characteristics of the material in general, as perceived by its taste, texture, smell, color, or temperature. Protected designation of origin. Official recognition by the European Commission of the existence of a group of products originating in a certain location,

Influence of the distribution chain on the quality of extra virgin olive oils Chapter | 7

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whose characteristics are bound to that environment and the human factors that form it. Quality of extra virgin olive oil. A set of traits that are typical of extra virgin olive oil and make it acceptable to the end consumer. Among the characteristics to be fulfilled are those determined by physicochemical means or those perceived by the senses that are included in the regulations in effect. Vegetable oils. Liquid oils at room temperature extracted from plants, or broadly defined without taking into account the state of the substance at a given temperature.

References 1. Palla M, Digiacomo M, Cristani C, et al. Composition of healthpromoting phenolic compounds in two extra virgin olive oils and diversity of associated yeasts. J Food Compos Anal. 2018;74:27
33. Available from: https://doi.org/10.1016/j.jfca.2018.08.008. 2. Bozzetto L, Alderisio A, Clemente G, et al. Gastrointestinal effects of extra-virgin olive oil associated with lower postprandial glycemia in type 1 diabetes. Clin Nutr. 2019;38(6):2645
2651. Available from: https://doi.org/10.1016/j.clnu.2018.11.015. 3. De Stefanis D, Scime` S, Accomazzo S, et al. Anti-proliferative effects of an extra-virgin olive oil extract enriched in ligstroside aglycone and oleocanthal on human liver cancer cell lines. Cancers (Basel). 2019;11(11). Available from: https://doi.org/10.3390/ cancers11111640. 4. Romani A, Ieri F, Urciuoli S, et al. Health effects of phenolic compounds found in extra-virgin olive oil, by-products, and leaf of Olea europaea L. Nutrients. 2019;11:1776. 5. Nocella C, Cammisotto V, Fianchini L, et al. Extra virgin olive oil and cardiovascular diseases: benefits for human health. Endocr, Metab Immune Disord: Drug Targets. 2017;18(1):4
13. Available from: https://doi.org/10.2174/1871530317666171114121533. 6. Sansone-Land A, Takeoka GR, Shoemaker CF. Volatile constituents of commercial imported and domestic black-ripe table olives (Olea europaea). Food Chem. 2014;149:285
295. Available from: https://doi.org/10.1016/j.foodchem.2013.10.090. 7. Stillitano T, Falcone G, De Luca AI, et al. A life cycle perspective to assess the environmental and economic impacts of innovative technologies in extra virgin olive oil extraction. Foods. 2019;8(6). Available from: https://doi.org/10.3390/foods8060209. 8. Torrecilla JS. In: Torrecilla JS, ed. Olive: Its Processing and Waste Management. New York: Nova Science Publishers; 2010. 9. Torrecilla JS, Vidal S, Aroca-Santos R, Wang SC, Cancilla JC. Spectroscopic determination of the photodegradation of monovarietal extra virgin olive oils and their binary mixtures through intelligent systems. Talanta. 2015;144. Available from: https://doi.org/ 10.1016/j.talanta.2015.06.042. 10. Aroca-Santos R, Lastra-Mejı´as M, Cancilla JC, Torrecilla JS. Intelligent modelling to monitor the evolution of quality of extra virgin olive oil in simulated distribution conditions. Biosyst Eng. 2018;172:49
56. Available from: https://doi.org/10.1016/j. biosystemseng.2018.05.007.

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11. Frankel EN. Nutritional and biological properties of extra virgin olive oil. J Agric Food Chem. 2011;59(3):785
792. Available from: https://doi.org/10.1021/jf103813t. 12. Bubonja-Sonje M, Giacometti J, Abram M. Antioxidant and antilisterial activity of olive oil, cocoa and rosemary extract polyphenols. Food Chem. 2011;127(4):1821
1827. Available from: https://doi. org/10.1016/j.foodchem.2011.02.071. 13. Cicerale S, Lucas LJ, Keast RSJ. Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil. Curr Opin Biotechnol. 2012;23(2):129
135. Available from: https://doi. org/10.1016/j.copbio.2011.09.006. 14. Serreli G, Deiana M. Extra virgin olive oil polyphenols: modulation of cellular pathways related to oxidant species and inflammation in aging. Cells. 2020;9:478. 15. Skiada V, Tsarouhas P, Varzakas T. Preliminary study and observation of “Kalamata PDO” extra virgin olive oil, in the Messinia region, southwest of Peloponnese (Greece). Foods. 2019;8(12). Available from: https://doi.org/10.3390/foods8120610. 16. Lastra-Mejias M, Izquierdo M, Torreblanca-Zanca A, et al. Cognitive fluorescence sensing to monitor the storage conditions and locate adulterations of extra virgin olive oil. Food Control. 2019;103:48
58. Available from: https://doi.org/10.1016/j.foodcont.2019.03.033. 17. Esposto S, Selvaggini R, Taticchi A, Veneziani G, Sordini B, Servili M. Quality evolution of extra-virgin olive oils according to their chemical composition during 22 months of storage under dark conditions. Food Chem. 2020;311:126044. Available from: https:// doi.org/10.1016/j.foodchem.2019.126044. 18. Meenu M, Cai Q, Xu B. A critical review on analytical techniques to detect adulteration of extra virgin olive oil. Trends Food Sci Technol. 2019;91:391
408. Available from: https://doi.org/ 10.1016/j.tifs.2019.07.045. 19. Lastra-Mejı´as M, Aroca-Santos R, Izquierdo M, Cancilla JC, Mena ML, Torrecilla JS. Chaotic parameters from fluorescence spectra to resolve fraudulent mixtures of fresh and expired protected designation of origin extra virgin olive oils. Talanta. 2019;195:1
7. Available from: https://doi.org/10.1016/j.talanta.2018.10.102. 20. He H, Lu W. High-throughput chemometric quality assessment of extra virgin olive oils using a microtiter plate reader. Sensors (Switzerland). 2019;19(19). Available from: https://doi.org/10.3390/s19194169. 21. Giuffre` AM, Capocasale M, Macrı` R, Caracciolo M, Zappia C, Poiana M. Volatile profiles of extra virgin olive oil, olive pomace oil, soybean oil and palm oil in different heating conditions. LWT. 2020;117. Available from: https://doi.org/10.1016/j.lwt.2019.108631. 22. Green HS, Li X, De Pra M, et al. A rapid method for the detection of extra virgin olive oil adulteration using UHPLC-CAD profiling of triacylglycerols and PCA. Food Control. 2020;107:106773. Available from: https://doi.org/10.1016/j.foodcont.2019.106773. 23. Izquierdo M, Lastra-Mejı´as M, Gonza´lez-Flores E, Cancilla JC, Aroca-Santos R, Torrecilla JS. Deep thermal imaging to compute the adulteration state of extra virgin olive oil. Comput Electron Agric. 2020;171:105290. Available from: https://doi.org/10.1016/j. compag.2020.105290. 24. Torrecilla JS, Mena ML, Ya´n˜ez-Seden˜o P, Garcı´a J. Application of artificial neural network to the determination of phenolic compounds in olive oil mill wastewater. J Food Eng. 2007;81(3). Available from: https://doi.org/10.1016/j.jfoodeng.2006.12.003. 25. Palancar MC, Arago´n JM, Migue´ns JA, Torrecilla JS. Application of a model reference adaptive control system to pH control. Effects of

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lag and delay time. Ind Eng Chem Res. 1996;35(11):4100
4110. Available from: https://doi.org/10.1021/ie9601301 . 26. Ayuso J, Haro MR, Escolar D. Simulation of the visible spectra for edible virgin olive oils: potential uses. Appl Spectrosc. 2004;58(4):474
480. Available from: https://doi.org/10.1366/ 000370204773580347. 27. Domenici V, Ancora D, Cifelli M, Serani A, Veracini CA, Zandomeneghi M. Extraction of pigment information from nearUV vis absorption spectra of extra virgin olive oils. J Agric Food Chem. 2014;62(38):9317
9325. Available from: https://doi.org/ 10.1021/jf503818k. 28. Aroca-Santos R, Cancilla JC, Matute G, Torrecilla JS. Identifying and quantifying adulterants in extra virgin olive oil of the picual

varietal by absorption spectroscopy and nonlinear modeling. J Agric Food Chem. 2015;63(23):5646
5652. Available from: https://doi.org/10.1021/acs.jafc.5b01700. 29. Palomar J, Torrecilla JS, Lemus J, Ferro VR, Rodrı´guez F. A COSMO-RS based guide to analyze/quantify the polarity of ionic liquids and their mixtures with organic cosolvents. Phys Chem Chem Phys. 2010;12(8):1991
2000. Available from: https://doi. org/10.1039/b920651p. 30. Crizel RL, Hoffmann JF, Zandona´ GP, Lobo PMS, Jorge RO, Chaves FC. Characterization of extra virgin olive oil from Southern Brazil. Eur J Lipid Sci Technol. 2020; 1900347:11
14. Available from: https://doi.org/10.1002/ ejlt.201900347.

Chapter 8

Spectroscopy to evaluate the quality control of extra-virgin olive oils Jose´ S. Torrecilla1, John C. Cancilla2, Ana M. Perez-Calabuig1 and Sandra Pradana-Lopez1 1

Department of Chemical and Materials Engineering, Complutense University of Madrid, Madrid, Spain, 2Scintillon Institute, San Diego, CA, United

States

Abbreviations EVOO HOMO LUMO NIR PDO UV
vis

extra-virgin olive oil highest occupied molecular orbital lowest unoccupied molecular orbital near-infrared protected designation of origin ultraviolet
visible

8.1 Introduction As in other business areas, quality control in the field of food is centered on the use of technologies based on the analysis of physical, chemical, microbiological, nutritional, or organoleptic properties with the aim of determining if a food is healthy, tasty, and meets the specifications indicated on their labels and expected by the consumer. Likewise, it should not be forgotten that quality control must pursue the fundamental objective of protecting the consumer from fraud as well as his or her health.1 Quality standards are established on the basis of organoleptic characteristics such as taste, color, and aroma or quantitative properties such as the content of certain compounds that are specific to the type of food analyzed or that are common to the rest of foods. Based on all of this, reactions and environments that deteriorate food, the packaging used, the maximum shelf life, and the type of consumer (e.g., stores, restaurants, or individuals) to whom the products are addressed are also established. Knowing these characteristics, it is relatively simple to determine which conditions can alter the quality of food within production or distribution, until the food is consumed by the final client.1 In some cases, these standards are set in food laws and regulations related to marketing, production, labeling, additives used, general manufacturing practices, and so on.

These regulations have not only been implemented recently, as already around 2500 BCE there were Egyptians who prevented the contamination of meat via regulations.2 Also, more than 2000 years ago, adulterations of cereals and edible fats were prohibited. At every point in history, attempts were made to protect food quality with the available tools.3 Nowadays, the control has improved as we have a greater understanding about technology that allows to analyze foods properly and enables estimating the loss of quality in each one of the stages at which already produced food can suffer from a quality loss until it reaches the consumer. What has been gained in recent years is the increase in supply, enabling the customer to choose those foods that best meet their needs or preferences. This is why under these circumstances, seeking and assuring quality is not only a health parameter, but also an economic one for companies immersed in supply and demand.1 To protect quality and safety a large number of studies have been carried out in order to identify those foods that are most subjected to fraudulent activities. The first seven foods that are mostly affected by illegal activities are the olive sector in the first place, followed by the dairy sector, the beekeeping sector, saffron, orange juice, coffee, and apple juice.4 That is why the objective of this chapter is vastly relevant, as it will focus on the product that suffers most from fraudulent activities: extra-virgin olive oil (EVOO). EVOO is one of the most demanded products by consumers worldwide, especially in countries belonging to the Mediterranean basin.5 This consumption is due not only to the organoleptic characteristics of EVOO but also to the healthy features that all EVOO varieties offer. This oil, as one of the main components of the Mediterranean diet, offers a high source of antioxidants that have shown to aid in the prevention of some psychological and cardiovascular

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00042-0 © 2021 Elsevier Inc. All rights reserved.

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diseases as well as cancers, among others.5
7 From a more economic point of view, according to the International Olive Council, it is expected that in the 2018
19 campaign, more than 3.4 million tons of EVOO will be produced worldwide. This reveals not only a great supply but also a corresponding demand, demonstrating that the olive sector is offering remarkable financial returns at a macroeconomic level.8 For the protection of the markets and the interests of all countries both at the production and consumption levels, the European Commission has implemented a Community legal framework requested by the European Parliament to protect the quality and safety of EVOO and its properties implementing regulation (EU) 2019/1604 or also to establish the labeling of olive oil following the Delegated Regulation (EU) 2018/1096. However, these controls are sometimes not sufficient to prevent fraud and damage to health caused by fraudulent practices. An example of these damages to health is the Spanish toxic oil syndrome from 1981, which affected over 20,000 people, causing serious diseases and, in some cases, death, and even today still presents long-lasting consequences.5,9 To combat these situations, in addition to the regulatory constraints established by the authorities, it is necessary to have tools and technologies that are capable of detecting fraudulent activities such as oils that do not meet the specifications required for sale or may be deleterious for the health of consumers. For this reason, it is necessary to use simple and rapid techniques to ensure the quality of EVOO. Thus, in addition to other techniques, spectroscopic pathways are good alternatives. These include ultraviolet
visible (UV
vis) spectroscopy, Fourier transform (FT) Raman spectroscopy, near-infrared (NIR) spectroscopy, or fluorescence spectroscopy.10 Many of these techniques are used not only for the detection and quality control of oils but also for the detection of various adulterants included in the EVOO itself.10,11 In the following sections, these spectroscopic techniques will be presented, as well as their applications within the EVOO sector.

8.1.1 Comparisons of olive oils with other edible oils One of the most widespread applications of spectroscopy in the oil sector is precisely to distinguish different types of oils of the same or different botanical origins. These differences are based on the fact that EVOOs and all related products lead to spectroscopic results that are completely different to those from any other seed oils. In general, these differences are based on the compounds that comprise each of the oils. Specifically, EVOO has compounds such as pigments, antioxidants, or vitamins that originate characteristic spectroscopic absorption.12 In

the case of seed oils, since they do not come from fruits, and chemical
physical operations of refining and extraction are carried out, their composition is totally different. They lack many of the valuable compounds or have reduced concentrations of, for example, phenolic compounds or vitamins, among others.13,14 Spectroscopy can also be used to classify types of oils and to detect adulterations, as differences still exist between different types or varietals of olive oil, as well as between pure and adulterated samples. When fraudulent activities such as adulterations are carried out by adding low concentrations of a cheaper or low-quality product, it is common to combine the spectroscopic results with linear and nonlinear algorithms for their detection and quantification.12,15,16 Therefore different spectroscopic techniques (vide infra), besides being valuable to characterize edible oils, can help in the fight against adulterations, protecting honorable producers as well as consumers.

8.1.2 Implications for human health and disease prevention EVOOs along with fish, vegetables, and fruits, among others, are healthy foods that form the Mediterranean diet. It is important to mention that a healthy and relatively disease-free population should include these foods among their regular intake.17 These are foods that are easily accessible to any stratum of society and that, when cooked properly and to the taste of the consumer, offer very appealing organoleptic characteristics, and, therefore, taking advantage of these healthy characteristics is very simple. Epidemiological data show that the Mediterranean diet in general has protective properties against certain pathologies such as cancer (colon, breast, skin, among others), coronary diseases, as well as helping during a normal aging process.17 These characteristics are linked to the composition of EVOO, which contains certain phenolic fractions (hydroxytyrosol, tyrosol, among others), with inherent antioxidant characteristics, as well as the popular unsaturated fats, namely, monounsaturated fatty acids, such as oleic acid, known for its beneficial properties, including the limitation of low-density lipoprotein cholesterol. If the antiinflammatory properties of EVOO are considered, protection can be found against diseases linked to inflammation such as several of the much feared and widespread neurodegenerative diseases, arthritis, and even cancer.18,19 Protection also extends to diseases of the digestive and respiratory systems due mainly to the antimicrobial characteristics of the phenolic compounds of the oil.18,20 However, much more research is needed in these areas in order to uncover all the healthy characteristics that EVOO undoubtedly has and are not yet sufficiently comprehended and even unknown.21

Spectroscopy to evaluate the quality control of extra-virgin olive oils Chapter | 8

Although the beneficial effects of EVOO on health are being heavily promoted, society is still not sufficiently aware of these characteristics and the value they have within our diet. Therefore it is necessary not only to advertise these characteristics but also to ensure that the oils continue to have these properties. Therefore we must continue to fight against adulteration and avoid becoming careless during production or distribution to not deteriorate both the actual EVOO and our perception toward it and its quality, so we can continue to benefit from its organoleptic and health properties.

8.2 Spectroscopy for quality control Spectroscopy studies the interaction of electromagnetic radiation with matter, depending on the wavelength. Electromagnetic radiation is therefore the fundamental pillar of spectroscopy and is categorized according to its wavelength (Fig. 8.1). This radiation is the combination of oscillating electric and magnetic fields that propagate from one point to another transporting energy. Unlike other radiations, the transport of this radiation does not require any means of propagation, that is, it can propagate in vacuum. The incidence of this radiation on matter can generate an absorption and/or emission of electromagnetic radiation and relate this exchanged energy to various levels of energies involved in their quantum transitions. All these effects generate a wide range of information about the matter under study. These applications are useful in a large number of work and research areas, both food and nonfood related. However, this large group of techniques that work with electromagnetic radiation offers a lot of qualitative and quantitative information. In recent years, one of the most requested applications is related to food quality control. Among the foods that benefit from these analyses is EVOO due to the inherent importance of its quality.22 Information about the composition of oils and also about potentially fraudulent activities that EVOO may have suffered during its production can be evaluated via spectroscopic techniques.22
24 Given that wavelength condition the energy supplied to the food under study, this provides different and often complementary information. For this reason, different spectroscopic methods will be covered in the next subsections.

Increasing energy and frequency Decreasing wavelength FIGURE 8.1 Electromagnetic radiation types according to wavelength.

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8.2.1 Ultraviolet
visible spectroscopy UV
vis spectroscopy is one of the simplest ways to evaluate the quality of food, because, in many cases, a simple change in visible colors, which is registered with this technique, reflects the evolution of the quality of the food itself.25 The Spanish research group AlgoReach has verified this, detecting adulterations and quality development of EVOO through the analysis of the absorption in this region of the spectrum (vide infra).23,25 UV
vis spectroscopy is usually considered a general technique because most molecules absorb in the wavelength range of 100
400 nm for UV and 400
700 nm for visible. Within these ranges, wavelengths between 100 and 200 nm correspond to deep UV. Since it is difficult to find sources that isolate either visible or ultraviolet radiation, UV
vis sources are usually used. Examples of sources in these ranges are deuterium lamps that produce radiation from 170 to 375 nm or a tungsten source for visible radiation covering 350
2500 nm.26,27 Radiation corresponding to the UV
vis has enough energy to make the electrons ascend from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The energy of the incident photon must exactly match the energy difference between the HOMO and the LUMO, which is also called the bandgap. This is why molecules with different chemical structures have different bandgaps and therefore specific resulting absorption spectra. The most common transitions in this energy range fall within the orbitals π (linkers) and π* (antilinkers), and, usually, these pi orbitals arise because of the presence of double bonds.26 For this reason, some of the best examples of molecules with clear ultraviolet absorption bands and spectra correspond to those containing double bonds. On the other hand, those molecules that absorb in the visible region are compounds that reflect a specific coloration, such as the greenish yellow tones of EVOO.26 Specifically, the research group AlgoReach has used UV
vis spectroscopy integrated with intelligent algorithms to determine the 3-month evolution and loss of quality of different types of EVOO during the distribution chain under different environmental conditions. This technique led to the development of light and cost-effective tools of easy use to monitor the evolution of the quality of oil.23 Likewise, the same research group developed another tool based on the combination of UV
vis spectroscopy and intelligent algorithms for the identification and quantification of typical and hard-to-detect EVOO adulterants such as inexpensive refined olive oil. Errors lower than 3% were obtained in the estimation of the concentration of the adulteration in EVOO.25 Also, this spectroscopic technique can be used to classify EVOO by geographic origin. An example of this application is

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presented by Alves et al., where the mentioned spectroscopy is integrated with mathematical tools to distinguish different EVOOs from the Mediterranean basin.28

process of olive pomace was monitored, thus giving answers to relevant questions in this regard.37

8.2.3 Raman spectroscopy 8.2.2 Near-infrared spectroscopy NIR spectroscopy in food analysis is routinely used for the determination of food composition (water, fat, protein, and so on). A great advantage of this methodology is that in many cases sample preparation is not necessary, so the analysis is simple and fast (less than 90 s) and in some cases can be carried out online in real time.29 One of the strengths of NIR spectroscopy is that it allows several constituents to be measured simultaneously. It can be used for quality and authenticity control in various areas such as in the fields of cereals, meat, fish, and dairy products.29 The NIR spectral region covers a wavelength range between 700 and 2500 nm, neighboring the region of visible radiation. Specifically, it covers the high wavelengths of the visible range and the low wavelengths of the IR region.29
31 One of the most important signals that NIR spectroscopy brings us is the response of the molecular bonds O
H, C
H, C
O, and N
H. In addition to its simplicity, these characteristics make this spectroscopy a suitable method to use in food control (cereals, meat, fish, dairy products, and so on) to monitor different compounds over time.29,32 For all these reasons, it has been used in applications related to agri-food, such as quality inspection of cereals, postharvest handling of fruits and vegetables, or food safety and authentication, among which EVOO should also be highlighted.29,31,33 This technique allows the analysis of their composition and humidity; therefore it is also possible to control if the samples have been adulterated.34 Among the different applications that are currently being developed, some interesting ones include EVOO quality control and for assistance in relatively complicated tasks in the olive sector. In the field of quality control, a large number of laboratories are carrying out control tasks by means of NIR spectroscopy. For instance, Mustorgy et al. integrated FT and NIR spectroscopic analysis for 106 types of EVOO from different Italian regions. For this purpose the equipment was calibrated for the analysis of fatty acid methyl esters and triacylglycerols.35 On the other hand, Casson et al. published a technique for evaluating the maturity of olives by means of NIR and visible infrared spectroscopy. These techniques were able to determine maturity parameters in a simple and fast way. As a reference, the humidity, the fat content, and the phenols present in the olives were taken.36 On the other hand, Altieri et al. published a technique based on NIR spectroscopy to determine the water and oil content in olive pomace. By means of this technique, the extraction

The always increasing computing power of new equipment has allowed Raman spectroscopy to gain popularity as an analytical tool in the food field. Using this spectroscopic technique, physicochemical information can be extracted from the food without destroying the sample.38 In particular, several applications can be found in various fields of the agricultural and food sector. The latter include oil, fruits, vegetables, meat, and dairy products.38 Raman spectroscopy is defined as a high-resolution photonic technique based on light scattering when a monochromatic light beam strikes an organic or inorganic material. This method provides chemical and structural information and is a nondestructive technique.39 A very important advantage is its simplicity and being harmless to the environment.22,38
40 Furthermore, this technique has been used to determine the CIS/TRANS isomers content of fatty acids from bands at 1656 and 1670 cm21 present in the EVOO. In this way, it can detect edible vegetable oils qualitatively and also quantitatively.22,41 As an example of application, Aykas et al. developed a quality control method based on Raman spectroscopy to authenticate EVOO using signals from vibrational spectra combined with pattern recognition algorithms. In this work, more than 150 samples of EVOO, virgin olive oil, and nonvirgin olive oil were used, in addition to adulterations consisting of other vegetable oils. Spectral data were collected using Raman spectroscopy (1064 nm excitation laser). Perfect sensitivity (100%) and high specificity (89%) were reported for the detection of EVOO.10

8.2.4 Fluorescent spectroscopy The application of fluorescence spectroscopy is widely used in the food sector.42 In most applications, no sample preparation is required, so it is a simple technique that can be applied for real-time analysis. It can be applied in the determination of changes affecting food quality either in the distribution chain or in the production center.24,42 This technique is currently being applied for the quality control of dairy products, meat, fish, egg products, oils, cereals, sugar, fruit, and vegetables. Fluorescence can also be used for the identification of bacteria of agro-food interest.42 The phenomenon of fluorescence is a type of luminescence that is characterized by the emission of electromagnetic radiation that a compound generates once it has absorbed energy from an incident radiation and then releases it in the form of a photon. Both electromagnetic radiations have different energies; the emitted radiation

Spectroscopy to evaluate the quality control of extra-virgin olive oils Chapter | 8

has lower energy or larger wavelengths than the initial radiation from the source. The typical mechanism of fluorescence involves three distinct sequential steps: first, the absorption of the electromagnetic radiation takes place; then, a nonradiative dissipation is produced; and finally, the emission of radiation of lower energy takes place.27 The three phases occur in a very short time, within the nanosecond scale, meaning that as soon as the light from the source ceases to impact the sample, the fluorescence stops as well. The AlgoReach research group has developed a portable device that includes supporting instruments built using a 3D printer and a cost-effective electromagnetic radiation source (light-emitting diode). This equipment takes advantage of fluorescence spectroscopy to achieve several applications regarding EVOO quality control. For example, the quality evolution of monovarietal EVOO samples was controlled during 2 months of storage and transport within different types of containers and under different storage conditions of temperature and exposure to light. The information collected was processed by intelligent algorithms, which using the information, collected via fluorescent spectroscopy, was able to distinguish adulterated from unadulterated samples, ones stored in dark environments from others exposed to light, or stored in transparent or colored bottles either made of glass or plastic, with high accuracy.24 Furthermore, the AlgoReach research group also employed this equipment for controlling the labeling of EVOOs with a protected designation of origin (PDO) by means of fluorescent spectroscopy and intelligent algorithms for pattern recognition. In this case the source used is an inexpensive laser diode. The complete experiment was developed by analyzing 254 mixtures of PDOs within the best-before date with other oils that were outside this time period. The models were capable of predicting the presence of other types of EVOO in concentrations even below 1.5% in weight.12

applications but also to detect adulterations and even to help solve production and olive grove issues.

Acknowledgment This work has been partially financed by the FEI program of the Universidad Complutense de Madrid under the references of the project FEI-EU-17-03 and FEI 18/10.

Mini-dictionary of terms G

G

G

G

G

G

G

8.3 Conclusion EVOO is one of the foods that have suffered more from fraudulent activities. For this reason the administrations of countries such as several from the European Union have been implementing measures for a long time to try to combat these fraudulent activities. In addition, other analytical techniques are being developed for the detection of adulterations and the control of the quality of these oils. In this work the focus has been given on spectroscopic techniques trying to give a simple, reliable, and cost-effective answer to these so denounced fraudulent activities. Among the routes that have been covered are UV
vis spectroscopy, infrared spectroscopy, Raman spectroscopy, and fluorescent spectroscopy. All these analytical routes are suitable alternatives to respond not only to quality control

95

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G

G

G

Chemometric. Chemistry discipline that focuses on the application of mathematical and/or statistical methods to chemical data Electromagnetic radiation. Type of variable electromagnetic field. Specifically, it is a combination of oscillating electric and magnetic fields, which propagate through space transporting energy from one place to another Fluorescence. A particular type of luminescence, which is characterized by the emission of a lower energy radiation as a result of an electromagnetic radiation exciting the matter under analysis Food quality. A set of traits that are specific to food and make it acceptable to end consumers. These qualities include both those determined by physicochemical pathways and those perceived by the senses. Food safety. It refers to the adequate availability of food, people’s access to it and the quality of it in order to fulfill the purposes established for it Light-emitting. diode (LED): A light source consisting of a semiconductor material. Specifically, it is a p
n junction diode, releasing energy in the form of photons (light) when activated Milling. Process by which grains or fruits are ground. In virgin olive oil, grinding is the process where the olive is broken down to form the paste from which the fat content present in the olives is extracted Near-infrared. Region of the shortest wavelength within the infrared spectrum, located between visible light and the mid-infrared, approximately between 700 and 2500 nm Quality control. A set of mechanisms and technologies used to detect the presence of nonconformities with respect to the purpose to be fulfilled prior to reaching the consumer Raman. Form of high-resolution vibrational spectroscopy or photonic technique based on light scattering Spectroscopy. Study of the interaction between electromagnetic radiation and matter. This interaction can carry out an absorption or emission of energy in the form of radiation

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UV
visible absorption. Measurement of the attenuation of radiation after it passes through a sample or is reflected by the surface of such sample

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17. Available from: https://doi. org/10.3390/foods9020221. 11. Iqdiam BM, Welt BA, Goodrich-Schneider R, Sims CA, Baker GL, Marshall MR. Influence of headspace oxygen on quality and shelf life of extra virgin olive oil during storage. Food Packag Shelf Life. 2020;23:100433. Available from: https://doi.org/10.1016/j. fpsl.2019.100433. 12. Torreblanca-Zanca A, Aroca-Santos R, Lastra-Mejı´as M, Izquierdo M, Cancilla JC, Torrecilla JS. Laser diode induced excitation of PDO extra virgin olive oils for cognitive authentication and fraud detection. Sens Actuators, B: Chem. 2019;280:1
9. Available from: https://doi.org/10.1016/j.snb.2018.10.014. 13. Pan F, Wen B, Luo X, et al. Influence of refining processes on the bioactive composition, in vitro antioxidant capacity, and their correlation of Perilla seed oil. Food Sci. 2020;85:1160
1166. Available from: https://doi.org/10.1111/1750-3841.15070. 14. Duman E. The influence of industrial refining stages on the physico-chemical properties, fatty acid composition and sterol contents in hazelnut oil. J Food Sci Technol. 2020;57. Available from: https://doi.org/10.1007/s13197-020-04285-w.

15. Palomar J, Torrecilla JS, Lemus J, Ferro VR, Rodrı´guez F. A COSMO-RS based guide to analyze/quantify the polarity of ionic liquids and their mixtures with organic cosolvents. Phys Chem Chem Phys. 2010;12(8). Available from: https://doi.org/10.1039/b920651p. 16. Palancar MC, Arago´n JM, Migue´ns JA, Torrecilla JS. Application of a model reference adaptive control system to pH control. Effects of lag and delay time. Ind Eng Chem Res. 1996;35(11). Available from: https://doi.org/10.1021/ie960130 1 . 17. Owen RW, Giacosa A, Hull WE, et al. Spacecrafts navigation signal research based on GNSS constellation. Lancet Oncol. 2000;1:107
112. Available from: https://doi.org/10.1007/978-3642-37404-3-4. 18. Cicerale S, Lucas LJ, Keast RSJ. Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil. Curr Opin Biotechnol. 2012;23(2):129
135. Available from: https://doi. org/10.1016/j.copbio.2011.09.006. 19. Serreli G, Deiana M. Extra virgin olive oil polyphenols: modulation of cellular pathways related to oxidant species and inflammation in aging. Cells. 2020;9:478. 20. Bubonja-sonje M, Giacometti J, Abram M. Antioxidant and antilisterial activity of olive oil, cocoa and rosemary extract polyphenols. Food Chem. 2011;127(4):1821
1827. Available from: https://doi. org/10.1016/j.foodchem.2011.02.071. 21. Frankel EN. Nutritional and biological properties of extra virgin olive oil. J Agric Food Chem. 2011;59(3):785
792. Available from: https://doi.org/10.1021/jf103813t. 22. Zhang XF, Zou MQ, Qi XH, Liu F, Zhang C, Yin F. Quantitative detection of adulterated olive oil by Raman spectroscopy and chemometrics. J Raman Spectrosc. 2011;42(9):1784
1788. Available from: https://doi.org/10.1002/jrs.2933. 23. Aroca-Santos R, Lastra-Mejı´as M, Cancilla JC, Torrecilla JS. Intelligent modelling to monitor the evolution of quality of extra virgin olive oil in simulated distribution conditions. Biosyst Eng. 2018;172:49
56. Available from: https://doi.org/10.1016/j. biosystemseng.2018.05.007. 24. Lastra-Mejias M, Izquierdo M, Torreblanca-Zanca A, et al. Cognitive fluorescence sensing to monitor the storage conditions and locate adulterations of extra virgin olive oil. Food Control. 2019;103:48
58. Available from: https://doi.org/10.1016/j.foodcont.2019.03.033. 25. Aroca-Santos R, Cancilla JC, Pariente ES, Torrecilla JS. Neural networks applied to characterize blends containing refined and extra virgin olive oils. Talanta. 2016;161:304
308. Available from: https://doi.org/10.1016/j.talanta.2016.08.033. 26. Muhammad SH, Akash KR. Essentials of Pharmaceutical Analysis. Singapore: Springer Nature; 2020. 27. Perkampus HH. UV-Vis Spectroscopy and Its Applications. Berlin: Springer-Verlag; 2013. 28. Alves FCGBS, Coqueiro A, Marc¸o PH, Valderrama P. Evaluation of olive oils from the Mediterranean region by UV
vis spectroscopy and independent component analysis. Food Chem. 2019;273:124
129. Available from: https://doi.org/10.1016/j. foodchem.2018.01.126. 29. Osborne BG. Near-infrared spectroscopy in food analysis. Encycl Anal Chem. 2000;1
14. Available from: https://doi.org/10.1002/ 9780470027318.a1018. 30. Siesler HW, Ozaki Y, Kawata S, Heise HM. Near-Infrared Spectroscopy: Principles, Instruments, Applications. Weinheim: Wiley-VCH; 2008.

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31. De Guzman GNA, Fang MH, Liang CH, Bao Z, Hu SF, Liu RS. Near-infrared phosphors and their full potential: a review on practical applications and future perspectives. J Lumin. 2020;219:116944. Available from: https://doi.org/10.1016/j.jlumin.2019.116944. 32. Cen H, He Y. Theory and application of near infrared reflectance spectroscopy in determination of food quality. Trends Food Sci Technol. 2007;18(2):72
83. Available from: https://doi.org/ 10.1016/j.tifs.2006.09.003. 33. Wang W, Paliwal J. Near-infrared spectroscopy and imaging in food quality and safety. Sens Instrum Food Qual Saf. 2007;1(4):193
207. Available from: https://doi.org/10.1007/s11694-007-9022-0. 34. Armenta S, Moros J, Garrigues S, de la Guardia M. The use of near-infrared spectrometry in the olive oil industry. Crit Rev Food Sci Nutr. 2010;50(6):567
582. Available from: https://doi.org/ 10.1080/10408390802606790. 35. Mustorgi E, Malegori C, Oliveri P, et al. A chemometric strategy to evaluate the comparability of PLS models obtained from quartz cuvettes and disposable glass vials in the determination of extra virgin olive oil quality parameters by NIR spectroscopy. Chemom Intell Lab Syst. 2020;199:103974. Available from: https://doi.org/ 10.1016/j.chemolab.2020.103974. 36. Casson A, Beghi R, Giovenzana V, Fiorindo I, Tugnolo A, Guidetti R. Environmental advantages of visible and near infrared spectroscopy for the prediction of intact olive ripeness. Biosyst Eng. 2020;189:1
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560. Available from: https://doi.org/10.1080/ 05704928.2011.593216. 39. Huang Y, Wang X, Lai K, Fan Y, Rasco BA. Trace analysis of organic compounds in foods with surface-enhanced Raman spectroscopy: methodology, progress, and challenges. Compr Rev Food Sci Food Saf. 2020;19:622
642. Available from: https://doi.org/ 10.1111/1541-4337.12531. 40. JIS da S de J, Lo¨benberg R, Bou-Chacra NA. Raman spectroscopy for quantitative analysis in the pharmaceutical industry. J Pharm Pharm Sci. 2020;23(1):24
46. Available from: https://doi.org/ 10.18433/jpps30649. 41. Zou MQ, Zhang XF, Xiao-Hua QI, et al. Rapid authentication of olive oil adulteration by Raman spectrometry. J Agric Food Chem. 2009;57(14):6001
6006. Available from: https://doi.org/10.1021/ jf900217s. 42. Karoui R, Blecker C. Fluorescence spectroscopy measurement for quality assessment of food systems—a review. Food Bioprocess Technol. 2011;4(3):364
386. Available from: https://doi.org/ 10.1007/s11947-010-0370-0.

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Chapter 9

Chemical composition of fermented green olives Alfredo Montan˜o and Antonio-Higinio Sa´nchez Food Biotechnology Department, Instituto de la Grasa-CSIC, Pablo de Olavide University Campus, Seville, Spain

Abbreviations IOOC SFAs MUFAs PUFAs TFAs MSG EAAs RDA EFSA

International Olive Oil Council saturated fatty acids monounsaturated fatty acids polyunsaturated fatty acids trans fatty acids monosodium glutamate essential amino acids recommended dietary allowance European Food Safety Authority

9.1 Introduction The worldwide production of table olives is around 2.7 3 106 t/year, the largest part of which is produced in Spain (21%), followed by Egypt (17%) and Turkey (15%).1 At the global level, three different styles (or preparations) of table olives stand out: Spanish-style green olives, natural green olives, and Californian-style black olives. The first two products are of special interest as they include a stage of fermentation, with the potential benefits that this implies from a nutritional and organoleptic point of view.2 Fermented green olives are the most popular fermented food in the European Union. Because of its organoleptic properties and its composition, the fermented green olive is identified in many countries with a high standard of living, where it is used as an appetizer with many kinds of alcoholic and nonalcoholic beverages and as a decorative or nutritional element of various dishes, such as salads, pastas, and pizzas. Of fermented green olives, alkali-treated ones in brine, also known as “Spanish-style green olives” or “pickled green olives,” are the most widely distributed and investigated type of table olive. However, there are other traditional preparations of fermented green olives that are of lesser

economic importance in the international market but highly appreciated by consumers in the Mediterranean region. One of these involves the direct brining of green olives without prior debittering with NaOH solution. This preparation is known as “untreated green olives in brine,” “naturally green olives,” “directly brined olives,” or “Sicilian-style green olives.”3,4 The taste of untreated green olives is completely different from that of alkalitreated fruits, mainly due to the residual bitterness they retain even after a long period of storage in brine. This olive type (green or turning-color) is one of the bases for many other commercial products, such as “seasoned” olives, which are very popular in Spain.4 When this preparation is made with natural black olives, it is known as Greek-style table olives, which have been extensively studied.5
10 This chapter reviews the two abovementioned processing types of fermented green olive—Spanish-style and untreated (green or turning-color) olives—with special emphasis on the physicochemical parameters and chemical composition, including the proximate composition and the most relevant minor components in relation to the health of the final products. It is introduced with a summary of information in the literature regarding the major components in the fresh fruit.5,11
19

9.2 Major components of raw olives The chemical composition of raw olives depends on several factors. Of these, the cultivar and the state of ripeness at the time of harvest are the most important. The main constituents of the olive flesh are water (60%
75%) and lipids (10%
25%). There is an inverse relationship between moisture content and lipid (oil) content, for the same degree of ripeness. Most of the lipids are triglycerides, but there are also diglycerides and free fatty acids.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00052-3 © 2021 Elsevier Inc. All rights reserved.

99

100

PART | 1 General Aspects of Olives and Olive Oil

Olive oil makes a significant contribution to the fact that table olives are considered a product of high biological and nutritive value. Soluble reducing and nonreducing sugars (3%
6% fresh pulp) are the most important components with regard to the fermentation and preservation stages in all the types of table olive processing. The main sugars in the flesh are glucose, followed by fructose, galactose, and mannitol and, at a lower level, sucrose. The distribution of the hexoses varies with the cultivar. During olive ripening, there is continuous decrease of sugars in the flesh. The protein content of the fresh pulp is relatively low, generally between 1% and 3%, and remains almost constant during growth and ripening of the fruits. It seems that the protein content is similar among cultivars and that its amino acid composition does not differ significantly, despite ecological and physiological differences. Crude fiber content varies between 1% and 4% of the fresh pulp, with the major components being cellulose, lignin, and hemicellulose. Phenolic compounds are important for the sensory characteristics of olives and have significant nutritional, physiological, and pharmaceutical effects on human health. Their levels range from 1% to 3% of the fresh pulp. The composition of the phenolic fraction in olive fruit is very complex, depending on cultivar, development stage, and season. The main polyphenol in raw olives is oleuropein, a glucoside ester of 3,4-dihydroxyphenyl ethanol (hydroxytyrosol) and elenolic acid; its concentration decreases with fruit ripening and olive tree irrigation. Other natural phenols that have been identified in the olive fruit are verbascoside, ligstroside, salidroside, rutin, luteolin 7-glucoside, cyanidin 3-glucoside, and cyanidin 3-rutinoside. Ash content varies from 0.6% to 1% of the fresh pulp, with the major elements being (in decreasing order of importance) K, Ca, P, Na, Mg, and S. Organic acids, with concentrations in raw olives between 0.5% and 1% fresh weight, are of great importance, owing to their buffering capacity during the fermentation stage and subsequent storage. Malic and citric acids are the two major acids in raw olives. The polysaccharides and pectic substances, major constituents of intercellular lamellae, have a cementing function and play an important role in the texture of the olive flesh. Levels of these compounds range between 0.3% and 0.6% of the fresh pulp.

9.3 Spanish-style green olives This processing method mainly includes an alkaline treatment with NaOH solution (lye) to chemically remove bitterness, a washing step to eliminate the excess of alkali,

and a stage in brine, where the fruits undergo the typical fermentation by lactic acid bacteria (Fig. 9.1). Information on the evolution of the physicochemical and microbiological characteristics, as well as of the main fermentation substrates and end products, during both spontaneous and controlled Spanish-style green olive fermentation is abundant.5,20
26 However, there are only a few systematic studies about the chemical composition of fermented product. This chapter is mainly based on chemical composition data of fermented green olives from the Spanish market. However, these data are of general interest because most of the Spanish-style green table olives commercialized in an international scale are from Spain.

9.3.1 Product in bulk The physicochemical characteristics and major compounds in brines along with the proximate composition of olive fruits from industrially fermented green olives of different Spanish cultivars (Manzanilla, Hojiblanca, and Gordal) are shown in Table 9.1.27,28 Mean values for pH, combined acidity, and salt content are practically identical to those found in Spanishstyle green olives marketed in Greece.29 However, the mean value for titratable acidity (TA) was considerably lower (0.53% vs 0.93%), which may be attributed to a lower sugar content of the raw material and/or differences in processing conditions. The major compounds in brine were lactic acid, acetic acid, ethanol, methanol, formic acid, and succinic acid. Other compounds, such as propanol, 2-butanol, and acetaldehyde, were also detected in smaller amounts. Residual fermentation substrates, namely, glucose, sucrose, mannitol, and citric acid, were detected in a limited number of samples, where their mean concentrations were 7.2, 23.9, 152.9, and 25.0 mg/100 mL, respectively. Propionic acid, which is characteristic of growth of Propionibacterium species, was also detected in a limited number of samples, in a concentration range of 3.0
81.4 mg/100 mL. Mean values of both the physicochemical characteristics and the major compounds are significantly (P , .05) affected by olive cultivar. Thus Hojiblanca cultivar usually shows higher values of pH and combined acidity than those in Manzanilla or Gordal cultivars. Researchers have paid particular attention to olive polyphenols because of their nutritional and sensory properties. Changes occurring in this class of compounds during Spanish-style processing have been extensively studied.19,26,30
32 The loss in total polyphenols during Spanish-style processing can reach some 90%.33 During the alkaline step in Spanish-style green olive processing, oleuropein is hydrolyzed into hydroxytyrosol and elenolic acid glucoside. During the fermentation step, there is a

Chemical composition of fermented green olives Chapter | 9

Spanish-style green olives

101

Untreated green olives in brine

Harvesting

Harvesting

Sorting

Sorting

Lye treatment

Brining

Washing

Culture addition (optional)

Brining Fermentation Culture addition (optional)

Grading

Fermentation

Packing (whole olives)

Packing with herbs (“seasoned” cracked olives)

Grading Packing (whole olives) Packing (pitted olives)

Cracking

Pitting

Stuffing

Packing (stuffed olives) FIGURE 9.1 Flow diagram of Spanish-style and untreated (natural) green table olives processing methods.

TABLE 9.1 Physicochemical characteristics, major compounds, and proximate composition of Spanish-style green olives in bulk.a Physicochemical characteristics and major compoundsb Range

Proximate composition (g/100 g pulp) Mean

Range

pH

3.65
4.40

4.04

Water

61.0
80.6

Titratable acidity (% lactic acid)

0.35
1.41

0.93

Fat

9.1
28.2

0.060
0.257

0.129

Protein

1.0
1.5

4.0
9.9

6.3

Ash

4.2
5.5

Lactic acid (g/100 mL)

0.49
2.21

1.24

Fiber

1.4
2.1

Acetic acid (g/100 mL)

Sugars

Combined acidity (N) Salt (% NaCl)

0.10
0.50

0.25

Ethanol (mg/100 mL)

21
244

97

Methanol (mg/100 mL)

1
115

61

Formic acid (mg/100 mL)

0
129

55

Succinic acid (mg/100 mL)

0
112

47

Hydroxytyrosol (mg/100 mL)

91
247

186

7
18

14

Tyrosol (mg/100 mL) a

Data adapted from Refs. [26
28]. Values in olive brine.

b

, 0.1

102

PART | 1 General Aspects of Olives and Olive Oil

slow acid hydrolysis of the elenolic acid glucoside, with the production of elenolic acid and glucose. Elenolic acid is comparatively unstable and tends to degrade due to the acid conditions of the medium. The glucose formed is used as substrate by the microorganisms present in the brine and maintains the microbial activity for a longer period of time. At the end, hydroxytyrosol and tyrosol are the major phenols in the fermented product. Their contents in brine (Table 9.1) vary in the range 91
247 mg/ 100 mL (hydroxytyrosol) and 7
18 mg/100 mL (tyrosol), depending on the olive cultivar.26 The beneficial effects of such phenols, such as antioxidant, antiinflammatory, and anticancer, have been extensively investigated during the last 20 years.34 Regarding the proximate composition of the fermented olives in bulk, the following observations can be made in comparison with the raw fruit: (1) sugars are practically absent, due to losses both by solubilization during the lye treatment and washing step and by fermentation; (2) the protein and fiber contents tend to decrease slightly; and (3) the ash content increases as a consequence of the alkaline treatment, fermentation, and storage in brine.

9.3.2 Packed product Prior to packing, green fermented olives must undergo a series of complementary operations (sorting, size-grading, washing, pitting, slicing, and stuffing) to adapt them to the different commercial presentations. Stuffing materials

are numerous: almond, cucumber, onion, garlic, caper, hazelnut, natural hot pepper, ham, lemon, cheese, formulated red pepper strips, formulated tuna strips, formulated anchovy strips, etc. Table 9.2 shows the ranges and mean values of the main physicochemical characteristics and proximate composition of packed Spanish-style green olives marketed in Spain. As expected, the physicochemical parameters showed lower mean values than those reported in Table 9.1 for the fermented product in bulk.35 Mean values for pH, TA, and salt content comply with those established by the International Olive Oil Council (IOOC)38 [the maximum pH value is established at 4.0 when the product is preserved by its own physicochemical characteristics, or 4.3 when it is preserved by pasteurization; the minimum value for TA, expressed as lactic acid, is established at 0.5% (w/v) for olives preserved by their own physicochemical characteristics, or 0.4% in the case of using preservatives or refrigeration; and the minimum NaCl content is established at 5% if olives are preserved by their own physicochemical characteristics, or 4% in the case of using preservatives or refrigeration]. Combined acidity (or residual lye) is an indicator of the buffer capacity of fermenting brines. As a general approach, the higher the combined acidity, the greater the amount of acid needed to reach a specific pH value. The mean value of combined acidity (0.046 N) is higher than that considered adequate for packed Spanish-style green olives (namely, 0.025 N or lower) in order to preserve the

TABLE 9.2 Range and mean values of the main physicochemical characteristics and proximate composition of commercial packed fermented green olives.a Spanish-style green olives

Untreated green olives

Range

Mean

Range

Mean

pH

3.25
4.19

3.69

3.46
4.07

3.92

Titratable acidity (% lactic acid)

0.42
1.77

0.64

0.27
1.26

0.70

Combined acidity (N)

0.021
0.095

0.046

0.030
0.074

0.044

Salt (% NaCl)

3.5
8.2

5.5

3.91
5.72

4.98

Water

65.2
83.4

75.7

58.5
73.0

68.7

Fat

8.7
23.2

15.1

18.0
28.0

21.8

Protein

0.7
3.7

1.2

0.9
1.4

1.2

Ash

2.5
7.0

4.5

2.5
5.0

3.8

Fiber

1.8
5.9

2.6

2.2
4.8

3.3

Physicochemical characteristic

b

Proximate composition (g/100 g edible portion)

a

Data adapted from Refs. [35
37]. Values in olive brine.

b

Chemical composition of fermented green olives Chapter | 9

product by its own physicochemical characteristics.5 This indicates that alternative preservation methods, such as pasteurization, are widely used today. Due to the consumption of sugars during the fermentation process and additional losses in later operations prior to packing, the content of sugars in the packed product is practically negligible.36 In addition, the organic acids present in raw olives are practically removed during processing. Therefore the main acids found in the packed product (0.3% lactic acid and 0.1% acetic acid, on average) are those produced during fermentation or storage or added to the cover liquid.36 The protein content in the packed product varies widely—the range (0.7%
3.7%) being higher than that for the product in bulk. This is not surprising considering many types of stuffing material or ingredient currently used in the packed product. Thus relatively high protein contents have been found in presentations with almond (3.4%
3.7%), hazelnut (1.8%), and those with stuffing materials of animal origin, such as ham (1.8%) or tuna (1.7%). Regarding the individual amino acids (Table 9.3), the most abundant ones are Glx (glutamic acid plus glutamine, 62
651 mg/100 g e.p.), Asx (aspartic acid plus asparagine, 60
299 mg/100 g e.p.), and leucine (46
209 mg/100 g e.p.).37 The range for the Glx/Asx ratio is quite close to the value of 1.0 in presentations based on olives alone. Higher values of this ratio could be indicative of monosodium glutamate (MSG) addition as a flavor enhancer. Values for total essential amino acids (EAAs) range from 41 to 57 g/100 g protein, which are higher than the reference protein value for adults (27.7) proposed by the WHO/FAO/UNU.44 As a result, the amino acid profile of packed Spanish-style green table olives shows a good balance of total EAA. More detailed information about the amino acid composition can be found elsewhere.37,45 The ranges of ash and fiber contents in packed olives (2.5%
7.0% and 1.8%
5.9%, respectively; Table 9.2) are also higher than those for green olives in bulk. It can be said that, on average, Spanish-style green olives have a moderate content (2.6%) of dietary fiber, which is interesting from a nutritional point of view. Fiber intake can decrease the incidence of various disorders, such as cardiovascular complications, gastrointestinal diseases, and hypercholesterolemia.46 The fiber content in green table olives is comparable to that reported for other vegetables or fruits and is exceeded by that in dried fruits (e.g., raisins and peaches) or nuts (e.g., almonds, hazelnuts, and peanuts).47 In the European Union (Reg. EC 1924/2006 and Reg. EU 1047/2012), it is possible to write on the label the claim “source of fiber” if the product contains at least 3 g of fiber/100 g e.p. Many commercial presentations have a content of fiber above this level, so they can be considered as a source of fiber.

103

Total polyphenol contents in the pulp of packed Spanish-style olives vary in the range of 200
1000 mg/ kg, depending on the olive cultivar.39 This concentration is similar to the total polyphenol concentration (330
500 mg/kg oil) in commercial Spanish virgin olive oils.48 The concentration of hydroxytyrosol in olive juice from commercial samples of Spanish-style green olives has been reported to range between 2068 and 7566 μM.39 This corresponds to 24
87 mg hydroxytyrosol/100 g e.p. (assuming a moisture content of 75% and a negligible amount of hydroxytyrosol in the oil phase), whereas the tyrosol content varies in the range of 4
14 mg/100 g e.p. Such contents of hydroxytyrosol and tyrosol are much higher than those of virgin olive oils (hydroxytyrosol, 24
87 mg/100 g e.p. vs 0.6
1.7 mg/100 g oil; tyrosol, 4
14 mg/100 g e.p. vs 0.5
1.3 mg/100 g oil). The fat concentration varies considerably, from 8.7 to 23.2 g/100 g e.p., due to the diverse fat concentrations in the cultivars devoted to this style and in the stuffing materials (Table 9.2). The most abundant fatty acids in table olives are oleic acid (C18:1), palmitic acid (C16:0), linoleic acid (C18:2n-6), and stearic acid (C18:0). In Spanish-style green olives, the concentrations of these fatty acids are within the following ranges (expressed in g/100 g e.p., Table 9.3): oleic acid, 6.91
10.65; palmitic acid, 1.17
2.53; linoleic acid, 0.52
1.05; and stearic acid, 0.22
0.40. More detailed information about the diverse fatty acid composition can be found elsewhere.40 Nutritional labeling requires the inclusion in the nutrition facts of information on total fat content (expressed as grams of triglycerides) and its different nutritional fractions: saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and trans fatty acids (TFAs). The latter fraction has a limited presence in table olives and its values per US serving (15 g e.p.) can always be expressed as 0. As in olive oil, the MUFA (due to the high content of oleic acid) is the main fraction of total fat accounting for about 60%
80% of the fat content of the different commercial presentations of table olives. The mean value of the index PUFA/ SFA, which is used to assess the nutritional quality of the lipid fraction in foods, is 0.41 in Spanish-style green olives. Nutritional guidelines recommend a PUFA/SFA ratio above 0.4
0.5.49 Sterols are important compounds both in olive oil and table olives. It has been known since the 1950s that plant sterols lower blood cholesterol levels.50 In Spanish-style green olives, the major sterols are β-sitosterol, Δ5-avenasterol, and campesterol with concentration ranges of 21.3
25.3, 1.0
1.5, and 0.8
0.9 mg/100 g e.p., respectively (Table 9.3). Other sterols, such as stigmasterol, clerosterol, and cholesterol, are also present, although in smaller amounts.41 The total sterol contents in table olives are similar to those found in many fruits and

104

PART | 1 General Aspects of Olives and Olive Oil

TABLE 9.3 Composition of the most relevant amino acids, polyphenols, fatty acids, sterols, tocopherols, and carotenoids in fermented green table olives.a Spanish-style green olives

Untreated green olives

Range

Mean

Range

Mean

Aspartic acid 1 asparagine (Asx)

60
299

98

70
97

89

Serine

29
120

45

34
54

44

Glutamic acid 1 glutamine (Glx)

62
651

188

75
103

93

Glycine

29
144

45

32
45

41

Histidine

14
69

23

16
23

20

Amino acids (mg/100 g e.p.)

Arginine

29
267

59

40
67

51

Threonine

26
94

42

33
46

41

Alanine

29
128

47

35
54

47

Proline

28
128

45

31
52

44

Tyrosine

22
93

36

25
38

33

Valine

32
136

52

39
56

51

Isoleucine

30
127

48

35
49

45

Leucine

46
209

74

54
76

70

Phenylalanine

29
156

48

33
50

43

Glx/Asx

0.9
5.2

2

1.0
1.1

1.1

24
87

49




97

4
14

8




13

Polyphenols (mg/100 g e.p.) Hydroxytyrosol Tyrosol b

Fatty acids (g/100 g e.p.) Palmitic acid (C16:0)

1.17
2.53

2.28

2.14
5.06

3.16

Stearic acid (C18:0)

0.22
0.40

0.35

0.38
0.65

0.51

Oleic acid (C18:1)

6.91
10.65

9.64

11.38
19.20

13.47

Linoleic acid (C18:2n-6)

0.52
1.05

0.96

1.65
3.64

2.09

SFA

2.08
3.08

2.77

2.40
5.99

3.86

MUFA

7.06
10.92

9.87

10.46
19.42

13.73

PUFA

0.64
1.23

1.14

0.52
3.87

2.34

TFA

0.12
0.21

0.18

0.14
0.44

0.27

β-Sitosterol

21.28
25.30

22.51

20.66
43.53

31.83

Δ -Avenasterol

1.04
1.47

1.28

0.89
5.24

2.78

Campesterol

0.77
0.87

0.86

0.93
1.97

1.30

0.95
5.73

2.96

0.94
5.50

3.15

0.20
1.39

0.30

0.04
0.73

0.20

Sterols (mg/100 g e.p.)

5

Tocopherols (mg/100 g e.p.) α-Tocopherol Carotenoids (mg/100 g e.p.) β-Carotene a

Data adapted from Refs. [37,39
43]. MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; TFA, trans fatty acid.

b

Chemical composition of fermented green olives Chapter | 9

vegetables but lower than those reported in nuts (e.g., almonds and peanuts) and virgin olive oil.51,52 Regarding cholesterol, its average content in Spanish-style olives is 0.5 mg/100 g e.p., although relatively high levels have been found in some presentations, such as Manzanilla olives stuffed with anchovy strip (3.4 mg/100 g e.p.). Bearing in mind that the serving size of olives for the United States and Canada is around 15 g and that this nutrient must be declared only if its content is above 2 mg in this portion, cholesterol can be always declared as 0, even in the presentations with the highest contents. The major forms of vitamin E found in table olives are α-tocopherol and γ-tocopherol, the latter in a markedly lower proportion. In Spanish-style green olives, the α-tocopherol content is present in relatively high amounts (0.95
5.73 mg/100 g e.p. with an average of 2.96 mg/100 g e.p.; Table 9.3).42 Since the recommended dietary allowance (RDA) for both men and women is 15 mg/day of α-tocopherol,53 one serving of 100 g of the edible portion of Spanish-style green olives containing 3 mg α-tocopherol/100 g can provide 20% of the RDA of vitamin E. A statistically significant relationship has been found between the α-tocopherol content and fat content, which explains that Gordal cultivar (due to its lower fat content) has lower levels of vitamin E than Manzanilla or Hojiblanca cultivars. The level of this vitamin is particularly high in olives stuffed with almond or hazelnut (3.8
5.2 mg/100 g e.p.) as a result of relatively high content of vitamin E in nuts. Plant carotenoids are the precursors of vitamin A found in the animal kingdom. The major dietary provitamin A carotenoids include α-carotene, β-carotene, and β-cryptoxanthin.54 No formal dietary recommendation for carotenoids has yet been established. Conclusions of many epidemiological studies revealed that a plasma level of 0.4 μmol/L β-carotene should be aimed at in order to benefit from the preventive health potential. This concentration can be achieved with consumption of 2
4 mg/day β-carotene. The only provitamin A carotenoid found in table olives is β-carotene, while α-carotene is also present in final products containing carrots. The content of β-carotene ranges between 144 and 1434 μg/100 g e.p. with an average of 300 μg/100 g e.p. (Table 9.3).43 Gordal cultivar shows the lowest β-carotene content (259 μg/100 g e.p., on average). The presence of stuffing material, or other ingredients, rich in carotenoids led to commercial presentations with high provitamin A contents, such as Manzanilla olives stuffed with hot pepper (  1.4 mg/100 g e.p.).

9.4 Untreated green olives in brine In this processing method the olives, after sorting and washing to remove surface dirt, are directly brined in

105

5%
10% of sodium chloride without any lye treatment (Fig. 9.1). The fermentation process is similar to that of untreated turning-color olives.5 Spontaneous fermentation is typically the result of growth of a complex microbial population, mainly constituted by lactic acid bacteria and yeasts. However, the indigenous flora of the fruits will vary depending on the quality of the raw material, harvesting conditions, and postharvest treatments, thus leading to variations in the sensory and organoleptic characteristics of the final product. Inoculation of the brine with a commercial starter culture of Lactobacillus pentosus or Lactobacillus plantarum reduces the probability of spoilage and helps one to achieve an improved and more-predictable fermentation process.55
57

9.4.1 Product in bulk An extensive survey on the chemical profile of the product in bulk has not been carried out to date. Both the fermentation process (which, in turn, is affected by NaCl concentration of the brine) and the olive cultivar have been demonstrated to affect the final chemical composition. As there is no previous lye treatment, the epidermis of the fruits remains intact and acts as a barrier, so that diffusion of sugar and polyphenols is slow. Consequently, levels of these compounds in the pulp after fermentation are higher in untreated green olives than in Spanish-style ones. The debittering process is mainly due to the activity of endogenous enzymes, esterase and β-glucosidase, on the oleuropein molecule during the first months of storage with the formation of hydroxytyrosol, oleoside 11-methyl ester, and decarboxymethyl elenolic acid linked to hydroxytyrosol. After that a slow chemical hydrolysis of oleuropein occurs.58 Lactic and acetic acids are the major fermentation end products. Citric, tartaric, and malic acids can also be detected in small amounts at the end of process.55 The final pH is 4.0 or lower.33,55,59

9.4.2 Packed product With regard to the chemical profile of the packed product, ranges and mean values for physicochemical characteristics and proximate composition of untreated (green and turning-color) olives sampled from the Spanish market are shown in Table 9.2. Mean values of physicochemical parameters for the packed product are similar to those for packed Spanish-style green olives.35 Limits for these parameters in untreated table olives have been established by the IOOC:38 the maximum pH is established at 4.3, irrespective of the preservation technique used; the minimum value for TA, expressed as lactic acid, is established at 0.3% (w/v), except for pasteurized olives; and the minimum salt content is established at 6% (w/v), except for pasteurized olives. Therefore the mean values of pH and

106

PART | 1 General Aspects of Olives and Olive Oil

TA shown in Table 9.2 are in compliance with the legislation, but salt content is below the limit. This may be a reflection of a tendency toward the use of pasteurization to stabilize this product, thus satisfying consumer demand for a foodstuff with a lower level of salt. As in the case of Spanish-style green olives, the sugar content is very low and the major organic acids found in the packed product (0.1% lactic acid and 0.2% acetic acid, on average) are those produced during fermentation or storage or added to the cover liquid.36 Presentations of untreated olives have lower moisture (  6% lower, on average) and higher fat (  7% higher, on average) contents than those of Spanish-style olives (Table 9.2). The fat content varies from 18 to 28 g/100 g e.p. The concentrations of the major fatty acids are significantly higher compared to those in Spanish-style olives (Table 9.3). The following ranges (g/100 g e.p.) have been found:40 oleic acid, 11.38
19.20; palmitic acid, 2.14
5.06; linoleic acid, 1.65
3.64; and stearic acid, 0.38
0.65. In addition, the averages of the nutritional fractions (SFA, MUFA, PUFA, and TFA) are higher in untreated green olives because of their greater fat content. The index PUFA/SFA in untreated green olives (0.61 on average) is significantly higher than in Spanish-style olives (0.41, on average). Therefore it appears that the untreated green olive is a healthier product based on the fatty acids composition. The mean protein content (1.2%) is the same as in Spanish-style green olives. The amounts of individual amino acids vary relatively little between presentations (Table 9.3). MSG is usually not added to this type of olives, which is reflected in the Glx/Asx ratio values that are quite close to 1.0. On average, the total EAA content is similar to that of Spanish-style green olive.37 The ash content ranges from 2.5% to 5.0%, with a mean value of 3.8%. Therefore ash values are lower in untreated olives than in treated green olives. This may be attributed not only to the lower concentrations of salt usually used in untreated olives but also to the lower permeability of the epidermis of the fruit or to a lower capacity of the olive tissue to absorb salts. On the other hand, the mean content of fiber is higher in untreated olives than in Spanish-style ones (Table 9.2). This can be attributed to the lye treatment, which affects the cell wall components of olives in the latter product.60 Similarly to what was mentioned earlier for olives in bulk, polyphenol content is higher in packed untreated than in packed treated olives.39 Hydroxytyrosol and tyrosol are the major phenols and concentrations of 9306 and 1395 μM, respectively, in olive juice (corresponding to 97 and 13 mg/100 g e.p.) which have been reported in packed untreated turning-color olives.39 These single phenols, especially hydroxytyrosol, make a significant contribution to the antioxidant activity of olives.61,62 Mean values of total and individual sterols are significantly higher in untreated green olives compared to

Spanish-style ones (Table 9.3). The following average concentrations of the major sterols have been found in Spanish commercial presentations of untreated green olives:41 β-sitosterol, 31.83 mg/100 g e.p.; Δ5-avenasterol, 2.78 mg/100 g e.p.; and campesterol, 1.30 mg/100 g e.p. The α-tocopherol contents of untreated green olives are in the range of 0.94
5.50 mg/100 g e.p. with an average of 3.15 mg/100 g e.p.42 This concentration is practically the same as in Spanish-style green olives (Table 9.3). The highest content of vitamin E is found in presentations made from Hojiblanca or Arbequina cultivars, whereas the Gordal cultivar has the lowest level, a similar trend to that observed in Spanish-style green olives. In general, the β-carotene content in untreated green olives (0.04
0.73 mg/100 g e.p.) is lower than in Spanish-style green olives (Table 9.3).43 This can be attributable to the decrease of the total carotenoid pigment concentrations in olives with maturation.63 Many of the commercial presentations in this type are “seasoned” olives and their β-carotene content is due not only to their olive content but also to the contribution of the ingredients, such as sliced carrots and pieces of pepper strips, which constitute an important part of the product. For example, “seasoned” olives from Verdial cultivar contain a high level of β-carotene ( . 0.7 mg/100 g e.p.) due in part to the olive (its maturation degree is green) and to the contribution of sliced carrots that constitute an important ingredient of this product.

9.5 Summary points G

G

G

G

G

In general, there are significant differences between the chemical composition of Spanish-style green olives and that of untreated (green or turning-color) ones. On average, untreated olives have lower moisture and ash contents but higher fat and fiber contents than treated olives. With regard to health and the prevention of disease, fermented green olives, both Spanish-style and untreated (green or turning-color) olives, have interesting properties, because of their moderate content of total dietary fiber and sterols, their favorable content of total essential amino acids and fatty acid fractions, and their relatively high vitamin E and polyphenol contents. Because of their higher contents of polyphenols, sterols, total fatty acid fractions, and fiber, untreated olives are nutritionally superior to treated olives. The consumption of fermented green olives in addition to virgin olive oil will be a more efficient means of obtaining bioactive components, such as polyphenols, tocopherols, and sterols, reinforcing the healthpromoting properties of the Mediterranean diet.

Chemical composition of fermented green olives Chapter | 9

Mini-dictionary of terms Carotenoids

Combined acidity

Essential amino acids (EAAs)

Monounsaturated fatty acids (MUFAs)

Polyphenols

Polyunsaturated fatty acids (PUFAs)

Carotenoids are fat-soluble plant pigments that have antioxidant properties and are responsible for bright red, yellow, and orange hues in many fruits and vegetables. Dietary carotenoids are thought to provide health benefits in decreasing the risk of disease, particularly certain cancers and eye disease. The term combined acidity (or residual lye) of an olive brine refers to the organic acid salts present in brine. It is an indicator of the buffer capacity of the medium and is obtained by potentiometric titration of the brine with hydrochloric acid to pH 2.6. As a general approach, the higher the combined acidity, the greater the amount of acid needed to reach a specific pH value. EAAs are those that are required in the diet, since the body cannot synthesize them in adequate amounts to maintain protein biosynthesis. Of the 21 amino acids common to all life forms, the 9 EAAs are: phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. MUFAs are chemically classified as fatty acids containing a single double bond. In the cis-configuration the hydrogen atoms are on the same side as the double bond, whereas in trans-configuration the hydrogen atoms and the double bond are present on opposite sides. The cisisomers are the predominant form of MUFA in food sources. The most common cis-configured MUFA in daily nutrition is oleic acid (18:1 n-9). No detrimental effects of MUFA-rich diets have been reported to date. Polyphenols are a heterogeneous group of substances that are found widely in the fruits, vegetables, cereals, and beverages. Polyphenols are secondary metabolites of plants and are generally involved in defense against ultraviolet radiation or aggression by pathogens. Recent evidence has suggested that polyphenolrich foods intake may be associated with decreased risk of chronic diseases. PUFAs are fatty acids that contain more than one double bond in their backbone. Two main compound groups can be distinguished among PUFAs: omega-3 and omega-6 families. The first double bond in the omega-3

Proximate composition

Recommended dietary allowance (RDA)

Saturated fatty acids (SFAs)

Sterols

Titratable acidity (TA)

Tocopherols

Trans fatty acids (TFAs)

107

family occurs at the third carbon from the methyl end of the chain, and in the case of the omega-6 family, the first double bond occurs at the sixth carbon from the methyl end of the chain. PUFAs have been linked to the reduction in cardiovascular diseases. Proximate composition is the term usually used in the field of feed/food and includes the six components of moisture, protein, fat, fiber, carbohydrate, and ash. The RDA is the estimated amount of a nutrient (or calories) per day considered necessary for the maintenance of good health. SFAs are straight-chain organic acids with an even number of carbon atoms. High levels of SFA intake have a negative effect on the blood lipid profile, including elevation of LDL cholesterol, a well-accepted biomarker for the risk of cardiovascular disease. Sterols, also known as steroid alcohols, are ringed lipids that occur in the membranes of plants, animals, and microorganisms. Cholesterol is a mammalian sterol. Plant sterols (or phytosterols) are found in significant quantities in vegetable oils, nuts, seeds, and leafy vegetables. They are normally poorly absorbed by the human intestine and inhibit cholesterol absorption. TA of an olive brine is defined as the amount of strong base (0.2 mol/L sodium hydroxide) needed to titrate the pH of the brine to pH 8.3 and is expressed as percentage of lactic acid. Tocopherols, the major forms of vitamin E, are a group of fat-soluble phenolic compounds with strong antioxidant properties. The major dietary sources of tocopherols are vegetable oils, such as corn, soybean, sesame, and cottonseed. Due to their antioxidant properties, tocopherols have been suggested to reduce the risk of cancer. TFAs by definition are geometric isomers of monounsaturated and PUFAs having at least one carbon
carbon double bond with hydrogens on opposite sides of the double bond (trans-configuration). A number of studies have shown an association of TFA consumption and increased risk of cardiovascular disease. This increased risk is because TFA increase the ratio of LDL-to-HDL cholesterol.

108

PART | 1 General Aspects of Olives and Olive Oil

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Chapter 10

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Paloma Rodrı´guez-Lo´pez1, Jesu´s Lozano-Sa´nchez1,2, Isabel Borras-Linares2, Tatiana Emanuelli3, Javier A. Menendez4 and Antonio Segura-Carretero2,5 1

Department of Food Science and Nutrition, University of Granada, Granada, Spain, 2Functional Food Research and Development Centre (CIDAF),

Health Science Technological Park, Granada, Spain, 3Department of Food Technology and Science, Center of Rural Sciences, Federal University of Santa Maria, Santa Maria, Brazil, 4ProCURE (Program Against Cancer Therapeutic Resistance), Catalan Institute of Oncology, Hospital Dr. Josep Trueta de Girona, Girona, Spain, 5Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain

Abbreviations 3,4-dihydroxyphenyl-ethanol or hydroxytyrosol 3,4-dihydroxyphenyl-ethanol acetate or hydroxytyrosol acetate 3,4-DHPEA-EA 3,4-dihydroxyphenyl-ethanol linked to elenolic acid 3,4-DHPEA-EDA 3,4-dihydroxyphenyl-ethanol linked to the dialdehydic form of elenolic acid EA elenolic acid EVOO extra-virgin olive oil LDL low-density lipoprotein p-HPEA p-hydroxyphenyl-ethanol or tyrosol p-HPEA-EA p-hydroxyphenyl-ethanol linked to elenolic acid p-HPEA-EDA p-hydroxyphenyl-ethanol linked to the dialdehydic form of elenolic acid VOO virgin olive oil 3,4-DHPEA 3,4-DHPEA-AC

10.1 Introduction: phenolic molecules in virgin olive oil Virgin olive oil (VOO) represents one of the typical lipidic source of the Mediterranean diet, which has been associated with a low incidence of several pathologies related with oxidative stress, such as cardiovascular and neurodegenerative diseases or even cancer1
3 and grand part of these benefits are associated with their richness in phenolic compounds.3 Virgin-olive oil phenolic compounds are characterized by a complex mixture of compounds belonging to different classes: secoiridoids, simple

and alcoholic phenols, lignans, hydroxychromans, and flavones. Among these compounds secoiridoids and alcoholic phenols are present in high amount in VOO. Secoiridoids are compounds that are usually bound to glycosides and produced from the secondary metabolism of terpenes. The secoiridoids, found only in plants belonging to the family of Oleaceae, which includes Olea europaea L., are characterized by the presence of elenolic acid (EA) in its glucosidic or aglyconic form in their molecular structure. In particular, they are formed from a phenyl ethyl alcohol (hydroxytyrosol and tyrosol), EA, and, eventually, a glucosidic residue. Oleuropein belongs to a specific group of coumarin-like compounds, the secoiridoids, which are abundant in Oleaceae (Fig. 10.1). The chemical structure of this compound is based on an ester of hydroxytyrosol (3,4-DHPEA) and the EA glucoside (an oleosidic skeleton common to the secoiridoid glucosides of Oleaceae)4
6 (Fig. 10.2). Olive oil secoiridoids in aglyconic forms derive from the glycosides in olives via the hydrolysis of endogenous glucosidases during crushing and malaxation. These newly formed amphiphilic substances are to be found both in the oily layer and the water although they are more concentrated in the latter fraction because of their polar functional groups. During the storage of VOO, hydrolytic mechanisms may be involved in the release of simple phenolics such as hydroxytyrosol and tyrosol from the more complex secoiridoids.7 The most abundant secoiridoids in VOO, identified for the first time by Montedoro et al.4
6,8 and confirmed by other authors,9
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Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00007-9 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 10.1 Total polyphenols content of 25 cultivated varieties.

FIGURE 10.2 Pathway of synthesis of oleuropein in Oleaceae.

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Chapter | 10

are the dialdehyde form of EA linked to hydroxytyrosol or tyrosol (p-HPEA), known respectively as 3,4-DHPEAEDA and p-HPEA-EDA, and an isomer of the oleuropein aglycon (3,4-DHPEA-EA) (Table 10.1). In 1999 another

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hydroxytyrosol derivative, hydroxytyrosol acetate (3,4DHPEA-AC), was found in VOO.12 With regard to phenolic acids, they are naturally occurring secondary aromatic plant metabolites found

TABLE 10.1 Phenolic compounds in virgin olive oil: compound name, general chemical structure and molecular weight. Compound

Substituent (MW)

Structure

Benzoic and derivative acids 5

3-Hydroxybenzoic acid

3-OH (138)

p-Hydroxybenzoic acid

4-OH (138)

3,4-Dihydroxybenzoic acid

3,4-OH (154)

Gentisic acid

2,5-OH (154)

Vanillic acid

3-OCH3, 4-OH (168)

Gallic acid

3,4,5-OH (170)

Syringic acid

3,5-OCH3, 4-OH (198)

6

4 1 3

2

5

6

COOH

Cinnamic acids and derivatives o-Coumaric acid

2-OH (164)

p-Coumaric acid

4-OH (164)

Caffeic acid

3,4-OH (180)

Ferulic acid

3-OCH3, 4-OH (194)

Sinapinic acid

3,5-OCH3, 4-OH (224)

COOH

4 1 3

2

5

6

Phenyl ethyl alcohols Tyrosol [(p-hydroxyphenyl)ethanol] or p-HPEA Hydroxytyrosol [(3,4-dihydroxyphenyl)ethanol] or 3,4-DHPEA

4-OH (138)

OH

4 1

3,4-OH (154) 3

2

5

6

Other phenol acids and derivatives p-Hydroxyphenylacetic acid

4-OH (152)

3,4-Dihydroxyphenylacetic acid

3,4-OH (168)

4-Hydroxy-3-methoxyphenylacetic acid

3-OCH3, 4-OH (182)

3-(3,4-Dihydroxyphenyl)propanoic acid

4

COOH

1 3

2

HO

COOH

HO

Dialdehydic forms of secoiridoids

R*

Decarboxymethyl oleuropein aglycon (3,4-DHPEA-EDA)

R1-OH (304)

Decarboxymethyl ligstroside aglycon (p-HPEA-EDA)

R1-H (320)

O

O

dialdehydic form of Elenolic Acid (EDA) CHO CHO

Compound

Substituent (MW)

Secoiridoid aglycons Oleuropein aglycon or 3,4-DHPEA-EA

R1-OH (378)

Ligstroside aglycon or p-HPEA-EA

R1-H (362) (Continued )

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TABLE 10.1 (Continued) Compound

Substituent (MW)

Aldehydic form of oleuropein aglycon

R1-OH (378)

Aldehydic form ligstroside aglycon

R1-H (362)

Structure

Structure

R1

O

O

OCH 3 C O

HO p-HPEA or 3,4-DHPEA

O

R*

OH Elenolic Acid (EA)

O

O

CH3 aldehydic form of Elenolic Acid (EA)

Compound

Substituent (MW)

Structure OH

Flavonols

OH

(1)-Taxifolin O

HO

OH OH

O

OH

Flavones Apigenin

R1-OH, R2-H (270)

Luteolin

R1-OH, R2-OH (286)

R2 O

HO

H R1

O

Lignans (1)-Pinoresinol

R-H (358)

(1)-1-acetoxypinoresinol

R-OCOCH3 (416)

(1)-1-Hydroxypinoresinol

R-OH (374)

OCH 3 OH

O H

R O

HO H3CO

Hydroxyisochromans 1-Phenyl-6,7-dihydroxyisochroman

R1,R2-H (242)

1-(30 -Methoxy-40 -hydroxy)phenyl-6,7-dihydroxy-isochroman

R1-OH,R2-OCH3 (288)

O R2 R1

HO OH

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Chapter | 10

widely throughout the plant kingdom.13,14 They contain two distinguishing constitutive carbon frameworks, the hydroxycinnamic and hydroxybenzoic structures. Their various contributions to plant life are currently being subject to intense scrutiny, one aspect of which deals specifically with their role in food quality.15,16 Phenolic acids have been associated with color and sensory qualities as well as with the health-related and antioxidant properties of foods.17 One impetus for analytical investigation has been the role of phenolics in the organoleptic properties (flavor and astringency) of foods.18,19 In addition, the content and profile of phenolic acids, their effect on fruit ripening, prevention of enzymatic browning, and their role in food preservation have been evaluated.20 Recent interest in phenolic acids stems from their potential protective role through fruit and vegetables against diseases that may be related to oxidative damage (coronary diseases, strokes, and cancers).21,22 In particular, several phenolic acids such as gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p- and o-coumaric, and ferulic and cinnamic have been identified and quantified in VOO (in quantities lower than 1 mg of analyte kg21 of olive oil). Two research groups have been involved in extensive analyses of VOO for these types of compounds.23
25 In one of their papers, the authors found that trans
cinnamic acid, sinapinic acid, caffeic acid, and 3,4-dihydroxyphenylacetic acid were present in several monovarietal VOOs of the six Spanish olive cultivars analyzed,24 and so these compounds might be potential markers of geographical origin or olive fruit variety. Concerning lignans, (1)-pinoresinol is a common component of the lignan fraction of several plants, such as Forsythia species26 and Sesamum indicum seeds, while (1)-1-acetoxypinoresinol and (1)-1-hydroxy-pinoresinol and their respective glucosides have been detected in the bark of O. europaea L. According to Owen et al.,27 the quantity of lignans in VOO may be as high as 100 mg/kg, but as with the simple phenolics and secoiridoids, considerable variation exists between different oils. As suggested by Brenes et al.,28 the quantity of lignans may be used as a varietal marker, and they reported a method to authenticate VOO produced by Picual olives based on the very low content of the lignan (1)-1-acetoxypinoresinol in these oils. Bianco et al.29 investigated the presence of hydroxyisochromans in VOOs. In fact, during the malaxation step of oil extraction, the quantities of hydroxytyrosol and carbonylic compounds are increased by hydrolytic processes via the activity of glycosidases and esterases, thus favoring the presence of all the compounds necessary for the formation of isochroman derivatives. Two hydroxyisochromans, formed by the reaction between hydroxytyrosol and benzaldehyde or vanillin, have been identified by HPLC
MS/MS and quantified in commercial VOOs.

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Flavonoids are widespread secondary plant metabolites. During the past decade an increasing number of publications highlighting the beneficial effects of flavonoids upon health has appeared, including some related to cancer and coronary diseases.30
32 Flavonoids are largely planar molecules, and their structural variation comes in part from the pattern of modification by hydroxylation, methoxylation, prenylation, or glycosylation. Flavonoid aglycones are subdivided into flavones, flavonols, flavanones, and flavanols, depending upon the presence of a carbonyl carbon at C-4, an OH group at C-3, a saturated single bond between C-2 and C-3, or a combination of a noncarbonyl at C-4 with an OH group at C-3. Several authors have reported that flavonoids such as luteolin and apigenin are also present in VOO.33
36 Luteolin may originate from rutin or luteolin-7-glucoside, and apigenin from apigenin glucosides. Some interesting studies have also been published describing how several flavonoids have been found in olive leaves and fruit.37
39

10.2 Why are the phenolic compounds in virgin olive oil so important? A few years ago, Boskou published an interesting review1 in which the sources of natural phenolic antioxidants were discussed, and the following idea was highlighted, “Widely distributed in the plant kingdom and abundant in our diet, plant phenolics are today among the most talked about classes of phytochemicals.” To answer the question, “Why are phenolic compounds so interesting?” the author of the review summarized several issues which have been studied in depth over the last decade: 1. The levels and chemical structure of antioxidant phenolics in different plant foods, aromatic plants, and various plant materials. 2. The probable role of plant phenolics in the prevention of various diseases associated with oxidative stress, such as cardiovascular and neurodegenerative diseases and cancer. 3. The ability of plant phenolics to modulate the activity of enzymes, a biological action not yet understood. 4. The ability of certain classes of plant phenolics such as flavonoids (also called polyphenolics) to bind to proteins. Flavonol
protein binding, such as binding to cell receptors and transporters, involves mechanisms that are not related to their direct activity as antioxidants. 5. The stabilization of edible oils, protection from formation of off-flavors, and the stabilization of flavors. 6. The preparation of food supplements. A recent review40 adds some outstanding aspects related to polyphenols, which emerged in recent years:

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1. The ability of plant phenolics to modulate the expression of genes that control numerous cellular functions in animals and humans, including inflammatory response, endogenous antioxidant defenses, lifespan, carbohydrate and lipid metabolism, cell cycle and apoptosis. 2. The ability of dietary phenolics to induce epigenetic changes. 3. The interplay between plant phenolics and gut microbiota that comprises the “prebiotic-like” effect of polyphenols that modulate gut microbiota by polyphenols and the biotransformation of plant phenolics by gut microbiota yielding low molecular weight compounds bearing increased biological activity.

10.3 Implications for human health and disease prevention VOO, which is obtained from ripe olives and is one of the symbols of the Mediterranean culture, is widely known for its health effects and beneficial properties. There is a strong association between the consumption of VOO as one of the main foods of the Mediterranean diet and the prevention of cardiovascular diseases.2 This relationship is due to the composition of the VOO, which has phenolic compounds, all of them highly related to health maintenance, in addition to its content in monounsaturated fatty acids such as oleic.3,41 In reference to the phenolic compounds, VOO phenolic compounds belong to three classes: simple phenols [tyrosol, hydroxytyrosol (3,4-DHPEA), and derivatives], secoiridoids (oleuropein aglycones, deacetoxy-oleuropein aglycone, oleocanthal, and secoiridoids derivatives), and lignan derivatives, which are extensively studied because of their high antioxidant capacity for metal-chelating and free radical scavenging activities.3,42,43 Polyphenols were determined to reduce morbidity, and they are especially related in the prevention of various diseases associated with oxidative stress, such as cardiovascular,2 neurodegenerative diseases, and cancer.1,3,43,44 Such effects are likely related to the decrease of low-density lipoprotein (LDL) levels and its oxidation, decreased oxidative stress, down-regulation of proinflammatory cytokines, as well as improved insulin sensitivity, endothelial function, and coagulation triggered by VOO polyphenols.45 Although glycoside polyphenols are poorly absorbed in the small intestine, oleuropein and mostly its aglycone forms have been found in the plasma after dietary intake and oleuropein was also shown to be able to cross plasma membrane of breast cancer cells.46 Hydroxytyrosol and tyrosol have been demonstrated to be the best absorbed phenolics in the intestinal tract after VOO intake and along with oleuropein have been shown to be able to reach tissues, including brain.46

Polyphenols, that are not absorbed in the small intestine, will reach the colon, where they can be fermented by gut microbiota. Recent data from a mice model revealed that VOO promotes desirable changes in the gut microbiota profile of mice as compared to refined olive oil.47 In vitro studies have shown VOO have capacity to prevent the oxidation of human LDLs, an ability that correlated with their content of minor polar phenolic compounds. Notable differences were observed between different varieties of oil in terms of quality (or quantity of phenolic compounds with antioxidant activity), higher LDL protective effects being observed for VOO rich in lignans than for VOO rich in simple phenols and secoiridoids.42 Similar results show that polyphenols from VOO are effective to prevent cytotoxic effects of reactive oxygen species. The protective ability of VOO is strictly parallels to their phenolic content.41 In addition to these properties, the antihypertensive effect has been attributed to the consumption of phenolic fraction extracted from olive oil. The recovered fractions showed different effects according to the amount and type of polyphenols,48 even polyphenols such an oleuropein aglycon extract from de olive leaf have notable effects on hypertensive markers, that is, a systolic and diastolic blood pressure and lipid profile. The antihypertensive properties of VOO are likely associated to its inhibitory activity against angiotensin Iconverting enzyme and calcium channel blocking activity.49 The hypoglycemic capacity of VOO has been also suggested by in vitro studies that revealed its ability to inhibit carbohydrate digesting enzymes.50 Moreover, hydroxytyrosol and oleuropein have been shown to mimic the effects of caloric restriction, namely, the activation of sirtuins (Sirt), which are NAD-dependent type-3 deacetylases that regulate lifespan and cellular metabolism by controlling the activity of several transcription factors. Sirt activation protects cells against oxidative stress and DNA damage and controls apoptosis, autophagy, cell proliferation, inflammation, protein synthesis, carbohydrate, and lipid metabolism.46 Thus effects of metabolic syndrome reduction could be attributed to olive oil phenolic compounds.50 The properties described before could attribute the beneficial health effects of VOO to the content of polyphenols, rather than to other bioactive compounds found in VOO. A randomized crossover clinical trial conducted in 2006 provided definitive evidence on the key role of VOO polyphenols to control heart disease risk factors. A 3-week daily dietary supplementation with 25 mL of olive oil increased high-density lipoprotein cholesterol levels and decreased oxidative stress markers, and these effects were linearly associated to the content of phenolics in the three olive oils evaluated (low-phenolics, 2.7 mg/kg; medium-phenolics, 164 mg/kg; and high-phenolics, 366 mg/kg).51 Olive oil polyphenols are also known for their anticancer activity on different cancer models. Hydroxytyrosol, tyrosol,

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Chapter | 10

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FIGURE 10.3 Phenolic compounds and their protective effect of different cancer types.

and their derivatives such as oleuropein, oleuropein aglycon, oleocanthal, and oleacein have been studied about their cancer prevention properties alone or associated to other anticancer drugs. Recent reviews have been published44,52; about olive oil polyphenols and their high potential as chemopreventive and anticancer agents in different cancer types (Fig. 10.3). Antiproliferative and proapoptotic effects have been implicated in the anticancer activity of olive oil polyphenols, but the detailed anticancer mechanisms remain to be elucidated. Studies reviewed comprise cell culture and preclinical approaches, which revealed that olive oil polyphenols are able to inhibit both initiation of carcinogenesis and metastasis. Clinical studies are lacking to confirm the anticancer properties of olive oil polyphenols and their contribution to the association of olive oil intake with reduced risk of cancer, as indicated by epidemiological studies. Regarding its inflammatory response, hydroxytyrosol (3,4-DHPEA) is able to inhibit the formation of proinflammatory cytokines, since this molecule can interfere in the metabolism of eicosanoids.53 Furthermore, other phenolic compound, oleocanthal, has a similar pharmacological effect and potency to ibuprofen in the blocking the biosynthetic pathway of prostaglandins by inhibiting cyclooxygenases 1 and 2.54 VOO can be considered as an example of a functional food containing a variety of components that may contribute to its overall health benefits. VOO is an integral ingredient of the Mediterranean diet, known worldwide, and its nutritional and health values and pleasant flavor have contributed to an increase in its consumption.55 The study of

other minor components such as phytosterols, carotenoids, tocopherols, and hydrophilic phenolics as part of the value of olive oil56 (Fig. 10.4) has gained prominence due to the multitude of beneficial effects added to those already known from the VOO.

10.4 Phenolic contribution to the oxidative stability of virgin olive oil Oxidation is an inevitable process that starts after the oil has been extracted and leads to a deterioration that becomes more pronounced during storage. Initially lipids are radically oxidized to hydroperoxides, which are odorless and tasteless57 and do not bring about any sensory changes. Nevertheless, decomposition occurs through homolytic cleavage of the hydroperoxide group, generating various volatile compounds known as secondary oxidation products, which are responsible for typical unpleasant sensory characteristics. Oxygen, light, temperature, metals, pigments, unsaturated fatty acid composition, and the quantity and kind of natural antioxidants are all factors that can influence the free-radical mechanism of the autoxidation process in different ways.58,59 Natural antioxidants behave in very different, complex ways at air
oil and oil
water interfaces, which significantly affect their relative activities in different lipid systems. The presence of hydrophilic phenolic compounds in VOO and their high antioxidant activity can be explained by the so-called polar paradox,60 which maintains that “polar antioxidants are more effective in

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PART | 1 General Aspects of Olives and Olive Oil

FIGURE 10.4 Relation between the olive oil composition and the health properties.

non-polar lipids whereas non-polar antioxidants are more active in polar lipid emulsions.” According to Frankel,61 in a bulk oil system hydrophilic antioxidants such as polar phenolics are located at the air
oil interface (a small quantity of air is always trapped in the oil) and thus protect more effectively against oxidation than lipophilic antioxidants such as tocopherols, which remain in solution in the oil. By studying the phenolic profiles of the oils after heating them to 180 C and then using HPLC
UV, HPLC
MS, and capillary electrophoresis
UV techniques, CarrascoPancorbo et al.62 found several hitherto unknown compounds that were probably linked to phenolic oxidation. In particular, seven peaks increased significantly when the thermal treatment was kept up for 1
3 h, and their presence has also been confirmed in refined olive oils. The concentrations of hydroxytyrosol, EA, 3,4-DHPEA-EDA, and 3,4-DHPEAEA decreased more rapidly with the thermal treatment than did other phenolic compounds present, confirming their high antioxidant power. Moreover, 3,4-DHPEA-AC and pHPEA-EA were more resistant to heat treatment, whereas the quantities of (1)-pinoresinol and (1)-1-acetoxypinoresinol remained almost unchanged (Fig. 10.5). A recent study by Lukic et al.63 evaluated the amount of phenols present in VOO according to the ripening degree, malaxation duration, and temperature on O.

europaea L. The results show that the highest variability was observed for ripening degree. Secoiridoids concentrations, especially oleuropein aglycon, decrease while the ripening degree increases, due to the increased hydrolytic activity of enzymes. Malaxation temperature also influences the phenol content. Although there is a controversy regarding the adequate extraction temperature and the maximum phenol content. Higher extraction temperature usually induces an increase in the phenol’s concentration as observed by Lukic et al.63 Regarding the oxidation, higher degree of transfer of phenols into oil phase brings better protection against oxidation and, in terms of quality, rancidity does not develop.63

10.5 Sensory properties affected by phenolics in virgin olive oil VOO is a natural fruit juice obtained directly from olives without any further refining process. Its flavor is characteristic and is markedly different from those of other edible fats and oils. The combined effects of taste, odor (directly via the nose or indirectly through the retronasal path via the mouth), and chemical responses (pungency, astringency, metallic, cooling, or burning) give rise to the sensation generally perceived as “flavor.”64 VOO, when extracted from fresh and

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Chapter | 10

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FIGURE 10.5 Correlations between OSI values (in h), phenol quantities and antioxidant activity (DPPH test) by spectrophotometric assays. (A) OSI versus total phenols (mg gallic acid kg21 VOO); (B) OSI versus DPPH (mmol trolox kg21 VOO); (C) OSI versus o-diphenols (mg gallic acid kg21 VOO). Analyses were carried out over three years; the number of samples is given in each figure (N). Three replicates were prepared and analyzed for each sample. OSI, oil stability index; VOO, Virgin olive oil.

healthy olives (O. europaea L.) and properly processed and stored, is characterized by a unique, highly appreciated combination of aroma and taste.65,66 The sensory aspect, due to the use of VOO as a seasoning in both cooked and raw foods, has great repercussions on its acceptability. Thus since sensory quality plays an important role in directing the preference of consumers, many attempts have been made to clarify the relationships between the sensory attributes in a VOO as perceived by assessors and its volatile and phenolic profiles, which are responsible for aroma and taste, respectively. The bitterness and pungency perceived by taste are positive attributes for a VOO. These two sensory characteristics are closely connected by the qualitative
quantitative phenolic profile of the product (Fig. 10.6). Generally, sensory attributes are in accordance with the chemical composition, and phenols have an essential role in the VOO organoleptic characteristics.

In addition to the bitterness and pungency, the antioxidant activity is responsible for VOO oxidative stability and shelf life. Changes in the sensory characteristics are observed in accordance with the ripening degree and, therefore, the phenolic content demonstrating once again phenols concentration and taste characteristics is strictly paralleling. 3,63

10.6 Comparisons of olive oils with other edible oils There are many edible oils from vegetables sources, and it is possible to make comparisons between them based on many features. Despite the benefits associated with the consumption of oleic acid, Ruı´z-Gutie´rrez et al.67 compared in 1996 the

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and soy, anthocyanins of black raspberries, resveratrol in grapes, or caffeic acids in coffee and other plants. All of these plants promote health benefits related with the reduction of morbidity and slow down the progression of cardiovascular, neurodegenerative, and cancer diseases. Olive oil is rich in hydroxytyrosol, oleuropein, and their derivatives, more than in others edible oils. They are powerful antioxidants with beneficial properties reported.43 Their biological effects, however, go far beyond the chelation of metals and scavenging of reactive species. In fact, olive polyphenols are able to modulate numerous key cellular pathways by regulating gene transcription factors and gene expression.68 These bioactive compounds are responsible for the beneficial effects and that suggest that the amount of polyphenols in each food is related with the qualitative characteristics compared to another food, such another edible oils.

Mini-dictionary of terms Polyphenols

Mediterranean diet FIGURE 10.6 Sensory profile and phenolic content of two different VOOs (HPh and LPh). (A) Sensory profiles of samples by QDA; the intensity of each descriptor is evaluated on a scale of 0
5; different perception routes: (1) orthonasal and (2) retronasal; (B) single and total phenolic content of samples; A, hydroxytyrosol; B, tyrosol; C, vanillic acid; D, unknown phenolic compound with a retention time of 30.69 min; E, unknown phenolic compound with a retention time of 36.27 min; F, 3,4DHPEA-EDA; G, (1)-pinoresinol; H, (1)-1-acetoxypinoresinol 1 pHPEA-EDA; I, 3,4-DHPEA-EA; L, p-HPEA-EA. HPh, High phenol oil; LPh, low phenol oil; QDA, quantitative descriptive analysis; VOO, virgin olive oil.

effect of two edible oils rich in oleic acid: olive oil and high-oleic sunflower oil and these authors reported that only the olive oil had effect in the reduction of blood pressure in hypertensive women, suggesting the importance of the minor olive oil components, such a polyphenols.67 As far as the properties related with the phenolic content are concerned, there are more benefits in oils rich in these compounds, even compared with other different types of olive oil with low-phenolic content. Depending on cultivars and environmental factors, the degree of ripeness of the olives and the extraction procedures, the polyphenols concentration may differ.3,41 In addition to VOO, there are many other plants rich in polyphenols: epigallocatechin and genistein, present in tea

Polyphenols are a group of chemical substances found in plants, characterized by the presence of more than one phenol unit or building block per molecule. Polyphenols are generally divided into hydrolyzable tannins (gallic acid esters of glucose and other sugars) and phenylpropanoids, such as lignins, flavonoids, and condensed tannins. Mediterranean Diet is the heritage of millennia of exchanges of people, cultures and foods from all the countries of the Mediterranean basin. It has been the basis of the eating habits of the countries of the region, originally based on Mediterranean agricultural and rural models and this was recognized by UNESCO in the Representative List of the Intangible Cultural Heritage of Humanity. The Mediterranean Diet follows a dietary pattern rich in plant foods (cereals, fruits, vegetables, legumes, nuts, seeds and olives), using olive oil as the main source of added fats, along with high to moderate intakes of fish and seafood, moderate consumption of eggs, poultry, and dairy products (cheese and yogurt), low consumption of red meat, and moderate intake of alcohol (mainly wine during meals). The pioneering study of the seven countries, together with a multitude of epidemiological studies have established the health benefits associated with adherence to the Mediterranean diet pattern (MDP), mainly in relation to the reduction of the risk of developing the metabolic syndrome, type diabetes 2, cardiovascular disease and some neurodegenerative diseases and cancers.69

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512.

Polyphenols in olive oil: the importance of phenolic compounds in the chemical composition of olive oil Chapter | 10

2. Estruch R, Ros E, Salas-Salvado´ J, Covas M, Corella D, Aro´s F. Primary prevention of cardiovascular disease with a Mediterranean diet supplemented with extra-virgin olive oil or nuts. N Engl J Med. 2018;378(25):e34. 3. Romani A, Ieri F, Urciuoli S, Noce A, Marrone G, Nediani C. Health effects of phenolic compounds found in extra-virgin olive oil, by-products, and leaf of Olea europaea L. Nutrients. 2019;11 (8). Available from: https://doi.org/10.3390/nu11081776. 4. Montedoro G, Servili M, Baldioli M, Miniati E. Simple and hydrolyzable phenolic compounds in virgin olive oil. 1. Their extraction, separation, and quantitative and semiquantitative evaluation by HPLC. J Agric Food Chem. 1992;40(9):1571
1576. 5. Montedoro G, Servili M, Baldioli M, Miniati E. Simple and hydrolyzable phenolic compounds in virgin olive oil. 2. Initial characterization of the hydrolyzable fraction. J Agric Food Chem. 1992;40 (9):1577
1580. 6. Montedoro G, Servili M, Baldioli M, Selvaggini R, Miniati E, Macchioni A. Simple and hydrolyzable compounds in virgin olive oil. 3. Spectroscopic characterizations of the secoiridoid derivatives. J Agric Food Chem. 1993;41(11):2228
2234. 7. Tsimidou M. Polyphenols and quality of virgin olive oil in retrospect [Olea europaea L.]. Ital J Food Sci. 1998;10. 8. Montedoro G, Cantarelli C. Indagine sulle sostanze fenoliche presenti nell’olio di oliva. Riv Ital Sost Grasse. 1969;46:115
124. 9. Cortesi N, Azzolini M, Rovellini P, Fedeli E. I componenti minori polari degli oli vergini di oliva: Ipotesi di struttura mediante LCMS. Riv Ital Sost Grasse. 1995;72:241
251. 10. Cortesi N, Azzolini M, Rovellini P, Fedeli E. Dosaggio dei componenti minori polari (CMP) in oli vergini di oliva. Riv Ital Sost Grasse. 1995;72:333
337. 11. Angerosa F, d’Alessandro N, Corana F, Mellerio G. Characterization of phenolic and secoiridoid aglycons present in virgin olive oil by gas chromatography-chemical ionization mass spectrometry. J Chromatogr A. 1996;736(1):195
203. Available from: https://doi.org/10.1016/0021-9673(95)01375-X. 12. Brenes M, Garcı´a A, Garcı´a P, Rios JJ, Garrido A. Phenolic compounds in Spanish olive oils. J Agric Food Chem. 1999;47 (9):3535
3540. 13. Prim N, Pastor FI, Diaz P. Biochemical studies on cloned Bacillus sp. BP-7 phenolic acid decarboxylase PadA. Appl Microbiol Biotechnol. 2003;63(1):51
56. 14. Exarchou V, Godejohann M, van Beek TA, Gerothanassis IP, Vervoort J. LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano. Anal Chem. 2003;75 (22):6288
6294. 15. Barthelmebs L, Divies C, Cavin JF. Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism. Appl Env Microbiol. 2000;66(8):3368
3375. 16. Ha¨kkinen S, Heinonen M, Ka¨renlampi S, Mykka¨nen H, Ruuskanen J, To¨rro¨nen R. Screening of selected flavonoids and phenolic acids in 19 berries. Food Res Int. 1999;32(5):345
353. Available from: https://doi.org/10.1016/S0963-9969(99)00095-2. 17. Maga JA, Katz I. Simple phenol and phenolic compounds in food flavor. Crit Rev Food Sci Nutr. 1978;10(4):323
372. 18. Harborne JB. The Flavonoids: Advances in Research Since 1980. Springer; 2013.

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19. Peleg H, Naim M, Rouseff RL, Zehavi U. Distribution of bound and free phenolic acids in oranges (Citrus sinensis) and grapefruits (Citrus paradisi). J Sci Food Agric. 1991;57(3):417
426. 20. Shahidi F, Naczk M. Food Phenolics Sources, Chemistry, Effects and Applications. Technomic Publishing Co.; 1995:281
313. 21. Robbins RJ. Phenolic acids in foods: an overview of analytical methodology. J Agric Food Chem. 2003;51(10):2866
2887. 22. Gomes CA, Gira˜o da Cruz T, Andrade JL, Milhazes N, Borges F, Marques MPM. Anticancer activity of phenolic acids of natural or synthetic origin: a structure-activity study. J Med Chem. 2003;46 (25):5395
5401. 23. Buiarelli F, Di Berardino S, Coccioli F, Jasionowska R, Russo MV. Determination of phenolic acids in olive oil by capillary electrophoresis. Ann Chim. 2004;94(9
10):699
705. 24. Carrasco Pancorbo A, Cruces-Blanco C, Segura Carretero A, Fernandez Gutierrez A. Sensitive determination of phenolic acids in extra-virgin olive oil by capillary zone electrophoresis. J Agric Food Chem. 2004;52(22):6687
6693. 25. Carrasco Pancorbo A, Segura Carretero A, Fernandez Gutierrez A. Co-electroosmotic capillary electrophoresis determination of phenolic acids in commercial olive oil. J Sep Sci. 2005;28(9
10):925
934. 26. Davin LB, Bedgar DL, Katayama T, Lewis NG. On the stereoselective synthesis of (1)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol. Phytochemistry. 1992;31 (11):3869
3874. Available from: https://doi.org/10.1016/S00319422(00)97544-7. 27. Owen RW, Mier W, Giacosa A, Hull WE, Spiegelhalder B, Bartsch H. Identification of lignans as major components in the phenolic fraction of olive oil. Clin Chem. 2000;46(7):976
988. 28. Brenes M, Garcıa A, Rios JJ, Garcıa P, Garrido A. Use of 1acetoxypinoresinol to authenticate Picual olive oils. Int J Food Sci Technol. 2002;37(6):615
625. 29. Bianco A, Coccioli F, Guiso M, Marra C. The occurrence in olive oil of a new class of phenolic compounds: hydroxy-isochromans. Food Chem. 2002;77(4):405
411. Available from: https://doi.org/ 10.1016/S0308-8146(01)00366-1. 30. Le Marchand L. Cancer preventive effects of flavonoids—a review. Biomed Pharmacother. 2002;56(6):296
301. 31. Kandaswami C, Lee LT, Lee PP, et al. The antitumor activities of flavonoids. In Vivo. 2005;19(5):895
909. 32. Nestel P. Isoflavones: their effects on cardiovascular risk and functions. Curr Opin Lipidol. 2003;14(1):3
8. 33. Rovellini P, Cortesi N, Fedeli E. Analysis of flavonoids from Olea europaea by HPLC-UV and HPLC-electrospray-MS. Riv Ital Sost Grasse. 1997;74(7):273
279. 34. Carrasco-Pancorbo A, Gomez-Caravaca AM, Cerretani L, Bendini A, Segura-Carretero A, Fernandez-Gutierrez A. Rapid quantification of the phenolic fraction of Spanish virgin olive oils by capillary electrophoresis with UV detection. J Agric Food Chem. 2006;54(21):7984
7991. 35. Morello JR, Vuorela S, Romero MP, Motilva MJ, Heinonen M. Antioxidant activity of olive pulp and olive oil phenolic compounds of the arbequina cultivar. J Agric Food Chem. 2005;53 (6):2002
2008. 36. Murkovic M, Lechner S, Pietzka A, Bratacos M, Katzogiannos E. Analysis of minor components in olive oil. J Biochem Biophys Methods. 2004;61(1):155
160. Available from: https://doi.org/ 10.1016/j.jbbm.2004.04.002.

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37. Bouaziz M, Grayer RJ, Simmonds MS, Damak M, Sayadi S. Identification and antioxidant potential of flavonoids and low molecular weight phenols in olive cultivar chemlali growing in Tunisia. J Agric Food Chem. 2005;53(2):236
241. 38. Ryan D, Prenzler PD, Lavee S, Antolovich M, Robards K. Quantitative changes in phenolic content during physiological development of the olive (Olea europaea) cultivar hardy’s mammoth. J Agric Food Chem. 2003;51(9):2532
2538. 39. Romani A, Pinelli P, Mulinacci N, Vincieri FF, Gravano E, Tattini M. HPLC analysis of flavonoids and secoiridoids in leaves of Ligustrum vulgare L. (Oleaceae). J Agric Food Chem. 2000;48(9):4091
4096. 40. Espı´n JC, Gonza´lez-Sarrı´as A, Toma´s-Barbera´n FA. The gut microbiota: a key factor in the therapeutic effects of (poly)phenols. Biochem Pharmacol. 2017;139:82
93. 41. Manna C, D’Angelo S, Migliardi V, Loffredi E, Mazzoni O, Morrica P. Protective effect of the phenolic fraction from virgin olive oils against oxidative stress in human cells. J Agric Food Chem. 2002;50(22):6521
6526. 42. Franconi F, Coinu R, Carta S, Urgeghe PP, Ieri F, Mulinacci N. Antioxidant effect of two virgin olive oils depends on the concentration and composition of minor polar compounds. J Agric Food Chem. 2006;54(8):3121
3125. 43. Gorzynik-Debicka M, Przychodzen P, Cappello F, KubanJankowska A, Marino Gammazza A, Knap N. Potential health benefits of olive oil and plant polyphenols. Int J Mol Sci. 2018;19(3). Available from: https://doi.org/10.3390/ijms19030686. 44. Toric J, Markovic AK, Brala CJ, Barbaric M. Anticancer effects of olive oil polyphenols and their combinations with anticancer drugs. Acta Pharm. 2019;69(4):461
482. 45. Schwingshackl L, Morze J, Hoffmann G. Mediterranean diet and health status: active ingredients and pharmacological mechanisms. Br J Pharmacol. 2020;177(6):1241
1257. 46. Rigacci S, Stefani M. Nutraceutical properties of olive oil polyphenols. An itinerary from cultured cells through animal models to humans. Int J Mol Sci. 2016;17(6). Available from: https://doi.org/ 10.3390/ijms17060843. 47. Martı´nez N, Prieto I, Hidalgo M, et al. Refined versus extra virgin olive oil high-fat diet impact on intestinal microbiota of mice and its relation to different physiological variables. Microorganisms. 2019;7(2):61. 48. Valls RM, Farras M, Suarez M, Fernandez-Castillejo S, Fito M, Konstantinidou V. Effects of functional olive oil enriched with its own phenolic compounds on endothelial function in hypertensive patients. A randomised controlled trial. Food Chem. 2015;167:30
35. 49. Susalit E, Agus N, Effendi I, Tjandrawinata RR, Nofiarny D, Perrinjaquet-Moccetti T. Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: comparison with captopril. Phytomedicine. 2011;18(4):251
258. 50. Loizzo MR, Di Lecce G, Boselli E, Menichini F, Frega NG. Inhibitory activity of phenolic compounds from extra virgin olive oils on the enzymes involved in diabetes, obesity and hypertension. J Food Biochem. 2011;35(2):381
399. 51. Covas M, Nyysso¨nen K, Poulsen HE, et al. The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med. 2006;145(5):333
341. 52. Shamshoum H, Vlavcheski F, Tsiani E. Anticancer effects of oleuropein. Biofactors. 2017;43(4):517
528.

53. Servili M, Esposto S, Fabiani R, Urbani S, Taticchi A, Mariucci F. Phenolic compounds in olive oil: antioxidant, health and organoleptic activities according to their chemical structure. Inflammopharmacology. 2009;17(2):76
84. 54. Beauchamp GK, Keast RS, Morel D, Lin J, Pika J, Han Q. Phytochemistry: ibuprofen-like activity in extra-virgin olive oil. Nature. 2005;437(7055):45
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424. 57. Frankel EN. Volatile lipid oxidation products. Prog Lipid Res. 1983;22(1):1
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26. 59. Min DB, Lee H-O. Chemistry of lipid oxidation. In: Flavor Chemistry. US: Springer; 1999:175
187. Available from: https:// doi.org/10.1007/978-1-4615-4693-1_16. 60. Porter WL, Black ED, Drolet AM. Use of polyamide oxidative fluorescence test on lipid emulsions: contrast in relative effectiveness of antioxidants in bulk versus dispersed systems. J Agric Food Chem. 1989;37(3):615
624. 61. Frankel EN. Antioxidants in lipid foods and their impact on food quality. Food Chem. 1996;57(1):51
55. 62. Carrasco-Pancorbo A, Cerretani L, Bendini A, Segura-Carretero A, Lercker G, Fernandez-Gutierrez A. Evaluation of the influence of thermal oxidation on the phenolic composition and on the antioxidant activity of extra-virgin olive oils. J Agric Food Chem. 2007;55(12):4771
4780. 63. Lukic I, Zanetic M, Jukic Spika M, Lukic M, Koprivnjak O, Brkic Bubola K. Complex interactive effects of ripening degree, malaxation duration and temperature on Oblica cv. virgin olive oil phenols, volatiles and sensory quality. Food Chem. 2017;232:610
620. 64. Kilcast D. Sensory science. Chem Brit. 2003;39. 62-62. 65. Kiritsakis A. Flavor components of olive oil
a review. J Am Oil Chem Soc. 1998;75(6):673
681. 66. Angerosa F, Mostallino R, Basti C, Vito R. Virgin olive oil odour notes: their relationships with volatile compounds from the lipoxygenase pathway and secoiridoid compounds. Food Chem. 2000;68 (3):283
287. Available from: https://doi.org/10.1016/S0308-8146 (99)00189-2. 67. Ruı´z-Gutie´rrez V, Muriana FJ, Guerrero A, Cert AM, Villar J. Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources. J Hypertens. 1996;14(12):1483
1490. 68. Gaforio JJ, Visioli F, Alarcon-de-la-Lastra C, et al. Virgin olive oil and health: summary of the III international conference on virgin olive oil and health consensus report, JAEN (Spain) 2018. Nutrients. 2019;11(9). Available from: https://doi.org/10.3390/ nu11092039. 69 Bach-Faig Anna, Berry Elliot M, Lairon Denis, et al. Mediterranean Diet Foundation Expert Group Mediterranean diet pyramid today. Science and cultural updates. Public Health Nutr. 2011;1475272714(12A):2274
2284. Available from: https://doi.org/10.1017/ S1368980011002515.

Chapter 11

Polyphenol oxidase and oleuropein in olives and their changes during olive ripening Francisca Ortega-Garcı´a1, Santos Blanco2, M. A´ngeles Peinado2 and Juan Perago´n1 1

Biochemistry and Molecular Biology Section, Department of Experimental Biology, University of Jae´n, Jae´n, Spain, 2Cell Biology Section, Department of Experimental Biology, University of Jae´n, Jae´n, Spain

Abbreviations DL-DOPA

HPLC IR Km PAGE PPO Vmax

DL-dihydroxyphenylalanine high-performance liquid chromatography fruit-ripeness index Michaelis constant polyacrylamide gel electrophoresis polyphenol oxidase maximum velocity

11.1 Introduction 11.1.1 Phenols, types, biological significance, and presence in olive Phenols are secondary metabolites of plants widely distributed throughout all plant organs and have important functions in the metabolism and physiology of plants. This complex group of substances has structures that vary from single-phenolic molecules such as hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] to highly polymerized compounds such as lignins. These compounds have diverse and important functions such as the maintenance of plant integrity (lignins), floral pigmentation (flavonoids), antibiotics (phytoalexins), and plant defense against pathogens or symbionts.1
3 The occurrence of these substances in food is broadly variable and reaches high levels in the olive fruit and oil.4 Currently, there is a keen interest in dietary polyphenols due to their antioxidant capacity and consequent benefits to human health.3,5,6 Oleuropein, the main phenol of the olive, is a heterosidic ester of β-glucosylated elenolic acid and hydroxytyrosol (Table 11.1). Many of the nutritional and organoleptic properties of olive oil depend on the content

of phenols in general and of oleuropein and hydroxytyrosol in particular. Oleuropein and hydroxytyrosol have a high antioxidant capacity with high free-radical scavenging activity.7 The amount of phenols in olive oil is considered as an index of the quality of this product,8 and in the case of table olives, a typical food in the Mediterranean diet, different procedures have been investigated and used to remove or eliminate the high concentration of oleuropein found in these olives.9,10

11.1.2 Ripening, polyphenol oxidase, structure, and biological properties Fruit ripening brings major changes in the content and composition of phenols. In this process, phenols oxidize to quinones. These compounds are highly reactive and polymerizable, bringing about the accumulation of browning pigments typical of mature fruits. The pigmentation of the fruit is often accompanied by a sensorial and nutritional change involving food-value loss. These oxidation reactions progress in the presence of oxygen and are catalyzed primarily by polyphenol oxidase (PPO, EC 1.10.3.1). PPO has been amply studied in many species of plants, fungi, and bacteria,11,12 and many of its properties have been extensively studied. Nevertheless, many aspects of its biological functions remain unknown. PPO has been located in chloroplast associated with the inner membrane of tylacoids, in the cytoplasm, and in vesicles between the membrane and the cell wall.13
15 A great variety of kinetic and molecular properties and isoforms of PPO have been described in different species.16 A comparative analysis of sequences of plant and fungal PPOs has shown several conserved structural features.12 It

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00048-1 © 2021 Elsevier Inc. All rights reserved.

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TABLE 11.1 Key features of polyphenol oxidase and oleuropein. 1. PPO is an enzyme that contains copper and catalyzes the hydroxylation of monophenols and the oxidation of o-diphenols to o-diquinones that accompany fruit ripening 2. Different isoforms of PPO have been found in different species of plants and seem resulting from conformational changes, association
dissociation phenomena, and covalent attachment of phenolic or carbohydrates 3. PPO has also been associated with the resistance of the plant against biotic and abiotic stress 4. Oleuropein is a heterosidic ester of β-glucosylated elenolic acid and hydroxytyrosol 5. Oleuropein is the main phenolic compound found in the olive and responsible for some of the nutritional properties of olive and olive oil 6. Oleuropein and hydroxytyrosol present a high antioxidant capacity with high free-radical scavenging 7. PPO and oleuropein have been linked to the chemical defense of the plant PPO, Polyphenol oxidase.

has been suggested that the different isoforms of PPO result from conformational changes,17 association
dissociation phenomena,18 covalent attachment of phenolic material,19 or possible attachment of carbohydrate.20 The molecular mass differs widely among species, with values of 2521
64 kDa.22 With respect to its biological function, PPO has been related to the ripening of fruits and to resistance against pathogens, herbivores, and different biotic as well as abiotic stress.11 In the olive, some studies have examined PPO but very few using the Picual variety. Ben-Shalom et al.23 reported the purification and properties of a catechol oxidase from green olives of cv. Manzanillo. Shomer et al.24 showed that this enzyme is located at the inner face of the thylakoids and in the mitochondria. Several works have described the time course of fruit PPO activity in several varieties during ripening,25
27 and Goupy et al.,26 related PPO activity to oleuropein concentration. To understand more about the biochemistry of phenols during olive-fruit maturation in this variety, we investigated the kinetics and molecular properties of PPO during fruit ripening in relation to total phenols and oleuropein concentration. Such information will help one to identify the optimal time for harvesting the olive of Picual variety and thereby optimize product quality. As a result of this work, we provide the kinetic and molecular characterization of polyphenol oxidase in the fruits and leaves of olive trees of the Picual variety.28 In this chapter, we summarize the most representative results found in this study. The study was made in olive trees (Fig. 11.1) located in an orchard at Torredonjimeno (Jae´n, Spain, 37 450 61vN, 3 570 12vW, 655 m a.s.l.). The trees were dryland farmed following traditional methods. Five trees selected in the orchard were sampled for leaves and fruits from all the positions on each tree. Five 25-cm-long branch samples with fruits near the apical end were collected, and all leaves and fruits from each branch were pooled. The samples were frozen in liquid nitrogen and

stored at 220 C until were analyzed. From August to November, seven samples of fruits (from F1 to F7) and leaves (from L1 to L7) were picked from the same trees with an interval of 30 days for the first and second samples and 15 days otherwise. The fruit-ripeness index (IR) was calculated as proposed by Uceda and Frı´as.29 Fig. 11.2 presents the aspect of the fruits and leaves used. Fruit and leaf acetone-powder extracts were used to assay PPO activity, protein quantification, polyacrylamide gel electrophoresis (PAGE), and Western blotting. Also, an immunohistochemical procedure in early October samples was used to determine the tissue location of PPO. In the same samples, phenolic extracts were obtained with 80% methanol. The phenolic fractions of fruit and leaves were analyzed by high-performance liquid chromatography using a reverse phase column (Spherisorb ODS-2) and a UV
vis and MS detector. Oleuropein was identified (Fig. 11.3) and quantified in fruits and leaves during ripening. The total phenolic content of the methanol extracts was also determined by a colorimetric assay.

11.2 Kinetic and molecular properties of PPO in the fruit and the leaf of olive trees of the Picual variety The study and characterization of PPO and oleuropein during fruit ripening is of great interest for a better characterization of fruit maturation in this Spanish cv. that constitutes 30% of the total olive-cultivation area in the world’s leading olive oil
producing country.30 In fruit and leaf a hyperbolic kinetic behavior was found when catechol, 4-methylcatechol, catechin, DL-DOPA, or chlorogenic acid was used as a substrate (Fig. 11.4). The highest specific activity was shown using catechol as the substrate. With practically all the substrates the specific activity found in the fruit was significantly higher than in the leaf. The values of Michaelis constant (Km),

Polyphenol oxidase and oleuropein in olives and their changes during olive ripening Chapter | 11

125

FIGURE 11.1 Photography of an olive tree used in the experiment.

1

IR:

0 Aug

2

3

0.28

1.02

Sep

4

5

2.69

3.82 Oct

6

7

4.79

4.93 Nov

FIGURE 11.2 Changes in the aspect and IR of Olea europaea L. cv. Picual over the experiment. IR, Fruit-ripeness index.

maximum velocity (Vmax), and catalytic efficiency for the different substrates in fruits differed significantly from the values in leaves, indicating that a different isoenzyme can be expressed in each organ.28 The relative molecular mass of the polypeptide that showed PPO activity in gel was 50 and 55 kDa in leaf and fruit, respectively (Fig. 11.5). Under denaturing conditions the molecular mass of this polypeptide determined by Western blotting was 27.7 kDa (Fig. 11.5). This indicated that the enzyme can have a molecular mass of 50
55 kDa with two subunits each of 27.7 kDa. The immunohistochemical location of PPO showed that, in the fruit, it is present in the epidermis below the cuticle. In the leaf, PPO immunoreactivity was located in epidermal and lagunal parenchyma

cells probably located in the plastids. Immunorreactivity was also shown in the vascular tissue in the zone around the phloem sieve cells and companion cells.28 All these results indicate that PPO is a protein extensively distributed in both organs, and probably it is expressed as two different isoenzymes in each tissue that also probably have different functions in the global metabolism of each organ.

11.3 Changes during ripening During ripening a significant and exponential increase of PPO activity was reported in the fruit (Fig. 11.5). The Vmax found at the last stage of ripening studied (F7) was eightfold higher than in the first stage (F1). The Km of PPO, when catechol was used as a substrate, also changes during ripening. The values increased from 8 mM (found in the samples F1, F2, and F3) to 25 mM (found in F5, F6, and F7).28 Coinciding with these changes in the Vmax and Km values, a higher activity was found when assayed by partially denaturing SDS
PAGE and developing with DLDOPA while a new band of 36 kDa was also reported in the samples F5, F6, and F7 (Fig. 11.5). This band can be used as a marker of the final phase of fruit maturation. The changes in the Km and the appearance of the new band in the last stages of ripening indicate that a new isoenzyme will also be present in these stages. Also, during ripening a significant rise in the protein expression level of PPO was found by Western blotting (Fig. 11.5). This result indicated that the synthesis of this protein is

PART | 1 General Aspects of Olives and Olive Oil

Intens. x 105

126

(C)

–MS2, 538.9 m/z

m/z -MS1, 51.4 min

Intens. x 105

(B)

(A)

Units of A280 nm

0.20

m/z

0.15 0.10 0.05 0 Oleuropein

0

10

20

30

40 50 60 Time of retention (min)

70

80

90

100

FIGURE 11.3 HPLC analysis of a methanol extract of fruit of Olea europaea L. cv. Picual to identify oleuropein. (A) Chromatograph obtained recording the absorbance at 280 nm, (B) MS analysis of the compound eluted at 51.4 6 1.3 min, and (C) MS
MS analysis of the fragment with m/z ratio 5 538.9. HPLC, High-performance liquid chromatography.

30

160

PPO-specific activity (units mg/protein)

(A)

(B)

120 20 80 10 40

0

0 0

0.05

0.1

0.15

0.20

0.25

(Substrate) (M)

0

0.05

0.1

0.15

0.20

0.25

0.30

(Substrate) (M)

FIGURE 11.4 Effect of the concentration of catechol (K), catechin (x), 4-methylcatechol (&), chlorogenic acid (’), and dl-DOPA (Δ) on the PPO-specific activity in fruit (A) and leaf (B) of Olea europaea L. cv. Picual. Results are expressed as mean 6 S.E.M. of five data.

stimulated in the last stages of fruit maturation. The combination of all these results indicated that two types of regulating mechanisms can coincide in this enzyme during fruit ripening: the induction of the synthesis of new molecules of protein and the differential expression of two isoenzymes. Both mechanisms would be part of a regulating

mechanism of the gene expression of PPO in the fruit of olive trees of the Picual variety during ripening. In the leaf, PPO activity also exponentially increased during ripening, although the changes were of a lower magnitude than in fruit. The Vmax value in H7 sample was twofold higher than in H1 sample whereas neither changes

Polyphenol oxidase and oleuropein in olives and their changes during olive ripening Chapter | 11

40 1000

20 500

0 0

(B)

FIGURE 11.5 Changes in oleuropein concentration, PPO activity, and protein expression level in fruit of Olea europaea L. cv. Picual during fruit ripening. (A) Evolution of oleuropein concentration (x, left y axis) and PPO-specific activity (K, right y axis). (B) Partially denaturing SDS
PAGE analysis of PPO activity developed with DL-DOPA in fruits samples with different maturity indexes. (C) Immunoblot analysis of PPO in fruits with different maturity indexes. Reprinted from Ortega-Garcı´a F, Blanco S, Peinado MA, Perago´n J. Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv. “Picual” trees during fruit ripening. Tree Physiol. 2008;28:45
54 with permission.

1500

20 F1

40 F2

F3

60 F4

80 F5

100 F6

PPO-specific activity (units mg/protein)

Oleuropein concentration (mg/g dry weight)

(A)

127

0 120 days

F7 55 kDa 36 kDa

(C) 27.7 kDa

in Km values nor the appearance of new bands has been reported.28 The ample distribution of PPO in leaf and its changes during ripening agree with the existence of an important function of this enzyme during the normal leaf metabolism, probably related to the protection of the plant against biotic and abiotic stress.31 In this sense, in an olive tree affected by a thermal stress by cold or freezing, a significant increase in leaf PPO-specific activity and oleuropein concentration has been found.32 The authors propose that PPO and oleuropein can help one to avoid oxidative damage induced by freezing and even can be considered elements for determining the recovery capacity and resistance to freezing of the different olive varieties.

11.4 Oleuropein concentration in fruit and leaf of olive during ripening Oleuropein concentration was also determined along ripening in the fruit and the leaf of olive trees of Picual variety.28 The results in the fruit are shown in Fig. 11.5. The oleuropein concentration significantly decreased in the fruit and increased in the leaf. The highest changes were found between the samples F1
F2 and L1
L2. By comparing the time courses of oleuropein concentration and PPO activity in both tissues, we found that the sharpest changes in oleuropein concentration during fruit ripening did not coincide with the increase in PPO activity. Thus there was no direct relationship between the changes in oleuropein concentration and the PPO activity.

11.5 Effects of the variety of cultivar For studying the effect of the variety, we repeat this study in fruit and leaf of Picual, Verdial, Arbequina, and Frantoio cv. located in the Agricultural Research and Training Centre “Estacio´n de Olivicultura y Elaiotecnia Venta del Llano,” Mengı´bar, Jae´n, Spain.33,34 These are four varieties characterized by different phenol concentrations and adapted to different regions. Frantoio, an Italian typical variety, showed, in fruit, the highest total phenol concentration and the lowest PPO activity and protein expression level. Verdial, an Andalucia and Extremadura Spanish typical variety, showed the lowest phenol concentration and the greatest PPO activity and protein expression level. Picual, major in Andalucı´a and Arbequina, in Catalun˜a, Spain, showed intermediate levels. These results are interpreted as a coordinated response between the synthesis and degradation of phenolics that are responsible of the specific levels in each variety. Significant increases in the PPO-specific activity and specific protein expression level were observed in all the varieties over ripening. Nevertheless, differential changes in Km, catalytic efficiency, and band pattern in partially denaturing SDS
PAGE were found for each variety. Comparing these results with the ripeness index of each variety, we concluded that the PPO activity is not the only element that determines the browning level reached by the olive fruit. With respect to the fruit phenolic profile, we found that each of these varieties showed a characteristic pattern

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that can be used for distinguishing between them. Oleuropein concentration showed a typical decrease along ripening, although with marked differences between varieties, leading to major dissimilarities in the concentration found at the last stages of ripening in which Picual showed the highest concentration followed by Arbequina, Frantoio, and Verdial. In the leaf the results reported by this new study confirm the previously described one.

11.6 Conclusion Oleuropein and PPO are present and respond with different intensities and characteristics in the fruits and leaves during ripening. Probably this different response agrees with the existence of a different function of both compounds in both organs. In the leaf, PPO and oleuropein can be related to the response against biotic and abiotic stress whereas in the fruit, PPO and oleuropein can be directly linked to the mechanism of fruit ripening. Analyzing the changes in both parameters during ripening, we find that an early harvest provides fruit with a higher phenol contents. The variety of olive trees also influences or determines the oleuropein concentration and PPO activity and its changes during ripening.

11.7 Summary points G

G

G

G

G

G

G

Phenols and oleuropein are minor components of olive and olive oil and have important antioxidant benefits for human health. Polyphenol oxidase catalyzed the oxidation of phenols to quinones that accompany the ripening of olives. Here, we describe the kinetic and molecular properties of PPO in the fruit and the leaf of olive of the Picual variety in relation to oleuropein concentration during ripening. Different PPO isoenzymes appear to be expressed in the fruit and the leaf during ripening. A high expression level of PPO was also found in the last stages of fruit maturation. The changes described in PPO expression are not responsible for the sharp fall in oleuropein concentration during the first stages of ripening. The results found in four varieties, Frantoio, Arbequina, Verdial, and Picual, indicated the existence of a coordinated response between oleuropein concentration and PPO.

References 1. Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism. Plant Cell. 1995;7:1085
1097.

2. Yedidia I, Shoresh M, Kerem Z, Benhamou N, Kapulnik Y, Chet I. Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Appl Environ Microbiol. 2003;69:7343
7353. 3. Ververidis F, Trantas E, Douglas C, Vollmer G, Kretzschmar G, Panopoulos N. Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health. Biotechnol J. 2007;2:1214
1234. 4. Brenes M, Garcı´a A, Garcı´a P, Rı´os JJ, Garrido A. Phenolic compounds in Spanish olive oils. J Agric Food Chem. 1999;47: 3535
3540. 5. Galli C, Visioli F. Antioxidant and other activities of phenolics in olives/olive oil, typical components of the Mediterranean diet. Lipids. 1999;34:S23
S26. 6. Dixon RA. Phytoestrogens. Annu Rev Plant Physiol Plant Mol Biol. 2004;55:225
261. 7. Visioli F, Poli A, Galli C. Antioxidant and other biological activities of phenols from olives and olive oil. Med Res Rev. 2002;22:65
75. 8. Angerosa F, Servili M, Selvaggini R, Taticchi A, Esposto S, Montedoro GF. Volatile compounds in virgin olive oil: occurrence and their relationship with the quality. J Chromatogr A. 2004;1054:17
31. 9. Ramirez E, Medina E, Brenes M, Romero C. Endogenous enzymes involve in the transformation of oleuropein in Spanish table olive varieties. J Agric Food Chem. 2014;62:9569
9575. 10. Brenes M, Ramirez E, Garcia P, Medina E, de Castro A, Romero C. New developments in table olive debittering. Acta Hortic. 2018;1199:483
487. 11. Mayer AM. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry. 2006;67:2318
2331. 12. Marusek CM, Trobaugh NM, Flurkey WH, Inlow JK. Comparative analysis of polyphenol oxidase from plant and fungal species. J Inorg Biochem. 2006;100:108
123. 13. Obukowicz M, Kennedy GS. Phenolic ultracytochemistry of tobacco cells undergoing the hypersensitive reaction to Pseudomonas solanacearum. Physiol Plant Pathol. 1981;18:339
344. 14. Sommer A, Ne’eman E, Steffens J, Mayer A, Harel E. Import, targeting and processing of a plant polyphenol oxidase. Plant Physiol. 1994;105:1301
1311. 15. Murata M, Tsurutani M, Hagiwara S, Homma S. Subcellular location of polyphenol oxidase in apples. Biosci Biotech Biochem. 1997;61:1495
1499. 16. Mayer AM, Harel E. Phenoloxidases and their significance in fruit and vegetables. In: Fox PF, ed. Food Enzymology. Vol. 1. London: Elsevier; 1991:373
393. 17. Lerner HR, Mayer AM, Harel E. Evidence for conformational changes in grape catechol oxidase. Phytochemistry. 1972;11: 2415
2421. 18. Jolley RL, Robb DA, Mason HS. The multiple forms of mushroom tyrosinase. J Biol Chem. 1969;244:1593
1599. 19. Gregory RPF, Bendall DS. ). The purification and some properties of the polyphenoloxidase from tea (Camellia sinensis L.). Biochem J. 1966;101:569
581. 20. Flurkey WH, Jen JJ. Purification of peach polyphenoloxidase in the presence of added protease inhibitors. J Food Biochem. 1980;4:29
41.

Polyphenol oxidase and oleuropein in olives and their changes during olive ripening Chapter | 11

21. Das JR, Bhat SG, Gowda LR. Purification and characterization of a polyphenol oxidase from the Kew cultivar of Indian pineapple fruit. J Agric Food Chem. 1997;45:2031
2035. 22. Marque`s L, Fleuriet A, Macheix JJ. Characterization of multiple forms of polyphenoloxidase from apple fruit. Plant Physiol Biochem. 1995;33:193
200. 23. Ben-Shalom NB, Kahn V, Harel E, Mayer AM. Catechol oxidase from green olives properties and partial purification. Phytochemistry. 1977;16:1153
1158. 24. Shomer I, Ben-Shalom N, Harel E, Mayer AM. The intracellular location of catechol oxidase in the olive fruit. Ann Bot. 1979;44:261
263. 25. Sciancalepore V, Longone V. Polyphenol oxidase activity and browning in green olives. J Agric Food Chem. 1984;32:320
321. 26. Goupy P, Fleuriet A, Amiot M-J, Macheix J-J. ). Enzymatic browning, oleuropein content, and diphenol oxidase activity in olive cultivars (Olea europaea L.). J Agric Food Chem. 1991;39:92
95. 27. Ebrahimzadeh H, Motamed N, Rastgar-Jazii F, MontasserKouhsari S, Shokraii EH. Oxidative enzyme activities and soluble protein content in leaves and fruits of olives during ripening. J Food Biochem. 2003;27:181
196. 28. Ortega-Garcı´a F, Blanco S, Peinado MA, Perago´n J. Polyphenol oxidase and its relationship with oleuropein concentration in fruits

29.

30.

31. 32.

33.

34.

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and leaves of olive (Olea europaea) cv. “Picual” trees during fruit ripening. Tree Physiol. 2008;28:45
54. Uceda M, Frı´as L. Harvest dates. Evolution of the fruit of content, oil composition and oil quality. In: Proceedings of II Seminario Oleı´cola Internacional (Consejo Oleı´cola Internacional Ed.). Co´rdoba; 1975:125
130. Barranco D. Variedades y patrones. In: Barranco D, Ferna´ndezEscobar R, Rallo L, eds. El Cultivo del Olivo. 2nd ed. Sevilla: Ediciones Mundi-Prensa, Junta de Andalucı´a; 1998:61
87. Shi Ch, Dai Y, Xu X, Xie Y, Liu Q. The purification of polyphenol oxidase from tobacco. Protein Expres Purif. 2002;24:51
55. Ortega-Garcı´a F, Perago´n J. The response of phenylalanine ammonia-lyase, polyphenol oxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual). J Sci Food Agric. 2009;89:1565
1573. Ortega-Garcı´a F, Perago´n J. Phenylalanine ammonia-lyase, polyphenol oxidase, and phenol concentration in fruits of Olea europaea L. cv. Picual, Verdial, Arbequina, and Frantoio during ripening. J Agric Food Chem. 2009;57:10331
10340. Ortega-Garcı´a F, Perago´n J. Phenol metabolism in the leaves of the olive tree (Olea europaea L.) cv. Picual, Verdial, Arbequina, and Frantoio during ripening. J Agric Food Chem. 2010;58: 12440
12448.

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Section 1.3

Stability, microbes, contaminants and adverse components and processes

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Chapter 12

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Jose´ Marı´a Landete1, He´ctor Rodrı´guez2, Jose´ Antonio Curiel3, Blanca de las Rivas4, Fe´lix Lo´pez de Felipe4 and Rosario Mun˜oz4 1

Food Technology Department, INIA-SGIT, Madrid, Spain, 2Inflammation and Macrophage Plasticity Lab, CICbioGUNE, Derio, Spain,

3

Functional Food Research and Development Center, Health Science Technological Park, Granada, Spain, 4Institute of Food Science,

Technology and Nutrition (ICTAN), CSIC, Madrid, Spain

List of abbreviations catechol Coleccio´n Espan˜ola de Cultivos Tipo (Spanish Collection of Type Cultures) EC ethyl catechol EG ethyl guaiacol EP ethyl phenol HcrAB hydroxycinnamate reductase (A and B subunits) HPLC high-performance liquid chromatography HT hydroxytyrosol LpdBCD gallate/protocatechuate decarboxylase (B, C, and D subunits) OMW olive mill wastewater P pyrogallol PAD phenolic acid decarboxylase, also known as PDC NAD nicotinamide adenine dinucleotide RT-PCR reverse transcription polymerase chain reaction SDS
PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis VC vinyl catechol VG vinyl guaiacol VP vinyl phenol VprA vinyl phenol reductase C CECT

12.1 Introduction The olive tree (Olea europaea L.) is one of the most important fruit trees in the Mediterranean countries. Their products, olive oil and also table olives, are important components of the Mediterranean diet and are largely consumed in the world. Olives are the major fermented

vegetables in Western countries. Homemade production of naturally fermented table olives is very common in Mediterranean countries, and the production methods vary according to local tradition. The fermentation is fundamental in order to have an appropriate decrease in pH and the development of a lactic acid microbiota that can outcompete and prevent the growth of spoilage microorganisms as well as potential pathogenic microbes. Lactic acid bacteria are recognized to play an important role in olive fermentation, and Lactobacillus plantarum and Lactobacillus pentosus are, in fact, regarded as the main species leading this process being often used as starter in guided olives fermentation.1
3 Although L. plantarum is present in small numbers in the fresh products, the traditional process consisting of lye treatment, washing, and brining of the fruits flavors its rapid and predominant growth over other microorganisms in the brines.3
6 Olive has been recognized as a source of biophenols. During the last years, and due to the great importance of phenolic compounds in the health beneficial properties of olive products, together with the prevalence of L. plantarum during olive fermentations, numerous studies have been carried out on the metabolism of olive phenolics by L. plantarum strains.

12.2 Phenolic compounds and Lactobacillus plantarum L. plantarum is a versatile bacterium found in a variety of ecological niches, ranging from vegetable and plant fermentations to the human gastrointestinal tract.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00028-6 © 2021 Elsevier Inc. All rights reserved.

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PART | 1 General Aspects of Olives and Olive Oil

L. plantarum cells are rods with rounded ends, straight, generally 0.9
1.2 μm wide per 3
8 μm long occurring singly, in pairs, or in short chains (Fig. 12.1). The genome of the lactic acid bacterium L. plantarum strain WCFS1 has been sequenced,7 and its size of 3.3 Mb is among the largest known for lactic acid bacteria.8,9 It is though that such genome length is related to the diversity of environmental niches in which L. plantarum is encountered. Nonetheless, this bacterium is most frequently found in the fermentation of plant-derived raw materials, which includes several industrial and artisan food and feed fermentations, such as must, olives, and a variety of vegetable fermentations. The effects of olive phenolic compounds on L. plantarum growth have been studied by several authors. Brines from nonalkali-treated green olives were tested for their antimicrobial properties against L. plantarum.10 They showed a marked bactericidal effect toward L. plantarum. Aqueous solutions of the total phenolics extracted from these brines had the same bactericidal effect. It has been demonstrated that diffused olive polyphenols exerted a significant inhibitory growth effect on L. plantarum during brining only when they were associated with NaCl.4 The viability of L. plantarum in the presence of single or combined fractions of isolated phenolic compounds from NaOH-treated and untreated olive brines was studied.11 When assayed at the concentrations founds in brines, only the single phenolic fraction containing hydroxytyrosol strongly inhibited L. plantarum. However, tyrosol, vanillic acid, verbascoside, and luteolin-7-glucoside, when assayed as single fractions, had no bactericidal effect against L. plantarum. Similar results were obtained by

FIGURE 12.1 Transmission electron micrograph of Lactobacillus plantarum CECT 748T grown in a defined media (12,000 3 ).

evaluating inhibitory activities of p-hydroxybenzoic, sinapic, syringic, protocatechuic, and cinnamic acids on L. plantarum growth.12 Hydroxytyrosol was the sole compound found to be bactericidal at low concentration.11 However, in the same study, the inhibitory combined effect of some olive phenolics was also clearly demonstrated.11 Several authors have studied the effects of oleuropein and its hydrolysis products on the survival of L. plantarum. The results reported were different depending on the antibacterial test method used. Oleuropein was bactericidal against L. plantarum strains isolated from green olive fermentations brines.13 Heat-treated oleuropein also demonstrated a strong bactericidal effect but not the alkali-treated oleuropein, which allowed survival of most of the L. plantarum strains tested. In addition, the inhibition of L. plantarum growth was always observed when oleuropein was associated with another phenolic fraction or added directly in brines. Contrarily, it was also reported that untreated oleuropein was not inhibitory to L. plantarum; however, when the aglycon was formed in the medium, cell viability decreased.14 Moreover, when oleuropein and NaCl were associated, the bactericidal effect on L. plantarum growth was very pronounced. The same authors also reported that the presence of glucose seems to delay the antimicrobial activity of these two compounds.14 The bactericidal effect of oleuropein was accompanied by changes in the typical bacillary structure of L. plantarum.13 The morphology of L. plantarum strains incubated in heat-treated or untreated oleuropein solutions changes in both size and shape. Cells become longer, often deformed in their bacillary structure and also become wider. This could indicate that oleuropein promotes peptidoglycan disruption, which could lead to cell death by destruction of the cell envelope. It has been described that oleuropein increased the leakage of inorganic phosphate, glutamic acid, and potassium from L. plantarum cells.15 In addition, brines from nonalkali-treated green olives showed a marked bactericidal effect toward L. plantarum.10 The bactericidal effect was shown by certain alterations at two different levels of the cellular ultrastructure, the cell wall, and the cytoplasmic membrane. Other changes in the cell ultrastructure were observed, such as the presence of mesosomal membranes. Fluorescence microscopy showed adsorption of phenolics to both whole cells and isolated cell walls from L. plantarum. These alterations possibly lead to the disruption of the cell envelope when the incubation time is extended, thus promoting cellular lysis.10 Recently, differential gene expression was used to increase the insights into the molecular mechanisms used by L. plantarum in the adaptation to oleuropein.16 According to transcriptomic data, L. plantarum reduces growth, remodels membrane phospholipids, and diminishes the expression of several ABC transporters. The controlled expression of all of these molecular

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Chapter | 12

players suggests that oleuropein could act as a signaling molecule in the plant
microbe interaction and facilitate the accommodation of beneficial microbes such as L. plantarum by the plant host, via controlled expression of bacterial molecular players involved in this reciprocal interplay.16 The effects of different phenolic compound concentrations on the fatty acid composition of L. plantarum isolated from traditional homemade olive brines were determined.17 They found that phenolic compounds altered L. plantarum fatty acid constitution. When caffeic and ferulic acids were added to the medium, lactobacillic acid production was reduced at the expense of unsaturated fatty acids, mainly palmitoleic acid. Consequently, in stationary-phase cultures an increase in the degree of unsaturation was found. The authors suggest that acidic phenols could modify the membrane fluidity, as a possible hypothesis for the changes observed in the unsaturated fatty acid content. The fatty acid biosynthetic pathway was thoroughly downregulated at the transcriptional level in response to olive oil challenge.18 A set of 230 genes were differentially expressed by L. plantarum to respond to olive oil. This response involved elements typical of the stringent response, as indicated by the induction of genes involved in stress-related pathways and downregulation of genes related to processes associated with rapid growth. A set of genes involved in the transport and metabolism of compatible solutes were downregulated, indicating that L. plantarum does not require osmoprotective mechanisms in presence of olive oil.18

12.3 Metabolism of phenolic compounds by Lactobacillus plantarum Table olives have a different qualitative and quantitative phenolic composition than the raw olive fruits from which they are prepared. There are also notable differences among the phenolic compounds present in olive fruit and oil. The phenolic compounds found in olives, in leaves, seed, pulp, as well as oil were identified.19 Different phenolic groups are present in some olive products, as phenolic acids, phenolic alcohols, flavonoids, and secoiridoids. Some of the phenolic acids present in olives are caffeic, p-coumaric, protocatechuic, vanillic, p-hydropxybenzoic, p-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, and ferulic acid. Among the phenolic alcohols, tyrosol and hydroxytyrosol are present in olive products. They can be found complexed to glucoside and acetate. The most frequently described flavonoids include luteolin 7-O-glucoside, rutin, and apigenin 7-O-glucoside, and the anthocyanins, cyaniding 3-O-glucoside and cyaniding 3-O-glucoside. The predominant secoiridoids of olive fruit pulp are oleuropein and ligstroside. Some oleuropein derivatives have also been described,

135

namely, demethyloleuropein, oleuropein aglycone, and elenoic acid. Table 12.1 summarizes the current knowledge on the metabolism of some phenolic compounds found in olive products by L. plantarum strains.

12.3.1 Phenolic acids The term “phenolic acids,” in general, describes phenols that possess one carboxylic acid functional group. The phenolic acids found in olive products contain mainly two distinguishing constitutive carbon frameworks: the hydroxycinnamic and the hydroxybenzoic structures. Fig. 12.2 summarizes the phenolic acids or related acids reported to be metabolized by L. plantarum strains.

12.3.1.1 Hydroxycinnamic acids Hydroxycinnamic acids are common in olive products. The presence of acids, such as p-coumaric, o-coumaric, caffeic, hydrocaffeic, ferulic, and sinapic acid, as well as cinnamic acid, has been reported in olive products.19 Several studies were performed in order to know if L. plantarum strains have the ability to degrade hydroxycinnamic acids. L. plantarum cultures were grown in presence of each acid, so, if L. plantarum cells are able to metabolize the hydroxycinnamic acid, the dead-end degradation products could be detected in the culture media. In addition, cell-free extracts containing all the L. plantarum soluble proteins were incubated in presence of the same acids in order to obtain information on the induction of the enzymes involved. Fig. 12.3 shows the high-performance liquid chromatography (HPLC) chromatograms of the hydroxycinnamic acids that are metabolized by L. plantarum CECT 748T cultures. As compared to the control [Fig. 12.3(1A)], supernatants obtained from cell cultures grown in p-coumaric acid showed the presence of vinyl- and ethyl phenols, resulting from the decarboxylation, and decarboxylation plus reduction of p-coumaric acid [Fig. 12.3(1B)]. A similar situation was observed in the caffeic acid sample as in the supernatants from the cultures, the products of the decarboxylation (4-vinyl catechol) as well as the decarboxylation plus reduction (4-ethyl catechol) of caffeic acid were identified [Fig. 12.3(2A) and (2B)]. Like p-coumaric and caffeic acid, ferulic acid was found to be metabolized by L. plantarum cell cultures [Fig. 12.3(3A) and (3B)]. Among the hydroxycinnamic acids assayed, only pcoumaric and caffeic acids were metabolized by both, cell cultures and cell-free extracts of L. plantarum CECT 748T; however, the extracts were unable to degrade ferulic acid. It was only partially modified by cell cultures. These results seem to indicate that the enzymes involved in ferulic acid metabolism need to be synthesized after their induction by the presence of this phenolic acid.

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PART | 1 General Aspects of Olives and Olive Oil

TABLE 12.1 Metabolism of phenolic compounds by Lactobacillus plantarum strains. Compound assayed

Compound produced

Enzymes involved

References

Caffeic acid

p-Vinyl catechol

PAD

[20,21,25,28,31]

Hydrocaffeic acid

HcrAB

[26]

Catechol

Not degraded

[30,31]

Chlorogenic acid

ND

[31]

Cinnamic acid

Not degraded

m-Coumaric acid

3-HPPA

HcrAB

[26]

o-Coumaric acid

Melilotic acid

HcrAB

[26]

p-Coumaric acid

p-Vinyl phenol

PAD

[20,21,25,28,31]

Phloretic acid

HcrAB

[26]

p-Vinyl guaiacol

PAD

[20,21,28,31]

Hydroferulic acid

HcrAB

[26]

Gallic acid

Pirogallol

LpdBCD

[31,32]

Hydroxybenzoic acid

Not degraded

[31]

Hydroxytyrosol

Not degraded

[12,38
40]

Oleuropein

Hydroxytyrosol

Phloretic acid

Not degraded

Protocatechuic acid

Catechol

Quercitin

Not degraded

Ferulic acid

[31]

β-Glucosidase, esterase

[12,38
40] [31]

LpdBCD

[31,32] [43]

Quinic acid

Catechol

Several enzymes

[30,35,36]

Shikimic acid

Catechol

Several enzymes

[30,35,36]

Sinapic acid

Hydrosinapic acid

HcrAB

[26]

Syringic acid

Not degraded

[31]

Tyrosol

Not degraded

[12]

Vanillic acid

Not degraded

[31]

p-Vinyl catechol

p-Ethyl catechol

VprA

[24]

p-Vinyl guaiacol

p-Ethyl guaiacol

VprA

[24]

p-Vinyl phenol

p-Ethyl phenol

VprA

[24]

ND, Not detected; HcrAB, hydroxycinnate reductase (A and B subunits); 3-HPPA, 3-(3-hydroxyphenyl)propionic acid; PAD, phenolic acid decarboxylase or PDC or PadA; LpdBCD, gallate decarboxylase (B, C, and D subunits); VprA, vinyl phenol reductase.

From these results, it could be concluded that uninduced L. plantarum cell-free extracts contained decarboxylases able to decarboxylate p-coumaric and caffeic acids into their vinyl derivatives. In fact a phenolic acid decarboxylase (PAD, also known as PDC) has been identified in L. plantarum strains. The gene encoding a p-coumarate decarboxylase (PAD) from L. plantarum LPCHL2 has been cloned and the recombinant protein overexpressed in Escherichia coli.20 The substrate specificity of the purified PAD by using several hydroxycinnamic acids (e.g., p-coumaric, o-coumaric, m-coumaric, ferulic, caffeic, hydrocaffeic, phloretic,

2-methoxycinnamic, and 3-methoxycinnamic acids) was analyzed.21 Only p-coumaric and caffeic acids were metabolized, at the same rate and KM. The authors concluded that only the acids with a para-hydroxyl group with respect to the unsaturated side chain and with a substitution
H or
OH in position meta were metabolized. In 2003 the complete genome sequence of L. plantarum WCFS1 was available.7 This strain possesses a PAD enzyme that differs, from the enzyme previously purified, mainly on its C-terminal region where the substrate specificity is determined. In addition, the PAD protein from the L. plantarum
type strain

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Chapter | 12

137

FIGURE 12.2 Chemical structure of the phenolic or related acids reported to be metabolized by Lactobacillus plantarum strains.

CECT 748T was crystallized, and its amino acid sequence is determined to be identical to that of L. plantarum WCFS1.22 The recombinant PAD from L. plantarum CECT 748T (Fig. 12.4) is able to decarboxylate exclusively the hydroxycinnamic acids, p-coumaric, caffeic, and ferulic acids.23 Kinetic analysis showed that the enzyme has a 14-fold higher KM value for p-coumaric and caffeic acids than for ferulic

acid. PDC catalyzes the formation of the corresponding 4vinyl derivatives (vinyl phenol, vinyl catechol, and vinyl guaiacol) from p-coumaric, caffeic, and ferulic acids, respectively.23 Subsequently, L. plantarum strains are able to reduce these vinyl derivatives into their corresponding ethyl derivatives (4-ethyl phenol, 4-ethyl catechol, and 4-ethyl guaiacol) by the action of the VprA protein (vinyl phenol reductase).24

138

PART | 1 General Aspects of Olives and Olive Oil

2000

150 (1A)

(1B)

mAU

100

1000 50 VP

EP

0

0

(2B)

(2A)

mAU

200

1000 100 VC

0

0 (3A)

2000

mAU

EC

(3B)

600 400

1000

VG

200

EG

0

0 0

20

40

60

80

Min

0

20

40

60

80

Min

FIGURE 12.3 HPLC chromatograms of the degradation of hydroxycinnamic acids by Lactobacillus plantarum. Chromatograms of supernatant from L. plantarum CECT 748T grown for 10 days in the presence of p-coumaric (1B), caffeic (2B), and ferulic acids (3B). The HPLC chromatograms from the control samples (1A, 2A, or 3A, respectively). EC, p-Ethyl catechol; EG, p-ethyl guaiacol; EP, p-ethyl phenol; HPLC, high-performance liquid chromatography; VC, p-vinyl catechol; VG, p-vinyl guaiacol; VP, p-vinyl phenol.

A mutant deficient in the PAD activity was constructed to study phenolic acid alternate pathways in L. plantarum.25 In culture media the main transformation of hydroxycinnamic acids is their decarboxylation; however, L. plantarum possesses an alternative pathway to transform hydroxycinnamic acids. Some of these acids could be reduced to their corresponding substituted phenylpropionic acids by the action of the HcrAB proteins (hydroxycinnamate reductase) recently described.26 mCoumaric, o-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids are also reduced by HcrAB to originate 3(3-hydroxyphenyl) propionic (3-HPPA), melilotic, phloretic, hydrocaffeic, hydroferulic, and hydrosinapic acids, respectively. These reduced acids are not further degraded by L. plantarum strains. These biotransformation pathways are the metabolic strategies followed by L. plantarum to tolerate the hostile environment generated by the presence of hydroxycinnamic acids. Transcriptional studies of the gene encoding

the PAD in L. plantarum LPCHL2 demonstrated that its transcription is phenolic acid
dependent. The strong, rapid, and inducible PAD enzyme synthesis following exposure to phenolic acids can be considered a specific chemical stress response to overcome phenolic acid toxicity. This was proved by disruption of the PAD encoding gene, which rendered the bacterium sensitive to these substrates and makes it unable to grown at a low pH in the presence of p-coumaric acid.25 To advance knowledge of the stress tolerance mechanisms of L. plantarum to hydroxycinnamic acids, whole-genome transcriptional profiling during challenge with p-coumaric acid was used.27 The transcriptional profile reveals a massive induction of genes involved in stress resistance and detoxification-related functions and a global shutdown of growth-associated processes. A specific oxidative stress response was induced, probably to counteract a p-coumaric-induced oxidative protein stress.27 Some of the induced genes were also responsive in this strain to other environmental stresses.

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Chapter | 12

12.3.1.2 Hydroxybenzoic acids Some hydroxybenzoic acids are found in olive products (Table 12.1). L. plantarum cells were grown in the presence of several hydroxybenzoic acids, such as gallic, syringic, p-hydroxybenzoic, protocatechuic, and vanillic acids. Among the hydroxybenzoic acids assayed, only gallic and protocatechuic acids were metabolized by both cell cultures and cell-free extracts from L. plantarum CECT 748T (Table 12.1). Fig. 12.5 shows the HPLC chromatograms obtained with L. plantarum cells grown in the presence of gallic acids. As compared to the control [Fig. 12.5(1A)], it could be observed that gallic acid was decarboxylated to pyrogallol [Fig. 12.5(1B)]. Pyrogallol was also obtained from gallic acid during the degradation of tannic acid by L. plantarum cell-free extracts.28 Previously, the occurrence of a gallate decarboxylase activity in L. plantarum strains was suggested.29 As early as 1971, Whiting and Coggins reported that L. plantarum cells grown in a medium containing protocatechuic acid completely metabolized it to catechol, and there was no indication of a further metabolism of catechol.30 Later, it was described that gallic and protocatechuic acids were decarboxylated to pyrogallol and catechol, respectively, by cultures of L. plantarum grown in presence of these hydroxybenzoic acids31 [Fig. 12.5 (2A) and (2B)]. These results indicate that pyrogallol and catechol are dead-end products of hydroxybenzoate degradation in L. plantarum cultures. The same five hydroxybenzoic acids (gallic, syringic, p-hydroxybenzoic, protocatechuic, and vanillic acids) were assayed by using recombinant hydroxycinammate decarboxylase (PAD) from L. plantarum CECT 748T. As expected, the phenolic acids that were not metabolized by L. plantarum cultures were not metabolized by the PAD enzyme. In addition, neither protocatechuic nor gallic acids were used as substrate by purified PAD.28 1

2

3

kDa 45 31 21.5 14.5 FIGURE 12.4 Expression and purification of the PAD protein from Lactobacillus plantarum CECT 748T. SDS
PAGE analysis of soluble Escherichia coli cell extracts. Lane 1: control plasmid. Lane 2: plasmidcontaining the L. plantarum PAD gene. Lane 3: fraction eluated after the affinity column. The 15% polyacrylamide gel was stained with Coomassie blue. The positions of molecular mass markers (Bio-Rad) are indicated on the left. The L. plantarum recombinant PAD protein is indicated by an arrow. PAD, Phenolic acid decarboxylase; SDS
PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis.

139

The decarboxylase enzyme responsible of the decarboxylation of gallic and protocatechuic acids by L. plantarum CECT 748T has been identified.32 This decarboxylase was identified as a nonoxidative aromatic acid decarboxylase composed of three different subunits (subunits B, C, and D). L. plantarum is the only bacterium in which the gene encoding subunit C (lpdC) is separated in the chromosome from the genes encoding subunits B (lpdB) and D (lpdD). LpdC is the only protein required to yield gallate/protocatechuate decarboxylase activity, although LpdB is also essential for activity.32 Purified LpdC showed decarboxylase activity only against two hydroxybenzoic acids, gallic, and protocatechuic acid. The effect of gallic acid on the expression of genes located in the gallate decarboxylase region of L. plantarum was analyzed by qPCR.33 Gallic acid greatly induced the expression of the lpdC gene, as well as that of GacP a transport protein, which is cotranscribed with it. Similarly, gallic acid induced the expression of lpdB, lpdD, and tanR, which encodes a transcriptional regulator. Taking these data together, a model for gallate decarboxylase regulation in L. plantarum was proposed.33 In this model the transcriptional regulator TanR is constantly present in the cell in a definite small amount under physiological conditions. The presence of gallic acid at very low concentrations is insufficient to induce the gallate decarboxylase. On external exposure to high gallic acid concentrations the level of gallic acid increases inside the cell by the diffusion of its uncharged form. TanR is positively autoregulated upon interaction with the inducer gallic acid. TanR protein could bind with DNA, and this binding results in the progression of the transcription of the gallate decarboxylase operon.33 Whole-genome transcription profiling was used to expand the insight into the molecular mechanism underlying the response of L. plantarum to a hydroxybenzoic acid and gallic acid.34 Gallic acid acts as a signal to trigger versatile responses, including nitrogen storage, tannin metabolism, or N-acetyl glucosamine utilization. The ability of L. plantarum to synthetize energy from gallic acid in the form of proton motive force and the gallic acid
mediated induction of nitrogen storage, utilization of carbon sources such as N-acetyl glucosamine or tannins, potentially confers competitive advantages to L. plantarum. Gene expression and organization of genes involved in gallic acid decarboxylation pointed toward a chemiosmotic mechanism of energy generation associated to gallic acid metabolism, which was experimentally supported by membrane potential and internal pH measurements.34

12.3.1.3 Phenolic-related acids As shown in Table 12.1, the metabolisms of two additional olive phenolic acids, such as phloretic and

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(1A)

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FIGURE 12.5 HPLC chromatograms of the degradation of hydroxybenzoic acids by Lactobacillus plantarum. Chromatograms of supernatant from L. plantarum CECT 748T grown for 10 days in the presence of gallic (1B), and protocatechuic acids (2B). The HPLC chromatograms from the control samples (1A, or 2A, respectively). C, Catechol; P, pyrogallol; SDS
PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis.

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chlorogenic acids, have been studied previously.31 However, chlorogenic acid was not detected by the chromatographic method used, and phloretic acid was not metabolized by cell cultures as well as by the cell-free extracts. In addition, a number of acids present in olives (such as cinnamic, elenoic, shikimic, and quinic acids) could be considered as phenolics on the basis of metabolic considerations, although they lack a phenolic group or even an aromatic ring. The metabolisms of quinic and shikimic acids by L. plantarum have been studied.30 L. plantarum reduced quinic acid and the related shikimic acid under anaerobic conditions in the presence of suitable hydrogen donors, including fructose, glucose, and lactates.35 L. plantarum shows two metabolic routes for these acids, one oxidative and one reductive. Both pathways proceed simultaneously under anaerobic conditions. The oxidative route gives catechol as end-product; there was no indication of further metabolism under anaerobic conditions. The pathway, which has been described to comprise 11 steps, has been elucidated by using several procedures, namely, the use of growing cells, washed cells grown with different substrates, cell-free extracts, and the separation and purification of a nicotinamide adenine dinucleotide
specific hydroaromatic dehydrogenase involved in five steps of the pathway.36 This enzyme, similarly to the enzymes catalyzing the second and sixth steps, was an inducible enzyme. In this pathway, both D- and L-lactates act indistinctly as hydrogen donors for the reductive steps, while lactate was simultaneously oxidized to acetate. These oxidoreductions proceeded under anaerobic conditions. However, L. plantarum not only reduces quinic acid but at the same time, even under anaerobic conditions, oxidizes a proportion and converts the product of dehydratase and decarboxylase action to catechol.30 It is not known whether

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dehydroshikimic acid is the branch point of the oxidative and reductive pathways or whether shikimic acid is oxidized to dehydroshikimate by a pyridine nucleotideindependent shikimate dehydrogenase found in cell-free extracts of this organism.30 The pathway suggested by these authors may be involved in raising the pH of their environment since all the metabolic changes involved bring about such an increase in pH.

12.3.2 Phenyl alcohols The presence of several phenyl alcohols (such as hydroxytyrosol, tyrosol, or catechol) has been described in olive products. Hydroxytyrosol and tyrosol are among the prevailing phenols in olive products. Hydroxytyrosol, which results from the hydrolysis of oleuropein, killed L. plantarum completely within 2 h.10 However, it has not been reported an antimicrobial effect for tyrosol.11,12 In relation to the bacterial metabolism of these phenols, it should be noted that none of them was degraded when L. plantarum strains were grown in the presence of hydroxytyrosol and tyrosol.12 Catechol was for the first time identified in natural black olive pulp in 2002.37 As mentioned in previous sections, no indication of a metabolism of catechol was reported.30,31

12.3.3 Glycosides Phenolic structural complexity is introduced by the common occurrence of certain phenolics in the O-glycosides form, glucose being the most commonly encountered sugar. Oleuropein is the main bitter secoiridoid glucoside present in olives. During crushing and malaxing processes, oleuropein and demethyloleuropein are hydrolyzed

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Chapter | 12

by endogenous glycosidases. The aglycons become soluble in the oil phase while the glycosides remain in the wastewater phase. Oleuropein was also found to be hydrolyzed by the β-glucosidase produced by L. plantarum strains.38 Cultures from L. plantarum
containing oleuropein showed a significant decrease of oleuropein content over time with a concomitant increase of aglycone derivatives, which were further degraded to hydroxytyrosol.39 Due to its instability in aqueous solution, the first compound of the enzymatic degradation of the oleuropein rearranges to several aglycone structures before transforming into stable final compounds. Therefore, at various steps of the enzymatic reaction, different chemical structures were determined (Fig. 12.6). The results obtained suggest that the L. plantarum activity on oleuropein involves a two-step process: (1) an enzymatic hydrolysis of the glycosidic linkage by a β-glucosidase action to release the aglycone, the first observable intermediate in the process; (2) after the β-glycosidic linkage is completely hydrolyzed, as deduced from the disappearance of oleuropein, other compounds originate, probably due to esterase activity that hydrolyzes the ester to an acid (elenoic acid) and an alcohol (hydroxytyrosol).39 Metabolism of oleuropein seems to be carried out by inducible enzymes since a cell-free extract from an L. plantarum culture grown in the absence of oleuropein was unable to metabolize it.12 It was reported that oleuropein consumption is concomitant with bacterial growth.40 The concentration of glucose in the medium is a critical variable affecting oleuropein breakdown in the olives. In the presence of 10
20 g/L glucose, oleuropein concentration decreased, but a 30%
40% residual was not hydrolyzed. The unhydrolyzed oleuropein concentration increased to 70% in the presence of 30
50 g/L glucose.40 Concurrent to the decrease of oleuropein, aglycone was detected only at trace levels since this derivative was further degraded to hydroxytyrosol. This indicated that the esterase activity involved in the biodegradation process of oleuropein was not affected by glucose.40 200

The presence of glycosidase activities was assayed in L. plantarum strains. The analyzed strains presented hydrolytic activity against α- and β-D-glucopyranoside and β-D-galactopyranoside.41 The presence of a β-glucosidase activity on L. plantarum strains has been also reported, and one active form of β-glucosidase in the culture filtrate of L. plantarum was identified.42 This enzyme was able to hydrolyze several β-linked glucose dimmers, with a hydrolysis rate order of (β-1,4) . (β-1,3) . (β-1,2) . (β-1,6). This protein seems to be a broad-specificity β-glucosidase since it can hydrolyze β-diglucosides and also aryl-β-glucosides. The enzyme had a molecular mass of about 40 kDa.42

12.3.4 Flavonols Among the flavonols whose presence in olive products has been generally detected, quercitin and its related glycoside, rutin (quercitin-3-rutinoside), are included.19 It was reported that quercitin was not degraded by L. plantarum strains.43 The fact that the quercitin is not degraded by L. plantarum strains after growth does not mean that it did not influence its growth performance. Among flavonols, quercitin showed high affinity by the lipid bilayer of membranes,44 therefore, could affect bacterial growth. It has been observed that in some L. plantarum strains, quercitin promotes quicker growth upon inoculation and higher growth rates.45 Quercetin improved several key fermentation traits for the performance of L. plantarum in food production, including accelerated fermentation of various sugars, and accelerated lactic acid production.45

12.4 Treatment of olive by-products by Lactobacillus plantarum Generally, the olive industry produces two residues, solids and olive mill wastewater (OMW). OMW is composed of the vegetation water of olives pulp tissue and oil in the form of a very stable emulsion. OMW is one of the most 200

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FIGURE 12.6 HPLC chromatograms of the degradation of oleuropein by Lactobacillus plantarum. Chromatograms of supernatant from L. plantarum CECT 748T grown for 10 days in the presence of oleuropein (B). The HPLC chromatogram from the control oleuropein sample is showed in (A). HT, Hydroxytyrosol; SDS
PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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complex plant effluents. The ecological problem of OMW is due primarily to the presence of phenolic compounds that make OMW toxic and resistant to biological degradation. Up to date, a large variety of methods have been suggested for the treatment of OMW; however, most of them aim the decomposition/destruction of the contained polyphenols and not their exploitation.46 Lactic acid bacteria with their capacity to reduce oxygen pressure, redox potential and pH, offer a new promising approach to the bioconversion of phenolic compounds present in olive wastes. In fact the effects of L. plantarum growth on the reductive decolorization and biodegradation of olive phenolic compounds were evaluated.47 OMW is unstable and turns black under aerobic conditions because of the autooxidation of phenolic compounds. L. plantarum growth on fresh OMW induced the depolymerization of phenolic compounds of high molecular weight, with a resultant decolorization of fresh OMW, and significant reduction of total phenols, in proportion to the dilution of OMW. They found that approximately 58% of the color, 55% of the chemical oxygen demand, and 46% of the phenolic compounds were removed when OMW was diluted 10 times. The removal of phenolic compounds was associated with the depolymerization, the partial adsorption on the cells, and the biodegradation of certain simple phenolic compounds. A similar phenolic depolymerization mechanism was observed in L. plantarum degrading tannins.28 In addition, the application of L. plantarum to the olive fruit during crushing could constitute a new microbiological process for olive oil quality improvement. The transformation of phenolic compounds contained in OMW into valuable products using L. plantarum was studied in order to increase their transportation from OMW to olive oil.48 Incubation of olive oil samples with fermented OMW by L. plantarum caused polyphenols to decrease in OMW and increase in oil. The lower total phenolic content in fermented OMW in comparison to OMW control resulted from the depolymerization of phenolic compounds of high molecular weight by L. plantarum. Fermentation with L. plantarum induced reductive depolymerization of OMW, which is more soluble in olive oil. The analysis of the phenolic compounds found in olive oil after storage showed that the application of L. plantarum favors the increase of all phenolic compounds in olive oil, especially by depolymerization and by reductive conversion of phenolic compounds of olive and oxygen fixation. The increase in phenolic content in olive oil was more prominent for simple polyphenols such as oleuropein, p-hydroxyphenylacetic, vanillic and ferulic acids, and tyrosol. In summary the authors concluded that olive oil mixed with the OMW and fermented by L. plantarum had a higher quality and stability because of a higher content of simple phenolic compounds.

Mini-dictionary of terms Lactobacillus plantarum: Lactobacillus plantarum (currently Lactiplantibacillus plantarum) is a species of lactic acid bacteria, which is mainly found in the fermentation of plant-derived raw materials. From the metabolism of sugars, it produces mainly lactic acid. Cells are rods with rounded ends, straight, generally 0.9
1.2 μm wide per 3
8 μm long occurring singly, in pairs or in short chains. L. plantarum cell extracts: L. plantarum cell-free extracts containing all the soluble cell proteins are obtained after disruption of the bacterial cells. Most of the enzymatic activities present in the cells are included in the cell extract. This is an adequate method to known the metabolic abilities of a specific bacterial strain. Inducible enzymes: Some enzymes are not always present in cell-free extracts because their synthesis is induced only in a specific growth situation. Extracts containing a specific inducible enzyme need to be prepared from cultures when the enzyme is induced as a response of a growth situation (e.g., L. plantarum growth in the presence of p-coumaric acid induces the synthesis of the enzyme p-coumarate decarboxylase). p-Coumarate decarboxylase (PAD): Enzyme responsible for the decarboxylation of several hydroxycinnamic acids (p-coumaric, ferulic, and caffeic acids), giving their corresponding vinyl derivatives (vinyl phenol, vinyl guaiacol, and vinyl catechol). This enzyme has been identified and characterized in L. plantarum strains. Vinyl phenol reductase (VprA): Enzyme involved in the reduction of the vinyl phenols (vinyl phenol, vinyl guaiacol, and vinyl catechol) originated by the action of PAD on several hydroxycinnamic acids (p-coumaric, ferulic and caffeic acids). Ethyl phenols (ethyl phenol, ethyl guaiacol, and ethyl catechol) are produced by the VprA action on vinyl phenols. The identification of L. plantarum VprA constitutes the first description of a bacterial enzyme possessing vinyl phenol reductase activity. Hydroxycinnamate reductase (HcrAB): Enzyme involved in the reduction of several hydroxycinnamic acids producing their corresponding substituted phenylpropionic acids. HcrAB action on caffeic, m-coumaric, ocoumaric, p-coumaric, ferulic, and sinapic acids originates hydrocaffeic, 3-(3-hydroxyphenyl) propionic, melilotic, phloretic, hydroferulic, and hydrosinapic acids, respectively. The identification of L. plantarum HcrAB constitutes the first description of a bacterial enzyme possessing hydroxycinnamate reductase activity. Gallate decarboxylase (LpdBCD) (also known as gallate/decarboxylase): Enzyme responsible for the decarboxylation of only two hydroxybenzoic acids (gallic and protocatechuic acids). The action of LpdBCD on gallic and protocatechuic acids originates pyrogallol and

Degradation of phenolic compounds found in olive products by Lactobacillus plantarum strains Chapter | 12

catechol, respectively. The identification of L. plantarum HcrAB constitutes the first description of a bacterial enzyme possessing gallate/protocatechuate decarboxylase activity. Effects of phenolic compounds on L. plantarum cells: The effects of phenolic compounds in L. plantarum cells are dose dependent. At higher concentration, these compounds produce growth inhibition. It has been described that changes in cell size and shape appeared at lower concentrations. Whole-genome transcription profile: In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles measure the entire genome simultaneously and can, for example, show how the cells react to a particular treatment. In L. plantarum the transcriptional response to olive oil, p-coumaric acid, gallic acid, and oleuropein presence has been described.

9. 10.

11.

12.

13.

14.

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2064. 2. Vega Leal-Sa´nchez M, Ruı´z-Barba JL, Sa´nchez AH, Rejano L, Jime´nez-Dı´az R, Garrido A. Fermentation profile and optimization of green olive fermentation using Lactobacillus plantarum LPCO10 as a starter culture. Food Microbiol. 2003;20:421
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68. Roze`s N, Peres C. Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum.. Appl Microbiol Biotechnol. 1996;45:839
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111. Esteban-Torres M, Revero´n I, Plaza-Vinuesa L, de las Rivas B, Mun˜oz R, Lo´pez de Felipe F. Transcriptional reprogramming at genome-scale of Lactobacillus plantarum WCFS1 in response to olive oil challenge. Front Microbiol. 2017;8:244. Ryan D, Robards K. Phenolic compounds in olives. Analyst. 1998;123:31R
44R. Cavin JF, Barthelmebs L, Divie`s C. Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization. Appl Environ Microbiol. 1997;63:1939
1944. Cavin JF, Barthelmebs L, Guzzo J, Van Beeumen J, Samyn B, Travers JF, Divie`s C. Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum. FEMS Microbiol Lett. 1997;147:291
295. Rodrı´guez H, de las Rivas B, Mun˜oz R, Manchen˜o JM. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum.. Acta Cryst. 2007;F63:300
303. Rodrı´guez H, Landete JM, Curiel JA, de las Rivas B, Manchen˜o JM, Mun˜oz R. Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748T. J Agric Food Chem. 2008;56:3068
3072. Santamarı´a L, Revero´n I, Lo´pez de Felipe F, de las Rivas B, Mun˜oz R. Ethylphenol formation by Lactobacillus plantarum:

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identification of the enzyme involved in the reduction of vinylphenols. Appl Environ Microbiol. 2018;84:e1064
18. Barthelmebs L, Divies C, Cavin JF. Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism. Appl Environ Microbiol. 2000;66:3368
3375. Santamarı´a L, Revero´n I, Lo´pez de Felipe F, de las Rivas B, Mun˜oz R. Unravelling the reduction pathway as an alternative metabolic route to hydroxycinnamate decarboxylation in Lactobacillus plantarum. Appl Environ Microbiol. 2018;84:e01123
18. Revero´n I, de las Rivas B, Mun˜oz R, Lo´pez de Felipe F. Genomewide transcriptomic responses of a human isolate of Lactobacillus plantarum exposed to p-coumaric stress. Mol Nutr Food Res. 2012;56:1848
1859. Rodrı´guez H, de las Rivas B, Go´mez-Cordove´s C, Mun˜oz R. Degradation of tannic acid by cell-free extracts of Lactobacillus plantarum.. Food Chem. 2008;107:664
670. Osawa R, Kuroiso K, Goto S, Shimizu A. Isolation of tannindegrading lactobacilli from human and fermented foods. Appl Environ Microbiol. 2000;66:3093
3097. Whiting GC, Coggins RA. The role of quinate and shikimate in the metabolism of lactobacilli. Ant Leeuw. 1971;37:33
49. Rodrı´guez H, Landete JM, de las Rivas B, Mun˜oz R. Metabolism of food phenolic acids by Lactobacillus plantarum CECT 748T. Food Chem. 2008;107:1393
1398. Jime´nez N, Curiel JA, Revero´n I, de las Rivas B, Mun˜oz R. Uncovering the Lactobacillus plantarum WCFS1 gallate decarboxylase involved in tannin degradation. Appl Environ Microbiol. 2013;79:4253
4263. Revero´n I, Jime´nez N, Curiel JA, Pen˜as E, Lo´pez, de Felipe F, de las Rivas B, Mun˜oz R. Differential gene expression by Lactobacillus plantarum WCFS1 in response to phenolic compounds reveals new genes involved in tannin degradation. Appl Environ Microbiol. 2017;83:e03387
16. Revero´n I, de las Rivas B, Matesanz R, Mun˜oz R, Lo´pez de Felipe F. Molecular adaptation of Lactobacillus plantarum WCFS1 to gallic acid revealed by genome-scale transcriptomic signature and physiological analysis. Microb Cell Fact. 2015;14:160. Whiting GC. Some biochemical and flavour aspects of lactic acid bacteria in ciders and other alcoholic beverages. In: Carr JG, Cutting CV, Whiting GC, eds. Lactic Acid Bacteria in Beverages and Food. London: Academic Press; 1975:69
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36. Whiting GC, Coggins RA. A new nicotinamide-adenine dinucleotide-dependent hydroaromatic dehydrogenase of Lactobacillus plantarum and its role in formation of (2)t-3,t-4-dihydroxycyclohexane-c-1-carboxylate. Biochem J. 1974;141:35
42. 37. Romero C, Garcı´a P, Brenes M, Garcı´a A, Garrido A. Phenolic compounds in natural black Spanish olive varieties. Eur Food Res Technol. 2002;215:489
496. 38. Ciafardini G, Marsilio V, Lanza B, Pozzi N. Hydrolysis of oleuropein by Lactobacillus plantarum strains associated with olive fermentation. Appl Environ Microbiol. 1994;60:4142
4147. 39. Marsilio V, Lanza B, Pozzi N. Progress in table olive debittering: degradation in vitro of oleuropein and its derivatives by Lactobacillus plantarum. JAOCS. 1996;73:593
597. 40. Marsilio V, Lanza B. Characterization of an oleuropein degrading strain of Lactobacillus plantarum Combined effects of compounds present in olive fermenting brines (phenols, glucose and NaCl) on bacterial activity. J Sci Food Agric. 1998;76:520
524. 41. Landete JM, Curiel JA, Rodrı´guez H, de las Rivas B, Mun˜oz R. Aryl glycosidases from Lactobacillus plantarum increase antioxidant activity of phenolic compounds. J Funct Foods. 2014;7: 322
329. 42. Sestelo ABF, Poza M, Villa TG. β-Glucosidase activity in a Lactobacillus plantarum wine strain. World J Microbiol Biotech. 2004;20:633
637. 43. Landete JM, Rodrı´guez H, de las Rivas B, Mun˜oz R. High-addedvalue antioxidants obtained from the degradation of wine phenolics by Lactobacillus plantarum. J Food Prot. 2007;70:2670
2675. 44. Nakayama T, Ono K, Hashimoto K. Affinity of antioxidant polyphenols for lipid bilayers evaluated with a liposome system. Biosc Biotechnol Biochem. 1998;62:1005
1007. 45. Curiel JA, Mun˜oz R, Lo´pez, de Felipe F. pH and dose-dependent effects of quercetin on the fermentation capacity of Lactobacillus plantarum. LWT—Food Sci Technol. 2010;43:926
933. 46. Arvanitoyannis IS, Kassaveti A, Stefanatos S. Current and potential uses of thermally treated olive oil waste. Int J Food Sci Technol. 2007;42:852
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845.

Chapter 13

Microbial colonization of naturally fermented olives E.Z. Panagou1, C.C. Tassou2 and G.-J.E. Nychas1 1

Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, School of Food and Nutritional

Sciences, Agricultural University of Athens, Athens, Greece, 2Hellenic Agricultural Organization DEMETER, Institute of Technology of Agricultural Products, Lykovrissi, Greece

Abbreviations CFU IOC LAB SEM

colony forming units International Olive Council lactic acid bacteria scanning electron microscopy

13.1 Introduction Fermentation is one of the oldest food processing/preservation technologies known to mankind, which is considered as an important determining factor to control microbial growth, improve digestibility and nutritional value of food, and enhance food safety.1,2 The popularity of fermented foods can be attributed to their extended shelf life, increased safety record, nutritional properties, and functional potential due to the presence of active microorganisms that belong to the technological microbiota. It has been suggested that the systematic consumption of these products could improve in the long run gastrointestinal health and provide other health benefits.3,4 Nowadays, fermentation processes associated with animal products (e.g., meat, fish, and dairy produce) have become increasingly sophisticated, in contrast with the fermentation of vegetables, particularly olives, which remains craft-based and empirical. This is surprising as the economic importance of olives in the European Union is undisputable, with c. 30% of world production originating from the member countries such as Spain, Italy, and Greece.5 After harvesting, olives are transported to the factory, sorted, washed, and finally brined in a salt solution.6 Currently, both green and black olives are fermented “naturally” on an industrial scale, without the addition of lactic acid bacteria (LAB) starter cultures. Natural (or spontaneous) fermentations typically result from the competitive activities of the indigenous microbiota,

together with a variety of contaminating microorganisms from fermentation vessels, pipelines, pumps, and other equipment in contact with the olives and brine.7,8 Those microorganisms best adapted to the food substrate and to technical control parameters eventually dominate the process. Today, fermentation control is limited to the maintenance of the olive ecosystem. In general the ecosystem in olives is influenced by the microbial association of harvested olives in the field,9
11 the range of intrinsic factors inherent to olives such as pH, water activity, availability of nutrients diffusing from the tissue, the structure of olive skin, the levels of indigenous antimicrobial compounds (phenolic compounds and organic acids),12 and the extrinsic factors such as temperature, storage conditions, and type of packaging.13,14 These intrinsic and extrinsic factors can influence the rate as well as the type of metabolism of the developing microbial association and its response to the external environment.15 The colonization (e.g., the spatial distribution of microorganisms on olives) is of great importance since the epidermis of the fruits is covered by a thick cuticle,16 which not only restricts the flow of nutrients from the fruit into the brine but also hinders the penetration of microorganisms from the brine into the product. The present review focuses on the topography (spatial distribution) of the population of yeasts and specific groups of bacteria, as well as on the effect of microbial colonization on various physicochemical characteristics of the fermented product.

13.2 Microbiota of olives Pseudomonas, Enterobacteriaceae, LAB, and yeasts/fungi are the principal members of the microbial association isolated from olive fruits (Table 13.1). The various

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00040-7 © 2021 Elsevier Inc. All rights reserved.

145

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PART | 1 General Aspects of Olives and Olive Oil

TABLE 13.1 Mean values (log10 CFU/g), range (min/max) of microorganisms of olive microbiota, and number of surveyed samples. Microorganism

Number of samples

Mean value

Range

Pseudomonads

31

6.08

4.0
8.50

Yeasts
Fungi

35

5.92

4.0
8.84

Lactic acid bacteria

26

5.03

3.6
7.35

Enterobacteriaceae

15

4.24

0.0
9.47

Pseudomonads

42

1.05

0.0
5.63

Yeasts
Fungi

42

4.95

2.7
6.41

Lactic acid bacteria

42

3.04

0.0
6.37

Enterobacteriaceae

42

0.68

0.0
4.32

First year

Second year

Microbial groups isolated from raw olives.9

bacterial and yeast groups isolated from olive fruits are shown in Tables 13.2 and 13.3, respectively.

13.2.1 Microbial diversity of raw olives The origin of this microbial flora can be traced back to olive leaves (Table 13.4) as well as on stored olives (Table 13.5) and physiological condition of the fruits (healthy or damaged olives) (Table 13.6). Pseudomonas savastanoi is a major microorganism, which has been studied in detail, causing the endemic disease olive knot or tubercle, which occurs in most regions of the world where the olive tree (Olea europaea) is cultivated. The disease takes its name from the woody outgrowths found most frequently on young stems and on branches and twigs as a consequence of wound infections. Extensive studies show that knot development involves colonization of the injection site, development of lysogenous cavities, and finally abnormal enlargement and proliferation of the host cells starting from the bacterial tissue. The development patterns of hypertrophy and hyperplasia suggest that they are induced by a substance with cytokinin-like activity, synthesized by P. savastanoi.18 The disease is related to a reduction of both olive yield and quality. P. savastanoi causes a similar disease on other plants, including Fraxinus excelsior (ash), Nerium oleander, and Ligustrum japonicum. Mesophilic heterotropic, aerobic, or facultative anaerobic bacteria were isolated and characterized by the leaves of olive trees of different ages for a period of 5 years.23 From the identified bacterial species a rich microbial biodiversity was revealed, which is dominated by Pseudomonas syringae (51%), followed by Xanthomonas campestris (6.7%), Erwinia herbicola (6%), Acetobacter aceti (4.7%),

Gluconobacter oxydans (4.3%), Pseudomonas fluorescens (3.9%), Bacillus megaterium (3.8%), Leuconostoc mesenteroides spp. dextranicum (3.1%), Lactobacillus plantarum (2.8%), Curtobacterium plantarum (2.2%), Micrococcus luteus (2.2%), Arthrobacter globiformis (1.4%), Klebsiella planticola (1.2%), Streptococcus faecium (1.2%), Clavibacter spp. (0.98%), Micrococcus spp. (0.82%), Serratia marcescens (0.81%), Bacillus subtilis (0.57%), Cellulomonas flavigena (0.4%), Erwinia spp. (0.37%), Zymomonas mobilis (0.3%), Bacillus spp. (0.29%), Alcaligenes faecalis (0.27%), Erwinia carotovora (0.08%), and Pseudomonas aeruginosa (0.04%). It is characteristic that the bacterial communities on olive leaves at a given time and age presented similar structure, but higher variability was observed for olive leaves of different ages sampled in the same year and for olive leaves of the same age sampled in different years. In a recent work the phyllosphere and carposphere of bacterial communities in olive trees subjected to two different soil management systems (sustainable and conventional) have been reported.11 Amplified DNA fragments of the 16S ribosomal RNA eubacterial gene (16S rRNA) of bacteria sampled from the surface of olive leaves and fruit were subjected to denaturing gradient gel electrophoresis. Results showed that the DNA sequences of isolated bacteria belonged to the phyla Proteobacteria, Actinobacteria, and Firmicutes. The most abundant bacteria belonged to the Enterobacteriaceae family, which is not unusual since this microbial group dominates on the aerial surfaces of plants as well as within healthy plant tissues and seeds. The bacterial species identified from raw olive fruit pulps (mesocarp) were Rahnella aquatilis, Kluyvera intermedia, Hafnia/Rahnella alvei, Serratia

Microbial colonization of naturally fermented olives Chapter | 13

147

TABLE 13.2 Bacterial groups isolated from raw olives.

TABLE 13.3 Yeasts isolated from olive fruits.

Pseudomonas spp.

Leuconostoc

Leucosporidium spp.

Savastanoi

Mesenteroides

Brettanomyces spp. Anomalus

Fluorescens

spp. mesenteroides

Metschnikowia pulcherrima

Aeruginosa

Dextranicum

Putida

Lactobacillus

Luteola

Delbrueckii

Candida Guilliermondi Olea Parapsilopsis var. intermedia Tenuis Utilis

Cepacia

Helveticus

Bacillus

Acidophilus

Subtilis

Casei spp. rhamnosus

Cryptococcus Albidus var. albidus Laurentii var. laurenti Macerans

Rhodotorula Aurantiaca Glutinis var. glutinis Mucilaginosa Rubra

Debaryomyces Hanseii Nicotianae

Saccharomyces Bayanus Cerevisiae var. ellipsoideus Chevalieri Exiguus Fermentati Globosus Italicus Kluyveri Oleaceus Rosei Rouxii Uvarum

Endomycopsis vini

Schizosaccharomyces spp. Octosporus

Hanseniaspora Osmophila Uvarum Valbyensis

Sporobolomyces roseus

Hansenula Anomala var. anomala Anomala var. schneggii Holstii

Torulopsis Candida Glabrata Holmi Magnoliae Stellata

Kloeckera Apiculata Corticis

Trichosporon pullulans

Kluyveromyces veronae

Pichia Fermentans Membranefaciens Pinus Polymorpha Terricola Vini var. vini

Megaterium

Casei spp. alactosus

Serratia marcescens

Xylosus

Erwinia carotovora

Plantarum

Klebsiella

Curvatus

Pneumoniae

Fermentum

Planticola

Brevis

Acetobacter aceti

Hilgardii

Arthrobacter globiformis

Leichmannii

Xanthomonas campestris

Coryniformis spp. coryniformis

Gluconobacter oxydans

Aeromonas spp.

Enterococcus faecium

Sobria

Zymomonas mobilis

Hydrophila

Cellulomonas flavigena Bacteria isolated from raw olives. Source: Adapted from Tassou CC. Microbiology of Olives With Emphasis on the Antimicrobial Activity of Phenolic Compounds [Ph.D. thesis]. University of Bath; 1993 and Panagou EZ. Fermentation, Preservation and Microbial Ecology of Fermented Table Olives [Ph.D. thesis]. Agricultural University of Athens; 2002.17

spp., Pantoea spp., Enterococcus spp., Curtobacterium spp., Methylobacterium spp., Frondihabitans suicicola, and Averyellaa dalhousiens. In the same work, it was also reported that insects could have a major contribution in the composition of plant-associated bacterial communities. Thus plant bacteria could use insects as vectors of dispersion, although it needs further elucidation whether the bacterial strains isolated from the digestive tract of insects originate from plants, as in the case of Serratia spp., or it is the opposite, as for Enterococcus spp. The diversity of bacterial communities in the olive tree phyllosphere was determined recently for two Portuguese olive tree cultivars using a culture-dependent approach taking into account the effect of cultivar and plant organ (leaf vs twig).24 The authors reported that the dominant phyla

Yeast species isolated from olives.9

were Proteobacteria, Actinobacteria, and Firmicutes. Bacterial diversity was impacted by tree cultivar and, to a lesser extent, by plant organs. It is characteristic that tree cultivar affects in a similar way the composition of the

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PART | 1 General Aspects of Olives and Olive Oil

TABLE 13.4 Bacteria isolated from olive leaves in two different years. Bacteria

Distribution (%) 1979

1991

67.86

51.00

Pseudomonas Savastanoi spp. syringae Delafieldi

0.11

Fluorescens

1.06

3.90

Aeruginosa




0.04

Bacillus spp.




0.29

Subtilis

0.34

0.57

Megaterium

4.02

3.80

Serratia marcescens

1.34

0.81

Erwinia spp.




0.37

Carotovora




0.08

Pantoea 8.50

Agglomerans

6.00

Klebsiella 1.40

Pneumoniae




Planticola

1.45

Lactobacillus plantarum


1.20

diversity associated with leaves, flowers, and fruits. This diversity was investigated by metabarcoding analysis of the ITS2 region followed by phylogenetic analysis of relevant genera with validated reference sequences.10 Results indicated that Ascomycota was the dominant phyla (93.6% of detected sequences) followed to a much lower extent by Basidiomycota (2.8%) and unidentified fungi (3.6%). With regard to plant pathogenic fungi, Colletotrichum spp. was the most abundant on ripe olive fruits represented by C. karstii, C. acutatum, and C. godetiae. High frequency was also observed for Pseudocercospora cladosporioides particularly in olive leaves compared to ripe fruits, whereas low isolation frequencies were recorded for other putative pathogens, including Fusarium spp., Alternaria spp., and Neofusicoccum spp. Among nonpathogenic species, Aureobacidium spp., Cladosporium spp., and Devriesia spp. were mostly represented. Higher incidence of these genera was recorded in the phyllosphere than in the carposhpere, indicating possible competitive action against fungal plant pathogens. Aureobacidium pullulans was one of the most abundant species in the olive canopy. This yeast-like fungus has been reported as one of the main colonizers of the phyllosphere and carposphere in different plant species, and it has been widely exploited as a biocontrol agent against other pathogenic fungi.25

2.80

Leuconostoc 1.12

Dextranicum




Mesenteroides


ssp. dextarnicum

3.10

Acetobacter aceti

1.23

4.70

Arthrobacter globiformis

2.07

1.40

Micrococcus luteus

3.63

2.20

Xanthomonas compestris

3.35

6.70

Gluconobacter oxydans

4.30

Curtobacterium plantarum

2.20

Enterococcus faecium

1.20

Clavibacter sp.

0.98

Micrococcus sp.

0.82

Cellulomonas flavigena

0.40

Zymomonas mobilis

0.30 0.27

Alcaligenes faecalis 9

Bacterial species isolated from olive leaves.

endophytic and epiphytic bacterial communities, whereas the plant organ has greater influence on epiphytic than on endophytic bacterial community structure. Apart from bacteria, olives are characterized by an extended fungal

13.2.2 Microbiota of olives related to olive oil production Early reports indicated the presence of microorganisms, primarily yeasts but also bacteria and molds, to a lesser extent, from heaps of pits (endocarps) from olive oil mills,19 whereas bacteria, yeasts, and molds have been isolated from the site of oviposition of Bactrocera oleae on olives (Table 13.5).22 During olive oil production, there is a close relationship between the composition of the microbiota on olives’ carposphere and that of newly extracted olive oil. Recent research has revealed that the microbiota of olive oil consists primarily of yeasts and fungi to a lesser extent.26 In a recent work the composition of microbiota during cv. Taggiasca extra-virgin olive oil production was investigated at different stages of production (wash water, olive paste malaxation, and extracted olive oil).27 The predominant yeast species identified were Kluyveromyces marxianus, Candida oleophila, Candida diddensiae, Candida norvegica, Wickerhamomyces anomalus, and Debaryomyces hansenii. The former yeast species was found only in the wash water samples whereas all other species were isolated from all three production stages. It needs to be noted that after six months storage of olive oil at room temperature only W. anomalus could be detected, indicating

Microbial colonization of naturally fermented olives Chapter | 13

149

TABLE 13.5 The microbiology of olives; stored olives (A), oviposition sites of Bactrocera oleae (B), and heaps of pits (C). A

B

C

Rhizopus nigricansa Actinomucora Oosporaa Aspergillus glaucusa Fusidiuma Geotrichum candiduma Penicillium purpurescensa Alternaria tenuisa Cheiranthia Pullalaria pullulansa Cladosporium avellaneuma Fusarium vasinfectuma

nd

Saccharomyces cerevisiaea Pichia fermentans Debaryomyces Kloeckeria Sybglobosusa Candida guillermondi var. membranefaciensa Intermediaa Parapsilopsisa Parapsilopsis var. intermediaa Pulcherrimaa Tenuisa Rhodotorula glutinisa Mucilaginosaa Trichosporon behrendii Cryptococcus laurentii

Rhodotorula mucilaginosaa Candida lipolyticaa Guillermondiia Parapsilopsis var. intermediaa Trichosporon

Micrococcus roseusa Flavusa Sarcina luteaa Bacillus cereusa Brevisa Megateriuma Pumilusa Subtilisa

nd

Molds Rhizopus Aspergillus Flavusa Glaucus Nigera Sydowia Terreusa Versicolora Fusarium Penicillium Notatuma Purpurogenum Alternaria Yeasts Pichia Fermentans Membranefaciens Saccharomyces Italicus Elegans Candida Cruseia Parapsilopsis var. intermediaa Trichosporon sericuma

Bacteria Aerobacter Escherichia Serratia marcescensa Plymuthicuma Pseudomonas aeruginosaa Achromobacter Bacillus subtilis Megaterium Cereus

Microflora isolated from different parts of olives. nd: Not detected. a Lipolytic strains. Source: Adapted from Verona O, Valleggi M. Il problema della conservazione delle sanse de oliva. Olearia. 1949;3:63919; Gonza´lez-Cancho F. Investigaciones sobre la conservacion de aceituna de molino. III. Poblacion microbiana de los trojes. Grasas Aceites. 1957;8:55; Gonza´lez-Cancho F. Investigaciones sobre la conservacion de la aceituna de molino. IV. Poblacion microbiana de los trojes. Grasas Aceites. 1957;8:25820,21; and Picci G. Ancora sulla microflora presenta nelle olive colpite de Dacus oleae. Ann Facol Agric. 1959;20:65.22

that the habitat of olive oil has a strong selective activity on yeast biota, allowing only few yeast species to survive during storage. It is also characteristic that a relatively high population of bacteria could be detected in wash water samples ranging from 3.77 to 4.30 log CFU/ g. However, bacteria were completely absent in the olive paste after malaxation indicating that this processing stage has a strong impact on the selection of the

surviving microorganisms coming from olives, eliminating mainly the bacterial component.

13.2.3 Microbiota of olives related to fermentation The natural fermentation of black and green olives involves a complex microbiota of LAB, yeasts,

150

PART | 1 General Aspects of Olives and Olive Oil

TABLE 13.6 The influence of the physiological condition of olives (damaged or healthy, green or black) and leaves on the microbiota in two different years. First year Microorganisms Olives

Yeasts

Lactic acid bacteria

Pseudomonads

Black healthy

5.9a (5b)

4.92 (4)

6.10 (4)

Green healthy

4.92 (4)

4.62 (5)

5.46 (3)

Black damaged

6.57 (10)

5.51 (8)

6.54 (10)

Green damaged

6.51 (7)

4.92 (2)

6.41 (7)

Leaves

5.08 (6)

4.56 (5)

5.27 (6)

ns

ns

c

F-Test

Second year Microorganisms Olives

Lactic acid bacteria

Yeasts

Green healthy

2.86 (22)

4.74 (19)

Geen damaged

3.26 (17)

4.39 (23)

Leaves

3.72 (15)

5.66 (15)

F-Test

ns

d

Microbial association of olive leaves and fruits (Tassou, 1993).9 ns: Not significant. a log10 CFU/g. b Number of samples surveyed. c Significant difference at 5% level. d Significant difference at 1% level.

(A)

FIGURE 13.1 Changes in (A) total viable counts (&), lactic acid bacteria (x), Pseudomonas spp. (W), Enterobacteriaceae (X), yeasts (%) and pH (B) in black olive fermentation. Population dynamics in olive fermentation.44

(B)

7

7 6

6 4

pH

log CFU/mL

5

3 5 2 1 4

0 0

8

16 24 32 40 48 56

0

8

16 24 32 40 48 56

Days

Pseudomonas, Staphylococcus, Clostridium and Gramnegative bacteria (Enterobacter, Citrobacter, Aeromonas, Escherichia, Klebsiella), and occasionally molds.28
31 The population dynamics of selected microbial groups in the brine during fermentation is well-established in the literature. The growth of Enterobacteriaceae is observed in the brine at the

onset of fermentation, but during the process the population of this microbial group decreases gradually, and it is not detected in the brine at the end of the process (Fig. 13.1). In addition, Pseudomonas and Clostridium can be found in the brine, but both microorganisms cannot survive in the brine due to the low pH value attained at the end of the process. So

Microbial colonization of naturally fermented olives Chapter | 13

these bacteria are not a serious problem in table olive fermentation under normal processing conditions. Finally, LAB and yeasts hold a substantial role in table olive fermentation and could be considered as the driving force of the process (Table 13.7). These groups have been reported previously for both green Conservolea and black Kalamata and Conservolea olives,32
34 as well as in the fermentation of green and black olives from Spanish, Italian, and Turkish varieties.35
39 Nowadays, the application of molecular methods based on both culture-dependent and culture-independent approaches has revealed that the microbial ecology of table olive fermentation in both spontaneous and inoculated processes is more complex, and the role of LAB and yeasts is of paramount importance to obtain high-quality products.40
43 The presence of aggregates of yeasts and bacteria on the surface and within fermented olives has been studied by scanning electron microscopy (SEM).44 On the surface of the olive epidermis, areas with a coating of epicuticular wax provide a site of attachment for both yeasts and bacteria (Fig. 13.2A). Very few microorganisms are present on the smooth epidermis (e.g., around stoma, Fig. 13.2B), although it is possible that they are removed during sample preparation. Each stomal opening is plugged with a mixed microbial colony, primarily of yeasts of elongate morphology, but also bacteria (Fig. 13.3A). The epidermis is easily detached from the mesocarp, allowing the microbial association in the substomal cavity to be observed. Yeasts of both spherical and elongate forms are present on the underside of the stomal opening, which is rich in epicuticular wax (Fig. 13.3A). The intercellular spaces of substomal cells are packed with bacteria in colonies (Fig. 13.3B). Microorganisms could not be detected deeper in the olive mesocarp.

13.2.4 Biochemical characteristics of microbial association 13.2.4.1 Effect on olive oil There is considerable information about the microbiota, especially its lipolytic activity on waste products from olive extraction mills. This lipolytic activity, especially with respect to determinant effect on the olive oil, has been studied in detail. Oil hydrolysis mediated by lipases and the resultant oxidation (rancidity) affects mainly the organoleptic properties of the final product. Olive fruit contains both a lipase and lipoxidase, the latter occurring in the endocarp.9 According to this researcher, the increase in titratable acidity of olive oil during storage is attributed to enzymic action rather than autocatalysis. Concurrently, Borbolla et al.46 could not verify the presence of a lipase or a lipoxidase in healthy olive. Consequently, they doubted the presence of these enzymes in nature and concluded that the lipolytic

151

TABLE 13.7 Microbial association during different fermentation stages of olives. Lactic acid bacteria

Spoilage microorganisms

Primary stage Streptococcus spp.

Aeromonas spp.

Pediococcus spp.

Aerobacter spp.

Leuconostoc mesenteroides

Enterobacter spp.

Leuconostoc dextranicum

Escherichia coli

Lactobacillus brevis

Pseudomonas spp.

Leuconostoc citreum

Bacillus polymyxa

Lactobacillus coryniformis

Bacillus macerans Clostridium spp. Flavobacterium spp.

Intermediate stage L. mesenteroides Lactobacillus plantarum L. brevis L. citreum L. coryniformis Final stage L. plantarum

Candida boidini

L. brevis

Pichia manshurica

Lactobacillus pentosus

Pichia kudriavzevii

Lactobacillus buchneri

Hansenula anomala

Lactobacillus paracasei

Rhodotorula minuta

L. mesenteroides

Rhodotorula rubra

L. citreum

Rhodotorula glutinis

L. coryniformis

Saccharomyces kluyveri Debaryomyces hansenii Saccharomyces oleaginosus Wickerhamomyces anomalus Cellulomonas spp. Propionibacterium spp.

Microbial association in olive fermentation. Source: Adapted from Garrido-Ferna´ndez A, Ferna´ndez-Dı´ez MJ, Adams MR. Table Olives: Production and Processing. London: Chapman & Hall; 1997; Panagou EZ. Fermentation, Preservation and Microbial Ecology of Fermented Table Olives [Ph.D. thesis]. Agricultural University of Athens; 2002; Arroyo-Lo´pez FN, Romero-Gil V, Bautista-Gallego J, et al. Yeasts in table olive processing: desirable or spoilage microorganisms?. Int J Food Microbiol. 2012;160:42
4945; Heperkan D. Microbiota of table olive fermentations and criteria of selection for their use as starters. Front Microbiol. 2013;4:143; Lanza B. Abnormal fermentation in tableolive processing: microbial origin and sensory evaluation. Front Microbiol. 2013;4:Article 91.

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PART | 1 General Aspects of Olives and Olive Oil

FIGURE 13.2 Micrograph of the microflora on the epidermis of naturally fermented black olives: (A) yeasts and bacteria form a biofilm in areas with epicuticular surface wax and (B) yeasts and bacteria in stomal aperture. Micrograph of olive epidermis.44

FIGURE 13.3 Micrograph of the microflora inside naturally fermented black olives: (A) yeasts and bacteria below a stomal opening and (B) bacteria colonizing the spaces between substomal cells. Microflora inside olives.44

enzymes were derived from microorganisms that grew in the olive paste at various stages of processing or even in the oil produced thereafter. However, despite the presence of endogenous lipase and lipoxidase in the olive fruit, many microorganisms growing on stored olives before processing, in the olive paste before extraction, on olives attacked by B. oleae, or in the final product are commonly lipolytic as suggested by previous studies.47
50 Lipolytic activity of microbial origin increases the acidity of oil produced from pits vis-a`-vis the virgin material. The yeasts isolated by these researchers are listed in Table 13.5. In 1954, Verona isolated a new genus Trichosporon spp. from pit samples from olive oil factories that produce lipase and split olive oil to glycerin and free fatty acids.51 Moreover, Tassou9 reviewed the microbiota at various stages of oil processing. She reports that molds, bacteria, and yeasts are mainly lipolytic, while the majority of molds isolated from heaps of pits produce lipase. Gonza´lezCancho20 isolated molds, yeasts, and bacteria from olives stored in heaps prior to oil production (Table 13.5). He noted that Serratia and Pseudomonas are the most lipolytic genera followed by Trichosporon and Pichia. The remaining microorganisms were lipase negative. In another study, Gonza´lez-Cancho observed geographical differences in the composition of the microbiota and isolated new species of bacteria, yeasts, and molds.21 The lipolytic strains are presented in Table 13.6. He concluded that the majority of the isolates produce lipase with the exception of Pichia fermentans, a strain of Cryptococcus laurentii, Trichosporon behrendii, and some fungal species of Penicillium and Alternaria. In another work, Gracia´n et al.52 analyzed chemically samples from olive pits, the same heap being sampled at various times before processing. They determined the quantity and quality of the oil by physical and chemical methods and found that by the time hydrolysis of fatty substances had occurred, a decrease in weight had also occurred. Both are attributed to microorganisms growing in and on the surface of the heap, splitting the glycerides to glycerin and free fatty acids. Microorganisms use the hydrolysis products for growth, thereby causing not only deterioration of the quality but also loss in the yield of the oil. The softening of olive is attributed partly to endogenous and microbial pectinolytic enzymes. The presence of pectinolytic enzymes, pectin methylesterase, and polygalacturonase has been confirmed in certain olive varieties,53
55 as well as the presence of a natural polygalacturonase inhibitor.56 The types of pectic enzymes and the changes they cause in pectic substances during ripening of the fruit are important and must be considered to determine the appropriate time of harvest and processing of certain table olive cultivars. The lipolytic enzymes (lipases) produced by many microorganisms on the raw product are found in the final product even after ultra high temperature (UHT) sterilization, and

Microbial colonization of naturally fermented olives Chapter | 13

consequently, they can cause spoilage of the sterile product. To act optimally the lipase needs an olive oil of pH 8.0-8.5 and a temperature of 40 C.57,58 There is high probability also of these enzymes surviving processing during the malaxation process of olives (carried out at 35 C-40 C) before oil extraction. Thus they can be transferred to the olive oil where they exacerbate the development of oxidative rancidity. Lipases from Candida rugosa,59 Saccharomycopsis lipolytica, and Micrococcus caseolyticus can hydrolyze olive oil to a great extent.60 The hydrolysis by the lipase of S. lipolytica is highly specific for oleic acid while that of M. caseolyticus is totally nonspecific.60 Olive oil stimulates growth of Achromobacter lipolytica, thereby increasing lipase production and hydrolysis.61 The same is observed for Pseudomonas mephitica.62 Olive oil stimulates lipase production initially by enhancing growth of Geotrichum candidum, but eventually the release of glycerol from the triglycerides diminishes lipase production.63,64 Lipases of Pseudomonas fragi, Staphylococcus aureus, Aspergillus niger can also hydrolyze olive oil.65
67 Small amounts of oleic acid activate, but higher concentrations, inhibit the lipase of Candida lipolytica.68 In a recent work, yeast biodiversity from oleic ecosystems (fresh olive fruits, olive paste, olive pomace) from Arbequina and Cornicabra varieties was investigated using molecular identification methodology together with their biotechnological properties.49 Fourteen different yeast species were identified in all belonging to seven genera, namely, Zygosaccharomyces spp., Pichia spp., Lachancea spp., Kluyveromyces spp., Saccharomyces spp., Candida spp., and Torulaspora spp. The most commonly isolated species were Pichia caribbica, Zygosaccharomyces fermentati, and Pichia holstii, followed by Pichia mississippiensis, Lachancea sp., Kluyveromyces thermotolerans, and Saccharomyces rosinii. In addition, the biotechnological properties of these yeast species were identified in terms of enzymatic activity (β-glucosidase, β-glucanase, carboxymethylcellulase, polygalacturonase, peroxidase, and lipase). It was reported that none of the isolated yeast species presented lipase activity, a few showed cellulose and polygalacturonase activities, whereas the majority presented β-glucosidase, β-glucanase, and peroxidase activities. The authors concluded that the quality of the olive oil could be affected by the presence of some yeasts with high enzymatic activities such as lipases, glucanases, cellulases, glucosidases, and polygalacturonases, whereas the presence of these strains in the olive byproduct (i.e., olive paste and olive pomace) could have potential application in industrial biotechnology for the production of enzymes. In another work undertaken by Italian researchers, it was demonstrated that the lipase activity of certain lipaseproducing yeasts in olive oil could be modulated by the water and polyphenol content of olive oil.69 Specifically, the inoculation of olive oil with the yeast species Candida

153

adriatica, C. diddensiae, and Yamadazyma terventina resulted in substantial increase in free fatty acidity, especially when olive oil was characterized by high water content and low polyphenol concentration. From the collected experimental data, it was inferred that the increasing content of total polyphenols could reduce the percentage of yeasts able to hydrolyze the triacylglycerols. The authors concluded that the quality of olive oil could be improved by controlling certain parameters such as the content of polyphenols and the separation of olive oil during the sedimentation stage from the settled material that is rich in water and microorganisms and could negatively affect the quality of the product. A positive contribution of yeasts could be related to the decrease of bitter taste of newly produced olive oil due to the enzymatic hydrolysis of oleuropein by β-glucosidase.70 These researchers showed that the yeasts Saccharomyces cerevisiae and Candida wickerhamii could hydrolyze oleuropein enzymatically through the production of β-glucosidase by these species. In addition the absence of lipases from these yeasts could have a positive impact on olive oil production by improving the sensory quality without affecting the composition of triglycerides.

13.2.4.2 Effect on fermentation The main metabolic products with significant presence in the brine during green and black olive fermentation are organic acids, namely, lactic, acetic, citric, tartaric, malic, and succinic acids.33,71 The accumulation of organic acids in the brine results in a gradual pH decrease to a value of 4.4 or lower (Fig. 13.1). which is necessary to ensure the microbiological safety of the fermented product during storage, distribution, and retailing. According to the Trade Standard Applying to Table Olives of the International Olive Council (IOC),72 natural olives should be characterized by 6% minimum NaCl concentration, 4.3 maximum pH value, and 0.3% minimum titratable acidity (expressed as lactic acid). LAB with homo and heterofermentative metabolism are the most important bacterial group in olives and could be characterized as the driving force of fermentation. The presence of Lactobacillus spp., Leuconostoc spp., and Pediococcus spp. is commonly reported in most table olive preparations.73,74 Their metabolic products contribute to the development of aroma and flavor sensory attributes, acidification of fermenting brines, and production of antimicrobial compounds and bacteriocins. Yeasts are also an important microbial group of the fermentation process that produces desirable volatile compounds and other metabolites that improve the organoleptic properties of the final fermented product, enhance the growth of LAB, and biodegrade phenolic compounds.75 Fermentation of black olives at high salt concentration is slow and further hampered by the availability of

154

PART | 1 General Aspects of Olives and Olive Oil

nutrients needed to initiate fermentation. The olive cuticle acts as a barrier to the diffusion of solutes into and out of the olive fruit, as there is no lye pretreatment to disrupt the cuticle, thus exchange can only take place through apertures on the surface of the fruit (stomata). Consequently, the diffusion of nutrients into the brine and the debittering of olives are slow, and fermentation is rather long. In fact the presence of residual fermentable material could be detected in the brine during the entire fermentation period.2 Substances that are leached from the olives include inorganic nutrients (minerals), sugars, pectic substances, sugar alcohols, vitamins, alkaloids, and phenolic compounds.9,47 In the early stages of olive fruit development, gaseous exchange takes place through the stomata, but later on some become occluded with wax, which is removed during the washing procedure before fermentation.76 The stomata of olive leaves are considered to be the site of entry of bacterial suspensions applied as sprays,77 and it is possible that some of the microorganisms on ripe olives might be associated with the stomata as well as the skin surface. Epicuticular waxes, present as a bloom on the surface of ripe olives, are known to influence the wettability of plant leaves78 and provide initially a site for microbial attachment and a substrate on which biofilms of yeasts and bacteria develop during fermentation. By the end of fermentation, yeasts and LAB are the dominant microorganisms in the brine. Our observations of the olives themselves show that there is a spatial differentiation between the two groups.17,44 Specifically, yeasts tend to predominate on the skin surface and right under the stomatal openings, whereas bacteria predominate in the intercellular spaces of the substomal cavities (Fig. 13.3). The spatial distribution of LAB and yeasts in olive stomatal openings has been verified in a recent work for cv. Conservolea natural black olive fermentation.79 In this work a mixed aggregation of LAB and yeasts embedded in extracellular polymeric matrix was observed in the area of stomata, whereas no adherent microorganisms could be observed on the surface of olives. Similar LAB/yeast aggregates have been reported in the case of Spanish-style green olive processing.80,81 However, in this processing style the spatial distribution of microorganisms was not restricted in stomatal openings, but it covered the whole surface of olives. This could be attributed to the fact that in Spanish-style processing the cuticle is disrupted by lye treatment, and hence, release of nutrients from olives takes place through the whole surface of the fruits. Consequently, microorganisms are in direct contact with the source of nutrients, and this could have a significant contribution in the development of microbial aggregates. In the light of these observations, it can be assumed that table olive fermentation consists of two different microbial ecosystems: one located in the

brine where microorganisms grow planktonically and the other on the surface of olives where microorganisms grow in the form of immobilized cells forming biofilms. These findings indicate that sampling of the brine alone will tend to underestimate the numbers of microorganisms present, and thus microbiological analyses should focus on the olives, instead of the brine, in order to describe more accurately the population dynamics during fermentation. Similarly, pH and organic acids levels in the brine may not be representative of the local conditions in the mesocarp. Vegetable tissue could buffer changes in pH in food materials, and the pH in the center of microbial colonies can be lower than at the edge.15,82,83 Organic acid end products are also affected by the extrinsic and intrinsic factors such as oxygen limitation, glucose and salt concentrations, and low pH prevailing at the beginning and end of fermentation.32,84,85

13.2.4.3 Dry-salted olives An important type of black olives is the so-called drysalted olives. The fruits are harvested at full ripeness and processed by coarse salt without adding any brine. As a result of the high osmotic pressure exerted by the salt, the fruits become gradually debiterred, shriveled, and eatable. The final physicochemical characteristics of the product are water activity (aw), 0.75
0.85; pH, 4.5
5.5; oil content, 35%
40%; moisture, 30%
35%; reducing sugars, 2.0%
2.5%; NaCl content, 4%
10%.86 Despite the relatively high pH, compared with fermented olives in brine, the low water activity/high salt content can ensure its microbiological safety during storage, although potential spoilage microorganisms, mainly fungi, can grow in these conditions.87 For this reason the Trade Standard Applying to Table Olives by the IOC defines a minimum salt concentration of 10%, expressed as 1 g of NaCl per 100 mL olive juice, to ensure the microbiological safety of this trade preparation.72 The initial microbiota consists of LAB, yeasts, pseudomonads, and enterobacteria (Table 13.8). Within the first 20 days, yeasts become the dominant species whereas no other microbial group survives for more than 20 days due to the low water activity of the product at that period (Fig. 13.4). The dominant yeast species is Candida famata, a yeast species that is characterized by high salt tolerance and is able to grow in sodium chloride concentrations up to 2.5 M.88 In another work the main microbiota of Thassos cv. black olives subjected to hot air dehydration under mild conditions (40 C for 24 h) consisted of yeasts, whereas LAB, Enterobacteriaceae, S. aureus, Bacillus spp., and Clostridium spp. were undetectable.89 The absence of LAB and the presence of yeasts were also confirmed by Spanish researchers during the debittering process of dry-salted olives of Manzanilla variety.90 However, in this work a high population of

Microbial colonization of naturally fermented olives Chapter | 13

155

TABLE 13.8 Microbial association (log10 CFU/g) during dry salting of black olives. Microorganism

Dry salting period (days) 0

20

40

60

80

Total viable counts

6.5 6 0.7

5.9 6 0.4

4.7 6 0.6

5.6 6 0.5

6.0 6 0.4

Lactic acid bacteria

4.1 6 0.3

,10

,10

,10

,10

Yeasts

5.7 6 0.6

5.6 6 0.2

4.7 6 0.5

5.6 6 0.4

6.0 6 0.5

Enterobacteria

3.7 6 0.9

,10

,10

,10

,10

Pseudomonads

4.0 6 0.5

,100

,100

,100

,100

Microbial association in dry salting. Results are expressed as mean 6 SD, n 5 3.86

FIGURE 13.4 Changes in water activity (K) and salt concentration (▲) in black olives during dry salting. Physico-chemical changes during dry salting.86

1.00

9 8

0.95

0.90

6 5

0.85 4 0.80

3

Water activity (aw)

NaCl (g/100 g)

7

2 0.75 1 0.70

0 0

10

20

30

40

50

60

70

80

Dry salting period (days)

Enterobacteriaceae could be detected at the end of the process, especially when the ratio of salt/olives was high (0.88). In this case a fast osmotic dehydration of olives took place that delayed the diffusion of salt in the olive flesh. Consequently, in the end of the process the percentage of salt was below the limit of 10% established by the IOC for this product, allowing the survival of this microbial group. It is worth noting that yeast species isolated from dry-salted olives could also have promising biotechnological applications. In this context the potential for extracellular lipase production by 97 D. hansenii isolates from dry-salted olives was investigated in an optimized synthetic medium.91 Results indicated that 20 isolates showed increased lipase activity ranging between 6.00 and 7.44 U/mL as well as increased biomass production (1.83
5.02 g/L, expressed as dry biomass) providing interesting perspectives for commercial exploitation. Study of the processed olives with SEM reveals the presence of a biofilm of yeasts covering almost the whole

FIGURE 13.5 Micrograph of olive epidermis after dry salting with yeast biofilm on the surface. Surface of dry salted olives.87

surface of olives (Fig. 13.5). In a cross section of the fruits, large intercellular spaces are observed under the epidermal cells (Fig. 13.6). The development of these spaces takes place due to the advanced stage of maturity

156

PART | 1 General Aspects of Olives and Olive Oil

FIGURE 13.6 Micrograph of dry-salted olives: (A) epidermal cells, (B) parenchyma cells, and (C) fungal hyphae. Cross section of dry salted olives.87

FIGURE 13.7 Micrograph of parenchyma cells of dry-salted olives with fungal growth. Cross section of dry salted olives.87

that the olives are harvested as well as the dry salting process. In this area an extensive network of fungal hyphae is observed that penetrates deeper into the parenchyma cells of the mesocarp at a prolonged storage of olives (Fig. 13.7). No fungal growth is observed on the surface of olives; instead extensive mycelium is evident in the mesocarp that is initially restricted in the intercellular spaces of parenchyma cells but finally penetrates and disintegrates the cells of olives. The presence of Penicillium and Aspergillus spp. has been identified in dry-salted olives stored under aerobic conditions, whereas modified atmosphere packaging (vacuum, 100% N2, 100% CO2, 40% CO2, 30% O2, 30% N2) could effectively control fungal growth.89,92 Fungi may negatively affect both the nutritional and esthetic values of olives. More importantly, there is increasing evidence linking fungal growth and mycotoxin production in table olives, but further research is needed to elucidate this issue.93

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160. 85. Bobillo M, Marshall VM. Effect of acidic pH and salt on acid endproducts by Lactobacillus plantarum in aerated, glucose-limited continuous culture. J Appl Bacteriol. 1992;73:67
70. 86. Panagou EZ. Greek dry-salted olives: monitoring the dry-salting process and subsequent physicochemical and microbiological profile during storage under different packing conditions at 4 and 20oC. Lebensm Wiss Technol. 2006; % 39:322
329. 87. Panagou EZ, Parker ML, Katsaboxakis KZ. Study of the indigenous microflora of dry-salted olives of the Thassos variety using scanning electron microscopy. Agric Res. 2003;26:3
10 (in Greek). 88. Dmytruk KV, Sibirny AA. Candida famata (Candida flareri). Yeast. 2012;29:453
458.

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89. Mantzouridou F, Tsimidou MZ. Microbiological quality and biophenol content of hot air-dried Thassos cv. table olives upon storage. Eur J Lipid Sci Technol. 2011;113:786
795. 90. Ramı´rez E, Garcı´a-Garcı´a P, de Castro A, Romero C, Brenes M. Debittering of black dry-salted olives. Eur J Lipid Sci Technol. 2013;115:1319
1324. 91. Papagora C, Roukas T, Kotzekidou P. Optimization of extracellular lipase production by Debaryomyces hansenii isolates from dry-

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salted olives using response surface methodology. Food Bioprod Process. 2013;91:413
420. 92. Panagou EZ, Tassou CC, Katsaboxakis KZ. Microbiological, physicochemical and organoleptic changes in dry-salted olives of Thassos variety stored under different modified atmospheres at 4 and 20 C. Int J Food Sci Technol. 2002;37:635
641. 93. Medina-Pradas E, Arroyo-Lo´pez FN. Presence of toxic microbial metabolites in table olives. Front Microbiol. 2015;6:873.

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Part 2

Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

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Section 2.1

General nutritional and health aspects

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Chapter 14

Overview of olive oil in vascular dysfunction Vasanti Suvarna and Dhvani Sharma Department of Pharmaceutical Chemistry and Quality Assurance, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Mumbai, India

Abbreviations alpha 2 adrenergic receptor angiotensin-converting enzyme blood pressure cardiovascular disease epithelial derived neutrophil activating peptide 78 endothelial nitric oxide synthase extra-virgin olive oil guanine nucleotide
binding protein (G protein), alpha inhibiting activity polypeptide-2 guanine nucleotide
binding protein (G protein), Gai3 alpha inhibiting activity polypeptide-3 HDL high-density lipoprotein—C-reactive protein c IFN-γ interferon gamma IL interleukin LDL low-density lipoprotein Med Diet Mediterranean diet MUFA monounsaturated fatty acid NO nitric oxide OL oleuropein OO olive oil OxLDL oxidized low-density lipoprotein PGI2 prostacyclin sE-selectin soluble endothelial selectin sP-selectin soluble platelet selectin TBX2 T-box transcription factor TNF-α tumor necrosis factor alpha TP/day table spoon per day USFDA The Food and Drug Administration VOO virgin olive oil

a2A/D ACE BP CVD ENA78 eNOS EVOO Gai2

14.1 Introduction Cardiovascular disease (CVD) is a type of a disease that centers around the heart. It includes coronary artery diseases such as myocardial infarction and angina (heart attack).1 It

also includes heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, venous thrombosis congenital heart disease, carditis, peripheral artery disease, thromboembolic disease, stroke, hypertensive heart disease, heart arrhythmia, and aortic disease.2,3 On an average about 17.9 million people died in 2015, that is, up to 32.1% around the globe due to CVD.4 In the Western world the high ratio of morbidity and mortality is due to CVD.5 Mortality caused due to CVD is approximately 48% in Europe and about 32.8% in the United States. According to Indian epidemiological records, 2222 out of 3034 subjects died due to CVD.6
9 The effect of CVD mortality depends on lifestyle, which adds up to 13.7% for smoking, 13.2% for less than stellar eating routine, and 12% for inert lifestyle. Unrestricted alcohol consumption, poor diet, and smoking are in charge of practically 40% of all demise.10 The risk factors for CVD are ethnicity, age, family history, sex, hypertension, lowdensity lipoprotein (LDL), elevated total cholesterol and triglycerides, overweight and type 2 diabetes, oxidative stress, omega-3 index, inflammation, arterial stiffness, and blood pressure (BP). The peril factors for CVD can be altered by lifestyle changes and diet.11 Preventive actions are being taken, such as campaign for low salt intake and smoking cessation and to boost exercise and physical training and activity, and also education on lifestyle management is being provided.5 The pharmacological preventive and treatment action of CVD has been tremendously improved over the previous decades, and a few powerful medications, for example, beta blockers, statins, antiplatelet, angiotensin-converting enzyme (ACE) inhibitors/ angiotensin receptor II antagonists, are presently accessible. Notwithstanding, physician-endorsed drugs, functional food, or the nutraceutical are by and large progressively included as supplement treatment of CVD or to avoid the probability of

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00030-4 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 14.1 The role of olive oil in the prevention of CVD. CVD, Cardiovascular disease.

Hypertenon

Other risk factors (plaque and hypercholesterolemia)

Vascular aging

Olive oil Endothelial dysfuncon and atherosclerosis

Dysglycemia

Inflammaon and redox imbalance

Prevent

Cardiovascular disease

risk associated with CVD, despite the fact that there are no published data that compare medicament versus nutraceutical and drug combination. Undoubtedly, foods containing polyphenols are blessed with extremely fascinating restorative

properties, the vast majority of which have just been exhibited in vitro.12 Medicinal services costs for CVD in the United States were assessed for 2011
12 to be over $315 billion and are likely to surpass $1 trillion yearly by 2030.11

Overview of olive oil in vascular dysfunction Chapter | 14

Since the ancient time, olive oil (OO) has been perceived for its nutritional characteristics, and by Greeks, it is regarded as “elixir of the youth and the health.” The main source of fat in the Mediterranean diet (Med Diet) is OO, thus acting as the key component. It has been acknowledged that OO predominantly virgin OO (VOO) plays a vital role as a cardioprotective impact of the Med Diet, ascribed to its beneficial and advantageous effects on many CVD risk factors including, but not limited to BP, lipid profile, insulin resistance, endothelial function, obesity, or inflammation. The minor segments of OO are of unique significance in cardiovascular well-being for its antioxidant and antiinflammatory effects and on the grounds that their properties positively tweak hemostatic factors and enhance the strength of arteriosclerotic plaque. USFDA in November 2004 claimed that having two tablespoons of OO daily was helpful in diminishing CVD because of the presence of monounsaturated fatty acids (MUFAs) (VOO—a Mediterranean dire essential). Specifically, the occurrence of CVD in European Mediterranean nations is generally low. A lot of information bolsters the proof that by hindering oxidative pressure, a routine Med Diet that consists of OO conceivably counteracts CVD (Fig. 14.1).13
15

14.2 Composition of olive oil OO consists of phenolic compounds, and unsaponifiable and saponifiable fractions. The phenolic compounds consist of ligans, oleuropein (OL), flavonoids, hydroxytyrosol, and tyroxol. Unsaponifiable fraction consists of pigments (chlorophyll), aliphatic and triterpene alcohols, carotenes, volatile compounds, hydrocarbons (squalene), and tocopherols. Saponifiable fraction consists of linolenic acid (max: 1%), stearic acid (0.5%
5%), palmitic acid (7.5%
20%), oleic acid (55%
85%), and linoleic acid (3%
21%).

14.2.1 Polyphenols Polyphenols are digestible and modify both endothelial dysfunction and inflammatory process involved in the improvement of CVD. Potential defensive impacts of polyphenols against CVD have been credited to a few highlights, including free-radical scavenging, antioxidant activity, inflammation related properties, and production of NO (nitric oxide). These effects lead to relaxation of blood vessels relaxing factors (vascular relaxing factors), for example, prostacyclin (PGI2) that leads to the suppression of vasoconstrictor endothelin-1 synthesis.16 Outcome of the PREDIMED randomized trial demonstrated that polyphenol-rich food regimes such as Med Diet supplemented with extra-virgin OO (EVOO) for 1 year. A remarkable increment in polyphenol levels was observed related with diminished danger of CVD.17 Oxidized LDL (OxLDL) is one of the leading CVD and atherosclerosis

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risk factors, and polyphenols present in EVOO inhibit the oxidation of LDL by binding to it. The probability of oxidation of LDL is based on the presence of fatty and antioxidant content attached to it.18
25 In EUROLIVE study a clinical trial was conducted with respect to consumption of OO that contains different concentrations of phenolic compounds, and the study showed a diminished level of oxidative stress marker, total cholesterol level, and triacylglycerol status. The study was conducted in a dose dependent manner.26
28

14.2.2 Triterpenes In vitro studies reveal that triterpenes present in EVOO have been shown to inhibit or hinder oxidation of LDL and arrest the LDL-mediated thrombin formation, thus averting antiatherogenic mice (developed atherosclerosis). When these mice were treated with triterpene, it showed a reduction in advancement of atherosclerosis.25,29

14.2.3 Sterols Sterols ought to inhibit the absorption of intestinal cholesterol, thus decreasing the concentration of circulatory cholesterol. This in turn decreases total plasma concentration of LDL cholesterol and thus helps in CVD. It not only decreases cholesterol but also decreases triglyceride levels thus aid in CVD.25,30

14.2.4 Oleacin Oleacin has an activity to reduce the release of myeloperoxidase from neutrophils, thus helping in stabilization of atherosclerotic plague.31 Oleacin also shows antihypertensive property by acting on ACE, thus preventing the conversion of angiotensin 1 to angiotensin, causing vasodilation.32,33 Patients suffering from acute myocardial infraction show an escalated level of neutral peptidase enzyme, oleacin, which inhibits this enzyme.25,34

14.2.5 Oleuropein OL shows antiatherogenic activity by inhibiting and hindering at the initial stage of atherogenesis. It blocks the endothelial activation and decreases the adhesion of monocytes to endothelial cell at mRNA level by decreasing the stimulated expression of lipopolysaccharide (vascular adhesion molecule 1).35 Proliferation of the cells of the vascular smooth muscle is impeded when treated with OL, related with decrease of extracellular controlled kinase 1/2 action.36 Manna et al. scrutinized the activity of OL in myocardial infraction and ischemia, and the data showed that OL reduced the level of creatinine kinase (CVD biomarker) and led to the oxidation of glutathione to oxidative stress, thus

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acting as cardioprotective.37 An in vivo study was conducted to analyze the impact of OL on hypercholesterolemic rabbits that were induced with ischemia and reperfusion. The treatment with OL for 3
6 weeks in normal and hypercholesterolemic rabbits led to a significant reduction in infarct size in normal rabbits and showed significant decrease at higher doses in hypercholesterolemic rabbits. Reperfused myocardium was protected by OL by decreasing the triglyceride and total cholesterol level.38 Hypochlorous acid can cause LDL peroxidation via chlorination of apoB-100. Hydroxytyrosol and OL shows scavenging activity toward hypochlorous acid, resulting in delay of atherosclerosis. LDL peroxidation is the rate limiting factor in the determination of atherosclerotic plaque. In recent times, the European Food Safety Authority has identified the beneficial effects of OO containing phenolic compounds on the oxidation of LDL and thus proving to be cardioprotective.39,40

14.2.6 Monounsaturated fatty acids EVOO’s lipid composition leads to a reduction in cholesterol level in plasma by decreasing LDL and very LDL (risk factor in CVD) and increases high-density lipoprotein (HDL) which acts as cardioprotective against the advancement of atherosclerosis.41
44 Apart from this, OxLDL activates inflammatory pathways and causes atherosclerosis thus, when switched to diet rich in oleic acid, inhibits the progression of atherosclerosis because LDLs rich in oleate are less prone to oxidation.45
47 Postprandial lipid level is reduced by the presence of lipids in EVOO as they decrease the levels of von Willebrand factor and t-box transcription factor (TBX2); malondialdehyde is released by platelets that will change the structure of LDL enabling the formation of foam cells by internalization with monocyte macrophage cells. Hence, the corelationship between CVD and platelet aggregation was established. MUFA has been analyzed to lower the BP than saturated fatty acid.48 The process by which BP is decreased by oleic acid is related to the composition of fatty acid in the membranes. When the concentration of oleic acid is increased, it leads to a decrease in the surface packing of phospholipid mainly the head groups. The altered structured controls the expression, localization, signaling, and activity of the molecules with respect to sympathomimetic receptor pathway, thus producing vasodilatory effect. In particular the activity of G protein is inhibited by oleic acid that in turn decreases the levels of Gai2, Gai3, Gaq/11, which leads to a significant change in a2A/D-adrenergic receptor activity, which are the important parameters in the control of both peripheral and central BP. In addition, it shows an inhibitory action on the activity of Gai proteins present in aorta.49 Minor components of OO such as polyphenols and tocopherols are believed to play a major role in treatment of hypertension. With this respect, high-phenolic content decreased the BP more significantly than did the low concentration.50

14.3 Effect of olive oil on cardiovascular disease risk factors 14.3.1 Hypertension Hypertension is caused when cardiac output and vascular resistance increase. It includes the failure of kidney to discharge sodium thus results in increased arterial pressure and blood, which increases cardiac output.51 Throughout the world, one of the leading causes of CVD mortality and morbidity is high BP or hypertension. A randomized study was conducted with two controls as placebo one with women who have mild hypertension and men, with stable condition of hypertension. The first study was conducted with respect to continuous consumption of OO rich in polyphenols (approximately 30 mg TP/day). It brought about a significant decrease in both systolic and diastolic BP when contrasted with the baseline value whereas OO free of polyphenols did not bring any significant change.52 In the second case, taking 7.4 mg TP/day of virgin OO for 3 weeks brought about a decrease in systolic BP when contrasted with 0.67 mg TP/day of EVOO.53 In 2015 Hohmann and coworkers conducted a metaanalysis study on healthy or CVD patients for checking the effectiveness of high-phenolic OO versus lowphenolic OO on CVD risk factor and discovered that it brought mellow impacts for bringing down systolic BP, yet no impact on diastolic BP.54,55

14.3.2 Vascular aging The hallmarks of vascular aging are vascular stiffness and endothelial dysfunction that leads to structural and functional changes in large arteries. Currently, few noninvasive techniques are used for assessing endothelial dysfunction. Randomized clinical trial was conducted with hypercholesterolemic subjects. They were subjected to preparation that contains a high level of OO-based polyphenols. It showed an enhancement of vasodilation of endothelium associated microvasculature, followed by a reduction in oxidative stress and an initial increase in the production of nitric oxide. These results were seen in the initial 4 h after taking the meal.52,55

14.3.3 Dysglycemia Diabetes mellitus or high glucose level in blood adds to about 3 million CVD-related death each year all around the world. Treating and preventing diabetes is of utmost importance as it connected with high risk of CVD. Moreover, dysglycemia is often associated with other CVD risks prompting the metabolic disorder, a condition with high tendency of atherosclerosis; control on the type of food intake is of the preventive measure. Wainstein

Overview of olive oil in vascular dysfunction Chapter | 14

and coworkers carried out a clinical trial with subjects who were suffering from type 2 diabetes mellitus. Consumption of olive leaf extract for 14 weeks altogether brought down fasting level and HbA1c level when contrasted with placebo. However, no effect was seen on postprandial insulin level.55,56

14.3.4 Inflammation and redox imbalance Identification and enhancement of biomarkers of oxidative stress and inflammation lead to the decrease in the occurrence of various risk factors associated with CVD. Moreover, there is a solid proof that polyphenols can exercise antiinflammatory or immunomodulatory effects.55

14.3.5 Oxidative stress
mediated endothelial dysfunction and atherosclerosis European subjects in the EUROLIVE study (randomized and crossover) were allotted in three groups that contained three different phenolic OO composition (high, medium, and low). The study was carried out for 3 weeks. The results showed an inverse relationship between phenolic OO concentration and OxLDL levels. In addition, it enhances the balance between reduced glutathione and oxidized glutathione and showed the production of plasma f2-isoprostanes, decrease in C18 hydroxy fatty acid. Apart from this, it also aids in the production of OxLDL autoantibody that has a protecting action against atherosclerosis.45,57,58 The inflammatory markers that are present in the vascular wall act as another important factor that helps in connecting the link between EVOO and CVD. The inflammatory markers include the levels of various cytokines [interleukin (IL)-1 beta, 5, 6, 7, 8, 12, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ)], high-sensitivity C-reactive protein, and plaque marker (IL-10, 13, and 18) levels. Med Diet, when supplemented with EVOO, enhanced the levels of these inflammatory markers, thus delaying atherosclerosis.59
61

14.3.6 Other risk factors Accumulation of lipid on the walls of the arteries results in the inflammation, which leads to the attraction of Tcells and monocytes leading to the developing lesions and narrowing of arteries due to the accumulation of LDL and leads to the formation of plaque. This plaque further narrows the arteries and causes myocardial infraction and ischemia after rupturing.51 Via oxidative stress mechanism, atherosclerotic stimulant (e.g., hypertension) may trigger the inflammatory response by producing a mononuclear leukocyte recruiting mechanism. This may bear legitimate oxidative stress on the walls of the artery causing atherosclerosis.62 Hypertension is caused due to

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disparity between vasorelaxing factor nitric oxide and vasoconstricting factor endothelin-1. When women (moderate hypertension and nonsmoker) for 1 year were subjected with Med Diet adjunct with EVOO, it led to an escalated nitric oxide level in the serum. In addition, alteration in gene expression that comprises (1) dilation of blood vessels due to upregulation of endothelial nitric oxide synthase (eNOS) occurred, which helps in protection of BP, (2) sterically hindering the enzymes by downregulating caveolin-2 by in-turn binds with eNOS.63 In addition, hypercholesterolemia is a CVD risk factor, and the components of Med Diet are related to decrease in non-HDL and LDL.64 A study was conducted by Hernae´z et al., for 1 year with Med Diet associated with VOO, and it resulted in the improvement of the HDL activity and function.65 It also improved other functions such as antiinflammatory or antioxidant property and cholesterol metabolism that are strongly associated with CVD risk.

14.4 Case studies A decrease in IL-6 and C-responsive protein (CRP) levels when contrasted with placebo66 was seen in young prehypertensive women who continuously consumed approximately 30 mg/day of OO rich in polyphenols for 2 months.66 In understanding a randomized trial with placebo with leaf extract for 2 weeks prompts fasting IL-6 in men who are middle aged and overweight. However, no effect was seen for TNF-α, ultrasensitive CRP, and IL-8.66 Polyphenolic OOs specifically Hydroxytyrosol have appeared to show in vivo, antithrombotic, and antiplatelet activity. A study with respect to dyslipidemic subjects consuming OO for 7 weeks prompted a reduction in the production of serum B2 thromboxane and an elevation of antioxidant capacity in plasma.67,68 A SUN cohort study was carried out with 18,118 Spanish subjects who were at a lower risk of CVD. A correlation between OO consumption and atrial fibrillation was analyzed for 10.1 years, and the cases that were confirmed with atrial fibrillation were 94. In a subgroup of overweight patients a nonsignificant and inverse relation was achieved. The final conclusion was that there is no association between OO consumption and atrial fibrillation.69 A current metaanalysis that included 3106 subjects exhibited that day-to-day consumption of OO in the range of 1
50 mg brought about a significantly increasingly articulated abatement in IL-6 and CRP.70 Another investigation included examinations surveying systolic and diastolic pulse, low as well as OxLDL, triglycerides, total cholesterol, and malondialdehyde. This investigation included 417 individuals with contrasting low versus highphenolic OO consumption. Average/medium impacts were noticed for bringing down systolic BP and little impacts or bringing down oxidative LDL. No results were found for

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the rest. Therefore this metaanalysis does not bolster the valuable impact of OO for diverse biomarkers.70 In another investigation contrasted the impact of OO consumption with other plant oils on the lipid levels in the blood demonstrated that OO consumption diminishes LDL, triglyceride, and total cholesterol, but when compared to other plant oils, it remarkably increased HDL. It had no appreciable effect seen on apoprotein A1 and B.71,72 Aggregation of proteins rich in triglycerides after meals leads to the development of CVD risk such as oxidative stress, LDL, endothelial dysfunction, and inflammation.73 In this regard, there is information on the impacts of the unsaponifiable fragment on human triglyceride-rich lipoproteins. When pomace OO was consumed, this lipoprotein level was seen to be reduced because of higher presence of minor nonpolar constituents when compared to refined OO that has a low concentration of nonpolar constituents.73 Deep-fried meals that are prepared using OO (which contains high concentration of phenolics) compared to sunflower oil have shown to decrease, in obese patients, oxidative stress thus may prevent CVD.74,75 A study demonstrated that a two-pointer increment in adhesion to Med Diet altogether led to 8% decrease in mortality rate and 10% decrease in mortality rate due to CVD.76 A Spanish study (Spanish cohort) was conducted in 2011 with 13,000 subjects and were subjected to high and low adherence to Med Diet. It was found that higher adherence to Med Diet led to a 20% decrease in CVD and overall 26% decrease in CVD risk factors.77 Another study called EPICNL study was conducted, which included 40,000 subjects and analyzed that when adherence to Med Diet is increased by 2 units, it showed a decrease in CVD and its associated risk factors.78 This was also confirmed by US cohort Northern Manhattan Study.79 Yet another study called The Lyon Diet Heart Study showed a decrease in 70% risk.80
82 In a PREDIMED study a large number of subjects were associated with Med Diet supplemented with EVOO. When the data were compared with control, it showed a 30% reduction in CVD risk.83
85 This study included 7447 subjects that at high risk of CVD were subjected to Med Diet containing mainly OO (approximately 42% fat). The study showed that there was a decrease in CVD risk when compared to control subjects that were subjected to diet which contained 37% fat. This milestone gave a proof that changing the nature of fat described with high MUFA and polyphenolics in the OO can be beneficial when compared to the fat obtained by animal.83,84,86 Another study, including 137 subjects who were above 64 years of age, showed that consuming Med Diet containing EVOO for 3 continuous months decreased the systolic BP. However, when the diet was continued for over a period of 6 months, there was an improvement of endothelial activity.87,88 Free sterols (sterol glycoside) when included in the diet regime will decrease the absorption of cholesterol

from the gut, thus diminishing the cardiovascular risk.89 Oxidized polyunsaturated fatty acid present in plasma lipoprotein is in charge for the commencement of atherosclerosis, thus consuming the high concentration of α-tocopherol will diminish the oxidation of polyunsaturated fatty acid thus preventing atherosclerosis. It also diminishes protein kinase C action and regulates few pathways associated with atherogenesis prevention.90 OO in these days is featured as superfood for the health benefits associated with it like the presence of different micronutrients and lowering lipid profile. Plus, since 2012, European Authority for Health Safety labeled OO with health claim and EU regulation no 432/2012 defined as “polyphenols present in OO leads to blood lipid protection against oxidative stress.”91 In any case, this claim is only valid if OO contains a minimum of 5 mg/20 g of hydroxytyosol (including its derivatives, e.g., tyrosol and OL complex) and OO. Furthermore, the label must bear the information for the consumer that the beneficial effect of OO is only obtained when 20 g OO is taken daily.92 A study was conducted to analyze the long-term effect of Med Diet with EVOO on 66 patients from Hospital Clinic of Barcelona. After a continuous study for 5 years, it was elucidated that it led to a decrease in the levels of inflammatory markers such as ENA78, IL-6, granulocyte macrophage colony stimulating factor, IL-8, granulocyte colony stimulating factor, macrophage chemotactic protein-1, IFN-γ, macrophage inflammatory protein-1β, TNF-α, IL-1β, IL-18, IL-5, IL-12p70, and IL-7. These markers have shown association with atherosclerosis.93 Another study included the intake of traditional Med Diet enriched with VOO in 210 subjects for 1 year resulted in decrease oxidative LDL and increase in resistance of LDL oxidation.65 Yet another study concluded that when 90 subjects with abdominal obesity are supplemented with strictly controlled Med Diet for 2 months, it led to a decrease in levels of CRP, sP-selectin, and sE-selectin.94 In case cohort design nested in the Prevencion con Dieta Mediterranean, randomized controlled trial with 231 subjects suffering from various kinds of CVD disease, when subjected Med Diet with EVOO for 1 year, increased the levels of tryptophan concentration in plasma which was closely associated with the decrease in CVD risk.95

14.5 Conclusion Med Diet containing OOs are associated with low risk of cardiovascular studies. The different components of OO especially phenolic compounds play a significant role to decrease the risk of CVD. All the studies have also shown the beneficial outcomes of Med Diet containing OOs that have fundamentally moved the consideration from the lipiddriven model that is portrayed by the ideal decrease of cholesterol levels to increasingly powerful focusing against

Overview of olive oil in vascular dysfunction Chapter | 14

aggravated risk factors of CVD. Epidemiological studies have demonstrated that the Med Diet can prevent the onset of chronic inflammatory disease such as CVD. Thus in the last few years, there has been a growing demand of instilling OO in their diet to decrease the CVD risk factor.

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S101. Available from: https://doi.org/10.1017/s0007114514003936. 27. Covas M, Konstantinidou V, Fito´ M. Olive oil and cardiovascular health. J Cardiovasc Pharmacol. 2009;54(6):477
482. 28. Fito´ M, Cladellas M, de la Torre R, et al. Anti-inflammatory effect of virgin olive oil in stable coronary disease patients: A randomized, crossover, controlled trial. Eur J Clin Nutr. 2008;62:570
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29. Acı´n S, Navarro MA, Perona JS, et al. Olive oil preparation determines the atherosclerotic protection in apolipoprotein E knockout mice. J Nutr Biochem. 2007;18(6):418
424. 30. Howell T, MacDougall D, Research PJ. Phytosterols partially explain differences in cholesterol metabolism caused by corn or olive oil feeding. J Lipid Res. 1998;38:892
900. 31. Czerwi´nska M, Kiss A, Chemistry MN. A comparison of antioxidant activities of oleuropein and its dialdehydic derivative from olive oil, oleacein. Food Chem. 2012;131(3):940
947. 32. Hansen K, Adsersen A, Christensen S, Jensen SR. Isolation of an angiotensin converting enzyme (ACE) inhibitor from Olea europaea and Olea lancea. Phytomedicine. 1996;2(4):319
325. 33. Somova L, Shode FO, Ramnanan P, Nadar A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies Africana leaves. J Ethnopharmacol. 2003;84 (2
3):299
305. 34. Czerwi´nska M, Kiss A, Chemistry MN. Inhibition of human neutrophils NEP activity, CD11b/CD18 expression and elastase release by 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, oleacein. Food Chem. 2014;153:1
8. 35. Carluccio M, Siculella L, Ancora MA. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler Thromb Vasc Biol. 2003;23(4). 36. Abe R, Beckett J, Nixon A, Rochier A. Olive oil polyphenol oleuropein inhibits smooth muscle cell proliferation. Eur J Vasc Endovasc Surg. 2011;41(6):814
820. 37. Manna C, Migliardi V, Golino P. Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion. J Nutr Biochem. 2004;15(8):461
468. 38. Andreadou I, Iliodromitis E. The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr. 2018;136(8):2213
2219. 39. Tijerina ACC, Frei B. Vitamin C protects against and reverses specific hypochlorous acid-and chloramine-dependent modifications of low-density lipoprotein. Biochem J. 2000;346(2):491
499. 40. Bulotta S, Celano M, Lepore SM, Montalcini T, Pujia A, Russo D. Beneficial effects of the olive oil phenolic components oleuropein and hydroxytyrosol: focus on protection against cardiovascular and metabolic diseases. J Transl Med. 2014;12(1):1
9. Available from: https://doi.org/10.1186/s12967-014-0219-9. 41. Mattson F, Research SG. Comparison of effects of dietary saturated, monounsaturated, and polyunsaturated fatty acids on plasma lipids and lipoproteins in man. J Lipid Res. 1985;26:194
202. 42. Martı´nez-Gonza´lez MA, Sa´nchez-Villegas A. The emerging role of Mediterranean diets in cardiovascular epidemiology: monounsaturated fats, olive oil, red wine or the whole pattern? Eur J Epidemiol. 2004;19(1):9
13. 43. Mensink RP, Katan MB. Effect of dietary fatty acids on serum lipids and lipoproteins. A meta-analysis of 27 trials. Arterioscler Thromb. 1992;12(8):911
919. Available from: https://doi.org/10.1161/01.ATV.12.8.911. 44. Soriguer F, Rojo-Martı´nez G, Goday A. Olive oil has a beneficial effect on impaired glucose regulation and other cardiometabolic risk factors. [email protected] study. Eur J Clin Nutr. 2013;67:911
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1224. Available from: https://doi.org/10.1002/mnfr.200600308. Reaven P, Parthasarathy S, Grasse BJ, et al. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects. J Clin Invest. 1993;91. Covas M. Olive oil and the cardiovascular system. Pharmacol Res. 2007;55(3):175
186. Yang Q, Alemany R, Casas J, Kitajka K, Lanier S. Influence of the membrane lipid structure on signal processing via G proteincoupled receptors. Mol Pharmacol. 2005;68(1):210
217. Ruiz-Gutie´rrez V, Muriana F, Guerrero A. Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources. J Hypertens. 1996;14(12):1483
1490. Zoltani CK. Nutraceuticals in Cardiovascular Diseases. Elsevier Inc; 2016. 10.1016/B978-0-12-802147-7.00004-8. Ruano J, Lopez-Miranda J, Fuentes F, et al. Phenolic content of virgin olive oil improves ischemic reactive hyperemia in hypercholesterolemic patients. J Am Coll Cardiol. 2005;46. Fito´ M, Cladellas M, Torre RD, et al. Antioxidant effect of virgin olive oil in patients with stable coronary heart disease: a randomized, crossover, controlled, clinical trial. Atherosclerosis. 2005;181(1):149
158. Hohmann CD, Cramer H, Michalsen A, et al. Effects of high phenolic olive oil on cardiovascular risk factors: a systematic review and meta-analysis. Phytomedicine. 2015;22(6):631
640. Available from: https://doi.org/10.1016/j.phymed.2015.03.019. Tome-Carneiro J, Phytomedicine FV. Polyphenol-based nutraceuticals for the prevention and treatment of cardiovascular disease: review of human evidence. Phytomedicine. 2016;23(11):1145
1174. Wainstein J, Ganz T, Boaz M, et al. Olive leaf extract as a hypoglycemic agent in both human diabetic subjects and in rats. J Med Food. 2012;15 (7):605
610. Available from: https://doi.org/10.1089/jmf.2011.0243. Castan˜er O, Fito´ M, Lo´pez-Sabater M, Nutrition HP. The effect of olive oil polyphenols on antibodies against oxidized LDL. A randomized clinical trial. Clin Nutr. 2011;30(4):490
493. Covas M, Nyysso¨nen K, Poulsen HE, et al. The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med. 2006;145(5). Casas R, Sacanella E, Urpı´-Sarda` M. Long-term immunomodulatory effects of a Mediterranean diet in adults at high risk of cardiovascular disease in the PREvencio´n con DIeta MEDiterra´nea (PREDIMED). J Nutr. 2016;146(9):1684
1693. Casas R, Sacanella E, Urpı´-Sarda` M, et al. The effects of the Mediterranean diet on biomarkers of vascular wall inflammation and plaque vulnerability in subjects with high risk for cardiovascular disease. A randomized trial. PLoS One. 2014;9(6). Available from: https://doi.org/10.1371/journal.pone.0100084. Casas R, Urpi-Sarda` M, Sacanella E, et al. Anti-inflammatory effects of the Mediterranean diet in the early and late stages of atheroma plaque development. Mediat Inflamm. 2017;2017. ´ , Toledo E, Aro´s F, et al. Extravirgin Martı´nez-Gonza´lez MA olive oil consumption reduces risk of atrial fibrillation. Circulation. 2014;130(1):18
26. Available from: https://doi. org/10.1161/circulationaha.113.006921.

Overview of olive oil in vascular dysfunction Chapter | 14

63. Storniolo CE, Casillas R, Bullo´ M, et al. A Mediterranean diet supplemented with extra virgin olive oil or nuts improves endothelial markers involved in blood pressure control in hypertensive women. Eur J Nutr. 2017;56(1):89
97. Available from: https://doi.org/ 10.1007/s00394-015-1060-5. 64. Reiner Z, Catapano AL, De Backer G, et al. ESC/EAS Guidelines for the management of dyslipidaemias: the Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). Eur Heart J. 2011;32(14):1769
1818. Available from: https://doi.org/10.1093/eurheartj/ehr158. ´ , Castan˜er O, Goday A, et al. The Mediterranean diet 65. Herna´ez A decreases LDL atherogenicity in high cardiovascular risk individuals: a randomized controlled trial. Mol Nutr Food Res. 2017;61 (9):1601015. Available from: https://doi.org/10.1002/mnfr.201601015. 66. Moreno-Luna R, Mun˜oz-Hernandez R. Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. Am J Hypertens. 2012;25 (12):1299
1304. 67. Visioli F, Caruso D, Grande S, et al. Virgin Olive Oil Study (VOLOS): vasoprotective potential of extra virgin olive oil in mildly dyslipidemic patients. Eur J Nutr. 2005;44(2):121
127. Available from: https://doi.org/10.1007/s00394-004-0504-0. 68. Visioli F, Bogani P, Grande S. Mediterranean food and health: building human evidence. J Physiol Pharmacol. 2005;56(1):37
49. 69. Bazal P, Gea A, de la Fuente-Arrillaga C, Barrio-Lo´pez MT, Martinez-Gonza´lez MA, Ruiz-Canela M. Olive oil intake and risk of atrial fibrillation in the SUN cohort. Nutr Metab Cardiovasc Dis. 2019;29(5):450
457. Available from: https://doi.org/10.1016/ j.numecd.2019.02.002. 70. Schwingshackl L, Christoph M, Nutrients GH. Effects of olive oil on markers of inflammation and endothelial function—a systematic review and meta-analysis. Nutrients. 2015;7(9):7651
7675. 71. Foscolou A, Critselis E, Panagiotakos D. Olive oil consumption and human health: a narrative review. Maturitas. 2018;118:60
66. Available from: https://doi.org/10.1016/j.maturitas.2018.10.013. 72. Ghobadi S, Hassanzadeh-Rostami Z, Mohammadian F, et al. Comparison of blood lipid-lowering effects of olive oil and other plant oils: a systematic review and meta-analysis of 27 randomized placebo-controlled clinical trials. Crit Rev Food Sci Nutr. 2018;59(13):2110
2124. Available from: https://doi. org/10.1080/10408398.2018.1438349. 73. Chan DC, Pang J, Romic G, Watts GF. Postprandial hypertriglyceridemia and cardiovascular disease: current and future therapies. Curr Atheroscler Rep. 2013;15(3):309. Available from: https://doi. org/10.1007/s11883-013-0309-9. 74. Covas MI, Fito´ M, de la Torre R. Minor Bioactive Olive Oil Components and Health: Key Data for Their Role in Providing Health Benefits in Humans. AOCS Press; 2015. 10.1016/B978-163067-041-2.50008-2. 75. Perez-Herrera A, Rangel-Zun˜iga O, Delgado-Lista J, et al. The antioxidants in oils heated at frying temperature, whether natural or added, could protect against postprandial oxidative stress in obese people. Food Chem. 2013;138(4):2250
2259. 76. Sofi F, Abbate R, Gensini GF. Accruing evidence on benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis. Am J Clin Nutr. 2010;92 (5):1189
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1464. 80. De Lorgeril M, Salen P, Martin J, Monjaud I, Circulation JD. Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation. 1999;99:779
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1459. 82. Trichopoulou A, Vasilopoulou E. Mediterranean Diet. Elsevier Inc.; 2015. 10.1016/B978-0-12-384947-2.00454-2. 83. Estruch R, Ros E, Salas-Salvado´ J, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med. 2013;368(14):1279
1290. Available from: https://doi.org/10.1056/ NEJMoa1200303. 84. Estruch R, Ros E, Salas-Salvado´ J, et al. Primary prevention of cardiovascular disease with a Mediterranean diet supplemented with extra-virgin olive oil or nuts. N Engl J Med. 2018;378(25):e34. Available from: https://doi.org/10.1056/NEJMoa1800389. 85. Catapano AL, Barrios V, Cicero AFG, Pirro M. Lifestyle interventions and nutraceuticals: guideline-based approach to cardiovascular disease prevention. Atheroscler X. 2019;1:100003. Available from: https://doi.org/10.1016/j.athx.2019.100003. 86. Saboo B, Singh RB, Bhardwaj K, et al. Fats and Oils for Health Promotion and Disease Prevention. Elsevier Inc; 2018. 10.1016/ b978-0-12-813148-0.00016-5. 87. Davis C, Hodgson J. A Mediterranean diet lowers blood pressure and improves endothelial function: results from the MedLey randomized intervention trial. Am J Clin Nutr. 2017;105 (6):1305
1313. 88. Gavahian M, Mousavi Khaneghah A, Lorenzo JM, et al. Health benefits of olive oil and its components: impacts on gut microbiota antioxidant activities, and prevention of noncommunicable diseases. Trends Food Sci Technol. 2019;88:220
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322. Available from: https://doi.org/10.3945/jn.116.241711.

Chapter 15

Olive in traditional Persian medicine: an overview Mohammad Mahdi Parvizi1, Maryam Saki2, Farhad Handjani3 and Mojtaba Heydari4 1

Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, 2Health System Research Center, Shiraz University of

Medical Sciences, Shiraz, Iran, 3Department of Dermatology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, 4

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Abbreviations DNA HIV PPARγ TGF-β1 TPM

deoxyribonucleic acid human immunodeficiency viruses peroxisome proliferator-activated receptor gamma transforming growth factor beta 1 traditional Persian medicine

15.1 Traditional Persian medicine Traditional Persian medicine (TPM) is a holistic medicine that dates back to 7000 years ago. Avicenna (CE 980
1037), the Persian philosopher and physician, is one of the key figures of TPM, because he was very instrumental in introducing and advocating this medicine in the world.1,2 The approach of TPM for treatment of various diseases originated from humoral medicine,3 in such a way that humors—including dam (blood), balgham (phlegm), safra (yellow bile), sawda (black bile)—define the temperament of the human body, animals, medicinal plants, fruits, foods, etc. Therefore according to TPM, each person has a specific temperament and he/she should consume the suitable medicinal plants, fruits, meats, and foods adhered to his/her temperament to stay healthy and prevent illness.4,5 The base of TPM is prevention from illness, and several remedies have been recommended for staying healthy.6

15.2 Olive in traditional Persian medicine 15.2.1 Olive temperament Olive is a highly used medicinal plant in TPM (Fig. 15.1). According to the concept of TPM, the temperament

(Mezaj) of the green mature olive is hot with a low grade, but the temperament of unripe olive is cold and dry. On the other hand, the temperament of black olive is hot and dry. Several parts of the olive, including oil, leaf, and extract, were used for health and treatment purposes. According to TPM sources, olive and olive-derived products were prescribed for the treatment of several diseases, including neurological disorders, oral cavity problems, gastrointestinal diseases, cardiovascular diseases, respiratory system disorders, skin diseases, urinary system disorders, and obstetrics and gynecology conditions as well as other maladies. The potential applications of olive in TPM in described in this chapter. In this endeavor, we have also tried to present relevant evidence about the traditional use of olive and olive-derived products from modern medicine.7
11

15.2.2 Comparison of olive oil with other edible oils Different edible herbal oils were used by traditional Persian physicians. They described different methods for the preparation and application of these oils. These methods included direct and indirect methods. The direct method was based on the extraction of oil from herbs via the compression of components or distillation of aromatic plant parts. The indirect method involved soaking the soft, fragrant aerial parts such as the flower, leaves, or fleshy fruits in vehicle oil.12,13 Olive oil was traditionally used as one of the most popular oils for soaking various herbs and plants. Besides olive oil, other edible oils such as almond and sesame oil were also used as vehicles for indirect oil extraction. The selection of these oils was based on their

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00002-X © 2021 Elsevier Inc. All rights reserved.

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FIGURE 15.1 The chapter on olive in Rhazes (CE 865
925) manuscript on nutrition entitled Benefits and Harms of Foods (in Arabic Manfe’ al aghzie va Mazareha), Iranian Parliament Library, Registration No. 6098.

known temperament characteristics. Olive oil was used if a cold and dry temperament was sought (i.e., treatment of hot swelling), while sesame oil was used when a warming effect was required (i.e., preexercise massage) and almond oil when a moisturizing effect was needed (i.e., for insomnia).5,12

15.3 Implications of olive for human health and disease prevention in traditional Persian medicine The main goal of TPM is human health and disease prevention. The essential healthy rules for prevention in TPM are six and are called settet-al-zaruriyyah. These rules are: (1) air, (2) eating and drinking, (3) sleeping and waking, (4) motion and stillness, (5) retention and evacuation, and (6) psychological
emotional experiences. In this concept, people can prevent illness by following the recommendations around these six rules. Air is necessary for human life, while food and drinks are important for human growth and help the human body to meet the required level of mineral elements, vitamins, proteins, etc. Moreover, wakefulness is important for humans to meet their needs and sleep is necessary to reinforce their faculties. Retention has the main role in providing stability and

homeostasis in humans; on the other hand, people need evacuation to remove wastes from their body. Lastly, psychological
emotional experiences, including grieve, shame, happiness, fear, and audacity, are important for humans to cope with their life, react to happenings, and deal with the external world.5,8,14,15 Moreover, olive is one of the few fruits mentioned in the holy Quran (cited seven times).16 Taking into account the above setting, olive was one of the fruits recommended for human health and disease prevention in TPM. According to Avicenna, unripe olive oil is the best type of olive oil for healthy individuals.8 The sages of TPM administered olive oil topically to keep the skin healthy, soft, and to deliver moisture to the skin and hair. Furthermore, according to TPM sages, consumption of olive and olive oil can increase the internal normal body temperature, called hararat-e-gharizi, that can lead to an increase in the level of happiness, as well as an increase in body metabolism. On the other hand, according to the concept of TPM, the consumption of olive before the main meals can facilitate fecal excretion, which could prevent many diseases. In this setting, olive can reduce abdominal distension, prevent dyspepsia, and improve digestion and, hence, increase the absorption of various foods by the body. On the other hand, it was suggested that the fume

Olive in traditional Persian medicine: an overview Chapter | 15

from burning black olives and its kernels is useful for the prevention and treatment of some lung disorders, such as asthma.4,8,9,11 In addition, olive kernel oil was recommended for the prevention of hair loss. Daily topical use of unripe olive oil was suggested to postpone the graying of hair. Moreover, the gum of the olive tree was suggested for cleaning the oral cavity, tongue, and teeth, while the wood of olive trees was recommended to be used as a toothbrush.8,9,11

15.4 Implications of olive in medicine based on traditional Persian medicine 15.4.1 Olive in dermatology There are several applications of olive for dermatological conditions in TPM.17,18 Topical use of olive was recommended for hozaz (seborrheic dermatitis), skins burns, and nail discoloration. Moreover, black olive was recommended for jomreh and namleh (acne), shera (urticaria), impetigo, alopecia areata, and tinea capitis. In addition, there is strong recommendation in using topical olive oil, fresh olive tree branches, and olive leaf powder for the acceleration of wound healing. Moreover, based on TPM sources, a natural medicine that contains olive kernels has a therapeutic effect on nail discoloration.7
11 In review of literature, topical use or ingestion of olive and its derivatives, including olive oil and olive leaf extract, have been proven effective in promoting the healing of chronic wounds,19 diabetic ulcers,20 second-degree burns,21
23 diaper dermatitis,24 wounds of sino-nasal surgeries,25 episiotomy wounds,26 and nonkeloid scars and striae.27 Olive oil squalene has been suggested for use in the treatment of irritated or sensitive skin.28 This product has also been shown to attenuate stress-induced ageing signs and pathologies29,30 and improve the skin barrier function of patients with senile xerosis.31 Olive oil has also been suggested to be useful in the prevention of pressure ulcers.32,33 Saponified olive oil and olive leaf extract have been proposed to have therapeutic effects on Pediculus humanus capitis34 and cutaneous leishmaniasis,35 respectively. As an antioxidant, olive oil has been suggested to have protective effects against oxidative stress on human epithelial cells.36 Moreover, one study has shown that olive oil dressing can help prevent bedsores in patients hospitalized in the intensive care unit.37

15.4.2 Olive in neurology and psychiatry The sages of TPM prescribed the gargling of decoction of the root of olive tree for headache. In addition, they recommended pouring the decoction of the root of the olive tree on the head of patients with nasal and postnasal

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discharge as well as pouring the decoction of all parts of the olive, including fruit, root, and leaf in patients with vertigo. Furthermore, according to TPM sources, olive and its derived products are very useful for memory improvement.7
11 According to current medical literature, olive oil has a therapeutic effect on headache.38 In this setting, patients with headache using olive oil for 2 months reported an 80%
90% improvement in the severity of migraine headache. It is possible that olive oil can improve migraine headache by affecting the lipid profile.39 Some studies have shown that olive oil has antiinflammatory properties, so this effect can also play a key role and act as an analgesic in patients with headache.40 In addition, there is evidence that shows that adding olive oil to the diet of patients with headache can prevent this condition.41 It has also been shown that the analgesic property of the topical application of the oil may be associated with the temperament of headache.42 This efficacy can also be associated with gastrointestinal disease
related headache.43,44 Olive and its derivatives, with their antioxidant,45
49 antiinflammatory,45,49,50 antiapoptotic,46,51 and antinociceptic properties,50,52,53 have been proposed to also have neuroprotective effects45
47,54,55 through various mechanisms. Modification of enkephalinase activity in the frontal cortex,56 modulation of the expression of genes and microRNAs involved in neuronal function and synaptic plasticity at the central level,57 increase of glutathione levels58 by group I and the reduction of glutamate release in synaptic spaces, and reactive oxygen species accumulation by group II metabotropic glutamate receptors55 are all the suggested mechanisms of the effect of olive derivatives on the neuronal system. In rodent models, dietary olive oil has been proven to affect cognitive functions,56,57 motor and emotional behavior,57 reduction of the risk of stroke,59 and decrease of damage to the brain when a stroke occurs.60
62 It has also been demonstrated that olive oil can have a protective effect on Alzheimer’s disease and cerebral amyloid angiopathy48,54,63 and to delay the development of amyotrophic lateral sclerosis,64 as well as pretreatment of ischemia via inducing some changes in lipid profile and decrease in brain edema.65 Enrichment of virgin olive oil in its phenolic compound is safe and has been suggested to be used for the treatment of chronic degenerative diseases such as Alzheimer’s disease.63,66 Hydroalcoholic extract of olive has been found to prevent the death of dopaminergic neurons in the rat model of Parkinson’s disease.51 Olive leaf extract has been investigated in the improvement of heroin-induced brain damage in mice.46 Application of short-term massage with olive oil seems to be effective in reducing the severity of uremic restless legs syndrome.67 Yogurt containing olive oil extract has been suggested to simulate fat-triggered sensations in the brain at the gustatory

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level.68 Oral administration of olive leaf extracts have been proposed to reduce anxiety-like behaviors induced by 6-hydroxydopamine in a rat model of Parkinson’s disease.69

15.4.3 Olive in ophthalmology Olive has been recommended for the treatment of dry eye and severe inflammation of the eyes in TPM. In addition, it has been suggested for orbital edema and treatment of eye discharge. Furthermore, it was suggested that the topical use of olive could lead to improvement of visual acuity.7,9,11 Olive and honey have also been used for the treatment of eye infections in TPM.70 Omega-3 and omega-6 fatty acids which are present in olive oil have been shown to be effective in treatment of dry eyes in multiple studies.71,72

15.4.4 Olive in urinary and reproductive system According to TPM, olive has a diuretic effect so it was recommended for conditions that would benefit from the administration of a diuretic. Moreover, olive was administered for renal stone patients and the proposed mechanism was a form of lithotripsy and increase in urine flow. Furthermore, it was mentioned that olive can increase libido, so it was administered for the patients with sexual dysfunction and problems with libido.7
11 Olive oil has been proven to reduce and prevent the growth of urinary stones in mice models.73 Olive leaf extract has been shown to provide protective properties against renal ischemic
reperfusion injury.74 It has also been suggested to alleviate cyclosporine75 and gentamycininduced76 nephrotoxicity. Olive oil has been suggested as a new medicinal product to prevent peritoneal fibrosis in patients with peritoneal dialysis.77 Olive oil has also been shown to increase urinary antioxidant capacity in middleaged and elderly individuals.78

15.4.5 Olive in obstetrics and gynecology TPM sages believed that enema with the decoction of olive, its fruit, and leaf extract was very effective for ulcerative ulcers of the uterus. Moreover, inserting vaginal suppositories of olive oil and olive leaf was considered helpful for vaginal discharge and bleeding. On the other hand, olive gum has been mentioned as a product that could facilitate menstrual bleeding.7
11 In rodents, extra-virgin olive oil and hydroalcoholic extract of olive have been shown to decrease serum malondialdehyde, resulting in lower blood pressure in pregnant mice with preeclampsia79 and reducing the degree of tissue damage in ovarian ischemia/reperfusion,80 respectively, through their antioxidant properties. Regarding the

antimicrobial properties of olive, the ethanolic extract of olive leaf might potentially treat Streptococcus agalactiae infection in pregnant women or newborns81 while topical use of ozonated olive oil can improve the clinical and paraclinical aspects of treatment of patients with vulvovaginal candidiasis.82 Olive leaf extract has been proven to suppress inflammatory cytokine production and NLRP3 inflammasomes in human placenta, leading to lower pregnancy complications.83 Moreover, several studies have shown the advantages of adding olive oil to the mothers’ diet during pregnancy. An in vivo study showed that an olive oil
enriched diet had preventive effects on hypertriglyceridemia in mothers with gestational diabetes mellitus. In addition, this regime had preventive effects in increasing peroxisome proliferator
activated receptor gamma (PPARγ) and peroxisome proliferator
activated receptor delta levels as well as overaccumulation of lipids in the liver of male fetus rats. This study also showed that olive oil had an inhibitory effect in increasing PPARγ levels in the liver of female fetus rats.84

15.4.6 Olive in rheumatology, rehabilitative medicine, and sports medicine According to TPM, decoction of olive was considered the medicine of choice for the treatment of gout and arthralgia. In addition, topical use of the leaf of olive was recommended for back ache and lumbar disk herniation. Topical administration of olive oil was considered for noninflammatory joint pains (defined as cold-temperament joint pains in TPM), extremity pain, and bone pain sensation.7
11 Studies have shown that using oral olive oil can increase serum calcium levels and decrease protein intake in severely obese adults.85 Having antioxidant86,87 and antiinflammatory properties88 as well as modulating effects on osteoblast physiology, olive oil can have a protective effect against bone pathologies89 and arthritic diseases90 through its phenolic compounds90 and maslinic acid.91 Olive and olive oil have been suggested to potentially control and prevent diseases associated with the overactivation of synovial fibroblasts, such as rheumatoid arthritis88,92,93 and help in the treatment of cartilage degeneration and recovery process in early osteoarthritis, if combined with physical activity.94,95 Topical application of olive oil has also been shown to attenuate inflammatory features of osteoarthritis.96 It has been proven that maslinic acid supplementation combined with moderate resistance training may increase upper muscle mass and grip strength and reduce knee pain, preventing mobilityrelated disability in elderly persons.97 Olive oil, as a constituent of a specific mixture, has also shown to cause relief from knee joint discomfort in healthy individuals.98 An olive oil
derived antioxidant mixture has been shown

Olive in traditional Persian medicine: an overview Chapter | 15

to ameliorate the age-related decline of skeletal muscle function.87 Olive oil supplemented with menaquinone-7 has been shown to significantly affect osteocalcin carboxylation and promote bone health.99 There are mixed data regarding the effect of olive oil on ovariectomy-induced osteoporosis.100,101 However, olive phenols have been suggested to prevent age-related and oxidative stressrelated processes of osteoporosis.102 Phonophoresis with virgin olive oil is an effective method for treatment of chronic lower back pain in female athletes.103 Olive oil, as a constituent of a beverage dietary supplementation, enhances a proinflammatory circulating environment in response to exercise, especially in young athletes.104 In active girls during exhaustive exercise, olive oil has been proven to prevent increasing inflammatory markers.105

15.4.7 Olive in gastroenterology According to TPM sources, the mature fruit of olive is one the medicinal plants that is highly recommended to be consumed at the same time along with the main meals. The sages of TPM believed that this recommendation can enhance the digestion process in the stomach, increase appetite, increase stool consistency, improve abdominal discomfort, and cleanse the inner surface of the stomach as well as have antibacterial and antiparasitic activity. In addition, there were recommendations to perform rectal enema by the decoction of olive for intestinal and rectal ulcers. Moreover, olive oil was suggested in TPM for the treatment of hemorrhoids. According to TPM sources, the olive leaf was useful in relieving pain sensation in splenic disorders.7
11,106 There is some controversy on the effects of olive and its derivatives on the liver. In most studies, however, olive oil,107
113 olive leaf extract,114
118 and olive fruit117,119 have been shown to have hepatoprotective effects120
123 in preeclampsia rats,107 older individuals108 especially those with high cardiovascular risk,109 sodium arsenate
induced hepatotoxicity in mice,110 deltamethrin-induced hepatotoxicity in mice,124 glucose-induced oxidative stress in liver cells,116 obesity-related liver inflammation,114,117 renal ischemic
reperfusion injury in liver tissue115 in ovariectomized rats,118 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced hepatotoxicity,111 carbon tetrachloride
induced hepatotoxicity,112 fluoride-induced toxicity in mice,119 and 2,4-Dinduced liver damage.113 These findings can be explained mainly through the antioxidant,30,45,109,110,113,118
120,122,124 antiapoptotic,124 and antiinflammatory properties109,124 of olive and its derivatives. Olive oil and its products have been proven to modulate gut microbiota and have a potential role in the prevention of inflammation.45,125
129 They improve the gastrointestinal inflammation and discomfort and augment the defensive role of the intestine.130
132 It has been specifically shown that olive oil can have

179

protective effects against pancreatitis133 and ulcerative colitis.134
137 Olive oil has been suggested to improve dyspeptic symptoms.138 Olive oil-based lipid emulsion has been recommended to be used in patients undergoing esophagectomy139 or hepatectomy,140 as it may promote the recovery process. Olive oil has been shown to be as effective as a mineral oil in the treatment of constipation in patients undergoing hemodialysis.141 Furthermore, studies have shown that the extra-virgin olive oil can exhibit preventive properties in gastrointestinal disorders and can increase β-diversity and the abundance of Bifidobacterium.142

15.4.8 Olive in lung and respiratory system According to TPM, the vapor of black olive was recommended for asthma, called “Rabv” in TPM,8,143 and lung diseases. Furthermore, olive was suggested for chronic cough and clearing the lung from sputum.7,9
11 It has been shown that while olive was not involved in reducing upper respiratory tract infections, olive leaf extract has been shown to decrease the duration of infection in high school athletes.144 In another study, olive extract has been suggested to have the therapeutic potential of Olea europaea in inhibiting transforming growth factor beta 1 (TGF-β1)
induced epithelial to mesenchymal transition and persistent inflammation in human nasal respiratory epithelial cells.145

15.4.9 Olive in endocrinology Olive oil has been shown to reduce the risk of developing diabetes in prediabetic patients.146 Olive and its derivatives have been proven to improve the glucose profile147 in healthy individuals148 or patients with diabetes149
154 or prediabetes.155 In addition, olive and its derivatives have been shown to prevent complications due to dyslipidemia156 and to improve metabolic dysfunction157 in the diabetic population. Olive oil has been suggested to improve the effect of aspirin on retinal vascular pattern in experimental diabetes mellitus.158 There is some controversy on the effect of olive and its derivatives on obesity and weight loss. In some studies, however, olive oil and olive leaf extract have been proposed to induce weight loss159,160 and reduce body fat.161
163 Olive oil has been proven to increase mitochondrial and body metabolism if combined with conjugated linoleic acid.164 Olive and its derivatives have been shown to exert beneficial effects on the male reproductive system, including positive effects on androgen hormonal profile of men,165 paraquat-induced oxidative stress in rat epididymis,166 busulfan-induced toxicity in rat testes,167 experimental model of penile fracture in rats168 and genetically modified soya bean
induced injury.169

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Olive oil has been suggested to lower the levels of oxidative stress markers, thyroid and reproductive hormones to the normal, in deltamethrin-induced oxidative stress.170 Specific olive polyphenol extract affects serum osteocalcin levels and may stabilize lumbar spine bone mineral density in postmenopausal women with osteopenia.171

15.4.10 Olive in infectious diseases Olive and its derivatives have been proven to have antimicrobial properties172
181 owing to the presence of secondary metabolites that act independently or in synergy,172 antioxidant activity,176 phenolic compounds,176,180 and active compounds such as oleanolic and maslinic acids179 and oleuropein. Olive leaf extract has been shown to have antimalaria activity.172 Olive oil polyphenol extract and olive leaf extract, as natural preservatives, have been suggested to exert antimicrobial activity against food-borne pathogens.173,175,182 Aqueous olive leaf extract has been used in the production of antimicrobial nanostructures that are effective on highly virulent multidrug-resistant Gram-negative strains of bacteria.174 Ozonized organic extra-virgin olive oil has been shown to be a growth inhibitor of giardia cysts177 Ethyl acetate and methanol extracts of olive leaf have been suggested to have antiAcanthamoeba activity.179 A phenolic derivative of olive has been shown to be effective against standard and clinical isolates of Staphylococcus species.180 Olive oil has also been recommended to be used in the treatment of diabetic foot.183,184

15.4.11 Olive in cardiology In the medical literature, there is some controversy regarding the cardiovascular effects of olive and its derivatives. In most studies, however, the antithrombotic,185 antiinflammatory,186
190 and antioxidant properties30,185
188,191
193 of olive and its derivatives have been proven to exert protective effects on the cardiovascular system.187,192,194
202 These effects include lowering blood pressure,30,128,163,203
210 reducing aging-related histopathological changes of the heart tissue,211 preventing severe exercise-induced cardiac hypertrophy,212 preventing185,186,213
217 and treating213 atherosclerosis, improving blood lipid profile,123,128,129,147,154,157,159,160,171,185,203,206,210,214,215,218
230 reducing postprandial lipid concentration,231 reducing lipid peroxidation,128,232 modulating vascular structure233 and function,188,189,233,234 protecting cardiomyocyte viability,235 increasing the antiinflammatory effect of high-density lipoprotein (HDL) and reducing the age-related decrease in antiatherogenic activity,190 and eliminating the deleterious consequences of ischemia and hypercholesterolemia,191 through various mechanisms. Olive leaf extract has been stated to have potential pharmacokinetic interactions with some antihypertensive drugs.236

Extra-virgin olive oil consumption improves the capacity of HDL to mediate cholesterol efflux.237 It has also been suggested to improve the fatty acid quality of egg yolk while lowering the egg yolk cholesterol level.238 Results from an in vivo study revealed that unfiltered virgin olive oil consists of low molecular weight peptides that can exert an antihypertensive effect possibly through the inhibition of angiotensin-converting enzyme activity.239

15.4.12 Olive in hematology and oncology Olive leaf tea may have more hematological health benefits over green tea and has been shown to provide preventive properties against anemia and other red blood cell disorders.240 Olive oil and olive leaf extract have been demonstrated to have antithrombotic effects via the prolongation of prothrombin and changes in thrombus morphology,241 protection against platelet activation,182,242 platelet adhesion,242 and possibly via antiinflammatory properties.242,243 Olive oil has been shown to exert antioxidant effects on blood cells30 such as human peripheral blood mononuclear cells244 and erythrocytes.245 Olive and its derivatives have been suggested to be used as new anticancer strategies246
259 against mouse mammary tumor growth,246 BRAF-mutated melanoma cells,247 7,12-dimethylbenz(a)anthrazene-induced carcinomas,248 249 glioblastoma progression, T24 bladder cells,251 some 250 breast cancer cell lines such as breast cancer MCF-7,254 skin cancer C32,254 colon cancer cells260 including HT-29 human colon adenocarcinoma cells,252,253 colon cancer SW420254 and SW620,253 benzo(a)pyrene [B(a)P]
induced colon tumors,256 human colon cancer cells (Caco-2),257 ulcerative colitis
associated colorectal cancer,258 and human liver cancer cell lines,255 through various mechanisms. Olive leaf is rich in oleuropein (Ole) which is a bioactive phenolic component with antitumor activity. This activity is related to the inhibitory effect of this bioactive component in performing aerobic glycolysis by tumor. Oleuropein exerts its inhibitory effect on tumor cell proliferation by decreasing glucose transporter-1, protein kinase isoform M2, and monocarboxylate transporter-4 expression. According to the literature, this effect has been shown in melanoma, colon carcinoma, breast cancer, and chronic myeloid leukemia.261 On the other hand, there is little evidence to show that feeding an enriched olive oil diet can induce tumorigenesis via the mechanism of cell growth and cell migration, in the xenograft model of cervical cancer in mice.262

15.4.13 Olive in immunology and allergy TPM sages recommended pouring the decoction of the root of olive trees on the head of the patients with nasal and postnasal discharge.7 Moreover, there are some suggestions

Olive in traditional Persian medicine: an overview Chapter | 15

about using olive leaves in the treatment of allergic rhinitis.263 Several studies have shown that virgin olive oil has antibacterial and antiinflammatory effects; therefore, it can be effective in patients with sinusitis and rhinitis.264 Olive oil has been suggested to be used as an immune adjuvant.202,265 Extra-virgin olive oil has been proven to be beneficial in exerting a preventive/palliative role in the management of systemic lupus erythematosus.254 Dried olive leaf extract has been proposed as a potential dietary agent for prophylaxis/treatment of autoimmune diabetes, and possibly other autoimmune diseases.266 Triacylglycerol-rich lipoproteins derived from healthy donors, who were fed different olive oils, demonstrated the modulation of cytokine secretion and cyclooxygenase-2 expression in macrophages.267 Olive oil, olive wine, and olive fruit extract have all been shown to decrease deoxyribonucleic acid oxidation and plasma inflammatory biomarkers,53,259,268
275 such as lowering high-sensitivity C-reactive protein in human immunodeficiency viruses-infected patients on antiretroviral treatment276 and reducing inflammation and oxidative stress in women with type 2 diabetes.277

15.4.14 Olive in poisonings According to TPM sources, the root of olive tree could be considered an excellent antidote for scorpion venom. Moreover, the topical use of some parts of olive and olive tree in the powder form was recommended for insect bites, scorpion bites, snake bites, etc. In addition, according to TPM, olive oil could be used to stimulate vomiting; hence, it could be helpful for washing and clearing the stomach in patients referred for taking toxic substances. Animal studies have shown that olive leaf extract is beneficial in lead poisoning thorough its antioxidant capacity and reducing apoptosis in the brain.278

15.5 Implication of olive in dentistry and oral cavity based on traditional Persian medicine The chewing of olive is recommended for the treatment of oral aphthous ulcers, toothache, and prevention of dental decay. In addition, the sages of TPM believed that gargling with saline in which olives had been soaked in is useful for strengthening gums and teeth.7,11 Olive and its derivatives can have various applications in dentistry due to its lubricating,279,280 antiinflammatory,281
284 antibacterial,279,285
289 antifungal,290 and antioxidant effects.283,284

15.5.1 Olive in preventive and restorative dentistry Olive oil and its ethanolic extract have shown to have antiplaque activity.279,285,291 This activity is attributed to the

181

modification of the pellicle structure and bioadhesion279 as well as antimicrobial effects.285 Olive oil has the potential to reduce enamel292
294 and dentin294,295 demineralization and aggregate remineralization of the tooth surface293 although in some studies it has been mentioned that it has less effect than fluoride containing products.292,294 By its lubricating effect, olive oil can substantially reduce the wear rates of enamel during tooth grinding.280 The adjunctive application of olive oil with calcium sodium phosphosilicate has a positive impact on the reversal of postsurgical root dentin hypersensitivity.296

15.5.2 Olive in endodontics Ozonated olive oil, either alone or in combination with chitosan nanoparticles, can prevent biofilm formation and eradicate resistant endodontic pathogens from the root canal and, therefore, increase the success rate of endodontic treatment. Ozonated olive oil can be effective when used as the intracanal medicament, root canal sealer, and irrigating solution.286 If used as an endodontic irrigate, a solution of 0.8% alcoholic extract of olive leaves can have a comparable antimicrobial effect to sodium hypochlorite.287 Olive oil can be a promising storage medium to maintain the periodontal ligament cell viability for the avulsed teeth.297,298

15.5.3 Olive in periodontics Olive oil can have preventive279,299,300 and therapeutic299 effects on gingivitis. Ozonated olive oil is proven to affect Gram-negative bacteria more than the Gram-positive ones.288 Mixed data are available about its antibacterial activity compared to chlorhexidine agents.288,300 If applied subgingivally, ozonated olive oil can be used as an adjunctive therapy as well as a monotherapy in improving periodontal conditions.301 Ozonated olive oil gel can be a promising adjunct to scaling and root planning in the treatment of aggressive periodontitis.293 Oleuropein, the most prominent phenolic compound in olive, can decrease alveolar bone loss in periodontal diseases as a result of decreased osteoclastic activity, inflammation, and apoptosis and increased osteoblastic activity.281 Although it neutralizes the antivolatile sulfide effect of triclosan,302 olive oil in a two-phase mouthwash formulation is also suggested to inhibit the production of volatile sulfide, leading to less oral malodor.303

15.5.4 Olive in oral medicine Olive leaf extract ointment and olive oil have healing effects on chemotherapy induced oral mucositis because of their antioxidant and antiinflammatory properties.283,302 Ozonized olive oil can be a new alternative for

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

the control of biofilm in patients with denture stomatitis.290,304,305 Due to its antioxidant functions and enhancement of cell-mediated immunity, extra-virgin olive oil has anticancer activity on lingual squamous cell carcinoma cell line283 and 7,12-dimethyl benzanthracene
induced salivary glands carcinogenesis.306 Ozonated olive oil can improve the wound size and epithelial healing of palatal wounds.307 The topical lycopene-enriched virgin olive oil is effective in treating patients with burning mouth syndrome.308 Daily use of olive oil containing agents can relieve symptoms of dry mouth.309

Olive oil is one of the constituents of zinc oxide eugenol cement and can reduce the irritating effect of eugenol in the cement.314

Avicenna: The most influential Iranian physician and philosopher of the premodern era (CE 980
1037). Humor: The wet fluid mass yielded from the first digestion of food.315 Temperament (Mezaj): A monolithic quality yielded by interaction between opposing qualities of elements.315 Hot temperament: The essence carrying heat as quality.315 Cold temperament: The essence carrying coldness as quality.315 Wet temperament: The essence carrying wetness as quality.315 Dry temperament: The essence carrying dryness as quality.315 Dam: Blood—one of the four bodily humors which is red, hot, wet, and very sweet and is responsible for body development and augmentation as well as keeping it hot and wet.315 Balgham: Phlegm—one of the four bodily humors, which is white, cold, wet, and slightly sweet and has the potential of turning into blood when necessary.315 Safra˜: Bile; chole; one of the four bodily humors which is hot and dry, pure red in color and is the foam of blood; it is light and swift and serves to stimulate intestinal evacuation.315 Sawda˜: Black bile, melanchole, one of the four bodily humors, which is cold and dry, and is a precipitate of blood with an acrid taste and serves to increase the appetite once it stimulates the cardia.315 Hara˜rat-e-gharizi: Innate heat.315 Settet-al-zaruriyyah: The six determinants of health which consists of (1) air, (2) eating and drinking, (3) sleep and wakefulness, (4) motion and stillness, (5) retention and evacuation, and (6) psychological
emotional experiences.315

15.6 Conclusion

References

15.5.5 Olive in orthodontics Ozonized olive oil gel in addition to standard oral hygiene regimens was found to be beneficial for orthodontic patients to prevent enamel decalcification.310 Through increased osteoblast and osteocyte counts and relative bone mineralization of the connective tissue layer forming alveolar bone, olive oil can reduce orthodontic relapse in rabbit models.311 Olive oil can increase TGF-β1 levels in pig models.312 Oleanolic acid acetate, which is richly found in olives, induces bone formation and remodeling through proper modulation of osteoblasts, osteoclasts, and inflammation with regulations of TGF-β and Wnt signaling.313 Ozonated olive oil can enhance secondary stability of mini-screw via a positive effect on cortical bone implant contact and increase in cortical bone area.291

15.5.6 Olive in prosthodontics

Olive is a highly used medicinal plant in TPM. Different parts of olive including fruit, leaves, and roots have been prescribed for medicinal purposes in TPM. The use of olive has been recommended for a wide variety of diseases in different organs including skin, brain, lung, heart, bowels, kidneys, and joints. Many recent studies support the traditional medicinal use of olives. However, more studies are needed to better elucidate the potential benefits of olive and its ingredients in the prevention and treatment of different diseases.

Mini-dictionary of terms Traditional Persian medicine (TPM): A holistic approach to medicine and treatment of various diseases originating from humoral medicine.315

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161. ¨ I¨, Ekici MAG, Alac¸am T, Barı E, Ulusoy C¸. Virgin olive 297. Ulusoy O oil, soybean oil, and Hank’s balanced salt solution used as storage media on periodontal ligament cell viability. Pediatric Dent. 2019;41(6):485
488. ¨ , Erdo˘gan Y. 16-Year follow-up of 298. Kırzıo˘glu Z, Erken Gu¨ngo¨r O an avulsed maxillary central incisor after replantation following 10-h storage: an unusual case. Spec Care Dent. 2017; 37(4):199
203. 299. Bansal A, Ingle NA, Kaur N, Ingle E, Charania Z. Effect of gum massage therapy with honey and olive oil on common pathogenic oral micro-organisms: a randomized controlled clinical trial. J Int Oral Health. 2015;7(11):63. 300. Younis SK, Salman FD. Effect or different concentrations of acidic olive leaves extractmouth rinse on plaque, gingivitis and periodontal pockets on adults. Tikret J Pharm Sci. 2008;4 (2):104
116. 301. Patel P, Patel A, Kumar S, Holmes J. Effect of subgingival application of topical ozonated olive oil in the treatment of chronic periodontitis: a randomized, controlled, double blind, clinical and microbiological study. Minerva Stomatol. 2012;61(9):381
398.

302. Young A, Jonski G, Ro¨lla G. A study of triclosan and its solubilizers as inhibitors of oral malodour. J Clin Periodontol. 2002;29 (12):1078
1081. 303. Yaegaki K, Sanada K. Effects of a two-phase oil-water mouthwash on halitosis. Clin Prev Dent. 1992;14(1):5
9. 304. Crastechini E, Koga-Ito CY, de Fa´tima Machado S, et al. Effect of ozonized olive oil on oral levels of Candida spp. in patients with denture stomatitis. Braz Dental Sci. 2018;21(1):111
118. 305. Al-Jader GH. Antimicrobial Activity of Olive Leaf Extract and Dental Gel Against Some Pathogenic Fungi and Bacteria. 2009. 306. Ziu M, Giasuddin A, Mohammad A. Anti-carcinogenic effect of virgin olive oil on DMBA-induced salivary glands carcinogenesis in rats. Med J Islamic World Acad Sci. 1993;6(4):306
310. 307. Patel PV, Kumar V, Kumar S, Gd V, Patel A. Therapeutic effect of topical ozonated oil on the epithelial healing of palatal wound sites: a planimetrical and cytological study. J Invest Clin Dent. 2011;2(4):248
258. 308. Cano-Carrillo P, Pons-Fuster A, Lo´pez-Jornet P. Efficacy of lycopene-enriched virgin olive oil for treating burning mouth syndrome: a double-blind randomised. J Oral Rehabil. 2014;41(4):296
305. 309. Ship J, McCutcheon J, Spivakovsky S, Kerr A. Safety and effectiveness of topical dry mouth products containing olive oil, betaine, and xylitol in reducing xerostomia for polypharmacy-induced dry mouth. J Oral Rehabil. 2007;34(10):724
732. 310. El-Tokhey SAGaM. In vivo study of the effectiveness of ozonized olive oil gel on inhibiting enamel demineralization during orthodontic treatment. J Am Sci. 2012;8(10). 311. Al-Hamdany AK, Al-Khatib AR, Al-Sadi HI. Influence of olive oil on alveolar bone response during orthodontic retention period: rabbit model study. Acta Odontol Scand. 2017;75(6):413
422. 312. Suparwitri S, Noviasari P. Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs. F1000Research. 2019;8(2028):2028. 313. Adhikari N, Neupane S, Aryal YP, et al. Effects of oleanolic acid acetate on bone formation in an experimental periodontitis model in mice. J Periodontal Res. 2019;54(5):533
545. 314. Jyoti BB. Phytotherapeutics in conservative dentistry & endodontics—a review. J Conserv Dent. 2005;8(2):31. 315. Shirzad M, Iran-Nejad S, Cheraqi Niroumand M, Shams Ardekani M. Iranian Traditional Medicine: A Dictionary. Tehran: Traditional Medicine and Materia Medica Research Center; 2014.

Chapter 16

The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Rafael de la Torre1,3, Montserrat Fito´2,3 and Marı´a-Isabel Covas4 1 2 3

Integrative Pharmacology and Systems Neurosciences, IMIM-Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, Spain, Cardiovascular Risk and Nutrition Research Groups, IMIM-Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, Spain, Biomedical Research Network Center on Obesity and Nutrition, Madrid, Spain, 4NUPROAS Handelsbolag (NUPROAS HB), Nacka˜, Sweden

Abbreviations apolipoprotein cholesterol efflux cardiovascular disease diastolic blood pressure European Food Safety Authority The effect of olive oil consumption on oxidative damage in European populations Hs-CRP high-sensitivity C-reactive protein HVAL homovanillyl alcohol MedDiet Mediterranean diet MUFA monounsaturated fatty acids OHTyr hydroxytyrosol OOPC olive oil phenolic compounds PON1 paraoxonase 1 PPARα peroxisome proliferator
activated receptor alpha PREDIMED The effect of Mediterranean diet on the primary prevention of cardiovascular disease SBP systolic blood pressure Tyr tyrosol VOO virgin olive oil Apo CE CVD DBP EFSA EUROLIVE

16.1 Background The beneficial effects of olive oil on cardiovascular risk factors are usually attributed to its high levels of monounsaturated fatty acids (MUFA), particularly to oleic acid. Olive oil is, however, more than a MUFA fat. Olive oil is an olive juice considered to be a functional food that, besides having a high MUFA level, contains minor components with biological properties.1 The content of the minor components of olive oil varies, depending on several conditions such as the

cultivar, climate, ripeness of the olives at harvesting, and the processing system employed to produce the olive oil. Three types of olive oil are currently present on the market: virgin, ordinary, or pomace.1 Virgin olive oils (VOOs) are produced by direct pressing or centrifugation of the olives, and among them, those with an acidity greater than or equal to 3.3 degrees (2 degrees in the European Union) are submitted to a refining process in which some components, mainly phenolic compounds, and to a lesser degree squalene, are lost.2 By mixing virgin and refined olive oils, an ordinary olive oil3 is produced and marketed. After VOO production the rest of the olive drupe and seed is processed and submitted to a refining process, resulting in pomace olive oil, to which a certain quantity of VOO is added before marketing. The minor components of VOO are classified into two types: the unsaponifiable fraction, defined as the fraction extracted with solvents after the saponification of the oil, and the soluble fraction that includes the phenolic compounds.1

16.2 Bioavailability of olive oil phenolic compounds 16.2.1 Absorption and disposition After olive oil ingestion, glycosides and aglycones (ligstroside and oleuropein) of tyrosol (Tyr) and hydroxytyrosol (OHTyr), the main olive oil phenolic compounds (OOPC),1,2 undergo rapid hydrolysis under gastric conditions with significant increases in the amount of Tyr and OHTyr free forms entering the small intestine.4 In in vitro models, both OHTyr and Tyr are able to cross human Caco-2 cell monolayers, via a bidirectional passive diffusion mechanism,5 and also rat

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00022-5 © 2021 Elsevier Inc. All rights reserved.

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segments of jejunum and ileum.4 In animal models an oral oily dose of Tyr and OHTyr promoted a recovery of the phenolics in 24 h urine greater (25%) than the obtained with an oral aqueous dose.6 In humans, there were also differences in Tyr or OHTyr recoveries according to the matrix in which OOPC are administered. Urinary OHTyr recovery was higher after VOO administration (44.2% of the OHTyr administered) than after the addition of OHTyr to a refined olive oil (23% of the OHTyr administered), or to a yogurt (5.8% of dose).7 Concerning oleuropein, data from animal models show it can be absorbed, albeit poorly, from isolated perfused rat intestine. The mechanism of absorption is unclear but may involve transcellular transport (sodium
glucose-linked transporter) via oleuropein-glycoside moiety or paracellular movement. However, the most plausible way for oleuropein to exert its biological activities seems to be through its conversion to OHTyr.8 This idea is supported by the results of bioavailability studies in rats, in which peak plasma concentrations after ingestion of high doses of oleuropein (100 mg/kg) were in the nanogram range, whereas those of OHTyr were highly increased.9, 10 These observations have been further confirmed in humans.11 In the process of crossing epithelial cells of the gastrointestinal tract, OOPC are subjected to an important firstpass metabolism. According to data of in vitro studies, about 10% of OHTyr is converted into homovanillyl alcohol (HVAL) by the catechol-O-methyltransferase.5 In addition to this O-methylated derivative, the glucuronides of OHTyr and Tyr have also been described.4 In contrast, there was no absorption of oleuropein as it was rapidly degraded by the colonic microflora resulting in OHTyr formation.4 In human hepatoma HepG2 cells, methylated and glucuronide forms of OHTyr were detected after 18 h of incubation, together with methyl-glucuronide metabolites. OHTyr acetate was largely converted into free OHTyr and subsequently metabolized, although small amounts of glucuronidated hydroxytyrosyl acetate were detected. Tyr was poorly metabolized, with ,10% recovered as glucuronide after 18 h. Minor amounts of free or conjugated phenols were detected in cell lysates. No sulfated metabolites were found.12 The pharmacokinetics of OHTyr intravenously administered to rats indicates a fast and extensive uptake of the molecule by the organs and tissues, with a preferential renal uptake.13 OHTyr was recovered mainly as sulfate conjugates. The recovery of OHTyr in urine was about 6% of the dose administered: 0.3% recovered as 3-methyl4-hydroxy-phenylethanol (HVAL or MOPET), 12.3% as 3,4-dihydroxyphenylacetic acid (DOPAC), 23.6% as homovanillic acid (3-methyl-4-hydroxy-phenylacetic acid, HVA), and 26% as 3,4-dihydroxyphenylacetaldehyde (DOPAL).13 In another study in rats the oral administration of increasing doses (1, 10, and 100 mg/ kg) of OHTyr (given in a refined olive oil matrix)

showed its accumulation in a dose-dependent manner not only in urine and plasma but also in the liver, kidney, and brain.14 Although nonabsorbable phenolic compounds can exert local antioxidant activities in the gastrointestinal tract, one of the prerequisites to assess OOPC physiological significance, however, is to have information regarding their bioavailability and metabolic disposition in vivo in humans. The first report on the bioavailability of OOPC in humans was provided by spiking Tyr and OHTyr to a refined olive oil (very low phenolic content) and administered to healthy volunteers. Phenolic compounds were dose dependently absorbed in humans, most of them being recovered in biological fluids as conjugates.15 Also in human studies, it was demonstrated that Tyr, OHTyr, and oleuropein were absorbed at the small intestine level.11 Phenolic compounds, particularly those bearing a catechol group, are typically biotransformed by three enzymatic systems: catechol-O-methyltransferase, sulfatases, and glucuronosyltransferases. Depending on the dose and the availability of cofactors, the proportion of methyl, sulfate, and glucuronide conjugates varies among subjects. Further studies on the OOPC bioavailability were performed with VOO in its natural form.16
18 After administering 25 mL of VOO (with an estimated content 1.2 mg of OHTyr), OHTyr plasma concentrations peaked at 30 min and those of its methylated metabolite, the HVAL at 50 min. Plasma peak concentrations were around 25 ng/mL for OHTyr and 4 ng/mL for HVAL. The estimated half-life for OHTyr was 3 h, reaching baseline concentrations after 8 h of the VOO ingestion (Fig. 16.1). More than 98% of both OHTyr and HVAL were in their conjugated forms, mainly glucuronates, confirming previous findings. In urine, OHTyr and HVAL concentrations peaked in the collection period 0
2 h.17 Despite the short half-life of Tyr and OHTyr, sustained consumption promotes an increase of OOPC in biological fluids (Fig. 16.2). Plasma and urinary levels of OHTyr and Tyr increase in a dose-dependent manner with the phenolic content of the olive oil administered (Fig. 16.2).18
22 Table 16.1 shows the urinary recoveries of OHTyr, Tyr, and HVAL after olive oil of medium (164 mg/kg) and high (466 mg/kg) phenolic content. With regard to the dose
effect relationship, 24 h urinary Tyr seems to be a better biomarker of sustained and moderate doses of VOO consumption than OHTyr.18 Both OHTyr and Tyr urinary concentrations are used in nutritional intervention studies as biomarkers of VOO ingestion.19
26 After ingestion of an enriched VOO with its OOPC, OHTyr sulfate was detected not only in plasma but also in erythrocytes.27 Concerning secoiridoids, when comparing the bioavailability of oleuropein-glycoside in ileostomy patients versus subjects with an intact colon, it was concluded that oleuropein was recovered mainly as OHTyr in healthy

The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Chapter | 16

195

9 8

LPC MPC HPC

Conc. (ng/mL)

7 6 5 4 3 2 1 0 0

24

48

72

96

h Without supplementation

After supplementation

FIGURE 16.1 Plasma hydroxytyrosol concentrations after ingestion of olive oil with low (LPC, 10 mg/kg), medium (133 mg/kg), and high (486 mg/kg) phenolic content. A single 25 mL dose was ingested before and after 4 days of olive oil supplementation (25 mL/day).19 (Adapted from Weinbrenner et al. (2004)).

individuals.11 Therefore at dietary doses the secoiridoids are essentially biotransformed by gut microflora, thus giving rise to the simple phenols OHTyr and Tyr. When oleuropein was administered at nondietary higher doses, a small fraction of this compound was detected in body fluids as oleuropein metabolites/derivatives,28 as has been previously observed in animal models.29

250

Percentage of change

Tyrosol Hydroxytyrosol 200

150

100

16.2.2 Metabolism

50

0

Low

Medium

High

Olive oil phenolic content FIGURE 16.2 Changes in urinary tyrosol and hydroxytyrosol after 3 weeks consumption of olive oil with high (366 mg/kg), medium (164 mg/kg), and low (2.7 mg/kg) phenolic content.1 (Adapted from Covas et al. (2006c)).

TABLE 16.1 Urinary recoveries (μmol) of olive oil phenolic compounds. Olive oil

Hydroxytyrosol

Tyrosol

HVAL

MPC

1.2 6 0.1

1.3 6 0.1

0.4 6 0.1

HPC

3.1 6 0.2

2.7 6 0.2

0.8 6 0.1

Values are expressed as mean 6 SD. HPC, High-phenolic content olive oil (466 mg/kg); HVAL, 3-methyl-4-hydroxyphenylethanol, the methylated biological metabolite of hydroxytyrosol; MPC, mediumphenolic content olive oil (164 mg/kg).

The number of OHTyr metabolites reported is increasing and so far more than 10 metabolites have been described. These include O-methylated forms,30 aldehydes and acids formed via oxidation of the aliphatic alcohol,13 sulfates,27, 31 glucuronides,32 acetylated and sulfated derivatives,33 as well as an N-acetylcysteine derivative.34 The latter was identified in a study in rats in which the dose-dependent metabolic disposition of OHTyr was investigated at three different doses (1, 10, and 100 mg/kg). Following OHTyr administration, dose-dependent variations in the recovery of all the metabolites evaluated were observed. At the lowest dose of 1 mg/kg the glucuronidation pathway was the most relevant (25%
30%), with lower recoveries for sulfation (14%). On the contrary, at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). The mercapturate conjugate of OHTyr (N-acetyl-5-Scysteinyl-OHTyr) was formed in a dose-dependent manner.34 Regarding the enzymes involved in OHTyr metabolism, we can differentiate between two major families. Those implicated in OHTyr phase I metabolism are cytosolic: non-microsomal alcohol and aldehyde dehydrogenases. The corresponding metabolites are 3,3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylacetic

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acid (DOPAC). The enzymes involved in OHTyr phase II reactions are sulfotransferases, uridine 50 -diphosphoglucuronosyl transferases, catechol-O-methyltransferases, and acetyltransferases.35 A metabolite resulting from methylation and glucuronidation has also been identified, although this represents a minor metabolic pathway.17, 32 A general representation of OHTyr metabolites is depicted in Fig. 16.3.

16.2.3 Endogenous sources of Tyr and OHTyr and endogenous bioconversion of Tyr into OHTyr Tyr and OHTyr are tyramine and dopamine oxidative metabolites, respectively, and ethanol consumption increases their endogenous formation as has been described both in animal studies36, 37 and in human clinical trials.38, 39 Also, preclinical studies have identified Tyr hydroxylation, mediated by cytochrome P450

isoforms CYP2A6 and CYP2D6, as an additional source of OHTyr.40 Ethanol plays a moderate inhibitory effect in this bioconversion.41 Tyr bioconversion into OHTyr in vivo in humans from Tyr enriched wine has been now assessed in human randomized clinical trials.41, 42 The most probable major mechanisms were (1) an increase of Tyr (and OHTyr) bioavailability due to ethanol, (2) an endogenous CYP-catalyzed conversion of Tyr into OHTyr (slightly inhibit by ethanol), and, to a lesser extent, (3) an ethanol-induced rise in both Tyr and OHTyr production following an alteration in tyramine and dopamine oxidative metabolisms.42

16.3 Bioactive effects of olive oil phenolic compounds in humans 16.3.1 Lipids and lipoproteins A review of eight studies indicated that the intake of high-phenol VOO did not promote significant increases in

FIGURE 16.3 HT metabolic pathways. ACT, O-Acetyl transferase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; COMT, catecholO-methyltransferase; CYP2A6 and CYP2D6, cytochrome P450 isoforms; GGT, c-glutamyl transferase; GlcA, glucuronic acid; HT, Hydroxytyrosol; HVAlc, homovanillyl alcohol; NAT, N-acetyl transferase; SULT, sulfotransferase; UGT, UDP-glucuronosyl transferase. (Adapted from Rodrı´guezMorato´ J, Boronat M, Kotronoulas A, et al. Metabolic disposition and biological significance of simple phenols of dietary origin: hydroxytyrosol and tyrosol. Drugs Met Rev. 2016;48:218
236; Boronat A, Mateus J, Soldevila-Domenech N, et al. Cardiovascular benefits of tyrosol and its endogenous conversion into hydroxytyrosol in humans. A randomized, controlled trial. Free Rad Biol Med. 2019;143:471
481).

The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Chapter | 16

total, low-density lipoprotein (LDL) or high-density lipoprotein (HDL) cholesterol level.43 However, in some clinical trials comparing the benefits of olive oils with differences in phenolic content, there was an increase in HDL cholesterol with the phenol-rich olive oil.19
22 In this regard, the increase in HDL cholesterol occurred in a dose-dependent manner with the phenolic content of the olive oil administered in the EUROLIVE study (The effect of olive oil consumption on oxidative damage in European populations), a multicenter clinical trial with 180 participants.22 In the EUROLIVE the total/HDL cholesterol and LDL/HDL cholesterol ratios decreased directly with the phenolic content of the olive oil administered from 366 to 2.7 mg/kg).22 Also, consumption of a high polyphenol
enriched VOO (500 mg/kg) improved the lipoprotein particle atherogenic ratios, such as total LDL particle/total HDL particle, small HDL/large HDL, and HDL cholesterol/HDL particle, and the subclasses profile versus a natural VOO in hypercholesterolemic individuals.44 OOPC have been shown to be able to upregulate the cholesterol metabolism-related gene expression pathways in vivo in humans. In this regard, peroxisome proliferator
activated receptor (PPAR) related, ABCA1, and SRB1 were upregulated in both, acute and sustained consumption of a VOO rich in phenolic compounds.45, 46 Sustained doses of functional olive oils (500 mg/kg of phenolics) enhanced the expression of key cholesterol efflux (CE) regulators particularly when mixed phenolics from olive oil and thyme (1:1) were involved.46 Data from a subsample of the EUROLIVE study showed that a natural high phenolic
content VOO (366 mg/kg) increased the HDL-mediated CE from macrophages compared with low-phenolic content olive oil in healthy subjects.47 In this study47 the improvement of HDL fluidity and a triglyceride-poor core could promote the HDL functionality. No improvement in HDL CE, however, was observed after olive oil enriched with its phenolic compounds (500 mg/kg) versus a natural VOO (80 mg/kg) in hypercholesterolemic individuals.48 Thus although data are promising, further randomized, controlled studies are required to establish the role of VOO and its phenolic compounds on HDL functionality.

16.3.2 Oxidative damage Oxidation of the lipid part, or directly of the apolipoprotein (Apo) B, of the LDL leads to changes in their conformation by which the LDL entry into the monocyte/ macrophage system of the arterial wall is enhanced and, due to this, atherosclerotic process developed.49 HDL protects LDL from oxidation.50 Also, HDL oxidation impairs HDL functionality rendering the lipoprotein less useful for the CE from macrophages.50 Consumption of VOO

197

rich in phenolic compounds has shown to provide additional protection, other than that provided by the oleic acid, in front of in vivo human HDL and LDL oxidation.22, 47 On November 2011 the European Food Safety Authority (EFSA) released a claim concerning the benefits of daily ingestion of olive oil rich in phenolic compounds, such as VOO.51 In order to bear the claim, 5 mg of OHTyr and its derivatives (e.g., oleuropein complex and Tyr) in olive oil should be consumed daily. These quantities, if provided by moderate amounts of olive oil, can be easily consumed in the context of a balanced diet. Here, we revise the main studies that supported this EFSA claim. Also, we will report the evidences concerning the protection of in vivo HDL oxidation in humans by phenol-rich VOO.

16.3.2.1 Postprandial effects Postprandial serum lipid levels have been found to correlate more closely to cardiovascular disease (CVD) than fasting lipids.52 Activation of PPARα suppresses postprandial lipemia through fatty acid oxidation in enterocytes.53 Functional olive oil enriched with its own phenolic compounds has shown to enhance the in vivo gene expression of PPARα in human peripheral blood mononuclear cells.45 Data on the effect of olive oil rich in phenolic compounds on the postprandial oxidative stress are difficult to compare. Some studies do not mention whether or no postprandial lipemia and/or hyperglycemia, which leads to oxidative stress, occurs after olive oil ingestion. In other studies, neither hyperlipidemia nor hyperglycemia occurs after olive oil ingestion. Ingestion of a 25 mL olive oil dose did not promote postprandial oxidative stress independently of the phenolic content of the olive oil,54 whereas single doses of 4021 and 50 mL55 did. With olive oil doses at which oxidative stress occurs, data from randomized, crossover, controlled human studies showed (1) an increase in the serum antioxidant capacity after VOO ingestion56 and (2) that the phenolic content of an olive oil modulated the degree of lipid and LDL oxidation, being the lipid oxidative damage lower after highthan after low-phenolic content olive oil.21, 57

16.3.2.2 Sustained consumption effects Controversial results have been obtained in small sample size (nB30), randomized, controlled human studies on the effect of sustained doses of OOPC on oxidative stress.23 Extensive differences existed among the studies in the experimental design, control of diet, sample population, age of participants, measurement or not of markers of the compliance of the intervention, as well as in the sensitivity and specificity of the oxidative stress biomarkers evaluated.

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The results of the EUROLIVE study, however, provided final evidence of the in vivo protective role of phenolic compounds from olive oil on lipid oxidative damage in healthy individuals, at real-life olive oil doses.22 The EUROLIVE study was a large, crossover, multicenter, clinical trial performed in 180 individuals from five European countries. Participants were randomly assigned to receive 25 mL/day of three similar olive oils, but with differences in their phenolic content (from 2.7 to 366 mg/kg of olive oil), in intervention periods of 3 weeks preceded by 2-week washout periods. All olive oils increased the HDL cholesterol and the ratio between the reduced and oxidized forms of glutathione. Consumption of mediumand high-phenolic content olive oil decreased lipid oxidative damage biomarkers such as plasma oxidized LDL, uninduced conjugated dienes, and hydroxy fatty acids. However, the most important results of the EUROLIVE study were that the increase in HDL cholesterol and the decrease in the lipid oxidative damage were linear with the phenolic content of the olive oil consumed. Also, in agreement with the decrease in oxidized LDL, an increase in autoantibodies against the oxidized LDL was observed in the frame of the EUROLIVE.58 The results of the EUROLIVE study provided first-level evidence that olive oil is more than a MUFA fat. Data from a subsample (n 5 990) of the PREDIMED study (The effect of Mediterranean diet on the primary prevention of cardiovascular disease) show that a Mediterranean diet (MedDiet), but only when enriched in VOO (316 mg/kg), decreased the LDL oxidation when compared with the control group (low-fat diet) after 3-month59 and 1-year interventions.24 A recent metaanalysis (N 5 13) reported that oxidized LDL levels decrease significantly after the consumption of high-phenolic VOO [n 5 300; mean effect: 20.25; CI: (20.50, 0.00); P 5 .05].43 Protective effects of OOPC on in vivo DNA oxidation, measured by the urinary excretion of 8-oxodeoxyguanosine, were found in healthy male subjects in a short-term study in which participants were submitted to a very low antioxidant diet.19 A protective effect on DNA oxidation, measured by the comet assay, was observed in postmenopausal women.60 Results of the EUROLIVE study, however, showed that consumption of 25 mg/day of olive oil during 3 weeks reduced DNA oxidation, measured by the 24 h urinary excretion of 8-oxodeoxyguanosine, in 182 healthy males irrespective of the phenolic content in olive oil.61 Differences in the type of population involved (with or without oxidative stress) could explain the differences among the results. The protective effects on oxidation markers in human trials are better displayed in oxidative stress conditions.1 Oxidative stress is linked to other pathological conditions present in chronic degenerative diseases such as inflammation, endothelial dysfunction, and hypertension. Here, we

revise the available information related to the role of OOPC-rich VOO on these issues.

16.3.3 Inflammation The protective mechanism of oleic acid-rich diets on inflammation has been attributed to a decrease in the LDL linoleic acid content. However, oleic acid is not the single responsible factor for the antiinflammatory properties of olive oil. Several studies have examined the antiinflammatory and vasculo-protective effect of OOPC in humans (Table 16.2). In these studies, VOO with high phenolic content has been shown to be effective in reducing the postprandial response of eicosanoid inflammatory mediators such as thromboxane B2 and prostaglandin 6-keto-PGF1α,56, 62, 63 the lipopolysaccharide, and the expression of proinflammatory genes,68 as well as other plasma inflammatory markers, such as high-sensitivity C-reactive protein (hs-CRP) or IL-6.25, 66 In a nonplacebo controlled study, extra-VOO consumption during 12 weeks reduced the age-related decrease in HDL and paraoxonase 1 (PON1) antiinflammatory activities in healthy volunteers.64 Concerning the effect on cell adhesion molecules, a decrease in serum intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at postprandial state after VOO ingestion when compared with refined olive oil ingestion has been reported.65 In sustained consumption studies a reduction in IL-6 and hs-CRP, but not in ICAM-1 or VCAM-1 levels, was reported related to the phenolic content of the olive oil in CVD patients.25 In patients with early atherosclerosis, however, a decrease in ICAM-1 in leukocytes was observed after VOO, but not after VOO enriched with epigallocatechin gallate.67 The effect of olive oil polyphenols modulating, toward a protective mode, the expression of proinflammatory genes is described in Section 16.4.2.

16.3.4 Endothelial function, blood pressure, and thrombosis Several studies reported beneficial effects of phenol-rich VOO on the endothelial function. An improved postischemic hyperemia via reduced oxidative stress and increased nitric oxide (NO) metabolites occurred after the acute intake of phenol-rich VOO in comparison with a low-phenol olive oil in hypercholesterolemic patients.57 Beneficial effects improving the endothelial function have been observed after a 4-month diet with polyphenol-rich olive oil in patients with early atherosclerosis.67 An enriched VOO improved the endothelial function in hypertensive patients by increasing ischemic reactive hyperemia,69 as has been previously described in

The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Chapter | 16

199

TABLE 16.2 Human randomized controlled studies on the effect of phenol-rich olive oil on inflammatory markers. Type of study

Interventions

Participants

Biomarkers

Results

References

Two consecutive periods, no washout diets ad libitum

VOO vs oleic acid
rich sunflower oil

12 postmenopausal women

TXB2 in PRP TXB2 in urine 6-keto-PGF1α

Lower in VOO Similar Similar

[62]

Randomized, crossover

VOO vs refined olive oil (intervention, 40 mL/day, 7 weeks; washout period, 4 weeks with usual diet)

Hyperlipidemic patients (12 men and 10 women)

Serum TBX2

Decrease with the phenolic content of the olive oil

[63]

Randomized, crossover postprandial

VOO vs refined olive oil (50 mL with potatoes)

12 healthy men

Plasma LTB4 Plasma TBX2

Decrease with the phenolic content of the olive oil

[56]

Randomized, crossover, postprandial

Fat meal with VOO vs refined olive oil, after 1 week of each (50 mg/m2 body surface)

Healthy (14) and hypertriglyceridemic (14) men

Serum ICAM-1 and VCAM-1 area under curve

Decrease with the phenolic content of the olive oil

[64,65]

Randomized, crossover

VOO vs refined olive oil (intervention period, 50 mL/day, 3 weeks; washout period 2 weeks with refined olive oil ad libitum)

28 men with CHD

hs-CRP, IL-6

Decrease with the phenolic content of the olive oil

[25]

Serum ICAM-1 and VCAM-11

No changes

Randomized, crossover

VOO vs oleic acid
rich sunflower oil

24 women with high-normal BP or stage 1 essential hypertension

ADMA hs-CRP

Decrease with the phenolic content of the olive oil

[66]

Randomized, parallel

VOO vs VOO 1 EGCG (30 mL/day, 4 months)

54 patients with early atherosclerosis

ICAM-1 White blood cells

Decrease with VOO

[65,67]

Randomized crossover

Breakfasts with VOO of high, intermediate, and low phenolic content

49 metabolic syndrome patients

Postprandial increase of lipopolysaccharide, NFKB, and other inflammatory genes

Repressed with high-phenol VOO

[68]

6-keto-PGF1α, 6-keto-prostaglandin 1α; ADMA, asymmetric dimethylarginine; CHD, coronary heart disease; hs-CRP, high-sensitivity C-reactive protein; ICAM-1, intercellular adhesion molecule-1; IL-1β, interleukin 1β; IL-6, interleukin 6; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; LTB4, leukotriene B4; MIF, macrophage migration inhibitory factor; NFκB, nuclear factor-kappa B; PRP, platelet-rich plasma; TNF-α, tumor necrosis factor alpha; TXB2, thromboxane B2; VCAM-1, vascular cell adhesion molecule-1.

hypertensive women after the consumption of VOO with a high PC content.66 Consumption of olive oil is known to reduce blood pressure. OOPC could play an additional beneficial role. A 2-month diet with olive oil rich in polyphenols decreased systolic and diastolic blood pressure (SBP and DBP) and improved endothelial function, in young women with mild hypertension compared with the same diet with low-polyphenol content olive oil.66 In this study,66 changes in blood pressure and endothelial

function were concomitant with markers related to vasodilatation, such as an increase in NO and a decrease in serum asymmetric dimethylarginine, as well as a reduction in oxidized LDL and hs-CRP. A decrease in SBP after VOO consumption, in comparison with refined olive oil, in hypertensive stable patients with CVD has been reported, together with a decrease in lipid oxidation.70 In a parallel study comparing the effect of VOO on blood pressure in diabetic patients and healthy individuals, a reduction in SBP was observed in both conditions.71 In a

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metaanalysis with 13 studies concerning the effects of high-phenolic VOO on cardiovascular risk factors, moderate effects for lowering SBP, but not DBP, were [mean effect: 20.52; CI: (20.77, 20.27); P , .01].43 Concerning thrombosis, the consumption of phenolrich VOO improved the postprandial prothrombotic state (activated coagulation factor VII, tissue factor, tissue plasminogen activator, plasminogen activator inhibitor type-1, and fibrinogen) in randomized controlled trials in both healthy and hypercholesterolemic subjects.72, 73 The role of phenolic compound-rich oils compared with a lowphenolic content oil on thromboxane B2, an inflammatory mediator and potent platelet aggregation inductor, has been discussed earlier (Table 16.2).

16.4 In vivo basic mechanisms assessed in human studies for explaining the bioactivity of olive oil rich in phenolic compounds There is a huge body of experimental studies concerning the basic mechanisms by which VOO and its phenolic compounds could exert their beneficial effects. From these, only two have been reported to occur in vivo in humans: (1) the increase in the antioxidant content of lipoproteins and (2) a nutrigenomic effect.

16.4.1 Increase in the antioxidant content of lipoproteins 16.4.1.1 Increase in the antioxidant content of low-density lipoprotein The susceptibility of LDL to oxidation depends not only on its fatty content but also on the LDL antioxidant content bound to the LDL.74 The total polyphenol content bound to human LDL increases in a dose-dependent manner with the phenolic content of the olive oil administered.21 OHTyr and Tyr metabolites bind human LDL after VOO ingestion, but not after refined olive oil ingestion, in an inverse relationship with plasma oxidized LDL.75 Phenolic compounds that can bind LDL are likely to perform their peroxyl scavenging activity in the arterial intima, where full LDL oxidation occurs.49

16.4.1.2 Increase in the antioxidant content of high-density lipoprotein An increase in the lipophilic antioxidant content of HDL, such as ubiquinol, beta-cryptoxanthin, lutein, and retinol, as well in hydrophilic antioxidants such as thymol sulfate, caffeic and hydroxyphenylpropionic acids sulfate, has been observed after VOO enriched with OOPC, and

OOPC plus thyme phenolics, when comparing with a controlled VOO (80 mg/kg).48 Data from a subsample of the EUROLIVE study show that OOPC biological metabolites, such as OHTyr and HVAL sulfate and glucuronide, bind to HDL in a dose-dependent manner of the phenolic content of the olive oil administered.47 ApoA
I and PON1 are main agents involved in HDL antioxidant capacity. PON1 is present in the circulation mainly linked to HDLs and is a key factor for the hydrolysis of oxidized lipids in plasma, a mechanism that also protects LDL from oxidation.76 In human studies, VOO consumption enhanced both PON1 plasma concentrations and PON1 antioxidant activity.27, 64

16.4.2 Nutrigenomic effect of virgin olive oil and its phenolic compounds Within the nutrigenomic field, we here focus on the transcriptomic data available from randomized, controlled human studies. Using microarray techniques, it has been reported that a breakfast based on OOPC-rich VOO (398 ppm) was able to postprandially repress the expression of proinflammatory genes when compared with a common olive oil
based breakfast (70 ppm) in metabolic syndrome individuals.77 The impact of OOPC, and also that OOPC mixed with thyme polyphenols, on CE-related genes has been referred to previously.45, 46, 48 In the frame of the PREDIMED trial, in one substudy,78 a 3-month intervention with VOO-enriched MedDiet prevented the increase in cyclooxigenase-2 and LDL receptor
related protein genes, as well as reduced the expression of monocyte chemoattractant protein 1 gene. This fact did not occur when the same MedDiet was supplemented with nuts, or low-fat diets were consumed. In a second study,79 and after 3-month intervention, functional annotation analyses showed that from 18 cardiovascular canonical pathways, 9 were modulated, toward a protective mode, by a MedDiet enriched with VOO, 4 when the MedDiet was enriched with nuts and none with a low-fat diet.79 Also, within the frame of a MedDiet, we have reported in healthy subjects that only after the supplementation of MedDiet with polyphenol-rich VOO, but not with polyphenol-low VOO, there was a significant decrease versus control group in the expression of inflammation-related genes.80 In an EUROLIVE substudy, we proposed for the first time an integrated scheme for the in vivo downregulation of the CD40/CD40L system and its downstream products promoted by the consumption of polyphenol-rich VOO.81 Also, within the EUROLIVE frame, the high-polyphenol VOO modulated the expression of genes related to the renin
angiotensin
aldosterone system, underlying the decrease in the systolic blood pressure observed.82

The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Chapter | 16

16.5 Conclusion From all referred previously, OOPC are bioavailable in human, their bioavailability improving when they are in olive oil. From data of randomized controlled trials comparing olive oils with differences in their OOPC content, and despite a lack of homogeneity among studies, OOPC reduce oxidative damage in a dose-dependent manner with the phenolic content present in the olive oil. Also, OOPC-rich VOO decreases inflammation and reduces blood pressure improving endothelial function in humans. Mechanisms by which OOPC can exert their protection “in vivo” in humans are linked to (1) an increase in the HDL and LDL lipoproteins antioxidant content and (2) to a nutrigenomic effect modulating toward a protective mode atherosclerosis-related gene.

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190. 19. Weinbrenner T, Fito´ M, de la Torre R, et al. Olive oils high in phenolic compounds modulate oxidative/antioxidative status in men. J Nutr. 2004;134:2314
2321. 20. Marrugat J, Covas MI, Fito´ M, et al. Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation. A randomized controlled trial. Eur J Nutr. 2004;43:140
147. 21. Covas MI, de la Torre K, Farre-Albaladejo M, et al. Postprandial LDL phenolic content and LDL oxidation are modulated by olive oil phenolic compounds in humans. Free Rad Biol Med. 2006;40:608
616. 22. Covas MI, Nyyssonen K, Poulsen HE, et al. The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med. 2006;145:333
341. 23. Covas MI, de la Torre R, Fito´ M. Virgin olive oil: a key food for cardiovascular risk protection. Br J Nutr. 2015;113:S19
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1203. 25. Fito´ M, Cladellas M, de la Torre R, et al. Anti-inflammatory effect of virgin olive oil in stable coronary disease patients: a randomized, crossover, controlled trial. Eur J Clin Nutr. 2008;62:570
574. 26 Estruch R, Ros E, Salas-Salvado´ J, et al. Primary prevention of cardiovascular disease with a mediterranean diet supplemented with extra-virgin olive oil or nuts. N Engl J Med. 2018;378(25):e34. 27. Pedret A, Ferna´ndez-Castillejo S, Valls RM, et al. Cardiovascular benefits of phenol-enriched virgin olive oils: new insights from the Virgin Olive Oil and HDL Functionality (VOHF) study. Mol Nutr Food Res. 2018;62(16):e1800456. 28. Garcı´a-Villalba R, Larrosa M, Possemiers S, Toma´s-Barbera´n FA, Espı´n JC. Bioavailability of phenolics from an oleuropein-rich olive (Olea europaea) leaf extract and its acute effect on plasma antioxidant status: comparison between pre- and postmenopausal women. Eur J Nutr. 2014;53:1015
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29. Serra A, Rubio´ L, Borra´s X, Macia` A, Romero MP, Motilva MJ. Distribution of olive oil phenolic compounds in rat tissues after administration of a phenolic extract from olive cake. Mol Nutr Food Res. 2012;56:486
496. 30. Caruso D, Visioli F, Patelli R, Galli C, Galli G. Urinary excretion of olive oil phenols and their metabolites in humans. Metabolism. 2001;50:1426
1428. 31. Tuck KL, Hayball PJ, Stupans I. Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats. J Agric Food Chem. 2002;50:2404
2409. 32. Khymenets O, Farre´ M, Pujadas M, et al. Direct analysis of glucuronidated metabolites of main olive oil phenols in human urine after dietary consumption of virgin olive oil. Food Chem. 2011;126:306
314. 33. Rubio´ L, Macia´ A, Valls RM, et al. A new hydroxytyrosol metabolite identified in human plasma: hydroxytyrosol acetate sulphate. Food Chem. 2012;134:1132
1136. 34. Kotronoulas A, Pizarro N, Serra A, et al. Dose-dependent metabolic disposition of hydroxytyrosol and formation of mercapturates in rats. Pharmacol Res. 2013;77:47
56. 35. Rodrı´guez-Morato´ J, Boronat M, Kotronoulas A, et al. Metabolic disposition and biological significance of simple phenols of dietary origin: hydroxytyrosol and tyrosol. Drugs Met Rev. 2016;48:218
236. 36. Davis VE, Walsh MJ. Alcohol, amines, and alkaloids: a possible biochemical basis for alcohol addiction. Science. 1970;167:1005
1007. 37. Tacker M, Creaven PJ, McIsaac WM. Alteration in tyramine metabolism by ethanol. Biochem Pharmacol. 1970;19:604
607. 38. De la Torre R, Covas MI, Pujadas MA, Fito´ M, Farre´ M. Is dopamine behind the health benefits of red wine? Eur J Nutr. 2006;45:307
310. 39. Pe´rez-Man˜a´ C, Farre M, Pujadas M, et al. Ethanol induces hydroxytyrosol formation in humans. Pharmacol Res. 2015;95
96:27
33. 40. Rodrı´guez-Morato´ J, Robledo P, Tanner JA, et al. CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol. Food Chem. 2017;217:716
725. 41. Pe´rez-Man˜a´ C, Farre´ M, Rodrı´guez-Morato´ J, et al. Moderate consumption of wine, through both its phenolic compounds and alcohol content, promotes hydroxytyrosol endogenous generation in humans. A randomized controlled trial. Mol Nutr Food Res. 2015;59:1213
1216. 42. Boronat A, Mateus J, Soldevila-Domenech N, et al. Cardiovascular benefits of tyrosol and its endogenous conversion into hydroxytyrosol in humans. A randomized, controlled trial. Free Rad Biol Med. 2019;143:471
481. 43. Hohmann CDD, Cramer H, Michalsen A, et al. Effects of high phenolic olive oil on cardiovascular risk factors: a systematic review and meta-analysis. Phytomedicine. 2015;22:631
640. 44. Ferna´ndez-Castillejo S, Valls RM, Castan˜er O, et al. Polyphenol rich olive oils improve lipoprotein particle atherogenic ratios and subclasses profile: a randomized, crossover, controlled trial. Mol Nutr Food Res. 2016;60:1544
1554. 45. Farra`s M, Valls RM, Ferna´ndez-Castillejo S, et al. Olive oil polyphenols enhance the expression of cholesterol efflux related genes in vivo in humans. A randomized controlled trial. J Nutr Biochem. 2013;24:1334
1339. 46. Farra`s M, Arranz S, Carrio´n S, et al. A functional virgin olive oil enriched with olive oil and thyme phenolic compounds improves the expression of cholesterol efflux-related genes: a randomized, crossover, controlled trial. Nutrients. 2019;11:1732. Available from: https://doi.org/10.3390/nu11081732.

47. Herna´ez A, Ferna´ndez-Castillero S, Farraˆs M, et al. Olive oil polyphenols enhance high density lipoprotein function in humans. A randomized controlled trial. Arterioscler Thromb Vasc Biol. 2014;34:2115
2119. 48. Farra`s M, Ferna´ndez-Castillejo S, Rubio´ L, et al. Phenol-enriched olive oils improve HDL antioxidant content in hypercholesterolemic subjects. A randomized, double-blind, cross-over, controlled trial. J Nutr Biochem. 2018;51:99
104. 49. Witzum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793
795. 50. Fogelman AM. When good cholesterol goes bad. Nat Med. 2004;10:902
903. 51. http://www.efsa.europa.eu/en/efsajournal/pub/2033.htm. 52. Roche HM, Gibney MJ. The impact of postprandial lipemia in accelerating atherothrombosis. J Cardiovasc Risk. 2000;7: 317
324. 53. Kimura R, Takahashi N, Murota K, et al. Activation of peroxisome proliferator-activated receptor-a (PPARa) suppresses postprandial lipidemia through fatty acid oxidationin enterocytes. Biochem Biophys Res Commun. 2011;410:1
6. 54. Weinbrenner T, Fito´ M, Farre´-Albaladejo M, et al. Bioavailability of olive oil phenolic compounds from olive oil and oxidative/antioxidative status at postprandial state in humans. Drugs Exp Clin Res. 2004;30:207
212. 55. Fito´ M, Gimeno E, Covas MI, et al. Postprandial and short-term effects of dietary virgin olive oil on oxidant/antioxidant status. Lipids. 2002;37:245
251. 56. Bogani P, Galli C, Villa M, Visioli F. Postprandial antiinflammatory and antioxidant effects of extra virgin olive oil. Atherosclerosis. 2007;190:181
186. 57. Ruano J, Lo´pez-Miranda J, Fuentes F, et al. Phenolic content of virgin olive oil improves ischemic reactive hyperemia in hypercholesterolemic patients. J Am Coll Cardiol. 2005;46:1864
1868. 58. Castan˜er O, Fito´ M, Lo´pez-Sabater MC, et al. The effect of olive oil polyphenols on antibodies against oxidized LDL. A randomized clinical trial. Clin Nutr. 2011;30:490
493. 59. Fito´ M, Estruch R, Salas-Salvado J, et al. Effect of the Mediterranean diet on heart failure biomarkers: a randomized sample from the PREDIMED trial. Eur J Heart Fail. 2014;16:543
550. 60. Salvini S, Sera F, Caruso D, et al. Daily consumption of a highphenol extra-virgin olive oil reduces oxidative DNA damage in postmenopausal women. Br J Nutr. 2006;95:742
751. 61. Machowetz A, Poulsen HE, Gruendel S, et al. Effect of olive oils on biomarkers of oxidative DNA stress in North and South Europeans. FASEB J. 2007;21:45
52. 62. Oubin˜a P, Sanchez-Muniz FJ, Rodenas S, Cuesta C. Eicosanoid production, thrombogenic ratio, and serum and LDL peroxides in normo- and hypercholesterolaemic postmenopausal women consuming two oleic acid-rich diets with different content of minor components. Br J Nutr. 2001;85:41
47. 63. Visioli F, Caruso D, Grande S, et al. Virgin olive oil study (VOLOS): vasoprotective potential of extra virgin olive oil in mildly dyslipidemic patients. Eur J Nutr. 2005;44:121
127. 64. Loued S, Berrougui H, Componova P, Ikhlef S, Helal O, Khalil A. Extra-virgin olive oil consumption reduces the age-related decrease in HDL and paraoxonase 1 anti-inflammatory activities. Br J Nutr. 2013;110:1272
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The bioavailability of olive oil phenolic compounds and their bioactive effects in humans Chapter | 16

65. Pacheco YM, Bermu´dez B, Lopez S, Abia R, Villar J, Muriana FJ. Minor compounds of olive oil have postprandial anti-inflammatory effects. Br J Nutr. 2007;98:260
263. 66. Moreno-Luna R, Mun˜oz-Hernandez R, Miranda ML, et al. Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. Am J Hypertens. 2012;25:1299
1304. 67. Widmer RJ, Freund MA, Flammer AJ, et al. Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis. Eur J Nutr. 2013;52:1223
1231. 68. Camargo A, Rangel-Zun˜iga OA, Haro C, et al. Olive oil phenolic compounds decrease the postprandial inflammatory 1 response by reducing postprandial plasma lipopolysaccharide levels. Food Chem. 2014;162:161
171. 69. Valls RM, Farra`s M, Sua´rez M, et al. Effects of functional olive oil enriched with its own phenolic compounds on endothelial function in hypertensive patients. A randomised controlled trial. Food Chem. 2015;167:30
35. 70. Fito´ M, Cladellas M, de la Torre R, et al. Antioxidant effect of virgin olive oil in patients with stable coronary heart disease: a randomised, crossover, controlled, clinical trial. Atherosclerosis. 2005;181:149
158. 71. Perona JS, Montero E, Sa´nchez-Domı´nguez JM, Can˜izares J, Garcia M, Ruiz-Gutie´rrez V. Evaluation of the effect of dietary virgin olive oil on blood pressure and lipid composition of serum and low-density lipoprotein in elderly type 2 diabetic subjects. J Agric Food Chem. 2009;57:11427
11433. 72. Pacheco YM, Lo´pez S, Bermu´dez B, Abia R, Muriana FJG. Extravirgin vs. refined olive oil on postprandial hemostatic markers in healthy subjects. J Thromb Haemost. 2006;4:1421
1422. 73. Ruano J, Lo´pez-Miranda J, de la Torre R, et al. Intake of phenol-rich virgin olive oil improves the postprandial prothrombotic profile in hypercholesterolemic patients. Am J Clin Nutr. 2007;86:341
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1013. 75. De la Torre-Carbot K, Cha´vez-Servı´n JL, Jauregui O, et al. Elevated circulating LDL phenol levels in men who consumed virgin rather than refined olive oil are associated with less oxidation of plasma LDL. J Nutr. 2010;140:501
508. 76. Soran H, Schofield JD, Liu Y, Durrington PN. How HDL protects LDL against atherogenic modification: paraoxonase 1 and other dramatis personae. Curr Opin Lipidol. 2015;26:247
256. 77. Camargo A, Ruano J, Fernandez JM, et al. Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil. BMC Genomics. 2010;11:253. 78. Llorente-Corte´s V, Estruch R, Mena MP, et al. Effect of Mediterranean diet on the expression of proatherogenic genes in a population at high cardiovascular risk. Atherosclerosis. 2010;208:442
450. 79. Castan˜er O, Corella D, Covas MI, et al. In vivo transcriptomic profile after a Mediterranean diet in high cardiovascular risk patients. A randomized controlled trial. Am J Clin Nutr. 2013;98:845
853. 80. Konstantinidou V, Covas MI, Mun˜oz-Aguayo D, et al. In vivo nutrigenomic effects of virgin olive oil polyphenols within the frame of the Mediterranean diet: a randomized controlled trial. FASEB J. 2010;24:2546
2557. 81. Castan˜er O, Covas MI, Khymenets O, et al. Protection of LDL from oxidation by olive oil polyphenols is associated with a downregulation of CD40-ligand expression and its downstream products in vivo in humans. Am J Clin Nutr. 2012;95:1238
1244. 82. Martı´n-Pela´ez S, Castan˜er O, Konstantinidou V, et al. Effect of olive oil phenolic compounds on the expression of blood pressurerelated genes in healthy individuals. Eur J Nutr. 2017;56:663
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Chapter 17

Mediterranean diet and role of olive oil Mana Shahbaz1, Emilio Sacanella1,2, Iasim Tahiri1 and Rosa Casas1,2 1

Department of Internal Medicine, Clinic Hospital, August Pi and Sunyer Biomedical Research Institute (IDIBAPS), University of Barcelona,

Barcelona, Spain, 2CIBER 06/03: Pathophysiology of Obesity and Nutrition, Institute of Health Carlos III, Madrid, Spain

Abbreviations body mass index blood pressure coronary heart disease C-reactive protein cardiovascular disease epithelial-derived neutrophil-activating peptide extra-virgin olive oil high-density lipoprotein high phenolic (extra) virgin OO hydroxytyrosol nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha IL interleukins LDL low-density lipoprotein LP(E)VOO low phenolic (extra) virgin OO MCP-1 monocyte chemotactic protein 1 MDP Mediterranean dietary pattern MedDiet Mediterranean diet MetS metabolic syndrome MMP-9 matrix metalloproteinase 9 MUFAs monounsaturated fatty acids NCD noncommunicable disease NF-κβ nuclear factor κβ NO nitric oxide OO olive oil oxLDL-C oxidized LDL cholesterol PUFA polyunsaturated fatty acids RCT randomized control trial ROO refined OO SO sunflower oil T2D type 2 diabetes TNF-α tumor necrosis factor α Tyr tyrosol WHO World Health Organization BMI BP CHD CRP CVD ENA78 EVOO HDL HP(E)VOO HTyr IκBα

17.1 Introduction According to the World Health Organization (WHO) and a variety of epidemiological studies in recent years, the

Mediterranean diet (MedDiet) plays a key role in the prevention of noncommunicable disease (NCD), all of them related to oxidative stress, chronic inflammation, and immune system diseases such as diabetes, cancer, and cardiovascular disease (CVD).1,2 The main factors implicated in the incidence of these NCDs are unhealthy dietary and lifestyle habits (sedentarism, tobacco or excess alcohol consumption, etc.) that may lead to these health problems, such as CVD, cancer, neurodegenerative diseases, metabolic syndrome (MetS), and diabetes.3 In addition, the populations living in the Mediterranean regions are known for their higher life expectancy compared to people in other parts of the world.3 The majority of the health benefits of this dietary pattern have been greatly attributed to the properties of extra-virgin olive oil (EVOO) and its bioactive compounds, because of its particular composition and high nutritional quality.2,4 Both in vivo and in vitro studies have demonstrated that the phenolic compounds of EVOO have positive effects on different physiological biomarkers such as nuclear factor κβ (NF-κβ), matrix metalloproteinase 9 (MMP-9), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and several interleukins (IL) related to atherosclerosis process.5
8 So, some of these positive effects include antimicrobial, antiinflammatory, and antioxidative properties of the phenolic compounds available in EVOO.5,9 The Mediterranean dietary pattern (MDP) is characterized by a high fat intake (40%
50% of total daily calories).10 Approximately 15%
25% of monounsaturated fatty acids (MUFAs) ingested in the MedDiet are from olive oil (OO), while saturated fats make up 8% of the fat content within the MedDiet.11 The antiinflammatory and antioxidant properties of this dietary pattern, due to its high content of EVOO are known to produce favorable effects on health status.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00043-2 © 2021 Elsevier Inc. All rights reserved.

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17.2 What is the Mediterranean diet? 17.2.1 Definition The concept of MedDiet was first introduced and defined by Keys as a diet high in vegetable oils and low in saturated fats, typical of Greece and Southern Italy.12 Since then, the definition has been advancing. The MDP has been the diet consumed by populations residing along the Mediterranean Sea. Due to the contribution of this diet to a better quality of life and an improved health status, this diet has become an important model for eating and dietary habits worldwide.13 The importance of the MedDiet has placed it on the UNESCO representative list of the Intangible Cultural Heritage of Humanity.14 The criteria not only includes the types of food consumed and their nutrient content but also the culinary practices used to prepare those foods, and the cultural and communal practices regarding food consumption in the Mediterranean area.14 There are multiple countries in the Mediterranean region with their own take on the MedDiet, depending on their food culture and production patterns.13 As a result, there is not one single traditional MedDiet, but instead, there are variations of diets that are based on the same specific foundation. This foundation consists of some basic characteristics, such as a high consumption of whole grain cereals, legumes, nuts, seeds, fruits, and vegetables. A moderate intake of fish and shellfish, eggs, white meat and dairy products, as well as alcohol, mostly in the form of wine and during meals, and a low intake of red and processed meats and sweets.13 In addition, the main fat consumed in the MedDiet diet is OO, a great source of MUFAs (15%
25% of total daily calories). OO is used for cooking, and as dressing for various dishes. The second most consumed source of fats in this diet is nuts, which are also good sources of unsaturated fats and polyunsaturated fatty acids (PUFAs).15

17.2.2 The pyramid The MDP has been presented to the world in the shape of a pyramid, with the base section representing the foods suggested to be consumed most frequently.16 As we go up the pyramid, the frequency of consumption decreases, meaning that the very top of it shows the foods that are suggested to be consumed the least frequently. This pyramid takes cultural and lifestyle elements into account, suggesting moderation, socialization, seasonality, physical activity, etc. For that reason the MedDiet is more than just a diet. It is a combination of dietary and lifestyle habits that affect individuals, both physically and mentally.16

that there is less of a constraint on the soil, the water, and sources of energy.17,18 In addition, the MedDiet encourages the consumption of locally grown, seasonal, and ecofriendly food that helps with preserving the environment.19 Since these locally grown products have to travel less, they promote saving of energy, reduce pollution, and greenhouse gas emissions.20 Considering the fundamental influence of OO on the MedDiet and its health benefits, it is important to note that olive trees are traditionally grown under rain-fed conditions. They do not need additional irrigation, which is a good contribution to sustainable farming. Nevertheless, there has been increasing interest in irrigation of olive orchard due to higher yields.21

17.3 Extra-virgin olive oil 17.3.1 Composition EVOO is obtained from olives only through mechanical or physical means, under conditions that do not alter the composition of the oil. Regular OO, however, comes from mixing EVOO and refined OO (ROO) (oil obtained from EVOO by refining methods).22 Approximately, 98%
99% of EVOO (the saponifiable fraction) is made up of triglycerides in the form of mostly MUFAs (oleic acid), considerable amounts of polyunsaturated fats (linoleic and α-linolenic), and small amounts of saturated fats (palmitic by 7.5%
20% and stearic acid by 0.5%
5%). The remaining 1%
2% (the unsaponifiable fraction) consists of tocopherols (α, β, γ, and δ-forms), sterols, polyphenols, pigments, hydrocarbons, aromatic and aliphatic alcohol, triterpene acids, waxes, and other minor components, which guarantee its high antioxidant potential.23 Some factors that can influence the composition of the minor components in EVOO include the type of cultivation, the climate, ripeness level of the olives at the time of harvest, and the processing system used for OO production.24 The latter refers to processes that differentiate regular OO from EVOO production. For example, Italian, Spanish, and Greek OOs are rich in oleic acid (B56%
83%) and present low concentrations in palmitic and linoleic acids in comparison to Tunisian OO, which is richer in palmitic and linoleic acids and poorer in oleic acid.23 In recent years it has become increasingly apparent that the cardioprotective effects of EVOO are not only due to the MUFA content. The minor components of EVOO, especially polyphenols, have shown to be significant contributors to these health benefits.25

17.2.3 Sustainability

17.3.2 Bioavailability of extra-virgin olive oil’s phenolic compounds

Since the MedDiet is a plant-centered diet, it reduces the number of animals produced and consumed. This means

The majority of research regarding the bioavailability of phenolic compounds in EVOO has focused on the three

Mediterranean diet and role of olive oil Chapter | 17

major EVOO phenolics: hydroxytyrosol (HTyr), tyrosol (Tyr), and oleuropein.2 In humans, their absorption has been evaluated to be about 55
66 mmol.%.23 These phenolic compounds also give EVOO its sensory characteristics, like the bitter and pungent or sweet taste.26,27 To understand the bioavailability of these polyphenols, we have to measure how much they are absorbed and metabolized in the human body. One way of doing that is to measure the presence of polyphenols in plasma or urine, after the ingestion of EVOO, using an analytical method based on rapid-resolution liquid chromatography coupled with mass spectrometric detection with a time-of-flight analyzer. By using that method, Garcı´a-Villalba et al. confirmed the presence of the majority of phenolic compounds of EVOO in the urine of their human participants, such as flavonoids and phenolic alcohols. This evidence suggests that the metabolization and absorption of EVOO polyphenols happen after ingestion.28 There are other polyphenols of EVOO that get poorly absorbed and do not get detected as clearly in the urine but can be detected in the epithelial cells of the intestine, as well as fecal material of humans. It has been suggested that these compounds may have local antioxidant activity that is limited to the gastrointestinal tract.29 It should be noted that phenolic compounds (polyphenols) of OO have high bioavailability and are among the most important components of EVOO because of their preventive effects on human health.30 Among other activities, EVOO has mainly been linked to antimicrobial, antioxidant, antiinflammatory effects and improvement in endothelial dysfunction and lipid profile.31 These make up 18%
37% of the unsaponifiable fraction of OO. The concentrations of polyphenol in EVOO can vary between 50 and 800 mg/kg.23 Besides, EVOO contains at least 36 different phenolic compounds that can vary depending on the type of EVOO.32 Most of health benefits such as the antioxidant capacity linked to EVOO can be attributed to this minor phenolic fraction. Among polyphenols (Fig. 17.1), it is possible to highlight phenolic acids and alcohols as well as flavonoids,

207

secoiridoids, and lignans. Tyrosol (Tyr) and hydroxytyrosol (HTyr) are the most abundant phenolic alcohols (90% of total content). Among phenolic acids, HTyr is the most potent antioxidant in the OO, reducing the oxidative stress, inhibiting the blood lipids oxidation. Indeed, the main “heart-health” claim reported by the European Food Safety Authority only is possible when 5 mg of HTyr and its derivatives in a daily intake of 20 g of EVOO.33

17.3.3 Effects of extra-virgin olive oil on chronic diseases 17.3.3.1 Obesity Multiple studies have demonstrated that consumption of EVOO may be helpful in prevention or management of weight gain, as well as the incidence of overweight and obesity. The SUN cohort study intended to address the concern that promotion of diets rich in EVOO would increase the obesity epidemic.34 They estimated the association of OO consumption and the likelihood of weight gain. The results, after a 28.5-month follow-up showed that individuals with higher intake of OO had higher total fat intake due to the high MUFA levels of OO. After multivariate adjustment, they found that higher OO consumption at baseline was associated with a lower likelihood of weight gain. They found no significant association between OO and the risk of obesity. Similarly, a systematic review of the MedDiet for long-term ( . 12 months) weight loss of multiple clinical trials concluded that the MedDiet was more effective compared to low-fat diets, partially due to its high EVOO content.35 A meta-analysis of 11 randomized control trials (RCTs) estimated the effects of EVOO consumption on the weight, body mass index (BMI), and waist circumference in adults without previous cardiovascular events.36 The results showed EVOO-rich diets reduced weight more than control diets 20.92 kg (95% CI 21.16 to 20.67; P 5 .1), reduced

FIGURE 17.1 An overview of the composition of EVOO. EVOO, Extra-virgin olive oil.

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waist circumference 20.60 cm (95% CI 21.17 to 20.04; P 5 .6), and reduced BMI 20.90 (95% CI 20.91 to 20.88; P , .001). The authors suggested that EVOO-rich diets can be an effective weight-control strategy in adults with no previous cardiovascular event. Also, Beulen et al. reported that a moderate high-fat MedDiet does not promote weight gain. After the replacement of 5% energy from saturated fatty acids with MUFA or PUFA, authors observed weight changes in the subjects of 20.38 and 20.5 kg, respectively. Also, the replacement of protein with MUFA or PUFA was associated with lower odds ratios for obesity.37

17.3.3.2 Hypertension One of the major risks for development of CVD is hypertension. Various human studies have demonstrated the protective effects of EVOO against hypertension. Though limited, the evidence from RCTs supports this claim. Perona et al. compared 31 hypertensive medically treated patients with 31 normotensive volunteers in an RCT.38 The participants consumed two different diets, 4 weeks each. One of them was a diet enriched in EVOO, the other one was a diet enriched in sunflower oil (SO). The results showed normalized systolic blood pressure (BP) in the hypertensive group with EVOO-enriched diet, but not in the SO-enriched diet. There were no significant differences observed in diastolic BP. In another RCT, 24 women diagnosed with high-normal BP or stage 1 essential hypertension at baseline participated.39 For homogenization, all of the participants followed an MDP for 4 months. After that, they were randomly assigned to either a MedDiet with polyphenol rich OO or with polyphenol free OO. They followed their assigned diets for 2 months. This was followed by a 4-month washout period, and then 2 months of the alternate diet. The results showed that after the polyphenol-rich OO diet, both systolic and diastolic BP decreased. All participants had a systolic BP of 140 mmHg or less, and 22 of the 24 participants had a diastolic BP of 99 mmHg or less. In contrast, after the polyphenol free OO diet, there were no changes observed in BP values compared to baseline. In the PREDIMED trial, after a 4-year intervention period of the MedDiet supplemented with EVOO (51 g/day) for men and women with CVD risk showed significant decrease in diastolic BP (21.53 mmHg), but not systolic BP.40 A substudy of the same trial that did a follow-up of 1 year revealed that the mean 24-hour ambulatory systolic and diastolic BP reduced by 2.3 and 1.2 mmHg, respectively. These results were observed in men and women who had been treated for hypertension.41 A recent meta-analysis carried out by Schwingshackl et al. (13 RCT included, 611 healthy participants, aged between 26 and 70 years and with a follow $ 3) compared

the effects of different types of OO on several cardiovascular risk factors such as lipid profile and BP and their antioxidant properties. OO analyzed were ROO, mixed OO, low phenolic (extra) virgin OO (LP(E)VOO), and high phenolic (extra) virgin OO (HP(E)VOO). Authors reported that HP(E)VOO might improve low-density lipoprotein (LDL) cholesterol levels as well as reduced systolic BP and improve oxidized LDL cholesterol (oxLDL-C).42 Nonetheless, George et al. did not find any significant effect on systolic BP for HP(E)VOO compared to LP(E) VOO [MD: 22.03 mmHg (95% CI 26.57 to 2.50); I2 5 79%; P 5 .38].43 Finally, a systematic review by Lopez et al. showed data from both human and animal studies regarding the beneficial health effects of EVOO on BP levels.44 The studies mentioned almost all agree on the BP lowering effects of EVOO, either due to the high oleic acid content of EVOO or due to the minor phenolic constituents of EVOO. These antihypertensive effects come from various mechanisms, including equilibrium between vasoconstrictors, vasodilators, and antiplatelet molecules. On the other hand, other mechanisms by which BP is decreased in individuals with hypertension might be explained by the positive correlation between EVOO intake and increase nitric oxide (NO) in plasma as well as in urine polyphenols levels.45,46

17.3.3.3 Dyslipidemia As a risk factor for stroke and coronary artery disease, dyslipidemia can affect CV morbidity and mortality.47 The American Heart Association has recommended the MedDiet rich in EVOO for improving the lipid profile and increasing high-density lipoprotein (HDL) cholesterol levels in individuals at risk for CVD.48 Khaw et al. measured changes in lipid profile, weight, and other metabolic markers in a group of men and women, after 4 weeks of consuming 53 g/day of three different dietary fats (extra-virgin coconut oil, EVOO, and butter).49 The results showed that butter significantly increased the total cholesterol/HDL cholesterol ratio compared to coconut oil (10.36, 95% CI 0.18
0.54) and EVOO (10.22, 95% CI 0.04
0.40). Butter also increased non-HDL cholesterol compared with coconut oil (10.39, 95% CI 0.16
0.62 mmol/L) and EVOO (10.39, 95% CI 0.16
0.62). Coconut oil did not significantly differ from EVOO for change in total cholesterol/HDL cholesterol ratio (20.14, 95% CI 20.33 to 0.05) or non-HDL cholesterol (0.002, 95% CI 20.23 to 0.24 mmol/L). Also, a study done in Brazil measured the effects of EVOO and fish oil on the lipid and oxidative stress parameters in patients with MetS.50 So, 102 patients were randomly assigned to one of the four groups. Patients in the first group were instructed to maintain their usual diet. Patients in the second group received a 3 g/day supplement of fish oil omega-3 fatty acids. The third group received 10 mL/day of EVOO at

Mediterranean diet and role of olive oil Chapter | 17

209

lunch and dinner. The fourth group (FOO) received a combination of 3 g/day fish oil omega-3 fatty acid, as well as 10 mL/day of EVOO. After 90 days the results showed a statistically significant decrease in total cholesterol, as well as LDL cholesterol levels, compared to the other three groups. Damasceno et al. compared the effects of EVOO, walnuts, and almonds on serum lipids and other markers of CVD.51 Using simple randomization, the individuals were divided into three isoenergetic diet sequences, each one lasting 4 weeks. In the EVOO group, virgin OO was a substitute for other common, refined types of oil used. In the other two groups, walnuts and almonds were substitutes for OO and other MUFA-rich foods, such as olives. The results of the study showed significant reductions of total cholesterol, LDL cholesterol, as well as the LDL/HDL cholesterol, compared to baseline. The mean reduction in LDL cholesterol in the EVOO group was 0.36 mmol/L (7.3%), in the walnut group 0.51 mmol/L (10.8%), and the almond group 0.61 mmol/L (13.4%). There were no changes in body weight, fasting blood glucose, or BP. Regarding the EVOO, the authors suggested that replacing common ROO with phenolic-rich EVOO would have a cholesterol lowering effect that is independent from the oils’ fatty acid content. A recent systematic review and meta-analysis (26 RCTs included) evaluated the effect of high versus low polyphenol OO on CVD risk factors in clinical trials.43 Authors reported that HP(E)VOO caused a higher improvement in oxLDL-C, total cholesterol, and HDL cholesterol levels than LP(E)VOO. Similar results were also reported by Schwingshackl et al.42 and Ghobadi et al.52 who pointed out a decrease in the LDL cholesterol levels, as well as significant improvements in the levels of oxLDL-C, total, and HDL cholesterol. Moreover, a systematic review on olive polyphenols and the MetS showed evidence of the antihyperlipidemic properties of EVOO due to both its high MUFA content as well as its phenolic compounds, based on multiple studies.53 Regardless of the root of antihyperlipidemic properties of EVOO (MUFA or phenolics), these studies agreed on EVOO consumption to decrease LDL cholesterol levels, as well as total cholesterol levels.

Secondary analyses of the PREDIMED trial showed that amongst elderly participants (3541 patients aged 55
80 years) with a high risk of CVD, there was a 40% decrease in the incidence of T2D in the intervention group following a MedDiet supplemented with EVOO after a median follow-up of 4.1 years.57 Using data from the Nurses’ Health Study, Guasch-Ferre et al. examined the association between OO intake and the risk of T2D in an American population (59,930 women aged 37
65 years from the NHSI and 85,157 women aged 26
45 years from NHII).58 After a 22-year follow-up period, it was observed that total OO consumption was associated with a substantially lower risk of T2D. In addition, the substitution of margarine, butter, and mayonnaise with the same amount of OO resulted in a lower risk of T2D among the older women of the sample population by 5%, 8%, and 15% lower risk of T2D, respectively. Also, BasterraGortari et al. in the framework of the PREDIMED trial (3230 participants with T2D at baseline) reported a significant reduction of starting a first glucose-lowering medication by 22% after 3.2 years of intervention with MedDiet supplemented with EVOO.59 In addition, evidence suggests that the type of dietary fat rather than the total amount of consumed fat is an influential component related to the risk of T2D.60 Several metaanalyses of RCTs demonstrated that replacing carbohydrates with MUFA has shown beneficial effects on health status of T2D patients, including improvement in glycemic control.61,62 With OO having a high content of MUFA (oleic acid), it has been proposed that the consumption of OO may have positive effects on the management of T2D. Schwingshackl et al. performed a meta-analysis of 29 RCTs (15,784 T2D cases between 2 weeks and 4.1 years of follow) to assess the association between intake of OO and risk of T2D.63 The results revealed that consumption of OO was inversely associated with risk of T2D. The dose
response meta-analysis showed that each 10 g daily increase in OO use was associated with a 9% decrease in risk of T2D (RR: 0.91; 95% CI 0.87
0.95; P , .01; I2 5 0%).

17.3.3.4 Diabetes

CVD is considered to be major public health issue worldwide, causing morbidity and mortality. Some metabolic risk factors for CVD include obesity, cholesterol, and smoking.64 Several interventional studies have reported that regular EVOO consumption may be associated with a reduced risk of CVD. As previously mentioned, one of the benefits of EVOO consumption is the antihypertensive effect of it, leading to the increase in the HDL/total cholesterol ratio. In 2012 the EPIC study of a Spanish population of 40,142 participants had a follow-up of 10.4 years.65 This cohort study aimed to find the association between EVOO

According to The International Diabetes Federation, diabetes is one of the most prevalent diseases worldwide, affecting between 400 and 500 million adults. In 2019 diabetes caused tremendous health expenditure, approximately 760 billion USD.54 Management of type 2 diabetes (T2D) requires dietary interventions, regular physical activity, medication, and insulin therapy.55 Regarding the dietary interventions, recommendations differ based on the ability of interventions in achieving weight loss, glycemic control, and a lipid profile that reduces risk of CVD.56

17.3.3.5 Cardiovascular disease and atherosclerosis

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consumption and incident coronary heart disease (CHD). The results showed a negative association between EVOO consumption and CHD (HR 0.78; 95% CI 0.59
1.03) for the top versus the bottom quartile intake ($28.9 vs 10 g/day). A study in the same EPIC population reported a 44% decrease in CVD-caused mortality associated with the intake of EVOO.66 Moreover, there was a dose
response effect, where every 10 g increase in the consumption of EVOO was associated with a 13% reduction in the risk of CVD-caused mortality. Atherosclerosis has been defined as “a low-grade chronic inflammatory disease of the vascular wall initiated by the accumulation of cholesterol-laden inflammatory cells (monocytes and T-lymphocytes) in the sub-endothelial space”.7 This inflammation at the plaque sites causes disturbances in the blood flow. The involvement of immune cells in atherosclerosis results in the production of various inflammatory mediators.67 Certain functional foods, such as EVOO, may improve endothelial function, cause a reduction in inflammatory molecules, and a reduction in CVD incidence and mortality.7,68 The main reason why following an MDP is known to be protective against atherosclerosis is due to EVOO’s phenolic compounds that are known to improve endothelial function and to have antiinflammatory and antioxidative properties.69 One of the potential ways that these phenolic compounds reduce the circulating inflammatory biomarkers is by the downregulation of NF-κβ.70 In this sense, Camargo et al. confirmed that after the consumption of MedDiet with EVOO, there was lower expression of NF-κβ, TNF-α, MCP-1, compared to the other two diets.5 In addition, there was lower expression of MMP-9, which is a metalloproteinase that contributes to plaque instability. Within the same participants, there was an increase in the expression of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which is an antiinflammatory gene. Also, EVOO might exert beneficial effects on endothelial function as well as markers of inflammation and endothelial function. Thus according to the results MedDiet with EVOO might promote downregulation of expression of several leukocyte molecules, as well as proinflammatory markers related to atherosclerosis progression [vascular cell adhesion molecule 1, intercellular adhesion molecule 1, E- and P-selectin, IL-1β, IL-6, IL-7, IL-8, IL-12p70, C-reactive protein (CRP), TNFR-60 and TNFR-80, MCP-1, regulated on activation, normal T cell expressed and secreted, epithelial-derived neutrophil-activating peptide (ENA78), TNF-α] and molecules related to atherosclerotic plaque instability (IL-10, IL-13, IL-18, MMP-9) in short (3 months), medium (1
3 years), and long term (5 years).7,8,71
74 Also, a meta-analysis of 30 clinical trials with 3106 participants, observed that EVOO containing interventions

(1
50 mg/day) showed a more significant decrease in CRP levels [MD: 20.64 mg/L (95% CI 20.96 to 20.31), P , .0001, I2 5 66%] compared to controls. Also, the EVOO intervention groups demonstrated more significant decreases in IL-6 levels [MD: 20.29 (95% CI 20.7 to 20.02), P , .04, I2 5 62%] compared to controls.75 Similar results were showed in several inflammatory markers (CRP, IL-6, IL-18, interferon gamma), after the inclusion of OO, regardless of its polyphenol content.76 On the other hand, EVOO might exert its cardioprotective effects by lowering oxidative stress. George et al. observed significant reductions of malondialdehyde (MD: 20.07 μmol/L, 95% CI 20.12, 20.02 μmol/L; I2: 88%; P 5 .004) and oxidized LDL (SMD: 20.44, 95% CI 20.78 to 20.10 μmol/L; I2: 41%; P 5 .01) after administering HP(E)VOO and LP(E)VOO.43

17.3.3.6 Cancer The WHO identifies cancer as the second leading cause of death worldwide, being responsible for approximately 9.6 million deaths in 2018.77 On the one hand, experimental evidence in vitro has suggested that, in addition to antioxidant and antiinflammatory properties of EVOO, its phenolic compounds exert molecule signaling pathways leading to apoptosis and cell growth inhibition in multiple cell lines.78 The PREDIMED study also showed significant reductions of the risk of breast cancer (68%) after analyzing 4152 women allocated to MedDiet supplemented with EVOO compared to the control group. In addition, authors observed a dose
response for each additional 5% of calories from EVOO.79 On the other hand, epidemiological studies have demonstrated that the populations within Europe that follow the MDP have significantly lower incidence of common cancers, especially compared to countries that follow a western diet such as the United States and the United Kingdom.80 A meta-analysis of 19 observational studies assessed the association between EOO consumption and the risk of various types of cancer.81 Overall, EVOO consumption showed to be inversely associated with the risk of cancer development. This conclusion was more prominent regarding breast cancer, as well as cancers of the digestive system. Interestingly, regarding the association between EVOO consumption and colorectal cancer, multiple case
control studies showed that the MUFA intake appears to be uninfluential. Yet, they found EVOO to have had a protective effect against colorectal cancer. This evidence may suggest that certain minor constituents of EVOO are the ones responsible for protection against initiation, promotion, and progression of cancer, regardless of EVOOs fatty acid content.82 In addition, a meta-analysis (3800 patients and 23,340 controls in 19 observational studies) exploring the effects of different diets on colorectal

Mediterranean diet and role of olive oil Chapter | 17

cancer concluded that proinflammatory diets showed association with an increased risk of colorectal cancer, while antiinflammatory diets that are rich in antiinflammatory food components (such as EVOO) showed protective effects against colorectal cancer.83 Similar results were shown by Pelucchi et al. who reported higher protection (38%) against risk of breast cancer in those women who were in the highest level compared to those who were in the lowest level of OO consumption.84

8.

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10.

17.4 Conclusion This review explains the major role that OO plays in making the MedDiet one of the healthiest diets known to this day. It also provides evidence regarding the physiological benefits of EVOO regarding health and disease. Depending on the composition of OO consumed, the different health benefits may vary. Further research is needed to better understand the mechanism of action of bioactive compounds of EVOO for the purposes of disease prevention and management.

11.

12.

13.

14.

Acknowledgments

15.

This research was funded by the Instituto de Salud Carlos III, Spain. CIBER OBN is an initiative of the Instituto de Salud Carlos III, Spain. 16.

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812. Available from: https://doi.org/ 10.1016/b978-0-12-374420-3.00085-1.

39. Moreno-Luna R, Mun˜oz-Herna´ndez R, Miranda ML, et al. Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. Am J Hypertens. 2012;25. Available from: https://doi.org/10.1038/ajh.2012.128. 40. Toledo E, Hu FB, Estruch R, et al. Effect of the Mediterranean diet on blood pressure in the PREDIMED trial: results from a randomized controlled trial. BMC Med. 2013;11(1). Available from: https://doi.org/10.1186/1741-7015-11-207. 41. Dome´nech M, Roman P, Lapetra J, et al. Mediterranean diet reduces 24-hour ambulatory blood pressure, blood glucose, and lipids. Hypertension. 2014;64(1):69
76. Available from: https:// doi.org/10.1161/hypertensionaha.113.03353. 42. Schwingshackl L, Krause M, Schmucker C, Hoffmann G, Ru¨cker G, Meerpohl JJ. Impact of different types of olive oil on cardiovascular risk factors: a systematic review and network meta-analysis. Nutr Metab Cardiovasc Dis. 2019;29(10):1030
1039. Available from: https://doi.org/10.1016/j.numecd.2019.07.001. 43. George ES, Marshall S, Mayr HL, et al. The effect of high-polyphenol extra virgin olive oil on cardiovascular risk factors: a systematic review and meta-analysis. Crit Rev Food Sci Nutr. 2018;59(17):2772
2795. Available from: https://doi.org/10.1080/10408398.2018.1470491. 44. Lopez S, Bermudez B, SM-DL Paz, Jaramillo S, Abia R, Muriana FJ. Virgin olive oil and hypertension. Curr Vasc Pharmacol. 2016;14(4):323
329. Available from: https://doi.org/10.2174/ 1570161114666160118105137. 45. Medina-Remo´n A, Tresserra-Rimbau A, Pons A, et al. Effects of total dietary polyphenols on plasma nitric oxide and blood pressure in a high cardiovascular risk cohort. The PREDIMED randomized trial. Nutr Metab Cardiovasc Dis. 2015;25(1):60
67. Available from: https://doi.org/10.1016/j.numecd.2014.09.001. 46. Storniolo CE, Casillas R, Bullo´ M, et al. Mediterranean diet supplemented with extra virgin olive oil or nuts improves endothelial markers involved in blood pressure control in hypertensive women. Eur J Nutr. 2015;56(1):89
97. Available from: https://doi.org/ 10.1007/s00394-015-1060-5. 47. Kopin L, Lowenstein CJ. Dyslipidemia. Ann Intern Med. 2017;167 (11). Available from: https://doi.org/10.7326/aitc201712050. 48. American Heart Association News. Mediterranean diet with virgin olive oil may be recipe for ‘good’ cholesterol, 2017. www.heart.org. ,https:// www.heart.org/en/news/2018/05/01/mediterranean-diet-with-virginolive-oil-may-be-recipe-for-good-cholesterol. Accessed 29.01.20. 49. Khaw K-T, Sharp SJ, Finikarides L, et al. Randomised trial of coconut oil, olive oil or butter on blood lipids and other cardiovascular risk factors in healthy men and women. BMJ Open. 2018;8 (3). Available from: https://doi.org/10.1136/bmjopen-2017-020167. 50. Venturini D, Sima˜o ANC, Urbano MR, Dichi I. Effects of extra virgin olive oil and fish oil on lipid profile and oxidative stress in patients with metabolic syndrome. Nutrition. 2015;31(6):834
840. Available from: https://doi.org/10.1016/j.nut.2014.12.016. 51. Damasceno NRT, Pe´rez-Heras A, Serra M, et al. Crossover study of diets enriched with virgin olive oil, walnuts or almonds. Effects on lipids and other cardiovascular risk markers. Nutr Metab Cardiovasc Dis. 2011;21. Available from: https://doi.org/10.1016/j.numecd.2010.12.006. 52. Ghobadi S, Hassanzadeh-Rostami Z, Mohammadian F, et al. Comparison of blood lipid-lowering effects of olive oil and other plant oils: A systematic review and meta-analysis of 27 randomized placebo-controlled clinical trials. Crit Rev Food Sci Nutr. 2018;59(13):2110
2124. Available from: https://doi.org/10.1080/10408398.2018.1438349.

Mediterranean diet and role of olive oil Chapter | 17

53. Saibandith B, Spencer JPE, Rowland IR, Commane DM. Olive polyphenols and the metabolic syndrome. Molecules. 2017;22(7):1082. Available from: https://doi.org/10.3390/molecules22071082. 54. International Diabetes Federation, 2019. ,https://www.idf.org/aboutdiabetes/what-is-diabetes/facts-figures.html. Accessed 22.01.20. 55. Evert AB, Boucher JL. New diabetes nutrition therapy recommendations: what you need to know. Diabetes Spectr. 2014;27(2):121
130. Available from: https://doi.org/10.2337/diaspect.27.2.121. 56. American Diabetes Association. Nutrition recommendations and interventions for diabetes: a position statement of the American Diabetes Association. Diabetes Care. 2007;31(suppl 1). Available from: https://doi.org/10.2337/dc08-s061. 57. Salas-Salvado´ J, Bullo´ M, Estruch R, et al. Prevention of diabetes with Mediterranean diets. Ann Intern Med. 2014;160(1):1
10. Available from: https://doi.org/10.7326/m13-1725. 58. Guasch-Ferre´ M, Hruby A, Salas-Salvado´ J, et al. Olive oil consumption and risk of type 2 diabetes in US women. Am J Clin Nutr. 2015;102(2):479
486. Available from: https://doi.org/10.3945/ ajcn.115.112029. 59. Basterra-Gortari FJ, Ruiz-Canela M, Martı´nez-Gonza´lez MA, et al. Effects of a Mediterranean eating plan on the need for glucoselowering medications in participants with type 2 diabetes: a subgroup analysis of the PREDIMED trial. Diabetes Care. 2019;42: dc182475. Available from: https://doi.org/10.2337/dc18-2475. 60. Perez-Martinez P, Garcia-Rios A, Delgado-Lista J, Perez-Jimenez F, Lopez-Miranda J. Mediterranean diet rich in olive oil and obesity, metabolic syndrome and diabetes mellitus. Curr Pharm Des. 2011;17(8):769
777. Available from: https://doi.org/10.2174/ 138161211795428948. 61. Qian F, Korat AA, Malik V, Hu FB. Metabolic effects of monounsaturated fatty acid
enriched diets compared with carbohydrate or polyunsaturated fatty acid
enriched diets in patients with type 2 diabetes: a systematic review and meta-analysis of randomized controlled trials. Diabetes Care. 2016;39(8):1448
1457. Available from: https://doi.org/10.2337/dc16-0513. 62. Schwingshackl L, Strasser B, Hoffmann G. Effects of monounsaturated fatty acids on glycaemic control in patients with abnormal glucose metabolism: a systematic review and meta-analysis. Ann Nutr Metab. 2011;58(4):290
296. Available from: https://doi.org/ 10.1159/000331214. 63. Schwingshackl L, Lampousi A-M, Portillo MP, Romaguera D, Hoffmann G, Boeing H. Olive oil in the prevention and management of type 2 diabetes mellitus: a systematic review and metaanalysis of cohort studies and intervention trials. Nutr Diabetes. 2017;7(4). Available from: https://doi.org/10.1038/nutd.2017.12. 64. Capewell S, Buchan I. Why have sustained increases in obesity and type 2 diabetes not offset declines in cardiovascular mortality over recent decades in Western countries? Nutr Metab Cardiovasc Dis. 2012;22(4):307
311. Available from: https://doi.org/10.1016/j. numecd.2012.01.005. 65. Buckland G, Travier N, Barricarte A, et al. Olive oil intake and CHD in the European Prospective Investigation into Cancer and Nutrition Spanish cohort. Br J Nutr. 2012;108(11):2075
2082. Available from: https://doi.org/10.1017/s000711451200298x. 66. Buckland G, Maye´n AL, Agudo A, et al. Olive oil intake and mortality within the Spanish population (EPIC-Spain). Am J Clin Nutr. 2012;96(1):142
149. Available from: https://doi.org/10.3945/ ajcn.111.024216.

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67. Hansson GK, Libby P, Tabas I. Inflammation and plaque vulnerability. J Intern Med. 2015;278(5):483
493. Available from: https:// doi.org/10.1111/joim.12406. 68. Tong TYN, Wareham NJ, Khaw K-T, Imamura F, Forouhi NG. Prospective association of the Mediterranean diet with cardiovascular disease incidence and mortality and its population impact in a non-Mediterranean population: the EPIC-Norfolk study. BMC Med. 2016;14(1). Available from: https://doi.org/ 10.1186/s12916-016-0677-4. 69. Moreno-Luna R, Mun˜ oz-Hernandez R, Miranda ML, et al. Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. Am J Hypertension. 2012;25. Available from: https://doi.org/ 10.1038/ajh.2012.128. 70. Perez-Herrera A, Delgado-Lista J, Torres-Sanchez LA, et al. The postprandial inflammatory response after ingestion of heated oils in obese persons is reduced by the presence of phenol compounds. Mol Nutr Food Res. 2012;56(3):510
514. Available from: https:// doi.org/10.1002/mnfr.201100533. 71. Estruch R, Martı´nez-Gonza´lez MA, Corella D, et al. Effects of a Mediterranean-style diet on cardiovascular risk factors. Ann Intern Med. 2006;145(1):1. Available from: https://doi.org/10.7326/00034819-145-1-200607040-00004. 72. Urpi-Sarda M, Casas R, Chiva-Blanch G, et al. The Mediterranean diet pattern and its main components are associated with lower plasma concentrations of tumor necrosis factor receptor 60 in patients at high risk for cardiovascular disease. J Nutr. 2012;142(6):1019
1025. Available from: https://doi.org/ 10.3945/jn.111.148726. 73. Mena MP, Sacanella E, Vazquez-Agell M, et al. Inhibition of circulating immune cell activation: a molecular antiinflammatory effect of the Mediterranean diet. Am J Clin Nutr. 2008;89(1):248
256. Available from: https://doi.org/10.3945/ajcn.2008.26094. 74. Casas R, Sacanella E, Urpı´-Sarda` M, et al. Long-term immunomodulatory effects of a Mediterranean diet in adults at high risk of cardiovascular disease in the PREvencio´n con DIeta MEDiterra´nea (PREDIMED) randomized controlled trial. J Nutr. 2016;146(9):1684
1693. Available from: https://doi.org/10.3945/jn.115.229476. 75. Schwingshackl L, Christoph M, Hoffmann G. Effects of olive oil on markers of inflammation and endothelial function—a systematic review and meta-analysis. Nutrients. 2015;7(9):7651
7675. Available from: https://doi.org/10.3390/nu7095356. 76. Tsartsou E, Proutsos N, Kampa E, Kampa M. Network metaanalysis of metabolic effects of olive-oil in humans shows the importance of olive oil consumption with moderate polyphenol levels as part of the Mediterranean diet. Front Nutr. 2019;6. Available from: https://doi.org/10.3389/fnut.2019.00006. 77. World Health Organization. Cancer. ,https://www.who.int/healthtopics/cancer#tab 5 tab_1. Accessed 21.01.20. 78. Casaburi I, Puoci F, Chimento A, et al. Potential of olive oil phenols as chemopreventive and therapeutic agents against cancer: a review of in vitro studies. Mol Nutr Food Res. 2012;57(1):71
83. Available from: https://doi.org/10.1002/mnfr.201200503. 79. Toledo E, Salas-Salvado´ J, Donat-Vargas C, et al. Mediterranean diet and invasive breast cancer risk among women at high cardiovascular risk in the PREDIMED trial: a randomized clinical trial. JAMA Intern Med. 2015;175(11):1752. Available from: https://doi. org/10.1001/jamainternmed.2015.4838.

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80. Owen RW, Haubner R, Wu¨rtele G, Hull WE, Spiegelhalder B, Bartsch H. Olives and olive oil in cancer prevention. Eur J Cancer Prev. 2004;13(4):319
326. Available from: https://doi.org/ 10.1097/01.cej.0000130221.19480.7e. 81. Psaltopoulou T, Kosti RI, Haidopoulos D, Dimopoulos M, Panagiotakos DB. Olive oil intake is inversely related to cancer prevalence: a systematic review and a meta-analysis of 13800 patients and 23340 controls in 19 observational studies. Lipids Health Dis. 2011;10(1):127. Available from: https://doi.org/ 10.1186/1476-511x-10-127.

82. Sotiroudis TG, Kyrtopoulos SA. Anticarcinogenic compounds of olive oil and related biomarkers. Eur J Nutr. 2008;47(S2):69
72. Available from: https://doi.org/10.1007/s00394-008-2008-9. 83. Shivappa N, Godos J, He´bert J, et al. Dietary inflammatory index and colorectal cancer risk—a meta-analysis. Nutrients. 2017;9 (9):1043. Available from: https://doi.org/10.3390/nu9091043. 84. Pelucchi C, Bosetti C, Negri E, Lipworth L, La Vecchia C. Olive oil and cancer risk: an update of epidemiological findings through 2010. Curr Pharm Des. 2011;17(8):805
812. Available from: https://doi.org/10.2174/138161211795428920.

Chapter 18

Probiotics from fermented olives H. Abouloifa1, Y. Rokni1, N. Ghabbour1, S. Karboune2, M. Brasca3, G. D’hallewin4, R. Ben Salah5, N. Ktari6, E. Saalaoui1 and A. Asehraou1 1

Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier University, Oujda, Morocco,

2

Department of Food Science and Agricultural Chemistry, Macdonald Campus, McGill University, Ste Anne de Bellevue, QC, Canada, 3Institute of

Sciences of Food Production, National Research Council of Italy, Milano, Italy, 4Institute of Sciences of Food Production, National Research Council of Italy, Sassari, Italy, 5Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax, Sfax, Tunisia, 6Laboratory of Enzyme Engineering and Microbiology, University of Sfax, National School of Engineering of Sfax (ENIS), Sfax, Tunisia

Abbreviations EFSA FDA LAB MD mN/m NaCl NaOH PFGE UNESCO WHO

European Food Safety Authority Food and Drug Administration lactic acid bacteria Mediterranean diet Newton-meters/meters sodium chloride sodium hydroxide pulsed-field gel electrophoresis United Nations Educational Scientific and Cultural Organization World Health Organization

18.1 Introduction Fermented olives and olive oil are considered the cornerstone of the Mediterranean diet (MD) pyramid. Because of its health benefits for the consumer, MD is inscribed since 2013 on the United Nations Educational Scientific and Cultural Organization’s (UNESCO’s) list of intangible cultural heritage of humanity in some Mediterranean countries (Cyprus, Croatia, Spain, Greece, Italy, and Morocco).1 This privilege is related to the positive effects of MD in reducing the risk of major chronic diseases, namely, heart diseases, Cancer, Alzheimer, and diabetes.2,3 Fermented olives are basically processed according to traditional and industrial processes.4 The industrial process (Spanish style) is based on alkali-treatment of olives, in sodium hydroxide solution of 2%
2.5% (w/v), followed by washings in tap water and brining at 10%
12% (w/v) of NaCl; while in the traditional process, olives (entire or cracked) are directly brined at around 5% of

NaCl. In both cases, olives are subject to a natural fermentation process, assured by the microbiota contaminating olives, containers, and the environment. The natural fermentation process of olives occurs in extreme environment (olive and brine), characterized by low nutrients and high polyphenols and sodium chloride contents. These conditions allow the microbiota surviving in this extreme environment, mainly composed of lactic acid bacteria (LAB) and yeasts,5 to become important source of probiotics. Therefore research on fermented olives, as a source of wild strains of LAB and yeast with probiotic and technological traits, constitutes a promising field of work.6 According to FAO/OMS,7 probiotics are live microorganisms that confer health benefits to the host, when administered in adequate amounts, particularly by assuring the inhibition of pathogens, stimulation of a protective immune response, and maintaining host health,8 and they are considered a very promising instrument for the treatment of immune diseases.9 The main objective of this chapter is to highlight the benefits of probiotics, obtained from fermented olives, in human health, bioprocessing, and biopreserving olives and other food products.

18.2 Probiotic microorganisms isolated from fermented olives The natural fermentation process of olives (green, turning, and black) is carried out mainly by LAB and yeasts.5 The main species isolated from fermented olives and previously cited for their probiotic properties are reported in Table 18.1.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00035-3 © 2021 Elsevier Inc. All rights reserved.

215

TABLE 18.1 Probiotic lactic acid bacteria and yeasts isolated from fermented olives. Species

Source (olive variety)

Country

Probiotic properties

Safety properties

Resistance to Low pH

Bile Salt

GJ

Panc

References

Agg

Hy

Ad

AA

AR

Hae

Gel

DNase

MD

Lactic acid bacteria Lactobacillus plantarum

Lactobacillus paraplantarum

Lactobacillus pentosus

Olea europaea

Italy

1

1

1

1

NT

NT

1

1

NT

NT

NT

NT

NT

[10]

Bella di Cerignola

Italy

1

1

NT

NT

NT

NT

1

1

NT

NT

NT

NT

NT

[11]

Halkidiki

Greece

1

1

NT

NT

NT

NT

1

2

1

2

NT

NT

NT

[12]

Nocellara Etnea

Italy

NT

NT

1

1

1

1

1

1

1

NT

NT

NT

NT

[13]

Campo Maior

Portugal

1

1

1

NT

1

1

1

1

1

2

NT

NT

2

[14]

Envendos

Portugal

1

1

1

NT

1

1

1

1

1

2

NT

NT

2

[14]

Moroccan Picholine

Morocco

1

1

NT

NT

1

1

NT

1

1

2

2

2

2

[15]

Manzanilla

Spain

1

1

1

1

1

NT

NT

NT

1

1

NT

NT

NT

[16]

Beja

Portugal

1

1

1

NT

1

1

1

1

1

2

NT

NT

2

[14]

Santare´m

Portugal

1

1

1

NT

1

1

1

1

1

2

NT

NT

2

[14]

Ladoeiro

Portugal

1

1

1

NT

1

1

1

1

1

2

NT

NT

2

[14]

Gordal

Spain

NT

NT

1

1

1

1

NT

1

1

2

NT

NT

NT

[17]

Manzanilla

Spain

NT

NT

1

1

1

1

NT

1

1

2

NT

NT

NT

[17]

Nocellara Etnea

Italy

NT

NT

1

1

1

1

1

1

1

NT

NT

NT

NT

[13]

Aloren˜a

Spain

1

1

1

NT

1

NT

1

1

NT

NT

NT

NT

NT

[18]

Nocellara del Belice

Italy

1

1

NT

1

NT

NT

1

1

2

NT

NT

NT

NT

[19]

Kalamata

Greece

1

1

NT

NT

NT

NT

1

2

1

1

NT

NT

NT

[12]

Kalamata

Greece

1

1

NT

NT

NT

NT

1

2

1

2

NT

NT

NT

[12]

Moroccan Picholine

Morocco

1

1

NT

NT

1

1

NT

1

1

2

2

2

2

[15]

Manzanilla and Gordal

Spain

1

1

1

1

1

NT

NT

NT

1

2

NT

NT

NT

[16]

Gordal

Spain

1

1

1

1

1

NT

NT

NT

1

2

NT

NT

NT

[16]

Lactobacillus paracasei subsp. paracasei

Halkidiki

Greece

1

1

NT

NT

NT

NT

1

2

1

2

NT

NT

NT

[12]

Lactobacillus brevis

Moroccan Picholine

Morocco

1

1

NT

NT

1

1

NT

1

1

2

2

2

2

[15]

Lactobacillus coryniformis

Nocellara del Belice

Italy

1

1

NT

1

NT

NT

1

1

2

NT

NT

NT

NT

[19]

Leuconostoc mesenteroides

Nocellara Etnea

Italy

NT

NT

1

1

1

1

1

1

2

NT

NT

NT

NT

[13]

Enterococcus spp.

Cypriot

Cyprus

1

1

NT

NT

NT

NT

NT

NT

NT

2

2

2

2

[20]

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Yeasts Saccharomyces cerevisiae Pichia kluyveri

Pichia manshurica

Bosana

Italy

NT

1

1

1

1

NT

NT

NT

NT

NT

NT

NT

NT

[22]

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Pichia guilliermondii Candida boidinii

Bosana

Italy

NT

1

1

1

1

NT

NT

NT

NT

NT

NT

NT

NT

[22]

Candida silvae

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Candida norvegica

Negrinha de Freixo

Portugal

NT

NT

1

1

1

NT

NT

1

NT

NT

NT

NT

NT

[23]

Rhodotorula mucilaginosa

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Metschnikowia pulcherrima

Conservolea

Greece

NT

NT

1

1

NT

NT

NT

NT

NT

NT

NT

NT

NT

[21]

Galactomyces reessii

Negrinha de Freixo

Portugal

NT

NT

1

1

1

NT

NT

1

NT

NT

NT

NT

NT

[23]

(2), Not detected; (1), presence; AA, antimicrobial activity; Ad, adhesion; Agg, auto-Aggregation; AR, antibiotic resistance; Gel, gelatinase; GJ, gastric juice; H, hydrophobicity; Hae, hemolysis; MD, mucin degradation; NT, not tested; Panc, pancreatic.

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18.2.1 Lactic acid bacteria The LAB strains isolated from fermented olives and selected for their probiotic potential belonged to the species of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactobacillus coryniformis, Leuconostoc mesenteroides and Enterococcus spp. (Table 18.1). Based on these previous works, L. plantarum and L. pentosus are the most isolated probiotic LAB from various Mediterranean fermented olive varieties and countries. Hence, L. plantarum was isolated from olive varieties of Italy (Bella di Cerignola, Nocellara Etna), Greece (Halkidiki), Portugal (Campo Maior, Envendos), Spain (Manzanilla), and Morocco (Moroccan Picholine), while L. pentosus was isolated from olive varieties of Spain (Gordal, Manzanilla, and Alorena), Italy (Nocellara Etna, and Nocellara del Belice), Greece (Kalamata), and Morocco (Moroccan Picholine). L. paraplantarum was isolated from Portuguese varieties (Beja, Santarem, and Ladoeiro).14 Other species, such as L. brevis, L. mesenteroides, and Enterococcus sp., were isolated from Moroccan Picholine (Morocco), Nocellara Etnea (Italy), and Cypriot (Cyprus) olive varieties, respectively. L. brevis and L. mesenteroides are not suitable in olive fermentation, because of their heterofermentative metabolism (gas production), associated with the risk of gas pocket spoilage in olive fruits, while Enterococcus sp. may be of health concern to the consumer because of its pathogenesis risk.24 The dominance of L. plantarum and L. pentosus in this wide range of olive varieties and different countries may be due to their tolerance to low nutrients and high sodium chloride and polyphenols contents occurring in olive brine. LAB strains, dominating in this extreme environment, could have important physiologic properties, which may be of health benefits to the consumer.

18.2.2 Yeasts Compared to LAB, few studies were conducted on the isolation of probiotic yeast strains from fermented olives (Table 18.1). The probiotic yeast strains isolated belonged to Saccharomyces cerevisiae obtained from Italian (Bosana) and Greek (conservolea) olive varieties.21,22 Strains of Pichia kluyveri, Pichia manshurica, Pichia guilliermondii, Candida silvae, Rhodotorula mucilaginosa, and Metschnikowia pulcherrima were isolated from Conservolea variety of Greece.21 Strains of Candida norvegica and Galactomyces reessii were isolated from olive variety Negrinha de Freixo of Portugal.23

18.3 Selection of probiotics from fermented olives The basic criteria of probiotics selection, adopted by the World Health Organization (WHO), include the

host-associated stress resistance, epithelium adhesion ability, and antimicrobial activity.7 Furthermore, the presence of antibiotic resistance and safety properties are the important criteria, recommended by European Food Safety Authority (EFSA), in selection of bacterial strains intended to food industry.25 LAB and yeasts, dominating during olive fermentation process, will be studied in this chapter.

18.3.1 Lactic acid bacteria The criteria used in the selection of probiotic strains of LAB are classified as probiotic and safety properties (Table 18.1). The probiotic properties mostly studied include the resistance to low pH and bile salt or the tolerance to gastric juice and pancreatic digestions, and in some cases all of them, with the main goal to achieve the survival of probiotics in gastrointestinal tract. The aggregation, hydrophobicity and adhesion capabilities, and antimicrobial activity were also studied. The safety properties mostly studied were antibiotic resistance and hemolysis activity. However, the gelatinase, DNase, and mucin degradation were studied in few works only.14,15,20 L. plantarum and L. pentosus were the most selected species as probiotics (Table 18.1). Strains of these species demonstrated their high tolerance to low pH (down to pH 1.5) and bile salts (up to 4%), production of exopolysaccharides, and good adherence to Caco-2 cells.10
12,17 For instance, L. pentosus GG2S-T2-168 strain showed not only high resistance to stress factors (low pH and bile salt) but higher values of auto-aggregation and hydrophobicity as well.17 Some other strains, of L. paraplantarum, L. paracasei subsp. paracasei, L. coryniformis, L. brevis, L. mesenteroides, and Enterococcus spp., were isolated from fermented olives with lower frequency, but with important probiotic properties.14,15,19,20 Strains of probiotic LAB demonstrated good inhibitory effect against pathogens (Listeria monocytogenes, Salmonella enteritidis, Escherichia coli O157:H7), no hemolytic activity, and no cytotoxicity to H4-1 human epithelial cells.17 Their antimicrobial activity against pathogens is mostly related to organic acids and bacteriocins production.18 Within LAB group, L. plantarum is considered one of the most suitable protective microorganism possessing the ability of producing antimicrobial compounds, such as plantaricin, but also very effective antifungal metabolites, such as PLA, 4-hydroxyphenyllactic acid, and cyclic dipeptides.26 In addition, certain probiotic Lactobacillus strains showed antifungal activity, associated with reduction of surface tension to 36.23
40.27 mN/m, against 41.03 mN/m obtained in noninoculated de Man, Rogosa, and Sharpe medium.15 The high auto-aggregation and hydrophobicity of these probiotic strains may be due to the production of

Probiotics from fermented olives Chapter | 18

bioactive molecules, particularly exopolysaccharides,14,15 assuring their development as biofilms on olives and containers. These acidic biofilms play important roles in protecting lactic bacteria cells in this toxic environment, rich of organic acids, NaCl, and polyphenols, against osmotic pressure and other competing microorganisms and assure the fixation of nutrients released from olives, allowing, consequently, their bioavailability to the bacterial cells.

18.3.2 Yeasts The criteria mostly used in selection of probiotic yeast strains, isolated from fermented olives, are reported on Table 18.1. The main criteria evaluated were tolerance to gastric juice and pancreatic digestion. Other probiotic properties, such as auto-aggregation, antioxidant activity, cholesterol removal, and antifungal activity, were studied in few works.23 In fact, research works on the characterization of potential probiotic yeasts, isolated from fermented olive, were recently initiated. To our knowledge the first work conducted on this subject was realized by Bonatsou et al.21 These authors characterized 12 yeast strains, isolated from fermented black Greek olive, for their tolerance to gastric and pancreatic digestions. Their results indicated that all of the yeast strains showed high resistance to NaCl, and most of them survived in gastric and pancreatic digestions. P. guilliermondii Y16 showed overall the highest resistance to NaCl and simulated digestions.21 Oliveira et al.23 isolated from natural fermentation of Negrinha de Freixo olive cultivar, S. cerevisiae 15A, Candida tropicalis 1A, C. norvegica 7A, and G. reessii 34A, as probiotic yeast strains. C. norvegica 7A and G. reessii 34A demonstrated antifungal activity against pathogenic yeast Cryptococcus neoformans, while S. cerevisiae 15A, C. tropicalis 1A, and C. norvegica 7A showed important values of auto-aggregation ( . 80% after 24 h) and antioxidant activity.23 The cholesterol removal capacity was demonstrated in probiotic strains of S. cerevisiae Sc24 and Candida boidinii Cb60, isolated from fermented Bosana olive.22 Deep studies on the other criteria should be conducted to find out other profitable probiotic properties from yeasts.

18.4 Safety properties of probiotics in human The safety assessment is the most important criteria, established by the European community (The European Union Novel Food regulation and Qualified Presumption of Safety), United States [Food and Drug Administration (FDA) and WHO], and Canada (Health Canada), for human use of probiotics, to avoid their unwanted

219

properties. The recommended safety criteria in probiotics selection include mainly isolation environment, taxonomic identification, and absence of virulence and transferable antibiotic resistance genes.27 The safety properties studied in probiotic LAB strains, isolated from fermented olive, are reported in Table 18.1. Most of these probiotic strains showed the absence of γ-hemolytic activity (i.e., lysis of red blood cells).12,14,15,17 However, virulence factors related to α-hemolytic and β-hemolytic activities were detected in some strains of L. pentosus and L. plantarum, respectively.16 The DNase and gelatinase activities, evaluated in few works (Table 18.1), were not detected in LAB strains.15,20 The mucin degradation and the cytotoxic activity against H4-1 human epithelial cells were not detected in probiotic strains isolated from fermented olives of Portugal, Italy, and Morocco.13
15 According to EFSA guidelines, the antibiotic resistance is the main safety criteria to evaluate in probiotic strains intended to human consumption.28 The studies indicated in Table 18.1 reported that the majority of LAB strains were resistant to antibiotics of the amino-glucoside group (i.e., kanamycin, gentamycin, and streptomycin) and glycopeptides (i.e., vancomycin), while they showed a sensitivity to other antibiotics (i.e., erythromycin and ampicillin).12
16,29 The resistance to the aminoglycosides is considered intrinsic in Lactobacillus.12 This intrinsic resistance was demonstrated to be nontransferable or nonacquired and may in part be due to chromosomally efflux-pumps genes (NorA, MepA, and MdeA).29 Basing bioinformatics analysis of L. pentosus MP-10 genome isolated from fermented olives, Abriouel et al.30 demonstrated the absence of acquired antibiotic resistance genes and confirmed that the antibiotic resistance exhibited by the strain is intrinsic and related to efflux-pump genes. It should be emphasized that the safety criteria used in previous works are limited to antibiotics resistance and hemolysis activity in LAB and absent in yeasts strains selection. However, the detection of β-hemolysis in some probiotic Lactobacillus strains indicates that the determination of other virulence factors (i.e., DNase, gelatinase, mucin degradation, and biogenic amine production) in LAB and yeasts selection is mandatory to avoid public health concerns.

18.5 Health-beneficial effects of probiotics from fermented olives Fermented olives, recognized as extreme environment, may be a good natural source of probiotics, which may be exploited to develop health-promoting functional products.5 In recent years, many works, based on in vitro or

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

in vivo tests, demonstrated various health beneficial effects of probiotic LAB from fermented olives, mainly the antibacterial, antifungal, antiadhesion, antiproliferative, anticancer, metals-detoxification, and immunestimulatory activities.19,31
33 Probiotic strains of L. brevis, L. pentosus, L. plantarum, L. paraplantarum, and L. coryniformis inhibited a wide spectrum of pathogenic bacteria (Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, E. coli, L. monocytogenes, Micrococcus luteus, Staphylococcus aureus, and Enterococcus faecalis).13
15 The antifungal activity, against Candida pelliculosa and Penicillium, was demonstrated in some potential probiotic strains of L. plantarum, L. pentosus, and L. brevis.15 Some Candida species (C. albicans and non-C. albicans) are known for their involvement in nosocomial diseases of fungal origin,34 while Penicillium is involved in food spoilage and in the production of mycotoxins, causing a serious public health concern.35 The antimicrobial probiotic Lactobacillus strains and/or their bioactive molecules can be used, as biological tools, in human health protection from nosocomial and foodborne pathogens (bacteria and fungi) and their toxins. The adhesion capacity on epithelial cells and Caco-2 colon cancer cells was demonstrated in L. pentosus D303.36 and L. coryniformis H307.6,19 and in L. pentosus B281 and L. plantarum B282, respectively.31 Some of these probiotic strains inhibited the adhesion of pathogenic bacteria on human intestinal cell,19 indicating their possible use in preventing gastrointestinal infection. The anticancer effect of L. pentosus B281 and L. plantarum B282, by inhibiting the proliferation of human colon cancer cells (Caco-2 and HT-29), was demonstrated by36 being the antiproliferative activity due to thermostable compounds.31 Furthermore, the biosequestration of various metals (iron, aluminum, cobalt, copper, zinc, cadmium, and mercury) was detected in the probiotic L. pentosus MP-10,33 indicating its possible use in reducing the metal toxicity exposure in human intestines. In vivo tests, conducted on the nematode Caenorhabditis elegans fed by probiotics L. pentosus D303.36 and L. coryniformis H307.6, showed an increase in longevity of the nematode to 18 days and 15 days, respectively, compared to the control (9.5 days), and displayed the protection of the nematode from pathogen infection.19 Some strains of L. pentosus and L. plantarum were selected for their immunomodulatory activity on mice by stimulating the production of proinflammatory and antiinflammatory, particularly interleukins factors (IL-1α, IL1β, IL-6, and IL-10)37 and chemokine ligands (CXCL-1, CXCL-2, CCL-3, and CCL-4).32,36 In fact, beneficial effects of L. pentosus LPG1 were demonstrated on mice health, particularly in reduction of weight loss, decrease of gut permeability, beneficial cytokine modulation, and tissue damage reduction.36

Probiotic strain of L. coryniformis, isolated from traditional goat cheese, was demonstrated for its healthbeneficial effects in mice and humans. L. coryniformis CECT5711 demonstrated endothelial-protective effect, suggesting its possible use in preventing vasculopathy in obese mice.38 In human, L. coryniformis CECT5711 increased the Hepatitis A virus antibodies compared to placebo, indicating its possible clinical benefits in protection from future infections39 and enhanced the immune response against influenza vaccine and protected against respiratory infections in elderly population.40

18.6 Technological properties of probiotics from fermented olives The technological properties that must meet probiotic strains of LAB and yeasts to dominate in the extreme environment of olive brine are mainly tolerance to NaCl and polyphenols (i.e., oleuropein), production of enzymes, and high acidification capacity. Lactic fermentation of olives (industrial process) is mostly initiated in brine of 10%
12% NaCl, leading to the natural selection of LAB strains tolerating this high salt concentration.4 This extreme environment is characterized also by high contents of oleuropein, the main natural bitter polyphenol of olive.41 LAB strains (L. plantarum, L. pentosus, L. paraplantarum, L. brevis, and Pediococcus sp.), isolated from fermented olives, demonstrated high tolerance to phenol (0.6%),15 and high biodegradation capacity of oleuropein (1%).14,42 The β-glucosidase activity, involved in oleuropein biodegradation, was detected in probiotic LAB isolated from various Mediterranean olive varieties,14,42
45 and also in yeasts,21,22 leading to the accumulation of hydroxytyrosol.42 The hydroxytyrosol, considered stable antioxidant, is highly desired in foods, because of its antiinflammatory, neuroprotective, immunomodulatory, nonmutagenic, nongenotoxic, and antifungal properties.46 The high acidification capacity of LAB is the most important criteria in the selection of starters intended to olive fermentation, to assure the biotransformation of olives, and to inhibit pathogenic and spoilage microorganisms.43,45 However, the low pH (pH , 4.5) in brine may increase the growth of yeasts and molds, involved in gaspocket spoilage of olives.47 Starters, composed of enzymes from yeasts (i.e., β-glucosidase), combined with oleuropeinolytic probiotic LAB may be of great interest in biological processing of fermented olives, from technological, functional, and environmental point of view. Probiotic starters may lead not only to avoid chemical debittering (NaOH) but also to reduce olive spoilages (i.e., gas-pockets), and to contribute to human health protection, thanks to enrichment of fermented olives with

Probiotics from fermented olives Chapter | 18

antioxidants (i.e., microorganisms.

hydroxytyrosol)

and

functional

18.7 Application of probiotics in olive fermentation Spontaneous fermentation of olives is essentially undergone by the natural microflora associated to olives, containers, and environment. The addition of probiotic LAB starters may offer advantages in health and technological aspects, during fermentation and preservation of olives. Studies, conducted on the application of autochthonous and nonautochthonous probiotic LAB in olive fermentation, are reported in Table 18.2.

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18.7.1 Application of autochthonous probiotics Argyri et al.48 obtained high levels of dominance of the probiotic strains (L. pentosus B281 and L. plantarum B282) used as starter in fermentation of heat-chocked olives, with survival rates of 94.7% and 100% for B281, and 55% and 58.8% for B282, at 8% and 10% NaCl, respectively. However, generally lower survival rates (81.2% and 93.3% for B281, and 83.3% and 0% for B282, respectively at 8% and 10% NaCl), were obtained in not heat-chocked olives.49 The heat-choking of green olive seems increase the permeability of olive flesh, leading to the release of nutrients important for microbial growth, which consequently increase the survival rate of Lactobacillus strains.

TABLE 18.2 Application of autochthonous and nonautochthonous probiotic lactic acid bacteria (LAB) as starters in olive fermentation. LAB strains

Source (olive variety)

Application (olive variety)

Probiotic properties

Conditions of application

Survival of the probiotics

References

Resistance to low pH and bile salts, good adherence to Caco-2 cells

Fermentation of heat shocked green olives

94.7% at 8% NaCl brines 100% at 10% NaCl brines

[48]

Autochthonous probiotic strains Lactobacillus pentosus B281

Kalamata

Halkidiki

L. pentosus B281

Kalamata

Halkidiki

Fermentation of green olive

81.3% at 8% NaCl brines 93.3% at 10% NaCl brines

[49]

Lactobacillus plantarum B282

Halkidiki

Halkidiki

Fermentation of heat shocked green olive

55% at 8% NaCl brines 58.8% at 10% NaCl brines

[48]

L. plantarum B282

Halkidiki

Halkidiki

Fermentation of green olive

83.3% at 8% NaCl brines 0% at 10% NaCl brines

[49]

L. pentosus TOMC LAB2

Gordal and Manzanilla

Manzanilla

Fermentation of green olive (pilot scale)

60% on olive surface

[50]

L. pentosus TOMC LAB4

Gordal and Manzanilla

Manzanilla

Fermentation of green olives (pilot scale)

53% on olive surface

[51]

L. pentosus TOMC LAB2

Gordal and Manzanilla

Manzanilla

Fermentation of green olives (industrial scale)

Dominance of the probiotic on olive surface

[52]

L. pentosus TOMC-LAB2

Gordal and Manzanilla

Manzanilla

Fermentation of green olive (pilot scale)

90% on olive surface 4.5% in spontaneously fermented olive

[50]

Resistance to gastric and pancreatic digestions, autoaggregation, and hydrophobicity properties

(Continued )

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 18.2 (Continued) LAB strains

Source (olive variety)

Application (olive variety)

Probiotic properties

Conditions of application

Survival of the probiotics

References

Survival in the stomach and small intestine and adhesion to intestinal cells

Fermentation of black olive

100% on olives

[53]

Fermentation of alkali-treated green olive

31.1% at 4% NaCl brines 41.1% at 8% NaCl brines at room temperature 0% at 4% NaCl brines at 4 C

[54]

Fermentation of nonalkali-treated green olive (inoculation after 60 days of brining)

High survival rate on olive

[55]

Fermentation of nonalkali-treated green olive (inoculation after 60 days of brining)

High survival rate on olive

[55]

Nonautochthonous probiotic strains Lactobacillus rhamnosus GG

Human

Hojiblanca

Lactobacillus paracasei IMPC2.1

Human

Bella di Cerignola

L. rhamnosus GG

Human

Giarraffa and Grossa di Spagna

L. rhamnosus H25

Cheese

Giarraffa and Grossa di Spagna

Survival during in vitro gastric and gastric plus duodenal digestion

Basing on RAPD PCR analysis, Rodriguez-Gomez et al.50 evidenced, in not-heated Manzanilla olives fermented with L. pentosus strains (TOMC LAB2 and TOMC LAB4), olives surface adhesion levels of 60% and 53% for TOMC LAB2 and TOMC LAB4, respectively. During large-scale fermentation of Manzanilla olives (Spanish-style), the dominance of L. pentosus TOMC LAB2 on the olives surface was higher during September and reduced during October to 70%
90%.51 Putative probiotic L. pentosus TOMC-LAB2, inoculated in traditional fermented green olives (Spanish style), showed a survival rate of 90% in olive fruit, against 4.5% obtained in spontaneously fermented olives (control), and the probiotic fermented olives were perceived by the consumers as similar to the traditional product.52 The probiotic LAB starters showed good effectiveness in traditional olive fermentation, in terms of probiotic and organoleptic properties of fermented olives, increasing consequently added value of traditional fermented olives.

18.7.2 Application of nonautochthonous probiotics Generally, the controlled fermentation of olives is conducted with starters composed of autochthonous strains of

LAB or yeasts. However, some probiotic strains (Lactobacillus rhamnosus GG and L. paracasei IMPC2.1) of human origin, tested as starters in olive fermentation, demonstrated their higher activity in this process. Hence, L. rhamnosus GG showed high survival rates in Hojiblanca (100%), and in Grossa di Spagna and Giarraffa olive varieties, after 3 and 4 months of fermentation, respectively.53,55 L. paracasei IMPC2.1, inoculated in debittered green olive brine of Bella di Cerignola variety, displayed, at room temperature, olive surface colonization levels of 31.1% and 41.1% at 4% and at 8% of NaCl, respectively, while at 4 C no survivals were observed on olive surface at 4% NaCl.54 L. rhamnosus H25, of cheese origin, used as starter in fermentation of debittered green olive, showed high survival rates in Grossa di Spagna and Giarraffa olives.55 These authors demonstrated that fermented olives, with L. rhamnosus GG and H25 strains, exhibited various volatile compounds and important sensorial traits, depending of the olive variety. These findings lead us to conclude that the probiotic LAB strains from other origins may be profitable in the fermentation process of different green olive varieties, providing multiple advantages, including the improvement of sensorial properties of the final product.

Probiotics from fermented olives Chapter | 18

18.8 Application of probiotics in biopreservation of fermented olives The biopreservation is more and more used in food preservation, because of the health concern of chemical additives. LAB has attracted increase interest of researchers in food biopreservation, because of their probiotic and safety properties.15,56 The applications of probiotic LAB in biopreservation of fermented olives are reported in Table 18.3. The inhibitory effect of probiotic LAB was demonstrated against pathogenic Gram-negative and Gram-positive bacteria (i.e., Yersinia enterocolitica, enterotoxigenic E. coli and

223

Listeria monocytogenes) and yeasts and molds involved in food spoilage (Penicillium sp. and C. pelliculosa).10,15,63 L. plantarum improved the hygienic and organoleptic quality and reduced the incidence of gas-pocket spoilage in inoculated fermented olive during storage.64,65 The probiotic L. pentosus B281 reduced drastically to undetectable limit, the survival of pathogens (E. coli O157:H7, S. enteritidis, and L. monocytogenes) after 40 days of storage of fermented olives Halkidiki, while the survival of B281 was not affected (100%).57 These fermented olives were stored in brine of high salt concentration (6%, w/v) and low pH (4.2), and initially

TABLE 18.3 Application of autochthonous probiotic lactic acid bacteria (LAB) as bio-preservative during storage of fermented olives. LAB strains

Source (olive variety)

Application (olive variety)

Probiotic properties

Resistance to low pH and bile salts, good adherence to Caco-2 cells

Conditions of application

Survival of the probiotics

References

Storage of fermented olive contaminated with pathogens

100% on olive after 40 days of storage

[57]

Lactobacillus pentosus B281

Halkidiki

Halkidiki

L. pentosus B281

Halkidiki

Halkidiki

Preservation of fermented green olive under modified atmosphere packaging

64.71% on olive after 6 months at 4 C 20% on olive after 6 months at 20 C

[58]

L. pentosus B281

Halkidiki

Halkidiki

Preservation of fermented green olives packed in polyethylene pouches at 4 C and 20 C

100% on olive after 6 months at 4 C 20% on olive after 6 months at 20 C

[59]

Lactobacillus plantarum B282

Kalamata

Halkidiki

Preservation of fermented green olives packed in polyethylene pouches at 4 C and 20 C

96% on olive after 6 months at 4 C 0% on olive after 6 months at 20 C

[59]

L. plantarum B282

Kalamata

Halkidiki

Preservation of fermented green olive under modified atmosphere packaging

94.12% on olive after 6 months at 4 C 0% on olive after 6 months at 20 C

[58]

(Continued )

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 18.3 (Continued) LAB strains

Source (olive variety)

Application (olive variety)

L. pentosus TOMC LAB2

Gordal and Manzanilla

Manzanilla

L. pentosus TOMC-LAB2 and L. pentosus TOMC-LAB4

Gordal and Manzanilla

L. pentosus TOMC-LAB2

Gordal and Manzanilla

Probiotic properties

Conditions of application

Survival of the probiotics

References

Preservation of fermented olives packed in brines with heat and not heat shocked

100% on olive in heatshocked 57.9% on olive in not heatshocked

[60]

Manzanilla

Preservation of packed and nonthermally treated fermented green olive

Survival on olive and brine in different package

[61]

Manzanilla

Preservation of fermented green olive in different packages and temperatures

100% on olive surface

[62]

Survival in gastric and pancreatic digestions, auto-aggregation, and hydrophobicity proprieties

contaminated with these pathogens at 7 log CFU/mL. After 6 months of storage of fermented green olives, under modified atmosphere packaging, the probiotic strains (L. pentosus B281 and L. plantarum B282) showed survival rates of 20% and 0% at 20 C, and 64.71% and 94.12% at 4 C, respectively.58 The survivals obtained for these two probiotics, in these conditions of packaging, were higher at 4 C than 20 C. The packed fermented olives showed better organoleptic properties after 6 months than 12 months of storage at 20 C and 4 C. In fermented green olives stored in polyethylene pouches and based on molecular analyze [pulsed-field gel electrophoresis (PFGE)], L. pentosus B281 and L. plantarum B282 showed higher survival rates of 100% and 96%, respectively, at 4 C, while for both strains the final survival at 20 C was less than 20%.59 Furthermore, L. pentosus B281 showed higher survival than L. plantarum B282, when inoculated in coculture in packed fermented olives and stored at both temperatures. During 200 days of storage of fermented green olives (Spanish style), Rodriguez-Gomez et al.60 obtained survival rates of the probiotic L. pentosus TOMC-LAB2 of 100% and 57.9% in heated (85 C/5 min) and nonheated olives, respectively. The fermented green olives of Manzanilla variety, based on Spanish-style process and inoculation with the probiotics L. pentosus (TOMC LAB2 and TOMC LAB4), were preserved in different packages and conditions.61 The results obtained, by the colony

counting method, showed that the survivals of LAB strains, at 22 C, were higher in olives fruits packed in plastic pouches with brine or modified atmosphere, and medium in olive fruits packed in glass jar with brine or in vacuum plastic pouches. However, the survival of LAB in olive brine, at 13 C and 22 C, was higher in glass jar and lower in plastic pouches. In all packages and conditions the color of the preserved olives was not affected during storage. In another work, based on molecular techniques analyses, L. pentosus TOMC-LAB2 exhibited high survival in fermented olive packed in plastic pouches under nitrogen atmosphere and in glass jars, during storage at 20 C.62 The fermented olive packed in plastic pouches, showed a degradation in their color, and no adherence of the probiotic to their surface, while in glass jar, the color of the olives was stable and showed 100% of adherence of the probiotic strain on their surface. These studies indicate that probiotic strains may survive in brine and olive surface during storage under different temperatures (13 C and 20 C) and packages (plastic pouches under modified atmosphere or vacuum pressed olives, and in glass jar), but with variable values. The optimization of storage conditions (packaging material and temperature) may increase the survival of the probiotics and improve their effectiveness in preservation and organoleptic properties of probiotic fermented olive.

Probiotics from fermented olives Chapter | 18

18.9 Application of probiotics from fermented olive in other foods fermentations The research works conducted on the application of probiotic LAB, isolated from fermented olive, in the production of other fermented foods are reported in Table 18.4. L. plantarum 33 strain, inoculated in olive paste, exhibited its effectiveness in stabilizing the pH during 30 days of storage, without affecting the sensorial and textural properties of the product.66 These authors demonstrated that microencapsulation improved the survival of L. plantarum 33 during storage (30 days) of olive paste at 4 C. Probiotic strains of L. plantarum (Lp S11T3E and Lp S2T10D) and L. pentosus Lps S3T60C, isolated from fermented olive and inoculated in pasteurized milk during the processing of Toma Piemontese Protected Denomination of Origin cheese, showed high survivals, without affecting the organoleptic properties of the final product.67 Saxami et al.68 demonstrated high survival of 6 log CFU/g after 35 days of storage at 4 C, of probiotics L. pentosus B281 and L. plantarum B282, isolated from fermented olive and inoculated in milk during yogurt processing, without affecting sensorial properties of final products. The probiotic LAB obtained from fermented olive, displayed their survival and effectiveness during processing and storage of other fermented foods (i.e., olive paste, cheese, and yogurt), without affecting the organoleptic properties of the products. These findings indicate the important probiotic characteristics of LAB strains, isolated from olive fermentation, profitable in other foods fermentations.

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18.10 Conclusion Olive fermentation process, occurring in extreme environment, is conducted mainly by LAB and yeasts. Microbial strains, dominating in this environment, characterized by low pH and nutrients contents, and high polyphenols and NaCl levels, should have important physiologic properties, allowing their use as probiotics. Probiotic L. plantarum and L. pentosus were the most isolated species from various fermented Mediterranean olive varieties and different countries. Some strains of these species demonstrated important probiotic properties, particularly their high tolerance to low pH, high bile salt concentrations, production of exopolysaccharides, auto-aggregation, hydrophobicity, good adherence to Caco-2 cells, and wide spectrum of antimicrobial activity against pathogenic bacteria, yeasts, and molds. The safety criteria mostly used are limited to antibiotic resistance and hemolysis; other virulence factors should be included in probiotics selection. Various beneficial health effects of probiotic LAB from fermented olive, mainly the antibacterial, antifungal, antiadhesion, antiproliferative, anticancer, metals detoxification, and immunostimulatory activities, were demonstrated using in vitro or in vivo tests on mice and humans. Probiotic yeast strains, isolated from this environment, belonged mostly to the genera of Saccharomyces, Pichia, Candida, Rhodotorula, Metschnikowia, and Galactomyces. However, fewer probiotic criteria were used in their selection, including resistance to gastric and pancreatic digestions, while their safety properties were not evaluated.

TABLE 18.4 Application of probiotic lactic acid bacteria (LAB) of fermented olive origin in other fermented foods. LAB strains

Origin (olive variety)

Application (fermented food)

Campo Maior

Olive paste

Storage of olive paste with probiotic strain

Survival during storage of olive paste at 4 C

[66]

L. plantarum (Lp S11T3E and Lp S2T10D) and Lactobacillus pentosus (Lps S3T60C)

Nocellara Etnea

Cheese (Toma Piemontese PDO)

Application of probiotics as adjunct cultures in pilotscale cheese process

High survival during cheese processing

[67]

L. pentosus B281 and L. plantarum B282

Kalamata and Halkidiki

Yogurt

Application of probiotics as starter adjuncts in yogurt production

6 log CFU/g in yogurt after 35 days of storage at 4 C

[68]

Lactobacillus plantarum 33

Conditions of application

Survival of the probiotics

References

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

Autochthonous probiotic starters composed of LAB (L. pentosus and L. plantarum) and enzymes (i.e., β-glucosidase) of yeasts may be used in olive fermentation, as well as their preservation during storage in various packages. These starters increased the functional properties of fermented olives, due to the accumulation of hydroxytyrosol, as a result of oleuropein biodegradation. Nonautochthonous probiotic LAB strains (L. paracasei IMPC2.1 and L. rhamnosus GG and H25), of human and cheese origins, showed their effectiveness in olive fermentation and storage, with good perception by the consumer. On the other hand, probiotic LAB of fermented olive origin demonstrated their effectiveness in other foods fermentations (olive paste, cheese, and yoghurt), without affecting organoleptic and sensorial properties of the final product. The optimization of probiotics selection and application conditions may enhance their health-beneficial effects, as well as their effectiveness in production and preservation of probiotic fermented olives and other food products.

Mini-dictionary of terms Mediterranean diet

Oleuropein Chemical debittering Oleuropeinolytic probiotic LAB Autochthonous strains Nonautochthonous strains

Survival rate

Health-beneficial effects of probiotics

Biosequestration of metals

“The Mediterranean diet involves a set of skills, knowledge, rituals, symbols, and traditions concerning crops, harvesting, fishing, animal husbandry, conservation, processing, cooking, and particularly the sharing and consumption of food” (UNESCO, 2013).1 The main natural polyphenol responsible of olive bitterness. Elimination of bitterness using chemicals (NaOH). Biodegradation of oleuropein by probiotic LAB. Strains isolated from fermented olives and inoculated in olive fermentation. Strains isolated from fermented olives and inoculated in other food products (olive paste, cheese, and yogurt), or isolated from other origins (human and cheese) and inoculated in olive fermentation. Percentage of surviving bacteria obtained under the conditions studied compared to the control. Antibacterial, antifungal, antiadhesion, antiproliferative, anticancer, metaldetoxification, and immune-stimulatory activities. Adsorption and accumulation of toxic metals (iron, aluminum, cobalt, copper, zinc, cadmium, and mercury) by probiotic

Natural fermentation process

LAB, leading to the reduction of metal toxicity exposure in human intestine. Spontaneous fermentation process occurring in olives brines.

Summary points G

G

G

G

G

G

Fermented green olives are the principal fermented product in the Mediterranean diet, which is known for its health benefits for the consumer. Natural fermentation process of green olives, occurring in extreme environment characterized by low nutrients and high polyphenols and sodium chloride contents, is an important source of probiotic LAB and yeasts, dominated by L. plantarum, L. pentosus, L. paracasei, L. rhamnosus, and L. coryniformis. Probiotic LAB strains, isolated from fermented olives, demonstrated high tolerance to low pH and high bile salt concentration; production of exopolysaccharides; auto-aggregation; hydrophobicity; good adherence to Caco-2 cells; wide spectrum of antimicrobial activity against pathogenic bacteria, yeasts, and molds; and no hemolysis, while antibiotic resistance is mostly related to intrinsic and nontransferable genes. Probiotic LAB strains exhibited numerous beneficial health effects, based in vitro and in vivo tests on mice and humans, particularly the antibacterial, antifungal, and antiadhesive effects against pathogens, the protective effect against virus infections (Hepatitis A and influenza), anticancer, and metal-detoxification activities. Probiotics starters composed of autochthonous or nonautochthonous LAB strains, from human and cheese origins, improved the fermentation process and storage conditions, as well as the functional properties of olives and other food products (olive paste, cheese, and yoghurt), with good perception of the products by the consumer. The optimization of probiotics selection and formulation may enhance their health-beneficial effects, as well as their effectiveness in production and preservation of probiotic fermented olives and other food products.

References 1. UNESCO. Mediterranean diet. ,https://ich.unesco.org/en/RL/mediterranean-diet-00884.; 2013 Accessed 08.11.19. 2. Singh B, Parsaik AK, Mielke MM, et al. Association of Mediterranean diet with mild cognitive impairment and Alzheimer’s disease: a systematic review and meta-analysis. J Alzheimers Dis. 2014;39(2):271
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1751. Bonatsou S, Tassou CC, Panagou EZ, Nychas GE. Table olive fermentation using starter cultures with multifunctional potential. Microorganisms. 2017;5(2):30. Peres CM, Peres C, Herna´ndez-Mendoza A, Malcata FX. Review on fermented plant materials as carriers and sources of potentially probiotic lactic acid bacteria
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M544. Argyri AA, Zoumpopoulou G, Karatzas KA, et al. Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiol. 2013;33(2):282
291. Botta C, Langerholc T, Cencic A, Cocolin L. In vitro selection and characterization of new probiotic candidates from table olive microbiota. PLoS One. 2014;9(4):e94457. Peres CM, Alves M, Hernandez-Mendoza A, et al. Novel isolates of lactobacilli from fermented Portuguese olive as potential probiotics. LWT-Food Sci Technol. 2014;59(1):234
246. Abouloifa H, Rokni Y, Bellaouchi R, et al. Characterization of probiotic properties of antifungal Lactobacillus strains isolated from traditional fermenting green olives. Probiotics Antimicrob Proteins. 2020;12:683
696. Benı´tez-Cabello A, Calero-Delgado B, Rodrı´guez-Go´mez F, Garrido-Ferna´ndez A, Jime´nez-Dı´az R, Arroyo-Lo´pez FN. Biodiversity and multifunctional features of lactic acid bacteria isolated from table olive biofilms. Front Microbiol. 2019;10:836. Bautista-Gallego J, Arroyo-Lo´pez FN, Rantsiou K, Jime´nez-Dı´az R, Garrido-Ferna´ndez A, Cocolin L. Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential. Food Res Int. 2013;50(1):135
142. Montoro BP, Benomar N, Lavilla Lerma L, Castillo Gutierrez S, Galvez A, Abriouel H. Fermented Alorena table olives as a source of potential probiotic Lactobacillus pentosus strains. Front Microbiol. 2016;7:1583.

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185. 28. EFSA. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J. 2012;10(6). 29. Casado Munoz Mdel C, Benomar N, Lerma LL, Galvez A, Abriouel H. Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturallyfermented Aloren˜a table olives throughout fermentation process. Int J Food Microbiol. 2014;172:110
118. 30. Abriouel H, Perez Montoro B, Casado Munoz MDC, Knapp CW, Galvez A, Benomar N. In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Alorena green table olives. PLoS One. 2017;12(6): e0176801. 31. Saxami G, Karapetsas A, Lamprianidou E, et al. Two potential probiotic Lactobacillus strains isolated from olive microbiota exhibit adhesion and anti-proliferative effects in cancer cell lines. J Funct Foods. 2016;24:461
471. 32. Saxami G, Karapetsas A, Chondrou P, et al. Potentially probiotic Lactobacillus strains with anti-proliferative activity induce cytokine/chemokine production and neutrophil recruitment in mice. Benef Microbes. 2017;8(4):615
623. 33. Abriouel H, Perez Montoro B, de la Fuente Ordonez JJ, Lavilla Lerma L, Knapp CW, Benomar N. New insights into the role of plasmids from probiotic Lactobacillus pentosus MP-10 in Alorena table olive brine fermentation. Sci Rep. 2019;9(1):10938. 34. Araujo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017;25 (1):62
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1082. Ben Salah R, Trabelsi I, Ben Mansour R, Lassoued S, Chouayekh H, Bejar S. A new Lactobacillus plantarum strain, TN8, from the gastro intestinal tract of poultry induces high cytokine production. Anaerobe. 2012;18(4):436
444. Toral M, Gomez-Guzman M, Jimenez R, et al. The probiotic Lactobacillus coryniformis CECT5711 reduces the vascular prooxidant and pro-inflammatory status in obese mice. Clin Sci (Lond). 2014;127(1):33
45. Redondo N, Nova E, Gheorghe A, Diaz LE, Hernandez A, Marcos A. Evaluation of Lactobacillus coryniformis CECT5711 strain as a coadjuvant in a vaccination process: a randomised clinical trial in healthy adults. Nutr Metab (Lond). 2017;14(2). Fonolla J, Gracian C, Maldonado-Lobon JA, et al. Effects of Lactobacillus coryniformis K8 CECT5711 on the immune response to influenza vaccination and the assessment of common respiratory symptoms in elderly subjects: a randomized controlled trial. Eur J Nutr. 2019;58(1):83
90. Montan˜o A, Sa´nchez AH, Lo´pez-Lo´pez A, de Castro A, Rejano L. Chemical composition of fermented green olives. In: Preedy V, Watson R, eds. Olives and Olive Oil in Health and Disease Prevention. Elsevier; 2010:291
297. Ghabbour N, Lamzira Z, Thonart P, Cidalia P, Markaoui M, Asehraou A. Selection of oleuropein-degrading lactic acid bacteria strains isolated from fermenting Moroccan green olives. Grasas Aceites. 2011;62(1):84
89. Abriouel H, Benomar N, Cobo A, et al. Characterization of lactic acid bacteria from naturally-fermented Manzanilla Alorena green table olives. Food Microbiol. 2012;32(2):308
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717. Abouloifa H, Rokni Y, Bellaouchi R, et al. Technological properties of potential probiotic Lactobacillus strains isolated from traditional fermenting green olive. J Microbiol Biotechnol Food Sci. 2020;9(5). Bertelli M, Kiani AK, Paolacci S, et al. Hydroxytyrosol: a natural compound with promising pharmacological activities. J Bacteriol. 2019;309:29
33. Asehraou A, Peres C, Brito D, Faid M, Serhrouchni M. Characterization of yeast strains isolated from bloaters of fermented green table olives during storage. Grasas Aceites. 2000;51 (4):225
229. Argyri AA, Nisiotou AA, Mallouchos A, Panagou EZ, Tassou CC. Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives. Int J Food Microbiol. 2014;171:68
76. Blana VA, Grounta A, Tassou CC, Nychas GJ, Panagou EZ. Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives. Food Microbiol. 2014;38:208
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50. Rodriguez-Gomez F, Romero-Gil V, Bautista-Gallego J, GarciaGarcia P, Garrido-Fernandez A, Arroyo-Lopez FN. Production of potential probiotic Spanish-style green table olives at pilot plant scale using multifunctional starters. Food Microbiol. 2014;44:278
287. 51. Rodriguez-Gomez F, Romero-Gil V, Arroyo-Lopez FN, et al. Assessing the challenges in the application of potential probiotic lactic acid bacteria in the large-scale fermentation of Spanish-style table olives. Front Microbiol. 2017;8:915. 52. Lopez-Lopez A, Moreno-Baquero JM, Rodriguez-Gomez F, Garcia-Garcia P, Garrido-Fernandez A. Sensory assessment by consumers of traditional and potentially probiotic green Spanish-style table olives. Front Nutr. 2018;5:53. 53. Lavermicocca P, Valerio F, Lonigro SL, et al. Study of adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food. Appl Environ Microbiol. 2005;71(8):4233
4240. 54. De Bellis P, Valerio F, Sisto A, Lonigro SL, Lavermicocca P. Probiotic table olives: microbial populations adhering on olive surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant. Int J Food Microbiol. 2010;140(1):6
13. 55. Randazzo CL, Todaro A, Pino A, et al. Giarraffa and Grossa di Spagna naturally fermented table olives: effect of starter and probiotic cultures on chemical, microbiological and sensory traits. Food Res Int. 2014;62:1154
1164. 56. Jimenez Diaz R, Rios-Sanchez RM, Desmazeaud M, Ruiz-Barba JL, Piard J-C. Plantaricins S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl Environ Microbiol. 1993;59(5):1416
1424. 57. Argyri AA, Lyra E, Panagou EZ, Tassou CC. Fate of Escherichia coli O157:H7, Salmonella enteritidis and Listeria monocytogenes during storage of fermented green table olives in brine. Food Microbiol. 2013;36(1):1
6. 58. Argyri AA, Nisiotou AA, Pramateftaki P, Doulgeraki AI, Panagou EZ, Tassou CC. Preservation of green table olives fermented with lactic acid bacteria with probiotic potential under modified atmosphere packaging. LWT - Food Sci Technol. 2015;62(1):783
790. 59. Blana VA, Polymeneas N, Tassou CC, Panagou EZ. Survival of potential probiotic lactic acid bacteria on fermented green table olives during packaging in polyethylene pouches at 4 and 20 degrees C. Food Microbiol. 2016;53(Pt B):71
75. 60. Rodriguez-Gomez F, Romero-Gil V, Garcia-Garcia P, GarridoFernandez A, Arroyo-Lopez FN. Fortification of table olive packing with the potential probiotic bacteria Lactobacillus pentosus TOMC-LAB2. Front Microbiol. 2014;5:467. 61. Rodrı´guez-Go´mez F, Romero-Gil V, Arroyo-Lo´pez FN, Bautista Gallego J, Garcı´a-Garcı´a P, Garrido-Ferna´ndez A. Effect of packaging and storage conditions on microbial survival, physicochemical characteristics and colour of non-thermally preserved green Spanish-style Manzanilla olives. LWT-Food Sci Technol. 2015;63 (1):367
375. 62. Rodriguez-Gomez F, Romero-Gil V, Garcia-Garcia P, ArroyoLopez FN, Garrido-Fernandez A. Effect of green Spanish-style Manzanilla packaging conditions on the prevalence of the putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2. Food Sci Nutr. 2016;4(2):181
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63. Lavermicocca P, Angiolillo L, Lonigro SL, et al. Lactobacillus plantarum 5BG survives during refrigerated storage bio-preserving packaged Spanish-style table olives (cv. Bella di Cerignola). Front Microbiol. 2018;9:889. 64. Asehraou A, Faid M, Akhartouf R. Pure culture fermentation of green olives by lactobacilli strains. Microbiol-Aliments-Nutr. 1993;11:221
228. 65. Ghabbour N, Rokni Y, Lamzira Z, et al. Controlled fermentation of Moroccan picholine green olives by oleuropein-degrading Lactobacilli strains. Grasas Aceites. 2016;67(2):e138.

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116. 68. Saxami G, Papadopoulou OS, Chorianopoulos N, Kourkoutas Y, Tassou CC, Galanis A. Molecular detection of two potential probiotic lactobacilli strains and evaluation of their performance as starter adjuncts in Yogurt production. Int J Mol Sci. 2016;17(5):E668.

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Chapter 19

Olive oil
contained phenolic compounds protect cells against H2O2-induced damage and modulate redox signaling by chelating intracellular labile iron Alexandra Barbouti1, Panagiotis Kanavaros1, Panagiotis Kitsoulis1, Vlasios Goulas2 and Dimitrios Galaris3 1

Department of Anatomy-Histology-Embryology, University of Ioannina Medical School, Ioannina, Greece, 2Department of Agricultural Sciences,

Biotechnology and Food Science, Cyprus University of Technology, Lemesos, Cyprus, 3Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina, Greece

Abbreviations DFO ERK GPxs HTyr JNK NOX Prx ROS Tyr

desferrioxamine extracellular signal
regulated kinase glutathione peroxidases hydroxytyrosol c-Jun NH2-terminal kinase nicotinamide adenine dinucleotide phosphate (NADPH) oxidase peroxiredoxin reactive oxygen species tyrosol

19.1 Introduction Epidemiological studies, performed during the second half of the previous century, showed that rates of coronary heart diseases were extremely low in Greece and Southern Europe compared to the rest of the investigated countries.1
3 These observations were attributed to differences in the dietary habits of these populations.1,2 Subsequent studies further supported the notion that Cretan diet, which subsequently was used as a basis to form the worldwide known “Mediterranean diet,” contributed to drastic reduction of additional diseases, such as cancer, stroke, and type 2 diabetes.4 The key component of this type of diet is olive oil,5 the healthful properties of which have been often related to its high levels of monounsaturated fatty acids, especially in the form of oleic acid.6 It has to be noted, however, that it is unlikely that oleic acid is exclusively responsible for the healthful effects of

olive oil, which apart from being the source of fatty acids contains also a remarkable amount of polar compounds, especially phenolics.6,7 It has been repeatedly proposed that uptake of phenolic compounds, present ubiquitously in Mediterranean diet, can provide resistance toward the so-called oxidative stress, which is regarded as a major contributor to the development of a plethora of chronic diseases.3,8,9 For a long time, it was generally believed that the abovementioned phenolic compounds exerted their beneficial effects by acting as strong antioxidants of free radical scavenger type.10 However, despite the initial optimistic expectations, the diet-derived antioxidants tested in clinical trials hitherto did not prove to be effective in preventing the development of oxidative stress
associated diseases or even slowing their progression. On the contrary, it was observed that they were harmful under some circumstances.11,12 In this presentation, we will focus our attention on elucidating molecular mechanisms by which phenolic compounds, present ubiquitously in Mediterranean diet and in olive oil, can exert their cytoprotective and cell signal modulating effects. In particular, an attempt will be made to reconcile the lack of benefit from classical free radical scavengers, in one hand, with the known causal role of oxidative stress in the initiation and progression of serious diseases, in the other. It is proposed that, under conditions of oxidative stress, concrete phenolic components of olive oil, such as hydroxytyrosol (HTyr), can provide health-promoting effects through chelation of intracellular “labile” (redox-active) iron. This small pool of intracellular iron is exclusively responsible for

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00049-3 © 2021 Elsevier Inc. All rights reserved.

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the interaction with hydroperoxides and the formation of extremely reactive free radicals, able to induce uncontrolled oxidations of basic cell components, such as DNA, proteins, and lipids. Furthermore, recent data will be discussed, indicating that iron chelation by HTyr can modulate specific intracellular redox signaling pathways and by this way can determine the outcome of cell fate in conditions of elevated oxidative stress.

19.2 The concept of oxidative stress The term “oxidative stress” was originally defined by Prof. Sies in order to denote the imbalance between prooxidants and antioxidants in favor of the former.13 It is well recognized today that the favorable aspects of aerobic life are also intimately connected with toxic oxidations, which are linked to numerous pathological conditions.14 Molecular oxygen, apart from being indispensable for maintaining life, represents also the main source for generation of reactive species in all types of aerobic cells. Apart from the majority of the consumed O2, which is reduced directly to H2O by the action of cytochrome oxidase in inner mitochondrial membrane, a small portion of it undergoes stepwise reduction by single electrons, leading to generation of intermediate reduction products (Scheme 19.1). Ο2

Ο2.–

SOD

Η2Ο2 1e

–

.

Cat, GPx, Prx

2e–

2H2O

Fe2+

OH

SCHEME 19.1 Schematic presentation of monovalent oxygen reduction and the formation of reactive intermediates.
 Reduction of molecular O2 with one electron to superoxide anion OU2 can be induced by a variety of 2 enzymatic and nonenzymatic mechanisms, as discussed in the text. The SOD family of enzymes, which are
present  ubiquitously in all aerobic cells, catalyzes the rapid conversion of OU2 to hydrogen peroxide (H2O2). The 2 latter can be reduced further, either enzymatically by Cat, GPxs, and Prxs to 2 H2O (2e2 reduction)
U or nonenzymatically by 1e to extremely reactive hydroxyl radicals OH . In the latter case the availability of labile iron in the form of Fe21 represents the critical point. Cat, Catalases; GPxs, glutathione peroxidases; Prxs, peroxiredoxins; SOD, superoxide dismutase.

These intermediates of O2 reduction, along with additional reactive species, produced by further interactions, are collectively called reactive oxygen species (ROS). It has been shown that ROS, such as OU2 2 and H2O2, are continuously generated and removed even under physiological conditions in all kinds of aerobic cells, thus creating a dynamic intracellular equilibrium. The steady state of this equilibrium varies among different types of cells or even in the same cell type under different conditions.14,15 Increased rates of monovalent reduction of O2 can be provoked by several means, such as induction of phagocytosis, mitochondria electron transfer, activation of NOX

enzymes, and redox cycling of exogenous compounds. Increased generation or decreased capacity to remove ROS leads to elevated steady-state levels of these species inside the cells.14,15 On the other hand, cells are capable of sensing even slight variations of this equilibrium and to trigger the appropriate responses. Thus depending on the intensity and the duration of the applied oxidative stress, basic cellular functions, such as cell proliferation and differentiation, are modulated. In addition, higher levels of stress can induce transient or permanent cell arrest or even cell death either by apoptosis or necrosis.15 Based on these observations, oxidative stress has been proposed to be implicated in the pathogenic molecular mechanisms of numerous diseases, including cardiovascular disease, ischemia/reperfusion syndrome, neurodegenerative diseases, cancer, and even the physiological process of normal aging.8,9,16
18 Intensive research work has been performed in order to elucidate the molecular mechanisms that are linked with pathological complications, with the ultimate aim to discover agents able to prevent or modulate the deleterious effects of oxidative stress under these conditions. It has been apparent by the time that an unlimited number of potentially protective agents are present in diet and especially in the “Mediterranean diet,” which has been proven to be able to prevent or impede the development of oxidative stress
associated diseases.2,19,20 The prevailing general idea is that antioxidants and free radical scavengers, present predominantly in this type of diet, are mainly responsible for the protection of cells and tissues against the deleterious effects of oxidative stress. It has to be acknowledged though that scientific support for this proposal is weak, and the exact molecular base for the action of the bioactive compounds in the Mediterranean diet remains elusive and needs further investigation.

19.3 Do free radical scavengers protect cells in conditions of oxidative stress? Since the introduction of the concept of oxidative stress in 1985, it has been shown that it is implicated in many human diseases, including cancer, neurodegenerative disorders as well as aging.8,9,16,18,21 Approximately the same period, epidemiological studies established the correlation of the traditional Mediterranean diet with beneficial health effects.1 Predominantly present in this type of diet are chemical compounds called “antioxidants,” which can scavenge reactive radicals in vitro. Thus it was assumed that they can also combat free radicals and oxidations in the body and prevent or delay oxidative stress
related diseases. The antioxidant (free radical scavenger) theory of health effects was dogmatically accepted, and antioxidants were considered from both scientists and the general

Olive oil
contained phenolic compounds protect cells against H2O2-induced damage Chapter | 19

population to be “miracle molecules” or “elixirs of life” that can protect from or cure numerous diseases. Unfortunately, the initial excitement diminished, since this assumption has never been proven. Several extensive animal and human prospective epidemiological studies that were designed in order to establish the antioxidant theory turned out to be inconclusive or even negative.22,23 Thus it may seem a paradox, although oxidative stress has been associated with many pathological conditions, largescale randomized clinical trials and metaanalysis have reported that supplementation of diet-derived antioxidants had little or no beneficial effects, while in some cases, it exerted adverse effects.11,12,24 It has to be stressed, however, that there is strong evidence indicating that diet-derived compounds can exert indirect “antioxidant” effects by (1) preventing the generation of reactive free radicals through the chelation of the necessary iron ions or (2) removing iron ions attached on positions of cellular macromolecules, thus making them insensitive against ensuing increase of oxidative stress.25
29

19.4 Intracellular “labile iron” as mediator of oxidative stress
induced effects It has to be stressed that OU2 2 and H2O2, the one- and twoelectron reduction products of oxygen, respectively, are not strong oxidizing agents and they can hardly interact with other cellular molecules. In spite of their low reactivity, OU2 2 is reduced to H2O2 by the action of superoxide dismutase, while H2O2 can rapidly metabolized to water by a network of antioxidant enzymes, such as catalase, glutathione peroxidase (GPx), and peroxiredoxin (Prx), which are expressed ubiquitously in all aerobic organisms. Nevertheless, in the presence of iron, the so-called Fenton-type reaction takes place,30 which involves the interaction between H2O2 and Fe21, producing the extremely reactive hydroxyl radicals ðHOU Þ, through intermediate formation of strongly reactive high oxidation states of iron (reaction 19.1). Fe

21

1 H2 O2 -½ferryl intermediates-Fe

31

1 HO 1 OH(19.1) U

The main factor that determines whether the relatively inert H2O2 will be reduced ezymatically to water or it will be converted to extremely reactive species is the presence of available redox-active iron, which represents the major catalysts for generation of reactive oxygen free radicals in aerobes.31,32 Noteworthy, since both ferryl intermediates and HOU are highly reactive, they cannot diffuse, but they react and oxidize target groups close to the vicinity of their formation (diffusion controlled interaction). Thus the potential locations of available iron ions

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determine also the specificity of H2O2-mediated oxidation.33 Fenton-type reactions can also be catalyzed by copper even more effectively than iron; however, iron represents the main catalyst in living cells, due mainly to its abundance biological systems.25,34 Considering its potential toxicity, it is not surprising that mammalian cells utilize sophisticated mechanisms to regulate iron homeostasis at both the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance.35,36 Moreover, most of the circulating and intracellular iron is securely stored in specific proteins, such as transferrin and ferritin, respectively, or within the active sites of enzymes. This sequestration keeps iron in a redox-inert state, restricted from reacting with oxygen reduction intermediates, and it is considered as one of the most important antioxidant defenses.31,37 It has to be noted that it is not the total amount of cellular iron that is responsible for ROS-induced toxicity, but rather a small fraction of it, often referred to as “labile iron.”38 Although poorly characterized, this pool of iron is redox-active, meaning that it contributes to the generation of extremely reactive free radicals from relatively unreactive peroxides. It is also chelatable, which means that it can be sequestrated by chelating compounds. Finally, it is exchangeable, that is it can be easily transferred among natural ligands and between cell compartments.39 Labile iron appears to be associated intracellularly with both low molecular weight compounds and macromolecules.40,41 Although it has been hitherto underestimated, strong accumulating experimental evidence suggests that labile iron dictates the ultimate outcome of oxidative stress
induced effects on cells, in conditions of oxidative stress.25,27,42,43

19.5 Olive oil
contained compounds prevent H2O2-induced DNA damage by chelating intracellular labile iron The requirement of labile iron to augment the reactivity of H2O2 and other organic peroxides against cellular macromolecules via the generation of HOU and ROU provides a rationale for the employment of iron chelating compounds to treat oxidative stress
associated pathologies,44 although supportive evidence from pilot studies in humans is currently limited.45 The most extensively clinically applied iron chelator is desferrioxamine (DFO), a siderophore of the hydroxamate class. However, in spite of its proven safety and efficacy, DFO is poorly absorbed in the gastrointestinal tract due to its hydrophilicity and has a short plasma half-life (B15 min). Moreover, it has been demonstrated that cells take up DFO only via fluidphase endocytosis, reaching intracellular compartments,

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such as endosomes and lysosomes.46 Nevertheless, treatment of cultured cells with DFO leads invariably to labile iron depletion in all cellular compartments.47 By using different types of human cell lines in culture, we recently attempted to evaluate the potential protective effects offered by chemical compounds to cells exposed to oxidative stress. It was observed that cellular DNA was extra sensitive under such conditions. Moreover, traditional antioxidants, such as ascorbate, α-tocopherol, trolox, N-acetyl-cystein, and α-lipoic acid, did not offer any protection, indicating the inability of free radical scavengers to prevent oxidative stress
induced cell damage.26 On the other hand, all iron chelators tested offered considerable protection, on the condition that they could penetrate plasma membrane and reach cell interior.25 It became evident from these results that the optimal protection under conditions of oxidative stress was offered by inhibition of the generation of reactive free radicals, rather than scavenging then following their production. In subsequent experiments with the same in vitro system, we used a variety of extracts derived from natural plant sources or isolated compounds from these extracts in order to evaluate their ability to protect DNA damage, when cells were exposed to oxidative stress. It was observed that polar extracts from olive oil offered considerable protection, indicating that they contained agents able to reach the intracellular space and to interrupt the process that leads to DNA damage.28 Noteworthy, extracts from olive oil mill wastewater also offered significant protection against H2O2-induced DNA damage, indicating that a significant amount of protective compounds present in olive fruit was of hydrophilic nature and ended up in the water phase rather than in oil phase.28 In an attempt to identify individual olive oil compounds able to protect DNA, we tested separately several of the main hydrophilic components of olive oil and olive mill wastewater. Prominent among them were HTyr, tyrosol (Tyr), and several flavonoids.28,29 It was observed that HTyr protected cells against H2O2-induced DNA damage, while Tyr, which lacks one hydroxyl group at the orthoposition compared to HTyr, was completely ineffective.28 This observation could not be explained on the basis of the antioxidant capacities of the tested compounds, but instead, was compatible with the hypothesis that phenolic constituents of olive oil exerted their protective effects through binding intracellular “labile iron” and in this way, preventing the generation of reactive hydroxyl radicals. In additional structure
activity studies, by using a large number of flavonoids, we were able to demonstrate a strong relationship between the cell protection and the iron-binding capacity of olive oil components and other plant-derived natural compounds.26
29,43 Additional indications, that intracellular chelation of loosely bound iron was responsible for the offered

protection of DNA, came from experiments in which the compounds used were gradually saturated with iron, before their addition in the culture medium. It was shown that the protective ability of flavonoids was decreased in parallel with their saturation with iron, indicating that their action was dependent on their capacity to bind iron inside the cells.29 Furthermore, by using an in situ method for estimation of the intracellular labile iron levels, called “calcein method,” we were able to estimate the iron-binding capacity of individual compounds in living cells and compare it directly to their protective capacity. It was observed that the potential protection offered by the individual compounds against H2O2-induced DNA damage was strongly correlated to their ability to diminish intracellular labile iron.27,29 On the other hand, no apparent correlation was observed between the antioxidant capacity of the compounds used (as estimated by common commercial methods) and their ability to protect cellular DNA.29 It was concluded from the abovementioned experiments that the chemical characteristics that contributed toward increasing the protective capacity of compounds were the presence of an ortho-dihydroxy group or a hydroxyl next to an alcoxy group in their chemical formula. Since this structural requirement is necessary for iron binding, it is clear that the protection offered by individual compounds contained in olive oil was relevant to their capacity to modulate the intracellular labile iron pool.

19.6 The role of iron in redox signaling For a long time, oxidative stress was regarded as a harmful condition for living cells and organisms. However, it was recently established that specific ROS and especially H2O2 were indispensable components of normal signal transduction mechanisms. This new example of signaling was conventionally named “redox signaling.”48 However, the question of whether labile iron was also implicated in this process had not been considered, until recently, when our group presented strong supporting evidence for this notion.42 The main obstacle in rationalizing the role of iron in oxidative stress
mediated signal transductions was the difficulty to reconcile for the underlying basic chemistry of this particular process. It was previously established that H2O2 exerts its multiple signaling actions by inducing the oxidation of sensitive cysteine residues on specific proteins to the corresponding sulfenic acid. This reaction represents a two-electron oxidation of the thiol group,49 while the hydroxyl radicals generated after the interaction of H2O2 with Fe21 oxidize cysteine thiol moieties by one single-electron to sulfur-centered free radical. To reconcile this contradiction, we speculated that ferryl or perferryl species, which are formed as intermediates during Fenton reaction (see reaction 19.1),

Olive oil
contained phenolic compounds protect cells against H2O2-induced damage Chapter | 19

should be considered as the actual oxidant in the case of H2O2-induced cysteine oxidation.42 The proposed model involves two-electron reaction steps, converting directly the sulfhydryl group of cysteine to sulfenic acid, as indicated in Scheme 19.2. P-S

–….

Fe2+ + H2O2

P-S

–….

[Fe=O]4+ + H2O

P-OH + Fe2+

SCHEME 19.2 Presentation of the molecular proposed mechanism for H2O2-induced and iron-catalyzed two-electron oxidation of thiolate anion in sensitive cysteine residues of proteins.

This particular model of interaction can take place only when the catalytic ferrous iron ion is attached to thiolate sulfur atom before the interaction with the peroxide. It was observed that depletion of intracellular labile iron before the exposure of the cells to H2O2 inhibited the observed H2O2-induced phosphorylation of MAP kinases in a highly specific mode. Thus labile iron influenced the late and sustained phases of c-Jun NH2-terminal kinase (JNK) and p38 phosphorylation but did not affect the early and transient phase of their phosphorylation, as well as the rapidly induced extracellular signal
regulated kinase (ERK) phosphorylation.42 These results predispose for persistent oxidative stress and elevated levels of intracellular labile iron in order to allow iron to be implicated in redox signaling mechanisms. This matches well with the corresponding rate constants of interaction of peroxides with Fe21 compared to those of specific proteins, such as Prxs and GPxs.50 As mentioned previously, since it is compatible to modulate labile iron levels by external intervention, it may be plausible to consider pharmacological iron depletion as means to modulate specific iron-dependent signaling processes, which are related to concrete pathological conditions.

19.7 Olive oil
contained compounds modulate redox signaling through chelation of labile iron The observation that iron chelation could modify cellularspecific signal transduction pathways, prompted us to examine whether components of olive oil with known iron chelating activity, should also be able to influence redox signaling. It was observed that HTyr, which we knew that, is able to bind and modulate intracellular labile iron, inhibited also the phosphorylation of JNK and p38 MAP kinases and prevented apoptosis, when cells were exposed to oxidative stress conditions.43 On the other hand, Tyr, which lacked the ortho-dihydroxy structure and thus was unable to chelate iron, was completely ineffective. It has to be stressed here that these particular components of extra virgin olive oil are phenolic alcohols which, in contrast to phenolic acids, are not charged and thus can diffuse easier through cell membranes.

235

19.8 Concluding remarks Oxidative stress has been proposed to be involved in the molecular mechanisms of almost any pathological condition in animals and humans.14
17,31,32 Consequently, intensive research work has been performed in order to discover agents able to prevent or cure the deleterious effects provoked under conditions of oxidative stress. Numerous such potentially protective agents are believed to be present in the so-called Mediterranean diet, which is indeed associated with reduced incidence of a number of pathophysiological complications, especially coronary heart diseases, diabetes, certain tumors, and the aging process.3,20 For a long time the prevailing idea was that antioxidants and free radical scavengers present predominantly in the Mediterranean diet are mainly responsible for the observed beneficial effects. It has to be stressed, however, that the experimental support for this postulation is still weak, and the exact molecular base for the action of bioactive compounds contained in the Mediterranean diet remains elusive. Based on experiments performed in our as well as other laboratories, we propose that the main mode of the cytoprotective action of natural bioactive agents, such as polyphenols, present abundantly in the Mediterranean diet in general and in olive oil in particular, is based mainly to their capacity: (1) to penetrate through biological membranes and (2) to chelate intracellular labile iron, thus preventing the formation of extremely reactive free radicals. Although the exact intracellular locations of labile iron ions attachment are not well defined at present, specific points on precious macromolecules, such as DNA, proteins, lipids, and carbohydrates, represent highly probable ligands. Consequently, iron-mediated oxidation of these particular ligands is likely to play an important role in oxidative stress
induced cell damage as well as in molecular signaling pathways that regulate basic cellular functions.31,42,43 Taking together the observations discussed earlier, it is tempting to speculate that specific olive oil
contained polyphenolic compounds can protect cells and modulate important signaling pathways in case of increased oxidative stress, just by binding and relocating intracellular labile iron ions. It is plausible to imagine that such an action should have profound effects on basic cellular responses that ultimately determine the final cell fate.

19.9 Summary points G

Oxidative stress is implicated in the molecular mechanisms of numerous diseases, including cardiovascular disease, ischemia
reperfusion syndrome, neurodegenerative diseases, cancer, and the physiological process of normal aging.

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G

G

G

PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

The Mediterranean diet contains an unlimited number of compounds that act in a beneficial way regarding the development of several such chronic diseases. Polar extract preparations from olive oil, which represents an important component of the Mediterranean diet, contain compounds able to protect cellular DNA in conditions of oxidative stress by modulating the levels of intracellular labile iron. The capacity of natural phenolic compounds contained in olive oil to diffuse through plasma membrane and chelate intracellular iron is mainly responsible for their cytoprotective and cell signal modulating properties.

References 1. Aravanis C, Corcondilas A, Dontas AS, Lekos D, Keys A. Coronary heart disease in seven countries. IX. The Greek islands of Crete and Corfu. Circulation. 1970;41:I88
I100. 2. Keys A, Menotti A, Karvonen MJ, Aravanis C, Blackburn H, Buzina R, Djordjevic BS, Dontas AS, Fidanza F, Keys MH. The diet and 15-year death rate in the seven countries study. Am J Epidemiol. 1986;124:903
915. 3. Keys A. Olive oil and coronary heart disease. Lancet. 1987;1:983
984. 4. Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med. 2003;348:2599
2608. 5. Visioli F, Bogani P, Grande S, Galli C. Mediterranean food and health, building human evidence. J Physiol Pharmacol. 2005;56:37
49. 6. Boskou G, Salta FN, Chrysostomou S, Mylona A, Chiou A, Andrikopoulos NK. Antioxidant capacity and phenolic profile of table olives from the Greek market. Food Chem. 2006;94:558
564. 7. Visioli F, Bernardini E. Extra virgin olive oil’s polyphenols: biological activities. Curr Pharm Des. 2011;17:786
804. 8. Di Domenico F, Barone E, Perluigi M, Butterfield DA. The triangle of death in Alzheimer’s disease brain: the aberrant cross-talk among energy metabolism, mammalian target of rapamycin signaling, and protein homeostasis revealed by redox proteomics. Antioxid Redox Signal. 2017;26:364
387. 9. Helfinger V, Schro¨der K. Redox control in cancer development and progression. Mol Aspects Med. 2018;63:88
98. 10. Visioli F, Bellomo G, Galli C. Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun. 1998;247:60
64. 11. Gutteridge JM, Halliwell B. Antioxidants: molecules, medicines, and myths. Biochem Biophys Res Commun. 2010;393:561
564. 12. Goodman M, Bostick RM, Kucuk O, Jones DP. Clinical trials of antioxidants as cancer prevention agents: past, present, and future. Free Radic Biol Med. 2011;51:1068
1084. 13. Sies H. Oxidative stress: introductory remarks. In: Sies H, ed. Oxidative Stress. London: Academic Press; 1985:1
8. 14. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 5th ed New York: Oxford University Press; 2015. 15. Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21:363
383. 16. Galaris D, Skiada V, Barbouti A. Redox signaling and cancer: the role of “labile” iron. Cancer Lett. 2008;266:21
29.

17. Griendling KK, Harrison DG. Dual role of reactive oxygen species in vascular growth. Circ. Res.. 1999;85:562
563. 18. Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat Med. 2004;10:S18
S25. 19. de Lorgeril M, Salen P. Modified Cretan Mediterranean diet in the prevention of coronary heart disease and cancer. An update. In: Simopoulos AP, Visioli F, eds. More on Mediterranean Diets. World Rev Nutr Diet. Basel: Karger; 2007:1
32. 20. Trichopoulou A, Lagiou P. Healthy traditional Mediterranean diet, an expression of culture, history, and lifestyle. Nutr Rev. 1997;55:383
389. 21. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239
247. 22. Warnholtz A, Mu¨nzel T. Why do antioxidants fail to provide clinical benefit? Curr Control Trials Cardiovasc Med. 2000;1:38
40. 23. Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, Peto R. Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N Engl J Med. 1996;334:1145
1149. 24. Halliwell B. The antioxidant paradox: less paradoxical now? Br J Clin Pharmacol. 2013;75:637
644. 25. Barbouti A, Doulias PT, Zhu BZ, Frei B, Galaris D. Intracellular iron, but not copper, plays a critical role in hydrogen peroxideinduced DNA damage. Free Radic Biol Med. 2001;31:490
498. 26. Barbouti A, Briasoulis E, Galaris D. Protective effects of olive oil components against hydrogen peroxide-induced DNA damage: the potential role of iron chelation. In: Preedy V, Watson R, eds. Olives and Olive Oil in Health and Disease Prevention. Oxford: Academic Press; 2010:1103
1109. 27. Kitsati N, Fokas D, Ouzouni MD, Mantzaris MD, Barbouti A, Galaris D. Lipophilic caffeic acid derivatives protect cells against H2O2-induced DNA damage by chelating intracellular labile iron. J Agric Food Chem. 2012;60:7873
7879. 28. Nousis L, Doulias PT, Aligiannis N, Bazios D, Agalias A, Galaris D, Mitakou S. DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide. Free Radic Res. 2005;39:787
795. 29. Melidou M, Riganakos K, Galaris D. Protection against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide, the role of iron chelation. Free Radic Biol Med. 2005;39:1591
1600. 30. Merkofer M, Kissner R, Hider RC, Brunk UT, Koppenol WH. Fenton chemistry and iron chelation under physiologically relevant conditions: electrochemistry and kinetics. Chem Res Toxicol. 2006;19:1263
1269. 31. Galaris D, Barbouti A, Pantopoulos K. Iron homeostasis and oxidative stress: an intimate relationship. Biochim Biophys Acta, Mol Cell Res. 2019;1866:118535. 32. Galaris D, Pantopoulos K. Oxidative stress and iron homeostasis, mechanistic and health aspects. Crit Rev Clin Lab Sci. 2008;45:1
23. 33. Chevion M. A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals. Free Radic Biol Med. 1988;5:27
37. 34. Mello-Filho AC, Meneghini R. Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals. Mutat Res. 1991;251:109
113. 35. Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys Acta. 2012;1823:1434
1443. 36. Pantopoulos K, Porwal SK, Tartakoff A, Devireddy L. Mechanisms of mammalian iron homeostasis. Biochemistry. 2012;51:5705
5724.

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contained phenolic compounds protect cells against H2O2-induced damage Chapter | 19

37. Gutteridge JMC, Halliwell B. Mini-review: oxidative stress, redox stress or redox success? Biochem Biophys Res Commun. 2018;502:183
186. 38. Cabantchik ZI. Labile iron in cells and body fluids: physiology, pathology, and pharmacology. Front Pharmacol. 2014;5:45. 39. Kakhlon O, Cabantchik ZI. The labile iron pool: characterization, measurement, and participation in cellular processes. Free Radic Biol Med. 2002;33:1037
1046. 40. Evans RW, Rafique R, Zarea A, Rapisarda C, Cammack R, Evans PJ, Porter JB, Hider RC. Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera. J Biol Inorg Chem. 2008;13:57
74. 41. Hider RC, Kong XL. Glutathione: a key component of the cytoplasmic labile iron pool. Biometals. 2011;24:1179
1187. 42. Mantzaris MD, Bellou S, Skiada V, Kitsati N, Fotsis T, Galaris D. Intracellular labile iron determines H2O2-induced apoptotic signaling via sustained activation of ASK1/JNK-p38 axis. Free Radic Biol Med. 2016;97:454
465. 43. Kitsati N, Mantzaris MD, Galaris D. Hydroxytyrosol inhibits hydrogen peroxide-induced apoptotic signaling via labile iron chelation. Redox Biol. 2016;10:233
242.

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44. Doll S, Conrad M. Iron and ferroptosis: a still ill-defined liaison. IUBMB Life. 2017;69:423
434. 45. Eid R, Arab NT, Greenwood MT. Iron mediated toxicity and programmed cell death: a review and a re-examination of existing paradigms. Biochim Biophys Acta, Mol Cell Res. 2017;1864:399
430. 46. Doulias PT, Christoforidis S, Brunk UT, Galaris D. Endosomal and lysosomal effects of desferrioxamine: protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest. Free Radic Biol Med. 2003;35:719
728. 47. Tenopoulou M, Doulias PT, Barbouti A, Brunk U, Galaris D. Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis. Biochem J. 2005;387:703
710. 48. Marinho HS, Real C, Cyrne L, Soares H, Antunes F. Hydrogen peroxide sensing, signaling and regulation of transcription factors. Redox Biol. 2014;2:535
562. 49. Roos G, Messens J. Protein sulfenic acid formation: from cellular damage to redox regulation. Free Radic Biol Med. 2011;51:314
326. 50. Winterbourn CC. The biological chemistry of hydrogen peroxide. Methods Enzymol. 2013;528:3
25.

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Chapter 20

Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors Morales-Martı´nez Adriana1, Montes Sergio2, Sa´nchez-Mendoza Alicia3, Quetzalli D. Angeles-Lo´pez1,4, Jime´nez-Go´mez Joel1, Martinez-Gopar Pablo Eliasib5 and Pe´rez-Severiano Francisca1 1

Laboratory of Molecular Neuropharmacology and Nanotechnology, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez,

Mexico City, Mexico, 2Department of Neurochemistry, National Institute of Neurology and Neurosugery Manuel Velasco Sua´rez, Mexico City, Mexico, 3Department of Pharmacology, National Institute of Cardiology Ignacio Cha´vez, Mexico City, Mexico, 4Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced studies of National Polithechnic Institute, Mexico City, Mexico, 5Department of Pharmacology, Center of Research and Advanced Studies of National Polytechnic Institute, Mexico

Abbreviations 3-NP 8-OHdG AA AD ALA ALS AvRM BDNF BBB CAT ChREBP/ MLX CNR1 COX-2 CRP DHA EPA GABA GGT GPX GSH GM HD HT LA LAB LC ω-3 LP LRRK2 LXRα MedDiets MPO MPTP

3-nitropropionic acid 8-hydroxy-deoxyguanosine arachidonic acid Alzheimer’s disease α-linolenic acid amyotrophic lateral sclerosis aversive radial maze brain-derived neurotrophic factor blood
brain barrier catalase carbohydrate regulatory element binding protein/ Max-like factor X cannabinoid type 1 receptor gene cyclooxygenase-2 C-reactive protein docosahexaenoic acid eicosapentaenoic acid gamma aminobutyric acid γ-glutamyl transpeptidase glutathione peroxidase reduced glutathione gut microbiota Huntington’s disease hydroxytyrosol linoleic acid lactic acid bacteria long-chain ω-3 lipid peroxidation leucine-rich repeat kinase 2 liver X receptors type α mediterranean diets myeloperoxidase 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MUFAs ND NMDA NO PD PINK1 PPARs PUFAs QUIN ROS SFA SOD

monounsaturated fatty acids not determined N-methyl D-aspartate nitric oxide Parkinson’s disease PTEN induced putative kinase 1 peroxisome proliferator
activated receptors polyunsaturated fatty acids quinolinic acid reactive oxygen species saturated fatty acids superoxide dismutase

20.1 Introduction Oils are fatty products extracted from animal or vegetable sources, and they have been widely used from engineering to the nutraceutical industry.1
5 Animal and vegetable oils are essential in the human diet, where their composition is crucial. The major components of edible oils are glycerol esters of fatty acids (96% in weight), free fatty acids, phospholipids, phytosterols, tocopherols, waxes, and other antioxidant substances as minor components. Dietary oils can help improving health depending on their fatty acid composition. According to the structure of their hydrocarbon chain, fatty acids can be saturated (SFAs), monounsaturated (MUFAs), or polyunsaturated (PUFAs), depending on the number of double bonds in the acyl chain.6
8 SFAs are characterized by the absence of double bonds. Short- and medium-chain SFAs9 are abundant in vegetable oils.10 MUFAs contain one double bond,11 while most abundant PUFAs are 18-, 20-, and 22-carbon fatty

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00024-9 © 2021 Elsevier Inc. All rights reserved.

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acids with two or more double bonds.12 Both MUFAs and PUFAs are found in high levels in animal oils.10 MUFAs resist oxidative breakdown for much longer periods of time than PUFAs,13 because accumulated double bonds are more susceptible to oxidation. Vegetable oils such as palm, palm kernel, and coconut oils contain higher amounts of unsaturated fatty acids than animal fats.14 Mammal cells can only desaturate fatty acids in a Δ9 position due to the lack of Δ12- and Δ15-desaturases. Therefore linoleic (LA; 18:2n-6) and α-linolenic (ALA; 18:3n-3) acids are essential for humans and must be supplied in the diet. Endogenously, those acids produce longchain PUFAs such as eicosapentaenoic acid (EPA; 20:5n3), docosahexaenoic acid (DHA; 22:6n-3), and arachidonic acid (AA; 20:4n-6)10,15 by the action of elongases and desaturases. On the other hand, the SFAs most commonly found in oils are myristic (C14:0), palmitic (C16:0), and palmitoleic (C18:0) acids, which confer structural rigidity to the plasmatic membrane.16 MUFAs such as oleic acid (C18:1n-9) are major components of oils, this acid being highly abundant in neuron membranes.9 PUFAs such as EPA, DHA, and AA are included in biological membranes, where the double bonds in their carbon chain alter membrane biophysical properties such as viscoelasticity and fluidity,17 a property that is been studied as a therapy for various diseases. Furthermore, fatty acids and their derivates have a key role incorporating phospholipids to the membrane, thus modifying its fluidity, signaling pathways, transport mechanisms, lipid raft ensemble, and the activity of membrane-bound enzymes.18 Several studies have stressed the importance of the consumption of fatty acids, either solid fats, oils, or nutraceutical supplements. The therapeutic usefulness of various types of oil has been demonstrated, improving the clinical picture and/or reducing the levels of noxious molecules in cardiovascular,9 neurodegenerative,19 inflammatory,20,21 respiratory, and even infectious disease. Dietary oils, especially fish and olive oils, have been proved to prevent or delay the onset of symptoms and signs of neurodegenerative diseases, due to its composition.22
24 It is known that olive oil is rich in ω-6 fatty acids, while fish oils are good sources of LC ω-3 fatty acids25; those fatty acids have demonstrated benefits to brain health in clinical trials as well as in vivo and in vitro studies.8 The specific effect of olive and fish oils in neurodegenerative models is discussed in this chapter, focusing on an in vitro synaptosomes model of Huntington’s disease (HD).

20.2 Fish oil Fish oils are most commonly extracted from capelin, sand eel, anchovy, horse mackerel, cod liver, pilchard, and

menhaden. Over 1 million tons of fish oils are produced and consumed yearly in the world.26 Oil composition depends on the fish source; PUFAs, predominantly DHA and EPA, account for 35%, in average.25 Fish oils are characterized for a high content of LC ω-3 fatty acids. Due to their unsaturation degree, they help to keep fluidity in biological membranes at low temperatures. These acids are produced by marine algae, which are consumed first by zooplankton and then by fish.27 There is strong evidence on the benefit of the consumption of LC ω-3 fatty acids to prevent coronary heart disease. European and American Heart Societies have incorporated EPA and DHA consumption into treatment guidelines for cardiac diseases.28 All fish oil types contain ω-3 fatty acids, but bluefishes (horse mackerel, salmon, and others) include more fatty acids in quantity and variety than white fish (conger, hake, and others).29 Fish oils also contain antioxidants such as selenium and vitamin E.30 Fish oils reduce cholesterol, triglyceride, and verylow-density lipoprotein (VLDL) levels, inhibit platelet aggregation, and can reduce blood pressure due to the membrane-stabilizing effect of ω-3 fatty acids.28,31,32 A link between dementia and lipid composition of the diet was reported in 2002, highlighting a lower dementia incidence in patients who ate bluefish at least once a week.33 A moderate consumption of fish oil has showed other health benefits due to its high content of PUFAs, especially ω-3 fatty acids.34

20.3 Olive oil The olive tree, Olea europaea, produces the olive fruit, a basic component of the Mediterranean diet. Components of the plant such as oleuropein, squalene, and hydroxytyrosol have been reported to modulate gene function.35 According to International Olive Council, 2019/20 season, nearly 3.67 million tons of olive oil is estimated to be produced. About 19 styles of olive oil are extracted in the main production regions around the world.36 NonMediterranean countries account for about 2.5% of the world production.37 Some olive oil components have been reported to possess anxiolytic, antidepressant, antioxidant, and antiinflammatory properties.38 Olive oil is the main source of oleic acid. Besides providing fluidity to biological membranes as mentioned before, oleic acid is known to directly regulate anorexigenic tone via the melanocortinergic system.19 Other olive oil components have shown therapeutic effects through antioxidant, antitumor, and antimicrobial activity, as well as a capacity to modulate gene function.35,39
42 Total fatty acid composition in typical olive and fish oils has been measured by our group, and we found oleic

Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors Chapter | 20

acid 81.69% and 35.06%, EPA 0.06% and 0.73%, and DHA 0.04% and 25.06% respectivelly.43

20.4 Implications for human health and disease prevention Extensive research has been conducted and efforts have been made to use oils as therapeutic agents in several conditions, with promising results. Their possible mechanisms of action have also been studied and are well established now. Various clinical trials on olive and fish oils, as well as the respective populations under study, are shown in Table 20.1. Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), HD, and amyotrophic lateral sclerosis (ALS) are known to involve oxidative damage as a pathogenic mechanism.44 The origin of these conditions is multifactorial, and in some cases, unknown, but they share neuronal damage as a

241

common trait in their pathogeny.45 The pathophysiology of these disorders includes cognitive, sensorial, and motor disabilities.46 Understanding the causal mechanisms underlying these diseases is necessary to develop more efficient therapies. Since no cure is known to date for neurodegenerative diseases, and the current treatments also fail to limit their progression, most therapeutic approaches have focused on alleviating patient’s symptoms. Various drug types are used in the clinic to treat psychiatric symptoms such as sleep disorders, as well as motor, genitourinary, and gastrointestinal signs.47 Novel therapeutic strategies have been proposed targeting the mechanisms of damage, when they are known. These treatments include immunotherapy, replacement or increased production of neurotransmitters, antiapoptotic agents, antioxidants, and membrane receptor antagonists.47,48 The protective effects of olive and fish oils in neurodegenerative models are described in Table 20.2. Olive oil has been the most

TABLE 20.1 Clinical trials and populations under study for therapies based on olive and fish oils. Disease or altered condition

Concentration or doses

Proposed mechanism

Improved parameter

Resource

Active component

References

Oxidative stress

50 mL/day/30 days

Improvement of antioxidant status in plasma and blood cells

m CAT, GPX (enzyme activity) m SOD, k CAT (gene expression)

Extra-virgin olive oil

HT

[86]

Metabolic syndrome

398 ppm

Reduction of postprandial inflammatory response

k NFκB activation

Virgin olive oil

HT

[87]

High cardiovascular risk subjects

Ad libitum

Reduced 24-h ambulatory BP, total cholesterol, and fasting glucose

MedDiets supplemented extra-virgin olive oil

ND

[88]

High cardiovascular risk subjects

21.4
56.9 g/ day

Reduced risks of cardiovascular disease and mortality

MedDiets supplemented extra-virgin olive oil

ND

[89]

Type 2 diabetes

30 g/day/8 weeks

Reduced CRP levels

k CRP

Olive oil

ND

[90]

Overweight subjects

40 g/day/3 months

High-quality extra-virgin olive oil decreased inflammation and oxidative stress both in normal and overweight subjects

k MPO k 8-OHdG m IL-10 m Adiponectin m LAB in GM

High-quality extra-virgin olive oil

ND

[91]

HIV 1 individuals

1.6 g/day/12 weeks

Reduced inflammation and gut permeability

k CD14

Fish oil

EPA, DHA

[92]

8-OHdG, 8-Hydroxy-deoxyguanosine; BP, blood pressure; CAT, catalase; CRP, C-reactive protein; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid, GPX, glutathione peroxidase; GM, gut microbiota; HT, hydroxytyrosol; LAB, lactic acid bacteria; MPO, myeloperoxidase; ND, not determined; SOD, superoxide dismutase.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

widely assayed, with interesting results on the experimental models and some mechanisms of action proposed.

20.5 Experimental models to study neurodegenerative diseases A number of experimental models have been developed to study and test new therapies to treat neurodegenerative

diseases. However, selecting a suitable model can be challenging. Depending on the objectives of the study, models ranging from cellular systems to isolated tissues or organs, to intact animals have been developed.46 The neurochemical traits of various diseases can be reproduced in animal models by administering toxins, either directly in the brain regions involved or systemically. For instance, the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA)

TABLE 20.2 Protective effects of olive and fish oil in models of neurodegeneration. Model

Dose

Proposed mechanism

Improved parameter

Resource

Active component

References

Huntington’s disease
like rat model

10% of caloric intake/14 days

The treatment exerted a brain antioxidant effect against the oxidative stress caused by 3-NP

k LP m GSH levels m Succinate dehydrogenase activity

Olive oil

ND

[93]

Ischemic brain injury Brain slices of Wistar rats

0.25 or 0.5 mL/kg/ 30 days

Virgin olive oil protected the brain from the effects of hypoxia-reoxygenation

k LP k Brain prostaglandin E2 k NO m GSH levels

Virgin olive oil

ND

[94]

Ischemic brain injury Brain slices of Wistar rats

0.25, 0.5, or 0.75 mL/ kg/30 days

Virgin olive oil protected the brain in a rat model of ischemia/reperfusion

k Infarct volume k Brain edema k BBB permeability m Neurologic deficit scores

Virgin olive oil

ND

[95]

Amyotrophic lateral sclerosis SOD1G93A mice

Chow diet enriched with 20% (w/w)/8 weeks

Extra-virgin olive oil ameliorated pathological outcomes and delayed the disease onset in SOD1G93A mice

m Survival rate m Motor coordination k ER stress k Autophagy k Muscle damage

Extravirgin olive oil

ND

[96]

Transient global cerebral ischemia Male Wistarrats

300 mg/kg/ day/8 days

Fish oil produced memory and cognitive recovery in the absence of neuronal rescue and/or hippocampal neurogenesis

AvRM improvement

Fish oil

ND

[97]

Huntington’s disease like rat model

15% w/w/ 20 days

Treatments exerts a neuroprotective effect preserved behavior function and preventing oxidative damage in striatal tissue.

Normal GABA levels kLP mPPARγ

Fish oil and olive oil

ND

[43]

Transient global cerebral ischemia

300 mg/kg/ day/7 days

Fish oil
mediated synaptic plasticity at the morphological (dendrite) and biochemical (protein) levels may be one mechanism that underlies the memory-protective effect of fish oil

m NeuN m BDNF m GAP-43

Fish oil

DHA

[98]

3-NP, 3-Nitropropionic acid; AvRM, aversive radial maze; BBB, blood
brain barrier; BDNF, brain-derived neurotrophic factor; CNR1, cannabinoid type 1 receptor gene; COX-2, cyclooxygenase-2, CAT, catalase, SOD, superoxide dismutase; DHA, docosahexaenoic acid; GSH, reduced glutathione; LP, lipid peroxidation; ND, Not determined; NO, nitric oxide; PPARγ, peroxisome proliferator-activated receptor gamma; GABA, gamma aminobutyric acid.

Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors Chapter | 20

in the substantia nigra of rats is widely used as a model for PD.49 The administration of excitotoxic substances such as kainic acid, ibotenic acid, quinolinic acid (QUIN), or 3nitropropionic acid (3-NP) induces neurodegeneration and mimics HD.50
53 Transgenic animal models have also been used to study neurodegenerative diseases, since these models develop some of the neuropathological hallmarks of these diseases. PD models include the leucine-rich repeat kinase 2 (LRRK2), parkin, protein deglycase DJ-1 also known as Parkinson disease protein 7, and putative kinase 1 (PINK1) genes,54 which mimic some features of the disease, such as a loss of dopaminergic neurons. On the other hand, the most widely used AD models are associated to the formation of amyloid plaques, such as PDAPP, Tg2576, APP23, and 3xTgAD.55 Finally, most transgenic animal strains used to study HD, such as R6/1, R6/2, N171-82Q, and BAC-HD, are based in a CAG abnormal extension to express and produce mutant huntingtin (htt).56 In vitro studies have been performed on cell lines, brain slices, brain homogenates, and brain-derived synaptosomes. The latter structures have raised interest, since they are capable of carrying out the synthesis, storage, and release of substances that participate in synaptic transmission.57

243

useful tool in neuronal research and have contributed to improve our understanding of neurodegenerative diseases involving the synaptic function.59
61 The main components of synaptosomes are plasmatic membranes; therefore the lipid composition of such membranes is a major target to study. It has been reported that they are composed of ceramides, sphingomyelins, and phospholipids, likely due to the presence of mitochondria.62 Previously, our team reported the fatty acid composition of whole brain
isolated synaptosomes (Table 20.3)43 from Wistar rats fed with an olive or fish oil
enriched diet. Synaptosomes from oil-fed animals showed higher concentrations of oleic acid and EPA than those isolated from control, standard diet-fed rats. On the other hand, striatum-derived synaptosomes showed a different composition with respect to whole brain
derived synaptosomes, with higher amounts of DHA, EPA, and oleic acid, while the levels of palmitic and palmitoleic acid were decreased in both fish and olive groups.63 The morphology of isolated synaptosomes was determined by electronic microscopy. As shown in Fig. 20.1A, synaptosomes contain small vesicles about 40
60 nm in diameter.64 We have also confirmed that isolated synaptosomes possess an active form of γ-glutamyl transpeptidase (GGT), a marker for the presence of a plasmatic membrane since this enzyme is associated with the membrane’s outer surface (Fig. 20.1B).

20.5.1 Synaptosomes as an in vitro model Synaptosomes are neuron synaptic terminals detached from the axon and dendrites. While not fully functioning neurons, they are still able to synthesize, store, and release neurotransmitters. Synaptosomes comprise a plasmatic membrane, N-methyl D-aspartate receptor, mitochondria, and vesicles.58 They have become an extremely

20.6 Huntington’s disease and oils as therapeutic agents HD is a hereditary neurodegenerative disorder caused by an abnormal expansion of repeated CAG in the first exon of the HD gene, which codes for a polyglutamine chain in

TABLE 20.3 Fatty acid synaptosome composition. Fatty acid

Control

Olive oil

Fish oil

C16:0

28.35 6 1.04

27.4 6 1.91

25.22 6 0.87

C16:1

0.35 6 0.03

0.48 6 0.04

0.29 6 0.05*

C18:0

23.89 6 0.69

24.15 6 0.47

24.84 6 0.67

C18:1n-9

18.55 6 0.56

21.31 6 0.34**

21.9 6 0.28***

C18:2n-6

0.78 6 0.04

0.9 6 0.04

0.99 6 0.15

C18:3n-3

0.97 6 0.1

1.37 6 0.27

1.32 6 0.16

C18:3n-6

0.26 6 0.11

0.16 6 0.03

0.04 6 0.01

C20:4n-6

11.64 6 0.57

11 6 0.64

10.09 6 0.34

C20:5n-3

0.01 6 0.007

0.02 6 0.008****

0.12 6 0.03***

C22:6n-3

15.13 6 1.0

13.3 6 1.21

15.14 6 0.88

Data are reported as % of total fatty acid composition in synaptosome fractions. *P , .05, fish oil diet versus olive oil diet; **P , .005; ***P , .001, significant differences to control group; and ****P , .005, significant differences in olive oil versus fish oil diet. Fatty acids that were modified by diets are highlighted in bold. Mean values 6 SEM of five rats per group are shown. One-way ANOVA followed by Tukey post hoc test.

Source: Reprinted from Morales-Martı´nez A, Sa´nchez-Mendoza A, Martı´nez-Lazcano JC, et al. Essential fatty acid-rich diets protect against striatal oxidative damage induced by quinolinic acid in rats. Nutr Neurosci. 2017;20:388
395, with permission.

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FIGURE 20.1 Isolated synaptosomes. (A) Representative electron micrographs of whole brain
derived synaptosomes at 20,000 3 , (B) GGT activity of brain striatal
derived synaptosomes from control, fish oil, and olive oil in animals. Data from six independent experiments (n 5 6). Enzyme activity was analyzed by one-way ANOVA followed by Tukey post hoc test, P 5 0.1121. ANOVA, analysis of variance; GGT, γ-Glutamyl transpeptidase.

the htt protein.65 HD patients suffer from behavioral and motor alterations; psychiatric symptoms and dementia are often observed in the adulthood, although juvenile cases have also been reported. Histologically, HD patients exhibit a 20%
30% decrease in brain mass with respect to healthy subjects.66 In the caudate nucleus
putamen region, also known as the dorsal striatum, loss rates range from 57% to 65%, while about 85% of striatal neurons suffer some degree of damage, which is more severe in medium spiny striatal neurons with neurochemical markers such as γ-aminobutyric acid (GABA), substance P, and enkephalin.50,67 At a microscopic level, the presence of intracellular deposits or inclusion bodies formed by polyglutamine aggregates of mutant htt is observed both in HD and transgenic models.68,69 To date, whether these aggregates are causative of the pathology or are a form of protection from the mutated protein is still under discussion.70
72 The administration of PUFAs has led to clinical, motor, and cognitive improvement both in HD patients and in transgenic models. MUFAs in olive oil, specifically oleic acid, stimulate peroxisome proliferator
activated receptors (PPARs). The β/δ and γ PPAR isoforms are found in different brain and spinal cord regions, and it has been suggested that they regulate the expression of genes involved in neurotransmission and neurodegeneration.73 To date, no treatment has been proved effective to cure or even mitigate the alterations affecting HD patients. Therefore the search for novel therapies in experimental models is an active research field. There is evidence that the use of fatty acids could have beneficial effects in HD. Vaddadi et al. demonstrated that the administration of PUFAs reduced involuntary movements induced by antipsychotic drugs in a psychiatric population.74,75 Linoleic acid (LA) and γ-LA showed antidyskinetic effects both in an experimental model and in patients suffering from motor disorders.76,77 The levels of n-6 and n-3 fatty acids were found to be decreased in

erythrocyte membranes of patients with late dystonia.78 In addition, significant clinical improvements in both motor and cognitive performances have been observed in an open study on HD patients after being orally supplemented with fatty acids, specifically n-6 LA and EPA.76 Furthermore, Clifford et al. conducted a study on a transgenic mouse strain for a HD mutation which showed agedependent oxidative damage. A significant protection against cellular oxidative stress and a clear improvement in behavior was observed in those HD mutated animals fed with a diet rich in LA, γ-LA, EPA, and dihomoγ-linolenic acid.79 On the other hand, there is evidence that dietary fats rich in n-6 (safflower), linolenic acid (mustard oil), and AA altered the profile of fatty acids in a stable neuronal plasma membrane.80 Gangliosides, sulfatides, and various types of glycosphingolipids are present in the plasma membrane and are particularly abundant in the nervous system. These lipids are highly concentrated in the outer surface of the plasma membrane, in specialized domains called lipid rafts, where they help regulate the spatial organization of membrane proteins.81 Overall, these findings provide valuable information on the impact of dietary lipids on cellular processes, emphasizing that the lipid composition of membranes is crucial for a good cellular function, especially in lipid bilayer parameters such as membrane fluidity. Membrane fluidity is a measurement of molecular mobility inside the lipid bilayer, which allows for lateral diffusion of embedded proteins; hence, biological membranes require both a high lateral fluidity and structural rigidity.82 In the neuronal membrane, where fluidity is a key property for an optimal receptor function,83 fatty acids account for about 50% of dry weight.84 Our research group has conducted studies on membrane fluidity of synaptosomes derived from either the whole brain (Fig. 20.2) or the striatum63 of Wistar rats fed with different diets. The animals were divided into three groups: (1) control, standard diet; (2) olive

Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors Chapter | 20

245

FIGURE 20.2 Membrane fluidity. Fluidity into the membrane by fatty acid enrichment of whole brain
derived synaptosomes from control, fish oil, and olive oil experimental groups. (A) DPH anisotropy, DPH fluorophore integrated in acyl chains of phospholipids into the membrane and (B) TMA-DPH anisotropy, TMA-DPH integrated in polar heads of phospholipids. Fluorescent anisotropy is reported as mean ( 6 standard error). DPH ***P , .001, significant differences to the control group. TMA-DPH *P , .05, significant differences to olive oil and control group. One-way ANOVA followed by Tukey post hoc test. ANOVA, analysis of variance; DPH, Diphenylhexatriene; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6phenyl-1,3,5-hexatriene.

FIGURE 20.3 Mechanisms of lipid action. (A) Physico-chemical: QUIN (NMDAr agonist) promotes an influx of calcium, increasing ROS formation, and consequently LP. Including fatty acids such as DHA, EPA, or OA into the membrane could prevent the damage caused by QUIN, decreasing ROS production and LP by changing the content of EPA, oleic, palmitic, and palmitoleic acids. As a result, membrane suffers mechanic changes, making them more fluid. (B) Signaling pathway: fatty acids such as DHA, EPA, or Oleic acid and their derivatives act as endogenous agonists of transcription factors such as PPARs, regulating target genes such as antioxidant (SOD, catalase), similarly to other exogenous agonists (glitazones, fibrates, L165041, GW501516). On the other hand, PUFAs produce metabolites such as lipoxins, eicosanoids, and resolvins, promoting an antiinflammatory response. DHA, Docosahexaenoic acid; EPA, eicosapentaenoic acid; LP, lipid peroxidation; QUIN, quinolinic acid; NMDA, N-methyl D-aspartate; PPARs, peroxisome proliferator
activated receptors; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; SOD, superoxide dismutase. Reprinted from Morales-Martı´nez A, Zamorano-Carrillo A, Montes S, et al. Rich fatty acids diet of fish and olive oils modifies membrane properties in striatal rat synaptosomes. Nutr Neurosci. 2019. doi:10.1080/1028415X.2019.1584692, with permission from Taylor & Francis.

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oil
enriched diet; and (3) fish oil
enriched diet. The composition of the lipids incorporated into synaptosomes is shown in Table 20.3. The levels of EPA and oleic acid were higher both in groups fed with olive and fish oils, while the levels of palmitoleic acid were decreased in the fish oil group. Then, membrane fluidity was measured in whole brain- and striatum-derived synaptosomes. Anisotropy in whole brain
derived synaptosomes is shown in Fig. 20.2. Anisotropy has an inversely proportional relationship with membrane fluidity, that is, decreased anisotropy values indicate a higher fluidity. As shown in Fig. 20.2A, those animals fed with oil-enriched diets showed higher membrane fluidity values than the control group into acyl chains of phospholipids.

20.7 Protective mechanism by polyunsaturated fatty acids in Huntington’s disease model The effects of fatty acids on membrane fluidity in neurodegenerative diseases can be studied in vivo or in vitro. Previous works have demonstrated the antioxidant effect of a diet rich in fatty acids in a QUIN-induced model of HD. While various mechanisms of action have been suggested by other studies, we focus on the changes in membrane fluidity resulting from the inclusion of specific fatty acids in the diet, and on synaptosomes as a model to measure this parameter and their influence on neurotransmission (Fig. 20.3). PUFAs stabilize membranes and modulate the levels of SFAs such as palmitic acid and MUFAs such as palmitoleic acid, which provide order and structural rigidity to the plasma membrane. Therefore decreased levels of palmitic acid and/or a higher DHA:palmitic acid ratio would result in a lower sensitivity to surface tension85 in the membrane. Although DHA levels did not change, the levels of EPA, a DHA precursor, were increased after fish oil supplementation, suggesting that this proportion could favor membrane fluidity and promote a smoother function. In addition, fatty acids could activate nuclear receptors and some transcription factors, including PPARs, liver X receptors type α (LXRα), the hepatic nuclear factor 4α, the sterol regulatory element binding protein-1 and -2, and the carbohydrate regulatory element binding protein/ Max-like factor X (ChREBP/MLX). PUFAs and their derivates also participate in the inflammatory response and antioxidant regulation.

20.8 Conclusion In conclusion, in this chapter we showed the relevance of the fatty acid composition in edible oils, since this composition will determine the effects of specific oils at a

membranal, cytosolic, and nuclear (signaling) level. Furthermore, we discussed the relevance of synaptosomes as a suitable model to study the effect of therapeutic interventions on neuron membranes in neurodegenerative diseases.

Mini-dictionary of terms Property of materials to exhibit variations in physical properties along different molecular axes. Edible oils Substances from vegetal or animal, the principal components of which are lipids; these are used as a nutritional supplement. Fatty acids Carboxylic acids with a hydrocarbon chain long or short. Membrane Anisotropic motions that contribute to the fluidity mobility of components into the biological membrane. MUFAs Fatty acids with one instauration into the hydrocarbon chain. Neurotransmission Transmission of a nerve impulse along to the nerve fiber. Phospholipids Amphiphilic lipids that are the main component of cell membranes and liposome structure. PUFAs Fatty acids with two or more instaurations into the hydrocarbon chain. SFAs Fatty acids with single bonds into the hydrocarbon chain. Synaptosomes Nerve terminals consisting mainly of membranes and capable of synthesizing, storing, and releasing neurotransmitters. Anisotropy

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88. 80. Srinivasarao P, Vajreswari A, Rupalatha PS, Narayanareddy K. Lipid composition and fatty acid profiles of myelin and synaptosomal membranes of rat brain in response to the consumption of different fats. J Nutr Biochem. 1997;8:527
534. 81. Bonetto G, Di Scala C. Importance of lipids for nervous system integrity: cooperation between gangliosides and sulfatides in myelin stability. J Neurosci. 2019;39:6218
6220. 82. Espinosa G, Lo´pez-Montero I, Monroya F, Langevin D. Shear rheology of lipid monolayers and insights on membrane fluidity. Proc Natl Acad Sci USA. 2011;108:6008
6013. 83. Egawa J, Pearn ML, Lemkuil BP, Patel PM, Head BP. Membrane lipid rafts and neurobiology: age-related changes in membrane lipids and loss of neuronal function. J Physiol. 2016;594:4565.

Synaptosomes as a model to study fish oil and olive oil effect as neuroprotectors Chapter | 20

84. Calder P. E-Mail The DHA content of a cell membrane can have a significant influence on cellular behaviour and responsiveness to signals. Ann Nutr Metab. 2016;8
21. 85. Carta G, Murru E, Banni S, Manca C. Palmitic acid: physiological role, metabolism and nutritional implications. Front Physiol. 2017;8:902. 86. Oliveras-Lo´pez MJ, Berna´ G, Jurado-Ruiz E, Lo´pez-Garcı´a de la Serrana H, Martı´n F. Consumption of extra-virgin olive oil rich in phenolic compounds has beneficial antioxidant effects in healthy human adults. J Funct Foods. 2014;10:475
484. 87. Camargo A, Rangel-Zun˜iga OA, Haro C, et al. Olive oil phenolic compounds decrease the postprandial inflammatory response by reducing postprandial plasma lipopolysaccharide levels. Food Chem. 2014;162:161
171. 88. Dome´nech M, Roman P, Lapetra J, et al. Mediterranean diet reduces 24-hour ambulatory blood pressure, blood glucose, and lipids: one-year randomized, clinical trial. Hypertension. 2014;64:69
76. 89. Guasch-Ferre´ M, Hu FB, Martı´nez-Gonza´lez MA, et al. Olive oil intake and risk of cardiovascular disease and mortality in the PREDIMED Study. BMC Med. 2014;12:78. 90. Atefi M, Pishdad GR, Faghih S. The effects of canola and olive oils on insulin resistance, inflammation and oxidative stress in women with type 2 diabetes: a randomized and controlled trial. J Diabetes Metab Disord. 2018;17:85
91. 91. Luisi MLE, Lucarini L, Biffi B, et al. Effect of Mediterranean diet enriched in high quality extra virgin olive oil on oxidative stress,

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inflammation and gut microbiota in obese and normal weight adult subjects. Front Pharmacol. 2019;10:1366. Zhang YG, Xia Y, Lu R, Sun J. Inflammation and intestinal leakiness in older HIV 1 individuals with fish oil treatment. Genes Dis. 2018;5:220
225. Tasset I, Pontes AJ, Hinojosa AJ, Torre R De, Tu´nez I. Olive oil reduces oxidative damage in a 3-nitropropionic acid-induced Huntington’s disease-like rat model. Nutr Neurosci. 2011;14:106
111. Gonza´lez-Correa JA, Mun˜oz-Marı´n J, Arrebola MM, et al. Dietary virgin olive oil reduces oxidative stress and cellular damage in rat brain slices subjected to hypoxia-reoxygenation. Lipids. 2007;42:921
929. Mohagheghi F, Bigdeli MR, Rasoulian B, Zeinanloo AA, Khoshbaten A. Dietary virgin olive oil reduces blood brain barrier permeability, brain edema, and brain injury in rats subjected to ischemia-reperfusion. Sci World J. 2010;10:1180
1191. Oliva´n S, Martı´nez-Beamonte R, Calvo AC, et al. Extra virgin olive oil intake delays the development of amyotrophic lateral sclerosis associated with reduced reticulum stress and autophagy in muscle of SOD1G93A mice. J Nutr Biochem. 2014;25:885
892. de Oliveira JN, Reis LO, Ferreira EDF, et al. Postischemic fish oil treatment confers task-dependent memory recovery. Physiol Behav. 2017;177:196
207. de Oliveira DV, Bernardi TC, de Melo SR, Godinho J, de Oliveira RMW, Milani H. Postischemic fish oil treatment restores dendritic integrity and synaptic proteins levels after transient, global cerebral ischemia in rats. J Chem Neuroanat. 2019;101:101683.

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Chapter 21

Olive oil and postprandial energy metabolism: implications for weight control Mario J Soares, MBBS, MSc, PhD and Kaveri Pathak, APD, PhD Nutrition and Dietetics, School of Public Health, Curtin University, Bentley Campus, Perth, WA, Australia

Abbreviations CCK DIT EVOO FOR MSD MUFA PUFA PYY RQ SFA SNS

cholecystokinin diet-induced thermogenesis extra-virgin olive oil fat oxidation rate Mediterranean-style diet monounsaturated fatty acids polyunsaturated fatty acids peptide YY respiratory quotient saturated fatty acids sympathetic nervous system

21.1 Introduction A traditional Mediterranean-type diet, with olive oil (OO) as the major source of monounsaturated fatty acids (MUFA), has been shown to be protective against cardiovascular disease and certain cancers.1,2 Diets high in MUFA are also beneficial to type 2 diabetics.3 Despite these health benefits, Mediterranean-type diets are not promoted for the management of obesity, due to concerns that their high-fat content may promote weight gain. A low-fat diet is the preferred option for better long-term reduction of metabolic risk. However, there is evidence that highMUFA diets are not predictive of weight gain.4
6 Extravirgin OO (EVOO) is high in MUFA but also consists of several minor components with potential biological properties.7 This chapter provides an overview of the postprandial metabolic handling of OO and its role in the regulation of body weight.

21.2 Body weight regulation and nutrient partitioning Weight and body energy content remain quite stable in most adults over a long period, despite daily fluctuations in energy intake and energy expenditure. This requires the presence of regulatory processes able to match fuel supply to energy requirements. In the main, energy balance is determined by the matching of macronutrient intake to energy expenditure and the ability to channel nutrients into oxidative versus storage pathways (nutrient partitioning). Most individuals reach a state of approximate weight maintenance in which the average composition of the fuels they oxidize matches the nutrient distribution in their diets.8 Protein and carbohydrate intakes elicit powerful autoregulatory adjustments in protein and carbohydrate oxidation, leading to near zero balance. In contrast, fat balance is less accurately regulated and more easily disrupted.8 Hence, the matching of fat oxidation to fat intake is a key determinant of fat balance and eventually, energy balance.

21.3 Can the type of fatty acid affect the rate of fat oxidation? Whole body measurements of the oxidation of dietary C18 fatty acids showed that oleic acid (C18:1) was oxidized 14 times more readily than stearic acid (C18:0).9 The fractional oxidation of chylomicron-derived oleic and palmitic acids also showed a significantly greater oxidation of oleic than palmitic acid.10 Delaney and colleagues11 placed volunteers on a weight-maintenance diet

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00004-3 © 2021 Elsevier Inc. All rights reserved.

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containing 40% of energy as fat and then fed them a variety of fatty acids labeled with 13C either in the methyl or carboxyl position. The author found that the oxidation of oleate was greater than palmitic or stearic acid. However, differences in fatty acid oxidation in humans also stemmed from differences in chain length, degree of unsaturation, the position, and stereo-isomeric configuration of their double bonds.11

21.4 Postprandial fat oxidation in humans There is considerable evidence from human studies, to indicate that the type of fat can also acutely influence its metabolic fate.9,11
16 Since the source of fat determines the overall fatty acid mix, we had tested meals based on whole foods to simulate a real-life situation.13,14 Following mixed meal ingestion in healthy humans, the action of insulin is to promote carbohydrate oxidation at the expense of fat oxidation. The whole body indirect calorimetry data showed that while there was a significant postprandial suppression of fat oxidation rate (FOR) following cream, this did not occur following EVOO breakfasts. Interestingly, the period of maximum suppression coincided with the time for fat digestion and absorption; 3
5 h after a meal.13,14 This would suggest that postprandial mix of fatty acids has significant effects on the body’s ability to channel this substrate into either storage or oxidative pathways. The minimal postprandial suppression of FOR following the OO meal would suggest that fat was the predominant nutrient being utilized during that meal. Such data support results obtained using stable isotopically labeled fatty acids10,11 and from formula diets where postprandial respiratory quotient and/or FORs were significantly higher following higher oleic acid intake.17 We have reviewed the available data on acute meal effects and acute on chronic diet-induced human trials that had tested OO against other oils on postprandial energy metabolism (Table 21.1). While the total number of studies retrieved was small, there was consistent evidence that OO has a role in increasing both diet-induced thermogenesis (DIT) and FOR; two factors that promote a lesser fat balance. Such effects need confirmation within a long-term dietary plan, for the beneficial physiology to have practice implications for obesity.

21.5 Is there a preferential effect of olive oil in abdominal obesity? Postprandial thermogenesis or DIT comprises two processes: an obligatory and a regulatory component. Meal size, meal composition, and the physiological characteristics of the individual largely determine the obligatory component, while the activity of the sympathetic nervous

system (SNS) fine-tunes the regulatory component. A higher DIT for a given meal would imply that less energy is available for storage, and hence such a meal would be less conducive to weight gain. Abdominal adipocytes are particularly sensitive to SNS-mediated lipolysis; a process whereby stored fat is broken down and mobilized. A decreased activation of the SNS has long been implicated in weight gain of both animals and man. We have reanalyzed our acute data to examine whether abdominal obesity, as judged by standard criteria of waist circumference, modified the response to OO (Table 21.2). Initial analysis indicated a greater FOR with OO, and in subjects with greater waist circumferences (data not shown). In the final model that included an interaction term, the effect was evident only for those men and women with higher waist following the EVOO meal (Table 21.2B). In contrast, carbohydrate oxidation rate was significantly lower following EVOO with no waist group 3 meal interaction (Table 21.2C). Overall, the reciprocal changes in substrate oxidation rates canceled out any effect on DIT. Such data make a case for trailing OO diets in those with abdominal obesity. Confirmation of such predictions would enhance practice guidelines for the current global crisis of obesity.

21.6 Olive oil, satiety, and food intake Food intake represents the other arm of the energy balance equation. The control of food intake is mediated through short- and long-term pathways. Hunger and satiety cues modify meal size, meal termination, and the intermeal interval. There are several gastrointestinal (meal-related) factors that regulate food intake. These include the physical form of food, gastric emptying, meal composition, and their resultant effects on several gastrointestinal hormones. There is some evidence that both the degree of saturation and chain length influence postingestive satiety,20 though this opinion is not shared by all.21 The hydrolysis of ingested fat results in monoglycerides and free fatty acids22 and pharmacological blockade of intraduodenal hydrolysis stimulates food intake in humans.23 This role for FA in food intake is ascribed to the longer chain fatty acids (C10 and greater), through their influence on cholecystokinin (CCK).23 Mediumchain fatty acids are without effect.23,24 Peptide YY (PYY) is an intestinal hormone that inhibits food intake, while ghrelin is secreted by the stomach and stimulates food ingestion. Interestingly, blockade of CCK release prevented the reduction in food intake seen with intraduodenal infusion of oleic acid compared to saline and was brought about by reversal of the normal rise in PYY and fall in ghrelin that accompanies oleic acid.24 The interplay between postprandial energy metabolism and food intake also needs to be considered, as there are

Olive oil and postprandial energy metabolism: implications for weight control Chapter | 21

253

TABLE 21.1 Randomized crossover studies of olive oil (OO) on postprandial energy expenditure and fat oxidation in humans. Author

Subjects

Intervention

Results

Comments

Casas-Agustench et al.15

Healthy men

Walnuts, OO, dairy-based SFA

DIT was 28% higher in walnut group, 23% greater in olive oil group as compared to SFA

No significant differences in SOR and satiety. Quality of fat may determine substrate utilization and DIT assisting in obesity management.

Jones et al.18

Healthy men

OO, flaxseed oil, and sunflower oil

DIT greater in olive oil compared to other two oils

Presence of oleic acid in addition to fatty acid chain length may have additional benefit. Increased DIT maybe useful for weight management.

Soares et al.14

Postmenopausal women

EVOO, cream

Postprandial COR rate decreased and FOR increased after OO consumption

Postprandial effects of OO consumption increase FOR and thus stimulate DIT in obese postmenopausal females.

Piers et al.13

Healthy men

EVOO, cream

Increased FOR and reduced COR postOO consumption

Participants with increased waist showed greater thermic effect to olive oil. This could translate as a weight loss strategy.

Healthy men

HF diet (50%)

Fasting metabolism—no effect

MUFA lowers RER and increased DIT in acute feeding. However, after 5 days both diets have similar metabolic responses.

OO, cottonseed oil

Postprandial metabolism: RER m PUFA versus MUFA

Duration of feed: 5 days

DIT k PUFA versus MUFA

OO, functional oil (MCT oil 1 OO 1 canola 1 flaxseed 1 coconut oil)

EE and FOR greater on day 2 but not different after 28 days

Acute studies

Acute on chronic Polley et al.16

Chronic studies St-Onge and Jones19

Overweight healthy men

Functional oil improved adiposity but without significant long-term metabolic effects, compared to OO.

Duration of feed: 28 days COR, Carbohydrate oxidation rate; CSO, cottonseed oil; DIT, diet-induced thermogenesis; EE, energy expenditure; EVOO, extra-virgin olive oil; FOR, fat oxidation rate; HF, high fat; MCT, medium-chain triglycerides; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; RER, resting energy requirements; SFA, saturated fatty acid; SOR, substrate oxidation rate.

strong relationships between substrate metabolism, appetite, and food intake.25,26 Given this frame work, one may expect the high oleic content of OO to influence food intake either directly, via CCK-mediated regulation of PYY and ghrelin, or indirectly, through its preferential stimulation of fat oxidation. Acute studies have involved manipulating MUFA intake through sources other than OO, and recording

satiety/food intake over that day and sometimes, the next. Sources of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) in these acute studies have also differed; hindering between-study comparisons of their outcomes. A study in normal-weight subjects showed that PUFA suppressed energy intake and ratings of motivation to eat on the trial day, when compared to MUFA and SFA.20 Another study on obese subjects indicated that

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 21.2 The modulatory effect of waist circumference on postprandial metabolism following olive oil
based breakfasts: a regression analysis.a Variable

Unstandardized β

SE of β

Standardized coefficient of β

t Value

P Value

2.281

.027

(A) Total postprandial energy expenditure over 5 h Constant

44.6

19.55

Fasting REE (kJ/h)

4.4

0.285

0.807

15.562

.000

Age (year)

20.54

0.174

20.100

23.114

.003

FFM (kg)

0.82

0.356

0.115

2.290

.026

22.989

.004

(B) Total postprandial fat oxidation over 5 h Constant

211.225

3.756

Fasting FOR (g/h)

2.055

0.454

0.497

4.527

.000

Fat intake (g)

0.610

0.169

0.408

3.604

.001

Waist group 3 meal interaction

3.588

1.372

0.243

2.615

.012

8.719

.001

(C) Total postprandial carbohydrate oxidation over 5 h Constant

57.5

6.59

Fasting COR (g/h)

1.55

0.536

0.334

2.883

.006

Age (year)

20.337

0.098

20.396

23.426

.001

Meal (0,1)

26.82

2.82

20.276

22.418

.019

Meal: 0 5 cream, 1 5 EVOO; waist group: 0 , 102/88, 1 . 102/88 cm based on gender. COR, Carbohydrate oxidation rate; FOR, fat oxidation rate; REE, resting energy expenditure. a Reanalysis of data from Piers et al. (2002) and Soares et al. (2004) comparing breakfast meals high in SFA or EVOO in n 5 26 men and women. All analyses forced the fasting value into the equation and then tested age, gender, site of study, FM, FFM, meal (0,1), and waist group (0,1) in a stepwise approach. Wherever gender and waist group made significant contributions, an interaction term of the two was then included.

linoleic acid suppressed fat but not energy intake, compared to oleic.27 However, these effects were not evident when the food intake data was compiled over 2 days. It appears that the source of fat has no acute influence on satiety index and food intake monitored over a day21. This area needs more work to gain greater clarity.

21.7 Mediterranean-style diets The truer test of whether edible oils regulate habitual food intake, energy expenditure, and body weight is through designs that focus on free-living intake over prolonged periods. Most high-fat foods are palatable, with a high energy density. This promotes passive overconsumption and can lead to obesity. Even during caloric restriction, animals with higher fat intakes display greater fat stores at the end of the trial.28 Traditionally, total fat intake on OObased diets is above the B30% levels recommended for health. The energy density of the diet is of particular importance in a free-living ad libitum setting, since apart from taste, it strongly affects satiety and daily energy intake.29,30 Not all Mediterranean dietary patterns are exactly the same, though some similarity within regions

can be identified.31 Increased fruit, vegetables, cereals, and legumes with moderate alcohol intake are classic features of this diet. The ingestion of bulky vegetable
rich dishes with high amounts of OO would reduce energy density, due to the dilutional effects of their considerable water and fiber content.4 The latter should aid long-term weight control. We have compiled 18 randomized human clinical studies in Table 21.3. The studies were conducted from 1994 to 2018 and had studied weight/fat loss, changes in measures of abdominal obesity measures as well as recorded food intake of their participants. Study durations varied from 4 weeks to 3 years, so we categorized them into short duration (,6 months, n 5 9), and long duration.

21.7.1 Food intake Table 21.3 suggests that when fed at same,45 or a higher percentage of energy as the control diet4,32,34 short-term studies of 4
12 weeks showed no difference in energy intake. Of the three studies lasting 6 months, only Sartorelli et al.39 observed a lower energy intake. The much longer interventions examined changes between 1.5

TABLE 21.3 Human intervention studies focusing on olive oil, and their effects on food intake, fat oxidation, and body weight. Authors

Study design

Duration of diet

Type of subjects

Control diet fat (% E)

MSD diet fat (%E)

Differences in energy intake

Differences in weight or fat loss

Short-term trials (n 5 9) Paniagua et al.32

Randomized crossover of 3 diets without washout; weight maintenance

4 weeks

Obese, insulin resistant

20-SFA, 47-SFA

38

NS

NS, but lower trunk: leg fat ratio on MSD versus 20SFA

Khaw et al.33

Randomized control design, three parallel intervention arms approximately equal in size: extravirgin coconut oil/butter/EVOO

4 weeks

Men/women

50 g/day

50 g/day

NS

Trend toward weight, fat, and waist loss, although NS

RodriguezVillar et al.34

Randomized crossover without washout; meal intake prescribed

6 weeks

T2DM

29

40

NS

NS

Candido et al.35

Randomized, double-blinded, placebo-controlled clinical trial; calorie restricted diet prescribed to both groups (22090 kJ/day)

9 weeks

Adult women with high body fat (B32%)

B32

B32

NS

Greater fat loss in EVOO group

Walker et al.4

Randomized crossover, with washout; meal intake prescribed

12 weeks

T2DM men /women

23

35

NS

NS, but lower upper: lower body fat ratio on MSD

Rozati36

Randomized, single-blinded, and placebo-controlled trial

12 weeks

Overweight/ obese men and women

B35%

B35%

Higher in OO group (P 5 .31)

No change in weight and waist

Rodrigues et al.37

Randomized, single-blinded, controlled, 3 arms parallel group [OO(control), DieTBra, and DieTBra 1 OO], clinical trial

12 weeks

Obese men/ women (BMI $ 35 kg m2)

B28%
35%

52 mL/day

Higher in only OO diet, lower in DieTBra and DieTBra 1 OO (P , .001)

Weight reduction was greater for DieTBra and DieTBra 1 OO than for OO. Fat mass reduction greater for DieTBra 1 OO than for OO

Long-term trials (n 5 9) Ferrara et al.38

Randomized crossover, without washout; meal intake prescribed

6 months

Hypertensive men/women

27

27

NS

NS

Sartorelli et al.39

Randomized, 2 arms parallel, dietary counseling only to MSD

6 months

Overweight/ obese men/ women







Lower on MSD

Greater weight loss and decreased waist circumference on MSD

Due et al.6

8-week weight loss followed by randomized controlled trial, 3-arm

6 months

Overweight/ obese women

23-low fat, 32-SFA

39

NS

Similar weight regain (Continued )

TABLE 21.3 (Continued) Authors

Study design

Duration of diet

Type of subjects

Control diet fat (% E)

MSD diet fat (%E)

Differences in energy intake

Differences in weight or fat loss

parallel design; ad libitum intake, all food provided Lasa et al.40

Post hoc analysis of the larger PREDIMED study, a parallel group, multicentric, randomized, controlled clinical trial with 3 arms: low-fat diet (control)/olive oil/nuts

12 months

Communitydwelling men and women

25 mL/day

25 mL/day

Higher on MSD (P 5 .001)

Greater weight loss and reduced WC on MSD than low-fat diet

Azadbakht et al.41

Randomized, 2-arm parallel design, weight loss diet prescribed

14 months

Overweight/ obese men/ women

20

30

NS

Greater weight loss and decreased waist circumference on MSD

McManus et al.42

Randomized, 2-arm parallel design; weight loss diet prescribed

18 months

Overweight men/women

20

35

Higher on MSD (P 5 .08)

Greater weight and fat loss on MSD

Esposito et al.43

Randomized, 2-arm parallel design; behavioral modification only to MSD

24 months

Obese women

28

28

Lower on MSD

Greater weight loss and lower WHR on MSD

Razquin et al.44

The PREDIMED study: large, parallelgroup, multicenter, randomized controlled trial

36 months

Men/women

38.19 6 5.9

41.84 6 5.1

Higher in OO group (P 5 .025)

NS

de Lorgeril et al.1

Randomized, 2-arm parallel design; dietary counseling only to MSD

48 months

Post-MI men/ women

33

30

Lower on MSD

NS

BMI, Body mass index; EVOO, extra-virgin olive oil; MI, myocardial infarction; MSD, Mediterranean-style diet; NS, nonsignificant; OO, olive oil; SFA, saturated fatty acid; T2DM, type 2 diabetes mellitus; WC, waist circumference; WHR, waist-to-hip ratio.

Olive oil and postprandial energy metabolism: implications for weight control Chapter | 21

and 4 years. While McManus et al.42 and Razquin et al.44 showed a trend for higher intake on the Mediterraneanstyle diet (MSD), the others indicated a significantly lower energy intake. One possible confounder was that three studies1,39,43 had specifically targeted the intervention group with their dietary/lifestyle changes, while the control group just received general instruction. Hence, there could be some bias in reporting free-living energy intake. Overall, there appears no convincing evidence to suggest that MSD reduced energy intake. This is not necessarily a negative conclusion, since in 9 of 18 studies (Table 21.3), the percentage of fat intake was higher on the MSD trials and a high-fat diet is expected to promote passive overconsumption of energy. A possible explanation is that MSD will lower the energy density through its water and fiber content.

promote long-term compliance to dietary change on such diets.

21.9 Summary G

G

G

G

G

21.7.2 Weight/fat loss Only two of the short-duration trials35,45 reported a greater weight/fat loss on the EVOO diet. Despite no difference in weight loss, Walker et al.4 had earlier found a change in body fat distribution that favored loss of abdominal fat. Piers et al.45 later confirmed this effect. The majority of short-term trials however did not show any differences in weight/fat loss32
34,36,45 except for two recent ones.35,37 In contrast, five of the nine trials that lasted 6 months or more demonstrated a great weight or fat loss on the EVOO diets.39
43 In four of these trials, a loss of abdominal obesity—measured as waist circumference or waist to hip ratios—was observed. There was no change in either weight or fat mass in hypertensive men/ women with long-term intervention,38 while weight regain patterns were found to be similar with either low fat, MUFA, or SFA intake.6

257

G

Postprandial thermogenesis accounts for the energy expenditure after a meal, while substrate oxidation rates determine the metabolic fate of the macronutrients ingested. Fat balance is governed by usual fat intake and the body’s ability to channel fatty acids into oxidative or storage pathways. Addressing fat balance is central to determining energy balance, and hence body weight regulation. Oleic acid and OO per se promote postprandial fat oxidation, indicating that lower amounts of this type of fat are channeled into storage. Abdominally obese individuals may derive a greater benefit from OO. Increasing OO intake as part of an MSD plan resulted in a greater weight or fat loss in over 50% of studies reviewed. Importantly, higher intakes of the oil did not promote overconsumption, and hence weight gain.

Acknowledgment The authors express gratitude to Dr. Leonard Piers, Dr. Karen Walker and Prof. Kerin O’Dea for their collaboration over the years, and to Curtin University for infrastructure support.

Mini-dictionary of terms 1. Substrate oxidation rates (SORs) refer to the amount of ingested alcohol, carbohydrate, protein, or fat that is oxidized to release energy from a meal. 2. Respiratory Quotient (RQ) is derived from indirect calorimetry and reflects the type of fuel being oxidised.

21.8 Conclusion There are several reasons to support the judicious use of OO in the formulation of diets for weight control. These include a higher postprandial thermogenesis and a shift of macronutrient utilization from carbohydrate to fat. OO, through its oleic content, also has the potential to influence hunger/satiety and to reduce food intake. Collectively, these factors ought to result in a lower body weight and, indeed, this is observed in over 50% of the studies that lasted .6 months. Moreover, in some trials there was a change in the distribution of body fat that favored lesser abdominal adiposity. Another observation from the data reviewed was that increasing OO intake, as part of an MSD, did not promote weight gain even when ingested in amounts above standard practice guidelines. This aspect is an added bonus in the battle of the bulge, since it would

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821. Soares MJ, Cummings SJ, Kenrick M, Mamo JCL, Piers LS. The acute effects of olive oil v. cream on postprandial thermogenesis and substrate oxidation in postmenopausal women. Br J Nutr. 2004;91:245
252. Casas-Agustench PL-UP, Bullo M, Ros E, Go´mez-Flores A, SalasSalvado J. Acute effects of three high-fat meals with different fat saturations on energy expenditure, substrate oxidation and satiety. Clin Nutr. 2009;28:39
45. Polley KR, Miller MK, Johnson M, et al. Metabolic responses to high-fat diets rich in MUFA v. PUFA. Br J Nutr. 2018;120:13
22. Kien CL, Bunn JY, Ugrasbul F. Increasing dietary palmitic acid decreases fat oxidation and daily energy expenditure. Am J Clin Nutr. 2005;82:320
326. Jones PJH, Jew S, AbuMweis S. The effect of dietary oleic, linoleic, and linolenic acids on fat oxidationand energy expenditure in healthy men. Metab. Clin. Exp.. 2008;57:1198
1203. St-Onge MP, Jones PJ. Greater rise in fat oxidation with mediumchain triglyceride consumption relative to long-chain triglyceride is associated with lower initial body weight and greater loss of subcutaneous adipose tissue. Int J Obes Relat Metab Disord.. 2003;27 (12):1565
1571. Lawton CL, Delargy HJ, Brockman J, Smith FC, Blundell JE. The degree of saturation of fatty acids influences post-ingestive satiety. Br J Nutr. 2000;83(5):473
482. MacIntosh CG, Holt SHA, Brand-Miller JC. The degree of fat saturation does not alter glycemic, insulinemic or satiety responses to a starchy staple in healthy men. J Nutr. 2003;133:2577
2580. Bonora E, Targher G, Formentini G, et al. The metabolic syndrome is an independent predictor of cardiovascular disease in type 2

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31 32

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diabetic subjects. Prospective data from the Verona Diabetes Complications Study. Diabet Med. 2004;21(1):52
58. Matzinger D, Degen L, Drewe J, et al. The role of long chain fatty acids in regulating food intake and cholecystokinin release in humans. Gut. 2008;46:688
693. Degen L, Drewe J, Piccoli F, et al. Effect of CCK-1 receptor blockade on ghrelin and PYY secretion in men. Am J Physiol Regul Integr Comp Physiol. 2007;292:R1391
R1399. Stubbs RJ, Ritz P, Coward WA, Prentice AM. Covert manipulation of the ratio of dietary fat to carbohydrate and energy density: effect on food intake and energy balance in free-living men eating ad libitum. Am J Clin Nutr. 1995;62:330
337. Ronnett GV, Kleman AM, Eun-Kyoung Kim E-K, Landree LE, Tu Y. Fatty acid metabolism, the central nervous system, and feeding. Obesity. 2006;14:201S
207S. Kamphuis MMJW, Westerterp-Plantenga MS, Saris WHM. Fat specific satiety in humans for fat high in linoleic acid vs fat high in oleic acid. Eur J Clin Nutr. 2001;55:499
508. Boozer CN, Brasseur A, Atkinson RL. Dietary fat affects weight loss and adiposity during energy restriction in rats. Am J Clin Nutr. 1993;58(6):846
852. Rolls BJ. Role of energy density in the overconsumption of fat. J Nutr. 2000;130:268S
271S. Ledikwe JH, Blanck HM, Kettel L, et al. Dietary energy density is associated with energy intake and weight status in US adults. Am J Clin Nutr. 2006;83(6):1362
1368. Noah A, Truswell AN. There are many Mediterranean diets. Asia Pac J Clin Nutr. 2001;10(1):2
9. Paniagua JA, Gallego de la Sacristana A, Romero I, et al. Monounsaturated fat-rich diet prevents central body fat distribution and decreases postprandial adiponectin expression induced by a carbohydrate-rich diet in insulin-resistant subjects. Diabetes Care. 2007;30(7):1717
1723. Khaw K-T, Sharp SJ, Finikarides L, et al. Randomised trial of coconut oil, olive oil or butter on blood lipids and other cardiovascular risk factors in healthy men and women. BMJ Open. 2018;8: e020167. Rodriguez-Villar C, Manzanares JM, Casals E, et al. Highmonounsaturated fat, olive oil-rich diet has effects similar to a highcarbohydrate diet on fasting and postprandial state and metabolic profiles of patients with type 2 diabetes. Metabolism. 2000;49: 1511
1517. Caˆndido FG, Valente FX, da Silva LE, et al. Consumption of extra virgin olive oil improves body composition and blood pressure in women with excess body fat: a randomized, doubleblinded, placebo-controlled clinical trial. Eur J Nutr. 2018;57: 2445
2455. Rozati M. Cardiometabolic and Immunological Impacts of Extra Virgin Olive Oil Consumption in Overweight and Obese Older Adults: A Randomized Controlled Trial. Lowell: Biomedical Engineering and Biotechnology, University of Massachusetts; 2014. Rodrigues APS, Rosa LPS, Silveira EA. PPARG2 Pro12Ala polymorphism influences body composition changes in severely obese patients consuming extra virgin olive oil: a randomized clinical trial. Nutr Metab. 2018;15(52). Ferrara LA, Raimondi AS, D’Episcopol, et al. Olive oil and reduced need for antihypertensive medications. Arch Intern Med. 2000;160: 837
842.

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39 Sartorelli DS, Sciarra EC, Franco LJ, Cardoso MA. Beneficial effects of short-term nutritional counselling at the primary healthcare level among Brazilian adults. Public Health Nutr. 2005;8(7): 820
825. 40 Lasa A, Miranda J, Bullo´ M, et al. Comparative effect of two Mediterranean diets versus a low-fat diet on glycaemic control in individuals with type 2 diabetes. Eur J Clin Nutr. 2014;68: 767
772. 41 Azadbakht L, Mirmiran P, Esmaillzadeh A, Azizi F. Better dietary adherence and weight maintenance achieved by a long-term moderate-fat diet. Br J Nutr. 2007;97:399
404. 42 McManus KAL, Sacks F. A randomized controlled trial of a moderate-fat, low-energy diet compared with a low fat, low-energy

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diet for weight loss in overweight adults. Int J Obes Relat Metab Disord. 2001;25:1503
1511. 43 Esposito K, Pontillo A, Di Palo C, et al. Effect of weight loss and lifestyle changes on vascular inflammatory markers in obese women: a randomised trial. JAMA. 2003;89(14):1799
1804. 44 Razquin C, Martinez JA, Martinez-Gonzalez MA, et al. A 3 years follow-up of a Mediterranean diet rich in virgin olive oil is associated with high plasma antioxidant capacity and reduced body weight gain. Eur J Clin Nutr. 2009;63(12):1387
1393. 45 Piers LS, Walker KZ, Esler MD, et al. Substitution of saturated with monounsaturated fat in a 4-week diet affects body weight and composition of overweight and obese men. Br J Nutr. 2003;90: 717
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Chapter 22

Effect of olive oil on metabolic syndrome Asavari Joshi and Anand Zanwar Centre for Innovation in Nutrition Health Disease, Interactive Research School for Health Affairs, Bharati Vidyapeeth (Deemed to be University), Pune, India

Abbreviations AA ACE ADMA ANP AP-1 Apo BP COX CRP CVD ET-1 EVOO FFA GLP-1 GLUT HDL-c HMGCR HT IL-6 LTB4 LDL-c MMP-9 MS MUFA NEP NF-κβ NO OA OO oxi-LDL RAAS ROS SREBP T2DM TG TLR-4 TNF-α VCAM-1 VLDL

arachidonic acid angiotensin-converting enzyme asymmetric dimethylarginine atrial natriuretic peptides activator protein-1 apolipoprotein blood pressure cyclooxygenase C-reactive protein cardiovascular disorder endothelin-1 extra-virgin olive oil free fatty acids glucagon-like peptide 1 glucose transporter high-density lipoprotein cholesterol 3-hydroxy-3-methyl-glutaryl CoA reductase hydroxytyrosol interleukin-6 leukotriene B4 low-density lipoprotein cholesterol matrixmetalloprotease-9 metabolic syndrome monounsaturated fatty acid neutral endopeptidase nuclear factor κβ nitric oxide oleic acid olive oil oxidized LDL renin
angiotensin
aldosterone system reactive oxygen species sterol regulatory element-binding proteins type 2 diabetes mellitus triglyceride Toll-like receptor 4 tumor necrosis factor-alpha vascular cell adhesion molecule-1 very low
density lipoprotein

22.1 Introduction Metabolic syndrome (MS) is a global health problem and has affected over a billion people in the world1 and the number is on rise. MS is a group of interconnected risk factors, which accelerate the risk for diseases such as type 2 diabetes mellitus (T2DM) and stroke along with other health issues. These risk factors mainly include insulin resistance, central obesity, dyslipidemia, and hypertension.2 Chronic inflammation plays critical role in the development and progression of MS.3 In addition, MS patients have higher oxidative stress and associated damage along with disturbed antioxidant defense system.4 Diet and sedentary lifestyle are the two modifiable causes basically responsible for the development and progression of MS. Individuals with MS have a fivefold higher risk for developing T2DM and a twofold higher risk for developing cardiovascular diseases (CVD). Altered carbohydrate metabolism is also considered as one of the risk factors for MS development.5 Here, Fig. 22.1 represents the risk factors associated with MS and associated health conditions. Mediterranean diet pattern is considered as one of the most health-promoting diet patterns. Mediterranean diet rich in virgin olive oil (OO) has been shown to positively influence health especially in case of MS.6 Many clinical studies have been conducted to evaluate the effect of OO (high or low phenolic content) in lipid disorders. One of the largest clinical studies with total 7216 participants of high-risk cardiovascular events [i.e., PREvencio´n con DIetaMEDiterra´nea (PREDIMED)—multicentric randomized, controlled, clinical trial] was carried out to study the association between intake of OO and risk of CVD and mortality. There was a significant correlation between increased OO consumption and reduced cardiovascular events; however, nonsignificant correlation was observed with respect to cancer and its associated mortality.7

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00038-9 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 22.1 Risk factors associated with metabolic syndrome and intimidated health conditions. Metabolic syndrome is a complex disease arising from combination of many risk factors. The principle risk factors are insulin resistance, dyslipidemia, central obesity, and hypertension. In majority of cases, metabolic syndrome leads to further health complications such as type 2 diabetes mellitus and/or cardiovascular diseases.

In Nurses’ Health Study, total 59,930 women were followed for 22 years to study the correlation of OO intake and (T2DM). Modestly lower risk of T2DM was noted when fat was replaced with OO.8 Network meta-analysis study (constituting of 30 clinical trials) concluded beneficial effects of OO on metabolic parameters such as glucose, triglycerides, and low-density lipoprotein (LDL) cholesterol (LDL-c) and high-density lipoprotein (HDL) cholesterol (HDL-c) through adherence to the Mediterranean diet.9 Various clinical studies have proved the observed effects of consumption of OO on CVD are via inflammatory markers and endothelial function.10 In randomized, crossover, and controlled trial, supplementation of OO improved various kinds of atherogenic indexes those are major risk factors for CVD.11 Further significant association of Mediterranean diet and its effect on gut microbiota has been reported/reviewed. Saturated fat replacement with monounsaturated and polyunsaturated fats along with polyphenols makes important changes in gut microbiota.12
14 Nutrigenomic studies have recorded modulation in expression of transcripts and miRNAs of lipid metabolism and inflammation indicating the positive role of olive phenols and oleic acid (OA) from OO owing to their antiinflammatory and antioxidant potentials.15 OO is obtained from the fruits of olive trees (Olea europaea). Maximum health benefits have been achieved by consuming extra-virgin OO (EVOO). The constituents of EVOO can be divided into two fractions. The saponifiable fraction is the principal fraction of OO that mainly consists of unsaturated and saturated fatty acids.16 OO is rich in monounsaturated fatty acid (MUFA), OA (55%
83%).

The unsaponifiable fraction (1%
2%) consists of phenolic compounds, hydrocarbons, sterols, tocopherols, pigments, and a few other minor compounds.17 Here, Fig. 22.2 represents the composition of EVOO. Phenolic compounds in the EVOO are shown to impart positive health effects. Oleocanthal, oleuropein, and its derivatives hydroxytyrosol (HT) and tyrosol are the phenolic compounds with significant biological activities.18 HT is considered as the major contributor to these observed activities.16 HT and tyrosol are absorbed up to 60%, mainly in the small intestine by passive diffusion in dosedependent manner in both, human and animals.5,19
21 Once absorbed, they undergo extensive metabolism that involves catechol-O-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, and phenolsulfotransferase.21,22 Here, we have reviewed pathways dysregulated in MS and modulation of these pathways by intervention of OO. Though traditionally health benefits of OO were attributed to its high OA content, epidemiological, clinical, and experimental data suggests the role of minor compounds especially phenolic compounds also.23

22.2 Olive oil and metabolic syndrome As mentioned earlier, OO contains OA (a major component of OO and minor components). It is very difficult to specifically assign observed clinical or experimental outcomes to either OA or minor components. Among the minor components, phenolic compounds those have been primarily studied are oleocanthal, oleuropein, HT, and tyrosol.24

Effect of olive oil on metabolic syndrome Chapter | 22

263

FIGURE 22.2 The composition of EVOO. Major fatty acid in EVOO is monounsaturated fatty acid, oleic acid. It also contents palmitic acid and stearic acid (saturated fatty acids) and linoleic acid and alpha-linolenic acid (polyunsaturated fatty acids). The unsaponifiable fraction of EVOO mainly consists of hydrocarbons, phenol compounds, sterols, tocopherols, and pigments. Both saponifiable and unsaponifiable fractions possess various biological activities. EVOO, Extra-virgin olive oil.

22.2.1 Olive oil and oxidative stress Many studies highlight the association of oxidative stress and chronic inflammatory condition with the development and progression of metabolic diseases. It can be considered as an early event in the disease pathology.25 Oxidative stress means loss of balance between the oxidative and antioxidative systems of the cells and tissues. It leads to the over production of oxidative free radicals. These free radicals are either reactive oxygen species (ROS) or reactive nitrogen species. Nutritional stress such as high-fat and/or high-carbohydrate diet (chronic hyperglycemia) also promotes oxidative stress. Free radicals can attack the cellular macromolecules such as proteins, lipids, and nucleic acids leading to the structural and functional changes. These alterations in the macromolecules lead to the decreased biological activity. Decreased biological activity may result in the loss of energy metabolism, altered cell signaling and transport, and other major functions that may further lead to cell death.26,27 In patients with MS, weak antioxidant defense is evident by depressed serum vitamin C and α-tocopherol concentrations, decreased superoxide dismutase activity, and increased lipid peroxidation, protein carbonyls, and xanthine oxidase activity.28 Both OA and phenolic compounds in EVOO are shown to diminish oxidative stress. OO is rich in OA. This cis-MUFA is less susceptible to oxidation than polyunsaturated fatty acids. High intake of OO results in proportionate incorporation of OA in the cellular membranes making them less susceptible to oxidative damage (i.e., lipid peroxidation). ROS induces arachidonic acid (AA) release from the membrane, which passes through enzymatic cascade to produce eicosanoids. Generally, AA-derived eicosanoids are inflammatory in

nature. It has been seen that OA reduces ROS-induced AA concentrations in various tissues.29 OO phenol compounds are amphiphilic in nature meaning they partition between lipid phase and water. It has been suggested that phenols and their metabolites enter the cellular compartments and exert their antioxidant activities.30 They can resolve oxidative stress by two different mechanisms. In first, they directly scavenge ROS and in second, they trigger the antioxidant defense mechanisms.31 The potency of antioxidant capacity, time required for detectable effect, and intracellular accumulation differs for these phenols. For example, HT has superior antioxidant capacity than the tyrosol. HT enters and exerts its effect within a few minutes of treatment. On the other hand, weak antioxidant tyrosol keeps accumulating within cell up to 12 h after treatment. Thus due to the presence of a combination of phenols, EVOO may be able to execute rapid as well as sustained antioxidant effect.32 In various studies, intervention with OO has been reported to lower oxidation of LDL along with decreasing trend of antibodies against the oxidized LDL (oxi-LDL) particles. These studies suggest that moderate phenolic content of OO is sufficient to lower the oxidation of LDL particles.9 Compared to linoleate-rich LDL particles, oleate-rich LDL particles are less susceptible to oxidation. In addition, compared to carbohydrate-rich diets, the particle size of LDL particles increases in OO-rich diets.33 Along with oleate enrichment of LDL particles by OO consumption, oxidation of these particles is prevented by direct or indirect interaction (through vitamin E) of phenols with these particles. Covas et al. have shown the direct binding of tyrosol to LDL particles and prevention of LDL oxidation.34 Phenols from the EVOO function as chain-breaking antioxidant for lipid peroxidation. Besides this, they also

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protect activity of other antioxidants such as vitamin E and impart sparing activity for antioxidants such as vitamin C.4 Oleuropein present in OO is a potent antioxidant. It acts as a scavenger for superoxide (O22) anion in neutrophils. It chelates the metal ions such as Cu21 and Fe31 those are involved in free radical generation.35 Rietjens et al. in their study have shown that HT and oleuropein are potent scavengers of hydroxyl radicals, peroxynitrite, and superoxide radicals but both these phenols along with homovanillic acid (metabolite of HT) and tyrosol are very poor scavengers of hypochlorous acid and hydrogen peroxide.36 Scientists have suggested that high antioxidant capacity of HT is probably due to the presence of catechol moiety in its structure.37,38 Exactly opposite activity of HT has been reported by Yvonne O’Dowd et al. They have shown that HT can scavenge hydrogen peroxide but not the superoxide anions released during the respiratory burst.39 Oxidative stress may result in damage to DNA. The damage can be in the form of oxidized DNA bases, DNA breaks, and 8-oxo deoxyguanosine in urine. OO intervention showed up to 62% reduction in the DNA oxidation markers.9 Proteomic studies have confirmed strengthening of antioxidant system by consumption of EVOO. Levels of glutathione are upregulated by enhancing the activities of glutamate dehydrogenase, glutathione synthase, and hydroxyacylglutathione hydrolase, along with downregulation of cysteinesulfinic acid decarboxylase. Other antioxidant enzymes are also shown to be upregulated at transcription level after EVOO treatment.40,41 Similar effects on transcription of enzymes involved in antioxidant defense were seen for tyrosol and its metabolites, tyrosol glucuronide, and sulfate. Both HT and tyrosol sulfate metabolites are shown to protect endothelial and intestinal cells from oxidative damage.42,43 Oxidative stress modifies cellular macromolecules in such a way that their functions are altered. Majority of these alterations result in the loss of function within cellular system. Through direct or indirect mechanisms, intervention with EVOO has been shown to bring down the oxidative stress. Lowered oxidative stress is evident by lower levels of oxidatively damaged macromolecules and strengthened antioxidant defense system.

22.2.2 Olive oil and hypertension Hypertension is a major risk factor for the CVD; stroke and myocardial infarction are among them. Central obesity, oxidative stress, insulin resistance, inflammation, and endothelial dysfunction are some of the key factors responsible for hypertension in MS. The antinatriuretic effect of insulin is retained in MS resulting in upregulated renal sodium reabsorption.44

Angiotensinogen is converted to angiotensin II through renin
angiotensin system by the action of angiotensinconverting enzyme (ACE). Angiotensin II is a strong mediator of renal sodium retention. Increased sodium reabsorption results in vascular stiffness due to volume expansion. On the other hand, atrial natriuretic peptides (ANP), released from the atria of the heart, enhances renal sodium excretion. In addition, ANP is degraded by neutral endopeptidase (NEP) and angiotensin II is inactivated by angiotensinase. Thus the activities of angiotensin II and ANP are balanced. All these factors further turn into the development of arterial hypertension.45 A large number of human trials and animal experiments have confirmed beneficial effect of OO on hypertension.46 Ferrara et al. in their double-blind, randomized crossover clinical trial conducted on 23 hypertensive patients showed that a diet rich in EVOO was associated with lower systolic and diastolic blood pressure (BP), and also with a reduced need for antihypertensive medication when compared with a diet rich in polyunsaturated fatty acids. The authors considered that high OA and phenolic compound content was responsible for the lowering of BP.47 SUN study has shown that men with a higher intake of OO had a lower risk of developing hypertension during the follow-up while this association was not present among women.48 Lockyer et al. have observed lowered vascular stiffness in human OO intervention study. Increased nitric oxide (NO) production resulting in improved vascular function has been suggested.49 In vitro cell-based studies support this assumption. In LPS-stimulated mouse macrophages, NO levels were elevated after OO phenols treatment probably through modulation of enzymes such as nicotinamide adenine dinucleotide phosphate oxidase and nitric oxide synthase.5 Based on the OO intervention study in the healthy individuals, Pela´ez et al. have concluded that EVOO with high content of phenols could modulate expression of genes in renin
angiotensin
aldosterone system (RAAS). Modulation of these genes might be responsible for the observed reduction in the BP.50 OA, HT, and other phenolic compounds act synergistically to inhibit ACE, block calcium channel activities, and lower the BP.5 Oleuropein has been shown to inhibit activity of NEP, thus protecting ANP resulting into lowering of hypertension.51 Endothelium plays very crucial role in regulating vascular tone through synthesis and release of potent vasodilator (NO) and vasoconstrictor (endothelin-1, ET-1). Insulin resistance leads hyperglycemia followed by flux of free fatty acids (FFA). Elevated levels of both glucose and FFA generate oxidative stress.52 ROS and hyperglycemia are the major contributors to the disturbances observed in the L-arginine
NO pathway resulting in the NO inactivation

Effect of olive oil on metabolic syndrome Chapter | 22

leading to elevation of plasma asymmetric dimethylarginine (ADMA). ADMA further inhibits NO synthesis and worsens oxidative stress. The ultimate outcome is vasoconstriction and development of atherosclerosis.53,54 In ECV304 endothelial cell model, it was shown that high glucose levels along with oxidative stress induced ET-1 synthesis and decreased intracellular NO levels. In this study, HT and other phenols present in EVOO were able to partially reverse these effects. The observed effects are probably through increased NO production by activating endothelial NO synthase, which might be through Akt, Erk, and p38. HT also increases [Ca21]i which further activates NO synthesis. Overall, these events lead to improvement in nitric oxide/ET-1 (NO/ET-1) proportions and endothelial functioning.52 Adrenergic receptors and downstream signaling are key factors for the central and peripheral controls of BP. Studies have showed that OA can regulate the activity of the adrenoreceptor signaling pathway. High consumption of EVOO increases the concentration of OA in the membranes. At this concentration, localization, activity, and expression of important signaling molecules in the adrenergic receptor pathway are modulated to enhance the production of vasodilatory stimuli such as cAMP and PKA and to restrict vasoconstriction pathways such as inositoltriphosphate, Ca21, diacylglycerol, and Rho kinase. Together all these factors result in lowering of BP.55 Marked improvement in the systolic and diastolic BP and endothelial functioning was seen in individuals with high normal BP or stage 1 essential hypertension, consuming high phenolic compounds containing OO. Authors have attributed this effect to high phenolic content of OO that increased plasma nitrites/nitrates ratio and decreased serum ADMA level.53,56 Oxidative stress, insulin resistance, and plasma high FFA levels lead to vasoconstriction, volume expansion, and disturbed vascular tone. Intervention of EVOO is shown to influence various pathways involved in BP regulation leading to improved vascular tone and imparting vasorelaxation resulting in lowering of hypertension.

22.2.3 Olive oil and inflammation Chronic low-grade inflammation has been associated with initiation and progression of MS and related complications. Data supports the association of proinflammatory state marked by elevated C-reactive protein (CRP) level and MS.57 High-sensitivity CRP, tumor necrosis factoralpha (TNF-α), fibrinogen, and interleukin-6 (IL-6) are the routine inflammatory markers associated with MS. Activity of cyclooxygenases (COXs), lipoxygenases, and thromboxygenases is enhanced in inflammatory conditions. AA is the principal substrate for inflammatory mediator synthesis. Though it is not clear how the

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inflammation leading to installation of MS is triggered, possible mechanisms are (1) secretion of inflammatory adipokines by adipose tissue, (2) insulin resistance, and (3) chronic positive nutrient supply.58 Chronic positive nutrient supply results in hypertrophy of adipocytes with generation of oxidative stress and overactivation of nuclear factor κβ (NF-κβ) leading to the development of systemic inflammation as this transcription factor regulates expression of genes coding for chemokines, cytokines, adhesion molecules, proinflammatory acute phase proteins, cox-2 enzyme to name some.59,60 Under inflammatory conditions, endothelial cells show elevated expression of adhesion molecules and promote further secretion of inflammatory cytokines by macrophages further rising the risk of CVD.59 The peripheral blood mononuclear cells in the individuals with chronic inflammation are also in the proinflammatory state and may get transformed into foam cells leading to atherosclerotic plaque development. Subsequent events are endothelial dysfunction, migration, inflammation, cell death, and atherosclerotic plaque development.61 Lee et al. have postulated that OA suppresses the activity of Δ5-desaturase, Δ6-desaturase, and elongase-5 leading to reduced synthesis of AA from linoleic acid. Lower AA levels may further lead to lower incorporation in the cell membranes and lower generation of AAderived inflammatory mediators.62 OA metabolite that is eicosatrienoic acid (20:3 n-9) has been shown to inhibit leukotriene B4 (LTB4) synthesis.23 OO phenols, oleuropein, and HT are shown to reduce activation of NF-κβ and activator protein-1 (AP-1) resulting into lowered expression of vascular cell adhesion molecule-1 (VCAM-1) by the stimulated endothelial cells. The VCAM-1 promoter contains various binding sites for transcription factors, such as NF-κβ and AP-1. Therefore reduced activation of NF-κβ and AP-1 results in reduced VCAM-1 promoter activity. Independent of the stimulus used, oleuropein and HT were able to downregulate expression of adhesion molecules such as VCAM-1, E-selectin, and intracellular adhesion molecule-1 dose dependently in endothelial cells.60,63 Oleocanthal has been shown to inhibit both COX-1 and COX-2 inflammatory enzymes in a dose-dependent manner. Its antiinflammatory mechanism is similar to ibuprofen. In vitro and animal studies show that HT inhibits expression of proinflammatory TNF-α, inducible nitric oxide synthase, and COX-2. In intestinal epithelial cells and Caco-2 cell line, treatment with these compounds resulted in attenuation of IL-8. In human studies, phenolic compounds of OO have been shown to decrease AAderived inflammatory mediators such as thromboxane b2,6-keto-PGF1a, high-sensitivity CRP, and IL-6.6 LTB4 generation was seen to be inhibited by oleuropein, tyrosol, HT, and caffeic acid in intact rat peritoneal

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leukocytes stimulated with calcium ionophore. This effect was by inhibiting the activity of 5-lipoxygenase.35,64 EVOO contains all the phenolic compounds mentioned earlier. So synergistically they function to inhibit both COX- and lipoxygenase-derived inflammatory mediators. Indeed, this was confirmed by Bogani et al. in their experiment.65 In zebra fish model, it was postulated that oleuropein may inhibit the activity of Toll-like receptor-4 (TLR-4) signaling cascade. Same phenol and HT have been shown to inhibit expression of matrixmetalloprotease-9 (MMP-9), further reducing the inflammation-induced angiogenesis. In THP-1 cell line, downregulation of TNF-α is reflected in lowered level of MMP-9, a crucial factor in atherosclerosis development.60 NF-κβ plays central role in the inflammatory pathways. Enzymes such as COX, lipoxygenases are involved in generation of inflammatory mediators especially from AA. EVOO has been shown to modulate the components of these inflammatory pathways in such a way that it also improves endothelial function.

22.2.4 Olive oil and insulin resistance It is well established that chronic inflammation represented by elevated TNF-α levels is associated with insulin resistance and diminishing antiinflammatory property of insulin. FFA and oxidative stress interfere with the insulin signaling leading to the development of insulin resistance.66 Insulin resistance results in impaired uptake and utilization of glucose in muscles and elevated release of nonesterified fatty acids from the adipose tissues. Insulin resistance also alters glucose homeostasis in the liver.67 Insulin resistance resulting in hyperinsulinemia stimulates activity of RAAS in endothelium leading to the production of angiotensin II and its proatherogenic effect. Angiotensin II acts through angiotensin I receptor inhibiting vasodilatation in blood vessels and glucose uptake in muscle cells by lowering glucose transporter-4 (GLUT4) levels.68,69 High levels of insulin and TNF-α are positively associated with high circulating ET-1 level in insulin resistant as well as normal individuals.44 All these factors link insulin resistance to atherosclerosis or CVD.61,70 Hamden et al. have mentioned various mechanisms for the hypoglycemic effect of HT.71 First, HT has insulinotropic effect, it enhances insulin secretion by pancreatic β cells by inhibiting K-ATP channel and activating the voltage-dependent calcium channel. Both the events are important for insulin secretion. Second, HT has been proposed to protect pancreatic β cells from ROS-induced cell death and/or enhance regeneration of these cells. Both these mechanisms are also supported by cell line
based data. When INS-1E cells were exposed to various concentrations of OO-derived phenols, most of them, including

HT, tyrosol increased glucose-induced insulin secretion but ferulic and sinapic acids inhibited glucose-induced insulin secretion. Though these compounds did not induce apoptosis at lower concentrations, higher concentrations were proapoptotic. Vanillic acid and vanillin induced apoptosis at all the concentrations tested.72 In another study in NIT-1 cells, tyrosol treatment resulted in attenuation of ER stress
mediated apoptosis by inhibiting JNK phosphorylation and cleavage of apoptosis executor molecules Caspase-3 and PARP.73 Third, HT and oleuropein were shown to enhance peripheral glucose uptake. This observation is supported by Lama et al. In their study, when high-fat diet
treated rats were fed with phenol rich virgin OO, liver showed enhanced uptake of glucose through glucose transporter-2 through Akt activation.18 Gene
nutrient interactions are possible in the case of high OA concentration. OA has been shown to interact with polymorphism of PPARc2 gene resulting improved insulin sensitivity.74 Fourth, HT enhances activities of enzymes involved in glycolysis pathway. Consumption of OO results in improved glycemic control. Glucagon-like peptide 1 (GLP1), a peptide produced in the small intestine, through its various actions imparts glycemic control. OO has been shown to enhance GLP-1 response even after 8 h of OO consumption.75 In lean Zucker male rats, higher stimulation of GLP-1 through glucose-independent insulinotropic peptide was observed when OO was fed rather than palm oil.74 Oxidative stress is a major factor for insulin resistance development in which both insulin secretion and sensitivity are hampered. EVOO probably shows ameliorating effects on insulin resistance by improving pancreatic β cell viability and enhancing insulin secretion and glucose uptake. EVOO may impart glycemic control at intestinal absorption level too.

22.2.5 Olive oil and dyslipidemia Increased flux of FFA, higher circulating levels of triglyceride (TG), LDL-c and smaller dense LDL-c, and lower HDL-c are among the dyslipidemia characteristics of MS. Genetic, environmental, and biological factors induce hypertrophy in the adipocytes. Hypertrophic adipocytes, in the presence of insulin resistance, are unable to incorporate FFA in the TGs and are released in the plasma. Insulin resistance and increased flux of FFA in liver lead to elevated assembly and secretion of very LDL (VLDL) particles from liver. Altered posttranslational regulations increase availability of Apo-B for VLDL assembly. Imbalanced proportion of HDL-c and LDL-c is observed because of overproduction of VLDL
ApoB particles and degradation of HDL
ApoAI particles.76 In the presence of oxidative stress, oxi-LDL-c particles are formed, which pass across endothelium. Oxi-LDL-c

Effect of olive oil on metabolic syndrome Chapter | 22

stimulates endothelial cells to synthesize and release proinflammatory cytokines followed by expression of adhesion molecules such as VCAM-1.66 Oxi-LDL-c is trigger for transformation of macrophages into foam cells and initiation of the cascade for atherosclerotic plaque development. TLR-4 connects dyslipidemia, inflammation, and MS. FFA can also induce inflammatory response through TLR-4.59 All these factors are the primary predictive of coronary heart disease.77 Similar to various mechanisms mentioned for hypoglycemic effect of OO phenols, mechanisms for the hypocholesterolemic action of OO phenols are mentioned. Accordingly, these phenols may inhibit dietary cholesterol absorption in the intestine, they may inhibit synthesis and secretion of cholesterol by liver or they may enhance excretion of excessive cholesterol by stimulating biliary secretion.71 OO phenols, especially HT show the modest effect on lowering TG levels. In patients with mild dyslipidemia, HT has been shown to lower hypertriglyceridemia by lowering TG levels. In another study, OO with high phenolic content has been shown to raise HDL-c levels.24 Administration of HT for a month in hyperlipidemic rats has been shown to improve lipid profile and antioxidant status. This effect is attributed to a lowered assembly and/or secretion of ApoB-containing lipoproteins.24 In a study by Sola et al., in hypercholesterolemic individuals, consumption of virgin OO has positive health impact. Upregulation of serum levels of ApoAI by OO is considered to play role for this observation.24 In MS, expression and activity of lipoprotein lipase is downregulated. Direct modulation of genes involved in lipid metabolism at transcription level has been suggested. In the study by Tsartsou et al., lipoprotein lipase level was significantly increased by high phenol OO intervention.9 OO is one of the rich dietary sources of squalene.30 Squalene is known to downregulate cholesterol synthesis by inhibiting activity of key enzyme involved in the cholesterol synthetic pathway, beta-hydroxy-beta-methylglutaryl-CoA reductase leading to lowered prenylation, and subsequent membrane translocation of ras oncogene. Animal studies have confirmed role of squalene in lowering cholesterol levels in serum. It has been shown that adipocyte differentiation was inhibited by both HT and oleuropein by downregulating adipogenesis-related genes, transcription factors, and downstream genes (PPAR, C/EBP and SREBP-1c, GLUT4, CD36, and FASN), thus reducing hypertrophy of adipocytes. In high-fat diet animal model, lipid deposits are seen in liver and peripheral organs such as skeletal muscles. At physiologically relevant concentrations, HT was able to reduce these lipid deposits by inhibiting lipogenic sterol

267

regulatory element-binding proteins (SREBP)-1c/FAS pathway, improving antioxidant defense and oxidative phosphorylation in mitochondria.6 Similar to squalene, in C6 glioma cells, OA decreased activity of acetyl-CoA carboxylase and 3-hydroxy-3methyl-glutaryl CoA reductase (HMGCR) at transcriptional and translational level. These are major regulatory enzymes of fatty acid and cholesterol synthesis, respectively. OA also affected the nuclear level of the SREBP1c and 2. Transcription factor SREBP-1c is essential for expression of genes involved in lipogenesis, while SREBP-2 is essential for the expression of genes involved in cholesterol synthesis.78 Sex-dependent effect of squalene on atherosclerosis and hepatosteatosis development has been postulated by Guille´n et al. They have shown reduced development of atherosclerosis in males but not in females. Opposite effects were observed in mice study lacking apolipoprotein E. In their experiment, HT at pharmacologically relevant dose of 10 mg/kg/mouse/day in low-cholesterol diets lowered ApoAI levels and enhanced the development of atherosclerosis.16 On low-cholesterol background, low percentages (5%
10%) of EVOO-enriched diets were able to stop the progression of induced atherosclerosis in rabbits and in female apolipoprotein E knockout (Apoe2/2) mice. But, on high-cholesterol background, higher percentages of EVOO failed to show a difference in atherosclerosis lesion development when compared with a carbohydraterich diet.40 Helal et al. have shown that in healthy individuals, EVOO consumption for 12 weeks improves the capacity of HDL to mediate cholesterol efflux (CE). This observation of improved CE after OO treatment was supported by a study in human monocyte-derived macrophages. Longterm EVOO consumption results in increased phospholipid layer flexibility of HDL particles along with increased expression of cholesterol transporters at transcriptional and translational levels (ABCA1 and ABCG1) leading to improved CE capacity of HDL.79 After HT treatment, in 3T3-L1 adipocytes, enhancement of mitochondrial functioning is shown by upregulated expression of mitochondrial complexes (at transcriptional level) and functionality. Improved mitochondrial function results in improved consumption of oxygen and lowered FFA content in the adipocytes. Similar effects on oxidative phosphorylation were seen in cultured human fibroblasts and retinal pigment epithelial cells.23,80 Thus intervention with EVOO may lead to enhanced HDL but lowered LDL assembly. Improved HDL levels may perform reverse CE efficiently that can be further excreted. Both OA and phenols inhibit lipogenesis and cholesterogenesis and may improve mitochondrial functioning as oxidative phosphorylation.

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22.3 Implications for human health with special reference to metabolic health Metabolic health encompasses systolic BP, TG levels, fasting blood glucose levels, HDL levels, and HOMO-IR in lean as well as obese individuals. All these parameters are adversely modulated in MS. A book entitled Olives and Olive Oil in Health and Disease Prevention, encompasses majority of the aspects of OO in health promotion and disease prevention.81 Many human studies (including healthy individuals and/or individuals with certain metabolic malfunctioning such as hypercholesterolemia or dyslipidemia) have highlighted usefulness of OO to modulate various markers of MS. Some of the important studies such as PREDIMED, EPIC (especially EPIC Spain cohort study), ATTICA cross-sectional study, and the SUN cohort have shown reduced incidences of CVDs and T2DM in healthy participants or improved markers of these diseases in patients participated. All these trials and few other trials have indicated beneficial role of EVOO consumption on MS risk factors. Fig. 22.3 represents various pathways leading to

risk factors of MS and action of EVOO to modulate these pathways to ameliorate the risk factors. The components of EVOO (i.e., OA and minor components especially phenolic compounds) have been shown to improve risk factors associated with MS that may further lead to CVD or T2DM development and progression. Both OA and phenolic components have been shown to ameliorate oxidative stress and inflammation owing to their strong antioxidant and antiinflammatory potentials. By reducing the oxidative stress and inflammation, they enhance functionality of adipocytes, pancreatic β cells, endothelial cells, hepatocytes, and peripheral muscle cells. Thus consumption of EVOO results in improved serum lipid profile, insulin level and sensitivity, antioxidant defense system, endothelial functioning, and vascular tone.

22.4 Summary Both OA and phenols are shown to impart positive effects on these risk factors. Plausibly, amelioration of inflammation and oxidative stress by EVOO as a whole is responsible for improved insulin secretion and sensitivity, vascular

FIGURE 22.3 Extra-virgin olive oil and metabolic syndrome. Pathways leading to oxidative stress, inflammation, hypertension, insulin resistance, dyslipidemia are dysregulated in MS. % represents components of these pathways modulated by EVOO. AA, Arachidonic acid; ACE, angiotensinconverting enzyme; AT1R, angiotensin type 1 receptor; AT2R, angiotensin type 2 receptor; EVOO, extra-virgin olive oil; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; MS, metabolic syndrome; RAAS, renin
angiotensin
aldosterone system; TG, triglyceride.

Effect of olive oil on metabolic syndrome Chapter | 22

269

TABLE 22.1 Mechanisms of extra-virgin olive oil (EVOO) on metabolic syndrome associated risk factors. Risk factor

EVOO component

Mechanism of action

Inflammation

Oleic acid

Change in membrane fluidity AA content in membranek Δ5-desaturase k, Δ6-desaturase k, elongasek NF-κβ activity k

Phenols

Plasma hs-CRP, IL-6, 7, and 18 levels k Expression of proinflammatory genesk Activities of cycloxygenases, lipoxygenases, and thromboxygenase k

Oleic acid

Lipid peroxidation k

Oxidative stress

Plasma oxi-LDL particle concentration k Phenols

Direct scavenging of ROS Lipid peroxidation k Sparing effect for other antioxidants Expression and activity of antioxidant defense machinery m Plasma oxi-LDL particle concentration k Oxidative DNA damage k Activity of xanthine oxidase k

Dyslipidemia

Oleic acid

Modulation of expression of genes involved in lipogenesis and cholesterol synthesis

Phenols

Intestinal cholesterol absorption k Synthesis and release of cholesterol by liverk Cholesterol efflux m Cholesterol excretion m Plasma TG level k Plasma LDL-c level k Plasma HDL-c level m Expression of adipogenesis-related genes k Mitochondrial functioning m

Endothelial dysfunction

Phenols

Plasma markers of endothelial activation k Vasodilatory response m

Insulin resistance

Oleic acid

Insulin secretion m Insulin sensitivity m

Phenols

Peripheral organs Glucose uptake m Viability of pancreatic β cells m Insulin secretion m

Oleic acid

Regulation of androgenic receptor functioning

Phenols

Nitrite/nitrate m Endothelial NO synthase activity m

Hypertension

The table summarizes the risk factors associated with metabolic syndrome. Effects of oleic acid and/or phenols on various pathways involved in the development and progression of metabolic syndrome are enlisted. HDL-c, High-density lipoprotein cholesterol; IL, interleukin; LDL-c, low-density lipoprotein cholesterol; NF-κβ, nuclear factor κβ; oxi-LDL, oxidized low-density lipoprotein; ROS, reactive oxygen species.

tone, and endothelial functioning. Taken together, intervention with EVOO for prolonged duration may lower the incidences of CVD in MS patients. Thus Table 22.1 summarizes the observed mechanisms of EVOO on MSassociated risk factors.

Mini-dictionary of terms Adipogenesis Angiogenesis

the process of preadipocyte differentiation into mature adipocytes the process of new blood vessel formation from the existing blood vessel

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Natriuretic effect excretion of sodium in the urine Apoptosis a process of programmed cell death Atherosclerosis a condition of plaque development in the arterial wall making the artery narrow and hard Central obesity excessive fat accumulation in abdominal area Cholesterogenesis biosynthesis of cholesterol Cholesterol The process of transfer of cholesterol from cells efflux to high-density lipoprotein (HDL) Dyslipidemia a lipoprotein disorder with decreased HDL-c and increased low-density lipoprotein cholesterol and triglycerides in serum. Free radicals any unstable molecular species with an unpaired electron in an atomic orbital Hepatosteatosis a condition in which intracellular accumulation of triacylglycerol in the liver is seen Insulinotropic stimulation of production, release, and/or effect activity of insulin Insulin resistance a condition in which normal insulin levels are incapable of achieving normal metabolic response or above normal insulin levels are required to achieve normal metabolic response Lipogenesis biosynthesis of lipids Oxidative stress imbalance between the production of free radicals and antioxidant defenses

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Section 2.2

Cardiovascular

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Chapter 23

Olive and olive oil: a one stop herbal solution for the prophylaxis and management of cardiovascular disorders Shanoo Suroowan, Bibi Sharmeen Jugreet, Nabeelah Bibi Sadeer and Mohamad Fawzi Mahomoodally Department of Health Sciences, Faculty of Science, University of Mauritius, Re´duit, Mauritius

Abbreviations AD Akt AO CAVI cl-CASP3 CHD CoO CVD Cyt-C DBP DXR ERK EVOO GO H9c2 HCHF HLD HNE HP HPCOO iNOS LP LPCOO MMP2 mRNA MUFA NF-κB OLE OLLE OO OOT p-Hsp27 PI3K p-MAPKAPK-2

Alzheimer’s disease protein kinase B avocado oil cardio-ankle vascular index cleaved caspase 3 coronary heart disease coconut oil cardiovascular disease cytochrome C diastolic blood pressure doxorubicin extracellular signal regulated kinases extra-virgin olive oil grapeseed oil cells (myocardial cell line), GRP78 and CHOP high carbohydrate high fat high-lipid diet hydroxynonenal high levels of olive oil phenolics high-polyphenol-content olive oil inducible nitric oxide synthase low levels of olive oil phenolics low-polyphenol-content olive oil metalloproteinase-2 messenger ribonucleic acid monounsaturated fatty acids nuclear factor κB oleuropein olive leaf extract olive oil onset oxidation temperature phospho-Hsp27 phosphoinositide-3-kinase phospho-MAPKAPK-2

PO PON1 p-SAPK/JNK PUFA RNA ROS SAMP8 mice SBP SI/R SIRT1 SO SOFE UNESCO VOO

high oleic peanut oil paraoxonase 1 phospho-SAPK/JNK polyunsaturated fatty acid ribonucleic acid reactive oxygen species senescence-accelerated mouse-prone 8 systolic blood pressure ischemia/reperfusion sirtuin 1 sunflower oil standardized olive fruit extract The United Nations Educational, Scientific and Cultural Organization virgin olive oil

23.1 Introduction Olive (Olea europaea L.) is an ancient species originating from the Mediterranean region. The most cherished product from O. europaea is olive oil (OO) which is produced from the fruit.1 Indeed, OO use is an integral part of the Mediterranean culture and has remained a fundamental ingredient added to food for ages.2 Given the popularity of the Mediterranean diet, in 2010, it was recognized as a Cultural Heritage of Humanity by UNESCO.3 Most of the OO available worldwide is produced in Greece, Italy, Morocco, Spain, Tunisia, and Turkey.4 Australia and the United States are also the manufacturers of OO,5 while Greeks are the number one consumer of OO in the world.6 Elucidation of the nutritional and health benefits of OO formed an area of interest and research as early as the 7th century BCE. Even Aristotle

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and Hippocrates recognized the potential benefits of OO and included it as a major ingredient in various medicinal preparations.1 In fact, OO is rich in triacylglycerols (98%
99%) of which around 80% is oleic acid.7 The oil is also rich in a plethora of bioactive phytochemicals including α- and γ-tocopherols, β-carotene, flavonoids, hydrophilic phenolic compounds, for example, hydroxytyrosol, oleocanthal, oleuropein (OLE), and tyrosol, phytosterols, and tocotrienols.8 While fats derived from animal origin are rich in saturated fatty acids, OO provides a healthy source of monounsaturated fatty acids (MUFA) and polyphenols.9,10 The widely acclaimed antioxidant properties of OO are due to its rich phenolic composition that has been validated by the European Food Safety Authority.11 Naturally, the use of OO as a culinary ingredient remains in vogue given its promising benefits on the cardiovascular and the central nervous systems. It acts against cancer and other noncommunicable diseases in which diet modification is indicated as a prominent line of action.12,13 Additional health benefits associated with the consumption of olive and its oil relate to their ability to prevent strokes and reduce overall mortality figures.14 This may be due to the fact that it averts the development of secondary risk factors associated with chronic degenerative disorders.15 OO is a complementary agent for the prophylaxis and management of various cancer types (gastric, colorectal, and breast), infections, metabolic syndrome, neurological conditions, and type II diabetes mellitus.16 An Italian study found an inverse relationship between children being overweight and OO consumption at home.17 In fact, in 2004, the Food and Drug Administration approved the claim on OOs labels indicating that the daily intake of two tablespoons of OO was beneficial in decreasing coronary heart disease (CHD) based on its high MUFA composition.18 Hence this book chapter aims to underscore the health benefits of olive and OO with particular emphasis on the cardiovascular system. It takes into account the historical uses associated with OO through the documentation of ethnobotanical studies. Moreover, it lays emphasis on laboratory-based in vitro, in vivo, as well as clinical studies to disseminate to the scientific community the valuable health assets of OO.

23.2 Ethnobotanical uses of Olea europaea L. Usage of olives and OO in traditional medicine in various parts of the world is centuries old. The leaves, fruits, and oil of olives have an ancient and remarkable history of nutritional, medicinal, and traditional uses. A total of 30 species are classified under the genus Olea. However, O. europaea is the only species used as a source of food19 and is fraught with medicinal values whereby a decoction

and infusion of oil of this species can be used to alleviate a panoply of ailments, including urinary tract infection, diabetes, high blood pressure, hair loss, diarrhea, gout, rheumatism, and can be used as a mouth and skin cleanser, among others.19,20 In the European and Mediterranean regions, O. europaea is commonly used as an emollient, febrifuge, tonic, antiinflammatory, diuretic, and hypotensive agent.21 In southeastern Morocco, the leaf, bark, and wood of O. europaea is used as an antifebrile to control high blood sugar level and to treat diarrhea and sores.22 The seed oil is taken orally as a laxative and an emollient as a daily skin care.23 In addition, when mixed with lemon juice, the oil
lemon emulsion acting as a cholagogue is consumed to treat gallstones. Hair loss, a stressful condition experienced by both men and women, can be prevented and treated by applying OO every night and shampooed the next morning.20 Infusion of the fresh and dried leaves is taken orally to treat hypertension, diabetes, and bronchial asthma and acts as an antiinflammatory agent.20,24 Virgin OO (VOO) is frequently consumed in Mediterranean countries and is known to enhance the endothelial protective capacity and promote antioxidant and antiinflammatory properties which subsequently reduce the risk of cardiovascular diseases (CVDs).25 Further traditional uses are summarized in Table 23.1.

23.2.1 Phytochemistry Polyphenolic compounds found in O. europaea, namely, oleuropeosides [OLE (1) and verbascoside (2)], flavonols [rutin (3)], flavones [luteolin-7-glucoside (4), apigenin-7glucoside (5), diosmetin-7-O-glucoside (6), luteolin (7), diosmetin (8)], flavan-3-ols [catechin (9)], substituted phenols [tyrosol (10), hydroxytyrosol (11), vanillin (12), vanillic acid (13), caffeic acid (14), oleoside (15)], and secoiridoid glycoside [oleuricine A (16) and oleuricine B (17)] are known to exert beneficial effects towards the cardiovascular system.38,39 The main active phenolic component of O. europaea is OLE (18), a natural product of the secoiridoid group responsible for the bitter taste of olive fruits and leaves.40,41 In addition to the bioactive compound OLE, dimethyl oleuropein (19), ligstroside (20), and oleoside 11-methyl ester (21) are reported as among the most abundant secoiridoids. In the leaves, OLE, flavonoids and luteolin 7-glucoside (22) are in a weight-byweight ratio of 19%, 1.8%, and 0.8%, respectively.

23.2.2 Pharmacology The pharmacological properties, namely, antiinflammatory, cardio- and neuro-protective of OLE isoforms, glycosidic forms, and aglycones are due to the presence of ortho-diphenolic group in the compounds, exhibiting potential radical scavenging properties.41 Indeed, several

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TABLE 23.1 Traditional uses of Olea europaea L. Country

Plant parts/ derivative

Preparation/route of administration

Ailments treated and/or therapeutic uses

References

Canary Islands

Leaf

Infusion (oral)

Hypotensive, hemorrhoid

Darias et al. (1996)26

Algeria

Leaf and fruit

Infusion (oral)

Hypoglycemic, hypotensive

Amel (2013)27

East Africa

Bark

Infusion (oral)

Tapeworm

Kokwaro (2009)28

Greece

Leaf

Infusion (oral)

High blood pressure

Lawrendiadis (1961)29

Palestine

Leaf and fruit

Decoction (oral)

Diabetes

Ali-Shtayeh et al. (2012)30

Italy

Oil

Oral

Renal lithiasis

De Feo et al. (1992)31

Topical

Burns, rheumatism, promote circulation

Tincture

Febrifuge

Leaf

Topical

Ingrown nails, restore epithelium

NI

Oil

Oral

Laxative

Al-Khalil (1995)23

NI

Leaf and fruit

Decoction (oral)

Diarrhea, respiratory, and urinary tract infections

Bellakhdar et al. (1991)32

NI

Oil

Topical

Hair loss

Hashmi et al. (2015)19

NI

Leaf

Infusion (oral)

Asthma

Hashmi et al. (2015)19

NI

Leaf

Infusion (oral)

Hypertension

Ribeiro et al. (1986)24

United States

Oil

Oral

Hypertension, agitation, laxative, vermicide

Hashmi et al. (2015)19

Japan

Leaf

Infusion (oral)

Stomach and intestinal diseases

Bellakhdar et al. (1991)32

Oil

Oral

Constipation and liver pain

Brazil

Leaf

Infusion (oral)

Diuretic, hypertension

Ribeiro et al. (1986)24

Tunisia

Leaf

Infusion (oral)

Bacterial infections namely gingivitis, otitis, icterus, and cough

Haloui et al. (2010)33

NI

Oil

Topical

Fractured limbs

Ghazanfar and Al-Al-Sabahi (1993)34

NI

Leaf

Infusion (oral)

Antipyretic

Hashmi et al. (2015)19

Morocco

Leaf

Decoction (oral)

Hypertension, diabetes

Tahraoui et al. (2007)22

NI

Fruit

Topical

Skin cleanser

Fujita et al. (1995)35

NI

Leaf

Infusion (oral)

Antiinflammatory, tonic

Ribeiro et al. (1986)24

Mediterranean

Leaf

Infusion (oral)

Gout

Flemmig et al. (2011)36

Leaf and fruit

Decoction (oral)

Hemorrhoids, rheumatism, and vasodilator

Su¨ntar et al. (2010)37

Leaf

Infusion

Eye infection

Hashmi et al. (2015)19

NI NI, Not indicated.

studies have shown that OLE possesses a wide range of pharmacologic and health-promoting properties, including antiarrhythmic, spasmolytic, immune-stimulant, cardioprotective, hypotensive, antiinflammatory, antioxidant, and antithrombotic effects.40 This chapter focuses mainly on the reported and scientifically validated benefits of olive and OO on the cardiovascular system.

23.2.3 Cardioprotective effects In this section, emphasis is laid on the various health benefits that have demonstrated distinct olive-derived extracts

and oils as cardioprotective agents. Various experimental models (in vitro, in vivo, and clinical trials) are regrouped in this section. Their mechanistic actions involved are also discussed where applicable.

23.2.4 In vitro investigations and cardioprotective mechanisms involved In vitro studies have vouched for the decrease in lowdensity lipoprotein (LDL) oxidation with an increase application of OO phenolic compounds to cells. Hydroxytyrosol or 3,4-dihydroxyphenyl-ethanol is one of

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the main components of OO and the most potent free radical scavenger among all other OO polyphenols.42 Olive leaf extract (OLLE) has been observed to exert cardioprotective action. This may be associated with the cellular redox modulating effects of its polyphenolic components. The ethanolic and methanolic extracts of olive leaves were compared in vitro to the effects of hydroxytyrosol, OLE, and quercetin used as a positive standard in a carbonyl compound [4-hydroxynonenal (HNE)]-induced model of oxidative injury in rat cardiomyocytes (H9c2). The extracts were found to inhibit 4-HNE-induced apoptosis which is characterized by a rise in the generation of reactive oxygen species (ROS), mitochondrial dysfunction, and an activation of proapoptotic cl-CASP3. They also caused the inhibition of HNE-induced phosphorylation of stress-activated transcription factors, and the effects of extracts on p-SAPK/JNK, p-Hsp27, and p-MAPKAPK-2 were found to be concentration-dependent and similar to hydroxytyrosol, OLE, and quercetin. Nonetheless, the downregulation of HNE-induced p-MAPKAPK-2 and p-cJun was more by methanolic than ethanolic extract, while lesser inhibitory effect was exhibited on HNE-induced pSAPK/JNK and p-Hsp27 by methanolic than ethanolic extract. Moreover, cl-CASP3 and p-Hsp27 were attenuated, particularly by quercetin. Furthermore, a predominant ROS inhibitory and mitochondrial protecting capacity at a concentration of 1
10 μg/mL for each extract, hydroxytyrosol, OLE, as well as quercetin was shown. The ethanolic extract that contained significant amounts of OLE, hydroxytyrosol, verbascoside, luteolin, and quercetin compared to the methanolic extract as identified by high performance liquid chromatography, displayed greater protection on cardiomyocyte viability than the methanolic extract or each phenolic compound against HNE-induced carbonyl stress and toxicity. This was likely due to the synergistic interactions of polyphenolics and other compounds found in ethanolic OLLEs.43 To understand the mechanism responsible for the cardioprotective effect of OLE against simulated ischemia/reperfusion (SI/R)
induced cardiomyocyte injury in vitro, Zhao and coworkers showed that OLE was able to decrease SI/ R-induced cell injury in neonatal rat cardiomyocyte.44 The compound was found to inhibit the excess production of ROS and stabilized mitochondrial membrane potential following SI/R. The results indicated that posttreatment with OLE, cellular apoptosis was inhibited, together with the attenuated expression of Cyt-C, cl-CASP3. Moreover, the Bcl-2/Bax ratio was increased and the phosphorylation of ERK1/2 and Akt was improved post-SI/R. However, the enhancement in phosphorylation was in part put to an end in the presence of a PI3K inhibitor (LY294002) and an ERK inhibitor (U0126). Thus the findings demonstrated that OLE’s protective effect against SI/R-induced injury

may be attributed partly as a result of the reduction of apoptosis via the activation of the PI3K/Akt and ERK1/2 signaling pathways. Figs. 23.1
23.3 illustrate the chemical structures of isolated compounds from olive and OO exhibiting potential benefits for the cardiovascular system.

23.2.5 In vivo studies and cardioprotective mechanisms involved In vitro investigations combined with in vivo studies provide deeper insights towards the validation of the purported effects of olive and OO. In an in vivo study in anesthetized rabbits the constituent OLE was found to exert antiischemic, hypolipidemic, as well as antioxidant effects.45 Administration of 10 or 20 mg of OLE/(kg/day) to eight groups receiving normal or hypercholesterolemic diets for a duration of 6 weeks or only 3 weeks at the higher dose was carried out. Rabbits consuming the normal diets were found to have reduced infarct size following the administration of 10 or 20 mg of OLE/(kg/day) for 3 or 6 weeks compared with the control group. However, in hypercholesterolemic rabbits, infarct size was reduced at only a high dose [20 mg/(kg/day)] for both 3 and 6 weeks in contrast to the cholesterol-fed control group. In addition, plasma lipid peroxidation product and protein carbonyl levels were decreased by OLE compared to the control groups, whereby an increase in these factors was observed relative to baseline as a result of ischemia and reperfusion. Red blood cell superoxide dismutase activity did not change during ischemia and reperfusion in OLEadministered rabbits. However, a significantly higher activity was noted in control groups that was then reduced following ischemia and reperfusion compared to baseline. Importantly, OLE treatment with both doses for 6 weeks resulted into decreased levels of cholesterol and triglyceride. A different profile of glycolysis metabolites was observed in OLE-treated groups compared with the controls. Thus, along with reduced infarct size, strong antioxidant protection and decreased circulating lipids were recorded with OLE treatment for 3 or 6 weeks. The investigation conducted by Poudyal et al. on obese and diabetic rat models reported that biocompounds such as OLE and hydroxytyrosol significantly reversed chronic inflammation and oxidative stress that triggers cardiovascular, hepatic and metabolic symptoms.46 In silico molecular docking showed that rutin, commonly found in O. europaea, has a higher binding affinity to HMGCoA compared to luteolin and cerivastatin (23). Subsequently, these findings suggested the development of an inhibitory agent from the leaves of O. europaea towards hypercholesterolemia and atherosclerosis.47 Also, reduced aortic ring reactivity, impaired glucose tolerance, abnormal plasma lipid profile, and hypertension

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FIGURE 23.1 Chemical structures of compounds 1
9 identified in olive and olive oil.

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FIGURE 23.2 Chemical structures of compounds 10
20 identified in olive and olive oil.

Olive and olive oil Chapter | 23

281

FIGURE 23.3 Chemical structures of compounds 21
24 identified in olive and olive oil.

were noted in those high carbohydrate high fat (HCHF) rats. Conversely, rats from the HCHF 1 OLLE group showed improved or normalized cardiovascular, hepatic, and metabolic signs with the exception of elevated blood pressure compared with HCHF rats. These findings suggested that an OLLE containing polyphenols was able to reverse chronic inflammation and oxidative stress that provoked cardiovascular, hepatic, and metabolic symptoms in this animal model of diet-induced diabetes and obesity. Atherosclerosis is a progressive disease characterized by the deposit of lipids and fibrous elements in large arteries, which is a major cause of heart disease and stroke. Atherosclerosis is not merely an inevitable degenerative outcome of aging, but rather a condition of chronic inflammation that can convert into an acute clinical condition as a result of plaque rupture and thrombosis.48 Olive and OO are rich in phenolic phytoantioxidants able to scavenge free radicals responsible mainly for the development of numerous chronic diseases, including atherosclerosis, stroke, chronic inflammation, among others.49 LDL is considered a prime risk factor to develop atherosclerosis and CVDs, including the formation of plaque. Wang et al. investigated the antiatherosclerosis activity following OLLE administration in rabbits with atherosclerosis. The animals were assigned randomly to three groups; control, high-lipid diet (HLD), and HLD

supplemented with OLLE group. Rabbits in the OLLE group developed less serious atherosclerotic lesions than that of the HLD group.50 Furthermore, the tunica intima was found to be reduced in both the size and thickness in OLLE group compared to HLD group. The level of malondialdehyde, a marker indicative of serum lipid peroxidation, was also reduced, suggesting the effect of OLLE on suppressing the production of lipid peroxidation. Besides, the levels of total cholesterol, triglyceride, and LDL cholesterol were also decreased in the OLLE group compared to the HLD group. Moreover, expression of inflammation factors were downregulated both at protein and messenger ribonucleic acid (mRNA) level with OLLE administration. Olmez et al. evaluated the effects of OLLE on serum lipid profile and early changes in atherosclerosis and endothelium-dependent relaxations in cholesterol-fed rats. The rats were given either 2% cholesterol-enriched or standard chow for a period of 8 weeks. In each group, some rats were also fed OLLE at doses of 50 or 100 mg/ (kg/day) (oral administration). Atorvastatin, as positive control, was given at a dose of 20 mg/kg of body weight daily. The levels of total cholesterol and LDL-cholesterol were found to increase in cholesterol-fed rats, while OLLE used at both doses and atorvastatin showed significant reductions in those levels. Hence, OLLE attenuated the increased levels of cholesterol, and in so doing,

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FIGURE 23.4 Effects of olive leaf phenolics on cardiovascular risk markers. [Adapted from Lockyer et al. (2012]

improved the atherogenic lipid profile in rats fed with high cholesterol diet, which, in turn, may have beneficial effects on atherosclerosis.51 Indeed, various other mechanisms have been elucidated and reported in scientific literature whereby OLLE has been found to exert remarkable cardioprotective effects and thus reduce the risk of CVDs52 (Fig. 23.4). In another in vivo experiment model the effects of chronic consumption of extra-VOO (EVOO), enriched in bioactive compounds derived from olive leaves and fruits, on blood pressure, endothelial function, inflammatory and oxidative status, including cholesterol level in circulation in spontaneously hypertensive rats (SHR) were investigated.53 Three groups consisting of a control-untreated SHR group, a SHR group [(1 mL/(rat/day)] supplemented with the control OO (17.6 mg/kg of phenolic compounds), and a SHR group [1 mL/(rat/day)] supplemented with an enriched EVOO (750 mg/kg of phenolic compounds) for 8 weeks. Prolonged administration of the enriched EVOO was found to decrease cardiac hypertrophy and systolic blood pressure (SBP) and enhanced the aortic endothelial dysfunction ex vivo measured in SHR. In enriched oilsupplemented group, diminished levels of plasma Angiotensin II, total cholesterol, and the urinary

endothelin-1 and oxidative stress biomarkers, whereas proinflammatory cytokines were unchanged. Thus treatment with EVOO enriched in bioactive compounds from the olive fruit and leaves may be useful in the reduction of cholesterol and blood pressure levels, alone or combined with pharmacological antihypertensive therapy. The antithrombotic activity of EVOO as a single dietary change in experimental thrombosis and primary hemostasis rat models has also been examined. Two different groups of animals were under investigation, for instance the control group which was fed a normal diet and the other one consisting of a diet enriched with EVOO (3%; w/w). Subsequent to 6 weeks of feeding, arterial thrombosis was elicited through the insertion of an artificial prosthesis (or “aortic loop”) into the aorta, while the induction of venous thrombosis was by the ligation of the inferior vena cava. “Template” bleeding time (BT), factor VII coagulant activity (FVII:C), and fibrinogen levels were then measured. The animals from the group fed with OO
enriched diet displayed a significant delay in the thrombotic occlusion of the “aortic loop,” a lower incidence of venous thrombosis along with a prolonged BT as opposed to the control group. Moreover, they showed lowered concentrations of plasma fibrinogen,

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although similar FVII:C levels were obtained; in spite of their lesser triglyceride levels. This in vivo study was the first to provide the experimental evidence of the thrombosis-preventing potential of OO, which may be mediated by reduced fibrinogen concentrations and the impairment of platelet/vessel wall interactions.54 In a study by Wu et al., both in vitro and in vivo experimental models were explored to evaluate the cardioprotective effects of OLLE and its main component hydroxytyrosol as well as to determine their corresponding mode of action. For instance, H9c2 cells (myocardial cell line) were treated with cobalt chloride (CoCl2) to induce hypoxia. It was revealed that hydroxytyrosol strikingly protected H9c2 cells against CoCl2-induced apoptosis. Moreover, hydroxytyrosol could lower the expression of mRNA and protein of GRP78 and CHOP induced by CoCl2 in vitro. Nevertheless, to induce myocardial infarction in rats (in vivo), isoproterenol was used for two consecutive days. Following pretreatment with OLLE for 1 month, a significant amelioration was noted in isoproterenol-induced decrease in ejection fraction and fractional shortening, increase in ratio of heart weight to body and development of infarction characterized by disordered cardiac muscle fibers and infiltration of inflammatory cells.55 Likewise, OLLE could as well reverse the increased expression of GRP78 and CHOP provoked by isoproterenol. In vivo experimental data demonstrated that the supplementation of purified hydroxytyrosol (4 mg/kg) improved blood lipids and antioxidant activity and reduced the size of atherosclerosis in a rabbit model of diet-induced atherosclerosis. The study concluded that hydroxytyrosol could be a natural antioxidant for the prevention of CVD.56 The cardioprotective effect of OLE in rats with acute myocardial infarction was assessed. The findings indicated that coronary ligation resulting into acute myocardial infarction characterized by impaired cardiac function was prevented by OLE pretreatment, partially via the reduction of oxidative stress and discharge of proinflammatory cytokines.57 The cardioprotective role of OLE in rats with doxorubicin (DXR)-induced cardiotoxicity was investigated.58 DXR-treated animals revealed very broad cytoplasmic vacuolization, while much fewer vacuolization was seen in OLE-treated groups. Furthermore, a significant increase in the release of cardiac enzymes into systemic circulation was observed. OLE administration at both doses [100 or 200 mg/(kg/BW)] and treatment protocols employed, that is before or even after DXR treatment, were found to reduce the DXR-elevated serum levels of creatine phosphokinase, creatine phosphokinaseMB, lactate dehydrogenase, aspartate- and alanineaminotransferase. Furthermore, DXR-induced lipid peroxidation, content of protein carbonyls, and nitrotyrosine concentration,

283

including inducible nitric oxide synthase induction in myocardial tissue were reduced. OLE was found to exert a protective effect by eliminating cardiotoxicity that was induced by DXR, as expressed by the changes in intracellular and peripheral markers. Thus these findings showed that acute DXR cardiotoxicity may be successfully treated with OLE through its antioxidative effects and the inhibition of nitrosative stress. Since the polyphenolic compounds present in OO exhibit potential antithrombotic and antiatherogenic activities, they may be a good pharmacological treatment for CHD and could also be used in cardiac surgeries and transplantations. In fact, the protective effect of OO was attributed to the elevated oleic acid content, OLE, and its derivatives.59 During oil extraction process, OLE is hydrolyzed by endogenous β-glucosidases into OLE aglycone, a bioactive compound responsible for the bitter and pungent taste of EVOO.41 OLE aglycone has the ability to confer cardioprotection through autophagy induction involving nuclear translocation and activation of the transcriptional factor EB. Data gathered by Miceli et al., provide solid evidence of the protective properties of OLE aglycone as a future nutraceutical agent against ageassociated aliments, including CVD.60 In the study of Bayram et al., SAMP8 mice (senescence-accelerated mouse-prone 8) were fed with semisynthetic diets along with 10% OO composed of either high (HP) or low levels of OO phenolics (LP) for a duration of 4.5 months. Mice ingesting the HP diet showed significantly reduced concentrations of the oxidative damage markers thiobarbituric acid
reactive substances and protein carbonyls in the heart, although proteasomal activity in both groups was same. Nrf2 as well as its target genes glutathione-S-transferase, γ-glutamyl cysteine synthetase, quinone oxidoreductase, and paraoxonase-2 (PON2) mRNA levels were significantly superior in heart tissue of the HP compared to the LP group. The HP-fed mice also demonstrated significantly higher PON1 activity in serum in contrast to those consuming the LP diet.61 Moreover, HP diets increased the relative sirtuin 1 (SIRT1) mRNA levels. In fact, the activation of SIRT1 by polyphenols has been reported to be beneficial for the control of oxidative stress, cellular senescence, and metabolism.62 In addition, cell culture experiments indicated that OO phenolic compound, hydroxytyrosol present in the HP oil may be accountable for the induction of Nrf2-dependent gene expression and the increase in PON activity. The antithrombotic effect of dietary supplementation with VOO administered in rabbits that were fed an atherogenic diet has also been investigated.63 In particular, the group of animals that were given saturated fatty acidenriched diet together with 15% of VOO showed an improvement in their lipid profile and a reduction of platelet hyperactivity and subendothelial thrombogenicity.

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In addition, the occurrence of severe morphological lesions in the endothelium and vascular wall was minimized in those rabbits. In a recent study, EVOO and fresh palm oil diets lowered blood pressure in rats. A reduced mean arterial pressure was seen in both groups as compared to the control. In furtherance the heart and kidney superoxide dismutase and catalase activities were increased in a significant manner in those groups. It was also evidenced that blood pressure reducing effects of both oils were through the Katp/Bkca ion channels. However, the greater effect achieved by EVOO was likely due to its high oleic acid (24) and polyphenol content.64

23.2.6 Clinical studies Interestingly, evidence from a number of clinical studies established OO intake to be inversely associated with CVD. In particular, OO and OLLEs polyphenols have been associated with several cardiovascular health benefits. For instance, Guasch-Ferre´ et al. investigated the association between total OO intake, its varieties (extra-virgin and common OO), and the risk of CVD and mortality in a Mediterranean population at an elevated risk of CVD. For this purpose, both men and women (55
80 years old) were randomized to one of three interventions; Mediterranean diets supplemented with nuts, EVOO, or a control low-fat diet. The median follow-up for the study was 4.8 years.65 CVD (myocardial infarction, stroke, and cardiovascular death) and mortality were established by medical records and National Death Index. Validated food frequency questionnaires were also used to evaluate OO consumption. For assessing the association between baseline and yearly repeated measurements of OO intake, CVD and mortality, multivariate Cox proportional hazards, and generalized estimating equations were employed. Throughout the follow-up, 277 cardiovascular events and 323 deaths were recorded. Participants in the highest energy-adjusted tertile of baseline total OO and EVOO consumption showed 35% and 39% decrease in risk of CVD, respectively, as opposed to the reference. Higher baseline total OO consumption was correlated with 48% reduced risk of cardiovascular mortality. Moreover, for each 10 g/day increase in the consumption of EVOO, both CVD and mortality risk were found to reduce by 10% and 7%, respectively. In addition, no significant associations were observed for cancer and allcause mortality. Unlike in the control group, the associations between cardiovascular events and the intake of EVOO were significant in the Mediterranean diet intervention groups. Thus from this study, OO consumption and, in particular, the extra-virgin variety was found to be

associated with reduced risks of CVD and mortality in individuals at high cardiovascular risk. The antihypertensive effect and the tolerability of OLE have also been evaluated and compared with captopril in patients with stage-1 hypertension in a clinical study.66 In addition, the hypolipidemic effects of OLLE in those patients were examined. The study comprised a 4-week run-in period followed by an 8-week treatment period. O. europaea leaf extract (EFLA943) was supplied orally (500 mg) two times a day in a flat-dose manner throughout the 8 weeks. Captopril was given at the dosage of 12.5 mg twice daily initially, then after 2 weeks, if needed; the dose of captopril was titrated to 25 mg twice daily accordingly based on patient’s response to treatment. While the primary efficacy endpoint was the reduction in SBP from baseline to week-8 of treatment, the secondary efficacy endpoints were SBP as well as diastolic blood pressure (DBP) changes at every time-point evaluation and the improvement of lipid profile. Blood pressure evaluation was conducted each week for 8 weeks of treatment, while that of lipid profile was done at an interval of 4-weeks. Interestingly, both groups demonstrated a significant reduction in SBP as well as DBP from baseline after 8 weeks of treatment. Besides, the reductions were not significantly different between the groups. The means of SBP from baseline to the end of the study were reduced in olive (211.5 6 8.5 mmHg) and captopril (213.7 6 7.6 mmHg) groups and those of DBP were 24.8 6 5.5 and 26.4 6 5.2 mmHg, respectively. A significant decline in the level of triglyceride was also observed in the olive-treated group, unlike in captopril group. Thus, it was found that OLLE at the dosage of 500 mg twice daily, was in the same way efficient in reducing SBP and DBP, as captopril given at its effective dose of 12.5
25 mg twice daily, in patients with stage-1 hypertension. Another randomized, placebo-controlled, crossover study involving patients with stable CHD was conducted to investigate the effect of two similar OOs, (VOO and refined OO), differing only in their concentration of phenolic compounds, on inflammatory markers.67 After intervention with VOO, both interleukin-6 and C-reactive protein were observed to decrease. No changes were seen in soluble intercellular and vascular adhesion molecules, glucose, as well as lipid profiles. It was, thus, implied that the intake of VOO could exert beneficial actions in patients with stable CHD as a supplementary intervention to pharmacological therapy. Similarly, in a randomized trial, Covas et al. assessed the effect of polyphenols in OO on heart disease risk factors. The researchers investigated whether OO’s phenolic content could further benefit the level of plasma lipid in comparison with monounsaturated acid content. For that purpose, the levels of plasma lipid and glucose, oxidative

Olive and olive oil Chapter | 23

damage to lipid levels including exogenous and endogenous antioxidants at baseline and before and after each intervention were measured. Participants of this study were assigned randomly to three sequences of daily administration of 25 mL of three OOs which contained low, medium, or high phenolic content (2.7, 164, and 366 mg/kg of OO, respectively), but were otherwise alike. The intervention periods were 3 weeks preceded by 2weeks washout periods. The levels of high-density lipoprotein (HDL) cholesterol were observed to increase linearly for low-, medium-, and high-polyphenol OO. A linear decrease in total cholesterol-HDL cholesterol ratio with respect to the phenolic content of the OOs was also obtained. Triglyceride levels were found to reduce for all OOs by an average of 0.05 mmol/L. Oxidative stress markers decreased linearly with increasing phenolic content. In addition, reduced mean changes were recorded for the levels of oxidized LDL with increasing polyphenol content of the OOs.68 In another similar study, polyphenol-rich OO diet was found to decrease blood pressure and enhance endothelial function in women of high-normal blood pressure or stage-1 essential hypertension.69 Two diets, a polyphenolrich OO (B30 mg/day) and a polyphenol-free OO diets, were used for this randomized, double-blind, crossover dietary-intervention study with each dietary period lasting for 2 months and a 4-week washout period in between diets. The results of the study revealed only the polyphenol-rich OO diet to lead to a significant decrease in both SBP and DBP. The serum asymmetric dimethylarginine, oxidized LDL, and plasma C-reactive protein also showed decreased levels. Besides, the polyphenol-rich OO diet induced a rise in plasma nitrites/nitrates as well as hyperemic area subsequent to ischemia. A direct relationship between CVDs and LDL oxidation is well-acknowledged, and oxidation of LDL particles has been strongly associated with the risk of CHD and myocardial infarction.70 In fact, in a human study, it was established that OO polyphenols consumption could lower plasma LDL concentrations and LDL atherogenicity in healthy young men. The volunteers ingested 25 mL/day raw lowpolyphenol-content OO (LPCOO, 366 mg/kg) or highpolyphenol-content OO (HPCOO, 2.7 mg/kg) for 3 weeks. Interventions were preceded by 2-weeks washout periods. Relative to baseline, plasma apolipoprotein B-100 concentrations and the number of total and small LDL particles were found to decrease after HPCOO intervention, which was different from the LPCOO group whereby significant increases were noted. On the other hand, LDL oxidation lag time was increased from baseline after the HPCOO intervention. An increase of 26% from baseline also occurred in the expression of lipoprotein lipase gene after the HPCOO intervention, while it remained unchanged after the LPCOO intervention.71

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A randomized, double-blind, crossover human trial study undertaken by Martı´n-Pela´ez et al. demonstrated that consumption of a diet consisting of an EVOO rich in phenolic compounds could modulate the expression of some of the genes related to the renin
angiotensin
aldosterone system that could be responsible for the decrease in SBP.72 Arterial stiffness is a significant cardiovascular risk factor and can be measured using cardio-ankle vascular index (CAVI). Accordingly, Pais et al. studied the impact of a proprietary standardized olive fruit extract (SOFE) in subjects at risk for arterial stiffness. Importantly, subjects who were given 500 mg SOFE showed the greatest reduction in mean CAVI scores and the strongest response in visual analogue scale energy intensity, while a significant decrease of 21.64% was reported for the mean triglyceride levels. Hence, the findings were indicative of enhanced arterial elasticity and that increasing HDL cholesterol and lowering triglycerides with SOFE could potentially lower the risk of developing atherosclerosis.73

23.3 Conclusion This chapter has undeniably underscored the benefits of olive and OO for the cardiovascular system. Inclusion of olive and OO in the diet is certainly a wise strategy for the prophylaxis of myocardial infarction and CHD. Indeed, in vitro, in vivo and clinical studies validate that phytoconstituents in olives and OO, notably OLE and hydroxytyrosol lower the risks of atherosclerosis and thrombosis. Besides these metabolites can help to manage the levels of LDLs and triglycerides, while they boost up high LDLs, reduce arterial stiffness, and enable high blood pressure control among other valuable benefits for the heart.

Mini-dictionary of terms Arterial stiffness

Atherosclerosis

Cardio-ankle vascular index

Cardiovascular disease

A consequence of biological aging and arteriosclerosis. Inflammation plays a major role in arteriosclerosis development, and consequently it is a major contributor in large arteries stiffening. A progressive disease characterized by the deposit of lipids and fibrous elements in large arteries, which is a major cause of heart disease and stroke. a new measure of arterial stiffness that reflects the stiffness from the ascending aorta to the ankle arteries and demonstrates little dependence on blood pressure during the evaluation. a class of diseases that involves the heart or blood vessels. This includes coronary artery diseases such as angina and myocardial infarction (commonly known as a heart attack) among others.

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Coronary heart disease Doxorubicin

Extra-virgin olive oil (EVOO)

Ischemia

Randomized trial

Visual analogue scale

Involves the reduction of blood flow to the heart muscle due to build-up of plaque in the arteries of the heart. A type of chemotherapy drug called anthracycline. It slows or stops the growth of cancer cells by blocking an enzyme called topoisomerase 2. EVOO is a typical ingredient in a Mediterranean diet, and its protective role is the result of its specific composition, including high proportions of MUFA (oleic acid), a balanced presence of polyunsaturated fatty acids, and other minor components, such as α-tocopherol and phenolic compounds, which guarantee its high antioxidant potential. A restriction in blood supply to tissues, causing a shortage of oxygen that is needed for cellular metabolism (to keep tissue alive). A type of scientific (often medical) experiment that aims to reduce certain sources of bias when testing the effectiveness of new treatments; this is accomplished by randomly allocating subjects to two or more groups, treating them differently, and then comparing them with respect to a measured response. A psychometric response scale that can be used in questionnaires. It is a measurement instrument for subjective characteristics or attitudes that cannot be directly measured.

Comparisons of olive oils with other edible oils Extra-virgin olive oil (EVOO) is composed of 98%
99% triglycerides and 1%
2% minor components. The fatty acids in the triglycerides are mainly represented by monounsaturates (oleic), with a small amount of saturates (palmitic and stearic), including an adequate presence of polyunsaturates (linoleic and α-linolenic). The minor components are α-tocopherol, phenol compounds, carotenoids (β-carotene and lutein), squalene, phytosterols, and chlorophyll, along with a large number of aromatic substances.74 Fatty acid composition (saturates, monounsaturates, polyunsaturates

Omega-6, and Omega-3) vary in dietary oils. In this regard, Viola and Viola reported the fatty acid composition of several dietary oils, notably, olive, peanut, maize, soya, and sunflower oils (SOs) (Table 23.2).74 Although vital for cell structure and function, some polyunsaturated fatty acids (PUFAs) with 18 carbon atoms, namely, linoleic, 18:2 ω-6 and α-linolenic, 18:3 ω-3, referred to as “essential,” or essential fatty acids (EFAs), cannot be synthesized by the body and must be consumed preformed in foods. Moreover, it is important that the two series ω-6 and ω-3 to be present in a correct ratio in the diet since a surplus of linoleic acid can cause the inhibition of the endogenous synthesis of the long chains of α-linolenic acid (eicosapentaenoic and docosahexaenoic acids) with subsequent damage to the body. In addition, the ratio between the ω-6 and ω-3 series should not be less than 10:1, particularly during growth because the long-chain ω-3 series are essential for the development of the brain and retina as well as has other vital functions such as antiplatelet aggregation, antiinflammatory, anticancer, and protection against skin dryness. While this recommended ratio is found in OO, the same is not the case for other vegetable oils, except for linseed and soy oils. OO’s balanced acid composition gives it a high nutritional value, given its EFA content (containing a balanced ratio between the two series). With regard to EVOO extracted from fruit, it has a significant value corresponding to the antioxidant power of its minor components. Besides, VOO contains 150
200 mg/kg α-tocopherol with an optimum ratio of vitamin E/PUFA (mg of vitamin E/g of polyunsaturates). This ratio, which should never be less than 0.5, is barely ever present in seed oils, but in EVOO, it is 1.5
2. On the other hand, in seed oils, the tocopherols present are mainly of β, γ, and δ types that are scarcely utilized by the body.74 Quite a number of studies have also been executed to assess the stability of edible oils during cooking process where they are exposed to heat and high temperature. At high temperatures, oils are subjected to significant changes due to the many chemical and physical reactions taking place, such as oxidation, hydrolysis, cyclization, isomerization, and polymerization.75

TABLE 23.2 Fatty acid composition of dietary oils. Dietary oils

Saturates

Monounsaturates

Polyunsaturates Omega-6

Omega-3

Olive oil

8
14

65
83

6
15

0.2
1.5

Peanut oil

17
21

40
70

13
28




Maize oil

12
28

32
35

40
62

0.1
0.5

Soya oil

10
18

18
30

35
52

6.5
9

Sunflower oil

5
13

21
35

56
66




Olive and olive oil Chapter | 23

In the study of Guillaume et al. the chemical and physical changes during heating of 10 commercial oils, namely, the high quality EVOO, VOO, OO, canola oil, SO, avocado oil (AO), rice bran oil, coconut oil (CoO), grapeseed oil (GO), and high oleic peanut oil (PO) were evaluated. In addition, different parameters, for instance, measurement of specific absorbance coefficient, free fatty acid, fatty acid profile, polar compound, oil stability index, and smoke point were used to predict the stability of the different edible oils when heated.76 In terms of fatty acid composition, GO showed the highest value of linoleic acid content (68.4%) followed by sunflower (50.4%), rice bran (32.4%), and canola (18.2%) oils. CoO showed the least value of oleic acid (7.9%) followed by GO (19.6%). High oleic acid peanut oil, which has been softly refined, displayed alike chemical and physical features to the different OO grades. Moreover, levels of trans-fat at initial conditions after reaching 240 C and after 6 h of heating at 180 C were determined. A remarkable difference between initial trans-fat content in refined oils and non-refined oils was observed. While grapeseed showed the highest amount of initial trans-fat content, EVOO and VOO demonstrated the lowest. These results were consistent with the oil production method, as refined oils are bleached and heated during the industrial process, while virgin oils such as EVOO, VOO, and avocado oil are produced only with mechanical processes and thus maintain a naturally lower level of trans fats. The oils’ oxidative stability decrement during time of heating at 180 C was also examined. PO, OO, SO, and AO showed the lowest values after 6 h of heating. Comparatively, CoO demonstrated high stability at the end of the induction time. Furthermore, even though the changes in the composition of fatty acids of the tested oils slightly differed for each oil and type of heating, a general trend was observed; whereby palmitic, stearic, and oleic relative’s percentages increased, whereas linoleic and linolenic levels decreased during heating. In GO, for example, after 360 min of heating at 180 C, oleic acid content increased by 26.83%, while linoleic and linolenic acids decreased by 9.81% and 66.48%, respectively. In addition, although by only 0.34%, oleic content was increased, while linoleic and linolenic contents decreased by 11.15% and 22.6%, respectively, in EVOO. Interestingly, reasonable predictors of an oil performance when heated were found to be oxidative stability, secondary products of oxidation, and total level of PUFAs. EVOO has demonstrated to be the most stable oil when heated, followed closely by CoO and other virgin oils such as avocado and high oleic acid seed oils.76 In the work of Lo´pez-Beceiro and colleagues, corn, sunflower, soybean oils, as well as four monovarietal OOs (arbequina, cornicabra, hojiblanca, picual) were compared using pressure differential scanning calorimetry and the thermooxidative stability of these oils were evaluated by

287

ASTM onset oxidation temperature (OOT) method. The samples were classified in decreasing order, with respect to their stability to oxidation, as follows: olive picual, olive cornicabra, olive hojiblanca, olive arbequina, corn, soybean, and sunflower oils. Although corn and arbequina oils showed very close OOT values, the oxidizing process was much faster in the corn oil. The contents in palmitic, oleic, and linoleic acids were found to be related to the OOT and thermal stability of the different vegetable oils. Thus the OOT parameter allowed the classification of the sunflower, soybean, corn, and OOs. A link was also found between some chemical components and the OOT results. In particular, linolenic, oleic, and palmitic acids were found to influence the thermooxidative stability of the oils to a great extent.13 Furthermore, the performance of OO during intermittent frying of potato slices and cod fillets was compared with corn, olive residue, and 50/50 w/w mixture of olive and corn oils.77 Based on the physicochemical changes, OO was observed to be more stable during frying, while corn oil was the least stable. Between the two foods used, cod caused a faster deterioration in the quality of oil because of the leaching of fish oil from the cod, which is rich in PUFAs that are rapidly oxidized. As for the mixed oil (olive/corn), it was found to be the second stable oil during frying. On the other hand, the stability of both the unsaponifiable and saponifiable fractions of AO, under a drastic heating treatment, was investigated and comparison was made with that of OO.78 Avocado oil and OO were characterized and compared at time 0 h and after different times of heating process (180 C). PUFA/SFA and ω-6/ ω-3 were higher in AO than in OO during the whole experiment. AO was reported to be richer than OO in total phytosterols at time 0 and 9 h of heating. Level of TBARs (thiobarbituric acid reactive substances) was higher in OO after 3 h, reaching the maximum values in both oils at 6 h of heating. Also, in OO, vitamin E was higher (35.52 vs 24.5 mg/100 g), while in AO, it disappeared earlier (at 4 vs 5 h). Nevertheless, the stability of AO was comparable to that of OO.

Implications in human health and disease prevention Olive oil is considered the healthiest oil in the world and its health benefits are supported by several lines of research. Both conventional and phytotherapy use leaf extracts of olive oil to treat or prevent hypertension. Clinical studies demonstrated that an oral administration of 500 mg (twice a day) of olive leaf extract significantly reduced blood pressure in patients with first-degree hypertension.66 Anticancer properties of olive oil have also been reported by many literatures due to its well

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characterized set of bio compounds, namely, oleuropein, tyrosol, hydroxytyrosol, verboscoside, ligustroide, and demethyleuropein. Antioxidant and antiatherogenic activities were also confirmed by several studies.79 Previous evidences suggested that olive oil is associated in reducing amyloid-β pathology and improving cognitive function in Alzheimer’s disease (AD) mouse models. A phenolic compound, oleocanthal, was reported to exhibit beneficial effect against AD pathology. Consuming olive oil as a medical food combined with donepezil offers an effective therapeutic approach by enhancing the noncholinergic mechanisms of donepezil and by providing additional mechanisms to reduce amyloid-β-related pathology in AD patients.80 Furthermore, a clinical trial showed that olive oil significantly decreased inflammatory markers and improved intestinal symptoms which suggest olive oil as a potential complementary medicine to treat ulcerative colitis.81 Olive oil is well-known for its antiaging properties. Indeed, in a study conducted by Romana-Souza et al. the traditional olive oil displayed significant activity in attenuating stress-induced ageing signs such as thinner dermis and collagen fiber loss in ex vivo human skin by suppressing MMP-2 expression, reactive oxygen species production, and extracellular signal regulated kinases 1/2 and cJUN phosphorylation.82 The oil can be used as a daily skin care regimen alone to delay aging, skin toxicity, and reduce acute radiation dermatitis.83 Interestingly, a controlled, randomized, double-blinded clinical trial reported that topical application of olive oil can prevent pressure ulcers in elderly people.84 Incorporating dietary supplements in the epigenetic therapy of cancer has become the focal interest of many researchers. Nanda et al. concluded in their study that olive oil plays a primordial role in the prevention of colon cancer by altering nuclear factor κB and apoptotic pathways via targeting noncoding RNAs and methylation machinery which as a result affect epigenome.85 Fig. 23.5 summarized the health benefits of olive oil in the management and prevention of ailments.

References 1

2

3 4

5 6

7

8

9

10

11

12

13

14

15

16 17

FIGURE 23.5 Health benefits of olive oil.

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258. 67 Fito´ M, Cladellas M, De la Torre R, et al. Anti-inflammatory effect of virgin olive oil in stable coronary disease patients: a randomized, crossover, controlled trial. Eur J Clin Nutr. 2008;62 (4):570
574. 68 Covas M-I, Nyysso¨nen K, Poulsen HE, et al. The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med. 2006;145(5):333
341. 69 Moreno-Luna R, Mun˜oz-Hernandez R, Miranda ML, et al. Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. Am J Hypertens. 2012;25(12):1299
1304.

70 Pandey KB, Rizvi SI. Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longevity. 2009;2(5): 270
278. ´ , Remaley AT, Farra`s M, et al. Olive oil polyphenols 71 Herna´ez A decrease LDL concentrations and LDL atherogenicity in men in a randomized controlled trial. J Nutr. 2015;145(8):1692
1697. 72 Martı´n-Pela´ez S, Castan˜er O, Konstantinidou V, et al. Effect of olive oil phenolic compounds on the expression of blood pressure-related genes in healthy individuals. Eur J Nutr. 2017;56(2):663
670. 73 Pais P, Villar A, Rull S. Impact of a proprietary standardized olive fruit extract (SOFE) on cardio-ankle vascular index, visual analog scale and C-reactive protein assessments in subjects with arterial stiffness risk. Drugs R D. 2016;16(4):355
368. 74 Viola P, Viola M. Virgin olive oil as a fundamental nutritional component and skin protector. Cldermatology. 2009;27(2):159
165. 75 Zribi A, Jabeur H, Flamini G, Bouaziz M. Quality assessment of refined oil blends during repeated deep frying monitored by SPME
GC
EIMS, GC and chemometrics. Int J Food Sci Technol. 2016;51(7):1594
1603. 76 Guillaume C, De Alzaa F, Ravetti L. Evaluation of chemical and physical changes in different commercial oils during heating. Acta Sci Nutr Health. 2018;2:02
11. 77 Chatzilazarou A, Gortzi O, Lalas S, Zoidis E, Tsaknis J. Physicochemical changes of olive oil and selected vegetable oils during frying. J Food Lipids. 2006;13(1):27
35. 78 Berasategi I, Barriuso B, Ansorena D, Astiasara´n I. Stability of avocado oil during heating: comparative study to olive oil. Food Chem. 2012;132(1):439
446. 79 Gorzynik-Debicka M, Przychodzen P, Cappello F, et al. Potential health benefits of olive oil and plant polyphenols. Int J Mol Sci. 2018;19(3):686. 80 Batarseh YS, Kaddoumi A. Oleocanthal-rich extra-virgin olive oil enhances donepezil effect by reducing amyloid-β load and related toxicity in a mouse model of Alzheimer’s disease. J Nutr Biochem. 2018;55:113
123. 81 Morvaridi M, Jafarirad S, Seyedian SS, Alavinejad P, Cheraghian B. The effects of extra virgin olive oil and canola oil on inflammatory markers and gastrointestinal symptoms in patients with ulcerative colitis. Eur J Clin Nutr. 2020;74(6):891
899. 82 Romana-Souza B, Monte-Alto-Costa A. Olive oil inhibits ageing signs induced by chronic stress in ex vivo human skin via inhibition of extracellular-signal-related kinase 1/2 and c-JUN pathways. Int J Cosmet Sci. 2019;41(2):156
163. 83 Chitapanarux I, Tovanabutra N, Chiewchanvit S, et al. Emulsion of olive oil and calcium hydroxide for the prevention of radiation dermatitis in hypofractionation post-mastectomy radiotherapy: a randomized controlled trial. Breast Care. 2019;14(6):394
400. 84 Dı´az-Valenzuela A, Garcı´a-Ferna´ndez FP, Carmona Ferna´ndez P, Valle Can˜ete MJ, Pancorbo-Hidalgo PL. Effectiveness and safety of olive oil preparation for topical use in pressure ulcer prevention: multicentre, controlled, randomised, and double-blinded clinical trial. Int Wound J. 2019;16(6):1314
1322. 85 Nanda N, Mahmood S, Bhatia A, Mahmood A, Dhawan DK. Chemopreventive role of olive oil in colon carcinogenesis by targeting noncoding RNAs and methylation machinery. Int J Cancer. 2019;144(5):1180
1194.

Chapter 24

Extra-virgin olive oils storage: Effect on constituents of biological significance Vita Di Stefano Department of Biological, Chemical, and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, Palermo, Italy

Abbreviations 3,4-DHPEA 3,4-DHPEA-EA 3,4-DHPEA-EDA DAc-10-OH Ole Agly EVOO MUFA p-HPEA p-HPEA-EA p-HPEA-EDA PUFA SFA

hydroxytyrosol oleuropein aglycon oleacin deacetoxy-oleuropein aglycon extra-virgin olive oil monounsaturated fatty acids tyrosol ligstroside aglycon oleocanthal polyunsaturated fatty acids saturated fatty acids

24.1 Introduction Extra-virgin olive oil (EVOO) is a particularly complex product. The control of its production chain requires not only the knowledge of the extraction of the oil contained in the olives but also of the phenomena of transformation of the chemical and functional components of the oil. In fact, high-quality EVOOs are obtained exclusively by mechanical and physical processes. EVOO sensory characteristics and chemical composition are influenced by different factors, including olive cultivars, pedoclimatic conditions, growing techniques, harvesting time, technological extraction process, and storage conditions.1
3 EVOO is composed by a main saponifiable fraction (up to the 99%), including triacylglycerols (98%) and diacylglycerols (1%). About 1%
2% of minor components (mixture of polar, nonpolar, and amphiphilic substances) are involved in the sensory and health-promoting properties of EVOO. Tocopherols, tocotrienols, flavonoids, lignans, and phenolic compounds belong to this small fraction and play a key role in the beneficial effects on human health attributed to EVOO, enhancing also the taste quality (bitter and pungent attributes).4
7

The hydrophilic phenolic compounds, generally indicated as polyphenols, even in small quantities in EVOO, are fundamental for protecting glycerides from oxidation and inhibiting oxidation through a variety of mechanisms based on radical scavenging, hydrogen atom transfer, and metal chelating.8
11 The main classes of EVOO polyphenols are the following: secoiridoids, phenolic acids, phenylethanoids, flavonoids, hydroxy-isocromans, and lignans. The phenolic acids (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p- and o-coumaric acid, ferulic acid, and cinnamic acid) were the first group of phenolic compounds found in EVOO; these compounds, together with phenyl-alcohols (tyrosol and hydroxytyrosol), hydroxy-isochromans, and flavonoids, are present in small amounts, while secoiridoids and lignans are the most prevalent ones.12,13 Secoiridoids are found within the family of Oleaceae and they are considered the main components (50%
70%) of the phenolic fraction. In olive oils the most abundant secoiridoid derivatives are the dialdehydic forms of elenoic acid linked either to hydroxytyrosol (3,4-dihydroxy-phenyl-ethanol) in oleacin (3,4-DHPEA-EDA) or to tyrosol (p-hydroxy-phenylethanol) in oleocanthal (p-HPEA-EDA), together with 3, 4-dihydroxyphenyl-ethanol linked to elenolic in oleuropein aglycon (3,4-DHPEA-EA) and p-hydroxyphenyl-ethanol linked to elenolic acid (p-HPEA-EA) in ligstroside aglycon. Among these, some have been studied for beneficial health properties; oleacin (3,4-DHPEA-EDA) has been used as a novel drug to prevent or reduce inflammation of endothelium, plays an important protective role against reactive oxygen species-induced oxidative injury due to its 5-lipoxygenase inhibitory effect,14 and showed angiotensin-converting enzyme inhibitory activity.15 Recent study demonstrates that oleocanthal (p-HPEAEDA), as well as naturally oleocanthal-rich EVOOs,

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00029-8 © 2021 Elsevier Inc. All rights reserved.

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induced cancer cell death via lysosomal membrane permeabilization in vitro and in vivo. Li et al. reported that oleocanthal inhibited tau fibrillization16 and in 2018, Batarseh and Kaddoumi described that high-oleocanthal EVOO reduced amyloid-β load and related toxicity in a mouse model of Alzheimer’s disease.17 The phenolic compounds, furthermore, strongly affect organoleptic properties of olive oils. In this respect the bitter taste is particularly related to the aglyconic forms of secoiridoids, whereas the presence of oleocanthal and oleacin has been linked to the pungency and bitterness of EVOO, characteristic of some EVOOs.18
20

24.2 Nutritional quality alteration of extra-virgin olive oil The assurance of the stability and quality of food products is a matter of great concern for producers and sellers. Olive oils like fats and lipid-containing foods undergo alterations in their chemical composition over time, which results in sensory and nutritional degradations. Lipases (triacylglycerol acylhydrolase) are the first hydrolytic enzymes of the degradation path of stored triacylglycerols in olive fruits. With fruit milling and during the malaxation phase, lipases come into contact with the oil, lipolysis starts, and free fatty acids (FFAs) are produced. The products of the lipolytic reaction are tasteless and odorless, and no sensory defects can be perceived. The oxidation of olive oil can be triggered from the earliest stages of olive processing, but then prevails during oil storage. Being an oxidative process, to prevent it, it would be sufficient to operate in the absence of oxygen. This alteration is catalyzed by enzymes called lipoxygenase and is favored by the possible competition of some environmental factors, including exposure to light and high temperature, the presence of peroxides, contact, or the presence of certain metals (iron, copper, and nickel). Lipid oxidation occurs through the interaction of triacylglycerols with molecular oxygen. The resulting reaction produces hydroperoxides by a free radical mechanism. Traces of metals or exposure to light are key factors in the initiation phase of the process; their presence, in fact, can determine the formation of free radicals necessary for the oxidation reaction.21 The activation energy of this reaction is high and can be accelerated by an increase in temperature. The decomposition of hydroperoxides, the primary oxidation products, produces a range of volatile and nonvolatile products. Some volatile components, mainly aldehydes, are the major cause of the sensory perception of the rancid defect in vegetable oils.22

The susceptibility of triacylglycerols to oxidation increases as the unsaturation level of its fatty acids increases. In fact, FFAs have a prooxidant effect; for this reason, it is important to have low levels of them in EVOO. It was demonstrated that the oxidative stability of neutralized oil decrease in proportion to increasing percentage of free oleic acid.23 Due to the oxidative degradation of triglycerides, a consistent increase was observed in peroxide number, K232 as expression of conjugated dienes in hydroperoxides and K270 as expression of derivatives from oxidative rancidification. In contrast, phenolic compounds and carotenoids decrease autoxidation in oil, while tocopherols, chlorophylls, and phospholipids demonstrate both antioxidant and prooxidant activity depending on the oil system and storage conditions.24 Olive oils contain a relatively rich variety of carotenoids (i.e., β-carotene, lutein, violaxanthin, neoxanthin, and other xanthophylls) and chlorophyll derivatives (i.e., chlorophylls A and B, pheophytins A and B, and other minor derivatives). Their relative composition in olive oil derives from the initial pigment composition of the olive fruits and from all chemical transformations occurring at different stages of olive oil production. Carotenoids and chlorophyll derivatives play an important role in oxidative stability of olive oils, related to their antioxidant nature in the dark and prooxidant activity in the light. The phenolic compounds are subjected to an evolution that starts in the fruit and ends in the bottle. In ripe olives, oleuropein and ligstroside are present, but during malaxation the action of endogenous β-glucosidases, leads to oleuropein and ligstroside aglycones (respectively 3,4-DHPEA-EA and p-HPEAEA). In EVOO, according to literature,25,26 the aglycones give the corresponding dialdehyde forms and the latter hydrolyze into the corresponding decarboxymethyl aglycon (3,4-DHPEA-EDA and p-HPEA-EDA). According to hydrolithic mechanisms still little known, the final compounds are formed: hydroxytyrosol (3,4DHPEA), tyrosol (p-HPEA), and elenolic acid. Aglycones and other secoiridoid derivatives, such as oleacin and oleocanthal, which have an antioxidant effect, are the main phenolic compounds of EVOO and are responsible for the shelf life of virgin olive oils. During storage, they undergo qualitative and quantitative changes. In this regard, it has been suggested to follow the shelf life of the EVOO by monitoring the oleuropein aglycon content as a favorable index for stability, together with the hydroxytyrosol content as a parameter expression of oil degradation during shelf life. Tyrosol and hydroxytyrosol concentration is usually low in fresh oils but increases during oil storage due to the hydrolysis of secoiridoids.

Extra-virgin olive oils storage: Effect on constituents of biological significance Chapter | 24

24.3 Storage of olive oil To maintain the phenol and volatile molecule content responsible for the highly appreciated organoleptic and nutritional properties in newly produced virgin olive oil during storage, it is essential to control all the factors that promote lipid oxidation. EVOO generally has a relatively long shelf life of approximately 16
18 months.27 This time frame for shelf life is appropriate as olive fruit used for olive oil production is harvested annually. Similar to other products that are produced in a limited period, but that are consumed throughout the year, EVOO must be stored. It is necessary to care for each step of the production process and of the factors that can affect its commercial life of olive oils. Some major factors influencing oxidative degradation of EVOO are the oxygen availability, the packaging materials, the storage temperature, exposure to light, and the fatty acid composition of EVOO (Fig. 24.1).

293

Frozen storage results in the best physicochemical parameters and antioxidant content (α-tocopherol and total phenols) and oxidative stability after 12 months of storage. As regards the organoleptic parameters, freezing preserved the best fruitiness and good levels of bitterness and pungency. Generally, low temperature storage not only can prevent oxidation processes from occurring, but they can even be usefully used to block them.34 Mulinacci et al.35 compared the effect of a long storage period on EVOO at 223 C with the same specimens at room temperature in dark conditions. The phenolic composition and aromatic profile were monitored during 18 months and a decrease of phenolic composition was observed for oils stored at room temperature starting from 3 months of storage. Conversely, the frozen oils showed negligible differences in aromatic profile until 12 months of storage. Unfortunately, storing oil at low temperatures is not always possible; ideal range for storage, however, stands between 10 C and 15 C.36,37

24.3.1 Storage temperature Numerous studies have been conducted on the effects of storage temperature on the quality of olive oil. Some authors have studied the shelf life of olive oil stored at high temperatures and have highlighted a sharp decline in olive oil quality indicators [peroxide value (PV), free acid value, and total phenols content].28
33 Other studies included low temperature in storage olive oil. Quality parameters (percentage of oleic acid, PV, K232, K270) in olive oil stored at low temperatures (24 C and 218 C) and in optimal conditions (15 C) appear to be closer to those found in fresh oil samples. Oxygen availability Storage temperature

Free fay acids

Pigments:

Light exposure

carotenoids and chlorophylls

Unsaturaon degree of fay acids

Tocopherols

Metal traces

Polyphenols

FIGURE 24.1 External factors that can influence storage oils.

24.3.2 Light and oxygen exposure EVOO must be protected from direct and indirect light. Small doses of UV radiations promote lipid oxidation in the presence of air. In the absence of air, however, direct sunlight causes a decrease in PV of the oil. In particular, in a study on several olive oil
packaging typologies, oil oxidation proceeded slowly in darkness, faster in diffused light, and even faster in direct sunlight. Prevention of light exposure during storage of virgin oil is absolutely necessary to extend shelf life, in fact in oils exposed to light, after only 2 months, lower phenols, tocopherol, carotenoid, and chlorophyll contents were found.38 In view of the statement, packaging techniques ensuring more effective protection from light should be recommended together with bottling procedures that do not jeopardize the quality of the oil. An enemy of EVOO is contact with oxygen. The great effect that oxygen availability has on the oxidation reaction rate is directly related to its partial pressure. Some technological manipulation such as centrifugation, decanting, and filtration can determine difference in the level of oxygen in EVOOs. The diffusion of oxygen into oil plays an important role on oxidation rate. A study of changes in total polar phenols, α-tocopherols, β-carotene, lutein, chlorophyll, and squalene contents during autoxidation indicated that most interactions may be suppressed if oxygen availability is limited.39 An effective solution to improve the stability of EVOO is to saturate the head space of storage container with inert gases, mainly nitrogen or argon. Both gases are extremely stable, odorless, and tasteless and reduce dissolved oxygen in oil.

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The uses of inert gases have been used in “tank blanketing” and nitrogen is applied to protect the oils.40 In the case of bottled or tank-stored olive oil, in which the surface
volume ratio in contact with the atmosphere is relatively small, the diffusion of oxygen into the bulk oil is a limiting parameter, and therefore the oxidation rate is controlled by diffusion.41 The technique involves the introduction of inert gas (taken from special cylinders suitable for food use) inside the tank and its discharge into the atmosphere through vent valves, thus maintaining an internal atmosphere always inert and under pressure constant. The products, thus, protected preserve their qualitative and organoleptic specificities.42 The influence of storage conditions and packaging materials (PET, HDPE, tin can, glass, and tetra brick) on olive oil quality has been considered in many papers.43
46 The ideal containers must repair the oil from the air, light, and high temperatures, without releasing unwanted substances, capable of altering the aromas and chemical composition of the product.47,48 Compared to glass, stainless steel containers have the advantage of totally protecting from light, as well as boasting much higher impact resistance, which translates into greater maneuverability. The storage silos used by the manufacturers are also built with this material, the best for large capacity containers. On a mechanical and practical level the tin has properties similar to steel, although being lighter and more prone to dents. It is a material that is hygienic and impenetrable to light—not surprisingly used for the classic 5- and 10-L containers of oil—but which in the long run can suffer contact with air. Glass represents a good barrier against moisture and gases, but transparent bottles cannot protect the olive oil from photooxidation. For this reason, glass containing specific additive to significantly reduce transmittance of light in the UV range have been developed.49 Traces of heavy metals in olive oils are known to have an effect on the rate of oxidation. Transition metals in olive oil may originate from the soil and fertilizers, or from contamination during processing and storage. In particular, metals such as iron and copper in EVOO can decompose hydrogen peroxide and lipid hydroperoxides into lipid peroxyl and alkoxyl radicals through a redox cycling pathway.24 Bendini et al.50 measured the oxidation products in EVOOs stored in presence and absence of copper. Drastic increases of free radicals were found in samples with metal traces. The results clearly demonstrate the ability of copper to promote autoxidation.

24.3.3 Fatty acids and polyphenols content The oxidative stability of olive oil with respect to other vegetable oils is mainly due to its fatty acid composition,

to the high monounsaturated fatty acids (MUFA)/polyunsaturated fatty acids (PUFA) ratio in particular, and to the presence of minor compounds (i.e., polyphenols, carotenoids) that play a main role in preventing oxidation.51 The expression of phenolic compounds in olive fruit is predominately driven by genetic factors, and large differences exist between olive cultivars.52 During storage the olive oil chemical composition is influenced mainly by balance between oxidative degradation and antioxidant activity due to the presence of both tocopherols and phenolic compounds. Fatty acid evolution reported as the sum of the saturated fatty acids (SFA), MUFA, and PUFA did not demonstrate a specific correlation with storage duration. These findings indicated their marginal involvement in the evolution of the quality of EVOOs, literature data shown the evolution of the polyphenol content of the EVOOs, after real-time storage monovarietal EVOOs in dark glass bottles, and at room temperature.26 Quality parameters (FFA, PV, K232, K270) after 18 months met all of the regulatory limits.53 The free acidity level increased from initial value of 0.30%
0.42% to a maximum value of 0.55%, but this increase is well below the limit of 0.8%. A significant increase in peroxide index was observed, but still below 20 mequiv of O2/kg. Measurements of absorbance at specific wavelengths (K232 and K270) in the UV region were used to provide information on the oxidative state. K232 values, after storage, showed values for all oils ranging from 1.90 to 2.21 without exceeding the limit (2.5) defined by the European regulations. Initial values of K270 were about 0.06
0.11 and the final values at the end of the experiment were 0.12
0.19 and then lower than their threshold value. Regarding acrylic composition, studied through GC
MS of FAMEs, the data showed a slow decrease during storage, without important differences between samples, in particular oleic acid ranging between 52.01% and 69.81% and linoleic acid ranging between 8.21% and 12.12%. Marked reduction of the most abundant secoiridoid derivatives corresponding to p-HPEA-EDA (oleocanthal), 3,4-DHPEA-EDA (oleacin), p-HPEA-EA (ligstroside aglycon), and DAc-10-OH Ole Agly (deacetoxy-10-hydroxy oleuropein aglycon) indicating a more active participation in the oxidative processes. The overall change in p-HPEA-EDA (oleocanthal) concentration between the different oils was about 10%
15% after storage. A similar pattern of degradation was noted for 3,4-DHPEA-EDA (oleacin), p-HPEA-EA (ligstroside aglycon), and DAc-10-OH Ole Agly (deacetoxy-oleuropein aglycon) between 20% and 27%.26 Contrary to the phenolics discussed thus far, an increase in hydroxytyrosol (10%
80%) was found after 18 months storage in oils of all cultivars. It is well established that

Extra-virgin olive oils storage: Effect on constituents of biological significance Chapter | 24

secoiridoids during aging of olive oil are transformed into hydrophilic substances such as hydroxytyrosol and tyrosol.26 Esposto et al.54 evaluated the autoxidation stability of several EVOOs with different phenol compounds content, after 22 months of storage. EVOOs with low (20
200 mg/ kg), medium (450
700), and high polyphenolic levels (750
1440 mg/kg) were studied. The authors found high correlations among PVs, K232, K270, and storage time. Compared to other legal parameters usually measured, the K232 spectrophotometric index demonstrated a higher positive correlation with the oxidative status of the EVOOs, which highlighted its potential use as an effective and cheap measure to monitor the legal quality evolution of such products during storage in dark conditions. This study also confirmed that the higher the initial quantity of polyphenols with radical scavenging properties, the longer duration of EVOO quality stability. Specifically, a higher initial concentration of these substances at the beginning of storage was associated with a lower loss until the end of the simulated shelf life experiment. During oil storage, secoiridoids undergo modifications (decomposition such as hydrolysis and oxidation reactions) that result in their decline and consequently to reduced intensity of the typical bitter taste and pungent note.55 Based on these studies, it can be assumed that the shelf life of an EVOO can be monitored by cheap and easy analyses. The valuation of initial antioxidant composition and particularly its polyphenol fraction can predict potential shelf life of EVOOs under determinate conditions (i.e., room temperature, dark conditions, and opportune packaging).

deterioration is a large economic concern in the food industry because it affects many quality characteristics such as off-flavors (rancidity), color, and the nutritive value. Some major factors that influence lipid oxidation in EVOOs are the oxygen availability, the packaging material, the storage temperature, exposure to light, and fatty acid composition. Oxidation is accelerated by the presence of FFAs and trace of metals. In contrast, decrease autoxidation in EVOOs tocopherols, chlorophylls, and polyphenols compounds. All quality parameters required by IOC (peroxide index, ultraviolet absorbance, and oleic acid percentage) have not undergone significant changes during storage. However, it is recognized that the oxidation process during storage involves modifications to the main and secondary components. Particularly important is the significance of the dialdehyde form compounds of decarboxymethyl oleoeuropein aglycon (3,4-DHPEA-EDA) and hydroxytyrosol (3,4-DHPEA) as indicators of the hydrolytic degradation of phenolic compounds: the first in inverse relation to degradation and the second directly related to degradation.

Highlights G

G

G

24.3.4 Tocoferols Vitamin E comprises tocopherols along with tocotrienols. The fat-soluble α-tocopherol, the analog having the highest biological activity, is the predominant representative of Vitamin E in virgin olive oil. In the initial stage of autoxidation a slightly prooxidant effect of α-tocopherol was observed. In the presence of phenolic antioxidants naturally occurring in olive oil, such as ortho-diphenols, α-tocopherol give rise to significant additional antioxidant effect.56 These results suggested that a loss of the principal antioxidants in EVOOs was always evident, but at different levels according to the initial concentration of the antioxidants. Specifically, a higher initial concentration of these substances at the beginning was associated with a lower loss during storage.

24.4 Conclusion Lipid oxidation is one of the major causes of quality deterioration in natural and processed foods. Oxidative

295

G

G

G

G

Extra-virgin olive oil (EVOO) has a relatively shelf life of approximately 16
18 months. Storage conditions are very important for the maintenance of nutrition quality of EVOOs. Fatty acid evolution in EVOOs, reported as the sum of the saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) did not demonstrate a specific correlation with storage. Change in MUFA and PUFA content after storage indicate their marginal involvement in the evolution of the quality of EVOOs. Beneficial effects of EVOO are linked to the minor components. EVOOs richest in secoiridoids derivatives showed a significant resistance to loss of oleuropein derivatives during storage. Oxygen availability, packaging material, the storage temperature, the exposure to light, and the fatty acid composition are important parameters for shelf life of EVOOs.

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1186. 25. Migliorini M, Cherubini C, Cecchi L, Zanoni B. Degradation of phenolic compounds during extra virgin olive oil shelf-life. Riv Ital delle Sostanze Grasse. 2013;90:71
80. 26. Di Stefano V, Melilli MG. Effect of storage on quality parameters and phenolic content of Italian extra-virgin olive oils. Nat Product Res. 2020;34(1):78
86. 27. Morello JR, Motilva MJ, Tovar MJ, Romero MP. Changes in commercial virgin olive oil (cv Arbequina) during storage, with special emphasis on the phenolic fraction. Food Chem. 2004;85:357
364. 28. Krichene D, Allalout A, Mancebo-Campos V, Salvador MD, Zarrouk M, Fregapane G. Stability of virgin olive oil and behaviour of its natural antioxidants under medium temperature accelerated storage conditions. Food Chem. 2010;121:171
177. 29. Farhoosh R, Hoseini-Yazdi SZ. Shelf-life prediction of olive oils using empirical models developed at low and high temperatures. Food Chem. 2013;141:557
565. 30. Velasco J, Dobarganes C. Oxidative stability of virgin olive oil. Eur J Lipid Sci Technol. 2002;104:661
676. 31. Garcı´a JM, Gutie´rrez F, Castellano JM, Perdiguero S, Morilla A, Albi MA. Influence of storage temperature on fruit ripening and olive oil quality. J Agric Food Chem. 1966;44:264
267. 32. Kalua CM, Bedgood DR, Bishop AG, Prenzler PD. Discrimination of storage conditions and freshness in virgin olive oil. J Agric Food Chem. 2006;54:7144
7151. 33. Go´mez-Alonso S, Mancebo-Campos V, Desamparados Salvador M, Fregapane G. Oxidation kinetics in olive oil triacylglycerols under accelerated shelf-life testing (25
75 C). Eur J Lipid Sci Technol. 2004;106(6):369
375. 34. Sa´nchez Gimeno AC, Abenoza M. Effect of low-temperature storage under optimal conditions on olive oil quality and its nutritional parameters. Riv Ital Delle Sostanze Grasse. 2015;XCII:243
251. 35. Mulinacci N, Ieri F, Ignesti G, et al. The freezing process helps to preserve the quality of extra virgin olive oil over time: a case study up to 18 months. Food Res Int. 2013;54(2):2008
2015. 36. Aparicio-Ruiz R, Aparicio R, Garcia-Gonzalez DL. Does “best before” date embody extra-virgin olive oil freshness? J Agric Food Chem. 2014;62(3):554
556. 37. Krichene D, Salvador MD, Fregapane G. Stability of virgin olive oil phenolic compounds during long-term storage (18 months)

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at temperatures of 5
50 C. J Agric Food Chem. 2015;63 (30):6779
6786. Caponio F, Bilancia MT, Pasqualone A, Sikorska E, Gomes T. Influence of the exposure to light on extra virgin olive oil quality during storage. Eur Food Res Technol. 2005;221:92
98. Blekas G, Psomiadou E, Tsimidou M, Boskou D. On the importance of total polar phenols to monitor the stability of Greek virgin olive oil. Eur J Lipid Sci Technol. 2002;104(6):340
346. Amirante P, Clodoveo ML, Dugo G, et al. The use of an inert gas during the olive oil bottling: shelf life evaluation. Ital J Food Sci. 2006;18(5):209
214. Yanishlieva-Maslarova NV. Inhibiting oxidation. In: Pokorny J, Yanishlieva N, Gordon M, eds. Antioxidants in Food. Cambridge: Woodhead Publishing Ltd; 2001:22. Garcı´a JM, Yousfi K. The postharvest of mill olives. Grasas Aceites. 2006;57(1):16
24. Cecchi T, De Marco C, Passamonti P, Pucciarelli F. Analytical definition of the quality of extra-virgin olive oil stored in polyethylene terephthalate bottles. J Food Lipids. 2006;13(3):251
258. Mendez AI, Falque E. Effect of storage time and container type on the quality of extra-virgin olive oil. Food Control. 2007;18 (5):521
529. Pristouri G, Badeka A, Kontominas MG. Effect of packaging material headspace, oxygen and light transmission, temperature and storage time on quality characteristics of extra virgin olive oil. Food Control. 2010;21(4):412
418. Samaniego-Sanchez C, Oliveras-Lopez MJ, Quesada-Granados JJ, Villalon-Mir M, Serrana HLG. Alterations in picual extra virgin olive oils under different storage conditions. Eur J Lipid Sci Technol. 2012;114(2):194
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47. Dabbou S, Gharbi I, Dabbou S, Brahmi F, Nakbi A, Hammami M. Impact of packaging material and storage time on olive oil quality. Afr J Biotechnol. 2011;10(74):16937
16947. 48. Tsimis DA, Karakasides NG. How the choice of container affects olive oil quality
a review. Packag Technol Sci. 2002;15(3):147
154. 49. Limbo S, Peri C, Piergiovanni L. Extra-virgin olive oil packaging. The Extra-Virgin Olive Oil Handbook. London: Wiley-Blackwell; 2014:179
199. 50. Bendini A, Cerretani L, Vecchi S, Carrasco-Pancorbo A, Lercker G. Protective effects of extra virgin olive oil phenolics on oxidative stability in the presence or absence of copper ions. J Agric Food Chem. 2006;54(13):4880
4887. 51. Bendini A, Cerretani L, Salvador M, Fregapane G, Lercker G. Stability of the sensory quality of virgin olive oil during storage: an overview. Ital Food Beverage Technol. 2010;LX:5
18. 52. Vinha AF, Ferreres F, Silva BM, et al. Phenolic profiles of Portuguese olive fruits (Olea europaea L.): influences of cultivar and geographical origin. Food Chem. 2005;89:561
568. 53. Reg. EC 640/2008, Reg. EEC 2015/1830, COI/T.15/NC No 3/Rev. 13 June 2019. 54. Esposto S, Selvaggini R, Taticchi A, Veneziani G, Sordini B, Servili M. Quality evolution of extra-virgin olive oils according to their chemical composition during 22 months of storage under dark conditions. Food Chem. 2020;311(1):126044. 55. Esti M, Contini M, Moneta E, Sinesio F. Phenolic compounds and temporal perception of bitterness and pungency in extra-virgin olive oils: changes occurring throughout storage. Food Chem. 2009;113(4):1095
1100. 56. Blekas G, Tsimidou M, Boskou D. Contribution of α-tocopherol to olive oil stability. Food Chem. 1995;52(3):289
294.

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Section 2.3

Oxidative stress

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Chapter 25

Antioxidants in olive oil phenolics: a focus on myoblasts Paraskevi Kouka, Aristidis S. Veskoukis and Demetrios Kouretas Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece

Abbreviations CAT HT Nrf2 OxS GSH RNS ROS SOD T

catalase hydroxytyrosol nuclear factor (erythroid-derived-2)-like 2 oxidative stress reduced form of glutathione reactive nitrogen species reactive oxygen species superoxide dismutase tyrosol

25.1 Introduction In the last few decades, polyphenols have attracted scientific attention due to their distinct biological roles. Numerous studies have reported their anticancer, antioxidant, antiatherogenic, antidiabetic, antiallergic, and antiinflammatory roles in animal and human health.1
3 Among the plant kingdom, olive tree parts, and especially olive oil (OO), play a prominent role in Mediterranean diet for centuries since it is associated with improvement of health status.4 The abovementioned important properties of polyphenols present in OO are mainly attributed to their potent antioxidant activity helping blood and tissues overcome the detrimental effects caused by free radicals and reactive species in general naturally produced in the organisms.5
7 When free radicals supersede a concentration threshold with the concomitant inability of tissue defense system to neutralize them, they cause detrimental oxidative modifications to biomolecules such as DNA, proteins, and lipids.8 As a result, many pathologies emerge, with cancer, diabetes, obesity, neurodegenerative, myogenic, and cardiovascular diseases being among them.9 Concerning all tissues in human body, muscles undergo the production of large amounts of reactive species, especially during exercise, a modality that alters

blood and tissue redox status and disrupts redox signaling.10 Moreover, overproduction of reactive species in muscle cells seems to be the main cause for many muscular pathological conditions, such as Duchenne muscular dystrophy, collagen VI
related muscular dystrophies, and RYR1-related myopathies.11 Considering the beneficial biological roles of OO polyphenols, they could putatively be used as agents of therapeutic interventions in order to alleviate some of the symptoms of the aforementioned pathological conditions.12 Based on the previous, in this chapter we try to holistically approach the biological role of OO polyphenols and their impact to the maintenance of muscle redox homeostasis.

25.2 Natural antioxidants: focus on olive oil constituents and their biological properties OO derives from the fruits of Olea europaea L., a tree that thrives in the Mediterranean basin, the countries of which account for more than 95% of the world OO production, estimated at approximately 2,000,000 t annually.13 Olives and OO have been essential constituents of the Mediterranean diet for more than 6000 years and they retain their economic and cultural significance until today.14 OO contains natural antioxidants such as vitamin E and polyphenols that act as natural preservatives delaying the onset of oxidation and rancidity and thus, increasing its life duration. In addition, these antioxidants have been associated with beneficial health effects.15 In recent years, many epidemiological and biological studies have reported that consumption of extra-virgin OO (EVOO) is associated with reduced oxidative damage, improved cardiovascular health, lower cancer prevalence, and healthier aging,16
19 especially on populations with increased risk factors, namely, smoking, hypertension,

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00009-2 © 2021 Elsevier Inc. All rights reserved.

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elevated LDL levels, overweight, obesity.20,21 These health benefits have been attributed to the chemical composition of EVOO, which however is largely affected by varying cultivar types, processing methods, and environmental factors.19,22,23 The main OO components are fatty acid triglycerides with the most prevalent being monounsaturated omega-9 fatty acids (oleic acid ranging between 55% and 83%), while linoleic and linolenic acids account for 3.5%
21% and 1%, respectively. In addition, there are more than 200 minor bioactive components in OO,24 namely, tocopherols, which prevent lipid oxidation in cellular membrane25 and hydrophilic or hydrophobic polyphenols, including phenolic acids and derivatives (e.g., vanillic and gallic acid), alcohols [e.g., tyrosol (T), hydroxytyrosol (HT)], secoiridoids (e.g., oleuropein, oleocanthal), lignans (e.g., pinoresinol), and flavones (e.g., luteolin).15 Those phenolic compounds are potent bioactive molecules displaying antioxidant, antiinflammatory, antimicrobial, antiviral, antiatherogenic, antithrombotic, antimutagenic, and hypoglycemic properties.26
28 The molecules with the most potent biological properties are HT, T, oleuropein, and oleocanthal.29
32 Their mechanism of action remains largely unknown but it has been scarcely reported that T and HT act as direct reactive oxygen species (ROS) scavengers,33
35 while oleocanthal is beneficial against several inflammatory diseases.36,37

25.3 Myoblasts and satellite cells: an overview It has been approximately 60 years since the identification of a type of cells residing on the surface of skeletal muscle fibers, termed “satellite cells.”38,39 Mauro and colleagues carried out electron microscopy experiments in the tibialis anticus muscle of frogs and noticed a cell type in between the muscle fibers and the surrounding membrane.38 They hypothesized that it could be a quiescent myoblast potentially with the ability to repair muscle tissue when it is required. These cells were named satellite cells due to their anatomical location. In parallel, Katz and colleagues also reported a cell-type residing in a similar location on muscle spindles. Today, satellite cells are considered as the components of myoblasts that contribute to muscle growth, homeostasis, and repair.40 The main cell types of skeletal muscles are myofibers, which are cylindrical, multinucleated cells responsible for muscle contraction. They are established during embryogenesis upon fusion of mononuclear myoblasts into immature myofibers, termed myotubes.41 It is noteworthy that myofiber nuclei are postmitotic and unable to reenter proliferation under physiological conditions. Therefore during postnatal life, the growth, homeostasis, and repair of myofibers rely solely on satellite cells.41 Typically,

satellite cells are quiescent and can be stimulated by muscle injury signals. Their functionality is required throughout life; however, it has been reported that the physiological procedure of aging decreases their number.41 Satellite cell properties render them a very intriguing research target and a very promising experimental practice is their cloning and generation of permanent muscle cell lines, able to provide myogenic cells.40 The first author to report the establishment of a permanent myogenic cell line was Yaffe back in 1968, who used newborn rats.42 Later on, Yaffe and Saxel isolated a control cell line (C2) from injured thigh muscle of 2-month-old C3H mice, which probably derived from satellite cells.43 In addition, C2 cells were recloned by Blau and colleagues leading to the production of the C2C12 cell line.44 Hauschka and colleagues displayed that single isolated cells from adult skeletal muscle could be expanded to muscle colonies following administration of the proper medium.45

25.4 Reactive species, the antioxidant defense system and redox homeostasis Reactive species are labile by nature, thus, their effects are largely influenced by multiple factors, such as the local environment and their concentration complicating the task to adopt a concentration threshold that defines their roles as physiological signaling molecules or detrimental agents.46,47 The primary free radical that is mainly produced in mitochondria is the superoxide anion radical (O2 2), which can be sequentially converted into “secondary” ROS, either directly or through enzyme/metal-catalyzed processes.48 For instance, O2 2 can be rapidly converted into hydrogen peroxide (H2O2) by superoxide dismutases (SODs), which, in turn, may undergo conversion to the hydroxyl radical (HO ) via the Fenton reaction in the presence of bivalent iron or monovalent copper.48
50 Alternatively, O2 2 may react with nitric oxide (NO ) to generate peroxynitrite (ONOO
). In order to maintain reactive species at physiological, nondeleterious levels, skeletal muscle and myogenic cells possess a variety of enzymatic [e.g., SOD, catalase (CAT)] and nonenzymatic antioxidant defenses.51 Acute exercise as a modality that alters skeletal muscle redox status and disrupts redox equilibrium may increase SOD activity specifically in the soleus muscle, which is characterized by a high mitochondrial density.52 On the contrary, SOD1-null mutations are associated with elevated oxidative stress (OxS) and accelerated age-dependent atrophy in skeletal muscle.53 Moreover, H2O2 can be decomposed by a variety of enzymes such as CAT or glutathione peroxidases (GPxs). Apart from enzymes, the antioxidant network includes nonenzymatic antioxidants such as vitamin A, C, G

G

G

G

G

Antioxidants in olive oil phenolics: a focus on myoblasts Chapter | 25

and E, biliverdin, and its derivate bilirubin, as well as GSH.51,54 The cellular responses to an electrophile stimulus are basically regulated by molecular mechanisms since they are mediated by at least four key transcription factors, nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), nuclear factor (erythroid-derived 2)-like 2-related factor (known as NFE2L2 or NRF2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α).51,54,55 All the previous mechanisms contribute to the development of redox homeostasis in muscle. According to the definition of homeostasis, it has been reported that it is the ability of a living system to maintain equilibrium.10 The most common examples of homeostasis include but are not limited to temperature regulation and the balance between acidity and alkalinity. The maintenance of homeostatic processes is a prerequisite for the normal function of organisms, while any disturbance is associated with various diseases.10 Redox homeostasis refers to the balance between oxidants [ROS or reactive nitrogen species (RNS)] and antioxidant mechanisms, which is finely regulated. Interestingly, low or moderate reactive species levels are essential for physiological signaling processes.10 However, when the production of oxidants exceeds the ability of the antioxidants to face them then a condition known as OxS is emerging and leads to the disruption of redox signaling.56

25.5 Oxidative
reductive stress and acute exercise Acute exercise has been extensively related with reactive species generation and OxS in an intensity-dependent manner due to various mechanisms,57,58 altering the muscle redox homeostasis.59 It is established that muscle damage after exercise recruits the relocation of inflammatory cells from the blood stream into the muscle fibers.60 Besides, ROS and cytokines produced by the inflammatory cells, when present in particular levels, can act as signaling molecules to facilitate muscle repair process following an exercise protocol.61 Consequently, inflammation or even moderate OxS is thought to be helpful in muscle healing.62 The overproduction of ROS during exercise has been established in the literature in the past 40 years, since it was first reported in 1978.63 In this study, increased levels of expired pentane, a marker of lipid oxidation, were observed after a 60-min cycle ergometer exercise at 50% VO2 max.64 In the following years, various research groups have examined the effects of several types of exercise on redox biomarkers, reporting elevated oxidation and depleted antioxidant molecules, such as GSH.64
67 While all these studies have assessed OxS by measuring the change in blood parameters, other studies have

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examined muscle biopsies as well.66 Interestingly, reactive species generated by exercise may have both beneficial and deleterious effects.64 ROS production can deteriorate muscle fiber integrity by damaging cellular macromolecules; however, increasing amounts of evidence suggest that moderate ROS concentration acts as stimuli on transcription factors that drive the expression of key antioxidant defense-related enzymes. This effect could be exploited by nutritional interventions (e.g., with antioxidants) aiming at increasing exercise performance, while simultaneously diminishing the muscle oxidative damage.68 The occurrence of reductive stress during exercise is an obscure event that has not been extensively studied; however, recent studies display its existence.59,69,70 Therefore until a decade ago, the idea in the field of redox biology that OxS and reactive species are damaging for normal tissue function is now disputed. On the one hand, it has been demonstrated that postexercise reductive stress is associated with beneficial effects.59,70,71 On the other, the low concentration of reactive species observed during reductive stress can disrupt redox homeostasis of eukaryotic cells, regarding the reduced and oxidized glutathione ratio.72 In addition, a recent study reported the diminished adaptation process of muscles in a reductive rather than oxidative state.73 According to the earlier, glutathione is situated at the spotlight as it is considered a major regulator of the cellular redox environment.74 Mitochondria are the basic source of free radicals in the cell; however, other ROS/RNS sources include NADPH oxidases, lipoxygenases, and xanthine oxidase (XO).54 Skeletal muscle, one of the largest tissues in the human body, is characterized by high energy requirements and excessive oxygen consumption to sustain their metabolic profile as well as their architecture, which is fine-tuned in order to facilitate contraction and force generation.75 Therefore muscle cells constantly produce moderate levels of ROS and RNS. During exercise, reactive species production is excessive and may overcome the intrinsic buffering ability of antioxidant mechanisms, leading to OxS.76
81 Interestingly, contrary to early reports, the rate of ROS production by mitochondria during exercise has been probably overestimated.55 Reactive species are involved in the modulation of muscle cell growth regulation, proliferation or differentiation,55,82,83 contractile performance during exercise,84,85 calcium signaling,86 glucose uptake,87 and mitochondrial biogenesis.55 Nevertheless, excessive reactive species levels are detrimental for muscle DNA, contractile proteins, and mitochondrial phospholipids. A common feature of early-onset myopathies is preand postnatal muscle weakness, suggesting a potential dysregulation in myogenesis. OxS seems to negatively affect myogenesis, although experimental data remains

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controversial. ROS deplete the intracellular GSH pool leading to NF-κB activation, thus, contributing to a reduction in MyoD expression.88 However, it remains obscure whether this reduction is a direct effect of ROS or a mere consequence of cell suffering.54 In the last years a common pathogenetic mechanism has emerged in many muscular pathological conditions (e.g., Duchenne muscular dystrophy, collagen VI
related muscular dystrophies, and RYR1-related myopathies), as they all have been associated with a disruption of redox homeostasis, pointing out to a potential therapeutic target.10

25.6 Olive extracts (mixtures) of bioactive compounds and their effects on myoblasts The potential bioactivity of OO polyphenols has been the focus of research with important findings, such as that some of the main polyphenolic compounds found in olive mill wastewater that are by-products of OO production (e.g., HT, T, and caffeic acid) act protectively against lipid peroxidation individually.89 However, recent studies have focused on the potential effects of polyphenolic mixtures, since an increasing amount of evidence implies a synergistic mode of action that surpasses the individual compound potential.90 In agreement with the aforementioned evidence, Kouka et al. have shown that different OO biophenolic extracts as well as olive floral extracts,91 with diverse biophenolic composition, exhibit strong antioxidant and antimutagenic activities in a variety of in vitro molecular tests.91 Specifically, improved redox status was evident in a C2C12 cell line upon administration of these extracts in terms of increased GSH levels in a dose-depended manner, in addition to lower levels of lipid and protein oxidation biomarkers, indicating a distinct antioxidant effect of OO biophenols.23,92,93 A noteworthy observation was that above a certain extract concentration threshold, the lipid and protein oxidation indices were increased indicating a prooxidant mode of action.92,93 Accordingly, OO biophenols demonstrated a dual effect driven by their concentration being antioxidant at lower and prooxidant at higher concentrations. This hypothesis was reinforced by the gradual increase and then decrease in GSH levels, as the concentration of OO extracts rises. In a study where olive mill wastes [i.e., olive pomace and OO mill wastewater (PEOP)] were utilized, it was shown that chronic kidney disease symptoms such as inflammation, OxS, and increased catabolism were diminished.94 PEOP contains a large percentage (90%) of antioxidant phenolic compounds, which implies that they could be potential therapeutic agents in patients with chronic kidney disease.94 Despite their well-established

antioxidant profile, polyphenolic compounds have also been found to display prooxidant action. For instance, a high dose of oleuropein was cytotoxic in a prostate cancer cell line by stimulating ROS production and, thus, OxS.95 Moreover, oleuropein’s and HT’s iron- and copperreducing activities may mediate their prooxidant effect, since it is known that these reduced metals may, in turn, catalyze the production of HO via Fenton reactions. In conclusion, the antioxidant/prooxidant effect of dietary polyphenols is influenced by a variety of factors, such as their levels and structure.96 Apart from their direct activities, polyphenols may also indirectly alter gene expression levels by interacting with certain transcription factors. For example, administration of HT in human retinal pigment epithelial cells enhanced GSH synthesis, the depression of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2), a transcription factor and master regulator of the antioxidant defense system.97
100 This particular transcription factor drives, among other genes, the expression of γ-glutamyl-cysteine ligase, the rate-limiting enzyme in the GSH biosynthesis pathway.101 Moreover, HT mediated the activation of Nrf2 via the NK-p62/SQSTM1 pathway.97 In various in vitro molecular tests, HT was a more potent antioxidant agent compared to T; however, this was not observed upon administration in myoblasts.23 According to the literature, both these OO compounds may activate the Keap1/Nrf2 pathway, thus improving redox status.7,102
104 Metabolically, T is a precursor of HT and it has been found that in J774 A.1 cells (mouse BALB/c monocyte macrophage), T accumulates intracellularly, thus exhibiting significant protective and antioxidant effects.105 On the contrary, HT intracellular levels were diminished rapidly.105 Furthermore, the maximal effect of HT following administration to Jurkat cells (human T lymphocytes) was observed after 5 min of incubation, followed by a gradual decrease over time up to 2 h suggesting a metabolic inactivation of HT.106 Therefore cellular metabolism may significantly affect the activity of polyphenolic extracts. In another study, HT acetate prevented tert-butyl hydroperoxide oxidative damage in the mitochondria of C2C12 cell line. According to the literature, several muscle disorders are caused by mitochondrial dysfunctions, such as muscle wasting, muscle atrophy, and degeneration.107 Oleuropein, another key OO polyphenol, enhanced insulin sensitivity in skeletal muscle by promoting the translocation of GLUT4 and the same effect was observed in the gastrocnemius muscle of mice fed with a high-fat diet.108 Taken together, these results indicate the potential of oleuropein as a promising treatment for type 2 diabetes.108 Similar potential therapeutic properties have been displayed by HT in skeletal muscle cells, adipocytes, hepatocytes, and pancreatic cells.109 Regarding skeletal G

Antioxidants in olive oil phenolics: a focus on myoblasts Chapter | 25

muscle cells in particular, apart from the increase of glucose uptake, HT enhances mitochondrial biogenesis, thus increasing the redox capacity and muscular health.110
112 In addition, a biologically relevant dose (10 μM) of oleuropein aglycone (3,4-DHPEA-EA) and oleuropein aglycone peracetylated [3,4-DHPEA-EA(P)] exerted beneficial effects on C2C12 cells treated with H2O2. Both compounds exerted cytoprotective ability against H2O2induced cell death, preventing the ROS-mediated degenerative process by functioning as efficient antioxidants.113

25.7 In vivo effects of olive oil rich in biophenols in muscle redox regulation In vivo studies carried out in piglets,89 broiler chickens,114 and lambs115 have demonstrated that the administration of feed supplemented with oil mill wastewater leads to lower levels of protein and lipid oxidation both in the blood and tissues and especially in the quadriceps muscle, a result with potential economic benefits. Moreover, OO’s biophenols from olive mill wastewater have been shown to increase GSH concentration also in human blood.116 Moreover, when administered in mice muscles, HT causes a drop in OxS biomarkers (nitric oxide and malondialdehyde) and an increase in the activity of antioxidant enzymes, including SOD, CAT, GSH,

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and GPx.111,117 In an interesting study where a range of HT doses (20
300 mg/kg/day) were administered in rats performing an exercise protocol for 10 weeks, the lowest dose decreased the running capacity, while the highest dose was able to maintain and even increase the running capacity. This effect might be attributed to a systemic prooxidant effect induced upon high HT dose administration during exercise training.118

25.8 Polyphenols and athletic performance As mentioned earlier, exercise has been linked to enhanced ROS production,119 and in turn to oxidative injury, muscle fatigue, and impaired exercise performance.120,121 Apart from the higher metabolic rate, oxygen consumption, and mitochondrial activity, ROS may be produced via macrophage infiltration, purine catabolism, and the activity of XO. Therefore it is essential that athletes develop a high antioxidant buffering capacity to avoid excessive muscle damage. Hypothetically, an antioxidant-rich dietary supplementation could prevent these negative effects from exercise bouts and to this end, several studies have been carried out testing different types of antioxidants with inconclusive results120 (Fig. 25.1). Typical treatment generally included vitamins

FIGURE 25.1 The biological roles of ROS/RNS on muscle redox homeostasis. RNS, Reactive nitrogen species; ROS, reactive oxygen species.

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FIGURE 25.2 The biological roles of polyphenolic compounds present in olive oil. It has been established that they exert antioxidant and antigenotoxic roles in vitro. However, their role in vivo as putative antioxidant agents is dual depending on the administered concentration and the time point of supplementation.

A, C, and/or E, at various dosages, time lengths, and combinations.122 In one case, 500 mg of vitamin C reduced exercise-induced lipid peroxidation and muscle damage in an untrained group of male athletes compared to the placebo group.12,122 Interestingly, numerous studies have reported negative effects upon antioxidant supplementation hypothesizing that the results varied due to differences in the analyzed population in each case.123 A turning point was the finding that exercise-induced ROS production can be beneficial. For instance, moderate exercise was defined as an antioxidant on the grounds that the mild burst of ROS acts as a stimuli for the activation of signaling pathways that lead to increased expression levels of antioxidant enzymes in humans124; therefore antioxidant supplementation could diminish these beneficial effects. Over the past few decades, a lot of scientific effort has been put on the endeavor to improve athletic performance through nutrition; however, the problem of whether or not athletes should use antioxidant supplements remains an important and highly debated topic to date (Fig. 25.2). Polyphenol supplementation outcome is currently controversial and the differences in study design among research groups (such as different exercise protocols, different training, and health history in subjects, as well as the use of various laboratory parameters to demonstrate these effects) further complicate the assessment of polyphenol effect on physical activity.123 Despite the differences, a common finding is that individuals with higher baseline levels of OxS can clearly benefit more from an antioxidant treatment, therefore, rendering an initial screening of a person’s redox state essential. Finally, potential interindividual differences regarding specific genetic variants in key enzymes for reactive species detoxification may be another important parameter that requires consideration.123,125

25.9 Conclusion Over the last decades, scientists try to improve athletic performance and also the outcome of myopathies through nutritional interventions that are rich in polyphenolic compounds, which have been established in the relevant literature as strong antioxidants. However, whether or not athletes or patients should use antioxidant supplements and whether polyphenolic administration is beneficial or detrimental on muscle repair remain important issues that are highly debated. The current knowledge shows that since the role of reactive species is dual (i.e., they are harmful or not depending on their concentration), the role of antioxidants and, thus, polyphenols is also twofold. Therefore novel sophisticated experimental approaches could offer insight in the aforementioned scientific queries.

Mini-dictionary of terms Free radicals

Reactive oxygen species (ROS) Oxidative stress (OxS)

Reductive stress

As a free radical can be called every molecular species capable of independent existence with an unpaired electron in an atomic orbital. ROS are oxygen-containing chemically reactive species. OxS describes a condition where the steadystate ROS concentration transiently or chronically supersedes a concentration threshold with the concomitant inability of tissue defense system to neutralize them, disturbing cellular redox signalling and its regulation and causing detrimental oxidative modifications to biomolecules such as DNA, proteins, and lipids. Reductive stress is the counterpart of OxS and defined as an abnormal increase of

Antioxidants in olive oil phenolics: a focus on myoblasts Chapter | 25

Glutathione (GSH)

Antioxidants

Polyphenols

Virgin olive oil

Myoblasts

reducing equivalents (GSH, NADH, NADPH) or overexpression of antioxidant enzymatic systems. GSH is the most abundant thiol in mammalian cells with antioxidant capacity. It works as glutathione peroxidases obligate cosubstrate leading to H2O2 elimination. GSH, in turn, is oxidized to GSSG. The recovering of GSH from GSSG is catalyzed by GR using NADPH as an electron donor. Moreover, GSH participates to cellular redox signaling (e.g., protein S-glutathionylation), regulation of cell proliferation and cell death, and detoxification of xenobiotics and their metabolites. An antioxidant can be defined as a molecule capable of inhibiting oxidation of other molecules through donating an electron to a free radical or other, more sophisticated mechanisms. Thus, an antioxidant delays or inhibits cellular damage. Antioxidants are produced during normal metabolism in the living organisms (e.g., GSH, uric acid) or they are obtained through diet. Polyphenols are an abundant class of nonvolatile secondary plant metabolites, characterized by the existence of one or more hydroxyl groups attached to an aromatic ring. Polyphenols have well-documented health benefits. Virgin OOs are the oils obtained from olives (Olea europaea L.) exclusively by mechanical or other physical means under conditions, mainly thermal conditions, that do not cause modifications in the oil, and which have not undergone any treatment other than washing, decantation, centrifugation, and filtration. Myoblasts are the embryonic precursors of muscle cells (myocytes). Myoblasts distinguish into muscle cells through myogenesis, where the myoblasts integrate into multinucleated myotubes, which later turn into the muscle fibers.

Comparisons of olive oils with other edible oils OO as the main culinary fat around the Mediterranean basin possesses a characteristic composition that consists of primarily MUFA, where the oleic acid embraces the 72%
79%. In contrast with PUFA, MUFAs are less vulnerable to oxidation providing an augmented availability of antioxidants in the dynamic form and better stability of OO to oxidation modifications.126 Moreover, OO consists of a minor portion of micronutrients named polyphenols,

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such as tyrosol, hydroxytyrosol, and oleocanthal, in a range of 250
500 mg/kg. Polyphenols are responsible for the astringency and bitter taste of oil and the health benefits attributed to OO consumption.126 Fats through diet play a crucial role into the organism due to their ability to improve the absorption of vitamins A, D, E, and K. Moreover, they are vital for brain and nerve utility. For these reasons the food industry utilizes the use of additional edible oils such as sunflower seed oil, fish oil, palm oil, and red palm oil, which lacks polyphenols126 and also coconut oil, sesame, camellia, walnut, soybean, and corn oil. These oils lack a great amount of polyphenols and MUFAs compared with OO and can release potentially harmful compounds when heated. Researchers have linked these compounds with various forms of cancer, Alzheimer’s and Parkinson’s disease, even though a lot of studies indicate that it may contribute somehow to human health. More specific, sesame oil127,128 consumption seems to improve lipid and triglyceride profile in human blood and also has antiinflammatory and antioxidant properties. Corn oil improves the plasma lipoprotein lipid profile in men and women with elevated cholesterol.129 Sunflower seed, fish, palm, and red palm oils have more favorable properties on cardiovascular health than harmful properties, if added to the diet in suitable quantities.126 Walnut oil improves lipid profile in type 2 diabetic patients130 and also had antitumor and antimetastatic effects in esophageal adenocarcinoma cells,131 while camellia oil displays hepatic and gastrointestinal ulcers protection against oxidative damage.132 A healthy, high-quality diet needs dietary fats. Oils deliver useful fatty acids. The different oils have different fatty acid profiles. Selecting oils with a higher portion of unsaturated fatty acids may offer greatest health benefits, although the different cooking practices may alter the beneficial properties of oils. EVOO has confirmed to be the most stable oil when heated, followed by coconut oil.133

Implications for human health and disease prevention Regularly consumption of OO according to Mediterranean diet pattern has been attributed to various positive health outcomes. Nevertheless, the last decade epidemiological investigations have established its protective character against chronic diseases, regarding the relationship among OO and mortality, CVD, diabetes, metabolic syndrome, obesity, and cancer, according to current cohort studies and dietary intervention trials.134,135 More specifically, OO consumption is associated with increased longevity, partially due to the OO’s cardioprotective role. There are indications on the benefits of OO

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for preventing several CVD risk factors, including diabetes, metabolic syndrome, and obesity.136,137 OO is also associated in preventing certain cancers, with favorable outcomes for breast and digestive tract cancers.138 These beneficial effects are documented by mechanistic evidence indicating that specific OO constituents possess antihypertensive, antithrombotic, antioxidant, antiinflammatory, and anticarcinogenic effects.134,139

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545. Available from: https://doi.org/10.1016/j.numecd.2006.03.005. Nousis L, Doulias P-T, Aligiannis N, et al. DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide. Free Radic Res. 2005;39(7):787
795. Available from: https://doi.org/10.1080/10715760500045806. Wang X, Li H, Zheng A, et al. Mitochondrial dysfunctionassociated OPA1 cleavage contributes to muscle degeneration: preventative effect of hydroxytyrosol acetate. Cell Death Dis. 2014;5. Available from: https://doi.org/10.1038/cddis.2014.473. Fujiwara Y, Tsukahara C, Ikeda N, et al. Oleuropein improves insulin resistance in skeletal muscle by promoting the translocation of GLUT4. J Clin Biochem Nutr. 2017;61. Available from: https:// doi.org/10.3164/jcbn.16-120. Vlavcheski F, Young M, Tsiani E. Antidiabetic effects of hydroxytyrosol: in vitro and in vivo evidence. Antioxidants (Basel, Switzerland). 2019;8(6). Available from: https://doi.org/10.3390/ antiox8060188. Friedel A, Raederstorff D, Roos F, Toepfer C, Wertz K. Hydroxytyrosol Benefits Muscle Differentiation and Muscle Contraction and Relaxation. Patent Application 13550972; 2013. Burattini S, Salucci S, Baldassarri V, et al. Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate. Food Chem Toxicol. 2013;55. Available from: https://doi.org/10.1016/j.fct.2012.12.049. Drira R, Sakamoto K. Modulation of adipogenesis, lipolysis and glucose consumption in 3T3-L1 adipocytes and C2C12 myotubes by hydroxytyrosol acetate: a comparative study. Biochem Biophys Res Commun. 2013;440. Available from: https://doi.org/10.1016/j. bbrc.2013.09.106. Nardi M, Bonacci S, Cariati L, et al. Synthesis and antioxidant evaluation of lipophilic oleuropein aglycone derivatives. Food Funct. 2017;8(12):4684
4692. Available from: https://doi.org/ 10.1039/c7fo01105a. Gerasopoulos K, Stagos D, Kokkas S, et al. Feed supplemented with byproducts from olive oil mill wastewater processing increases antioxidant capacity in broiler chickens. Food Chem Toxicol. 2015;82:42
49. Available from: https://doi.org/10.1016/j. fct.2015.04.021. Makri S, Kafantaris I, Savva S, et al. Novel feed including olive oil mill wastewater bioactive compounds enhanced the redox status of lambs. Vivo. 2018;32(2):291
302. Available from: https:// doi.org/10.21873/invivo.11237. Visioli F, Wolfram R, Richard D, Abdullah MICB, Crea R. Olive phenolics increase glutathione levels in healthy volunteers. J Agric Food Chem. 2009;57(5):1793
1796. Available from: https://doi. org/10.1021/jf8034429.

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117 Cao K, Xu J, Zou X, et al. Hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice. Free Radic Biol Med. 2014;67. Available from: https://doi.org/10.1016/j.freeradbiomed.2013.11.029. 118 Al Fazazi S, Casuso RA, Arago´n-Vela J, Casals C, Huertas JR. Effects of hydroxytyrosol dose on the redox status of exercised rats: the role of hydroxytyrosol in exercise performance. J Int Soc Sports Nutr. 2018;15. Available from: https://doi.org/10.1186/ s12970-018-0221-3. 119 Halliwell B, Gutteridge J. Free radicals in biology and medicine. Oxford University Press, New York; 2015. 120 Bast A, Haenen G. Nutritional antioxidants: it is time to categorise. Antioxidants in Sport Nutrition. Taylor & Francis; 2014. 10.1201/b17442-3. 121 Reid MB, Haack KE, Franchek KM, Valberg PA, Kobzik L, West MS. Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol. 1992;73. Available from: https://doi.org/10.1152/jappl.1992.73.5.1797. 122 Wagner K-H. Antioxidants in sport nutrition: all the same effectiveness? Antioxidants in Sport Nutrition. CRC Press/Taylor & Francis; 2014. 10.1201/b17442-5. 123 D’Angelo S. Polyphenols and athletic performance: a review on human data. Plant Physiological Aspects of Phenolic Compounds. IntechOpen; 2019. 10.5772/intechopen.85031. 124 Gomez-Cabrera MC, Domenech E, Romagnoli M, et al. Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance. Am J Clin Nutr. 2008;87. Available from: https://doi. org/10.1093/ajcn/87.1.142. 125 Spanidis Y, Veskoukis AS, Papanikolaou C, et al. Exercise-induced reductive stress is a protective mechanism against oxidative stress in peripheral blood mononuclear cells. Oxid Med Cell Longev. 2018;2018. Available from: https://doi.org/10.1155/2018/3053704. 126 Bester D, Esterhuyse AJ, Truter EJ, Van Rooyen J. Cardiovascular effects of edible oils: a comparison between four popular edible oils. Nutr Res Rev. 2010;23. Available from: https://doi.org/ 10.1017/S0954422410000223. 127 Namayandeh SM, Kaseb F, Lesan S. Olive and sesame oil effect on lipid profile in hypercholesterolemic patients, which better? Int J Prev Med. 2013;4. 128 Hsu E, Parthasarathy S. Anti-inflammatory and antioxidant effects of sesame oil on atherosclerosis: a descriptive literature review. Cureus. 2017;9. Available from: https://doi.org/10.7759/ cureus.1438.

129 Maki KC, Lawless AL, Kelley KM, Kaden VN, Geiger CJ, Dicklin MR. Corn oil improves the plasma lipoprotein lipid profile compared with extra-virgin olive oil consumption in men and women with elevated cholesterol: results from a randomized controlled feeding trial. J Clin Lipidol. 2015;9. Available from: https://doi.org/10.1016/j.jacl.2014.10.006. 130 Zibaeenezhad MJ, Farhadi P, Attar A, Mosleh A, Amirmoezi F, Azimi A. Effects of walnut oil on lipid profiles in hyperlipidemic type 2 diabetic patients: a randomized, double-blind, placebocontrolled trial. Nutr Diabetes. 2017;7. Available from: https://doi. org/10.1038/nutd.2017.8. 131 Batirel S, Yilmaz AM, Sahin A, Perakakis N, Kartal Ozer N, Mantzoros CS. Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells. Clin Nutr. 2018;37. Available from: https://doi.org/10.1016/j.clnu.2017.10.016. 132 Cheng YT, Lu CC, Yen GC. Beneficial effects of camellia oil (Camellia oleifera Abel.) on hepatoprotective and gastroprotective activities. J Nutr Sci Vitaminol (Tokyo). 2015;61. Available from: https://doi.org/10.3177/jnsv.61.S100. 133 Alzaa DF. Evaluation of chemical and physical changes in different commercial oils during heating. Acta Sci Nutr Health. 2018;. 134 Buckland G, Gonzalez CA. The role of olive oil in disease prevention: a focus on the recent epidemiological evidence from cohort studies and dietary intervention trials. Br J Nutr. 2015;113. Available from: https://doi.org/10.1017/S0007114514003936. 135 Caramia G. Virgin olive oil. From legend to scientific knowledge of the nutraceutical aspects. Pediatr Med Chir. 2006;28. 136 Ruiz-Canela M, Martı´nez-Gonza´lez MA. Olive oil in the primary prevention of cardiovascular disease. Maturitas. 2011;68. Available from: https://doi.org/10.1016/j.maturitas.2010.12.002. 137 Perez-Martinez P, Garcia-Rios A, Delgado-Lista J, Perez-Jimenez F, Lopez-Miranda J. Mediterranean diet rich in olive oil and obesity, metabolic syndrome and diabetes mellitus. Curr Pharm Des. 2011;17. Available from: https://doi.org/10.2174/ 138161211795428948. 138 Visioli F, Grande S, Bogani P, Galli C. The role of antioxidants in the Mediterranean diets: focus on cancer. Eur J Cancer Prev. 2004;13. Available from: https://doi.org/10.1097/01.cej. 0000137513.71845.f6. 139 Bergouignan A, Momken I, Schoeller DA, Simon C, Blanc S. Metabolic fate of saturated and monounsaturated dietary fats: the Mediterranean diet revisited from epidemiological evidence to cellular mechanisms. Prog Lipid Res. 2009;48. Available from: https://doi.org/10.1016/j.plipres.2009.02.004.

Chapter 26

Antioxidant activity in olive oils Gamze Guclu1, Hasim Kelebek2 and Serkan Selli1 1

Department of Food Engineering, Faculty of Agriculture, Cukurova University, Adana, Turkey, 2Department of Food Engineering, Faculty of

Engineering, Adana Alparslan Turkes Science and Technology University, Adana, Turkey

Abbreviations 3,4-DHPEA ABTS CHD CVDs DPPH EVOO HPLC LDL MUFAs p-HPEA p-HPEAEDA PUFAs ROS TEAC VOO

hydroxytyrosol 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) coronary heart disease cardiovascular diseases 1,1-diphenyl-2-picrylhydrazyl extra-virgin olive oil high performance liquid chromatography low-density lipoproteins monounsaturated fatty acids tyrosol oleocanthal polyunsaturated fatty acids reactive oxygen species Trolox equivalent antioxidant capacity virgin olive oil

26.1 Introduction The essential ingredient of the Mediterranean diet is olive oil and its cultivation and consumption go back to the ancient ages.1 The components of the Mediterranean diet can differ according to cultures, yet olive oil is the common one presumably due to this geographical area constituting approximately 90% of world’s olive oil production.2 As it is well known, Spain, Italy, Greece, Tunisia, and Turkey are the major producer countries; however, the cultivation of this vegetable oil is not limited to only these countries and has been expanding all over the world.3 This essential product is highly appreciated for its unique nutritional, health, and sensory properties. Recently, people’s expectation from their diet includes not only maintaining life but also supporting physical and mental health by consuming functional foods. As the trend of consuming functional foods increases daily, the search for health-beneficial ingredients have come into

prominence. In this manner, virgin olive oil (VOO) is a versatile product fulfilling all the conditions and, as it is reported to have many favorable effects on human health, is attributed as an important functional food.4 Many kinds of research are investigating the health effects of olive oil. To sum these up, olive oil can be attributed to the reduction of cardiovascular mortality, blood pressure, inflammation, oxidative damage, hemostasis, and a reduced risk of neurodegenerative diseases and cancer.2,5
10 Extensive knowledge is available that provides evidence of the benefits of olive oil consumption on cardiovascular diseases (CVDs) and oxidative degradation, which has mainly resulted from the different antioxidant compounds of the product. The prevention of CVDs and certain types of cancer by olive oil consumption is related to its significant constituents. Olive oils are mainly composed of fatty acids which include, particularly oleic acid, and some minor constituents with bioactive properties such as phenolic compounds, tocopherols, carotenoids, and sterols.11 Among the fatty acids the high content of monounsaturated fatty acids (MUFAs) differentiates olive oil from other edible vegetable oils. All of these bioactive components make olive oil a valuable functional food having a high level of antioxidants and beneficial health effects and also a being product of significant economic and social importance in the Mediterranean region. Antioxidant activity can be defined as a limitation or inhibition of nutrient oxidation (especially lipids and proteins) by restraining oxidative chain reactions. Compounds displaying antioxidant activity have become highly important as they have an effective role in the inhibition of oxidative stress
related diseases when they are consumed with foods. The imbalance between free radicals (singlet and reactive oxygen, hydroxyl, and superoxide radicals) and antioxidants causes the formation of oxidative stress. As a result of this stress formation, diseases such as cancer, Alzheimer’s and Parkinson’s diseases, CVDs, and diabetes may occur in the long term.12 Due to these disease-prevention effects,

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functional foods, including bioactive compounds, have attracted attention from all platforms. Olive oil, as mentioned before, includes a wide variety of antioxidant compounds, and the concentrations of these compounds are relatively higher than in other edible vegetable oils. This mainly is due to the absence of the refining process that has a detrimental effect on these components and is obligatory in other vegetable oils. These significant compounds in VOO have been investigated intensely because of their actions as strong antioxidants and radical scavengers.13 Thus in this chapter, bioactive compounds and their functionality in olive oil are reported and discussed.

26.2 Natural antioxidants found in olive oil Antioxidants are defined as the compounds that can decelerate or prevent the oxidation of foods containing fatty substances and oils in particular, by halting the initialization of autoxidation reactions.14 With this information, antioxidant-rich foods received remarkable attention as agents that can act against oxidative degeneration. Olive oil is a particularly important product not only because of its nutritional value but also as the minor compounds in its content are mainly responsible for the beneficial health aspects. The composition has fatty acids comprising nearly 98% of all content, with the minor components forming the remaining 2%. These valuable components are the reason olive oil is attributed as the healthiest among all vegetable oils. The common edible vegetable oils are produced from seeds (such as sunflower, rapeseed, and peanut) yet olive oil is obtained from the olive fruit. Thus these minor compounds are claimed to be found in relatively higher amounts in olive oil due to the physical and chemical processes applied in the refining procedure for seed oils. Minimal processing allows these compounds to be able to remain in even greater numbers in extra-VOO (EVOO).15,16 The unsaponifiable fraction comprises phenolic compounds, tocopherols, phytosterols, hydrocarbons, and pigments in which phenolics were reported to domain 27% of the minor compounds.17 All these compounds display effective antioxidant activity and health-beneficial properties. Each group of components is explained broadly in the following subsections.

26.2.1 Phenolic compounds Phenolic compounds are the most widely dispersed secondary metabolites in the plant kingdom mainly synthesized from the shikimic acid and phenylpropanoid pathways.18 These compounds are defined as substances with benzene rings to which one or more hydroxyl (
OH) groups are attached.19 Olive oil is a rich source of

phenolics, and increasing evidence presents the rising value of phenolic compounds due to the related potent antiinflammatory, antimicrobial, and antioxidant aspects that support highly the health benefits of olive oil
dominant nutrition.20 Phenolic compounds as food ingredients are again of importance in many aspects such as having different biological activities that have positive effects on human health, affecting taste and odor formation, participating in the color formation and change, displaying antimicrobial and antioxidant effects, causing enzyme inhibition, and being used as purity control criteria in some foods.21 Apart from their nutritional benefits, they mainly contribute to the bitter taste and astringency of the olive fruit and allow olives to be consumed without some form of processing such as fermentation or brining.22 A large part of the phenolic compounds, found mostly in the fruit pulp, passes through to the olive oil during processing. Numerous studies have already revealed that more than 30 phenolics have been determined in olive oil with a high variation in the range of composition (0.02 g21 g/kg).20,23
27 The distribution of these structurally distinct compounds varies extremely between olive oil samples, and the present evidence shows that they are dependent on an array of critical production parameters. These can be specified as preharvest conditions such as olive cultivar, geographical origin, climate and irrigation, or the maturity of olives and postharvest parameters such as oil extraction, processing, and storage techniques.28
36 Olive oil phenolics can be classified according to their structural similarities as phenolic acids, phenolic alcohols, secoiridoids, hydroxy-isocromans, flavonoids, and lignans. The classification and subgroups of phenolics are displayed in detail in Fig. 26.1. Phenolic acids such as caffeic, vanillic, syringic, p-coumaric, o-coumaric, and p-hydroxybenzoic are reported as the first group of phenolics detected in olive oil,37,38 while the most abundant phenolic compounds are embodied by those in the secoiridoid group.39 Oleuropein is the most representative secoiridoid comprising up to 14% of olive dry weight followed by ligstroside, yet as these compounds are hydrophilic and their aglycone forms are generally found in olive oils.40 Throughout the oil extraction, hydrophilic secoiridoids are hydrolyzed with the activity of glucosidase enzymes resulting in the production of more lipophilic derivatives passing to the olive oil. These conditions are the reason for the difference in phenolic composition between olive fruits and oil.39 In lipophilic secoiridoids the decarboxymethyl aglycone of ligstroside, namely, oleocanthal (p-HPEA-EDA) stands out with its effect on both sensorial properties and the health benefits of olive oils. This compound is mainly responsible for the bitter taste of oil.41 Oleocanthal is known to be heat resistance; hence, it can maintain its biological activity while the other phenolics may undergo

Antioxidant activity in olive oils Chapter | 26

FIGURE 26.1 The classification and representative compounds of phenolics found in olive oils.

degradation.42 In addition, this compound was reported to inhibit oxidation of low-density lipoproteins (LDL). In the study conducted by Coni et al.,43 rabbits were fed a special formulation diet, including olive oil and oleocanthal. According to the results, the addition of olive oil increased the resilience of LDL to oxidation and decreased the levels of cholesterol. Following from oleocanthal, other abundant secoiridoids are the dialdehydic form of elenolic acid linked to hydroxytyrosol and tyrosol (shown as 3,4DHPEA-EDA and p-HPEA-EDA, respectively). Phenolic acids found in olive oil are generally the derivatives formed from benzoic and cinnamic acids. The total concentration of phenolic acids is slightly lower compared with other phenolic alcohols or flavonoids, while olives originally have higher amounts.44 Protocatechuic acid, vanillic acid, syringic acid, gallic acid as hydroxybenzoic acids and caffeic acid, p-coumaric acid, cinnamic acid, and ferulic acid as hydroxycinnamic acids are the most commonly identified phenolic acids in a wide variety of olive oils.24
27,45
47 Countless studies have reported the total concentration of these compounds in the range of 0.5
13 mg/kg of oil. These phenolic acids are of importance due to their ability to be utilized as potential indicators of geographical origin and olive cultivars.48,49 In addition, most of the phenolic acids are known to display antioxidant activity, and this activity occurs with the aid of the OH groups in their structures. It was reported that antioxidant activity increases when more

315

OH groups are found and the weaker their bond is in the structure. Phenolic acids having these conditions were reported to have a higher Trolox equivalent antioxidant capacity (TEAC) value obtained from the ABTS (2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (1,1-diphenyl-2-picrylhydrazyl) antioxidant activity methods.50,51 Following phenolic acids, flavonoids are probably the most studied phenolics as they constitute the largest group of plant-derived phenolic compounds. These compounds can be classified into two groups, namely, flavonols and flavones. This group is formed from compounds containing two benzene rings linked by a linear three-carbon chain.52 Apigenin and luteolin are the representatives of this group in olive oils (Fig. 26.2); in addition, lower amounts of diosmetin, luteolin-7-glucoside, and rutin were also quantified in several studies.24
27 Apigenin was attributed as the substrate of the hydroxylase enzyme and lead to the formation of luteolin, while diosmetin is formed as a methoxy derivative of luteolin.53 Accumulating studies reported the total amount of flavonoids in a broad array of olive oil samples from Portugal, Tunisia, Albania, and Turkey varying between 0.1 and 15.9 mg/kg.23
25,54 In recent years the biological properties of flavonoids have evidently been understood with the increasing studies in this field. According to the data present in the literature, flavonoids were reported to have a decreasing effect on the oxidation of LDL and, therefore, the related thrombosis risk. These abilities play a crucial role in CVDs. As these compounds can deactivate damaging free radicals, it is safe to emphasize them as highly effective antioxidants.11,55 In addition, the antioxidant activity of flavonoids was claimed to depend on the aromatic hydroxyl group location and the abundance of them in the structure.56 As mentioned before, phenolic acids and flavonoids are substantial in olive oil chemistry, yet the most intriguing group is phenolic alcohols. The former groups are widely found in most varieties of fruits and vegetables; however, olive oil earns its specialty from its phenolic alcohol content among vegetable oils. The major compounds of this group are represented by hydroxytyrosol

FIGURE 26.2 Chemical structures of the main flavonoids found in olive oil.

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(3,4-DHPEA) and tyrosol (p-HPEA).57 These two compounds can be found in all olive products such as table olives, olive oil, and olive leaves. It was revealed that the amounts of these compounds increase in the storage period of oils mainly caused by the degradation of secoiridoids. Especially, hydroxytyrosol is synthesized as the main product of the degradation reactions of oleuropein. The reaction takes place as follows: (1) the sugar moiety (glucopyranose) in oleuropein is first subjected to hydrolysis with the activity of β-glucosidase enzyme, (2) the aglycone form of oleuropein is obtained, (3) a final hydrolysis of oleuropein aglycone is realized and hydroxytyrosol and elenolic acid are obtained as final products. Another way is the acid hydrolysis of oleuropein that directly forms hydroxytyrosol and elenolic acid.58 The schematic representation of this transformation is given in Fig. 26.3. All these compounds are highly bioavailable and they are absorbed from olive oil independent doses which are related to their antioxidant effects.59 Tyrosol was reported to exhibit long-term efficiency in inhibiting excessive production of reactive oxygen species (ROS). Meanwhile, hydroxytyrosol displays higher radical scavenging activity possibly due to the catechol moiety in its structure. Although the activity is higher, hydroxytyrosol was reported to metabolize quickly; thus, it is effective in the short term.60
62

The working principle of hydroxytyrosol is realized in two different ways. The compound scavenges the stress-produced superoxide anion and hydroxyl groups, and on the other side, it has an activating role in signaling different pathways in cells, which results in the increase of defenses against oxidative stress. By means of all these activities, hydroxytyrosol exhibits antioxidative defenses.63,64 There is also a health claim that was approved by the European Food Safety Authority (EFSA), and this claim includes only olive oil phenolics. Briefly, the claim reports that olive oil polyphenols are the protectors of blood lipids from oxidative stress and hence olive oil should be in the daily diet. The authority also set a minimum limit and advised the consumption of at least 5 mg of hydroxytyrosol and its derivatives (e.g., tyrosol and oleuropein complex).65

26.2.2 Tocopherols Tocopherols are amphipathic compounds comprising a hydrophilic chromanol ring along with a hydrophobic side chain and are found in various combinations in plant tissues.66 This group of constituents is generally named Vitamin E that is actually subclassified into two groups of, in total, eight compounds; tocopherols and

FIGURE 26.3 The schematic representation of hydroxytyrosol formation from oleuropein.

Antioxidant activity in olive oils Chapter | 26

tocotrienols. Both tocopherols and tocotrienols have four compounds (specifically α-, β-, γ-, and δ-isomers) differing only in the position of methyl and hydrogen groups on the aromatic ring. These homologues differ in polarity properties because of their number of methyl groups, with δ-, β-, γ-, and α-tocopherols containing 1, 2, 2, and 3 methyl groups, respectively. In addition, β- and γ-tocopherols both contain two methyl groups but in different positions.67 The tocopherol homologues have different antioxidant activity in oil
water emulsions and bulk oils, which are often explained by their differences in polarity. It was elucidated that antioxidant activity of more polar δ-tocopherol was better than α-tocopherol in menhaden oil in water emulsions, while in bulk menhaden oil, α-tocopherol was a more effective antioxidant than δ-tocopherol.68 Tocopherols are considered one of the most important free radical scavenging antioxidant compounds in foods and other biological tissues. These compounds are the second most studied minor compound of olive oil primarily because of their essential role as antioxidants. Similar to the phenolics, these compounds react to and are oxidized by free radicals and ROS, thereby preventing oxidation. They particularly scavenge radicals in cellular membranes and lipoprotein structures. The reaction of tocopherol radical formation is initiated through the donation of a hydrogen atom from the hydroxyl group of tocopherols resulting in the termination of acids oxidation reactions of polyunsaturated fatty acids (PUFAs). This tocopherol radical formation is of high importance as the reaction allows tocopherol to participate in multiple peroxidation-inhibiting events. Researches concerning the occurrence and activity of tocopherols reported that one tocopherol molecule can protect up to 108 PUFAs from oxidation with this multiple-reaction ability.66,69 The studies about the antioxidant activity of tocopherols found in olive oils have augmented in the last decades. Olive oil possesses all four isomers of tocopherols with α-tocopherol being the dominant form. α-Tocopherol (Fig. 26.4) content forms approximately 90% of all tocopherols followed by β- and γ-forms. δ-Isomers are also detected yet in minor amounts in olive oil. The antioxidant effects of isomers are in the same order as their occurrence amounts, with the highest activity displayed by α-forms. The total concentration of these compounds is reported to vary within the range of 50
785 mg/kg in

FIGURE 26.4 Chemical structure of α-tocopherol.

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olive oils from Portugal, Spain, Italy, Turkey, and Greece.70
75 In addition, research has indicated the olive oil obtained from early harvest olives contain higher amounts of tocopherols.76 This fluctuation in the concentrations of compounds is derived from many variables including olive cultivars, geographical conditions, and maturation indices. Tocopherols are also investigated as authenticity markers and as a purity criterion.77,78 The separation, identification, and quantitative determination of tocopherols in oils and other sources have been sufficiently performed using either normal- or reversed-phase high performance liquid chromatography (HPLC). To summarize, the key role of tocopherols is to inhibit lipid peroxidation with the aid of their high antioxidant activities. This feature increases their importance in the prevention of CVD and cancer formation.79
81 In addition, vegetable oils provide a noteworthy part of the vitamin E requirement of humans and the absorption of tocopherols is known to be higher if the diet is rich in MUFA and PUFAs as they are lipophilic components. In this manner, olive oil offers an optimum ratio of α-tocopherol to unsaturated fatty acids which makes it a great source of vitamin E in nutrition.82 Moreover, in the study investigating the relationship between vitamin E and heart diseases, tocopherols were reported to act associatively with other antioxidants.83 Thus the existence of natural antioxidants such as phenolics and carotenes might enhance the efficiency of tocopherols. Olive oil again has an abundance of antioxidants and has become prominent amongst all vegetable oils.

26.2.3 Squalene The representative hydrocarbon in olive oil is an unsaturated 30-carbon triterpene compound, namely, squalene. The hydrocarbon fraction is comprised almost completely of squalene, and the occurrence of this compound was first reported more than five decades ago. The first determination of squalene was in shark liver oil; olive and amaranth oil followed as rich sources of this hydrocarbon.84 Squalene is an important intermediary in the production of sterols and a crucial precursor in the synthesis of phytosterols and cholesterol in all humans, animals, and plants.85 It is synthesized by the activity of the squalene synthase converting farnesyl pyrophosphate into squalene. It is also found abundantly in human skin lipids and adipose tissue. Numerous health-beneficial properties have been attributed to squalene, and the sources of this compound can be classified roughly according to commercial and dietary importance. The occurrence of squalene is reported to be related to the lipid composition of plants that indicates the differences of its content in vegetable oils. Olive oil stands out with its squalene

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content, while other oils extracted from fruits such as palm and avocado possess much lower amounts.82 Amaranth oil and the wastes of the olive oil industry are the commercial sources of squalene as the former includes 6
8 g/100 g oil, while olive oil has a mean value between 150 and 870 mg/100 g. Recently, Salvo et al.86 proposed a single step solid phase extraction procedure and reported the squalene content could be obtained in even higher amounts. The study investigated 33 different EVOO samples from Turkey, Tunisia, Spain, Portugal, Greece, the United States, Slovenia, Israel, Albania, and Italy, the squalene content varied within the range of 1448
7474 mg/kg. The rest of the vegetal sources comprised peanut oil, avocado oil, rice bran oil, and palm oil. These oils contain a far less amount, and, hence, are only used in dietary supplies.87 Squalene is of high importance as it is related to the prevention of certain cancer types. Specifically, this hydrocarbon is very resilient to the oxidation reactions and moreover functions as a quenching molecule of reactive oxygen on the surface of human skin under sunlight exposure. In addition, the consumption of squalene in the diet is reported to have a role in the regular increase of triglycerides in the blood, repressing the plasma cholesterol increase. Squalene also is attributed to a high radical scavenging activity.82 The detoxification and chemopreventive effects of squalene were also confirmed by in vivo and in vitro researches.88,89 This was also supported by the trials run over sharks having a high amount of squalene in their livers; the findings displayed a lower chance of cancer occurrence in sharks.90,91 Moreover, even if squalene is an intermediary in cholesterol synthesis in humans, daily consumption was reported not to increase cholesterol levels.91 Under the light of its importance in skin protection from tumor formation, squalene attracted remarkable attention from cosmetic industry. In addition to this feature, the compound was reported to also act in skin hydration, damaged skin repair, and aging skin rejuvenation. These properties increased the demand for squalene usage in cosmetic formulations including creams, hair, and makeup products.92

26.2.4 Pigments There are two main pigments responsible for the color of olive oils. The pigments are identified as chlorophylls and carotenoids. Chlorophylls are formed from chlorophylls a and b and their derivatives and give green color. These compounds are sensitive to environmental aspects and are reported to degrade under light exposure resulting in the bleaching of oil. In addition, maturation has a decreasing effect on these pigments. Cichelli and Pertesana93 reported that oils obtained from black olives have chlorophyll

amount 30% lower than of green. The production process of olive oils also has a degradation effect on the chlorophyll fraction with the loss of acids in the structure. This transformation may be related to the cooxidation of lipid peroxides and the lipoxygenase enzyme.94 Thus chlorophyll was reported to be detected only in freshly squeezed oils.95 The second group of chloroplast pigments comprises carotenoids which contribute to the yellowness of olive oils. Most carotenoids have 40 carbon atoms in their structures. The major carotenoids found in olive oil are β-carotene and lutein which originate in the olives and pass through to the oil. The other forms of this group are minor olive-originated xanthophylls, namely, neoxanthin, β-cryptoxanthin and violaxanthin, and other xanthophylls formed via the extraction process (luteoxanthin, neochrome, and auroxanthin).96,97 Carotenoids are of prime importance as they are known to be the precursors of vitamin A. The main compounds displaying this provitamin A property are α-, β-, and γ-carotene along with β-cryptoxanthin.98 Along with the effects on color, chlorophylls and carotenoids have a crucial role in the oxidative stability of olive oil. Interestingly, these compounds can act as antioxidants in the dark while displaying a prooxidant activity in the light.99 Carotenoids having more than nine double bonds act as quenchers of singlet oxygen. Specifically, β-carotene (Fig. 26.5) minimizes the oxidation of lipids exhibiting a filtration of light under light exposure.100 Lutein also is gaining attention in relation to eye health; specifically, it is reported to have demanded effects in the prevention of cataracts and age-related macular degeneration.101
103 In addition, carotenoids are implied to work synergistically with other antioxidants in olive oils such as phenolics and tocopherols and together they provide higher oxidative stability and more effective anticarcinogenic activity. Moreover, as a practical application, the determination of carotenoid composition can be performed as the criteria of authenticity. The common procedure to determine chlorophyll and carotene content is by the application of HPLC, while a rough total estimation of these pigments can be performed using an ultraviolet
visible spectrophotometer. The content of these pigments varies highly in olive oils depending on olive cultivar, climatic conditions, irrigation applications, and most importantly the maturation stages. The total pigment concentration was reported to be

FIGURE 26.5 Chemical display of β-carotene.

Antioxidant activity in olive oils Chapter | 26

between 5.1 and 19.4 ppm for Italian olive oils, 3.9 and 8.6 ppm for Croatian olive oils, 15.5
45.7 ppm for Spanish olive oils.104
106 Pheophytin has an abundance of total pigments with a value of 75% and concentrations between 2 and 37 mg/kg in olive oil.

26.2.5 Sterols Sterols make up a large portion of the unsaponifiable fraction in olive oils. These bioactive compounds are reported to be found in almost every vegetable oil, whereas the major compound in animals is cholesterol. Sterols are synthesized from squalene.107 The representative form of sterols in olive oils is β-sitosterol covering approximately 90% of the total sterol content. The remaining compounds are δ-5-avenasterol and campesterol with 5%
20% and 2%
4% of the total composition, respectively108 (Fig. 26.6). The European Union Commission109 has set a lower limit of 1000 ppm of total sterol concentration for all olive oils (EVOO, VOO, and refined olive oil). Thus the total sterol contents of oils are generally in the range of 1000
3000 ppm with the majority being β-sitosterol. Other sterols including stigmasterol, cholesterol, δ-7-campesterol, sitostanol, δ-7-stigmastenol, and δ-7-avenasterol are also found yet in minor amounts.110 All previously mentioned compounds have an active role in maintaining olive oil stability by the inhibition of polymerization reaction during the frying process.53 The sterol content is of prime importance as it is investigated to detect adulteration in olive oils. Each variety of olives and oils is reported to have a peculiar profile of sterols; therefore, this property can be used as a crucial parameter in the authentication. Olive oil is generally adulterated with cheaper hazelnut, sunflower, cotton, and canola oils in order to reduce expenses. In this manner the determination

FIGURE 26.6 Important phytosterols found commonly in olive oils.

319

of fraud in olive oils can be carried out by the analysis of the sterol profile. The studies concerning the detection of olive oil adulteration have recently been compiled, and researchers have developed both chromatographic and spectroscopic methods of detection.67,111,112 As mentioned earlier, the sterol profile is substantial and specific for each type of oil. Indeed, the concentrations of phytosterols also differ even within the oils obtained from the same cultivar of olives. Numerous factors are known to have an effect on sterol content similarly to other bioactive compounds in olive oil. Olive cultivar is the main factor followed by the maturity index, oil-extraction process, and storage—all parameters having an enormous effect on sterol composition.113 Phytosterols are in the spotlight as their bioactive properties have been recognized by various studies. These compounds are reported to possess antioxidant,114 antiinflammatory,115 and antibacterial116 capabilities. Furthermore, these phytosterols, precisely β-sitosterol, are proven to display chemopreventive and anticancer potential against the most common types, including breast, colon, prostate, and lung cancers.117
120 In addition, studies revealed that β-sitosterol can restrain cell viability of tumors by attacking and corrupting the structural integrity of cell microtubules. Moreover, this bioactive compound was proven to inhibit the growth of cancer cells with its ability to scavenge ROS. This ability allows β-sitosterol to also prevent metastasis by halting the migration of tumor cells.121 Phytosterols also perform a critical role in decreasing cholesterol levels in plasma and cell membranes which benefit cardiovascular health.122

26.3 Implications for human health and disease prevention Consumption of olive oil in the diet has been proven after numerous researches to have beneficial effects on human health. Especially in the last decade, accumulated data obtained from epidemiological studies confirmed that olive oil has the ability to regulate and inhibit specific chronic diseases. The schematic display of diseases affected positively by olive oil consumption is summarized in Fig. 26.7.123
128 The most investigated topic in this scheme is CVD.2,5,7,129 The defensive activity of olive oil against CVDs is mainly caused by the high ratio of MUFA/PUFA in addition to the presence of bioactive compounds.28,130 This composition is unique to olive oil, and other vegetable oils are not as rich in these bioactive compounds. Martinez-Gonzalez et al.129 conducted a comprehensive study investigating the relation of olive oil consumption, coronary heart disease (CHD), and heart stroke. More than 100,000 cases participated in the study, and according to the results, the consumption of olive oil at certain doses displayed a crucially protective effect. The

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FIGURE 26.7 Diseases regulated by the consumption of olive oil.

authors claimed that an increase in consumption of olive oil by 25 g/day reduced CHD risk by 4% and the risk of a stroke by up to 26%. In addition, these findings were supported by numerous studies within the literature, all having the common result of the cardio-protective activity of olive oil.123,131
133 Researches mainly carried out in the last decade have proven the effects of olive oil consumption on type 2 diabetes, metabolic syndrome, and obesity.134
136 These diseases are all attributed to risk factors of CVDs. The most extensive study in this topic investigated more than 10,000 diabetes cases.137 The outcome of this study indicated the relation between olive consumption and diabetes risk formation. The results implied that the chance of developing diabetes is much lower in cases with a high Mediterranean score compared with the low scores. In addition, the same conclusion was obtained in other studies investigating a broad array of patients.138
140 In another 2-year cohort research, it was found that the daily consumption of 8 g of olive oil reduced insulin resistance and improved endothelial function in 90 cases.141 Based on this increasing evidence, it is safe to conclude that the Mediterranean diet, rich in olive oil, has an improving effect on glucose metabolism and regulates the risk of diabetes. There are accumulating epidemiological studies suggesting the beneficial effects of olive oil consumption in the reduction of cancer risk. Indeed, the antioxidant activities exerted mainly by phenolic compounds in olive oil

have a reducing effect on the occurrence of cancer.23 The studies generally focused on the effects of olive oil consumption on breast and digestive system cancers. A systematic review was conducted by Psaltopoulou et al.142 investigating the association of developing the risk of cancer. The highest reduction in the risk of the disease related to olive oil consumption was observed in breast and the digestive system (stomach, colon, rectal, and pancreas) cancers. These results were supported also by researches testing patients from different countries.143
145 However, these obtained results still cannot explain the prevention mechanism of olive oil on diseases clearly.142 In addition, the anticancer effects of single phenolics extracted from olive oil, including hydroxytyrosol, tyrosol ligstroside aglycone, and oleuropein aglycone in human breast cells, were investigated. Of all the tested fractions, aglycones of oleuropein and ligstroside were reported to induce strong antitumor activities. The outcome of the study highlighted that the phenolics naturally found in olive oil had a reversible effect on breast cancer.146 In summary, all mounting epidemiological researches indicate that the consumption of an olive oil
rich diet has a clearly protective effect on CVDs clearly and has a high prevention ratio in diseases such as diabetes, breast and digestive system cancers, and inflammation diseases. As the disease-prevention mechanism of olive oil bioactive compounds cannot be determined precisely, further studies should be designed. In addition, although the

Antioxidant activity in olive oils Chapter | 26

consumption of olive oil above the required daily intake does not cause any induction of diseases, it is advised for consumers to avoid excessive eating of any kind of food.

26.4 Conclusion This chapter aimed to elucidate the contents of the main bioactive compounds (phenolics, tocopherols, squalenes, pigments, and sterols) and effect on the antioxidant capacity of olive oils. These compounds have a valuable antioxidant activity endowing olive oil with high stability during the storage period, as well as their benefits to human health and on the sensory properties of olive oil. Moreover, due to high levels of bioactive compounds particularly phenolic compounds, olive oil can be proposed as a promising edible oil in preventing the risk of cardiovascular and cancer diseases, diabetes, and inflammation cases. Therefore the consumption of olive oil, especially of EVOO, can be recommended to consumers because of the highest content of antioxidant compounds related to an antioxidant activity which have positive effects on human health.

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68. Kiritsakis A, Shahidi F. Olive oil quality and its relation to the functional bioactives and their properties. Olives and Olive Oil as Functional Foods: Bioactivity, Chemistry and Processing. Hoboken, NJ: Wiley; 2017:205. Carpentier S, Knaus M, Suh MY. Associations between lutein, zeaxanthin, and age-related macular degeneration: an overview. Crit Rev Food Sci Nutr. 2009;49:313
326. Cho E, Hankinson SE, Rosner B, Willett WC, Colditz GA. Prospective study of lutein/zeaxanthin intake and risk of age-related macular degeneration. Am J Clin Nutr. 2008;87:1837
1843. Schalch W, Cohn W, Barker FM, et al. Xanthophyll accumulation in the human retina during supplementation with lutein or zeaxanthin
the LUXEA (lutein xanthophyll eye accumulation) study. Arch Biochem Biophys. 2007;458:128
135. Roca M, Gandul-Rojas B, Gallardo-Guerrero L, Mı´nguezMosquera MI. Pigment parameters determining Spanish virgin olive oil authenticity: stability during storage. J Am Oil Chem Soc. 2003;80(12):1237
1240. Borello E, Domenici V. Determination of pigments in virgin and extra-virgin olive oils: a comparison between two near UV-Vis spectroscopic techniques. Foods. 2019;8(1):18. ˇ Saroli´ c M, Gugi´c M, Friganovi´c E, Giovanni Tuberoso CI, Jerkovi´c I. Phytochemicals and other characteristics of Croatian monovarietal extra virgin olive oils from Oblica, Lastovka and Levantinka varieties. Molecules. 2015;20:4395
4409. Goodwin TW. Biosynthesis of sterols. In: Stumpf PK, Conn EE, eds. The Biochemistry of Plants, Lipids: Structure and Function. Vol. 4. London: Academic Press; 1980:485
507. Lukic M, Lukic I, Krapac M, Sladonja B, Pilizota V. Sterols and triterpene diols in olive oil as indicators of variety and degree of ripening. Food Chem. 2013;136:251
258. European Union Commission. Regulation characteristics of olive oil and pomace oils and their analytical methods. J Eur Commun L. 1989/2003;295:57
66. Yorulmaz HO, Konuskan DB. Antioxidant activity, sterol and fatty acid compositions of Turkish olive oils as an indicator of variety and ripening degree. J Food Sci Technol. 2017;54 (12):4067
4077. Li Y, Fang T, Zhu S, Huang F, Chen Z, Wang Y. Detection of olive oil adulteration with waste cooking oil via Raman spectroscopy combined with iPLS and SiPLS. Spectrochim Acta A Mol Biomol Spectrosc. 2018;189:37
43. Meras ID, Manzano JD, Rodriguez DA, De la Pena AM. Detection and quantification of extra virgin olive oil adulteration by means of auto fluorescence excitation-emission profiles combined with multi-way classification. Talanta. 2018;178:751
762. Ferna´ndez-Cuesta A, Leo´n L, Velasco L, De la Rosa R. Changes in squalene and sterols associated with olive maturation. Food Res Int. 2013;54(2):1885
1889. Cercaci L, Passalacqua G, Poerio A, Rodriguez-Estrada MT, Lercker G. Composition of total sterols (4-desmethyl-sterols) in extra virgin olive oils obtained with different extraction technologies and their influence on the oil oxidative stability. Food Chem. 2007;102(1):66
76. Bouic PJ. Sterols and sterolins: new drugs for the immune system? Drug Discov Today. 2002;7(14):775
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Awad AB, Downie A, Fink CS, Kim U. Dietary phytosterol inhibits the growth and metastasis of MDA-MB-231 human breast cancer cells grown in SCID mice. Anticancer Res. 2000;20(2 A):821
824. Choi YH, Kong KR, Kim Y, et al. Induction of Bax and activation of caspases during β-sitosterol-mediated apoptosis in human colon cancer cells. Int J Oncol. 2003;23(6):1657
1662. Vundru SS, Kale RK, Singh RP. β-Sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells. BMC Complement Altern Med. 2013;13(1):1
9. Mendilaharsu M, De Stefani E, Deneo-Pellegrini H, Carzoglio J, Ronco A. Phytosterols and risk of lung cancer: a case-control study in Uruguay. Lung Cancer. 1998;21(1):37
45. Awad AB, Fink CS, Williams H, Kim U. In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells. Eur J Cancer Prev. 2001;10(6):507
513. Baskar AA, Ignacimuthu S, Paulraj GM, Al Numair KS. Chemopreventive potential of β-sitosterol in experimental colon cancer model-an in vitro and in vivo study. BMC Complement Altern Med. 2010;10(1):24. Choudhary PS, Tran SL. Phytosterols: perspectives in human nutrition and clinical therapy. Curr Med Chem. 2011;18(29):4557
4567. Covas MI, Konstantinidou V, Fito M. Olive oil and cardiovascular health. J Cardiovasc Pharmacol. 2009;54:477
482. Lucas L, Russell A, Keast R. Molecular mechanisms of inflammation. Anti-inflammatory benefits of virgin olive oil and the phenolic compound oleocanthal. Curr Pharm Des. 2011;17:754
768. Martin-Pelaez S, Covas MI, Fito M, Kusar A, Pravst I. Health effects of olive oil polyphenols: recent advances and possibilities for the use of health claims. Mol Nutr Food Res. 2013;57:760
771. Vitaglione P, Savarese M, Paduano A, Scalfi L, Fogliano V, Sacchi R. Healthy virgin olive oil: a matter of bitterness. Crit Rev Food Sci Nutr. 2013;55:1808
1818. Cardeno A, Sanchez-Hidalgo M, Alarcon-de-la-Lastra C. An update of olive oil phenols in inflammation and cancer: molecular mechanisms and clinical implications. Curr Med Chem. 2013;20:4758
4776. Barbaro B, Toietta G, Maggio R, et al. Effects of the olivederived polyphenol oleuropein on human health. Int J Mol Sci. 2014;15:18508
18524. Martı´nez-Gonza´lez MA, Dominguez LJ, Delgado-Rodrı´guez M. Olive oil consumption and risk of CHD and/or stroke: a metaanalysis of case-control, cohort and intervention studies. Br J Nutr. 2014;112(2):248
259. Medeiros DM, Hampton M. In: Wildman REC, ed. Olive Oil and Health Benefits Handbook of Nutraceuticals and Functional Foods. 2nd ed. Boca Raton, FL: CRC Press; 2007:297
308. Ruiz-Canela M, Martinez-Gonzalez MA. Olive oil in the primary prevention of cardiovascular disease. Maturitas. 2011;68:245
250. Sofi F, Abbate R, Gensini GF. Accruing evidence about benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis. Am J Clin Nutr. 2010;92:1189
1196. Estruch R, Ros E, Salas-Salvado J. Mediterranean diet for primary prevention of cardiovascular disease. N Engl J Med. 2013;369:676
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134. Perez-Martinez P, Garcia-Rios A, Delgado-Lista J. Mediterranean diet rich in olive oil and obesity, metabolic syndrome and diabetes mellitus. Curr Pharm Des. 2011;17:769
777. 135. Esposito K, Maiorino MI, Ceriello A. Prevention and control of type 2 diabetes by Mediterranean diet: a systematic review. Diabetes Res Clin Pract. 2010;89:97
102. 136. Buckland G, Bach A, Serra-Majem L. Obesity and the Mediterranean diet: a systematic review of observational and intervention studies. Obes Rev. 2008;9:582
593. 137. Romaguera D, Guevara M, Norat T. Mediterranean diet and type 2 diabetes risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) study: the InterAct project. Diabetes Care. 2011;34:1913
1918. 138. Martinez-Gonzalez MA, de la Fuente-Arrillaga C, Nunez-Cordoba JM. Adherence to Mediterranean diet and risk of developing diabetes: prospective cohort study. Br Med J. 2008;336:1348
1351. 139. Salas-Salvado J, Bullo M, Babio N. Reduction in the incidence of type 2-diabetes with the Mediterranean diet: results of the PREDIMED-Reus Nutrition Intervention Randomized Trial. Diabetes Care. 2010;34:14
19. 140. Salas-Salvado J, Bullo M, Estruch R. Prevention of diabetes with Mediterranean diets: a subgroup analysis of a randomized trial. Ann Intern Med. 2014;160:1
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1446. 142. Psaltopoulou T, Kosti RI, Haidopoulos D. Olive oil intake is inversely related to cancer prevalence: a systematic review and a meta-analysis of 13,800 patients and 23,340 controls in 19 observational studies. Lipids Health Dis. 2011;10(127):14. 143. Pelucchi C, Bosetti C, Negri E. Olive oil and cancer risk: an update of epidemiological findings through 2010. Curr Pharm Des. 2011;17:805
812. 144. Braga C, La Vecchia C, Franceschi S. Olive oil, other seasoning fats, and the risk of colorectal carcinoma. Cancer. 1998;82:448
453. 145. Buckland G, Travier N, Agudo A. Olive oil intake and breast cancer risk in the Mediterranean countries of the European Prospective Investigation into Cancer and Nutrition study. Int J Cancer. 2012;131:2465
2469. 146. Menendez JA, Vazquez-Martin A, Garcia-Villalba R, et al. AntiHER2 (erbB2) oncogene effects of phenolic compounds directly isolated from commercial extravirgin olive oil (EVOO). BMC Cancer. 2008;8:377
399.

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Section 2.4

Cancer and immunology

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Chapter 27

Olives and olive oil compounds active against pathogenic microorganisms Manuel Brenes, Eduardo Medina, Pedro Garcı´a, Concepcio´n Romero and Antonio de Castro Food Biotechnology Department, Instituto de la Grasa (IG-CSIC), Seville, Spain

Abbreviations EDA dialdehydic form of decarboxymethyl elenolic acid HyEDA EDA linked to hydroxytyrosol TyEDA EDA linked to tyrosol

27.1 Introduction Olive oil and olive leaf extracts have been used in folk medicine since ancient times. Romans and Greeks employed olive extracts to treat many diseases and an extract of boiled olive leaves was administered as a drink to malaria patients during the 19th century. Hence, the Mediterranean countries have cultivated the olive tree (Olea europaea L.) to produce olive oil, table olives, and olive leaf extracts for centuries. At present, both olive oil and table olives are important components of the Mediterranean diet and are largely consumed throughout the world. In addition, there are many enterprises that commercialize olive leaf extracts to treat a myriad of diseases, many of them caused by microorganisms. Features of the main food-related bacteria pathogens investigated in relation to olive antimicrobials are presented in Table 27.1. Nevertheless, it was not until the middle of the 20th century that researchers started to look for antimicrobial compounds in olive products, especially in table olives. Juven et al.1 stated that oleuropein was the inhibitory substance of lactic acid bacteria growth during the fermentation of the Spanish-style green olives. Later, Fleming et al.2 and Kubo et al.3 demonstrated that the aglycone of oleuropein was more inhibitory than oleuropein against several bacteria, including certain lactic acid bacteria. More recently, it was demonstrated that the main phenolic compound in olive brines is hydroxytyrosol, a component of the oleuropein moiety, which inhibited the growth of

Lactobacillus plantarum in a model system,4 but its presence in the brines of both olives nontreated and treated with NaOH did not lead to an explanation for the lack of lactic acid fermentation in the former olive brines. There is controversy over the antimicrobial activity of hydroxytyrosol. Fleming et al.2 did not find any inhibitory activity in this substance against several bacteria, Capasso et al.5 detected a very limited activity against Pseudomonas savastanoi and Corynebacterium michiganensis, and Obied et al.6 reported that pure hydroxytyrosol was less active against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa than a phenolic olive extract containing this substance. By contrast, Bisignano et al.7 studied the in vitro susceptibility of several human intestinal or respiratory track pathogens to hydroxytyrosol and oleuropein, and the results of this study suggested a high antimicrobial activity of hydroxytyrosol against these microorganisms. Thus this substance was proposed as useful for the treatment of intestinal or respiratory tract infections in humans. Recently, the antimicrobial activity exerted by hydroxytyrosol has been discussed by Medina-Martı´nez et al.8 and minimum inhibitory concentrations (MICs) values were calculated for several bacterial strains. In most of the cases, hydroxytyrosol showed a limited inhibition. A concentration above 1000 μg/L was needed to exert bactericidal effect, contrary to results previously obtained by other researchers. Canal et al.9 investigated the antimicrobial activity of single phenolic compounds, including tyrosol, hydroxytyrosol, oleuropein, luteolin, and apigenin, against two yeast species, Saccharomyces cerevisiae and Aureobasidium pullulans, and they found an effective antimicrobial activity with concentration higher than 200 μg/L. Both oleuropein and hydroxytyrosol have also been identified as effective against HIV viral fusion and integration,10 and the secoiridoid glucoside oleuropein has shown antiviral activity against several pathogens in vitro.11 Indeed, many

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00027-4 © 2021 Elsevier Inc. All rights reserved.

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TABLE 27.1 Main food-related bacterial pathogens which have been investigated in relation to olive antimicrobials. Species

Important traits

Salmonella enterica sv. Enteritidis

Very common food poisoning organism. Salmonellosis mortality is low, but symptoms may be severe. Poultry, eggs, and meat are the products usually involved.

Escherichia coli 0157:H7

This strain causes hemorrhagic colitis, particularly in vulnerable groups. Associated with raw or undercooked meat, improperly processed dairy products, and vegetables.

Shigella sonnei

Normally transmitted by food handlers. Shigellosis varies from asymptomatic infection to severe illness. Salads and contaminated water or food are the main sources of infection.

Yersinia enterocolitica

Gastroenteritis caused by this organism can be confused with appendicitis. Milk and milk products are implicated foods, but person-to-person transmission has also been reported.

Helicobacter pylori

Linked to a majority of peptic ulcers and to some types of gastric cancer. Its transmission of water or food is still under research.

Staphylococcus aureus

Forms heat-stable enterotoxin that causes common food poisoning worldwide. Present in recontaminated cooked meat and poultry, cream-filled pastries, milk, and other foods.

Listeria monocytogenes

Listeriosis may cause abortion in pregnant women, and mortality in immunocompromised individuals may reach 40%. Ubiquitous organism: salads, raw milk cheese, meat, and other products.

enterprises that commercialize olive leaf extracts claim antiviral activity for their products because of their content in oleuropein and elenolic acid, a component of the oleuropein moiety. The antiviral activity of synthetic calcium elenolate was extensively studied in vitro by pharmaceutical companies during the 1960s and 1970s,12 but experiments in vivo failed. More recently, olive leaf extract enterprises have defended the antiviral properties of their products on the basis of a supposed different isomeric form of elenolic acid in these natural products than that of the synthetic calcium elenolate, but a clear scientific demonstration of this statement is not available. However, researchers have been reporting antifungal and antibacterial activity in olive leaf extracts for years. Gullo´n et al.13 and Medina et al.14 found antibacterial activity in olive leaf extracts against S. aureus, E. coli, Listeria innocua, P. aeruginosa, and Salmonella enterica. Palmeri et al.15 discovered a strong bactericidal effect of olive leaf extracts against Bacillus cereus and mesophilic bacteria, and Shialy et al.16 have recently reported the in vitro inhibition of the growth of different molds belonging to Candida and Aspergillus species by these extracts. All these studies have pointed out the phenolic compounds, in particular oleuropein and its derivatives, as the active agent in the extracts. Furthermore, some reports have shown that phenolic compounds can also be active against phytopathogenic microorganisms,5,17,18 and several simple phenols were tested in vitro against fungi and bacteria. Capasso et al.5 found that the most active phenolic compound in the olive oil mill wastewaters against P. savastanoi and Clavibacter michiganensis was methylcatechol, whereas

Baidez et al.17 proposed quercetin and luteolin as the most antifungal phenolic compounds in the olive plant. Recently, Brenes et al.19 correlated the antimicrobial activity of table olive wastewaters with the presence of olive glutaraldehyde-like compounds, which showed a noticeable bactericidal effect against species of Erwinia, Clavibacter, Agrobacterium, and Pseudomonas, and antifungal activity against species of Phytophthora, Colletotrichum, Alternaria, Botrytis, and Pestalotiopsis. Therefore results are contradictory in many cases, and researchers have studied the antimicrobial activity of olives and their derived products by using commercial phenolic compounds (oleuropein, hydroxytyrosol, luteolin, quercetin, and others) because of the difficulty to isolate pure substances. Besides, some of these substances, such as oleuropein, luteolin, and quercetin, are absent or found in a very low concentration in olive oil and table olives.

27.2 Main antimicrobial compounds in olive oil In recent years, several studies have been focused on the antimicrobial activity of olive oil and the identification of its active compounds.20,21 A comparison of this activity was tested in vitro among different edible vegetable oils (Fig. 27.1). Virgin olive oil had strong bactericidal activity against a broad spectrum of microorganisms, this effect being higher in general against Gram-positive than Gram-negative bacteria. Moreover, this activity was higher in virgin olive oils, followed by that of olive oils and

Olives and olive oil compounds active against pathogenic microorganisms Chapter | 27

331

FIGURE 27.1 Comparison of the antimicrobial activity of virgin olive oil and other edible vegetable oils (sunflower, corn, rapeseed, soybean, and cotton). N0, CFU/mL inoculated; N1, CFU/mL after 1 h.

pomace olive oils, which is in accordance with their content in phenolic compounds. By contrast, this effect was not observed for other edible vegetable oils (corn, sunflower, soybean, rapeseed, and cotton). Olive oil exerted bactericidal activity in vitro against the enteric microorganisms E. coli and Clostridium perfringens but also against the beneficial bacteria Lactobacillus acidophilus and Bifidobacterium bifidum.20 Zampa et al.22 have tested in vitro the antimicrobial activity of hydroxytyrosol and an olive phenolic extract against a variety of microorganisms of the gut microbiota. Strains of Lactobacillus salivarius, Bifidobacterium adolescentis, and Bacteroides vulgatus were highly sensitive to both hydroxytyrosol and the phenolic extract, while the strains of C. perfringens, Clostridium clostridioforme, and Enterococcus faecalis showed moderate or no susceptibility to them. Hence, it seems that the consumption of olive oil could modulate the growth of intestinal microbiota, although it would depend on the amount of oil ingested and the absorption of polyphenols before they reach the colon. A strong bactericidal effect of olive oil against several foodborne pathogens such as Listeria monocytogenes, S. aureus, Yersinia sp., Salmonella Enteritidis, and Shigella sonnei20 has also been demonstrated in vitro. These findings confirmed the previous results obtained by Radford et al.23 They made egg mayonnaise with virgin olive oil and inoculated it with Salmonella Enteritidis, and the number of microorganisms reduced to an undetectable level after 48 h. Medina et al.21 have confirmed these results inoculating egg and milk mayonnaises made with different types of vegetable oils with Salmonella Enteritidis and lettuce salad with L. monocytogenes (Fig. 27.2). Consequently, these results open up the possibility of using olive oil as a food preservative to prevent the growth of foodborne pathogens or to delay the onset of food spoilage. Several food commodities have been attributed to antimicrobial activity. Among them, wine and tea are the two most reported products with bactericidal activity. Survival of Salmonella Typhimurium, S. sonnei, and E. coli in common beverages (cola, beer, milk, and wine) has been

FIGURE 27.2 Survival of Salmonella Enteritidis in egg mayonnaises and Listeria monocytogenes in lettuce salad elaborated with different oils and initially inoculated with 2 3 103 CFU/g.

evaluated,24 and wine showed the highest bactericidal effect. Many other researchers have confirmed the inactivation of bacteria in wine and its killing effect when consumed with a meal, the activity being assigned to its content in ethanol, organic acids, the polymeric phenolic fraction, simple phenols, and low pH.25,26 Likewise, catechins purified from green and black tea inhibited the growth of many bacterial species.27 Coffee extracts also exhibited strong bactericidal action against a broad spectrum of microorganisms,28 and vinegar is a wellrecognized bactericidal foodstuff because of its high content of acetic acid.29 Taking into account all these previous data, Medina et al.21 ran some experiments to compare the survival of pathogenic bacteria (S. aureus, L. monocytogenes, Salmonella Enteritidis, E. coli, S. sonnei, and Yersinia sp.) in olive oil extracts and several common beverages after 5 min of contact. The main conclusion of this work is reflected in Fig. 27.3. All tested pathogens survived in peach, pineapple and orange juices, cow’s milk, yogurt drink, coffee extracts, beer with and without alcohol, and Coca-Cola. By contrast, green and black tea showed slight bactericidal activity, followed by red and white wine. Vinegar exerted the strongest bactericidal effect, followed by the aqueous extracts of virgin olive oil. It was, therefore, confirmed that compounds in olive

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

FIGURE 27.3 Comparison of the antimicrobial activity of virgin olive oil extracts and other foodstuffs against Staphylococcus aureus, Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, Shigella sonnei, and Yersinia sp.

oil possess a strong killer effect against pathogens, which is higher than reported for many other foodstuffs such as coffee, wine, or tea. The question is “which are these bactericidal compounds?” Very few studies have tried to correlate the antimicrobial activity of olive oil with specific compounds. Radford et al.23 and Keceli and Robinson30 attributed this activity to the high acidity of the oil as well as simple phenols such as hydroxytyrosol and tyrosol. However, the main phenolic compounds in olive oil are the secoiridoid aglycons of oleuropein and ligustroside, and the lignans.31 Finally, Medina et al.20 have correlated the antimicrobial activity of olive oil with the following phenolic compounds: the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA) and tyrosol (TyEDA), and free hydroxytyrosol and tyrosol. They isolated each phenolic compound from virgin olive oil by HPLC and tested their antibacterial activity against L. monocytogenes. Each compound was tested at its concentration found in oil and at a similar constant concentration for all of them. It was confirmed that the compounds with strong bactericidal activity were TyEDA followed by HyEDA (Fig. 27.4). No significant effect was observed for hydroxytyrosol and tyrosol and the oleuropein and ligustroside aglycons. It seems that the oleosidic part of the active molecule is very important to exert the killer action. These facts were subsequently confirmed by Karaomanoglu et al.,32 who found a high antimicrobial activity for Turkish virgin olive oils, in particular with certain simple phenolic compounds, against E. coli O157: H7, L. monocytogenes, and Salmonella Enteritidis, finding no effect for refined oils. Likewise, Yakhlef et al.33 attributed the bactericidal effect of Algerian virgin olive oils to their phenolic content and especially to HyEDA and TyEDA compounds, reducing more than three logarithmic units the initial inoculum of tested pathogenic

FIGURE 27.4 Structures of the main antimicrobial compounds in olive oil and table olives.

microorganisms in only 30 min contact. Accordingly, the use of virgin olive oils in the preparation of foods might be beneficial in terms of preventing foodborne diseases that arise from pathogenic bacteria. Helicobacter pylori is responsible for most peptic ulcers and gastric cancer, and the infection is currently eradicated with antibiotics although this therapy fails in 10%
30% of patients.34 Many herbal extracts, essential oils, and foodstuffs have exhibited inhibitory activity against H. pylori in vitro35,36 although experiments in vivo failed.37 The in vitro activity of olive oil polyphenols against H. pylori has also been studied.38 These researchers have discovered a strong anti-H. pylori activity exerted by olive oil extracts rich in phenolic compounds. This activity was even effective against some antibiotic-resistant strains and, more importantly, a very low concentration of the olive oil extract was necessary. Again, the compounds that showed a stronger bactericidal activity against H. pylori were TyEDA and HyEDA. Thus it has been confirmed for many microorganisms that these two substances are responsible for most of the bactericidal activity in olive oil. Indeed, TyEDA accounts for most of the anti-H. pylori activity and exerted its bioactivity at a very low concentration in vitro (,1.5 μg/mL). TyEDA and HyEDA are complex phenolic compounds present in most virgin olive oils in concentrations up to 250 mg/kg oil that can be hydrolyzed during olive oil storage.18 The antibacterial treatment of H. pylori is difficult because of the habitat occupied by the organism below the layer of mucus adherent to the gastric mucosa. Thus it is necessary for the antibacterial compounds to diffuse from the oil to the gastric juice and be stable during digestion. Corona et al.39 have suggested that these secoiridoids are hydrolyzed at the low pH of the gastric juice, but Romero et al.38 have demonstrated under simulated gastric conditions that both HyEDA and TyEDA are

Olives and olive oil compounds active against pathogenic microorganisms Chapter | 27

stable for hours at a pH as low as 2. The latter researchers have also reported that almost half of the phenolic compounds diffused from the oil into the simulated gastric juice; the more polar the compound, the more complete the diffusion reached. Hydroxytyrosol and tyrosol totally diffused into the acidic water, whereas the diffusion yield of lignans, flavones, and oleuropein and ligustroside aglycons was lower. It was concluded that approximately half of the HyEDA and TyEDA are present in the oil diffused into the simulated gastric juice. Therefore it was demonstrated that this anti-H. pylori substances can diffuse from the oil to the gastric juice during digestion, are stable for hours in the acidic environment of the gastric juice, and could exert their bioactivity against the microorganism. This bactericidal activity of olive oil against H. pylori was also studied in vivo by Castro et al.40 Olive oil was supplied to infected patients, and an eradication rate of 11%
23% was observed after 1 year of intervention, although a higher partial suppression (19%
43%) was observed during the duration of treatment. However, these promising results must be confirmed in more trials with longer duration, different doses, and types of oils. Thus these results could open the possibility of considering virgin olive oil a chemopreventive agent for peptic ulcers or gastric cancer.

27.3 Main antimicrobial compounds in table olives Table olives were the first olive product that investigators started for the search for antimicrobials.41,42 Many researchers have tried to explain the inhibition of the lactic acid fermentation in olive brines because of their content in oleuropein and hydroxytyrosol.1,4 Other researchers have reported a potential antimicrobial activity of table olive extracts, but they have not assigned this activity to any compounds. However, Medina et al.43 have recently demonstrated that the main antimicrobial compounds in table olives are the dialdehydic form of decarboxymethyl elenolic acid (EDA) (Fig. 27.4), HyEDA, and an isomer of oleoside 11-methyl ester. In fact, both EDA and HyEDA can explain most of the antimicrobial activity found in olive brines. It must be noted that TyEDA was not detected in the olive brines. These investigators stored olives of the Manzanilla and Gordal variety in an acidified brine under aseptic conditions for 2 months and inoculated the brines with a strain of Lactobacillus pentosus. The microorganism died in the Manzanilla brines and survived in the Gordal. Subsequently, the content of the brines in phenolic and oleosidic compounds was analyzed, and all these substances were isolated by HPLC and their bactericidal activity against L. pentosus was tested. It can be observed

333

TABLE 27.2 Effect of isolated phenolic and oleosidic compounds from olive brines on the viability of Lactobacillus pentosus. Compound log CFU/mL Hydroxytyrosol

.8

Hydroxytyrosol 1-glucoside

.8

Hydroxytyrosol 4-glucoside

.8

Oleoside

.8

Tyrosol

.8

Secoxyloganin

.8

Secologanoside

.8

Oleoside 11-methyl ester

3

EDA

3.5

HyEDA

,1.2

Log CFU/mL after 48 h incubation of Gordal brines enriched with the isolated compounds, and inoculated with 6 log CFU/mL cells. EDA, dialdehydic form of decarboxymethyl elenolic acid; HyEDA, dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol.

TABLE 27.3 Effect of oleuropein (5.8 mM), hydroxytyrosol (5.2 mM), and the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA), (0.5 mM) on the viability of several microorganisms present in table olives. Log CFU/mL after 48 h incubation Microorganism oleuropein hydroxytyrosol HyEDA Enterobacter aerogenes

5.2

2.9

1.6

Escherichia coli

4.4

1.3

2.1

Enterococcus faecium

5.6

5.5

1.3

Enterococcus faecalis

5.3

4.0

,1.0

Leuconostoc mesenteroides

.8.0

5.0

2.0

Lactobacillus pentosus

.8.0

.8.0

,1.0

Lactobacillus plantarum

.8.0

.8.0

3.0

Saccharomyces cerevisiae

.6.0

.6.0

.6.0

Pichia membranaefaciens

.6.0

.6.0

.6.0

Initial inoculations were 6 and 3.5 log CFU/mL for bacteria and yeasts, respectively.

in Table 27.2 that only the three abovementioned compounds significantly reduced the number of inoculated cells, although HyEDA was the strongest bactericidal compound detected in the olive brines and it could alone

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FIGURE 27.5 Formation of antimicrobial compounds during table olive processing.

explain the inhibition of lactobacillus growth in Manzanilla brines. However, an additive effect was also observed in the three antimicrobial compounds identified, and depending on their concentration, a synergistic effect of all inhibitors must not be ruled out to explain lactic acid bacteria growth inhibition in olive brines. In addition, the antimicrobial activity of oleuropein, hydroxytyrosol, and HyEDA against several microorganisms found in table olives was studied (Table 27.3). It must be noted that the concentration of HyEDA was 10 times lower than that of the other two substances. Oleuropein was ineffective against most microorganisms and hydroxytyrosol allowed for the growth of L. plantarum and L. pentosus. It was again confirmed that HyEDA exerted the highest activity against all bacteria tested. These findings have been confirmed at a pilot plant scale with olives processed according to the Spanish-style green olive method.44 It is well-known that these olives do not properly ferment by lactic acid bacteria when an insufficient NaOH treatment is undertaken, and there was no explanation for this phenomenon until now. Medina et al.44 have demonstrated that an alkaline treatment with a low NaOH strength and insufficient alkali penetration allowed for the presence of EDA and HyEDA in brines (Fig. 27.5), and they inhibited the growth of L. pentosus. By contrast, a more intense alkaline treatment gave rise to an abundant growth of the microorganism without any antimicrobials in brines. Likewise, both HyEDA and EDA were always found in Manzanilla brines of olives nontreated with NaOH. Subsequently, Medina et al.45 showed that the effect of these olive antimicrobial compounds had the same bactericidal activity as commercial glutaraldehyde and o-phthalaldehyde disinfectants against Pseudomonas fluorescens, S. aureus, E. faecalis, and E. coli. This bactericidal activity was due to the presence of a dialdehydic structure in its molecule. Therefore olive brines can be a food source of antimicrobial compounds such as HyEDA and EDA, in particular, those brine with olives nontreated with NaOH. Although table olives have a strong safety record, the presence of pathogenic bacteria in the brine of table olives has been investigated.46 Grounta et al.47 reported that Greek natural black olives inhibited the

growth of different pathogenic microorganisms inoculated within the first 2 days of storage. Medina et al.48 inoculated E. coli, S. enterica, L. monocytogenes, and S. aureus into several industrial olive brines and observed a reduction of 5 log unit of initial population between 5 min and 17 days depending on the conditions. This antimicrobial activity was correlated with the presence of phenolic and oleosidic substances in the olive brines. This correlation has been recently confirmed in commercial presentation of natural green olives49 and in natural black olives50 confirming the adverse habitats of table olives for foodborne pathogenic microorganisms.

Acknowledgment The work was supported by the projects AGL2016-76820-R and RTI2018-093994-J-100, AEI/FEDER, UE.

References 1. Juven B, Samish Z, Henis Y, Jacoby B. Identification of oleuropein as a natural inhibitor of lactic acid fermentation of green olives. J Agric Res. 1968;18:137
138. 2. Fleming HP, Walter WM, Etchells JL. Antimicrobial properties of oleuropein and products of its hydrolysis. Appl Microbiol. 1973;26:777
782. 3. Kubo I, Matsumoto A, Takase I. A multichemical defense mechanism of bitter olive Olea europeaea (Oleaceae). Is oleuropein a phytoalexin precursor? J Chem Ecol. 1985;11:251
263. 4. Ruiz-Barba JL, Brenes M, Jime´nez R, Garcı´a P, Garrido A. Inhibition of Lactobacillus plantarum by polyphenols extracted from two different kinds of olive brines. J Appl Bacteriol. 1993;74:15
19. 5. Capasso R, Evidente A, Schivo L, Orru G, Marcialis M, Cristinzo G. Antibacterial polyphenols from olive oil mill waste water. J Appl Bacteriol. 1995;79:393
398. 6. Obied HK, Bedgood DR, Prenzler PD, Robards K. Bioscreening of Australian olive mill waste extracts: biophenol content, antioxidant, antimicrobial and molluscicidal activities. Food Chem Toxicol. 2007;47:1238
1248. 7. Bisignano G, Tomaino A, lo Cascio R, Crisafi G, Uccella N, Saija A. On the “in vitro” antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol. 1999;51:971
974. 8. Medina-Martı´nez MS, Truchado P, Castro-Iban˜ez I, Allende A. Antimicrobial activity of hydroxytyrosol: a current controversy. Biosci Biotechnol Biochem. 2016;80:801
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Olives and olive oil compounds active against pathogenic microorganisms Chapter | 27

9. Canal C, Ozen B, Baysal H. Characterization of antimicrobial activities of olive phenolics on yeasts using conventional methods and mid-infrared spectroscopy. J Food Sci Technol. 2019;56: 149
158. 10. Yu S, Zhao G. Development of polyphenols as HIV-1 integrase inhibitors: a summary and perspective. Curr Med Chem. 2012;19: 5536
5561. 11. Ma SC, He ZD, Deng XL, et al. In vitro evaluation of secoiridoid glucosides from the fruits of Ligustrum lucidum as antiviral agents. Chem Pharm Bull. 2001;49:1471
1473. 12. Renis HE. In vitro antiviral activity of calcium elenolate. Antimicrob Agents Chemother. 1969;9:167
172. 13. Gullo´n B, Gullo´n P, Eibes G, et al. Valorisation of olive agroindustrial by-products as a source of bioactive compounds. Sci Total Environ. 2018;645:533
542. 14. Medina E, Romero C, Garcı´a P, Brenes M. Characterization of bioactive compounds in commercial olive leaf extracts, and olive leaves and their infusions. Food Funct. 2019;10:4716
4724. 15. Palmeri R, Parafati L, Trippa D, et al. Addition of olive leaf extract (OLE) for producing fortified fresh pasteurized milk with an extended shelf life. Antioxidants. 2019;8:255. 16. Shialy Z, Zarrin M, Sadeghi Nejad B, Yusef Naanaie S. In vitro antifungal properties of Pistacia atlantica and olive extracts on different fungal species. Curr Med Mycol. 2015;1:40
45. 17. Baidez AG, Go´mez P, del Rı´o JA, Ortun˜o A. Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten. Phyisiol Mol Plant Pathol. 2006;69: 224
229. 18. Brenes M, Garcı´a A, Garcı´a P, Garrido A. Acid hydrolysis of secoiridoid aglycons during storage of virgin olive oils. J Agric Food Chem. 2011;49:5609
5614. 19. Brenes M, Garcı´a A, de los Santos B, et al. Olive glutaraldehydelike compounds against plant pathogenic bacteria and fungi. Food Chem. 2011;125:1262
1266. 20. Medina E, de Castro A, Romero C, Brenes M. Comparison of the concentration of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem. 2006;54:4954
4961. 21. Medina E, Romero C, Brenes M, de Castro A. Antimicrobial activity of olive oil, vinegar, and various beverages against foodborne pathogens. J Food Prot. 2007;70:1194
1199. 22. Zampa A, Silvi S, Servili M, Montedoro GF, Orpianese C, Cresci A. In vitro modulatory effects of colonic microflora by olive oil iridoids. Microb Ecol Health Dis. 2006;18:147
153. 23. Radford SA, Tassou CC, Nychas GJE, Board RG. The influence of different oils on the death rate of Salmonella enteritidis in homemade mayonnaise. Lett Appl Microbiol. 1991;12:125
128. 24. Sheth NK, Wisniewski TR, Frason TR. Survival of enteric pathogens in common beverages: an in vitro study. Am J Gastroenterol. 1988;83:658
660. 25. Møretrø T, Daeschel MA. Wine is bactericidal to foodborne pathogens. J Food Sci. 2004;69:251
257. 26. Rhodes PL, Mitchell JW, Wilson MW, Melton LD. Antilisterial activity of grape juice and grape extracts derived from Vitis vinifera variety Ribier. Int J Food Microbiol. 2006;107:281
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27. Friedman M, Henika PR, Levin CE, Mandrell RE, Kozukue N. Antimicrobial activities of tea catechins and theaflavins and tea extracts against Bacillus cereus. J Food Prot. 2006;69: 354
361. 28. Okabe Y, Yamamoto Y, Yasuda K, Hochito K, Ishii N. The antibacterial effects of coffee on Escherichia coli and Helicobacter pylori. J Clin Biochem Nutr. 2003;34:85
87. 29. Entani E, Asai M, Tsujihata S, Tsukamoto Y, Ohta M. Antibacterial action of vinegar against foodborne pathogenic bacteria including Escherichia coli O157:H7. J Food Prot. 1998;61:953
959. 30. Keceli T, Robinson RK. Antimicrobial activity of phenolic extracts from virgin olive oil. Milchwissenschaft. 2002;57:436
440. 31. Brenes M, Garcı´a A, Garcı´a P, Rios JL, Garrido A. Phenolic compounds in Spanish olive oils. J Agric Food Chem. 1999;47:3535
3540. 32. Karaosmanoglu H, Soyer F, Ozen B, Tokatli F. Antimicrobial and antioxidant activities of Turkish extra virgin olive oils. J Agric Food Chem. 2010;58:8238
8245. 33. Yakhlef W, Arhab R, Romero C, Brenes M, de Castro A, Medina E. Phenolic composition and antimicrobial activity of Algerian olive products and by-products. LWT—Food Sci Technol. 2018;93:323
328. 34. Cavallaro L, Egan B, O
Morain C, Di Mario F. Treatment of Helicobacter pylori infection. Helicobacter. 2006;11:36
39. 35. Ho CY, Lin YT, Labbe RG, Shetty K. Inhibition of Helicobacter pylori by phenolic extracts of sprouted peas (Pisum sativum L.). J Food Biochem. 2006;30:21
34. 36. Nohynek LJ, Alakomi H, Ka¨hko¨nen MP, et al. Berry phenolics: antimicrobial properties and mechanisms of action against sever human pathogens. Nutr Cancer. 2006;54:18
32. 37. Zhang L, Ma J, Pan K, Go VL, Chen J, You W. Efficacy of cranberry juice on Helicobacter pylori infection: a double-blind randomized placebo-controlled trial. Helicobacter. 2005;10:139
145. 38. Romero C, Medina E, de Castro A, Brenes M. In vitro activity of olive oil polyphenols against Helicobacter pylori. J Agric Food Chem. 2007;2007(55):680
686. 39. Corona G, Tzounis X, Dessi MA, et al. The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation. Free Radic Res. 2006;40:647
658. 40. Castro M, Romero C, de Castro A, et al. Assessment of Helicobacter pylori eradication by virgin olive oil. Helicobacter. 2012;17:305
311. 41. Juven B, Henis Y. Studies on the antimicrobial activity of olive phenolic compounds. J Appl Bacteriol. 1970;33:721
732. 42. Federici F, Bongi G. Improved method for the isolation of bacterial inhibitors from oleuropein hydrolysis. Appl Environ Microbiol. 1983;46:509
510. 43. Medina E, Brenes M, Romero C, Garcı´a A, de Castro A. Main antimicrobials in table olives. J Agric Food Chem. 2007;55:9817
9823. 44. Medina E, Romero C, de Castro A, Brenes M, Garcı´a A. Inhibitors of lactic acid fermentation in Spanish-style green olive brines of the Manzanilla variety. Food Chem. 2008;110:932
937. 45. Medina E, Brenes M, Garcı´a A, de Castro A. Bactericidal activity of glutaraldehyde-like compounds from olive products. J Food Prot. 2009;72:2611
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46. Medina-Pradas E, Arroyo-Lo´pez FN. Presence of toxic microbial metabolites in table olives. Front Microbiol. 2015;6:873. 47. Grounta A, Nychas GJE, Panagou EZ. Survival of food-borne pathogens on natural black table olives after post-processing contamination. Int J Food Microbiol. 2013;161:197
202. 48. Medina E, Brenes M, Romero C, Ramı´rez E, de Castro A. Survival of foodborne pathogenic bacteria in table olive brines. Food Control. 2013;34:719
724.

49. Romero-Gil V, Medina E, Garrido-Fernandez A, Arroyo-Lopez FN. Foodborne pathogen survival in commercial Aloren˜a de Ma´laga table olive packaging. Front Microbiol. 2018;9:2471. 50. Medina E, Garcı´a-Garcı´a P, Romero C, de Castro A, Brenes M. Aerobic industrial processing of Empeltre cv. natural black olives and product characterization. Int J Food Sci Technol. 2020;55:534
541. Available from: https://doi.org/10.1111/ijfs.14282.

Chapter 28

Olive oil in the prevention of breast and colon carcinogenesis Aliza Hannah Stark and Zecharia Madar Robert H. Smith Faculty of Agriculture, Food and Environment, School of Nutritional Sciences, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel

Abbreviations Akt BC COX-2 CRC DAO DMBA DMH DNA DOA EMT ER EVOO HER HIF-1α IL-6 IFN-γ IL-17A MedDiet NF-κB P3IK p53 PPARγ PR RCT RT-PCR Sfrp4 TGFβ TNF-α

protein kinase B breast cancer cyclooxygenase-2 colorectal cancer diamine oxidase 7,12-dimethylbenz[a]anthracene dimethylhydrazine deoxyribonucleic acid secoiridoid decarboxymethyl oleuropein aglycone epithelial
mesenchymal transition estrogen receptor extra-virgin olive oil human epidermal growth factor receptor hypoxia-inducible factor 1-alpha interleukin 6 interferon gamma interleukin 17A Mediterranean diet nuclear factor kappa B phosphatidylinositol-3-kinase cellular tumor antigen/tumor suppressor proliferator-activated receptor gamma progesterone receptor randomized control trials reverse transcription polymerase chain reaction secreted frizzled-related protein 4 transforming growth factor beta tumor necrosis factor alpha

28.1 Introduction Colorectal cancer (CRC) and breast cancer (BC) are listed in the top 10 causes of death in the developed world.1 Consequently, cancer prevention has become one of the most significant public health challenges of our times.

Primary prevention is thought to be the most effective way to fight cancer2 as it is well known that prevention is better than cure. In order to successfully reduce cancer incidence, modifiable risk factors must be identified. Thus diet and lifestyle changes are at the forefront of cancer prevention, as genetics and age cannot be altered.3 It is widely accepted that diet plays a significant role in cancer development and treatment. In order to establish which dietary components impact cancer, many different scientific approaches/models have been used. However, the vast majority of studies done in human populations have not been able to definitively show that any specific dietary component protects against cancer development. This is due to the nature of epidemiological studies that can only find associations between a nutrient, a food or a dietary pattern, and cancer risk.4,5 Furthermore, it is extremely difficult to detect and nullify confounding factors. Statistical control is a mathematical manipulation of data and cannot entirely neutralize physiological and metabolic factors. It must also be taken into consideration that, to date, genetic profiles have not been included in most cancer-related studies. Currently, randomized control trials (RCT) are the most reliable sources of evidence showing a causal relationship between diet and cancer development or progression.6 Unfortunately, because of the high-cost and long-term nature of these studies, evidence of this variety is scarce. These methodological challenges have led scientists to use animal and cell models, in attempts to reproduce human cancer development. Results from these models have provided excellent hypotheses, describing underlying mechanisms for the prevention of various types of cancer. The paramount limitation of these studies is that the experimental induction of cancer, or the use of cancer cell lines, does not replicate the true etiology of cancer in the human body. Thus, extrapolation of these results to humans has led to

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00047-X © 2021 Elsevier Inc. All rights reserved.

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much debate regarding the modification of cancer risk and mechanisms of action through diet. Understanding these constraints leads us to conclude that it is premature to declare that a specific food, like olive oil, has cancerprotective properties. However, the existing evidence points the way to further research on the subject. This chapter will critically present the current, state of the knowledge for olive oil as a cancer preventive agent. The chemical makeup of olive oil is described extensively in other chapters of this book. It should be noted that minimally processed extra-virgin olive oil (EVOO) is rich in numerous compounds such as monounsaturated fatty acids, polyphenols, squalene, and phytosteroids all with the potential to provide protection against the development of cancer.7

28.2 Breast cancer and olive oil Data from the World Cancer Research Fund report that BC is the second most common cancer globally and the most common cancer in women.8 In 2018 nearly two million new cases of BC were diagnosed worldwide. The impact of diet on BC incidence has been extensively researched. A recent meta-analysis indicated that following a healthy dietary pattern significantly decreased the risk of BC, especially in the case of postmenopausal, hormone receptor
negative disease.9 Yet, no consistent associations have been established between specific dietary components and BC with the exception of alcohol.10 There is reason to believe that dietary components can interact with numerous biological pathways associated with cancer development. This includes carcinogen bioactivation, cell signaling, angiogenesis, hormone regulation, and inflammation.11,12 Consequently, attempts are being made to identify specific dietary patterns, specific foods such as olive oil, and specific nutrients that impact BC development.

28.2.1 Human studies Individuals do not usually consume isolated foods or nutrients, making it very difficult to examine the potential therapeutic properties of any single dietary component, including olive oil. In addition, interactions occur between individual components of the diet and may have synergistic or antagonistic effects. Thus much of the recent nutritional research focuses on dietary patterns, which appears to be a more accurate way of studying healthy diets.13 A strong inverse relationship between adherence to the Mediterranean diet (MedDiet) and cancer has been documented in numerous studies.14 This dietary pattern is associated with reduction in oxidative stress, inflammation, cell proliferation, angiogenesis, and deoxyribonucleic acid (DNA) damage.14,15 This has led to the assumption that following the MedDiet will reduce cancer development with olive oil as the main source of added

dietary fat. Past studies have also documented significantly lower rates of BC in Mediterranean countries in comparison to other European countries or the United States.16 Although this is commonly attributed to adherence to the MedDiet, data are far from conclusive. In a recent meta-analysis of observational studies (n 5 11), Schwingshackl et al. documented an 8% reduction in BC incidence [95% confidence interval (CI) 20.89 to 296] in those that had good adherence to the Mediterranean dietary pattern.17 The European Prospective Investigation into Cancer and Nutrition (EPIC) study followed 335,062 women recruited from 1992 to 2000, in 10 European countries for 11 years.18 In premenopausal women adhering to the MedDiet, no association was found with lessened risk of BC. In postmenopausal women, greater adherence to the MedDiet was inversely associated with the overall risk of BC but the association was much stronger in women with estrogen receptor (ER) negative cancer. The MedDiet was found to reduce risk of BC by 6% overall, and by 7% in postmenopausal women. For tumors lacking the estrogen or progesterone receptors (PR), the diet reduced risk by 20% in postmenopausal women. Similar results were reported from a cohort in Holland,19 in which women who were high adherers to the MedDiet were compared to women with low adherence. A statistically significant inverse association between consuming a Mediterranean-type diet and risk of ER negative BC was reported with a hazard ratio of 0.60 (95% CI 0.39
0.93). However, there were only nonsignificant weak inverse associations with ER positive or total BC risk. When the same authors looked at other data and carried out a metaanalysis, the summary of hazard ratios for high versus low adherence to the MedDiet indicated protection of only 0.94 for total postmenopausal BC. However, comparison of hazard ratios indicated that ER positive cancer 5 0.98, while ER negative had a 0.73 ratio. The findings support an inverse association between the MedDiet and receptor negative BC. Following a Mediterranean dietary pattern appears to provide some protection against BC development, but it is not possible to attribute this effect specifically to olive oil or any of its components. It must also be taken into consideration that there may be synergistic interactions among the many components of the diet that leads to decreased morbidity. Case
control studies are not considered to provide highquality evidence in the hierarchy of proof.20 Nevertheless, in a meta-analysis of predominately case
control studies Psaltopoulou and colleagues found that olive oil consumption was associated with lower odds of developing BC (95% CI 20.78 to 20.12).21 This included several studies from Mediterranean countries such as Greece, Spain, Italy, and France. In the same meta-analysis the authors were unable to establish if monounsaturated fat was the protective factor in olive oil, or if other components were responsible for its protection against BC. To the best of our knowledge, only a few prospective studies have investigated the association between olive oil

Olive oil in the prevention of breast and colon carcinogenesis Chapter | 28

intake and the risk of BC. Buckland et al. reported the results from the Spanish population within the EPIC study.22 They were unable to show any significant association between olive oil and cancer mortality. However, a serious limitation of the study was that the variety of olive oil consumed was not specified. EVOO, using cold press extraction, is rich in polyphenols and may have a very different impact on health than refined olive oils produced with solvent extraction. Secondary analysis of the PREDIMED trial (Prevencion con Dieta Mediterranea) included examining the effects of the MedDiet supplemented with EVOO on BC.23 The study was a 1:1:1 randomized, single-blind, controlled field trial in Spain. Women aged 60
80, with no prior history of BC (n 5 4152), were followed for a median of 4.8 years. Participants were randomly allocated to one of three diets: a MedDiet supplemented with EVOO, a MedDiet supplemented with mixed nuts, or a control diet. The multivariableadjusted hazard ratios for the MedDiet with EVOO group versus the control group were 0.31 (95% CI 0.13
0.77). Analyses with yearly cumulative updated dietary exposures showed that the hazard ratio for each additional 5% of calories from EVOO was 0.72 (95% CI 0.57
0.90). Finally, a 62% lower risk of malignant BC was reported in comparison to women in the control group. PREDIMED is considered the first randomized trial finding a beneficial effect of long-term dietary supplementation with EVOO on BC incidence. The major limitations of the study were that BC was not the primary end point and that there were relatively few observed BC cases. But, the observed risk reduction of BC in women consuming high levels of EVOO as part of the MedDiet appears to be sufficiently robust that even with a small number of cases, differences were significant. Arguments in favor of olive oil’s contribution to BC prevention often focus on specific nutrients found in EVOO which can act as antioxidants and are considered to have diverse effects on cellular function.24 Compounds in olive oil have been shown to interact with cellular proteins to modulate intracellular signals, downregulate inflammation, modify kinase activity, and control cell cycle progression. However, implications for human health must be made with caution. The compounds may have low bioavailability and undergo chemical modification into numerous metabolites in the body.25 Along with the numerous chemically active compounds found in olive oil, the unique fatty acid makeup may be an important factor in any possible role in the protection against human BC development. The assumption that oleic acid, found in high concentrations in olive oil, may have anticancer properties is not new. With a single double bond the potential for oxidation is limited. In addition, the relatively low n-6 PUFA/n-3 PUFA ratio found in olive oil may also be important.26 Specific mechanisms are not fully understood, although lipids can impact all the stages of carcinogenesis, alter

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hormonal status, modify the structure and function of cell membranes, modulate cell signaling transduction pathways, regulate gene expression, and influence the immune system.27 Despite the numerous mechanisms proposed for a protective effect of oleic acid on BC, it has not been possible to provide sufficient evidence to confirm these hypotheses in humans.21

28.2.2 Cell culture models Cell culture models are useful for identifying biologically active compounds and investigating possible mechanisms of action. Numerous studies, using a wide range of cellular models, indicate that compounds found in olive oil have anticarcinogenic effects. One model for BC research uses BC stem cells. The BC stem cells are known to be particularly aggressive malignant cells defined in terms of functional traits of self-renewal, differentiation, therapy resistance, and tumor/metastasis-initiating capacity.28 Isolated phenolic compounds from EVOO targeted tumor-initiating, drug-resistant populations of BC stem cells.29 In vitro screening revealed that the secoiridoid decarboxymethyl oleuropein aglycone (DOA) was responsible for suppression of the functional traits and could potently block the formation of multicellular tumorspheres. This protective effect was consistent in a variety of genetically diverse BC models. An additional study by Cruz-Lozano et al. examined chemopreventive benefits of hydroxytyrosol, another phenolic phytochemical from olives with antioxidant properties.30 This particular compound is considered a powerful natural antioxidant and its oxygen radical absorbing capacity value is one of the highest found in plants.31 Hydroxytyrosol has been shown to protect against oxidative DNA damage in human breast cell lines but it did not impact cell proliferation or increase apoptosis.32 Hydroxytyrosol had a greater impact when tested in a cell model of triple-negative BC.30 BC cells/tumors are commonly categorized by the presence of hormone-receptors, estrogen (ER), progesterone (PR), and HER2 (human epidermal growth factor receptor). The triple-negative tumors represent a very aggressive form of cancer characterized by poor survival rates, high proliferation, metastases, and drug resistance.33 Cruz-Lozano’s group showed that hydroxytyrosol acted directly on the BC cells, reducing their number and aggressiveness as well as inhibiting their capacity to multiply. Decreased tumor cell migration in this model was attributed to interruption in the epithelial
mesenchymal transition, Wnt/β-catenin, and TGFβ signaling pathways. It has been suggested that hydroxytyrosol may be the most important anticancer agent found in diets rich in olive oil. Hydroxytyrosol exerts anticancer activity through several different mechanisms. It reduces levels of proinflammatory cytokines, thus quenching the inflammation that underlies the cancer disease process. It also inhibits cell proliferation, a key characteristic of cancer, and promotes apoptosis (cell death) of several types of cancer cells, including BC cells.34

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Based on numerous research findings indicating an anticancer effect of hydroxytyrosol, a clinical trial is currently being conducted at the Houston Methodist Hospital, which studies the effects hydroxytyrosol has on the prevention of BC in high-risk patients.35 Ciancarelli et al. note that the bioavailability of polyphenols in humans is limited and their concentrations within the systemic circulation and tissues are very low in comparison with endogenous antioxidants, such as uric acid or ascorbic acid.15 Therefore concentrations in the blood could be well below those needed to achieve the biological effects observed in vitro or in laboratory animals. Notably, once ingested, polyphenolic molecules undergo extensive metabolism that reduces their antioxidant activity; they appear in the bloodstream as a mixture of compounds, bioactivity of which in the systemic circulation has yet to be demonstrated.24 Components of functional foods such as olive oil are thought to have the capability of enhancing the natural antioxidant defense system by scavenging reactive oxygen and nitrogen species, protecting and repairing DNA damage, as well as modulating signal transduction pathways and gene expression. Major pathways affected include the proinflammatory pathways regulated by nuclear factor kappa B (NFκB), as well as those associated with cytokines and chemokines.11 However, confirmation in humans remains elusive.

28.2.3 Animal models Numerous animal models have been developed to research BC. For many years, our laboratory studied olive oil using a 7,12-dimethylbenz[a]anthracene (DMBA) rat model. Intragastric intubation of DMBA was used to induce carcinogenesis, predominately in the mammary tissue, and within several weeks tumorigenesis occurred.36
38 One of the most striking results from our work was that when healthy pregnant rats were fed olive oil as 7% of their diet, 21 days following birth they had lower 17β estradiol levels than animals fed 7% corn oil.38 Because exposure to estrogen is associated with increased risk of BC,39 this finding is of great interest. BC was induced in the female offspring of the same rats. All offspring were fed 7% corn oil diets. A marked reduction in tumor size was found in rats that had been exposed to 7% olive oil in utero.38 More recent studies in animal models have focused on specific compounds from olive oil such as oleuropein and hydroxytyrosol. Elamin et al. used a xenograft nude mice model (BALB/c OlaHsd-foxn1) and injected MDA-MB-231 human BC cells into the animal’s right flank.40 Once tumors appeared, mice were given various treatments. Tumor size was smaller following the injection of oleuropein that significantly inhibited growth compared to controls (79 vs 173 mm3, respectively). Smaller tumors were accompanied by a reduction in NF-κB expression, a transcription factor

central to cell proliferation. Oleuropein also triggered apoptosis in more than 40% of the MDA-MB-231 cells compared with 6% in the control group. When combined with doxorubicin, an anticancer drug, a strong synergistic effect was observed in inhibition of tumor growth along with a significant increase in apoptosis. The impact of hydroxytyrosol on mammary carcinoma was also investigated by Granados-Principal et al. using the classic DMBA rat model.41 Following treatment, tumor growth and proliferation rate were inhibited. Gene expression was determined by microarray assay and quantitative RT-PCR on the biopsied mammary carcinomas. Hydroxytyrosol modified gene expression for pathways associated with apoptosis, cell cycle, proliferation, differentiation, survival, and transformation. High expression of secreted frizzled-related protein 4 (Sfrp4) was also found. Sfrp4 is considered to be an antagonist of the Wnt pathway and categorized as a tumor suppressor gene. Another study carried out by Corominas-Faja et al. tested DOA, an additional phenolic compound isolated from EVOO.29 Using a model where mice were orthotopically injected with cancer stem cells, BC cell populations pretreated with DOA did not lead to tumor formation for several months. It appears reasonable to suggest that DOA reduced tumor-initiating states. Compounds from olive oil may impact tumorigenicity by modifying controllers of cell fate involving both the downregulation of transcription factors and the reactivation of epigenetically suppressed differentiation pathways.

28.3 Colorectal cancer and olive oil CRC involves transformation of healthy epithelial cells in the colon or rectum into adenocarcinomas.42 This is attributed to genetic and epigenetic modifications along with changes in the microenvironment in the digestive tract. Among cancers, it has the third highest global incidence rate.8 In contrast to BC, there is significantly greater biological plausibility that dietary factors play a central role in CRC development, as the digestive track is constantly exposed to foods, food additives, and food contaminants. Numerous epidemiologic studies have linked CRC with food intake.43
45 In the past, it was estimated that CRC could be reduced by 60%
70% if individuals complied with dietary guidelines.46 In fact, it was estimated that almost 75% of all the sporadic cases of CRC were directly affected by dietary intake.47 One can argue regarding the exact extent of the impact of dietary exposures, but it is clear that what one eats is linked to the risk of developing colon cancer. The question is then asked, can we isolate the impact of EVOO, rich in antioxidants and biologically active compounds, on CRC development? Stoneham et al. reported that olive oil consumption was negatively associated with CRC based on the analysis of epidemiological studies.48

Olive oil in the prevention of breast and colon carcinogenesis Chapter | 28

However, 20 years later, conclusive evidence to support this hypothesis is not available. Olive oil undergoes many changes as it moves through the digestive tract. Exposure to acidic conditions in the stomach, contact with enzymes, and interaction with the microbiota modify its chemical structures and produce new metabolites.24 In addition, it is thought that olive oil and its components have the ability to alter microbiota composition in the colon.49 It has been proposed that changes in gut microbes through diet may be an important factor in the development of CRC.50 Alterations of microbial composition have the potential to impact the carcinogenic process and immune response during tumor development. However, this is a new area of study and specific results on exposure to olive oil are not available. Additionally, there is no consensus regarding the optimal make-up of the microbiome to prevent CRC nor how diet-based strategies can modify CRC risk via manipulation of gut bacteria. Long before scientists had the technology to identify the human microbiome, Stoneham et al. proposed the “diamine oxidase (DAO) hypothesis” linking olive oil, gut microbes, and CRC.48 The DAO hypothesis suggests that olive oil inhibits the production of deoxycholic acid, a secondary bile acid with tumorigenic properties produced by bacterial enzymes. Reduction of deoxycholic acid may influence polyamine metabolism, an important regulator in the colonic mucosa. This interaction could modulate mucosal turnover, polyp formation, and carcinogenesis.

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higher levels of olive oil consumption had a decreased risk of log OR 20.36 (95% CI 20.50, 20.21), or 30% lower probability of developing cancer. Despite the seemingly conclusive results of this meta-analysis, the authors clearly state the limitations of case
control studies and the numerous methodological constraints when interpreting the data. For example, causality cannot be determined. Comparing extreme categories (highest vs lowest intake levels) is problematic, and olive oil consumption may be a proxy for numerous other factors (i.e., high vegetable intake or low beef consumption). Unfortunately, no RCT or large prospective cohort studies have specifically looked at the effects of EVOO on CRC. Several years ago, two intervention studies were done in human subjects who consumed phenol-rich olive oil for a short time period.52,53 Exposure to olive oil resulted in a reduction in oxidative DNA damage. However, this is not sufficient evidence to assume a significant reduction in cancer incidence. Gaforio et al. in their consensus report from the III International Conference on Virgin Olive Oil held in Spain, 2018, note that a substantial obstacle to obtaining satisfactory evidence in the prevention of carcinogenesis in humans is the scarcity or even the absence of surrogate markers for cancer that foods could modulate.54 Because cancer development often occurs over a period of years, it makes well-designed intervention studies almost impossible. Hence, published scientific studies looking at the interactions of olive oil and colon pathologies focus almost entirely on possible mechanisms of action using cell culture and animal studies.

28.3.1 Human studies The MedDiet is considered to be protective against CRC.14 The Mediterranean region traditionally has lower rates of CRC in comparison to other developed countries. 16 Commonly, this is associated with diet. In 11 observational studies, 6 cohort studies and 5 case
control studies that looked at adherence to the MedDiet and CRC, the risk ratio/odds ratio reported was 0.82 (95% CI 0.75
0.88); 0.86 (95% CI 0.80
0.92); and 0.71 (95% CI 0.57
0.88), respectively.17 The overall hazard ratio was 0.86 (95% CI 0.80
0.92). Results have been strongest using data from case
control studies. In a large study from Spain (n 5 1629 cases and n 5 3509 controls), it was reported that a high adherence to the MedDiet led to a 30%
40% decreased risk of colon cancer.51 However, similar to data on BC, it is impossible to isolate the impact of olive oil, per se, from these findings. Psaltopoulou et al. in a meta-analysis of 13,800 patients and 23,340 controls in 19 observational studies looked specifically at olive oil consumption.21 They concluded that higher intake of olive oil in comparison to lower levels of consumption was associated with a 34% lower likelihood of having any type of cancer. Eight of the studies included data for cancers of the gastrointestinal tract. Individuals with

28.3.2 Cell culture models Colon cancer research is often conducted in cell culture models, and numerous components of olive oil have demonstrated in vitro antitumor activity.49 Reduced proliferation and increased apoptosis are the archetypical responses expected in response to exposure to isolated compounds from EVOO. Squalene, oleanolic acid, and polyphenols from olive oil all have chemopreventive properties in cell culture models of colon cancer.7 Any decrease in initiation, promotion, and progression of cancer cell growth is considered beneficial. This is achieved via mechanisms that impact the redox balance in the cells, regulate cellular processes, and modify critical signaling pathways relevant to cancer development. For example, in Caco-2 adenocarcinoma cells, hydroxytyrosol acetate has been shown to inhibit cell proliferation, arrest cell cycle, and upregulate the transcription of genes involved in apoptosis.55 Ca´rdeno et al. examined anticancer molecular mechanisms induced by oleuropein and hydroxytyrosol in HT-29 cells, another human colon adenocarcinoma cell line.56 Although oleuropein was less potent, both compounds induced changes in cell cycle and increased apoptosis. Specific changes in protein expression were observed with a decrease in hypoxia-inducible factor 1-alpha

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(HIF-1α) and upregulation of p53. Hydroxytyrosol also significantly upregulated peroxisome proliferator-activated receptor gamma (PPARγ) expression. The authors proposed that PPARγ upregulation was a principal mechanism involved in tumor shrinkage, and induction of apoptosis was carried out via p53 pathway activation adapting the HIF-1α response to hypoxia. It is interesting to note that more recent studies not only have included hydroxytyrosol but also investigated its metabolites/catabolites formed by gut microbiota (phenylacetic, hydroxyphenylpropionic).57 These compounds produced cell cycle arrest and promoted apoptosis in the Caco-2 and HT-29 colon cancer cell lines. Rigacci et al. summarized possible anticancer properties of olive oil polyphenols. They included a long list of mechanisms such as induction of apoptosis, cell cycle delay, reduced cell proliferation, and reduced cell viability.58 They also cite many possible signaling pathways and modification of a wide range of proteins beyond those mentioned here. Overall, a large number of in vitro studies exploring the antiproliferation properties of olive oil and olive oil extracts in different colon cancer cell models have been carried out. Consistently they show protective effects against cancer development. Up-to-date extensive reviews are available.49 It is important to note that Borzı` et al. emphasized the fact that most of the concentrations used in these studies are not considered physiological and cannot be achieved in vivo.49 Furthermore, the limited capacity of these compounds to be absorbed, along with their rapid transformation to other metabolites, raises the question of their chemopreventive capacities in humans.49,58 Despite positive results, cell culture models cannot predict in vivo responses and some contradictory results have been reported, including prooxidative activity of specific compounds under certain circumstances.58

receiving DMH injections with or without three weekly oral doses of EVOO.60 In this model, olive oil treatment was able to lower tumor incidence and inhibit tumor development. Olive oil decreased inflammatory and angiogenic markers and restored the proapoptotic expression of markers reduced by exposure to DMH. The EVOO played an important role in epigenetic therapy by altering NF-κB and apoptotic pathways by targeting noncoding RNAs and methylation machinery. Dietary exposure to olive oil was able to modify epigenetic mechanisms resulting in a reduction of the tumor burden. Diverse animal models of CRC have been used to explore the chemopreventive impact of olive oil and its components. Giner et al. studied the role of oleuropein in C57BL/6 mice exposed to azoxymethane (AOM)/dextran sulfate sodium.61 This model is inflammation driven and results in CRC. It is considered to better reflect the etiology of CRC resulting from inflammatory bowel disease which is highly associated with carcinogenesis in humans.62 Oleuropein was dissolved in drinking water and given daily from day 7 until the end of the experiment. Oleuropein was able to suppress the growth and multiplicity of colonic tumors. Treatment with oleuropein also reduced intestinal inflammatory cytokine concentrations (IL-6, IFN-γ, TNF-α, and IL-17A) and decreased cyclooxygenase-2 (COX-2), which is considered an important mediator of angiogenesis and tumor growth. Bax levels were restored by oleuropein indicating increased apoptosis. Downregulation of proteins involved in proliferation pathways [NF-κB, Wnt/β-catenin, phosphatidylinositol-3-kinase (P3IK)/Akt] was observed. Levels of signal transducers and activators of transcription were also reduced. The authors concluded that oleuropein could be a promising protective agent against colitisassociated CRC.

28.3.3 Animal models

28.4 Conclusion: implications for human health and disease prevention

Animal models provide substantial evidence that olive oil and its many constituent compounds have the ability to impact CRC. Our early work, some 20 years ago, involved repeated injections of dimethylhydrazine (DMH) to induce colon cancer in rats.59 Healthy female rats were fed high levels of olive oil (15% w/w). DMH was injected to their male offspring, but the previous in utero exposure to olive oil had little impact on tumor yield and size. However, when offspring continued to consume the same 15% olive oil diet given to their mothers, the response to DMH was modified with a significant reduction in proliferative activity of the colon epithelial cells and an increase in T-killer lymphocytes in and around tumors. The same DMH model is still in use today. Nanda et al. examined differences in epigenetic factors in male rats

Olive oil is considered one of the healthiest oils for human consumption. It is an integral part of the MedDiet and there is evidence that it has numerous therapeutic properties as a functional food. However, the current available evidence lacks a unified, controlled approach regarding its role in carcinogenesis. The question of olive oil’s role as a chemopreventive agent has not been resolved. Is olive oil truly able to reduce induction, prevent or slow cancer progression, or possibly reverse carcinogenesis at a premalignant stage? In a recent narrative review, Foscolou et al. examined the role of olive oil in human health.63 They concluded that olive oil “exhibits a protective role against overall and particularly BC occurrence.” Wani et al. also surmise that hydroxytyrosol may prevent cancer.64 This supposition is indisputably premature, and possibly imprecise, due to the

Olive oil in the prevention of breast and colon carcinogenesis Chapter | 28

issues of absorption and metabolism of biologically active compounds in olive oil. In contrast, Grosso et al. using the same available data and standardized methods of systematic review and metaanalyses raised questions regarding the strength of the data associating diet and cancer.65 They clearly state that there is “a potential role of diet in certain cancers, but the evidence is not conclusive and may be driven or mediated by lifestyle factors.” Making the case for olive oil as a chemopreventive agent is ongoing, and the jury is still out. Identifying causal relationships between a single food or nutrient in chronic disease is extremely difficult. Although olive oil may indeed help one to prevent breast and colon cancer, it is our opinion that, at this time, there is insufficient rigorous scientific evidence to make health claims in this area.

Mini-dictionary of terms Apoptosis Carcinogenesis

programmed cell death. the transformation of normal, healthy cells into cancer cells. Chemoprevention the use of substances to stop cancer from developing. Colorectal abnormal growth of cells in the colon or rectum cancer that leads to tumor formation. Extra-virgin the highest grade of cold-pressed olive oil with low olive oil levels of free oleic acid (not more than 0.8 g/100 g). Hydroxytyrosol a phenolic compound found in olives and olive leaves with biological activity. Mediterranean a dietary pattern based on the traditional eating diet habits of countries such as Italy, Spain, and Greece. Oleuropein a glycosylated phenolic compound with biological activity found in olives with a bitter taste. PREDIMED trial A large-scale, prospective, randomized, control trial carried out in Spain that looked at the impact of nuts and olive oil on disease incidence.

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149. 23. Toledo E, Salas-Salvado J, Donat-Vargas C, et al. Mediterranean diet and invasive breast cancer risk among women at high cardiovascular risk in the PREDIMED trial: a randomized clinical trial. JAMA Intern Med. 2015;175:1752
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95. Escrich E, Solanas M, Moral R, Escrich R. Modulatory effects and molecular mechanisms of olive oil and other dietary lipids in breast cancer. Curr Pharm Des. 2011;17:813
830. Bakhshinyan D, Adile AA, Qazi MA, et al. Introduction to cancer stem cells: past, present, and future. Methods Mol Biol. 2018;1692:1
16. Corominas-Faja B, Cuya`s E, Lozano-Sa´nchez J, et al. Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells. Carcinogenesis. 2018;39:601
613. Cruz-Lozano M, Gonza´lez-Gonza´lez A, Marchal JA, et al. Hydroxytyrosol inhibits cancer stem cells and the metastatic capacity of triple-negative breast cancer cell lines by the simultaneous targeting of epithelial-to-mesenchymal transition, Wnt/β-catenin and TGFβ signaling pathways. Eur J Nutr. 2019;58:3207
3219. Granados-Principal S, Quiles JL, Ramirez-Tortosa CL, SanchezRovira P, Ramirez-Tortosa MC. Hydroxytyrosol: from laboratory investigations to future clinical trials. Nutr Rev. 2010;68:191
206. Warleta F, Quesada CS, Campos M, Allouche Y, Beltra´n G, Gaforio JJ. Hydroxytyrosol protects against oxidative DNA damage in human breast cells. Nutrients. 2011;3:839
857. Jitariu AA, Cıˆmpean AM, Ribatti D, Raica M. Triple negative breast cancer: the kiss of death. Oncotarget. 2017;8:46652
46662. Imran M, Nadeem M, Gilani SA, Khan S, Sajid MW, Amir RM. Antitumor perspectives of oleuropein and its metabolite hydroxytyrosol: recent updates. J Food Sci. 2018;83:1781
1791. National Institutes of Health. Clinical trials. Internet: ,https:// clinicaltrials.gov/ct2/show/NCT02068092.; 2020 Accessed February, 2020. Kossoy G, Yarden G, Ben-Hur H, et al. Comparative effects of dimethylbenz(a)anthacene and a 15% olive-oil diet on cellular components and expression of apoptosis-related proteins in the spleen and mammary gland tumors of rats. Oncol Rep. 2001;8:435
439. Kossoy G, Yarden G, Benhur H, et al. Transplacental effects of a 15% olive-oil diet on chemically-induced tumorigenesis in offspring. Oncol Rep. 2000;7:1145
1148. Stark AH, Kossoy G, Zusman I, Yarden G, Madar Z. Olive oil consumption during pregnancy and lactation in rats influences mammary cancer development in female offspring. Nutr Cancer. 2003;46:59
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1135. Elamin MH, Elmahi AB, Daghestani MH, et al. Synergistic antibreast-cancer effects of combined treatment with oleuropein and doxorubicin in vivo. Altern Ther Health Med. 2019;25:17
24. Granados-Principal S, Quiles JL, Ramirez-Tortosa C, et al. Hydroxytyrosol inhibits growth and cell proliferation and promotes high expression of sfrp4 in rat mammary tumours. Mol Nutr Food Res. 2011;55(suppl 1):S117
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644. 45. Fliss-Isakov N, Kariv R, Webb M, et al. A healthy lifestyle pattern has a protective association with colorectal polyps. Eur J Clin Nutr. 2020;74. Available from: https://doi.org/10.1038/ s41430-019-0481-2. 46. Donaldson MS. Nutrition and cancer: a review of the evidence for an anti-cancer diet. Nutr J. 2004;3:19. 47. Johnson IT. New approaches to the role of diet in the prevention of cancers of the alimentary tract. Mutat Res. 2004;551:9
28. 48. Stoneham M, Goldacre M, Seagroatt V, Gill L. Olive oil, diet and colorectal cancer: an ecological study and a hypothesis. J Epidemiol Community Health. 2000;54:756
760. 49. Borzı` AM, Biondi A, Basile F, Luca S, Vicari ESD, Vacante M. Olive oil effects on colorectal cancer. Nutrients. 2018;11(1):32. 50. Song M, Chan AT. Diet, gut microbiota, and colorectal cancer prevention: a review of potential mechanisms and promising targets for future research. Curr Colorectal Cancer Rep. 2017;13:429
439. 51. Castello´ A, Amiano P, Ferna´ndez de Larrea N, et al. Low adherence to the Western and high adherence to the Mediterranean dietary patterns could prevent colorectal cancer. Eur J Nutr. 2018;58:1
11. 52. Machowetz A, Poulsen HE, Gruendel S, et al. Effect of olive oils on biomarkers of oxidative DNA stress in Northern and Southern Europeans. FASEB J. 2007;21:45
52. 53. Salvini S, Sera F, Caruso D, et al. Daily Consumption of a highphenol extra-virgin olive oil reduces oxidative DNA damage in postmenopausal women. Br J Nutr. 2006;95:742
751. 54. Gaforio JJ, Visioli F, Alarco´n-de-la-Lastra C, et al. Virgin olive oil and health: summary of the III international conference on virgin olive oil and health consensus report, JAEN (Spain) 2018. Nutrients. 2019;11:2039. 55. Mateos R, Pereira-Caro G, Bacon JR, et al. Anticancer activity of olive oil hydroxytyrosyl acetate in human adenocarcinoma Caco-2 cells. J Agric Food Chem. 2013;61:3264
3269. 56. Ca´rdeno A, Sa´nchez-Hidalgo M, Rosillo MA, Alarco´n de la Lastra C. Oleuropein, a secoiridoid derived from olive tree, inhibits the proliferation of human colorectal cancer cell through downregulation of HIF-1α. Nutr Cancer. 2013;65:147
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Chapter 29

The effects of olive oil and other dietary fats on redox status on breast cancer Marı´a Jesu´s Ramı´rez-Expo´sito1, Marı´a Pilar Carrera-Gonza´lez1,2 and Jose´ Manuel Martı´nez-Martos1 1

Experimental and Clinical Physiopathology Research Group CTS1039, Department of Health Sciences, School of Experimental and Health Sciences,

University of Jae´n, Campus Universitario Las Lagunillas, Jae´n, Spain, 2Department of Nursing, Pharmacology and Physiotherapy, Faculty of Medicine and Nursing, University of Cordoba, IMIBIC, Co´rdoba, Spain

Abbreviations CAT EVOO GPx GSH GSSG MDA MUFAs NMU OAESO PUFAs SO SOD TAC TBARS

catalase extra-virgin olive oil glutathione peroxidase reduced glutathione oxidized glutathione malondialdehyde monounsaturated fatty acids N-methyl-nitrosourea oleic acid-enriched sunflower oil polyunsaturated fatty acids sunflower oil superoxide dismutase total antioxidant capacity thiobarbituric acid-reactive substances

29.1 Introduction Breast cancer is a disease with multiple etiology, linked to genetic, environmental, social demographic, behavioral, psychological, and hormonal factors.1,2 Of the risk factors, nutritional components can be associated to 30%
40% of disease cases.3,4 Several studies have been carried out to clarify which elements in the diet could participate positively or negatively in the development and/or progression of breast cancer,5 as well as the mechanisms through which those nutrients could be involved in their progression, recurrence, or mortality.6 In addition, nutritional components might be directly related to the generation of reactive species in the body, triggering oxidative stress, causing cell oxidative damage and, therefore, increasing the risk of disease. Also, estrogen deficit could be responsible of the decreased activities of some enzymatic antioxidant defense systems acting as positive

signals in gene control on antioxidant mRNA enzymes expression,7 and breast cancer is an illness driven by estrogen. Dietary fat is one of the most investigated nutrients in relation to breast cancer in epidemiological, experimental, and clinical studies,8,9 and it has been already proven to be the positive association between high fat intake and carcinogenesis. Also, high dietary fat can stimulate lipid peroxidation, thus favoring oxidative stress in cancer patients.10 Some studies have also shown that reduced fat intake is associated to lower recurrence rates and longer survival after breast cancer diagnosis.6,11 However, most of the studies related to the role of dietary fat in breast cancer have been performed using animal models, wherein these animals are fed with high levels of dietary fats (15%
20%).12 Such high fat feeding in animals try to closely resemble the fat intake in humans, but it also could inadvertently exacerbate some of the physiopathological mechanisms that might already be affected. In fact, these percentages of fat imply an extremely high amount of fat for rodents, which is very far from their normal/physiological intake.13 Therefore we have analyzed the influence of several dietary fats with normolipidic content (4% fat) on oxidative stress parameters and antioxidant defense systems in animals with breast cancer induced by the administration of N-methyl-nitrosourea (NMU). We have tested the use of extra-virgin olive oil (EVOO), rich in monounsaturated fatty acids (MUFAs), mainly oleic acid (18:1 n-9), and the main source of fat in Mediterranean countries, refined sunflower oil (SO), rich in n-6 polyunsaturated fatty acids (PUFAs), and oleic acid-enriched SO (OAESO), with an equilibrated content between both types of fatty acids. In addition, the hormonal status of the animals have been also examined

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00046-8 © 2021 Elsevier Inc. All rights reserved.

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because breast cancer is an illness driven by estrogen,14 because, as stated before, estrogen deficit could be responsible for the decreased activities of some enzymatic antioxidant defense systems. In fact, these hormones act as positive signals in mRNA expression of antioxidant enzyme genes.7 This has allowed increasing our knowledge on the influence of several types of dietary fats on the redox status, the nonenzyme and the enzyme antioxidant defense systems, the hormonal status, and their relationship with the carcinogenesis process under more physiological dietary conditions.

29.2 Dietary fat and carcinogenesis parameters Dietary fats have been hypothesized to play a key role in the etiology of breast cancer malignancy.15,16 Thus high dietary intake of n-6 PUFAs has demonstrated a strong stimulating effect on mammary carcinogenesis, whereas high levels of MUFAs have a protective role in breast cancer.15,16 However, these studies, that have used animal models of mammary tumors, employed high levels of dietary fats (15%
20%), which purposively highlight the processes affected. However, although these high percentages of fat are comparable to the intake in humans to a certain extent, they imply an extremely high amount of fat for rodents which is very far from their natural/physiological intake. Therefore the use of normolipidic dietary fat is necessary. In these conditions the administration of different dietary fats with normolipidic content does not modify the carcinogenesis parameters in rats with breast cancer (Table 29.1). Thus latency period, defined as the days between the first NMU injection and the appearance of the first tumor, was similar in animals fed with SO and EVOO. In animals fed with OAESO, latency period is slightly shorter, but without statistical significance.

Tumor incidence, defined as the percentage of rats bearing at least one malignant mammary tumor at sacrifice, was of 81% in animals fed with EVOO, of 62% in animals fed with SO, and of 56% in animals fed OAESO. These differences were also not statistically significant. Regarding total tumor yield, defined as the cumulative total number of malignant mammary tumors in each dietary group at sacrifice, a similar nonstatistical significance was obtained between groups, with the highest values in animals fed with SO (2.2 6 0.5), the lowest in animals fed with OAESO group (1.6 6 0.3), and intermediate similar values in animals fed with EVOO (1.8 6 0.1). Finally, animals fed with OAESO also showed the highest incidence of mortality.

29.3 Dietary fat and histopathology of breast tumors Histological analysis (Fig. 29.1) of the carcinomas of the rat mammary gland of animals fed with the different dietary fats has showed papillary and cribriform patterns, extensive solid areas, and tumoral necrosis. Wellcircumscribed carcinomas with nodular appearance and zones with infiltrative pattern with intense desmoplastic reaction have been also seen.17,18 However, histopathological results of the morphological analysis (Fig. 29.2) have showed a decrease in the percentage of tubules in tumors of animals fed with SO and OAESO when compared to animals fed with EVOO. In the same way a decrease in the percentage of mitosis has been observed in the tumors of animal fed with EVOO and OAESO. Finally, significant differences were found in the final grading score between groups, with animals fed with EVOO showing 81% of type I and 19% of type II tumors, animals fed with SO showing 84% of type I and 16% of type II tumors, and animals fed with OAESO showing 100% of type I tumors. Therefore histological

TABLE 29.1 Parameters of the carcinogenesis in N-methyl-nitrosourea treated rats fed with diet containing 4% of fat constituted by extra-virgin olive oil (EVOO), refined sunflower oil (SO), and refined sunflower oil enriched with 50% oleic acid [oleic acid-enriched SO (OAESO)]. Dietary fat EVOO

SO

OAESO

Latency period (in days; mean 6 SEM)

102.2 6 3.8

100.1 6 5.9

87.3 6 4.3

Tumor incidence (%)

81.2

62.5

56.2

Mortality incidence (%)

0.0

10.0

33.3

Total tumor yield

24

22

15

Tumor yield per rat (mean 6 SEM)

1.8 6 0.1

2.2 6 0.5

1.6 6 0.3

The effects of olive oil and other dietary fats on redox status on breast cancer Chapter | 29

349

FIGURE 29.1 Histopathological characteristics of mammary tissue from healthy animals (A) and animals with breast cancer induced by NMU fed with dietary fat constituted by (B) EVOO, (C) refined SO, and (D) refined sunflower oil enriched with 50% oleic acid (OAESO). EVOO, Extra-virgin olive oil; NMU, N-methyl-nitrosourea; OAESO, oleic acid-enriched sunflower oil; SO, sunflower oil.

FIGURE 29.2 Histopathological results of the morphological analysis of NMU-induced mammary tumors in rats from the different dietary groups, displayed as the percentage of tumors from each dietary group exhibiting the different categories of the histopathological feature analyzed. NMU, N-Methyl-nitrosourea.

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analysis does demonstrate differential effects of each dietary fat at the cellular level.

29.4 Dietary fat and redox status 29.4.1 Oxidative stress To understand the relationship between these histopathological changes and dietary fat, we have analyzed the redox and hormonal status promoted by a normolipidic fat intake, since it has been extensively demonstrated that high consumption of dietary fat is linked to oxidative stress due to the production of reactive species, which promotes lipid peroxidation, high levels of DNA adducts,19 and mutations in the p53 tumor suppressor gene, which are among the most frequent alterations in breast cancer20 and are responsible for cell cycle regulation, apoptosis, facilitating DNA repair, and antagonizing angiogenesis in both human and rodent models.19,20 As a whole, they lead to genomic instability, thereby promoting carcinogenesis.6,19 We have observed that in the NMU model, no changes occur either in total antioxidant capacity (TAC), lipid peroxidation, or protein oxidation circulating biomarkers, independently of the source of dietary fat (Fig. 29.3). These data do not agree with results obtained in human studies. In women with breast cancer, changes in several circulating markers of oxidative stress, including plasma TAC,21,22 lipid peroxidation products such as malondialdehyde (MDA),6,22
24 and carbonyl group content21,22 have been found. In fact, increased level of plasma lipid peroxidation in breast cancer patients is in accordance with most of the previous findings of elevated levels of different lipid peroxidation products in circulation of breast cancer women, such as thiobarbituric acid-reactive substances (TBARS) in serum and erythrocytes, and MDA in serum, plasma, erythrocytes, and blood.22,25 Also, it has been described21 that advanced breast cancer patients showed enhanced lipid peroxidation, increased carbonyl protein content, and a reduction in TAC, indicating that advanced breast cancer patients display specific free radical oxidation mechanism involved in the propagation of inflammation consistent with cancer progression.26,27 Furthermore it has been found several similarities between advanced and early disease breast cancer patients, suggesting that the maintenance of these parameters could be necessary to ensure disease progression. It has also been described that breast carcinoma is related to an increase in lipid peroxidation in plasma with concomitant decrease in antioxidant defense capacity in blood cells, which becomes more pronounced during aging of patients.28,29 All these data support the prevalence of oxidative stress in breast cancer patients.30,31 Therefore in the NMU model, the

changes that occur in the oxidative stress parameters are not strong enough to be reflected at the systemic level using standard biomarkers.

29.4.2 Nonenzyme antioxidant defense systems On the contrary, nonenzyme antioxidant defense system mediated by reduced glutathione (GSH) and oxidized glutathione (GSSG) (Fig. 29.4) have showed a significant decrease in circulating levels of GSH in animals with mammary tumors fed with SO as the source of fat. However, no significant changes have been found in animals without mammary tumors fed with EVOO or OAESO as source of dietary fats. Furthermore, animals of all dietary groups with mammary tumors have showed increased circulating levels of GSSG, with higher percent of increase in those animals fed with SO than in animals fed with EVOO or OAESO as source of fat. As a whole, more sensitive circulating oxidative stress biomarkers such as GSH and GSSG are effectively modified with breast cancer in the NMU animal model. In fact, animals of all dietary groups with breast cancer showed increased levels of GSSG, although the animals fed EVOO and OAESO showed the lowest increase against SO diet. However, only animals fed with the SO diet have showed changes in the levels of GSH, which reflects a major protection against tumorigenesis for dietary EVOO and OAESO. When comparing with humans, Panis et al.21 have also described an initial decrease in GSH content in early disease breast cancer patients that later increase to reach control levels in advanced breast cancer patients. This data suggests that the antioxidant status in breast cancer patients (demonstrated as an increase in GSH levels) is indicative of a response to enhanced lipoperoxidation.24,32
34 Our data partially support this hypothesis. GSH levels remain low in our animals, probably due to the fact that their clinicopathological characteristics are more comparable to an early than an advanced stage. Furthermore, reduced levels of GSH are also in accordance with other authors, and it has also been hypothesized that GSH status is inversely related to malignant transformation.35,36 As a whole, breast cancer is accompanied by the decrease in the main player of the nonenzyme antioxidant defense systems (GSH), and the type of dietary fat is able to modulate GSH levels, showing a protective role for EVOO and OAESO.

29.4.3 Enzyme antioxidant defense systems Regarding enzyme antioxidant defense system (Fig. 29.5), we have shown an important decrease in superoxide dismutase (SOD) activity in animals with mammary tumors independently of the source of dietary

The effects of olive oil and other dietary fats on redox status on breast cancer Chapter | 29

351

FIGURE 29.3 TAC (A), TBARS (B), and carbonyl group content (C) in serum of animals with breast cancer induced by NMU fed with dietary fat constituted by EVOO, refined SO, and refined sunflower oil enriched with 50% oleic acid (OAESO). Results are expressed in trolox equivalents (μmol)/ mg of protein for TAC, in mg/mL for TBARS, and in nmol/mg of protein for protein carbonyls (mean 6 SEM). EVOO, extra-virgin olive oil; NMU, N-methyl-nitrosourea; OAESO, oleic acid-enriched sunflower oil; SO, sunflower oil; TAC, total antioxidant capacity; TBARS, thiobarbituric acid reactive substances.

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FIGURE 29.4 GSH (A) and GSSG (B) contents in serum of animals with breast cancer induced by NMU fed with dietary fat constituted by EVOO, refined SO, and refined sunflower oil enriched with 50% oleic acid (OAESO). Results are expressed in μM/mL (mean 6 SEM; *P , .05; **P , .01). EVOO, Extra-virgin olive oil; GSH, Reduced glutathione; GSSG, oxidized glutathione; OAESO, oleic acid-enriched sunflower oil; NMU, N-methyl-nitrosourea; SO, sunflower oil.

fat, although this decrease has been higher in animals fed a diet with SO than in animals fed with EVOO or OAESO as source of dietary fat. Also, catalase (CAT) activity has been decreased in animals with mammary tumors fed a diet with SO or OAESO, whereas no changes have been found in animals fed with EVOO as source of dietary fat. Finally, glutathione peroxidase (GPx) activity has been found decreased in animals with mammary tumors fed a diet with EVOO or SO, whereas no changes were found in animals fed with OAESO as source of dietary fat. Several reports have shown a reduction in the activities of SOD, CAT, and GPx in breast cancer, which becomes insufficient for antioxidant protection, resulting in an elevated level of lipid peroxidation and protein oxidation

mediated by the increased production of H2O2. Further decrease in antioxidant defenses caused by the reduced level of GSH with breast cancer induces more pronounced lipid peroxidation and protein oxidation.22,25,28,29,37 Oxidative damage caused by decreased capacity for H2O2 elimination is related to decrease activities of SOD and CAT, as well as to suppress direct antioxidant action of GSH. This is in agreement with previous findings that CAT has a more significant role than GPx in protecting against oxidative stress, and that CAT is at least as important as GSH in cellular defense against H2O2-mediated damage.38,39 Therefore dietary EVOO maintains unchanged CAT activity, which also supports a limited protective role of this dietary fat against breast cancer as opposed to other dietary fats, including OAESO with a high content in oleic acid.

The effects of olive oil and other dietary fats on redox status on breast cancer Chapter | 29

353

FIGURE 29.5 SOD (A), CAT (B), and GPx (C) activities in serum of animals with breast cancer induced by NMU fed with dietary fat constituted by EVOO, refined SO, and refined sunflower oil enriched with 50% oleic acid (OAESO). Results are expressed in U/mL (mean 6 SEM; *P . .05; **P , .01). CAT, Catalase; EVOO, extra-virgin olive oil; GPx, glutathione peroxidase; OAESO, oleic acidenriched sunflower oil; NMU, N-methyl-nitrosourea; SO, sunflower oil; SOD, superoxide dismutase.

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29.5 Dietary fat and hormonal status in breast cancer We have also analyzed circulating levels of estradiol and progesterone in the different groups analyzed according to dietary fat (Fig. 29.6). We have found decreased circulating levels of estradiol and increased circulating levels of progesterone in animals with breast cancer depending on the source of dietary fat, with the animals fed EVOO, the only ones which maintain their estrogen levels unchanged. The antioxidant effect of estrogens has been related not only to a direct free radical scavenging activity40,41 but also to the upregulation of antioxidant enzymes.42,43 It is widely described in vitro and in vivo that estrogens correlate positively with antioxidant enzyme expression throughout the menstrual cycle.40,41,44 Bellanti et al.7

have recently described that estrogen deficit could be responsible for the decreased activities of SOD and GPx but not CAT, which probably is not regulated by estrogens, at least in humans. Also, estrogen acts as a positive signal in gene control of antioxidant mRNA enzyme expression which, in turn, modulates redox balance. It seems that the regulatory effects of estrogen are mediated by transcriptional activation of estrogen-responsive genes, involving intracellular estrogen receptors.45 As liganddependent transcriptional factors, the hormone-bound estrogen receptors interact with estrogen response elements to stimulate several genes in estrogen-responsive tissues and to regulate gene transactivation.46 Estrogens in vitro activate MAPK and NFκB,47 driving the expression of the antioxidant enzymes SOD and GPx.42 CAT is especially important in the case of limited GSH content

FIGURE 29.6 Estradiol (A) and progesterone (B) levels in serum of animals with breast cancer induced by NMU fed with dietary fat constituted by EVOO, refined SO, and refined sunflower oil enriched with 50% oleic acid (OAESO). Results are expressed in pg/mL for estradiol and ng/mL for progesterone (mean 6 SEM; **P , .01). EVOO, Extravirgin olive oil; OAESO, oleic acid-enriched sunflower oil; NMU, N-methyl-nitrosourea; SO, sunflower oil.

The effects of olive oil and other dietary fats on redox status on breast cancer Chapter | 29

or reduced GPx activity and plays a significant role in the development of tolerance to oxidative stress in the adaptive response of cells.48 Bellanti et al.7 have also hypothesized that the regulation of CAT is not influenced by estradiol levels despite the variations in GSH and GPx expression; their study showed that circulating redox status is closely correlated to estrogen levels. Further studies are required to much better understand the influence of estrogens on the circulating antioxidant defense systems in rodents. However, these findings strongly suggest that sex hormones may control, almost in part, antioxidant gene expression and, therefore, their corresponding enzyme antioxidant defense systems.

29.6 Conclusion Normolipidic content of several dietary fats do not influence carcinogenesis parameters in the NMU model of rat breast cancer but modify the histopathology. However, although serum oxidative stress biomarkers such as TAC, lipid peroxidation, and protein oxidation are not able to demonstrate the existence of an altered redox status in animals with mammary tumors induced by NMU, more sensitive circulating biomarkers such as GSH and GSSG showed both changes with breast cancer and the influence of the source of dietary fat. Furthermore, circulating enzyme antioxidant defense systems are also affected by breast cancer, and the source of dietary fat specifically influences the enzyme activities that are modified. Therefore particular changes in the antioxidant defense systems are mechanisms that link dietary fat to breast cancer, and EVOO shows limited beneficial effects. Also, each dietary fat could show a different influence (positive or negative) on the several stages of the promotion, development, and/or progression of breast cancer.

Mini-dictionary of terms Angiogenesis

Apoptosis

Cancer

Cribriform

Tumor angiogenesis is the growth of new blood vessels that tumors need to grow. This process is caused by the release of chemicals by the tumor and by host cells near the tumor. A form of cell death in which a programmed sequence of events leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis plays a crucial role in developing and maintaining the health of the body by eliminating old cells, unnecessary cells, and unhealthy cells. Term used to indicate malignant neoplasms, which usually are invasive, may metastasize, and recur after attempted removal. Morphologic structure that is pierced by numerous small holes.

Desmoplastic

Monounsaturated fatty acids Necrosis

N-methylnitrosourea

Oxidative stress

Papillary

Polyphenols

Polyunsaturated fatty acids Reactive oxygen species

355

This term refers to the growth of fibrous or connective tissue. Some tumors elicits a desmoplastic reaction, the pervasive growth of dense fibrous tissue around the tumor. An unsaturated fatty acid whose carbon chain has one double or triple valence bond per molecule. The death of cells or tissues from severe injury or disease, especially in a localized area of the body. A member of the class of N-nitrosoureas that is urea in which one of the nitrogens is substituted by methyl and nitroso groups. It has a role as a carcinogenic agent, a mutagen, a teratogenic agent, and an alkylating agent. A condition of increased oxidant production in cells characterized by the release of free radicals and resulting in cellular degeneration. A tumor that looks like long, thin “finger-like” growths. These tumors grow from tissue that lines the inside of an organ. Compounds found in many plant foods that can be grouped into flavonoids, phenolic acid, polyphenolic amides, and other polyphenols. They act as antioxidants and protect cells and body chemicals against damage caused by free radicals that contribute to tissue damage in the body. An unsaturated fatty acid whose carbon chain has more than one double or triple valence bond per molecule. A type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell. A build-up of reactive oxygen species in cells may cause damage to DNA, RNA, and proteins, and may cause cell death. Reactive oxygen species are free radicals.

Implications for human health and disease prevention Most of the beneficial effects suggested for EVOO on cancer prevention have been attributed not only to its particular fatty acid composition but also to its content in polyphenols such as tyrosol and hydroxytyrosol.49
51 However, the content of these compounds in EVOO is pretty variable, not only between the types of olive fruit used but also in the same type of olive fruit depending on the geographical distribution and/or the year of recollection. For example, we have found a content of tyrosol and hydroxytyrosol between 3.0
11.9 and 4.2
22.0 mg/kg, respectively, in several Picual-type EVOOs, between 0.9
5.8 and 1.1
13.3 mg/kg, respectively, in several Arbequina-type EVOOs and between 4.6
10.6 and 10.1
18.7 mg/kg, respectively, in several Hojiblancatype EVOOs. Therefore our results support that if the

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putative beneficial effects of dietary EVOO are due, at least in part, to its antioxidant content, these benefits would not be applicable to all types of EVOO, but only to those EVOOs particularly rich in polyphenols. Therefore the information regarding the main antioxidant molecule content, or alternatively, the TAC of the EVOOs should be included in the consumer information labels of all commercially available EVOOs, as an index of the antioxidant quality of the product, which could also increase their commercial value. This will allow the consumer to choose dietary fats with real, but not marketing-related, beneficial effects of these oils on health.

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123. 4. Xu L, Peterson LL. The impact of diet on breast cancer outcomes. Curr Nutr Rep. 2019;8(3):212
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S45. 6. Makarem N, Chandran U, Bandera EV, Parekh N. Dietary fat in breast cancer survival. Annu Rev Nutr. 2013;33:319
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536. 11. Rock CL, Demark-Wahnefried W. Nutrition and survival after the diagnosis of breast cancer: a review of the evidence. J Clin Oncol. 2002;20(15):3302
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13. Reeves PG, Nielsen FH, Fahey Jr. GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123(11):1939
1951. 14. Aupperlee MD, Zhao Y, Tan YS, et al. Puberty-specific promotion of mammary tumorigenesis by a high animal fat diet. Breast Cancer Res. 2015;17(1):138. Available from: https://doi.org/ 10.1186/s13058-015-0646-4 [published Online First: Epub Date]|. 15. Escrich E, Moral R, Grau L, Costa I, Solanas M. Molecular mechanisms of the effects of olive oil and other dietary lipids on cancer. Mol Nutr Food Res. 2007;51(10):1279
1292. 16. Escrich E, Solanas M, Moral R, Escrich R. Modulatory effects and molecular mechanisms of olive oil and other dietary lipids in breast cancer. Curr Pharm Des. 2011;17(8):813
830. 17. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19(5):403
410. 18. Solanas M, Moral R, Escrich E. The stimulating effect of a high-fat n-6 polyunsaturated diet on rat DMBA-induced mammary tumors is not related to changes in c-Ha-ras1 mRNA tumor expression. Nutr Res. 2001;21:1261
1273. 19. Kang DH. Oxidative stress, DNA damage, and breast cancer. AACN Clin Issues. 2002;13(4):540
549. 20. Gasco M, Shami S, Crook T. The p53 pathway in breast cancer. Breast Cancer Res. 2002;4(2):70
76. 21. Panis C, Victorino VJ, Herrera AC, et al. Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer. Breast Cancer Res Treat. 2012;133(3):881
888. 22. Ramirez-Exposito MJ, Sanchez-Lopez E, Cueto-Urena C, et al. Circulating oxidative stress parameters in pre- and postmenopausal healthy women and in women suffering from breast cancer treated or not with neoadjuvant chemotherapy. Exp Gerontol. 2014;58C:34
42. 23. Gonenc A, Erten D, Aslan S, Akinci M, Simsek B, Torun M. Lipid peroxidation and antioxidant status in blood and tissue of malignant breast tumor and benign breast disease. Cell Biol Int. 2006; 30(4):376
380. 24. Rajneesh CP, Manimaran A, Sasikala KR, Adaikappan P. Lipid peroxidation and antioxidant status in patients with breast cancer. Singap Med J. 2008;49(8):640
643. 25. Sharma A, Rajappa M, Saxena A, Sharma M. Antioxidant status in advanced cervical cancer patients undergoing neoadjuvant chemoradiation. Br J Biomed Sci. 2007;64(1):23
27. 26. Badid N, Ahmed FZ, Merzouk H, et al. Oxidant/antioxidant status, lipids and hormonal profile in overweight women with breast cancer. Pathol Oncol Res. 2010;16(2):159
167. 27. Negre-Salvayre A, Coatrieux C, Ingueneau C, Salvayre R. Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. Br J Pharmacol. 2008;153(1):6
20. 28. Kasapovic J, Pejic S, Stojiljkovic V, et al. Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages after chemotherapy with 5-fluorouracil, doxorubicin and cyclophosphamide. Clin Biochem. 2010;43(16
17):1287
1293. 29. Kasapovic J, Pejic S, Todorovic A, Stojiljkovic V, Pajovic SB. Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages. Cell Biochem Funct. 2008;26(6):723
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30. Mannello F, Tonti GA, Pagliarani S, et al. The 8-epimer of prostaglandin F(2alpha), a marker of lipid peroxidation and oxidative stress, is decreased in the nipple aspirate fluid of women with breast cancer. Int J Cancer. 2007;120(9):1971
1976. 31. Tas F, Hansel H, Belce A, et al. Oxidative stress in breast cancer. Med Oncol. 2005;22(1):11
15. 32. Franco R, Cidlowski JA. Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ. 2009;16(10):1303
1314. 33. Ravuri C, Svineng G, Pankiv S, Huseby NE. Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of gamma-glutamyltransferase expression. Free Radic Res. 2011;45(5):600
610. 34. Zuccari DPD, Jardim BV, Moschetta MG, et al. The prognostic value of glutathione (GSH) and glutathione peroxidase (GPX) in breast cancer. Eur J Cancer. 2012;48:S215. 35. Kumaraguruparan R, Subapriya R, Kabalimoorthy J, Nagini S. Antioxidant profile in the circulation of patients with fibroadenoma and adenocarcinoma of the breast. Clin Biochem. 2002;35(4):275
279. 36. Yeh CC, Hou MF, Tsai SM, et al. Superoxide anion radical, lipid peroxides and antioxidant status in the blood of patients with breast cancer. Clin Chim Acta. 2005;361(1
2):104
111. 37. Kopanski Z, Grabowska M, Kosiniak-Kamysz A, et al. The influence of antineoplastic chemotherapy on the glutathione enzymes activity in the blood. Biofactors. 2004;22(1
4):79
82. 38. Mueller S, Riedel HD, Stremmel W. Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes. Blood. 1997;90(12):4973
4978. 39. Glorieux C, Sandoval JM, Dejeans N, et al. Evaluation of potential mechanisms controlling the catalase expression in breast cancer cells. Oxid Med Cell Longev. 2018;. Available from: https://doi.org/10.1155/ 2018/5351967. Artn 5351967. [published Online First: Epub Date]|. 40. Bednarek-Tupikowska G, Tupikowski K, Bidzinska B, et al. Serum lipid peroxides and total antioxidant status in postmenopausal women on hormone replacement therapy. Gynecol Endocrinol. 2004;19(2):57
63. 41. Bednarek-Tupikowska G, Tworowska U, Jedrychowska I, et al. Effects of oestradiol and oestroprogestin on erythrocyte antioxidative enzyme system activity in postmenopausal women. Clin Endocrinol (Oxf). 2006;64(4):463
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42. Borras C, Gambini J, Lopez-Grueso R, Pallardo FV, Vina J. Direct antioxidant and protective effect of estradiol on isolated mitochondria. Biochim Biophys Acta. 2010;1802(1):205
211. 43. Chang SP, Yang WS, Lee SK, Min WK, Park JS, Kim SB. Effects of hormonal replacement therapy on oxidative stress and total antioxidant capacity in postmenopausal hemodialysis patients. Ren Fail. 2002;24(1):49
57. 44. Baltgalvis KA, Greising SM, Warren GL, Lowe DA. Estrogen regulates estrogen receptors and antioxidant gene expression in mouse skeletal muscle. PLoS One. 2010;5(4):e10164. 45. McDonnell DP, Wijayaratne A, Chang CY, Norris JD. Elucidation of the molecular mechanism of action of selective estrogen receptor modulators. Am J Cardiol. 2002;90(1A):35F
43F. 46. Green S, Chambon P. Nuclear receptors enhance our understanding of transcription regulation. Trends Genet. 1988;4(11):309
314. 47. Ruiz-Ramos R, Lopez-Carrillo L, Rios-Perez AD, De VizcayaRuiz A, Cebrian ME. Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins, NF-kappaB activation and cell proliferation in human breast cancer MCF-7 cells. Mutat Res. 2009;674(1-2):109
115. 48. Goyal MM, Basak A. Human catalase: looking for complete identity. Protein Cell. 2010;1(10):888
897. 49. Carrera-Gonzalez MP, Ramirez-Exposito MJ, Mayas MD, Martinez-Martos JM. Protective role of oleuropein and its metabolite hydroxytyrosol on cancer. Trends Food Sci Tech. 2013; 31(2):92
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Chapter 30

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Eva Batanero and Mayte Villalba Deparment of Biochemistry and Molecular Biology, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain

Abbreviations 3D aa CaBP CtD Cys GST His MS N-glycan NHBE nsLTP NtD OA OAS PBMC pI PLG PME PPIA PR SCIT SLIT SOD SSP TLP

three-dimensional amino acid Ca21-binding protein C-terminal domain cysteine residue glutathione S-transferase histidine mass spectrometry asparagine-linked glycan normal human bronchial epithelial nonspecific lipid-transfer protein N-terminal domain occupational asthma oral allergy syndrome peripheral blood mononuclear cell isoelectric point poly(lactide-co-glycolide) pectin-methylesterase peptidyl prolyl cis
trans isomerase A or cyclophilin A pathogenesis-related subcutaneous immunotherapy sublingual immunotherapy superoxide dismutase seed storage protein thaumatin-like protein

30.1 Introduction Respiratory allergy is a major public health problem that affects the quality of life of millions of children and adults, and its prevalence has dramatically increased in many countries throughout the last few decades, particularly in those industrialized, where it affects approximately 30% of the population. This

disorder is characterized by raised IgE-antibody levels to otherwise harmless environmental antigens (so-called allergens), which are responsible for the clinical symptoms such as asthma, eczema, rhinoconjunctivitis, or anaphylactic shock, due to the activation of cells in latter encounters between the allergen and the body (Fig. 30.1). In Mediterranean countries, olive tree (Olea europaea) pollen constitutes one of the most important causes of pollinosis, being the main cause of allergic sensitization in southern Spain and some regions of Italy where this tree is extensively cultivated.1 The importance of olive pollinosis in Spain is evidenced in a study conducted in 2914 allergic patients from all over the country, in which approximately 52% of rhinoconjunctivitis patients and 37% of asthmatic patients were sensitized to olive pollen.2 Today, the olive tree cultivation has spread worldwide for its olive oil, its good-quality wood, and the olive fruit and frequently is used as ornamental plant. For this reason the number of olive pollen allergic patients is increasing in some areas of America, Australia, Japan, China, and South Africa.1 This species sheds its pollen in high concentrations (reaching a weekly average of 500 gr/m3 and exceptional daily peaks higher than 5000 gr/m3 in some areas of southern Spain) during pollination season, leading to allergic symptoms from seasonal rhinoconjunctivitis to asthma in susceptible individuals. The onset of allergic symptoms in pollen-sensitive patients is often related to the number of airborne pollen grains. For olive the threshold level of airborne pollen required to elicit clinical symptoms in sensitized patients is extremely high, around 400 gr/m3, compared to 50 gr/m3 for patients allergic to grass pollens.3 However, the threshold level appears to vary among sensitive patients and during pollination season.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00041-9 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 30.1 Mechanism of type-I allergy. Allergy involves two temporally different processes: sensitization and provocation. In an initial exposure to the allergen of susceptible individuals, the uptake of allergen by professional APCs leads to the activation of allergen-specific Th2 cells that produce key cytokines. These cytokines are involved in the class switching of B-cells to IgE synthesis. These antibodies specifically bind to a highaffinity receptor on effector cells (mast cells and basophils), resulting in allergic sensitization. Subsequent encounters with the allergen cause crosslinking of effector cell-bound IgE (provocation stage), leading to the cell activation and the rapid secretion of a wide array of mediators responsible for the allergic symptoms. APCs, Antigen-presenting cells; Th2, T helper 2.

FIGURE 30.2 Allergogram of olive pollen. Olive pollen proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extract and stained with Coomassie blue (CB panel) or detected with individual sera from patient allergic to olive pollen (IgE panel). The IgEbinding patterns were selected as representative for variability and complexity of responses in individuals suffering from olive pollinosis. Major and minor allergens are indicated by a color code. Molecular masses of standard proteins are indicated in kDa.

Over the last few years, intense efforts have been made to define the allergenic components (allergogram) of olive pollen,1,4 after pioneering studies in the 1980s.5 The knowledge of the complete olive pollen allergogram would enable us to understand the mechanisms involved in the development of pollinosis as well as to design

novel strategies for an accurate diagnosis and a safer and more effective immunotherapy. Standard laboratory techniques have shown that olive pollen extract has a very complex and heterogeneous allergogram, containing at least 20 protein bands with allergenic activity (Fig. 30.2). To date, 14 allergens from olive pollen have already been

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Chapter | 30

identified, isolated, and characterized—named Ole e 1
15 according to the recommendations published by the World Health Organization and International Union of Immunological Societies (WHO/IUIS). However, new allergens are waiting to be detected and identified. In this sense the advances in both genomic and proteomic tools—for example, two-dimensional electrophoresis in combination with mass spectrometry (MS)—have allowed the detection and identification of four novel allergens of olive pollen with clinical relevance: Ole e 11,6 Ole e 12,7 Ole e 14,8 and Ole e 15.9 Several olive pollen allergens have been defined as major allergens, because they exhibit prevalence values higher than 50%, such as Ole e 1, in contrast to the minor allergens (e.g., Ole e 3), which are recognized by less than 50% of sensitized patients. However, the prevalence of olive pollen allergens (major vs minor allergens) is related with the levels of airborne pollen, and therefore, with the geographical area of the sensitized population.3,10,11 While Ole e 1 seems to be the only clinical relevant allergen involved in olive pollinosis in areas of low/intermediate pollen exposure, minor allergens that rarely are recognized by allergic patients in normally pollen-exposed areas become major allergens (e.g., Ole e 6, Ole e 7, Ole e 9, Ole e 10, and Ole e 11) in locations with high levels of pollen exposure (Fig. 30.2). According to this fact, it has been suggested that allergic patients from areas with extremely high levels of pollen exposure exhibit different and more complex allergogram, as determined by molecular diagnosis, when compared with patients living in areas with lower pollen count. Finally, it should be noted that Ole e 15 is more prevalent among child population with olive pollinosis.9 Regarding allergogram heterogeneity, 45 different allergograms were observed when 8 olive pollen allergens were tested in 156 patients from Jae´n with olive pollinosis.3 The main features of olive pollen allergens are summarized in Table 30.1. Olive pollen allergens have been classified into 13 of the 134 AllFam families, according to their biochemical functions.12 Olive pollen allergens reveal to be restricted to a small number of taxonomically diverse plant families such as Ole e 1 or are ubiquitous (panallergens) such as Ole e 2 (profilin), Ole e 3 (polcalcin), and Ole e 15 (cyclophilin). Pan-allergens constitute families of homologous and structurally related proteins from different species responsible for extensive IgE crossreactivity among a variety of allergic sources. Interestingly, the prevalence of pan-allergens Ole e 2 and Ole e 3 is usually low (around 20%), indicating that they might not be clinical relevant olive pollen allergens and that the sensitization to them might be caused by other sources. Biochemical and molecular studies to characterize olive pollen allergens have shown that polymorphism is a general feature. Ole e 1, Ole e 5, and Ole e 7 present a high degree of polymorphism. For Ole e 9 a relatively low, although still

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significant, degree of polymorphism has been detected. Allergen polymorphism is closely related to the cultivar origin of olive pollen,13 as it has been described for other sources of plant allergens such as date palm (Phoenix dactylifera) or birch (Betula pendula) pollens and apple (Malus domestica). In this context, it has been speculated that a broad polymorphism could be involved in the physiology of the olive tree reproductive system, including the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance.13 Regarding the clinical implications of allergen polymorphism, the differences in allergen composition in cultivars, particularly in Ole e 1, are responsible for important differences in the allergenic activity.13 The concentration of Ole e 9 has also been reported to vary several hundred times between different pollen batches. Preliminary studies regarding the levels of expression for Ole e 2, Ole e 3, Ole e 5, and Ole e 6 in the major olive cultivars indicate the presence of significant differences.13 This is a major concern for clinicians since reliability of pollen extracts used for clinical purposes is required for an accurate diagnosis, and effective and safe immunotherapy. Pollen extracts should imitate as much as possible the composition of the panel of allergens to which patients are normally exposed and are reactive. Therefore the quantification of both major and minor allergens must be an integral part of the standardization of olive pollen extracts used in clinical applications. Furthermore, it has been reported that olive pollen allergens are quickly released in different rates from pollen, having an impact on the sensitization and the elicitation of allergic symptoms. High yields of Ole e 1, Ole e 6, and Ole e 7 are obtained after 15 min of pollen hydration in mild saline buffers, and others such as Ole e 3, Ole e 9, and Ole e 10 are extracted after 3 h. This property facilitates the accessibility of the proteins once the pollen grains come in contact with respiratory mucosa, explaining the fast induction of allergic symptoms. Olive pollen allergy, such as an example of allergic respiratory disorders, is a multifactorial disease that results from the interaction between both environmental and genetic factors. A strong association between HLA class II antigens DR7 and DQB2 and the IgE response to Ole e 1, Ole e 2, and Ole e 3 have been reported in unrelated populations.3,14 HLA-DR2 antigen is associated with the IgE response to Ole e 10. Many other factors related to the developed-country lifestyle, including atmospheric pollution, exposure to tobacco smoke, diet, and hygiene habits, may also have a considerable effect on respiratory allergy. In this sense, numerous studies suggest that the functional state of the airway epithelium— the first line of defense against inhaled substances, including respiratory virus, air pollutants, and aeroallergens— can influence the host immune response and clinical outcome.15 Thus olive pollen allergy represents an interesting

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 30.1 Olive pollen allergens and their main features. Olive pollen allergens Allergen

MW (kDa)a

pIb

Prevalence (%)

Cross-reactivity

Family

Recombinant expression

Ole e 1

16.3/18.5*

5.5
6.5*

55
90

Oleaceae

Ole e 1-like

Escherichia coli Pichia pastoris Arabidopsis thaliana

Ole e 2

14
16**

5.1**

24

Pollens, plant foods, and latex

Profilin

E. coli

Ole e 3

9.2*

4.3*

20
30

Pollens

Polcalcin

E. coli A. thaliana

Ole e 4

32**

4.6
5.1*

80

ND

Degradation product of Ole e 9




Ole e 5

16**

5.7**

35

ND

Superoxide dismutase

E. coli

Ole e 6

5.8*

5.8**

10
55

Oleaceae (PD)

Ole e 6

P. pastoris

Ole e 7

9.8
10.3*

$ 9*

47

Pollens (Oleaceae), plant foods (mainly peach and pear)

Nonspecific lipidtransfer protein

P. pastoris

Ole e 8

18.8*

4.5**

5

Oleaceae (PD)

Ca21-binding protein

E. coli A. thaliana

Ole e 9

46.4*

4.8
5.4*

65*

Pollens, plant foods, and latex

1,3-β-Glucanase

P. pastoris (NtD and CtD)

Ole e 10

10.8*

5.8*

55*

Pollens, palnt foods, and latex

Carbohydratebinding module 43

P. pastoris S. frugiperda

Ole e 11

39.6*

6.3
9.3*

55.9
75.6

Oleaceae (PD)

Pectin methylesterase

P. pastoris

Ole e 12

34.2
34.9*

4.8
5.8*

33

Pollens and peach

Isoflavone reductase

E. coli

Ole e 14

46.5**

5.9
6.5*

19

Pollens

Polygalacturonase

E. coli

Ole e 15

19.4***

8.7**

12.5
14.3

Pollens, plant foods, and animal extracts

Cyclophilin

E. coli

c

ND, Not determined; PD, preliminary data. a Molecular mass determined by mass spectrometry (*) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (**). For Ole e 1, molecular masses of nonglycosylated/glycosylated forms are shown. (***) Molecular mass of His6-tagged recombinant Ole e 15 determined by mass spectrometry. b Isoelectric point (pI) determined experimentally (*) or deduced from the amino acid sequence (**). c Data from allergic patients living in a region with high exposure to olive pollen.

model to study the allergic response; therefore the identification of allergens of olive pollen will be required for the development of rational strategies for standardization, patient diagnosis, and therapy, which may increase the quality of life of allergic patients.

30.2 Ole e 1 as a marker for sensitization to Oleaceae pollens Ole e 1 is the main allergen of olive pollen with a prevalence ranging from 55% to 90%, depending on the

geographical areas. It is the most abundant protein of olive pollen, representing up to 20% of the total protein content of pollen in the most profuse varieties. Ole e 1 contributes significantly to the total allergenicity of the olive pollen extracts, and its concentration correlates well with the total allergenic potency of the extracts. Moreover, it also represents the main sensitizing agent within the protein family of Ole e 1-like proteins, and it is a diagnosis marker for sensitization Oleaceae pollens.16 Ole e 1 is a polymorphic and acidic (isoelectric point, pI, 5.5
6.5) glycoprotein of 145 amino acid (aa) residues

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Chapter | 30

with a glycan at position asparagine-111 (N-glycan).17,18 Because of the presence of the glycan, Ole e 1 exhibits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a pattern of multiple bands with two main components, a glycosylated form of 20 kDa (85% of the whole allergen) and a nonglycosylated variant of 18.5 kDa (10%). Two minor variants corresponding to the hyperglycosylated (22 kDa) and the 20 kDa dimer (40 kDa) are frequently present in Ole e 1 preparations. Its single polypeptide chain contains six cysteine residues that are involved in three disulfide bridges: Cys19
Cys90, Cys22
Cys131, and Cys43
Cys78.19 Ole e 1 belongs to a large family of homologous proteins (Ole e 1-like proteins) that are specifically expressed in pollen tissue, and it has been suggested to be involved in fertilization events: pollen hydration and/or pollen germination.13,20 This is in agreement with Ole e 1 localization in the endoplasmic reticulum, pollen wall, and tapetum and in the outer region of the pollen exine. This family comprises allergenic members such as Ole e 1, Fra e 1 (Fraxinus excelsior), Lig v 1 (Ligustrum vulgare), Syr v 1 (Syringa vulgaris)—members of Oleaceae family—Pla l 1 (Plantago lanceolata), Che a 1 (Chenopodium album), Lol p 11 (Lolium perenne), Phl p 11 (Phleum pratense), Aca f 1 (Acacia farnesiana), Cros s 1 (Crocus sativu), Pro j 1 (Prosopis juliflora), and Sal k 5 (Salsola kali), as well as nonallergenic members such as BB18 from Betula verrucosa. Other members of the family are known through their corresponding nucleotide sequences, and their derived mature proteins have not been isolated or characterized; therefore their potential allergenicity has not been explored: LAT52 (Lycopersicon esculentum), Zmc13 (Zea mays), and OSPG (Oryza sativa), and putative proteins from Phalaris coerulescens, Sambucus nigra, and Arabidopsis thaliana. The position of the six Cys is conserved in all members, suggesting similar three-dimensional (3D) structures. In this sense the 3D structure of Pla l 1—the Ole e 1-like protein from plantain pollen—has been solved, being formed by a seven-stranded β-barrel structure stabilized by three disulfide bonds.21 In addition, all these proteins exhibit a putative N-glycosylation site, which seems to be conserved among members of the same taxonomic family. This fact could be related to a potential role for the N-glycan in modulation of the biological function and/or discrimination between species. A high degree of cross-reactivity has been described among Oleaceae pollens, and Ole e 1-like proteins are one of the molecules involved in such process.10,22 It has been demonstrated that epitopes of Ole e 1 are only present in Oleaceae pollens but not in unrelated ones.16 This could be explained by comparing the aa sequence of the members of this family: identity values ranging from 86% to 89% are obtained for the Oleaceae counterparts, whereas low but significant identities (32%
39%) are displayed in the remaining members. Moreover, isoforms of Ole e 1 and Ole e 1-like allergens from Oleaceae, which differ only in a few aa, show

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differences in both IgE- and IgG-reactivity. Finally, ash pollen is an important cause of allergy in Central Europe, whereas privet and lilac—two ornamental plants—have been described as elicitors of allergic symptoms in conditions of local exposure.23 Thus the study of Ole e 1-like allergens will provide knowledge of the molecular organization of allergenic epitopes, which is invaluable in terms of allergen standardization and diagnostic as well as in the designing of novel allergen-specific immunotherapy. T-cell24 and B-cell25 epitopes of Ole e 1 have been analyzed. Ca´rdaba et al.24 have defined the regions 91
102 and 109
130 of Ole e 1 as immunodominant T-cell epitopes but display no IgE-binding capacity. At least four B-cell epitopes have been defined by means of 12-aa overlapping synthetic peptides and recombinant large fragments, being the C-terminal an immunodominant region and the tyrosine at position 141 a critical residue for IgE binding.25 Interestingly, Wildner et al.26 reported that the in vitro endolysosomal degradation of Ole e 1 with the cathepsin S protease generated a peptide profile that substantially overlapped with the T-cell epitopes identified for this allergen by Ca´rdaba et al.24 Finally, Ole e 1 has been used as a model aeroallergen for studying the role of the functional state of bronchial epithelium in the allergic response.27 For this purpose, primary normal human bronchial epithelial (NHBE) cells from two healthy donors were cultured in air
liquid interface conditions on transwell supports that allow one to obtain a pseudostratified epithelium with a mucociliary phenotype, which mimics human airway epithelium in vivo. Exposure of differentiating NHBE cells to Ole e 1 neither impaired the epithelial barrier establishment nor disrupted the apical junctional complex formation as indicated by transepithelial electrical resistance measurements, ultrastructural studies, and western blot analyses. However, the exposure to the allergen altered the cytokine secretion pattern of NHBE cells, which showed a strong dependence of both the differentiation state of the bronchial epithelial cells at the moment of exposure and the donor features. In addition, Ole e 1 has been used as aeroallergen model to describe the interfacial activity of an aeroallergen, once it reach the epithelial lining fluid. In this sense, Ole e 1 tended to interact with liquid-ordered and liquid-condensed domains, generated in the context of lipid monolayers as models of eukaryotic cell membrane and pulmonary surfactant, using Langmuir-derived balances.28

30.3 Ole e 2 and Ole e 10, two allergens associated with asthma Quiralte et al.3 reported that Ole e 2 and Ole e 10 show a statistically significant association with asthma.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

Ole e 2, an acidic (pI 5.1) 14
16 kDa protein, belongs to the well-known pan-allergen family of profilins.29 Its molecular and immunological properties did not differ from those of other profilins. Ole e 2 exhibits polymorphism, with important implications for the 3D structure of the molecule. Profilins contain a large number of members from plant and animal tissues which are involved in the assembly of actin filaments, being recognized as pan-allergens from fruits, vegetables, pollens, and latex. Interestingly, the comparison of plant profilins with homologous from fungi and vertebrates revealed that they form a highly conserved family with sequence identities between 70% and 85% among each other, but low identities (30%
40%) compared with nonallergenic profilins from other eukaryotes, including human beings. This explains their implication in crossreactivity between profilins from different vegetable sources, and their low clinical relevance (around 20% of prevalence). However, because of sensitivity to thermal denaturalization and gastric proteolysis, they are involved in the cross-reactivity between pollens and plant foods—also known as oral allergy syndrome (OAS) or pollen
fruit syndrome—whose symptoms are elicited after eating raw foods and limited to the oropharyngeal mucosa. Ole e 10, a Cys-rich small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as major allergen from olive pollen in high exposure areas (prevalence of 55%).30 Ole e 10 shows identity with deduced sequences from A. thaliana genes (42%
46% identity), with the noncatalytic C-terminal domain (CtD) of plant 1,3-β-glucanases (27%
53% identity) such as Ole e 9, and with Cys-box domains from three families of 1,3-β-glycosyltransferases involved in yeast development: Epd1, Gas1p, and Phr2 families (23% identity).30 However, it is important to remark that Ole e 10 is an independent protein that defines a novel family of carbohydrate-binding modules, so-called CBM43.31 The ability of Ole e 10 to bind specifically 1,3-β-glucans, its localization within the mature pollen grain inside Golgi-derived vesicles, and its colocalization with callose (1,3-β-glucans) in the growing pollen tube suggests a role for this protein in the pollen tube wall reformation during germination.31 Regarding its allergenic activity, Ole e 10 is an allergen per se that can act as a primary sensitizer agent: it defines a new family of pan-allergens that shows notable intra- and interspecies cross-reactivity and is a powerful candidate for pollen
latex
fruit syndrome.30 Although it is a relevant allergen, Ole e 10 exhibits low intrinsic antigenicity because no specific IgG antibodies were elicited after different attempts to immunize animals with pure antigen.30 This fact, added to its biological activity and retardation release from pollen after hydration allow us to speculate that this allergen could be expelled from the pollen linked to particles such as 1,3-β-glucans—it has been shown that

this glycan promote airway allergic response in humans. In this way the allergenic potential of Ole e 10 could be increased, contributing to eliciting asthma in allergic patients.

30.4 Ole e 3 and Ole e 8: Ca21 -binding allergens Ole e 3 and Ole e 8 are two Ca21-binding proteins (CaBPs) of the EF-hand family. Ole e 3 is a small (9.2 kDa) and acidic protein (pI 4.3) with a single polypeptide chain which does not contain cysteines.32 Ole e 3 belongs to the widespread polcalcin family, which is characterized by its specific expression in pollen and the presence of two EF-hand motifs.33 Polcalcins belong to the buffering-type CaBP subfamily and may have a role as inhibitors of cytoplasmic streaming of Ca21 in growing pollen tubes.33 The reported prevalence for this family of pan-allergens varies between 5% and 46%, and it is around 20% and 30% for Ole e 3. Members of this family show low or no polymorphism and sequence identities ranging from 64% to 92%, which explains their strong cross-reactivity. Thus diagnosis of polcalcin-sensitized patients could be achieved whatever polcalcin used, whereas for immunotherapy, the polcalcin that acts as primary sensitizer agent should be used. Ole e 8 (18.8 kDa and pI 4.5) seems to be the first member of a new family of CaBPs with four EF-hand motifs present in the pollen tissue: it is a calcium-sensor protein that should display a regulatory function and perhaps may involve it in signal transduction pathways.34 This allergen is present at very low levels in the pollen (0.02%
0.05% of total protein) and shows low prevalence (5% of prevalence).35 In addition, all sera reactive to Ole e 8 also recognize Ole e 3.34 Ole e 8 shows a low sequence identity with Ole e 3 and other CaBPs out of the EF-hand sites. Moreover, this allergen seems not to have counterparts with conserved aa sequence in other taxonomically nonrelated allergenic pollens, as significant cross-reactivity was observed only with Oleaceae.35 It is important to note that EF-hand motifs of the Ca21-binding allergens have a nonsignificant role in their IgE and IgG epitopes, and cross-reactivity; however, their IgE-binding capacity is affected by the conformational change induced by the binding/releasing of Ca21 ions.36 This property would allow designing hypoallergenic derivatives (hypoallergens) to be used in immunotherapy.

30.5 Ole e 7, a nonspecific lipid-transfer protein, and its clinical significance Ole e 7 is a basic protein (pI $ 9) with a molecular mass around 10 kDa.37 Although it is a minor allergen, its

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Chapter | 30

prevalence increases up to 47% in populations exposed to high levels of olive pollen counts (over 5000 gr/m3), such as those from the southern Spain. Interestingly, a large number of adverse reactions are recorded in patients sensitized to Ole e 7, as well as to Ole e 9, in particularly during immunotherapy; these patients are less tolerant to immunotherapy at the recommended allergen doses.38 Moreover, Ole e 7 belongs to the nonspecific lipidtransfer protein (nsLTP) family of around 9 kDa ubiquitously distributed through the plant kingdom, and whose members show sequence identities from 47% to 92%.39 The aa sequence of Ole e 7 was obtained by de novo peptide sequencing by MS after in-gel tryptic digestion.40 All nsLTPs conserve a pattern of eight cysteines, forming four disulfide bridges, which are essential for the compact fold of nsLTPs in the characteristic all-α-type structure and, therefore, the lipid binding and allergenic properties of these proteins. Interestingly, other major groups of plant food allergens, the 2S albumins and cereal α-amylase/trypsin inhibitors, show 3D structures close to nsLTP fold; these three groups constitute the prolamin superfamily. nsLTPs bind different types of lipids and are involved in plant defense mechanisms against phytopathogenic bacteria and fungi—becoming members of the pathogenesis-related 14 (PR-14) protein family—and probably in the assembly of hydrophobic protective layers of surface polymers such as cutin. Recently, it has been shown that Ole e 7 preferentially binds negatively charged phospholipids, and the lipid binding has not effect on its allergenicity.41 Several members of nsLTP family have been identified as relevant allergens in plant foods and even latex and pollens.39 Because of its ubiquitous distribution in different plant species and tissues, nsLTPs have been proposed as a novel pan-allergen with a potential spectrum of cross-reactivity comparable to that reported for profilins. Interestingly, nsLTP sensitization shows an unexpected pattern throughout Europe, with high prevalence in the Mediterranean area, but a low incidence in Northern and Central Europe. Genetic factors, differences in dietary habits, and level and composition of pollen exposure could account for such differences. Food nsLTPs are considered to be “true” food allergens that sensitize directly via oral route and are responsible of the induction of severe symptoms in many patients. These features seem to be related to their high resistance to both heat treatment and digestive proteolysis. Moreover, cross-reactivity among food nsLTPs allergens from botanically related and unrelated species has been described.39 However, concerns exit whether pollen nsLTP allergens can act as primary sensitizer agent itself via the respiratory tract leading to food allergy because of cross-reactivity to food nsLTPs. In this sense, Oeo-Santos et al.41 demonstrated that Ole e 7 cross-reacted with the

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peach nsLTP Pru p 3 (its main allergen); suggesting that Ole e 7 acts as the primary sensitizer agent, which promotes secondary sensitization to peach. This fact would explain the clinical manifestation of cosensitized to both Ole e 7 and Pru p 3 in several patients living in regions with high olive pollen-exposure such as the southern Spain.42

30.6 Ole e 9 and pollen
latex
fruit syndrome Ole e 9 is a 1,3-β-glucanase belonging to PR-2 protein family, enzymatic activity of which has been shown.43 It exhibits low sequence identity (32%
39%) to long 1,3β-glucanases from plants. It is an acidic (pI 4.8
5.4) and polymorphic glycoprotein (46.4 kDa) composed of two structurally and immunologically well-defined domains that are connected by a segment of 10
15 aa.44,45 The N-terminal domain (NtD) of 334 aa contains the 1,3-β-glucanase activity, and its 3D modeling fits well to a triose-phosphate isomerase (TIM)
barrel structure common to all known 1,3-β-glucanases. The CtD, with around 100 aa, is a CBM43 that shows sequence identity with 1,3-β-glucanases from plant tissues, the Epd1/Gas-1p/Phr2 protein families and Ole e 10.44 Its capacity to bind 1,3-β-glucans suggests a role in the binding of the substrate.22 The disulfide bridges of the molecule have been determined at positions Cys14
Cys76, Cys33
Cys94, and Cys39
Cys48.44 Its 3D structure has been resolved, representing a novel type of allergen folding which consists of two parallel α-helices, a small antiparallel β-sheet, and a 3
10 helix, all connected by long coil segments.46 Moreover, the CtD-epitope mapping shows that B-cell epitopes are mainly located on the loops.46 Ole e 9 is a major allergen in populations living in highly exposed areas with a prevalence of 65%.43 The ubiquity of 1,3-β-glucanases in higher plants suggests that they could be involved in the pollen
latex
fruit syndrome. This is supported by a study showing the involving of NtD in cross-reactivity among pollens, vegetable foods, and latex.45 Moreover, it has been shown that NtD may be a useful marker of disease in diagnosis of pollen allergic patients at risk of developing allergic symptoms to other vegetable sources.47 CtD has been described as a marker for patients who could develop asthma.47 Moreover, Ole e 9 has been identified as the causative agent of occupational rhinitis in a researcher.48

30.7 Other allergens from olive pollen: Ole e 4, Ole e 5, and Ole e 6 Ole e 4 is a partially characterized polymorphic allergen with an apparent molecular mass of 32 kDa and an acidic

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pI (4.6
5.1).49 However, it is not clear whether Ole e 4 is a genuine allergen or a proteolytic degradation product of Ole e 9, since all of the peptides obtained for the protein aligned with segments of Ole e 9. Ole e 5 (16 kDa, pI 5.7) is the Cu/Zn-superoxide dismutase (SOD) of olive pollen, with an 80%
90% sequence identity with Cu/Zn-SODs from other plants.49,50 SODs catalyze the dismutation of superoxide anions into molecular oxygen and hydrogen peroxide. It has been suggested that pollen antioxidant systems such as Ole e 5 could play a role in pollen
stigma interactions and defense because of the constitutive accumulation of reactive oxygen species/H2O2 in angiosperm stigmas.13 Ole e 5 is a minor allergen with a prevalence around 35%; however, it could be involved as a putative crossreactive allergen in the pollen
latex
fruit syndrome due to the ubiquity of this enzyme family and its sequence identity with the allergenic Cu/Zn-SOD from tomato fruit. Moreover, it has been suggested that the allergenic MnSOD from latex (Hev b 10) could be involved in crossreactivity with SODs from related and unrelated species. Ole e 6 is a small (5.8 kDa), acidic (pI 5.8), and Cysrich allergen, prevalence of which is very dependent on the degree of pollen exposure, ranging from 10% to 55%.51 It displays a peculiar twice repeated cysteine motif (Cys-X3-Cys-X3-Cys) which is also present in the aa sequence deduced (107 aa) from Tap1, a stamen-specific gene from snapdragon (Antirrhinum majus).51 The 3D structure of Ole e 6 has been resolved and consists of two parallel α-helices joined together by three disulfide bridges.52 Ole e 6 shows 59% sequence identity with a Cys-rich pollen surface molecule (Ntp-CysR, 63 aa) from Nicotiana tabacum expressed almost exclusively in developing anthers and highly abundant in pollen. It has been suggested that Ntp-CysR could interact with pistil factors. Concerning cross-reactivity, an Ole e 6-like protein has been reported in ash pollen,22 and it is expected that homologous allergens exist in other Oleaceae members.

30.8 New approaches for new allergens: Ole e 11, Ole e 12, Ole e 14, and Ole e 15 In order to complete the allergogram of olive pollen, novel approaches have been used to identify new allergens from this source. A pectin-methylesterase (PME) was identified as a new olive pollen allergen—named Ole e 11—by means of two-dimensional electrophoresis in combination with MS analysis.6 PMEs are enzymes widely used in the food processing industry; thus it is important to determine their allergenic potency. Despite that PMEs have been identified in different allergenic foods such as peach, carrot,

tomato, kiwi, and strawberry, these enzymes do not exhibit allergenicity. Update only PMEs from olive (Ole e 11), birch, and S. kali (Sal k 1) pollens have been described as allergens. Ole e 11 is a polymorphic glycoprotein with a molecular mass of about 40 kDa and a pI ranging from 6.3 to 9.3. Ole e 11 has been described as a major allergen from olive pollen in population exposed to high levels of pollen (55.9%
75.6% of prevalence). Ole e 11 shows low cross-reactivity with other members of this protein family. A similar approach was used to identify the minor allergen named Ole e 12 in olive pollen.7 Ole e 12, an acidic (pI 4.8
5.8) polymorphic protein of 34.2
34.9 kDa, is an isoflavone reductase, an enzyme that seems to be involved in plant response to stress. Ole e 12 counterparts have been identified in several plant foods (including pear, soybean, pistachio, and avocado) and pollens (such as birch, plane tree, or cypress). Although it is a minor allergen (33% of prevalence), its relevance lies in its association with sensitization to peach. The recently identified allergen Ole e 14 is a polygalacturonases, an enzyme that hydrolyzes the α-1,4-glycosidic bonds between galacturonic acid residues in the polygalacturonan, the main component of pectin from the plant cell wall.8 Ole e 14 is an acidic (5.9) polymorphic protein of 46.5 kDa, which has been produced as a recombinant histidine (His)-tagged fusion protein in Escherichia coli. It is a minor allergen with a prevalence up to 19% in a total of 482 olive-pollen-sensitized patients from different Spanish regions. This low rate is presumed to be due to the hypoallergenic isoform used in the study. In addition, it has been shown that Ole e 14 is highly implicated in cross-reactivity among pollens from different families;8 however, no studies have been done about its role in pollen-plant food cross-reactivity. The sequencing of the wild olive tree (Olea oleaster) genome in 2017 allowed San Segundo-Acosta et al.9 to delineate the olive pollen proteome and, thus, to complete its allergogram by using a high-performance bottom-up liquid chromatography
tandem MS. After blasting the identified proteins against the Allergome database, a total of 203 proteins, which are distributed into 47 allergen families, were detected in olive pollen. Among them, four potential allergens (cyclophilin, enolase, and both cytosolic and mitochondrial malate dehydrogenases) were expressed in E. coli as His6-tagged N-terminal proteins to assess their IgE-binding capacity. Only cyclophilin, a basic (pI 8.7) protein of 19 kDa, was identified as a new relevant allergen of olive pollen and named Ole e 15 according to the WHO/IUIS. Cyclophilins exhibit peptidyl-prolyl cis
trans isomerase activity, playing an important role in protein folding due they catalyze the interconversion cis
trans isomer of peptide bonds

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Chapter | 30

between proline residues and another aa residues. These enzymes are present in all cell types and they have been described as allergens in pollen, plant foods, molds, and animals. Due to the high sequence identity among members of this family, Ole e 15 and other cyclophilin allergens are involved in cross-reactivity in plants, molds, and animals (including humans); thus these allergens can be defined as pan-allergens. The prevalence of Ole e 15 was around 12.5%
14.3% in two Spanish populations, mainly among child population with olive pollinosis. It is worth mentioning that, for decades, the detection of Ole e 15 in the extract by standard methods (immunoblotting using IgE from allergic patients) has been masked by the nonglycosylated form of Ole e 1, which exhibits a similar molecular mass (18.5 kDa). Finally, eight Ole e 15-PPIA (peptidyl prolyl cis
trans isomerase A or cyclophilin A) chimeras were generated by fusing segments of the human cyclophilin A (PPIA) to Ole e 15 and used to identify the IgEbinding sites of the pollen allergen.53 Interestingly, three chimeras (4, 15, and 17) exhibited very low IgE-binding capacity, making them potential hypoallergens. Finally, the combination of T7 phage display and protein microarray technologies has allowed to identify 10 unique allergenic peptides or mimotopes in olive pollen by using only few microliters of sera from allergic patients.54 Phage display technology allows expression of heterologous peptides fused to a coat protein of a phage that infects E. coli. On the other hand, a protein microarray is a high-throughput tool for simultaneous screening and rapid analysis of a large number of samples. In this study, one peptide (OL14) was selected for expressing as fusion protein to the N-terminal end of His6
GST (glutathione S-transferase) protein, and it was found that OL14 displays low allergenicity: it was recognized by 8 out of the 92 tested sera. However, this study highlights the feasibility of using phage display technology in combination with protein microarrays in allergy research.

30.9 The role of N-glycans in olive pollen allergy Even though allergologists are skeptic about the clinical significance of glycan-related IgE-reactivity, increased number of reports have demonstrated that glycans, as part of an allergen, can elicit IgE antibodies in susceptible individuals. Many of these glycoepitopes behave as panepitopes, leading to extensive cross-reactivity among \pollens, plant foods, and insect venoms because of the presence of common monosaccharide components at specific positions along the chain.55 Thus they are so-called crossreactive carbohydrate determinants. The N-glycan of Ole e 1 is involved in antigenic and allergenic activities, being responsible for cross-reactivity

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FIGURE 30.3 N-glycans of Ole e 1. Structures of the major N-glycans isolated from Ole e 1 were determined by 1H NMR and MALDI
TOF. Minor α(1,3)-fucosylated N-glycans were also detected. αn and βn indicate α(1,n) and β(1,n) linkages (n 5 2
6).

between Ole e 1 and nonrelated glycoproteins.18,56,57 The primary structure of the N-glycan of Ole e 1 has been determined in two different studies, and the presence of glycoforms (different but closely related to glycan structures at a single N-glycosylation site) has been described. Interestingly, Castro et al.58 demonstrated by MS analysis that the N-glycan moiety displays an important variability between olive tree cultivars. Ole e 1 has a major “complex” N-glycan (GlcNAcMan3XylGlcNAc2) and one major “high mannose” N-glycan (Man7GlcNAc2) (Fig. 30.3). Also a minor “complex” N-glycan carrying an α(1,3)-fucose residue attached to the proximal glucosamine residue has also been detected.56,57 It was demonstrated that both β(1,2)-xylose and α(1,3)-fucose are involved in IgE binding, being the involvement of “high mannose” structures in IgE binding unlikely.56,57 It has been suggested that both monosaccharides could account for the immunogenicity of plant glycans in humans, since they are absent in human N-glycans. In addition, the free N-glycan of Ole e 1 is able to induce in vitro histamine release from basophils of olive pollen sensitized patients, confirming the allergenic character of this glycan.56 The role of the N-glycan of Ole e 9 in the allergenicity of the molecule has not been studied so far.

30.10 Pollensomes: natural vehicles for pollen allergens Allergic sensitization and elicitation of symptoms in susceptible individuals require the release of the allergen

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from pollen, since the intact pollen grain cannot access to the lower airways due to its aerodynamic diameter, which is usually greater than 20 μm. Numerous evidences have shown the presence in the air of both pollen-derived submicronic (,10 μm) and paucimicronic (,1 μm) particles that carry allergens, which includes mainly starch granules and polysaccharide particles; these pollen-derived particles are absent or scarce in olive pollen.59 In 2014 Prado et al.60 reported a new mechanism by which olive pollen allergens are carried in the environmental respirable aerosol based on pollensomes. Pollensomes are nanosized vesicles (28
60 nm) that are released by fresh olive pollen during in vitro germination and pollen tube growth (Fig. 30.4). These nanovesicles are enriched in pectin, one of the main component of pollen tube wall, that regulates the extensibility and stiffness of the cell wall during pollen tube growth. Further MS analysis revealed the presence of different type of proteins in pollensomes,

including, among others, proteins involved in (1) metabolism and signal transduction (i.e., glyceraldehyde-3phosphate dehydrogenase); (2) proteins synthesis and folding (i.e., PIPP), and cell wall expansion (i.e., PME); (3) cytoskeleton network (e.g., actin); (4) membrane transport (e.g., H1-ATPase 6); and (5) defense/stress response (e.g., PCBER and heat shock protein 70). Some of the identified pollensome proteins have been described as allergens, including Ole e 1, Ole e 11 (PME), Ole e 12 (PCBER), and Ole e 15 (PIPP).60 The presence of Ole e 1, Ole e 11, and Ole e 12 allergens in pollensomes has been confirmed by dot blot using specific antibodies, being the first report that describes allergens as a cargo of secreted nanovesicles.60 Pollensomes may represent a widespread vehicle for pollen allergens, since they have been isolated from other clinically relevant pollens, including both gymnosperm—pine (Pinus sylvestris)— and angiosperm—birch (B. verrucosa) and ryegrass (L. perenne)—species.61 In addition, Ole e 12 has been defined as a protein marker of pollensomes due to the detection of Ole e 12-like proteins in the four studied pollens. The allergenic activity of pollensomes has been shown according to IgE binding, human basophil activation, and skin prick test studies performed in olive pollen allergic patients.61 The assumption that secretion of pollensomes is a naturally occurring phenomenon is supported by the fact that airborne pollensomes—containing Ole e 1 and Ole e 12 allergens—were isolated from stage 7 filters of a cascade impactor collector.61 Thus the presence of pollensomes in the environmental respirable aerosol could explain both the severe asthmatic symptoms associated with olive tree pollination season, and the high airborne allergen levels during periods when no pollen or very low content of pollens are present in the air.62 However, the question that remains unanswered is how much airborne pollensomes contribute to the initiation and/or exacerbation of the allergic response in susceptible individuals. Finally, the nature of pollensomes makes its interaction with pollutants possible, which would increase the allergenic potential of these nanovesicles.

30.11 Recombinant olive pollen allergens as diagnostic and therapeutic tools

FIGURE 30.4 Pollensomes are natural vehicles for pollen allergens. (A) Scanning electron micrograph of an olive pollen grain. (B) Olive pollen was germinated in germination medium for 16 h at 30 C in the dark. (C) Transmission electron micrograph of olive pollensomes released during in vitro germination and pollen tube growth.

Many of the disadvantages associated with the use of allergen extracts from biological sources for diagnosis and treatment of allergy—for example, difficult allergen standardization, risk of new sensitization and anaphylactic side effects, and endotoxin contamination—can be overcome with the use of recombinant allergens. Various heterologous systems are currently used for the production of recombinant allergens equaling the natural ones as defined molecules in consistent quality and high amount.

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Most of the olive pollen allergens have been obtained using these methods. Ole e 2,63 Ole e 3,33 Ole e 5 (as a fusion protein with GST),50 Ole e 8,34 Ole e 12,7 Ole e 14 (as a His-tagged fusion protein),8 and Ole e 15 (as a His-tagged fusion protein)9 allergens have been produced in bacteria E. coli as soluble high-yield recombinant proteins, whereas the production of recombinant Ole e 1 as a fusion protein with GST in this system rendered a poor-quality product. It was mainly obtained as insoluble inclusion bodies.20 The yeast Pichia pastoris has been used as a successful expression system for olive pollen allergens with posttranslational modifications (such as glycosylation) and complex folding (including disulfide bridges): Ole e 1,64 Ole e 6,65 Ole e 7,40 the NtD and CtD of Ole e 9,44,45 and Ole e 116 have been produced with high yields in P. pastoris, and the recombinant proteins are molecular and immunological equivalent to the natural allergens. For Ole e 10, better yields and lower degradation of the soluble and functional recombinant protein have been achieved using baculovirus in host insect cell (Spodoptera frugiperda) system66 than with the yeast P. pastoris.67 Finally, Ole e 3 and Ole e 8 have also been produced in stable transgenic plants of A. thaliana.68 These transgenic plants could constitute important tools to design edible vaccines for allergy. Recombinant allergens allow us for the first time to determine the precise sensitization profile (allergogram) of patients, which is a prerequisite to select the allergens for patient-tailored immunotherapy. It is well established that a panel of a few recombinant allergens is sufficient to diagnosis most of pollen allergic patients because of the extensive cross-reactivity. Thus the first step for this procedure is the selection of the most relevant allergens that contain most of the important B- and T-cell epitopes and represent the originally sensitizing agents within a crossreacting group, for example, for Oleaceae pollinosis Ole e 1, the main sensitizing agent of Ole e 1-like family, could be used as diagnostic marker.16 Moreover, recombinant allergens can be engineered to produce hypoallergens or hypoallergenic derivatives that exhibit reduced or no allergenic activity but preserved T-cell epitopes and immunogenicity for safer forms of immunotherapy. Several recombinant DNA strategies, as well as synthetic peptide chemistry, have been employed to convert allergens into hypoallergenic derivatives: fragments, oligomers, point or deletion mutants, hybrids, and T- or B-peptides. In vitro and in vivo evaluation of hypoallergens is then required to identify the best vaccines for immunotherapy before clinical application. Clinical trials performed with hypoallergenic derivatives as well as recombinant wild-type allergens have shown that these molecules can be used in immunotherapy in a near future. Hypoallergenic derivatives of Ole e 1 have been engineered on the basis of the disruption of the

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immunodominant IgE epitope of the C-terminal of the molecule by producing one point and two deletion mutants.69 In addition, a peptide T of Ole e 1 has been designed on the basis of epitope mapping studies.70 To select the most suitable derivatives for immunotherapy, they have been tested in vitro and in vivo.69,70 Interestingly, a natural hypoallergenic isoform of Ole e 14 has been produced in E. coli.8 Besides its potential application in clinic, this type of molecules could be useful to determine the features that make an allergen an allergen. In addition, recombinant allergens can be used to study the properties of olive pollen allergens, for example, determination of the 3D structures of Ole e 652 and CtD of Ole e 9,46 assignment of disulfide bridges of Ole e 119 and Ole e 9 CtD,44 epitope mapping of Ole e 125 and Ole e 9,44
46 or epidemiological studies.3,11,38,47 Recombinant Ole e 1 and Ole e 9, and natural Ole e 7 were included in the MEDALL (Mechanisms of the Development of Allergy) microarray that was used in the first systematic study, which compared the IgE and IgG responses toward 47 respiratory and food allergens in 340 sera of the EGEA (Epidemiological study of the Genetics and Environment and Asthma, bronchial hyperresponsive and atopy) cohort of individuals recruited in five French regions.71 The authors found that exposure to an allergen via respiratory route induces preferentially IgE sensitization, whereas the exposure via oral route and possibly via the skin promotes an IgG response. So far, rOle e 1 is the only olive pollen recombinant allergen used in prick-test with patients in a study performed a few years ago, since current Spanish legislation does not allow the use of recombinant molecules on humans, although they may improve diagnosis and the treatment of olive tree pollinosis.

30.12 New concepts for specific immunotherapy using Ole e 1 as a model Specific subcutaneous immunotherapy (SCIT) with olive pollen extract is a well-established and the most common curative treatment for patients with olive pollen allergy. Even though this treatment can offer protection, it has several disadvantages, including long duration, anaphylactic side effects, and limited efficacy. During the last two decades, sublingual immunotherapy (SLIT)—based on the administration of tablets or liquid drops—has been accepted in clinical practice as a safe and an effective alternative treatment to SCIT for allergic rhinitis and allergic asthma.72 Few studies have evaluated the use of SLIT for the treatment of patients with olive pollinosis, using standardized olive pollen extract or allergoids.73
76 Despite the great promise of SLIT, the main disadvantages of this therapy remain the efficacy, the side effects, and the lack of standardization of allergen extracts.

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Therefore the design of novel therapeutic approaches for allergy is required. In this sense, mucosal tolerance induction with nasal vaccines based on free or encapsulated hypoallergenic derivatives is a promising alternative strategy to conventional immunotherapy. A mouse model of IgE sensitization to Ole e 1 mimicking the human B- and T-cell responses has been established for preclinical testing of new nasal vaccines against allergy.70 Four prophylactic approaches were conducted to investigate whether nasal tolerance induction with vaccines based on Ole e 1 or derivatives could prevent sensitization in mice. In a first approach, low doses of a nasal vaccine based on a deletion mutant of Ole e 1 were able to protect mice against sensitization to the allergen.4 In addition, Marazuela et al.70 have demonstrated that prophylactic intranasal administration of an immunodominant peptide T of Ole e 1 (109
130 aa) may substitute for the whole protein in protecting mice against subsequent sensitization to the allergen. Moreover, specific protection for long

term was maintained. In a third study, it was shown that intranasal administration of micrograms of this peptide T of Ole e 1 encapsulated in poly(lactide-co-glycolide) (PLG) microparticles as carrier vaccines prevented subsequent sensitization to the allergen.77 In a previous work, PLG microparticles were described as a suitable vehicle vaccine for Ole e 1 that elicit a specific Th1-type response in mice, thus becoming a promising concept for allergy vaccine. During the last years, exosome-based vaccines have been proposed as a novel strategy for the treatment of human diseases, including allergy. Exosomes are nanovesicles that are released in the extracellular environment by a variety of cell types and showed immunomodulatory properties. Our group has observed that intranasal administration of tolerogenic exosomes protects mice against sensitization to Ole e 1 (Fig. 30.5).78 In this respect, although the four mentioned approaches (using the same prophylactic protocol) suppress the most important clinical features of allergy—specific-IgE antibodies in serum, Th2-response,

FIGURE 30.5 Intranasal pretreatment with tolerogenic exosomes protects mice against allergic sensitization. Exosomes were isolated from BALF from mice that were tolerized by respiratory exposure to Ole e 1. Exosomes isolated from naı¨ve mice were used as controls. These exosomes were assayed as a preventive vaccine in a mouse model of allergy induced by i.p. sensitization to Ole e 1 followed by airway allergen challenge. Mice were i.n. pretreated for 3 consecutive days with tolerogenic exosomes 1 week prior sensitization/challenge with the allergen, and the allergic response was analyzed. Pretreatment with tolerogenic exosomes inhibits both airway inflammation and specific-IgE production. (A) Representative lung section stained with hematoxylin-eosin of tolerogenic exosomes-pretreated mice shows a reduced inflammatory cell infiltration compared to sham-pretreated mice that received control exosomes. Magnifications, 3 20 (ExoTol and ExoCon), 3 10 (Naı¨ve). (B) Serum IgE levels were determined by ELISA. Data are expressed as means 6 standard error (n 5 15 mice/group) from three independent experiments. *P , .001. BALF, Bronchoalveolar lavage fluid; ExoCon, animals pretreated with control exosomes; ExoTol, mice pretreated with tolerogenic exosomes; i.n., intranasal; i.p., intraperitoneal; Naı¨ve, no treated mice. Based on data from Prado N, Marazuela EG, Segura E, et al. Exosomes from bronchoalveolar fluid of tolerized mice prevent allergic reaction. J Immunol. 2008;181:1519
1525.

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and airway inflammation—exosomes have advantages over the previously reported vaccines. They are acellular and stable structures containing a wide array of cellular proteins, some of which modulate immune responses. Since exosomes are natural antigen-transferring units between immune cells, they allow cross-presentation and contribute to amplify immune responses reducing the dose of antigen required to induce an immune response. Finally, “exosome display technology” permits manipulation of their protein composition and tailoring for different functions. These studies emphasize the high potential of nasal vaccines against allergy. Despite the clinical relevance to test the therapeutic effects of these vaccines, the possibility of using prophylactic vaccines for early prevention in atopic individuals or children at risk has been proposed. On the other hand, Calzada et al.79 have employed DNA microarrays for the differential expression analysis in peripheral blood mononuclear cells (PBMCs) from olive pollen allergic patients after in vitro stimulation with four Ole e 1-derived peptides containing T-cell epitopes. They found that the peptide T (10
31 aa) of Ole e 1 regulated the expression of 51 genes involved mainly in the maintenance of peripheral T-cell tolerance and inflammatory response. In another study published in 2019, the same authors analyzed the immunomodulatory capacity and safety of five dodecapeptides derived from Ole e 1, using PBMCs from olive pollen allergic patients.80 Interestingly, two of the peptides inhibited the proliferative response against the olive pollen extract and induced the secretion of the regulatory cytokines IL-10 and IL-35. Moreover, none of the peptides induced the activation of basophils derived from olive pollen allergic patients. This study provides important information about the potential therapeutic tools for the treatment of olive pollen allergy.

used for predicting the B-cell and T-cell epitopes of Ole e 13 in comparison to other allergenic TLPs from plant foods and pollens.82 In addition, the role of 16 TLPs— including Ole e 13—in cross-reactivity between plant foods and pollens has been addressed using a microarray immunoassay and 329 allergic patient’s sera from seven Spanish regions.83 Despite that only 6 out of 16 proteins were identified as relevant allergens because of studied population, TLPs should be considered in OAS diagnostic and treatment. It is worth to mention that Ole e 13 is degraded during the processing of raw olives for human consumption.84 This could explain the low number of allergic reactions caused by ingestion of the olive fruits. Finally, during the last years olive oil manufactures are exploring the use of olive seeds as superfood because their enrichment in antioxidant, polyphenols, and fiber of high quality. Thus the potential allergenicity of olive seed was assayed, and 11S and 7S (vicilins) seed storage proteins (SSPs) have been detected.85 SSPs are among the main food allergen families.

Mini-dictionary terms Term Allergen

Allergogram

Allergy

30.13 Olive fruit: a new source of olive allergens In Mediterranean countries, olive fruit is consumed either directly or through processed products as olive oil. Allergic reactions to olive fruit and its derived products have rarely been documented in literature, and in most of the cases involves occupational allergy: few cases of contact dermatitis on workers after exposure to olive oil, and occupational rhinitis, occupational asthma (OA), contact urticaria, and anaphylactic shock to olive fruits have been reported. Ole e 13, a thaumatin-like protein (TLP), has been identified as the major causative allergen of OA to olive fruits in olive-oil mill workers.81 To date, Ole e 13 is the only food allergen identified in olive fruit. TLPs belong to the PR-5 family, members of which exhibit molecular masses ranging from 20 to 30 kDa and play a role in the plant defense system against biotic and abiotic stresses. Several bioinformatics approaches have been

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Cross-reactivity

Epitope Hypoallergen Major allergen

Definition Harmless environmental antigens, foreign to the body, causing an IgE-mediated hypersensitivity reaction in susceptible individuals. Common allergen sources are pollens, house dust mites, molds, pets, insect venoms, foods, and drugs. The allergen profile from a biological source recognized by serum IgE from a particular allergic patient. A hypersensitivity reaction mediated by IgE antibodies (type I hypersensitivity). It is an inflammatory disease caused by dysregulated type 2T helper cell-biased immune response to allergens of complex genetic and environmental origins. Typical allergic symptoms include asthma, rhinoconjunctivitis, gastrointestinal symptoms, atopic dermatitis, and anaphylaxis. The ability of an antibody raised against an epitope on an antigen to react with an identical or similar epitope on a different antigen. The molecular basis of allergenic cross-reactivity is the occurrence of common epitopes in both the primary sensitizer and the cross-reactive allergen source. A site of an antigen that is recognized by an antibody, B-cell, or T-cell. A derivative form of an allergen with reduced or no allergenic activity. An allergen that is recognized by more than 50% of serum IgE from patients

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allergic to the biological source containing the allergen. Mimotope A molecule—usually a peptide—that mimics an epitope present in an antigen, and therefore, it can induce a desired immune response. Antibodies and T-cells can recognize it. Moreover, mimotopes are good candidates to use in immunotherapy. They are generated by phage display technology. Minor allergen An allergen from a biological source that is recognized by less than 50% of serum IgE from patients allergic to this source. Pan-allergen An allergen widely distributed in the nature with conserved structural properties, which is responsible for cross-reactivity between different allergen sources. Phage display An in vitro screening technique for studying technology protein
ligand interactions, and other macromolecules, which is based on the expression of peptide or protein libraries linked to the coat proteins of a phages. The most common phages used for this technique are filamentous phages that infect Escherichia coli (f1, fd, M13); however, T4, T7, and λ phages can also be used. Phage display technology represents a valuable tool for basic and clinical researches, due to its wide range of application such as epitope mapping, identification of new ligands and drug discovery. Pollensomes Nanovesicles released by pollen during germination and pollen tube growth that act as vehicle for pollen allergens in the environmental respirable aerosol. Pollinosis Allergic reaction to pollen. Subcutaneous Conventional allergen-specific immunotherapy (SCIT) immunotherapy is based on repeated subcutaneous injections of increasing doses of disease-eliciting allergen in the form of natural allergen-containing extract to sensitized patients with the aim of inducing a state of clinical tolerance to the allergen. Sublingual It is an alternative allergen immunotherapy (SLIT) immunotherapy to treat allergy based on the administration of small doses of the allergen as liquid formulation or as tablets under the tongue. Tolerance A state of specific immunological unresponsiveness.

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46. 41. Oeo-Santos C, Navas A, Benede´ S, et al. New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: new role of allergen Ole e 7. Allergy. 2019;75 (Epub ahead of print, http://doi.org/10.1111/all.14086). 42. Palacı´n A, Go´mez-Casado C, Rivas LA, et al. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study. PLoS One. 2012;7: e50799. 43. Huecas S, Villalba M, Rodrı´guez R. Ole e 9, a major olive pollen allergen is a 1,3-β-glucanase. Isolation, characterization, amino acid sequence, and tissue specificity. J Biol Chem. 2001;276:27959
27966. 44. Palomares O, Villalba M, Rodrı´guez R. The C-terminal segment of the 1,3-β-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization. Biochem J. 2003;369:593
601. 45. Palomares O, Villalba M, Quiralte J, Polo F, Rodrı´guez R. 1,3β-glucanases as candidates in latex-pollen-vegetable food crossreactivity. Clin Exp Allergy. 2005;35:345
351. 46. Trevin˜o MA, Palomares O, Castrillo I, et al. Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen. Protein Sci. 2008;17:371
376. 47. Palomares O, Villalba M, Quiralte J, Rodrı´guez R. Allergenic contribution of the IgE-reactive domains of the 1,3-β-glucanase Ole e 9: diagnostic value in olive pollen allergy. Ann Allergy Asthma Immunol. 2006;97:61
65. 48. Palomares O, Ferna´ndez-Nieto M, Villalba M, Rodrı´guez R, Cuesta-Herranz J. Occupational allergy in a researcher due to Ole e 9, an allergenic 1,3-β-glucanase from olive pollen. Allergy. 2008;63:784
785. 49. Boluda L, Alonso C, Ferna´ndez-Caldas E. Purification, characterization, and partial sequencing of two new allergens of Olea europaea. J Allergy Clin Immunol. 1998;101:210
216. 50. Butteroni C, Afferni C, Barletta B, et al. Cloning and expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase. Int Arch Allergy Immunol. 2005;137:9
17. 51. Batanero E, Ledesma A, Villalba M, Rodrı´guez R. Purification, amino acid sequence and immunological characterization of Ole e

374

52.

53.

54.

55. 56.

57.

58.

59.

60.

61. 62.

63.

64.

65.

66.

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6, a cysteine-enriched allergen from olive tree pollen. FEBS Lett. 1997;410:293
296. Trevin˜o MA, Garcı´a-Mayoral MF, Barral P, et al. NMR solution structure of Ole e 6, a major allergen from olive tree pollen. J Biol Chem. 2004;279:39035
39041. San Segundo-Acosta P, Oeo-Santos C, Navas A, Jurado A, Villalba M, Barderas R. Ole e 15 and its human counterpart-PPIA-chimeras reveal an heterogeneous IgE response in olive pollen allergic patients. Sci Rep. 2019;9:15027. San Segundo-Acosta P, Garranzo-Asensio M, Oeo-Santos C, et al. High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components. J Immunol Methods. 2018;456:44
53. Altmann F. The role of protein glycosylation in allergy. Int Arch Allergy Immunol. 2007;142:99
115. Batanero E, Crespo JF, Monsalve RI, Martı´n-Esteban M, Villalba M, Rodrı´guez R. IgE-binding and histamine release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1. J Allergy Clin Immunol. 1999;103:147
153. Van Ree R, Cabanes-Macheteau M, Akkerdaas J, et al. β(1,2)Xylose and α(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem. 2000;275:11451
11458. Castro AJ, Bednarczyk A, Schaeffer-Reiss C, Rodrı´guez-Garcı´a MI, Van Dorsselaer A, Alche´ JD. Screening of Ole e 1 polymorphism among olive cultivars by peptide mapping and N-glycopeptide analysis. Proteomics. 2010;10:953
962. Rodrı´guez-Garcı´a MI, M’rani-Alaoui M, Ferna´ndez MC. Behaviour of storage lipids during development and germination of olive (Olea europaea L.) pollen. Protoplasma. 2003;221:237
244. Prado N, Alche´ JD, Casado-Vela J, et al. Nanovesicles are secreted during pollen germination and pollen tube growth: a possible role in fertilization. Mol Plant. 2014;7:573
577. Prado N, De Linares C, Sanz ML, et al. Pollensomes as natural vehicles for pollen allergens. J Immunol. 2015;195:445
449. De Linares C, Nieto-Lugilde D, Alba F, Dı´az de la Guardia C, Gala´n C, Trigo MM. Detection of airborne allergen (Ole e 1) in relation to Olea europaea pollen in Spain. Clin Exp Allergy. 2007;37:125
132. Asturias JA, Arilla MC, Go´mez-Bayo´n N, Martı´nez J, Martı´nez A, Palacios R. Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive pollen. J Allergy Clin Immunol. 1997;100:365
372. Huecas S, Villalba M, Gonza´lez EM, Martı´nez-Ruiz A, Rodrı´guez R. Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris. Eur J Biochem. 1999;261:539
546. Barral P, Tejera ML, Trevin˜o MA, et al. Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast. Detection of partial oxidation of methionine by NMR. Protein Expr Purif. 2004;37:336
343. Barral P, Serrano AG, Batanero E, Pe´rez-Gil J, Villalba M, Rodrı´guez R. A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system. J Biotechnol. 2006;121:402
409.

67. Barral P, Batanero E, Villalba M, Rodrı´guez R. Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: evidence of post-translational modifications. Protein Expr Purif. 2005;44:147
154. 68. Ledesma A, Moral V, Villalba M, Salinas J, Rodrı´guez R. Ca21binding allergens from olive pollen exhibit biochemical and immunological activity when expressed in stable transgenic Arabidopsis. FEBS J. 2006;273:4425
4434. 69. Marazuela EG, Rodrı´guez R, Barber D, Villalba M, Batanero E. Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines. Clin Exp Allergy. 2007;37:251
260. 70. Marazuela EG, Rodrı´guez R, Ferna´ndez-Garcı´a H, Garcı´a MS, Villalba M, Batanero E. Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen. Mol Immunol. 2008;45:438
445. 71. Siroux V, Lupinek C, Resch Y, et al. Specific IgE and IgG measured by the MeDALL allergen-chip depend on allergen and route of exposure: the EGEA study. J Allergy Clin Immunol. 2017;139:643
654. 72. Tankersley M, Han JK, Nolte H. Clinical aspects of sublingual immunotherapy tablets and drops. Ann Allergy Asthma Immunol. 2020;S1081-1206(19):31532
315327. 73. Vourdas D, Syrigou E, Potamianou P, et al. Double-blind, placebocontrolled evaluation of sublingual immunotherapy with standardized olive pollen extract in pediatric patients with allergic rhinoconjunctivitis and mild asthma due to olive pollen sensitization. Allergy. 1998;53:662
672. 74. Leonardi S, Arena A, Bruno ME, et al. Olea sublingual allergoid immunotherapy administered with two different treatment regimens. Allergy Asthma Proc. 2010;31:e25
29. 75. Irani C, Saleh RA, Jammal M, Haddad F. High-dose sublingual immunotherapy in patients with uncontrolled allergic rhinitis sensitized to pollen: a real-life clinical study. Int Forum Allergy Rhinol. 2014;4:802
807. 76. Al-Asad K, Al-Nazer S, Al-Faqih A, Hashem MJ. Evaluation of a sublingual immunotherapy solution in olive-induced respiratory allergy in Jordan: a retrospective observational study. J Asthma Allergy. 2017;10:23
30. 77. Marazuela EG, Prado N, Moro E, et al. Intranasal vaccination with poly(lactide-co-glycolide) microparticles containing a peptide T of Ole e 1 prevents mice against sensitization. Clin Exp Allergy. 2008;38:520
528. 78. Prado N, Marazuela EG, Segura E, et al. Exosomes from bronchoalveolar fluid of tolerized mice prevent allergic reaction. J Immunol. 2008;181:1519
1525. 79. Calzada D, Aguerri M, Baos S, et al. Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1derived peptides. Mol Immunol. 2015;64:252
261. ´ , et al. Therapeutic 80. Calzada D, Cremades-Jimeno L, Pedro MA potential of peptides from Ole e 1 in olive-pollen allergy. Sci Rep. 2019;9:15942. 81. Palomares O, Alca´ntara M, Quiralte J, Villalba M, Garzo´n F, Rodrı´guez R. Airway disease and thaumatin-like protein in an olive-oil mill worker. N Engl J Med. 2008;358:1306
1308. 82. Jime´nez-Lo´pez JC, Robles-Bolivar P, Lo´pez-Valverde FJ, LimaCabello E, Kotchoni SO, Alche´ JD. Ole e 13 is the unique food

Olive pollen allergens: an insight into clinical, diagnostic, and therapeutic concepts of allergy Chapter | 30

allergen in olive: structure-functional, substrate docking, and molecular allergenicity comparative analysis. J Mol Graph Model. 2016;66:26
40. 83. Palacı´n A, Rivas LA, Go´mez-Casado C, et al. The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray. PLoS One. 2012;7: e44088.

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168. 85. Jime´nez-Lo´pez JC, Zafra A, Palanco L, Florido JF, Alche´ JD. Identification and assessment of the potential allergenicity of 7s vicilins in olive (Olea europaea L.) seeds. Biomed Res Int. 2016;2016:4946872.

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Chapter 31

Cancer preventive role of olives and olive oil via modulation of apoptosis and nuclear factor-kappa B activation ˘ 5, Vaishali Aggarwal1, Gaurav Kumar2, Diwakar Aggarwal3, Mu¨kerrem Betu¨l Yerer4, Ahmet Cumaoglu 6 7 8 3 9 Manoj Kumar , Katrin Sak , Sonam Mittal , Hardeep Singh Tuli and Gautam Sethi 1

Department of Histopathology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, 2Department of

Biochemistry, Delhi University, South Campus, New Delhi, India, 3Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Ambala, India, 4Department of Pharmacology, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey, 5Department of Biochemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey, 6Department of Chemistry, Maharishi Markandeshwar University, Sadopur, Ambala, India, 7 NGO Praeventio, Tartu, Estonia, 8School of Biotechnology, Jawaharlal Nehru University, New Delhi, India, 9Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Abbreviations AKT AOM Bak Bax Bcl-2 BH3 B-Raf COX-2 CRC DSS ERβ/ ERα EVOO FasL IKK IL-1β IL-6 JNK MAPK NF-κB OLE p-ERK PGE2 PI3K ROS TNF TNF-α TNFR1

AKT serine threonine kinase 1 azoxymethane Bcl-2 antagonist killer Bcl-2-associated X, apoptosis regulator B-cell lymphoma/leukemia type 2 borane B-Raf proto-oncogene cyclooxygenase-2 colorectal cancer dextran sulfate sodium estrogen receptor α/β extra-virgin olive oil Fas ligand IκBs kinase interleukin-1β interleukin-6 c-Jun NH-2 terminal kinase mitogen-activated protein kinases nuclear factor kappa-light-chain-enhancer of activated B cells olive leaf extract phosphorylated-extracellular regulated MAP kinase prostaglandin E2 phosphatidylinositol 3-kinase reactive oxygen species tumor necrosis factor tumor necrosis factor alpha tumor necrosis factor receptor superfamily member 1

31.1 Introduction The health benefits of olives and olive oil, especially extra-virgin olive oil (EVOO), are widely recognized throughout the world.1 Olives are the fruits of Olea europaea L. (olive tree) which is native of Syria, Lebanon, Jordan, Israel, and Palestine; nowadays it is widely cultivated also in the entire Mediterranean region, particularly in Spain, Greece, and Italy.2,3 Based on the historical evidence, the use of olive tree fruits dates back to the 7000
5000 BCE, with their healing and salutary effects known since ancient times.3 Most importantly, consumption of olive-derived products has been associated with protection against inflammation-related and cancerous diseases but used also with respect to their blood pressure and cholesterol lowering and cardioprotective properties.1,2,4,5 These health-promoting effects are probably derived from a variety of bioactive constituents contained in olives, including mono-saturated fatty acids (especially oleic acid, but also stearic acid, linoleic acid, palmitic acid, and palmitoleic acid), phenolics (hydroxytyrosol, tyrosol, oleuropein, some phenolic acids, flavonoids, and lignans), and tocopherols.1
6 Although the exact content of these compounds has been shown to considerably vary depending on the variety and age of olive tree cultivars, geographical location, cultivational conditions, and agronomic factors, all these agents are well known for their antioxidant, antiinflammatory, antiviral, antimicrobial, chemopreventive,

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00005-5 © 2021 Elsevier Inc. All rights reserved.

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and/or anticancer properties, contributing to protection against malignant transformation, promotion, progression, and metastasis.1,2,4 Numerous epidemiological studies have indeed shown reduced incidence and mortality rate of several malignancies in the regions where people are adherent to the customary Mediterranean food diet, of which olives and its products (olive oil, etc.) are the key components.6
9 This chapter presents the current knowledge about different anticancer activities of various chemical components of olives, with a special focus to their potential action on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and apoptotic death in different malignancies.

31.2 Chemistry and sources With regard to the chemical composition of olive oil, its main constituents are triacylglycerols (triglycerides or fats). An array of lipids such as hydrocarbons, free fatty acid, glycerol, phosphatides (fatty acid esters of glycerol phosphate with a nitrogen base), pigments, flavor compounds, aliphatic and aromatic alcohols, tocopherols, sterols, and microscopic bits of olive is also present. Triacylglycerol’s are mainly the molecules that are derived from esterification of glycerol molecule with three fatty acid moieties. The glycerol molecule is mainly in the form of an “E-shaped” molecule, while the fatty acids resemble hydrocarbon chains (14
24 carbon atoms in length). The fatty acids mainly present in olive oil include palmitic (C16:0), stearic (C18:0), linoleic (C18:2), palmitoleic

FIGURE 31.1 Structural formula of sterols present in olive oil.

(C16:1), oleic (C18:1), and linolenic (C18:3). Eicosanoic, heptadecanoic, and myristic acids (C14:0) can be found in trace quantities. The major tocopherols present in virgin olive oils include 90% of α-homolog of the eight known “E-vitamers” (Fig. 31.1). This α-tocopherol is basically present in the free form, while the aromatic and aliphatic alcohols are found in esterified form. Of these the most significant ones are diterpene and fatty alcohols. In addition, the main pigment-containing compounds are chlorophylls and carotenoids. The main carotenoids present in olive oil are lutein and β-carotene responsible for virgin olive oil yellow hues color, whereas the presence of chlorophylls contributes to the green shade of olive oil. Chlorophylls are encountered as pheophytins. The major odorants that contribute to the particular aroma of olive oil include esters, aldehydes, acids, and alcohols.

31.3 Cancer prevention mechanisms 31.3.1 Activation of B-cell lymphoma type 2 (Bcl-2)-associated X, apoptosis regulator and Bcl-2 antagonist killer apoptotic signals The apoptotic roles of olive oil and its polyphenols and triterpenoids have been very well studied in different malignancies and have illustrated with positive effect results that have also been summarized in the recent scientific literature highlighting the role of olive oil in view of its anticancer effects via modulating different cellular processes.3,10
14 In a recent review by Ziberna et al.,15

Cancer preventive role of olives and olive oil Chapter | 31

the authors reflected in detail upon the role of oleanolic acid, an olive oil triterpenoid in inducing apoptosis via augmentation in B-cell lymphoma/leukemia type 2 (Bcl-2)-associated X, apoptosis regulator (Bax)/Bcl-2 expression ratio and stimulation of apoptosis signalregulating kinase 1/reactive oxygen species (ROS)/p38 MAPK (mitogen-activated protein kinases) pathway.15 In another study, derivatives of virgin olive oil mainly colonic metabolites and hydroxytyrosol were reported to induce apoptosis in human Caco-2 colon cancer cells.16 Interestingly, administration of 5 μM hydroxytyrosol was reported to downregulate p53, cytochrome c, Bax, caspase 3 along with upregulation of metallothionein and nuclear factor erythroid 2-related factor 2, the prosurvival proteins in human IMR-32 neuroblastoma cells.17 In addition, oleocanthal and oleacein extracts of EVOO led to apoptosis in nonmelanoma skin cancer cells via inhibition of Erk and Akt phosphorylation along with downregulation of B-Raf (B-Raf proto-oncogene) expression.18 Moreover, EVOO phenols were also shown to block cell cycle progression and induced apoptosis via arresting cells prior to mitosis in G2/M phase in T24 and 5637 human bladder cancer cell lines.19 High EVOO diet also induces metabolic adaptations with increased glycolysis, elevated cytotoxic T cells infiltration, and decreased transforming growth factor beta 1 which eventually increased apoptosis in rat mammary tumors.20 Peluso et al. also highlighted that stress activated p38 MAPKs and c-Jun NH-2 terminal kinase (JNK) which can be the potential targets of olive-derived nutraceuticals.12 Also, Barone et al. reported that omega3-polysaturated fatty acids and olive oils can inhibit fatty acid synthase and β-hydroxy β-methylglutaryl-CoA reductase gene expression via elevated ERβ/ERα (estrogen receptor α/β) ratio and prevented the growth of intestinal polyps.21 Oleuropein, a polyphenol of olive oil, was also reported to cause apoptosis via Bax and Bcl-2-mediated activation of p53-dependent signaling pathway in MCF-7 breast cancer cells.22 In another study carried out to study apoptotic effect of oleuropein, it was seen to inhibit phosphatidylinositol 3-kinase/AKT serine threonine kinase 1 (PI3K/AKT) pathway thereby activating caspase-facilitated apoptosis in HepG2 human hepatocellular carcinoma cells.23 These findings were also supplemented by Llor et al. in Caco-2 and HT-29 colorectal cancer (CRC) cells wherein they reported supplementation with fish oil and olive oil resulted in apoptosis induction via downregulation of Bcl-2 and cyclooxygenase-2 (COX-2) expression.24 Besides these signaling cascades, the chemopreventive role of olive oil has also been illustrated to be attributed to its potential to target methylation machinery and noncoding RNAs in colon cancer via alteration of NF-κB and apoptotic pathways.25

379

31.3.2 Modulation of tumor necrosis factor and Fas ligand expression/activity In recent years a lot of emphasis is being given to explore molecules from natural origins with the therapeutic potential for cancer treatment, since they may be safer with fewer side effects.3,26
31 Most of these molecules from natural origins are derived from commonly consumed foodstuff, including the olive and olive oil.32 Taking into account the lower incidence of mortality rates from cancer and cardiovascular diseases which is mainly attributed to the consumption of the diet in Mediterranean region, many scientists investigated the potential role of olive and olive oil in treatment as well as protection from several chronic diseases, including cancer.33,34 This part will focus on its role in inflammatory processes that might have crucial importance in tumor initiation as well as progression. Fas ligand (FasL), a tumor necrosis factor (TNF) superfamily member, leads to the induction of apoptosis via cross-linking of Fas (Apo-1/CD95) that, in turn, contributes to cell-mediated cytotoxicity and immune homeostasis.35 TNF alpha (TNF-α) can induce apoptosis in malignant cells via ligation of the TNF receptor superfamily member 1 (TNFR1).36,37 The reports on the role of TNF-α in cancer progression revealed that it plays a major role in inhibiting antitumor immune response. This is mediated via direct leukocyte modulation along with their survival and activation, which alters the tumor cell phenotype.38
42 Accumulated experimental, clinical, and epidemiological data that reveal the basic healthy roles of the Mediterranean diet are mainly shown to come from olive and olive oil, which has different components such as triterpenoids (erythrodiol and uvaol),43 flavonoids, and phenolic components (apigenin-7-glucoside oleuropein, luteolin-7-glucoside, verbascoside, and tyrosol)44 that are even shown to have antineoplastic activity in different cancer types in vitro43,45
47 (Table 31.1) and in vivo (Table 31.2).45,48 The chemopreventive effect of olive oil is not only due to the fatty acids but also the flavonoids and polyphenols (the phenolic compounds).46 Furthermore, the causal relationship established between tumor development and chronic inflammatory microenvironment has led to novel strategies in inflammatory process in cancer treatment strategies, and the active components of O. europaea have shown to play immune-regulatory33,49 and protective roles on some chronic diseases such as gastritis which may lead to gastric cancer induced by ethanol over these antiinflammatory roles.50 Al-Quraishy et al.50 have shown that olive leaf extract (OLE) decreased the inflammatory response by reducing TNF-α, COX-2, and NF-κB expression, along with downregulation of interleukin-1β (IL-1β) and inducible nitric oxide synthase in gastric

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 31.1 Overview of various in vitro studies conducted on the effect of olive oil and associated phenolics in prevention of cancer. Type of cancer

Cell line used

Phenolic constituents/supplements associated with olive oil/dose

Effects/mechanism

Ref.

Colon cancer

LoVo

IC50—66.70 and 30.47 μmol/L

kTumor growth, cell cycle arrest at the G2/M phase, apoptosis (mAPAF-1)

[100]

Caco-2

0.2%, 0.5%, and 1% olive oil vol/vol dissolved in culture medium

Upregulation of AIF and CELF1 protein expression

[101]

HT-29, CaCo2, WiDr

100 μM

kTumor cell growth (mEGFR degradation: EGFR phosphorylation at pY1045 and mCbl)

[102]

DLD1, HCT116, HCT8, HT29, SW48

20 mmol/L

Apigenin and ABT-263 synergistic effect on apoptosis with decreased Mcl-1 and ERK/AKT

[68]

COLO 320 DM

IC50 266.2 μM

kTumor growth, kβ-catenin, kPCNA, chemopreventive

[103]

SW480, HCT15

20
40 μM/apigenin

kCell proliferation, kWnt/β-catenin signaling pathway

[104]

HT29

IC50 was 37.5 6 0.9 μg/mL/maslinic acid

Apoptosis, kBcl-2, Bax, mcaspase-9 and -3

[70]

HT115

25 μg/mL HT (3,4dihydroxyphenylethanol), tyrosol (phydroxyphenylethanol), pinoresinol and caffeic acid

Inhibiting several stages in carcinogenesis, including metastasis. kTotal cell number

[105]

Caco-2

30 and 400 μM

mApoptosis, ktumor development

[24]

Liver cancer

HepG2, Huh7, Hep3B

4.81 μg/mL, corresponding to 5 μM oleocanthal, 9 μM ligstroside aglycone, and 9.62 μg/mL containing 10 μM oleocanthal, 18 μM ligstroside aglycone

Antiproliferative effects, induction of autophagy, proinflammatory, kcell proliferation, mcell death

[106]

Urinary bladder cancer

T24, 5637

10
100 μg/mL (combination with paclitaxel or mitomycin)

kIn proliferation, clonogenic ability, growth arrest in G2/M phase

[19]

T-24

10
100 μg/mL/extra-virgin olive oil phenols

kProliferation and motility. Cytotoxic effect on tumor cells without affecting normal urothelial fibroblasts

[107]

Breast cancer

MCF-7

10
75 μM/HT and OL

kOf E2-dependent activation of extracellular-regulated kinase, chemopreventive role via attenuation of estrogen-dependent rapid signals involved in uncontrolled tumor cell growth

[108]

SKBR3

200 μg/mL/olive leaf extract

Cytotoxic activity, inhibiting cell proliferation

[109]

JIMT-1

Olive leaf extract, 7 μg/mL

Induced apoptosis, cell cycle arrest at G1 phase, inactivation of MAPKproliferation pathway

[110]

MCF-7

200 μg/mL of OL, 50 μg/mL of HT

kCell viability of MCF-7 cells. mPercentage of cells in G0/G1 phase

[111]

MDA human breast cancer cell line

200 μg/mL of OL

Antimetastatic effects, TIMP1 and TIMP3 gene expression was significantly increased. Thus kthe MMPs gene expression

[112]

(Continued )

Cancer preventive role of olives and olive oil Chapter | 31

381

TABLE 31.1 (Continued) Type of cancer

Prostatic cancer

Pancreatic cancer

Leukemia (blood cancer)

Cell line used

Phenolic constituents/supplements associated with olive oil/dose

Effects/mechanism

Ref.

BT-474, SK-Br3

200 mg/mL/trastuzumab

kHer-2/neu-coded, p185 Her-2/neu oncoprotein, oleic acid synergistically enhanced trastuzumab ability to induce downregulation of p185 Her-2/neu

[113]

MCF-10A, MDAMB-231, MCF-7

200 μM of OL

Delaying cell cycle at S phase. mCyclindependent inhibitor p21 inhibited antiapoptosis and proproliferation protein NF-κB and cyclin D1

[114]

HMLER, SUM-159

20 μmol/L/olive oil

Blocked the formation of multicellular tumorspheres, kof SAM cofactorbinding pocket of DNMTs and ATPbinding kinase domain site of mTOR

[115]

DU 145 cells

1
100 μmol/L/ellagic acid

Apoptosis (kATP, pro-MMP-2/-9, VEGF), antiproliferative activity

[116]

LNCaP (androgenresponsive) and DU145 (androgennonresponsive) cells

100
500 μM/OL

Antiproliferative effect induces apoptosis, kcell viability. Induction of thiol group modifications, reactive oxygen species, γ-glutamylcysteine synthetase, heme oxygenase-1, and pAkt leading to prooxidant effect on tumor cells

[117]

Mia-PaCa-2

100 and 200 μg/mL

Antiproliferative activity increased antioxidant activity

[118]

PC3

20 μM/oleocanthal

Antiinflammatory and neuroprotective benefits, kcancer cell viability, ktumor burden, induced lysosomal membrane permeabilization rapid necrotic cell death

[119]

K562

Olive leaf extract, 100, 125, and 150 μg/mL

kK562 cell proliferation. Cell cycle arrest at G0/G1 and G2/M phases. Apoptosis induction and K562 cell differentiation to monocyte lineage

[47]

AIF, Apoptosis-inducing factor; AKT, AKT serine threonine kinase 1; Bax, Bcl-2-associated X, apoptosis regulator; Bcl-2, B-cell lymphoma/leukemia type 2; DNMT, DNA methyltranferase 1; EGFR, epidermal growth factor receptor; ERK, extracellular regulated MAP kinase; HT, hydroxytyrosol; MAPK, mitogenactivated protein kinases; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; OL, oleuropein; SAM, S-adenosyl-L-methionine; VEGF, vascular endothelial growth factor.

mucosa. Oleuropein (a phenolic secoiridoid) found in the O. europaea leaves and olive drupes can exert antiinflammatory effects in several models of inflammation.51
53 Therefore the antiinflammatory effects of oleuropein has also been investigated in chronic colitis, where it was reported to favor milieu supporting CRC initiation, growth, progression, and metastasis.54,55 Furthermore, Giner et al.56 have revealed that oleuropein prevents colonic neoplasia in azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC in mice. They target the inflammation-associated processes in colon and inhibit activation of the transcription factors involved (signal transducer and activator of transcription 3, PI3K/AKT, NF-κB, and β-catenin) and by

reducing the Th17 response related to interleukin-6 (IL-6) and TNF-α secretion in AOM/DSS-induced CRC. In anticancer therapy, Fas/FasL system is the main signaling pathway that plays a role in apoptosis induction.35 Martin et al.43 have investigated the anticancer effect of two major triterpenoids from olive, erythrodiol and uvaol, on astrocytoma over Fas/FasL cell death pathway related to TNFR1 and CD40, finding that both triterpenes increased ROS along with the loss of mitochondrial membrane potential which caused the induction of this apoptotic pathway. Lamy et al.46 have examined the properties of three phenolic compounds (hydroxytyrosol, oleuropein, and tyrosol) and oleic acid (a monounsaturated fatty acid)

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 31.2 Anticancer effects of olive oil and its phenolic constituents in in vivo studies. Phenolic constituent/ olive oil

Experimental models

Effects

Mechanism

Dose

Ref.

Luteolin

BalbC nude mice inoculated with LoVo cells

Apoptosis

mAPAF-1, mcell cycle arrest—G2/ M phase, ktumor growth

20
40 mg/kg

[100]

Hydroxytyrosol

Mice with HT-29 xenografts

Reduced malignant cell growth

EGFR phosphorylation at pY1045, mCbl—mEGFR degradation

10 mg/kg (200 μL)

[102]

Apigenin

CB-17 SCID mice implanted with HCT116 cells

Apoptosis

kMcl-1, AKT, and ERK

25 mg/kg

[68]

Olive oil

Wistar rats inoculated with 1,2dimethylhydrazine

Chemoprevention ktumor growth

kβ-Catenin and PCNA

10
20 mg/kg

[103]

Female athymic nude mice inoculated subcutaneously with SUM-159 cells

Reduced subsequent tumorforming capacity

Mice orthotopically injected with CSC-enriched BC cells treated with DOA—tumor-free for several months

20 μmol/L

[115]

Swiss albino mice with spontaneous soft tissue sarcomas

kCell proliferation

p53 pathway activation, kHIF-1α

0.005%
0.1%

[45]

Azoxymethane/dextran sulfate sodium-induced CRC in C57BL/6 mice

Chemoprevention, modulatory effect on the Th17 response

kCD4 1 , Rorγt 1 , IL-17 1 , IFNγ 1 T cells in the lamina propria

50
100 mg/kg

[56]

RIP-Tag mice

Antiinflammatory and neuroprotective benefits

kCancer cell viability, ktumor burden, induced lysosomal membrane permeabilization

5 mg/kg

[119]

Oleuropein

Oleocanthal

AKT, AKT serine threonine kinase 1; BC, breast cancer; CRC, colorectal cancer; CSC, cancer stem cell; DOA, docetaxel; EGFR, epidermal growth factor receptor; ERK, extracellular regulated MAP kinase; IL, interleukin; PCNA, proliferating cell nulcear antigen.

on TNF-α-induced COX-2 expression in glioblastoma cells. In this study, these compounds significantly decreased the COX-2, PGE2 (prostaglandin E2), p-NF-κB, p-ERK (phosphorylated-extracellular-regulated MAP kinase), and p-JNK expression. Also, on the basis of olive cultivars type the concentration of the phenols in EVOO could range from 50/800 mg/kg57 and the plasma concentration of phenolic compounds in humans has been found to be in between 0.1 and 59 μM.58 In a clinical trial with OLE, it has shown to decrease the TNF-α levels in the salivary glands of cancer patients with oral mucositis after chemotherapy. In addition, phenolic compounds of olive oil (vanillic acid, p-coumaric acid, homovanillic acid, caffeic acid, kaempferol, oleuropein, tyrosol) reduced the TNF-α, IL-1β, and IL-6 and inhibited the PGE2 significantly in human whole blood cultures.59 In conclusion, many components of olive and olive oil might play important roles against chronic inflammatory

responses by effectively targeting various members of the TNF superfamily.

31.3.3 Inhibition of cell survival proteins (B-cell lymphoma/leukemia type 2) Bcl-2 belongs to the Bcl-2 family proteins that mediate both either pro- or antiapoptotic signaling. Overexpression of Bcl-2 and other prosurvival Bcl-2 family proteins has been reported in various cancers with few exceptions and their expression is usually correlated with lesser susceptibility to drug treatment and resistance to chemo/ radiotherapies. The alteration in Bcl-2 expression may occur through chromosomal translocation, increased transcription/translation, increased protein stability, and gene amplification.60
64 Antiapoptotic Bcl-2 protein interacts

Cancer preventive role of olives and olive oil Chapter | 31

with the proapoptotic Bax and Bcl-2 antagonist killer (Bak) proteins to induce oncogenesis by inhibiting cell death. Bcl-2 protein binds to the borane (BH3) dimerization domain of Bax and Bak proteins.65 Thus targeting Bcl-2 prosurvival function has increased the interest among scientific community to develop novel drug molecule as potential anticancer therapeutic agents. Molecules mimicking the proapoptotic BH3 domains highlight a direct treatment approach against prosurvival effects of Bcl-2.63,66 Various scientific studies have reported the inhibitory potential of olive oil on Bcl-2 overexpression and functions. Previously, it has been observed that apigenin (subclass of flavonoids in olive oils) inhibited Bcl-2 functions and induced apoptosis in colorectal carcinoma in synergism with ABT-263, a BH-3 mimetic.67
69 Similarly, olive oil fatty acids (linoleic and oleic acids) lead to the cell differentiation and apoptosis by mediating downregulation of Bcl-2 expression.24 In another study, maslinic acid, a triterpene found in olives had shown the growth inhibitory effects against colon cancer. Exposure of HT29 colon-cancer cells to maslinic acid induced apoptotic cell death by attenuating Bcl-2 expression thereby stimulated the release of cytochrome c and activated caspases.70 Similarly, the mechanism of action of oleanolic acid

383

(triterpene from olives) on growth inhibition has also been explored in various cancer cells such as nonsmallcell lung cancer, lung adenocarcinoma, B16F10 melanoma cells, breast cancer, and colon cancer. It inhibited the growth of tumor cells via cell cycle arrest and apoptosis by the activation of caspase-3 along with high expression of Bax and reduced expression of Bcl-2.71
74 Hence, inhibition of Bcl-2 expression as well as its function by components of olive oil could be used as potential therapeutics against cancers.

31.3.4 Regulation of nuclear factor kappa-lightchain-enhancer of activated B cells activation NF-κB is a transcription factor that is known for a plethora of signaling cascades associated with cancer. A variety of cellular molecules such as chemokines, cytokines, cell adhesion molecules, growth factors, and apoptotic proteins act as potent targets of NF-κB.75
86 The cellular activity of NF-κB is under the strict control of IκBs kinase (IKK complex) which, in turn, has two catalytic (IKKα/β) and one regulatory (IKKγ/NF-κB essential modulator) subunits. IκBs (including IκBα, IκBβ, IκBε, and Bcl-3) act as inhibitors of NF-κB, where phosphorylation events can be done by IKK complex, so as to provoke the ubiquitination

FIGURE 31.2 Mechanism of regulation of NF-κB pathway via olive oil and its bioactive components. NF-κB, Nuclear factor kappa-light-chainenhancer of activated B cells.

384

PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

and thereby, the proteasomal degradation of NF-κB, resulting in rapid activation and nuclear translocation of NF-κB.87
89 Nuclear NF-κB can, in turn, govern a variety of cellular inflammatory pathways.90
94 A substantial amount of positively published data have supported the inhibitory impact of olive oil on translocation and/or activation of NF-κB.95,96 Previously, the inhibitory effect of olive oil polyphenols on NF-κB has been reported in an in vitro study with human monocytes/ macrophages.48 Similarly, another in vitro study with healthy volunteer monocytes derived from macrophages demonstrated a concentration-dependent inhibitory effect of EVOO extract on the translocation of NF-κB (Table 31.1).97 Moreover, recently, an in vitro study with human THP-1 monocytes demonstrated a significant suppression in NF-κB expression as a consequence of the treatment of hydroxytyrosol, one of the components in olive oil. This study also claimed the neuroprotective effects of hydroxytyrosol in early brain injury as a consequence of NF-κB inhibition.98 More recently, Serra et al. demonstrated antioxidative effects of EVOO in human colon adenocarcinoma Caco-2 cells as a consequence of NF-κB inhibition. Hence, inhibition of NF-κB activation as well as its nuclear translocation by components of olive oil is associated with cell survival and improving the cellular redox status (as detailed in Fig. 31.2).99

31.4 Conclusion and future perspectives Olive oil and phenols have recently emerged as promising supplemental compounds owing to their promising anticancer effects. The scientific data from different experimental groups have highlighted the antitumorigenic potential of olive oil and its components in various models (Table 31.2). The results from different studies have highlighted the role of olive oil as well as its components in the inhibition of malignant cells at different phases from tumor initiation to invasion to tumor progression. However, we still need to cumulatively analyze the findings from separate research groups to understand the precise mechanism of action of olive oil in mitigating tumorigenesis in malignant cell populations. Hence, the present literature supplements the supporting scientific evidence for olive oil and its derivatives to present with promising anticancer effects. However, for detailed understanding of the mechanism of action of olive oil, well-designed experimental studies are needed both to understand the chemopreventive potential of olive oil in malignant cell populations and identify various compounds with therapeutic potential. Further research to carry out intervention-based studies on cancer patients are also needed to decipher the anticancer actions of olive oil in conjunction with standardized chemotherapeutic regimes.

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529. 103. Baskar AA, Ignacimuthu S, Paulraj GM, Al Numair KS. Chemopreventive potential of beta-sitosterol in experimental colon cancer model—an in vitro and in vivo study. BMC Complement Altern Med. 2010;10:24. 104. Xu M, Wang S, Song YU, Yao J, Huang K, Zhu X. Apigenin suppresses colorectal cancer cell proliferation, migration and invasion via inhibition of the Wnt/beta-catenin signaling pathway. Oncol Lett. 2016;11:3075
3080. 105. Hashim YZ, Rowland IR, McGlynn H, et al. Inhibitory effects of olive oil phenolics on invasion in human colon adenocarcinoma cells in vitro. Int J Cancer. 2008;122:495
500. 106. De Stefanis D, Scime S, Accomazzo S, et al. Anti-proliferative effects of an extra-virgin olive oil extract enriched in ligstroside aglycone and oleocanthal on human liver cancer cell lines. Cancers (Basel). 2019;11. 107. Coccia A, Bastianelli D, Mosca L, et al. Extra virgin olive oil phenols suppress migration and invasion of T24 human bladder cancer cells through modulation of matrix metalloproteinase-2. Nutr Cancer. 2014;66:946
954. 108. Sirianni R, Chimento A, De Luca A, et al. Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation. Mol Nutr Food Res. 2010;54: 833
840. 109. Quirantes-Pine R, Zurek G, Barrajon-Catalan E, et al. A metabolite-profiling approach to assess the uptake and metabolism of phenolic compounds from olive leaves in SKBR3 cells by HPLC-ESI-QTOF-MS. J Pharm Biomed Anal. 2013;72: 121
126. 110. Barrajon-Catalan E, Taamalli A, Quirantes-Pine R, et al. Differential metabolomic analysis of the potential antiproliferative mechanism of olive leaf extract on the JIMT-1 breast cancer cell line. J Pharm Biomed Anal. 2015;105:156
162. 111. Han J, Talorete TP, Yamada P, Isoda H. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology. 2009;59: 45
53. 112. Hassan ZK, Elamin MH, Daghestani MH, et al. Oleuropein induces anti-metastatic effects in breast cancer. Asian Pac J Cancer Prev. 2012;13:4555
4559. 113. Menendez JA, Vellon L, Colomer R, Lupu R. Oleic acid, the main monounsaturated fatty acid of olive oil, suppresses Her-2/ neu (erbB-2) expression and synergistically enhances the growth inhibitory effects of trastuzumab (Herceptin) in breast cancer cells

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with Her-2/neu oncogene amplification. Ann Oncol. 2005;16: 359
371. 114. Elamin MH, Daghestani MH, Omer SA, et al. Olive oil oleuropein has anti-breast cancer properties with higher efficiency on ERnegative cells. Food Chem Toxicol. 2013;53:310
316. 115. Corominas-Faja B, Cuyas E, Lozano-Sanchez J, et al. Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells. Carcinogenesis. 2018;39:601
613. 116. Losso JN, Bansode RR, Trappey 2nd A, Bawadi HA, Truax R. In vitro anti-proliferative activities of ellagic acid. J Nutr Biochem. 2004;15:672
678.

117. Acquaviva R, Di Giacomo C, Sorrenti V, et al. Antiproliferative effect of oleuropein in prostate cell lines. Int J Oncol. 2012; 41:31
38. 118. Goldsmith CD, Vuong QV, Sadeqzadeh E, Stathopoulos CE, Roach PD, Scarlett CJ. Phytochemical properties and antiproliferative activity of Olea europaea L. leaf extracts against pancreatic cancer cells. Molecules. 2015;20:12992
13004. 119. Goren L, Zhang G, Kaushik S, Breslin PAS, Du YN, Foster DA. (2)-Oleocanthal and (2)-oleocanthal-rich olive oils induce lysosomal membrane permeabilization in cancer cells. PLoS One. 2019;14:e0216024.

Chapter 32

Immune system and olive oil Seyede Sanaz Seyedebrahimi Medical University of Kurdistan, Sanandaj, Islamic Republic of Iran

Abbreviations COX-2 EVOO HT IBD IFN IL iNOS IκB-α LPS MAPK MMP MS MUFA NF-κB Nrf2 OA VOO

cyclooxygenase 2 extra-virgin olive oil hydroxytyrosol inflammatory bowel disease interferon interleukin inducible nitric oxide synthase I-kappa-B-α lipopolysaccharide mitogen-activated protein kinases metalloproteinase multiple sclerosis monounsaturated fatty acid nuclear transcription factor kappa-light-chain-enhancer of activated B cells nuclear factor (erythroid-derived 2)-like 2 oleanolic acid fraction virgin olive oil

32.1 Introduction The growing interest on the relationship between Mediterranean diet (Med Diet) and human health hinges on the increasing number of published experimental, clinical, and epidemiological studies that are based upon its effect on the human health status.1
3 Several studies have shed new light on an array of potential benefits of Med Diet pattern on several malignancies, immunological and inflammation conditions4
6. Interestingly, olive oil is responsible for the effects of Med Diet remarkable healthy benefits as well as its immunomodulatory properties proved with epidemiological study on Mediterranean area people with olive oil daily consumption that is estimated as 30 g/day.7
9 Olive oil is a composite of saponifiable fraction and unsaponifiable fraction (UF), which is a minor fraction.10
12 The multilateral effects of nutrition on immune system are undoubtable13 and so does the olive oil. Chronic inflammation and oxidative stress are

involved in pathogenesis of many chronic degenerative diseases such as autoimmune disease and inflammatory conditions.14 Olive oil due to its composition is capable of modulating several immune functions because of their anti-inflammatory and antioxidant properties.15,16

32.2 Effects of olive oil components on immune responses Consumption of some foods as well as certain nutrients can help to preserve good health or to increase the risk of developing chronic degenerative disease. While diet influences all the bodily systems and tissues, the immune system is no exception.17
19 The major function of immune system is to response to cell injury elicited by trauma or infection to protect the host by the inflammatory process that is mediated by immune cells, blood vessels, and molecular mediators. Immune system deficiency also persists in inflammatory response, and oxidative conditions in the body are responsible for many diseases. Inflammation is a normal biological response to protect the body. Sometimes tissue health is not restored so inflammation becomes a chronic condition. This change is capable to erode surrounding tissues and becomes a systemic disorder.20,21 Interestingly, chronic inflammation produces oxidative stress through cellular damages and death due to proteins, lipids, and DNA/RNA hydrolysis oxidative stress process.22,23 Oxidative stress is the result of an imbalance between oxidant and antioxidant systems that leads to increase in the formation of reactive oxygen species (ROS) or free radicals and reactive nitrogen species, although they are involved in maintaining of cell homeostasis and functions such as receptors activations, gene expression, and signal transduction in limited quantity in normal conditions.24 Recent studies demonstrated that in oxidant conditions, ROS and its enzymatic and nonenzymatic sources, including NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase),

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00006-7 © 2021 Elsevier Inc. All rights reserved.

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cyclooxygenase (COX), and lipoxygenase, exist in different mammalian cells25,26 that can be implicated in pathogenesis of various diseases such as cancer, inflammatory disorders, cardiovascular, and metabolic diseases.27
30 During the last decades, clinical studies demonstrate that olive oil
rich diet decreases the proinflammatory level in sera of interleukin (IL)-18, IL-7, and IL-6. Twomonth consumption of monounsaturated fatty acid (MUFA)-rich diet significantly decreased intracellular adhesion molecule 1 in the peripheral blood mononuclear cells (PBMCs) compared to a normal diet. The principal phenolic compound is oleuropein that, in the intestinal tract, is hydrolyzed to hydroxytyrosol (HT) (3,4-dihydroxyphenyl-ethanol) as a main end product of extra-virgin olive oil (EVOO) metabolism.31
33 Polyphenols finally come in the blood but not in the initial biological form.34 Several studies investigated minor compounds of olive oil that have some antiinflammatory and antibiotic properties.35,36 HT is a phenolic compound that can downregulate immunological response and reduce inflammation and edema as well as proinflammatory cytokine such as IL-1β and tumor necrosis factor-α (TNF-α).37 It inhibits lipoxygenase activity and lowers the level of reactive oxygen metabolites that are responsible for diseases such as gastrointestinal disease and atherosclerosis.38
40 HT prevents oxidative damage in human erythrocytes.41 Lipopolysaccharide (LPS)-stimulated peritoneal macrophages isolated from mice demonstrate that UF of EVOO has antiinflammatory and antioxidant effects by inhibiting LPS-induced intracellular ROS and nitrite production as well as COX-2 and inducible nitric oxide synthase (iNOS) protein expression. These effects were related to modulate different intracellular signaling pathways from nuclear transcription factor kappa-light-chainenhancer of activated B cells (NF-κB) to mitogenactivated protein kinase (MAPK) through the modulation of cellular redox.42,43 Anti-inflammatory properties of maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes. They are found in the nonglyceride fraction of olive pomace oil (orujo olive oil). Mononuclear cells treated with olive pomace oil revealed that uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1β and IL-6 production at 100 μM, but at 10 μM, uvaol and oleanolic acid enhanced the generation of TNF-α with no significant change of cytokine expression by Maslinic. All four triterpenes inhibited production of I-309(ccl1), at 50 and 100 μM. However, uvaol enhanced I-309 production at 10 μM. The triterpene dialcohols had a similar effect on interferon (IFN)-γ (MIG/CXCL9) production. This study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and antiinflammatory properties depending on chemical structure and dose and may be useful in modulating the immune response.44

Polyphenols suppress TNF-α and may activate the transcription factor, nuclear factor (erythroid-derived 2)like 2 (Nrf2). Nrf2 plays a key role in cellular protection against oxidative stress and inflammation.45 HT, taxifolin, and oleic acid (OA) as olive oil compounds affect endothelial cell functions essential for angiogenesis and can inhibit specific autophosphorylation sites of VEGFR-2 and also its signaling pathway.46 VOO contains micronutrients [UF and squalene (SQ), phenolic fraction (PF) and HT] on visfatin. Visfatin is a biomarker of inflammation47 that is secreted by activated leukocytes as a pre
B cell colony
enhancing factor in bone marrow stroma with antiapoptotic activity and in inflammation regulation.48
50 Martin et al. declared the effect of VOO micronutrients (UF and SQ, PF and HT) in LPS induces inflammation on primary human monocytes.51 They found that the cell cultures of monocytes with different concentrations of UF and PF from VOO have antiinflammatory properties through downregulation of visfatin exit from cells, probably due to HT.51 Micronutrients of VOO impact on gene regulation of sirtuin 1 (SIRT1) and its downstream target PPARγ.52 SIRT1 expression is critical to restore normal condition after activation of macrophage and its cytokine production.53 So in SIRT1 deficiency situations, systemic and possibly vascular wall inflammation can occur.54 VOO micronutrients reupregulate suppressed SIRT1 gene expression caused by LPS induction in monocytes and also increase PPARγ gene expression. UF in VOO is capable of restoring mRNA levels of SIRT1 and PPARγ genes. VOO micronutrients, especially HT, reduce the proinflammatory genes expressions such as IL-1β, IL-6, and TNF-α and increase antiinflammatory cytokine IL-10 gene expression.51 Other compounds of olive such as oleanolic acid and related triterpenoids can inhibit visfatin production in murine 3T3-L1 adipocytes possibly through blocking PPARγ activation besides suppressible effects on cytidine
cytidine
adenosine
adenosine
thymidine/ enhancer binding protein α.55 In late pregnancy compared to women who are not pregnant, there is a higher level of free radicals and lipid peroxides product in the body. However, during pregnancy, antioxidant is upregulated to restore the balance of biological oxidative stress. In pathological pregnancies such as Pre-eclampsia (PE), the production of ROS increased by heat shock protein (Hsp) has long been known to protect cells from oxidative stress, and PE condition shows decrement Hsp70 in placental.56,57 Hsp70 repairs damaged proteins and, in fact, minimizes cell death due to stress agents; Hsp70 sera level in PE seems to reflect the occurrence of systemic inflammation, oxidative stress.58,59 Recent study focused on the survey of Hsp70 level in PE pregnant white rat model serum fed

Immune system and olive oil Chapter | 32

different doses of EVOO.60 Olive oil in form of EVOO is antioxidant-rich component owing to tocopherols (vitamin E) that ability to maintain the levels of Hsp70 has been proven.61 Different doses of EVOO, starting at a low (0.45 g/bw/day) to high (1.8 g/bw/day), suppressed the induction of Hsp70 in the blood, and from the lowest dose induce antiapoptotic effect in cells undergoing oxidative stress in PE.60 Animal models were conducted to show the effects of olive oil on inflammatory conditions in pregnancy. Olive oil induces antiinflammatory effect in late pregnancy and during lactation in sows and their piglets more than fish oil. Besides the effect of olive oil on milk fat content, it leads to significantly decreased IL-1β, IL-6, and TNF-α concentrations in milk in sows also lowered the plasma IL-1β and TNF-α levels in piglets.62 Combination of EVOO with nanoherbal andaliman (Zanthoxylum acanthopodium) increased the expression of Hsp70 in placental of PE rat model.63 It decreased the level of necrosis in hepatocyte cells in PE rat models,64 which is a multisystem disorder associated with chronic immune activation in prenatal condition.65 Olive oil
based lipid emulation [olive oil (80%) and soybean oil (20%)] known as ClinOleic as a parenteral nutrition (PN) shows some difference. Soybean oil
based lipid emulsions only are approved lipid formulation for clinical use in PN. A transient but significant decrease in systolic blood pressure is experienced in infusing olive oil
based PN compared to soybean-based PN. Olive oil resulted in abatement in immune functions, including granulocyte and monocyte phagocytosis, oxidative burst activity of granulocyte (ROS generation) and also decrease in the circulating levels of inflammatory markers [C-reactive protein (CRP), IL-6, and TNF-α] and oxidative stress such as redox potential of glutathione/glutathione disulfide couple and redox potential of cysteine/ cysteine couple with no significant difference with soybean oil
based lipid.66

32.3 Olive oil and immune-mediated inflammatory diseases Immune-mediated inflammatory disease (IMID) is a term used to define a group of chronic clinically heterogeneous diseases that result from abnormal activity or overreactions of the body’s immune system.67 IMID condition encompasses several subsets such as allergic disease, autoimmunity diseases, and some human immunodeficiency virus (HIV)-associated disease.67
69 It shares diverse epidemiological and clinical features such as existence of inflammation followed from immune dysregulation and activation of inflammatory cytokines such as IL2, IL-6, and TNF-α, which can cause pathological

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consequences.70
72 IMID prevalence has been raised in well-developed industries of different countries.73,74 Lifestyle and diet affect the promotion and maintenance of health status during the entire life course.75,76 Metabolized substances from the digestion and absorption of EVOO are distributed throughout the body through the bloodstream. They are useful for improving systemic and local inflammatory condition in IMID.77

32.3.1 Allergy Allergic diseases are a number of conditions caused by hypersensitivity of the immune system to the typical harmless substances in environment.78 Sarcoidosis is a type of antiallergic sentences.79 A study was conducted on HT effects of antiinflammatory activity by costimulation of PBMCs of healthy donors with Parietaria judaica pollen 1 (Parj 1) allergen and HT.80 Parj 1 allergen is capable of activating components of the innate and adaptive immune system during the telegenic response and at last induces INF-γ and IL-10 producing cells.81,82 HT modulates an allergenic-specific immune response potentiating a suppressive immune response toward an allergen through the induction of IL10 production.80 Common allergies are atopic dermatitis (AD) and allergy asthma that had improved in treatment with olive oil.

32.3.1.1 Atopic dermatitis (AD) Natural oils are advocated and extensively used worldwide as a moisturizer and natural skin care in AD or acute conditions. But there is an absence of evidence to support their efficiency and safety profile. The properties of these natural oils to treat or prevent dermatological conditions largely depend on the unique characteristics of the phytochemical composition.83,84 Especially the ratio of the oleic acid (OA) to linoleic acid (LA) determines the effect on the skin. Positive effects are generally associated with low OA and high LA ratios. High LA concentrations have been shown to accelerate skin barrier development and repair, skin hydration and suppress the severity of AD and be steroid sparing.85,86 Recent study clarifies differences of sunflower seed oil that is a natural oil with high LA/OA ratio with olive oil with lower ratio of LA/OA. While sunflower seed oil leads to hydration and preserve stratum corneum integrity, olive oil significantly damages the skin barrier and induces erythema by disrupting the lipid structure of stratum corneum and inhibiting homeostasis.85 AD-like lesions in a mouse model, topical treatment with olive oil had a protective effect, significantly damage the barrier and develop the AD. The usage of sea buckthorn (SBT) oil in contrast with olive oil blocks NF-κB/

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signal transducer and activator of transcription 1 (STAT1) signaling pathway activation and suppresses production of Th2 chemokine cells. SBT besides OA is composed of higher palmitic acid in comparison with olive oil.87 Olive oil can impair skin barrier, but it is important to mention that allergic contact dermatitis from olive oil is rare.88,89

32.3.1.2 Allergy asthma Asthma is a chronic inflammatory disease and it is characterized by inflammation in bronchial caused by aberrant responses of lung to normally innocuous environmental allergens. This results severe inflammatory cascade so airways become narrow and patients experience coughing, shortness of breath, and wheezing.90,91 Environmental and genetic factors together play an important role in the etiology of asthma.92 A study was conducted to show the effect of Med Diet as well as olive oil to asthma disease. Adherence to the Mediterranean-type diet is associated with lower prevalence of asthma symptoms among school-aged children.93 n 2 3 fatty acids (MUFA) which exist in olive oil have antiinflammatory properties in asthma.94 Olive oil on its own significantly inhibited histamine induction via its receptor of guinea pig ileum cells because olive oil acts as H1 receptors antagonist95 such antiasthmatic activity may be useful in the management of bronchial asthma as an alternative drug therapy. Olive oil use orally in asthma patients leads to inhibit the milkinduced leukocytosis and eosinophilia significantly, whereas the milk intoxicant group showed a significant increase in the eosinophil and leukocytes count.96 Interestingly, olive oil foot massage increased the adequacy of asthma control as well as improved the daily activities and decreased shortness of breath and wheeze in asthma patients.97 Together these studies mentioned that the potential of olive oil to cure asthma symptoms and further studies must be done.

Maslinic acid treatment on LPS-induced RAW 264.7 cells, an arthritis model, suppressed TNF-α activation as well as the phosphorylation of I-kappa-B-α (IκB-α). Maslinic acid exerted antiinflammatory and antiarthritic effects as shown by the suppression of paw edema, arthritis score, inflammatory cells, and destruction of synovium in knee joints.101 Rosillo et al. worked on RA by DBA-1/ J mice collagen type II
induced RA mouse (collageninduced arthritis) model. Mice were fed HT acetate (HTyAc), an EVOO. Polyphenol and histological and biological analyses were conducted. The HTy-Ac diet significantly repressed development of arthritis edema and decreased serum IgG1 and IgG2a. Moreover, serum levels of cartilage oligomeric matrix protein (COMP) and MMP-3 declined. These effects are due to dramatic reduction in proinflammatory cytokines level in sera such as TNF-α, IL-1β, and IL-17 as well as relevant transduction signaling pathways molecules Janus kinase (JAK)/STAT, MAPKs, and NF-κB, besides enhancement in expression of heme oxygenase-1 (HO-1) and Nrf2.102 Dietary EVOO leads to reduction in joint edema and cartilage destruction. COMP and MMP-3 levels, as well as proinflammatory cytokines levels (TNF-α, IFN-γ, IL-1β, IL-6, and IL17A). The activation of JAK/STAT, MAPKs, and NF-κB pathways was drastically ameliorated. Nrf2 and HO-1 protein expressions were significantly upregulated in mice fed with EVOO phenolic compounds.103 Refined olive oil (ROO) with HT usage in rodent model of RA induced by carrageenan showed a significant impact on both chronic and acute inflammation status through decreasing in bone resorption degree along with osteophyte formation. Paw edema, soft tissue swelling, and histological damage were improved in addition to reduction in COX-2 and iNOS expression.104 The high Med Diet pattern could reduce RA risk in ever smoking women.105 All of these studies show that olive oil supplements might be capable of providing a basis for developing a new dietary and therapeutic strategy for the prevention of RA.

32.3.2 Autoimmunity 32.3.2.1 Rheumatoid arthritis

32.3.2.2 Inflammatory bowel disease

Rheumatoid arthritis (RA) is one of the common autoimmune diseases. RA is a chronic multistep and progressive disease that leads to systemic inflammatory disorder.98 Different studies demonstrated the anti-inflammatory effect of olive oil components in RA disease. Bitler et al. proved that administration of hydrolyzed olive vegetation water in LPS-treated BALB/c mice and THP-1 cells (treat with LPS) as a model of joint inflammation leads to TNFα abatement,15 and in other mice model, oleuropein fraction was capable to reduce chemokines such as MIP-1 and MIP-2, myeloperoxidase activity, metalloproteinase (MMP)-1, and MMP-3 along with leukocyte infiltration to joint.99,100

Inflammatory bowel disease (IBD) encompasses a range of multifactorial chronic relapsing immune-mediated inflammatory condition, ulcerative disease, and Crohn’s disease (CD) constitutes the two main subtypes. Similar to several other autoimmune and chronic inflammatory conditions, the incidence and prevalence of IBD are increasing worldwide specially in Western countries.106,107 Several genetics and environmental factors produce immune dysregulation, which are responsible for IBD condition. EVOO can induce inhibitory effects on proinflammatory protein expression and suppress oxidative events in dextran sulfate sodium
induced mice model.108,109 Oxysterols present in food are the oxidized

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of dietary cholesterol. They interfere with homeostasis of human digestive tract and may lead to IBD or colon cancer.110,111 Serra et al. show the effect of EVOO on oxysterol proinflammatory actions.112 Phenolic extract components of EVOO are capable to inhibit iNOS induction of oxysterols. There was no increase in nitric oxide concentration and remained at normal level. They have potency to modulate directly phosphorylation of p38 and JNK1/2 and activation of NF-κB while interfering in IκB phosphorylation.112 Moreover, oxysterols proinflammatory actions such as increases level in IL-8 and IL-6 concentration besides decrease of GSH level, can be modulated by EVOO as an important antioxidant activity.112
114 Therefore chronic intestinal inflammation was suppressed.112 Several studies were conducted to evaluate olive oil fractions on IBD. Polyphenolic compounds of olive oil such as HT, oleuropein, and OA result to attenuate in STAT3, TNF-α levels and downregulate JNK phosphorylation, and also depression of several inflammatory mediator expressions such as COX-2, iNOS, IL-8, IL-6, and IL-1β and increase in IL-10 as an antiinflammatory cytokine was observed.109,115,116 All of these studies show a positive effect of olive oil in inflammation status of intestine.

32.3.2.3 Systemic lupus erythematous Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic inflammatory that can effect multiple organs.117 Olive oil
treated pristane-induced SLE mice models reduced renal damage association with adverse reduction in microsomal prostaglandin E synthesa-2 due to pristane and also inhibit the activation of NF-κB, MAPK, and JAK/STAT signaling pathway as an antiinflammatory effect. EVOO-diet intake significantly abates activation of STAT3 and MAPK at transcriptional level.118 Surprisingly, it repressed IL-10 and IL-17 production through interfere in JNK-p38-pERK-MAPK and STAT3 signaling pathway. Suppression in proinflammatory cytokine production in splenocytes such as IL-6, IL1β, and TNF-α was observed. Serum level of matrix MMP-3 reduced beside. Kidney expression of HO-1 and Nrf2 was upregulated.118 In a recent study, EVOO phenolic extract show adverse antiinflammatory and immunomodulatory effects in phytohemagglutinin (PHA)-stimulated PBMC from SLE patients and also in healthy donor. T cell ERK1/2 signaling pathway has been decreased by PE and also decreased PHA-induced phosphorylation of ERK1/2. CD691 T cells were increased in healthy donor but were opposed with PE that frequently decreased. Although IL1β level has no difference in SLE and healthy donors, PE treatment could reduce its level in both models. PE leads to reduction in IL-6 and INF-γ, in which they are

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favorable for severity of SLE condition.119,120 PE suppressed drastically PHA-stimulated inhibitory protein of NF-κB (IκB-α) degradation. PHA leads to increase of IκB-α degradation and activates NF-κB in both healthy and patient models; also, PE can suppress both similarly, which may be an exponent for the capability of diet on immunoregulatory. TNF-α and IL-10 productions in SLE PBMCs cultures were much more compared to healthy donors with PHA, and it is clear that TNF-α is associated with kidney destruction.121 Anti-dsDNA antibody production and IL-10 are very important in SLE pathogenicity via Th1 suppression, and consequently its survival, proliferation, and differentiation of B cells.122 But PE, significantly in cell culture, inhibits IL-10 and TNF-α production, but IL-10 was increased in healthy cell culture that declares its antiinflammatory and immunomodulatory effects of PE in human PBMCs.121

32.3.3 Other complications 32.3.3.1 Atherosclerosis and cardiovascular disease The Med Diet supplemented with olive oil products is a protective preventing cardiovascular disease even in highrisk persons not only by improving classical risk factors but also by promoting an antiinflammatory effect in meatbased or Westernized diets are favorable for improving inflammatory conditions.123
125 Atherosclerosis is an inflammatory condition and the main cause of cardiovascular disease. The idea of being an IMID condition came from abundant immune cells and their products specially proinflammatory cytokines in atherosclerotic lesions.126
128 Many studies demonstrated that food ingredients or vitamins can influence on atherosclerotic biomarkers, worthy of note, diets based on olive oil that is characterized by high content of antioxidant polyphenols. These are associated with low prevalence of cardiovascular diseases and atherosclerotic vascular disease.129
131 EVOO consumption can provide beneficial effects on inflammatory markers such as IL-6 and CRP in coronary disease patients.132 Monocyte recruitment is crucial in pathogenesis of atherosclerosis.133 Olive oil phenolic extract compounds are capable to impair NF-κB signaling in monocytes that besides their antiinflammatory consequences and can suppress MMP-9 stimulation.134 Katsarou et al. used high oleic sunflower oil that is containing EVOO
polar phenolic compounds (10% of EVOO phenols) in the cardiovascular (hypercholesterolemia) rat model.135 This oil attenuated increases in malondialdehyde (MDA) and TNF-α levels. This induces endothelial-leukocyte adhesion molecule 1 (E-selectin) and vascular cell adhesion protein 1 levels, but no difference in glutathione and IL-6 levels was observed.

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Antiinflammatory and antioxidant of EVOO refer to these phenols.135 Liver analysis of rats fed to VOO evinces reduction in protein oxidative damage markers.136 Inflammatory angiogenesis play a key role in atherosclerosis pathogenesis and tightly regulated by the proinflammatory enzyme COX-2 and MMPs.137,138 HT from EVOO has antiangiogenic ability by reducing matrix MMP-2. VOO tyrosol (T) and (HT) has antioxidant role in Phorbol 12-myristate 13-acetate (PMA)-stimulated monocytes (THP-1 cell lines) as an atheroma model. HT decreases anion superoxide and tyrosol fully prevented ROS overproduction in both T- and HT-treated cells expression of MMP-9 was reduced.139 Oleuropein and HT influence inflammatory angiogenesis in endothelial cells stimulated by PMA through inhibition of COX-2 protein expression and prostanoid production, MMP-9 protein release, and gelatinase activity.140 HT treatment of human PBMCs and U937 monocytes blunted monocyte matrix invasive potential and reduced MMP-9 release and expression without affecting tissue inhibitor of MMP-1 also can inhibit phorbol ester-induced NF-κB activation. Repressive effect of HT on NF-κB protein kinase C (PKC)α and PKCβ1 activation leads to downregulation of prostaglandin E2 production and COX-2 expression, without affecting COX1140 olive oil can be a therapeutic agent for atherosclerosis in human and animal models131,141 to improve the health status of peoples suffering from heart and vascularmediated disease.142

32.3.3.2 Human immunodeficiency virus
associated disease Acquired immunodeficiency syndrome (AIDS) is caused by HIV. Patients with AIDS represent progressive failure of the immune system that allows life-threatening opportunistic for several immunological disease to thrive. EVOO and ROO were evaluated on HIV-infected people.143 To prolong life expectancy and increase quality of HIV patients antiretroviral therapy (ART) was developed. However, persons successfully treated with ART also have an increased risk for developing many chronic conditions such as atherosclerosis. In this study atherosclerosis biomarkers (e.g., IL-6, ox-LDL, superoxide dismutase (SOD), GSH-Px, and MDA), erythrocyte sedimentation rate (ESR), high-sensitivity CRP (hsCRP), and IL-6 were evaluated. Atherosclerosis biomarkers had no changes but the level of ESR and hsCRP decreased even in EVOO more than ROO.143 In a cross-sectional study based on Med Diet, intake was conducted for 227 HIV-infected patients. Adherence to a Mediterranean dietary pattern is an effective strategy to reduce cardiovascular risk affection in HIV-infected patients with fat redistribution.144 Olive leaf extract

(OLE) has anti-HIV effect in a dose-dependent manner. Uninfected MT2 cells were cocultured with HIV-1infected H9 T lymphocytes and OLE.145 Results demonstrate that OLE inhibits acute infection and cell-to-cell transmission of HIV-1 from T-infected lymphocytes to MT2 cells. Evaluation of p24 expression in infected H9 cells indicates that OLE is capable of HIV-1 replication in cells. OLE induces ant apoptotic properties in infected cells by upregulation in apoptosis inhibitor proteins (IAP1/2), IL-2, IL-2Rα signaling pathways.145 Collective evidence from previous studies on AIDS suggests that olive oil products and components have antiviral activity. It can be used as an alternative agent.

32.4 Conclusion Growing evidence suggests that olive and its products such as olive oil can modulate immune function and affect overall health status of patients.

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325. 104 Silva S, Sepodes B, Rocha J, et al. Protective effects of hydroxytyrosol-supplemented refined olive oil in animal models of

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1413. Sa´nchez-Fidalgo S, Villegas I, Cardeno A, et al. Extra-virgin olive oil-enriched diet modulates DSS-colitis-associated colon carcinogenesis in mice. Clin Nutr. 2010;29(5):663
673. Deiana M, Calfapietra S, Incani A, et al. Derangement of intestinal epithelial cell monolayer by dietary cholesterol oxidation products. Free Radic Biol Med. 2017;113:539
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2057. Serra G, Incani A, Serreli G, et al. Olive oil polyphenols reduce oxysterols-induced redox imbalance and pro-inflammatory response in intestinal cells. Redox Biol. 2018;17:348
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1037.

Section 2.5

Other effects, uses and diseases

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Chapter 33

Effect of olive oil on the skin Diana Badiu1 and Rajkumar Rajendram2,3,4 1

Faculty of Medicine, Ovidius University of Constanta, Constanta, Romania, 2Department of Medicine, King Abdulaziz Medical City, King Abdullah

International Medical Research Center, Ministry of National Guard—Health Affairs, Riyadh, Saudi Arabia, 3College of Medicine, King Saud bin Abdulaziz University of Health Sciences, Riyadh, Saudi Arabia, 4Nutritional Sciences Research Division, School of Life Sciences, King’s College London, London, United Kingdom

Abbreviations 15-HETE AA ALA COX DEI DHA DNA ECM EFAs EPA ERK 1/2 FAs GAGs GLA IL LA LOX LTB4(5) MEs MHBO MMP-2 MUFAs NO OA PGE2(3) PUFAs ROS SDA SSD TGA UV VCAM-1 VEGF

hydroxyeicosatetraenoic acid arachidonic acid α-linolenic acid cyclooxygenase dermal
epidermal interface docosahexaenoic acid deoxyribonucleic acid extracellular matrix essential fatty acids eicosapentaenoic acid extracellular signal-regulated kinase 1/2 fatty acids glycosaminoglycans gamma-linolenic acid interleukins linoleic acid lipoxygenase leukotriene B4(5) microemulsions mixture of honey, beeswax, and olive oil metalloproteinase-2 monounsaturated fatty acids nitric oxide oleic acid prostaglandin E2(3) polyunsaturated fatty acids reactive oxygen species stearidonic acid silver sulfadiazine triacylglycerols ultraviolet vascular cell adhesion molecule 1 vascular endothelial growth factor

33.1 Introduction The skin, the largest organ of the human body, performs many essential functions. It is a complex organ that regulates heat and water loss from the body while preventing the entry of environmental toxins and microorganisms. The skin is also a sensory organ that detects touch, pressure, pain, and temperature. In most developed countries, life-expectancy has increased over the last century. The appearance and structure of skin change with longevity. Aging skin is a very visible and, for many people, an undesirable sign of seniority.1 The process of aging and the appearance of wrinkles generally increase with age, being caused primarily by the reduction of collagen content. Xerosis can result in complications such as pruritis, inflammation, and infection of the skin, particularly if left untreated. In cases of severe xerosis, excoriations and bruising may develop anywhere on the body. These secondary lesions may hide the primary pathology (i.e., xerosis). Therefore the skin should be monitored periodically for dryness, especially in the elderly. However, topical application of plant oils, such as olive oil, could reduce inflammation by providing other antiproliferative metabolites [e.g., collagen and glycosaminoglycans (GAGs)] and/or stimulate fibroblasts. This can maintain the strength of the tissues.2 There is significant interest in the possible use of olive oil, in clinical practice, in humans. The effect of direct application of olive oil to the skin has been studied. But, the limited evidence that is available suggests that, in

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00032-8 © 2021 Elsevier Inc. All rights reserved.

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some cases, this may be detrimental.3 For example, in healthy volunteers, topical application of olive oil for 4 weeks reduced stratum corneum integrity and caused mild erythema.3 This chapter reviews the main beneficial effects of skin, especially on wrinkles, xerosis, and pruritis.

33.2 Skin: a natural barrier. Structure and physiology The skin is a highly efficient self-repairing barrier. It keeps “the insides in and the outside out.” The skin consists of several layers of cells that are constantly being turned over (i.e., the stratum corneum, viable epidermis, underlying dermis, and the innermost subcutaneous fat layer, hypodermis). The cycle of shedding and regeneration occurs approximately once every 30 days in vivo. Although often thought to be a simple physical barrier, the skin is metabolically active and has immunological and histological responses to assault. The outermost and toughest layer of the skin is the stratum corneum (the “horny layer”). It consists of only 10
15 layers of nonviable corneocytes (cornified keratinocytes); the same dead cells that fingernails are made from. The structure of the stratum corneum can be described in terms of a “brick-and-mortar” construction. The large, dead, anucleate, keratinized cells (bricks) are tightly packed together like bricks. The small spaces between the cells are filled with lipids (mortar). The corneocytes prevent the passage of foreign material, while the lipids ensure that water is retained. The “dead” corneocytes on the surface of healthy skin detach from neighboring cells and are shed into the environment. These are replaced by younger cells from deeper layers. This orderly process of desquamation is controlled by corneodesmosomes and lipids.4 Corneodesmosomes bind the corneocytes together maintaining intercellular cohesion and tissue integrity. Tissue thickness is maintained by the intercellular actions of these two components. When the stratum corneum is dry, it is approximately 10 μm thick, a significant depth in dermatological terms. When wet, it can swell to several times this size. This thin membrane regulates water loss from the body, preventing desiccation while simultaneously restricting the entry of toxins and microorganisms. The viable epidermis is about 80 μm thick and the second layer, dermis that is several millimeters thick, contains capillaries and nerve endings.4

33.2.1 Skin care products, exfoliation, and the transdermal passage of molecules Skin accounts for 10% of body mass and has an average area of 1.7 m2. So, the skin should, theoretically, provide

multiple potential sites for the administration of medication for both local and systemic diseases. However, it is surprisingly tough and functions much more like a barrier than a membrane. So, most skin-care products moisturize only the “dead” layer of the skin (i.e., stratum corneum). Hydration of this layer prevents the sensation of dry skin. However, this is often deleterious, as the stratum corneum is easily waterlogged. The transfer of foreign material across the skin is restricted by both the “dead” stratum corneum and the “live” dermis and epidermis. Generally, only small water-soluble molecules can diffuse slowly into the skin. However, physical exfoliation of approximately 10 μm of skin increases the penetration of the active ingredients of topical skin-care products and medications across the stratum corneum.

33.2.2 Oxidative stress, inflammation, and metabolism of fatty acids in the skin The metabolic activity of the skin may cause oxidative stress. Toxic reactive oxygen species (ROS) alter the internal structure of the skin, affecting the proliferation of cells. This causes aging, xerosis, and pruritis. The skin also produces inflammatory prostaglandins and leukotrienes from arachidonic acid (AA) via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. However, skin does not produce some important fatty acids [FAs; e.g., linoleic acid (LA), α-linolenic acid (ALA), AA (20:4 omega-6), eicosapentaenoic acid (EPA, 20:5, omega-3), and docosahexaenoic acid (DHA, 22:6 omega-3)].5 These FAs must be obtained from exogenous sources (e.g., food). The ratio between omega-6 and omega-3 FAs should be maintained in favor of omega-3 FAs. This is because the omega-3 FAs, especially EPA and DHA, have more antiinflammatory effects.6 FAs maintain skin homeostasis, preventing the development of conditions such as hemorrhagic spots, erythema, “perspiratio insensibilis,” dehydratation, xerosis (dry skin), and pruritis (itchiness).

33.3 Clinical features and pathophysiology of aging conditions: wrinkles, pruritis, and xerosis 33.3.1 Wrinkles Wrinkles result from major cellular changes that occur within the skin. Cell turnover and regeneration gradually slows between the ages of 35 and 45 years, the subclinical phase of aging. The process of aging and the appearance of wrinkles generally increase after the age of 45, the clinical phase of aging. Very few people over the age of

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50 have escaped wrinkles. The difference is only in the degree of the blemish. With increasing age the skin thins as cell turnover and regeneration slows. The normally undulating, ridge-like dermal
epidermal interface (DEI) flattens. This reduces the surface area available for the exchange of nutrients between the dermis below and the epidermis above. Thus aging reduces the supply of nutrients to the epidermis, accelerating the degeneration of epidermal cells. Although the metabolism of epidermal cells slows, the elimination of the waste products of cellular metabolism (e.g., free radicals) is also reduced. Intracellular accumulation of free radicals can induce genetic mutations, which may ultimately cause cancer. Simplistically, wrinkles are caused by a reduction in the collagen content of the skin.7 While wrinkles are a very visible sign of aging, age-related loss of collagen and other molecules from skin, cartilages, or ligaments results in significant structural defects throughout the body.8 There are 19 forms of collagen which account for almost 20% of total body protein.9 The most abundant is type I collagen.9 Types IV and VII are particularly important at the dermal
epidermal junction.10 Type VII collagen is anchored to sheets of type IV collagen, which forms a basal layer. The progressive loss of nutrients at the DEI reduces the circulation of the various factors that stimulate and support collagen synthesis. A vicious cycle ensues. Without sufficient collagen, the skin sags further, exacerbating the deficiency of nutrients.11. In addition to the loss of skin thickness due to lack of collagen, aging skin is looser and lacks elasticity. These are hallmarks of wrinkles. Matured aging skin contains more elastin than younger skin.12 The elastin fills the space left by the deficiency of collagen. Unfortunately, the elastin in aging skin is fragmented, calcified, and contains excessive amounts of lipids.12 GAGs form a water-saturated gel in which hydrophilic molecules, hormones, peptides, and ions circulate. In the process of aging, collagen is gradually replaced by the weaker GAGs.13 Collagen provides a much better support structure than GAGs. The reduction in the amount of collagen and replacement with weaker macromolecules results in thicker skin that is less elastic.13 With age the GAG gel tends to sag, further compromising cellular metabolism and mitosis. Fibroblasts synthesize and maintain the extracellular matrix (ECM). Fibroblasts provide a structural framework for the skin and play a critical role in wound healing. The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuous secretion of collagen, the main constituent of the ECM. Fibroblasts in aged tissue produce less collagen.14 However, fibroblasts isolated from aged tissue can still

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produce significant amounts of collagen when stimulated by endogenous factors such as transforming growth factor-beta.15 Type I collagen is the predominant collagen in reconstructed healthy skin which is based on a good DEI with abundant types IV and VII collagen.16 Stimulation of fibroblasts to produce more collagen and GAGs in vivo could potentially rejuvenate aging skin. If so, wrinkles would disappear and skin thickness could be increased. Unsurprisingly, there is significant interest in the dermo-restitutive effects of olive oil. These may be able to reverse signs of aging and treat diseases of the skin.

33.3.2 Xerosis and pruritis Xerosis and pruritis are the two most common dermatological conditions worldwide. Sadly, these dermatological phenomena are now global public health concerns. Pruritis may be caused by any of a number of infectious, hepatic, metabolic, and hematological diseases. There are also many dermatological causes of pruritis, the most common of which is xerosis.17 In xerotic skin, corneodesmolysis is inefficient, so corneodesmosomes persist, disturbing the orderly process of desquamation.4 As described earlier, effective desquamation requires breakdown of corneodesmosomes by corneodesmolysis. In healthy skin, corneodesmolysis effectively eliminates the corneodesmosomes. However, free water is required for corneodesmolysis. Dry, dehydrated skin cannot provide this water. Lipids retain water in the skin and so are also required for effective corneodesmolysis.18 Therefore dehydration and/or a deficiency in lipid content may result in xerosis.19
21 Xerosis is most commonly caused by water loss. Other causes include cold air, exposure to heaters, exposure to alkaline soap or various toxins, chronic illness, and systemic disease. Xerosis can often be observed on the legs, which may develop a “cracked porcelain” appearance.22 The symptoms and signs of xerosis (e.g., itching, bleeding, dermatitis, and dry skin) can persist for years without treatment.23 Xerotic skin produces many proinflammatory metabolites (e.g., AA). 15-Hydroxyeicosatetraenoic acid (15HETE) is the product of metabolism of AA by 15lipoxygenases. 15-HETE inhibits leukotriene B4-induced chemotaxis in human polymorphonuclear leukocytes.24 Prostaglandins D, E, and F are produced when AA is metabolized by COX. At low concentrations, these prostaglandins maintain skin homeostasis and so are essential metabolites. However, at higher concentrations prostaglandins induce keratinocyte inflammation.25 So, replacing those substances with natural, healthy fats such as polyunsaturated

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FAs (PUFAs; i.e., EPA and DHA) may reduce inflammation.26 PUFAs play a major role in skin homeostasis. LA, for example, is metabolized to molecules with antiproliferative properties by 15-lipoxygenase. So, LA deficiency can cause scaly skin as noted in hairless mice.27 PUFAs could also be involved in decreasing nonspecific markers of inflammation (e.g., C-reactive protein)28 and in the regulation of hormone synthesis (e.g., estriol).29 So, PUFAs are now included in many antipruritic ointments and creams. However, excessive intake of PUFAs may, paradoxically, increase production of ROS. So, intake of PUFAs should be restricted to the recommended daily amounts.30 Olive oil has been called “liquid gold” since the time of Homer. The benefits of olives, a particularly nutritious fruit, and olive oil are even mentioned in the Quran.31 Centuries ago, this “liquid gold” was a luxurious rarity; today, olive oil is readily available throughout the world. Yet, olive oil is still highly desirable, not only for cooking but also for its beneficial effects on health.

33.4 General beneficial properties and constituents of olive oil The incidence of cardiovascular and neurodegenerative diseases in the inhabitants of the Mediterranean basin is low.32 This is thought to be related to the Mediterranean diet that is rich in olives. The effect of olive oil on cardiovascular disease has, therefore, been extensively investigated. For example, Guasch-Ferre´ et al.32 studied the association between total intake of olive oil and the risk of cardiovascular disease in 7216 men and women in the Prevencio´n con Dieta Mediterra´nea (PREDIMED) study. The effect of olive oil consumption on stroke, myocardial infarction, cardiovascular death, and mortality was assessed using a validated food questionnaire (i.e., measuring intake of olive oil). Those with the highest total olive oil and extra-virgin olive oil consumption had 35% and 39% reduction of cardiovascular disease risk, respectively. Olive oil may also provide protection from pathogens after ingestion of contaminated food. Medina et al.33 assessed the survival of foodborne pathogens in aqueous extracts of olives, virgin olive oil, vinegar, and several beverages. They found that olive oil, especially in mayonnaises and salads, reduces the numbers of Salmonella, Enteritidis, and Listeria monocytogenes present. Which of the many constituents of olive oil are responsible for these beneficial effects remains unclear. This has been difficult to determine because the natural compounds in any oil vary with the cultivar, time of harvesting, and extraction process.

The extraction of oil usually involves crushing fruits or vegetables and then separating the sediment (i.e., pulp) under different pressures.34 The preparation of olive oil initially involves crushing olives into a paste and then malaxing (mixing) the paste.34 The paste is then centrifuged to separate the oil. The oil is left in tanks where further gravity-driven separation (racking) occurs. The oil may then be filtered and bottled. Extra-virgin olive oil is mechanically extracted from pure olives, without any chemicals. Olive oil produced this way has a much darker color and contains more monounsaturated FAs (MUFAs) and PUFAs.35 Interestingly, removal of free FAs from olive oil lightens its color.36
38 After the mechanical and chemical extraction processes, regular olive oil must be further refined to be fit for consumption. This refinement process removes any residual pulp and can enrich the extracted oils’ content of essential FAs (EFAs), vitamins, polyphenols, and phytosterols.38 The polyphenol content of olive oils varies from 50 to 1000 mg/kg, depending on the extraction processes, packaging, and storage.39
42 It was thought that the so-called extra-virgin olive oil contains more polyphenols than refined olive oil.36,37,43 However, filtration of olive oil results in significant loss of polyphenols.44,45 Olives have high triacylglycerols (TGAs) content. TGAs include many esters and various FAs. The predominant FA, TGAs are monounsaturated [e.g., oleic acid (OA) up to 83% w/w, palmitic acid, LA, stearic acid, and palmitoleic acid]. Other important components are amphiphilic compounds; the phytosterols, squalene, tocopherols, and phenols.46
48 Peroxides are the main products of olive oil oxidation. Other constituents of olive oil may enhance the production of epoxides and/or other oxides from drugs,49 which accumulate in tissues.50
52 The oxide content is often either disregarded or simply assumed to be low in studies of unsaturated dietary oils. Peroxides, in particular, are not known to have any therapeutic value; it is therefore important to demonstrate that oils used to treat skin diseases have low peroxide contents. Antioxidants protect dietary oils from oxidation in situ and also prevent lipid peroxidation in vivo.53,54

33.5 The effects of olive oil on the skin Olive oil can be used to treat wrinkles, xerosis, and pruritis.55 Topical administration of olive oil improves the symptoms of pruritus in patients with renal failure treated with hemodialysis.55 The improvement of pruritis in patients on hemodialysis was also noted with topical administration of fish and safflower oils.55 However, pruritis in patients on maintenance hemodialysis is often due

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to dehydration of the stratum corneum.56 Even simple emollients may relieve this.56 So, olive oil may simply act as an emollient in this situation. Dietary ingestion of olive oil by atopic patients resulted in significant changes in plasma FAs profiles.57 All lipid fractions are affected. The changes in plasma LA, ALA, and gamma-linolenic acid (GLA) are particularly significant.57 Stearidonic acid (SDA), a rare omega3 FA, can be detected in plasma, but the amounts present are usually below thresholds for quantification.57 Administration of SDA increases production of EPA in vivo more than ALA.58 Dietary supplementation with SDA (0.75
1.50 g) increases levels of EPA in both erythrocytes and plasma phospholipids.58 However, daily intake of 0.60 g SDA is not sufficient to significantly increase the production of EPA or phospholipids.58 Ceramides may have an important role in the barrier function of skin, and reduced levels of ceramides in the stratum corneum may be relevant in the etiology of atopic dermatitis. LA is esterified to ceramide 1 but OA is not. OA could function as a molecular rivet in the stabilization of lipid lamellar sheets. Stabilization of lipid lamellar sheets in the skin could reduce the loss of water from the skin, especially in the elderly.59 In view of the significant amount of OA present in olive oil, it is surprising that consumption of olive oil has little effect on the plasma profile of OA. OA is not an EFA and so is not required for health. OA is not taken up as aggressively into plasma lipids as PUFAs. Overall changes in fatty cholesterol esters were less robust than those for triglycerides or phospholipids. However, if symptoms of atopic dermatitis are more dependent on membrane function than eicosanoid production, then such an increase in PUFAs in phospholipids bilayers could effectively increase membrane fluidity and function.57 External damage to the skin may be caused by ultraviolet (UV) radiation which also causes free radical formation. UVA radiation damages deoxyribonucleic acid (DNA) and PUFAs.60 Exposure to more than 30 min of UV radiation reduces α-tocopherol in the skin.61 So, α-tocopherol, either applied topically or taken orally, may reduce damage to the skin induced by UV radiation.61 A study of rats exposed to UV radiation and fed either safflower oil or olive oil found that rats fed with olive oil had less severe lesions.62 The UV light
induced skin changes mimicked those of normal aging.62 Rats fed with olive oil had better skin than those fed with safflower oil.63 So, people who consume olive oil should have younger looking skin than those who consume corn oil or safflower oil. Although olive oil has only recently been included in modern cosmetics, this pleiotropic oil has been used on the skin for thousands of years. Egyptian pharaohs used olive oil to moisturize their skin and hair and the Romans used the healing properties of olive oil to treat wounds.

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Individuals who have used olive oil for several years report that the strength of their finger nails increased within months and the thickness of their hair increased within a few years.64 Improvements in skin conditions occurred within weeks of starting to use olive oil.64 The times taken for these effects to manifest correspond to the time required for newly formed cells to become physically apparent. These cell lines of the skin, fingernails, and hair are developed from dermal stem cells using the FAs available at the time of their formation.64 Thus the formation of these tissues from dermal stem cells is complex and dependent on dietary FAs. The beneficial effects of dietary olive oil on skin, hair, and nails could be due, at least in part, to the large amounts of PUFAs present ( . 80%).65 These PUFAs have a metabolically favorable omega-6/omega-3 ratio of approximately 2:1.66 These FAs have important roles in the immune response.67 The FAs profile of olive oil is very similar to blackcurrant seed oil, which has been reported to improve immune function.68 The presence of both GLA and SDA in olive oil (the metabolic products of the EFAs, LA, and ALA, respectively) bypasses delta6-desaturase the rate-limiting enzyme in production of EPA from ALA.58 This could explain the improvement of atopic symptoms with the use of olive oil. Healing of the skin is a highly ordered systemic process that requires coordination of cellular proliferation and migration as well as tissue remodeling.69 The skin reacts to messenger molecules (e.g., cytokines and chemokines) by producing biological response modifiers (e.g., monoclonal antibodies, recombinant cytokines, or fusion proteins) which activate enzymes (e.g., collagenase and gelatinase) that recognize and degrade damaged cells and nonfunctional tissues.70 This process supports the immune system and repair mechanisms to treat injuries (i.e., cuts, bruises, abrasions, acne lesions, scars, wounds, and burns), dysfunction (i.e., keratosis, keloid and hypertrophic scars, excess dryness), and prevent reactions such as immune-mediated inflammatory skin diseases (i.e., inflammatory acne, rosacea, eczema, and most dermatitis) that damage the skin.

33.5.1 Antioxidant and antiinflammatory properties of olive oil Extracts from olive oil can scavenge hydroxyl radicals better than other oils.71 In addition to the direct antioxidant effects of olive oil, extracts are also higher inhibitors of xanthine oxidase activity. The nonsaponifiable fraction of olive oil contains significantly more squalene than seed oils. Skin sebum also contains large amounts of squalene. Squalene protects the skin, probably by scavenging singlet oxygen72 generated by UV light.73 Inhibiting UV-induced

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peroxidation in this way would provide the skin with photoprotection.74,75 Virgin olive oil applied to the skin after sunbathing could protect against skin cancer by decreasing the growth of tumors.76 These substances, like other antioxidants found in olives (e.g., vitamin E, polyphenols, and phytosterols) have antiinflammatory properties. These compounds may delay aging of the skin. Vitamin E and hydroxytyrosol, in particular, can restore skin smoothness, by protecting skin from UV light and free radical damage.77 A study by Romana-Souza and Monte-Alto-Coste78 found that administration of various antioxidants, such as those present in olive oil, could reduce stress-induced skin aging. After treatment with high levels of epinephrine or olive oil for 13 days, olives showed a highepinephrine-level-induced reduction in both epidermis and dermis of the ex vivo human skin. Higher levels of epinephrine increase ROS and malondialdehyde. This effect was attenuated by olive oil in ex vivo human skin. Other damaging conditions correlated with skin sensitivity include stressful experiences (e.g., aggression of nerves, ligaments, and muscles) which activate the sympathetic nervous system.79,80 Olives attenuated epinephrine-induced increase in extracellular signalrelated kinase 1/2 (ERK 1/2) and protein matrix metalloproteinase-2 (MMP-2) expression in ex vivo human skin.81 This is supported by the fact that olives attenuate many of the signs of stress-induced aging in ex vivo skin by reducing MMP-2 level, ROS, or ERK 1/2.78 Romana-Souza and Monte-Alto-Coste81 also showed that stress-induced oxidative damage plays an important role in the aging of skin. Supplementation with olive oil could inhibit their effects.81 The dermal and epidermal thickness of mice exposed to chronic stress was reduced in comparison to that in nonstressed mice. The organization of collagen fibers was not affected. Olive oil inhibited the effect of chronic stress on dermal and epidermal thickness.

healing process. These observations may provide the foundation for the future development of pharmaceuticals to promote healing. Kazemi-Darabadi et al.84 compared the effects of olive oil and lime water with SSD on the healing of thirddegree burns on 63 adult male Bulb/C mice. There were three groups. The control group received topical normal saline solution. The second group received SSD ointment daily. Topical olive oil and lime water was applied daily to the mice from the third group. Each group was subdivided into three subgroups. The topical treatments or saline were applied to each subgroup for 7, 14, or 21 days, respectively. After the second week, there was more collagen synthesis in the mice in the two treatment groups than in the saline-treated mice. The epidermis of the group treated with olive oil and lime water was significantly thicker than that of the group treated with SSD in the second week. Thus the combination of olive oil and lime water may improve healing of third-degree burns compared to SSD. Although lime water has cytotoxic effects, in combination with olive oil its adverse effects were negated. We also investigated the antiinflammatory and dermorestitutive effects of olive oil in Wistar rats in vivo using a model of induced thermal burn injury.85 Histological and hematological analyses were performed. Olive oil reduced the time taken for the wounds to heal 14
16 days85 (Fig. 33.1). Of the seven hematological parameters assessed, three (RBC, hemoglobin, and hematocrit) were normal in the animals treated with olive oil.85 Histological analysis demonstrated partial reconstruction of the basal stratum from the epidermis, the blood vessels, collagen, and fibroblasts of the dermis and hypodermis85 (Fig. 33.2). The results of this study suggest that the antiinflammatory and dermo-regenerative properties of olive

33.5.2 Wound healing Many studies have showed that plants (e.g., olives) have beneficial effects on the skin, especially on damaged skin (e.g., burned).82 Moustafa and Atiba83 studied the healing of burns treated with silver sulfadiazine (SSD) or a mixture of honey, beeswax, and olive oil (MHBO). After receiving a standardized second-degree burn, the dogs were divided into three groups [two treatment groups (MHBO, SSD 1%) and a control group]. The control group did not receive any treatment. The healing process was observed until complete healing of the wounds. The inflammation and exudate were much less in the MHBO group than the SSD and control groups. So MHBO accelerated the

FIGURE 33.1 High-power photomicrograph of the skin of Wistar rats treated with fish oil. This photomicrograph shows the skin of Wistar rats treated with fish oil. Stained with hematoxylin and eosin. Medium power ( 3 100).

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FIGURE 33.2 High-power photomicrograph of the skin of Wistar rats treated with olive oil. This photomicrograph shows the skin of Wistar rats treated with olive oil. Stained with hematoxylin and eosin. Medium power ( 3 100).

oil were similar to those of two fish oils described in our previous study.85 However, direct application of oil to the skin is relatively inefficient, erratic, and undesirable in the 21st century. So, constituents of plant and fish oils have therefore been studied to determine which molecules are most beneficial to wound healing. For example, the effects of oleuropein, one of the most important constituents of olive oil, were studied by Mehraein et al.86 A 1 cm long full-thickness incision was made on the back of necks of 16-month-old Balb/c mice under anesthesia.86 The incision was left unsutured and the mice received intradermal injections of either oleuropein (50 mg/kg once daily) or distilled water for 7 days.86 On days 3 and 7 after making the incision and starting the injections, some mice were sacrificed. Western blotting was performed to measure vascular endothelial growth factor (VEGF) protein expression. Oleuropein reduced cell infiltration at the wound site on days 3 and 7 postincision and significantly increased collagen deposition and VEGF protein expression.86 The precise mechanisms of the effects of olive oil and its constituents remain unclear but it is possible that their effects on capillary blood flow and endothelial function could become relevant.

33.5.3 Olive oil and endothelial function Very few studies have addressed the effects of long-term olive oil consumption on endothelial function. The Mediterranean diet, rich in olive oil, improved endothelial function in patients with diabetes or hypercholesterolemia,87 as assessed by measuring endothelium-dependent vasoreactivity. In a randomized crossover trial, Perona et al.88 demonstrated an improvement in endothelial

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function in 22 hypercholesterolemic subjects fed with a Mediterranean diet. However, the results were more significant when the dietary olive oil was partially replaced with walnuts. Perona et al.89 observed a greater improvement in flow-mediated dilatation in patients on fluvastatin who were also advised to eat a Mediterranean-style diet. Esposito et al.90 conducted a randomized controlled trial in 180 subjects with the metabolic syndrome who were instructed to eat a Mediterranean-style diet, including olive oil. After 2 years of follow-up, they observed improved endothelial function as measured by reduction in blood pressure and platelet aggregation response to Larginine, the precursor of nitric oxide (NO). They also reported a significant reduction of markers of systemic vascular inflammation, such as C-reactive protein and the interleukins (IL), IL-6, IL-7, and IL-18 as well as reduction in total cholesterol and low-density lipoprotein cholesterol. However, the mechanisms by which dietary olive oil produces these effects and the actual components of the oil responsible for these effects are unclear. The current understanding of the effects of the components of olive oil on endothelial activation is described later. It has been suggested that lipolytic enzymes are upregulated as energy requirements increase during acute inflammation.91 This releases free FAs from circulating lipoproteins for use by tissues, including the endothelium. The increase in endothelial free FA concentrations decreases endothelial NO bioactivity due to both superoxide generation and reduced endothelial NO synthase activity.92 Studies in vitro suggest that PUFAs are more proinflammatory than MUFAs and saturated FAs.93 In fact, LA (18:2, omega-6) has greater capacity to induce oxidative and inflammatory stress than other FAs. Incubation of LA with endothelial cells promotes nuclear factor kappa B activation and transcriptional activity.94 This effect is attenuated by vitamin E.94 In addition, exposure of endothelial cells to LA induces the production of cytokines such as IL-6 and IL-8 that are involved in the initiation and progression of atherosclerosis.95 Conversely, omega-3 PUFAs are believed to exert a protective effect on the endothelium. Particularly, DHA that decreases the expression of vascular cell adhesion molecule 1 (VCAM-1) on the vascular endothelium reduces monocyte adhesion96 and EPA which increases NO production. Although the results from in vitro studies are promising, the results of in vivo studies are more controversial. There was no reduction in soluble adhesion molecules in patients receiving omega-3 FAs after 6 weeks of treatment.76 However, intercellular adhesion molecule 1 and E-selectin were reduced after 7-month treatment with omega-3 FAs.76 Male smokers treated with omega-3 FAs for 6 weeks had reduced prothrombogenic von Willebrand factor but increased VCAM-1 and E-selectin.97 Their results were corroborated by Johansen et al.98

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The key inflammatory mediators released by the endothelium include the eicosanoids which are derived from the omega-6 PUFAs, AA. Prostaglandin E2 (PGE2) can cause pain and vasodilation and leukotriene B4 (LTB4) is a chemoattractant and activates neutrophils. PGE2 and LTB4 are formed from AA via COX and LOX pathways, respectively. However, EPA is also a potential COX substrate and can compete with AA, leading to formation of PGE3. However, the synthesis of PGE3 from EPA is very inefficient.99 EPA is also a substrate for LOX, forming LTB5, with less inflammatory activity than LTB4.58 Thus increasing the omega-3 FAs content in the diet can shift the eicosanoids produced to those with less inflammatory effects. The skin is at high risk of oxidative damage from ROS for at least two reasons. First, it is exposed to oxygen from a rich blood flow and the air at the surface of the skin. Second, a number of the physiological processes that occur in the skin, including cellular metabolism and differentiation, are photosensitive.100 The compounds in the skin which absorb light and act as photosensitizers generate ROS in the presence of UV light. Oxidative damage to epidermal cells101 and underlying connective tissue is recognized cosmetically as aging of the skin. The question is whether food components can modulate this actinic damage. Candidate nutrients that might prevent or reduce actinic damage include those that are present in sufficient quantity in skin and are either oxidizable, antioxidant or indirectly influence these activities. The FAs composition of the epidermis is 25% unsaturated, relatively chemically unstable and susceptible to ROS.102 There is considerable interest in the use of natural compounds in skin protection. Topical application of antioxidants, experimentally, indicates that they may usefully decrease photodamage and associated inflammation.101 The use of various antioxidants such as vitamin C,17,103 vitamin E104 alone, and in combination105 as topical photo-protectants has been investigated. It has also been shown that hydroxytyrosol in extravirgin olive oil is highly protective against DNA damage caused by peroxynitrite, which is formed by superoxide radicals and NO.106

33.5.4 Use of olive oil in the clinical treatment of foot ulcers Foot ulceration is increasing in incidence. This is a common complication of trauma and diabetes mellitus and can lead to infection, gangrene, and even amputation. Nasiri et al.107 conducted a double-blind randomized clinical trial study of the effect of olive oil on foot ulcers (Wagner’s ulcer grade 1 and 2 lesions) in human patients with diabetes. They reported that olive oil in combination with routine care was more effective than routine care

alone. This observation suggests that olive oil can be used directly on broken human skin, without harmful effects. In contrast to its positive role in wound healing, topically applied olive oil has a detrimental effect on the integrity of the stratum corneum and the function of the skin as a barrier.3 So, there is increasing interest in extracting constituents of olive oil and their formulation into cosmeceuticals and pharmaceuticals that do not harm the skin.

33.5.5 Delivery of constituents of olive oil to the skin Ozone is a well-known oxidant that absorbs most of the UV radiation from the Sun. Ozone has been used in health care since ancient times.108 Although ozone is inherently unstable, incorporation of ozone into vegetable oil may preserve its properties.109 Several molecules present in oils, especially olive oil (e.g., triglycerides and PUFAs), can retain ozone, extending its activity. The more PUFAs present, the greater the amount of ozone that can be retained.110 Carata and contributors111. found similar results with ozonated extra-virgin olive oil. This combined the beneficial properties of extra-virgin olive oil with those of ozone. Although combination with ozone may increase the clinical efficacy of olive oil, more modern methods of delivery of the constituents of “liquid gold” are highly desirable. In this context, microemulsions (MEs) are promising formulations for the delivery of both pharmaceutical and cosmeceuticals.112 Chaiyana et al.113 studied olive oil incorporated into MEs. The antioxidant properties were assessed by the acid, iodine, saponification index, and pseudoternary phase diagram characteristics. After analyzing two MEs (i.e., the first made from olive oil, Tween 85, propylene glycol, and water and the second from olive oil, Tween 85, ethanol, and water), the in vitro antioxidant and skin moisturizing features achieved were comparable to native olive oil. So, MEs can provide antioxidants and moisturize the skin. MEs can be easily incorporated into products for topical application and drug delivery.

33.6 Conclusion The understanding of the value of olive oil for the treatment of skin diseases is increasing exponentially. The direct application of olive oil to the skin, especially after bathing, reduces dryness and itching. Furthermore, olive oil and its constituents can be used to enhance wound healing in clinical practice. The most beneficial constituents of olive oil include polyphenols and PUFAs. These have significant antioxidant effects and decrease ROS levels, protecting the skin from oxidative stress. They also modulate the immune

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system and have an important role in white blood cell proliferation and cytokine production. Until recently, these effects had mainly been described in vitro. Regardless, epidemiological data associating the consumption of olive oil with reduced risk of cardiovascular or a neurodegenerative disease is encouraging. So, in addition to its beneficial effects on the skin, olive oil has widespread effects that protect the whole body. To confirm these beneficial effects, clinical trials are required. However, for several reasons, these studies are unlikely to be conducted. On the basis of strength of the currently available data, we therefore support and encourage the use of olive oil for nutritional and medicinal purposes.

Mini-dictionary of terms The most common extracellular protein in connective tissues matrixes. Collagen is often used in skin creams and in plastic surgery. Erythema Redness of the skin or mucous membranes. It is caused by hyperemia (increased blood flow) in superficial capillaries. It is caused by inflammation. Homeostasis The ability of living organisms to resist external changes to maintain a relatively stable internal state. Lipids Hydrocarbon compounds that do not dissolve in water but are soluble in nonpolar solvents (e.g., ethanol). Lipids include oils and fats. Pathogens An organism that may cause disease. Pathogens include bacteria and viruses. Polyunsaturated These FAs contain more than one double bond FAs in their structure. Polyunsaturated fats and oils are made mainly from vegetable oils. They are considered to be healthier than those made from saturated fats. Prostaglandins These are physiologically active lipid compounds (eicosanoids) derived from arachidonic acid. Their effects are diverse and hormone like. Pruritis Commonly known as itch. It produces a sensation of discomfort and a desire to rub or scratch to ease it. Toxins A poisonous substance that causes disease. Wrinkles Creases in the skin. A feature of aging or solar damage. Xerosis A skin condition characterized by excessively dry skin. Collagen

Comparisons of olive oils with other edible oils Various oils derived from plants have been used for centuries for antiinflammatory, antioxidant, and antibacterial effects, promoting wound healing.

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Trigonella foenum-graecum L. (Fenugreek), which belongs to the Fabaceae family, is commonly used as a spice. Fenugreek seeds seem to have beneficial effects on the digestive system.114 Fenugreek oil is also used in the treatment of diabetes.115 Sesamum indicum L. (Sesame) belongs to the Pedaliaceae family from Africa. Sesame has a higher implication on human nutrition. This oilseed has ethnobotanical and pharmaceutical medical applications. It has laxative and emollient properties.116 Seed oil from Vitis vinifera L. (grapes) which belongs to the Vitaceae family has a high unsaturated FAs content, especially LA.117 One study reported that avocado oil has beneficial effects when used in salads.118 The lipid content of avocado improved carotenoid absorption.118 Nevin and Rajamohan119 showed that coconut oil lowered serum and tissue lipid in rats. Another study found that regular topical application of sunflower oil in comparison with olive oil or no oil improved the skin of newborn babies.120 Camellia oil has an antiinflammatory effect.121 This is thought to be mediated by triterpene alcohols that reduce induced inflammation in mouse skin.121 As triterpene compounds from plant seed oils may inhibit two-stage chemical carcinogenesis in mouse skin,122 it is possible that Camellia oil may protect against photocarcinogenesis.

Implications for human health and disease prevention Olives and olive oil has been consumed by the inhabitants of the Mediterranean region for centuries. However, it has only been relatively recently that olive oil has been the subject of intense laboratory-based, epidemiological, and clinical studies. These data have validated the intuition of inhabitants of the Mediterranean basin and demonstrated many benefits of olive oil to human health and the prevention of disease. The understanding of the biological value of the constituents of olive oil has thereby been greatly increased. The main health benefits of olive oil are due to MUFAs, PUFAs, tocopherols, phospholipids, and phenolics.123 These compounds also give olive oil its unique taste and flavor. The composition of olive oil differs between cultivars, harvesting regimes, and agroclimatic features.124 Although olive oil reduces the risk of atherosclerosis and malignancy, the best recognized uses of olive oil are culinary and for the skin. Using olive oil directly on the body after bathing is particularly complementary to the skin. The fats in olive oil are predominantly responsible for these beneficial effects, especially on the skin, nervous system, and lipoproteins.

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As well as providing precursors of important hormones, olive oil consumption increases the absorption of liposoluble vitamins. Many nutritionists recommend consumption of a variety of lipids but limiting total consumption to only 30% of calorific intake; this is hard achieve without olive oil.125 The vital roles of the constituents of olive oil include antioxidant and antimicrobial functions. Some experimental data have shown that regular consumption of natural oils reduces the risk of diseases such as myocardial infarction and stroke. So it is possible that novel therapies for these life-threatening diseases may be developed from constituents of olive oil.31 More than a culinary delight, the “liquid gold” that is olive oil is being increasingly recognized for its holistic therapeutic benefits.

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Human skin, the largest organ of the human body, is a highly efficient self-repairing barrier. Wrinkles, xerosis (dry skin), and pruritis (itchness) are the main pathologies that affect skin. Olive oil can be used to treat wrinkles, xerosis, and pruritis. Some constituents of skin and olive oil have antioxidant and antiinflammatory properties. These perform important protective roles against toxins and pathogens. Although olive oil has been used since Roman times, the properties of this pleiotropic oil are still being investigated. Besides many beneficial effects of olive oil on the skin, olive oil can also improve health by preventing cardiovascular and neurodegenerative diseases.

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76. 123. Covas MI, Nyyssonen K, Poulsen HE. The effect of polyphenols in olive oil on heart disease risk factors. Ann Int Med. 2006;145:333
431. 124. Covas MI. Bioactive effects of olive oil phenolic compounds in humans: Reduction of heart disease factors and oxidative damage. Inflammopharmacology. 2008;16:216
218. 125. Violaa P, Viola M. Virgin olive oil as a fundamental nutritional component and skin protector. Clin Dermatol. 2009;27:159
165.

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Chapter 34

Extra-virgin olive oil, cognition and brain health Elisabetta Lauretti1, Luigi Iuliano2 and Domenico Pratico`1 1

Alzheimer’s Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, United States, 2Department of Medico-Surgical

Sciences and Biotechnologies., Sapienza University of Rome, Latina, Italy

Abbreviations AD ATP CNS CPLX1 DNA EVOO GLT-1 IL-1β IL-6 IL-10 LTP MED PSD-95 ROS SOD Tg TNF-α

Alzheimer’s disease adenosine triphosphate central nervous system complexin 1 deoxyribonucleic acid extra-virgin olive oil glutamate transporter 1 interleukin 1 β interleukin-6 interleukin 10 long-term potentiation Mediterranean diet postsynaptic protein 95 reactive oxygen species superoxide dismutase transgenic tumor necrosis factor α

34.1 Introduction Recent demographic trends show that aging of the global population is now a reality. Although this process is more prominent in Europe and North America where there is a decline in fertility and improvement in survival, over the past decades, in general, the life expectancy has been increasing significantly in most countries around the world.1 The downside of this trend is that as the average age of populations continues to rise, also the number of older adults with age-associated health problems will increase. Among all the age-related conditions, cognitive decline is definitely one of the most common and feared. Currently, dementia is one of the prevailing causes of disability with 47 million people diagnosed with it worldwide, bearing a huge social and economic impact.2 For

these reasons, recently, the attention of the scientific community has been shifted toward the understanding of the age-dependent anatomical and pathophysiological changes that normally occur in the central nervous system (CNS) with the goal of promoting healthy brain aging and ultimately a better quality of life. Brain aging is a process associated with significant increase in oxidative stress, inflammation, loss of gray and white matter across most brain regions, and reduced levels of synaptic proteins and synaptic spines, which often results in alteration of cognitive functions.3 Although reduced level of cognitive functions may become evident later in life, the changes leading to neuronal damage are progressive and start years before the symptoms become apparent giving reasons to believe we can delay or even prevent their onset. Observational epidemiological studies suggest a relationship between lifestyle and dietary habits with brain health and cognitive performance. Therefore a large number of research studies have been investigating the effect of specific dietary regimens on late-life memory and cognition providing interesting results.4 Given the well-documented benefits of the Mediterranean diet (MED) on cardiovascular diseases and longevity, intensive research has begun to evaluate its efficacy also in terms of brain health. Human studies have consistently indicated that strong adherence to the MED and consumption of extra-virgin olive oil (EVOO), one of its major components, is associated with enhanced memory functions and lower risk of developing mild cognitive impairment and Alzheimer’s disease (AD).5
9 The claimed beneficial properties of EVOO have been well documented also in vitro and in vivo in several aging models. The results of most of these studies show that the EVOO health benefits are related to its polyphenolic

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00018-3 © 2021 Elsevier Inc. All rights reserved.

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components such as oleuropein, hydroxytyrosol, and oleocanthal. These phenolic compounds are widely recognized antioxidant and antiinflammatory factors that have been shown to reverse oxidative damage, enhance brain energy metabolism, and ameliorate age-dependent deficits in spatial, working memory, and motor functions.5
9 This chapter summarizes the literature on this topic and reviews the possible mechanisms explaining the action of EVOO on brain health, cognition, and memory (Table 34.1). Overall the neuroprotective effect of EVOO on age-associated cognitive decline proves that nutritional approaches may be an efficient strategy to prevent or slow cognitive decline with a great impact on public health.

34.2 Cognition, memory, and brain aging A normal aging brain goes through a series of neuroanatomical and neurophysiological changes.3 By comparing the brains of healthy young and healthy elderly individuals, functional magnetic resonance imaging studies have shown loss of gray matter, across most brain regions, including cortex, cerebellum, hippocampus, amygdala, and nucleus accumbens. In addition, in the aging brain, we also see reduced levels of synaptic proteins and synaptic spines as well as reduced length of myelinated axons. An interesting study using deoxyribonucleic acid (DNA) microarray technology investigated how gene expression varies in the frontal cortex of normal individual ranging from 26 to 106 years of age. The analysis of more than 11,000 genes revealed that 4% of these genes were significantly changed with age.24 The majority of these changes were related to genes involved in synaptic plasticity, vesicular transport, mitochondrial function, DNA repair and genes modulating inflammation, and oxidative stress pathways.25 One of the first theories about aging was elaborated by Harman in 1956 and involves oxidative stress.26 According to this theory, aging is considered a progressive, inevitable process in part dependent on the accumulation of oxidative damage in nucleic acids, lipids, proteins, and/or carbohydrates. Starting from this original concept, over the years, many reports have shown that during normal brain aging but even more in neurodegenerative diseases, there is an increase in oxidized proteins and lipid contents, DNA damage, and mitochondrial dysfunction.27 Given its high levels of oxygen consumption and polyunsaturated fatty acids composition, the brain is considered an organ very susceptible to the accumulation of free radicals and reactive oxygen species (ROS) that damage organelles and irreversibly alter important cell function.27 All the research done implementing in vitro and in vivo systems consistently confirmed the detrimental effects exerted by free radicals and ROS

hyperproduction on neurons and glial cells. In particular, the production of excessive ROS has been linked to neuronal death,28 synaptic loss, and decreased performance in cognitive functions.29,30 In fact, mice overexpressing prooxidant enzymes such as the superoxide dismutase 1 (SOD-1) display impaired hippocampus-dependent spatial memory function and deficient long-term potentiation (LTP).30 Moreover, a large body of literature has described a close relationship between aging, oxidative stress, and inflammation. ROS are known to promote inflammation, which in turn is known to be increased with age.31,32 Several studies suggest that genetic or environmental factors that trigger inflammation promote an aging profile.33 Rodent models of aging display signs of chronic inflammation such as activation of microglia, astrocytes, and elevated levels of several proinflammatory and decrease of antiinflammatory cytokines in certain areas of the brain, including cortex, hippocampus, and hypothalamus, all key players in memory and learning.34
36 By middle age, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interlukin-1β (IL-1β) are upregulated in the brain of aged mice and humans, whereas the levels of the antiinflammatory IL-10 are reduced.37,38 Therefore it is possible that in the CNS an overly exaggerated activation of the immune system associated with age might negatively impact neuronal and synaptic function and neuronal survival that underlie some of the observed changes in cognitive functions. Although the mechanisms accountable for the observed change are not completely understood, the most common and the most feared readout of all these alterations is cognitive and memory decline. In pathological brain aging, attention deficits, reduction in working memory, long-term memory, episodic memory, and processing speed are frequently seen.25 As life expectancy has increased, in recent years, researchers have been devoted to the study of factors, both genetic and environmental, that can control or influence how fast these changes occur with the hope to halt or slow down this process and ultimately promote healthy brain aging.

34.3 Evidence of beneficial effects of extra-virgin olive oil on brain health and cognition in human Recently, because the average age of the population worldwide continues to raise, considerable attention has been shifted toward the influence that modifiable factors such as lifestyle and dietary habits have on brain health, cognition, and dementia. Originally the Seven Countries Study39 provided the first, solid scientific evidence that dietary habit has profound

Extra-virgin olive oil, cognition and brain health Chapter | 34

417

TABLE 34.1 In vitro and in vivo studies demonstrating the effect of extra-virgin olive oil (EVOO) and its components on Alzheimer’s disease pathological markers. Compound

Species

Activity

References

Human

Reduces decline in visual memory and verbal fluency

Berr et al.6

Human

Reduces risk of developing dementia

Lefe`vre-Arbogast et al.7

Human

Improves global cognition

Martı´nez-Lapiscina et al.9,10

MED 1 EVOO

Human

Improves memory and global cognitive scores

Valls-Pedret et al.8

EVOO

Wistar rats

Reduces step-through latency (light
dark box test) Increases glutathione reductase activity and expression

Pitozzi et al.11

Improves contextual memory Improves motor coordination Reduces lipid peroxidation Reduces expression of COX2 and GFAP

Pitozzi et al.12

SAMP8 mice

Improves working memory and memory retention Increases glutathione reductase activity Increases superoxide dismutase activity Decreases brain lipid and protein oxidation

Farr et al.13

NMRI mice

Improves spatial working memory Increases ATP levels

Reutzel et al.14

SH-SY5Y cells

Increases ATP levels

Reutzel et al.14

Oleocanthal

TgSwDI mice

Reduces astrocytes activation and IL-1β Improves hippocampal-dependent burrowing test

Qosa et al.15

Hydroxytyrosol

In vitro

Protects from Fe21- and NO-induced loss of ATP

Beauchamp et al. 16

EVOO

3 3 Tg mice

Rescues contextual, spatial, and working memory Increases SYP expression Reduces GFAP

Lauretti et al.17

5XFAD mice

Increases expression of PSD-95 and GLT-1 Reduces IL-1β and GFAP

Batarseh et al. 18

hTau mice

Rescues contextual, spatial, and working memory Improves basal synaptic activity and plasticity Improves short-term plasticity Increases expression of CPLX1

Lauretti et al.19

TgCRND8 mice

Rescues cognitive impairment in the step-down test

Grossi et al.20 Pantano et al.21

TgCRND8 mice

Facilitates hippocampal synaptic activity and longterm plasticity

Luccarini et al.22

Restores posttetanic potentiation and LTP

Pu et al.23

EVOO

C57Bl/6J mice

Oleuropein, hydroxytyrosol, and oleurosid

Oleuropein

Rats

ATP, Adenosine triphosphate; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter 1; IL-1β, interlukin-1β; LTP, long-term potentiation; MED, Mediterranean diet; SYP, synaptophysin.

effects on health in general. The study was conducted in seven countries, that is, Finland, Greece, Italy, Japan, the United States, Yugoslavia
Serbia, and the Netherlands, and enrolled 12,763 individuals. The intent of the study was to investigate the overall (i.e., coronary and cancer) death rates

as a function of geographical residence and the aim of discovering preventable risk factors.40 The lowest mortality rates found in countries of the Mediterranean area resulted associated with dietary factors, rather than ethnicity. Since then a large number of

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studies, including clinical trials and metaanalyses, confirmed the beneficial effects that adherence to the MED has on multiple health outcomes.5,41 On the other hand, there is accumulating evidence that the MED, and olive oil in particular, could confer protection against the inexorable surge of dementia. Evidence for a reduced risk of developing mild cognitive impairment and AD in subjects with higher adherence to MED comes from a number of studies.42
44 Solfrizzi et al.45 reported on an elderly population, aged 65
84 years, in southern Italy with a typical MED and a high daily consumption of EVOO (46 g/day), in which they showed a direct relationship between macronutrient intake and age-related changes in global cognitive function, selective attention, and episodic memory assessed by test batteries. In addition, in a longitudinal study conducted in southern Italy with over 8.5 years follow-up, which included 704 nondemented subjects aged 65
84 years, the intake of monounsaturated fatty acids, which is typically linked to olive oil consumption, was associated with a protective effect on agerelated cognitive decline in an 8.5-year follow-up.46 As the main component of the MED and source of polyphenols and other antioxidants, the neuroprotective properties of the EVOO with regard to the aging process have been also extensively investigated. In the report of Berr et al., 6947 individuals, age 65 and older, were selected to be included in a multicenter cohort study, involving three French cities—Bordeaux, Montpellier, and Dijon—with the goal to examine the association between EVOO consumption and the risk of cognitive decline.6 For the first time, this study showed that in a large nondemented elderly population, the intake of EVOO correlates with reduced chances of decline in visual memory and verbal fluency. Later, a cohort of 1329 participants without a diagnosis of dementia was followed up to 12 years. In this case, high intake of EVOO in combination with other polyphenols was found to lower the risk of developing dementia by 50% confirming the health effect of this plant-based oil.7 Another study evaluated cognitive function in 334 participants enrolled in the largest dietary intervention study (PREDIMED trial) and consuming a MED supplemented with EVOO over a 4-year period. Cognitive change over time was assessed by a comprehensive battery of neuropsychological test that revealed better memory, frontal function, and global cognitive scores compared to those on a control diet.8 Cognitive function was also assessed in 522 participants at high vascular risk from the same PREDIMED trial after 6.5 years of dietary intervention.9 Once more, the EVOO diet resulted in significantly improved global cognition independently from other cofactors such as ApoE, family history, physical activity, and vascular risk factors. Finally, a parallel study following other 285 participants from the same PREDIMEDNAVARRA trial further supported the previous outcomes.10

In summary, all of these reports provide evidence for the protective effect of EVOO against cognitive decline in the elderly population and justify the need for further research and clinical trials devoted to explore the extent of its therapeutic properties in a larger cohort; to analyze the influence that gender, race, comorbidity, and risk factors may have on its in vivo effect; and to identify the specific level of intake of EVOO that is necessary to obtain the protective effect on brain health and cognition in this population.

34.4 Evidence of beneficial effects of extra-virgin olive oil on cognition and neuroinflammation in aging rodents Given the large body of evidence in favor of the beneficial effects exerted by EVOO in terms of improved cognitive functions in the elderly, much research has been conducted also in vivo using aging models to determine the biological activity of EVOO and its phenolic components. One of the first works trying to understand how EVOO improves brain health and reduces cognitive decline is the study from Pitozzi et al.11 In this study, 10-month old male Wistar rats were fed with an EVOO diet rich in phenolic antioxidants for 12 months, then motor and cognitive functions were assessed at the end of the treatment. Although the authors did not observe significant improvements upon EVOO consumption in the specific tasks performed, they reported a reduced step-through latency in the light
dark box test and decreased brain glutathione reductase activity and expression. Since the light
dark preference test is one of the most common models of anxiety used in rodents and glutathione reductase activity has been also associated with anxiety, the authors speculated that EVOO might have an effect on emotional behavior. In a follow-up study from the same group, under similar experimental conditions, C57Bl/6J mice were fed from middle age to senescence with EVOO rich in polyphenols. Once again behavioral tests were performed to assess memory, motor, and emotional behavior. This time the EVOO group showed improved contextual memory and motor coordination compared to controls. The latter effect was explained by significant reduction of lipid peroxidation in the cerebellum.12 The effect of EVOO on learning and memory was also tested in a model of accelerated senescence, the SAMP8 mice. In these animals, EVOO significantly ameliorated working memory and memory retention as assessed by Tmaze and one-trial novel object recognition. Consistently with the previous study, EVOO-treated mice also displayed increased glutathione reductase and SOD activity, while decreased brain lipid and protein oxidation suggesting that EVOO by mediating an antioxidant effect might reverse oxidative damage in the aging brain.13 Finally, the effect of

Extra-virgin olive oil, cognition and brain health Chapter | 34

long-term administration of purified olive secoiridoids, including oleuropein, hydroxytyrosol, and oleurosid, was evaluated in a different aging model, the NMRI (Naval Medical Research Institute) mice.47 These mice show agedependent deficits in spatial, working memory and motor functions. In this study, beside behavioral assessment, the authors also investigated the expression of genes linked to longevity, mitochondrial biogenesis and function, synaptic plasticity, and oxidative stress. In agreement with previous findings, EVOO-fed mice showed better spatial working memory; however, in comparison to controls, no differences were found in the activity of respiratory chain complex I and IV and in the activity of the investigated antioxidant enzymes. Interestingly, in the brain of the treated animals, levels of adenosine triphosphate (ATP) were increased suggesting that the beneficial effect on memory could be mediated by an increase in ATP, the main source of energy in eukaryotic cells. This theory was later confirmed in vitro, in SH-SY5Y cells, where incubation with the secoiridoids resulted in augmented basal ATP levels.14 These observations are particularly relevant for an organ such as the brain, characterized by such a high energy demand. In fact, the high oxygen consumption combined with the high level of polyunsaturated fatty acid makes the brain more vulnerable to oxidative stress and consequent oxidative damage48 Oxidative stress can also trigger inflammation. For instance, the aging brain shows activation signs for several inflammatory factors such as ILs and glial fibrillary acidic protein (GFAP), marker of astrocytosis. In recent years, many reports have demonstrated the antiinflammatory properties of EVOO and its phenolic components in brain tissue, especially oleocanthal and hydroxytyrosol, which have been shown to reduce astrocytes activation and IL-1β production in the brain.15 Moreover, hydroxytyrosol, a free radical scavenger, has been shown to protect from Fe21- and NO-induced loss of ATP.16,49
51 Collectively, the body of evidence from the literature demonstrates the EVOO potential in counteracting many features of pathological brain aging and neurodegenerative diseases such as cognitive decline, oxidative stress, reduction of ATP levels, and brain energy metabolism. The overall evidence supports the hypothesis that EVOO intake could be seen as a medicinal food to reduce the risk of developing dementia in old age.

34.5 Evidence of beneficial effects of extra-virgin olive oil on Alzheimer’s disease
associated memory and cognitive impairment Besides the beneficial effect against cognitive decline normally associated with age, EVOO showed also great potential in preventing memory loss in AD. Currently, 44

419

million people worldwide are living with AD or a related form of dementia, a number estimated to increase in the next years due to a longer life expectancy for the general population. Given the lack of a cure and the high prevalence of this disorder among the elderly, AD represents a serious economic and social burden for our society justifying the urgent need for the development of novel preventative and or curative therapies. In recent years, accumulating evidence has described the positive effect of EVOO long-term treatment in several mouse models of the disease. EVOO and its polyphenol extracts have shown to significantly ameliorate hippocampal-dependent memory, including fear, spatial, and working memory, in transgenic animals when administrated in the early phase of the disease.15,17,19
21 Administration of an EVOO-rich diet for 6 months completely restored memory performance in the triple transgenic mouse model with plaques and tangles (i.e., 3 3 Tg mice) and in the hTau mice (a model of primary tauopathy) when tested in the Y-maze, fear conditioning, and Morris water maze tasks.17,19 Moreover, the oleuropein extract from EVOO improved cognitive performance in TgCRND8 animals to the level of wild-type control mice in the step-down avoidance test.15,20,21

34.6 Extra-virgin olive oil and synaptic proteins Although the protective role of EVOO on brain health and cognition is widely recognized, the molecular mechanism underlying this biological effect is still elusive. Few studies have attempted to investigate the effect of EVOO treatment on synaptic function and synaptic proteins. For example, one study showed that when EVOO is given to the 5XFAD mice in combination with donepezil, the expression of postsynaptic density protein 95 (PSD-95), an important marker of postsynaptic integrity, and glutamate transporter 1 (GLT1), an integral membrane protein essential for terminating the postsynaptic action of glutamate are increased suggesting better synaptic glutamate regulation.18 No differences in PSD-95 expression were instead observed in 3 3 Tg mice upon EVOO treatment. However, this AD animal model fed with EVOO-rich diet displayed a statistically significant increase in the levels of synaptophysin, a marker of presynaptic integrity, when compared with the control group confirming an effect of EVOO on synaptic function.17 Furthermore, in a more recent study, chronic administration of EVOO resulted in upregulation of complexin 1 (CPLX1) in the hippocampus of the tauopathy model, the hTau mice. CPLX1 is a SNARE complex
binding protein located on the presynaptic side, which regulates vesicle release.52 Downregulation of CPLX1 has been linked to increased risk of developing AD and Parkinson’s disease,53,54 since its loss

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of function can impair basal synaptic strength and shortterm synaptic plasticity.55 In conclusion, these results provide evidence for an additional biological property of EVOO in neurodegenerative disorders associated with hippocampal synaptic dysfunction and support EVOO as promising therapeutic for promoting synaptic health in AD.

34.7 Extra-virgin olive oil and long-term potentiation Learning and memory formation strongly relies on changes in synaptic strength and its efficiency since they allows enhancement of neuronal associations and facilitates synaptic transmission.56 Assessment of LTP response, which is often described as a long-lasting change in electrical response to a transient input, currently represents the best model to explain how long-lasting memory-associated synaptic changes are likely to occur in the brain. In fact, biochemical events that occur after induction of LTP such as gene expression, cell signaling, and de novo protein synthesis are similar to the ones occurring during memory acquisition. Moreover, inhibition of hippocampal LTP has been shown to block hippocampal learning and retention of tasks.56,57 For these reasons, besides the behavioral test paradigms, LTP analysis is widely implemented to study memory and learning function in vivo, in particular in relation to hippocampus plasticity. Therefore to evaluate whether the enhanced memory associated with EVOO consumption correlated with changes in hippocampal synaptic activity and plasticity, our group performed LTP analysis of hippocampal slices from EVOO-fed hTau mice and compared it to controls. Our study revealed that compared with controls the EVOO-treated mice had an elevation in basal synaptic activity and in two forms of short-term plasticity: pairedpulse facilitation and posttetanic potentiation. Interestingly, these data are in agreement with two other recent studies, which demonstrated that oleuropein facilitates hippocampal synaptic activity and long-term plasticity and restores posttetanic potentiation and LTP in the TgCRND8 mice.22,23 Collectively, these findings have important pathophysiological implications because for the first time they provide a cellular mechanism for the observed beneficial effect exerted by EVOO on cognition in vivo and explain the related protection from cognitive decline associated with its chronic consumption.

34.8 Conclusion Early epidemiological studies have consistently reported the beneficial effect of EVOO as the main component of the MED for healthy brain aging and cognition. These observations have been confirmed and supported by a

large body of evidence ranging from in vitro to in vivo investigations, which highlight the various medicinal actions of EVOO and in particular its polyphenolic components. The proposed mechanisms for the underlying effect of EVOO on brain health are reduction of brain oxidative stress, lower neuroinflammation, improvement in synaptic basal activity and neuronal function, and increased brain energy metabolism. Overall the findings reviewed here support the efficacy of EVOO in terms of neuroprotection and prevention of cognitive decline, which is an important cause of disability in both elderly and in AD patients. However, despite this compelling evidence in favor of EVOO’s potent antibrain aging activity the extent to which this could be applied in terms of therapeutic intervention and the molecular mechanisms whereby EVOO evokes these multiple responses need further investigations.

34.9 Mini-dictionary of terms Alzheimer’s disease: A chronic neurodegenerative disease characterized by brain deposition of amyloid β peptides and phosphorylated tau protein leading to progressive neuronal loss. Brain aging: The changes that occur in brain structures and functions as result of the increasing chronological age. Mediterranean diet: A diet implemented originally in countries around the Mediterranean Sea (i.e., Greece, Italy), which among other food emphasizes the daily consumption of fruits, vegetables, oilve oil, and whole grains, and it includes less dairy and meat than a typical Western diet. Randomized clinical trial: A type of clinical study that aims to reduce certain sources of bias when testing the effectiveness of new treatments. This goal is accomplished by randomly allocating subjects to two or more groups, treating them differently, and then comparing them with respect to a measured response. Preclinical study: A study to test a drug, a procedure, or another medical treatment mostly in animal models of human diseases. The aim of a preclinical study is to collect data in support of the efficacy and safety of the new treatment. Preclinical studies are required before clinical trials in humans can be started.

34.10 Comparisons of olive oil with other edible oils As discussed earlier, regular daily olive oil consumption is considered as the most important and integral component of MED and as having a major role in the brain health benefit of this diet. This concept has been the propeller for some health organizations in non-Mediterranean

Extra-virgin olive oil, cognition and brain health Chapter | 34

countries to promote a MED and the usage of olive oil as the main source of dietary fat. However, this policy has not always been very successful since adopting this type of oil could be more expensive in comparison with other edible oils in these populations. For this reason, in recent years, these countries have been looking for a potential alternative to the olive oil. Among them, canola oil has gained increasing attention as a suitable substitute to olive oil especially in countries that lack the primary source for it: the olive tree. As result, canola oil consumption is now quite high in many of these countries because of its lower price compared with olive oil, but also and most importantly because there is a diffuse perception that the canola oil is a healthy choice. Most of the studies so far investigating the relationship between canola oil consumption and health benefits have shown limited evidence of beneficial effects or neutral action on biomarkers of risk factors for cardiovascular diseases. On the other hand, studies have provided conflicting results depending on the experimental model implemented, the length of the treatment, and the particular end point considered. However, no much data are available on the biological effects that chronic exposure to dietary canola oil may have on cognitive function and the development of the AD—phenotype that typically includes memory impairments, dysruption of synaptic integrity, Aβ, and tau neuropathology. To address this scientific question, we chronically administered canola oil to the 3 3 Tg mice, which manifest all these aspects, including memory deficits , Aβ deposits, and tau tangles pathology. At the end of the treatment, we found that exposure to the canola oil-rich diet resulted in a significant increase in body weight and impairments in their working memory together with decreased levels of PSD-95, a marker of synaptic integrity, and an increase in the ratio of insoluble Aβ 42/40. Taken together, our findings do not support a beneficial effect of chronic canola oil consumption on two important aspects of the AD pathophysiology, which include memory impairments and synaptic integrity. While more studies are needed, our data do not justify the current trend aimed at replacing olive oil with canola oil or other edible oils.

34.11 Implications for human health and disease prevention For the last two decades, solid evidence has been accumulating in support of the health benefits of dietary consumption of MED on cardiovascular diseases and longevity. More recently, new and intensive research has also begun to evaluate its efficacy also in terms of brain health. Human studies have indicated that strong adherence to the MED and consumption of EVOO, one of its

421

major components, is associated with enhanced memory functions and lower risk of developing mild cognitive impairment and AD. The beneficial properties of EVOO have been well documented also in vitro and in vivo in several aging models. The results of most of these studies show that the EVOO health benefits are related to the antioxidant and antiinflammatory properties that have been shown to reverse oxidative damage, enhance brain energy metabolism, and ameliorate age-dependent deficits in spatial, working memory, and motor functions. Collectively, the available data provide strong clinical and preclinical support on the concept that EVOO should be considered as a beneficial agent not only for preventing pathological brain aging but also for halting the development of AD and related dementia.

References 1

World Population Ageing. Department of Economic and Social Affairs. 2017. 2 Alzheimer’s Association. 2020 Alzheimer’s disease facts and figures. Alzheimer’s Dement. 2020;16:391
460. 3 Glisky EL. Chapter 1. Changes in cognitive function in human aging. Frontiers in Neuroscience. Brain Aging: Models, Methods, and Mechanisms. CRC Press/Taylor & Francis; 2007. 4 Dominguez LJ, Barbagallo M. Nutritional prevention of cognitive decline and dementia. Acta Biomed. 2018;89(2):276
290. Available from: https://doi.org/10.23750/abm.v89i2.7401. 5 Roma´n GC, Jackson RE, Reis J, Roma´n AN, Toledo JB, Toledo E. Extra-virgin olive oil for potential prevention of Alzheimer disease. Rev Neurol (Paris). 2019;175(10):705
723. Available from: https://doi.org/10.1016/j.neurol.2019.07.017. 6 Berr C, Portet F, Carriere I, et al. Olive oil and cognition: results from the three-city study. Dement Geriatr Cogn Disord. 2009;28 (4):357
364. Available from: https://doi.org/10.1159/000253483. 7 Lefe`vre-Arbogast S, Gaudout D, Bensalem J, et al. Pattern of polyphenol intake and the long-term risk of dementia in older persons. Neurology. 2018;90(22):e1979
e1988. Available from: https://doi. org/10.1212/WNL.0000000000005607. 8 Valls-Pedret C, Sala-Vila A, Serra-Mir M, et al. Mediterranean diet and age-related cognitive decline: a randomized clinical trial. JAMA Intern Med. 2015;175(7):1094
1103. Available from: https://doi. org/10.1001/jamainternmed.2015.1668. 9 Martı´nez-Lapiscina EH, Clavero P, Toledo E, et al. Mediterranean diet improves cognition: the PREDIMED-NAVARRA randomised trial. J Neurol Neurosurg Psychiatry. 2013;84(12):1318
1325. Available from: https://doi.org/10.1136/jnnp-2012-304792. 10 Martı´nez-Lapiscina EH, Clavero P, Toledo E, et al. Virgin olive oil supplementation and long-term cognition: the PREDIMEDNAVARRA randomized, trial. J Nutr Health Aging. 2013;17 (6):544
552. Available from: https://doi.org/10.1007/s12603-0130027-6. 11 Pitozzi V, Jacomelli M, Zaid M, et al. Effects of dietary extravirgin olive oil on behaviour and brain biochemical parameters in ageing rats. Br J Nutr. 2010;103(11):1674
1683. Available from: https://doi.org/10.1017/S0007114509993655.

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12 Pitozzi V, Jacomelli M, Catelan D, et al. Long-term dietary extravirgin olive oil rich in polyphenols reverses age-related dysfunctions in motor coordination and contextual memory in mice: role of oxidative stress. Rejuvenation Res. 2012;15(6):601
612. Available from: https://doi.org/10.1089/rej.2012.1346. 13 Farr SA, Price TO, Dominguez LJ, et al. Extra virgin olive oil improves learning and memory in SAMP8 mice. J Alzheimers Dis. 2012;28(1):81
92. Available from: https://doi.org/10.3233/JAD2011-110662. 14 Reutzel M, Grewal R, Silaidos C, et al. Effects of long-term treatment with a blend of highly purified olive secoiridoids on cognition and brain ATP levels in aged NMRI mice. Oxid Med Cell Longev. 2018;2018:4070935. Available from: https://doi.org/10.1155/2018/ 4070935. 15 Qosa H, Mohamed LA, Batarseh YS, et al. Extra-virgin olive oil attenuates amyloid-β and tau pathologies in the brains of TgSwDI mice. J Nutr Biochem. 2015;26(12):1479
1490. Available from: https://doi.org/10.1016/j.jnutbio.2015.07.022. 16 Beauchamp GK, Keast RS, Morel D, et al. Phytochemistry: ibuprofen-like activity in extra-virgin olive oil.. Nature. 2005;437 (7055):45
46. doi:10.1038/437045a. 17 Lauretti E, Iuliano L, Pratico` D. Extra-virgin olive oil ameliorates cognition and neuropathology of the 3xTg mice: role of autophagy. Ann Clin Transl Neurol. 2017;4(8):564
574. Available from: https://doi.org/10.1002/acn3.431. 18 Batarseh YS, Kaddoumi A. Oleocanthal-rich extra-virgin olive oil enhances donepezil effect by reducing amyloid-β load and related toxicity in a mouse model of Alzheimer’s disease. J Nutr Biochem. 2018;55:113
123. Available from: https://doi.org/10.1016/j. jnutbio.2017.12.006. 19 Lauretti E, Nenov M, Dincer O, Iuliano L, Pratico` D. Extra virgin olive oil improves synaptic activity, short-term plasticity, memory, and neuropathology in a tauopathy model. Aging Cell. 2019;19(1): e13076. Available from: https://doi.org/10.1111/acel.13076. 20 Grossi C, Rigacci S, Ambrosini S, et al. The polyphenol oleuropein aglycone protects TgCRND8 mice against Aß plaque pathology. PLoS One. 2013;8(8):e71702. Available from: https://doi.org/ 10.1371/journal.pone.0071702. 21 Pantano D, Luccarini I, Nardiello P, Servili M, Stefani M, Casamenti F. Oleuropein aglycone and polyphenols from olive mill waste water ameliorate cognitive deficits and neuropathology. Br J Clin Pharmacol. 2017;83:54
62. Available from: https://doi.org/ 10.1111/bcp.12993. 22 Luccarini I, Grossi C, Rigacci S, et al. Oleuropein aglycone protects against pyroglutamylated-3 amyloid-ß toxicity: biochemical, epigenetic and functional correlates. Neurobiol Aging. 2015;36 (2):648
663. 23 Pu FF, Yin S, Chen HY, et al. Oleuropein improves long term potentiation at perforant path-dentate gyrus synapses in vivo. Chin Herb Med. 2005;7(3):255
260. Available from: https://doi.org/ 10.1016/S1674-6384(15)60047-8. 24 Lu T, Pan Y, Kao SY, et al. Gene regulation and DNA damage in the ageing human brain. Nature. 2004;429(6994):883
891. 25 Fjell AM, Walhovd KB. Structural brain changes in aging: courses, causes and cognitive consequences. Rev Neurosci. 2010;21:187
221. 26 Herman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11(3):298
300.

27 Mariani E, Polidori MC, Cherubini A, Mecocci P. Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview. J Chromatogr B: Anal Technol Biomed Life Sci. 2005;827(1):65
75. Available from: https://doi.org/10.1016/j.jchromb.2005.04.023. 28 Valencia A1, Mora´n J. Reactive oxygen species induce different cell death mechanisms in cultured neurons. Free Radic Biol Med. 2004;36(9):1112
1125. 29 Mast JD, Tomalty KM, Vogel H, Clandinin TR. Reactive oxygen species act remotely to cause synapse loss in a Drosophila model of developmental mitochondrial encephalopathy. Development. 2008;135(15):2669
2679. Available from: https://doi.org/10.1242/ dev.020644. 30 Massaad CA, Klann E. Reactive oxygen species in the regulation of synaptic plasticity and memory. Antioxid Redox Signal. 2011;14 (10):2013
2054. Available from: https://doi.org/10.1089/ars.2010.3208. 31 Lynch MA. Age-related neuroinflammatory changes negatively impact on neuronal function. Front Aging Neurosci. 2010;1:6. Available from: https://doi.org/10.3389/neuro.24.006.2009. 32 Gabuzda D, Yankner BA. Inflammation links ageing to the brain. Nature.. 2013;497(7448):197
198. 33 Denver P, McClean PL. Distinguishing normal brain aging from the development of Alzheimer’s disease: inflammation, insulin signaling and cognition. Neural Regen Res. 2018;13(10):1719
1730. Available from: https://doi.org/10.4103/1673-5374.238608. 34 Ye SM, Johnson RW. Increased interleukin-6 expression by microglia from brain of aged mice. J Neuroimmunol. 1999;93 (1
2):139
148. 35 Maher CG, Sherrington C, Elkins M, Herbert RD, Moseley AM. Challenges for evidence-based physical therapy: accessing and interpreting high-quality evidence on therapy. Phys Ther. 2004;84 (7):644
654. 36 Godbout JP, Chen J, Abraham J, et al. Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. FASEB J. 2005;19(10):1329
1331. 37 Sparkman NL, Johnson RW. Neuroinflammation associated with aging sensitizes the brain to the effects of infection or stress. Neuroimmunomodulation. 2008;15(4
6):323
330. Available from: https://doi.org/10.1159/000156474.e. 38 Johnson SM, An RW. age-related decline in interleukin-10 may contribute to the increased expression of interleukin-6 in brain of aged mice. Neuroimmunomodulation. 2001;9(4):183
192. 39 Keys A. Coronary heart disease in seven countries. Summary. Circulation. 1970;41(4 suppl):I186
I195. 40 Keys A. Seven countries: a multivariate analysis of death and coronary heart disease. Cambridge: Harvard University Press; 1980. 41 Dinu M, Pagliai G, Casini A, Sofi F. Mediterranean diet and multiple health outcomes: an umbrella review of meta-analyses of observational studies and randomised trials. Eur J Clin Nutr. 2018;72 (1):30
43. 42 Psaltopoulou T, Sergentanis TN, Panagiotakos DB, Sergentanis IN, Kosti R, Scarmeas N. Mediterranean diet, stroke, cognitive impairment, and depression: a meta-analysis. Ann Neurol. 2013;74 (4):580
591. Available from: https://doi.org/10.1002/ana.23944. 43 Singh B, Parsaik AK, Mielke MM, et al. Association of Mediterranean diet with mild cognitive impairment and Alzheimer’s disease: a systematic review and meta-analysis. J Alzheimers Dis. 2014;39(2):271
282. Available from: https://doi. org/10.3233/JAD-130830.

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44 Lourida I, Soni M, Thompson-Coon J, et al. Mediterranean diet, cognitive function, and dementia: a systematic review. Epidemiology. 2013;24(4):479
489. Available from: https://doi. org/10.1097/EDE.0b013e3182944410. 45 Solfrizzi V, Panza F, Torres F, et al. High monounsaturated fatty acids intake protects against age-related cognitive decline. Neurology. 1999;52(8):1563
1569. Available from: https://doi.org/ 10.1212/WNL.52.8.1563. 46 Solfrizzi V1, D’Introno A, et al. Unsaturated fatty acids intake and all-causes mortality: a 8.5-year follow-up of the Italian Longitudinal Study on Aging. Exp Gerontol. 2005;40(4):335
343. Available from: https://doi.org/10.1016/j.exger.2005.01.003. 47 Gower AJ1, Lamberty Y. The aged mouse as a model of cognitive decline with special emphasis on studies in NMRI mice. Behav Brain Res. 1993;57(2):163
173. 48 Angeloni C, Malaguti M, Barbalace MC, Hrelia S. Bioactivity of olive oil phenols in neuroprotection. Int J Mol Sci. 2017;18 (11):2230. Available from: https://doi.org/10.3390/ijms18112230. 49 Visioli F, Poli A, Gall C. Antioxidant and other biological activities of phenols from olives and olive oil. Med Res Rev. 2002;22:65
75. Available from: https://doi.org/10.1002/med.1028. 50 Petroni A, Blasevich M, Salami M, Papini N, Montedoro GF, Galli C. Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thromb Res. 1995;78:151
160. Available from: https://doi.org/10.1016/0049-3848(95)00043-7. 51 Gonza´lez-Correa JA, Navas MD, Mun˜oz-Marı´n J, et al. Effects of hydroxytyrosol and hydroxytyrosol acetate administration to rats on

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7876. Available from: https://doi.org/ 10.1021/jf801502z. Lai Y, Diao J, Cipriano DJ, et al. Complexin inhibits spontaneous release and synchronizes Ca2 1 -triggered synaptic vesicle fusion by distinct mechanisms. eLife. 2014;3:e03756. Available from: https://doi.org/10.7554/eLife.03756. ¨ mu¨r O ¨ , et al. Biomarkers in prodromal PARK4 Lahut S, Gispert S, O and rapid eye movement sleep behavior disorder show role of complexin 1 loss for risk of Parkinson’s disease. Model Mech. 2017;10 (5):619
631. Available from: https://doi.org/10.1242/dmm.028035. Ramos-Miguel A, Sawada K, Jones AA, et al. Presynaptic proteins complexin-I and complexin-II differentially influence cognitive function in early and late stages of Alzheimer’s disease. Acta Neuropathol. 2017;133(3):395
407. Available from: https://doi. org/10.1007/s00401-016-1647-9. Chang S, Reim K, Pedersen M, Neher E, Brose N, Taschenberger H. Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse. J Neurosci. 2015;35(21):8272
8290. Available from: https://doi.org/ 10.1523/JNEUROSCI.4841-14.2015. Lynch MA. Long-term potentiation and memory. Physiol Rev. 2004;84(1):87
136. Morris RG, Anderson E, Lynch GS, Baudry M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature. 1986;319:774
776.

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Chapter 35

The foundation for the use of olive oil in skin care and botanical cosmeceuticals Edmund M. Weisberg1 and Leslie S. Baumann2 1

The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, United States, 2Baumann

Research and Cosmetic Institute, Miami, FL, United States

Abbreviations 8-OHdG AA AD FDA MED MMP TPA UV

8-hydroxy-deoxyguanosine arachidonic acid atopic dermatitis Food and Drug Administration minimal erythema dose matrix metalloproteinase 12-O-tetradecanoylphorbol acetate ultraviolet

cutaneous damages (see Table 35.2).1,7 In addition, olive oil shows promise as a potential photoprotective agent. This chapter focuses on the chemical constituents of olives and olive oil believed to confer salutary effects as

TABLE 35.1 Key facts about botanical cosmeceuticals. G

G

35.1 Introduction Olive oil, derived from the fruits of the olive tree (Olea europaea), is the primary source of fat in the Mediterranean diet, known to be one of the world’s healthiest, and has long been considered one of the most important of the natural essential oils. For millennia, as long as it has been part of the human diet, people have also used olives and olive oil for various nonculinary purposes, including for its beneficial effects on the skin (see Table 35.1). The ancient Egyptians and Romans used olive oil in food, cosmetics, massage oil for athletes, anointing oil, and salve for soothing wounds, and the ancient Greeks bathed with olive oil.1 In addition, the leaves and fruits of the olive plant have been used as external emollients to treat skin ulcers and inflammatory wounds.2 In the contemporary world, olive oil is popular worldwide, gaining widespread use throughout Europe, Asia, North America, and Latin America.3
6 Also in recent years, the topical application of olive oil has been reported to be an effective option in treating xerosis; rosacea; psoriasis; atopic dermatitis (AD); contact dermatitis (particularly diaper dermatitis); eczema (including severe hand and foot eczema); seborrhea; pruritus; and various inflammations, burns, and other

G

G

G

Uses of many herbs in modern products date back centuries or millennia to ancient or traditional cultures Technological advances have enabled us to more carefully study the biochemistry of botanical extracts and thus verify traditional uses and identify novel therapeutic applications or potential Natural products pose fewer risks than chemical-based products in terms of toxicity and adverse effects Careful screening for a substance’s inflammatory potential is necessary before including a natural ingredient in skin care formulations Before using a natural ingredient as a therapeutic tool, laboratory benefits must also be translated to human benefits through extensive clinical testing

TABLE 35.2 Cutaneous indications for olive oil. G G G G G G G G G G

Atopic dermatitis Burns Contact dermatitis (especially diaper dermatitis) Eczema (particularly severe cases in hands and feet) Pruritus Psoriasis Rosacea Seborrhea Various inflammations Xerosis

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00044-4 © 2021 Elsevier Inc. All rights reserved.

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well as the research supporting the consumption and topical application of olive oil to promote cutaneous health.

35.2 Chemistry 35.2.1 Constituents Olive oil contains several active ingredients, including polyphenols, squalene, fatty acids (particularly the monounsaturated fat oleic acid), triglycerides, tocopherols, carotenoids, sterols, and chlorophylls (see Table 35.3).1 The phenols in virgin olive oil have been found to scavenge reactive oxygen and nitrogen species implicated in human disease,1 but it has not yet been established whether these olive oil compounds impart an effect beyond those on extracellular sources of nitric oxide.8 Squalene, which acts as an antioxidant in olive oil, is present in unusually high concentrations in comparison to other fats and oils typically found in the human diet.9
11 The major constituents of the unsaponifiable fraction of virgin olive oil include erythrodiol, beta-sitosterol, and squalene; the key components of the polar fraction include the polyphenols oleuropein, tyrosol, hydroxytyrosol, and caffeic acid, of which antioxidant properties have been clearly identified (Table 35.3).12 Beta-sitosterol and tyrosol have been found to modulate reactive oxygen species and nitric oxide synthesis, arachidonic acid (AA) release, and the production of AA metabolites.13 Antioxidants act to neutralize free radicals, also known as reactive oxygen species. Free radicals develop when oxygen molecules combine with other molecules, leaving an odd number of electrons. An oxygen molecule with paired electrons is stable, but oxygen with an unpaired electron is considered “reactive,” because it searches for and absorbs electrons from surrounding vital components rendering them damaged.14

TABLE 35.3 Active ingredients of olive oil. Primary active ingredients

Unsaponifiable fraction

Polar fraction

Carotenoids

Beta-sitosterol

The polyphenols caffeic acid, hydroxytyrosol, oleuropein, and tyrosol

Chlorophylls

Erythrodiol

Fatty acids (especially oleic acid)

Squalene

35.2.2 Properties A study of the topical application of virgin olive oil on edema in mice induced by AA or 12-O-tetradecanoylphorbol acetate (TPA) was designed to assess the antiinflammatory effects of the unsaponifiable and polar fractions. Assays with both classes of compounds revealed antiinflammatory effects exhibited by both groups, with beta-sitosterol and erythrodiol demonstrating a potent antiedematous effect in the TPA model and oleuropein, tyrosol, hydroxytyrosol, and caffeic acid showing a significant inhibitory effect. The unsaponifiable fraction more strongly inhibited AA, and oleuropein was a potent inhibitor among the polar components. The investigators concluded that the antiinflammatory activity ascribed to both groups of compounds may be influential in the healthy effects attributed to virgin olive oil.12 Other studies have also shown that the polyphenolic constituents of olive oil exhibit protective activity against inflammation,1,15 which plays a significant contributory role in the majority of dermatologic disorders. Importantly, while olive oil gains traction as an antiinflammatory agent, it is also generally considered as very weakly irritant, with adverse side effects to its topical use rarely reported, though it is deemed unsuitable or contraindicated in patients with venous insufficiency and related eczema on the lower extremities.16 The main phenolic compounds contained in olive oil, all of which display significant antioxidant activity, include simple phenols (hydroxytyrosol and tyrosol), secoiridoids (oleuropein, the aglycone of ligstroside, and their respective decarboxylated dialdehyde derivatives), and the lignans [(1)-]-acetoxypinoresinol and pinoresinol (see Table 35.4).17 Lignans, specifically, have been found to be potent antioxidants that inhibit cellular growth in skin, breast, colon, and lung cancers as well as inhibit in vivo lipid peroxidation.18 Notably, the high consumption of extra-virgin olive oil, which is rife with antioxidant characteristics derived from these three polyphenol groups as well as squalene and oleic acid, is thought to confer protection against oxidative stress and some of its manifestations, such as aging, as well as skin and other

TABLE 35.4 Polyphenolic constituents in olive oil. Simple phenols

Secoiridoids

Lignans

Squalene

Hydroxytyrosol

Oleuropein

Acetoxypinoresinol

Sterols

Tyrosol

Ligstroside

Pinoresinol

Polyphenols

Tocopherols

10-Hydroxyoleuropein

Triglycerides

10-Hydroxyligstroside

The foundation for the use of olive oil in skin care and botanical cosmeceuticals Chapter | 35

cancers.17 Consistent and heavy consumption of olive oil, along with vegetables and legumes, has also been found to impart protection against actinic damage.19 While oleic acid is clearly an important constituent of olive oil, the fact that it is contained in widely eaten animal foods such as poultry and pork suggests that it is not the main component responsible for the favorable impact ascribed to olive oil.20

35.2.3 Oleocanthal Recently, Segura Palacios et al. conducted a quasiexperimental pilot study to assess the topical efficacy of an oily fluid enriched with oleocanthal extract as compared to a conventional oily fluid to mitigate the inflammatory reaction following photodynamic therapy. Oleocanthal (decarboxy methyl ligstroside aglycone), a phenolic substance found in extra-virgin olive oil, in an oily emollient was applied three times daily for 1 week to 23 patients diagnosed with actinic keratosis/field cancerization in the forehead and/or scalp, who were treated with photodynamic therapy. The first cohort of consecutive patients, 24 total, received a conventional emollient oily fluid. The investigators observed that 48 h after treatment, patients who were treated with the oleocanthal fluid experienced significantly greater amelioration in inflammation. In addition, 60.9% of the oleocanthal group achieved a complete response 3 months after photodynamic therapy compared to 29.2% of the conventional oil group, with this difference approaching statistical significance.21

35.2.4 Oleuropein Oleuropein, the most abundant phenolic compound found in olive leaves and oil, has been shown to display antioxidant and free radical-scavenging activities.22 Also present in the stems and flowers of the olive plant, oleuropein, an ester of elenolic acid and 3,4-dihydroxyphenyl ethanol and the primary glycoside in olives,23 is believed to be the main constituent accounting for its antioxidant and antimelanogenesis properties.24 Notably, olive leaves, which contain a copious supply of oleuropein, are associated with imparting significantly more antioxidant activities than olive fruit.25 Hydroxytyrosol, an ortho-diphenolic compound and essential constituent of oleuropein, has been demonstrated in vitro to prevent apoptotic cell death caused by ultraviolet (UV) B in HaCaT cells.26,27 Both oleuropein and hydroxytyrosol deliver dynamic anticancer activity at the initiation, promotion, and metastasis stages and provide protection against several cancers, including skin tumors.23 The antioxidant activity of both compounds, measured as more robust than that of vitamin E, is attributed to their phenolic content.28,29 Notably in the skin

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care realm, oleuropein and lipophilic olive mill wastewater derivatives have been identified as active ingredients for stabilizing cosmetic products.30

35.2.5 Protection against ultraviolet damage Just over a decade ago, Kimura and Sumiyoshi conducted a study with hairless mice revealing that olive leaf extract and its primary constituent oleuropein protected the skin against chronic UVB-induced damage and carcinogenesis as well as tumor growth likely by lowering cutaneous cyclooxygenase-2 levels thus hindering the expression of vascular endothelial growth factor, and various matrix metalloproteinases (MMPs), specifically MMP-2, MMP9, and MMP-13.31 In 2010 they examined the potential protective effects of olive leaf extract and oleuropein on acute damage engendered by UVB exposure in C57BL/6J mice. Both oral extract (300 or 1000 mg/kg) and oral oleuropein (25 or 85 mg/kg) inhibited skin thickness increases caused by daily doses of UVB (120 mJ/cm2 for 5 days, then every other day for 9 days). Olive leaf extract and oleuropein also prevented increases in Ki-67- and 8-hydroxy-20 -deoxyguanosine-positive cell numbers, melanin granule area, and MMP-13 expression.32 Skin tumor formation has also been prevented through the preinitiation with oleuropein in a two-stage carcinogenesis model in mice, likely due to the antioxidant and antiapoptotic properties of the olive protein, according to the researchers.33 Using an emulsion and emulgel containing oleuropein and vitamin E as a reference compound, Perugini et al. found that the botanical ingredient accounted for diminished erythema (22%), transepidermal water loss (35%), and blood flow (30%). The investigators concluded that the use of oleuropein in cosmetic formulations merits additional study for its potential to contribute to limiting UV damage.22

35.2.6 Wound healing In a 2011 assessment of the wound healing activity of O. europaea leaf extracts using in vivo wound models and the reference ointment Madecassol (Bayer, Istanbul, Turkey) for comparison, Koca et al. found that the aqueous extract did indeed display wound healing properties, with oleuropein (4.6059%) identified as the main active constituent.2 Three years later, Mehraein et al. divided 24 aged male Balb/c mice into control and experimental groups in a skin wound healing investigation. Collagen fiber deposition was significantly increased and reepithelialization more advanced in the oleuropein group on days 3 and 7 after incision, and the researchers noted reduced cell infiltration. They concluded that oleuropein accelerates

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cutaneous wound healing in mice and presents potential for clinical applications in the surgery setting for human wound healing.34 In a separate study, Mehraein et al. also found that the phenolic compound again speeded reepithelialization, enhancing collagen fiber production and improving blood flow to wound areas through upregulating vascular endothelial growth factor protein expression.35

35.2.7 Hair growth Tong et al. have reported that topically applied oleuropein promoted the anagen hair growth phase in telogenic C57BL/6N mouse skin.36 In addition, an O. europaea subcutaneous immunotherapy has resulted in diminished cutaneous reactivity while emerging as safe and tolerable to patients with rhinoconjunctivitis.37

35.3 Dietary protection The notion that one’s diet can impact the health and appearance of the skin might seem intuitive, but it was somewhat undermined by a couple of studies around 1970 related to acne and various potential dietary triggers. While cogently arguing for abandoning the traditional belief expressed by the dermatology community, promulgated by these studies, that diet does not contribute to acne etiology, Cordain contended that the dogma claiming that diet and acne are unrelated was based on two fundamentally flawed studies from 1969 by Fulton et al. and 1971 by Anderson. He argued that these studies lacked control groups, statistical data treatment, as well as blinding and/or placebos and were characterized by inadequate sample sizes and insufficient or absent baseline diet data, among other deficiencies.38 Such studies likely contributed to diverting the dermatology community from a deeper appreciation of the role that nutrition can play in skin health, other than offering the healthy suggestions of eating more fruits and vegetables and specifically trying to identify food allergens with cutaneous manifestations or food triggers of skin conditions (particularly rosacea). A plethora of more recent research and, likely, current investigations underway, on the direct effects on health from the consumption or supplementation of various nutrients points us in a different direction, however. Much of this work focuses on the potential benefits imparted to the skin as a result of the intake of certain foods or supplements. For example, in 2003, investigators conducted a cross-sectional study of 302 healthy men and women, collecting data on serum concentrations of nutrients, dietary consumption of nutrients, and various cutaneous measurements (including hydration, sebum content, and surface pH). The study demonstrated statistically significant relationships between serum vitamin A and cutaneous sebum

content and surface pH as well as between skin hydration and dietary consumption of total fat, saturated fat, and monounsaturated fat. The authors concluded that such findings suggest that changes in an individual’s baseline nutritional status can affect the condition of the skin.39 This is but one example to buttress the argument that oral delivery of nutrients must be considered as well as systemic administration and topical application of medication and cosmetics when evaluating and trying to promote health skin.

35.3.1 Monounsaturated fats For optimum health the chief dietary fat should be derived from foods and oils rich in monounsaturated fat. When monounsaturated fats predominate, saturated fats, transfatty acids, and omega-6 polyunsaturated fatty acids are counterbalanced, and the ratio of omega-6 to omega-3 fatty acids improves as the proportion of omega-3 acids increases. This is notable particularly in the West, where the ratio of omega-6 to omega-3 fatty acid consumption has grown from 10:1 during the mid-1990s,40 to approximately 15:1
16.7:1 now,41 far from what is thought to be the healthy ratio closer to 4:1.40 Both of these omega groups are essential for healthy human growth and development. A higher risk for depression and various inflammatory conditions has been associated with a high ratio of omega-6 to omega-3 fatty acids.42 Olive oil and olives, along with nuts (except for walnuts and butternuts), peanuts (a legume), avocados, canola oil, high-oleic sunflower oil, and high-oleic safflower oil, all contain significant levels of monounsaturated fats and are considered influential in ameliorating dry skin (see Table 35.5). An interesting study of a potential link between dietary intake and skin wrinkling in sun-exposed areas was conducted by Purba et al. in 2001. They used food frequency questionnaires and cutaneous microtopographic measurements to evaluate diet and skin wrinkling in 177 Greek-born individuals living in Melbourne, Australia, 69 Greek subjects residing in rural Greece, 48 Anglo-Celtic

TABLE 35.5 Dietary ingredients high in omega-3 monounsaturated fats, which confer positive effects on the skin. G G G G G G G

Avocados Canola oil Nuts (except walnuts and butternuts) Olive oil and olives Peanuts Safflower oil Sunflower oil

The foundation for the use of olive oil in skin care and botanical cosmeceuticals Chapter | 35

Australian elderly individuals living in Melbourne, Australia, and 159 Swedish elderly participants living in Sweden. The Swedish elderly were found to manifest the least wrinkling in sun-exposed areas, followed by the Greek-born in Melbourne, rural Greek elderly, and then Anglo-Celtic Australians. Correlation and regression analyses yielded data that prompted the investigators to conclude that diet may very well have an impact on skin wrinkling. Generally, they found that individuals who consumed more vegetables (especially green leafy vegetables, spinach specifically, as well as asparagus, celery, eggplant, garlic, and onions/leeks), olive oil, monounsaturated fat, and legumes as well as lower levels of milk and milk products, butter, margarine, and sugar products exhibited fewer wrinkles in sun-exposed skin. High consumption of olive oil, legumes, vegetables, fish, and cereal was especially protective against photodamage. Significantly, the authors suggested that diets high in monounsaturated acids may increase the levels of monounsaturated fatty acids in the epidermis, which resist oxidative damage, as opposed to epidermal polyunsaturated fatty acids, which are more susceptible to oxidation. Further, they speculated that such a phenomenon may explain their findings of a correlation between monounsaturated olive oil and less wrinkling as well as the higher level of wrinkling associated with the consumption of polyunsaturated margarine.19 A previous study found that olive oil appeared to be important in potentiating the antioxidant activity of plasma lycopene after the consumption of tomato products along with olive oil.43

35.3.2 An olive oil
rich diet In 2001 Moreno et al. performed a study to ascertain the effect of a diet rich in olive oil on important inflammation mediators, namely, oxidative stress and prostaglandin synthesis. In the process, they compared the effects on rats of an olive oil
rich diet to those of corn oil
rich and fish oil
rich diets. Both olive oil and fish oil were found to decrease AA release and the ensuing synthesis of AA metabolites, but olive oil was more efficient in reducing oxidative stress. Prostaglandin E2 levels were measured to be lower in the rats fed the olive oil or fish oil diets as compared to the corn oil diet.44

35.4 Photoprotection 35.4.1 Ultraviolet B Twenty years ago, investigators assessed the capacity of extra-virgin olive oil to combat reactive oxygen species and skin tumors induced by UV light exposure. Topical application of the oil to hairless mice before or after repeated exposure to UVB radiation led to delays in the

429

onset of skin tumors as compared to exposures in control mice. Differences between control mice and the mice pretreated with olive oil declined with additional exposures to UVB. Interestingly, mice treated with olive oil following UVB exposure exhibited significantly fewer tumors than mice in the control group. Researchers concluded that topical application of olive oil after UVB exposure is effective in retarding the onset and suppressing the number of murine skin tumors caused by UVB exposure.11 Based on previous evidence showing that topically applied vitamin E and epigallocatechin gallate extracted from green tea had delayed the onset of UV-induced skin cancer in mice, as well as the reportedly potent in vitro antioxidant activity of olive oil, several of the same investigators sought to examine the effects of topically applied olive oil, particularly in delaying the onset and decreasing the number of UV-induced skin cancers in mice. Results comparable to the Budiyanto et al. study were obtained insofar as extra-virgin olive oil topically applied immediately following UVB radiation exposure did, in fact, significantly lower the number and delay the onset of skin cancer lesions as well as reduce the formation in murine epidermis of the free radical-induced 8-hydroxydeoxyguanosine (8-OHdG). However, no effects on skin cancer formation were associated with extra-virgin olive oil topically applied prior to UV exposure or the pre- or postexposure application of regular olive oil. The authors speculated that the mechanism of action of extra-virgin olive oil in this context may work by reducing 8-OHdG formation, which is known to be involved in gene mutation. They concluded that the daily topical use of extravirgin olive oil following sun exposure may have the same impact on human skin, potentially slowing the development of UV-induced skin cancer.45 An in vivo investigation in 32 volunteers to ascertain the effects of various topical agents, including olive oil, on the transmission of UVB radiation phototherapy has also provided information regarding the usefulness of olive oil for the skin. The minimal erythema dose (MED) necessary to cause skin reddening for each volunteer was identified through a phototest and then was repeated using each test formulation. The effects on MED of each product were assessed after 24 h. Of the agents tested, olive oil and glycerin had no impact on MED and were thus deemed suitable for use prior to phototherapy.46

35.4.2 Ultraviolet A Hydroxytyrosol, one of the key polyphenolic components of the polar fraction of olive oil, has been studied for its effects on UVA-induced cell damage in the human melanoma cell line M14. Investigators identified a dosedependent protective effect exerted by the phenol, which prevented an increase in the usual markers of oxidative

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stress. The arrest of melanoma cell proliferation was observed at higher hydroxytyrosol concentrations. The olive oil constituent also displayed a dose-dependent apoptotic effect on melanoma cells. In addition, it was metabolized by catechol-O-methyltransferase into methylated derivatives, which bear more responsibility than the original molecule in manifesting in vivo antioxidant activity in cosmetics and functional foods containing olive oil. Further, such derivatives have the potential to interfere with the activity of certain methyltransferases involved in the catabolism of various pharmacological compounds. The investigators concluded that hydroxytyrosol prevents protein damage engendered by UVA in melanoma cells but does not completely counteract the effects of irradiation, and that the dose-dependent differential impact on melanoma cells of hydroxytyrosol should be considered in relation to the selection of cosmetic and food products containing olive oil and potential interactions.47 Previously, hydroxytyrosol was studied for its effects on the proliferation, apoptosis, and cell cycle of human leukemia (HL60) and colon adenocarcinoma cells (HT29 and HT29 clone 19A). The olive oil antioxidant displayed a capacity to inhibit cell proliferation in both cancer types, more successfully in HL60. It was also found to induce apoptosis in HL60 cells after 24 h of incubation and arrest the HL60 cells in the G0/G1 phase of the cell cycle. Investigators concluded that hydroxytyrosol appears to exhibit protective properties against cancer and may be a major factor in the reputed anticancer activity of virgin olive oil.48 Hydroxytyrosol, present abundantly in olive leaves and oil, makes its way to the human diet primarily through extra-virgin olive oil. In this form, this phenol has been found to display antiinflammatory, antitumor, antiviral, antibacterial, and antifungal activity as well as antioxidant properties and, as the most actively investigated natural phenolic compound, is believed to have substantial potential as a pharmacological agent.49

35.5 Topical applications for dermatologic conditions In contemporary times, olive oil is not generally known as a first-line treatment for cutaneous disorders, but it has come to be considered an effective therapeutic option for several conditions. A brief review follows of the literature on such applications.

35.5.1 Olive oil and dry skin In 2005 investigators compared the cutaneous effects and stability against oxidation of olive oil and hemp-seed oil. Both of these unsaturated fatty acids are thought to ameliorate xerosis and related manifestations of aging. The

researchers observed that olive oil was more stable than hemp-seed oil against peroxidation, and that the chlorophyll found in extra-virgin olive oil exhibited greater photostability than that included in hemp-seed oil, which they speculated might be the result of a higher concentration of antioxidants in olive oil. After preparing emulsions with the two oils, they concluded that some of the gelemulsion preparations were appropriate for spraying on the skin.50 It is worth noting that the unusual stability of extra-virgin olive oil, as well as its pungent taste, has been attributed to the polyphenolic antioxidant constituents hydroxytyrosol and oleuropein.20 In addition, these olive oil components have been demonstrated to display more potent antioxidant properties than vitamin E and the food preservative butylhydroxytoluene.20 In a study comparing the biophysical properties on the skin of olive oil and sunflower seed oil, Danby et al. enlisted 19 adult volunteers with and without an AD history. The results of the two randomized forearmcontrolled mechanistic trials revealed that topically applying olive oil for 4 weeks yielded significant declines in stratum corneum integrity and engendered mild erythema regardless of AD history. The authors concluded that the use of olive oil should be discouraged for treating dry skin and in infant massage.51 More research seems warranted to determine the relative safety and effectiveness of olive oil in this context.

35.5.2 Olive oil and dermatitis In a randomized controlled trial to test the cutaneous effects of two different topical ointments on the skin of premature infants conducted from October 2004 to November 2006, investigators prospectively enrolled 173 infants between 25 and 36 weeks of gestation admitted into a neonatal intensive care unit and randomly scheduled them for daily therapy with a water-in-oil emollient cream, an olive oil cream (70% lanolin, 30% olive oil), or a control ointment. After a maximum of 4 weeks of treatment, statistically less dermatitis was observed in the neonates treated with olive oil cream as compared to the emollient cream, with beneficial effects enduring throughout the trial and both test groups achieving better outcomes than the control group.6 However, some studies point to the inferiority of olive oil as compared to other moisturizing agents. A doubleblind controlled trial with 26 adult subjects conducted by Verallo-Rowell et al. in 2008 compared virgin coconut oil and virgin olive oil for moisturizing dryness and removing Staphylococcus aureus from colonized atopic skin. After 4 weeks of randomized twice-daily treatments, only one subject taking virgin coconut oil remained positive, whereas half of those taking virgin olive oil remained positive.52

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Nevertheless, a British study in 2011 indicated that approximately 52% of newborn nursery units recommend the use of topical olive oil in the skin care of newborns.3,53 In response to these and other contradictory findings, the prevailing contention is that topical olive oil can inhibit skin barrier function and exacerbate AD symptoms.3 Vaughn and colleagues acknowledge that several current studies are flawed insofar as they do not identify how the olive oil was processed or delineate what other chemicals might be active in topical olive oil preparations.3

35.5.3 Olive oil and wound healing In 2016 Donato-Trancoso et al. studied the antiinflammatory and antioxidant impact of olive oil administration on wound healing of pressure ulcers through daily treatment of male Swiss mice. They observed improvement in cutaneous wound healing through the increased production of reactive oxygen species and nitric oxide, leading to diminished oxidative damage and inflammation and enhanced dermal reconstruction and wound closure.54,55 An earlier study by Edraki et al. using 60 rats tested sea buckthorn, olive oil, a sea buckthorn/olive oil mixture, silver sulfadiazine, and a saline control on full-thickness burn wounds.55,56 They noted that the use of sea buckthorn, olive oil, and the mixture of the two led to more rapid wound contraction as compared to the silver sulfadiazine and saline control. The rats treated with the sea buckthorn/olive oil combination experienced more effective exudate control than the other groups, and more advanced reepithelialization. The silver sulfadiazine group, by contrast, manifested immature granulation, ulceration, and necrotic tissue. The investigators concluded that olive oil and sea buckthorn, alone and in combination, appear to be effective ingredients in dressings for full-thickness burns.56

35.5.4 Antifungal properties of olive oil At least one study has suggested an antimicrobial function for olive fruit and olive oil. Specifically, researchers studied the antifungal activity of the aliphatic aldehydes in olives—hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, and (E)-2-nonenal—against six strains of Trichophyton mentagrophytes, one strain of Microsporum canis, and seven strains of Candida spp. The tested aldehydes inhibited the growth of T. mentagrophytes and M. canis but had no impact on any of the Candida spp. strains. In a concentration-dependent fashion, elastase activity was inhibited by (E)-2-octenal and (E)-2-nonenal, which is significant insofar as elastase is an enzyme that breaks down elastin, one of the integral building blocks of the dermis. Investigators suggested that their findings support the use of olives and olive oil in the topical treatment of cutaneous diseases, particularly fungal skin infections.57

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35.6 Olive oil in combination 35.6.1 Atopic dermatitis and psoriasis Olive oil has shown promise in combination with other ingredients for the treatment of dermatoses. A traditional Iranian ointment combining honey, olive oil, and sesame, called Olea, that has recently been used to heal burns was investigated by Zahmatkesh et al. in a randomized controlled trial, in which 30 hospitalized patients were treated (10 with Olea and 20 with Acetate Mafenide 8.5%). Debridement surgery was subsequently indicated for 13 patients in the Acetate Mafenide group, but none in the Olea group. The researchers reported that only one patient (10%) in the Olea group had positive cultures after 7 days, whereas 19 (95%) had positive cultures in the Acetate Mafenide group. They concluded that the ointment combining olive oil, honey, and sesame is an effective burn treatment as it speeds tissue repair, prevents infections, and promotes debridement.58 In earlier work, Al-Waili conducted a partially controlled, single-blind study to evaluate the effects of a honey, olive oil, and beeswax mixture (1:1:1) on 21 patients with AD and 18 patients with psoriasis. Eleven of 21 AD patients were instructed to use topical betamethasone esters and 10 of 18 psoriasis patients used clobetasol propionate. Eight of the 10 AD patients in the honey/olive oil/beeswax treatment group displayed significant improvement in the evaluated symptoms (i.e., erythema, scaling, lichenification, excoriation, indurations, oozing, and pruritus) after 2 weeks. Over the same time period, five of the eight psoriasis patients in the honey/olive oil/ beeswax group also demonstrated significant improvement in the assessed symptoms (i.e., redness, scaling, thickening, and pruritus). In addition, the symptoms in five of the 11 AD patients treated with betamethasone and five of the 10 psoriasis patients treated with clobetasol propionate remained stable after their treatments were replaced with the honey/olive oil/beeswax mixture combined with corticosteroid representing a 75% reduction in their original dose. Al-Waili concluded that the honey/ olive oil/beeswax mixture appeared to be effective in the management of these skin disorders.59

35.6.2 Fungal and bacterial infections The following year, Al-Waili tested this same mixture in 37 patients as a treatment for the cutaneous fungal infections pityriasis versicolor, tinea cruris, tinea corporis, and tinea faciei. The honey/olive oil/beeswax mixture was applied to the various lesions three times daily for up to 4 weeks. The author observed a clinical response in terms of erythema, scaling, and pruritus in 86% of pityriasis versicolor patients, 78% of tinea cruris patients, and 75%

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of tinea corporis patients, with mycological cure achieved in a significant percentage of patients (75%, 71%, and 62% of patients with pityriasis versicolor, tinea cruris, and tinea corporis, respectively). In addition, after 3 weeks of therapy, the lone patient with tinea faciei exhibited clinical as well as mycological resolution. Al-Waili suggested that these findings indicate that the honey/olive oil/beeswax mixture appears effective for treating these cutaneous fungi and that future-controlled trials evaluating this compound are warranted.60 In 2005 Al-Waili assessed the effects of the same honey/olive oil/beeswax mixture on the growth of S. aureus and Candida albicans isolated from humans and found that both the honey mixtures as well as honey alone were effective in suppressing bacterial growth, whereas mild-tomoderate growth was observed on media containing olive oil or beeswax.61 That same year, Al-Waili tested the honey/olive oil/beeswax mixture on 12 infants experiencing diaper dermatitis. The infants were treated four times daily for 7 days, and erythema was assessed on a five-point scale. A significant reduction in the mean lesion score was observed from baseline (2.91 6 0.79) to day 7 (0.66 6 0.98). In addition, C. albicans was isolated in four patients prior to the treatment regimen, but only two patients after the 1 week of treatment. Overall, the honey/olive oil/beeswax compound was deemed to be a safe as well as clinically and mycologically effective therapy for diaper dermatitis.62

35.6.3 Anal fissure and hemorrhoids The next year, Al-Waili et al. conducted a prospective pilot study to assess the therapeutic effects of topical application of the mixture on patients with anal fissure or hemorrhoids. Fifteen consecutive patients who presented with anal fissure or first- to third-degree hemorrhoids were treated with a 12-h application of the honey/olive oil/beeswax ointment. The symptoms were scored at baseline and on a weekly basis up to 4 weeks. The natural mixture allayed itching and significantly decreased bleeding in hemorrhoid patients and similar improvements were noted in the anal fissure patients, with no side effects reported in any patients. The authors concluded that the honey/olive oil/beeswax compound is clinically effective and safe for the treatment of hemorrhoids and anal fissure and warrants additional testing in randomized double-blind trials.63

35.7 Cosmeceuticals The designation “cosmeceutical” has still not been codified or officially recognized by regulatory bodies such as the US Food and Drug Administration (FDA), although it has been debated for decades. In other words, it has no legal meaning. Importantly, though, the term is increasingly part

of the public lexiconyet it might not be clearly understood. The word “cosmeceuticals” refers to products that are known to have a biologic action but are regulated as cosmetics (e.g., products containing retinol) rather than as drugs. This means that manufacturers, by not listing certain ingredients as active or the main ingredients, can more readily market products. In particular, this allows for the introduction of products touted for their inclusion of specific ingredients that are not listed as active or as the main ingredients. While the FDA does not regulate such products, consumers purchase cosmeceuticals with the hope that some tangible differences will be achieved through their use. There are several such products on the market that include olive oil, such as bar and liquid soaps, bath oils, soaks for nails, lip balms, massage oils, shampoos, moisturizers, as well as after-sun products, many of which feature olive oil as the main active ingredient. These products likely vary widely in quality and effectiveness.

35.8 Conclusion The Mediterranean diet, thought to be one of the healthiest around the world, has garnered significant attention in recent years as a result of a lower incidence of coronary heart disease as well as breast and colon cancers in the peoples living in that region. Olive oil, as the primary source of healthy fat in this diet, is considered one of the chief components of the Mediterranean diet, along with plant foods and fish. The wider benefits of olive oil, like many other botanically derived products, were appreciated in the ancient world and are now actively in the process of being rediscovered and more deeply elucidated. In terms of contemporary dermatologic medicine, olive oil appears to be an effective therapeutic option for several conditions, shows promise for future inclusion in photoprotective products, and has been incorporated into a wide array of over-the-counter topical products. The fact that several constituents in olive oil are known to exhibit significant antioxidant activity along with the results of recent studies yielding evidence of antiinflammatory and anticarcinogenic effects conferred by olive oil provides reasons for future research and optimism regarding the expansion of medical and dermatologic applications. If olive oil is found to be even remotely as well regarded an antioxidant, antiinflammatory, and photoprotective agent in topical formulations as it is a healthy food and cooking oil, it might be bumped to the head of the growing list of potent herbal ingredients used and studied in dermatology.

35.9 Summary points G

Olive oil has been used for dermatologic purposes for thousands of years, since the times of the ancient Egyptians, Greeks, and Romans.

The foundation for the use of olive oil in skin care and botanical cosmeceuticals Chapter | 35

G

G

G

G

G

Recent epidemiologic evidence suggests an association between olive oil consumption and a lower incidence of cardiovascular disease and certain cancers. The antiinflammatory and antioxidant activity exhibited by olive oil is also thought to have implications for dermatologic applications. Use as a photoprotective agent is an important potential dermatologic application of olive oil. Olive oil is currently used in topical applications for the treatment of several skin conditions, including dry skin, itch, and inflammation as well as disorders such as rosacea. Olive oil appears to be better suited to addressing wound healing and, perhaps, photoaging and skin cancer than other cutaneous conditions in which the integrity of the stratum corneum may be further compromised by its application.

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301. 22. Perugini P, Vettor M, Rona C, et al. Efficacy of oleuropein against UVB irradiation: preliminary evaluation. Int J Cosmet Sci. 2008;30 (2):113
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1791. 24. Kishikawa A, Ashour A, Zhu Q, Yasuda M, Ishikawa H, Shimizu K. Multiple biological effects of olive oil by-products such as leaves, stems, flowers, olive milled waste, fruit pulp, and seeds of the olive plant on skin. Phytother Res. 2015;29(6):877
886. 25. Zheng J, Wei JT, Liu JF, Lieu YW. Research progress on biological activities of Olea europaea leaf extract. Zhongguo Zhong Yao Za Zhi. 2016;41(4):613
618. 26. Salucci S, Burattini S, Battistelli M, et al. Tyrosol prevents apoptosis in irradiated keratinocytes. J Dermatol Sci. 2015;80(1):61
68. 27. Jeon S, Choi M. Anti-inflammatory and anti-aging effects of hydroxytyrosol on human dermal fibroblasts (HDFs). Biomed Dermatol. 2018;2:21. 28. Visioli F, Bellomo G, Galli C. Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun. 1998;247 (1):60
64. 29. Poliˇsak N, Kreft S, Koˇcevar Glavaˇc N. Vegetable butters and oils in skin wound healing: scientific evidence for new opportunities in dermatology. Phytother Res. 2020;34(2):254
269. 30. Aissa I, Kharrat N, Aloui F, Sellami M, Bouaziz M, Gargouri Y. Valorization of antioxidants extracted from olive mill wastewater. Biotechnol Appl Biochem. 2017;64(4):579
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31. Kimura Y, Sumiyoshi M. Olive leaf extract and its main component oleuropein prevent chronic ultraviolet B radiation-induced skin damage and carcinogenesis in hairless mice. J Nutr. 2009;139(11):2079
2086. 32. Sumiyoshi M, Kimura Y. Effects of olive leaf extract and its main component oleuropein on acute ultraviolet B irradiation-induced skin changes in C57BL/6J mice. Phytother Res. 2010;24(7):995
1003. 33. John DNS, Mamat THT, Surien O, Taib IS, Masre SF. Preinitiation effect of oleuropein towards apoptotic and oxidative stress levels on the early development of two-stage skin carcinogenesis. JKIMSU. 2019;8(1):43
51. 34. Mehraein F, Sarbishegi M, Aslani A. Evaluation of effect of oleuropein on skin wound healing in aged male BALB/c mice. Cell J. 2014;16(1):25
30. 35. Mehraein F, Sarbishegi M, Aslani A. Therapeutic effects of oleuropein on wounded skin in young male BALB/c mice. Wounds. 2014;26(3):83
88. 36. Tong T, Kim N, Park T. Topical application of oleuropein induces anagen hair growth in telogen mouse skin. PLoS One. 2015;10(6): e0129578. 37. Saenza De San Pedro B, Mur MP, Valverde L, et al. Clinical assessment of tolerability, immunological and cutaneous reactivity effects of an abbreviated schedule with Olea europaea native extract of subcutaneous immunotherapy. Eur Ann Allergy Clin Immunol. 2019;10.23822. [Epub ahead of print]. 38. Cordain L. Implications for the role of diet in acne. Semin Cutan Med Surg. 2005;24:84
91. 39. Boelsma E, van de Vijver LP, Goldbohm RA, Klo¨pping-Ketelaars IA, Hendriks HF, Roza L. Human skin condition and its associations with nutrient concentrations in serum and diet. Am J Clin Nutr. 2003;77:348
355. 40. Sugano M. Characteristics of fats in Japanese diets and current recommendations. Lipids. 1996;31:S283
S286. 41. Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother. 2006;60:502
507. 42. Kiecolt-Glaser JK, Belury MA, Porter K, Beversdorf DQ, Glaser R. Depressive symptoms, omega-6:omega-3 fatty acids, and inflammation in older adults. Psychosom Med. 2007;69:217
224. 43. Lee A, Thurnham DI, Chopra M. Consumption of tomato products with olive oil but not sunflower oil increases the antioxidant activity of plasma. Free Radic Biol Med. 2000;29:1051
1055. 44. Moreno JJ, Carbonell T, Sa´nchez T, Miret S, Mitjavila MT. Olive oil decreases both oxidative stress and the production of arachidonic acid metabolites by the prostaglandin G/H synthase pathway in rat macrophages. J Nutr. 2001;131:2145
2149. 45. Ichihashi M, Ahmed NU, Budiyanto A, et al. Preventive effect of an antioxidant on ultraviolet-induced skin cancer in mice. J Dermatol Sci. 2000;23:S45
S50. 46. Fetil E, Akarsu S, Ilknur T, Kusku E, Gu¨nes AT. Effects of some emollients on the transmission of ultraviolet. Photodermatol Photoimmunol Photomed. 2006;22:137
140. 47. D’Angelo S, Ingrosso D, Migliardi V, et al. Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells. Free Radic Biol Med. 2005;38:908
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48. Fabiani R, De Bartolomeo A, Rosignoli P, Servili M, Montedoro GF, Morozzi G. Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis. Eur J Cancer Prev. 2002;11:351
358. 49. Bertelli M, Kiani AK, Paolacci S, et al. Hydroxytyrosol: a natural compound with promising pharmacological activities. J Biotechnol. 2020;309:29
33. 50. Sapino S, Carlotti ME, Peira E, Gallarate M. Hemp-seed and olive oils: their stability against oxidation and use in O/W emulsions. J Cosmet Sci. 2005;56:227
251. 51. Danby SG, AlEnezi T, Sultan A, et al. Effect of olive and sunflower seed oil on the adult skin barrier: implications for neonatal skin care. Pediatr Dermatol. 2013;30(1):42
50. 52. Verallo-Rowell VM, Dillague KM, Syah-Tjundawan BS. Novel antibacterial and emollient effects of coconut and virgin olive oils in adult atopic dermatitis. Dermatitis. 2008;19(6):308
315. 53. Cooke A, Cork M, Danby S. A national survey of UK maternity and neonatal units regarding the use of oil for baby skincare. Br J Midwifery. 2011;19:354
362. 54. Donato-Trancoso A, Monte-Alto-Costa A, Romana-Souza B. Olive oil-induced reduction of oxidative damage and inflammation promotes wound healing of pressure ulcers in mice. J Dermatol Sci. 2016;83(1):60
69. 55. Lin T-K, Zhong L, Santiago JL. Anti-inflammatory and skin barrier repair effects of topical application of some plant oils. Int J Mol Sci. 2017;19(1):70. 56. Edraki M, Akbarzadeh A, Hosseinzadeh M, Tanideh N, Salehi A, Koohi-Hosseinabadi O. Healing effects of sea buckthorn, olive oil, and their mixture on full-thickness burn wounds. Adv Skin Wound Care. 2014;27(7):317
323. 57. Battinelli L, Daniele C, Cristiani M, Bisignano G, Saija A, Mazzanti G. In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit. Phytomedicine. 2006;13:558
563. 58. Zahmatkesh M, Manesh MJ, Babashahabi R. Effect of Olea ointment and Acetate Mafenide on burn wounds
a randomized clinical trial. Iran J Nurs Midwifery Res. 2015;20(5):599
603. 59. Al-Waili NS. Topical application of natural honey, beeswax and olive oil mixture for atopic dermatitis or psoriasis: partially controlled, single-blinded study. Complement Ther Med. 2003;11: 226
234. 60. Al-Waili NS. An alternative treatment for pityriasis versicolor, tinea cruris, tinea corporis and tinea faciei with topical application of honey, olive oil and beeswax mixture: an open pilot study. Complement Ther Med. 2004;12:45
47. 61. Al-Waili NS. Mixture of honey, beeswax and olive oil inhibits growth of Staphylococcus aureus and Candida albicans. Arch Med Res. 2005;36:10
13. 62. Al-Waili NS. Clinical and mycological benefits of topical application of honey, olive oil and beeswax in diaper dermatitis. Clin Microbiol Infect. 2005;11:160
163. 63. Al-Waili NS, Saloom KS, Al-Waili TN, Al-Waili AN. The safety and efficacy of a mixture of honey, olive oil, and beeswax for the management of hemorrhoids and anal fissure: a pilot study. Sci World J. 2006;6:1998
2005.

Chapter 36

Olive oil and male fertility Germa´n Domı´nguez-Vı´as1,2, Ana Bele´n Segarra1, Manuel Ramı´rez-Sa´nchez1 and Isabel Prieto1 1

Unit of Physiology, Department of Health Sciences, University of Jae´n, Jae´n, Spain, 2Department of Physiology, Faculty of Health Sciences, Ceuta,

University of Granada, Granada, Spain

Abbreviations ACE ACEi AngII AngIII AngIV AT1 cAMP CD26 CD36 DPP4 GGT HFDs KatA LH MUFA NOx OS RAS ROS

angiotensin converting enzyme angiotensin converting enzyme inhibitor angiotensin II angiotensin III angiotensin IV angiotensin II receptor type 1 cyclic adenosine monophosphate cluster of differentiation 26 cluster of differentiation 36 dipeptidyl peptidase-4 γ-glutamyl transferase high-fat diets catalase luteinizing hormone monounsaturated fatty acids nitrogen oxide oxidative stress renin
angiotensin system reactive oxygen species

36.1 Diet and male fertility Infertility has demonstrated to be an important health problem that affect more than 10% of couples all over the world,1 and approximately 50% of cases are related to male infertility.2 Besides other lifestyle factors such as physical activities, chemicals contaminant, or genetic determinants, diet plays an important role in the development of these alterations offering an opportunity for prevention and therapeutic approach. Several dietary components have been linked to a decrease in male fertility; however, these studies usually are controversial. At present, the most convincing results relate to the high intake of saturated fatty acids with morbid obesity as detrimental for male fertility.1 The majority of nutritional factors that have suggested to improve male fertility are included in the Mediterranean

dietary pattern, characterized by the use of virgin olive oil as the main fat source. A diet rich vegetables and fruits provide a high amount of vitamins and others antioxidant nutrients with beneficial effects on spermatogenesis and reproductive function. However, several studies relate food frequency questionnaires and fertility parameters showing contradictory results. In a case-control study, subfertile men consumed less raw vegetables and fruits,3 but in other study4 that analyzed healthy volunteers, authors did not stablish significant relation between fruit and vegetable consumption and fertility. More recently, Chiu et al.5 also did not found correlation when related total fruit and vegetables in the diet with semen parameters, but when the concentration of pesticide was considered, the total sperm amount decreased by 49%. In fat, pesticides contribute to impair spermatogenesis, diminishing sperm amount and motility.6 In addition, the presence of metabolites of pesticides in urine has been demonstrated to be high in subjects consuming raw vegetables at least five times in a week.7,8 However, when fruit and vegetables without pesticides were considered, the intake of 2
3 units/day increased the production of sperm by more than 150%.9 These results indicate that fruit and vegetables (free of pesticides) may improve men’s fertility. It has been suggested that these beneficial effects could be associated to the high intake of polyphenols and others antioxidants. Similar contradictory results have been found when the effects of coffee and caffeine consumption were analyzed. In vitro studies with human Sertoli cells demonstrated different responses depending on caffeine dosage. Low or moderate caffeine concentration improves cells survival, but with higher concentration of caffeine, the antioxidant capacity of cells drops, and the levels of oxidative stress (OS) increase.10 The same nonlineal response to caffeine has been found in previous clinical studies.11,12 Other recent studies also document a reduction in fertility when men consume high doses of caffeine.13,14

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00057-2 © 2021 Elsevier Inc. All rights reserved.

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Another food group that has been widely studied about its relationship with reproductive potential is milk and dairy products. In fact, milk is an important source for endocrine active molecules15 that could affect the reproductive health. For example, it has been estimated that approximately 70% of estrogens in diet are derived from milk,16 and these exogenous hormones have been suggested as a cause of altered spermatogenesis.15 Nevertheless, important differences have been described between whole and low fat milk. Although full fat dairy products stablished an inverse relationship with sperm morphology and motility.17 low fat dairy products correlated positively with high sperm concentration and motility.18 Thus the deleterious effects of whole dairy on male fertility seem to be more related to high saturated fat concentration than estrogens presence. Finally, the influence of meat and fish intake has also been examined. The consumption of different types of meat affects the outcomes of assisted reproductive technology.19 Poultry meat is correlated to higher fertilization rates, and this rate decreased progressively with the intake of processed meats,18
20 probably according to the increased saturated fat intake. Fish consumption improves impressively sperm count and motility, probably linked to a change in the balance between the different polyunsaturated fatty acids (ratio ω-3/ω-6).18,20,21 However, similar to fruit and vegetal, the beneficial effects of fish intake would be countered by the intake of chemical contaminants, as methyl-mercury.22 Therefore, to date, the information we have about the diet
male fertility link indicates that the main effects are related to the quality of dietary fatty acids and the balance between antioxidants/OS levels.

36.2 Dietary lipid and male fertility The formation of sperm in mammalian testicles is a complex process that includes the production of gametes and posterior maturation in a multistep process. During the final steps, sperm cells lose most of their cytosolic organelles, decrease the adaptive response to stress, and increase the sensitivity to the environment.23 In this sense the so-called Western diet has demonstrated to exert a negative impact on male fertility, sperm count, and quality, mainly related with changes in plasma lipid (dyslipidemia) and systemic OS. The sperm maturation depends on the composition of the epididymal fluid that is selectively filtered from the blood. Therefore plasma composition and the integrity of epididymal epithelium are both important factors that determine male fertility.24 Several studies have demonstrated the negative impact of lipid metabolism on male fertility, but the molecular mechanism involved remains unknown. The effect of dietary and plasma cholesterol has been one of the most parameters studied until now in animal models. Rabbits are an excellent model because their lipid

metabolism is similar to the human being. When rabbits are feed with diets rich in cholesterol (more than 2%), total plasma cholesterol and very low-density lipoprotein particles increase significantly. Previous studies had demonstrated that a diet enriched in cholesterol (0.05%) decreases the ability of sperm to undergo acrosomal reaction and changes their membrane composition.25,26 Although plasma cholesterol was higher when rabbits feed the cholesterol-enriched diet, no differences were found in seminal plasma or sperm cholesterol amount. Surprisingly, all these effects were restored when the animals were fed with a diet supplemented with 7% of olive oil.27
29 This beneficial effect of olive oil in hypercholesterolemic rabbits could be determined by the specific characteristic of oleic acid and the high amount of antioxidants in this oil. The studies carried out with rodents demonstrated that the epididymis is very sensible to circulating factors, with effects similar to described in hypercholesterolemic rabbits.30
32 Nevertheless, the possible relation between dietary cholesterol and fertility is not clear in human, although data from pathological conditions such as obesity and metabolic syndrome indicate a link with infertility.33,34 Different studies tried to analyses the effects of cholesterollowering treatment, but the results were contradictory.35,36 Other dietary important factor is the quality of fat intake. Spermatozoa present a high concentration of polyunsaturated fatty acids, with a pivotal role in fertility, and the dietary fatty acids affect spermatozoa composition and sperm quantity and quality. The high concentration of saturated fatty acids (palmitic) or the low concentration of polyunsaturated fatty acids (docosahexaenoic acid) is the main factor that compromises male fertility.37 The fatty acids are accumulated in testis cells through two processes: passive diffusion in the plasmatic membrane or active transport mediated by CD36, a membrane protein expressed in Sertoli cells.38 The high docosahexaenoic acid concentration in spermatozoa is supported by Sertoli cells.39 On the other hand, the metabolic transformation of fatty acids and the desaturation and elongation activity in the testis is high,40 and it is regulated by the pattern of fatty acids composition in the sperm39,40 and the activity of different hormones as luteinizing hormone (LH) and adrenocorticotropic hormone.41 Several studies have demonstrated the positive effect of diet supplementation with different sources of vegetal polyunsaturated acids on spermatozoa development and function, for example, with walnuts.42 Fish oil supplementation in the diet significantly affects sperms’ fatty acid profile, but the improvement in sperm quantity and quality in infertile men is not clear and seems to need the synergic effects of vitamins. Several mechanisms have been suggested for the effects of dietary polyunsaturated acids on spermatogenesis which

Olive oil and male fertility Chapter | 36

are similar to the ones exerting in cardiovascular cells such as hormone activity, changes in membrane fluidity, and in the activity of membrane proteins or alterations in nuclear receptors and membrane ion channels.43 Although monounsaturated fatty acids (MUFAs) seem to be less important in sperm functionality, oleic acid is the major MUFA that influences spermatozoa, and the ratio C18:1/C18:0 (oleic/palmitic) has demonstrated to be a good index of membrane fluidity.44 In membranes of spermatozoa, palmitic acid is transformed into oleic acid by desaturase in order to maintain fluidity,44 and this parameter could be used as a predictor of sperm quality.

36.3 Male fertility and oxidative stress The OS is an imbalance between the levels of reactive oxygen species (ROS) and antioxidants, being mainly caused by genetic, epigenetic, lifestyle-related factors (overconsumption of alcohol, smoking, radiation, obesity, infection, inflammation, and also varicocele) which are the main causes involved in the pathophysiology of male infertility.45,46 ROS are an assemblage of molecules including oxygen-centered radicals, which are provided with one or more unpaired electrons and nonradical oxygen derivatives such as superoxide anion (GO22), proxyl (GROO), hydroxyl radicals (GOH), nitrogen oxide (NOx), and hydrogen peroxide (H2O2).47,48 All of them are normal derivatives in various metabolic and physiologic processes at low levels of ROS, being necessary for sperm capacitation, hyperactivation, sperm maturation, chemotaxis, binding to zona pellucida, acrosomal reaction, and sperm
ovule fertilization.49
51 Their excessive production results in the phenomenon of OS as a consequence of antioxidants neutralization in the seminal plasma.52,53 The main causes of infertility in men are due to direct damage of ROS at the nucleus and mitochondria DNA of the sperm, with high levels of DNA fragmentation and modification.54
57 Other types of DNA damage that can occur are telomere shortening, epigenetic alterations, and Y chromosomal microdeletions.55 In addition, DNA fragmentation has shown to be a powerful indicator of fertility, even more than conventional semen parameters.57 Along with DNA damage the influence of ROS on other factors, such as lipid peroxidation of sperm58
60, must also be taken into account, although its content of polyunsaturated fatty acids decreases during maturation.61 The axonemal injury seems to be also important, with a marked deterioration in adenosine triphosphate production and repair mechanisms.62 The defensive antioxidant systems are abundant in plasma or natural sperm cells63 Mainly, they include the γ-glutamyl transferase (GGT), superoxide dismutase, catalase (KatA), glutathione-S-transferase, and glutathione peroxidase enzyme families, as well as combination of

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nonenzymatic antioxidants that include vitamins (B12, C, E), astaxanthin, coenzyme Q10, glutathione, pyruvate, taurine, hypotaurine, L-carnitine, urate, albumin, beta-carotenes, ubiquinol, melatonin, cytochrome C, zinc, among other ions, and herbal medicine extracts.64
73 Many studies received positive results about the improvement of the poor characteristics of sperm function and several fertilization results and pregnancy rates after antioxidant supplementation.74
77 The administration of antioxidant supplements in vitro and in patients with male infertility tends to improve sperm functions, such as DNA integrity and sperm motility. Notably, the treatment with different antioxidants allows to restore redox homeostasis; however, the benefit of these therapies is still unclear because it is possible that an excess of oral antioxidants interferes with sperm fertilization, induces other pathologies derived from reducing stress,78
80 and the benefit is also questioned because of the limited size of the samples.81,82 More recently, new data propose a revision of the influence of OS within the male reproductive tract. The theory that the ROS produced by the sperm cells themselves are a leading cause of sperm dysfunction is questioned. It is because the probes used to measure ROS are inaccurate, demonstrating that neither the superoxide anion nor other free radicals that cause the production of the formation of the lipid peroxidation product, 4-hydroxynonenal, are related to the loss of sperm motility during incubation in vitro.83,84 The ROS alter the hormonal profile in male,85 through the damage to Leydig cells and others endocrine structures, such as the hypothalamic
pituitary
adrenal axis. The excessive production of ROS increases the releasing of cortisol, with a decrease of LH, follicle-stimulating hormone, and testosterone. Lower level of testosterone also decreases the ability of sperm to defend against oxidative damage.86 The effect of ROS on the hypothalamic-pituitary-testis (HPT) axis also affects other hormonal system, for example, the secretion of insulin from the pancreas, the release of leptin from the adipocytes, and the production of gonadotropin-releasing hormone from the hypothalamus.85

36.4 Mediterranean diet, olive oil, and male fertility Although lipids have been the main nutrients related to male fertility, other components of the diet have also been studied. In animal models, a restriction in food intake decreased the levels of testosterone and LH in serum, the weight of epididyme, and the degeneration of spermatocytes.87 The same effect over testosterone has been demonstrated in humans.88 On the other hand, the obesity associated to an increase in energy intake has also been related to deleterious effects on male fertility.89 Zinc deficiency causes decreased testosterone and increased apoptosis in the testis of rats.90 In men the

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combination of folate and zinc supplementation increases sperm concentration.91 High doses of copper decrease sperm concentrations in rats92 and in humans, seminal copper levels correlate negatively with sperm concentrations.93 Other micronutrients, such as iron and manganese, also have been demonstrated to play an important role in testicular function.94,95 Therefore the quality of the diet may have a more marked effect on spermatogenesis than total caloric intake. The dietary pattern, characterized by high intake of fruit and vegetables, fish, legumes, and whole grains, has a beneficial effect on sperm motility, but it does not seem to be related with concentration and morphology.96 While the consumption of a Western dietary pattern was unrelated to conventional semen quality parameters, different studies have demonstrated an association between the adherence to a healthy diet, the semen quality, and the fertility rates in humans.97 The Mediterranean diet is a dietary pattern with wellestablished health benefits, related mainly with cardiovascular function, mainly due to the presence of antiinflammatory and antioxidative compounds.98 However, the reproductive benefits of this dietary pattern are less clear. High adherence to the Mediterranean diet is positively associated with total sperm count,99 lower DNA fragmentation, higher sperm motility,100 higher sperm concentration, and higher levels of testosterone,101 and this association is not dependent of other factors. The beneficial effects of Mediterranean diet on male fertility could be mediated through several mechanisms. This dietary pattern is rich in nutrients with antiinflammatory properties that may affect male glands.102 On the other hand, compared with other cells, sperm and testicular cells have a high concentration of long-chain polyunsaturated fatty acids, then the high intake of ω-3 fatty acids from fish could explain the positive effects of this diet. Furthermore, a high proportion of fat-soluble vitamins, that play a crucial role in fertilization, characterize seafood. Finally, Mediterranean diet provides a lot of antioxidant compounds.103 Until now, however, not many studies have been conducted in order to analyze the possible role on male fertility as one of the most characteristic components of Mediterranean diet: the olive oil. High-fat diets (HFDs) have demonstrated marked detrimental effects on male fertility. One of the most useful animal models for the study about dietary fat and semen quality are rabbits. In previous studies, it was demonstrated that hypercholesterolemic white New Zealand rabbits (0.05% cholesterol) significantly increased its serum and sperm membrane cholesterol, and this change altered membrane-coupled sperm specific functions, such as osmotic resistance, acrosomal reaction, and sperm capacitation.104 Surprisingly, the addition of olive oil (7% v/w) to the diet of these animals blocked the harmful effects on sperm quality and

functionality. However, the supplementation of the diet only with olive oil did not cause any significant changes in fertility parameters. These results probably are related to the decrease in serum cholesterol and to alterations in the sperm membrane lipids that interfere with the intracellular cascades.27 Olive oil contains until 80% MUFA and less than 14% saturated fatty acids. MUFAs increase the membrane fluidity and are less easily damaged by lipid peroxidation. More recently, Kacel and Iguer-Ouada105 have demonstrated that compared to the standard diet of male roosters, provided with 0.4 mL of olive oil, showed a significantly improved sperm concentration, sperm viability, and sperm motility. Finally, the high content of antioxidant compounds in virgin olive oil has demonstrated a positive effect on sperm viability. Hydroxytyrosol, the most important antioxidant present in virgin olive oil composition, has demonstrated to improve significantly sperm viability and to decrease sperm DNA oxidation and ROS levels during human sperm in vitro incubation.106

36.5 The local renin
angiotensin system in the testis, dietary olive oil, and male fertility The renin
angiotensin system (RAS) has been widely implicated in the homeostatic control of water balance and electrolytes, as in the control of blood pressure. Alterations in systemic and local RAS have been related to the development of hypertension and others metabolic diseases, as obesity and type 2 diabetes.107 Similar to other tissues such as brain, kidney, white adipose tissue, and liver, a local RAS has been described in testis, with relevant functions in male fertility.108
110 This local RAS inhibits the production and release of testosterone during steroidogenesis.111 Angiotensin II (AngII), the main peptide into the system, inhibits the Leydig cell function and the activity of adenylate cyclase in the Leydig cell membranes, and reduces the level of chorionic gonadotropinstimulated cyclic adenosine monophosphate (cAMP).112 AngII also take part in the paracrine regulation of seminiferous tubules. Through the AT1 receptor, induce contraction and increase of intracellular calcium in peritubular myoid cells. Several aminopeptidase activities implicated in the metabolism of angiotensin peptides (glutamyl, alanyl, aspartyl, and cystinyl aminopeptidase activities) have been described in testis.108 This local RAS is influenced by dietary fatty acids. Several studies have demonstrated an inhibitory effect of HFDs on the expression of several components of RAS in testis, included renin, angiotensin converting enzyme (ACE), and the AT1 receptor. Interestingly, these effects could be reversed by the inhibitors of ACE (ACEi) and

Olive oil and male fertility Chapter | 36

AT1 blockers.110 Antihypertensive drugs (captopril and propranolol) selectively modify angiotensinase activities in the testis of genetic hypertensive rats (SHR) at the same time that decrease systolic blood pressure and testosterone levels in plasma.109 The main enzymatic activity implicated in the metabolism of AngII in the testis (angiotensinase A, glutamyl aminopeptidase activity) is altered during the mice development by dietary fatty acids and cholesterol. Angiotensinase A selectively hydrolyzes the N-terminal Asp residue of AngII and release angiotensin III (AngIII), a peptide with lower vasoconstrictor effect.107 Compared with saturated fat sources (lard or coconut), unsaturated fat sources (sunflower, fish, and olive oil) showed higher membranebound activity and, thus, an increased metabolism of AngII.113 On the other hand, dietary fatty acids also affect the levels of testosterone and Angiotensinase A activity in the plasma of male rats, and these changes are related to the blood pressure values.114 AngIII is hydrolyzed by angiotensinase M (alanyl aminopeptidase) to angiotensin IV (AngIV). This peptide (AngIV) binds to the receptor AT4, also, namely, insulin-regulated aminopeptidase or oxytocinase. Diverse fat sources have demonstrated to modify oxytocinase activity in the testis of mice. Compared to fish oil, rich in ω-3 fatty acids, lard or coconut oils decreased soluble and membrane-bound oxytocinase activity. Oxytocin is involved in the local control of androgen biosynthesis by decreasing testosterone concentration and increasing 5α-dihydrotestosterone, and, therefore, changes in oxytocinase activity could have repercussion in male fertility.115 Compared to butter, a diet enriched with virgin olive oil did not alter glutamyl aminopeptidase activity in testis, and these enzymatic values correlated with total plasma cholesterol and triglycerides values. This result demonstrated a relationship between the alteration in plasma lipid profile caused by dietary saturated fatty acids and cholesterol, and the local RAS in testis, and the protective role of MUFAs from virgin olive oil.65 Moreover, the intake of a diet rich in virgin olive oil also improved the oxidative status of testis in male Wistar rats. This effect was demonstrated by the lower activity of GGT (γ-glutamyl transferase), an important enzyme involved in glutathione metabolism, and the higher activity of dipeptidyl peptidase-4 (DPP4/CD26), with an important immunoregulatory role in testes which contributes to the maintenance of normal spermatogenesis.65

36.6 Implications for human health and disease prevention Infertility has demonstrated to represent a relevant health problem, and closely to 50% of cases are related to male

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reproductive dysfunction. Diseases associated with poor dietary habits, such as obesity or metabolic syndrome, also indicate a link between diet and infertility. Therefore trying to develop therapeutic and prevention approaches appears to be a priority in order to decrease male infertility. Different diet components have been linked to problems in male fertility, mainly diets rich in saturated fatty acids and cholesterol. Conversely, several components of the Mediterranean diet have demonstrated to improve male fertility. To date, the results indicate that the main effects of diet on male fertility depend of the quality of dietary fatty acids and the balance between OS and antioxidants intake. Fat intake has a pivotal role in fertility, sperm quantity, and quality, because spermatozoa presents high amount of unsaturated fatty acids in their membranes. However, the OS seems to be the main cause involved in the pathophysiology of male infertility. ROS directly damage the nucleus and mitochondrial DNA and initiate the peroxidation of fatty acids in the sperm. In fact, antioxidant supplementation shows improvement in the characteristic of semen. Public health strategies to improve the quality of fat in the diet and the intake of antioxidant could help to avoid the global incidence of male infertility.

36.7 Comparisons of olive oils with other edible oils The Western diet, characterized by a high intake of saturated fatty acids and cholesterol, has demonstrated to exert a negative impact on male fertility, mainly related with an increase in plasma cholesterol and levels of OS. In animals models, a diet enriched in cholesterol changes the lipid composition on spermatozoa membranes and decreases its ability to undergo acrosomal reaction. However, these deleterious effects are avoided when the diet of the animals is supplemented with olive oil. The positive effect of olive oil on male fertility seems to be related with both its ratio C18:1/C18:0 (oleic/palmitic), an index of membrane fluidity, and the high amount of antioxidants into its composition. In the same way, different studies have demonstrated an association between the adherence to Mediterranean diet, the semen quality, and the fertility rates in human. The beneficial effects of Mediterranean diet on male fertility could be mediated through several mechanisms, related with its content in antioxidants and antiinflammatory compounds. Besides, the intake of olive oil provides more than 80% MUFAs and less than 14% saturated fatty acids. MUFAs contribute to lower plasma cholesterol, increase the membrane fluidity in the spermatozoa, and are less easily damaged by lipid peroxidation.

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A local RAS has been described in testis, with relevant functions in male fertility. Several studies have demonstrated an inhibitor effect of diet rich in fats on the expression of several components of this local RAS. Compared with saturated fats, unsaturated fats increase the activity of peptidases than metabolize angiotensin peptides, and these changes are related to the levels of plasma testosterone and the values of systolic blood pressure. Other hormone with important role in testis is oxytocin. Oxytocin is involved in the local control of androgen biosynthesis. Compared with saturated fat sources, diet rich in unsaturated fatty acids increase oxytocinase activity. High amount of saturated fatty acids and cholesterol in diet has demonstrated to affect angiotensinase activity in testis, and these changes correlated with the levels of plasmatic lipid. However, a diet rich in olive oil do not affect angiotensinases and support the protective role of MUFAs. The intake of olive oil also improves the oxidative status of testis.

Mini-dictionary of terms G

G

G

G

G

G

G

Mediterranean diet. Definitions of the Mediterranean diet vary across some settings. Usually, Mediterranean diet includes the traditional dietary habits in the Mediterranean area. This includes the high intake of fruit and vegetable, and the use of virgin olive oil as the main fat source. Virgin olive oil. Virgin olive oil is the oil produced from the fruit of the Olea europaea L. by only physical (mechanical) means. This oil must be free acidity expressed as oleic acid (not more than 0.8 g/100 g). Western diet. The Western diet is a dietary pattern that is characterized by high intakes of red meat, whole fat dairy, eggs, butter, and processed foods; a low intake of fruit, vegetable, and whole grains. Metabolic syndrome. Metabolic syndrome, insulinresistance syndrome, or syndrome X is the cooccurrence of metabolic risk factors for both type 2 diabetes and cardiovascular disease (abdominal obesity, hyperglycemia, dyslipidemia, and hypertension). Oxidative stress. OS is defined as a disturbance in the balance between the production of ROS (free radicals) and antioxidant defenses into the body. OS occurs when excess oxygen radicals are produced in cells, which could overload the normal antioxidant capacity. Reactive oxygen species. ROS, also called oxygen radicals or free radicals, are a type of unstable molecule that easily reacts with other molecules in a cell. The ROS in cells may cause damage to DNA, RNA, and proteins and may cause several pathologies. Antioxidants. Antioxidants are substances able to protect the cells from the damage caused by free radicals.

G

G

G

G

Antioxidants include beta-carotene, lycopene, vitamins (A, C, E), polyphenols, and other substances. Sperm capacitation. Sperm capacitations correspond to some physiological changes of the spermatozoa in the female genital tract before they are capable of penetrating and fertilizing the eggs. It is considered a preparation for the acrosome reaction. Acrosomal reaction. Acrosomal reactions correspond to the release of different enzymes from the acrosome outside of the sperm (exocytosis of the acrosome). This process causes the sperm to penetrate the zona pellucida of the egg and begin fertilization. When the acrosome’s proteolytic enzymes are released, they begin to degrade the egg’s protein-rich protective coat. Renin
angiotensin system. The RAS is a hormonal physiological system in which main functions are the regulation of blood pressure and fluid balance. This system includes several enzymes and peptides. Angiotensinases. Angiotensinases are the enzymes included in the RAS implicated in the metabolism of different angiotensin peptides, such as AngII and AngIII.

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38. Rato L, Alves MG, Cavaco JE, et al. High-energy diets: a threat for male fertility? Obes Rev. 2014;15:996
1007. Available from: https://doi.org/10.1111/obr.12226. 39. Saether T, Tran TN, Rootwelt H, et al. Essential fatty acid deficiency induces fatty acid desaturase expression in rat epididymis, but not in testis. Reproduction. 2007;133(2):467
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532. Available from: https://doi.org/10.1016/s1357-2725(01)00152-2. 42. Robbins WA, Xun L, FitzGerald LZ, et al. Walnuts improve semen quality in men consuming a Western-style diet: randomized control dietary intervention trial. Biol Reprod. 2012;87(4):101
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1640. Available from: https://doi. org/10.1093/humrep/der132. 48. Wagner H, Cheng JW, Ko EY. Role of reactive oxygen species in male infertility: an updated review of literature. Arab J Urol. 2018;16 (1):35
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166. Available from: https://doi.org/10.1016/ 0891-5849(93)90006-G. 50. Ford WC. Regulation of sperm function by reactive oxygen species. Hum Reprod Update. 2004;10(5):387
399. Available from: https://doi.org/10.1093/humupd/dmh034. 51. Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa: Physiology and pathology. Int J Androl. 1997;20(2):61
69. Available from: https://doi.org/10.1046/j.1365-2605.1997.00044.x. 52. Agarwal A, Sharma RK, Nallella KP, et al. Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril. 2006;86(4):878
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54. Agarwal A, Allamaneni SS, Nallella KP, et al. Correlation of reactive oxygen species levels with the fertilization rate after in vitro fertilization: a qualified meta-analysis. Fertil Steril. 2005;84(1): 228
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895. Available from: https://doi.org/10.1111/j.1461-0248.2011.01653.x. 65. Domı´nguez-Vı´as G, Segarra AB, Martı´nez-Can˜amero M, et al. Influence of a virgin olive oil versus butter plus cholesterolenriched diet on testicular enzymatic activities in adult male rats. Int J Mol Sci. 2017;18(8):1701. Available from: https://doi.org/ 10.3390/ijms18081701. 66. Moilanen J, Hovatta O. Excretion of alpha-tocopherol into human seminal plasma after oral administration. Andrologia. 1995;27 (3):133
136. Available from: https://doi.org/10.1111/j.14390272.1995.tb01083.x. 67. Shang X, Huang Y, Ye Z, et al. Protection of melatonin against damage of sperm mitochondrial function induced by reactive oxygen species. Zhonghua Nan Ke Xue. 2004;10(8):604
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Olive oil and male fertility Chapter | 36

68. Sies H, Stahl W, Sundquist AR. Antioxidant functions of vitamins: vitamins E and C, beta-carotene, and other carotenoids. Ann N Y Acad Sci. 1992;669(1):7
20. Available from: https://doi.org/ 10.1111/j.1749-6632.1992.tb17085.x. 69. Tavilani H, Goodarzi MT, Vaisi-raygani A, et al. Activity of antioxidant enzymes in seminal plasma and their relationship with lipid peroxidation of spermatozoa. Int Braz J Urol. 2008;34(4):485
491. Available from: https://doi.org/10.1590/s1677-55382008000400011. 70. Tavilani H, Goodarzl MT, Doosti M, et al. Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa. Reprod Biomed Online. 2008;16 (5):649
656. Available from: https://doi.org/10.1016/S1472-6483 (10)60478-6. 71. Terai K, Horie S, Fukuhara S, et al. Combination therapy with antioxidants improves total motile sperm counts: a preliminary study. Reprod Med Biol. 2019;19(1):89
94. Available from: https://doi. org/10.1002/rmb2.12308. 72. Waheed MM, Gouda EM, Khalifa TA. Impact of seminal plasma superoxide dismutase and glutathione peroxidase on cryopreserved buffalo spermatozoa. Anim Reprod Sci. 2013;142(3
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188. Available from: https://doi.org/10.4103/jrpp.jrpp_18_100. 74. Chi HJ, Kim JH, Ryu CS, et al. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum Reprod. 2008; 23(5):1023
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571. Available from: https:// doi.org/10.1016/j.fertnstert.2008.07.1715. 76. Greco E, Romano S, Iacobelli M, et al. ICSI in cases of sperm DNA damage: Beneficial effect of oral antioxidant treatment. Hum Reprod. 2005;20(9):2590
2594. Available from: https://doi.org/ 10.1093/humrep/dei091. 77. Tunc O, Thompson J, Tremellen K. Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online. 2009;18(6):761
768. Available from: https://doi.org/10.1016/ S1472-6483(10)60024-7. 78. Bjelakovic G, Nikolova D, Gluud LL, et al. Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases. Sao Paulo Med J. 2015;133(2):164
165. Available from: https://doi.org/10.1590/1516-3180.20151332T1. 79. Me´ne´zo YJ, Hazout A, Panteix G, et al. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online. 2007;14(4):418
421. Available from: https://doi. org/10.1016/S1472-6483(10)60887-5. 80. Verma A, Kanwar KC. Human sperm motility and lipid peroxidation in different ascorbic acid concentrations: an in vitro analysis. Andrologia. 2009;30(6):325
329. Available from: https://doi.org/ 10.1111/j.1439-0272.1998.tb01178.x. 81. Akmal M, Qadri JQ, Al-Waili NS, et al. Improvement in human semen quality after oral supplementation of vitamin C. J Med Food. 2006;9(3):440
442. Available from: https://doi.org/10.1089/ jmf.2006.9.440.

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82. Donnelly ET, McClure N, Lewis SE. Antioxidant supplementation in vitro does not improve human sperm motility. Fertil Steril. 1999;72(3):484
495. Available from: https://doi.org/10.1016/ S0015-0282(99)00267-8. 83. Netherton JK, Hetherington L, Ogle RA, et al. Mass spectrometry reveals new insights into the production of superoxide anions and 4-hydroxynonenal adducted proteins in human sperm. Proteomics. 2020;20(2):e1900205. Available from: https://doi.org/10.1002/ pmic.201900205. 84. Nixon B, Bernstein IR, Cafe SL, et al. A kinase anchor protein 4 is vulnerable to oxidative adduction in male germ cells. Front Cell Dev Biol. 2019;7:319. Available from: https://doi.org/10.3389/fcell.2019.00319. 85. Darbandi M, Darbandi S, Agarwal A, et al. Reactive oxygen species and male reproductive hormones. Reprod Biol Endocrinol. 2018;16 (1):87. Available from: https://doi.org/10.1186/s12958-018-0406-2. 86. Turner TT, Bang HJ, Lysiak JJ. Experimental testicular torsion: reperfusion blood flow and subsequent testicular venous plasma testosterone concentrations. Urology. 2005;65(2):390
394. Available from: https://doi.org/10.1016/j.urology.2004.09.033. 87. Rehm S, White TE, Zahalka EA, et al. Effects of food restriction on testis and accessory sex glands in maturing rats. Toxicol Pathol. 2008;36(5):687
694. Available from: https://doi.org/10.1177/ 0192623308320275. 88. Cangemi R, Friedmann AJ, Holloszy JO, et al. Long-term effects of calorie restriction on serum sex-hormone concentrations in men. Aging Cell. 2010;9(2):236
242. Available from: https://doi.org/ 10.1111/j.1474-9726.2010.00553.x. 89. Ha˚konsen LB, Thulstrup AM, Aggerholm AS, et al. Does weight loss improve semen quality and reproductive hormones? Results from a cohort of severely obese men. Reprod Health. 2011;8:24. Available from: https://doi.org/10.1186/1742-4755-8-24. 90. Omu AE, Al-Azemi MK, Al-Maghrebi M, et al. Molecular basis for the effects of zinc deficiency on spermatogenesis: an experimental study in the Sprague-Dawley rat model. Indian J Urol. 2015;31(1):57
64. Available from: https://doi.org/10.4103/09701591.139570. 91. Raigani M, Yaghmaei B, Amirjannti N, et al. The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men. Andrologia. 2014;46(9):956
962. Available from: https://doi.org/ 10.1111/and.12180. 92. Sakhaee E, Emadi L, Abshenas J, et al. Evaluation of epididymal sperm quality following experimentally induced copper poisoning in male rats. Andrologia. 2012;44(suppl 1):110
116. Available from: https://doi.org/10.1111/j.1439-0272.2010.01147.x. 93. Li P, Zhong Y, Jiang X, et al. Seminal plasma metals concentration with respect to semen quality. Biol Trace Elem Res. 2012;148(1):1
6. Available from: https://doi.org/10.1007/ s12011-012-9335-7. 94. Soliman A, Yassin M, De Sanctis V. Intravenous iron replacement therapy in eugonadal males with iron-deficiency anemia: effects on pituitary gonadal axis and sperm parameters: a pilot study. Indian J Endocrinol Metab. 2014;18(3):310
316. Available from: https:// doi.org/10.4103/2230-8210.131158. 95. Bonke E, Zwicker K, Dro¨se S. Manganese ions induce H2O2 generation at the ubiquinone binding site of mitochondrial complex II. Arch Biochem Biophys. 2015;580:75
83. Available from: https:// doi.org/10.1016/j.abb.2015.06.011.

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96. Ricci E, Al-Beitawi S, Cipriani S, et al. Dietary habits and semen parameters: a systematic narrative review. Andrology. 2018;6 (1):104
116. Available from: https://doi.org/10.1111/andr.12452. 97. Salas-Huetos A, Bullo´ M, Salas-Salvado´ J. Dietary patterns, foods and nutrients in male fertility parameters and fecundability: a systematic review of observational studies. Hum Reprod Update. 2017;23 (4):371
389. Available from: https://doi.org/10.1093/humupd/dmx006. 98. Trichopoulou A, Costacou T, Bamia C, et al. Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med. 2003;348(26):2599
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106. Kedechi S, Zribi N, Louati N, et al. Antioxidant effect of hydroxytyrosol on human sperm quality during in vitro incubation. Andrologia.. 2017;49(1). Available from: https://doi.org/10.1111/ and.12595. 107. Ramı´rez M, Prieto I, Alba F, et al. Role of central and peripheral aminopeptidase activities in the control of blood pressure: a working hypothesis. Heart Fail Rev. 2008;13:339
353. Available from: https://doi.org/10.1007/s10741-007-9066-6. 108. Prieto I, Arechaga G, Ramı´rez-Expo´sito MJ, et al. Aminopeptidases in the gonads of male and female rats. Fertil Steril. 2002;77:802
804. Available from: https://doi.org/10.1016/ s0015-0282(01)03242-3. 109. Segarra AB, Prieto I, Villarejo AB, et al. Effects of antihypertensive drugs on angiotensinase activities in the testis of spontaneously hypertensive rats. Horm Metab Res. 2013;45:344
348. Available from: https://doi.org/10.1055/s-0032-1329988. 110. Alves-Pereira JL, Frantz ED, Ramos Cd, et al. Beneficial effects of renin-angiotensin system blockers on testicular steroidogenesis. J Urol. 2014;192:1878
1883. Available from: https://doi.org/ 10.1016/j.juro.2014.05.093. 111. Dufau ML, Khanum A, Tsai-Morris CR. Multistep regulation of Leydig cell function. J Steroid Biochem. 1987;27:343
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291. Available from: https://doi.org/10.1055/s-2008-1046800. 115. Segarra AB, Arechaga G, Prieto I, et al. Effects of dietary supplementation with fish oil, lard, or coconut oil on oxytocinase activity in the testis of mice. Arch Androl. 2002; 48(3):233
236. Available from: https://doi.org/10.1080/ 01485010252869333.

Chapter 37

Revealing the molecular mechanism of Olea europaea L. in treatment of cataract Farid A. Badria1 and Abdullah A. Elgazar2 1

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt, 2Department of Pharmacognosy,

Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt

Abbreviations AGEs ADR CP-1 CAT GSH GSH-Px HBA HT HIF-1 LEC TGR5 MIC MUFA NSAID OLE PPARs SOD

advanced glycation end products aldose reductase calpain-1 catalase glutathione glutathione peroxidase hydroxybenzoic acids hydroxytyrosol hypoxia induced factor 1 lens epithelium cells membrane-type receptor for bile acids minimum inhibitory concentration monounsaturated fatty acid nonsteroidal antiinflammatory drugs olive leaves extract proliferator peroxisome
activated receptors superoxide dismutase

37.1 Introduction Natural products, their derivatives, and traditional remedies have an uprising attention in drug discovery approaches. Natural products are characterized by their structural diversity, so they could be utilized as biological function modifiers. This opened the door for the development of new technologies in discovering new drugs based on natural products’ screening, because they are a rich wealth of bioactive compounds. These bioactive compounds have a disease inhibiting abilities with many advantages over the synthetic ones such as lower toxicity with more effectiveness. Natural products’ ethno-pharmacological uses and pure compound isolation from their crude extracts are the basis for developing a lead compound through natural products drug discovery.1 The liver research laboratory (FAB-Lab, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt) presented several approaches for a better utilization of

natural products as potential therapeutic agents especially that deal with enzymes as drug targets by designing of enzyme inhibitors from commonly available natural products. Later, modulation of different biological activities via semisynthesis of commonly available natural products was extensively studied by Badria’s group (Table 37.1). All the previous examples are about using one compound that targets single enzyme (the on-target approach), with high selectivity to prevent any undesired effects raised form off-targeting. Previously, it was undesirable for drugs to inhibit many targets, because of the adverse effects. Though, the complexity of the current hopeless pathologies has demonstrated clearly that monopharmacology is insufficient to achieve the required therapeutic outcome. In parallel, it was found that molecules targeting more than one protein may have a safer profile when compared to the single-targeted.17,18 Disclosure the mechanism of action of certain natural compounds on any biological systems need to shed more lights on the plant or herb contains these constituents. Having enough evidences to prove the effect of olive leaves extract (OLE) on cataract via inhibition of aldose reductase (ADR) lead us to thorough study investigation of on olive (Olea europaea L.) from different aspects. The olive (O. europaea L.) is a small tree, belonging to the family Oleaceae that is native to warm temperate and tropical regions of the world. The commercial importance of the tree comes from its fruit that is known as olive, it is typically distributed in the coastal areas of the eastern Mediterranean basin, northern Africa, the coastal areas of southeastern Europe; hence, the Mediterranean region is considered as the main source of olive oil. Historical evidences showed that olives were cultivated for commercial purposes as far back as 3000 BCE. Also, olive oil was used for health maintenance by ancient Egyptian for treatment of several illnesses and was found as one of the components used for mummification. Also, olive tree was considered as blessed plant as

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00008-0 © 2021 Elsevier Inc. All rights reserved.

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TABLE 37.1 Development of natural product as potential therapeutic agents in FAB-Lab. No.

Studies for utilization of natural products as potential therapeutic agents

References

1

Anti-herpes activity of isolated compounds from frankincense

[2]

2

Chemistry and immunomodulatory activity of frankincense oil

[3]

3

Mirazid: a new schistosomicidal drug

[4]

4

Is man helpless against cancer? An environmental approach: antimutagenic agents from Egyptian food and medicinal preparations

[5]

5

Free-B-Ring flavonoids as potential lead compounds for colon cancer therapy

[6]

6

Immune-modulatory potentials of anti-neoplaston A-10 in breast cancer patients

[7]

7

Potential utility of anti-neoplaston A-10 levels in breast cancer

[8]

Studies for discovery of enzyme inhibitors from natural products 1

Olive and ginkgo extracts as potential cataract therapy with differential inhibitory activity on aldose reductase

[9]

2

Flavonoids containing an alpha-keto group as a new type of tyrosinase inhibitors from natural products as potential treatments for hyperpigmentation

[10]

3

Cycloartane glycoside: a new lactate dehydrogenase inhibitor, from the aerial part of agriculture waste of watermelon

[11]

Studies for chemical modification of structure natural products and their bioactivities 1

Betulinic acid analogues as potent topoisomerase inhibitors

[12]

2

Synthesis, docking, cytotoxicity, and LTA4H inhibitory activity of new gingerol derivatives as potential colorectal cancer therapy

[13]

3

Design and pharmacophore modeling of bi-aryl methyl eugenol analogs as breast cancer invasion inhibitors

[14]

4

Approach for chemo-sensitization of cisplatin-resistant ovarian cancer by cucurbitacin B

[15]

5

Derivatization, molecular docking and in vitro acetylcholinesterase inhibitory activity of glycyrrhizin as a selective anti-Alzheimer agent

[16]

mentioned in several religious scriptures such as the Bible and Holy Quran.19,20 Besides the nutritional values of olive and its oil, it was proven by several studies that they possess potential medicinal functions. Epidemiological studies have linked decreased incidence of cardiovascular diseases and some types of cancers in areas consuming olive oil as the main dietary fat,21,22 and also the oil has antioxidant, antimicrobial, and antiinflammatory effects and is used in several cosmetic and skin products.23 It is now explained that high content of monounsaturated fatty acid (MUFA) and other phytochemicals such as tocopherols, phospholipids, phenolics, and triterpenes are responsible for the reported biological activities.24 Consequently, the quality of olive and olive oil constituents alters according to the level of these components, which is affected by numerous factors such as cultivar, harvesting time, and the used processing techniques.25 So, it was logical that olive-based products grab the attention of both scientist and public as a functional food that would help to maintain health state and protect against several diseases. Several laboratories around the

world started a campaign to investigate the pharmacological effect of other parts to extrapolate the benefits from this unique tree.26,27 In this chapter, we will give a brief summary about the chemistry and pharmacology of olive leaves, shed the light on the pathogenesis of cataract and the limitations of the current therapy, emphasize on the reported studies discussing the role of bioactive molecules in olive leaves in treatment of cataract by interfering with molecular targets associated with cataract formation, also report an in silico study that would give insights on possible molecular mechanisms that could be considered in the future for the development of more advantageous therapies.

37.2 Olive leaves, chemistry, biology, and therapeutics 37.2.1 Chemistry of olive leaves O. europaea leaves chemical composition has been investigated by several studies and showed the presence of variable phytochemical classes such as phenolics, iridoids,

Revealing the molecular mechanism of Olea europaea L. in treatment of cataract Chapter | 37

triterpenes, and MUFAs, the chemical analysis of the leaves in comparison to fruit and the oil showed similar profile; however, there are significant differences in their concentrations. So, olive leaves are considered as important source for the isolation of highly valuable pharmaceuticals and nutraceuticals.

37.2.1.1 Polyphenolic compounds in olive leaves Phenolics are aromatic compounds substituted by one or more hydroxyl substituents that can range from simple acids such as hydroxybenzoic acids (HBA) and phenyl alcohol to polymeric compounds such as lignins and tannins. HBA are derivative of benzoic acid, while hydroxycinnamates are derivatives of cinnamic acid and considered as the main skelta of other analogues such as caffeic, ferulic, p-coumaric, and sinapic acids. The major caffeic acid derivative in olives is verbascoside, a heterosidic ester of caffeic acid, rutinose, and 3,4-dihydroxyphenylethanol (Fig. 37.1A). Phenyl alcohol includes molecules containing the C6
C2 pattern represented in tyrosol or to hydroxytyrosol (HT)28 (Fig. 37.1B). Flavonoids, another class of phenolic compounds, were found in olive leaves as aglycone (apigenin,

447

diosmetin, luteolin, and quercetin) or glycoside (luteolin5-O-glucoside, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, quercetin-7-O-rutinoside)29 (Fig. 37.1C).

37.2.1.2 Secoiridoids in olive leaves Secoiridoids are a type of iridoids, which is characterized by the presence of cyclopentanodihydropyran ring system. Different substitutions of carboxylic iridoids after fissure of cyclopentane ring give rise to analogues of elenolic. While they are not phenolic compounds, they tend to be found as esters of tyrosol and HT. Oleuropein, an ester of elenolic acid with HT, is found in high percentage among phenolic compounds in olives especially immature fruits. Other examples of elenolic acid derivatives found in high concentrations are ligstroside, an ester of elenolic acid with tyrosol, demethyloleuropein, and the nonphenolic glycoside oleoside, and the phenolic glycoside nuezhenide30 (Fig. 37.1D).

37.2.1.3 Lignans in olive leaves Lignans are products of the dimerization of two phenylpropene or phenylpropene precursors (C6
C3 dimers), a study using HPLC
ESI
TOF
MS/IT
MS reveals the presence of syringaresinol, pinoresinol, and acetoxypinoresinol31 (Fig. 37.1E).

FIGURE 37.1 Chemical structure of different phytochemicals in olive leaves extract: (A) phenolic acids, (B) phenolic alcohols, (C) flavonoids, (D) secoiridoids, (E) lignans, and (F) triterpene.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

37.2.1.4 Triterpenes in olive leaves Several triterpenoids along with β-amyrin have been isolated from O. europaea leaves such as oleanolic acid, maslinic acid, erythrodiol, uvaol, and ursolic acid32,33 (Fig. 37.1F).

37.2.2 Pharmacology of olive leaves 37.2.2.1 Antidiabetic activity The hypoglycemic effect of O. europaea leaves has been demonstrated in alloxan-induced diabetic rabbits. Oleuropein was isolated from olive leaves and administrated at a dose of 20 mg/kg for up to 16 weeks. The blood glucose level was comparable to that observed in normal control. It was observed that the level of blood glucose along with the antioxidant-related markers was comparable to the normal control rabbits. The study demonstrated the impact of oleuropein as an antihyperglycemic and antioxidative agent.34 Oleuropein also increased glucose-stimulated insulin secretion in β-cells, by activating extracellular signal-regulated kinases/mitogen-activated protein kinase signaling pathway. Moreover, it protected against cytotoxicity induced by amylin amyloids that was found to be related to the 3-HT moiety of oleuropein.35 Oleanolic acid, another abundant triterpene in olive leaves, was found as agonist of TGR5 that controls various metabolic pathways, accordingly, this might explain the demonstrated reduced blood glucose, increased insulin levels, and glucose tolerance in high-fat diet
induced hyperglycemia model.36 Moreover, oleanolic acid showed protective effect against diabetic nephropathy in streptozotocin-induced diabetic nephropathy in rats by reducing oxidative stress in kidneys.37 In vitro studies were done to investigate the ability of olive-derived extracts or compound to inhibit molecular targets associated with diabetes where two cultivars of O. europaea fruit and 14 phenolic compounds were found to be effective inhibitors of α-amylase and α-glucosidase enzymes.38 Khlif et al. reported an isomer of oleanolic acid from the stem of O. europaea that significantly inhibited amylase and lipase enzymes in vitro.39

cell line by inducing DNA damage,43 also maslinic acid showed cytotoxic effect on HT-29 cell line by decreasing the expression of Bcl-2 and activating caspases 3 and 9.44

37.2.2.3 Antihypertensive and cardioprotective activity Oleanolic and ursolic acids isolated from the leaves of O. europaea from different cultivars were investigated for their antihypertensive. The triterpenes oleanolic acid and ursolic acid protected against hypertension and atherosclerosis and improved the insulin resistance of the test rats.45 OLEs were found to be effective in treatment of stage1 hypertension when given at the dosage of 500 mg two times daily, by lowering systolic and diastolic blood pressure.46 This result is consistent with another study, which confirmed the hypotensive and hypocholesterolemic effects of administration of 500 or 1000 mg/day of OLE on borderline hypertensive patient.47 OLE s showed antagonistic effects on Ca21 channel ex vivo and in vitro by reducing systolic left ventricular pressure, heart rate, and significant increase in the relative coronary flow. This activity could be attributed to the presence of oleuropein that inhibited L-type calcium channel directly and reversibly.48

37.2.2.4 Antiinflammatory and antinociceptive activities The antiinflammatory activity of OLE was evaluated using carrageenan-induced paw edema and formalin test, and in both cases the extract showed significant dose-dependent analgesic and antiinflammatory effects. Mahjoub et al, reported that such activity is more prominent in case of chloroform extract rather than methanolic extract.49,50 In agreement to these results, maslinic acid, ursolic acid, oleuropein, and hydroxytyrosol demonstrated antiinflammatory and antinociceptive activities in different assays such as acetic acid
induced writhing, formalininduced pain, carrageenan-induced paw edema, and capsaicin-induced mechanical allodynia in mice.51
54

37.2.2.2 Anticancer activity OLE and its constituents demonstrated cytotoxic effect against several cancer cell lines. Fares et al. reported the antiproliferative effect of ethanolic extract of olive leaves against leukemia by induction of apoptotic pathways.40 Oleuropein showed cytotoxic activity against HeLa cervical cancer cell line through mechanisms involving apoptotic mitochondrial cascade41 and induces apoptosis in HT-29 through p53 activation and downregulation of HIF-1.42 Oleanolic acid and its derivative erythrodiol, maslinic acid, and uvaol inhibit the proliferation of MCF-7

37.2.2.5 Antimicrobial activity Olive leave extracts from different cultivars were tested against several bacterial and fungal strains by Edziri et al. Chetoui cultivar showed significant inhibitory action against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Candida albicans, Enterococcus faecalis, and Escherichia coli, with percentages of inhibition ranged from 83% to 93% at 2 3 minimum inhibitory concentration values, also it prevented biofilm formation that could be attributed to the high content of phenolics especially apigenin 7-O-glucoside and oleuropein in comparison to other cultivar.55

Revealing the molecular mechanism of Olea europaea L. in treatment of cataract Chapter | 37

37.2.2.6 Antioxidant activity The extract of olive leaves was assessed for their antioxidant effect using microwave-heated soybean oil model. Interestingly, the extract increased the availability of vitamin E in the samples and decreased the loss of polyunsaturated fatty acids and the formation of oxidation products.56 HPLC analysis of extracts of leaves, fruits, and seeds of olive different cultivars from Portugal showed that the presence of phenolic compounds such as oleuropein, HT verbascoside, luteolin-7-glucoside, and rutin may explain the antioxidant effect exhibited by the extract.57 This is consistent with the antioxidant exhibited by olive extract and compounds such as caffeic acid, rutin, and oleuropein in DPPH reported by Lee and Lee.58

37.3 Cataract: pathogenesis and current treatment Cataract is one of the most important factors causing blindness globally. Cloudiness of crystalline fibers in eye lens obstructs the passage of light and causes cataract. About 17 million cataract cases have been worldwide per year. Epidemiological studies showed that women are more affected by it compared to men us.59 Cataract formation is usually a slow process. It can be observed by following symptoms such as blurred vision, nearsightedness while initiation of cataract, glare from lights, double vision in the affected eye, and frequently changes in eyeglasses or contact lenses.

449

2. Congenital cataract: A congenital cataract is associated with developmental abnormalities of the eye. It is responsible for about 10% of childhood blindness.60 3. Pharmacologic cataract: Some drugs such as steroids lead to posterior subcapsular cataract, and also psychotropic medications such as thioridazine and chlorpromazine are associated with cataract formation.61 4. Traumatic cataract: Traumatic cataract can occur at any part of eye lens because all the parts of eye lens are subjected to trauma. This type of cataract can be induced by radiation, surgical procedures for treatment of glaucoma, vitrectomy, injection of medications in the eye, or retinal detachment repair.62
65 5. Metabolic cataract: Cortical, nuclear, and posterior subcapsular cataracts at early age are linked to the incidence of metabolic abnormalities specifically dyslipidemia, hyperglycemia, central obesity, and hypertension.66 Fig. 37.2 summarize factors responsible for deposition of cataract.

37.3.2 Molecular mechanisms behind cataract formation Several studies have reported the molecular mechanisms behind the formation of cataract; hence a preventive therapeutic strategy could be developed by inhibiting one or more of these pathways, and these mechanisms could be enumerated as the following.

37.3.2.1 Oxidative stress 37.3.1 Etiology of cataract 1. Senile cataract: It is an age-related cataract also known as senile cataract where the vision is impaired due to gradual progressive opacity of the eye lens.

FIGURE 37.2 Factors contributing in development of cataract.

Oxidative stress is an imbalance between reactive oxygen species (ROS) production and antioxidant defense systems.67 ROS are produced in mitochondria of lens epithelium cells (LEC) and in the lens fiber cells. These ROS are very reactive molecules and can affect proteins, lipids,

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

and DNA structure and function in living cells resulting mutagenesis and cell death.68 The LECs maintain ROS at low levels either by activation antioxidant enzymes such as catalase (CAT), glutathione (GSH), peroxidase (GSH-Px), superoxide dismutase (SOD) or DNA repair enzymes to protect lipids, proteins, and nucleic acids. These antioxidant enzymes are found in all lens epithelial cells and lens fiber cells and protect the lens from oxidative damage by ROS to maintain lens clarity.69,70 The levels of SOD, CAT, and GSH-Px are found to be decreased in cataract patient, which leads to the interaction of ROS with macromolecules leading to lipid peroxidation, formation of insoluble protein aggregates, DNA and membrane pump systems damage, consequently activating necrotic and apoptotic mechanism loss that finally lead to cataract.71 Proliferator peroxisome
activated receptors (PPARs) are nuclear receptors that modulate the activity of several transcriptional factors, their activation plays important role in glucose level control, downregulation of inflammatory cytokines, and upregulation of several antioxidant enzymes, hence they are considered as attractive targets for developing new therapy for ocular diseases, especially PPAR-α that is highly expressed in retinal pigment epithelia.72
74

37.3.2.2 Nonenzymatic glycation Hyperglycemic environment is characterized by excess glucose that reacts nonenzymatically with proteins, tissues, or blood constituents; therefore nonenzymatic glycation is increased leading to the formation of amadori products, which in later stage give rise to the reactive different dicarbonyl compounds, that is, glyoxal, methylglyoxal, and deoxyglucosone that finally produce advanced glycation end products (AGEs) causing chronic, irreversible abnormalities affecting chromosomal DNA, eye lens crystallins, and extracellular matrix. So, AGEs were used by several experimental protocols for studying cataractogenesis.75

37.3.2.3 Polyol pathway The increase of polyols in the lens is a primary cause in the formation of cataract. In hyperglycemic conditions, glucose diffuses passively from the aqueous humor into the lens. ADR, the enzyme within the lens converts glucose to sorbitol, galactose and galactitol. These polyols cannot diffuse passively out of the lens and accumulate or converted to fructose. The accumulation of polyols results in osmotic gradient, which promotes diffusion of water from the aqueous humor dragging sodium to the lens, contributing in electrolyte imbalances and finally cataract formation.76

37.3.2.4 Calpain activation Calpain is superfamily of cysteine protease that is activated once intracellular Ca21 level is enough. Since

hyperglycemia promotes Ca21 release from endoplasmic reticulum, overactivated calpain converts crystallins into insoluble deformed crystallins. It also lyses lens cytoskeletal proteins that in turn increase lenticular Ca21, hence accelerating calpain overactivation and cataract formation, this was supported by clinical study that found high level of expression of calpain in diabetic patients compared to nondiabetic group.77

37.3.3 Current strategies for treatment and prevention of cataract Once cataract is diagnosed, there is no pharmacological treatment that can resolve the issues, and surgical intervention is needed to the cataractous lens with artificial one. In the meantime, the determination of risk factor groups and possible molecular mechanisms associated with cataract formation could be helpful for the prevention of cataract deposition. As previously mentioned, controlling oxidative stress, AGE formation, blocking polyol pathway in the eye could be a helpful strategy to protect against cataract development and could be classified as follows: 1. ADR inhibitors: These compounds decrease the accumulation of sorbitol in the lens, in hyperglycemic patients; however, developed synthetic agents have been terminated due to severe adverse effects. 2. Nonsteroidal antiinflammatory drugs (NSAID): It has been found that NSAID administration decreases the incidence of cataract in diabetic patient, through several mechanisms involving inhibition of nonenzymatic glycation of lens proteins, and other mechanisms related to oxidative stress modulation also have been reported. 3. Agents acting on glutathione (GSH): Recent studies indicated that vitamin E and N-acetylcysteine amide and melatonin could protect against cataract by maintaining antioxidative mechanism directly or indirectly by increasing levels of GSH.78

37.4 Plausible molecular mechanism of Olea europaea in treatment of cataract In our lab, we investigated the link between the hypoglycemic effect of OLE and its ability to protect against diabetic cataract by inhibiting the key enzyme in the polyol pathway. A validated model based on ADR of rabbit eye lenses was done, which revealed the ability of OLE to produce a noncompetitive ADR inhibition at IC50 5 65 μg/mL; however, our study did not address which of the active components of OLE are responsible for such activity, or if there would be other mechanisms should be considered, also there is no adequate analysis

Revealing the molecular mechanism of Olea europaea L. in treatment of cataract Chapter | 37

for the available data to establish an evidence-based concept for prevention and treatment of cataract by OLE or its components.79 In order to evaluate the significance of the in vitro inhibition of ADR by OLE, the effect of these extracts against cataract formation was tested ex vivo (unpublished data). Rabbit lenses incubated with medium supplemented by high glucose (30 mM) became opaque and lost its clearness, while the morphology of the lenses treated with 30 mM glucose along with 65 μg/mL of OLE appeared relatively similar to that of control lenses incubated with a normal glucose (5.5 mM) concentration along with fructose (30 mM) as shown in Fig. 37.3. Several studies reported a plethora of bioactivities of OLE and its component in the last decades, so it would be quite challenging to go through such enormous amount of data without a proper guide, here is where chemoinformatic tools could be beneficial. For example, molecular docking has been used for identifying bioactive compounds against set of molecular target, which is known as target fishing,80,81 so we decided to study the ability of compounds isolated from olive leaves to interact with distinctive molecular targets associated with cataract formation in silico, then we will discuss these findings in the light of related studies. The target fishing studies were done by building a database of 27 compounds found in O. europaea leaves based on KNApSAcK Metabolite Activity Database (http://www.knapsackfamily.com/knapsack_jsp/top.html) and LC/MS profile of the plant; the compounds were downloaded from PubChem in mol2 format and loaded in iGemdock, a virtual screening software,82 for molecular

451

docking of three molecular targets, crystal structures of the molecular targets were download from PDB as 3et1, 3rx3, and 2g8j representing proliferator peroxisome
activated receptor alpha (PPAR-α), ADR, and calpain-1 (CP1), respectively. The active site of each target was determined as sphere with radius 8A around the cocrystallized ligand. The compounds were docked to the active site using iGEMDOCK software by setting the parameters as the following: population size 200, generations 70, and number of solutions was set to 2. Best poses and types of interaction between the compound and the binding site were assessed for postdocking analysis, and also Pharmascore, another scoring function, was used for reranking the docked compounds based on their ability to afford significant interactions with active sites.83 Finally, compounds that have binding energy equal to or lower than that of cocrystallized ligand were selected for poses visualization using Discovery Studio ligand interaction visualizer. The results of the molecular docking against PPAR-α showed that five compounds, namely, oleuropein, acteoside, peonidin 3-(6v-p-coumarylglucoside), cycloolivil, and oleoside dimethyl ester achieved comparable binding affinity to the cocrystallized ligand as shown in Table 37.2, their interaction with active site is depicted in Fig. 37.4A. This is agreed with previous studies that showed that oleuropein reduces triglyceride levels through PPAR-α activation.84 Acetoside is a phenylpropanoid consisted of three moieties of caffeic acid, tyrosol linked by rutinose, while caffeic acid itself is known as PPAR-α

TABLE 37.2 Binding energy of top-ranked compounds of olive leaves docked in proliferator peroxisome
activated receptor active site.

FIGURE 37.3 Protective effect of olive leaves extract against cataract induced by incubation of isolated rabbit eye lens in high glucose medium: (A) eye lens incubated in normal level of glucose, (B) eye lens incubated in high level of glucose, (C) eye lens incubated in high level of glucose and treated by 65 μg/mL of olive leaves extract.

Compound

Energy

E (pharma)

Oleuropein

2154.3

2179.1

Acteoside

2145.4

2175.4

Standard activator

2126.6

2154.4

Peonidin 3-(6v-pcoumarylglucoside)

2136.2

2148.7

Cycloolivil

2118.7

2146.4

Oleoside dimethyl ester

2120.5

2141.6

Rutin

2118.4

2136.9

Secologanoside

2108.6

2131.3

1-Acetoxypinoresinol

2113.2

2130.7

8(1)-Hydroxypinoresinol

2100

2129.1

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

FIGURE 37.4 Molecular interactions of top-ranked compounds docked in the active site of (A) PPAR-α: oleuropein, acetoside, cycloolivil, peonidin 3-(6v-p-coumarylglucoside); (B) ADR: acetoside, oleuroside, oleuropein; and (C) calpain-1: acetoside, oleuroside, oleuropein. ADR, Aldose reductase; PPARs, proliferator peroxisome
activated receptors.

agonist.85 Acetoside itself exerted its antiinflammatory effect by PPAR-mediated mechanism.86 The anthocyanin peonidin 3-3-(6v-p-coumarylglucoside) consists of cyanidin linked to p-coumaric acid by glucose; interestingly, anthocyanins and cyanidins were reported to be agonist to PPAR-α.87,88 In the case of cycloolivil and oleoside dimethyl ester, there were no available studies addressing their interaction with PPAR-α; however, iridoids similar to the later were reported to inhibit adipocyte differentiation by activation of PPAR-α.89 In the case of ADR, 14 compounds of the compounds were able to achieve better binding affinity than the cocrystallized ligands as shown in Table 37.3, and the interaction of the top 5 compounds is represented in Fig. 37.4B. These compounds could be classified to seco-iridoids, phenolic compounds, pentacyclic triterpenes, and lignans, and again there were several reports that support these findings, for example, acetoside was found to be uncompetitive inhibitor for ADR,90 which is agree with our kinetic study for determination of inhibition mode of OLE, also oleuropein was found to be ADR inhibitor in diabetic model91 since oleuroside, oleoside dimethyl ester, ligstroside, and secologanoside are structurally related, it could be safe to assume their ability to exert the same activity. Rutin is quercetin glycoside that represents one of the major classes of ADR inhibitors of ADR, and achieved IC50 5 3 μM for ADR isolated from rat lenses also cornoside were also reported to possess the same activity but to

TABLE 37.3 Binding energy of top-ranked compounds of olive leaves docked in aldose reductase active site. Compound

Energy

E (pharma)

Acteoside

2175.8

2244.7

Oleuropein

2164.2

2226.9

Oleuroside

2153.6

2219.7

Peonidin 3-(6v-pcoumarylglucoside)

2171.1

2216.3

Oleoside dimethyl ester

2146.5

2212.1

Ligstroside

2160.4

2208.3

Cycloolivil

2140.2

2207.7

Bryonolic acid

2143

2199.4

Tormentic acid

2136.5

2194.4

Cornoside

2142.9

2191.1

Maslinic acid

2132.5

2190

Rutin

2145.5

2184.3

1-Acetoxypinoresinol

2120.6

2180.3

Secologanoside

2131.4

2176.3

Standard inhibitor

2115.6

2174.3

Revealing the molecular mechanism of Olea europaea L. in treatment of cataract Chapter | 37

much lower extend.92 There were no available studies for the inhibitory effect of maslinic, bryonolic, and tormentic acids to ADR; however, structurally similar triptotriterpenic acid A was found to inhibit ADR-2.93 Finally, there were no studies reporting the effect of the 2 lignans 1-acetoxypinoresinol and cycloolivil. Regarding the docking of the compounds in the binding site of CP-1, six compounds achieved less binding energy or equal to the cocrystallized ligands, as shown in Table 37.4 and their interaction with active site is demonstrated in Fig. 37.4C. There is scarce in the data available TABLE 37.4 Binding energy of chemical constituents of olive leaves docked in calpain-1 active site. Compound

Energy

E(pharma)

Oleuroside

2121.9

2160.8

Acteoside

2131

2159.2

Oleuropein

2121.6

2152

Rutin

2125.2

2151.6

Secologanoside

2110.7

2143.3

Standard inhibitor

2114.8

2140.1

8-Hydroxypinoresinol

2111.9

2140.1

453

about natural inhibitor of this enzyme, among the topranked compounds, only acteoside and rutin were previously reported to interact with this target where a recent study designed inhibitors based on caffeic acid scaffold, which is found also in acteoside and another study revealed the ability of rutin to inhibit the enzyme at IC50 5 96.2 μM.93,94 Besides that it is worthy to note that several components of O. europaea were found to decrease the formation of (AGEs), Kontogianni et al. showed that methanolic olive leaf extract but not the aqueous extract prevented the formation of fluorescent AGEs in vitro, and the phytochemical profiling of this extract showed the presence of phenolic compounds such as flavonoid glycosides of luteolin and tyrosol, also the seco-iridoid oleuropein.95 This is also supported by several in vivo studies that demonstrated the ability of these compounds to reduce serum AGEs in hyperglycemia-induced experimental models.96 This effect could be exerted by polyphenolic through different mechanisms such as free radical scavenging properties of these molecules or their ability to trap the carbonyl species the precursor of AGEs by competing with lysine or arginine amino acid in the target protein leading to the formation of monoglyoxal and/or diglyoxal adducts and halting the formation of AGEs. The later mechanism was mainly noticed in compounds bearing FIGURE 37.5 Protective effect of OLE and its component against cataractogenesis. OLE activates PPAR, which reduces inflammatory response and oxidative stress by upregulation of antioxidant enzymes and decrease gene expression of inflammatory cytokines; moreover, PPAR-α activation leads to control of glucose level in the case of hyperglycemia. OLE inhibits ADR preventing the formation of AGEs and formation of polyols, OLE also could exert its effect by inhibiting the activation of calpain1, which affect the function of crystallins and promote the formation of cataract. ADR, Aldose reductase; AGEs, advanced glycation end products; OLE, Olea europaea leaves extract, PPAR, proliferator peroxisome
activated receptor.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

dihydroxy phenol such as tyrosol, cinnamic acid derivatives, and quercetin analogues, which can explain the high inhibitory activity of acteoside against AGEs formation.97
99 Potential mechanism of action of different compounds found in olive leaves is depicted in Fig. 37.5.

37.5 Conclusion and future perspective O. europaea is an important medicinal plant with plethora of bioactivities, while there is great attention to the nutritional and medical benefits of olive oil and olives, and olive leaves were found to contain a variety of phytochemicals that would help prevent and manage chronic diseases and their complications. Cataract one of the chronic diseases that affect millions of people, yet there is no effective pharmacological treatment till now, and in this chapter, we showed how active components of OLE exert their action for prevention and treatment of cataract on the molecular level as speculated from in vitro, ex vivo, in silico studies and reported previous studies. We suggest that the multitargeting properties of major compounds such as oleuropein, acteoside, and their derivatives would achieve better therapeutic outcomes for treatment of metabolic disease; however, a well-designed in vivo experiment is needed, which would allow subsequently prepare standardized extract that can be used safely in future clinical trials.

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798. 37 Dubey VK, Patil CR, Kamble SM, et al. Oleanolic acid prevents progression of streptozotocin induced diabetic nephropathy and protects renal microstructures in Sprague Dawley rats. J Pharmacol Pharmacother. 2013;4(1):47
52. 38 Dekdouk N, Malafronte N, Russo D, et al. Phenolic compounds from Olea europaea L. possess antioxidant activity and inhibit carbohydrate metabolizing enzymes in vitro. Evid Based Complement Altern Med. 2015;2015:684925. 39 Khlif Ines, et al. A new triterpene from Olea europea stem with antidiabetic activity. Chemistry of Natural Compounds. 2012;48(5):799
802. Available from: https://doi.org/10.1007/s10600-012-0386-y. 40 Fares R, Bazzi S, Baydoun SE, Abdel-Massih RM. The antioxidant and anti-proliferative activity of the Lebanese Olea europaea extract. Plant Foods Hum Nutr. 2011;66(1):58
63. 41 Yao J, Wu J, Yang X, Yang J, Zhang Y, Du L. Oleuropein induced apoptosis in HeLa cells via a mitochondrial apoptotic cascade associated with activation of the c-Jun NH2-terminal kinase. J Pharmacol Sci. 2014;125(3):300
311. 42 Ca´rdeno A, Sa´nchez-Hidalgo M, Cortes-Delgado A, Alarco´n, de la Lastra C. Mechanisms involved in the antiproliferative and proapoptotic effects of unsaponifiable fraction of extra virgin olive oil on HT-29 cancer cells. Nutr Cancer. 2013;65(6):908
918. 43 Allouche Y, Warleta F, Campos M, et al. Antioxidant, antiproliferative, and pro-apoptotic capacities of pentacyclic triterpenes found in the skin of olives on MCF-7 human breast cancer cells and their effects on DNA damage. J Agric Food Chem. 2011;59(1):121
130. 44 Reyes-Zurita FJ, Rufino-Palomares EE, Lupia´n˜ez JA, Cascante M. Maslinic acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway. Cancer Lett. 2009;273(1):44
54. 45 Somova LI, Shode FO, Ramnanan P, Nadar A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated

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258. Perrinjaquet-Moccetti T, Busjahn A, Schmidlin C, Schmidt A, Bradl B, Aydogan C. Food supplementation with an olive (Olea europaea L.) leaf extract reduces blood pressure in borderline hypertensive monozygotic twins. Phytother Res. 2008;22 (9):1239
1242. Scheffler A, Rauwald HW, Kampa B, Mann U, Mohr FW, Dhein S. Olea europaea leaf extract exerts L-type Ca(2 1 ) channel antagonistic effects. J Ethnopharmacol. 2008;120(2):233
240. Esmaeili-Mahani S, Rezaeezadeh-Roukerd M, Esmaeilpour K, et al. Olive (Olea europaea L.) leaf extract elicits antinociceptive activity, potentiates morphine analgesia and suppresses morphine hyperalgesia in rats. J Ethnopharmacol. 2010;132(1):200
205. Mahjoub RC, Khemiss M, Dhidah M, Dellaı¨ A, Bouraoui A, Khemiss F. Chloroformic and methanolic extracts of Olea europaea L. leaves present anti-inflammatory and analgesic activities. ISRN Pharmacol. 2011;2011:564972. Impellizzeri D, Esposito E, Mazzon E, et al. The effects of oleuropein aglycone, an olive oil compound, in a mouse model of carrageenan-induced pleurisy. Clin Nutr. 2011;30(4):533
540. Gong D, Geng C, Jiang L, Cao J, Yoshimura H, Zhong L. Effects of hydroxytyrosol-20 on carrageenan-induced acute inflammation and hyperalgesia in rats. Phytother Res. 2009;23(5):646
650. Nieto FR, Cobos EJ, Entrena JM, Parra A, Garcı´a-Granados A, Baeyens JM. Antiallodynic and analgesic effects of maslinic acid, a pentacyclic triterpenoid from Olea europaea. J Nat Prod. 2013;76 (4):737
740. Ikeda Y, Murakami A, Ohigashi H. Ursolic acid: an anti- and proinflammatory triterpenoid. Mol Nutr Food Res. 2008;52(1):26
42. Edziri H, Jaziri R, Chehab H, et al. A comparative study on chemical composition, antibiofilm and biological activities of leaves extracts of four Tunisian olive cultivars. Heliyon. 2019;5(5): e01604-e. Malheiro R, Rodrigues N, Manzke G, et al. The use of olive leaves and tea extracts as effective antioxidants against the oxidation of soybean oil under microwave heating. Ind Crops Prod. 2013;44:37
43. Silva S, Gomes L, Leitao F, Coelho A, Boas LV. Phenolic compounds and antioxidant activity of Olea europaea L. fruits and leaves. Food Sc Technol Int. 2006;12(5):385
395. Lee O-H, Lee B-Y. Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol. 2010;101(10):3751
3754. Foster A. Cataract—a global perspective: output, outcome and outlay. Eye (Lond). 1999;13(Pt 3b):449
453. Moore AT. Understanding the molecular genetics of congenital cataract may have wider implications for age related cataract. Br J Ophthalmol. 2004;88(1):2
3. Li J, Tripathi RC, Tripathi BJ. Drug-induced ocular disorders. Drug Saf. 2008;31(2):127
141. Mohammadpour M, Hashemi H, Jabbarvand M, Babaei N. Clinicopathologic report of anterior subcapsular cataracts after combined administration of corticosteroids and cyclosporine following renal transplantation. JCRS Online Case Rep. 2013;1(2):e37
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63 Mochizuki T, Masai I. The lens equator: a platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens. Dev Growth Differ. 2014;56(5):387
401. 64 Dong X, Ayala M, Lo¨fgren S, So¨derberg PG. Ultraviolet radiationinduced cataract: age and maximum acceptable dose. Invest Ophthalmol Vis Sci. 2003;44(3):1150
1154. 65 Feng H, Adelman RA. Cataract formation following vitreoretinal procedures. Clin Ophthalmol. 2014;8:1957
1965. 66 Park S, Lee E-H. Association between metabolic syndrome and age-related cataract. Int J Ophthalmol. 2015;8(4):804
811. 67 Sies H. What is oxidative stress. In: Keaney JF, ed. Oxidative Stress and Vascular Disease. Boston, MA: Springer; 2000:1
8. 68 Huang L, Yappert MC, Jumblatt MM, Borchman D. Hyperoxia and thyroxine treatment and the relationships between reactive oxygen species generation, mitochondrial membrane potential, and cardiolipin in human lens epithelial cell cultures. Curr Eye Res. 2008;33(7):575
586. 69 Berthoud VM, Beyer EC. Oxidative stress, lens gap junctions, and cataracts. Antioxid Redox Signal. 2009;11(2):339
353. 70 Brennan LA, Kantorow M. Mitochondrial function and redox control in the aging eye: role of MsrA and other repair systems in cataract and macular degenerations. Exp Eye Res. 2009;88(2):195
203. 71 Ozmen B, Ozmen D, Erkin E, Gu¨ner I, Habif S, Bayindir O. Lens superoxide dismutase and catalase activities in diabetic cataract. Clin Biochem. 2002;35(1):69
72. 72 Khatol P, Saraf S, Jain A. Peroxisome proliferated activated receptors (PPARs): opportunities and challenges for ocular therapy. Crit Rev Ther Drug Carrier Syst. 2018;35(1):65
97. 73 Moran E, Ding L, Wang Z, et al. Protective and antioxidant effects of PPARα in the ischemic retina. Invest Ophthalmol Vis Sci. 2014;55(7):4568
4576. 74 van Raalte DH, Li M, Pritchard PH, Wasan KM. Peroxisome proliferator-activated receptor (PPAR)-α: a pharmacological target with a promising future. Pharm Res. 2004;21(9):1531
1538. 75 Bansal S, Kare PK, Tripathi AK, Madhu SV. Advanced glycation end products: a potential contributor of oxidative stress for cardiovascular problems in diabetes. In: Chakraborti S, Dhalla NS, Ganguly NK, Dikshit M, eds. Oxidative Stress in Heart Diseases. Singapore: Springer Singapore; 2019:437
459. 76 Badria FA, Elimam DM, Elabshihy MS, Ibrahim AS. Aldose reductase inhibitors from nature: a new hope for treatment of cataract. JOJ Ophthal. 2017;5. 77 Ahn YJ, Kim MS, Chung SK. Calpain and caspase-12 expression in lens epithelial cells of diabetic cataracts. Am J Ophthalmol. 2016;167:31
37. 78 Gupta S, Selvan VK, Agrawal S, Saxena R. Advances in pharmacological strategies for the prevention of cataract development. Indian J Ophthalmol. 2009;57(3):175. 79 Elimam DMA, Uddin Ibrahim AS, Liou GI, Badria FA-EA. Olive and ginkgo extracts as potential cataract therapy with differential inhibitory activity on aldose reductase. Drug Discov Ther. 2017;11(1):41
46. 80 Moumbock AF, Li J, Mishra P, Gao M, Gu¨nther S. Current computational methods for predicting protein interactions of natural products. Comput Struc Biotechnol J. 2019;17:1367
1376. 81 Elgazar AA, Knany HR., Ali MS. Insights on the molecular mechanism of anti-inflammatory effect of formula from Islamic traditional medicine: an in-silico study. J Tradit Complement Med. 2019;9(4):353
363. 82 Hsu KC, Chen YF, Lin SR, Yang JM. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological

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interactions and post-screening analysis. BMC Bioinformatics. 2011;12(suppl 1):S33. Hsu K-C, Chen Y-F, Lin S-R, Yang J-M. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis. BMC Bioinformatics. 2011;12(1):S33. Malliou F, Andreadou I, Gonzalez FJ, et al. The olive constituent oleuropein, as a PPARα agonist, markedly reduces serum triglycerides. J Nutri Biochem. 2018;59:17
28. Kim B, Kim JE, Kim H-S. Caffeic acid induces keratinocyte differentiation by activation of PPAR-α. J Pharm Pharmacol. 2014;66 (1):84
92. Esposito E, Mazzon E, Paterniti I, et al. PPAR-alpha contributes to the anti-inflammatory activity of verbascoside in a model of inflammatory bowel disease in mice. PPAR Res. 2010;2010:917312. Jia Y, Kim J-Y, Jun H-J, et al. Cyanidin is an agonistic ligand for peroxisome proliferator-activated receptor-alpha reducing hepatic lipid. Biochim Biophys Acta. 2013;1831(4):698
708. Du C, Shi Y, Ren Y, et al. Anthocyanins inhibit high-glucoseinduced cholesterol accumulation and inflammation by activating LXRα pathway in HK-2 cells. Drug Des Devel Ther. 2015;9:5099. Bai N, He K, Ibarra A, et al. Iridoids from Fraxinus excelsior with adipocyte differentiation-inhibitory and PPARalpha activation activity. J Nat Prod. 2009;73(1):2
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8 [Online ahead of print]. Li HM, Kim JK, Jang JM, Cui CB, Lim SS. Analysis of the inhibitory activity of Abeliophyllum distichum leaf constituents against aldose reductase by using high-speed counter current chromatography. Arch Pharm Res. 2013;36(9):1104
1112. ´ , Manzanaro S. Aldose reductase inhibitors from de la Fuente JA natural sources. Nat Prod Rep. 2003;20(2):243
251. Yoo YJ, Nam DH, Jung SY, et al. Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants and calpain inhibitors. Bioorg Med Chem Lett. 2011;21(10):2850
2854. Kontogianni VG, Charisiadis P, Margianni E, et al. Olive leaf extracts are a natural source of advanced glycation end product inhibitors. J Med Food. 2013;16(9):817
822. West BJ, Deng S, Uwaya A, et al. Iridoids are natural glycation inhibitors. Glycoconj J. 2016;33(4):671
681. Khan M, Liu H, Wang J, Sun B. Inhibitory effect of phenolic compounds and plant extracts on the formation of advance glycation end products: a comprehensive review. Food Res Int. 2020;130:108933. Yu SY, Lee IS, Jung SH, et al. Caffeoylated phenylpropanoid glycosides from Brandisia hancei inhibit advanced glycation end product formation and aldose reductase in vitro and vessel dilation in larval zebrafish in vivo. Planta Med. 2013;79(18):1705
1709. Liu Y-H, Lu Y-L, Han C-H, Hou W-C. Inhibitory activities of acteoside, isoacteoside, and its structural constituents against protein glycation in vitro. Bot Stud. 2013;54(1):6.

Chapter 38

Olive leaf, DNA damage and chelation therapy ˇ ˇ Andrea Cabarkapa-Pirkovi c´ 1, Lada Zivkovi c´ 1, Dragana Dekanski2, Dijana Topalovic´ 1 and 1 Biljana Spremo-Potparevic´ 1

Department of Pathobiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia, 2Biomedical Research, R&D Institute, Galenika a.d.,

Belgrade, Serbia

Abbreviations DPPHG EDTA EFSA HT LD50 LIP NOAEL OL OLE ROS Pb Cd H2O2 ABTS-2,20

2,2-diphenyl-1-picrylhydrazyl ethylenediaminetetraacetic acid the European Food Safety Authority hydroxytyrosol median lethal dose labile iron pool no-observed-adverse-effect level olive leaf olive leaf extract reactive oxygen species lead cadmium hydrogen peroxide Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

38.1 Olive leaf Diet rich in olives and olive oil, Mediterranean diet, has become a gold standard in disease prevention. Olive (Olea europaea L.) products consumption is associated with numerous health benefits and with reduced incidence of many pathological conditions, such as coronary heart diseases, diabetes, and certain tumors.1 The observed beneficial effects are ascribed to the polyphenol compounds found in olives, namely, to the phenolics and flavonoids. The polyphenol content in various products derived from the olive plant depends on the variety, climate, and maturity during harvest and processing technology.2 Although the main products of O. europaea L. are fruits and oil, studies have shown that olive leaves and secondary byproducts from olive oil production can be precious sources from which bioactive compounds can be recovered and used for biomedical applications.3 The main

bioactive component of olive leaf (OL) and olive oil is considered to be oleuropein, which is responsible for the majority of their observed biological effects. In 1960 the first report on the hypotensive effect in animals treated with oleuropein has initiated interest in the OL.4 Since then, olive leaves have found their commercial use in several fields, including pharmacological and food industries. The traditional use of olive leaves as a remedy for preservation and health is old for centuries, ever since the Egyptians used olive leaves as a good preservative in the mummification process and as an antimalaria drug.5 As a folk medicine, olive leaves were used as a tea, powder, or extract for the treatment of hypertension, arteriosclerosis, rheumatism, gout, diabetes.6 Over the past 20 years the effects of olive leaves and OL extracts (OLEs) have been examined in a number of studies, both in vitro and in vivo, showing marked health effects such as radioprotective, antiproliferative, cytotoxic effects on cancer cells; antifungal activity; antiatherosclerotic, antihypertensive, hypoglycemic, and cardioprotective effects as well as benefits in fighting obesity.7,8 OL and OLE are a rich source of antioxidants. There are five groups of phenolic compounds in the OL: oleuropeosides (oleuropein and verbascoside); flavones (luteolin-7-glucoside, apigenin-7-glucoside, luteolin); flavonols (routines); flavanols (catechin); and substituted phenols (tyrosol, hydroxytyrosol (HT), vanillic acid, and caffeic acid).9 Oleuropein is the dominant active substance in OL, and it is an ester of elenolic acid and HT and belongs to the class of secoiridoids. There are qualitative differences in total polyphenols and oleuropein content, depending on the kind of leaf (fresh, refrigerated, dried, frozen, or lyophilized), cultivar, sampling time, and production area. The study from 2016, examined several

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00021-3 © 2021 Elsevier Inc. All rights reserved.

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extracts from OL and found that polyphenol content varied from 7.87 to 34.21 mg/g, depending on the extraction temperature and drying process.10 Analyses of the presence of metabolites in urine, after consumption of OLE in high single dose or continued use for 28 days as a supplement in healthy subjects, showed that the only active component of OLE, which reaches the systemic circulation and whose metabolites can be found in urine within 6 h after ingestion, is, in fact, oleuropein.11 The same study showed that the applied concentration of the extract and the oleuropein content were crucial parameters for its metabolic presence, not the length of administration. Also, a study that showed reduced inflammation in subjects who consumed a modified Mediterranean diet enriched with olive oil for 4 weeks indicates that shortterm interventions with increased concentrations of components present in olive oil can produce a biologically significant effect.12 Our study confirmed this assumption, by achieving relevant improvement in patients with rheumatoid arthritis who used supplementation with dry OLE for 6 weeks.13 A key factor in achieving the biological effects of natural substances from dietary sources is bioavailability. It has been shown that the bioavailability of phenolic components can be significantly influenced by age and hormonal status of individuals.14 Phenolic components from olive and its derivatives have shown rapid absorption after ingestion and widespread distribution in internal organs such as liver, kidney, brain, and spleen.15 A pharmacokinetic study by Garcia-Villalba et al.14 showed that after ingestion of OLE, its metabolites HT, oleuropein aglycone, elenolic acid, luteolin, and apigenin can be found in plasma and urine within a few hours. Visioli et al.16 and Vissers et al.17 have shown that the absorption of phenolic components from olive products is dose dependent and that at least 55%
66% of the ingested amount is successfully absorbed. There are several preclinical safety data for various OLEs and some toxicological data concerning the toxicity of oleuropein. The LD50 of an extract of OL was not precise when it was given intraperitoneally (i.p.) as a single dose of 1300 mg/kg or as a dose of 3000 mg/kg orally in mouse.18 The LD50 of a standardized aqueous olive pulp extract with HT as the major constituent of biological significance was also reported to be greater than 2000 mg/ kg. Moreover, in a subchronic study, the no-observedadverse-effect level (NOAEL) of the same extract in rats was found to be 2000 mg/kg/day. In developmental and reproductive toxicity studies, the studied extract did not cause toxicity at levels up to 2000 mg/kg daily.19 Further toxicological evaluation on HT in rats, in which NOAEL was 500 mg/kg/day was reported by Aun˜on-Calles et al.20 The results of our acute oral toxicity study were consistent with previous findings and confirmed that oleuropein-rich, standardized OLE (EFLA 943, from

Frutarom) is a safe material when administered via oral gavage to rats in a single dose of 2000 mg/kg. Clinical signs and gross findings of treatment-related adverse effects were not observed in the experimental rats.21 Reviewing the literature about the safety and the genotoxic and mutagenic effects of OL and OLE, we found that phenolics from olive products in some previous investigations showed that they can themselves be genotoxic and cytotoxic when applied in very high doses and can act in a prooxidant or an antioxidant way, dependent of the concentration and duration of exposure.22
24 Our in vitro studies on human peripheral blood lymphocytes have shown a genotoxic effect of OLE for concentrations above 1 mg/mL.25 The safety of using OLE as a supplement was confirmed in a clinical study of hypertensive patients who took a dose of 500 mg orally twice per day, with a total daily dose of 1000 mg, which had an effect on decrease in systolic and diastolic blood pressure but did not cause any side effects.26 Moreover, OLE has been recently approved by the European Food Safety Authority (EFSA) as a safe food additive. There is a prevailing idea that predominantly antioxidants and free radical scavengers are responsible for the beneficial effects of the Mediterranean diet. However, it should be emphasized that the many underlining mechanisms of its effects are still insufficiently examined and the exact molecular pathways of action of bioactive compounds dominating in the Mediterranean diet remain to be elucidated.

38.2 Antioxidant effects of olive leaf, scavenging, and chelation Almost any oxidative process that normally goes on in our cells will consequently produce some by-product free radicals. Free radicals are unstable products of cellular metabolism seeking to achieve electronic stability by reacting with the first adjacent stable molecule and removing its electron (reaction of initiations). Further, as a chain reaction of substrate oxidation (propagation) continues, biochemical, structural, functional changes of biomolecules arise. This reaction continues until their neutralization and the prevention of further propagation by endogenous or exogenous antioxidants.27 Free radicals have many physiological roles and are continuously generated and removed within the cells under normal physiological conditions. Cells have specific sensory systems that are responsive to even minor variations in the cellular redox equilibrium, and which initiate the redox signaling transduction to fine-tune the metabolism. If their production is uncontrolled and exceeds the antioxidant capacity of cells, a pathological state in the cell is being developed, and oxidation of membrane lipids, cellular proteins,

Olive leaf, DNA damage and chelation therapy Chapter | 38

and DNA can occur. The two most common mechanisms of primary antioxidant protection are free radicals scavenging through proton donation and neutralization of radicals and the binding of transition metals to an inactive form, known as chelation. The primary antioxidant protection system includes exogenous antioxidants originating from food (e.g., vitamin C, vitamin E, and polyphenols), endogenous cellular antioxidants (thiols, Nacetyl-cysteine, NADPH and NADH, ubiquinone), and enzymes (superoxide dismutase, catalase, glutathione redox cycle enzymes) as well as metal-binding proteins that chelate free iron and copper ions, which act as catalysts in the oxidizing reactions in cells. Phenolics and flavonoids, the main components of OL, are widely recognized for their antioxidant activity and free radical scavenging capacity.28
31 Because of the remarkable antioxidant properties of flavonoids and their actions in a variety of ways, they have high pharmacological importance as therapeutic agents. Mechanisms by which flavonoids prevent oxidative damage to biomolecules include (1) direct reactive oxygen species (ROS) scavenging, (2) metal-chelating activity, (3) activation of antioxidant enzymes, (4) inhibition of oxidases, (5) mitigation of nitrosative stress, and (6) increase in antioxidant properties of low molecular antioxidants.24 In this text, we will address the two most common mechanisms of antioxidant action, ROS scavenging and metal chelation. Many flavonoids are powerful antioxidants, and they exceed or are comparable to the antioxidant capacities of vitamins C and α-tocopherol.32,33 There is a distinct relationship of their molecular structure with their properties.34 Namely, the flavonoid antioxidant activity depends on the arrangement of hydroxyl aromatic groups and from the configuration and the total number of hydroxyl groups on its core structure. These arrangements substantially influence the mechanism of antioxidant activity.35 Strong radical scavenging activity of certain flavonoids has been directly correlated with the presence of an ortho-dihydroxy (catechol) structure in the B ring, hydroxyl groups at positions 3 and 5 in the C ring, and the presence of 2,3double bond in C ring.24 OL is an abundant source of such structural compounds. Namely, the previous phytochemical analysis of OLE showed high oleuropein content (almost 20%), followed by HT, the flavone-7-glucosides of luteolin and apigenin, verbascoside, quercetin, and caffeic acid, all of them known to be efficient radical scavengers.29,36 OLE exhibited pronounced antioxidant properties in different in vitro and in vivo systems.29,37
39 So far, the protective effects of OLE on oxidative cell damage in the lymphocytes, erythrocytes, skin cells, cardiomyocytes, hepatocytes, neurons, etc. have been shown.39
42 The radical scavenging ability is considered its main mode of action, and it can be directly attributed to its phenolic constituents.29,43 In our previous study the

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high antioxidative potential of the oleuropein-rich, standardized dry OLE was confirmed in vitro using the 2,2diphenyl-1-picrylhydrazyl (DPPH ),38 while the study of Benavente-Garcia et al.29 also showed efficient radical scavenging properties against 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS1  radical. Although these tests are standard tests for in vitro antioxidant evaluation, these radicals are not present in cellular biochemical oxidative processes. Lins et al.41 demonstrated OLE as a scavenger of superoxide anion, hypochlorous acid, and nitric oxide species that are produced in cells in vivo. By analyzing the content and the antioxidant activity of individual OL components, Benavente-Garcia et al.29 concluded that flavonols, flavans-3-ols, and flavones with catechol structures were the most efficient radical scavengers for the ABTS. Further, several studies showed that the total antioxidant capacity of joined components in the OL has shown a more potent effect than its isolated individual components, indicating a synergistic antioxidant effect of the OL components.29,43 Although studies on antioxidant properties of individual components of OL showed that caffeic acid, rutin, and luteolin have better antioxidant properties then oleuropein, which is the main component of OL, the antioxidant properties of the entire extract of olive leaves can still be ascribed mainly to the high oleuropein content and less to the amount of flavonoids.29,31 Many flavonoids display potent antioxidant activity in vitro, while they have poor effects in vivo due to their extensive biotransformation in the metabolism after they undergo phase I deglycosylation of flavonoid glycosides and phase II metabolism, including glucuronidation, sulfation, and O-methylation of resulting aglycones.24 These metabolic changes lead to the blocking of free radical scavenging phenolic hydroxyl groups in the metabolites of flavonoids that can be found circulating as well as intracellular. That is why most of the metabolites have greatly reduced in vitro antioxidant activity when compared to their originating compound, which is, for example, observed for O-methylated and -glucuronidated derivatives of quercetin, (2)-epicatechin, catechin, and luteolin.24 Interestingly, the antioxidant properties of the OL are only mildly reduced following the gastric and intestinal digestions, compared to the antioxidant capacity of the fresh extract.44 These antioxidant features were attributed to the oleuropein metabolites that can maintain a strong antioxidant activity in vivo even after digestion.44 The main metabolite of OL is the HT, elenolic acid ester of oleuropein that contains an ortho-diphenolic group. HT was identified as a compound with high antioxidant capacity and metal-chelating and free radical scavenging activities.45 Studies showed that conjugated metabolites of HT are the primary metabolites recovered in plasma and urine of subjects at 24 h after OLE postingestion.46 In G

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terms of antioxidant properties, HT appears to be only slightly less reactive than oleuropein.31 The study that examined five extracts of olive oil containing different amounts of HT showed that the HT-rich extracts were the strongest antioxidant and antigenotoxic agents in vitro.47 This study also indicated one very important finding that cell metabolism can alter the activity of administered olive oil extracts. A similar finding was described in the paper of Gordon et al.48 who emphasized that the antioxidant activity of HT changes in different hydrophilic and hydrophobic phases, influencing the radical scavenging properties and stability, which is an important notice to consider. When they are found in the intracellular space, flavonoids and polyphenols may protect cells from oxidative stress by several different mechanisms. The free radical scavenging ability is currently the most explored mechanism and the widespread theory for the beneficial effects of flavonoids and polyphenols represented in vivo. However, there are not many studies in vivo that established a direct correlation between the protective efficiencies and the scavenging capacities of these compounds. Now there is an increasing number of studies that support the alternative explanation that the intracellular binding of redox-active iron is the basis for their protective capacity. Thus the prevention of reactive free radical formation, rather than scavenging of free radicals after these have been generated, is the new theory for their protective role.49 The chelation of the metal ions in oxidative reactions, using a transition metal ion such as copper or iron, is the second most common mechanism attributed to the antioxidative activity of phenolic compounds.33 Transition metals, such as iron, have many fundamental roles as protein cofactors and are required for numerous biochemical processes in the cells and appear largely sequestered in forms that are unable to catalyze a free radical reaction. However, if not tightly bound in the active sites of enzymes or securely stored in a redox-inert state, they participate in deleterious oxidative reactions as the major catalyst for a generation of reactive free radicals through the Fenton reaction in the presence of hydrogen peroxide (H2O2).50 These products of the Fenton reaction are short lived, but their high reactivity causes them to oxidize surrounding molecules in their vicinity such as nucleic acids, proteins, membrane lipids, carbohydrates. Out of all transition metals, iron plays a major role in H2O2-induced toxicity, but also in redox signaling due to its abundance in cells and organisms. The fraction of unshielded iron, found in an unbound form in cells, is called “labile iron pool” (LIP).51 This LIP is redox active and can catalyze the production of a highly toxic hydroxyl radical via the Fenton/ Haber
Weiss reaction cycle. On the other hand, it is also chelatable and can be bound by chelating compounds. Petrat et al. showed that there is an existence of an LIP in

the cell nucleus.52 Its concentration and state can vary depending on the prevailing conditions in the cells. Excessive iron-mediated oxidation was implicated in the etiology of diseases such as rheumatoid arthritis, Parkinson’s disease, atherosclerosis, cancer, Alzheimer’s disease, and postischemic reperfusion injury.53 A key question is whether olive-rich Mediterranean diet or olive leaves contain such iron-chelating components, and to which extent can these compounds reach intracellular compartments and protect cellular components from oxidation by iron binding, occupation of all-metal coordination, and prevention of metal redox cycling. Galaris et al.54 experimentally addressed these questions and showed that the Mediterranean diet contains a great number of ironchelating molecules, especially phenolic compounds with an ortho-dihydroxyl group, which are protective against iron-mediated oxidative stress. Another study, by Andjelkovi´c et al.,55 showed that the phenolic acids bearing catechol or galloyl groups (caffeic acid, gallic acid, protocatechuic acid, and chlorogenic acid) together with HT can form a complex with ferrous iron at physiological pH (7.4) and chelate iron. Catechol moiety in the B ring has been shown to be essential for the chelation, and it is the main contributory site for the metal chelation.33 However, it should be addressed that a prerequisite for effective chelating action of a particular compound is its ability to penetrate the plasma membrane in order to act in the interior of the cells. The protective effects are more efficient when the lipophilicity of the compounds is increased, allowing them to diffuse through the cell membrane and reach the intracellular space.56 The phenolic acids, such as caffeic acid that are negatively charged at neutral pH, are able to chelate iron but unable to diffuse through the plasma membrane, thus they are largely ineffective in cells. For such compounds, longer incubation periods are required and relatively higher concentrations to be protective.56 In vitro study by Kitsati et al.57 showed that HT, which is the main metabolite of OL, can penetrate cells and modulate intracellular labile iron through the chelating action of the ortho-dihydroxy group of this compound and reduce DNA damage in the cells. Further, it was shown that the complexation of flavonoids with ferrous, ferric, and cupric ions can enhance their antioxidant activity. Namely, it was found flavonoid
metal complexes of Cu21, Fe21, or Fe31 to increase their antioxidant activity and more effective at preventing hypoxia
reoxygenation injury than the corresponding compounds alone.58

38.3 Effects of the olive leaf on the DNA damage The most of research done on antioxidant activity of flavonoids and polyphenols has been performed in cell-free

Olive leaf, DNA damage and chelation therapy Chapter | 38

systems and relatively rarely using living cells. Thus there still is only partial and to some extent contradicting knowledge about their ability to protect mammalian cells and their structural molecules from oxidative damage.59 The DNA molecule is more prone to oxidation compared to other cellular components, and thus it is the most sensitive biomarker for oxidant-induced damage in cells. Specifically, there is evidence to suggest the association of elevated ROS production, oxidative stress triggering, and the onset of genomic instability with the malignant cell transformation.60 Under normal physiological conditions, primary oxidative DNA damage is removed or repaired by the activity of the DNA repair mechanisms.61 Unless removed or repaired, primary oxidative damage can lead to mutations and potential development of carcinogenesis especially if mutations arise in parts of the genes responsible for regulating cell growth (proto-oncogenes, tumor suppressors, genes regulators of DNA repair).60 Consequently, there were a large number of studies performed in the past decade in search of compounds that would be able to protect nuclear DNA against oxidative damage. Flavonoids and polyphenols have been extensively studied in this context. Conflicting results were obtained, with both genotoxic and antigenotoxic effects being reported for compounds originating from an OL or other olive products.23,28 Antioxidant/prooxidant properties of flavonoids were associated with their antigenotoxic/genotoxic activities.24,49 Namely, flavonoids that are abundant in OL have previously been reported for both antioxidant and prooxidant effects, apoptotic, antiproliferative, and cytotoxic and genotoxic activities as well as antigenotoxic effects in cells, dependent on the system used for the evaluation and the concentration of the compounds.23,62,63 Studies showed that there is a range of concentrations between protective and cytotoxic effects to the cells for each flavonoid compound and that their activity is dependent on the local environment, such as the presence of transition metals in cells.59 This phenomenon was described as a “quercetin paradox” on the example of the flavonoid quercetin, which is one of the compounds of the OL as well. Namely, quercetin is an effective radical scavenger and protects cells against H2O2-induced DNA damage, but during this process, quercetin gets oxidized and becomes reactive.64 By accumulating these reactive intermediates, at very high concentrations of quercetin, in the end, it leads to cytotoxicity. Namely, after scavenging ROS, flavonoids form flavonoid phenoxyl radicals (Fl-OG), a highly reactive species with a very short lifetime. These flavonoid radicals are subject to further oxidation, forming the more stable flavonoid quinones that undergo conjugation with nucleophiles, such as GSH, cysteine, or DNA24 (see Fig. 38.1). These flavonoid
DNA complexes have a strong affinity for metal chelation, forming metal
flavonoid
DNA

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complexes.65 Such metal
flavonoid complexes that efficiently intercalate into the double helix of the DNA have previously been described for quercetin.66 The intercalated metal
flavonoid
DNA complexes have been ascribed for the potential antitumor activity of flavonoids.67 Namely, the works of Yamashita et al.68 and Amrani et al.69 showed that transition metal
flavonoid complexes that directly intercalate to DNA molecule can produce site-specific oxidative DNA damage or hydrolytic cleavage in the place of their conjugation. On the other hand, different authors showed quercetin
metal complexes that bind DNA have better antioxidant properties than quercetin itself70,71 and that bindings of quercetin to DNA can protect against induction of oxidative DNA damage.72 The presence of transition metals at neutral pH can render flavonoids such as quercetin to undergo an autooxidation, accompanied by rapid accumulation of H2O2, which means that at high concentrations of flavonoids can themselves cause oxidative stress in the presence of transition metals.73 Other authors confirmed that the genotoxic effect of flavonoids in human cells is based on their prooxidant properties and it seems to be concentration dependent.49,74 It is clear that these complexes between flavonoids and metals have a dual role, they can act as more potent antioxidants than the parent flavonoids, but they as well may be prooxidants and enhance the oxidative damage of biomolecules.67 Both cellular metal levels and the concentration of ROS in cells determines whether flavonoids will act as a DNA protective or DNA damaging molecules, and it may be cell type dependent as well as dose dependent.23,59,74,75 We have previously evaluated the genotoxic and antigenotoxic potential of OLE in human peripheral blood cells. The concentrations of OLE above 1000 μg/mL displayed genotoxic effects, while the equal concentrations or less than 1000 μg/mL exhibit antigenotoxic effects.25 This is in accordance with findings of other studies who also reported that relatively low concentrations of extracts from olive oil (up to 75 μg/mL) offered strong protection against DNA damage, while at concentrations of 100 μg/mL or higher, they exerted genotoxic effect.23 It should be stated that this genotoxic concentration is very high and it would be difficult to achieve it intracellularly by any amount of OL ingestion. Interestingly, a study by Anter et al.28 showed that OLE and luteolin, but not oleuropein, induced DNA fragmentation. Also, the research of Aun˜on-Calles et al.20 indicated that the main metabolite of oleuropein, HT, is nongenotoxic and nonmutagenic at concentrations that far exceed those attainable after intake. Antigenotoxic properties of OL and its compounds have been extensively explored, and several proposed mechanisms have been addressed in the literature. Different inducers of oxidative DNA damage were used

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FIGURE 38.1 Oxidation of phenolic compounds with catechol moiety identified within leaves of Olea europaea L. in the presence of Fe31 and production of O2G2 (R2 is the OH group in hydroxytyrosol or elenolic acid group in oleuropein aglycone).

in these tests, with hydrogen peroxide being the most commonly used chemical for the production of ROS via Fenton’s reaction and causing oxidative DNA damage.76 Even xenobiotics and certain hormones were used to affect DNA integrity, and to evaluate the antigenotoxic effects of OL.25,42,76 OLE has exhibited protective effects against induced H2O2-oxidative DNA damage but also from hormone-induced oxidative DNA damage.25,28,42 Oleuropein was also found to decrease H2O2-induced DNA damage.23,28 Four different mechanisms were proposed for the antigenotoxic effects of OL and its compounds: ROS scavenging, chelation of metals, induction of antioxidant enzymes, and stimulation of DNA repair.25,28,42,49 In these studies, different protocols, pretreatment and posttreatment, with olive compounds were used and their administration exhibited DNA protective effects under both experimental conditions. In pretreatment protocol, cells preincubated with olive compounds were able to increase the antioxidant capacity by stimulating the antioxidant enzymes that help maintain redox homeostasis when exposed to oxidants. This makes them more resistant to genotoxic effects. Abo Ghanema and Sadek,77 and the study of El-Damrawy78 showed that OLE can significantly increase the activity of antioxidant enzymes in the blood. Further, other studies found that polyphenolic substances, such as oleuropein, can increase the gene expression of antioxidant enzymes superoxide dismutase and catalase.79 Our study with thyroxine-induced DNA damage had a better protective outcome in cells preincubated with OLE because L-thyroxine causes oxidative stress mainly by lowering the antioxidant capacity of cells.42 A better outcome in pretreatment can be also be explained by another possible mechanism, the ability of OL compounds acting as a chelating agent that binds intracellular iron and preventing redox reactions. Melidou et al.49 showed that a short incubation of flavonoids before exposure to H2O2 can protect nuclear DNA and ascribed protective effect to the chelating abilities. Their study showed a direct correlation of metal-chelating abilities with DNA protective effects. They excluded other flavonoid-induced mechanisms of protection, for example, altered gene expression, because a period of exposure to flavonoids was very short (15 min). An important feature that could also enable the antigenotoxic effect in pretreatment is its ability of flavonoids to bind to DNA via intercalation, which would be able to protect DNA from the effects of ROS.72

Conversely, in the study where oxidative DNA damage was caused by redox-active catechol-adrenaline or, the H2O2 application of OLE in posttreatment was more effective in reducing DNA damage than in pretreatment.25 This effect can be explained by the fact that in posttreatment the antigenotoxic effect is a consequence of OL acting at intervention level, mainly as a free radical scavenger. The scavenging ability of OL can be attributed to its phenolic constituents.29 A second possible mechanism by which OL compounds could decrease the level of H2O2 and adrenaline-induced DNA damage in posttreatment could be by stimulating the mechanisms of DNA repair as we reported earlier in our research.25 However, this mechanism of OL action is less probable due to experimental conditions used, where cells were incubated for a short time, only 30 min.25 This period is not long enough to alter gene expression since Chiaramonte et al.80 showed that significant DNA damage repair occurred within 1 h after exposure to an oxidative agent. All the abovementioned effects of OLE in cells are described mainly in in vitro studies, and the exact mechanisms of potential genotoxicity and their ability to protect mammalian cells against oxidative DNA damage in vivo are still poorly understood and this topic clearly requires further studies.

38.4 Chelation therapy and olive leaf Chelation therapy is commonly used in clinical treatment for the removal of lead (Pb) and cadmium (Cd) in exposed individuals with toxic metals burden above intervention limits.81 Cadmium and lead are prominent environmental contaminants with long-lasting effects in the body, inducing renal, neuronal as well as cardiovascular toxicity.82,83 Oxidative damage has been strongly implicated in the mechanisms of Pb-induced toxicity.81,84 Toxic metals are favoring enhanced ROS production and disruption of antioxidant balance in cells leading to the damage of cellular components such as lipids, proteins, and DNA.85,86 Previous studies demonstrated elevated DNA damage following Pb exposure.84,87 Ethylenediaminetetraacetic acid (EDTA) is the most commonly used agent for the removal of lead from cells. EDTA can bind toxic metals and create stable complexes with Pb and Cd that are mobilized from cells and eliminated in vivo via urine.81 Besides removing lead, EDTA

Olive leaf, DNA damage and chelation therapy Chapter | 38

is able to reduce ROS production in cells and the increase of total antioxidant capacity in the blood.81,88 Namely, because of its relative lack of specificity, EDTA chelation results with mobilization of not only heavy metals, but also of essential metals such as zinc, copper, iron, cobalt, and manganese. Decreasing metal load in cells should provide benefits in terms of reduction of metal-mediated oxidative stress. That is the reason why the effect of chelation was explored in humans with various oxidative stress-linked pathologies but produced conflicting results.89
91 The study by Roussel et al.88 demonstrated that EDTA chelation therapy can exert in vivo antioxidant protection against oxidative damage to the DNA and lipids in healthy humans. Similarly, our study on Pbexposed workers also demonstrated that the administration of CaNa2EDTA chelation treatment was able to decrease the levels of DNA damage in peripheral blood lymphocytes.81 We proposed that chelation therapy improved oxidant/antioxidant balance in the cells by decreasing metal-dependent formation of ROS and preventing biomolecule degradation. It was also previously shown that uptake of certain natural compounds such as flavonoids can provide protection from the lead-induced damage and helps to restore the imbalanced prooxidant/ antioxidant ratio.92 There is a common practice to add antioxidants to chelation treatment, as an adjuvant of the treatment.93 Guided by this, we explored the possible combination of EDTA chelation treatment with the effect of a natural antioxidant such as dry OLE, against the oxidative DNA damage caused by lead exposure.94 Unexpectedly, when the lymphocytes of Pb-exposed workers were incubated with OLE in vitro, the increase of

463

DNA damage was observed, indicating it is prooxidant rather than antioxidant effect (Fig. 38.2). After the patients followed a standard 5-day chelation treatment, the entire experiment was repeated and the levels of DNA damage were measured again. Although the overall level of damage was decreased after CaNa2EDTA chelation, when OLE was added to the cells, the DNA fragmentation was elevated again. A similar effect was previously observed in the combination of EDTA chelation with vitamin C, where the addition of the vitamin C to the standard chelation cocktail produced an increase of oxidative damage to the DNA, lipids, and proteins.95 Namely, it was shown that the addition of high doses of vitamin C to the standard EDTA chelation therapy can produce prooxidant effects while in the absence of ascorbate, there were no signs of oxidative damage.95 The effects we observed with OLE producing damage in cells of Pb-exposed subjects can be explained by the fact that the elevated concentration of metals in Pb-exposed cells is a suitable environment for the molecules such as flavonoids and vitamin C to be able to autoxidize.96,97 It is well known that Pb interferes with iron metabolism, and one of the molecular toxic effects of Pb is ionic displacement. Studies have shown that Pb is readily able to substitute other bivalent cations such as Fe21, increasing their contents in the serum.92 Indeed, our previous analyses of the serum trace elements in the group of Pbexposed workers confirmed elevated trace elements as well as an increased level of Fe among others.84 Excess iron in the cell is known to catalyze the Fenton reaction and cause the generation of ROS, and polyphenols and flavonoids are particularly known to exhibit prooxidant

FIGURE 38.2 The example of cell preparation of Pb-exposed worker before chelation therapy: non-treated cells (A) and DOLE-treated cells of the same subject (B) showing increase of DNA migration after DOLE treatment, by comet assay. DOLE, Dry olive leaf extract. Taken from our paper: ˇ ˇ ˇ ´ Cabarkapa Cabarkapa A, Dekanski D, Zivkovi c´ L, MilanovicM, Bajic´ V, Topalovic´ D, Giampieri F, Gasparrini M, Battino M, Spremo-Potparevic´ B. Unexpected effect of dry olive leaf extract on the level of DNA damage in lymphocytes of lead intoxicated workers, before and after CaNa2EDTA chelation therapy. Food Chem Toxicol. 2017;106(Pt B):616
623.

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activity in the presence of free iron and copper.97 The oxidation mechanisms of flavonoids and OL compounds in the presence of transition metals such as iron have been described in detail previously in this chapter. It seems plausible that in the presence of increased free iron in the serum of workers exposed to Pb, OLE could have exhibited prooxidant effects by the formation of metal
flavonoid complexes with Fe. In support of this, Andjelkovi´c et al.55 previously described that HT, the main metabolite of OL, possesses strong iron-binding properties. Moreover, a supplementation regimen with HT was shown to significantly reduce iron stores.98 These flavonoid
metal complexes can be formed in the nucleus, in the vicinity of a DNA molecule. Further, these flavonoid
metal complexes bind to DNA via intercalation, which would be able to affect DNA integrity.65 Thus the application of flavonoids such as OLE in lead intoxicated individuals should be carefully assessed considering the demonstrated prooxidant and genotoxic effects. It is most probable that the addition of the flavonoid-rich dry OLE to the EDTA-chelating agent would not provide an additional advantage to the effect of chelation, at least not in terms of DNA protection from oxidative damage. Moreover, these studies indicate that when treating metal
associated pathologies or heavy metal
induced toxicity, the concomitant use of flavonoids should be very carefully considered.

Mini-dictionary of terms DNA damage

Single-strand breaks (SSBs) Botanical extracts Reactive oxygen species Oxidative stress

Free radical scavenger Transition metal Heavy metals

Chelation therapy

an alteration in the chemical structure of DNA, such as a strand break, a base missing from the DNA backbone, or a chemically changed base that introduce deviations from its normal, intact structure discontinuities (breaks) in one strand of the DNA double helix concentrated plant materials, which have been extracted through a variety of methods reactive molecules and free radicals derived from molecular oxygen an imbalance between free radicals production and antioxidant defenses that detoxify the reactive intermediates a chemical substance that removes or deactivates free radicals and unwanted reaction products by donating an electron metallic element that has variable valency, the atom of which has a partially filled d subshell any metallic chemical element with high atomic mass and relatively high densities, which are toxic even at very low concentrations a medical procedure with the administration of chelating agents to mobilize and remove heavy metals from the body

Chelator

Labile iron pool (LIP)

a binding agent that suppresses chemical activity by forming chelates-bonding of ions and molecules to metal ions a cellular pool of chelatable and redox-active iron, which is transitory and made up of iron ions bound to low-affinity ligands

Comparisons of olive oils with other edible oils Edible oils are sources of dietary fats that play an essential role in the body, satisfying nutritional needs, growth and are necessary for proper functioning of brain and nerve system as well as the endocrine environment. Beyond this role, they can be very rich sources of micronutrients and bioactive substances such as alkaloids, carotenoids, and polyphenols. Since edible oils are one of the main components of our diet, it is important to compare their biological effects. There is a variety of different edible oils on the market and most common vegetable oils include soybean oil, sunflower, corn, olive, and coconut oils. Each of these oils has different characteristics and most of the recommendations on adding certain oils to the diet are based on their total fat content (including saturated, polyunsaturated, monounsaturated, and trans fat) to help support a healthy body weight and reduce the risk of disease. However, secondary metabolites found in edible oils need to be considered as important dietary agents. Especially, the involvement of polyphenols and related compounds has become a hot topic in human nutrition research, based on the evidence from epidemiological and human intervention studies showing the protective effects of various (poly)phenol-rich foods against neurodegenerative diseases, cancer, and cardiovascular disease.99 Recently, a database named Phenol-Explorer was developed, providing information on the nature and quantities of polyphenols found in the main foods consumed with our diet, including 10 different edible oils.100 To determine polyphenol intake in populations and study their effects on health, it is essential to have thorough data on their content in foods.101 Searching this database on edible oils and comparing olive oil to sunflower oil, corn oil, soy oil, rapeseed oil, and sesame seed oil components, we can observe that olive oil has by far the largest variety of different polyphenol components in one oil compared to all other oils. While components such as hydroxycinnamic and hydroxybenzoic acids can be found in a variety of edible oils, flavones such as apigenin and luteolin and tyrosols, such as hydroxytyrosol and tyrosol, and lignan pinoresinol can be found only in olive oil. The health benefits of polyphenols from edible oils were ascribed to their antioxidant activity, preventing mitochondrial misfunctioning and oxidative stress, both of them having a

Olive leaf, DNA damage and chelation therapy Chapter | 38

relevant role in age-related pathological alterations. However, it should be noted that the concentrations at which most of the polyphenols exhibit such antioxidant activity are unlikely to be achieved in vivo as many of them have very limited bioavailability and are extensively metabolized.99 As previously mentioned in this chapter, a pharmacokinetic study by Garcia-Villalba et al.14 showed that out of all olive oil metabolites, HT, oleuropein aglycone, elenolic acid, luteolin, and apigenin can be found in plasma and urine within a few hours following the ingestion. These are the exact compounds that are ascribed to the beneficial effects of olive oil in most studies. It was shown that diets with polyphenol-rich edible oils after prolonged use may affect the heart physiology, bone density as well as brain health.102
104 These animal studies provided evidence of edible oils (virgin olive oil and fish oil) having especial importance in prevention of certain pathologies and showed how edible oils varying in lipid profile and polyphenol content affected the heart and the brain during aging. Moreover, the latest studies observed that animals fed for life on virgin olive oil, sunflower oil, or fish oil had different life spans, with longer life span observed in animals fed on virgin olive oil or fish oil than those fed on sunflower oil.105 This effect was attributed to the ability of oil components to affect mitochondrial function, oxidative stress, and telomere length.104,106 Gene expression changes related to aging in animals fed with virgin olive oil or fish oil were related mostly to improved mitochondrial function and oxidative stress pathways, accompanied by cell cycle control and increased telomere length, while sunflower oil-rich diet showed no gene expression changes related to age.106 Also, study by Santos-Gonza´lez et al.107 demonstrated how diet rich with olive oil after 24 months consumption can lead to a markedly different plasma proteome in comparison to sunflower oil
fed animals. These studies explained that olive oil consumption provides mitochondrial maintaining turnover probably through increased biogenesis of new mitochondria and elimination of damaged mitochondria by increasing autophagy, and those olive oil components might be also able to induce the corresponding antioxidant systems to counteract age-related oxidative stress.106,108 It is certain that polyphenols from olive oil may affect several key metabolic pathways and promising therapeutic approaches require further research on the exact mechanisms and polyphenols involved. Blending of different edible oils with olive oil could be a good strategy combining the potency and offering the proper balance of fatty acids and antioxidants to enhance their oxidative and thermal stability. This may lead to replacement of commonly consumed oils and the development of next-generation functional foods to achieve dietary recommendations, as well as for slowing down the mechanisms contributing to normal and pathological

465

aging, including oxidative stress, inflammation, and vascular dysfunction.

Implications for human health and disease prevention Many single components of olive (O. europaea L.) are known to have positive effects on health, reducing oxidative stress, inflammation, and other important risk factors of age-related diseases. The research on pharmacological properties of the bioactive components of this medicinal treasure is very active and could lead to the formulation of functional foods and nutraceuticals. A clinical trial has reported that the daily consumption (50 mL) of oleuropein-rich extra-virgin olive oil increased the total antioxidant capacity in plasma of healthy elderly people.109 The results of the same study also show a significant increase in catalase in erythrocytes and a decrease in superoxide dismutase and glutathione peroxidase activity.109 Metaanalysis showed that consumption of olive oil rich in polyphenols and monounsaturated fat (MUFA) reduces all-cause mortality (11%), cardiovascular mortality (12%), cardiovascular events (9%), and stroke.110 The same study showed that significant associations could only be found between higher intakes of olive oil and reduced risks of all-cause mortality, cardiovascular events, and stroke, respectively, while the group only on MUFA diet did not reveal any significant risk reduction.110 This indicated the possibility that polyphenol components in olive oil and not MUFA content are of the crucial significance for the observed effect. Indeed, the PREDIMED trial showed that olive oil consumption, specifically the extra-virgin variety, is associated with reduced risks of cardiovascular disease and mortality.111 Further, Tresserra-Rimbau et al.112 used data from the PREDIMED study and found that a relative reduction in all-cause mortality is directly related to total polyphenol intake from olive oil, and those who reported a high polyphenol intake, especially of stilbenes and lignans, showed a reduced risk of overall mortality compared to those with lower intakes. There are new findings showing that even in lower amounts, which are usually attained in the diet, polyphenols may exert bioactive properties by affecting the mechanisms in the cells beyond the classic antioxidant scavenging, such as modulation of intracellular signaling cascades.113,114 Apart from olive oil, other olive products such as OL also contain large amounts of the same polyphenols found in the olive oil. Phenolic compounds from olive mill wastewater extract are already suggested for inclusion into food and beverages due to their biological effects.115 OL contains many of the same phenolics as the olive oil but in much higher concentration.9,116 The main active compound of olive oil as well as the OL is

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oleuropein, but both of them also contain flavonoids such as luteolin, apigenin, and quercetin, tannins and caffeic acid.36 Good absorption and bioavailability of active constituents enhance the beneficial properties of OL extract (OLE).46 Although less explored and much less consumed than olive oil, oleuropein-rich OLE has potential to be utilized as a new functional food material with antioxidant action, considering its numerous health benefits and costeffectiveness. It is plausible that its antioxidative compounds act by dual functions, as they can comprise both iron-binding and free radical scavenging properties in the same molecules. The consumption of such polyphenolrich foods throughout life holds a potential to limit development of age-related dysfunctions caused by oxidative damage. However, the therapeutic and pharmacological potential of OL still remains to be fully translated in humans and tested in different clinical trial conditions and needs to be evaluated with caution until rigorous controlled trials determine whether OL and/or its in vivo metabolites have the same benefits and efficacy as they showed in animals and in vitro studies.

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377. 37. Luo S, Jiang X, Jia L, et al. In vivo and in vitro antioxidant activities of methanol extracts from olive leaves on Caenorhabditis elegans. Molecules. 2019;24(4):704. 38. Dekanski D, Selakovi´c V, Piperski V, Radulovi´c Z, Koreni´c A, Radenovi´c L. Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils. Phytomedicine. 2011;18 (13):1137
1143. ¨ ztezcan S, Do˘gru-Abbaso˘glu S, Bingu¨l I, Ye¸sil-Mizrak 39. C ¸ oban J, O K, Uysal M. Olive leaf extract decreases age-induced oxidative stress in major organs of aged rats. Geriatr Gerontol Int. 2014;14 (4):996
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1600. Welch KD, Davis TZ, Van Eden ME, Aust SD. Deleterious ironmediated oxidation of biomolecules. Free Radic Biol Med. 2002;32 (7):577
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92. Petrat F, Rauen U, de Groot H. Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology. 1999;29(4):1171
1179. Reilly CA, Aust SD. Biological oxidations catalyzed by iron released from ferritin. In: Rhodes CJ, ed. Toxicology of the Human Environment. The Critical Role of Free Radicals. London: Taylor and Francis; 2000:155
190. Galaris D, Barbouti A, Pantopoulos K. Iron homeostasis and oxidative stress: an intimate relationship. Biochim Biophys Acta—Mol Cell Res. 2019;1866(12):118535. Andjelkovi´c M, Van Camp J, De Meulenaer B, et al. Iron-chelation properties of phenolic acids bearing catechol and galloylgroups. Food Chem. 2006;8(1):23
31. Kitsati N, Fokas D, Ouzouni MD, Mantzaris MD, Barbouti A, Galaris D. Lipophilic caffeic acid derivatives protect cells against H2O2-induced DNA damage by chelating intracellular labile iron. J Agric Food Chem. 2012;60:7873
7879. Kitsati N, Mantzaris MD, Galaris D. Hydroxytyrosol inhibits hydrogen peroxide-induced apoptotic signaling via labile iron chelation. Redox Biol. 2016;10:233
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253. Seibert H, Maser E, Schweda K, Seibert S, Gu¨lden M. Cytoprotective activity against peroxide-induced oxidative damage and cytotoxicity of flavonoids in C6 rat glioma cells. Food Chem Toxicol. 2011;49:2398
2407. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem. 2004;266:37
56. Lu AL, Li X, Gu Y, Wright PM, Chang DY. Repair of oxidative DNA damage: mechanisms and functions. Cell Biochem Biophys. 2001;35(2):141
170. Cotelle N. Role of flavonoids in oxidative stress. Curr Top Med Chem. 2001;1:569
590. Mijatovic SA, Timotijevic GS, Miljkovic DM, et al. Multiple antimelanoma potential of dry olive leaf extract. Int J Cancer. 2011;128:1955
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64. Boots AW, Li H, Schins PF, et al. The quercetin paradox. Toxicol Appl Pharmacol. 2007;222:89
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copper(II) complex and DNA with the use of the neutral red dye fluorophor probe. Anal Chim Acta. 2007;584(1):19
27. 67. Kasprzak MM, Erxleben A, Ochocki J. Properties and applications of flavonoid metal complexes. RSC Adv. 2015;5:45853
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115. 69. El Amrani FBA, Perello L, Real JA, et al. Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato) (methanol) iron (III) complex. J Inorg Biochem. 2006;100:1208
1218. 70. Zhou J, Wang L, Wang J, Tang N. Synthesis, characterization, antioxidative and antitumor activities of solid quercetin and rare earth (III) complexes. J Inorg Biochem. 2001;83(11):41
48. 71. Raza A, Xu X, Xia L, Xia C, Tang J, Ouyang Z. Quercetin-iron complex: synthesis, characterization, antioxidant, DNA binding, DNA cleavage, and antibacterial activity studies. J Fluoresc. 2016;26:2023
2031. 72. Kanakis P, Tarantilis A, Polissiou MG, Diamantoglou S, TajmirRiahi HA. DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin. J Biomol Struct Dyn. 2005;22(6):719
724. 73. El Hajji H, Nkhili E, Tomao V, Dangles O. Interactions of quercetin with iron and copper ions: complexation and autoxidation. Free Radic Res. 2006;40:303
320. 74. Yen GC, Duh PD, Tsai HL, Huang SL. Pro-oxidative properties of flavonoids in human lymphocytes. Biosci Biotechnol Biochem. 2003;67:1215
1222. 75. Xiao L, Luo G, Tang Y, Yao P. Quercetin and iron metabolism: what we know and what we need to know. Food Chem Toxicol. 2018;114:190
203. 76. Turkez H, Togar B, Polat E. Olive leaf extract modulates permethrin induced genetic and oxidative damage in rats. Cytotechnology. 2012;64(4):459
464. 77. Abo Ghanema II, Sadek KM. Olive leaves extract restored the antioxidant perturbations in red blood cells hemolysate in streptozotocin induced diabetic rats. World Acad Sci Eng Technol. 2012;64:159
165. 78. El-Damrawy SZ. Alleviate the oxidative stress in aged rabbit bucks by using olive leave extract. Egypt Poult Sci. 2011;31:737
744. 79. Masella R, Vari R, D’Archivio M, et al. Extra virgin olive oil biophenols inhibit cell-mediated oxidation of LDL by increasing the mRNA transcription of glutathione-related enzymes. J Nutr. 2004;134:785
791. 80. Chiaramonte R, Bartolini E, Riso P, et al. Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes. J Cell Biochem. 2001;82:437
444. ˇ ˇ 81. Cabarkapa A, Borozan S, Zivkovi´ c L, et al. CaNa2EDTA chelation attenuates cell damage in workers exposed to lead-a pilot study. Chem Biol Int. 2015;242:171
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813. Ercal N, Gurer-Orhan H, Aykin-Burns N. Toxic metals and oxidative stress. Part I: Mechanisms involved in metal-induced oxidative damage. Curr Top Med Chem. 2001;1(6):529
539. Loyd DR, Philips DH. Oxidative DNA damage mediated by copper (II), iron(II) and nickel(II) Fenton reactions: evidence for sitespecific mechanisms in the formation of double-strand breaks, 8hydroxydeoxyguanosine and putative intrastrand cross-links. Mutat Res. 1999;424:23
36. Manikantan P, Balachandar V, Sasikala K. DNA damage in workers occupationally exposed to lead, using comet assay. Inst Biol J. 2010;2(1):103
110. Roussel AM, Hininger-Favier I, Waters RS, Osman M, Fernholz K, Anderson RA. EDTA chelation therapy, without added vitamin C, decreases oxidative DNA damage and lipid peroxidation. Altern Med Rev. 2009;14(1):56
61. Knudtson ML, Wyse DG, Galbraith PD, et al. Chelation therapy for ischemic heart disease: a randomized controlled trial. JAMA. 2002;287:481
486. Fulgenzi A, De Giuseppe R, Bamonti F, Ferrero ME. Improvement of oxidative and metabolic parameters by cellfood administration in patients affected by neurodegenerative diseases on chelation treatment. Biomed Res Int. 2014;2014:281510. Born T, Kontoghiorghe CN, Spyrou A, Kolnagou A, Kontoghiorghes GJ. EDTA chelation reappraisal following new clinical trials and regular use in millions of patients: review of preliminary findings and risk/benefit assessment. Toxicol Mech Methods. 2013;23(1):11
17. Flora G, Gupta D, Tiwari A. Toxicity of lead: a review with recent updates. Interdiscip Toxicol. 2012;5:47
58. Rozema TC. The protocol for safe and effective administration of EDTA and other chelating agents for vascular disease, degenerative disease and metal toxicity. J Adv Med. 1997;10:11
17. ˇ ˇ Cabarkapa A, Dekanski D, Zivkovi´ c L, et al. Unexpected effect of dry olive leaf extract on the level of DNA damage in lymphocytes of lead intoxicated workers, before and after CaNa2EDTA chelation therapy. Food Chem Toxicol. 2017;106(Pt B):616
623. Hininger I, Waters R, Osman M, et al. Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant benefits of therapy. Free Radic Biol Med. 2005;38(12):1565
1570. Miller DM, Buettner GR, Aust SD. Transition metals as catalysts of “autoxidation” reactions. Free Radic Biol Med. 1990;8:95
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1557. Navarro-Hortal MD, Ramı´rez-Tortosa CL, Varela-Lo´pez A, et al. Heart histopathology and mitochondrial ultrastructure in aged rats fed for 24 months on different unsaturated fats (virgin olive oil, sunflower oil or fish oil) and affected by different longevity. Nutrients. 2019;11:2390. Varela-Lo´pez A, Ochoa JJ, Llamas-Elvira JM, et al. Loss of bone mineral density associated with age in male rats fed on sunflower oil is avoided by virgin olive oil intake or coenzyme Q supplementation. Int J Mol Sci. 2017;18:1397. Ramirez-Tortosa CL, Varela-Lo´pez A, Navarro-Hortal MD, et al. Longevity and cause of death in male Wistar rats fed lifelong diets based on virgin olive oil, sunflower oil or fish oil. J Gerontol A Biol Sci Med Sci. 2020;75(3):442
451. Varela-Lopez A, Pe´rez-Lo´pez MP, Ramirez-Tortosa CL, et al. Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver

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358. Bullon P, Battino M, Varela-Lopez A, et al. Diets based on virgin olive oil or fish oil but not on sunflower oil prevent age-related alveolar bone resorption by mitochondrial-related mechanisms. PLoS One. 2013;8(9):e74234. Oliveras-Lo´pez MJ, Molina JJM, Mir MV, Rey EF, Martı´n F, de la Serrana HL-G. Extra virgin olive oil (EVOO) consumption and antioxidant status in healthy institutionalized elderly humans. Arch Gerontol Geriatr. 2013;57(2):234
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Chapter 39

Olive polyphenols and chronic alcohol protection Carla Petrella1, Giampiero Ferraguti2, Luigi Tarani3, George N. Chaldakov4, Mauro Ceccanti5, Antonio Greco6, Massimo Ralli6 and Marco Fiore1,6 1

Institute of Biochemistry and Cell Biology, IBBC-CNR, Rome, Italy, 2Department of Experimental Medicine, Sapienza University of Rome,

Rome, Italy, 3Department of Pediatrics, Medical Faculty, Sapienza University of Rome, Rome, Italy, 4Department of Anatomy and Cell Biology, Medical University, Varna, Bulgaria, 5Centro Riferimento Alcologico Regione Lazio, ASL Roma 1, Rome, Italy, 6Department of Sense Organs, Sapienza University of Rome, Rome, Italy

Abbreviations ADH ALDH2 BDNF CHD CYP2E1 DNA ELISA FAS FASD FORD FORT NAD NGF

alcohol dehydrogenase aldehyde dehydrogenase brain-derived neurotrophic factor coronary heart disease cytochrome P450 2E1 deoxyribonucleic acid enzyme-linked immunosorbent assay fetal alcohol syndrome fetal alcohol spectrum disorders free oxygen radicals defense free oxygen radicals test nicotinamide adenine dinucleotide nerve growth factor

39.1 Alcohol consumption: effects and mechanisms Chronic alcohol consumption is a social and public health problem in many countries. Scientific research constantly focuses on finding solutions not only for the management of psychological dependence but also for the treatment of the numerous pathologies deriving from alcohol abuse1
3 and from abstinence after sudden interruption.4
6 What emerges from numerous scientific studies is that the damage related to alcohol intake is not exclusively induced by its chronic consumption. Indeed, it has been widely demonstrated that, for example, occasional alcohol intake during pregnancy is responsible for the onset of various fetal disorders related to alcohol exposure, collectively called the “spectrum of fetal-alcoholic disorders” (FASD).7,8 Among these, fetal alcohol syndrome (FAS) is

classified as the most serious permanent disability that occurs in the fetus exposed, during intrauterine life, to alcohol.9,10 Children with FAS exhibit specific physical peculiarities, especially of the head and face. Characteristic signs in the face are folds at the corners of the eyes, narrow eye slits, strabismus, short and flat nose, thin and vermillion upper lip, elongated and flat nasolabial sulcus, long and narrow forehead, and maxillary and mandibular hypoplasia.11
13 Even the skeletal system suffers the consequences of exposure to alcohol. Growth retardation was observed as evidenced by lower than average height, body weight, and head circumference values. Another clinical manifestation commonly associated with FAS is the presence of a variable degree of microcephaly, that is, a reduced circumference of the skull, which also represents the safest evidence of the presence of brain damage. The other anomalies recorded in the brain are related to the reduction in the size of the cerebral and cerebellar vault, the basal ganglia, and the diencephalon.14,15 Cardiac malformations are also present, particularly represented by ventricular septal defects.16 A high percentage of patients, exposed to alcohol during gestation, which exhibit the typical characteristics of FAS in growth and face, present rather significant behavioral and cognitive deficits. Among the neurological and neuropsychological disorders that compose the clinical picture of FAS, we find sleep disturbance and reduced sucking reflex, delayed mental development, intellectual deficit, attention and memory disorders, fine motor disorders, hyperactivity and impulsivity, and speech and hearing disorders.17,18 Behavioral and cognitive anomalies can be detected through age-specific psychometric tests, useful

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00003-1 © 2021 Elsevier Inc. All rights reserved.

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not only to establish the diagnosis but also to organize an ad hoc treatment plan, aimed at reducing, controlling, and in less serious cases to recover the observed deficits.19 It has been also shown that even the consumption of alcohol by the future father during the conception period can negatively influence the development of the unborn child.20 The mechanism by which paternal alcohol consumption influences the development of the unborn child has yet to be fully established. However, it has been hypothesized that this occurs concerning mutations in paternal deoxyribonucleic acid (DNA) due to alcohol (direct route) or through epigenetic mechanisms (indirect route).21 Paternal exposure is also capable of inducing greater sensitivity in children to the rewarding effects of alcohol, which could lead to an increased risk of abuse of this substance in adult life.20,22 As for the effects of uncontrolled and continuous alcohol consumption on the body, it is known that it can cause various clinical conditions, including cirrhosis of the liver, chronic pancreatitis, epilepsy, polyneuropathy, heart disease, nutritional deficiencies and neuro-behavioral problems.23
27 These morbid conditions are centered in multiple tissue areas targeted by the toxic action of alcohol. At the level of the central nervous system, chronic alcohol intake causes alterations in memory, attention span, working memory, inhibition, and executive functioning.4,6,26,28 The mechanisms that play a role in neurotoxic actions induced by chronic alcoholic treatment have not yet been identified. Several studies have hypothesized that alcohol intake (not only chronic, but also acute, or gestational/preconceptual) may interrupt the synthesis of neurotrophins,29
31 which are a family of proteins that play a crucial role in cognitive function, including learning and memory processes.32
34 Certainly, the alcohol-induced increase in cell death or the reduced cell proliferation in the different regions of the brain related to these functions (hippocampus, extrahippocampal cortex, and in the anterior brain)35,36 play a role in determining these central function deficits. Chronic alcohol consumption causes metabolic and pathological changes in the liver, documented both in humans and in experimental models,27,37 due to the metabolism of ethanol, which occurs mainly in this organ.37 In humans, ethanol is oxidized to acetaldehyde by NAD 1 , mainly through the liver enzyme alcohol dehydrogenase (ADH). When the quantity of ethanol consumed exceeds the catabolic capacities of the dehydrogenases, ethanol is metabolized by other enzymes that are part of the superfamily of the cytochrome P-450, in particular by CYP2E1. The production of superoxide radicals resulting from the activity of the enzyme induces the oxidation of polyunsaturated fatty acids with the production of aldehyde toxicants. Acetaldehyde is a highly unstable compound and rapidly forms free radicals that are highly toxic if not

extinguished by antioxidants.38,39 In human embryos and fetuses,38,40 ADH enzymes are not yet expressed in significant quantities (induction of ADH begins only after birth). As a result, the fetal liver cannot metabolize ethanol or other xenobiotics. In various human fetal tissues, CYP2E1 expression and activity were detected after the start of organogenesis (at about 50 days of gestation). Exposure to ethanol promotes the induction of this enzyme in fetal and adult tissues. CYP2E1 contributes significantly to the oxidative system of microsomal ethanol, and its activity in fetal tissues contributes significantly to the toxicity of maternal consumption of ethanol. These free radicals can damage embryonic neural crest cells and can lead to severe birth defects, mental retardation, and physical abnormalities in newborns.41,42 In chronic alcoholics, prolonged exposure of the kidney and liver to these compounds can lead to serious harm. Acetaldehyde is transformed into acetic acid by aldehyde dehydrogenase (ALDH2). Finally, acetic acid is transformed into acetyl-CoA from acyl-CoA synthase and acetyl-CoA synthase 2 located in the mitochondria. Once formed acetyl-CoA, it enters the normal citric acid cycle. In human adults the excess of acetate and NADH cofactor inhibits the normal aerobic metabolism of the Krebs cycle, moving toward lipid metabolism, with the synthesis of triglycerides, leading to steatosis of the liver.43 These alterations are generally associated with the oxidation process of alcohol and, in particular, with oxidative stress with consequent production of free radicals and lipid peroxidation.39,44 Oxidative stress in cells, derived from alcohol consumption and associated with altered lipid metabolism, also induces morphological and functional alterations (enzymatic inactivation, interference with DNA repair mechanisms, and depletion of antioxidant systems) in the different regions of the brain than in peripheral tissues.38

39.2 Polyphenols: a brief overview Polyphenols45,46 are a structural class of mainly natural, but also synthetic or semisynthetic organic chemicals, characterized by the presence of multiple phenolic groups. Polyphenols are characterized by the presence of multiples of phenolic structural units. The number and characteristics of these phenolic structures are the basis of the unique physical, chemical, and biological (metabolic, toxic, therapeutic, etc.) properties. In nature, polyphenols are produced by the secondary metabolism of plants, where, in relation to the chemical diversity that characterizes them, they play different roles: defense against herbivorous animals (impart unpleasant flavor) and pathogens (phytoalexins), mechanical support (lignin) and barrier against the microbial invasion, attraction for pollinators and the dispersion of the fruit (anthocyanins),

Olive polyphenols and chronic alcohol protection Chapter | 39

growth inhibitors of competing plants. The traditional Mediterranean diet is characterized by a high intake of foods rich in phenols, including extra virgin olive oil, walnuts,47,48 vegetables, fruit, legumes, and whole grains, chocolate, as well as in drinks such as red wine, coffee, and black tea.49
52 Some polyphenols are specific to particular foods, for example, isoflavones in soybeans or flavanones in citrus fruits, while others are found in all vegetable products, such as quercetin present in vegetables, fruit, cereals, legumes, wine, and tea. Generally, however, as in the case of olive oil, there are complex mixtures of polyphenols.53

39.2.1 Polyphenols in human health Despite the presence in nature of many types of polyphenols, characterized by a marked structural and functional variability, in general, the activity of polyphenols can be summarized as follows: G

G

Antioxidant and antiinflammatory: Several studies have demonstrated that flavonoids can inhibit regulatory enzymes or transcription factors important for controlling mediators involved in inflammation.54,55 Anticancer activity: Several experimental studies supported anticancer activities of major polyphenol classes (flavonoids, phenolic acids, lignans, and stilbenes).56,57 The mechanisms of action mainly included the modulation of molecular events and signaling pathways associated with cell survival, proliferation, differentiation, migration, angiogenesis, hormone activities, detoxification enzymes, immune responses, etc. Notably, the anticancer effects of polyphenols varied with cancer types, cell lines, and doses by modulating angiogenesis, apoptosis, inflammation, metastasis, tumor cell proliferation.58

However, clinical trials about the anticancer actions of polyphenols are limited, and not always conclusive. Therefore the use of these polyphenols in cancer treatment should be cautious.59

39.2.2 Polyphenols in olive oils Extra virgin olive oil is one of the representative products of the tradition and culture of the Mediterranean countries. For years the consumption and marketing of this food has mainly concerned areas with olive growing traditions, such as Spain, Portugal, Greece, Italy, Turkey, Tunisia, Syria, and Morocco; but today olive cultivation is developing above all where the consumption of virgin olive oil has always been very modest as in Australia, Argentina, South Africa, Chile, and Argentina.60 Virgin olive oil is obtained only by mechanical extraction and can be consumed directly, without any further

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physical
chemical refining or rectification treatment. Its chemical composition is represented by a fat-soluble fraction and the components that make up the water-soluble fraction. In the hydrophilic fraction, we find, in particular, the so-called natural antioxidants of extra-virgin olive oils represented by carotenes, tocopherols, and hydrophilic phenolic substances.61
63 These antioxidants are those most correlated with the health-giving properties of extravirgin olive oil. It should also be remembered that tocopherols and carotenoids might be found in other vegetable fats or animal fats, while hydrophilic phenolic substances are present exclusively in olive oil. Many agronomic practices (climatic conditions, cultivar, fruit ripening) used in the production of extra-virgin olive oil influence qualitatively and quantitatively its phenolic composition. On the other hand, even technological aspects can have a significant impact on the composition of extra-virgin olive oil. The collection method, the conservation of the drupes, the pressing can influence the optimal extraction of phenols. In particular, during the pressing process, all the phenolic compounds of an oil originate, making this phase a critical point in the production of quality oil.64,65 The phenolic concentration in extra-virgin olive oil is closely related to the activity of the endogenous enzymatic patrimony in the olive fruit. During the pressing process, there is the activation of the enzyme ßglucosidase contained in the epicarp of the fruit that catalyzes the hydrolysis of oleuropein, demethyloleuropein, and ligustroside contained in the fruit of the olive.66 Extra-virgin olive oil contains in addition to monounsaturated fatty acid at least 36 structurally distinct phenolic compounds. The main phenolic compounds contained in olives and extra virgin olive oil are tyrosol and hydroxytyrosol (which origin both from the antioxidant oleuropein), whereas oleuropein is mostly present in the olive leaves. However, among different extra-virgin olive oils, there is a huge variation in the composition and concentration of phenolic compounds.67,68 Several scientific studies conducted in this regard have highlighted its beneficial effects on health. In fact, many researchers have shown that some compounds, mainly phenolic substances, are related to a long series of positive effects on human health69
71: they intervene as direct antioxidants, lower cholesterol levels and inhibit peroxidation of low-density lipoprotein, slow down tumor growth inhibit some chemical carcinogens, slow down platelet aggregation, and finally carry out hypoallergenic and antiinflammatory activities.72
74

39.2.3 Olive polyphenols and alcohol drinking Uncontrolled and continuous ingestion of alcohol has dramatic consequences on the entire organism. Scientific

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research has constantly focused on finding solutions not only for the management of psychological dependence but also for the treatment of the numerous pathologies resulting from the abuse of alcohol. The oxidative imbalance is one of the most important mechanisms leading to alcohol-induced toxicity. The metabolism of ethanol is closely related to free radicals’ generation and oxidative stress, with the consequence to alter lipids and proteins, inducing cellular damages. Strategies to reduce oxidative stress are believed to be of great use in managing the damage caused by alcohol abuse. In this context the use of polyphenols, as diet supplements, could be of great interest in the prevention/reduction of free radical
induced cell damage. Several studies discussed the relationship between the way of drinking in the Mediterranean diet and longevity.75,76 Moderate red wine consumption, in particular, seems to elicit beneficial cardiovascular effects,77 mainly, but not exclusively, due to the presence of resveratrol, a nonflavonoid phenol, naturally produced by grapes, that shows favorable properties, like antiinflammatory, vessel/endothelium protection, and antioxidant.78 Its beneficial properties could explain at least in part the French paradox,79,80 the paradoxical epidemiological observation that French people have a relatively low incidence of coronary heart disease (CHD) while having a diet relatively rich in saturated fats, in apparent contradiction to the widely held belief that the high consumption of such fats is a risk factor for CHD. Indeed, it was speculated that the greater consumption of wine, mainly the red one, could bring greater antioxidant content, in particular, by resveratrol, capable of playing a protective antioxidant role and, therefore, fundamental for the prevention of cardiovascular diseases. Concerning, in particular, in olive oil polyphenols,81,82 it has been demonstrated that the administration of a blend of pomace polyphenols, in the mouse, increased the levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in crucial areas of the limbic system and olfactory bulbs, which play a key role in learning and memory processes and in the proliferation and migration of endogenous progenitor cells present in the rodent and human brain.81,83
87 Moreover, in the serum of mice treated with the polyphenol’s blend decreased also the levels of reduced glutathione.82 However, another study carried out with a different blend of olive leaf polyphenols containing mostly oleuropein by a different method of extraction elicited a quite toxic response.88 A recent study conducted in a mouse model of alcohol addiction demonstrated that polyphenols supplementation prevented ethanol-related oxidative stress increase.89 Polyphenol’s blend used in the study, derived from a natural standardized olive pulp extract, contained a high percentage of hydroxytyrosol (30%) and other

hydroxytyrosol derivatives (20%). The mixture was administered (per os—oral administration) for 2 months at a dose of 20 mg/kg in alcoholic adult male mice. This was the first study to demonstrate that olive polyphenols’ supplementation in a mouse model of ethanol addiction may confer protection against ethanol-induced oxidative stress by reducing serum-free oxygen radicals but not affecting the free oxygen radicals defense (FORD). Another study investigated the effect of olive polyphenols supplementation containing mostly hydroxytyrosol (50 mg/day for 15 consecutive days) in men affected by alcohol use disorders during withdrawal, by evaluating serum BDNF and NGF, known to be involved in alcohol addiction. BDNF and NGF were measured by enzymelinked immunosorbent assay (ELISA) on days 1, 3, 7, and 15 of the detoxification period.90 Some parameters of oxidative stress were analyzed too as FORD and free oxygen radicals test (FORT). No differences in oxidative status due to polyphenols were found. However, withdrawal elicited the expected increase in BDNF over 2 weeks that was counteracted on day 3 by polyphenols. As for NGF, no effects of polyphenols supplementation were discovered to antagonize the NGF serum elevation during withdrawal. In conclusion, the present data may indicate that monitoring serum BDNF and/or NGF in alcoholics undergoing detoxification could contribute to characterize alcohol dependence profiles to improve recovery processes throughout also antioxidant compounds. Monitoring serum levels of neurotrophins could be important to characterize alcohol dependence profiles to improve recovery processes throughout antioxidant compounds supplementation.

39.3 Conclusion The use of natural substances as remedies to alleviate the symptoms related to various pathologies is becoming increasingly widespread. The growing number of studies in this field provides scientific support for the evidence that the Mediterranean diet is a valid support for the prevention of conditions in which oxidative stress is altered. As for alcohol use disorders,91,92 polyphenols could represent a valid tool to alleviate the physical consequences related to free radicals/reactive oxygen species production (Fig. 39.1). On the other hand, it is necessary to remember that the quantities required to obtain a beneficial effect should be considered “pharmacological” doses since the supplementation obtained exclusively from the diet is obviously not sufficient to significantly counteract already established diseases. Further investigations on polyphenols are absolutely encouraged, and in the near future, they will be able to increase current knowledge not only on the efficacy but also on the mechanism of action of these interesting natural compounds.

Olive polyphenols and chronic alcohol protection Chapter | 39

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FIGURE 39.1 Schematic representations of the beneficial effects of the consumption of extra-virgin olive oil on the damage induced by the abuse of alcoholic beverages.

Acknowledgments Authors thank Regione Lazio, Sapienza University of Rome, ASL-Roma 1, and IBBC-CNR for supporting their study.

Disclaimer Nothing to disclose.

Conflicts of interest All the authors do declare no conflicts of interest.

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Chapter 40

Olive oil diet and amyloidosis: focus on Alzheimer’s disease Elisabetta Lauretti Alzheimer’s Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, United States

Abbreviations AD Aβ NFTs APP MD MCI EVOO CTFα CTFβ CTFγ PS1 APH1 PEN2 BBB NEP IDE MMP P-gp LRP1 PARP1 SIRT1 FOXO ATG LC3B1/2 WNT

Alzheimer’s disease amyloid β neurofibrillary tangles amyloid precursor protein Mediterranean diet mild cognitive impairment extravirgin olive oil C-terminal fragment α C-terminal fragment β C-terminal fragment γ presenilin 1 anterior pharynx defective presenilin enhancer 2 blood
brain barrier neprilysin insulin degrading enzyme matrix metalloproteinases P-glycoprotein low-density lipoprotein receptor
related protein-1 poly(ADP-ribose) polymerase 1 sirtuin 1 forkhead box protein O1 autophagy gene microtubule-associated protein 1/2 light chain 3 beta wingless-related integration site

40.1 Introduction Amyloidosis is a pathological condition associated with the presence of intracellular or extracellular accumulation of amyloid deposits composed by aggregated peptides and proteins. These proteinaceous species are organized into fibrils, which share a common beta-sheet core. Among the human amyloidosis, Alzheimer’s disease (AD) is by far the most common and studied disorder.1 As the most prevalent form of neurodegenerative dementia, AD

constitutes a major health-care burden. Worldwide, the estimated number of people currently living with AD is 44 million, a number that will continue to increase rapidly in coming years due to aging of the baby boom generation.2 Clinically, AD is defined by a noticeable decline of memory and cognitive functions that worsen over a period of years leading to impaired communication skills, confusion, behavioral changes, poor judgment, passiveness, and depression.3 AD neuropathology is characterized by extracellular accumulation of amyloid-β (Aβ) plaques and intracellular aggregation of neurofibrillary tangles (NFTs). NFTs are composed by hyperphosphorylated tau, a microtubule binding protein important for microtubules assembly and stabilization. Aβ plaques instead contain deposits of fibrillary Aβ peptides generated by enzymatic cleavage of the Aβ precursor protein (APP). Aβ peptides are predominantly observed in neocortex and hippocampus (the main cognitive and memory structures of the brain) and in the cerebrovasculature of AD patients4 where their progressive accumulation contributes to several downstream toxic effects, including oxidative stress, neuroinflammation, synaptic loss, and cellular death.4,5 Although the underlying causes leading to Aβ deposits are currently unclear, several lifestyle factors (including diet) have been found to possibly reduce or delay the onset of AD. In this regard, epidemiological data have shown that adherence to the Mediterranean diet (MD) is associated with reduced risk of developing AD and improved memory and cognitive functions.6
9 As the key component of the MD, extravirgin olive oil (EVOO) consumption has been extensively investigated for its multimodal effects on AD pathology. High EVOO intake has been consistently associated with lower risk of developing AD, mild cognitive impairment (MCI), and dementia in older individuals.10 Furthermore, randomized clinical trials showed that long-term intervention with an EVOO-rich

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00013-4 © 2021 Elsevier Inc. All rights reserved.

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diet provides protection against AD resulting in better cognitive performances, especially across fluency and memory tasks, and less MCI as compared to controls.11 In addition, preclinical and in vitro studies have described multiple positive effects of EVOO and its phenolic compounds, oleocanthal and oleuropein aglycon, on APP processing and Aβ clearance.12 The present chapter summarizes the existing evidences supporting EVOO supplementation as potential nonpharmacological approach to delay the onset and/or reduce the severity of AD pathology with particular emphasis on its ability to modulate Aβ peptides neurobiology and pathophysiology.

40.2 Amyloid-β biology and function The Aβ plaques are mainly composed by Aβ 1
40 and 1
42 peptides, derived by proteolytic cleavage of the type 1 transmembrane glycoprotein APP.13 Full-length APP can undergo to nonamyloidogenic and/or amyloidogenic processing (Fig. 40.1). In the nonamyloidogenic pathway, which as the name states does not lead to Aβ production, APP is first cleaved by the α-secretase, resulting in the secreted soluble APP fragment (secreted (s)APPα) and in the membrane-bound C-terminal fragment α (CTFα).14 CTFα is successively processed by an enzymatic complex consisting of presenilin 1 (PS1), nicastrin, anterior pharynx defective (APH1), and presenilin

enhancer 2 (PEN2) called γ-secretase to generate P3 and C-terminal fragment (CTFγ).15 In the amyloidogenic pathway, instead, APP is initially cleaved by β-secretase [mainly β-site APP cleaving enzyme 1 (BACE1) in neurons], with generation of s-APPβ and the membranebound C-terminal fragment β (CTFβ). The C-terminal fragment is finally truncated by γ-secretase complex to give Aβ peptides which aggregate to form monomers, oligomers, fibrils and eventually Aβ plaques.16 Aβ biogenesis is usually counterbalanced by its degradation and active transport across the blood
brain barrier (BBB). The major pathways involved in Aβ catabolism are the autophagy system and several degrading enzymes such as angiotensin converting enzyme, neprilysin (NEP), insulin degrading enzyme (IDE), cathepsin B, plasmin, and matrix metalloproteinases (MMP). Among them, NEP, which levels are decreased in the AD brain, particularly in vulnerable regions such as the hippocampus, is considered one of the most important for the control of Aβ levels. In fact, this enzyme can degrade both monomeric and oligomeric extracytoplasmatic Aβ peptides, and its overexpression consistently reduces Aβ level in vivo in APP transgenic mice.17 Impaired autophagic processes have also been linked to Aβ and to AD pathology.18 Defective autophagy directly influences the levels of both intracellular and extracellular Aβ in vivo and in vitro. Several studies have established that either pharmacological induction of autophagy or genetic overexpression of

FIGURE 40.1 Aβ biogenesis.; Human APP is processed by the nonamyloidogenic and amyloidogenic pathways. In the nonamyloidogenic pathway, APP is cleaved by the α-secretase generating the secreted (s)-APPα and the membrane-bound CTFα. CTFα is successively processed by the γ-secretase [PS1, nicastrin, anterior pharynx defective (APH1), and PEN2] to generate P3 and C-terminal fragment (CTFγ). In the amyloidogenic pathway, APP is cleaved by the β-secretase (BACE1) with generation of s-APPβ and membrane-bound CTFβ. CTFβ is finally truncated by γ-secretase to generate Aβ peptides. APP, Amyloid precursor protein; Aβ, amyloid-βCTFα, C-terminal fragment α; CTFβ, C-terminal fragment β; PEN2, presenilin enhancer 2; PS1, presenilin 1.

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important autophagy genes (ATGs) prevent Aβ accumulation and toxicity by promoting its clearance in several transgenic AD models.19 When secreted into the extracellular space, Aβ can also be transported from the brain into the blood. This transport across the BBB is carrier-[apolipoprotein E (apoE) and P-glycoprotein (P-gp)] or receptor-mediated [advance glycation end product (RAGE) and low-density lipoprotein receptor
related protein 1 (LRP1)] and is very important for the homeostatic regulation of Aβ levels in the CNS.20 Although there are not known physiological function attributed to Aβ peptides, several studies have implied a neuroprotective role for APP. Once synthetized, APP is transported to the synaptic sites where it seems to be involved with synaptogenesis. Moreover, APP can modulate GABAergic synaptic strength via interaction with Cav1.2 L-type calcium channel and consequent alteration of calcium homeostasis.13 In support of these observations, loss of functions studies carried out on APP KO mice have revealed that, although not lethal, genetic deletion of APP is associated with reduced brain weight, locomotor activity, impaired long term potentiation (LTP), Morris water maze task, abnormal GABAergic transmission and reactive gliosis.13

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40.3 Amyloid-β pathophysiology Although Aβ is normally produced and secreted from neurons, there is not Aβ deposition in the brain of young individuals suggesting that under normal conditions, Aβ is efficiently cleared by cells. Aβ species are highaggregation-prone peptides; thus the sustained elevation of Aβ levels due to either increase in Aβ production or decrease in Aβ degradation is the primary reason for Aβ accumulation in the AD brain. There are three distinct forms and steps in the Aβ aggregation process. Aβ peptides first aggregates as monomers, then these monomers form Aβ soluble oligomers which gradually organize into insoluble antiparallel-β-plated sheets of filaments and amyloid plaques. Which one of these forms is the most toxic is still a topic of intense debate. While clinical trials have shown that removal of Aβ plaques is not enough to rescue cognitive function in AD, recent research proposes the Aβ oligomeric state as the main culprit for neuronal damage.19 Soluble Aβ oligomers have been reported to exert their neurotoxicity through several mechanisms (Fig. 40.2). Aβ40
42 was shown to insert into the plasma membrane and alter its integrity by forming pores or channels, thus increasing membrane permeability and disrupting calcium homeostasis. Both the aspects are crucial

FIGURE 40.2 Aβ toxicity in Alzheimer’s disease.; Soluble Aβ oligomers can interact with multiple receptors activating downstream pathways leading to WNT signaling inhibition, synaptic and LTP dysfunction, ROS production and apoptosis. Alternatively, Aβ oligomers can insert into the plasma membrane and create ion channels or pores that result in increased membrane permeability and disruption of calcium homeostasis with consequent alteration of the normal synaptic activity. Finally, Aβ oligomers cause inflammatory response by the activation of both microglia and astrocytes. Aβ, Amyloid-β WNT, wingless-related integration site.

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for normal synaptic function and plasticity.21 Other studies have also shown that Aβ oligomers can promote rapid neuronal depolarization interacting with the NMDAR signaling pathway. Moreover, Aβ oligomers were shown to bind to the cellular prion protein, the nerve growth factor receptor, and to the frizzled receptor to induce synaptic dysfunction, neuronal cell death, and inhibition of the Wnt signaling pathway, respectively (Fig. 40.2).22 Finally, increased Aβ levels promote neuroinflammation, another constant feature of the AD pathology, by inducing proinflammatory cytokines production and release by microglia and astrocytes.22

40.4 Impact of extravirgin olive oil on amyloid-β pathology Long-term EVOO intake is associated with lower risk of developing AD, MCI, and dementia in the elderly,10 and recent clinical trials suggested that supplementation with an EVOO-rich diet provides protection against AD and ameliorates cognitive performances.11 In vitro and in vivo studies in animal models of the disease support the epidemiological reports and shed light into the multiple mechanisms by which EVOO exerts its therapeutic effect.12, 23 In fact, chronic consumption of EVOO seems to be able not only to reduce the symptoms of AD but also to delay or prevent its onset. While EVOO can modify several aspects of AD pathology such as tau, inflammation, synaptic plasticity, and neurogenesis, it can also specifically impact Aβ neurobiology.12,23,24 Collectively, data available strongly suggest that EVOO can act

simultaneously on APP processing, Aβ clearance, and as inhibitor of Aβ peptide aggregation in vitro and in vivo (Fig. 40.3).

40.5 Extravirgin olive oil inhibits amyloid-β peptide production and aggregation One of the main EVOO biological activities is the modulation of Aβ production. In human APP-expressing HEK695 cells and SKNSH neuroblastoma cells, oleuropein, a polyphenol found in EVOO, enhances the nonamyloidogenic pathway of APP processing in a dosedependent manner, resulting in a reduction in Aβ oligomers release.25 These effects were independent from changes in α-secretase and β-secretase expression but instead were due to the enhanced activity of matrix metallopeptidase 9 (MMP9), which can act as an α-secretase enzyme. Further, several studies in AD transgenic mice (TgSwDI and 5xFAD) have demonstrated that chronic consumption of EVOO is associated with a significant reduction of the APP amyloidogenic processing, in favor of the nonamyloidogenic processing.24
29 In these animal models an EVOO-rich diet resulted in increased sAPPα and reduced sAPPβ levels, which correlated with decreased brain Aβ peptide accumulation.25
27,30 Interestingly, oleuropein and oleocanthal (another natural phenol found in EVOO) may also prevent the aggregation and subsequent neurotoxicity of Aβ peptides. Oleuropein strongly associates with the monomeric form of Aβ40 and the oxidized AβMet35 isoform in three FIGURE 40.3 The potential mechanisms of action underlying the beneficial effects of EVOO and its components.; Experimental evidence provided indicate multiple potential mechanisms of action underlying EVOO beneficial effects that can be summarized into the inhibition of Aβ peptides synthesis, inhibition of Aβ peptides aggregation, promotion of Aβ peptides proteolytic cleavage, promotion of Aβ clearance through the BBB, autophagy induction, and inhibition of oxidative stress. Aβ, Amyloid-β; BBB, blood
brain barrier EVOO, extravirgin olive oil.

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regions involved in Aβ fibrillation suggesting that this polyphenol can also interfere with Aβ peptide aggregation.31,32 Importantly, Rigacci et al. demonstrated that OLE can not only prevent Aβ fibrillation but also remodel preexisting Aβ fibrils, reducing the release of toxic fragment in vitro.33 Finally, oleuropein was also shown to counteract the generation of pE3-Aβ, a specific neurotoxic N-truncated Aβ peptide highly prone to aggregation in SH-SY5Y neuroblastoma cells and in 12-month TgCRND8 mice.34 Although data are less compelling compared to oleuropein, Pitt et al.35 have shown that when oleocanthal is added to neurons before Aβ oligomeric treatment, it promotes conversion of Aβ monomers to oligomers. This transition though does not leads to the formation of fibrillary structures, the oleocanthal-modified peptides show a reduced ability to bind to the cell membrane and synapses, resulting in less synaptic damage.35 Collectively, the available in vitro data on both oleuropein and oleocanthal provide mechanistic support to the observed antiaggregative effect of EVOO in vivo and suggest that long-term consumption of EVOO starting at early age could significantly prevent Aβ pathological aggregation and subsequent neurotoxicity.

40.6 Extravirgin olive oil induction of amyloid-β proteolytic cleavage and blood
brain barrier clearance In AD, the reduction of Aβ clearance efficiency is one of the major causes of Aβ accumulation and consequent development of neuronal and synaptic dysfunctions. As mentioned earlier, proteolytic cleavage of Aβ plays an important role in this process, and for these reasons, upregulation of NEP or IDE has been proposed to protect the brain against Aβ accumulation and cognitive decline.24 Several reports have described that besides its effect on Aβ biogenesis, EVOO-rich diet can also enhances Aβ degradation. Abuznait et al. demonstrated that reduced Aβ burden following oleocanthal administration is associated with increased expression of IDE and NEP in brain microvessels of WT animals treated with 125I-Aβ40.27 In vivo data were also confirmed in vitro, in bEnd3 endothelial cells. When treated with oleocanthal, these cells consistently showed increase in the expression of both enzymes, thus supporting the hypothesis that EVOOinduced Aβ degradation and clearance is mostly due to the induction in the expression of IDE and NEP.24 In addition to reducing total Aβ levels by promoting upregulation of Aβ degrading enzymes, EVOO and oleocanthal enhances Aβ clearance across the BBB.25,28 TgSwDI mice receiving EVOO-enriched diet showed upregulation of the ApoE pathway. The proposed

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mechanism responsible for the improved efficiency of the ApoE-dependent pathway is the significant increase in the expression of ApoE, ABCA1 proteins, and their transcription regulators peroxisome proliferator-activated receptor gamma and liver X receptor in the animal receiving the diet compared to control group. In addition, the same transgenic mice displayed higher expression of two major Aβ transport proteins: P-gp and LRP1 in brain microvessels.25,28 EVOO supplementation is an ideal nonpharmacologic intervention for AD; it is well tolerated, without any side effects reported; thus EVOO could be combined with other therapies without the risk of overlapping toxicities. In this regard the recent study showing the enhance effect of a combination therapy that consists of daily intake of EVOO together with donepezil, a specific inhibitor of acetylcholinesterase is very promising. Using an early intervention approach, EVOO was shown not only to improve donepezil therapeutic effect against Aβ pathology in the 5XFAD mouse model of AD but also to provide additional health benefits such as reduced cerebrovascular deposition of Aβ and brain inflammation.30

40.7 Extravirgin olive oil induction of autophagy activation and amyloid-β proteolytic clearance Impaired autophagic processes can directly influence the levels of both intracellular and extracellular Aβ and have also been linked to Aβ plaques generation.19 Autophagy dysfunction is known to occur in AD pathology where the accumulation of autophagosomes and lysosomes in dystrophic neurites of AD brains has been consistently reported.36 The reduction of beclin-1, a proautophagic factor, is also observed in AD, and PS1 mutation associated with the familial form of AD was shown to impair autophagosome acidification and cathepsin activation.37,38 As one of the major pathways involved in the maintenance of cellular and proteins homeostasis, if the autophagy efficiency is compromised, cells, and in particular neurons, accumulate damaged organelles and harmful protein aggregates such as Aβ and tau, leading to reactive oxygen species (ROS) production and cell death.36 In this regard, several studies have established that either pharmacological induction of autophagy or genetic overexpression of important ATGs prevent Aβ accumulation and toxicity by promoting its clearance in several transgenic AD models.36 Therefore the enhancement of the autophagy-lysosomal system could represent a potential therapeutic approach for AD. Several lines of evidence have demonstrated that EVOO also acts as an autophagy inducer in both cultured

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cells and in transgenic models of AD.25,39
41 One of the first works to show autophagy induction by the EVOO polyphenol oleuropein in AD mice is the study of Grossi. In this study, young and old TgCRND8 mice fed with oleuropein displayed significantly less Aβ deposition and plaques formation together with an increase in the abundance of autophagic vesicles. Induction of autophagy was further supported by upregulation of beclin-1 expression and increased LC3BII/I ratio found in the cortex of the same animals.25 The proposed molecular mechanism underlying this effect was the inhibition of the mTOR signaling pathway, which was downregulated in N2A neuroblastoma cells treated with oleuropein.25 Oleuropein can also modulate mTOR via the activation of the CaCAMKK-AMPK-mTOR axis.41 In SH-SY5Y cultured cells, oleuropein treatment triggers a rapid release of calcium from intracellular stores, which activates CaMKK kinase.41 This kinase, in turn, phosphorylates and activates AMPK, a negative regulator of mTOR activity. The involvement of AMPK in the modulation of mTOR signaling pathway was further confirmed by in vivo studies. Additionally, we have recently shown that besides oleuropein also the consumption of EVOO ameliorate memory, cognitive impairments, amyloid, and tau pathology through the activation of cell autophagy in another mouse model of AD pathology, the 3 3 Tg mice.39 Finally, it has been suggested that oleuropein can modulate autophagy via additional mechanisms. TgCRND8 mice fed with oleuropein present reduced poly (ADP-ribose) polymerase 1 (PARP1) activation.41,42 The activity of this enzyme is linked to the depletion of NAD and inhibition of the deacetylase sirtuin 1 (SIRT1). SIRT1 can induce autophagy either through the deacetylation of many transcription factors including FOXO and p53 or via direct deacetylation of ATGs ATG5, ATG7, and LC3B.43
45 In support of this hypothesis, oleuropein treatment was found to reduce PARP1 activation and promote SIRT1 and beclin-1 expression in N2A cells.41,42

40.8 Conclusion The beneficial effect of EVOO against aging and neurodegenerative diseases has been widely recognized. Epidemiologic studies have consistently suggested that chronic EVOO consumption, as part of the MD, is associated with reduce risk of developing AD, which among other things is characterized by brain amyloidosis. For these reasons, in the past decade, research has been focusing on the anti-Aβ effects of specific EVOO phenolic components and their potential mechanisms of action. Collectively, the multiple mechanisms underlying EVOO beneficial effects on brain amyloidosis can be categorized in the following groups: inhibition of Aβ peptides synthesis, inhibition of Aβ peptides aggregation, promotion of

Aβ peptides proteolytic cleavage, and clearance through the BBB and autophagy induction. It is intuitive that what makes EVOO so interesting from a clinical point of view is its wide spectrum of action and the absence of toxicity and adverse effects. In this regard the recent study showing the enhanced effect of a combination therapy that consists of daily intake of EVOO together with donepezil is very promising. The relevance of this study lays in the fact that EVOO not only enhances donepezil effects but also provides additional benefits and further supports the idea that future AD therapeutic approaches should probably always include EVOO supplementation. Clinical trial in patients with early AD symptoms using olive oil, oleocanthal, or oleuropein alone and/or in combination with classical and FDA-approved AD drugs are needed in order to confirm the positive outcomes obtained in preclinical studies.

Mini-dictionary of terms Amyloidosis is a term that describes a pathological condition characterized by the accumulation of deposits of abnormal proteins in body tissues and organs which impairs their functions. Alzheimer’s disease: A chronic neurodegenerative disorder characterized by toxic deposition of amyloid β peptides and phosphorylated tau protein in the brain leading to neuroinflammation, neuronal dysfunction, and neuronal loss. Mediterranean diet is one of the healthiest eating plan based on the Italian and Greek traditional cuisine. Main components of this diet include vegetables, fruits, whole grains, beans, fish, eggs, nut, and olive oil. Moderate intake is instead recommended for dairy products and meat. Autophagy or self-eating is a physiological process crucial during development and for the maintenance of cellular homeostasis in which misfolded or aggregated proteins and damaged organelles are removed from the cytosol. In the case of stress stimuli such as nutrient deprivation, autophagy can also be induced as an adaptive response of the cells to promote survival. Dysfunctional autophagy is frequently observed in neurodegenerative diseases. Amyloid beta oligomers refer to an intermediate state occurring during Aβ fibrillization. Soluble Aβ oligomers are organized into different structures ranging from dimers, trimers, tetramers, pentamers, and decamers. Recent evidence suggests that these oligomeric forms are indeed the most toxic. Proteolytic cleavage is the hydrolysis of the peptide bonds between amino acids in proteins catalyzed by specific enzymes: peptidases, proteases, or proteolytic cleavage enzymes.

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Protein fibrillation defines a phenomenon by which misfolded proteins or peptides sharing a common cross-β sheet structure self-associate forming linear aggregates. This process is not limited to the amyloid beta peptide. It has been observed for prion protein, α-synuclein, insulin, glucagon, and β2-microglobulin. Aβ fibrillation involves three main steps: nucleation, oligomerization, and fibril formation. The nucleation phase is when free Aβ peptides initially interact with each other to form small units. Oligomerizations instead, refer to the aggregation of these units with the formation of prefibrillar soluble Aβ oligomers. Fibrillization is when these oligomers assemble ultimately resulting in the fibrillation of Aβ. The exact mechanism responsible for this phenomenon is not completely understood. Neurofibrillary tangles (NFTs), one of the primary hallmark of Alzheimer’s disease, are intracellular filamentous aggregates composed by hyperphosphorylated and misfolded microtubule-associated protein tau. Preclinical study refers to a required stage of research that precedes clinical trials in which a drug, a medical device or a medical procedure is tested in animals. The aim of a preclinical study is to evaluate the safety profile of the new treatment before it can be tested in humans.

cardiovascular diseases. Specifically, there is a paucity of data on the biological effects of chronic canola oil consumption in relation to the development of Aβ and tau neuropathology and cognitive dysfunction. In view of the interest in the potential benefits of canola oil, we recently investigated its effect in a mouse model of AD, the 3 3 Tg mice, which manifest all the aspects of AD pathology (memory impairments, Aβ plaques, and tau tangles). Interestingly, our findings do not support any health effects of this oil, since canola inhibited memory, decrease levels of PSD-95, a marker of synaptic integrity, increase the ratio of insoluble Aβ 42/40, and led to weight gain in AD mice. Apart from canola, recent literature has suggested that the use of coconut oil may lower plasma cholesterol, blood pressure, and blood glucose levels, all risks factors for AD and cardiovascular disease. In addition, coconut oil is rich in phenolic compounds recognized for their antioxidant properties. Despite the premises, currently there is no evidence showing that this oil may be used to reduce the risk of developing AD. Only two small randomized clinical trials have been conducted. However, at this time, no clinical data support the use of coconut oil in relation to AD.

Comparisons of extravirgin olive oils with other edible oils

Implications for human health and disease prevention

As discussed in this chapter, as one of the pillar of the MD, extravirgin olive oil (EVOO) and its polyphenolic components are accountable for many of the health benefits associated with this diet. Recognized by the World Health Organization as one of the healthiest and sustainable eating plan, MD and consumption of EVOO have been promoted worldwide as an antiaging and diseasefighting elixir. EVOO health claims have greatly stimulated the scientific community to expand this research also to additional plant-based oils, including coconut and canola, which in certain countries, where the olive oil tree is not available, are the principal substitute to the EVOO. However, each of these oils has different characteristics in terms of saturated fats, antioxidant, and chemical properties; thus we need to be careful before advocating their use as alternative source of fat to prevent AD or cognitive decline. Canola oil consumption is quite high in many countries because it widely available and inexpensive. However, at this time, the majority of the studies have provided conflicting results depending on the length of the treatment, the extraction method, the quality of the oil, and the experimental model adopted. Thus there are only limited evidences in support of canola beneficial effects on brain aging and biomarkers of risk factors for

Alzheimer’s disease is a multifactorial disorder with several mechanistic pathways contributing to its etiology and, risk factors including genetic, age, lifestyle, cardiovascular, and metabolic diseases which can significantly influence an individual’s chances of developing this disease. The current scientific literature suggests that a healthy dietary pattern may play an important role in preventing or slowing down AD. In particular, many studies have provided solid evidence in support of the health benefits of EVOO on longevity and brain function. Results from in vitro and in vivo studies, in several AD/aging models, have consistently documented the neuroprotective and disease-modifying properties of EVOO which has been shown to ameliorate Aβ and tau pathology, age-dependent deficits in spatial, working memory and motor functions, reverse oxidative damage, and enhance brain energy metabolism. Altogether, clinical and preclinical data strongly endorse the preventative or therapeutic effects of EVOO intake on Alzheimer’s disease. After more than a century from its discovery by Alois Alzheimer, we still do not have a cure for this devastating disorder. Thus in this scenario, It must be emphasized how important can prevention be. Focusing on health factors that can be easily modified may represent a great opportunity at the

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moment for preventing or delaying AD from developing in the first place.

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32. Bazoti FN, Bergquist J, Markides K, Tsarbopoulos A. Localization of the noncovalent binding site between amyloid-beta-peptide and oleuropein using electrospray ionization FT-ICR mass spectrometry. J Am Soc Mass Spectrom. 2008;19(8):1078
1085. 33. Rigacci S, Guidotti V, Bucciantini M, Nichino D, Relini A, Berti A, Stefani M. Aβ(1-42) aggregates into non-toxic amyloid assemblies in the presence of the natural polyphenol oleuropein aglycon. Curr Alzheimer Res. 2011;8(8):841
852. 34. Luccarini I, Grossi C, Rigacci S, Coppi E, Pugliese AM, Pantano D, la Marca G, Ed Dami T, Berti A, Stefani M, Casamenti F. Oleuropein aglycone protects against pyroglutamylated-3 amyloidß toxicity: biochemical, epigenetic and functional correlates. Neurobiol Aging. 2015;36(2):648
663. 35. Pitt J, Roth W, Lacor P, Smith 3rd AB, Blankenship M, Velasco P, De Felice F, Breslin P, Klein WL. Alzheimer’s-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal. Toxicol Appl Pharmacol. 2009;240(2):189
197. 36. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol. 2005;64:113
122. 37. Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, Small S, Spencer B, Rockenstein E, Levine B, WyssCoray T. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J Clin Invest. 2008;118(6):2190
2199. Available from: https://doi.org/10.1172/JCI33585. 38. Chong CM, Ke M, Tan Y, Huang Z, Zhang K, Ai N, Ge W, Qin D, Lu JH, Su H. Presenilin 1 deficiency suppresses autophagy in human neural stem cells through reducing γ-secretase-independent ERK/CREB signaling. Cell Death Disease. 2018;9:879.

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39. Lauretti E, Iuliano L, Pratico` D. Extra-virgin olive oil ameliorates cognition and neuropathology of the 3xTg mice: role of autophagy. Ann Clin Transl Neurol. 2017;4(8):564
574. Available from: https://doi.org/10.1002/acn3.431. 40. Menendez JA, Joven J, Aragone`s G, Barrajo´n-Catala´n E, Beltra´nDebo´n R, Borra´s-Linares I, Camps J, Corominas-Faja B, Cufı´ S, Ferna´ndez-Arroyo S, Garcia-Heredia A, Herna´ndez-Aguilera A, Herranz-Lo´pez M, Jime´nez-Sa´nchez C, Lo´pez-Bonet E, LozanoSa´nchez J, Luciano-Mateo F, Martin-Castillo B, Martin-Paredero V, Pe´rez-Sa´nchez A, Oliveras-Ferraros C, Riera-Borrull M, Rodrı´guez-Gallego E, Quirantes-Pine´ R, Rull A, Toma´s-Menor L, Vazquez-Martin A, Alonso-Villaverde C, Micol V, SeguraCarretero A. Xenohormetic and anti-aging activity of secoiridoid polyphenols present in extra virgin olive oil: a new family of gerosuppressant agents. Cell Cycle. 2013;12(4):555
578. 41. Rigacci S, Miceli C, Nediani C, Berti A, Cascella R, Pantano D, Nardiello P, Luccarini I, Casamenti F, Stefani M. Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight. Oncotarget. 2015;6(34):35344
35357. 42. Cordero JG, Garcı´a-Escudero R, Avila J, Gargini R, Garcı´aEscudero V. Benefit of oleuropein aglycone for Alzheimer’s disease by promoting autophagy. Oxid Med Cell Longev. 2018;2018:5010741. 43. Ng F, Tang BL. Sirtuins’ modulation of autophagy. J Cell Physiol. 2013;228(12):2262
2270. 44. Luccarini I, Pantano D, Nardiello P, Lapucci A, Miceli C, Nediani C, Berti A, Stefani M, Casamenti F. The polyphenol oleuropein aglycone modulates the PARP1-SIRT1 interplay: an in vitro and in vivo study. J Alzheimer’s Disease. 2016;54(2):737
750. 45. Chung S, Yao H, Caito S, Hwang JW, Arunachalam G, Rahman I. Regulation of SIRT1 in cellular functions: role of polyphenols. Arch Biochem Biophys. 2010;501(1):79
90.

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Chapter 41

Benefits and challenges of olive biophenols: a perspective Hassan Rasouli, Mehdi Hosseini Mazinani and Kamahldin Haghbeen Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Abbreviations COX ERK EVOO HIF-1α MAPK mTOR OOBPs PPs VOO

cyclooxygenase extracellular signal
regulated kinase extra-virgin olive oil hypoxia-inducible factor 1α mitogen-activated protein kinase mammalian target of rapamycin olive oil biophenols polyphenols virgin olive oil

41.1 Introduction Today, natural products have received much attention from both academia and industries by sharing most promising pharmaceutical features for enhancing the quality of daily lifestyle and life expectancy.1 Many long ago, Hippocrates, a well-known Greek philosopher, inserted a great emphasis on the potential health benefits of natural foods by expressing this magic statement: “Let food be thy medicine and medicine be thy food.”2 Later on, ancient generations have learned to modify natural edible substances for production of diverse categories of foods, spices and supplements. Up to now, thousands of natural substances from both terranean and marine organisms have been isolated or identified and these elements have possessed spectacular medical values.3 The term polyphenols (PPs) is referred to a diverse category of bioactive secondary metabolites widely distributed in fruits, vegetables, beverages, and plant-based foods, identified by a phenolic scaffold (an aromatic benzene ring saturated with
OH groups), which have a considerable functionality in plant defense system and physiological processes.2
4 PPs are chemically different metabolites, structurally classified into simple phenolic

compounds with C6 scaffolds, flavonoids, and phenolic polymers (e.g., lignins and phlobaphenes). PPs have been known for their antioxidant potential to scavenge toxic free radicals and reactive oxygen species (ROS) or reactive nitrogen species. Each class of PPs has a specific biological property by which the strength or weakness of their biochemical potential can be judged.2
4 Hydroxylation, glycosylation, methylation, and acetylation are common (bio)chemical modifications that may naturally or synthetically change the chemical scaffolds of PPs.5 The accumulating body of evidence suggests that PPs are not only key metabolites to regulate the basic molecular/biochemical process of plant cells or tissues, these compounds also exhibit potential health-promoting effects.4 Environmental stimulants, expression of particular genes, plant cultivars, climate adaptation, and soil and irrigation water quality are determinant factors to affect the production of PPs within plant tissues.6 Among Mediterranean trees, olive (Olea europaea L.) is thought to be one of the most sacred and oldest trees in this region because its fruits, oil, and leaves have broadly been utilized for many purposes from cooking to table application.7 Various cultivars of olive trees have presently planted all over the world and studies reported that virgin olive oil (VOO) is an enriched source of phenolic compounds, monounsaturated fatty acids, and other beneficial natural products.8 Romeo and Uccella in 1996 introduced the term “biophenols” for OOBPs to highlight a more comprehensive and chemically accurate explanation to refer various classes of plant phenolic constituents.9 The “bio” prefix of biophenols showed the biological origin of these metabolites. Considerably, various types of PPs are not naturally existed in plant tissues but produced during harvesting processes or transporting plant tissues. Therefore the term of biophenols covered all phenolic compounds derived from botanical sources.9 In

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00045-6 © 2021 Elsevier Inc. All rights reserved.

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this chapter, we used both terms to refer to generally discussed materials in the available literature. The biosynthesis of olive PPs and related signaling pathways have not entirely been understood because the function of involved enzymatic reactions may change when the trees would be under the pressure of environmental tensions or harvesting steps.10 Furthermore, a better understanding of biosynthetic pathways of olive biophenols requires long-term studies on olive cultivars planted in different regions.10 This chapter briefly sets out to review the functionality of OOBPs for improving human health using both literature searches and gene ontology analysis. Indeed, discussed are also the major limitations of OOBPs either for clinical studies or cosmetic industries.

41.2 An overview of plant polyphenols Phenolic compounds are a diverse category of plant secondary metabolites widely unraveled a broad spectrum of biological functionalities. They have a moderate-to-high molecular weight, are generally responsible for plant
environment interactions such as pigmentation and defensive mechanisms.11 PPs are potent antioxidants. However, they may also display “prooxidant” activity under certain conditions.12 Presently, several different classification systems were suggested to categorize PPs; however, classifying system based on PPs, chemical backbone is the most common one (Fig. 41.1A). Regardless of PPs chemical backbone variation, shikimic acid, and phenylpropanoid pathways are deemed to be the most important routes of PPs biosynthesis, though the exact biochemical pathway(s) has not been discovered. Some metabolites such as phenylalanine, tyrosine, and tryptophan are important amino acids participated in the biosynthesis of PPs.2
6,11 Biologically, two enzymes, including tyrosine-ammonia lyase and phenylalanine ammonialyase, may play functional roles in the regulation of PPs production within the plant cells.6 However, there are many uncertainties in the biosynthetic process of PPs, and the sophistication of their backbone may ultimately determine the number of enzymes and chemical reactions in each metabolomic path.4,6,13 Physiological parameters such as developmental stages of plant growth, flowering, ripening, postharvest modifications may also be effective in the alteration of PPs appearance in plant organs or tissues.6 There are specific types of phenolic compounds in olive tissues called phenolic secoiridoids. The metabolomic pathway of secoiridoid biophenols is varying from other phenolic compounds.14 Chemical substitution of functional groups of PPs backbone could change their binding mode to catalytic or binding residues of receptor active sites. It seems quite obvious that the distribution of quantum molecular

orbitals around PPs backbone moieties would determine the chemical hardness or softness of PPs to accept or donate electrons to construct chemical bridges between their atoms and amino acids side chain elements (Fig. 41.1C). Both simple and complex PPs have unique patterns of quantum molecular orbitals by which their potency to show inhibitory activity should be judged.3

41.2.1 Extraction and purity of phenolic compounds The obtainment of PPs from their sources required specific extraction methods. Accordingly, several different protocols are available to isolate PPs from fruits, vegetables, and botanical origins.15 Fundamentally, PPs are moderately water-soluble molecules, though some categories may display high resistance to water solubility.2,4 Therefore the ability of applied extraction methods to separate and identify final phenolic content is depending on the chemical essence of PPs and potential solubility in specific buffers. Utilization of the wrong extraction methods only wastes the time and budget of laboratory. Solvent extraction, ultrasound-assisted extraction, and supercritical fluid extraction procedures are common routes for PPs extraction.16 Chemically, molecular weight of PPs is ranging from 180 (for simple phenolic acids) to 4000 Da (for phenolic polymers), possessing several numbers of
OH groups ( . 12), naturally occurring as glycosidic forms. Chemical modification of PPs backbone would affect their purity in extraction solutions.17 The final purity of PPs would determine their utilization for various applications. For clinical application the required purity quality of PPs should be $ 95%, whereas basic nutrition needed a purity equal to 50
80%. In reality, for cosmetic industries the required PPs purity is thought to be # 40%18 because most of the PPs in such forms would be combined with a mixture of chemical ingredients. High-throughput purification methods should simultaneously apply to prepare a considerable quantity of PPs. The ultimate price of purified PPs is depending on application of these compounds for pharmaceutical, laboratory, or nutrition purposes. Basically, highly purified PPs for laboratory/drug design goals are among the most expensive plant metabolites. For example, the price of ellagic acid with purity $ 95% prepared by high-performance liquid chromatography (HPLC) for high-quality academic experiments is about h116.21 per gram whereas the cost of unpurified forms of ellagic acid mixed with gelatin or polyvinylpyrrolidone (purity , 90%) is less than $20 per 500 mg ellagic acid powder. The low purified form of ellagic acid is mainly used for dietary applications. Similarly, the commercial concentration of tyrosol, a

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491

FIGURE 41.1 Classification of PPs. (A) Flowchart of main classes of PPs, (B) Examples of phenolic compounds, and (C) Distribution of molecular chemical quantum orbitals around functional groups of a flavonoid backbone. During interaction of PPs and target active site catalytic residues, chemical change of molecular orbitals in each donor or acceptor site can affect the total affinity PPs to incline toward residues side chains. Pie charts display the contribution of molecular orbitals around chemical scaffold of studied flavonoid and
OH groups. HOMO, High occupied molecular orbitals; LUMO, low occupied molecular orbitals; PPs, polyphenols.

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simple phenol extracted from olive oil, with absolute purity of $ 98% valued h230 per 20 mg and it would be used as reference metabolite in chemical analytical investigations.

41.2.2 Polyphenols biological functions PPs unraveled a wide-spectrum of protective functionalities. As reviewed,2,4,6 both flavonoid and nonflavonoid metabolites could reduce the expression of inflammatory cytokines or chemokines; improve blood glucose level; ameliorate neurodegenerative anomalies; alleviate blood pressure; placate cardiovascular dysfunction; modulate cell apoptosis; balance antioxidant status; improve vision and eye function, regulation of signaling pathways; inhibit angiogenesis and cell malignancy; modulate/regulate gene expression pattern; scavenge toxic free radicals and ROS molecules; shield skin tissues from ultraviolet waves; increase skin health; prevent obesity and weight gain; and improve mental health and cognitive function.2,4,6 PPs could interact with their cellular targets directly or indirectly. For direct interaction a variety of enzymes, signaling pathways, and even genes’ promoters may be targeted. Nevertheless, the direct effects of PPs in vivo are not understood owing to low bioavailability of these metabolites.19 Rapid metabolism of PPs and their instability are two determinant factors to influence their biological actions within the human body. Therefore direct interaction of PPs with cellular components may include transient effects. In contrast, long-term and regular consumption of specific doses of PPs alone or combined with other metabolites/food ingredients may provide considerable health benefits than intermittent consumption of PPrich products. In such conditions, human body can receive certain concentration of PPs on the basis of daily, thereby leading to healthy life-style.20

41.2.3 Normal and clinical consumption of polyphenols Selecting an optimized and healthy diet is a challenging issue since ancient times. Foods and natural ingredients are essential elements to guarantee human health.4 Consumption of PPs occurs every day when the diet comprised a part of grains, vegetables, fruits, juices, red wine, chocolate, coffee, black and green tea, extra-VOO (EVOO), and processed phenolic-enriched substances. Many people believe that overconsumption of plant-based foods could provide higher concentrations of promising ingredient for their body. This is absolutely wrong because there are many differences between normal and clinical nutrition. In this regard, the regular consumption of PPs (or foods enriched with PPs) by eating various

types of fruits or vegetables may improve human health.2,21 On the other hand, clinical consumption of PPs has only dealt with application the certain quantities of phenolic metabolites on the basis of daily. Interestingly, the daily requirement of human body to intake PPs is not determined completely. This means that consumption of 400
1000 mg of PPs per day may improve metabolic process of human body but large-scale clinical studies will be required to draw a comprehesnive conclusion regarding the health benefits of PPs. Studies speculated that almost 5%
10% of PPs would be intake from small intestine and the rest were excreted into urine or feces.22,23 As depicted in Fig. 41.2, administration of PPs for both normal and clinical purposes has its own benefits and demerits. Normal or sporadic consumption of PPsenriched fruits, vegetables, or functional foods would only provide short-term invisible health benefits. In contrast, clinically controlled administration of PPs would provide strong visible long-term effects on human health. As shown in this illustration, recommendation of PPs for different applications should pass at least three phases of bioassays. This means that reaching successful results in in silico or in vitro experiments should not be a reason to exaggerate functionalities of PPs for marketing, clinics, or other benefited areas, though promising results were observed in primary studies. Presently, the literature over-highlighted the potential benefits of PPs while enough evidence-based data from long-term multinational clinical trials are not comprehensively provided. Antioxidant activity of PPs is the main reason for recommending these metabolites for marketing. Controversially, some clinical trials failed to approve health benefits of antioxidant therapies. A well-known example of clinical failures for antioxidant therapy attributed to vitamin E.24 Similarly, failures for higher doses of resveratrol (5 g) administration were also reported.25

41.3 Olive status in Iran and worldwide statistics Olives are important trees in the Mediterranean basin and widely their fruits processed for oil extraction and table uses. Olive trees have almost grown all over the world. In Iran, several different olive cultivars are mainly planted in mountainous, semi-arid and salinized parts of country.7,26 This variation demonstrates that native Iranian olives possessed particular types of physiological adaption systems by which a broad spectrum of environmental changes could be tolerated. Today, worldwide request for olive oil and fruit has fastened the plantation of olive commercial cultivars in

Benefits and challenges of olive biophenols: a perspective Chapter | 41

493

FIGURE 41.2 Graphical illustration of experimental assays of PPs of recommending these metabolites for large-scale administrations. The merits and demerits of both normal and clinical nutrition have been shown. Without considering the discussed challenges herein, PPs should not be recommended for global marketing. PPs, Polyphenols.

favorite regions. The given opportunity keeps encouraging on several countries such as Australia, Peru, Chile, China, Argentina, Iran, and Turkey to develop more olive orchards. However, olives moderately tolerated the harsh condition of surrounded environment but various factors such as soil quality and chemico-physical properties, irrigation water, salinity, chilling, drought, pest, and disease would determine the stability and adaptation potential of this tree to many regions around the world.27 In comparison to commercial olive cultivars such as Picual, Arbosana, Frantoio, Arbequina, and Koroneiki, Iranian varieties such as Zard, Mari, and Rowghani could produce considerable amount of both table olive and virgin oil either for local consumption or international marketing. Other cultivars such as Dezful, Direh, and Tokhme-Kabki were also deemed to be the best olive candidates for further breeding programs. Over the past decade, olive cultivation has received much attention from agricultural departments and other olive experts so that some important sites of olive propagation and cultivation expanded around Iran.

Internationally, in both Northern and Southern Hemispheres, the adaptation of olive trees to climate change and the total amount of precipitation per year are depending on the physiological/botanical potency of candidates’ cultivars. Torres et al.28 studied the functional roles of environmental parameters on olive growth in Southern Hemisphere countries and reported that temperature, irrigation, and soil quality are three determinant factors to strengthen the growth of olive cultivars or impeding them from reaching the acme of growth and sustainable yielding. Accordingly, an increase in temperature may decrease the availability of flowers for pollination and producing fruits. In contrast, chilling may be benefited the flowering power of olives but its physiological and/or molecular aspects have not understood.28 Both Mediterranean and non-Mediterranean regions have their own merits and demerits for cultivation of commercial olive cultivars, and the attribution of commercial olives to each region should be critically investigated.27 Countries such as Spain, Italy, Portugal, and Greece are major producers of olive oil and table olive.

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According to estimations, more than 4.5 million hectares are devoted to cultivation of commercial olive cultivars in Europe, which lead to extraction of more than 2 million tons of olive oil in this continent. Similarly, the total olive oil production of Iran in 2019 was considered to be . 7000 t and altogether more than 100,000 ha devoted to the cultivation of olives. In comparison to Australia, Iran has a greater potential to expand its modern olive orchards for a higher production of olive products. Per capita olive oil consumption in Iran is less than 0.5 L by 2019. Contrary, Greece occupied the first rank of olive oil consumption by possessing more than 12 L per capita. After that, Spain ( . 9 L) and Italy ( . 5 L) are top countries to consume olive oil. According to Scopus updates (https://www.scopus. com/home.uri) from 2000 to the end of January of 2020, Spain, Italy, the United States, and China are top countries involved in the research on olive oil by occupying 20%, 16%, 9%, and 5% of all published data. Other countries such as Greece, Turkey, India, Britain, and France ranked in the next positions. In contrast, Iran possessed only 3% of all publications on olive oil or similar products and most of the published papers in Iran endowed to research on the quality and phytochemical constituents of olive oil obtained from various cultivars. The most of published data on olive oil were in the field of agriculture, biological science and biochemistry, and medicine. Italy, Spain, Greece, Tunisia, the United States, and France were the pioneer countries to research on olive phenolic content. Comparably, Iran only occupied 2% of all researches in this area and co-ranked with other countries such as Japan, Germany, Tunisia, and Australia. According to the literature searches, four olive biophenols, including tyrosol, hydroxytyrosol (Htyrosol), oleuropein, and oleocanthal, were most studied OOBPs (Fig. 41.3).

41.3.1 Olive databases Studies indicated that the business horizon of olive industry is tightly linked to online multimedia broadcasting either for sharing horticultural experiences and molecular information with multinational olive breeders and researchers. Presently, the information of more than 500 PPs isolated from .400 foods plus their biological functions and chemical information is listed in Phenol-Explorer database (http://phenolexplorer.eu/).29 The number of olive scientific databases for sharing its genetic materials, metabolites, and productivity is limited,30 and only 15 databases covered the information for olive secondary metabolites, genetic variation, germplasms, and sequenced genomes. OLEUM project consortium (http://www.oleumproject.eu/partners/consortium) is one example of olive database developers for two reasons, including unifying the current knowledge of olive studies and large-scale analysis of olive biological data. Table 41.1 represents a list of recently developed olive databases.

41.3.2 Olive phenolic metabolites Chemical studies reported that various parts of olive aerial tissues, fruits, leaves, stem bark, roots, and flowers have shown to be the location of biosynthesis of several different types of secondary metabolites. Olmo-Garcı´a et al.31 comprehensively studied the distribution of secondary metabolites of O. europaea L. using liquid chromatography and gas chromatography approaches and reported that PPs, free fatty acids, sterols, tocopherols, dialcohols, and triterpenic acids were the most abundant secondary substances. Other studies have also shown that several lignan metabolites such as pinoresinol, syringaresinol, and acetoxypinoresinol were found in olive oil or its aerial tissues. Both flavonoids and nonflavonoid metabolites were identified in olive tissues.32,33 Olive oil consists of B500 mg/kg biophenols.34 Carluccio et al.35 reported that olive oils generated from various olive cultivars may comprise a range of 40
100 mg/kg phenolic compounds. Secoiridoids were other important chemical metabolites of olive. These metabolites occurred in aglycone or glycosidic forms and their biosynthesis would be affected by both physiological environmental factors.36 Metabolomics investigations showed that the accumulation of secoiridoids was occurred during the middle and late stages of olive fruits development. Considerably, secoiridoids are common secondary metabolites among Oleaceae and some families of dicotyledonous plants.37 Oleuropein, oleuroside, demethyloleuropein, ligstroside, and closest aglycone relatives are most ostentatious olive secoiridoids. Alagna et al.38 reported the biosynthesis of olive biophenols originated from several metabolic routes, including mevalonate diphosphate, phenylpropanoid, and secoiridoid pathways. In olive, especially during fruits shaping or development, secoiridoid pathways will determine the amount of total biophenols produced within olive tissues. It is worth to voice that oleuropein is one of the most important factors to determine olive oil bitterness.14 Interestingly, the concentration of olive biophenols may change tree by tree. Olive cultivars such as Cornicabra, Coratina, Koroneiki, Manzanillo, Maurino, Mission, Picual, Ogliarola, Picholine, and Moraiolo have total biophenols content equal to 600 mg/kg oil, whereas biophenol profile of Arbequina, Casaliva, Nocellara, Picudo, Sevillano, Tanche, Itrana, Taggiasca, and Biancolilla is deemed to be less than 200 mg/kg olive oil. In contrast, phenolic content of Frantoiano, Arbosana, Carolea, Hojiblanca, and Ogliarda is more than 300 mg/kg olive oil, respectively.39 Studies reported that various parts of olive aerial tissues have different types of phenolic compounds. For example, metabolites such as oleuropein, Htyrosol, caffeic acid, apigenin, tyrosol, and verbascoside are major chemical components of EtOH extract of olive leaves.39 HPLC analysis of olive leaves extract prepared by maceration

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495

FIGURE 41.3 (A) International statistics on olive cultivation and olive oil production, (B) per capita olive oil consumption for Iran and other countries, (C) total proportion of worldwide countries on olive oil studies, (D) ranking of top countries to study olive biophenols, and (E) chemical backbone of most studied olive biophenols. The given data in this figure may be changed during the next few years,

method have also exhibited different numbers of phenolic compounds such as coumaric acid, rutin, syringic acid, freulic acid, gallic acid, luteolin, quercetin, protocatechuic acid, caffeic acid, tyrosol, oleuropein, and hydroxyltyrosol, respectively.40 Talhaoui et al.41 studied biophenols composition of six different cultivars of olives cultivated under same agronomical condition and reported a significant change of olive biophenols ratio from fruits to oil. Accordingly, the highest ratio of biophenols changes was observed for secoiridoids and flavonoids, whereas the highest amount of secoiridoids was found in the Picual cultivar oil. These outcomes suggested that in addition to environmental factors, genetic variation of olive cultivars is another

determinant factor to modify the balance of produced biophenols within the extra-virgin oil or fruits. A study by Servili et al.42 has shown that the irrigation pattern had minor effects on fatty acid composition of VOO. In contrast, irrigation regime may markedly affect the concentrations of olive alcohols, esters, C6-saturated and unsaturated aldehydes.42 As discussed, simple biophenols such as tyrosol and hydroxyltyrosol were also found in EVOO, exhibited considerable antioxidant activity.43 Another important EVOO metabolite is oleacein in which its accumulation depends on the genetic composition of olive cultivars. Both wild and commercial varieties of olive trees could produce oleacein biophenol but in a different concentration.

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TABLE 41.1 An updated list of olive databases up to January 2020. Database

Public access

Country

Germplasm

Genetics

Metabolome

OLEA

Yes

Italy

K







OLEA SSR marker

Yes

Italy

K

K




WOGBC

No

Spain/Morocco/ Turkey

K

K




National Clonal Germplasm Repository—Tree Fruit & Nut Crops & Grapes

Yes

United States

K

K




Istrian olive database

Yes

Croatia/Slovenia

K

K




CRA-OLI collection

No

Italy

K

K

K

Australian DNA Fingerprints of Olive Cultivars

No

Australia




K




Certolio

Yes

Italy




K




ReprOlive

Yes

Spain




K




Olive Genetic Diversity Database (OGDD)

Yes

Tunisia




K

K

FATG-DB04

No

French







K

Oli monovarietali italiani

Yes

Italy







K

SIAN ICQR

Yes

Italy







K

Italian National Database of PDO/PGI Extra Virgin Olive Oils

No

Italy







K

EuroFIR

Yes

Belgium







K

OliveNet

Yes

Australia







K

Olive Genome

Yes

Turkey/Spain/China/ United States




K




ReprOlive

Yes

Spain

K

K




OliveIran Data

No

Iran

K

K




SIAN, Sistema Informativo Agricolo Nazionale.

Oleacein scavenged toxic free radicals with a higher potential in comparison to oleuropein and other biophenols.44 Like other OOPs, phenylethanoid compound oleocanthal [or (
)-decarboxymethyl ligstroside aglycone] is widely found in EVOO. Approximately 36 distinguish phenolic compounds were isolated from VOO that possessed promising biological profile. Most considerably, oleocanthal is responsible for almost a large proportion of olive bitterness and pungency and it caused throat burn process.44,45 A swelling volume of scientific investigations showed that among EVOO biophenols, oleocanthal is very sensitive to light and oxygen, and owing to the decomposability potential, it is not found in olive oil in higher concentrations. Interestingly, synthetic forms of oleocanthal showed antiinflammatory properties.45 Coumarin metabolites such as scopoletin and esculetin were also found in EVOO. Indeed, secoiridoids are also

coumarin-like biophenols widely biosynthesized from secondary metabolism of terpenes and specifically attributed to genus of Oleaceae family.46 Studies reported that in addition to the mentioned coumarins, another compound called scopolin and several different classes of lignans and lignin-like metabolites such as (2)-olivil, (1)-1hydroxypinoresinol-4v-O-methyl ether have also been extracted from bark tissues of olive trees. The highest distribution of flavonoids and secoiridoids (or glycosidic forms) was found to be in olive fruits and seeds. Rutin, vicenin, quercetin and quercetin glycosides, glycosidic forms of apigenin and luteolin were also isolated from both olive pulps and fruits.47 Oleuropein, oleoside, secologanoside, ethanoic acid methyl esters, and ligstrosides are extensively abundant in olive leaf tissues. Olive trunk and branches wood is also enriched with a diverse category of biophenols. Ligstrosides are well-known examples of everywhere

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present olive biophenols, which accumulated in different concentrations between fruit, seed, and wood tissues. EVOO is a unique source of biologically activated phenolic compounds such as vanillic acid, oleuropein, coumaric acid, lignans metabolites, oleuropein aglycon, flavonoids, and many other biophenols that are commercially valuable, and their marketing becomes an excellent field to reach eye-catching financial sources.48 Hashmi et al.49 systematically reviewed the phytochemical essence of olive secondary ingredients and highlighted promising metabolites for further studies. In addition to phenolic content, several different pigments, including β-carotene, lutein, chlorophyll a and b, pheophytin α, pyropheophytin α, pheophorbide b, and xanthophylls such as neoxanthin, violaxanthin, and other pigments, were also isolated from tissues of olive fruits. A wide range of monosaccharides, disaccharides, and sugar carboxylic acids or alcohols is also isolated from various parts of olive tissues.48,50 By and large, agronomical traits and olive cultivar, climate changes, soil quality, rainfall pattern, soil organic materials, biotic tensions, irrigation water quality, salinity, chilling, higher temperature, altitude and longitude, ripening stage, extraction quality, intrinsic oxidative reactions, storage place, and time are important parameters to determine the total amount of olive biophenols.

41.4 Pharmacological functionalities of olive biophenols Olive phenolic content showed a wide range of biological functionalities by which these metabolites could affect various cellular targets. The benefits of VOOPs are for various scopes such as antimicrobial, antiviral, antiinflammatory, anticancer, antidiabetic, anticardiovascular diseases in vitro and in vivo.39,49 Accordingly, consuming olive biophenolenriched foods is a unique source of functional supplementary ingredients to improve human lifestyle.50 According to our literature searches and gene ontology analyses, four phenolic compounds—tyrosol, oleuropein, Htyrosol, and oleocanthal—were most studied olive biophenols to affect cellular pathways. Based upon the available data, olive biophenols could regulate total secretion of inflammatory markers [e.g., interleukins (ILs) and tumor necrosis factors (TNFs)] under in vitro or animal models but may be less effective against human inflammatory targets and their molecular mechanisms covered another aspect of cellular responses to occurred abnormalities. Our results also showed that Htyrosol and oleuropein were common to affect four targets, including TNF-α, IL-1β, Nrf2, and NF-κB signaling cascades. Htyrosol and oleocanthal could affect iNOS pathway with similar mode of action. Indeed, Htyrosol, oleuropein,

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and oleocanthal together could significantly modulate the expression pattern of cyclooxygenase (COX)-1 cascade. Generally, COXs are quintessential regulators of prostaglandins, typical inducers of inflammation, and sever pains.51 Although antiinflammatory effect of olive biophenols is not visible for long term, however, the evidence postulates that such metabolites play a protective role against complications of inflammations.52 Our data have also shown that tyrosol, Htyrosol, and oleuropein were modulated mitogen-activated protein kinase (MAPK) pathway, a versatile cellular signaling pathway53 to regulate cellular process such as cell proliferation, cell division and differentiation, apoptosis, inflammation, cell cycle arrest, and the development of endothelial cells.53 Accordingly, a direct correlation between MAPK and several human diseases such as cancer, type 2 diabetes, and Alzheimer was reported.53 COX-2 gene expression was also modulated by tyrosol and Htyrosol metabolites. As shown in Fig. 41.4A, Htyrosol alone could regulate considerable numbers of signaling pathways or gene expression patterns (B18 cases). One of the most important targets identified herein was hypoxia-inducible factor 1α (HIF-1α) that plays a critical role in the development of cancerous cells. Convincing pieces of evidence have provided the strong correlation between the expression of HIF-1α and tumor metastasis, angiogenesis, proliferation, and development, and therefore this target has been considered as promising drug target in the cornerstone of cancer treatment.54 Martı´nez-Lara et al.55 studied the effects of Htyrosol on HIF-1α pathways and confirmed that Htyrosol may be considered as a preventive therapeutic approach to decrease stress and oxidative damage linked to HIF-1α expression and cancerous cell potency. Other pathways such as PPARγ, PI3K, AKT, flavin-containing monooxygenases, EGFR, Fas pathway, and forkhead box class O3A were also regulated by Htyrosol.56,57 Our gene ontology searches also showed that extracellular signal
regulated kinase (ERK) pathways were also affected by tyrosol and oleuropein. ERK pathway is an important and major part of MAPK signaling route, involved in a variety of cellular process, including proliferation and differentiation.58 Interestingly, two pathways, including 50 -AMP-activated protein kinase or AMPK and mammalian target of rapamycin, were two signaling cascades that modulated by both oleuropein and oleocanthal (Fig. 41.4). Alkhatib et al.59 reviewed the possible health benefits of olive oil nutraceuticals against diabetes and reported that most of the OOBPs could modulate inflammatory responses of diabetic complications. Antioxidant activity, antiinflammation, and body metabolism augmentation were the most visible biological mode of action of OOBPs.59 de Bock et al.60 reported that obese patients that consumed capsules comprised 51.1 mg oleuropein

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FIGURE 41.4 (A) Comparative profile of covered signaling pathways by OOBPs, (B) the identified regulatory network for molecular action of OOBPs, and (C) the percentage of most important involved signaling pathways for gene/term ontology searches. OOBPs, Olive oil biophenols.

and 9.7 mg Htyrosol increased β-cells function and IL-6, whereas the coadministration of these biophenols has no effects on IL-8 and TNF-α and C-reactive protein. However, similar to these studies, our results were also shown that NRF2 and TNF signaling cascades were the most potential cellular targets that OOBPs could be involved in their regulatory processes. Elevated blood glucose generated from overactivity of carbohydrate digestive enzymes is one of the most important complications of diabetic patients. The inhibition of two enzymes, including α-amylase and α-glucosidase, were deemed to be effective in the primary prevention of type 2 diabetes. VOO phenolic extracts have inhibited these enzymes in a dose-dependent manner and apparently, secoiridoids were more successful rather than flavonoids.61 Hadrich et al.62 showed that oleuropein (400 μM) and Htyrosol (150 μM) could significantly deter 75%

activity of α-glucosidase enzyme in vitro. Accordingly, it should be noted that diabetes mellitus is a complex metabolic syndrome63 and long-term broad-spectrum clinical studies will be required to approve the quality of olive biophenols for diabetes therapy perspectives. The beneficial properties of OOPs on cardiovascular complications and risk factors are now identified and both of monounsaturated fatty acids and biophenols could placate the abnormalities of heart diseases. Covas et al.64 reported that olive oil phenolic content could provide benefits for protecting cells from oxidative damage and lipid peroxidation. As shown in Fig. 41.4B or C, olive biophenols were involved in cellular responses to ROS and oxidative damages and clinical outcomes also approved their protective effects for cellular targets. High phenolic EVOO was also displayed to be effective against some

Benefits and challenges of olive biophenols: a perspective Chapter | 41

cardiovascular risk factors in comparison to low phenolic EVOO and refined oil.65 Owing to the lack of extensive volume of public data on beneficial effects of olive oil phenolic content on cardiovascular complications, there are overall few certainties of evidence to confirm the widespread application of these products for this case.

41.5 Recycling olive by-products for cosmetic industries Cosmetic application of olive oil and biophenols provided an excellent groundwork for developing financial marketing in this area. Presently, a variety of olive oil-based cosmetic products existed locally and internationally in markets. It seems quite ostentatious that the worldwide demand for these products has dramatically surged up. Most of these cosmetic commodities are developed for protecting skin from environmental damages, and the rest are produced for improving the growth of hairs, body hydration, air conditioning, muscle function, and

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clearness of skin. As shown in Fig. 41.5, the variation of olive-based cosmetic ingredients is enough to find the target products based upon the desire of customers. Rodrigues et al.50 reported that discharging of olive oil producing factories into surrounded environment could cause significant problems for the nearby ecosystems mainly owing to the presence of toxic elements in the produced wastewaters. According to their SWOT model for olive oil by-products, converting and recycling waste materials of olive oil producing factories may support cosmetic industries for developing new formulations of olive-based commodities. Olive leaves, stones, mill wastewater, and pomace have considerable amount of bioactive phenolic content, and recycling these materials may bring out economic benefits for oil producers instead of threw out them into nature and causing problem for ecological biosystems.66 Management olive oil by-products recycling challenges through developing high-quality extraction methods, using modern installations and tools, chemical modification, and detoxification of olive mill wastewater active substances may

FIGURE 41.5 A number of cosmetic and food supplementary commodities produced from olive oil or its biophenols content. (The purpose of this illustration is to show some examples of olive-based ingredients not for advertising their marketing value.)

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be considered as recommended strategies to decrease the amount of environmental pollutants of olive oil extraction factories. However, stabilization, valorization, and bioconversion of olive oil by-products using up-to-date mechanized systems into more useful healthy commodities will facilitate recycling networks to reduce linked environmental concerns effectually.67 Long-term following of such programs requires financial supports from local and international sectors, and consequently, international olive oil producers should be aware of such updates for improving sustainable growth of their economy.

41.6 Limitations of polyphenols for clinical applications There are some critical limitations with PPs that should be discussed before large-scale utilization of these metabolites. According to the literature, three distinct features, including reducing inflammatory responses, improving cellular metabolism balance, and modulatory effects against signaling cascades, gave PPs enough permission to affect numerous numbers of tissues or cellular targets.2,4 However, one of the most important reasons that PP-based drugs have not been developed yet is that PPs bioavailability is not considerable as well as many drugs. This means that PPs are not stable metabolites in human body to entirely effect on certain targets.68,69 Some of olive biophenols such as oleuropein and Htyrosol are well absorbed in the gastrointestinal tracts, but later they are not bioavailable inside bloodstream owing to postmodification of their chemical backbone mainly in the form of sulfate and glucuronide conjugates.70 Studies on bioavailability of olive biophenols suggested that about 5% of administrated biophenols were excreted into urine as simple phenols such as tyrosol or Htyrosol, and the absorption ratio of OOPs is thought to be between 55% and 66%.71 Despite literature recommendation on anti-Alzheimer effects of PPs, the available in vitro or in vivo models have shown that only a few categories of flavonoids were able to reach central nervous system and others could not pass the blood
brain barrier, a physical hindrance between brain tissues and bloodstream.72 Nutritionists have considered PPs as supplementary food ingredients in the cornerstone of disease prevention. More importantly, attributing the term of “functional food” to PPs may require evidence-based clinical data. Accordingly, considering PPs as a subclass of vitamins may fill out this gap owing to vitamin-like molecular mode of action of phenolic ingredients.4 Carried out clinical trials were not recruited considerable number of participants, and more importantly, the duration of considered interventions is not long enough to unravel all aspect of PPs molecular mode of action.73,74

However, demanding for olive oil consumption yearly increased, and owing to extensive use of olive products in food and pharmacies,75 it is necessary to understand olive biophenols molecular mode of action more comprehensively. Therefore elucidation of involved cellular signaling pathways in the enzymatic changes of olive phenolic compounds within gastrointestinal tract and other parts of human body would provide invaluable information about their ultimate destiny in cell to develop further delivery systems for improving the potential benefits of these miracle metabolites. As discussed, olive oil is considered as a functional food to improve human health indices.76 Owing to many proven benefits of EVOO, health experts recommended the regular consumption of olive oil in dietary regimens.77 Recommending clinical effectiveness of olive oil consumption required long-term programs in target countries, and keep encouraging on people may increase demands for olive oil gradually. However, accumulated data ostentatiously display that the manifold health properties of olive oil originated from its chemical bioactive components and establishing more investigational programs might strengthen the power of evidence-based data for local and international consumers.

41.7 Conclusion EVOO comprised a variety of secondary metabolites functionally showed biological activity in the concentration of micromolar. Studies demonstrated that OOBPs are promising supplementary ingredients to ameliorate the expression of inflammatory markers, particularly those cytokines that functionally are involved in the pathogenesis of metabolic disorders. It seems obesity, cancer, diabetes mellitus, and inflammation were the major human diseases that OOBPs could deal with their signaling pathways. By and large, the available data could not demystify the accurate molecular effects of OOBPs and further high-quality efforts are needed to unravel their pharmacokinetics and pharmacodynamic properties. Therefore using further clinical studies the effectiveness of OOBPs should be investigated. Time will tell what changes the game. . .

Mini-dictionary of terms Active ingredient: A chemical substance in a pharmaceutical or cosmetic product that is biologically (or biochemically) active. Biophenols: Biophenols are biologically active phenolic compounds isolated from natural sources. Clinical nutrition: A very specific type of nutrition prescribed by clinics or medical experts to maintain patient’s health.

Benefits and challenges of olive biophenols: a perspective Chapter | 41

Database: A database is a seriously organized set of data stored at a computer and its information can be browsed, compared, or analyzed with similar datasets or together. Environmental stress: An abiotic or biotic process that potentially can postpone the sustainable growth of living cells. Functional food: Foods that offer special health benefits beyond their nutritional value. Gene ontology: Gene ontology is a bioinformatic process to describe the function of gene products among all species. Normal nutrition: A process by which individuals take in and consume food substances on the basis of daily. Olive mill wastewater: Wastewater produced during olive oil extraction in olive mills. Signaling pathways: A cellular signaling pathway is a set of biochemical reactions by which a cell reacts to inward/outward stimulants to regulate molecular status of itself and nearby cells.

Acknowledgments None.

Conflict of interest statement The authors declare no potential conflict of interest.

Funding None.

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Chapter 42

Treatment and valorization of olive mill wastewater Parvin Mohammadnejad, Kamahldin Haghbeen and Hassan Rasouli Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Abbreviations AOP BOD COD CPE CSTR DPPH HT LDL MF NF OBP OMW OL ROPO ROS SPE SFT TSS TY TOC UAF UF UASB

advanced oxidation process biological oxygen demand chemical oxygen demand cloud point extraction complete stirred tank reactor 2,2-diphenyl-1-picrylhydrazyl hydroxytyrosol low-density lipoprotein microfiltration nanofiltration olive biophenols olive mill wastewater oleuropein refined olive-pomace oil reactive oxygen species solid-phase extraction supercritical fluid technology total suspended solids tyrosol total organic compounds up-flow anaerobic reactor filter ultrafiltration up-flow sludge blanket reactor

42.1 Introduction Olive oil owes some of its magic health-beneficial properties to the presence of phenolic compounds (PCs), which are also called olive biophenols (OBP). However, during olive oil production, a significant amount of these compounds remains in the olive oil by-products. This is why, there is now a growing interest in treatment and valorization of the olive oil industry wastes. In addition to the traditional method, two common processes of olive oil extraction including two-phase and three-phase systems are practiced. During this agro-

industrial activity a large amount of resources is consumed and a great deal of solid and liquid residues, possessing severe threats to the environment, is concomitantly produced.1 For instance, two main wastewater streams of olive washing wastewater and olive mill wastewater (OMW) are inevitably formed. Undoubtedly, the latter is an important by-product of olive oil production from both environmental and valorization potential points of views. OMW is an environmentally problematic effluent due to its high organic load, increased biologicalto-chemical oxygen demand ratio (BOD/COD), low biodegradability, and high levels of recalcitrant and phytotoxic substances.2 Despite the contribution of OMW phenolic content to the environmental pollutants, they conversely have diverse bioactivities, which seem to be beneficial to human health. Accordingly, in addition to finding economically viable eco-friendly solutions for OMW treatment to limit its environmental impacts, exploitation of this liquid via sustainable strategies to recover its potential bioactive compounds and use them in development of nutraceuticals, pharmaceuticals, and cosmeceuticals is the focus of current research.3 To obtain an overview of the recent advances in this field, this chapter reviews: G G

G

G

olive oil production processes; OMW source, its physical properties and chemical composition analysis; developments in treatment and valorization of OMW; and exploitation of OMW potentials as valuable source of nutraceutical.

42.2 Olive oil production processes Olive-oil industry is a strategic socioeconomic sector in Mediterranean countries, which accounts for about 95%

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00058-4 © 2021 Elsevier Inc. All rights reserved.

505

506

PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

FIGURE 42.1 The output of olive mills working with traditional and modern processes.

of the global production. Owing to the nutritional value and health benefits of olive products, the industry is rapidly growing. Countries such as Argentina, Australia, Iran, and South Africa as emergent producers are promoting olive cultivation and processing.4 Olive oil extraction encompasses the basic operations, followed by three major steps of crushing, malaxation, and separation (Fig. 42.1).5 Reception stage is the first step common to all oil mills. Cleaning, rinsing, fruit quality control including weight, acidity, fat yield, and the storage of olives all occur at this stage. Then, milling is carried out by traditional or modern installations followed by a beating at a proper temperature to favor oil extraction. Finally, the separation of phases is completed by means of various extraction techniques involving either traditional discontinuous batch (pressing) or continuous centrifugation (two- and three-phases) of the olive pulp. Until the advent of modern centrifugation methods, traditional system of press (occasionally with minor modifications) was the common way of olive oil extraction.6 This was a discontinuous system because of three separate stages of charge formation,a pressing, and removal of the pressing mats. With natural decantation of the liquid phase, the oily part is separated from the water phase.

a b

This process yields the following products and byproducts (on average per 1 ton of olives): G G

G

“virgin olive oil”b (200 kg); “spent olives,” a solid residue with 25% water and 6% oil (400 kg); and “vegetable water” as liquid waste; a mixture of the olive juice with water added through the extraction process (0.4
0.6 m3).

In the press system the quality of produced oil gradually diminishes with increase in extracting pressure. Continuous (three- and two-phase) systems with horizontal centrifuges (decanters) were invented to increase the productivity of oil mills and improve the quality of the resulting oil. As demonstrated in Fig. 42.1, the oil, solid olive pomace (with 98%
99% of the phenols initially found in the olive fruit), and wastewater fraction are generated at the end of three-phase process. A second oil extraction (using organic solvents, generally hexane) from obtained pomace is needed, owing to its high moisture level (65%
70%). The crude olive-pomace oil (OPO) is the solventextracted crude oil product and refined OPO (ROPO) obtained from crude pomace oil by refining methods. OPO as a mixture of ROPO, and virgin olive oil is fit for

The ensemble of wagon, needle, and pressing mats piled up with the paste receives the name of “charge.” The oil directly obtained from ripe olive fruits without any further refining process.

Treatment and valorization of olive mill wastewater Chapter | 42

human consumption. On average, 200 kg oil, 500
600 kg pomace (with 4% oil), and 1
1.2 ton of OMW (with 1% oil) per 1 ton of olives is produced through three-phase system.1 The amount of OMW produced in three-phase system is three-times of that produced through pressing approach. The rapid expansion of olive oil industry and illegally dumping of wastewaters of olive oil mills generated during both traditional and three-phase systems raised demands for legislation regarding the treatment of this environmentally hazardous waste and encouraged the development of new technologies. With this regard a new eco-friendly two-phase centrifugation system was introduced to the market in the early 1990s, which could save water during the extraction stage, reducing OMW production to 75% as compared to three-phase approach.7 This technology produces two fractions of liquid olive oil and semisolid pomace (or “alperujo” with more than 60% moisture and approximately 80% of the olive weight), which needs to be dried before second oil extraction/utilizations in management systems (Fig. 42.1). Typically, from processing of 1 ton of olives, 200 kg oil, 800
950 kg semisolid pomace, and 0.085
1.1 m3 OMW (with higher concentration of PCs8) is produced in this system. However, some manufacturers resist to shift to this eco-friendly system, claiming that the quality of oil obtained from three-phase approach is better.5 A comparative overview of these processes is presented in Table 42.1.

507

42.3 Source of olive mill wastewater, its physical properties and chemical composition The most detrimental ecological impact of olive oil production relates to the accumulation of massive quantities of OMW deriving from pressing and centrifugation systems. Total estimated volumes of OMW produced annually is about 10
30 million m3 in the Mediterranean area (2.5 L for each liter of olive oil), which is equal to 98% of worldwide production. It exerts negative effects on plants, soil properties, aquatic ecosystems, and air.9 The composition and characteristics of OMW varies greatly with regard to numerous factors (i.e., manufacturing process, variety and maturity of olive, region of origin, climatic factors, as well as cultivation methods and storage time).10 Generally, OMW is a mixture of water (83%
94%), organic (4%
18%) and inorganic substances (0.4%
2.5%) with a pH of 3
6.11 The organic substances could be lipids (1.0%
1.5%), pectin, mucilage, lignin, and tannins (1.0%
1.5%), carbohydrates (2.0%
8.0%), and free sugars of glucose, fructose, galactose, mannose, and sucrose traces (1.0%
4.5%), along with poly alcohols, amino acids and polyphenols, which participate in its high values of COD (37
318 g/L) and BOD (15
135 g/L).12,13 OMW has also high total suspended solids (TSS 6.0
69 g/L) and high concentrations of mineral fraction especially potassium and phosphorus.2 Carbon:nitrogen:phosphorus ratio of OMW from traditional pressing and continuous centrifugation

TABLE 42.1 A comparative overview of three processes of olive oil production. Method

Input

Output

Description

Press

Olives (1 ton)

Oil (200 kg)

Washing water (0.1
0.12 m3)

Solid waste (400 kg)

Less water (about 40%) Less wastewater Low capital cost Concentrated/highly polluted wastewater

Energy (40
63 kWh)

Wastewater (0.4
0.6 m3)

Threephase

Olives (1 ton) Washing water (0.1
0.12 m3) Decanter water (0.5
1 m3) Oil polishing water (0.01 m3) Energy (90
117 kWh)

Oil (200 kg) Olive pomace (500
600 kg) OMW (1
1.2 m3)

High water requirement (500 L/ton of olive paste) High OMW

Twophase

Olives (1 ton)

Oil (200 kg)

Washing water (0.1
0.12 m3)

Semisolid pomace (800
950 kg)

Energy (,90 kWh)

OMW (0.085
1.1 m3)

Eco-friendly Less OMW Save water and energy (by 80% and 20%, respectively) Cheaper than three-phase (25%) Difficult treatment of semisolid pomace

Centrifugation

OMW, Olive mill wastewater.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 42.2 Characteristics of olive mill wastewater. Parameter

Unit

Value

Parameter

Unit

Value

Conductivity

mS/cm

5
81

Carbohydrate

g/100 g

2
8

pH




4.7
5.7

Pectin

g/100 g

1
1.5

Water

g/100 g

83
94

Pb

μg/L

6.7 2 10

BOD

g/L

13.5
46

Cd

μg/L

0.03 2 1

COD

g/L

16.5
190

Fe

μg/L

6.5 2 23

Organic compounds

g/100 g

4
18

Zn

μg/L

2.94 2 4.98

Inorganic compounds

g/100 g

0.4
2.5

Cu

μg/L

2.4 2 2.96

Dry residue

g/L

11.5
90

Mn

μg/L

0.9 2 20

Total phenol

g/100 g

0.5
24

Mg

g/L

0.03 2 0.19

Total Solid

g/100 g

32
300

Ca

g/L

0.03 2 1.1

Total nitrogen

g/100 g

0.3
1.5

K

g/L

0.73 2 8.6

O&G

g/100 g

0.2
10

Na

g/L

0.05 2 0.8

Lipids

g/L

0.58
7

P

g/100 g

0.06
0.32

Sugars

g/L

1.3
4.3










BOD, Biological oxygen demand; COD, chemical oxygen demand.

methods are 100:1.77:0.94 and 100:1.34:0.70, respectively.1 The dry residue of OMW varies from 4.1% to 16.4%.14 According to PCs polarity, which are rather hydrophilic with partition coefficients (oil/water) of 6 3 1024
1.5, approximately 53% of their initial concentration in olive fruit (1%
3% of the fresh pulp weight) is transferred into OMW (about 1.0%
1.8% w/v) with only 2% ending up in olive oil (0.05% w/w).3 The degradation of PCs and high concentrations of lignin and tannins (52.27
180 g/L Pt
Co unitsc) give OMW a cloudy reddish-brown to black color and a fetid odor.4 More details about the chemical composition of OMW have been collected in Table 42.2. The overall amount of OMW produced in top 10 producer countries according to the International Olive Council in 2016 has also been demonstrated in Fig. 42.2. The OBP of OMW (0.5
80 g/L) are greatly diverse in nature depending on varietal, physiological, geographical, seasonal, environmental, and pathological factors, as well as the type of olive mill applied for the oil extraction.15 More than 100 different OBP have been characterized in olive products, with more than 50 found in OMW.16 They either inherently exist in olive fruit or generated through extraction procedures. Phenolic profile of OMW covers various types of simple phenols, phenolic alcohols in

c

particular, phenolic acids, flavonoids, isochromans, lignans, secoiridoids, and high molecular weight compounds. The most abundant phenyl alcohols in OMW are tyrosol (TY) and hydroxytyrosol (HT) followed by cornoside and catechol.16 Vanillic, p-coumaric, sinapic, syringic, and caffeic acids are the most frequently reported phenolic acids in OMW. Oleuropein (OL), an ester of elenolic acid and HT, is the characteristic substance of Oleaceae. It has also been found in OMW. Among other identified secoiridoids in fragmentation profile of OMW are ligstroside, elenolic and elenolic acid glucoside, HT glucoside, and verbascoside. Some derivatives of secoiridoid such as di-aldehyde of 3,4dihydroxyphenyl-elenolic acid bound to HT, hydroxytyrosyl acyclodihydroelenolate (HT-ACDE), and comselogoside are also identified in OMW. Flavonoids (known by C6
C3
C6 skeleton) with a variety of hydroxylated, methoxylated, and glycosylated derivatives are among the other reported OBP in OMW. This includes quercetin, anthocyanin, apigenin, hesperidin, and cyanidin flavone as the most important and successfully isolated ones from OMW. Some studies have also reported the presence of OBP with molecular weights of 0.6
5.0 kDa in OMW mixture possessing good antioxidant properties. Polymerin is a complex metal polymeric organic

The Platinum
Cobalt (Pt
Co) scale is a color scale introduced in 1892 to evaluate pollution levels in wastewater.

Treatment and valorization of olive mill wastewater Chapter | 42

509

FIGURE 42.2 OMW production and its total phenolic content by Mediterranean countries. OMW, Olive mill wastewater.

mixture, as a derivative of humic acid, with strong antioxidant capacity that has also been found in OMW.17

42.4 Developments in treatment and valorization of olive mill wastewater The polluting power of 1 m3 of OMW equals 200 m3 of domestic sewage18 as a result of the physicochemical properties of acidic OMW, especially its high COD and BOD values, as well as the existence of a variety of complex low-degradable substances. Due to lack of strict regulations, uncontrolled discharge of OMW (produced at high flow rates) into water streams is considered a serious environmental issue. On the other hand, the results of different studies are in favor of potential useful applications of different OMW phenols. Consequently, an extensive research is underway aiming at either to detoxify or to recover the valuable compounds of OMW. The examined methods including physicochemical and biological including integrated processes are briefly described in Tables 42.3 and 42.4, respectively.

42.4.1 Removal of phenolic compounds 42.4.1.1 Physical methods Physical methods are usually applied as common pretreatment approaches for the removal of solids. Investigations

have established that 95% of PCs are removed through applying sand filtration.19 Separation of compounds, based on their molecular weight through micro-, ultra-, and nanofiltration, applied in series, removed 99% of COD.20 Up to 70% removal of COD and 95%
99% lipid (such as oleic acid) removal could be achieved using sedimentation, filtration, and centrifugation methods, separately.21 In addition, these methods (except to sedimentation) were able to remove 10%
13.1% of polyphenols.22 Combination of the physical methods have also been examined. Applying a combination of centrifugation/filtration/activated carbon adsorption achieved 94% and 83% PCs and total organic compounds (TOC) removal, respectively.23 Thermal treatment of OMW (i.e., evaporation and distillation), aiming at condensation of the waste materials, removed 20%
80% of COD.24 However, due to high-energy demand and operating costs, these methods are known as ineffective approaches.21 To reduce the energy consumption and waste volumes, irreversible thermal options (combustion, cocombustion, and pyrolysis) have been investigated,25 but emission of toxic substances and requirement of expensive installations could limit the large-scale application of these methods too. Lime treatment as a relatively cheap process has been assessed for OMW treatment. More than 40% COD, 95% oil and grease, and 62%
73% removal of PCs has been reported via lime treatment in a range of 10
40 g/L (lime dose).26 Addition of lime or bentonite (beneficial for

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

TABLE 42.3 Nonbiological based technologies for treatment of olive mill wastewater. Category

Description

Result

Sedimentation Filtration Flotation Centrifugation

Total/partial solids removal

70% COD removal 30% oil recovery

Microfiltration Ultrafiltration Nanofiltration, Reverse osmosis

Separation of compounds based on their molecular weight

99% COD removal

Adsorption/desorption

Nonionic XAD4, XAD16, and XAD7HP resins were used

A final concentration of 378 g/L in gallic acid equivalents was reached

Evaporation distillation

Decomposition

Toxic gases production and high cost

Solar distillation

Waste elimination

Combined

Membrane distillation

Physical

Thermal

Physicochemical Neutralization Precipitation Adsorption

Addition of FeCl3, Ca(OH)2/MgO, Na2SiO3, adsorption on activated carbon

30%
50% COD removal 80%
95% COD removal by combining precipitation with adsorption

Oxidation advanced oxidation processes Chemical oxidation (Fenton reaction) Extraction Coagulation Combined

Ozonolysis, wet oxidation, O3/H2O2 photolysis, photocatalysis Ferric chloride catalyst was used for the activation of H2O2 Fenton-like reaction using FeCl3 as catalyst AOPs (O3/UV, H2O2/UV) Photo-Fenton WHPCO Ozonation process Liquid
liquid extraction/ethyl acetate CPE/Genapol X-080 CPE/Triton X-114 Electrocoagulation/NaCl Electrocoagulation/aluminum electrodes Ferric chloride coagulation, lime precipitation, electrocoagulation and Fenton’s reagent

40%
60% COD removal using simple oxidation practices 86% COD removal 99.8% phenols removal 99% phenols removal 97.44% phenols removal System operating at 50 C reduced considerably the COD, color, and total phenolic contents 80% total phenol removal 90% total phenol removal 89.5% total phenol removal . 96% total phenol removal 97% total phenol removal

AOP, Advanced oxidation process; COD, chemical oxygen demand; CPE, cloud point extraction; WHPCO, wet hydrogen peroxide photocatalytic oxidation.

removal of PCs with Mw larger than 1 kDa) after separation processes have increased selective removal efficiency of lipid and polyphenols by 99.5% and 43%, respectively.27 Electrocoagulation, a process based on the in situ currentinduced generation of coagulant, is considered a pretreatment step for biological techniques. Application of this approach with an aluminum anode achieved 91% of PCs removal and 52%
76% reduction in COD after 25
30 min reduction time at optimal pH of 4
6 and current density of 75 mA/ cm2. The COD reduction was 42% in case of iron anode.

The elimination rate of COD was proportional to current density, reducing also almost 96% of OMW color. After addition of NaCl (2 g/L) to OMW, 97% of PCs were removed by current density of 250 mA/cm2.28

42.4.1.2 Physicochemical methods To remove PCs from OMW, various advanced oxidation processes, based on in situ generation of hydroxyl radicals (HO ) with highly oxidation capacity, such as Fe (II)/H2O2, Fe(II)/H2O2/UV, ozonation (O3), O3/H2O2, G

Treatment and valorization of olive mill wastewater Chapter | 42

511

TABLE 42.4 Biological based technologies for treatment of olive mill wastewater (OMW). Category

Description

Result

Anaerobic processes

Dilution, nutrients addition, and alkalinity regulation Rhodotorula mucilaginosa CH4 Aspergillus niger P6 Two-phase anaerobic digester reactors

60%
80% COD removal for 2
5 digestion days 90% COD removal for 25 digestion days 83.45% total polyphenols removal 94.58% total polyphenols removal 70%
78% phenols removal

Aerobic processes

OMW cocomposting with sesame bark

5%
75% COD removal for few days of digestion

Biofilm, activated sludge

80% COD for longer period

CW, Lactobacillus paracasei

22.7% phenols removal

Pretreatment by Candida tropicalis

54% phenols removal

Mixing and digestion

Together with other agricultural wastes

75%
90% COD removal Nutrients and pH adjustment by combining wastes

Enzymatic

Laccase Trametes versicolor, Pleurotus spp. Strains Lentinula edodes

89% polyphenols removal69%
70% phenols removal90% polyphenols removal

Phenol oxidase Pleurotus ostreatus

90% polyphenols removal

Oxidation and biological processes

75% phenols 80%
99% COD removal

Pleurotus sajor-caju and Trametes versicolor, adsorption

From 85.3% to 88.7% phenols removal

Biodegradation, diffusion, photocatalytic degradation (TiO2), and adsorption processes (powdered activated carbon sorbent)

87% polyphenols removal

Electro-Fenton, anaerobic digestion, ultrafiltration

95% phenols removal

Filtration, adsorbent resins (XAD16 and XAD7HP), evaporation

99.99% polyphenols removal

Settling, centrifugation, filtration, and activated carbon adsorption

94% phenols removal

Combined

Electro-Fenton process, anaerobic digestion

100% polyphenols removal

Lime treatment, coagulation/flocculation/sedimentation/filtration

62%
73% phenols removal

COD, Chemical oxygen demand; CW, cheese whey’s.

H2O2/UV, hetero/homogeneous Fenton-like processes, TiO2/UV, and ZnO2/UV, have been investigated.29,30 Treatment of OMW with Fenton-like reaction using FeCl3 as a catalyst resulted in 99.8% removal of PCs, and applying O3/UV and H2O2/UV methods removed over 99% of PCs. Through photo-Fenton homo/heterogeneous photocatalytic oxidation (TiO2/UV, ZnO2/UV), 87% of COD, 84% of TOC, 97.44% of lignin, and 98.31% of TSS were removed from OMW.21 Among oxidation methods, ozonation has been the most selective one with 94.3% PCs and 77.8% COD removal owing to the strong oxidizing power of ozone.31,32 In addition, a combination of the mentioned physical and

physicochemical methods has been performed to maximize the COD and phenols removal.4,21

42.4.1.3 Biological treatments The OMW is characterized by low biodegradability due to the presence of phenols, particularly lignin-like polymers, which cause critical problems for chemical coagulant, H2O2, and filtration methods.33,34 Thereby, application of microorganisms such as bacteria, fungi, and archaea in different aerobic and anaerobic environmentally friendly processes have been suggested as advantageous biological remediation with biogas production.

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PART | 2 Nutritional, Pharmacological and Metabolic Properties of Olives and Olive Oil

However, due to the inhibitory effects of OMW, originates from its acidic pH and PCs, selection of the appropriate microorganisms seems to be the key step. Low sludge generation, less energy requirement, methane gas production, and easy restart of the system are among the benefits of these technologies.35 42.4.1.3.1 Aerobic digestion Aerobic digestion is used as a pretreatment step to reduce OMW toxicity through the reduction of PCs and improve the efficiency of anaerobic method. Many studies have investigated the application of different adapted species in pretreatment of OMW such as cultures of Aspergillus niger, Aspergillus terreus, Azotobacter chroococcum, and Geotrichum candidum.36 The given results have proved their positive effects with up to 80%
85% COD removal, 59%
87% reduction in toxicity, 65%
95% removal of total phenols, and 77% BOD removal.37 Treatment of OMW with indigenous microorganisms have achieved 56% and 90% of polyphenols removal by day 1 and 7, respectively.38 Selective removal (100%) of certain phenols by A. terreus has also been reported.39 It is claimed that aerobic treatment requires multiple dilution presteps to reduce COD level. 42.4.1.3.2 Anaerobic digestion In last 20 years, most of OMW treatment studies focused on anaerobic conversion of organic load into methane (a source of energy) and volatile fatty acids, using different reactors including anaerobic sequencing batch rector, periodic anaerobic baffled reactor, up-flow anaerobic reactor filter (UAF), up-flow sludge blanket reactor (UASB), and hybrid reactors, along with complete stirred tank reactor (CSTR) as the most famous and broadly used suspended-biomass reactors in treatment of OMW. Less sludge production, high consistency of microorganisms, generation of biogas energy, and reactivation are among the advantages of anaerobic digestion over the expensive aerobic treatment.38 Reduction of COD, PCs and TSS by 50%, 20%, and 10.3% has been achieved, respectively, by anaerobic digestion of OMW using CSTRs.38 UAFs, considered biofilm reactors using activated carbon, is a superior method operated for OMW treatment with noticeable adsorption capacity for PCs.40 UASB reactor can effectively operate under high organic load of OMW with high treatment performance and 85%
95% of COD removal at laboratory scale, but because of high energy consumption, the reactor is not suggested for industrial scale application.38,41 About 78% of PCs were removed from OMW, using twophase anaerobic digester reactors. Treatment of OMW through cocomposting using sesame bark (OMW sludge with sesame bark) has diminished TOC by 52.72%, and

water-soluble phenols by 72%.42 Filtration followed by anaerobic digestion and addition of CaCO3 reduced 88.7% COD and eliminated 60% of TOC, and 20% of polyphenols.43 42.4.1.3.3 Enzymes As environmentally sustainable tools, application of lignin degrader enzymes (i.e., phenol oxidase, peroxidase, and laccase) from white rot basidiomycetes has gained sufficient removal results in the treatment of OMW. For instance, 90% polyphenol reduction using Lentinula edodes laccase, 90% PCs removal by Pleurotus ostreatus phenol oxidase, 70% removal by laccase from Pleurotus spp. strains, and reduction of 87% phenols by Trametes versicolor have been reported.44
46 Industrial application and effectiveness of enzymes is limited due to their high cost and reliance on the environmental factors that affect both their optimal functions and stability.

42.4.1.4 Integrated techniques The efficiency of biological methods in treating OMW is low because of high organic content; thereby, complementary steps are required to maximize PCs removal. Within this line, various combination of methods such as centrifugation, filtration, adsorption, oxidation, and microbial technologies have led to great removal and even recovery of potential compounds. As a successful example, reduction of total PCs has reached from 85.3% to 88.7% by combined Pleurotus sajor-caju and T. versicolor, adsorption, and diffusion techniques.47 Removal of 87% of total polyphenols by photocatalytic degradation and adsorption processes;48 95% monophenolic compounds by electro-Fenton, anaerobic digestion, and ultrafiltration (UF);49 99.99% polyphenols through filtration, adsorbent resins (XAD16 and XAD7HP), and evaporation;50 as well as 100% reduction of total PCs by integrated electro-Fenton process and anaerobic digestion51 are among other effectual combined operations. ElectroFenton process with total phenols removal by 66% improved the performance of anaerobic digestion. Other integrated strategies, such as Phanerochaete chrysosporium/UF,34 anaerobic digestion/ozonation,52 as well as ozone/UV radiation,21 have successfully depolymerized and removed total PCs from OMW. Although all the studied methodologies are promising ways for OMW treatment, a few of them seem to be economically viable in industrial scales.

42.4.2 Recovery of phenolic compounds In spite of its hazardous environmental impact, OMW is also referred to as a great source of functional natural substances, in particular OBP. OMW phenols with high

Treatment and valorization of olive mill wastewater Chapter | 42

antioxidant activity are the object of interest to pharmaceutical, cosmetic, antiaging, and antiwrinkle sectors, as well as food industries.13 Accordingly, recovery of these valuable constituents has been the focus of recent research. As reactive chemical species, OBP are vulnerable to oxidation, hydrolysis, and polymerization mainly because of OMW characteristics, which can work as a reaction medium (containing enzymes, organic acids, and metals as catalysts, and proteins, polysaccharides, and phenols themselves as substrates). As a result, extraction and recovery of theses bioactive compounds is a tough analytical operation.53

42.4.2.1 Extraction In plants, phenols usually are in the forms of esters or glycosides. This phenomenon participates in distinguishing phenols based on the polarity variance. Accordingly, various extraction techniques including liquid
liquid extraction, solid
liquid extraction, and solid-phase extraction (SPE) with different extraction solvents such as water, ethanol, methanol, ethyl acetate, and acetone under variable time and temperature can be applied for selective or total PCs recovery. Simple PCs are more easily soluble in polar protic medium. Compounds such as gallic, cinnamic, and coumaric acids prefer water, dichloromethane, and acetone, respectively. Consequently, conventional, simple, and convenient solvent extraction of phenols from OMW is initially carried out with a polar protic solvent (i.e., food grade hydro-ethanolic or methanolic mixtures), followed by the extraction by solvents with low polarity.54 Recovery (up to 90%) of phenolic monomers of low and medium molecular weight has been carried out via liquid
liquid extraction method with ethyl acetate as the most appropriate solvent.55 The extraction power of solvents for HT follows ethyl acetate . methyl isobutyl ketone . methyl ethyl ketone . diethyl ether.56 A good selective recovery of HT (85.46%) was achieved using continuous counter-current liquid
liquid extraction, purification by chromatographic systems, yielding 1.225 g HT/L of OMW.35 Liquid
liquid extraction as a pretreatment of membrane filtration is successfully used to recover polyphenols, but attention has been drawn to the development of other techniques due to the high volumes of solvent needed in liquid
liquid extraction, being timeconsuming process, and the negative impacts on health and environment.35,57 As an alternative to solid
liquid extraction, simple and clean SPE has achieved a recovery of OBP (60%) from OMW using diethyl ether.35 To specifically recover HT (1 g/L of OMW), reversed phase SPE has been performed. Cloud point extraction (CPE), as a clean technology using only 4%
12% surfactant volumes, has obtained 89.5% and 100% recovery of total PCs and

513

tocopherols, respectively, with Genapol X-080.58 A 96% successive recovery of individual phenols is also achieved using CPE with a total of 4%
6% Triton X-114.59 Nonetheless, all these methods are economically inappropriate enough to be ignored for industrial scale use. Several high-efficient and cheap nonconventional methodologies such as sub- and supercritical fluid extractions and mechanical pressurized liquid extraction (with reduction in extraction time, process temperature, and solvent consumption), as promising tools for the recovery of valuable compounds (polyphenols, fatty acids, and tocopherols) from olive by-products have been examined. Green supercritical fluid technology (SFT) by the omission of organic solvent and using supercritical fluids as the result of high pressure and temperature possessing liquid-like solvent power and gas-like diffusivity has been used as a clean and nontoxic solvent to recover potential compounds. As intriguing procedure with significant reduction in oxidation, SFT applies CO2 as the most common nonexplosive supercritical fluid with relatively low viscosity and low surface tension of the system. Alteration in density through adjusting the pressure and temperature, as well as addition of cosolvents could change the systems’ extraction selectivity. Fast pressurized liquid extraction of PCs from OMW based on applying organic solvents at high temperature and pressure has also been performed using reduced volumes of methanol/ ethanol
water as the most efficient solvent.53 However, expensive instrumentation is required to provide high pressure, which makes it not cost-effective at industrial scale. There are some other technologies such as laser ablation, high voltage electrical discharge, and pulsed electric field used for nutraceutical recovery from agricultural wastes but not in the case of OMW, which require to be investigated.

42.4.2.2 Membrane technology Membrane (filtration) technology is the most promising system for the recovery of biophenols at industrial scale.60 This safe, cheap, and efficient system separates PCs selectively based on their molecular mass. Various filtration techniques of microfiltration (MF), UF, direct contact membrane distillation process with polytetrafluoroethylene, fluoropolymer membrane, nanofiltration (NF), vacuum membrane distillation, osmotic distillation, and reverse osmosis have been studied to recover phenols and remediate OMW. Three different fractions of PCs with concentrations of 2.5
5.3, 1.4
3.1, and 1.4
3.1 g/L were recovered via MF, UF, and NF, respectively. Combination of MF, NF, micellar-enhanced UF with an anionic surfactant/a hydrophobic polyvinylidene fluoride membrane (74% rejection rate of polyphenols using SDS under optimum pH of 2) and hydrophobic polypropylene

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membrane contactors with Cyanex 923 (97%
99% recovery of phenols in contact time of 5
6 min) have also been examined for phenol extraction.21,61 A combination of enzymatic pretreatment/three-phase membrane system and A. niger β-glucosidase/MF/UF have been successfully employed for the recovery of OMW polyphenols and HT, respectively.62

42.4.2.3 Adsorption Adsorption technology is one of the most commonly applied processes for the recovery of polyphenols.63 This approach is known as a low cost, simple, and reusable system, minimizing the transformation of the target compounds. By using synthetic resins, it is possible to selectively recover PCs from OMW. Commercially available resins such as Amberlite XAD or Duolite, Amberlite XAD/Sephadex LH20, and Amberlite XAD/Lewatit EP have been used to recover HT from OMW.64 Specifically, 77% recovery of HT was accomplished by means of integrated adsorption
desorption using Isolute ENV 1 resin. Low-cost ecofriendly adsorbents, such as banana peel, were demonstrated to have adsorption capacity up to 80% for phenols.65 Various procedures based on adsorption capacity of the stationary phase such as continuous counter current extraction (1.0 g of purified HT per 1 L of OMW), middle-pressure liquid chromatography, and preparative scale thin layer chromatography (91 mg/L of OMW) with 80% purity have been investigated to recover HT from OMW. High stability of HT at harsh conditions (200 C
220 C and 1.0%
1.5% sulfuric acid catalyst) is advantages for recovery processes.66 Sometimes some chemical treatments are applied prior to separation. For instance, HT was obtained through the hydrolysis of OL by esterases, or acid hydrolysis of secoiridoid derivatives and verbascoside.67

42.5 Exploitation of olive mill wastewater potentials as valuable source of nutraceutical Although the OBPs contribute to OMW environmental hazards, they conversely have diverse bioactivities with recognized health attributes, including antioxidant, antiinflammatory, cardiovascular, immunomodulatory, gastrointestinal, endocrine, and respiratory, as well as antimicrobial and chemotherapeutic effects along with the effects on central nervous system.67,68 Results of many in vitro, ex vivo, and in vivo studies support these pharmacological activities of OBPs, which relates to prevention of diseases and promotion of human health. It has been reported that OBPs represent potential specific and nonspecific mechanism of actions through binding to various molecular targets at cellular and subcellular levels. However, there are also some evidences

about the prooxidant capacity of some OBP, which deserves further investigations.16

42.5.1 Antioxidant activity Under oxidative stress resulting from the production of reactive oxygen species (ROS) during physiological activities, the function of biological macromolecules is disrupted, which may trigger degenerative diseases including but not limited to cancer, diabetes mellitus, and neurodegenerative disorders. It has been reported that OBP, orthodiphenolic compounds in particular, from OMW can significantly decrease the negative effects of ROS through antioxidant mechanisms.66 No doubt that methoxylated derivatives of catechols have improved lipid solubility that makes them more effective antioxidants in lipid systems.69,70 Antioxidant capacity of OBP has been assessed by different methods such as low-density lipoprotein (LDL) oxidation, DPPH radical scavenging activity, and superoxide anion scavenging.9 The antioxidant activity of caffeic acid, an important hydroxycinnamic acid derivatives from OMW, was investigated in vitro against tertbutyl hydroperoxide induced oxidative stress in U937 human monocytic cells (showing an increased resistance to oxidative challenge)71 and in vivo against reactive species of oxygen and nitrogen.72 Vanillic acid, verbascoside, elenolic acid, OL, and p-coumaric acid are among other compounds of OMW, which have been evaluated for their antioxidant associated health benefits. Extracted HT from OMW showed antioxidant activity in the plasma and liver of rats. It worked as hypoglycemic and antioxidant agents, hence alleviated oxidative stress and enhanced enzymatic defense in diabetic rats.73 In a different study, it was shown that HT could activate the mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells and protect human peripheral blood mononuclear cell against oxidative stress and DNA damage mediated by 2,3,7,8-tetrachlorodibenzodioxin.74 HT inhibits lipid oxidation in food matrices during chilling storage.67,75 Vanillic acid exhibited an alkylperoxyl radicalscavenging activity comparable to rutin, up to ninefold more than that of quercetin and 1.5-fold of α-tocopherol.76 OBPs also exert their antioxidant activity via induction of endogenous antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, γ-glutamylcysteine synthetase, and quinone reductase through the activation of antioxidant responsive elements.77

42.5.2 Antimicrobial effects Antibacterial activity of OMW correlates to its phenolic content, being more effective on Gram-positive bacteria. A large number of studies have extensively investigated a

Treatment and valorization of olive mill wastewater Chapter | 42

vast array of in vitro, in vivo, and in clinical trials antibacterial, antifungal, antiviral, and antiprotozoal activities of PCs from OMW. The compounds OL, HT, 4hydroxybenzoic acid, vanillic acid, elenolic acid, and pcoumaric acid are among the most active bactericidal.16 Phytotoxic catechol from OMW has also shown antimicrobial activity against plant pathogens. There is evidence that as the major OBP of OMW, HT is active against Pseudomonas savastanoi but represents no activity against Bacillus subtilis, Saccharomyces cerevisiae, or Escherichia coli.78 The existing results introduce OL, vanillic acid, and caffeic acid as the most active antifungals.67 Antiviral OBP act through interference with the viral amino acid production and prevent virus shedding, replication, and its entry to cells, thereby stimulate phagocytosis.79 Among OBP of OMW, antibacterial elenolic acid (calcium elenolate) has been reported to act also as antiviral acid.80,81

42.5.3 Antiinflammatory activity Oxidative stress increases progression of cancer via enhancing inflammation because of the activated nuclear factor kappa B (NF-κB). The OBP represent antiinflammatory activity through the inhibition of proinflammatory enzymes and downregulation of proinflammatory cytokines. Reduction of carrageenan-induced inflammation in rats and postprandially suppression of vascular inflammation markers are among antiinflammatory activities of OBP.82 OL extracted from OMW has been suggested as antiinflammatory compound due to its ability to inhibit 5lipoxygenase and identified in human whole blood cultures.75 HT has inhibited leukocytes leukotriene B4, and prostaglandin sparing.83 Caffeic acid from OMW through inhibition of 5-lipoxygenase (more active than TY and less active than HT and OL) can serve as an antiinflammatory compound. Verbascoside is also as an antiinflammatory agent of OMW, which acts through multiple mechanisms.16,84

42.5.4 Cardiovascular effects Oxidative stress is also a momentous factor in hypertension. The OBP possess efficient antihypertensive impacts, control blood pressure through the proposed mechanisms of dilatations of the blood vessels, and inhibition of calcium channels. Both in vitro and in vivo studies showed the antiplatelet effects of OBP (especially HT and TY) in rats via inhibition of TNF-α, cAMP-phosphodiesterase.85 In vitro studies showed that TX-B2 production was reduced by HT from OMW. It also reversed vascular dysfunction caused by enhancing levels of ROS due to hypercholesterolemia in an in vitro study.16,85 Extracted HT from OMW, as an approved nutraceutical, possess the

515

ability of maintaining healthy LDL cholesterol values, lipid antioxidation, and cardio-protective effect in human cells.86 Decrease in superoxide anion production by cultured human promonocyte cells (THP-1) has proved antiatherogenic activity of TY.87 Antiatherogenic activity of OBP, especially HT, TY, and OL, has broadly studied both in vitro and in vivo. They cause an increase in the resistance of LDL to oxidation and high-density lipoprotein levels.88 Among odiphenols, HT, and OL are the most bioactive phenols of OMW with protective activity against LDL oxidation shown in the in vitro studies.89 OL has also exhibited antihypertensive (vasodilator), antibacterial, antimycoplasmal, and cardio-protective effect on rat heart.75 OL by the inhibition of LDL oxidation and platelets aggregation, fatty acid composition of rat heart, enhancing nitric oxide production, acts as antiatherogenic and cardio-protective biocompound.75,90 The hypoglycemic activity of OL in normal and diabetic rats has also been reported. Verbascoside from OMW was shown to act as an antihypertensive, angiotensin-converting enzyme inhibitor agent. It is also a cardioactive (chronotropic, inotropic, and coronary vasodilator mediated through cAMP). Verbascoside acts as antiatherogenic through plasma lipid peroxidation. It moderates oxidative stress and erythrocyte membrane fluidity.91 Protection against the development of left ventricular systolic dysfunction, suppression of the cardiotoxic activities of doxorubicin, in vivo antiarrhythmic, cardioprotective activities, inotropic, chronotropic activities, and increased coronary perfusion rate are among other reported cardioprotective properties of OBP.16

42.5.5 Immunomodulatory effects Oxidation-sensitive immune cells require a redox balance to regulate immune response. Oxidized macromolecules (such as DNA and proteins) as immunogenic agents lead to autoimmune disorders. Studies have shown that odiphenols, HT, and OL protect human erythrocytes and DNA against oxidative damages. There is evidence related to adjustment of immune functions by OBP (especially HT and TY) through enhancing the levels of cytosolic calcium in lymphomonocytes, protection of human neutrophils by scavenging H2O2, reduction of proinflammatory cytokines in blood, inhibition of IFN-γ and IL-17 production, dose-dependent production of protective oxidized LDL autoantibodies, as well as inhibition of calcineurin.16

42.5.6 Gastrointestinal effects The potential chemo-preventive activities of OBP of OMW have been investigated. Studies have proved HT

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and OL as chemo-preventive against different strains of Helicobacter pylori. Inhibition of peroxynitrite-dependent DNA damage, induction of cytochrome c-dependent apoptosis, along with inhibition of tumor cells proliferation are among chemo-preventive activities of HT, beneficial against peptic ulcers and gastric cancer.67 In vivo investigation of OBP extracted from OMW has revealed their modulatory impact on digestive enzymes. Inhibition of α-amylase activity by HT, activation of pepsin and inhibition of trypsin, lipase, glycerol, glycerokinase, dehydrogenase, and glycerol-3-phosphate dehydrogenase by OL are among the reported gastrointestinal activities of extracted OBP. Besides, OL possesses endocrinal activity by thyroid stimulation and modulation of hypolipidemic
hypoglycemic activity.67

42.5.7 Endocrine effects Oxidative stress plays a major part in diabetes pathogenesis. Extracted OBP from OMW act as antidiabetic agents through the inhibition of carbohydrate digestive enzymes (α-amylase and α-glucosidase).16,92 OBP also possess secretagogue activity and protect β-cells against oxidative stress. Inhibition of ROS generation, reduction of blood glucose levels in diabetic rats, hypoglycemic activity, and TGR5 (a receptor active in combating obesity) agonist activity are reported in vivo bioactivities of extracted HT and OL from OMW.73 Lower prevalence of osteoporosis in ovariectomized rats (an experimental model of postmenopausal osteoporosis) as the result of hormonal changes has linked to OBP, in particular, HT, TY, and OL.93 In vitro reduction of adipocyte differentiation at the gene expression level is among reported impacts of OL.94

42.5.8 Chemo-preventive effects Cellular responses to oxidative stress to reinstate redox balance lead to a wide range of consequences from apoptosis to necrosis. At oncogenesis stage, PCs act efficiently through induction of apoptosis and inhibition of proliferation. They control cell growth and alleviate respiratory tract, upper gastrointestinal, breast, and colorectal cancers. Studies have shown the inhibition of H2O2induced DNA damage and proliferation in gastric and colon cancer cell lines treated by OMW extracts, indicating the presence of antiproliferative agents in OMW.95 Phytotoxic catechol has shown antimicrobial activity against plant pathogens, acted as carcinogenic in rat stomach, possessing antioxidant and anticancer activities. Rutin is another antioxidant OBP found in OMW with hepatoprotective, antiatherogenic (less active than quercetin), and antiinflammatory (only in chronic inflammation) activities. Studies have shown that this OBP works as a

blocking agent for heterocyclic amine-induced rat liver carcinogenicity proving its chemo-preventive activity.16,67

42.5.9 Respiratory effects Inflammatory lung diseases are associated with oxidative stress. As an interesting example, the effect of HT from OMW in the prevention of consequences of passive smoke
induced oxidative stress in rats has been demonstrated.96

42.6 Safety concerns The safety of natural biophenols as xenobiotic compounds, with the ability to cause adverse effects, is under investigation. Inducing oxidative stress, reversible gastrointestinal and renal irritation, cytotoxic activity, acute amyloid leukemia, antinutrient properties, impairment of carbohydrate absorption, inhibition of heme iron absorption in Caco-2 cells are among the suspected potential adverse effects of OBP,16,97,98 which need to be better evaluated. Moreover, based on synergistic, antagonistic, and additive interaction of OBP, along with drug interference with natural products, there is need to clinically evaluate the drug-OBP interactions.

42.7 Concluding remarks Although progress in developing improved processes for olive oil production is challenging, especially in terms of oil quality and quantity, the advances in treatment and valorization of the wastes and residues of this agroindustrial activity have reached new horizons. Various biological and nonbiological techniques have been examined, separately and in combination, for both treatment and recovery of biophenols from olive oil wastewater. The results can be categorized based on efficacy and feasibility. Nonetheless, most of these techniques are in queue to be applied in large-scale plans mainly due to the lack of cost-effectivity. Yet, research is underway to address the economical points of these solution and shed more light on the real value of OMW and its components.

Acknowledgment Dr. K. Haghbeen appreciates the financial support provided by the National Institute of Genetic Engineering and Biotechnology of I.R. Iran (project 713).

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86. Fki I, Ghorbel H, Allouche N, Sahnoun Z, Sayadi S. Phenolic extract and purified hydroxytyrosol recovered from olive mill wastewater prevent in vivo low-density lipoprotein oxidation and modulates lipid metabolism in rats fed a cholesterol-rich diet. J New Sci. 2015;. 87. Nakbi A, Dabbou S, Champion S, et al. Modulation of the superoxide anion production and MMP-9 expression in PMA stimulated THP-1 cells by olive oil minor components: tyrosol and hydroxytyrosol. Food Res Int. 2011;44:575
581. 88. Jemai H, Fki I, Bouaziz M, et al. Lipid-lowering and antioxidant effects of hydroxytyrosol and its triacetylated derivative recovered from olive tree leaves in cholesterol-fed rats. J Agric Food Chem. 2008;56:2630
2636. 89. Amarowicz R, Pegg RB. The potential protective effects of phenolic compounds against low-density lipoprotein oxidation. Curr Pharm Des. 2017;23:2754
2766. 90. Daccache A, Lion C, Sibille N, et al. Oleuropein and derivatives from olives as Tau aggregation inhibitors. Neurochem Int. 2011;58:700
707. 91. Liu M-J, Li J-X, Guo H-Z, Lee K-M, Qin L, Chan K-M. The effects of verbascoside on plasma lipid peroxidation level and erythrocyte membrane fluidity during immobilization in rabbits: a time course study. Life Sci. 2003;73:883
892. 92. Hamden K, Allouche N, Jouadi B, et al. Inhibitory action of purified hydroxytyrosol from stored olive mill waste on intestinal disaccharidases and lipase activities and pancreatic toxicity in diabetic rats. Food Sci Biotechnol. 2010;19:439
447. 93. Puel C, Mardon J, Agalias A, et al. Major phenolic compounds in olive oil modulate bone loss in an ovariectomy/inflammation experimental model. J Agric Food Chem. 2008;56:9417
9422. 94. Santiago-Mora R, Casado-Dı´az A, De Castro M, Quesada-Go´mez J. Oleuropein enhances osteoblastogenesis and inhibits adipogenesis: the effect on differentiation in stem cells derived from bone marrow. Osteoporos Int. 2011;22:675
684. 95. Obied HK, Prenzler PD, Konczak I, Rehman A-u, Robards K. Chemistry and bioactivity of olive biophenols in some antioxidant and antiproliferative in vitro bioassays. Chem Res Toxicol. 2009;22:227
234. 96. Visioli F, Galli C, Plasmati E, et al. Olive phenol hydroxytyrosol prevents passive smoking
induced oxidative stress. Circulation. 2000;102:2169
2171. 97. Ma Q, Kim E-Y, Han O. Bioactive dietary polyphenols decrease heme iron absorption by decreasing basolateral iron release in human intestinal Caco-2 cells. J Nutr. 2010;140:1117
1121. 98. Lambert JD, Sang S, Yang CS. Possible controversy over dietary polyphenols: benefits vs risks. Chem Res Toxicol. 2007;20:583
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Part 3

Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

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Section 3.1

Tyrosol and hydroxytyrosol

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Chapter 43

Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil M. Emı´lia Juan and Joana M. Planas Departament of Biochemistry and Physiology, Facultat de Farma`cia i Cie`ncies de l’Alimentacio´ and Institut de Recerca en Nutricio´ i Seguretat Alimenta`ria (INSA-UB), Universitat de Barcelona (UB), Barcelona, Spain

Abbreviations ACF AOM DSS DMH FAP IL-6 IL-10 MAM MDF ROS TNF-α

aberrant crypt foci azoxymethane dextran sulfate sodium 1,2-dimethylhydrazine familial adenomatous polyposis interleukin 6 interleukin 10 methylazoxymethanol mucin depleted foci reactive oxygen species tumor necrosis factor α

43.1 Introduction Colorectal cancer has been reported as the third most common malignancy worldwide with an incidence of a 10.2% of all diagnosed cancers and a mortality of a 9.2% in both genders.1 Around 5%
15% of patients show genetic predisposition to developing the disease due to inherited gene mutations, namely, the familial adenomatous polyposis (FAP) or the hereditary nonpolyposis colorectal cancer.2 However, most cases are consequence of sporadic gene mutations linked to environmental risk factors, such as tobacco, alcohol, diet, or physical exercise,3 meaning that they might be partly prevented by way of adequate lifestyle and dietary habits. In this context, several epidemiological studies have provided evidence that adherence to the Mediterranean diet is associated with a moderate reduction of colorectal cancer risk.4
6 This dietary pattern is characterized by a high consumption of

vegetables, legumes, cereals, fruits, pasta, bread, nuts and seeds, moderate amounts of fish, white meat, dairy products and eggs, as well as small ingestion of red meat and wine.7 Noteworthy, fresh vegetables and fruits are a source of a vast array of minor bioactive components that have demonstrated to exert a protective role in front of colorectal cancer, such as trans-resveratrol from grapes,8 sulforaphane from cruciferous vegetables,9 or luteolin from celery and parsley,10 to mention only a few examples. For this reason, chemoprevention with natural compounds is currently considered an intervention strategy for the control and constraint of carcinogenesis.11 Table olives and olive oil are key components of the Mediterranean diet, as demonstrated by the fact that these commodities constitute a regular food in the different countries and regions bordering the Mediterranean Sea. The fruit of Olea europaea L. is a versatile product with uses ranging from direct intake as a snack to an ingredient in different recipes, while olive oil is employed for both cooking and seasoning.7 Table olives and olive oil not only contain important nutrients but also minor components with nutraceutical value, due to its unique composition, rich in monounsaturated fatty acids, mostly oleic acid and minor bioactive compounds such as tocopherols, squalene, polyphenols, and pentacyclic triterpenes.12 Among the latter group of compounds, stands out maslinic acid (Fig. 43.1), which is a gaining interest in the recent years, due to its lack of harmful effects13 along with its numerous health protective activities, namely, antidiabetic, antioxidant, antiinflammatory, cardioprotective, neuroprotective, and antitumoral.14

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00026-2 © 2021 Elsevier Inc. All rights reserved.

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43.2 Maslinic acid, a pentacyclic triterpene from Olea europaea L Although maslinic acid was first isolated in 1927 from the leaves of Crataegus oxyacantha L, the identification of this compound in Olea europaea L. did not come until 1961,15 and in 1994 was established as the main pentacyclic triterpene in table olives.16 The synthesis of maslinic acid in Olea europaea L. has been comprehensively evaluated by Stiti and coworkers17 that postulated a biosynthetic pathway from 2,3-oxidosqualene. In the olive tree the generation of pentacyclic triterpenes from 2,3-oxidosqualene requires the formation of different carbocationic intermediates (Fig. 43.2). The oleanyl cation lead to ß-amyrin, and different oxidation steps catalyzed by cytochrome (CYP)

FIGURE 43.1 Chemical structure of maslinic acid.

FIGURE 43.2 Synthesis of maslinic acid in Olea europaea L.

450 enzymes produces, in first place, the alcohol erythrodiol, then oleanolic acid, and a further hydroxylation yield maslinic acid.17 Maslinic acid is present in the waxes of the fruits and leaves of Olea europaea L. where it develops a physical barrier that acts as a first defense against pathogens as well as preventing water loss.18 Therefore table olives hold important concentrations of maslinic acid, although the content depends not only on different factors, such as the variety, cultivar, climate, degree of ripening on the time of harvesting but also on the method of elaboration of olives and postfermentation conditions. For these reasons, their content in this food (Table 43.1) could range from 287.1 6 66.6 mg/kg in the Manzanilla variety19 to 2508 6 68.8 mg/kg in Arbequina.21 Concerning olive oils, since they are obtained with processes involving pressing which disrupt the surface waxes, part of maslinic acid is transferred to the oil. Nevertheless, the concentration of maslinic acid in the oil is much lower than in the fruit and depends on its quality (Table 43.1). Virgin and extra virgin olive oil contain values ranging from 19 to 251 mg/kg.21 Noteworthy that maslinic acid has not been detected in any other edible oil, except in the ones obtained from the fruits of Olea europaea L.

Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil Chapter | 43

TABLE 43.1 Content of maslinic acid in Olea europaea L. Food

Maslinic acid

References

527

the regulation of cell cycle progression are the most common feature of transformed cells, the antiproliferative activities described for this compound from table olives and olive oil demonstrate its potential use as a chemopreventive agent against colon cancer.

Table olives (mg/kg fresh weight) Manzanilla, plain black

287.1 6 66.6

[19]

Manzanilla, plain green

384.1 6 50.0

[19]

43.3.2 Studies on apoptosis

Hojiblanca, plain green

904.7 6 259.6

[19]

Gordal, plain green

414.2 6 89.3

[19]

Kalamata, plain natural black

1318 6 401

[19]

Marfil

1740 6 60

[20]

Empletre

1862 6 183

[21]

2508 6 68.8

[21]

Apoptosis is an intrinsic cell death program that has a crucial role during normal mammalian development and in regulating tissue homeostasis by eliminating unwanted cells. Dysregulation of apoptosis leads to various human diseases such as cancer and autoimmunity.27 Apoptosis may be prompted by several molecular pathways, of which the extrinsic and intrinsic pathways are the best characterized.28 The extrinsic or death receptor pathway is mediated by a subgroup of the TNF receptor superfamily called death receptors (TNFR1, FAS, and TRAIL) (Fig. 43.3). Receptor-mediated cell death is initiated by recruitment of adaptor proteins, like FADD, which bind to pro-caspases to generate a death-inducing signaling complex (DISC) that leads to the activation of caspase-8. Caspase-8 directly cleaves and activates caspase-3, the executioner enzyme of apoptosis. The intrinsic pathway also known as mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, or Smac. The release of cytochrome c into the cytosol triggers caspase-3 activation through the formation of the cytochrome c/APAF1/ pro-caspase-9-containing apoptosome. Smac promotes caspase activation through the neutralization of the inhibitory effects of IAPs. The receptor and the mitochondrial pathway can be interconnected at different levels, for example, through Bid, a protein of the Bcl-2 family. Activation of caspases is negatively regulated at the receptor level by FLIP, which blocks caspase-8 activation, and at the mitochondria by Bcl-2 family proteins and by IAPs. Effector caspases cause cell-wide specific proteolysis and dysfunction, including the labeling of the cell with “eat me” signals, thus allowing the apoptotic cell to be recognized and engulfed by phagocytic cells.27 Defects in apoptosis play a key role in tumor pathogenesis, allowing neoplastic cells to survive beyond their normally intended lifespan, subverting the need for exogenous survival factors, providing protection from hypoxia and oxidative stress as tumor mass expands, and allowing time for accumulative genetic alterations that deregulate cell proliferation, interfere with differentiation, promote angiogenesis, and increase cell motility and invasiveness during tumor progression. Consequently, compounds that can eliminate aberrant cell clones by the induction of apoptosis may have a chemopreventive or even a therapeutic potential.23

Arbequina

Olive oil (mg/kg) Olive oil, extra virgin

19
98

[22]

Olive oil, virgin

145
251

[22]

43.3 Cancer chemopreventive activity of maslinic acid in colon cancer cells in vitro 43.3.1 Studies on cell proliferation Impairment in the regulation of cell proliferation is a key event in the initiation stage of colorectal cancer, since once the cells start to divide, the development of more defects in key genes induce the persistence in cell growth,23 hence the relevance in finding compounds that elicit antiproliferative effects in transformed cells.23 The exposure for 72 h of increasing concentrations of maslinic acid to the human colorectal adenocarcinoma cell line HT-29 demonstrated that this pentacyclic triterpene was able to reduce cell growth in a dose-dependent manner with an IC50 of 101.2 6 7.8 μM without inducing any sign of nonspecific cytotoxicity.24 Reyes et al.25 reported similar results after the incubation of maslinic acid for 72 h in HT-29 and Caco-2 cells which resulted in an inhibition of growth with IC50 of 61 6 1 and 85 6 5 μM, respectively. Further experiments conducted by the same authors indicated that maslinic acid elicited this inhibition of proliferation by arresting cell cycle. The cell population was significantly increased in the G0/G1 phases, whereas the one in the S phase was reduced.25 Recently, the suppression of cell growth has also been reported for other cell lines such as HCT116 and SW480 with the use of a cell viability assay (MTT) that reported a dose-dependent inhibition of the viability with IC50 values of approximately 20 μM for both cell lines.26 Given that defects in

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 43.3 Schematic representation of the extrinsic and intrinsic pathways involved in caspase activation leading to apoptosis.

The antitumoral activity of maslinic acid on apoptosis was investigated in HT-29 human colorectal carcinoma cells. In the intrinsic cell death pathway the production of reactive oxygen species (ROS) in the mitochondria constitutes a mechanism of apoptosis induction.28 Therefore the effect of maslinic acid over ROS production at several time points was evaluated in HT-29 (Fig. 43.4A). The incubation of HT-29 cells with 150 μM of maslinic acid for 4 h showed a marked enhancement of the production of superoxide anion radical (O22 ) in the mitochondria.24 The generation of ROS may be restricted to 4 h after incubation with this pentacyclic triterpene, as ROS was detected neither at 2 h nor at 6 h. The production of ROS G

in the mitochondria only occurs in an early phase of apoptosis; thus this event suggests a role of ROS as secondary messengers that triggers a rapid release of cytochrome c from mitochondria into the cytosol, thus, in turn, activating procaspase-9 and the downstream effectors.24 In the inducement of apoptosis the activation of caspase-3 is an important achievement since this protease represents the converging point of different caspasedependent apoptosis pathways.28 Maslinic acid was able to enhance the activity of capase-3 in a time and dosedependent manner (Fig. 43.4B). Incubation of HT-29 cells with 150 μM of maslinic acid from 3 to 48 h demonstrated that this pentacyclic triterpene was able to increase

Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil Chapter | 43

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FIGURE 43.4 Effects of maslinic acid on apoptosis in HT-29 cells. (A) Detection of superoxide radicals in the mitochondria of HT-29 cells. Cells were incubated with the medium alone (control) and with 150 μM of maslinic acid. Time-dependent (B.1) and dose-dependent (B.2) caspase-3 activity in HT-29 cells. Results are mean 6 SEM (n 5 3). Data was evaluated by 1-way ANOVA and post hoc Tukey’s multiple comparison tests. Asterisks indicate different from control: *P , .05.

the activity of caspase-3 in a 334% with respect of control cells at 12 h. At 24, 36, and 48 h the enhancement of caspase-3 with respect to control cells was approximately of 1500%.24 In view of the response of maslinic acid at

24 h, this experimental time was chosen for the evaluation of the proapoptotic activity of this pentacyclic triterpene at concentrations.24 Incubations of HT-29 cells to increasing concentration of maslinic acid showed that already

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 43.5 Early (A) and late (B) apoptosis events in human HT-29 cells incubated without (control) or with 150 μM of maslinic acid. Cells that accumulated Hoechst 33342 dye due to membrane disintegration (A) were counted at 24 h and expressed as percentages relative to cell grown with and without maslinic acid. Cells that displayed nuclear fragmentation (B) were counted and expressed as the percentage of apoptotic cells. Arrows indicate apoptotic cells due to membrane disintegration (early apoptosis) or nuclear fragmentation (late apoptosis). Results are expressed as mean 6 SEM (n 5 3). Data was evaluated by Student’s t test. Asterisks indicate different from the control group: *P , .05.

25 μM was able to activate caspase-3 in a 329% with respect to the control cells.24 Once caspases are triggered, numerous target proteins broadly distributed throughout the cell are cleaved resulting in the morphological changes that are hallmark of programmed cell death.28 Hence, the achievement of apoptosis beyond the activation of caspase-3 lead to the disintegration of the plasma membrane is shown in Fig. 43.5. HT-29 cells incubated with 150 μM of maslinic acid for 24 h accumulated Hoechst 33342 dye in a 49.4% 6 2.9% with respect to the control group.24 Fig. 43.5 displays the confirmation with Hoechst 33258 staining of the full execution of apoptosis. When HT-29 cells were incubated with 150 μM of maslinic acid for 24 h, a nuclear fragmentation of a 30.8% 6 2.9% was observed.24 Subsequently, these results were further confirmed in HT-29 cells associating the activation of caspases with the involvement of the JNK-Bid signaling pathway via the activation of p53.29 Conversely, maslinic acid induced the activation of apoptosis in Caco-2 cells through a death receptor
mediated apoptotic mechanism.30 Consequently, the capacity of activating both pathways suggest a versatility in the mechanism of action of maslinic acid that could be of great use as a chemopreventive agent for colon cancer.

43.4 Cancer chemopreventive activity of maslinic acid in animal models in vivo The use of animal models constitutes an essential step in the assessment of the activity of natural bioactive compounds.

Although different experimental models of colorectal cancer have been described, they can be grouped into carcinogeninduced or genetic-based animal models.31,32 Both can mimic the alteration of different signaling pathways involved in colorectal cancer in humans, thus allowing an accurate investigation of the different steps that takes place in this disease.31,32 Noteworthy, the most common cancer is the nonfamiliar colorectal type and in rodents can be replicated with the use of carcinogens.32 Conversely, the use of transgenic mice has proven as a powerful tool to investigate the mechanisms underlying familial cancers such as human FAP and hereditary nonpolyposis colon cancer.31

43.4.1 Studies with experimental models induced by carcinogens In 1963, Laqueur tried to induce amyotrophic lateral sclerosis with the nuts of Cycas circinalis and found by chance that the rats suffered colon cancer.33 The analysis of the components of the nuts revealed cycasin as the active carcinogen. Then, in 1967, Druckrey and coworkers were able to establish that a structurally similar molecule, 1,2-dimethylhydrazine (DMH) was able to selectively induce the development of tumors in the colon of rats.34 Although since then, several other compounds like nitrosamines, heterocyclic amines or aromatic amines have also been proposed as colonotropic carcinogens, DMH and its metabolite azoxymethane (AOM) are the most widely used for this purpose.32 The DMH and AOM model holds a multistep

Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil Chapter | 43

531

FIGURE 43.6 ACF observed under a light microscope after staining of the colon with methylene blue. The images show the whole mount colon of control rats (A) and DMH-injected animals showing a topographic view of ACF with three crypts (indicated by a white arrow) (B). Original magnification, 3 10. The graphs display the effects of maslinic acid at the doses of 0, 5, 10, and 25 mg on the number of ACF in total colon (C) as well as ACF distribution in colonic segments (D). Results are expressed as mean 6 SEM, n 5 6
8. Data was assessed by the nonparametric Kruskal
Wallis test, followed by Dunn’s multiple comparison analysis. Asterisks indicate different from the control group: *P , .05; **P , .01. ACF, Aberrant crypt foci; DMH, 1,2-dimethylhydrazine.

development that displays morphological and molecular features such as human sporadic colorectal cancer.35 Hence, this valid and highly praised model is widely used for the evaluation of environmental, dietary, and chemopreventive compounds. After subcutaneous or intraperitoneal injection, DMH undergoes metabolization to AOM, which is further hydroxylated in the liver to methylazoxymethanol (MAM) through the activity of the inducible cytochrome P-450 isoform CYP2E1.35 MAM is relatively stable, with a half-life of approximately 12 h and reaches the colon where it transforms to methyldiazonium ion. This last carcinogenic metabolite is a highly reactive molecule that produces free radicals that bind to DNA leading to mutations.35 Consequently, DMH and AOM are effective colonotropic carcinogens that can be used to mimic human sporadic colorectal cancer and are usually applied either in short-term studies (6
12 weeks) where the animals are scored for aberrant crypt foci (ACF) and mucin depleted foci (MDF) or long-term studies (40 weeks) for the evaluation of the number of colonic tumors. In both types of approaches the chemoprevention can begin prior to the use of the carcinogen to assess the effect on the initiation phase or be applied during the promotion or progression phases.32,35 In a short-term study, ACF are the first morphological lesions in the development of colorectal cancer and are characterized by a marked hyperplasia.36 These biomarkers appear within 2
3 weeks after the application of the carcinogen and can be identified on the surface of the whole

mount colon mucosa after staining with methylene blue (Fig. 43.6) as crypts with increased size, thicker epithelial lining, enlarged pericryptal zone, and slit-like luminal opening.36 Another reliable biomarker, in these short-term studies, is MDF that constitute a more advanced staged than ACF. MDF are ACF with scarce or absent production of mucins secreted by goblet cells, which is a common feature of severe dysplasia (Fig. 43.7). Moreover, these lesions have been acknowledged in humans at high risk of colorectal cancer thus being considered a hallmark of malignant potential.37 The assessment of MDF also is carried out on the mucosa of whole mount colon after staining with high-iron diamine alcian blue (HID-AB) which dyes crypts with mucin production.37 The chemopreventive activity of maslinic acid was evaluated in a short-term study (7 weeks) in which early biomarkers of colon carcinogenesis were induced by three intraperitoneal injections of DMH (20 mg/kg).38 Under these experimental conditions, ACF and MDF appeared in the positive control groups (DMH1 /MA2 ) following a regional distribution along the colon, being present mainly in the middle and distal regions.35,36 The oral daily administration of maslinic acid at 5, 10, and 25 mg/kg starting 1 week prior to the injection of the carcinogen efficiently diminished the development of ACF in total colon in a 15%, 18%, and 33% (Fig. 43.6C). When the effect of maslinic acid was analyzed in the different segments, it is worth mentioning the fact that the three doses were more

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 43.7 MDF assessed after dying the colon with high-iron diamine/alcian blue/neutral red staining (HID-AB) and analyzed under a light microscope. Topographic features of the colonic epithelium of control animals (A) and DMH-treated rats displaying MDF with four crypts (B). Original magnification, 3 10. The graphs show the effects of maslinic acid at the doses of 0, 5, 10, and 25 mg on the number of MDF in total colon (C) as well as MDF distribution in colonic segments (D). Results are expressed as mean 6 SEM, n 5 6
8. Data was assessed by the nonparametric Kruskal
Wallis test, followed by Dunn’s multiple comparison analysis. Asterisks indicate difference from the control group: *P , .05; **P , .01. DMH, 1, 2-Dimethylhydrazine; MDF, mucin depleted foci.

active in the distal part of the colon were inhibitions of 25%, 22%, and 42% were found (Fig. 43.6D).38 Concerning MDF, maslinic acid elicited an important halt in the development of these dysplastic lesions even at the low doses of 5 mg/kg that showed an inhibition in total colon of 27% that reached a 51% when maslinic acid was administered at 25 mg/kg (Fig. 43.7C). The chemopreventive activity of maslinic acid was higher in the distal colon with reductions of 29%, 26%, and 45% observed after treatments with maslinic acid at 5, 10, and 25 mg/kg (Fig. 43.7D).38 Altogether, these results provide unequivocal evidence of the chemopreventive potential of maslinic acid in the initiation phase of colon cancer. The experimental model of colorectal cancer induced by DMH activated mutations in the DNA that altered different genes. In the first step of colorectal cancer, when hyperplastic ACF develops, the mutation of K-ras appears with a frequency ranging from 20% to 40%.37,39 This mutation triggers the activation of Ras and its downstream signaling pathways, namely, Raf/MEK/MAPK and PI3K/Akt/PKB.37,39 Moreover, in dysplastic MDF, mutations of the β-catenin gene activate the Wnt signaling pathways leading to cell proliferation.40 In addition, mutations of both K-ras and β-catenin have been suggested to be implicated in the upregulation of NF-κB, cyclin D1, COX-2, and iNOS which, in turn, leads to an enhancement of cell proliferation and a reduction of apoptosis.23 Considering all the processes involved in the early stages of the colorectal cancer

development, one plausible activity of maslinic acid could be the antiinflammatory properties described for this compound41,42 that could attenuate the depletion of mucins observed in animals induced with DMH.23 The antiinflammatory activities of maslinic acid were associated with its ability to constrain the transcription factor NF-κB associated to colorectal cancer since it was described to downregulate the expression of COX-2 and iNOs which were found in MDF41,42. The inflammation of the mucosa of the colon induced by the activation of the Wnt/β-catenin pathway triggered the activation of c-Myc, c-Jun and cyclin-D1 linked to cell proliferation as well as Bcl-2, Bcl-xl, and p53, associated to apoptosis.23 Hence, the inhibition of maslinic acid toward the development of hyperplastic ACF and dysplastic MDF suggest that this pentacyclic triterpene could elicit an important role in the initiation steps of colorectal cancer. The AOM/dextran sulfate sodium (DSS) model mimics the colitis-related colorectal carcinogenesis and has also been employed to evaluate the chemopreventive activity of maslinic acid in an assay that scored tumors as endpoint of the study.26 In this experimental model, AOM at 10 mg/kg was administered by intraperitoneal injection and 1 week after mice were given DSS at 2.5% in drinking water for 1 week. After 10 days the cycle of administration of AOM/DSS was repeated three more times. Under these experimental conditions, tumors developed in the middle and distal regions of the colon and rectum.

Cancer chemopreventive activity of maslinic acid, a pentacyclic triterpene from olives and olive oil Chapter | 43

Treatment of mice with maslinic acid at 10 and 30 mg/kg, starting 1 week after the first AOM intraperitoneal injection, was able to reduce the development of adenomas and adenocarcinomas. Hence, the oral administration of maslinic acid for 10 weeks was able to suppress colitis as well as the tumor development.26 Maslinic acid was able to downregulate interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) which are pro-inflammatory cytokines and at the same time an upregulation of interleukin 10 (IL-10) was observed. Those results confirmed the antiinflammatory activity of maslinic acid, which in this animal model exerted an important role in reducing the inflammation induced by DSS that acts as a trigger not only of the ulcerous colitis but also of the associated development of colorectal tumors induced by AOM.26 Other factors are also involved in colorectal cancer, such as adenosine monophosphate (AMP)-activated protein kinase (AMPK), that have been demonstrated to be a regulator of tumorigenesis, since the activation has been linked to a proapopototic activity.43 Maslinic acid administration was able to regulate the AMPK-mTor signaling, thus confirming the involvement of this pathway in the effects of this compound inhibiting cell proliferation and restoring apoptosis.26

43.4.2 Studies with genetic-based models of colorectal cancer The antitumoral activity of maslinic acid has also been evaluated in transgenic mice. The ApcMin/1 mice harbors a mutation of the adenomatous polyposis (Apc) gene which is the same gene involved in human FAP. Patients of FAP suffer from numerous adenomas in the colon that develop to colon cancer, and in many cases, they also undergo adenomas in the small intestine.31 Therefore the ApcMin/1 mice constitutes a widely used model to evaluate chemopreventive agents against human FAP since these mice hold numerous intestinal tumors, in both the small and the large intestine, from an early age.31 The antitumoral activity of maslinic acid was evaluated in 4-week old ApcMin/1 mice after the administration of 100 mg/kg of diet.44 Consumption of maslinic acid for 6 weeks significantly suppressed the formation of polyps in the small intestine in a 45%.44 Regarding the distribution along the small intestine, this pentacyclic triterpene was able to suppress the development of polyps in a 69%, 4%, and 28% in the proximal, medial, and distal segments.44 Sa´nchez-Tena and coworkers44 investigated the molecular mechanism involved in the antitumoral activity of maslinic acid by comparing control ApcMin/1 mice with the ones treated with the pentacyclic triterpene. The analysis of microarray expression allowed the identification of the genes implied

533

both in cell proliferation as well as in apoptosis. Concerning cell proliferation, this genetically modified mice contains a mutation in the Apc gene yielding to the degradation ß-catenin in the Wnt/ß-catenin pathway.31,45 Accumulation of ß-catenin in the intestinal cells facilitates the activation of different genes involved in survival, such as c-Myc, Cyclin D1, and COX-2.31 Maslinic acid downregulated the expression of GSK3ß gene which is constitutively activated in colon cancer cells and plays an important role in the Wnt/ß-catenin pathway and prosurvival signaling.44 Cell cycle is a strictly controlled by an intricate network of signaling pathways that converge in the regulation of cyclin-dependent kinase (CDK) complexes.46 The administration of maslinic acid at 100 mg/kg of diet for 6 weeks was able to downregulate the gene expression of Cyclin D that conducts the G1 phase progression, as well as Cdk4 and Cdk6 which inhibits the CDK complexes.44 These changes eventually induce G1-phase cell cycle arrest, and the ensuing decrease on the spontaneous tumor formation observed in the transgenic animals. The antitumoral activity observed after the treatment of ApcMin/1 mice with maslinic acid at 100 mg/kg of diet could also be due to the induction of apoptosis via the mitochondrial apoptosis pathway, as suggested by the observed downregulation of the antiapoptotic genes Bcl-2 and Bcl-xL. Moreover, the proapoptotic activity of maslinic acid was also confirmed by the upregulation of Bax which is a tumor suppressor gene.44 As previously stated, chronic inflammation has been associated with the promotion and progression of colon cancer, not only in the most common cancer, which is the nonfamiliar colorectal type, but also in human FAP.47 Hence the antiinflammatory activities described for maslinic acid which involves the suppression of NFkB42 may also be involved in the antitumoral effect described for maslinic acid in ApcMin/1 mice. Finally, maslinic acid was also responsible of metabolic changes in ApcMin/1 mice that could be associated with a protective role against intestinal tumorigenesis.44 In this sense the treatment with maslinic acid at 100 mg/kg of diet for 6 weeks showed antihyperlipidemic and antihyperglycemic effects.44 Considering that hyperglycemia and obesity have been linked to a higher risk of developing colorectal cancer, the observed role of maslinic acid in metabolism could also contribute in the chemopreventive action described for this pentacyclic triterpene.44

43.5 Implications for human health and disease prevention Maslinic acid appears as an interesting bioactive molecule for the prevention of colorectal cancer, given the results obtained in vitro studies that reveal this compound as a potent molecule in inhibiting cell proliferation and restoring apoptosis in cancer cells, but more importantly in the in vivo data, that confirms the chemopreventive activity of

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

this pentacyclic triterpene. Different factors are involved in the risk of developing colorectal cancer, of them, physical activity, inflammation, obesity, and dietary habits play key role. A dietary pattern that ensures a high consumption of fiber, vegetables, and fruits has been correlated with a lower incidence of colorectal cancer.4
6 The study of the chemopreventive activity of maslinic acid in an experimental model induced by the carcinogens DMH showed that 5 mg/kg already halted the development of preneoplastic lesions.38 This low dose of maslinic acid could be achieved following a diet rich in foods with a high concentration of this compound. The consumption of 125 g, which is considered a serving size of lentils,48 chickpeas,48 eggplants,49 and spinaches49 will provide 4.9, 7.7, 8.4, and 14.2 mg of maslinic acid, respectively. The content of maslinic acid in table olives is noteworthy, since the consumption of eight small-sized Arbequina olives would contribute with 25 mg of maslinic acid, and five medium-sized Empeltre would supply with 19 mg.21 Therefore the results summarized in the present chapter strengthen the evidence that maslinic acid exerts a protective role against colorectal disease and could contribute to develop dietary recommendations of the food sources of this pentacyclic triterpene for the prevention of this disease.

Mini-dictionary of terms The earliest identifiable neoplastic lesions in the colon carcinogenetic model. Apoptosis Form of cell death in which a programmed sequence of events leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis plays a crucial role in developing and maintaining the health of the body by eliminating old cells, unnecessary cells, and unhealthy cells. Bioactive compound A compound present (in food) that exerts reproducible biological effects at dietary levels (including metabolites postconsumption). Chemopreventive A compound, either natural or synthetic, that agent prevents, reverses, or blocks the development of cancer. Cell proliferation The process that results in an increase of the number of cells and is defined by the balance between cell divisions and cell loss through cell death or differentiation. 1,2Carcinogen that acts as a DNA methylating Dimethylhydrazine agent and used to induce colon tumors in experimental animals. Familial An autosomal dominant inherited condition adenomatous in which numerous adenomatous polyps polyposis form mainly in the epithelium of the large intestine. While these polyps start out benign, malignant transformation into colon cancer occurs when they are left untreated. Aberrant crypt foci

Mucin depleted foci

Nutraceutical

Phytochemical

Aberrant crypt foci with absent or scant mucous production, correlated with carcinogenesis and proposed as endpoints to study the modulation of colon carcinogenesis in short-term studies. A compound or mixture of compounds present in food or food supplements intended to exert a therapeutic effect. A compound present in plants (a plant metabolite).

Acknowledgments This research was funded by Ministerio de Ciencia e Innovacio´n, grant numbers AGL2009-12866 and AGL2013-41188 and Generalitat de Catalunya, grant number 2017SGR945.

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13. Sa´nchez-Gonza´lez M, Lozano-Mena G, Juan ME, Garcı´a-Granados A, Planas JM. Assessment of the safety of maslinic acid, a bioactive compound from Olea europaea L. Mol Nutr Food Res. 2013;57:339
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75. 21. Moreno-Gonza´lez R, Juan ME, Planas JM. Profiling of pentacyclic triterpenes and polyphenols by LC-MS in Arbequina and Empeltre table olives. LWT
Food Sci Technol. 2020;126:109310. 22. Pe´rez-Camino MC, Cert A. Quantitative determination of hydroxy pentacyclic triterpene acids in vegetable oils. J Agric Food Chem. 1999;47:1558
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43. 25. Reyes FJ, Centelles JJ, Lupia´n˜ez JA, Cascante M. (2α,3β)-2,3dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europaea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells. FEBS Lett. 2006;580:6302
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390. 28. Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev. 2007;87:99
163. 29. Reyes-Zurita FJ, Pacho´n-Pen˜a G, Liza´rraga D, Rufino-Palomares EE, Cascante M, Lupia´n˜ez JA. The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53dependent mechanism. BMC Cancer. 2011;11:154
167. 30. Reyes-Zurita FJ, Rufino-Palomares EE, Garcı´a-Salguero L, et al. Maslinic acid, a natural triterpene, induces a death receptormediated apoptotic mechanism in Caco-2 p53-deficient colon adenocarcinoma cells. PLoS One. 2016;11:e0146178
e0146194. 31. Jackstadt R, Sansom OJ. Mouse models of intestinal cancer. J Pathol. 2016;238:141
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163. 34. Druckrey H, Preussmann R, Matzkies F, Ivankovic S. Selective production of intestinal cancer in rats by 1,2-dimethylhydrazine. Naturwissenschaften. 1967;54:285
286. 35. Perˇse M, Cerar A. Morphological and molecular alterations in 1,2 dimethylhydrazine and azoxymethane induced colon carcinogenesis in rats. J Biomed Biotechnol. 2011;2011:473964
473978. 36. Raju J. Azoxymethane-induced rat aberrant crypt foci: relevance in studying chemoprevention of colon cancer. World J Gastroenterol. 2008;21:6632
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123. 38. Juan ME, Lozano-Mena G, Sa´nchez-Gonza´lez M, Planas JM. Reduction of preneoplastic lesions induced by 1,2-dimethylhydrazine in rat colon by maslinic acid, a pentacyclic triterpene from Olea europaea L. Molecules. 2019;24:E1266. 39. Takahashi M, Wakabayashi K. Gene mutations and altered gene expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer Sci. 2004;95:475
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86. 42. Hsum YW, Yew WT, Hong PL, et al. Cancer chemopreventive activity of maslinic acid: Suppression of COX-2 expression and inhibition of NF-B and AP-1 activation in Raji cells. Planta Med. 2011;77:152
157. 43. Song X, Kim SY, Zhang L, et al. Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer. Cell Death Dis. 2014;5:e1504. 44. Sa´nchez-Tena S, Reyes-Zurita FJ, Dı´az-Moralli S, et al. Maslinic acid-enriched diet decreases intestinal tumorigenesis in ApcMin/1 mice through transcriptomic and metabolomic reprogramming. PLoS One. 2013;8:e59392
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762.

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Chapter 44

Hydroxytyrosol, olive oil, and use in aging Mercedes Cano1, Mario Mun˜oz2,3, Antonio Ayala3, Rafael Medina1 and Sandro Argu¨elles1 1

Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain, 2Center for Neuroscience and Cell Biology, University of

Coimbra, Coimbra, Portugal, 3Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Seville, Spain

Abbreviations AMPK C/EBPβ CAT CNR-1 DDR ESC EVOO HO-1 HT IFNγ IGF-1 IL-1RA iPSC JAK/ STAT mTOR NF-κB Nrf2/ Keap1 PARPs ROS SASP SIRT1 SOD TERT TGF-β1 TNF-α VF

AMP-activated protein kinase CCAT/enhancer binding protein beta catalase cannabinoid receptor-gene type 1 DNA damage response embryonic stem cells extra-virgin olive oil hemeoxygenase-1 hydroxytyrosol interferon gamma insulin-like growth factor 1 interleukin 1 receptor antagonist induced pluripotent stem cells Janus kinase/signal transducers and activators of transcription mammalian target of rapamycin nuclear factor κB nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein poly(ADP-ribose) polymerases reactive oxygen species senescence-associated secretory phenotype sirtuin 1 superoxide dismutase telomerase reverse transcriptase transforming growth factor tumor necrosis factor alpha vascular fibroblast

44.1 Introduction Aging is a complex process that cannot be easily defined. Generally, a simple definition could be the loss of optimal functions of an organism throughout the years in a progressive and inevitable way. This process can be either accelerated or delayed, the molecular and cellular consequences of aging being clearly identified. Aging promotes the dysregulation of metabolic signaling pathways,

impairment of mitochondrial functions, reduction of proteostasis, deficiency of stem cell (SC) regenerative capacity, increased cellular senescence, and release of senescence-associated secretory phenotype (SASP), as well as enhanced continued inflammation, increased production of harmful reactive oxygen species (ROS), and genomic instability, a result of telomere erosion, and, finally, chromatin and epigenetic alterations.1
3 Theories that have tried to explain the causes of aging have never been fully satisfactory.4 In 1990 Medvedev compiled the currently known aging theories and found around 300 different models.5 Two main categories classify these theories: programmed and damage theories.6,7 The “programmed” theories suggest that aging is genetically encoded by our DNA, such deliberate gene expression direct or indirectly induces cell senescence by suppressing repair and maintenance mechanisms.8
11 Authors who support damage theories hypothesize that internal and/or external factors such as radiations, chemical harmful, environmental factors, free-radical production, immunological dysfunctions, and telomere shortening can induce cumulative damage throughout a lifetime.12
17 Nonetheless, other authors agree with a combined theory between programmed and damage aging.18,19 It is highly likely that there is not a single and easy cause of aging, but the debate is still ongoing. Life expectancy has been steadily increasing in all regions of the world in the last century.20,21 There will soon be more older people than children and more people over the age of 100 years.22 According to World Population Prospects 2019,21 16% of the worldwide population will be over the age of 65 by 2050; this percentage in Europe and North America will increase as much as up to 25%. The fact that people are living much longer will add substantially to future health and socioeconomic problems. Aging is the main risk factor for chronic pathologies, including neurodegenerative diseases and cancer23 along with the increased multimorbidity associated with these diseases.24

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00050-X © 2021 Elsevier Inc. All rights reserved.

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The research challenge approach will be to ameliorate aging or to prevent new old-age diseases and morbidity. To accomplish that, a deeper understanding of the molecular mechanisms of aging, as well as the translation of this knowledge into therapeutic approaches that could control aging and prevent chronic age-related pathologies, is crucial.25 Various strategies are used to control the effects of aging of normal organism functions. Antioxidants, diet, exercise, caloric restriction, and even activating enzymes of phase II detoxification appear to delay the decay function to a greater or lesser extent.2 Regarding diet, metaanalyses of observational studies and randomized clinical trials revealed that the Mediterranean diet (MD) reduced the risk of overall mortality, cardiovascular diseases, cancer incidence, and neurodegenerative diseases.26,27 One characteristic of the MD is the use of olive oil as the main source of fat used in both food and cooking,28 which is rich in oleic acid, biophenols, and vitamin E.29 Theses olive oil macrocomponents have been extensively studied showing interesting health benefits29; however, it has recently been discovered that the microcomponents contained in olive oil such as hydroxytyrosol have shown antiinflammatory, antimicrobial, neuroprotection, and the antitumor effect.30,31 This chapter focuses on the beneficial properties of hydroxytyrosol in aging.

44.2 Cellular and molecular mechanism of aging It is well known that aging is a complex biological process. However, during the previous decade, as a result of increased aging research, it has become possible to elucidate several mechanisms that at physiological levels contribute to the promotion of cell survival while their dysregulation with age accelerates the loss of biochemical and physiological functions. In this context, recently we summarized the main hallmark or mechanism of aging, at both molecular and cellular levels.2 The molecular and cellular mechanisms of aging include dysregulation of metabolic signaling pathways, impairment of mitochondrial function, reduced proteostasis, deficiency of SC regenerative capacity, increased cellular senescence and release of SASP, enhanced continued inflammation, increased production of harmful ROS and genomic instability as a result of telomere erosion, and chromatin and epigenetic alterations.1
3

44.2.1 Dysregulation of energy metabolic signaling pathways It is known that adequate regulation of energy metabolic signaling pathways is essential to promote cell survival.

In context of aging the main signaling pathways associated with energy metabolism are 50 adenosine monophosphate-activated protein kinase (AMPK); mammalian target of rapamycin (mTOR); insulin-like growth factor (IGF-1) and sirtuins 1 (SIRT1). Their dysregulation leads to inadequate response to energy, nutrient, and growth factors, which contribute to acceleration of aging and age-related diseases. In fact, using several aging experimental models (yeast, worms, flies, rodents, and primates), it has been suggested that while activation of mTOR and insulin/IGF-1 pathways stimulates proliferation, inhibit autophagy, and promote the aging process, moderate/short-term activation of AMPK and SIRT1 pathways inhibits proliferation, induce autophagy, promote adequate stress response and longevity.1
3

44.2.2 Impairment of mitochondrial function An effective mitochondrial function is essential for promoting cell survival and longevity under adverse conditions such as nuclear or mitochondrial DNA damage (genomic instability) and uncontrolled cellular stress. Results of experimental models of aging suggest that enhanced mitochondrial function can promote longevity and healthy aging, while impairment of the mitochondrial function led to aberrant production of ROS promoting aging and age-related diseases.1,2,32 The main modulators of mitochondrial function include ROS, Ca21, and the cellular AMP/ATP ratio. Key factors controlling mitochondrial function are the pro- and antiaging pathways. For example, induction of insulin/IFG-1 and mTOR proaging pathways increases proliferation, promotes the inhibition of AMPK, SIRT1, autophagy/mitophagy, and FOXO-related gene expression, responsible for stress response and immune defenses.32

44.2.3 Reduced proteostasis Protein homeostasis or “proteostasis” is an essential biological process responsible for maintenance of the cellular proteome through three mechanisms: protein synthesis and folding, conformational maintenance, and degradation.33 An important group of proteins contributing to proteostasis are molecular chaperones, which are responsible for folding, refolding, and disaggregation events inside the cell. Under physiological conditions, the two major cellular systems associated with degradation of misfolded proteins and aggregates are the ubiquitin
proteasome and the autophagosomal
lysosomal.1,33 However, the increase of misfolded proteins during aging overloads the capacity of chaperones to function properly. In fact, diminished proteostasis is a hallmark of aging and promote age-related diseases, and both reduction of proaging pathway (insulin/IGF-1 and

Hydroxytyrosol, olive oil, and use in aging Chapter | 44

mTOR) and induction of antiaging pathway (AMPK and SIRT1) extend the life span by improving proteostasis.1,33

44.2.4 Deficiency of stem cell regenerative capacity Multipotency and the capacity to self-renew are the hallmark of SCs. Under physiological conditions, SCs promote proliferation, cell growth, and tissue repair. However, aberrant activity of SCs can induce cancer, while their loss promotes aging and age-related diseases.1,34 Regenerative medicine suggests that SCs can be used to restore or rejuvenate tissues. In fact, several laboratories have reported that using heterochronic parabiosis young blood was able to enhance regeneration in old animals, decrease cardiac damage, and increase cognitive function.34 Upregulation of the cell-cycle inhibitor p16INK4a and inhibition of p38 mitogen-activated protein kinases (MAPK) and JAK/STAT improve the regenerative capacity, while inhibition of mTOR and Cdc42 restores hematopoiesis in old mice.34 Common approaches to replacement damaged cells and enhanced tissue homeostasis include differentiation of induced pluripotent SCs, mesenchymal SCs, or embryonic SCs.34

44.2.5 Cellular senescence and release of senescence-associated secretory phenotype Permanent state of cell cycle arrest, also called cellular senescence, is an important mechanism that during development and in young organisms stimulate optimal tissue repair. Cellular senescence is also a potent tumor suppression mechanism.2,35,36 In old organisms, cellular senescence contributes to biological aging. In fact, this state is promoted by signaling pathways whose dysregulation is characteristic of the aging process such as energy metabolic, DNA damage/repair response, oxidative and endoplasmic reticulum stress, secretion of proinflammatory factors, and/or induction of antiapoptotic genes.35
37 Senescent cells produce and release “SASP” that consists in proinflammatory cytokines, chemokines, growth factors, and matrix proteases. The transcription factors associated with the induction of SASP include NF-κB, GATA4, and C/EBPβ.36 The main type of senescence includes replicative senescence (shortening of telomeres); DNA damage
induced senescence (ionizing and UV); oncogene-induced senescence (activation of Ras or BRAF, or inactivation of PTEN); oxidative stress-induced senescence (endogenous or exogenous oxidizing products); chemotherapy-induced senescence (multiple anticancer drugs); epigenetically induced senescence (inhibitors of DNA methylases or histone deacetylases); mitochondrial dysfunction
associated senescence and

539

paracrine senescence (produced by SASP). Cellular senescence can be controlled by specifically targeting senescent cell with senolytic compounds such as ABT263, ABT737, FOXO4-DRI, quercetin, 2-DG, dasatinib, or indirectly by targeting NF-κB or other pathways controlling the secretory phenotype.36 Recently, we have summarized how targeting prosenescent enzymes (MAPK) with acacetin, baicalein, chlorogenic acid, epigallocatechin-3-gallate, fisetin, gallic acid, genistein, quercetin, resveratrol, and tripotolide can reduce senescent cells and improve longevity.37

44.2.6 Increased production of harmful reactive oxygen species Under physiological conditions, aerobic living cells yield ROS. ROS molecules produce oxidative stress (imbalance between oxidants and antioxidants in favor of the oxidants). Depending on its levels, ROS can promote cell survival or induce cell death. In fact, (1) basal oxidative stress stimulates cellular antioxidant capacity and exerts adaptive response by inducing cell signaling; (2) intermedium oxidative stress stimulates organelle and biomolecule damage, autophagy, apoptosis or senescence, and finally; and (3) high oxidative stress promotes irreversible cell damage leading to cell death.2,38,39 One of the most important mechanisms available to prevent oxidative stress damage is the modulation of Nrf2 (nuclear factorE2-related factor 2)/Keap1 (Kelch-like ECH-associated protein 1) or NF-κB (nuclear factor κB). Nrf2 stimulates several antioxidant and detoxication enzymes, and NF-κB promotes the upregulation of genes associated with inflammatory, immune, and acute phase responses.38 Oxidative stress has been linked with aging, and persistent and uncontrolled oxidative stress is considered one of higher risk factors for developing common pathological processes such as cancer, diabetes, cardiovascular and liver disease as well as Alzheimer’s and Parkinson’s diseases.40 In fact, recently it has been reported that longlived individuals (centenarians) have lower oxidative damage, especially lower plasma lipid peroxidation biomarkers, than control individuals.41

44.2.7 Enhanced continued inflammation Inflammation is an essential process used by the immune systems to preserve cell and tissue homeostasis. Uncontrolled inflammation promotes cellular damage because of increased production of proinflammatory molecules including interferon (IFN) γ, tumor necrosis factor alpha (TNF-α), growth factor, and ROS. In the aging contest, it is well known that if the cell cannot control the molecular mechanisms of aging, the result will be a permanent inflammatory state, which is common in

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older persons.35 Moreover, persistent inflammation is the major risk factor in the development of chronic inflammatory associated diseases such as neurodegenerative diseases, cancer, cardiovascular diseases, and diabetes.42 In fact, successful aging (longevity) is encouraged by an efficient response antiinflammatory, in part, due to upregulation of antiinflammatory mediators including adiponectin, transforming growth factor (TGF)-β1, interleukin (IL) 1 receptor antagonist (IL-1RA), and cortisol. The control of inflammation during aging (inflammaging) is a potent approach to reduce age-related diseases and improve longevity.43

44.2.8 Genomic instability Precise regulation of biological processes such as protein synthesis and gene expression depends on DNA integrity. All these processes are essential in the maintenance of genomic stability. Uncontrolled DNA damage throughout life can lead to genomic instability and has an important role on cellular aging.2,35,44 The major factors responsible of DNA damage include ROS, replication stress, alkylating agents, UV, ionizing radiation, and genomic drugs. To control DNA damage, cells have protective mechanisms including antioxidant and enzymatic systems that neutralize ROS responsible for increased DNA damage, as well as DNA damage response (DDR) machinery.44,45 Depending on the agent responsible for promotion of DNA damage, specific DDR mechanisms are activated, for example, nucleotide excision repair (NER), base excision repair, single-strand DNA breaks repair, double-strand DNA breaks repair, or mismatch repair. Some of the most essential proteins involved in DDR are poly (ADP-ribose) polymerases, which also have a key role in the transcriptional regulation of lipid metabolism, mitochondrial biogenesis, and antioxidant defence.44 Telomere erosion, and chromatin and epigenetic alterations also contribute to genomic instability and cellular aging. Telomerase is an RNA-protein complex that can elongate a telomeric DNA chain through telomerase reverse transcriptase.46 The cellular aging is associated with reduction of telomerase activity. A progressive telomere shortening with increased cell divisions reported in somatic normal human cells promotes replicative senescence (aging).35,46 Recently, it has been reported that the telomere shortening rate, but not the initial telomere length alone, can be a potent predictor of a species life span. Moreover, some of the hallmarks of aging such as cellular senescence and decreasing DDR, which are promoted by short telomeres, can be used to measure species longevity.47 However, excessive activation or upregulation of telomerase lead to immortalization and the cells become fully malignant.35,46 Gene expression is dynamically influenced by chromatin and epigenetic alterations, which facilitates access of transcriptional machinery (enzymes that modify DNA directly or the core

histones) to DNA. Reduction of histones or imbalance of histone modification coupled with DNA methylation changes, local and global chromatin remodeling, breakdown of nuclear lamina as a consequence of environmental stimuli, and nutrient availability have been linked to cellular aging.1,35,48

44.3 Beneficial effects of hydroxytyrosol and olive oil on molecular and cellular mechanisms of aging The MD has been defined since the 1960s, and it is characterized by high in vegetable oils and low intake of saturated fats. Other nutrients are currently included as specific components of this diet, such as extra-virgin olive oil (EVOO) (cold pressed), fruits, vegetables, cereals, nuts, and legumes. Recently, the “Prevencio´n con Dieta Mediterranea” (PREDIMED) study has shown that an MD supplemented with nuts or EVOO (key components of the MD) can modulate inflammatory markers, decrease the incidence of cardiovascular events, or prevent the onset of chronic diseases such as metabolic diabetes, metabolic syndrome, or cancer. Also, the MD has a preventive role in cognitive dysfunctions and neurodegenerative disorders.49
51 Moreover, a large number of studies have related the MD with prevention of other age-related diseases and greater survival.52
54 These effects of MD are associated to the high intake of foods rich in polyphenolic compounds such olive oil which is an essential component of this diet.55
58 In the composition of olive oil, two fractions are distinguished, one saponifiable (98%
99% of the total weight) that contains principally oleic acid and another nonsaponifiable that is rich in polyphenols among other compounds. The phenolic fraction of olive oil includes both hydrophilic and lipophilic phenols. The hydroxytyrosol (HT) or 2-(3,4dihydroxyphenyl) ethanol and their derivatives are the major hydrophilic phenols compound in EVOO.59,60 These compounds are well known for their biological properties as antioxidants, antiinflammatory, or neuroprotective, among others.61
66 A proposal of The European Food Safety Authority Panel is the intake of 5 mg/day of HT and its derivatives to get these effects. These amounts can easily be achieved consuming moderate amounts of olive oil. Currently, the use of natural compounds containing bioactive molecules with high antiaging potential is of growing interest. In this context, we present evidence on how HT can target several of the distinctive signs of aging.

44.3.1 Effects of hydroxytyrosol on metabolic regulation Cellular survival and longevity improvement depend on the correct regulation of the pathways responsible for

Hydroxytyrosol, olive oil, and use in aging Chapter | 44

energy metabolism. In response to fasting the most known proaging pathways, mTOR and IGF, are negatively regulated, while the antiaging pathways, AMPK and SIRT1, are stimulated.

44.3.1.1 Effects of hydroxytyrosol on adenosine monophosphate-activated protein kinase The AMPK pathway is responsible for regulating the energy balance in the cells based on available nutrients and metabolic stress that activate when the energy available to the cells begins to run out. Its deregulation is associated with accelerated aging.67 Different studies have shown that HT stimulates the AMPK pathway. Thus studies in db/db mice (which present hyperglycemia and obesity) treated with HT showed increased survival and neuronal protection through the activation of this pathway.68 On the other hand, hepatocyte experiments show that HT reduced the synthesis of lipids through the activation of AMPK, which decreases the activity of enzymes involved in lipogenesis, this avoiding the accumulation of liver lipids.69 Moreover, in adipocytes HT reduced the levels of free fatty acids, increased the phosphorylationdependent inhibition of ACC, and increased the activity of AMPK.70 Interestingly, in vitro studies with endothelial epithelial cells have shown that HT increased catalase (CAT) expression through the AMPK
FOXO3a pathway, reducing intracellular ROS levels.71

541

expression of mTOR is pathologically high. In this context, it has recently been reported that in several cancer cells, the negative regulation induced by HT of PI3K/ AKT (best known activator of mTOR) promotes the G2/ M cell cycle arrest and apoptosis in vitro, as well as decreased tumor growth and angiogenesis in vivo.77

44.3.2 Effects of hydroxytyrosol on oxidative stress It is known that an excess of oxidative stress accelerates the aging processes such as inhibition of autophagy or mitochondrial dysfunction.1,3 One of the most studied properties of HT is its ability to reduce oxidative stress. In fact, several studies have shown that HT treatment increases the activity of genes necessary for the defense of oxidative stress such as FOXO3a, Nrf2 or its target glutathione peroxidase (GPx), glutathione reductase, hemeoxygenase-1 (HO-1), CAT, among others.78 Thus in healthy volunteers who ingested regularly HT improved the antioxidant defense [e.g., superoxide dismutase (SOD) or total antioxidant status].79 In addition, this protective effect of HT has also been reported in adipose tissue of mice fed with high-fat diet supplemented with HT, in which oxidative stress, the oxidation of proteins, and lipids decreased, increasing their antioxidant defenses such as CAT, SOD, or GPx, among others, contributing to better control of inflammatory status and lipogenesis.80

44.3.1.2 Effects of hydroxytyrosol on sirtuins 1 Another signaling pathway that is involved in the regulation of the aging process is SIRT1 pathway. Different studies have shown that this pathway is also a target of HT. Thus experiments performed on vascular fibroblasts showed that HT induced an Akt/mTOR suppression mediated by an increase in SIRT1 that inhibited the inflammatory response in the VAF.72 Moreover, in senescence-accelerated mouse-prone 8 (SAMP8) model, HT produced an upregulation of SIRT1 that resulted in a decrease of oxidative stress and an increase of antioxidant response. The authors suggest that this protective effect is due to an increase of genes Nrf2 and Nrf2-target gene such as γ-glutamyl cysteine synthetase (γ-GCS) and glutathione-S-transferase.73 The same potentiating effect of HT on SIRT1 was found in other studies. For example, HT stimulated the nuclear location of SIRT1-inducing autophagy in primary chondrocytes.74

44.3.1.3 Effects of hydroxytyrosol on mammalian target of rapamycin mTOR is involved in the regulation of cell proliferation and survival under physiological conditions, but also it is related to proaging pathway, and its inhibition increases the life expectancy.75,76 In several types of cancer the

44.3.3 Effects of hydroxytyrosol on mitochondria dysfunction One of the characteristics of aging is mitochondrial dysfunction, which results in an increasing of oxidative stress and accumulation of damage by the production of mitochondrial ROS (mtROS) that accelerates the aging and associated pathological states.1,3 Several studies, both in vitro and in vivo, have shown a protective effect of HT on mitochondrial dysfunction. For example, in the brain of rats with arsenic-induced mitochondrial dysfunction, the administration of HT recovered activity of the antioxidant enzymes and mitochondrial complex (I, II, and IV).81 Moreover, under inflammatory conditions, HT prevented the mitochondrial dysfunction improving endothelial function by increasing mitochondrial biogenesis and ATP production. In addition, the authors reported that HT decreased mtROS production, oxidative damage, inflammatory cytokines, and endothelial adhesion molecules.82 In this context, experiment in cardiomyocytes showed that HT protected mitochondrial electron transport chain complexes I
IV, decreasing apoptosis-inducing factors and oxidative stress, and improving the integrity of complex III.83 Interestingly, maternal HT administration

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diminished mitochondrial dysfunction of prenatally stressed offspring by upregulating complexes I
V components, increased SOD2 and FOXO1/3.84

44.3.4 Effects of hydroxytyrosol on autophagy The mechanism of autophagy is essential to maintain cellular homeostasis, contributing to reduce age-related diseases and extend longevity.2,3 Several studies have shown that HT promotes autophagy by upregulating SIRT1. For example, in vascular adventitial fibroblasts stimulated with TNF-α, HT induced autophagic flux stimulating SIRT1, inhibiting AKT/mTOR and promoting autophagy markers (Beclin and LC3).72 However, a recent study reported that HT still induces autophagy in SIRT1depleted cells inducing other pathways such as p62.74 The autophagy markers (p62 and LC3II) and the activation of AMPK increased in human THP-1 monocytes treated with HT, which intensified the inhibition of mTOR/AKT pathways.85 In inflammatory models with LPS, HT not only stimulated autophagy but also decreased proinflammatory molecules such as TNF-α, IL-10, IL-1β, IL-6, and monocyte chemoattractant protein 1 (MCP-1).85,86

44.3.5 Effects of hydroxytyrosol on DNA damage/repair DNA repair is fundamental for maintaining genomic stability and improving longevity.1,3 In this context, there are trials to test the protective effect of HT on DNA repair. For example, hydroxytyrosol decreased DNA damage caused by oxidative stress in both normal and cancerous cells using the comet test to evaluate the DNA chain.87 Similarly, HT protection was found in response to different DNA damage inducers, such as UVB radiation or acrylamide exposure, in blood cells88 or tumor cell lines.89 In addition to the known antioxidant activity of HT as a possible mechanism responsible for preventing DNA damage, studies of the protective impacts of HT on other mechanisms of reparation of DNA such as NER or homologous recombination would be necessary.

44.3.6 Effects of hydroxytyrosol on epigenetic regulation Regarding the epigenetic mechanisms that contribute to the aging process, HT has been shown to affect DNA methylation and the modulation of microRNAs expression. HT regulates the endocannabinoid system (related to control of oxidative stress, inflammation, and the immune system) in human Caco-2 through epigenetic mechanisms reducing the methylation levels of the cannabinoid receptor gene type 1 (CNR1), thus increasing its expression and inhibiting the

proliferation of these cancer cells.90 On the other hand, the effect of nutritionally relevant amounts of HT has also been described on the intestinal expression of several miRNAs suggesting that the modulation of miRNAs’ action through HT consumption might partially explain its healthful activities.91

44.4 Conclusion Research on prolongevity therapies provides an effective contribution to ensure healthy lives and promotes well-being for everyone at all ages (notably the elderly, a major societal challenge). This is aligned with one of the recent goals in the UN 2030 agenda. A better understanding of aging mechanisms will help to design therapeutic and prevention strategies to ameliorate the morbidity associated to new old diseases. It is well established that the excellent properties of nutrients contained in the MD help prevent ageassociated diseases. In the seeking of new approaches to fight age-associated diseases, many researchers focus their efforts on the nutrients the MD offers, especially those micronutrients contained in the nonsaponifiable fraction such as HT. These compounds are well known for their biological properties as antioxidants, antiinflammatory, or neuroprotective, among others. The role of HT has been well established in aging by promoting prolongevity molecular signaling pathways (AMPK and SIRT1), mitochondrial biogenesis, DNA repair, autophagy, ROS clearance, and epigenetic regulation (Fig. 44.1). However, we are far from understanding completely all HT and hydrophilic phenols properties. It will be a scientific challenge to decipher the characteristics and the beneficial effects of these micronutrients and the benefits of the MD.

Mini-dictionary of terms Aging: Aging is a complex process responsible of decrease optimal physiological and biochemical functions of an organism throughout the years in a progressive and inevitable way. Autophagy: Autophagy is an essential biological process responsible to eliminate/degrade and recycle intracellular damaged molecules or cells component within the lysosome/vacuole. Ineffective autophagy can contribute to accelerate the aging process. Hydroxytyrosol (HT): HT is a phenolic microcomponent contained in olive oil with reported antiinflammatory, antimicrobial, neuroprotection, and the antitumor effect. Inflammaging: Inflammaging is a biological process as a result of persistent inflammation in our body that increase with age, promote chronic age-related pathologies, and decrease longevity. Life expectancy: Life expectancy is the average period that a person may expect to live. Measure of life expectancy indicate how healthy is the population.

Hydroxytyrosol, olive oil, and use in aging Chapter | 44

543

FIGURE 44.1 Properties of hydroxytyrosol. Hydroxytyrosol is a phenolic compound obtained from a nonsaponifiable fraction (1%
2% of total) derived from Olea europaea olive oil. Its properties have been demonstrated beneficial in (1) metabolism regulation promoting prolongevity pathways by activation of AMPK and SIRT1 and inhibition of mTOR and IGF; (2) mitochondrial dysfunction promoting mitochondrial biogenesis; (3) DNA repair, preventing the damage of harmful molecules as ROS or UVB radiation; (4) promoting the clearance of ROS by upregulation of antioxidant proteins such as CAT, SOD, and GPx; (5) inducing autophagy by activation of AMPK, SIRT1, and p62; and (6) controlling epigenetic regulation by reducing the methylation levels of DNA. AMPK, AMP-activated protein kinase; CAT, catalase; GPx, glutathione peroxidase; IGF, insulin-like growth factor; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; SIRT1, sirtuin 1; SOD, superoxide dismutase.

Mediterranean diet (MD): The MD is historical and cultural patrimony of Mediterranean countries characterized by high intake of extra-virgin olive oil, fruits, vegetables, cereals, nuts, fish, and legumes and low intake of saturated fats. The MD reduced the risk of overall mortality, cardiovascular diseases, cancer incidence, and neurodegenerative diseases. Mitochondrial dysfunction: Mitochondrial dysfunction is a process in which the mitochondria decrease the efficiency to yield the energy necessary for the appropriate function of the cells, and it associated with accelerated aging and pathological stages. Oxidative stress: Oxidative stress is an increase of production of free radicals and decrease of antioxidants defense in the cells, which could result in biomolecules damage, cells and tissue damage. Uncontrolled oxidative stress is associated with accelerate aging and high risk of age-related pathologies.

Proteostasis: Proteostasis also called protein homeostasis is a significant biological process responsible for preservation of the cellular proteome through three mechanisms: protein synthesis and folding, conformational maintenance, and degradation. Decrease of proteostasis is associated with accelerated aging and high risk of age-related pathologies. Senescence: Senescence is a biological event responsible of decreasing cell division by stimulation of stable growth arrest without inducing cell death mechanisms. Senescence is a key factor linked with accelerate aging and high risk of age-related pathologies.

Comparisons of olive oils with other edible oils It is known that an MD reduces the risk of overall mortality, and several common chronic diseases such as cardiovascular

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diseases, cancer, and neurodegenerative diseases. In fact, it has been reported that the MD promotes healthy aging and longevity. Given that a key component of the MD is extravirgin olive oil, which is the main source of fat used in both food and cooking, a considerable number of research have shown its health benefits. Despite additional oils (sunflower, canola, soybean, peanut, coconut, corn, avocado, etc.) are available in the food markets, extra-virgin olive oil has shown considerable health benefits and improved nutritional profile with higher antioxidants and antiinflammatory effect. Moreover, microcomponents contained in extra-virgin olive oil such as HT have shown antiinflammatory, antimicrobial, neuroprotection, and the antitumor effect. In fact, HT has one of the highest antioxidant capacities reported, and there is no report showing similar effect in other types of food oils. Another oil with similar extra-virgin olive oil nutritional benefits is the avocado oil; however, so far few data are supporting its positive effect on longevity.

Implications for human health and disease prevention Aging is a high risk factor for chronic pathologies, the prevalence of which is expected to increase in the next two decades. These pathologies include, for example, neurodegenerative diseases, diabetes, osteoporosis, cancer, or cardiovascular disease. Theatrically, it is expected that approaches to control aging process could be able to delay the onset of multiple age-related pathologies. The first step to design a successful “antiaging” therapy is increasing our understanding of the cellular and molecular mechanisms, the dysregulation of which promotes the loss of biochemical and physiological functions with age. In this context, here we have summarized the main mechanism of aging, and because the HT has beneficial effects in all these mechanisms, it is possible use HT, or other olive oil components, in combination with nonpharmacological approaches with “antiaging” effect, such as caloric restriction or exercise, to provide an effective approach against aging and age-related pathologies.

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155.

Chapter 45

Hydroxytyrosol and hydroxytyrosyl fatty esters: occurrence and anti-inflammatory properties Pierluigi Plastina Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy

Abbreviations AA b.w. COX-2 CXCL10 EVOO HFD Hty HtyAce HtyBut HtyOle HtySte IBD IL iNOS IRF-1 LPS MAPK MCP-1 MMP MPO MT NF-κB NO Nrf2 NSAID OO PBMC

arachidonic acid body weight cyclooxygenase-2 CXC motif chemokine 10 extra-virgin olive oil high-fat diet hydroxytyrosol hydroxytyrosyl acetate hydroxytyrosol butyrate hydroxytyrosyl oleate hydroxytyrosyl stearate inflammatory bowel diseases interleukin inducible nitric oxide synthase interferon regulatory factor-1 lipopolysaccharide mitogen-activated protein kinase monocyte chemotactic protein-1 matrix metalloproteinase myeloperoxidase malaxation temperature nuclear factor-kappa B nitric oxide nuclear factor erythroid 2
related factor 2 nonsteroidal anti-inflammatory drug olive oil peripheral blood mononuclear cell

FIGURE 45.1 Structure of (A) hydroxytyrosol (Hty) and (B) hydroxytyrosyl esters.

PGE2 PKC PMA PPAR TLR TNF-α TPA

prostaglandin E2 protein kinase C phorbol myristate acetate peroxisome proliferator
activated receptor Toll-like receptor tumor necrosis factor α 12-O-tetradecanoylphorbol acetate

45.1 Introduction Phenolic compounds present in olive and extra-virgin olive oil (EVOO) are assumed to contribute to the health benefits associated with the consumption of EVOO.1,2 The Regulation 432/2012 of the European Union authorized the claim “olive oil polyphenols contribute to the protection of blood lipids from oxidative stress” on the basis of the scientific opinion of the European Food Safety Authority that “a daily intake of 20 g of olive oil, which contains at least 5 mg of hydroxytyrosol and its derivatives (e.g., oleuropein and tyrosol) provides the expected beneficial effects.”3,4 Hydroxytyrosol [2-(3,4dihydroxyphenyl)ethanol, Hty, Fig. 45.1A] is one of the major phenolic components of olive and EVOO. It is present in the esterified forms, as acetate (hydroxytyrosyl acetate, HtyAce, Fig. 45.1B with n 5 0) or as secoiridoids derivatives (oleuropein, its aglycone and oleacein), as well as in the free form. Hydroxytyrosol displays a wide range of potential health benefits, including antioxidant, cardioprotective, anticancer, and anti-inflammatory effects.5
7 In addition, its use as a dietary supplement as well as an additive in functional foods has been reported.8
10 However, Hty shows low oral bioavailability and fast elimination in humans, mainly due to its hydrophilic character.11 In

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00010-9 © 2021 Elsevier Inc. All rights reserved.

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view of this an increasing interest has been observed for lipophilic derivatives of Hty,12,13 and in particular for those bearing a lipid moiety (lipophenols). Indeed, hydroxytyrosyl esters bearing fatty acid acyl chain retain and in some cases boost the antioxidant properties of Hty. Moreover, they do not show some of drawbacks described earlier because of their higher lipophilicity with respect to Hty.14
23 Moreover, some Hty esters, including those of Hty with n-3 polyunsaturated fatty acids, have been synthesized with the aim of stabilizing oil matrices and emulsions.24
32 Although these compounds have been mainly known as semisynthetic lipophenols of Hty with enhanced antioxidant activities, some Hty esters have been recently identified in olive oil (OO), EVOO, as well as in by-products, and some of their potential health benefits have been investigated.33
37 This chapter focuses on Hty and this emerging class of Hty esters. Their occurrence in OO as well as their anti-inflammatory properties have been reviewed.

45.2 Occurrence Several simple phenolics were first identified in EVOOs in the 1970s and 1980s, but the most important phenolic compounds, including Hty and oleuropein, were discovered in the 1990s.38 HtyAce has been identified for the first time in Spanish OOs.39 More recently, a lipophilic derivative of Hty bearing oleic acid fatty chain, hydroxytyrosyl oleate (HtyOle, Fig. 45.2), has been identified in EVOO and OO.33 This result was further confirmed as HtyOle was found in a monovarietal EVOO produced from olives of Carolea cultivar in the campaign 2017/ 2018, and its amount was quantified as 4.9 mg/kg of oil.34 HtyOle has been also identified in OO by-products, namely, pomace and olive mill waste waters.35 The amount of Hty in OOs depends on many factors, including cultivar and geographical origin of olive plant, pedoclimatic conditions, production procedures, and OO quality.40,41 Among these factors, the cultivar (genotype) has a preponderant influence on phenolic composition.42 Several researches have been focused on a limited number of cultivars, with particular emphasis on traditional cultivars with regional importance in terms of EVOO production. A high variability in the amount of Hty (0.6
55.2 mg/L) was found in EVOOs obtained from 10 Tuscany (Italy) cultivars in the campaign 1998
99. The highest amount was found for Ginestrino cultivar.43 Tura

FIGURE 45.2 Structure of hydroxytyrosyl oleate (HtyOle).

et al. reported that cultivar (18 cultivars assessed for 4 years in the same place) had a limited influence on Hty amount that was found in the range 0.398
3.380 mg/kg. A high variability was found due to environmental influence by macroclimate conditions (three cultivars from three different regions for 3 years), and Hty ranged between 1.587 and 6.167 mg/kg of oil.44 Baiano et al. studied the effects of genotype and growing location on the quality of monovarietal EVOOs obtained from olives of Coratina, Nocellara, Ogliarola, and Peranzana cultivars picked in four locations of the Apulia region (Italy). The concentration of Hty was found in the range 0.4
0.9 mg gallic acid equivalent kg21 of oil.45 The amount of Hty and HtyAce in EVOOs from three cultivars (Roggianella, Sinopolese, and Ottobratica) grown in Calabria (Southern Italy) was found in the ranges 2.57
3.28 and 1.23
32.99 mg/kg, respectively.46 Bellumori et al. analyzed over 100 samples of Italian EVOOs produced in Tuscany and Apulian regions in 2017 and 2018 from 13 olive cultivars. The concentration of Hty (after acidic hydrolysis) was found in the range 0.29
8.90 mg per 20 g of oil.47 Monovarietal EVOOs obtained from olives from seven Italian cultivars (Frantoio, Ottobratica, Pendolino, Leccino, Maurialo, Maurino, and Coratina) introduced in southwestern province of Pakistan were investigated, and Hty was found in the range 1.79
9.16 mg/kg of oil.48 Go´mez-Rico et al. investigated EVOOs from six Spanish varieties as regards the content of major phenolics. Hty was found in the range 0.4
4.9 mg/kg of oil for EVOOs obtained from the unripe fruits and 0.6
5.0 mg/kg of oil for EVOOs obtained from the ripe fruits. The highest values were found in the Picudo variety.49 Sa´nchez de Medina et al. investigated the phenolic profile of monovarietal EVOOs obtained from olive fruits collected at intermediate ripening from seven different cultivars located in different areas of the Southern Spain in the campaign 2013
14. The concentration of Hty was found in the range 1.42
33.89 mg/mL of oil.50 Becerra-Herrera et al. characterized the phenolic profile of a total of 50 Spanish EVOO samples collected and analyzed from the years 2011
12 and from nine different protected designations of origin and seven OO cultivars. The concentration of Hty was found in the range 6.939
72.71 mg/kg of oil.51 Twenty-seven samples of monovarietal Portuguese EVOOs produced from olives from nine different varieties collected over the period October to November 2018 were analyzed. The concentration of Hty varied in the range 0.651
5.259 mg per 20 g of oil.52 In the work of Rodrigues et al., 28 centenarian olive trees were selected and oils were extracted and their phenolic fraction characterized during four consecutive seasons (2014
17). The concentration of Hty was found in the range 1.1
7.1 mg of tyrosol equivalents kg21 of oil.53 Arslan et al.

Hydroxytyrosol and hydroxytyrosyl fatty esters: occurrence and anti-inflammatory properties Chapter | 45

investigated the variation of phenolic compounds in the EVOOs produced from olives of Sarıulak variety cultivated in three different locations in the southern region of Turkey. The concentration of Hty was found in the range 1.27
2.14 mg/kg of oil.54 Seventy-eight EVOO samples collected from 10 different countries (Mediterranean and South American countries) in the production years 2009
11 were investigated as regards both qualitative profile and quantitative composition of the phenolic fraction. Hyt was found in almost all studied samples, and the concentration was found in the range 0.14
58.45 mg/kg of oil.55 The study of Miho et al. characterized the phenolic profile of a representative panel of monovarietal EVOOs from 80 olive cultivars from 15 countries representing the main EVOO-producing areas worldwide. With the aim of avoiding agroclimatic and production differences, the olive cultivars were grown in the same orchard (Cordoba, Spain), and their oils were extracted by application of the same protocol, to allow an unbiased characterization of the influence of cultivar on EVOO phenolic profiles. Hty was found in the ranges 0.46
4.89 and 0.28
7.57 mg/kg of oil for 2014/15 and 2015/16 campaigns, respectively.56 The phenolic profile of EVOOs is also influenced by the oil extraction process. Malaxation induces the activation of several endogenous fruit enzymes, such as β-glucosidases and esterases, which promote the hydrolysis of secoiridoids leading to an increase of their corresponding derivatives.57 It has been reported that the content of phenolic compounds in EVOOs was much more affected by the malaxation temperature (MT) than the kneading time. The concentration of Hty as well as other phenolics has been found increased as the MT rose from 20 C to 40 C, probably due to an increase in the partition coefficient of the phenolics between the oily and water phases of the olive paste.58 By contrary, oxidation of phenolic compounds catalyzed by endogenous fruit enzymes, such as phenoloxidases and peroxidases, occurs during the extraction process and leads to a reduced phenolic concentration in EVOOs.59 In the study of Miho et al. the effects of recommended MT (28 C) and the absence of oxygen during malaxation in the phenolic profiles of eight monovarietal EVOOs have been investigated. The concentrations of Hty determined in EVOOs obtained under vacuum were found higher than those observed in standard condition for all the samples. By contrary, the effect of malaxation times was less clear.60 The content of Hty has been found to increase during olive ripening as well as storage of OO as a result of the hydrolysis of secoiridoids.61 Benito et al. studied the evolution of some antioxidants, including Hty in OOs obtained from Arbequina cultivar throughout the harvesting period with weekly sampling. The content of Hty

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ranged from 0.07 to 0.36 mg/kg of oil. However, there was not a clear trend in the content of this compound across the harvest dates.62 The effect of ripening stage on the phenolic profile of the OOs obtained from Ottobratica cultivar was studied by Sicari et al. during the campaign 2007
08. The concentration of Hty was found to reach its highest value (3.88 mg/kg of oil) in the oil obtained from the olives at the intermediate ripening. Parallel to this, the amount of HtyAce was found to decrease from the initial value (7.39 kg21 of oil).63 The effect of storage on quality parameters and phenolic content of Italian monovarietal EVOOs in initial conditions and after storage has been investigated. In particular, an increase in Hty (10%
80%) was found after 18 months of storage in oils from all cultivars.64 A decrease in secoiridoids and an increase of Hty and tyrosol content were also observed in EVOOs from five Greek cultivars stored for 24 months.65

45.3 Anti-inflammatory properties Inflammation is essential to maintain homeostasis and provide a protective response to damaged cells and tissues, irritants or pathogens, involving immune cells and molecular mediators.66 However, under certain pathological conditions, this mechanism is imbalanced, and a chronic low-grade inflammatory condition is associated with several human degenerative diseases, including Alzheimer’s disease, Parkinson’s disease, diabetes, cancer, and inflammatory bowel diseases (IBD).67
69 During inflammation, microbial products such as lipopolysaccharide (LPS), inflammatory cytokines, or interferon-γ prompt the upregulation of inducible isoform of nitric oxide synthase (iNOS) leading to high concentrations of nitric oxide (NO), which acts as a key mediator in several inflammatory disorders.70 Transcription factors such as nuclear factor-kappa B (NF-κB) mediate the expression of iNOS and other inducible genes such as cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in immune and inflammatory responses.71 However, many anti-inflammatory agents, including statins, corticosteroids, and nonsteroidal anti-inflammatory drugs (NSAIDs), can cause side effects and fail to deal with the underlying inflammatory state.72 In this view, EVOO has been proposed as a key functional food for the prevention of inflammatory diseases, and several literatures have shown the anti-inflammatory properties of Hty in both in vitro and in vivo models.73

45.4 In vitro studies Maiuri et al. found that Hty downregulated the expression of iNOS and COX-2 at mRNA and protein level in J774 murine macrophages stimulated with LPS, by blocking

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the activation of NF-κB, signal transducer and activator of transcription-1α, and interferon regulatory factor-1 (IRF-1).74 Hty was also found to inhibit the production of NO, prostaglandin E2 (PGE2) as well as proinflammatory ILs, TNF-α, and chemokines [CXC motif chemokine 10 (CXCL10) and monocyte chemotactic protein-1 (MCP-1)] in LPS-stimulated RAW 264.7 murine macrophages. Inhibition was found to take place at a transcriptional level, as gene expression of iNOS, IL1α, CXCL10, macrophage inflammatory protein-1β, matrix metalloproteinase (MMP)-9, and prostaglandin E2 synthase were reduced by Hty, and the effects were partly mediated via the NF-κB pathway.75 Treatment of LPSstimulated murine peritoneal macrophages with Hty suppressed iNOS gene expression and NO production through NF-κB-independent pathway.76 Hty has been also reported to inhibit degranulation in murine peritoneal mast cells.77 The ability of Hty to inhibit proinflammatory cytokines and NO production, iNOS and COX-2 expression by blocking NF-κB activation has been reported also in human THP-1 monocytic leukemia cells stimulated with LPS.78,79 Treatment of human monocytes from peripheral blood mononuclear cells (PBMCs) with Hty inhibited the production of O22, reduced the expression of COX-2 at both the mRNA and protein levels and the release of PGE2 in a concentrationdependent way.80 Further studies reported that Hty was able to suppress the activity and expression of MMP-9 and COX-2 enzymes in phorbol myristate acetate (PMA)
stimulated human monocytes from PBMC and U937 human monocytic cell line, by the inhibition of NFκB and activation of protein kinase C (PKC)α and PKCβ1.81 Later, the same group reported on the antiinflammatory properties of Hty in human Simpson
Golabi
Behmel syndrome adipocytes. Hty was found to inhibit TNF-α-induced mRNA levels of several pro-inflammatory genes and microRNAs via NF-κB inhibition.82 At nutritionally relevant concentrations, Hty was found to mitigate oxidative burst and CD11b expression in human granulocytes and monocytes stimulated with PMA and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.83 Calabriso et al. reported that the pretreatment with Hty of PMA-stimulated endothelial cells suppressed inflammatory angiogenesis by reducing mitochondrial superoxide production and lipid peroxidation and increasing superoxide dismutase activity.84 Positive effects of Hty have been reported also in neuroinflammation as Hty reduced the inflammation induced by two different stimuli in BV2 microglia cells, through NF-κB-dependent (for LPS) and independent (α-synuclein) pathways.85 Moreover, Hty reduced CD86 (marker of pro-inflammatory or M1 phenotype) expression and increased M2 marker CD206 (marker of anti-

inflammatory or M2 phenotype) expression in BV2 and primary microglia cells. Moreover, Hty suppressed the LPS-induced Toll-like receptor 4 (TLR4) in BV2 microglia cells.86 Hty and HtyAce have been shown to possess anti-inflammatory and joint protective properties in a murine model of rheumatoid arthritis, as both compounds inhibited the production of MMPs and proinflammatory cytokines, including TNF-α and IL-6 in IL-1β-induced human synovial cells, through the inhibition of mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation.87 HtyAce had been previously reported to inhibit NO production and iNOS and COX-2 protein expression in LPS-stimulated murine peritoneal macrophages via NF-κB modulation.88 Moreover, a protective effect of HtyAce against inflammation in vascular endothelial cells partly through the SIRT6-mediated PKM2 signaling pathway has been reported.89 Anti-inflammatory properties of Hty fatty acid esters have been recently disclosed. Hty esters with short, medium, and long acyl chains were evaluated for their ability to reduce NO production in LPS-stimulated RAW 264.7 macrophages. Among the compounds investigated, hydroxytyrosyl stearate (HtySte, Fig. 45.1B with n 5 16) and HtyOle were found to decrease NO production in a concentration-dependent way, while the other compounds, including Hty and HtyAce, were ineffective in the tested concentration range (0.5
5 μM). HtyOle suppressed PGE2 production and decreased the expression of iNOS, COX-2, and IL1β.35 In the same cell line, hydroxytyrosol butyrate (HtyBut, Fig. 45.1B with n 5 2) has been found to exert the highest inhibition (IC50 7.0 μM) among the tested compounds against NO production.90 The lipophilic character of HtyOle suggested its potential use as active compound in epidermal and dermal formulations for the treatment of the inflammation of the cutaneous stratus. Indeed, antioxidant and skin regenerative properties of HtyOle have been reported in human keratinocytes.34 Pretreatment with Hty or HtyAce of primary human keratinocytes stimulated with IL-1β or TLR3 ligand attenuated thymic stromal lymphopoietin in a concentration-dependent manner. Moreover, the expression of several inflammationrelated genes was downregulated by both compounds through modulation of the NF-κB pathway.91

45.5 In vivo studies In vivo anti-inflammatory potential of Hty was first investigated as a topical remedy on edema induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA) in mice. Hty was found to reduce AA- and TPA-induced edema and to inhibit the enzyme myeloperoxidase (MPO), a marker of the neutrophil influx in the inflamed tissues.92 Anti-inflammatory properties of Hty

Hydroxytyrosol and hydroxytyrosyl fatty esters: occurrence and anti-inflammatory properties Chapter | 45

were further assessed on carrageenan-induced acute inflammation and hyperalgesia in rats. Moreover, Hty decreased pro-inflammatory cytokines IL-1β and TNF-α without affecting mRNA expression of anti-inflammatory cytokine of IL-10.93 The anti-inflammatory properties of Hty as a potential COX-2 inhibitor were further assessed in a carrageenaninduced rat paw edema model. Despite being less effective than a selective COX-2 inhibitor and a representative NSAID, Hty did not cause gastric damage, which is a typical adverse side effect of many anti-inflammatory drugs.94 Hty exerted anti-inflammatory effects by suppressing TLR 2 and its downstream pathways in Staphylococcus aureus-induced mastitis in mice. Hty attenuated mammary tissue inflammatory injury, suppressed the activity of MPO, and inhibited the expression of IL-1β, IL-6, and TNF-α.95 Dietary supplementation of Hty decreased paw edema and histological damage and reduced COX-2 and iNOS expression,96 while administration of HtyAce inhibited MAPKs and NF-κB signaling pathways in collagen-induced arthritis animal models.97 Hty has been reported to reduce inflammatory markers COX-2 and TNF-α in a mouse model of systemic inflammation at the dose of 80 mg/kg body weight (b.w.) in Balb/c mice stimulated by intraperitoneal injection of LPS.98 Administration of Hty reduced the levels of some LPS-induced neuro-inflammatory mediators and microglia/astrocyte activation in mouse brain.86 Hty and HtyAce have investigated in IBD models. Diets supplemented with EVOO enriched with Hty decreased mortality, reduced pro-inflammatory TNF-α, COX-2, and iNOS expression, and increased antiinflammatory IL-10, while HtyAce inhibited COX-2 protein expression and NF-κB activation in dextran sulfate sodium
induced acute colitis in mice.99
101 Diets supplemented with Hty and HtyAce had anti-inflammatory effects, decreasing plasma levels of TNF-α in rats fed a cholesterol-rich diet. In adipose tissue, supplementation with HtyAce decreased MCP-1 and IL-1β below control levels.102 Supplementation with Hty improved the white adipose tissue dysfunction in mice-fed high-fat diet (HFD) through the modulation of key transcription factors, including NF-κB, nuclear factor erythroid 2
related factor 2 (Nrf2), sterol regulatory element
binding protein 1, and peroxisome proliferator
activated receptor gamma (PPAR-γ) as well as their target genes.103 Hty at the dose of 10 mg/kg b.w./day prevented metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of nonalcoholic fatty liver disease induced by HFD.104 Hty supplementation in HFD-fed mice attenuated the metabolic and inflammatory alterations produced by HFD, activating transcription factors PPAR-α and Nrf2, and attenuating NF-κB activation.105

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Dietary supplementations of Hty and HtyAce were investigated in pristane-induced systemic lupus erythematous, a multisystemic autoimmune chronic disease. Dietary supplementation with Hty and HtyAce reduced pro-inflammatory cytokines and prevented renal damage with a considerable blockage of different inflammatoryrelated pathways in mice.106

Mini-dictionary of terms are molecules containing a sugar and a nonsugar moiety (aglycone). Lipophenols are phenolics bearing a lipid moiety. Hydroxytyrosyl fatty ester contains hydroxytyrosol head group and a fatty acyl chain. Monovarietal oils are obtained from olives of a single cultivar or genotype. Functional foods are foods that offer health benefits beyond their nutritional value. Bioavailability is the percentage of an administered drug or a nutrient that is available in systemic circulation. M1 macrophages are macrophages characterized by a proinflammatory phenotype. M2 macrophages are macrophages characterized by an antiinflammatory phenotype. Cytokines are small proteins secreted and released by cells that are important for the interactions and communications between cells. Lipopolysaccharide is the major component of the outer membrane of Gram-negative bacteria consisting of a lipid and a polysaccharide moiety. Carrageenan is a high molecular weight linear polysaccharide. Glycosides

Comparisons of olive oils with other edible oils Phenolic compounds are present in all vegetable oils and play a key role for the oxidative stability of the polyunsaturated fatty acids contained therein. However, the presence of Hty has not been reported in cold-pressed oils from the seeds of soy, sunflower, rapeseed, corn, grapeseed, hemp, flaxseed, rice bran, pumpkin, and peanut.107,108 Hty was found to be under the detection limits in a variety of cold-pressed gourmet oils, including grapeseed oil, canola oil, avocado oil, coconut oil, or palm oil.109 By contrary, Hty has been found in date seed oil in a concentration range 6.94
10.22 mg/kg of oil, depending on the variety.110 Hty esters have not been reported in other edible oils thus far.

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77. Persia FA, Mariani ML, Fogal TH, Penissi AB. Hydroxytyrosol and oleuropein of olive oil inhibit mast cell degranulation induced by immune and non-immune pathways. Phytomed. 2014;21:1400
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Chapter 46

Influence of olive oil on pancreatic, biliary, and gastric secretion: role of gastrointestinal peptides Maria Dolores Yago, Maria Alba Martinez-Burgos, Namaa Audi, Mariano Man˜as and Emilio Martinez-Victoria Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain

Abbreviations BA CCK CSI FFAR FO GPR MUFA PP PUFA PYY SO T2D VOO

bile acid cholecystokinin cholesterol saturation index free fatty acid receptor fish oil G protein
coupled receptor monounsaturated fatty acids pancreatic polypeptide polyunsaturated fatty acids peptide YY sunflower oil type 2 diabetes virgin olive oil

46.1 Introduction Both at the level of the popular media and the scientific literature, it has been the relationship between the intake of fat and cardiovascular disease that has received the most attention. In contrast, the effects of the amount or type of dietary fat on gastrointestinal function are poorly known. It was for this reason that we decided to concentrate our efforts on the relation between the prolonged intake of a specific type of dietary fat and changes in digestive function, particularly secretory function. This is what we call “adaptation to dietary fat type.” We have studied in different species the adaptation of biliary, gastric, and, mainly, exocrine pancreatic secretion to dietary fat type. Moreover, with the purpose of elucidating the mechanisms involved in adaptation, we have used experimental models ranging from the whole subject (either conscious or anesthetized) to ex vivo preparations (viable pancreatic acinar cells) and continuous cell lines.

In our in vivo studies, most of them described in this chapter, we have tested the effects of two dietary fats that are used preferentially in our geographic area: virgin olive oil (VOO) and sunflower oil (SO). While the health and therapeutic benefits of olive oil on the gastrointestinal system were mentioned already by Hippocrates in ancient times, we sought to provide scientific support for the actions of this major component of the Mediterranean diet.

46.2 Olive oil and digestive secretion in dogs 46.2.1 Exocrine pancreatic secretion Since the findings of Pavlov in the early 1900s, the adaptation of the exocrine pancreas to dietary changes has been studied in various species, especially the rat. The consensus of these studies is that, given an adequate dietary protein supply, the major digestive enzymes, proteases, amylase, and lipase, change in both pancreatic tissue and its secretion proportionally to the amount of their respective substrates in the diet. The physiological significance of this adaptation would be to optimize the digestion and utilization of that substrate. In contrast to the general agreement about the influence of the amount of dietary fat, there is a considerable controversy over the effects of the type of fat. On the other hand, the greatest number of studies on the pancreatic adaptation to diet has centered the attention on the changes in tissue enzyme content, but very few reports exist concerning the overall secretory pancreatic response to endogenous or exogenous stimulation after medium- or long-term intake of a specific diet. These reasons moved us to undertake the following investigations in dogs.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00039-0 © 2021 Elsevier Inc. All rights reserved.

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Weaning mongrel dogs were randomly assigned to one of two isoenergetic and isoproteic diets (33% energy as fat) that differed only in the nature of the fat source: VOO or SO. Expressed as a percentage of total fatty acids, oleic (18:1 n-9) and linoleic (18:2 n-6) acids comprised, respectively, 60.9% and 15.3% in VOO diet, whereas corresponding values in SO diet were 25.5% (oleic) and 56.3% (linoleic). After 8 months on the diets, animals were fitted with permanent pancreatic cannula in order to collect fasting and postprandial samples of pure pancreatic juice (see Fig. 46.1). The most striking results in this study1 were observed after food ingestion. Dogs fed on the SO diet responded to food with marked increases in pancreatic flow rate, whereas an attenuated response was observed in VOO animals. The secretion of bicarbonate, total protein, amylase, lipase, and chymotrypsin followed the same pattern as the pancreatic flow rate. Parallel experiments conducted in these very same animals during the adaptation period revealed that, while there were no differences between groups VOO and SO in fat digestibility,2 an improved digestive and metabolic utilization of protein was actually evidenced in VOO animals.3 Hence, pancreatic adaptation to diets containing olive oil as dietary fat source seems to represent a key advantage in terms of exocrine pancreatic economy, at least in this species. Our findings raised the suggestion that prolonged intake of VOO or SO might result in different circulating levels of some of the gastrointestinal hormones that control exocrine pancreatic secretion (Table 46.1). Oleic acid is a strong stimulus for the release of cholecystokinin (CCK),4,5 a major pancreatic secretagogue,6 but it is also a very effective releaser of peptide YY (PYY)5 and pancreatic polypeptide (PP),7,8 two gastrointestinal peptides

that inhibit exocrine pancreatic secretion.6 We sought confirmation of this hypothesis by submitting dogs to similar diets (35% energy as either VOO or SO) and feeding periods (6 months from weaning). Our data9 revealed that fasting and postprandial values for plasma PYY (Fig. 46.2) and fasting values for plasma PP were significantly higher in animals fed on the VOO diet, which explains the attenuated secretory activity found in this group in our previous study.1 We also examined10 if these changes were related not only to the type but also to the amount of fat. For this purpose, we fed weaning dogs for 6 months with diets containing 20% energy as fat (VOO or SO). Once more, we found a reduced pancreatic response to food in the VOO group. Interestingly, a comparison of secretory parameters obtained after high olive oil feeding1 (33% energy) and medium olive oil feeding10 indicated that the higher the content of VOO in the diet, the more attenuated the postprandial pancreatic response was. Considering the ability of oleic acid to elevate in the long term the blood levels of such inhibitory factors as PYY and PP, our results suggest a close inverse relationship between the content of oleic acid in the habitual diet and exocrine pancreatic response to a meal.

46.2.2 Bile secretion We used the same diets and dietary protocol than in the above study1 of exocrine pancreatic secretion. Following the adaptation period to diets, a permanent cannula was implanted in the common bile duct to allow collection of bile samples. No differences in bile flow rate were appreciated between VOO and SO animals in fasting conditions.11 FIGURE 46.1 Experimental design of the studies on long-term (8 months) adaptation of exocrine pancreatic secretion to dietary fat type in dogs. Dogs were allowed 1 week of recovery before the experiments were begun. Fasting (F) and postprandial (P) samples of pure pancreatic juice were collected at hourly intervals. The dotted area represents the postprandial period. SO, Sunflower oil; VOO, virgin olive oil.

Influence of olive oil on pancreatic, biliary, and gastric secretion: role of gastrointestinal peptides Chapter | 46

559

TABLE 46.1 Key facts of selected gastrointestinal peptide hormones. Gastrin

G

G

CCK

G

G

PYY

G

G

PP

G G

Somatostatin

G G

G

Stimulates ECL cells of the gastric mucosa to release histamine which, in turn, acts in a paracrine fashion to increase acid secretion from parietal cells Trophic factor for the gastric mucosa Stimulates pancreatic growth and exocrine pancreatic secretion, gallbladder contraction, and relaxation of the sphincter of Oddi; inhibits indirectly acid secretion via gastric somatostatin; and delays gastric emptying Reduces food intake Inhibits gastric acid secretion, exocrine pancreatic secretion, and intestinal motor and secretory functions; delays gastric emptying Reduces food intake Inhibits exocrine pancreatic secretion and gallbladder contraction; delays gastric emptying Reduces food intake Important paracrine mediator Inhibits gastrin and histamine release and gastric acid secretion, pepsinogen secretion, exocrine pancreatic secretion, and biliary secretion of fluid and bicarbonate General inhibitory action on secretion, absorption, and release of gastrointestinal hormones

This table lists a selection of peptide hormones of the gastrointestinal tract involved in the regulation of digestive secretions and/or motility, and their major functions. CCK, Cholecystokinin; ECL, enterochromaffin-like; PP, pancreatic polypeptide; PYY, peptide YY.

FIGURE 46.2 Plasma PYY concentration in dogs fed for 6 months on diets containing either VOO or SO as the fat source (35% energy). F represents the fasting situation. The arrow denotes the time of food ingestion. Values are means 6 SEM (n 5 4 for both groups). #P , .05 between the two groups at specific time points. PYY, Peptide YY; SO, sunflower oil; VOO, virgin olive oil. Reprinted from Yago MD, et al. Plasma peptide YY and pancreatic polypeptide in dogs after long-term adaptation to dietary fats of different degrees of saturation: olive and sunflower oil. J Nutr Biochem. 1997;8(9):504, with permission of Elsevier Ltd.

This parameter increased immediately in response to food intake in both dietary groups, but the temporal pattern was different. In VOO dogs, this rise lasted until the second postprandial hour, whereas at this time, bile flow rate in SO group had returned to premeal values and even a further, significant, decrease was observed.

Bile acid (BA) concentration in bile secreted by fasting dogs was significantly higher in those animals longterm fed the SO diet. In this group, food intake did not modify initially the values for this parameter, but a significant decrease was evident from the second postprandial hour onward. Opposite, the meal evoked in VOO dogs a rapid, significant, and transient increase in the concentration of BA. In both groups, changes in BA secretion (output, Fig. 46.3) were parallel to those in BA concentration. The marked elevation in BA concentration and secretion after eating suggests a greater involvement of the gallbladder in animals fed with the diet rich in VOO. This enhanced postprandial response in BA would increase the BA circulating pool, the efficiency of the enterohepatic circulation of these anions, and hepatic choleresis which, in turn, would account for the prolonged bile flow found in VOO animals as compared to SO ones. Enhanced gallbladder emptying in animals fed on olive oil diets is consistent with the ability of oleic acid to strongly stimulate the release of CCK. This peptide causes gallbladder contraction and relaxation of the sphincter of Oddi,12 thus allowing concentrated bile (rich in BA) to flow into the duodenum.

46.3 Olive oil and digestive secretion in humans Eighteen subjects with gallstones in the gallbladder and scheduled for elective surgery (cholecystectomy) were assigned to a VOO group or an SO group. They were instructed to consume their habitual diets (at home) for the 30-day period before surgery, with these requirements: (1) the only cooking fat had to be VOO or SO and

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FIGURE 46.3 Bile acid secretion in dogs fed for 8 months on diets containing either VOO or SO as the fat source (33% energy). F represents the fasting situation. The arrow denotes the time of food ingestion. Values are means 6 SEM (n 5 4 for both groups). For the VOO group, *P , .05 as compared with the respective fasting value; for the SO group, 1P , .05 as compared with the respective fasting value; #P , .05 between the two groups at specific time points. SO, Sunflower oil; VOO, virgin olive oil. Reprinted from Ballesta MC, et al. Adaptation of biliary response to dietary olive oil and sunflower-seed oil in dogs. Br J Nutr. 1992;68(1):179, with permission of Cambridge University Press.

(2) avoidance of eating food items high in other types of dietary fat (e.g., saturated fat). Compliance with the diets was checked by completion of four 7-day dietary records. Dietary assessment showed that intake of energy, protein, carbohydrates, and total fat was similar in both groups, with the main difference concerning the intake of monounsaturated fatty acids (MUFA, mean value of 40.1% of total fatty acids in VOO group vs 26.2% in SO group) and polyunsaturated fatty acids (PUFA, 8.3% in VOO group vs 19.8% in SO group).

Samples of gallbladder bile were obtained during cholecystectomy. In addition, a postcholecystectomy study was conducted 48 h after surgery, once it had been confirmed that subjects could tolerate oral feeding. The test meals were isoenergetic and isonitrogenous, contained no measurable amounts of cholesterol and phospholipids and were composed of 17% energy as protein, 30% as fat, and 53% as carbohydrate, in addition to vitamins and minerals. VOO was added to the meal given to the VOO group, and SO to the SO group. The content of MUFA and PUFA, expressed as a percentage of total fatty acids, was, respectively, 63.1% and 8.2% for the VOO liquid meal, and 29.7% and 44.6% for the SO liquid meal. For the postcholecystectomy study the participants were intubated with a nasoduodenal tube that enabled separate aspiration of gastric and duodenal contents. Each subject was studied on 2 consecutive days. On the first one, after a minimum 8 h fast, samples were taken to establish basal values and then the subjects were given the test meals (200 mL ingested over 30 min). Postprandial sampling took place at 30, 60, 120, and 180 min after beginning the ingestion of the meal (see Fig. 46.4). At every point, samples of blood and duodenal and gastric content were collected. The complete procedure was repeated on the second experimental day. The following sections describe the main findings of this study.

46.3.1 Plasma profile of gastrointestinal peptides The ingestion of the liquid meal13 led to significantly higher levels of plasma CCK in VOO subjects compared with SO subjects throughout the 30
120 min postprandial period (Fig. 46.5A), which seems reasonable given the potency of oleic acid as a CCK releaser.

FIGURE 46.4 Experimental design of the studies on adaptation to dietary fat type in humans. Subjects with gallstones in the gallbladder were kept on diets that contained either VOO or SO as the main source of fat for 30 days before elective cholecystectomy. Samples of gallbladder bile were obtained at surgery. Forty-eight hours after cholecystectomy, fasted subjects were given mixed liquid meals with 30% energy of the corresponding oil. Samples of blood and gastrointestinal contents were taken at the indicated times. The complete procedure was repeated next day. F and P denote, respectively, fasting and postprandial samples. Meals were ingested over 30 min (black area). The dotted area represents the rest of the postprandial period. SO, Sunflower oil; VOO, virgin olive oil.

Influence of olive oil on pancreatic, biliary, and gastric secretion: role of gastrointestinal peptides Chapter | 46

We also determined the circulating levels of the inhibitory peptides PYY14 and PP.15 In the fasting state, plasma PYY concentration in group VOO was significantly greater than in group SO. Following the test meal, PYY levels in VOO subjects remained consistently higher (Fig. 46.5B). Although feeding did not evoke in either group a marked elevation in plasma PYY, values tended to increase, slowly, during the postprandial period (Fig. 46.5B). Investigations in humans have shown a delayed PYY response to food that depends on the size of the meal and its fat content. Fatevoked PYY release is in part due to a direct action of unabsorbed fat digestion products reaching the endocrine L cells at the ileocolonic mucosa.16 In our conditions, the size, fat content, and administration rate of the meal, together with the absorptive processes along the tube, may have resulted in a delayed arrival of a small amount of such products to the distal intestine. Postprandial release of PYY is also stimulated by a number of gastrointestinal peptides, including CCK.16 Again, this is coherent with our results, since postprandial levels of CCK and PYY were higher in group VOO, where we found a significant positive correlation between both parameters.17

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As for PP, our data15 evidence in the two dietary groups the typical response to a meal, that is, an early peak followed by a sustained elevation for several hours (Fig. 46.5C). The peak is derived from vagal cholinergic activity and the, second, intestinal phase from a complex interaction between nerves and hormones.6,18 Our experiments revealed that circulating PP levels were significantly greater in VOO group for the entire postprandial period (Fig. 46.5C), in agreement with intestinal perfusion studies showing MUFA as strong stimulants for human PP secretion.7,8 Moreover, the fact that both CCK and PP plasma levels were higher in VOO subjects is consistent with the role of CCK as a hormonal mediator in the intestinal phase of PP release.6,18 Of note, the higher concentrations of the inhibitory peptides PYY and PP associated with the habitual intake of olive oil confirm our previous results in dogs. This has a special relevance if we consider that the 30-day period before surgery cannot be strictly considered as an adaptation period to the diet, at least with the meaning that this word receives in the animal studies, where the experimental subjects can be fed on chemically defined, controlled diets for long periods of time.

FIGURE 46.5 Time-course evolution of plasma concentrations of CCK (A), PYY (B), and PP (C) in cholecystectomized subjects after the administration of a liquid meal containing either VOO or SO. Subjects were kept on diets that contained VOO or SO as the main source of dietary fat for 30 days before surgery and thereafter were given meals that included the corresponding oil. F represents the fasting situation. The black bar denotes the duration of meal ingestion (30 min). Values are means 6 SEM (n 5 18 for both groups). For the VOO group, *P , .05 as compared with the respective fasting value; for the SO group, 1P , .05 as compared with the respective fasting value; #P , .05 between the two groups at specific time points. CCK, Cholecystokinin; PP, pancreatic polypeptide; PYY, peptide YY; SO, sunflower oil; VOO, virgin olive oil. Reprinted from (A) Yago MD, et al. Pancreatic enzyme secretion in response to test meals differing in the quality of dietary fat (olive and sunflowerseed oils) in human subjects. Br J Nutr. 1997;78(1):32, with permission of Cambridge University Press; (B) Serrano P, et al. Influence of type of dietary fat (olive and sunflower oil) upon gastric acid secretion and release of gastrin, somatostatin, and peptide YY in man. Dig Dis Sci. 1997;42(3):630, with permission of Springer Nature; (C) Serrano P, et al. Influence of the type of dietary fat upon the plasma levels of secretin and pancreatic polypeptide in cholecystectomized humans. Biog Amines. 1998;14(4), with permission of International Medart-Brill Academic Publishers.

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46.3.2 Exocrine pancreatic secretion In the same subjects, we determined the activity of pancreatic amylase, trypsin, chymotrypsin, lipase, and colipase in samples of duodenal content.13 No differences became apparent between the groups, with the exception of postprandial lipase activity, which was significantly higher in VOO subjects. Overall, our results do not reveal a great influence of dietary fat type on human pancreatic enzyme secretion, except for lipase. One explanation is that longer periods of time may be needed for the human pancreas to be affected by a modification in dietary fat composition. Yet, our observation of clear differences in the plasma profile of gastrointestinal peptides in these subjects (CCK, PYY, PP), together with the fact that we do not have data of true secretory rates (output) due to the limitations associated with human research, obliges us not to exclude the possibility that the type of fat can affect human pancreatic enzyme secretion to a greater extent than is evident from this study.

46.3.3 Gastric secretion Since the 19th century, many authors have demonstrated that the presence of fat at different segments of the gastrointestinal tract inhibits acid secretion. In our study,14 gastric pH in fasted subjects did not differ between the dietary groups (Fig. 46.6A). Using physiological stimulation by test liquid meals (pH 6.33) that were permitted to seek its natural pH, we found a prompt pH increase after food ingestion in both groups, followed by a decline due to the continuous secretion of acid by parietal cells of the stomach. In group VOO, pH remained elevated until the first postprandial hour and then this parameter showed a slow decrease toward the fasting value, whereas in group SO the return to baseline was completed within 1 h and a further decline was recorded, so values significantly lower than the fasting ones were reached at the end of the experimental period (Fig. 46.6A). Accordingly, H1 concentration (mEq/L) was significantly greater in SO volunteers at 60 min postprandially (data not shown). These results provide evidence in humans that a 30-day period of VOO diets results in attenuated intragastric acidity in response to a meal when compared with diets containing SO. The effect of olive oil on gastric acid secretion involved the suppression of serum gastrin14 (Fig. 46.6B), the main hormonal mediator of the postprandial increases in gastric acid secretion in humans.19 Given that the only nutrients that stimulate gastrin secretion are small peptides and amino acids resulting from protein digestion,12 a similar increase in serum levels should have been expected in both dietary groups after ingestion of two test meals that were identical except for the type of fat added.

We suggest that gastrin suppression in VOO subjects is associated with some hormonal factors released by oleic acid, with the ability to reduce gastrin secretion from G cells. A candidate is CCK, which displayed higher postprandial levels in VOO subjects. CCK has been described as a typical enterogastrone (see the “Minidictionary of terms” section), and this effect is not direct, but mediated through the release of somatostatin from gastric D cells.19 Somatostatin, in turn, inhibits not only gastrin release, but also histamine release from enterochromaffin-like cells and acid secretion from parietal cells.20 It should be mentioned here that, as part of our study, we measured plasma somatostatin and found similar values in groups VOO and SO.14 Nonetheless, this does not rule out our hypothesis, since most of somatostatin action on gastric acid secretion is exerted through paracrine pathways.20 We also propose PYY, higher as well in the VOO group, as a mediator for the action of olive oil on acid secretion, since this peptide is a well-established inhibitor of gastric acid secretion.12,21 Reduction of gastric acidity by VOO may contribute, together with other mechanisms,22 to the beneficial effects of VOO in the prevention and healing of peptic ulcers.

46.3.4 Biliary lipid composition and bile lithogenicity Our experimental design (see the beginning of Section 46.3) made possible the collection of gallbladder bile at surgery. In addition, the postcholecystectomy experiments allowed us to study the composition of fasting and postprandial hepatic bile. This was important, since the analysis of only gallbladder bile could have masked the influence of the dietary intervention because of the occurrence of preexisting gallstones. We collected hepatic bile samples by duodenal intubation. Although duodenal sampling does not permit the measurement of biliary lipid secretion (output), it provides a mechanism by which the proportion of biliary lipid classes (relative to total lipids) in hepatic bile can be quantified, offering data that would otherwise be difficult to obtain in humans in vivo. Intake of VOO or SO diets for 30 days before cholecystectomy23 did not affect the cholesterol saturation index (CSI) in gallbladder bile (mean values of 1.056 in VOO group and 1.013 in SO group; CSI calculated according to Carey24), a somehow expected finding considering the presence of established gallstones in the gallbladder. In the postcholecystectomy study,23 we found no differences between our groups in molar percentages of lipids or cholesterol saturation at fasting. CSI was

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VOO diet, whereas hepatic bile remained supersaturated until the end of the experiment in those given SO (Fig. 46.7). It is difficult to interpret these results without data of secretory rates, but two mechanisms could explain these differences. On the one hand, the ingestion (chronic, acute, or both) of the two types of fat might have produced different feeding motility patterns through differential stimulation of humoral agents, in turn affecting choleresis. A second possibility is that patients in the SO group have normal BA secretion in response to food but that this is coupled with a supranormal cholesterol secretion rate, which is in line with the hypocholesterolemic effect of n-6 PUFA.26 The difficulty and risks of sample collection and a big disparity in experimental protocols have probably made that the very few studies on the influence of dietary fat on bile composition and lithogenicity in humans have yielded equivocal results. Epidemiological research27 indicates a negative role for saturated fat in lithogenicity and gallstone formation. In contrast, a beneficial effect of n-3 PUFA through decreasing mucin production has lately been confirmed in animals.28 While the influence of specific types of unsaturated fats (MUFA, n-6-PUFA and n3-PUFA) on bile composition and cholesterol saturation awaits further investigation, it should be highlighted that more recent epidemiological work29 shows that higher adherence to healthy dietary patterns, as measured by three different diet-quality scores, is associated with lower risk of symptomatic gallstone disease.

FIGURE 46.6 Time-course evolution of gastric pH (A) and serum gastrin concentration (B) in cholecystectomized subjects after the administration of a liquid meal containing either VOO or SO. Subjects were kept on diets that contained VOO or SO as the main source of dietary fat for 30 days before surgery and thereafter were given meals that included the corresponding oil. F represents the fasting situation. The black bar denotes the duration of meal ingestion (30 min). Values are means 6 SEM (n 5 18 for both groups). For the VOO group, *P , .05 as compared with the respective fasting value; for the SO group, 1P , .05 as compared with the respective fasting value. SO, Sunflower oil; VOO, virgin olive oil. Reprinted from Serrano P, et al. Influence of type of dietary fat (olive and sunflower oil) upon gastric acid secretion and release of gastrin, somatostatin, and peptide YY in man. Dig Dis Sci. 1997;42 (3):629,630, with permission of Springer Nature.

indicative of cholesterol supersaturation in both of them, a physiological phenomenon after overnight fasting, which interrupts the enterohepatic circulation of BA causing bile to supersaturate with cholesterol. Afterward, meal-evoked recirculation of BA increases BA output, and hepatic bile reverts to a less saturated state, a phenomenon that has been observed in healthy, lithiasic, and cholecystectomized subjects. Surprisingly, we found this typical postprandial response only in subjects given the

46.4 Adaptation of digestive function and gastrointestinal peptides to dietary fat type: final considerations 46.4.1 Pancreas Although it is a fact30 that the digestive system, including its neural and hormonal networks, adapts to diet, literature over the past few years on this topic, and particularly on the adaptation of secretions to dietary components, has been largely absent. When it comes to the exocrine pancreas, it is only worth mentioning an article that examines the adaptive changes of pancreatic protease secretion to a short-term vegan diet31 and some research performed in ruminants, both wild32 and domestic.33 Nonetheless, and despite it is indirectly related to our investigations, recent work34 has been done that shows that dietary fat not only affects exocrine pancreatic secretion but can indeed modify the pancreatic structure necessary for optimal function of this organ. The authors34 performed a comparative analysis of the pancreases of aged rats that had been fed throughout their lives (2 years) with balanced isocaloric diets with different fat sources: VOO, SO, or fish oil (FO). This model would reflect

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FIGURE 46.7 Time-course evolution of the CSI in duodenal samples from cholecystectomized subjects after the administration of a liquid meal containing either VOO or SO. Subjects were kept on diets that contained VOO or SO as the main source of dietary fat for 30 days before surgery and thereafter were given meals that included the corresponding oil. F represents the fasting situation. The black bar denotes the duration of meal ingestion (30 min). Values are means 6 SEM (n 5 18 for both groups). CSI was calculated in these samples according to the method of Metzger et al.25 For the VOO group, *P , .05 as compared with the respective fasting value; #P , .05 between the two groups at specific time points. CSI, Cholesterol saturation index; SO, sunflower oil; VOO, virgin olive oil. Reprinted from Yago MD, et al. Effect of the type of dietary fat on biliary lipid composition and bile lithogenicity in humans with cholesterol gallstone disease. Nutrition. 2005;21(3):345, with permission of Elsevier Ltd.

what happens in humans who consume the same type of dietary fat throughout their lives. The highlight of this study is that the type of fat can affect the exocrine and endocrine compartments of the pancreas differently. Moreover, FO and SO evoked histological changes that were absent in VOO-fed rats. Thus lifelong consumption of the FO diet mainly affected the exocrine pancreas and was associated with acinar fibrosis and macrophage infiltrates in peri-insular regions, all histological changes typically observed in pancreatic fibrosis of the elderly. On the other hand, SO-rich diets mainly led to endocrine alterations with higher β-cell number and twice the insulin content. These signs have also been described in relation to glucose intolerance, insulin resistance, or prediabetes.35 Although SO-group animals were normoglycemic and normoinsulinemic, the results were suggestive of the onset of a prepathological state where insulin resistance triggers an adaptive response of the pancreas which, among other strategies, may include hyperplasia or hypertrophy of β cells and increased insulin secretion rate in order to maintain glycemia within the normal range. In short, this study34 shows that the composition of dietary fat can play an important role in pancreatic aging and supports a beneficial effect of VOO consumption in maintaining the microstructure of the gland. This is reinforced, within the context of obesity rather than aging, by findings from a

study36 in mice with diet-induced obesity and type 2 diabetes (T2D), where VOO showed to very effectively reverse many of the alterations in glycemia homeostasis by, in part, improving pancreatic β cell function. Mechanisms underlying enhanced performance of the pancreatic islets after prolonged intake of different dietary fats may be complex. In this way, although some of the abovementioned studies compare VOO with other fats that mainly differ in fatty acid composition, the beneficial effects of VOO can be also ascribed to many other bioactive compounds present in the unsaponifiable fraction of VOO, as confirmed for tyrosol in in vitro research using NIT-1 pancreatic β cells.37 Optimal functions of pancreatic β cells and glucose homeostasis are also related to pancreatic cancer. With no doubt, this cancer has been the focus of late research as far as the relationship between dietary fat and pancreas is concerned, which seems justifiable considering its mortality rate (less than 3% of patients survive more than 5 years). The risk of this cancer is increased in T2D. Decreased peripheral sensitivity to insulin, hyperglycemia, hyperinsulinemia, and persistent inflammation are all involved in the mechanism of T2D-induced pancreatic cancer.38 Hyperinsulinemia is associated with the onset of many types of cancer, but the deleterious effects of insulin are exerted at their maximum expression in the pancreas, as the peptide concentrates locally (20-fold as compared to plasma). The beneficial effects of VOO on metabolic risk factors in T2D patients,39 which are likely exerted at multiple levels, including direct effects on the endocrine pancreas, may be at least in part due to oleic acid, as shown by a prospective cohort study with more than 20,000 participants recruited into EPIC-Norfolk.40

46.4.2 Gastrointestinal peptides The past decade has seen an explosion of research into the field of physiological regulation of food intake, where peptides such as CCK, PYY, and PP have a main role as anorexigenic signaling molecules.12,41 This has been accompanied, on the one hand, by great advances in the knowledge of how the enteroendocrine cells of the digestive tract respond to the components of the diet (the socalled nutritional chemosensing) and, on the other, to the realization of investigations aimed at examining whether different types of macronutrients (e.g., fat) differentially affect appetite and the secretion of these gastrointestinal peptides, in an attempt to find new strategies for better obesity management. Fat-induced release of CCK and PYY from I cells in the proximal gut and L cells in the distal gut, respectively, is mediated by G protein
coupled receptors (GPRs) that show certain degree of specificity. Thus triacylglycerols are not ligands for these receptors, which imply that fat

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digestion is a requisite in order to CCK and PYY to be secreted, something known for some time now. Among the products obtained after the digestion of fat, it is fatty acids with carbon chain length $ 12 that have the ability to trigger CCK release from I cells via activation of receptors GPR40 (also known as free fatty acid receptor 1 or FFAR1) and GPR120 (FFAR4).42,43 In L cells the receptor that has been suggested as most probably involved in dietary fat
induced PYY response is GPR119, which is activated by monoacylglycerols and some fatty acids derivatives (acylethanolamides).42,43 Nevertheless, L cells also express GPR120,42 so a direct action of long-chain fatty acids has to be considered as well. In fact, intraduodenal administration of oleic acid stimulates PYY release in humans.5 Despite great advances in mechanisms of fatty acid sensing by gut endocrine cells and the discovery of some tumor lines, such as the CCK-secreting line STC-1, discrepancy still exists regarding which type of dietary fat elicits the greatest CCK and PYY responses in terms of plasma level elevation. In the past years a number of studies have examined this topic in humans in relation to appetite and food intake regulation.44
50 However, probably due to the variability of experimental designs, the results obtained are discrepant, there being studies that show no differences in the secretion of CCK and PYY after the intake of several fats, while in other cases, those differences do exist, but they are not always in favor of the same dietary fat. The same reason makes it very difficult to compare the results of this research with ours. An important aspect is the length of the exposure to dietary fat, since the intestinal fat sensing systems can be influenced by habitual dietary fat intake.51 Most studies involve just acute meal challenges,5,44
46,50 and only a few of them examine plasma hormone levels after several days (maximum 7) of ingesting the experimental fats.47
49 In our investigations in humans13
15 the approach was to achieve an adaptation as closely as possible to that obtained in our study in dogs,1,9
11 which were fed with the two experimental fats from weaning and over 6
8 months. For that purpose, our patients13
15 were allocated to the experimental groups, VOO and SO, according to the type of dietary fat habitually consumed (olive oil or SO, information gained from a dietary history interview at the beginning of the study), and then, they were submitted correspondingly to the 30-day VOO and SO diets (see earlier) under our supervision. Hence, our results are more likely to reflect a longterm adaptation to habitual diet and are by no means comparable with those found after an acute test or following the consumption for a few days of a particular fat. This could explain, in part, the discrepancies between our findings and those by the group of Cooper,45,46,49 who compares the effects of MUFA and n-6 PUFA and shows that the latter elicit greater postprandial PYY45,46 and CCK49 responses.

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Additional inconsistencies among the studies relate the physical state of the test meal (solid vs liquid), the mode of delivery (oral44
46,49 vs intraintestinal5,50), and, importantly, the percentage of energy supplied by total fat and the fatty acid composition of the meal. To focus on the studies which, like ours, used oral test meals containing either MUFA or n-6 PUFA,44
46 it should be said that test meals contained between 50% and 70% energy as fat, much higher than our test meals which comprised 30% kcal. Also, the only source of fat in our test meals was either VOO (rich in MUFA) or SO (rich in n-6 PUFA), chosen for being the most representative dietary oils used in our area, whereas in the abovementioned studies,44
46 various dietary fats were mixed so that the percentage of energy coming from the fatty acid of interest was similar between test meals. Differences in peptide results could also be accounted for by these factors.

46.5 Summary points Compared with SO, habitual intake of VOO may be beneficial for the gastrointestinal system. The following points summarize our main conclusions: G

G

G

G

Long-term adaptation to diets high in VOO attenuates postprandial exocrine pancreatic secretory activity in dogs in a manner proportional to its amount in the diet. This effect is accompanied by elevation in the circulating levels of PYY and PP and is exerted without derangement of nutrient utilization, hence representing a key advantage in terms of pancreatic economy. Patterns of biliary response to food in dogs fed for 8 months with diets containing VOO are indicative of a greater involvement of the gallbladder. A 30-day adaptation period to a diet containing VOO results in reduced gastric acidity following a liquid meal with the same oil in humans. Olive oil, then, may be a useful component in the nutritional therapy of those gastrointestinal diseases requiring a limitation of acid secretion. The type of dietary fat habitually consumed can influence bile composition in humans. In our study, this influence was noted especially after cholecystectomy, when a physiological postprandial decrease in hepatic bile lithogenicity occurred in patients given VOO meals but not in those receiving SO meals. Provided they are confirmed in healthy subjects, these results together with a very efficient gallbladder emptying as suggested by our investigations in dogs would indicate a beneficial role of olive oil in the pathogenesis of gallstone disease.

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The abovementioned effects in humans are mediated, at least in part, by changes in the fasting and/or postprandial release of several gastrointestinal hormones: intake of VOO results in higher plasma levels of CCK, PYY, and PP and lower levels of gastrin compared with SO diets.

Mini-dictionary of terms Anorexigenic signaling molecules: Peptides that reduce the appetite. Beta (β) cells: Produce insulin, a hormone that controls the level of glucose in blood (i.e., glycemia). These cells are located in the endocrine portion of the pancreas. Cholecystectomy: Surgical procedure to remove the gallbladder. Cholesterol saturation index (CSI): The lithogenic index or CSI is a number obtained taking into account the proportion of cholesterol, bile acids, and phospholipids in a bile sample. It is often estimated using the critical tables proposed by Carey (see Ref. [24]). A CSI value greater than 1.0 is indicative of bile saturated in cholesterol. Endocrine regulation: It refers to cells or endocrine glands secreting molecules (hormones) that enter the bloodstream to subsequently reach their target cells and exert their actions. Enterogastrone: A circulating factor released from the intestine that inhibits gastric acid secretion. Enterohepatic circulation: The circulation of biliary acids, bilirubin, drugs, or other substances from the liver to bile, followed by entry into the proximal intestine (duodenum), reabsorption in the distal intestine (ileum) and transport back to the liver. EPIC: European Prospective Investigation into Cancer and Nutrition. It is one of the largest epidemiological studies in the world, with more than half a million participants recruited across 10 European countries and followed for almost 15 years. Fat digestibility: The percentage of fat ingested that is actually absorbed in the intestine. Hypocholesterolemic effect: Cholesterol (plasma)-lowering effect. Lithogenic bile: Bile that favors gallstone production. This may be associated with several conditions, the most important being increased secretion of cholesterol in the bile. Paracrine: Regulatory action exerted on nearby cells, with the regulatory molecule diffusing through the extracellular fluid, without entering the bloodstream. Postprandial: After eating a meal. Unsaponifiable fraction: The unsaponifiable fraction of a fat product includes all of the components that, after a process called alkaline hydrolysis (saponification), are

barely soluble in aqueous solutions but are soluble in organic solvents. The unsaponifiable fraction of virgin olive oil contains small amounts of bioactive compounds, many of them with antioxidant activity.

Comparisons of olive oils with other edible oils Throughout the entire chapter, virgin olive oil (VOO) has been compared mainly with sunflower oil. In our experimental conditions, our results do support (see the “Summary points” section) long-term consumption of VOO as an added fat for attaining optimal digestive functionality, best within the context of the Mediterranean diet as a whole. However, it is clear that more studies are needed, preferably in humans, that incorporate experimental designs and protocols carefully elaborated, in which diets and meals administered to the study subjects contain relevant and reasonable amounts of fat, from a nutritional and physiological point of view, and that not only include acute meal challenges.

Implications for human health and disease prevention Regulatory mechanisms of food intake, which underlie the appearance of hunger and fullness sensations, have been studied for many years, although in the past decade, these investigations have greatly intensified due to their direct relationship with obesity and, certainly, great advances have been made in their knowledge. The nutrients that reach the lumen of the gastrointestinal tract are broken by digestive enzymes and the products obtained are specifically detected (i.e. chemosensed) by different enteroendocrine cells located in the mucosa that, then, secrete signaling peptide molecules (gut peptides) that can act on nearby cells and also give rise to responses in organs and tissues traveling through the blood or, alternatively, through the generation of electrical signals in nerve fibers. By these different ways, gut peptides manage to regulate digestive secretions and motility patterns so that these are consistent with the amount and type of nutrients ingested, thereby optimizing the processes of digestion and absorption. In addition, with the participation of the nervous system, gut peptides inform the brain about the amount of food that has been ingested and the energy that is being absorbed. These processes fall into what is known as the gut
brain axis and are crucial for energy homeostasis. Once informed of the characteristics of the food being ingested, the brain generates orders aimed at finishing the meal (fullness or satiation) so excessive energy intake is avoided. Some gut peptides, when elevated in blood, also determine the phenomenon of satiety

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(i.e., how long it will take until the next meal). Hours after the meal have finished, the plasma levels of all these anorexigenic peptides return back to resting values, causing the hunger signal to fire. Taking all the above together, it is easy to understand why more and more research is focused on ascertaining whether particular components of the diet give rise to greater or lesser satiation/satiety and what are the gut peptides involved. From previous sections of this work, it is clear that there is still a lot of work to do regarding the influence of the type of dietary fat, as there is still much disagreement. Conducting additional research to relate the intake of different fats with circulating levels of peptides will clarify these discrepancies. Elucidating which receptors are expressed in the different types of enteroendocrine cells and what exactly is their specificity with respect to fatty acids or other fat digestion products will also be of great help. Meanwhile, adherence to a highquality food and lifestyle pattern, such as the Mediterranean one, is the best option, since it has been inversely associated with obesity according to epidemiological studies. This association, obviously, may be explained by the action of different components and mechanisms (e.g., fiber intake and satiety), but if we take into account that olive oil in the Mediterranean areas has traditionally provided up to 40% of daily energy, it is hard to believe that this dietary fat produces less satiation/ satiety than others, at least in the context of habitual consumption. Scientific evidence will have to confirm this point. Another relevant aspect of the binomial virgin olive oil (VOO) health refers to glycemic control and risk of type 2 diabetes (T2D) mellitus. Insulin secretion from pancreatic β cells is stimulated not only by nutrients absorbed from the digestive tract (mainly glucose) but also by several peptides released by enteroendocrine cells. These peptides are collectively called incretins. The best known incretins are glucagon-like peptide-1 and glucosedependent insulinotropic peptide. Although the beneficial actions of VOO in T2D are attributed to increased insulin sensitivity in peripheral tissues and better β cell functionality (see earlier), incretin-mediated mechanisms could also intervene. Moreover, evidence from animal experimentation suggests that both cholecystokinin and peptide YY could incorporate in a near future into the incretin group. All this reinforces the interest in the study of how dietary fat influences the circulating levels of all these peptides. Finally, it is increasingly accepted that antioxidant and antiinflammatory bioactive compounds in the unsaponifiable fraction of VOO may have a beneficial role in digestive conditions such as ulcerative colitis and Crohn’s disease. The therapeutic potential of some of these compounds is currently being explored.

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17. Yago MD, Man˜as M, Gonza´lez MV, Martı´nez-Victoria E, Pe´rez MT, Mataix J. Plasma levels of cholecystokinin and peptide YY in humans: response to dietary fats of different degrees of unsaturation (olive and sunflower oil). Biog Amines. 1997;13:319
331. ´ 18. Sliwi´ nska-Mosso´n M, Marek G, Milnerowicz H. The role of pancreatic polypeptide in pancreatic diseases. Adv Clin Exp Med. 2017;26:1447
1455. 19. Rehfeld JF. Premises for cholecystokinin and gastrin peptides in diabetes therapy. Clin Med Insights Endocrinol Diabetes. 2019;12:1
6. 20. Schubert ML, Rehfeld JF. Gastric peptides—gastrin and somatostatin. Compr Physiol. 2019;10:197
228. 21. Lafferty RA, Flatt PR, Irwin N. Emerging therapeutic potential for peptide YY for obesity-diabetes. Peptides. 2018;100:269
274. 22. Lombardo L, Grasso F, Lanciano F, Loria S, Monetti E. Broadspectrum health protection of extra virgin olive oil compounds. Stud Nat Prod Chem. 2018;57:41
77. 23. Yago MD, Gonza´lez V, Serrano P, et al. Effect of the type of dietary fat on biliary lipid composition and bile lithogenicity in humans with cholesterol gallstone disease. Nutrition. 2005;21:339
347. 24. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res. 1978;19:945
955. 25. Metzger AL, Heymsfield S, Grundy SM. The lithogenic index
a numerical expression for the relative lithogenicity of bile. Gastroenterology. 1972;62:499
501. 26. No Authors Listed. Fats and fatty acids in human nutrition. Report of an expert consultation. FAO Food Nutr Pap. 2010;91:1
166. 27. Tsai CJ, Leitzmann MF, Willett WC, Giovannucci EL. Long-chain saturated fatty acids consumption and risk of gallstone disease among men. Ann Surg. 2008;247:95
103. 28. Jang SI, Fang S, Kim KP, et al. Combination treatment with n-3 polyunsaturated fatty acids and ursodeoxycholic acid dissolves cholesterol gallstones in mice. Sci Rep. 2019;9:12740. 29. Wirth J, Song M, Fung TT, et al. Diet-quality scores and the risk of symptomatic gallstone disease: a prospective cohort study of male US health professionals. Int J Epidemiol. 2018;47:1938
1946. 30. Covasa M. Deficits in gastrointestinal responses controlling food intake and body weight. Am J Physiol Regul Integr Comp Physiol. 2010;299:R1423
R1439. 31. Walkowiak J, Ma˛dry E, Lisowska A, et al. Adaptive changes of pancreatic protease secretion to a short-term vegan diet: influence of reduced intake and modification of protein. Br J Nutr. 2012;107:272
276. 32. Guilloteau P, Vitari F, Metzinger-Le Meuth V, et al. Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe deer. BMC Vet Res. 2012;8:70. 33. Kowalik B, Majewska MP, Miltko R, Beł˙zecki G. The effect of supplementing sheep with rapeseed and linseed oils on the activity of pancreatic digestive enzymes. J Anim Physiol Anim Nutr. 2018;102:1194
1198. 34. Roche E, Ramı´rez-Tortosa CL, Arribas MI, et al. Comparative analysis of pancreatic changes in aged rats fed life long with sunflower, fish, or olive oils. J Gerontol A Biol Sci Med Sci. 2014;69:934
944. 35. Jones HB, Nugent D, Jenkins R. Variation in characteristics of islets of Langerhans in insulin-resistant, diabetic and non-diabeticrat strains. Int J Exp Pathol. 2010;91:288
301.

´ lvarez-Amor L, Varela LM, et al. Extra virgin 36. Jurado-Ruiz E, A olive oil diet intervention improves insulin resistance and islet performance in diet-induced diabetes in mice. Sci Rep. 2019;9:11311. 37. Lee H, Im SW, Jung CH, Jang YJ, Ha TY, Ahn J. Tyrosol, an olive oil polyphenol, inhibits ER stress-induced apoptosis in pancreatic β-cell through JNK signaling. Biochem Biophys Res Commun. 2016;469:748
752. 38. Li Y, Bian X, Wei S, He M, Yang Y. The relationship between pancreatic cancer and type 2 diabetes: cause and consequence. Cancer Manag Res. 2019;11:8257
8268. 39. Schwingshackl L, Lampousi AM, Portillo MP, Romaguera D, Hoffmann G, Boeing H. Olive oil in the prevention and management of type 2 diabetes mellitus: a systematic review and metaanalysis of cohort studies and intervention trials. Nutr Diabetes. 2017;7:e262. 40. Banim PJ, Luben R, Khaw KT, Hart AR. Dietary oleic acid is inversely associated with pancreatic cancer
data from food diaries in a cohort study. Pancreatology. 2018;18:655
660. 41. Holzer P, Reichmann F, Farzi A. Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut-brain axis. Neuropeptides. 2012;46:261
274. 42. Raka F, Farr S, Kelly J, Stoianov A, Adeli K. Metabolic control via nutrient-sensing mechanisms: role of taste receptors and the gut-brain neuroendocrine axis. Am J Physiol Endocrinol Metab. 2019;317:E559
E572. 43. Roura E, Depoortere I, Navarro M. Review: chemosensing of nutrients and non-nutrients in the human and porcine gastrointestinal tract. Animal. 2019;13:2714
2726. 44. Robertson MD, Jackson KG, Fielding BA, Morgan LM, Williams CM, Frayn KN. Acute ingestion of a meal rich in n-3 polyunsaturated fatty acids results in rapid gastric emptying in humans. Am J Clin Nutr. 2002;76:232
238. 45. Kozimor A, Chang H, Cooper JA. Effects of dietary fatty acid composition from a high fat meal on satiety. Appetite. 2013;69:39
45. 46. Stevenson JL, Clevenger HC, Cooper JA. Hunger and satiety responses to high-fat meals of varying fatty acid composition in women with obesity. Obesity. 2015;23:1980
1986. 47. Stevenson JL, Paton CM, Cooper JA. Hunger and satiety responses to high-fat meals after a high-polyunsaturated fat diet: a randomized trial. Nutrition. 2017;41:14
23. 48. Cooper JA, Watras AC, Paton CM, Wegner FH, Adams AK, Schoeller DA. Impact of exercise and dietary fatty acid composition from a high-fat diet on markers of hunger and satiety. Appetite. 2011;56:171
178. 49. Polley KR, Kamal F, Paton CM, Cooper JA. Appetite responses to high-fat diets rich in mono-unsaturated versus poly-unsaturated fats. Appetite. 2019;134:172
181. 50. Maljaars J, Romeyn EA, Haddeman E, Peters HP, Masclee AA. Effect of fat saturation on satiety, hormone release, and food intake. Am J Clin Nutr. 2009;89:1019
1024. 51. Cvijanovic N, Isaacs NJ, Rayner CK, Feinle-Bisset C, Young RL, Little TJ. Duodenal fatty acid sensor and transporter expression following acute fat exposure in healthy lean humans. Clin Nutr. 2017;36:564
569.

Chapter 47

Effects of virgin olive oil on fatty acid composition of pancreatic cell membranes: modulation of acinar cell function and signaling, and cell injury Maria Alba Martinez-Burgos 1 , Maria Dolores Yago 1 , Belen Lopez-Millan 1 , Jose Antonio Pariente 2 , Emilio Martinez-Victoria 1 and Mariano Man˜ as 1 1

Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain,

2

Department of Physiology, Faculty of Sciences, University of Extremadura, Badajoz, Spain

Abbreviations 21

[Ca ]c CCK CCK-8 DAG DHA EPA GSH IP3 LCPUFA MUFA NFκB PUFA PYY SFA VOO

21

cytosolic Ca concentration cholecystokinin cholecystokinin-octapeptide diacylglycerol docosahexaenoic acid (C22:6 n-3) eicosapentaenoic acid (C20:5 n-3) glutathione (reduced form
SH) inositol trisphosphate long-chain polyunsaturated fatty acids monounsaturated fatty acids nuclear factor kappa B polyunsaturated fatty acids peptide YY saturated fatty acids virgin olive oil

47.1 Introduction The involvement of diet in human health and in the prevention and treatment of disease is a well-known topic, recognized and accepted by international institutions for a long time. Over recent years, many functional foods and ingredients have been developed and characterized. Beyond adequate nutritional effects, their biological actions are relevant to an improved state of health and well-being and/or reduction of risk of chronic disease. It is known that Mediterranean countries have a lower prevalence of cardiovascular diseases, compared to Northern Europe and other developed countries, and life

expectancies that are among the highest. Consequently, the Mediterranean diet has been described as a cultural model for dietary improvement. There is a need for sound nutritional and scientific information to support the many claims for the health-enhancing effects of the Mediterranean diet, and the generation of new data on the health benefits of the components of this diet is important. Used as the major culinary fat, virgin olive oil (VOO) is an integral ingredient of the Mediterranean diet (which provides up to 40% of calories as fat), and evidence about its health benefits keeps accumulating. In fact, VOO appears to be an example of a functional food, with varied components that may contribute to its overall therapeutic characteristics, especially its high levels of monounsaturated fatty acids (MUFA) and other minor components (e.g., phenolic compounds, mainly hydroxytyrosol) with antioxidant actions. The type of dietary fat is more closely related to the incidence of some chronic diseases than the level of dietary fat.1 Thus habitual consumption of dietary fats with different fatty acid profiles may have different physiological consequences and effects on health. Mammalian cellular and subcellular membranes consist of a lipid bilayer, with functional proteins either bound to the surface or embedded into the bilayer. The lipid bilayer is composed of phospholipids and cholesterol. Polar head groups of these lipids occupy the outer surfaces of the bilayer, while the inner hydrophobic core consists primarily of long fatty acyl chains. It is generally accepted that biological membranes do not have

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00012-2 © 2021 Elsevier Inc. All rights reserved.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

a constant composition. Some factors, such as age, physiologic state, cell type, antioxidant capacity, or metabolic activity can modulate the structure and function of cell membranes.2 In addition, the diet constitutes a major exogenous determinant. Thus dietary differences in fatty acid intake can alter the fatty acid composition of membrane phospholipids. Many authors have investigated the adaptation of human and animal cells to dietary fat, and the results indicate that the response is tissue specific. After a change in dietary fat composition, membranes of the intestinal epithelial cells seem to be the first to adapt.3 This adaptation involves a modification in membrane fluidity and cell function, particularly the transport capacity and metabolic activity of the membranes.2 The liver is also very sensitive to dietary-induced changes.4 In contrast, other tissues such as the brain5 or skeletal muscle4 seem to show relatively minor adaptive alterations in the lipid composition of their membranes. The biological effects of dietary fatty acids are partly due to their incorporation into the cellular structures of organs and tissues. Changes in membrane fatty acid composition may, in turn, influence cell function.6 This is not an unexpected finding, since there is growing evidence that fatty acids can participate in intracellular processes as diverse as signal transduction or the regulation of gene expression.7 Briefly, modulation of cell function by membrane fatty acids can be achieved by some of the following mechanisms: G

G

G

An indirect effect through structural changes, which affect either bulk lipid fluidity or specific lipid domains. The conformation of transporters, receptors, and membrane-linked enzymes is probably sensitive to changes in the structure of their lipid microenvironment, leading to changes in their activity. Phospholipid fatty acids participate themselves in signaling pathways. Examples are found in the hydrolysis of phosphatidylinositol phosphates by phospholipases or eicosanoid production. Consequently, these pathways may be strongly influenced by dietary fat. A direct link between dietary fatty acid consumption, gene expression, and tissue proliferation is provided by the fact that many transcription factors are regulated by fatty acids.

47.2 Dietary lipids and pancreatic secretion Since the findings of Pavlov in the early 1900s many authors have investigated in various species the adaptation of the exocrine pancreas to the type of food available. The general consensus of these studies is that, for dietary components such as carbohydrates, protein, and lipid,

there is a positive relationship between their level in the diet and the tissue content of pancreatic enzymes necessary for their breakdown. Concerning lipids, both the amount and type seem to have an influence,8 although a controversy over the effects of the fatty acid composition of dietary fat on the adaptive process of pancreatic enzymes is unresolved. Previous investigations of the in vivo pancreatic response after medium- or long-term intake of diets differing in the fat source indicated that pancreatic adaptation to dietary fat type is mediated, at least in part, by changes in the circulating levels of some gastrointestinal hormones.9,10 So, the first option to explain the mechanisms of the pancreatic adaptation to dietary fat involves the existence of hormonal mediators (see Chapter 46 in this book). However, a second possibility to explain the mechanisms of pancreatic adaptation is that dietary fat composition is affecting directly the secretory activity of pancreatic acinar cells by changing their responsiveness to circulating secretagogues and/or altering intracellular signaling as a consequence of the modification of the fatty acid composition of the pancreatic membranes. As mentioned previously, there is evidence in different tissues that the lipid profile of the diet can influence the fatty acid composition of cell membranes, this being associated with a modification of cell function.6 Regarding the exocrine pancreas, information on this topic is very limited11 but supports the abovementioned view. Besides, it should be mentioned that the most frequent methodological approach has consisted of the analysis of the enzyme content of the gland. A different approach is to examine, after long-term intake of specific diets, the secretory response in the whole animal or in isolated pancreatic acini, studying the influence of the modification of the lipid profile of pancreatic membranes. In this chapter, we will describe the effect of dietary fat type on the fatty acid composition of rat pancreatic membranes and the interaction with the exocrine pancreatic secretion in anesthetized animals, in preparation of isolated rat pancreatic acini and in the AR42J cell line. Responses to both physiological and pathological stimuli will be described. In the in vivo studies, VOO and sunflower oil (SO) were compared. In AR42J cells, we used (1) oleic acid (18:1 n-9, MUFA), major fatty acid in VOO; (2) linoleic acid [18:2 n-6, n-6 polyunsaturated fatty acid (PUFA) or n-6 PUFA], major fatty acid in SO; and (3) n-3 long-chain PUFA (n-3 LCPUFA), present in fish oils.

47.3 Pancreatic secretion in anesthetized rats Male weaning Wistar rats were fed over an 8-week period with two semipurified, isoenergetic, and isonitrogenous

Effects of virgin olive oil on fatty acid composition of pancreatic cell membranes Chapter | 47

diets that were essentially AIN-93G diets except that total fat content was increased from 7 to 10 wt.% at the expense of carbohydrate.12 The two diets differed only in the nature of the fat source: VOO or SO. At the end of the 8-week feeding period, we examined the exocrine pancreatic secretion, both at rest and following stimulation with cholecystokinin (CCK)-octapeptide (CCK-8). The protein and amylase content of the pancreas was also analyzed. Furthermore, to confirm a direct effect of the type of dietary fat, the fatty acid composition of pancreatic membranes was determined after feeding the respective diets. This investigation12 showed that the diets did not affect food intake and body weight gain during the 8-week feeding period. We also failed to find any difference between our groups in the weight of pancreases. The analysis of pancreatic cell membranes revealed that our dietary protocol was satisfactory, since a direct effect of the type of dietary fat on the pancreatic gland was confirmed. The fatty acid profile of pancreatic cell membranes reflected the composition of the lipid component of the experimental diets. After 8 weeks on the diets, pancreatic membranes in the VOO group had significantly higher levels of oleic acid (43.09 6 2.19, expressed as percentage of total fatty acid content) and total MUFA (48.30 6 2.52), whereas a higher level of PUFA (31.14 6 2.04), particularly n-6 PUFA such as linoleic acid (18.58 6 1.20), was found in the SO group.12 These data are consistent with those obtained by others.11,13 The proportion of total saturated fatty acids (SFA) was similar in the membranes of the two groups. In fact, except for 18:0 values, no further differences could be detected. This resistance of the SFA fraction to dietaryinduced alterations is a common feature in different tissues.11 In addition, feeding diets rich in VOO or SO did not alter significantly the SFA/unsaturated fatty acid ratio or the unsaturation index. This suggests that membranes display a considerable degree of homeostasis with respect to these parameters, and that changes in the proportion of several major fatty acids are associated with some metabolic compensation in order to keep plasma membrane fluidity within a certain range of values. By using the method of direct cannulation of the pancreatic duct, we were able to confirm in this study12 a modification of the secretory output as a function of the type of dietary fat. Under resting conditions, pancreatic flow rate and amylase output were significantly higher in rats on the SO diet than in those on the VOO one (0.68 6 0.054 vs 0.37 6 0.021 μL/min and 61.79 6 6.03 vs 35.84 6 3.55 mU/min, respectively). The mechanism underlying this effect is, however, far from clear. In the whole animal, exocrine pancreatic secretion is regulated by a complex integration of hormonal and neural mechanisms, with stimulatory factors

571

being balanced by a variety of inhibitory regulators, including peptide YY (PYY). In the pancreas, PYY has been shown to inhibit not only the secretion stimulated by secretin and/or CCK but also basal secretion. In previous studies conducted in dogs and humans, we found that medium-/long-term intake of diets containing VOO as the major fat source evoked a significant elevation of plasma PYY in resting conditions as compared to the levels measured after SO feeding.10 Thus it is tempting to speculate that the diminished exocrine secretion observed in resting conditions in the VOO group of the present study might reflect an augmentation of the resting PYY levels. Changes in exocrine pancreatic secretion following intravenous infusion of CCK-8 suggest that the two groups responded differently to this secretagogue, with the time course of changes in the secretory parameters during and after the infusion of CCK-8 showing a different pattern in each group.12 The existence in the VOO group, but not in the SO one, of a positive direct correlation between flow rate and both protein concentration and amylase activity lends further support for an influence of the type of dietary lipids on the secretory activity of the exocrine pancreas. In conclusion, the present investigation shows that chronic intake of diets differing only in the type of fat added (VOO or SO) influences the exocrine pancreatic secretion in anesthetized rats. The major differences between the dietary treatments concerned the magnitude of the secretion of fluid and amylase in resting conditions as well as the time course of changes in all major secretory parameters evoked by a continuous intravenous infusion of CCK-8.12 At the present time, there is no definite explanation for this behavior of the gland. Clearly, the observed differences in pancreatic secretion from anesthetized rats are not the consequence of a variation in tissue content of protein or amylase because we could not find a clear effect of the type of dietary lipids on protein content and amylase activity in pancreatic homogenates after feeding the experimental diets for 8 weeks. Considering the dietary-induced changes in the fatty acid composition of pancreatic membranes, the possibility exists that in vivo data are reflecting a distinct modulatory effect of the type of dietary fat on the secretory activity at the cellular level.

47.4 Experiments in isolated pancreatic acini The above idea was reinforced by our results14 in viable pancreatic acinar cells isolated from rats kept on identical dietary protocol as in the previous study. Diets containing either VOO or SO were given to separate groups of rats for 8 weeks. Acinar cell function was assessed by

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

determining basal and CCK-8-stimulated amylase release in suspensions of fresh viable acini. We also examined the effect of these diets on the mobilization of intracellular free Ca21, a key mediator of CCK-8-evoked enzyme secretion. That our dietary protocol was appropriate for our purposes was also confirmed by the membrane fatty acid analyses showing, like in previous work,12 the sensitivity of the pancreas to dietary fat changes. After 8 weeks on the diets, we found again that the membrane fatty acids were profoundly affected by the diets; the rats fed the VOO diet had higher levels of 18:1 n-9 and total MUFA compared with the animals fed the SO diet. Reciprocally, the SO diet resulted in greater levels of total and n-6 PUFA than the VOO diet. Adaptation to the diet did not modify the concentration
response curve for CCK-8-induced amylase release in pancreatic acinar cells (Fig. 47.1). Our results are consistent with those in the literature15 since, in our conditions, the strongest secretory effect was observed at 0.1 nM (10210 M) CCK-8, and a characteristic decrease occurred after the addition of higher concentrations of the secretagogue. This pattern was followed in cells from both groups, but quantitatively marked differences were revealed between them. Thus values for basal amylase release in acini from rats fed the VOO diet were similar to those reported by most authors,15 whereas release in cells from the SO-fed rats was markedly higher. In contrast to the observations in basal conditions, net amylase secretion in response to all concentrations of CCK-8 was drastically reduced after SO feeding (Fig. 47.1). This diminished secretory activity may be explained in part by the attenuation of CCK-8-evoked Ca21 responses in the SO group14 (data not shown). Moreover, the fact that not only the absolute value of the cytosolic Ca21 concentration ([Ca21]c) peak but also the peak increase over basal were lower in cells from the SO-fed rats suggests a reduction in the filling state of CCK8-releasable Ca21 pools and/or a limitation in the production or effectiveness of the mediators that participate in the Ca21-signaling pathways. Parallel experiments undertaken by our group16 confirmed the effects of VOO by using an inverted fluorescence microscope attached to a continuous perfusion system to study cellular Ca21 homeostasis at a single-cell level. A group fed a commercial chow was used as control. Feeding diets rich in VOO did not significantly alter the resting [Ca21]c values or basal amylase secretion. However, both the Ca21 oscillations and the large Ca21 transients in response, respectively, to low and high concentrations of CCK-8 were significantly enhanced by the VOO diet (Fig. 47.2A and B) compared with the control one (Fig. 47.2C and D). These effects on Ca21 mobilization correlated, to a great extent, with CCK-8-evoked amylase secretory activity.

The differences in acinar secretory activity and Ca21 mobilization in our studies are most probably related to the dietary-induced changes in cell membrane composition. Many steps of the stimulus
secretion coupling process are membrane dependent; differential enrichment in certain fatty acids may influence the accessibility of the CCK receptor, the interaction with G proteins, or the functionality of enzymes such as phospholipases and protein kinase C, which interact with cell membranes during their activation. Occupation of CCK receptors on pancreatic acinar cells is linked to phospholipase C activation and subsequent production of inositol trisphosphate (IP3), which initiates the Ca21 signal, and diacylglycerol (DAG). The membrane modifications could reasonably involve an alteration in the phosphoinositide turnover and a change in the supply of inositol lipid precursors of IP3. Increased production of IP3 in acini from rats fed VOO may explain the enhancement of intracellular Ca21 mobilization in response to CCK-8, because the initial increase in Ca21 transients is due mainly to Ca21 released from IP3-sensitive internal stores. Alternatively, it is tempting to speculate that DAG, abundantly generated by phospholipase C and possibly with different acyl moieties as a consequence of changes in the membrane, may have resulted in our study in differential activation of protein kinase C, a crucial modulator of the secretory machinery of acinar cells. This is strongly supported by the finding in guinea pig epidermis that DAG with an 18:2 n-6 metabolite at the 2-position inhibited protein kinase C isozymes compared with 1,2-dioleylglycerol.17 In conclusion, the type of dietary fat influences significantly the fatty acid profile of rat pancreatic membranes, and this fact is associated with modulation of cell function in fresh acinar cells as assessed by intracellular Ca21 mobilization and amylase release in response to CCK-8.

47.5 AR42J studies Recent research in nutritional science is trying to elucidate the effect of dietary lipids on cell function in health and disease conditions, and cell culture appears to be a good approach for investigating the molecular aspects of this problem. A much wider array of modifications is possible in cultured cells than in intact animals, and the environmental conditions can be controlled better. Another valuable advantage, in terms of budget and time economy, is that animals require long periods of adaptation to dietary fats, whereas adaptation of cultured cells is achieved in a few days. The AR42J cell line is the only currently available cell line that maintains many characteristics of normal pancreatic acinar cells, such as the synthesis and secretion of digestive enzymes. AR42J cells show receptor expression and signal transduction mechanisms parallel to those of

Effects of virgin olive oil on fatty acid composition of pancreatic cell membranes Chapter | 47

FIGURE 47.1 Effects of dietary fat type on amylase secretion in rat pancreatic acinar cells. The figure shows net amylase release (increase above basal) stimulated by CCK-8 in viable pancreatic acini isolated from rats fed for 8 weeks with diets containing either VOO or SO as the fat source (10 wt.%). Amylase released during the incubation (30 min) of acini with CCK-8 is expressed as a percentage of the initial total cell content. Acini exposed to the incubation solution alone served as unstimulated controls for the determination of basal release. Results are mean 6 SEM (n 5 15
37 separate experiments). Mean values were significantly different between the dietary groups: **P , .005, ***P , .001. CCK-8, Cholecystokinin-octapeptide; SO, sunflower oil; VOO, virgin olive oil. Reprinted from Yago MD, et al. Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling. Br J Nutr. 2004;91 (2):230, with permission of Cambridge University Press.

normal pancreatic acinar cells, and they are being widely used to study secretion, cell signaling, cytoskeleton function, apoptosis, and pancreatitis responses of the exocrine pancreas.18
20 Thus we thought that this cell line could be a suitable in vitro model for our purpose of examining the molecular mechanisms of the effect of dietary lipids on membrane composition and cell function. Surprisingly, we found that, in spite of the wide usage in recent years, no information was available on the baseline lipid composition of AR42J cell membranes, and there were no attempts made in recorded scientific literature to modify the fatty acid composition of AR42J cells in culture. Our aim was, then, to determine the membrane fatty acid composition of AR42J cells, to investigate whether these cells adapt their membranes after exposure to different fatty acids in the culture medium, and to confirm if this process is similar to the adaptation of the rat exocrine pancreas that occurs when dietary fat intake is modified.14,21 In addition, we explored whether membrane changes associate with modified cell function and, also, if provides differential protection against harmful stimuli.

573

In our studies the addition of oleic acid (AR42J-O group) or linoleic acid (AR42J-L) to the culture medium for 72 h profoundly influenced the fatty acid composition of AR42J cell membranes22 (see Table 47.1). The pattern and direction of changes were parallel to those found in rats fed VOO or SO.14 Both VOO in rats and oleic acid in AR42J cells evoked a significant increase in membrane MUFA (due to oleic acid) at the expense of SFA and PUFA, and we also observed that both SO in rats and linoleic acid in AR42J cells produced significant increases in total and n-6 PUFA at the expense of SFA and MUFA. Variations in the other fatty acid indices followed the same trend after feeding the oils in vivo or growing the cells with the respective major fatty acid. We also attempted23 to increase the content of n-3 LCPUFA in AR42J cell membranes. For this purpose, we used the same protocol as described for oleic and linoleic acids, but, instead, a 60:40 mixture of eicosapentaenoic acid (EPA, 20:5 n-3) plus docosahexaenoic acid (DHA, C22:6 n-3) was added to the culture medium (AR42J-n3 group). Membrane fatty acid analysis showed EPA and DHA values of 4.37% 6 0.36% and 7.08% 6 0.11% of total fatty acids, respectively. These values are markedly higher than those found in any of the other groups (see Table 47.1). Compared with control cells (not modified membranes, AR42J-C), the increase in membrane n-3 LCPUFA was accompanied by a decrease in the content of oleic and palmitic acids. We also studied in AR42J cells if, similar to pancreatic acini after in vivo feeding, the changes in membrane profile were associated with a modulation of the secretory activity. This investigation showed that, for any concentration of the secretagogue, net amylase secretion in AR42J-L cells was lower than in the AR42J-O group (Fig. 47.3), although statistical significance was reached only at 1028 and 1027 M CCK-8. These results agree with those described earlier in pancreatic acinar cells from rats adapted to diets containing either VOO or SO.14 Net amylase secretion in the AR42J-n3 group was significantly lower compared with the other two groups for 10210 M and higher CCK-8 concentrations. In conclusion, enrichment of culture medium with oleic or linoleic acid modifies the fatty acid spectrum of AR42J cell membranes in a manner that resembles the pattern found in pancreatic membranes of rats fed diets rich in VOO or SO, respectively. Moreover, although we cannot compare with in vivo data, n-3 LCPUFA also incorporates into membranes of this cell line. These changes in membrane lipid profile are associated with a modulation of the secretory activity. Therefore this rat pancreatoma cell line constitutes a suitable model to conduct studies aiming to investigate the modulatory influence of membrane compositional changes on acinar cell function in health and disease conditions.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 47.2 Effect of VOO on Ca21 mobilization in rat pancreatic acinar cells. Fluorescence microscopy original chart recordings showing CCK8-evoked changes in cytosolic Ca21 concentration ([Ca21]c) in fura-2-loaded single acinar cells of rats fed for 8 weeks with either a diet containing VOO as the fat source (A and B) or with a standard chow (C and D). Cells were perfused (black line) with 10 nM CCK-8 (A and C) or 20 pM CCK-8 (B and D). Traces are representative of 120 pancreatic acinar cells taken from 6 to 10 rats (A and B) and from 5 to 9 rats (C and D). CCK-8, Cholecystokinin-octapeptide; VOO, virgin olive oil. Reprinted from Martı´nez MA, et al. Dietary virgin olive oil enhances secretagogue-evoked signaling in rat pancreatic acinar cells. Nutrition. 2004;20(6):538, with permission of Elsevier Ltd.

47.6 AR42J cell model of acute pancreatitis The next step in our research was to explore whether modification of the membrane fatty acid profile of AR42J cells modulates damage induced by different noxious compounds. One of them is cerulein, a CCK analog that is used to induce in rats mild acute pancreatitis characterized by relatively high apoptosis and low necrosis. Cerulein triggers, both in rat pancreatic acinar cells and in AR42J cells,24
26 the same alterations that are described in early stages of acute pancreatitis: abnormal calcium (Ca21) signaling, secretory blockade and intracellular activation of zymogens, mitochondrial dysfunction, and endoplasmic reticulum stress, all accompanied by alterations in certain cytoprotective activities, the appearance of oxidative stress, stimulation of the synthesis of

proinflammatory mediators, and activation of cell death pathways (for more information about the cerulein model of acute pancreatitis, see Chapter 48 in this book). In this series of experiments, we chose to enrich AR42J membranes with either oleic acid or n-3 LCPUFA for their role as functional ingredients of the Mediterranean diet. As a control group, we used AR42J cells with their original, not modified, membranes. Similar to previous experiments, fetal calf serum containing the specific fatty acids was added to the culture medium during the differentiation period (72 h). The fatty acids were then retired and 1028 M cerulein was added for a further 24 h. Next, we examined normal cell dynamics in response to CCK-8 (secretory activity and Ca21 signaling) and determined a number of parameters relative to oxidative and inflammatory status and cell death.

Effects of virgin olive oil on fatty acid composition of pancreatic cell membranes Chapter | 47

575

TABLE 47.1 Selected fatty acids in membranes from differentiated AR42J cells cultured for 72 h in medium containing unmodified fetal calf serum (AR42J-C), serum enriched in oleic acid (AR42J-O), or serum enriched in linoleic acid (AR42J-L). Fatty acid

AR42J-C

AR42J-O

AR42J-L

14:0

3.40 6 0.20a

4.58 6 0.08b

4.43 6 0.09b

16:0

28.05 6 0.21a

27.39 6 0.10a

28.14 6 0.38a

16:1 n-7

3.47 6 0.20a

3.85 6 0.19a

3.70 6 0.16a

18:0

16.41 6 0.26a

12.35 6 0.33b

13.04 6 0.30b

18:1 n-9

25.69 6 0.41a

32.40 6 1.11b

15.95 6 0.22c

18:2 n-6

3.33 6 0.12a

2.39 6 0.03a

17.03 6 0.21b

18:3 n-3

3.83 6 0.20

b

5.11 6 0.36

3.78 6 0.24a

20:4 n-6

3.80 6 0.11a

2.01 6 0.11b

1.86 6 0.06b

20:5 n-3

0.58 6 0.02a

0.36 6 0.02b

0.39 6 0.06b

22:6 n-3

1.87 6 0.10a

1.08 6 0.06b

0.88 6 0.07b

a

Membrane fatty acid modifications in AR42J cells were evoked during the 72 h differentiation period toward an exocrine phenotype (100 nM dexamethasone) by the addition of 18:1 n-9 (AR42J-O cells), 18:2 n-6 (AR42J-L cells), or vehicle (AR42J-C cells). Results are expressed as percentage of total fatty acid content. Values are means 6 SEM (AR42J-C: n 5 18, from six batches of cells; AR42J-O: n 5 15, from five batches; AR42J-L: n 5 15, from five batches). For a particular row, values with different superscript letters are significantly different at P , .05. Source: Reprinted from Audi et al. Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids. Exp Biol Med. 2007;232(4):538, with permission of Sage Publishing.

47.6.1 Cell function (amylase secretion and Ca21 homeostasis) Pretreatment with cerulein decreased the net secretion stimulated by CCK-8 in all cells, regardless of their fatty acid profile (Fig. 47.4). The only difference that should be noted is that in cells with membranes enriched in n-3 LCPUFA, the decrease was apparently of lesser magnitude (Fig. 47.4C), but this is because cells not treated with cerulein (n-3 group) showed an already attenuated secretion and cerulein did not decrease the secretion further (n-3-Cer), except for the lowest concentrations of CCK-8. Ca21 homeostasis in cells pretreated or not with cerulein was also investigated. To better assess the Ca21 responses to the secretagogue CCK-8 (1029 M), we calculated both the peak increase above basal (Fig. 47.5A) and the corresponding area under the curve (Fig. 47.5B). In AR42J cells not injured by cerulein, the higher Ca21 responses were observed in cells with membranes rich in oleic acid (O group, see insets in Fig. 47.5A and B). However, this response was greatly reduced by

FIGURE 47.3 Effects of modification of membrane fatty acid profile on amylase secretion in AR42J cells. AR42J cells were cultured in medium containing fetal calf serum enriched with different fatty acids: oleic acid (AR42J-O), linoleic acid (AR42J-L), or a 60:40 mixture of eicosapentaenoic plus docosahexaenoic acids (AR42J-n3). Serum was added during the 72-h differentiation period. Next, amylase secretion was determined. The curves show net amylase release (increase above basal) stimulated by CCK-8 after incubation (50 min) of cells with the secretagogue. Cells exposed to the incubation solution alone served as unstimulated controls for the determination of basal release. Results are expressed as a percentage of the initial total cell content of amylase and are means 6 SEM. For a given concentration of CCK-8, n 5 14
24 observations using at least three different batches of cells. *P , .05 for AR42J-n3 versus the other two groups at specific CCK-8 concentrations; #P , .05 between the three groups at specific CCK-8 concentrations. CCK-8, Cholecystokinin-octapeptide.

cerulein (O-Cer group), as was the case in cells with unmodified membranes (C-Cer vs C) and in those with membranes rich in n-3 LCPUFA (n-3-Cer vs n-3). The same patterns were found regardless of whether the results were expressed as peak increase above basal (Fig. 47.5A) or as area under the curve (AUC) (Fig. 47.5B), with the only exception of cells enriched in n-3 LCPUFA. In these cells, AUC showed a very low value (n-3 group), and cerulein could not decrease it much further (n-3-Cer).

47.6.2 Secretion of inflammatory mediators Although leucocytes are the main source of cytokines in acute pancreatitis, there is strong evidence that the inflammatory cascade is initiated in the acinar cells. Very soon after the insult, intraacinar NFκB is activated, leading to augmented expression of cytokines,

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 47.4 Effects of cerulein pretreatment on amylase secretion in AR42J cells with different membrane fatty acid profile. AR42J cells were cultured in medium containing unmodified fetal calf serum (A), serum enriched with oleic acid (B), or serum enriched with a 60:40 mixture of eicosapentaenoic plus docosahexaenoic acids (C). Serum with (or without) the corresponding fatty acids was added to the culture medium during the 72-h differentiation period. Next, medium fatty acids were retired and cells were cultured for an additional 24 h with 1028 M cerulein to reproduce acute pancreatitis damage (C-Cer, O-Cer, n-3-Cer) or vehicle (C, O, n-3). Afterward, amylase secretion was determined. The curves show net amylase release (increase above basal) stimulated by CCK-8 after incubation (50 min) of cells with the secretagogue. Cells exposed to the incubation solution alone served as unstimulated controls for the determination of basal release. Results are expressed as a percentage of the initial total cell content of amylase and are means 6 SEM. For a given concentration of CCK-8, n 5 8
24 observations using at least three different batches of cells. *P , .05 between groups pretreated or not with cerulein at specific CCK-8 concentrations. CCK-8, Cholecystokinin-octapeptide.

chemokines, and adhesion molecules that recruit and mediate infiltration of immune cells into the site of injury.27 After pretreatment for 24 h with vehicle or with cerulein, the concentration of cytokines IL-6 and IL-10 in the culture medium was analyzed by Luminex multiplex assay, and the ratio IL-10/IL-6 calculated, since low values have been reported to be indicative of cell damage.28 In cells with unmodified membranes, the presence of cerulein markedly decreased the ratio IL-10/IL-6 from values of 2.05 6 0.83 (C group, mean 6 SEM) to 0.81 6 0.17 (C-Cer). In contrast, this index was not modified by pretreatment with cerulein either in cells with oleic acid
enriched membranes (O group: 1.83 6 0.36; O-Cer group: 2.16 6 0.46) or in those with n-3 LCPUFAenriched membranes (n-3 group: 1.71 6 0.35; n-3-Cer group: 1.65 6 0.28). These results are in good agreement with the wellknown beneficial effects of both oleic and n-3 fatty acids in inflammatory conditions such as rheumatoid arthritis and cardiovascular diseases.29
32

47.6.3 Antioxidant defenses As an index of the intracellular nonenzymatic antioxidant defenses, we measured the concentration of GSH in cell lysates from AR42J cells by ELISA. Depletion of GSH in pancreatic tissue is a hallmark of acute pancreatitis.33,34

In cells not damaged with cerulein, we observed that the enrichment of AR42J cell membranes with either oleic acid or n-3 LCPUFA diminished the cellular content of GSH (Fig. 47.6A). Pretreatment for 24 h with cerulein affected this parameter differentially. Thus GSH decreased 21% in group C-Cer compared with C group (Fig. 47.6B), whereas in the other groups an increase was apparent, being especially remarkable that found in oleic acid
enriched cells (114.4% increase in O-Cer as compared with O group).

47.6.4 Cell viability and apoptosis As part of our study, we were interested in examining how the changes in membrane profile affected the cells after the noxious influence of cerulein. For that purpose, we determined cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and estimated the percentage of apoptotic cells by flow cytometry (Annexin V). In cells not pretreated with cerulein (see insets in Fig. 47.7A and B), the highest viability (MTT) and lower apoptosis rate were found in cells with unmodified membrane (C group). Free fatty acids are toxic to cells in culture. Although the fatty acids added to the culture medium were previously complexed with proteins of the fetal calf serum, some small amount of free fatty acids could have remained in the medium, giving rise to these

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577

differences. Of note, 1028 M cerulein decreased cell viability in all groups except in the group with membranes enriched with oleic acid (O-Cer), in which this parameter remained constant (Fig. 47.7A). As for the apoptosis index, it increased significantly in C-Cer group as compared with C group, a typical feature in cerulein-induced acute pancreatitis, including the AR42J model.35 Conversely, it did not change in the oleic acid groups (O-Cer vs O group) nor in the n-3 groups (n3-Cer vs n-3 groups). Taken into account these parameters in cells treated or not with cerulein, it seems that membranes enriched in oleic acid provided AR42J cells with a higher level of protection.

47.7 Summary points G

G

G

FIGURE 47.5 Effects of cerulein pretreatment on intracellular Ca21 homeostasis in AR42J cells with different membrane fatty acid profile. AR42J cells were cultured in medium containing unmodified fetal calf serum (C and C-Cer groups), serum enriched with oleic acid (O and O-Cer groups), or serum enriched with a 60:40 mixture of eicosapentaenoic plus docosahexaenoic acids (n-3 and n-3-Cer groups). Serum with (or without) the corresponding fatty acids was added to the culture medium during the 72-h differentiation period. Next, medium fatty acids were retired and cells were cultured for an additional 24 h with 1028 M cerulein to reproduce acute pancreatitis damage (C-Cer, O-Cer, n-3-Cer) or vehicle (C, O, n-3). Afterward, cells were loaded with fura-2 AM and changes in cytosolic Ca21 concentration ([Ca21]c) in response to 1029 M CCK-8 were determined by spectrofluorimetry. The Ca21 response was analyzed in terms of peak increases above basal (A) and area under the curve (B). To calculate the AUC in each individual Ca21 experiment, basal [Ca21]c values before the addition of CCK-8 were averaged to estimate the baseline, and this was subtracted from each of the stimulation values. The numbers obtained in this way were then summed (the same number of data points was used for all experiments). Data are means 6 SEM of n 5 6 observations using at least two different batches of cells. *P , .05 between groups treated or not with cerulein. The inset depicts the influence of membrane fatty acid profile in groups not treated with cerulein (unlike superscript letters denote P , .05). AUC, Area under the curve; CCK-8, cholecystokinin-octapeptide.

G

Chronic (8 weeks) intake of diets containing virgin olive oil (VOO) (10 wt.%) as fat source diminishes the secretion of fluid and amylase in resting conditions compared with diets containing sunflower oil (SO). According to previous studies of our group in other species, this effect could be related to the ability of VOO to elevate the resting blood levels of certain inhibitory gastrointestinal hormones such as peptide YY. Adaptation to VOO or SO diets also influences the timecourse changes of all pancreatic secretory parameters in the anesthetized rat preparation. The differences between dietary groups cannot be accounted for by changes in the gland enzyme content. However, the finding that pancreatic cell membranes enrich in those fatty acids most abundant in the fat ingested suggests that in vivo data may reflect a direct modulatory effect of the type of dietary fat on the secretory activity at the cellular level. Indeed, in subsequent studies with viable pancreatic acinar cells of rats kept on identical dietary protocol (i.e., 8 weeks with diets containing 10 wt.% of either VOO or SO), we could confirm that dietary-induced changes in the fatty acid profile of pancreatic membranes are associated with modulation of pancreatic cell function as assessed by intracellular Ca21 mobilization and amylase release in response to cholecystokinin-octapeptide (CCK-8). The effects on the Ca21 response were noted not only in the magnitude of large Ca21 transients evoked by high concentrations of CCK-8 but also in the amplitude and frequency of Ca21 oscillations induced by low, more physiological, concentrations of the secretagogue. The AR42J pancreatoma cell line shows that supplementation of culture media with oleic acid, linoleic acid, or a mixture of eicosapentaenoic acid plus docosahexaenoic acid modifies in 72 h the membrane fatty acid spectrum of these cells. For oleic and linoleic acids the pattern and direction of changes are parallel to those found in rats fed diets enriched in VOO or SO. In addition, these changes are accompanied by a

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 47.6 Effects of cerulein pretreatment on GSH content in AR42J cells with different membrane fatty acid profiles. AR42J cells were cultured in medium containing unmodified fetal calf serum (C and C-Cer groups), serum enriched with oleic acid (O and O-Cer groups), or serum enriched with a 60:40 mixture of eicosapentaenoic plus docosahexaenoic acids (n-3 and n-3-Cer groups). Serum with (or without) the corresponding fatty acids was added to the culture medium during the 72-h differentiation period. Next, medium fatty acids were retired and cells were cultured for an additional 24 h with 1028 M cerulein to reproduce acute pancreatitis damage (B) or vehicle (A). Afterward, GSH in cell lysates was determined by an ELISA kit. Values are means of n 5 4 observations from at least two different batches of cells. (A) GSH levels are expressed as nmol/mg protein. Unlike superscript letters denote P , .05. (B) The effect of cerulein pretreatment is shown as percentage variation relative to the corresponding membrane group not treated with cerulein. GSH, Glutathione (reduced form
SH).

G

modulation of the secretory activity that involves Ca21 signaling. Therefore AR42J cells seem to be a suitable model in physiological and pathophysiological studies aiming to assess the effect of membrane compositional changes on acinar cell function. Enrichment of pancreatic AR42J cells with oleic acid or n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) differentially affects cell function and oxidative
inflammatory parameters: (1) In both cases cells are protected against cerulein-induced changes in inflammatory status; (2) cells with membranes enriched in oleic acid show the best performance relative to apoptotic death, since the apoptotic rate in the absence of cerulein is not as high as that observed in n-3 LCPUFA cells and, on the other hand, they are not affected by cerulein; and (3) amylase secretion stimulated by CCK-8 is greatly affected in n-3 LCPUFA cells, especially after the cerulein insult.

Mini-dictionary of terms Acute pancreatitis model: Acute pancreatitis is an inflammatory process that is accompanied by significant organ damage and death of pancreatic cells. Due to the impossibility of obtaining samples of human pancreatic tissue during an episode of acute pancreatitis, research on this disease must be done with animal or cellular models that mimic the alterations found in the disease. One of the most used models of acute pancreatitis is the cerulein one, which can be applied to animal experimentation and also to in vitro research with cells, such as the AR42J cells. Cell death: Damaged cells have three types of death: (1) apoptosis: programmed death, consisting of a

regulated sequence of events that leads to cell removal without releasing any compound to the environment; (2) necrosis: rapid and uncontrolled destruction of the cell, normally associated with a significant ATP deficit, and where the discharge of cellular contents after the rupture of the cell membrane evokes the appearance of an inflammatory process; and (3) autophagic cell death: highly regulated process by which cells degrade their own macromolecules and organelles through the lysosomal system (lysosomal enzymes). It is characterized by intracellular accumulation of autophagic vacuoles. For more detailed information, see Ref. [36]. Cellular signaling: Part of any communication process that rules basic activities of cells and coordinates multiple-cell actions. The capability of cells to sense and correctly respond to their microenvironment is the basis of development, tissue and organ repair, and immunity, as well as normal tissue homeostasis. Deficiencies in signaling and cellular information processing may cause diseases. Oxidative stress: It is the consequence of an imbalance between the systemic manifestation of reactive oxygen species and the ability of biological systems to detoxify the reactive intermediates or to repair the resulting injury. In humans, oxidative stress can cause disturbances in normal mechanisms of cellular events and is thought to be involved in the development of several diseases.

Comparisons of olive oils with other edible oils Several edible oils and fats are continuously mentioned and referenced in the text, such as sunflower oil, rich in

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FIGURE 47.7 Effects of cerulein pretreatment on viability and apoptotic cell death in AR42J cells with different membrane fatty acid profile. AR42J cells were cultured in medium containing unmodified fetal calf serum (C and C-Cer groups), serum enriched with oleic acid (O and O-Cer groups), or serum enriched with a 60:40 mixture of eicosapentaenoic plus docosahexaenoic acids (n-3 and n-3-Cer groups). Serum with (or without) the corresponding fatty acids was added to the culture medium during the 72-h differentiation period. Next, medium fatty acids were retired and cells were cultured for an additional 24 h with 1028 M cerulein to reproduce acute pancreatitis damage (C-Cer, O-Cer, n-3-Cer) or vehicle (C, O, n3). Afterward, assays were performed. (A) Cell viability was determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed as percentage relative to the untreated group (C group). Values are means 6 SEM of n 5 30
140 observations using at least three different batches of cells. (B) Percentage of apoptotic cells (relative to total cells) was determined by flow cytometry (Annexin V). Values are means 6 SEM of two samples per group (10,000 events/sample). For both (A) and (B): *P , .05 between groups treated or not with cerulein. Insets depict the influence of membrane fatty acid profile in groups not treated with cerulein (unlike superscript letters denote P , .05).

linoleic acid [n-6 polyunsaturated fatty acid (PUFA) (n-6 PUFA)], or fish oil, which contains abundant long-chain n-3 PUFA, always in comparative terms with virgin olive oil, the main fatty acid of which is oleic acid (n-9 monounsaturated fatty acid).

modification of the lipid profile of cell membranes, which may condition the response of the cell both in physiological conditions and in situations of cellular damage (inflammatory, oxidative, etc.). The results may be of interest for the prevention of various diseases that have an inflammatory base or present high levels of oxidative stress.

Implications for human health and disease prevention

References

Virgin olive oil (VOO) is the main added fat in the Mediterranean diet. Numerous studies confirm the role of this food pattern in the prevention of different chronic noncommunicable diseases. Thus the PREDIMED study shows that Mediterranean dietary pattern supplemented with either VOO or nuts decreases the incidence of cardiovascular diseases. There is also evidence that associates VOO with the prevention of type 2 diabetes mellitus, cancer, overweight, obesity, etc. This chapter includes the most relevant findings of studies examining the influence of VOO through

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2769. 3. Stenson WF, Seetharam B, Talkad V, Pickett W, Dudeja P, Brasitus TA. Effects of dietary fish oil supplementation on membrane fluidity and enzyme activity in rat small intestine. Biochem J. 1989;263:41
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482. 6. Quiles JL, Huertas JR, Man˜as M, Ochoa JJ, Battino M, Mataix J. Dietary fat type and regular exercise affect mitochondrial composition and function depending on specific tissue in the rat. J Bioenerg Biomembr. 2001;33:127
134. 7. Fuentes NR, Kim E, Fan YY, Chapkin RS. Omega-3 fatty acids, membrane remodeling and cancer prevention. Mol Aspects Med. 2018;64:79
91. 8. Yago MD, Martinez-Victoria E, Huertas JR, Man˜as M. Effects of the amount and type of dietary fat on exocrine pancreatic secretion in dogs after different periods of adaptation. Arch Physiol Biochem. 1997;105:78
85. 9. Yago MD, Gonzalez MV, Martinez-Victoria E, et al. Pancreatic enzyme secretion in response to test meals differing in the quality of dietary fat (olive oil and sunflower seed oils) in human subjects. Br J Nutr. 1997;78:27
39. 10. Yago MD, Martinez-Victoria E, Man˜as M, Martinez MA, Mataix J. Plasma peptide YY and pancreatic polypeptide in dogs after longterm adaptation to dietary fats of different degrees of saturation: olive and sunflower oil. J Nutr Biochem. 1997;8:502
507. 11. Soriguer FJ, Tinahones FJ, Monzan A, et al. Varying incorporation of fatty acids into phospholipids from muscle, adipose and pancreatic exocrine tissues and thymocytes in adult rats fed with diets rich in different fatty acids. Eur J Epidemiol. 2000;16:585
594. 12. Diaz RJ, Yago MD, Martinez-Victoria E, Naranjo JA, Martinez MA, Man˜as M. Comparison of the effects of dietary sunflower oil and virgin olive oil on rat exocrine pancreatic secretion in vivo. Lipids. 2003;38:1119
1126. 13. Begin ME, Ells St. G, Jean P, Vachereau A, Beaudoin AR. Fatty acid and enzymatic compositional changes in the pancreas of rats fed dietary n-3 and n-6 polyunsaturated fatty acids. Int J Pancreatol. 1990;6:151
160. 14. Yago MD, Diaz RJ, Ramirez R, Martinez MA, Man˜as M, Martı´nez-Victoria E. Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling. Br J Nutr. 2004;91:227
234. 15. Lajas AI, Pozo MJ, Salido GM, Pariente JA. Effect of basic fibroblast growth factor on cholecystokinin-induced amylase release and intracellular calcium increase in male rat pancreatic acinar cells. Biochem Pharmacol. 1998;55:903
908. 16. Martinez MA, Lajas AI, Yago MD, et al. Dietary virgin olive oil enhances secretagogue-evoked signaling in rat pancreatic acinar cells. Nutrition. 2004;20:536
541. 17. Cho Y, Ziboh VA. Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol. J Lipid Res. 1994;35:913
921. 18. Guo HY, Cui ZJ. Extracellular histones activate plasma membrane toll-like receptor 9 to trigger calcium oscillations in rat pancreatic acinar tumor cell AR4-2J. Cells. 2018;8:3. 19. Ameur FZ, Mehedi N, Kheroua O, Saı¨di D, Salido GM, Gonzalez A. Sulfanilic acid increases intracellular free-calcium concentration, induces reactive oxygen species production and impairs

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440. Yago MD, Diaz RJ, Martinez MA, et al. Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells. J Nutr Biochem. 2006;17:242
249. Audi N, Mesa MD, Martı´nez MA, Martinez-Victoria E, Man˜as M, Yago MD. Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids. Exp Biol Med. 2007;232:532
541. Lo´pez-Milla´n B. Respuestas celulares a modificaciones en el perfil lipı´dico y a la presencia de un antioxidante del aceite de oliva en un modelo in vitro de pancreatitis. Aspectos Inflamatorios [Ph.D. thesis]. Granada: University of Granada; 2011. ISBN: 9788469469347. Chen WD, Zhang JL, Wang XY, Hu ZW, Qian YB. The JAK2/ STAT3 signaling pathway is required for inflammation and cell death induced by caerulein in AR42J cells. Eur Rev Med Pharmacol Sci. 2019;23:1770
1777. Song EA, Lim JW, Kim H. Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in caerulein-stimulated pancreatic acinar cells. Int J Biochem Cell Biol. 2017;88:60
68. Ben-Dror K, Birk R. Oleic acid ameliorates palmitic acid-induced ER stress and inflammation markers in naive and caerulein-treated exocrine pancreas cells. Biosci Rep. 2019;39:14. Jakkampudi A, Jangala R, Reddy BR, Mitnala S, Nageshwar Reddy D, Talukdar R. NF-κB in acute pancreatitis: mechanisms and therapeutic potential. Pancreatology. 2016;16:477
488. Hoene M, Weigert C. The role of interleukin-6 in insulin resistance, body fat distribution and energy balance. Obes Rev. 2008;9:20
29. Nocella C, Cammisotto V, Fianchini L, et al. Extra virgin olive oil and cardiovascular diseases: benefits for human health. Endocr Metab Immune Disord Drug Targets. 2018;18:4
13. Berbert AA, Kondo CR, Almendra CL, Matsuo T, Dichi I. Supplementation of fish oil and olive oil in patients with rheumatoid arthritis. Nutrition. 2005;21:131
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592.

Chapter 48

Hydroxytyrosol: features and impact on pancreatitis Belen Lopez-Millan, Maria Alba Martinez-Burgos, Mariano Man˜as, Emilio Martinez-Victoria and Maria Dolores Yago Department of Physiology and Institute of Nutrition and Food Technology “Jose Mataix”, University of Granada, Granada, Spain

Abbreviations AP AUC CCK ELISA ER FAEE GSH HT IL-1β IL-6 MD NF-κB PMCA PPAR-γ ROS SERCA TNF-α UPR

acute pancreatitis area under the curve cholecystokinin enzyme-linked immunosorbent assay endoplasmic reticulum fatty acid ethyl ester glutathione (reduced
SH
form) hydroxytyrosol interleukin-1β interleukin-6 Mediterranean diet nuclear factor kappa B plasma membrane Ca21-ATPase peroxisome proliferator
activated receptor-γ reactive oxygen species sarco/endoplasmic reticulum Ca21-ATPase tumor necrosis factor-α unfolded protein response

by the presence of hemorrhage and extensive necrosis, and in which local inflammation evolves toward an uncontrolled systemic response with multiorgan failure and a mortality rate that can reach 20%
50%. Except for the most severe cases, that require antibiotics and even surgery, the current guidelines for AP treatment include keeping the pancreas at rest (with fasting and administration of somatostatin analogues), correction of metabolic abnormalities, and use of intravenous fluids, analgesics, antiinflammatory drugs, and protease inhibitors.2 Unfortunately, these standard therapies, in addition to being nonspecific, show limited clinical efficiency and/or cause serious side effects, all of which underscores (1) the importance of prevention and (2) the need to find novel molecules that improve both the symptoms and the evolution of the disease.

48.2 Acute pancreatitis: key aspects 48.1 Introduction Acute pancreatitis (AP) is an inflammatory process of the exocrine pancreas associated with important parenchymal death which globally affects approximately 34 per 100,000 individuals per year.1 Currently, AP is considered a multifactorial disease, in which metabolic/environmental factors able to damage the acinar cells interact with genetic variants to make individuals more prone to developing the disease. Gallstones blocking the common bile duct and alcohol abuse are the most common cause of AP, followed by hypertriglyceridemia. About 70%
80% AP patients suffer a mild edematous form of the disease, which usually remits without sequelae, but the remaining 20%
30% experiences a serious process that is characterized locally

It is clearly established that injury to the pancreatic acinar cell is the starting point for AP. Therefore investigation of the pathogenesis of this disease has focused on studying the cellular events that occur in response to that injury, among which the most important are abnormal calcium (Ca21) signaling, secretory blockade and intracellular activation of zymogens, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, all accompanied by alterations in certain cytoprotective activities, the appearance of oxidative stress, stimulation of the synthesis of proinflammatory mediators, and activation of cell death pathways. These events, which have been examined in experimental models due to the impossibility of obtaining samples of human pancreatic tissue during an episode of AP, have been shown to be directly triggered by nonoxidative

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00025-0 © 2021 Elsevier Inc. All rights reserved.

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metabolites of alcohol (fatty acid ethyl esters—FAEEs) and bile acids.3 Also, they can be indirectly evoked by changes in intraductal conditions, such as obstructioninduced increased pressure and acidification caused by ductal cell exposure to bile acids.4 This is relevant, because it is still debated whether or not bile acids are able to reach the acinar cells in gallstone AP. Of note, they are also reproduced when acinar cells are hyperstimulated with cerulein,3 a cholecystokinin (CCK) analogue, as will be seen throughout this chapter. Finally, the release of a variety of molecules by damaged acinar cells leads to infiltration of defense cells and initiation of an inflammatory process that, at first, remains confined in the pancreas but, occasionally, can be amplified at systemic level.

48.2.1 Abnormal Ca21 signaling and mitochondrial dysfunction Cytosolic Ca21 homeostasis plays a dominant role in both physiological and pathological responses in the acinar cell.5 Following physiological stimulation of acinar cells with such secretagogues as the neurotransmitter acetylcholine or the hormone CCK, release of Ca21 from the ER occurs, which causes small increases in the concentration of cytosolic Ca21 ([Ca21]c) in the apical pole of the cell. These Ca21 elevations drive the exocytosis of zymogen granules. Nearby mitochondria buffer these increases by capturing Ca21 which, together with the pumping activity of plasma membrane and ER Ca21-ATPases [plasma membrane Ca21-ATPase (PMCA) and sarco/ endoplasmic reticulum Ca21-ATPase (SERCA), respectively], contributes to the termination of the Ca21 signal. In addition, Ca21 entry into the mitochondria stimulates ATP output necessary to carry out zymogen secretion and to keep ATPases functioning. On the contrary, common acinar cell toxins (such as FAEEs and bile acids) and hyperstimulation of CCK receptors evoke a massive release of Ca21 from the ER stores causing their depletion, which, in turn, induces the influx of external Ca21 through plasma membrane channels, a process known as store-operated Ca21 entry. As a consequence, abnormal, prolonged elevation of cytosolic Ca21 appears. This pathological Ca21 signal is a central event that mediates many AP features, one of them being the mitochondrial dysfunction. Since mitochondria act as cellular Ca21 buffers, the pathological, sustained elevations in [Ca21]c raise mitochondrial Ca21 leading to Ca21-dependent generation of reactive oxygen species (ROS) that promote apoptotic cell death, a protective local mechanism. However, as the level of the insult increases, mitochondrial matrix overloads with Ca21, which causes mitochondrial depolarization and reduced ATP synthesis. The decrease of ATP impairs de Ca21

clearance activity of PMCA and SERCA pumps and, therefore, contributes to the maintenance of the abnormal Ca21 signal. With the persistence of Ca21 overload, ATP is completely depleted and cells are forced into necrotic cell death.6 Thus the cell death pattern seems to depend on the duration and severity of the disturbance of cytosolic Ca21 levels in pancreatic acinar cells.

48.2.2 Endoplasmic reticulum stress and impairment of cytoprotective-associated responses Pancreatic acinar cells have copious ER in accordance with an extremely high rate of protein production, which make them particularly vulnerable to ER stress. This is the name given to that condition in which the capacity of the ER to efficiently process proteins is overwhelmed, thus leading to accumulation of misfolded and/or unfolded proteins within the ER lumen. When ER stress happens, cells initiate a cytoprotective response, the unfolded protein response (UPR), which involves the activation of intracellular pathways that culminate in (1) increasing the folding capacity of the ER by the enlargement of the ER, enhanced synthesis of foldases and other ER enzymes, or even cell hypertrophy and (2) stimulating degradation of abnormal proteins by the process of autophagy. In the face of severe ER stress, these mechanisms fail to restore homeostasis, and UPR pathways are deviated to promote apoptosis and activate the nuclear factor kappa B (NF-κB). ER stress is found to be an early event in all models of experimental AP,7 which may occur through increased protein synthesis (zymogens and lysosomal enzymes) and/or attenuated cytoprotective responses (UPR) due to mitochondrial dysfunction.

48.2.3 Zymogen activation Intracellular protease activation, which is dependent on pathological Ca21 elevation, is one of the earliest events of AP. The most accepted mechanism involves colocalization of zymogens with lysosomal hydrolases, resulting in trypsinogen activation by cathepsin B. Active trypsin damages the membrane of colocalization organelles resulting in the leakage of cathepsin B into the cytosol. Then, cathepsin B, and not trypsin, induces cell death, which will be apoptosis or necrosis depending on the cytosolic cathepsin B levels.8

48.2.4 Secretory blockade In AP, apical-regulated secretion is inhibited. This phenomenon has been studied especially in the hyperstimulation AP model. It has long been known that

Hydroxytyrosol: features and impact on pancreatitis Chapter | 48

hyperstimulation of the pancreas results in inhibition of the secretion rate, both in the whole animal and in isolated pancreatic acinar cells. In the latter case, cerulein concentrations greater than 1029 M (which evokes maximum amylase secretion) cause a marked decrease. The exact mechanisms of the secretory blockade are not yet clear. Disturbances of the actin apical cytoskeleton (intimately involved with zymogen granule exocytosis) and defective formation of fusion pore during exocytosis due to ATP shortage (as discussed previously, mitochondria dysfunction) have been proposed.9 It may also be associated with trypsin activation (colocalization) and impaired autophagy.8

48.2.5 Oxidative stress ROS have become in recent years one of the key mediators of the pathogenesis and progression of AP, whatever its etiology. Current data suggests that there are two critical stages in the generation of ROS. In a first stage, very early, ROS are produced by the acinar cell itself; later, this role preferably falls on the neutrophils infiltrated in the damaged gland.10 ROS are generated in aerobic cells as a by-product of different metabolic reactions, but under normal conditions, they are rapidly counteracted by a whole system of enzymatic and nonenzymatic antioxidants located inside the cell. Reduced glutathione (GSH) is the major nonprotein thiol in mammalian cells and its depletion in pancreatic tissue is a hallmark in AP.11 Loss of GSH in AP may not only be due to consumption during ROS detoxification but also to hydrolysis by activated intracellular zymogens.10 When ROS production exceeds these cell-defense mechanisms, as occurs in AP, oxidative attack on proteins, DNA, and lipids elicits serious structural damage and functional alterations, one of them being anomalous dynamics of the actin cytoskeleton.12 In addition, redox unbalance is also involved in intracellular signaling, and this seems to be relevant in at least two aspects of the pathogenic mechanisms of AP: G

G

ER and store-operated Ca21 channels in the plasma membrane are sensitive to redox status, which is directly related to the fact that ROS increases both the intracellular release and the entry of Ca21 to the acinar cell, thereby magnifying cell Ca21 overload.5 ROS act as proinflammatory mediators and promote inflammatory cell infiltration and activity in the pancreas during AP (see next).

48.2.6 Nuclear factor kappa B activation Although leucocytes are the main source of cytokines in AP, there is strong evidence that the inflammatory

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cascade is initiated in the acinar cells. Very soon after the insult, intraacinar NF-κB is activated, leading to augmented expression of cytokines, chemokines, and adhesion molecules that recruit and mediate infiltration of immune cells into the site of injury.8,13 Later, injured and necrotic cells release a variety of molecules known as “damage-associated molecular patterns,” which extend inflammation via specific receptors on the immune cells.4 The precise mechanisms responsible for intraacinar NF-κB activation have not been completely elucidated, but pathologic Ca21 signaling, activation of protein kinase C isoforms, and ROS seem to be involved.13 Trypsinogen activation takes place in parallel, but it is an independent event.8 Among proinflammatory mediators released by acinar cells are TNF-α, IL-6, IL-1β, and several chemokines.13

48.3 Lifestyle, Mediterranean diet, hydroxytyrosol, and acute pancreatitis The increasing incidence of AP, together with the high mortality of severe cases and the absence of effective therapy, leads us to seek new strategies aimed at the prevention and treatment of this pathology. Regarding prevention, and if etiology is taken into account, it seems reasonable to assume that several lifestyle factors can modify the risk of AP or affect the disease outcome. Thus regional differences in lifestyle may underlie the variability in AP incidence across Europe, with highest values usually reported from eastern and northern regions.14 The study by Roberts et al.14 also shows that the highest ratio of alcohol-to-gallstone AP is found in eastern Europe and the lowest one in southern European countries. Although information on the role of type and pattern of alcohol consumption and risk of AP is too scarce to draw conclusions, it seems that from the point of view of AP prevention, the Mediterranean drinking pattern (regular but moderate intake of alcohol, primarily in the form of wine and generally during meals) can possibly be considered a reasonable way of consuming alcohol. Another modifiable risk factor may be the diet. However, research examining associations between dietary habits and AP is very limited, probably due to the lack of knowledge about the pathogenesis of the disease. A prospective cohort study performed in Sweden15,16 showed that a high vegetable and fish consumption may reduce the risk of nongallstone-related AP, and a more recent study reported that biliary AP associates positively with intake of saturated fat and cholesterol but negatively with intake of fiber.17 As far as we know, there have been no studies evaluating the association between the Mediterranean diet (MD)

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and AP. However, it is well known that this eating pattern protects against many inflammatory diseases. In addition, adherence to Mediterranean lifestyle has been found to be inversely associated with overweight and obesity.18 This is noteworthy, since obesity increases both AP incidence (mainly through associated augmentation in gallstone and hypertriglyceridemia) and severity (potentially through a number of metabolic and inflammatory pathways).19 In fact, the obesity pandemic may partly explain that AP admissions have escalated over the last decades worldwide. Considering all the earlier discussions, it is reasonable to think that the MD can be beneficial in AP from a preventive point of view. Moreover, although health advantages of the MD have been attributed to the overall food pattern, together with sociocultural and lifestyle factors, there is no doubt that, due to its abundance in phytochemicals, the MD can possibly provide very interesting molecules for the treatment of AP once the disease has already been established. Interest in health-promoting actions of phytochemicals has grown exponentially in the last decade, and this has been reflected in the publication of numerous articles examining their pharmacological potential in different diseases, including AP (see the “Comparisons of olive oils with other edible oils” section). It must be said, however, that the majority of plantderived products investigated in relation to this disease come from remedies used in traditional medicine from Asian countries such as China and India. All these considerations led us to carry out research to know the modulating role of some typical components of the MD on the cellular mechanisms involved in the development of AP. The components we have studied have been the following: 1. Oleic acid and n-3 PUFA. Our own work has revealed that dietary-induced modification of membrane fatty acid composition in different tissues20,21 is associated with marked differences in susceptibility to oxidation20 and with functional changes that, in the case of the exocrine pancreas, involves both intracellular Ca21 signaling and secretory activity.22
24 This, together with abundant literature on the modulatory properties of both oleic and n-3 PUFA in oxidative and inflammatory processes led us to explore these aspects in AP (see Chapter 47 in this book). 2. Hydroxytyrosol (HT), present in the unsaponifiable fraction of virgin olive oil, is possibly one of the most actively investigated natural phenols at this moment. Many health advantages, besides its antioxidant capacity,25 have been attributed to HT, namely, antiinflammatory and anticancer effects,26
28 improvement of endothelial and vascular function,29 and amelioration of ER stress30 and mitochondrial function.31 Some of

these benefits, scientifically proved in a large number of clinical studies, made the European Food Safety Authority (EFSA) authorize in 2011 a health claim on olive oil polyphenols in relation to protection of lowdensity lipoproteins (LDL) from oxidative damage. However, to the best of our knowledge, no study has so far examined a possible beneficial role of HT in AP. To carry out our research, we chose to hyperstimulate AR42J cells with cerulein. Among the different experimental models of AP, the cerulein one is most often used. As mentioned earlier, it reproduces all the early cellular events central to the pathogenesis of AP.32 Cerulein, a CCK analogue, when given to a rat in doses that are 10
100 times greater than a physiologic equivalent, causes mild AP characterized by relatively high apoptosis and low necrosis. The advantages of this model are its inexpensiveness, rapid induction, and wide reproducibility and applicability, with a major disadvantage being that only a mild form is developed, and thus the clinical relevance is limited. It can also be applied to in vitro research (either freshly isolated pancreatic acinar cells or pancreatic cell lines). Given that we were seeking to examine the very early intraacinar events in the pathogenesis of AP, we opted for an in vitro model, the AR42J cell line, which our group had used before33
39 to investigate whether modification of the membrane fatty acid profile of these cells modulates damage induced by different noxious compounds, including bile acids, interleukins, and cerulein. AR42J cells have been taken as the basis for developing several AP cell models.40
42 As in previous work,33
39 AR42J cells were used following treatment with 100 nM dexamethasone for 72 h to induce differentiation toward the exocrine phenotype. Afterward, AR42J cells were incubated with HT (50 μM) during 2 h in order to evaluate the protective effect of HT on our AP model. Then, cerulein was added and cells were incubated, with HT still present, for a further 24 h (see Fig. 48.1), except for the measurement of NF-κB activation, where cells were exposed to cerulein for only 30 min. The role of HT on AP was assessed by studying its effect on oxidative-inflammatory damage induced by cerulein at the early stage. The following subsections describe the most relevant results.

48.3.1 Hydroxytyrosol improves AR42J antioxidant defenses As an index of the intracellular nonenzymatic antioxidant defenses, we measured43,44 the concentration of GSH in cell lysates from AR42J cells by ELISA. We observed that 1028 M cerulein induced a decrease in GSH

Hydroxytyrosol: features and impact on pancreatitis Chapter | 48

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FIGURE 48.1 Experimental design. AR42J cells were used following treatment with 100 nM dexamethasone for 72 h to induce differentiation toward the exocrine phenotype. Afterward, AR42J cells were incubated with HT (50 μM) during 2 h in order to evaluate the protective effect of HT on our AP model. Then, cerulein was added and cells were incubated, with HT still present, for a further 24 h, except for the measurement of NF-κB activation, where cells were exposed to cerulein for only 30 min. AP, Acute pancreatitis; HT, hydroxytyrosol; NF-κB, nuclear factor kappa B.

concentration (Fig. 48.2), similar to observed in pancreatic tissue in AP.11 It has been shown that GSH depletion may contribute to the progression from mild to severe AP.10 Surprisingly, HT not only prevented the ceruleintriggered drop in GSH values but in fact led to significantly higher levels compared to control (untreated) cells. Therefore HT could prevent the pancreatic injury in AP through its effect on antioxidant machinery. These findings are consistent with those in other models. Thus in human keratinocytes treated with HT, Benincasa et al.25 reported a decrease of glutathione-S-transferase, an enzyme known for their ability to catalyze the conjugation of the reduced form of GSH. Also, Xie et al.45 observed an increase of hepatic GSH content in mice treated with HT-clofibrate.

48.3.2 Suppressive effect of hydroxytyrosol on nuclear factor kappa B activation and cytokine release Early intraacinar NF-κB activation and subsequent secretion of cytokines may be crucial in AP given that promotes the recruitment of inflammatory cells. In this way, we studied44,46 the influence of HT on NF-κB activation, and IL-6 secretion evoked by 1028 M cerulein (Fig. 48.3). For NF-κB activation assessment, we examined NF-κB binding to DNA in nuclear extracts (TransAMTM NF-kB Chemi p65/p50/p52 ELISA kit, Active Motif). IL-6 in culture medium was analyzed by Luminex Multiplex assay. Our results confirm that cerulein activated NF-κB, as shown by increased p65 DNA-binding (Fig. 48.3A), and augmented the secretion of IL-6 into the culture medium (Fig. 48.3B). The presence of HT prevented both NF-κB activation and IL-6 secretion, indicating a clear in vitro efficacy on these inflammatory parameters. The activation of oxidative-sensitive transcription factors, such as NF-κB, plays a critical role in the pathogenesis of AP,8,13 and many studies have confirmed that, in addition to inflammatory cells infiltrated in pancreatic tissue, the acinar cells themselves act as a source of cytokines.4,8,13 This may be relevant, given that the proinflammatory

FIGURE 48.2 Effect of HT on oxidative status in cerulein-stimulated AR42J cells. Bars represent GSH levels in cell lysates from AR42J cells. Values are means of duplicates of three independent experiments, with their standard errors represented by vertical bars. a,b,c: mean values with unlike superscript letters were significantly different (P , .05). Cerulein concentration: 1028 M; HT concentration: 50 μM. See Fig. 48.1 for details about the experimental design. GSH, Reduced glutathione; HT, hydroxytyrosol.

cytokine IL-6 has been consolidated not only as a marker of AP severity but also as a direct agent that causes pancreatic damage.47 Numerous studies show an antiinflammatory effect of HT26,27 in other tissues or cells. The inhibition of NF-κB activation by HT has been recently reported28 in human hepatocellular carcinoma.

48.3.3 Hydroxytyrosol protects against the cell death induced by cerulein Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay46 indicated that 1027 M cerulein induced a significant loss of cell viability in AR42J cells after 24-h incubation (Fig. 48.4A). However, when cerulein was added in the continuous presence of HT, cell viability resulted in values similar to control (92.70% 6 2.62%). To go further in the examination of the cell death pattern, apoptosis after 24-h treatment with 1027 M cerulein was studied46 by flow cytometry (Annexin V-FITC and propidium iodide). The assay

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FIGURE 48.3 Effect of HT on cerulein-induced activation of NF-κB in AR42J cells. (A) p65 DNA-binding activity. (B) IL-6 concentration in culture medium of AR42J cells. Values are means of three independent experiments, with their standard errors represented by vertical bars. a,b: mean values with unlike superscript letters were significantly different (P , .05). Cerulein concentration: 1028 M; HT concentration: 50 μM. See Fig. 48.1 for details about the experimental design. HT, Hydroxytyrosol; IL-6, interleukin-6; NF-κB, nuclear factor kappa B.

FIGURE 48.4 Effect of HT on viability and cell death after cerulein treatment of AR42J cells. (A) Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay and expressed as percentage relative to the untreated group. Values are means, with their standard errors represented by vertical bars. a,b: mean values with unlike superscript letters were significantly different (P , .05). (B) Percentage of apoptotic cells as determined by flow cytometry (Annexin V). Low panel: representative dot-plot diagrams displaying cell populations. Cerulein concentration: 1027 M; HT concentration: 50 μM. See Fig. 48.1 for details about the experimental design. HT, Hydroxytyrosol.

showed that cerulein-induced cell death was mediated by apoptosis and that HT is able to prevent it (Fig. 48.4B). A protective role of HT against toxic effects has been recently indicated by Tortora et al.48 in human erythrocytes. The authors observed that the treatment of erythrocytes with HT prevents apoptotic death evoked by lysophosphatidic acid.

48.3.4 Hydroxytyrosol restores physiological Ca21 signaling and secretory pattern impaired by cerulein Because the function of pancreatic acinar cells is to perform exocytosis of digestive enzymes, it was appropriate to examine both cytosolic Ca21 as a key intracellular

Hydroxytyrosol: features and impact on pancreatitis Chapter | 48

messenger controlling pancreatic secretion and the ability to secrete amylase (Fig. 48.5). First, we analyzed46 Ca21 homeostasis in healthy (control) AR42J cells. After baseline recording, fura-2-loaded cells were exposed to the secretagogue CCK-8 in a final concentration of 1029 M. Cells showed the typical response of pancreatic acinar cells to this CCK-8 concentration, characterized by a sharp increase followed by a slow decrease toward a value close to the prestimulation level (Fig. 48.5A). This physiological pattern was disturbed by pretreatment for 24 h with 1028 M cerulein: the initial rise was not so prompt and the maximal [Ca21]c at the peak was lower (Fig. 48.5A), all of which led to a statistically significant decrease in the Ca21 area under the curve (Fig. 48.5B). Nevertheless, the impairment of Ca21 signaling induced by cerulein was completely restored by HT, both in terms of temporal pattern and magnitude (Fig. 48.5A and B). Then, we evaluated amylase secretion in our AP model studying the concentration
response curves for CCK-8stimulated net amylase release (i.e., above basal).46 We can see (Fig. 48.5C) that, similar to those reported in fresh acinar cells from rodent pancreas,12 amylase release curves in response to CCK-8 are also biphasic in differentiated AR42J cells, with maximum amylase release occurring in response to 1029 M CCK-8. This ability to secrete amylase was injured by cerulein, reducing significantly amylase release evoked by the same concentrations of CCK-8 (Fig. 48.5C). Finally, as observed on Ca21 signaling, this cerulein-induced damage was counteracted by HT, indicative of a protective effect against the noxious stimulus at a functional level. This is of noticeable

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importance, given that altered Ca21 and diminished secretion (secretory blockade) are key features in AP.

48.4 Summary points G

G

We have studied the role of HT in AP. In our in vitro model of AP using differentiated AR42J cells treated with cerulein (1028 or 1027 M), cells displayed an oxidative-inflammatory damage similar to that evoked by cerulein hyperstimulation in vivo. By using this model, we present the first evidence that the presence of HT may protect acinar cells against injurious effects of cerulein. This effect is exerted through different mechanisms: G Improvement of cell antioxidant defenses (GSH) G Suppression of NF-κB activation and cytokine release (IL-6) G Decreased apoptosis G Restoration of physiological Ca21 signaling and secretory pattern

Mini-dictionary of terms Apoptosis: Cell death genetically programmed or induced by external stimuli. It is characterized by a highly controlled cellular process that culminates in the formation of apoptotic bodies, which can be easily and cleanly eliminated by phagocytic cells. This process contrasts with necrosis, where destruction of the cells occurs. In necrotic death the rupture of the plasma membrane allows cell FIGURE 48.5 Effect of HT and cerulein on functional status of AR42J cells. (A) Time-course changes in [Ca21]c evoked by 1029 M CCK-8 as determined by fluorescence spectrophotometry in fura-2-loaded cells. (B) AUC of the CCK-8-evoked Ca21 response. a,b: mean values with unlike superscript letters were significantly different (P , .05). (C) Net amylase secretion stimulated by increasing the concentrations of the secretagogue CCK-8. Amylase secretion is expressed as a percentage of total initial content. Values are means with their standard errors represented by vertical bars. * Mean values significantly different (P , .05) from those in the other two groups. Cerulein concentration: 1028 M; HT concentration: 50 μM. See Fig. 48.1 for details about the experimental design. AUC, Area under the curve; CCK, cholecystokinin; HT, hydroxytyrosol.

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contents to be discharged into the extracellular space thereby causing an inflammatory reaction. Autophagy: Intracellular digestion of molecules, structures, and organelles of the cell. Chemokines: A family of small cytokines (see next terms). Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are, thus, chemotactic cytokines. Chemotaxis: Property of some cells to move in response to a chemical stimulus. Cytokines: A large group of proteins, peptides, or glycoproteins that function as signaling molecules and mediate and regulate immunity, inflammation, and hematopoiesis. Cytokines are produced throughout the body mainly, but not only, by cells of the immune system. Endoplasmic reticulum: A multifunctional organelle that stores calcium and, among other metabolic functions, is responsible for protein folding and processing in cells. European Food Safety Authority (EFSA): A European agency. It was set up in 2002, following a series of food crises in the late 1990s, to be a source of scientific advice and communication on risks associated with the food chain. The work of EFSA covers all subjects with a direct or indirect impact on food and feed safety, including animal health and welfare, plant protection, and human nutrition. Intraductal: Within a duct. Pancreatic tissue is organized in acini and ducts. Acini are formed by acinar cells, which are responsible for secreting a zymogen-rich solution that, afterward, flows through the pancreatic ducts to finally reach the duodenum. Epithelial cells lining the ducts (ductal cells) also contribute by adding water and electrolytes to the zymogen-rich solution. Lysosomal enzyme: An enzyme in an organelle (a little organ within the cell) called the lysosome. Lysosomal enzymes degrade (break down) macromolecules (large molecules) and other materials (such as bacteria) that have been taken up by the cell during the process of endocytosis. Mitochondrial depolarization: Alteration in mitochondrial membrane potential. The latter refers to a difference in electrical charges across the mitochondrial membrane, with a small excess of negative charges inside and positives outside. The maintenance of mitochondrial membrane potential is crucial for ATP synthesis. Certain stimuli can cause the membrane potential to be altered (depolarization), which means that the mitochondrion, as a functional entity, “dies.” NF-κB (nuclear factor κB): Protein complex that controls the transcription of DNA (i.e., a transcription factor). Under normal conditions it is inactivated in the cytosol of the cells, but certain stimuli can activate it, in which case some of its components, such as P65 or P50, separate from the complex and go to the nucleus of the cells to

modify the expression of genes. Specifically, it regulates genes related to cell proliferation and survival and induces the expression and synthesis of inflammatory factors. Phenotype: In genetics the phenotype is the composite of the observable characteristics of the organism. The term covers morphology or physical form and structure, its developmental processes, its biochemical and physiological properties, its behavior, and the products of behavior. The phenotype results from two basic factors: the expression of genetic code (genotype) and the influence of environmental factors. Both factors may interact, further affecting phenotype. Phytochemicals: Chemical compounds produced by plants. As a term, phytochemicals are generally used to describe plant compounds that are under investigation, whose health effects are not fully established and thereby cannot be scientifically defined as essential nutrients. Reactive oxygen species (ROS): A type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell. The reduction of molecular oxygen (O2) produces superoxide (GO22), which is the precursor of most other ROS. Zymogen: Enzyme precursor requiring some biochemical change (such as a hydrolysis reaction or conformational modification) to become active. Zymogens accumulate in small organelles called zymogen granules. Following appropriate stimulation of the cell, zymogen granules undergo exocytosis, which makes it possible for the granule content to be released into the extracellular space. Among others, zymogen granules are found in pancreatic acinar cells. Pancreatic zymogens are precursors of digestive enzymes such as the protease trypsin. They are secreted into the pancreatic juice and, in normal conditions, will be activated only when they reach the duodenum. In this way, pancreatic structures and cells are protected from digestion.

Comparisons of olive oils with other edible oils Since this chapter does not focus on olive oil as such, but on one of its constituents, hydroxytyrosol (HT), it may be more relevant to briefly review in this section the actions of other phytochemicals, instead of other oils, in acute pancreatitis (AP). A large number of phytochemicals have been explored for preventing or treating AP (for a review, see Refs. [49,50]). Most studies have been conducted in rats, where these compounds have shown to play a beneficial role in experimental AP induced by ethanol, bile acids, cerulein, pancreatic duct ligation, or L-arginine. There is quite a lot of variability in experimental designs, so that in some studies the phytochemical is administered before the

Hydroxytyrosol: features and impact on pancreatitis Chapter | 48

insult, while in others, it is done during the AP induction protocol or once this has been completed. Making an exhaustive description of the effectiveness, mechanism of action and potential therapeutic value of these molecules would take us far beyond the scope of this chapter. However, for comparison purposes with HT, we will summarize the most relevant information about two very well-known phenols, curcumin and resveratrol. Curcumin is a natural polyphenolic compound derived from the long-nourishing rhizome, otherwise known as Indian turmeric, grown in India, China, and in Southeast Asia. Curcumin has shown potential preventive/therapeutic effects in AP by blocking the activation of NF-κB and inhibiting the release of proinflammatory mediators, reducing oxidative damage and promoting antioxidant cell-defenses, attenuating trypsin activation and causing apoptosis. It should be mentioned here that in the models of severe AP, where necrosis predominates, apoptosis induction is considered a beneficial effect. Another proven mechanism of curcumin-evoked protective pancreatic effects is mediated through upregulation of peroxisome proliferator-activated receptor gamma (PPAR-γ), which in turn produces beneficial antiinflammatory and antioxidant effects. Resveratrol is a natural phenolic compound, occurring among others in grapes and red wine. Resveratrol alleviates AP by reversing the abnormal Ca21 signal. It also appears to inhibit the NF-κB pathway and the subsequent expression of proinflammatory mediators. It has been shown to protect the pancreas against harmful toxicity of reactive oxygen species via increases in endogenous antioxidants such as reduced glutathione and acting itself as a scavenger. As for the effects of resveratrol on the pancreatic cell death pattern, both promotion and decline of apoptosis have been reported. In both cases, resveratrol favorably influenced the evolution of AP according to several severity scores. As described in previous sections, many of these effects have also been confirmed for HT in our cellular AP model. Furthermore, all the three compounds (HT, curcumin, and resveratrol) share an important feature, that is, multiple targets and mechanisms of action.

Implications for human health and disease prevention Data discussed in this chapter show, in a cellular model, a beneficial action of hydroxytyrosol (HT) in the early events of mild AP, mediated by antiinflammatory and cytoprotective effects, together with restoration of normal cell function (Ca21 homeostasis and secretory activity) and promotion of antioxidant defenses. These results are novel since, so far, no one has examined the potential of

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HT in this pathology. However, further investigations, both in experimental animals and in humans, are needed to fully ascertain the preventive or therapeutic role of HT in AP. Animal studies should be done in pancreatitis models of different severity. Furthermore, our results are mainly indicative of a preventive effect of HT, given that the phenol had been in the culture medium for 2 h when the cell aggression was initiated. Therefore while confirming this preventive effect, new experiments in vivo should also explore HT efficiency once the disease is established. In human research, several important aspects must be taken into account: G

G

HT has been studied in cellular, animal, and human models that demonstrate it is a safe compound. However, it is known that HT is absorbed well in the digestive tract, but quickly undergoes metabolization phenomena. Therefore the doses to be used should be carefully studied, as well as the possibility of using pharmaceutical formulations that may improve the bioavailability of the product. In clinical pancreatitis, it will be difficult to test the preventive effect of HT, since it could only be administered once the pathogenic process has begun and the patient has experienced signs and symptoms that have made him go to the hospital. The only possibility may be to test its protective role in endoscopic retrograde cholangio-pancreatography. This is a procedure to help diagnose the condition of the liver, bile ducts, pancreas, or gallbladder, which, occasionally, triggers an episode of AP. In fact, some phytochemicals and other drugs are being currently tested with a preventive purpose.

While these investigations are being carried out, the recommendation that derives from our results, as well as from different clinical trials, is the adoption of a healthy eating and lifestyle pattern, such as the Mediterranean one, in order to reduce the risk for conditions that are well-known established causes of AP (excessive alcohol consumption of high graduation, biliary lithiasis, and hypertriglyceridemia). Moreover, an additional advantage of adhering to this food pattern as a whole is the possibility of positive interactions between HT and many other phytochemicals supplied by fruits and vegetables. Results available (see “Comparisons of olive oils with other edible oils” section) support this notion.

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2. Lankisch PG, Apte M, Banks PA. Acute pancreatitis. Lancet. 2015;386:85
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1346. 7. Biczo G, Vegh ET, Shalbueva N, et al. Mitochondrial dysfunction, through impaired autophagy, leads to endoplasmic reticulum stress, deregulated lipid metabolism, and pancreatitis in animal models. Gastroenterology. 2018;154:689
703. 8. Saluja A, Dudeja V, Dawra R, Sah RP. Early intra-acinar events in pathogenesis of pancreatitis. Gastroenterology. 2019;156:1979
1993. 9. Petersen OH, Tepikin AV, Gerasimenko JV, Gerasimenko OV, Sutton R, Criddle DN. Fatty acids, alcohol and fatty acid ethyl esters: toxic Ca21 signal generation and pancreatitis. Cell Calcium. 2009;45:634
642. 10. Pe´rez S, Pereda J, Sabater L, Sastre J. Redox signaling in acute pancreatitis. Redox Biol. 2015;5:1
14. 11. Lutgendorff F, Trulsson LM, van Minnen LP, et al. Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2008;295:G1111
G1121. 12. Rosado JA, Gonza´lez A, Salido GM, Pariente JA. Effects of reactive oxygen species on actin filament polymerisation and amylase secretion in mouse pancreatic acinar cells. Cell Signal. 2002;14:547
556. 13. Jakkampudi A, Jangala R, Reddy BR, Mitnala S, Nageshwar Reddy D, Talukdar R. NF-κB in acute pancreatitis: mechanisms and therapeutic potential. Pancreatology. 2016;16:477
488. 14. Roberts SE, Morrison-Rees S, John A, Williams JG, Brown TH, Samuel DG. The incidence and aetiology of acute pancreatitis across Europe. Pancreatology. 2017;17:155
165. ˚ , Wolk 15. Oskarsson V, Sadr-Azodi O, Orsini N, Andre´n-Sandberg A A. Vegetables, fruit and risk of non-gallstone-related acute pancreatitis: a population-based prospective cohort study. Gut. 2013;62:1187
1192. 16. Oskarsson V, Orsini N, Sadr-Azodi O, Wolk A. Fish consumption and risk of non-gallstone-related acute pancreatitis: a prospective cohort study. Am J Clin Nutr. 2015;101:72
78. 17. Setiawan VW, Pandol SJ, Porcel J, et al. Dietary factors reduce risk of acute pancreatitis in a large multiethnic cohort. Clin Gastroenterol Hepatol. 2017;15:257
265. 18. Katsagoni CN, Psarra G, Georgoulis M, Tambalis K, Panagiotakos DB, Sidossis LS, ευζην Study Group. High and moderate adherence to Mediterranean lifestyle is inversely associated with overweight, general and abdominal obesity in children and adolescents: the MediLIFE-index. Nutr Res. 2019;73:38
47.

19. Khatua B, El-Kurdi B, Singh VP. Obesity and pancreatitis. Curr Opin Gastroenterol. 2017;33:374
382. 20. Mataix J, Quiles JL, Huertas JR, Battino M, Man˜as M. Tissue specific interactions of exercise, dietary fatty acids, and vitamin E in lipid peroxidation. Free Radic Biol Med. 1998;24:511
521. 21. Dı´az RJ, Yago MD, Martı´nez-Victoria E, Naranjo JA, Martı´nez MA, Man˜as M. Comparison of the effects of dietary sunflower oil and virgin olive oil on rat exocrine pancreatic secretion in vivo. Lipids. 2003;38:1119
1126. 22. Martı´nez MA, Lajas AI, Yago MD, et al. Dietary virgin olive oil enhances secretagogue-evoked calcium signaling in rat pancreatic acinar cells. Nutrition. 2004;20:536
541. 23. Yago MD, Diaz RJ, Ramirez R, Martinez MA, Man˜as M, Martinez-Victoria E. Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling. Br J Nutr. 2004;91:227
234. 24. Yago MD, Dı´az RJ, Martı´nez MA, et al. Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells. J Nutr Biochem. 2006;17:242
249. 25. Benincasa C, La Torre C, Plastina P, et al. Hydroxytyrosyl oleate: improved extraction procedure from olive oil and by-products, and in vitro antioxidant and skin regenerative properties. Antioxidants (Basel). 2019;8(7):233. 26. Zhi LQ, Yao SX, Liu HL, Li M, Duan N, Ma JB. Hydroxytyrosol inhibits the inflammatory response of osteoarthritis chondrocytes via SIRT6-mediated autophagy. Mol Med Rep. 2018;17 (3):4035
4042. 27. Yonezawa Y, Miyashita T, Nejishima H, Takeda Y, Imai K, Ogawa H. Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells. J Vet Med Sci. 2018;80(12):1801
1807. 28. Zhao B, Ma Y, Xu Z, et al. Hydroxytyrosol, a natural molecule from olive oil, suppresses the growth of human hepatocellular carcinoma cells via inactivating AKT and nuclear factor-kappa B pathways. Cancer Lett. 2014;347(1):79
87. ´ , Lo´pez de Las Hazas MC, Rubio´ L, et al. Protective 29. Catala´n U effect of hydroxytyrosol and its predominant plasmatic human metabolites against endothelial dysfunction in human aortic endothelial cells. Mol Nutr Food Res. 2015;59:2523
2536. 30. Giordano E, Davalos A, Nicod N, Visioli F. Hydroxytyrosol attenuates tunicamycin-induced endoplasmic reticulum stress in human hepatocarcinoma cells. Mol Nutr Food Res. 2014;58:954
962. 31. Zheng A, Li H, Xu J, et al. Hydroxytyrosol improves mitochondrial function and reduces oxidative stress in the brain of db/db mice: role of AMP-activated protein kinase activation. Br J Nutr. 2015;113:1667
1676. 32. Gorelick FS, Lerch MM. Do animal models of acute pancreatitis reproduce human disease? Cell Mol Gastroenterol Hepatol. 2017;4:251
262. 33. Audi N, Mesa MD, Martı´nez MA, Martı´nez-Victoria E, Man˜as M, Yago MD. Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids. Exp Biol Med (Maywood). 2007;232:532
541. 34. Audi N, Martinez MA, Mesa MD, Martinez-Victoria E, Man˜as M, Yago MD. Interleukin-6 effects on amylase secretion and calcium signalling in pancreatic AR42J cells: modulation by membrane fatty acid composition. Proc Nutr Soc. 2008;67:25.

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35. Santana C, Lo´pez-Milla´n MB, Martı´nez-Burgos MA, Man˜as M, Martı´nez-Victoria E, Yago MD. Influence of membrane fatty acid composition on cell viability and lipid peroxidation in a cell model (AR42J) of cerulein-induced acute pancreatitis. Proc Nutr Soc. 2010;69(OCE3):E313. 36. Lo´pez-Milla´n MB, Santana C, Audi N, et al. Modification of membrane fatty acid composition of pancreatic AR42J cells influences bile acid-induced Ca21 responses. Acta Physiol. 2009;195 (s667):154
155. 37. Lopez-Millan MB, Santana C, Yago MD, Martinez-Burgos MA, Man˜as M, Martinez-Victoria E. Membrane enrichment with specific fatty acids (oleic acid or w3 PUFA) modifies the inflammatory response in cerulein-stimulated AR42J cells. Acta Physiol. 2012;206(s693):P125. 38. Lopez-Millan MB, Santana C, Yago MD, Martinez-Burgos MA, Martinez-Victoria E, Man˜as M. Impairment of calcium homeostasis and secretory response in AR42J cells after cerulein treatment. Influence of changes in membrane fatty acid profile. Acta Physiol. 2012;206(s693):P127. 39. Lopez-Millan MB, Santana C, Martinez-Burgos MA, MartinezVictoria E, Man˜as M, Yago MD. Modification of membrane fatty acid composition affects cell death and antioxidant defences in cerulein-stimulated AR42J cells. Acta Physiol. 2012;206(s693):P128. 40. Chen WD, Zhang JL, Wang XY, Hu ZW, Qian YB. The JAK2/ STAT3 signaling pathway is required for inflammation and cell death induced by cerulein in AR42J cells. Eur Rev Med Pharmacol Sci. 2019;23:1770
1777. 41. Yang Z, Yang W, Lu M, et al. Role of the c-Jun N-terminal kinase signaling pathway in the activation of trypsinogen in rat pancreatic acinar cells. Int J Mol Med. 2018;41:1119
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42. Lugea A, Gerloff A, Su HY, et al. The combination of alcohol and cigarette smoke induces endoplasmic reticulum stress and cell death in pancreatic acinar cells. Gastroenterology. 2017;153:1674
1686. 43. Santana-Ojeda C. Aspectos oxidativos en la respuesta celular de un modelo in vitro de pancreatitis. Papel del hidroxitirosol y de la modificacio´n del perfil lipı´dico de membrana [Ph.D. thesis]. Granada: University of Granada; 2011. 44. Lo´pez Milla´n MB, Santana C, Yago MD, et al. Actividad funcional del hidroxitirosol en ce´lulas AR42J tratadas con ceruleina. Nutr Hosp. 2012;27:1719. 45. Xie YD, Chen ZZ, Li N, et al. Hydroxytyrosol nicotinate, a new multifunctional hypolipidemic and hypoglycemic agent. Biomed Pharmacother. 2018;99:715
724. 46. Lo´pez-Milla´n B. Respuestas celulares a modificaciones en el perfil lipı´dico y a la presencia de un antioxidante del aceite de oliva en un modelo in vitro de pancreatitis. Aspectos inflamatorios [Ph.D. thesis]. Granada: University of Granada; 2011. ISBN: 9788469469347. 47. Staubli SM, Oertli D, Nebiker CA. Laboratory markers predicting severity of acute pancreatitis. Crit Rev Clin Lab Sci. 2015;52 (6):273
283. 48. Tortora F, Notariale R, Lang F, Manna C. Hydroxytyrosol decreases phosphatidylserine exposure and inhibits suicidal death induced by lysophosphatidic acid in human erythrocytes. Cell Physiol Biochem. 2019;53(6):921
932. 49. Anchi P, Khurana A, Bale S, Godugu C. The role of plant-derived products in pancreatitis: experimental and clinical evidence. Phytother Res. 2017;31:591
623. 50. Tarasiuk A, Fichna J. Effectiveness and therapeutic value of phytochemicals in acute pancreatitis: a review. Pancreatology. 2019;19:481
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Chapter 49

The effects of extra-virgin olive oil minority compounds hydroxytyrosol and oleuropein on glioma Marı´a Jesu´s Ramı´rez-Expo´sito1, Marı´a Pilar Carrera-Gonza´lez1,2 and Jose´ Manuel Martı´nez-Martos1 1

Experimental and Clinical Physiopathology Research Group CTS1039, Department of Health Sciences, School of Experimental and Health Sciences,

University of Jae´n, Campus Universitario Las Lagunillas, Jae´n, Spain, 2Department of Nursing, Pharmacology and Physiotherapy, Faculty of Medicine and Nursing, University of Cordoba, IMIBIC, Co´rdoba, Spain

Abbreviations CAT DNA ENU GPx GSH HTX MDA OLEU ROS SOD TBARS

catalase deoxyribonucleic acid N-ethyl-N-nitrosourea glutathione peroxidase reduced glutathione hydroxytyrosol malondialdehyde oleuropein reactive oxygen species superoxide dismutase thiobarbituric acid-reactive substances

49.1 Introduction Tumor formation is a multistep process that involves several molecular and cellular alterations, which promotes the transformation of a normal cell into a malignant one. Free radicals, mainly reactive oxygen species (ROS), are involved in that process triggering lipid peroxidation of the cellular membranes and oxidation of proteins and deoxyribonucleic acid (DNA). These alterations result in genetic mutations and/or alteration of cell growth.1
4 Furthermore, ROS generation is a constant feature of oxygen metabolism in cells, and the imbalance between ROS generation and the efficient antioxidant mechanisms leads to oxidative damage.1,5,6 In fact, tumor development has been associated not only with oxidative stress but also with a reduced response of antioxidant defense systems. In this way the central nervous system is especially vulnerable to this free radical
induced damage.7 However, ROS may also kill cancer cells through the block of key

steps in the cell cycle and promoting apoptosis through mechanisms that remain to be discovered.8 In this sense, several chemotherapy drugs that enhance apoptotic killing of cancer cells act by lowering antioxidant levels, and their action ceases if antioxidant compounds are administered concomitantly.9 Thus administration of exogenous antioxidants has received particular attention due to their potential to modulate oxidative stress and to act as putative antitumor compounds, both alone or in combination with various anticancer drugs.10 Several nutritional intervention trials have shown that antioxidants are not obviously effective in preventing cancer,8,11,12 whereas other epidemiological evidence has indicated that increased consumption of fruits and vegetables containing antioxidants is associated with health improvements in terms of cancer risks.13 Phenolic compounds derived from olives and virgin olive oils, especially oleuropein (OLEU) and its major metabolite hydroxytyrosol (HTX), exert important antiinflammatory, cardioprotective, and anticancer activities both in vitro and in vivo due to their antioxidant properties,2,3,14
16 potentially reducing the risk of mutagenesis and carcinogenesis.17 Thus, in the present chapter, we summarize the in vivo antitumor properties and their effects on oxidative stress biomarkers, on nonenzymatic and on enzymatic antioxidant defense systems, of the phenolic compounds OLEU and HTX in two animal models of glioma, the C6 glioma implanted at the subcutaneous region and the transplacental N-ethyl-N-nitrosourea (ENU)-induced glioma tumors. Rat C6 glioma models are widely used to evaluate the effects of novel therapies. The implantation of C6 gliomas into a rat brain is a popular and proper animal model mimicking human central

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00023-7 © 2021 Elsevier Inc. All rights reserved.

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nervous system tumors, and thus many investigations using this model have been reported.18 To overcome the disadvantages of the intracerebral C6 glioma model,19 in other studies, C6 glioma cells were implanted into the rat abdominal subcutaneous region. The growth pattern and pathological findings in the subcutaneous region were compared with those cells implanted in the cerebral region, and it was demonstrated that the subcutaneous model was a useful tool.20,21 By other hand, transplacental ENU-induced tumors of nervous system have also been widely used as an experimental brain tumor model.22
26 This model shows high rate of tumor induction (normally of 100%) and the appearance of multiple tumors per brain.24 In addition, the profile and time course of tumor progression in this experimental model have been extensively documented24,27,28 (Fig. 49.1). Thus both the C6implanted glioma tumors and the ENU models have allowed us to analyze the oxidative status and redox processes in brain and their relationship with the mechanisms

underlying tumor development and the effects of OLEU and HTX as antioxidants.

49.1.1 Extra-virgin olive oil minority compounds as potent antioxidants Several reports have shown that antioxidants may be useful in treating primary brain tumors because a dynamic relationship exists between oxidative stress and brain tumor appearance. In fact, the tumor microenvironment is a key player in the neoplastic process. However, cancer cells also can be destroyed by ROS, which alter the cell cycle and promote cell death.1,8 Cancer cells largely driven by the RAS and MYC oncogenes are among the most difficult to treat due to their high levels of ROSdestroying antioxidants. Furthermore, several chemotherapy drugs that enhance the apoptotic killing of cancer cells act by lowering antioxidant levels, and their actions FIGURE 49.1 Representative specimens of the C6 glioma cells implanted at the subcutaneous region-induced tumor (A) and transplacental ENU-induced brain tumor (B). Microscopic view with H&E staining using 10 3 magnification. ENU, N-Ethyl-N-nitrosourea.

The effects of extra-virgin olive oil minority compounds hydroxytyrosol and oleuropein on glioma Chapter | 49

cease if they are administered along with antioxidant compounds.9 Therefore a very precise balance must occur between prooxidant and antioxidant activities, which is the key to promoting either cell proliferation or cell death.1 The established beneficial effects of extra-virgin olive oil in the context of the Mediterranean diet have been mainly attributed to minor though highly bioactive components, including polyphenols.29
31 The primary polyphenols are OLEU, HTX, and α-tocopherol. The OLEU hydrolyzes to the catechol HTX and functions as a hydrophilic phenolic antioxidant that is oxidized to its catechol quinone during redox cycling. We have described that the IC50 values of OLEU and HTX for C6 glioma cells in vitro were comparable with those of other well-known natural antioxidant substances, such as resveratrol or melatonin (at a millimolar level), with OLEU being more potent than HTX.32,33 Our experimental designs in vivo include both subcutaneous and oral administration of OLEU and HTX. In the first case a short-term intervention (5 days of daily subcutaneous injections of 100 μg of OLEU or 100 μg of HTX dissolved in saline solution) was enough to show the effects of both compounds on animals with C6-implanted gliomas. On the second case, oral administration of OLEU and HTX (50 and 25 mg/L, respectively) in drinking water ad libitum was maintained during 30 weeks in animals with ENU-induced gliomas. This long-term intervention was also adapted to the course of tumor development in the ENU-induced glioma model. It must be also taken into account that, in the stomach, OLEU undergoes acid hydrolysis with the formation of different metabolites, including HTX, so that only a small amount of unchanged OLEU not only reaches the systemic circulation32 but also crosses the blood
brain barrier.34

49.1.2 Effects of oleuropein and hydroxytyrosol on tumor growth Both animal models showed that HTX treatment decreased tumor volumes whereas the treatment with OLEU did not have any effect on tumor growth (Fig. 49.2). This lack of effects of OLEU could suggest that we had not used an adequate amount of OLEU or that we had used it during too short time. Therefore we also carried out experiments using higher doses of OLEU and/or longer administration periods. However, no inhibition of tumor growth was found in any situation. Moreover, we observed a stimulatory effect on tumor growth under those conditions (data not shown). The mechanisms underlying the lack of stimulatory effects of OLEU depending on the dose used on tumor growth remain to be discovered. OLEU has been

595

FIGURE 49.2 Tumor volumes in animals with C6 glioma cells implanted in the subcutaneous region and animals with transplacental ENU-induced tumors before and after the treatments with vehicle (control), OLEU, and HTX. Results are expressed in percentage versus control (mean 6 SEM; ***P , .001). ENU, N-Ethyl-N-nitrosourea; HTX, hydroxytyrosol; OLEU, oleuropein.

described as a potent scavenger of oxygen-free radicals35 and nitrogen-based free-radical species.36 In addition, it plays an important role in the prevention of DNA damage, thus inhibiting mutagenesis and carcinogenesis.37 Furthermore38, it has been demonstrated that the antitumor effect of OLEU may be exerted by the disruption of actin filaments in tumor cells. However, to date, the antitumor effects of OLEU and HTX have been mainly described by in vitro studies, including our previous one.32 We propose three hypotheses to explain the stimulatory effects of OLEU in our in vivo glioma model: (1) OLEU inhibits the immune response against tumors (very potent in this animal model); (2) OLEU could have structural similarities with growth factors or other types of stimulators, acting as an agonist and thus leading to an increase in tumor growth by mechanisms not related to its antioxidant properties; and (3) OLEU, via its potent antioxidant properties, inhibits the destruction of cancer cells via ROS and promotes tumor growth. In fact, tumor cells with high levels of ROS-destroying antioxidants are among the most difficult to treat. In addition, several chemotherapy drugs that act by lowering antioxidant levels cease their actions if antioxidant compounds are concomitantly administered.8,9 Therefore further research is necessary to solve these issues. With regard to the inhibitory effects of HTX administration on tumor growth, a growing number of in vitro studies have demonstrated that it has antiproliferative effects against several tumor cell lines, likely involving inactivation of the AKT and nuclear factor-kappa B (NF-κB) pathways.39 In vivo, HTX seems to induce cell cycle arrest and apoptosis.40 Although the in vivo effects of HTX are highly dependent on the dose used41, both animal models with different treatment and administration protocols showed similar inhibition rates.

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49.1.3 Effects of oleuropein and hydroxytyrosol on oxidative stress parameters Several authors have shown that OLEU and HTX inhibit oxidative stress.2,3,14,17 Therefore we have studied the effects of OLEU and HTX on circulating oxidative stress parameters such as lipid peroxidation [through the measurement of thiobarbituric acid-reactive substances (TBARS) content] and protein oxidation (through the measurement of carbonyl groups content) in both models of glioma tumors. We found increased levels of lipid peroxidation in animals with C6 glioma tumors treated with OLEU or HTX, whereas no changes were found in animals with ENU-induced gliomas treated with OLEU or a significant decrease was found in animals treated with HTX (Fig. 49.3A). On the contrary, only the treatment with OLEU-modified protein oxidation levels in animals with C6 gliomas, whereas no changes were found after the treatment with HTX. Furthermore, no changes in

FIGURE 49.3 Circulating TBARS (A) and carbonyl groups content (B) in animals with C6 glioma implanted at the subcutaneous region and animals with transplacental ENU-induced tumors treated with vehicle (control), with OLEU and with HTX. Results are expressed in percentage versus control (mean 6 SEM; **P , 0.01; ***P , 0.001). ENU, NEthyl-N-nitrosourea; HTX, hydroxytyrosol; OLEU, oleuropein; TBARS, thiobarbituric acid reactive substances.

protein oxidation were found in animals with ENUinduced gliomas after the treatment with OLEU or HTX (Fig. 49.3B). Lipid peroxidation is an early biomarker of oxidative damage because of the increased propagation of free radicals associated with it. Elevations in oxidative stress in cells can lead to modifications of a number of cellular targets, causing cellular damage and a subsequent lack of cellular repair processes associated with carcinogenesis.1,4,42 Cancer cells are under increased and persistent oxidative stress due to the elevated generation of intracellular free radicals, leading to promotion of the carcinogenic process through mechanisms such as oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction.1,43 The magnitude of this oxidative damage depends not only on free-radical levels but also on the efficiencies of the antioxidant mechanisms. The disruption of this delicate oxidant/antioxidant balance seems to play a causative role in carcinogenesis.5 Therefore high levels of oxidative stress result in peroxidation of membrane lipids and generation of peroxides that can decompose into multiple mutagenic aldehyde products, mainly malondialdehyde (MDA), which is involved in cancer progression.44 Previous results of our lab4 agree with others45 that have observed increased TBARS levels in tumor tissue samples compared with those obtained from peritumoral areas, which could have been attributed to an increased formation or inadequate clearance of free radicals by cellular antioxidants. In astrocytomas, meningiomas, metastatic tumors, and other tumor types, TBARS levels were significantly higher compared with the corresponding peritumoral adjacent tissues. In addition, differences were observed when the astrocytoma tumor group was compared with other tumor groups. When the TBARS levels of the low- and highgrade tumors were compared, lipid peroxidation was significantly higher in the high-grade tumors. Elevated levels of lipid peroxidation products support the hypothesis that tumor cells produce a large amount of free radicals and that a relationship between free-radical activity and carcinogenesis exists. Furthermore, previous studies have also shown that the lipid peroxidation state depends on the tumoral area studied. An estimation of lipid peroxidation in low- and high-grade astrocytomas45 has revealed that TBARS levels in low-grade astrocytomas are significantly elevated compared with those in malignant lesions, particularly at the tumor surface. Further, examinations of energetic and oxidant metabolic processes in low-grade gliomas obtained from the centers and peripheries of tumors46 have shown that lipid peroxidation increases at the periphery compared with the center. It has been studied lipid peroxidation levels in serum as well as in tissue samples obtained from patients with high- and low-grade glial tumors,47 revealing that those with high-grade

The effects of extra-virgin olive oil minority compounds hydroxytyrosol and oleuropein on glioma Chapter | 49

tumors have higher MDA levels in both their sera and tissues compared with patients with low-grade tumors and controls. We have also shown increased levels of TBARS in serum of animals with C6 gliomas, although no differences were found in animals with ENU-induced gliomas. In this model the systemic lipid peroxidation status does not seem to reflect the oxidative changes that occur at tissue level, and only HTX treatment inhibited lipid peroxidation in our study, which correlated with the inhibition of tumor growth. Several products of lipid peroxidation are responsible for protein oxidation.48 We have not found changes in circulating carbonyl groups content either in animals with C6-implanted glioma or ENU-induced glioma after the treatment with HTX, but an increase in protein oxidation levels was found after the treatment with OLEU in animals with C6-implanted glioma. In tumor tissue, we had previously described increased carbonyl groups content in animals with ENU-induced glioma, also suggesting that changes detected in tumor tissue are not clearly reflected at circulating levels. In the same way, OLEU but not HTX seems to promote an increase in carbonyl groups content at circulating level in animals with C6-implanted gliomas.

49.1.4 Effects of oleuropein and hydroxytyrosol on nonenzymatic antioxidant defense systems The magnitude of the oxidative damage depends on not only free radicals production but also the efficiencies of the antioxidant mechanisms.49 Cellular antioxidants and free-radical scavengers protect the cell against toxic levels of oxygen radicals and include reduced glutathione (GSH), which is an important nonprotein thiol. A significant depletion of GSH has been found in astrocytomas, meningiomas, metastatic tumors, and other types of brain tumors compared with their peritumoral tissues.45,50 It has been reported a significant decrease in GSH in high-grade compared with low-grade tumors.45 Here, we also showed a decrease in total GSH with the treatment of OLEU or HTX in both animal models (Fig. 49.4), being more exacerbated in animals with ENU-induced gliomas. These discrepancies could be due not only to the obvious differences between the animal models but also to the differences in the route of administration and the dose of OLEU and HTX administrated. Nevertheless, in both models, GSH depletion is probably related to an enhanced prooxidant milieu, which correlates with the increase in circulating lipid peroxides observed in the C6 glioma model but not with the ENU-induced model. Our results also agree with those of Navarro et al.,51 who have shown that changes in GSH status and the antioxidant system in blood and cancer cells are associated with tumor growth

597

FIGURE 49.4 Total circulating GSH content in animals with C6 glioma implanted at the subcutaneous region and animals with transplacental ENU-induced tumors treated with vehicle (control), with OLEU, and with HTX. Results are expressed in percentage versus control (mean 6 SEM; **P , 0.01; ***P , 0.001). ENU, N-Ethyl-N-nitrosourea; GSH, glutathione; HTX, hydroxytyrosol; OLEU, oleuropein.

in vivo. GSH depletion is due to increased H2O2 production by tumors, as well as to changes in glutathione peroxidase (GPx) activity. In our animal models the treatment with OLEU or HTX had important effects on this nonenzymatic antioxidant defense system. Furthermore, GSH emerges as an important participant in the delicate equilibrium between oxidant and antioxidants in brain tumors.1,33,52

49.1.5 Effects of oleuropein and hydroxytyrosol on enzymatic antioxidant defense systems During prolonged oxidative stress, changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and GPx occur. These enzymes normally act to prevent or decrease the tissue damage caused by free radicals. Thus SOD metabolizes free radicals and dismutates superoxide anions (O2 2) to H2O2 and protects cells against O2 2-mediated lipid peroxidation, CAT converts H2O2 into H2O and O2, and GPx reduces hydrogen peroxide and other organic peroxides.53 We found that the highest levels of SOD activity occurred in animals with C6-implanted glioma tumors treated with OLEU but not with HTX. On the contrary, animals with ENU-induced gliomas showed decreased levels of SOD after the treatment with both OLEU and HTX, being slightly higher the effects of HTX (Fig. 49.5A). It has been described that SOD levels are decreased in brain tumors compared with normal controls.54,55 However, other authors56 have reported relatively higher SOD activity levels in human glioma cells compared with other tumor types, which seems to contradict the general observation of low SOD activity in tumor cells.56 This exception may be explained by the unique characteristics of the G

G

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FIGURE 49.5 Circulating superoxide dismutase (A), catalase (B), and glutathione peroxidase (C) specific activities in animals with C6 glioma implanted at the subcutaneous region and animals with transplacental ENU-induced tumors treated with vehicle (control), with OLEU and with HTX. Results are expressed in percentage versus control (mean 6 SEM; **P , 0.01; ***P , 0.001). ENU, N-Ethyl-N-nitrosourea; HTX, hydroxytyrosol; OLEU, oleuropein.

brain, which is a high oxygen-consuming organ. There is an increased production of superoxides during normal aerobic metabolism in brain cells. Thus relatively high levels of SOD and other antioxidant enzymes are required to remove high levels of free radicals to protect against damage to brain tissues. Although SOD plays an important role in protecting against oxidative damage, it is likely

that a balance of antioxidant enzymes is more important than their levels, which may influence intracellular oxidative states. In any case, most studies have indicated a significant reduction in SOD activity in several brain tumor types, such as gliomas, meningiomas, and metastatic tumors. A reduction in SOD activity with an increase in MDA levels in both the blood and tumor tissues of meningioma patients has been also reported.57 An increase in SOD activity levels, therefore, should play a protective role against tumor growth, but our results showed that the animals with C6-implanted glioma with larger tumors had high SOD levels, which also seemed to be promoted by OLEU but not by HTX. This could have been an indicator of the existence of high levels of superoxide radicals that promoted the development and proliferation of glioma tumors in this animal model. Regarding CAT activity, similar results were obtained. Thus OLEU increased CAT activity, whereas no changes were observed following the HTX treatment in animals with C6-implanted glioma. On the contrary, in animals with ENU-induced gliomas, both OLEU and HTX decreased circulating levels of CAT activity (Fig. 49.5B). Several authors have described increased CAT activity in brain tumors.5,55,58 Therefore the treatment with HTX could benefit an antitumor status through the inhibition of CAT activity, at least in animals with ENU-induced gliomas. In addition, the level of CAT activity could be a reflect of those of SOD activity. In this way the lower SOD activity observed with the treatments would promote a decrease in H2O2 production and a lower CAT activity may be necessary to catalyze the reaction from H2O2 to H2O and O2. Finally, and to complete the study of the redox state, we have found higher levels of GPx activity after the treatment with OLEU or HTX in animals with C6implanted gliomas, whereas no changes were detected after the treatment with both compounds in animals with ENU-induced gliomas (Fig. 49.5C). However, we have described a significant increase in GPx activity in brain tumor tissue of animals with ENU-induced gliomas, although this change was not reflected at circulating levels.4 This increase in GPx activity could compensate the effect of lower CAT activity in the removal of H2O2. Although CAT and GPx act removing H2O2, their behavior in animals with ENU-induced tumors is different. Antioxidant proteins with similar enzymatic activity may have different effects after modulation due to the different localizations within cells. Both GPx and CAT remove H2O2, but their contribution varies depending on the amount and the localization of H2O2 production.59 On the contrary, several authors have found diminished levels of GPx in brain tumors.54,60 Moreover, when the authors separated the cases according to their histopathological tumor type, GPx markedly decreased as the tumors

The effects of extra-virgin olive oil minority compounds hydroxytyrosol and oleuropein on glioma Chapter | 49

became more malignant.54 The low levels of antioxidants in brain tumors could be the result of this increased oxidative damage, or these low levels may have aggravated the free-radical damage and increased the chance of developing cancer, supporting the role of antioxidants in the prevention of cancer and the role of oxidative injury in its development. However, the putative role of free radicals in the destruction of cancer cells must also be considered as well as the importance of an adequate balance between prooxidants and antioxidants. Depending on the imbalance present, either protection against tumor growth or its promotion could be favored. Therefore it seems that the overall effects of the antioxidant defense system depend on the intracellular equilibrium between the several antioxidant enzymes rather than of a single component.49 The imbalance in the adequate expression and/or activity of antioxidant enzymes can promote the generation of oxidative stress.49,61 This evidence implied that the balance of SOD, CAT, and GPx is more important than the level of each one to prevent or to induce the tumor growth or its promotion. Therefore the ability to scavenge oxygen-free radicals seems to be impaired in the animals with tumors because of the reduced levels of antioxidant defenses, which may predispose them to cancer progression, whereas HTX, but not OLEU, is able to restore and/or enhancing those defenses in a certain degree and promoting the decrease of tumor growth.

49.2 Conclusion The treatment with HTX, but not with OLEU for both short and long periods and independently of the route of administration, led to the significant inhibition of tumor growth in our in vivo glioma models via redox-related mechanisms involving endogenous enzymatic and nonenzymatic antioxidant defense systems. However, the exact mechanisms involved could depend on the animal model used. In any case, the ability to ROS scavenging seems to be impaired in the animals with tumors because of the reduced levels of antioxidants, which may predispose them to cancer progression, and at least HTX, could not only to avoid this progression but also to inhibit it.

Mini-dictionary of terms Akt

Apoptosis

A group of enzymes involved in several processes related to cell growth and survival. Akt enzymes help to transfer signals inside cells. An Akt enzyme is a type of serine/threonine protein kinase and also called protein kinase B. A form of cell death in which a programmed sequence of events leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis plays a crucial role in developing and maintaining the health of

Cancer

Free radicals

Glioma

N-Ethyl-Nnitrosourea

NF-κB

Oxidative stress

Polyphenols

Reactive oxygen species

599

the body by eliminating old, unnecessary, and unhealthy cells. Term used to indicate malignant neoplasms, which usually are invasive, may metastasis, and recur after attempted removal. A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. The presence of an unpaired electron results in certain common properties that are shared by most radicals. Many radicals are unstable and highly reactive. A brain tumor that begins in a glial, or supportive, cell, in the brain or spinal cord. Malignant gliomas are the most common primary tumors of the central nervous system (the brain and spinal cord). A member of the class of N-nitrosoureas that is urea in which one of the nitrogens is substituted by ethyl and nitroso groups. The chemical is an alkylating agent and acts as a potent mutagen by transferring the ethyl group of ENU to nucleobases (usually thymine) in nucleic acids. Nuclear factor kappa-light-chain-enhancer of activated B cells. It is a protein complex that controls transcription of DNA, cytokine production, and cell survival. A condition of increased oxidant production in cells characterized by the release of free radicals and resulting in cellular degeneration. Compounds found in many plant foods that can be grouped into flavonoids, phenolic acid, polyphenolic amides, and other polyphenols. They act as antioxidants and protect cells and body chemicals against damage caused by free radicals that contribute to tissue damage in the body. A type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell. A buildup of reactive oxygen species in cells may cause damage to DNA, RNA, and proteins and may cause cell death. Reactive oxygen species are free radicals.

Implications for human health and disease prevention Most antineoplastic drugs are not selective against tumor cells but also affect normal cells, leading to a wide variety of adverse events in several tissues. These adverse events are due to the mechanism of action of these drugs. Numerous studies, including ours, conducted with hydroxytyrosol (HTX), have demonstrated its antioxidant and antiinflammatory effects, as well as its role in the prevention and modulation of cancer, including glioma. Many of these antitumor properties of HTX may be due to its

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ability to modulate the antioxidant defense systems. Apart from its possible role as an antioxidant food supplement, and despite the precautions to be taken because of the importance of maintaining redox balances in the body, the use of chemotherapy treatments supplemented with HTX should be explored to select the best combination of doses and routes of administration for improving the prognosis of patients with glioma.

Acknowledgments This study was supported by Junta de Andalucı´a (PAIDI group CTS1039), Consejerı´a de Innovacio´n, Ciencia y Empresa through Proyecto de Excelencia Motriz (grant CVI2009
4957M), and Universidad de Jae´n through Plan Propio de Apoyo a la Investigacio´n.

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6526. de la Puerta R, Martinez Dominguez ME, Ruiz-Gutierrez V, et al. Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission. Life Sci. 2001;69(10):1213
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9313.

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Section 3.2

Oleuropein

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Chapter 50

The usage of oleuropein on myocardium Maria Tsoumani1, Ioulia Tseti2 and Ioanna Andreadou1 1

Laboratoty of Pharmacology, School of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece, 2Uni-Pharma S.A., Athens,

Greece

Abbreviations AMPK ATP Bax Bcl-2 cl-CASP3 CHOP CK CMs CVDs DXR EAM EF ER ERK1/2 GRP78 GSH H2O2 IHD IL-1β IRIs LC3-II LDH LVDP LVEDP MAO-A MDA MI mTOR NF-κB NO p-c-JUN p-HSP27 PIKKs pMAPKAPK2 p-SAPK/ JNK RISK ROS

adenosine monophosphate
dependent protein kinase adenosine triphosphate BCL2-associated X protein B-cell lymphoma 2 cleaved caspase-3 C/EBP homologous protein creatine kinase cardiomyocytes cardiovascular diseases doxorubicin experimental autoimmune myocarditis ejection fraction endoplasmic reticulum extracellular signal
regulated kinase 1/2 glucose-regulated protein 78 glutathione hydrogen peroxide ischemic heart disease interleukin-1β ischemia
reperfusion injuries microtubule-associated protein 1a/1b-light chain 3 lactate dehydrogenase left ventricular developed pressure left ventricular end-diastolic pressure monoamine oxidase-A malondialdehyde myocardial infarction mammalian target of rapamycin nuclear factor kappa-B nitric oxide phosphorylated c-Jun phosphorylated heat shock protein 27 phosphatidylinositol-3 kinase
related kinases phosphorylated mitogen
activated protein kinase
activated protein kinase 2 phosphorylated stress-activated protein kinase reperfusion injury salvage kinase reactive oxygen species

SI/R SOD STAT3 STEMI MI, TBARS TFEB TNF-α ULK1 4-HNE

simulated ischemia/reperfusion superoxide dismutase signal transducer and activator of transcription 3 ST segment elevation thiobarbituric acid reactive substances transcriptional factor EB tumor necrosis factor-α Unc-51-like autophagy activating kinase 1 4-hydroxynonenal, 8-iso-PGF2α; 8-iso-prostaglandin F2α

50.1 Introduction Cardiovascular diseases (CVDs), especially those related to ischemic heart disease (IHD), namely, acute myocardial infarction (MI) (ST segment elevation MI 5 STEMI, and non-STEMI 5 NSTEMI), stable angina, and ischemic cardiomyopathy still remain the main cause for mortality worldwide.1 MI can be defined as a lack of coronary blood flow with dramatic consequences for the myocardium.2 Dietary factors are considered to be important contributors to cardiovascular risk, either directly, or most commonly, through their effects on cardiovascular risk factors such as dyslipidemia, hypertension, and diabetes mellitus. Natural nutritional compounds termed as “nutraceuticals” can play crucial role as preventive medicines or as treatments of CVD. Several classes of nutraceuticals have been proposed to have potential benefits in the treatment of CVD.3 Among them the usage of oleuropein on myocardium has been the subject of research for many years. Oleuropein possesses many biological activities such as antioxidant, vasodilatatory, antithrombotic, antiatherogenic, and antiinflammatory and show beneficial effect on several aspects of CVD.4,5 Evidence from experimental studies make oleuropein as an ideal compound for preventing damages caused by ischemia
reperfusion injuries (IRIs). In the present chapter, we provide an update regarding the effects of oleuropein on the myocardium in order to

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00016-X © 2021 Elsevier Inc. All rights reserved.

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gain a more complete understanding of the role of this compound as a cardioprotective agent against CVDs.

50.2 The effect of oleuropein on cardiomyocytes The adult mammalian heart is composed of many cell types, the most abundant (B70%
85%) are cardiomyocytes (CMs).6 Although nonmyocytes cells being fibroblasts, endothelial cells, and perivascular cells occupy a relatively small volume fraction, they are essential for normal heart homeostasis, providing the extracellular matrix, intercellular communication, and vascular supply needed for efficient CM contraction and long-term survival.6,7 It has been investigated if oleuropein can protect H9c2 CMs in vitro against 4-hydroxynonenal (4-HNE) stressinduced toxicity and apoptosis.8 HNE inhibited viability and accelerated apoptosis in a concentration-dependent manner, whereas oleuropein, similar to hydroxytyrosol and quercetin, exerted significant antiapoptotic effects against HNE-induced toxicity in H9c2 cells. The protective mechanisms of action were attributed to a decrease of the induction of the apoptotic stress markers [phosphorylated mitogen
activated protein kinase
activated protein kinase 2 (p-MAPKAPK-2), phosphorylated stressactivated protein kinase (p-SAPK/JNK), phosphorylated heat shock protein 27 (p-HSP27), phosphorylated c-Jun (p-c-JUN), and cleaved caspase-3 (cl-CASP3)]. In addition, the protective effect of oleuropein was attributed to its antioxidant action measured by the reduction of reactive oxygen species (ROS) production and to amelioration of the mitochondria membrane potential.8 Another study from Zhao et al. was designed in order to clarify the cardioprotective effect of oleuropein against simulated ischemia/reperfusion (SI/R)-induced CM injury in vitro and the underlying mechanisms related to antiapoptotic properties. The cardioprotective effect of oleuropein in neonatal rat CMs was confirmed in vitro using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTT dye was reduced after neonatal rat CMs were subjected to SI/R injury, whereas treatment with oleuropein (100, 200, and 400 μg/mL) significantly increased the MTT dye reduction in a dosedependent manner. Meanwhile, the compound also reduced in a dose-dependent manner lactate dehydrogenase (LDH) activity in the culture medium. Oleuropein treatment markedly decreased apoptosis rate in a dosedependent manner and stabilized mitochondrial membrane potential after SI/R. Furthermore, oleuropein attenuated the expression of cytochrome c, c-caspase-3, and c-caspase-9; increased the B-cell lymphoma 2 (Bcl-2)/BCL2associated X protein (Bax) ratio; and enhanced the

phosphorylation of extracellular signal
regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) after SI/R providing that the antiapoptotic effect of oleuropein was mediated by the reperfusion injury salvage kinase (RISK) pathway. However, the phosphorylation enhancement was partially abolished in the presence of LY294002 [phosphoinositide-3-kinase (PI3K) inhibitor)] and U0126 (ERK1/2 inhibitor). All these findings indicate that oleuropein is protective against SI/R-induced injury, and its effect may be partly due to the attenuation of apoptosis via the activation of the PI3K/Akt and ERK1/2 signaling pathways.9 Recently, Miceli et al.10 studied the effect of oleuropein as an autophagy enhancer in CMs with overexpression of monoamine oxidase-A (MAO-A), an enzyme that its expression and activity are increased in cardiac diseases.11 Overexpression of MAO-A by degrading catecholamine and serotonin in the heart produces ammonia and H2O2, which causes oxidative stress, autophagic flux blockade, and cell necrosis. Oleuropein can counteract the cytotoxic effects of MAO-A through autophagy activation as reflected by the increase of autophagic vacuoles and autophagy-specific markers [Beclin1 and microtubuleassociated protein 1a/1b-light chain 3 (LC3-II)]. Also, the cardioprotective effect of oleuropein is exerted through restoration of defective autophagic flux. In this mechanism, nuclear translocation and activation of the transcriptional factor EB (TFEB) is crucial since the transcriptional regulation of autophagy under the effect of oleuropein was correlated with a significant cell death decrease, and to mitochondrial functionality recovery. These improvements disappeared after TFEB silencing, leading to the hypothesis that TFEB activation was crucial for the protective effects of oleuropein against MAO-Ainduced autophagy dysfunction. In addition, TFEB translocation and autophagy recovery induced by oleuropein did not affect ROS status in CMs, further highlighting its peculiarity as an autophagy inducer.10 The protective effects of oleuropein against the acrolein-inducing CM H9c2 cell apoptosis through endoplasmic reticulum (ER) stress and Bcl2/Bax pathways have been investigated.12 ER stress is linked to the pathogenesis of CVDs, including MI, hypertrophy, and heart failure. It is assumed that acrolein and related aldehyde consumption increased the myocardial sensitivity to ischemia. The results of the study demonstrated that the two major components of ER stress, glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), as well as Bax expression were upregulated, while Bcl2 expression was downregulated both at the protein and mRNA levels, when the H9c2 cells were treated with acrolein for 12 h. The altered expression of GRP78, CHOP, Bcl2, and Bax was alleviated by oleuropein. However, the mRNA expression of GRP78 and CHOP

The usage of oleuropein on myocardium Chapter | 50

was not reversed by oleuropein, a finding that requires further investigation. Conclusively, these data support that oleuropein, along with hydroxytyrosol, inhibits the cytotoxicity of acrolein in CM H9c2 cells through ER stress and Bcl2/Bax pathways.12 In summary, the majority of the studies have shown that oleuropein increases CMs’ viability against various conditions that cause cell toxicity, indicating a direct action on CMs. The mechanisms responsible for the oleuropein’s protective effect include modulation of the apoptotic process by decreasing apoptotic signaling proteins, stabilization of mitochondrial membrane potential, and autophagy activation. However, investigation of the effects of oleuropein on hypoxia/reoxygenation in other cell types beyond CMs has not yet been performed.

50.3 Oleuropein’s cardioprotective effect against myocardial ischemia
reperfusion injury It is well known that reperfusion, the restoration of blood flow in the ischemic myocardium, is the most effective strategy to treat the ischemic injury. However, the paradox is that the reperfusion itself can deteriorate the ischemic damage and cause irreversible injury. Myocardial tissue damage due to IRI primarily depends on the extent of blood flow reduction. Overproduction of ROS, intracellular calcium overload, inflammatory cell infiltration, and energy metabolism disorders are suggested to be the most important features of IRI. The manifestations of reperfusion injury include myocardial necrosis, incidence of lifethreatening arrhythmias, myocardial stunning, and endothelial dysfunction, in addition to significant CM death.13 Administration of antioxidants during reperfusion may reduce the severity of IRI. Oleuropein acts as antioxidant molecule, providing one alternative aspect among the therapeutic approaches to overcoming the oxidative stress associated with IHD. The antioxidant characteristics of oleuropein are attributed to its structure. It contains an ortho-diphenolic group that provides the ability to scavenge ROS through hydrogen donation and to stabilize oxygen radicals through the formation of an intramolecular hydrogen bond between the free hydrogen of the hydroxyl group and its phenoxyl radicals. Particularly, an o-diOH substitution confers a high antioxidant property, whereas single hydroxyl substitutions, for example, tyrosol, provide none.4 The studies conducted in the last decade were based on the previous numerous studies that have provided evidence for antioxidant properties for oleuropein. These data concern a scavenging effect of oleuropein against hypochlorous acid and NO, similar to those exerted by common antioxidants such as ascorbic acid (vitamin C)

607

and α-tocopherol (vitamin E)14,15 and also the effect of oleuropein on lipid oxidation. Oleuropein has a protective effect in counteracting lipid peroxidation such as oxidized low-density lipoprotein, malondialdehyde (MDA), and 4HNE. In addition, oleuropein supplementation in healthy volunteers decreased in a dose-dependent manner, the urinary excretion of 8-iso-prostaglandin F2α (8-iso-PGF2α), indicating inhibition of in vivo lipid peroxidation.16,17 Studies in isolated hearts (ex vivo studies) and in vivo studies have been conducted in order to investigate the protective effect of oleuropein against myocardial IRI and the underlying molecular mechanisms (Table 50.1).

50.3.1 The effect of oleuropein in ex vivo models In isolated rat hearts pretreated with oleuropein at a dose of 50 μmol/L (corresponding to 20 μg/g of wet weight) for 15 min and then subjected to no-flow global ischemia and reperfusion, creatine kinase (CK), glutathione (GSH) release, and oxidized GSH were reduced in the perfusate. The antioxidant effect of oleuropein was attributed by stimulation of the expression of intracellular antioxidant enzymes and by increasing the level of nonenzymatic antioxidants such as GSH, α-tocopherol, β-carotene, and ascorbic acid.18 Also, lipid peroxidation assessed by measuring the thiobarbituric acid reactive substances (TBARS) was evaluated, and it was observed that oleuropein can be completely protective since no significant increase in TBARS was determined in the oleuropeintreated hearts compared to the sham ones.18 Esmailidehaj et al. investigated whether perfusion of isolated rat hearts with 10 and 50 μg/g heart oleuropein 5 min before the induction of ischemia or at the beginning of the reperfusion had any effect on the hemodynamic parameters, namely, heart rate, left ventricular (LV) enddiastolic pressure (LVEDP), LV developed pressure (LVDP), rate of rise of LV pressure ( 6 dp/dt) and coronary outflow, infarct size and biochemical factors following IRI. All cardiac hemodynamic parameters significantly improved in all groups that received oleuropein compared to the control groups independently of the time of administration. Also, the infarct size was smaller and the coronary outflow levels of CK and MDA were lower in the oleuropein treated groups in comparison with the control groups.19 The same group also explored the effect of the pretreatment of rats with a single dose of intraperitoneal oleuropein (100 mg/kg) 1, 3, 6, 12, 24, and 48 h before their hearts were subjected to 30 min ischemia and 120 min reperfusion under Langendorff apparatus. They observed that oleuropein exerts cardioprotection by reducing infarct size and reperfusion-induced cardiac dysfunction, ischemic, and reperfusion arrhythmias for at least 3 h. Importantly, they have pointed out that the

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TABLE 50.1 Effects and mechanisms involved in the cardioprotective effect of oleuropein in myocardial ischemia/ reperfusion injury. Experimental model

Oleuropein (dose, route, and time of administration)

Effect

Mechanism

Ref.

Isolated rat hearts: global ischemia/reperfusion (30/ 60 min)

50 μmol/L (20 μg/g) i.p., before induction of ischemia

Cardiac markers: decrease in CK release

Oxidative stress: decrease in GSH release Lipid peroxidation: decrease in TBARS

[18]

Isolated rat hearts: global ischemia/reperfusion (30/ 90 min)

10 μg/g and 50 μg/g i.p., (1) before induction of ischemia or (2) for 5 min at the beginning of the reperfusion

Cardiac hemodynamic parameters: no difference in heart rate, decrease in LVEDP, increase in LVDP, coronary outflow, and contractility index Infarct size: decrease

Lipid peroxidation: decrease in MDA

[19]

Isolated rat hearts: regional ischemia/ reperfusion (30/120 min)

100 mg/kg (20 μg/g) i.p., 1, 3, 6, 12, 24, and 48 h before induction of ischemia

Cardiac hemodynamic parameters: decrease in LVEDP, increase in LVESP and LVDP in 1- and 3-h treatment groups Ischemic-induced arrhythmias: decrease in VT and VF in 1- and 3-h treatment groups Infarct size: decrease

Not described

[20]

Reperfusion-induced arrhythmias in rats: regional ischemia/ reperfusion of LAD (5/ 15 min)

10 or 50 mg/kg, i.v., (1) 2 min before ischemia (prophylaxis) (2) 2 min before reperfusion (treatment)

Heart rate: no change Mean arterial blood pressure: decrease at 50 mg/kg

Not described

[22]

I/R in vivo rat model: global ischemia/ reperfusion (30/180 min)

20 mg/kg, i.p. for 2 consecutive days

Cardiac markers: decrease in CK-MB, LDH Infarct size: decrease

Inhibition of caspase-3 activity Decrease in p53, p-MEK, and p-ERK protein expression Decrease in pIκBα protein expression Increase in pSTAT3 protein expression Oxidative stress: increased levels of SOD and GSH Lipid peroxidation: decrease in MDA concentration Inflammation: decrease of TNF-α, IL-1β, and IL-6

[24]

CK, Creatine kinase; CK-MB, creatine kinase myocardial band; GSH, glutathione; IL-1β, interleukin 1β; IL-6, interleukin 6; LAD, left anterior descending coronary artery; LDH, lactate dehydrogenase; LVDP, left ventricular diastolic pressure; LVEDP, left ventricular end-diastolic pressure; LVESP, left ventricular end systolic pressure; MDA, malondialdehyde; p-ERK, phosphorylated extracellular-signal-regulated kinase; p-IκBα, phosphorylated inhibitor of nuclear factor kappa B; p-MEK, phosphorylated mitogen
activated protein kinase/ERK kinase; p-STAT3, phosphorylated signal transducer and activator of transcription 3; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TNF-α, tumor necrosis factor-α; VF, ventricular fibrillation; VT, ventricular tachycardia.

The usage of oleuropein on myocardium Chapter | 50

cardioprotective effect of oleuropein stops 6 h later.20 This study raises a question that until now it has not been answered, how long do the cardioprotective effects of oleuropein last for.

50.3.2 The effect of oleuropein in in vivo models The first publication regarding the in vivo cardioprotective effects of oleuropein against IRI was released by our group in 2006.21 Recent studies have investigated the antiarrhythmic effects of oleuropein when was administered intravenously (10 or 50 mg/kg) in rats prior to coronary artery ligation (as prophylaxis) or before and during reperfusion (as treatment). Oleuropein as a prophylaxis had no effect on heart rate compared to the control group, but it significantly reduced the mean arterial blood pressure at the dose of 50 mg/kg. Similarly, oleuropein administration during reperfusion significantly reduced blood pressure at the dose of 50 mg/kg indicating hypotensive effect. The antiarrhythmic effect of oleuropein against reperfusioninduced arrhythmia was evident with delayed initiation of arrhythmias and decreased incidence of irreversible fibrillation (or mortality).22 The sudden reperfusion of acutely ischemic myocardium in STEMI patients undergoing percutaneous coronary intervention may be accompanied by ventricular arrhythmias such as ventricular fibrillation.23 Thus the effect of oleuropein protecting heart against reperfusion-induced arrhythmias is of high importance. Jin et al. in an in vivo study reported that oleuropein administered in rats at the dose of 20 mg/kg for 2 consecutive days significantly inhibited the extent of MI size and reduced serum levels of CK-MB and LDH compared with the controls.24 Regarding the molecular mechanism of the cardioprotective properties of oleuropein, this study focused on key mediators of reperfusion injury such as apoptosis, inflammatory response, and oxidative stress.24 Apoptosis has been shown to play an important role in determining infarct size, extent of LV remodeling, and development of early symptomatic heart failure after acute MI.25 Thus the inhibition of myocardial apoptosis may be implicated in cardiac function recovery following acute MI.24 Oleuropein administration for 2 consecutive days significantly inhibited caspase-3 activity, p53, pMEK, and p-ERK protein expression compared with the control group.24 Importantly, there are data suggesting that the cardioprotection of oleuropein is not diminished in animals with comorbidities such as hypercholesterolemia, hypertension, and diabetes. Our group has shown that oleuropein reduces myocardial infarct size in hypercholesterolemic rabbits.21 Nekooeian et al., indicated that 4-week administration of oleuropein in rats with simultaneous type 2 diabetes and renal hypertension demonstrates antihypertensive, antidiabetic, and cardioprotective effects characterized by decreased blood

609

pressure and fasting blood glucose, improved contractility, and decreased cardiac damages. It was assumed that such beneficial effects might be related to oleuropein’s antioxidant activity characterized by increased erythrocyte levels of superoxide dismutase (SOD), which may partly explain oleuropein’s antihypertensive activity, as well.26

50.3.3 Oleuropein as a conditioning mimetic in order to protect the damaged myocardium Myocardial ischemia and the subsequent reperfusion injury can be mitigated after implementation of brief episodes of myocardial ischemia and reperfusion. These episodes applied directly to the myocardium by occlusion/ reperfusion of the coronary artery or by implementation at a remote noncardiac site can salvage the myocardium from prolonged ischemia/reperfusion by activating molecular defense mechanisms (known as conditioning mechanisms).27 The underlying mechanisms of preconditioning involve the activation of PI3K/Akt, ERK1/2 axis, generation of low, sublethal levels of ROS, and opening of mitochondrial KATP channels, whereas in reperfusion further phosphorylation of PI3K and Akt kinases, and inhibition of the mitochondrial permeability transition pore opening take place.28 Pharmacological preconditioning represents an ideal alternative to ischemic preconditioning, and recently, nutritional preconditioning is considered a form of pharmacological preconditioning mediated by the intake of nutraceuticals through diet.29 Esmailidehaj et al. investigated if oleuropein exerts preconditioning-like effects. In this study, oleuropein was administered as a single dose (100 mg/kg) before ischemia to induce preconditioning in isolated rat hearts. The results of the study proved that oleuropein confers cardioprotection by limiting the infarct size, but it has no preconditioning effects in rat hearts.20 Our group compared the effects of oleuropein administered at nutritional dose in anesthetized rabbits and preconditioning in terms of the cell signaling and myocardial metabolic profile changes.30 The results of our study indicated that oleuropein exerts cardioprotection in vivo through activation of intracellular signaling, which are also involved in the preconditioning mechanism. Specifically, oleuropein treatment in both normal- and cholesterol-fed animals resulted in significant activation of the PI3 and Akt kinases, phosphorylation of eNOS, as well as in activation of signal transducer and activator of transcription 3 (STAT3) to the same degree as preconditioning. In addition, oleuropein protected through inhibition of the oxidative stress during reperfusion, reduced the circulating cholesterol and LDL levels in cholesterolfed animals and increased myocardial adenosine triphosphate (ATP) content and total adenine nucleotides.30

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50.4 Molecular understanding of the protective role of oleuropein Although studies have investigated the cardioprotective effect of oleuropein against IRI, these studies have mostly investigated its antioxidant, antiapoptotic, and antiinflammatory activities as mechanisms of its cardioprotective action. The molecular understanding of the cardioprotective effect of oleuropein will suggest the direction for future, more intensive, research in order to use oleuropein as treatment for MI. One of the modern therapeutic approaches for CVDs is the mechanistic target of proteins.31 Mammalian target of rapamycin (mTOR) is a serine/threonine protein that belongs to the family of phosphatidylinositol-3 kinase
related kinases (PIKKs) that forms the catalytic subunit of two distinct protein complexes, known as mTOR complex 1 (mTORC1) and 2 (mTORC2). The physiological role of mTOR involves many cellular processes, such as cell growth, metabolism, proliferation, survival, transcription, translation, apoptosis, motility, and autophagy. It is a general consensus that mTOR exerts protective effects modulating apoptosis and autophagy processes as well as favoring tissue repair under oxidative stress. Inhibition of mTOR has beneficial effects on CVD (effect against atherosclerosis, cardiac hypertrophy, and heart failure), and also in extending the life span.32 Many studies have reported that oleuropein activates the phosphorylation of adenosine monophosphate
dependent protein kinase (AMPK) and antagonizes mTORC1 at the functional level. The investigation of the role of oleuropein in chronic doxorubicin (DXR)-induced cardiomyopathy in a rat model revealed that the cardioprotection of oleuropein against the histopathological, structural, functional, and cardiac alterations induced by subchronic DXR exposure is attributed in a process mediated by activation of AMPK and suppression of inducible nitric oxide synthase.33 The crucial role of AMPK was confirmed in a subsequent publication of our group investigating the effects of oleuropein in rabbits subjected to myocardial ischemia followed by reperfusion. Indeed, the results evidenced that the mechanism of action of oleuropein was related also to the activation of AMPK.30 In a recent study, oleuropein reduced H2O2-induced mesenchymal stem cells autophagy by modulating autophagy-related death signals, including mTOR, unc51-like autophagy activating kinase 1 (ULK1), Beclin-1, AMPK, and LC3. This study provides evidence that the short-term cell priming with oleuropein might enhance the therapeutic effect of mesenchymal stem cells against ischemic vascular diseases, providing an important potential improvement for emerging therapeutic strategies.34 In a similar study evaluating the antioxidant effect of oleuropein on c2c12 myoblast cells, it was explored that

pretreatment with oleuropein at 200 and 400 μM led to a significant decrease in ROS levels by 15.7% and 35.8%, respectively, in comparison with the elevated levels of ROS after treatment with H2O2. The results of this study demonstrated that oleuropein activates AMPK in a dosedependent manner but has no stimulatory effect on PI3/ Akt pathway.35 Conclusively, signaling analyses in cultured cells and in vivo studies provide strong evidence for the important role of the AMPK/mTOR axis providing further knowledge to the molecular basis of oleuropein’s cardioprotection.

50.5 The role of oleuropein in other cardiovascular disorders Ischemic cardiomyopathy is the most common cause of heart failure and can arise from remodeling after a STEMI from multiple small nontransmural infarctions or chronic repetitive ischemia in the absence of infarction.36 Janahmadi et al. examined the effects of oleuropein pretreatment in rats with MI by determining echocardiographic and hemodynamic parameters, serum concentrations of oxidative stress, and inflammatory markers.37 The authors found that oleuropein (10, 20, or 30 mg/kg) provides cardioprotection in rats characterized by impaired cardiac function [reduced LV developed and systolic pressures, 6 dp/dt, stroke volume, ejection fraction (EF), and cardiac output] caused by ligation of left main coronary artery. The ligation of coronary artery was associated with increased level of serum MDA and decreased levels of serum SOD and GSH reductase. Pretreatment with oleuropein for 7 days prevented the reduction of the abovementioned antioxidant biomarkers. Also, oleuropein reduced serum concentration of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) indicating that pretreatment prevents cardiac impairment partly by reducing oxidative stress and release of proinflammatory cytokines.37 The same group examined the effects of oleuropein on the progression of heart failure induced by permanent ligation of the coronary artery in rats.38 Oleuropein administered at 10 or 20 mg/kg/day for 5 weeks after permanent ligation attenuated the progression of heart failure by mitigating the decrease of systolic blood pressure, LVSP, 1 dp/dt, and 2 dp/dt and the increase of LVEDP and infarct size in coronary artery
ligated rats.38 These findings proved that oleuropein had cardioprotective effects in these models. Such a conclusion received support from previous studies of our group demonstrating that oleuropein was cardioprotective against acute and chronic DXR-induced cardiotomyopathies.33,39 DXR, an anthracycline, which is used in chemotherapy is cardiotoxic and may lead to dose-dependent cardiomyopathy

The usage of oleuropein on myocardium Chapter | 50

and heart failure.40 In a subchronic setting, oleuropein administration (1000 and 2000 mg/kg, i.p.) prevented DXR-induced cardiomyopathy by preserving LV contractility. DXR treatment groups had significantly lower fractional shortening compared to controls and lower LV wall thickness. Importantly, oleuropein prevented this cardiotoxic insult.33 Last, the effects of oleuropein on experimental autoimmune myocarditis (EAM) were evaluated.41 Myocarditis is a heterogeneous group of cardiovascular disorders defined by inflammation of the heart muscle. The primary clinical manifestations of myocarditis are heart failure and sudden death in children and young adults. Myocarditis is usually caused by several factors, such as viral and bacterial infections, drug toxicity, and autoimmune reactions.42 EAMs in rats were induced by subcutaneous injections of porcine cardiac myosin. Cardiac function parameters, myocardial pathology, myocardial inflammatory cell infiltration, and nuclear factor kappa-B (NF-κB) expression were measured. Data showed that oleuropein treatment improved cardiac functions in EAM rats, and the LVSP, LVDP, rate of decrease of LV pressure (2dp/dt) were significantly reduced, whereas 1 dp/dt and EF were significantly increased. Histological analysis revealed extensive myocardial injuries with inflammatory cell infiltration, a finding that was significantly improved by oleuropein therapy. Moreover, oleuropein decreased the serum production of TNF-α, IL1β, and IL-6 and inhibited the NF-κB signaling pathway in myocardial tissue, suggesting that oleuropein effectively prevents the development of myocarditis, at least in part, by inhibiting the NF-κB-mediated inflammatory responses.41

clinical research should be conducted in order to support the intake of oleuropein in patients.

Mini-dictionary of terms Adenosine monophosphate
dependent protein kinase

Apoptosis Autophagy

Conditioning mechanisms

Heart failure

Ischemia
reperfusion injury

Myocardial infarction

50.6 Conclusion The present chapter discussed the potential therapeutic role of oleuropein against IRI. Oleuropein increases CMs viability and considerably reduces the infarct size in experimental models ex vivo and in vivo. Furthermore, it successfully provides protection in other aspects of CVD such as heart failure and myocarditis. However, we must point out that until now, there are no studies that have investigated the cardioprotective effects of oleuropein when it is administered during ischemia and at reperfusion in order to define its potential as a therapeutic cardioprotective agent. Studies conducted in the last decade have given us greater insight into mechanistic details regarding the cardioprotective effect of oleuropein. However, in order to develop novel therapeutic strategies based on oleuropein, dose and time of administration (i.e., before or during ischemia) should be standardized as well as mechanism of action should be clarified. Further

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Nutraceuticals Nutritional preconditioning

Reperfusion injury salvage kinase (RISK) pathway

Highly conserved intracellular kinase that regulates mitochondrial biogenesis, increases oxidative mitochondrial metabolism, decreases apoptosis through inhibition of mTOR signaling, and increases autophagy. Caspase dependent, genetically controlled form of cell death. Intracellular lysosomal
mediated catabolic process in which senescent or damaged proteins and organelles are degraded by lysosomes. Endogenous cardioprotective strategies by application of short cycles of ischemia and reperfusion either before or after the sustained ischemic insult. A chronic, progressive condition in which the heart muscle is unable to pump enough blood to meet the body’s needs for blood and oxygen. Tissue ischemia with inadequate oxygen supply followed by successful reperfusion initiates a wide and complex array of biochemical responses that may aggravate local injury as well as induce impairment of remote organ function. Inadequate blood supply to the heart, causing death to a portion of the heart muscle. Medicinally or nutritionally functional foods. Regular intake of natural nutrients as part of daily diet that offers cardioprotection. Group of prosurvival protein kinases (including Akt and Erk1/2), which confer powerful cardioprotection, when activated specifically at the time of myocardial reperfusion.

Comparisons of olive oils with other edible oils Dietary supplementation with edible oils, namely, olive oil, sunflower seed oil, fish oil, pulp oil, and palm oil may play a vital role in reducing the mortality rate due to heart disease.43 This chapter is devoted to the

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

cardioprotective effects of oleuropein found in olive oil. Red palm oil has shown beneficial effects on cardiac recovery from IRI.43 The tocotrienols such α-tocotrienol and γ-tocotrienol present in palm oil have been shown to offer cardioprotection due to their antioxidant properties. They decrease infarct size by increasing the phosphorylation of Akt protein. Seabuckthorn pulp oil obtained from the fruits of seabuckthorn [Hippophae rhamnoides L. (Elaeagnaceae)] attenuates IRI by stabilizing hemodynamic parameters, increasing antioxidant defense system, decreasing inflammatory markers, and upregulating the antiapoptotic proteins that were mediated by augmentation of Akt/eNOS signaling pathway. Also, it increased NO availability and activity.44 To our knowledge, there are no studies regarding the effects of sunflower oil and fish oil on IRI.

Implications for human health and disease prevention Currently, we have strong evidence that persons at high risk for cardiovascular events assigned to a Mediterranean diet, supplemented with extra-virgin olive oil or nuts, had a lower rate of major cardiovascular events than those assigned to a reduced-fat diet.45 Preclinical studies suggest that oleuropein has beneficial effects in the prevention and progression of CVDs; however, the levels of oleuropein are relatively low in virgin olive oil.46 Therefore clinical studies in cardiovascular patients should be conducted using virgin olive oils or nutraceuticals with defined concentration of oleuropein.

References 1. Townsend N, Wilson L, Bhatnagar P, et al. Cardiovascular disease in Europe : epidemiological update 2016. Eur Heart J. 2016;37: 3232
3245. 2. Heusch G. Myocardial ischemia: lack of coronary blood flow, myocardial oxygen supply-demand imbalance, or what? Am J Physiol Hear Circ Physiol. 2019;316(6):H1439
H1446. 3. Sosnowska B, Penson P, Banach M. The role of nutraceuticals in the prevention of cardiovascular disease. Cardiovasc Diagn Ther. 2017;7(suppl 1):S21
S31. 4. Nediani C, Ruzzolini J, Romani A, et al. Oleuropein, a bioactive compound from Olea europaea L., as a potential preventive and therapeutic agent in non-communicable diseases. Antioxidants. 2019;8(12):578. 5. Efentakis P, Iliodromitis EK, Mikros E, et al. Effects of the olive tree leaf constituents on myocardial oxidative damage and atherosclerosis. Planta Med. 2015;81:648
654. 6. Zhou P, Pu WT. Recounting cardiac cellular composition. Circ Res. 2016;118(3):368
370. 7. Andreadou I, Cabrera-Fuentes HA, Devaux Y, et al. Immune cells as targets for cardioprotection: new players and novel therapeutic opportunities. Cardiovasc Res. 2019;115(7):1117
1130.

8. Bali EB, Ergin V, Rackova L, et al. Olive leaf extracts protect cardiomyocytes against 4-hydroxynonenal-induced toxicity in vitro: comparison with oleuropein, hydroxytyrosol, and quercetin. Planta Med. 2014;80(12):984
992. 9. Zhao Q, Bai Y, Li C, et al. Oleuropein protects cardiomyocyte against apoptosis via activating the reperfusion injury salvage kinase pathway in vitro. Evid-Based Complement Altern Med. 2017;2017. 10. Miceli C, Santin Y, Manzella N, et al. Oleuropein aglycone protects against MAO-a-induced autophagy impairment and cardiomyocyte death through activation of TFEB. Oxid Med Cell Longev. 2018;2018. 11. Kaludercic N, Mialet-Perez J, Paolocci N, et al. Monoamine oxidases as sources of oxidants in the heart Nina. J Mol Cell Cardiol. 2014;0:34
42. 12. Xu Y, Wu L, Chen A, et al. Protective effects of olive leaf extract on acrolein-exacerbated myocardial infarction via an endoplasmic reticulum stress pathway. Int J Mol Sci. 2018;19(2):1
15. 13. Kalogeris T, Baines CP, Krenz M, et al. Cell biology of ischemia/ reperfusion injury. 1st ed. International Review of Cell and Molecular Biology. Vol. 298. Elsevier Inc.; 2012:229
317. 14. Visioli F, Bellomo G, Galli C. Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun. 1998;247 (1):60
64. 15. De La Puerta R, Domı´nguez MEM, Ruı´z-Gutı´errez V, et al. Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission. Life Sci. 2001;69(10): 1213
1222. 16. Visioli F, Bellomo G, Montedoro GF, et al. Low density lipoprotein oxidation is inhibited in vitro by olive oil constituents. Atherosclerosis. 1995;117(1):25
32. 17. Visioli F, Caruso D, Galli C, et al. Olive oils rich in natural catecholic phenols decrease isoprostane excretion in humans. Biochem Biophys Res Commun. 2000;278(3):797
799. 18. Manna C, Migliardi V, Golino P, et al. Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion. J Nutr Biochem. 2004;15(8):461
466. 19. Esmailidehaj M, Bajoovand S, Rezvani ME, et al. Effect of oleuropein on myocardial dysfunction and oxidative stress induced by ischemic-reperfusion injury in isolated rat heart. J Ayurveda Integr Med. 2016;7(4):224
230. 20. Esmailidehaj M, Rasulian B, Rezvani ME, et al. The anti-infarct, antistunning and antiarrhythmic effects of oleuropein in isolated rat heart. EXCLI J. 2012;11:150
162. 21. Andreadou I, Iliodromitis EK, Mikros E, et al. The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr. 2006;136:2213
2219. 22. Baharvand B, Esmailidehaj M, Alihosaini J, et al. Prophylactic and therapeutic effects of oleuropein on reperfusion-induced arrhythmia in anesthetized rat. Iran Biomed J. 2016;20(1):41
48. 23. Sattler SM, Skibsbye L, Linz D, et al. Ventricular arrhythmias in first acute myocardial infarction: epidemiology, mechanisms, and interventions in large animal models. Front Cardiovasc Med. 2019;6:158. 24. Jin HX, Zhang YH, Guo RN, et al. Inhibition of MEK/ERK/ STAT3 signaling in oleuropein treatment inhibits myocardial ischemia/reperfusion. Int J Mol Med. 2018;42(2):1034
1043. 25. Teringova E, Tousek P. Apoptosis in ischemic heart disease. J Transl Med. 2017;15(1):1
7.

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26. Nekooeian A, Khalili A, Khosravi M. Oleuropein offers cardioprotection in rats with simultaneous type 2 diabetes and renal hypertension. Indian J Pharmacol. 2014;46(4):398
403. 27. Cho YJ, Kim WH. Perioperative cardioprotection by remote ischemic conditioning. Int J Mol Sci. 2019;20:19. 28. Heusch G. Molecular basis of cardioprotection signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res. 2015;116(4):674
699. 29. Efentakis P, Rizakou A, Christodoulou E, et al. Saffron (Crocus sativus) intake provides nutritional preconditioning against myocardial ischemia
reperfusion injury in Wild Type and ApoE(2/ 2 ) mice: involvement of Nrf2 activation. Nutr Metab Cardiovasc Dis. 2017;27(10):919
929. 30. Andreadou I, Benaki D, Efentakis P, et al. The natural olive constituent oleuropein induces nutritional cardioprotection in normal and cholesterol-fed rabbits: comparison with preconditioning. Planta Med. 2015;81(8):655
663. 31. Jay SM, Lee RT. Protein engineering for cardiovascular therapeutics: untapped potential for cardiac repair. Circ Res. 2013;113(7): 933
944. 32. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism and disease. Cell. 2017;168(6):960
976. 33. Andreadou I, Mikros E, Ioannidis K, et al. Oleuropein prevents doxorubicin-induced cardiomyopathy interfering with signaling molecules and cardiomyocyte metabolism. J Mol Cell Cardiol. 2014;69:4
16. 34. Ji ST, Kim YJ, Jung SY, et al. Oleuropein attenuates hydrogen peroxide-induced autophagic cell death in human adipose-derived stem cells. Biochem Biophys Res Commun. 2018;499(3): 675
680. 35. Hadrich F, Garcia M, Maalej A, et al. Oleuropein activated AMPK and induced insulin sensitivity in C2C12 muscle cells. Life Sci. 2016;151:167
173.

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36. Lindsey ML, Bolli R, Canty JM, et al. Guidelines for experimental models of myocardial ischemia and infarction. Am J Physiol
Hear Circ Physiol. 2018;314(4):H812
H838. 37. Janahmadi Z, Nekooeian AA, Moaref AR, et al. Oleuropein offers cardioprotection in rats with acute myocardial infarction. Cardiovasc Toxicol. 2015;15(1):61
68. 38. Janahmadi Z, Nekooeian AA, Moaref AR. Oleuropein attenuates the progression of heart failure in rats by antioxidant and antiinflammatory effects. Naunyn-Schmiedeberg’s Arch Pharmacol. 2017;390:245
252. 39. Andreadou I, Sigala F, Iliodromitis EK, et al. Acute doxorubicin cardiotoxicity is successfully treated with the phytochemical oleuropein through suppression of oxidative and nitrosative stress. J Mol Cell Cardiol. 2007;42(3):549
558. 40. Prathumsap N, Shinlapawittayatorn K, Chattipakorn SC, et al. Effects of doxorubicin on the heart: From molecular mechanisms to intervention strategies. Eur J Pharmacol. 2020;866:172818. 41. Zhang JY, Yang Z, Fang K, et al. Oleuropein prevents the development of experimental autoimmune myocarditis in rats. Int Immunopharmacol. 2017;48(208):187
195. 42. Jensen LD, Marchant DJ. Emerging pharmacologic targets and treatments for myocarditis. Pharmacol Ther. 2016;161:40
51. 43. Bester D, Esterhuyse AJ, Truter EJ, et al. Cardiovascular effects of edible oils: a comparison between four popular edible oils. Nutr Res Rev. 2010;23:334
348. 44. Suchal K, Bhatia J, Malik S, et al. Seabuckthorn pulp oil protects against myocardial ischemia-reperfusion injury in rats through activation of Akt/eNOS. Front Pharmacol. 2016;29(7):155. 45. Estruch R, Ros E, Salas-Salvado´ J, et al. Primary prevention of cardiovascular disease with a mediterranean diet supplemented with extra-virgin olive oil or nuts. N Engl J Med. 2018;378:e34. 46. Souza PAL, Marcadenti A, Portal VL. Effects of olive oil phenolic compounds on inflammation in the prevention and treatment of coronary artery disease. Nutrients. 2017;9:10.

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Chapter 51

Oleuropein and skin cancer Siti Fathiah Masre Centre for Toxicology and Health Risk Studies, Faculty of Health Sciences, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Abbreviations AKT BCC CDKN2A COX DMBA DNA HH MAPK MMP NF-κβ NMSC PI3K PTCH SCC TNF-α TPA TUNEL UV VEGF

protein kinase B basal cell carcinoma cyclin-dependent kinase inhibitor 2A cyclooxygenase 7,12-dimethylbenz(a)anthracene deoxyribonucleic acid hedgehog mitogen-activated protein kinase matrix metalloproteinase nuclear factor-kappa β nonmelanoma skin cancer phosphoinositide 3-kinase patched squamous cell carcinoma tumor necrosis factor-alpha 12-O-tetradecanoylphorbol-13-acetate terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling ultraviolet vascular endothelial growth factor

51.1 Introduction The use of natural products in cancer prevention research studies is due to their being applied since ancient times, less toxic, rich antioxidant properties, and widely consumed as a food sources. In many studies, natural products have shown to prevent skin cancer formation by inhibiting, delaying, and/or reversing the process of carcinogenesis. Oleuropein, one of the natural products derived from olive plants, is a phenolic compound that is abundantly found in the olive leaves. Oleuropein gives a bitter taste to olives and olive oil. Several studies have exhibited that the usage of oleuropein may have a potency effect in preventing skin cancer development of melanoma and nonmelanoma cancers. There are various mechanistic actions of oleuropein on different types of skin cancer. Hamdi and Castellon

suggested that the underlying reason regarding the anticancer effects of oleuropein may due to its ability to specifically inhibit cancer cells with no adverse effects on normal cells.1 Oleuropein induced inhibition in proliferation and migration of melanoma skin cancer cells with no toxicity effects on normal cells.1 Indeed, a previous study has reported that oleuropein acts as a protective agent by restoring normal cell function and significantly reducing DNA damage against chemotherapeutic drug-induced toxicity.2 Thus the selective action of oleuropein on cancer cells offers an effective way of targeting multiple steps in skin carcinogenesis events leading to tumor regression.

51.2 Skin cancer Skin is a part of the integument system that acts as a protective barrier to humans. It is the outermost tissue in the human body and is extremely vulnerable to various harmful agents, including ultraviolet (UV) radiation, viruses, and carcinogenic substances. Long exposure to UV radiation emitted by sunlight is a main causal factor of skin cancer which allows several genetic alterations for cancer formation. Besides, lifestyle factors such as smoking, diet, and alcohol consumption also contribute to skin cancer development. The skin cancer incidence has increased so fast in the last decades that it was considered the silent epidemic of the 20th century.3 Skin cancer can be divided into two types that are melanoma and nonmelanoma. Nonmelanoma accounts for more than 90% of all skin cancer cases and melanoma for only 4% of cases.4 Although melanoma has the lowest incidence, it causes the most deaths among all skin cancers. Approximately 300,000 cases of melanoma were reported worldwide in 2018 and is the 19th incidence among all cancer rankings. Nonmelanoma skin cancer (NMSC) ranks as fifth cancer in both men and women, worldwide.4 NMSC can be further divided into two major

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00001-8 © 2021 Elsevier Inc. All rights reserved.

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forms that are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Studies in cell culture, animal model, and pathological analysis of human neoplasms have confirmed that cancer development involves several stages in which each stage is required to form a malignant tumor. The three important stages in the carcinogenesis process are initiation, promotion, and progression.5 The initiation stage is the first step in carcinogenesis, and this stage is irreversible. The initiated cells undergo further proliferation to develop neoplastic tissue upon exposure to a promoter agent. Then the progression stage can happen when the neoplastic tissue changes to form malignancy which may further lead to tumor invasion and metastasis.6

51.2.1 Melanoma skin cancer Melanoma is the most aggressive type of skin cancer due to its ability to metastasize. Its incidence keeps rising particularly in developed countries. There are several genetic and environmental factors involved in the pathogenesis of melanoma skin cancer. The genetic factor includes the germline or somatic mutation. The environmental factor involves skin type or skin color, sunlight exposure, and nevi (mole) appearance.7 Among the symptoms of melanoma is a mole that has changed in size, color, shape, and causes itching or bleeding. Studies have shown that the most common mutated gene in malignant melanoma is B-Raf which happens in almost 60% of cases, followed by N-Ras mutation with 30% of cases.8,9 B-Raf is under Raf family members which play a key role in the mitogen-activated protein kinase (MAPK) pathway that coordinates various cellular functions.10 Past studies demonstrated that B-Raf mutation is not a feature of malignant melanoma alone but also showed a high frequency in benign and atypical melanocyte lesions.11,12 N-Ras is one of the isoforms in the Ras family, which is under the MAPK and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways.13 Thus the majority of genetic abnormalities in melanoma involve with MAPK pathway. It is a core in melanoma formation and can affect various biological events such as cell proliferation, apoptosis, and cell cycle.14 There are also other genes that contributed to melanoma invasiveness and metastatic features. The upregulation of matrix metalloproteinases (MMPs) promotes the metastatic power of melanoma and resulted in excessive tumor cell invasion and infiltrate into other tissue areas.14,15 An increase of MMPs expression was also mediated by changes in the tumor microenvironment and genetic alterations of certain inflammatory signaling pathways.14 Furthermore, mutations in the cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor suppressor gene, or in a gene that involved in cell cycle regulation such as

cyclin-dependent kinase 4 can lead to the formation of melanoma skin cancer.

51.2.2 Nonmelanoma skin cancer BCC and SCC of the skin are the two most common forms of NMSC.16 The incidence of skin BCC is three times more than skin SCC. However, skin SCC cases contributed to the vast numbers of NMSC-related deaths.17,18 NMSC is also known as a keratinocyte cancer, and various etiological factors can lead to the development of NMSC with UV radiation exposure being prominent. The other factors include virus infections, long-term immunosuppression, and chronic skin inflammation.16 BCC of the skin is the most common type of skin cancer that occurs in humans worldwide. BCC is less likely to spread as compared to skin SCC, which can metastasize and increase the mortality rate. Malignant BCC cells develop from the outer hair root sheath and the interfollicular epidermis in the basal epithelial layer.19 Two main subtypes are nodular and superficial BCC which typically occur on the face and neck parts. BCC is known to grow slowly, destroys the surrounding tissues, but rarely spreads or results in mortality. BCC recurrence acts as the main challenge for its treatment. Li et al. reported that Hedgehog (HH) signaling has been established via in vivo, in vitro, and clinical trial studies as a key genetic mutation in BCC formation.20 The growth of BCC is associated with a mutation in the gene that upregulates HH signaling such as PTCH, a tumor suppressor gene. In many BCC cases, there is at least one mutation in the PTCH gene that is responsible for the activation of HH signaling.21 Deactivation of tumor suppressor gene PTCH with UV radiation exposure is considered essential in the BCC pathogenesis process. SCC of the skin arises from epithelial keratinocyte cells, and it is the second most common skin cancer. Cumulative exposure to UV radiation, chronic wounds, carcinogenic contact, and human papillomavirus infection are some of the risk factors of SCC.22 Although skin SCC lesions can be treated by surgery, some lesions continue to progress and metastasize leading to mortality.23 The metastatic potential of skin SCC may be related to its high genomic alterations.22 In most cases of skin SCC, it frequently exerts a mutation in the tumor suppressor gene p53, followed by NOTCH, CDKN2A, Ras/MAPK, and PI3K/AKT pathways.24 Mutations in the p53 gene can be caused by exposure to UV light radiation. As a consequence, mutated p53 may lead to dysregulation of the cell cycle and induce epithelial cells to grow uncontrollably. Skin SCC commonly grows like a hard lump with a scaly or ulcer appearance. Skin cancer can be prevented by avoiding excess UV exposure from outdoor activities, wearing protective clothing, and applying sunblock cream. High consumptions of

Oleuropein and skin cancer Chapter | 51

fruits and vegetables also have the potential to reduce the risk of cancer. Natural products from plants have been used to prevent several diseases including cancer for centuries.25 Moreover, compounds such as polyphenol and flavonoid from plants have shown their potency as a chemopreventive agent in various types of cancer. Lately, there have been extensive in vivo and in vitro studies on natural phytochemical agents toward cancer due to various health benefits and fewer side effects.26,27

51.3 Beneficial properties of oleuropein Olive (Olea europaea L.) is one of the most important plants in Mediterranean countries. Although there are more than 800 million olive trees worldwide, 98% of them are cultivated in Mediterranean countries. Olives and olive oil have been consumed as food or medicine for centuries. Furthermore, olive leaves have also been used since ancient times due to their medicinal properties. Olive and its derivatives are gaining widespread attention as important components in a healthy diet due to its high phenolic content. Secoiridoid and its derivative, oleuropein, are known to be the main phenolic compounds in olive, which form most of the polyphenols of olive. Oleuropein which belongs to the Oleaceae family can be found predominantly in various parts of the olive plant such as in leaves, seeds, and pulps. Oleuropein can also be found in olive oil.28 However, oleuropein is found in high quantities in the leaves part which can reach up to 90 mg/g (of leaf dry weight). Several studies have reported on the biological roles of oleuropein as an antioxidant, antiproliferation, proapoptosis, antiinflammatory, and antiviral.29 Oleuropein that exhibits many biological properties makes it a novel therapeutic agent, which has the potential to serve as a treatment for several diseases including cancer. Moreover, oleuropein has been widely studied through in vitro and in vivo models due to its preventive and anticancer properties. The antioxidant property by oleuropein has exerted beneficial outcomes in various biological activities.1 They prevent DNA damage upon exposure to hydrogen peroxide, which is an oxidant agent in human leukemia and blood mononuclear cells.30 The underlying mechanism behind this oleuropein activity has yet to be explored, but it could be due to its antioxidant property that impedes the formation of oxidative stress. Indeed, pretreatment of oleuropein acts as a cardioprotective agent against the ischemic heart model in rats, likely due to its antioxidant effect.31 In the experiment by Manna et al., oleuropein has significantly reduced lipid peroxidation and oxidized glutathione release in ischemic hearts, thus preventing oxidative stress.31 Several studies have demonstrated the antiproliferation activity of oleuropein in cancer cells.32,33 Oleuropein

617

inhibited the growth of colorectal cancer cells by significantly reduced Ki-67 proliferative marker expression.32 Moreover, the proliferation of colon adenocarcinoma cells was also inhibited by blocking the G2/M cell cycle phase upon treatment with oleuropein. This antiproliferation effect of oleuropein may be due to inhibition of p38 and cAMP response element-binding activation which is responsible for tumor progression.33 In regards to the proapoptotic effect of oleuropein, Elamin et al. demonstrated a significant reduction in BCL-2 and surviving, antiapoptotic markers and increased apoptotic markers of caspase-3, and Bax in oleuropeintreated breast cancer cells.34 Similarly, a study by Cardeno et al. reported that oleuropein treatment induced apoptotic activity in colon cancer cells (HT-29) by upregulating p53 expression that mediates Bax activation.35 Giner et al. also demonstrated that colorectal cancer in mouse model treated with oleuropein showed the activation of p53 and increased in Bax expression mediating apoptosis.32 Oleuropein has shown an antiinflammatory effect in several pathological conditions.36 The antiinflammatory activity of oleuropein was reported in cisplatin-induced renal injury in mice, in which oleuropein suppressed phosphorylated p65, nuclear factor-kappa β (NF-κβ), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX-2) inflammatory markers expression.37 Also, oleuropein has prevented chronic inflammatory microenvironment in human glioblastoma cells (U-87MG) by significantly suppressed TNF-α, COX-2, and NF-κβ.38

51.3.1 The effects of oleuropein on cancerassociated mechanisms Several epidemiological studies have shown that the occurrence of cancer among people in Mediterranean countries is much lower in comparison to other countries. This is strongly associated with diet in the Mediterranean countries.39 Randomized clinical trial research has revealed the benefits of the Mediterranean diet, which was capable of reducing the mortality rate and cancer risk by about 56% and 61%, respectively, in the consecutive 4 years.40,41 This interesting evidence has discovered the consumption of olives and olive oil as a major contributing factor of the Mediterranean diet health benefits.42 The most abundant phenolic compounds in olives are oleuropein in several parts of the olive, including the leaves, fruits, roots, and oils.1,36,43 A large number of in vitro studies (Table 51.1) have documented the anticancer effect of oleuropein in various types of cancer cell lines, including breast, bladder, leukemia, liver, cervical, thyroid, colorectal, lung, prostate, renal, brain, blood vessel, and skin.36,59,60 In accordance with the in vitro studies (Table 51.2), several animal

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

TABLE 51.1 In vitro studies on anticancer effect of oleuropein. Cancer

Action

References

Skin

Antiproliferation, antimigration, prevented motility and invasion, disrupted actin filament

[1]

Proapoptosis, antiproliferation

[44]

Proapoptotic effect

[34]

Reduced cell viability, proapoptosis, and inhibited HER2 oncogene

[45]

Proapoptosis

[46]

Antiproliferation and proapoptotic effects

[47]

Bladder

Antiproliferation

[48]

Leukemia

Antiproliferation, prevented motility, and invasion

[1]

Antiproliferation and proapoptosis

[49]

Liver

Inhibited cell viability and proapoptosis

[50]

Cervix

Proapoptotic and antiproliferation

[51]

Thyroid

Antiinflammatory, antiproliferation, and antioxidant

[52]

Colorectal

Proapoptotic and antiinflammatory

[53]

Antiproliferation

[33]

Proapoptosis

[54]

Antiinflammatory

[32]

Antiproliferation and proapoptosis

[35]

Lung

Antiproliferation, antimigration, antioxidant

[55]

Prostate

Reduced cell viability, induced necrosis, and antioxidant

[56]

Renal

Antiproliferation and antimigration

[1]

Brain

Induced autophagy

[57]

Antimigration

[1]

Antiangiogenesis, antiproliferation, antimigration

[58]

Breast

Blood vessel

TABLE 51.2 In vivo studies on anticancer effect of oleuropein. Cancer

Action

References

Skin

Reduced skin thickness and increased skin elasticity, antiproliferation, antiinflammatory, antiangiogenesis

[61]

Antiproliferation

[1]

Antiangiogenesis and antilymphangiogenesis

[43]

Proapoptotic, antioxidant

[62]

Reduced skin thickness, proapoptotic, and antioxidant

[63]

Antiproliferation, antiinflammatory, and proapoptosis

[32]

Reduced tumor progression and DNA damage

[64]

Brain

Induced autophagy and antiinflammatory

[57]

Tongue

Regressed tumor progression and antiproliferation

[65]

Breast

Antiproliferation and antimetastasis

[66]

Colorectal

Oleuropein and skin cancer Chapter | 51

models have also reported the ability of oleuropein to prevent skin, breast, tongue, brain, and colorectal cancers.67 Thus various anticancer effects of oleuropein can potentially develop it as an effective chemotherapy approach. Indeed, a recent study by Goldsmith et al. has indicated the proapoptotic effect of oleuropein in pancreatic cancer cells, while showing a protective effect on noncancer cells.60 Similarly, Samara et al. showed that oleuropein analog 24 has a significant cytotoxicity effect on several solid tumor cell lines and nontoxic to normal cells.68 Interestingly, oleuropein reduced the viability and changed the morphology of cancer cells, yet the surrounding noncancer cells were not affected, presenting the selectivity effect of oleuropein as a potential cancer-preventive agent.60

51.3.2 Oleuropein and melanoma skin cancer There are a small number of studies that have addressed the effect of oleuropein on melanoma skin cancer. Hamdi and Castellon conducted an experimental study to see the effect of oleuropein on seven different advanced grade tumor cell lines, including human malignant melanoma cells (RPMI-7951).1 Growth of the melanoma cells was significantly inhibited in a dose-dependent manner of oleuropein. In the radial migration assay, they showed complete inhibition of cell motility by oleuropein. Hamdi and Castellon also reported on the antiangiogenic effect of oleuropein using a tube-formation assay.1 Interestingly, oleuropein can disrupt the actin cytoskeleton in melanoma cells within 2 h by inducing cell rounding formation1. However, they stated that the glucose moiety in oleuropein could be the reason for oleuropein to enter into the cancer cells and cause cell rounding. Yet, their other findings also showed that the ability of oleuropein to induce cell rounding and inhibit cell proliferation was not fully obliterated even with excess glucose and β-galactosidase treatment. Thus oleuropein may have other routes to enter the melanoma cancer cells and is not dependent on glucose transporter.1 In a melanoma allograft mouse model, Song et al. demonstrated the effects of oleuropein in suppressing tumor growth, angiogenesis, and lymph node metastasis.43 Song et al. used a C57BL/6N mouse that was fed with a high-fat diet (with and without oleuropein) for 16 weeks before being subcutaneously injected with B16F10 melanoma cell lines and growth for additional 3 weeks.43 The high fat diet
induced obesity promotes tumor progression and lymph node metastasis in melanoma cancer cells.69 Song et al. reported that dietary oleuropein
inhibited B16F10 tumor proliferation by lowering the expression of Ki67 and, at the same time, showed a proapoptotic activity by significantly increasing the expression of TUNELpositive cells and cleaving poly(ADP-ribose)polymerase

619

in the melanoma allograft model.43 They showed that oleuropein intake decreased the angiogenesis event in both B16F10 tumor tissues and lymph nodes of the mouse model by reducing all vascular endothelial growth factor (VEGF) markers expression. Similarly an in vitro study on human umbilical vein endothelial cell tube formation assay revealed direct inhibition of oleuropein toward the endothelial cells. However, Song et al. not only reported on the potential of oleuropein to suppress tumor formation in melanoma cell-bearing mice but oleuropein can also inhibit adipogenesis that reduces fat accumulation via in vitro and in vivo studies.43 Decrease in adipogenesis is in accordance with the decrease in B16F10 tumor proliferation43. Thus dietary intake of oleuropein can potentially overcome the overweight and obesity issues and most importantly inhibit melanoma tumor progression associated with obesity. Can oleuropein alone or in combination with a chemotherapeutic drug fight against melanoma? Ruzzolini et al. initially demonstrated a cytotoxic effect of oleuropein, extracted from the leaves of O. europaea L. plants on A375 melanoma cell lines.44 To validate that the cytotoxic effect on melanoma cells was fully attributed by oleuropein, they have incubated the oleuropein in A375 cells. Indeed, the outcome revealed the presence of oleuropein in the cells for 72 h without being metabolized.44 They showed that oleuropein suppressed A375 cell proliferation by inhibiting pAKT/mTOR pathway, one of the crucial pathways related to cancer survival and growth.44 Moreover, by using matrigel, they have confirmed that oleuropein treatment inhibited melanoma cells invasion as compared to untreated cells. Ruzzolini et al. have combined oleuropein with chemotherapeutic drugs, including vemurafenib, dacarbazine, and everolimus, to improve the treatment efficacy for advanced melanoma.44 This combination was important to prevent the drug resistance issue and to reduce the medical side effects of the drug. A combination of oleuropein with vemurafenib showed no efficacy effect on the A375 cells. However, the combination of oleuropein with dacarbazine and oleuropein with everolimus showed inhibitory activity against A375 cells, particularly by downregulating the pAKT/mTOR pathway. Through investigation, they found out that a combination of oleuropein and everolimus has more efficacies on A375 cells as compared to oleuropein and dacarbazine, and oleuropein alone.44 Oleuropein and everolimus showed the best combination to induce apoptosis and reduce cell viability through MTT and colony-forming unit assays. A wide type of melanoma cases has developed resistance to vemurafenib drug.44 Vemurafenib is used as a targeted drug for BRAF V600E mutation in melanoma. To overcome the vemurafenib resistance, Ruzzolini et al. have used A375 vemurafenib-resistant melanoma cell

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

lines to validate the efficacy of oleuropein and everolimus combination.44 Resistant melanoma cells demonstrated an increased percentage of dead cells, together with a significant reduction of pAKT/S6 and pERK pathways (which have been linked to cancer development) upon combination treatment with oleuropein
everolimus as compared to a single treatment.44 Overall, Ruzzolini et al. have shown that oleuropein has great potential as a therapeutic agent to enhance the efficacy of current chemotherapy drugs for melanoma cells and BRAF-resistant melanoma cells.44 However, further determination on the best administration route for oleuropein together with its effective dose is suggested to be done in the next study.

51.3.3 Oleuropein and nonmelanoma skin cancer We have examined the pretreatment effects of oleuropein in the early stage of skin SCC in vivo.62,63 The model of two-stage skin carcinogenesis induced by initiating agent, 7,12-dimethylbenz(a)anthracene and promoting agent, 12O-tetradecanoylphorbol-13-acetate (DMBA/TPA) on ICR mice was used. Oleuropein was pretreated via topical application to the mice twice per week before the DMBA/ TPA induction for 10 weeks. The studies were divided into two stages which are tumor initiation and tumor promotion. For the preinitiation effect of oleuropein the mouse was pretreated daily for 2 weeks prior to DMBA/ TPA induction.62 Whilst for the prepromotion effect of oleuropein, the mouse was induced with DMBA first, then pretreated with oleuropein prior to TPA induction.63 Histopathological examination of the skin tissues of preinitiation and prepromotion stages discovered a combination of mild hyperplasia and normal single epidermal layer, as compared to the DMBA/TPA control group which showed thick hyperplasia with messy epithelial layers.62,63 Indeed, the measurement analysis revealed that pretreatment of oleuropein has significantly reduced the epidermal thickness compared to the DMBA/TPA control group. There was no significant difference in the measurement of skin thickness between the oleuropeintreated group and vehicle-untreated group63 showing the efficacy of oleuropein to reverse the skin carcinogenesis to have a formation like a normal single epithelial layer. Pretreatment of oleuropein significantly induced proapoptotic activities via increased activated caspase-3 labeling at each initiation and promotion stage.62,63 Hence, the precancerous cell death stimulated by oleuropein may be the mechanism behind the prevention of skin SCC development. Again, past studies by Elamin et al. and Goldsmith et al. have demonstrated a selective proapoptotic effect of oleuropein on cancer cells.34,60 Evaluation of oxidative stress activity showed a significant decrease in lipid peroxidation (malondialdehyde) levels and increased

antioxidant superoxide dismutase upon pretreatment of oleuropein at each stage. Thus the effect of oleuropein as a protective agent against oxidative damage could be high due to its antioxidant potency. Kimura and Sumiyashi have observed the development of NMSCs upon chronic exposure of UVB radiation (310 nm) on hairless mice for 30 weeks.61 Oleuropein was given orally after 3 weeks of UVB radiation with a significant reduction in skin thickness and increased skin elasticity as compared to the cancer group. Also, the oleuropein-treated group has significantly prevented tumor incidence and tumor growth, together with a reduction of blood vessel diameters.61 The oleuropein-treated group showed a significant decrease in the expression of MMP-2, MMP-9, COX-2, VEGF, Ki-67, and CD31 than in chronically UVB-radiated skin. Reduction of blood vessel diameters by oleuropein in their study may be supported with the inhibition of VEGF and CD31 expression, which is essential to induce blood vessel growth and vascular permeability. Moreover, the inhibition of MMPs and COX-2 activities as inflammatory modulators and Ki-67 expression to promote tumor growth may be linked to the preventive mechanisms of oleuropein in UVB-irradiated skin cancer. Kimura and Sumiyashi also compared the effect between oleuropein and olive leaf extract in the UVB-induced nonmelanoma skin carcinogenesis. Both compounds have the same effect to prevent tumor development.61 The concentration of oleuropein in olive leaf is greater compared to olive oil and olive fruit. Thus they claimed that the preventive effect of olive leaf extract may be high due to the potency effect of oleuropein. By using the same UVB-induced NMSC model, Ichihashi et al. and Budiyanto et al. have depicted the photopreventive ability of olive oil.70,71 Both studies demonstrated the inhibitory effect of olive oil on tumor incidence and DNA damage. In regards to treatment time, both studies reported that olive oil treatment after UVB exposure showed a significant effect in preventing tumor incidence as compared to pretreatment of olive oil prior to UVB exposure which revealed no photopreventive effect. However, knowing that hydroxytyrosol and tyrosol are highly presented in olive oil with a minor amount of oleuropein, the photopreventive effects may not be solely contributed by the oleuropein.61 Ichihashi et al. also claimed that oleuropein in the olive oil reduces DNA damage in the UVB-irradiated mice.72 Hence, detail studies are required to examine the effects of a specific compound in olive oil and compare it with oleuropein, olive leaf extract, or olive fruit extract. Using Swiss albino mice research has reported the effect of oleuropein on a rare type of NMSC, Kaposi sarcoma.1 They revealed that 1% oleuropein given in drinking water to the mice has induced complete tumor regression in 9
12 days. Hamdi and Castellon added that

Oleuropein and skin cancer Chapter | 51

oleuropein showed a similar regression effect in mice with single or multiple tumors.1 However, they reported that the increased number of tumors in the mouse may have delayed the regression time. Moreover, in the early developmental stage of Kaposi sarcoma, they discovered that oleuropein treatment has altered tumor appearance in which the tumors were nonadhesive and crumbly as compared to the solid appearance of tumors in untreated mice. Even after cessation of oleuropein treatment in the drinking water, the mice stayed living throughout their life span without tumor formation.

51.4 Conclusion Continuous increment of skin cancer cases over time has been a great challenge for researchers with many unwanted side effects and drug resistance issues associated with the current cancer treatment such as chemotherapy and radiotherapy. Alternative cancer treatment from natural products is one of the increasingly popular approaches to reduce cancer burden worldwide. Oleuropein has shown promising pharmacological roles such as antiinflammatory, antioxidant, antiproliferation, proapoptosis, and antiangiogenesis on melanoma and NMSCs. Treatment with oleuropein has no adverse effects. Finally, further specific studies to determine the best dose and route of administration for oleuropein need to be done before venturing into human studies. Oleuropein can be potentially developed as an active anticancer agent or combined with current conventional drugs for complete inhibition of skin cancer.

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6. 4. Bray F., Ferlay J., Soerjomataram I., Siegel R.L., Torre L.A., Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424. doi: 10.3322/caac.21492 5. Neagu M, Caruntu C, Constantin C, et al. Chemically induced skin carcinogenesis: updates in experimental models (review). Oncol Rep. 2016;35(5):2516
2528. 6. Robertson FM. Skin carcinogenesis. In: Schwab M, ed. Encyclopedia of Cancer. 2011. Berlin: Springer; 2011:293.

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260. 11. Uribe P, Wistuba II, Gonzales S. BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin. Am J Dermatopathol. 2003;25(5):365
370. 12. Pollock PM, Cohen-Solal K, Sood R, et al. Melanoma mouse model implicates metabotropic glutamate signalling in melanocytic neoplasia. Nat Genet. 2003;34(1):108
112. 13. Chappell WH, Steelman LS, Long JM, et al. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health. Oncotarget. 2011;2:135
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43. Song H, Lim DY, Jung JI, et al. Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat dietinduced solid tumor growth and lymph node metastasis. Oncotarget. 2017;8(19):32027
32042. 44. Ruzzolini J, Peppicelli S, Andreucci E, et al. Oleuropein, the main polyphenol of Olea europaea leaf extract, has an anti-cancer effect on human BRAF melanoma cells and potentiates the cytotoxicity of current chemotherapies. Nutrients. 2018;10(12):1950. 45. Menendez JA, Vazquez-Martin A, Colomer R, et al. Olive oil’s bitter principle reverses acquired autoresistance to trastuzumab (Herceptin) in HER2-overexpressing breast cancer cells. BMC Cancer. 2007;7:80. 46. Hassan ZK, Elamin MH, Omer SA, et al. Oleuropein induces apoptosis via the p53 pathway in breast cancer cells. Asian Pac J Cancer Prev. 2014;14(11):6739
6742. 47. Han J, Talorete TPN, Yamada P, Isoda H. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology. 2009;59(1):45
53. 48. Goulas V, Exarchou V, Troganis AN, et al. Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells. Mol Nutr Food Res. 2009;53(5):600
608. 49. Abaza L, Talorete TPN, Yamada P, Kurita Y, Zarrouk M, Isoda H. Induction of growth inhibition and differentiation of human leukemia HL-60 cells by Tunisian gerboui olive leaf extract. Biosci Biotech Biochem. 2007;71:1306
1312. 50. Yan CM, Chai EQ, Cai HY, Miao GY, Ma W. Oleuropein induces apoptosis via activation of caspases and suppression of phosphatidylinositol 3-kinase/protein kinase B pathway in HepG2 human hepatoma cell line. Mol Med Rep. 2015;11(6):4617
4624. 51. Yao J, Wu J, Yang X, Yang J, Zhang Y, Du L. Oleuropein induced apoptosis in HeLa cells via a mitochondrial apoptotic cascade associated with activation of the c-Jun NH2-terminal kinase. J Pharmacol Sci. 2014;125:300
311. 52. Bulotta S, Corradino R, Celano M, et al. Antioxidant and antigrowth action of peracetylated oleuropein in thyroid cancer cells. J Mol Endocrinol. 2013;51:181
189. 53. Llor X, Pons E, Roca A. The effects of fish oil, olive oil, oleic acid and linoleic acid on colorectal neoplastic processes. Clin Nutr. 2003;22:71
79. 54. Notarnicola M, Pisanti S, Tutino V, et al. Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells. Genes Nutr. 2011;6(1):63
69. 55. Mao W, Shi H, Chen X, et al. Anti-proliferation and migration effects of oleuropein on human A549 lung carcinoma cells. Lat Am J Pharm. 2012;32:1217
1221. 56. Acquaviva R, Di Giacomo C, Sorrenti V, et al. Antiproliferative effect of oleuropein in prostate cell lines. Int J Oncol. 2012;41 (1):31
38. 57. Rigacci S, Miceli C, Nediani C, et al. Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight. Oncotarget. 2015;6(34):35344
35357. 58. Lamy S, Ouanouki A, Be´liveau R, Desrosiers RR. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation. Exp Cell Res. 2014;322(1):89
98. 59. Imran M, Nadeem M, Gilani SA, Khan S, Sajid MW, Amir RM. Antitumor perspectives of oleuropein and its metabolite hydroxytyrosol: recent updates. J Food Sci. 2018;83(7):1781
1791.

Oleuropein and skin cancer Chapter | 51

60. Goldsmith CD, Bond DR, Jankowski H, et al. The olive biophenols oleuropein and hydroxytyrosol selectively reduce proliferation, influence the cell cycle, and induce apoptosis in pancreatic cancer cells. Int J Mol Sci. 2018;19(7):1937. 61. Kimura Y, Sumiyoshi M. Olive lead extract and its main component oleuropein prevent chronic ultraviolet B radiation-induced skin damage and carcinogenesis in hairless mice. J Nutr. 2009;139 (11):2079
2086. 62. Masre SF, Izzuddeen A, John DNS, Hamid ZA. The effects of oleuropein on apoptotic rate and oxidative stress profiles during tumour promotion stage in the mouse skin carcinogenesis model. Sains Malays. 2019;48(2):347
352. 63. John DNS, Mamat THT, Surien O, Taib IS. Pre-initiation effect of oleuropein towards apoptotic and oxidative stress levels on the early development of two-stage skin carcinogenesis. JKIMSU. 2019;8(1):43
51. 64. Sepporta MV, Fuccelli R, Rosignoli P, et al. Oleuropein prevents azoxymethane-induced colon crypt dysplasia and leukocytes DNA damage in A/J mice. J Med Food. 2016;19(10):983
989. 65. Grawish ME, Zyada MM, Zaher AR. Inhibition of 4-NQO-induced F433 rat tongue carcinogenesis by oleuropein-rich extract. Med Oncol. 2011;28:1163
1168.

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66. Sepporta MV, Fuccelli R, Rosignoli P, et al. Oleuropein inhibits tumour growth and metastases dissemination in ovariectomised nude mice with MCF-7 human breast tumour xenografts. J Funct Foods. 2014;8:269
273. 67. Fabiani R. Anti-cancer properties of olive oil secoiridoid phenols: a systematic review of in vivo studies. Food Funct. 2016;7 (10):4145
4159. 68. Samara P, Christoforidou N, Lemus C, et al. New semi-synthetic analogs of oleuropein show improved anticancer activity in vitro and in vivo. Eur J Med Chem. 2017;137:11
29. 69. Jung JI, Cho HJ, Jung YJ, et al. High-fat diet-induced obesity increases lymphangiogenesis and lymph node metastasis in the B16F10 melanoma allograft model: roles of adipocytes and M2macrophages. Int J Cancer. 2015;136:258
270. 70. Ichihashi M, Ahem NU, Budiyanto A, Osawa T. Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice. J Dermatol Sci. 2000;23:S45
S50. 71. Budiyanto A, Ahmed NU, Wu A, et al. Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice. Carcinogenesis. 2000;21(11):2085
2090. 72. Ichihashi M, Ueda M, Budiyanto A, et al. UV-induced skin damage. Toxicology. 2003;189:21
39.

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Chapter 52

Oleuropein, olive, and insulin resistance Tomoko Ishikawa1 and Yoko Fujiwara2 1

Institute for Human Life Innovation, Ochanomizu University, Tokyo, Japan, 2Graduate School of Humanities and Sciences, Ochanomizu University,

Tokyo, Japan

Abbreviations ACC AICAR Akt ALT AMPK ANOVA AST CC CVD DPP-4 EVOO GLP-1 GLUT4 GYS HFD HOMA-IR HT HT-Ac HT-Et IR IRS LDL NAFLD ND OGTT PI3K RCT ROS TG TNF T2D

acetyl-CoA carboxylase 5-aminoimidazole-4-carboxamide ribonucleoside protein kinase B alanine aminotransferase AMP-activated protein kinase analysis of variance aspartate aminotransferase compound C cardiovascular disease dipeptidyl peptidase-4 extra-virgin olive oil glucagon-like peptide 1 glucose transporter 4 glycogen synthase high-fat diet homeostasis model assessment of insulin resistance hydroxytyrosol hydroxytyrosyl acetate ethyl hydroxytyrosyl ether insulin receptor insulin receptor substrate low-density lipoprotein nonalcoholic fatty liver disease normal diet oral glucose tolerance test phosphoinositide 3-kinase randomized controlled trial reactive oxygen species triglyceride tumor necrosis factor type 2 diabetes

52.1 Introduction Insulin, the sole hormone that exhibits a hypoglycemic effect, is responsible for dietary glucose consumption by promoting uptake of postprandial blood glucose into myocytes and adipocytes and regulating metabolism of

hepatocytes and other target cells. Chronic lifestyle-related imbalances between energy intake and expenditure continually increase insulin levels, reduce insulin sensitivity of target cells, and result in insulin resistance. Insulin resistance is a state in which insulin sensitivity is reduced and the effect of insulin cannot be sufficiently exerted. Genetic predisposition, obesity, lack of exercise, high-fat diets, and stress are known as inducers of insulin resistance. In other words, insulin resistance is a dysfunctional condition closely related to one’s lifestyle. Insulin resistance is also closely related to various physiological disorders, such as type 2 diabetes (T2D), nonalcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD), and Alzheimer’s dementia. Therefore improving insulin resistance is an effective strategy for medical treatment and prevention of lifestyle-related diseases, of which morbidity is currently increasing worldwide. Mediterranean eating habits with high olive oil intake have attracted wide interest as a healthy diet to reduce T2D and cardiovascular events. In particular, olive oil polyphenols such as oleuropein and its metabolite hydroxytyrosol (HT) have been suggested to contribute to the prevention and improvement of insulin resistance (Table 52.1). In this chapter, we will review findings, including our own recent study, on the effects and mechanisms of oleuropein and olive on insulin resistance.

52.2 The mechanism of insulin-induced hypoglycemia The mechanism of hypoglycemia by insulin has been elucidated in detail in hepatocytes.1,2 Under insulin-sensitive conditions, insulin signals in adipocytes downregulate adipose triglyceride (TG) lipase and hormone-sensitive lipase, which then inhibit lipolysis and promote lipogenesis. As a result, the supply of nonesterified fatty acids and glycerol from adipose tissues via blood circulation is reduced and gluconeogenesis is suppressed in hepatocytes. Insulin also stimulates hepatocytes directly and leads to sequential

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00011-0 © 2021 Elsevier Inc. All rights reserved.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

TABLE 52.1 Effects of olive oil polyphenols and its metabolites related to insulin resistance. Component

Subject

Condition

Dose

Effects related to insulin resistance

Extra-virgin olive oil

Human

Healthy

10 g

G G

Olive leaf extract

Olive leaf extract

Human

Mouse

Obese

Obese

51.1 mg oleuropein, 9.7 mg HT/day

G

0, 1, 10, and 25 mg/kg (10% oleuropein)/day

G

G

G

G G

G

G

Olive-tree leaves extract

Oleuropein

3T3-L1derived adipocyte cell

High glucose

400, 600, and 800 μg/mL

C2C12derived myotube cell

Normal, H2O2 treated

0, 200, and 400 μM

G G

G G

G

G

Oleuropein

Oleuropein

Oleuropein

C2C12derived myotube cell, mouse

Normal, obese

Rat skeletal muscle (ex vivo)

5 mM glucose

Chick skeletal muscle cell

Normal

0, 1, 10, 100 μM, HFD containing 0.038% oleuropein

G G G G

0, 0.5, 1, 1.5, and 3 mM

G G G

0, 5, and 10 μM

G G

G

Hydroxytyrosol

Review

2

2

G G G G

Hydroxytyrosol

Rat

NAFLD

10 mg/kg/day

G

G

G G

Hydroxytyrosol

Mouse

Diabetes

50 mg/kg/day in HFD diabetic mice, 10 mg/kg/ day in db/db diabetic mice

G G

Reference

Postprandial blood glucose k Postprandial blood LDL cholesterol k

Violi et al.4

Insulin sensitivity m (OGTT) Blood IL-6, IGFBP-1, IGFBP-2 m

de Bock et al.5

Body weight gain, blood glucose k Insulin resistance k (IPGTT: glucose tolerance test by intraperitoneal injection, HOMA-IR) Adipose tissue mass k Adipose PPAR, adiponectin, leptin receptor mRNA m Liver TNF-α, IL-1β, IL-6, Juk-1 mRNA k Liver PPARγ, Glut-4, Ampk mRNA m

Vezza et al.10

Lipid accumulation k AMPK phosphorylation m

Jime´nezSa´nchez et al.12

Glucose consumption m H2O2-induced ROS, TBARS increase k AMPK, ACC, ERk phosphorylation m GLUT4 protein level m

Hadrich et al.14

GLUT4 translocation m AMPK phosphorylation m Fasting blood glucose k HOMA-IR k

Fujiwara et al.16

Glucose uptake m GLUT4 translocation m AMPK phospholyration m

Alkhateeb et al.17

Mitochondrial biogenesis m Mitochondrial superoxide production k Sirt1, NRF1, TFAM, ATP5a1 mRNA m

Kikusato et al.25

Fasting blood glucose k Insulin resistance k Glucose tolerance m Insulin sensitivity m

Peyrol et al.27

Liver fat accumulation, ballooning, inflammation k Fasting blood glucose, insulin level k Insulin sensitivity m (OGTT) Blood ALT, AST, cholesterol, insulin, HOMA-IR k

Pirozzi et al.28

Insulin sensitivity m (OGTT) Blood glucose, insulin, HOMAIR, leptin, adiponectin, IL-6, CRP k

Cao et al.30

(Continued )

Oleuropein, olive, and insulin resistance Chapter | 52

627

TABLE 52.1 (Continued) Component

Subject

Condition

Dose

Effects related to insulin resistance G G

G

Hydroxytyrosol, hydroxytyrosyl acetate, ethyl hydroxytyrosyl ether

Rat

Highcholesterol diet

0.04 g /100 g dry weight of diet

G G

G G

Reference

Liver TG, oxidative stress k Skeletal muscle oxidative stress k Skeletal muscle mitochondrial complex activity m Blood glucose, insulin, leptin k Oxygen radical absorbance capacity (ORAC) m Lipid oxidation k Adipose inflammation k

Tabernero et al.31

ACC, Acetyl-CoA carboxylase; ALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; AST, aspartate aminotransferase; GLUT4, glucose transporter 4; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance.; LDL, low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; OGTT, oral glucose tolerance test; PPAR, peroxisome proliferator
activated receptor; ROS, reactive oxygen species; TG, triglyceride; TNF, tumor necrosis factor.

phosphorylation of insulin receptor (IR), IR substrate (IRS), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt). These insulin signals promote hepatic glycogen synthesis by activating glycogen synthase (GYS) and suppress gluconeogenesis by downregulating gluconeogenic enzymes, resulting in the increase of concentrationdependent blood glucose uptake into hepatocytes. Furthermore, insulin promotes protein synthesis and lipogenesis in hepatocytes to consume postprandial blood glucose. In skeletal muscle, insulin leads to sequential phosphorylation of IR
IRS
PI3K
Akt, as in hepatocytes. These stimulations accelerate trafficking of glucose transporter 4 (GLUT4)-containing vesicles into the cell membrane, enhance blood glucose uptake into myocytes, and then increase glycogen storage by activating GYS. The skeletal muscle is a major organ that plays a key role in insulin sensitivity. A study to investigate how insulin resistance arises elucidated that elderly individuals were markedly insulinresistant as compared with young controls, and this resistance was attributable to reduced insulin-stimulated muscle glucose metabolisms with mitochondrial dysfunctions.3 Therefore improving skeletal muscle function is an attractive target for overcoming insulin resistance.

52.3 Implications for human health and disease prevention Although few randomized controlled trials (RCTs) have been performed in humans, there are reports claiming that diets containing extra-virgin olive oil (EVOO) improve glucose tolerance in healthy subjects,4 and that olive leaf

polyphenols improve insulin sensitivity in middle-aged obese men.5

52.3.1 Healthy subjects The effects of EVOO on postprandial glycemic and lipid profiles were investigated in 25 healthy subjects by a randomized crossover design in a report by Violi et al.4 Two hours after from a typical Mediterranean lunch with or without 10 g EVOO, blood concentrations of glucose, insulin, incretin [glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide], dipeptidyl peptidase-4 (DPP-4), and DPP-4 activity were measured. In subjects with EVOO intake, the increases in postprandial blood concentrations of glucose were significantly lower than in subjects without EVOO intake. At this time, the blood insulin and incretin concentrations in subjects with EVOO intake were significantly higher, and the DPP-4 protein level and activity were significantly lower than in subjects without EVOO intake. Compared to corn oil, EVOO significantly reduced postprandial blood glucose concentrations and DPP-4 activity and significantly increased blood insulin and GLP-1 concentrations. A previous report by Carnevale et al. and Violi has demonstrated that a Mediterranean-type meal with EVOO is associated with reduced postprandial oxidative stress.6 Chronic exposure to oxidative stress has been reported to cause insulin resistance, β-cell dysfunction, impaired glucose tolerance, and mitochondrial dysfunction.7 A previous review has also mentioned that experimental and clinical data suggest an inverse association between insulin sensitivity and ROS levels. The previous findings suggest that EVOO reduced postprandial oxidative stress, which may have resulted in the increase of insulin secretion and the decrease of postprandial blood glucose.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

52.3.2 Obese subjects In a randomized, double-blinded, placebo-controlled, crossover trial of 46 middle-aged men who are likely to be insulin-resistant due to being overweight, it was shown that olive leaf extract administration improves insulin sensitivity.5 A daily dose of 51.1 mg oleuropein and 9.7 mg HT suspended in safflower oil was taken as four capsules every day. After 12 weeks a 75 g oral glucose tolerance test (OGTT) was performed. The olive leaf extract supplementation led to a reduction in the area under the curve for both glucose and insulin and improved the Matsuda Index8 for insulin sensitivity by 15%. This result is comparable to the finding that metformin, a common antidiabetic medication, improves insulin sensitivity in overweight nondiabetic patients by 17%.9 The mainstream of medication research for diabetes is drugs, such as DPP4 antagonists and GLP agonists, that improve only β-cell secretion capacity. In contrast, the olive leaf extract supplementation was expected to contribute to improving insulin resistance by facilitating both insulin secretion of pancreatic β cells and insulin sensitivity.

52.4 Oleuropein and olive on insulin resistance 52.4.1 Effects of olive leaf extract and oleuropein Recently, the effects of olive leaf extract containing 12% (w/w) phenolic compound and 10% oleuropein have been investigated in high-fat diet (HFD)-induced obese mice.10 Male C57BL/6J mice were fed a standard chow diet (13% calories from fat) or HFD (50% energy from fat) for 5 weeks. The extract was administrated (0, 1, 10, and 25 mg/kg) daily by oral gavage. At the fourth week, glucose tolerance test was performed by intraperitoneal injection. The extract administration reduced body weight gain, blood glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR). The olive leaf extract also improved glucose tolerance, plasma lipid profiles, and inflammatory status in adipose tissue and liver, which are associated with obesity. Furthermore, the extract administration was able to counteract the intestinal dysbiosis and ameliorated the aortic endothelial dysfunction of obese mice. In obesity, excessive storage of lipid in adipocytes leads to hypoxia and inflammation in adipose tissues and further enhances insulin resistance.11 The effects of olivetree leaves extract have also been examined in an insulinresistant adipocyte model.12 The 3T3-L1 preadipocyte cell line was differentiated and cultured with high glucose medium containing 4.5 mg/mL insulin. In this model, it is known that the hypertrophic adipocytes induce insulin resistance and oxidative stress and indicate high levels of

cytoplasmic lipid accumulation. A volume of 800 μg/mL of crude olive-tree leaves extract significantly decreased intracellular lipid accumulation through AMP-activated protein kinase (AMPK)-dependent mechanisms in hypertrophic and insulin-resistant adipocytes. Our unpublished data have shown that oleuropein significantly suppresses intracellular lipid accumulation during the differentiation of 3T3-L1 cells (Fig. 52.1).

52.4.2 Promotion of glucose uptake under insulin-resistant state Regarding the mechanism to improve insulin resistance by oleuropein, several groups have investigated mainly in the skeletal muscles. Interestingly, these reports suggest that the promotion of glucose uptake via GLUT4 translocation by oleuropein is not dependent on the IR/IRS/Akt pathway, but rather through activation of AMPK. In 1999 Kim et al. showed that the reduction of glucose disposal rate in T2D occurred independently of Akt phosphorylation in human skeletal muscles.13 To determine whether impaired Akt activation could play a significant role in insulin resistance in T2D patients, the authors investigated in vivo insulin-stimulated glucose metabolism and activation and phosphorylation of Akt isoforms in biopsy skeletal tissues. Glucose disposal rates associated with insulin stimulation were significantly reduced, in decreasing order of lean, obese nondiabetic, and diabetic subjects. In diabetic patients, insulinstimulated IRS-1- and IRS-2-associated PI3K activities were significantly lower, but Akt1/2 and Akt3 protein levels and phosphorylation were increased as normal, similarly to lean subjects.13 These results suggest that Akt is unlikely to be a major factor in insulin resistance in skeletal muscles of obese and T2D subjects. However, the detailed mechanism of the regulation of glucose consumption without Akt phosphorylation was not elucidated in this research. In 2016 Hadrich et al. showed important insights into the mechanism of glucose consumption in skeletal muscles in a study examining the effects of oleuropein as a therapeutic agent in diabetes.14 Myotubes, differentiated from mouse myoblast C2C12 cells, were treated with 0, 200, and 400 μM oleuropein for 24 h, and the quantity of glucose consumed by the cells was measured. Oleuropein promoted glucose consumption in differentiated myotubes in a dose-dependent manner and significantly enhanced the phosphorylation of AMPK/acetyl-CoA carboxylase (ACC), but not PI3K/Akt phosphorylation. Total GLUT4 protein levels were upregulated by oleuropein. These results agree with previous reports that AMPK/ACC pathways may facilitate the translocation of the glucose transporter.15 Unfortunately, Hadrich et al. could not show

Oleuropein, olive, and insulin resistance Chapter | 52

FIGURE 52.1 Oleuropein suppressed intracellular lipid accumulation during differentiation of 3T3-L1 cells. Representative microscopic images stained with oil red O (A) and measurement results of lipid accumulation (B). 3T3-L1 preadipocytes were cultured with differentiation medium containing oleuropein (0, 1, 10, and 100 μM; Ctrl, Ole1, Ole10, and Ole100) for 8 days. The cells were stained with oil red O. After microscopic observation, oil red O was extracted with isopropyl alcohol, and the absorbance was measured as a relative lipid concentration. All values are expressed as mean 6 SD (n 5 3). Statistical analysis was performed using ANOVA followed by Tukey
Kramer’s test for multiple comparisons. Values not sharing a common Roman letter are significantly different (P , .05). ANOVA, Analysis of variance.

conclusive evidence that oleuropein-induced AMPK phosphorylation promotes GLUT4 membrane translocation. We have demonstrated that oleuropein promotes membrane translocation of GLUT4 in skeletal muscle cells both in vitro and in vivo.16 The differentiated C2C12 cells were preincubated with 0, 1, 10, 100 μM oleuropein and 100 nM insulin for 60 min, and then glucose incorporation was examined for 30 min. Oleuropein (10, 100 μM) enhanced glucose uptake into the myotubes, similarly to insulin stimulation. In coexistence with insulin, no further enhancement of glucose uptake by oleuropein was observed (Fig. 52.2). The expression levels of GLUT4 mRNA in the myotubes were not significantly different among control, oleuropein, and insulin treatment. However, the GLUT4 protein levels in the plasma

629

membrane fraction were significantly increased with 10 μM oleuropein treatment, similarly to insulin treatment (Fig. 52.3). The oleuropein treatment also activated the phosphorylation of AMPK (Thr 172), but not Akt (Ser 473) (Fig. 52.4). As a model of insulin resistance induced by obesity, the effects of oleuropein on glucose tolerance were examined under lipotoxic conditions. Male C57BL/ 6J mice were fed a normal diet (ND, 13% energy from fat), HFD (50% energy from fat), or HFD containing 0.038% oleuropein for 12 weeks. Administration of oleuropein significantly reduced the fasting blood glucose and improved HOMA-IR. The GLUT4 translocation in the gastrocnemius muscles was examined by immunofluorescent staining. The GLUT4 signals in HFD-fed mice (HFD) were weak and observed in both the cell membrane and cytoplasm. On the contrary, in oleuropein administrated mice (OLE), GLUT4 signals were more frequently observed. GLUT4 mainly translocated to cell membranes, and those localized in the cytoplasm exhibited distinct granular distributions (Fig. 52.5). These results suggest that oleuropein improves insulin resistance by translocation of GLUT4 via a pathway different from the Insulin/IR/PI3K/Akt signals, and AMPK might be involved in this pathway. The previous findings have been further validated ex vivo by another group.17 The investigation of the beneficial effects of oleuropein on glucose uptake and parameters relevant to the normal homeostatic mechanisms of glucose regulation was carried out using isolated Wistar rat skeletal muscles. The soleus muscles were incubated with 1.5 mM oleuropein for 0, 3, 6, 12, and 18 h in a time-course experiment and incubated with 0, 0.5, 1, 1.5, and 3 mM oleuropein for 12 h in a dose
response experiment. Oleuropein increased glucose uptake in the soleus muscles in dose- and time-dependent manners. The phosphorylation of AMPK was significantly increased with oleuropein treatment, similar to 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a well-known activator of AMPK, and blocked with compound C (CC), an AMPK inhibitor. The PI3K phosphorylation was not affected by both oleuropein and wortmannin, a PI3K inhibitor. Oleuropein also did not change the total GLUT4 content in skeletal muscle but significantly increased the GLUT4 content of the plasma membrane fraction. This translocation of GLUT4 induced by oleuropein was reset by CC. These findings indicated that oleuropein may induce GLUT4 translocation via the AMPKmediated pathway. Interestingly, in T2D patients, AMPK-mediated GLUT4 translocation in skeletal muscle has been reported to be promoted by exercise and contributes to improvement of insulin resistance.18 In T2D and severe obesity, insulin-stimulated GLUT4 translocation is impaired, which may be mainly attributed to defective insulin/IR/

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

IRS/PI3K signaling. However, many studies have suggested that AMPK signaling is not disturbed under insulin-resistant states.19 An intervention study has been conducted on seven T2D patients and eight healthy subjects.20 After acute exercise for 45 min, blood glucose concentrations were significantly decreased than before exercise in T2D patients. AMPK α2 activity in the skeletal muscles was significantly increased in T2D compared to in the healthy controls. These results have supported the hypothesis that exercise increases glucose uptake through an insulin-independent mechanism mediated by AMPK in T2D patients. Another interventional, RCT study for insulin resistance in nonobese and nondiabetic subjects has been performed in 12 young, lean, insulinresistant individuals.21 Upon insulin resistance in skeletal muscles, myocytes are unable to take in postprandial glucose and store it as glycogen. As a result, dietary glucose accumulates as liver fat due to hepatic de novo lipogenesis, which is one risk factor for NAFLD. A single bout of exercise for 45 min rescued the above insulin resistance to improve postprandial glycogen synthesis in skeletal muscles, suppress postprandial hepatic de novo lipogenesis, and decrease hepatic TG concentration. Thus both exercise and orally administered oleuropein improve insulin resistance, both of which likely involve AMPK activation. Although the mechanism of AMPK activation may be different between exercise and oleuropein, improving skeletal muscle insulin resistance is a promising strategy for the prevention and treatment of metabolic syndrome and T2D.

52.4.3 Improvement of mitochondrial dysfunction under insulin-resistant state

FIGURE 52.2 Oleuropein enhanced glucose uptake into C2C12 myotube cells independently of insulin. After 5 days of differentiation the myotube cells were incubated with oleuropein (0, 1, 10, and 100 μM; Ctrl, Ole1, Ole10, and Ole100) or 100 nM insulin (Ins) for 60 min. The cells were washed with phosphate buffer saline twice and preincubated with Krebs
Ringer bicarbonate buffer for 40 min. Then, the cells were incubated with 20 μM 2-[3H] deoxy-D-glucose (2 kBq/mL) for 30 min. Radioactivity of the cell lysate was measured using a scintillation counter, and uptake was adjusted by protein concentration measured by Bradford assay (A). Amounts of glucose incorporated into cells were evaluated by calculating the decrease in glucose concentrations in the medium after 24 h incubation (B). Glucose uptake was measured with or without insulin (C). All values are expressed as mean 6 SD (n 5 3). Statistical analysis was performed using ANOVA followed by Tukey
Kramer’s test for multiple comparisons. Values not sharing a common Roman letter are significantly different (P , .05). ANOVA, Analysis of variance.

Improvement of mitochondrial dysfunction may also reduce oxidative stress and contribute to the amelioration of insulin resistance.22 Mitochondrial dysfunction induces oxidative stress in skeletal muscles, liver, and adipose tissues.11 Increase of the reactive oxygen species (ROS) level promotes further production of mitochondrialmediated superoxide and lipid peroxidation, which ultimately results in progression of insulin resistance.23 In a recent study24 a mitochondria-targeted antioxidant MitoQ partially prevented the adiposity and the reduction of GLUT4 protein levels involved in insulin signaling observed in adipose tissue of obese rats. MitoQ also prevented the downregulation of adiponectin and GLUT 4 in a tumor necrosis factor (TNF)α-induced insulin-resistant 3T3-L1 adipocyte model. It has been reported that oleuropein treatment increased uncoupling protein gene expression and induced mitochondrial biosynthesis in primary culture of skeletal muscle cells.25 Though the mitochondrion is considered to be a

Oleuropein, olive, and insulin resistance Chapter | 52

631

FIGURE 52.3 Oleuropein enhanced GLUT4 translocation in plasma membrane independently of insulin. C2C12 cells were incubated with oleuropein or insulin for 2 h, and GLUT4 mRNA expression was measured by real-time RT-PCR (A). The plasma membrane of C2C12 cells was isolated by ultracentrifugation and sucrose gradient. GLUT4 protein level was analyzed by Western blotting and normalized by the plasma membrane protein detected by N 1 /K 1 ATPase antibody (B). Values are given as mean 6 SD (n 5 3). Statistical analysis was performed using ANOVA followed by Tukey
Kramer’s test for multiple comparisons. Values not sharing a common Roman letter are significantly different (P , .05). ANOVA, Analysis of variance; GLUT4, glucose transporter 4.

generator of ROS, oleuropein treatment also suppressed superoxide generation per mitochondrion. Therefore it can be hypothesized that oleuropein administration may have a beneficial effect on mitochondrial function and cellular oxidative metabolism. Bo´dis and Roden examined the link of the mitochondrial function of white adipose tissues with lipogenesis and lipolysis in insulin-sensitive and insulin-resistant humans.26 They have indicated that abnormal mitochondrial function in human white adipose tissues consequently induces inflammation, enhances the release of cytokines and lipid metabolites (free fatty acids and glycerol), and promotes ectopic lipid accumulation and decreases glucose uptake in liver and skeletal muscles, which are linked to systemic insulin resistance. Although the effects of oleuropein on adipocytes are poorly studied, improving mitochondrial function and/or GLUT4mediated glucose uptake would be very promising therapeutic strategies for improving and preventing systemic insulin resistance.

52.5 Effects of metabolites of oleuropein on insulin resistance HT, a major metabolite of oleuropein, is an olive component that may be involved in the low incidence of CVD and T2D in Mediterranean countries.27 In clinical, animal, and cell culture studies, HT and its lipophilic derivatives have been reported to improve lipid profiles, blood glucose levels, and insulin sensitivity through antioxidative and antiinflammatory effects.

In HFD-induced insulin-resistant NAFLD model rats, HT has been shown to protect the liver from damage associated with fat accumulation and improve glucose tolerance.28 In this study, three groups of rats were served either a standard diet, HFD, or HFD with HT administration (10 mg/kg/day). The OGTT at the fifth week indicated that HT treatment significantly reduced blood glucose concentrations at 120 min after glucose loading. After 6 weeks, fasting serum levels of insulin, HOMA-IR, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cholesterol were remarkably reduced by HT treatment. HT also increased peroxisome proliferator
activated receptor alpha, fibroblast growth factor-21, carnitine palmitoyltransferase 1 mRNA, and ACC phosphorylation in the metabolically impaired liver. HT and other olive oil phenols have been reported to reduce the plasma glucose level in alloxan-induced diabetic rats by reducing free radicals and moderating oxidative stress.29 In HFD-induced obese mice and db/db diabetic mice, HT (10 mg/kg/day) administration significantly reduced the fasting glucose concentrations, similarly to a traditional antidiabetic drug, metformin (225 mg/kg/day).30 HT treatment remarkably reduced oxidative stress markers in liver and skeletal muscle and significantly reduced fasting serum TG, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein (LDL) cholesterol, whereas metformin had no apparent effect on the oxidative stress and the lipid profiles. HT dose dependently decreased fatty acid synthase and sterol regulatory element-binding protein 1c levels. It was suggested that HT may be a beneficial factor in regulating cholesterol and fatty acid. For more information on hepatic fatty acid and cholesterol synthesis.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

The physiological effects of HT and its lipophilic derivatives, hydroxytyrosyl acetate (HT-Ac), and ethyl hydroxytyrosyl ether (HT-Et) were compared in hypercholesterolemic rats.31 Supplementation of HT or its derivatives improved serum glucose, insulin, leptin, and

malondialdehyde levels. Interestingly, HT-Ac, which is a natural component of olive oil, was the most effective derivative. These results have suggested that the effects of olive oil polyphenols on insulin resistance might be due to antioxidant activity that suppresses oxidative stress, which is widely associated with diabetes and its complications. However, a study that validates utilization has suggested that HT plasma level after ingestion of 25 mL of EVOO is quite low, at about 50
160 nM, and that bioactivity should be carefully evaluated.32 For more information on bioavailability and bioactive effects.

52.6 Comparisons of olive oils with other edible oils Oleuropein is a polyphenol specific to olive oil and is hardly contained in other edible oils. There are reports that other edible oils improve insulin resistance. In a randomized controlled trial the administration of flaxseed oil to patients with gestational diabetes mellitus reduced insulin levels and insulin resistance compared to placebo.33 These effects are reported to be attributed to α-linolenic acid, an n-3 fatty acid contained in flaxseed oil. It has been reported that supplementation of fish oil34 and crustacean egg oil,35 which are rich in n-3 fatty acids, also improves insulin resistance. However, these two oils are not as commonly used in daily diets as olive oil.

52.7 Conclusion

FIGURE 52.4 Oleuropein activated the phosphorylation of AMPK but not Akt. Lysates from the C2C12 cells treated with oleuropein (1, 10, and 100 μM; Ctrl, Ole1, Ole10, Ole100) or 100 nM insulin (Ins) for 20 min were analyzed by Western blotting using anti-Akt and antiphosphor-Akt (Ser 473) antibodies (A). Lysates from C2C12 cells treated with 0, 1, 10, and 100 μM oleuropein or 500 μM AICAR, the AMPK activator, for 24 h were analyzed using anti-AMPK and antiphosphor-AMPK (Thr 172) antibodies (B). Values are given as mean 6 SD (n 5 3). Statistical analysis was performed using ANOVA followed by Tukey
Kramer’s test for multiple comparisons. Values not sharing a common Roman letter are significantly different (P , .05). AMPK, AMP-activated protein kinase; ANOVA, analysis of variance.

Olive oil polyphenols, especially oleuropein, contribute to postprandial nutrient metabolism, including insulin sensitivity. It has been revealed that the mechanism of action is not the phosphorylation of Akt in the insulin signaling pathway but the regulation of AMPK-mediated translocation of GLUT4 to the membrane. Unfortunately, how oleuropein regulates the recruitment of cytoplasmic GLUT4-containing vesicles to the plasma membrane is poorly understood. Interestingly, both oleuropein and exercise promote glucose uptake into skeletal muscles via phosphorylation of AMPK. Eating habits and exercise are core elements for improving lifestyle-related diseases. Even if exercise cannot be performed satisfactorily for various reasons, the ingestion of olives can be expected to FIGURE 52.5 Oleuropein-induced GLUT4 translocation in skeletal muscle of mice with HFD-induced obesity. Representative confocal fluorescence images with anti-GLUT4 antiserum in ND-, HFD-, and oleuropein-fed mice (ND, HFD, OLE). FITC-labeled secondary antibody was used to detect GLUT4, and nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). GLUT4, Glucose transporter 4; HFD, high-fat diet; ND, normal diet.

Oleuropein, olive, and insulin resistance Chapter | 52

provide an alternative effect to exercise on insulin resistance. Improvement of insulin resistance is one of the most effective health-contributing mechanisms of olive and oleuropein. It is hoped that future studies will result in more beneficial evidence for elderly and preclinical people.

Mini-dictionary of terms

Randomized controlled trial (RCT)

Oleuropein

Oleuropein is a polyphenol mainly contained in olive leaves and is well known to have high antioxidant activity. Oleuropein and its metabolites are the major components of polyphenols in olive oil. Hydroxytyrosol (HT) HT, a major metabolite of oleuropein, is one of the most likely contributors to enhancing the healthiness of the Mediterranean diet. Insulin sensitivity Insulin, secreted from pancreatic β cells, is a hormone that acts on target organs such as skeletal muscle, adipose tissue, and liver to promote glucose uptake into cells. Insulin sensitivity is the ability of target organs to perceive and respond to insulin. Insulin resistance Insulin resistance is a condition in which insulin sensitivity is reduced and the effects of insulin are not fully exerted despite sufficient blood insulin concentration. Insulin resistance is known to be associated with various physiological disorders. Type 2 diabetes (T2D) Diabetes is divided into several types according to the cause. T2D is caused by a decrease in insulin secretion and sensitivity due to lifestyle and genetic factors. Type 1 diabetes, on the other hand, is caused by disruption of pancreatic β cells and very little or no secretion of insulin. Nonalcoholic fatty liver disease NAFLD encompasses a spectrum (NAFLD) of diseases ranging from simple hepatic steatosis (NAFL) to nonalcoholic steatohepatitis (NASH), with increasing levels of inflammation and fibrosis. It is known that when NASH becomes severe, it progresses to cirrhosis and hepatic cancer. Glucose transporter 4 (GLUT4) Glucose transporters are membrane proteins that take up glucose into cells by facilitated

Oral glucose tolerance test (OGTT)

Homeostasis model assessment of insulin resistance (HOMAIR)

633

diffusion. GLUT4 is expressed in adipose tissues, skeletal muscles, and cardiac muscles. For glucose uptake into cells, the recruitment of cytoplasmic GLUT4containing vesicles to the cell membrane is essential. A randomized controlled trial is one of the best ways of keeping the bias of the researchers out of the data and making sure that a study gives the fairest representation of several clinical interventions. In evidence-based medicine, this is a research method with a high level of evidence, following metaanalysis in which multiple randomized controlled trials are collected and analyzed. A test to determine the ability to maintain normal blood glucose levels. After oral administration of glucose, blood is collected at regular intervals to examine changes in blood glucose and insulin levels. HOMA-IR is an index indicating the degree of insulin resistance. It is calculated from the concentrations of insulin and glucose in fasting plasma.

References 1. Roden M, Shulman GI. The integrative biology of type 2 diabetes. Nature. 2019;576(7785):51
60. Available from: https://doi.org/ 10.1038/s41586-019-1797-8. 2. Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest. 2000;106. Available from: https://doi.org/10.1172/JCI10842. 3. Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science (80-). 2003;300(5622):1140
1142. Available from: https://doi.org/ 10.1126/science.1082889. 4. Violi F, Loffredo L, Pignatelli P, et al. Extra virgin olive oil use is associated with improved post-prandial blood glucose and LDL cholesterol in healthy subjects. Nutr Diabetes. 2015;57:e172. Available from: https://doi.org/10.1038/nutd.2015.23. 5. de Bock M, Derraik JGB, Brennan CM, et al. Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: a randomized, placebo-controlled, crossover trial. PLoS One. 2013;8(3). Available from: https://doi.org/10.1371/journal. pone.0057622. 6. Carnevale R, Pignatelli P, Nocella C, et al. Extra virgin olive oil blunt post-prandial oxidative stress via NOX2 down-regulation. Atherosclerosis. 2014;235(2):649
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7. Rains JL, Jain SK. Oxidative stress, insulin signaling, and diabetes. Free Radic Biol Med. 2011;50(5):567
575. Available from: https://doi.org/10.1016/j.freeradbiomed.2010.12.006. 8. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462
1470. Available from: https://doi.org/10.2337/diacare.22.9.1462. 9. Ou HY, Cheng JT, Yu EH, Wu T. Metformin increases insulin sensitivity and plasma beta-endorphin in human subjects. Horm Metab Res. 2006;38(2):106
111. Available from: https://doi.org/10.1055/ s-2006-925128. 10. Vezza T, Rodrı´guez-Nogales A, Algieri F, et al. The metabolic and vascular protective effects of olive (Olea europaea L.) leaf extract in diet-induced obesity in mice are related to the amelioration of gut microbiota dysbiosis and to its immunomodulatory properties. Pharmacol Res. 2019;150:104487. Available from: https://doi.org/ 10.1016/j.phrs.2019.104487. 11. Hussain T, Tan B, Murtaza G, et al. Flavonoids and type 2 diabetes: evidence of efficacy in clinical and animal studies and delivery strategies to enhance their therapeutic efficacy. Pharmacol Res. 2020;152:104629. Available from: https://doi.org/10.1016/j. phrs.2020.104629. 12. Jime´nez-Sa´nchez C, Olivares-Vicente M, Rodrı´guez-Pe´rez C, et al. AMPK modulatory activity of olive-tree leaves phenolic compounds: bioassay-guided isolation on adipocyte model and in silico approach. PLoS One. 2017;12:e0173074. Available from: https:// doi.org/10.1371/journal.pone.0173074. 13. Kim Y-B, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB. Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest. 1999;104:733. 14. Hadrich F, Garcia M, Maalej A, et al. Oleuropein activated AMPK and induced insulin sensitivity in C2C12 muscle cells. Life Sci. 2016;151:167
173. Available from: https://doi.org/10.1016/j. lfs.2016.02.027. 15. Lee YM, Lee JO, Jung J-H, et al. Retinoic acid leads to cytoskeletal rearrangement through AMPK-Rac1 and stimulates glucose uptake through AMPK-p38 MAPK in skeletal muscle cells. J Biol Chem. 2008;283(49):33969
33974. Available from: https://doi. org/10.1074/jbc.M804469200. 16. Fujiwara Y, Tsukahara C, Ikeda N, et al. Oleuropein improves insulin resistance in skeletal muscle by promoting the translocation of GLUT4. J Clin Biochem Nutr. 2017;61(3):196
202. Available from: https://doi.org/10.3164/jcbn.16-120. 17. Alkhateeb H, Al-Duais M, Qnais E. Beneficial effects of oleuropein on glucose uptake and on parameters relevant to the normal homeostatic mechanisms of glucose regulation in rat skeletal muscle. Phytother Res. 2018;32(4):651
656. Available from: https:// doi.org/10.1002/ptr.6012. 18. Hayashi T, Wojtaszewski JFP, Goodyear LJ. Exercise regulation of glucose transport in skeletal muscle. Am J Physiol. 1997;273. Available from: https://doi.org/10.1152/ajpendo.1997.273.6.e1039. 6 36-6. 19. Kennedy JW, Hirshman MF, Gervino EV, et al. Acute exercise induces GLUT4 translocation in skeletal muscle of normal human subjects and subjects with type 2 diabetes. Diabetes. 1999;48(5):1192
1197. Available from: https://doi.org/10.2337/diabetes.48.5.1192.

20. Musi N, Fujii N, Hirshman MF, et al. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes. 2001;50(5):921
927. Available from: https://doi.org/10.2337/diabetes.50.5.921. 21. Rabøl R, Petersen KF, Dufour S, Flannery C, Shulman GI. Reversal of muscle insulin resistance with exercise reduces postprandial hepatic de novo lipogenesis in insulin resistant individuals. Proc Natl Acad Sci USA. 2011;108(33):13705
13709. Available from: https://doi.org/10.1073/pnas.1110105108. 22. Gonzalez-Franquesa A, Patti M-E. Insulin resistance and mitochondrial dysfunction. Adv Exp Med Biol. 2017;982:465
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12072. Available from: https://doi.org/10.1096/fj.201900347RR. 25. Kikusato M, Muroi H, Uwabe Y, Furukawa K, Toyomizu M. Oleuropein induces mitochondrial biogenesis and decreases reactive oxygen species generation in cultured avian muscle cells, possibly via an up-regulation of peroxisome proliferator-activated receptor γ coactivator-1α. Anim Sci J. 2016;87(11):1371
1378. Available from: https://doi.org/10.1111/asj.12559. 26. Bo´dis K, Roden M. Energy metabolism of white adipose tissue and insulin resistance in humans. Eur J Clin Invest. 2018;48(11). Available from: https://doi.org/10.1111/eci.13017. 27. Peyrol J, Riva C, Amiot MJ. Hydroxytyrosol in the prevention of the metabolic syndrome and related disorders. Nutrients. 2017;9(3). Available from: https://doi.org/10.3390/nu9030306. 28. Pirozzi C, Lama A, Simeoli R, et al. Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD. J Nutr Biochem. 2016;30:108
115. Available from: https://doi.org/10.1016/j. jnutbio.2015.12.004. 29. Hamden K, Allouche N, Damak M, Elfeki A. Hypoglycemic and antioxidant effects of phenolic extracts and purified hydroxytyrosol from olive mill waste in vitro and in rats. Chem Biol Interact. 2009;180(3):421
432. Available from: https://doi.org/10.1016/j. cbi.2009.04.002. 30. Cao K, Xu J, Zou X, et al. Hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice. Free Radic Biol Med. 2014;67:396
407. Available from: https://doi.org/10.1016/j.freeradbiomed.2013.11.029. 31. Tabernero M, Sarria´ B, Largo C, et al. Comparative evaluation of the metabolic effects of hydroxytyrosol and its lipophilic derivatives (hydroxytyrosyl acetate and ethyl hydroxytyrosyl ether) in hypercholesterolemic rats. Food Funct. 2014;5(7):1556
1563. Available from: https://doi.org/10.1039/c3fo60677e. 32. Miro-Casas E, Covas MI, Farre M, et al. Hydroxytyrosol disposition in humans. Clin Chem. 2003;49(6 Pt 1):945
952. 33. Jamilian M, Tabassi Z, Reiner Z, et al. The effects of omega-3 fatty acids from flaxseed oil on genetic and metabolic profiles in patients with gestational diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Br J Nutr. 2020;. Available from: https:// doi.org/10.1017/S0007114519003416.

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34. Martins A, Crisma A, Masi L, et al. Attenuation of obesity and insulin resistance by fish oil supplementation is associated with improved skeletal muscle mitochondrial function in mice fed a high-fat diet. J Nutr Biochem. 2018;55:76
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35. Hu S, Wang J, Yan X, et al. Egg oil from portunus trituberculatus alleviates insulin resistance through activation of insulin signaling in mice. Appl Physiol Nutr Metab. 2019;44 (10):1081
1088. Available from: https://doi.org/10.1139/ apnm-2018-0718.

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Section 3.3

Oleic acid

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Chapter 53

Oleic acid—the main component of olive oil on postprandial metabolic processes Sergio Lopez1, Beatriz Bermudez1, Sergio Montserrat-de la Paz, Yolanda M. Pacheco, Almudena Ortega-Gomez, Lourdes M. Varela, Ana Lemus-Conejo, Maria C. Millan-Linares, Maria A. Rosillo, Rocio Abia and Francisco J.G. Muriana Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Campus Universitario Pablo de Olavide, Seville, Spain

List of abbreviations AUC CHD FFA FVIIa G GTTTM HOMA I IGI IR IRS ISI MCP-1/ CCL2 MUFA NF-κB OA OGIS OGTT OPG PA PAI-1 RANK RANKL ROO rQUICKI SFA TF TG tPA

area under the curve coronary heart disease free fatty acids activated factor VII glucose glucose and triglyceride tolerance test meal homeostatic model assessment insulin insulinogenic index insulin resistance insulin receptor substrate insulin sensitivity index monocyte chemoattractant protein-1/chemokine C-C motif ligand 2 monounsaturated fatty acids nuclear factor kappa-light-chain-enhancer of activated B cells oleic acid oral glucose insulin sensitivity oral glucose tolerance test osteoprotegerin palmitic acid plasminogen activator inhibitor-1 receptor activator of NF-κB RANK ligand refined olive oil revised-quantitative insulin sensitivity check index saturated fatty acids tissue factor triglycerides tissue plasminogen activator

53.1 Introduction Olive oil is a fruit oil obtained from the olive (Olea europaea; family Oleaceae along with lilacs, jasmine, and ash trees), a traditional tree crop of the Mediterranean Basin, and can be consumed in the natural state known as extra and/or virgin olive oil, or as a refined product. One of the main differences of olive oil with other oils is its high content in oleic acid (OA) (Tables 53.1 and 53.2), which makes up 56%
84% of total fatty acids complexed in the form of triglycerides (TG). Evidence from epidemiological studies suggests that a higher proportion of monounsaturated fatty acids (MUFA), notably OA, in the diet is linked with a reduction in the risk of coronary heart disease (CHD). To achieve this benefit, olive oil as a major source of OA is to replace a similar amount of saturated fat and not increase the total number of daily calories. The biochemical bases of the ameliorative effect of OA are thought to be modification of cell membranes,1 plasma lipid and lipoprotein concentrations,2,3 inhibition of coagulation,4,5 improvement of glucose homeostasis,6,7 blood pressure,8 and attenuation of inflammation9,10 and oxidative status11,12 in fasting conditions. Some experimental studies have also noticed the potential bioactivity of OA against the dysfunctional liver13 and white adipose tissue,14 and to fight acute ischemic stroke15 and chronic neurodegeneration by its beneficial role on membrane fluidity of synaptosomes located in the subcortical region of the forebrain16 and on function of mitochondria and peroxisomes in microglia.17 In addition, mice fed on a high-fat diet enriched in OA but not in palmitic acid (PA)

1. To be considered as equal first author. Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00034-1 © 2021 Elsevier Inc. All rights reserved.

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TABLE 53.1 Key facts of oleic acid. 1. Oleic acid is the most widely distributed and plentiful of all fatty acids. It is called a monounsaturated omega-9 fatty acid because of the single double bond at the carbon 9 position. 2. Oleic acid is essential to the human body. It is involved in cellular energy production but also in a myriad of metabolic functions. 3. Oleic acid lowers the risk of a heart disease and aids in cancer and neurodegenerative disease prevention. 4. Current dietary recommendations require that oleic acid content in an optimized healthy diet is maintained at about 20% of total energy, while the average contribution of polyunsaturated and saturated fatty acids to dietary energy is reduced to no more than about 5% and 10%, respectively. 5. The olive oil pressed from the ripe fruit of the olive (Olea europaea) is the best-known natural source of oleic acid. This table lists the key facts of oleic acid including the basic concept of oleic acid, the role of oleic acid in maintaining health, the nutritional recommendations of oleic acid, and the primary dietary source of oleic acid.

TABLE 53.2 Structure and some properties of oleic acid. Oleic acid

IUPAC name

(9Z)-octadec-9-enoic acid

Other names

(9Z)-octadecenoic acid; (Z)-Octadec-9-enoic acid; cis-9-octadecenoic acid; cis-Δ9-octadecenoic acid; 18:1 cis-9

CAS number

[112-80-1]

SMILES

CCCCCCCC\C 5 C/CCCCCCC(OH) 5 O

Molecular formula

C18H34O2

Molar mass

282.4614 g/mol

Appearance

Pale yellow or brownish yellow oily liquid with lard-like odor

Density

0.895 g/mL

Melting point

13 C
14 C (286K)

Boiling point

360 C (633K) (760 mmHg)

Solubility in water

Insoluble in water; soluble in methanol

This table summarizes the chemical structure and property data of oleic acid in its standardized state (at 25 C, 100 kPa).

protects from reduced mesolimbic dopamine function during obesity.18 Close to these exciting observations, impaired cognitive function can be reversed by lowering the ingestion of PA via substitution with OA in young women.19

More recently, a body of evidence has grown that supports the hypotheses that postprandial metabolism of dietary fats plays a causal role in the pathogenesis and progression of CHD. However, the relevance of OA

Oleic acid—the main component of olive oil on postprandial metabolic processes Chapter | 53

TF netAUC (ng h/mL)

OA/PA

40 TF (ng/mL)

regarding saturated fatty acids (SFA), notably PA, in dietary fats to influence postprandial metabolic processes is only partially understood. In this chapter, we describe that the proportion of OA and PA in olive oil, compared to other dietary fats, could provide benefits and be considered as a nutritional determinant, at least, for the regulation of coagulation, glucose homeostasis, and inflammation during postprandial state.

30

0

2

4

30

6

8

r = −0.967

20

r = −0.998

10 0

0

2

4

6

8

7

8

20

ROO =

The extrinsic clotting cascade is thought to play a crucial role in the shift of the hemostatic balance, and it is triggered by tissue factor (TF) bound to or shed from blood cells and the disrupted endothelium. TF is a small integral transmembrane glycoprotein that acts as a cofactor in the proteolytic activity of factor VII/VIIa toward factor IX and factor X. Increases in the levels of TF are reflected by the increase in plasma fibrinogen levels. In contrast, plasminogen and its activators, including tissue plasminogen activator (tPA), mediate the proteolytic degradation of fibrin. Indeed, the endothelial release of tPA is considered as a primary endogenous defense mechanism against thrombosis. Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily, is not only the main physiologic inhibitor of tPA in the fibrinolytic system but also PAI-2, PAI-3, α2-macroglobulin, and C1 esterase inhibitor. Thus endogenous tPA is rapidly neutralized by PAI-1, which binds to the active site of tPA and forms a stable 1:1 stoichiometric (tPA/ PAI-1) complex. Despite earlier observations indicating that thrombotic complications might be mediated by high levels of circulating TF20 and accelerated during postprandial lipemia,21 there is little available data regarding the postprandial effects of dietary fats on TF. In contrast, much more information exists regarding the effects of dietary fat composition on postprandial levels of activated factor VII (FVIIa).22 Activation of FVII by TF represents a critical event in thrombogenesis. Indeed, the transient rise in FVIIa after a fat-rich meal is generally detectable 2
3 h postprandially and it persists for at least 8 h, thus displaying dose
response characteristics similar to those reported for TF in healthy subjects (Fig. 53.1). The increase in FVIIa is correlated with fasting TG levels, but not to the postprandial levels of TG.23 Interestingly, the main dietary determinants of postprandial changes in FVIIa are OA and PA,24 which agrees with the inverse correlation between the postprandial response for TF and the OA-to-PA ratio (MUFA/SFA) in dietary fats.25 The high content of OA in olive oil determines significantly lower TF expression than other dietary fats.

40

MUFA/SFA

10

53.2 Oleic acid on postprandial thrombogenesis

641

0

0

1

OA/PA MUFA/SFA 6.83 5.43

2

3

4 5 Time (h)

6

FIGURE 53.1 Postprandial levels of TF after the ingestion of a meal containing ROO in healthy men. The ingestion of a meal containing ROO elicits a postprandial response (peaking at 2 h) for TF in healthy men. This effect, by means of the TF netAUC, correlated with OA-toPA or MUFA-to-SFA ratio in dietary fats, including ROO (x), highpalmitic sunflower oil (’), and butter (&) (see inset). Values are means 6 SD (n 5 14). MUFA, Monounsaturated fatty acids; netAUC, net area under the curve; OA, oleic acid; PA, palmitic acid; ROO, refined olive oil; SFA, saturated fatty acids; TF, tissue factor.

53.3 Oleic acid on postprandial fibrinolysis Circulating PAI-1 is derived from a variety of sources, including the vascular endothelium, adipose tissue, and liver, and it is involved in the onset of obesity, diabetes, and CHD. It was described that high-fat meals could induce a peak of PAI-1 2 h postprandially in healthy subjects.25 Similar to TF, the postprandial response for PAI-1 was significantly correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats. Olive oil, providing higher OA/PA and MUFA/SFA ratios than butter, induced a lower PAI-1 net area under the curves (netAUC) (Fig. 53.2). It was found that a temporary increase in postprandial levels of PAI-1 antigen leads to an impaired fibrinolytic activity after SFA (PA)-enriched meals,26 whereas improved fibrinolytic activity has been associated with a decline in postprandial PAI-1 after high-MUFA (OA) meals.27 Together these observations could have important clinical consequences, as the morning peak in PAI-1 antigen corresponds with the circadian peak in the incidence of acute myocardial infarction. In prospective clinical and multicenter studies of angina pectoris and postinfarction patients, the tPA antigen could serve to predict subsequent acute coronary syndromes. Encoded by a gene on chromosome 8, tPA is a 68 kDa serine protease of 530 amino acids and the endothelium is its principal site of generation. Acute release of tPA and PAI-1 antigens has a U-shaped characteristic, according to their strong diurnal variations in the net fibrinolytic activity. There is evidence that the ingestion of high-fat meals leads to a marked decline in postprandial tPA levels in healthy subjects.25 However, the postprandial response of tPA

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40

PAI–1 (μg/L)

OA/PA MUFA/SFA 30 20

–76.13

6.83

5.43

–64.95

2.36

2.42

–55.17

0.82

0.48

10 ROO

0 0

1

2

3

4

5

6

7

8

PAI-1 netAUC (μg h/L)

Time (h) FIGURE 53.2 Postprandial levels of PAI-1 after the ingestion of a meal containing ROO in healthy men. The ingestion of a meal containing ROO elicits a postprandial response (peaking at 2 h) for PAI-1 in healthy men, though a marked decline from 0 to 8 h denotes the strong diurnal (characteristic U-shaped) variations in net fibrinolytic activity. The values for PAI-1 netAUC decreased when increased the OA-to-PA or MUFA-to-SFA ratio in dietary fats, following the order: ROO (white bar) . high-palmitic sunflower oil (gray bar) . butter (black bar). Values are means 6 SD (n 5 14). MUFA, Monounsaturated fatty acids; netAUC, net area under the curve; OA, oleic acid; PA, palmitic acid; PAI-1, plasminogen activator inhibitor-1; ROO, refined olive oil; SFA, saturated fatty acids.

appears to be not directly dependent on dietary MUFA (OA) or SFA (PA) content, but on postprandial TG levels elicited by dietary fats. Remarkably, fasting tPA antigen is linked to the metabolic syndrome, and it is positively related to fasting TG and insulin, as well as to the waist-to-hip ratio in familial combined hyperlipidemia.28 This component of the endogenous fibrinolytic system also predicts first CHD events in the Framingham Heart Study.29 These data are consistent with the view that the OAto-PA ratio (MUFA/SFA) in dietary fats may have a regulatory influence on certain thrombogenic and fibrinolytic markers during the postprandial state in healthy subjects. A decrease in the postprandial levels of TF (antithrombotic effect) and PAI-1 (profibrinolytic effect) was observed after the ingestion of olive oil when compared to other dietary fats, such as butter or high-palmitic sunflower oil. We suggest that the Mediterranean diet can reduce cardiovascular events in part due to the high OA content in olive oil (high-OA-to-PA and MUFA/SFA ratios) and its impact on postprandial hemostatic system.

53.4 Oleic acid on postprandial β-cell function and insulin sensitivity The loss of β-cell function and insulin sensitivity is known to contribute to the development of diabetes, a metabolic disorder that develops over a course of months to years. It has been hypothesized that insulin-resistance syndromes might be a postprandial phenomenon linked to acute dietary fat metabolism, as suggested from frequently sampled intravenous glucose tolerance test data.30 Exaggerated postprandial triglyceridemia is indeed an inherent feature of diabetic dyslipidemia and it is frequently found even in diabetic patients with normal fasting TG.31 There is also strong evidence that SFA selectively desensitizes the response of peripheral tissue to insulin, whereas unsaturated fatty acids

(including OA) may counteract this effect.32 Such phenomena would be in accordance with studies linking the nature of dietary fats to dysfunctions in insulin secretion and the frequency of type 2 diabetes. However, it remains unclear to what extent dietary OA (MUFA) and PA (SFA) influence the postprandial control of insulin secretion and resistance, even in healthy subjects. Here, we show that empirical indices of β-cell function and insulin sensitivity (Box 53.1) could postprandially discriminate the influence of dietary fats supplied simultaneously to a single individual in conjunction with a relatively low-carbohydrate load from a test meal similar to the oral glucose tolerance test (OGTT), which was named the glucose and TG tolerance test meal (GTTTM).33 This novel test was relatively low in carbohydrates to circumvent the confounding and masking effects of adding carbohydrates to a high-fat meal. In healthy subjects with similar fasting insulin sensitivity assessed by HOMA-IR, rQUICKI, and basalBelfiore indices for glycemia and blood free fatty acids (FFA), olive oil (MUFA)- and butter (SFA)-enriched meals elicited superimposable postprandial glucose responses.33 However, the higher content of OA in olive oil decreased the postprandial insulinemic peak and the AUC for insulin and FFA (Fig. 53.3). Even a high-fat meal increased postprandial β-cell function when assessed by the insulinogenic index (IGI), IGI/HOMA-IR, AUCI/ AUCG and HOMA-B, and decreased postprandial insulin sensitivity as assessed by ISGTTTM, OGISGTTTM, and the postprandial Belfiore indices for glycemia and blood FFA, subjects became less insulin resistant postprandially as the proportion of OA versus PA increased in dietary fats (Fig. 53.4). It was also discovered that when the early postprandial insulin response was used as a measure of β-cell function, it decreased according to the high OA-toPA and MUFA/SFA ratios in olive oil. These observations were corroborated in subjects with high fasting TG concentrations.34 A more detailed analysis on clustering

Oleic acid—the main component of olive oil on postprandial metabolic processes Chapter | 53

643

BOX 53.1 Empirical indices of β-cell function and insulin sensitivity. Among the empirical indices of β-cell function, the insulinogenic index (IGI) is a surrogate measure of first-phase insulin secretion, and it was calculated as the difference between the postprandial insulin peak (t 5 60 min) and the basal insulin in relation to the difference in glucose (IGI 5 ΔI0
60 /ΔG0
60). We also calculated the ratio of the IGI to the homeostasis model assessment of insulin resistance (IGI/HOMA-IR), which gives an adjusted measure of β-cell function that accounts for variations in insulin sensitivity. The ratio of the insulin to glucose areas under the curve (AUC I /AUC G ) is significantly correlated with glucose sensitivity and it is a sophisticated parameter to describe the β-cell secretory process (Mari et al., 2001). Finally, the homeostasis model assessment of β-cell function (HOMA-B) as I0 3 3.33/(G0 2 3.5) was also used, and we assessed β-cell function during GTTTM by extending the values of HOMA-B to those at 60 min.We used three surrogate measures of insulin-mediated glucose disposal: (1) the homeostasis model assessment of insulin resistance (HOMAIR 5 I0 3 G 0 divided by 22.5) (Matthews et al., 1985); (2) the revised-quantitative insulin sensitivity check index (rQUICKI 5 1/[log I 0 1 log G0 1 log FFA 0 ]) (Perseghin et al., 2001); and (3) the basal insulin sensitivity (IS) index (ISI0 ) for glycemia and blood FFA proposed by Belfiore et al. (1998; see later).The basal IS indices (ISI 0) for glycemia and blood FFA were calculated according to the following equations (Belfiore et al., 1998): ISIðGÞ0 5

2 ðI0 UG0 Þ 1 1

ISIðFFAÞ0 5

2 ðI0 UFFA0 Þ 1 1

where I0, G0, and FFA0 refer to fasting levels (t 5 0) of insulin, glucose, and FFA, respectively, and which are expressed by making the mean normal value equal to 1. For convenience, fasting values after our control diet were considered as the mean normal values.The classical minimal model of glucose kinetics described by an equation to define the rate of appearance of glucose in systemic circulation after the ingestion of a mixed meal (Caumo et al., 2000), and that primarily yields the following expression for IS (ISGTTTM): ISGTTTM 5


t0  
480 



480 480 f UGDU AUC ðGt0 2G0 Þ=Gt0 0 2 AUC ðG480 2Gt0 Þ=G480 t0 = AUCðGt0 2G0 Þt0 2 GEUAUC ðGt 2G0 Þ=Gt 0 0 2 AUCðG480 2Gt0 Þt0 480 AUCðIt0 2I0 Þt0 0 2 AUCðI480 2It0 Þt0

because glycemia and insulinemia displayed an undershoot below the basal level during part of the test. The values for t0 can be evaluated in each individual by linearly interpolating the glucose or insulin samples that immediately precede and follow the crossing of the baseline glucose or insulin level. The AUC of ΔG/G, ΔG, and ΔI is calculated separately in the intervals 0 2 t0 and t0 2 N. The negative AUC calculated in the interval t0 2 N is subtracted from the positive AUC calculated in the interval 0 2 t0. In this equation, f is the fraction of ingested glucose that actually appears in systemic circulation (0.87); GD is the dose of ingested glucose; AUC denotes the area under the curves of glycemia (G, mg/mL) and insulinemia (I, μU/mL) in the frame from t 5 0 to the end of the test (t 5 480 min); GE is the glucose effectiveness (3.7 3 1022 min21  dL  kg21), which is obtained as the product of SG (fractional GE) 5 0.028 min21 and V (glucose distribution volume) 5 1.34 dL/kg. The AUC of ΔG/G and ΔG is calculated separately in the intervals 0 2 t0 and t0 2 480 min. Estimates of t0, the time when the glucose or insulin concentration crosses and falls below the baseline level. The IS index, ISGTTTM, has the same units (min21  dL  kg21/ μU/mL) as the analogous OGTT.The OGIS index (OGISGTTTM) was derived by using the 3-h OGIS equation (Mari et al., 2001). This method provides an IS index that closely correlates with insulin-stimulated glucose metabolism (the M-value) during euglycemic clamps. The OGIS (mL/m2/min) is a dynamic descriptor of IS and it is calculated according to the following model-derived formula:  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 B 1 B2 1 4p5p6ðG120 2 GCLAMPÞClGTTTM OGISGTTTM 5 2 where p5 and p6 are fixed rate constants (11.5 3 1023 and 117, respectively, in SI units); G120 is the plasma concentration of glucose measured at 120 min during GTTTM; GCLAMP is the clamp glucose concentration (normally 90 mg/dL); ClGTTTM (the glucose clearance during the test) and B are obtained by means of the following equations:


 


p1 UGD 2 V ðG180 2 G120 Þ=60 =G120 1 p3 =G0 ClGTTTM 5 p4 B 5 p5 ðG120 2 GCLAMP Þ 1 1 UClGTTTM I120 2 I0 1 p2 where GD is the dose of glucose ingested (in g/m2 ); V represents the total glucose distribution volume (10 L/m 2); p 1, p 2, p 3 , and p 4 are fixed rate constants of 2.89, 1618, 779, and 2642, respectively; G0 , G120, and G180 are the plasma concentrations of glucose measured at 0, 120, and 180 min; whereas I0 and I 120 are the levels of plasma insulin measured at 0 and 120 min during GTTTM. The procedure to calculate the OGISGTTTM was downloaded from http://www.ladseb.pd.cnr. it/bioing/ogis/home.html.The ISI GTTTM was calculated according to equations proposed by Belfiore et al. (1998). Because insulin sensitivity is inversely proportional to the increase in both insulin and glucose, this index combines the changes in glucose and insulin into one value [ISI(G) GTTTM]. The previous considerations also apply to a formula which should (Continued )

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

BOX 53.1 (Continued) express the insulin sensitivity of blood FFA, in which case the two factors to be combined are blood insulin and FFA [ISI (FFA) GTTTM ]. The following equations were applied: ISIðGÞGTTTM 5

2 ðAUCG UAUCI Þ 1 1

ISIðFFAÞGTTTM 5

2 ðAUCFFA UAUCI Þ 1 1

where AUCG and AUCI are the area under the curves of glycemia (mmol/L), and insulinemia (μU/mL), in the frame from t 5 0 to the end of the test (t 5 480 min), taking the mean normal value as the unit value. For convenience, postprandial responses after our control meal were considered as the mean normal values. These formulae give values around 1 in subjects with normal IS, and they have been favorably compared with other IS indices obtained from OGTT and euglycemic clamps with good results (Belfiore et al., 1998). References: Belfiore, F., Iannello, S., Volpicelli, G., 1998. Insulin sensitivity indices calculated from basal and OGTT-induced insulin, glucose, and FFA levels. Mol. Genet. Metab. 63, 134
141. Caumo, A., Bergman, R.N., Cobelli, C., 2000. Insulin sensitivity from meal tolerance tests in normal subjects: a minimal model index. J. Clin. Endocrinol. Metab. 85, 4396
4402. Mari, A., Pacini, G., Murphy, E., Ludvik, B., Nolan J.J., 2001. A model-based method for assessing insulin sensitivity from the oral glucose tolerance test. Diabetes Care 24, 539
548. Matthews, D.R., Hosker, J.P., Rudenski, A.S., Taylor, B.A., Treacher, D.F., Turner, R.C., 1985. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412
419. Perseghin, G., Caumo, A., Caloni, M., Tesolin, G., Luzi, L., 2001. Incorporation of the fasting plasma FFA concentration into QUICKI improves its association with insulin sensitivity in nonobese individuals. J. Clin. Endocrinol. Metab. 86, 4776
4781. This table shows some empirical indices of β-cell function and insulin sensitivity, and how they can be calculated.

OA/PA MUFA/SFA ROO = Butter =

6.83 0.82

Insulin netAUC FFA netAUC (pmol h/L) (μmol h/L)

5.43 0.48

494 626

82 73

3420 4610

379 326

Insulin (pmol/L)

250 200 150 100

53.5 Possible mechanisms by which oleic acid is acting on postprandial glucose homeostasis

50 0 1000

FFA (μmol/L)

of postprandial β-cell function and insulin sensitivity by using high-fat meals containing a panel of SFA (PA and stearic acid), MUFA (palmitoleic acid and OA), and PUFA (linoleic and α-linolenic acids) revealed the specific involvement of OA and PA, including a successful discrimination between beneficial effects of OA and adverse effects of PA, on postprandial glycemic control, both in healthy subjects and in those with hypertriglyceridemia at fasting.35

800 600 400 200 0

0

1

2

3

4

5

6

7

8

Time (h) FIGURE 53.3 Postprandial levels of insulin and FFA after the ingestion of a meal containing ROO or butter in healthy men. The ingestion of a meal containing ROO or butter elicits a postprandial response (peaking at 2 h) for insulin in healthy men. As expected, FFA levels were suppressed following meal ingestion (ROO . butter); however, FFA clearance was faster with ROO when compared to butter. The values for insulin and FFA netAUC decreased when OA-to-PA or MUFA-to-SFA ratio in dietary fats was increased. Values are means 6 SD (n 5 14). FFA, Free fatty acids; MUFA, monounsaturated fatty acids; netAUC, net area under the curve; OA, oleic acid; PA, palmitic acid; ROO, refined olive oil; SFA, saturated fatty acids.

Dietary fats, particularly those containing SFA, are known to potentiate insulin secretion and insulin resistance. Indeed, β-cells are particularly sensitive to the degree of unsaturation of the fatty acids.36 It is likely that OA and PA could compete at the level of the β-cell,37 in line with a previous model to explain the capacity of fatty acids to trigger insulin secretion by glucose-responsive TG/FFA cycling.38 The islet tissue, which expresses lipoprotein lipase, could access postprandial TG as a source of FFA, such that the FFA type and concentration in the immediate vicinity of the β-cells is likely to be dependent on the nature of dietary fats. This system could be linked to the local promotion of both intracellular TG lipolysis and fatty acid esterification. FFA deprivation in islet tissue has indeed been reported to impede glucose-stimulated insulin secretion, a process rapidly reversed by replacement with exogenous FFA.39 OA was found to elicit half the insulinotropic potency of PA or stearic acid. Thus we

Oleic acid—the main component of olive oil on postprandial metabolic processes Chapter | 53

Indices of β-cell function 0

100

200

300

Indices of insulin sensitivity 400

200

150

100

50

0

IGI 0–60 minGTTTM

ISGTTTM

0

50

100

150

340

320

300

280

260

240

IGI/HOMA-IR 0–60 minGTTTM

OGISGTTTM

0

10

20

30

0.9

0.8

0.7

0.6

AUCI/AUCG 0–120 minGTTTM

ISI(G)GTTTM

0

40

80

120

160

0.8

0.6

HOMA-B 60 minGTTTM

0.4

0.2

645

FIGURE 53.4 Empirical indices of β-cell function and IS after the ingestion of a meal containing ROO or butter in healthy men. ROO reduces the values for postprandial empirical indices of β-cell function and IR (reverse to IS). Units: IGI: pmol/mmol; IGI/HOMA-IR: L/ mmol; AUCI/AUCG: pmol/mmol; HOMA-B: pmol/mmol; ISGTTTM: min21 3 dL 3 kg21/μU/mL; OGISGTTTM: mL 3 m22 3 min21; ISI(G)GTTTM and ISI(FFA)GTTTM have no units. Values are means 6 SD (n 5 14). AUCG, Area under the curve for glucose; AUCI, area under the curve for insulin; FFA, free fatty acids; GTTTM, glucose and triglyceride tolerance test meal; HOMA, homeostatic model assessment; IGI, insulinogenic index; IR, insulin resistance; IS, insulin sensitivity; ISI, insulin sensitivity index; OGIS, oral glucose insulin sensitivity; ROO, refined olive oil.

0

ISI(FFA)GTTTM

ROO = Butter =

OA/PA MUFA/SFA 6.83 5.43 0.82 0.48

hypothesize that when compared to SFA (PA), OA might moderate postprandial hyperactivity of β-cells, although whether this maintenance of glucose tolerance during feeding periods could prevent or delay the development of overt type 2 diabetes has to be elucidated. Artificial elevation of plasma FFA in healthy humans was known to induce adverse structural and functional changes in muscles, and significant suppression of insulinmediated glucose disposal 4
5 h after lipid/heparin infusion. Through the GTTTM, it was observed a significant increase in the postprandial values for FFA several hours after ingestion of the SFA-rich meal (Fig. 53.3). They were similar to the usual fasting FFA levels observed in obese and type 2 diabetic subjects40 and to those reached artificially and shown to significantly impair insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity associated with IRS-1, and Akt serine phosphorylation in skeletal muscle.41 When compared to OA, SFA (PA) dramatically decreases postprandial

insulin sensitivity toward blood FFA [i.e., reducing postprandial ISI(FFA)GTTTM] (Fig. 53.4), suggesting that lower ratios of OA to PA (MUFA/SFA) in dietary fats could profoundly affect the antilipolytic action of insulin.

53.6 Oleic acid on postprandial inflammation Inflammation accounts for a range of physiological processes that caused by microbial, allergic, autoimmune, and physical injuries may also stem as a metabolic response from the ingestion of high-fat meals.42,43 The intensity, persistence, and/or preference for certain anatomical sites of acute inflammatory reactions enable the switch from physiological to pathological inflammation; the latter condition is then named chronic inflammation, which plays a major role in the pathogenesis of metabolic syndrome and other inflammatory disorders, including CHD. Therefore it may be crucial to know the potential of dietary fatty acids in promoting

646

PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

an undesired future condition of chronic inflammation in healthy subjects or its worsening in patients at risk via abnormal repeated waves of postprandial inflammation. When compared to the ingestion of a meal enriched in PA, meals enriched in OA (olive oil) have been shown to minimize postprandial activation of the proinflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) not only in blood cells from healthy subjects but also in those from patients with metabolic syndrome.44 In agreement, postprandial plasma from individuals subjected to a high-MUFA (OA) meal also had a lower concentration of monocyte chemoattractant protein-1 (MCP-1, also referred to as chemokine C-C motif ligand 2, CCL2). This biomarker is often used to assess systemic inflammation and progression of atherosclerosis by its functional linkage to regional recruitment of immune cells in inflamed tissues.45 Occurring more frequently in advanced plaques, arterial calcification is the consequence of an inflammatorydriven osteogenic program that can predict plaque instability and thereby the risk of acute myocardial infarction or stroke. This local tissue biomineralization resembles bone development with the formation of calcium deposits by osteoblast-like cells and resorption of calcium deposits by osteoclasts.46 Our team has discovered that two important factors of bone and vascular calcification, such as receptor activator of NF-κB (RANK) ligand (RANKL) and osteoprotegerin (OPG) as a decoy receptor for RANKL that compete with RANK to inhibit monocyte differentiation into mature and functional osteoclast, can be modulated by the type of predominant fatty acid in high-fat meals during the postprandial state in healthy subjects.47 When compared to a high-SFA (PA) meal, a high-MUFA (OA) meal based on olive oil significantly decreases postprandial RANKL/OPG ratio in plasma. We found that lipoproteins of intestinal origin obtained from volunteers proved to be metabolic entities with regulatory capacity on monocyte differentiation into monocytederived osteoclasts. Due to the importance of the delicate mineral balance in bone and vessels, often opposed, more research is needed to establish the contribution of these postprandial changes to osteoclastogenesis in a tissuespecific manner. Complement C3 is an emerging biomarker for cardiovascular risk prediction. Increased levels of plasma C3 parallel with adipose tissue inflammation and insulin resistance. Recent studies have found that MUFA in a meal induces significantly lower postprandial levels of C3 than SFA in normal-weight and overweight women.48 This complement has been also associated with obesitymediated eye diseases, such as age-related macular degeneration.49 Interestingly, the potent oxidative and inflammatory response induced by postprandial lipoproteins rich in PA (i.e., those obtained at the postprandial triglyceridemic peak from healthy volunteers after the ingestion of

% DNA in tail

Positive control

>90

TRL-PA

25

TRL-OA

5

Negative control

0

FIGURE 53.5 Evaluation by comet assay of DNA damage in human monocytes exposed to camptothecin (positive control), postprandial lipoproteins rich in PA (TRL-PA), postprandial lipoproteins rich in OA (TRL-OA) or nothing (negative control). The ingestion of a meal containing ROO produces postprandial lipoproteins that have a little, if any, impact on DNA integrity of monocytes. OA, oleic acid; PA, palmitic acid; ROO, Refined olive oil; TRL-OA, triglyceride-rich lipoproteins obtained at the postprandial triglyceridemic peak (B2 h) following the ingestion of a meal containing ROO; TRL-PA, triglyceride-rich lipoproteins obtained at the postprandial triglyceridemic peak (B2 h) following the ingestion of a meal containing butter.

a high-SFA meal) in human retinal pigment epithelial cells is not seen by postprandial lipoproteins rich in OA obtained after the ingestion of a high-MUFA meal (olive oil) from the same donors.50 In tune with these studies, when meals rich in SFA or MUFA (olive oil) are compared, the postprandial oxidative stress by means of plasma biomarkers is lower with olive oil in elderly people.51 An excessive postprandial oxidative stress could induce DNA damage in circulating cells.52 On this topic, we have observed that postprandial lipoproteins rich in OA, in contrast to those rich in PA, cause little if any DNA damage in human monocytes.53 Representative images of the single-cell electrophoresis
based method, also known as the comet assay, are depicted in Fig. 53.5.

53.7 Conclusion These data highlight the need to study the mechanisms and pathways that might account for transient but repetitive (daily) dietary fat-induced postprandial hyperinsulinemia and heightened inflammation. Such phenomena would imply a greater propensity toward new-onset

Oleic acid—the main component of olive oil on postprandial metabolic processes Chapter | 53

G

SFA

Palmitic acid

Coagulation Insulin resistance Inflammation

G

Oleic acid

647

Postprandial inflammation inversely correlated with the presence of OA (instead of PA) in dietary fats in healthy subjects and in individuals at risk of CHD. Olive oil, due to its high OA-to-PA and MUFA-toSFA ratios, had the healthiest effect on these postprandial metabolic processes.

Olive oil MUFA Postprandial homeostasis FIGURE 53.6 Dietary fatty acids may have influence on postprandial metabolic processes. The high content of oleic acid (MUFA) in olive oil contributes to counterbalance dietary SFA (palmitic acid)-induced postprandial disturbances of metabolic processes related to blood coagulation, glucose homeostasis, and inflammation. MUFA, Monounsaturated fatty acids; SFA, saturated fatty acids.

diabetes and inflammatory disorders as the insulinemic, inflammatory, and lipemic postprandial responses become more pronounced (exaggerated and prolonged). This largely subclinical and silent condition further suggests a new role for lifestyle as diabetes and inflammatory risk factors and links postprandial metabolism of (saturated) dietary fats with diabetogenic and inflammatory disorders. We conclude that olive oil, mainly due to its high content in OA (MUFA), is useful when designing optimal dietary fat intake to counteract hypercoagulability postprandial states and to reduce postprandial insulin levels and inflammation by the recovering of peripheral insulin sensitivity and the maintenance of tissue homeostasis (Fig. 53.6).

53.8 Summary points G

G

G

G

G

Postprandial response for TF inversely correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats in healthy subjects. Postprandial response for PAI-1 inversely correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats in healthy subjects. Postprandial response for insulin (and FFA) and insulinemic peaks inversely correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats in subjects with normal and high fasting concentrations of TG. Postprandial hyperactivity of β-cells inversely correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats in subjects with normal and high fasting concentrations of TG. Postprandial insulin resistance inversely correlated with the OA-to-PA ratio (MUFA/SFA) in dietary fats in subjects with normal and high fasting concentrations of TG.

53.9 Acknowledgments This study was supported by grants from the MCYT and MEC (AGL2001-0584, AGL2004
04958, AGL2011
29008, and AGL2016
80852-R) and by a grant from the Fundacio´n CEAS (Centro de Excelencia en Investigacio´n sobre Aceite de Oliva y Salud). MAR acknowledges financial support from the Spanish Research Council (CSIC)/Juan de la Cierva (FJCI-2017
33132).

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29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

activation and relation to metabolic syndrome. Atherosclerosis. 2004;175:345
351. Tofler GH, Massaro J, O’Donnell CJ, et al. Plasminogen activator inhibitor and the risk of cardiovascular disease: The Framingham Heart Study. Thromb Res. 2016;140:30
35. Pedrini MT, Niederwanger A, Kranebitter M, et al. Postprandial lipaemia induces an acute decrease of insulin sensitivity in healthy men independently of plasma NEFA levels. Diabetologia. 2006;49:1612
1618. Tentolouris N, Stylianou A, Lourida E, et al. High postprandial triglyceridemia in patients with type 2 diabetes and microalbuminuria. J Lipid Res. 2007;48:218
225. Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function. Diabetes. 2003;52:726
733. Lopez S, Bermudez B, Pacheco YM, Villar J, Abia R, Muriana FJG. Distinctive postprandial modulation of β-cell function and insulin sensitivity by dietary fats, MUFA versus SFA. Am J Clin Nutr. 2008;88:638
644. Lopez S, Bermudez B, Ortega A, et al. Effects of meals rich in either monounsaturated or saturated fat on lipid concentrations and on insulin secretion and action in subjects with high fasting triglyceride concentrations. Am J Clin Nutr. 2011;93:494
499. Bermudez B, Ortega-Gomez A, Varela LM, et al. Clustering effects on postprandial insulin secretion and sensitivity in response to meals with different fatty acid compositions. Food Funct. 2014;5:1374
1380. Nemecz M, Constantin A, Dumitrescu M, et al. The distinct effects of palmitic and oleic acid on pancreatic beta cell function: the elucidation of associated mechanisms and effector molecules. Front Pharmacol. 2019;9:1554. Liu X, Zeng X, Chen X, et al. Oleic acid protects insulin-secreting INS-1E cells against palmitic acid-induced lipotoxicity along with an amelioration of ER stress. Endocrine. 2019;64:512
524. Nolan CJ, Madiraju MS, Delghingaro-Augusto V, Peyot ML, Prentki M. Fatty acid signaling in the beta-cell and insulin secretion. Diabetes. 2006;55:S16
S23. Stein DT, Esser V, Stevenson BE, et al. Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat. J Clin Invest. 1996;97:2728
2735. McGarry JD. Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes. 2002;51:7
18. Belfort R, Mandarino L, Kashyap S, et al. Dose-response effect of elevated plasma free fatty acid on insulin signaling. Diabetes. 2005;54:1640
1648. de Vries MA, Klop B, Eskes SA, et al. The postprandial situation as a pro-inflammatory condition. Clin Investig Arterioscler. 2014;26:184
192. Teeman CS, Kurti SP, Cull BJ, Emerson SR, Haub MD, Rosenkranz SK. Postprandial lipemic and inflammatory responses to high-fat meals: a review of the roles of acute and chronic exercise. Nutr Metab. 2016;13:80
94. Cruz-Teno C, Perez-Martinez P, Delgado-Lista J, et al. Dietary fat modifies the postprandial inflammatory state in subjects with metabolic syndrome: the LIPGENE study. Mol Nutr Food Res. 2012;56:854
865.

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45. Bianconi V, Sahebkar A, Atkin SL, Pirro M. The regulation and importance of monocyte chemoattractant protein-1. Curr Opin Hematol. 2018;25:44
51. 46. Aldi S, Eriksson L, Kronqvist M, et al. Dual roles of heparanase in human carotid plaque calcification. Atherosclerosis. 2019;283:127
136. 47. Naranjo MC, Garcia I, Bermudez B, et al. Acute effects of dietary fatty acids on osteclastogenesis via RANKL/RANK/OPG system. Mol Nutr Food Res. 2016;60:2505
2513. 48. Lopes LL, Rocha DMUP, Silva AD, Peluzio MDCG, Bressan J, Hermsdorff HHM. Postprandial lipid response to high-saturated and high-monounsaturated fat meals in normal-weight or overweight women. J Am Coll Nutr. 2018;37:308
315. 49. Natoli R, Fernando N, Dahlenburg T, et al. Obesity-induced metabolic disturbance drives oxidative stress and complement activation in the retinal environment. Mol Vis. 2018;24:201
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50. Montserrat-de la Paz S, Naranjo MC, Bermudez B, et al. Postprandial dietary fatty acids exert divergent inflammatory responses in retinal-pigmented epithelium cells. Food Funct. 2016;7:1345
1353. 51. Meza-Miranda ER, Camargo A, Rangel-Zun˜iga OA, et al. Postprandial oxidative stress is modulated by dietary fat in adipose tissue from elderly people. Age. 2014;36:507
517. 52. Brown M, McClean CM, Davison GW, Brown JCW, Murphy MH. Preceding exercise and postprandial hypertriglyceridemia: effects on lymphocyte cell DNA damage and vascular inflammation. Lipids Health Dis. 2019;18:125. 53. Lopez S, Jaramillo S, Varela LM, et al. p38 MAPK protects human monocytes from postprandial triglyceride-rich lipoprotein-induced toxicity. J Nutr. 2013;143:620
626.

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Chapter 54

Oleic acid and olive oil polyphenols downregulate fatty acid and cholesterol synthesis in brain and liver cells Antonio Gnoni1, Serena Longo2, Fabrizio Damiano2, Gabriele Vincenzo Gnoni2 and Anna Maria Giudetti2 1

Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari “Aldo Moro”, Bari, Italy, 2Department of Biological and

Environmental Sciences and Technologies, University of Salento, Lecce, Italy

Abbreviations ACC DGAT DNL EVOO FAS GAE HMG-CoA HMGCR HPE HTyr LPE NAFL NAFLD NASH OA Ole Tyr TAG VLDL

acetyl-CoA carboxylase diacylglycerol acyltransferase de novo lipogenesis extra virgin olive oil fatty acid synthase gallic acid equivalents 3-hydroxy-3-methyl glutaryl-CoA 3-hydroxy-3-methyl glutaryl-CoA reductase high phenolic extract hydroxytyrosol low phenolic extract nonalcoholic fatty liver nonalcoholic fatty liver disease nonalcoholic steatohepatitis oleic acid oleuropein tyrosol triacylglycerol very-low-density lipoprotein

54.1 Introduction Extra virgin olive oil (EVOO) is universally recognized as a symbol of the Mediterranean diet. EVOO intake is associated with a low incidence of different degenerative pathologies such as cardiovascular diseases, neurological disorders including Parkinson’s and Alzheimer’s diseases, and cancer.1
4 The nutritional and healthy values of EVOO have historically been ascribed to its high content in monounsaturated fatty acids, mainly oleic acid (OA) (C18:1, ω-9), representing 49%
83% of the total fatty acids in EVOO. OA

consumption was claimed to promote the prevention of cardiovascular diseases and to influence the expression of metabolic genes that protect tissues from oxidative and inflammatory damages associated with different diseases.5,6 Current epidemiological and experimental studies strongly support the fact that the beneficial effects of EVOO are also due to its minor bioactive components, such as phenols. The main phenolic compounds found in EVOO include hydroxytyrosol (HTyr), tyrosol (Tyr), and several products of the conjugation of these phenols with oleanolic acid, such as oleuropein (Ole) (for chemical structure see Fig. 54.1). EVOO phenolic compounds are believed to exert beneficial effects as a consequence of their antioxidant, antimicrobial, and antiinflammatory activities.7,8 However, OA and phenolic compounds play other biological actions that are yet poorly understood. Several lines of evidence indicate that EVOO and its components, besides improving blood pressure9 and endothelial function,2,10 also ameliorate lipid profile.11 Brain and liver are tissues in which lipid metabolism is particularly active. This chapter is focused on the effects of OA and EVOO polyphenol compounds on the de novo fatty acids and cholesterol syntheses in different tissues and cell lines.

54.1.1 Pathways of de novo lipogenesis and cholesterol synthesis De novo lipogenesis (DNL or de novo fatty acid synthesis) is a cellular process that leads to the synthesis of saturated fatty acids, mainly palmitic acid (C16:0). It is finely tuned in response to nutritional and hormonal states.12

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00036-5 © 2021 Elsevier Inc. All rights reserved.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

FIGURE 54.1 Chemical structures of hydroxytyrosol (A), tyrosol (B), and oleuropein (C).

FIGURE 54.2 De novo lipogenesis and cholesterol synthesis from glucose. ACC, Acetyl-CoA carboxylase; ACLY, ATP-citrate lyase; CiC, citrate carrier; FAS, fatty acid synthase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PDH, pyruvate dehydrogenase; PyC, pyruvate carrier.

DNL is considered the formation of fatty acids mainly from glucose (Fig. 54.2). Glucose oxidation through glycolysis produces pyruvate which, after entering mitochondria through the pyruvate carrier, becomes a substrate for pyruvate dehydrogenase generating acetyl-CoA, which can be oxidized into the Krebs cycle. However, when the energetic status of the cells is high, acetyl-CoA is exported in the form of citrate by the citrate carrier from mitochondria to the cytosol. Here, by the action of ATP-citrate lyase, citrate is transformed in oxaloacetate and acetyl-CoA, which represents the starter molecule for both DNL and cholesterol synthesis. By the action of acetyl-CoA carboxylase

(ACC), the key enzyme of DNL, acetyl-CoA undergoes carboxylation to form malonyl-CoA which, in the presence of fatty acid synthase (FAS), leads to the final major product: palmitic acid. In the cytosol, acetyl-CoA is also the precursor of cholesterol synthesis, which starts with the sequential condensation of three acetyl-CoA molecules to form 3-hydroxy3-methyl glutaryl-CoA (HMG-CoA). This compound, by the action of HMG-CoA reductase (HMGCR), the key regulatory enzyme of cholesterol synthesis, leads to the formation of mevalonate. After several enzymatic steps, cholesterol is formed.

Oleic acid and olive oil polyphenols downregulate fatty acid and cholesterol synthesis in brain Chapter | 54

54.1.2 Effect of extra virgin olive oil components on lipid synthesis in brain cells The brain is an organ with a high content of lipids, which are the fundamental components of neuronal membranes and are essential for brain function. Alterations in lipid metabolism are the cause of or are associated with many neurological diseases.13,14 Attenuation of age- and disease-associated cognitive decline by EVOO active components has been demonstrated in cellular, animal, and human models.15,16 While the antioxidant potential of EVOO has been well documented, only a few studies have reported the effects of EVOO components on lipid synthesis in the brain, even though brain cholesterol and fatty acids are primarily supplied by de novo synthesis due to the prevention of lipoprotein uptake in the brain, from the circulation, by blood
brain barrier.14,17 In a previous study from our laboratory,18 carried out in C6 glioma cells, considered a useful model to study cerebral dysfunction, we found that comparing the effects of OA, polyunsaturated fatty acids, and saturated fatty acids, OA was the most effective in reducing DNL and cholesterol synthesis. In a subsequent study,19 it has been reported that the decrease of cholesterol synthesis was more pronounced in C6 cells simultaneously incubated with OA and HTyr. In the same way, incubation of C6 cells with OA or HTyr, individually or in association, determined a reduction of fatty acids production as well as of their incorporation into complex lipids. The inhibition of DNL and cholesterologenesis in

653

OA/HTyr-treated C6 cells has been indicated to be a consequence of ACC and HMGCR reduced activity, respectively. Transcriptional mechanisms are involved in the action of EVOO components since it has been shown that OA and HTyr can reduce ACC and HMGCR expression both at the level of mRNA and protein synthesis.19 This decrease was more evident when OA was added to C6 cells together with HTyr, indicating an additive effect of these EVOO components. No significant effect was observed on FAS activity, mRNA, and protein level. Altogether, these findings suggest a rapid and direct effect of EVOO components on lipid synthesis in C6 cells.

54.1.3 Effects of extra virgin olive oil phenols on hepatic lipid synthesis Evidence reports that EVOO phenols, either in the form of extracts or as pure compounds, may offer indirect protection against atherosclerosis by reducing hepatic and blood lipid levels.20 However, studies on these effects have produced controversial results in animal and human experiments.21
23 We recently reported24 that treatment (4 h) with Tyr, HTyr, and Ole (25 μM) significantly inhibit, with respect to control cells, fatty acid, and cholesterol synthesis, in isolated rat hepatocytes as shown by the decrease in the labeled acetate incorporation into both fatty acids and cholesterol (Fig. 54.3). ACC activity, markedly reduced by HTyr and FIGURE 54.3 Effects of pure phenols, HPE and LPE on fatty acids and cholesterol synthesis. Primary rat hepatocytes were incubated for 4 h with [1-14C] acetate and 25 μM of Tyr HTyr and Ole or with 0.1 μL/mL of HPE or LPE of olive oil. Labeled acetate incorporation into fatty acids (panel A) or cholesterol (panel B) was followed. The data, expressed as nmol [1-14C] acetate incorporated/h/mg protein, are mean 6 SD of five experiments. *Values significantly different at P , .05 versus untretated hepatocytes (control, CTR). HPE, High phenolic extract; HTyr, hydroxytyrosol; LPE, low phenol extract; Ole, oleuropein; Tyr, tyrosol.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

Ole, was poorly affected by Tyr addition to the hepatocytes. Vice versa, no significant change in FAS activity was observed by any EVOO phenol. The activity of HMGCR, the key enzyme of cholesterol synthesis, was reduced by Tyr, HTyr, and Ole with respect to control cells. Besides, HTyr, Tyr, and Ole can reduce triacylglycerol (TAG) synthesis by inhibiting the activity of diacylglycerol acyltransferase (DGAT), a key enzyme in TAG synthesis. The effect of a high phenolic extract (HPE) obtained from EVOO of cultivar Coratina of Apulia region (Italy) or a low phenolic extract (LPE), from commercial refined olive oil, on hepatic lipid synthesis was also reported.25 The total phenol content was found to be 10 times higher in HPE with respect to LPE [548 6 42 mg gallic acid equivalents (GAE)/kg of oil vs 48 6 4 mg GAE/kg of oil]. Results obtained after HPE or LPE (0.1 μL/mL) addition to isolated hepatocytes showed that only HPE addition to cells induced a noticeable inhibition of both fatty acids and cholesterol synthesis, with respect to untreated cells. No significant effect was instead reported after LPE addition. ACC, HMGCR, and DGAT specific activities were markedly reduced in isolated hepatocytes, with respect to the untreated cells, after HPE but not LPE addition. No effect was reported for FAS activity upon any treatment (Fig. 54.4). Concerning the insensitivity of FAS activity to phenol or the phenol extract addition, it is worth recalling that while ACC is regulated by both short- and longterm mechanisms, only the latter are involved in FAS regulation.24

54.1.4 Effects of olive oil phenols on liver steatosis and steatohepatitis Nonalcoholic fatty liver (NAFL) disease (NAFLD) is a major cause of liver-related morbidity and mortality. Without excess alcoholic consumption the simple steatosis, named NAFL, may progress to nonalcoholic steatohepatitis (NASH), with hepatocytes injury and lobular and portal inflammation,26 which may, in turn, progresses to cirrhosis, hepatocarcinoma, and liver failure.27 Fig. 54.5 shows the main pathways involved in the development of hepatic steatosis. Hepatic steatosis occurs when there is an imbalance between lipid synthesis (DNL and TAG synthesis) and lipid clearance [fatty acid β-oxidation and very-low-density lipoprotein (VLDL) secretion]. Thus, in NAFLD, a shift of cellular metabolism from fatty acid β-oxidation to TAG accumulation occurs.28 DNL is emerging as an important contributor to hepatic steatosis.29 Increased DNL can not only cause hepatic steatosis and/or hypertriglyceridemia but may also

FIGURE 54.4 Effect of pure phenols, HPE and LPE on ACC, FAS and HMGCR activities. After 4 h of incubation with 25 μM of Tyr, HTyr, and Ole or with 0.1 μL/mL of HPE or LPE of olive oil, activities of (A) acetyl-CoA carboxylase (ACC), (B) fatty acid synthase (FAS), and (C) 3-hydroxy-3-methyl glutaryl-CoA reductase (HMGCR) were assayed in digitonin-permeabilized rat hepatocytes. Values expressed as a percentage of the control are mean 6 SD of five experiments. Control specific activity: ACC and FAS 0.22 6 0.05 and 0.71 6 0.08 nmol [1-14C] acetylCoA inc./min/mg protein, respectively; HMGCR 43.0 6 5.8 pmol [3-14C] HMG-CoA inc./min/mg protein. *Values significantly different at P , .05 versus untretated hepatocytes (control, CTR). ACC, AcetylCoA carboxylase; FAS, fatty acid synthase; HMGCR, 3-hydroxy-3methyl glutaryl-CoA reductase; HPE, high phenolic extract; HTyr, hydroxytyrosol; LPE, low phenol extract; Ole, oleuropein; Tyr, tyrosol.

induce steatohepatitis, as saturated fatty acids, such as palmitate, can cause inflammation and apoptosis.30 The importance of DNL in NAFLD is further supported by studies showing that in obese patients with NAFLD, about 26% of hepatic TAG was derived from DNL.31,32

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FIGURE 54.5 Contribution of various metabolic pathways to hepatic steatosis.

A study33 carried out in rats with NAFLD showed that olive oil consumption decreased liver TAG accumulation by improving insulin resistance and increasing the secretion of hepatic TAG as VLDL. Reduced lipid accumulation was also observed in different models of steatotic rats fed on a balanced olive oil
rich diet.34,35 In mice fed a high-fat diet (HFD), Ole administration attenuated dietinduced liver steatosis36 and reduced free fatty acidinduced lipogenesis via lowered extracellular signalregulated kinase activation.37 Moreover, Ole exerted preventive effect toward NASH progression to hepatic fibrosis.38 HTyr supplementation to rats with HFD-induced NAFLD showed a protective effect against liver damage caused by HFD.39

54.2 Conclusion The increasing interest in the Mediterranean diet hinges on its beneficial properties. EVOO is the main source of dietary fat and, due to the high content of OAs and polyphenols, EVOO is considered a key feature of the healthy property of the Mediterranean diet. In addition to the well-known EVOO phenol antioxidant effects, results here reported show that EVOO health effects could be related, at least in part, to the downregulation of fatty acid and cholesterol syntheses by EVOO components, observed in rat-cultured hepatocytes and glioblastoma cells.

The findings reported in this chapter increase the knowledge of the regulation of lipid synthesis by EVOO components, highlighting a new approach for preventing and treating disorders of lipid metabolism and its complications. However, the significance of these results should be confirmed in vivo in appropriate cell animal models and in humans.

Mini-dictionary of terms Acetyl-CoA carboxylase (ACC) is the first and ratelimiting enzyme of de novo fatty acid biosynthesis that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA, for fatty acid biosynthesis. C6 glioma cells is an experimental model used to study the mechanism of tumor metabolism and growth and in the design and evaluation of anticancer therapies. De novo lipogenesis (DNL) is the biochemical process of synthesizing fatty acids from acetyl-CoA, mainly coming from carbohydrate sources. Fatty acids are carboxylic acids with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28. Fatty acid synthase (FAS) is a multienzymatic complex that catalyzes fatty acid synthesis. 3-Hydroxy-3-methyl glutaryl-CoA reductase (HMGCR) is the rate-controlling enzyme of the pathway of cholesterol synthesis.

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Isolated hepatocytes are primary cultured hepatocytes representing an in vitro model system helping to investigate (patho)physiological processes of the liver. Mitochondrial carriers are proteins from a solute carrier family that transfer hydrophilic molecules across the inner mitochondrial membrane. Nonalcoholic steatohepatitis (NASH) is a pathological liver condition characterized by a build-up of fat in the liver with associated inflammation and damage that can lead to the scarring of the liver. Olive oil phenols are compounds that are naturally present in extra virgin olive oil that exhibits significant beneficial effects in many human diseases.

Comparison of olive oils with other edible oils Extra virgin olive oil is made from cold-pressed olives, while other oils are extracted from both processed and pressed seeds. Other edible oils are highly processed, and the resultant oil must then be refined, often using chemicals, to make them fit for human consumption.

Implications for human health and disease prevention Consumption of olive oil improves plasma lipid profiles and reduces dyslipidemia through an improvement in the quantity and function of high-density lipoprotein cholesterol and in the reduction of low-density lipoprotein cholesterol. In addition, olive oil decreases blood pressure, improves endothelial function, and reduces inflammation. Mediterranean diet rich in extra virgin olive oil decreases the risk of diabetes onset, and it is inversely correlated with coronary heart diseases, stroke, and cardiovascular mortality.

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Section 3.4

Oleocanthal

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Chapter 55

Olive oil oleocanthal and estrogen receptor expression Nehad M. Ayoub Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology (JUST), Irbid, Jordan

Abbreviations AF AMPK AP-1 cAMP COX DBD ER ERE EVOO GPER1 HSP HCC IL-6 LBD LPS LMP MET MIP-1α mTOR NTD PARP p-HPEA-EDA ROS STAT3 TPF

activation function adenosine monophosphate
activated protein kinase activator protein-1 cyclic adenosine monophosphate cyclooxygenase DNA-binding domain estrogen receptor estrogen response element extra-virgin olive oil G protein
coupled estrogen receptor heat shock protein human hepatocellular carcinoma interleukin-6 ligand-binding domain lipopolysaccharide lysosomal membrane permeabilization hepatocyte growth factor receptor macrophage inflammatory protein-1α mammalian target of rapamycin N-amino-terminal domain poly (ADP-ribose) polymerase p-hydroxyphenylethanol-elenolic acid dialdehyde reactive oxygen species signal transducer and activator of transcription 3 total polyphenolic fraction

55.1 Introduction Chemically, olive oil is composed of major and minor components. Fatty acids are considered the major components of olive oil representing 95%
97% of its composition.1 Fatty acids are mostly composed of monounsaturated fatty acids, while polyunsaturated fatty acids and saturated fatty acids are presented to a lesser extent.2 The minor components of olive oil constitute 1%
2% of the total content of the oil.2
5 Among the minor components, phenolic compounds

are particularly essential for the beneficial health effects attributed to olive oil. Olive oil contains more than 30 different phenolic compounds.6 In general, phenolic compounds found in olive oil can be classified into three major categories that are simple phenols, secoiridoids, and lignans.7,8 Simple phenols include compounds such as tyrosol and hydroxytyrosol, while secoiridoids include compounds such as oleuropein, oleacein, and oleocanthal.7,9 Tyrosol, hydroxytyrosol, and their secoiridoid derivatives constitute most of the minor phenolic fractions of olive oil. The concentration of these compounds ranges from 100 and 300 mg/kg in extra-virgin olive oil (EVOO) and concentrations up to 1000 mg/kg have been also reported.2,10
12 The concentration and composition of phenolic compounds of olive oil are determined by several factors, including environmental and cultivation factors, extraction and processing factors, and storage conditions and duration.5,13
15 Among phenolic compounds of EVOO, oleocanthal had gained recent attention for its several favorable health effects.

55.2 Oleocanthal In 1993 oleocanthal was first isolated from EVOO along with other secoiridoids by Montedoro and colleagues.16 Subsequently, Beauchamp and coworkers demonstrated the antiinflammatory activity of oleocanthal in 2005.17 Regularly, oleocanthal counts for 0.02% by weight of EVOO,2,18 therefore corresponding to 10% of the total phenolic content of EVOO.19 Oleocanthal is the principal component of EVOO responsible for the throat irritating sensation and the pungent properties associated with the ingestion of olive oil.4,20
22

55.2.1 Chemical structure of oleocanthal Oleocanthal is the elenolic acid ester of tyrosol, a common olive phenolic alcohol.23 Beauchamp and colleagues

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00053-5 © 2021 Elsevier Inc. All rights reserved.

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55.2.3 Biological activity of oleocanthal

FIGURE 55.1 Chemical structure of the natural olive oil phenolic compound, oleocanthal.

assigned oleocanthal its name in which oleo- stands for olive, -canth- for its sting sensation, and -al corresponding for the presence of aldehyde groups in the chemical structure of the compound.17 Other chemical names of oleocanthal include decarboxymethyl ligstroside aglycone,21 dialdehydic form of deacetoxy-ligstroside aglycone,17 deacetoxy-dialdehydic ligstroside aglycone,22 and p-hydroxyphenylethanol-elenolic acid dialdehyde (p-HPEA-EDA).6 Naturally existing oleocanthal is presented in the S-configuration of chiral carbon, while synthetic oleocanthal is presented in the R-configuration.12,17 Several research groups were able to synthesize both oleocanthal enantiomers and further identify its stereochemistry.24
26 Fig. 55.1 shows the chemical structure of the natural S-(2)-oleocanthal.

55.2.2 Pharmacokinetics of oleocanthal Studies on the pharmacokinetics and bioavailability of oleocanthal are limited. Corona et al. conducted a detailed investigation of the absorption and metabolism for the phenolic compounds hydroxytyrosol, tyrosol, and its conjugated forms.27 Exposure to gastric acidity promoted rapid and time-dependent hydrolysis of the conjugated forms of tyrosol and hydroxytyrosol resulting in a remarkable increase for the amounts of free tyrosol and hydroxytyrosol entering the small intestine.27 In addition, both compounds were found to be subject to absorption through rat segments of jejunum and ileum and were further metabolized through classic phase I/II biotransformation reactions.27 In line with this, Romero et al. showed the stability of oleocanthal and other olive polyphenolic compounds in acidic conditions for up to 4 h.28 More than 50 different metabolites were identified in human urine for oleocanthal and other polyphenols after the ingestion of EVOO. Most of these metabolites were detected in urine after 2 h of ingestion and metabolites were obtained through phase I and phase II biotransformation reactions.29 Oleocanthal was mostly metabolized via phase I reactions, including hydrogenation, hydroxylation, and hydration. However, some of the hydrogenated metabolites of oleocanthal were further subject to phase II glucuronidation reactions.29 Additional studies on biotransformation and pharmacokinetics of oleocanthal will be mandatory to better address these gaps in the literature.

Oleocanthal has several biological activities. Oleocanthal exerts antiinflammatory, antioxidant, anticancer, and neuroprotective pharmacologic activities. Despite structural dissimilarity, oleocanthal exhibited similar antiinflammatory properties to ibuprofen, a nonsteroidal antiinflammatory drug.17,30 Oleocanthal inhibited inflammatory cyclooxygenase (COX) enzymes 1 and 2 in a dose-dependent manner. It was also more potent than ibuprofen in inhibiting COX activity at equimolar concentrations.17,30 Besides, oleocanthal has been shown to inhibit the 5-lipoxygenase enzyme responsible for the production of inflammatory leukotrienes further contributing to the antiinflammatory and antioxidant properties of the phenolic compound.31 Oleocanthal inhibited gene expression of the cytokines macrophage inflammatory protein-1α (MIP-1α) and interleukin-6 (IL-6) in murine macrophages and chondrocytes that are known mediators of inflammation in rheumatic disease.32,33 Further, oleocanthal blocked lipopolysaccharide (LPS)-mediated osteoarthritis in human primary chondrocytes by suppressing LPS-induced inflammatory response, matrix metalloprotease-13, and ADAMTS-5 via MAPK/NF-kB pathways.34 In a recent study by Montoya et al., oleocanthal inhibited canonical and noncanonical inflammasome signaling pathways involved in LPS-stimulated murine peritoneal macrophages.35 These antiinflammatory effects were accompanied by the reduction of reactive oxygen species (ROS) and nitrites levels in oleocanthal-treated cells along with reduced protein expression of p38, JNK, and ERK.35 Oleocanthal demonstrated remarkable antioxidant effects mediated by a plethora of cellular mechanisms. Oleocanthal has been shown to counteract oxidative stress by increased H2O2 scavenging activity, reducing ROS production, and increasing intracellular levels of reduced glutathione.36
38 In addition, oleocanthal inhibited the activity of nicotinamide adenine dinucleotide phosphate oxidase and reduced the intracellular level of superoxide anion in isolated human monocytes.39 In a proteomic analysis conducted by Giusti and coworkers, the antioxidant activity of oleocanthal upregulated the expression of proteins related to the proteasome, the chaperone heat shock protein (HSP) 90, the glycolytic enzyme pyruvate kinase, and the antioxidant enzyme peroxiredoxin 1.37 Further, oleocanthal protection against oxidative stress was mediated by AKT activation.37 The anticancer properties of oleocanthal have been extensively described in the literature. Oleocanthal demonstrated antiproliferative, apoptotic, antimigratory, antiinvasive, and antiangiogenesis activities in different types of solid cancers. Oleocanthal treatment inhibited proliferation and induced apoptosis in human hepatocellular carcinoma (HCC) cells in vitro and suppressed tumor growth in an orthotopic HCC animal model.40 These effects of oleocanthal were attributed to inhibiting signal transducer and activator of transcription 3

Olive oil oleocanthal and estrogen receptor expression Chapter | 55

(STAT3) activity leading to reduced epithelial
mesenchymal transition.40 Gu et al. also revealed that oleocanthal anticancer activity in melanoma was mediated by inhibition of STAT3 phosphorylation leading to reduced STAT3 nuclear localization and its transcriptional activity.41 Fogli et al. showed the in vitro antiproliferative activity of oleocanthal against human malignant melanoma cells was mediated by inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression.42 Oleocanthal inhibited proliferation and colony formation of hepatocellular and colorectal carcinoma cells.43 It also induced apoptosis as indicated by activation of caspase 3/7, poly (ADP-ribose) polymerase (PARP) cleavage, and chromatin condensation. Oleocanthal inhibited proliferation and induced apoptosis in neuroblastoma cells mediated by oxidative stress.44 Oleocanthal also suppressed the proliferation of multiple myeloma cells in vitro by inhibiting MIP1α expression and secretion and inducing apoptosis signaling pathways.45 LeGendre et al. described a novel mechanism for the anticancer effects of oleocanthal through induction of necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP).46 In line with this, a recent report by Goren et al. showed the ability of oleocanthal to induce LMP leading to cellular toxicity in cell culture and animal model of pancreatic neuroendocrine tumors.47 Oleocanthal also showed activity in leukemia cells through the induction of cell differentiation mediated by increased oxidative superoxide ions.48 Mechanistically, oleocanthal anticancer activities have been further attributed to inhibition of the mammalian target of rapamycin (mTOR) and the HSP 90 in solid and hematologic cancers.49,50 Khanal et al. reported that the anticancer activity of oleocanthal is mediated by activation of adenosine monophosphate
activated protein kinase (AMPK) in human colorectal adenocarcinoma HT-29 cells.51 The activation of AMPK inhibited the activity of COX2 and the activator protein (AP)-1 resulting in tumor suppression both in vitro and in vivo.51 Oleocanthal also demonstrated the ability to inhibit the phosphorylation and subsequent activation of the hepatocyte growth factor receptor, MET, a receptor tyrosine kinase known for its oncogenic effects in several solid cancers.52 Polini et al. showed that EVOO extract containing oleocanthal and oleacein reduced viability, migration, and clonogenic potential of nonmelanoma skin cancer cells through inhibition of ERK and AKT phosphorylation and suppression of B-RAF expression.7 Recently, Diez-Bello et al. showed that oleocanthal downregulated expression of TRPC6 channels in both triple-negative MDA-MB-231 and hormonedependent MCF-7 breast cancer cell lines.53 Oleocanthal inhibited proliferation of liver cancer HepG2, Huh7, and Hep3B cells in a dose- and time-dependent manner.54 The antiproliferative effects of oleocanthal in liver cancer cells were mediated by induction of autophagy and were further enhanced by exposure to the cytokine tumor necrosis factor α (TNFα).54 It is worth mentioning that oleocanthal

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anticancer activity demonstrated selectivity toward cancer cells with reduced or lack of apparent cytotoxicity in nontumorigenic cell lines.5,42,50,53 Table 55.1 summarizes the anticancer activities attributed to oleocanthal and the molecular targets described for the compound. The neuroprotective effects of oleocanthal have been well addressed in literature. Several lines of evidence support the beneficial activity of oleocanthal in degenerative neurologic disorders such as Alzheimer’s disease.38,55
57 Limited studies assessed the cardiovascular benefits of oleocanthal. Intake of oleocanthal-rich EVOO induced acute antiplatelet activity in adults according to a randomized crossover study.58

55.3 Estrogens and estrogen receptors Estrogen is the predominant reproductive steroidal hormone in females. Estrogens are primarily synthesized in the ovaries and minor amounts are produced in the adrenal glands and adipose tissues.59 Chemically, estrogens comprise four different hormones, including estradiol, estrone, estriol, and estetrol.60 Several physiological functions are regulated by estrogens, including the menstrual cycle and the reproductive system, bone density, neuroendocrine function, cholesterol mobilization, and cardiovascular system.60,61 In females, estradiol promotes epithelial cell proliferation of the uterine endometrium and mammary glands starting in puberty.62 Estradiol, the predominant circulating estrogen in humans, is mainly secreted by ovaries and is the most potent estrogen in circulation.63 The biologic functions of estrogens are mediated by binding to estrogen receptors (ERs). Classic ERs belong to the nuclear receptor superfamily, which act as transcription factors known to modulate gene expression in target tissues. These include ERα and ERβ.60,64 Recently, a new membrane ER was discovered in target cells and is known as the G protein
coupled estrogen receptor, GPER1.65 A growing evidence showed that estradiol binds to GPER1 and mediates fast physiological responses to estrogens.64,65 All four estrogens bind to both nuclear and membrane ERs, with variable affinity and strength of the response.60 ERα and ERβ are encoded by two distinct genes, ESR1 and ESR2, respectively. These genes are located on different chromosomes, and several mRNA splice variants are known to exist for both receptors in normal and diseased tissues.66 ERα is largely expressed in gonadal organs such as the mammary gland, ovary, uterus, as well as male reproductive organs, the prostate, and testes.64,67 ERα is also expressed at lower levels in adipose tissue, liver, and bones.64 Alternatively, ERβ is primarily expressed in nongonadal tissues.64,68 ERβ is mainly found in the colon, bladder, bone marrow, vascular endothelium, and lung.64,68 Despite the tissuespecific distribution of each receptor, both receptors are expressed in the central nervous system and

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TABLE 55.1 Anticancer activities of oleocanthal. Study model

Disease

Biologic effect

Molecular target

References

In vitro/ in vivo

Melanoma

Antiproliferative Antimigratory Antiinvasive Apoptotic

Inhibition of STAT3 phosphorylation and nuclear translocation Reduced Ki-67 and CD 31 levels

[41]

In vitro

Melanoma

Antiproliferative

Inhibition of ERK1/2 and AKT phosphorylation Downregulation of Bcl-2 expression

[42]

In vitro

Nonmelanoma skin cancer

Antiproliferative Antimigratory Inhibiting colony and spheroid formation

Inhibition of ERK and AKT phosphorylation Suppression of B-RAF expression

[7]

In vitro/ in vivo

Hepatocellular carcinoma

Antiproliferative Antimigratory Antiinvasive Apoptotic

Reduced STAT3 activation and nuclear translocation

[40]

In vitro

Hepatocellular and colorectal carcinoma

Antiproliferative Apoptotic Inhibition of colony formation

Induction of PARP cleavage Activation of caspase 3/7 Chromatin condensation Increased intracellular ROS production

[43]

In vitro

Hepatocellular carcinoma

Antiproliferative Induction of autophagy

Effect enhanced by TNFα

[54]

In vitro

Breast and prostate cancer

Antiproliferative Antimigratory Antiinvasive

Inhibition of MET phosphorylation Reduced CD 31 levels

[52]

In vitro

Breast, pancreatic, and prostate cancer

Apoptotic

Induction of LMP Inhibiting ASM activity

[46]

In vitro

Breast cancer

Antiproliferative

Inhibition of mTOR

[49]

In vitro

Breast cancer

Antiproliferative Antimigratory

Downregulation of TRPC6 channel expression

[53]

In vitro/ in vivo

Colorectal cancer

Antiproliferative Apoptotic Inhibition of colony formation

Inhibition of COX2 Activation of AMPK Activation of caspase 3

[51]

In vitro

Multiple myeloma

Antiproliferative Apoptotic

Inhibition of MIP-1α Downregulation of AKT and ERK1/2

[45]

In vitro

Neuroblastoma

Antiproliferative Inhibition of neurite growth Cytotoxic

Increased oxidative stress

[44]

In vitro/ in vivo

Pancreatic neuroendocrine tumors

Antiproliferative

Induction of LMP

[47]

In vitro

Promyelocytic leukemia

Antiproliferative Induction of differentiation Apoptotic

Production of superoxide ions

[48]

ASM, Acid sphingomyelinase; LMP, lysosomal membrane permeabilization; mTOR, mammalian target of rapamycin; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor.

cardiovascular system.67 Overall, ERα has dominant roles in the mammary gland and uterus development, preservation of bone density, and regulation of metabolism.67 ERβ has prominent effects on neuronal function and immune system.67 Importantly, ERβ has been shown to suppress proliferation and invasion of ERα-positive breast cancer cells.67,69 These effects are attributed to

the ability of ERβ to inhibit ERα selective target gene expression in breast tissue.69 The structures of ERα and ERβ are composed of several domains and both receptors have common structural regions. Being members of the nuclear hormone receptors superfamily of transcription regulators, both receptors are composed of functionally distinct domains termed A/B, C, D, and E/F

Olive oil oleocanthal and estrogen receptor expression Chapter | 55

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FIGURE 55.2 Schematic representation of classic estrogen receptors. Both ERα and ERβ are composed of six different structural and functional domains. These domains are the A/B domain that contains the N-terminal and the AF1 transactivation domains, the DBD (domain C), the hinge region (domain D), and the E/F domains at the C-terminal region that contain both LBD and the AF2 transactivation domain. ERα is encoded by ESR1 gene located on the long arm of chromosome 6, while ERβ is encoded by ESR2 gene located on the long arm of chromosome 14. The percentage homology for the two ERs is indicated. AF1, Activation function domain1; AF2, activation function domain 2; DBD, DNA-binding domain; ER, estrogen receptor; LBD, ligand-binding domain; NTD, N-amino-terminal domain.

presenting in full-length receptors.63 The A/B domain represents the N-amino-terminal domain (NTD) involved in gene transcription and transactivation and contains a zinc finger that mediates binding to target gene sequences.60 The C region is the DNA-binding domain (DBD) that contributes to ER dimerization and binding to specific sequences in the chromatin known as estrogen response elements (EREs).60,63 The D domain is a flexible hinge region that links the C region to the multifunctional carboxyl-terminal (E) domain. The D region also contains the nuclear localization signal that is unmasked upon estrogen binding which subsequently allows the translocation of the ligand
receptor complex to nucleus.60 The carboxyl-terminal E/F region, also known as the ligand-binding domain (LBD), represents the binding site of estrogen along with coactivators and corepressors.60,63 In addition, two activation function (AF) domains, AF1 and AF2, are known to regulate ER transcriptional activity and exist within NTD and LBD, respectively.60 Estradiol binds with equal affinity to both ERα and ERβ, and both receptors bind to the same EREs.70,71 Fig. 55.2 represents the different domains of classic ERs. Based on molecular pathways regulating gene expression, signaling through ERs can be divided into genomic and nongenomic. Genomic signaling is associated with alterations to gene transcriptional activities and is further classified into direct or indirect.64 Direct genomic signaling represents the classical pathway of ER signaling in which nuclear ERs act as transcription factors upon binding to their ligand, estradiol.72 As a result, ligandactivated ERs dimerize in the cytoplasm, and subsequently, the ligand
receptor complex is translocated to the nucleus where it binds DNA directly at specific EREs resulting in transcriptional regulations of target genes.72

EREs are located within or close to the promoter region of target genes.72 Over 70,000 EREs have been identified through genome-wide screening that further enhanced the prediction of genes controlled by estrogens in humans.73 Interestingly, estradiol has been shown to regulate transcription of several genes that lack EREs in their promoter regions suggestive of indirect genomic signaling of ERs. In the latter pathway, estradiol
ER complexes regulate gene transcription through protein
protein interactions with other transcription factors without direct DNA binding. For this, indirect genomic signaling is also known as “transcriptional cross talk” and is responsible for the regulation of several genes lacking EREs.60,74 Nongenomic signaling of estrogens is mediated by GPER1. Activation of this receptor stimulates signaltransduction mechanisms associated with activation of intracellular second messengers such as cyclic adenosine monophosphate (cAMP) and the subsequent activation of multiple protein kinases.75 Protein kinase cascades activated in response to GPER1 include the mitogen-activated protein kinase (MAPK), the phosphoinositide 3-kinase (PI3K), the phospholipase C (PLC)/protein kinase C (PKCs) signaling pathway along with activation of membrane receptor tyrosine kinases.60,75 Collectively, the activation of these kinases allows phosphorylation of transcription factors that could ultimately alter gene transcription.75

55.4 Impact of oleocanthal on estrogen receptor Lately, ER has been introduced as a potential molecular target to oleocanthal favorable biological activities. Few studies in literature had investigated the impact of

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oleocanthal on ERs in target cells and tissues. The following part summarizes evidence from the literature on the ability of the phenolic compound, oleocanthal, to bind ERs and modulate the expression of target genes and the molecular effects resulting from such activity.

55.4.1 Binding of oleocanthal to estrogen receptor The ability of oleocanthal to bind ERs was first demonstrated in literature by Keiler et al. using a competition binding assay.76 The binding affinities for oleocanthal were determined toward purified human ERα and ERβ and fluorescein-labeled 17β-estradiol. In this competition binding assay the binding affinities of oleocanthal were calculated relative to that of 17β-estradiol. Though oleocanthal was reported to bind both ERs, oleocanthal had a 6.7-fold greater affinity toward binding to ERα compared to ERβ with relative ER binding affinity of 0.102% and 0.0166% relative to that of 17β-estradiol for both receptors, respectively.76 In addition, docking studies showed that oleocanthal can orient, in its best energetically favored conformation, at the ligand-binding pocket of ERα by two types of interactions: hydrogen bonding and van der Waals. Furthermore, the overlay of the 3D structures of oleocanthal and 17β-estradiol showed a high degree of overlapping.77 Interestingly, the binding modes of tamoxifen, a gold-standard selective ER modulator, and its active metabolite, 4-hydroxytamoxifen, were different from that observed with oleocanthal when these compounds were docked to the same crystal structure of ERα.77

55.4.2 Oleocanthal modulates estrogen receptor gene expression Keiler et al. investigated the effect of an extract of EVOO enriched in phenolic constituents, named total polyphenolic fraction (TPF) on ER gene expression in animal models. Exposure to TPF extract in ovariectomized rats reduced uterine expression of ESR1 and ESR2 mRNA levels and increased mRNA levels of the estrogen response markers C3 and Cabp9 compared to ovariectomized animals not exposed to the polyphenolic extract.76 The uterine gene expression pattern supported an estrogen-agonistic effect for the polyphenolic extract of EVOO on both ERs. This study also revealed the ability of oleocanthal to bind ERs that could explain the impact of the TPF on estrogen response genes.76 Oleocanthal has been shown to selectively modulate reporter gene activity of ER genes. In this regard, oleocanthal reduced reporter gene activity in ERα-expressing breast cancer
derived MVLN cells while inducing a weak ERα transactivation in bone-derived U2OS cells.78 These effects could be explained by differences of coregulators expressed in the different tissues. However,

oleocanthal treatment did not stimulate the luciferase activity of the ERβ-expressing cells.78

55.4.3 Molecular effects of oleocanthal mediated via estrogen receptor targeting The effect of TPF extracted from EVOO regarding its potential bone-sparing effects was examined in animal models.76 Ovariectomized female rats of about 12 months old were used as an experimental model for postmenopausal osteoporosis. The removal of ovaries of these mature rats produced a state of deficient estrogen levels, which is comparable to postmenopausal status in human subjects. TPF extract was administered orally to ovariectomized rats for a duration of 12 weeks. TPF was found to be rich in tyrosol, hydroxytyrosol, oleocanthal, oleacein, ligstroside, and oleuropein; however, oleacein, oleocanthal, and ligstroside were at the highest levels corresponding to 8% of the dry weight. The concentration of the polyphenolic extract used to feed the rats was 800 mg/kg diet that approximately contained 150 mg/kg diet of oleocanthal. At the end of this study, the hormonal impact of estrogen depletion in animals was confirmed by a remarkable decrease of uterine weight in ovariectomized animals compared to sham animals. Though not statistically significant, ovariectomized rats fed on oleocanthal-rich TPF showed some uterotrophic effects compared to ovariectomized animals who did not receive TPF.76 In order to assess bone-sparing effects for the TPF extract, the microarchitecture of the proximal tibia metaphysis has been analyzed by micro-computed tomography. Several parameters were measured such as bone volume fraction, connectivity density, trabecular number, trabecular thickness, and trabecular separation. Compared to sham animals, ovariectomized rats revealed a significant drop in bone volume fraction and density in response to 3-month estrogen depletion. Nevertheless, no changes were observed in the bone microarchitecture for all above-measured parameters in ovariectomized animals exposed to dietary TPF.76 Oleocanthal showed anticancer activity in hormonedependent ER-positive breast cancer cells in culture and animal models.77 Exposure to oleocanthal reduced viability of luminal A MCF-7 and T-47D breast cancer cells in a dose-dependent manner in both mitogen-free and 17β-estradiol-supplemented media (10 nM). Similarly, the growth of luminal B cells, BT-474 was suppressed upon treatment with oleocanthal in a dose-dependent fashion in the presence or absence of 17β-estradiol as the mitogen. In BT-474 cells, oleocanthal treatment remarkably reduced levels of ERα at 20 and 40 μM as demonstrated by Western blot and immunofluorescent staining in both mitogen-free and 17β-estradiol-containing media. In addition, a combination of oleocanthal treatment with

Olive oil oleocanthal and estrogen receptor expression Chapter | 55

tamoxifen produced synergistic growth inhibition in all three ER-positive breast cancer cells. The effect of the combined treatment was assessed using combination index, dose-reduction index, and isobologram analysis.77 The effect of oleocanthal treatment on ER expression was further examined in vivo using an orthotopic athymic mice model bearing BT-474 tumor xenografts.77 Animals were treated with intraperitoneal oleocanthal in two different concentrations (5 and 10 mg/kg) for a duration of 6 weeks. Oleocanthal significantly reduced BT-474 tumor weight and volume in treated animals compared to the vehicle-treated group. Oleocanthal treatment inhibited 17β-estradiol-mediated BT-474 tumor growth by 97% compared to vehicletreated control animals. These effects were also associated with reduced total levels of ERα in oleocanthal-treated groups compared to vehicle-treated control animals.77

55.5 Conclusion Oleocanthal is a phenolic olive oil compound with several biologic activities reported in the literature. The beneficial effects of olive oil consumption could be attributed to its content of polyphenolic compounds as oleocanthal. Multiple molecular mechanisms have been explored for the antiinflammatory, antioxidant, anticancer, and neuroprotective effects of the compound. Recently, ER has been also recognized as a target of oleocanthal. Oleocanthal has been shown to bind ERs and modify their expression in different cell types. Oleocanthal bone-sparing effects and anticancer activity can be mediated, at least in part, by its selective modulation of ER activity and gene expression. Further investigation of oleocanthal
ER interactions can provide greater insights into the biologic effects of the compound and its potential application in cardiovascular disease, rheumatology, and hormone-dependent cancers.

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1420. 11. Monti SM, Ritieni A, Sacchi R, Skog K, Borgen E, Fogliano V. Characterization of phenolic compounds in virgin olive oil and their effect on the formation of carcinogenic/mutagenic heterocyclic amines in a model system. J Agric Food Chem. 2001;49(8):3969
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658. 28. Romero C, Medina E, Vargas J, Brenes M, De Castro A. In vitro activity of olive oil polyphenols against Helicobacter pylori. J Agric Food Chem. 2007;55(3):680
686. 29. Garcia-Villalba R, Carrasco-Pancorbo A, Nevedomskaya E, et al. Exploratory analysis of human urine by LC-ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols. Anal Bioanal Chem. 2010;398(1):463
475. 30. Lucas L, Russell A, Keast R. Molecular mechanisms of inflammation. Anti-inflammatory benefits of virgin olive oil and the phenolic compound oleocanthal. Curr Pharm Des. 2011;17(8):754
768. 31. Vougogiannopoulou K, Lemus C, Halabalaki M, et al. One-step semisynthesis of oleacein and the determination as a 5lipoxygenase inhibitor. J Nat Prod. 2014;77(3):441
445. 32. Scotece M, Gomez R, Conde J, et al. Further evidence for the antiinflammatory activity of oleocanthal: inhibition of MIP-1alpha and IL-6 in J774 macrophages and in ATDC5 chondrocytes. Life Sci. 2012;91(23-24):1229
1235. 33. Iacono A, Gomez R, Sperry J, et al. Effect of oleocanthal and its derivatives on inflammatory response induced by lipopolysaccharide in a murine chondrocyte cell line. Arthritis Rheum. 2010;62 (6):1675
1682. 34. Scotece M, Conde J, Abella V, et al. Oleocanthal inhibits catabolic and inflammatory mediators in LPS-activated human primary osteoarthritis (OA) chondrocytes through MAPKs/NF-kappaB pathways. Cell Physiol Biochem. 2018;49(6):2414
2426. 35. Montoya T, Castejon ML, Sanchez-Hidalgo M, Gonzalez-Benjumea A, Fernandez-Bolanos JG, Alarcon de-la-Lastra C. Oleocanthal modulates LPS-induced murine peritoneal macrophages activation via regulation of inflammasome, Nrf-2/HO-1, and MAPKs signaling pathways. J Agric Food Chem. 2019;67(19):5552
5559. 36. Galvano F, La Fauci L, Graziani G, et al. Phenolic compounds and antioxidant activity of Italian extra virgin olive oil Monti Iblei. J Med Food. 2007;10(4):650
656. 37. Giusti L, Angeloni C, Barbalace MC, et al. A proteomic approach to uncover neuroprotective mechanisms of oleocanthal against oxidative stress. Int J Mol Sci. 2018;19(8). 38. Angeloni C, Giusti L, Hrelia S. New neuroprotective perspectives in fighting oxidative stress and improving cellular energy metabolism by oleocanthal. Neural Regen Res. 2019;14(7):1217
1218. 39. Rosignoli P, Fuccelli R, Fabiani R, Servili M, Morozzi G. Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes. J Nutr Biochem. 2013;24(8):1513
1519.

40. Pei T, Meng Q, Han J, et al.
)-Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma. Oncotarget. 2016;7(28):43475
43491. 41. Gu Y, Wang J, Peng L.
)-Oleocanthal exerts anti-melanoma activities and inhibits STAT3 signaling pathway. Oncol Rep. 2017;37(1):483
491. 42. Fogli S, Arena C, Carpi S, et al. Cytotoxic activity of oleocanthal isolated from virgin olive oil on human melanoma cells. Nutr Cancer. 2016;68(5):873
877. 43. Cusimano A, Balasus D, Azzolina A, et al. Oleocanthal exerts antitumor effects on human liver and colon cancer cells through ROS generation. Int J Oncol. 2017;51(2):533
544. 44. Unsal UU, Mete M, Aydemir I, Duransoy YK, Umur AS, Tuglu MI. Inhibiting effect of oleocanthal on neuroblastoma cancer cell proliferation in culture. Biotech Histochem. 2019;1
9. 45. Scotece M, Gomez R, Conde J, et al. Oleocanthal inhibits proliferation and MIP-1alpha expression in human multiple myeloma cells. Curr Med Chem. 2013;20(19):2467
2475. 46. LeGendre O, Breslin PA, Foster DA.
)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization. Mol Cell Oncol. 2015;2(4):e1006077. 47. Goren L, Zhang G, Kaushik S, Breslin PAS, Du YN, Foster DA.
)-Oleocanthal and (
)-oleocanthal-rich olive oils induce lysosomal membrane permeabilization in cancer cells. PLoS One. 2019;14(8):e0216024. 48. Fabiani R, De Bartolomeo A, Rosignoli P, et al. Virgin olive oil phenols inhibit proliferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation. J Nutr. 2006;136(3):614
619. 49. Khanfar MA, Bardaweel SK, Akl MR, El Sayed KA. Olive oilderived oleocanthal as potent inhibitor of mammalian target of rapamycin: biological evaluation and molecular modeling studies. Phytother Res. 2015;29(11):1776
1782. 50. Margarucci L, Monti MC, Cassiano C, et al. Chemical proteomicsdriven discovery of oleocanthal as an Hsp90 inhibitor. Chem Commun (Camb). 2013;49(52):5844
5846. 51. Khanal P, Oh WK, Yun HJ, et al. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis. Carcinogenesis.. 2011;32(4):545
553. 52. Elnagar AY, Sylvester PW, El Sayed KA.
)-Oleocanthal as a cMet inhibitor for the control of metastatic breast and prostate cancers. Planta Med. 2011;77(10):1013
1019. 53. Diez-Bello R, Jardin I, Lopez JJ, et al.
)-Oleocanthal inhibits proliferation and migration by modulating Ca(21) entry through TRPC6 in breast cancer cells. Biochim Biophys Acta Mol Cell Res. 2019;1866(3):474
485. 54. De Stefanis D, Scime S, Accomazzo S, et al. Anti-proliferative effects of an extra-virgin olive oil extract enriched in ligstroside aglycone and oleocanthal on human liver cancer cell lines. Cancers (Basel). 2019;11(11). 55. Pitt J, Roth W, Lacor P, et al. Alzheimer’s-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal. Toxicol Appl Pharmacol. 2009;240 (2):189
197. 56. Abuznait AH, Qosa H, Busnena BA, El Sayed KA, Kaddoumi A. Olive-oil-derived oleocanthal enhances beta-amyloid clearance as a potential neuroprotective mechanism against Alzheimer’s disease: in vitro and in vivo studies. ACS Chem Neurosci. 2013;4(6):973
982.

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57. Angeloni C, Malaguti M, Barbalace MC, Hrelia S. Bioactivity of olive oil phenols in neuroprotection. Int J Mol Sci. 2017;18(11). 58. Agrawal K, Melliou E, Li X, et al. Oleocanthal-rich extra virgin olive oil demonstrates acute anti-platelet effects in healthy men in a randomized trial. J Funct Foods. 2017;36:84
93. 59. Simpson ER. Sources of estrogen and their importance. J Steroid Biochem Mol Biol. 2003;86(3
5):225
230. 60. Fuentes N, Silveyra P. Estrogen receptor signaling mechanisms. Adv Protein Chem Struct Biol. 2019;116:135
170. 61. Lee HR, Kim TH, Choi KC. Functions and physiological roles of two types of estrogen receptors, ERalpha and ERbeta, identified by estrogen receptor knockout mouse. Lab Anim Res. 2012;28(2):71
76. 62. Koos RD. Minireview: putting physiology back into estrogens’ mechanism of action. Endocrinology. 2011;152(12):4481
4488. 63. Yasar P, Ayaz G, User SD, Gupur G, Muyan M. Molecular mechanism of estrogen-estrogen receptor signaling. Reprod Med Biol. 2017;16(1):4
20. 64. Cui J, Shen Y, Li R. Estrogen synthesis and signaling pathways during aging: from periphery to brain. Trends Mol Med. 2013;19 (3):197
209. 65. Molina L, Figueroa CD, Bhoola KD, Ehrenfeld P. GPER-1/GPR30 a novel estrogen receptor sited in the cell membrane: therapeutic coupling to breast cancer. Expert Opin Ther Targets. 2017;21 (8):755
766. 66. Deroo BJ, Korach KS. Estrogen receptors and human disease. J Clin Invest. 2006;116(3):561
570. 67. Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F. Estrogen receptors alpha (ERalpha) and beta (ERbeta): subtype-selective ligands and clinical potential. Steroids. 2014;90:13
29. 68. Bottner M, Thelen P, Jarry H. Estrogen receptor beta: tissue distribution and the still largely enigmatic physiological function. J Steroid Biochem Mol Biol. 2014;139:245
251.

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69. Rizza P, Barone I, Zito D, et al. Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines. Breast Cancer Res. 2014;16(1):R21. 70. Starkey NJE, Li Y, Drenkhahn-Weinaug SK, Liu J, Lubahn DB. 27-hydroxycholesterol is an estrogen receptor beta-selective negative allosteric modifier of 17beta-estradiol binding. Endocrinology. 2018;159(5):1972
1981. 71. Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD. Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects. Circulation. 2000;101(15):1792
1798. 72. Bjornstrom L, Sjoberg M. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol. 2005;19(4):833
842. 73. Bourdeau V, Deschenes J, Metivier R, et al. Genome-wide identification of high-affinity estrogen response elements in human and mouse. Mol Endocrinol. 2004;18(6):1411
1427. 74. Aranda A, Pascual A. Nuclear hormone receptors and gene expression. Physiol Rev. 2001;81(3):1269
1304. 75. Vrtacnik P, Ostanek B, Mencej-Bedrac S, Marc J. The many faces of estrogen signaling. Biochem Med (Zagreb). 2014;24(3):329
342. 76. Keiler AM, Zierau O, Bernhardt R, et al. Impact of a functionalized olive oil extract on the uterus and the bone in a model of postmenopausal osteoporosis. Eur J Nutr. 2014;53(4):1073
1081. 77. Ayoub NM, Siddique AB, Ebrahim HY, Mohyeldin MM, El Sayed KA. The olive oil phenolic (
)-oleocanthal modulates estrogen receptor expression in luminal breast cancer in vitro and in vivo and synergizes with tamoxifen treatment. Eur J Pharmacol. 2017;810:100
111. 78. Keiler AM, Djiogue S, Ehrhardt T, et al. Oleocanthal modulates estradiol-induced gene expression involving estrogen receptor alpha. Planta Med. 2015;81(14):1263
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Chapter 56

Neuroprotective effects of oleocanthal in neurological disorders Yazan S. Batarseh1, Sweilem B. Al Rihani2, Euitaek Yang2 and Amal Kaddoumi2 1

Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan,

2

Department of Drug Discovery and Development, Harrison School of Pharmacy, Pharmacy Research Building, Auburn University, Auburn, AL,

United States

Abbreviations amyloid-β amyloid-β oligomers a disintegrin and metalloproteinase Alzheimer’s disease adenosine monophosphate (AMP)-activated protein kinase ApoE apolipoprotein E BBB blood
brain barrier CAA cerebral amyloid angiopathy COX cyclooxygenase CSF cerebrospinal fluid EVOO extra-virgin olive oil FTD frontotemporal dementia GFAP glial fibrillary acidic protein HO-1 heme oxygenase-1 IDE insulin degrading enzyme IL-1β interleukin-1β LPS lipopolysaccharide LRP1 LDL lipoprotein receptor-related protein-1 MAPK mitogen-activated protein kinase MMP matrix metalloproteinase Nep neprilysin NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NFTs neurofibrillary tangles NLRP3 NACHT, LRR, and PYD domain-containing protein 3 Nrf-2 nuclear factor (erythroid-derived 2)-like 2 NSAIDs nonsteroidal antiinflammatory drugs P-gp P-glycoprotein PPARγ peroxisome proliferator
activated receptor-γ PSD-95 postsynaptic density protein 95 ROS reactive oxygen species Aβ Aβo ADAM AD AMPK

SNAP25 SOD TNF-α ULK1

synaptosome-associated protein-25 superoxide dismutase tissue necrosis factor-α Unc-51-like autophagy activating kinase

56.1 Introduction Several clinical studies have reported extra-virgin olive oil (EVOO) as one component of the Mediterranean diet to improve cognitive function and slow progression of memory impairment.1
4 EVOO is a high-quality olive oil obtained from the first pressing of olive fruit by mechanical means.5 Ninety-five percent of EVOO content is composed of glycerol fraction with the remaining nonglycerol fraction representing the phenolic compounds that account for EVOO resistance to oxidative rancidity.6,7 The total nonglycerol content of EVOO represents about 500 mg/kg with over 30 chemical substances that belong to different classes, including alcohols, sterols, hydrocarbons, and volatile compounds.8 The most abundant phenolic compounds in EVOO are tyrosol, hydroxytyrosol, and other complex ester secoiridoids; these compounds share the hydroxytyrosol or tyrosol moiety. Among EVOO phenolics, oleocanthal, a dialdehydic form of deacetoxyligstroside glycoside, is a naturally occurring phenolic secoiridoid that has related chemical structure to the secoiridoids ligstroside and oleuropein aglycone, which are common in EVOO. Oleocanthal is responsible for the bitter and pungent taste of EVOO and has antiinflammatory and antioxidant properties similar to the nonsteroidal antiinflammatory drug (NSAID) ibuprofen.9

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00055-9 © 2021 Elsevier Inc. All rights reserved.

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Findings from animal and in vitro studies have shown that oleocanthal possesses a neuroprotective activity against Alzheimer’s disease (AD) and related disorders. AD is one of the most common neurodegenerative diseases, affecting the elderly population worldwide with estimates of 60%
80% of identified cases.10 These prevalence percentages translate to a significant number of AD patients with incidence rates increasing greatly after reaching the age of 65 years. Currently, there is more than 50 million patients with AD globally, and the number is expected to increase that could lead to a worldwide crisis.10
13 AD has a distinctive diagnosable clinical presentation profile spanning from loss in memory and cognition to personality changes and aggression. The molecular pathophysiology of AD includes amyloid-β (Aβ) aggregation leading to Aβ plaques deposition, and phosphorylated tau protein fragments accumulated in the form of neurofibrillary tangles (NFTs).14
16 In addition, AD brains are characterized with oxidative stress and neuroinflammation leading to astrocytes and microglial activation, increase in reactive oxygen and nitrogen species, and elevated levels of reactive cytokines [such as interleukin-6, interleukin-1β, and tissue necrosis factor-α (TNF-α)].17 Another key hallmark in AD pathology is the compromised blood
brain barrier (BBB) integrity.12,13,18 Emerging evidence suggests that AD has a major vascular component where BBB injury represents the initial hit followed by Aβ accumulation and NFT formation.12,13,18 Moreover, Aβ deposition on the vasculature of the BBB leads to a distinctive abnormality called cerebral amyloid angiopathy (CAA).19,20 Majority of patients suffering from AD also have CAA as a comorbid condition with estimates approaching 85%
95% of the cases.21 Both AD and CAA have major amyloidogenic components that require interventional efforts to overcome their drastic downstream complications. The significant rise in the prevalence of AD and related disorders, and the lack of options for its effective treatment urge the search for novel therapeutic drugs and strategies to prevent and/or delay the onset of AD. In this chapter, we will review reported findings with oleocanthal and oleocanthal-rich EVOO and summarize its therapeutic potential to prevent and/or delay AD and related disorders, including CAA and tauopathy in several cellular and animal models with main focus on oleocanthal effect on Aβ clearance, BBB function, neuroinflammation, oxidative stress, and tau aggregation.

56.2 Oleocanthal induces brain amyloidβ clearance A number of in vitro and in vivo studies have shown that oleocanthal and oleocanthal-rich EVOO have important

neuroprotective effects that could prevent and reduce the risk of developing AD and related disorders.22
25 While the exact neuroprotective mechanism(s) of action of oleocanthal against AD is not fully examined, several potential pathways for oleocanthal have been demonstrated, which could protect against and/or reduce the risk of AD. Initial in vitro studies demonstrated the effect of oleocanthal on key hallmarks of AD, namely, Aβ and tau aggregation,16,26
28 which contribute significantly to neurodegeneration and memory loss.16 In these in vitro studies, oleocanthal disrupted Aβ and tau aggregation by altering soluble Aβ42 oligomerization state that was associated with significantly reduced synaptic loss,26 and by locking tau into the unfolded form preventing its aggregation.27,28 Aβ deposition in brain parenchyma, and in and around cerebral blood vessels play a central role in a series of response mechanisms that lead to compromises in BBB integrity and AD pathology.29
32 However, the underlying mechanisms for Aβ accumulation are not well understood. Aβ is produced by the proteolytic cleavage of amyloid precursor protein via the coordinated action of βand γ-secretases.33,34 The increase in Aβ levels during AD and CAA is thought to be mediated in part by an imbalance between Aβ biosynthesis and clearance pathways, which ultimately favors Aβ accumulation.35 As Aβ is continuously produced within the brain and its accumulation is deleterious,36 efficient clearance mechanism(s) are essential to prevent its accumulation and subsequent aggregation in the brain. Clearance of Aβ from the brain takes place by multiple pathways, including (Fig. 56.1) (1) transport across the BBB,20 (2) enzymatic degradation by specific Aβ-degrading enzymes, namely, neprilysin (NEP) and insulin degrading enzyme (IDE),37 (3) lysosomal degradation (autophagy),38 (4) apolipoprotein E (ApoE)-mediated clearance,39 and (5) the bulk flow of the interstitial fluid into the cerebrospinal fluid (CSF).40 To test the effect of oleocanthal on Aβ clearance by modulating previously listed pathways, in vitro and in vivo studies were performed. When tested in mouse brain endothelial cells treated with oleocanthal, findings demonstrated that oleocanthal significantly upregulated the expression of Aβ major transport proteins, namely, Pglycoprotein (P-gp) and LDL lipoprotein receptor-related protein-1 (LRP1), which play key role in Aβ clearance across the endothelial cells of the BBB, in a concentration-dependent manner.24 When tested in vivo in wild-type and AD mice models,22
25 similarly, oleocanthal significantly increased Aβ clearance and reduced cerebrovascular and parenchymal Aβ levels. Oleocanthal reduced Aβ accumulation in mice brains by increasing the expression of endothelium-P-gp and LRP1, determined in isolated brain microvessels, which increased Aβ clearance across the BBB,22
25 and by increasing Aβ degradation

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FIGURE 56.1 Clearance pathways of Aβ from the brain. Aβ cleared from the brain interstitial fluid by transport across the BBB mediated by P-gp and LRP1, enzymatic degradation by NEP and IDE, ApoE-clearance pathway and autophagy, and through the bulk flow of CSF. ApoE, Apolipoprotein E; Aβ, amyloid-β; BBB, blood
brain barrier; IDE, insulin degrading enzyme; LRP1, LDL lipoprotein receptor related protein-1; NEP, neprilysin; P-gp, P-glycoprotein.

enzymes NEP and IDE in mice brains.22
25 Furthermore, oleocanthal reduced brain Aβ accumulation in AD mice brains by inducing ApoE-dependent clearance pathway through peroxisome proliferator
activated receptor-γ (PPARγ) activation.23,25 When tested in TgSwDI, a CAA mouse model, mice treatment with oleocanthal-rich EVOO for 3 months significantly reduced brain Aβ levels by enhancing autophagy by activating adenosine monophosphate-activated protein kinase/Unc-51-like autophagy activating kinase 1 (AMPK/ULK1) pathway.22 Autophagy impairment has been associated with impaired clearance of Aβ, hyperphosphorylated tau, and damaged cellular organelles, which could lead to their aggregation and hence accumulation and subsequent neurotoxicity.41 Collectively, and based on the previous findings, oleocanthal reduced Aβ brain levels by upregulating pathways vital for Aβ clearance.

56.3 Oleocanthal enhances blood
brain barrier integrity and function Alongside the major hallmarks of AD, Aβ plaques, and NFT, BBB dysfunction is thought to be a major driver for neurodegeneration and AD progression by reducing Aβ clearance, increasing Aβ accumulation, and associated neuroinflammation.12,13,42,43 Therefore maintaining a healthy and functional BBB, or restoring BBB function could be a key strategy to prevent or slow the progression of AD.44 The BBB is a highly specialized structural and biochemical barrier that regulates the entry of blood
borne molecules and cells into brain and preserves ionic

homeostasis within the brain microenvironment.45,46 The BBB functions to maintain brain homeostasis and normal neuronal function. The uniqueness of BBB is given by the endothelial cells, with adherens junctions as cell
cell interaction stabilizers, and tight junctions that regulate BBB paracellular permeability, limit paracellular transport of water, ions, and larger molecules into the brain, and organize the cell membrane in luminal and abluminal sides.46,47 The molecular transport at the BBB takes place mostly by transcellular pathways, including (1) gradient diffusion, (2) transporters (e.g., amino acids, glucose, and Aβ), and (3) receptor-mediated transcytosis (e.g., leptin, insulin, transferrin, and Aβ). Passage of all other hydrophilic compounds, including drugs, is highly restricted under normal conditions. These endothelial cells have highly controlled permeability toward plasmatic compounds and ions and have high transendothelial electrical resistance.48 In addition, the BBB has transport proteins and receptors that assist or limit molecules entry into the brain thus maintaining brain homeostasis and normal neuronal function.46 Some of these transporters expressed at the BBB are specialized in active efflux or uptake of different molecules, including nutrients, brain waste products, and drugs.49 Recent experimental evidence highlights BBB integrity and function in onset and progression of amyloid pathogenesis
related disorders.50
53 These studies support an association between Aβ, tight junctions, and BBB properties, correlation between Aβ accumulation and microvascular impairment in the AD brain.54 Pharmacological targeting of the BBB permeability and function is expected to restore BBB function by limiting unwanted molecules brain entry and efficient

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removal of Aβ from the brain. Beside increased brain Aβ clearance, oleocanthal was able to improve BBB function and integrity. Several studies demonstrated that the treatment of wild-type, AD, and CAA mouse models with oleocanthal or oleocanthal-rich EVOO significantly improved the BBB integrity and function.22
25 For example, when tested in TgSwDI and 5XFAD mice, which represent a CAA and AD mouse models, respectively, mice treatment with oleocanthal-rich EVOO for 3 months, added to mice diet, significantly reduced BBB leakiness as determined by immunostaining to evaluate the extravasation of the endogenous BBB permeability marker, namely, immunoglobulin-G in the cortex and hippocampus regions of mice brains.22,25 Enhanced BBB integrity was associated with significant increase in endothelialtight junction proteins, including ZO-1, claudin-5, occludin, and JAM1.22,25 In addition to increased BBB integrity, oleocanthal enhanced BBB function by increasing the expression of Aβ major transport proteins, P-gp and LRP1, expressed on brain microvessels as described earlier.22
25 The important effect of oleocanthal on BBB function and Aβ clearance across the BBB was further confirmed by inhibition studies with valspodar, as a P-gp-specific inhibitor, and receptor-associated protein (RAP), as an LRP1 inhibitor, where results demonstrated that both inhibitors diminished oleocanthal-induced BBB function as measured by brain efflux index assay.24

56.4 Oleocanthal reduces neuroinflammation and oxidative stress Aggregation of aberrant proteins, including Aβ and NFT, not only directly interferes with neuronal function but also starts a cascade of events comprising several inflammatory and oxidative pathways leading to the production and release of an array of harmful inflammatory oxidative stress mediators.14 Inflammation due to Aβ accumulation could lead to alteration in the brain environment on the cellular level; for instance, astrocytes may neglect their neuro-supportive role and convert to inflammatory reactive cells.55 Although acute inflammatory response may be beneficial at the beginning, chronic inflammation as the one seen in AD may disturb the delicate balance in the brain leading to altered neuronal function and in extreme situations neuronal death.56 Besides, dysregulation of free radical detoxification due to increased levels of reactive oxygen species (ROS) and/or decreased antioxidant capacity could result in oxidative stress.57 In the brain, neurons contain oxidizable polyunsaturated fatty acids that could generate radicals triggering oxidative stress. In addition, in AD brains, glial cells activation could contribute to increased levels of ROS via activation of NADPH oxidase.58
60 ROS

accumulation in the membrane of neurons could trigger neurodegeneration and neuroinflammation by disrupting membrane permeability, which could ultimately result in cognition impairment.60 Targeting inflammation as a therapeutic venue for neurodegeneration represents a novel approach with many hopeful expectations.61 Beauchamp et al. investigated whether oleocanthal antiinflammatory effect resembles that of ibuprofen, an NSAID, widely used in modern medicine as analgesic, antipyretic, and antiinflammatory drug.9 Ibuprofen, an inhibitor of cyclooxygenase-1 (COX1) and COX-2, interferes with arachidonic acid pathway to deliver its antiinflammatory action.62 Similar to ibuprofen, oleocanthal demonstrated partial inhibition of COX enzymes, suggesting that oleocanthal may have a long-term protective effect against inflammation-related medical abnormalities such as neurodegeneration and cardiovascular diseases.9 Oleocanthal displays its antiinflammatory effect by targeting multiple mechanisms. For example, Carpi et al. investigated the therapeutic role of oleocanthal and its natural analog hydroxy oleocanthal on fully differentiated Simpson
Golabi
Behmel syndrome adipocytes challenged with TNF-α,63 a well-known mediator of inflammation and irritation in adipose tissue.64 Their results showed that adipocytes pretreatment with either phenol was able to reduce TNF-α-induced activation of inflammation by targeting nuclear factor kappalight-chain-enhancer of activated B (NF-κB) pathway.63 NF-κB pathway inhibition by oleocanthal and hydroxy oleocanthal reduced the expression of several inflammatory and oxidative stress markers as follows. Oleocanthal and hydroxy oleocanthal reduced inflammatory cytokines [e.g., interleukin-1β (IL-1β)], COX-2, angiogenesis factors involved in inflammatory pathways such as vascular endothelial growth factor receptor 2 and matrix metalloproteinase-2 (MMP2), oxidative markers such as nicotinamide adenine dinucleotide phosphate oxidase, antioxidant-involved enzymes [e.g., superoxide dismutase (SOD) and glutathione peroxidase], and chemotaxis and infiltration markers (e.g., monocyte chemoattractant protein-1, chemokine C-X-C motif ligand-10 (CXCL10), macrophage colony-stimulating factor), while the expression of the antiinflammatory and metabolic effector PPARγ was enhanced.63 Furthermore, Scotece et al. investigated the antiinflammatory effect of oleocanthal on primary chondrocytes treated with lipopolysaccharide (LPS) to induce inflammation.65 Chondrocytes were collected from human biopsies of cartilages from patients with symptoms of osteoarthritis. The authors of this study reported that the effect of oleocanthal was safe and reduced LPS-induced inflammatory cytokines and oxidative stress markers as determined by reduced nitric oxide synthase-2 and COX-2 protein and their mRNA expressions and inhibited MMP13 and a disintegrin

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and metalloproteinase (ADAM) with thrombospondin type 1 Motif-5.65 Moreover, it was exhibited in the same study that these effects were attributed to the modulation of mitogen-activated protein kinase (MAPK)/P38/NF-κB pathways providing additional evidence on oleocanthal antiinflammatory effect. Moreover, Montoya and colleagues evaluated oleocanthal effect on both the canonical and noncanonical inflammasome; canonical-associated proteins include NACHT, LRR, and PYD domaincontaining protein 3 (NLRP3), ASC, and pro- and cleaved caspase-1, and noncanonical inflammasome is mediated by caspase-11 activation.66 Findings from this study confirmed oleocanthal-inhibited LPS-induced inflammation in peritoneal macrophages isolated from mice by downregulating the expression of proteins associated with canonical and noncanonical inflammasome activation, an effect that was mediated by MAPK and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2)/heme oxygenase-1 (HO-1) signaling pathways. Besides, treatment with oleocanthal exhibited reduced oxidation stress as shown by a reduction in the levels of ROS and nitrites, which collectively reduced significant levels of inflammatory cytokines, thus, contributing to its overall antiinflammatory and antioxidant profile.66 When tested in the immortalized human neuronal SHSY5Y cells, oleocanthal in the absence or presence of hydrogen peroxide, added to trigger oxidative stress, demonstrated oleocanthal significantly increased cells viability and rectified the toxic effect of hydrogen peroxide
induced oxidative stress by increasing glutathione production.67 To investigate the molecular events related to oleocanthal protective effect, the authors of this study implemented a proteomic analysis; their finding indicated that in response to ROS insult, oleocanthal modulated a set of proteins, including proteasome, ubiquitin, and chaperone proteins, which play important role in maintaining equilibrium between protein synthesis, folding, trafficking, secretion, and degradation in different cell compartments.67 These results support oleocanthal role as an antioxidant molecule, thus, providing neuroprotective effect. In AD, while the means by which oleocanthal provides its neuroprotective role remain to be well elucidated, several studies presented evidence on the antiinflammatory, antioxidant, and neuroprotective effects of oleocanthal in vitro and in vivo in models of AD.22
25 Findings from our laboratory established oleocanthal treatment for 1 month in TgSwDI mice significantly reduced astrocytes activation as demonstrated by reduced immunoreactivity of glial fibrillary acidic protein (GFAP), an intermediate filament protein found in astrocytes, suggesting reduced inflammation.23 These results were further confirmed by significant reduction in cytokines level represented by IL1β, one of the major extracellular reactive immunomodulators overexpressed in AD brains.23 Furthermore,

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Batarseh et al. reported oleocanthal effect on modulating the pathological events of Aβ oligomers (Aβo) in neurons and astrocytes to further evaluate oleocanthal neuroprotective effect.68 Study findings showed oleocanthal blocks Aβo-induced synaptotoxicity,68 which are consistent with the findings of Pitt et al.26 The synaptic markers monitored for integrity were synaptosome-associated protein-25 (SNAP-25) and postsynaptic density protein 95 (PSD-95).68 Increased expression of these synaptic markers by oleocanthal treatment represents a noteworthy observation for the protective role of oleocanthal in neurons as a consequence of reduced neuroinflammation mediated by reduced Aβo-induced astrogliosis.68 Oleocanthal reduced Aβo-induced inflammation markers, namely, IL-6 and GFAP, and restored the neurosupportive function of astrocytes by increasing glutamate transporter-1 and glucose transporter-1 expressions.68 To further confirm the protective antiinflammatory effect of oleocanthal, in vivo studies in CAA and AD mouse models were performed using oleocanthal-rich EVOO added to mice diet.22,25 Batarseh and Kaddoumi investigated the effect of oleocanthal-rich EVOO, as medical food, on enhancing the effect of donepezil to reduce Aβ levels and related pathology in 5xFAD mouse model of AD.25 Donepezil is an acetylcholine esterase inhibitor approved by FDA for use for all AD stages (mild-to-severe),69 which has some noncholinergic activity.70 Oleocanthalrich EVOO consumption separately or in combination with donepezil significantly reduced Aβ levels. In addition, oleocanthal-rich EVOO combined with donepezil enhanced synaptic markers and increased the BBB integrity and function, an effect that was associated with reduced inflammation and increased antioxidant capacity.25 These findings were further confirmed by Al Rihani and colleagues who investigated the effect of oleocanthalrich EVOO on Aβ-related pathology and neuroinflammation in another mouse model, namely, TgSwDI.22 Similarly, mice consumption of oleocanthal-rich EVOO for 3 months starting at 6 months of age demonstrated significant reduction in Aβ and related pathology in mice brains and reduced neuroinflammation and oxidative stress, which collectively resulted in improved learning and memory function. Oleocanthal-rich EVOO inhibited the activation of NLRP3 inflammasome that was associated with enhanced autophagy through AMPK/ULK1 pathway, thus, providing further evidence on the antiinflammatory protective effect of oleocanthal in CAA and AD that could directly and/or indirectly reduce Aβ-related pathology.22 Moreover, oleocanthal-rich EVOO significantly reduced oxidative stress markers, including protein carbonyl that is an important detectable marker of protein oxidation and has been detected in AD brains,71 and increased SOD levels in mice brains.22 SOD acts as an antioxidant enzyme that catalyzes the dismutation of

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superoxide anion free radical into molecular oxygen and hydrogen peroxide; and in AD, SOD levels are reduced suggesting oxidative stress.72 However, more studies are necessary to elucidate the mechanism(s) by which oleocanthal exerts its antioxidant effect in brains of AD and related disorders.

56.4.1 Oleocanthal inhibits tau fibrillization In addition to AD, frontotemporal dementia (FTD) and tauopathy are other types of dementia and are characterized by tau aggregation and NFTs formation in neurons.73 Tau is a microtubule-associated protein; it is highly expressed in neuronal axons acting as a microtubule stabilizer, regulating neurite outgrowth and other microtubuledependent functions.74 Besides genetic mutations and unknown environmental factors, multiple mechanisms have been proposed to cause tau fibrillization and NFTs formation such as posttranslational modifications.75 Initial in vitro studies investigated oleocanthal inhibitory effect on tau aggregation.26,28 In these studies the authors mixed oleocanthal with the hexapeptide within the third repeat of tau, which is important for tau fibrillization, and demonstrated that oleocanthal through its aldehyde groups formed an adduct with the lysine residue via Schiff base reaction locking tau into its naturally unfolded form and thereby inhibiting tau fibrillization.27,28 When tested in vivo in TgSwDI mouse model of CAA and AD fed with oleocanthal-rich EVOO for 3 months, oleocanthal significantly reduced total tau and tau phosphorylation at amino acid threonine position 231. While additional studies are necessary to explain the mechanism of such

reduction in tau and its phosphorylated form in mice brains, enhanced autophagy and lysosomal degradation could contributed to this effect.22,76

56.5 Conclusion In conclusion, oleocanthal exhibits several neuroprotective properties. Reported beneficial effects of oleocanthal against AD and related disorders are summarized in Fig. 56.2 and include the following: (1) counteract Aβ-related pathology by increasing its clearance via targeting multiple pathways (see Fig. 56.1) and inhibit tau fibrillization; (2) enhance BBB function and integrity; (3) have antiinflammatory effect with characteristics similar to ibuprofen, and ability to modulate several neuroinflammatory regulators such as Nrf-2/ HO-1, MAPKs, and NF-κB; and (4) reduce oxidative stress. While mechanisms related to oleocanthal antiinflammatory effect are well established, further studies are necessary to elucidate the mechanism(s) by which oleocanthal modulates proteins related to Aβ clearance, BBB function, and oxidative stress. Moreover, additional studies are necessary to evaluate the effect of oleocanthal on tau-related disorders such as FTD and tauopathy, and other neurological diseases, including Parkinson disease and amyotrophic lateral sclerosis. Collectively, and based on reported findings, oleocanthal demonstrated a neuroprotective effect against AD and CAA and thus could be developed as a novel therapeutic molecule to prevent, hold progression, and/or treat AD and possibly other related neurodegenerative disorders. In addition to AD the scope of oleocanthal activity can be extended to include several other inflammatory conditions such as cardiovascular disorders, cancer, and osteoarthritis.

FIGURE 56.2 Neuroprotective effect of oleocanthal and oleocanthal-rich EVOO by modulating key hallmarks of AD and CAA. AD, Alzheimer’s disease; CAA, cerebral amyloid angiopathy; EVOO, extra-virgin olive oil.

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19S. 63 Carpi S, Scoditti E, Massaro M, et al. The extra-virgin olive oil polyphenols oleocanthal and oleacein counteract inflammationrelated gene and miRNA expression in adipocytes by attenuating NF-kappaB activation. Nutrients. 2019;11(12). Available from: https://doi.org/10.3390/nu11122855. 64 Cawthorn WP, Sethi JK. TNF-alpha and adipocyte biology. FEBS Lett. 2008;582(1):117
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Chapter 57

S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor and its application to synergize targeted therapies and prevent breast cancer recurrence Khalid A. El Sayed School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, United States

Abbreviation ALDH1 ASCs Akt ALK AMPK 50 ATP BC Bcl2 Brk CA 15-3 CD31 CD44 CDK-6 EGFR EVOO EMT ERK1/2 ER FAK Fas FDA GAB2 GRB2 HER2 HGF HSP HVS JAK Ki-67

aldehyde dehydrogenase-1 adipose-derived mesenchymal stem cells protein kinase B anaplastic lymphoma kinase AMP (adenosine monophosphate)-activated protein kinase adenosine triphosphate breast cancer B-cell lymphoma-2 breast tumor kinase cancer antigen 15-3 cluster of differentiation 31 cluster of differentiation 44 cyclin-dependent kinase-6 epidermal growth factor receptor extra-virgin olive oil epithelial-to-mesenchymal transition extracellular signal-regulated kinase1/2 estrogen receptors focal adhesion kinase apoptosis antigen 1 Food and Drug Administration GRB2-associated-binding protein 2 growth factor receptor-bound protein 2 human epidermal growth factor-2 hepatocyte growth factor heat shock protein homovanillyl sinapate Janus kinase antigen KI-67

Km

Michaelis
Menten constant, the substrate concentration that produces half-maximal velocity LP lapatinib MAPK mitogen-activated protein kinase MBD multisubstrate
multifunctional docking/binding domain MEK mitogen-activated protein kinase kinase MET mesenchymal-epithelial transition factor receptor MIP-1α macrophage inflammatory protein 1-α MMP matrix metalloproteinase mTOR mammalian target of rapamycin Muc-1 mucin 1, cell surface associated MVD microvessel density NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NK4 natural killer cell transcript-4 (IL-32) NSCLC nonsmall cell lung cancer OC S-(2)-oleocanthal PDB protein database PDK pyruvate dehydrogenase kinase PI3K phosphatidyl-inositol 3-kinase PLC phospholipase C PTB phosphotyrosine binding Rac1 Ras-related C3 botulinum toxin substrate 1 Ras rat sarcoma kinase RET rearranged during transfection proto-oncogene RON Recepteur d’Origine Nantais (MST1R) ROS ROS proto-oncogene RTK receptor tyrosine kinase SH2 Src homology 2 SPSB1 ryanodine receptor domain and SOCS box containing 1 STAT3 signal transducers and activator of transcription 3 TNBC triple negative breast cancer

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00037-7 © 2021 Elsevier Inc. All rights reserved.

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tyrosol sinapate vehicle control vascular endothelial growth factor maximum velocity seen at enzyme saturating substrate concentration) of the enzymatic reaction

57.1 Introduction 57.1.1 Receptor tyrosine kinases-background knowledge The receptor tyrosine kinases (RTKs) are highly selective mammalian cell surface receptors that are critical for several growth factors, cytokines, and hormones regulation.1
7 RTKs are members of the protein tyrosine kinases family, and they possess a transmembrane domain. There are more than 90 tyrosine kinase genes identified in the human genome, out of which 58 encode RTKs.1,2 All RTKs share closely related molecular architecture, including ligand binding extracellular region, single transmembrane helix, intracellular regulatory domain (juxtamembrane), and cytoplasmic tyrosine kinase domain.1,2 Growth factor and natural ligand binding to the extracellular domain induce dimerization of monomeric RTKs, leading to a favorable molecular confirmation that facilitate their cytoplasmic tyrosine kinase domains activation through auto-phosphorylation, generating binding sites for a series of cytosolic adaptor proteins such as Src homology 2 (SH2), which are recruited to the phosphorylated tyrosine residues on the activated receptor.3 These activated protein complexes subsequently lead to diverse cellular responses, including proliferation, migration, differentiation, survival, neovascularization, and tissue repair, which enable the irreversible cells’ entry to the S-phase of the cell cycle.2,3 RTKs have been shown not only to be key regulators of normal cellular processes but also they play critical role in the development and progression of many malignancy types.2
4 In normal cells, the RTK activity is strictly regulated by the action of tyrosine kinase phosphatases, enzymes that dephosphorylate phosphotyrosine residues in the cytoplasmic kinase domain of RTKs, thus turning-off their action. However, dysregulation or constitutive activation of RTKs frequently associated with a wide range of cancers. Deregulated activation of RTKs can be caused by gain-of-function point mutations, genomic amplification or rearrangement of the corresponding genes, overexpression or abnormal autocrine, endocrine, or paracrine stimulation of both receptor and ligand.5,6 Dysregulation of RTKs in cancer cells may promote tumorigenesis by multiple mechanisms, including enhanced cell survival, inhibition of cell death, and sometimes they contribute to various steps of tumor development and progression, including metastasis.6 Due to the

selective alteration of RTKs in cancer cells and its implication in many aspects of the malignancy phenotypes, selective pharmacological targeting of these signaling pathways presents promising intervention opportunities for targeted cancer therapies with minimal expected offtarget effects. Selective blocking of aberrant RTK signaling by inhibiting its catalytic activity with small-molecule inhibitors, which compete with adenosine triphosphate (ATP) at its binding site in kinase domain, is a common drug discovery and development strategy that resulted several Food and Drug Administration (FDA)-approved targeted therapies.

57.1.2 c-MET as a potential molecular target in oncology c-MET, a RTK proto-oncogene, is the only known highaffinity receptor for its mammalian agonistic ligand hepatocyte growth factor (HGF), the scatter factor.8,9 c-MET belongs to the MET (MNNG HOS transforming gene) family and is expressed on the surfaces of various cells, like other numerous RTKs.8
10 c-MET is expressed in epithelial, melanocytes, hematopoietic, neuronal, microglial, and endothelial cells. c-MET and HGF are required for normal cellular development and homeostasis. Activation occurs after binding of mature HGF to the cMET receptor extracellular ligand binding domain prior to c-MET dimerization.8
10 This dimerization triggers multiple trans- and auto-phosphorylation events of the cMET cytoplasmic tail intracellular kinase domain tyrosines 1234 and 1235, which induce conformational changes that promote phosphorylation of c-MET C-terminal multisubstrate
multifunctional docking/binding domain (MBD).11 The MBD distinguishes c-MET from any other RTK pathways.11
13 MBD is composed of two tyrosine-containing motifs (HY1349VHVNAT) and (Y1356VNVK), at which it dimerizes with the SRC family kinases, and other signaling proteins such as GRB2 and phospholipase C (PLC), which activate focal adhesion kinase (FAK) and mediates extensive downstream motility cascades.11,12 c-MET signaling also is implemented via GAB1, an adaptor protein specific to c-MET, which mediates most biologically relevant c-MET-dependent signaling.12 Tyrosines 1349 and 1356 offer proper docking platform to recruit other important signaling proteins such as SH2 and PTB (phosphotyrosine binding) domains, and SRC family kinases required for motility activation (Fig. 57.1).10,11 Subsequently this process activates multiple downstream signal transducers like phosphatidylinositol 3-kinase (PI3K), signal transducers and activator of transcription 3 (STAT3), and extracellular signalregulated kinase1/2 (ERK1/2).8
12,14 This activation also initiates additional downstream signaling pathways,

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FIGURE 57.1 Overview of c-MET structural elements and its downstream effectors.

including mitogen-activated protein kinase (MAPK), Akt, and NFκB, promoting diverse cellular and functional responses, including cell proliferation, survival, motility, differentiation, epithelial-to-mesenchymal transition (EMT), scattering, angiogenesis, invasion, and invasive growth during embryo development (Fig. 57.1).9
14 Several membrane surface proteins, including epidermal growth factor receptor (EGFR), β-catenin, MUC-1, CD44, VEGFA, plexin B, FAK, HER, integrin α6β4, Fas, and others proven to interact with c-MET and contribute to its biological response dynamics. c-MET is critical for the control of tissue homeostasis under normal physiological conditions; however, its aberrant activation by mutation, amplification, or protein overexpression is common in human cancers.8,9 c-METmediated invasive growth is quiescent in nonpathological conditions and normally only fully active during wound healing and tissue regeneration.14 Dysregulation of cMET correlates with aggressive proliferation, invasive character, and pathological motility, especially in triple negative breast cancer (TNBC).8
14 Aberrant c-MET activation is associated with the development and progression of diverse cancer types, including hereditary papillary renal cell carcinoma, lung, head and neck, breast, prostate, pancreatic, and gastric cancers.12,13,15
18 c-MET amplification also correlates with multiple tumor cells escape from the anticancer effects of several targeted and

chemotherapies.18
22 Resistance development of non
small cell lung cancer (NSCLC) for the first-generation EGFR inhibitor gefitinib is mediated via c-MET overexpression, which induce MAPK, STAT3, and ERK1/2 pathways, bypassing the EGFR signaling need.20,21 Multiple tumors were reported to escape the HER family targeted therapies through dysregulated c-MET and crosstalk between c-MET and HER family members.20
27 c-MET overexpression promoted acquired resistance to BC cells treated with the anti-HER2 targeted therapies lapatinib (LP) and trastuzumab.23
26 MET maintains NSCLC cells survival through its heterodimerization with the trans-phosphorylated EGFR, HER2, HER3, and RET, making HER and RET targeted therapies ineffective.28 EGFR can stimulate c-MET pathway through MAPK to enhance NSCLC invasion and brain metastasis.29 EGFR induction of c-MET is known to be independent of its natural ligand HGF in multiple malignancies.30 The recent validation of the crosstalk regulatory association between HGF/c-MET axis and noncoding RNA (ncRNAs) in multiple tumors qualified c-MET to play a critical role in all stages of cancer development, including recurrence.31 The adipose-derived mesenchymal stem cells (ASCs) promote BC recurrence via HGF/c-MET signaling.32 The correlation between c-MET and ALDH1 support the survival and tumor-sphere formation of ALDH1-positive BC stem cells and, therefore, c-MET

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was proven to contribute to dormant tumor cells’ activation and disease relapse.33 Meanwhile the c-MET pathway supported the self-renewal and tumorigenicity of stem-like cells of multiple malignancies including the head and neck squamous cell carcinoma.34 The promoting role of c-MET for BC recurrence was mediated by the suppressor of cytokine signaling protein SplA/ryanodine receptor domain and SOCS box containing 1 (SPSB1).35 More than 280 small-molecule c-MET inhibitors are currently in different clinical trial phases and would seem to have potential to delay the progression of a wide array of MET-dependent malignancies.7
10 Crizotinib (Xalkori) is a c-MET, ALK, and ROS1 RTK inhibitor approved by the FDA for ALK-driven lung cancer.7
10 Cabozantinib is a dual MET and VEGFR2 inhibitor approved by the FDA in 2012 for medullary thyroid cancer.7
10 Crizotinib significantly impaired the angiogenesis and reduced the TNBC burden in C3(1)-Tag mouse model but had no effect in prophylactic mode.36 Clinical trials proved the ability of the c-MET inhibitor tivantinib to extend the progression-free survival and tripled patients’ survival in multiple metastatic cancers.37 The documented efficacies of these marketed agents, as well as of promising ones still undergoing clinical trials, validate c-MET as important molecular target in cancer therapy.7
10 Current strategies employed to interrupt c-MET signaling involve blocking the interaction between c-MET and HGF via neutralizing antibodies directed against either HGF or cMET, or truncated HGF forms with antagonistic activity on c-MET such as NK4 or c-MET biologics, for example, ribozymes, dominant-negative receptors, decoy receptors, and peptides.7
10 The latter concepts do not address the ligand-independent c-MET activation and may only be effective for HGF-driven cancers, reversing cancerassociated phenotypes such as motility, invasion, and tumor growth.7
11 Acceleration of c-MET degradation triggered by HSP90 inhibition was also used.7
10 Interfering with c-MET kinase domain active site with small molecules is another common c-MET-targeting strategy.7
10 Most of former approaches are better suited to block ligand-mediated c-MET activity, while the small-molecule c-MET inhibitors can inhibit the

HGF-dependent cancers as well as tumors driven by other c-MET-dependent mechanisms, such as receptor amplification and activating mutations.7
11 c-MET kinase (ATP binding site) domain topographical features (Fig. 57.2) include (1) the hinge region represented by Met1160 and Pro1158. Interactions at this site are highly characteristic for all c-MET ATP competitive inhibitors targeting the kinase domain.38,39 (2) The central hydrophobic region. (3) Two hydrophobic subpockets. (4) The c-MET activation loop represented by Asp1222Lys1245. Small-molecule ATP-competitive c-MET inhibitors may adopt either type-I or type-II binding modes.11,38 Type-I inhibitors assume a U-shape geometry via the interactions with both the hinge region Met1160 and activation loop residue Tyr1230 (Fig. 57.2). Type II inhibitors adopt extended orientation, binding at the activation loop side only, DFG-out or protein conformations with various degrees of “DFG-out”.

57.1.3 The extra-virgin olive oil phenolic S-(2)-oleocanthal biological activities Epidemiological and clinical studies compellingly documented the Mediterranean diet ability to reduce the incidence of breast and colon cancers, cardiovascular diseases, slow the decline of cognitive functions with aging, and slow the Alzheimer’s disease progression.40
50 Olive oil is a key ingredient of the Mediterranean diet, wherein it represents the main fat source.41
53 The antiinflammatory activity of the phenolic secoiridoid S(2)-oleocanthal (OC, Fig. 57.3) is well documented and proven to target in vitro and in vivo COX1-3,5-lipoxygenase, NO, iNOS, IL-6, and macrophage inflammatory protein 1-α (MIP-1α) expression and activation.54
59 Thus, OC proved to show NSAID-like activity comparable to ibuprofen.54,55,59 OC-inhibited cell viability and COX-2 expression by activation of AMPK-mediated pathways in HT-29 cells, justifying its preventive and therapeutic effects against colon cancers.57 OC-inhibited angiogenesis,38 MIP-1α, IL-6 expression and secretion,58 and 5lipoxygenase.59 OC also inhibited HSP90, an essential FIGURE 57.2 Topographical binding mode (A) and MOLCAD surface interactions (B) of (2)-oleocanthal at the c-MET kinase domain.38

S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor Chapter | 57

O 7

3'

HO

6'

1'

O

S 5 3

O (–)–Oleocanthal(OC)

1

O

9

E

O

O

OH

OH 8

HO

O

O O

O O

HO O

Tyrosol sinapate (TS)

685

O

Homovanillyl sinapate (HVS)

FIGURE 57.3 Chemical structures of S-(2)-oleocanthal and its synthetic c-MET inhibitory bioisosteres.

molecular chaperone involved in various cancer hallmarks,60 inhibited ATPase activity, and changed HSP90 oligomerization state.60 OC induced both primary necrotic and apoptotic cell death through the enhancement of lysosomal membrane permeabilization by inhibiting acid sphingomyelinase activity, which destabilizes the interaction between proteins necessary for lysosomal membrane stability.61 OC also inhibited mammalian target of rapamycin (mTOR) activation, with an IC50 value of 708 nM, downregulating p-mTOR in the TNBC MDA-MB-231 cells.62 Chemical proteomics proved HSP70 and HSP90, as major OC interactors in living systems.63 OC inhibited the proliferation, cell cycle progression, migration, invasion, and induced apoptosis in hepatocellular carcinoma in vitro, suppressed its growth and metastasis to lung in vivo via reducing STAT3 nuclear translocation and DNA binding activity, and its downstream effectors.64 OC showed in vitro activity against the human melanoma cells at low μM doses, with significant inhibition of ERK1/2 and Akt phosphorylation and downregulation of Bcl-2 expression.65 OC also showed activities against pancreatic, prostate, and non-melanoma skin cancers.38,61,65 Unlike other phenolic natural products, OC tends to spontaneously interacts in vivo with lysine and other amino acids-containing protein, forming highly stable Schiff’s bases, rendering the prediction of its oral bioavailability highly challenging.47 Based on its anticancer activities, (2)-oleocanthal was virtually screened for binding ability and mode against the crystal structures of several cell survival and motility controlling kinases including CDK1, CDK2, PKA, PKC, EGFR, GSK-3β, MEK1, JNK1, KIT, and c-MET using the SYBYL-X package program Surflex-Dock.38 The docking results showed high binding scores for OC toward the c-MET’s ATP binding site in multiple crystal structures, including the protein database (PDB) numbers 3I5N, 1R0P, and 2RFS (Fig. 57.2).38 Oleocanthal showed hydrogen bond (HB) interactions between its phenolic hydroxyl group and both of the cMET hinge region Pro1158 and Met1160 in PDB 3I5N (Fig. 57.2).38 Additional HB interactions noted between the OC’s C-1 aldehydic group with the activation loop Tyr1230 and Arg1086 (Fig. 57.2). The MOLCAD visualization of OC docked pose showed its perfectly complete

fitting at the c-MET’s ATP pocket (Fig. 57.2).38 Oleocanthal filled the space between the hinge region and activation loop Pro1158/Asn1167-Tyr1230, while its ester moiety was not playing direct binding role but caused a favorable alignment of other binding pharmacophores to the target critical amino acids. Similar or better binding modes observed for OC in other c-MET wild-type crystal structure 1R0P, and the mutant-type structure 2RFS, which was relatively similar to the binding of the known experimental c-MET inhibitor SU11274.38 (2)-Oleocanthal exerted a dose-dependent inhibitory effect against the wild-type c-MET kinase phosphorylation with an IC50 value of 4.8 μM in the cell-free Z0 -LYTE kinase assay-Tyr2 Peptide.38 OC at 10 μM inhibited 70% of the c-MET kinase phosphorylation in this assay.38 OC inhibited the in vitro proliferation, migration, and invasion of the human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with IC50 range of 10
20 μM.38 OC also demonstrated antiangiogenic activity via downregulating the expression of the microvessel density (MVD) marker CD31 in endothelial colony forming cells, with an IC50 value of 4.4 μM.38

57.1.4 Hit-to-lead validation of oleocanthal as a c-MET inhibitor Hit-to-lead optimization of OC started by assessing and confirming OC in vitro ability to inhibit the growth of several additional human BC cell lines MDA-MB-231, MCF7, and BT-474, in the presence and absence of 40 ng/mL mitogenic HGF, while similar treatment doses showed no effect on the non-tumorigenic human mammary epithelial cells MCF10A cell growth.66 The inhibition of mammary cancer cell growth was associated with the suppression of the c-MET receptor activation in response to its natural ligand HGF in MDA-MB-231, MCF-7, and BT474 BC cell lines.66 (2)-Oleocanthal treatment dose-dependently inhibited HGF-induced cell migration, invasion, and G1/S cell cycle progression in the aforementioned BC cell lines. OC BC suppressive effects proved associated with the blockade of EMT and suppression of the cellular motility.66 OC treatment restored the expression of the epithelial markers Ecadherin and Zo-1 in MDA-MB-231 cells and suppressed

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

the expression of the mesenchymal marker vimentin.66 OC stabilized the expression of E-cadherin and Zo-1 in MCF-7 and BT-474 cells.66 OC treatment also significantly reduced the scattering, motility, invasion, and consequently stabilized several BC cell lines cell-cell adhesion.66 Transfection of c-MET-targeted siRNA decreased c-MET expression by about 90% and increased the caspase-8 and RIP cleavage, without affecting caspase-9 and cytochrome c levels.66 cMET depletion resulted a pattern of apoptosis identical to 25 μM OC treatment, which sensitized MDA-MB-231 cells by inducing caspase-8-dependent intrinsic apoptosis by downregulating c-MET expression.66 OC 25 μM treatment enhanced the caspase-8 and caspase-3 activation and apoptosis in MDA-MB-231 cells.66 The caspases inhibitor ZVAD-FMK treatment completely abolished the OC-induced apoptosis in MDA-MB-231 cells, confirming OC-induced caspases and subsequent apoptotic mechanism.66 Three weeks of (2)-oleocanthal 5 mg/kg, intraperitoneal treatment, three times a week suppressed 60% of the TNBC MDAMB-231/GFP cells growth in an orthotopic xenograft model of BC in athymic nude mice.66 OC treatment decreased the cancer cell proliferation as indicated by the reduction of Ki67 and CD31 staining in treated nude mouse tumors and significantly reduced c-MET phosphorylation in comparison with the vehicle control (VC) mouse tumors. This study was the first documentation of the in vivo OC activity via c-MET suppression, which promoted OC as a viable anticancer lead.66

57.1.5 Structure
activity relationship study and optimization of oleocanthal bioisostere c-MET inhibitors Studies directed to understand OC structure
activity relationship as a c-MET inhibitor through designing synthetic bioisosteres.67
69 Optimizations indicated that neither the carbamate nor amide functionalities improved the c-MET

activity versus the linear OC ester.67 This highlights the uniqueness of the OC ester functionality as optimal pharmacophore for c-MET binding affinity. Efforts toward finding the best esterifying acid replacing the elenolic acid in OC identified sinapic acid as a potential bioisostere to maintain c-MET inhibitory activity.67 Tyrosol sinapate (TS, Fig. 57.3) was, therefore, the first OC synthetic ester bioisotere generated by using the Mitsunobu chemoselective esterification of tyrosol with sinapic acid.67 TS inhibited the c-MET phosphorylation in the Z0 -LYTE Kinase Assay-Tyr6 Peptide kit cell-free assay, with an IC50 value of 13.7 μM. The IC50 of OC in this assay was 5.5 μM.67 The Omnia kinase Assay-Tyr12 peptide kit was used to assess the kinetic modality of TS and OC as c-MET kinase ATP competitive inhibitors.67 In this assay the Michaelis
Menten parameters Km (Michaelis
Menten constant, the substrate concentration that produces half-maximal velocity) and Vmax (maximum velocity seen at enzyme saturating substrate concentration) of the enzymatic reaction in presence of various concentrations of TS and OC were studied versus the positive drug control SU11274, the standard ATP-competitive cMET inhibitor.67 Both TS and OC did not affect the Vmax while the Km was increased.67 This pattern proved their cMET ATP-competitive inhibitory effects, which was similar to SU11274.67 Extensive optimizations of the tyrosol alcohol part in TS included the atom linker number between the two aromatic groups, unsaturation and double bond geometry, various substitutions at the linker and tyrosol side aromatic ring. These efforts identified homovanillyl sinapate (HVS, Fig. 57.3) as a promising c-MET inhibitory ester inspired by the chemistry of OC.68,69 Impressively, the 6atoms linker connecting both aromatic rings, similar to that of OC, was optimal for the c-MET inhibitory potency (Fig. 57.4).68 HVS showed improved potency against wild-type c-MET and its oncogenic variant in cell-free Z0 LYTE assays.68,69 HVS showed IC50 of 1.0 6 0.2 μM FIGURE 57.4 Optimization of tyrosol sinapate to homovanillyl sinapate.68,69

S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor Chapter | 57

versus the IC50 of 5.2 6 0.8 μM for OC against the wildtype c-MET.68,69 HVS also showed an IC50 of 0.9 6 0.4 μM versus an IC50 3.9 6 1.2 μM for OC against the mutant-type c-MET M1250T.68,69 Both HVS and OC were not active against the mutant c-MET Y1230C and Y1235D.69 HVS significantly suppressed the c-METmediated growth of several BC cells, without adversely affecting the non-tumorigenic mammary epithelial cells growth. HVS dose-dependently inhibited HGF-induced, but not EGF-induced cell scattering, in addition to HGFmediated migration, invasion, and 3-dimensional proliferation of multiple tumor cell spheroids including the BC MDA-MB-231, MDA-MB-468 and the advanced prostate cancer DU145 cells (Fig. 57.4).68,69 HVS showed good selectivity for c-MET and Abelson murine leukemia viral oncogene homolog 1 (ABL1) when profiled against a panel of c-MET structurally close kinases. Intra-peritoneal HVS 10 mg/kg, 3 3 /week, for 3 weeks, suppressed the MDA-MB-231 tumor growth in nude mouse xenograft model by 92% on the final study day, compared to the vehicle-treated control group, without adversely affecting the treated mice’s normal body weight or their gross phenotype.68,69 HVS also showed excellent pharmacodynamics by significantly inhibiting the phosphorylation of c-MET kinase in collected treated MDA-MB-231 xenograft tumors compared to the vehicletreated control group, as shown by Western blot analysis of the isolated tumor tissues, without any change in total c-MET levels. HVS also significantly reduced the levels of the expression of Ki-67 and the tumor microvessel density (MVD), calculated by new vessel formation using CD-31 staining.68,69

57.1.6 Combination studies of oleocanthal with targeted therapies and estrogen modulators The crosstalk between c-MET and the HER family members, beside the c-MET involvement in resistance to antiHER2 targeted therapies, supported hypothesizing OC to synergize with the dual EGFR-HER2 inhibitor lapatinib (LP), which commonly used in clinical practice for HER2-EGFR-dependent breast and lung cancers.23
26,70 The study started with investigating the activity of OC and LP monotherapies against the viability of the HER2positive BC cell lines BT-474 and SK-BR-3 cells.70 Monotherapy of each of OC and LP treatments showed dose-dependent inhibition of the proliferation of BT-474 and SK-BR-3 cells, with IC50 values of 25.1 μM, 27.3 μM, 123.0 nM, and 117.3 nM, respectively.70 OC showed minimal effects on the viability of the nontumorigenic mammary epithelial MCF12A cells at IC50 of 82.6 μM and peripheral neuronal Schwann CRL-2765 cells at IC50 of 68.9 μM over 48 h treatment.70 To identify

687

combined synergism, BT-474 and SK-BR-3 cells were treated with fixed OC each of 5, 10, and 15 μM treatment doses and combined with increasing LP 30
90 nM treatment doses for 48 h.70 LP combined with OC showed IC50 values in the range of 31.5
33.2 nM for BT-474 cells and 37.7
102.8 nM for SK-BR-3 cells, respectively. Isobologram analysis of OC-LP combination treatment in both cell lines indicated synergistic antiproliferative pattern. OC-LP combination index (CI) further indicated synergism with values ,1.0 in both BT-474 and SK-BR-3 cells.70 In BT-474 cells, the CI value was 0.74 for 30 nM LP combined with 12 μM OC treatment, while in SK-BR3 cells, the CI was 0.87 for 60 nM LP when combined with 15 μM OC treatment.70 The dose reduction index (DRI) values for combined OC-LP treatments showed multiple-fold reductions for both compounds.70 OC-LP combined treatment reduced c-MET, HER2, and EGFR activation phosphorylation without significantly changing their total levels. OC-LP combined treatment also reduced multiple downstream signaling proteins activation, including PDK, Akt, and mTOR. Concurrently, several mitogenic signaling pathways were also inhibited by combined OC and LP treatment, including the RASMAPK and JAK-STATs pathways in both BT-474 and SK-BR-3 cells.70 Combined OC-LP treatment resulted in downregulated cyclins D1 and D3 and total levels of cyclin-dependent kinase-6 (CDK-6) in both BT474 and SK-BR-3 cells, in addition to increased total levels of the cell cycle arrest proteins p21 and p27 in treated cancer cells, compared to vehicle-treated controls.70 Microarray analysis of BT-474 cells treated with LP-OC combination showed suppressed phosphorylation of multiple molecular targets, including HER-2, FAK, JAK1, and MEK2.70 The in vivo antitumor effect of combined OC-LP synergy was assessed in a nude mouse xenograft model against the luminal B BC BT-474 cells.70 OC was used at 5 and 10 mg/kg doses, ip, 3 3 /week, while LP was used orally at 12.5 and 50 mg/kg doses, 5 3 /week.70 The mean tumor weight in vehicle-treated control group was 1,515.3 6 273.1 mg at the end of treatment duration.70 The mean tumor weight for OC-treated groups was 178.9 6 37.0 and 103.9 6 40.9 mg for the 5 mg/kg and 10 mg/kg treatments, respectively.70 The OC 5.0 and 10.0 mg/kg treatments resulted in 92.42% and 97.86% tumor growth reduction, respectively, versus VC.70 The mean tumor weight for LP-treated groups were 213.4 6 19.9 and 153.2 6 2.4 mg for the 12.5 and 50.0 mg/kg treatments, respectively. LP treatment at 12.5 and 50.0 mg/kg resulted in 89.2% and 95.3% inhibition of tumor growth, respectively, versus VC.70 Combined OC 10 mg/kg, ip, 3 3 /week with LP 12.5 mg/kg, oral, 5 3 /week resulted in the greatest tumor growth inhibition, compared to the VCs with a mean

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

tumor weight of 46.6 6 27.1 mg, representing over 99% tumor growth reduction, without adversely affecting body weight in treated mice.70 Western blot analysis of tumors collected at the study end showed significant downregulation of total and phosphorylated levels of each of EGFR and c-MET most notable among animals treated with OC-LP combination.70 This study highlighted the OC translational potential as a component in extra-virgin olive oil (EVOO) rich in phenolics or as a possible future pure nutraceutical product, to be applied as a targeted monotherapy treatment or combined with other HERtargeting RTKI therapies for the control of HER-positive tumors. (2)-Oleocanthal also showed antiestrogenic activity and significant synergy with the selective estrogen receptor (ER) modulator tamoxifen, but this topic is covered in a separate chapter topic in this book.71 A very important progress made in the oleocanthal research was the discovery and patenting of a novel simplified method for OC and other olive phenolics isolation based on liquid
liquid extraction and the first validation of significant OC in vivo oral activity against the TNBC in a nude mouse orthotopic xenograft model at 10 mg/kg daily dose for 30 days.72,73

57.1.7 Oleocanthal as a novel first-in-class breast cancer recurrence inhibitor Research efforts toward the discovery of BC recurrence and metastasis inhibitors are modest because clinical trials on these treatment endpoints may need large patient numbers, long treatment period, and, therefore, ultimately these studies are not financially feasible.74 Thus seeking a natural product such as OC as a recurrence inhibitor based on its validated c-MET RTK as a main molecular target, which is strongly involved in dormant tumor activation is well justified. As a nutraceutical, OC translational development for use by BC patients and survivors should be significantly shorter and cost-effective compared to medicinal agents classified as drugs. Thus, OC ability to inhibit the TNBC and HER21/ER1 BC locoregional recurrences after primary tumor surgical excision was recently validated.74
76 After subcutaneously xenografting the luminal B BT-474 BC cells, all mice (n 5 10) developed primary breast tumors. These tumors were surgically excised once the average tumor volume reached 400 mm3.74 Mice were then randomly assigned into two groups, n 5 5 each. One group was treated with VC and the other group with 10 mg/kg oral OC daily for 21 days.74 At the study end, 4 out of 5 mice developed locoregional tumor recurrence (80%) in the VC group, while only 2 out of 5 mice developed recurrent tumors (40%) in the OC-treated group. The mean tumor weight of

vehicle-treated and OC-treated groups was 1.5 6 0.9 and 0.2 6 0.1 g, respectively. OC induced 95% recurrent tumor growth inhibition.74 A similar experiment was conducted using the TNBC MDA-MB-231 cells. Oral daily OC (10 mg/kg) treatments continued for 21 days, and recurrent tumor growth was compared with the VC group.74 OC did not prevent TNBC locoregional recurrence since 5 out of 5 mice developed recurred tumors. Yet, OC significantly suppressed the recurred TNBC growth since the mean weight for tumors at the experiment end was 2.03 6 0.8 and 0.92 6 0.4 g in VC and OCtreated groups, respectively. Though OC did not inhibit tumor recurrence in this model of TNBC, OC significantly suppressed tumor growth, by 58%, as compared to VC-treated animals.74 When this experiment was repeated after formulating OC as a 1:7 solid dispersion formulation in (1)-xylitol, a formulation coded OC-X, the OC-Xtreated group showed 60% TNBC recurrence prevention since 2 out of 5 OC-X-treated mice recurred tumors after surgery versus 5 out of 5 mice developed TNBC recurrence in VC group.75 This clearly showed the potential of OC-delivery method to improve its clinical outcomes. In both TNBC and luminal B BC recurrence xenograft models, OC treatment promoted an epithelial phenotype, as indicated by markers of EMT, which correlates with BC recurrence.74 For instance, OC treatment stabilized Ecadherin and concomitantly reduced vimentin expression in the BT-474 and MDA-MB-231 primary tumors that did recur after surgical excision.74 It is well documented that recurrent tumors are usually more aggressive, resistant, and show low expression of E-cadherin and high expression of vimentin.74,75 c-MET is a key regulator of EMT and its activation downregulates the expression of the epithelial markers E-cadherin and cytokeratins 8/18 and upregulates the mesenchymal protein vimentin.66 Cancer cells undergoing EMT acquire an aggressive invasive profile.66 Signaling downstream of c-MET enhances breakdown of cell
cell junctions and promotes cell invasion.63 Activated c-MET and HER2 (BT-474 only) levels were significantly lowered.74 The serum levels of the cancer antigen 15-3 (CA 15-3) at the study end were significantly higher in control-treated mice versus the OCtreated mice in both TNBC and luminal B BC recurrence models.74,76 CA 15-3 is a predictive marker for monitoring the postoperative recurrence and metastasis risk in cancer patients.74,76 OC also significantly suppressed HER21/ER1 BC recurrence after completion of LP neoadjuvant regimen, followed by primary tumor surgical excision.74 After inoculating BT-474 cancer cells, all mice (n 5 10) developed primary breast tumors. LP neoadjuvant treatment (50 mg/kg) started when tumors averaged 30 mm3 in volume and continued for 12 days. Primary breast tumors were surgically excised when tumor volume averaged 400 mm3, or upon completion of

S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor Chapter | 57

the LP treatment regimen, whichever was earlier.74 Mice were then randomly assigned into two groups (n 5 5). One group treated with VC (LP-VC) and the other group started a daily oral OC treatment at 10 mg/kg (LP-OC).74 At study end, 4 out of 5 mice (80%) in the VC group developed recurrent tumors, while only 3 out of 5 mice (60%) developed recurrent tumors in the OCtreated group.74 Tumor weight means for vehicle-treated and OC-treated groups were 1.3 6 0.7 and 0.2 6 0.2 g, respectively.74 The OC-treated group (LP-OC) showed 89% tumor growth inhibition, versus vehicle-treated control group (LP-VC).74 Taste masked formulations of OC [OC solid dispersion with (1)-xylitol and OC effervescent formulation] further supported the c-MET, HER2, and locoregional recurrence suppressive activities against TNBC and HER21/ER1 BC in nude mouse models.75,76 Although the OC anti-BC recurrence studies were focused on locoregional and not the distant recurrence, literature strongly correlate locoregional with distant recurrence, especially in patients with lymph-node-positive BC.77,78 EVOO, which contains OC at variable ingredient levels, has been used as food and remedy throughout human history and evidenced epidemiologically as a key component of the Mediterranean diets.74 Thus, the direct advantages of developing OC as a nutraceutical for BC recurrence prevention by cancer survivors include its expected longterm safety profile based on historical human consumption of olive oil, cost-effectiveness based on the sustained plant supply source, relative ease and economic viability of commercial production, and recent preliminary safety study in a mouse model in which OC exhibited good oral safety margin with a toxic dose approaching 25-fold its therapeutic dose.79

57.2 Conclusion (2)-Oleocanthal is a bioactive monophenolic secoiridoid occurring in EVOO. The positive health benefits of the olive oil
rich Mediterranean diet may be attributed—in part—to (2)-oleocanthal, which showed a wide margin of biological activities at the cardiovascular, neuroprotection, and oncology directions. The anticancer activities of oleocanthal may largely correlate with its c-MET targeting effects at the molecular level. This important molecular target and optimal safety profile qualified oleocanthal to become a viable nutraceutical for the prevention and treatment of multiple malignancies, specifically BC. (2)-Oleocanthal also identified as a first-in-class BC recurrence inhibitor and thus proposed for long-term use by BC survivors’ use to prevent disease relapse. (2)-Oleocanthal is another unique and impactful natural product lead with documented and progressive anticancer applications.

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1011. Shalaby SY, Sumpio BJ, Sumpio BE. Olive oil: molecular mechanisms and cardiovascular protective role. In: De Leonardis A, ed. Virgin Olive Oil. New York: Nova Science Publisher; 2014: 211
232. Toledo E, Salas-Salvado J, Donat-Vargas C, et al. Mediterranean diet and invasive breast cancer risk among women at high cardiovascular risk in the PREDIMED trial: a randomized clinical trial. JAMA Intern Med. 2015;175:1752
1760. Psaltopoulou T, Kosti RI, Haidopoulos D, Dimopoulos M, Panagiotakos DB. Olive oil intake is inversely related to cancer prevalence: a systematic review and a meta-analysis of 13800 patients and 23340 controls in 19 observational studies. Lipids Health Dis. 2011;10:127. Available from: https://doi.org/10.1186/ 1476-511X-10-127. Schwingshackl L, Schwedhelm C, Galbete C, Hoffmann G. Adherence to Mediterranean diet and risk of cancer: an updated systematic review and meta-analysis. Nutrients. 2017;9:E1063. Available from: https://doi.org/10.3390/nu9101063. Escricha E, Morala R, Solanas M. Olive oil, an essential component of the Mediterranean diet, and breast cancer. Public Health Nut. 2011;14:2323
2332. Qosa H, Mohamed LA, Batarseh YS, et al. Extra-virgin olive oil attenuates amyloid-β and tau pathologies in the brains of TgSwDI mice. J Nutr Biochem. 2015;26:1479
1490. Monti MC, Margarucci L, Tosco A, Riccio R, Casapullo A. New insights on the interaction mechanism between tau protein and oleocanthal, an extra-virgin olive-oil bioactive component. Food Funct. 2011;2:423
428.

S-(2)-Oleocanthal as a c-Met receptor tyrosine kinase inhibitor Chapter | 57

48. Batarseh YS, Mohamed LA, Al Rihani SB, et al. Oleocanthal ameliorates amyloid-β oligomers toxicity on astrocytes and neuronal cells: in-vitro studies. Neuroscience. 2017;352:204
215. 49. Qosa H, Batarseh YS, Mohyeldin MM, El Sayed KA, Keller JN, Kaddoumi A. Oleocanthal enhances amyloid-β clearance from the brains of TgSwDI mice and in vitro across a human blood-brain barrier model. ACS Chem Neurosci. 2015;6:1849
1859. 50. Abuznait AH, Qosa H, Busnena BA, El Sayed KA, Kaddoumi A. Olive-oil-derived oleocanthal enhances β-amyloid clearance as a potential neuroprotective mechanism against Alzheimer’s disease: in vitro and in vivo studies. ACS Chem Neurosci. 2013;4:973
982. 51. Oliveras-Ferraros C, Ferna´ndez-Arroyo S, Vazquez-Martin A, et al. Crude phenolic extracts from extra virgin olive oil circumvent de novo breast cancer resistance to HER1/HER2-targeting drugs by inducing GADD45-sensed cellular stress, G2/M arrest and hyperacetylation of histone H3. Int J Oncol. 2011;38:1533
1547. 52. Michelle Q, Fabino ND. Mediterranean diet for cancer prevention: a review of the evidence and a guide to adherence. Nat Med J. 2017;9. 53. Escricha E, Morala R, Solanas M. Olive oil, an essential component of the Mediterranean diet, and breast cancer. Public Health Nut. 2011;14:2323
2332. 54. Beauchamp GK, Keast RSJ, Morel D, et al. Phytochemistry: ibuprofenlike activity in extra-virgin olive oil. Nature. 2005;437:45
46. 55. Francisco V, Ruiz-Fernandez C, Lahera, et al. Natural molecules for healthy lifestyles: oleocanthal from extra virgin olive oil. J Agric Food Chem. 2019;67:3845
3853. 56. Pang KL, Chin KY. The biological activities of oleocanthal from a molecular perspective. Nutrients. 2018;6:5. Available from: https:// doi.org/10.3390/nu10050570. 57. Khanal P, Oh WK, Yun HJ, et al. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis. Carcinogenesis. 2011;32:545
553. 58. Scotece M, Go´mez R, Conde J, et al. Oleocanthal inhibits proliferation and MIP-1α expression in human multiple myeloma cells. Curr Med Chem. 2013;20:2467
2475. 59. Scotece M, Conde J, Abella V, et al. New drugs from ancient natural foods. Oleocanthal, the natural occurring spicy compound of olive oil: a brief history. Drug Disc Today. 2015;20:406
410. 60. Margarucci L, Monti MC, Cassiano C, Mozzicafreddo M, Angeletti M. Chemical proteomics-driven discovery of oleocanthal as an Hsp90 inhibitor. Chem Commun (Camb). 2013;49:5844
5846. 61. LeGendre O’, Breslin PAS, Foster DA. (2)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization. Mol Cell Oncol. 2015;2:e1006077. 62. Khanfar MA, Bardaweel SK, Akl MR, El Sayed KA. Olive oilderived oleocanthal as potent inhibitor of mammalian target of rapamycin: biological evaluation and molecular modeling studies. Phytother Res. 2015;29:1776
1782. 63. Cassiano C, Casapullo A, Tosco A, Monti MC, Riccio R. In-cell interactome of oleocanthal, an extra virgin olive oil bioactive component. Nat Prod Commun. 2015;10:1013
1016. 64. Pei T, Meng Q, Han J, et al. (2)-Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma. Oncotarget. 2016;7:43475
43491. 65. Fogli S, Arena C, Carpi S, et al. Cytotoxic activity of oleocanthal isolated from virgin olive oil on human melanoma cells. Nutr Cancer. 2016;68:873
877.

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66. Akl MR, Busnena BA, Mohyeldin MM, et al. Olive phenolics as cMet inhibitors: (2)-oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models. 2014. PLoS One. 2014;9:e97622. 67. Busnena BA, Foudah AI, Melancon T, El Sayed KA. Olive secoiridoids and semisynthetic bioisostere analogues for the control of metastatic breast cancer. Bioorg Med Chem. 2013;21:2117
2127. 68. Mohyeldin MM, Busnena BA, Akl MR, Dragoi AM, Cardelli JA, El Sayed KA. Novel c-Met inhibitory olive secoiridoid semisynthetic analogs for the control of invasive breast cancer. Eur J Med Chem. 2016;118:299
315. 69. Mohyeldin MM, Akl MR, Dragoi AM, Cardelli JA, El Sayed KA. The oleocanthal-based homovanillyl sinapate as a novel c-Met inhibitor. Oncotarget. 2016;7:32247
32273. 70. Siddique AB, Ebrahim HY, Akl MR, et al. (2)-Oleocanthal combined with lapatinib treatment synergized against HER-2 positive breast cancer in vitro and in vivo. Nutrients. 2019;11:412. 71. Ayoub NM, Siddique AB, Ebrahim HY, Mohyeldin MM, El Sayed KA. The olive oil phenolic (2)-oleocanthal modulates estrogen receptor expression in luminal breast cancer in vitro and in vivo and synergizes with tamoxifen treatment. Eur J Pharmacol. 2017;810:100
111. 72. Siddique A, Ebrahim HE, Btarsah Y, et al. Novel liquid-liquid extraction and self-emulsion methods for simplified isolation of extra-virgin olive oil phenolics with emphasis on (2)-oleocanthal and its oral anti-breast cancer activity. PLoS One. 2019;14: e0214798. Available from: https://doi.org/10.1371/journal. pone.0214798. 73. El Sayed KA, Siddique A, Ebrahim H. Oleocanthal Isolation and Cancer Treatment. PCT/US2017/043308, WO2018/017967. 2018. ,https://patentscope.wipo.int/search/en/detail.jsf?docId 5 WO2018 017967&_cid 5 P22-KEKKGC-85983-1.. 74. Siddique AB, Ayoub NM, Tajmim A, Meyer SA, Hill RA, El Sayed KA. (2)-Oleocanthal prevents breast cancer locoregional recurrence after primary tumor surgical excision and neoadjuvant targeted therapy in orthotopic nude mouse models. Cancers (Basel). 2019;11:637. Available from: https://doi.org/10.3390/ cancers11050637. 75. Qusa MH, Siddique AB, Nazzal S, El Sayed K. Novel olive oil phenolic (2)-oleocanthal (1)-xylitol-based solid dispersion formulations with potent oral anti-breast cancer activities. Int. J. Pharm. 2019;569:118596. Available from: https://doi.org/10.1016/j. ijpharm.2019.118596. 76. Tajmim A, Siddique A, El Sayed KA. Optimization of tastemasked (
)-oleocanthal effervescent formulation with potent breast cancer progression and recurrence suppressive activities. Pharmaceutics. 2019;11:515. Available from: https://doi.org/ 10.3390/pharmaceutics11100515. 77. Wright JL, Takita C, Reis IM, et al. Predictors of locoregional outcome in patients receiving neoadjuvant therapy and postmastectomy radiation. Cancer. 2013;119:16
25. 78. Buonaguro FM, Pauza CD, Tornesello ML, Hainaut P, Franco R, Tommasino M. Cancer diagnostic and predictive biomarkers 2016. Biomed Res Int. 2017;2017:7362721. 79. Siddique AB, King JA, Meyer SA, Abdelwahed K, Busnena B, El Sayed K. Safety evaluations of single dose of the olive secoiridoid S-(2)-oleocanthal in Swiss albino mice. Nutrients. 2020;12:314. Available from: https://doi.org/10.3390/nu12020314.

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Chapter 58

Phenolic compounds in olive oil mill wastewater Jose´ S. Torrecilla1 and John C. Cancilla2 1

Department of Chemical and Materials Engineering, Complutense University of Madrid, Spain, 2Scintillon Institute, San Diego, CA, United States

Abbreviations BOD5 COD EVOO OMW

biochemical oxygen demand in 5 days chemical oxygen demand extra-virgin olive oil olive oil mill wastewater

58.1 Introduction Extra-virgin olive oil (EVOO) is a well-known and key component of the Mediterranean diet. This olive juice presents a series of unique characteristics,1 including the presence of phenolic compounds.2 These compounds are known to protect against oxidative stress and contribute to the fact that EVOO is a healthy food that has been shown to possess beneficial effects against cardiovascular pathologies, different types of cancer, and other diseases.2
4 According to the International Olive Oil Council, during the 2017
18 campaign, more than 3.4 million tons of EVOO were produced worldwide while also revealing an expected decrease for the 2019
20 campaign of 0.2

million tons. As for table olives, worldwide, during the 2017
18 campaign, more than 2.6 million tons were collected, with a small increase expected for the 2019
20 campaign to 2.9 million tons. In both cases, production is almost entirely centered within the European Union. These high yields not only entail a health and economical benefit but also pose a significant environmental problem.5,6 This is due to the generation of high quantities of waste with a considerable environmental impact, such as pomace and pruning waste. Furthermore, and specifically, more than 30 million m3 of olive oil mill wastewater (OMW) per year in the European community are generated.7 After the two-phase manufacturing process of EVOO, the resulting OMW has a dark brown color and high humidity from a physicochemical point of view. Its average composition shown in Table 58.1 should be highlighted. Due to the presence of dissolved salts in the OMW, its electrical conductivity is relatively high. Likewise, the waste is of an acidic nature mainly due to the presence of fatty acids. This nature prevents or limits biological activity to a great extent. It is worth noting that the high humidity

TABLE 58.1 Average physicochemical properties of olive oil mill wastewater as well as present compounds.7 Compound

Concentration (g/L)

Parameter

Quantity

Ammonium

1.8

BOD5 (g/L)

37.5

Calcium

0.7

COD (g/L)

197.0

Magnesium

2.6

Electrical conductivity (mS/cm)

9.2

Potassium

5.9

pH

4.8

Sodium

1.7

Total dissolved salt (g/L)

16.5

Water content (%)

79.0

BOD5, Biochemical oxygen demand in 5 days; COD, chemical oxygen demand.

Olives and Olive Oil in Health and Disease Prevention. DOI: https://doi.org/10.1016/B978-0-12-819528-4.00051-1 © 2021 Elsevier Inc. All rights reserved.

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

of the waste makes its subsequent treatment difficult. Publications indicate that OMW can present a relative humidity of up to 94%.8 From an environmental point of view, it is also worth noting that the exceptionally high chemical oxygen demand (COD) values are derived from high organic matter content.7 Different ways of producing EVOO generate distinct amounts and compositions of waste. The most efficient production process considering the energy and environmental points of view is the pressing method, followed by the two-phase method, and then the three-phase method.9 However, parameters such as biochemical oxygen demand in 5 days (BOD5), COD, and the percentage of total solids are lower in the case of production technologies based on centrifugation (two or three phases) than by pressure.10 Phenolic compounds are fundamentally in the olive pulp that is transferred to the EVOO during its production process, fundamentally in the beating phase. It must be considered that antioxidant compounds are mostly more soluble in aqueous media than in oily ones, so those that are not transferred to the EVOO will be found in the aqueous remains (waste).11,12 Therefore a large part of the composition of antioxidants (hydroxytyrosol, tyrosol, caffeic acid, etc.) is found in the residues of the production process.13,14 Depending on the solubility and the partition coefficients between oil and water, different compositions of the phenolic fractions within the waste itself will be obtained. In Table 58.2 the main phenolic compounds and general composition of these wastes are shown. TABLE 58.2 Main phenolic compound composition of olive oil mill wastewater.11 Compound

Quantity (mg/L)

Vanillic acid

52.64

Ferulic acid

8.75

p-Coumaric acid

5.41

Oleuropein-aglycone mono-aldehyde (3,4DHPEA-EA)

1.05

Ligstroside-aglycone di-aldehyde (p-HPEAEDA)

0.90

Hydroxytyrosol

57.29

Tyrosol

14.52

Other phenolic compounds

97.94

Total phenols

238.50

Some sources indicate higher amounts of phenolic compounds; even up to 24 g/L of OMW, revealing the great variability regarding different olive wastes.8 The phenolic compounds that are present in EVOO, and also in the aqueous residues that have been mentioned, lead to higher protection rates against oxidation. This occurs because the first oxidation that takes place is in fact of these phenolic compounds, instead of the lipid substrate, thus protecting the stability of the product. Phenolic compounds provide this stability through mechanisms among which we can highlight the search for radicals and the transfer of hydrogen atoms as well as metals.15,16

58.2 Phenolic compounds in olive oil mill wastewater Phenolic compounds possess one or more aromatic rings, one or more hydroxyl groups, and various functional chains. Within the waste produced in the olive oil sector, more than 50 phenolic compounds have been identified and isolated. The different compounds are classified according to their molecular weight or chemical structure, among other properties.1,16 For example, according to their chemical structure, three main groups of phenolic compounds can be distinguished (Fig. 58.1): (1) cinnamic acids, (2) benzoic acid derivatives, and (3) tyrosol-related compounds.17 However, as mentioned earlier, it is necessary to bear in mind that the characteristics of OMWs are variable. They depend on a large number of factors, such as the method of oil extraction, the ripeness and variety of the olives harvested, their geographic origin, and climatic conditions.8 The major phenolic component of both the pulp of many varieties of olives and the leaves of the olive tree is oleuropein. This biophenol is precisely what gives the bitter taste to some EVOOs. Oleuropein is the compound designated according to IUPAC as [(4S,5E,6S)-4-[2-[2(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2tetrahydropyranil]oxy]-4H-pyran-3-carboxyl. Likewise, during the process of extracting EVOO, hydroxytyrosol is produced by the action of the enzyme esterase. This phenolic compound is also found in the OMW. Hydroxytyrosol is one of the most powerful antioxidants. Low doses of this polyphenol have been shown to reduce the consequences of oxidative stress in rats.18 Hydroxytyrosol has become commercially available for research purposes and costs between US $1000 and $2000/g.19 Also, in view of its applicability in the health FIGURE 58.1 Fundamental molecule from which the main groups of phenolic compounds in OMW derive: (A) cinnamic acid, (B) benzoic acid, and (C) tyrosol. OMW, Olive oil mill wastewater.

Phenolic compounds in olive oil mill wastewater Chapter | 58

and food fields, several procedures have been proposed to recover hydroxytyrosol from OMW. Among other methods, this compound was recovered from OMW, with a yield over 85%, using a three-stage continuous liquid
liquid extraction unit. A total of 1.225 g of hydroxytyrosol was extracted per liter of OMW, and then 1 g was purified by chromatographic methods.19

58.2.1 Health benefits of extra-virgin olive oil phenolic compounds As presented in this chapter, both EVOO and the residues that come from the production of this juice contain a high quantity and variety of phenolic compounds. The richness and structures of these phenolic compounds will depend on the origin, variety, cultivation techniques, and production of the EVOO. It should not be forgotten that the distribution chain will also affect the concentrations of these compounds within the EVOO. These phenolic compounds, in addition to other characteristics, are beneficial to health as they provide a positive effect on various human pathologies, among which three major groups should be highlighted: (1) antimicrobial, (2) antioxidant, and (3) antiinflammatory, Fig. 58.2.20 1. Regarding antimicrobial properties, phenolic compounds inhibit the growth of microorganisms and act as therapeutic agents in some cases of infectious diseases. In particular, hydroxytyrosol and tyrosol, among others, have shown a beneficial effect in the case of treatment of diseases of the respiratory and digestive systems.20,21 2. With regard to antioxidant characteristics, it is important to highlight the action of phenolic compounds against free radicals that can cause oxidative damage to molecules such as lipids and even DNA. This aspect notably favors diseases of the circulatory system, cancer, degenerative diseases, etc. It has been shown that the presence of phenolic compounds has a beneficial effect against the oxidation of lipid compounds and also against oxidative damage to DNA and, therefore, has a subsequent positive effect on the appearance of the abovementioned diseases.20,21

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3. To trace the antiinflammatory characteristics of phenolic compounds, it is necessary to view the negative influence that chronic inflammation has on various diseases such as cancer, cardiovascular diseases, arthritis, or several neurodegenerative diseases. In tests carried out in vivo, it has been highlighted that the intake of EVOO, due to its concentrations of phenolic compounds, has a mitigating effect on the body’s inflammatory responses, thus reducing the risk of chronic inflammatory disease.20,22 In view of all these characteristics, it is necessary to properly manage these compounds that are present both in EVOO and in the waste generated.

58.2.2 Phenolic compounds in different types of oil EVOO is one of the few oils that comes from a fruit, and its extraction is only done by physical means without using any chemical process or solvent. Likewise, the temperature at which this oil is produced is controlled so that it does not degrade the compounds that make the oil a healthy product and organoleptically desirable for the consumer.23 Other advantages of this oil are that as it comes from a fruit, no refining stage is necessary, enabling it to be marketed once it has been milled, preserving its aroma and its composition in vitamins, polyphenols, etc.24 These characteristics condition that a large number of varieties of EVOO with different characteristics and properties exist, being totally different from oils extracted from seeds. Seed oils are different as it is necessary to use chemical solvents for extraction, high temperatures during this process, and the need to refine the oil produced by different routes makes seed oil lose valuable compounds that would make them healthier for consumption, such as phenolic compounds or vitamins, among other compounds.25,26 This refining operation makes the oil absolutely flat and has very few organoleptic properties. It is also necessary to consider the large amount of resources that are necessary to treat the waste generated during the production of these seed oils.27 All these differences mean that, in general, EVOO is more beneficial than any other seed oil. Furthermore, even refined olive oils are generally more valuable than those from seeds.

58.3 Olive oil mill wastewater management

FIGURE 58.2 Positive effects of phenolic compounds in EVOO.

In this section, different methods and procedures will be presented to obtain an economic return and environmental protection from the waste generated in the production of EVOO in the mill itself. The wastewater generated in

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

the production process possesses two well-differentiated traits. On one hand, it is water with a high environmental impact and, on the other, within its composition; relevant compounds can be found for nutritional and industrial purposes. The high toxicity is well known regarding certain phenolic compounds such as tyrosol, hydroxytyrosol, catechol, protocatechuic acid, or caffeic acid in different seeds (Cucumis sativus, Lepidium sativum, Sorghum bicolor, etc.) and different organisms (Daphnia magna, Thamnocephalus platyurus, Brachionus calyciflorus, Pseudokirchneriella subcapitata, etc.).28 This is why the incorporation of this type of waste into urban wastewater for unified treatment can generate serious effects on microorganisms.29 The two most toxic phenolic compounds present in OMWs are catechol and hydroxytyrosol. Therefore the elimination of these phenolic compounds from the wastewater of the oil mill would notably reduce their environmental impact and would also enrich the value of the sector, since these polyphenolic compounds when isolated are very attractive for the pharmaceutical market.8 The treatment methods that will be discussed here will focus mainly on physical, biological, chemical, natural, and combined processes to extract phenolic compounds to reduce the organic content of the waste.

58.3.1 Olive oil mill wastewater treatment As with any other waste, it is important to stress that reducing the global amount generated is the best way to achieve a reduced environmental impact of the production process. One step toward this is accomplished by replacing the three-phase centrifugal method with the twophase one to produce olive oil. However, over the last few years, a large number of processes have been proposed for the treatment of OMW. Composting processes have been developed to enrich farmland using the residues. For more complicated processes where the applications of physicochemical or biological treatments are required, in some cases, it is necessary to implement a pretreatment. Specifically, these processes are mainly classified as solids separation or evaporation by thermal methods.8 The big problem of these energy exchanging pretreatments is their high cost which, in some cases, makes them unviable. It should be considered that although the final objective is the treatment of the waste, it would be advisable to select those processes that are more cost-effective or that generate a greater positive impact on the sector. Once the pretreatments have been carried out, taking into account the experience that exists in the treatment of urban wastewater, among other treatment methodologies, flocculation, flotation, sedimentation, dilution, evaporation ponds, and, in some cases, incineration should

be highlighted. There are also other perhaps more sophisticated methods such as reverse osmosis, oxidation, and various chemical and electrochemical treatments.8,30 Also, these OMWs, based on their composition, and after carrying out an appropriate conditioning, could be employed as animal feed. However, none of the processes mentioned have resulted in a global and optimal treatment method for the waste. Therefore depending on the situation, one methodology or another should be used to accomplish the main objective of reducing the environmental impact of the waste (see below). In this context a more complex alternative will also be looked into, which is the separation of those valuable compounds present in the waste itself in order to obtain a greater economic return that will have repercussions on an increase in the added value of both the olive and the commercial sector in general.

58.3.2 Phenolic compound isolation Although OMWs are a source of valuable molecules, phenolic compounds are worth highlighting as the most relevant. Their isolation and applicability within the olive sector and others are being studied.30 Among the main phenolic compounds, hydroxytyrosol is one of the most interesting and studied ones, as it is a well-known antioxidant. It is necessary to take into account that to treat the generated OMWs and extract phenolic compounds with the degree of purity required to be used for commercial applications, it is necessary to implement a series of processes. In general, these treatments must be preceded by processes where the OMW receives energy for the separation of compounds that can generate difficulties down the road. In general, these pretreatments are usually based on matter and energy exchange processes (e.g., thermal concentration or lyophilization), physical separation processes (e.g., microfiltration, ultrafiltration, and nanofiltration or liquid membranes), and more.8,30,31 It is worth noting that processes for separating phenolic compounds from OMW were carried out with results close to 100% by means of a distillation process with contact membranes made of polytetrafluoroethylene for 8 h.32 Separation alternatives combining nanofiltration and reverse osmosis operations have also been proposed. In particular, a polysulfone membrane (25 kDa) was used to partially remove the heavier fragments of hydroxycinnamic acid derivatives and flavonols at the same time.33 Cost-effective separation alternatives would be those based on adsorption and filtration, using sand as a filter medium followed by treatment with active carbon, leading to a reported 95% removal rate of phenolic compounds. This way, these authors stated that 65% COD of the OMW is also reduced.34 Considering regeneration and the influence of temperature on the process, zeolites are considered to be the materials that are most useful in

Phenolic compounds in olive oil mill wastewater Chapter | 58

reducing the organic load of the OMW (COD) as well as phenolic content.8 In general, technologies based on filtration (micro-, ultra-, and nano-variants) have been used in other sectors, and the experience gathered suggests that these techniques are more viable economically speaking when compared to other approaches while still being technologically sound.8 Concerning the phenolic extraction with solvents, it is necessary to understand the chemical structure of these compounds in order to find the solvents that are the most suitable to separate them from the rest of the OMW matrix. In general, phenolic compounds are more soluble in polar media such as alcohols or esters. For example, ethyl acetate is a suitable solvent for the extraction of phenolic compounds of average molecular weight, with a corresponding recovery rate of up to 90%.35 Other studies showed that supercritical CO2 extraction can be an efficient approach for the recovery of phenolic compounds from OMW.36 The commercial production of OMW-extracted hydroxytyrosol represents a strong secondary economic return for the olive oil sector. It is carried out through three general patented steps: (1) an initial treatment with acid; (2) an incubation process to increase the concentration of the phenolic compound to be extracted, converting the oleuropein into hydroxytyrosol, followed by a supercritical extraction; and (3) finally, a lyophilization of the resulting product.37 Furthermore, in this way, pure hydroxytyrosol (99.5%) is gathered from OMW using chromatographic columns filled with two resins.8 In the food sector, OMW is degreased and concentrated as a step prior to the extraction of phenols using ethanol in combination with an organic acid. Thereafter, the separation of phenols and food fibers is performed by precipitation of the condensed alcohol.33 The phenolic extract is already used as an additive in chocolates, pastries, and general bakery goods.

58.3.3 Phenolic compound removal Within the techniques used to reduce the environmental impact of OMW, we can highlight physicochemical (oxidation, coagulation, etc.), biological, and natural treatment routes, among others. All of them will lead to the reduction of the organic load of the waste which increases the biodegradability of the waste. The most common techniques are the so-called advanced oxidation processes of the OMW. Due to the acidity of OMW, the Fenton and photo-Fenton processes (advanced oxidation process in which highly reactive radicals are produced) have been considered as appropriate technologies for their treatment.8,38,39 It has been reported that phenolic compounds are more efficiently removed by the photo-Fenton treatment than by biological or enzymatic treatments.8,40 On the other hand, another

697

advanced oxidation process is based on the reaction of ozone with OMW. In particular, phenolic compounds present in OMW react strongly with ozone, leading to their oxidation in a more selective fashion than the previously mentioned advanced oxidation processes.41 Regarding coagulation, one of the most recommended techniques for the elimination of phenolic compounds is the electrocoagulation process. This technique is based on a circulating electric current that corrodes the anode and allows the removal of contaminants in the cathode by flotation.42,43 Abdelwahab and collaborators obtained a remarkable 97% reduction of phenolic compounds from OMW after a 2-h treatment.43 Consequently, electrocoagulation is considered a promising alternative technology to existing methods. It can also be applied as an OMW pretreatment phase.8 On the other hand, biological treatments are compelling alternatives that take advantage of the “abilities” of certain microorganisms, both aerobic and anaerobic. Microorganisms such as bacteria and fungi have been tested to treat OMW. Aerobic bacteria have been employed mainly as an approach for the removal of phytotoxic compounds. On the other hand, fungi have proven to be effective in reducing organic matter and especially lowering toxicity.44 On the other hand, several yeasts, in particular Candida tropicalis or Lactobacillus paracasei used as cosubstrates, via aerobic digestion, can degrade phenolic compounds by 45% and 23%, respectively.8 In contrast, anaerobic digestion is a process consisting of a series of microbial transformations of organic compounds into light hydrocarbons such as methane or volatile esters such as acetate, propionate, butyrate, isobutyrate, valerate, or isovalerate.8,45 Therefore anaerobic digestion cannot deal adequately with high organic loads present in mill residues. It therefore needs to be diluted several times before treatment. Furthermore, the presence of some inhibitors and toxic compounds within the OMW, that is, polyphenols and lipids, makes these wastes unsuitable for direct biological treatment. Therefore pretreatments aimed at lowering the concentration of phenolic compounds have been developed to treat OMW by anaerobic digestion.46 In the case of establishing pretreatments before applying biological methods, the pretreatment of OMW with sand and activated carbon filtration to partially eliminate the phenols present in the waste should be highlighted.8 Alternatively, by using two-phase anaerobic digestion reactors operated at mesophilic temperature, phenol removal efficiencies are 70%
78%.47 In many occasions, to favor the elimination of phenolic compounds, combined techniques at the cost of an increase of the prices are implemented. An example of this is the combination of centrifugation and filtration as well as activated carbon adsorption. All these lead to an elimination of phenols (94% reduction) and organic

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PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

matter (83% reduction).48 On the other hand, Khoufi and collaborators developed a pilot-scale process for the treatment of OMWs through the combination of electroFenton, anaerobic digestion, and ultrafiltration.45 This process resulted in a 95% decrease of phenolic compounds, particularly, monophenolic ones. It should also be noted that the technology of ultrafiltration as a posttreatment can adapt the OMW waste to the conditions of the riverbed where it could be discharged. On the other hand, Duarte and collaborators proposed three treatment steps: adsorption, fungal biodegradation, and dissemination of the biodegradation products generated. Over a period of 29 days the organic load was reduced by up to 67% and phenolic compounds were reduced by up to 89%.49

58.4 Conclusion The production of EVOO is accompanied by a significant environmental problem, as a large amount of hazardous waste is originated. One of the main issues of the OMW is the toxicity of phenolic fractions and their slow biodegradation. Therefore it is key to reduce the environmental impact of this waste, which can be done while taking advantage of it by extracting valuable molecules such as phenolic compounds that can result in an added value or resource for the olive sector. These elimination and treatment approaches can range from those capable of extracting the compounds with high added value present in the OMW (filtration, extraction, etc.) to others that reduce the organic load and eliminate the phenolic compounds by means of physicochemical, biological, or combined treatments. In general, researchers are working actively to propose treatment techniques that are both effective in treatment while being economically beneficial.

Mini-dictionary of terms Biochemical oxygen demand in 5 days: Chemical oxygen demand:

Conductivity Olive oil mill wastewater: Three-phase method:

Two-phase method:

Quantity of dissolved oxygen consumed in 5 days by biological processes breaking down organic matter. Commonly used to indirectly measure the amount of organic compounds or organic pollutants in water. This test measures the quality of the water. Measure of the ability of a material or fluid to conduct an electric current. Watery residue generated during the olive crushing process. Olive milling process in which three phases are generated (olive oil, solid residue, and watery residue). Olive milling process in which two phases are generated (olive oil and solid
liquid residue).

Aerobic digestion:

Anaerobic digestion:

Biodegradation:

Bacterial process where these microorganisms consume organic matter and convert it to CO2. This type of process is carried out in the presence of oxygen. Process in which microorganisms break down biodegradable material in the absence of oxygen. Process by which organic substances are broken down by the enzymes produced by living organisms.

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4110. Available from: https://doi.org/10.1021/ie960130 1 . Justino C, Marques AG, Duarte KR, et al. Degradation of phenols in olive oil mill wastewater by biological, enzymatic, and photoFenton oxidation. Environ Sci Pollut Res. 2010;17(3):650
656. Available from: https://doi.org/10.1007/s11356-009-0256-8. Chedeville O, Debacq M, Porte C. Removal of phenolic compounds present in olive mill wastewaters by ozonation. Desalination. 2009;249(2):865
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700

PART | 3 Specific Components of Olive Oil and Their Effects on Tissue and Body Systems

42. Hanafi F, Assobhei O, Mountadar M. Detoxification and discoloration of Moroccan olive mill wastewater by electrocoagulation. J Hazard Mater. 2010;174(1
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46. Stamatelatou K, Kopsahelis A, Blika PS, et al. Anaerobic digestion of olive mill wastewater in a periodic anaerobic baffled reactor (PABR) followed by further effluent purification via membrane separation technologies. J Chem Technol Biotechnol. 2009;84(6): 909
917. Available from: https://doi.org/10.1002/jctb.2170. 47. Fezzani B, Cheikh R Ben. Two-phase anaerobic co-digestion of olive mill wastes in semi-continuous digesters at mesophilic temperature. Bioresour Technol. 2010;101(6):1628
1634. Available from: https://doi.org/10.1016/j.biortech.2009.09.067. 48. Paraskeva P, Diamadopoulos E. Technologies for olive mill wastewater (OMW) treatment: a review. J Chem Technol Biotechnol. 2006;81:1475
1485. 49. Duarte KR, Freitas AC, Pereira R, et al. Treatment of olive oil mill wastewater by silica-alginate-fungi biocomposites. Water Air Soil Pollut. 2012;223(7):4307
4318. Available from: https://doi.org/ 10.1007/s11270-012-1193-6.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AA. See Arachidonic acid (AA) Abdominal obesity, preferential effect of olive oil in, 252 Aberrant crypt foci (ACF), 530
531, 531f Abelson murine leukemia viral oncogene homolog 1 (ABL1), 687 Abnormal Ca21 signaling and acute pancreatitis, 582 Absorption, 193
195 changes in urinary tyrosol and hydroxytyrosol, 195f plasma hydroxytyrosol concentrations, 195f Aca f 1 (Acacia farnesiana), 363 ACC. See Acetyl-CoA carboxylase (ACC) ACE. See Angiotensin-converting enzyme (ACE) ACEi. See Inhibitors of ACE (ACEi) Aceituna Aloren˜a de Ma´laga PDO, 20 Aceituna de Mallorca PDO, 20 Acetaldehyde, 472 Acetobacter aceti, 146 Acetoside, 451
452, 452f Acetyl-CoA carboxylase (ACC), 628
629, 652 Acetylation, 489 ACF. See Aberrant crypt foci (ACF) Achromobacter lipolytica, 152
153 Acinar cell toxins, 582 ACP. See Acyl carrier protein (ACP) Actinobacteria, 146
148 Activation function (AF), 664
665 Activator protein (AP), 663 AP-1, 265, 302
303 Acute exercise, 303
304 Acute myocardial infarction, 605 Acute pancreatitis (AP), 581
583 abnormal Ca21 signaling, 582 AR42J cell model of, 574
577 comparisons of olive oils with other edible oils, 588
589 endoplasmic reticulum stress, 582 hydroxytyrosol, 583
587 improves AR42J antioxidant defenses, 584
585 protects against cell death induced by cerulean, 585
586 restores physiological Ca21 signaling and secretory pattern, 586
587 suppressive effect on NF-kB and cytokine release, 585

impairment of cytoprotective-associated responses, 582 implications for human health and disease prevention, 589 lifestyle, 583
587 Mediterranean diet, 583
587 mitochondrial dysfunction, 582 NF-kB activation, 583 oxidative stress, 583 secretory blockade, 582
583 zymogen activation, 582 Acyl carrier protein (ACP), 32
35 AD. See Alzheimer’s disease (AD); Atopic dermatitis (AD) Adenosine monophosphate
activated protein kinase (AMPK), 538, 610, 628 HT effects on, 541 Adenosine triphosphatase (ATPase), 41
42 Adenosine triphosphate (ATP), 418
419, 682 ADH. See Alcohol dehydrogenase (ADH) Adipose-derived mesenchymal stem cells (ASCs), 683
684 ADMA. See Asymmetric dimethylarginine (ADMA) ADR. See Aldose reductase (ADR) Adrenocorticotropic hormone, 436 Adsorption, 514, 697
698 Adulterated EVOO detection using IT, 81
82 Adulteration, 79 Advanced glycation end products (AGEs), 450 Advanced oxidation processes, 697 Aerobic digestion, 512 Aerobic living cells, 539 Aeromonas, 149
151 AF. See Activation function (AF) AFLP. See Amplified fragment length polymorphism (AFLP) Agents acting on GSH, 450 AGEs. See Advanced glycation end products (AGEs) Aging, 401
402, 416, 537 brain, 415
416 cellular and molecular mechanism, 538
540 cellular senescence and release of SASP, 539 deficiency of stem cell regenerative capacity, 539 dysregulation of energy metabolic signaling pathways, 538

enhanced continued inflammation, 539
540 genomic instability, 540 impairment of mitochondrial function, 538 increased production of harmful reactive oxygen species, 539 reduced proteostasis, 538
539 of global population, 415 hydroxytyrosol effects on autophagy, 542 on DNA damage/repair, 542 on epigenetic regulation, 542 on metabolic regulation, 540
541 on mitochondria dysfunction, 541
542 on molecular and cellular mechanisms, 540
542 on oxidative stress, 541 implications for human health and disease prevention, 544 olive oil effects on molecular and cellular mechanisms, 540
542 Aglycone, 134, 292, 447 Agrobacterium, 330 AICAR. See 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) Akt. See Protein kinase B (Akt) ALA. See α-linolenic acid (ALA) Alanine aminotransferase (ALT), 631 Alatsolies, 20 Alcaligenes faecalis, 146 Alcohol consumption, 471
472 drinking, 473
474 use disorders, 474 Alcohol dehydrogenase (ADH), 472 enzymes, 472 Aldehyde dehydrogenase (ALDH2), 472 ALDH2. See Aldehyde dehydrogenase (ALDH2) Aldose reductase (ADR), 445 inhibitors, 450 Allergens, 359 Allergogram, 360
361, 360f, 369 Allergy, 391
392 asthma, 392 atopic dermatitis, 391
392 olive in, 180
181 α-linolenic acid (ALA), 402 α-tocopherol, 106, 378 ALS. See Amyotrophic lateral sclerosis (ALS)

701

702

Index

ALT. See Alanine aminotransferase (ALT) Alternaria, 146
148, 152
153, 330 Alzheimer’s dementia, 625 Alzheimer’s disease (AD), 177
178, 241, 415
416, 479, 672 Aβ toxicity in, 481f EVOO and AD
associated memory, 419 AM. See Association mapping (AM) Amino acid composition, 100 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), 629 AMP-activated protein kinase/Unc-51-like autophagy activating kinase 1 pathway (AMPK/ULK1 pathway), 672
673 AMPK. See Adenosine monophosphate
activated protein kinase (AMPK) Amplified fragment length polymorphism (AFLP), 32
35 Amylase secretion, 575 Amyloid-β (Aβ), 479, 672, 673f biology and function, 480
481 EVOO comparisons with other edible oils, 485 impact on Aβ pathology, 482 induction of autophagy activation and Aβ proteolytic clearance, 483
484 induction of Aβ proteolytic cleavage and BBB clearance, 483 inhibits Aβ production and aggregation, 482
483 implications for human health and disease prevention, 485
486 pathophysiology, 481
482 toxicity in AD, 481f Amyloidosis, 479 Amyotrophic lateral sclerosis (ALS), 241 Anaerobic digestion, 512 Anal fissure, olive oil for, 432 Ang III. See Angiotensin III (Ang III) AngII. See Angiotensin II (AngII) Angina, 165 Angiotensin II (AngII), 264, 438 Angiotensin III (Ang III), 439 Angiotensin IV (AngIV), 439 Angiotensin-converting enzyme (ACE), 264, 438
439 inhibitors, 165
166 Angiotensinase A, 439 Angiotensinogen, 264 AngIV. See Angiotensin IV (AngIV) Animal models BC and olive oil, 340 cancer chemopreventive activity in, 530
533 CRC and olive oil, 342 ANP. See Atrial natriuretic peptides (ANP) ANS. See Anthocyanidin synthase (ANS) Anterior pharynx defective (APH1), 480 Anthocyanidin synthase (ANS), 40 Anthocyanin peonidin, 452 Anticancer activity of oleocanthal, 662, 664t of olive leaves, 448

Antidiabetic activity of olive leaves, 448 Antifungal properties of olive oil, 431 Antihypertensive activity of olive leaves, 448 Antihypertensive drugs, 438
439 Anti-inflammatory activity of olive leaves, 448 of OMW, 515 Antimicrobial activity of olive leaves, 448 Antimicrobial compounds in olive oil, 330
333 in table olives, 333
334 Antimicrobial effects of OMW, 514
515 Antinociceptive activities of olive leaves, 448 Antioxidants, 232, 406, 426, 437, 472 activity implications for human health and disease prevention, 319
321 natural antioxidants, 314
319 of olive leaves, 449 in olive oils, 313
314 of OMW, 514 content of lipoproteins, 200 defenses, 302
303, 576 in olive oil phenolics acute exercise, 303
304 implications for human health and disease prevention, 307
308 in vivo effects of olive oil rich in biophenols in muscle redox regulation, 305 myoblasts, 302 natural antioxidants, 301
302 olive extracts of bioactive compounds, 304
305 oxidative
reductive stress, 303
304 polyphenols and athletic performance, 305
306 reactive species, 302
303 satellite cells, 302 property, 617 Antiretroviral therapy (ART), 394 Antirrhinum majus.. See Snapdragon (Antirrhinum majus) AOM. See Azoxymethane (AOM) AOM/DSS. See Azoxymethane/dextran sulfate sodium (AOM/DSS) AP. See Activator protein (AP); Acute pancreatitis (AP) APH1. See Anterior pharynx defective (APH1) Apigenin, 315, 315f, 382t, 494
495 Apigenin-7-glucoside, 67, 135, 276 Apigenin-7-glucoside oleuropein, 379 Apo-1/CD95. See Apoptosis via cross-linking of Fas (Apo-1/CD95) Apolipoprotein (Apo), 197 Apolipoprotein E (apoE), 481, 672 Apoptosis, 576
577 regulator, 378
379 Apoptosis via cross-linking of Fas (Apo-1/ CD95), 379 APP. See Aβ precursor protein (APP) AR42J cells, 584, 585f AR42J studies, 572
573

model of acute pancreatitis, 574
577 antioxidant defenses, 576 cell function, 575 cell viability and apoptosis, 576
577 fatty acids in membranes, 575t inflammatory mediators secretion, 575
576 Arabidopsis thaliana, 363 Arachidonic acid (AA), 263, 265, 402, 426, 550
551 Arbequina, 494 Arbosana, 494 Archaic method, 16
18 Arogenate dehydrogenase (DH), 40
41 Aroma, 91 ART. See Antiretroviral therapy (ART) Arthrobacter globiformis, 146 Ascomycota, 146
148 ASCs. See Adipose-derived mesenchymal stem cells (ASCs) Ash pollen, 363 Aspartate aminotransferase (AST), 631 Aspergillus, 330 A. niger, 152
153, 512 A. terreus, 512 Association mapping (AM), 32
35 studies, 32
35 AST. See Aspartate aminotransferase (AST) Asthma, 392 olive pollen allergens associated with, 363
364 Asymmetric dimethylarginine (ADMA), 264
265 AT1 blockers, 438
439 ATGs. See Autophagy genes (ATGs) Atherosclerosis, 167, 209
210, 281, 393
394 Atopic dermatitis (AD), 391
392, 425
426 olive oil for, 431 ATP. See Adenosine triphosphate (ATP) ATPase. See Adenosine triphosphatase (ATPase) Atrial natriuretic peptides (ANP), 264 Aureobacidium spp., 146
148 Aureobasidium pullulans, 146
148, 329 Autochthonous probiotics application, 221
222 lactic acid bacteria LAB, 223t Autoimmunity IBD, 392
393 RA, 392 SLE, 393 Autophagy activation, 483
484 hydroxytyrosol effects on, 542 Autophagy genes (ATGs), 481 Autoxidation processes, 57 Averyellaa dalhousiens, 146
148 Azotobacter chroococcum, 512 Azoxymethane (AOM), 342, 530
531 Azoxymethane/dextran sulfate sodium (AOM/DSS), 379
381, 532
533 Aβ. See Amyloid-β (Aβ) Aβ oligomers (Aβo), 675
676 Aβ precursor protein (APP), 479, 480f

Index

B B-cell epitopes, 363 B-cell lymphoma type 2 (Bcl-2), 533, 606 antagonist killer apoptotic signals, 378
379 Bcl-2-associated X activation, 378
379 BACE1. See β-site APP cleaving enzyme 1 (BACE1) Bacillus spp., 146 B. cereus, 448 B. megaterium, 146 B. subtilis, 146, 329, 514
515 Bacterial infections, olive oil for, 431
432 Bacteroides vulgatus, 331 Bactrocera oleae.. See Olive fly (Bactrocera oleae) Basal cell carcinoma (BCC), 615
616 Basidiomycota, 146
148 BBB. See Blood
brain barrier (BBB) BC. See Breast cancer (BC) BCC. See Basal cell carcinoma (BCC) Bcl-2. See B-cell lymphoma type 2 (Bcl-2) Bcl-xL gene, 533 BDNF. See Brain-derived neurotrophic factor (BDNF) β-carotene, 106 chemical display of, 318f β-cell function, 643b β-glucosidases (β-GLUs), 39, 141 β-glucosylated elenolic acid, 123 β-site APP cleaving enzyme 1 (BACE1), 480 Betula pendula.. See Birch (Betula pendula) Betula verrucosa.. See Birch (Betula pendula) Biancolilla, 494 Bifidobacterium, 179 B. adolescentis, 331 B. bifidum, 331 Bile acids, 581
582 Bile lithogenicity, 562
563 Bile secretion in dogs, 558
559, 560f Biliary lipid composition, 562
563 Bioactive components, 313 Bioactive ingredients in olive leaves anticancer properties, 73 cardioprotective activity, 71
73 diabetes, 74 extraction procedures, 68
71 postharvest treatment, 67
68 respiratory diseases, 74 sampling, 65
67 Bioactivity of oleocanthal, 662
663 of OLf extracts, 71 Bioavailability of EVOOs phenolic compounds, 206
207 of olive oil phenolic compounds absorption and disposition, 193
195 bioactive effects of olive oil phenolic compounds, 196
200 endogenous sources of Tyr and OHTyr, 196 in vivo basic mechanisms, 200 metabolism, 195
196 Biodegradation process, 141 Biofilms, 154

Biological significance in olive, 123 Biological-to-chemical oxygen demand ratio (BOD/COD), 505 Biophenols, 495
496 Biopreservation of fermented olives, 223
224 Birch (Betula pendula), 361, 367
368 Bitterness of olives, 7, 10, 15 Black olives, 5
6 in dry salt, 11 Bladder cancer, 380t Bleeding time (BT), 282
283 Blood pressure (BP), 165, 198
200, 208 evaluation, 284 Blood
brain barrier (BBB), 481, 672 clearance, 483 BMI. See Body mass index (BMI) BOD/COD. See Biological-to-chemical oxygen demand ratio (BOD/COD) Body mass index (BMI), 207
208 Body weight regulation, 251 Bone-sparing effects, 666 Botanical cosmeceuticals, 425t, 432 Botrytis, 330 BP. See Blood pressure (BP) Brachionus calyciflorus, 696 Brain aging, 415
416 amyloidosis, 484 brain cells, EVOO components on lipid synthesis in, 653 brain health, EVOO and, 416
418 Brain-derived neurotrophic factor (BDNF), 474 Breast cancer (BC), 337, 347, 380t dietary fat and carcinogenesis parameters, 348 and histopathology, 348
350 and hormonal status, 354
355 and redox status, 350
353 implications for human health and disease prevention, 355
356 and olive oil, 338
340 animal models, 340 cell culture models, 339
340 human studies, 338
339 “Brick-and-mortar” construction, 402 Brining, 10
11 BT. See Bleeding time (BT)

C c-Jun NH-2 terminal kinase (JNK), 235, 379 c-MET inhibitor, 685
686 as potential molecular target in oncology, 682
684, 683f 2-C-methyl-D-erythritol 2,4-cyclo-PP synthase (OeMECPS), 39 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (OeCDPMES), 39 2-C-methyl-D-erythritol 4-phosphate (MEP), 39 C-reactive protein (CRP), 210, 265, 404 C-responsive protein (CRP), 169 C-terminal fragment α (CTFα), 480 C-terminal fragment β (CTFβ), 480 C-terminal fragment γ (CTFγ), 480

703

C/EBP homologous protein (CHOP), 606
607 C4H. See Cinnamate 4-hydroxylase (C4H) Ca21 homeostasis, 575 Ca21-binding allergens, 364 Ca21-binding proteins (CaBPs), 364 CAA. See Cerebral amyloid angiopathy (CAA) Caco-2 cells, 527 Cadmium, 462
463 Caenorhabditis elegans, 220 Caffeic acid, 276, 494
495 Calcein method, 234 Californian-style black olives, 11, 99 Calpain activation and cataract formation, 450 cAMP. See Cyclic adenosine monophosphate (cAMP) CaNa2EDTA chelation, 463
464 Cancer, 210
211, 596
597 cancer-associated mechanisms, 617
619 prevention mechanisms anticancer effects of olive oil, 382t apoptosis regulator, 378
379 Bcl-2 antagonist killer apoptotic signals, 378
379 Bcl-2-associated X activation, 378
379 Fas ligand expression/activity, 379
382 nuclear factor kappa-light-chain-enhancer regulation of activated B cells activation, 383
384 tumor necrosis factor modulation, 379
382 Cancer chemopreventive activity in animal models in vivo, 530
533 studies with experimental models, 530
533 studies with genetic-based models of colorectal cancer, 533 in colon cancer cells in vitro extrinsic and intrinsic pathways, 528f studies on apoptosis, 527
530 studies on cell proliferation, 527 Candida spp., 152
153, 220, 330, 431 C. adriatica, 152
153 C. albicans, 448 C. diddensiae, 148
149, 152
153 C. famata, 154
155 C. lipolytica, 152
153 C. norvegica, 148
149, 218 C. oleophila, 148
149 C. rugosa, 152
153 C. silvae, 218 C. tropicalis, 697 C. wickerhamii, 152
153 Cannabinoid receptor gene type 1 (CNR1), 542 Captopril, 438
439 Carbohydrate, 251 Carbohydrate regulatory element binding protein/Max-like factor X (ChREBP/ MLX), 246 Carcinogenesis parameters, 348t dietary fat and, 348 process, 616 Carcinogens, 530
533 Cardiac malformations, 471
472

704

Index

Cardio-ankle vascular index (CAVI), 285 Cardiology, olive in, 180 Cardiomyocytes (CMs), 606 oleuropein effect on, 606
607 Cardioprotective activity, 71
73 evidence on cardiovascular protective effect, 72t of olive leaves, 448 Cardiovascular disease (CVD), 65, 165, 166f, 197, 205, 209
210, 262, 284, 313, 393
394, 605, 625 oleuropein against, 610
611 OO effect on CVD risk factors, 169 dysglycemia, 168
169 hypertension, 168 inflammation and redox imbalance, 169 OS
mediated endothelial dysfunction and atherosclerosis, 169 vascular aging, 168 Cardiovascular effects of OMW, 515 Care products, 402 Carolea, 494 Carotenoids, 87
88, 105, 313, 318 Cartilage oligomeric matrix protein (COMP), 392 Casaliva, 494 Caspase-3, 528
530 Caspase-8, 527 “Cassanese” unripe fruit, 42
43 Castelvetrano-style green olives, 10 Catalase (CAT), 302
303, 450, 597
599 Cataract, 449
450 etiology, 449 factors contributing in development, 449f molecular mechanisms behind cataract formation, 449
450 of O. europaea in cataract treatment, 450
454 protective effect of olive leaves extract against, 451f strategies for treatment and prevention, 450 Catechin. See Flavan-3-ols Catechol, 124
125, 126f, 140 Catechol-O-methyltransferase, 194 ß-Catenin, 533 Cathepsin B, 481 CAVI. See Cardio-ankle vascular index (CAVI) CBM43, 364 CC. See Compound C (CC) CCK. See Cholecystokinin (CCK) CCK-octapeptide (CCK-8), 570
572, 586
587 CD. See Crohn’s disease (CD) CDK. See Cyclin-dependent kinase (CDK) CDKN2A. See Cyclin-dependent kinase inhibitor 2A (CDKN2A) CE. See Cholesterol efflux (CE) Cell culture models BC and olive oil, 339
340 CRC and olive oil, 341
342 Cell cycle, 533 Cell function, 575 Cell viability, 576
577

Cellular mechanism on aging, 538
540 hydroxytyrosol and olive oil effects on, 540
542 Cellular senescence, 539 Cellular senescence and release of SASP, 539 Cellulomonas flavigena, 146 Central nervous system (CNS), 415 Central obesity, 264 Ceramides, 405 Cerasiformis, 28 Cereals, 79 Cerebral amyloid angiopathy (CAA), 672 Cerulein, 586
587 CH3 genes. See 3-Hydroxylase-encoding genes (CH3 genes) Chalcone isomerase (CHI), 40 Chalcone synthase (CHS), 40 CHD. See Coronary heart disease (CHD) Che a 1 (Chenopodium album), 363 Chelating intracellular labile iron, 233
234 Chelation, 458
459 antioxidant effects, 458
460 of labile iron, 235 Chelation therapy cell preparation of Pb-exposed worker before, 463f and olive leaf, 462
464 Chemical oxygen demand (COD), 693
694 Chemo-preventive effects of OMW, 516 Chenopodium album.. See Che a 1 (Chenopodium album) CHI. See Chalcone isomerase (CHI) Chlorogenic acid, 139
140 Chlorophylls, 318, 378 pigments, 87
88 Cholecystectomy, 559
560 Cholecystokinin (CCK), 252
253, 558, 559t, 561f, 564, 570
571, 581
582 receptors, 572 Cholesterol efflux (CE), 197, 267 Cholesterol saturation index (CSI), 562 Cholesterol synthesis, 651
652 CHOP. See C/EBP homologous protein (CHOP) ChREBP/MLX. See Carbohydrate regulatory element binding protein/Max-like factor X (ChREBP/MLX) Chronic diseases, EVOO effects on cancer, 210
211 CVD and atherosclerosis, 209
210 diabetes, 209 dyslipidemia, 208
209 hypertension, 208 obesity, 207
208 Chronic hyperglycemia, 263 Chronic inflammation, 261, 389 CHS. See Chalcone synthase (CHS) CI. See Confidence interval (CI) Cinnamate 4-hydroxylase (C4H), 40 Cinnamic acid, 140 Cis-MUFA, 263 Citrobacter, 149
151 4CL. See 4-Coumarate-CoA ligase (4CL)

cl-CASP3, 278 Cladosporium spp., 146
148 Clavibacter spp., 146, 330 C. michiganensis, 330 ClinOleic, 391 Clostridium, 9, 149
151 C. botulinum, 21
22 C. clostridioforme, 331 C. perfringens, 331 Cloud point extraction (CPE), 513 CMs. See Cardiomyocytes (CMs) CNNs. See Convolutional neural networks (CNNs) CNR1. See Cannabinoid receptor gene type 1 (CNR1) CNS. See Central nervous system (CNS) Cobalt chloride (CoCl2), 283 COD. See Chemical oxygen demand (COD) Cognition, 416 EVOO in aging rodents, 418
419 EVOO in human, 416
418 Cognitive impairment, EVOO and, 419 Collagen, 401 Colletotrichum, 146
148, 330 Colletotrichum acutatum, 146
148 Colletotrichum godetiae, 146
148 Colletotrichum karstii, 146
148 Colon cancer, 380t cancer chemopreventive activity in colon cancer cells, 527
530 Color, 91 Colorectal cancer (CRC), 337, 525 and olive oil, 340
342 animal models, 342 cell culture models, 341
342 human studies, 341 studies with genetic-based models of, 533 Comet assay. See Single-cell electrophoresis
based method COMP. See Cartilage oligomeric matrix protein (COMP) Complete stirred tank reactor (CSTR), 512 Complexin 1 (CPLX1), 419
420 Compound C (CC), 629 Comselogoside, 508
509 Conditioning mechanisms, 609 Confidence interval (CI), 338 Congenital cataract, 449 Constituents of olive oil, 426 Consumer packs, natural table olives in, 22 Convolutional neural networks (CNNs), 81 Coratina, 494 Cornicabra, 494 Coronary heart disease (CHD), 209
210, 276, 319
320, 473
474, 639
640 Corticosteroids, 549 Corynebacterium michiganensis, 329 Cosmeceuticals. See Botanical cosmeceuticals 4-Coumarate-CoA ligase (4CL), 40 Coumarin metabolites, 496 Council for Agricultural Research and Economics-Research Centre for Olive, Fruit and Citrus Crops (CREA-OFA), 28

Index

COX. See Cyclooxygenase (COX) CPE. See Cloud point extraction (CPE) CPLX1. See Complexin 1 (CPLX1) CRC. See Colorectal cancer (CRC) CREA-OFA. See Council for Agricultural Research and Economics-Research Centre for Olive, Fruit and Citrus Crops (CREA-OFA) Crocus sativu.. See Cros s 1 (Crocus sativu) Crohn’s disease (CD), 392
393 Cros s 1 (Crocus sativu), 363 CRP. See C-reactive protein (CRP); Cresponsive protein (CRP) Crude fiber, 100 Cryptococcus laurentii, 152
153 Cryptosporidium, 21
22 CSI. See Cholesterol saturation index (CSI) CSTR. See Complete stirred tank reactor (CSTR) CTFα. See C-terminal fragment α (CTFα) CTFβ. See C-terminal fragment β (CTFβ) CTFγ. See C-terminal fragment γ (CTFγ) Cucumis sativus, 696 Cultivar variety, effects of, 127
128 Cultivated olive (var. europaea), 28 Curcumin, 589 Curtobacterium spp., 146
148 C. plantarum, 146 Cuspidata, 28, 31 CVD. See Cardiovascular disease (CVD) Cyclic adenosine monophosphate (cAMP), 438
439, 664
665 Cyclin-dependent kinase (CDK), 533 Cyclin-dependent kinase inhibitor 2A (CDKN2A), 616 Cycloolivil, 452f Cyclooxygenase (COX), 265, 389
390, 402, 408, 662 COX-1, 265, 674
675 cascade, 497 COX-2, 265, 342, 379, 390, 617, 674
675 Cyclophilin. See Ole e 15 CYP2E1 expression, 472 CYP450 enzymes. See Cytochrome 450 enzymes (CYP450 enzymes) Cytochrome 450 enzymes (CYP450 enzymes), 526 Cytochrome c, 527 Cytoprotective-associated response impairment, 582

D Damage theories, 537 Damage-associated molecular patterns, 583 DAO hypothesis. See Diamine oxidase hypothesis (DAO hypothesis) Daphnia magna, 696 Darkening, 12 DArT. See Diversity arrays technology (DArT) Date olives, 20 Date palm (Phoenix dactylifera), 361 DBD. See DNA-binding domain (DBD) DBP. See Diastolic blood pressure (DBP) DDR. See DNA damage response (DDR)

De novo lipogenesis, 651
652, 652f Deacetoxyligstroside glycoside, 671 Death receptors, 527 Death-inducing signaling complex (DISC), 527 Debaryomyces hansenii, 148
149 Debittering process, 105 Decarboxylase enzyme, 139 Decarboxymethyl elenolic acid (EDA), 333
334 Decarboxymethyl oleuropein aglycone (DOA), 339
340 Defect predominantly perceived intensity (DPP intensity), 9 Deficiency of stem cell regenerative capacity, 539 Dehydrated olives, 8 DEI flattens. See Dermal
epidermal interface flattens (DEI flattens) Dementia, 415 Demethyloleuropein, 135, 494 Dentistry, preventive and restorative, 181 Dentistry and oral cavity based on TPM, 181
182 endodontics, olive in, 181 oral medicine, olive in, 181
182 orthodontics, olive in, 182 periodontics, olive in, 181 preventive and restorative dentistry, olive in, 181 prosthodontics, olive in, 182 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (OeDXR), 39 1-Deoxy-D-xylulose-5-phosphate synthase (DXS), 39
41 Deoxyribonucleic acid (DNA), 338, 405, 416, 472, 593
594 Dermal
epidermal interface flattens (DEI flattens), 403 Dermatologic conditions, topical applications for, 430
431 antifungal properties of olive oil, 431 olive oil and dermatitis, 430
431 olive oil and dry skin, 430 olive oil and wound healing, 431 Dermatology, olive in, 177 Desferrioxamine (DFO), 233
234 Desquamation, 402 Devriesia spp., 146
148 Dextran sulfate sodium (DSS), 342, 532
533 Dextranicum, 146 DFO. See Desferrioxamine (DFO) DFR. See Dihydroflavonol 4-reductase (DFR) DGAT. See Diacylglycerol acyltransferase (DGAT) DH. See Arogenate dehydrogenase (DH) DHA. See Docosahexaenoic acid (DHA) 3,4-DHPEA-EA. See Oleuropein aglycon (3,4DHPEA-EA) 3,4-DHPEA-elenolic acid dialdehyde (3,4-DHPEA-EDA), 39
40, 57 Diabetes, 74, 168
169, 209 Diabetes mellitus. See Diabetes Diacylglycerol acyltransferase (DGAT), 654

705

Diamine oxidase hypothesis (DAO hypothesis), 341 Diastolic blood pressure (DBP), 199
200, 284 Diet, 177 and male fertility, 435
436 rich vegetables and fruits, 435 Diet-induced thermogenesis (DIT), 252 Dietary factors, 605 Dietary fat, 347
348 and carcinogenesis parameters, 348 on gastrointestinal function, 557 and histopathology of breast tumors, 348
350 and hormonal status in breast cancer, 354
355 and redox status, 350
353 Dietary fatty acids, 647f Dietary ingestion of olive oil, 405 Dietary lipids and male fertility, 436
437 and pancreatic secretion, 570 Dietary oils, 239
240 Dietary protection, 428
429 monounsaturated fats, 428
429 Differential scanning calorimeter (DSC), 81 Diffusion, 28
30 Digestive secretion in dogs bile secretion, 558
559, 560f exocrine pancreatic secretion, 557
558 in humans, 559
563 biliary lipid composition and bile lithogenicity, 562
563 exocrine pancreatic secretion, 562 gastric secretion, 562 plasma profile of gastrointestinal peptides, 560
561 Diglycerides, 99
100 Dihydroflavonol 4-reductase (DFR), 40 3,4-Dihydroxyphenyl-ethanol (3,4-DHPEA). See Hydroxytyrosol (HT) (2-(3,4-Dihydroxyphenyl)ethanol (Hty). See Hydroxytyrosol (HT) 3,4-Dihydroxyphenylacetaldehyde (DOPAL), 194
196 3,4-Dihydroxyphenylacetic acid (DOPAC), 194
196 7,12-Dimethylbenz[a]anthracene (DMBA), 340 Dimethylhydrazine (DMH), 342, 530
531 Diosmetin, 276 Diosmetin-7-O-glucoside, 276 Dipeptidyl peptidase-4 (DPP-4), 439, 627 2,2-Diphenyl-1-picrylhydrazyl (DPPHG), 459 Directly brined olives, 99 DISC. See Death-inducing signaling complex (DISC) Disposition, 193
195 chain, 85 DIT. See Diet-induced thermogenesis (DIT) Diversity arrays technology (DArT), 32
35 DJ-1. See Protein deglycase (DJ-1) DMBA. See 7,12-Dimethylbenz[a]anthracene (DMBA) DMH. See Dimethylhydrazine (DMH)

706

Index

DNA. See Deoxyribonucleic acid (DNA) DNA damage response (DDR), 540 DNA damage/repair, 437 enzymes, 450 hydroxytyrosol effects on, 542 DNA-binding domain (DBD), 664
665 DOA. See Decarboxymethyl oleuropein aglycone (DOA) Docosahexaenoic acid (DHA), 402, 436 DOPAC. See 3,4-Dihydroxyphenylacetic acid (DOPAC) DOPAL. See 3,4-Dihydroxyphenylacetaldehyde (DOPAL) Dose reduction index (DRI), 687 Doxorubicin (DXR), 283, 610 DXR-induced lipid peroxidation, 283 DPP intensity. See Defect predominantly perceived intensity (DPP intensity) DPP-4. See Dipeptidyl peptidase-4 (DPP-4) DPPHG. See 2,2-Diphenyl-1-picrylhydrazyl (DPPHG) DRI. See Dose reduction index (DRI) Dry salt, partial dehydration of olives using, 21 Dry skin, olive oil and, 430 Dry-salted olives, 154
156, 156f micrograph of olive epidermis, 155f parenchyma cells of dry-salted olives with fungal growth, 156f DSC. See Differential scanning calorimeter (DSC) DSS. See Dextran sulfate sodium (DSS) DXR. See Doxorubicin (DXR) DXS. See 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) Dysglycemia, 168
169, 208
209 olive oil and, 266
267

E E-selectin. See Endothelial-leukocyte adhesion molecule 1 (E-selectin) “E-shaped” molecule, 378 E-vitamers, 378 EA. See Elenolic acid (EA) EAAs. See Essential amino acids (EAAs) EAM. See Experimental autoimmune myocarditis (EAM) ECM. See Extracellular matrix (ECM) EDA. See Decarboxymethyl elenolic acid (EDA) Edible vegetable oils, 85 EDTA. See Ethylenediaminetetraacetic acid (EDTA) EFSA. See European Food Safety Authority (EFSA) EGEA. See Epidemiological study of Genetics and Environment and Asthma (EGEA) EGF. See Epidermal growth factor (EGF) EGFR. See Epidermal growth factor receptor (EGFR) Eicosapentaenoic acid (EPA), 402, 408 Electrocoagulation, 510, 697 Electromagnetic radiation, 93 Electrospray ionization mode, 65 Elenoic acid, 135, 140

Elenolic acid (EA), 100
102, 111
113 Elevated blood glucose, 498 ELISA. See Enzyme-linked immunosorbent assay (ELISA) EMT. See Epithelial-to-mesenchymal transition (EMT) Endocrine effects of OMW, 516 Endocrinology, olive in, 179
180 Endodontics, olive in, 181 Endogenous bioconversion of Tyr into OHTyr, 196 Endogenous sources of Tyr and OHTyr, 196 Endoplasmic reticulum stress (ER stress), 581 and acute pancreatitis, 582 Endothelial dysfunction, 167, 264 Endothelial function, 198
200 olive oil and, 407
408 Endothelial nitric oxide synthase (eNOS), 169 Endothelial-leukocyte adhesion molecule 1 (Eselectin), 393
394 Endothelin-1 (ET-1), 264
265 Endothelium, 264
265 Energy metabolic signaling pathway dysregulation, 538 Enhanced continued inflammation, 539
540 Enkephalinase activity, 177
178 eNOS. See Endothelial nitric oxide synthase (eNOS) Enteritidis, 404 Enterobacter, 149
151 Enterobacteriaceae, 145
146 Enterococcus spp., 146
148, 216t, 218 E. faecalis, 220, 331, 334, 448 ENU. See N-ethyl-N-nitrosourea (ENU) Enzymatic antioxidant defense systems, effects of OLEU and HTXon, 597
599 Enzymatic hydrolysis, 141 Enzyme-linked immunosorbent assay (ELISA), 474 Enzymes, 512 antioxidant defense systems, 350
353 EPA. See Eicosapentaenoic acid (EPA) EPIC. See European Prospective Investigation into Cancer and Nutrition (EPIC) Epidemiological study of Genetics and Environment and Asthma (EGEA), 369 Epidermal growth factor (EGF), 682
683 Epidermal growth factor receptor (EGFR), 682
683 Epigenetic regulation, hydroxytyrosol effects on, 542 Epithelial-to-mesenchymal transition (EMT), 682
683 ER stress. See Endoplasmic reticulum stress (ER stress) EREs. See Estrogen response elements (EREs) ERK 1/2. See Extracellular signal-related kinase 1/2 (ERK 1/2) ERK pathways. See Extracellular signal
regulated kinase pathways (ERK pathways) ERs. See Estrogen receptors (ERs) Erwinia spp., 146, 330 E. carotovora, 146

E. herbicola, 146 Erythrocyte sedimentation rate (ESR), 394 Erythrodiol, 30, 379, 390, 448 ERβ/ERα ratio. See Estrogen receptor α/β ratio (ERβ/ERα ratio) Escherichia coli, 21
22, 149
151, 218, 220, 329
331, 334, 448, 514
515 E. coli 0157:H7, 330t ESR. See Erythrocyte sedimentation rate (ESR) Essential amino acids (EAAs), 103 Estradiol, 354f Estrogen, 339
340, 347, 663
665 modulators, 687
688 Estrogen receptor α/β ratio (ERβ/ERα ratio), 379 Estrogen receptors (ERs), 663
665, 665f, 688 impact of oleocanthal on, 665
667 binding of oleocanthal to ER, 666 molecular effects of oleocanthal mediated via ER targeting, 666
667 oleocanthal modulates ER gene expression, 666 Estrogen response elements (EREs), 664
665 ESTs. See Expressed sequence tags (ESTs) ET-1. See Endothelin-1 (ET-1) Ethanoic acid methyl esters, 496
497 Ethyl hydroxytyrosyl ether (HT-Et), 632 Ethylenediaminetetraacetic acid (EDTA), 462
463 EU. See European Union (EU) Europaea, 28 European Food Safety Authority (EFSA), 197, 218, 316, 458, 584 Panel, 540 European Prospective Investigation into Cancer and Nutrition (EPIC), 338 European Union (EU), 5 EVOO. See Extra virgin olive oil (EVOO) Exfoliation, 402 Exocrine pancreatic secretion, 562 in dogs, 557
558 Experimental autoimmune myocarditis (EAM), 611 Expressed sequence tags (ESTs), 32
35 Extensive knowledge, 313 Extra virgin olive oil (EVOO), 30, 79, 85, 91, 167, 178, 181, 205, 207f, 251
252, 262, 266, 282, 291, 301
302, 314, 337
338, 347
348, 377
378, 390
391, 404, 415
416, 426
427, 473, 479
480, 526, 547, 627, 651, 661, 671, 687
688, 693. See also Olive oils (OO); Virgin olive oil (VOO) antithrombotic activity, 282
283 beneficial effects, 482f of consumption, 475f bioavailability of EVOOs phenolic compounds, 206
207 comparisons between olive oils and edible oils, 80 with other edible oils, 420
421, 485 components, 268 on lipid synthesis in brain cells, 653 composition of, 206, 263f

Index

detection of adulterated EVOO using IT, 81
82 diets, 257 effects on chronic diseases, 207
211 evidence of beneficial effects on AD
associated memory and cognitive impairment, 419 on brain health and cognition in human, 416
418 on cognition and neuroinflammation in aging rodents, 418
419 human health and disease prevention, 80 implications and disease prevention, 421, 599
600 impact on Aβ pathology, 482 induction of autophagy activation and Aβ proteolytic clearance, 483
484 of Aβ proteolytic cleavage and BBB clearance, 483 inhibiting Aβ production and aggregation, 482
483 in vitro and in vivo studies, 417t IT, 80
81 and long-term potentiation, 420 minority compounds as potent antioxidants, 594
595 nutritional quality alteration, 292 OLEU and HTX effects on enzymatic antioxidant defense systems, 597
599 on nonenzymatic antioxidant defense systems, 597 on oxidative stress parameters, 596
597 on tumor growth, 595 phenolic compounds, 262, 695, 695f phenolic profile of, 549 phenolic S-(2)-oleocanthal biological activities, 684
685 phenols on hepatic lipid synthesis, 653
654 quality, 85
88 comparisons of olive oils with edible oils, 85
86 laboratory quality control, 86
87 olive oils for human health and disease prevention, 86 real-time quality control, 87
88 storage, 293
295 fatty acids and polyphenols content, 294
295 light and oxygen exposure, 293
294 temperature, 293 tocoferols, 295 and synaptic proteins, 419
420 Extracellular matrix (ECM), 403 Extracellular signal-related kinase 1/2 (ERK 1/ 2), 278, 406, 682
683 Extracellular signal
regulated kinase pathways (ERK pathways), 235, 497 Extraction, 68
71, 513 application of response surface methodology, 69t liquid
liquid, 513

oil extraction process, 58 of phenolic compounds, 490
492

F

F3050H. See Flavonol 30 50 -hydrogenase (F3050H) F30H. See Flavonol 30 -hydrogenase (F30H) F3H. See Flavanone 3-hydroxylase (F3H) FAB-Lab, 445 natural products as potential therapeutic agents in, 446t Factor VII coagulant activity (FVII:C), 282
283 FAD2
1 gene, 38 FADD, 527 FADS2 gene. See Fatty acid desaturases 2 gene (FADS2 gene) FAEEs. See Fatty acid ethyl esters (FAEEs) FAK. See Focal adhesion kinase (FAK) Familial adenomatous polyposis (FAP), 525 FAP. See Familial adenomatous polyposis (FAP) Farnesyl diphosphate synthase (FPPS), 40
41 FAS. See Fatty acid synthase (FAS) FAs. See Fatty acids (FAs) FAS. See Fetal alcohol syndrome (FAS) Fas ligand (FasL), 379 expression/activity, 379
382 Fas/FasL system, 381
382 FASD. See Spectrum of fetal-alcoholic disorders (FASD) FasL. See Fas ligand (FasL) Fat balance, 251, 257 loss, 257 Fat oxidation rate (FOR), 252 Fat-evoked PYY release, 561 Fatty acid affect rate of fat oxidation, 251
252 Fatty acid desaturases 2 gene (FADS2 gene), 32
35 Fatty acid ethyl esters (FAEEs), 581
582 Fatty acid synthase (FAS), 652 Fatty acids (FAs), 53
56, 239
240, 402, 426, 436, 661 content, 294
295 evolution, 294 metabolism of, 402 range values reported by literature, of VOO, 54t synaptosome composition, 243t FDA. See Food and Drug Administration (FDA) Fenton/Haber
Weiss reaction cycle, 460 Fermentation, 8
9, 19
20, 99, 145 microbial association effect on, 153
154 microbiota of olives related to, 149
151 changes in total viable counts, 150f microbial association during different fermentation stages, 151t micrograph of microflora inside naturally fermented black olives, 152f micrograph of microflora on epidermis, 152f

707

Fermented green olives, chemical composition of components of raw olives, 99
100 Spanish-style green olives, 100
105 untreated green olives in brine, 105
106 Fermented olives, 215 probiotic lactic acid bacteria and yeasts isolated from, 216t probiotic microorganisms isolated from, 215
218 probiotics application in biopreservation, 223
224 in foods fermentations, 225 Fetal alcohol syndrome (FAS), 471
472 FFA. See Free fatty acids (FFA) Fibrils, 479 Fibrinogen, 265 Fibroblasts, 403 Firmicutes, 146
148 Fish oil, 240, 241t, 242t Flavan-3-ols, 276 Flavan-3-ols, 124
125 Flavanols, 457
458 Flavanone 3-hydroxylase (F3H), 40 Flavones, 276, 379, 457
458 Flavonoids, 115, 135, 291, 315, 315f, 377
379, 447, 459
463, 465
466, 496 antioxidant activity, 459 flavonoid
DNA complexes, 461 glycosides, 453 Flavonol 30 -hydrogenase (F30H), 40 Flavonol 30 50 -hydrogenase (F3050H), 40 Flavonol synthase (FLS), 40 Flavonols, 135, 141, 276, 452
453, 457
458 FLS. See Flavonol synthase (FLS) Fluidity, 244 Fluorescent spectroscopy, 94
95 Focal adhesion kinase (FAK), 682
683 Food and Drug Administration (FDA), 219, 682 Food intake, 252
257 Food-related bacterial pathogens, 330t Foot ulcers, olive oil use in clinical treatment of, 408 FOR. See Fat oxidation rate (FOR) FORD. See Free oxygen radicals defense (FORD) FORT. See Free oxygen radicals test (FORT) Fourier transform (FT), 92 Raman spectroscopy, 92 FPPS. See Farnesyl diphosphate synthase (FPPS) Fra e 1, 363 Frantoiano, 494 Fraxinus excelsior, 146, 363 Free fatty acid receptor 1 (FFAR1). See GPR40 Free fatty acid receptor 4 (FFAR4). See GPR120 Free fatty acids (FFA), 99
100, 264
265, 292, 378, 642
644, 644f Free oxygen radicals defense (FORD), 474 Free oxygen radicals test (FORT), 474 Free radicals, 263, 389
390, 403, 426

708

Index

Free radicals (Continued) scavengers protect cells in conditions of OS, 232
233 Frondihabitans suicicola, 146
148 Frontotemporal dementia (FTD), 676 Fruit-ripeness index (IR), 124 FT. See Fourier transform (FT) FTD. See Frontotemporal dementia (FTD) Functional food, 500 Fungal biodegradation, 697
698 Fungal infections, olive oil for, 431
432 Fusarium spp., 146
148 FVII:C. See Factor VII coagulant activity (FVII:C)

G

G 3 E interaction. See Genotype 3 environment interaction (G 3 E interaction) G protein
coupled receptors (GPRs), 564
565 G10H. See Geraniol-10-hydroxylase (G10H) GABA. See γ-aminobutyric acid (GABA) GAE. See Gallic acid equivalents (GAE) GAGs. See Glycosaminoglycans (GAGs) Galactomyces reessii, 218 Gallate decarboxylase, 139 Gallic acid, 139 Gallic acid equivalents (GAE), 654 Gallstones, 581 γ-aminobutyric acid (GABA), 243
244 γ-glutamyl transferase (GGT), 437 γ-glutamyl transpeptidase, 243 γ-linolenic acid (GLA), 405 γ-secretase, 480 GAP. See Good agricultural practices (GAP) Gastric damage, 551 Gastric secretion, 562 Gastrin, 559t Gastroenterology, olive in, 179 Gastrointestinal effects of OMW, 515
516 Gastrointestinal peptides, 564
565 adaptation of digestive function and, 563
565 digestive secretion in dogs, 557
559 digestive secretion in humans, 559
563 hormones, 559t GBS. See Genotyping by sequencing (GBS) Gene ontology (GO), 41
42 Genetic diversity, 28 Genetic resources, 28
30 Genome sequencing, 32 Genome-wide association study (GWAS), 32
35 Genomic instability, 540 Genotype 3 environment interaction (G 3 E interaction), 31 Genotyping by sequencing (GBS), 32
35 Geotrichum candidum, 152
153, 512 Geraniol synthase (GES), 40
41 Geraniol-10-hydroxylase (G10H), 40
41 Geranylgeranyl pyrophosphate synthase (GGPS), 40
41 GES. See Geraniol synthase (GES) GFAP. See Glial fibrillary acidic protein (GFAP)

GGPS. See Geranylgeranyl pyrophosphate synthase (GGPS) GGT. See γ-glutamyl transferase (GGT) GHP. See Good hygienic practices (GHP) GLA. See γ-linolenic acid (GLA) Glass, 294 Glial fibrillary acidic protein (GFAP), 675
676 GLT-1. See Glutamate transporter 1 (GLT-1) Glucagon-like peptide 1 (GLP-1), 627 Gluconobacter oxydans, 146 Glucose, 262 Glucose and TG tolerance test meal (GTTTM), 642 Glucose transporter 4 (GLUT4), 266, 627 Glucose uptake, 627 under insulin-resistant state, 628
630 Glucose-regulated protein (GRP78), 606
607 Glucuronosyltransferases, 194 Glutamate transporter 1 (GLT-1), 419
420 Glutamyl aminopeptidase activity, 439 Glutathione (GSH), 303, 350, 352f, 450, 583 agents acting on, 450 Glutathione peroxidase (GPx), 233, 302
303, 350
352, 598
599 enzyme families, 437 Glutathione-S-transferase, 437 Glycerol, 378 Glycogen synthase (GYS), 625
627 Glycosaminoglycans (GAGs), 401, 403 Glycoside, 140
141, 447 HPLC chromatograms of degradation, 141f Glycosylation, 489 GMP. See Good manufacturing processes (GMP) GO. See Gene ontology (GO) Good agricultural practices (GAP), 15 Good hygienic practices (GHP), 15 Good manufacturing processes (GMP), 15 GPR40, 564
565 GPR119, 564
565 GPR120, 564
565 GPRs. See G protein
coupled receptors (GPRs) GPx. See Glutathione peroxidase (GPx) Gramnegative bacteria, 149
151 Green olives, 5, 8
10 Castelvetrano-style green olives, 10 Picholine-style green olives, 10 Spanish-style green olives, 8
9 Green table olives, 103 Growing area, 53 GRP78. See Glucose-regulated protein (GRP78) GSH. See Glutathione (GSH) GSSG. See Oxidized glutathione (GSSG) GTTTM. See Glucose and TG tolerance test meal (GTTTM) GWAS. See Genome-wide association study (GWAS) Gynecology, olive in, 178 GYS. See Glycogen synthase (GYS)

H HACCP. See Hazard analysis and critical control point (HACCP) Hafnia/Rahnella alvei, 146
148 Hair growth, 428 Hazard analysis and critical control point (HACCP), 15 HB interactions. See Hydrogen bond interactions (HB interactions) HBA. See Hydroxybenzoic acids (HBA) HCC. See Human hepatocellular carcinoma (HCC) HCHF. See High carbohydrate high fat (HCHF) HD. See Huntington’s disease (HD) HDL. See High-density lipoprotein (HDL) HDL-c. See High-density lipoproteincholesterol (HDL-c) Health benefits of EVOO phenolic compounds, 695 health-beneficial effects of probiotics from fermented olives, 219
220 Healthy subjects, 627 Heat shock protein (HSP), 390
391, 662 Heat treatment, preservation of natural table olives with, 22 nonthermal pasteurization of natural table olives, 22 preservation of natural table olives by pasteurization, 22 Heating, partial dehydration of olives by, 21 Hedgehog signaling (HH signaling), 616 Helicobacter pylori, 330t, 332
333, 515
516 Hematology, olive in, 180 Hemeoxygenase-1 (HO-1), 541, 674
675 Hemorrhoids, olive oil for, 432 Hepatic lipid synthesis, 653
654 Hepatocyte growth factor (HGF), 682
683 Hepatocytes, 653
654 HER2, 339
340 HFD. See High-fat diet (HFD) 15-HETE. See 15-Hydroxyeicosatetraenoic acid (15-HETE) HGF. See Hepatocyte growth factor (HGF) HH signaling. See Hedgehog signaling (HH signaling) HHP. See High hydrostatic pressure (HHP) HID-AB. See High-iron diamine alcian blue (HID-AB) HIF-1α. See Hypoxia-inducible factor 1α (HIF1α) High carbohydrate high fat (HCHF), 278
281 High EVOO diet, 379 High hydrostatic pressure (HHP), 22 High phenolic (extra) virgin OO (HP(E)VOO), 208 High phenolic extract (HPE), 653f, 654 High-density lipoprotein (HDL), 168, 180, 196
197, 208, 262, 284
285 antioxidant content of, 200 High-density lipoprotein-cholesterol (HDL-c), 262 High-fat diet (HFD), 438, 628, 655 HFD-fed mice, 629

Index

High-iron diamine alcian blue (HID-AB), 531 High-lipid diet (HLD), 281 High-performance liquid chromatography (HPLC), 135, 138f, 317 of degradation of hydroxybenzoic acids, 140f High-polyphenol-content OO (HPCOO), 285 High-sensitivity Creactiv protein (hs-CRP), 198, 265, 394 High-throughput purification methods, 490
492 Highest occupied molecular orbital (HOMO), 93 Hit-to-lead validation of oleocanthal as c-MET inhibitor, 685
686 HIV. See Human immunodeficiency virus (HIV) HLA-DR2 antigen, 361
362 HLD. See High-lipid diet (HLD) HMG-CoA. See 3-Hydroxy-3-methyl glutarylCoA (HMG-CoA) HMGCR. See 3-Hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) 4-HNE. See 4-Hydroxynonenal (4-HNE) HO-1. See Hemeoxygenase-1 (HO-1) Hojiblanca, 494 HOMO. See Highest occupied molecular orbital (HOMO) Homovanillyl alcohol (HVAL), 194 Hormonereceptors, 339
340 Horny layer, 402 HP(E)VOO. See High phenolic (extra) virgin OO (HP(E)VOO) HPCOO. See High-polyphenol-content OO (HPCOO) HPE. See High phenolic extract (HPE) HPLC. See High-performance liquid chromatography (HPLC) 3-HPPA. See (3-Hydroxyphenyl) propionic acid (3-HPPA) hs-CRP. See High-sensitivity Creactiv protein (hs-CRP) HSP. See Heat shock protein (HSP) HT. See Hydroxytyrosol (HT) HT-29 cells, 527
530 maslinic acid effects on apoptosis in, 529f HT-Ac. See Hydroxytyrosyl acetate (HT-Ac) HT-ACDE. See Hydroxytyrosyl acyclodihydroelenolate (HT-ACDE) HT-Et. See Ethyl hydroxytyrosyl ether (HT-Et) HTX. See Hydroxytyrosol (HT) HtyAce. See Hydroxytyrosyl acetate (HT-Ac) HtyOle. See Hydroxytyrosyl oleate (HtyOle) HTyr. See Hydroxytyrosol (HT) Htyrosol, 494
495, 497 Human bioactive effects of olive oil phenolic compounds in lipids and lipoproteins, 196
197 oxidative damage, 197
198 intervention studies focusing on olive oil, 255t polyphenols in human health, 473 randomized controlled studies on effect of phenol-rich olive oil, 199t

safety properties of probiotics in, 219 Human hepatocellular carcinoma (HCC), 662
663 Human immunodeficiency virus (HIV), 391 HIV
associated disease, 394 Human studies BC and olive oil, 338
339 CRC and olive oil, 341 n vivo basic mechanisms assessed in, 200 increase in antioxidant content of lipoproteins, 200 nutrigenomic effect of virgin olive oil and phenolic compounds, 200 Humoral medicine, 175 Huntington’s disease (HD), 240 and oils as therapeutic agents, 243
246 protective mechanism by polyunsaturated fatty acids, 246 HVAL. See Homovanillyl alcohol (HVAL) Hybrid reactors, 512 Hydrocarbons, 314, 378 Hydrogen bond interactions (HB interactions), 685 Hydrogen peroxide (H2O2), 302
303, 437, 461
462 Hydrophilic phenolic compounds, 291 Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (OeHMBPPS), 39 3-Hydroxy-3-methyl glutaryl-CoA (HMGCoA), 652 3-Hydroxy-3-methyl-glutaryl CoA reductase (HMGCR), 267, 652 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (OeHMBPPR), 39 Hydroxybenzoic acids (HBA), 139, 447 Hydroxycinnamic acids, 135
138 8-Hydroxydeoxyguanosine (8-OHdG), 429 6-Hydroxydopamine (6-OHDA), 242
243 15-Hydroxyeicosatetraenoic acid (15-HETE), 403 Hydroxyl radical (HOG), 302
303, 437 3-Hydroxylase-encoding genes (CH3 genes), 32 Hydroxylation, 489 4-Hydroxynonenal (4-HNE), 278, 606 (3-Hydroxyphenyl) propionic acid (3-HPPA), 138 Hydroxytyrosol (HT), 15, 30
31, 106, 111
113, 116
117, 123, 134, 140, 193
194, 206
207, 231
232, 262
264, 266, 276, 278, 291, 315
316, 329
330, 332
334, 339
340, 377
378, 382t, 390, 415
416, 429
430, 438, 459
460, 473, 540, 547
548, 583
587, 593
595, 625, 651, 652f, 661 anti-inflammatory properties, 549 comparisons of olive oils with other edible oils, 544, 551 effects on autophagy, 542 on DNA damage/repair, 542 on epigenetic regulation, 542 on metabolic regulation, 540
541

709

on mitochondria dysfunction, 541
542 on molecular and cellular mechanisms, 540
542 on nonenzymatic antioxidant defense systems, 597 on oxidative stress, 541 on oxidative stress parameters, 596
597 on tumor growth, 595 endogenous sources of Tyr and, 196 improves AR42J antioxidant defenses, 584
585 in vitro studies, 549
550 in vivo studies, 550
551 occurrence, 548
549 properties of, 543f protects against cell death, 585
586 restores physiological Ca21 signaling and secretory pattern, 586
587 suppressive effect on NF-kB and cytokine release, 585 Hydroxytyrosyl acetate (HT-Ac), 547, 551, 632 Hydroxytyrosyl acyclodihydroelenolate (HTACDE), 508
509 Hydroxytyrosyl oleate (HtyOle), 548, 548f HyEDA. See Hydroxytyrosol (HT) Hypercholesterolemia, 169 Hypercholesterolemic rabbits, 167
168 Hyperglycemia, 264
265 Hyperinsulinemia, 564 Hypertension, 168, 208, 264 olive oil and, 264
265 Hypertrophic adipocytes, 266 Hypoxia-inducible factor 1α (HIF-1α), 497

I I-kappa-B-α (IkB-α), 392 IBD. See Inflammatory bowel disease (IBD) ICAM-1. See Intercellular adhesion molecule-1 (ICAM-1) IDE. See Insulin degrading enzyme (IDE) IFN-γ. See Interferon gamma (IFN-γ) iGEMDOCK software, 451 IGF-1. See Insulin-like growth factor (IGF-1) IGI. See Insulinogenic index (IGI) IHD. See Ischemic heart disease (IHD) IkB-α. See I-kappa-B-α (IkB-α) IKK complex. See IκBs kinase (IKK complex) ILs. See Interleukins (ILs) Immune responses, olive oil component effects on, 389
391 Immune-mediated inflammatory disease (IMID), 391
394 olive oil and, 391
394 allergy, 391
392 atherosclerosis, 393
394 autoimmunity, 392
393 cardiovascular disease, 393
394 HIV
associated disease, 394 Immunology, olive in, 180
181 Immunomodulatory effects of OMW, 515 In vitro studies on anticancer effect of oleuropein, 618t of HT, 549
550 in vitro model, synaptosomes as, 243

710

Index

In vivo basic mechanisms assessed in human studies, 200 In vivo effects of olive oil rich in biophenols in muscle redox regulation, 305 In vivo studies on anticancer effect of oleuropein, 618t of HT, 550
551 Indian turmeric, 589 Inducible nitric oxide synthase (iNOS), 390 Ineffective approaches, 509 Infectious diseases, olive in, 180 Infertility, 435, 439 Inflammation, 198, 264, 389
390, 402, 539
540, 549 olive oil and, 265
266 and redox imbalance, 169 Inflammatory bowel disease (IBD), 392
393 Inflammatory mediators secretion, 575
576 Inflammatory process, 167 Infrared thermography (IT), 80
81 detection of adulterated EVOO using IT, 81
82 Inhibitors of ACE (ACEi), 438
439 iNOS. See Inducible nitric oxide synthase (iNOS) Insulin, 625 Insulin-induced hypoglycemia mechanism, 625
627 sensitivity, 642
644 Insulin degrading enzyme (IDE), 481, 672 Insulin receptor (IR), 625
627 Insulin receptor substrate (IRS), 625
627, 645 Insulin resistance, 264, 625, 626t insulin-resistant state, 630
631 metabolites effects of oleuropein on insulin resistance, 631
632 oleuropein and olive on effects of olive leaf extract and oleuropein, 628 mitochondrial dysfunction under insulinresistant state, 630
631 promotion of glucose uptake under insulin-resistant state, 628
630 olive oil and, 266 Insulin-like growth factor (IGF-1), 538 Insulinogenic index (IGI), 642
644 Integrated techniques, 512 Inter-SSR (ISSR), 32
35 Intercellular adhesion molecule-1 (ICAM-1), 198 Interferon gamma (IFN-γ), 169 Interleukins (ILs), 169, 205, 497 IL-1β, 379
381, 390, 416 IL-6, 265, 390, 394, 416, 532
533, 662 IL-7, 390 IL-18, 390 International Olive Council (IOC), 5, 56, 153 International Olive Oil Council (IOOC), 28
30, 85, 102
103 Intracellular labile iron as mediator of oxidative stress
induced effects, 233 Intracellular protease activation, 582 IOC. See International Olive Council (IOC)

IOOC. See International Olive Oil Council (IOOC) IR. See Fruit-ripeness index (IR); Insulin receptor (IR) Iridoids, 446
447, 452 Iron chelators, 234 role in redox signaling, 234
235 IRS. See Insulin receptor substrate (IRS) Ischemic heart disease (IHD), 605 Isolated pancreatic acini, experiments in, 571
572 Isolated phenolic and oleosidic compounds effect, 333t Isolated synaptosomes, 244f ISSR. See Inter-SSR (ISSR) IT. See Infrared thermography (IT) Italian virgin olive oils, chemical composition of comparisons of olive oils with edible oils, 59 fatty acids, 53
56 implications for human health and disease prevention, 59 Italian olive groves surfaces, 52f Italian olive oil production, 53f phenolic compounds, 57
58 SQ, 57 sterols and triterpenic alcohols, 56
57 tocopherols, 58
59 Itrana, 494 IκBs kinase (IKK complex), 383
384

J JAK/STAT, 539 JNK. See c-Jun NH-2 terminal kinase (JNK)

K K232 (absorption of ultraviolet light at a wavelength of 232 nm), 87, 292
293 K270 (absorption of ultraviolet light at a wavelength of 270 nm), 292
293 Kalahari olives, 20 KatA. See Superoxide dismutase, catalase (KatA) Kinetic and molecular properties of PPO, 124
125 Klebsiella, 149
151 Klebsiella planticola, 146 Kluyvera intermedia, 146
148 Kluyveromyces spp., 152
153 K. marxianus, 148
149 K. thermotolerans, 152
153 Koroneiki, 494

L LA. See Linolenic acid (LA) LAB. See Lactic acid bacteria (LAB) Labile iron, 233 chelation of, 235 Labile iron pool (LIP), 460 Laboratory quality control of EVOO, 86
87 Lachancea sp., 152
153

Lactate dehydrogenase (LDH), 606 Lactic acid bacteria (LAB), 8
9, 142, 145
146, 215, 218
219, 221t Lactobacillus, 153, 219 L. acidophilus, 331 L. brevis, 216t, 218, 220 L. coryniformis, 216t, 218, 220 L. paracasei, 697 L. paracasei subsp. paracasei, 216t, 218 L. paraplantarum, 216t, 218, 220 L. pentosus, 8
9, 133, 216t, 218, 220, 333
334 L. plantarum, 8
9, 133
135, 146, 216t, 218, 220 metabolism of phenolic compounds by, 135
141 phenolic compounds and, 133
135 treatment of olive by-products by, 141
142 L. salivarius, 331 Lapatinib (LP), 683 Large-scale gene expression, 35 LAT52, 363 LBD. See Ligand-binding domain (LBD) LD. See Linkage disequilibrium (LD) LDH. See Lactate dehydrogenase (LDH) LDL. See Low-density lipoprotein (LDL) LDL-c. See Low-density lipoproteincholesterol (LDL-c) Lead (Pb), 462
463 Lens epithelium cells (LEC), 449
450 Lens fiber cells, 449
450 Lentinula edodes laccase, 512 Lepidium sativum, 696 Leucine-rich repeat kinase 2 (LRRK2), 243 Leuconostoc spp., 153 L. mesenteroides, 146, 216t, 218 Leukotriene B4 (LTB4), 408 Luteolin-7-glucoside, 379 LH. See Luteinizing hormone (LH) Life-expectancy, 401, 537 Lig v 1 (Ligustrum vulgare), 363 Ligand-binding domain (LBD), 664
665 Light exposure for EVOO storage, 293
294 Lignans, 291, 377
378, 426
427, 447 Lignins, 123, 447, 489 Ligstroside, 494, 496
497 Ligustroside, 15 Ligustrum japonicum, 146 Ligustrum vulgare.. See Lig v 1 (Ligustrum vulgare) Lime treatment, 509
510 Linkage disequilibrium (LD), 32
35 Linolenic acid (LA), 244, 377
378, 391, 402 LIP. See Labile iron pool (LIP) Lipases, 292 Lipids, 196
197 action mechanisms, 245f oxidation, 292 peroxidation, 596
597 synthesis in brain cells, 653 Lipopolysaccharide (LPS), 390, 662, 674
675 Lipoprotein receptor
related protein 1 (LRP1), 481, 672
673

Index

Lipoproteins, 196
197 antioxidant content of, 200 Lipoxygenase (LOX), 265, 303, 389
390, 402, 408 Liquid gold. See Olive oils (OO) Liquid
liquid extraction, 513 Listeria innocua, 330 Listeria monocytogenes, 220, 330t, 331, 334, 404 Liver analysis, 393
394 cancer, 380t steatosis, 654
655 Liver X receptors type α (LXRα), 246 LMP. See Lysosomal membrane permeabilization (LMP) Lol p 11, 363 Lolium perenne.. See Ryegrass (Lolium perenne) Long terminal repeat (LTR), 32 Long-term potentiation (LTP), 416 EVOO and, 420 Low phenolic (extra) virgin OO (LP(E)VOO), 208 Low phenolic extract (LPE), 654 Low-density lipoprotein (LDL), 53, 116, 165, 196
197, 262, 277
278, 314
315, 514, 631 antioxidant content of, 200 Low-density lipoprotein-cholesterol (LDL-c), 262 Low-polyphenol-content OO (LPCOO), 285 Lowest unoccupied molecular orbital (LUMO), 93 LOX. See Lipoxygenase (LOX) LP. See Lapatinib (LP) LP(E)VOO. See Low phenolic (extra) virgin OO (LP(E)VOO) LPCOO. See Low-polyphenol-content OO (LPCOO) LPE. See Low phenolic extract (LPE) LPS. See Lipopolysaccharide (LPS) LRP1. See Lipoprotein receptor
related protein 1 (LRP1) LRRK2. See Leucine-rich repeat kinase 2 (LRRK2) LTB4. See Leukotriene B4 (LTB4) LTP. See Long-term potentiation (LTP) LTR. See Long terminal repeat (LTR) LUMO. See Lowest unoccupied molecular orbital (LUMO) Lung system, olive in, 179 Lupeol synthase (LUS), 32
35, 40
41 LUPS. See Lupeol synthase (LUS) Lutein, 318 Luteinizing hormone (LH), 436 Luteolin, 276, 315, 315f, 382t Luteolin-7-glucoside. See Flavones Luteolin 7-O-glucoside, 67, 135 LXRα. See Liver X receptors type α (LXRα) Lycopersicon esculentum, 363 Lysosomal membrane permeabilization (LMP), 662
663

M Macaronesia, 28 Macrophage inflammatory protein-1α (MIP1α), 662 Major “complex” N-glycan (GlcNAcMan3XylGlcNAc2), 367 Malaxation temperature (MT), 549 Male fertility comparisons of olive oils with other edible oils, 439
440 diet and, 435
436 dietary lipid and, 436
437 implications for human health and disease prevention, 439 Mediterranean diet, olive oil, and, 437
438 oxidative stress and, 437 Malondialdehyde (MDA), 210, 350, 393
394, 596
597 Malus domestica.. See Pollens and apple (Malus domestica) MAM. See Methylazoxymethanol (MAM) Mammal cells, 240 Mammalian cellular and subcellular membranes, 569
570 Mammalian target of rapamycin (mTOR), 538, 610, 663, 684
685 HT effects on, 541 Man7GlcNAc2. See One major “high mannose” N-glycan (Man7GlcNAc2) Manzanilla olives, 103
105 Manzanillo, 494 MAO-A. See Monoamine oxidase-A (MAO-A) MAPK. See Mitogen-activated protein kinase (MAPK) Marker-assisted selection (MAS), 32
35 Maslinic acid (MA), 390, 448, 526 cancer chemopreventive activity in animal models in vivo, 530
533 in colon cancer cells in vitro, 527
530 chemical structure, 526f content in O. europaea L., 527t effects on apoptosis in HT-29 cells, 529f implications for human health and disease prevention, 533
534 synthesis in O. europaea L., 526f Matrix metalloproteinases (MMPs), 427, 481, 616 MMP-1, 392 MMP-2, 406 MMP-3, 392 MMP9, 205, 482 Maurino, 494 MBD. See Multifunctional docking/binding domain (MBD) MCI. See Mild cognitive impairment (MCI) MCP-1. See Monocyte chemoattractant protein1 (MCP-1) MD. See Mediterranean diet (MD) MDA. See Malondialdehyde (MDA) MDF. See Mucin depleted foci (MDF) MDP. See Mediterranean dietary pattern (MDP) Mechanisms of Development of Allergy (MEDALL), 369

711

MED. See Mediterranean diet (MD) Med Diet. See Mediterranean diet (MD) MEDALL. See Mechanisms of Development of Allergy (MEDALL) MedDiet. See Mediterranean diet (MD) Mediterranean basin, 28 Mediterranean diet (MD), 111, 167, 198, 205
206, 215, 231, 262, 338, 341, 379, 389, 392
394, 415
416, 457, 460, 473, 479, 525, 538, 569, 583
584 and acute pancreatitis, 583
587 EVOO, 206
211 and olive oil and male fertility, 437
438 pattern, 261 pyramid shape, 206 sustainability, 206 Mediterranean dietary pattern (MDP), 205 Mediterranean-style diets (MSD), 251, 254
257 food intake, 254
257 weight/fat loss, 257 Medium-chain fatty acids, 252
253 Melanoma skin cancer, 616, 619
620 Membrane fluidity, 244, 245f technology, 513
514 Memory, 416 MEP. See 2-C-methyl-D-erythritol 4-phosphate (MEP) Messenger ribonucleic acid (mRNA), 42, 281 Metaanalysis, 465
466 Metabolic cataract, 449 Metabolic pathways to hepatic steatosis, 655f Metabolic regulation, hydroxytyrosol effects on, 540
541 Metabolic syndrome (MS), 205, 261, 262f implications for human health with special reference to metabolic health, 263
264 olive oil and, 262
267 Metabolites effects of oleuropein on insulin resistance, 631
632 Metal
flavonoid complexes, 461 Metalloproteinase, 210 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 242
243 Methylation, 489 Methylazoxymethanol (MAM), 530
531 4-Methylcatechol, 124
125 Methylobacterium spp., 146
148 MetS. See Metabolic syndrome (MS) Metschnikowia pulcherrima, 218 MF. See Microfiltration (MF) MHBO. See Mixture of honey, beeswax, and olive oil (MHBO) Micro-RNAs (miRNAs), 42 Microbial association biochemical characteristics of, 151
156 dry-salted olives, 154
156 effect on fermentation, 153
154 effect on olive oil, 151
153 during dry salting of black olives, 155t Microbial ecology, 149
151 Microbiota of olives, 145
156

712

Index

Microbiota of olives (Continued) biochemical characteristics of microbial association, 151
156 mean values of microorganisms, 146t microbial diversity of raw olives, 146
148, 148t, 149t, 150t related to fermentation, 149
151 related to olive oil production, 148
149 yeasts isolated from olive fruits, 147t Micrococcus spp., 146 M. caseolyticus, 152
153 M. luteus, 146, 220 Microfiltration (MF), 513
514 Micronutrients, 437
438 of VOO, 390 Microsporum canis, 431 Microvessel density (MVD), 685 Microwaves, partial dehydration of olives using, 21 Mild cognitive impairment (MCI), 479
480 Milk, 436 MIP-1α. See Macrophage inflammatory protein-1α (MIP-1α) miRNAs. See Micro-RNAs (miRNAs) Mission, 494 Mitochondrial dysfunction and acute pancreatitis, 582 hydroxytyrosol effects on, 541
542 under insulin-resistant state, 630
631 Mitochondrial function impairment, 538 Mitogen-activated protein kinase (MAPK), 390, 497, 539, 616, 665, 674
675, 682
683 Mixture of honey, beeswax, and olive oil (MHBO), 406 MMPs. See Matrix metalloproteinases (MMPs) Molecular chaperones, 538
539 Molecular characterization, 32
35 Molecular effects of oleocanthal mediated via ER targeting, 666
667 Molecular markers, 32
35 Molecular mechanism on aging, 538
540 behind cataract formation, 449
450 calpain activation, 450 nonenzymatic glycation, 450 oxidative stress, 449
450 polyol pathway, 450 hydroxytyrosol and olive oil effects on, 540
542 of O. europaea in cataract treatment, 450
454 Monoamine oxidase-A (MAO-A), 606 Monocyte chemoattractant protein-1 (MCP-1), 205, 645
646 Monocyte chemotactic protein 1. See Monocyte chemoattractant protein-1 (MCP-1) Monosodium glutamate (MSG), 103 Monounsaturated fatty acids (MUFAs), 30, 53, 92, 103, 167
168, 193, 205, 239
240, 251, 262, 276, 294, 313, 347
348, 390, 392, 404, 428
429, 437
438, 446
447, 465
466, 561, 569, 639, 661 Monovarietal EVOOs, 548
549

Moraiolo, 494 MPO. See Myeloperoxidase (MPO) MPTP. See 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) mRNA. See Messenger ribonucleic acid (mRNA) MS. See Metabolic syndrome (MS) MSD. See Mediterranean-style diets (MSD) MSG. See Monosodium glutamate (MSG) MT. See Malaxation temperature (MT) mTOR. See Mammalian target of rapamycin (mTOR) mTOR complex 1 (mTORC1 and mTORC2), 610 Mucin depleted foci (MDF), 530
531, 532f MUFAs. See Monounsaturated fatty acids (MUFAs) Multifunctional docking/binding domain (MBD), 682
683 MVD. See Microvessel density (MVD) MYC oncogene, 594
595 Myeloperoxidase (MPO), 550
551 Myoblasts, 302 Myocardial infarction, 165 Myocardial ischemia
reperfusion injury, 607
609 oleuropein’s cardioprotective effect against, 607
609 oleuropein’s effects and mechanisms in cardioprotective effect, 608t Myofibers, 302

N n-3 polyunsaturated fatty acids (n-3 PUFAs), 339, 563, 584 n-6 polyunsaturated fatty acids (n-6 PUFAs), 347
348 N-acetylcysteine derivative, 195
196 N-amino-terminal domain (NTD), 664
665 N-ethyl-N-nitrosourea (ENU), 593
594 N-glycans of Ole e 1, 367f role in olive pollen allergy, 367 N-methyl-nitrosourea (NMU), 347
348 morphological analysis of NMU-induced mammary tumors, 349f parameters of carcinogenesis, 348t N-terminal domain (NtD), 365 NACHT, LRR, and PYD domain-containing protein 3 (NLRP3), 675
676 NADPH oxidase. See Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) NAFL. See Nonalcoholic fatty liver (NAFL) NAFLD. See Nonalcoholic fatty liver disease (NAFLD) Nanoherbal andaliman (Zanthoxylum acanthopodium), 391 NASH. See Nonalcoholic steatohepatitis (NASH) Natural antioxidants, 117
118, 301
302, 314
319 Natural fermentation process, 215 Natural green

olives, 99 Sicilian table olives, 20 Natural oils, 391 Natural olives, 8, 10
11 Natural products, 445, 489 as potential therapeutic agents in FAB-Lab, 446t Natural table olives, 15
16 bulk preservation of, 22 in consumer packs, 22 processing, 16
21, 18t by archaic method, 16
18 by fermentation, 19
20 olive cultivars used in, 17t by partially dehydration, 20
21 secondary processing of, 21 Natural vehicles for pollen allergens, 367 Naturally green olive, 99 Naturally processed table olives, 15
16 nutritional and health-related aspects, 22
23 preservation and storage methods for, 21
22 processing, 16
21 secondary processing of natural table olives, 21 NCD. See Noncommunicable disease (NCD) ncRNAs. See Noncoding RNA (ncRNAs) Near-infrared spectroscopy (NIR spectroscopy), 92, 94 Neofusicoccum spp., 146
148 NEP. See Neutral endopeptidase (NEP) Neprilysin (NEP), 481 NER. See Nucleotide excision repair (NER) Nerium oleander, 146 Nerve growth factor (NGF), 474 Network meta-analysis study, 262 Neurodegeneration, 243 Neurodegenerative diseases, 241 experimental models to study, 242
243 synaptosomes as in vitro model, 243 Neurofibrillary tangles (NFTs), 672 Neuroinflammation, 674
676 EVOO on and, 418
419 Neurology, olive in, 177
178 Neuroprotective effects of oleocanthal in neurological disorders, 676f enhancing BBB integrity and function, 673
674 inducing brain amyloid-β clearance, 672
673 reducing neuroinflammation and oxidative stress, 674
676 Neutral endopeptidase (NEP), 264 Next-generation sequencing techniques (NGS techniques), 32 NF-kB. See Nuclear factor kappa B (NF-kB) NFE2L2. See Nuclear factor (erythroid-derived 2)-like 2-related factor (NFE2L2) NFTs. See Neurofibrillary tangles (NFTs) NG-nitro-L-arginine-methyl ester, 71
73 NGF. See Nerve growth factor (NGF) NGS techniques. See Next-generation sequencing techniques (NGS techniques)

Index

NHBE cells. See Normal human bronchial epithelial cells (NHBE cells) Nicastrin, 480 Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), 303, 389
390 NIR spectroscopy. See Near-infrared spectroscopy (NIR spectroscopy) Nitric oxide (NO), 198
199, 208, 264, 302
303, 407 synthesis, 426 Nitric oxide/ET-1 (NO/ET-1), 264
265 Nitrogen, 294 Nitrogen oxide (NOx), 437 3-Nitropropionic acid (3-NP), 243 NLRP3. See NACHT, LRR, and PYD domaincontaining protein 3 (NLRP3) NMSC. See Nonmelanoma skin cancer (NMSC) NMU. See N-methyl-nitrosourea (NMU) NO. See Nitric oxide (NO) No-observed-adverse-effect level (NOAEL), 458 NO/ET-1. See Nitric oxide/ET-1 (NO/ET-1) NOAEL. See No-observed-adverse-effect level (NOAEL) Nocellara, 494 Nonalcoholic fatty liver (NAFL), 654 Nonalcoholic fatty liver disease (NAFLD), 625, 654 Nonalcoholic steatohepatitis (NASH), 654 Nonalkali-treated green olives, 134
135 Nonautochthonous probiotics application, 222 Noncoding RNA (ncRNAs), 683 Noncommunicable disease (NCD), 65, 205 Nonenzymatic antioxidant defense systems, effects of OLEU and HTX on, 597 Nonenzymatic glycation and cataract formation, 450 Nonenzyme antioxidant defense systems, 350 Nonmelanoma skin cancer (NMSC), 615
617, 620
621 Non
small cell lung cancer (NSCLC), 683 Nonspecific lipid-transfer protein. See Ole e 7 Nonspecific lipidtransfer protein (nsLTP), 364
365 Nonsteroidal anti-inflammatory drugs (NSAID), 450, 549, 671 Nonthermal pasteurization of natural table olives, 22 Normal human bronchial epithelial cells (NHBE cells), 363 Normolipidic dietary fat, 348 3-NP. See 3-Nitropropionic acid (3-NP) NRF2. See Nuclear factor (erythroid-derived 2)-like 2-related factor (NFE2L2) NSAID. See Nonsteroidal anti-inflammatory drugs (NSAID) NSCLC. See Non
small cell lung cancer (NSCLC) nsLTP. See Nonspecific lipidtransfer protein (nsLTP) NTD. See N-amino-terminal domain (NTD) NtD. See N-terminal domain (NtD)

Nuclear factor (erythroid-derived 2)-like 2related factor (NFE2L2), 302
304, 390, 674
675 Nuclear factor kappa B (NF-kB), 205, 265
266, 302
303, 377
378, 383
384, 515, 582, 617 activation, 583 mechanism of regulation, 383f Nuclear factor kappa-light-chain-enhancer regulation of activated B cells activation, 383
384 Nuclear transcription factor kappa-light-chainenhancer of activated B cells (NF-kB), 390 Nucleotide excision repair (NER), 540 Nurses’ Health Study, 262 Nutrient partitioning, 251 Nutrigenomic effect of virgin olive oil and phenolic compounds, 200 Nutritional and health-related aspects of table olives, 22
23 Nutritional stress, 263

O OA. See Occupational asthma (OA); Oleic acid (OA) OAESO. See Oleic acid-enriched SO (OAESO) OAS. See Oral allergy syndrome (OAS) Obese subjects, 628 Obesity, 207
208, 251 OBP. See Olive biophenols (OBP) Obstetrics, olive in, 178 Occupational asthma (OA), 371, 405 OeCDPMES. See 2-C-methyl-D-erythritol 4phosphate cytidyltransferase (OeCDPMES) OeDXR. See 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (OeDXR) OeHMBPPR. See 4-Hydroxy-3-methylbut-2enyl diphosphate reductase (OeHMBPPR) OeHMBPPS. See Hydroxy-2-methyl-2-(E)butenyl 4-diphosphate synthase (OeHMBPPS) OeMECPS. See 2-C-methyl-D-erythritol 2,4cyclo-PP synthase (OeMECPS) Ogliarda, 494 Ogliarola, 494 OGTT. See Oral glucose tolerance test (OGTT) 6-OHDA. See 6-Hydroxydopamine (6-OHDA) 8-OHdG. See 8-Hydroxydeoxyguanosine (8OHdG) OHTyr. See Hydroxytyrosol (HT) Oil extraction process, 58 Oil hydrolysis, 151
152 Oil mill wastewater (OMW), 693, 694f OL. See Olive leaf (OL) OLE. See Oleuropein (OLE); Oleuropeosides (OLE) Ole e 1, 361 concepts for specific immunotherapy, 369
371 as marker for sensitization to Oleaceae pollens, 362
363

713

N-glycans of, 367f Ole e 2, 361, 363
364 Ole e 3, 364 Ole e 4, 365
366 Ole e 5, 365
366 Ole e 6, 361, 365
366 Ole e 7, 361, 364
365 Ole e 8, 364 Ole e 9, 361, 365 Ole e 10, 361, 363
364 Ole e 11, 366
367 Ole e 12, 366
367 Ole e 14, 366
367 Ole e 15, 361, 366
367 Olea europaea L., 15, 111
113, 179 Olea genus, 28 Oleaceae, 111
113 pathway of synthesis of oleuropein in, 112f Oleacein, 30
31 Oleacin, 167, 291 Oleanolic acid, 65, 390, 448 Oleaster, 28 Oleate-rich LDL particles, 263 Oleic acid (OA), 92, 251
252, 257, 262, 377
378, 390, 558, 584, 639, 640t, 651 acting on postprandial glucose homeostasis, 644
645 on postprandial fibrinolysis, 641
642 on postprandial inflammation, 645
646 on postprandial thrombogenesis, 641 on postprandial β-cell function and insulin sensitivity, 642
644 structure and properties of, 640t Oleic acid-enriched SO (OAESO), 347
348 Oleocanthal, 30
31, 265, 276, 291
292, 314
315, 382t, 415
416, 427, 497, 661
663 anticancer activities of, 664t bioisostere c-MET inhibitors, 686
687 biological activity of, 662
663 as c-MET inhibitor, 685
686 chemical structure of, 661
662 enhancing BBB integrity and function, 673
674 inducing brain amyloid-β clearance, 672
673 inhibiting tau fibrillization, 676 modulation of ER gene expression, 666 molecular effects, mediated via ER targeting, 666
667 impact of oleocanthal on ER, 665
667 novel first-in-class breast cancer recurrence inhibitor, 688
689 pharmacokinetics of, 662 reducing neuroinflammation and oxidative stress, 674
676 with targeted therapies and estrogen modulators, 687
688 S-(
)-Oleocanthal c-MET as potential molecular target in oncology, 682
684 EVOO phenolic S-(2)-oleocanthal biological activities, 684
685

714

Index

S-(
)-Oleocanthal (Continued) hit-to-lead validation of oleocanthal as cMET inhibitor, 685
686 oleocanthal novel first-in-class breast cancer recurrence inhibitor, 688
689 with targeted therapies and estrogen modulators, 687
688 RTKs, 682 structure
activity relationship study, 686
687 Oleoside, 276, 496
497 OLEU. See Oleuropein (OLE) OLEUM project consortium, 494 Oleuricine A, 276 Oleuricine B, 276 Oleuropein (OLE), 5, 7
9, 15, 74, 116, 123, 124t, 127f, 134, 140
141, 167
168, 180, 264, 266, 276, 304
305, 314
315, 329
330, 377
378, 382t, 415
416, 427, 448, 452, 452f, 457
459, 482
484, 494
497, 508
509, 593
595, 615, 652f activated phosphorylation of AMPK, 632f aglycone, 135 beneficial properties, 617
621 on cancer-associated mechanisms, 617
619 and melanoma skin cancer, 619
620 and nonmelanoma skin cancer, 620
621 cardioprotective effect against IRI, 607
609 concentration in fruit and leaf of olive during ripening, 127 as conditioning mimetic, 609 derivatives, 30
31 effects on cardiomyocytes, 606
607 in ex vivo models, 607
609 in in vivo models, 609 and mechanisms in cardioprotective effect, 608t on nonenzymatic antioxidant defense systems, 597 on oxidative stress parameters, 596
597 on tumor growth, 595 enhanced glucose uptake into C2C12 myotube cells, 630f enhanced GLUT4 translocation in plasma membrane, 631f in vitro studies on anticancer effect, 618t in vivo studies on anticancer effect, 618t molecular understanding of protective role, 610 oleuropein-induced GLUT4 translocation, 632f and olive on insulin resistance, 628
631 role in other cardiovascular disorders, 610
611 suppressed intracellular lipid accumulation, 629f Oleuropein aglycon (3,4-DHPEA-EA), 73, 116
118, 291
292, 304
305 Oleuropeosides (OLE), 276, 457
458 Oleuroside, 452f, 494

OLf. See Olive leaf (OL) Olive (Olea europaea L. ), 5, 28
30, 36t, 65, 133, 146, 240, 275, 301, 359, 377
378, 425, 445, 457, 489
490, 617 by-products treatment by L. plantarum, 141
142 cardioprotective effects, 277 chemical structures of compounds, 279f, 280f, 281f cultivars, 493 cultivation, 51 darkened by oxidation, 8, 11
12 effects of olive leaf phenolics on cardiovascular risk markers, 282f in endodontics, 181 ethnobotanical uses, 276
285 extracts of bioactive compounds, 304
305 fruits, 57, 371 genetics CREA-OFA olive germplasm, 29f olive genomics as tool for olive oil quality, 32
43 origin, diffusion, and genetic resources, 28
30 phenotypic variability and breeding programs for olive oil quality, 30
31 worldwide olive germplasm, 29f genomics as tool for olive oil quality genome sequencing, 32 molecular characterization, quantitative trait loci analysis, 32
35 small nuclear RNA, 42
43 transcriptomics for olive oil quality, 35
42 for human health and disease prevention in TPM, 176
177 on insulin resistance, 628
631 kernel oil, 177 maslinic acid content, 527t synthesis, 526f in medicine based on TPM in cardiology, 180 in dermatology, 177 in endocrinology, 179
180 in gastroenterology, 179 in hematology and oncology, 180 in immunology and allergy, 180
181 in infectious diseases, 180 in lung and respiratory system, 179 in neurology and psychiatry, 177
178 in obstetrics and gynecology, 178 in ophthalmology, 178 in poisonings, 181 in rheumatology, rehabilitative medicine, and sports medicine, 178
179 in urinary and reproductive system, 178 molecular mechanism in cataract treatment, 450
454 in oral medicine, 181
182 in orthodontics, 182 in periodontics, 181 pharmacology, 276
277 phenols, 123, 262

phytochemistry, 276 polyphenols, 473
474 in preventive and restorative dentistry, 181 probiotics application in fermentation, 221
222 in prosthodontics, 182 in Rhazes, 176f status in Iran and worldwide statistics, 492
497 olive databases, 494, 496t olive phenolic metabolites, 494
497 styles and suggested olive cultivars, 19t temperament, 175 in TPM olive oil with edible oils, 175
176 olive temperament, 175 traditional uses, 277t Olive biophenols (OBP), 505, 508, 514 Olive fly (Bactrocera oleae), 41
42, 148
149 Olive leaf (OL), 65, 66f, 457
458 antioxidant effects, 458
460 chelation therapy and, 462
464 chemistry of, 446
448 binding energy of chemical constituents, 453t binding energy of top-ranked compounds, 451t, 452t chemical structure of phytochemicals, 447f lignans in, 447 polyphenolic compounds, 447 secoiridoids in, 447 triterpenes in, 448 comparisons of olive oils with other edible oils, 464
465 effects on DNA damage, 460
462 implications for human health and disease prevention, 465
466 pharmacology, 448
449 anticancer activity, 448 antidiabetic activity, 448 antihypertensive and cardioprotective activity, 448 antiinflammatory and antinociceptive activities, 448 antimicrobial activity, 448 antioxidant activity, 449 Olive leaf extract (OLLE), 278, 283, 329, 379
381, 394, 445, 448, 457
458, 465
466 aglycone, 283 cardioprotective effect of, 283 and oleuropein, 628 Olive mill wastewater (OMW), 141
142, 505 biological based technologies for treatment, 511t characteristics, 508t developments in treatment and valorization, 509
514 exploitation of OMW potentials, 514
516 antiinflammatory activity, 515 antimicrobial effects, 514
515 antioxidant activity, 514 cardiovascular effects, 515

Index

chemo-preventive effects, 516 endocrine effects, 516 gastrointestinal effects, 515
516 immunomodulatory effects, 515 respiratory effects, 516 nonbiological based technologies for treatment, 510t olive oil production processes, 505
507, 507t output of olive mills, 506f phenolic compounds recovery of, 512
514 removal of, 509
512 production and total phenolic content, 509f source and physical properties and chemical composition, 507
509 Olive oil biophenols (OOBPs), 497
498, 498f cosmetic and food supplementary commodities, 499f pharmacological functionalities, 497
499 Olive oil phenolic compounds (OOPC), 193
194 Olive oil phenolics (OOPs), 498
500 Olive oil
contained compounds modulate redox signaling, 235 prevent H2O2-induced DNA damage, 233
234 Olive oils (OO), 80, 167, 193, 205, 240
241, 251
254, 262, 267, 275, 292, 301, 313
314, 329, 382t, 389, 404, 425, 547
548, 639. See also Extra virgin olive oil (EVOO); Virgin olive oil (VOO) active ingredients, 426t antimicrobial compounds, 332f cardioprotective effects, 277 case studies, 169
170 chemical structures of compounds, 279f, 280f, 281f clinical studies, 284
285 in combination anal fissure and hemorrhoids, 432 atopic dermatitis and psoriasis, 431 fungal and bacterial infections, 431
432 comparisons with other edible oils, 307 composition, 167
168 constituents, 426 cutaneous indications, 425t diseases regulated by consumption, 320f with edible oils, 175
176 effect on CVD risk factors, 168
169 on molecular and cellular mechanisms, 540
542 free radical scavengers protect cells in conditions of OS, 232
233 hair growth, 428 for human health and disease prevention, 86 human intervention studies, 255t intracellular labile iron as mediator of OS
induced effects, 233 in vitro investigations and cardioprotective mechanisms, 277
278 microbial association effect, 151
153

microbiota of olives related to production, 148
149 oleocanthal, 427 oleuropein, 427 olive oil
contained compounds modulate redox signaling, 235 prevent H2O2-induced DNA damage, 233
234 oxidative stress, 232 pharmacology, 276
277 phenol compounds, 263 phenolics, 314
315 phenols on liver steatosis and steatohepatitis, 654
655 polyphenolic constituents, 426t polyphenols, 116
117, 198, 473 preferential effect in abdominal obesity, 252 production, 275
276 properties, 426
427 protection against ultraviolet damage, 427 quality olive genomics as tool, 32
43 phenotypic variability and breeding programs, 30
31 randomized crossover studies, 253t role of iron in redox signaling, 234
235 secoiridoids, 111
113 squalene, 177 wound healing, 427
428 Olive OMW, 693, 693t, 694t management, 695
698 phenolic compound isolation, 696
697 phenolic compound removal, 697
698 treatment, 696 Olive pollen allergens allergogram, 360
361, 360f features, 361, 362t intranasal pretreatment with tolerogenic exosomes, 370f Ole e 1 concepts for specific immunotherapy, 369
371 as marker for sensitization to Oleaceae pollens, 362
363 Ole e 2, 363
364 Ole e 3, 364 Ole e 4, 365
366 Ole e 5, 365
366 Ole e 6, 365
366 Ole e 7, 364
365 Ole e 8, 364 Ole e 9, 365 Ole e 10, 363
364 Ole e 11, 366
367 Ole e 12, 366
367 Ole e 14, 366
367 Ole e 15, 366
367 olive fruit, 371 pollen
latex
fruit syndrome, 365 pollensomes, 367
368, 368f recombinant, 368
369 Olive pollen allergy, 361
362 N-glycans role in, 367 Olive pomace oil (OPO), 79, 506
507

715

Olive ripening changes during ripening, 125
127 effects of variety of cultivar, 127
128 kinetic and molecular properties of PPO, 124
125 oleuropein concentration in fruit and leaf of olive during ripening, 127 phenols, types, biological significance, and presence in olive, 123 ripening, polyphenol oxidase, structure, and biological properties, 123
124 Olive skins (alambrado), 9 Olive tree. See Olive (Olea europaea L. ) OLLE. See Olive leaf extract (OLLE) ω-3 fatty acids, 240 Omega-3 monounsaturated fats, 428t Omega-3 PUFAs, 407 OMW. See Oil mill wastewater (OMW); Olive mill wastewater (OMW) Oncology, olive in, 180 One major “high mannose” N-glycan (Man7GlcNAc2), 367 OO. See Olive oils (OO) OOBPs. See Olive oil biophenols (OOBPs) OOPC. See Olive oil phenolic compounds (OOPC) OOPs. See Olive oil phenolics (OOPs) OPG. See Osteoprotegerin (OPG) Ophthalmology, olive in, 178 OPO. See Olive pomace oil (OPO) Oral allergy syndrome (OAS), 364 Oral glucose tolerance test (OGTT), 628, 642 Oral medicine, olive in, 181
182 Organic acids, 145 Organoleptic characteristics, 91 Orthodontics, olive in, 182 Oryza sativa, 363 OS. See Oxidative stress (OS) OSPG, 363 Osteoprotegerin (OPG), 646 Oxidative damage, 197
198 endothelial function, blood pressure, and thrombosis, 198
200 inflammation, 198 postprandial effects, 197 sustained consumption effects, 197
198 Oxidative degradation, 313 Oxidative stability of VOO, 117
118 Oxidative stress (OS), 111, 231
232, 264, 266, 302
303, 350, 389
390, 402, 435, 515, 583, 674
676 and cataract formation, 449
450 effects of OLEU and HTX on OS parameters, 596
597 free radical scavengers protect cells in conditions of, 232
233 hydroxytyrosol effects on, 541 male fertility and, 437 monovalent oxygen reduction, 232f olive oil and, 263
264 OS
induced effects, 233 OS
mediated endothelial dysfunction and atherosclerosis, 169 Oxidative
reductive stress, 303
304

716

Index

Oxidative
reductive stress (Continued) biological roles of ROS/RNS, 305f Oxidized glutathione (GSSG), 350, 352f Oxidized LDL (OxLDL), 167 Oxidized LDL cholesterol (oxLDL-C), 208 Oxidized macromolecules, 515 OxS. See Oxidative stress (OS) Oxygen exposure for EVOO storage, 293
294 Ozonated olive oil, 181
182

P p-coumaric acid, 135 3
3-(6v-p-coumarylglucoside), 452 p-ERK. See Phosphorylated-extracellularregulated MAP kinase (p-ERK) P-glycoprotein (P-gp), 481, 672
673 P-gp. See P-glycoprotein (P-gp) p-Hsp27, 278 p-hydroxyphenylethanol-elenolic acid dialdehyde (p-HPEA-EDA), 291
292, 294, 661
662 PA. See Palmitic acid (PA) Packed product, Spanish-style green olives, 102
105 composition of relevant amino acids, polyphenols, fatty acids, 104t range and mean values of main physicochemical characteristics, 102t Packed product, untreated green olives in brine, 105
106 PAD. See Phenolic acid decarboxylase (PAD) PAGE. See Polyacrylamide gel electrophoresis (PAGE) PAI-1. See Plasminogen activator inhibitor-1 (PAI-1) PAL. See Phenylalanine ammonia lyase (PAL) Palmitic acid (PA), 377
378, 639
640 Palmitoleic acid, 377
378 Pan-allergens. See Ole e 1 Pancreas, 563
564 Pancreatic acinar cells, 582 Pancreatic secretion in anesthetized rats, 570
571 AR42J cell model of acute pancreatitis, 574
577 AR42J studies, 572
573 comparisons of olive oils with other edible oils, 578
579 dietary lipids and, 570 experiments in isolated pancreatic acini, 571
572 implications for human health and disease prevention, 579 Pantoea spp., 146
148 Paraoxonase-2 (PON2), 283 Parietaria judaica pollen 1 (Parj 1), 391 Parj 1. See Parietaria judaica pollen 1 (Parj 1) Parkin, 243 Parkinson’s disease (PD), 177
178, 241 PARP. See Poly(ADP-ribose)polymerase (PARP) Partially dehydration, natural table olive processing by, 20
21 of olives by heating, 21

of olives on tree, 20 of olives using dry salt, 21 of olives using microwaves, 21 Pasteurization, 22 Paternal exposure, 472 Pathogenesis-related 14 protein family (PR-14 protein family), 364
365 Pathogenic microorganisms, 330 antimicrobial compounds in olive oil, 330
333 in table olives, 333
334 effect of isolated phenolic and oleosidic compounds, 333t PBMCs. See Peripheral blood mononuclear cells (PBMCs) PC. See Phenolic compounds (PC) PD. See Parkinson’s disease (PD) PDB. See Protein database (PDB) PDO. See Protected designation of origin (PDO) PE. See Phenolic extract (PE); Preeclampsia (PE) Peanut, 314 Pectic substances, 100 Pectin-methylesterase (PME), 366 Pediculus humanus capitis, 177 Pediococcus spp., 153 PEN2. See Presenilin enhancer 2 (PEN2) Penicillium, 152
153 Pentacyclic triterpenes, 525 Peonidin 3-3-(6v-p-coumarylglucoside), 452, 452f PEOP, 304 Peptide YY (PYY), 252
253, 558, 559t, 561f, 564, 571 Periodontics, olive in, 181 Peripheral blood mononuclear cells (PBMCs), 371, 390 Peroxidases (PRX), 39
40, 450 Peroxides, 404 Peroxiredoxin (Prx), 233 Peroxisome proliferator
activated receptors (PPARs), 197, 244, 450 PPAR-α, 451 PPARγ, 178, 341
342, 672
673 Persistent inflammation, 539
540 Pestalotiopsis, 330 PF. See Phenolic fraction (PF) PGC-1α. See PPAR gamma coactivator 1-alpha (PGC-1α) PGE2. See Prostaglandin E2 (PGE2) PGI. See Protected geographical indication (PGI) PHA. See Phytohemagglutinin (PHA) Phalaris coerulescens, 363 Pharmacokinetics of oleocanthal, 662 Pharmacologic cataract, 449 Phenolic acid decarboxylase (PAD), 136
137, 139f Phenolic acids, 135
140, 137f, 291, 314
315, 377
378, 460 hydroxybenzoic acids, 139 hydroxycinnamic acids, 135
138 phenolic-related acids, 139
140

Phenolic components, 379 Phenolic compounds (PC), 57
58, 100, 133
135, 145, 200, 291, 313
316, 426, 490, 505, 547, 551, 615 extraction and purity of, 490
492 isolation, 696
697 and L. plantarum, 133
135 metabolism by L. plantarum, 135
141, 136t flavonols, 141 glycosides, 140
141 phenolic acids, 135
140 phenyl alcohols, 140 in olive OMW, 694
695 health benefits of EVOO phenolic compounds, 695 olive OMW management, 695
698 phenolic compounds in types of oil, 695 oxidation, 462f recover, 512
514 adsorption, 514 extraction, 513 membrane technology, 513
514 removal, 697
698 biological treatments, 511
512 integrated techniques, 512 physical methods, 509
510 physicochemical methods, 510
511 in VOO, 113t, 115
116 Phenolic extract (PE), 393 Phenolic fraction (PF), 390 Phenolics, 377
378, 446
447 contribution to oxidative stability of VOO, 117
118 molecules in VOO, 111
115 total polyphenols content, 112f phenolic-related acids, 139
140 profile of EVOOs, 549 in VOO, 118
119 Phenols, 123, 263
264 Phenotypic variability and breeding programs for olive oil quality, 30
31 Phenyl alcohol, 140, 447 Phenylalanine ammonia lyase (PAL), 40 Phl p 11 (Phleum pratense), 363 Phlobaphenes, 489 Phloretic acid, 139
140 Phoenix dactylifera.. See Date palm (Phoenix dactylifera) Phonophoresis, 178
179 Phosphatides, 378 Phosphatidylinositol 3-kinase. See Phosphoinositide 3-kinase (PI3K) Phosphatidylinositol 3-kinase/AKT serine threonine kinase 1 pathway (PI3K/AKT pathway), 379 Phosphatidylinositol-3 kinase
related kinases (PIKKs), 610 Phosphoinositide 3-kinase (PI3K), 616, 625
627, 665, 682
683 Phospholipase C (PLC), 665, 682
683 Phospholipid fatty acids, 570 Phospholipids, 446 Phosphorylated-extracellular-regulated MAP kinase (p-ERK), 381
382

Index

Photoprotection ultraviolet A, 429
430 ultraviolet B, 429 Photosynthesis, 38 Physical methods for PC removal, 509
510 Physicochemical methods for PC removal, 510
511 Phytohemagglutinin (PHA), 393 Phytophthora, 330 Phytosterols, 314, 319, 319f PI3K. See Phosphoinositide 3-kinase (PI3K) PI3K/AKT pathway. See Phosphatidylinositol 3-kinase/AKT serine threonine kinase 1 pathway (PI3K/AKT pathway) PI3K/Akt/PKB, 532 Pichia spp., 152
153 P. caribbica, 152
153 P. fermentans, 152
153 P. guilliermondii, 218 P. holstii, 152
153 P. kluyveri, 218 P. manshurica, 218 P. mississippiensis, 152
153 Picholine, 494 Picholine-style green olives, 10 Pickled green olives, 99 Picual, 494 Picudo, 494 Pigments, 314, 318
319 PIKKs. See Phosphatidylinositol-3 kinase
related kinases (PIKKs) Pine (Pinus sylvestris), 367
368 PINK1. See Putative kinase 1 (PINK1) Pinus sylvestris.. See Pine (Pinus sylvestris) PKCs. See Protein kinaseC (PKCs) Pla l 1 (Plantago lanceolata), 363 Plasma membrane Ca21-ATPase (PMCA), 582 Plasma profile of gastrointestinal peptides, 560
561 Plasmin, 481 Plasminogen activator inhibitor-1 (PAI-1), 641 PLC. See Phospholipase C (PLC) Pleurotus ostreatus, 512 PLG. See Poly(lactide-co-glycolide) (PLG) PMCA. See Plasma membrane Ca21-ATPase (PMCA) PME. See Pectin-methylesterase (PME) Poisonings, olive in, 181 Polcalcin. See Ole e 3 Pollen
fruit syndrome, 364 Pollen
latex
fruit syndrome, 365 Pollens and apple (Malus domestica), 361 Pollensomes, 367
368, 368f Pollinosis, 359 Poly(ADP-ribose)polymerase (PARP), 662
663 PARP1, 484 Poly(lactide-co-glycolide) (PLG), 370 Polyacrylamide gel electrophoresis (PAGE), 124 Polymerin, 508
509 Polymorphisms, 32
35 Polyol pathway and cataract formation, 450 Polypeptide (PP), 558, 559t, 561f, 564

Polyphenol oxidase (PPO), 39
40, 123
124, 124t kinetic and molecular properties, 124
125 Polyphenolic(s), 115 compounds, 16 in olive leaves, 447 constituents, 426t Polyphenoloxidase. See Polyphenol oxidase (PPO) Polyphenols (PPs), 23, 100
102, 106, 167, 235, 276, 390, 426, 465
466, 472
474, 489
492, 525 alcohol drinking, 473
474 and athletic performance, 305
306 biological functions, 492 classification, 491f content, 294
295 experimental assays, 493f extraction and purity of phenolic compounds, 490
492 in human health, 473 limitations for clinical applications, 500 normal and clinical consumption, 492 in olive oils, 473 human health and disease prevention, 116
117 olive oils with edible oils, 119
120 phenolic compounds in VOO, 115
116 phenolic contribution to oxidative stability of VOO, 117
118 phenolic molecules in VOO, 111
115 sensory properties affected by phenolics in VOO, 118
119 olive polyphenols, 473
474 Polysaccharides, 100 Polyunsaturated fatty acids (PUFAs), 30, 53, 103, 206, 239
240, 253
254, 294, 316
317, 404, 436, 661 in HD model, 246 Pomace olive oils, 330
331 PON2. See Paraoxonase-2 (PON2) Postharvest treatment, 67
68 Postprandial effects, 197 Postprandial energy metabolism body weight regulation and nutrient partitioning, 251 fatty acid affect rate of fat oxidation, 251
252 Mediterranean-style diets, 254
257 food intake, 254
257 weight/fat loss, 257 olive oil, satiety, and food intake, 252
254 postprandial fat oxidation in humans, 252 preferential effect of olive oil in abdominal obesity, 252 Postprandial fat oxidation in humans, 252 Postprandial fibrinolysis, OA on, 641
642, 642f Postprandial glucose homeostasis, 644
645 Postprandial inflammation, 645
646 Postprandial thermogenesis, 252 Postprandial thrombogenesis, OA on, 641 Postprandial β-cell function, 642
644

717

Postsynaptic density protein 95 (PSD-95), 419
420, 675
676 Poultry meat, 436 PP. See Polypeptide (PP) PPAR gamma coactivator 1-alpha (PGC-1α), 302
303 PPARs. See Peroxisome proliferator
activated receptors (PPARs) PPO. See Polyphenol oxidase (PPO) PPs. See Polyphenols (PPs) PR. See Progesterone (PR) PR-14 protein family. See Pathogenesis-related 14 protein family (PR-14 protein family) PREDIMED study. See Prevencio´n con Dieta Mediterranea study (PREDIMED study) Preeclampsia (PE), 390
391 Presenilin 1 (PS1), 480 Presenilin enhancer 2 (PEN2), 480 Preservation, 9 Preservation and storage methods for naturally processed table olives, 21
22 bulk preservation of natural table olives, 22 natural table olives in consumer packs, 22 preservation of natural table olives with heat treatment, 22 Prevencio´n con Dieta Mediterranea study (PREDIMED study), 261, 339, 404, 418, 465
466, 540 Pro j 1, 363 Probiotics application of autochthonous probiotics, 221
222 in biopreservation of fermented olives, 223
224 from fermented olive in foods fermentations, 225 of nonautochthonous probiotics, 222 in olive fermentation, 221
222 health-beneficial effects, 219
220 LAB, 225t microorganisms isolated from fermented olives, 215
218 LAB, 218 yeasts, 218 safety properties of probiotics in human, 219 selection, 218
219 LAB, 218
219 yeasts, 219 technological properties, 220
221 Profilin. See Ole e 2 Progesterone (PR), 339
340, 354f Programmed theories, 537 Propionibacterium, 9, 100 Propranolol, 438
439 Prosopis juliflora, 363 Prostaglandin E2 (PGE2), 381
382, 408 Prosthodontics, olive in, 182 Protected designation of origin (PDO), 51
52, 95 Protected geographical indication (PGI), 51
52 Protein, 251 Protein database (PDB), 685

718

Index

Protein deglycase (DJ-1), 243 Protein homeostasis. See Proteostasis Protein kinase B (Akt), 278, 625
627 Protein kinaseC (PKCs), 665 Proteobacteria, 146
148 Proteolytic clearance, 483
484 Proteolytic cleavage, 480, 483 Proteostasis, 538
539 reduced proteostasis, 538
539 Provocation, 360f Proximate composition, 99 Proxyl (GROO), 437 Pruritis, 402
404 PRX. See Peroxidases (PRX) Prx. See Peroxiredoxin (Prx) PS1. See Presenilin 1 (PS1) PSD-95. See Postsynaptic density protein 95 (PSD-95) Pseudocercospora cladosporioides, 146
148 Pseudokirchneriella subcapitata, 696 Pseudomonas, 145
146, 149
153, 330 P. aeruginosa, 146, 220, 329
330, 448 P. fluorescens, 146, 334 P. fragi, 152
153 P. mephitica, 152
153 P. savastanoi, 146, 329
330, 514 P. syringae, 146 Psoriasis, olive oil for, 431 Psychiatry, olive in, 177
178 PUFAs. See Polyunsaturated fatty acids (PUFAs) Putative kinase 1 (PINK1), 243 PYY. See Peptide YY (PYY)

Q QDA. See Quantitative descriptive analysis (QDA) QTL. See Quantitative trait loci (QTL) Quantitative descriptive analysis (QDA), 9 Quantitative trait loci (QTL), 32
35 analysis, 32
35 Quercetin, 460
461 paradox, 460
461 QUIN. See Quinolinic acid (QUIN) Quinic acid, 140 Quinolinic acid (QUIN), 243

R RA. See Rheumatoid arthritis (RA) RAAS. See Renin
angiotensin
aldosterone system (RAAS) Raf/MEK/MAPK, 532 Rahnella aquatilis, 146
148 Raisin olives, 20 Raman spectroscopy, 94 Random amplification of polymorphic DNA (RAPD), 32
35 Randomized control trials (RCT), 168, 207
208, 337
338, 627 research, 617 Randomized crossover studies of olive oil, 253t RANK. See Receptor activator of NF-kB (RANK)

RANK ligand (RANKL), 646 RAPD. See Random amplification of polymorphic DNA (RAPD) Rapeseed, 314 RAS. See Renin
angiotensin system (RAS) Rat C6 glioma models, 593
594 Raw olives, 99
100 microbial diversity of, 146
148 RCT. See Randomized control trials (RCT) RDA. See Recommended dietary allowance (RDA) Reactive nitrogen species (RNS), 263, 302
303 Reactive oxygen species (ROS), 59, 232, 263
265, 278, 315
316, 389
390, 402, 416, 426, 437, 449
450, 489, 514, 537, 582, 593
594, 630, 662, 674 increased production of harmful, 539 molecules, 539 Reactive species, 302
303 Real-time quality control of EVOO, 87
88 Receptor activator of NF-kB (RANK), 646 Receptor tyrosine kinases (RTKs), 682 Recombinant olive pollen allergens, 367
368 Recommended dietary allowance (RDA), 105 Recycling olive by-products for cosmetic industries, 499
500 Redox homeostasis, 302
303 imbalance, 169 Refined olive oil (ROO), 79, 206, 392 Refined OPO (ROPO), 506
507 Regulation 432/2012 of European Union, 547 Rehabilitative medicine, olive in, 178
179 Renin, 438
439 Renin
angiotensin system (RAS), 438
440 oncogene, 594
595 Renin
angiotensin
aldosterone system (RAAS), 264 Reproductive system, olive in, 178 Respiratory allergy, 359 Respiratory diseases, 74 Respiratory effects of OMW, 516 Respiratory system, olive in, 179 Response surface methodology (RSM), 68
71 Restorative dentistry, 181 Restriction fragment length polymorphism (RFLP), 32
35 Resveratrol, 589 RFLP. See Restriction fragment length polymorphism (RFLP) Rheumatoid arthritis (RA), 178
179, 392 Rheumatology, olive in, 178
179 Rhodotorula mucilaginosa, 218 16S ribosomal RNA eubacterial gene (16S rRNA), 146
148 Ripe olives, 11 Ripening, 123
124 changes during, 125
127 oleuropein concentration in fruit and leaf of olive during, 127 RNS. See Reactive nitrogen species (RNS) ROO. See Refined olive oil (ROO) ROPO. See Refined OPO (ROPO)

ROS. See Reactive oxygen species (ROS) RSM. See Response surface methodology (RSM) RTKs. See Receptor tyrosine kinases (RTKs) Rutin. See Flavonols Ryegrass (Lolium perenne), 367
368

S Saccharomyces spp., 152
153 S. cerevisiae, 152
153, 218, 329, 514
515 S. rosinii, 152
153 Saccharomycopsis lipolytica, 152
153 Sal k 5 (Salsola kali), 363 Salmonella, 21
22, 404 S. enterica, 330, 334 S. enterica sv. Enteritidis, 330t S. enteritidis, 218, 331 Salsola kali.. See Sal k 5 (Salsola kali) Sambucus nigra, 363 SAMP8 model. See Senescence-accelerated mouse-prone 8 model (SAMP8 model) Sarco/endoplasmic reticulum Ca21-ATPase (SERCA), 582 Sarcoidosis, 391 SASP. See Senescence-associated secretory phenotype (SASP) Satellite cells, 302 Satiety, 252
254 Saturated fatty acids (SFAs), 30, 103, 239
240, 253
254, 294, 640
641, 661 SBP. See Systolic blood pressure (SBP) SBT oil. See Sea buckthorn oil (SBT oil) SC. See Stem cell (SC) Scanning electron microscopy (SEM), 151 SCAR. See Sequence-characterized amplified regions (SCAR) Scavenging, antioxidant effects of, 458
460 SCC. See Squamous cell carcinoma (SCC) SCIT. See Subcutaneous immunotherapy (SCIT) Scopolin, 496 SCPL.. See Serine carboxypeptidase-like (SCPL) SDA. See Stearidonic acid (SDA) Sea buckthorn oil (SBT oil), 391
392 “Seasoned” olives, 99 Secoiridoid glycoside. See Oleuricine A Secoiridoids, 30
31, 111
113, 291, 494, 496 accumulation, 39 in olive leaves, 447 Secologanoside, 496
497 Secondary oxidation products, 117 Secondary processing of natural table olives, 21 Secreted frizzled-related protein 4 (Sfrp4), 340 Secretory blockade and acute pancreatitis, 582
583 SEM. See Scanning electron microscopy (SEM) Semiripe olives, 11 Semisynthetic lipophenols, 547
548 Senescence-accelerated mouse-prone 8 model (SAMP8 model), 541

Index

Senescence-associated secretory phenotype (SASP), 537, 539 cellular senescence and release of, 539 Senile cataract, 449 Sensitization, 360f Sensory properties affected by phenolics in VOO, 118
119, 120f Sequence-characterized amplified regions (SCAR), 32
35 SERCA. See Sarco/endoplasmic reticulum Ca21-ATPase (SERCA) Serine carboxypeptidase-like (SCPL), 41
42 Serratia spp., 146
148, 152
153 S. marcescens, 146 Sertoli cells, 436 Sesame (Sesamum indicum), 32 Sevillano, 494 Sevillian-style green olives. See Spanish-style green olives SFAs. See Saturated fatty acids (SFAs) Sfrp4. See Secreted frizzled-related protein 4 (Sfrp4) SFT. See Supercritical fluid technology (SFT) SH-SY5Y cultured cells, 483
484 SH2. See Src homology 2 (SH2) Shelf life, 292
293 Shigella sonnei, 330t, 331 Shikimic acid, 140 Short interfering RNAs (siRNAs), 42 SHR. See Spontaneously hypertensive rats (SHR) Shriveled olives, 8, 20 SI/R. See Simulated ischemia/reperfusion (SI/ R) Sicilianstyle green olives, 99 Signal transducer and activator of transcription 1 (STAT1), 391
392 Signal transducer and activator of transcription 3 (STAT3), 662
663, 682
683 Silver sulfadiazine (SSD), 406 Simple sequence repeats (SSRs), 32
35 Simply black olives, 11 Simulated ischemia/reperfusion (SI/R), 278 Single-cell electrophoresis
based method, 646, 646f siRNAs. See Short interfering RNAs (siRNAs) Sirtuin 1 (SIRT1), 484, 538 HT effects on, 541 Skeletal muscle, 303, 627 Skin, 401 beneficial properties and constituents of olive oil, 404 cancer, 615
617 melanoma skin cancer, 616 NMSC, 616
617 care constituents, 426 hair growth, 428 oleocanthal, 427 oleuropein, 427 products, 402 properties, 426
427 protection against ultraviolet damage, 427 wound healing, 427
428

dietary ingredients in omega-3 monounsaturated fats, 428t exfoliation, 402 inflammation, 402 metabolism of fatty acids, 402 olive oil effects, 404
408 antioxidant and anti-inflammatory properties, 405
406 delivery of constituents of olive oil, 408 olive oil and endothelial function, 407
408 olive oil use in clinical treatment of foot ulcers, 408 wound healing, 406
407 oxidative stress, 402 pruritis, 403
404 structure and physiology, 402 transdermal passage of molecules, 402 of Wistar rats, 406f wrinkles, 402
403 xerosis, 403
404 SLE. See Systemic lupus erythematosus (SLE) SLIT. See Sublingual immunotherapy (SLIT) Smac, 527 Small nuclear RNA (snRNA), 32, 42
43 SNAP-25. See Synaptosome-associated protein25 (SNAP-25) Snapdragon (Antirrhinum majus), 366 snRNA. See Small nuclear RNA (snRNA) SNS. See Sympathetic nervous system (SNS) SO. See Sunflower oil (SO) SOCS box containing 1 (SPSB1), 683
684 SOD. See Superoxide dismutase (SOD) SODs. See Superoxide dismutases (SODs) SOFE. See Standardized olive fruit extract (SOFE) Soleus muscle, 302
303 Solid-phase extraction (SPE), 513 Somatostatin, 559t Sorghum bicolor, 696 Spanish-style green olives, 8
9, 99
105 packed product, 102
105 physicochemical characteristics, compounds, and proximate composition, 101t product in bulk, 100
102 SPE. See Solid-phase extraction (SPE) Spectroscopy to evaluate quality control of EVOOs human health and disease prevention, 92
93 olive oils with edible oils, 92 for quality control, 93
95 fluorescent spectroscopy, 94
95 NIR spectroscopy, 94 Raman spectroscopy, 94 UV
vis spectroscopy, 93
94 Spectrum of fetal-alcoholic disorders (FASD), 471
472 Spermatozoa, 436 Spontaneously hypertensive rats (SHR), 282 Sports medicine, olive in, 178
179 SPSB1. See SOCS box containing 1 (SPSB1) Squalene (SQ), 57, 317
318, 390, 426, 525 Squalene synthase (SQS), 40
41

719

Squamous cell carcinoma (SCC), 615
616 Src homology 2 (SH2), 682 SREBP. See Sterol regulatory element-binding proteins (SREBP) SSD. See Silver sulfadiazine (SSD) SSRs. See Simple sequence repeats (SSRs) Standardized olive fruit extract (SOFE), 285 Staphylococcus, 149
151, 180 Staphylococcus aureus, 21
22, 152
153, 220, 329
331, 330t, 334, 448 S. aureus oxacillin resistant, 74 Staphyloelies, 20 STAT1. See Signal transducer and activator of transcription 1 (STAT1) Statins, 549 Statistical control, 337
338 Stearic acid, 251
252, 377
378 Stearidonic acid (SDA), 405 Steatohepatitis, 654
655 Stem cell (SC), 537 Sterol regulatory element-binding proteins (SREBP), 267 Sterols, 56
57, 103
106, 167, 313, 319 Stratum corneum, 402 Streptococcus agalactiae, 178 Streptococcus faecium, 146 Stroke, 261 Structure
activity relationship study, 686
687 Subcutaneous immunotherapy (SCIT), 369
370 Sublingual immunotherapy (SLIT), 369
370 Substituted phenols, 457
458 Substituted phenols. See Tyrosol (Tyr) Sucrose transporter 1 (SUT1), 32
35 Sulfatases, 194 SUN study, 264 Sunflower, 314 Sunflower oil (SO), 208, 347
348, 557, 570
571 Supercritical fluid technology (SFT), 513 Superoxide anion (GO22), 437 Superoxide anion radical (O2G2), 302
303 Superoxide dismutase (SOD), 350
352, 450, 541, 597
598 SOD-1, 416 Superoxide dismutase, catalase (KatA), 437 Superoxide dismutases (SODs), 302
303 Sustainability of Mediterranean diet, 206 SUT1. See Sucrose transporter 1 (SUT1) Sympathetic nervous system (SNS), 252 SNS-mediated lipolysis, 252 Synaptic plasticity, 416, 418
419 Synaptic proteins, EVOO and, 419
420 Synaptosome-associated protein-25 (SNAP-25), 675
676 Synaptosomes as in vitro model, 243 as model to study fish oil and olive oil effect experimental models to study neurodegenerative diseases, 242
243 fish oil, 240 HD and oils as therapeutic agents, 243
246

720

Index

Synaptosomes (Continued) human health and disease prevention, 241
242 olive oil, 240
241 protective mechanism by polyunsaturated fatty acids, 246 Synovial fibroblasts, 178
179 Syr v 1, 363 Syringa vulgaris, 363 Systemic lupus erythematosus (SLE), 181, 393 Systolic blood pressure (SBP), 199
200, 282

T t-box transcription factor (TBX2), 168 T-cell epitopes, 363 T2D. See Type 2 diabetes (T2D) TA. See Titratable acidity (TA) Table olives, 5, 7t, 153 antimicrobial compounds in, 332f, 333
334 composition of final products, 12
13 nutritional and health-related aspects of, 22
23 and olive oil, 525 processing methods, 8
12 production, exportation, importation, and consumption, 6t ripeness, 5
6 trade preparations, 7
8 TAC. See Total antioxidant capacity (TAC) Taggiasca, 494 TAGs. See Triacylglycerols (TAGs) Tanche, 494 Tank blanketing, 294 Tannins, 447 Target fishing, 451 TBARS. See Thiobarbituric acid-reactive substances (TBARS) TBX2. See t-box transcription factor (TBX2) TEAC. See Trolox equivalent antioxidant capacity (TEAC) Telomerase, 540 TEs. See Transposable elements (TEs) Testis, 439 12-O-Tetradecanoylphorbol acetate (TPA), 426, 550
551 TF. See Tissue factor (TF) TFAs. See Trans fatty acids (TFAs) TFEB. See Transcriptional factor EB (TFEB) TFs. See Total flavonoids (TFs) TG. See Triglyceride (TG) Thamnocephalus platyurus, 696 Thaumatin-like protein (TLP), 371 Thermograms, 81 Thiobarbituric acid-reactive substances (TBARS), 350, 596, 596f, 607 Thrombosis, 198
200 Thromboxygenases, 265 Throumbes, 20 Thrubolea, 20 Thrumba-style olives, 20 TIM. See Triose-phosphate isomerase (TIM) Tissue factor (TF), 641, 641f Tissue necrosis factor-α (TNF-α), 379, 672 Tissue plasminogen activator (tPA), 641

Titratable acidity (TA), 100 TLP. See Thaumatin-like protein (TLP) TLR-4. See Toll-like receptor-4 (TLR-4) TNBC. See Triple negative breast cancer (TNBC) TNF. See Tumor necrosis factor (TNF) TNF receptor superfamily member 1 (TNFR1), 379 TNF-α. See Tissue necrosis factor-α (TNF-α) TNFR1. See TNF receptor superfamily member 1 (TNFR1) TOC. See Total organic compounds (TOC) Tocoferols. See Tocopherols Tocopherols, 58
59, 106, 117
118, 291, 295, 301
302, 313
314, 316
317, 317f, 377
378, 446, 525 Tocotrienols, 291 Toll-like receptor-4 (TLR-4), 265, 267 Torulaspora spp., 152
153 Total antioxidant capacity (TAC), 350 Total flavonoids (TFs), 65
67 Total organic compounds (TOC), 509 Total phenols (TPs), 65
67 Total polyphenolic fraction (TPF), 666 TPA. See 12-O-Tetradecanoylphorbol acetate (TPA) tPA. See Tissue plasminogen activator (tPA) TPF. See Total polyphenolic fraction (TPF) TPM. See Traditional Persian medicine (TPM) TPs. See Total phenols (TPs) Traditional Persian medicine (TPM), 175 olive in, 175
176 in dentistry and oral cavity based on, 181
182 for human health and disease prevention in, 176
177 in medicine based on, 177
181 Trametes versicolor, 512 Trans fatty acids (TFAs), 103 Trans-resveratrol, 525 Transcriptional factor EB (TFEB), 606 Transcriptomics for olive oil quality, 35
42 Transdermal passage of molecules, 402 Transposable elements (TEs), 32 Traumatic cataract, 449 Treated olives, 7
8 Tree, partial dehydration of olives on, 20 Triacylglycerol acylhydrolase, 292 Triacylglycerols (TAGs), 30, 80, 404, 654 Trichosporon spp., 152
153 T. behrendii, 152
153 T. mentagrophytes, 431 Triglyceride (TG), 170, 262, 266, 625
627, 639 Triose-phosphate isomerase (TIM), 365 Triple negative breast cancer (TNBC), 683 Triterpenes, 167, 446
447 in olive leaves, 448 Triterpenic alcohols, 56
57 Triterpenoids, 65, 379 Trolox equivalent antioxidant capacity (TEAC), 315 True-to-type cultivars, 31 Tumor growth, effects of OLEU and HTX on, 595

Tumor necrosis factor (TNF), 379, 497, 630
631 modulation, 379
382 TNF-α, 169, 205, 265, 390, 416, 532
533, 617, 663 Turning-color olives, 5 TyEDA. See Tyrosol (Tyr) Type-I allergy, 360f Type I collagen, 403 Type 2 diabetes (T2D), 209, 261, 625 Type 2 diabetes mellitus (T2DM). See Type 2 diabetes (T2D) Type VII collagen, 403 Tyrosol (Tyr), 30
31, 106, 111
113, 116
117, 193
194, 206
207, 234, 262, 276, 315
316, 332
333, 377
379, 473, 494
495, 497, 651, 652f, 661 endogenous sources of OHTyr and, 196 sinapate to homovanillyl sinapate, 686f

U UA. See Ursolic acid (UA) UAF. See Up-flow anaerobic reactor filter (UAF) UASB. See Up-flow sludge blanket reactor (UASB) UDP-glucose: anthocyanin: flavonoid glucosyltransferase (UFGT), 40 UF. See Ultra-filtration (UF); Unsaponifiable fraction (UF) Ultra-filtration (UF), 512 Ultraviolet A (UVA), 429
430 Ultraviolet B (UVB), 429 Ultraviolet radiation (UV radiation), 405, 615 Ultraviolet
visible spectroscopy (UV
vis spectroscopy), 92
94 Unfolded protein response (UPR), 582 United Nations Educational Scientific and Cultural Organization (UNESCO), 215 Unsaponifiable fraction (UF), 389 50 Untranslated region (50 UTR), 32
35 Untreated green olives in brine, 99, 105
106 packed product, 105
106 product in bulk, 105 Up-flow anaerobic reactor filter (UAF), 512 Up-flow sludge blanket reactor (UASB), 512 UPR. See Unfolded protein response (UPR) Urinary cancer, 380t Urinary system, olive in, 178 Ursolic acid (UA), 68, 448 UV radiation. See Ultraviolet radiation (UV radiation) UVA. See Ultraviolet A (UVA) Uvaol, 30, 379, 390, 448 UVB. See Ultraviolet B (UVB) UV
vis spectroscopy. See Ultraviolet
visible spectroscopy (UV
vis spectroscopy)

V Valorization, 509
514 Vanillic acid, 276 Vanillin, 276

Index

Varietal-dependent factors, 53 Vascular aging, 168 Vascular cell adhesion molecule-1 (VCAM-1), 198, 265, 407 Vascular endothelial growth factor (VEGF), 407, 619 VC. See Vehicle control (VC) VCAM-1. See Vascular cell adhesion molecule-1 (VCAM-1) Vegetable oil market, 85
86 VEGF. See Vascular endothelial growth factor (VEGF) Vehicle control (VC), 685
686 Verbascoside, 65, 276, 379, 494
495, 515 Very-low-density lipoprotein (VLDL), 266, 654 Vinegar, 331
332 Vinyl phenol reductase, 136
137 Virgin olive oil (VOO), 51, 111, 167, 193, 200, 276, 313, 330
331, 390, 473, 489
490, 526, 557, 563
564, 569
571, 579. See also Extra virgin olive oil (EVOO); Olive oils (OO) comparison of antimicrobial activity, 332f phenolic compounds in, 115
116 phenolic contribution to oxidative stability of, 117
118

phenolic molecules in, 111
115 sensory properties affected by phenolics in, 118
119 Vitamin E, 23, 295, 316
317 VLDL. See Very-low-density lipoprotein (VLDL) VOO. See Virgin olive oil (VOO) VOOPs, 497

W Water/weak salt brine curing, 16
18 Western diet, 436, 438
439 WHO. See World Health Organization (WHO) WHO/IUIS. See World Health Organization and International Union of Immunological Societies (WHO/IUIS) Wickerhamomyces anomalus, 148
149 World Health Organization (WHO), 80, 205, 218 World Health Organization and International Union of Immunological Societies (WHO/IUIS), 360
361 Wound healing, 406
407, 427
428 olive oil and, 431 Wrinkles, 402
403

721

X Xanthine oxidase (XO), 303 Xanthomonas campestris, 146 Xenograft nude mice model, 340 Xerosis, 401
404

Y Yamadazyma terventina, 152
153 Yeasts, 10
11, 145
146, 218
219 Yersinia sp., 331 Y. enterocolitica, 223, 330t

Z Zanthoxylum acanthopodium.. See Nanoherbal andaliman (Zanthoxylum acanthopodium) Zapaterı´a, 9 Zea mays, 363 Zinc deficiency, 437
438 Zmc13, 363 Zygosaccharomyces spp., 152
153 Z. fermentati, 152
153 Zymogen activation and acute pancreatitis, 582 Zymomonas mobilis, 146