Pathology: Oxidative Stress and Dietary Antioxidants [1 ed.] 0128159723, 9780128159729

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PATHOLOGY

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PATHOLOGY Oxidative Stress and Dietary Antioxidants

Edited by

VICTOR R. PREEDY Department of Nutrition and Dietetics, School of Life Course Sciences, King’s College London, London, United Kingdom

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 © 2020 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: 978-0-12-815972-9 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Tari Broderick Editorial Project Manager: Timothy Bennett Production Project Manager: Poulouse Joseph Cover Designer: Christian Bilbow Typeset by MPS Limited, Chennai, India

Contents

List of contributors Preface

Molecular mechanisms of diabetic retinopathy Oxidative stress and reactive oxygen species Relationship between oxidative stress and other biochemical pathways Oxidative stress and structural and functional alterations in diabetic retinopathy Oxidative stress-related epigenetic modifications in diabetic retinopathy Conclusion Summary points References

xi xv

I Oxidative Stress and Pathology 1. Free radicals: what they are and what they do ´ AND FILIP BENKO EVA TVRDA

List of abbreviations Introduction Free radicals: general characteristics Oxidative chain reactions Selected free and nonfree radicals: characteristics and behavior Sources of free radicals Oxidative stress Free radicals in health and disease Conclusion Applications to other areas of pathology Summary points Acknowledgment References

3 3 4 4 4 7 8 10 11 11 11 12 12

33 33 35 35 35

´ VERO COSTA, JE´SSICA LEITE GARCIA, MARIANE RO ´ GULA DE ALMEIDA SILVA, CAROL CRISTINA VA ANA PAULA COSTA RODRIGUES FERRAZ, FABIANE VALENTINI FRANCISQUETI-FERRON, ARTUR JUNIO TOGNERI FERRON AND CAMILA RENATA CORREˆA

List of abbreviations Introduction Mitochondrial production of reactive species Role of oxidative stress in cardiovascular disease Summary points References

39 39 40 41 46 47

5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

ASHOK AGARWAL, KRISTIAN LEISEGANG AND PALLAV SENGUPTA

15 15 16 16 22 23 24 24 24 25

MAHMOOD A. AL-AZZAWI

List of abbreviations Introduction Chronic obstructive pulmonary disease overview Oxidative stress The role of oxidative stress in chronic obstructive pulmonary disease pathogenesis Environmental reactive species and chronic obstructive pulmonary disease Cellular reactive species and chronic obstructive pulmonary disease Applications to other areas of pathology Summary points References

3. Oxidative stress and diabetic retinopathy KEMAL TEKIN AND MERVE INANC TEKIN

List of abbreviations Introduction

32

4. Pathological bases of oxidative stress in the development of cardiovascular diseases

2. Oxidative stress in pathologies of male reproductive disorders List of abbreviations Introduction Redox biology in male reproduction Oxidative stress and male factor infertility Assessment of oxidative stress in the male reproductive system Use of antioxidants in male infertility treatment Applications to other areas of pathology Conclusion Summary points References

29 30

29 29

v

49 49 49 50 50 56 58 62 62 62

vi

Contents

6. Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies ¨ ZTAS¸ AND ˙IFFET ˙IPEK BOSGELMEZ YE¸sIM O ¸

List of abbreviations Introduction Epidemiology and global burden of sickle cell disease Clinical manifestations of sickle cell disease and treatment options Pathophysiology of sickle cell disease Oxidative stress in sickle cell disease pathogenesis Agents targeting antioxidant defense in sickle cell disease patients Future prospects Applications to other areas of pathology Summary points References

65 65 66 67 67 69

10. The regulation of intracellular redox homeostasis in cancer progression and its therapy

72 72 73 73 73

PRITAM SADHUKHAN AND PARAMES C. SIL

7. Nrf2 and oxidative stress OSAMU WADA-HIRAIKE

List of abbreviations Introduction Nrf2: function Nrf2: relation with disease Dietary compounds that could activate the Nrf2 and Nrf2/ARE pathway Applications to other areas of pathology Summary points References

77 77 77 80 81 84 84 84

8. Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy 87 87 87 88 88 89 89 90 90 92 93 94 94 94

9. Paraoxonase 1 as antioxidant enzyme in children MAXIMILIANO MARTI´N, VALERIA HIRSCHLER, ELIANA BOTTA AND FERNANDO BRITES

List of abbreviations

List of abbreviations Introduction Role of oxidative stress in carcinogenesis Targeting oxidative stress in cancer therapeutics Conclusion Applications to other areas of pathology Summary points References

97 98 100 102 102 103 103

105 105 107 110 112 112 113 113

II Antioxidants and Pathology 11. Antioxidant and chelator cocktails to prevent oxidative stress under iron-overload conditions SIRINART KUMFU, SIRIPORN CHATTIPAKORN AND NIPON CHATTIPAKORN

ARISTIDIS S. VESKOUKIS

List of abbreviations Introduction Oxidative stress: a short historical overview Redox signaling: definition and mechanisms A paradox Redox signaling as a determinant for pathogen elimination in the context of oxidative stress Redox signaling as a determinant for pathogen thriving in the context of oxidative stress The perception shift The intriguing dual role of antioxidants Association of reactive species and antioxidants with putative therapy against pathogens Future perspectives in therapy against pathogens Applications to other areas of pathology Summary points References

Introduction Paraoxonase 1: general characteristics Paraoxonase 1 in pediatric populations Conclusion Applications to other areas of pathology Summary points References

97

List of abbreviations Introduction The effects of the combination of an iron chelator with vitamin c on oxidative stress and iron status in iron-overloaded conditions The effects of a combination of an iron chelator with silymarin on oxidative stress and iron status in iron-overloaded conditions The effects of a combination of an iron chelator with Nacetylcysteine on oxidative stress and iron status in ironoverloaded conditions The effects of a combination of an iron chelator with vitamin E on oxidative stress and iron status in iron-overloaded conditions The effects of the combination of an iron chelator with curcumin or idebenone on oxidative stress and iron status in iron-overloaded conditions The effects of a combination of an iron chelator with other antioxidants and multiantioxidants on oxidative stress and iron status in iron-overloaded conditions Applications to other areas of pathology Summary points Acknowledgments References

117 117

118

118

119

120

121

123 123 124 124 124

Contents

12. Ac¸aı´ (Euterpe oleracea Martius) as an antioxidant PRISCILA OLIVEIRA BARBOSA, MELINA OLIVEIRA DE SOUZA, DANIELA PALA AND RENATA NASCIMENTO FREITAS

List of abbreviations Introduction Ac¸aı´ antioxidants Antioxidant activity of ac¸aı´: in vitro bioassays Antioxidant activity of ac¸aı´: animal model assays Antioxidant activity of ac¸aı´: human studies Applications to other areas of pathology Summary points References

127 127 128 129 130 130 131 132 132

13. Amaryllidaceae alkaloids and neuronal cell protection NATALIE CORTES, RAFAEL POSADA-DUQUE, GLORIA PATRICIA ´ MEZ, JAUME BASTIDA AND EDISON OSORIO CARDONA-GO

List of abbreviations Introduction Neurodegenerative diseases and neuronal cell damage Oxidative stress related with neuronal cell damage Current pharmacotherapy of dementias (mainly Alzheimer’s disease) Amaryllidaceae alkaloids Efficacy of amaryllidaceae alkaloids in oxidative stress and neuronal cell damage Conclusion Applications to other areas of pathology Summary points Acknowledgments References

135 135 136 137 138 139 139 141 142 142 142 142

14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology JUNICHI R. SAKAKI, MELISSA M. MELOUGH AND OCK K. CHUN

List of abbreviations Introduction Oxidative stress and inflammation Bone physiology and pathology under oxidative stress Dietary anthocyanins as antioxidants Cell studies Animal bone disease models Human studies Conclusion Applications to other areas of pathology Summary points References

145 145 146 146 147 147 149 155 156 156 156 156

15. Ascorbic acid as an antioxidant and applications to the central nervous system

Role of ascorbic acid in neuropsychiatric disorders Conclusion Applications to other areas of pathology Funding Summary points References Further reading

vii 161 163 164 164 164 164 167

16. Artichoke leaf extract and use in metabolic syndrome as an antioxidant KHATEREH REZAZADEH AND MEHRANGIZ EBRAHIMI-MAMEGHANI

List of abbreviations Introduction Oxidative stress and metabolic syndrome Antioxidants and metabolic syndrome Artichoke leaf extract: bioactive compounds, pharmacokinetics, and safety Artichoke leaf extract, oxidative stress, and metabolic syndrome Conclusion Applications to other areas of pathology Summary points References

169 169 170 171 171 173 175 175 175 176

17. Bilberry anthocyanins as agents to address oxidative stress JERRY T. THORNTHWAITE, SETH P. THIBADO AND KYLE A. THORNTHWAITE

List of abbreviations Introduction Other areas of pathology Chemistry of bilberry anthocyanins Why plants contain anthocyanins Why humans and animals must consume anthocyanins Bilberry anticancer activity Bilberry protective effect against chemotherapy and radiation therapy Bilberry’s importance in the prevention and treatment of cardiovascular disease Bilberry’s importance in the prevention and treatment of diabetes Bilberry and macular degeneration (vision) Bilberry’s importance in preventing and curing dementia and Alzheimer’s disease Summary points Acknowledgments References

179 179 179 180 181 181 181 182 183 183 184 184 185 185 185

18. Clitoria ternatea beverages and antioxidant usage SIRICHAI ADISAKWATTANA, PORNTIP PASUKAMONSET AND CHAROONSRI CHUSAK

´ CIA S. RODRIGUES MORGANA MORETTI AND ANA LU

Introduction Ascorbic acid applications in neurodegenerative diseases

159 160

List of abbreviations Introduction Use of Clitoria ternatea flowers

189 189 190

viii Bioactive components Antioxidant properties of Clitoria ternatea flowers Toxicity Human study Food uses Applications to other areas of pathology Conclusions Summary points References

Contents

190 190 193 193 193 193 194 195 195

19. Curcumin and related antioxidants: applications to tissue pathology CAROLINA ALVES DOS SANTOS, MAHENDRA RAI, ´ DEL FIOL, JOSE´ MARTINS DE OLIVEIRA JR, FERNANDO DE SA ROGERIO AUGUSTO PROFETA, DENICEZAR BALDO AND MARCO VINI´CIUS CHAUD

List of abbreviations Introduction Antiinflammatory activity Antitumoral properties Activity in myocardial diseases Antineurodegenerative effect As an antimicrobial agent Applications to other areas of pathology Therapeutic challenges Conclusion and future perspectives Summary points References

197 197 198 200 200 201 201 202 202 203 203 203

20. Dacryodes edulis: protective antioxidant effects on diabetes pathology

ROBERTA MASELLA

List of abbreviations 225 Introduction 225 Olive oil characteristic and extraction procedures that influence EVOO antioxidant properties 226 Extra virgin olive oil polyphenols 226 Applications in other areas of pathology 231 Summary points 231 References 231

23. Ginkgo biloba extract as an antioxidant in nerve regeneration ¨ NAY NAHIDE EKICI-GU

List of abbreviations Introduction Peripheral nerve injury Neurodegeneration and neuroregeneration Intracellular signaling and angiogenesis in neuronal regeneration Oxidative stress in neurodegenerative processes Ginkgo biloba Summary points References

235 235 235 236 237 238 238 243 243

¨ L ANLAR AND MERVE BACANLI HATICE GU

205 205 207 207 209 209 209 210 210

21. Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases

List of abbreviations Introduction Chemistry and sources Pharmacokinetics and bioavailability Antioxidant effects Cancer Cardiovascular diseases Hepatic and renal diseases Conclusion Summary points References

247 247 248 248 249 249 250 250 252 252 252

25. Mediterranean diet: the role of antioxidants in liver disease LUDOVICO ABENAVOLI, LORENZO ROMANO, PAOLA GUALTIERI, GEMMA LOU DE SANTIS AND ANTONINO DE LORENZO

PUNNIYAKOTI VEERAVEEDU THANIKACHALAM, SRINIVASAN RAMAMURTHY, ANOOP KUMAR, MEENAKSHI GUPTA AND GARIMA BANSAL

List of abbreviations Introduction Summary points References

ANNALISA SILENZI, CLAUDIO GIOVANNINI, BEATRICE SCAZZOCCHIO, ROSARIA VARI`, MASSIMO D’ARCHIVIO, CARMELA SANTANGELO AND

24. Lycopene as an antioxidant in human health and diseases

OLAKUNLE SANNI, OCHUKO L. ERUKAINURE AND MD. SHAHIDUL ISLAM

Introduction Phytochemistry of Dacryode edulis Diabetic pathogenesis Protective effects of D. edulis in diabetes pathology Proposed protective mechanism of diabetes pathology of D. edulis Conclusion Conflict of interests Acknowledgment References

22. Extra virgin olive oil polyphenols: biological properties and antioxidant activity

213 213 221 221

List of abbreviations Introduction Applications in other areas of disease Nonalcoholic fatty liver disease Alcoholic liver disease

255 255 257 258 260

Contents

Conclusions Summary points References

262 262 263

26. Melatonin, antioxidant capacity, and male reproductive function

Type 2 diabetes Therapeutic applications of phenolics and obesity Conclusion Conflict of interest Acknowledgment Summary points References

ix 301 301 306 306 306 306 306

FAHIMEH MOHAMMADGHASEMI

List of abbreviations Introduction Concluding remarks Summary points References

265 265 272 273 273

27. Methylsulfonylmethane as an antioxidant and its use in pathology MATTHEW BUTAWAN, RODNEY L. BENJAMIN AND RICHARD J. BLOOMER

List of abbreviations Introduction Overview of antioxidant effects Methylsulfonylmethane in oxidative stress pathology Conclusion Summary points References

277 277 279 281 286 286 286

28. Improving antioxidant capacity of foods: adding mushroom powder to pasta LIWEN WANG, MARGARET ANNE BRENNAN AND CHARLES STEPHEN BRENNAN

List of abbreviations Introduction Oxidative stress Major antioxidants of mushrooms Antioxidant mechanisms in different models Applications in other areas of pathology Summary points References

289 289 289 290 292 295 295 295

MARI´A DESAMPARADOS SALVADOR, ROSA M. OJEDA-AMADOR AND GIUSEPPE FREGAPANE

List of abbreviations Introduction Applications to pathology Beneficial lipidic components Antioxidant vitamins (ACE) Antioxidant minerals (Se, Cu, Zn, Mn) Polar phenolic compounds Summary points References Further reading

309 309 311 312 313 314 315 316 317 320

31. Sambucus ebulus L., antioxidants and potential in disease ´ ALEKSANDRA CVETANOVIC

List of abbreviations Introduction Traditional use Chemical composition Antioxidant potential Anticancer activity Other biological activities of Sambucus ebulus Application in other areas of pathology Conclusion Summary points Acknowledgment References

321 321 322 323 326 328 329 331 331 331 331 331

32. Selenium usage and oxidative stress in Graves’ disease and Graves’ orbitopathy

29. Phenolics: therapeutic applications against oxidative injury in obesity and type 2 diabetes pathology

` , GIULIA LANZOLLA, GIOVANNA ROTONDO MICHELE MARINO DOTTORE AND CLAUDIO MARCOCCI

VERONICA F. SALAU, OCHUKO L. ERUKAINURE AND MD. SHAHIDUL ISLAM

List of abbreviations Introduction Phenolics Phenolic acids Flavonoids Lignans and neolignans Tannins Oxidative stress: reactive species and antioxidants Obesity

30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

297 297 298 298 298 299 299 299 300

List of abbreviations Introduction Applications to other areas of pathology Oxidative stress in Graves’ hyperthyroidism Oxidative stress in Graves’ orbitopathy Use of antioxidants in the management of Graves’ orbitopathy Selenium Use of selenium in Graves’ hyperthyroidism Use of selenium in Graves’ orbitopathy Summary points References Further reading

335 335 336 336 337 338 339 339 340 343 343 344

x

Contents

33. Antioxidant potential of Stevia rebaudiana (Bertoni)

Toxic effects of thymoquinone Conclusion Summary points References

KASHIF AMEER, GUI-HUN JIANG, RAI MUHAMMAD AMIR AND JONG-BANG EUN

List of abbreviations Introduction Applications in other areas of pathology Effectiveness against cancer Effectiveness against cariogenesis Effectiveness against atherosclerosis Effectiveness against hypertension Effectiveness against obesity, diabetes, and in blood glucose homeostasis Antioxidant potential against free radicals and stevia potential Conclusion and future perspective Summary points References Further reading

376 376 376 376

345 345 346 346 346 347 347

36. Yacon (Smallanthus sonchifolius) use as an antioxidant in diabetes

347

List of abbreviations Introduction: from oxidative stress to antioxidants acting on diabetes Antioxidants and diabetes mellitus Antioxidant activity in hyperglycemia and cardiomyopathy Smallanthus sonchifolius: origins and ethnobotanical characteristics The nutritional characteristics and phytochemical profile of Smallanthus sonchifolius Smallanthus sonchifolius nutrients and its interaction in diabetes Applications in other areas of pathology Summary points References

347 355 355 355 356

34. Tea antioxidants in terms of phenolic and nonphenolic metabolites PROTIVA RANI DAS AND JONG-BANG EUN

List of abbreviations 357 Introduction 357 Oxidative stress associated pathogenesis 358 Role of antioxidants in oxidative stress
induced pathogenesis 359 Conclusions 363 Application in other areas of pathology 364 Summary points 365 References 365

ANA PAULA COSTA RODRIGUES FERRAZ, JE´SSICA LEITE GARCIA, ´ GULA DE ALMEIDA, ´ VERO COSTA, CAROL CRISTINA VA MARIANE RO CRISTINA SCHIMITT GREGOLIN, PEDRO HENRIQUE RIZZI ALVES, FABIANA KUROKAWA HASIMOTO, CAROLINA B. BERCHIERI-RONCHI, KLINSMANN CAROLO DOS SANTOS AND CAMILA RENATA CORREˆA

379 379 380 380 380 381 383 384 384 384

III Techniques and Resources 37. Screening procedures and tests for antioxidants JAN BORLINGHAUS, JANA REITER, MICHAEL RIES AND MARTIN C.H. GRUHLKE

35. Thymoquinone: the active compound of black seed (Nigella sativa) ¨ L ANLAR AND MERVE BACANLI HATICE GU

List of abbreviations Introduction Bioavailability and kinetics Thymoquinone and health Antioxidant activity Antidiabetic activity Anticancer activity Cardiovascular activity Effects on gastrointestinal system Hepatoprotective effects Nephroprotective activity Effects on pulmonary system Effects on the nervous system Antiinflammatory activity Applications in other areas of pathology

369 369 370 370 370 370 371 372 372 373 373 374 374 374 375

List of abbreviations Introduction Summary points References

389 389 394 394

38. Recommended resources for pathology: oxidative stress and dietary antioxidants RAJKUMAR RAJENDRAM, VINOOD B. PATEL AND VICTOR R. PREEDY

List of abbreviations Introduction Resources Acknowledgments Summary points References

Index

397 397 397 401 401 401

403

List of contributors

Ludovico Abenavoli Department of Health Sciences, University “Magna Graecia”, Catanzaro, Italy

Richard J. Bloomer School of Health Studies, The University of Memphis, Memphis, TN, United States

Sirichai Adisakwattana Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand

Jan Borlinghaus Department of Plant Physiology, Worringer Weg 1, Aachen, Germany; LumiBioSciences, c/o Worringer Weg 1, Aachen, Germany

Ashok Agarwal American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

Eliana Botta Laboratory of Lipids and Atherosclerosis, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina ˙ ˙ Iffet Ipek Bo¸sgelmez Department of Toxicology, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey

Mahmood A. Al-Azzawi Clinical Biochemistry & Molecular Biology, College of Dentistry, Al-Ayen University, Al-Nasiriyah, Iraq Pedro Henrique Rizzi Alves Institute of Bioscience, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Charles Stephen Brennan Lincoln University, Department of Food Science, Lincoln, New Zealand; Tianjin University of Commerce, Biotechnology and Food Science, Tianjin, P.R. China; Riddet Institute, Palmerston North, New Zealand

Kashif Ameer Institute of Food and Nutritional Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan; Department of Food Science and Technology and BK 21 Plus Program, College of Agriculture & Life Sciences, Chonnam National University, Gwangju, South Korea

Margaret Anne Brennan Lincoln University, Department of Food Science, Lincoln, New Zealand Fernando Brites Laboratory of Lipids and Atherosclerosis, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina

Rai Muhammad Amir Institute of Food and Nutritional Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan Hatice Gu¨l Anlar Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Zonguldak Bulent Ecevit University, Zonguldak, Turkey

Matthew Butawan School of Health Studies, The University of Memphis, Memphis, TN, United States

Merve Bacanli Department of Pharmaceutical Toxicology, Gu¨lhane Faculty of Pharmacy, University of Health Sciences, Ankara, Turkey

Gloria Patricia Cardona-Go´mez Cellular and Molecular Neurobiology Area, Group of Neuroscience of Antioquia, Faculty of Medicine, Universidad de Antioquia UdeA, Medellı´n, Colombia

Denicezar Baldo Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil

Nipon Chattipakorn Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Garima Bansal Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga, India Priscila Oliveira Barbosa Nucleus of Research in Biological Sciences (NUPEB), Federal University of Ouro Preto, Minas Gerais, Brazil

Siriporn Chattipakorn Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Jaume Bastida Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farma`cia i Cie`ncies de l’Alimentacio´, Universitat de Barcelona, Barcelona, Spain Rodney L. Benjamin United States

Marco Vinı´cius Chaud Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil Ock K. Chun Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

Bergstrom Nutrition, Vancouver, WA,

Filip Benko Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Charoonsri Chusak Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand

Carolina B. Berchieri-Ronchi Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Camila Renata Correˆa Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

xi

xii

List of contributors

Natalie Cortes Grupo de Investigacio´n en Sustancias Bioactivas, Facultad de Ciencias Farmace´uticas y Alimentarias, Universidad de Antioquia UdeA, Medellı´n, Colombia Mariane Ro´vero Costa Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Jong-Bang Eun Department of Food Science and Technology and BK 21 Plus Program, Graduate School of Chonnam National University, Gwangju, South Korea; Department of Food Science and Technology and BK 21 Plus Program, College of Agriculture & Life Sciences, Chonnam National University, Gwangju, South Korea

Aleksandra Cvetanovi´c Department of Biotechnology and Pharmaceutical Engineering, Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

Ana Paula Costa Rodrigues Ferraz Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Massimo D’Archivio Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy

Artur Junio Togneri Ferron Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Protiva Rani Das Department of Food Science and Technology and BK 21 Plus Program, Graduate School of Chonnam National University, Gwangju, South Korea

Fabiane Valentini Francisqueti-Ferron Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Carol Cristina Va´gula de Almeida Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Giuseppe Fregapane Department of Food Science and Technology, Faculty of Chemistry, University of Castilla-La Mancha, Ciudad Real, Spain

Antonino De Lorenzo Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

Renata Nascimento Freitas Nucleus of Research in Biological Sciences (NUPEB), Federal University of Ouro Preto, Minas Gerais, Brazil; School of Nutrition, Federal University of Ouro Preto, Minas Gerais, Brazil

Jose´ Martins de Oliveira Jr Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil Fernando de Sa´ Del Fiol Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil Gemma Lou De Santis Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Melina Oliveira de Souza School of Nutrition, Federal University of Ouro Preto, Minas Gerais, Brazil Giovanna Rotondo Dottore Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Pisa, Italy Carolina Alves dos Santos Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil

Je´ssica Leite Garcia Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil Claudio Giovannini Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy Cristina Schimitt Gregolin Medical School, Mato Grosso State University (UFMT), Sinop, MT, Brazil Martin C.H. Gruhlke Department of Plant Physiology, Worringer Weg 1, Aachen, Germany Paola Gualtieri Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Meenakshi Gupta Department of Pharmacology, ISF College of Pharmacy, Moga, India

Klinsmann Carolo dos Santos Clinical Research Centre (CRC), Lund University Diabetes Centre, Lund University, Malmo¨, Sweden

Fabiana Kurokawa Hasimoto Institute of Bioscience, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Mehrangiz Ebrahimi-Mameghani Nutrition Research Center, Department of Nutrition & Biochemistry, School of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

Valeria Hirschler Laboratory of Lipids and Atherosclerosis, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina

Nahide Ekici-Gu¨nay Department of Clinical Biochemistry, University of Health Sciences, Kayseri City Training and Research Hospital, Kayseri, Turkey

Md. Shahidul Islam Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa

Ochuko L. Erukainure Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa; Department of Pharmacology, School of Clinical Medicine, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

Gui-Hun Jiang School of Public Health, Jilin Medical University, Jilin, P.R. China Anoop Kumar Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER-R), Ministry of Chemical & Fertilizers, Govt. of India, Lucknow, India

List of contributors

xiii

Sirinart Kumfu Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Rogerio Augusto Profeta Technology & Environmental Process Graduate Course, University of Sorocaba, Sorocaba, Brazil

Giulia Lanzolla Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Pisa, Italy

Mahendra Rai Basic Science Research Faculty Fellow (UGC), Department of Biotechnology, SGB Amravati University, Amravati, India

Kristian Leisegang School of Natural Medicine, University of the Western Cape, Cape Town, South Africa

Rajkumar Rajendram College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; Department of Nutrition and Dietetics, School of Life Course Sciences, King’s College London, London, United Kingdom

Claudio Marcocci Department of Clinical and Experimental Medicine, Endocrinology Unit II, University of Pisa and University Hospital of Pisa, Pisa, Italy; Endocrinology Unit, University of Pisa and University Hospital of Pisa, Pisa, Italy Michele Marino` Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Pisa, Italy Maximiliano Martı´n Laboratory of Lipids and Atherosclerosis, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina

Srinivasan Ramamurthy College of Pharmacy and Health Sciences, University of Science & Technology of Fujairah, Fujairah, UAE Jana Reiter Department of Plant Physiology, Worringer Weg 1, Aachen, Germany; LumiBioSciences, c/o Worringer Weg 1, Aachen, Germany Khatereh Rezazadeh Nutrition Research Center, School of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

Roberta Masella Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy

Michael Ries Department of Plant Physiology, Worringer Weg 1, Aachen, Germany; LumiBioSciences, c/o Worringer Weg 1, Aachen, Germany

Melissa M. Melough Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

Ana Lu´cia S. Rodrigues Department of Biochemistry, Center of Biological Sciences, Federal University of Santa Catarina, Santa Catarina, Brazil

Fahimeh Mohammadghasemi Cellular & Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran Morgana Moretti Department of Biochemistry, Center of Biological Sciences, Federal University of Santa Catarina, Santa Catarina, Brazil Rosa M. Ojeda-Amador Department of Food Science and Technology, Faculty of Chemistry, University of CastillaLa Mancha, Ciudad Real, Spain Edison Osorio Institute of Biology, Faculty of Exact and Natural Sciences, Universidad de Antioquia UdeA, Medellı´n, Colombia ¨ zta¸s Department of Biochemistry, Faculty of Ye¸sim O Medicine, Hacettepe University, Ankara, Turkey Daniela Pala School of Nutrition, Federal University of Ouro Preto, Minas Gerais, Brazil Porntip Pasukamonset Thailand

Life Center, Q House, Bangkok,

Vinood B. Patel School of Life Sciences, University of Westminster, London, United Kingdom Rafael Posada-Duque Institute of Biology, Faculty of Exact and Natural Sciences, Universidad de Antioquia UdeA, Medellı´n, Colombia; Cellular and Molecular Neurobiology Area, Group of Neuroscience of Antioquia, Faculty of Medicine, Universidad de Antioquia UdeA, Medellı´n, Colombia Victor R. Preedy Department of Nutrition and Dietetics, School of Life Course Sciences, King’s College London, London, United Kingdom

Lorenzo Romano Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Pritam Sadhukhan Division of Molecular Medicine, Bose Institute, Kolkata, India Junichi R. Sakaki Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States Veronica F. Salau Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa Marı´a Desamparados Salvador Department of Food Science and Technology, Faculty of Chemistry, University of Castilla-La Mancha, Ciudad Real, Spain Olakunle Sanni Department of Biochemistry, School of Life Sciences, University of Kwazulu-Natal (Westville Campus), Durban, South Africa Carmela Santangelo Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy Beatrice Scazzocchio Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy Pallav Sengupta Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Selangor, Malaysia Parames C. Sil Division of Molecular Medicine, Bose Institute, Kolkata, India

xiv

List of contributors

Annalisa Silenzi Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy

Eva Tvrda´ Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Carol Cristina Va´gula de Almeida Silva Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

Rosaria Varı` Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy

Kemal Tekin Ophthalmology Department, Ercis State Hospital, Van, Turkey

Aristidis S. Veskoukis School of Health Sciences, Department of Biochemistry and Biotechnology, University of Thessaly, Mezourlo, Greece

Merve Inanc Tekin Ophthalmology Department, Ercis State Hospital, Van, Turkey Punniyakoti Veeraveedu Thanikachalam GRT Institute of Pharmaceutical Education and Research, Tiruttani, India Seth P. Thibado Department of Chemistry, Union University, Jackson, TN, United States Jerry T. Thornthwaite Cancer Research Institute of West Tennessee, Henderson, TN, United States Kyle A. Thornthwaite Cancer Research Institute of West Tennessee, Henderson, TN, United States

Osamu Wada-Hiraike Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Liwen Wang Lincoln University, Department of Food Science, Lincoln, New Zealand; Tianjin University of Commerce, Biotechnology and Food Science, Tianjin, P.R. China; Riddet Institute, Palmerston North, New Zealand

Preface

or otherwise. However, in this book the science of oxidative stress is not described in isolation but in concert with other processes such as apoptosis, cell signaling, receptor mediated responses, and so on. This approach recognizes that diseases are often multifactorial and oxidative stress is a single component of this. In the book, each chapter has a section that describes other areas of pathology so that there is holistic coverage and applicability of the information. The book imparts holistic information with a structured format.

Pathology is the study and science of disease and usually encompasses specific subfields from cells to the whole body. In the clinical setting, pathologists are specialists who may focus on one or more areas. For example, The Royal College of Pathologists has over 23 subspecialties, including histopathology. neuropathology, cellular pathology, molecular pathology, cytopathology, pediatric pathology, forensic pathology, clinical biochemistry (chemical pathology), hematology immunology, medical microbiology, virology, clinical cytogenetics, molecular genetics, oral-maxillofacial pathology, toxicology, and clinical embryology. Some areas of pathology overlap and the affected individual may have more than one manifestation of the disease, requiring attention by more than one practitioner. Very often, oxidative stress is a feature of pathologies and affects different cells and organs in a number of ways. These can be manifested as changes in biomarker profiles (clinical biochemistry) or stained tissue sections of biopsies pre-or postmortem (histopathology). Oxidative stress can also arise due to nutritional imbalance before the onset of disease, at the point of diagnosis, and as a result of therapy. There is thus a fundamental need to understand the processes inherent in the oxidative stress of disease. This is important as oxidative stress can be ameliorated with pharmacological, nutraceutical, or natural agents. Food-based antioxidants, however, circumvent the side effects associated with pharmacological antioxidants or supplements. While pathologists and clinical workers understand the processes in disease, they are less conversant in the science of nutrition and dietetics. On the other hand, nutritionists and dietitians are less conversant with the detailed clinical background and science of pathology. Thus pathologists, healthcare workers, food scientists, and nutritionists are separated by divergent skills and professional disciplines that need to be bridged in order to advance medical science. Hitherto, this transdisciplinary divide has been difficult to bridge. Pathology: Oxidative Stress and Dietary Antioxidants aims to cover, in a single volume, the science of oxidative stress in disease and the potentially therapeutic usage of antioxidants in the diet, food matrix, plants,

Part I: Oxidative Stress and Pathology Part II: Antioxidants and Pathology Part III: Techniques and Resources Each chapter has a section called “Applications to Other Areas of Pathology.” Part I, Oxidative Stress and Pathology, covers free radicals (what they are), various disease, and cellular biochemistry. In Part II, Antioxidants and Pathology, we describe numerous agents and their actions. The caveat of the chapters in Part II is that there needs to be further in-depth analysis of these components in terms of safety and efficacy as some material is exploratory or preclinical. A cautionary and critical approach is needed. Nevertheless, the material in Part II can provide the framework for further in-depth analysis or studies. This would be via well-designed clinical trials or via the analysis of pathways, mechanisms, and components in order to devise new therapeutic strategies. Part III, Techniques and Resources, is more practically based. The book is designed for nutritionists, dietitians, food scientists, pathologists, pathology residents and professionals, physicians, medical students, healthcare workers, the healthcare industry, and research scientists. The academic level ranges from students, undergraduate, post graduates, lecturers, and professors. Contributions are from leading national and international experts, including those from world-renowned institutions. Victor R. Preedy

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P A R T

I

Oxidative Stress and Pathology

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C H A P T E R

1 Free radicals: what they are and what they do Eva Tvrda´ and Filip Benko Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

List of abbreviations
SH 1 O2 1 Δg 1 εg 4-HNA 8-oxoG As CAT Cd CH2 Co Cr Cu Fe FR G3PDH GPx H2O2 Hg HOCl L• LOO• LPO MDA Mn NADPH NO• NOS NS O2•2 O3 OH• ONOO2 ONOOCO22 ONOOH OONO2 OS Pb RNS RO• ROO• ROOH

ROS SOD

sulfhydryl group singlet oxygen delta state sigma state 4- hydroxynonenal 7, 8-dihydro-8-oxo-guanosine arsenic catalase cadmium methylene cobalt chromium copper iron free radical glyceraldehyde-3-phosphate dehydrogenase glutathione peroxidase hydrogen peroxide mercury hypochlorous acid lipid radical lipid peroxyl radical lipid peroxidation malondialdehyde manganese nicotinamide adenine dinucleotide phosphate nitric oxide nitric oxide synthases nitrosative stress superoxide radical ozone hydroxyl radical peroxynitrite nitroso peroxo carboxylate peroxynitrous acid peroxynitrite oxidative stress lead reactive nitrogen species alkoxyl radical peroxyl radical lipoperoxide

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00001-9

reactive oxygen species superoxide dismutase

Introduction Oxygen is a crucial element for life on Earth as it plays a dominant role in the controlled oxidation of molecules containing carbon, subsequently leading to the generation of energy essential for the survival of all aerobic systems. Interestingly, its oxidative properties make oxygen exhibit conflicting behavior in that it is essential for life while it can also aggravate cellular damage by a variety of oxidative events.1 Aerobic cells are persistently facing this so-called oxygen paradox: while oxygen is imperative to sustain aerobic life, at the same time, it may become highly toxic to cell survival.2 This “dark side” of oxygen relates directly to the fact that each oxygen atom has one unpaired electron in its outer valence shell, while molecular oxygen has two unpaired electrons. The reductive properties of the cellular environment provide numerous opportunities for oxygen to undergo unexpected univalent reduction. As such, aerobic metabolism leads to the generation of a diversity of by-products called free radicals (FRs),1
3 which, under normal circumstances, are necessary for a normal cell survival.4 On the other hand, if FR levels become too high, either because of their overproduction or due to a low antioxidant capacity, oxidative stress (OS) emerges that often has fatal consequences on cell function.2,5 Prior to the 1970s, FRs were widely overlooked either because of lack of evidence concerning their existence in a living system or because they were considered to be generally insignificant. Regardless of little attention received from the scientific community for

3

© 2020 Elsevier Inc. All rights reserved.

4

1. Free radicals: what they are and what they do

decades, their existence, formation, and importance in a diversity of diseases is now widely accepted.5

reactions in living systems.2
5 An overview of the most common ROS and RNS is provided in Table 1.1.

Free radicals: general characteristics

Oxidative chain reactions

A FR is defined as any atom, molecule, or a fragment of an atom or molecule that contains at least one unpaired electron and exhibits, to a certain extent, an independent existence.2 FRs may be created by the (1) addition of a single electron to a neutral atom or molecule, or by the (2) loss of one electron from a neutral atom or molecule:

Much of the potency of FRs stems from their natural tendency to be involved in oxidative processes occurring during a chain reaction. FR reactions have three distinct identifiable phases1:

A-minus one electron-A1 B-plus one electron-B2 Additionally, FRs may also be created by the (3) homolysis of covalent bonds that may lead to the creation of intermediate reactive by-products. This process requires energy such as heat, UV light, or ionizing radiation. FRs may have positive, negative, or neutral charges.3,4 The presence of an unpaired electron results in certain common properties shared by most radicals. FRs are generally unstable and highly reactive. They can either donate an electron to, or accept an electron from, other molecules, therefore behaving as oxidants or reductants, respectively.2,5 Since they have an unpaired electron, they are electrophilic and attack sites of increased electron density, which are usually present in compounds with nitrogen atoms (proteins, nucleic acids) and carbon
carbon double bonds (phospholipids, polyunsaturated fatty acids (PUFAs)).4 During such an electron interchange, new radicals are formed from previously nonradical molecules and chain reactions may occur. In cells, one-electron modification of molecules can yield sulfur-, oxygen-, carbon-, and nitrogen-centered FRs.4 Furthermore, ions of transition metals have a radical nature (Cu21, Fe21, etc.).2 The majority of FRs have a very short half-life, which is why it is very difficult to synthesize, store, quantify, or use them in experimental or clinical settings.2 The most common and important FRs related to biology are oxygen-centered radicals, called reactive oxygen species (ROS) and nitrogen-centered molecules, called reactive nitrogen species (RNS).3 ROS and RNS can be further classified into two groups of compounds; radicals and nonradicals. Radicals are species that contain at least one unpaired electron in the outer shells around the nucleus and are capable of existing independently. Nonradical species are not FRs per se, but can easily lead to oxidative

1. Initiation: initial creation of FRs. Usually it is a hemolytic cleavage with the assistance of heat, UV, or metal catalysts. 2. Propagation: FRs are generated and regenerated repeatedly as a result of the chain reaction. 3. Termination: two FRs react with each other to form a stable, nonradical product.

Selected free and nonfree radicals: characteristics and behavior Superoxide radical (O2•2) The recognition that living systems produce significant amounts of superoxide through normal metabolic pathways and that enzymes, particularly superoxide dismutase (SOD), help protect cells against the potential toxicity of this FR,6 represents the pillars of why FR-associated biology was considered to be “superoxide-centric” for decades. O2•2 is the principal FR generated as a result of a monovalent reduction of oxygen and the addition of one electron4,5 by enzymatic processes, autooxidation, or by nonenzymatic electron transfers.2 O2 1 e2 -O2 2 O2•2 is mostly produced within the mitochondria by xanthine oxidase,7 lipoxygenase, cyclooxygenase,8 or NADPH-dependent oxidase. Superoxide does not react with most molecules in aqueous solutions. Its reactivity with nonradical molecules is pH-dependent. The radical is a stronger reductant than oxidant.2 As a reducing agent, O2•2 reacts with iron complexes such as cytochrome and reduces Fe31 to Fe21: O2 1 e2 -O2 2 O2 1 Fe12 -Fe13 1 O2 2 ðauto 2 oxidationÞ As an oxidant, O2•2 can oxidize transition metal ions (Cu31, Fe41, Mn31), dopamine, adrenaline, or ascorbate, leading to the formation of hydrogen peroxide.2 Superoxide radicals react with another superoxide radical in a dismutation reaction, in which one radical

I. Oxidative Stress and Pathology

5

Selected free and nonfree radicals: characteristics and behavior

TABLE 1.1 Reactive oxygen and nitrogen species with relevance in biology and medicine. Reactive oxygen species (ROS) Radical species

Nonradical species

Name

Formula

Name

Formula

Superoxide

O2•2

Hydrogen peroxide

H2O2

Hypochlorous acid

HOCl

Hypobromous acid

HOBr

•

Hydroxyl radical

OH

•

Peroxyl radical

ROO •

Alkoxyl radical

RO

Ozone

O3

Hydroperoxyl radical

HO2•

Singlet oxygen

1

Lipid peroxide

LOOH

Lipid peroxyl radical

•

LOO

O2

Reactive nitrogen species (RNS) Radical species Name

Nonradical species Formula •

Name

Formula

Nitric oxide

NO

Nitrous acid

HNO2

Nitrogen dioxide

NO2•

Nitrosyl cation

NO1

Nitroxyl anion

NO2

Dinitrogen tetroxide

N2O4

Dinitrogen trioxide

N2O3

Peroxynitrite

ONOO2

Peroxynitrous acid

ONOOH

Nitronium (nitryl) cation

NO21

Nitryl chloride

NO2Cl

Alkyl peroxynitrite

ROONO

is oxidized to oxygen while the other is reduced to hydrogen peroxide.9 O2 1 O2 2 1 2H2 O-H2 O2
1 O2 catalyzed by Cu; Zn; Mn 2 SOD The primary toxicity of O2•2 is associated with its ability to initiate oxidative chain reactions, followed by the generation of other reactive metabolites such as hydrogen peroxide, hydroxyl radical, singlet oxygen, or peroxynitrite (ONOO2).2,5

Hydrogen peroxide (H2O2) Hydrogen peroxide is a two-electron product of oxygen reduction that is able to participate in signal transduction, cell proliferation, differentiation, and response to stress.2 H2O2 is primarily formed in a dismutation reaction catalyzed by SOD, but it can also be produced by other enzymes such as xanthine, glucose monoamine, and D-amino acid oxidases, or during ascorbate and

polyphenol oxidation.1 The molecule has no unpaired electrons and, hence, is not a radical in nature,2 but it can cause significant damage to cells even at low concentrations (10 μM). H2O2 is soluble in water and has the ability to easily penetrate through biological membranes.5 Furthermore, it can directly damage some enzymes such as glyceraldehyde-3-phosphate dehydrogenase (G3PDH) through oxidation of the sulfhydryl (
SH) group.10 Within the cell, H2O2 has the ability to accumulate within the cell and interact with iron and copper ions leading to significant damage to lipids, deoxyribonucleic acid (DNA), and proteins through the hydroxyl radical emerging from interactions between H2O2 and transition metals.2,5

Hydroxyl radical (OH•) Hydroxyl radicals are defined as the neutral form of the hydroxide ion and are highly reactive FRs2 that can aggressively react with organic and inorganic

I. Oxidative Stress and Pathology

6

1. Free radicals: what they are and what they do

molecules and cause more severe damage to the cell than any other FR.10 OH• is created in the Fenton reaction as a result of interactions between H2O2 and metal ions (Fe12 or Cu1) often bound in complex forms with different proteins such as ferritin or ceruloplasmin.11 Under stress conditions, excessive O2•2 may release these metal ions from their respective complexes. OH• may also be created by the Haber
Weiss reaction comprising O2•2 and H2O2 interactions.12 Fe12 1 H2 O2 -Fe13 1 OH 1 OH2 ðFenton reactionÞ 2 O2 1 H2 O2 -O2 1 OH 1 OH2 ðHaber 2 Weiss reactionÞ Other sources of OH• are also known, such as reactions with hypochlorous acid, quinones, and semiquinones.5 Hydroxyl radicals are particularly dangerous because of their ability to reduce disulfide bonds in proteins, above all fibrinogen, resulting in their unfolding and scrambled refolding into abnormal spatial configurations. Consequences of such reactions may be translated into numerous disorders, such as atherosclerosis, cancer, and neurological pathologies.13

Alkoxyl (RO•) and peroxyl (ROO•) radicals Alkoxyl and peroxyl radicals are products of lipid peroxidation (LPO) following the decomposition of lipoperoxides (ROOH) by UV and/or heat in the presence of transition metal ions.2 ROOH 1 Fe31 -ROO 1 Fe21 1 H1 ROOH 1 Fe21 RO 1 Fe31 1 OH2

These observations suggest that 1O2 may be a significant biochemical intermediate in a number of biological processes. Moreover, 1O2 may be generated from ozone and cysteine, methionine, glutathione, albumin, ascorbic acid, and NAD(P)H.10,16 1 O2 is a highly powerful oxidizing agent that can cause DNA17 and tissue damage,15 particularly in skin and eyes. Exposure to light containing high levels of 1 O2 may lead to the accumulation of porphyrins in the skin thereby causing swelling, blistering, eruptions, and scarring.5,10,14

Ozone (O3) Ozone is a powerful oxidant that may be formed by catalyzed water oxidation, which plays important roles in inflammation.18 O3 has the ability to generate other reactive intermediates by oxidizing specific molecules and functional groups such as lipids, nucleic acids, and amines, alcohols, aldehydes, and sulfhydryl present in proteins.19 Exposure to O3 may also cause chromosomal aberrations due to a direct attack by O3 or by other FRs generated as a result of its reactions.2,19

Hypochlorous acid (HOCl) Hypochlorous acid is a major oxidant created from hydrogen peroxide and chloride within the activated neutrophils at the site of inflammation. The reaction is catalyzed by the enzyme myeloperoxidase.2,5 H2 O2 1 Cl2 -HOCl 1 OH

10

Both organic radicals are relatively good oxidants with the ability to subtract hydrogen from other lipid molecules, leading to the branching of LPO. Such chain reactions may also proceed to generate new FRs (e.g., singlet oxygen) and can promote tumor development.14

Singlet oxygen (1O2) Singlet oxygen is a highly excited, metastable, nonradical state of molecular oxygen and a highly reactive and toxic ROS.2 1O2 can exist in two states: (1) upon activation, the molecular oxygen is excited to the first delta state (1Δg) and, then, (2) to an even higher excited singlet state, the sigma state (1εg). The 1Δg state is extremely reactive and is normally produced by the activation of neutrophils and eosinophils.15 It is also formed during selected enzymatic reactions catalyzed by enzymes such as lipoxygenases, dioxygenases, and lactoperoxidase.16 HOCl 1 H2 O2 -1 O2 1 H2 O 1 Cl2

HOCl is a potent ROS involved in oxidation and chlorination processes. It can pass through membranes, causing damage to membrane proteins and lipids as well as intracellular biomolecules. It has the ability to oxidize thiols, urate, and ascorbate.20 HOCl chlorinates amines to give chloramines; tyrosyl residues creating ring chlorinated products, cholesterol, and unsaturated lipids to form chlorohydrins.20,21 It also chlorinates pyrimidine bases in DNA and tyrosine units in proteins, leading to protein and DNA fragmentation by a variety of mechanisms.10 Furthermore, HOCl produced by activated neutrophils may interact with superoxide, leading to the formation of OH•. Reactions between HOCl and H2O2 may lead to 1O2 generation.2

Nitric oxide (nitrogen monoxide; NO•) NO• is a colorless gas that is synthetized by various nitric oxide synthases (NOS) while converting L-arginine to L-citrulline.22 There are three types of NOS

I. Oxidative Stress and Pathology

Sources of free radicals

isoforms: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS; NOS2), and endothelial NOS (eNOS, NOS3), all of which are involved in the formation of NO•. L 2 Arginine 1 O2 1 NADPH-L 2 Citrulline 1 NO
1 NADP1 catalyzed by NOS The molecule is soluble in both water and lipids, which is why it is able to readily diffuse through the plasma membrane and cytoplasm.23 Under physiological conditions NO• is an important intracellular second messenger that is involved in smooth muscle relaxation within blood vessels. NO• also plays important roles in the regulation of cellular redox states and enzymatic activities by protein nitrosylation.23,24 Moreover, it is involved in numerous other biological processes such as blood pressure and immune regulation, platelet aggregation, lung vasodilatation, as well as neurotransmission and defensive mechanisms.24 To a certain extent NO• may be regarded as an FR scavenger as it interacts with hydroxyl, peroxyl, or thiyl radicals leading to the generation of nitric acid, nitrosoperoxyl, or nitrosothiol, respectively, with the latter being responsible for the inhibition of LPO by NO•. However, at high concentrations ( . mmol/L), NO• may be involved in the pathology of septic shock, chronic inflammation, increased risk for oncogenesis, transplant rejection, asthma, epilepsy, and neurodegenerative diseases.10

Other reactive nitrogen species Peroxynitrite (oxoperoxonitrate; OONO2) emerges from the reaction between O2•2 and NO•. OONO2 may diffuse to more distant places in the cell where lipids, proteins, or DNA can be oxidized and nitrated.2 The molecule is highly toxic25 and can directly react with CO2 to form highly reactive nitroso peroxo carboxylate (ONOOCO22) or peroxynitrous acid (ONOOH). ONOOH undergoes homolysis to form OH• and NO2, or rearranges to form NO3. OONO2 may oxidize lipids, methionine, and tyrosine residues in proteins and DNA to form nitroguanine.2
5 Tyrosine nitration in proteins is generally used as a biomarker of peroxynitrite formation.

Sources of free radicals FRs have long been proposed to play only negative roles in organisms, particularly when produced in large amounts, at the wrong place, or when the defense mechanisms of the organism designed to counteract their toxic effect fail.26

7

FRs may originate from endogenous or exogenous sources. The most prominent endogenous sources include organelles that have high requirements for oxygen and are illustrated in Fig. 1.1.

Mitochondria Mitochondria are the main source of intracellular FRs. Superoxide as a by-product of normal mitochondrial metabolism is produced at two major sites within the electron transport chain, specifically complex I (NADH dehydrogenase) and complex III (ubiquinone cytochrome c reductase). The generation of superoxide is nonenzymatic and increases with the metabolic rate.27 O2•2 is subsequently converted to H2O2 by the mitochondrial SOD. H2O2 can be further detoxified by catalase (CAT) and glutathione peroxidase (GPx). Other mitochondrial components that may contribute to FR generation include monoamino oxidase, α-ketoglutarate dehydrogenase, glycerol phosphate dehydrogenase, and p66shc.2,5,26,27

Peroxisomes The peroxisomal respiratory pathway involves the transfer of electrons from various metabolites, primarily fatty acids, to oxygen and resulting in the formation of H2O2.28 However, energy is released in the form of heat. Other FRs produced in peroxisomes include O2•2 OH• and NO•. Different peroxisomal enzymes such as acyl CoA oxidases, D-amino acid oxidase, L-α-hydroxy oxidase, urate oxidase, xanthine oxidase, and D-aspartate oxidase have also been shown to produce different FRs.5,28

Endoplasmic reticulum Various enzymes such as cytochrome p-450, b5, and diamine oxidase contribute to FR generation within the endoplasmic reticulum. A considerate proportion of H2O2 is created by the action of the important thiol oxidase enzyme, Erop1p, which catalyzes the transfer of electrons from dithiols to molecular oxygen.5,29 Other relevant endogenous sources of FRs include metal-catalyzed reactions, prostaglandin synthesis, autoxidation of adrenalin, reduced riboflavin, FMNH2, FADH2, xanthine oxidase, cascade of arachidonic acid, immune cells, stress, excessive exercise, cancer, aging, ischemia-reperfusion, atherogenesis, hemodialysis, and so forth.1
3 On the other hand, FRs may enter biological systems from diverse exogenous sources,1
5,26 among which the most notable ones are outlined in Fig. 1.2.

I. Oxidative Stress and Pathology

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1. Free radicals: what they are and what they do

FIGURE 1.1 Typical endogenous sources of free radicals. FIGURE 1.2 Most common exoge-

Exogenous sources of free radicals

nous sources of free radicals.

Air polution, automobile exhausts and fumes

UV and microwave radiation, X-rays, gamma rays

Smoking

Free radicals

Alcohol

Forest fires, volcano activities

Water polution

Oxidative stress A living organism is a well-organized and sophisticated system where FRs act under strict control and at specific locations only. If FRs have already been generated and/or have caused damage, the organism’s intricate system designed to protect it against the toxic effects of reactive metabolites readily recognizes damaged molecules and quickly reacts to remove any structures that had

been irreparably altered. However, if one of the protective mechanisms of the organism against FRs fails, their action becomes uncontrollable, causing damage to molecules and cells, thereby leading to the death of the organism.2,14,26 When there is an imbalance between the FR production and/or concentration (ROS/RNS) and antioxidant defense systems, the former will be produced at higher levels leading to OS and/or nitrosative stress (NS). Since FRs are highly reactive, their targets comprise all

I. Oxidative Stress and Pathology

Oxidative stress

Xenobiotics

Pathological disorder

Free radicals

9

FIGURE 1.3 Circumstances leading to free radical/ROS overproduction and their implications on the living system.

Metabolic processes Reactive oxygen species Antioxidants Oxidated nucleic acids Lipid peroxides Oxidated nucleotides

Alkanes Aldehydic products

Oxidated amino acids

Conjugated dienes

important classes of biological molecules including proteins, lipids, and nucleic acids.10,14 The impact of OS/NS depends on the type of oxidant, the location and intensity of its production, the composition and activities of relevant antioxidants, and the ability of repair systems.2,5 A summary of causes and consequences of FR and/or ROS overproduction are provided in Fig. 1.3.

Proteins Protein oxidation may be caused by a variety of radical species such as O2•2, OH•, RO•, ROO•, and nonradical species such as H2O2, O3, HOCl, 1O2, and OONO2. FR oxidize different amino acids, causing formation of protein cross-links, protein denaturation, and the loss of their functionality, decreased enzymatic activity, and altered function of receptors and transport proteins.30 Amino acids containing sulfur such as methionine and cysteine are more susceptible to oxidation and may be easily converted to disulfides and methionine sulfoxide.30,31 Oxidative insults to amino acids may result in the formation of different oxidation products such as nitrotryptophan, kynurenine, 2-, 3-, and 4-hydroxyphenylalanine; tyrosine-tyrosine cross-links, 2-oxohistidine, asparagine, aspartic acid, glutamic semialdehyde, and 2-amino-3ketobutyric acid.31 Moreover, oxidative damage to amino acid residues such as lysine, proline, threonine, and arginine leads to carbonyl derivatives. The presence of protein carbonyls has been proposed as a marker of FR-mediated protein oxidation.32 Other specific markers of protein oxidation comprise O-tyrosine (a marker for OH•) and 3-nitrotyrosine (a marker for RNS). Increased levels of protein carbonyls may be observed in a number of pathological conditions such as Alzheimer’s disease and Parkinson’s disease, muscular dystrophy, cataracts, diabetes, and atherosclerosis.32
35

Deoxyribonucleic acid ROS and RNS can oxidatively damage nucleic acids. Generally speaking, mitochondrial DNA is more susceptible to oxidative insults than nuclear DNA as it is located in close proximity to the primary source of the FR. ROS, most importantly OH•, directly interacts with all the components of DNA, causing a number of alternations including singleand double-stranded breaks. The radical is able to extract hydrogen to create a number of modified purine and pyrimidine by-products as well as DNAprotein cross-links. The most commonly observed pyrimidine adducts comprise thymine glycol, uracil glycol, and hydantoin. The purine adducts frequently observed under OS/NS conditions include 8hydroxydeoxy guanosine, 8-hydroxy deoxy adenosine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Predominant adducts from the sugar moiety in DNA include glycolic acid, 2-deoxytetrodialdose, and erythrose.36 8-hydroxy deoxyguanosine is considered to be the leading biomarker of oxidative DNA damage and is frequently involved in mutagenesis, carcinogenesis, and ageing.37 On the other hand, RNS, most importantly OONO2, interacts with guanine to create nitrative and oxidative DNA lesions such as 8-nitroguanine and 8oxodeoxyguanosine, respectively.5,36,37 Accordingly, 8nitroguanine is believed to be a mutagenic DNA lesion involved in carcinogenesis.

Ribonucleic acid Ribonucleic acid (RNA) is more prone to oxidative damage than DNA due to its single-stranded structure and lack of effective repair mechanisms and protection. Furthermore, cytoplasmic RNAs are located in close proximity to the mitochondria where significant amounts of ROS are produced.38 7, 8-dihydro-8-oxo-guanosine

I. Oxidative Stress and Pathology

10

1. Free radicals: what they are and what they do

FIGURE 1.4 Most common disease states triggered, or accompanied, by free radical overproduction.

Cancer

Joints

Brain

rheumatoid anthritis psoriatic arthritis

Alzheimer's disease Parkinson's disease

Lungs

Heart

asthma allergies

heart attack stroke high blood pressure

Kidney

Blood vessels

chronic kidney disease renal nephritis

atherosclerosis hypertension varicose veins

Reproduction infertility

(8-oxoG) is the most extensively studied RNA damage product and its levels are elevated in numerous pathological conditions including Alzheimer’s disease, Parkinson’s disease, hemochromastosis, atherosclerosis, and myopathies.39
41

Lipids Membrane lipids, particularly PUFA residues of phospholipids, are the substrate of choice for oxidation. LPO results in the loss of membrane functionality that is often accompanied by decreased fluidity and the inactivation of membrane-bound enzymes and receptors.2,5 LPO is initiated when FRs abstract hydrogen from a methylene group (CH2) in a fatty acid, which results in the formation of a carbon-centered lipid radical (L•). The lipid radical can easily interact with molecular oxygen to create a lipid peroxyl radical (LOO•). Subsequently, LOO• undergoes cyclic rearrangements to create endoperoxides that form malondialdehyde (MDA) and 4-hydroxyl nonenal (4-HNA), which may cause damage to DNA and proteins. Such lipid radicals may further propagate the peroxidation process by abstracting hydrogen from other lipid molecules.42

Free radicals in health and disease FR overproduction and the subsequent OS have been associated with a wide variety of pathologies, such as diabetes mellitus43 and metabolic syndrome44; atherosclerosis and cardiovascular diseases45; skin and

tumor diseases46,47; neurodegenerative diseases, including Alzheimer’s and Parkinson’s48; autoimmune disorders49; male and female infertility50,51; as well as psychological impairments such as bipolar disorder, schizophrenia, or attention deficit hyperactivity disorder.52 The most prominent pathologies triggered or accompanied by OS are provided in Fig. 1.4. Nevertheless, FRs and reactive intermediates are necessary for a number of cell functions and their deleterious effects come into place only when produced in excess. As such, FRs as well as their antioxidant counterparts may play various roles in the living system, some being beneficial while others are harmful. It must be emphasized that a complete suppression of FR formation is not desirable, which is why, under physiological conditions, controlled and moderate FR production is required.2,14,26,53,54 A short summary of the most prominent physiological roles of FRs is provided in Table 1.2. Depending on the type and concentration of FR, exposure time, and activation of antioxidant defense mechanisms and repair systems, cells exposed to oxidative insults may respond in three ways: 1. Adaptation: cells will upregulate their antioxidant defense systems. 2. Tissue injury: increased OS may cause damage to all molecular targets. The exact point of attack may often be unclear as injury mechanisms tend to overlap. Cells may respond in different ways; increased proliferation, halted cell cycle and senescence are often observed. 3. Cell death: this can occur by two mechanisms, necrosis and apoptosis. In necrotic cell death, the

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Summary points

TABLE 1.2 Physiological roles of reactive oxygen/nitrogen species in the regulation of cellular function. Physiological function

Reactive species involved

Phagocytosis (macrophages)

Superoxide, hydrogen peroxide, nitric oxide

Respiratory burst (neutrophils): immune response, protection against bacterial infection

Superoxide, hydrogen peroxide, peroxynitrite, hypochlorous acid, hypochlorite

Biochemical reactions (hydroxylation, carboxylation, peroxidation, reduction of ribonucleotides)

Hydroxyl radical, hydrogen peroxide

Spermatogenesis, capacitation, acrosome reaction, fertilization

Superoxide, hydrogen peroxide

Control of vascular tone and platelet adhesion

Nitric oxide

Signal transduction and intercellular communication

Superoxide, nitric oxide

Cellular growth, differentiation and proliferation

Nitric oxide, hydrogen peroxide

Apoptosis

Hydrogen peroxide

Acclimation to stress conditions

Superoxide, hydrogen peroxide

cell swells, ruptures, and releases its contents into the surrounding area significantly affecting neighboring cells. The intracellular content may comprise antioxidants such as catalase or glutathione, as well as oxidants such as copper and iron ions. Even if a cell enters necrosis by other than FR-mediated mechanisms, necrotic cell death usually leads to further oxidative damage to the adjacent tissue. In apoptosis, the cell’s intrinsic “suicide mechanisms” are activated; apoptotic cells do not release their contents and, thus, apoptosis does not, in general, cause further damage to surrounding cells.2,3,5,10,14 Based on the currently available information on FRs and the redox state of organisms, we may say that generated FRs and antioxidants play major roles in the cellular oxidative
reduction processes, thereby constantly changing the redox state of the cells. Such changes stimulate or inhibit the activities of communication molecules and regulatory proteins, leading to changes in the ability of signal pathways to control cellular fate. While an oxidative environment is more suitable for cell death by apoptosis or necrosis, a reducing milieu supports cell survival.2,5,54

Conclusion In conclusion, the oxidative state of a cell cannot be perceived as just “black or white.” FRs as well as antioxidants have regulatory functions in diverse physiological and pathological processes, precisely via a continuous modulation of the redox state of the organism. Nevertheless, currently we do not know where the exact border between their beneficial and harmful effects is.

What such effects are depend on how we may benefit from modulating the effects of FRs, reactive metabolites, and antioxidants by regulating such determinants and their balance within the cellular redox state.

Applications to other areas of pathology Understanding the chemistry and behavior of FRs has illuminated the nature of OS and its consequences for the organism. This, in turn, has already revealed possibilities to improve human health either by avoiding FR overproduction or by using antioxidants. Unraveling the specific nature of the cellular redox balance may offer new clues to define the fine line dividing the effects of FRs into positive and negative consequences. Antimicrobial and cytotoxic effects of FRs may be exploited particularly in anticancer therapies such as radiotherapy, chemotherapy, and immunotherapy. Since tumor cells contain fewer FR scavengers, it might be of interest to look for a way to produce large amounts of FRs in tumor tissues for future anticancer therapy. Furthermore, elucidation on the susceptibility of specific FRs toward antioxidant mechanisms of action may lead to the discovery of more disease-specific, target-directed, highly bioavailable antioxidants applicable for the management and/ or treatment of a variety of diseases associated with OS.

Summary points • Because of its unique characteristics, oxygen exhibits a double-edged behavior in aerobic cells. • FRs are by-products of normal aerobic metabolism.

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1. Free radicals: what they are and what they do

• FRs play important roles in fertilization, immune response, and cell signaling. • An imbalance between FR production and antioxidant defense mechanisms leads to OS with detrimental effects on cell function and survival. • It is crucial to understand the exact boundary between positive and negative effects of FRs in health and disease.

Acknowledgment This chapter was supported by the Slovak Research and Development Agency grant no. APVV-15
0544 and by the KEGA grant no. 009SPU-4/2018.

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14. 21. Prutz WA. Hypochlorous acid interactions with thiols, nucleotides, DNA, and other biological substrates. Arch Biochem Biophys 1996;332:110
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304. 30. Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997;324:1
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C H A P T E R

2 Oxidative stress in pathologies of male reproductive disorders Ashok Agarwal1, Kristian Leisegang2 and Pallav Sengupta3 1

American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States 2School of Natural Medicine, University of the Western Cape, Cape Town, South Africa 3Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Selangor, Malaysia

List of abbreviations 3βHSD 4-HNE AR ATP BMI CAT CVD CYP17 DNA ED FR GnRH GPx H2O2 HIV HPG IFN-Ɣ IL-1β JNK LPO LDL MDA MMP MNDs mtDNA MTHFR NADPH NO NOS O2 dO22 ORP OS P450scc PUFA Redox ROS

SDF SOD StAR T2DM TAC TBARS TH-1 TH-2 TNF-α WHO

3β-hydroxysteroid dehydrogenase 4-hydroxynonenal acrosome reaction adenosine triphosphate body mass index catalase cardiovascular disease 17α-hydroxylase/17, 20-lyase deoxyribose nucleic acid erectile dysfunction free radical gonadotropin releasing hormone glutathione peroxidase hydrogen peroxide human immunodeficiency virus hypothalamic-pituitary-gonadal interferon-gamma interleukin 1-beta c-Jun N-terminal kinase lipid peroxidation low density lipoprotein malondialdehyde mitochondrial membrane potential micronutrient deficiencies mitochondrial DNA methyl tetrahydrofolatereductase nicotinamide adenine dinucleotide phosphate nitric oxide nitric oxide synthase oxygen superoxide radical oxidation-reduction potential oxidative stress P450 cholesterol side-chain cleavage polyunsaturated fatty acid reduction
oxidation reaction reactive oxygen species

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00002-0

sperm DNA fragmentation superoxide dismutase steroidogenic acute regulatory type 2 diabetes mellitus total antioxidant capacity thiobarbituric acid assays T-helper lymphocyte type 1 T-helper lymphocyte type 2 tumor necrosis factor-alpha World Health Organization

Introduction Male factor infertility contributes to almost half of the global prevalence of infertility.1 The etiology of male infertility is still not completely understood. Some of the most deleterious factors that affect male fecundity include environmental, physiological, genetic, and epigenetic changes, dietary habits, and lifestyle factors. A common underlying mechanism by which most of these factors disrupt male fertility is by disturbing the intricate balance between the production and scavenging of reactive oxygen species (ROS). This imbalance of increased ROS generation and reduced antioxidant capacity leads to oxidative stress (OS).2 ROS, the toxic derivates of oxygen metabolism, mediate some important male reproductive functions such as sperm capacitation, hyperactivation, and acrosome reactions.3 However, excess generation of ROS impairs redox balance in the reproductive tract, leading to cellular and molecular damage.4 Spermatozoa are not adequately equipped for cell repair and thus are excessively vulnerable to OS due to the unique

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© 2020 Elsevier Inc. All rights reserved.

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2. Oxidative stress in pathologies of male reproductive disorders

structure of spermatozoa with very little cytoplasmic content.5 They are readily affected by OS-induced lipid peroxidation (LPO) due to their high polyunsaturated fatty acid (PUFA) content in the sperm membrane.6 This culminates in the overall deterioration of semen quality, rendering men subfertile or infertile.7

Redox biology in male reproduction Oxygen (O2) is a paradoxical element, essential for adenosine triphosphate (ATP) generation through oxidative phosphorylation, thereby generating potentially cytotoxic ROS as a by-product of cellular respiration. Additional cellular sources of ROS include endoplasmic reticulum and peroxisomes where there is significant O2 demand and consumption. Common endogenous ROS include hydroxyl radical (dOH), hydrogen peroxide (H2O2), and superoxide radical (dO22). Through regulatory phosphorylation/dephosphorylation (redox) switches, ROS have important cellular signaling mechanisms, mediated through adaption to physiological concentrations.8 This is further regulated through endogenous defense mechanisms called antioxidants that evolved to regulate increased ROS production through the rise of aerobic respiration and the mitochondria. Some of the important antioxidants include the superoxide dismutase (SOD) family, glutathione peroxidase (GPx), and catalase (CAT) peroxiredoxin and thioredoxin.9 A state of OS drives cellular injury and molecular pathophysiology similarly to the general adaption syndrome of cellular stressors.10 This results in an accumulation of cellular injury associated with ageing, and noncommunicable chronic diseases including obesity and metabolic syndrome, cardiovascular diseases (CVDs), type II diabetes mellitus, numerous malignancies, and neurodegeneration.11 With the discovery of ROS in the ejaculate, it was considered to be toxic to the spermatozoa. However, ROS have critical important physiological and regulatory functions in male reproduction12 and have been shown to be critical in male fertility, including regulation of spermatogenesis.13 Through Sertoli cell and macrophage mediated early mitotic divisions of germ cells, subsequent meiotic divisions produce spermatids. These cells undergo chromatin condensation and associated membrane remodeling as part of their transformation into spermatozoa. Germ cells are a significant source of ROS that are critical for the sperm maturation process alongside other signaling functions.14 Following spermatogenesis, ROS are important in epididymal transport and maturation associated mostly with motility. ROS further mediate

postejaculation maturation in the female reproductive tract, in which case redox switches are required for capacitation, acrosome reaction, sperm-zona binding, and oocyte fusion for successful fertilization.13 ROS are generated from spermatids and mature spermatozoa directly through a mechanism of ROSinduced ROS generation. H2O2 has been investigated as a significant mediator of redox biology and switches relevant to spermatogenesis and oocyte binding.15 These have been summarized in Fig. 2.1.

Oxidative stress and male factor infertility The etiopathogenesis of male infertility involve numerous factors. This includes lifestyle and environmental factors, genetic and epigenetic alterations, as well as other physiological factors. Most of these factors, either individually or in combination, interfere with male reproductive functioning through OS.2 This affects the cellular environment by damaging biomolecules (carbohydrates, lipids, proteins, and nucleic acids).4 Spermatozoa have diminutive cytoplasm and thus lack adequate intracellular antioxidant capacity and cell repair machineries. This structural peculiarity render spermatozoa very vulnerable to damage through OS.5 Moreover, spermatozoa cell membranes are rich in PUFA that are susceptible to OS-induced LPO. OS-mediated oxidative damage to spermatozoa leads to impairments of structural and functional integrity that pave the way to male infertility.6,7 Sources of excessive ROS in the male reproductive tract are illustrated in Fig. 2.2.

Sources of reactive oxygen species in the male reproductive tract Endogenous reactive oxygen species and antioxidants Leukocytes Seminal plasma contains a substantial quantity of peroxidase-positive leukocytes (polymorphonuclear leukocytes, chiefly neutrophils, 50%
60%) as well as activated macrophages (20%
30%). These find their origin in the accessory glands like the prostate and seminal vesicles. If the number of leukocytes in the seminal plasma exceeds 1 3 106 WBC/mL of semen, the condition is referred to as leukocytospermia.16 In a state of inflammation, leukocytes generate 100-times greater ROS than that produced in normal state. This response is triggered as a defense mechanism that also induces nicotinamide adenine dinucleotide phosphate (NADPH) production via the hexose monophosphate

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FIGURE 2.1 The redox regulatory roles of reactive oxygen species (ROS) in male spermatogenesis and post-ejaculation maturation of spermatozoa.18
21

FIGURE 2.2 Sources of reactive oxygen species (ROS) and resulting oxidative stress (OS) in the male reproductive tract.

(HMP) shunt.4 Leukocyte-mediated immune response against male urogenital inflammation involves ROS generation as the prime mechanism and is thus responsible for the induction of OS.17 The concomitant

increase in levels of proinflammatory mediators (IL-8, IL-6, and TNFα) together with a decrease in antioxidant capacity during inflammation can trigger respiratory burst, leading to OS.18

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2. Oxidative stress in pathologies of male reproductive disorders

Immature spermatozoa Maturing spermatozoa normally extrude off their cytoplasm while becoming structured for fertilization. Abnormalities in spermiogenesis often result in excess cytoplasm retention in the mid-piece of matured spermatozoa, referred to as excess residual cytoplasm. More cytoplasm renders the spermatozoa with excess regulatory enzymes of glucose metabolism, such as the glucose-6-phosphate dehydrogenase. Greater metabolism leads to increased NADPH production that initiates more ROS generation by the sperm membrane NADPH oxidase.19 Hence sperm with excess cytoplasm, or teratozoospermic sperm, generate greater amounts of ROS than what is produced by morphologically normal spermatozoa. The key enzyme for ROS production in sperm, NADPH oxidase, differs from those in leukocytes, the former being calcium dependent, while in leukocyte it is protein kinase-C dependent.20 Exogenous reactive oxygen species and antioxidants Radiation Evidence increasingly suggests that mobile phone ionizing radiation has adverse effects on semen quality. It elevates ROS generation in the male reproductive tissues, induces sperm deoxyribose nucleic acid (DNA) fragmentation and affects sperm vitality, motility and count, thereby leading to male subfertility or infertility.5 Radiofrequency electromagnetic waves breach the synchronized intracellular electron transfer in the spermatozoa membrane and disrupt their normal functions.21 It affects male reproductive functions via thermal and nonthermal mechanisms. Temperature regulation of testis occurs mainly through surface conduction and, thus, is vulnerable to changes in electromagnetic energy. Scrotal temperature may get elevated, even by just 1 C, thereby disrupting normal spermatogenesis. Radiation may also induce OS or interfere with the sperm membrane potential to cease germ cell development and induce apoptosis. Sperm DNA fragmentation (SDF), various epigenetic modifications of spermatozoa, impairment of steroidogenesis, and degeneration of Leydig cells may result from chronic exposure to radiation.21 Lifestyle factors Unhealthy lifestyle adoptions such as an improper diet, lack of exercise, prolonged working hours in a restricted posture, sleep deprivation, smoking, alcoholism, and other factors that comply with hectic, modern lifestyles cumulate in physical and psychological stress.22 Lifestyle factors are responsible for an array of physiological disorders such as obesity, hypertension,

diabetes, and others, which further leads to the development of chronic diseases.23 The worldwide prevalence and increase in poor lifestyle factors are associated with the global deteriorating trend of male fertility.22,23 Smoking elevates ROS levels beyond endogenous antioxidant capacity. Smoking may be responsible for a 48% increase of leukocytes in the seminal plasma, increasing seminal ROS levels by 107%, and reducing antioxidants with increased 8-hydroxy-20
deoxyguanosine (8-OHdG) as a potent OS biomarker.21 Smoking also increases blood and semen concentrations of cadmium and lead, which further increases production of ROS.21 Chronic smoking, which induces germ cell apoptosis, sperm DNA damage, and impaired sperm motility, lead to compromised male fecundity.24 Excess consumption of alcohol may initiate uncontrolled production of ROS and the rapid depletion of antioxidant capacity. Acetaldehyde, a by-product of ethanol metabolism, reacts with sperm cytoplasmic components to yield ROS and thereby decreases the percentage of functional spermatozoa.25 Toxins With the advancement of sociocultural and economic development, industrialization and domestic sophistication, the use of endocrine disruptors and environmental toxins are rapidly escalating. Accumulation of these environmental toxins may induce overproduction of testicular ROS. This excessive production of FRs through oxidative damage impairs sperm morphology, functions, and drive germ cell apoptosis. Use of plastics, heavy metals, and pesticides affect semen quality and the rate of spermatogenesis by generating OS and interfering with the functions of germ cells and other testicular cells.21

Oxidative stress in male reproductive pathology Genitourinary tract infections One of the most common genitourinary tract infections is prostatitis.26 Prostate infection may incur via bacteria that originate in the urinary tract or through sexual transmission, such as in the case of Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vagilalis.27 Common bacteria that are non-sexually transmitted include streptococci (S. viridans and S. pyogens), gram-negative bacteria (Escherichia coli, Proteus mirabilis), atypical mycoplasma strains (Ureaplasma urealyticum), Mycoplasma hominis, and coagulasenegative staphylococci (S. haemolyticus, S. epidermidis). The microbial agents promote acute inflammatory reactions through the influx of a substantial number of leukocytes in the reproductive tract. The infiltrated

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leukocytes, in turn, induce excess ROS generation leading to OS.28 Oxidative damage to spermatozoa can also occur through viral infections, including Epstein
Barr virus, cytomegalovirus, herpes simplex virus, and others. Semen of infertile men have shown traces of herpes simplex DNA.29 Systemic infections Numerous chronic systemic infections are associated with increased OS in the male urogenital system. The number of seminal leukocytes significantly increase in human immunodeficiency virus (HIV) infection and is a potent source of endogenous ROS.30 Infections caused by hepatitis B and C also induce OS in the liver and male reproductive tract.31 Systemic OS and that in the male reproductive system is also caused by other chronic infections such as tuberculosis,32 malaria,33 and Chagas disease.34 Autoimmune/inflammatory conditions Non-infective or non-bacterial chronic prostatitis is a very common prostate pathology resulting in high seminal OS.35 This accounts for more than 90% of all prostatitis cases, affecting 10% of all men worldwide.26 These conditions generally trigger autoimmune responses against self-antigens of a prostate or seminal origin. These exaggerated responses induce proinflammatory mediators and trigger increased seminal leukocytes, thereby generating excessive ROS and leading to OS.35 A speculated mechanism that evokes this autoimmune response is probably the polymorphism of interleukin 10 (IL-10), which is an antiinflammatory TH-2 cytokine.36 A decrease in IL-10 may initiate the TH-1 dependent immune responses that activate Tlymphocytes against prostate antigens. Inflammatory cytokines like TNF-α, IFN-γ, and IL-1β initiate chemotaxis and leukocytes activation. These leukocytes serve as major sources of seminal OS, leading to severe disruption of sperm membrane integrity and significantly reduces semen quality.37 OS is also incurred by vasectomy reversal via damage to the blood
testis barrier. This triggers immune responses against the spermatozoa38 and leads to a high influx of leukocytes, cytokines, and the production of more ROS.38

19

insufficiency in the testis result in OS, which induces oxidative damage and testicular dysfunction.40 High OS-related parameters in infertile varicocele patients compared to normal fertile men in recent studies suggest OS-mediated impairment of male reproductive functions in varicocele patients. Varicocele-induced increased production of ROS parallels with decreased sperm DNA integrity.41 Cryptorchidism, characterized by hypospermatogenesis, also significantly contributes to male factor infertility. In cryptorchidism, gonocytes fail to mature into type-A spermatogonial.42 Even if cryptorchidism patients undergo surgical interventions with orchidoplexy in early stages, these men will still have high sperm
ROS generation and an increased rate of SDF.13 Male fecundity can also be affected by spermatic cord torsions. A prolonged testicular ischemia due to surgery or spontaneous restoration of testicular blood flow can lead to a sudden influx of activated leukocytes.13 This triggers FR generation that exceeds the capacity of the endogenous antioxidants.5 Other chronic diseases Diabetes is one of the most prevalent chronic diseases with innumerable systemic effects. It greatly impairs male reproductive functions affecting spermatogenesis as well as erectile capacity. Diabetic men have shown higher sperm
DNA fragmentation induced by OS compared to normal men.42 Chronic kidney diseases also lead to systemic inflammation and induction of OS.43 Hemoglobinopathies like β-thalassemia involve obligatory multiple blood transfusions that can cause iron overload. This, in turn, induces systemic OS and, in men, oxidative damage to sperm may render it nonfunctional.44 Homocysteines may act as reproductive disruptors and OS inducers via accrued toxins.44 Hyperhomocysteinaemia results mainly through less remethylation of homocysteine by the enzyme methyl tetrahydrofolatereductase (MTHFR) to methionine, due to either dietary folate deficiency or (as mostly reported in infertile men) a single nucleotide polymorphism in gene encoding MTHFR.45 Men with such conditions are at high risk of homocysteine-induced OS.

Cryptorchidism and varicocele Varicocele is a major cause of male infertility and is reported to affect about 40% of male partners of infertile couples. It is a pathological state that entails the abnormal dilatation of the pampiniform plexus around the spermatic cord.39 Among the hypothesis that have been put forward to relate varicocele with male infertility, testicular hyperthermia and hypoxia seem to be most accepted mechanisms. Hyperthermia and oxygen

Oxidative stress in erectile dysfunction Adequate sexual and erectile functionality is critical for men. Men engaged in sexual activities have improved quality of life and live longer.46 However, erectile dysfunction (ED) is a common complaint in males and there is a 40% chance of ED in men aged 40 or older.46 This is mediated through complex psychological, neurological, endocrine, immune, and vascular

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factors acquired through lifestyle, environmental exposure, and underlying pathology.47 Vascular failure of the penis is a common final mechanism in ED, driven by dysfunction and loss of relevant corporal smooth muscle cells in the corporal sinusoids. This increasingly appears to be mediated and accelerated through intracellular OS, resulting in increased endothelial cell dysfunction, smooth muscle cell dysfunction, and increased apoptosis rates.46 Importantly, ED is associated as a risk factor as well as a diagnostic predictor of OS-related chronic morbidity including visceral adiposity, hypertension, dyslipidemia, male hypogonadism, Type 2 diabetes mellitus (T2DM), atherosclerosis, and subsequent CVD.47 Age is a significant risk factor for ED, where OS is a central mechanism in ageing and age-related pathologies including ED.11 Adequate erection requires nitric oxide (NO) through transcription of nitric oxide synthase (NOS) for vasodilation and penile engorgement with blood.47 In this context, inflammation and OS are common mediators of vascular failure in ED via reduction in NOS and subsequent NO.47 Inflammation-associated increase in cellular adhesion molecules and dysfunctional endothelial cells promote local arteriosclerosis and stiffening of the vasculature, which further leads to local inflammation and OS.47 This, in turn, is associated with an increased collagen ratio and fibrosis, further stiffing in the vasculature to prevent vasodilation and engorgement. MPO-dependent oxidized low density lipoprotein (LDL) has been shown to reduce endothelial NOS mRNA expression and is correlated with poor International Index of Erectile Function (IIEF) scores, alongside increased inflammatory markers including IL8.48 OS related to chronic pelvic pain syndrome (prostatitis) induced ED have shown increased MDA levels, decreased cGMP related transcription of NOS and NO, decreased SOD activity, and increased smooth muscle cell apoptosis in the corpus cavernosum of rats.49 Increased ROS, specifically dO22, has been suggested to result in inability for penile vasodilation within the corpus cavernosum and progressive chronic vasculopathy, resulting in ED.50 OS, therefore, appears to mediate numerous risk factors, including age, associated with ED, through endothelial and smooth muscle cell dysfunction, and reduced NO synthesis. This is a common mediator in peripheral vascular pathology associated with obesity, CVD, and T2DM. Oxidative stress in hypogonadism OS is known to inhibit Leydig cell steroidogenesis, mediated through the reduced transcription of steroidogenic acute regulatory (StAR) protein and the subsequent transfer of cholesterol into the inner

mitochondrial membrane for metabolism into 17hydroxypregnenalone.51 ROS, particularly H2O2 derived from Leydig cells as well as testicular macrophages, result in mitochondrial dysfunction mediated steroidogenesis collapse through decreased transcription of steroidogenic enzymes, particularly P450 cholesterol side-chain cleavage (P450scc), 17α-hydroxylase/17, 20lyase (CYP17), and 3β-hydroxysteroid dehydrogenase/ Δ5
Δ4 isomerase (3βHSD).52 OS in Leydig cells is further correlated with reduced cellular SOD, GPx, and CAT, alongside the initiation of apoptosis.53 The resulting hypogonadism is an important mechanism in aging, obesity, metabolic syndrome, and subsequent comorbidities driven by systemic and local chronic inflammation and OS.54 Hypothalamic inflammation is increasingly associated with hypogonadotropic hypogonadism, particularly in obesity, metabolic syndrome, and anorexia.55 This subclinical inflammation and associated OS correlates with systemic inflammation and may precede the onset of obesity due to established unfavorable alterations in energy intake and expenditure. Inflammatory cytokines and ROS activate mitochondrial and endoplasmic reticulum stress through the c-Jun N-terminal kinase (JNK) pathways.56 Importantly in men, this OS results in reduced gonadotropin releasing hormone (GnRH), negatively modulating the hypothalamicpituitary-gonadal (HPG) axis contributing to OSinduced hypogonadism.56 Male hypogonadism is a relatively common finding in infertility and ageing males, and is also a predictor of chronic diseases including obesity, metabolic syndromes, CVD, and T2DM.57

Mechanisms of oxidative stress-induced reproductive dysfunction Lipid peroxidation Sperm plasma membrane is rich in PUFAs with unconjugated double bonds within their methylene groups. The carbon
hydrogen bond is weekend by the presence of this double bond and the hydrogen becomes vulnerable to oxidative attack. Unregulated increase in sperm intracellular ROS aids a progressive reaction cascade inducing LPO (Fig. 2.3).58 As this is a self-propagating autocatalytic reaction, it accounts for 60% of sperm membrane fatty acids damage and, thus, sperm membrane fluidity is compromised. The sperm membrane becomes nonspecifically permeable, its receptors and enzymes are disrupted, and these lead to impaired sperm functions.21,58 Induction of LPO follows a progressive destructive chain of reactions till it terminates. “Initiation” of oxidative damage is via hydrogen atom isolation from the

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Oxidative stress and male factor infertility

21

FIGURE 2.3 The effect of excessive reactive oxygen species (ROS) and oxidative stress (OS) on spermatozoa.

loosened bond with carbon. This is followed by further FRs and lipid radical production. The FRs interact with oxygen giving rise to peroxyl radicals58 that further take hydrogen atoms from membrane lipids. This autocatalytic reaction continues in the “propagation” phase of oxidative damage, rapidly damaging cellular components. The existing FRs keep attacking the succeeding lipids by yielding toxic aldehydes through degradation of hydroperoxide.17,19,58 Cytotoxic peroxyl and alkyl radicals are repeatedly produced till formation of a stable end product, malondialdehyde (MDA), that marks the “termination” of the peroxidation chain.58 Hydrophilic 4-hydroxynonenal is also a crucial LPO product that affects proteomics and genomics of spermatozoa.21 DNA integrity OS is associated with increased SDF (Fig. 2.3). The underlying mechanisms include chromatin crosslinking, random modification of DNA base-pairs, and sperm chromosomal microdeletions.21,58 Excess ROS induced LPO results in the deletion of adenine and pyridine nucleotides.21 The etiopathogenesis of SDF is still not completely understood and may include urogenital infection, advanced age, varicocele, improper lifestyle factors, heat stress, environmental factors, radiation, and error in protamination.59 It is stipulated that SDF is commonly induced by OS via uncontrolled ROS generation and LPO. Testicular SDF lead to abortive germ cell apoptosis, impaired sperm maturation.60 Recent research in reproductive medicine proposed

treatment of such pathological conditions with potent antioxidants that seem to decrease ROS and SDF.61 Mitochondrial dysfunction Mitochondrial ATP generation is provided through protein complexes, namely complex 1 (NADH dehydrogenase), quinine pool, complex III (bc1), and cytochrome c, with complex IV (terminal oxidase) as an end point.62 For appropriate cellular respiration, an electrical gradient across the mitochondrial membrane is required, whereby
136 to
140 mV is considered an optimal mitochondrial membrane potential (MMP).63 ROS are prominently generated through aerobic respiration, and excessive ROS or reduced antioxidants result in mitochondrial dysfunction.64 This dysfunction can be assessed through MMP, where ROS-induced variations result in a down-regulation of ATP formation and, subsequently, further increase ROS production.63 Mitochondrial dysfunction is increasingly found to be associated with the molecular pathophysiology of numerous diseases, including obesity, metabolic syndrome, T2DM, CVD, malignancy, and dementia.11 This further negatively affects spermatogenesis and steroidogenesis.64 The role of mitochondrial dysfunction in ageing and human pathology are not fully understood; however, this has emerged as an important area of further study on the role of mitochondrial in physiology and pathophysiology.64 The standard semen analysis is generally limited in diagnostic interpretation. This includes sperm concentration, mobility, viability and morphology, and seminal volume, pH, and leukocyte concentration where

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22

2. Oxidative stress in pathologies of male reproductive disorders

indicated. The non-standard functional capacity of spermatozoa is important, particularly DNA fragmentation and MMP. MMP is an important parameter that reflects mitochondrial function in the spermatozoa, critically important for motility and high metabolic energy demands. A prominent source of endogenous ROS is the mitochondria. Through the electron transport chain, dO22 is released and then metabolized by SOD2 into H2O2 by SOD2.65 H2O2 moves out of the mitochondria into the cytosol and metabolized by GPx, although it is also metabolized by mitochondrial CAT and peroxiredoxin.66 OS damages macromolecules, and accumulates over time. This affects the cell membrane, cellular DNA, and mitochondrial DNA (mtDNA) (Fig. 2.3),67 while the resulting mitochondrial dysfunction causes additional mitochondrial ROS production, leading to poor spermatogenesis and sperm concentration, reduced motility and normal morphological forms, damaged somatic and mitochondrial DNA with associated reduced MMP with impaired ATP synthesis and metabolic functioning, risking increased apoptosis and infertility.13,68 Furthermore, as with numerous other cells, Leydig cells mitochondrial function and MMP are critical for steroidogenesis. Mitochondrial dysfunction inhibits cholesterol uptake and metabolism into 17-OH-pregnenalone within the mitochondrial membrane, mediating male hypogonadism.52,54 Apoptosis ROS regulate numerous cellular metabolic and molecular activity, with critical functionality in immune regulation and inflammatory response, healing and repair, ion transport, and genomic function. Importantly, ROS mediates apoptosis pathways in most cells, either within a physiological or pathological context.69 DNA damage induced through excessive ROS results in upregulation of germ cell apoptosis, with subsequent impairment of spermatogenesis(Fig. 2.3).70 Increased H2O2 upregulates caspase-3, caspase-7, and annexin-V in germ cells in the process spermatogenesis. With the tight protective compaction of spermatozoa DNA protamines in chromatin condensation, DNA repair mechanisms are reduced to further allow accumulation of damage through the entire spermatogenesis process and resulting in downstream apoptosis and reduced fertilization potential.71

Assessment of oxidative stress in the male reproductive system Assessment of seminal ROS, and potentially serum OS and inflammatory markers, is increasingly

indicated in the assessment of male reproductive dysfunction and infertility. This investigation is important for diagnostic and prognostic assessments as well as to guide management options and therapeutic improvements.72 Moreover, poor seminal quality relevant to OS, DNA fragmentation, and epigenetic alterations are emerging as important predictors of pregnancy complications, recurrent pregnancy loss, and impaired health in the next generation.73 However, seminal ROS assessment methods and interpretation remain varied, and further standardization is required for improved clinical use and predictive value. Currently there are numerous direct and indirect assessment options for seminal fluid, including breakdown products of oxidized macromolecules. LPO can be assessed by MDA, thiobarbituric acid assays (TBARS), or 4-hydroxynonenal (4-HNE). Evidence of oxidized protein molecules and DNA damage can be assessed through protein carbonyl, a highly sensitive marker of protein oxidation in seminal fluid, and apoptotic markers including increased seminal caspase-3 or annexin V.74 More generically, an assessment of total ROS and total antioxidant capacity (TAC) can be gained for insight into redox balance. However, more recently there has been validation and clinical standardization of the seminal oxidation-reduction potential (ORP), measured by the MiOXSYS system, that can indicate either a shift to oxidation or reduction through electrochemical gradients.13 Importantly, functional analysis of semen as a standard approach is increasingly evident in the literature, specifically for diagnostic and prognostic purposes. These include DNA fragmentation and various assessments of OS in the male reproductive tract and even in serum.72 Currently many of these methods are not suitable for clinical practice due to laboratory requirements, costs, and the need for validation and standardization in clinical interpretation. Furthermore, many of these systems do not evaluate a full OS spectrum, but different components or biochemical products of OS.13 The use of the ORP as a validated and standardized marker in seminal fluid for diagnostic and management guidelines has become increasingly evident. ORP directly measures OS or reductive stress (RS) in seminal fluid, incorporating known and unknown ROS and antioxidants, thereby reflecting tissue pathophysiology and damage if not within normal range. Furthermore, increased ORP (as in OS) is correlated with poor fertility and ART outcomes and poor parameters on standard semen analysis. The ORP can be considered as the most sensitive and validated assessment for comprehensive, rapid (within minutes) and cost-effective clinical assessment for fresh or cryopreserved seminal fluid.13

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Use of antioxidants in male infertility treatment

Use of antioxidants in male infertility treatment Nutritional sources Nutritional therapy is important for patient management, particularly in pathology mediated by inflammation and OS. Nutritional therapy should be clinically applied, particularly in cases where OS is evident, and can be through direct laboratory assessment (investigation) or indirectly through an assessment of clinical history relevant to lifestyle and environmental exposure that promotes OS and male infertility. This can further be expanded to micronutrient deficiencies (MNDs) in malnourished patients with excess adiposity or being underweight.70 Avoidance of harmful exposure and habits detrimental to OS and fertility is recommended, and include tobacco and alcohol consumption, pollution and smog, poor nutritional choices, drugs and other environmental toxins (e.g., oestrogenmimetics, pesticides, heavy metals), and radiation exposure. Nutritional therapy and consideration of supplementation to reduced inflammation and OS through improved antioxidant intake to prevent excessive ROS activity are further important considerations.9 The neutralization of excessive ROS in a setting of OS can be achieved through exogenous antioxidant intake. This includes a variety of micronutrients, including carotenes, tocopherols, ascorbic acid, glutamine, zinc, and selenium.75 Nutritional habits are an important consideration in male infertility assessments. This is particularly significant in the context of obesity and metabolic syndrome. It has been increasingly established that nutritional intake modulates spermatogenesis.76 There are currently clearly established relationships with obesogenic foods and increased visceral adiposity and poor fertility parameters.76 Unfavorable foods associated with adiposity include increased fat intake, red and processed meats, refined sugars, soy-based foods, dairy, and alcohol.77 Beneficial foods for male infertility include antioxidant rich fruits and vegetables, fiber, PUFAs, antioxidants, micronutrients, and various phytonutrients.76 This can be further sourced through appropriate seafood intake, vegetable and fish oils, nuts, seeds, and short-term supplementation.77 Adherence to the Mediterranean diet and similar dietary habits improve standard semen parameters in normal and overweight men.78 These nutritional habits are further associated with reduced inflammation and OS, and a correlated reduction of DNA fragmentation index.79 Although nutrition is an important cornerstone in weight management, reduced risk of chronic disease, and improved reproductive health and fertility parameters, this is aided by appropriate moderate exercise.

23

A combination of an appropriate diet with exercise improves fertility parameters with a reduction in seminal OS and DNA fragmentation in spermatozoa.80

Supplementary antioxidants The use of nutritional supplementation has increased significantly in recent years, and contain vitamins, minerals, amino acids, and phytonutrients with significant antioxidant potential.81 Increasingly, evidence suggests the benefits of antioxidant use for the management of male infertility, particularly when OS is apparent. However, there are currently no clear guidelines for the type, dosage, and duration of supplementation for male infertility.72 Importantly, the correct concentrations of various antioxidants are required to reach the reproductive tract to exert potential benefits for male patients. The intake of antioxidant supplementation and micronutrients has a beneficial effect on DNA fragmentation, improves fertility and ART outcomes, as well as reducing the risk of pregnancy complications and embryonic development.82 This has also been observed in obesity patients across a body mass index (BMI) range and improving fertility parameters even without changes in weight adiposity distribution.83 Various antioxidants have been investigated for male infertility management, used alone or in various combinations. More wellestablished antioxidant supplements for male infertility include vitamin A and other carotenes, vitamin C, vitamin E (tocopherols), selenium, zinc, copper, glutathione, cysteine, alpha-lipoicacid, coenzyme Q10, and lycopene.13 Further randomized trials are needed to elucidate the appropriate application in clinical practice, including dosages and duration of treatment, although evidence suggests that clinical use should be advised for OS-related infertility in males. This should be done following appropriate clinical assessment, including DNA fragmentation in spermatozoa and seminal OS or ORP evaluation, where possible, and currently short-term (3
6 months) duration is recommended to avoid as yet unknown adverse effects.84

Phytonutrients Extractions of traditional medicinal plants have increasingly been established to have multiple biological activities and modulating physiology, and are potentially useful in pathophysiology. Plant extractions can be obtained through nutrition (e.g., tea, fruit, vegetables) or targeted extractions of medicinal plants.85 The ongoing investigation and clinical application of traditional medicines and phytotherapy is recommended by the World Health Organization (WHO)

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2. Oxidative stress in pathologies of male reproductive disorders

alongside the increasing scientific and clinical interest relevant to drug discovery.86 Importantly, many of these nutrients, at least in part, exert effects through antioxidant activity and/or activation of endogenous antioxidant defense mechanisms in male infertility due to a range of compounds including flavonoids, polyphenols, and catechins.85 The few existing studies with relevant human evidence suggest phytotherapy to address numerous disorders, including poor spermatogenesis, poor libido, ED, and hypogonadism. This includes Lepidium meyenii,87 Eurycoma longifolia,88 Tribulus terrestris, Asparagus recemosus and Withania somnifera,89 Mucuna pruriens,90 Andrographis paniculata, and Acanthopanax senticos.91 Further phytomedicines that improve OS in male reproduction as well as showing benefits for semen parameters, include green tea (epigallocatechin gallate),92 turmeric (curcuminoids),93 and grape seed (resveratrol).94 These and other traditional herbal remedies require further investigation for novel applications in the management of OS-associated male sexual dysfunction through preclinical mechanistic studies and well-designed and controlled randomized trials.95 As with antioxidant supplementation, appropriate extraction methods, standardized quality markers, dosage, and duration of treatment requires significant further investigation.84

Antioxidant paradox and reductive stress With increased ROS exposure and/or reduced antioxidant capacity, OS is detrimental to cellular activity. Conversely, RS is where excessive antioxidants and/or ROS depletion can result in a shift toward RS.96 This is a shift from redox balance for biological activity to a reduction state, which is also detrimental for cells as physiological redox switches and signaling is disrupted.96 RS is considered as significant as OS in cellular pathology, and further is associated with metabolic syndrome cardiac disease (e.g., cardiomyopathy), neurodegeneration and Alzheimer’s disease, and dysregulation of embryogenesis.97 Importantly, RS may arise from excessive consumption of antioxidants, including vitamins, minerals, and phytonutrients, conversely diminishing life expectancy in what is termed the antioxidant paradox.96 Within male reproduction, RS may inhibit chromatin condensation, epididymis maturation, capacitation, and the acrosome reaction.

Applications to other areas of pathology Male infertility is associated with an increase in allcause morbidity and mortality. Semen quality is,

therefore, suggested to be a predictor and health marker for future disease.98 ED is associated with chronic morbidity, particularly atherosclerosis and CVD.47 It is likely that these outcomes share common mediators through OS. Male hypogonadism is a relatively common finding in infertility and ageing males, and is also a predictor of chronic diseases including obesity, metabolic syndrome, CVD, and T2DM.57 Testosterone therapy improves inflammatory and OS markers in these males, although it is reported to be detrimental to fertility in most of the cases.99 It can be argued that an assessment of testosterone, male fertility parameters, SDF, seminal OS, and ORP require clinical testing relevant to all causes of morbidity and mortality in males. Furthermore, systemic dysfunction and pathology, including obesity, metabolic syndrome, diabetes, CVD, accelerated ageing, and neurodegeneration, are predominantly an increased risk of infertility in males and poor pregnancy as well as adverse effects to the offspring’s health.73 Therefore, poor seminal quality relevant to OS, DNA fragmentation, and epigenetic alterations are emerging as important predictors of pregnancy complications, recurrent pregnancy loss, and impaired health in the next generation.73

Conclusion OS serves as a common pathway through which numerous factors act upon, and adversely affect, male reproductive functions. ROS is produced endogenously by immature spermatozoa and seminal leukocytes, and at normal level it takes part in vital male reproductive functions. OS is induced by the production of excessive ROS that supersedes endogenous antioxidant capacity. Excess ROS initiates a chain of reactions, disrupting cellular components and rendering spermatozoa as merely functional. Various pathological conditions participate in the common path of induction of OS via which an array of inflammatory reactions take place to disrupt male reproductive functions. Treatment and management strategies of OSinduced male infertility are still emerging with the most advent in recent years.

Summary points • ROS are important physiological mediators of male fertility, sperm maturation, and fertilization. • Excessive ROS disrupt spermatogenesis, semen quality, steroidogenesis, and sexual functionality. • Endogenous ROS in the male reproductive track predominantly arise through immature spermatozoa and leukocytes.

I. Oxidative Stress and Pathology

References

• Exogenous ROS resulting in reproductive tract OS and male infertility include exposure to various endocrine disruptors, radiation, unhealthy lifestyle, and MNDs. • Male reproductive pathology causing infertility through OS include varicocele, cryptorchidism, and genital tract infections (including prostatitis) • Systemic pathology causing infertility through OS include visceral adiposity, diabetes, systemic inflammatory disease, and autoimmunity. • Antioxidant therapy shows some evidence of improving reproductive tract OS and fertility outcomes.

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9. Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal 2011;14:367
81. Deepinder F, Cocuzza M, Agarwal A. Should seminal oxidative stress measurement be offered routinely to men presenting for infertility evaluation? End Pract 2008;14:484
91. McPherson NO, Lane M. Male obesity and subfertility, is it really about increased adiposity? Asian J Androl 2015;17:450. Shamsi M, Venkatesh S, Tanwar M, Talwar P, Sharma R, Dhawan A, et al. DNA integrity and semen quality in men with low seminal antioxidant levels. Mut Res/Fund Mol Mech Mut 2009;665:29
36. Ho¨hn A, Weber D, Jung T, Ott C, Hugo M, Kochlik B, et al. Happily (n) ever after: aging in the context of oxidative stress, proteostasis loss and cellular senescence. Redox Biol 2017;11:482
501. Danielewicz A, Przybyłowicz K, Przybyłowicz M. Dietary patterns and poor semen quality risk in men: a cross-sectional study. Nutrients 2018;10:1162. 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. Human Reprod Update 2017;23:371
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78. Ricci E, Al-Beitawi S, Cipriani S, Alteri A, Chiaffarino F, Candiani M, et al. Dietary habits and semen parameters: a systematic narrative review. Andrology 2018;6:104
16. 79. Vujkovic M, de Vries JH, Dohle GR, Bonsel GJ, Lindemans J, Macklon NS, et al. Associations between dietary patterns and semen quality in men undergoing IVF/ICSI treatment. Human Reprod 2009;24:1304
12. 80. Vaamonde D, Garcia-Manso JM, Hackney AC. Impact of physical activity and exercise on male reproductive potential: a new assessment questionnaire. Revista Andaluza de Medicina del Deporte 2017;10:79
93. 81. Radimer K, Bindewald B, Hughes J, Ervin B, Swanson C, Picciano MF. Dietary supplement use by US adults: data from the National Health and Nutrition Examination Survey, 1999
2000. Am J Epidemiol 2004;160:339
49. 82. Mora-Esteves C, Shin D. Nutrient supplementation: improving male fertility fourfold. Semin Reprod Med 2013;31:293
300. 83. Montanino Oliva M, Minutolo E, Lippa A, Iaconianni P, Vaiarelli A. Effect of myoinositol and antioxidants on sperm quality in men with metabolic syndrome. Int J Endocrinol 2016;2016:1674950. 84. Sheweita SA, Tilmisany AM, Al-Sawaf H. Mechanisms of male infertility: role of antioxidants. Curr Drug Metab 2005;6:495
501. 85. Nantia E, Moundipa P, Monsees T, Carreau S. Medicinal plants as potential male anti-infertility agents: a review. Basic Clin Androl 2009;19:148. 86. World Health Organization. The Selection and Use of Essential Medicines: Report of the WHO Expert Committee, 2015 (including the 19th WHO Model List of Essential Medicines and the 5th WHO Model List of Essential Medicines for Children). World Health Organization; 2015. 87. Gonzales GF, Cordova A, Vega K, Chung A, Villena A, Go´n˜ez C, et al. Effect of Lepidium meyenii (MACA) on sexual desire and its absent relationship with serum testosterone levels in adult healthy men. Andrologia 2002;34:367
72. 88. De Andrade E, De Mesquita AA, de Almeida Claro J, De Andrade PM, Ortiz V, Paranhos M, et al. Study of the efficacy of

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4. Devi PR, Laxmi V, Charulata C, Rajyalakshmi A, editors. “Alternative medicine”—a right choice for male infertility management. International Congress Series. Elsevier; 2004. Shukla KK, Mahdi AA, Ahmad MK, Jaiswar SP, Shankwar SN, Tiwari SC. Mucuna pruriens reduces stress and improves the quality of semen in infertile men. Evidence-Based Compl Alt Med 2010;7:137
44. Mkrtchyan A, Panosyan V, Panossian A, Wikman G, Wagner H. A phase I clinical study of Andrographis paniculata fixed combination Kan Jangt versus ginseng and valerian on the semen quality of healthy male subjects. Phytomedicine 2005;12:403
9. Mosbah R, Yousef MI, Mantovani A. Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: the protective effects of green tea extract. Exp Toxicol Pathol 2015;67:253
9. Hu Y, Zhang J, Kong W, Zhao G, Yang M. Mechanisms of antifungal and anti-aflatoxigenic properties of essential oil derived from turmeric (Curcuma longa L.) on Aspergillus flavus. Food Chem 2017;220:1
8. Cui X, Jing X, Wu X, Yan M. Protective effect of resveratrol on spermatozoa function in male infertility induced by excess weight and obesity. Mol Med Rep 2016;14:4659
65. Bella AJ, Shamloul R. Traditional plant aphrodisiacs and male sexual dysfunction. Phytother Res 2014;28:831
5. Pe´rez-Torres I, Guarner-Lans V, Rubio-Ruiz ME. Reductive stress in inflammation-associated diseases and the pro-oxidant effect of antioxidant agents. Int J Mol Sci 2017;18:2098. Brewer AC, Mustafi SB, Murray TV, Rajasekaran NS, Benjamin IJ. Reductive stress linked to small HSPs, G6PD, and Nrf2 pathways in heart disease. Antioxid Redox signal 2013;18:1114
27. Eisenberg ML, Li S, Behr B, Cullen MR, Galusha D, Lamb DJ, et al. Semen quality, infertility and mortality in the USA. Hum Reprod 2014;29:1567
74. Winter AG, Zhao F, Lee RK. Androgen deficiency and metabolic syndrome in men. Transl Androl Urol 2014;3:50.

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C H A P T E R

3 Oxidative stress and diabetic retinopathy Kemal Tekin and Merve Inanc Tekin Ophthalmology Department, Ercis State Hospital, Van, Turkey

List of abbreviations AGE DAG Dnmt DM DR eNOS GAPDH GSH GSSG ICAM-1 NADPH NF-kB NO Nox miRNA OS PKC RAGE ROS SIRT SOD-2 VEGF

prevalence of cardiovascular diseases, peripheral arterial disease, and stroke, while damage to the microvascular structure causes complications in kidneys, nerves, and eyes. Diabetic patients are susceptible to multiple ocular complications including refractive deviations, ocular surface disorders, cataract formation, and glaucoma. However, one of the most severe ocular complications is diabetic retinopathy (DR), which is one of the major microvascular complications of DM and is the leading cause of acquired blindness.2

advanced glycation end product diacylglycerol DNA methyltransferase diabetes mellitus diabetic retinopathy endothelial nitric oxide synthase glyceraldehyde 3-phosphate dehydrogenase glutathione oxidized glutathione intercellular adhesion molecule-1 nicotinamide adenine dinucleotide phosphate nuclear factor kappa B nitric oxide nicotinamide adenine dinucleotide phosphate oxidase microRNA oxidative stress protein kinase C receptor for advanced glycation end products reactive oxygen species sirtuin protein superoxide dismutase-2 vascular endothelial growth factor

Molecular mechanisms of diabetic retinopathy The pathogenesis of DR is multifactorial, and the precise mechanisms are not clear yet. Hyperglycemia plays a central role in the development of DR; prolonged exposure of the cells to high glucose levels is shown to cause alterations in cellular metabolism. Before the clinical diagnosis of DR, the cells of the retina start to respond to this hyperglycemic environment by modifying metabolism. A number of clinical trials reveal that even with strict glycemic control, complications including DR still affect many patients with DM, suggesting that stressors of diabetic complications continue beyond the point when glycemic control has been achieved.3,4 Besides hyperglycemia, several interconnecting biochemical mechanisms have been proposed, including inflammation, increased accumulation of advanced glycation end products (AGEs), enhanced polyol pathway, overactivation of hexosamine pathway, and activation of protein kinase C (PKC) (Fig. 3.1).5
8 All these mechanisms appear to be associated with mitochondrial overproduction of reactive oxygen species (ROS).9

Introduction Diabetes mellitus (DM), a life-long progressive degenerative disease characterized by high circulating glucose, is the result of the body’s inability to either produce enough insulin or use insulin effectively. According to the International Diabetes Federation, in 2014 8.2% of adults between the ages of 20 and 79 years (387 million people) have DM and this number is expected to exceed 592 million in 2035.1 In DM, sustained high circulating glucose levels damage the vascular structures causing micro- and macrovascular complications throughout the body. Damage to the macrovascular structures increases the

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00003-2

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© 2020 Elsevier Inc. All rights reserved.

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3. Oxidative stress and diabetic retinopathy

FIGURE 3.1 Hyperglycemia-induced four classical mechanisms described in the pathogenesis of diabetic retinopathy (DR): increased polyol pathway flux, accumulation of AGEs, PKC pathway activity and activation of the hexosamine pathway. Excess superoxide partially inhibits the glycolytic enzyme GAPDH and diverts metabolites from glycolysis into pathways of glucose overutilization, resulting in increased flux of dihydroxyacetone phosphate (DHAP) to DAG, an activator of PKC, and of triose phosphates to methylglyoxal, the major intracellular AGE precursor. Increased conversion of fructose-6-phosphate to UDP-N-acetylglucosamine increases modification of proteins by GlcNAc, and increased glucose flux through the polyol pathway expends NADPH and consumes GSH.

Oxidative stress and reactive oxygen species Free radicals are produced continuously by normal cellular mechanisms. The body utilizes approximately 95% of them for metabolism and 5% of the oxygen is converted to ROS. ROS produced during normal oxidative metabolism are eliminated by an efficient scavenging system, but an imbalance between excess production and/or inefficient removal of ROS can result in excessive levels of either molecular oxygen or ROS, thus resulting in increased “oxidative stress” (OS). Increase in OS in DM and its role in the development of diabetic complications is now well accepted. The retina is the most metabolically active tissue in the body and is therefore susceptible to OS owing to its hypermetabolic state.10 ROS are produced by both enzymatic and nonenzymatic mechanisms; nonenzymatic production is mainly the consequence of mitochondrial oxidative phosphorylation and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is the primary enzyme responsible for their production.11,12

Nonenzymatic production of reactive oxygen species Mitochondria are the major endogenous source of superoxide. Glucose oxidation increases during hyperglycemia, resulting in an increase in voltage gradient across the mitochondrial membrane. When a critical threshold is reached in voltage gradient, electron transfer in the complex III of the electron transport chain is blocked. The electrons accumulate at coenzyme Q that then interact with molecular oxygen to form superoxide anions (Fig. 3.2).13 Hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain is considered to activate the major pathways of hyperglycemic damage by inhibiting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity, and to induce mutations in mitochondrial DNA resulting in defective subunits of the electron transport complexes and eventually causing increased superoxide production at physiological concentrations of glucose.9,14 In accordance with these data, the levels of transcription of mitochondrial DNA encoded NADH dehydrogenase 1 and 6 of complex I and cytochrome b of complex III have been reported to be subnormal in the retinas of diabetic

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FIGURE 3.2 Electron transport in the mitochondrial respiratory chain and the production of superoxide. Increased hyperglycemiaderived electron donors from the TCA cycle (NADH and FADH2) generate a high mitochondrial membrane potential by pumping protons across the mitochondrial inner membrane. This inhibits electron transport at complex III. When complex III cannot receive electrons from coenzyme Q (CoQ), the electrons would be accepted by O2, which could produce ROS and result in OS.

patients.15,16 Furthermore, cytosolic ROSs, via activation of matrix metalloproteinases, damage the retinal mitochondrial membrane and further aggravate mitochondrial dysfunction.17,18

Enzymatic production of reactive oxygen species Nox, a multiprotein enzyme, catalyzes oxygen to produce superoxide and/or hydrogen peroxide by accepting electrons from NADPH. In diabetes, increased activity of Nox is observed in several tissues, including pancreatic beta cells and the retina, suggesting it to be one of the major sources of ROS generation.19,20 The Nox system is divided into different isoforms. Nox 1, Nox 2, and Nox 4 isoforms are highly expressed in the vascular system.21 The increased activity of Nox 2 was shown in the retinas of diabetic mice. This increase is associated with increased production of ROS and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF).22 Arginase is another important promoter of OS. The increased arginase in the retina has been shown to be associated with endothelial cell dysfunction in diabetes.23 The increased activity of arginase causes endothelial nitric oxide synthase (eNOS) uncoupling due to reduced levels of L-arginine, and uncoupled eNOS generates superoxide that reacts with nitric oxide (NO) to form peroxynitrite, which can enhance the activity of NADPH oxidase, and

FIGURE 3.3 Oxidative stress
mediated signaling pathway in the development of diabetic retinopathy. Hyperglycemia increases OS, and ROS activate NF-κB. Together, this increases nitric oxide (NO) production via activating the inducible form of nitric oxide synthase (iNOS). NO and superoxide react to form peroxynitrite (OONO 2 ). Increased superoxide radicals inhibit GAPDH. This results in apoptosis of retinal capillary cells, and ultimately culminates in the development of retinopathy.

further fuel increased ROS production (Fig. 3.3). Furthermore, acyl-CoA dehydrogenase and glycerol phosphate dehydrogenase also contribute to the generation of ROS.24

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Relationship between oxidative stress and other biochemical pathways Four typical hyperglycemia-induced mechanisms are described in the pathogenesis of DR: increased polyol pathway flux, accumulation of AGEs, PKC pathway activity, and activation of the hexosamine pathway. Biochemical abnormalities in the retina that are aggravated in DM can also induce OS. However, it is still not clear how these mechanisms lead to increased OS and the development of DR.

Enhanced polyol pathway Activation of polyol pathway increases OS by both, decreasing intracellular antioxidation and increasing ROSs. Hyperglycemia activates polyol pathway. Aldose reductase, the rate-limiting enzyme of the polyol pathway, reduces unused glucose to sorbitol by oxidizing NADPH to NADP 1 . NADPH provides the production of glutathione (GSH) from oxidized glutathione (GSSG), whereas NADPH deficiency will also cause GSH deficiency. GSH is an important scavenger of ROS; the decrease in GSH can induce or exacerbate OS.25 Increased use of NADPH by aldose reductase is associated with decreased GSH levels, and increased availability of NADPH for Nox, results in increase of ROS.26 Moreover, increased intracellular ratio of NADH/NAD 1 leads to inhibition of GAPDH and increases intracellular triose phosphate concentrations that induce the formation of methylglyoxal, a precursor of diacylglycerol (DAG) and AGEs, thereby leading to the activation of the other two important pathways, the AGE and PKC pathways.14,27

activation of proinflammatory pathways, and NF-κB activation. For all these reasons, there is a close cycle between AGEs and OS.

Activation of protein kinase C PKC is a family of serine/threonine protein kinases that plays an important role in controlling the function of other proteins and in several signal transduction cascades. PKC enzymes are activated by signals, including increased concentration of DAG or calcium ions (Ca21). Although OS and the activation of PKC are initially defined as independent biochemical outcomes of hyperglycemia, evidence is increasing that the abnormalities are, to some extent, related to each other. Hyperglycemia and increased OS can activate PKC, and the modification of PKC can further contribute to redox-mediated signaling events, while prolonged oxidant exposure could result in sustained PKC activation, which stimulates the formation of DAG.30 PKC activation also contributes to ROS production and OS by increasing the activity of Nox.31 Other effects triggered by PKC activation are: inhibition eNOS expression in endothelial cells32; increased expression of VEGF in vascular smooth muscle cells33; decreased NO production in smooth muscle cells34; activation of NF-κB.35 Furthermore, inhibition of PKC can inhibit the hyperglycemia-induced increase in free radical production.36 Thus it is possible that retinal PKC activation seen in diabetes could also be, in part, the result of increased OS.

Activation of hexosamine pathway Increased accumulation of advanced glycation end products AGEs are proteins, lipids, or nucleic acids that become nonenzymatically glycated after exposure to sugars. Increased accumulation of AGEs has been implicated in changes in the structure, function, and activity of extracellular and intracellular proteins, including proteins involved in the regulation of gene transcription by chemical rearrangements and the formation of cross-links.28 In the late stage, AGEs are irreversibly formed and accumulate within retinal capillary cells and bind with receptor for advanced glycation end products (RAGE) and initiate a sequence of events that eventually lead to retinal damage in DR.29 Activation of NAPDH oxidase is speculated to be the primary mechanism by which AGEs-RAGE induces OS, which, in turn, transduces multiple signals, eventually resulting in cytokine formation,

Hyperglycemia-induced OS also contributes to the pathogenesis of diabetes by increasing the flux of fructose-6-phosphate into the hexosamine pathway. The inhibition of GAPDH by ROS causes the diversion of all glycolytic metabolites to the hexosamine pathway in which fructose-6-phosphate is converted to glucosamine 6-phosphate that is subsequently converted to uridine diphosphate N-acetyl glucosamine, a substrate used for the posttranslational modification of intracellular factors, including transcription factors results in endothelial cell and retinal neuron apoptosis.37 Furthermore, OS induces the release of proinflammatory mediators including cytokines, NO, and prostaglandins by the activation of nuclear factor kappa B (NF-kB), and the levels of NF-kB, IL-1β, IL-6, and IL-8 were found to be high in the retina and vitreous of patients with proliferative DR as well as in animal models of DR.38,39 The OS might directly or indirectly

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FIGURE 3.4 Oxidative stress
mediated metabolic disturbances in diabetic retinopathy. Biochemical abnormalities in the retina that are aggravated in DM can also induce OS.

induce upregulation of several inflammatory mediators in DM, and DR is currently considered as a lowgrade chronic inflammatory disease.40

Oxidative stress and structural and functional alterations in diabetic retinopathy Biochemical changes induced by OS cause both functional and structural alterations in the retinal microvasculature (Fig. 3.4). ROS triggers these changes with both direct and indirect mechanisms and by effector molecules.

Basement membrane thickening OS induces AGEs formation on collagen leading to cross-linking28 that causes basement membrane thickening.41 Thickening of the basement membrane limits the transport of growth factors results in pericyte and endothelial cell loss. Moreover, ROS has been shown to cause blood
retinal barrier destruction and alterations in retinal blood flow.

Alterations in retinal microvasculature The interaction between AGEs and their cell surface receptor mediates the generation of ROS, the activation of NF-κB, and the expression of VEGF.42 Due to activation of NF-kB, the expression of the inducible form of NOS is increased, resulting in increased NO production. Increased levels of nitrogen species, including NO and peroxynitrite, promote leukocyte adhesion to retinal vessels. This contributes to hyperpermeability and the breakdown of the blood
retinal barrier, ultimately leading to angiogenesis and thrombosis.43 ROS also stimulate endothelin-1, a potent vasoconstrictor.

Apoptosis of retinal cells The OS induced mechanisms are complex, but most probably include changes in membrane lipid peroxidation, oxidative damage to macromolecules necessary for cellular functions, changes in signal transduction, and expression of genes.44,45 It has been shown that exposure of pericytes and endothelial cells to high glucose lead to increased OS, caspase-3 activity, NF-κB activity, and other transcription factors leading to capillary cell death (Fig. 3.5).39,46 These mediators, in turn, further increase ROS production and lead to DNA damage and apoptosis. ROS increase permeability of mitochondrial pores that induces the release of cytochrome c and other proapoptotic factors from retinal mitochondria to initiate apoptosis by the activation of caspases.47 Caspases, a group of cysteine proteases, play a role in apoptosis.48 Activation of caspase-9 initiates a cascade of events that activates executioner caspase-3 that is responsible for fragmenting DNA.49 Another mediator of apoptosis is the NF-kB. Activation of NF-kB leads to initiation of a proapoptotic program, which, in turn, leads to induction of proinflammatory mediators including tumor necrosis factor alpha and inducible NOS in retinal endothelial cells and pericytes.38,50

Oxidative stress-related epigenetic modifications in diabetic retinopathy In DR the expression of many genes is altered in the retina related to metabolic anomalies, including OS.51 The major epigenetic modifications are DNA methylation, histone modification, and the effects of miRNAs and sirtuin proteins (SIRTs).

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FIGURE 3.5 Color fundus photography of a patient with nonproliferative diabetic retinopathy. Hard exudates and hemorrhages which are indicators of pericyte loss and endothelial cell damage were observed.

DNA methylation DNA methylation plays an important role in the process of DR and metabolic memory. Normally, CpG islands, rich in promoter regions, contain unmethylated cytosines. Methylation of the CpG changes protein-DNA interactions leading to alterations in chromatin structure resulting in gene suppression.52 The methylation process is done with DNA methyl transferases (Dnmts). Diabetes activates Dnmts in the retina, and CpG islands are hypermethylated in nuclear DNA and mtDNA.53 mtDNA transcription and electron transport chains are disrupted due to hypermethylation.

Histone modification Another effective factor is histone modification in DR. Histones are the proteins that can regulate the structure of DNA and the expression of various genes. There are five main histone families: H1/H5, H2A, H2B, H3, and H4. The N-terminal sequences of histones can be modified mainly by acetylation and methylation.51 The acetylation of histones at lysine residues generally occurs with transcriptionally active genes, whereas the methylation of lysine is related to gene activation or repression.54 Histones at the promoter of the enzyme critical in biosynthesis of the intracellular antioxidant GSH are also modified in DM, resulting in decreased binding of the transcriptional factor Nrf2, and decreasing its transcription.55,56 Furthermore, the promoter and enhancer regions of superoxide

dismutase-2 (SOD-2) change in diabetes,57 and histone lysine acylation and methylation in monocytes at inflammation-related and diabetes-related gene loci can be induced by hyperglycemia.58,59 It was shown that hyperglycemia and hydrogen peroxide treatment increase the expression of coactivator associated arginine methyltransferase 1 via histone 3 arginine 17 asymmetric demethylation in RPE cells, suggesting that OS-mediated cell damage is associated with histone modification.60

MicroRNAs MicroRNAs (miRNAs) are tiny noncoding RNAs regulating posttranscriptional gene expression by binding to their target messenger RNAs. Circulating miRNA levels are altered in diabetes and it was shown that upregulation of VEGF is associated with the downregulation of miRNA-126, miRNA-146a, and miRNA-200b in DR animal models.61,62 Moreover, the expression of miRNA-26b-5p increases after exposure to high levels of glucose,63 and miRNA-26b-5p facilitates cardiomyocyte apoptosis by increasing the production of ROS,64 suggesting its contribution to endothelial apoptosis in DR.

Sirtuin proteins Sirtuin proteins (SIRTs) are a family of histone deacetylases that play key roles in the regulation of metabolism, OS, and DNA repair.65,66 Humans have seven SIRTs, of these SIRT1, which regulates OS response

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References

As described in DR, the contribution of mitochondrial damage due to OS has been shown in Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis.77 One of the most important regulators of inflammation is the transcription factor nuclear factor-κB (NFκB), because activation of NF-κB as a pathological mechanism has been shown in several diseases including atherosclerosis, septic shock, and pulmonary diseases.78
80

Summary points

FIGURE 3.6

Schematic representation of diabetes-induced metabolic disturbances, reactive oxygen species (ROS), and epigenetic modifications. Increase in ROS degrades biomolecules and epigenetic modifications alter gene expression of proteins associated with maintain a redox balance. Apoptosis is accelerated resulting in the development of diabetic retinopathy.

and RPE cell apoptosis via the deacetylation of cytoplasmic p53, is closely related with DM and its complications.67,68 It has been shown that SIRT1 activity decreases in the retinas of DM patients.69 In conclusion, DM is one of the OS states in which ROS increase and/or antioxidant mechanisms are inhibited. OS has been implicated as a contributor to the onset and the progression of DR. OS causes retinal damage by inducing endothelial cell dysfunction, pericyte apoptosis, and angiogenesis. Metabolic abnormalities induced by hyperglycemia—including polyol, AGE, PKC, hexosamine related pathways, and epigenetic modifications—are involved in the etiopathogenesis of DR and seem to be influenced by OS (Fig. 3.6).

Conclusion This chapter describes the role of OS in the pathogenesis of DR. There is growing scientific interest in connecting OS with a variety of pathological conditions including DM and other human diseases such as hypertension, neurodegenerative diseases, asthma, rheumatoid arthritis, and chronic renal failure.70
76

• DM is one of the OS states in which ROS increase and/or antioxidant mechanisms are inhibited. • The retina is the most metabolically active tissue in the body and is, therefore, highly susceptible to OS owing to its hypermetabolic state. • Hyperglycemia-induced metabolic abnormalities and OS seem to be inter-related. • In DR, the expression of many genes is altered in the retina and are related to metabolic anomalies including OS. • Biochemical changes induced by OS cause functional and structural alterations in the retinal microvasculature.

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9. Nakamura M, Barber AJ, Antonetti DA, LaNoue KF, Robinson KA, Buse MG, et al. Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons. J Biol Chem 2001;276:43748
55. Romeo G, Liu WH, Asnaghi V, Kern TS, Lorenzi M. Activation of nuclear factor-kappaB induced by diabetes and high glucose regulates a proapoptotic program in retinal pericytes. Diabetes 2002;51:2241
8. Kowluru RA, Koppolu P, Chakrabarti S, Chen S. Diabetesinduced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants. Free Radic Res 2003;37:1169
80. Kern TS. Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy. Exp Diabetes Res 2007;2007:95103. Khan ZA, Chakrabarti S. Cellular signaling and potential new treatment targets in diabetic retinopathy. Exp Diabetes Res 2007;2007:31867. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 2006;114:597
605. Yamagishi S, Nakamura K, Matsui T, Inagaki Y, Takenaka K, Jinnouchi Y, et al. Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression. J Biol Chem 2006;281:20213
20. Matsura T, Kai M, Fujii Y, Ito H, Yamada K. Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation. Free Radic Res 1999;30:73
83. Kaneto H, Kajimoto Y, Miyagawa J, Matsuoka T, Fujitani Y, Umayahara Y, et al. Beneficial effects of antioxidants in diabetes: possible protection of pancreatic β-cells against glucose toxicity. Diabetes 1999;48:2398
406. Kowluru RA, Tang J, Kern TS. Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 2001;50:1938
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47. Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radic Biol Med 2001;31:367
73. 48. Alnemri ES. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 1997;64:33
42. 49. Kristal BS, Koopmans SJ, Jackson CT, Ikeno Y, Park BJ, Yu BP. Oxidant-mediated repression of mitochondrial transcription in diabetic rats. Free Radic Biol Med 1997;22:813
22. 50. Dutta J, Fan Y, Gupta N, Fan G, Gelinas C. Current insights into the regulation of programmed cell death by NF-κB. Oncogene 2006;25:6800
16. 51. Kowluru RA, Kowluru A, Mishra M, Kumar B. Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy. Prog Retin Eye Res 2015;48:40
61. 52. Gravina S, Vijg J. Epigenetic factors in aging and longevity. Pflug Arch 2010;459:247
58. 53. Tewari S, Zhong Q, Santos JM, Kowluru RA. Mitochondria DNA replication and DNA methylation in the metabolic memory associated with continued progression of diabetic retinopathy. Invest Ophthalmol Vis Sci 2012;53:4881
8. 54. Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 2005;6:838
49. 55. Mishra M, Zhong Q, Kowluru RA. Epigenetic modifications of Nrf2-mediated glutamate-cysteine ligase: implications for the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression. Free Radic Biol Med 2014;75:129
39. 56. Zhong Q, Mishra M, Kowluru RA. Transcription factor Nrf2mediated antioxidant defense system in the development of diabetic retinopathy. Invest Ophthalmol Vis Sci 2013;54:3941
8. 57. Zhong Q, Kowluru RA. Epigenetic modification of Sod2 in the development of diabetic retinopathy and in the metabolic memory: role of histone methylation. Invest Ophthalmol Vis Sci 2013;54:244
50. 58. Miao F, Wu X, Zhang L, Yuan YC, Riggs AD, Natarajan R. Genome-wide analysis of histone lysine methylation variations caused by diabetic conditions in human monocytes. J Biol Chem 2007;282:13854
63. 59. Yun JM, Jialal I, Devaraj S. Epigenetic regulation of high glucose-induced proinflammatory cytokine production in monocytes by curcumin. J Nutr Biochem 2011;22:450
8. 60. Kim DI, Park MJ, Lim SK, Choi JH, Kim JC, Han HJ, et al. Highglucose-induced CARM1 expression regulates apoptosis of human retinal pigment epithelial cells via histone 3 arginine 17 dimethylation: role in diabetic retinopathy. Arch Biochem Biophys 2014;560:36
43. 61. Mastropasqua R, Toto L, Cipollone F, Santovito D, Carpineto P, Mastropasqua L. Role of microRNAs in the modulation of diabetic retinopathy. Prog Retin Eye Res 2014;43:92
107. 62. Silva VA, Polesskaya A, Sousa TA, Correˆa VM, Andre´ ND, Reis RI, et al. Expression and cellular localization ofmicroRNA-29b and RAX, an activator of the RNA-dependent protein kinase

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40. Dey N, Bera A, Das F, Ghosh-Choudhury N, Kasinath BS, Choudhury GG. High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression. Cell Signal 2015;27:1276
85. Suh JH, Choi E, Cha MJ, Song BW, Ham O, Lee SY, et al. Upregulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3β protein expression. Biochem Biophys Res Commun 2012;423:404
10. D’Onofrio N, Vitiello M, Casale R, Servillo L, Giovane A, Balestrieri ML. Sirtuins in vascular diseases: emerging roles and therapeutic potential. Biochim Biophys Acta 2015;1852:1311
22. Colak Y, Ozturk O, Senates E, Tuncer I, Yorulmaz E, Adali G, et al. SIRT1 as a potential therapeutic target for treatment of nonalcoholic fatty liver disease. Med Sci Monit 2011;17:HY5
9. Jing E, Emanuelli B, Hirschey MD, Boucher J, Lee KY, Lombard D, et al. Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production. Proc Nati Acad Sci U S A 2011;108:14608
13. Bhattacharya S, Chaum E, Johnson DA, Johnson LR. Age-related susceptibility to apoptosis in human retinal pigment epithelial cells is triggered by disruption of p53-Mdm2 association. Invest Ophthalmol Vis Sci 2012;53:8350
66. Kubota S, Ozawa Y, Kurihara T, Sasaki M, Yuki K, Miyake S, et al. Roles of AMP-activated protein kinase in diabetes-induced retinal inflammation. Invest Ophthalmol Vis Sci 2011;52:9142
8. Caramori G, Papi A. Oxidants and asthma. Rev Thorax 2004;59:170
3. Ceriello A. Possible role of oxidative stress in the pathogenesis of hypertension. Rev Diabetes Care 2008;31:S181
4. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr 2000;71:621S
9S. Galle J. Oxidative stress in chronic renal failure. Nephrol Dial Transplant 2001;16:2135
42. Guo C, Sun L, Chen X, Zhang D. Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural Regen Res 2013;8:2003
14. Liu SF, Malik AB. NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 2006;290:L622
45. MacNee W. Oxidative stress and lung inflammation in airways disease. Eur. J. Pharmacol 2001;429:195
207. Mahajan A, Tandon VR. Antioxidants and rheumatoid arthritis. J. Indian Rheumatol. Ass 2004;12:139
42. Singh RP, Sharad S, Kapur S. Free radicals and oxidative stress in neurodegenerative diseases: relevance of dietary antioxidants. JIACM 2004;5:218
25. Wright JG, Christman JW. The role of nuclear factor kappa B in the pathogenesis of pulmonary diseases: implications for therapy. Am J Respir Med 2003;2:211
19. Yu XH, Zheng XL, Tang CK. Nuclear factor-κB activation as a pathological mechanism of lipid metabolism and atherosclerosis. Adv Clin Chem 2015;70:1
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C H A P T E R

4 Pathological bases of oxidative stress in the development of cardiovascular diseases Mariane Ro´vero Costa, Je´ssica Leite Garcia, Carol Cristina Va´gula de Almeida Silva, Ana Paula Costa Rodrigues Ferraz, Fabiane Valentini Francisqueti-Ferron, Artur Junio Togneri Ferron and Camila Renata Correˆa Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil

ONOO 2 oxLDL PAMPs RAGE RNS ROS RS Sirt1 SOD TGF-β TNF-α UT-II VCAM-1 XO

List of abbreviations 4-HNE ACR ADP AGE ALE ATP AT-II Bcl-2 BH4 CVD DAMP DNA ETC ET-1 eNOS GPX GO H2O2 ICAM-1 IL-1 IL-18 IL-6 IRI LDL MDA MGO MPO NADPH NF-κB NK NO NOS Nox Nrf2 O2 2

4-hydroxy-nonenal acrolein adenosine diphosphate advanced glycation end-products advanced lipoxidation end-products adenosine triphosphate angiotensin II B-cell lymphoma 2 tetrahydrobiopterin cardiovascular disease damage-associated molecular pattern deoxyribonucleic acid electron transport chain endothelin-1 endothelial NO synthase glutathione peroxidase glyoxal hydrogen peroxide intercellular adhesion molecule 1 interleucin-1 interleucin-18 interleucin-6 ischemia-reperfusion injury low-density lipoprotein malondialdehyde methylglyoxal mieloperoxidase nicotinamide adenine dinucleotide phosphate nuclear factor kappa B natural killer nitric oxide NO synthase NADPH oxidases nuclear factor erythroid 2
related factor 2 superoxide

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00004-4

peroxynitrite oxidized LDL pathogen-associated molecular pattern receptors for AGE reactive nitrogen species reactive oxygen species reactive species sirtuin 1 superoxide dismutase transforming growth factor beta tumor necrosis factor urotensin II vascular cell adhesion protein 1 xanthine oxidase

Introduction Cardiovascular disease (CVD) is an ordinary term for a group of disorders of the heart and blood vessels, including coronary, cerebrovascular, peripheral arterial, rheumatic, congenital heart disease, and venous thromboembolism. These diseases are the main cause of death in Western countries, totaling 17.9 million deaths per year and representing 31% of the global mortality.1 Of these deaths, over 75% happen in lowand middle-income countries and almost equally in males and females.2 CVD is multifactorial, involving behavioral aspects such as unhealthy eating habits, physical inactivity, and smoking as well as environmental and genetic factors.1 All these causes are associated with a phenomenon called oxidative stress (OS), characterized by the redox system imbalance that occurs when the

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4. Pathological bases of oxidative stress in the development of cardiovascular diseases

FIGURE 4.1 Cardiovascular disease and its mechanisms. Cardiovascular disease (CVD) is an umbrella term including several diseases of multifactorial etiology and several mechanisms which have oxidative stress in common. Reactive species (RS) are involved in physiologic processes (proliferation, growth, differentiation, apoptosis, migration, contraction, and cytoskeletal regulation) playing a key role in both health and disease by acting as signaling molecules. However, when in excess, RS can trigger the development of pathologic conditions (chronic inflammation and autoimmune diseases, sensory impairment, CVD, cancer, fibrotic disease, obesity, insulin resistance, neurological disorders, and infectious diseases).

production of reactive species (RS) exceeds the defense antioxidant capacity, or vice versa (Fig. 4.1).3 In the context of CVD, OS occurs mainly due to the overproduction of RS.3 These species include reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can damage molecules—such as DNA, lipids, carbohydrates, and proteins—by modifying their structures.4 RS can oxidize macromolecules generating oxidation products such as malondialdehyde (MDA), glyoxal (GO), acrolein (ACR), 4-hydroxy-nonenal (4HNE), and methylglyoxal (MGO), responsible for the formation of carbonylated proteins including advanced glycation end-product (AGE) and advanced lipoxidation end-products (ALE), which are important agents in the development of pathologies associated with OS.5 In the pathophysiology of CVD, several cellular compartments and systems, including enzyme systems, immune cells and mitochondrial dysfunction, are involved with the production of RS and OS. These factors are closely linked to the development of cardiac changes considering that the heart is a highly metabolic organ due to its high energy demand, which promotes a favorable scenario for oxidative damage.6

Mitochondrial production of reactive species Mitochondria is an organelle composed of a double membrane and whose main function is the synthesis

of adenosine triphosphate (ATP).7 Mitochondria comprises 30% to 40% of myocyte volume and represent the largest source of ROS in the cardiovascular system. The constant movements of systole and diastole require a high demand of energy produced through oxidative phosphorylation.8 This process is based on the transfer of electrons through the electron transport chain. Under physiological conditions, the respiratory chain works efficiently, using over 98% of the electron transport for ATP synthesis. Only 1%
2% of electrons are released to generate superoxide radical (O2 2 ) that is decomposed by superoxide dismutase (SOD).3 The ATP synthesis process is based on the transfer of electrons through the mitochondrial respiratory chain coupled with transporting protons (H1) from the matrix to the intermembrane space to generate the proton motive force which is used for the conversion from adenosine diphosphate (ADP) to ATP.9 During this process, electrons escape from the electron transport chain and react with oxygen (O2) forming O2 2 , which is impermeable to the mitochondrial membrane. Thus it is converted into hydrogen peroxide (H2O2) by SOD. Glutathione peroxidase (GPx) is responsible for the reduction of H2O2 into H2O.10 In excess, H2O2, due to its neutral character, can easily leave mitochondria and can be reduced by the Fenton or Haber-Weiss reactions forming hydroxyl radical (OH•), a highly reactive molecule that leads to a significant oxidative damage (Fig. 4.2).11

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Role of oxidative stress in cardiovascular disease

41 FIGURE 4.2 Mitochondrial reactive oxygen species production. Reactive oxygen species (ROS) production by mitochondria occurs physiologically through the electron transport chain (ETC). In pathological conditions such as in CVD, an important production of ROS occurs leading to damage in mitochondrial DNA, production of impaired proteins and lipid membrane lipid peroxidation, triggering the change in membrane permeability. These damages are associated with impairment of the ability of mitochondria to produce ATP. In summary, mitochondrial dysfunction, activation of apoptotic mechanisms, and necrosis.

The production of ROS by the mitochondria represents a continuous cycle and is responsible for changes in cellular energetic demand. The mitochondria respond rapidly to fluctuations in energy requirements in order to maintain cellular levels of ATP.12 However, excessive stimulation of the mitochondria by the excess of substrate leads to ROS overproduction and the depletion of antioxidants, resulting in an imbalance between the oxidant production and the antioxidant defense system, which constitutes OS.13 The mitochondria are sensitive to changes in the supply of nutrients and oxygen and adapt to cellular environment changes. Functional abnormalities of cardiac mitochondria in CVD lead to enhanced OS, reduced production of ATP and energy supply, increased cell apoptosis, and impaired autophagic mechanisms.14

Role of oxidative stress in cardiovascular disease CVD can be triggered by OS in several situations such as ischemia and reperfusion, atherosclerosis, hyperlipidemia, inflammation, and peripheral arterial hypertension.

Ischemia and reperfusion Ischemia is a phenomenon caused by reduced, or obstructed, arterial blood flow that is responsible for a significant imbalance of oxygen and metabolic substrates.15 Nevertheless, the arterial blood-flow restoration and reoxygenation are followed by the

exacerbation of tissue damage and inflammatory response. This process is known as ischemiareperfusion injury (IRI).16 IRI is importantly associated with tissue damage in a range of clinical situations such as myocardial infarction, stroke, and organ transplantation.17 The mechanisms involving the IRI development are still being studied; however, some pathophysiological processes that contribute to the formation of lesions in the ischemia are associated with decreased ATP levels and intracellular pH that results in cell death by necrotic, and the activation of apoptotic and autophagic mechanisms.18 However, in reperfusion, with the increase of oxygen availability, there is an increase in S formation, representing the trigger of the tissue injury.17 The increase of RS can cause tissue damage through the direct injury of macromolecules and/or DNA, or through the activation of pathways related to various mechanisms. Among them, we point out the induction of the inflammatory response through the activation of nuclear factor kappa B (NF-κB), resulting in the expression of proinflammatory cytokines and prohypertrophic proteins.15 The NF-κB also leads to fibrosis through the activation of profibrotic mediators promoting the differentiation process from cardiac fibroblasts to myofibroblasts, increasing the production of extracellular matrix and disturbing the calcium-handling capacity that interferes in the autophagy process. All these events which are modulated by ROS orchestrate the development of cardiac failure (Fig. 4.3).19 These processes can act separately or simultaneously, and although their order varies depending on the tissue, they all entail two crucial

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4. Pathological bases of oxidative stress in the development of cardiovascular diseases

FIGURE 4.3 The phenomenon of ischemia and reperfusion. Ischemia results in decreased ATP production and, consequently, a perturba-

tion of the ion pump leading to the intracellular accumulation of Na1, Ca21, and H1. In addition, there is a shift in the production of energy through anaerobic glycolysis, which also contributes to the intracellular acidification. During ischemia, the activation and the increase in the expression of ROS-producing enzymes and the electron transport chain (ETC) dysfunction occur. As the oxygen returns to the ischemic tissue, on reperfusion, ROS production is intensified. ROS represents a proinflammatory stimulus that contributes to the magnitude of oxidative stress (OS) by activating the cells of the immune system. Through direct action, the ROS lead to DNA damage and proteins and lipid peroxidation. These events lead to cellular death.

steps: permanent mitochondrial lesion and redox signaling disruption.16

Hyperlipidemia and atherosclerosis Atherosclerosis is the main cause of coronary heart, cerebral, and peripheral vascular disease as well as infarction. Several factors may increase the risk of these diseases, such as smoking, hyperlipidemia, metabolic syndrome, diabetes mellitus, obesity, and sedentary lifestyle.20 Atherosclerosis is a chronic inflammatory disease characterized by the formation of atheromas (plaques of fat, cholesterol, and other substances) inside the blood vessels, causing their narrowing and obstruction.21 This process is closely related to the prooxidant environment in which RS participate in the atherogenesis by the oxidation of low-density lipoprotein (LDL). The oxidized LDL (oxLDL) is engulfed by the vascular wall macrophages

to form foamy cells, which are among the initial signatures of the atherogenesis.22 Thereafter, an inflammatory process is installed in order to eliminate these particles, beginning with the migration of monocytes from the blood stream targeting injured vascular tissues where these cells differentiate into macrophages. Monocyte-derived macrophages uptake oxLDL and become lipid-rich foam cells, leading to the overproduction of ROS, mainly by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, to digest the fat. These cells release proinflammatory cytokines contributing to the amplification of inflammation and migration of new inflammatory cells, contributing to the increase of the thickness of the endothelium forming the atheroma plaque (Fig. 4.4).23 Atheroma plaques may also appear in specific situations, such as diabetes mellitus. High levels of blood glucose induce continuous irreversible glycation and oxidation of proteins and lipids, which lead to the formation of AGE in the arterial wall.24 AGE can bind to

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Role of oxidative stress in cardiovascular disease

43

FIGURE 4.4 Atheroma plaque formation and advanced glycation end-product (AGE). The prooxidant environment associated with conditions such as smoking, diabetes, obesity and hyperlipidemia favors endothelial dysfunction. ROS derived from advanced glycation endproduct (AGE) response are present in the circulation oxidize low-density lipoprotein (LDL) and injure the endothelium by triggering an inflammatory response. Adherence of monocytes to the endothelium and infiltration of macrophages in the vessel intima occur. The macrophages phagocyte the oxidized LDL (oxLDL) forming foam cells and contributing to the formation of ROS. Platelet activation also occurs.

the receptors for AGE (RAGE) on the cell surface, which is expressed in many tissues including the vascular system. The binding of AGE to RAGE causes intracellular signaling that leads to the production of ROS, mainly via activation of NF-κB and Nox.24 This activation also leads to the downregulation of sirtuin 1 (Sirt1) and nuclear factor erythroid 2
related factor 2 (Nrf2), which are ways of producing antioxidant enzymes.25 Subsequently, there is an increase in the production of proinflammatory cytokines, chemokines, apoptosis regulators such as B-cell lymphoma 2 (Bcl-2), Fas and vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), as well as macrophages and platelets activation.24 Interestingly, ROS production by RAGE activation causes positive feedback, upregulating the RAGE expression and, thus, leading to the exacerbation of the inflammatory processes.22

Hypertension Hypertension is a multifactorial disorder involving perturbations of the vasculature, kidney, and central

nervous system. Its etiology depends on several factors including smoking, diet, genetics, family history, and preexisting pathologies.9 In addition to the complex etiology of this disease, oxidative and nitrosative stresses appear to be a common feature within hypertensive disorders. Indeed, 95% of reported high blood pressure cases are deemed “essential hypertension,” and at the molecular level, OS seems to be a common feature of hypertensive states.26 At the vascular level, ROS and RNS are responsible for the regulation of the physiological and pathological signaling. In physiological conditions, RS are responsible for maintaining adequate blood flow and dilatation of blood vessels.27 During OS, they promote the vascular damage observed in chronic conditions.28 Evidence supports the role of the vascular wall as a major source of ROS that are generated by enzymatic and nonenzymatic systems existing in blood vessels. Several of these systems are known to be predominant in pathologic processes. The best-characterized source of ROS is NADPH oxidase (Nox). Other sources of ROS are xanthine oxidase (XO), NO synthase (NOS), and mitochondrial enzymes (Fig. 4.5).29

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4. Pathological bases of oxidative stress in the development of cardiovascular diseases

FIGURE 4.5 Reactive species and hypertension. Some factors induce the production of reactive species (RS) by activating mitochondrial production and NADPH oxidases (Nox). Another RS source is the enzyme xanthine oxidase (XO). Regardless the source, these species lead to the activation of nuclear factor kappa B (NF-kB) and, therefore, to inflammatory response. In addition, RS are associated with lipid peroxidation and mitochondrial dysfunction. All these factors lead to mitochondrial dysfunction and are considered the mainstays of the development of hypertension based on oxidative stress (OS).

Nicotinamide adenine dinucleotide phosphate oxidase NADPH oxidases are a family of transmembrane proteins that are oxygen- and NADPH-dependent oxidoreductases which produce O2 2 and/or H2O2 in various cell types and tissues. Nox is the primary biochemical source of ROS in the vasculature, particu30 larly of O2 2 . The mammalian Nox family includes seven isoforms: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2. Nox isoform expression varies among different cell types of renal and systemic vasculatures, often with more than one isoform expressed in various cell types.30 The kidney and vasculature are rich sources of Nox-derived ROS, which, under pathological conditions, play an important role in renal dysfunction and vascular damage.31 Several factors such as hormones, growth factors, immune mediators, and some vasoactive peptides as angiotensin II (AT-II), endothelin-1 (ET-1), and urotensin II (UT-II) could activate the NADPH system, leading to hypertension. This occurs since the RS produced

in response to these factors are capable of inactivating the nitric oxide (NO) forming peroxynitrite (ONOO 2 ), leading to an impaired endothelium vasodilation and an uncoupling of the endothelial NO synthase (eNOS), thereby producing additional 29,31 In this way, the process is related to the O2 2 . development of hypertension through the increase of vasoconstriction and extracellular matrix accumulation.30 Xanthine oxidase XO is another important source of ROS and RNS in the vascular endothelium. It catalyzes the last two steps of purine metabolism (i.e., the conversion of hypoxanthine to xanthine and then into uric acid). In this reaction, the formation of O2 and H2O2 occurs 2 from the consumption of oxygen and these RS contribute to the development of hypertension through endothelial dysfunction.31 Thus eating habits have important consequences for cardiovascular health, since excessive consumption of foods such as animal protein (e.g., meat and seafood) as well as alcohol and

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Role of oxidative stress in cardiovascular disease

fructose lead to an increase in the activity of this enzyme with the consequent increase in OS.32 Another factor that stands out in the development of arterial hypertension is the increase of serum levels of uric acid, a metabolite that at high concentrations has prooxidant properties by favoring lipid oxidation with an evident inhibition of the reverse cholesterol transport and important proinflammatory effects at tissue level.33 Therefore the hyperuricemia has several pathophysiological consequences to vascular health.34 The two prooxidant factors, XO activity and hyperuricemia, lead to vascular injury through ROS cytotoxic action, especially by the increased synthesis of ONOO 2 , leading to a decrease of NO, resulting in endothelial dysfunction, and a rapid progression of atherosclerotic disease. These alterations are associated with platelet aggregation, vasoconstriction, proliferation of smooth muscle cells, fibrotic remodeling, and vascular inflammation, which all contribute to the development of CVD.34 Uncoupled endothelial nitric oxide synthase eNOS represents the main source of NO in the vasculature, which is produced in the endothelial cells in the inner surface of the blood arteries. The NO is known as an antihypertensive and antithrombotic factor since it regulates vasodilation, adhesion, and plaquetary aggregation. eNOS transfer electrons from NADPH to the oxygenase domain, which is also the binding site for tetrahydrobiopterin (BH4), oxygen, and L-arginine; the electrons are used to reduce O2 and to oxidize L-arginine to L-citrulline and NO.35 In pathological conditions such as in the absence of BH4 and L-arginine cofactors or in the presence of OS, the eNOS may cease producing NO and start generating O2 2 . The process in which eNOS loses its physiological property is called “eNOS uncoupling.”36 As a result, there is a compromise in the formation of NO, and an increase in the eNOS-mediated generation of 36,37 O2 2 contributing to enhance preexisting OS. This event of OS associated with eNOS uncoupling is characteristic of clinical conditions commonly related to CVDs such as diabetes mellitus, atherosclerosis, cerebral ischemia, and hypertension.38 Mitochondrial dysfunction in hypertension Mitochondria may be involved in the genesis of hypertension, and hypertension itself may promote mitochondrial dysfunction.39 Hypertension is associated with structural mitochondrial changes such as decreased mass and density, swelling, cristae remodeling and fragmentation. Obviously, these structural alterations are accompanied by an impairment of mitochondrial metabolic and bioenergetic functions,

45

including ROS overproduction and diminished ATP production.40 Moreover, hypertension has been associated with cardiolipin peroxidation, which is a phospholipid exclusively found in the inner mitochondrial membrane and is necessary for an adequate cristae formation. The cardiolipin oxidation triggers apoptosis mechanisms and ROS decreases mitochondrial membrane fluidity and energy production, creating a cycle of oxidative damage.40

Inflammation Inflammation is a protective answer against insults or injuries, essential to eliminate or neutralize organisms and foreign bodies.41 This response includes the participation of plenty of immune cell types such as granulocytes, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and T and B cells.42,43 However, the chronic activation of the inflammatory processes is related to a major action of cytokines and to the promotion of OS.44 The cytokines secreted by the immune cells act as intracellular signals and during the recruitment and activation of phagocytic cells contribute to the exacerbation of the inflammatory process.45 The activation of phagocytes in response to damage-associated molecular pattern (DAMP) and pathogen-associated molecular pattern (PAMP), and the binding of cytokines to their receptors, is responsible for the production of RS through Nox and myeloperoxidase (MPO). This production occurs by oxidoreduction reactions with several objectives, including the magnification of the inflammatory response and the destruction of microorganisms and cell debris. However, given the excessive stimulation of these immune cells, cellular and tissue damages occur (Fig. 4.6).46 These RS are important intracellular signals within the cells of the immune system, involving the activation of NF-κB, a major transcription factor involved in the signaling of inflammatory cytokines.47 Thus we can establish a link between the production of RS, the immune system, and inflammation.48 Inflammation is a central process in the development of CVD since it is involved in several pathological conditions including diabetes, hypertension, dyslipidemia, and obesity.49,50 In the latter, inflammation represents a link between visceral adiposity and CVD due to the greater production of adipokines by the adipose tissue that reaches other tissues, resulting in systemic inflammation. This whole process is related to CVD through some cytokines, including tumor necrosis factor (TNF-α), transforming growth factor beta (TGF-β), and different interleukins such as

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4. Pathological bases of oxidative stress in the development of cardiovascular diseases

FIGURE 4.6 Inflammation and the production of reactive oxygen species (ROS). Damageassociated molecular pattern (DAMP) and pathogen-associated molecular pattern (PAMP) activate phagocytes by binding to their receptors. Subsequently, NADPH oxidase (Nox) produces superoxide (O2 2 ) that undergoes an action of superoxide dismutase (SOD) and is converted into hydrogen peroxide (H2O2), and in the latter is converted into H2O and O2. However, in order to neutralize the aggressive agent, myeloperoxidase (MPO) converts H2O2 into hypochlorous acid. This acid, in addition to acting on the phagosome, is released into the extracellular medium. A continuous or exacerbated stimulation of this mechanism leads to the development of the increased production of the reactive species (RS).

interleucin-1 (IL-1), interleucin-6 (IL-6), and interleucin-18 (IL-18). These adipokines are involved in the stimulation of fibroblast proliferation, collagen and matrix production, and metalloproteinases expression that lead to tissue remodeling.51 It is important to emphasize that OS and inflammation are mutually dependent processes according to the condition in which they are initiated. Applications to other areas of pathology OS plays a role in the pathophysiology of various diseases, not only in CVD. It is the pivot of multiple disorders and can be used to enlighten the individual redox status by measuring lipid peroxidation and glycoxidation products, antioxidant enzymes, and vitamin levels.52 Although this practice has not been used in clinical routine, OS biomarkers have been widely used in experimental and clinical research.52
54 For instance, they have been effectively employed to monitor the stages of numerous diseases such as the neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s diseases), type II diabetes, some types of cancer, IRI and rheumatoid arthritis.54 These biomarkers have been measured in human studies at tissue and systemic level, such as skeletal muscle, plasma or blood cells, urine, and saliva.55,56 In this context, the measurement of OS biomarkers represents a promising approach for

predicting the onset of a disease, assessing its progression, and evaluating the effect of treatments.57

Summary points • Factors associated with cardiovascular disease (CVD): behavioral aspects, unhealthy eating habits, physical inactivity, smoking, and environmental and genetic factors. • Reactive species: RS include reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can damage molecules such as DNA, lipids, carbohydrates, and proteins. • Pathophysiology of CVD: several cellular compartments and systems are involved with the production of RS and OS, enzyme system, immune cells, and mitochondrial dysfunction. • Mitochondrial ROS: mitochondria comprise 30% to 40% of myocyte volume and represent the largest source of ROS in the cardiovascular system. • Mitochondrial dysfunction: this plays a key role in triggering OS in CVD • CVD mechanisms: ischemia and reperfusion, atherosclerosis, hyperlipidemia, inflammation, and peripheral arterial hypertension can trigger CVD through a common phenomenon, namely oxidative stress.

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References

References 1. World Health Organization. Cardiovascular diseases (CVDs). Geneva, Switzerland: World Health Organization; 2017. Available from: ,http://www.who.int/en/news-room/factsheets/detail/cardiovascular-diseases-(cvds).. 2. American Heart Association. Heart Disease and Stroke Statistics. Dallas, TX: American Heart Association; 2017. Available from: ,https://www.ahajournals.org/doi/pdf/ 10.1161/CIR.0000000000000485.. 3. Chistiakov DA, Shkurat TP, Melnichenko AA, Grechko V, Orekhov NA. The role of mitochondrial dysfunction in cardiovascular disease: a brief review. Ann Med 2018;50(2):121
7. 4. Semchyshyn HM. Reactive carbonyl species in vivo: eeneration and dual biological effects. Sci World J 2014;2014:27
31. 5. Aldini G, Dalle-Donne I, Colombo R, Facino RM, Milzani A, Carini M. Lipoxidation-derived reactive carbonyl species as potential drug targets in preventing protein carbonylation and related cellular dysfunction. Chem Med Chem 2006;1(10):1045
58. 6. Tocchi A, Quarles EK, Basisty N, Gitari L, Peter S. Mitochondrial dyfunction in cardiac ageing. Biochim Biophys Acta 2016;1847(11):1424
33. 7. Friedman JR, Nunnari J. Mitochondrial form and function. Nature 2014;505(7483):335
43. 8. Grivennikova VG, Vinogradov AD. Mitochondrial production of reactive oxygen species. Biochemistry 2013;78(13):1490
511. 9. Dikalov SI, Dikalova AE. Contribution of mitochondrial oxidative stress to hypertension. Curr Opin Nephrol Hypertens 2017;25 (2):73
80. 10. Quintana-cabrera R, Bolan˜os JP. Glutathione g-glutamylcysteine hydrogen peroxide detoxification, Methods Enzymol. 2013;527:129
44. 11. Jeˇzek J. Reactive oxygen species and mitochondrial dynamics: the Yin and Yang of mitochondrial dysfunction and cancer progression. Antioxidants 2018. 12. Long Q, Yang K, Yang Q. Regulation of mitochondrial ATP synthase in cardiac pathophysiology. Am J Cardiovasc Dis 2015;5 (1):19
32. 13. Nita MB, Grzybowski A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev 2016;2016. 14. Schrepfer E, Scorrano L. Mitofusins, from mitochondria to metabolism. Mol Cell 2016;61(5):683
94. 15. Eltsching HK, Eckle T. Ischemia and reperfusion-from mechanism to translation. Nat Med 2014;17(11). 16. Rodrı´guez-lara SQ, Cardona-mun˜oz EG, Ramı´rez-lizardo EJ, Totsuka-sutto SE, Castillo-romero A, Garcı´a-Cobia´n TA, et al. Alternative interventions to prevent oxidative damage following ischemia / reperfusion. Oxid Med Cell Longev 2016;2016. 17. Vries DK, Kortekaas KA, Tsikas D, Wijermars LGM, Van Noorden CJF, Suchy MT, et al. Oxidative damage in clinical ischemia / reperfusion injury. Antioxid Redox Sign 2013;19(6):536
45. 18. Bellanti F. Ischemia-reperfusion injury: evidences for translational research. Ann Transl Med 2016;4(7):3
5. 19. Kurian GA, Rajagopal R, Vedantham S, Rajesh M. The role of oxidative stress in myocardial ischemia and reperfusion injury and remodeling: revisited. Oxid Med Cell Longev 2016;2016. 20. Rudolf J, Lewandrowski KB. Cholesterol, lipoproteins, highsensitivity c-reactive protein, and other risk factors for arterosclerosis. Clin Lab Med 2014;34:113
27. 21. Gimbrone Jr MA, Garcı´a-Carden˜a G. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 2017;118 (4):620
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22. Kattoor AJ, Pothineni NVK, Palagiri D, Mehta JL. Oxidative stress in atherosclerosis. Curr Atheroscler Rep 2017;19(42):1
11. 23. Jacinto TA, Meireles GS, Dias AT, Aires R, Porto ML, Gava AL, et al. Increased ROS production and DNA damage in monocytes are biomarkers of aging and atherosclerosis. Biol Res 2018;51 (33):1
13. 24. Mcnair E, Qureshi FM, Cls C, Prasad K, Pearce FC. Atherosclerosis and the hypercholesterolemic AGE
RAGE axis. Int J Angiol 2016;1(212):110
16. 25. Chen XJ, Wu WJ, Qi Z, Jie JP, Chen X, Wang F, et al. Advanced glycation end - products induce oxidative stress through the Sirt1/Nrf2 axis by interacting with the receptor of AGEs under diabetic conditions. J Cell Biochem 2018;1
12. 26. Loperena R, Harrison DG. Oxidative stress and hypertensive diseases. Med Clin North 2018;101:169
93. 27. Touyz RM, Schiffrin EL. Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol 2004;122:339
52. 28. Sorriento D, De Luca N, Trimarco B, Iaccarino G. The antioxidant therapy: new insights in the treatment of hypertension. Front Physiol 2018;9(258):1
11. 29. Ramon R, Gonza´lez J, Paoletto F. The role of oxidative stress in the pathophysiology of hypertension. Hypertens Res 2016;1
10. 30. Qi-An S, Marschall SR, Nageswara RM. Oxidative stress, NADPH oxidases, and arteries. Hamostaseologie 2017;36(2):77
88. 31. Brito R, Castillo G, Valls N, Rodrigo R. Oxidative stress in hypertension: mechanisms and therapeutic opportunities. Exp Clin Endocrinol Diabetes 2015. 32. Bhatti JS, Bhatti GK, Hemanchandra R. Mitochondrial dysfunction and oxidative stress in metabolic disorders — a step towards mitochondria based therapeutic strategies. Mol Basis Dis 2017;1863(5):1066
77. 33. Gustafsson D, Unwin R. The pathophysiology of hyperuricaemia and its possible relationship to cardiovascular disease, morbidity and mortality. BMC Nephrology 2013;14(64):1
9. 34. Cicero AFG, Fogacci F, Bove M, Veronesi M, Rizzo M, Giovannini M, et al. Short-term effects of a combined nutraceutical on lipid level, fatty liver biomarkers, hemodynamic parameters, and estimated cardiovascular disease risk: a doubleblind, placebo-controlled randomized clinical trial. Adv Ther 2017;34(8):1966
75. 35. Feng C. Mechanism of nitric oxide synthase regulation: electron transfer and interdomain interactions. Coord Chem Rev 2012;256 (3-4):393
411. 36. Li Q, Yon J, Cai H. Mechanisms and consequences of eNOS dysfunction in hypertension. J Hypertens 2016;33(6):1128
36. 37. Forte M, Conti V, Damato A, Ambrosio M, Puca AA, Sciarretta S, et al. Targeting nitric oxide with natural derived compounds as a therapeutic strategy in vascular diseases. Oxid Med Cell Longev 2016;2016. 38. Li H, Forstemann U. Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease. Curr Opin Pharmacoloy 2018;13:161
7. 39. Rubattu S, Stanzione R, Volpe M. Mitochondrial dysfunction contributes to hypertensive target organ damage: lessons from an animal model of human disease. Oxid Med Cell Longev 2016;2016. 40. Eirin A, Lerman A, Lerman LO. Enhancing Mitochondrial Health to Treat Hypertension. Curr Hypertension Rep 2018;20 (89):1
8. 41. Minihane AM, Vinoy S, Russell WR, Baka A, Roche HM, Tuohy KM, et al. Low-grade inflammation, diet composition and health: current research evidence and its translation. Br J Nutr 2015;114(07):999
1012.

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42. Nicholson LB. The immune system. Essays Biochem 2016;60 (3):275
301. 43. Freire MO, Van Dyke TE. Natural resolution of inflammation. Periodontol. 2014;63(1):149
64. 44. Pashkow FJ. Oxidative stress and inflammation in heart disease: do antioxidants have a role in treatment and/or prevention? Int J Inflamm 2011;201:1
9. 45. Neri M, Fineschi V, Di Paolo M, Pomara C, Riezzo I, Turillazzi E, et al. Cardiac oxidative stress and inflammatory cytokines response after myocardial infarction. Curr Vasc Pharmacol 2015;13:26
36. 46. Paiva CN, Bozza MT. Are reactive oxygen species always detrimental to pathogens? Antioxid Redox Signal 2014;20(6):1000
37. 47. Filippin LI, Vercelino R, Marroni NP, Xavier RM. Redox influence on the inflammatory response in rheumatoid arthritis. Rev Bras Reumatol 2008;48(1):17
24. 48. Xie M, Burchefield JS, Joseph A. Pathological ventricular remodeling: mechanisms: Part 1 of 2. Circulation 2014;128(4):388
400. 49. Srivastava KK, Kumar R. Stress, oxidative injury and disease. Ind J Clin Biochem 2015;30(1):3
10. 50. Biswas SK. Does the interdependence between oxidative stress and inflammation explain the antioxidant paradox? Oxid Med Cell Longev 2016;2016:17
19.

51. Bartekova M, Radosinska J, Jelemensky M, Dhalla NS. Role of cytokines and inflammation in heart function during health and disease. Heart Fail Rev 2018;23. 52. Marrocco I, Altieri F, Peluso I. Measurement and clinical significance of biomarkers of oxidative stress in humans. Oxid Med Cell Longev 2017;2017:1
32. 53. Siti HN, Kamisah Y, Kamsiah J. The role of oxidative stress, antioxidants and vascular in fl ammation in cardiovascular disease (a review). Vasc Pharmacol 2015;71:40
56. 54. Frijhoff J, Winyard PG, Zarkovic N, Davies SS, Stocker R, Cheng D, et al. Clinical relevance of biomarkers of oxidative stress. Antioxid Redox Signal 2015;23(14):1144
70. 55. Schwedhelm E, Bo¨ger RH. Application of gas chromatographymass spectrometry for analysis of isoprostanes: their role in cardiovascular disease. Clin Chem Lab Med 2003;41(12):1552
61. 56. Su H, Gornitsky M, Velly AM, Yu H, Benarroch M, Schipper HM. Salivary DNA, lipid, and protein oxidation in nonsmokers with periodontal disease. Free Radic Biol Med 2009; 46(7):914
21. 57. Margaritelis NV, Cobley JN, Paschalis V, Veskoukis AS, Theodorou AA. Going retro: oxidative stress biomarkers in modern redox biology. Free Radic Biol Med 2016;98:2
12.

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C H A P T E R

5 Pathological association between oxidative stress and chronic obstructive pulmonary disease Mahmood A. Al-Azzawi Clinical Biochemistry & Molecular Biology, College of Dentistry, Al-Ayen University, Al-Nasiriyah, Iraq

List of abbreviations Aβ ICS mtDNA NADPH oxidase NF-κB RONS

is often underestimated, it has been considered the major incentive of COPD because it could lead to further strengthening of the other three mechanisms.3 In this chapter, we highlight the mechanisms that explain the development of oxidative stress and its role in causing COPD and some associated diseases.

amyloid beta inhaled corticosteroids mitochondrial DNA nicotinamide adenine dinucleotide phosphate oxidase. nuclear factor kappa-light-chain-enhancer of activated B cells reactive oxygen and reactive nitrogen species

Chronic obstructive pulmonary disease overview COPD is a long-term respiratory disorder accompanied by serious systemic features and related diseases that have adverse effects on the nature of life. It is an inflammatory disease and is characterized by lung inflammation and limited airflow through the airways that cannot be fully reversed, which, in turn, leads to an accelerated drop in lung function. COPD is closely associated with pulmonary arterial hypertension, hypoxemia, and hypercapnia, leading to greatly increased risk of cardiorespiratory intricacies and increased exacerbation of patients’ condition.4 Though the incidence rate of COPD is likely to intensify in both developed and developing countries, COPD poses a heavier burden on the Asia-Pacific and African regions where smoking is still widespread and gradually increasing.5 Cigarette smoking is undoubtedly a significant contributing factor for COPD.2 Exposure to biomass fuels and air pollutants are considered other predisposing factors.6 Interestingly, there is a long transition period in general that separates the exposure to smoke and the onset of the disease, which explains why there is a high rate of COPD in the elderly and former smokers. This transition period may last for a number of years. Once the symptoms of the

Introduction Chronic obstructive pulmonary disease (COPD) is an extremely widespread disease worldwide. The main characteristic of this disease is a constant airflow restriction, which is often developing and correlated with an increased chronic inflammatory response in the airways and lungs to deleterious gases and particles.1 The most significant aspect in the pathogenesis of COPD and its related concurrent diseases is the progressive effect of oxidative stress. Present styles of curing for COPD are typically inefficient, The evolution of dynamic therapies for COPD has been seriously restricted as the mechanisms and intermediaries that motivate the initiation and development of chronic inflammation, lung dysfunction, deficient lung immunity, emphysema, and other extrapulmonary comorbidities features are not clearly defined.2 All that is known so far is that four major mechanisms are engaged in the pathophysiological variation noticed in COPD: inflammation, apoptosis, protease
antiprotease imbalance, and oxidative stress. While oxidative stress

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00005-6

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

disease are present in patients, the damage becomes irreversible.7 Unfortunately, the disease is not confined to pulmonary manifestations of COPD; many systemic disorders arise in the form of comorbid diseases, in particular, lung carcinoma, skeletal muscle degeneration, cardiac impairment, and osteoporosis.8

Oxidative stress Oxidative stress is described as an unbalance between oxidant and antioxidant levels in favor of a prooxidant environment in cells and tissues. It results in disturbances of redox signaling and control and/or gives rise to the biomolecules destruction.9 Oxidative stress is characterized by an extreme accretion of toxic molecules comprising an oxygen atom with an unpaired electron in its outer shell, creating the final product of the normal oxygen metabolism, termed reactive oxygen species (ROS) (Table 5.1). The major origin of ROS is the electron transport chain in the mitochondrial membrane.10 Changes in the natural oxidoreduction state of cells can lead to toxic impacts through the creation of free radicals and peroxides that can seriously destroy the proteins, fats, and DNA of the cell.11 Also, species that contain nitrogen, referred to as reactive nitrogen species (RNS) (Table 5.1), contain nitric oxide (NO∙), which is somewhat unreactive, and its by-product peroxynitrite (ONOO2), a strong oxidant capable of destroying numerous biological TABLE 5.1 Major reactive oxygen and nitrogen species (RONS). Reactive oxygen species (ROS) Superoxide anion (radical)

(O2•2)

Reactive nitrogen species (RNS) Peroxynitrite (ONOO2)

Hydroxyl ion (radical) (•OH2)

Nitrogen dioxide (radical) (NO2•2)

Singlet oxygen (1O2)

Alkyl peroxynitrite (ROONO) •2

Peroxyl (radical) (ROO )

Dinitrogen tetroxide (N2O4)

Alkoxy (radical) (RO•2)

Dinitrogen triroxide (N2O3)

Hydrogen peroxide (H2O2)

Nitrate/nitrite (NO32/NO22)

Hypochlorite (HOCl)

Nitryl chloride (NO2Cl)

Hydroperoxyl (radical) (HO2•)

Nitric oxide (radical) (NO•)

Ozone (O3)

Nitrous acid (HNO2) Nitrosyl cation/anion (NO1/NO2)

RONS can be categorized into two sets; radicals and nonradicals. Radicals are the species that contain one or more unpaired electron in the outer orbits round the atomic nucleus and are able of autonomous existence. The singular number of the electron(s) of free radicals makes it unstable, short-term, and highly reactive. Due to this extreme reactivity, they can strip electrons from other molecules in order to stabilize. Therefore the assaulted molecule loses its electron and becomes a free radical, causing a series of interactions that ultimately damage the living cell. The nonradicals are not free radicals, but they can easily trigger free radical interactions in living cells.

molecules. Similarly as ROS, RNS function a double role, where they can be deleterious or useful for living systems. Nitric oxide, now known as the organizer of serious physiological pathways, can intermediate cellular toxicity that damages metabolic enzymes and producing peroxynitrite by interacting with superoxide. While the role of ROS and RNS (together referred to as RONS) in cellular destruction and signal transduction is obvious, many dialectical queries are still being raised. At low concentrations, RONS perform a substantial role as regulatory intermediaries in signaling operations; however, at average or high concentrations, they damage organisms and can deactivate many cellular functions. This suggests that the levels of the reactive species set whether they are harmful or useful, but the exact levels in which this distinction occurs are generally unknown.12 It is obvious that oxidative stress acts as a significant role in the progression of most chronic diseases, influencing human health during the entire lifespan.11 The oxidant is a species that creates or reinforces oxidation, whereas the antioxidant molecule suppresses either the production of oxidants or blocks the oxidation itself. Oxidative stress results from the failure of innate antioxidant mechanisms to counteract oxidants produced internally or externally, resulting in an imbalance between oxidants and antioxidants.2 Oxidative stress can be produced from accretion in the production of oxidants (in the form of free radicals or RONS), low levels of antioxidants, or low-antioxidant enzyme activity.13 The exhaustion of dietary antioxidants (e.g., vitamins E, C, D, carotenoids, and flavonoids) and micronutrients (e.g., iron, copper, zinc, and selenium) can also participate in oxidative stress as they are essential for the favorable performance of antioxidant enzymes.14 In chronic inflammatory cases like COPD, oxidative stress is mostly caused by raising production of ROS as a result to vulnerability to toxins (e.g., cigarette smoke, infection) and the uninterrupted stimulation of endogenic enzymes (e.g., NADPH oxidases).15

The role of oxidative stress in chronic obstructive pulmonary disease pathogenesis The continuous raise in oxidative stress is a key factor in promoting airway and systemic inflammation in COPD, and has a major role in the emergence and development of COPD and associated diseases16 (Fig. 5.1). Oxidation caused by increased levels of RONS causes direct damage, which, in turn, leads to pulmonary and systemic inflammation.17 Increased oxidative stress markers (such as, superoxide, and malondialdehyde) in the air spaces, phlegm, breathe, lungs, and blood in patients with COPD, and may also

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51

FIGURE 5.1 The role of oxidative stress in the pathogenesis of COPD. The generation of exogenous and endogenous activated species, in addition to a significant depletion in the antioxidant defense system, can lead to the development of COPD and its comorbidities. COPD, Chronic obstructive pulmonary disease; NO synthase, nitric oxide synthase; RNS, reactive nitrogen species; ROS, reactive oxygen species.

contribute to the pathogenesis of many human diseases, reveal that there are higher levels of ROS in the airways.18 Results of studies exhibited that patients

with COPD manifested increased oxidative stress levels, escorted by a decrease in several endogenous enzymatic antioxidants18
22 (Figs. 5.2 and 5.3).

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

(A)

Lipid peroxide (mmol/L)

40 35

31.57

COPD vs smoker control: P-value< .001*** COPD vs nonsmoker controls: P-value < .001*** Smoker controls vs nonsmoker controls: P-value = .492 (NS)

30 25 20 14.3

15

13.38

10 5 0 COPD

(B)

Smoker controls

Nonsmoker controls

Superoxide dismutase (U/mL)

16 14

14.33

COPD vs smoker control: P-value < .001*** COPD vs nonsmoker controls: P-value < .001*** Smoker controls vs nonsmoker controls: P-value < .001***

12 10

8.19

8 5.33 6 4 2 0 COPD

Smoker controls

Nonsmoker controls

FIGURE 5.2 Levels of oxidant and antioxidant in COPD patients versus control group. COPD patients showed a significant increase in the oxidative stress marker (lipid peroxide) (Fig. 5.2A) and a significant decrease in endogenous enzymatic antioxidant levels (superoxide dismutase, reduced glutathione, and catalase) (Fig. 5.2B
D) versus healthy controls. COPD, Chronic obstructive pulmonary disease; NS, nonsignificant. Source: Modified from Al-Azzawi MA, Ghoneim AH, Elmadbouh I. Evaluation of vitamin D, vitamin D binding protein gene polymorphism with oxidant
antioxidant profiles in chronic obstructive pulmonary disease. J Med Biochem 2017;36:331
40 with permission.

Oxidative stress can be stimulated by exposure to exogenous sources of ROS like air pollution, alcohol, tobacco smoke, heavy metals, or endogenously liberated ROS from leukocytes and macrophages engaged in the inflammatory system.3 Releasing damaging mediators such as neutrophil elastase and matrix metalloproteinases makes them play a key role in inflammatory processes and have been involved in the development, and evolving, of all of the pulmonary characteristics of COPD. Furthermore, pulmonary

neutrophilic inflammation is a characteristic of cigarette smoking, but considerably for COPD patients, it is persistent, even for those who have quit smoking.23,24 Neutrophils and macrophages, which are considered as activated immune cells, release ROS as part of the inflammatory system.25 Even though the ideal level of ROS production is obligatory for normal oxidative metabolism, enormous ROS has a detrimental effect on cell continuity and can destroy other essential molecules such as lipids, proteins, DNA, mtDNA, and

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The role of oxidative stress in chronic obstructive pulmonary disease pathogenesis

(C) 30 25

53

Reduced glutathione (mg/dL) COPD vs smoker control: P-value < .001*** COPD vs nonsmoker controls: P-value < .001*** Smoker controls vs nonsmoker controls: P-value = .217 (NS)

24.17

22.32

20 15 8.08

10 5 0

COPD

(D)

Smoker controls

Nonsmoker controls

Catalase (mmol/min/mL) 25 20

COPD vs smoker control: P-value = .348 (NS) COPD vs nonsmoker control: P-value < .001*** Smoker control vs nonsmoker: P-value < .001***

18.95

15 10

7.13

6.11

5 0 COPD

Smoker controls

Nonsmoker controls

FIGURE 5.2 (Continued)

RNA. Disorders in the activity of mtDNA function lead to epithelial cell damage and death, which result in the progression of COPD.15,26 Anionic ROS hypochlorite (OCl2) or its conjugate acid, hypochlorous acid (HOCl), is synthesized during the respiratory burst, where neutrophil myeloperoxidase (MPO) catalyzes the oxidation of chloride ions via hydrogen peroxide. HOCl is extremely reactive, quickly interacts with different biomolecules and cannot reach distant intracellular targets.15

Nevertheless, numerous more stable chloramines, which can diffuse over larger distances, can be generated by the reaction of HOCl with amines. It has been detected that a scarce number of low-molecular weight amines (in particular, nicotine in cigarette smoke) can create a chloramine that can navigate through cellular membranes and mediate the damage of intracellular protein via HOCl.24 At the molecular level, ROS may stimulate lipid peroxidation and produce malondialdehyde, which can deactivate many

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

(A) 140 120

Superoxide anion (mmol/L) Control vs admission: P-value < .001 *** Control vs discharge: P-value < .001 *** Admission vs discharge: P-value = .562 (NS)

97.2

100.6

100 80

43.58 60 40 20 0 Control group

(B) 90 80

Admission

Discharge

Advanced oxidation protein products (mmol/L) Control vs admission: P-value < .001*** Control vs discharge: P-value < .001*** Admission vs discharge: P-value = .279 (NS)

59 55

70 60 50 40 30

22

20 10 0 Control group

Admission

Discharge

FIGURE 5.3 Oxidative stress and antioxidative defense status in COPD patients versus control group. In this study, the oxidative stress and antioxidative defense parameters have been measured in patients with severe COPD exacerbation. All parameters were assessed in patients at two time points: First, 1 day after admission and, second, after 7
10 days or when they were clinically stable enough to be discharged. The levels of oxidative stress parameters; superoxide anion (O2•2) and advanced oxidation protein products (AOPP) were significantly higher in COPD patients at both time points (admission and discharge) versus control group (Fig. 5.3A and B). At discharge, the patients had significantly higher levels of the oxidative stress parameters; malondialdehyde (MDA) and total oxidant status (TOS) compared to admission (Fig. 5.3C and D). While the levels of antioxidative defense parameters; superoxide dismutase (SOD) was significantly lower in patients at both time points (admission and discharge) versus the control group (Fig. 5.3E). No significant difference in paraoxonase PON1 activity (POase) and diazoxonase PON1 activity (DZOase) between patients and control group was noted (Fig. 5.3F and G). At discharge, total antioxidant status (TAS) was significantly lower in patients versus control group (Fig. 5.3H). Finally, prooxidative
antioxidative balance (PAB) levels were higher in patients than in the control group at both time points (Fig. 5.3I). Source: Modified from Stanojkovic I, Kotur-Stevuljevic J, Milenkovic B, Spasic S, Vujic T, Stefanovic A, et al. Pulmonary function, oxidative stress and inflammatory markers in severe COPD exacerbation. Respir Med 2011;105(Suppl. 1):S31
S37 with permission.

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The role of oxidative stress in chronic obstructive pulmonary disease pathogenesis

(C) 2 1.8

55

Malondialdehyde (mmol/L) Control vs admission: P-value = .0941 (NS) Control vs discharge: P-value< .01 ** Admission vs discharge: P-value< .001***

1.6

1.60

1.07

1.4

0.94

1.2 1 0.8 0.6 0.4 0.2 0

Control group

(D)

Admission

Discharge

Total oxidant status (mmol/L)

12 Control vs admission: P-value = .337 (NS) Control vs discharge: P-value < .01 ** Admission and discharge: P-value < .05 *

5.74

10

8

3.02

6 2.68 4

2

0 Control group

Admission

Discharge

FIGURE 5.3 (Continued)

cellular proteins by producing protein crosslinkages.27 This may induce pulmonary inflammation, reinforcing alveolar wall devastation and emphysema emergence. A further product of lipid peroxidation,

which has many cytotoxic effects, is 4-hydroxy-2,3nonenal (4-HNE)28 that may appear to cause the accumulation of cytoplasmic Ca21, stimulate the proinflammatory cytokines and NF-κB expression,

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56

5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

(E)

Superoxide dismutase (IU/L)

180

Control vs admission: P-value < .001 *** Control vs discharge: P-value < .001 *** Admission vs discharge: P-value = .894 (NS)

118 160 140 120 100 80 60 40

20

19

20 0 Control group

(F)

Admission

Discharge

Paraoxonase PON1 activity (IU/L) 900 800

Control vs admission: P-value = .0983 (NS) Control vs discharge: P-value = .07786 (NS) Admission vs discharge: P-value = .119 (NS)

447 417

700 600

330

500 400 300 200 100 0 Control group

Admission

Discharge

FIGURE 5.3 (Continued)

mitochondrial malfunction, and apoptosis. Oxidation of proteins could result in stimulation of NF-κB, p38 MAPK, induction of inflammatory genes, and suppression of the efficacy of endogenous antiproteases, which may participate in COPD pathogenesis.29,30

Environmental reactive species and chronic obstructive pulmonary disease Inhalant oxidants in the environmental air, involving cigarette smoke, ozone, nitrogen dioxide,

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Environmental reactive species and chronic obstructive pulmonary disease

57

Diazoxonase PON1 activity (IU/L)

(G) 18000

Control vs admission: P-value = .079 (NS) Control vs discharge: P-value = .072 (NS) Admission vs discharge: P-value = .206 (NS)

16000

11549

11134

14000 9618 12000 10000 8000 6000 4000 2000 0 Control group

Admission

Discharge

Total antioxidant status (mmol/L)

(H) 1.2

1

Control and admission: P-value = .0955 (NS) Control and discharge: P-value < .01 ** Admission and discharge: P-value = .1077 (NS)

0.815

0.74 0.685

0.8

0.6

0.4

0.2

0 Control group

Admission

Discharge

FIGURE 5.3 (Continued)

acid aerosols, and fuel combustion, are wellconfirmed reasons for oxidative stress. Cigarette smoke involves high contents of toxic free radicals ( . 1016 molecules/puff) comprising RONS31 and more than 4700 greatly reactive chemical

complexes, in particular, aldehydes and quinones.32 Numerous vital cellular processes comprising inflammation, proliferation, differentiation, survival, and metabolism have been influenced by oxidative stress, which results from exposure to free radicals

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

(I)

Prooxidative–antioxidative balance (arb. U)

200 180

Control vs admission: P-value < .001*** Control vs discharge: P-value < .001*** Admission vs discharge: P-value < .05*

144 126

160 140 120 100

72

80 60 40 20 0 Control group

Admission

Discharge

FIGURE 5.3 (Continued)

of cigarette smoke. Oxidative stress/ damage producing by vulnerability to these free radicals is probably a significant mechanism by which smoking induces many diseases such as cancer, circulatory diseases, and COPD (Fig. 5.4). The nature of ROS in cigarette smoke differs from short lifetime oxidant inorganic radicals, for example, the superoxide radical (O2•
) and the nitric oxide radical (NO•), to long lifetime organic radicals, for example, semiquinones that can undergo redox cycling within the epithelial lining fluid of smokers for some considerable period of time.33 It is wellindicated that lung and protein carbonyls formation responds to lipid peroxides/carbonyls caused by cigarette smoke, and this response in some way plays a role in causing COPD.34 Cigarette smoking also contributes to elevated levels of neutrophilic MPO, which is an oxidizing agent that develops HOCl and turns tyrosine to tyrosyl radical.35 An association between the content of neutrophilic MPO and the extent of the pulmonary function disorder has been noticed in COPD patients, and indicated by some studies.36 It has been established that cigarette smoke can induce the alveolar macrophages activation; this is actually what has been noted in the bronchoalveolar lavage fluid (BALF) from the lungs of COPD patients and smokers, but not in nonsmokers.2

Cellular reactive species and chronic obstructive pulmonary disease Cellular reactive oxygen species A portion of the inflammatory
immune response toward inhaled pathogens, smoke components, and/or inflammatory and epithelial cells within the lungs produce enzymatically, the cellular-derived ROS.37 Oxidants existing in cigarette smoke promote the generation of ROS via phagocytes conducive to the releasing of inflammatory intermediators.4 Within a cell, there are some origins for ROS generation, but the essential ROS producer is the NADPH oxidase (NOX)—which is an enzyme with different isoforms NOX1, NOX2, NOX3, NOX4, and NOX5—and two related enzymes, DUOX1 and DUOX2, have been reported, and most, if not all, isoforms were targeted to cellular membranes.12,38 In human individuals, NOX1 and NOX2 are important ROS producers, consisting of an enzyme compound in both phagocytic and nonphagocytic cells, including macrophages, neutrophils, epithelial cells, endothelial cells, and skeletal muscles.39 NOX can exist in numerous cell types, but mainly forms in neutrophils in accordance with usual conditions.40 Activation of neutrophils and macrophages makes them produce ROS through the NADPH oxidase mechanism, causing

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FIGURE 5.4 A diagram depicting a series of events underlying biological effects of cigarette smoking. Passive or active exposure to cigarette smoke results in the generation of oxidative stress followed by cell signaling, activation of protein kinase cascades and transcription factors, and releasing of inflammatory mediators. The end effect of this sequence of reactions is pulmonary and systemic inflammation and apoptosis. In case of extended response, it can lead to fibrotic and neoplastic damage. Oxidative stress induced by cigarette smoke starts a set of inflammatory reactions which comprise ciliotoxicity, enhanced mucous secretion, and minimize clearance, which results in mucous detention in the airways, proper preparation for bacterial colonization and infection, some of the ingredients of cigarette smoke are irritants, may cause cell damage or death as well as local inflammation. In addition, oxidants in cigarette smoke incite an influx of neutrophils and monocytes into the lungs. Due to the high amounts of circulating ROS produced by these cells, these oxidants may cause oxidative damage and modifications or destruction of cells and extracellular matrix compositions of the lungs. This will ultimately lead to airway and lung inflammation, which may develop into chronic bronchitis, pulmonary emphysema, and COPD. ROS, reactive oxygen species; RNS, reactive nitrogen species; GR, glucocorticoid receptor; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; CREB, cyclic AMP response element binding protein; COPD, chronic obstructive pulmonary disease.

an additional increase of oxidative stress in the lungs of COPD patients and smokers. It has been indicated for a long time that ROS produced via NOX have a significant part in the causing of a several of various chronic lung dysfunction that leads to obstructive physiology, especially, cystic fibrosis, asthma, and COPD.2 Also, neutrophils activation in COPD causes generation and excretion of enzymes (i.e., neutrophil elastase, proteinase 3, and cathepsin G) that are somewhat related to the stage of damage that occurs in extracellular matrixes in the airways of COPD patients.41 Different sorts of neutrophilic behaviors have been examined in COPD patients. Phagocytosis by neutrophils is a critical part of the immune system.42 However, independent studies have indicated that phagocytosis has been reduced in neutrophils that

originated from COPD.43 ROS are oxidative outburst molecules that have been boosted after identification and activation of immunity. ROS intermediate the oxidation of biological constituents of the host cell. Thus, it is predictable that the liberation of ROS is one of the main etiologies of tissue damage in the airways of COPD patients.44 By comparing smokers with regular lung function and nonsmokers, increased amounts of ROS, which is estimated by NADPH oxidase, were detected in neutrophils of COPD patients.45 Also, it has been detected that increased ROS generation of blood and sputum obtained from COPD patients were related to the COPD exacerbation.46 Fig. 5.5 shows the production of ROS in the mitochondrial respiratory chain by physiologic cellular metabolism or by the impact of extrinsic agents.

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

FIGURE 5.5 Production of ROS in the mitochondrial respiratory chain by physiologic cellular metabolism or by the impact of extrinsic agents. Generation of ROS is a physiological activity, and these species may have a useful or deleterious impact. ROS products are produced under regular physiological circumstances by cause of the partial reduction of molecular oxygen. ROS, that is, superoxide anion (O2•2), hydroxyl radical (•OH2), hydrogen peroxide (H2O2), and singlet oxygen (1O2) form by different processes as a product of the mitochondrial respiratory chain in some photochemical and enzymatic reaction, as a result of the exposure to UV light, ionizing radiation, xenobiotics, chemotherapeutic drugs, or heavy metal ions as well as other factors such as inflammation, ageing, reoxygenation injury, and respiratory acidosis. Due to the natural protective mechanism that comprises enzymatic antioxidants, normal cells constantly invalidate the effect of oxygen derivatives produced. Under special effects, the intensity of generated ROS, either during cellular metabolism or from environmental stimuli, may exceed the normal capacity of the cells to eliminate them. The turbulent balance produces a condition termed oxidative stress, which is responsible for damaging biomolecules. The incompetent reform mechanism may eventually provoke mutations and encourage of neoplastic transformation or cell death. O2, Molecular oxygen; Po2, partial oxygen tension; ROO•2, peroxyl radical.

Cellular reactive nitrogen species Even though elevated ROS generation is the main mechanism of oxidative stress in COPD and other chronic and acute lung diseases, strict clues indicate that RNS is also engaged in COPD. RNS involve nitric oxide (NO•) and its derivatives such as nitrogen dioxide and peroxynitrite. Equally with ROS, besides the endogenous production, RNS exists in cigarette smoke and other air pollutants in NO• form, and has many of the identical deleterious impacts as ROS.2 Endogenously, NO• is related to a lot of signaling pathways of functional and pathological processes in mammals; but at high levels, it causes significant damage to the surrounding tissue and can interact with superoxide anions (O2•2) producing peroxynitrite radical (ONOO2), a more reactive species and more deleterious.47 Nitric Oxide: NO performs a critical part in maintaining the activity of the airways and the vascular systems and is produced on three isoforms of NO synthases (NOS); neuronal (nNOS), inducible (iNOS), and

endothelial (eNOS). Specifically, iNOS is not essentially expressed, but is provoked by some stimulus involving endogenous mediators (chemokines and cytokines) and exogenous factors (viral infection, environmental pollutants, bacterial toxins, etc.).48 The levels of exhaled nitric oxide (eNO) has been examined in COPD patients, asthmatic patients, and healthy individuals. COPD patients had higher levels of eNO than controls, but less than asthmatic patients. Also, eNO levels in COPD patients was in reverse relation with lung function criteria, such as forced exhalation volume at 1 sec (FEV1) and the ability to diffuse carbon monoxide.49 Through some studies established to differentiate between alveolar and bronchial NO by evaluating the eNO at multiple expired flows, it was found that there is a relationship between COPD and elevated alveolar NO.50 It was observed in bronchial smooth muscle cells in COPD patients that there was an increased expression of iNOS and this increase is associated with the degree of restricted airflow.51 It was observed that eNO in the airway and alveolar chambers were not affected by high doses of ICS,

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TABLE 5.2 Environmental and cellular sources of reactive oxygen and nitrogen species (RONS). Environmental sources

Cellular sources

• UV light/ionizing radiation • Air and water pollutants

• ROS Production by Mitochondria: Respiratory chain oxidases

• Redox-cycling chemicals (insecticide, chemotherapeutic drugs, bleomycin, doxorubicin, etc.)

• ROS Production by Endoplasmic reticulum: Microsomal oxidation, flavoproteins, CYP enzymes

• Alcohol

• ROS Production by Peroxisomes: Oxidases, flavoproteins

• Ozone • Tobacco smoke

• ROS Production by Plasma membrane: Lipooxygenases, prostaglandin synthase, cyclooxygenase, NADPH oxidase

• Cooking (smoky meat, consumed oil, fat). • Infectious agents

• ROS Production by Cytoplasm: Xanthine oxidase, uncoupled NOS, thiols, hydroquinones, catecholamines, flavins

• Toxic metals (e.g., Hg, Cd, As, Pb, Cu, Fe, Cr, Co)

• ROS Production by Lysosomes: Myeloperoxidases (phagocytes)

• Industrial solvents • High temperature

• ROS Production by Metal-catalyzed reactions (e.g., Fe21, Cu21, Zn21, Al31)

• Engineered nanoparticles • CCl4

• RNS Production: NO∙ is produced from the metabolism of the amino acid, L-arginine, and catalyzed by nitric oxide synthases (NOS)

• Drugs (e.g., cyclosporine, tacrolimus, gentamycin, halothane, ethanol, paracetamol, metronidazole) The extracellular sources of RONS include factors that stimulate reactive species, such as environmental pollutants, radiation exposure, microbial infection, and prolonged exposition to engineered nanoparticles. The major origins of intracellular RONS are mitochondria, peroxisomes, endoplasmic reticulum (ER) stress, microsomes, inflammatory cells, cellular-metabolizing enzymes, and the NOX family of NADPH oxidases in cell membranes. Predominately, mitochondria carry out the major source of RONS production during the electron transport chain. NADPH oxidase is the predominant origin of the O2•2 that is constituted by the one-electron reduction of molecular oxygen with electrons provided by NADPH through internal respiration. The majority of the O2•2 is dismutated enzymatically by SOD or spontaneously into H2O2. H2O2 is not a free radical because it has no unpaired electrons, but it is able to form the highly reactive ROS hydroxyl ion (OH•) through the Fenton or Haber
Weiss reaction. Highly reactive hydroxyl radicals directly eliminate electrons from any molecule in its path; converting that molecule into a free radical, and react particularly with proteins and phospholipids in cell membranes. In neutrophils, in the existence of chloride and MPO, H2O2 can turn into hypochlorous acid and this in itself a ROS specifically destructive to cellular proteins. NO• is generated from L-arginine by major three isoforms of NOS: eNOS concerned with vasodilation and vascular regulation, nNOS controls intracellular signaling pathways, and iNOS is stimulated by responding to cytokine signals or various endotoxins. Lastly, ONOO2 which is a highly strong oxidant, formed by the reaction of O2•2 with NO•. Al, Aluminum; As, arsenic; Br, bromine; CCl4, carbon tetrachloride; Cd, cadmium; Co, cobalt; Cr, chromium; Cu, copper; CYP enzymes, cytochrome P450 enzymes; eNOS, epithelial NOS; Fe, iron; Hg, mercury; H2O2, hydrogen peroxide; iNOS, inducible NOS; MPO, myeloperoxidase; NADPH oxidases, nicotinamide adenine dinucleotide phosphate oxidases; nNOS, neuronal NOS; NO•, nitric oxide radical; NOS, nitric oxide synthase; NOX, NADPH oxidases; O2•2, radical superoxide anion; ONOO2, peroxynitrite; Pb, lead; SOD, superoxide dismutase.

demonstrating that iNOS in COPD is not a major exporter of NO. However, a different study found that the expression and the impact of nNOS increased in COPD accordingly to the severity of the disease, indicating that the increased NO in these patients may be originated from nNOS.47 Peroxynitrite: ONOO2 anion, a highly strong oxidant, but in contrast to NO and O2•, is not a free radical, and is formed by the rapid reaction between of the two free radicals (O2•2, NO•). ONOO2 is a potent oxidant and nitrating agent. NO, ONOO2 can spread over a long range of cells and even cross over cell membranes due to its relative constancy. This molecule can destroy cells’ biological molecules, which are proteins, lipids, and DNA. Comorbidities of COPD have a certain relation to the ONOO2 formation because of its formation (i.e., ONOO2), includes the consuming of NO, decreasing the biological availability of NO for physiological functions.52 Peroxynitrite levels also increase in the breath and sputum macrophages of

COPD patients. The lungs of COPD patients had elevated extents of nitric oxide and this appears to be linked to increased expression of inducible and neural nitric oxide synthases (NOS2 and NOS1).16 During the simultaneous presence of iNOS and arginase expressing macrophages in patients with COPD, it was observed that a higher level of nitrotyrosine (a derivative of peroxynitrite) exists in the sputum macrophages of COPD patients, which, in turn, is accompanied by a considerable decline of lung function for these patients.53 By studying the levels of peroxynitrite in COPD patients and healthy controls (smokers/nonsmokers), peroxynitrite levels were significantly more elevated in patients than in healthy controls.54 ONOO2 can result in nitration (addition of NO2) in majority categories of biological molecules. ONOO2 could thus induce suppression of mitochondrial respiration, protein malfunction, and destruction to cell membranes and DNA.55 Another study proposed that ONOO2 caused nitration in primary

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5. Pathological association between oxidative stress and chronic obstructive pulmonary disease

isolated alveolar epithelial cells, pulmonary vascular endothelial cells, and pulmonary arterial smooth muscle cells from resistance vessels.16 Thus supplementary researches are required to emphasize and scout the molecular mechanisms of ONOO2. Table 5.2 shows the main sources of environmental and cellular RONS.

Applications to other areas of pathology Biogenic substances are exposed to oxidative destruction by increased generation of free radicals, causing many incurable diseases like cancer, atherosclerosis, cardiovascular disease, diabetes, chronic inflammations, stroke, septic shock, and other neurodegenerative diseases in humans.56 Compatible and decisive evidence has indicated that oxidative stress is engaged in the pathogenesis of Parkinson’s disease. The brain needs increasing concentrations of the metal ion to do its tasks. Even so, the brain has a weak ability to deal with oxidative stress and show low-regenerative capability, making it more prone to oxidative damage. The brain undergoes a high rate of oxidative activity that generates a large number of free radicals; the brain nerve tissue also contains a comparatively low level of antioxidants. In addition, catecholamines that are present in almost all brain areas are especially susceptible to free radicals production. Oxidative stress, which mediates the neurotoxicity, induced by the unusual cumulation of tau and amyloid beta (Aβ) proteins in certain brain areas involved in memory, may enhance Aβ production and aggregation as well as support tau hyperphosphorylation and polymerization, further augmenting a variety of neurotoxic effects involving ROS generation, thus, forming a risk pathway that would encourage the onset and development of Alzheimer’s disease.57 Recent studies have shown that hyperglycemia is linked with growing cellular and systemic oxidative stress. Antioxidant therapy indicated the possibility of repressing or decelerating of diabetic neuropathy in animal models, confirming the significant role of ROS in the pathogenesis of diabetic neuropathy.58

Summary points • Incessant oxidative stress, that is, free radicals
antioxidant imbalance is the hallmark of the lungs of COPD patients. • The continuous rise in oxidative stress is a key factor in promoting airway and systemic inflammation in COPD, and plays a major role in the emergence and development of COPD and associated diseases. Oxidation caused by increased levels of RONS causes direct damage, which, in

turn, leads to pulmonary and systemic inflammation. • Oxidative stress is engaged in many changes and disorders that will cause disruption in physiological lung functions and in the pathogenesis of COPD. • These changes and disorders include suppression of endogenous antiproteases, excessive mucus secretion, membrane lipid peroxidation, alveolar epithelial damage, and reconstructive of the extracellular matrix. They stimulate the proinflammatory cytokines and NF-κB expression, mitochondrial malfunction, and apoptosis. • Definitive and harmonious evidence suggest that oxidative stress is implicated in the pathogenesis of many intractable diseases such as cancer, atherosclerosis, cardiovascular disease, diabetes, chronic inflammation, stroke, septic shock, and neurodegenerative diseases in humans.

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C H A P T E R

6 Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies ¨ zta¸s1 and ˙Iffet ˙Ipek Bo¸sgelmez2 Ye¸sim O 1

Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey 2 Department of Toxicology, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey

List of abbreviations ACS CAT EMA FDA GPx GSH Hb HbF HbS LC-MS/MS NADPH NO Prx-II RBC ROS SCA SCD SOD VOC

One of the earliest records of SCD has been traced back to 1670. Konotey-Ahulu, an expert in this field, could define his relatives with SCD from his familytree spanning three centuries.1 However, the available history of the disease, as illustrated briefly in Fig. 6.2, dates back to 1846 with the presentation of an “asplenic patient.” The first report on SCD in medical literature was published in 1910 by Herrick who named the disease after evaluating clinical records describing the “peculiar, elongated, sickle-shaped” erythrocytes (red blood cells; RBCs) in a peripheral smear of a WestIndian student suffering from severe anemia.2 In 1915, the possibility of inheritance was recognized after the third case of SCD, and with observation of abnormal RBCs in one of the parent’s peripheral blood smear.3 In the following years, several reports pointed out the link between abnormality of hemoglobin (Hb) and reversible sickling after reoxygenation,4 irreversible sickling in some RBCs after reoxygenation,5 and structural Hb-change induced by deoxygenation.6 The report of abnormal electrophoretic mobility of Hb from sickle erythrocytes led Pauling to describe SCD as the “first molecular disease” in 1949.7 Then, the role of sickle Hb (HbS) polymers in SCD pathology was defined by Harris,8 as well as the insoluble nature of deoxygenated HbS defined by Perutz and Mitchison.9 Another pivotal work was in 1957 by Ingram and Hunt who revealed that insolubility and change of electrophoretic mobility of HbS were due to molecular alteration of beta-globin chain at the 6th position (i.e., glutamate was exchanged with valine).10 In 1984, allogenic stem cell transplantation was established as the

acute chest syndrome catalase European Medicines Agency United States Food and Drug Administration glutathione peroxidase reduced glutathione hemoglobin fetal hemoglobin sickle hemoglobin liquid chromatography tandem mass spectrometry nicotinamide adenine dinucleotide phosphate nitric oxide peroxiredoxin 2 red blood cell, erythrocyte reactive oxygen species sickle cell anemia sickle cell disease superoxide dismutase vaso-occlusive crisis

Introduction Sickle cell disease (SCD) is among the most prevalent hemoglobinopathies with prominent morbidity and mortality, affecting millions of people worldwide. For many years, different tribes in Africa have described this inherited disease under local names such as “chwechweechwe” and “nwiiwii” that correspond to “recurrent pain", the most burdening symptom of SCD. The increasing number of publications related to “sickle cell disease” and “oxidative stress” reflects its global importance (Fig. 6.1). Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00006-8

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6. Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies

FIGURE 6.1 The number of publications in PubMed per year related to search terms “sickle cell disease” or the combination of “sickle cell disease” and “oxidative stress.” As of April 27, 2019, the combination “sickle cell disease” and “oxidative stress” hit 17 publications in 2019.

FIGURE 6.2 The available history of sickle cell disease (SCD).

first successful curative option for SCD.11 In 1995, findings of a multicenter, randomized double-blind placebocontrolled trial showed that hydroxyurea could reduce painful crises in adult patients.12 The United States Food and Drug Administration (FDA) approved hydroxyurea in 1998 and for two decades hydroxyurea remained the sole drug for treatment of SCD in adults. European Medicines Agency (EMA) approval was not until 2007. The damage on sickle RBCs depends on several factors including oxidative stress. In recent years, promising studies on antioxidant agents such as L-glutamine

in patients have been published. In 2017, in view of available data, FDA approved L-glutamine for the prevention of acute vaso-occlusive crises (VOC).13

Epidemiology and global burden of sickle cell disease The global incidence of SCD has been estimated to be more than 300,000 infants born each year and projections indicate that the annual number may exceed

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Pathophysiology of sickle cell disease

400,000 by 2050.14 Inheritance of HbS genes is particularly common among people who live in, or whose ancestors come from, regions with malaria endemicity such as sub-Saharan Africa, the western coast of the Arabian peninsula, India, and some Mediterranean countries including Greece, Italy, and Turkey.15 The “malaria hypothesis” proposed by Haldane almost seven decades ago suggests that the high frequency of inherited Hb disorders in the malaria-endemic regions16 is not coincidental, indeed sickle-trait may serve as a protective mechanism against malaria.17 Distribution of HbS allele has spread far beyond its origins owing to substantial population movements that made it prevalent in Europe18 and some regions of South and Central America,19 resulting in SCD becoming a worldwide health issue. The World Health Organization global health estimates in 2018 highlight the critical point of some countries including India, the Democratic Republic of Congo, Nigeria, and Bangladesh.20 Population estimates in the United States have revealed a total of around 100,000 patients, and it has been suggested that SCD affects nearly 1 out of every 365 AfricanAmerican newborns and 1 in 16,300 HispanicAmerican newborns.21 A recent metaanalysis stated that the global metaestimate for birth prevalence of homozygous SCD is 111.91 per 100,000 live births; however, due to the regional factors, birth prevalence was 1125.49 in Africa and 43.12 per 100,000 in Europe.22 Since SCD presents challenges to healthcare systems, effective use of newborn screening programs may provide a viable tool for timely preventive measures. Moreover, it may contribute to a significant reduction in childhood mortality.18

Clinical manifestations of sickle cell disease and treatment options SCD presents with acute and chronic complications. Common acute complications include acute pain, acute chest syndrome (ACS), and stroke. Chronic complications such as vaso-occlusion can lead to damage in all organs. Patients typically suffer from anemia, chronic inflammation, as well as VOC. Clinical features arising from the presence of sickle cells affect almost every system of the body (Table 6.1). Management guidelines present valuable approaches23; however, it is obvious from the clinical experiences that patients with SCD may admit with distinct clinical presentations, and respond variably to therapeutic or preventive efforts. Since polymerization of HbS triggers pathological conditions leading to diverse clinical complications, curative treatment options, including hematopoietic stem cell transplantation and gene

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therapy, target the elimination of HbS production. On the other hand, the aim of disease-modifying therapies is either to increase fetal Hb (HbF) levels via hydroxyurea or to elevate HbA levels through transfusion. In addition, the FDA approved L-glutamine for prevention of acute VOC in 2017. A number of preventive and supportive options such as immunization, antibiotic prophylaxis, and NSAID use have increased the quality of life; however, the impact on overall life expectancy has been limited.13 Therefore there is still an unmet demand for new therapies. In the United States National Library of Medicine clinical trials website, our query with the term “sickle cell” retrieved 676 studies, 485 of which were interventional (i.e., clinical studies). Among these trials, 210 studies were completed, 121 are recruiting, 58 were terminated, 41 not yet recruiting/active-not recruiting, 22 withdrawn, and the rest were classified under other status (accessed 23.04.2019) (https://clinicaltrials.gov/).

Pathophysiology of sickle cell disease The life span of sickle RBCs is shortened to almost 10 days and pragmatic changes appear regarding cell functions as well as its shape. Sickle cells lose their “deformability”, the essential feature of RBCs, which enables them to travel through capillaries. Contrary to the complex outcome, the reason for these effects is unexpectedly easy to understand. Only 1 point mutation in which the hydrophilic amino acid “glutamate” is replaced by hydrophobic “valine.” The mutant Hb will lose its solubility under hypoxia, acidosis, and dehydration. The resultant Hb polymerization will form long fibers inside RBC, distorting its shape into a sickle cell. The major molecular, cellular, and biophysical processes in SCD pathophysiology may be briefly outlined as: polymerization of HbS, the vaso-occlusion (via impaired biorheology, increased adhesion of sickle RBC), hemolysis-mediated endothelial dysfunction, and inflammation.24 While chronic hemolysis is the main cause of anemia, hypersplenism also contributes to excessive removal of sickle RBCs in most patients. In addition, iron deficiency may aggravate the condition. As a result, to compensate for anemia, stress erythropoiesis becomes a common feature in SCD releasing young cells into circulation.25 Obviously, the RBC membrane is prone to oxidative damage induced by heme and free-iron released from HbS. Lipid peroxidation and increased production of oxysterols (oxidized cholesterol species) adversely affect membrane packaging. Recent studies report alterations of membrane phospholipid metabolism26 as well as increased acid sphingomyelinase activity

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6. Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies

TABLE 6.1 A summary of complications of sickle cell disease, health maintenance, and management of complications. Acute complications Prevention and/or treatment options

Chronic complications Clinical presentation

Prevention and/or treatment options

Target

Clinical presentation

Hematopoietic

Aplastic crisis, splenic sequestration crisis, hyperhemolytic crisis

Pulmonary

ACS, pulmonary thromboembolism, asthma, pneumonia

For ACS: hydroxyurea, judicious use of IV fluids; cephalosporin (IV), macrolide antibiotic (oral), supplemental oxygen

Pulmonary hypertension, sleep Supplemental oxygen, disordered breathing, chronic diuretics, pulmonary restrictive lung disease vasodilators

Pain

Acute VOC

Hydroxyurea, hydration, nonsteroidal antiinflamatory drugs (NSAIDs), opioids

Chronic pain (tissue infarction), osteonecrosis, ulcers

Infections

Sepsis, meningitis

Penicillin prophylaxis, pneumococcal vaccination

Cardiovascular

Myocardial infarction, dysrhythmia, sudden cardiac death, autonomic dysfunction, venous thromboembolism

Nervous system

Ischemic/hemorrhagic stroke, seizures, transient ischemic attack

Renal

Acute renal failure, Renal function monitoring, nephrotic syndrome, renal avoiding nephrotoxic agents, infarction, medication hemodialysis toxicity, hematuria

Compensated hemolytic anemia, asplenia, chronic hypersplenism

NSAIDs, opioids

Diastolic dysfunction, heart failure

For stroke: transcranial Doppler (TCD); chronic transfusion; hydroxyurea for those with normal MRI and TCD after 1 year of transfusions.

Musculoskeletal Dactylitis, avascular necrosis

Silent cerebral infarcts, cognitive/behavioral issues

Hypertension (due to vasoocclusions at microvasculature), chronic renal failure, concentrating defect, renal medullary carcinoma

Regular assessment of organ function, ACEinhibitor or ARB for proteinuria, transfusion or EPO for anemia, dialysis

Osteoporosis, osteomyelitis, ulcers (esp. leg ulcers), osteonecrosis

Local or systemic antibiotics for leg ulcers

Hepatobiliary

Hepatic sequestration crisis, cholecystitis, liver injury, acute intrahepatic cholestasis

Hydration, surgical interventions Gallstones

Ocular

Retinal artery occlusion, retinal detachment, posthyphemia glaucoma

Annual dilated fundal examinations

Retinopathy, blindness, vitreous hemorrhage

Dilated fundal examinations, laser therapy

Genitourinary

Priapism

Hydration, exercise, and hydroxyurea; analgesia, sympathomimetic injection, shunt/ aspiration

Erectile dysfunction, sperm abnormalities, hypogonadism, recurrent priapism, impotence

Urology consultation, analgesia, hormone therapy

Endocrine Obstetric

Delayed puberty, growth retardation Fetal and maternal complications

Compilation from: National Heart, Lung, and Blood Institute. Evidence-based management of sickle cell disease: expert panel report. 2014. http://www.nhlbi. nih.gov/health-pro/guidelines/sickle-cell-disease-guidelines/.23 UpToDate content on sickle cell disease for additional information on screening, diagnosis, and management. 2019. https://www.uptodate.com/contents/image? imageKey 5 HEME%2F108318&topicKey 5 HEME%2F7119&source 5 outline_link.

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Oxidative stress in sickle cell disease pathogenesis

in sickle RBCs, contributing to inflammatory microparticle generation.27 Although the vaso-occlusion in SCD had long been linked to Hb polymerization, sickling, and adhesion of RBCs, the critical role of endothelial injury and concurrent sterile vascular inflammation in VOC has only recently been understood.28 Leukocyte gene-expression profiling of steadystate (without crisis) SCD patients demonstrated an intense inflammatory response,29 moreover a relative increase of systemic inflammatory markers (e.g., soluble adhesion molecules, endothelin-1, and cytokines) both in crisis and steady-state patients compared to healthy controls.30 Last, but not least, hemolysis-induced release of Hb into circulation ends up with consumption of local nitric oxide (NO) in the vasculature leading to vasoconstriction that contributes to vaso-occlusions.31

Oxidative stress in sickle cell disease pathogenesis The physiology of red blood cells in coping with oxidative stress Erythrocytes, especially in view of extreme oxidative stress during their life span, are designed to cope with challenging conditions; thus they have to minimize the effects of reactive species arising from either endogenous or exogenous sources. These cells, acting like mobile-detoxifying systems, are equipped with effective protective systems containing nonenzymatic and enzymatic antioxidants. Several low-molecularweight components including reduced glutathione (GSH) and ascorbic acid are readily available. The enzymatic group comprises mainly superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin 2(Prx-II). Regarding RBC’s redox-enzymes, Prx-II, CAT, and SOD have been reported to account for 43% of the non
Hb proteins in cytosol.32

The pathology of sickle red blood cells due to increased oxidative stress Nonenzymatic production of reactive oxygen species (ROS) in sickle RBCs by HbS auto-oxidation and iron-mediated Fenton chemistry reactions are wellknown sources of ROS production in sickle RBCs compared to normal RBCs (Fig. 6.3). In addition, a lower content of GSH, which is a pivotal endogenous antioxidant and a key component of the enzymatic antioxidant system, is evident in SCD patients.33 This significant ROS production and lower GSH content may not be limited to RBCs, and possibly the case

69

might be similar in platelets and neutrophils of these patients.34 Recently, an additional ROS generating source within RBCs has been proposed.35 This enzymatic mechanism of ROS production mediated by NADPH-oxidase that appears to be regulated via both intracellular (protein kinase C and RacGTPases, and calcium signaling) and extracellular factors (cytokines, such as transforming growth factor b1 and endothelin1). Since young cells produce more ROS than older ones due to higher enzymatic activity, the cumulative oxidative load in these patients may become more pronounced. Another striking report was regarding mitochondria-retaining RBCs, which may be associated with excessive ROS levels in SCD.36 An intriguing point is the regulation conferred by nuclear factor erythroid 2-related factor 2 (Nrf2). Normally Nrf2 is under control of repressor proteins such as Kelch-like ECH-associated protein 1 (Keap1). When oxidative stress is induced, Nrf2 is released from cytoplasm to the nucleus for coordinated upregulation of antioxidant defense. In the case of SCD, a recent experimental-model study on Nrf2 activation via knock-down of Keap1 showed alleviation of ROSmediated organ injury and inflammation. Moreover, Nrf2 activation via a synthetic compound resulted in a similar effect by decreasing organ injury.37 Nrf2 has also been proposed as a modulator of HbF expression in erythroid progenitors.38

Clinical consequences of oxidative stress in sickle cell disease The role of increased oxidative stress in the pathogenesis of SCD has been pointed out by diverse studies (Table 6.2). ROS have been suggested to promote the vicious circle of accelerated hemolysis, endothelial dysfunction, reduced NO bioavailability, inflammation, and hypercoagulability in SCD.48 During vasoocclusions, ischemia/reperfusion injury also produces free radicals that induce oxidative damage accompanied by a decrease in antioxidant capacity, for example, decreased GSH levels in the RBC and in the plasma.49 Since GSH is essential for maintenance of redox in RBCs and vascular integrity, any dysregulation can become detrimental. The proteomic analysis of sickle RBCs has revealed that HSP27, HSP70, and Prx-II were differentially expressed in sickle cells compared to normal RBCs. Presence of iron has been linked with a marked increase in Prx-II membrane binding in sickle RBC fractions than controls. It has been proposed that the monomers and dimers of Prx-II have been increased in sickle cells compared to normal RBCs, possibly as a mechanism against membrane damage. In addition, a confirmation test in transgenic

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6. Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies

FIGURE 6.3 Sources of oxidative stress in SCD and possible targets of available antioxidants. NAD, Nicotinamide adenine dinucleotide; GSH, reduced glutathione.

sickle mouse models demonstrated a possible novel role of Prx-II as a molecular chaperone.44 In SCD, activation of NADPH-oxidases has been suggested to produce ROS, as well as enable downstream proinflammatory signaling and subsequent endothelial activation. Thus proinflammatory cytokine production, endothelial activation, leukocyte adhesion, and vaso-occlusion become inevitable. As a result, if an intervention in the cascade can be utilized; via Tolllike receptor blockade, NADPH-oxidase inhibition may be a potential therapeutic target.50 Plasma from SCD patients contains cell-free ferrous Hb that leads to consumption of significant amounts of NO. The diminished effect of NO on homeostatic vascular function may result in deleterious vascular complications.31 Moreover, hemolysis may also contribute to the release of erythrocyte arginase, which limits arginine-bioavailability. Since L-arginine is the substrate for NO synthesis, and typically higher than normal arginase activity in immature-RBCs, reticulocytes and plasma, may result in limited NOavailability.51 Thus these events impair NO-dependent beneficial effects on vascular function.

Iron-overload either via chronic transfusion or hemolysis in SCD results in the generation of free radicals that damage macromolecules including proteins, lipids, and DNA.52 Previous studies stated that plasma lipids and proteins are potential targets of oxidative damage in SCD, since lipid peroxidation53 and protein oxidation54 in the plasma of patients were increased. Oztas et al., reported increased protein carbonyl and decreased total sulfhydryl contents in steady-state sickle cell anemia (SCA)-patients compared to the controls. This study also showed that these patients had higher plasma iron and hemolysate copper than carriers and controls. Moreover, plasma protein oxidation was positively-correlated with plasma iron, and negatively-correlated with hemolysate zinc levels.54 Plasma proteins exposed to increased oxidative stress in SCD demonstrated various posttranslational modifications. Albumin, the major plasma protein, was shown to have malondialdehyde-adducts in SCD patients with pulmonary hypertension.42 An altered electrophoretic mobility of sickle-plasma albumin has been shown.55 SCD patients were also reported to have methemalbumin in their plasma.56 Not only

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71

Oxidative stress in sickle cell disease pathogenesis

TABLE 6.2 A summary of representative studies on the role of oxidative stress on sickle cell disease. Study design/endpoints

Results

Highlights of the study

References

Contents of ROS, protein carbonyl, lipid peroxidation (TBARS), total-thiols (T-SH), GSH, CAT, myeloperoxidase (MPO), xanthine oxidase (XO), nucleotide metabolites in patients (n 5 15) and controls (n 5 30)

Levels of ROS, TBARS, T-SHm activities of CAT and MPOm, GSH levelk, hypoxanthinem xanthinek, uric acidk, XO activityk

Impairment of cellular redox homeostasis and alteration of purine metabolite via oxidative stress in SCA

Castilhos et al.39

Assessment of clinical and hematological features of β-globin gene haplotypes with plasma lipid peroxidation (LPO), plasma nitrite and nitrate (NOx), RBC contents of GSH, GSSG, and activities of GPx, glutathione reductase (GRd), and SOD in pediatric SCD patients (n 5 95) and healthy children (n 5 40)

Plasma LPO and NOxm, activities of GRd and SODk GSH / GSSG ratiok, GPx activitym

High HbF related with low LPO, while with high GRd activity and NOx levels

Rusanova et al.40

Oxidative stress biomarkers, NO metabolites, endothelin-1, and hematological parameters were evaluated in children/adolescents with SS(n 5 20) and SC(n 5 23) vs controls (n 5 12)

Oxidative stress and antioxidant enzyme activitiesm in SS and SC children compared to AA group Correlation between microvascular function and oxidative/nitrosative stress in SS children

Severity of oxidative stress: SS . SC . AA.

Analysis of purified albumin by matrix-assisted laser-desorption/ ionization-time-of-flight mass spectrometry LC-MS/MS, dot-blotting and Western blotting

A differential malondialdehyde (MDA)modified albumin peptide in SCApatients with pulmonary hypertension

Possible link between pathogenesis of pulmonary hypertension of SCA with oxidative posttranslational modification of serum albumin

Odhiambo et al.42

Blood samples from healthy donors (AA) and homozygous (SS) SCD patients; 2D-gel electrophoresis (2D-DIGE), LC-MS/MS

Peroxiredoxin 3 isoform bm peroxiredoxin 1m CATm protein repairm

Adaptive response of sickle cells to oxidative conditions via proteins that scavenge oxygen radicals, refold damaged proteins, or turnover of oxidatively damaged proteins

Kakhniashvili et al.43

2D-DIGE and MS/MS, Western blotting

Differentially expressed 65 proteins; major clusters (membrane-cytoskeleton proteins, metabolic enzymes, ubiquitinproteasome system, flotillins, chaperones)

Proposed sensitivities: Biondani HSP27-to hypoxia, et al.44 HSP70-to iron; Prx-II membrane-binding more sensitive to iron

In vitro protein profiling of 13 sickle RBC membrane samples exposed to 50 μM hydroxyurea versus control. Methods: 2D-gel electrophoresis, LCMS/MS, immunoblotting

In SS RBC membranes exposed to hydroxyurea, tyrosine phosphorylation of CATm twofold

Adaptive response to oxidative damage; CATm, thioredoxin peroxidase-1m, flavin reductasem

Ghatpande et al.45

Comparison of levels of polyubiquitination, function of proteasomes and effect of hydroxyurea therapy in RBCs

In RBC samples from untreated SCD patients, ROS and poly-ubiquitinated proteinsm versus hydroxyurea-treated SCD patients and healthy controls

A possible mechanism of action for hydroxyurea may be degradation of oxidatively damaged proteins via ubiquitin-proteasome system

Warang et al.46

Plasma 8-isoprostane, protein carbonyl and nitrotyrosine levelsm; a significant positive correlation of freeHb with protein carbonyl and nitrotyrosine levels

Released Hb may generate oxidative stress and decrease NO bioavailability, leading to vasoconstriction, thrombosis, and inflammation

Kupesiz et al.47

In patients with high HbF levels; LPO levelsk, NOx levelsm, GRd activitym

An association between high LPO levels, low SOD plus GRd activities with increased severity of clinical outcomes

Mo¨ckesch et al.41

Hemolysis and nitrotyrosine levelm in SS compared to SC (and AA for nitrotyrosine)

Methods: flow cytometry, Western blot, proteasomal activity assays Evaluation of role of hemolysis on markers of plasma protein and lipid oxidation in 25 SCD patients

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6. Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies

plasma proteins, but also lipids and lipoproteins as part of cellular defense acting in scavenging free radicals, are targets of oxidative stress.

Agents targeting antioxidant defense in sickle cell disease patients The common disease-modifying strategies for SCD treatment are focused on either supplying nonsickle RBCs (blood transfusion) or increasing HbF-content (hydroxyurea); however, promising data on mitigation of oxidative stress by various antioxidant agents, both in experimental and clinical trials, have been documented or under evaluation (Table 6.3). Amelioration of oxidative damage by antioxidants, such as N-acetyl34 L-cysteine, vitamin C, and vitamin E has been shown. Interestingly, besides induction of HbF production, hydroxyurea has also been suggested to act on modulation of NO-signaling pathway, RBC rheology, and oxidative stress.62 Moreover, potentially beneficial combined use of antioxidants with hydroxyurea has also been proposed.63 Possibly the most oxidative stress targeted agent available is L-glutamine. The rationale behind L-glutamine studies arises from the report underlining the possible role of three amino acids including glutamine in inhibition of sickling of RBCs. Data regarding the decrease in nicotinamide

adenine dinucleotide (NAD) redox potential in sickle RBCs, as well as the significant increase in rate of glutamine transport (a precursor for NAD) have led to clinical studies in SCD patients. Oral supplementation of L-glutamine has not only increased NADH content, but also elevated the NAD-redox potential.58 In addition, glutamate, which is a by-product of glutamine in NAD synthesis, may contribute to synthesis of GSH (γ-glutamyl-cysteinyl-glycine).

Future prospects HbS is a major source of oxidative stress in sickle RBCs that has also depleted its antioxidant defense. Chronic hemolysis of sickle RBCs is another source of oxidative stress induced by free-heme released into plasma. Continuous heme degradation depletes endothelial NO, and vasculature becomes prone to vasoconstriction that makes the clogging easier by sickle RBCs. Chronic vaso-occlusions trigger a chronic sterile inflammation of the vasculature that contribute to ROS production in circulation. Therefore biomolecules are under tremendous oxidative stress in RBC and plasma milieu of SCD patients. The oxidative modifications in biomolecules should be demonstrated by novel techniques such as mass spectrometry and omic technologies. Understanding SCD-induced oxidative modifications

TABLE 6.3 Some agents targeting antioxidant defense in sickle cell disease. Agents/strategies

Comments

Hydroxyurea

• Inhibition of iron-induced LPO and MetHb formation in an RBC-model • Blockage of t-butyl hydroperoxide mediated LPO, and changes in membrane • Dose-dependent inhibition of iron-mediated hydroxyl radical generation

58 L-glutamine

• NAD-redox potentialm • NADH levelm possibly via glutathione recycling

N-acetyl-L-cysteine (NAC)59,60

Daily 1200 or 2400 mg NAC (6 weeks)

57

• • • •

whole blood GSHm phosphatidylserine-exposure of RBC outer membranek plasma advanced glycation end-productsk cell-free Hbk

600, 1200, or 2400 mg of NAC (per oral, divided 3 times a day) • In 2400 mg dose group: GSH content in RBCm, • Dense cellsk, • VOCk α-Lipoic acid (200 mg)

α-lipoic acid61

• CATm in HbAS (SCD trait group), • MDA and carbonyl levelsk in HbAA (controls), • No significant effect in HbSS (SCD patients) LSD1-inhibition plus 36

mTOR-inhibition

Improvement in sickle RBC survival by • LSD1 inhibitor (RN-1) and mTOR inhibitor (sirolimus) • Via preventing ROS production

I. Oxidative Stress and Pathology

References

in plasma and RBCs may also help us explore novel targeted-therapeutic strategies.

Applications to other areas of pathology This chapter describes the role of oxidative stress in the pathogenesis of SCD. Distribution of this hemoglobinopathy has spread far beyond its origins as a result of significant population movements. Despite the current depth of knowledge on the molecular basis of SCD and current strategies primarily focusing on prevention of HbS formation, demand for novel and more effective therapies have remained to be fulfilled. The instability of RBCs and some other components triggers oxidative stress owing to various mechanisms. HbS is prone to hemolysis and has a short life span. The resulting oxidative events, as also reported in other Hb-related diseases, may benefit from antioxidant strategies. Among antioxidant-agent based attempts, a successive example is L-glutamine, which has been recently approved as a drug for SCD treatment. Thus evaluation of oxidative stress-related events and proposed antioxidant protection may be of use in other hemolytic disorders and hemoglobinopathies.

Summary points • This chapter focuses on the role of oxidative stress in SCD. • SCD is a life-threatening hereditary hemoglobinopathy that affects millions of people worldwide. • Under various conditions such as deoxygenation, dehydration or acidosis, Hb that includes mutant sickle β-globin subunit (HbS) polymerizes to form a sickle shape. • Patients generally suffer from anemia, chronic inflammation, and vaso-occlusive crisis. • Damage on RBCs arises due to various factors, including oxidative stress induced by cell-free iron released from heme. • In recent years, antioxidant agents such as L-glutamine per se or in combination with hydroxyurea have become promising treatment approaches, by increasing the antioxidant defense in SCD patients.

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50. Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC, Sachdev V, et al. Dysregulated arginine metabolism, hemolysisassociated pulmonary hypertension, and mortality in sickle cell disease. JAMA 2005;294(1):81
90. Coates TD. Physiology and pathophysiology of iron in hemoglobin-associated diseases. Free Radic Biol Med 2014;72: 23
40. Oztas Y, Sabuncuoglu S, Unal S, Ozgunes H, Ozgunes N. Hypocholesterolemia is associated negatively with hemolysate lipid peroxidation in sickle cell anemia patients. Clin Exp Med 2011;11(3):195
8. Oztas Y, Durukan I, Unal S, Ozgunes N. Plasma protein oxidation is correlated positively with plasma iron levels and negatively with hemolysate zinc levels in sickle-cell anemia patients. Int J Lab Hematol 2012;34(2):129
35. Ozgunes N, Oztas Y, Unal S, Yaman H. Structural modification of plasma albumin in sickle cell anemia. Acta Haematol 2015;133 (1):67
9. Hanson MS, Piknova B, Keszler A, Diers AR, Wang X, Gladwin MT, et al. Methaemalbumin formation in sickle cell disease: effect on oxidative protein modification and HO-1 induction. Br J Haematol 2011;54(4):502
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References

57. Agil A, Sadrzadeh SM. Hydroxyurea protects erythrocytes against oxidative damage. Redox Rep 2000;5(1):29
34. 58. Niihara Y, Zerez CR, Akiyama DS, Tanaka KR. Oral Lglutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell NAD redox potential. Am J Hematol 1998;58(2):117
21. 59. Nur E, Brandjes DP, Teerlink T, Otten HM, Oude Elferink RP, Muskiet F, et al. N-acetylcysteine reduces oxidative stress in sickle cell patients. Ann Hematol 2012;91(7):1097
105. 60. Pace BS, Shartava A, Pack-Mabien A, Mulekar M, Ardia A, Goodman SR. Effects of N-acetylcysteine on dense cell formation in sickle cell disease. Am J Hematol 2003;73(1):26
32.

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61. Martins VD, Manfredini V, Peralba MC, Benfato MS. Alphalipoic acid modifies oxidative stress parameters in sickle cell trait subjects and sickle cell patients. Clin Nutr 2009;28(2):192
7. 62. Nader E, Grau M, Fort R, Collins B, Cannas G, Gauthier A, et al. Hydroxyurea therapy modulates sickle cell anemia red blood cell physiology: impact on RBC deformability, oxidative stress, nitrite levels and nitric oxide synthase signalling pathway. Nitric Oxide 2018;81:28
35. 63. Iyamu EW, Fasold H, Roa D, delPilar Aguinaga M, Asakura T, Turner EA. Hydroxyurea-induced oxidative damage of normal and sickle cell hemoglobins in vitro: amelioration by radical scavengers. J Clin Lab Anal 2001;15(1):1
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C H A P T E R

7 Nrf2 and oxidative stress Osamu Wada-Hiraike Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

List of abbreviations Aβ ARE bZIP CNC DGR DHA DMF EPA GSS HO-1 Keap1 Neh Nrf2 OGG1 OS ROS SOD

been nominated as a primary target for research involving neuroprotection, neurodegenerative disease, cancer prevention and treatment, autoimmune diseases, and endocrinological diseases. Nrf2 is an inducible stress-response gene that maintains appropriate cellular OS levels. Kelchlike ECH-associated protein 1 (Keap1; Fig. 7.1), a negative regulator of Nrf2 (Fig. 7.2), is known to degrade Nrf2 in a protein ubiquitination-dependent manner (Fig. 7.3). If organs are exposed to OS, reactive OS factors are produced, and Nrf2 is activated and translocates to the nucleus where it binds to antioxidant response elements (AREs). Thereafter, downstream genes of the AREs, including antioxidants and survival factors, are induced in a process known as the canonical regulatory mechanism.2 ARE-regulated factors include as many as approximately 250 genes involved in cellular homeostasis that primarily serve as cytoprotective factors, antioxidant factors, detoxifying enzymes, and drug transporters4 (Fig. 7.4). Aside from the involvement of Nrf2 in this canonical pathway (Fig. 7.5), the functions of Nrf2 are far more diverse than originally envisioned because of their complex regulatory mechanisms. This complexity presents new experimental challenges and opportunities for scientists. In this chapter, the mechanisms of Nrf2 in OS are presented and the dietary chemical compounds that can potentially activate Nrf2 are discussed in detail. Numerous natural compounds affect Nrf2 expression, translocation to the nucleus, and ARE binding.

amyloid-beta antioxidant response element basic leucine zipper cap “n” collar glycine repeat domain docosahexaenoic acid dimethyl fumarate eicosapentaenoic acid glutathione synthetase hem oxygenase-1 kelch-like ECH-associated protein 1 Nrf2-ECH homology nuclear factor-E2-related factor 2 8-oxoguanine DNA glycosylase oxidative stress reactive oxygen species superoxide dismutase

Introduction Oxygen is required to maintain cellular functions because it is indispensable for energy metabolism. During the process of energy metabolism, superoxide anions, including hydrogen peroxide and hydroxyl radicals (O22, H2O2, OH2, ONOO2), are inevitably produced and are known as reactive oxygen species (ROS). ROS primarily produce less harm at lowcellular concentrations; in fact, low levels of ROS are thought to be necessary for cellular homeostasis.1 However, aberrant increases in ROS lead to a state of oxidative stress (OS) in cells, thereby inducing damage to cellular components such as DNA, RNA, lipids, and proteins.2 Most eukaryotic organisms can mount an OS-inducible response called the phase 2 response. Nuclear factor-E2-related factor 2 (Nrf2) plays a central role in maintaining and modulating cellular OS levels. The transcription factor Nrf2 was identified in 1994,3 and more than 20 years after its discovery, Nrf2 has Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00007-X

Nrf2: function Nrf2 is a member of the cap “n” collar (CNC) and basic leucine zipper (bZIP) transcription factor families. The predicted amino acid sequence of Nrf2 is

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© 2020 Elsevier Inc. All rights reserved.

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7. Nrf2 and oxidative stress

FIGURE 7.1 Protein structure of Nrf2. The protein structure of Nrf2 is illustrated. The Nrf2 protein possesses seven NRF2-ECH homology (Neh) domains. The Neh1 domain contains the CNC-bZIP motif, which is crucial for interactions with small MAF proteins. The Neh2 domain is responsible for the interaction with Keap1 E3 ligase, and other ubiquitin ligases, including CRIF1 and WDR23, bind to Nrf2 through this domain. Lysine residues located in the Neh2 domain are targeted by polyubiquitination and contribute to the degradation of Nrf2 by the 26S proteasome. The Neh3, Neh4, and Neh5 domains are important for the transcriptional activation function of Nrf2. The Neh3 domain contains a nuclear localization signal sequence. CBP, a histone acetyltransferase protein, and a CDH6 protein coactivator, is recruited at Neh3. CBP, p300, and RAC3 bind to Nrf2 through the Neh4 and Neh5 domains. βTrCP ubiquitin ligase binds to Nrf2 through the Neh6 domain.

FIGURE 7.2 Protein structure of Keap1. A broad complex-tramtrack-bric-a-brac (BTB) domain, an intervening region (IVR), and six Kelch domains are representative and specific domains that confer Keap1 with unique functions. The BTB domain is responsible for the dimerization of Keap1 and its interaction with CUL3. Keap1 binds to Nrf2 as a dimer, and the C-terminal Kelch domain has been shown to be a binding surface for the Nrf2 protein.

FIGURE 7.3 Ubiquitination regulation of Nrf2 by Keap1. Under physiological and unstressed conditions, Nrf2 is downregulated by its negative regulator Keap1 because Nrf2 is sequestered by the negative regulator complex involving Keap1. Nrf2 protein is degraded through the ubiquitination system and proteasomic complex. After the occurrence of cellular stress, modification of cysteine residues of Keap1 promotes the release of Keap1.

605 aa long, but due to variations in acidic residues and posttranscriptional modifications, the molecular weight of Nrf2 varies from 96 to 118 kDa.3 Nrf2 was originally identified as an activator of β-globin gene

expression,5 but further analysis of Nrf2 revealed its main function as a master regulator of OS in cells. Nrf2 possesses several functional domains, including the CNC and bZip domains and seven Nrf2-ECH

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Nrf2: function

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FIGURE 7.4 Intranuclear mechanism of Nrf2 transcription. Nrf2 forms a heterodimer complex with small Maf proteins and then binds to antioxidant response elements. The complex thereafter binds to activator complex including p300/CBP, RAC3, CHD6, and HDAC3. p300/ CBP, RAC3, and CHD6 might acetylate Nrf2 and HDAC3 deacetylates Nrf2. Various downstream genes are activated, and transcribed Nrf2 might function as positive and negative regulator.

FIGURE 7.5 Overview of Nrf2 function. In cytoplasm, Nrf2 is regulated by degradation complex including Keap1. Nrf2 protein is also degraded through the autophagic system. Th endoplasmic reticulum system produces HRD1, resulting in the downregulation of Nrf2 function. After the occurrence of cellular stress, modification of cysteine residues of Keap1 promotes the release of Nrf2 and Nrf2 translocates to the nucleus. Nrf2 forms a transcriptional Figcomplex and various downstream genes are activated, and cytoprotection is achieved. Nrf2 is acetylated by CBP and SUMOylated by Ubc9. Transcriptional activity of Nrf2 is repressed by Keap1, which is translocated to the nucleus through KPNA6, dissociates Nrf2 from ARE sequences and both Nrf2 and Keap1 are nuclear exported by Crm1.

homology (Neh) domains.6 The Neh1 domain is involved in binding to the DNA of the ARE (50 GTGACNNNGC-30 ) via bZIp7 and in heterodimerization with Maf proteins.8 In addition, the Neh1 domain

interacts with the E2 ubiquitin-conjugating enzyme UbcM2, which is proposed to be a central negative regulator of the Nrf2 protein. The Neh1 domain possesses a nuclear localization signal. Thus the Neh1 domain is

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essential for Nrf2 to function as a transcription factor. The N-terminal Neh2 domain is responsible for the interaction between Nrf2 and Keap1 because the 29 DLG31 and 79ETGE82 motifs in the Neh2 domain have been proven to be Keap1 binding sites.9 The Neh6 domain is also responsible for the degradation of Nrf2, because the Neh6 domain binds with βTrCP through its 343DSGIS347 and 382DSAPGS387 motifs.10 Therefore Neh2 and Neh6 are both involved in Nrf2 maintenance and in protecting Nrf2 from ubiquitination. The Neh3, Neh4, and Neh5 domains contribute to associations with other transcription factors, including CHD6, CBP, and RAC3. The role of the Neh7 domain is relatively unknown, but the domain has been implicated in the repression of Nrf2 by RXRα11 (Fig. 7.2). The main function of Nrf2 is thought to be as a transcription factor as Nrf2 directly binds to the cis-acting ARE sequence after translocation to the nucleus.7 It is estimated that approximately 1055 genes possess AREs in the promoter region, including genes associated with antioxidation and detoxification responses, cellular proliferation, metabolism, the immune response, signaling, cell survival, and the cell cycle. Thus the physiological functions of Nrf2 could be quite diverse (Fig. 7.4). Although Nrf2 has diverse functions, it is considered to be the master regulator of the cellular redox state because deletion of Nrf2 or decreases in Nrf2 during ageing are associated with increases in OS.2 While Nrf2 is ubiquitously expressed in human tissues,3 during normoxia, Nrf2 is held in the cytoplasm where it forms a complex with the inhibitory protein Keap1 and is maintained at low levels. The protein structure of Keap1 is characterized by six domains, namely, three broad complex-tramtrack-bric-a-brac domains, one intervening region, and two glycine repeat domains (DGR).6 The DLG motif in Nrf2 is important for complex formation with Keap1, and Nrf2 is primarily targeted for proteasomal ubiquitination and degradation.12 Under OS, Nrf2 is released from the DGR domain in Keap1, thus blocking Nrf2 ubiquitination and degradation. Furthermore, the binding of Nrf2 to the DGR domain is competitively inhibited by proteins such as p62 and BRCA2. Thus Nrf2 is able to respond to autophagy and DNA damage signaling. After sensing OS, Nrf2 dissociates from Keap1 and subsequently translocates into the nucleus, which ultimately results in its binding to genomic DNA ARE sequences and in the efficient transcription of downstream target genes. This stress-inducible mechanism is postulated to be the most important defense mechanism against OS, and appropriate functioning of the Nrf2/Keap1 pathway is indispensable for counteracting OS and protecting multiple organs and cells through antioxidant activity. Antioxidants,

including superoxide dismutase (SOD), catalase, and 8-oxoguanine DNA glycosylase (OGG1) are elevated and play cytoprotective roles under OS.13,14 The described mechanism of the Nrf2/ARE pathway is known to be the canonical mechanism, but it should be noted that the protein interaction between Nrf2Keap1 can be disrupted by p62, DPP3, PALB2, p21, BRCA1, and BRCA2. Recently another regulatory mechanism of Nrf2 has been proposed. Aside from Keap1, βTrCP, Hrd1, and WDR23 are also regarded as candidates to induce Nrf2 ubiquitination and subsequent proteasomal degradation. Nrf2 protein expression is regulated by various factors such as NF-κB, Sp-1, c-Jun, p53, c-Myc, BRCA1,15 and even Nrf2 itself.16 Nrf2 is also regulated by mTORC1 in a cap-dependent process under stable conditions, but the regulatory mechanism of Nrf2 in the presence of ROS differs. This mechanism is carried out in a cap-independent manner by an internal ribosomal entry site mechanism.17 Furthermore, miRNAs can regulate Nrf2 synthesis at the posttranscriptional level.18 Modification of CpG islands in the Nrf2 promoter, H3 histone methylation, and H4 histone acetylation are believed to be other transcriptional regulatory mechanisms for Nrf2.19

Nrf2: relation with disease The pathophysiologies of several chronic diseases are linked to OS. Representative conditions include cerebrovascular disease, neurodegenerative diseases, chronic pulmonary lung disease, diabetes mellitus, and autoimmune diseases. The mechanism that worsens these diseases is postulated to be an initial inflammation reaction with subsequent excessive ROS production. Excessive ROS might cause extensive cellular damage that necessitates redox adjustment by the antioxidant system. One representative example of this phenomenon occurs in the pancreas. Hyperglycemia leads to OS in the pancreas, and poor antioxidant status in pancreatic islets plays a detrimental role in the pathogenesis and progression of diabetes.20 In contrast, antioxidants have been shown to resolve diabetes and associated complications. The pathogenesis of Nrf2-related diseases is best characterized in neuron systems because the central nervous system is known to be sensitive to OS.21 Disruption of the redox system ultimately results in neuronal damage. Brain injury is generally divided into two types: primary and secondary brain injuries. After hemorrhage in brain tissue, pooled blood from hematomas causes the formation of masses, and these masses induce neuronal cell death within a few hours after the onset of

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Dietary compounds that could activate the Nrf2 and Nrf2/ARE pathway

intracerebral hemorrhage.22 Traumatic brain injury (TBI) is a major cause of death and disability. TBI can be classified as a type of primary brain damage. OS plays a central role in the pathogenesis of TBI,23 and it has been demonstrated that activation of Nrf2 could reduce brain damage caused by TBI. Following TBI, Nrf2 expression in brain tissue increases, and expression of phase II enzymes is elevated as a result of Nrf2/ARE pathway activation. Secondary brain injury is caused by hematoma-associated mechanisms and progresses over a few days.24 Hematoma-associated mechanisms induce neuronal damage, which subsequently leads to the development of OS. Unless the OS is resolved, the brain tissue suffers from additional OS-related effects. An animal model of stroke using middle cerebral artery occlusion revealed that Keap1 levels were decreased and this decrease was paralleled by increases in Nrf2 and its downstream proteins, such as thioredoxins, glutathione synthetase (GSS), and hem oxygenase-1 (HO-1).25 This phenomenon was accompanied by increased ROS. Activation of the Nrf2 pathway is also critical for scavenging ROS in Alzheimer’s disease (AD),26 and histological findings of AD include cerebral deposition of amyloid-beta (Aβ) peptides in senile plaques and neurofibrillary tangles of hyperphosphorylated tau aggregates. The Aβ protein is known to regulate cellular fate because it induces neuronal cell death in AD. In addition, Aβ is produced in cultured cells, demonstrating that OS plays a fundamental role in AD pathophysiology. Mitochondrial ROS production increases Aβ production,28 and the levels of ROS-related genes, including NQO-1, HO-1, p62, and glutathione redox system genes are altered in the hippocampus and cerebellum in AD. Thus Nrf2 activation has been regarded to play positive roles in inhibiting the development of AD. To support this hypothesis, an activator of the Nrf2/ARE pathway, 2cyano-3,12-dioxooleana-1,9-dien-28-oic acid-methyl amide was used to treat transgenic AD mice and the drug improved memory and decreased senile plaque formation and Aβ accumulation.29 Several studies have suggested that Nrf2 also plays protective roles in Parkinson’s disease (PD) because the substantia nigra of PD patients exhibits elevated levels of Nrf2 downstream genes, such as NQO-1 and HO-1,30 and administration of Nrf2 activators ameliorates the PD phenotype in experimental mouse models,31 indicating that astrocytic Nrf2 is critical for neuroprotection in PD models. Multiple sclerosis (MS) is a chronic inflammatory disease occurring in the central nervous system. MS has several causative factors, such as genetic, immunological, and environmental factors, and elevation of OS markers and decreases in antioxidant capacity are regarded as hallmarks of MS.32,33 MS is associated with persistent inflammation and lesions in

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white and gray matter with cortical demyelination and degeneration might be caused by malfunction of Nrf2.34 Consistent with the case in other tissues, activation of Nrf2 reduces ROS production and decreases OS, resulting in reduced apoptosis and survival of motor neurons among mouse and rat neuronal stem or progenitor cells. Dimethyl fumarate (DMF) activates Nrf2 because it dampens the regulatory mechanism of Keap1; however, the exact mechanism of DMF action in MS remains to be elucidated.35 DMF is hydrolyzed by esterases in the small intestine and serves as a protective agent in the central nervous system. DMF has been approved by the United States Food and Drug Administration as a new first-line oral drug for the treatment of relapsing MS.36,37 Because it intervenes in the function of Keap1, DMF can reduce ROS production and elevate antioxidant levels.14,37 Therefore the therapeutic potential of DMF has been investigated in OS and chronic inflammatory diseases. Aside from DMF, dietary compounds that contain ingredients sufficient to activate Nrf2 are currently being extensively investigated because these compounds generally do not exert adverse effects on human homeostasis. In next section, representative chemicals that could activate Nrf2 are summarized.

Dietary compounds that could activate the Nrf2 and Nrf2/ARE pathway I have described findings indicating that activation of Nrf2 can result in improvement of neurological diseases, chronic pulmonary disease, endocrinological diseases, and cancer. It has also been noted that several dietary compounds can activate Nrf2 ARE systems through specific mechanisms. Thus several bioactive compounds derived from foods can be novel and safe candidates for the treatment of chronic diseases. I here present several promising candidates and briefly summarize the possible mechanisms underlying their activation of Nrf2 (Table 7.1). Olive oil contains several dietary chemicals that could activate Nrf2.38 The representative phenolic compounds hydroxytyrosol, tyrosol, oleuropein, ligstroside, oleacein, and oleocanthal have been investigated in vitro and in vivo, and potential beneficial effects of these compounds have been reported. Several studies have demonstrated beneficial effects of hydroxytyrosol using animal and in vitro models39; hydroxytyrosol has diverse biological functions, including antioxidant activity, cardiovascular protective effects and neuroprotective effects. Hydroxytyrosol has been shown to activate the Nrf2/ARE pathway and some studies have indicated that hydroxytyrosol can inhibit cancer cell

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TABLE 7.1 Food-derived chemicals and their relationships with Nrf2 activation. Molecular mechanisms that relate to Nrf2 function Compound

Category

Nrf2 expression

Nrf2 nuclear translocation

Nrf2 binding to ARE

Expression of ARE genes

Phosphorylation

3H-1,2-dithiole-3thione

m

m

m

3-O-caffeoyl-1methylquinic acid

m

m

m

m

m

m

m

m

Allyl sulfides

Isothiocyanate

Brazilin

Isoflavonoid

Cafestol

Furan diterpenoid

Carnosol

Diterpene

m

m m

m

Capsaicin m

Chaocone Chlorophyllin

m

m m

Chlorophyll m

Cinnamaldehyde Curcumin

m

m

m

m

m

m

Epigallocatechin gallate

Polyphenol

Eupatilin

Flavone

m

(
)-Epicatechin

Polyphenol

m

m

Fisetin

Flavonoid

m

m

Hydroxytyrosol

Polyphenol

m

m

Isoorientin

Flavone

Kahweol

Diterpenoid

Oleuropein

Glycoside

m

Omega-3 fatty acids

m

m

m

m m

m

m

m

m

m

m

m

m

Alkaloid

Plumbagin

Quinoid

Quercetin

Polyphenol

m

Resveratrol

Polyphenol

m

m

m

m

m

m

Tyrosol

Polyphenol

Xanthohumol

Flavonoid

Zerumbone

Sesquiterpene

m

m

Piperine

Sulforaphane

m m

m m

m

m

m m

Various phytochemicals are known to activate several Nrf2 functions. The numerous roles of these chemicals in the activation of Nrf2 have been demonstrated using animal and in vitro models. Possible molecular mechanisms of these compounds are depicted.

growth and induce apoptosis.40 Tyrosol is another phenolic compound that lacks the ortho-diphenolic chemical structure present in hydroxytyrosol. Tyrosol is present in olive oil and alcoholic beverages such as wine and beer. Tyrosol has antioxidant properties similar to hydroxytyrosol, but its antioxidant ability is

weaker than that of hydroxytyrosol. However, several in vivo and in vitro experiments have suggested that tyrosol might possess neuroprotective properties.41 Although tyrosol has been demonstrated to activate the Nrf2/ARE pathway,42 its precise mechanism remains to be elucidated.

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Dietary compounds that could activate the Nrf2 and Nrf2/ARE pathway

Oleuropein is a glycoside compound found in immature and unprocessed olives and it been reported to exhibit protective effects in cardiovascular disease models. Oleuropein activates Nrf2 and is expected to have protective effects against neurodegenerative diseases. Relatively high concentrations of oleuropein (1
10 μM) can protect endothelial cells against functional impairment due to angiotensin-induced cell senescence.43 Oleuropein-treated cells exhibit striking features; after activation of Nrf2 and HO-1 expression, increases in proliferation and telomerase activity, and decreases in senescence and ROS levels are observed. In addition, oleuropein can improve the migration, adhesion, and tube formation of endothelial progenitor cells in a dose-dependent manner. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are present in fish oil and flaxseed. Numerous studies have indicated the possible positive effects of ω-3 PUFAs in protecting against adverse physiological effects. An in vitro study indicated that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) activate Nrf2/ARE signaling in a dose-dependent manner using a luciferase assay. Altogether, ω
3 PUFAs reduce ROS generation in a dose-dependent manner under basal conditions and in H2O2-treated cells, and significantly increase GSS, GCL, and GPx4 levels.44 Polyphenols consist of a group compounds with aromatic rings that are characterized by the presence of one or more hydroxyl groups with various structural complexities. Polyphenols that are able to activate Nrf2 have been investigated because the anti
OS function of polyphenols is potent. Among the group of polyphenols, resveratrol (trans-3,5,40-trihydroxystilbene), epicatechin, catechin, and epigallocatechin gallate have been investigated as possible activators of Nrf2. Resveratrol is synthesized by plants as a phytoalexin and can be found in berries, nuts, and some medicinal plants. Resveratrol is abundant in the skin of grapes and in red wine and is known to enhance several factors related to longevity and antioxidant, antiinflammatory, and growth-inhibiting processes. Resveratrol can potentiate beneficial effects in neurological diseases by quenching OS, scavenging free radicals, and activating the endogenous antioxidant system. Resveratrol treatment of neuronal stem cells can protect the cells from OS damage by promoting the expression of Nrf2, thereby promoting Nrf2 downstream genes such as NQO-1 and HO-1. In addition, resveratrol treatment increases SOD and GSS activity in a dose-dependent manner and reduces malondialdehyde levels in cells.45 Quercetin is a dietary flavonoid present in red kidney beans, capers, radishes, onions, fruits, nuts, tea, and red wine. Quercetin is polyphenol and is thought to have functions including anti
OS effects. Quercetin

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has been shown to enhance Nrf2 expression and to inhibit Nrf2 ubiquitination by promoting the degradation of Keap1. Nrf2 subsequently tends to translocate to the nucleus and bind to AREs, elevating the expression of Nrf2 downstream genes.46 Epigallocatechin gallate is one of the most abundant catechin polyphenols in green tea and is a bioactive compound because of its strong antioxidant and antiinflammatory properties. Several molecular mechanisms by which epigallocatechin gallate stimulates the Nrf2/ ARE pathway have been revealed in in vitro studies.47 Epigallocatechin gallate can activate the canonical Nrf2 pathway and interact with kinases such as ERK, PI3K, PKC, and JNK,48 thereby causing the dissociation of the Nrf2/Keap1 complex. Epigallocatechin gallate induces activation of Akt and ERK1/2 in endothelial cells, whereas this compound induces activation of p38 MAPK and Akt in B lymphoblasts. Neuroprotective effects of epigallocatechin gallate have been reported in a rat model of ischemia/reperfusion49 and in cultured neurons and AD models. Protection of neurons by epigallocatechin gallate is mediated by activation of the Nrf2/ARE pathway. This mechanism has been supported by observations of increased expression of GSS and HO-1, which decreases ROS and results in reductions in OS-induced cell death.50 Epigallocatechin gallate also suppresses Aβ-induced cytotoxicity by reducing activation of the MAPK signal transduction cascade.51 (
)-Epicatechin is also a natural product found at high concentrations in green tea and cocoa, and has been reported to protect cells from OS. (
)-Epicatechin has been shown to increase Nrf2 and GSS expression in treated cortical astrocytes. High doses of (
)-epicatechin exert neuroprotective effects in embryonic cortical neuronal cells by decreasing OS and promoting cell viability. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione) is a main component of natural turmeric and is included in the group of polyphenols. Curcumin has long been used in curry spices and is also used in Chinese and Japanese foods. Curcumin is gaining global attention because it has been shown to have diverse physiological effects, including antiangiogenic, antiinflammatory, anticancer, and antioxidative effects, thus showing a variety of beneficial properties. However, the bioavailability of curcumin is relatively poor, the effect of curcumin on the Nrf2/ARE pathway is highly dose-dependent, and activation of the Nrf2/ ARE pathway requires doses in the order of μM. It has been reported that curcumin treatment at doses of 15
30 μM can activate Nrf2 function, but the activation of Nrf2 by curcumin is complicated because curcumin exerts its protective effects in both Nrf2-dependent and Nrf2-independent manners.

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Lycopene is a representative carotenoid present in tomatoes and carrots. This phytochemical agent is a polyunsaturated hydrocarbon and tends to accumulate in specific organs, such as the testes, adrenals, liver, and prostate. Lycopene exhibits antioxidant activity by stimulating the Nrf2/ARE pathway, as indicated by an earlier study using HepG2 and MCF-7 cells that demonstrated that lycopene promotes Nrf2 nuclear translocation and upregulates the ARE system. Furthermore, lycopene also exerts neuroprotective effects; Aβinduced mitochondrial OS is quenched and apoptosis is partially reversed by lycopene treatment in cultured rat cortical neurons. Cafestol and kahweol are present in coffee and these compounds are known to promote the binding of Nrf2 to AREs. One study demonstrated that mice expressing Nrf2 express NQO-1, glutathione S-transferase class alpha 1, UDP-glucuronosyl transferase 1A6, and the glutamate cysteine ligase catalytic subunit, which are known to be antioxidant genes, at higher levels than Nrf2-knockout mice.52 Induction of cancer chemopreventive enzymes by coffee is mediated by the transcription factor Nrf2. There is also evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein. Sulforaphane (1-isothiocyanato-4-(methylsulfinyl) butane) is present in vegetables, including cauliflower, broccoli, and Brussels sprouts. This isothiocyanate compound has been shown to activate the Nrf2 pathway. Thus ARE downstream genes are elevated after sulforaphane treatment. The group of allyl sulfides includes diallyl sulfide, diallyl disulfide, and diallyl trisulfide. These compounds, which are organosulfur compounds found abundantly in garlic, are thought to activate Nrf2, and the underlying mechanism is known for diallyl trisulfide. Diallyl trisulfide directly affects Keap1-Nrf2 binding, causing Nrf2 to be released and translocate to the nucleus to facilitate the expression of ARE downstream genes. Another mechanism by which allyl sulfides activate Nrf2 has also been proposed. Allyl sulfides influence kinases that could activate Nrf2 and lead to the dissociation of the Nrf2-Keap1 complex. Cinnamaldehyde present in the bark of Cinnamomum species is a commonly used food flavoring that possesses multiple functional properties, including anticancer, antiinflammatory, and antioxidant properties. The mechanism by which cinnamaldehyde activates Nrf2 is thought to be the canonical pathway. Thus cinnamaldehyde treatment potentiates Nrf2 translocation to the nucleus and elevates HO-1 levels.53 This detailed explanation of oil and phytochemical compounds is not exhaustive, because numerous other dietary phytochemicals not discussed here might possess the ability to activate the Nrf2/ARE pathway. In summary, plants often produce substances to protect

themselves from attack by microorganisms, and these responses involve detoxification and redox homeostasisrestoring mechanisms. Within these stress responses, some chemicals can regulate the Nrf2/ARE pathway; such chemicals can be utilized as possible candidate drugs for humans.

Applications to other areas of pathology Oxidative stress is a balance between reactive oxygen species (ROS) and scavenger of ROS. Excessive ROS is primary supposed to be harmful for maintaining homeostasis and Nrf2/Keap1 system is one of the most major pathways for the reaction of ROS in human body. The Nrf2/Keap1 system is becoming a therapeutic target to improve harmful conditions of the human body, and naturally derived compounds can be utilized as therapeutic modalities.

Summary points • This chapter focuses on the properties of nuclear factor-E2-related factor 2 (Nrf2), which is an oxidative stress
inducible transcription factor. • Nrf2 is a master regulator that maintains appropriate oxidative stress levels in cells. • The physiological role of Nrf2 remains to be elucidated, because excessive Nrf2 activity is suspected to be carcinogenic. • Nrf2 functions have been characterized in various tissues, but the central nervous system might be a novel target tissue of Nrf2 because it is highly susceptible to oxidative stress. • Regulation of Nrf2 function is regarded as a therapeutic target, and various phytochemicals are currently being investigated for their Nrf2regulating effects. • Phytochemicals present in oil and wine have been well characterized as modulators of Nrf2 function.

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56. 9. McMahon M, Thomas N, Itoh K, Yamamoto M, Hayes JD. Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron. J Biol Chem 2004;279 (30):31556
67. 10. Chowdhry S, Zhang Y, McMahon M, Sutherland C, Cuadrado A, Hayes JD. Nrf2 is controlled by two distinct beta-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. Oncogene 2013;32(32):3765
81. 11. Wang H, Liu K, Geng M, et al. RXRalpha inhibits the NRF2ARE signaling pathway through a direct interaction with the Neh7 domain of NRF2. Cancer Res 2013;73(10):3097
108. 12. Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol 2004;24(24):10941
53. 13. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007;47:89
116. 14. Akino N, Wada-Hiraike O, Terao H, et al. Activation of Nrf2 might reduce oxidative stress in human granulosa cells. Mol Cell Endocrinol 2018;470:96
104. 15. Kang HJ, Hong YB, Kim HJ, et al. Detoxification: a novel function of BRCA1 in tumor suppression? Toxicol Sci 2011;122(1):26
37. 16. Kwak MK, Itoh K, Yamamoto M, Kensler TW. Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element-like sequences in the nrf2 promoter. Mol Cell Biol 2002;22(9):2883
92. 17. Li W, Thakor N, Xu EY, et al. An internal ribosomal entry site mediates redox-sensitive translation of Nrf2. Nucl Acids Res 2010;38(3):778
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601. 19. Yu S, Khor TO, Cheung KL, et al. Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice. PLoS One 2010;5(1):e8579. 20. Forbes JM, Coughlan MT, Cooper ME. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 2008;57 (6):1446
54. 21. Liu Z, Zhou T, Ziegler AC, Dimitrion P, Zuo L. Oxidative stress in neurodegenerative diseases: from molecular mechanisms to clinical applications. Oxid Med Cell Longev 2017;2017:2525967. 22. Siaw-Debrah F, Nyanzu M, Ni H, et al. Preclinical studies and translational applications of intracerebral hemorrhage. Biomed Res Int 2017;2017:5135429. 23. Yan W, Wang HD, Hu ZG, Wang QF, Yin HX. Activation of Nrf2-ARE pathway in brain after traumatic brain injury. Neurosci Lett 2008;431(2):150
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908. 25. Sugiyama T, Imai T, Nakamura S, et al. A novel Nrf2 activator, RS9, attenuates secondary brain injury after intracerebral hemorrhage in sub-acute phase. Brain Res 2018. 26. Tramutola A, Lanzillotta C, Perluigi M, Butterfield DA. Oxidative stress, protein modification and Alzheimer disease. Brain Res Bull 2017;133:88
96. 27. Chen Z, Zhong C. Oxidative stress in Alzheimer’s disease. Neurosci Bull 2014;30(2):271
81. 28. Bhat AH, Dar KB, Anees S, et al. Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight. Biomed Pharmacother 2015;74:101
10. 29. Dumont M, Wille E, Calingasan NY, et al. Triterpenoid CDDOmethylamide improves memory and decreases amyloid plaques in a transgenic mouse model of Alzheimer’s disease. J Neurochem 2009;109(2):502
12. 30. Lastres-Becker I, Ulusoy A, Innamorato NG, et al. alphaSynuclein expression and Nrf2 deficiency cooperate to aggravate protein aggregation, neuronal death and inflammation in early-stage Parkinson’s disease. Hum Mol Genet 2012;21 (14):3173
92. 31. Burton NC, Kensler TW, Guilarte TR. In vivo modulation of the Parkinsonian phenotype by Nrf2. Neurotoxicology 2006;27 (6):1094
100. 32. Choi IY, Lee P, Adany P, et al. In vivo evidence of oxidative stress in brains of patients with progressive multiple sclerosis. Mult Scler 2018;24(8):1029
38. 33. Ferreira KPZ, Oliveira SR, Kallaur AP, et al. Disease progression and oxidative stress are associated with higher serum ferritin levels in patients with multiple sclerosis. J Neurol Sci 2017;373:236
41. 34. Zhang Q, Li Z, Wu S, et al. Myricetin alleviates cuprizoneinduced behavioral dysfunction and demyelination in mice by Nrf2 pathway. Food Funct 2016;7(10):4332
42. 35. Suneetha A, Raja Rajeswari K. Role of dimethyl fumarate in oxidative stress of multiple sclerosis: a review. J Chromatogr B Analyt Technol Biomed Life Sci 2016;1019:15
20. 36. Prosperini L, Pontecorvo S. Dimethyl fumarate in the management of multiple sclerosis: appropriate patient selection and special considerations. Ther Clin Risk Manag 2016;12:339
50. 37. Smith MD, Martin KA, Calabresi PA, Bhargava P. Dimethyl fumarate alters B-cell memory and cytokine production in MS patients. Ann Clin Transl Neurol 2017;4(5):351
5. 38. Martinez-Huelamo M, Rodriguez-Morato J, Boronat A, de la Torre R. Modulation of Nrf2 by olive oil and wine polyphenols and neuroprotection. Antioxidants (Basel) 2017;6(4). 39. Tasset I, Pontes AJ, Hinojosa AJ, de la Torre R, Tunez I. Olive oil reduces oxidative damage in a 3-nitropropionic acid-induced Huntington’s disease-like rat model. Nutr Neurosci 2011;14(3):106
11. 40. Peng S, Zhang B, Yao J, Duan D, Fang J. Dual protection of hydroxytyrosol, an olive oil polyphenol, against oxidative damage in PC12 cells. Food Funct 2015;6(6):2091
100. 41. Bu Y, Rho S, Kim J, et al. Neuroprotective effect of tyrosol on transient focal cerebral ischemia in rats. Neurosci Lett 2007;414 (3):218
21. 42. Wang WC, Xia YM, Yang B, et al. Protective effects of tyrosol against LPS-induced acute lung injury via inhibiting NF-kappaB and AP-1 activation and activating the HO-1/Nrf2 pathways. Biol Pharm Bull 2017;40(5):583
93. 43. Parzonko A, Czerwinska ME, Kiss AK, Naruszewicz M. Oleuropein and oleacein may restore biological functions of endothelial progenitor cells impaired by angiotensin II via activation of Nrf2/heme oxygenase-1 pathway. Phytomedicine 2013;20(12):1088
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44. Zgorzynska E, Dziedzic B, Gorzkiewicz A, et al. Omega-3 polyunsaturated fatty acids improve the antioxidative defense in rat astrocytes via an Nrf2-dependent mechanism. Pharmacol Rep 2017;69(5):935
42. 45. Shen C, Cheng W, Yu P, et al. Resveratrol pretreatment attenuates injury and promotes proliferation of neural stem cells following oxygen-glucose deprivation/reoxygenation by upregulating the expression of Nrf2, HO-1 and NQO1 in vitro. Mol Med Rep 2016;14(4):3646
54. 46. Tanigawa S, Fujii M, Hou DX. Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin. Free Radic Biol Med 2007;42(11):1690
703. 47. Bahia PK, Rattray M, Williams RJ. Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent antioxidant response element activity and up-regulates glutathione in cortical astrocytes. J Neurochem 2008;106(5):2194
204. 48. Andreadi CK, Howells LM, Atherfold PA, Manson MM. Involvement of Nrf2, p38, B-Raf, and nuclear factor-kappaB, but not phosphatidylinositol 3-kinase, in induction of hemeoxygenase1 by dietary polyphenols. Mol Pharmacol 2006;69(3):1033
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49. Han J, Wang M, Jing X, Shi H, Ren M, Lou H. (-)-Epigallocatechin gallate protects against cerebral ischemiainduced oxidative stress via Nrf2/ARE signaling. Neurochem Res 2014;39(7):1292
9. 50. Romeo L, Intrieri M, D’Agata V, et al. The major green tea polyphenol, (-)-epigallocatechin-3-gallate, induces heme oxygenase in rat neurons and acts as an effective neuroprotective agent against oxidative stress. J Am Coll Nutr 2009;28(Suppl):492S
9S. 51. Cheng-Chung Wei J, Huang HC, Chen WJ, Huang CN, Peng CH, Lin CL. Epigallocatechin gallate attenuates amyloid betainduced inflammation and neurotoxicity in EOC 13.31 microglia. Eur J Pharmacol 2016;770:16
24. 52. Higgins LG, Cavin C, Itoh K, Yamamoto M, Hayes JD. Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein. Toxicol Appl Pharmacol 2008;226(3):328
37. 53. Kim NY, Ahn SG, Kim SA. Cinnamaldehyde protects human dental pulp cells against oxidative stress through the Nrf2/HO-1dependent antioxidant response. Eur J Pharmacol 2017;815:73
9.

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C H A P T E R

8 Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy Aristidis S. Veskoukis School of Health Sciences, Department of Biochemistry and Biotechnology, University of Thessaly, Mezourlo, Greece

List of abbreviations AA GSH H2O2 HO• mTOR NAC Nf-κB NOX2 Nrf2 O2 2 O2 OS ROS RS SOD TLR

Moreover, the interesting shift toward a novel perception of the roles of RS and antioxidants on the regulation of pathogen burden will be discussed. It should be noted that, following the literature, the focus is mostly on bacteria with less information presented concerning viruses and protozoans.

amino acid(s) reduced form of glutathione hydrogen peroxide hydroxyl radical Mammalian target of rapamycin N-acetylcysteine nuclear factor kappa-light-chain-enhancer of activated B cells NADPH oxidase 2 nuclear factor (erythroid-derived 2)-like 2 superoxide anion molecular oxygen oxidative Stress reactive oxygen species reactive species superoxide dismutase toll-like receptor

Oxidative stress: a short historical overview OS was defined by Helmut Sies in 1985 as “a disturbance in the prooxidant
antioxidant balance in favor of the former.”1 This was the most influential definition of this biological phenomenon, which, until 15 years ago, was thought to be well characterized. However, the uncertainty dictated by science itself highly disputed this fact. The evolution of ideas in redox biology has demonstrated that the 1985 definition of OS is not adequate. Therefore it was expanded by Sies as “a disturbance in the prooxidant/antioxidant balance in favor of the former, leading to potential damage.”2 This definition is indicative of the ideas prevailing in that premature era of redox biology. According to them, prooxidants (hereafter, they will be referred to as RS) are considered as totally negative entities and harmful agents for biomolecules, cells, tissues, and, finally, organisms. However, we know today that this is far from reality. The presence of RS is mandatory for cell normal function and survival. Two studies published in 1995 and 1997 demonstrated that H2O2 is essential for signal transduction associated with plateletderived growth factor3 and epidermal growth factor.4

Introduction During the past few decades, the idea that redox biology is inextricably linked to pathogeny induced by microorganisms to the host has gained many adherents due to the accumulation of relevant experimental evidence. Redox—a compound word for reductions and oxidations, that is, gain or loss of electrons, respectively, which are chemical reactions that occur concomitantly—is associated with terms such as reactive species (RS), oxidative stress (OS), antioxidants, as well as redox signaling. In this chapter, these concepts will be described and their connections with disease and putative therapies against pathogens will be presented.

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00008-1

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8. Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy

Later, distinguished researchers expanded this knowledge, formulating the currently accepted opinion that the most fundamental cellular processes depend mostly on redox signaling.5,6 To this end, a new definition of OS emerged in 2006 as “a disruption of redox signaling and control.”7 This is considered the most accurate and acceptable definition to date since it designates that OS depends on redox signaling.

Redox signaling: definition and mechanisms Redox signaling is defined in this chapter as “the process of communication between cells that coordinates their function and is dependent on reductions and oxidations between proteins that transduce signals and RS that act as the second messengers.” It has been proposed that reactive oxygen species (ROS), such as • H2O2, O2 2 and HO are signaling molecules, although they do not all act directly as second messengers.8,9 The field of redox biology has been hugely expanded over the past 30 years; however, the mechanisms that redox signaling pathways obey have not been fully elucidated. We know that redox signaling is fundamental in preserving cell and microorganism survival and, importantly, many diseases have been linked to loss of signaling control.9,10 The paradigm that defines RS as signaling molecules has been enriched lately and new molecules [e.g., amino acids (AA) that are not RS]

have been found capable to transduce (in)directly redox signals. Indeed, covalent modification of AA is a mechanism for translating a redox signal to a cellular response. Cysteine is the most interesting AA of this category due to its exciting chemical property to obtain a wide range of oxidation states (i.e., 22 to 16). Thus it is a second messenger that, depending on the oxidation degree, can be reversibly or irreversibly converted to persulfides and sulfinic acids, respectively.11 Another important trait of cysteine is that it is fundamental for redox signaling in bacteria and, hence, regulates the response of the host to several microbeinduced diseases.10 This usually occurs via OxyR, a transcription factor that is activated by the formation of a disulfide bond between two cysteine residues, and is a H2O2 sensor.12 This means that it regulates the expression of antioxidant genes in an OS context. SoxR acts also by controlling the expression of antioxidant enzymes, but it is triggered by enhanced O2 2 concentration (Table 8.1).13

A paradox The atmosphere of the Earth was enriched with O2 due to the photosynthetic activity of cyanobacteria, approximately 2.3 billion years ago. This fact is called the “Great Oxidation Event,” which in terms of semiotics indicates how important process oxidation is

TABLE 8.1 The most well characterized signaling mechanisms and their functions. Function Signaling mechanism

Pathogens

Outcome to the host

Transcription factors

OxyR

Interacts with RNA polymerase and induces transcription of genes that enhance resistance of bacteria to antibiotics. It is a H2O2 sensor.

Enhanced immune response is required in order to cope with pathogens that have fortified defense against reactive species

SoxR

Controls expression of genes related to oxidative stress.

NF-κB

Its activation by the pathogens induces adaptive responses against immune response of the host.

Nrf2

Regulates the expression of antioxidant enzymes after its activation by the generation of reactive species.

H2O2

Activates transcription factors, such as OxyR.

O2 2

Indirect role, a precursor of H2O2.

Reactive species

•

Amino acids

OH

Triggers the activation of Nrf2. Indirect role.

Cysteine

Its oxidation products such as sulfenic acids trigger the activation of redox-related transcription factors (i.e., Nrf2).

Arginine

The pathogens suffer from AA starvation and face serious problems

Asparagine

Regulate the host innate immunity

Tryptophan This table presents some of the most widely analyzed mechanisms of signal transduction in pathogenic microorganisms. They are categorized as transcription factors, reactive species, and amino acids. Furthermore, their function in pathogens and host organisms on the grounds of disease onset is described.

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Redox signaling as a determinant for pathogen thriving in the context of oxidative stress

considered by researchers of different fields.14 The new composition of atmosphere led microbes to develop defensive mechanisms against increased generation of RS, thus mitigating the adverse effects of OS. Given that the host immune system utilizes RS during its effort to confront infections (i.e., oxidative burst), several pathogenic microorganisms have evolved strategies to survive and, even thrive, in the host environment by adequately facing RS generation and the subsequent OS. One of them, probably the most sophisticated, is the escape from the phagocyte toward the cytosol of the host.15 The host immune system seems to be much more elaborate. It manages to confront microbes through biological procedures, such as oxidation of ferritin by H2O2 producing free iron that induces RS generation via Fenton reaction.16 It therefore becomes evident that the production of RS in the host environment is the essential defensive process for microorganism scavenging. Thus someone could aptly consider that antioxidant administration is a practice that does not help the host to confront pathogens. However, this does not always hold true since there is experimental evidence that antioxidants may be proven effective against microbe invasion in some cases.17 This paradox, according to which some microbes are eliminated by RS, whereas others thrive in OS, can be clarified on the basis of redox signaling of microbes and the host. Such mechanisms have been described and will be presented as examples.18

Redox signaling as a determinant for pathogen elimination in the context of oxidative stress The perception that used to dominate the scientific community until recently supports the notion that RS are detrimental for pathogen survival. This is the reason why oxidative burst has been assigned by nature to ROS production, especially through activation of NADPH oxidase. Although there are hundreds of studies backing up this aspect, only a few of them will be referred to in this section. The author has no intention to underestimate the significance of these groundbreaking observations. Nevertheless, he believes that the findings pointing to the novel point of view fit better to the aim of the chapter. Redox signaling, through various representatives, plays key roles in killing microorganisms. It has been shown that Salmonella typhimurium, which causes typhoid fever, is eliminated by macrophage-induced ROS. According to this mechanism, ROS are generated by NOX2 that is triggered by the stimulation of TLR.19 Furthermore, autophagy is a common procedure for pathogen killing by the host. Its biological basis has not been fully elucidated, but it appears that it applies to a wide spectrum of

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microorganisms, namely bacteria (e.g., Porphyromonas gingivalis implicated in periodontal diseases), viruses (e.g., HSV-1 causing herpes), and protozoans (e.g., Toxoplasma gondii responsible for toxoplasmosis).18,20 Another important redox-related pathway for controlling virulence is the inhibition of mTOR. In particular, ROS that are excessively generated during infections inhibit mTOR, which has been shown to block infection development by human cytomegalovirus.18 These examples are indicative of the beneficial action of ROS against pathogen burden elimination. Fig. 8.1 summarizes some of the main redox-related paths that are activated when RS kill pathogens. It is worth mentioning, though, that the in-depth mechanisms that promote ROS-induced killing of microbes need to be elucidated.

Redox signaling as a determinant for pathogen thriving in the context of oxidative stress In this section, a few examples with respect to the ability of some bacteria to diminish host defense by adopting very clever mechanisms are presented. As it has been proposed a defensive mechanism of the host after the infection-induced oxidative burst is the import of GSH in high concentration (i.e., 10 mM) in the cytosol of phagocytes.21 Several infectious microorganisms sense the threatening increase in host GSH levels and manage to survive. An interesting example is that of Listeria monocytogenes, which causes listeriosis. It has been recently reported that GSH binds allosterically to the transcription factor PrfA that regulates the pathogen virulence by activating the appropriate genes.22 Another example indicating that bacteria have developed elaborate practices in order to surpass the host defense comes from S. typhimurium. During inflammation followed by the bacterial invasion, phagocyte activity induces the production of the oxidizing agent tetrathionate by thiosulfate. The fact that S. typhimurium uses this molecule as its own terminal electron acceptor shows that inflammation, which is normally the procedure for bacteria elimination, aids the growth of the microorganism.23 A commonly observed mechanism applied by the host against pathogens is to withhold iron, thus it cannot be used by microbes. Intriguingly, there are transcription factors (i.e., DtxR and Fur) that help pathogens to overcome that depletion by regulating their response. To this end, and due to DtxR, Corynebacterium diphtheriae (the bacterium that causes diphtheria) secrets its toxin in an iron-free environment as is the case for Fur that regulates alpha toxin and leukotoxin expression by Staphylococcus aureus (responsible for several serious infections).24 Moreover, Helicobacter pylori, another

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FIGURE 8.1 Redox-related mechanisms of pathogen elimination. Phagocytosis is the biological process during which the infected host eliminates pathogenic microorganisms. The role of ROS, which are excessively generated as an outcome of oxidative burst, in this procedure is decisive. Hence the killing of pathogens is mediated by ROS, whose action is regulated through redox signaling pathways. This figure summarizes a few fundamental, redox-related mechanisms leading to the decrease of the pathogen burden.

common pathogen that is the main cause of ulcers, utilizes inflammation to thrive. Specifically, calprotectin that is secreted by phagocytes sequesters zinc, a process that reduces inflammation and, hence, increases pathogen burden.25 Antioxidants also seem to be harmful for the host. It seems that resveratrol, which is a potent naturally occurring antioxidant, enhances replication and, thus, the virulence of hepatitis C virus.26 Fig. 8.2 provides some additional examples regarding the impact of the oxidative environment on the thriving of commonly encountered bacteria and viruses. Particularly, the cases of Mycobacterium abscessus,27 Bacillus anthracis,28 Trypanosoma cruzi,29 P. gingivalis,30 H. pylori,31 influenza A,32 and Porcine circovirus33 are presented. The examples point out that there are specific redox signaling pathways assisting pathogens to overcome the detrimental outcome of host-induced antioxidant generation or inflammatory response. Thus the therapeutic strategies need to be much more intellectual since many bacteria are able to survive the excessive ROS production during oxidative burst, which is the fundamental weapon of hosts against bacteria.

thought, antioxidant administration should be applied with caution because they could potentially impair ROS production and reduce their effectiveness against microbes. However, based on the controversial results analyzed in the previous sections, it is apparent that we are in the middle of a shift regarding how effective ROS and antioxidants can be on pathogen elimination.18 Such shifts in scientific ideas usually bring to the surface a “paradigm shift,” a term that was introduced by the philosopher Thomas Kuhn in his The Structure of Scientific Revolutions.34 Kuhn states that a paradigm shift is defined on the basis of strict criteria and, in particular, it is followed by changes in the constitution of the scientific community such as alterations in its members or the practices and experimental tools used. Although in our case there is definitely a countermarch to the dominating point of view, it is not sure yet that we are in front of a paradigm shift as proposed by Kuhn. Therefore the change in the current notion is more conservatively characterized by the author as a “perception shift.” This is the term that will be adopted in this manuscript and it is conceptually demonstrated in Fig. 8.3.

The perception shift

The intriguing dual role of antioxidants

Until recently, the prevailing idea was that ROS are entities generated in the phagocytes during oxidative burst mitigating the killing of pathogens. In this line of

Over the past 15 years, there has been a shift in the established notion that antioxidants are mainly beneficial for human health. The idea prevailing a

I. Oxidative Stress and Pathology

The intriguing dual role of antioxidants

91 FIGURE 8.2 Experimental findings supporting the beneficial role of oxidative environment on microbe thriving. Over the past 15 years there is scarce evidence that ROS and antioxidants might act differently and not as it had been thought previously. This idea has gained many adherents recently due to the multitude of relevant findings. This is a figurative demonstration of experimental data indicating that OS is not an obstacle in pathogen survival. On the contrary, some microorganisms are favored by ROS, whereas antioxidants act as inhibitory factors for their development.

FIGURE 8.3 The perception shift regarding the effects of reactive species and antioxidants on disease onset induced by microorganisms and putative therapies. This is a conceptual model illustrating the countermarch to the notion that ROS are detrimental biological entities for pathogens and that antioxidants should be administered with caution since they may protect microbes, and thus increase pathogen burden (i.e., the “old perception”). On the contrary, recent evidence has challenged this idea. Therefore it is currently believed that, in some cases, ROS promote thriving of microbes and antioxidants are beneficial for the host by decreasing the infection burden (i.e., the new perception). However, in this chapter this phenomenon is not characterized as a paradigm shift since this is a highly specific term with especial traits described by the eminent science historian and philosopher Thomas Kuhn. Alternatively, the term “perception shift” is adopted by the author because he believes that there is not enough evidence to do otherwise. Still, it appears that we are in the middle of a process that could potentially alter the opinion of researchers about the biology of infections and, thus, lead to the discovery of novel therapeutic approaches.

few decades ago was insightfully expressed by Gey in 1986 as the “antioxidant hypothesis,” which states that antioxidants, either being physical or chemical compounds, exert their benefits in human health

via elimination of OS.35 As is obvious from this hypothesis, the excessive production of RS (i.e., induction of OS) is considered deleterious and each antioxidant molecule that lowers their concentration is

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8. Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy

FIGURE 8.4 A mathematical illustration of the “new perception.” Although there is a perception shift, there is no uniformity with respect to the impact of antioxidants and (pro)oxidants on pathogen burden. The administration or depletion of antioxidants and (pro)oxidants, at least on the basis of the currently available data, does not seem to act against all pathogenic microorganisms in the same manner. Thus as indicated by the level of statistical significance randomly put at P 5 .8 and P 5 .9 for administration and depletion, respectively (with the level of significance set at P 5 .05), the pathogen burden measured in arbitrary units (i.e., AU) is not affected uniformly.

advantageous or, in some cases, even a panacea. Numerous studies in the following years have asserted the beneficial action of antioxidants on different research topics, such as nutrition,36
39 toxicology,40 and pathology induced by microorganisms.15 This idea has been challenged recently, and there is increasing evidence that the action of antioxidants does not seem to be that clear due to several factors. Indicatively, publications in high-impact journals have reported that exogenous antioxidants, and especially NAC as well as vitamins C and E, which are key players in the arsenal of human antioxidant defense, accelerate lung cancer progression and increase melanoma in mice.41,42 Furthermore, a metaanalysis has ended up with the intriguing finding that antioxidants increase mortality in humans.43 In addition, antioxidants act differently in in vitro and in vivo models,37,44 whereas their action appears to depend on the system studied,36,45
48 denoting how tricky this research topic is. The most interesting idea that has emerged stipulates that antioxidants are effective under a personalized regimen. In lay terms this means that antioxidants act differently when they are administered in individuals that suffer from deficiencies in specific intrinsic antioxidants,49 and it has been implied that such individuals constitute the most suitable cohort for monitoring the antioxidant activity of diverse compounds.50

The same pattern follows the evolution in the research findings regarding the effects of antioxidants on pathogenic microorganisms per se and on the host health status.15 Several studies have reported that antioxidants might be adequate defensive modulations since there are several microorganisms that thrive during OS, while there is evidence that they may also act in the opposite way.15,18,51 The dual role of antioxidants and, consequently of (pro)oxidants, has not allowed the establishment of a clear direction concerning their action on microbes. Fig. 8.4 illustrates the result of the controversial findings that have led to the perception shift in the form of a mathematical conceptual model as designed by the author (i.e., it has not been published in an original paper but it is used in this chapter as an exemplar).

Association of reactive species and antioxidants with putative therapy against pathogens It has been demonstrated that antibacterial drugs predominantly act by inhibiting DNA replication and repair, protein synthesis, and cell-wall turnover.52 They are divided in two major categories, namely bactericidal that kill bacteria and bacteriostatic that inhibit bacteria growth. Kohansky et al.53 through a

I. Oxidative Stress and Pathology

Future perspectives in therapy against pathogens

groundbreaking study mostly contributed to the formulation of the until-recently prevailing paradigm that antibiotics kill bacteria via excessive RS generation. In particular, the researchers showed that three major classes of antibiotics are effective because they lead to the production of HO•, which is the final product of NADH depletion mediated by Krebs cycle and the Fenton reaction. Furthermore, their bactericidal activity is reversed by iron chelators and thiourea, which act as antioxidants by preventing Fenton reaction and by scavenging RS.53 On the same page, other eminent articles have reported that antibiotic effectiveness is enhanced when mutations inactivate the antioxidant defense of bacteria,54 whereas antibiotic-induced RS oxidatively modify DNA accompanied by lethality.55 However, new experimental findings in high-ranking journals have challenged this notion.56 Indicatively, antibiotics can be bactericidal in the absence of O2 when no ROS are formed,57 while bacteria that lack catalase and peroxidase activity are not more susceptible to diverse antibiotics.58 Furthermore, although iron chelators and thiourea prevent antibiotic action, they can be active during anaerobic conditions as well.59 There is a great debate in the literature whether ROS are the major contributors through which antibiotics kill bacteria. There is evidence toward both directions, but it is not that clear whether antioxidants as ROS scavenging compounds or metal chelators could be beneficial. Fig. 8.5 illustrates the dual role of antioxidants on the reduction of pathogen burden. It is noteworthy that the vast amount of experimental data

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have been produced using in vitro models and clinical approaches are missing. Therefore the following question arises: What will be the paradigm/perception if we study the activity of antibiotics following a real infection inside the human body?

Future perspectives in therapy against pathogens In this chapter, the association of the basic ideas of redox biology with the defense and the putative therapy against pathogenic microorganisms was presented and discussed. The idea that RS are harmful and, hence, antioxidants could potentially mitigate in microbe survival is currently being challenged. There is evidence that some pathogens thrive in the context of OS and, as a consequence, the beneficial role of RS seems to be under question. Therefore therapeutic approaches need to be adapted appropriately by taking into account the perception shift observed in the field. Regarding redox-related therapeutic regimens, it is important to target specific signaling pathways with known participation in pathogen elimination and to adopt the correct biomarkers in order to monitor the outcome of medicines on infectious diseases.40 The measurement of biomarkers that are functionally, and even structurally, clustered enhances their translational potency, as has been reported previously.60 After adopting the biomarkers that can adequately monitor the progression of a pathogen-induced disease, it will

FIGURE 8.5 The putative role of antioxidants as therapeutic agents. This figure illustrates the controversial role of antioxidant administration on pathogen burden. It is evident that specific antioxidant compounds are beneficial against infection induced by some microorganisms, whereas others seem to be ineffective, or even harmful, for the host. Thus due to lack of consistency in the experimental data there is no possibility to generalize regarding the putative therapy of infections on the grounds of antioxidant supplements.

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8. Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy

be much easier to develop the proper medicine and to administer it under the most effective regimen.

Applications to other areas of pathology Redox biology has been a rapidly expanding field over the past few decades. The accumulated knowledge has led to the development of novel notions regarding the biological role of RS and antioxidants. In parallel to the perception shift presented in this chapter, it appears that there is a pure paradigm shift in this research area according to which RS and antioxidants can be both detrimental and beneficial. Moreover, it is now established that RS are key elements of several pathologies (e.g., cancer, diabetes, neurodegenerative diseases), although their exact role remains to be elucidated. This is also the case for antioxidants. Through the discussion in this chapter, it is evident that redox signaling is a major contributor regarding the action of RS and antioxidants on pathogen-related diseases. Therefore an in-depth study could potentially provide useful information about the development of novel therapeutic approaches. Following a similar practice with respect to research in the field of pathology, the etiology of several redoxrelated diseases could be more easily revealed by investigating the biological background of redox signaling pathways. Toward this research direction, the selection of the appropriate biomarkers should gain much attention because, ideally, they need to form a network with common functional and structural traits in order to have enhanced translational potency. This practice aids the monitoring of disease onset and the adoption of proper therapeutic regimens.

Summary points • This chapter focuses on the role of redox signaling and antioxidants on pathogen-induced disease and therapy. • RS exert both harmful and beneficial actions. • RS are key signaling molecules. • Antioxidants act dually, being potentially beneficial and detrimental in the context of disease. • Some pathogens are eliminated by RS and others thrive in OS. • Redox signaling is a determinant factor for microbe development or killing. • Novel therapeutic approaches should follow the perception shift observed in the field and presented in this chapter. • Selecting the proper biomarkers for monitoring disease onset is essential for adopting the right therapy.

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C H A P T E R

9 Paraoxonase 1 as antioxidant enzyme in children Maximiliano Martı´n, Valeria Hirschler, Eliana Botta and Fernando Brites Laboratory of Lipids and Atherosclerosis, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina

levels.1,2 These findings were further confirmed by the direct association between low-density lipoprotein cholesterol (LDL-C), the principal lipoprotein transporting cholesterol in blood, and CVD.3 It is noteworthy that high LDL-C levels detected in young adults constitute a strong predictor of CVD later in adulthood.4 Currently, CVD is considered an inflammatory pathology, characterized by the formation of atherosclerotic lesions. These lesions occur principally in large and medium-sized arteries and can lead to ischemia of the heart, brain, or extremities, resulting in infarction.5 Besides these, they may be present throughout a person’s lifetime. In fact, the earliest type of lesion, called fatty streak, which is common in infants and young children, is a pure inflammatory lesion.6 In persons with hypercholesterolemia, the influx of inflammatory cells is preceded by the extracellular deposition of amorphous and membranous lipids.7 Interestingly, different autopsy-based studies have shown the presence of fatty streaks in children and adolescents,8 thus confirming that atherosclerosis is a disorder of slow progression that begins very early in life. In addition to high total cholesterol and LDL-C levels, other CVD risk factors and risk behaviors can be detected in childhood, and the extent of their presence has been linked to the severity of atherosclerosis in adulthood.8 In fact, several studies have suggested that CVD risk factors could be tracked to various degrees into adulthood.8
10 As a result, many authors have proposed individual detection and intervention on CVD risk factors and behavior throughout childhood and adolescence as a strategy to reduce CVD risk in adulthood.8 In 2012 the National Heart, Lung, and Blood Institute (NHLBI) recommended the

List of abbreviations Apo ARE CETP GSPx HDL IDL LCAT LDL LOOH Lp-(P)LA2 METS OS PLTP PON PS ROS S1P SAA T1D VLDL

apoprotein arylesterase cholesteryl-transfer protein gluthation peroxidase high-density lipoprotein intermediate-density lipoprotein lecithin cholesteryl acyl transferase low-density lipoprotein lipid hydroperoxide lipoprotein-associated phospholipase A2 metabolic syndrome oxidative stress phospholipid transfer protein paraoxonase phosphatidyl serine reactive oxygen species sphingosine 1 phosphate serum amyloid A type 1 diabetes mellitus very low-density lipoprotein

Introduction Cardiovascular disease (CVD) is the leading cause of death globally. In fact, 17.7 million people died from CVD in 2015, which represents 31% of all deaths worldwide. There are several recognized risk factors for CVD, including sedentary lifestyle, smoking, overweight/obesity, hypertension, Type 2 diabetes, and dyslipidemia. In particular, dyslipidemia has been shown to be strongly associated with CVD. This is supported by several epidemiological studies showing either a strong relation between serum total cholesterol and CVD risk, or changes in CVD incidence rate, as a result of interventions aimed at lowering cholesterol

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universal screening of CVD risk factors during childhood.11 Atherosclerosis is a complex process that begins in areas where disturbed blood flow induces partial endothelial activation. Activated endothelial cells produce adhesion molecules, including E-selectin, Pselectin, and vascular cell adhesion molecule-1 (VCAM-1), causing monocytes to attach to the endothelium and move into the arterial intima. Monocytes are then transformed into macrophages and further activated by encounters with pathogen-associated molecular patterns, damage-associated molecular patterns, and various proinflammatory cytokines.12 A key step in the development of the atherosclerotic plaque is the engulfment by endocytosis of LDL particles by macrophages, leading to the formation of foam cells that, together with activated endothelial cells, secrete a host of proinflammatory cytokines, matrix metalloproteinases, and cathepsins, fragilizing the plaque.12 Chemical modification of LDL is necessary to induce the formation of foam cells. Such proatherogenic modifications of LDL include aggregation, enzymatic digestion, and oxidation. LDL oxidation dramatically increases the affinity of the particle for macrophage scavenger receptors, leading to rapid uptake of the modified lipoproteins.13 This process begins when, following transcytosis to the subendothelial space,14 LDL particles are retained in the arterial wall through specific interactions between apolipoprotein (apo) B and arterial proteoglycans.15 Oxidative stress (OS) in the subendothelial space is responsible for the formation of oxidized LDL (oxLDL) via oxidants with enzymatic and nonenzymatic origins.13 Indeed, LDL oxidation is negligible in the circulation because of the presence of multiple antioxidant defense systems that involve antioxidant enzymes, such as superoxide dismutase, catalase, and glutathioneperoxidase, but equally in hydrosoluble antioxidants, such as ascorbate, urate, and bilirubin.16 Additionally, liposoluble vitamin E compounds, mainly α-tocopherol, are transported by LDL particles together with other lipophilic antioxidants, such as ubiquinol-10, and hence protect LDL from oxidation not only in plasma, but also in the arterial wall.17 Exposure to cell-derived oxidants is necessary for the oxidative modification of LDL. These oxidants can be free radicals (one-electron) and nonfree radicals (two-electron) and mainly originate from reactions catalyzed by 12/15-lipoxygenase, myeloperoxidase, nitric oxide synthases, and NADPH oxidases, as well as those mediated by transition metals, heme, and hemoglobin.18 Two-electron oxidants (hypochlorite and peroxynitrite) modify LDL almost instantly, primarily attacking apo B. In contrast, high-density lipoprotein (HDL) constitutes the only lipoprotein fraction with antiatherogenic

properties.19 It has been known for decades that HDL possess the ability to promote cholesterol efflux through transporters or receptors like ATP-binding cassette A1 or scavenger receptor B-I.19 Nevertheless, it is currently understood that HDL particles may exert several other functions, including antioxidant, antiinflammatory, antiapoptotic, antithrombotic, and antiglycative activities.19 In particular, HDL antioxidant activity has received much attention in recent years. This mainly refers to the ability of HDL particles to inhibit LDL oxidation.20 Given the role that LDL oxidation plays in the initiation and progression of atherosclerosis, this property of HDL is considered fundamental for the inhibition and reversion of plaque formation. Several HDL protein components contribute to its antioxidant activity (Table 9.1). These components include apolipoprotein (apo) A-I, apo A-II, apo A-IV, apo C-II, apo C-III, apo D, apo E, apo F, apo J, apo L-I, apo M, glutathione peroxidase 3 (GPX 3), paraoxonase (PON) 1, PON 3, lipoprotein associated phospholipase A2 (Lp-PLA2), lecithin-cholesterol acyl transferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP), among others.19 In addition, some of HDL lipid components are also involved in its antioxidant properties, mainly sphingosine-1-phosphate and phosphatidylserine.20 In particular, PON1, the main HDL-associated hydrolase, has attracted much interest and created significant controversy (Fig. 9.1).

Paraoxonase 1: general characteristics PON1 is a glycoprotein enzyme that is almost exclusively associated with HDL in the circulation. It is mainly secreted by the liver, though local synthesis occurs in several tissues.21 PON1 is capable of hydrolyzing a wide variety of substrates such as lactones, glucuronide drugs, thiolactones, arylesters, cyclic carbonates, organophosphorus pesticides, and nerve gases.22,23 Its name is derived from the ability to hydrolyze the pesticide paraoxon.24 PON1 activity is frequently evaluated employing two different substrates, paraoxon (PON activity) and phenylacetate (arylesterase; ARE, activity). Both measurements are complementary given that PON1 activity better reflects the enzyme antioxidant activity, while ARE activity represents an estimate of its concentration.24 PON1 activity toward some substrates, including paraoxon, is influenced by genetic polymorphisms. The most studied PON1 gene polymorphisms result from amino acid substitutions at positions 192 (Gln-Arg) and 55 (Leu-Met) in the coding region of the gene. Alleles at the 192 (Q and R alleles) loci have been associated

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TABLE 9.1 Major HDL-associated components contributing to its antioxidant activity. Protein Main source

Mechanism of antioxidative activity

Comments

Apo A-I

Liver, small intestine

Reduction of LOOH to redox-inactive LOH, ROS scavenging, PON1 activation

Largely determined by oxidation status of Met residues

Apo A-II

Liver

Reduction of LOOH to redox-inactive LOH

Largely determined by oxidation status of Met residues

Apo A-IV

Intestine

Removal of oxidized lipids from cells and lipoproteins, ROS scavenging, PON1 activation

Primarily in a lipid-free form

Apo D

Brain, testes

Inhibition of lipid peroxidation by reduction of LOOH

Binds to LCAT Favors HDL to LDL association

Apo E

Liver

Inhibition of lipid peroxidation

Binds PON1 Activity is allele specific

Apo F

Liver

Modulator of CETP activity

Apo J

Brain, testes, ovary, liver, pancreas

Removal of oxidation products

Apo L1

Pancreas, lung, prostate, liver, placenta, spleen

Unknown

Apo M Liver, kidney

Unknown

CETP

Enhanced transfer of oxidized lipids between LDL and HDL

Exchanges CE for TG between VLDL, LDL and HDL

GSPx-3 Kidney

Reduction of LOOH

Glutathione-dependent activity

LCAT

Liver

Hydrolysis of short-chain oxidized PLs

PAFAH

Macrophages

Hydrolysis of short-chain oxidized PLs

PLTP

Placenta, pancreas, lung, kidney, heart, liver, muscle, brain

Unknown

PON1

Liver

Hydrolysis of short-chain oxidized PLs

PON3

Liver, kidney

Lactonase activity

PS

Multiple tissues

Sequestering of transition metals

Interacts with PON1

S1P

Multiple tissues

Inhibition of lipid peroxidation

Binds to apo M

SAA

Liver

Binding of LOOH

Liver, adipose tissue

Strong activity toward LOOH

Weak activity toward LOOH?

Apo, apolipoprotein; CETP, cholesteryl-ester transfer protein; GSPx 3, glutathione selenoperoxidase 3; LCAT, lecithin cholesteryl-acyl transferase; PAF-AH, platelet activating factor acetyl hydrolase; PLTP, phospholipid transfer protein; PON, paraoxonase; PS, Phosphatidylserine; S1P, sphingosine 1 phosphate; SAA, serum amyloid A; ROS, reactive oxygen species; LOOH, lipid hydroperoxide; LOH, lipid hydroxide; PL, phospholipids; CE, cholesteryl ester; TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein. Reference: A. Kontush, Chapman, M. High-density lipoproteins: structure, metabolism, function and therapeutics. John Wiley and Sons, Hoboken, 2012.

with enzyme activity. The RR-genotype exhibits a high paraoxonase activity (high activity phenotype), while the QQ-genotype exhibits low paraoxonase activity (low activity phenotype).25 On the other hand, alleles at the 55 (L and M alleles) position have been linked to enzyme concentration. Indeed, individuals of the LL genotype display the highest PON1 concentration.26 Traditionally, hydrolysis of oxidized phospholipids (oxPLs) has been considered to represent the major function of this enzyme. This activity involves hydrolysis of phosphatidylcholine-based oxPL and generation of bioactive lysophosphatidylcholine.22 Furthermore, several studies have provided evidence

linking PON1 to atherosclerosis. In vitro experiments first showed that the PON1-containing fraction of HDL was the most effective at preventing the accumulation of lipid peroxides on LDL.27 Animal models have provided further evidence for the antioxidative role of PON1. Knockout mice, which lacked PON1, presented impaired antioxidative activity in HDL, in addition to increased susceptibility to organophosphate poisoning and atherosclerosis.28 Consistent with this finding, mice over-expressing human PON1 showed reduced generation of reactive oxygen species (ROS) and decreased foam-cell formation.29 Different epidemiological studies have reported an inverse association

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FIGURE 9.1 The atherogenic process. Atherogenesis begins when low-density lipoprotein (LDL) particles cross to the subendothelial space becoming oxidized. Oxidized LDLs activate endothelial cells causing the secretion of adhesion molecules and monocyte chemotactic protein 1 (MCP-1), leading to the recruitment of monocytes that differentiate to macrophages in the intima. Macrophages secrete a host of proinflammatory cytokines that lead to further recruitment of monocytes. Additionally, activated macrophages recognize and engulf oxidized LDL, transforming into foam cells. Finally, foam cells together with activated endothelial cells secrete growth factors and metalloproteinases that degrade extracellular matrix and fragilize the atherogenic plaque.

between serum PON1 activity and coronary events.30 In particular, the largest single cohort study to date (Cleveland Clinic GeneBank study) involving 3668 patients following coronary angiography revealed a greater than twofold risk of new cardiovascular events in the lowest quartile of serum PON1 activity compared with the highest one.31 Regarding genetic evidence, studies have shown that PON1 192QQ homozygotes display better protection from LDL oxidation.32 Nevertheless, other studies have questioned the relevance of PON1 as an antioxidant enzyme. In this regard, PON1 failed to hydrolyze oxPLs in several in vitro experiments.33 Moreover, PON1 is weakly reactive toward lipid hydroperoxides (LOOH).34 Indeed, Kersonsky et al., suggested that lactones and not oxPLs constitute physiological substrates of PON1.23 An interesting possibility derives from a recent work by Huang et al.,35 who proposed that PON1 can form a tertiary complex with HDL and myeloperoxidase that can modulate the activity of the latter. However, it is worth noting that it is not PON1 antiatherogenic function that is argued, but whether it plays this role by preventing lipid oxidation. In fact, PON1 is involved in several HDL atheroprotective properties (Table 9.2). As a consequence, PON1 remains a target of interest in studies exploring the association between HDL function and CVD (Fig. 9.2).

Paraoxonase 1 in pediatric populations Evidence regarding PON1 in pediatric populations is scarce. Nonetheless, a few studies have explored PON1 status in children and adolescents, particularly in those with cardiometabolic risk, which is intimately associated with OS. Agirbasli et al.,36 found that body mass index (BMI) was the main determinant of PON activity in children who presented either obesity or insulin resistance. Moreover, Krzystek-Korpacka et al.,37 reported lower ARE activity in obese children as a result of OS and inflammation. In this regard, Cayir et al.,38 reported both lower PON and ARE activities in obese children. Interestingly, these alterations were reversed after 6-month treatment with metformin. Similarly, Adhe-Rojekar et al.,39 found that ARE and lactonase activities were significantly reduced in adolescents with metabolic syndrome (METS). Consistently, Ferre et al.,40 observed lower lactonase activity in obese children with METS compared to obese children without METS and healthy controls. In agreement, Koncsos et al.,41 found that PON and ARE activities were lower in obese children, apart from being positively associated with adiponectin and negatively with leptin levels. Accordingly, Alegrı´a-Torres et al., suggested that PON1 Q192R polymorphism was a marker for insulin resistance in children.42 Moreover, Rupe´rez et al.,43 observed

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TABLE 9.2 PON1 antiatherogenic functions. Function

Mechanism

Reverse cholesterol transport

Promotion of macrophage cholesterol efflux, binding and upregulation of ABCA-1, binding, and upregulation of SR-BI.

Antioxidant activity

Prevention of cell membrane oxidation, inhibition of lipoperoxide accumulation in LDL, reduction of macrophage oxidative status, prevention of LCAT oxidative inactivation.

Antiinflammatory activity

Reduction of biological effects of oxLDL, prevention of monocyte-macrophage differentiation, inhibition of macrophage cytokine synthesis, ROS production, and phagocytic activity.

Antiapoptotic activity

Protection of endothelial cells from oxLDL-induced apoptosis and removal of toxic apoptotic products.

Antiglycative activity

Inhibition of LDL glycation, prevention of diabetes development and preservation of β-cell insulin secretion.

Antithrombotic activity Preservation of endothelial function, stimulation of eNOS-dependent NO production. PON1, paraoxonase 1; ABCA1, ATP binding cassette A1; SR-BI, scavenger receptor BI; LDL, low density lipoprotein; LCAT, lecithin-cholesteryl acyl transferase; ox-LDL, oxidized LDL; ROS, reactive oxygen species; eNOS, endothelial nitric oxide synthase; NO, nitric oxide. Reference: M. Mackness, B. Mackness. Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles. Gene. 567, 12
21.

FIGURE 9.2 Paraoxonase 1 and its multiple activities and substrates. Paraoxonase 1 (PON1) is capable of hydrolyzing multiple substrates. The figure shows PON1 activity towards in vitro substrates, such as organophosphorus compounds and arylesters, and in vivo substrates, like oxidized phospholipids and lactones.

that intronic PON1 SNP rs854566 was negatively associated with childhood obesity and positively associated with PON activity. Furthermore, Roest et al.,44 reported that the L-variant of the L55M SNP was associated with carotid artery intima-media thickness in children with familiar hypercholesterolemia. In another study, Craciun et al.,45 observed lower lactonase activity in adolescents with type 1 diabetes mellitus (T1D). Furthermore, Fekih et al.,46 proposed both L55M and Q192R PON1 polymorphisms as genetic markers for the development of nephropathy in children and adolescents with T1D. Moreover, Kordonouri et al.,47

and Kao et al.,48 found a direct association between the L/L genotype of PON1 L55M polymorphism and the development of retinopathy in adolescents with T1D. Nevertheless, other studies contradict these findings. In a recent study, Ramı´rez-Jime´nez et al.,49 observed no differences in either PON or ARE activities between obese, overweight, and healthy children. Furthermore, they found L55M and Q192R PON1 polymorphisms to be similarly distributed between the groups. Moreover, Eren et al.,50 reported that obese children and children with METS had higher PON activity compared with healthy children.

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In addition to metabolic diseases, PON1 status in children has been studied in other pathologies and conditions that involve OS. Ayar et al.,51 found lower ARE activity in children with sepsis and proposed ARE as a potent biomarker in this critical situation. Similarly, Hashemi et al.,52 reported that children in the active phase of nephrotic syndrome displayed lower PON and ARE activities than children in the remission phase. Furthermore, Kahraman et al.,53 found that PON activity was lower in children and adolescents who were exposed to passive smoking and presented high levels of OS. Similarly, Ece et al.,54 observed lower PON activity and high levels of OS in malnourished children. Moreover, Andersen et al.,55 found that children prenatally exposed to pesticides who possessed the PON1 192R-allele presented an adverse cardiovascular risk profile at school age. This profile was characterized by higher waist circumference, body fat content, BMI Z-scores, blood pressure, and serum concentrations of leptin and insulin growth factor (IGF)-I. None of these alterations were present in children with the PON1 192QQ genotype who were also prenatally exposed to pesticides. It is worth noting that, in direct contradiction with these findings, Huen et al.,56 reported that PON1192QQ children had a 9.3-fold (at age two) or 2.5-fold (at age five) higher probability of being obese compared with PON1192RR children, although none of these children were exposed to pesticides. In a study performed by our group,57 we found that children living in San Antonio de los Cobres (SAC), located 3700 m above sea level, presented lower PON activity than children who lived at sea level. High altitude is associated with hypobaric hypoxia and OS, which could explain these findings. Interestingly, SAC children also showed higher ARE activity and apo A-I levels, which appears to be associated to high altitude. Similarly, in another study (unpublished data), we observed that SAC children also displayed lower PON activity than children from Chicoana, located at 1400 m above sea level. These results highlight the importance of PON 1 status in children subjected to different conditions associated with OS. Nevertheless, this relationship between OS and PON1 activities was not confirmed in another study performed by our group58 in which we found no differences in either PON or ARE activities in children and adolescents who were classified according to the intensity of their regular physical activity regularly.

Conclusion The link between PON1 and CVD risk has been known for years. Multiple studies have shed light on

the role that this enzyme could potentially play in the prevention of atherogenesis and its consequences. Several mechanisms have been proposed to explain PON1 function. These mechanisms go beyond the traditionally suggested hydrolysis of oxPLs. Indeed, PON1 seems to play an important role in multiple HDL atheroprotective properties, besides its antioxidant activity. Nevertheless, despite the abundant literature on this topic, the evidence regarding pediatric populations remains scarce. In this chapter, we have summarized the studies that have analyzed PON1 status in children. These studies highlight the intimate association between PON1 status and OS, which can begin in childhood and continue into adulthood. Furthermore, some authors have proposed PON1 polymorphisms to be risk factors for different complications associated with cardiometabolic diseases. All these alterations can increase the risk of premature atherosclerosis. Further longitudinal studies should explore the mechanisms that could link PON1 alterations in childhood to cardiometabolic diseases in early adulthood.

Applications to other areas of pathology It is well-known that PON1 status plays a remarkable role in the progression of pathologies characterized by OS in pediatric populations. Moreover, PON1 has been identified as an important factor in the evolution of different pathologies in adults. Epidemiological studies have reported an inverse association between serum PON1 activity and coronary events.30,31 In fact, genetic studies have shown that homozygotes for the 192QQ genotype, which possesses the highest PON1 activity, display better protection to LDL from oxidation and, in consequence, lower CVD risk.32 Furthermore, PON1 has also been proposed as a relevant factor in other pathologies such as neurodegenerative diseases. In particular, Castellazzi et al., found that low-ARE activity was a common denominator in mild cognitive impairment, late-onset Alzheimer’s disease, vascular dementia, mixed dementia, and multiple sclerosis.59 Alterations in PON1 activity are also present in different types of cancer. Both PON and ARE activities have been reported to be lower in breast, ovarian, endometrial, colorectal, gastric, esophagic, stomach, oral, laryngeal, thyroid, and lung cancers.60 Finally, ARE activity, but not PON activity, was decreased in bladder cancer and acute myeloid leukemia.60 These studies shed light on the role that PON1 plays in different pathologies beyond its well-known association to CVD risk.

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References

Summary points • PON 1 is an antioxidant enzyme mostly associated in plasma to HDL. • PON1 is mainly synthesized by the liver, although synthesis in different tissues also occurs. • PON1 is capable of hydrolyzing different substrates both in vivo (lactones and oxidized phospholipids) and in vitro (organophosphorus compounds and arylesters). • PON1 activity toward some substrates is influenced by different polymorphisms, the most studied of which is 192 (Gln-Arg). • The PON1-containing fraction of HDL is the most effective at preventing the accumulation of lipid peroxides on LDL. • PON1 activity is altered in children suffering from pathologies and conditions associated to OS.

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77. 21. Mackness B, Durrington PN, Mackness MI. Human serum paraoxonase. Gen Pharmacol 1998;31:329
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6. 23. Khersonsky O, Tawfik DS. Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 2005;44:6371
82. 24. Nevin DN, Zambon A, Furlong CE, Richter RJ, Humbert R, Hokanson JE, et al. Paraoxonase genotypes, lipoprotein lipase activity, and HDL. Arterioscler Thromb Vasc Biol 1996;16:1243
9. 25. Humbert R, Adler DA, Disteche CM, Hassett C, Omiecinski CJ, Furlong CE. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet 1993;3:73
6. 26. Garin MC, James RW, Dussoix P, Blanche´ H, Passa P, Froguel P, et al. Paraoxonase polymorphism Met-Leu54 is associated with modified serum concentrations of the enzyme. A possible link between the paraoxonase gene and increased risk of cardiovascular disease in diabetes. J Clin Invest 1996;99:62
6. 27. Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 1991;286:152
4. 28. Ng DS, Chu T, Esposito B, Hui P, Connelly PW, Gross PL. Paraoxonase-1 deficiency in mice predisposes to vascular inflammation, oxidative stress, and thrombogenicity in the absence of hyperlipidemia. Cardiovasc Pathol 2008;17:226
32. 29. She ZG, Zheng W, Wei YS, Chen HZ, Wang AB, Li HL, et al. Human paraoxonase gene cluster transgenic overexpression represses atherogenesis and promotes atherosclerotic plaque stability in ApoE-null mice. Circ Res 2009;104:1160
8. 30. Wang M, Lang X, Cui S, Zou L, Cao J, Wang S, et al. Quantitative assessment of the influence of paraoxonase 1 activity and coronary heart disease risk. DNA Cell Biol 2012;31:975
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arylesterase activities with cardiovascular risk. Arterioscler Thromb Vasc Biol 2012;32:2803
12. Bhattacharyya T, Nicholls SJ, Topol EJ, Zhang R, Yang X, Schmitt D, et al. Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk. JAMA 2008;299:1265
76. Connelly PW, Draganov D, Maguire GF. Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite. Free Radic Biol Med 2005;38:164
74. Teiber JF, Draganov DI, La Du BN. Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH. J Lipid Res 2004;45:2260
8. Huang Y, Wu Z, Riwanto M, Gao S, Levison BS, Gu X, et al. Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex. J Clin Invest 2013;123:3815
28. Agirbasli M, Tanrikulu A, Erkus E, Azizy M, Sevim BA, Kaya Z, et al. Serum paraoxonase-1 activity in children: the effects of obesity and insulin resistance. Acta Cardiol 2014;69:679
85. ´ Krzystek-Korpacka M, Patryn E, Hotowy K, Czapinska E, Majda J, Kustrzeba-Wo´jcicka I, et al. Paraoxonase (PON)-1 activity in overweight and obese children and adolescents: associationwith obesity-related inflammation and oxidative stress. Adv Clin Exp Med 2013;22:229
36. ˙ Gurbuz F, Kurt N, Yildirim A. The effect of C ¸ ayır A, Turan MI, lifestyle change and metformin therapy on serum arylesterase and paraoxonase activity in obese children. J Pediatr Endocrinol Metab 2015;28:551
6. Adhe-Rojekar A, Mogarekar MR, Rojekar MV. Paraoxonase activity in metabolic syndrome in children and adolescents. Casp J Intern Med 2018;9:116
20. Ferre´ N, Feliu A, Garcı´a-Heredia A, Marsillach J, Parı´s N, Zaragoza-Jordana M, et al. Impaired paraoxonase-1 status in obese children. Relationships with insulin resistance and metabolic syndrome. Clin Biochem 2013;46:1830
6. Koncsos P, Seres I, Harangi M, Illye´s I, Jo´zsa L, Go¨nczi F, et al. Human paraoxonase-1 activity in childhood obesity and its relation to leptin and adiponectin levels. Pediatr Res 2010;67:309
13. Alegrı´a-Torres JA, Garcı´a-Domı´nguez ML, Cruz M, AradillasGarcı´a C. Q192R polymorphism of paraoxonase 1 gene associated with insulin resistance in Mexican children. Arch Med Res 2015;46:78
83. Rupe´rez AI, Lo´pez-Guarnido O, Gil F, Olza J, Gil-Campos M, Leis R, et al. Paraoxonase 1 activities and genetic variation in childhood obesity. Br J Nutr 2013;110:1639
47. Roest M, Rodenburg J, Wiegman A, Kastelein JJ, Voorbij HA. Paraoxonase genotype and carotid intima-media thickness in children with familial hypercholesterolemia. Eur J Cardiovasc Prev Rehabil 2006;13:464
6. Craciun EC, Leucuta DC, Rusu RL, David BA, Cret V, Dronca E. Paraoxonase-1 activities in children and adolescents with type 1 diabetes mellitus. Acta Biochim Pol 2016;63:511
15. Fekih O, Triki S, Rejeb J, Neffati F, Douki W, Ommezzine A, et al. Paraoxonase 1 polymorphisms (L55M and Q192R) as a genetic marker of diabetic nephropathy in youth with type 1 diabetes. Endokrynol Pol 2017;68:35
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47. Kordonouri O, James RW, Bennetts B, Chan A, Kao YL, Danne T, et al. Modulation by blood glucose levels of activity and concentration of paraoxonase in young patients with type 1 diabetes mellitus. Metabolism 2001;50:657
60. 48. Kao Y, Donaghue KC, Chan A, Bennetts BH, Knight J, Silink M. Paraoxonase gene cluster is a genetic marker for early microvascular complications in type 1 diabetes. Diabet Med 2002;19:212
15. 49. Ramı´rez-Jime´nez R, Martı´nez-Salazar MF, Almenares-Lo´pez D, Ya´n˜ez-Estrada L, Monroy-Noyola A. Relationship between paraoxonase-1 and butyrylcholinesterase activities and nutritional status in mexican children. Metab Syndr Relat Disord 2018;16:90
6. 50. Eren E, Abuhandan M, Solmaz A, Ta¸skın A. Serum paraoxonase/arylesterase activity and oxidative stress status in children with metabolic syndrome. J Clin Res Pediatr Endocrinol 2014;6:163
8. ¨ . Effects of paraoxonase, 51. Ayar G, Atmaca YM, Alı¸sık M, Erel O arylesterase, ceruloplasmin, catalase, and myeloperoxidase activities on prognosis in pediatric patients with sepsis. Clin Biochem 2017;50:414
17. 52. Hashemi M, Sadeghi-Bojd S, Raeisi M, Moazeni-Roodi A. Evaluation of paraoxonase activity in children with nephrotic syndrome. Nephrourol Mon 2013;5:978
82. ¨ zer O ¨ F. ˘ 53. Kahraman FU, Torun E, Osmanoglu NK, Oruc¸lu S, O Serum oxidative stress parameters and paraoxonase-1 in children and adolescents exposed to passive smoking. Pediatr Int 2017;59:68
73. 54. Ece A, Gu¨rkan F, Celik F, Bo¸snak M, Yel S, Balik H, et al. Paraoxonase, total antioxidant activity and peroxide levels in marasmic children: relationships with leptin. Clin Biochem 2007;40:634
9. 55. Andersen HR, Wohlfahrt-Veje C, Dalga˚rd C, Christiansen L, Main KM, Nellemann C, et al. Paraoxonase 1 polymorphism and prenatal pesticide exposure associated with adverse cardiovascular risk profiles at school age. PLoS One 2012;7:e36830. 56. Huen K, Harley K, Beckman K, Eskenazi B, Holland N. Associations of PON1 and genetic ancestry with obesity in early childhood. PLoS One 2013;8:e62565. 57. Hirschler V, Martı´n M, Oestreicher K, Molinari C, Tetzlaff W, Botta E, et al. Activity of the antioxidant enzyme paraoxonase in Argentinean children living at high altitude. Redox Rep 2018;23:35
40. 58. Verona J, Gilligan LE, Gime´nez C, Verona MF, Lombardo SM, Baenz A, et al. Physical activity and cardiometabolic risk in male children and adolescents: the Balcarce study. Life Sci 2013;93:64
8. 59. Castellazzi M, Trentini A, Romani A, Valacchi G, Bellini T, Bonaccorsi G, et al. Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of neuroinflammatory and neurodegenerative diseases. Int J Biochem Cell Biol 2016;81:356
63. 60. Arenas M, Rodrı´guez E, Sahebkar A, Sabater S, Rizo D, Pallise´ O, et al. Paraoxonase-1 activity in patients with cancer: a systematic review and meta-analysis. Crit Rev Oncol Hematol 2018;127:6
14.

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C H A P T E R

10 The regulation of intracellular redox homeostasis in cancer progression and its therapy Pritam Sadhukhan and Parames C. Sil Division of Molecular Medicine, Bose Institute, Kolkata, India

List of abbreviations AKT/PKB ASK1 Bax Bcl2 Bim CAT EGFR ERK FOXO GCLC GPx GRIM19 HIFs ICAM-1 IKK IL iNOS JNK Keap1 KRAS MAPK MMPs mTOR NADPH NF‑κB NOS NOX Nrf2 OS PAHs PDC-E2 PI3K RNS ROMO1 ROS

protein kinase B apoptosis signal-regulating kinase 1 Bcl2 associated X protein B-cell lymphoma 2 Bcl2-like protein 11 catalase epidermal growth factor receptor extracellular signal-regulated kinases forkhead box protein O glutamate-cysteine ligase catalytic subunit glutathione peroxidase gene associated with retinoid-IFN-induced mortality 19 hypoxia-inducible factors intercellular adhesion molecule 1 IκB kinase interleukins inducible nitric oxide synthase c-Jun N-terminal kinases kelch-like ECH-associated protein 1 kirsten rat sarcoma viral oncogene homolog mitogen activated protein kinase matrix metalloproteinases mammalian target of rapamycin nicotinamide adenine dinucleotide phosphate hydrogen nuclear factor kappa B nitric oxide synthase NADPH oxidase nuclear factor (erythroid-derived 2)-like 2 oxidative stress poly aromatic hydrocarbons pyruvate dehydrogenase multienzyme complex phosphatidylinositol 3-kinase reactive nitrogen species reactive oxygen species modulator 1 reactive oxygen species

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00010-X

SHC-1 SOD STAT proteins VCAM-1 VEGF

src homology 2 domain containing transforming protein 1 superoxide dismutase signal transducer and activator of transcription vascular cell adhesion protein 1 vascular endothelial growth factor

Introduction Numerous catabolic and anabolic reactions take place in a living system to produce energy and maintain normal cellular homeostasis. The driving forces for these biochemical reactions are several intracellular free radicals. These free radicals are produced as a byproduct of these biochemical reactions involving oxygen and localized in various cellular compartments, such as mitochondria, endoplasmic reticulum, and peroxisomes. Mitochondrion, being the most reactive cellular organelle, is regarded as the major source of reactive radicals. These biochemical reactions facilitate the metabolism and growth of a living system. Oxygen-containing free radicals present in the living system are collectively termed reactive oxygen species (ROS) because of the presence of oxygen moiety.1 Intracellular ROS comprised mainly of superoxide (O2 2 ) and hydrogen peroxide (H2O2). Besides these, singlet oxygen, ozone (O3), hydroxyl radical (HO•), peroxyl radicals (LOO•), and organic peroxides (ROOR0 ) also act as ROS. Several reports suggest that in mitochondria, approximately 2% of the consumed oxygen is converted into superoxides through redox reactions.2 Different intracellular ROS exhibit contrasting biological effects depending on the cellular

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localization, concentration, and antioxidant status of the cells as ROS can both facilitate cellular proliferation and death by differentially regulating the associated signaling cascades. Peroxisomes play a critical role in the production and removal of ROS through different metabolic reactions. The oxidizing environment of the endoplasmic reticulum facilitates protein folding through the formation of disulfide bonds and can also elicit intracellular ROS levels through the process of protein oxidation.3,4 These reactive species are mostly unstable, with a short half-life. They mainly act as secondary messengers in different signaling cascades and regulate almost every important life process. Apart from ROS, reactive nitrogen species (RNS) are also present in different cellular compartments, and play significant roles in normal cellular functioning.5 In the intracellular environment, ROS can be generated by different enzymatic and nonenzymatic processes. Enzyme-catalyzed reactions are carried out by different respiratory enzymes such as NADPH oxidase, endothelial nitric oxide synthase (eNOS), arachidonic acid, xanthine oxidase, and various metabolic enzymes such as the lipoxygenase, cyclooxygenase, and cytochrome P450 enzymes, and play a major role in the production of intracellular ROS.6 Apart from enzyme-mediated ROS production, reactions of the electron transport chain are a major source of ROS produced in a nonenzymatic manner.7 The level of intracellular ROS can induce several metabolic functions. Therefore the regulation of ROS production and removal is critical for cellular homeostasis. A very low to moderate level of intracellular ROS (specifically H2O2) is beneficial for cellular homeostasis as it can facilitate the progression of several cellular

processes such as proliferation, differentiation, and migration (Fig. 10.1). Apart from this, ROS can also regulate the production of different proinflammatory cytokines (such as IL-1, IL-6, IL-8 and IL-10) by regulating the stress-responsive transcription factor, nuclear factorκB (NF‑κB), and mediated signaling pathway.8
10 However, during cellular stress, ROS accumulate in different cellular compartments and cause damage to cellular biomolecules like protein, lipids, carbohydrates, and nucleic acids11 (Fig. 10.2). To attenuate the harmful effect of ROS, endogenous enzymatic and nonenzymatic ROS scavenging mechanisms exist in different intracellular compartments (and will be discussed in following sections) for tight regulation of intracellular ROS.12,13

Sources of intracellular ROS There are many reasons for the production of intracellular ROS, ranging from normal cellular metabolism to environmental pollution. The reasons behind ROS exposure are broadly classified as (1) endogenous sources or (2) exogenous sources. These are elaborately described in the following sections (Fig. 10.3). 1. Endogenous sources In various biochemical reactions, the activity of different enzymes such as NADPH oxidases, xanthine oxidase, cyclooxygenase, lipoxygenase, and other free ions (such as iron and copper) contribute to the production of ROS. Functioning of the mitochondrial electron transport chain mostly contributes to the intracellular generation of superoxide. Peroxisomes, lysosomes, endoplasmic

FIGURE 10.1 Effect of free radical accumulation on biomolecules. A schematic diagram representing the damaging effect of free radical accumulation in different cellular compartments. Accumulation of free radicals lead to damage the intracellular biomolecules such as proteins, carbohydrates, nucleic acids, and lipids. It causes structural and functional changes in biomolecules and impairs several signaling cascades.

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several mitochondrial proteins such as mTOR, Bcl2, SHC-1, ROMO1, NOS, and various uncoupling proteins are regulated for the production of ROS and to maintain normal cellular proliferation rate, growth, differentiation, and function.14 Different transcription factors such as HNF-α, Nrf2, and others control the uptake and storage of extracellular metal ions and other xenobiotics from the environment or neighboring cells via connexin channels.15
17

Regulation in catabolism of intracellular ROS FIGURE 10.2 Effect of intracellular ROS content on cellular homeostasis. A diagramatic representation of the intracellular ROS level on different cellular functions and their fate.

reticulum, and other cellular organelles that require oxygen for their activity also contribute to the intracellular ROS pool. Apart from these, the cellular activity involving immune responses like the activation of macrophages and production of cytokines generate different reactive radicals, including superoxide, nitric oxide, hydroxyl radical, and hydrogen peroxide.6 2. Exogenous sources Apart from several endogenous ROS inducers, there exist a number of ways of ROS generation in which certain external sources including environmental pollution and drug exposure play a major role. Ultraviolet radiation, polycyclic aromatic hydrocarbons (PAHs), and γ-irradiation are the major exogenous source of intracellular ROS. Therapeutic ingestion of different drugs like cisplatin, doxorubicin, paclitaxel, metformin, atorvastatin, and others also contribute to the elevation of intracellular ROS. Exposure to different exogenous ROS inducer reacts with different intracellular biomolecules leading the genotoxicity and several other pathophysiological conditions including cancer.6

Regulation of intracellular ROS production To regulate intracellular ROS levels, different endogenous pathways are present in mammalian cells. Since NADP oxidase (NOXs) are a major source of ROS, there are several location specific checkpoints to regulate the production of ROS. The activity of the apoprotein, Ca21 signaling and activity of small G proteins are tightly regulated to restrict the activity of NOXs and production of ROS. Mitochondria is another potential source of endogenous ROS. The activity of

Until a few decades ago, superoxide dismutase, catalase, and other enzymes of glutathione metabolism were thought to be the only way for the endogenous elimination of intracellular ROS (Fig. 10.3). These enzymes can directly or indirectly react with specific ROS and give rise to stable unreactive molecules. Recent research reports have identified several other antioxidant enzymes such as thioredoxins, peroxynitrite reductases, thioredoxin reductases, methionine sulfoxide reductase, and others.18,19 These enzymes largely maintain the level of reduced glutathione. Under oxidative stress (OS) conditions, the activity of glycolytic enzyme pyruvate kinase gets attenuated (by oxidation at Cys358) and inhibits the further production of ROS through the catabolic reactions of glucose. This leads to the reduction of GSSG to normalize the cellular level of ROS and increases the production of NADPH.20 Intracellular ROS level can also be regulated by exogenous administration of several antioxidant molecules. These molecules can directly scavenge ROS or activate several signaling pathways to attenuate the effect of increasing ROS level.21,22

Role of oxidative stress in carcinogenesis The role of intracellular ROS in different cellular processes is understood from several research reports, but the role of ROS in the development of tumor or carcinogenesis is still not clear. It has been shown that administration of insulin results in the accumulation of intracellular ROS and increased proliferation of tumor cells. To cope up with the high proliferation efficiency, cancer cells are transformed with the capability of high ATP catabolism, which leads to the increase in production and accumulation of ROS. Different reports suggested dual roles of ROS depending on the environment, in that at times they act as tumor suppressers, but can also act as oncogenes. At low to moderate levels, ROS can activate the mitogen activated protein kinase (MAPK) dependent signaling cascade

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10. The regulation of intracellular redox homeostasis in cancer progression and its therapy

FIGURE 10.3 Different sources of ROS and its intracellular fate. A schematic representation of different exogenous and endogenous sources of ROS and its effect on cellular fate upon accumulation. The panel on the right represents the most predominant intracelular antioxidant machinary and its functioning.

by regulating the activity of ERK, JNK, and other cell cycle regulatory proteins. Thus ROS can promote cell survival and proliferation. ROS-mediated modulation of the MAPK signaling cascade also facilitates tumor initiation and progression. Accumulation of ROS has been shown to facilitate tumor progression through the modulation of the tumor suppressor proteins PTPs and PTEN.23 PTPs are also reported to upregulate the expression level of several antioxidant genes and facilitate cancer cells to sustain the increased oxidative load, thereby facilitating their proliferation and progression. High levels of ROS are also associated with the metastasis of cancer cells as ROS induces the detachment of cells from the matrix. Therefore high levels of intracellular ROS is regraded to have a critical role in each step of carcinogenesis, starting from initiation to migration.24 Different studies on cellular mutation also suggest the occurrence of chronic inflammatory diseases (such as colitis and gastric ulcer) associated to the induction of OS and are also a critical regulatory factor in the initiation of cellular transformation.25 Cumulatively, it can be concluded that ROS can affect the cellular metabolism at a very primary level by affecting the functions of different biomolecules. Specifically, it has genotoxic effects on the nuclear material. ROS can also modulate different regulators of key signaling pathways, thereby activating oncogenes and inactivating tumor suppressors. Therefore ROS can be regarded as a potential cause for the development of cancer progression (Fig. 10.4). The detailed

effect of ROS in cancer initiation and progression are discussed in the following sections.

Modulation of mitogen activated protein kinase Mitogen activated protein kinase (MAPK) family proteins are of the most important regulatory proteins that promote cell survival and drive the progression of cancer. Some of the important proteins of this group are ERK1/2, p38 MAPK, and JNK. MAPK family proteins are activated by redox signaling through activity of upstream kinases (such as MAPKKK, MAPKK, and MAPK) and are inactivated by specific phosphatases.26 MAPK family proteins possess dual functionality depending on the stimulus and cellular localization. In the presence of ROS, ERK1/2 promotes the survival of cancer cells, whereas activation of JNK promotes autophagy and apoptosis. Under persistent ROS insult, overexpression of receptor tyrosine kinase and oncogenic mutation in the RAS gene cause constitutive activation of ERK1/2 in the cytoplasm.27 This leads to the activation of cytoprotective pathways facilitating the progression of cancer. Furthermore, ROS can activate EGFR, leading to nuclear translocation of ERK1/2, which facilitates the proliferation of cancer cells. An oxidative environment leads to ASK1 dependent activation of p38 MAPK and can attenuate the growth stimulatory effects of ERK1/2, but promote the dormancy of the cancer cells.28 A number of research reports confirmed the downregulation of p38 MAPK in cancer pathology.

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Role of oxidative stress in carcinogenesis

FIGURE 10.4

109

Role of ROS in cancer development. A diagramatic representation of the effects of ROS in cancer initiation and

progression.

Modulation of Keap1-Nrf2 pathway The Keap1-Nrf2 mediated signaling cascade is critical to maintain intracellular redox balance. Recent reports suggest that Nrf2 possess dual roles in cancer. At initial stage of cancer, Nrf2 expression ameliorate the damaging effect of intracellular ROS accumulation and, thus, can attenuate the initiation of cancer-cell transformation.29 It acts as a transcription factor and can stimulate several cytoprotective signaling cascades to detoxify different carcinogens and other mutagenic molecules. To the contrary, in the later stage of the disease, Nrf2 facilitates cancer cells to survive against the high oxidative load and to escape the cellular death, thereby promoting pathways by activating different metabolic and cytoprotective genes.30 During the initial stage of cancer development, ROS-induced hyperactivation of ERK mediate growth promoting signaling pathways and downregulate the expression of Nrf2. Thus it facilitates the accumulation of intracellular ROS and induces tumor formation.31 On the other hand, researchers investigating the regulation Nrf2 have shown constitutive expression of Nrf2 in diseased tissue. In cancer tissues, Nrf2 overexpression has been found to be associated with the somatic mutation of the Nrf2 and Keap1, downregulation of Keap1 expression, miRN- mediated regulation of these two proteins, and oncogenic activation of Nrf2 genes. Activation of Nrf2 is also found to be associated with the stimulation of chemoresistance by modulating the activity of SOD, HO-1, GCLC, and GSH.29

Modulation of FOXO transcription factors and p53 The transcription factors in the FOXO family and the tumor suppressor p53 play critical roles in the

downstream MAPK signaling cascade by primarily regulating the progression of cell cycle and proapoptotic pathways. These molecules can induce apoptosis by activating both caspase-dependent intrinsic apoptotic pathways and TNFα-FasL
mediated extrinsic apoptotic pathways.32 Apart from these, FOXO and p53 molecules can upregulate several antioxidant genes such as SOD, CAT, and GPx in response to OS stimuli. The two transcription factors are downregulated (through phosphorylation) by the accumulation of intracellular ROS and activity of PI3K-mediated signaling pathways, thus promoting the proliferation of the cells. The IKK/ NFκB molecules induce phosphorylation of the FOXO proteins, thereby promoting its nuclear exclusion and proteasomal degradation. FOXO family proteins can stimulate cancer-cell survival by facilitating the regulatory genes of migration and invasion. At the same time, oncogenic mutation in the p53 protein induces the loss of the wild type p53 molecule and dysregulate the homeostasis between cell cycle and apoptosis regulatory proteins p21, Bax, Bcl2, and Bim.33 The crosstalk between p53 and FOXO family proteins is critical for cell survival as well as death. FOXO family proteins can significantly regulate the p53-induced activation of IGF-1 mediated signaling pathways determining the fate of the cell. p53 can also regulate the posttranslational activation of different mammalian FOXO family proteins.34,35

Modulation of NF-κB NF-κB is a transcription factor in the cytoplasm that translocates to the nucleus in an activated form. It can regulate the expression of more than 200 genes, mainly regulating the production of cytokines, cell adhesion

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molecules, and cell survival and death. Occurrence of chronic inflammation has a direct relationship with carcinogenesis and since NF-κB is the chief regulator for the production of cytokines, researchers have predicted a direct relationship of NF-κB with carcinogenesis. Under OS conditions, NF-κB can be activated by thioredoxin in an ROS-dependent manner.36 In mammalian cells, NF-κB can stimulate tumorigenesis by regulating several oncogenes. NF-κB can promote cellular transformation by regulating c-myc and stimulate drug resistance by activating MDR-1. NF-κB can also regulate the activity of several antiapoptotic genes (i.e., Bcl2, XIAP, survivin) and other cell cycle regulatory proteins such as cyclin D.37,38 It can promote migration and invasion in the tumor environment by regulating the expression of E-selectin, VCAM-1, ICAM-1, iNOS, and MMPs. In the presence of IL-1, NF-κB can also regulate HIF-1α and VEGF by modulating the activity of COX-2, thereby facilitating the survival of cancer cells under stressed conditions.39

Modulation of STAT family proteins STAT family proteins such as STAT 3 and STAT 5 are one of the major regulators of cell survival and proliferation. Several reports showed constitutive expression of STAT3/5 in different types of cancer. These transcription factors are the regulators of different oncogenes and can stimulate several signaling cascades in the nucleus and cytoplasm. Accumulation of cytoplasmic ROS is directly linked with the activation of STAT family proteins and other proteins (such as JAK) in the upstream. Moreover, STAT family proteins can interfere with the intracellular oxidative

metabolism.40 Activated STAT proteins can modulate the activity of NOXs and other mitochondrial enzymes (i.e., GRIM19, PDC-E2) involved in the ETC, thereby stimulating the accumulation of ROS in the cytoplasm. In addition, STAT 3 can elevate intracellular ROS level by modulating Rac1, thereby promoting DNA damage, mutagenesis, and genomic instability. To the contrary, other reports also suggest that unphosphorylated STAT 5 has ROS metabolizing ability in cancer cells. Activated STAT family proteins are also known to increase the expression of different antioxidant genes (mainly SOD2) and HIF2α, thereby increasing the ROS sustenance ability of tumor cells and the rate of energy production through glycolysis, respectively.41

Targeting oxidative stress in cancer therapeutics Research reports over the past few decades confirmed the deleterious effect of ROS in the development of different pathophysiological conditions, including cancer (Fig. 10.5). In cancer, ROS is a critical component in the initiation and progression of the disease by regulating several cellular processes, including cell proliferation, angiogenesis, and metastasis. In modern medical management, most of conventional therapeutic approaches (e.g., radiotherapy, chemotherapy, photodynamic therapy) against cancer stimulate the production of ROS as it can trigger cell-death pathways. However, as ROS can both trigger and suppress cellular death, in recent times therapeutic strategies are being proposed considering different prooxidants and antioxidants. Prooxidant and antioxidant therapeutic strategies have their own advantages and

FIGURE 10.5 Different ROS-induced oncogenic pathways. A comprehensive representation of different ROS sensitive cellular pathways predominant in the transformed cells.

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FIGURE 10.6 Possible therapeutic strategies against cancer. An overview of cancer therapeutic strategies targeting ROS and/or intracelluar antioxidants and their effects on cellular redox status.

disadvantages, which can be attributed to the complex regulation of ROS in malignant and normal cells. Various efforts have been made to discuss the paradox in the rationales of the opposite ROS-manipulation strategies in cancer therapy6,42 (Fig. 10.6).

Antioxidant-mediated therapeutic strategy Since ROS act as secondary messengers in different cell proliferative signaling pathways and induce oncogenic mutations, several transcription factors like Nrf2 act as tumor suppressors by upregulating the level of endogenous antioxidant molecules. Several antioxidant molecules are also under consideration as they can directly neutralize free radicals or can stimulate ROS detoxifying signaling cascades. Antioxidants like vitamin C, tocopherol, and NAC can potentially attenuate the tumor progression by downregulating the hypoxiainducible factors. In various preclinical studies, overexpression of different antioxidant genes such as SOD1, SOD3, CAT, HO-1, and Nrf2 have been reported to be beneficial against different types of human cancer. Different epidemiological reports suggest that supplementation of several antioxidants such as vitamin E, β carotene, and selenium could attenuate cancer initiation. In a recent report, ellagic acid was found to be advantageous against metastasis, invasion, and angiogenesis in prostate and bladder cancers. Apart from this, various naturally occurring antioxidant molecules like curcumin, mangiferin, resveratrol, and quercetin have been found to modulate different signaling molecules, including NFκB, MAPK, IKK, MMP-9, COX-2, ICAM, VCAM, VEGF, and FGF in attenuating dissimilar cancers in various experimental models.43,44 Dietary intake of antioxidant-rich food or food containing nutraceuticals (with antioxidant properties) such as vitamins A and D, genistein, choline, and theanine

have been shown to control the anomalous proliferation of cancer stem cells in different types of cancer.43

Upregulation of intracellular ROS levels Beyond a threshold level, intracellular ROS can induce cell-death pathways by activating different signaling molecules. Therefore clinicians developed many therapeutic strategies for different types of cancer (e.g., lung cancer, breast cancer, promyelocytic leukemia) to increase the accumulation of intracellular ROS. Due to higher metabolic activity, cancer cells possess more oxidative load than the normal cells and for their sustenance cancer cells are adapted higher antioxidant activity. Therefore cancer cells are more susceptible to further ROS insult than normal cells. Several chemotherapeutic drugs like cisplatin, oxaliplatin, doxorubicin, docetaxel, paclitaxel, arsenic trioxide, and 5fluorouracil have been chemically synthesized and have been conventionally used for cancer treatment over the past two decades. These kinds of drugs can induce severe toxicity in different cellular organelles such as mitochondria, peroxisomes, and endoplasmic reticulum. Acute toxicity in these organelles leads to irreversible impairment of different intracellular biomolecules and cause impairment in many life processes. It is noteworthy that in mitochondria, these kinds of drugs interfere with the signaling pathway involved with ATP synthesis and generate superoxide radicals. Moreover, these drugs can also induce mitochondrial dysfunction and lower the mitochondria membrane potential, thereby releasing cytochrome C in the cytoplasm and activate caspase-dependent apoptotic pathways.5 Ingestion of these kinds of drugs induces lipid peroxidation and protein carbonylation, and downregulate the activity of different intracellular antioxidant enzymes (e.g., SOD, CAT, GPx, GST) and

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other metabolites (e.g., β-carotene, vitamin E, GSH).33 There are drugs (e.g., bortezomib, thapsigargin, celecoxib, nelfinavir) that induce stress-responsive pathways in endoplasmic reticulum. These kinds of drugs can impair the protein folding process and upregulate the unfolded protein responses leading to the accumulation of misfolded proteins and activation of apoptosis. Different reports proved the anticancer efficacy of these prooxidant drugs; however, these drugs have severe cytotoxic effects in other vital organs (e.g., kidney, liver, heart) of the body. Therefore scientists are now focusing on synthesizing prooxidant drugs with pharmacophore from natural sources. These kinds of synthetic derivatives have shown selective toxicity in cancer cells without causing significant toxicity in normal cells. Recently, a series of 22 novel bislawsone derivatives were preclinically tested in our laboratory and promising therapeutic efficacy was observed in vitro and in vivo.33 The incorporation of specific functional groups enhances the bioavailability and pharmacokinetic characteristics of the molecule.

Target antioxidants to decrease ROS scavenging capacity The major drawback of prooxidant therapy is that over prolonged treatment, few cancer cells gain resistance to the increased oxidative environment by escalating the endogenous antioxidant machinery. These antioxidants happen to be the key weapon for cancer cells to deal with the increasing oxidative load and in development of drug resistance. The upregulation of different antioxidants such as SOD, Nrf2, and CAT is driven by the activity of different oncogenes such as oncogenic KRAS, EGFR, and NFκB.45 Cancer cells are more highly dependent on antioxidant machinery (for survival) compared to normal cells. Therefore several preclinical researchers have investigated targeting different endogenous antioxidants for cancer therapy with promising results. Targeting antioxidants (e.g., sulfiredoxin) that are conditionally expressed in cancer cells and not in normal cells was found to be beneficial in selective killing of cancer cells.46 Several agents such as phenethyl isothiocyanate, L-buthionine sulfoximine, and imexon have been identified to target intracellular GSH and downregulate its synthesis.47 Various natural products such as curcumin, piperlongumine, mangiferin, quercetin, and resveratrol were found to directly or indirectly attenuate the activity of several antioxidants pertaining to anticancer effects.48,49 Recently, drug delivery systems have been designed by considering the increased GSH concentration in cancer cells compared to normal cells. Drug-loaded mesoporous silica nanoparticles (MSNs) are being capped with gold

nanoparticles (GNPs) in such a manner that at high concentrations of GSH, GNPs would detach from the MSNs and release the preloaded drug candidate in the appropriate cancer environment. This kind of approach will lead to more specific ways to treat cancer.50

Conclusion In this chapter the role of ROS in initiation and progression of cancer has been discussed in detail and insight is given on the therapeutic strategies targeting OS. Although several reports suggest that accumulation of intracellular ROS can alter cellular metabolism and thus play a critical role in the pathogenesis of cancer, many questions regarding the role of ROS in cellular transformation persist. Different modern, highthroughput molecular biology techniques and advanced bioinformatics tools may be of great help in deciphering the critical threshold level and nature of ROS responsible for the initiation of cancer-cell transformation. In recent times most cancer patients are supplemented with dietary antioxidants along with conventional chemotherapies. However, the big controversy yet to be resolved is whether antioxidants supplementation will suppress cancer progression, and which among the applications of prooxidants or downregulation of antioxidants are the most effective way to treat cancer.

Applications to other areas of pathology Apart from cancer, accumulation of intracellular ROS plays a causative role in the pathogenesis of several organ pathophysiologies including diabetes. This can arise due to several reasons, but mainly because of exposure to environmental pollutants and drugs. In diabetes, increased levels of ROS induce the accumulation of advanced glycation end products and causes severe metabolic impairment. Occurrence of OSassociated pathophysiology is easily identifiable by the estimation of different tissue-specific serum biomarkers. OS also plays a major role in the development and progression of different neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis.51 In each neurodegenerative disorder, persistent OS conditions severely impair the cognitive functions of an individual. In these kinds of pathophysiological conditions, different naturally occurring antioxidants such as curcumin, mangiferin, genistein, morin, resveratrol, silibinin, ferulic acid, quercetin, and others have been found to

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References

be beneficial.52 The ameliorative activity of these natural products is mainly attributed to their radical scavenging activity, nontoxic nature at physiologically relevant doses, and ability to modulate different signaling cascades. These molecules are active in different prosurvival signaling cascades by mainly activating MAPK, PI3K/AKT, and JAK/STAT pathways and downregulating the death-signaling molecules.53,54 These molecules can also upregulate the activity of different antioxidant molecules (e.g., SOD, CAT, GST, GR, GSH) in a stress-responsive manner.53,55

11. 12. 13.

14.

15.

Summary points

16.

• ROS play an important role in normal cellular growth, differentiation, and function. • Accumulation of ROS in different cellular compartments leads to damage of biomolecules and impaired cellular signaling. • Increased ROS levels stimulate several oncogenic signaling cascades, resulting in tumor metastasis, invasion, and angiogenesis. • Several preclinical studies suggest that ROS induction and ROS scavenging could be beneficial therapeutic tools to treat human cancers. • In recent times, targeting antioxidants molecules was found to be a plausible therapeutic option.

17.

18. 19. 20.

21.

22.

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96. 8. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, et al. ROS and ROS-mediated cellular signaling. Oxid Med Cell Longev 2016;2016:4350965. 9. Ghosh S, Basak P, Dutta S, Chowdhury S, Sil PC. New insights into the ameliorative effects of ferulic acid in pathophysiological conditions. Food Chem Toxicol. 2017;103:41
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57. Lu J, Holmgren A. The thioredoxin antioxidant system. Free Rad Biol Med 2014;66:75
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52. Das J, Roy A, Sil PC. Mechanism of the protective action of taurine in toxin and drug induced organ pathophysiology and diabetic complications: a review. Food Funct 2012;3(12):1251
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91. Zhang X, Wang X, Wu T, Li B, Liu T, Wang R, et al. Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation. Sci Rep 2015;5:12579. Poillet-Perez L, Despouy G, Delage-Mourroux R, Boyer-Guittaut M. Interplay between ROS and autophagy in cancer cells, from tumor initiation to cancer therapy. Redox Biol 2015;4:184
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18. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011;475(7354):106.

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31. Liu J, Ma L, Chen X, Wang J, Yu T, Gong Y, et al. ERK inhibition sensitizes cancer cells to oleanolic acid-induced apoptosis through ERK/Nrf2/ROS pathway. Tumor Biol. 2016;37(6):8181
7. 32. Shrestha A, Nepal S, Kim MJ, Chang JH, Kim SH, Jeong GS, et al. Critical role of AMPK/FoxO3A axis in globular adiponectininduced cell cycle arrest and apoptosis in cancer cells. J Cell Physiol 2016;231(2):357
69. 33. Sadhukhan P, Saha S, Sinha K, Brahmachari G, Sil PC. Selective pro-apoptotic activity of novel 3, 30 -(aryl/alkyl-methylene) bis (2-hydroxynaphthalene-1, 4-dione) derivatives on human cancer cells via the induction reactive oxygen species. PLoS One 2016;11 (7):e0158694. 34. Zou Z, Chang H, Li H, Wang S. Induction of reactive oxygen species: an emerging approach for cancer therapy. Apoptosis 2017;22(11):1321
35. 35. De Brache`ne AC, Demoulin JB. FOXO transcription factors in cancer development and therapy. Cell Mol Life Sci 2016;73(6):1159
72. 36. De Simone V, Franze E, Ronchetti G, Colantoni A, Fantini MC, Di Fusco D, et al. Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth. Oncogene 2015;34(27):3493. 37. Li W, Li J, Wang R, Xie H, Jia Z. MDR1 will play a key role in pharmacokinetic changes under hypoxia at high altitude and its potential regulatory networks. Drug Metab Rev 2015;47(2):191
8. 38. Liu Y, Gao X, Deeb D, Zhang Y, Shaw J, Valeriote FA, et al. Mycotoxin verrucarin A inhibits proliferation and induces apoptosis in prostate cancer cells by inhibiting prosurvival Akt/NFκB/mTOR signaling. J Exp Ther Oncol 2016;11(4):251
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11 Antioxidant and chelator cocktails to prevent oxidative stress under iron-overload conditions Sirinart Kumfu, Siriporn Chattipakorn and Nipon Chattipakorn Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

List of abbreviations 4-HDA 4-HNE 8-OH-dG AD4 ALP ALT AST BP β-TI β-TM [Ca21]i DFO DFP DFX GPx GSH Hb H2O2 IFNγ IL-4 LIC LIP LVEF MDA MRI NAC NTBI OS OSI PBMC PMN ppm PS RBC ROS SOD TAC TBARS TIBC

4-hydroxyalkenals 4-hydroxynonenal 8-hydroxy-20
deoxyguanosine N-acetylcysteine amide alkaline phosphatase alanine aminotransferase aspartate aminotransferase blood pressure beta-thalassemia intermedia beta-thalassemia major intracellular Ca21 deferoxamine deferiprone deferasirox glutathione peroxidase reduced glutathione hemoglobin hydrogen peroxide interferon gamma interleukin liver iron concentration labile iron pool left ventricular ejection fraction malondialdehyde magnetic Resonance Imaging N-acetyl cysteine nontransferrin-bound iron oxidative stress oxidative stress index peripheral blood mononuclear cells polymorphonuclears parts per million phosphatidyl serine red blood cell reactive oxygen species superoxide dismutase total antioxidant capacity thiobarbituric acid reactive substances total iron binding capacity

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00011-1

TOS TNF-α UIE

total oxidant status tumor necrosis factor urinary iron excretion

Introduction Hereditary hemochromatosis is the primary form of iron overload resulting from an autosomal disorder, leading to abnormal iron metabolism and resulting in increased intestinal iron absorption in affected patients.1 High parenteral iron administration is the main cause of the secondary form of iron overload that is often seen in patients with transfusion-dependent, hereditary, or acquired anemias.2 Importantly, inherited hemoglobinopathies are very common genetic disorders in humans, in which all β-thalassemia major (β-TM) patients and approximately 20% of those with sickle cell disease are transfusion-dependent.2 Under iron-overload conditions, the excess iron is released into the blood circulation, which can result in it exceeding the serum transferrin iron-binding capacity (TIBC), leading to the appearance of highly reactive nontransferrin-bound iron (NTBI) and increased serum iron, ferritin, and iron deposition in many tissues.3,4 The labile iron form of NTBI leads to the formation of reactive oxygen species (ROS), in particular, highly toxic hydroxyl radicals via the Fenton reaction.4 Under physiological conditions, numerous endogenous antioxidants prevent the harmful effects of oxidative stress (OS). These antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH), and catalase.5 However, in iron-overload conditions, cellular antioxidant capacity is exceeded, resulting in excess ROS that can damage membrane lipids, proteins, nucleic acids, mitochondria,

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and other cellular components.3,4 The aldehyde products from lipid peroxidation such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) can form covalent links to proteins, resulting in protein dysfunction.6 In addition, under conditions of iron overload these mechanisms are activated causing fibrosis, apoptosis, and inflammation initiating complex pathophysiologies and leading to organ dysfunction.3,4 Iron chelation therapy is a standard treatment for iron-overload patients especially in transfusiondependent thalassemia patients.7 The main chelators used in clinical practice are deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX).7 Although iron chelation therapies are standard treatment, serious side effects have been experienced and unsuccessful treatment has been reported in some patients treated with a single iron chelator.7 Growing evidence indicates that antioxidant compounds decrease OS, increase antioxidant levels, decrease tissues iron deposition, and cause organ dysfunction in iron-overloaded animal models.8
14 The mechanisms involving the antioxidant therapies that are responsible for the attenuation of organ dysfunction induced by iron overload conditions could be due to their antioxidant abilities and iron-chelating property. Antioxidant therapies include N-acetylcysteine (NAC),15 silymarin,16,17 and curcumin.18 It has been proposed that the combination of an iron chelator with one of these antioxidants may exert greater efficacy than monotherapy in decreasing the iron accumulation in organs and reducing OS, leading to improved organ function under iron overload conditions. In this chapter, the combined effects of an iron chelator with several antioxidants on OS, antioxidants, and iron status in iron-overloaded conditions are comprehensively summarized and discussed.

The effects of the combination of an iron chelator with vitamin c on oxidative stress and iron status in iron-overloaded conditions β-TM patients have significantly lower levels of vitamin C in comparison to healthy individuals.19,20 However, vitamin C administration may induce iron toxicity by interfering with the reduction of Fe31 to Fe21 (a toxic form), resulting in increased gastrointestinal iron absorption and increased iron release from the reticuloendothelial system.21,22 In an animal study using rats, the addition of vitamin C combined with DFO or DFP reduced the serum iron, myocardial OS markers, myocardial tissue damage, and increases myocardial glutathione in comparison to those rats administered with DFO or DFP alone (Table 11.1).11 In addition, a combination of vitamin C with DFX modulates the release of iron from the reticuloendothelial system,

which correlates positively with the efficiency of DFX chelation therapy, resulting in decreased hepatic iron concentration in osteogenic dystrophy rats (ascorbatedeficient rats) (Table 11.1).23 In addition, a combined iron chelator with vitamin C increased urinary iron excretion (UIE) in several models of iron-overload patients (Table 11.1).24
27 In β-TM patients receiving daily DFO, DFP, or DFX supplemented with vitamin C, increased cardiac magnetic resonance imaging (MRI) T2 and hemoglobin levels and decreased systemic iron-overload were observed without adverse effects (Table 11.1).19 Also, a combined treatment of DFO and vitamin C increased left ventricular ejection fraction in adult nonthalassemic patients in iron-overload conditions (Table 11.1).28 However, large-scale cohort studies are needed to verify their use in iron-overload patients.

The effects of a combination of an iron chelator with silymarin on oxidative stress and iron status in iron-overloaded conditions Silymarin is a flavonoid isolated from the Silybum marianum plant that has several beneficial properties including antioxidant, antiinflammatory, and ironchelating effects.16,17 The medicinal properties of silymarin have been studied in the treatment of Alzheimer’s disease, Parkinson’s disease, sepsis, burns, osteoporosis, diabetes, cholestasis, hypercholesterolemia, and thalassemia.16,17 A combined DFO plus silymarin treatment for 1 week did not provide any protective effects against the liver, kidney, and heart iron deposition in ironoverloaded rats (Table 11.2).9 However, the short duration of silymarin treatment could be responsible for this negative result. A study using a combined DFO plus silymarin treatment for 2 weeks resulted in a protective effect regarding iron overload
induced hepatotoxicity and led to decreased serum iron in iron-overloaded rats (Table 11.2).8 Also, treatment with silymarin led to restoration of GSH levels and the proliferation of peripheral blood mononuclear cells (PBMC) in thalassemia patients who received DFO treatment (Table 11.2).29 In clinical studies, the combined iron chelation and silymarin therapy was effective for improving the iron status in patients with β-TM. The improvements were quantified by decreased serum ferritin, serum iron, and liver iron concentration (LIC) and increased TIBC, glutathione, and liver function (Table 11.2).17,30
33 In addition, combined DFO plus silymarin restored the immune response defects in patients with β-thalassemia major, identified by decreased levels of inflammation markers and increased antiinflammatory cytokines (Table 11.2).34,35 Silymarin stimulates the cell-mediated immune response in β-thalassemia major, by a direct action on cytokine-producing mononuclear cells

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The effects of a combination of an iron chelator with N-acetylcysteine on oxidative stress and iron status in iron-overloaded conditions

TABLE 11.1 Summary of the effects of the combination of an iron chelator with vitamin C on oxidative stress and iron status in ironoverload conditions. Model

Iron overload induction

Adult male albino rats

Osteogenic dystrophy rats (ascorbate-deficient rats)

Intervention

Results

References

2% carbonyl-iron diet

DFO 200 mg/kg or DFP 75 mg/ kg 1 vitamin C 200 mg/day

k Serum iron, myocardial TBARS, 8-OH-dG, myocardial tissue damage

[11]

6 months

2 weeks

m Myocardial glutathione

Iron dextran injections DFX 75 mg/kg/day 1 vitamin C 150, of 200 mg/kg 900, 2250 ppm 10 weeks

k Hepatic iron

[23]

12 weeks

13 β-TM and 1 with congenital sideroblastic anemia patients

DFO given over 12 h 1 ascorbic acid 750 mg

m UIE

[24]

12 untreated idiopathic hemochromatosis patients

8-h 1 g DFO i.v. infusion 1 vitamin C 1 or 2 g orally 2 h

m UIE

[25]

4 myelodysplasia and 4 β-TM patients

DFP 100 mg/kg/day 1 ascorbic acid 200 mg

m UIE

[26]

mm UIE

[27]

12 or 24 h 1 Congenital red cell aplasia and 3 β-TM patients

DFO 1 vitamin C 1000 mg/day

180 β-TM (vitamin C-deficient) patients

DFO 40 mg/kg/day, DFP

k Serum iron, ferritin, LIC [19]

75 mg/kg/day, or DFX

m Hb, cardiac MRI T2

m Stool iron excretion

25 mg/kg/day 1 vitamin C 100 mg/day 1 year Adult nonthalassemic patients including 11 myelodysplastic syndrome, 3 chronic hemolysis, 1 red cell aplasia (Diamond -Black fan syndrome), 1 acute myeloid leukemia

Transfusional iron overload

DFO 2 g/day 1 vitamin C 200 mg/day

m LVEF

[28]

12 months

8-OH-dG, 8-hydroxy-2ʹ
deoxyguanosine; β-TM, beta-thalassemia major; DFO, deferoxamine; DFP, deferiprone; DFX, deferasirox; Hb, hemoglobin; LIC, liver iron concentration; LVEF, left ventricular ejection fraction; MRI, Magnetic Resonance Imaging; ppm, parts per million; TBARS, Thiobarbituric acid reactive substances; UIE, urinary iron excretion.

(Table 11.2).34 The possible mechanisms of the silymarin therapies that enable them to improve iron-overload conditions, immune status, and liver function are probably due to their antioxidant abilities and immunomodulatory, antiinflammatory, and iron-chelating properties.

The effects of a combination of an iron chelator with N-acetylcysteine on oxidative stress and iron status in iron-overloaded conditions N-acetylcysteine (NAC) is a potent antioxidant and a precursor of glutathione synthesis.15 NAC is a scavenger of free radicals and could chelate chromium, boron, and possibly iron.15,36 These properties of NAC may enable it to reduce OS and tissue-iron concentration in organs. In an iron-overloaded condition, NAC

exerted protective effects against cardiac, liver, brain injury, and dysfunction in iron-overloaded rodent models.12
14,37
39 Interestingly, a combined iron chelator therapy of DFP plus NAC exerted greater efficacy in reducing cardiac and brain iron deposition, OS, and apoptosis than did a monotherapy, resulting in restoration of heart and brain function in iron-overloaded rodent models (Table 11.3).12
14,38,39 In clinical studies, a combination of an iron chelator plus the amide form of N-acetylcysteine (AD4) exerted greater efficacy in reducing OS markers in blood cells from thalassemia patients than a combination of an iron chelator plus NAC (Table 11.3).40 Moreover, combined iron chelator plus NAC therapy effectively reduced serum OS, increased Hb levels, and reduced DNA damage in β-TM children (Table 11.3).41 Due to limited information, further clinical studies are needed to warrant the

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TABLE 11.2 Summary of the effects of a combination of an iron chelator with silymarin on oxidative stress and iron status in ironoverload conditions. Model Adult male Wistar rats

Iron overload induction

Intervention

Results

References

Iron dextran 100 mg/kg IP

DFO 50 mg/kg/day 1 silymarin

m Serum ferritin

[9]

2 weeks

200 mg/kg/day

2 Iron deposition in kidney, liver and heart

1 week Adult male Wistar rats

Iron dextran 100 mg/kg IP

DFO 50 mg/kg 1 silymarin

k Serum iron, ALT, necrotic hepatocytes

2 weeks

200 mg/kg

m Serum AST

[8]

2 weeks PBMC from 28 β-TM patients

DFO treatment with patients 50 mg/kg 1 PBMC were treated with silymarin (0, 5, 10, or 20 μg/ml)

m GSH, PBMC proliferation

[29]

60 β-TM patients

DFO 40
50 mg/kg 1 silymarin tablet (140 mg) 3 times a day

k serum ferritin, liver ALP

[30]

20 β-TM children patients

40 β-TM children patients

49 β-TM patients

82 β-TM patients

25 β-TM patients

13 β-TM patients

m RBC glutathione

3 months

2 Hb

DFX 20
40 mg/kg/day 1 silymarin tablet (140 mg) 3 times a day

k Serum iron, serum ferritin

6 months

2 Hb

DFP 75 mg/kg/day 1 silymarin tablet (140 mg) 3 times a day

k Serum iron, serum ferritin

9 months

m TIBC

DFO 40 mg/kg 1 silymarin tablet (140 mg) 3 times a day

k Serum iron, serum ferritin, TIBC

9 months

m Liver function

DFO or DFP or DFX 1 silymarin tablet (140 mg) 3 times a day

k Serum iron, serum ferritin, LIC

26 weeks

m TIBC

DFO 40 mg/kg/day 1 silymarin tablet (140 mg) 3 times a day

k TNF-α

12 weeks

m IFNγ, IL-4

DFO 40
50 mg/kg 1 silymarin tablet (140 mg) 3 times a day

k IL-10

6 months

2 TGF-β, IL-17, IL-23

[31]

m TIBC

[32]

[33]

[17]

[34]

[35]

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, Aspartate aminotransferase; β-TI, beta-thalassemia intermedia; β-TM, beta-thalassemia major; DFO, deferoxamine; DFP, deferiprone; DFX, deferasirox; Hb, hemoglobin; IFNγ, interferon gamma; IL-4, interleukin; LIC, liver iron concentration; PBMC, peripheral blood mononuclear cells; RBC, red blood cell; TIBC, total iron-binding capacity; TNF-α, tumor necrosis factor.

use of this combined therapy, especially its impact on organ function in iron-overloaded patients.

The effects of a combination of an iron chelator with vitamin E on oxidative stress and iron status in iron-overloaded conditions The vitamin E family are potent antioxidants, acting by scavenging free radicals, and donating hydrogen

atoms to free radicals, thereby reducing their impact.42 Oral treatment with vitamin E improves the antioxidant
oxidant balance, reduces OS in beta-thalassemia intermedia patients,43,44 and can attenuate lipid peroxidation in patients on hemodialysis receiving intravenous iron.45 A combination of an iron chelator with vitamin E effectively reduces serum OS, increase Hb levels, and decrease GPx activity in β-TM patients (Table 11.4).41,46 In addition, the combination of DFP with an antioxidant cocktail (vitamin E and NAC) can

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The effects of the combination of an iron chelator with curcumin or idebenone on oxidative stress and iron status in iron-overloaded conditions

TABLE 11.3 Summary of the effects of the combination of an iron chelator with n-acetylcysteine on oxidative stress and iron status in iron-overload conditions. Model

Iron overload induction

Intervention

Adult C57/BL6 male wildtype (WT) and heterozygous

0.2% ferrocene/ kg of diet

DFP 75 mg/kg/day 1 NAC 100 mg/ k Plasma MDA, NTBI, kg/day cardiac iron & MDA, apoptosis

βKO type (muβth-3/1, [HT])

4 months

1 month

Male Wistar rats

0.2% ferrocene/ kg of diet

DFP 50 mg/kg/day 1 NAC 100 mg/ k Plasma MDA, brain iron & MDA, kg/day mitochondrial ROS

8 weeks

2 months

0.2% ferrocene/ kg of diet

DFP 75 mg/kg/day 1 NAC 100 mg/ k NTBI, brain iron, mitochondrial ROS, kg/day apoptosis and Alzheimer’s related protein

4 months

2 months

0.2% ferrocene/ kg of diet

DFP 75 mg/kg/day 1 NAC 100 mg/ k Plasma MDA, NTBI, cardiac kg/day iron&MDA, mitochondrial ROS

4 months

for 2 months

0.2% ferrocene/ kg of diet

DFP 75 mg/kg/day 1 NAC 100 mg/ k Plasma MDA, NTBI, cardiac iron & kg/day MDA

4 months

2 months

m [Ca21]i transients, Cardiac function

DFO treatment on patient 1 blood cells were treated with

m GSH (RBC, platelets, PMN)

1 mM NAC or AD4

k ROS (RBC, platelets, PMN), RBC lysis

DFX 20
30 mg/kg/day, or DFP 75 mg/kg/day, or DFO 35
40 mg/ kg/day 1 NAC 10 mg/kg

k Serum TOS, OSI, DNA damage

3 months

m TAC, Hb

Male Wistar rats

Male Wistar rats

Male Wistar rats

Blood cells from 2 β-TI 4 β-TM patients

25 Children with β-TM patients

Results

References [12]

m Cardiac function [13]

m mitochondrial function, brain function [14]

m Spine density, mitochondrial function [39]

m Mitochondrial function, cardiac function [38]

[40]

[41]

AD4, N-acetylcysteine amide; β-TM, beta-thalassemia major; [Ca ]i, Intracellular Ca ; DFO, deferoxamine; DFP, deferiprone; DFX, deferasirox; GSH, reduced glutathione; Hb, hemoglobin; MDA, Malondialdehyde; NAC, N-acetyl cysteine; NTBI, nontransferrin bound iron; OSI, oxidative stress index; PMN, polymorphonuclears; RBC, red blood cell; ROS, reactive oxygen species; TAC, total antioxidant capacity; TOS, total oxidant status. 21

improve anemia, iron overload, and OS in HbE-β-thalassemia patients (Table 11.4).47 These findings are promising, but further clinical studies are needed to determine the effect of the combination of an iron chelator and vitamin E on tissue-iron accumulation and organ function in iron-overloaded patients.

The effects of the combination of an iron chelator with curcumin or idebenone on oxidative stress and iron status in ironoverloaded conditions Curcumin is a natural herb containing polyphenol compounds that have been used in traditional medicine for thousands of years.18 It has extensive potentially useful biological attributes including antioxidant, antiinflammatory, antiangiogenic, antitumor, antiaging, and iron-chelating properties.18 Accumulating

21

evidence indicates that curcuminoids (a mixture containing curcumin) are effective in decreasing plasma NTBI and myocardial iron as well as increasing lipid peroxidation and improving cardiac function in ironloaded thalassemic mice.48 It is also effective in removing NTBI in thalassemic patients.49 The combination of tetrahydrocurcumin (THU) and DFP exerted a greater efficacy than either as a monotherapy in reducing systemic iron overload, OS, hypertension, and vascular dysfunction in iron-overloaded mice (Table 11.5).10 Moreover, a combination of the iron chelator with curcumin effectively reduced OS; however, it did not cause any changes in Hb, serum iron, and ferritin in β-TM patients (Table 11.5).50 Interestingly, treatment with an iron chelator and two antioxidant cocktails (curcuminoids and NAC) has been shown to improve anemia and iron-overload conditions, and reduce OS in patients with HbE-β-thalassemia (Table 11.5).47 These findings suggest that the combination of an iron

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11. Antioxidant and chelator cocktails to prevent oxidative stress under iron-overload conditions

TABLE 11.4 Summary of the effects of a combination of an iron chelator with vitamin e on oxidative stress and iron status in ironoverload conditions. Model

Iron overload induction

30 β-TM patients

25 children with β-TM patients

19 HbE-β-thalassemia patients

Intervention

Results

References

DFO 1 vitamin E 400 mg/day

k GPx activity

[46]

3 months

2 SOD activity, TAC

DFX 20
30 mg/kg/day, or DFP 75 mg/kg/day, or DFO 35
40 mg/kg/day 1 vitamin E 10 U/kg

k TOS, OSI

3 months

m TAC, Hb

DFP 50 mg/kg/day 1 vitamin E 400 IU/day 1 NAC 200 mg/day

k NTBI, ferritin, RBC (MDA, ROS, GPx)

12 months

m Hb, RBC GSH

[41]

[47]

β-TM, beta-thalassemia major; DFO, deferoxamine; DFP, deferiprone; GPx, glutathione peroxidase; GSH, reduced glutathione; Hb, hemoglobin; MDA, malondialdehyde; NAC, N-acetyl cysteine; NTBI, non-transferrin bound iron; OSI, oxidative stress index; ROS, reactive oxygen species; SOD, superoxide dismutase; TAC, total antioxidant capacity; TOS, total oxidant status.

TABLE 11.5 Summary of the effects of a combination of an iron chelator with curcumin or idebenone on oxidative stress and iron status in iron-overload conditions. Model Adult male ICR mice

Iron overload induction

Results

Iron sucrose, i.p. DFP 50 mg/kg/day 1 k Serum iron, NTBI, ferritin, BP, 10 mg/kg/day Tetrahydrocurcumin (THU) 50 mg/kg/day MDA (plasma, heart, kidney, liver) 8 weeks

31 β-TM patients

Intervention

8 weeks

m GSH, vascular responsiveness

DFO 40
50 mg/kg 1 curcumin 2 500 mg/ day

k MDA, total and direct bilirubin

12 weeks

m TAC

References [10]

[50]

2 Hb, serum iron, ferritin 16 HbE-β-thalassemia patients

Periodontal ligament cells from 3 Friedreich’s ataxia patients and 3

DFP 50 mg/kg/day 1 curcuminoids 500 mg/day 1 NAC 200 mg/day

k NTBI, ferritin, RBC (MDA, ROS, GPx)

12 months

m Hb, RBC GSH

DFP 25 μM 1 idebenone 10 μM

m Catalase, SOD, frataxin

1 day

k Active-caspase3

DFP 20 mg/kg/day 1 idebenone 20 mg/ kg/day

Improvement in the kinetic functions, neurologic gait and posture scores

11 months

k Heart hypertrophy

[47]

[51]

Healthy individuals 20 Friedreich’s ataxia patients

[52]

Parameters and iron deposits in dentate nucleus 5 Friedreich’s ataxia patients

DFP 20 mg/kg/day 1 idebenone 5, 15 mg/ kg/day

m Neurological function

10
24 months

k Cardiac hypertrophy

[53]

BP, blood pressure; β-TM, beta-thalassemia major; DFO, deferoxamine; DFP, deferiprone; GPx, glutathione peroxidase; GSH, reduced glutathione; Hb, hemoglobin; MDA, Malondialdehyde; NAC, N-acetyl cysteine; NTBI, non-transferrin bound iron; RBC, red blood cell; ROS, reactive oxygen species; SOD, superoxide dismutase; TAC, total antioxidant capacity.

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Applications to other areas of pathology

chelator and antioxidant cocktails could be more useful in treating iron-overload patients than treatment with curcumin alone. Idebenone is a synthetic analog of coenzyme Q10 that has been shown to be useful in the treatment of Alzheimer’s disease and Friedreich’s ataxia.54 Interestingly, combined iron chelator DFP with idebenone therapy is relatively safe, can increase antioxidant enzymes, decrease apoptosis, and might improve neurological function and cardiac hypertrophy (Table 11.5).51
53 Due to limited studies, further studies are needed to warrant the use of this combined therapy clinically.

The effects of a combination of an iron chelator with other antioxidants and multiantioxidants on oxidative stress and iron status in ironoverloaded conditions

summarized in Table 11.6.55
59 The combined treatment of DFO with melatonin and related pineal products showed a decrease in OS markers in iron-overloaded rat brain homogenates (Table 11.6).55 A combined treatment with an iron chelator plus fermented papaya preparation (FPP) or hydroxyurea also showed a significant reduction in OS parameters and increased GSH, but without a significant improvement in the hematological parameters (Table 11.6).56
58 A combined iron chelator with multivitamin therapy also improved the antioxidant
oxidant balance, LIC, hepatic fibrosis, and increased Hb in β-TM patients (Table 11.6).59 Further studies are needed to investigate the effects of combined treatments with iron chelators and these promising antioxidants on tissue-iron accumulation or organ function in iron-overloaded patients.

Applications to other areas of pathology The effects of the combination of an iron chelator with other antioxidants and multiantioxidants on OS and iron status in iron-overloaded conditions are

This chapter focuses on the impact of the antioxidants and chelator cocktails on preventing OS and

TABLE 11.6 Summary of the effects of a combination of an iron chelator with other antioxidants and multiantioxidants on oxidative stress and iron status in iron-overload conditions. Model Brain homogenates from male Sprague
Dawley rats

Iron overload induction

Intervention

Results

References

5 mM H2O2

DFO (10, 50, 100, 200, 400,

k MDA 1 4-HDA

[55]

60 min

600, 800 nM)

DFO 1 Fermented papaya preparation (FPP) 3 g 3 3 times/day

k ROS

[56]

3 months

m GSH

DFO 1 Fermented papaya preparation (FPP) 3 g 3 3 times/day

k ROS, lipid peroxidation, PS externalization

3 months

mGSH

1 Melatonin 100 μM or 1 5-Methoxytryptophol 100 μM or 1 pinoline 10 μM RBC, platelets, PMN from 2 β-TI and 3 β-TM patients

Peripheral blood cells from 2 β-TI and 4 β-TM patients

[57]

2 Hb 17 β-TI and 4 HbEβ-thalassemia patients

DFX 1 Hydroxyurea 10
20 mg/kg/day

k Serum ferritin, LIP, PS externalization, ROS

12 months

m GSH

[58]

2 Hb 39 β-TM patients

DFP 75
100 mg/kg/day 1 vitamin E (400 or 600 mg/day) 1 vitamin C (100 mg/ day) 1 vitamin A (25000 IU/week)

k MDA, ferritin, serum transaminase, SOD, LIC

12 months

m Vitamins, GSH, Hb, glutathione reductase

[59]

4-HDA, 4-hydroxyalkenals; 4-HNE, 4-hydroxynonenal; β-TI, beta-thalassemia intermedia; β-TM, beta-thalassemia major; DFO, deferoxamine; DFP, deferiprone; DFX, deferasirox; GSH, reduced glutathione; Hb, hemoglobin; H2O2, Hydrogen peroxide; LIC, liver iron concentration; LIP, Labile iron pool; MDA, Malondialdehyde; PMN, polymorphonuclears; PS, Phosphatidyl serine; RBC, red blood cell; ROS, reactive oxygen species; SOD, superoxide dismutase.

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11. Antioxidant and chelator cocktails to prevent oxidative stress under iron-overload conditions

improving iron status under iron overload conditions. Besides hereditary hemochromatosis and transfusiondependent thalassemia and sickle cell disease patients, excess iron accumulation is also found in several diseases including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, Friedreich’s ataxia, myelodysplastic syndromes, ischemia-reperfusion injury, and aging.2,60 Since iron chelator therapy has for some time been the main choice for treating ironoverload patients with extensive evidence to support its use, the use of an iron chelator may provide beneficial effects on those diseases with excess iron accumulation. Excess iron accumulation leads to excess ROS generation that can damage several cellular components. This damage can serve as the underlying basis of many diseases.3,4,60 Since many antioxidants have been shown to confer beneficial effects on the treatment of several diseases via their ability to reduce OS, the combination of an iron chelator with an antioxidant may exert greater therapeutic efficacy by decreasing iron accumulation in organs and attenuating OS, ultimately leading to improved organ function under ironoverload conditions. This chapter comprehensively summarizes the beneficial effects of the use of the combination of an iron chelator with several major antioxidants including vitamin C, NAC, silymarin, vitamin E, curcumin, idebenone, and some other antioxidants under ironoverload conditions. There is growing evidence from previous studies to indicate that combined therapy might exert greater beneficial effects than monotherapies on reducing OS, and improving the iron status and organ function in β-thalassemia and Friedreich’s ataxia patients. Therefore the use of antioxidant and chelator cocktails could be promising novel strategies to combat these pathological conditions due to iron overload. Nevertheless, further studies are needed to warrant their use in a clinical setting.

Summary points • This chapter focuses on the beneficial effect of the combination of an iron chelator with several antioxidants, individually and together, under ironoverload conditions. • The increase in labile iron under conditions of iron overload leads to an increase in oxidative stress that can damage several cellular components, ultimately leading to organ dysfunction. • Iron chelation therapy is a standard treatment for iron-overload patients. • Several antioxidants have been shown to decrease oxidative stress, increase antioxidant levels,

decrease iron deposition in tissues, and improve organ dysfunction in iron-overloaded animal models. • Studies using the combined treatment of an iron chelator with an antioxidant showed promising enhanced therapeutic benefits when compared to outcomes from a single therapy under iron-overload conditions. • Larger and longer cohort studies are needed to verify their uses in iron overload patients.

Acknowledgments This work is supported by the NSTDA Research Chair Grant from the National Science and Technology Development Agency (NC) and the Thailand Research Fund grant RTA6080003 (SCC) and MRG6180239 (SK).

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18. Wongjaikam S, Kumfu S, Khamseekaew J, Chattipakorn SC, Chattipakorn N. Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine. Sci Rep 2017;7:44460. Wongjaikam S, Kumfu S, Khamseekaew J, Sripetchwandee J, Srichairatanakool S, Fucharoen S, et al. Combined iron chelator and antioxidant exerted greater efficacy on cardioprotection than monotherapy in iron-overloaded rats. PLoS One 2016;11:e0159414. Amer J, Atlas D, Fibach E. N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells. Biochim Biophys Acta 2008;1780:249
55. Ozdemir ZC, Koc A, Aycicek A, Kocyigit A. N-Acetylcysteine supplementation reduces oxidative stress and DNA damage in children with beta-thalassemia. Hemoglobin 2014;38:359
64. Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy. Free Radic Biol Med 2014;72:76
90. Tesoriere L, D’Arpa D, Butera D, Allegra M, Renda D, Maggio A, et al. Oral supplements of vitamin E improve measures of oxidative stress in plasma and reduce oxidative damage to LDL and erythrocytes in beta-thalassemia intermedia patients. Free Radic Res 2001;34:529
40. Pfeifer WP, Degasperi GR, Almeida MT, Vercesi AE, Costa FF, Saad ST. Vitamin E supplementation reduces oxidative stress in beta thalassaemia intermedia. Acta Haematol 2008;120:225
31. Roob JM, Khoschsorur G, Tiran A, Horina JH, Holzer H, Winklhofer-Roob BM. Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J Am Soc Nephrol 2000;11:539
49. Rashidi M, Aboomardani M, Rafraf M, Arefhosseini SR, Keshtkar A, Joshaghani H. Effects of Vitamin E and Zinc Supplementation on Antioxidants in Beta thalassemia major Patients. Iran J Pediatr 2011;21:8
14. Yanpanitch OU, Hatairaktham S, Charoensakdi R, Panichkul N, Fucharoen S, Srichairatanakool S, et al. Treatment of betathalassemia/hemoglobin e with antioxidant cocktails results in

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decreased oxidative stress, increased hemoglobin concentration, and improvement of the hypercoagulable state. Oxid Med Cell Longev 2015;2015:537954. Thephinlap C, Phisalaphong C, Lailerd N, Chattipakorn N, Winichagoon P, Vadolas J, et al. Reversal of cardiac iron loading and dysfunction in thalassemic mice by curcuminoids. Med Chem 2011;7:62
9. Srichairatanakool S, Thephinlap C, Phisalaphong C, Porter JB, Fucharoen S. Curcumin contributes to in vitro removal of nontransferrin bound iron by deferiprone and desferrioxamine in thalassemic plasma. Med Chem 2007;3:469
74. Nasseri E, Mohammadi E, Tamaddoni A, Qujeq D, Zayeri F, Zand H. Benefits of curcumin supplementation on antioxidant status in beta-thalassemia major patients: a double-blind randomized controlled clinical trial. Ann Nutr Metab 2017;71:136
44. Quesada MP, Jones J, Rodriguez-Lozano FJ, Moraleda JM, Martinez S. Novel aberrant genetic and epigenetic events in Friedreich’s ataxia. Exp Cell Res 2015;335:51
61. Velasco-Sanchez D, Aracil A, Montero R, Mas A, Jimenez L, O’Callaghan M, et al. Combined therapy with idebenone and deferiprone in patients with Friedreich’s ataxia. Cerebellum 2011;10:1
8. Elincx-Benizri S, Glik A, Merkel D, Arad M, Freimark D, Kozlova E, et al. Clinical experience with deferiprone treatment for friedreich ataxia. J Child Neurol 2016;31:1036
40.

54. Parkinson MH, Schulz JB, Giunti P. Co-enzyme Q10 and idebenone use in Friedreich’s ataxia. J Neurochem 2013;126(Suppl 1):125
41. 55. Ortega-Gutierrez S, Garcia JJ, Martinez-Ballarin E, Reiter RJ, Millan-Plano S, Robinson M, et al. Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates. Neurosci Lett 2002;323:55
9. 56. Amer J, Goldfarb A, Rachmilewitz EA, Fibach E. Fermented papaya preparation as redox regulator in blood cells of betathalassemic mice and patients. Phytother Res 2008;22:820
8. 57. Fibach E, Tan ES, Jamuar S, Ng I, Amer J, Rachmilewitz EA. Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-betathalassemia following administration of a fermented papaya preparation. Phytother Res 2010;24:1334
8. 58. Italia K, Chandrakala S, Ghosh K, Colah R. Can hydroxyurea serve as a free radical scavenger and reduce iron overload in beta-thalassemia patients? Free Radic Res 2016;50:959
65. 59. Elalfy MS, Adly AA, Attia AA, Ibrahim FA, Mohammed AS, Sayed AM. Effect of antioxidant therapy on hepatic fibrosis and liver iron concentrations in beta-thalassemia major patients. Hemoglobin 2013;37:257
76. 60. Dusek P, Schneider SA, Aaseth J. Iron chelation in the treatment of neurodegenerative diseases. J Trace Elem Med Biol 2016;38:81
92.

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C H A P T E R

12 Ac¸aı´ (Euterpe oleracea Martius) as an antioxidant Priscila Oliveira Barbosa1, Melina Oliveira de Souza2, Daniela Pala2 and Renata Nascimento Freitas1,2 1

Nucleus of Research in Biological Sciences (NUPEB), Federal University of Ouro Preto, Minas Gerais, Brazil 2 School of Nutrition, Federal University of Ouro Preto, Minas Gerais, Brazil

List of abbreviations CAT GPx LDL MDA OS PON ROS SOD TBARS

catalase glutathione peroxidase low-density lipoprotein malondialdehyde oxidative stress paraoxonase reactive oxygen species superoxide dismutase thiobarbituric acid reactive substances

Introduction Nutrition is an ancient science that aims to study the interactions among food, nutrients, and human health. As Hippocrates, considered the father of the modern medicine, famously said: “Let food be thy medicine and medicine be thy food.” Over the past few decades, several studies have attributed to certain foods the ability to provide benefits to human health that go beyond macronutrient content, by being able to act on cellular signaling pathways through the control of oxidative stress (OS), and reducing the incidence of many chronic diseases, including obesity, Type 2 diabetes, cardiovascular diseases, cancer, and neurodegenerative diseases.1,2 OS has been proposed as a pathogenic mechanism related to many conditions, including atherosclerosis, inflammation, certain cancers, and the process of aging. OS occurs due to the imbalance between the activity of the antioxidant defense system and the production of prooxidants, that is, between the production Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00012-3

and the removal of free radicals, mainly by the reactive oxygen species (ROS).3 ROS are formed in our body under physiological conditions and its amount is controlled by antioxidant defenses, which may be food sources, such as polyphenols.4 ROS are capable of attacking molecular targets such as the nucleus and cell membranes, and their overproduction can cause oxidative damage to lipids, proteins, and nucleic acids. In some disease conditions, an imbalance between these systems or a loss of its control can take place. In this way, the increase of OS is involved in the emergence and evolution of several nontransmissible chronic diseases.5 Epidemiological studies show a correlation between a diet rich in vitamins, minerals, fibers, and phenolic compounds with the prevention of diseases. Parallel to this, there has been growing recognition and interest in the consumption of exotic tropical fruits.6,7 Ac¸aı´ is the fruit of Euterpe oleracea Martius, a palm native to the Amazon region. It is small, round, and dark purple in color.8 In the northern region of Brazil, the purple ac¸aı´ pulp is mostly consumed with manioc flour and roasted fish or shrimp. In other regions, it is usually served frozen mixed with other ingredients such as banana, granola, and guarana syrup. Frozen dessert, “cream,” and jelly, among other traditional preparations, are made from ac¸aı´ broth.9 Ac¸aı´ has been at the center of the traditional diet of populations in the Amazonian estuary for centuries. Due to its nutritive and energetic properties, it became famous in Brazil in the 1990s and consumption of ac¸aı´ has been increasing ever since. According to Embrapa (a Brazilian agency for agricultural research), ac¸aı´

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exportation began in 2000, with fruit pulp processing companies exporting 32 tonnes to the United States. Export has increased exponentially, from 1,136 tonnes in 2002 to 14,431 tonnes in 2010. International demand began in the United States— promoted by its nutritional and chemical composition being rich in antioxidants, fiber, and good fats—and spread to regions around the world, such as Australia, Europe, and Japan. In 2004, 80% of the production of ac¸aı´ was in Para´, in the Amazon estuary. It is estimated that the Brazilian market increased from 8.527 tonnes in 2001 to 11.231 tonnes in 2002, an increase of 32%. The main import markets are Rio de Janeiro, Sa˜o Paulo, and Belo Horizonte.11 The increasing popularity, production, and exportation of ac¸aı´, and its consumption worldwide, also increased the interest of researchers to investigate the possible effects of ac¸aı´ in oxidative metabolism. These effects have been attributed to the peculiar bioactive and nutritional characteristics of the fruit. Its phytochemical composition displays a variety of phenolic compounds and, regarding the nutritional composition, the ac¸aı´ has an expressive amount of lipids (monounsaturated and polyunsaturated), fiber, and phytosterols.10 Considering the increase in consumption of ac¸aı´ and its phytochemical composition and nutritional value, research into its properties has attracted the attention of several research groups around the world. This chapter reviews the studies on the possible effects of ac¸aı´ on the oxidative state.

Ac¸aı´ antioxidants Dietary antioxidants are naturally present in foods, especially in fruits and vegetables, and are derived from secondary plant metabolism.11 They are able to stabilize free radicals and disrupt the cascade of oxidative reactions generated by reactive species. Ascorbic acid, α-tocopherol, and β-carotene are among the most well-known antioxidants,12 while other molecules found in plant foods also act as nonenzymatic antioxidants, such as phenolic compounds.13 Polyphenols or phenolic compounds are characterized by having one or more aromatic rings attached to hydroxyl groups and can be classified into different subgroups: phenolic acids, flavonoids, stilbenes, coumarins, and tannins. Flavonoids make up the largest subgroup of polyphenols, and more than 4000 different flavonoid types have been identified. The chemical structure of flavonoids involves a fundamental nucleus consisting of fifteen carbon atoms arranged in three rings (C6
C3
C6), two substituted phenolic rings (A and B) and one pyran (heterocyclic C chain)

coupled to ring A (Fig. 12.1). Flavonoids can be subdivided into different groups according to their chemical structure: flavones, flavanols, flavanones, isoflavones, and anthocyanins. Perhaps the main interest in studying ac¸aı´ is due to the presence of flavonoids with antioxidant capacity. Ac¸aı´ phytochemical composition is characterized by the presence of five main flavonoids of the class of anthocyanins: cyanidin-3-rutinoside, cyanidin-3-glucoside, cyanidin-3-sambubioside, peonidin-3-glucoside, and peonidine-3-rutinoside (Fig. 12.2).10 In addition to these phytochemicals, other phenolic compounds are found in lower concentrations, such as ferulic acid, epicatechin, p-hydroxybenzoic acid, gallic acid, protocatechuic acid, catechin, ellagic acid, vanillic acid, p-coumaric acid, and lignans.14,15 Although ac¸aı´ has a very interesting phytochemical composition, it is still unknown how these compounds are absorbed and metabolized in the human body, and more studies are needed in order to clarify the ability of these dietary antioxidants in a complex physiological system. The antioxidant potential of flavonoids is complex and involves different levels of action.16 These compounds act directly through the elimination of free radicals; however, the sequestration of these radicals is not a unique biochemistry action. Oxidation of lipids, for example, involves three stages: (1) initiation (free radicals remove a hydrogen atom from a polyunsaturated fatty acid to form a lipid radical); (2) propagation (lipid radicals and molecular oxygen form lipid peroxide radicals, which break down into more radicals); and (3) terminus (the new radicals react with each other or with antioxidants to eliminate radicals).17 Flavonoids can act at any of these stages. These compounds can block initiation by sequestering primary radicals, may react with peroxide radicals to retard propagation, or may react with the other radicals formed during propagation, accelerating the termination process.18 Flavonoids may also interact with transition metals, performing the process of chelation. Metals such as iron can catalytically form reactive free radicals, and

FIGURE 12.1 General structure of flavonoids.

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FIGURE 12.2 Structure of the five major flavonoids found in ac¸aı´ (Euterpe oleracea Martius). Source: PubChem ,https://pubchem.ncbi.nlm.nih. gov/..

some flavonoid structures have the chemical ability to chelate metals in a state that is reactivity inhibited.19 In addition, flavonoids can also act by inhibiting prooxidant enzymes. The most prominent example is the inhibition of xanthine oxidase, which may in certain states, produce superoxide radicals.20 There is another possible indirect antioxidant action of flavonoids. Recently, studies have evaluated the effects of these compounds on activating the Nrf2-Keap1 pathways, allowing Nrf2 to be translocated to the nucleus and binding to the promoter region of the antioxidant response element.21 This mechanism allows the transcription of genes encoding detoxifying antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). The high content of polyphenols presents in ac¸aı´ and its possible effects on human health have aroused the interest of researchers. A growing body of in vitro and in vivo research and clinical studies have provided evidence of the potential antioxidant effect of ac¸aı´. Research has achieved considerable progress and some of the most relevant work regarding the antioxidant capacity of ac¸aı´ will be discussed next.

Antioxidant activity of ac¸aı´: in vitro bioassays Through the quantification and identification of the phenolic compounds present in ac¸aı´, several in vitro assays have evaluated its potential antioxidant effect.14,22
26 Most studies evaluated the ability of ac¸aı´ to neutralize reactive species of oxygen and nitrogen (i.e., ROS and RNS). Some studies have used specific tests to evaluate the ability of ac¸aı´ to absorb oxygen and nitrogen radicals. In addition, studies evaluating the antioxidant effect of ac¸aı´ on several cell lines are also described.27
29 Schauss et al.22 verified the antioxidant capacity of freeze-dried ac¸aı´ pulp and found a value of 1027 μmol Trolox equivalent/g and the total phenolic content reported was 13.9 mg gallic acid equivalents/g. The study also evaluated the ability of ac¸aı´ through the assay of oxygen radical absorbance capacity with fluorescein as the fluorescence probe (ORACFL): hydrophilic (HORACFL), lipophilic (L-ORACFL), peroxynitrite radical averting capacity (NORAC), and hydroxyl radical averting capacity (HORAC). The values found were 997 μmol Trolox equivalents/g for H-ORACFL; 30 μmol Trolox equivalents/g for L-ORACFL; 34 μmol Trolox

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12. Ac¸aı´ (Euterpe oleracea Martius) as an antioxidant

equivalents/g for NORAC, and 52 μmol Trolox equivalents/g for HORAC. The values found for the ac¸aı´ pulp were superior to other berries studied thus far. Several in vitro studies using different cell lines confirm the promising antioxidant effect of ac¸aı´. Machado et al.27 evaluated the effect of an ac¸aı´ extract in neuroblasts (SH-SY5Y) and found a reduction of ROS and lipid peroxidation. Ac¸aı´ was also studied in astrocytes to evaluate the protection it provides against the accumulation of manganese (Mn), a vital element for human health, but neurotoxic in high concentrations. The anthocyanins derived from ac¸aı´ were effective in protecting the cells against the neurotoxicity of Mn, preventing OS, and restoring the reduced glutathione and oxidized glutathione ratio.28

Antioxidant activity of ac¸aı´: animal model assays Over the past 15 years, several studies have evaluated the effects of ac¸aı´ supplementation in different animal models. Some studies sought to examine the antioxidant effect of the fruit on the prevention or progression of metabolic diseases, such as hypercholesterolemia, diabetes, renal ischemia, and nonalcoholic fatty liver disease.30
32 Another study investigated the effects of ac¸aı´ in retinal impairment induced by methyl-mercury (MeHg), a biohazard pollutant released into the aquatic environment by gold mining or industrial activity.33 There has also been research evaluating the protective antioxidant effect of diets supplemented with ac¸aı´ in a model of alcoholic liver diseases (ALD).34 The analysis of biomarkers of OS in hypercholesterolemic rats receiving ac¸aı´ in their diet indicated a reduction in the concentration of carbonyl proteins, a biomarker for protein damage caused by oxidized amino acid residues under stressed conditions.30 In addition, hypercholesterolemic rats supplemented with ac¸aı´ in their diet showed significantly increased concentration of sulfhydryl groups, which can be physiological free radical scavengers. The concentrations of thiobarbituric acid reactive substances (TBARS), an OS marker of lipid peroxidation, were also evaluated in different models.32,34 These studies have shown that there is a decrease in TBARS in animals that received ac¸aı´ as a preventive approach or as treatment. Ac¸aı´ intake had a positive effect on the activity of the antioxidant enzyme SOD and on the activity and expression of paraoxonase (PON), suggesting that ac¸aı´ could function to reduce the stressful environment caused by a hypercholesterolemic diet.30,35 Regarding the GPx, ac¸aı´ promoted an increased expression of this enzyme in the liver of diabetic rats32 and in an ALD injury model.34 Guerra et al.32 evaluated the possible antioxidant protective effects of ac¸aı´ on the production of ROS by

neutrophils in streptozotocin-induced diabetic rats. The study showed that diabetic rats receiving a diet supplementation with 2% ac¸aı´ presented a reduction in the production of ROS. The authors believe that ac¸aı´ can be a dietary strategy to reduce the occurrence of diabetic complications, by exerting an important role in the improvement of antioxidant status, mainly because it is able to neutralize ROS. An elegant study using Caenorhabditis elegans as a model,36 showed that the ac¸aı´ increased both oxidative and osmotic stress resistance by reducing intracellular ROS accumulation and preventing the reduction of protein sulfhydryl groups under stressed conditions. Gene expression analysis indicated that stress resistance by inducing antioxidant enzymes, mediated by ac¸aı´, is dependent on the DAF-16 and OSR-1/UNC-43/SEK-1 osmotic stress pathways. The antioxidant effect of ac¸aı´ was studied in a Drosophila melanogaster model, an organism in which genetic and pharmacological modifications of stress resistance and lifespan mechanisms have been well elucidated. In the model of OS-dependent and chemically-induced aging, ac¸aı´ can be effective against multiple sources of OS and is very effective in prolonging the lifespan of the oxidative challenge, acting primarily by decreasing the transcription of the GSTD1 gene that encodes the detoxifying enzyme glutathioneS-transferase37 induced in adult flies during normal aging and when the flies are over-regulated with OS. In SOD1 mutant flies, it was found that ac¸aı´ supplementation at the later-life stage, promotes the survival of SOD1 knockdown flies. These results indicate that ac¸aı´ affect a range of biological processes involved in stress response and maintenance of cellular and tissue homeostasis.38 Based on the studies carried out on animal models, we believe that the antioxidant effect of ac¸aı´ can occur at two levels: (1) direct scavenging of free radicals, decreasing the endogenous ROS production, and (2) increasing the expression of endogenous ROS scavenging enzymes.

Antioxidant activity of ac¸aı´: human studies Although the antioxidant capacity of ac¸aı´ has been described in several in vitro and animal studies, human studies are still limited, and have mainly associated the effect of ac¸aı´ consumption on long-term oxidative metabolism. A study evaluated 30 healthy women who consumed ac¸aı´ pulp for 4 weeks. Ac¸aı´ intake increased CAT activity and reduced the production of ROS in polymorphonuclear cells (Fig. 12.3) and increased the

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Applications to other areas of pathology

FIGURE 12.3 Reduction in ROS after supplementation with 200 g of ac¸aı´ pulp for 4 weeks in healthy woman.39 Data are expressed as the mean and SD. ROS, reactive species oxygen; SD, standard deviation. Source: I thank Elsevier and the Journal Nutrition for permission to use this figure.

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cholesterol esters to high-density lipoprotein, emphasizing the positive effect of ac¸aı´ on lipid metabolism and confirming the effect of ac¸aı´ on oxidative metabolism in humans.40 Table 12.1 contains a summary of the results found in the published studies discussed in this chapter. Although ac¸aı´ has shown a highly antioxidant effect and its involvement in the redox state, few human studies have been performed. Therefore more research is needed to elucidate the mechanisms of the fruit, if indirectly or directly, on oxidative metabolism. Considering that the imbalance of oxidative status is a process underlying pathological mechanisms of some important chronic diseases such as diabetes, cancer and cardivascular diseases, it is important to establish safe dietary recommendations for humans.

Applications to other areas of pathology

FIGURE 12.4 The increase in total antioxidant capacity in polymorphonuclear cells after supplementation with 200 g of ac¸aı´ pulp during 4 weeks in healthy woman.39 Data are expressed as the mean and SD. SD, standard deviation. Source: I thank Elsevier and the journal Nutrition for permission to use this figure.

total antioxidant capacity in serum. In addition, there was an increase in total antioxidant capacity in the polymorphonuclear cells of volunteers (Fig. 12.4), the serum concentration of protein carbonyl was reduced, and the total sulfhydryl groups increased. These findings suggest that the daily intake of ac¸aı´ was able to increase antioxidant capacity and protect cells from oxidative damage.39 The same group also reported that the intake of ac¸aı´ was able to decrease serum concentrations of oxidized low-density lipoprotein (LDL) and malonic dialdehyde, both products of OS. There was also a serum increase of PON enzyme activity and of the transfer of

It is well established that OS plays a central role in the pathogenesis and development of numerous diseases. Maintaining the homeostasis of the redox system is important to ensure control of OS, and can reduce the risk of developing chronic diseases through various mechanisms. Ac¸aı´ has been shown to prevent OS via antioxidant activity in various models. Furthermore, ac¸aı´ has also been found to have antiinflammatory, anticarcinogenic, and antiproliferative effects.41
45 Several studies have focused on the role of bioactive compounds of ac¸aı´ on the microbiome. It is known that intestinal microbiota play an important role on the absorption of components of the diet and can be modified by the consumption of different nutrients. In fact, ac¸aı´ polyphenols may be degraded during the digestion process, but not completely destroyed. One part of the polyphenols is absorbed in the colon, modifying physiological functions on the intestinal microbiota, promoting changes in groups of bacteria, and increasing the production of short-chain fatty acids.46 Thus, adding ac¸aı´ to the regular diet may promote health bennefits. However there is still a lack of human studies evaluating the bioavailability of nutrients and phytochemicals present in this fruit. In addition, it is necessary to know which molecular pathways are modified or regulated by ac¸aı´, for understanding of how the consumption of this fruit can lead to the beneficial effects reported by the studies. We believe that the effects of ac¸aı´ found in the literature make this fruit an important ally to human health.

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12. Ac¸aı´ (Euterpe oleracea Martius) as an antioxidant

Summary of several studies evaluating the antioxidant effects of ac¸aı´ (Euterpe oleracea Martius).

TABLE 12.1 Study

Experimental model

Ac¸aı´

Results

Schauss et al. Polymorphonuclear cells Freeze-dried ac¸aı´ pulp (2006)

Decreased ROS

Souza et al. (2010)

Hypercholesterolemic rats

Ac¸aı´ pulp (200 g added in the diet during 6 weeks)

Decreased the levels of carbonyl proteins and increased levels of sulfhydryl groups in the serum

Guerra et al. (2011)

Streptozotocin-induced diabetic rats

Ac¸aı´ pulp (200 g added in the diet during 30 days)

Increasing glutathione content and reduction of TBARS levels in the liver

VrailasMortimer et al. (2012)

Drosophila melanogaster

Ac¸aı´ extracts

Reduction of the enzyme GSTD1 transcription

Laslo et al. (2013)

Drosophila melanogaster— Freeze-dried ac¸aı´ pulp Model SOD1 mutant

Extended lifespan

Bonomo et al. Caenorhabditis elegans (2014)

Lyophilized ac¸aı´

Reduced ROS production and of sulfhydryl group levels

Santos et al. (2014)

Rat primary astrocytes

Ac¸aı´ extract from freeze-dried ac¸aı´ powder

Prevented Mn-induced OS by restoring the GSH/GSSG ratio and reducing lipid peroxidation

El Morsy et al. (2015)

Rats—Model renal ischemia/reperfusion (I/R) injury

Ac¸aı´ extract (500 or 1000 mg/kg Reduced in MDA renal level for 15 days by oral gavage)

Barbosa et al. Healthy women (2015)

Ac¸aı´ pulp (intake 200 g during 4 weeks)

Increased CAT and decreased ROS in polymorphonuclear cells In the serum, increased the total antioxidant capacity and the total sulfhydryl groups and reduced protein carbonyl levels

Pereira et al. (2016)

Hypercholesterolemic rats

Ac¸aı´ pulp (200 g added in the diet during 6 weeks)

Increased PON activity and mRNA expression in the liver

Zhou et al. (2018)

Rats—Model de alcoholic liver diseases (ALD)

Ac¸aı´ fruit (1 mL/100 g was In the liver increased in the level of glutathione and the activity of administered by oral gavage for SOD and reduction the content of MDA 8 weeks)

Pala et al. (2018)

Healthy women

Ac¸aı´ pulp (intake 200 g during 4 weeks)

Summary points • This chapter focuses on the antioxidant activity of ac¸aı´ fruit (Euterpe oleracea Martius). • Antioxidant properties of the ac¸aı´ are attributed to the presence of polyphenols, mainly anthocyanins. • Studies have shown that ac¸aı´ improves the oxidative status both in vitro and in vivo. • Research was carried out on people to endorse the effect of the ac¸aı´ on oxidative metabolism. • More studies are necessary to investigate the molecular mechanisms by which the antioxidant effects from ac¸aı´ happen.

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6. 26. Pacheco-Palencia LA, Talcott ST, Safe S, Mertens-Talcott S. Absorption and biological activity of phytochemical-rich extracts from acai (Euterpe oleracea Mart.) pulp and oil in vitro. J Agric Food Chem 2008;56(10):3593
600. 27. Machado AK, Andreazza AC, da Silva TM, et al. Neuroprotective effects of acai (Euterpe oleracea Mart.) against rotenone in vitro exposure. Oxid Med Cell Longev 2016;2016:8940850. 28. da Silva Santos V, Bisen-Hersh E, Yu Y, et al. Anthocyanin-rich acai (Euterpe oleracea Mart.) extract attenuates manganeseinduced oxidative stress in rat primary astrocyte cultures. J Toxicol Environ Health A 2014;77(7):390
404. 29. Wong DY, Musgrave IF, Harvey BS, Smid SD. Acai (Euterpe oleraceae Mart.) berry extract exerts neuroprotective effects against beta-amyloid exposure in vitro. Neurosci Lett 2013;556:221
6. 30. de Souza MO, Silva M, Silva ME, Oliveira Rde P, Pedrosa ML. Diet supplementation with acai (Euterpe oleracea Mart.) pulp improves biomarkers of oxidative stress and the serum lipid profile in rats. Nutrition 2010;26(7-8):804
10. 31. El Morsy EM, Ahmed MA, Ahmed AA. Attenuation of renal ischemia/reperfusion injury by acai extract preconditioning in a rat model. Life Sci 2015;123:35
42.

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32. Guerra JF, Magalhaes CL, Costa DC, Silva ME, Pedrosa ML. Dietary acai modulates ROS production by neutrophils and gene expression of liver antioxidant enzymes in rats. J Clin Biochem Nutr 2011;49(3):188
94. 33. Brasil A, Rocha FAF, Gomes BD, et al. Diet enriched with the Amazon fruit acai (Euterpe oleracea) prevents electrophysiological deficits and oxidative stress induced by methyl-mercury in the rat retina. Nutr Neurosci 2017;20(5):265
72. 34. Zhou J, Zhang J, Wang C, et al. Acai (Euterpe oleracea Mart.) attenuates alcohol-induced liver injury in rats by alleviating oxidative stress and inflammatory response. Exp Ther Med 2018;15(1):166
72. 35. Pereira RR, de Abreu IC, Guerra JF, et al. Acai (Euterpe oleracea Mart.) upregulates paraoxonase 1 gene expression and activity with concomitant reduction of hepatic steatosis in high-fat dietfed rats. Oxid Med Cell Longev 2016;2016:8379105. 36. Bonomo LF, Silva DN, Boasquivis PF, et al. Acai (Euterpe oleracea Mart.) modulates oxidative stress resistance in Caenorhabditis elegans by direct and indirect mechanisms. PLoS One 2014;9(3): e89933. 37. Vrailas-Mortimer A, Gomez R, Dowse H, Sanyal S. A survey of the protective effects of some commercially available antioxidant supplements in genetically and chemically induced models of oxidative stress in Drosophila melanogaster. Exp Gerontol 2012;47(9):712
22. 38. Laslo M, Sun X, Hsiao CT, Wu WW, Shen RF, Zou S. A botanical containing freeze dried acai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies. Age (Dordr) 2013;35(4):1117
32. 39. Barbosa PO, Pala D, Silva CT, et al. Acai (Euterpe oleracea Mart.) pulp dietary intake improves cellular antioxidant enzymes and biomarkers of serum in healthy women. Nutrition 2016;32(6):674
80. 40. Pala D, Barbosa PO, Silva CT, et al. Acai (Euterpe oleracea Mart.) dietary intake affects plasma lipids, apolipoproteins, cholesteryl ester transfer to high-density lipoprotein and redox metabolism: a prospective study in women. Clin Nutr 2018;37(2):618
23. 41. Feio CA, Izar MC, Ihara SS, et al. Euterpe oleracea (acai) modifies sterol metabolism and attenuates experimentally-induced atherosclerosis. J Atheroscler Thromb 2012;19(3):237
45. 42. Holderness J, Schepetkin IA, Freedman B, et al. Polysaccharides isolated from Acai fruit induce innate immune responses. PLoS One 2011;6(2):e17301. 43. Noratto GD, Angel-Morales G, Talcott ST, Mertens-Talcott SU. Polyphenolics from acai (Euterpe oleracea Mart.) and red muscadine grape (Vitis rotundifolia) protect human umbilical vascular Endothelial cells (HUVEC) from glucose- and lipopolysaccharide (LPS)-induced inflammation and target microRNA-126. J Agric Food Chem 2011;59(14):7999
8012. 44. Machado DE, Rodrigues-Baptista KC, Alessandra-Perini J, et al. Euterpe oleracea extract (Acai) is a promising novel pharmacological therapeutic treatment for experimental endometriosis. PLoS One 2016;11(11):e0166059. 45. Da Silva BJM, Souza-Monteiro JR, Rogez H, Crespo-Lopez ME, Do Nascimento JLM, Silva EO. Selective effects of Euterpe oleracea (acai) on Leishmania (Leishmania) amazonensis and Leishmania infantum. Biomed Pharmacother 2018;97:1613
21. 46. Alqurashi RM, Alarifi SN, Walton GE, Costabile AF, Rowland IR, Commane DM. In vitro approaches to assess the effects of acai (Euterpe oleracea) digestion on polyphenol availability and the subsequent impact on the faecal microbiota. Food Chem 2017;234:190
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C H A P T E R

13 Amaryllidaceae alkaloids and neuronal cell protection Natalie Cortes1, Rafael Posada-Duque2,3, Gloria Patricia Cardona-Go´mez3, Jaume Bastida4 and Edison Osorio2 1

Grupo de Investigacio´n en Sustancias Bioactivas, Facultad de Ciencias Farmace´uticas y Alimentarias, Universidad de Antioquia UdeA, Medellı´n, Colombia 2Institute of Biology, Faculty of Exact and Natural Sciences, Universidad de Antioquia UdeA, Medellı´n, Colombia 3Cellular and Molecular Neurobiology Area, Group of Neuroscience of Antioquia, Faculty of Medicine, Universidad de Antioquia UdeA, Medellı´n, Colombia 4Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farma`cia i Cie`ncies de l’Alimentacio´, Universitat de Barcelona, Barcelona, Spain

List of abbreviations DPPH ABTS 4-HNE AMPA AChE ATP AD AAs Aβ BBB BuChE CNS ER FRAP HAT LDH MDA MAPK NADPH NMDA NVU nAChR NOS ORAC OS RNS ROS SET NR1 NR2B-D

1,1-diphenyl-2-picrylhydrazyl 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 4-hydroxy-2-trans-nonenal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid acetylcholinesterase adenosine triphosphate Alzheimer’s disease Amaryllidaceae alkaloids amyloid-β blood
brain barrier butyrylcholinesterase central nervous system endoplasmic reticulum ferric reducing ability of plasma hydrogen atom transfer lactate dehydrogenase malondialdehyde mitogen-activated protein kinase pathway nicotinamide adenine dinucleotide phosphate N-methyl-D-aspartic acid neurovascular unit nicotinic acetylcholine receptor nitrous oxide systems oxygen radical absorbance capacity oxidative stress reactive nitrogen species reactive oxygen species single electron transfer subunit of the N-methyl-D-aspartate receptor subunit of the N-methyl-D-aspartate receptor

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00013-5

Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease and the most prevalent cause of dementia, being responsible for approximately 70% of cases.1 An irreversible neurological disorder, AD is characterized by memory failure, cognitive dysfunction, behavioral disturbances, difficulties in carrying out daily activities, and disorientation of time and place.2 The pathogenesis of AD is linked with the accumulation of extracellular β-amyloid plaques, progressive intracellular tau pathology, mitochondrial abnormalities, and neuroinflammatory processes, which slowly affect neurons, resulting in loss of memory and the ability to store new information.1,3 The main cause of AD is thought to be the generation of reactive oxygen species (ROS) as a result of oxidative stress (OS).4 In pathological conditions, such as AD, various factors are responsible for the accumulation of both ROS and reactive nitrogen species (RNS). Free radicals can be generated by an imbalance of neurotransmitters, when there is also an increase in the intraneuronal concentration of Ca21, by microglial activation, mitochondrial biochemical reactions, and by β amyloid plaques.5 Due to a high lipid content, the brain is particularly vulnerable to damage caused by ROS and RNS, and brain membranes are the first

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© 2020 Elsevier Inc. All rights reserved.

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13. Amaryllidaceae alkaloids and neuronal cell protection

target.6 The reaction products of lipid peroxidation lead to the decomposition of polyunsaturated fatty acids and the formation of reactive aldehydes, such as 4-hydroxy-2-trans-nonenal (4-HNE) and malondialdehyde (MDA), which react with deoxyribonucleic acid and certain proteins, damaging their structure and impairing their function.7 Therefore OS could be considered as a therapeutic target in AD treatment. There are currently no disease-modifying therapies for AD. Available therapeutic approaches to AD provide only modest symptomatic relief and are aimed at cognitive symptoms, such as disturbances in memory and perception.1,8 These drugs are classified into two groups, N-methyl-D-aspartic acid (NMDA) receptor antagonists and acetylcholinesterase (AChE) inhibitors.9 Memantine is the only NMDA receptor antagonist drug on the market for AD treatment, while AChE inhibitors (including galanthamine, donepezil, and rivastigmine), which increase the availability of acetylcholine at central cholinergic synapses, predominate. However, the problem with currently approved AD drugs is that in addition to being only symptomatic therapies, they only work for a short period of time.1,8 Therefore the development of new strategies for AD therapy is one of the timeliest challenges in modern medicine. Given the importance of increased OS in the pathogenesis of AD, new treatments could focus on neuronal cell protection through antioxidant actions. Natural compounds provide a variety of structural features and biological activities and are an attractive source for developing therapeutic drugs. The potent AChE inhibitor derived from natural sources, galanthamine, was the first Amaryllidaceae alkaloid (AA) to be approved as a prescription drug in the treatment of AD.10,11 Its effectivity has prompted the screening of other AAs of different structural types for their biological activity, and results have shown that neuroprotective effects might be associated, above all, with lycorine- and crinine-type alkaloids. AChE enzyme inhibitory activity is related to galanthamine- and lycorine-type alkaloids.9,12
15 However, very little research has been published on the protective effects of AAs against oxidative neural cell injury. In a recent study,16 it is suggested that the potential of AAs as neuroprotective agents against oxidative stimulus is due to their structural versatility. This chapter focuses on the available data on the antioxidant activity of AAs and its relationship with their neuroprotective effects.

Neurodegenerative diseases and neuronal cell damage Dementia is defined as a clinical syndrome characterized by a progressive deterioration of cognitive

functions involving all synaptic functions.17 By the year 2001, around 25 million people were affected by dementia and it is estimated that the number would grow to about 81 million by 2040.18 AD is the most common type of dementia, accounting for 50%
70% of cases worldwide, and is therefore a major public health problem.19 Other types of brain conditions have been associated with the development of neurodegenerative processes that trigger dementia, including cerebrovascular accidents, traumas, and metabolic diseases.20 Notably, the coexistence of these comorbidity factors and neurodegenerative diseases is known to be strongly linked with the development of dementia, suggesting a reciprocal interaction between ischemia, diabetes, head trauma, and neurodegeneration.21 Epidemiological studies indicate that AD and cerebrovascular accidents share the same risk factors, and this fact has redirected interest in neurovascular factors as fundamental elements in the pathogenesis of neurodegenerative diseases.21,22 At the molecular level, the development of neuronal injury is largely due to an increase in the release of excitatory neurotransmitters to the synaptic cleft, mainly glutamate. This process, known as “excitotoxicity,” is a canonical response to pump failure, ion energy deficit, and insufficient neurotransmitter uptake mechanisms by astrocytic cells.23 Glutamate overload leads to prolonged stimulation of ionotropic glutamate receptors of AMPA (α-amino acid-3hydroxy-5-methyl-4-isoxazolepropionic acid) type and NMDA receptors, drastically increasing the influx of Ca21, Na1, K1, and water in neurons. The excessive accumulation of ions and the simultaneous deregulation of several signaling pathways mediated by proteases, lipases, and nucleases alter neuronal function and lead to cell death.24 After cerebral ischemia or AD-type neurodegeneration, a very broad spectrum of pathophysiological mechanisms is activated in the neurovascular unit (NVU). Historically, these mechanisms can be divided into excitotoxic, free radical and proapoptotic pathways, in which the imbalance of Ca21 could be involved in most of these multifactorial signals. It has been assumed that the accumulation of intracellular Ca21 and generalized ionic imbalance are the major mediators of cell death in a neurodegenerative process induced by ischemia and AD.25,26 At the cellular level, the generation of ROS, nitric oxide, peroxynitrites, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress leads to ionic deregulation and metabolic stress. In the case of vascular dementia, interruption of blood supply to the brain during ischemia results in deprivation of oxygen and glucose and, therefore, a reduction in available energy for the functioning of

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Oxidative stress related with neuronal cell damage

brain cells.27 Neurons in particular become unable to maintain the ionic gradients necessary for cellular function and homeostasis.28 This results in excessive neuronal depolarization, increased release of excitatory neurotransmitters, and reduced reuptake of these neurotransmitters from the extracellular space. This positive feedback induces a simultaneous excessive accumulation of Ca21 and Na1 ions, with the spreading of multiple signaling pathways, activating the catabolic processes, which affect the NVU.27,28 Increased concentration of glutamate in the central nervous system (CNS) occurs in various pathologies including ischemia and AD. It results in the hyperactivation of glutamatergic receptors.29,30 The different components of the NVU are susceptible to this toxicity process, which in neurons is called excitotoxicity and in glial cells, gliotoxicity.31,32 Endothelial cells are known to recognize high levels of glutamate as toxic, specifically in the cerebral microvasculature.33,34 In neurons, glutamate excitotoxicity results in reduced adenosine triphosphate (ATP) synthesis, ROS activation, calpains, ER stress, activation of MAPK and NOS, and consequent deterioration of the actin and microtubule cytoskeleton, dendrite loss, and cellular death.35,36 Some studies show that gliotoxicity induced by glutamate facilitates the influx of intracellular Ca21 and the exit of reservoirs of ER and mitochondria, increasing the release of LDH, generation of ROS, and a decrease of glutathione and ATP, which are necessary for the coupling with neuronal activity.31,32 Also, it has been shown in vivo that when endothelial cells of the cerebral microvasculature, adult or neonatal, are exposed to glutamate, the blood
brain barrier (BBB) is permeabilized, which can induce activity of the tissue plasminogen activator.33,34 However, in investigations conducted in brain microvasculature cells, it has been shown that this cell type expresses NMDA receptors, such as NR1 and NR2B-D, which apparently have a crucial role in the integrity of the BBB.37 The exposure of high concentrations of glutamate increases the RNS (e.g., peroxynitrites) and decreases cell viability, an effect that is reversed by dizocilpine, a noncompetitive antagonist of the NMDA receptor, especially in neurovascular components.33,34

Oxidative stress related with neuronal cell damage ROS production under normal conditions is directly linked to well characterize biochemical pathways. It is an inevitable consequence of respiration and also occurs through other processes that generate multiple signaling molecules and species related to antioxidant defense. However, the brain is especially susceptible to

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oxidative imbalance due to its high-energy demand, high-oxygen consumption, and an important concentration of easily oxidizable molecules. Therefore it must compensate continuously for the products of the high metabolism of oxygen, which are easily susceptible to changes in the blood flow. This balance of ROS depends on a series of antioxidant mechanisms consisting mainly of the enzymes superoxide dismutase, glutathione peroxidase, glutaredoxins, thiodoroxins, and catalase, which are tissue- and cell-specific and maintain the homeostasis of these reactive species. If these normal mechanisms of compensation and regulation become ineffective in resolving cell damage, characterized by the imbalance between ROS production and elimination, deregulated mechanisms of the pathological process of the disease are initiated. The exacerbation of OS decreases the ability to eliminate excess ROS in the cell, which leads to reactions of lipid peroxidation of plasma and mitochondrial membranes, and the oxidation of structural and enzymatic proteins. The subsequent irreversible modification of their structure and tertiary function38 is followed by apoptosis.39 In this way, OS becomes a cyclic process where the ROS produced destroy the biomolecules, which eventually leads to a greater accumulation of these reactive species.5 Although AD has been characterized mainly through molecular mechanisms of neuronal dysfunction, considerable evidence indicates that ROS and OS contribute to associated dementias through multiple mechanisms.39 The neurotoxic oligomer amyloid-β (Aβ), which is the main neuropathological diagnostic criteria of the disease, together with the hyperphosphorylated protein τ, are the principal mediators of neurodegeneration and there is a causal relationship between the imbalance of ROS and these misfolded proteins. Several lines of evidence have shown that the formation of Aβ promotes the production of ROS, since Aβ activates the NADPH-dependent oxidase enzyme, leading to the production of O2•
.40 In addition, H2O2 is generated directly during the Aβ aggregation process and this oligomer can simultaneously convert molecular oxygen into H2O2 by reducing the divalent metal ions (Fe21, Cu21). On the other hand, the Aβ oligomers are inserted into the membrane bilayers, resulting in the production of ROS followed by the oxidation of nucleic acids and intracellular proteins. Among the products of lipid peroxidation, 4-HNE is highly reactive and occurs mainly in the brain as a consequence of the lipid peroxidation of arachidonic acid, a very abundant component of polyunsaturated fatty acid omega-6 neuronal membranes.40 Aβ also promotes neurotoxicity by interfering with Ca21 homeostasis, since Aβ-induced 4-HNE impairs membrane Ca21 pumps and promotes its entry through

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13. Amaryllidaceae alkaloids and neuronal cell protection

voltage-dependent and ligand-dependent calcium channels. The excess intracellular calcium plays a central role in excitatory neurotoxicity, triggering neurodegenerative processes characterized by the alteration of synaptic plasticity, imbalance in the production and clearance of neurotransmitters, inflammation, alterations in dendritic projections, and substantial synaptic loss that consequently leads to neuronal death.38

Current pharmacotherapy of dementias (mainly Alzheimer’s disease) To date, although none of the drugs approved for the treatment of AD by the US Food and Drug Administration completely protects the brain from the degenerative process characteristic of the disease,41 current pharmacotherapeutic approaches improve the quality of life of patients due to their effect on the cholinergic and glutamatergic systems (Fig. 13.1). Commercial drugs work by regulating the levels of certain neurotransmitters in the brain, mainly acetylcholine and glutamate. Although initially only the inhibition of the enzyme AChE was considered as the main therapeutic strategy, the synergism presented with memantine, a NMDA receptor blocker prescribed in advanced stages of AD, is a more robust approach to treat the symptoms of late-stage AD patients.42

Galanthamine, the only drug of natural origin for the symptomatic treatment of Alzheimer’s disease Unlike the semisynthetic donepezil and rivastigmine, and synthetic memantine, galanthamine is an isoquinolinic alkaloid produced by plants of the Amaryllidaceae family. This compound was discovered and isolated for the first time from the bulbs of Galanthus nivalis (common snowdrop) by the Bulgarian chemist D.S. Paskov and his team in 1956.43 Although in its therapeutic beginnings, galanthamine was used to treat poliomyelitis, as an anesthetic, and for the treatment of neuropathic pain,44 its effective action on the CNS promoted it as a medicine for the palliative treatment of AD. Presenting a dual mode of action on the cholinergic system, either as a reversible and competitive inhibitor of the AChE enzyme or as an allosteric modulator of the nicotinic acetylcholine receptor (nAChR), galanthamine is a well-established drug for the treatment of AD, by modulating the signaling of acetylcholine. Galanthamine can be effective not only by itself, but can also interact with other molecular targets, thereby enhancing the effect of other drugs on the CNS. For example, the activity of P-glycoprotein, a multidrug resistance transporter present in the vascular endothelium of the brain, decreases after interaction with galanthamine.45 In addition, galanthamine increases

FIGURE 13.1 Drugs approved for the symptomatic treatment of Alzheimer’s Disease.

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Efficacy of amaryllidaceae alkaloids in oxidative stress and neuronal cell damage

the protective effect of rofecoxib (an antiinflammatory inhibitor of cyclooxygenase 2) and caffeic acid against mitochondrial dysfunction induced by neurotoxicity, oxidative damage, and cognitive impairment in rats.46 In this biological context, galanthamine, in addition to facilitating the antioxidant activity of other molecules, acts on its own in oxidative processes that are the protagonists of the neurodegenerative process of AD. In fact, it intervenes in mitochondrial dysfunction, modulating the changes in the mitochondrial membrane potential and the neuronal morphology induced by the treatment with different oxidative agents such as Aβ peptides or hydrogen peroxide.47 In addition to interacting with proteins related to OS, galanthamine can also stabilize reactive molecules due to the presence of the enol group in its structure.16 The set of modulatory and reactive activities of galanthamine in the pathogenesis of AD opens new perspectives on the potential of molecules with a similar biosynthetic origin. The chemical diversity that accompanies the different biological activities of AAs is an interesting point in the search for new therapeutic alternatives for the palliative and modulatory treatment of AD.

Amaryllidaceae alkaloids AAs are a key chemotaxonomic feature of the Amaryllidoideae subfamily of the Amaryllidaceae plant family, renowned for its alkaloid-containing species. This subfamily includes more than 800 perennial bulb species classified into 59 genera that are found throughout the tropics and temperate regions worldwide. Since Gerrard’s isolation of lycorine from Narcissus pseudonarcissus in 1877, more than 550 AAs have been chemically described. Besides the predominant tertiary bases, N-oxides, salts, and dimer alkaloids have also been found. Although it is unusual to find other alkaloid types in the Amaryllidoideae subfamily, Sceletium alkaloids have been reported as well as some tetrahydroisoquinoline alkaloids, such as capnoidine and bulbocapnine from various Galanthus species. The identification of new alkaloids of diverse skeleton types has become easier due to advances in increasingly sensitive analytical techniques, with gas chromatography
mass spectrometry and nuclear magnetic resonance (1D and 2D) currently being the methods most used for structural characterization. The structurally varied AAs are grouped into nine main skeleton types, represented by norbelladine, lycorine, homolycorine, crinine, haemanthamine, narciclasine, tazettine, montanine, and galanthamine (Fig. 13.2). Ghosal’s numbering system is used here for all the skeleton types.48

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Plants of the Amaryllidaceae plant family have been used for thousands of years as natural remedies, and the alkaloids from their extracts have been the target of chemical research for nearly 200 years. Over the past three decades many alkaloids have been isolated, synthesized, and screened for biological properties. The wide range of biological and pharmacological activities exhibited by AAs, including AChE/BuChE inhibitory, antitumoral, cytostatic, antivirus, and antifungal, have attracted growing research interest. As already mentioned, galanthamine has been commercialized for the treatment of AD, while other lycorine-, haemanthamine-, and narciclasine-type alkaloids have been used as lead molecules in anticancer research.49
51 The biological and pharmacological activities of AAs have been extensively reviewed (e.g., Refs. 51,52) and the alkaloids of the most studied genera, such as Crinum,48,53 Narcissus,54,55 Galanthus,56 Hippeastrum,57 Lycoris,58 Pancratium,59 and Zephyranthes,60 have been the subject of monographic chapters. Our understanding of the biosynthesis of AAs (Fig. 13.3) has been enhanced by state-of-the-art omics technologies. Transcriptome analysis of the coexpression of biosynthetic genes has enabled the identification and characterization of norbelladine 40 -O-methyltransferase, which catalyzes the methylation of norbelladine,61 and CYP96T1, which catalyzes the para-para0 phenol coupling.62 The current knowledge of AA biosynthesis has been reviewed.63

Efficacy of amaryllidaceae alkaloids in oxidative stress and neuronal cell damage AAs have been reported as molecules with interesting pharmacological properties, including neuroprotective,12,14,15 apparently due to an ability to stabilize free radicals generated in excitotoxic processes.16 The antioxidant effects reported for Amaryllidaceae species are shown in Table 13.1, although the antioxidant and/or antiradical activity of extracts, fractions, or molecules cannot be based on a single test because of the different mechanisms by which a reactive molecule or product of OS can be stabilized. The mechanisms reported for the stabilization of these species involve hydrogen atom transfer (HAT), single electron transfer (SET), and the ability to chelate transition metals.69 HAT assays include the ORAC and inhibition of lipoperoxidation assays. SET methods are composed of FRAP, DPPH, and ABTS, among others. Certain plants of the family Amaryllidaceae, although not evaluated for their antioxidant activity, show neuroprotective properties against toxic stimuli that generate cellular OS. Extracts of Crinum jagus (J. Thomps.) Dandy, Hippeastrum elegans (Spreng.)

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12

11

OH 3

HO

4

HO

2

MeN

2

1

4 3

3

HO

4′

5′

10

6′

5

1 6

HO

3′

2′

9

O

β′

7

MeO

9

H

H 10b

10a

2 1

H

12

MeO

6

Norbelladine

10

11

N

6a

8

4

4a

H

α

NH

1

O

β

H

10b 10a

4a

Lycorine

8

6a

O

6

7

Homolycorine

O OH

2 1

2

OH

3

11

11 10

10

O

10a

9

10b

H O

8

6a

7

O

4

4a

9

10a

H

12

N

O

8 7

9

N

O

OH

4

OH

10a 10b

8

6a

6

4a

NH

6

7

OH

Haemanthamine

Crinine

3

H

10

O

12

6a

6

1

4

4a

10b

2

OMe OH

3

1

O

Narciclasine

OMe 2 10

O O

11

O

12

3

10a

O

8

4a 11

6

7

Montanine

N

6a

8

6

Tazettine

10a

12

N H

6a 7

4

10b

10 9

4

12

6

O MeO

OH

4a

3

1

2

11 11a

10 9

OH

O

6a 7

1

H NMe

4a

1

10b

8

OMe

4

10a

9

OH

2

3

Galanthamine

FIGURE 13.2 The main types of Amaryllidaceae alkaloids. HO

NH2 HO

HO

COOH

L-Phe

CHO

HO

Protocatechuic aldehyde

H2N

Tyramine

L-Tyr

H2N HO

HO

HO HO

HO N

HO

H

NH

O

N

H

Schiff's base (isomeric structures in solution)

HO HO RO

Norbelladine: R=H O-metilnorbelladine: R=Me Otho-para' Types

OH

4'

NH

HO

Para-para' Types

Lycorine Homolycorine

FIGURE 13.3 Biosynthetic pathway of the Amaryllidaceae alkaloids.

Crinine Haemantamine Tazettine Narciclasine Montanine

COOH

Para-ortho' Types Galanthamine

Me

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Conclusion

TABLE 13.1

Antioxidant and/or neuroprotector effects of amaryllidaceae species.

Plant name

Part(s) of plant evaluated

Type of extract(s) used

Models/ Tests performed

Type of biological model

Antioxidant alkaloids and/or neuroprotectors

References

Roots and leaves

Chloroformic

ORAC, FRAP, TBARS

In vitro, in silico

Lycoramine, Anhydrolycorine, 8-Odemethylmaritidine, lycorine

16

Crinum asiaticum L.

Leaves

Ethanolic

DPPH, TBARS

In vitro, ex vivo

Lycorine

64

Crinum jagus (J. THOMPS.) DANDY.

Bulbs

Methanolic

DPPH

In vitro

Nonidentified

65

Crinum latifolium L.

Bulbs

Ethanolic

ABTS1, DPPH

In vitro

4,8-dimethoxy-cripowellin, 9-methoxycripowellin, 4-methoxy-8-hydroxy-cripowellin, cripowellin

66

Crinum ornatum (L.f.) Herb.

Bulbs

Ethanolic

DPPH

In vitro

Lycorine, crinamine, heamanthamine, hamayne, ornamine

67

Eucharis bonplandii (Kunth) Traub.

Bulbs

Chloroformic

ORAC, FRAP, TBARS

In vitro, in silico

Galanthamine, sanguinine, vittatine, 8-Odemethylmaritidine, lycorine

16

Eucharis caucana Meerow.

Bulbs

Chloroformic

ORAC, FRAP, TBARS

In vitro, in silico

Vittatine, 8-O-demethylmaritidine, tazettine

16

Hippeastrum morelianum Lem.

Root, leaves and bulbs

Ethanolic

DPPH

In vitro

Lycorine

68

Hippeastrum psittacinum Herb.

Bulbs

Ethanolic

DPPH

In vitro

Lycorine

68

Hippeastrum santacatarina (Traub) Dutilh

Bulbs

Ethanolic

DPPH

In vitro

Lycorine

68

Narcissus broussonetii Lag.

Bulbs

Ethanolic

DPPH

In vitro

Tazettine, ismine, homolycorine, lycorine, 8-Odemetilhomolicorine, 3-epimacronine

69

Clivia miniata (Lindl.) Bosse

H.E. Moore, Lycoris aurea (L’He´r.) Herb, Phaedranassa lehmannii Regel, and Zephyranthes carinata Herb are rich in crinine, galanthamine, lycorine, and tazettine alkaloids, nucleus with potential antiradical activity.12,14,16 In addition, in order to corroborate in vitro results, in silico trials have been integrated to evaluate the theoretical free radical stabilizing capacity of the alkaloids. The energies needed to remove an electron and release a hydrogen atom are thermodynamic parameters that can be used to evaluate the most probable mechanism by which the alkaloids are acting, and in this way explain their in vitro activity.16 Thus extracts and/or fractions rich in galanthamine, crinine, tazettine, and/or lycorine alkaloids have a potential neuroprotective activity via stabilization of radical species. However, it cannot be ruled out that they are simultaneously activating other biochemical pathways related to the activation of the endogenous antioxidant system or neuronal survival routes.

Conclusion An important feature of AD, the most common form of dementia worldwide, is the presence of oxidative damage at the neuronal level that is thought to be mainly due to the overproduction of ROS.5,6 To date, two classes of drugs have been approved to treat AD, although therapeutic strategies remain essentially symptomatic. AD is increasingly considered as a multifactorial disease that needs to be treated with drugs covering multiple targets, including the inhibition of OS. Nevertheless, to date no clinical trial has clearly demonstrated that a specific antioxidant intervention prevents AD progression. Therefore more extensive research is required to develop alternative therapeutic strategies. Among alkaloids with multiple targets, AAs have been reported as molecules with interesting pharmacological properties, including neuroprotective effects that are apparently due to an ability to stabilize

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13. Amaryllidaceae alkaloids and neuronal cell protection

free radicals generated in excitotoxic processes. These molecules may therefore play a role in the development of new classes of drugs. Further studies are required to determine whether AAs could be potential drug candidates for multifunctional treatment of AD, including antioxidant therapy, as well as to elucidate the complex relationships among AAs, oxidation, and AD.

• This chapter provides an overview of neurodegenerative diseases and the efficacy of AAs in treating neuronal cell damage.

Acknowledgments The authors express their gratitude to the Iberian-American Programme for Cooperation and Development (CYTED) (Ref.416RT0511)
BIFRENES Thematic Network.

Applications to other areas of pathology The treatment of many diseases is highly dependent on natural products. Among these, alkaloids are a particular group of nitrogen-containing compounds with low-molecular weight, and active at different cellular levels in the neurodegenerative diseases.10,44 Some alkaloids have demonstrated multiple biological activities in the CNS, and those with potential therapeutic value are grouped according to various pathophysiological mechanisms.44 The antioxidant potential of alkaloids has also been gaining ground, due to their ability to stabilize free radicals generated by a neuronal excitotoxic process. However, the precise mechanism of action of alkaloids on OS is not always clear and could include more mechanisms. While this chapter provides an overview of neurodegenerative diseases and neuronal oxidative cell damage in the main dementia (AD). It is important to revisit others neurodegenerative illness associated with dementia including amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease, and their relationship with antioxidant alkaloids.

Summary points • This chapter focuses on AAs, which are a series of alkaloids unique to Amaryllidaceae plants. • Galanthamine, is the first AA to be approved as a prescription drug in the treatment of AD. • Galanthamine is a potent AChE inhibitor that increases the availability of acetylcholine at central cholinergic synapses. • However, under pathological conditions, in AD there is an imbalance of the neurotransmitters. • This imbalance leads to excitotoxic processes, which causes accumulation of reactive oxygen and nitrogen species. • New treatment strategies to ameliorate AD could be centered on the area of oxidative neural cell injury. • The antioxidant potential of AAs has been gaining ground due to their ability to stabilize free radicals generated by a neuronal excitotoxic process.

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11.

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C H A P T E R

14 Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology Junichi R. Sakaki, Melissa M. Melough and Ock K. Chun Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

List of abbreviations m k 2 ALP BALP BFR BMC BMD Bmp4 BS BV Ca CAT CFA COL CRP CSF-1 CTX DPD DXA ERK FFQ FRAP GPx hs-CRP IFN-γ IGF-1 IL iNOS LPS MAR MCP-1 MDA MIP1α MMP MSC NFATc1 NF-κB NHANES NO

increase decrease no significant difference alkaline phosphatase bone-specific alkaline phosphatase bone formation rate bone mineral content bone mineral density bone morphogenetic protein 4 bone surface bone volume calcium catalase complete Freund’s adjuvant collagen type 1 C-reactive protein colony stimulating factor 1 C-terminal telopeptide of type 1 collagen deoxypyridinoline dual-energy X-ray absorptiometry extracellular signal-regulated kinases food frequency questionnaire free radical antioxidant power glutathione peroxidase high sensitivity C-reactive protein interferon gamma insulin-like growth factor 1 interleukin inducible nitric oxide synthase lipopolysaccharide mineral apposition rate monocyte chemoattractant protein 1 malondialdehyde macrophage inflammatory protein 1 alpha matrix metalloproteinase mesenchymal stem cell nuclear factor of activated T-cells, cytoplasmic 1 nuclear factor kappa B National Health and Nutrition Examination Survey nitric oxide

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00014-7

NTX Ob.S/BS Oc Oc.S/BS OcN OPG ORX OS OVX P1NP PGE2 PPAR-γ RANK RANKL RCT ROS Runx2 SOD sRANKL TAC TBARS TbN TbS TbSp TbT TNF-α TRAP TRAP 1 TRAP5b

N-terminal telopeptide of type 1 collagen osteoblast surface/bone surface osteocalcin osteoclast surface/bone surface osteoclast number osteoprotegerin orchidectomized oxidative stress ovariectomized procollagen type 1 N-terminal propeptide prostaglaindin E2 peroxisome proliferator-activated receptor gamma receptor activator of nuclear factor kappa B receptor activator of nuclear factor kappa B ligand randomized controlled trial reactive oxygen species runt-related transcription factor 2 superoxide dismutase soluble receptor activator of nuclear factor kappa B ligand total antioxidant capacity thiobarbituric acid reactive substances trabecular number trabecular separation trabecular spacing trabecular thickness tumor necrosis factor alpha tartrate-resistant acid phosphatase tartrate-resistant acid phosphatase positive tartrate-resistant acid phosphatase 5b

Introduction Bone health is becoming a growing concern as the population grows older. Based on data from the National Health and Nutrition Examination Survey (NHANES) III (1988
94), it was estimated that in the United States over 40 million adults aged 50 years or older had osteoporosis or low-bone mass.1 Oxidative stress

145

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14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology

(OS) and inflammation are major contributors to agerelated bone loss.2 These pathways are closely associated with one another and are affected by various health conditions as well as modifiable factors such as nutrition. While pharmaceutical treatment is available, poor compliance limits its efficacy as only 40% of patients with age-related bone loss taking medications maintain compliance for the first year, and 20% remain compliant for the second year of treatment.3 Additionally, medications such as bisphosphonates may have deleterious side effects.4 Consequently, dietary treatments to promote bone health have gained considerable attention. Polyphenols are bioactive compounds found in plants and have been investigated for their role in the treatment of chronic diseases such as cardiovascular disease, cancer, and osteoporosis. Anthocyanins represent a subclass of polyphenols and are abundantly found in fruits such as blueberries, blackberries, plums, and grapes. Anthocyanins exhibit powerful antioxidant properties5,6; thus, given the involvement of OS in the development of bone diseases, anthocyanins may improve bone health. The purpose of this chapter is to examine existing literature to identify the roles that anthocyanins and anthocyanin-rich foods play in inhibiting the progression of bone loss and to elucidate the underlying mechanisms at work.

Oxidative stress and inflammation OS results from an imbalance in the redox system, favoring prooxidants over antioxidants. Excessive reactive oxygen species (ROS) and free radicals react with various biomolecules, inducing damage to proteins, lipids, and DNA.7,8 Antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) inhibit the damage induced by prooxidants by scavenging ROS. Thus a reduction in the antioxidant defense system’s capability or an increase in oxidative compounds induces OS. The inflammatory pathway is closely related to OS in that they promote each other. Under inflammatory conditions, proinflammatory agents such as interferon gamma (IFN-γ) and lipopolysaccharide (LPS) induce neutrophils and phagocytes to produce ROS. Additionally, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are produced and promote the production of other inflammatory mediators such as ROS, nitric oxide, and prostaglandin E2.7 Hydrogen peroxide activates nuclear factor kappa B (NF-κB), a transcription factor responsible for regulating the genetic expression of proinflammatory compounds such as cytokines, chemokines, and adhesion molecules,9 repeating the cycle of inflammation and OS.

Bone physiology and pathology under oxidative stress Bone is a metabolically active tissue that adapts to mechanical demands and is capable of self-repair. In order to maintain homeostasis, it undergoes continuous remodeling in which old bone tissue is resorbed by osteoclasts and new bone tissue is formed by osteoblasts. The balance between bone resorption and formation dictates the direction of net bone metabolism: when formation outpaces resorption, the net result is anabolic, and when resorption outpaces formation, the net result is catabolic. Osteoclasts are terminally differentiated myeloid cells that resorb bone by removing bone mineral matrix,10 and can be identified by their expression of tartrate-resistant acid phosphatase (TRAP).11 Osteoclast precursors differentiate into osteoclasts in the presence of the osteoclastogenic cytokines colony stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL).12 Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and thus acts as a negative regulator for osteoclast differentiation.12 The ratio between RANKL and OPG can be interpreted as a measure of osteoclast differentiation and function.13 Osteoclastogenic transcription factors such as c-fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) direct myeloid precursors away from the fate of macrophages and toward osteoclasts.10 Osteoblasts are derived from pluripotent mesenchymal stem cells through the direction of osteoblastogenic transcription factors such as runt-related transcription factor 2 (Runx2) and are responsible for the formation of bone tissue through bone mineralization and the production of bone mineral proteins.10 During bone formation, some osteoblasts become trapped in the mineralized matrix that they secrete, becoming osteocytes. Osteocytes occupy cavities within the bone called lacunae and form a dendritic network between other osteocytes and osteoblasts on the bone surface, which extends throughout the bone. Osteocytes are engaged in maintaining the turnover of the bone matrix and are thought to initiate and direct bone remodeling.10 Under OS, the abundance of ROS contributes to enhanced osteoclast activity and thus increased bone resorption. ROS acts as a signaling mediator for osteoclast differentiation.14 ROS also promotes the expression of RANKL that, after binding to RANK, activates transcription factors such as NFATc1 that promote the expression of osteoclastogenic genes.14,15 Furthermore, the oxidation of lipids generates ligands activating peroxisome proliferator-activated receptor (PPAR-γ) that inhibit Wnt signaling.2 As Wnt signaling is crucial in promoting osteoblast differentiation and activity and inhibiting osteoblast apoptosis, this leads to reduced

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Cell studies

bone formation. ROS also inhibits osteoblast differentiation through its involvement in extracellular signalrelated kinases (ERK) and ERK-dependent NF-κB activation.16

Dietary anthocyanins as antioxidants Since oxidative damage promotes bone resorption, it has been suggested that antioxidant defense protects against bone loss. Antioxidant enzymes catalyze reactions that transform ROS into harmless by-products.8,14 Therefore increasing antioxidant activity may mitigate OS-induced bone resorption. Anthocyanins are plant compounds that are primarily consumed through fruits and fruit products such as berries, juice, and red wine. Table 14.1 displays the anthocyanin content of several foods included in this review.17 Anthocyanins have been investigated for their antioxidant properties.18
21 In animal and cell studies, anthocyanins are studied as individual compounds such as delphinidin or malvidin, which may be bound to a glycoside or found as an aglycone, as a fruit extract or as whole fruit. In human observational studies, anthocyanin intake is typically estimated from a food frequency questionnaire and a polyphenol database, whereas in intervention studies subjects are often provided with whole fruit. A limitation of using whole fruit or whole fruit extracts is that fruits are known to possess other bioactive compounds that may affect redox balance or bone turnover, such as vitamins C and K, potentially confounding the effects of anthocyanins.22 However, these studies still provide valuable insight as they model realistic dietary conditions in which anthocyanins are consumed in their naturally occurring forms as parts of whole foods and entire diets. TABLE 14.1 Food Apple

Cell studies Berry extracts Several cell studies have been conducted to examine the effects of extracts of various berries on indices of bone health (Table 14.2). In RAW 264.7 osteoclast precursor macrophages, bilberry, blackcurrant,23 and ac¸aı´ berry extracts24 reduced osteoclastogenesis, and ac¸aı´ berries also reduced hydroxyapatite resorption, indicating reduced osteoclast activity. Ac¸aı´ berries appeared to induce these effects by inhibiting the production of proinflammatory cytokines and promoting the production of antiosteoclastogenic cytokines.24 In preosteoblast MC3T3-E1 cells, blueberry extract suppressed TNF-αand IL-1-induced NO production, an osteoblast function inhibitor.25

Black rice extract In multipotent C3H10T1/2 cells, black rice extract induced osteogenesis as measured by increased ALP activity.26,27 Black rice extract also increased the mRNA expression of osteoblastogenic markers ALP, Runx2, osteocalcin, and osterix, and increased intracellular calcification.26,27

Dried plum Bu et al., reported that dried plum reduced cyclooxygenase and NFATc1 expression, resorption pit formation, and number of TRAP 1 cells in RAW 264.7 macrophages, indicating reduced osteoclast differentiation.28 The production of inflammatory mediators was reduced, indicating an antiinflammatory effect. This same group reported that dried plum increased

Anthocyanin content in commonly consumed foods (in mg per 100 g or per 100 mL). Total anthocyanins

Cyanidin

Petunidin

Delphinidin

Malvidin

Peonidin

Pelargonidin










42.69

97.59

39.22










1.09

16.69

0.66

1.17

0.02




20.45




1.59

1.57

Bilberry

285.21

85.26

Black raspberry

686.79

669.01

Blackcurrant

157.78

62.46

3.87

89.62




Blueberry

163.3

8.46

31.53

35.43

67.59

20.29




Cranberry

101.05

43.46




7.67

0.44

49.16

0.32

Grape

48.04

1.16

1.97

2.27

39

3.62

0.02

Plum

6.98

4.73




0.02




2.21

0.02

19.27

0.19

1.98

2.01

13.84

1.25




Red wine

The contents of individual and total anthocyanins of commonly consumed fruits and red wine are shown. Data are obtained from the USDA Database for the Flavonoid Content of Selected Foods, Release 3.2.17.

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14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology

TABLE 14.2 Reference

Cell studies assessing the effect of anthocyanin-rich foods and compounds on bone health.

Design

Treatment

Outcomes

Berry extracts Moriwaki et al.23

RAW 264.7 (preosteoclast-like) cells

0.01, 1, and 100 mL21 bilberry and blackcurrant extracts 3 1 h

Bilberry and blackcurrant extracts k osteoclastogenesis (k TRAP 1 cells)

Brito et al.24

RAW 264.7 cells

25, 50 and 100 μg/mL ac¸aı´ berry extract 3 5 days

k Osteoclastogenesis (k TRAP 1 cells) and osteoclast activity (hydroxyapatite resorption) k Proinflammatory cytokine production (IL-1α, IL-6, TNF-α) m Antiosteoclastogenic cytokine production (IL-3, IL-4, IL-13, IFN-γ)

Zhang et al.25

MC3T3-E1

10 and 100 μg/mL blueberry extract 3 24 h

k NO production 2 Expression of RANKL

Black rice extract Jang et al.26

C3H10T1/2 (multipotent) and primary bone marrow cells

10, 20, 40, and 80 μg/mL black rice extract 3 10 days

m ALP activity and m expression of osteoblastogenic markers (ALP and osterix)

Kim et al.27

C3H10T1/2 cells

10 and 40 μg/mL black rice extract 3 16 days

m ALP activity and mRNA expression of osteoblastogenic markers (Runx2, ALP, osteocalcin) and increased intracellular calcification deposition

RAW 264.7 cells

Dried plum polyphenols (10, 20, 30 μg/mL) 3 24 h

k Expression of osteoclast precursor (cyclooxygenase) and proinflammatory enzyme (iNOS)

Dried plum Bu et al.28

k Production of inflammatory mediators (NO and TNF-α) k Osteoclast differentiation (k TRAP 1 cells, NFATc1 expression, and resorption pit formation) Bu et al.29

MC3T3-E1 cells

Dried plum polyphenols (2.5, 5, 10, and 20 μg/mL) 3 24 h

5
20 μg/mL m bone formation marker (ALP) 5 μg/mL m bone formation marker (IGF-1) m Osteoblast activity and function marker (mineralized nodule formation) m Expression of bone formation enzyme (lysyl oxidase) m Runx2, Osterix, and IGF-1 mRNA expression k TNF-α-induced upregulation of RANKL 2 OPG

Anthocyanin compounds Saulite et al.30

Human MSC (multipotent cells)

25
200 μM malvidin chloride, cyanidin chloride, and delphinidin chloride 3 3 days

Malvidin m accumulation of calcium in osteocytes; cyanidin and delphinidin k Malvidin m expression of osteogenic transcription factors (Bmp2, Runx2) Cyanidin and delphinidin m chrondrogenic markers Col2a1 and aggrecan

Moriwaki et al.23

RAW 264.7 (preosteoclast-like) cells

0.25, 1, 5, and 20 μg/mL delphinidin, cyanidin and peonidin 3 1 h

Delphinidin k osteoclastogenesis (k TRAP 1 cells) and osteoclastogenic transcription factors (k NF-κB, c-Fos, NFATc1)

Dou et al.31

RAW 264.7 cells and bone marrow macrophages

0.1
30 μg/mL cyanidin 3 2
3 days

,1 μg/mL cyanidin m osteoclastogenesis (k TRAP 1 cells), while .10 μg/mL k

(Continued)

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Animal bone disease models

TABLE 14.2

149

(Continued)

Reference

Design

Treatment

Outcomes

Cheng et al.32

RAW 264.7 cells and bone marrow macrophages

0.5
20 μM cyanidin chloride 3 5
7 days

k Osteoclastogenesis (k TRAP 1 cells) and osteoclast activity (hydroxyapatite resorption)

Casati et al.33

MC3T3-E1 (preosteoblast-like) cells

10211
1025 M delphinidin3-rutinoside 3 24 h

m Expression of osteoblastogenic markers (type 1 collagen, ALP and osteocalcin) Prevented oxidative stress-induced osteoblast dysfunction

Data from cell studies assessing the effects anthocyanin-rich foods and individual compounds on bone health parameters are shown.

osteoblast formation and activity via the increased protein production of ALP and IGF-1, the mRNA expression of Runx2, osterix, IGF-1, and lysyl oxidase, and mineralized nodule formation in MC3T3-E1 cells.29 Additionally, dried plum inhibited osteoclast differentiation by suppression of RANKL.29

Anthocyanin compounds Moriwaki et al., observed that delphinidin inhibited osteoclastogenesis and osteoclastogenic transcription factors in RAW 264.7 macrophages.23 Further, Casati et al., observed that delphinidin-3-rutinoside increased expression of osteoblastogenic markers in MC3T3-E1 cells.33 Malvidin increased calcium accumulation in osteocytes and increased the expression of osteogenic transcription factors.30 Cyanidin chloride treatment in RAW 264.7 macrophages inhibited osteoclastogenesis and osteoclast activity.32 However, Dou et al., reported that while a high dose of cyanidin inhibited osteoclastogenesis, a low dose promoted it.31 Furthermore, cyanidin, in addition to delphinidin, reduced the calcium accumulation in osteocytes.30 Given these results, it appears that in vitro treatment with the isolated anthocyanin compounds delphinidin and malvidin show some indications of bone promotion, while the effects of cyanidin are still unclear.

Animal bone disease models Perhaps the most commonly used model to study osteoporosis in animals is hormone deficiency via the removal of the sex organs—ovariectomy (OVX) in females or orchidectomy (ORX) in males. This depletes the associated sex hormone (estrogen in ovaries, testosterone in testes), mimicking the sex-hormone depletion observed with aging. The decreased production of estrogen by the ovaries after menopause often causes deterioration of the trabecular bone, increases endocortical resorption, and increases cortical porosity, resulting in the development of osteoporosis and increased

risk of fragility fracture.34 Removing the sex organ induces hormone deficiency without aging, enabling the usage of both young and adult animals. The use of an inflammatory model can be another useful tool in investigating bone disease and elucidating key mechanistic pathways. Rheumatoid arthritis is characterized by chronic inflammation, joint destruction, and bone loss35 and can be induced by transgenic breeding or by administration of a proinflammatory compound such as complete Freund’s adjuvant (CFA) or sRANKL. The diet-induced obesity model provides another perspective into the associations between obesity, inflammation, OS, and bone health. Obesity is closely associated with chronic low-grade inflammation and OS; therefore, low antioxidant capacity and inflammation may increase bone resorption. Finally, aged, but otherwise healthy, animals allow researchers to investigate the effects of natural aging on bone health. This is particularly useful as it most naturally reflects the decrease in bone mass and weakening of the microarchitecture that is brought on by aging.36

Hormone deficiency model Berries such as blueberries, blackberries, and blackcurrants are abundant in flavonoids, particularly the anthocyanins cyanidin, delphinidin, and malvidin. The effects of various berries and berry juice on bone health in OVX or ORX animals was evaluated (Table 14.3). Consumption of a 5% blackberry diet, but not 10%, was found to improve BMD and trabecular microarchitecture in OVX rats, suggesting that a moderate intake of blackberries is beneficial for bone health.37 Similarly, blackcurrants improved BMD and trabecular bone mass through inhibition of bone resorption activity.39 Blueberries were also found to improve BMD by increasing bone formation (ALP and osteocalcin) and decreasing bone resorption (CTX and urinary DPD).40,41 Interestingly, Zhang et al., reported that young rats consuming blueberries prior to the OVX procedure prevented OVX-induced bone loss in adulthood by increasing osteoblasts’ cytoskeletal organization,

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150 TABLE 14.3 Reference

14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology

Animal studies assessing the effect of anthocyanin-rich foods and compounds on bone health. Design

Treatment/Diet

Outcomes

5% and 10% blackberry extract 3 100 days

5% blackberry extract m BMD and improved trabecular BV, TbN, and TbS

Hormone deficiency model

Berry and berry extracts Kaume et al.37

OVX Sprague-Dawley rats

2 Plasma antioxidant enzyme activity (TBARS, GPx, SOD, CAT) or TBARS 2 Bone metabolism biomarkers (serum ALP, Oc, and IGF-1; urinary DPD) Shimizu et al.38

OVX Sprague-Dawley rats

500 mg/kg/d bilberry extract 3 2 months

2 OVX-induced bone loss

Zheng et al.39

OVX C57BL/6J mice

1% blackcurrant extract 3 1, 2, and 3 months

m BMD and trabecular BV 2 Serum markers of bone formation (P1NP) or resorption (CTX) k Number of TRAP 1 osteoclast-like cells and k bone resorption activity

Zhang et al.40

OVX Sprague-Dawley rats

10% blueberry 3 14 days 1

Blueberry consumption prior to OVX surgery prevented OVX-induced bone loss in adulthood m Serum bone formation markers (ALP activity and Oc); k Serum bone resorption marker (CTX) m Myosin gene expression

Devareddy et al.41

OVX Sprague-Dawley rats

5% blueberry 3 100 days

m BMD k ALP, COL, and TRAP femoral mRNA expression m Bone formation marker (serum ALP activity); 2 serum Oc; 2 bone resorption marker (urinary DPD)

Villarreal et al.42

ORX rats

27% and 45% cranberry juice concentrate 3 4 months

m Plasma antioxidant capacity (TAC) 2 Bone quality in femur, fourth lumbar vertebra, or tibia 2 Bone formation marker (plasma IGF-1)

Dried plum Smith et al.43

OVX Sprague-Dawley rats

5%, 15%, 25% dried plum 3 6 weeks

15% and 25% dried plum m BMD, trabecular BV, and cortical thickness k Bone turnover markers P1NP and urinary DPD m Cancellous BFR, MAR and mineralizing surface Upregulated expression of bone formation proteins Bmp4 and IGF-1 Down-regulated expression of osteoclast differentiation marker NFATc1

Deyhim et al.44

OVX Sprague-Dawley rats

5%, 15%, 25% dried plum 3 2 months

m BMD and quality (overall yield and ultimate force) m Improved trabecular microarchitecture (BV, connectivity) 2 Serum markers of bone formation (IGF-1, ALP) or resorption (TRAP)

(Continued)

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151

Animal bone disease models

TABLE 14.3

(Continued)

Reference

Design

Treatment/Diet

Outcomes

Rendina et al.45

OVX C57BL/6J mice

5%, 15%, 25% dried plum 3 4 weeks

25% dried plum: m BMD and BMC Improved bone morphometric parameters (BV, TbN, TbSp, TbT) and m Bone strength and stiffness 15% and 25% dried plum: inhibited osteoclastogenesis (restored OVX-induced suppression of granulocytes and committed monocyte populations and increased lymphoblast populations in bone marrow) 15% and 25% dried plum: kconcanavalin-induced TNF-α production in splenocytes; 2 IL-6

Bu et al.46

ORX Sprague-Dawley rats

25% dried plum 3 3 months

m BMD and improved BV, TbN, TbS, and cortical thickness Non-significant tendency to decrease urinary DPD and Ca 2 Serum ALP or Oc

Franklin et al.47

ORX Sprague-Dawley rats

5%, 15%, and 25% dried plum 3 3 months

15% and 25% dried plum m BMD, ultimate load, compressive force and stiffness 25% dried plum m trabecular BV m TbN and TbSp 2 Serum bone formation markers Oc or ALP; m IGF-1 k Bone resorption marker urinary DPD Down-regulated RANKL and OPG mRNA expression and production

Other foods/mixed Pawlowski et al.48

OVX Sprague-Dawley rats

Extracts of grape seed (0.25% and 1.2%), plum (9% and 20%), blueberry (9% and 25%) and grape (0.6% and 3%)

Plum m urinary Ca retention

Hohman and Weaver49

OVX Sprague-Dawley rats

25% grape 3 2 months

m Net bone Ca retention

1.2% Grape seed extract and 3% grape k urinary NTX; plum 2

m Femoral cortical thickness and breaking strength 2 BMD, trabecular microarchitecture, or reference point indentation

Jang et al.26

Rendina et al.18

OVX Sprague-Dawley rats

100 and 200 mg/kg/d black rice extract gavage 3 2 months

200 mg/kg/d black rice extract m BMD

OVX C57BL/6 mice

25% dried plum, apple, apricot, grape, and mango 3 2 months

Dried plum, apricot and grape m BMD

100 mg/kg/d black rice extract m bone strength

Dried plum and apricot m trabecular BV and TbN and prevented bone loss Dried plum and apple down-regulated gene expression of osteoclast differentiation (NFATc1) Dried plum, apple and mango upregulated gene-expression of osteoblast activity (COL) All treatments 2 ALP or Oc gene expression Dried plum, grape and mango m plasma GPx activity

(Continued)

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152 TABLE 14.3 Reference

14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology

(Continued) Design

Treatment/Diet

Outcomes

Anthocyanin compounds Cheng et al.32

OVX C57BL/6 mice

Cyanidin chloride (5 mg/kg) intraperitoneally injected every 2 days 3 6 weeks

m Trabecular BV and TbN

Moriwaki et al.23

OVX C57BL/6 mice

1, 3 and 10 mg/kg delphinidin gavage 3 28 days

m Trabecular BV, TbT and TbN

CFA-induced arthritis male Sprague-Dawley rats

10, 20, and 40 mg/kg/d cherry anthocyanins 3 14 days

40 mg/kg/d cherry anthocyanins k inflammatory markers (serum TNF-α, PGE2 in paws)

C57BL/6 transgenic mice modeling rheumatoid arthritis (over-expressing human TNF)

20% dried plum 3 4 weeks

sRANKL-induced osteoporosis female C57BL/6 mice

10 mg/kg/d delphinidin gavage 3 14 days

Inflammation-induced model He et al.19

Mirza et al.35

Moriwaki et al.23

40 mg/kg/d cherry anthocyanins m serum markers of antioxidant capacity (TAC) and enzyme activity (SOD); k decreased oxidative stress (MDA) Dried plum slowed onset of arthritis and k joint bone erosions; protected articular cartilage and reduced synovitis Dried plum k osteoclastogenesis (TRAP 1 cells)

Attenuated sRANKL-induced bone loss Improved Trabecular BV, TbN and TbS; k OcN/BS

Diet-induced obesity model HF diet-induced obese male C57BL mice

5% blueberry, 6.3% blackberry and 5.7% blackcurrant 3 3 months

Blackcurrant m plasma ALP

Halloran et al.50

Adult and old male C57BL/6 mice

15% and 25% dried plum 3 6 months

25% dried plum m distal femoral metaphysis BV in both adult and old mice, whereas 15% only increased BV in adult mice

Sakaki et al.51

Young and old female C57BL/6 J mice

1% blackcurrant 3 4 months

In young mice, m Trabecular BV and GPx, and k CTX

Lee et al.20

All treatments 2 BMC, BMD, plasma antioxidant (FRAP, SOD) and bone formation (IGF-1, ALP)

Aging model

m Ob.S/BS in old mice Borderline-significant k TNF-α in old mice

Healthy animal model Smith et al.21

Adult male C57BL/6 mice

25% dried plum 3 4 or 12 weeks

m BMC, BMD, trabecular BV, and cortical thickness k Serum marker of bone formation (P1NP) at 4 weeks; slightly k at 12 weeks k Serum marker of bone resorption (pyridoline) at 12 weeks m Serum antioxidant enzyme activity (GPx) at 12 weeks

Shahnazari et al.52

Young and adult male C57BL/6 mice

25% dried plum 3 1, 2, or 4 weeks

m BV and TbT; k Oc.S/BS k Serum bone resorption marker (CTX) k Osteoclast precursor pool k Ob.S/BS and BFR k Proinflammatory cytokines (interleukins, TNF-α and MCP-1)

Cardoso et al.53

Young female Wistar rats

15 mL/d whole grape juice, 10 mL/d red wine, or 15 mL 4% resveratrol solution 3 2 months; all groups consumed a high-fat diet and were subjected to physical training

Red wine m BMC, femoral mass and distance between epiphyses Red wine and resveratrol m BMD and diaphysis midpoint width

Data from animal studies assessing the effects of anthocyanin-rich foods and individual compounds on bone health parameters are shown.

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Animal bone disease models

revealing that early blueberry consumption helped protect osteoblast function post-OVX.40 Conversely, Pawlowski et al., reported that neither low- nor high-dose blueberry diets affected the bone turnover markers urinary calcium and NTX excretion.48 Neither bilberry38 nor cranberry juice42 positively impacted bone health, although cranberry juice improved plasma antioxidant capacity. Dried plums (Prunus domestica L.) are rich in anthocyanins as well as neochlorogenic acid, vitamin K, and magnesium.44,45,54 In OVX and ORX animal studies, dried plums in doses ranging between 5% and 25% consistently improved BMD and trabecular microarchitecture.18,43,44,46,47,54 The underlying mechanisms suggested by these studies included the inhibition of osteoclastogenesis and bone resorption. When measured, urinary DPD, a bone resorption marker, decreased with dried plum consumption.43,46,47 Dried plum also decreased the genetic expression of osteoclast-promoting NFATc118,43 and RANKL,47 decreased urinary calcium excretion,46 and increased the granulocyte, lymphoblast, and committed monocyte populations in bone marrow.54 Additionally, dried plum appeared to promote the formation of bone by increased expression of IGF-1 and Bmp4.43,47 With regard to osteoblast activity, dried plum upregulated the COL expression, but did not affect serum ALP and osteocalcin,44,46,47 suggesting that dried plum’s bone-strengthening mechanism is driven more so by inhibited osteoclast function rather than increased osteoblast activity. Finally, dried plum reduced TNF-α production in splenocytes54 and increased plasma GPx activity,18 demonstrating antiinflammatory and antioxidant effects. Besides berries and dried plums, other anthocyaninrich fruits such as grapes, apples, apricots, and mangoes have been evaluated for their effect on bone health in OVX and ORX animals. Grape demonstrated favorable effects on bone as measured by improved BMD,18 increased bone calcium retention, cortical thickness, and breaking strength,49 and decreased bone resorption.48 Plasma antioxidant activity also increased with grape consumption.18 An extract of black rice, a grain rich in cyanidin,55 similarly improved BMD and increased bone strength.26 A diet supplemented with 25% apricot for 2 months improved BMD and trabecular bone volume (BV), while equal diets of apple and mango did not.18 Lastly, the effects of individual anthocyanin compounds on OVX mice were evaluated. Both a cyanidin chloride intraperitoneal injection for 6 weeks32 and a delphinidin gavage for 1 month resulted23 in increased trabecular microarchitecture. These results support the notion that the beneficial effects of certain foods on bone health are, at least in part, attributed to anthocyanins. In summary, the results of these studies suggest that anthocyanin-rich foods positively impact bone

153

health in OVX and ORX animals. Dried plum consistently demonstrated favorable effects such as increased BMD, but other foods such as berries and grapes also demonstrated bone-promoting properties. The underlying mechanisms appear to be related to decreased osteoclast differentiation or activity, and increased osteoblast activity.

Inflammatory bone disease model Dried plum slowed the onset of and attenuated the symptoms of arthritis in transgenic mice modeling rheumatoid arthritis, and also inhibited osteoclastogenesis.35 In a CFA-induced model of arthritis, a diet supplemented with cherry anthocyanins for 14 days decreased serum markers of inflammation, increased serum antioxidant capacity, and reduced OS.19 Moriwaki et al., reported that a delphinidin gavage delivered over 2 weeks attenuated bone loss by improving trabecular microarchitecture in sRANKL-induced osteoporotic mice. These results indicate that anthocyanins and anthocyanin-rich food reduce inflammation and, thereby, mediate secondary symptoms such as osteoporosis and eroded joints in arthritis. Additionally, they improve the redox balance, which may directly contribute to improved inflammatory conditions.

Diet-induced obesity model Lee et al., utilized mice induced with obesity with a high-fat diet and fed diets supplemented with blueberry, blackberry, or blackcurrant for 3 months and found that while blackcurrant increased plasma ALP activity, no treatment affected BMD, BMC, plasma antioxidant status, or other markers of bone formation.20 These results reveal that anthocyanin-rich berries may have potential in affecting bone turnover in a high-fat diet obesity animal model, but warrants further investigation.

Aging model Aging is associated with increased bone turnover, becoming particularly evident postmenopause. A diet supplemented with 15% dried plum for 6 months increased trabecular BV in adult mice, but a 25% dose was able to increase BV in both adult and old mice, demonstrating a more potent response to a higher dose in old mice.50 In contrast, a diet supplemented with 1% blackcurrant for 4 months increased trabecular BV in young mice, but not in old mice. This was associated with an increase in GPx activity, suggesting improved antioxidant activity.51 In summary, a large dose of dried plum demonstrates capability in improving trabecular

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14. Anthocyanins and anthocyanin-rich food as antioxidants in bone pathology

bone structure in an aging mouse model, whereas blackcurrant showed improvements only in mice that were not of advanced age.

Healthy animals Lastly, three studies evaluated the effects of either dried plum, grape juice, or wine on bone health in healthy young or adult mice and rats. Dried plum was reported to improve several bone health parameters, TABLE 14.4 Reference

including BMD and BMC,21 and trabecular BV.21,52 This was accompanied by a reduction in both bone resorption and formation markers; however, this also indicates that dried plum may impart its benefits by reducing overall bone turnover, but inhibits resorption more than formation. Additionally, antioxidant activity increased21 and inflammation decreased,52 suggesting their involvement in the bone remodeling process. In young rats consuming a high-fat diet (but not obese) and subjected to physical activity, red wine increased

Human studies assessing the effect of anthocyanin-rich foods and compounds on bone health. Design

Measurements/treatments

Outcomes

Randomized controlled trials Hooshmand et al.22

12-month RCT Osteopenic postmenopausal women (n 5 160)

Effect of dried plum (100 g/d) versus dried m BMD in ulna and spine apple (75 g/d) on BMD in whole body, k Serum markers of bone formation (BALP) and spine, forearm and hip resorption (TRAP5b) Assessed serum BALP and TRAP5b All subjects received supplemental 500 mg Ca 1 400 IU vitamin D daily

Arjmandi et al.56

3-month RCT

Simonavice et al.57

6-month RCT

Kaume et al.5

9-month RCT

Postmenopausal women (n 5 58)

Breast cancer survivors (n 5 23)

Postmenopausal smokers (n 5 65)

Effect of dried plum (100 g/d) on serum and urinary markers of bone formation and resorption

m Serum bone formation markers (IGF-1 and BALP) 2 Bone resorption makers (serum TRAP and helical peptide; urinary DPD)

Effect of dried plum (total 90 g/d) on BMD 2 BMD in whole body, spine, femur and forearm 2 Serum bone formation (BALP) or resorption (TRAP5b) All subjects received supplemental 450 mg markers Ca 1 800 IU vitamin D daily, in addition to 2 Serum inflammatory marker (CRP) resistance training Effect of blackberries (45 g/d) and blueberries (45 g/d) on BMD and BMC in smokers

Blackberry, but not blueberry, m BMD Neither berry group affected markers of bone resorption or formation, inflammation or oxidative stress

Assessed serum TBARS, hs-CRP, BALP and Oc; urinary DPD and creatinine

Cohort studies Effect of habitual dietary flavonoid intake (FFQ) on BMD in hip, spine and femoral neck

Anthocyanin, in addition to total flavonoid and flavone, intake was positively associated with BMD

Fairweather- 1993
2004 Cohort study Effect of various dietary patterns on BMD Tait et al.59 in hip, femoral neck and spine 2646 Postmenopausal women from the Twins UK registry

Moderate positive association between wine intake and BMD

Welch et al.58

1996
2000 Cohort study 3160 women aged 18
79 years from the Twins UK registry

Cross-sectional studies Zheng et al.60

2012
13 Cross-sectional study Middle-aged and elderly Chinese subjects (2239 women, 1078 men)

Effect of habitual dietary flavonoid intake (FFQ) on BMD in whole body, femoral neck, and lumbar spine

Total flavonoid, flavanol, flavan-3-ol, flavone and proanthocyanidin, but not anthocyanin, intakes were associated with higher BMD in women No association between flavonoid intake or any subclass and BMD in men

Data from human studies assessing the effects of anthocyanin-rich foods and overall anthocyanin intake on bone health parameters are shown.

II. Antioxidants and Pathology

Human studies

BMD and BMC, while grape juice failed to do so after 2 months.53 In summary, dried plum and red wine improved bone health in healthy animals, and the determining mechanism may be due to a stronger inhibition of bone resorption than bone formation.

Human studies

155

such as the participants’ health conditions and demographics at baseline, treatment duration, and outcome variables. With regard to berries, Kaume et al., reported that blackberries but not blueberries improved BMD in postmenopausal smokers.5 However, neither treatment affected bone resorption or resorption, nor did they alter indicators of inflammation or OS, leaving much unanswered with regard to identifying potential mechanisms.

Randomized controlled trials The relationship between anthocyanins and bone health in humans is summarized in Table 14.4. Dried plum demonstrates potential in improving bone health, but not all results are consistent. In a 12-month randomized controlled trial (RCT) in postmenopausal osteopenic women, a 100 g/d intervention of dried plum increased BMD and decreased bone formation and resorption markers when compared to a daily intervention of dried apples.22 However, Arjmandi et al., reported that in a 3-month RCT in postmenopausal women, the same dose of dried plum increased serum bone formation markers, but did not affect bone resorption markers.56 Results from the former study suggest that overall bone turnover is suppressed, with a stronger inhibition of resorption over formation, while the results from the latter study indicate that bone formation is increased. Simonavice et al., reported no effect of dried plum on BMD, bone formation, or resorption in breast cancer survivors.57 These discrepancies may relate to differences in study populations and designs

Epidemiologic studies There is a paucity of literature describing the associations between anthocyanin or anthocyanin-rich food and bone health in epidemiologic studies. In cohort studies, Welch et al., evaluated the effect of habitual dietary flavonoids on BMD in the Twins UK cohort and reported that anthocyanin intake was positively associated with BMD.58 In a postmenopausal subset of the same cohort, Fairweather-Tait et al., reported a moderately positive association between wine intake and BMD.59 In a cross-sectional study of middle-aged and elderly Chinese subjects, anthocyanin intake was not associated with BMD.60 The discrepancies observed in anthocyanin’s association with BMD between the studies by Welch et al.,58 and Zhang et al,60 may derive from key differences in study design: a cohort study measures exposure and outcome in chronological order, thus is able to distinguish between cause and effect; while a cross-sectional study measures events only at a

FIGURE 14.1 Potential mechanism of anthocyanins on inflammation and oxidative stress as they relate to bone health. Illustration of the mechanism of anthocyanins on bone health. Under excessive OS, ROS upregulates inflammatory mediators such as IL-6, TNF-α, IFN-γ, or LPS, which further induces OS. ROS also upregulates osteoclastogenic transcription factors and mediators such as RANKL, NFATc1, TRAP, cfos, and CSF-1, promoting bone resorption. ROS also downregulates osteoblast differentiation and activity by inhibiting IGF-1, ALP, osterix, Runx2, and osteocalcin, inhibiting bone formation. Anthocyanins function as antioxidants, reducing OS and interrupting the inflammatory-OS cycle. Anthocyanins inhibit the action of ROS on osteoclasts and osteoblasts, ultimately promoting bone formation and reducing bone resorption. The net result is increased bone strength and bone mineral density, and reduced risk of fragility fractures. The red line indicates inhibition and the green arrow indicates promotion.

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single point in time, thus is unable to determine causality. Therefore the lack of association between anthocyanin intake and BMD in the cross-sectional study60 does not disprove anthocyanin’s protective effect on bone. Additionally, other causes of inconsistencies may arise from differences in study populations and measurement and categorization of anthocyanin intake. Thus interpretations from these data should remain conservative until additional studies are performed.

Conclusion In conclusion, the current evidence supports the notion that anthocyanins and anthocyanin-rich foods have strong potential in improving certain key indicators of bone health through the modulation of redox balance and inflammation. The evidence in animal and cell studies consistently suggests that anthocyanin treatment inhibits osteoclast activity and differentiation, while promoting osteoblast activity and formation. Data from human studies are not fully consistent, yet suggest the potential for beneficial effects of anthocyanins on bone health. Populations with, or at risk for, bone deterioration seem to gain the most benefit from anthocyanin-rich foods, but the small number of available studies should be confirmed and extended in future studies.

Applications to other areas of pathology With regard to applications to other areas of pathology, the results indicate that anthocyanins and anthocyanin-rich foods may also favorably affect other chronic diseases in which OS and inflammation are implicated, such as cardiovascular disease, cancer, neurodegenerative disease, and pulmonary disease. By preventing and repairing damage induced by OS, these natural antioxidants may see effectiveness in treating symptoms, delaying or even preventing the incidence of such maladies (Fig. 14.1).

Summary points • OS and inflammation promote bone loss, which is becoming an increasing concern as the population ages. • Anthocyanins exhibit antioxidant properties and have recently been investigated for their bonepromoting properties. • Cell and animal studies consistently demonstrate favorable effects of anthocyanins in upregulating bone formation and inhibiting bone resorption.

• Human studies support the potential benefit of anthocyanins in bone health, though the findings are not as consistent. • Mechanisms underlying the bone-protective effects of anthocyanins appear to be related to their antioxidant and antiinflammatory properties.

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12. Casati L, Pagani F, Fibiani M, Lo Scalzo R, Sibilia V. Potential of delphinidin-3-rutinoside extracted from Solanum melongena L. as promoter of osteoblastic MC3T3-E1 function and antagonist of oxidative damage. Eur J Nutr 2018;0:1
14. Va¨a¨na¨nen H, Ha¨rko¨nen P. Estrogen and bone metabolism. Maturitas 1996;23:S65
9. Mirza F, Lorenzo J, Drissi H, Lee FY, Soung DY. Dried plum alleviates symptoms of inflammatory arthritis in TNF transgenic mice. J Nutr Biochem 2018;52:54
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25.

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C H A P T E R

15 Ascorbic acid as an antioxidant and applications to the central nervous system Morgana Moretti and Ana Lu´cia S. Rodrigues Department of Biochemistry, Center of Biological Sciences, Federal University of Santa Catarina, Santa Catarina, Brazil

Introduction Ascorbic acid is a water-soluble vitamin that exerts several physiological functions, including neuromodulation in the brain, where it is concentrated and participates in the regulation of central nervous system (CNS) homeostasis.1 Ascorbic acid is a reducing agent, and several physiological functions of this compound depends on its oxidation-reduction properties.2 The dry form of ascorbic acid is stable, but it quickly oxidizes in solution or during exposure to air or heat. In mammals, at physiological pH, deprotonation occurs at the C3 hydroxyl group, producing the dehydroascorbate anion, the endogenous form of this compound,3 as shown in Fig. 15.1. As a reducing agent, ascorbate protects against the harmful effects of free radicals generated during physiological processes. Most of these free radicals have an unpaired electron that can be neutralized by the one-electron oxidation of ascorbate. In the aqueous phase of tissue, it can scavenge hydroxyl- and peroxyl-radicals, peroxynitrite, singlet oxygen, and superoxide. Since ascorbate can prevent the oxidation of vitamin E, it also blocks the oxidation of cellular membranes, being an important molecule in the cellular antioxidant system.4 After being absorbed by a family of sodiumdependent transporters in the gut,1 ascorbate reaches the bloodstream and is distributed throughout the body. Ascorbate enters the CNS directly through the choroid plexus via type 2 sodium-dependent transporters (SVCT2). Dehydroascorbate can enter the CNS via glucose transporters (GLUT1) across blood
brain barrier endothelium. Once in the cerebrospinal

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00015-9

fluid, ascorbate or dehydroascorbate are incorporated by neurons through SVCT2 or GLUT1, respectively. Dehydroascorbate can be reduced to ascorbate inside the neuron or released by GLUT1. Glial cells obtain ascorbate by the reduction of dehydroascorbate, which is uptaked by GLUT1. SVCT2 does not participate in this process.5 Reactive oxygen species (ROS), including free oxygen radicals and nonradical oxidants like hydrogen peroxide (H2O2) are produced by regular cellular metabolism. At physiological concentration, H2O2 does not injure cells, indeed under this condition it acts as an intracellular signaling molecule for cell survival.6 However, when transition metals are present, it produces hydroxyl radicals via the Fenton oxidation mechanism. If the antioxidant defense system is deficient, this condition can lead to cellular oxidative damage that can affect lipids, proteins, and nucleic acids. Importantly, cellular redox imbalance is a central feature of neurodegenerative diseases and psychiatric disorders.7,8 It has been described that several antioxidant enzymes and nonenzymatic antioxidants exert positive effects against oxidative stress (OS) in neurodegenerative and psychiatric conditions.9,10 Ascorbate is present at millimolar concentrations in neuron-rich areas and exerts important neuromodulatory functions, particularly in the hippocampus and amygdala.11 This raises the hypothesis that ascorbate could protect these brain areas against oxidative injury and elicit therapeutic roles in neuropathologies. In this chapter we present evidence on the role of ascorbic acid in neurodegeneration and psychiatric disorders, focusing on its antioxidant mechanism.

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FIGURE 15.1 Ascorbic acid oxidation process. At physiological pH, the formation of dehidroascorbate occurs after deprotonation of ascorbic acid at the C3 hydroxyl group. This is a two-step, reversible process. First, the ascorbyl radical is produced through either the loss of a hydrogen atom or electron transfer, followed by rapid deprotonation. Although it may react with other radicals, it generally decays to dehydroascorbate via deprotonation.

Ascorbic acid applications in neurodegenerative diseases Alzheimer’s disease The antioxidant effect of ascorbic acid has been investigated in animal models of Alzheimer’s disease. A 6-month treatment with ascorbic acid recovered behavioral deficits and decreased Aβ oligomers in mice. This effect was associated with decreased brain oxidative damage and other markers of disease progression.12 Consistent with these findings, rats subjected to hippocampal injection of fibrillar Aβ presented reduced OS markers and proinflammatory cytokines after ascorbic acid administration.13 This compound also abrogated the increased intracellular calcium and cell death induced by Aβ in PC12 cells.14 The treatment with ascorbic acid also significantly decreased behavioral deficits in a rat model of Alzheimer’s disease induced by repeated administration of aluminum chloride (AlCl3).15 The therapeutic effect of ascorbic acid was associated with its antiproteolytic and antioxidant properties. Interestingly, the administration of ascorbic acid at lower doses (200 and 400 mg/kg for 21 days) elicited an antioxidant effect and a higher dose (600 mg/kg) induced prooxidant/ neurotoxic effects in the colchicine-induced Alzheimer’s disease model. The antioxidant effect of lower doses was accompanied by recovery of memory impairments and prevention of neurodegeneration and neuroinflammation in the hippocampus of colchicine-treated rats.16 Additionally, it was demonstrated that ascorbate deficiency exacerbates mitochondrial OS in APP/PSEN1 mouse model for Alzheimer’s disease. Remarkably, ascorbate supplementation alleviated ROS production and supported mitochondrial health.17 Some clinical studies have also explored the association between Alzheimer’s disease and ascorbic acid intake or plasma ascorbate concentrations. It was found that plasma ascorbate levels are lower in

individuals with Alzheimer’s disease,18 and that intake of vitamin C was associated with reduced incidence of Alzheimer’s disease,19
21 as well as decreased risks of cognitive impairment and dementia.19 Higher blood vitamin C concentration was associated with reduced risk for cognitive decline in women with the apolipoprotein E (APOE) gene, which is a strong genetic risk indicator for Alzheimer’s disease.22 Two recent metaanalyses confirmed these findings and suggested a reduced systemic availability of dietary antioxidants, including ascorbic acid, in individuals with Alzheimer’s disease.23,24 Conversely, Morris et al.25 showed that intake of ascorbic acid was not significantly associated with Alzheimer’s disease risk during a mean follow-up period of 3.9 years.

Parkinson’s disease Parkinson’s disease is associated with the formation of pathological protein aggregates known as Lewy bodies, mainly composed by α-synuclein. The overexpression of α-synuclein seems to increase the sensitivity of dopaminergic neurons to oxidative damage, leading to mitochondrial dysfunction.26 Studies examining the properties of ascorbic acid in Parkinson’s disease models have shown a significant effect of this compound on α-synuclein. Wang et al.27 described that α-synuclein-Cu21 (which accelerates the formation of toxic aggregates) is reduced to α-synuclein-Cu1 by ascorbate, a process associated with decreased cellular redox species generation. Furthermore, the prevention of excessive ROS formation by ascorbic acid reduces the formation of α-synuclein in Saccharomyces cerevisiae cells.28 The neuroprotective effect of ascorbate, especially along with clinically used iron chelator, and its association with antioxidant properties was recently demonstrated by Sun et al.29 Some studies investigated the association between Parkinson’s disease and ascorbate levels in serum or

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FIGURE 15.2 Neuroprotective effect of ascorbic acid. Neurodegenerative diseases are associated with increased brain OS and inflammation. The neuroprotective effect of ascorbic acid is likely associated with its antioxidant and antiinflammatory properties.

lymphocytes. Ascorbate levels were reported to be higher30 or lower31 in Parkinson’s disease patients compared to control subjects or patients at less severe stages, respectively. No association was also found by Fernandez-Calle et al.32. Some authors also reported that intake of antioxidant vitamins, including ascorbic acid, were not associated with Parkinson’s disease risk.33
36 On the other hand, combined administration of high doses of tocopherol and ascorbic acid reduced Parkinson’s disease progression,37 although the individual contribution of ascorbic acid is unknown. Ascorbic acid also improved functional performance of patients with Parkinson’s disease who experienced on
off effects, but did not change the pattern of on
off effects, severity of parkinsonism/dyskinesia, or self-assessment ratings.38

Multiple sclerosis and amyotrophic lateral sclerosis OS along with demyelination and neurodegeneration are important features of multiple sclerosis, a chronic inflammatory disease of the CNS. Intrahippocampal injection of ascorbic acid improved memory in a rat model of multiple sclerosis induced by ethidium bromide.39 It was also demonstrated that L-ascorbyl-2phosphate, a stable form of vitamin C, promoted oligodendrocytes generation and remyelination and provided significant therapeutic effect in a cuprizone-mediated demyelination animal model.40 Besler et al.41 reported that serum levels of ascorbate were decreased in multiple sclerosis patients compared with healthy sex- and age-matched individuals, an effect associated with increased lipid peroxidation

products. Ghadirian et al.42 showed that higher serum ascorbate was associated with decreased risk of multiple sclerosis. However, others found no association between the intake of fruits and vegetables rich in vitamin C and the risk of multiple sclerosis.36,43
45 Despite no direct evidence of its antioxidant effects, ascorbic acid also has neuroprotective properties in amyotrophic lateral sclerosis, a progressive neurodegenerative disease of the spinal cord motor neurons. It was demonstrated that trientine (a copper chelate) plus ascorbic acid delayed the onset of neurological signs and the time to reach total paralysis in familial amyotrophic lateral sclerosis transgenic mice.46 In the same model, ascorbate administered alone and before the onset of the disease significantly extended survival.47 The intake of ascorbic acid was not associated with the risk of amyotrophic lateral in an epidemiological study, but higher intake of food rich in antioxidants protected against the development of this disease.48 Fig. 15.2 illustrates the possible neuroprotective mechanisms of ascorbic acid.

Role of ascorbic acid in neuropsychiatric disorders Schizophrenia Schizophrenia is a mental illness that comprises positive, negative, and cognitive symptoms. Hyperactive dopaminergic signal transduction in the mesolimbic pathway,49 and OS,50 among other factors, are associated with the neurobiology of schizophrenia. Cabungcal

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et al.51 reported that glutathione deficit during brain development induced a schizophrenic phenotype, and the synthesis of ascorbate was found to be increased to compensate the glutathione deficit. Heiser et al.52 demonstrated that administration of antipsychotic drugs increased formation of ROS in the blood of rats, an effect counteracted by the administration of ascorbic acid, suggesting that this vitamin could be useful as a therapeutic adjuvant in schizophrenia. The antioxidant effects of ascorbic acid were not directly evaluated in the available studies, but several authors demonstrated that it exerts positive effects against schizophrenic-like behavior53 and human psychosis,54 probably, due to the antagonism of dopaminergic function. Epidemiological studies have shown that patients with schizophrenia have decreased levels of ascorbate in serum, leukocytes, and plasma,55 lower fasting ascorbate levels, and lower urinary ascorbate excretion after ascorbic acid load,56 as well as decreased ascorbate urinary excretion.56 Moreover, clinical studies revealed substantial benefits from the addition of ascorbic acid to conventional treatment for schizophrenic patients.57
59 Interestingly, the administration of placebo plus antipsychotics or ascorbic acid plus antipsychotics to schizophrenic patients decreased serum malondialdehyde and increased plasma ascorbate levels.59 Even with some controversial results,60 a beneficial effect of ascorbic acid in the treatment of schizophrenia has been suggested. The molecular mechanisms underlying this effect remain poorly understood, but an overall reduction of OS may account for it.

Major depressive disorder Several studies have demonstrated that ascorbic acid has valuable effects in depression, a psychiatric illness associated with high morbidity and mortality worldwide. It was shown that this compound acts as neuromodulator and induces an antidepressant-like behavior in the tail suspension test (TST, a predictive test for depression).61
64 This antidepressant-like effect in the TST is associated with its ability to elicit modulatory effects on several targets, that is, activation of PI3K and mTOR signaling, GSK-3β inhibition,65 activation of GABAA receptors, and the possible inhibition of GABAB receptors.66 Ascorbic acid also modulates the opioid system, inhibits NMDA receptors and nitric oxide-cGMP pathways,64 and modulates monoaminergic systems.61 The antidepressant-like effect of ascorbic acid in the TST is also dependent on induction of heme oxygenase-1, which is crucial in protecting cells against inflammatory damage and oxidative or nitrosative stress.65

Particularly interesting in the context of this chapter, is that ascorbic acid reversed the depressive-like behavior induced by acute restraint stress in mice, an effect comparable to that was obtained with the conventional antidepressant fluoxetine. It was found that the stress procedure increased lipid peroxidation in the cerebral cortex and hippocampus and enhanced antioxidant defenses (likely as a compensatory response). Besides showing an antidepressant-like effect, ascorbic acid reduced lipid peroxidation to control levels and restored the activity of superoxide dismutase, glutathione reductase, and glutathione peroxidase.67 A similar profile was described in a model of depression induced by chronic unpredictable stress, in which repeated administration of ascorbic acid reversed the depressive-like behavior and restored the oxidative damage induced by stress with efficacy comparable to fluoxetine.68 The possible mechanisms involved in the antidepressant-like effect of ascorbic are illustrated in Fig. 15.3. Contrary to preclinical studies, which suggest a robust antidepressant-like effect of ascorbic acid, inconsistent results were found in humans. Poor ascorbate status is associated with increased depressive symptoms69
72 and high vitamin C plasmatic concentration is associated with elevated mood.73 Interestingly, plasma ascorbate levels in depressed individuals increased significantly after 4 and 12 weeks of antidepressant therapy compared to baseline levels.70 A negative association between the intake of ascorbic acid and depressive symptoms was also reported.74
76 Besides having a positive effect on the mood of healthy individuals,77 clinical studies revealed that ascorbic acid administration alone,78 or combined with antidepressants,79,80 reduced depressive symptoms in pediatric and adult patients; however, no direct role of its antioxidant property was evaluated in these studies. Conversely, other clinical studies did not find any significant effect of ascorbic acid in patients with major depressive disorder.81,82

Bipolar disorder Bipolar disorder is a mood disorder characterized by dramatic changes in mood, energy, and behavior. Its pathophysiology involves an excessive dopaminergic/glutamatergic transmission and decreased cholinergic muscarinic signaling.83 Bipolar disorder is also associated with an activation of GSK-3β-mediated signaling, which can lead to neuronal cell death. Drugs with mood-stabilizing properties, such as lithium and valproate can inhibit GSK-3β-mediated signaling pathways.84,85 Although the lack of preclinical studies investigating the putative mood-stabilizing effect of ascorbate, Huang et al.86 showed that this compound

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Conclusion

FIGURE 15.3

Antidepressant-like effect of ascorbic acid. Besides its antioxidant action, the antidepressant-like effect of ascorbic acid involves activation of dopaminergic, serotonergic, and noradrenergic systems. It also inhibits NMDA receptors and potassium channels, activates GABAA receptors, and possibly inhibits GABAB receptors. The antidepressant-like effect of ascorbic acid also involves activation of PI3K/Akt pathways, inhibition of GSK-3β, and stimulation of mTOR and its target proteins.

prevented GSK-3β activation in the cell line by a mechanism dependent on a reduction of ROS levels. Regarding clinical studies, ascorbic acid administration was reported to reduce depressive, manic, and paranoid symptoms, besides causing enhancement in general personality functioning.87 Similarly, ascorbic acid intake rapidly (3
6 hours after a single administration) reduced the severity of both manic and depressive symptoms of bipolar individuals.88 On the other hand, combined ascorbic acid and ethylene diamine tetra acetic acid improved depression, but not mania, in a double-blind study.89

Anxiety disorders Anxiety disorders are among the most common psychiatric illnesses, with a lifetime prevalence of almost 30%.90 Several studies have shown that OS is closely related to anxiety. Anxious mice had increased OS in neuronal and glial cells in the cerebellum and hippocampus, neurons of the cerebral cortex, and peripheral leukocytes.91 Steenkamp et al.92 investigated the relationship between OS, anxiety, and depression symptoms in medication-free individuals with major depressive disorder and found that depressed individuals with more severe anxiety, but not subjects with more severe depression, showed increased OS, after controlling for age, sex, body mass index, and

smoking. Moreover, OS markers were increased in patients with anxiety, as reviewed by Hovatta et al.93 The antioxidant property of ascorbic acid may be an important mechanism associated with its anxiolytic effect. A double-blind, randomized, placebo-controlled study94 revealed that a 14-day oral supplementation with ascorbic acid reduced anxiety levels in a sample of high school students. Oral supplementation with ascorbic acid also decreased anxiety scores in a randomized single blind placebo-control study.81 A double-blind, randomized, placebo-controlled study showed that a single oral dose of ascorbic acid produced an anxiolytic effect in high anxiety subjects.95 Animal studies reported similar effects; ascorbic acid administration was effective to cause anxiolytic effect in mice in an elevated plus maze, open-field, test and light
dark preference test.96 Anxiety-like behavior also decreased in rats under prolonged ascorbic acid consumption.97

Conclusion Studies dealing with the role of ascorbic acid in neurodegenerative and psychiatric diseases are still in initial stages. The current knowledge allows us to suggest that it may have a more robust effect for the management of Alzheimer’s disease and depression than previously thought. As shown in Fig. 15.4, ascorbic

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Summary points

FIGURE 15.4 Role of ascorbic acid in neurodegenerative and psychiatric diseases. Ascorbic acid has neuromodulatory and antioxidant properties that contribute to its beneficial effects against neurodegeneration and psychopathologies.

acid’s neuromodulatory functions and ability to decrease either OS or formation of protein aggregates may contribute to its beneficial effects against the behavioral and neurochemical impairments observed in certain neuropathologies. Since ascorbic acid is a low-cost compound that has an attractive risk-benefit profile, it could be an interesting future approach, particularly as add-on treatment for these diseases. The antioxidant and neuromodulatory roles of ascorbic acid for the management of other CNS diseases remains to be further explored to validate the existing results and accelerate knowledge in this field.

Applications to other areas of pathology This chapter confirms that ascorbic acid protects against the behavioral and neurochemical damages observed in psychiatric and neurodegenerative conditions, effects that are associated, at least in part, with its antioxidant properties. The antioxidant effects of ascorbic acid also seem to play a protective role in patients with sepsis and septic shock,98 skin diseases,99 and cancer.100

Funding Dr. Rodrigues’ studies were supported by grants from the National Council of Technological and Scientific Development (CNPq, Brazil) [grant number 310113/2017-2] and CAPES.

• This chapter focuses on ascorbate and its biological mechanisms in neurodegenerative diseases and psychiatric disorders. • Ascorbate has neuroprotective roles in animal models of Alzheimer’s and Parkinson’ diseases, multiple sclerosis, and amyotrophic lateral sclerosis. • There is an inverse association between ascorbate status and Alzheimer’s disease symptoms, and ascorbate supplementation has protective roles in this disease. • Clinical studies investigating the effects of ascorbate in Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis show inconsistent results. • Ascorbic acid exerts beneficial effects in animal models of schizophrenia and depression, whereas in bipolar disorder models its effects have not been established. • A beneficial effect of ascorbic acid for the treatment of patients with schizophrenia and depression has been reported. • Ascorbate decreases OS, reduces the formation of protein aggregates, and exerts neuromodulatory functions that may contribute to the reduction of the impairments observed in these neuropathologies.

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9. Dadheech G, Mishra S, Gautam S, Sharma P. Oxidative stress, alpha-tocopherol, ascorbic acid and reduced glutathione status in schizophrenics. Indian J Clin Biochem 2006;21:34
8. Suboticanec K, Folnegovic-Smalc V, Turcin R, Mestrovic B, Buzina R. Plasma levels and urinary vitamin C excretion in schizophrenic patients. Hum Nutr Clin Nutr 1986;40:421
8. Beauclair L, Vinogradov S, Riney SJ, Csernansky JG, Hollister LE. An adjunctive role for ascorbic acid in the treatment of schizophrenia? J Clin Psychopharmacol 1987;7:282
3. Dakhale GN, Khanzode SD, Khanzode SS, Saoji A. Supplementation of vitamin C with atypical antipsychotics reduces oxidative stress and improves the outcome of schizophrenia. Psychopharmacology 2005;182:494
8. Sandyk R, Kanofsky JD. Vitamin C in the treatment of schizophrenia. Int J Neurosci 1993;68:67
71. Bentsen H, Osnes K, Refsum H, Solberg DK, Bohmer T. A randomized placebo-controlled trial of an omega-3 fatty acid and vitamins E 1 C in schizophrenia. Transl Psych 2013;3:e335. Binfare RW, Rosa AO, Lobato KR, Santos AR, Rodrigues AL. Ascorbic acid administration produces an antidepressant-like effect: evidence for the involvement of monoaminergic neurotransmission. Prog Neuropsychopharmacol Biol Psych 2009;33: 530
40. Moretti M, Budni J, Freitas AE, Neis VB, Ribeiro CM, Balen OG, et al. TNF-alpha-induced depressive-like phenotype and p38 (MAPK) activation are abolished by ascorbic acid treatment. Eur Neuropsychopharmacol 2015;25:902
12. Moretti M, Budni J, Ribeiro CM, Rodrigues AL. Involvement of different types of potassium channels in the antidepressant-like effect of ascorbic acid in the mouse tail suspension test. Eur J Pharmacol 2012;687:21
7. Moretti M, Freitas AE, Budni J, Fernandes SC, Balen GO, Rodrigues AL. Involvement of nitric oxide-cGMP pathway in the antidepressant-like effect of ascorbic acid in the tail suspension test. Behav Brain Res 2011;225:328
33. Moretti M, Budni J, Freitas AE, Rosa PB, Rodrigues AL. Antidepressant-like effect of ascorbic acid is associated with the modulation of mammalian target of rapamycin pathway. J Psychiatr Res 2014;48:16
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66. Rosa PB, Neis VB, Ribeiro CM, Moretti M, Rodrigues AL. Antidepressant-like effects of ascorbic acid and ketamine involve modulation of GABAA and GABAB receptors. Pharmacol Rep 2016;68:996
1001. 67. Moretti M, Budni J, Dos Santos DB, Antunes A, Daufenbach JF, Manosso LM, et al. Protective effects of ascorbic acid on behavior and oxidative status of restraint-stressed mice. J Mol Neurosci 2013;49:68
79. 68. Moretti M, Colla A, Balen GO, dos Santos DB, Budni J, de Freitas AE, et al. Ascorbic acid treatment, similarly to fluoxetine, reverses depressive-like behavior and brain oxidative damage induced by chronic unpredictable stress. J Psychiatr Res 2012;46:331
40. 69. Chang CW, Chen MJ, Wang TE, Chang WH, Lin CC, Liu CY. Scurvy in a patient with depression. Dig Dis Sci 2007;52:1259
61. 70. Khanzode SD, Dakhale GN, Khanzode SS, Saoji A, Palasodkar R. Oxidative damage and major depression: the potential antioxidant action of selective serotonin re-uptake inhibitors. Redox Rep 2003;8:365
70. 71. DeSantis J. Scurvy and psychiatric symptoms. Perspect Psychiatr Care 1993;29:18
22. 72. Gariballa S. Poor vitamin C status is associated with increased depression symptoms following acute illness in older people. Int J Vitam Nutr Res 2014;84:12
17. 73. Pullar JM, Carr AC, Bozonet SM, Vissers MCM. High vitamin C status is associated with elevated mood in male tertiary students. Antioxidants 2018;7. 74. Oishi J, Doi H, Kawakami N. Nutrition and depressive symptoms in community-dwelling elderly persons in Japan. Acta Med Okayama 2009;63:9
17. 75. Payne ME, Steck SE, George RR, Steffens DC. Fruit, vegetable, and antioxidant intakes are lower in older adults with depression. J Acad Nutr Diet 2012;112:2022
7. 76. Prohan M, Amani R, Nematpour S, Jomehzadeh N, Haghighizadeh MH. Total antioxidant capacity of diet and serum, dietary antioxidant vitamins intake, and serum hs-CRP levels in relation to depression scales in university male students. Redox Rep 2014;19:133
9. 77. Brody S. High-dose ascorbic acid increases intercourse frequency and improves mood: a randomized controlled clinical trial. Biol Psych 2002;52:371
4. 78. Cocchi P, Silenzi M, Calabri G, Salvi G. Antidepressant effect of vitamin C. Pediatrics 1980;65:862
3. 79. Amr M, El-Mogy A, Shams T, Vieira K, Lakhan SE. Efficacy of vitamin C as an adjunct to fluoxetine therapy in pediatric major depressive disorder: a randomized, double-blind, placebocontrolled pilot study. Nutr J 2013;12:31. 80 Aburawi SG, Ghambirlou FA, Attumi AA, Altubuly RA, Kara AA. Effect of ascorbic acid on mental depression drug therapy: clinical study. J Psychol Psychother 2014;4:131. 81. Mazloom Z, Ekramzadeh M, Hejazi N. Efficacy of supplementary vitamins C and E on anxiety, depression and stress in type 2 diabetic patients: a randomized, single-blind, placebocontrolled trial. Pak J Biol Sci 2013;16:1597
600. 82. Sahraian A, Ghanizadeh A, Kazemeini F. Vitamin C as an adjuvant for treating major depressive disorder and suicidal behavior, a randomized placebo-controlled clinical trial. Trials 2015;16:94. 83. Muneer A. Staging models in bipolar disorder: a systematic review of the literature. Clin Psychopharmacol Neurosci 2016;14:117
30. 84. Sani G, Napoletano F, Forte AM, Kotzalidis GD, Panaccione I, Porfiri GM, et al. The wnt pathway in mood disorders. Curr Neuropharmacol 2012;10:239
53. 85. Gould TD, Dow ER, O’Donnell KC, Chen G, Manji HK. Targeting signal transduction pathways in the treatment of

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Further reading

mood disorders: recent insights into the relevance of the Wnt pathway. CNS Neurol Disord Drug Targets 2007;6:193
204. 86. Huang L, Wu S, Xing D. High fluence low-power laser irradiation induces apoptosis via inactivation of Akt/GSK3beta signaling pathway. J Cell Physiol 2011;226:588
601. 87. Milner G. Ascorbic acid in chronic psychiatric patients
a controlled trial. Br J Psych 1963;109:294
9. 88. Naylor GJ, Smith AH. Vanadium: a possible aetiological factor in manic depressive illness. Psychol Med 1981;11:249
56. 89. Kay DS, Naylor GJ, Smith AH, Greenwood C. The therapeutic effect of ascorbic acid and EDTA in manic-depressive psychosis: double-blind comparisons with standard treatments. Psychol Med 1984;14:533
9. 90. Kessler RC, Berglund P, Demler O, Jin R, Merikangas KR, Walters EE. Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psych 2005;62:593
602. 91. Rammal H, Bouayed J, Younos C, Soulimani R. Evidence that oxidative stress is linked to anxiety-related behaviour in mice. Brain Behav Immun 2008;22:1156
9. 92. Steenkamp LR, Hough CM, Reus VI, Jain FA, Epel ES, James SJ, et al. Severity of anxiety- but not depression- is associated with oxidative stress in Major Depressive Disorder. J Affect Disord 2017;219:193
200. 93. Hovatta I, Juhila J, Donner J. Oxidative stress in anxiety and comorbid disorders. Neurosci Res 2010;68:261
75. 94. de Oliveira IJ, de Souza VV, Motta V, Da-Silva SL. Effects of oral vitamin C supplementation on anxiety in students: a

95.

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double-blind, randomized, placebo-controlled trial. Pak J Biol Sci 2015;18:11
18. Moritz B, Schwarzbold ML, Guarnieri R, Diaz AP, Rodrigues AL, Dafre AL. Effects of ascorbic acid on anxiety state and affect in a non-clinical sample. Acta Neurobiol Exp 2017;77:362
72. Puty B, Maximino C, Brasil A, Gouveia A, Oliveira KR, Batista J, et al. Ascorbic acid protects against anxiogenic-like effect induced by methylmercury in zebrafish: action on the serotonergic system. Zebrafish 2014;11:365
70. Fraga DB, Olescowicz G, Moretti M, Siteneski A, Tavares MK, Azevedo D, et al. Anxiolytic effects of ascorbic acid and ketamine in mice. J Psychiatr Res 2018;100:16
23. Marik PE. Hydrocortisone, ascorbic acid and thiamine (HAT therapy) for the treatment of sepsis. focus on ascorbic acid. Nutrients 2018;10(11). pii: E1762. Wang K, Jiang H, Li W, Qiang M, Dong T, Li H. Role of vitamin C in skin diseases. Front Physiol 2018;9:819. El Halabi I, Bejjany R, Nasr R, Mukherji D, Temraz S, Nassar FJ, et al. Ascorbic acid in colon cancer: from the basic to the clinical applications. Int J Mol Sci 2018;19(9). pii: E2752.

Further reading Hughes et al., 2011 Hughes RN, Lowther CL, van Nobelen M. Prolonged treatment with vitamins C and E separately and together decreases anxiety-related open-field behavior and acoustic startle in hooded rats. Pharmacol Biochem Behav 2011;97:494
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C H A P T E R

16 Artichoke leaf extract and use in metabolic syndrome as an antioxidant Khatereh Rezazadeh1 and Mehrangiz Ebrahimi-Mameghani2 1

Nutrition Research Center, School of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran 2 Nutrition Research Center, Department of Nutrition & Biochemistry, School of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

List of abbreviations ABTS ALE CA CAT CVD DDPH DHCA DHFA FA FFA FRAP GPx GR HDL-C IFA IDF MDA NADPH NCEP ATPIII NOS Nrf2 OS Ox-LDL PTEN ROS RNS SOD T2DM TAC VSMC WHO

2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt artichoke leaf extract caffeic acid catalase cardiovascular disease 2,2-diphenyl-1-picrylhydrazyl dihydrocaffeic acid dihydroferulic acid ferulic acid free fatty acids ferric reducing ability of plasma glutathione peroxidase glutathione reductase high-density lipoprotein cholesterol isoferulic acid International Diabetes Federation malondialdehyde nicotinamide adenine dinucleotide phosphate National Cholesterol Education Program Adult Treatment Panel III nitric oxide synthase nuclear factor E2
related factor 2 oxidative stress oxidized low-density lipoprotein phosphatase and tensin homolog reactive oxygen species reactive nitrogen species superoxidase dismutase type 2 diabetes mellitus total antioxidant capacity smooth muscle cells World Health Organization

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00016-0

Introduction Globe artichoke, Cynara cardunculus L. subsp. Scolymus (L.) Hayek, is a herbaceous perennial plant that belongs to the Asteraceae family (Compositae). Artichoke is widely grown in the Mediterranean area, but is also cultivated in other parts of the world. The head, or capitula, and bracts that encircle the flower heads are the edible portions of artichokes, which are consumed as a healthy food worldwide.1 Traditionally, artichoke leaves have been used in herbal medicine as a choleretic agent to treat hepato-biliary disease.2 In experimental and human studies, the leaf and head extract of artichoke has exhibited several therapeutic properties including hepatoprotective, hypoglycemic, hypocholesterlomic, antimicrobial, antifungal, as well as antioxidant effects.3
12 The antioxidant activity of artichoke is attributable to its polyphenolic compounds including mono- and dicaffeoylquinic acids (e.g., cynarin and chlorogenic acid) and flavonoids content (e.g., apigenin, luteolin, and their glucosides and rutinosides) as well as sesquiterpene lactones.13 Metabolic syndrome is a cluster of risk factors associated with an increase in the risk of Type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), which occur together more frequently than would be expected by chance alone. Various criteria have been suggested by different organizations for a clinical diagnosis of metabolic syndrome. Clinical definitions developed by the National Cholesterol Education Program

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© 2020 Elsevier Inc. All rights reserved.

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16. Artichoke leaf extract and use in metabolic syndrome as an antioxidant

Adult Treatment Panel III (NCEP ATPIII), the International Diabetes Federation (IDF), and World Health Organization (WHO) have become popular over the past decade. However, there is some controversy concerning the components and cut points used in these definitions. In 2009, therefore, a meeting between several major organizations including the International Diabetes Federation Task Force on Epidemiology and Prevention National Heart, Lung, and Blood Institute; American Heart Association, World Heart Federation, International Atherosclerosis Society, and International Association for the Study of Obesity agreed on one unified definition,14 as shown in Table 16.1. It defined hyperglycemia, hypertension, dyslipidemia (lowered high-density lipoprotein cholesterol [HDL-C] and raised triglycerides), and abdominal obesity as the main criteria. A person with 3 out of 5 of these features is defined to have metabolic syndrome. In addition, the 2009 joint interim statement proposed that national or regional waist circumference cut points should be used to determine abdominal obesity. Metabolic syndrome has been indicated to have a strong association with OS; a condition which defined as the imbalance between production of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and antioxidant capability.15 Recent evidence showed that OS is not only a risk factor of metabolic syndrome, but also an important contributor to the development of metabolic syndrome, because of the pathologic role of ROS in various features of metabolic syndrome, including lipoprotein metabolism, endothelial function, visceral adiposity, and insulin resistance.16 Some bioactive compounds found in foods and herbs have been proposed as a potential therapeutic strategy for restoration of impaired redox balance in patients with metabolic syndrome to limit increasing prevalence of metabolic syndrome and its comorbidities.17 Artichoke, as an antioxidant-rich plant, has been indicated to have antioxidant activity in TABLE 16.1

experimental studies and some human studies.3,4,8,18
39 Our recent meta-analysis demonstrated the antioxidant property of ALE in animal models.40 It was found that artichoke extract could decrease production of oxidants such as ROS and lipid peroxidation products and increase antioxidative defense, such as the activity of enzymatic and nonenzymatic antioxidants. It is noted that prevention and treatment of metabolic syndrome could be an important approach to reduce the burden of CVD and T2D in the general population. In this chapter, we will focus on evidence confirming the antioxidant effects of ALE in the prevention and treatment of metabolic syndrome, while the link between OS and metabolic syndrome will also be emphasized.

Oxidative stress and metabolic syndrome OS, a disturbance in the balance of oxidative and antioxidative systems of cells and tissues, leads to the excess production of ROS and RNS.41 Although, free radicals and ROS play an essential role in the regulation of some cell functions and also act as intercellular and intracellular signals at normal and moderate concentrations, they could attack all major cell structures, including proteins, lipids, and nucleic acids at high concentrations. Therefore excessive ROS produced could alter cell signaling, cell cycle control, cell structure, and cellular transport mechanisms. The major sites of ROS production are mitochondrial electron-transport systems; however, ROS are also generated by the activity of enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, lipooxygenases, cyclooxygenases, and uncoupled nitric oxide synthase (NOS). Since, mitochondria are targeted by cellular ROS, overproduction of ROS may significantly affect mitochondrial

Unified clinical criteria for diagnosis of metabolic syndrome.

Risk factor

Defining level

Abdominal obesity, given as waist circumferencea

Elevated waist circumference based on population- and country-specific definitions

Triglycerides

$ 150 mg/dL (1.7 mmol/L) and/or drug treatment for elevated triglycerides

HDL-C Men

, 40 mg/dL (1.0 mmol/L)

Women

, 50 mg/dL (1.3 mmol/L) and/or drug treatment for reduced HDL-C

Blood pressure

$ 130 mm Hg systolic and/or $ 85 mm Hg diastolic and/or antihypertensive drug treatment

Fasting glucose

$ 100 mg/dL and/or drug treatment for elevated glucose

a

It is suggested that either the AHA/NHLBI or IDF cutoff be used for Europeans and the IDF cutoff used for non-Europeans until more data are available. AHA/NHLBI, American Heart Association/National Heart, Lung, and Blood; IDF, International Diabetes Federation; HDL-C, high-density lipoprotein cholesterol.

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Artichoke leaf extract: bioactive compounds, pharmacokinetics, and safety

functions and promote OS. Mitochondrial dysfunction is obviously implicated in the development and progression of metabolic syndrome components and complications. Antioxidant defenses of the human body contribute to regulating ROS levels. The antioxidant system consists of two major groups: (1) endogenous enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx); and (2) nonenzymatic antioxidants including dietary components (vitamins C, A, and E, carotenoids, polyphenols, flavonoids, folic acid, selenium, zinc) and various circulating biomolecules (ceruloplasmin, albumin, pyruvate, nitric oxide, glutathione, uric acid, melatonin, N-acetycysteine, coenzyme Q10, bilirubin). One of the defects in metabolic syndrome and its related disorders is decreased antioxidant defense protection and increased cellular and systemic OS, and subsequent oxidative damage to cell structures. Notably, there is ongoing debate about whether OS is the cause or the consequence of metabolic syndrome. The strong relationship of OS with various constituents of metabolic syndrome has been investigated recently.16 In addition, OS along with chronic lowgrade inflammation may contribute to the development of clinical complications of metabolic syndrome including CVD and T2DM.42 Inflammatory agents, prompted by OS, could participate in ROS production and the oxidative process. Patients with metabolic syndrome have been shown to have increased oxidative injury, as evidenced by diminished some antioxidants, including vitamins E and C, and SOD, and increased biomarkers of protein oxidation, and lipid peroxidation such as malondialdehyde (MDA).16,43,44 Moreover, elevated concentration of oxidized-LDL (ox-LDL), a product of lipid peroxidation, was associated with increased development of metabolic syndrome.45 Mechanistic studies indicated that overproduction of ROS in patients with metabolic syndrome may to be a main underlying mechanism for mitochondrial dysfunction that is clearly involved in the pathogenesis of metabolic syndrome. Mitochondria-derived OS can be implicated in the development of metabolic syndrome components including obesity, insulin resistance, dyslipidemia, and hypertension. Several mechanisms have been suggested to explain the link between OS and metabolic syndrome. One of these mechanisms is the elevated level of plasma-free fatty acids (FFA) in metabolic syndrome that stimulate ROS production through unknown pathways. A high level of FFA may increase free radicals and ROS in endothelial and vascular smooth muscle cells (VSMC), as well as trigger ROS augmentation in adipose

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tissue.46 Another mechanism for the association between OS and metabolic syndrome has been related to the regulatory role of transcription factor p53, a critical tumor suppressor which could regulate the expression of GPx and SOD. It was shown that suppression of p53 in pathophysiological conditions such as metabolic syndrome may down-regulate GPx and SOD expression and subsequently stimulate ROS generation. Furthermore, induction of p53 in metabolic syndrome could inactivate tumor suppressor phosphatase and tensin homolog (PTEN), members of phosphoinositide phosphatase family, and, thereby, affect the PI3K/Akt signaling cascade and insulin sensitivity.47

Antioxidants and metabolic syndrome Based on evidence indicating that the association between OS and metabolic syndrome, there is great interest in developing strategies that aim to reduce ROS, for the treatment of metabolic syndrome. Antioxidant therapy has been suggested as a promising therapeutic approache for the restoration of impaired redox states in patients with metabolic syndrome. Medicinal herbs are important sources of natural antioxidants. Substantial evidence indicated that nutritional antioxidant could be used to prevent and treat metabolic syndrome and its health complications.46 The underlying mechanisms for free radical scavenging activity of most bioactive compounds found in medicinal herbs are not fully elucidated. The structure of the compound, interactions with other agents, and redox status of the body play key roles in determining their antioxidant efficacy. ROS scavenging antioxidants could normalize mitochondrial functions and result in preventing metabolic syndrome development and its subsequent complications Artichoke, as an antioxidant-rich herb, indicated antioxidant properties in animal and in vitro studies and limited clinical trials, and may be helpful in the prevention and treatment of several diseases resulting from OS, including metabolic syndrome.40

Artichoke leaf extract: bioactive compounds, pharmacokinetics, and safety Bioactive compounds The main bioactive components of ALE are (1) polyphenolic compounds and (2) triterpenes and sesquiterpene lactones (Figs. 16.1
16.3). 1. Polyphenolic compounds of ALE are comprised mostly of (a) caffeoylquinic acids (mono- and diesters) and (b) flavonoids.

II. Antioxidants and Pathology

FIGURE 16.1 Structures of main caffeoylquinic acids identified in artichoke leaf extract (ALE). Chlorogenic acid (5-O-caffeoylquinic acid), 1,5-O-dicaffeoylquinic acid, and cynarin (1,3-O-dicaffeoylquinic acid) are the main caffeoylquinic acids in ALE. ALE, Artichoke leaf extract.

FIGURE 16.2 Structures of main flavonoids identified in artichokes. Flavones (apigenin, luteolin and naringenin) and anthocyanidins (cyanidin) are the main flavonoids in ALE. ALE, Artichoke leaf extract.

FIGURE 16.3 Structures of main sesquiterpene lactones identified in artichokes. Cyanoropicrin and grosheimin are the main sesquiterpene lactones in ALE. ALE, Artichoke leaf extract.

Artichoke leaf extract, oxidative stress, and metabolic syndrome

a. Caffeoylquinic acids are structured of one or two caffeic acid moieties and one quinic acid molecule, which is composed by a broad range of derivatives. The most abundant caffeoylquinic derivatives identified in ALE are chlorogenic acid (5-O-caffeoylquinic acid) and 1,5-Odicaffeoylquinic acid. Cynarin (1,3-Odicaffeoylquinic acid) is the most well-known caffeoylquinic derivative found in ALE, even though it is not the most abundant component. Cynarin and chlorogenic acid constitute about 1.5% and 39% of the caffeoylquinic acid derivatives of artichoke, respectively.1 b. Flavonoids are structured by three rings, two of them are benzene rings (ring A and B), joined by an oxygenated pyrane ring (ring C). The main flavonoids identified in ALE are flavones (apigenin, luteolin, naringenin) and anthocyanidins (cyanidin, peonidin, pelphinidin). Luteolin and apigenin glycosides have been found in the leaves and heads of artichoke, even though anthocyanin pigments are identified only in the capitula. The most representative flavone glycosides found in artichoke are luteolin-7-Oglucoside, luteolin-7-O-rutinoside, apigenin-7-Oglucoside, apigenin-7-O-rutinoside, naringenin-7O-rutinoside, and naringenin-7-O-glucoside. In addition, the main anthocyanin detected in artichoke heads or capitula are cyanidin 3-Oglucoside, cyanidin 3,5-diglucoside, cyanidin 3,5malonyldiglucoside, cyanidin 3-(3v-malonyl) glucoside (VIII), and cyanidin 3-(6v-malonyl) glucoside. Several minor anthocyanins were also found in artichoke such as peonidin and delphinidin derivatives.1,48 2. Triterpenes and sesquiterpene lactones have been detected in artichoke as the main component of lipophilic fraction. The basic structure of triterpenes is formed by six isoprene units and the chemical structure of sesquiterpene lactones are composed of three isoprene units and one lactone ring. Triterpenes are found in low amounts in the leaves of artichoke, while sesquiterpenes are present mostly in the leaves, and in lesser amounts in the capitula. Cyanoropicrin is the most abundant sesquiterpene in artichoke. Cynarascolosides A, B, and C, grosheimin, deacylcynaropicrin, lupenyl acetate, and ψ-taraxasteryl acetate were also detected in artichoke.48

Pharmacokinetics The evaluation of the pharmacokinetics of artichoke extract in human volunteers confirmed the bioavailability of metabolites derived from bioactive

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compounds. The metabolites derived from caffeoylquinic acids are caffeic acid (CA), ferulic acid (FA) (methylated derivates of CA), isoferulic acid (IFA), dihydrocaffeic acid (DHCA), and dihydroferulic acid (DHFA). Although it was shown that none of the genuine target extract components in plasma or urine could be found, they were detected as sulfates or glucuronides in plasma and urine with the exception of DHFA. Luteolin was also identified as sulfate or glucuronide in plasma and urine.49

Safety The leaf of artichoke is usually considered as a safe herbal medicine, and has been approved for use by the German Commission E Monographs for dyspepsia.50 In clinical trials, ALE revealed good tolerability and low incidence of mild and transient adverse events.51,52 Recently, an in vivo evaluation of mutagenic and genotoxic activity of artichoke leaf aqueous extract showed no mutagenic effects. However, only at 2000 mg/kg dose, the highest dose tested, was genotoxic activity observed.53

Artichoke leaf extract, oxidative stress, and metabolic syndrome The review of experimental and clinical studies supports the potent antioxidant capacity of ALE through the protection of cells and tissues against OS induced by various oxidants or toxins. Our recent systematic review study showed that ALE demonstrates antioxidant activity in animal studies, in vitro systems and clinical trials, and the meta-analysis of findings from animal studies confirmed the free-radical scavenging activity of ALE.40 Furthermore, our literature review indicated that ALE could be effective as an antioxidant in preventing or treating OS-related diseases. It is known that OS is increased in metabolic syndrome, as well as T2DM and CVD. Previous studies have shown increased ROS production, biomarkers of protein oxidation and lipid peroxidation, and decreased enzymatic and nonenzymatic antioxidants in patients with metabolic syndrome.15,16 A recent clinical trial reported that ALE supplementation in patients with metabolic syndrome decreased ox-LDL levels significantly with no significant effect on MDA, SOD, GPx, and the total antioxidant capacity (TAC) concentrations4 (Table 16.2). As shown in Fig. 16.4, ALE could be therefore be effective in the prevention and/or treatment of metabolic syndrome through the suppression of OS. Free-radical scavenging potential of ALE was assayed in vitro by FRAP (ferric reducing ability of plasma), ABTS (2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)

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174 TABLE 16.2

16. Artichoke leaf extract and use in metabolic syndrome as an antioxidant

Effects of artichoke leaf extract (ALE) on oxidative/antioxidative status biomarkers in metabolic syndrome patients. ALE group (n 5 33)

Placebo group (n 5 35)

MD (95% CI), p

Befored

2.95 (0.85)

2.73 (0.52)

0.23 (0.17 to 20.12), 0.194a

Afterd

2.90 (0.91)

2.65 (0.62)

0.14 (20.49 to 0.77), 0.647b

MD (95% CI), pc

20.04 (20.26 to 0.32), 0.836

20.07 (20.35 to 0.19), 0.563

Befored

1.75 (0.28)

1.83 (0.37)

20.08 (20.25 to 0.09), 0.370a

Afterd

1.75 (0.27)

1.80 (0.39)

0.03 (20.07 to 0.13), 0.540b

MD (95% CI), pc

0.00 (20.05 to 0.05), 0.880

20.03 (20.07 to 0.00), 0.115

Befored

1745.86 (377.40)

1752.50 (266.10)

26.64 (2166.45 to 153.17), 0.935a

Afterd

1772.57 (308.91)

1767.10 (307.3)

96.21 (244.70 to 237.13), 0.174b

MD (95% CI), pc

30.74 (268.77 to 130.25), 0.533

31.48 (221.20 to 84.17), 0.233

Befored

42.75 (13.04)

41.64 (12.58)

1.11 (25.19 to 7.41), 0.726a

Afterd

42.20 (11.66)

41.81 (11.55)

0.19 (26.26 to 6.65), 0.951b

MD (95% CI), pc

20.55 (24.72 to 3.61), 0.789

0.16 (23.29 to 3.63), 0.922

Befored

5914.28 (965.28)

5623.87 (996.77)

290.41 (2222.25 to 802.28), 0.261a

Afterd

5647.42 (1031.93)

5494.35 (924.05)

2443.94 (2850.63 to 237.25), .033b

MD (95% CI), pc

2266.8 (2505.69 to 228.02), .030

2129.51 (2346.39 to 87.36), 0.232

Variable MDA (nmol/mL)

TAC (mmol/L)

SOD (U/gHb)

Gpx (U/gHb)

Ox-LDL (U/L)

a

Independent t-test. Analysis of covariance (adjusted for change of intake of energy, vitamins A, E, and C, zinc and selenium, and baseline values). c Paired t-test. d Mean (SD). P-values of statistical significance (P , .05) are presented in bold. ALE, artichoke leaf extract; ox-LDL, oxidized low-density lipoprotein; MDA, malondialdehyde; SOD, superoxidase dismutase; GPx, glutathione peroxidase; TAC, total antioxidant capacity. Source: We obtained copyright permission from Elsevier to reuse this table.4 b

diammonium salt), and DDPH (2,2-diphenyl-1-picrylhydrazyl) methods. The results indicated high-antioxidant activity for ALE. In addition, the comprehensive antioxidant food database lists artichoke as an antioxidant-rich herb.54 The antioxidant effects of ALE appear to be mediated by three mechanisms: (1) suppressing the formation of free radicals by inhibiting some enzymes or chelating metal ions involved in free-radical generation; (2) scavenging ROS produced under OS; and (3) protecting or upregulating antioxidant defences.55 Moreover, antioxidants could be modulating ROSdependent cell functional signaling through intercepting ROS at the level of critical signaling pathways involving several phosphatase, protein kinases, and transcription factors.56 It has been hypothesized that

bioactive compounds of ALE may protect tissues from oxidative damage and lipid peroxidation by acting as hydrogen donors, reducing agents, metal chelator, and singlet-oxygen quenchers. In addition, bioactive components of ALE may act as radical scavengers. Most of the antioxidant properties of ALE have been assigned to polyphenolics compounds (caffeoylquinic acids and flavonoids) and sesquiterpenes. Cynarin and chlorogenic acid, as the utmost important caffeoylquinic derivatives in ALE, may be partly responsible for its antioxidant capacity.57 Although flavonoids are found in minor concentrations in artichoke (about 10% or less of phenolic compounds), they indicated strong antioxidant capacity. Luteolin and luteolin-7-O-glucoside are considered as power antioxidants that protect low-density lipoprotein

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Summary points

metabolic syndrome components and prevent metabolic syndrome
related complications.

Applications to other areas of pathology

FIGURE 16.4 Role of ALE in prevention and treatment of metabolic syndrome. ALE could act as a therapeutic agent for patients with metabolic syndrome through inhibition of oxidative stress. ALE, Artichoke leaf extract; MetS, metabolic syndrome; ROS: reactive oxygen species.

cholesterol against oxidation. Cyanoropicrin, a major class of sesquiterpene, could inhibit ROS production by activating the nuclear factor E2
related factor 2 (Nrf2), a transcription factor that modulates antioxidant response.

This chapter describes the antioxidant effects of ALE in prevention and treatment of metabolic syndrome, and also defines the link between oxidative stress and metabolic syndrome. The pathophysiological implications of OS have been documented in a variety of conditions including obesity, diabetes mellitus, dyslipidemia, hypertension, and cardiovascular disease as well as neurodegenerative diseases of the nervous system and cancer. Finding from in vitro studies showed that ALE may exhibit apoptosis and chemopreventive properties in breast cancer cells and hepatoma cells by increased ROS production.26,58 In addition, in vitro and animal studies revealed that ALE could protect the liver and kidney against freeradical damage induced by toxins or generator of ROS.59,60

Summary points Conclusion OS involvement in the multifaceted pathophysiology of metabolic syndrome and its complications has been widely accepted. This pathophysiological condition requires further investigation into the potential natural antioxidants are capable of preventing or treating metabolic syndrome and related disorders such as CVD and T2DM. There is a growing body of evidence that herbal antioxidants play an important role in the neutralization of ROS formed during oxidative damage, and improving the antioxidant defense system. ALE from the antioxidant-rich plant have been found to confer beneficial antioxidant effects in animal models, in vitro systems and in some clinical trials. The finding of a meta-analysis of animal studies reported convincing evidence for antioxidant activity of ALE. In patients with metabolic syndrome, supplementation of ALE could lead to slight improvement in antioxidant systems. Nevertheless, antioxidant mechanisms of ALE are complicated and multifactorial. Currently, there is limited information concerning the type and dosage of ALE that are required to establish the beneficial antioxidant effects in metabolic syndrome patients. Further, additional double-blind, placebo-controlled, randomized, clinical trials are warranted to determine the duration needed to alleviate

• Oxidative stress (OS) is an imbalance between the production of ROS or RNS and the body’s antioxidant capability. • OS involvement in pathophysiology of metabolic syndrome has been widely accepted. • Mitochondrial dysfunction is obviously implicated in the development and progression of metabolic syndrome components and complications. • Some natural antioxidants found in medicinal herbs have been proposed as a potential therapeutic strategy for restoration of impaired redox balance in patients with metabolic syndrome. • Artichoke as an extract from the antioxidant-rich plant indicated antioxidant activity in experimental studies and some human studies. • The antioxidant activity of artichoke is attributable to its polyphenolic compounds (monoand dicaffeoylquinic acids and flavonoids) and sesquiterpene lactones. • In patients with metabolic syndrome, supplementation of ALE could lead to the slight improvement of antioxidant systems. • Future clinical trials are warranted to clarify the type and dosage of artichoke extract and study duration needed to alleviate metabolic syndrome components and prevent metabolic syndrome
related complications.

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16. Artichoke leaf extract and use in metabolic syndrome as an antioxidant

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C H A P T E R

17 Bilberry anthocyanins as agents to address oxidative stress Jerry T. Thornthwaite1, Seth P. Thibado2 and Kyle A. Thornthwaite1 1

Cancer Research Institute of West Tennessee, Henderson, TN, United States 2Department of Chemistry, Union University, Jackson, TN, United States

List of abbreviations AD APP CVD DOX IL NNS OS ROS T2D VEGF

Alzheimer’s disease amyloid precursor protein processing cardiovascular disease doxorubicin interleukin nutrananospheres oxidative stress reactive oxygen species type 2 diabetes vascular endothelial growth factor

Introduction Bilberry (Vaccinium myrtillus L.) is one of the richest natural sources of anthocyanins, which are polyphenols comprising soluble glycoside pigments producing blue to red (purple) coloring in flowers, fruits, and vegetables. These polyphenolic components have highantioxidant content, and are believed to be the key compounds responsible for the many reported health benefits of eating berries (Fig. 17.1). The daily intake of anthocyanins in the average US diet is estimated to be between 180 and 215 mg, whereas the intake of other dietary flavonoids such as genistein, quercetin, and apigenin is only 20
25 mg/day.1 Epidemiologic studies suggest that the consumption of anthocyanins lowers the risk of cardiovascular disease (CVD), diabetes, arthritis, vision diseases, and cancer due, at least in part, to their antioxidant and antiinflammatory activities.2 Research advancing current scientific understanding of the health benefits of berries continues to increase. The Berry Health Benefits Symposium (BHBS) is a biennial meeting highlighting the most recent berry Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00017-2

health benefits research from all over the world.3 Excellent reviews on bilberry were published in 20114 and 2014.5 Berries are a rich source of a wide variety of bioactive compounds such as anthocyanins, which have potent antioxidant, anticancer, antimutagenic, antimicrobial, antiinflammatory, and antineurodegenerative properties, and, at least in the case of cancer, can be directly cytotoxic in vitro.6,7 This chapter presents a comprehensive and critical review that addresses the antioxidant activities of the anthocyanins, especially with bilberry. Antioxidants found in berries are natural gifts for the prevention and treatment of a variety of diseases, as shown in Fig. 17.1. We prepared a video to explain the general aspects of the antioxidant process, and may be viewed at https://www.youtube.com/watch?v 5 KCF6prDSrHE

Other areas of pathology This chapter describes the use of bilberry as an antioxidant to neutralize the devastating effects of oxidation on a variety of diseases. While we have tried to cover the major disease states, other areas such as weight loss, exercise physiology, and skin care are not covered. There is a host of websites that address these applications and can be researched by Googling bilberry and the subject of interest. While we consider these not to be life threatening issues, we are all interested in the effects of antioxidants to help with weight loss, exercise physiology, and antiaging of our skin. This chapter does not go into great detail about the oxidant
antioxidant biochemical mechanisms of

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17. Bilberry anthocyanins as agents to address oxidative stress

FIGURE 17.1 Examples of berry sources and preventive and treatment benefits of anthocyanins for cancer treatment, heart disease, diabetes, vision including macular degeneration, and brain diseases such as dementia and Alzheimer’s disease. The mg content of anthocyanins per 100 g of berries are shown. References for anthocyanins content: Bilberry and blueberries (www.ncbi.nlm. nih.gov/books/NBK92770/); Blackberry and raspberry (https://www.sciencedirect.com/science/article/pii/S0889157509002622); Pomegranate (http://docsdrive.com/pdfs/ansinet/ pjbs/2013/636-641.pdf); Cherries (https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC1082898/pdf/S1110724304404136.pdf); Ac¸aı´ and strawberries (www.immunehealthscience. com/anthocyanins.html).

action. Two very good reviews4,5 may expand some of the areas we do not cover. Finally, we have recently published studies showing the anticancer effects using bilberry and other nutraceuticals to kill cancer cells in vitro directly.6,7 Furthermore, we have developed assay systems for measuring antioxidation8 and cell viability6,7 using flow cytometry, which have many applications for pathology medical research.

Chemistry of bilberry anthocyanins Anthocyanins are plant pigments that give fruits, leaves, flowers, stems, and roots their rich colors that span the visible light spectrum. About 560 different anthocyanins have been identified using analytical chemistry. Capillary zone electrophoresis, nuclear magnetic resonance, and mass spectroscopy have been used in identifying at least 15 anthocyanins in bilberry extracts.9,10 Six of the major anthocyanin’s structures in bilberry with their primary colors found over the visible light spectrum are shown in Fig. 17.2. The anthocyanin content of fresh bilberries is over 50% higher than in fresh blueberries, and both are over 50% higher

than commercially available juices. The daily intake of anthocyanins by Americans is estimated to be about 200 mg, about ninefold higher than that of other dietary flavonoids.11 Genes important for detoxification and antioxidant defense induced by mild stress may provide health benefits. These defenses have been attributed to anthocyanins, which are 25% of the mass of bilberries. Antioxidants, such as glutathione and other components of the detoxification systems, biochemically function by the transactivation of genes, such as gamma-glutamylcysteine synthetase, containing electrophile response elements within their promoters.12 The conjugated bonds in the anthocyanins absorb light at about 500 nm and are the basis for the bright red, blue, and purple colors of fruits and vegetables, as well as the autumn foliage of trees when the supply of chlorophylls in foliage is decreased due to the lack of sun, thus revealing anthocyanin components resulting in a deciduous event.13 Most naturally occurring anthocyanins are derived from six aglycones, namely, pelargonidin, cyanidin, delphinidin, pelargonidin, petunidin, peonidin, and malvidin. In aqueous solutions, they exist in various equilibrium forms depending on the pH. In highly acidic media, red flavylium cations predominate, in

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Bilberry anticancer activity

FIGURE 17.2 Spectral absorbance ranges for the six most-abundant anthocyanins in bilberries.

slightly acidic to neutral solutions blue quinonoidal bases are formed by deprotonation, while colorless hemiketal forms are produced by hydration.14 Anthocyanins span the color spectrum (Figs. 17.1 and 17.2) of raspberries, strawberries, blueberries, blackberries, ac¸aı´ berries, and the deep purple of bilberries.15 Quantitatively for bilberry, cyanidin was found in the highest quantities (mean amount 0.053 μg/mL). Delphinidin and petunidin were found in quantities 2.5-fold lower than cyanidin.16

Why plants contain anthocyanins Plants must protect themselves from radiationinduced damage such as ultraviolet light from the sun. Human consumption of natural products from plants may also provide protection from radiation damage.17 Furthermore, defense against herbivores is a beneficial strategy for plants and well worth the allocation of cellular resources from growth and reproduction to apply chemical elicitors to induce defense responses.17,18

Why humans and animals must consume anthocyanins Numerous studies indicate that a diet rich in berries positively affects human health. Regular consumption of fruits may delay aging processes and reduce the risk of various illnesses, such as cancer, cardiovascular,

rheumatoid arthritis, vision, dementia, and Alzheimer’s or Parkinson diseases.19
23 Bilberry and black currant juices show high antiadhesive and antibacterial activity against food-spoiled bacteria belonging to the genus Asaia.14,17,18 In particular, bilberry juice is characterized by a high content of polyphenols including A-type proanthocyanidin, which shows strong antibacterial properties. The high content of bioactive compounds with proven health-promoting properties makes them a valuable supplement in consumer drinks, as well as alternatives to artificial additives to maintain the antimicrobial stability of the final products.14 Only 18% of the US population met the recommendation of at least 2 cups of fruit per day. Children aged 2
5 years consumed the most total fruit, of which about half was juice and only 4% were berries, thus significantly reducing the antioxidant activity. The highest berry consumption in adults was by those 65 years and older, non-Hispanic Whites, with high education and income levels. Education is critical in making people aware of the importance good health and dietary guidance, as well as the availability of fruit in the home. Fruit consumption is about twice the amount in these home environments compared to other groups.24 Furthermore, participation in sports gives the child a coach or mentor who can reinforce the parents’ emphasis on nutrition.

Bilberry anticancer activity Data from numerous cell culture and animal models indicate that berry anthocyanins are potent

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anticarcinogenic agents and are protective against genomic instability for a number of sites in the carcinogenic pathway. Significant clinical research studies indicate that anthocyanins exhibit anticancer activity and the evaluation of bilberry anthocyanins for cancer prevention is becoming important.25,26 Natural compounds and their derivatives are now playing an increasingly important role, in that over 60% of therapeutic drugs used in the treatment of cancer are from natural compounds.27 Anthocyanins upregulate tumor suppressor genes, induce apoptosis in cancer cells, repair and protect genomic DNA stability by reducing age-related oxidative stress (OS), and improve neuronal cognitive brain function.24
27 By increasing their bioavailability, bilberry anthocyanins can be directly cytotoxic to cancer cells in vitro.7 To increase their bioavailability, bilberry was encapsulated in 5.52 nm mean diameter liposomal micelles, called NutraNanoSpheres (NNS).7 In general, anthocyanins and other nutraceuticals purified from natural products such as curcumin, genistein, resveratrol, artemisinin, and vitamin C have been shown to exhibit anticarcinogenic activity against K562 cancer cells in vitro.6,7 Cancer chemopreventive activities of anthocyanins reveal radical scavenging activity, antiangiogenesis, apoptosis, stimulation of detoxifying enzymes, and antiinflammation. Anthocyanins modulate the expression and activation of multiple genes associated with these cellular functions, which include, in part, the PI3K/Akt, ERK, JNK, and MAPK pathways.25 Data from numerous cell culture and animal models indicate that berry components such as anthocyanins are potent anticarcinogenic agents and are protective against genomic instability for a number of sites in the carcinogenic pathway. These anticarcinogenic mechanisms include inhibition of cell invasiveness and metastasis, decreased DNA binding of the carcinogen modulation of carcinogen activation and detoxification, inhibition of oxidative DNA damage, and altered cell signaling for malignant transformation.26,27 Dietary, regular, consumption of anthocyanins such as from bilberries, vitamins (i.e., A, C, D, E, B2), carotenoids, flavonoids, curcumins, resveratrol, magnesium, and omega-3 fatty acids show protective effects against lung function loss, help mitigate the DNA damage plus epigenetic changes from lung cancer or pulmonary disease, and provide dietary intervention early in life for maintaining healthy lung function.28 Colorectal cancer, after lung and prostate cancers, is the third-most common diagnosed cancer in men worldwide. For women, colon cancer is second to breast cancer worldwide and is 18% higher in developed countries, which may be related to the Western

diet and lifestyle.29 The chemoprotective activity of anthocyanin-rich extracts from bilberry caused a significant decrease in rat colon carcinogenesis as measured by the COX-2 mRNA gene expression.30,31 Numerous epidemiological, preclinical, and clinical studies strongly support anthocyanins for preventing and treating colon cancer.32 Bilberries prevented the formation and growth of colorectal cancer development and growth in chemically induced tumors of an azoxymethane/dextran sodium sulfate mouse model.33 Vascular endothelial growth factor (VEGF) plays a crucial role for the vascularization of tumors. The vasculature in adult skin remains normally quiescent. Each of the berry samples studied significantly inhibited both peroxide as well as TNF alpha
induced VEGF expression by the human keratinocytes.20,34 Concerning breast cancer, Syzygium cumini (jamun) has the same diversity of anthocyanins as bilberry and other fruit, including blueberry and black raspberry.20 These berries are antagonistic against 17β-estrogenmediated breast cancer with a reduction in mammary carcinogenicity.19,20,35

Bilberry protective effect against chemotherapy and radiation therapy A problem of cancer chemotherapy is the high cytotoxicity toward normal, rapidly proliferating, immune cells, especially in the bone marrow. To mitigate these side effects, modified therapeutic regimens using antioxidants, such as combination therapies, have been introduced. Doxorubicin (DOX) is a commonly used chemotherapeutic agent that has serious dose-limiting cardiotoxicity due to the generation of reactive oxygen species (ROS). Bilberry supplementation reduced OS in many tissue injuries due their high content of antioxidants. The potential protective effect of bilberry extract against DOX-induced cardiotoxicity in rats has been investigated.36 Bilberry extracts have shown significant protective capability against cisplatin cytotoxicity in rats against hearing loss by the impaired cochlea, the spiral tube shaped like a snail shell forming part of the inner ear, which is essential for hearing.37 Cisplatin is one of the most effective chemotherapeutic agents; however, injury may occur at higher doses. The aim of this study was to investigate the effect of bilberry on cisplatin-induced toxic effects in the rat ovary. Bilberry administration reduced the cisplatin-induced ovarian toxicity by alleviating freeradical damage, but did not protect all of the ovary tissue.38 Radiation therapy has been a mainstay of cancer treatment for decades. Ionizing radiation (IR) is used

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Bilberry’s importance in the prevention and treatment of diabetes

for the destruction of cancer cells and shrinkage of tumors. However, the increase of radiation resistance in cancer cells and radiation toxicity to normal tissues are serious concerns. The exposure to radiation generates intracellular ROS, which leads to DNA damage by lipid peroxidation, removal of thiol groups from cellular and membrane proteins, strand breaks, and base alterations39 Anthocyanin supplementation coupled with standard radiation therapy may improve the efficacy of cancer therapies by increasing tumor response and decreasing toxicity.40 In cancer patients treated with radiotherapy to the abdominopelvic region may benefit from dietary modifications and the use of functional foods (i.e., fortified food with added ingredients to provide specific health-improving benefits, such as antioxidants).41

Bilberry’s importance in the prevention and treatment of cardiovascular disease Some epidemiological studies suggest that increased consumption of anthocyanins is associated with lower risk of CVD and hypertension. Bilberry extracts (concentration-dependent) inhibited mitochondrial stage 3 respiration (by 23%
61%) with pyruvate plus malate, mildly (by 1.2
1.3-fold) uncoupled the oxidative phosphorylation and increased (by 30%
87%) the state 4 respiration.42 Furthermore, animal and in vitro studies strongly indicate that bilberry and blueberry have the potential to ameliorate cardiometabolic CVD.43 Pretreatment with bilberry significantly guarded against DOX-induced increase in serum activities of lactate dehydrogenase, creatine phosphokinase, and creatine kinase-MB, as well as the level of troponin I. Bilberry alleviated ECG changes in rats treated with DOX and attenuated its pathological changes. Bilberry protects against DOX-induced cardiotoxicity in rats.44 Consumption of fresh bilberry has an important influence on the prophylaxis and progression of CVD due to its antioxidant properties and antiplatelet activity.43,45 Berries can assist in the restoration of morphology and functions of the heart and vessels after injury by inhibiting platelets function, protecting vascular endothelial cells, regulating lipids metabolism, modulating blood pressure, alleviating ischemia/reperfusion injury, suppressing thrombosis, reducing OS, and reducing inflammation.46 Furthermore, bilberry anthocyanins modulate the inflammation processes by significantly decreasing blood concentrations of C-reactive protein, interleukin (IL)-6, IL-15, and increasing tumor nuclear factor-alpha, which targets NFkappa-B, a transcriptional factor that is crucial in inflammatory responses.47 These studies suggest that bilberry anthocyanins modulate inflammation

183

and is a significant supplement for the prevention and treatment of heart disease.

Bilberry’s importance in the prevention and treatment of diabetes Dietary strategies for alleviating health complications associated with Type 2 diabetes (T2D) are being studied as supplements and/or alternatives to pharmaceutical interventions. Bilberries are rich in anthocyanins that may influence carbohydrate absorption and digestion by affecting low-blood sugar after a meal (postprandial glycemia), because of their antioxidant and antiinflammatory properties.48 Fasting serum hippuric acid, which is used as a biomarker for anthocyanin consumption, is increased after a diet of anthocyanin-rich bilberries and may contribute to the beneficial effect of bilberry consumption through its associations with better glycemic control and β-cell function.49 Antioxidants richly present in bilberry fruits are believed to have significant effects on diabetes-related brain dysfunctions, mainly due to their abilities to modulate neurotransmitter release that lead to reduction of the negative impact of free radicals on cognitive processes. It is well-known that diabetes is a risk factor that may trigger brain atrophy, cognitive dysfunction, and, finally, lead to memory loss. Bilberry anthocyanins influence the morphology of, and probably exhibit beneficial and neuroprotective effects on, hippocampal neurons during diabetes.50,51 Consumption of anthocyanin-rich bilberries is associated with a lower risk of developing T2D. The antidiabetic role of anthocyanins is based on cellular and molecular mechanisms, and animal and in vitro studies strongly indicate that bilberry and blueberry have the potential to help cure T2D and its cardiometabolic outcomes.43,50 In a study of 180 patients with T2D, analysis of clinical, functional, and morphological changes during treatment with antioxidant and angio-protective anthocyanins proved effective in T2D patients with diabetic retinopathy, and can be considered not only as a preventive, but also as a therapeutic, measure in T2D patients.52 Bilberry enhances hyperglycemia and insulin sensitivity by activation of AMP-activated protein kinase, thus reducing the blood glucose concentration and enhanced insulin sensitivity. These findings provide a biochemical basis for the use of bilberries and have important implications for the prevention and treatment of T2D via activation of AMP-activated protein kinase.53 Furthermore, bilberry anthocyanins may exert their beneficial effects in diabetes by increasing insulin secretion and reducing apoptosis, causing increased proliferation of pancreatic β-cells, regulating glucose

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17. Bilberry anthocyanins as agents to address oxidative stress

metabolism, and reducing inflammation in muscle.54 Therefore a diet rich in anthocyanins from bilberries and other berry sources may have beneficial effects in the efforts to prevent T2D in high-risk persons.48
55

Bilberry and macular degeneration (vision) Bilberry anthocyanins significantly ameliorate the neuroprotective effects by reducing the ROS found in photostressed murine retinas.56 The extract also suppressed photoinduced apoptosis in the photoreceptor cell layer and the shortening of outer segments of rod and cone photoreceptors. N-acetyl-L-cysteine, a well-known antioxidant, also suppressed ROS species.56 Excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration.57 Blue light is a high-energy or short-wavelength visible light that induces retinal diseases such as age-related macular degeneration and retinitis pigmentosa. Bilberry showed protective effects against blue light retinal photoreceptor cell damage through inhibition of ROS production and activation of proapoptotic proteins. Therefore

bilberry is a potential source of bioactive compounds that potentially serve as a therapeutic approach for age-related macular degeneration.58
60 Bilberry anthocyanins and blueberries61 promote rhodopsin synthesis and regeneration, increase retinal sensitivity to changes in light intensity, improve visual acuity and dark adaptation, as well as improve the blood supply to the retina. Therefore anthocyanins should have a significant effect on agerelated macular degeneration, diabetic retinopathy, primary open-angle glaucoma, and night blindness.60
63 In summary, excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration.64 Interestingly, bilberry supplementation improved some of the objective and subjective parameters of eye fatigue induced by research patients staring at a video monitor all day.57

Bilberry’s importance in preventing and curing dementia and Alzheimer’s disease The pathogenesis of Alzheimer’s disease (AD) is strongly correlated with the aggregation and deposition

FIGURE 17.3 Summary of the antioxidant effect of anthocyanins acting as free-radical scavengers, chemistry of bilberry polyphenols, and prevention and curing of disease examples. Source: Elsevier is accounted for 16% of the world market in science, technology, and medical publishing.

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References

of the amyloid beta (Aβ1-42) peptide in fibrillar form. Significant studies have shown that bilberry anthocyanin extracts inhibit Aβ1 accumulation.65 Bilberry anthocyanins cause neuroprotection in transgenic AD mice. Bilberry may help to improve memory and learning capability in chemically induced Alzheimer’s disease in experimental animal models.66 The increase in β-amyloid production is due, in part, to altered amyloid precursor protein processing (APP), a key pathogenic feature of AD. Bilberry anthocyanins cause neuroprotection in transgenic AD mice by decreased APP C-terminal fragment levels in the cerebral cortex compared to APdE9 mice on the control diet.67 Furthermore, there is strong evidence that OS participates in the etiology of neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases, and even emotional stress. The protective effect of anthocyanins on the cerebral OS was studied using the whiskers-cut model. In mice, such treatment causes psychological or emotional distress leading to OS in tissues. Bilberry anthocyanins were active in the brain, suppressing stress-induced cerebral OS and dopamine abnormalities in distressed mice. These effects of anthocyanin treatment suggest their possible usefulness for the treatment of cerebral disorders related to OS.68 Similar results were obtained from rats where berries were beneficial in the prevention of memory deficits.69 A summary of the role of bilberries in the prevention and treatment of human and animal diseases is shown in Fig. 17.3. Much of what is reported in this chapter is based on our experience in the development of immunological formulations and techniques to measure antioxidation8 and tumor cytotoxicity.6,7 Fig. 17.3 describes the free-radical scavenging abilities of anthocyanins and the importance of these in the examples presented in this chapter on the antioxidant effects of bilberry.

Summary points • This chapter focuses on bilberry, which contains anthocyanins as polyphenols that act as antioxidants. • Anthocyanins span the visible color spectrum, and bilberry contains predominately cyanidin, one of six of the most studied anthocyanins. • Bilberries contain over 50% more anthocyanins than blackberries, and both lose 50% of their antioxidants when the fresh berries are processed into a drink. • Anthocyanins upregulate tumor suppressor genes, induce apoptosis in cancer cells, repair and protect genomic DNA stability by reducing age-related oxidative stress, and improve neuronal cognitive brain function.

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• By increasing the bioavailability, bilberry anthocyanins can be directly cytotoxic to cancer cells in vitro. • Bilberry has a protective effect for normal cells against chemotherapy and radiation therapy. • Antioxidants richly present in bilberry fruits are believed to have significant effects on diabetesrelated brain dysfunctions, mainly due to their abilities to modulate neurotransmitter release that leads to a reduction of the negative impact of free radicals on cognitive processes. • Increased consumption of anthocyanins is associated with lower risk of CVD and hypertension. • Bilberry anthocyanins significantly ameliorate the neuroprotective effects by reducing the reactive oxygen species found in photostressed murine retinas. • Oxidative stress participates in the etiology of neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases, and in emotional stress.

Acknowledgments All work for this chapter was conducted at the Cancer Research Institute of West Tennessee, under the direction of Dr. Jerry Thornthwaite, Ph.D., who provided all the materials and equipment used. This research was supported in part by generous donations from Mr. Henry Respess, The Shumard Foundation and the Carter Family Trust. We thank Dr. Tony Kirk and Bonita Thornthwaite for reviewing this manuscript. Special thanks goes to Patty O’Neal for her assisting in both the office and laboratory preparations for this study.

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42. Choi YH, Kwon HS, Shin SG, Chung CK. Vaccinium uliginosum L. improves amyloid β protein-induced learning and memory impairment in alzheimer’s disease in mice. Prev Nutr Food Sci. 2014;19(4):343
7. Vepsa¨la¨inen S, Koivisto H, Pekkarinen E, Ma¨kinen P, Dobson G, McDougall GJ, et al. Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer’s disease. J Nutr Biochem. 2013;24(1):360
70. Rahman MM, Ichiyanagi T, Komiyama T, Sato S, Konishi T. Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status. J Agric Food Chem. 2008;56(16):7545
50. Ramirez MR, Izquierdo I, do Carmo Bassols Raseira M, Zuanazzi JA, Barros D, Henriques AT. Effect of lyophilised Vaccinium berries on memory, anxiety and locomotion in adult rats. Pharmacol Res. 2005;52(6):457
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C H A P T E R

18 Clitoria ternatea beverages and antioxidant usage Sirichai Adisakwattana1, Porntip Pasukamonset2 and Charoonsri Chusak1 1

Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 2Life Center, Q House, Bangkok, Thailand

List of abbreviations AAPH DPPH FICP FRAP HRSA MDA NF-κB OS ORAC ROS SRSA TBARS TEAC

2,20 -azobis-2-methyl-propanimidamide dihydrochloride 1,1-diphenyl 2-picrylhydrazyl ferrous ion chelating power ferric reducing antioxidant power hydroxyl radical scavenging activity malondialdehyde nuclear factor kappa B oxidative stress oxygen radical absorbance capacity reactive oxygen species superoxide radical scavenging activity thiobarbituric acid reactive substances trolox equivalent antioxidant capacity

Introduction Over the past two decades the deleterious effects of oxidative stress (OS) have become one of the most serious issues for biological researchers all over the world. OS is the condition arising from the imbalance between prooxidant and antioxidant homeostasis in the body. This imbalance in homeostasis leads to an increased production of toxic reactive oxygen species (ROS), such as hydrogen peroxide, nitric oxide, superoxide, and hydroxyl radicals.1 It has been shown that ROS are generated from mitochondrial metabolism, cytochrome P450s, various cytosolic enzyme systems, and exogenous sources.2 These excessive ROS then interact with cellular molecules within cells, causing oxidative damage and modification to proteins, lipids,

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00018-4

and DNA. Recently, OS has been implicated in the development and progression of several illnesses such as Alzheimer’s and Parkinson’s diseases, hypertension, diabetes, cardiovascular diseases, autoimmune disorders, and cancer.2 Given their critical health effects, antioxidants from natural sources have been studied to ameliorate free radical
induced oxidative damage. Extensive experimental and clinical studies have focused on the antioxidant activity of edible plants through scavenging free radicals, inhibiting the initiation or propagation of chain reactions and preventing damage to cellular molecules.3 Clitoria ternatea L. is an edible flowering plant which belongs to the Fabaceae family. It is an important plant that is widely cultivated in tropical and temperate regions worldwide, including Asia, Southeast Asia, the Caribbean, and Central and South America (Fig. 18.1). The flower of C. ternatea, known as butterfly pea, blue pea, Cordofan Pea, and Asian pigeonwings, has several colors ranging from dark blue, light blue, to white. There are several reports in recent literature on the pharmacological properties of C. ternatea, including antihistaminic,4 antihyperglycemic,5 wound healing,6 antiinflammatory,7 and hepatoprotective activities.8 Interestingly, most research has focused on the antioxidant effect of C. ternatea flower extract in vitro, animal, and human studies. This chapter summarizes evidence of the protective role of C. ternatea flower extract against oxidative damages of biological molecules related to its antioxidant activity. We also discuss the use of C. ternatea flowers as an antioxidant in foods and beverages.

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FIGURE 18.1 Flowers of Clitoria ternatea L. The common name of Clitoria ternatea is butterfly pea, blue pea, Cordofan pea, and Asian pigeonwings. This plant belongs to the Fabaceae family.

Use of Clitoria ternatea flowers Traditionally, the flowers of C. ternatea have been used as an alternative treatment for snakebite and scorpion sting in India.9 An infusion of C. ternatea flowers promotes menstruation and induces uterine contractions.10 In Thailand, the flower of C. ternatea is used for stimulating hair growth and coloring hair. The blue color of C. ternatea flowers is commonly used as a natural source of food coloring for the preparation of various delicacies. For example, it is often used to make blue glutinous or jasmine rice. A tea made with the dried flowers of C. ternatea is a popular herbal beverage in Southeast Asia.

Bioactive components The scientific literature has documented that the flowers of C. ternatea contain several potentially bioactive components identified through a variety of analytical processes. The flowers of C. ternatea have a variety of phytochemical compounds such as flavonoids, and the major colorant molecules are anthocyanins, derived from the basic classes of delphinidin. It also contains ternatin anthocyanins, namely, A1
A3, B1
B4, C1, and D1
D3. The color of anthocyanins, ranging from deep blue to magenta, is dependent on the pH of the plant’s surroundings. C. ternatea flowers also contain flavonoids and glycosides such as quercetin, quercetin-3-rutinoside (rutin), quercetin 3-O-dirhamnoside myricetin, myricetin3-rutinoside, kaempferol, kaempferol-3-rutinoside, and kaempferol-3-neohesperidoside.7,11
14 In addition, phytosterols such as campesterol, stigmasterol, β-sitosterol, and sitostanol were also identified in the flower.11 Identified flavonoids belong to a group of antioxidants that may play a key role in the bioactive functions of C. ternatea flowers.

Antioxidant properties of Clitoria ternatea flowers Studies evaluating C. ternatea flower extract have provided valuable information about antioxidant activity. The crude extracts from C. ternatea flowers have shown antioxidant activity in various model systems. Chayaratanasin et al., investigated the antioxidant activity of C. ternatea flower extract using various in vitro assay systems, including 1,1-diphenyl 2picrylhydrazyl (DPPH) scavenging activity, trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), hydroxyl radical scavenging activity (HRSA), superoxide radical scavenging activity (SRSA), and ferrous ion chelating power (FICP). The findings suggest that the extract of C. ternatea flowers produced less-potent antioxidant activity when compared to antioxidant compounds (ascorbic acid, Trolox, and EDTA).15 Similarly, C. ternatea flower extract could scavenge free radicals generated from DPPH, and the IC50 value of the extract was higher than ascorbic acid.16 A comparative study of different extraction solvent systems demonstrated that aqueous C. ternatea flower extract exhibited a stronger DPPH scavenging activity than ethanol extract.17 Activated macrophages are one of the leading causes of chronic inflammation by producing and releasing ROS in response to phagocytosis or stimulation with various agents. It is known that lipopolysaccharide (LPS) or endotoxin could induce a variety of immune responses related to the regulation of macrophage functions and the induction of inflammatory cytokines.18 Classical signal transduction of macrophages induced by LPS requires binding to toll-like receptors, leading to trigger generation of ROS from NADPH oxidase and mitochondria.18 Furthermore, ROS-mediated redux alteration activates the nuclear translocation of transcription factor NF-κB and then mediates the LPS-induced cyclooxygenase-2 (COX-2)

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Antioxidant properties of Clitoria ternatea flowers

expressions. The LPS-induced macrophage activation also triggers the amplification of inducible nitric oxide synthase (iNOS) expressions.19,20 Nair et al., reported the protective role of C. ternatea flower extract against LPS-induced inflammation in macrophage cells.7 The isolated fraction containing quercetin glycosides inhibited cyclooxygenase-2 (COX-2) activity, and it demonstrated partial ability to suppress ROS-mediated NFκB activation. The study also found that the isolated fraction containing ternatin anthocyanins showed different mechanisms through a non
ROS suppression pathway, including the inhibition of NF-κB translocation, protein expression of iNOS, and nitric oxide production.7 The inhibitory activity exhibited by C. ternatea flower extract suggested its use in the protection of inflammatory processes related to the excessive production of proinflammatory mediators from macrophage cells. It has long been recognized that free radical
induced lipid peroxidation in biological membranes is thought to contribute to the initiation and progression of pathological events and aging.21 Erythrocytes are highly susceptible to free radical
induced endogenous oxidative damages, which eventually causes hemolysis. The evidence suggests that lipid peroxidation in erythrocyte membranes is associated with the impairment of erythrocyte deformability.21 In vitro studies have also been utilized to investigate the protective ability of C. ternatea

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flower extract against AAPH-induced hemolysis and oxidative damage of canine erythrocytes.22 C. ternatea flower extract protected AAPH-induced erythrocyte hemolysis and reduced membrane lipid peroxidation and protein oxidation in erythrocytes. Also, the extract could maintain normal glutathione levels in erythrocytes with oxidative damage induced by AAPH. It was suggested that the extract of C. ternatea quenches the peroxyl radicals before these radicals attack the erythrocyte membrane, leading to reduction of hemolysis and prevention of lipid peroxidation and GSH depletion. Consequently, C. ternatea flower extract also attenuated AAPH-induced morphological alterations of erythrocytes (Fig. 18.2). Protein glycation is a nucleophilic addition reaction between the amino group of a protein and the carbonyl group of a reducing monosaccharide. Further modifications of the early stage protein glycations are rearrangement, oxidation, polymerization, and cleavage, which result in irreversible conjugates called advanced glycation end-products (AGEs).23 The excessive formation of AGEs and their accumulation in the tissues is an important contributor to diabetic complications such as retinopathy, nephropathy, and neuropathy. The aqueous extract of C. ternatea flowers has been demonstrated to be an effective inhibitor of protein glycation.15 The extract showed inhibitory properties against fructose-induced protein glycation and AGEs in bovine serum albumin (BSA). During

FIGURE 18.2 The effect of Clitoria ternatea on AAPH-induced morphological changes of erythrocytes. Erythrocytes were preincubated for 5 min at 37 C with Clitoria ternatea extract (50
400 μg/mL) and trolox (100 μg/mL), followed by incubation with or without 50 mM AAPH solution for 4 h. Untreated erythrocytes showed normal biconcave shapes (A), whereas AAPH caused numerous extrusion protuberances on erythrocyte surfaces (B). The morphological changes induced by AAPH were slightly prevented when erythrocytes were incubated with (C) 50 μg/mL, (D) 100 μg/mL, and (E) 200 μg/mL Clitoria ternatea extract. The presence of 400 μg/mL Clitoria ternatea extract (F) or 100 μg/mL trolox (G) maintained the normal biconcave shape of erythrocytes. Scanning electron microscope (SEM) images of erythrocytes originally appeared in Ref. [22]. Source: Reproduced with permission from Elsevier (License number 4454000483592).

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FIGURE 18.3

Calcium alginate beads of Clitoria ternatea. Calcium alginate beads were comprised of 10% Clitoria ternatea extract, 1.5% alginate, and 3%CaCl2 (w/v). Calcium alginate bead images (A) without and (B) with Clitoria ternatea.

TABLE 18.1

Antioxidant activity of Clitoria ternatea (CT) and its calcium alginate beads after gastrointestinal digestion.

Preparation

ABTS (mg Trolox equivalents)

FRAP (mg FeSO4 equivalents)

6.60 6 0.03

3.56 6 0.04

Simulated gastric fluid (SGF) CT CT beads



3.23 6 0.01

1.23 6 0.05

5.52 6 0.02

2.47 6 0.04

Simulated intestinal fluid (SIF) CT CT beads



3.39 6 0.01

6.33 6 0.03

Clitoria ternatea extract and its microbeads, with equal amounts of polyphenol contents (0.802 mg gallic acid), were added into gastric phase solution and incubated for 1 h at 37 C. The gastric digesta were adjusted to the pH 4.5 and followed by addition of intestinal phase solution (pH 7.2). The reaction was incubated for 2 h at 37 C. Finally, the antioxidant activity of digesta was determined using FRAP and ABTS assays. Data are expressed as mean 6 S.E.M, n 5 4.  P , .05 compared to CT. Data appeared in Ref. [29]. Copied with permission from Elsevier (License number 4454010609341).

hyperglycemia, albumin undergoes oxidative structural modification and functional alteration by increased protein glycation related to the early occurrence of vascular complications.24 The aqueous extract of C. ternatea flowers also prevented oxidationdependent damages to the BSA by decreasing protein carbonyl formation and protein thiol depletion.15 The results of this study suggest that C. ternatea might suppress formation of AGEs and oxidative modification to protein through its free radical scavenging activity. Substantial evidence reveals that ROS in humans, induced by ultraviolet (UV) radiation, are strongly involved in the occurrence of skin damage, leading to wrinkles, hyperpigmentation, and sagging.25 A recent study proved that pretreatment with aqueous extract of C. ternatea flowers reduced H2O2-induced cytotoxicity and UV-induced mitochondrial DNA damage in human HaCaT keratinocytes.26 The findings suggest that the mechanisms for this protective effect may be through the free radical scavenging activity of flavonol glycosides and anthocyanin, thus resulting in reduced OS arising from H2O2 and UV exposure.26 Moreover,

C. ternatea flower extract acts as a UV filter due to its ability to absorb UVB (290
320 nm) and UVC (200
290 nm). Several studies have been published regarding the decrease in stability and antioxidant activity of polyphenols in edible plants after gastrointestinal digestion.27 In spite of low bioavailability and biological activity in the gastrointestinal tract, this leads to promising results for preventing diseases of polyphenols.28 It has been found that C. ternatea flower extract presents instability and low-antioxidant capacity under the alkaline environment of the intestinal medium.29 The problem related to instability of polyphenols in C. ternatea flower extract has been resolved with the use of the calcium alginate microencapsulation technique (Fig. 18.3). Antioxidant activity of C. ternatea flower extract immobilized within the alginate beads was increased (Table 18.1), while the phenolic content was preserved, after simulated gastric fluid and intestinal fluid digestion.29 Consequently, C. ternatea flower extract microbeads increase the ability to bind bile acid and inhibit pancreatic α-amylase.

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Applications to other areas of pathology

Toxicity

Food uses

Considering its worldwide food uses, it is important to determine the toxicological effects of the C. ternatea flower. In one study, no observed changes were reported for hematological and biochemical parameters in rats treated with C. ternatea flower extract (2000 mg/kg) for 24 hours and 14 days.30 The study dealing with the possible testicular toxicity indicated no effect in rats treated with C. ternatea flower extract (100 mg/kg) for 28 days. The extract also could reduce ketoconazole-induced testicular damage in rats.16

Lipid peroxidation in foods is a major concern for consumers because it produces undesirable odor and flavor and poorer food quality. In meat products, the heating process induces lipid and protein oxidation in muscle tissues, leading to rancidity and changes in the meat’s odor, flavor, and color.36 The protective effect of C. ternatea against protein and lipid peroxidation was tested in a cooked meat model.37 Spray-dried C. ternatea powder (0.02%
0.16% w/w) was added to raw pork patties cooked in a fan-assisted oven and compared with Butylated hydroxytoluene (BHT), a common synthetic antioxidant additive. The addition of C. ternatea powder lessened lipid and protein oxidation and increased the value of TEAC in the pork patties during a 6-day storage period. Incorporation of CTE to cooked pork patties demonstrated good color, sensory attributes, and overall acceptability after storage. The researchers suggest that CTE (0.08%
0.16%) could potentially inhibit oxidative rancidity as well as 0.02% BHT. In the bakery manufacturing process, fats normally enhance the softer, final texture of cakes, but they are prone to lipid peroxidation by generating ROS and free radicals when exposed to oxygen and heat.38 Replacing wheat flour with spray-dried C. ternatea flower powder (5%
20%) was found to decrease the level of thiobarbituric acid reactive substances (TBARs) in sponge cake.39 It is important to note that the polyphenol content and antioxidant capacity of sponge cake were markedly increased by the addition of C. ternatea flower powder. The researchers indicated that partial replacement of wheat flour with 5% C. ternatea in sponge cake is recommended with respect to the overall acceptance score and physicochemical properties. In addition to the use of C. ternatea in sponge cake, spray-dried C. ternatea powder could reduce the starch hydrolysis of different types of flours.40 Bread incorporated with 5% w/w C. ternatea was found to show a decreased rate of starch hydrolysis.40

Human study It has been shown that acute consumption of beverages causes an increase in plasma antioxidant capacity in humans.31,32 Studies examining the acute in vivo plasma antioxidant effect of C. ternatea flower extract have been performed with herbal beverages.33 Chusak et al., prepared a 400 mL single dose of beverage from 1 or 2 g of spray-dried C. ternatea powder (Fig. 18.4). In a crossover study using 15 healthy subjects, significant increases in the plasma FRAP, ORAC, and TEAC values were observed 30 minutes after drinking the C. ternatea beverage.33 Interestingly, ingestion of the C. ternatea beverage did not affect plasma glucose levels in the fasting state, suggesting that consumption of C. ternatea beverages causes an increase in plasma antioxidant capacity without hypoglycemia. This study supports the evidence that in vitro antioxidant effects of C. ternatea flower extract translate to demonstrable benefits in humans. The more frequent cause of postprandial OS is the metabolism of a high-carbohydrate meal.34 Postprandial OS is thought to be involved in triggers of the inflammatory process in atherosclerosis, endothelial dysfunction, hypercoagulability, and sympathetic hyperactivity.35 Acute consumption of sucrose beverages (50 g/400 mL) resulted in a significant reduction in plasma antioxidant activity (FRAP, TEAC, and ORAC values) and significantly increased plasma MDA concentration in healthy subjects.33 Postprandial OS was attenuated when the C. ternatea beverage was supplied with sucrose. Volunteers who drank C. ternatea beverages (1 g or 2 g/400 mL) containing sucrose exhibited a reduction in plasma lipid peroxidation (MDA), with a concomitant increase in plasma FRAP, TEAC, and ORAC. These data suggest that consumption of C. ternatea beverages protected sucrose-induced postprandial OS due to the antioxidant activity of C. ternatea flowers. Further evidence from long-term human intervention studies is required to investigate the antioxidant role of C. ternatea beverages in biological systems.

Applications to other areas of pathology It is evident that C. ternatea acts as an antioxidant, thereby preventing free radical
induced biological molecule damage. Interestingly, C. ternatea flowers have been demonstrated to possess antidiabetic activity. For example, C. ternatea flower extract inhibited intestinal α-glucosidase and pancreatic α-amylase.41 The combination of C. ternatea extract with Hibiscus sabdariffa (Roselle) produced additive inhibition against

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FIGURE 18.4 The effect of Clitoria ternatea beverages on antioxidant capacity in healthy subjects. (A) The spray-dried Clitoria ternatea extract (CTE). (B) Healthy men consumed five different beverages, (1) 50 g sucrose; (2) 1 g CTE; (3) 2 g CTE; (4) 50 g sucrose and 1 g; and (5) 50 g sucrose and 2 g CTE in 400 mL water. (C) FRAP, (D) TEAC, (E) ORAC, and (F) MDA changes area under the curves (AUCs) of the five different beverages after 2 h of consumption. Values are means 6 SEM, n 5 15. The various beverages has significantly different results (P , .05). Figures (C
F) were originally appeared in Ref. [33]. Source: Reproduced under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

pancreatic α-amylase.41 In addition, oral administration of C. ternatea extract decreased blood glucose and glycosylated hemoglobin in alloxan-induced diabetic rats via increasing hepatic glucokinase activity and decreasing hepatic glucose-6-phosphatase activity.42 However, there is a need to conduct well-planned clinical studies to evaluate the efficacy and safety of C. ternatea beverages for people with Type 2 diabetes.

Conclusions The flowers of C. ternatea have potent antioxidant activity that can protect biological molecules against oxidative damage. Consumption of C. ternatea flower beverages increases plasma antioxidant capacity and reduces lipid peroxidation induced by sucrose in healthy subjects. Adding C. ternatea flower powder to

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References

meat or bakery products strongly decreases lipid peroxidation associated with an increase in antioxidant capacity. Further studies on the long-term consumption of C. ternatea beverages are needed to confirm these potentially beneficial effects in humans.

Summary points • Aqueous extract of C. ternatea flower contains phenolic compounds which exhibit antioxidant activity using various in vitro assay systems. • The major colorant compounds of C. ternatea flowers are anthocyanins, derived from the basic classes of delphinidin such as ternatin A1
A3, B1
B4, C1 and D1
D3. Other flavonoids present in the flower are quercetin, myricetin, and kaempferol and their glycosides. • Recent evidence based on cell culture and cell-free studies indicates that the ability of C. ternatea flower extract to inhibit oxidative damages to biomolecules is related to their antioxidant activity. • Consumption of C. ternatea flower beverages, with or without sucrose, induced a significant rise in plasma antioxidant capacity, with a concomitant reduction of by-product of lipid peroxidation (TBARs) in humans. • C. ternatea flower extract has been shown to reduce lipid peroxidation and increase antioxidant capacity in meat and bakery products.

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35. O’Keefe JH, Gheewala NM, O’Keefe JO. Dietary strategies for improving post-prandial glucose, lipids, inflammation, and cardiovascular health. J Am Coll Cardiol 2008;51(3):249
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7. 37. Pasukamonset P, Kwon O, Adisakwattana S. Oxidative stability of cooked pork patties incorporated with Clitoria ternatea extract (blue pea flower petal) during refrigerated storage. J Food Process Pres 2017;41(1):e12751. 38. Lu T-M, Lee C-C, Mau J-L, Lin S-D. Quality and antioxidant property of green tea sponge cake. Food Chem 2010;119 (3):1090
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9. 40. Chusak C, Henry C, Chantarasinlapin P, Techasukthavorn V, Adisakwattana S. Influence of Clitoria ternatea flower extract on the in vitro enzymatic digestibility of starch and its application in bread. Foods 2018;7(7):102. 41. Adisakwattana S, Ruengsamran T, Kampa P, Sompong W. In vitro inhibitory effects of plant-based foods and their combinations on intestinal α-glucosidase and pancreatic α-amylase. BMC Complement Altern Med 2012;12:110. 42. Daisy P, Santosh K, Rajathi M. Antihyperglycemic and antihyperlipidemic effects of Clitoria ternatea Linn. in alloxan-induced diabetic rats. Afr J Microbiol Res 2009;3:287
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C H A P T E R

19 Curcumin and related antioxidants: applications to tissue pathology Carolina Alves dos Santos1, Mahendra Rai2, Jose´ Martins de Oliveira Jr1, Fernando de Sa´ Del Fiol1, Rogerio Augusto Profeta3, Denicezar Baldo1 and Marco Vinı´cius Chaud1 1

Faculty of Pharmaceutical Sciences, University of Sorocaba (UNISO) Brazil, Sorocaba, Brazil 2Basic Science Research Faculty Fellow (UGC), Department of Biotechnology, SGB Amravati University, Amravati, India 3Technology & Environmental Process Graduate Course, University of Sorocaba, Sorocaba, Brazil

List of abbreviations COX CUR FDA ILs LOX ROS PAHO TNF-α TPA VEGF

cyclooxygenase curcumin Food and Drug Administration interleukins lipoxygenase oxidative stress Pan American Health Organization Tumour necrosis factor 12-O-tetradecanoylphorbol-13-acetate vascular endothelium growth factor

Introduction Curcumin (diferuloylmethane) extracted from the roots of Curcuma longa is an active component with a polyphenol ring structure.1 Curcumin (CUR) was first isolated in 1815, while its chemical structure was determined in 1973 by Roughley and Whiting.2,3 Despite some controversies regarding the bioavailability of curcumin, curcuminoids are agents of natural origin with a variety of applications described as anticancer, antiinflammatory, antioxidant, and antibacterial.4 CUR is a pleiotropic substance, that is, the molecule is capable of interacting with multiple targets involved in the inflammatory reaction such as tumors, necrosis factor α (TNF-α), and interleukins (ILs).5 In nature curcumin is a naturally hydrophobic molecule, therefore,

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00019-6

its bioavailability is impaired, limiting its use in therapeutics. Its chemical constitution is based on two polyphenolic rings that are replaced by methoxy ether in ortho, and its tautomerization changes the relative concentrations of the cis
trans forms which vary according to temperature, the polarity of solvent, and pH. Though there are some toxicity studies, the Food and Drug Administration (FDA) has declared curcumin as generally safe (Fig. 19.1).6 The metabolic process of CUR and related compounds is determinant of its biological effects against a large variety of tissue pathologies. In gastrointestinal absorption, bioconjugation is the main process acting in CUR degradation. When administrated by intravenous or intraperitoneal route, a reduction in subproducts with therapeutic action is necessary to guarantee bioactivity and chemical stability. In a pathological process, the imbalance between free radicals and the immunological system against oxidative stress (OS), is controlled by CUR being antioxidant CUR can reduce reactive oxygen species (ROS) production, remove free radicals, and increase antioxidant enzymes (Fig. 19.2).7 CUR can restore bacterial susceptibility to antibiotics, therefore, the antibacterial action of CUR is synergistic when associated with other antibiotic compounds such as cefixime, vancomycin, and tetracyclines.8,9 Bacterial cells treated with CUR have permeable membranes that allow greater entry or greater release

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of fluorescein complex. The rupture of the membrane leading to the uptake of fluorescein complex or propidium iodide (PI) is further supported by flow cytometry and steady-state fluorescence. Fluorescence microscopy studies using a LIVE/DEAD kit confirmed entry of PI molecules into Staphylococcal cells due to membrane damage caused by CUR.9,10 Numerous studies have suggested different metabolites of CUR being biotransformed into dihydrocurcumin and tetrahydrocurcumin (THC). Subsequently, these products are converted into conjugated monoglucuronide. In another study, the major bile metabolites

of CUR have been reported to be THC and hexahydrocurcumin conjugates.11 The experimental results suggest that major metabolites of CUR in mice are curcumin-glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide, and THC.12 Sirtuins are highly conserved nicotine adenine dinucleotide
dependent proteins that regulate the activity of target enzymes and transcription factors by deacetylation. Due to its antiinflammatory and antioxidant properties, CUR promotes the increase of sirtuins (SIRT1). This increase generates protective effects against a variety of neurological disorders including glutamate excitotoxicity, β-amyloid-induced cell death in cortical neurons, cerebral ischemic damage, and stroke. Activation of adenosine 50 monophosphate-activated protein kinase and SIRT1 by CUR has also been observed to mediate the protective effects of CUR against cardiac injury, diabetes, and abnormalities of lipid metabolism. These protective effects of SIRT1 activation are partially mediated by the deacetylation of p53 and the reduction of apoptosis. Mehta et al.13 reported a wide range of properties of CUR compounds describing their capacity to inhibit mononuclear blood cells, neutrophil activation, lymphocyte reactions, and antiproliferation of muscles cells. Related with its antioxidant activity it is a potent scavenger of reactive species, protecting blood cells and DNA (Table 19.1; Fig. 19.3).

Antiinflammatory activity Inflammation is a cell-mediated chemical mechanism of the immune system as a defense mechanism against deleterious microorganisms or stimuli. Acute inflammation activates immune system cells as lymphocytes,

FIGURE 19.1 Curcumin in different forms.

FIGURE 19.2 Different administration routes of curcumin.

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Antiinflammatory activity

TABLE 19.1

Curcumin cell activation mechanisms.

Curcumin therapeutic effects

Cells enrolled

Mechanism activated

Reference

Antiinflammatory

TNF-α, TNF-kB, IL-6, COX, LOX

Immunologic system

18

Antitumoral

D1 cyclin, caspase 8, poly ADP cleavage, TPA

Apoptosis factors, elimination of tumor initiating cells

13

TNF-α, COX-2, T NF-κβ, STAT proteins

Immunologic molecular reactions

7

Cardiac effects

p53 NFkB

Inhibition and secretion of VEGP genes

27

Neurodegenerative

Cytokines, TNF, Interleukin, Interferon

Akt/Nrf2 pathways, Nrf2

34

Antimicrobial

MDA, NO, MPO activity, TNF-α

MDA levels reduction

37

FIGURE 19.3 Factors determining the therapeutical properties of curcumin.

while chronic inflammation is caused by proinflammatory mediators manifested by a variety of diseases such as rheumatoid, cardiovascular, and cerebral arthritis.14 CUR has been proved to have strong antiinflammatory activity due to antiinflammatory cytokines6 (Table 19.2). The antiinflammatory and antioxidant properties of CUR prevent the prolonged presence of free oxygen radicals, which is one of the factors that suppresses the healing process.15 CUR has demonstrated in vitro and in vivo antiinflammatory effects, however, bioavailability and instability problems in biological fluids limit its use in therapy. CUR analogs have been developed to improve the bioavailability and bioactivity of CUR as mono carbonyl analogs demonstrate excellent pharmacokinetic profiles, stability at physiological pH, and have great ability to inhibit proinflammatory cytokines TNF-α and IL-6.16,17

According to Dudics et al.,18 the mechanism for the antiinflammatory properties of CUR is not restricted to TNF-kappa B activity, which is able to reduce proinflammatory cytokines such as TNF-α-induced adhesion of monocytes to endothelial cells and regulate cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, suppressing proinflammatory mediators and inflammation. Fan et al.19 proposed the use of nanomicelles with hyaluronic acid (HA), the main component of the extracellular matrix, and CUR to overcome bioavailability and the lubricating action between joints. In a study carried out by Akyuz et al.,20 it was reported that the positive antiinflammatory effects in rats induced middle ear infection. A positive effect in epithelial proliferation and inflammatory infiltration was recorded after CUR administration. However, no positive effect was observed in GSH-PX activity, but the effects in lipid peroxidation showed by the multidrug

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19. Curcumin and related antioxidants: applications to tissue pathology

Antiinflammatory curcumin properties.

Activity

Mechanism

Reference

Rheumatic disorders and, in general, inflammation

Regulates cyclooxygenase and lipoxygenase

18

Management of chronic anterior uveitis

Inhibits arachidonic acid metabolism, cyclooxygenase, lipoxygenase, cytokines nuclear factor-kB, and release of steroidal hormones

29

Modulating inflammatory responses in diseased kidney

Up-regulation of peroxisome proliferatoractivated receptor-γ (PPAR-γ) activation

31

Idiopathic inflammatory orbital pseudotumors

Sensitizes the tissues to endogenous corticoids and to increase adrenal steroidogenesis

29

Suppress colonic inflammation

Blocks the upstream of NF-κB and IκB kinases

31

administration levels were considered better after CUR administration (30 mg/kg/day). The mechanism reported by Ghoneim et al.21 related to doses of 50
100 mg/kg with no changes in biochemical parameters, but doses under 200 mg/kg were shown to reduce oxidative injury. The dose 30 mg/kg is enough for the inhibition of the kβ factor.

Antitumoral properties Resistance to some antitumoral cancer therapies created the necessity of new alternative compounds that are able to prevent and act as growth inhibitors against tumoral cells including those resistant to traditional compounds. Due to the versatile properties of CUR and its related compounds that can target and interact with multiple molecules, it seems more rational and interesting as a mono target therapy. Pisano et al.22 reported on the mechanisms that are involved in the anticancer properties of CUR. These are related with suppression mechanisms of D1 cyclin, inducing apoptosis of tumoral cells by activating caspase 8, releasing cytochrome c and other caspases, and the activation of poly ADP cleavage. CUR plays an important role in the immunological modulation involved in the tumoral mechanism in metastatic cancer cells, transcription factors, and angiogenesis. Conney et al.23 reported on the antioxidant properties of CUR for the inhibition of tumoral 12-O-tetradecanoylphorbol-13-acetate (TPA) responsible for tumors in mouse skin. In a pediatric treatment where the therapeutic protocol is based on drugs currently used in adult treatment, the incorporation of natural compounds have been reported in the treatment of different tumors such as bone, liver, and brain. This mechanism consists of the inhibition of DNA topoisomerase inducing

apoptosis, the decrease of teloisomerase, and the elimination of tumor initiating cells.24 In this case, CUR is particularly interesting in brain tumor treatment due its lipophilic character that makes molecules permeable to the blood brain barrier. The activity of CUR on neuroblastoma tumors is due to the enhanced rate of reactive species, decreasing mitochondrial potential and increasing proapoptotic factors.24 In gastric cancer, CUR inhibits proliferation and invasion via the expression of cyclin D and kinase K1. Mehta et al.13 reported CUR as an inhibitory agent by interfering with certain signal transduction pathways that are critical for cell growth directly related with anticancer properties. Ciftci et al.25 reported the toxic effects of CUR and the ability of this natural compound to inhibit the growth of tumoral cells in colon carcinogenesis. Other kinds of tumors are also suppressed such as prostate, biliary, oral, and uterine. Molecular reaction mediates all these events such as TNF-α, COX-2, and metalloproteinases being targets of CUR that includes NF-κβ and signal transducer and activator of transcription proteins. Additionally, CUR induces the apoptotic mechanism of tumoral cells, prevents radiationinduced other cancer steps treatment for activation of the NF-κB pathway, and increases sensitivity to ionizing radiation. It is also important to note that CUR has the ability to suppress metastasis and the invasion progress of cancer cells in different types of tumors.7

Activity in myocardial diseases Reports from the Pan American Health Organization (PAHO) in 2017 showed that cardiac diseases were the most common cause of death worldwide. Comorbidities such as fat, diabetics, and hypertension increase the death rates. Emphasis has been placed on the properties of natural compounds

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As an antimicrobial agent

for the treatment of cardiac diseases. CUR seems to be associated with antidiabetic effects in cardiomyopathy diseases.26 Several studies provide evidence that CUR is beneficial in cardiac diseases, particularly in multiple pathological conditions. The potential of CUR interaction modulates a number of symptoms by activating different pathways involved in myocardial protection and as a preventer of tumorigenesis with respect to stabilization and apoptotic regulation. The p53 NF-κB interaction with p300 as a regulator of cardiac cells and regeneration in hypertrophy are important bioactivities of curcumin.27 Saberi-Karimian et al.28 reported the effects of CUR on angiogenesis, which seems to be related with expression inhibition and secretion of vascular endothelium growth factor (VEGF) genes. The inhibition promoted by CUR plays an important role in angiogenesis both in normal and pathological angiogenesis. Liu et al.29 reported CUR as a cardioprotective agent. The authors studied the efficacy of CUR in rat models for the regulation of autophagy and mammalian target of rapamycin (mTOR) signaling in cardiac hypertrophy and fibrosis. The hypertrophy was induced by injecting isoprenaline (5 mg/kg/day), which was treated with or without CUR (200 mg/kg/ day). They reported that CUR targeted mTOR/autophagy was able to reduce cardiac hypertrophy and fibrosis.

compounds such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found in fish, CUR, and others act in the inflammatory system. Interestingly, Mazzanti and Giacomo et al.32 reported an increased expression level of the genes involved in synaptic plasticity, and as a result special memory was improved in female rats. Cognitive deficits are directly associated with higher levels of ROS and reactive nitrogen species (RNS), directly involved in senile plaques, as the brain possesses a fragility to oxidative damage. CUR can bind to reactive metals, activating glutathione S-transferase, restoring glutathione content in the brain, and inducing antioxidant enzymes, thus, avoiding brain damage.32 Considering the complexity in the treatment of neurodegenerative diseases, the mechanisms involved in this pathology have still not been elucidated. Therapy that can act at different levels shows an interesting alternative for AD treatment.33 Wu et al.34 reported that CUR can significantly reduce OS levels in the middle cerebral in rats, protecting neurons from ischemic injury involved in Akt/ Nrf2 pathways and Nrf2 involved in neuroprotective effects caused by OS. The effects of CUR in neurodegenerative disease such as Huntington’s disease were also reported with positive results in the hypothesis that the mitochondrial cell integrity was maintained, which was evidenced in data that correlate the neurodegenerative process with defects of mitochondrial functions in biological material from sick patients.35

Antineurodegenerative effect The accumulation of proteins and peptides is the base of neurodegenerative diseases such as Alzheimer’s disease (AD) and sclerosis diseases. The primordial proteins involved in all these processes are amyloid-β, which are the main targets in CUR brain effects. Cells from the immune system that misfolded proteins and digest pathogenic proteins, peptides, and others pathogens. People with neurodegenerative diseases seem to show low capacities for the phagocytoses of Aβ from the brain. Epidemiological studies have shown evidences of CUR protecting against AD.30 Besides these, a number of additional disorders are risk factors for chronic inflammation that are an associated risk factor for AD.31 The most important biological alterations promoted by AD is the higher inflammatory character and OS and this uncontrolled state results in serious complications. Proinflammatory delivery is related with the release of cytokines such as TNF, IL, and interferon in macrophages. In the brain, the main mediator enrolled in the neuroinflammatory state are microglial cells. Dietary components may modify the chronic inflammation process by modulating cells signaling. Evidences have shown that some

As an antimicrobial agent The increasing scenario of antimicrobial resistance to available antibiotics has become a catastrophic risk. The wide use of antimicrobials in medicine, food, and crops along with its irresponsible misuse in therapy has generated this alarming situation. CUR and other naturally occurring bioactives provide a good alternative to antimicrobials. The pleiotropic nature of the CUR molecule allows for the interaction of CUR compounds with different parts of microorganisms and also in the immunological systems of sick persons. In this sense, the combined effect of CUR compounds in antimicrobial therapy is promising. In addition, the wide spectrum of activity against bacteria (Gram positive and Gram negative), viruses, and fungi has shown its versatility in applications. Rai et al.27 reported that in the United States and Europe about 23,000 and 25,000 deaths annually can be attributed to MDR microorganisms respectively. Zorofchian et al.36 claimed that the antimicrobial potential of CUR depends on its solubility, bioactivity, and bioavailability. Many formulations and strategies can be used to

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19. Curcumin and related antioxidants: applications to tissue pathology

increase these parameters for proper therapeutic effects. Its antibacterial activity was demonstrated against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. However, CUR in nano form seems to be more effective against Gram positive than Gram negative bacteria. Another interesting approach in antimicrobial treatment is the possibility of combining existing antibiotics with CUR to potentialize action and avoid resistance. Zhou et al.37 studied the efficacy of CUR and erythromycin against S. aureus in osteomyelitis. They evaluated CUR, erythromycin, and a combination of both the compounds. The authors found no inhibitory effect of erythromycin, but CUR as a monotherapy and combined with erythromycin was able to reduce proinflammatory cytokines and suppress the growth of S. aureus. The combination of these two compounds exhibited strong evidence in MRSA treatment. Rai et al.38 attributed the inhibitory potential of CUR to link in FtsZ target inhibiting bacteria cell proliferation.

effectiveness against different pathologies. In hepatobiliary pathology, Hu et al.41 reports that CUR shows protective effects in hepatobiliary conditions in nonalcoholic fatty disease, cholestasis conditions, and carcinomas. In hepatic fibrosis, CUR has shown attenuation, the inhibition of cellular senescence involved in sclerosing cholangitis with significate reductions in liver lipid accumulation, and the reduction of cholesterol, triglyceride, and free fatty acids. The mechanisms involved in this process are mitochondrial protection, inhibition of apoptosis, and protection against OS. Zhang et al.16 report the use of CUR in an intraperitoneal delivery in a model for colorectal peritoneal carcinomatosis a controlled release of CUR was obtained showing promising application in this pathology. In a mechanism involving antiinflammatory cytokines (MCP-1, NF-κB, TNF-α, IL-1β, COX-2, and cav-1), CUR also reduced fibrosis exposed antifibrotic effects, a factor that also contributes to renal functions and renal pathology conditions as reported by Sun et al.6

Applications to other areas of pathology

Therapeutic challenges

Mogharbel et al.39 report the utilization of CUR in cell therapy exploring the fluorescent properties of CUR in cell tracking. CUR-loaded polycaprolactone nanoparticles (NPC) were prepared. Studied have shown the promising ability of using the florescence of CUR in cell therapy after considering the safety parameters. In a similar approach Li et al.40 studied the ability of CUR to target cancer stem cells (CSCs), the system responsible for initiating and maintaining cancer and contributing to recurrence and drug resistance. In this approach, CUR can target CSCs in mechanisms involved in self-renewal pathways (Wnt/b-catenin, Notch, sonic hedgehog) and specific microRNAs, showing the chemoprevention and therapy potential of CUR. In a gastrointestinal evaluation, CUR has shown

Teow et al.42 discussed some studies that show limitations to CUR application in therapy. CUR induces the potential aberrations in chromosomes and DNA at 10 μg/mL. DNA damage increases the carcinogenic toxic effects of CUR. The potential ROS induced by CUR enforces the tumorigenic effects. Beside all the carcinogenic potential, the chelator can affect minerals absorption and interfere in drugs metabolism. In an interesting approach, Xu et al.7 suggested exploring the beneficial potential of CUR and CUR compounds in a monotherapy and combined with other compounds. The advancement in different formulations and drug delivery systems can also support the safe use of CUR, reducing solubility problems and thereby improving its bioavailability in the human body (Table 19.3).

TABLE 19.3

Curcumin alternative formulations for challenges in therapy implementation.

Curcumin challenges

Possible curcumin alternative formulations

Solubility

Solid dispersion, solid lipid nanoparticles, liposomes, liquid crystal, dendrimers, SEEDS, pickering

Biodisponibility

Oral formulations technology, parenteral administration, cutaneous permeability

Chemical stability

Nanoparticulate systems, emulsion-based systems

Target delivery systems

Specific colon delivery, specific dosage according to tissue delivery pathology, thermo-responsive, pH-responsive, and enzymatic-responsive systems

II. Antioxidants and pathology

References

Conclusion and future perspectives Curcumin (CUR) is a polyphenol, a constituent of C. longa, which has been used for culinary purposes and also in biomedical applications in Asian countries. Since ancient times, people have used CUR for inflammation, wounds, and other microbial infections. It has already demonstrated its potential as antiinflammatory, anticancerous, antineurodegenerative, antimicrobial, and effect against myocardial diseases. There exist enormous possibilities to use turmeric in general and CUR in particular as a novel therapeutic agent. The bioavailability of CUR is a major problem, however, it can be solved, if CUR is used in nano form or with fats imparting solubility to it. The toxicity of CUR has not been proven yet, except for some reports. For this purpose, in-depth studies are required.

Summary points • This chapter focuses on the antioxidant effects of CUR against tissue pathologies. • CUR is a pleiotropic substance extracted from the roots of C. longa; curcuminoids are agents of natural origin with a variety of applications. • Its chemical constitution is based on two polyphenolic rings that are replaced by methoxy ether in ortho, and its tautomerization changes the relative concentrations of cis
trans forms. • Experimental results suggest that the major metabolites of CUR in mice are curcuminglucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and THC. • This chapter focuses on the application of CUR against numerous pathologies, namely inflammatory, tumoral, myocardial, neurodegenerative, and microbial diseases. • The metabolic process of CUR and its related compounds is determinant of its biological effects against a large variety of tissue pathologies. • In a pathological process, an imbalance between free radicals and the immunological system against OS, is controlled by curcumin being an antioxidant compound. • CUR can reduce ROS production, remove free radicals, and increase antioxidant enzymes.

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9. 5. Alves TF, Chaud MV, Grotto D, et al. Association of silver nanoparticles and curcumin solid dispersion: antimicrobial and antioxidant properties. AAPS PharmSciTech 2017. Available from: https://doi.org/10.1208/s12249-017-0832-z. 6. Sun J, Chen F, Braun C, et al. Role of curcumin in the management of pathological pain. Elsevier GmbH 2018. Available from: https://doi.org/10.1016/j.phymed.2018.04.045. 7. Xu XY, Meng X, Li S, Gan RY, Li Y, Li HB. Bioactivity, health benefits, and related molecular mechanisms of curcumin: current progress, challenges, and perspectives. Nutrients 2018;10. Available from: https://doi.org/10.3390/nu10101553. 8. Kali A, Bhuvaneshwar D, Charles PV, Seetha K. Antibacterial synergy of curcumin with antibiotics against biofilm producing clinical bacterial isolates. J Basic Clin Pharm 2016;7:93. 9. Tyagi P, Singh M, Kumari H, Kumari A, Mukhopadhyay K. Bactericidal activity of curcumin I is associated with damaging of bacterial membrane. PLoS One 2015;10:1
15. 10. Pi C, Yuan J, Liu H, et al. In vitro and in vivo evaluation of curcumin loaded hollow microspheres prepared with ethyl cellulose and citric acid. Int J Biol Macromol 2018;115:1046
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7. 12. Lin J-K, Shiau S-YL-S. Mechanisms of cancer chemoprevention by curcumin. Proc Natl Sci Counc 2001;25:59
66. 13. Mehta K, Pantzis P, McQueen T, Aggarwal B. Antiproliferative effect of curcumin against human breast tumor cell lines. Anticancer Drugs 1997;8:470
81. 14. Chen L, Deng H, Cui H, et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 2018;9:7204
18. 15. Dang LH, Nguyen TH, Tran HLB, Doan VN, Tran NQ. Injectable nanocurcumin-formulated chitosan-g-pluronic hydrogel exhibiting a great potential for burn treatment. J Healthc Eng 2018;2018. Available from: https://doi.org/10.1155/2018/5754890. 16. Zhang X, Chen M, Zou P, et al. Curcumin analog WZ35 induced cell death via ROS-dependent ER stress and G2/M cell cycle arrest in human prostate cancer cells. BMC Cancer 2015;15:1
11. 17. Zhang P, Bai H, Liu G, et al. MicroRNA-33b, upregulated by EF24, a curcumin analog, suppresses the epithelial-tomesenchymal transition (EMT) and migratory potential of melanoma cells by targeting HMGA2. Toxicol Lett 2015;234:151
61. 18. Dudics S, Langan D, Meka RR, et al. Natural products for the treatment of autoimmune arthritis: their mechanisms of action, targeted delivery, and interplay with the host microbiome. Int J Mol Sci 2018;19. Available from: https://doi.org/10.3390/ ijms19092508. 19. Fan N, Ma P, Wang X, et al. Storage stability and solubilization ability of HPMC in curcumin amorphous solid dispersions formulated by Eudragit E100. Carbohydr Polym 2018;199:492
8. 20. Akyuz S, Turan F, Gurbuzler L, Arici A, Sogut E, Ozkan O. The anti-inflammatory and antioxidant effects of curcumin in middle ear infection. J Craniofac Surg 2016;27:e494
7. 21. Ghoneim OM, Bill A, Dhuguru J, Szollosi DE, Edafiogho IO. Design, synthesis and biological evaluation of piperazinoenaminones as novel suppressants of pro-inflammatory cytokines. Bioorg Med Chem 2018;26:3890
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22. Pisano M, Pagnan G, Dettori MA, et al. Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells. Mol Cancer 2010;9:1
12. 23. Conney AH, Lyst T, Ferraro T, et al. Inhibitory effect of curcumin and some related dietary compounds on tumor promotion and arachidonic acid metabolism in mouse skin. Sci Y 1979;14:421
4. 24. Ferrucci V, Boffa I, De Masi G, Zollo M. Natural compounds for pediatric cancer treatment. Naunyn Schmiedebergs Arch Pharmacol 2016;389:131
49. 25. Ciftci G, Aksoy A, cenesiz S, et al. Therapeutic role of curcumin in oxidative DNA damage caused by formaldehyde. Microsc Res Tech 2015;78:391
5. 26. Karuppagounder V, Arumugam S, Giridharan VV, et al. Tiny molecule, big power: multi-target approach for curcumin in diabetic cardiomyopathy. Nutrition 2017;34:47
54. 27. Ray A, Rana S, Banerjee D, et al. Improved bioavailability of targeted curcumin delivery efficiently regressed cardiac hypertrophy by modulating apoptotic load within cardiac microenvironment. Elsevier B.V; 2016. Available from: https://doi.org/10.1016/j.taap.2015.11.011. 28. Saberi-Karimian M, Katsiki N, Caraglia M, Boccellino M, Majeed M, Sahebkar A. Vascular endothelial growth factor: an important molecular target of curcumin; 2017. ,https://doi.org/10.1080/ 10408398.2017.1366892.. 29. Liu R, Zhang H-B, Yang J, Wang J-R, Liu J-X, Li C-L. Curcumin alleviates isoproterenol-induced cardiac hypertrophy and fibrosis through inhibition of autophagy and activation of mTOR. Eur Rev Med Pharmacol Sci 2018;22:7500
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6. 31. Young NA, Bruss MS, Gardner M, et al. Oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced NFkB signaling and macrophage migration. PLoS One 2014;9. Available from: https://doi.org/10.1371/journal.pone.0111559.

32. Mazzanti G, Di Giacomo S. Curcumin and resveratrol in the management of cognitive disorders: what is the clinical evidence? Molecules 2016;21:1
27. 33. Davinelli S, Sapere N, Zella D, Bracale R, Intrieri M, Scapagnini G. Pleiotropic protective effects of phytochemicals in Alzheimer’s disease. Oxid Med Cell Longev 2012;2012. Available from: https://doi.org/10.1155/2012/386527. 34. Wu J, Li Q, Wang X, et al. Neuroprotection by curcumin in ischemic brain injury involves the Akt/Nrf2 pathway. PLoS One 2013;8. Available from: https://doi.org/10.1371/journal. pone.0059843. 35. Sandhir R, Halder A, Sunkaria A. Mitochondria as a centrally positioned hub in the innate immune response. Biochim Biophys Acta - Mol Basis Dis 2017;1863:1090
7. 36. Zorofchian Moghadamtousi S, Abdul Kadir H, Hassandarvish P, Tajik H, Abubakar S, Zandi K. A review on antibacterial, antiviral, and antifungal activity of curcumin. Biomed Res Int 2014;2014. Available from: https://doi.org/10.1155/2014/186864. 37. Zhou Z, Pan C, Lu Y, et al. Combination of erythromycin and curcumin alleviates Staphylococcus aureus induced osteomyelitis in rats. Front Cell Infect Microbiol 2017;7:1
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C H A P T E R

20 Dacryodes edulis: protective antioxidant effects on diabetes pathology Olakunle Sanni1, Ochuko L. Erukainure1,2 and Md. Shahidul Islam1 1

Department of Biochemistry, School of Life Sciences, University of Kwazulu-Natal (Westville Campus), Durban, South Africa 2Department of Pharmacology, School of Clinical Medicine, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

Introduction

Dacryodes edulis

Several studies have implicated oxidative stress (OS) in the etiology of degenerative diseases such as diabetes, cancer, and cardiovascular diseases.1 OS is the result of an imbalance between the free radicals generating system and the free radicals scavenging system in favor of the former. This could be as a result of an increase in free radicals production coupled with decreasing activity in both the endogenous (e.g., catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) and exogenous (e.g., trace metals and vitamins A, C, and E) antioxidant defense mechanisms.2 Hyperglycemia has been reported to induce free radicals through various mechanisms such as the polyol pathway, glucose autooxidation, activation of protein kinase C, hexosamine pathways, and the production of advanced glycation end products.3 There are multiple mechanisms through which phytochemicals combat hyperglycemia such as the inhibition of glucose absorption,4,5 β-cells regeneration and insulin releasing activity,6 increased insulin sensitivity,5 aldose reductase pathway inhibition,7 and antioxidant defense activity.8 Reports show that there is a decreased incidence of human disease in relation to the dietary intake of antioxidant-rich foods9; amongst these antioxidant-rich foods is Dacryodes edulis. (D. edulis) has a wide range of medicinal applications due to its high antioxidant activity. A number of its pharmacological activities have been reported.10,11 Its antioxidant activity has been attributed to the presence of phenols and flavonoids.10

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00020-2

D. edulis, a fruit tree native to Africa, belongs to the family Burseraceae. It is commonly known as the African pear or butter fruit tree in Africa, safoutier in France and the eben tree in the United States.12 The folklore use of D. edulis has been documented including the use of the stem bark in the treatment of coughs13 and the leaves in the treatment of malaria, labor pains, retarded growth, and epilepsy in children.12 The fruit is edible, either cooked or uncooked, and its nutritional value has been reported.14

Phytochemistry of Dacryode edulis Preliminary phytochemistry The preliminary phytochemical screening of D. edulis leaves, fruit, and stem bark revealed the presence of saponins, tannins, alkaloids flavonoids, and phenols as summarized in Table 20.1. Flavonoids Flavonoids are a group of hydroxylated phenols that contain natural benzo-γ-pyrone derivates.21 They are the most abundant group of phenols and largely responsible for the biological activities of most plants.22 Their pharmacological abilities are well documented and linked to the arrangement functional groups about the nuclear structure (benzo-γ-pyrone)

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© 2020 Elsevier Inc. All rights reserved.

206 TABLE 20.1

20. Dacryodes edulis: protective antioxidant effects on diabetes pathology

Studies on the preliminary phytochemistry of D. edulis.

Phytochemicals

Plant part

Solvent for extraction

References

Flavonoids

Fruit and pulp

Methanol

10,15,16

Stem exudates

Freeze-dried

17

Stem bark

Aqueous

18

Seed

Hexane

19

Saponins

Seed, stem bark

Aqueous

17,20

Alkaloids

Stem bark

Aqueous

18

Fruit skin Tannins

TABLE 20.2

Bioactive compounds of D. edulis identified by GC-MS, HPLC, and LC-MS.

Class of phytochemical

Compounds

Plant part

Extract

References

Phenol

Gallic acid

Fruit/seed

Hexane

16

Methanol

32

Gallate Catechol Methyl gallate

Flavonoid

Methyl 3,4,5-trihydroxybenzote

Stem bark

Methanol

33

Quercetin

Fruit

Methanol

15,16

Quercetin rhamnoside Saponins

Tannins

16

Sitosterol

Leaves, stem bark

Ethanol, methanol

4,33

Urs-20-en-3-ol acetate

Stem bark

Ethanol

4

Ascorbic acid 2,6-dihexodecanoate

Leaves

Ethanol

4

such that they are able to scavenge reactive oxygen species (ROS)23 and inhibit lipid peroxidation4 and cyclooxygenase, thereby attenuating inflammation.24 Erukainure et al.4 revealed the presence of flavonoids in the ethanol extract of D. edulis leaves, while Omoregie and Okugbo25 also identified flavonoids in the methanol extract. Flavonoids in the fruits and stem bark of D. edulis have also been documented.15,17,18,20 Tannins Tannins are water-soluble polyphenols widely distributed in almost all plant foods.26 Relatively, they are of high molecular weight with the ability to form complexes with carbohydrate and proteins.27 For example, they form complexes with starch and digestive enzymes including α-amylase, α-glucosidase, and β-galactosidase,26 resulting in the formation of less digestible complexes, thereby slowing the postprandial blood glucose. This correlates to the findings of Erukainure et al.4 that extracts of D. edulis inhibit α-amylase and α-glucosidase.

Saponins Saponins are a class of polyphenols consisting of glycosides linked to a hydrophobic aglycone (sapogenin), which may be triterpenoid or steroid.28 The amphipathic characteristic of saponins has been linked to many of their pharmacological activities, which include increased intestinal mucosal permeability, thereby facilitating the uptake of substances into the cells.29 Several studies have shown the glucose uptake enhancing ability of plant products, which is linked to the presence of saponins.5,30,31

Bioactive compounds of D. edulis Gas chromatography-mass spectrometry (GC-MS), analytical high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS) studies on various extract of different parts of D. edulis have revealed the presence of compounds that are of biological importance as shown in Table 20.2.

II. Antioxidants and Pathology

Protective effects of D. edulis in diabetes pathology

Diabetic pathogenesis Dietary carbohydrate is the major source of exogenous glucose in the body. Due to the importance of glucose to meet the energy requirements of the cells, mammals have developed an advance mechanism to maintain the glucose level within a certain threshold in the blood during the fast and fed states. This mechanism involves the hormonal modulation of glucose production by the liver (during the fast state) or glucose uptake and utilization by the muscle and the peripheral tissues (during the fed state). Insulin is the main source of hormonal regulation of the energy metabolism. It is produced by the β-cells of the pancreas in response to elevated blood glucose. In vitro and in vivo studies have shown that the release of insulin is stimulated by the exposure of the pancreatic islet to elevated glucose concentrations.34 The primary effect of insulin is to facilitate the uptake of glucose by the glucose transporter (GLUT4), which is predominant in the skeletal muscle and adipose tissues. An impairment in maintaining the blood glucose threshold leads to the multifactorial physiological state called hyperglycemia. Chronic exposure of β-cells to fatty acid and glucose beyond the physiological concentrations (hyperglycemia and hyperlipidemia) causes glucolipotoxicity, which leads to a cascade of events resulting in β-cell dysfunction and insulin resistance.35 Both β-cell dysfunction and insulin resistance are the underlying pathology of type 2 diabetes, which accounts for 90%
95% of diabetic cases.36

Hyperglycemia-induced oxidative stress and diabetic complications Insulin secretion is highly connected to glucose regulation, glucose, therefore, poses as a principal and critical determinant of β-cell functioning.37 Persistence hyperglycemia causes a progressive decline of β-cell functioning, leading to β-cell exhaustion and eventually to β-cell demise and dysfunction. A decrease in β-cell mass by .60% has been reported to be parallel to the extent of a reduction in glucose-stimulated insulin secretion (GSIS).38 In diabetes, glucose homeostasis is impaired either by insufficient insulin secretion or failure of action of insulin on insulin-target tissues or both. Consequently, the glucose concentrations in the blood remain high and will result in an influx of glucose into insulinindependent tissues. Hyperglycemia increases the reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD 1) ratio,39 which in turn inhibits pyruvate dehydrogenase,

207

thus causing the accumulation of glycolytic intermediates, glycerol-3-phosphate, and dihydroxyacetone. Since diabetes is also characterized with the elevation of free fatty acid (FFA), there is increased de novo synthesis of diacylglycerol (DAG). DAG has been demonstrated to increase free radicals production via the activation of NADH oxidase in both endothelial cells and smooth muscle cells.40 NADH is a substrate for NADH oxidase, which generate reactive oxygen. Aldose reductase, an enzyme responsible for the conversion of excess glucose to sorbitol in the polyol pathway, competes with reduced glutathione for NADPH, as a cofactor, hence, decreasing the reduced glutathione. However, the excessive production of oxidants coupled with the depletion of the antioxidant defense system results in OS. OS increases the release of cytokines such as tumor necrosis factor alpha (TNF-α), which may decrease the autophosphorylation of the insulin receptor in a cascade event for the proliferation of GLUT4 in the muscle cells, thereby causing insulin resistance.41 Likewise, DAG abates insulin signaling, thus, increasing insulin resistance. In addition, prolonged OS causes oxidative damages, thereby causing both micro- and macrovascular complications. OS affects cell functioning by interfering with the expression of genes that are crucial for cell functions. For instance, cyclooxygenases (COX2) have been implicated in many diabetic complications such as retinopathy, nephropathy, atherosclerosis, and neuropathy.42 COX2 has been expressed in the sciatic nerve,43 renal cortex,44 and renal medulla42 in diabetic rats.

Protective effects of D. edulis in diabetes pathology The understanding of the metabolic process of the diabetes pathology has opened novel therapeutic approaches in the treatment of diabetes. Researches have grown over the years using phytochemicals in the development of new types of therapeutics for the treatment of diabetes and its complications. Reports have demonstrated the protective effect of D. edulis in diabetes pathology and this will be discussed under the several approaches, namely carbohydrate digestion and absorption, β-cell regeneration and insulin secretion, glucose uptake and utilization, lipid metabolism, and antioxidant effects.

Carbohydrate digestion and absorption The digestion of carbohydrate to glucose and its subsequent absorption by the intestinal membrane to the blood stream is a key factor of postprandial blood

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glucose. Inhibition of this process is crucial in the management of diabetes and its complications. D. edulis has been demonstrated to inhibit carbohydrate digestive enzymes in vitro. Erukainure et al.4 demonstrated a significant inhibition of both α-glucosidase and α-amylase activities by the ethyl acetate, ethanol, and aqueous extract of the leaves. In another study, the aqueous fruit extract of both raw and roasted D. edulis demonstrated dose-dependent inhibitory activities for both α-glucosidase and α-amylase.45 The sodium-dependent glucose transporter (SGLT1) and GLUT2 are important pathways of glucose transport across the intestinal brush border. There are evidences that the expressions of these transporters are increased in diabetic subjects, thus, increasing postprandial glucose and hyperglycemia.46 Quercetin has been demonstrated to inhibit the intestinal glucose transporter,47 thus, suppressing postprandial blood glucose. The methanol stem bark extract of D. edulis has been shown to contain quercetin.15,16,33 Glycemia and gastric emptying are bidirectionally related. Delayed gastric emptying decreases postprandial glucose and, subsequently, hyperglycemia. Dietary fibers are known to delay gastric emptying.48 The fruit pulp of D. edulis has been reported to have a high dietary fiber content.49

β-cell regeneration and insulin secretion Increases in postprandial blood glucose stimulate the pancreas to increase the proliferation of β-cells in response to the increases demand for insulin. However, as hyperglycemia persists, it leads to a decline in β-cell functioning as a result of β-cell exhaustion and β-cell demise. A recent report demonstrated the pancreatic β-cell regeneration potential of the hexane extract of D. edulis fruit in diabetes rats.50 The result was substantiated by the histological examination of the pancreatic microarchitecture of the diabetic rats orally treated with 1600 mg/kg of the extract, which was found to be similar to those of the normal control rats.

Glucose uptake and utilization Primarily, skeletal muscle, adipose tissue, and the liver play a dominant role in glucose utilization. GLUT4 is responsible for the glucose uptake in adipose tissue and skeletal muscle. The mechanism of action involves a series of cascade pathways when insulin binds to the insulin receptor resulting in the activation of signaling enzymes (phosphatidylinositol3 kinase and AKT serine/threonine kinase).51 Consequently, the GLUT4 is translocated from the intracellular pool to the plasma membrane where it

translocates glucose into the cells. Phytochemicals have been found to improve glucose uptake through various mechanisms. For instance, gallic acid induces GLUT4 translocation to the membrane and glucose uptake activity in 3T3-L1 cells.52 Also, sistosterol53 and quercetin54 have been reported to significantly increase the phosphorylation of AMP-activated protein kinase alpha (AMPKα) and glucose uptake in L6 myotube cells lines. Gallic acid, sitosterol, and quercetin are bioactive compounds found in different parts of D. edulis as shown in Table 20.2. On the other hand, the liver utilizes glucose through glycolysis or stores it as glycogen when the blood glucose levels are elevated. Insulin, thus, increases the activity of glucokinase (hexokinase) and decreases the activity of key enzymes in the gluconeogenesis pathway. Erukainure et al.4 demonstrated a significant decrease in the activity of glucose-6-phosphatase in a dose-dependent manner when liver tissue was incubated with ethyl acetate, ethanol, and aqueous extract of D. edulis. Also, catechin gallate present in the hexane extract of D. edulis fruit16 has been demonstrated to suppress the gluconeogenesis enzyme expression in H4IIE rat hepatoma cells.55

Lipid metabolism It has been shown that the flavanol epigallocatechin3-gallate inhibits fatty acid synthase (FAS) activity and lipogenesis.56

Antioxidant protective effect As previously illustrated, hyperglycemia generates free radicals and, at the same time, depletes the endogenous antioxidant defense system, thereby leading to OS. OS is the major cause of both macro- and microvascular complications in diabetes. The mechanism of the D. edulis antioxidant protective effect can be categorized into two pathways, namely (1) the scavenging of hyperglycemia-induced free radicals and (2) the improvement of the antioxidant defense system. Scavenging of hyperglycemia-induced free radicals by D. edulis The strong antioxidant ability of D. edulis has been linked with its phytochemical constituents. Polyphenols quench free radicals through numerous mechanisms, namely (1) through electron donating ability, (2) through proton donation, (3) by chelating metal ions, and (4) by improving endogenous antioxidant enzyme activity. 1. Proton-donating mechanisms: The mechanism involving phenolic hydrogen as a proton-donating radical is as a result of the

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Conflict of interests

formation of a stable radical, usually by the presence of a O-dihydroxy structure in one of the polyphenol rings.57 Also, the concentration of polyphenol must be relatively low compared to the substrate to be oxidized.58 An in vitro 2,20 -diphenyl1-picrylhydrazyl (DPPH) assay involves the protonation of unstable DPPH• radical and forms an antioxidant radical59 using this mechanism. The DPPH radical scavenging activities of D. edulis have been documented. Onocha et al.60 reported the DPPH radical scavenging activities of the hexane fraction of the leaves of D. edulis to be 93%, while Erukainuire et al.4 reported ethyl acetate, ethanol, and aqueous extracts of the leaves with IC50 values of 54.90, 1.83, and 44.57 μg/mL respectively. The activity of the seed, pulp, and the fruit peels have also been reported.16,61,62 2. Electron donating ability: The electron donating mechanism provides for an electron to be donated by the polyphenol group to the ROS-forming polyphenol radical. The polyphenol radical is less reactive and stabilizes as a result of its ability to distribute the radicals all over the molecules.63 A ferric reducing antioxidant power (FRAP) assay is based on the electron donating ability of polyphenol. It measures the ability of polyphenol to reduce Fe21 to Fe31. The FRAP activities of D. edulis have been elucidated. Ogunmoyole et al.62 revealed the high reducing power of the aqueous extract of the seed of D. edulis. Omoregie et al.25 likewise reported that the methanol extract of the leaves had no significant difference when compared with ascorbic acid. Also, Erukainure et al.4 revealed the high reducing power of the ethyl acetate, ethanol, and aqueous extracts of the leaves of D. edulis with the ethyl acetate extract showing the highest activity. 3. Metal ion chelation: This mechanism involves the formation of a stable complex compound when polyphenols chelate metal ions (usually Fe21), thus, preventing the complex from partaking in reactions that generate hydroxyl radical (OH•).63 The Fe21 chelating ability of polyphenol is usually measured by determining the malondialdehyde (MDA) level in lipid peroxidation. Fe21 can catalyze one-electron transfer reactions that generate OH• in Fenton reactions.5 It has been reported that OH• accounts for increased lipid peroxidation, hence, cell damage.64 Decreases in MDA levels by D. edulis have been reported. Erukainure et al.4 reported that the ethanol extract of the leaves of D. edulis decreased the MDA level in the pancreatic tissue. Ogunmoyole et al.62 also reported the same for the aqueous extract of the fruit.

Improving endogenous antioxidant enzyme activity The activities of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase, etc., involved in the removal of ROS play an important role in abating OS. Several studies have reported the ability of polyphenols to improve the activities of antioxidant enzymes in disease states.5,30,65,66 The ability of D. edulis to mitigate OS in disease states has been reported. Omonhinmin and Agbara11 reported a significant increase in the catalase activity and reduced glutathione (GSH) level in both the liver tissue and the serum of rats pretreated with ethanol extract of D. edulis when compared to untreated CCl4induced rats. Erukainure et al.4 also reported an improved GSH level and catalase and SOD activity in Fe21-induced OS both in pancreatic and hepatic tissues after treatment with the ethanol extract of the leaves of D. edulis. The significant increase in the activities of these enzymes indicates the antioxidative potential of D. edulis.

Proposed protective mechanism of diabetes pathology of D. edulis Based on the processes involved in diabetes pathology as discussed in this chapter, D. edulis may bring about its protective activities by the reduction of postprandial glucose, the modulation of the redox homeostasis, and the increased activity of the antioxidant defense system, thereby attenuating OS. This process can be summarized into five groups as shown in Fig. 20.1.

Conclusion Studies carried out on D. edulis demonstrate its antioxidant therapeutic potentials in diabetes. These potentials are attributed to its phytochemical constituents, particularly polyphenols. Thus, D. edulis may be employed as a potent functional food and/or nutraceutical in the treatment and management of diabetes and its complication. However, more studies are required on the molecular mechanisms involved in its antioxidant and antidiabetic activity.

Conflict of interests The authors report no conflict of interests.

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20. Dacryodes edulis: protective antioxidant effects on diabetes pathology

FIGURE 20.1 Proposed protective mechanism of diabetes pathology of D. edulis. Group A (A1, A2, and A3): Glucose metabolism and absorption A1 5 Inhibition of carbohydrate digestion enzymes A2 5 Delay in gastric emptying A3 5 Inhibition of glucose absorption Group B (B1and B2): Increased β-cell and insulin secretion B1 5 Protection and regeneration of pancreatic β-cell B2 5 Increased insulin secretion Group C: (C1 and C2): Increased action of insulin C1 5 Increase uptake of glucose by adipose tissue C2 5 Increase glucose utilization by the liver and decreased glucose hepatic production Group D: (D1 and D2): Increased antioxidant defensive mechanism D1 5 Scavenging and decreased production of free radicals D2 5 Increased antioxidant defense activity

Acknowledgment

References

This work was supported by a Competitive Research Grant from the Research Office, University of KwaZulu-Natal, Durban; and a Grant Support for Women and Young Researchers, National Research Foundationa (NRF), Pretoria, South Africa. The first author also received a Freestanding, Innovation, and Scarce Skills Scholarship from the National Research Foundation (NRF), Pretoria, South Africa.

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60. Choi D-Y, Lee Y-J, Hong JT, Lee H-J. Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer’s disease. Brain Res Bullet 2012;87:144
53.

II. Antioxidants and Pathology

C H A P T E R

21 Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases Punniyakoti Veeraveedu Thanikachalam1, Srinivasan Ramamurthy2, Anoop Kumar3, Meenakshi Gupta4 and Garima Bansal5 1

GRT Institute of Pharmaceutical Education and Research, Tiruttani, India 2College of Pharmacy and Health Sciences, University of Science & Technology of Fujairah, Fujairah, UAE 3Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER-R), Ministry of Chemical & Fertilizers, Govt. of India, Lucknow, India 4Department of Pharmacology, ISF College of Pharmacy, Moga, India 5Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga, India

List of abbreviations AAC AD ADHD ANP APP AP-1 ATP CAT CCL2 COMT CoQ COX-2 CPK CRP CTGF CVD DHLA DNMTs EGCG FN GPx GSH GSSG HDL HO-1 H2O2 ICAM IL iNOS JNK

abdominal aortic constriction Alzheimer disease attention-deficit hyperactivity disorder atrial natriuretic polypeptide amyloid precursor protein-α activator protein 1 adenosine triphosphate catalase C-C motif chemokine ligand 2 catechol-O-methyltransferase coenzyme Q cyclooxygenase 2 creatine phosphokinase C-reactive protein connective tissue growth factor cardiovascular disease dihydrolipoic acid DNA methyltransferase epigallocatechin gallate fibronectin glutathione peroxidase glutathione oxidized glutathione high-density lipoprotein cholesterol heme oxygenase-1 hydrogen peroxide intracellular adhesion molecule interleukin inducible nitric oxide synthase c-Jun-N-terminal kinase

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00021-4

LA LDL-C LOX LXR α/β MAPK MCP MPO MPTP NF-κB nNOS NOX-4 OS PD PUFAs RNS ROS SODs SREBP-1 TC TNF-α VEGF

alpha-lipoic acid low-density lipoprotein cholesterol lipooxygenase liver X receptor mitogen-activated protein kinase monocyte chemoattractant protein myeloperoxidase 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nuclear factor- kappa B neuronal nitric oxide synthase NADPH oxidase 4 oxidative stress Parkinson’s disease polyunsaturated fatty acids reactive nitrogen species reactive oxygen species superoxide dismutases sterol regulatory element-binding protein 1 total cholesterol tumor necrosis factor alpha vascular endothelial growth factor

Introduction What is oxidative stress? Oxidative stress (OS) arises due to an imbalance between the systemic expression of reactive oxygen species (ROS) and a biological system’s capacity to promptly detoxify the receptive intermediates or to fix

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21. Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases

the subsequent damage. Imbalance in the redox state of the cells generates toxic effects via peroxides and free radicals production. Such toxic effects damage the integral cell components such as nucleic acids, lipids, and proteins. OS caused by a disturbance in the oxidative metabolism is the source of base damage and strand breaks in DNA. Moreover, ROS are the major reason for base damage (indirect) in DNA. Further, some ROS act as cellular messengers in redox signaling.1 Substantial evidence has demonstrated that OS in humans has been found to be associated with the occurrence of various types of diseases such as cancer, Alzheimer’s disease, Parkinson’s disease, sickle-cell disease, myocardial infarction, heart failure, lichen planus, vitiligo, Lafora disease, diabetes, attention-deficit hyperactivity disorder, atherosclerosis, fragile X syndrome, autism, infection, chronic fatigue syndrome, and depression, etc.2
4 ROS are involved with immune system activity by attacking and killing pathogens. ROS are produced by mitohormesis, which is crucial for the prevention of aging.

Reactive oxygen species In biological terms, ROS are generated as a byproduct in oxygen metabolism and have a significant function in homeostasis and cell signaling. Whereas environmental stress (heat or UV exposure) increases the ROS levels, which might cause serious damage to cell structures. Various reactive species are represented in Table 21.1.

TABLE 21.1 

O 22 , superoxide anion

OH, hydroxyl radical

ROOH, organic hydroperoxide •

Antioxidants are compounds that compensate for the deleterious effect of oxidants by acting against ROS and reactive nitrogen species (RNS) and, hence, ultimately protect the tissues from damage caused by various stresses.1 There are two types of antioxidant systems, namely (1) endogenous antioxidants, and (2) exogenous antioxidants. Thiols, ascorbic acid, or polyphenol molecules are among the various antioxidants that act as reducing agents by inhibiting the oxidation of many molecules by being self-oxidized. Antioxidants like glutathione and vitamin C, A and E along with enzymes like catalase (CAT), superoxide dismutase (SOD), and many peroxidases are maintained by complex systems in plants and animals.5 There are basically two lines of defense involved with the antioxidant activity in the cell, where the first line consists of coenzyme Q, β-carotene, and vitamin E, and these are found at the fat-soluble membrane of the cell. Among these, vitamin E is the main chainbreaking antioxidant of the cell membrane. However, water-soluble antioxidant scavengers are found inside the cell, some examples include superoxide dismutases (SODs), glutathione peroxidase (GPx), vitamin C, and catalase (CAT). In dietary supplements, antioxidants are widely used and studies are conducted to find out the protective role of dietary supplements against various types of diseases including coronary heart disease, cancer, immunological disorders, neurodegenerative disease, and even altitude sickness.

List of various reactive species.

H2O2, hydrogen peroxide •

Antioxidants

•

It is formed by the electron transport chain in many autoxidation reactions. It is unreactive, but can release Fe21 from iron
sulfur proteins and ferritin. It is involved in the formation of H2O2 through dismutation or by enzymatic catalysis and is a precursor for metal-catalyzed hydroxyl radical (•OH) formation. It is formed by the dismutation of  O 22 or by direct reduction of O2, H2O2, and lipid-soluble and, thus, possess diffusibility across membranes. It is formed by Fenton reaction and the decomposition of peroxynitrite. Highly reactive; will attack most cellular components. Formed by radical reactions with cellular components such as lipids and nucleobases.

RO , alkoxy and ROO , peroxy radicals

Oxygen-centered radicals participate in lipid peroxidation reactions produced in the presence of oxygen either by radical addition across double bonds or hydrogen abstraction.

HOCl, hypochlorous acid

Formed by the action of myeloperoxidase from H2O2. Lipid-soluble and highly reactive. Readily oxidizes protein constituents such as thiol groups, amino groups, and methionine.

ONOO2, peroxynitrite

Formed through a rapid reaction between  O 22 and NO•. The reactivity is similar to HOCl and lipid-soluble in nature. It forms peroxynitrous acid upon protonation, which subsequently forms hydroxyl radical and nitrogen dioxide through homolytic cleavage.

II. Antioxidants and Pathology

Introduction

Endogenous antioxidants Endogenous protein antioxidants Superoxide dismutases As the first-line defense, SODs convert extremely reactive superoxide radicals (O2 (2) or  O 22 ) into hydrogen peroxide (H2O2) and molecular oxygen via dismutation. Four isozymes of SODs have been identified. SOD1 (associated with Cu/Zn) along with CAT and GPx convert these superoxide radicals into H2O2.5 Catalase CAT is another endogenous antioxidant enzyme that protects the cell against the harmful effects of ROS. CAT is a tetrameric porphyrin
containing enzyme that is located in peroxisomes and catalyzes the H2O2 conversion (in two steps) into water (H2O) and molecular oxygen.5,6 First: Catalase-FeðIIIÞ 1 H2 O2 -Compound I Second: Compound I 1 H2 O2 -Catalase-FeðIIIÞ 1 2H2 O 1 O2 Glutathione peroxidase GPx as an enzyme that exists in two different forms, namely seleniumdependent and selenium-independent. Each of these forms has different subunits and active sites. This enzyme catalyzes the conversion of H2O2 or
ROOH (organic peroxide) into H2O or alcohol. During the course of the process, glutathione (GSH) is oxidized to GSSG (oxidized glutathione).5,6 This reaction protects the polyunsaturated fatty acids (PUFAs) of cell membranes, where the GPx serves as a part of the multicomponent antioxidant defense system within the cell and, hence, such reactions are considered significant. Endogenous nonprotein antioxidants Glutathione GSH with the chemical formula C10H17N3O6S is composed of three amino acids, namely glutamate, cysteine, and glycine (L-γ-glutamylL-cysteinyl-glycine). GSH is a nonprotein thiol that is active in its reduced form and is generally found in most cell types. GSH as an antioxidant provides defense against ROS and RNS along with detoxication enzymes such as GSH peroxidases and GSH-Stransferases.4,5 In animals, GSH/glutathione disulfide is considered as the main redox couple. Alpha-lipoic acid Alpha-lipoic acid (α-LA) along with its potential in recharging enzymes of the mitochondria, performs antioxidant activity in most of the sub-cellular compartment of the body.7 α-LA (oxidized) generally in its polar and nonpolar forms provides such antioxidant effects. Coenzyme Q Coenzyme Q (CoQ) is naturally found in humans with high levels in the liver, heart,

215

and kidneys, etc. CoQ is a benzoquinone derivative and is localized at the mitochondrial respiratory chain along with some other internal membranes. CoQ is directly involved in the transportation of electrons in the respiratory chain and also couples it with oxidative phosphorylation. For such roles, it is also found to be associated with energy transduction and aerobic adenosine triphosphate (ATP) production. CoQ is an endogenously synthesized lipid-soluble antioxidant, found in most internal membranes and, hence, its protective function is found to be extended to lipids, proteins, and DNA.8 Ferritin Ferritin is a blood protein that consists of iron. Ferritin having a cytosolic form consists of two subunits, namely H and L. The apoferritin shell is formed by the association of 24 ferritin subunits, where each apoferritin molecule takes up iron atoms. Ferritin limits the iron(II) availability for participation in the production of ROS.9 Uric acid In the purine degradation pathway, uric acid is an intermediate product. However, the uricase enzyme acts as an inhibitor for uric acid, but researchers have reported that such enzymes (uricase) have been inactivated during the evolution of humans and apes. Previous studies have reported that high uric acid levels in blood protect from oxidative injury in neural, vascular, and cardiac cells.10 Bilirubin The heme group in the form of a substrate is used in the production of iron, biliverdin, and carbon monoxide by the heme oxygenase-1 (HO-1) enzyme. Ultimately, the heme is degraded for the reduction of biliverdin to bilirubin by the biliverdinreductase enzyme. Bile pigments as potent in vitro scavengers of free radicals are an important inducible antioxidant system that protects against various cellular stresses including oxidative damage. Bilirubin protects neurons against damages caused by H2O2 and also has a protective role against ischemic injury in isolated hearts.11 Exogenous antioxidants The body obtains essential antioxidants like vitamin E and C as dietary supplements. Vitamin E intercalated with phospholipids is supposed to give protection to PUFAs. PUFA forms a double bond structure as it is interrupted by methylene and, hence, is susceptible to free radical-based oxidation. Vitamin C is a significant antioxidant due to the formation of relatively stable ascorbyl free radical by the donation of an electron. Ascorbate is a scavenger of ROS and is found to be significant against H2O2, singlet oxygen, superoxide radical anion, and hydroxyl radical. Vitamin C also scavenges RNS to prevent the nitrosation of

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21. Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases

target molecules. Many plant pigments including β-Carotene and various other flavonoids have antioxidant property12 and plant phenols are seen to inhibit lipid peroxidation and lipoxygenase enzymes (in vitro).

Epigallocatechin gallate The leaves from Camellia sinensis are used in making black or green tea.13 Black tea is produced by the overoxidation of tea leaves and this makes it different from green tea and, hence, green tea possesses a higher polyphenol content.14 Such polyphenols contain many phenolic rings and EGCG is observed to be the most abundant compound (around 40%) and also represents the most active chemical component of this family.13 Based on previous studies, polyphenols are regarded as one of the most potent antioxidants and chemopreventive agents. Polyphenols perform the neutralization of free radicals and, hence, decrease the inflammation and inhibit the development of tumors.15,16 Pharmacological actions of epigallocatechin gallate EGCG has been found to exhibit a protective effect in cardiovascular disease (CVD; cardiac arrest, atherosclerosis, and cardiac hypertrophy), metabolic syndromes (diabetes and obesity), neurodegenerative disease (Parkinson’s disease, Alzheimer’s disease), aging, and cancer (Tables 21.2
21.4). Epigallocatechin gallate and cardiovascular diseases EGCG as a polyphenol constitutes about B30% of the total composition of green tea. Previous studies have shown an association between green tea consumption and a reduced risk for CVD, but the actual mechanism behind the role is still not known. In a study by Aneja et al., treatment with EGCG (10 mg/ kg) was confirmed to limit myeloperoxidase activity, IL-6 plasma level, creatine phosphokinase content, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) DNA binding in rodents having ischemiareperfusion injury
based heart attacks.17 Sheng et al. showed the significance of EGCG in an abdominal aortic constriction (AAC) model of cardiac hypertrophy; the outcomes suggested a depletion in the plasma endothelin content, atrial natriuretic polypeptide, heart weight indices, hydroxyproline content, and the expression of a proliferating cell nuclear antigen found in the hypertrophic myocardium.18 Whereas Cai et al. reported that treatment with EGCG (50 mg/kg) inhibited NF-κB generation and connective tissue growth factor overexpression induced by AAC or angiotensin II. EGCG has also been reported to reduce collagen

synthesis and fibronectin (FN) expression along with the proliferation of rat cardiac fibroblasts induced by angiotensin II or AAC. These results suggest that EGCG are significant in the prevention of remodeling accompanied by pressure overload-induced hypertrophy.19 In the same year, another study by the same group showed that EGCG-based treatment (0.02%) inhibited inflammatory mediators such as LOX-1, MCP, ICAM-1, CCL2, iNOS, and CRP and markers of OS (NOX-4, p22phox) and improved mRNA expression of antioxidant enzyme (HO-1) in an atherosclerotic animal model.20 EGCG inhibits cancer-inducing molecules and OS and, hence, provides defense against isoprenaline-induced myocardial damage and ultimately provides protection to the heart.21 Wang et al. demonstrated the antiatherosclerotic effect of EGCG through the attenuation of plaque development, depletion of plasma concentration of total cholesterol (TC), LDL, proinflammatory cytokines (TNF-α, IL-6) and improved levels of antiinflammatory cytokine (IL-10) and HDL in ApoE2/2 mice.22 Additionally, EGCG controls the genes associated with lipid metabolism (LXRα/β and SREBP-1). Oyama et al. have reported that heart/muscle-specific MnSOD-deficient mice (H/ M-SOD22/2) that had received EGCG tended to have a lower mortality rate and showed less inflammation and, thereby, preserved cardiac function and telomere biology (Table 21.2).23 Another study indicated that EGCG treatment (10 μM) was able to lower MI via infarct volume (33.5% 6 4.1%) depletion in Langendroff-perfused rat hearts by acting on the adenosine and opioid receptors24 (Table 21.3). Based on a study involving 82 patients with early atherosclerosis, it was shown that the consumption of EGCG (30 mL) supplemented with olive oil remarkably increased endothelial activity by decreasing the concentration of leukocytes.25 A study by Widlansky et al. indicated that EGCG (300 mg for initial dose followed by 150 mg twice daily for 2 weeks) increased endothelial activity by improving the brachial artery flow
based dilation among 42 subjects with coronary artery disease26 (Table 21.4). Another randomized study on adults ( . 20 years, without any disease) showed the potential role of EGCG in the prevention of CVD development by decreasing the low-density lipoprotein cholesterol (LDL-C) levels.27 Epigallocatechin gallate and neurodegenerative disease Alzheimer’s and Parkinson’s disease (AD and PD, respectively) are associated with protein misfolding and β-sheet-rich amyloid fibrils formation or aggregation. A study by Bieschke et al. showed polyphenol (
)-EGCG mediated inhibition of α-synuclein and amyloid-β fibrillogenesis.28 Moreover, in another

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217

Introduction

TABLE 21.2

Summary of in vivo studies of the protective effect of epigallocatechin gallate on various diseases.

Disease model

Dose

Effect

References

Atherosclerosis (hypercholesterolemic diet Wistar rats)

100 mg/kg

Reduced TC, TG, LDL, and VLDL cholesterol levels

48

Atherosclerosis (ApoE2/2 fed with HFD)

40 mg/kg

Attenuated plaque formation, reduced plasma levels of TC, LDL, and proinflammatory cytokines (TNF-α and IL-6), and increased HDL level and antiinflammatory cytokines (IL-10) level Modulated genes involved in lipid metabolism (LXRα/β and SREBP-1)

22

Atherosclerosis (ApoE2/2 mice)

0.8 g/L

Reduced aortic weights, aortic cholesterol, and aortic TG

49

Atherosclerosis (Porphyromonas gingivalis-induced ApoE2/2 mice)

0.02% Solution

Reduced CRP, MCP-1, CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22phox, and iNOS gene expression levels Increased expression of HO-1 mRNA

20

Atherosclerosis (atherogenic-dietfed Wistar rats)

100 mg/kg

Reduced CRP and hematological inflammatory markers (erythrocyte sedimentation rate, leucocyte, and platelet count)

50

Cardiac hypertrophy (AACinduced rats)

25, 50, 100 mg/kg

Inhibition of telomere shortening and loss of TRF2 Reduced MDA contents, heart weight indices, apoptosis, and ANP, plasma endothelin, and hyp levels Decreased the proliferating cell nuclear antigen expression in the hypertrophic myocardium Increment of nitrite concentrations was observed

18,51

Cardiac hypertrophy (Ang II and AAC-induced Sprague-Dawley rats)

50 mg/kg

Inhibited NF-κB and CTGF overexpression Reduced collagen synthesis, cardiac fibroblasts proliferation, and FN expression

19

Cardiac hypertrophy (AACinduced Sprague-Dawley rats)

0.02%, 0.04%, and 0.08%

Suppressed increase in heart weight by 69%. Attenuated myocyte cell size and fibrosis

52

Heart failure (H/M-SOD22/ 2 mice)

10, 100 mg/L

Reduced myocardial oxidative stress and FFA 23 Inhibited NOS2, NT, fatty acid synthase, TLR4, and Sirt1 expressions

Heart failure (Ageing C57BL/6 mice)

50 mg/kg/day

Improved cardiac diastolic function by upregulating cTnI and reducing histone acetylation 1 and 3

53

MI (Ang II-induced adult Wistar rats)

50 mg/kg

Attenuated endoglin expression from binding to AP-1 transcription in the cardiac fibroblasts

54

MI (male Wistar rats)

10 mg/kg

Attenuation in architectural derangement was observed throughout 17 the distribution area of the left coronary artery Reduced plasma CPK activity (399 6 40.23 U/L from 1100 6 112.58 U/L) Reduced plasma IL-6 levels (494 6 92.3 pg/mL from 1312.83 6 224.6 pg/mL) Reduced myeloperoxidase activity (18.73 6 1.8 U/100 mg tissue from 29.30 6 2.29 U/100 mg tissue) In addition, reduced NF-κB and AP-1 DNA binding

Diabetes (STZ-induced rats)

25, 50 mg/kg

Reduced TC, TG, LDL cholesterol, and glucose levels. No significant changes in plasma HDL cholesterol

55

Type 2 diabetes (nicotinamide and STZ)

2 mg/kg

Significantly reduced plasma glucose, HbA1c, HOMA-1R and lipid profile Increased plasma insulin level and antioxidant enzymes (SOD, CAT, and GSH) and decreased apoptotic markers Bax, caspase 3, 9. Improved myocardial function by reducing inflammatory markers

56

Antioxidant effects (NMDAinduced neurotoxicity in mice)

30, 60 mg/kg/day

Decreased ROS production

57

Mice NMDA toxicity model




Enhanced behavioral and neurotoxic effects of NMDA Decreased ROS production

58

(Continued)

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218 TABLE 21.2

21. Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases

(Continued)

Disease model

Dose

Effect

References

MPTP- and DA-induced neurodegeneration in mice and rats MPTP- and 6-OHDA- induced toxicity in male C57BL mice

2 mg/kg/day

Prevented the accumulation of iron and α-synuclein in the substantia 59 nigra Decreased Bax, caspase-6, gadd45, and TRAIL expression levels

Alzheimer’s disease (PS2 transgenic mice model of AD)

1.5 or 3 mg/kg body weight in drinking water

Enhanced memory function Induced α-secretase activity Reduced β- and γ-secretase activities

60

6-OHDA, 6-Hydroxydopamine; AP-1, activator protein 1; Bax, Bcl-2-associated X; BCL2, B-cell lymphoma protein 2 (Bcl-2); CAT, catalase; CCL2, C-C Motif Chemokine Ligand 2; CPK, creatine phosphokinase; CRP, C-reactive protein; CTGF, connective tissue growth factor; cTnI, troponin I; DA, dopamine; FFA, free fatty acid; FN, fibronectin; gadd45, growth Arrest and DNA Damage-inducible 45; GSH, glutathione; GSK-3, glycogen Synthase Kinase-3; H2O2, hydrogen peroxide; HbA1c, hemoglobin A1C; HO-1, heme oxygenase; HOMA-1R, Homeostasis Model Assessment; HSP60, heat shock protein 60; ICAM-1, intracellular adhesion molecule 1; IL, interleukin; iNOS, inducible nitric oxide synthase; LDL, low-density lipoprotein; LOX-1, lipooxygenase 1; LPS, Lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein 1; MDA, malondialdehyde; MMP-9, matrix matellaoproteinase 9; MPO, myeloperoxidase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NF-κB, nuclear factor-kappa light chain enhancer of activated B cells; NMDA, N-methylD-aspartate; NOS2, nitrix oxide synthase 2; NOX-4, NADPH Oxidase 4; NT, nitrotyrosine; ROS, reactive oxygen species; SOD, superoxide dismuatase; TC, total cholesterol; TG, triglyceride; TLR4, toll-like receptor 4; TRAIL, TNF-related apoptosis-inducing ligand; TRF2, telomeric repeat-binding factor 2; VCAM-1, vascular cell adhesion molecule 1; VLDL, very low-density lipoprotein.

TABLE 21.3

Summary of in vitro studies of the protective effect of epigallocatechin gallate on various diseases.

Disease model

Dose

Effect

References

Atherosclerosis (HUVECs with ox-LDL)

50 μM

Protected against ox-LDL-induced endothelial dysfunction

61

Atherosclerosis (THP-1-derived macrophages)

10 μM

Reduced macrophage cholesteryl ester content and MCP-1 mRNA

62

Cardiac hypertrophy (PE-induced H9C2 cardiomyocytes)

100 μM

Activated AMPK by reducing Nppa, BNP mRNA and decreased cell surface area in H9C2 cardiomyocytes

63

Heart failure (ventricular muscle strips from Mybpc-3 targeted knock-in and WT mice)

1.8 μM

Diastolic sarcomere length and fractional sarcomere shortening were not affected

64

0.01, 0.1, 1, 10 μM

Regulated electrophysical characteristics of the left atrium Dose-dependent reduction of action potential duration Suppressed ISO-induced atrial arrhythmogenesis via inhibition of Ca21/calmodulin or cGMP-dependent protein kinase

65

Cardiac arrhythmogenic activity (ISO-induced mice cardiomyocytes)

MI (Langerdorff-perfused rat heart)

10 μM

Reduced infarct volume by acting on ADR and OPR

24

6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neuroblastoma SH-SY5Y cells

50 μM

Increased expression levels of Bax, bad, gadd45, fas, fas ligand, and caspase 3, 6, and 10

66

1 μM

Decreased expression levels of Bcl-2, Bcl-XL, and Bcl-W.

N2a cells stably transfected with “Swedish” mutant human APP Inflammatory response induced by IL-1β and Aβ in human astrocytoma, U373MG cells (AD) Head and neck squamous cell carcinoma (HNSCC) cells and breast carcinoma cell lines

Human prostate carcinoma LNCaP cells

5
80 μM

Elevated active ADAM10 protein Increased APP α cleavage and α-secretase activity with no alteration in β-or γ-secretase activities

0.2
20 μM Modulated MAPK signaling pathway 10, 30 μg

Decreased the levels of Bcl-2 and Bcl-XL proteins Increased Bax protein and activation of caspase 9 Inhibited phosphorylation of EGFR, Stat3, ERK, cfos, and cyclin D1

20
80 μM Decreased expression of the proapoptotic protein Bcl-2 Activated p21/WAF1, Bax, and caspase 3.

67

35 68

69

ADAM10, A disintegrin and metalloproteinase domain-containing protein 10; ADR, adenosine receptor; AMPK, 50 AMP-activated protein kinase; APP, amyloid precursor protein; Bax, Bcl-2-associated X; Bcl-2, B-cell lymphoma protein 2; Bcl-XL, B-cell lymphoma-extra-large; c-JNK, c-Jun-N-terminal kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HUVEC, human umbilical vein endothelial cells; ISO, isoproterenol; LDL, lowdensity lipoprotein; MAPK, Mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein 1; OPR, opioid receptor; ox-LDL, oxidized low-density lipoprotein; Stat3, signal transducer and activator of transcription3; THP, human acute monocytic leukemia cell line.

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Introduction

TABLE 21.4

Efficacy of epigallocatechin gallate (EGCG) on various diseases in clinical trials.

Disease

Study population

Dose

Effect

References

Atherosclerosis (doubleblind, randomized trial)

82 patients with early atherosclerosis

30 mL supplemented with olive oil

Improved endothelial function by reducing the number of leukocytes.

25

Coronary artery disease (double-blind, randomized trial)

42 patients with coronary artery disease

300 mg initial dose followed by 150 mg for 2 weeks

Improved endothelial function through dilation of the brachial artery.

26

Cardiac hypertrophy

25 male patients with wildtype transthyretin amyloid cardiomyopathy

600 mg for 12 months

Decreased LV myocardial mass and TC. LV wall thickness and mitral annular plane systolic excursion remained unchanged.

70

Obesity (double-blind, randomized and parallel design study)

100 overweight or obese male subjects, aged 40
65 years

400 mg twice daily for 8 weeks

Reduction of diastolic blood pressure. More positive mood than the control group.

71

Radiation-induced breast cancer (interventional, randomized, parallel)

49 participants

Sprayed three times a day at 0.05 mL/cm2 to the whole radiation

EGCG reduced the pain and feelings of burning, itching, pulling, and tenderness.

72

Acne vulgaris (randomized, double-blind)

35 participants

17 subjects treated with 1% EGCG 18 subjects treated with 5% EGCG

NA

73

Inflammation (randomized, split-face trial)

35 (17 men and 18 women, mean age: 22.1)

1% or 5% topical solution twice a day

Significantly reduced inflammation by inhibiting NF-κB and AP-1 pathways. Induced cytotoxicity of human sebocytes via apoptosis and decreased the viability of P. acnes.

74

AP-1, Activator protein 1; LV, left ventricular; NF-κB, nuclear factor-kappa light chain enhancer of activated B cells; TC, total cholesterol.

study, Raneva et al. demonstrated the oral administration of EGCG (150 mg/kg) leads to the enhanced antioxidative potential of rat plasma by α-tocopherol preservation.29 The prevention of iron and α-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice by EGCG was reported in a study conducted by Mandel et al.30 (Table 21.2). Furthermore, these activities were found to be because of the antioxidant potential and iron-chelating functions of EGCG respectively. Choi et al. showed reduced expression of neuronal nitric oxide synthase (nNOS) in their MPTP-induced PD mouse model.31 The role of EGCG in the modulation of amyloid precursor protein (APP) cleavage and reduction of cerebral amyloidosis in AD transgenic mice was determined.32 In addition, a study demonstrated the protective role of EGCG against β-amyloid-induced neurotoxicity in cultured hippocampal neurons, showing significant antioxidant activity33 and enhanced cell survival through decreased MDA levels and apoptotic signaling markers respectively. In an in vitro study on liver cytosol homogenates, the significant inhibitory effect of EGCG on the catechol-O-methyltransferase activity at a low IC50

concentration (0.2 μM) was shown, hence, suggesting their potential role in the treatment of PD.34 Kim et al. demonstrated that the inflammatory response induced by IL-1β and β-amyloid fragment (25
35) in human astrocytoma cells was suppressed by EGCG through inhibiting the activation of NF-κB and expression of VEGF, COX-2, phosphorylation of MAPK and JNK, which further supports the significance of EGCG in the treatment of various neurological disorders35 (Table 21.3). Epigallocatechin gallate and cancer Oncogenesis is considered as a composite and several-step procedure where modifications take place at the cellular and molecular levels.36 Cancer formation can be categorized into three stages. These include (1) initiation, the rapid phase, where a carcinogen interacts for the first time with the DNA and further gets distributed and transported to the specific tissue where its metabolic activation takes place, leading to genotoxic damage; (2) promotion, which is a reversible process where the proliferation of mutated cells takes place and further undergoes replication and may also originate a focus of preneoplastic cells; and (3) the

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21. Protective role of epigallocatechin gallate, a dietary antioxidant against oxidative stress in various diseases

progression stage, known as the neoplastic transformation stage where uncontrolled growth leads to tumor formation. This stage involves the formation of an abnormal mass of tissue (neoplastic) from the premalignant cells with improved potential of metastasis, invasiveness, and angiogenesis. Research on cancer cells has reported the significant potential of EGCG against oncogenesis. Chen et al. reported successful cell growth inhibition by EGCG by inducing apoptosis and modulation of gene expression.37 Paschka et al. reported the active potential of EGCG (via green tea consumption) in the inhibition of cell proliferation and also suggested its potential in the induction of apoptosis while studying its effect on prostate cancer. Studies on EGCG have shown that it can inhibit DNA methylation through the inactivation of DNA methyltransferase. EGCG has also been shown to possess significant antioxidant and radical scavenging activities.38 In another study (in vivo), EGCG was found to inhibit platelet-derived growth factor
induced apoptosis and to regulate cell cycle pathways of vascular smooth muscle cells, which ultimately influenced the inhibition of tumor growth, angiogenesis, and metastasis.39 EGCG in another study was reported to cause deregulation of the cell cycle and apoptosis of cancer cells, which might be regulated via NF-κB inhibition.40 Shankar et al. have shown the potential of EGCG in

the regulation of gene expression of the set of genes responsible for cancer progression, invasion, metastasis, and angiogenesis and, hence, can be a significant agent for chemoprevention against pancreatic cancer.41 Epigallocatechin gallate and aging The process of aging is distinguished by the escalating loss of tissue and organ function42 and OS
dependent studies are based on the assumption that age-related loss of function is because of a collection of ROS/RNS-based injuries. In parallel, OS was found to be associated with several age-related conditions such as cardiovascular and neurological disorders, chronic obstructive pulmonary disease, chronic kidney disease, and cancer as well as sarcopenia and frailty.43 The exploitation of EGCG for the development of a potent antiaging agent has been suggested by Han et al.44 Advancement in the application EGCG has shown dose-dependent protection against UV-Binduced injury on hairless mouse skin.45 Chen et al. have reported the potential role of EGCG as an antiaging agent on an aging mouse model induced by Dgalactose.46 Kim et al. suggested the futuristic role of EGCG as a cosmetic component with impacts on moisture retention, wrinkle formation, and skin hydration along with a radical scavenging function and melanin degeneration.47

FIGURE 21.1 The schematic pathway involved in the protective role of EGCG in various pathological diseases.

II. Antioxidants and Pathology

References

Summary points • This chapter focuses on the protective role EGCG, a dietary antioxidant, against OS in various diseases. • The significant involvement of OS in various pathophysiological conditions including CVD, neurodegenerative disease, cancer, and aging, etc., has been indicated. • Two types of antioxidant (endo- and exogenous) systems have been discussed here that have been shown to compensate for the deleterious effects of various insults arising due to OS. • As discussed in this chapter, substantial evidence from preclinical and clinical studies has demonstrated that EGCG is found to show an effective role in the treatment of cardiovascular disorders such as MI, cardiac hypertrophy and atherosclerosis, neurodegenerative disorders like PD and AD, and metabolic syndromes like diabetes, cancer, and aging through the modulation of various cellular and molecular mechanisms (Fig. 21.1). • This chapter attempts to enhance the basic understanding of the molecular aspects of EGCG in various diseases.

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C H A P T E R

22 Extra virgin olive oil polyphenols: biological properties and antioxidant activity Annalisa Silenzi, Claudio Giovannini, Beatrice Scazzocchio, Rosaria Varı`, Massimo D’Archivio, Carmela Santangelo and Roberta Masella Gender-Specific Prevention and Health Unit, Centre for Gender-specific Medicine, Italian National Institute of Health, Rome, Italy

List of abbreviations 3,4-DHPEA-EDA 3,4-HPEA-EDA COX CVD EGFR EVOO HT or 3,4-DHPEA IMID MD MUFA NCDs Nrf1 OS PAF ROS Tfam Tyr or 3,4-HPEA

dialdehyde form of decarboxymethyl-elenolic acid bound to HT dialdehyde form of decarboxymethyl-elenolic acid bound to Tyr cyclooxygenase cardiovascular disease epidermal growth factor receptor extra virgin olive oil hydroxytyrosol immune-mediated inflammatory disease Mediterranean diet monounsaturated fatty acids chronic noncommunicable diseases nuclear respiratory factor 1 oxidative stress platelet-activating factor reactive oxygen species mitochondrial transcription factor A tyrosol

Introduction This chapter aims to describe the properties of extra virgin olive oil (EVOO) on human health, focusing on the biological and antioxidant activities of the polyphenols it contains. EVOO is universally recognized as a symbol of the Mediterranean diet (MD) and its effects on human health have been largely demonstrated by relevant intervention studies.1
3 EVOO has been shown to be one of the main resources against the development of

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00022-6

cancer, neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease, metabolic syndrome, and, generically, chronic noncommunicable diseases (NCDs).4 Although the characteristics of a healthy diet depend on many personal variabilities, availability, and eating habits, some basic “ingredients” have been identified to exert positive effects. Numerous studies have shown that a healthy diet should contain vegetables, fruit, legumes, wholegrains, and nuts, and less than 10% and 30% of the total intake of energy from sugars and total fat respectively; all features characterizing the MD, long accredited for its beneficial effects on health, which recommends a daily intake of 30
50 g of EVOO.5,6 The merit of the beneficial properties of EVOO is given by its peculiar composition. In addition to a high content of monounsaturated fatty acids (MUFAs), first of all oleic acid, it contains, in fact, a series of bioactive compounds with an enormous variability in the different types of olives and in the EVOO-based products available for human consumption.7 These compounds including polyphenols and vitamin E are well known to exert antioxidant, antiinflammatory, insulinsensitizing, cardioprotective, antiatherogenic, neuroprotective, immunomodulatory, and anticancer activities.8 The beneficial effects of the polyphenols in EVOO have been recognized by the American Food and Drug Administration (FDA)9 and by the European Food Safety Authority (EFSA). The latter recommends a daily consumption of about 20 g EVOO daily, which

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is two tablespoons, to prevent the onset of cardiovascular diseases (CVD) and inflammation, and to counteract oxidative stress (OS) caused by free radicals10; this quantity is in agreement with those of the MD. The beneficial effects observed in subjects consuming EVOO as their main seasoning fat appear to be associated, especially, with its polyphenol content. The quantity of ingested oil polyphenols is of the order of 4
9 mg/day. However, the benefit these compounds can bring to human health is the result of a synergy of several factors including concentration and composition, extent of absorption and metabolism, and bioavailability in target tissues.11,12 The bioavailability of EVOO phenolics has been determined in several studies by measuring the concentration of phenolic compounds and their metabolites in biological fluids, mainly plasma and urine, after ingestion of pure compounds or olive oil, either pure or enriched with phenolics.13 It must be noted that some polyphenols can undergo a rapid metabolism carried out by human and intestinal microflora enzymes, producing a number of metabolites that can be found in biological fluids instead of the ingested forms, and might represent biologically active species. Moreover, to fully understand what the beneficial effects of EVOO polyphenols on health may be, it is necessary to evaluate many other variables such as different gender responses when taking EVOO and the variation of intestinal microbiota, which besides varying considerably among individuals, can, in turn, be modified by polyphenols.14,15 Finally, considering the consumption of EVOO as a part of a diet made also of other food, the interactions that can be created between the polyphenols contained in EVOO and other dietary components cannot be underestimated.

Olive oil characteristic and extraction procedures that influence EVOO antioxidant properties More than 30 hydrophilic phenolic compounds have been identified in the oil derived from the fruit of the olive tree (Olea europaea L., family of Oleaceae), most of which are responsible for the organoleptic properties, flavors and bitter aromas, pungent sensation, and oxidative stability of the oil.16 However, the amount of these compounds depends on several factors such as the olive cultivars, the fruit ripening phase, some environmental factors (altitude, cultivation practices, and irrigation quantity), the extraction conditions (heating, water addition, extraction systems used to separate the oil from the olive paste), and the storage conditions due to spontaneous oxidation and the deposition of

suspended particles that can range from 50 to 800 mg/kg.7 The cultivar, that is, the variety of the olive, is the main factor in differentiating the quality of the oils obtained. Each cultivar has a specific organoleptic profile characterized by aromatic substances, number of polyphenols, and specific composition of sterols (two components of the so-called unsaponifiable fraction of the oil that accounts for 3% of the total and that plays an important role from the health and organoleptic points of view). Each cultivar also records differences in the composition of fatty acids (the so-called saponifiable fraction, which represents 97%
99% of the oil). Considering the content of polyphenols, an important variable is the production procedure of the oil. This process plays a fundamental role in the quality of the final product. Different factors, in fact, influence the phases of grinding and preparation of the mixture and the separation of solids and liquids.17 The olive oil mechanical extraction process begins with the crushing of the olives and the separation of the oil from the fruit pulp under high pressure. Olive oil can be extracted, postpressurized, and re-pressed with or without the use of hot water. This process leads to the production of an oil characterized by a greater intensity of color, a weaker aroma, and a higher content of free fatty acids and polyphenols.18 The oil obtained by chemical extraction, on the contrary, can be used for consumption only after refining, an important process that aims to purify the oil chemically extracted of any residual solvent and other impurities. Refined olive oil is free of vitamins, polyphenols, phytosterols, and other low molecular weight ingredients.18 Another interesting feature affecting virgin olive oil properties is filtration. Unfiltered olive oil preserves additional polyphenols of higher polarity that are typically lost with small amounts of water, which are removed upon filtration The oil classified as EVOO is derived only from mechanical extraction and, due to its chemical and sensorial parameters, has a lower yield and a higher cost than other oils, but it contains the highest level of polyphenols. However, due to multiple technological processes, the content of polyphenols may vary in EVOO19 (Fig. 22.1).

Extra virgin olive oil polyphenols Chemistry Polyphenols are organic compounds that are derived from plants and have aroused great interest in

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Extra virgin olive oil polyphenols

FIGURE 22.1 Olive oil extraction processes. TIn the absence of faults perceived by sensory analysis and with an acidity lower than 0.8%, the oil can be defined as extra virgin (EVOO). EVOO and virgin olive oil are obtained by mechanical extraction, while refined olive oil by chemical extraction. The extraction process influences the polyphenol content and, thus, the antioxidant properties. Olive oil is a mixture of reined and virgin olive oil.

nutrition in the past few decades. All classes of polyphenols are contained in different percentages in food of vegetable origin such as fruit, vegetables, tea, cocoa, and EVOO.20 The different polyphenols can be classified as flavonoids, phenolic acids, phenolic alcohols, stilbenes, and lignans, according to their chemical structure.12 The most abundantly occurring polyphenols in food are flavonoids and phenolic acids, which account for 60% and 30% of dietary polyphenols respectively. Among the most represented phenolic molecules in EVOO are the phenyletanoids such as oleocanthal, hydroxytyrosol (HT; 3,4-DHPEA), and tyrosol (Tyr; 3,4-HPEA) whose concentrations increase during oil storage because of the hydrolysis of secoiridoids, the other representative class of EVOO polyphenols. Secoiridoids include several compounds such as oleacein, oleuropein, the dialdehyde form of decarboxymethyl-elenolic acid bound to HT (3,4DHPEA-EDA) or to Tyr (3,4-HPEA-EDA), and isomers of oleuropein and ligstroside aglycons. Another class is that of phenolic acids divided in hydroxybenzoic acid derivatives (such as gallic and protocatechuic acids) and hydroxycinnamic acid derivatives (such as caffeic and coumaric acids). Flavonoids such as luteolin and apigenin are present in much lower levels than other phenols (see Table 22.1 for more details).18,21,22 It is worth noting that a remarkable number of polyphenols has been found in different olive-derived matrices (i.e., paste, pomace, aqueous extract) that contain flavonoids such as luteolin-7-glucoside and rutin. Olive leaves contain HT, luteolin-7-glucoside,

apigenin-7-glucoside, and a relevant amount of oleuropein (1%
14%); much more than olive oil (0.005%
0.12%).18

Antioxidant and antiinflammatory properties An increasing number of preclinical, clinical, and epidemiological studies indicate that the consumption of polyphenols can play a vital role in maintaining human health through the regulation of metabolism, weight control, chronic diseases, and cell proliferation.8,23 For example, some intervention studies on healthy human subjects have shown a beneficial effect of EVOO polyphenols on body weight control and a reduction of fat mass with an increase in muscle mass, suggesting a possible effect of the long-term intake of oil on the variation of body mass. In this study it was observed that the intake of polyphenolic compounds was able to improve the antioxidant status present in the subjects in about 30 days of treatment.24 It is important to note that polyphenols are able to intervene in cellular physiological processes such as proliferation and apoptosis, carrying out contrasting activities. Specifically, they can improve survival and protect cells from cytotoxicity by inhibiting apoptosis or, on the contrary, they are able to induce apoptosis and prevent/block tumor growth, depending on the concentration and the cellular system studied.25,26 The biological activity of polyphenols is strongly related to their antioxidant and antiinflammatory properties since they might be able to reduce the pool of reactive

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228 TABLE 22.1 Secoiridoidsa

22. Extra virgin olive oil polyphenols: biological properties and antioxidant activity

Main polyphenol classes contained in EVOO. Lignans

Phenylethanoids

Flavonoids

Hydroxy-isocromans

• 3,4-DHPEA- • (1)-1• Oleocanthal • Luteoin • 1-Phenyl-6,7EDA acetoxypinoreinol • HT (3,4• Apigenin dihydroxydihydroxypheny • p-HPEA-EDA • (1)-1-pinoresinol isochroman -ethanolor3,4• Oleacein • 1-(39-methoxy-49DHPEA) hydroxy)-phenyl• Oleuropein • 3,4-DHPEA• Tyr (p6,7- dihydroxyEA hydroxyphenyl isochroman ethanol or p• p-HPEA-EA HPEA)

Phenolic acids • Hydroxybenzoic acid derivatives (gallic acid, protocatechuic acid, phydroxybenzoic acid, vanillic acid, syringic acid) • Hydroxycinnaic acid derivatives (caffeic acid, p-and o-coumaric acid, ferulic acid, and cinnamic acid)

a

Main classes.

oxygen species (ROS), to neutralize potentially carcinogenic metabolites, and to counteract the inflammatory processes associated with the onset of several pathological conditions.27 For this reason, polyphenols have been considered as preventive and/or therapeutic agents against NCDs (such as CVD, type 2 diabetes, neurodegenerative disorders, and cancer as well as obesity, a main risk factor for NCDs),8,23 which are characterized by the onset of OS and increased free radicals production. In addition, there is a body of research showing the ability of polyphenols to modulate the human immune system by influencing the proliferation and activity of white blood cells as well as the production of cytokines or other factors that participate in immunological defense.28 It has been suggested that the EVOO polyphenolic compounds, once ingested and metabolized, could counteract local and systemic inflammatory environments such as in the immune-mediated inflammatory diseases (IMIDs).24,29,30 However, all the biological activities of polyphenols such as their ability to prevent the onset of various diseases are closely linked to the antioxidant action they can exert at the level of cells, tissues, and organs of the body. OS, in fact, represents a common factor in the pathogenesis of a number of diseases.

Antioxidant activities In oxidative reactions, oxygen is the last electron acceptor in an electron flow system that produces adenosine triphosphate (ATP). When this flow and energy production are disjointed, ROS are formed. ROS are normally produced within the body in limited quantities that are required to regulate physiological processes including the maintenance of cellular homeostasis and functions such as signal transduction, gene expression, and receptor activation.31 Nevertheless, when ROS are produced uncontrollably or are increased by extracellular agents, overcoming

FIGURE 22.2 EVOO polyphenol antioxidant activity. External agents such as ionizing and UV radiations and toxicants as well as products of aerobic metabolism can cause OS. EVOO polyphenols counteract the intracellular overproduction of ROS, thus, inhibiting the onset of OS and macromolecular damage.

the antioxidant capacity of the cell, intracellular redox homeostasis is altered and OS follows, inducing cell damages (membrane and macromolecular alterations).21,32
34 An increasing number of studies show how OS is associated with different diseases including cell transformation and cancer, atherosclerosis, CVD, and central nervous system disorders such as familial amyotrophic lateral sclerosis, adolescent convulsions related to glutathione peroxidase, Parkinson’s disease, and Alzheimer’s dementia as well as to a variety of age-related disorders.35,36 As antioxidant compounds, polyphenols can act as chain breakers, radical scavengers, and metal chelators depending on their chemical structures, which also influence their antioxidant power12,37 (Fig. 22.2). However, a main antioxidant mechanism of polyphenols resides in their ability to improve the human endogenous antioxidant defense system by influencing the pathways that regulate antioxidant/electrophile response element

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Extra virgin olive oil polyphenols

229 FIGURE 22.3 EVOO polyphenols induce phase II gene expressions through ARE activation. EVOO polyphenols cause the strengthening of intracellular antioxidant defenses by activating antioxidant enzyme genes. The molecular mechanism responsible is supported by a number of studies. Polyphenols (1) modify the capability of the inhibitor Keap1 in sequestering the transcription factor Nrf2; and/or (2) activate MAPK proteins (ERK, JNK, and p38) probably involved in Nrf2 stabilization. Thus Nrf2 is allowed to translocate in the nucleus where it transactivates the ARE/EpRE-containing promoter of phase II genes.

FIGURE 22.4

Polyphenol mechanisms of action. As antioxidant compounds, polyphenols can act as chain breakers, radical scavengers, and metal chelators. Moreover, it has been shown that they can enhance cellular antioxidant defenses by modulating gene expression, signal transduction, and enzyme activities. They can interact with receptors, interfering with cell proliferation and cell death.

(ARE/EpRE) activation, and, consequently, inducing the expression and transcription of phase II detoxifying and antioxidant enzyme genes (Fig. 22.3).38 Emerging results suggest that polyphenols, irrespective of their conventional antioxidant activities, can exert potentially more broad-spectrum beneficial effects, taking part in regulatory molecular mechanisms such as modulating directly the activities of receptors or enzymes involved in signal transduction pathways, which in the end influence the fate and function of cells39 (Fig. 22.4).

Antioxidant activities of the main extra virgin olive oil polyphenols The beneficial activity of EVOO polyphenols is systemic and influences different parts of the organism; in fact, the preventive and/or therapeutic effects against different pathologies have been studied. However, the mechanism of action mainly refers to their antioxidant activity. The main bioactive compounds of EVOO, namely HT, Tyr, and oleuropein have demonstrated their protective power in various diseases including CVD22,40 and metabolic diseases3 (Fig. 22.5).

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FIGURE 22.5 Hydroxytyrosol, tyrosol, oleuropein, and oleocanthal. Chemical structures of the main EVOO polyphenols, namely tyrosol, hydroxytyrosol, oleuropein, and oleocanthal.

Hydroxytyrosol The free radical scavenging properties of HT have been convincingly confirmed in some rat studies, demonstrating a beneficial effect in the prevention of diabetes mellitus and during antidiabetic therapy.41 Studies carried out in the 3T3-L1 adipocyte cell line have shown that HT is able to stimulate mitochondrial biosynthesis, which often decreases in diabetes mellitus. The most reliable hypothesis is that HT modulates this biosynthesis through the upregulation of PGC-1α. Relatively low concentrations of HT in adipocytes increase the expression of all mitochondrial respiratory chain complexes including ATP synthase. HT, therefore, protects mitochondria from the reduction of mitochondrial DNA synthesis and modulates the activity of critical transcription factors such as Nrf1 (nuclear respiratory factor 1) and Tfam (mitochondrial transcription factor A).42 Other important effects of HT have been found in colon cancer. In some studies, in fact, it has been found that HT is able to reduce the level of epidermal growth factor receptor (EGFR) by promoting its degradation. EGRF is one of the key receptors that activate colon carcinogenesis as it regulates proliferation, apoptosis, angiogenesis, and tumor cell invasion.43 Furthermore, HT was found to be an effective cytotoxic agent in breast cancer cell models, inhibiting the cell cycle in the G0/G1 phase by decreasing the level of cyclin D1. Oleuropein Oleuropein appears to play an important role in CVD as it seems to reduce the number of blood vessels showing antiangiogenic properties.4 Other important effects attributed to it are the inhibition of low density

lipoproteins (LDL) oxidation mediated by macrophages34 and the protection of β-cells producing insulin (INS-1) against the deleterious effect of cytokines.44 Also, the protective and therapeutic effects cannot be underestimated under neoplastic conditions. There are numerous studies that confirm the antitumor activity of oleuropein observed in human tumor cell lines such as mammary adenocarcinoma,45 melanoma, carcinoma of the urinary bladder, colorectal adenocarcinoma,46,47 prostate carcinoma,45 lung carcinoma,48 renal cell adenocarcinoma,45 and glioma.49 One of the protective mechanisms studied in HT-29 cells seems to be involved in the activation of intrinsic apoptotic pathway through peroxisome proliferator-activated receptor gamma (PPARγ) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFκB) signaling pathways coming up to cyclooxygenase-2 (COX-2) downregulation and modulating p53 suppressor protein levels.46 Tyrosol The biological effects of Tyr and its derivatives include an antioxidant and protective action on circulating low-density lipoproteins, which are easy targets of free radicals attack.33,50 In fact, it has been demonstrated that Tyr is able to counteract the oxidative modifications of LDL incubated with macrophage cell J774 A.1, although at concentrations much higher than for hydroxytyrosol. Tyr has been able to significantly reduce the increase in thiobarbituric acid reactive substances (TBAR) and relative electrophoretic mobility (REM) compared to untreated cells, although to a lesser extent than HT, which has a greater capacity for scavenging.33 Studies in humans show that the oxidation of LDL is inversely proportional to the dose of ingested Tyr. Tyr is able to bind human LDL lipoproteins and to exert its protective activity probably through the elimination of peroxyl in the intima arterial, where the oxidation of LDL mainly occurs. It was observed that plasma incubation with extracts of virgin olive oil led to an increase in phenolic compounds previously linked to LDL. The increase in the total phenolic content of LDL in a dose-dependent way with the phenolic content of olive oil administered was also shown in postprandial studies.51 Antiplatelet activity was also highlighted due to the modulation of cAMP phosphodiesterase and platelet-activating factor (PAF). From the neuroprotective point of view, Tyr has shown a protective effect in the cerebral ischemia of rats through the reduction of brain infarct volume and neurological dysfunctions that follow transient middle cerebral artery occlusion (MCAo).52

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Oleocanthal Oleocanthal, the compound responsible for the pungent and spicy taste of EVOO, has been studied for its antiinflammatory and antineoplastic effects. The results of these studies show that, in fact, this compound is able to inhibit cyclooxygenase (COX)-1 and -2 in a dose-dependent manner, imitating the antiinflammatory action of synthetic non-steroidal antiinflammatory drugs (NSAID) ibuprofen. Furthermore, it was able to significantly inhibit COX-1 and COX-2 enzymes at equimolar level concentrations. For example, oleocanthal (25 μM) inhibits 41%
57% of the COX activity in comparison to ibuprofen (25 μM), which inhibits 13%
18% of the COX activity.53 Due to these properties, oleocanthal is becoming a compound of interest in cancer research. In fact, the inflammatory enzymes it attenuates, COX-1 and COX-2, are responsible for the conversion of arachidonic acid into prostaglandins and tromboxanes, which are produced in response to inflammatory or toxic stimuli and implicated in the pathogenesis of various tumors, both in humans and animals.54,55 Hydroxytyrosol and tyrosol glucuronides and sulfates In recent years, researchers have also focused on sulfate and glucuronide metabolites during the evaluation of in vitro and in vivo experiments. Peyrol et al.56 showed that HT glucuronide exerted the same antioxidant activity of HT in endothelial cells; however, this was due to the intracellular β-glucuronidase action that favored deconjugation, leading to the formation of free HT. Moreover, Atzeri et al.57 observed that HT and Tyr sulfate entered intestinal Caco-2 cells after 30 minutes of incubation and underwent an extensive metabolization, giving rise to a pool of metabolites, mainly sulfate and methyl-sulfate HT and Tyr. Thus, it is conceivable that though being deconjugated before entering the cells, HT and Tyr metabolites can be reformed inside the cell environment so acting as conjugated forms together with HT and Tyr free forms.

Applications in other areas of pathology Evidences that the appropriate intake of foods containing important bioactive compounds are always greater and are continually confirmed1 and EVOO is given a key role in health and for a better quality of life, thanks to its polyphenol content. The mechanism of action of EVOO polyphenols is pleiotropic; however, it mainly relates to their antioxidant activity. The polyphenols present in EVOO reduce the level of ROS by protecting biomolecules from oxidative damage

and by inhibiting a number of intracellular signaling pathways associated with the activation of inflammatory, proliferating, and cytotoxicity processes. They are also found to modulate the human immune system, influencing the proliferation of white blood cells and the production of cytokines. The main compounds present in EVOO, which is a cornerstone of the MD, are HT, Tyr, oleuropein, and oleocanthal, which are powerful antioxidants that have widely shown antitumor, antiangiogenic, and antiinflammatory properties. On this basis, in November, 2018, the USFDA announced the possibility of including the “qualified health claim” on EVOO bottle labels.58

Summary points • EVOO is universally recognized as a symbol of the MD. • EVOO, due to its antioxidant properties, has been shown to be one of the main resources against several diseases characterized by OS. • The merit of EVOO beneficial properties is given by its peculiar composition, first of all the content of antioxidant polyphenols. • The biological activity of EVOO polyphenols is strongly related to their antioxidant and antiinflammatory properties. • The main compounds present in EVOO are HT, Tyr, oleuropein, and oleocanthal. • EVOO polyphenols show high free radical scavenging properties. • Polyphenols are able to counteract the oxidative modifications of LDL. • EVOO is increasingly considered an important component of a healthy diet.

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C H A P T E R

23 Ginkgo biloba extract as an antioxidant in nerve regeneration Nahide Ekici-Gu¨nay Department of Clinical Biochemistry, University of Health Sciences, Kayseri City Training and Research Hospital, Kayseri, Turkey

in the central and peripheral nerve systems.1,2 This chapter is focused on OS mechanisms in the etiopathogenesis of nerve injuries and neurodegenerative disease. In addition, the neuroprotective and neuroregenerative effects of GB, an antioxidant known to have antiinflammatory, antiviral, and anticarcinogenic activities, are discussed.

List of abbreviations AD ALS CNS GB HD MS OS PD ROS

Alzheimer’s disease Amyotrophic lateral sclerosis central nervous system Ginkgo biloba Huntington’s disease multiple sclerosis oxidative stress Parkinson disease reactive oxygen species

Peripheral nerve injury Introduction It is known that dietary foods have an important role in brain physiology and functionality as well as that neurotrophic activity is stimulated by dietary foods. Nerve injuries and neurodegenerative processes lead to the recruitment of neuroregeneration mechanisms by activating reflex defense mechanisms in order to maintain neuronal functionality. In neurodegenerative and neuroregenerative processes, treatment outcomes are closely related to the management of disrupted redox signaling and oxidative stress (OS). Chronic exposure to OS and concomitant neuroinflammation are accepted as primary causes of the neurodegenerative process. In peripheral nerve injury, the restoration of oxidative balance is the determining factor in the recovery of functional synaptic transmission during healing processes. Although several studies have been conducted to investigate regenerative responses to Ginkgo biloba (GB), known to be a potent polyvalent free radical scavenger and antioxidant, in different tissues, the majority of these studies on experimental models are associated with regeneration Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00023-8

The nerves consist of small cells, namely neurons, which help motor movements and senses by connecting the brain and spinal cord to other body regions. Data arising from the brain and spinal cord are transmitted to other body regions as signals and vice versa. Central nervous system (CNS) disorders, comprising a wide spectrum, lead to the loss of sensorial, motor, and cognitive functions as exemplified in Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS), and peripheral traumatic nerve injuries as well as retinal disorders and visual defects associated with age-related macular degeneration. Peripheral nerve injury can hinder the functioning of relevant muscle by disrupting signaling from and to the brain and sensorial loss in the area innervated by the nerve. Nerve injury is associated with some consequences in the neuronal cell body, dorsal root ganglia, and distal segment neuronal elements, leading to apoptotic cell death.3 Programmed neuronal death is triggered after injury or impairment in neuronal growth factors, hormones, or extracellular matrix factors followed by the formation of reactive oxygen or reactive nitrate species (ROS or RNS), mitochondrial damage,

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activation of the Bcl protein family, and the release of apoptosis-inducing factors to the cytosol.4,5 The inflammatory response accompanying neuronal injury includes controlled effector molecules with counter effects and multiple cell types.6 It has been proposed that the brain cells show greater predisposition to oxidative injury due to the relative scarcity of antioxidant systems when compared to other organ systems.

Neurodegeneration and neuroregeneration Several mechanisms can contribute to CNS damage including nerve degeneration, injuries resulting in apoptotic or necrotic neuronal cell death, excitotoxic factors, inflammatory-demyelinating disorders, and oxidative damage caused by hypoxia among others.7 Regenerative processes are rather comprehensive in neurodegenerative disorders, which are associated with the progressive loss of neuronal cells and, thus, functional loss in the nervous system. Neuroregeneration is defined as the repair or renewal of nerve tissues, cells, or cell products. Neuroregeneration involves the formation of novel neurons, glial cells, axons, myelin, or synapses. The term neuroprotection denotes the recovery of neurons and nonneuronal cells (i.e., glial cells and endothelial cells) in order to maintain normal function. Peptides released from swelled nerve endings at proximal to an axonal transection lead to local changes

that affect microcirculation in damaged environments. The molecular changes seen in the distal segment include neutrophil recruitment, release of neural cell adhesion molecules, cytokines, and other soluble factors, and the upregulation of their receptors. These changes result in a cascade of events, known as Wallerian degeneration, in the distal segment of a neuron. Neuronal regeneration following Wallerian degeneration is highly affected by cytokines released from immune cells. Following axonal cut and loss of neurotrophic factors, neurons are subjected to OS by the effects of these cytokines, while antioxidant protective factors are involved at this point (Fig. 23.1). The CNS including the brain, spinal cord, and retina has spontaneous regeneration capacity albeit limited. Since nerve remyelination was first shown in humans8 there has been an increase in the number of studies on therapeutic agents that support spontaneous regeneration. There is a critical need for pharmacological therapies and regenerative strategies that delay the progression of CNS disorders and support tissue regeneration. It seems important to control inflammatory reactions, which are caused by both injury and biomaterials used for regeneration for success of neuronal treatment. After neuronal injury, signals generated promote alterations in gene expression, ensuring a regenerative state for neurons.9

FIGURE 23.1 Schematic representation of the role of oxidative nitrosative stress in peripheral nerve regeneration. Following axonal cut and loss of neurotrophic factors, neurons are subjected to oxidative stress by the effects of these cytokines and antioxidant protective factors are involved.

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Intracellular signaling and angiogenesis in neuronal regeneration

Depending on the severity of the neuronal injury, the activation of OS by ROS or RNS can markedly affect the neuronal death mechanism and degree.10 Inflammation is an important event in nerve degeneration and regeneration. Timing and degree of inflammation can improve or hamper the regeneration process. As in central neuronal diseases such as motor neuron disorders, progressive dementia, and cognitive disorders, the control of inflammatory reactions in peripheral neurons caused by trauma and regeneration plays an important role as it determines treatment success. It is reasonable to think that the neurodegenerative mechanisms driving neuroinflammatory processes can be blocked by various synthetic agents, thus, delaying the progression of disability. There are some challenges in the development of synthetic compounds in the treatment of neurodegenerative diseases or neuroprotection. These compounds have failed to meet expectations and often show a number of adverse effects.11 Given the multiple mechanisms involved and partial benefit from the modulation of a single specific pathway, natural supplement therapies involving multiple functions have become increasingly important in neuroregenerative processes. The nervous system is classified into two components including the CNS consisting of the brain, spinal cord, and related ganglia and the peripheral nervous system consisting of the cranial and spinal nerves. The peripheral nervous system has a greater ability for autonomous regeneration when compared to the CNS. Myelin residues created by damaged axon is rapidly removed by macrophages in the peripheral nervous system, however, macrophages are lacking in the CNS. In CNS injury, astrocytes are recruited to the damaged environment causing scar tissue formation, which is considered as a barrier for regeneration. There are inhibitory proteins (such as Nogo, MAG, or OMgp) at the myelin sheath that prevent neuronal sprouting, but such inhibitory proteins are lacking in the peripheral nervous system.12 Due to selective neuronal vulnerability, neurons residing in different brain regions show varying sensitivities to OS, which is also true for the peripheral nervous system. Several intrinsically different types are present for different neurons, even in the brain structures that are seemingly homogenous. The damage caused by OS can be determined by this variation in neurons.13

Intracellular signaling and angiogenesis in neuronal regeneration The mechanisms involved in redox signaling required to convert cellular signals to chemical

237

modulation are highly complex. This complexity causes cell death and the development of certain diseases by contributing risk to undesirable results. Caspase/calpain activity, phosphoinositide 3-kinase (PI3K)/AKT pathway, and mitogen-activated protein kinase (MAPK) signaling are intracellular, calciummediated regulatory pathways required for neuronal regeneration. Among these, PI3K/AKT signaling is the main neuronal prosurvival mechanism that plays an important role in axonal regeneration. Therapeutic approaches modulating these pathways either in positive or negative manners can contribute to regenerative processes. The mammalian target of rapamycin (mTOR) signaling network, a serine/threonine kinase in mammalians, is one of the mechanisms used to regulate cell behavior during transition between catabolic and anabolic states. The mTOR activation occurs along with altered PI3K/AKT signaling and insulin resistance in the brain in Alzheimer’s disease which is a neurodegenerative condition and known to be associated with change in redox signaling.14 It was shown that neurodegeneration can be reduced by the inhibition of mTOR activity in AD, PD, cerebral stroke, and Huntington’s disease (HD).15,16 Also, it has been proposed that corticospinal and peripheral neuronal regeneration could be regulated via the mTOR pathway.17 In neural tissues, the transcription factor Nrf2 (nuclear factor erythroid 2-related factor) is activated when the redox state is shifted toward oxidation; then, the activated Nrf2 leads to the upregulation of phase 2 antioxidant enzymes by binding to the nucleus. The Nrf2/antioxidant response element (ARE) signaling pathway is the primary mechanism in the defense against OS. The deletion of Nrf2 gene expression results in an elevation in OS and relatively lesser axonal regeneration.18 Thus enhancing nuclear Nrf2 expression and, therefore, OS inhibition, has been proposed as an important approach for preventing oxidative neuronal damage and supporting the repair process after peripheral nerve injury. Several enzymatic and nonenzymatic antioxidants with effects on signaling pathways serve to remove excessive ROS in cells and tissues and to protect against oxidative injury. Exogenous and endogenous antioxidants contributing to the removal of free radicals have been used to promote neuronal regeneration. Neovascularization is also important for regeneration in neural tissues. The formation of de novo microvessels within the body of peripheral nerves affects the success of axonal sprouting and regeneration.19 Appropriate angiogenesis during neuronal regeneration prevents graft rejection via the formation of a fibrous capsule surrounding the biomaterial used.20

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Undoubtedly, tissue OS and the removal of local inflammation will have positive effects on angiogenesis and neuronal regeneration.

Oxidative stress in neurodegenerative processes Chemical reduction and oxidation balance (redox) are essential to maintain normal physiology of cells. Several physiological and pathological responses are regulated in distinct cell tissues and biological systems through this balance.21 Both the central and peripheral nervous systems are highly sensitive to alterations in the redox state.22 The nervous system, particularly the brain, lacks effective mechanisms to remove the pro-oxidative molecules that get accumulated. Given that the brain consumes 20% of the oxygen used in the whole body, although it comprises 2% of the total body mass, the risk of oxidation caused by O2 metabolites is inevitable due to generation at excessive amounts in a restricted area.23 Basal ganglions are rich in iron.24 Fe12, the redoxactive form of iron, catalyzes the transformation of all ROS to their most reactive form, namely hydroxyl alkyl, via the Fenton and Haber
Weiss reactions of hydrogen peroxide.25 All these factors make the nervous system, particularly the brain, a vulnerable target for oxidative species. Brain aging and loss of mitochondrial function are considered to be other underlying mechanisms in neurodegenerative diseases. Neuronal oxidative/nitrative damage is associated with a reduction in the energy transmission systems due to a loss in mitochondrial function.26 Oxidative stress is known as one of the primary causes of neural damage following injury.27 It was shown that neural damage induces ROS and nitric oxide production in axotomized neurons. It is thought that OS plays an adverse role in the functional recovery of neurons caused by peripheral injury similarly to neurodegenerative disorders.28 As in the case of the CNS, both the ischemic and inflammatory processes are induced when an injury occurs in peripheral nerves.29 It was shown that ROS are generated in axotomized neurons in the case of injury.30 The inhibition of OS formation expedites the repair process and physiological recovery following nerve injury.31 The brain has the second-most extensive concentration of lipids.32 In both the central and peripheral nervous systems, 70%
85% of myelin dry mass consists of lipids, which is an electrical isolator and axonal transmission facilitator.33 Polyunsaturated fat acids (linoleic acid and arachidonic acid) are major targets of oxidation, which are extensively present in neuronal membranes.34Together with protein and lipid

modifications, lipid peroxidation products are associated with the progression of neurodegeneration either directly or indirectly. Lipid peroxidation is also involved in the neuroinflammatory condition where microglial activation accompanied by abnormal dopamine metabolism is triggered.35 Inflammation and OS have close interactions.36 The activation of neuroinflammatory cells exacerbates neurodegenerative processes. The continuous stress during the neuroinflammatory state suppresses cellular defenses and induces neurotoxicity emerging at the onset or during the progression of brain injury.37 Phospholipase-A2 hydrolyzes membrane phospholipids to arachidonic acid and lysophospholipids. The arachidonic acid is metabolized into eicosanoids (prostaglandins, leukotrienes, thromboxanes) while the lysophospholipids are converted to platelet-activating lipid mediator forms. These lipid mediators have a critical role in the induction, continuation, and regulation of neuroinflammation and OS.

Ginkgo biloba GB or maidenhair tree, also termed ginkgo or gingko in texts, is known as a “living fossil” as it is the only existing member of the Ginkgoaceae family. The name refers to “Gingko,” which is derived from the name Yin-Kuo meaning “silver apricot” in Chinese and “biloba” indicating its fan-shaped leaves. Its use as an herbal medicine dates back to the late 15th century in China. The extract of GB leaves is widely used as an herbal medicine and nutritional supplement in both Europe and the United States. GB extract contains approximately 300 chemical substances, however, the exact role is unclear for each individual component. It has antiinflammatory, antiviral, and anticarcinogenic activities in addition to antioxidant properties. It has been proposed that GB extract has several beneficial effects on CNS function. It was shown that flavonoids and terpenoids are the most active components found in EGb761 extract. The standardized formulation contains 24% flavonoid glycosides (primarily hydrolyzed to flavone aglycones such as quercetin, kaempferol, and isorhamnetin) and 6% terpene lactones (2.8%
3.4% ginkgolides A, B, and C, and 2.6%
3.2% bilobalide)38 (Fig. 23.2). The compounds in the extract (flavonoid, bilobalide, ginkgolides) exert pharmacological effects by crossing the blood
brain barrier39 (Fig. 23.3). The flavonoids are herbal polyphenolic compounds with low molecular weight that comprise a large family, exerting antioxidant effect by neutralizing peroxyl, perhydroxyl, and

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Ginkgo biloba

FIGURE 23.2 The chemical structures of major components of Ginkgo biloba extract (EGb761). (A) Flavonoids: Kaempherol: R1~H, R2~OH; Quercetin: R1, R2~OH; Isorhamnetin: R1~OCH3, R2~OH. (B) Bilobalide. (C) Gingkolides: A: R1, R2, R3~OH; B: R1~OH, R2, R3~H; C: R1, R2~OH, R3~H.

FIGURE 23.3 An overview of the pharmacological effects of GB components. The general effects of Ginkgo biloba components are summarized.

hydroxyl radicals. Ginkgolic acid, a toxic phenolic compound in EGb761, has a plasma half-life of 4
5 hours and is implied in allergic and immunotoxic effects; thus, it is recommended that ginkgolic acid levels should not exceed 5 μg/g in the preparation by the German Federal Institute for Drugs and Medical Devices Commission E. In humans, it is proposed that standard GB extracts used at doses of # 240 mg/day are safe and have no interactions with nonphytomedications clinically.40 However, the doses used in in vivo and in vitro experimental studies of GB are markedly higher than the maximum doses proposed for humans. Future studies should focus on how dose tolerability can be supported to a dose level in human models by contribution of GB and its compounds to neuroregenerative effects. In this chapter, the effects of GB extract are evaluated on the recovery of cerebral failure, AD, multiinfarct dementia, peripheral arterial occlusive disease, tinnitus, vestibular disorders, myocardial ischemia, traumatic brain injury, macular degeneration, autism, and neurotoxicity caused by hypertension and chemotherapy through OS mechanisms and the neuroprotective/neuroregenerative effects of GB extract are also addressed (Fig. 23.4).

Neuroprotective and nerve regenerative mechanisms of action of Ginkgo biloba GB extract has a potent antioxidant pharmacological effect in the protection of vascular endothelia cells from OS injury by the removal of free radicals. In addition to antioxidant features, direct activation of the nervous system via neurons, increased cerebral and peripheral blood flow through blood flow modulation, decreased vascular permeability, and decreased platelet aggregation through blockade of biochemical reactions in platelets enhance neuroprotective effect of Ginkgo Biloba. Its antiinflammatory effect is associated to cyclooxygenase (COX) and lipoxygenase inhibition by flavonoids in the GB. Ginkgetin (a biflavon from GB leaves) inhibits phospholipase-A2, which hydrolyzes membrane glycerophospholipids and COX2-dependent prostaglandin D phase to release arachidonic acid (a precursor for eicosanoids), prostaglandins, and leukotrienes. In addition, ginkgolide A and B (GB flavonoids) inhibit proinflammatory cytokines such as TNF-α and interleukin-1.41 The platelet-activating factor (PAF) stimulates leukotriene synthesis involved in the pathogenesis of

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FIGURE 23.4 Oxidative stress-related neurodegenerative diseases that can be managed with GB extract. Neuronal cells are particularly sensitive to oxidative stress elements due to their cellular composition. In neurodegenerative diseases, oxidative stress plays a decisive role in the vicious cycle of the process, both as a cause and as a result of the disease. Therefore GB extract alleviating or preventing oxidative damage may be a potential therapeutic nutraceutical for neurodegenerative diseases.

inflammatory processes. Ginkgolide B (BN-52021), a diterpene in EG761, is a potent PAF receptor antagonist that is considered to have neuroprotective effects in the CNS. In experimental models, pretreatment with BN-52021 markedly decreased eicosanoid, thromboxane B, and leukotriene levels in cerebrospinal fluid (CSF).42 It was shown that GB activates the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is known to be a major molecular mechanism in protection against OS and deletion of its gene expression inhibits neuronal regeneration by stimulating phase II genes through Kelch-like ECH-associated protein-1 (Keap1)43 (Fig. 23.5).

Ginkgo biloba in experimental neuronal models and diseases The major neurodegenerative disorders in which oxidative/nitrosative stress plays a role include HD, AD, PD, MS, glaucoma, seizures and epilepsy, diabetes mellitus and peripheral diabetic neuropathy, lysosomal storage diseases, excitotoxicity, traumatic nerve and brain injury, cerebral experimental allergic encephalitis, depression, bipolar disorders, schizophrenia, autism, and traumatic brain injury.44 The native immune response depends on the synthesis of ROS at physiological levels by NADPH

oxidases (NOXs). In chronic granulomatous disorder associated with point mutations in NOX proteins, cognitive impairment, and decreased IQ accompanied by impaired immune responses were attributed to specific neuronal changes resulting from NOX activity.45 Taken together, the mentioned data indicate that the basal synthesis of physiological reactive species is essential for normal cellular functioning including the regulation of neurotransmission, and that shifts in redox balance lead to neurological diseases due to OS. Cerebral failure Tissue ATP levels were measured in brain tissues by inducing temporary middle cerebral artery occlusion in rats having received or not received bilobalide, the active substance of GB. It was shown that bilobalide prevented ATP loss.46 The bilobalide provided protection against the interruption of oxidative phosphorylation by enhancing the expression of the COXIII subunit of cytochrome oxidase, which harbors the mitochondrial DNA code. This increased the respiratory control rate of mitochondria, resulting in elevated APT levels. The therapeutic effect of GB on normal aging, degenerative dementia, and age-related impairment in other cognitive functions has been attributed to increased neuronal ATP.39 In an experimental rat study, it was shown that pretreatment with EGb761 for a week prevented neuronal death caused

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FIGURE 23.5 Molecular effects mechanism of GB extract (EGb761) in peripheral nerve injury and neurodegenerative diseases. It was shown that GB activated the Nrf2 signaling pathway, which is known to be a major molecular mechanism in protection against oxidative stress and deletion of its gene expression inhibits neuronal regeneration by stimulating phase II genes through Keap1.

by 8-minute cerebral global ischemia through antioxidant and antiinflammatory mechanisms.47 Axonal restoration Jang et al. showed that intraperitoneal injection of GB extract promoted nerve regeneration in an experimental model of facial nerve crush.48 It was shown that doses .100 mg/kg/day promoted electrophysiological and functional nerve regeneration in rats that underwent sciatic nerve transection and repair.49 In a similar study, it was shown that biomaterials filled with gelatin-containing bilobalide for nerve defect bridging in rats supported axonal regeneration at doses of only 10
50 mg/mL due to the antioxidant property of GB.50 Alzheimer’s disease AD is a progressive neurodegenerative disorder in which disease etiology and progression are closely related to OS. In AD, there are defects in electron transport due to a severe reduction in mitochondrial enzyme activity. Extraneuronal senile beta-amyloid (Aß) plaques and intraneuronal tau protein (τ) lead to an excessive deposition of neurofibrillary tangles and apoptosis. In fact, it is proposed that the plaques and tangles in AD are formed as a result of OS and even

they serve as a primary antioxidant defense line rather than being promoters of pathogenesis. GB provided greater protection against free radicals in mutation-induced models of AD when compared to those in wild-type nonstressed physiological conditions.51 In a rat model of AD, it was shown that GB and its components, bilobalide and quercetin, markedly increased cell proliferation in hippocampal neurons in a dose-dependent manner. In addition, bilobalide and quercetin elevated neurotrophic factor levels by increasing the phosphorylation of cyclicAMP response element binding protein (CREB). In the same study, the restoration of synaptic losses and important dendritic processes were shown by immunofluorescence staining52 (Fig. 23.6). In type II diabetes mellitus, it was shown that redox balance was impaired, and that the resultant OS was associated to comorbidity and neurodegenerative disease, and that it was predisposed to the development of AD.53 Bacterial neurotoxins (such as lipopolysaccharides; LPS) were found in brain tissues involved in AD. It was shown that EGb761 reduced neuroinflammatory activity through the COX/PEG2 pathway when primary microglial cell activation was provided by LPS.54

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FIGURE 23.6

Antineuroinflammatory effects of GB extract (EGb761) in neurodegenerative diseases. GB extract (EGb761) has shown antineuroinflammatory activity in LPS-activated primary microglia cells. Also, EGb761 blocks microglial activation in terms of attenuation of both mPGES-1 protein expression and concomitant PGE2 production. EGb761 is able to inhibition neuroinflammatory activation by targeting the COX/PGE2 pathway. This suggests that GB may affect the mTOR pathway in PD, cerebral stroke, and HD, and especially in AD.

Parkinson’s disease PD is a neurodegenerative disease leading to movement disorders due to the progressive loss of dopaminergic neurons and dopamine deprivation in basal ganglions. In PD, nigral neuronal death emerges by genetic predisposition and environmental toxins. Oxidative stress is one of the mechanisms underlying nigral dopaminergic cell death. Increased OS leads to the overactivation of the ubiquitin-proteasome system, which, in turn, causes the accumulation of damaged and misfolded proteins. In PD, the use of synthetic antioxidants is not encouraged due to suspected carcinogen promoter activities. It was shown that GB, by its antioxidant effect, provided neuroprotection/neurorecovery against induced damage in mid-brain dopaminergic neurons in animal models of PD. GB is suggested as an alternative in the treatment of PD in the future.55

Huntington’s disease HD is an autosomal dominant, neurodegenerative disorder characterized by progressive loss of motor dysfunction, unusual sequence repeats in the Huntington gene, and the onset of distinct neuropsychiatric symptoms. In HD, the misfolding of polyQ proteins due to defective genetic encoding leads to the formation of protein aggregates that cannot be sufficiently removed. Although its molecular mechanisms haven’t been fully elucidated, mitochondrial dysfunction and defects in bio-energy contribute to the disease course. In HD, some antioxidant enzymes (superoxide dismutase and glutathione reductase) were elevated.56 It was shown that GB regulated the accumulation of polyQ proteins by regulating proteasome impairment, and it was recommended as a potential therapeutic.57

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References

Summary points

Amyotrophic lateral sclerosis No weakness or motor neuron loss occurred in rat models of motor neuron degeneration where superoxide dismutase-1 (SOD-1) gene, an enzyme of the antioxidant system, was completely abolished. It was shown that mutant SOD-1 protein caused toxicity in healthy motor neurons. Animal models of amyotrophic lateral sclerosis (ALS) were developed based on these studies.58 It was shown that oral EGb761 use prolonged survival via a gender-specific neuroprotective effect in mutant transgenic rat models of ALS (G93A), and that GB extract could be an effective treatment in patients with ALS.59 Pheochromocytoma It was shown that quercetin-aglycone, a flavonoid in GB, protects neurons against lipid hydroperoxide attack in pheochromocytoma PC-12 cells.60 It was shown that pretreatment with ginkgolide K ameliorated OS induced by H2O2 through the recovery of mitochondrial membrane potential and inhibition of caspase-3 activity, thus, preserving pheochromocytoma PC-12 cells in rats.61 Glaucoma In glaucoma, glial cells in the retina and lamina cribrosa (Mueller cells, astrocytes, and microglia) lead to the formation of neurotoxic substances such as nitric oxide and TNF-α due to increased intraocular pressure. This causes a secondary OS surge and neuronal injury. There are studies on visual field defects and contrast sensitivity in human models of normaltension glaucoma in which GB was used via oral route, revealing contradictory results. In rat models of optic nerve injury, intraperitoneal use of GB provided a higher survival rate in retinal ganglion cells when compared to the oral route. It was shown that oxidative damage, considered as a potential mechanism in the progression of age-related macular degeneration, is terminated by oral GB use.62 Multiple sclerosis Oxidative stress plays a major role in the pathogenesis of MS. ROS are implicated in demyelization and axonal damage in both human and animal models of MS. There are several studies on the use of GB in MS, revealing contradictory results. It was shown that GB at a dose of 240 mg/day (p.o.) had mild benefits on depression, anxiety, fatigue, and quantitative assessments of symptom severity and functional performance in MS patients.63 However, in another study, GB (120 mg) failed to improve cognitive performance in MS patients.64

• This chapter focuses on the effects of GB, an antioxidant, on neuroregenerative processes. • Oxidative stress (OS) is inevitable in neuronal injuries and neurodegenerative disorders. • OS drives neuronal changes related to aging and the pathologies of neurodegenerative diseases. • The management of OS is the most important factor in determining the success of neuronal regeneration. • GB favors nerve regeneration by its antioxidant properties. • GB causes the upregulation of Nrf2 activation, a transcription factor for phase II antioxidant enzymes in antioxidant signaling pathways, in addition to its antioxidant capacity, making it a potent fighter against OS. • It will be important to support surgery and medical therapies for nerve regeneration by readily available, noninvasive dietary factors without adverse effects. • Individual evaluation of GB components in neuroregenerative processes will improve its therapeutic effectiveness. • The fact that doses used in experimental models are higher than maximum doses recommended in humans indicates that future studies should focus on how dose tolerability can be supported in human models.

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59. Ferrante RJ, Klein AM, Dedeoglu A, Beal MF. Therapeutic efficacy of EGb761 (Ginkgo biloba extract) in a transgenic mouse model of amyotrophic lateral sclerosis. J Mol Neurosci 2001;17:89
96. 60. Shirai M, Kawai Y, Yamanishi R, Kinoshita T, Chuman H, Terao J. Effect of a conjugated quercetin metabolite, quercetin 3glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells. Free Radic Res 2006;40:1047
53. 61. Ma S, Liu X, Xun Q, Zhang X. Neuroprotective effect of Ginkgolide K against H2O2-induced PC12 cell cytotoxicity by ameliorating mitochondrial dysfunction and oxidative stress. Biol Pharm Bull 2014;37:217
25. 62. Bagetta G, Nucci C. New trends in basic and clinical research of glaucoma: a neurodegenerative disease of the visual system
part B, volume 221 (Progress in Brain Research)”. 1st ed. USA: Elsewier B. V.; 2015. 63. Johnson SK, Diamond BJ, Rausch S, Kaufman M, Shiflett SC, Graves L. The effect of Ginkgo biloba on functional measures in multiple sclerosis:a pilot randomized controlled trial. Explore (NY.) 2006;2:19
24. 64. Lovera JF, Kim E, Heriza E, Fitzpatrick M, Hunziker J, Turner AP, et al. Ginkgo biloba does not improve cognitive function in MS: a randomized placebo-controlled trial. Neurology. 2012;79:1278
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C H A P T E R

24 Lycopene as an antioxidant in human health and diseases Hatice Gu¨l Anlar1 and Merve Bacanli2 1

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Zonguldak Bulent Ecevit University, Zonguldak, Turkey 2Department of Pharmaceutical Toxicology, Gu¨lhane Faculty of Pharmacy, University of Health Sciences, Ankara, Turkey

List of abbreviations BDL CAT FDA GPx GRAS GST LDL OTA ROS SD SOD

bile duct ligation catalase Food and Drug Administration glutathione peroxidase generally recognized as safe glutathione-S-transferase low-density lipoprotein Ochratoxin A reactive oxygen species standard deviation superoxide dismutase

Introduction It is well known that reactive oxygen species (ROS) play important roles in the pathogenesis of important chronic diseases including cancer, diabetes, cardiovascular and neurologic diseases, and Alzheimer’s disease.1 Antioxidants are compounds that can scavenge ROS to protect biological systems from oxidative damage. Various compounds may destroy the balance between the antioxidant defense system and free radicals. This alteration can result in damage in the DNA, lipids, and/or proteins.2 Phytochemicals derived from natural plants have been used commonly for the prevention and/or treatment of different diseases due to their important antioxidant properties. The therapeutic importance of various plant-derived compounds have been quoted in the ancient cultures and traditions of many countries and societies and they are believed to be cost-effective

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00024-X

and safe.3,4 Phenolic compounds are the main compounds responsible for the antioxidant properties of plants. Phenolic compounds are secondary metabolites that are derivatives of the pentose phosphate, shikimate, and phenylpropanoid pathways.5 In many studies, it is shown that fruit and vegetable
rich diets are associated with a decreased risk of chronic diseases.6 The intake of phytochemicals can range between 50 and 800 mg/day, depending on the consumption of vegetables, fruit, and specific beverages.7 The most-studied phytochemicals are carotenoids, which are the lipophilic pigments responsible for the yellow, orange, and red colors of plant foods and the red color of some fish (e.g., salmon) and crustaceans. Their effects against chronic diseases have been attributed to their antioxidant activity.8 More than 700 carotenoids have been identified, about 40 of which are present in the human diet, and about 20 have been identified in the blood and tissues (Fig. 24.1).9,10 Carotenoids are classified according to their bioefficacies. β-carotene has provitamin A activity,11 lycopene has been associated with the protective effects on diseases,10 and other carotenoids such as lutein and zeaxanthin have been related with the reduced risk of age-related macular degeneration12,13 and cataract.14 Lycopene (Fig. 24.2) or C40H56, an aliphatic hydrocarbon carotenoid present in tomatoes, has been demonstrated to possess health protective effects. It is naturally synthesized by plants and it gives a red color to fruit and vegetables, although not all red-colored plants contain lycopene.15 There is growing interest in lycopene due to its high antioxidant capacity.16

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24. Lycopene as an antioxidant in human health and diseases

FIGURE 24.1 Essential carotenoids. The figure shows five essential carotenoids for human life.

FIGURE 24.2 Chemical structure of lycopene. The figure shows that lycopene is an aliphatic hydrocarbon carotenoid.

This chapter mainly focuses on the general properties, chemistry, sources, mechanism of action, pharmacokinetics, and protective effects of lycopene against oxidative stress
related chronic diseases.

Chemistry and sources Lycopene has an open-chain hydrocarbon containing 11 conjugated and nonconjugated double bonds arranged in a linear array. These bonds can undergo isomerization from trans- to mono- or poly cis-isomers during chemical reactions, light, and thermoenergy. The molecular formula of lycopene is C40H56 and it has all-trans, 5-cis, 9-cis, 13-cis, and 15-cis isoforms (Fig. 24.3).18 The isomeric form of lycopene gives the color.19 The trans-isoform of lycopene is commonly found in the human diet and the cis-isoform is found in human blood, plasma, milk, and tissue samples.19
22 The antioxidant capacity of lycopene changes according to the isoforms: 5-cis . 9-cis . 7cis . 13-cis . 15-cis . 11-cis . all-trans.23 Lycopene, a nutritional supplement, is classified as generally recognized as safe (GRAS) by the United States Food and Drug Administration (USFDA).24 For many years, this phytochemical has been used without any problems. Besides, no toxic effects were observed from the consumption of two synthetic crystalline lycopene samples (BASF lycopene 10 CWD and Lyco Vit

10%, each containing 10% lycopene),25 and lycopene derived from fungal biomass of Blakeslea trispos.18 There are two types of lycopene, namely natural and synthetic (Fig. 24.4).26 In previous studies, it was found that lycopene intake levels varied from 9 to 55 mg/day from different lycopene sources such as tomatoes, tomato juice, tomato sauce, and nutritional supplements (Table 24.1).27,28 The dietary intake of lycopene varies in different cultures and countries. The highest dietary intake of lycopene is found in Italy with an average intake of 7.4 mg/day.29

Pharmacokinetics and bioavailability Lycopene is absorbed by passive diffusion like lipids. In various studies, it was found that lycopene absorption may be facilitated by other transporters. Lycopene absorption might vary by age, sex, hormonal status, body mass and composition, blood lipid levels, smoking, alcohol consumption, and competition with other carotenoids or cholesterol.30 The liver, testes, adrenal glands, and adipose tissues are the target organs of lycopene distribution.31 5,6-Dihydroxy-5,6-dihydro lycopene is the main metabolite of lycopene in human plasma. Lycopene may undergo in vivo metabolism to form epoxides and these epoxides may be converted to the polar 5,6dihydroxy-5,6-dihydro lycopene metabolite.32

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Cancer

FIGURE 24.3 Isomers of lycopene. The figure shows seven isomers of lycopene.17

In previous studies, it was found that lycopene from thermally processed tomato products was more bioavailable than that sourced from fresh tomato. Its absorption can be affected by dietary composition. A fat sourced diet increases the bioavailability of lycopene. The bioavailability of trans isomers in food is less than that of all-trans isomers.10

Antioxidant effects

FIGURE 24.4 Two types of lycopene. The figure shows two types of lycopene, which are natural and synthetic lycopene.

Lycopene is the most effective antioxidant in vitro among all carotenoids. This carotenoid has strong 1O2 and ROS scavenger properties.1,17 It can protect DNA, lipids, and other macromolecules with this effect.10 In another study with healthy subjects, 15 days of lycopene treatment decreased oxidative stress.33

Cancer TABLE 24.1

Lycopene content of various tomato products.

Lycopene content (mg/100 g) 6 SD

Source

55.45 6 4.33

Tomato paste

17.98 6 1.47

Tomato sauce

17.23 6 2.18

Ketchup

16.67

Tomato puree

15.99 6 0.90

Spaghetti sauce

10.77 6 1.07

Tomato juice

9.27 6 1.02

Whole tomatoes

The table shows the lycopene content of various tomato products.27

There are many studies related to the beneficial effects of lycopene against cancer. In the Mediterranean diet, tomato and tomato products consumption is very high. For this reason, the cancer incidence is lower in these regions.34 Lycopene shows anticancer activity with different mechanisms. It decreases the levels of antiapoptotic protein Bcl-2 and increases the levels of Bax, which is the regulator protein of apoptosis.35 Lycopene also increases cell cycle arrest in the G0-G1 phase.36 Lycopene prevents changes in p53 expression.24 Among all types of cancer, lycopene shows the most protective effect against prostate cancer. In prostate cancer cell lines (DU145 and LNCaP), lycopene

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24. Lycopene as an antioxidant in human health and diseases

FIGURE 24.5

DNA damage in the kidney of rats induced by OTA (with and without supplementation of lycopene). DNA damaged was shown as length in (A), as tail moment in (B), and as tail intensity in (C). The figure shows DNA damage in the kidney of rats induced by OTA (with and without supplementation of lycopene).44 Control Group I (C7); 1 mL of corn oil by intragastric lavage (i.g.) for 7 days; Control Group II (C14) received 1 mL of corn oil (i.g.) for 14 days; Lycopene Control Group I (L7) received 5 mg/kg/day lycopene (i.g.) for 7 days; Lycopene Control Group II (L14) received 5 mg/kg/day lycopene (i.g.) for 14 days; OTA Group (OTA) received 0.5 mg/kg/day OTA (i.g.) for 14 days; OTA and Lycopene 7 Group (OTA 1 L7) received 0.5 mg/kg/day OTA (i.g.) for 14 days and 5 mg/kg/day lycopene (i.g.) for last 7 days; OTA and Lycopene 14 Group (OTA 1 L14) received 0.5 mg/kg/day OTA (i.g.) and 5 mg/kg/ day lycopene (i.g.) for 14 days. Bars that do not share same letters (superscripts) are significantly different from each other (P , .05).

reduced the growth of cells.37,38 In a cohort study, Seventh-day Adventist men who consumed high levels of tomato products more than five times per week showed significantly decreased risk of prostate cancer compared with men who consumed lower amounts of these products.39 In gastric cancer, lycopene consumption reduced oxidative stress with the regulation of glutathione levels and glutathione-S-transferase (GST) and glutathione peroxidase (GPx) enzyme activities.10 In a study with 449 subjects from Belgium, it was found that low consumption of tomatoes resulted in an increase in gastric cancer risk.40 In many epidemiological studies, it was concluded that lycopene reduced the risk of breast, lung, colorectal, and ovarian cancers.10

Cardiovascular diseases Low-density lipoprotein (LDL) oxidation is very important in cardiovascular diseases. Lycopene may affect the oxidation of LDL due to its antioxidant effects.10 In a study, 19 healthy volunteers took a placebo, tomato juice (50.4 mg lycopene), spaghetti sauce (39.2 mg lycopene), and tomato oleoresin (75 mg lycopene) daily for a week. A significant decrease was seen in serum lipid peroxidation and LDL oxidation in the lycopene treated groups.41 When 24 subjects consumed lycopene containing tomato products (40 mg/day lycopene), it was observed that triglyceride and LDL levels were decreased in the lycopene treated subjects.42

Hepatic and renal diseases Yilmaz et al. studied the beneficial effects of lycopene against aflatoxin B1-induced renal and cardiac damage in rats. They found a significant decrease in the activities of antioxidant enzymes (GST, GPx, superoxide dismutase (SOD), and catalase (CAT)) and the nonenzymatic antioxidant system in these rats, and lycopene had protective effects against these unwanted changes.43 Ochratoxin A (OTA) is one of the most prevalent mycotoxins in the world as it has nephrotoxic and hepatotoxic properties. In a study using male Sprague
Dawley rats, OTA (0.5 mg/kg bw/day) was administered by gavage for 14 days, whereas lycopene (5 mg/kg/day) was applied on the last 7 days or for 14 days of the feeding period with OTA treatment. Genotoxicity was evaluated by comet assay. It was shown that OTA caused marked increases in tail length, tail moment, and tail intensity compared to the control, both in the kidney and liver cells, but not in the lymphocytes. Lycopene administration alone for 7 and 14 days did not provide any significant changes in DNA damage of the lymphocytes, renal, and hepatic cells compared to the controls. However, lycopene for both 7 and 14 days, with OTA exposure in renal and hepatic cells, supplied significant decreases in tail length, tail moment, and tail intensity compared to the OTA-exposed rats (Fig. 24.5).44 Aydın et al.45 studied the beneficial effects of lycopene against obstructive jaundice, a frequently observed condition caused by obstruction of the bile duct or its flow and seen in many clinical situations,

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Hepatic and renal diseases

TABLE 24.2

Oxidative stress parameters in hepatic tissues in rats with obstructive jaundice. Group I

Group II

251.3 6 79.4

753.0 6 219.1

266.4 6 38.2b

GSH (nmol/mg tissue)

3.1 6 1.2

0.9 6 0.8a

2.5 6 0.9b

NO (nmol/mg tissue)

68.6 6 8.4

112.3 6 16.6a

73.0 6 3.4b

CAT (U/mg protein)

164.8 6 34.9

93.4 6 23.1a

174.9 6 38.5b

SOD (U/mg protein)

86.1 6 6.0

50.9 6 4.7a

77.5 6 7.8a,b

GST (U/mg protein)

0.4 6 0.1

0.2 6 0.1a

0.4 6 0.1b

MDA (pmol/mg protein)

Group III a

P , .05, group I compared with group II and group III. P , .05, group II compared with group III. The results are given as mean 6 SD. Group I (the sham group) was subjected to a sham operation and treated once daily with 0.5 mL of maize oil orally. Group II (the BDL group) was subjected to BDL and was treated once daily with 0.5 mL of maize oil orally. Group III (the BDL 1 Lycopene group) was subjected to BDL and treated once daily with 100 mg/kg body weight of lycopene in 0.5 mL of maize oil orally. The table shows oxidative stress parameters in hepatic tissues of rats.45

a

b

FIGURE 24.6 Histopathologic section of hepatic tissue of rats with acute cholestasis. The figure shows histopathological findings of the liver of rats.46 (A) Normal liver histology from the sham group; (B) a portal tract from the sham group; (C) portal inflammation including neutrophil leukocytes and bile ductal proliferation in the bile duct ligation (BDL) group; (D) a necrotic focus near the portal tract in the BDL group; (E) portal inflammation and bile ductal proliferations (arrowhead) in the BDL 1 Lycopene group.

and which may end in serious complications like sepsis, immune depression, coagulopathy, wound breakdown, gastrointestinal hemorrhage, and hepatic and renal failures. Daily doses of 100 mg/kg/bw lycopene were given to the bile duct ligation (BDL) group orally for 14 days and after that genotoxic damage, liver functions, and oxidative stress parameters were evaluated. Their study showed that lycopene significantly recovered the oxidative stress parameters in hepatic and renal tissues (Table 24.2) in addition to its ameliorative effects on the liver function parameters and DNA damage in the animals used.

Another study by the same group revealed that lycopene also has protective effects against acute cholestasis. Lycopene treatment (10 mg/kg/bw) significantly ameliorated the liver function parameters, DNA damage, and oxidative stress. It also alleviated histological changes of the liver (Fig. 24.6).46

Gastrointestinal diseases It is concluded that lycopene has antiulcer effects. Jain and Katti demonstrated that lycopene (2 mg/kg) and hesperidin (100 mg/kg) decreased gastric

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24. Lycopene as an antioxidant in human health and diseases

secretion and total acidity and increased gastric pH in ˘ ulcer-induced rats.47 In addition, Boyacıoglu et al. showed that lycopene treatment protected rats against indomethacin-induced gastric ulcers.48 It is also known that lycopene has significant protective effects against Helicobacter pylori infections.10

and it has a high antioxidant capacity. Data from a literature search demonstrates the animal, epidemiology, and cell culture studies about the beneficial health effects of lycopene. However, more clinical data are needed to support these findings and the mechanism of action of lycopene.

Neurodegenerative diseases The nervous system has an extremely high lipid content and extremely low antioxidant capacity. Thus this system is sensitive to oxidative damage.49 In an animal study, lycopene showed protective effects against rotenone-induced Parkinson’s disease.50 Besides, this carotenoid has beneficial effects on myeloid β
induced neurotoxicity,51 3-nitropropionic acid
induced mitochondrial oxidative damage,52 and trimethyltin-induced neurotoxicity in cultured rat hippocampal neurons.53 Liu et al. observed that lycopene treatment improved cognitive deficits and showed protective effects on inflammation caused by β-amyloid in rats.54 In another study with C57BL/6J mice, lycopene inhibited lipopolysaccharide-induced memory loss and accumulation of amyloid-β with a 5-week treatment.55 It is also concluded that low lycopene levels might have a role in the increased risk of psychiatric disorders.56

Applications in other areas of pathology Oxidative stress and decreased antioxidant status have been suggested as important factors in male infertility. It is also known that lycopene is highly concentrated in the testes. Thus low lycopene levels in men might have a role in male infertility.10 The beneficial effects of lycopene on bone health were demonstrated in various cell cultures. The proliferation of osteoblast-like SaOS-2 cells was stimulated by lycopene.57 Additionally, Park et al. demonstrated that lycopene had an inhibitory effect on the proliferation of osteoblastic cells (MC3T3).58 ˙ On the other hand, Icel et al. showed that lycopene was importantly effective in the prevention of inflammation and oxidative stress on eye tissue associated with diabetes.59

Conclusion The intake of an antioxidant vegetable and fruit
rich diet is associated with a decreased risk of chronic diseases caused by oxidative stress. Lycopene is the most important carotenoid in the human diet

Summary points • ROS are the main reason for the most important chronic diseases. • Natural antioxidant compounds are known to be effective in the prevention and treatment of these diseases. • Lycopene is an aliphatic hydrocarbon carotenoid present in red-colored plants, especially tomatoes. • Lycopene is a strong antioxidant and it can protect DNA, lipids, and other macromolecules against oxidative stress. • Lycopene plays a role in increasing the antioxidant status and in lowering the oxidative damage in several pathological conditions. • It ameliorates the levels of GSH and the activities of antioxidant enzymes such as GPx, GST, SOD, and CAT. • The beneficial effects of lycopene against cancer, cardiovascular, gastrointestinal, neurodegenerative, hepatic, and renal diseases were shown. • However, more clinical data are needed to support these findings and the mechanism of action of lycopene.

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C H A P T E R

25 Mediterranean diet: the role of antioxidants in liver disease Ludovico Abenavoli1, Lorenzo Romano2, Paola Gualtieri2, Gemma Lou De Santis2 and Antonino De Lorenzo2 1

Department of Health Sciences, University “Magna Graecia”, Catanzaro, Italy 2Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

List of abbreviations AAH ALD ASH BMI DXA MAI MD NACCP NAFLD NASH

acute alcoholic hepatitis alcoholic liver disease alcoholic steatohepatitis body mass index dual-energy X-ray absorptiometry Mediterranean Adequacy Index Mediterranean diet nutrient hazard analysis and critical control point nonalcoholic fatty liver disease nonalcoholic steatohepatitis

Introduction The suboptimal quality of dietary patterns and nutrients is the main modifiable cause of morbidity and mortality.1 The analysis of food patterns evaluates the different combinations of foods, and best reflects the effectiveness of the diet in the prevention of diseases.2 Only by exploring food patterns it is possible to evaluate the additive effects between foods, the manifestation of multiple effects, and to overcome the confusing aspects of diet-therapy. The Mediterranean diet (MD) is a model characterized by the main consumption of plant-based foods and fish and reduced consumption of meat and dairy products with the exception of milk, yogurt, and aged cheeses (Fig. 25.1). Alcoholic beverages are also moderately permitted, especially red wine during meals. The intake of lipids must be elevated up to 40% of the total energy intake. In fact, the ratio of saturated and monounsaturated fats will be high because of the source of lipids

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00025-1

representing a distinctive pattern of the MD.3 Interest in antioxidants and polyphenols has increased over time, as epidemiology has indicated an inverse association between the presence of nutrients rich in antioxidants and the risk of mortality from noncommunicable diseases.4 For decades, the antioxidant compounds present in some nutrients have been considered only powerful “scavengers of free radicals.” Then, their action was related to several biological effects such as antiinflammation action, inhibition of tumor proliferation, cholesterol absorption, and modulation of different enzymes including telomerase and others implicated in redox reactions. Within a Mediterranean food pattern, the presence of antioxidants is synonymous with longevity and health.2 For example, in carcinogenesis there are potential chemopreventive mechanisms such as the modulation of the energy metabolism of a carcinoma, pathway regulation, and inhibition of cell proliferation and apoptotic induction capacity. Furthermore, in vitro, in vivo, and epidemiological studies have shown the ability of these bioactive molecules to positively influence the critical passages in atherogenesis including the oxidation of low-density lipoprotein, the release of nitric oxide, inflammation, oxidative stress, chemotaxis, cell adhesion, foam cell formation, smooth muscle cell proliferation, and platelet aggregation.2,4 A powerful antioxidant activity in vitro may be absent in humans due to poor bioavailability. If a potent bioactive compound, even if in large quantities, does not reach its site of action, it is not able to exert its beneficial effects on the target tissues. Therefore for

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25. Mediterranean diet: the role of antioxidants in liver disease

FIGURE 25.1 The Mediterranean diet pyramid.

the prevention of diseases and the improvement of human health it is necessary to know the biological capacities, the bioavailability, and the metabolites of the polyphenols contained in foods in order to choose the best dietary pattern.5 In the MD, foods mainly rich in polyphenols are fruit, vegetables, red wine, wild herbs, spices, nuts, and also olives, especially extra virgin olive oil. Not least, today, there are other elements in the Mediterranean pattern very rich in polyphenols such as turmeric, spices, coffee, green tea, and chocolate from the influence of other cultures.6,7 Some bioactive molecules are specific to certain nutrients such as isoflavones in soy. In general, mixtures of polyphenols with different concentrations in the same category are contained in different foods. In red wine there are flavonols, flavanols, proanthocyanidins, anthocyanins, phenolic acids, hydroxycinnamates, and stilbenes, in particular resveratrol.8 Even in extra virgin olive oil there is a great variability in concentration and composition, in fact, there are up to 36 phenolic compounds and the total concentration varies between 0.02 and 600 mg/kg.9 The intake of antioxidants, in particular of polyphenols, has been correlated with a reduction in mortality rates.4 A systematic review of the use of antioxidant supplementation in healthy subjects and subjects affected by noncommunicable diseases has concluded that these are not associated with a reduction in mortality assessed for all causes. Indeed, in the analysis of single

individual studies it was found that vitamin antioxidants were associated with higher mortality from all causes.9 The causes of this phenomenon are the reduced intake of micronutrients, less assimilable by the supplements than the phytocompound. In this way, it has been highlighted that the adequacy of food choices in the Mediterranean pattern is an essential objective to maintain a state of health.10 In the era of evidence-based medicine, the MD represents the gold standard in preventive medicine, probably due to the harmonic combination of many elements with antioxidant and anti-inflammatory properties, which overwhelm any single nutritive or alimentary element. The role and effectiveness of the MD, supplemented with extra virgin olive oil, nuts, and red wine, in the prevention of cardiovascular events in high risk subjects has been confirmed.11 In light of the various studies, the Mediterranean pattern has proven itself in epidemiological studies to reduce mortality, probably due to the strength of the phytocompound and its modulation also on the microbiome.1 Furthermore, the presence of antioxidants is a highly qualitative and nutritional standard index and in the choice of food, the origin, whether conventional or organic, of fruit and vegetables must be taken into account. Therefore it is necessary to select vegetables of organic origin since in the absence of pesticides or insecticides it produces antioxidant bioactive molecules useful for growth in a competitive environment.12

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Applications in other areas of disease

Ultimately it is necessary to ensure a quota and a prudent quality of antioxidants in the diet in order to prevent metabolic syndromes, associated complications, and carcinogenesis.13 Contextually, a controlled food chain is necessary in order to ensure the absence of contaminants and suitable antioxidants in the diet. Otherwise, the risk is to consume suboptimal, harmful, and poor food for human health. The nutrient analysis critical control point (NACCP) process represents the correct way of monitoring the nutritional quality of foods of the selected dietary pattern in order to have the highest antioxidant content. In fact, it is possible to monitor the entire production chain and identify where the critical points are to intervene and improve the quality of nutrients.14 Moreover, special attention must be paid to measurement standards for the quality and quantity of antioxidants. However, it remains clear that adherence to the Mediterranean pattern increases life quality and life expectancy by reducing the risk of death, also linked to the higher content of antioxidants and polyphenols mainly present in foods rich in fiber. This leads to a modulation of the bacterial flora that influences, and is influenced by, polyphenols and the foods that contain them. In particular, flora is able to metabolize and make certain substances absorbable. Furthermore, phytocompounds, in terms of yield, on the absorption and action of polyphenols and antioxidants are superior to isolated compounds. This phenomenon is attributable to the components present in the food that may increase in synergy the bioavailability and the metabolic action of probiotics.15 In this chapter, we want to clarify the intake of antioxidants, typical of the MD and evaluate their effect on nonalcoholic and alcoholic liver disease (ALD), the main hepatic injury influenced by dietetic profile.

Applications in other areas of disease In healthy patients, several trials have shown the efficacy of integrations with antioxidant foods/drinks on gene expression, metabolic and inflammatory pathways,6,8 suggesting that antioxidant status is a consequence of the antioxidant content of foods.16 Several diseases, like as cardiovascular, cancer, neurodegenerative and eye diseases, show altered pathways or excess of oxidative stress.16 For this reason, it is necessary to identify therapeutic strategies that contemplate the use of antioxidants. As already mentioned, fruit and vegetables are the main source of these molecules, particularly low molecular weight antioxidants that perform a protective

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function from oxidative stress at the cellular level. Alpha-tocopherol, ascorbic acid, and beta-carotene would seem to have an effect on the reduction of the risk of many diseases probably due to a synergistic effect rather than a single antioxidant. In general, the MD, with its high intake of antioxidants, contributes substantially to the reduction of cardiovascular risk and, in particular, to the reduction of the incidence of thrombosis, hypertension, type 2 diabetes mellitus, and obesity as demonstrated by the PREDIMED study.17 On the other hand, regarding neurodegenerative diseases, it has been shown that oxidative stress acts on cellular function and structure. Due to the antioxidants taken in from the diet, in particular those belonging to the polysaccharide class from vegetables, fruit, cereals, legumes, tea, nuts, mushrooms, and probiotics, it is possible to achieve an improvement in diseases. They would seem to reduce cognitive and motor decline, intervening in mitochondrial function, the antioxidant pathway, and protein misfolding as well as acting as scavengers against free radicals. In this case, polysaccharides seem to show a double effect of oxidative stress reduction and protection against the related diseases. In obesity, seen in its phenotype spectrum and defined by specific metabolic characteristics, antioxidants can limit the inflammatory and oxidative state of subjects, reducing the progression of cardiometabolic comorbidities.18 As for cancer, however, it is true that several studies have highlighted the role of antioxidants,19 but it is always necessary to pay attention to the absolute data. For example, in the case of polyphenols, their poor bioavailability has highlighted the need for further studies toward understanding the mechanisms of action in different tissues as well as the influence of genetic variability. Moreover, with a low bioavailability it is difficult to think of an effective ability to modulate pathways regulating cell growth. There are theories that consider a pro-oxidant action of some phytochemicals. These can be oxidized and lead to the formation, in turn, of molecules that are then responsible for the transcription of genes for the antioxidant defense as a quercetin. Embracing these theories, it would be possible to recognize a nonactive role in the prevention or treatment of cancer of antioxidants, but at the end of a series of reactions.19 However, it has been demonstrated that synthetic antitumor drugs, although effective in treatment, are toxic. On the other hand, phytochemicals have zero or minimal side effects, so much so that they have seen considerably increased use in recent years.20

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Nonalcoholic fatty liver disease Definition Nonalcoholic fatty liver disease (NAFLD) refers to a quantitatively significant presence of fat in the liver, in the absence of other liver disease or alcohol abuse.21 Within NAFLD there are two clinical pictures called simple steatosis and nonalcoholic steatohepatitis (NASH).

Etiopathogenesis Steatosis is a benign accumulation of fat and only rarely progresses, while NASH presents steatosis, necroinflammation, and fibrosis, with progression to cirrhosis and hepatocellular carcinoma. Both have the same risk factors shared with cardiovascular diseases, namely obesity, insulin resistance, dyslipidemia, metabolic syndrome, and diabetes.22 As already noted, the listed risk factors are secondary to suboptimal nutrient quality, excess of calories, sedentary lifestyle, environmental contaminants, and genetic predisposition (Table 25.1). These factors make the highest incidence of NAFLD in western and developing countries in which eating habits have been progressively subverted by the western diet with “junk food.”23 In addition, the growth in prevalence, up until now only in adults, is increasing in obese children and NALFD has become the most common liver disease. The average prevalence of NAFLD ranges between 20% and 30% and that of NASH from 3% to 16% and these are higher in western countries and growing in developing countries.24 The pathogenesis of NAFLD leads back to the theory of “multiple hits.”22 Responsible for the “first hit” are metabolic syndromes that cause the accumulation of fat in the liver and insulin resistance. This makes the liver vulnerable to “second hits” leading to NASH such as oxidative stress, mitochondrial dysfunction, TABLE 25.1 liver disease.

Risk factors for progression of nonalcoholic fatty

age $ 50 years Western diet Genetic predisposition BMI $ 28/kg/m2 Hepatic necroinflammatory activity Insulin resistance Hypertriglyceridemia Metabolic syndrome components

inflammation, and xenobiotics. Today, among the “second hits” there is an increase in cytokines produced by intestinal permeability, a frequent condition in hepatopathic patients with small intestine bacterial overgrowth.24 Intestinal microflora represent the first source of endotoxin, especially in conditions of overgrowth or unbalance between species, that can influence the progression from steatosis to steatohepatitis.25 It is emerging that the changes of intestinal flora have multiple roles in the pathogenesis of many inflammatory and chronic diseases including obesity, insulin resistance, and dyslipidemia; all conditions associated with NALFD.19 Oxidative stress also plays a key role in the progression of NALFD. In fact, patients with progression in NASH present lower levels of antioxidants.25 Central obesity, identified as an excess of abdominal fat, is frequently associated with NAFLD and increases the risk of the progression of the disease as well as a high body mass index (BMI). The increase of visceral fat, evaluable with body composition and anthropoplicometry, is associated with low adiponectin levels and insulin resistance in NAFLD. Regardless of BMI, the determination of abdominal fat, for example, with dual-energy X-ray absorptiometry (DXA), is essential to identify normal weight or overweight in risk subjects and to follow the effectiveness of treatment over time.26

Treatment Currently, NAFLD has become an emerging health problem worldwide due to its high prevalence. Efforts to diagnosis, prevent, and treat it are important and must remain so in the future since NALFD can progress to cirrhosis or cancer and is related to cardiovascular disease. Based on the risk factors underlying the development of NALFD, the treatment that showed the best result was a multidisciplinary one. Nutritional intervention that drives the adhesion to a Mediterranean dietary pattern and physical activity is more effective than any single pharmacological option.22 Treatment for NAFLD involves a change in lifestyle habits. Literature data and international guidelines highlight the health benefits of weight loss and exercise. In this way, the MD seems to be perfect for patients with NAFLD due to its effectiveness on the liver status, leading to an improvement of insulin sensitivity and lipid profile, but also as a primary form of prevention against related diseases.27 The recent position paper of the Italian Association for the Study of the Liver suggests the use of a low carbohydrate and low saturated fat diet and an increase in the consumption of fruit and vegetables with the exclusion of drinks enriched with

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Nonalcoholic fatty liver disease

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FIGURE 25.2 Influence of Mediterranean diet on nonalcoholic fatty liver disease.

fructose.28 The use of the MD in patients with NAFLD resulted in an improvement in clinical and biochemical data with a reduction of steatosis and improvement of metabolic parameters in the patients (Fig. 25.2). Subsequently, the efficacy of antioxidant supplementation in the MD to improve treatment was analyzed.29
31 Indeed, the combined effect of the MD and the physical activity approach are able to improve biochemical and anthropometric parameters, hepatic fat accumulation, and the Kotronen index.29 Based on a pathogenesis linked to stress, antioxidant supplements were used in combination with the MD and these made an improvement in insulin sensitivity.31 The combination of probiotics with the MD and physical activity has a synergistic action in maintaining liver health. Among the many species of bacteria, Lactobacillus seems to be the most promising.32 Probiotics and commensal bacteria are able to modulate the gut microbiota and to give health benefits to the host (Fig. 25.3). Probiotics themselves, their vitality, their balance, and their action must be supported through prebiotics, plant fibers, and polyphenols; all molecules present in the MD. At the same time, industrial foods and foods contaminated by pesticides such as glyphosate must be avoided. Glyphosate has a direct action on the metabolism of soil and plant bacteria and indirectly on the microbiome. Accordingly, organic foods from a controlled supply chain should be preferred.14 The definition of MD is complex. Over

time, research has shifted attention from the single nutrients to the alimentary pattern and defined it in reference to the postwar food model of Nicotera.33
35 To evaluate and compare the food model of Nicotera, the Mediterranean adequacy index (MAI) was developed. This is obtained from the ratio of the sum of the percentage of total energy from Mediterranean foods and the total energy from nonMediterranean foods.36 In particular, a meal that has an MAI . 7 reduces postprandial inflammation, reduces oxidative stress, and positively modulates the expression of inflammatory genes.37 In general, it was observed that populations with a high MAI food pattern had a low mortality risk. The reduction in mortality, inflammation, and oxidative stress is attributed to the bioactivity of fibers, monounsaturated fatty acids, in particular ω
3, vitamin A, C, and E, and polyphenols.16 However, longitudinal studies on eating habits have shown a reduction of MAI in regions of Italy, and to get a health benefit it is necessary to encourage the population to have a target daily score .5.36 The temple of the MD improves the understanding of the balanced combination of fruit, vegetables, fish, legumes, cereals and polyunsaturated fats of extra virgin olive oil, with a reduced consumption of meat and dairy products and a moderate consumption of alcohol, mainly red wine.33 The carbohydrates should be complex and starchy from unrefined cereals, bread and pasta, legumes, and vegetables, and only 5% must

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25. Mediterranean diet: the role of antioxidants in liver disease

FIGURE 25.3 Relationship between healthy dietetic regimen and gut microbiota modulation.

be simple from fruit or red wine. The energy derived from ethyl alcohol, mainly wine consumed during meals, can be included in acceptable quantities with two glasses a day for men and one for women. Vegetables are important elements of the MD as the main source of phytosterols, which reduce cholesterol and cardiovascular risk. The general recommendations for the diet must be personalized and dependent on body weight.

Alcoholic liver disease Definition ALD refers to different liver disease states caused by the excessive consumption of alcohol. It is a degenerative process characterized by three disease stages, each progressively more severe, and frequently overlapping with alcoholic steatohepatitis (ASH), acute alcoholic hepatitis (AAH), and alcoholic cirrhosis.

produced. In ethanol catabolism, microsomal oxidation by cytochromes results in the formation of ROS and of acetaldehyde, which are both associated with liver damage by the activation of inflammatory processes (Fig. 25.4). However, the excessive production of ROS can be eliminated by antioxidants.39 In ASH, there are morphological alterations of the liver with ectopic fat filling without symptoms, even for many years. Often transient pains are present in the right hypochondrium and epigastrium. ASH may precede AAH due to excessive and prolonged alcohol abuse with liver tissue inflammation, necrosis, and fibrosis with reduced function. AAH describes both a precise histopathological picture and a clinical syndrome characterized by jaundice and liver failure in individuals who consume large amounts of alcohol.40 This develops within a complex framework of pathological alcohol consumption. In the same way, the definition of nutritional status is essential to start treatment quickly.

Etiopathogenesis

Epidemiology

The ethanol metabolism is first to be transformed into acetaldehyde and then into acetic acid due to NAD 1 dependent dehydrogenases. The necessity of NAD 1 limits the metabolism of high quantities of ethanol, thus, requiring, in the case of excess consumption, the intervention of the enzyme CYP2E1, which is part of the cytochrome P450 complex.38 In addition to alcohol, many xenobiotics engage cytochrome enzymes and can compete with it such as drugs, toxins, and steroids. This enzyme is induced and more expressed in chronic alcohol consumers in order to increase the detoxifying capacity of the liver, but as a result greater quantities of reactive oxygen species (ROS) are

Data on the consumption of alcoholic beverages are determined by estimate since it is difficult to document average consumption and excess consumption. Currently, alcohol consumption is higher in the United States, Europe, and some western Pacific regions; areas where there is a high presence of AAH.40 In addition, the incidence of ALD has increased in eastern regions such as China due to the rapid socio-cultural changes that also affect the consumption habits of alcoholic beverages, especially among young people. Traditionally, the consumption of alcohol occurred during meals and excesses, if any, took place during the weekend. Today there is talk, instead, of “binge

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Alcoholic liver disease

FIGURE 25.4 Pathogenetic mechanisms involved in alcoholic liver disease.

FIGURE

25.5 Binge drinking definition by National Institute of Alcohol Abuse and Alcoholism (NIH), World Health Organization (WHO), and Alcohol Use Disorders Identification Test (AUDIT).

drinking” or the excessive consumption of alcohol outside of meals, with over five drinks being consumed in about two hours and increased use of spirits during several days of the week (Fig. 25.5). Given the toxic and damaging nature of alcohol and its metabolites, this causes damage, both acute and chronic, capable of causing liver disease, but also malnutrition, gastrointestinal disorders, psychosis, and other systemic diseases.38

Treatment At the base of treatment for ALD, there is abstention from alcohol and according to the stage and the complications of the disease, nutritional, pharmacological, and surgical therapies are proposed.41 ASH represents the first step of ALD and is an already advanced stage in which there is an excessive accumulation of fat in the liver due to alcohol consumption .20
30 g/day, without other etiological causes of liver disease. In the

clinical management of ASH, the patient must be guided and invited to follow counseling to succeed in alcohol withdrawal so that the liver damage is reduced and the progression of cirrhosis is slowed. Nutritional intervention is the next step. Based on the pathogenesis induced by oxidative damage and the possible overlapping of malnutrition by defect or excess, it is necessary to set up a personalized diet plan. In general, it is essential to ensure optimal nutritional intake with 30
45 kcal/day/kg of body weight with 1
1.5 g of protein per kilogram, and to correct any deficiencies of micronutrients.42 In addition to these general indications, diet-therapy must be tailored to the patient’s clinical and nutritional needs through the careful assessment of their nutritional status and subsequent follow-up. The MD can reflect the characteristics indicated for nutritional support. Moreover, following the Mediterranean pattern using foods rich in antioxidants and fibers, it is possible to act on the pathogenetic mechanisms of oxidative damage. Extra virgin olive

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25. Mediterranean diet: the role of antioxidants in liver disease

oil, organic and subjected to a controlled supply chain, must be a major component of the diet-therapy. This is an important source of energy for the presence of mainly monounsaturated triglycerides such as oleic acid and polyunsaturated acids such ω
3 and ω
6. It is also rich in fat-soluble vitamins, tocopherols and β-carotene, phytosterols, and polyphenols, such as hydroxytyrosol. In addition to the choice of the most effective nutrients belonging to the Mediterranean area, patients undergoing nutritional treatment should be evaluated and monitored using body composition techniques. During nutritional treatment, the determination of body mass allows for the evaluation of the effectiveness, safety, and personalization of macronutrients such as proteins based on the represented lean mass. Finally, the determination of water status is pivotal to avoid a confounding effect in weight since patients with protein malnutrition are subject to extracellular fluid expansion. Simultaneously with the evaluation of the nutritional and water statuses, it is necessary to monitor the functionality through biochemical tests and the protein pool reserve. Recently, the role of microbiota in ASH has been highlighted. These patients are subject to low-quality diets that damage and alter the balance of the intestinal flora. This involves a workload to the liver and it has been observed that the same bacterial flora when altered produces alcohol, which can in turn damage the hepatocytes. The health of bacterial flora in these patients must be improved through the MD, which, as seen, contains all the foods and nutrients essential to the balance of the flora. In addition, probiotics can be administered in combination with pre- and postbiotics and polyphenols, present in the phytocomplex that is food, to improve health, balance, and intestinal permeability.43

Malnutrition Malnutrition has a high incidence and a varying severity in patients with ALD, and there are also deficiencies of microelements, vitamins and minerals, and proteins. The presence of obesity or excess weight should not be confused since in alcohol consumers this is a frequent condition and often overlaps with protein-energy malnutrition. The severity of malnutrition directly influences both short-term and long-term mortality. For mild malnutrition the mortality rate is 14%, compared to 76% for severe malnutrition, one year after diagnosis.42 Therefore diet-therapy and nutritional support are essential in the management of patients with severe forms of ALD, to correct the protein-calorie malnutrition, and as stated by the American Association for the Study of Liver

Diseases.44 In general, oral/enteral nutrition is preferable to parenteral nutrition, which alone has proven to be inadequate. Guidelines suggest a regular diet containing respectively 1
1.5 g of protein/kg and 30
40 kcal/kg of body weight. Supplementation with branched amino acids is indicated in patients at risk of encephalopathy intolerant to proteins as it improves nitrogen balance, reduces hepatic steatosis, and improves nutritional indices. If the patient is unable to eat due to anorexia or altered mental status, then a tube for enteral feeding should be considered. Since the pathogenesis of ALD concentrates on oxidative stress induced by alcohol and the consequent inflammation, strategies based on drugs and nutraceutical extracts with antioxidant power have been explored.41 Currently, there are several synthetic hepatoprotective drugs such as bifendate that are effective in the treatment of liver damage, but their use is strongly limited due to their side effects. Therefore in the condition of abstinence for the treatment of ALD, different antioxidant molecules were studied, in combination with corticosteroids and without them. Nacetylcysteine, S-adenosyl-L-methionine, and combinations of antioxidants like β-carotene, vitamin A, C, E, and selenium have been described. In general, the combination of more antioxidants with glucocorticoids can influence mortality rates.41,44

Conclusions Finally, waiting for further investigation, it can be concluded that in ALD, in addition to abstinence and medical therapy as indicated, it is necessary to set up a nutritional support. A diet-therapy is preferentially administered orally, and in the case of enteral anorexia with a tube if tolerated, consisting of 30
45 kcal/day/ kg with 1
1.5 g of protein per kilogram of body weight respectively, preferably with the administration of an evening snack of about 500 kcal and the use of branched amino acids in case of intolerance to proteins.

Summary points • The MD is a model characterized by the main consumption of plant-based foods and fish and reduced consumption of meat and dairy products. • In the era of evidence-based medicine, the MD represents the gold standard in preventive medicine, probably due to the harmonic combination of many elements with antioxidant and anti-inflammatory properties that overwhelm any single nutritive or alimentary element.

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• This alimentary regimen, with its high intake of antioxidants, contributes substantially to the reduction of the onset of many chronic diseases such as cardiovascular diseases, hypertension, type 2 diabetes mellitus, obesity, and cancer. • In this chapter, the effects of antioxidant use in the presence of liver pathologies are described.

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31. Abenavoli L, Greco M, Milic N, Accattato F, Foti D, Gulletta E, et al. Effect of Mediterranean diet and antioxidant formulation in non-alcoholic fatty liver disease: a randomized study. Nutrients 2017;9:870. 32. Abenavoli L, Scarpellini E, Rouabhia S, Balsano C, Luzza F. Probiotics in non-alcoholic fatty liver disease: which and when. Ann Hepatol 2013;12:357
63. 33. Fidanza F. The Mediterranean Italian diet: keys to contemporary thinking. Proc Nutr Soc 1991;50:519
26. 34. Kromhout D, Menotti A, Bloemberg B, Aravanis C, Blackburn H, Buzina R, et al. Dietary saturated and trans fatty acids and cholesterol and 25-year mortality from coronary heart disease: the Seven Countries Study. Prev Med 1995;24:308
15. 35. Alberti-Fidanza A, Fidanza F. Mediterranean Adequacy Index of Italian diets. Public Health Nutr 2004;7:937
41. 36. Alberti A, Fruttini D, Fidanza F. The Mediterranean Adequacy Index: further confirming results of validity. Nutr Metab Cardiovasc Dis 2009;19:61
6. 37. Menotti A, Alberti-Fidanza A, Fidanza F. The association of the Mediterranean Adequacy Index with fatal coronary events in an Italian middle-aged male population followed for 40 years. Nutr Metab Cardiovasc Dis 2012;22:369
75. 38. Abenavoli L, Masarone M, Federico A, Rosato V, Dallio M, Loguercio C, et al. Alcoholic hepatitis: pathogenesis, diagnosis and treatment. Rev Recent Clin Trials 2016;11:159
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39. Song X, Liu Z, Zhang J, Zhang C, Dong Y, Ren Z, et al. Antioxidative and hepatoprotective effects of enzymatic and acidic-hydrolysis of Pleurotus geesteranus mycelium polysaccharides on alcoholic liver diseases. Carbohydr Polym 2018;201:75
86. 40. Masarone M, Rosato V, Dallio M, Abenavoli L, Federico A, Loguercio C, et al. Epidemiology and natural history of alcoholic liver disease. Rev Recent Clin Trials 2016;11:167
74. 41. Rosato V, Abenavoli L, Federico A, Masarone M, Persico M. Pharmacotherapy of alcoholic liver disease in clinical practice. Int J Clin Pract 2016;70:119
31. 42. European Association for the Study of the Liver. EASL Clinical Practice Guidelines on nutrition in chronic liver disease. J Hepatol 2019;70:172
93. 43. De Lorenzo A, Costacurta M, Merra G, Gualtieri P, Cioccoloni G, Marchetti M, et al. Can psychobiotics intake modulate psychological profile and body composition of women affected by normal weight obese syndrome and obesity? A double blind randomized clinical trial. J Transl Med 2017;15:135. 44. O’Shea RS, Dasarathy S, McCullough AJ, Practice Guideline Committee of the American Association for the Study of Liver Diseases, Practice Parameters Committee of the American College of Gastroenterology. Alcoholic liver disease. Hepatology 2010;51:307
28.

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C H A P T E R

26 Melatonin, antioxidant capacity, and male reproductive function Fahimeh Mohammadghasemi Cellular & Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran

List of abbreviations ABP AFMK AMK ARE E2 GPxX GSR GST HPG Keap1 MDA MT Nrf2 RNS ROS SCN SOD STAR TAC TBARS

reproductive tract can produce high amounts of ROS just by itself. There are several factors that increase the levels of ROS in the male reproductive system including life style, obesity, aging, radiation, smoking, infections,2,3 chemotherapy,4,5 drugs,6,7 irradiation,8,9 testicular torsion/detorsion, varicocele,2 high concentrations of some metals such as iron, cadmium, and lead in the testes,10,11 cryptorchidism, varicocele,11 endocrinological disorders, hyperthyroidism,12 non-alcoholic fatty liver disease,13 and diabetes2,3 (Fig. 26.1).

androgen binding protein N-acetyl-N-formyl-5-methoxykynurenamine N-acetyl-5-methoxykynuramine antioxidant-response element 17-b-estradiol glutathione peroxidase glutathione reductase glutathione-S-transferase hypothalamo-pituitary-gonadal Kelch-like ECH-associating protein1 malondialdehyde melatonin receptors nuclear factor erythroid 2-related factor 2 reactive nitrogen species reactive oxygen species suprachiasmatic nucleus super oxide dismutase steroidogenic acute regulatory total antioxidant capacity thiobarbituric acid reactive substances

The antioxidant system in the male reproductive tract

Introduction Oxidative stress and male reproduction Any imbalance between the production of reactive oxygen species (ROS) and the antioxidant system results in oxidative stress.1 The overproduction of ROS may result in a variety of diseases and infertility.1 ROS is produced in high amounts in the male reproductive tract. Due to the presence of unsaturated fatty acids in high amounts and also the high levels of germ cell proliferation and metabolism in the testes, it is apparent that the testes represent the perfect environment to react with ROS.2 In addition, seminal fluid is the perfect environment for the aggregation of ROS. Therefore the male

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00026-3

The reproductive system of male mammals has two types agents, namely enzymatic and non-enzymatic antioxidant agents, to reduce the level of oxidants and associated side effects. The enzymatic agents involved are superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase, and glutathione-S-transferase (GST). There are many non-enzymatic agents including vitamins A, E, C, and B-complex, glutathione, carnitine, co-enzyme 10, resveratrol, melatonin, and some minerals including copper, selenium, zinc, and chrome,1,11 hypotaurine, taurine, albumin, and uric acid.14 Furthermore, there are several strategies for increasing the activity of the antioxidative system including physical exercise, the treatment of diseases, alterations in life style, and the consumption of antioxidants. Studies have indicated that the consumption of various antioxidants from different origins can repair or ameliorate testicular damage induced by injuries, drugs, chemotherapy, and toxic agents. There are various sources of antioxidants including vitamins, minerals, medicinal drugs, vegetables and

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26. Melatonin, antioxidant capacity, and male reproductive function

FIGURE 26.1 Diagram showing side effects of ROS on male fertility.

fruit extracts, which are rich in phenols and flavonoids, and also melatonin as a pineal hormone. One of the most common, important, and powerful materials that is used as an antioxidative and beneficial agent against various types of damage induced in the testes is melatonin.1,4
10,15
32

Melatonin Melatonin is an important hormone produced by the pineal gland and is secreted in a rhythmic manner. At first, melatonin was studied for its effects upon reproduction.33 Melatonin is produced in pinealocytes by enzymic reactions, which are depicted in Fig. 26.2. The production of melatonin is dependent upon the light/dark cycle.34 Melatonin can also be synthesized by other tissues and cells such as the Harderian gland, the gastrointestinal tract,34 testes,35 epithelial cells, bone marrow cells, lymphocytes, retina, placenta, the liver, and the ovaries.10,36 Mitochondria have also been shown to produce melatonin,37 thus, explaining why the level of melatonin is higher in mitochondria than in the cytosol and blood circulation.38 In other words, mitochondrial sources of melatonin can be found in every living organism.39,40 Melatonin is distributed by body fluids such as blood, Cerebrospinal fluid (CSF), ovarian follicles, and bile. The concentration of melatonin in these fluids depends on the time of day, shift work, an individual’s general health and diet, the ingestion of certain types of diet and

fruit, stress, age, drugs, and reproductive status.40 Chemically, melatonin is an indolamine. The presence of indol in melatonin is essential for the scavenging of free radicals and reducing ROS. If indol is removed or replaced by similar structures such as benzofurane then the antioxidant activity will be reduced. The indol in melatonin has two side chains located at C3 (3-amid) and C5 (5-methoxy).41 These two functional groups in melatonin are responsible for both its antioxidative ability and amphiphilic characteristics.41 For the reasons mentioned, the antioxidative effects of melatonin are several times higher than those of vitamin E and C.41

The antioxidative properties of melatonin The ability of melatonin to scavenge free radicals is very high. Tan was the first to describe the radical scavenging effect of melatonin.42 Subsequently, many studies have been carried out to confirm this effect both in vitro and in vivo.34,43
45 Melatonin also stimulates the upregulation of antioxidant enzymes, especially GPx, glutathione reductase and glutathatione,34,46 but also SOD, and rarely catalase. Melatonin can also neutralize both ROS and reactive nitrogen species (RNS).23 Furthermore, there is a positive correlation between the antioxidant activity of melatonin and serum melatonin levels during the light/dark cycle.47 Melatonin suppresses the activity of peroxidase enzymes including 5- and 12lipooxygenases and NO synthases.23 In addition, melatonin’s metabolites (three generations) including c3CHM,

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FIGURE 26.2 Diagram showing the synthesis steps of melatonin in pinealocytes.

N-acetyl-N-formyl-5-methoxykynurenamine (AFMK), and N-acetyl-5-methoxykynuramine (AMK) have powerful radical scavenging activity.34,48 In these reactions, melatonin can neutralize up to ten radical products. However, other free radical scavengers just act upon one molecule.34 Melatonin has amphiphilic characteristics and can, therefore, pass through any lipidic or aqueous molecule and can cross any membrane and act upon any cell or subcellular parts. It protects lipids, proteins, and DNA against oxidative stress.38 By binding to metals such as aluminum, cadmium, copper, iron, lead, and zinc, melatonin reduces the formation of ROS.49 Melatonin also acts upon SIRT3, a member of the silent information regulator 2 family located in the mitochondrial matrix to reduce oxidative stress.50 Melatonin also reduces oxygen consumption in the mitochondria resulting from the formation of low levels of ROS and can, therefore, reduce the rate of apoptosis.23 The antioxidative effects of melatonin occur through two mechanisms, namely direct and indirect. Melatonin scavenges free radicals via a direct effect. However, melatonin can also act indirectly through melatonin receptors located in the cell membranes or organelles to activate antioxidant enzymes. Via the indirect mechanism, melatonin shows significant antioxidative effects even at low concentrations. It is possible that signal transduction pathways associated with receptors amplify this response.40 The exact mechanisms related to antioxidant enzymic activity in response to melatonin remain unknown. It is possible that melatonin regulates the transcription factor Nrf2,40,51 which is critical for the regulation of various antioxidant genes.51,52 Nrf2 is found in vertebrates,52 is located in the cytoplasm, and is attached to the Keap1

protein. By upregulating Nrf2, melatonin can protect cells and increase the expression of antioxidant enzymes53 (Fig. 26.3).

Melatonin and the male reproductive system Due to the detection of melatonin receptors in the human hypothalamus and pituitary gland, it was concluded that melatonin regulates the production of gonadotropin-releasing hormones.10 Melatonin exerts many of its roles via its receptors. Melatonin (MT) receptors are located both in the brain, in the suprachiasmatic nucleus, to regulate the circadianpacemaker and in the peripheral organs. There are three types of this receptor, namely MT1, MT2, and MT3. Melatonin receptors belong to the superfamily of G-protein coupled receptors. Melatonin receptors are located on the plasma membrane, cytosol, nucleus, and mitochondrial membrane. It is possible that receptors in the mitochondria can provide protection against the high levels of ROS produced in this organelle.38 Exogenous melatonin increases the expression of MT1 and MT2 receptors in the testes in response to testicular damage.19,27 Furthermore, MT receptors been detected in both the female and male reproductive tract.54 Melatonin receptors have also been detected in the testes, epididymis, prostate, vas deferens, and seminal vesicles.55 In the testes, all types of germ cells, Leydig cells, and Sertoli cells express MT receptors.55,56 Melatonin is also known to exert effects upon the testes and the differentiation of male germ cells.19 The precise effects of melatonin on sex hormones varies and depends on both species and physiological

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26. Melatonin, antioxidant capacity, and male reproductive function

FIGURE 26.3 Various roles of melatonin in cells.

conditions. In rodents, during long days, the expression of androgen receptors and ABP is reduced by melatonin. However, the administration of melatonin to short-day breeders has been shown to have beneficial effects on their gonads.56 Melatonin affects the production of testosterone in Leydig cells both in a cAMP-dependent and a cAMPindependent pathway manner.10,57 Furthermore, in a dose-dependent manner, melatonin has been shown to inhibit the secretion of testosterone in rat Leydig cells.58 Melatonin has also been shown to inhibit the production of androgens via MT1 receptors in hamster Leydig cells.59 In mouse ma-10 Leydig tumor cells, melatonin inhibits STAR protein and acts through both MT1 and MT2 receptors.35 In rat Sertoli cells, melatonin has been shown to reduce both the expression and activity of lactate dehydrogenase.57 Melatonin also increases the expression of cyclin D1 and cyclin E along with occludin and claudin in Sertoli cells, which regulate spermatogenesis.57,60 In contrast to many studies that focus on the beneficial effects of melatonin on the testes and male fertility, there are several reports relating to the suppressive effects of melatonin on the testes. For example, the administration of melatonin to prepubertal mice for 6 weeks resulted in the reduced diameters of the seminiferous tubules and an increased proportion of aspermic tubules.61 Furthermore, the application of

exogenous melatonin to aged mice reduced the germinal epithelium thickness in the seminiferous tubules and spermatogenesis index.62 Also, the injection of melatonin suppressed the testes of male hamsters. Melatonin has also been shown to inhibit the production of androgens in hamster testes.23 These observations suggest that the age of the animal and the species itself may have an important effect in the role of melatonin on male reproduction.

Melatonin: antioxidative properties and testicular tissue protection Over the past few decades, extensive studies have been performed on the beneficial effects of melatonin on the testes including a range of chemical, pharmaceutical, disease, and toxic agents (Table 26.1). Melatonin has been shown to protect the testes against 2bromopropane,24 dexamethasone,27 bisphenol A,28 cadmium,10 gentamicin,7 microwave radiation,25 radiofrequency,9 ischemic reperfusion,63 Co γ-ray,8 testicular torsion
detorsion,64 formaldehyde,29 epilepsy,30 diabetes,22 cigarette smoking,15 nicotine,26 acetyl salicylic acid,20 di-2-ethylhexyl phatalate,18 chemotherapy with doxorubicin,4 procarbazine,6 cisplatin,16 cyclophosphamide,5 busulfan,21,19,10 and obesity.17 The most important beneficial effect of melatonin in these studies was

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TABLE 26.1

A summary of the results of beneficial and antioxidative effects of melatonin on testis damaged by various toxicants. Main results after co-treatment with toxic agent and melatonin

Species

Toxic agent

Melatonin dose

Reference

Rat

2-bromopropane

5 mg/kg a single dose

Ns body weight m Testis weight m Sperm quality m Testis GSH Ns sperm GSH k Apoptosis index k Caspase 3 activity

24

Golden hamster

Dexamethasone

10 mg/kg/d for 7 days

m Body weight m Testis weight m SOD, catalase, and GSH-PX k MDA k Apoptosis index k Bax/BCL2 and caspase 3 m MT1 receptor in testis

27

Rat

Bisphenol A

10 mg/kg/d for 3 and 6 weeks

k Oxidative stress m Glutathione, SOD, CAT in Testis and sperm MDA, H2O2 in testis and sperm m Bcl2 expression k Apoptosis percent m Serum testosterone

28

Mouse

Cadmium

10 mg/kg for 2 days

Ns testis weight m Sperm mitochondrial Membrane potential m Acrosomal integrity Ns sperm number m Sperm motility k Abnormal sperm k Germ cell apoptosis m SOD, GSH, in testis k MDA in testis k TNFα and IL-1 in testis m Serum testosterone m Serum inhibin and LH k Serum FSH

10

Rat

Gentamicin

15 mg/kg for 6 days

m Sperm count and motility Ns sperm morphology m Spermatogonia number m GSH, GST, catalase in testis Ns MDA in testis

7

Rat

Microwave radiation

2 mg/kg

Ns protein carbonyl content Ns alkaline DNAase activity Ns catalase activity k MDA level k Xanthine oxidase activity k Acid DNAse activity

31

Rat

Ischemic reperfusion (IR)

10 mg/kg 1 h before and After (IR)

k Abnormal sperm m Spermatogenic index

63

Mouse

Radiation radiofrequency

5 mg/kg for 35 days

m GSH and SOD level in testis k TBARS in testis m Sperm parameters k Sperm DNA fragmentation Spermatogonia number m Spermatid number

9

(Continued)

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270 TABLE 26.1

26. Melatonin, antioxidant capacity, and male reproductive function

(Continued) Main results after co-treatment with toxic agent and melatonin

Species

Toxic agent

Melatonin dose

Reference

Mouse

60 Co γ-ray

100 mg/kg, 30 min before radiotherapy

m Sperm parameters m Testicular TAC m Spermatogonia/Sertoli ratio

8

Rat

Testicular torsion
detorsion

17 mg/kg, 15 min before detorsion

m Serum inhibin m Spermatogenesis index k MDA in testis

64

Rat

Formaldehyde

25 mg/kg every other day for 1 month

k MDA in testis m SOD and GSH-PX in testis k Bax in germ cells and Leydig cells

29

Rat

Procarbazine

10 mg/kg 5 days/week for 4 weeks

Ns testis and body weight Ns sperm parameters Ns seminiferous tubule thickness Ns serum FSH and LH Ns serum testosterone Ns SOD and NO2/N3 in testis Ns MDA in testis m GPX in testis

6

Rat

Doxorubicin

15 mg/kg/d for 10 days

Ns body weight Ns sperm morphology Ns catalase in testis Ns GSH in testis k MDA in testis m GR, GST, SOD in testis

4

Rat

Cyclophosphamide and cisplatin

10 mg/kg (single dose)

m Sperm count and motility k MDA in testis m GSH and GSH-PX in testis m Serum testosterone m Spermatogenic index

5

Rat

Cisplatin

10 mg/kg for 5 days

m Testis weight after 5 days Ns testis weight after 50 days m Sperm count after 5 days Ns sperm count after 50 days m Sperm motility after 5 and 50 days

16

Rat

Cigarette smoke

25 mg/kg 5 days/week for 4 weeks

m Spermatocyte number Ns spermatogonia number

15

Rat

Streptozotocin

5 mg/kg for 5 days

m Spermatogenesis index k Seminiferous tubule diameter k Germ cell apoptosis k Basement membrane thickness

22

Mouse

Di-(2-ethylhexyl) phthalate

10 mg/kg for 14 days

Ns body and testis weight m Serum testosterone k Serum LH m Testicular TAS level Ns GSH in testis m SOD and catalase in testis k MDA and NO in testis k TNF-α and IL-1 beta in testis

18

Rat

Microwave radiation

2 mg/kg/d for 45 days

Ns testis weight k Protein carbonyl content in testis k ROS in testis k Xanthine oxide in testis k MDA in testis k Apoptosis in sperm

25

(Continued)

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Introduction

TABLE 26.1 Species

(Continued) Toxic agent

Melatonin dose

Main results after co-treatment with toxic agent and melatonin

Reference

m Serum testosterone m LDH-X activity in testis Mouse

Acetyl salicylic acid

10 mg/kg for 14 days

m Sperm parameters m Sperm chromatin integrity k Sperm DNA fragmentation index k Germ cell apoptosis m Serum TAC m Serum testosterone

20

Mouse

Nicotine

10 mg/kg for 30 days

Ns spermatogonia number m Leptotene spermatocyte number m Pachytene spermatocyte number m Round spermatid number m Halo sperm (%) m Sperm DNA fragmentation index k Germ cell apoptosis m Sperm parameters Ns serum LH m Serum testosterone

26

Mouse

Busulfan

10 mg/kg in vivo 10-7 m in vitro

k ERS (endoplasmic reticulum stress) k ERS apoptotic proteins

19

C18-4 cell line Ns, not significant; m increase; k decrease.

the reduction of oxidative stress and, therefore, the reduction of male germ cell apoptosis, improvements in sperm quality, and consequent better spermatogenesis. In most previous studies on melatonin, doses of 5 and 10 mg/kg were able to show antioxidative activity and, therefore, had beneficial effects on testicular function and histopathology (Table 26.1). However, other doses such as 15, 17, 20, 25, and 100 mg/kg have also shown such effects (Table 26.1). Even at a low dose of 2 mg/kg, melatonin was able to show antioxidative effects in the testes.31,25 It is possible that at low doses, melatonin acts indirectly via its receptors, MT1 and MT2, in the testes.40 A previous study showed that just one hour after the administration of melatonin at a dose of 2
5 mg/kg, serum levels reached pharmacological values and diminished gradually to basal values over the course of 8
12 hours.65 A single dose of melatonin at a low dose against 2-bromopropane,24 cyclophosphamide, and cisplatin,5 and prior to testicular detorsion,64 or even a single high dose of 100 mg/kg before radiotherapy,8 can show antioxidative properties in the testes and is, therefore, useful for spermatogenesis. In vitro models have shown that melatonin has prooxidant effects at high doses. It is possible that at high doses, melatonin can alter the function of the mitochondria or activate calmodulin. Therefore in normal cells, melatonin has antiapoptotic effects. However, in cancer cells, melatonin can show pro-apoptotic effects.66

In many earlier studies, melatonin was shown to increase testicular Glutathione (GSH), SOD, catalase, GSH-PX, and GST activity. In addition, melatonin is able to reduce H2O2, MDA, and TBARS activity in the testes. Consequently, melatonin can result in better spermatogenesis and sperm parameters (Table 26.1). In some reports, testicular MDA,6,7 catalase,4,31 SOD,6 GSH activity, 4,18 and NO2/NO3 6 activity in the testes did not change following melatonin treatment. These data indicate the complexity in the arrangement and organization of these key enzymes.23 In other words, these reports suggest that species, duration and dose of melatonin treatment, the type of toxic agent, and the type of tissue are critical in the responsivity of the testes to exogenous melatonin. By increasing TAC in the testes or serum, melatonin can have useful effects on spermatogenesis against radiotherapy,8 di-2-(ethylhexyl) phthalate,18 and acetyl salicylic acid.20 TAC is described as a set of reactions between different antioxidant elements and their effects on the antioxidant-pro-oxidant balance. TAC reflects the low molecular weights of antioxidants and does not reflect the antioxidant enzyme activity.1 Various doses of melatonin can also reduce the apoptosis index and markers in the testes against different toxic agents that can induce damage in the testes (Table 26.1). By reducing oxidative stress, melatonin is able to reduce the sperm DNA fragmentation index or can increase the numbers of halo sperms. In other words

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26. Melatonin, antioxidant capacity, and male reproductive function

it is able to increase the integrity of sperm chromatin.9,20,26 It is possible that by acting on both Leydig cells and Sertoli cells, which have melatonin receptors, melatonin could protect the testes against pathological and toxic agents. In this regard it has been shown that melatonin affects the endocrine activity of Leydig cells. Melatonin can also influence cell growth, proliferation, and oxidant/antioxidant state in the testes via the Sertoli cells.57 The HPG-axis, and also the Leydig cells, are the main sources of testosterone production. Therefore the reduction of ROS may result in modifications to the sex hormones. An increase in serum testosterone has been documented following the administration of melatonin to rats treated with bisphenol A,28 mice treated with cadmium,10 rats treated with cyclophosphamide 1 cisplatin,5 mice treated with busulfan,21 mice treated with di-2-ethylhexyl phatalate,18 rats treated with radiotherapy,25 mice treated with acetyl salicylic acid,20 and mice treated with nicotine26 (Table 26.1).

Melatonin, semen, and sperm quality Melatonin is found in the fluid that forms the semen. However, there is no evidence to suggest that the seminal vesicles produce melatonin locally. It appears that the origin of the melatonin in the semen is actually the blood. This is why the concentration of melatonin in semen is much lower than in the blood. The protective role of melatonin against oxidative stress is both ambiguous and controversial. In this regard, it has been proposed that the antioxidative function of melatonin in human semen is not particularly beneficial because the levels of melatonin in the semen are very low, usually at the nanomolar level.67 The incubation of human spermatozoa with 1 mM of melatonin for 30 minute was shown to increase sperm motility.68 In other words, low levels of semen melatonin are associated with high levels of sperm abnormality. In this regard, it has been reported that patients with erectile dysfunction have lower serum melatonin level than that of controls.69 In azoospermic and teratozoospermic patients, the levels of melatonin in the semen are lower in comparison with fertile men.1 In hamsters, melatonin treatment activates motility of the sperm flagella and, therefore, results in better penetration of sperm into the oocyte.1 Melatonin treatment in mice in which testicular damage had been incurred by cadmium, led to an increase in sperm mitochondrial membrane potential, acrosome integrity, and sperm motility, and promoted normal morphology. It is believed that these effects occurred due to the elevation of SOD and GSH activity in the testes.10 By affecting the production of ATP, stabilizing the inner membrane, and modifying the electron transport

chain in mitochondria, melatonin may increase sperm motility.9,68 In testes damaged by radiation, melatonin is known to improve sperm parameters and by reducing ROS also reducing the levels of sperm DNA fragmentation.9 Melatonin ameliorates the toxic effects of chemotherapy on sperm parameters.4,5,16,21 The reduction of sperm DNA fragmentation index, increased sperm chromatin integrity, and improved sperm count, morphology, and motility have been reported following the administration of melatonin in mice treated with acetyl salicylic acid20 and nicotine.26 Therefore the administration of melatonin as a powerful antioxidant may enhance sperm quality. In contrast to the studies mentioned, there are also some contradictory results. The long-term oral administration of melatonin at a daily dose of 3 mg/kg for 6 months to eight healthy young males resulted in no changes in semen quality, serum testosterone, E2, Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and melatonin levels in six of the males. However, in the two remaining males, sperm count, motility, and E2 levels were reduced. It is possible that this was due to aromatase inhibition during the long period of administration, or due to the inhibitory effects of melatonin on gonadotropins.65 Melatonin may also act upon tubulins and suppress the motility of the sperm flagella.65 It has also been claimed that the presence of melatonin in the seminal plasma has inhibitory effects on sperm motility and sperm count.70

Application in other areas of pathology The antioxidative and protective effects of melatonin are observed in other pathological conditions. The neuroprotective properties of melatonin have been shown in aging and Alzheimer’s disease animal models.71 Melatonin regulates the production of ROS in diabetic pancreas models.66 In fibrosis and fatty liver models, melatonin also has protective effects.66 Melatonin ameliorates damages induced by ischemic reperfusion in the heart and brain both in animals and in humans.34 Melatonin has various roles in cells. For example, melatonin has antioxidant and anti-inflammatory properties, prevents tumors from spreading, regulates the circadian rhythm and sleep,34 regulates apoptosis and cell proliferation,19 but also exerts functions including antidiabetes,72 the regulation of body weight,32 and antiaging actions.40

Concluding remarks Melatonin is an antioxidant that has beneficial effects on spermatogenesis and male fertility. These

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References

beneficial effects are evident at low and high doses and also over acute and chronic time courses. However, some reports describe side effects on male reproduction, but it is evident that the reported data depend on the dose and duration of melatonin administration, physiological and pathological conditions, species, age, and differences in protocol. There have been many experiments conducted on the effects of melatonin on the testes and the reproductive system of male animals. However, only few experiments have been carried out in this regard in humans. Therefore specific pharmacological studies should be carried out in humans in the future to investigate the potential benefits and risks of melatonin.

10.

11. 12.

13.

14.

15.

Summary points • This chapter focuses on melatonin, a powerful antioxidant, in relation to male reproduction. • The presence of indol in melatonin is essential for the scavenging of free radicals and reducing ROS. • The antioxidative effects of melatonin are several times higher than those of vitamin E and C. • The antioxidative effects of melatonin occur through direct and indirect mechanisms. • Melatonin protects male fertility against chemotherapy, drugs, diseases, and toxic agents

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7. Atilgan D, Parlaktas BS, Uluocak N, Erdemir F, Kilic S, Erkorkmaz U, et al. Weight loss and melatonin reduce obesityinduced oxidative damage in rat testis. Adv Urol 2013;2013. Bahrami N, Goudarzi M, Hosseinzadeh A, Sabbagh S, Reiter RJ, Mehrzadi S. Evaluating the protective effects of melatonin on di (2-ethylhexyl) phthalate-induced testicular injury in adult mice. Biomed Pharmacother 2018;108:515
23. Cui Y, Ren L, Li B, Fang J, Zhai Y, He X, et al. Melatonin relieves busulfan-induced spermatogonial stem cell apoptosis of mouse testis by inhibiting endoplasmic reticulum stress. Cell Physiol Biochem 2017;44(6):2407
21. Emami NH, Lafout FM, Mohammadghasemi F. Administration of melatonin protects against acetylsalicylic acid-induced impairment of male reproductive function in mice. Iran J Basic Med Sci 2018;21(2):124. Ghasemi FM, Faghani M, Khajehjahromi S, Bahadori M, Nasiri E, Hemadi M. Effect of melatonin on proliferative activity and apoptosis in spermatogenic cells in mouse under chemotherapy. Zhonghua Shengzhi Yu Biyun Zazhi (Chinese J Reprod Contracept.) 2010;21(2):79
94. Guneli E, Tugyan K, Ozturk H, Gumustekin M, Cilaker S, Uysal N. Effect of melatonin on testicular damage in streptozotocininduced diabetes rats. Eur Surg Res 2008;40(4):354
60. Hardeland R. Atioxidative protection by melatonin. Endocrine 2005;27(2):119
30. Huang F, Ning H, Xin Q-Q, Huang Y, Wang H, Zhang ZH, et al. Melatonin pretreatment attenuates 2-bromopropane-induced testicular toxicity in rats. Toxicology 2009;256(1
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91. Mohammadghasemi F, Khajeh Jahromi S. Melatonin ameliorates testicular damages induced by nicotine in mice. Iran J Basic Med Sci 2018;21(6):639
44. Mukherjee A, Haldar C, Vishwas DK. Melatonin prevents dexamethasone-induced testicular oxidative stress and germ cell apoptosis in golden hamster, M esocricetus auratus. Andrologia 2015;47(8):920
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76. 30. Shokri S, Kazemi M, Firouzjaei M, Hemadi M, Moayeri A, Ganjkhani M, et al. Melatonin protects testes against lithiumpilocarpine-induced temporal lobe epilepsy in rats: a time course study. Andrologia 2015;47(3):343
53. 31. Sokolovic D, Djordjevic B, Kocic G, Stoimenov TJ, Stanojkovic Z, Sokolovic DM, et al. The effects of melatonin on oxidative stress parameters and DNA fragmentation in testicular tissue of rats exposed to microwave radiation. Adv ClExp Med Off Organ Wroclaw Med Univ 2015;24(3):429
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9. 45. Sewerynek E, Melchiorri D, Ortiz GG, Poeggeler B, Reiter RJ. Melatonin reduces H2O2-induced lipid peroxidation in homogenates of different rat brain regions. J Pineal Res 1995;19(1):51
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47. Benot S, Gobema R, Reiter RJ, Garcia-Maurin˜o S, Osuna C, Guerrero JM. Physiological levels of melatonin contribute to the antioxidant capacity of human serum. J Pineal Res 1999;27 (1):59
64. 48. Schaefer M, Hardeland R. The melatonin metabolite N1-acetyl5-methoxykynuramine is a potent singlet oxygen scavenger. J Pineal Res 2009;46(1):49
52. 49. Limson J, Nyokong T, Daya S. The interaction of melatonin and its precursors with aluminium, cadmium, copper, iron, lead, and zinc: an adsorptive voltammetric study. J Pineal Res 1998;24(1):15
21. 50. Zhai M, Li B, Duan W, Jing L, Zhang B, Zhang M, et al. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT 3-dependent regulation of oxidative stress and apoptosis. J Pineal Res 2017;63(2):e12419. 51. Ding K, Wang H, Xu J, Li T, Zhang L, Ding Y, et al. Melatonin stimulates antioxidant enzymes and reduces oxidative stress in experimental traumatic brain injury: the Nrf2
ARE signaling pathway as a potential mechanism. Free Radic Biol Med 2014;73:1
11. 52. Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul 2006;46:113
40. 53. Sun B, Yang S, Li S, Hang C. Melatonin upregulates nuclear factor erythroid-2 related factor 2 (Nrf2) and mediates mitophagy to protect against early brain injury after subarachnoid hemorrhage. Med Sci Monit 2018;24:6422. 54. Hemadi M, Saki G, Shokri S, Ghasemi FM. Follicular dynamics in neonate vitrified ovarian grafts after host treatment with melatonin. Folia Morphol (Wars.) (Engl Transl.) 2011;70(1):18
23. 55. Gonza´lez-Arto M, Aguilar D, Gaspar-Torrubia E, Gallego M, Carvajal-Serna M, Herrera-Marcos L, et al. Melatonin MT1 and MT2 receptors in the ram reproductive tract. Int J Mol Sci 2017;18(3):662. 56. Yu K, Deng S-L, Sun T-C, Li Y-Y, Liu Y-X. Melatonin regulates the synthesis of steroid hormones on male reproduction: a review. Molecules 2018;23(2):447. 57. Frungieri M, Calandra R, Rossi S. Local actions of melatonin in somatic cells of the testis. Int J Mol Sci 2017;18(6):1170. 58. Valenti S, Guido R, Giusti M, Giordano G. In vitro acute and prolonged effects of melatonin on purified rat Leydig cell steroidogenesis and adenosine 30 , 50 -monophosphate production. Endocrinology 1995;136(12):5357
62. 59. Frungieri MB, Mayerhofer A, Zitta K, Pignataro OP, Calandra RS, Gonzalez-Calvar SI. Direct effect of melatonin on Syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and interaction with the local corticotropin-releasing hormone system. Endocrinology 2005;146 (3):1541
52. 60. Yang W-C, Tang K-Q, Fu C-Z, Riaz H, Zhang Q, Zan L-S. Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2. Anim Reprod Sci 2014;147(1-2):10
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45. 62. Mehraein F, Negahdar F. Morphometric evaluation of seminiferous tubules in aged mice testes after melatonin administration. Cell J 2011;13(1):1. 63. Kurcer Z, Hekimoglu A, Aral F, Baba F, Sahna E. Effect of melatonin on epididymal sperm quality after testicular ischemia/ reperfusion in rats. Fertil Steril 2010;93(5):1545
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C H A P T E R

27 Methylsulfonylmethane as an antioxidant and its use in pathology Matthew Butawan1, Rodney L. Benjamin2 and Richard J. Bloomer1 1

School of Health Studies, The University of Memphis, Memphis, TN, United States, 2Bergstrom Nutrition, Vancouver, WA, United States

List of abbreviations BBB CAT CCl4 DMSO GI GRAS GPx GR GSH GSSG GST H2O2 HIV IBD IL iNOS LPS MPO MSM NADP1 NADPH NFκB NLRP3 NO NOAEL NOX Nrf2 O22 OH2 ONOO2 OPD PUFA ROS SOD TAC TEAC TLR

TNFα Trx/Prx

blood
brain barrier catalase carbon tetrachloride dimethylsulfoxide gastrointestinal generally recognized as safe glutathione peroxidase glutathione reductase reduced glutathione oxidized glutathione glutathione S-transferase hydrogen peroxide human immunodeficiency virus inflammatory bowel disease interleukin inducible nitric oxide synthase lipopolysaccharide myeloperoxidase methylsulfonylmethane oxidized nicotinamide adenine dinucleotide diphosphate reduced nicotinamide adenine dinucleotide diphosphate nuclear Factor Kappa-light-chain-enhancer of activated B cells Nucleotide-binding domain, leucine-rich repeat family pyrin domain containing 3 nitric oxide no-observed-adverse-event-level NADPH oxidase nuclear factor (erythroid-derived 2)-like 2 superoxide anion hydroxyl radical peroxynitrite radical O-phenylenediamine polyunsaturated fatty acid reactive oxygen species superoxide dismutase total antioxidant capacity Trolox equivalent antioxidant capacity toll-like receptor

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00027-5

tumor necrosis factor-α thioredoxin/peroxiredoxin system

Introduction Methylsulfonylmethane (MSM) is an organosulfur compound of the Earth’s sulfur cycle along with dimethylsulfide and dimethylsulfoxide (DMSO). Beginning in the early 1960s, researchers began experimenting with DMSO as a cryopreservant for organ transplantation with much success. MSM later gained attention after Williams and colleagues reported that DMSO was oxidized to MSM in rabbits.1 Combined with the fact that MSM did not have the malodorous side effects that DMSO possessed, MSM gained traction as the more commonly used supplement. Since these early investigations, the biological effects of MSM have expanded greatly with much research demonstrating the antiinflammatory, immunomodulatory, and antioxidant actions of MSM. With these early investigations leading to the use of MSM as a dietary supplement came the sponsorship of multiple private industry toxicity reports. Table 27.1 displays MSM toxicity summaries from both sponsored toxicity reports and published in vivo and in vitro data. The data suggest that MSM is nontoxic at relatively high dosages, for example, the no-observed-adverse-eventlevel (NOAEL) is listed at 5 g/kg when given orally or intraperitoneally to mice or rats.2 In fact, to our knowledge, only a single mortality has occurred in any MSM toxicity study using a dosage of 15,380 mg MSM/kg of body weight. This study reported the acute oral LD50 of $ 17,020 mg MSM/kg of body weight. That said, doses used in humans are much lower, with most doses being

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© 2020 Elsevier Inc. All rights reserved.

278 TABLE 27.1

27. Methylsulfonylmethane as an antioxidant and its use in pathology

MSM toxicity summary.

Cell line

Incubation time

Dosage (IC50)

Human smooth muscle

96 h

1% (reversible at 1%; partially reversible at 2%
3%; irreversible at 4%)

Human endothelial

96 h

1.8% (completely reversible up to 4%)

SK-BR3 (human breast cancer)

24 h

300 mM

MDA-MB231 (human breast adenocarcinoma)

24 h

300 mM

AGS (human gastric adenocarcinoma)

72 h

297.8 mM

HepG2 (human liver carcinoma)

72 h

232.3 mM

KYSE-30 (human esophageal carcinoma)

72 h

297.2 mM

YD-38 (human gingival carcinoma)

24 h

200 mM

Species

Route

Duration

NOAEL

Acute # 15 days Mice

Oral

Not stated (acute)

5 g/kg BW

Mice

Intraperitoneal

Not stated (acute)

5 g/kg BW

Mice

Oral gavage

14 days

2 g/kg BW

Mice

Oral gavage

15 days

5.0 g/kg BW

Rat

Intragastric

6 days

20 g/kg BW

Intragastric

14 days

10.25 g/kg BW

a

Rat

LD50 $ 17.02 g/kg BW Rat

Intraperitoneal

Not stated (acute)

5 g/kg BW

Rat

Oral gavage

14 days

5.0 g/kg BW

Rat

Oral gavage

14 days

2 g/kg BW

Rat

Serial intranasal injections

Not stated (B3 days)

0.6 ml/kg BW

Rat

Oral gavage

15 days

2 g/kg BW

Rabbit

Dermal

14 days

5.0 g/kg BW

Dog Subacute Gestating Rat

Oral

Not stated

2 g/kg BW

Oral gavage (14 days)

21 days

1 g/kg BW/day

Cow Subchronic Rats

Oral

30 days

1.2 g/kg BW/day

Oral gavage

90 days

1.5 g/kg BW/day

Mice

Oral

91 days

1.5 g/kg BW

Rat

Oral

90 days

1.5 g/kg BW

Horse

Oral syringe

84 days

0.04 g/kg BW/day

a

Study with reported mortality. In vitro and in vivo toxicity studies summarized from peer-reviewed studies and industry-sponsored reports.

between 1 and 6 g/day. Aided by these toxicity reports, Bergstrom Nutrition’s OptiMSM was awarded the generally recognized as safe (GRAS) status by the United States Food and Drug Administration (USFDA). Pharmacokinetic studies in rats using MSM with radiolabeled sulfur have yielded interesting results. In one such study, MSM was shown to be primarily eliminated in the urine while the remaining MSM was

widely distributed throughout bodily tissues.3 The following study elaborated on these findings by quantifying the amount of MSM reaching various organs.4 An overview of the findings from these studies is displayed in Fig. 27.1. Work in humans indicate that plasma levels appear to stabilize after about 4 weeks of daily supplementation, with stable trough concentrations being dose-dependent.5 The tissue distribution

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Overview of antioxidant effects

279

FIGURE 27.1 Tissue distribution and bioavailability of MSM. Elimination and tissue distribution of MSM in rats 48 h postoral ingestion. (A) Approximately 90% of orally ingested MSM is eliminated via urine or feces with approximately 10% remaining in the body 48 h after ingestion; percentages displayed from Otsuki et al.3 data; (B) The remaining 10% appears to be roughly evenly distributed throughout tissues; percentages adapted from Magnuson et al.4 data.

in humans is expected to be similar to those observed in rats as MSM has been shown to cross the blood
brain barrier (BBB) in humans as well.6 Currently, MSM is most commonly used as a treatment for arthritis7,8 and interstitial cystitis.9 However, with its broad bodily distribution and pleiotropic effects, MSM may have many other potential preventative applications. In the following sections, the potential cellular and tissue effects of MSM as an antioxidant will be discussed along with the possible influences on pathophysiology.

Overview of antioxidant effects Biophysical properties The unique physicochemical properties of MSM lend themselves to interesting biophysical effects, particularly on membrane interactions. To better understand the antioxidant functions of MSM, the biophysical effects of MSM and the more heavily researched DMSO should be discussed as these may influence the function of the double membrane
bound organelle and primary producer of reactive oxygen species (ROS), the mitochondria. Mitochondrial dysfunction is believed to be a major contributor to the pathogenesis of many disease states.10 With respect to membrane interactions, DMSO has previously been shown to modulate the structure and properties of cell membranes11 and the adjacent water ordering12 in a concentration-dependent manner. At low concentrations, DMSO causes the membrane to thin. As the concentration of DMSO increases, pores form as

DMSO interacts with polar phospholipids. Beyond a concentration threshold around 25% molar concentration for DMSO, the membrane disintegrates in the hygroscopic medium,11 while concentrations above 0.1 molar DMSO fraction result in dehydration of the lipid membranes and reduced intermembrane distances.12 Together these biophysical effects may influence the electrochemical gradient and could have implications in vesicle secretion as these often involve a change in membrane potential or depolarization. Madji et al. reported that DMSO increases exocytotic neurotransmitter release.13 Alternatively, these effects could also influence the mitochondrial membrane potential, and, in fact, DMSO has previously been shown to impair mitochondrial membrane integrity and alter the mitochondrial membrane potential in vitro.14 The mentioned biophysical effects of DMSO may be similar for MSM as both are commonly used organic solvents and effective co-transporters. Moreover, MSM may reduce the mitochondrial membrane potential of both cancerous15 and noncancerous cells16 through similar actions.

Antioxidant enzyme production/activity ROS are essential components of cell signaling, but when produced in excess can damage lipids, proteins, and nucleic acids. That said, a homeostatic range of intracellular ROS must be maintained by balancing ROS production and both nonenzymatic and enzymatic antioxidants. Nuclear factor erythroid 2-related factor 2 (Nrf2) is emerging as a key modulator of oxidative stress by sensing redox status and regulating

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280

27. Methylsulfonylmethane as an antioxidant and its use in pathology

antioxidant-related gene expression. Upon activation of Nrf2 by ROS, Nrf2 translocates to the nucleus and upregulates superoxide dismutase (SOD)-3, peroxiredoxin, thioredoxin, thioredoxin reductase, and many glutathione-related genes. MSM has previously been demonstrated to attenuate toxin-induced reductions in antioxidant enzymatic activities, specifically that of SOD17
19 and catalase (CAT)17
20 in multiple tissues. These alterations may be attributed to an increase in the translocation of Nrf2 to the nucleus, an effect previously observed in a neuroblastoma cell line.21 Increased activities of SOD and CAT would improve the neutralization of superoxide radical (O2 2 ) and hydrogen peroxide (H2O2) respectively, and possibly hydroxyl radical (OH2) production from the dissociation of H2O2. The diverse cellular effects of MSM can theoretically result in numerous changes in antioxidant enzyme production and activity. Presently, pretreatment with MSM has only been shown to attenuate toxin-induced reductions in SOD, CAT, and glutathione peroxidase (GPx)19 as well as reduce myeloperoxidase (MPO) activity.17,20,22 Though Nrf2 is known to upregulate glutathione-related genes, these effects do not always translate to changes in enzymatic activity. For example, increased translocation of Nrf2 to the nucleus does not change the activity of either GPx or glutathione S-transferase (GST).21 If an increased production in glutathione synthesis genes is accomplished, an increase in intracellular glutathione concentrations could greatly enhance the redox hub activity, which will be discussed in the following section.

Redox hub The redox hub is the inter-cycling of molecules between reduced and oxidized forms. This cycling can occur via enzymatic or nonenzymatic reactions. At the center of the hub is the cycling of reduced glutathione (GSH) and oxidized glutathione (GSSG). The interconversion of GSH and GSSG plays a central role as it can also affect the cycling of vitamin C between its reduced form, ascorbate, and its oxidized forms, monodehydroascorbate (mDHA) and dehydroascorbate (DHA) as well as convert lipid radicals to less reactive lipid hydroxides or hydroxy peroxide. Fig. 27.2 displays a simplified diagram of the redox hub with enzymatic and nonenzymatic reactions. In addition to regulating reduced and oxidized forms of certain molecules, the redox hub also contributes to the metabolic state of the cell as enzymes such as glutathione reductase (GR) oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the oxidized form (NADP1). These molecules are highly involved in multiple biosynthesis pathways.

FIGURE 27.2 Redox hub. The redox hub represents intracellular small molecules that can take on either reduced or oxidized conformations. The oxidation/reduction of glutathione appears to interact with the reduction/oxidation of other intracellular small molecules. MSM may be able to influence the GSH:GSSG ratio. ASC, Ascorbate; H2O, water; Prx, peroxiredoxin; TR, thioredoxin reductase; TXSH, reduced thioredoxins; TXSSXT, oxidized thioredoxins.

The ratio of GSH/GSSG is often used as an indicator of oxidative status. In a previous in vitro study, MSM pretreatment reportedly restored the GSH/GSSG ratio following human immunodeficiency virus type 1 (HIV-1)-Tat exposure.21 In terms of GSH, Tat exposure with MSM pretreatment was significantly greater than the levels from Tat exposure alone, while GSSG levels were significantly lower in Tat exposure with MSM versus Tat alone. Similar results have been found in a variety of tissues, such that GSH with MSM treatment was significantly increased17,19,20,22
24 and GSSG levels were significantly decreased.19 However, given the dynamic nature of these markers, these results are not always observed.25
27

Free radical scavenging Free radical scavenging was a proposed antioxidant mechanism of MSM following a single cell
free experiment in which MSM showed the ability to reduce the quenching activity of hypochlorite on O-phenylenediamine (OPD) oxidation, though this effect was much less pronounced than with DMSO.28 It is plausible that MSM may have been reduced to DMSO by OPD, thereby providing the quenching power as sulfoxides can then be oxidized to sulfones through reactions with hypochlorite. No other models of free radical scavenging have been proposed or tested. Therefore

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Methylsulfonylmethane in oxidative stress pathology

this is one of the less likely mechanisms of antioxidant function for MSM.

Methylsulfonylmethane in oxidative stress pathology Inflammation Chronic low-grade inflammation is a component of nearly all disease pathologies. Inflammation and oxidative stress have an interdependent relationship, whereby excess ROS can induce proinflammatory cytokine production and/or cytokine signaling can include ROS production. At the center of this interdependent relationship usually lie inflammasomes, multiprotein complexes responsible for the activation of the inflammatory response. The NLRP3 inflammasome has been linked to the pathogenesis of several diseases.29 In short, a proinflammatory priming signal triggers NLRP3 monomer transcription and translation. Upon activation by one of several signals including ROS generation, NLRP3 monomers oligomerize to form the activated NLRP3 inflammasome, which can then promote the maturation and secretion of proinflammatory cytokines. Additionally, activated NLRP3 may also degrade the antioxidant enzyme regulator Nrf2. MSM may modulate oxidative stress
inflammation crosstalk at the priming and/or activation step(s). During the priming step, MSM may reduce the activity of the proinflammatory NFκB transcription factor. This reduces the transcription of the NLRP3 monomer, thus, resulting in impaired NLRP3 inflammasome

281

assembly and reduced suppression on Nrf2. Fig. 27.3 displays the potential actions of MSM, ultimately leading to a reduction in ROS available to act as a stimulatory signal for both NFκB nuclear translocation and NLRP3 inflammasome assembly. Previous MSM studies using lipopolysaccharide (LPS)induced toll-like receptor (TLR)-4 activation support this. For example, Kim et al.30 reported that MSM reduced the nuclear translocation of NFκB. Furthermore, Ahn et al. reported that NLRP3 inflammasome activation was inhibited with less intracellular ROS.31 By mediating this crosstalk between inflammation and oxidative stress, MSM may alleviate the chronic low-grade inflammation associated with the pathogenesis of many chronic disorders.

Brain In addition to cognitive processing, the human brain indirectly coordinates numerous peripheral organ functions through the activation of various neuroendocrine axes such as the hypothalamic-pituitary-adrenal and the hypothalamic-pituitary-thyroid axes. These dynamic functions require the brain to constantly remain plastic. In order to handle all of these functions, the brain has an extremely high energy and oxygen demand. In fact, the brain consumes B20% of the body’s total oxygen consumption to support this demand. This large oxidative metabolism makes the brain susceptible to the overproduction of ROS. Major brain sources of O2 2 are derived from NADPH oxidase (NOX) activity and complex I of the mitochondrial energy metabolism.32 Within the brain, H2O2 is an important ROS/signaling molecule FIGURE 27.3 MSM mediation of oxidative stress and inflammation. MSM has been demonstrated to influence both the priming and activation steps of NLRP3 inflammasome assembly. Green arrows display the suggested net effect of MSM treatment. IκB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; Keap1, Kelch-like ECH associated protein 1.

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282

27. Methylsulfonylmethane as an antioxidant and its use in pathology

as it’s critically involved in synaptic plasticity, but can also be catalyzed to the highly reactive OH2. OH2 are very reactive with polyunsaturated fatty acids (PUFAs), substrates found in abundance within the brain. The thioredoxin/peroxiredoxin antioxidant system (Trx/Prx) of brain mitochondria appears to be a preferred method of H2O2 degradation rather than CAT.32 Overwhelming of the brain antioxidant systems is implicated in the pathology of neurodegenerative disorders and traumatic brain injuries as ROS induce damage to cellular components such as PUFAs, thereby affecting membrane integrity and causing mitochondrial DNA damage, which can affect mitochondrial function. MSM crosses the BBB where it is equally distributed between white and gray matter.6 Previously MSM has been shown to decrease neuronal cell NO and ROS secretion while restoring the GSH/GSSG ratio upon dosing with HIV-1 Tat. This is believed to be mediated by the improved nuclear translocation of Nrf221 with MSM treatment. More recently, MSM has also been shown to reduce ROS and RNS following HIV-1 Tat exposure. This was accompanied by increased medial frontal cortex GSH levels and improved behavior in mice exposed to HIV-1 Tat.33 Together, these studies suggest MSM can affect the cycling of the redox hub within the brain.

Cardiopulmonary The cardiopulmonary system, inclusive of the heart, lungs, and vasculature, is tasked with extracting oxygen and circulating nutrients to all cells of the body while removing metabolic waste. To accomplish this, continuous, rhythmic contractions of the heart are required to pump blood to the lungs for oxygen diffusion and elsewhere. This constant requirement for energy by the cardiac muscle and exposure to ambient air and oxygen in the lungs puts this system at risk of the overproduction of ROS, in particular when oxygen demands are significant. In the heart, ROS from cardiac mitochondria, NOX2, and/or uncoupled endothelial nitric oxide synthase can target the sarcoplasmic reticulum or transverse tubule and disrupt calcium handling. Disruptions in calcium release can result in arrhythmia and if left untreated for an extended period of time can further lead to cardiac remodeling and eventually heart failure.34 Within the vasculature, excess NO production from increased inducible nitric oxide synthase (iNOS) expression may result in impaired vasoconstriction. Because the lungs are readily exposed to environmental air, alveolar macrophage and goblet cell functions are important to prevent infections and produce mucus for gas diffusion respectively. An overproduction of ROS and lipid peroxide

byproducts like isoprostanes within the lungs can affect nearby smooth muscle and provoke bronchoconstriction and vasoconstriction.35 Within the heart and lungs, the most important antioxidant systems include mitochondrial CAT and the Trx/Prx and GSH/GSSG systems; while the most important feature in the vasculature is the regulation of iNOS expression. The overproduction of ROS is implicated in the pathogenesis of pulmonary hypertension, bronchopulmonary dysplasia, and many other cardiopulmonary disorders. To date, few studies have investigated the effects of MSM on the heart. An in vitro study of cardiomyocytes indicated no oxidative stress related changes with MSM treatment,26 however, MSM did exhibit an antiinflammatory effect in response to tumor necrosis factor-α (TNFα). In contrast, MSM may be an effective preventative treatment for lung injuries. MSM has previously been shown to protect against pulmonary hypertension by reducing mean arterial pressure and right ventricular systolic pressure while attenuating the monocrotoline-induced reductions of CAT, GPx, and SOD activities and GSH levels.19 Backflow of blood is commonly seen with pulmonary hypertension and, thus, increases the right ventricular systolic pressure. If left untreated, this often causes heart failure. In another study, mice pretreated with MSM prior to paraquat-induced acute lung injury displayed increased SOD and CAT activities in the lungs along with improved oxidative stress markers such as increased GSH and decreased malondialdehyde (MDA), TNFα, and MPO activity.17 Similar effects were not observed by DiSilvestro et al.23 when mice were treated with carbon tetrachloride (CCl4). These results suggest MSM may be an effective indirect antioxidant within these tissues. MSM appears to increase the antioxidant enzyme activities of SOD, CAT, and GPx. These enzymes may also influence the cycling of the redox hub and affect GSH/GSSG ratios and Trx/Prx systems within these tissues, which could further promote protection against lipid radicals.

Kidney In order to remove water soluble toxins and regulate the osmolarity and osmolality of the blood, the proximal tubules of glomeruli must generate enough energy to biosynthesize transporters and power ATPrequiring transporters. Thus, the cells comprising the proximal tubules are most susceptible to oxidative stress.36 Oxidative stress, primarily from OH2 or peroxynitrite (ONOO2) radicals, can target membrane lipids and impair membrane integrity and permeability, which can then lead to acute tubular necrosis. Resulting tubule damage ultimately reduces the

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Methylsulfonylmethane in oxidative stress pathology

glomerular filtration rate and can cause kidney injury. To mediate these damaging effects, the main antioxidants that the kidneys rely on are vitamins C and E, GSH, SOD, CAT, and GPx.37 MSM has been shown to be protective against glycerol-induced acute renal failure in rats.38 This was accompanied by significantly lower blood urea and serum creatinine levels compared to those receiving glycerol alone; moreover, GSH levels were markedly increased after MSM treatment. Though much additional research is needed, results from this study suggest MSM may be an effective antioxidant by mediating the cycling of the redox hub within the kidneys.

Liver The liver serves multiple metabolic functions from regulating glucose and lipid stores and secretion to degrading metabolic waste, drugs, and chemicals into excretable compounds. In addition to mitochondrialderived ROS, cytochrome p450 enzymes located on the endoplasmic reticulum catalyze the degradation of toxins and produce O22 as a byproduct. Because of the multifunctional role of hepatocytes and, thus, numerous potential sources of ROS, an overproduction of ROS is more likely than for other cell types. A change to the redox state of the hepatocyte often affects redoxsensitive transcription factors and can be responsible for altered protein expression within a stressed cell. Oxidative stress appears to be an important component in the pathogenesis of a number of liver-related injuries including nonalcoholic fatty liver disease, hepatic encephalopathy, hepatic fibrosis, and other liver diseases.39 MSM has been demonstrated to protect against acetaminophen-induced hepatotoxicity by attenuating SOD activity and GSH levels, while reducing MPO activity and MDA levels.22 In another study, mice pretreated with MSM prior to paraquat-induced acute liver injury displayed increased SOD and CAT activities in the liver along with improved oxidative stress markers such as increased GSH and decreased MDA, TNFα, and MPO activity.17 In another study, rats treated with MSM and CCl4 showed reduced MDA, TNFα, and interleukin-6 (IL-6) levels and increased SOD and CAT activities.18 Mitochondrial effects were also suggested through a reduction in Bax:Bcl2 ratio. Liver enzymes ALT, AST, and CYP2e1 expression were also increased. DiSilvestro et al.23 demonstrated that mice treated with MSM and CCl4 produced a significant increase in GSH and this was accompanied by improved liver enzymes. These results suggest MSM may effectively modulate antioxidant enzyme activity and redox hub cycling within the liver.

283

Gastrointestinal tract The gastrointestinal tract regularly encounters new pathogens and toxins that are often inadvertently ingested with food. As such, gastrointestinal (GI) tissue has many local immune cells that can contribute to the local ROS from epithelia encountering pathogens/ toxins or from gut flora.40 An overproduction of ROS within the epithelia may target tight junctions and affect the permeability of the GI tract and/or activate the local immune cells and contribute to inflammation.41 Alternatively, the microbiome has been suggested to affect a number of different disease pathologies in the gut and other organs.42 Several MSM studies have demonstrated a protective effect of MSM on the GI tract. Two studies have utilized a model of acetic acid-induced colitis in rats.20,43 Treatment with MSM daily for 4 days resulted in lower colonic levels of MDA and MPO activity, but increases in GSH and CAT activity. Both studies demonstrated significant decreases in inflammation, one using IL-1β, and the other through histological evaluation. Additional studies have shown the ability of MSM to protect against gastric mucosal injury from acidified ethanol and bisphosphonates.44,45 Interestingly, MSM was previously shown to be one of the strongest serum biomarkers for patients with inflammatory bowel disease (IBD) based on nuclear magnetic resonance fingerprinting,46 as IBD patients had far lower serum MSM concentrations than patients without IBD. This may suggest a lack of MSMproducing bacteria within the gut microbiome. Others have suggested microbiome sulfur metabolism may influence host-metabolism and oxidative stress.47 These changes may be mediated by microbiome production of antiinflammatory short chain fatty acids. Currently, MSM effects on the GI tract are limited, but show promise via changes to antioxidant enzyme activities and the cycling of the redox hub. Future studies may focus on the GI effects of MSM supplementation from a microbiome perspective as well as changes at the tissue level.

Musculoskeletal system and exercise Physical inactivity is suggested to be a major contributor to chronic disease development.48 As such, most healthcare providers often encourage physical exercise as a preventative measure despite the fact that strenuous physical exercise has the potential to generate ROS and lead to an acute state of oxidative stress. While this is the case, particularly when exercise is performed by those who are unaccustomed to such a stressor (i.e., untrained), the degree of oxidative stress is generally low and transient. Regardless, many individuals seek

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methods of lessening the oxidative burden and turn to dietary supplements to aid in this quest. Evidence indicates that preloading with MSM prior to strenuous exercise can have promising results in blunting exercise-induced oxidative stress. Following aerobic exercise, MSM effectively reduces MDA,27,49 protein oxidation,27,49 and bilirubin27,50 postexercise and increases total antioxidant capacity (TAC).27,50 Glutathione levels are not as consistent; one study reported an increase to GSH and decrease in GSSG with MSM preloading and exercise,49 while another reported no significant differences.27 Similar discrepancies are also noted following anaerobic exercise, where one study reported an increase in Trolox equivalent antioxidant capacity (TEAC),25 while another study found no differences.25 In addition to acute exercise, many individuals have used MSM to lessen the pain resulting from arthritis. In fact, arthritis is likely the most commonly cited reason for using MSM. Chronic low-grade systemic inflammation appears to drive the overproduction of ROS within the arthritic joint.51 By doing so, degeneration of the cartilage and synovium within the joint space contribute to the pathology of arthritis. This arthritic pain is often a hindrance to individuals trying to partake in physical activity52,53 and evidence supports the use of MSM to provide relief from the pain of arthritis.7,8

Applications in other areas of pathology MSM may be able to indirectly influence ROS production by improving glucose homeostasis. In mouse models of diet-induced obesity and genetically obese diabetic animals, MSM treatment reduced blood glucose, hepatic triglyceride, and cholesterol levels and made mice hypersensitive to insulin.54 Improvements to measures such as these may indirectly provide protection from the overproduction of ROS as many of these variables are known to stimulate mitochondrial activity and, thus, ROS generation. Interestingly, much research has focused on the anticancer effects of MSM. Caron and colleagues have suggested that MSM treatment of metastatic cancer cells is able to revert the metastatic phenotype.55
58 Other studies have shown MSM can inhibit cancer cell viability at concentrations of approximately 300 mM. Because MSM shows broad tissue distribution, it may be able to alleviate tissue injuries at many levels. It is important to keep in mind that each of these tissues can greatly affect other tissues, either positively or negatively. For instance, liver failure can cause renal failure, which can cause metabolic acidosis resulting in oxidative stress within the brain and encephalopathy. A generalized overview of these interacting pathways is provided in Fig. 27.4.

FIGURE 27.4 Simplistic scheme of interorgan pathologies. Because MSM can penetrate multiple tissues, it may influence the pathophysiological state of one tissue, which can then affect the pathological state of other tissues. This interrelationship may span throughout the entire organism making defining an underlying mechanism nearly impossible.

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Methylsulfonylmethane in oxidative stress pathology

TABLE 27.2 Study type

Overview of oxidative stress markers effected by MSM.

Stimulus/tissue

Human Exercise-induced oxidative stress

Result

Reference

MDA

MSM blunted the postexercise rise in MDA 2 and 24 h postexercise

GSH/GSSG

Increase in plasma GSH and decrease in GSSG in MSM group postexercise; GSH/GSSG ratio preexercise, immediately postexercise, 30 min, and 2 h postexercise

PC

Significant decrease postexercise

MDA

Pretreatment with oral MSM resulted in reduced MDA levels preexercise and 2 h postexercise despite not inducing overall increase

GSH/GSSG

No differences

TAC

Maintained TAC elevation 24 h postexercise

PC

Significant decrease 2 and 24 h postexercise

Bilirubin

Significantly lower immediately after exercise

Uric acid

Significantly reduced serum uric acid levels postexercise

Human Exercise-induced oxidative stress

MDA

No change

59

Human Osteoarthritis

MDA

MSM group significantly lowered levels of MDA in urine at 12 weeks versus placebo

8

Human Exercise-induced oxidative stress

TAC

Significant increase in TAC 2 and 24 h after exercise

50

Bilirubin

Significantly lower 2 and 24 h after exercise

Human Exercise-induced oxidative stress

GSH/GSSG

No differences

TEAC

TEAC increased significantly following exercise

Human Exercise-induced oxidative stress

SOD

No change

TEAC

No change

Animal HIV-1 Tat

GSH/GSSG

Increased GSH towards baseline; reduced GSSG towards baseline; increased GSH/GSSG

ROS

Reduced ROS/RNS

Animal HIV-1 Tat

ROS

Reduced ROS/RNS

33

Animal Glycerol-induced renal failure

GSH/GSSG

Attenuated reduction in GSH

38

Urea

Significantly reduced

MDA

Pretreatment with oral MSM attenuated rise in MDA

GSH/GSSG

Increased GSH levels of colonic tissues; prevented colonic depletion of GSH

CAT

Elevation in CAT activity

MPO

Decrease in MPO activity

MDA

Pretreatment with oral MSM attenuated rise in MDA

GSH/GSSG

Attenuated GSH loss in liver; pretreatment prevented the reduction of hepatic GSH content by paraquat

CAT

Elevation in CAT activity

SOD

Elevation in SOD activity

MPO

MPO activity decreased in lung and liver

MDA

Pretreatment with oral MSM attenuated rise in MDA dose-dependently (maximal suppression of 60% in response to 400 mg/kg/day)

Human Exercise-induced oxidative stress

Animal Acetate-induced colitis

Animal Paraquat-induced lung and liver injury

Animal Monocrotoline-induced pulmonary hypertension

49

27

25

60

21

20

17

19

(Continued)

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27. Methylsulfonylmethane as an antioxidant and its use in pathology

TABLE 27.2 Study type

(Continued)

Stimulus/tissue

Result

Reference

GSH/GSSG

Significantly restored levels of GSH and GSSG. Value of GSH/GSSG ratio significantly lower in PAH, but recovered to control levels after the treatment and further enhanced at a higher dose

CAT

Elevation in CAT activity

SOD

Elevation in SOD activity

GPx

Dose-proportional increase in GPX activity

MDA

Pretreatment with MSM attenuated rise in MDA

CAT

Elevation in CAT activity

SOD

Elevation in SOD activity

MDA

Pretreatment with MSM attenuated rise in MDA

GSH/GSSG

Pretreatment with MSM led to GSH reduction in hepatic tissue

SOD

Elevation in SOD activity

MPO

Attenuation of MPO activity

Lipid radicals

Reduced levels of lipid peroxidases

GSH/GSSG

Increase in GSH; result potentiated by vitamin C

NO

Reduced NO plasma levels after several weeks of intense exercise

CO

Reduction in CO release

Animal CCl4-induced acute liver injury

GSH/GSSG

Increased GSH in liver; no change in lung or skeletal muscle

23

In vitro LPS-induced inflammation of macrophages

ROS

Significant reduction

31

In vitro TNF-α-induced inflammation of cardiomyocytes

GSH/GSSG

No differences

26

TAC

No difference

Animal CCl4-induced acute liver injury

Animal Acetaminophen-induced hepatotoxicity

Animal Exercise-induced oxidative stress

18

22

24

A summary of peer-reviewed studies including antioxidant markers is shown above.

Conclusion The unique properties of MSM allow for broad tissue distribution throughout the body. By affecting the redox status of one or several systems, MSM can potentially influence the pathogenesis of numerous disorders. MSM likely provides protection from tissue injury by mediating the crosstalk between oxidative stress and inflammation. Additional research is needed to determine the entirety of therapeutic benefits owing to MSM (Table 27.2).

Summary points

• MSM is widely distributed throughout bodily tissues. • MSM alters activities of superoxide dismutase, catalase, and myeloperoxidase antioxidant enzymes in a variety of tissues. • MSM appears to affect the cycling of reduced and oxidized glutathione, ultimately affecting the redox hub and redox status. • In addition to antioxidant activity, MSM also demonstrates an antiinflammatory effect suggesting it can mediate the crosstalk between these processes.

References

• MSM is naturally occurring, commonly found in onions and garlic, and nontoxic.

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C H A P T E R

28 Improving antioxidant capacity of foods: adding mushroom powder to pasta Liwen Wang1,2,3, Margaret Anne Brennan1 and Charles Stephen Brennan1,2,3 1

Lincoln University, Department of Food Science, Lincoln, New Zealand 2Tianjin University of Commerce, Biotechnology and Food Science, Tianjin, P.R. China 3Riddet Institute, Palmerston North, New Zealand

List of abbreviations ABTS CAT DMH DPPH GPx GR GSH GSSG GST H2O2 MDA NO_ NSCs O2 2  OH ORAC RNS ROS SOD t-BHP

polyphenols can lower the risk of cardiovascular diseases and lung cancer.2 There is also a growing tendency for consumers to willingly increase antioxidant intake on the belief that this improves their overall nutrition, however, further evidence needs to be supplied explaining how naturally occurring antioxidants affect underlying biological effects. Mushrooms have been demonstrated to possess health-promoting properties; some common edible mushrooms are shown in Fig. 28.1. Many mushroom species have been proven to be effective, acting in antiinflammatory, antitumor, and antibacterial roles, and involved in immunomodulating therapies.3 Thus summarizing the mechanisms of antioxidants in mushrooms when incorporating them into food aimed at increasing antioxidant intake is increasingly important.

2,2v-azinobis-3-ethylbenzothiazoline-6-sulfonic acid catalase 1,2-dimethylhydrazine 2,2-diphenyl-1-picrylhydrazyl glutathione peroxidase GSH reductase glutathione oxidized glutathione GSH S-transferase hydrogen peroxide malondialdehyde nitric oxide neural stem cells superoxide anion hydroxyl radical oxygen radical absorbance capacity reactive nitrogen species reactive oxygen species superoxide dismutase tert-butyl hydroperoxide

Oxidative stress

Introduction Antioxidants are considered an effective way to treat chronic diseases such as obesity, type 2 diabetes, cardiovascular disease, rheumatoid arthritis, neurological disorders, and even cancer.1 They play an essential role in regulating the metabolic system and associated molecular signaling pathways. Many compounds such as polysaccharides, polyphenols, and vitamins have been found to possess bioactive and antioxidant abilities. Epidemiological and metaanalysis data strongly support the benefits of longterm consumption of food rich in antioxidants as plant Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00028-7

Oxidative stress is the imbalance between reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defenses, leading to the damage of important biomolecules and cells with subsequent detrimental effects, which may involve chronic disease, inflammation, and cancer initiation and progression.4 Antioxidants act as defense agents against these reactive species by preventing their production or converting them into harmless products by scavenging ROS and quenching excess energy.5 Antioxidants are either enzymatic such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) or non-enzymatic such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), phenols, and other antioxidants.1

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mass), Agaricus bisporus contains less chitin in its fruiting body than its mycelia. By contrast, the chitin content is significantly higher in the fruiting body than in the mycelia of Flammulina velutipes.11 Other species have comparable amounts of chitin in their fruiting bodies and mycelia. Along with beta-glucan, chitin is the dominant component of the fungal cell wall. It is a water-insoluble structural N-containing polysaccharide and is characterized by beta-(1-4)-branched N-acetylglucosamine units.12 Chitosan, the deacetylated form of chitin, has been found to have a number of biomedical and biotechnological applications.11 The contents of chitin and chitosan in a variety of mushrooms have been quantified as shown in Table 28.1. Chitosan was detected only in some of the samples, which indicates that most of the amino groups of the glucosamine units are acetylated.

Phenolic compounds FIGURE 28.1 Commonly eaten mushrooms. (A) White button mushroom (Agaricus bisporus) (B) Portobello/Swiss brown mushroom (Agaricus bisporus) (C) Wood ear mushroom (Auricula polytricha). Source: Photo courtesy M.A. Brennan.

Major antioxidants of mushrooms Polysaccharides Polysaccharides are essential for the enhancement of immunity and the modulation of defensive responses. They are components of mushrooms’ hyphae, and include trehalose, glucans, chitin, and chitosan (partially acetylated chitin forms).6 Trehalose is a non-reducing disaccharide in which two glucose units are linked by an α, α-1, 1-glycosidic bond. The trehalose content varies from 0.16% to 8.01% of fresh weight in several edible mushrooms (Table 28.1).7 The major structural feature of mushroom betaglucan is a beta-1,3-D-glucan main chain with beta-1,6D-glucosyl branches along the main chain. Previous research has suggested that beta-glucan concentrations range from 0.21 to 0.53 g/100 g on a dry basis using the method of McCleary and Holmes.8,9 However, the latest research evaluating the beta-glucan content of nine culinary mushrooms including Lentinula edodes (shiitake) and five different Pleurotus species, found the range to be between 15 and 22 g/100 g (dry mass) (Table 28.2).10 Meanwhile, in Table 28.2, higher glucan contents have been showed to be present in the stipe of a mushroom compared to the mushroom caps, which provides support for the potential use of mushroom byproducts.10 With 4.6 g of chitin per 100 g (dry

Polyphenols are considered to be an extensive and complex group of plant substances. They arise biogenetically from two central synthetic pathways, namely the shikimate pathway and the acetate pathway.13 Contents of phenols in edible mushrooms Mushrooms contain between 1 and 6 mg of phenolic compounds per gram of dried mushroom, depending on the species.3 The main phenolic compounds found in mushrooms are phenolic acids and flavonoids. Phenolic acids can be separated into two major groups, namely hydroxybenzoic acids and hydroxycinnamic acids, which are derived from the non-phenolic molecules benzoic and cinnamic acid respectively. Table 28.3 illustrates the phenolic acid compounds found in several mushroom species.3,14 The main antioxidant phenolic compounds found in mushrooms are gallic, protocatechuic, hydroxybenzoic, p-coumaric, cinnamic, and caffeic acids.15 Homogentisic acid is the only free phenolic acid found in mushrooms, although the content varies considerably among species. A. bisporus contains between 3 and 4 mg/g of homogentisic acid, while Pleurotus ostreatus contains less than 1 mg/g. Gallic acid is the second main component of phenolic acids in mushrooms (0.1
0.3 mg/g) and is possibly present in its conjugated form. Flavonoids are assumed to be produced only in plants, not animals and fungi. As such, research has shown the absence of flavonoids in many Portuguese wild mushrooms.16 However, more recent research has suggested the presence of flavonoids in some edible/cultivated mushrooms.3,17 When comparing phenolic compound contents, it should be borne in mind that the composition of

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Major antioxidants of mushrooms

TABLE 28.1

Trehalose, chitin, and ergosterol of a variety of edible mushrooms. Agaricus bisporus Lentinula edodes (button mushroom) (Shiitake)

Pleurotus eryngii Pleurotus ostreatus (king oyster (oyster mushroom) mushroom)

Flammulina velutipes (golden needle mushroom)

Trehalose (g/100 g FW)

0.16

3.38

4.42

8.01

2.64

Chitin content (g/100 g DW) in mycelia

9.60

2.49

0.82

3.56

1.21

Chitin content (g/100 g DW) in fruiting body

4.69

1.87

0.76

3.16

9.83

Ergosterol (mg/100 g FW)

56.30

84.90

68.00

na

na

Vitamin D2 (μg/100 g FW)

0.11

0.44

0.72

na

na

DW, Dried weight; FW: fresh weight; na, not available. Trehalose is a disaccharide metabolized by the body, it varies between mushroom species. Variation of chitin content in mycelia and fruiting body varies between species, indicating a possible reason to make use of mushroom co-products. Ergosterol is the precursor to vitamin D2. From: Reis FS, Barros L, Martins A, Ferreira IC. Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study. Food Chem Toxicol 2012;50:191
7; Nitschke J, Altenbach HJ, Malolepszy T, Molleken H. A new method for the quantification of chitin and chitosan in edible mushrooms. Carbohydr Res 2011;346:1307
10; Phillips KM, Ruggio DM, Horst RL, Minor B, Simon RR, Feeney MJ, Byrdwell WC, Haytowitz DB. Vitamin D and sterol composition of 10 types of mushrooms from retail suppliers in the United States. J Agric Food Chem 2011;59:7841
53.

TABLE 28.2

Glucan content and distribution of a variety of edible mushrooms. All glucans

α-Glucans

Cap

10.05

1.55

8.61

Stalk

14.96

2.67

12.30

Cap

12.35

3.51

8.84

Stalk

14.65

4.57

10.08

Cap

20.54

0.76

19.78

Stalk

26.75

1.44

25.31

Pleurotus ostreatus (oyster mushroom)

Whole

25.64

1.41

24.23

Pleurotus eryngii (king oyster mushroom)

Whole

19.24

3.92

15.32

Mushrooms Agaricus bisporus (white button mushroom)

Agaricus bisporus (brown button mushroom)

Lentinula edodes (Shiitake)

β-Glucans

Expressed as grams of glucans per 100 gram of dried weight of mushrooms. Beta-glucan is the predominant glucan in all mushrooms shown, it is also more predominant in the stalk, rather than the cap, of A. bisporus and L. edodes. From Sari M, Prange A, Lelley JI, Hambitzer R. Screening of beta-glucan contents in commercially cultivated and wild growing mushrooms. Food Chem 2017;216:45
51.

TABLE 28.3

Phenolic acids and cinnamic acid content of a variety of edible mushrooms, expressed as μg per gram of dried mushroom.

Mushrooms

Agaricus bisporus Agaricus bisporus Lentinula edodes Pleurotus ostreatus Pleurotus eryngii (button mushroom) (brown button mushroom) (Shiitake) (oyster mushroom) (king oyster mushroom)

Gallic acid

94.90

nd

nd

290.34

nd

Caffeic acid

15.54

82

nd

nd

nd

Protocatechuic acid

16.21

nd

0.36

19.32

0.06

p-Hydroxybenzoic acid

15.39

nd

1.57

4.69

0.10

p-Coumaric acid

10.38

nd

nd

11.15

1.04

0.38

0.09

0.02

0.23

0.20

nd

nd

629.86

nd

Cinnamic acid Homogentisic acid

3444.30

nd, Not detected. Homogentisic acid is a free phenolic acid and is the most abundant, however, its content varies greatly. Gallic acid, the second most abundant phenolic acid, may be bound. From: Palacios I, Lozano M, Moro C, D’Arrigo M, Rostagno MA, Martı´nez JA, Garcı´a-Lafuente A, Guillamo´n E, Villares A. Antioxidant properties of phenolic compounds occurring in edible mushrooms. Food Chem 2011;128:674
8; Reis FS, Martins A, Barros L, Ferreira ICFR. Antioxidant properties and phenolic profile of the most widely appreciated cultivated mushrooms: a comparative study between in vivo and in vitro samples. Food Chem Toxicol 2012;50:1201
07.

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28. Improving antioxidant capacity of foods: adding mushroom powder to pasta

mushrooms may be associated with cultivation or with natural environmental factors of wild mushrooms. For example, Woldegiorgis found that a higher amount of caffeic acid might be due to the coffee waste substrate used for growing P. ostreatus mushroom.18 The relationship of phenolic compound structures and antioxidant ability Since polyphenolic compounds play an important role in antioxidant activity, many studies have attempted to investigate the relationship of the structural and functional activities of these compounds. The overall effectiveness of natural phenolic antioxidants is dependent on the involvement of the phenolic hydrogen in radical reactions, the stability of the natural antioxidants’ radical formed during reactions, and the chemical substitutions present in the structure.19 Substitutions in structure are believed to be the most significant contribution to the capability of natural antioxidants. For instance, hydroxyl substitutions enhance antioxidant activity, and caffeic acid has been reported to be a better antioxidant than both ferulic acid and p-coumaric acid, possibly due to the presence of a second hydroxyl group (Fig. 28.2). The relative antioxidant ability of cinnamic acid and its derivatives has been ranked as caffeic acid . p-coumaric acid . cinnamic acid.19,20 This reasoning also explains the incremental rise in the antioxidant ability (gallic acid . protocatechuic acid . hydroxybenzoic acid) between hydroxybenzoic acid and its derivatives. Acid protons appear to have little impact since both caffeic acid and chlorogenic acid are effective in lipid oxidation. The allylic group ensures that cinnamic acid has greater H-donating ability, and subsequent radical stabilization, than the carboxylate group in benzoic acids, which could explain why caffeic acid is more active than protocatechuic acid.20

Vitamins The vitamins found in mushrooms include ascorbic acid, carotenoids, and tocopherols. Vitamins of the B group including thiamine, riboflavin, biotin, and pyridoxine are commonly found in the fruiting body of A. bisporus.21 Vitamin D deficiency is linked to the development of metabolic syndrome and diabetes, hypertension, intestinal inflammation, and certain cancers.22 Table 28.1 illustrates the content of ergosterol (vitamin D precursor) (mg/100 g) and vitamin D2 (μg/100 g fresh weight) in three mushroom species.23

Other antioxidants of mushrooms Other bioactive ingredients in mushrooms such as indole, free fatty acids, and amino acids also show effective bioactive capacities. For example, A. bisporus exhibited antioxidant effects that were linked to the

presence of ergothioneine, an essential amino acid for humans.24 It demonstrated effective intrinsic antihydroxyl, antiperoxyl, and antiperoxynitrite radical antioxidant activity as compared to classic antioxidant molecules, which reduced glutathione (GSH), uric acid, and trolox.25

Antioxidant mechanisms in different models Cell lines model It is well documented that trehalose accumulation in stressed cells plays a significant role in protecting the cellular constituents from oxidative damage. For instance, when Saccharomyces cerevisiae cells were exposed to hydrogen peroxide (H2O2), it caused oxidative damage to the cellular protein. Trehalose reduced the degree of damage to the proteins by scavenging the free radicals.26 In murine macrophage RAW 264.7 cells, trehalose directly reduced oxidative stress by suppressing H2O2-primed arachidonic acid release and hemolysate-induced lipid peroxidation generation.27 Beta-glucan is believed to be the major bioactive polysaccharide of mushrooms.28 Research has shown that beta-glucan-rich extracts from Geastrum saccatum mushroom inhibit lipid peroxidation at a dose of 0.27 mg/mL (59.1%) and can protect against oxidative stress by scavenging hydroxyl (OH; 77%) and superoxide (O2 2 ; 88.4%) radicals.29 Beta-glucan isolated from a hot water extract of the fruiting body of the edible mushroom Entoloma lividoalbum stimulated the production of macrophages, splenocytes, and thymocytes and exhibited OH and O2 2 scavenging activities and reducing properties.30 Additionally, different beta-glucan extraction methods may affect antioxidant ability. Ultrasonically extracted Ganoderma lucidum beta-D-glucan has a higher molecular weight, an optimal degree of branching (ratio of (1,6)β-D-glucosyl branches of (1,3; 1,6)-β-D-glucans) polysaccharides, and better in vitro antioxidant activity.31 Phenolic compounds from the fruiting body of mushrooms have been proven to have antioxidant properties both in vitro and in vivo.32 Gallic acid has been described as a potent natural antioxidant able to scavenge ROS. In B16 melanoma cells, gallic acid downregulated ROS generation and increased the GSH/ GSSG (oxidized glutathione) ratio, which implied an enhanced intracellular reducing power.33 Caffeic acid is considered to have high antioxidant activity and great radical scavenging activity due to it being present in a high concentration (approximately 15 μg/g dried weight) in A. bisporus. When comparing hydroxycinnamic acids (caffeic, ferulic, and p-coumaric acid), only caffeic acid (5 μM) was shown to confer a significant inhibitory effect on human low-density lipoprotein oxidation in the analysis of three different

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Antioxidant mechanisms in different models

293 FIGURE 28.2 Diagrammatic representation of some phenolic acid compounds found in edible mushrooms. These chemical diagrams show the increasing presence of OH groups in both the hydroxybenzoic acid family and the cinnamic acid family, this appears to be related to the trend of increasing antioxidant ability.

systems (cupric ions, 2,20 -azobis (2-amidinopropane)hydrochloride (AAPH), and mouse peritoneal macrophages).34 Protocatechuic acid is a water-soluble monomeric phenolic acid with slightly lower free radical scavenging effects than caffeic acid. Protocatechuic acid acts as a potential neuroprotective agent, partly by promoting endogenous antioxidant enzymatic activities and by inhibiting free radical generation. In PC-12 cells, protocatechuic acid prevented H2O2-induced reduction and attenuated apoptotic cell death. In these cells, the activity of GSH and CAT were augmented.35 In rat neural stem cells (NSCs), obtained from 13.5-day-old rat embryos, protocatechuic acid increased the cellular viability of NSCs and stimulated cell proliferation, preventing NSC apoptosis by depressing the level of intracellular ROS significantly under normal conditions.36 p-Coumaric acid has also been shown to have antioxidant activities against free radicals.20 In an in vitro study comparing the reduction potential and free radical scavenging capacity of 16 antioxidants using 2,2diphenyl-1-picrylhydrazyl (DPPH) and 2,2v-azinobis3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and OH and O2 radical scavenging assays, p-coumaric acid 2

exhibited lower scavenging capacity for DPPH•, ABTS1•, and O2 2 radicals, and lower ferric reducing power than caffeic acid and ferulic acid,37 which is consistent with the structure analysis above. In food systems and cells, p-coumaric acid exhibits a similar antioxidant activity as caffeic acid and ferulic acid. In the omega-3 fatty acid emulsion system, p-coumaric acid showed a similar antioxidant capacity to caffeic acid and ferulic acid for reducing H2O2, thiobarbituric acid reactive substances, and malondialdehyde.38 p-Coumaric acid was also found to exert similar effects of eliminating ROS as caffeic acid and ferulic acid in colon adenocarcinoma (HT29-D4) cells or an even higher ability than ferulic acid in human lung (A549) cells at concentrations of 50
200 μmol/L.39 These results indicate that p-coumaric acid may perform differently in single-component systems compared to complicated systems such as foods and cells. Homogentistic acid is an intermediate in the metabolism of tyrosine, and it has been reported to have antioxidant activities against free radicals. In lung WI 38 fibroblasts cells, homogentistic acid was found to scavenge ROS and DPPH radicals to prevent H2O2induced lipid peroxidation.40

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Evidence of the protective role of mushroom antioxidants on diseases using animal models Many studies have focused on the use of mushroom polysaccharides in animal model systems to explore the mechanisms by which these compounds protect against diseases. For instance, trehalose treatment abrogated lipid peroxidation in the presence of blood in a rat femoral artery model.27 High-fat-diet obese C57BL/6J mice, treated with beta-glucan-rich extract from Pleurotus sajorcaju and metformin showed protection against the oxidative damage caused by enzymatic antioxidant (SOD, CAT, and GPx) activities in the kidney and liver.41 Gallic acid has been found to reduce oxidative stress and GSSG content and enhance the levels of GSH, GPx, GSH reductase (GR), and GSH S-transferase (GST) in the hepatic tissue of rats with high fat diet-induced obesity. GSH is the main non-protein antioxidant in the cell and provides electrons for the enzyme GPx, which then reduces H2O2 to H2O. These results demonstrate that the intake of gallic acid can be beneficial for the suppression of high-fat-diet-induced dyslipidemia, hepatosteatosis, and oxidative stress in rats.42 Gallic acid also exerted a significant chemopreventive effect on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Wistar rats by playing an essential role in lipid peroxidation and the antioxidant defense system. Oral supplementation of gallic acid (50 mg/kg body weight) elevated a series of antioxidant levels, namely SOD, CAT, GSH, GR, and GPx levels in tissues (liver, intestine, proximal colon, distal colon, and cecum), which were diminished in DMH-treated groups.43 Endothelial cells are at high risk of oxidant injury due to acting as an element of the vascular system and having close contact with flowing blood and xanthine oxidase.44 In parallel with the inhibitory effect of ECV304 endothelial cells after damage by oxygen radicals, caffeic acid also exerted a hypertension suppressive effect in a mice model. In both stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats, caffeic acid inhibited angiotensin II-induced cell proliferation in vascular smooth muscle cells by decreasing the genera45 tion of ROS (O2 2 production). Consistent with its neuroprotective effect in a cell model, protocatechuic acid has also been found to possess a neuroprotective capacity by ameliorating oxidative stress in the brains of aged rats. In a Y-maze behavioral test, protocatechuic acid improved the cognition of aged rats, which were injected intraperitoneally, after 7 days. It also reduced the content of lipid peroxide and increased the activity of GPx and SOD. These results suggest that protocatechuic acid might be a potential neuroprotective agent and its effects were achieved at least partly by promoting endogenous antioxidant enzymatic activities and inhibiting free radical generation.46

Protocatechuic acid also protects against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in rats through its antioxidant and antiinflammatory characteristics. Pretreatment of rats with protocatechuic acid (50
100 mg/kg) by gavage for 5 days before a single dose of t-BHP significantly reduced the incidence of liver lesions and moderated oxidative stress of the liver by regulating the levels of GSH and the lipid peroxidation marker malondialdehyde (MDA).47

How in vitro and in vivo methods are related to potential benefits derived from functioned food All the previous discussion has been based on evidence using in vitro and in vivo models to substantiate the benefits of using isolated compounds to protect against oxidative stress and disease. However, consumers have become focused on using their diets to improve their intake of antioxidants. Examples of these include the use of plant bioactive ingredients rich in phenolic compounds and antioxidants to improve the nutritional status of foods. For instance, mushrooms have been incorporated into noodles and pasta as a functional supplement to improve their nutritional profiles. The effects of the supplementation of plant proteins from mushroom powder on the nutritional quality of pasta have been studied. On the basis of cooking and sensory quality, pasta in combination with 8% mushroom powder resulted in a more nutritious pasta.48 Other research showed that different kinds of mushroom powder such as shiitake, porcini, and white button mushroom could be incorporated into pasta. However, mushroom powder increased the cooking loss as well as firmness and resistance of pasta uniaxial tension.49 The incorporation of mushroom into noodles can improve the radical scavenging ability of pasta significantly. Total phenolic content, DPPH, and ORAC assays were conducted to assess the potential antioxidant values. The antioxidant abilities of pasta supplemented with three mushrooms were significantly higher than the control and the order of antioxidant ability was porcini mushroom . white button mushroom . shiitake mushroom.50 Since mushrooms are natural products rich in fiber, they are also considered an excellent choice to be added to other food products including bread, cakes, and extrusion food. For example, mushroom mycelium could be incorporated into bread to increase its antioxidant content. Extruded snack food with black ear mushroom (Auricularia auricula) showed increased total phenolic concentration and a higher percentage of free radicals scavenging effect in a DPPH assay. An ORAC assay also found that black ear inclusion gave a high

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References

antioxidant activity. An in vitro starch digestion of the snack products showed that the black ear mushroom attenuated starch digestion, suggesting a lower glycemic response when consumed.51 Although some work with mushroom food products has been carried out and the consumption of foods rich in mushroom bioactive compounds appears to be beneficial, the claim that a functionalized food will bring beneficial effects needs more in-depth study. Bioaccessibility and bioavailability studies are required due to the animate nature of the digestive tract, which cannot easily be replicated in vitro. It is known that some bioactive compounds, for example, high-molecular-weight food polyphenols, are not bioaccessible in the human gut. They do, however, remain associated with the food matrix and pass undissolved and unaltered through the upper intestine.52 Despite the fact that the bioavailability of different phenolic compounds is variable, it is recognized that after passing through the upper intestine the phenolic compound concentration is relatively low.53 It has been noted that insoluble and soluble dietary fiber interfere with the bioavailability/absorption of macronutrients and biomolecules, especially fat, some minerals, and trace elements.52 Therefore as mushrooms are rich in fiber, this may interfere with the absorption of phenolic compounds and other antioxidants. More studies are needed in order to understand the role of fiber in nutrient uptake. However, even if the absorption of antioxidant compounds appears to be reduced, increased consumption of antioxidants is still an advantage because it promotes an antioxidant environment within the gut.52

Applications in other areas of pathology The chapter has covered areas in which natural ingredients could be used in food systems to affect the diet of patients and, hence, their nutritional well-being (in relation to obesity and diabetes). However, evidence has shown a relationship between plant polysaccharides and polyphenols with microbial fermentation and intestinal health. It is possible that in the next 3
5 years researchers will illustrate that the gut
brain axis may be controlled by selective polysaccharide and phenolic substances so that gene regulation and rates of expression can be achieved to manipulate neurological disorders (such as Alzheimer’s and Parkinson’s diseases) and the overall pathology and development of the brain.

Summary points • Mushroom material is a rich source of antioxidant material in the form of polyphenols.

295

• Mushroom antioxidant compounds appear to be associated with the non-starch polysaccharide components of mushrooms. • These substances can manipulate oxidative stress and, therefore, have benefits in terms of obesity, diabetes, liver disease, and coronary heart diseases to name a few. • Their utilization in food systems is an effective way to enhance the nutritional quality of our diets. • In vitro and in vivo analyses of isolated compounds have been conducted, however, there is still a paucity of research on how combinations of biologically active materials function in complex food systems.

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8. 38. Pei K, Ou J, Huang J, Ou S. p-Coumaric acid and its conjugates: dietary sources, pharmacokinetic properties and biological activities. J Sci Food Agric 2016;96:2952
62. 39. Nasr BN, Kilani JS, Kovacic H, Chekir-Ghedira L, Ghedira K, Luis J. The effects of caffeic, coumaric and ferulic acids on proliferation, superoxide production, adhesion and migration of human tumor cells in vitro. Eur J Pharmacol 2015;766:99
105. 40. Kang KA, Chae S, Lee KH, Zhang R, Jung MS, You HJ, et al. Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells. Biotechnol Bioprocess Eng 2005;10:556. 41. Kanagasabapathy G, Malek SNA, Mahmood AA, Chua KH, Vikineswary S, Kuppusamy UR. Beta-glucan-rich extract from Pleurotus sajor-caju (Fr.) singer prevents obesity and oxidative stress in C57BL/6J mice fed on a high-fat diet. Evid Based Complement Altern Med 2013;2013:10. 42. Hsu C-L, Yen G-C. Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Br J Nutr 2007;98:727
35. 43. Giftson JS, Jayanthi S, Nalini N. Chemopreventive efficacy of gallic acid, an antioxidant and anticarcinogenic polyphenol, against 1,2-dimethyl hydrazine induced rat colon carcinogenesis. Investig New Drugs 2010;28:251
9. 44. Beyer G, Melzig MF. Effects of selected flavonoids and caffeic acid derivatives on hypoxanthine-xanthine oxidase-induced toxicity in cultivated human cells. Planta Med 2003;69:1125
9. 45. Li P-G, Xu J-W, Ikeda K, Kobayakawa A, Kayano Y, Mitani T, et al. Caffeic acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II in stroke-prone spontaneously hypertensive rats. Hypertension Res 2005;28:369. 46. Shi G-F, An L-J, Jiang B, Guan S, Bao Y-M. Alpinia protocatechuic acid protects against oxidative damage in vitro and reduces oxidative stress in vivo. Neurosci Lett 2006;403:206
10. 47. Liu C-L, Wang J-M, Chu C-Y, Cheng M-T, Tseng T-H. In vivo protective effect of protocatechuic acid on tert-butyl hydroperoxide-induced rat hepatotoxicity. Food Chem Toxicol 2002;40:635
41. 48. Kaur G, Sharma S, Nagi H, Ranote P. Enrichment of pasta with different plant proteins. J Food Science Technol 2013;50:1000
5. 49. Lu X, Brennan MA, Serventi L, Mason S, Brennan CS. How the inclusion of mushroom powder can affect the physicochemical characteristics of pasta. Int J Food Science Technol 2016;51:2433
9. 50. Lu X, Brennan MA, Serventi L, Liu J, Guan W, Brennan CS. Addition of mushroom powder to pasta enhances the antioxidant content and modulates the predictive glycaemic response of pasta. Food Chem 2018;264:199
209. 51. Valle´e M, Lu X, Narciso JO, Li W, Qin Y, Brennan MA, et al. Physical, predictive glycaemic response and antioxidative properties of black ear mushroom (Auricularia auricula) extrudates. Plant Foods Hum Nutr 2017;72:301
7. 52. Quiro´s-Sauceda A, Palafox-Carlos H, Sa´yago-Ayerdi S, AyalaZavala J, Bello-Perez LA, Alvarez-Parrilla E, et al. Dietary fiber and phenolic compounds as functional ingredients: interaction and possible effect after ingestion. Food Funct 2014;5:1063
72. 53. Heleno SA, Martins A, Queiroz MJRP, Ferreira ICFR. Bioactivity of phenolic acids: metabolites versus parent compounds: a review. Food Chem 2015;173:501
13.

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C H A P T E R

29 Phenolics: therapeutic applications against oxidative injury in obesity and type 2 diabetes pathology Veronica F. Salau1, Ochuko L. Erukainure1,2 and Md. Shahidul Islam1 1

Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa 2Department of Pharmacology, School of Clinical Medicine, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

List of abbreviations AMPK Bcl-2 BMI C/EBPβ C/EBPα COX-2 CRP DGAT1 DNA ERK FABP4 FAS GLUT4 GPx GSH GST HBA HbA1c HCAs HDL-c HMGCoA reductase IFN-γ IL-10 IL-1β IL-6 iNOS JNK LDL-c LOX LPAATθ MCP-1 m-TOR NF-κB

50 AMP-activated protein kinase B-cell lymphoma 2 body mass index CCAAT enhancer-binding proteins beta CCAAT enhancer-binding proteins alpha cyclooxygenase-2 C-reactive protein diglyceride acyltransferase deoxyribonucleic acid extracellular signal-regulated kinase Fatty acid binding protein 4 fetal alcohol syndrome glucose transporter 4 glutathione peroxidase glutathione glutathione S-transferase hydroxybenzoic acid hemoglobin A1c hydroxycinnamic acids high-density lipoproteins c 5-hydroxy-3-methylglutaryl-coenzyme A reductase interferon-gamma interleukin-10 interleukin-1 beta interleukin-6 inducible nitric oxide synthase Jun N-terminal Kinase low-density lipoproteins lipoxygenases lysophosphatidic acid acyltransferase monocyte chemoattractant protein 1 mammalian target of rapamycin nuclear factor kappa B

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00029-9

NO OS p38MAPK p70S6K PAL PGC1α PPAR-γ ROS Sirt1 SOD SREBP-1c T2D TG TNF-α

nitric oxide oxidative stress p 38 Mitogen activated protein kinase p70 S6 kinase phenylalanine ammonia-lyase peroxisome proliferator-activated receptor gamma coactivator 1-alpha peroxisome proliferator-activated receptor gamma reactive oxygen species sirtuin 1 superoxide dismutase sterol response element-binding protein 1c type 2 diabetes triglyceride tumor necrosis factor-alpha

Introduction The efficacy of phenolic compounds on human health has made them highly attractive and has continued to gain much attention publicly including in the science and health sectors.1 This group of bioactive compounds are dominant secondary metabolites found in various parts of plants and are supplied to the body when considerable amounts of phenolic-rich fruit, vegetables, and beverages are consumed.1,2 Several studies have elucidated a positive correlation between the consumption of fruit, vegetables, and beverages rich in plant phenolics and their protective effect against noncommunicable diseases such as obesity and type 2 diabetes (T2D) among others owing to their

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29. Phenolics: therapeutic applications against oxidative injury in obesity and type 2 diabetes pathology

antioxidative characteristics.3 Oxidative stress (OS), which occurs as a result of an imbalance in the body’s prooxidant and antioxidant levels, has been associated with several diseases. The incidence of obesity, which is a risk factor for the occurrence of other T2D health challenges, is on the rise, creating enormous social problems worldwide. It is characterized by a build-up of excess fat resulting from an imbalanced energy intake and energy expenditure. Adipokines secreted by the adipose tissues participate in the generation of OS in an obese condition and this plays an important role in the development of T2D.4 T2D represents a sustained blood glucose level that results from impaired insulin secretion and/or insulin action. Hyperglycemia induces the generation of reactive oxygen species (ROS). OS is induced when the generated ROS surpass the cell’s antioxidant defense system. Phenolic compounds, which are natural components of food plants, are dietary antioxidants that help to protect against oxidative injury. This chapter focuses the antioxidative and protective properties of phenolic compounds on oxidative damage involved in T2D and obesity.

biosynthetic process.8 For instance, the substitution of cinnamate by hydroxyl or methoxyl groups at carbon 3 and 5 positions yields caffeic, ferulic, and sinapic acids. Other processes may include chain shortening and lengthening without ring formation, which produce benzoic acid and other derivatives and condensation reactions with malonyl residues, which yield flavonoids, etc.9 A variety of phenolics ranging from simple- to complex-structured phenolics are, therefore, produced from this pathway with each being identified by their basic skeleton such as cinnamic acids (C6-C3), benzoic acids (C6-C1), flavonoids (C6-C3-C6), proanthocyanidins [(C6-C3-C6)n], lignans (C6-C3-C3C6), and lignins [(C6-C3)n], etc. (Fig. 29.1).8

Classes of phenolics Phenolics are often grouped into four main classes, namely phenolic acid, flavonoids, lignans and neolignans, and tannins.

Phenolic acids Phenolics Phenolics are a group of secondary metabolites ubiquitously dispersed in plants that play pivotal roles in plant physiology and cellular metabolism such as structure, insect and predator resistance, pollination, growth and development, reproduction, etc.1 Numerous qualities of plant-derived foods are subject to phenolic compounds such as color, taste, and flavor.5 A variety of vegetables and fruit are now highly attractive for their therapeutic impact on human health owing to the presence of phenolics with their natural antioxidative power. This amazing group of plant bioactive compounds work synergistically with endogenous antioxidant enzymes to combat various diseases.1,6 An obvious structural feature of phenolics is the presence of a benzene ring with one or more functional hydroxyl groups; their structures range from simple phenolic molecules such as benzoic acids to complex phenolic polymers such as tannins.6 Phenolic compounds are biogenetically synthesized from either the shikimate/phenylpropanoid acid pathway, which basically produces phenylpropanoids, or the acetate/ malonate pathway, where simple phenols are mainly produced.1,7 A larger portion of existing phenolics are synthesized by the phenylpropanoid pathway, which are commonly from phenylalanine or tyrosine (amino acids) origin. Phenylalanine is first converted to cinnamic acid by phenylalanine ammonia-lyase (PAL), which then enters into the pathway. The addition of one or more hydroxyl groups is a crucial step in the

This class consists of two subgroups, namely hydroxybenzoic acid (HBA) and hydroxycinnamic acid (HCA). They are naturally present as conjugates, although their presence as free acids has been reported in fruit.10 HBAs have general C6-C1 skeletons. Their structure consists of methoxylations and hydroxylations at the aromatic ring. Common HBAs include 4-hydroxybenzoic, gallic, p-hydroxybenzoic, vanillic, syringic, and protocatechuic acids. While HCAs consist of a nine carbon (C6-C3) skeleton and a side chain double bond (with cis or trans configuration).11 Caffeic, o-coumaric, p-coumaric, mcoumaric, ferulic, and cinnamic acids are amongst the most common HCAs.

Flavonoids Flavonoids are the most abundant phenolics and the largest group of natural products with a C6-C3-C6 skeleton.12 They are widely distributed in fruit and vegetables. This class is subdivided into the flavonoids (2-phenylbenzopyrans), isoflavonoids (3-benzopyrans), and neoflavonoids (4-benzopyrans). This subdivision is dependent on the position of the linkage of the aromatic ring to the benzopyrano (chromano) moiety with a common chalcone precursor.12 Common flavonoids include luteolin, apigenin, catechin, and kaempferol.

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Oxidative stress: reactive species and antioxidants

FIGURE 29.1 Chemical phenolic compounds.

Lignans and neolignans These are large groups of phenolics with (C6-C3) 2 skeletons. They are produced by the oxidative dimerization of two phenylpropanoid units. When the two units (C6-C3) are β,β0 -linked these are lignans, while neolignans are linked by m,m0 ; γ,γ0 ; β,m0 .13 Compounds belonging to this class include syringaresinol, syringaresinol 4-O-β-D-glucopyranoside, syringaresinol 4-O-β-Dglucopyranosyl-(1-6)-β-D-glucopyranoside, liriodendrin, 5-methoxylariciresinol 40 -O-β-D-glucopyranoside, and dehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside.14

Tannins These are water-soluble phenolic compounds that can form strong complexes with polysaccharides and proteins. They are subdivided into hydrolyzable tannins and condensed tannins. Such compounds include chebulinic acid, chebulagic acid, ellagitannin, and leucocyanidin.

Oxidative stress: reactive species and antioxidants In normal regulation of the body’s physiological function, the generation of reactive species is expected during cellular aerobic metabolism and its homeostasis

structures

of

some

is maintained by the presence of cellular enzymatic and nonenzymatic antioxidants. However, elevated levels of reactive species can be generated from intrinsic origins such as infections, inflammation, and mental stress among others and extrinsic origins such as alcohol consumption, smoking, environmental stress, and medication to mention but a few. OS is a condition where there is a suppression in the capacity of the antioxidant system of the cells to mop off or detoxify excessive reactive species. These accumulated reactive species in the body play a key role in the pathogenesis of many noncommunicable diseases such as diabetes, heart diseases, obesity, and retinopathy among others.15,16 Free radicals are chemically reactive species with one or more unpaired electron in their outermost shell that are capable of modifying nearby biological molecules.17 ROS, which are derived from aerobic metabolism, include free oxygen radicals such as hydroxyl U (OHU ), superoxide (OU2 2 ), peroxyl (ROO ), lipid peroxyl • (LOO ), and alkoxyl (RO_) radicals and nonradicals such as hypochlorous acid (HOCl2), ozone (O3), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Reactive nitrogen species (RNS), which are nitrogencontaining oxidants, include free radicals such as nitric oxide (NOU ), nitrogen dioxide (NOU2 ) and nonradicals such as peroxynitrite (ONOO2) and dinitrogen trioxide (N2O3).15,18 Free radical reactions typically occur in steps with each step activating the next step, thereby producing a chain reaction. The three steps of reactions are initiation, propagation, and termination.18 For example,

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29. Phenolics: therapeutic applications against oxidative injury in obesity and type 2 diabetes pathology

the initiation of lipid peroxidation leads to the propagation of radical chain reactions, which terminates with products that oxidatively react with close biological molecules.19 The accrual of reactive species causes oxidative damage to cellular membranes and cellular structural components such as lipids, protein, and DNA. This is exemplified by oxidative modifications to the cell membrane and lipoprotein by process of lipid peroxidation, the formation of DNA lesions resulting from oxidative DNA damage, changes in protein configuration, and defective enzyme activities due to protein oxidative damage.15 When biological cells and tissues are subjected to the deleterious effects of radicals, the processes of cellular antioxidant defenses are activated; these antioxidants are molecules that prevent or detoxify free radical reactions and inhibit or repair oxidative cellular injury.20 Antioxidants can be categorized as endogenous and exogenous. Endogenous antioxidants can be basically classified into enzymatic or nonenzymatic antioxidants and are naturally produced by cellular metabolism, while exogenous antioxidants basically belong to nonenzymatic groups and are supplied to the body from the consumption of fruit and vegetables.15 Antioxidant actions can include prevention, which is usually the first line of defense, or chain breaking. The prevention method usually involves lowering the chain initiation step, and the chain breaking method involves the stabilization of the free radicals formed in the chain reaction process or by simply neutralizing them.15,21 Endogenous antioxidant enzymes include superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GRx), and peroxidoxins (Prx) present in different compartments in the cell. For example, SOD is located in the mitochondria and cytosol where it catalyzes the reduction of superoxide anion OU2 2 into H2O2 and oxygen, H2O2 is further neutralized by GPx located in the cytoplasm and extracellular matrix to water or by catalase present in the peroxisome to water and oxygen. Endogenous nonenzymatic antioxidants also participate in the first line of defense basically by scavenging radicals and include lowmolecular-weight substances such as glutathione (GSH), ferritin, bilirubin, coenzyme Q, uric acid, lipoic acid, and metal-chelating proteins, etc.15,20,21 Phenolic compounds, which are the focus of this chapter, belong to the exogenous nonenzymatic group of antioxidants. Other examples include ascorbic acid, α-tocopherol, and carotenoids, etc.21 They work synergistically with endogenous antioxidants. Increased consumption of exogenous antioxidants is necessary to boost the activities of intrinsic antioxidants since sustained spikes in free radical levels causes oxidative damages, which have been implicated in diseases and aging.21,22

Obesity Obesity as defined by the World Health Organization is an excessive accumulation of fat that poses a risk to health.23 A simple way of classifying obesity is by measuring the body mass index (BMI), which is a measure of body weight in kilograms in relation to height in meters squared. A BMI of greater than or equal to 30 kg/m2 is categorized as obesity, while that of greater than or equal to 25 kg/m2 but less or equivalent to 30 kg/m2 is categorized as overweight.23 Obesity basically represents a disproportionate energy intake and energy expenditure balance resulting in excessive fat accumulation.4,24 Its usual causes include changes in lifestyle and dietary habits, physical inactivity, social-economic status, and genetic and environmental factors. The prevalence of obesity is on the rise; its epidemic proportions are not only confined in developed countries anymore, but are now a global issue.23 It is a major global health issue associated with high mortality and increased chances of developing other chronic diseases such as T2D, heart diseases, hypertension, certain cancers, and sleep apnea, etc.,25 and these in turn decrease life expectancy.26 It is a disease found within all age brackets and is widely accepted as a major risk factor for the development of T2D.23,27 Between 1975 and 2016, the occurrence of obesity cases almost tripled. In 2016, over one-third of the world’s 1.9 billion overweight adults were classified as obese, while over 340 million children and teenagers between the ages of 5 and 19 were recorded as overweight or obese, and over 41 million children below 5 years of age had an overweight or obese record.23 The alarming rise in global obesity observed in the past three decades will make obesity a major cause of global medical expenditure in the next 25
50 years.28

Oxidative stress and obesity Adipose tissue plays a major role in the generation of OS and proinflammation in obesity.4 Apart from the function of adipose tissue as fat storage tissue, white adipose tissues contain preadipocytes and mature adipocytes that are primary sources of adipokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), plasminogen activator inhibitor-1 (PAI-1), leptin, and adiponectin, which maintain homeostatic physiological conditions in normal-weight individuals.24,29 However, the elevated levels of these proinflammatory cytokines seen in obese individuals gives rise to OS because they are known to be strong activators of ROS and RNS, which can damage several organs.24 Consumption of a calorie-dense diet stimulates rapid growth of adipocytes that can in turn initiate the production of ROS leading to OS;28,29 this is evident in the

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Therapeutic applications of phenolics and obesity

production of ROS load such as superoxide radical from adipocyte’s nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation.24,30 Uncontrolled fat intake causes increased adiposity, which has a typical characteristic of lipolysis, and the free fatty acids (FFAs) produced become prone to oxidation as seen in lipid peroxidation causing damages to cellular membranes, therefore, leading to OS, lipotoxicity, mitochondrial dysfunction, and cellular damage.4,24 Increased metabolic efficiency, as seen in obesity, leads to the formation of excess ROS such as OU2 2 formed from an increased cellular mitochondrial function in an obese individual.29,31 Other factors such as reduced antioxidant levels, activation of ROS-generating enzymes such as xanthine oxidase and NADPH oxidase, chronic inflammation, mitochondrial dysfunction, and irregular postprandial ROS production are contributors to OS generation24,29,32

Type 2 diabetes Over 90% of all diabetic sufferers are type 2 diabetics, this makes it the most common type of diabetes associated with health problems and death.33
35 T2D is a heterogenous disorder represented by chronic elevated blood glucose resulting from a deficient insulin secretion associated with resistance to insulin action.33,36 It majorly develops from being overweight/ obese owing to a sedentary lifestyle, unhealthy eating habits, and physical inactivity along with other factors including family history, stress, and advancing age.37 The consequential effects of the disease comprise neuropathy, nephropathy, cardiovascular diseases, diabetic toe/foot amputation, and eventual death.33 The number of T2D cases is increasing globally causing increased global medical expenditure and health concerns. In 2017, an estimated 425 million adults were living with diabetes and this is projected to increase by 48% by 2045.38 In 2016, diabetes was the seventh leading cause of death with over 1.6 million cases of death23 and this rose to 4 million in 2017 with a total sum of USD727 billion estimated for healthcare expenditure.38

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consequently, causing cellular damage with the eventual development of T2D-related micro- and macrovascular complications.35,39,41 Excessive ROS depletes pancreatic β-cells, thus, decreasing insulin secretion with concomitant increase in blood glucose. Insulin resistance is a common connection between T2D and obesity such that increased adiposity stimulates the release of excess FFAs and the production of adipokines, this in addition with OS impairs β-cell function leading to insulin resistance. Pancreatic β-cells compensate for this by hypersecretion of insulin in turn causing impaired glucose tolerance, β-cell destruction, and the eventual development of T2D.27 Currently available antiobesogenic and antidiabetic drugs are known for their various side effects and high cost. Orlistat is a common antiobesogenic drug that decreases fat absorption from the diet by inhibiting pancreatic lipase; its side effects include bloating, oily or leaky stool, headaches, flatulence, etc.28 Weight gain and headaches are common side effects of sulfonylureas, which are widely used antidiabetic drugs that promote insulin secretion.41 The therapeutic use of phenolics, which can regulate both weight gain and glycemic indices, are, therefore, safer and cheaper approaches to the management of these global epidemics.

Therapeutic applications of phenolics and obesity The therapeutic effect of phenolics on obesity has been reported in several studies. These studies employed in vitro, in vivo, and in silico methods in arriving at their conclusions. The mechanisms by which these phenolics bring about their antiobesogenic activities include inhibition of lipid metabolizing enzymes,42 downregulation of lipogenic proteins expression,43 induction of adipocyte apoptosis,44 inhibition of lipid accumulation,45 modulation of lipid homeostasis,46 and attenuation of OS and inflammation.46 The antiobesogenic activities and mechanisms of action of selected phenolics are summarized in Table 29.1.

Phenolics and type 2 diabetes Oxidative stress and type 2 diabetes Oxidative stress (OS) is a major participant in cellular injury and diabetic complications caused by chronic elevated blood glucose.39 OS usually results from several mechanisms such as glucose autoxidation, nonenzymatic glycosylation of proteins, and intrinsic antioxidant enzymes.40 Chronic hyperglycemia has been implicated in the generation of excessive ROS, which overpower the intrinsic antioxidant defenses leading to OS and,

Phenolics have been reported for their antidiabetic properties in both basic and clinical studies. Their antidiabetic mechanisms are mostly linked to their ability to scavenge hyperglycemia-generated free radicals as well as arrest other oxidative-mediated activities such as β-cell dysfunction, apoptosis of pancreatic β-cells, and insulin resistance. The antidiabetic activities of selected phenolics and their mechanisms of action are summarized in Table 29.2.

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TABLE 29.1 Antiobesogenic properties of selected phenolics and their mechanism of action. Compounds

Structure

Antiobesogenic activity

Mechanism of action

Reference

5-Hydroxy-3,6,7,8,30,40hexamethoxyflavone

Adipocyte apoptosis

Increased intracellular calcium level with concomitant increase 44 of calpain and caspase-12 activities

Nobiletin

Inhibition of hepatic and intestinal lipid accumulation

Increased hepatic β-oxidation Reduced expression of C/EBPβ and PPARg in adipocytes

45

Hesperetin

Anti-inflammation in adipocytes Inactivation of hormone sensitive lipase (HSL) Inhibition of lipid accumulation

Inhibition of NFkB; TNF-α, IL-6 Deactivation of ERK pathway

47

Inhibition of lipase activity Hypolipidemia

Suppression of serum cholesterol and triglyceride levels

42

Naringenin

Kaempferol

Quercetin

Inhibition of lipase activity Inhibition of lipid accumulation and obesity-induced inflammation Inhibition of TG synthesis Inhibition of adipogenesis

Suppresses C/EBPβ, C/EBPα, PPARγ, and FABP4 protein levels Reduces protein levels of lipin1, DGAT1, and LPAATθ Downregulates the activation of m-TOR and p70S6K Inhibits ERK1/2, JNK, and p38MAPK and MCP-1, TNF-α, IL1β, and IL-6 activities Stimulates IL-10

42,43

Caffeic acid

Inhibition of lipid accumulation Inhibition of adipogenesis

Suppresses PPARγ protein expression Modulates adipogenic, lipolytic and β-oxidation genes expression

48

Inhibits HMGCoA reductase, acetyl carboxylase, and FAS Increases the phosphorylation of AMPK

Hydroxytyrosol

Gallic acid

Inhibition of lipid homeostasis Modulation of redox imbalance

Increased serum levels of HDL-c with concomitant reduction of total cholesterol, LDL-c, and TG levels Increases GPx, GRd, and GST activities as well as GSH level Inhibits serum leptin levels

48

TABLE 29.2 Antidiabetic properties of selected phenolics and their mechanism of action. Compounds Structure

Antidiabetic activity

Mechanism of action

Reference

Quercetin

Inhibition of key enzymes linked to type 2 diabetes Inhibition of inflammatory markers of diabetes Muscle glucose uptake Hypoglycemic activity Prevention of polyol accumulation

Inhibits α-glucosidase and α-amylase activities Inhibits COX-2, NF-κB, NO, LOX, TNF-α, IL-6, IL-1β, IFN-γ, and iNOS Modulates GLUT4 Arrest of gluconeogenesis Inhibits lens aldose reductase

49

Kaempferol

Arrest of pancreatic β-cell apoptosis Attenuation of pancreatic redox imbalance Improves muscle glucose uptake

Downregulates the expression of PPAR-γ and SREBP-1c Depletes β-cell caspase-3 activity Increases antioxidant activities Modulates GLUT 4 and AMPK

50

Resveratrol

Muscle glucose uptake Attenuation of redox imbalance and inflammation Improvement of glucose homeostasis

Upregulates GLUT 4 and AMPK Inhibits NF-κB, COX-1, TNF-α, CRP, and IL-6 Reduces insulin resistance Suppresses postprandial glucagon responses Improves HbA1c level

51

Naringenin

Inhibition of carbohydrate metabolizing enzymes Improvement of glucose homeostasis Attenuation of oxidative stress

Inhibits α-glucosidase activity Increased SOD activity Improves insulin sensitivity and glucose tolerance

52

Luteolin

Attenuation of oxidative stress and inflammation Suppression of hepatic lipogenesis Improvement of glucose homeostasis

Reduces mast cell and macrophage infiltrations Inhibits inflammatory cytokines Increases SOD activity Reduces the expression of CREB-binding protein/p300 gene Decreases FAS activity and SREBP-1c expression Improved glucose tolerance Improves insulin sensitivity

50

Gallic acid

Attenuation of oxidative stress and inflammation Modulation of glucose homeostasis Improves muscle glucose uptake

Activation of the AMPK/Sirt1/PGC1α pathway Improves antioxidant enzyme activities, while depleting inflammatory cytokines

53

Catechin

Ameliorates oxidative stress and inflammation Modulates glucose homeostasis Inhibition of carbohydrate metabolizing enzymes

Inhibits α-amylase and α-glucosidase Increased antioxidant enzyme activity Improves insulin sensitivity Improves glucose tolerance Decreases levels of white blood cells, monocytes, lymphocytes, and platelets Downregulates C/EBPα and PPAR-γ

54

Rutin

Improves glucose homeostasis Attenuates oxidative stress and inflammation Arrest pancreatic β-cell apoptosis

Improves insulin secretion Inhibits inflammatory cytokines Downregulates caspase-3 and upregulates Bcl-2 protein expressions

50

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29. Phenolics: therapeutic applications against oxidative injury in obesity and type 2 diabetes pathology

Conclusion In conclusion, phenolics are well documented for their antioxidant properties, which have been attributed to their chemical structures. These antioxidant properties have been explored as therapeutics against obesity and T2D and their complications. Phenolics may also serve as major ingredients in the development and production of functional foods and nutraceuticals, which can serve as adjuncts in the treatment and management of obesity and T2D.

Conflict of interest The authors report no conflict of interest.

Acknowledgment This study was supported by a competitive research grant from the Research Office, University of KwaZulu-Natal (UKZN), Durban; an incentive grant for rated researchers and a grant support for women and young researchers from the National Research Foundation (NRF), Pretoria, South Africa.

Summary points • Phenolics are a group of secondary metabolites widely distributed in plants and are involved in plant cellular metabolism and physiology. • This group of compounds has been reported to work with endogenous antioxidants to fight against various diseases. • The chemical structure of phenolics, which include an aromatic ring and hydroxyl groups with or without other functional groups, are crucial for their antioxidant properties.

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9. 27. Bailey CJ. The challenge of managing coexistent type 2 diabetes and obesity. BMj 2011;342:d1996. 28. Mopuri R, Islam MS. Medicinal plants and phytochemicals with anti-obesogenic potentials: a review. Biomed Pharmacother 2017;89:1442
52. 29. Savini I, Catani M, Evangelista D, Gasperi V, Avigliano L. Obesity-associated oxidative stress: strategies finalized to improve redox state. Int J Mol Sci 2013;14(5):10497
538. 30. Brasier AR, Recinos III A, Eledrisi MS. Vascular inflammation and the renin-angiotensin system. Arterioscler Thromb Vasc Biol 2002;22(8):1257
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31. Khan N, Naz L, Yasmeen G. Obesity: an independent risk factor for systemic oxidative stress. Pakistan J Pharm Sci 2006;19(1):62
5. 32. Sies H, Stahl W, Sevanian A. Nutritional, dietary and postprandial oxidative stress. J Nutr 2005;135(5):969
72. 33. Islam MS, Choi H. Comparative effects of dietary ginger (Zingiber officinale) and garlic (Allium sativum) investigated in a type 2 diabetes model of rats. J Med Food 2008;11(1):152
9. 34. Atlas D, International Diabetes Federation. IDF diabetes atlas. 7th ed. Brussels: International Diabetes Federation; 2015. 35. Erukainure OL, Narainpersad N, Singh M, Olakunle S, Islam MS. Clerodendrum volubile inhibits key enzymes linked to type 2 diabetes but induces cytotoxicity in human embryonic kidney (HEK293) cells via exacerbated oxidative stress and proinflammation. Biomed Pharmacother 2018;106:1144
52. 36. Ozougwu J, Obimba K, Belonwu C, Unakalamba C. The pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. J Physiol Pathophysiol 2013;4(4):46
57. 37. Srinivasan K, Viswanad B, Asrat L, Kaul C, Ramarao P. Combination of high-fat diet-fed and low-dose streptozotocintreated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol Res 2005;52(4):313
20. 38. Cho N, Shaw J, Karuranga S, et al. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pract 2018;138:271
81. 39. Tiwari BK, Pandey KB, Abidi A, Rizvi SI. Markers of oxidative stress during diabetes mellitus. J Biomarkers 2013;2013:378790. 40. Matough FA, Budin SB, Hamid ZA, Alwahaibi N, Mohamed J. The role of oxidative stress and antioxidants in diabetic complications. Sultan Qaboos Univ Med J 2012;12(1):5. 41. Vinayagam R, Jayachandran M, Xu B. Antidiabetic effects of simple phenolic acids: a comprehensive review. Phytother Res 2016;30(2):184
99. 42. Sergent T, Vanderstraeten J, Winand J, Beguin P, Schneider Y-J. Phenolic compounds and plant extracts as potential natural anti-obesity substances. Food Chem 2012;135(1):68
73. 43. Seo M-J, Lee Y-J, Hwang J-H, Kim K-J, Lee B-Y. The inhibitory effects of quercetin on obesity and obesity-induced inflammation

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by regulation of MAPK signaling. J Nutr Biochem 2015;26 (11):1308
16. Sergeev IN, Li S, Ho C-T, Rawson NE, Dushenkov S. Polymethoxyflavones activate Ca2 1 -dependent apoptotic targets in adipocytes. J Agric Food Chem 2009;57(13):5771
6. Mulvihill EE, Huff MW. Protection from metabolic dysregulation, obesity, and atherosclerosis by citrus flavonoids: activation of hepatic PGC1α-mediated fatty acid oxidation. PPAR Res 2012;2012:857142. Hsu C-L, Yen G-C. Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Br J Nutr 2007;98(4):727
35. Assini JM, Mulvihill EE, Burke AC, et al. Naringenin prevents obesity, hepatic steatosis, and glucose intolerance in male mice independent of fibroblast growth factor 21. Endocrinology 2015;156(6):2087
102. Lutfi E, Babin PJ, Gutie´rrez J, Capilla E, Navarro I. Caffeic acid and hydroxytyrosol have anti-obesogenic properties in zebrafish and rainbow trout models. PLoS one 2017;12(6):e0178833. Chen S, Jiang H, Wu X, Fang J. Therapeutic effects of quercetin on inflammation, obesity, and type 2 diabetes. Mediat Inflammation 2016;2016:340637. Vinayagam R, Xu B. Antidiabetic properties of dietary flavonoids: a cellular mechanism review. Nutr Metab 2015;12(1):60. Nanjan MJ, Betz J. Resveratrol for the management of diabetes and its downstream pathologies. Eur Endocrinol 2014;10(1):31. Hasanein P, Fazeli F. Role of naringenin in protection against diabetic hyperalgesia and tactile allodynia in male Wistar rats. J Physiol Biochem 2014;70(4):997
1006. Abdel-Moneim A, Yousef AI, El-Twab SMA, Reheim ESA, Ashour MB. Gallic acid and p-coumaric acid attenuate type 2 diabetes-induced neurodegeneration in rats. Metab Brain Dis 2017;32(4):1279
86. Samarghandian S, Azimi-Nezhad M, Farkhondeh T. Catechin treatment ameliorates diabetes and its complications in streptozotocin-induced diabetic rats. Dose-Response 2017;15(1). 1559325817691158.

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C H A P T E R

30 Pistachio nut, its virgin oil, and their antioxidant and bioactive activities Marı´a Desamparados Salvador, Rosa M. Ojeda-Amador and Giuseppe Fregapane Department of Food Science and Technology, Faculty of Chemistry, University of Castilla-La Mancha, Ciudad Real, Spain

at an average of 3500
4000 MT annually.11 Iran has been a major pistachio producer for thousands of years and is currently one of the main growers (315,151 tons/ year) alongside the United States (406,646 tons/year), and Turkey (170,000 tons)12. In central Spain, this crop is rapidly growing, reaching a production of 2418 tons/ year (10th world position). Pistachios are widely consumed as snacks (roasted, natural, or salted-roasted) and as components of several edible products.13

List of abbreviations DRI ROS TPP VPO

dietary reference intake reactive oxygen species total polar phenolics virgin pistachio oil

Introduction Nuts are considered a great source of biologically active compounds, mainly due to their high content of unsaturated and essential fatty acids (EFAs) and phenolic compounds.1
8 Indeed, the first health claim specific to nuts and the reduction of the risk of heart disease, “scientific evidence suggests but does not prove that eating 1.5 oz (42.5 g) per day of most nuts, such as pistachios, as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease,” was approved in July 2003 by the United States Food and Drug Administration (USFDA).9 The pistachio (Pistacia vera L.) is a small tree and member of the Anacardiaceae family, native to central and western Asia and estimated to be B80 million years old. It was introduced in Europe before Roman times and later spread to all regions around the world with a Mediterranean ecosystem like parts of the United States and North Africa. Nowadays, P. vera L. is cultivated as an agricultural crop in the Middle East, California, and Mediterranean Europe, being in continuous expansion due to the presence of large geographical areas with favorable climatic conditions and to the existence of new varieties.10 The world pistachio production is estimated Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00030-5

Pistachios are highly nutritious Pistachio nuts are a dry (3%
5% moisture) and nutritious-dense food principally due to its elevated lipid (48%
63%) and protein content (18%
22%), as well in dietary fiber (8%
12%; Table 30.1).14
16 Indeed, the recommended daily consumption of nuts such as pistachios (1.5 oz equivalent to 42.5 g9) reaches approximately 15% of the dietary reference intake (DRI; Table 30.1) for proteins, 11% and 18% of the DRI for males and females respectively for fiber, and 24% of the DRI for fat. The latter nutrient, although present in high amounts, possesses a balanced content of mono- (56%
77%) and polyunsaturated (14%
33%; Table 30.2) fatty acids, which could help in the reduction of low-density lipoprotein (LDL)-cholesterol levels and, therefore, the risk of coronary heart disease.7,14,19 Furthermore, a comprehensive phytochemical analysis of pistachio nuts has revealed the presence of a variety of biologically active polar and nonpolar constituents such as tocopherols, phytosterols, and phenolic compounds,6,15 being among the top 50 foods with a high antioxidant potential.20

309

© 2020 Elsevier Inc. All rights reserved.

310 TABLE 30.1

30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

Content and nutritional value of macronutrients in raw and roasted pistachios. Raw (1.5 oz)

Water (g)

% DRI

1.9 (3%
5%)

Energy (kcal)

238

Protein (g)

Roasted (1.5 oz)

% DRI

RDI

11.1

2400a

0.8 10.8

243

8.6 (18%
22%)

15.3

9.0

16.0

56

19.3 (48%
63%)

24.1

19.5

24.4

, 80b

Carbohydrates (g)

4.0 (8%
11%)

3.1

3.9

3.0

130

Of which sugars

3.2

5.9

6.0

55

Fiber (g)

4.5 (8%
12%)

11.5

38/25

Lipid (g)

11.9

3,3 4.4

a

As average between male and female requirements. , 30% of energy requirements (2400 kcal/day). DRI, Dietary reference intakes (adult males, 19
50 years).18 1.5 oz 5 42.5 g. Data are from Tsantili E, Takidelli C, Christopoulos MV, Lambrinea E, Rouskas D, Roussos PA. Physical, compositional and sensory differences in nuts among pistachio (Pistachia vera L.) varieties. Sci Horticulturae 2010;125(4):562
8; Bullo´ M, Juanola-Falgarona M, Herna´ndez-Alonso P, Salas-Salvado´ J. Nutrition attributes and health effects of pistachio nuts. Br J Nutr 2015;113(2):S79
93; Schulze-Kaysers N, Feuereisen MM, Schieber A. Phenolic compounds in edible species of the Anacardiaceae family
a review. RSC Adv 2015;5:73301
14; USDA. National nutrient database for standard reference. Release April 2018.14
17 b

TABLE 30.2

Content and nutritional value of lipidic components in raw and roasted pistachios. Raw (1.5 oz)

% DRI

Roasted (1.5 oz)

% DRI

19.3 (48%
63%)

26.3

19.5

26.6

80a

Saturated (g)

2.5 (10%
16%)

9.4

2.4

9.0

, 27b

Monounsaturated (g)

9.9 (515
81%)

37.1

10.4

39.1

. 27b

Polyunsaturated (g)

6.1 (14%
33%)

22.9

5.7

21.3

, 27b

18:2 n-6 (LNA) (g)

6.0 (13%
31%)

35.2

5.6

32.8

17

18:3 n-3 (ALA) (g)

0.13 (1%
4%)

7.7

0.09

5.6

EPA/DPA/DHA

0

Lipid (g)

Phytosterols (mg)

91

DRI

1.6

0 18.2

94.4

18.9

500

, 30% of energy requirements (2400 kcal/day). 10% of energy requirements (2400 kcal/day). DRI, Dietary reference intakes (adult males, 19
50 years).18 1.5 oz 5 42.5 g. Data are from Tsantili E, Takidelli C, Christopoulos MV, Lambrinea E, Rouskas D, Roussos PA. Physical, compositional and sensory differences in nuts among pistachio (Pistachia vera L.) varieties. Sci Horticulturae 2010;125(4):562
8; Bullo´ M, Juanola-Falgarona M, Herna´ndez-Alonso P, Salas-Salvado´ J. Nutrition attributes and health effects of pistachio nuts. Br J Nutr 2015;113(2):S79
93; Schulze-Kaysers N, Feuereisen MM, Schieber A. Phenolic compounds in edible species of the Anacardiaceae family
a review. RSC Adv 2015;5:73301
14; USDA. National Nutrient Database for Standard Reference; 2018. Release April 2018.14
17 a

b

Consumption and uses of pistachios Nuts are preferably consumed roasted since this treatment improves their desirable flavor, color, crispiness, and crunchy texture.21
23 Nevertheless, the roasting process involves decreases in moisture content, lipid modifications, and changes in color as well as the formation of compounds responsible for the typical roasted nut flavor, mainly due to Maillard reaction products.24
26 Currently, the main uses of pistachio seeds are as snack followed by the pastry and ice-cream industries, all of them requiring a high nut quality. Furthermore, it is also

a valuable product for cosmetic care formulations such as lotions, soaps, skin creams, lip balms, shampoos, and hair conditioner,27,28 and also for therapeutic products with antiinflammatory properties.29 However, other alternatives are being explored for pistachios that do not satisfy these high-quality nut specifications and among these the production of its virgin nut oil with outstanding sensory qualities should be highlighted.30
32 Virgin nut oils can be extracted using mechanical systems, which obtains oils with great organoleptic and nutritional values, or by employing organic solvents, which requires a downstream refining process to make them edible.33
41

II. Antioxidants and Pathology

311

Applications to pathology

With the increasing demand for novel edible oils, virgin oils obtained from nuts are receiving particular attention due to their potential nutritional properties, generally denominated as “healthy oils,” and their attractive and peculiar sensory characteristics “gourmet oils,” which provide added value to the consumer as compared to traditional refined vegetable oils.42 Cold-pressed oils are defined by the FAO-WHO Codex STAN 210 (1999)43 as natural products obtained without altering the nature of the oil, by mechanical procedures. Pistachio oil is not yet described by the current Codex Alimentarius on Fats and Oils (FAO-WHO), however, a project for amending the Standard for Named Vegetable Oils (Codex STAN 210-1999)44 is under study and consideration with the purpose of incorporating composition standards for cold-pressed oils including virgin nut oils (e.g., walnut, pistachio, hazelnut as well as avocado fruit oil). Commercial pistachio oil products, prized as a specialty oil owing to its beneficial effects on human health, are sold in several Middle Eastern and European countries, but not as yet being very extended due to its high market price.

Reactive oxygen species and oxidative stress Reactive oxygen species (ROS) are byproducts of aerobic metabolism, which includes superoxide anions, hydrogen peroxide, and hydroxyl radicals. Low or moderate concentrations of ROS are also involved in physiological responses as part of signaling processes and defense mechanisms against infectious agents.45 An excessive production of ROS by exogenous redox chemicals, physical agents, bacterial or viral infections, or under abnormal pathophysiologic conditions induces a serious imbalance or mismatched redox equilibrium between the production of ROS and the ability of cells to defend against them. This last situation is known as oxidative stress. ROS not only induce direct damage to critical biomolecules, but also indirectly alter or dysregulate cellular signaling.46 The pathophysiological consequences of such oxidative injury include cardiovascular and neurodegenerative disorders, cancer, and rheumatoid arthritis, etc.46,47 Oxidative stress is characterized by an increased production of cellular oxidants that can attack lipid, protein, and nucleic acid simultaneously in living cells. The preservation of the redox status of the cell is vital for survival. The endogenous antioxidant enzymatic system is responsible for protecting body cells against systemic oxidation.48 It is composed of enzymatic and nonenzymatic components, which can be classified into several categories, namely (1) metal chelators

capable of preventing free radical formation by inhibiting metal catalyzed reactions such as Fenton reaction; (2) low-molecular weight antioxidants (e.g., glutathione (GSH) and ascorbic acid); (3) enzymes synthesizing or regenerating the reduced forms of antioxidants such as glutamate-cysteine ligase (GCL) and glutathione reductase (GR); and (4) ROS-interacting enzymes such as superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalases (CAT).48

Applications to pathology As already mentioned, nuts are a well-established fundamental component of a healthy diet. They are rich in protein and fat with a balanced content of mono- and polyunsaturated fatty acids (Table 30.2), and contain several bioactive compounds such as antioxidants that can beneficially impact health outcomes.3,42 Indeed, tree nuts have been referred to as a natural functional food due to the synergistic interactions amongst their many bioactive constituents, which may favorably influence human physiology.7 The main known health-promoting effects of pistachio consumption are summarized in Table 30.3. Evidence suggests that nuts and nut oils can lower LDL-cholesterol levels and, hence, reduce the risk of coronary heart disease due to lipid composition and secondary metabolites, denominated as phytochemicals, with diverse bioactivities.19,67 This has been confirmed by the PREDIMED study (Prevention with Mediterranean Diet) and other epidemiological and clinical trials, which have also indicated that a high intake of nuts (approx. 40 g daily) can lower the incidence of hypertension, metabolic syndrome, diabetes, cancer, other inflammatory conditions, and total mortality.2,64 Among nuts, pistachios (Pistacia spp.) exhibit interesting nutritional properties because they contain cardioprotective constituents such as a high oleic acid content, phytosterols, phenolics, and tocopherols, leading to a potential high antioxidant and antiinflammatory food product.15,54 These dietary components may contribute to antioxidant defense and counteract oxidative damage and oxidative stress by scavenging and neutralizing free radicals or by providing compounds that can induce the gene expression of endogenous antioxidants.56,57 A comprehensive review on the effects of pistachio consumption on health outcomes has been published,8 in which the main intervention studies performed are discussed; including those that show improvement in glucose metabolism65,66 and of the vascular function and systemic hemodynamics.50,51

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312

30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

TABLE 30.3

Main health-promoting effects and biological activities of pistachios.

Health/biological activity

Component and/or mechanism involved

Cardioprotective

LDL-cholesterol lowering:

Reference

• mono- and polyunsaturated fatty acids;

[7,14,19]

• phytosterol

[49]

Vascular function and hemodynamics

[50,51]

Reduced LDL oxidation: vitamin C

[52]

Selenium

[53]

Enhanced immune function

[15,54]

γ-Tocopherol

[55]

Oxidative stress reduction

Scavenging and neutralizing free radicals and/or gene expression of endogenous antioxidants

[56,57]

Antioxidant

Tocopherols and tocotrienols: protect membrane lipids from oxidation

[58]

γ-Tocopherol: lipid peroxidation inhibition

[59]

Vitamin C: provides stability to ROS

[52]

Se: selenoproteins and selenoenzymes

[53,60]

Polar phenolics Trolox Equivalent Antioxidant Capacity: 8
35 mmol/kg DPPH and 50
330 mmol/kg ORAC

[61
63]

PREDIMED study (Prevention with Mediterranean Diet)

[2,64
66]

Antiinflammatory

Other pathologies: hypertension, metabolic syndrome, diabetes, cancer

Beneficial lipidic components The average oil content found in pistachio seeds is about 55%, ranging from 48% in Avdat cultivar to 63% in Kastel.40,68,69

Oleic and essential fatty acids of pistachios Several studies have reported on the fatty acid (FA) profile of the lipidic fraction of the pistachio nut, which is greatly dependent on cultivar. It shows a high content of oleic acid (C18:1, n-9) of between 51% and 81% (Table 30.2), which is close to olive oil70 and higher than many other seed oils.43 Kerman cultivar presents the lowest amount in this healthy fatty acid, highlighting, on the other hand, Sridique cultivar as the one with the highest oleic acid content. Intermedia content (62%
75%) is found in other varieties such as Larnaka, Mateur, and Sirora.14,39,40 The recommended daily consumption of nuts such as pistachios (1.5 oz equivalent to 42.5 g9) provides a high amount of oleic acid; more than the 40% of the DRI (Table 30.2). The adequate intake of oleic acid shows a beneficial effect in cholesterol reduction71 and, moreover, exerts an antiinflammatory cellular effect.72 Thus clear evidence exists that pistachio consumption (25
84 g/day) may be beneficial to ameliorate the

lipid profile and attenuate the inflammatory markers and blood pressure in individuals with overweightassociated risk factors.8,50,65,66,73,74 The essential linoleic acid (C18:2, n-6, LNA) is the second most abundant fatty acid (13% 2 31%; Table 30.2) found in pistachios, with the greatest proportion (B30%) found in Kerman40 as expected by its low oleate value. More than one-third of the daily DRI is reached by the consumption of 1.5 oz of this kind of nut. A much lower content (1%
4%)—though nutritionally significant (6%
8% of DRI; Table 30.2)—for the essential α-linolenic acid (C18:3, n-3, ALA) is found.32,39,40,75 The observed wide variation in the FA profile is relevant regarding the selection of pistachio cultivars or their oils with differentiated nutritional values. Essential Fatty Acids (EFAs), LNA and ALA, play an important role in the human diet, both for their biological functions and prevention and therapy of different pathologies.76 EFAs improve the lipidic pattern and the excitability of myocardium cells and are, therefore, useful in the prevention of cardiovascular diseases and postinfarction arrhythmias. They are precursors of prostaglandins and leukotrienes, which are involved in inflammation and immune response. Moreover, they could prevent several hormonedependent tumors (like breast and prostatic cancer).

II. Antioxidants and Pathology

Antioxidant vitamins (ACE)

EFAs are also involved in anatomic and functional central nervous system development.

Other beneficial lipidic components: phospholipids and phytosterols Phospholipids (PL) possess higher nutritional interest as compared to glycerolipids, however, they have been rarely studied in virgin pistachio oils (VPOs). Nevertheless, VPOs are recognized as having one of the highest phospholipid content among nut oils, showing phosphatidylcholine in the greatest content (3000 mg/kg oil), followed by phosphatidylserine (2800 mg/kg), and phosphatidylinositol (1200 mg/kg).77 A high content of PL (18,824 mg/kg) is observed in the pistachio kernel, with phosphatidic acid, phosphatidylinositol, and phosphatidylcholine being its main components with 5289, 4909, and 3637 mg/kg respectively.78 Furthermore, among nuts, pistachio has one of the highest phytosterol contents,1,79 which is able to reduce blood cholesterol49 as well as to decrease the risk of certain types of cancer and enhance immune function.7,80
82 A high phytosterol content (2100 2 7600 mg/kg) as compared to virgin olive oil (1100 2 2100 mg/kg) 83 as well as to several other vegetable oils 43 is found in VPOs. A remarkable difference is observed among varieties with Sirora showing the lowest amount (3600 mg/kg), Aegina, Kastel, and Kerman a similar intermediate content (3900 2 4110 mg/kg), and Avdat, Larnaka, Mateur, and Napoletana possessing the highest levels (7300 2 7600 mg/kg40). As observed in other vegetable oils, β-sitosterol is the predominant phytosterol also in this kind of nut oils, ranging from 50% to 88%; and a campesterol content of 2.2% 2 5.1% is found.37,40,84,85 Several studies have demonstrated a dose-response reduction of cholesterol mediated by phytosterols, even at low levels similar to those found in plant-based diets with pistachios.49 Although 500 mg of phytosterols per serving are needed to support the health recommendations of the Food and Drug Administration (FDA), the high content of phytosterols in pistachio nuts (close to 20% of the DRI; Table 30.2) may be sufficient to play a synergistic role with the unsaturated FA and low content of saturated FA in helping to maintain normal cholesterol levels.6 In addition, lipophilic pistachio fractions containing a combination of β-sitosterol and PUFAs showed significant reduction in lipid accumulation in mature adipocytes.86

Antioxidant vitamins (ACE) Vitamins with recognized antioxidant activities include carotenoids, beta-carotene in particular

313

(provitamin A), vitamin C (ascorbic acid), and vitamin E (tocopherols), which are sometimes denominated as ACE. Among these, vitamin E is the most abundant vitamin found in most nuts; whereas, in general, nuts are not good sources of vitamins A and C.6

Carotenoids (provitamin A) exert chain-breaking activity and perform a range of functions in human health Nuts do not contain retinol, the preformed form of vitamin A, since it is only found in food of animal origin, especially liver and egg yolk. In contrast, betacarotene is widely found in fruit and vegetables. The concentrations of carotenoids (provitamin A) measured in the different nut types are highest in pistachios along with values for β-carotene of about 200 μg/ 100 g and for lutein/zeaxanthin of about 2760 μg/100 g. These levels are several folds higher as compared to hazelnuts (26 μg/100 g for β-carotene), which exhibited the highest content of carotenoids after pistachios.87 However, the consumption of the recommended 42.5 g (1.5 oz) of pistachios reaches only 2.4% and 1.3% for raw and roasted nuts respectively (Table 30.4) of the RDI for vitamin A (6 μg of β-carotene are equivalent to 1 μg of retinol equivalents; RE). β-Carotene primarily exerts antioxidant effects preventing the initiation of FA peroxidation chain reactions and has a provitamin A function.88 Moreover, individual carotenoids may also act through other mechanisms; for example, lutein/zeaxanthin constitute macular pigment in the eye and are recognized as important modulators of infant and child visual and cognitive development.89 There is evidence that carotenoids, in addition to beneficial effects on eye health, also produce improvements in cognitive function and cardiovascular health and may help to prevent some types of cancer.88,89 The content of carotenoids in pistachios and its virgin oil greatly depends on variety, degree of ripeness, environmental conditions, and geographical origin40,90 with their main constituents being lutein, β-carotene, neoxanthin, and luteoxanthin.

Pistachios are rich in different forms of tocopherols and tocotrienols (vitamin E) Nuts are generally an excellent source for tocopherols; for instance, 42.5 g of almonds or hazelnuts provide up to 75% and 73% of the DRI for vitamin E (15 mg/day of α-tocopherol equivalents (TE) recommended for adults)91, possessing mainly the α-tocopherol homologue ( . 80%). Other nuts contain

II. Antioxidants and Pathology

314 TABLE 30.4

30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

Content and nutritional value of antioxidant vitamins in raw and roasted pistachios. Raw (1.5 oz)

Vitamin A, RE (μg) Retinol (μg) Carotene, beta (μg) Lutein/zeaxanthin (μg)

% DRI

21.6

2.4

Roasted (1.5 oz) 11.3

0

0

129.7

67.6

1235

% DRI

DRI

1.3

900

493

Vitamin C (mg)

2.4

2.7

1.3

1.4

90

Vitamin E, TE (mg)

2.1

13.9

1.9

12.8

15

α-Tocopherol (mg)

1.2

8.1

0.9

6.2

15

γ-Tocopherol (mg)

8.7

10.0

DRI, Dietary reference intakes (adult males, 19
50 years). 1.5 oz 5 42.5 g. RE; retinol equivalents; TE, alpha-tocopherol equivalents. Data are from Tsantili E, Takidelli C, Christopoulos MV, Lambrinea E, Rouskas D, Roussos PA. Physical, compositional and sensory differences in nuts among pistachio (Pistachia vera L.) varieties. Sci Horticulturae 2010;125(4):562
8; Bullo´ M, Juanola-Falgarona M, Herna´ndez-Alonso P, Salas-Salvado´ J. Nutrition attributes and health effects of pistachio nuts. Br J Nutr 2015;113(2):S79
93; Schulze-Kaysers N, Feuereisen MM, Schieber A. Phenolic compounds in edible species of the Anacardiaceae family
a review. RSC Adv 2015;5:73301
14; USDA. National Nutrient Database for Standard Reference; 2018. Release April 2018.14
17 18

much lower amounts of vitamin E. Pistachios and walnuts, which contain almost exclusively the γ-tocopherol form ( . 90%;10 mg of this homologue is equivalent to 1 mg of TE) cover about 13%
14% of the RDI for this vitamin by a portion of 42.5 g (Table 30.4). As expected, roasting decreases the amounts of β-carotene significantly in most nut varieties (e.g., 20% in pistachios) and is associated with a significantly lower level of α-tocopherol (54% in almonds and 20% in hazelnuts) and γ-tocopherol (30% in hazelnuts and 56% in walnuts), but affects to a lesser extent the αand γ-tocopherols contents in pistachios.87 As expected, a high content of γ-tocopherol is observed in VPO (162
804 mg/kg).13,40,85,92,93 Among cultivars, VPO from the Kerman variety showed the highest content (719 mg/kg) compared with the rest of the varieties (B600 mg/kg).40 Pistachios are rich in different forms of tocopherols and tocotrienols, all together referred to as vitamin E, possessing a powerful lipid-soluble antioxidant activity that protects cell membrane lipids from oxidation.58 Furthermore, α-tocopherol has shown to be a potent modulator of gene expression and γ-tocopherol appears to be highly effective in preventing cancer-related processes.94 γ-Tocopherol is also considered an important functional compound and demonstrates a similar bioavailability that of α-tocopherol,95 acting both in vivo and in vitro as an antioxidant and being an even more efficient antioxidant in food lipid matrices.96 Moreover, although studies are limited, non α-tocopherol and tocotrienols have important functions. γ-tocopherol has been shown to be an effective inhibitor of peroxynitrite-induced lipid peroxidation.59 Also, γ-tocopherol is effective at inhibiting inflammatory reactions and, therefore, the beneficial role of pistachios in

inflammatory-related diseases may also be explained by the relatively high amount of γ-tocopherol they contain.55 Tocotrienols possess excellent antioxidant activity in vitro and have been suggested to suppress ROS more efficiently than tocopherols97 along with being found to be more effective at reducing the ageing process and age-related diseases.

Ascorbic acid (vitamin C) reacts with several types of reactive oxygen species Vitamin C is found only in a few food products, mainly vegetables and fresh fruit, whereas in dehydrated products like nuts, its content is low. In fact, only 1.5%
3.0% of the DRI for vitamin C is provided by the consumption of the recommended portion of pistachios (42.5 g; Table 30.4). The antioxidant effect of vitamin C is well known, it easily gives up electrons to provide stability to reactive species such as ROS.52 It therefore protects cellular components from free radical damage and membranes against lipid peroxidation damage by eliminating peroxyl radicals and free radicals in aqueous body compartments (cells, blood, and lymph). Furthermore, vitamin C is also effective in regenerating vitamin E by reducing tocopheroxyl radical and coenzyme Q10 and reduces the oxidation of LDL implicated in the pathogenesis of atherosclerosis.52

Antioxidant minerals (Se, Cu, Zn, Mn) Pistachios are rich in several minerals such as potassium (K), magnesium (Mg), calcium (Ca), copper (Cu),

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Polar phenolic compounds

and manganese (Mn), and, therefore, could play a beneficial role in blood pressure regulation or in bonerelated diseases.15 Moreover, they contain significant amounts of zinc (Zn) and selenium (Se), both minerals with recognized antioxidant effects that are involved in the prevention of cardiovascular diseases and some types of cancer.98,99 As expected, the roasting process does not modify the chemical structure of these elements and, therefore, they generally slightly concentrate during this treatment due to the reduction of the water content (Table 30.5).

According to the recommended consumption of nuts (1.5 oz daily9), between 5.4% and 7.7%, for raw and roasted pistachio respectively, of the daily DRI for Se is reached (Table 30.5). Pistachios are, therefore, a significant source of this antioxidant mineral, but not in a high content ( . 20% DRI). In this sense, Brazil nut serves as an excellent source of this mineral compound; one kernel (approx. 5 g) supplies 174% of the DRI for Se.1,91

Selenium proteins are well known for protecting cells from oxidative stress damage

Other antioxidant minerals involved in the antioxidant cellular defense mechanism that fight against ROS (e.g., hydroxyl radicals, superoxide, hydrogen peroxide) and prevent oxidative stress, especially in the liver, are Cu, Zn, and Mn. Cu and Zn play a role in the cytosolic enzyme superoxide dismutase, which converts superoxide to hydrogen peroxide.52 Whereas Mn and Zn are necessary for the activity of the mitochondrial enzyme superoxide dismutase that converts superoxide to hydrogen peroxide. Furthermore, Cu is required for the activity of the antioxidant protein ceruloplasmin, which may prevent Cu and Zn from participating in oxidation reactions.52 The intake of these antioxidant minerals provided by the consumption of 45 g of pistachios is very high for Cu (61% of the DRI, Table 30.5) and Mn (22%
23%) and significant for Zn (8%
9%).

Se is an essential trace element, and its low status in humans has been linked to increased risk of various diseases such as cancer and heart diseases.52,53 It is most specifically related to antioxidant function due to its important role in selenoproteins and selenoenzymes such as glutathione peroxidases (GPx), thioredoxin reductase (TrxR), and iodothyronine deiodinases (IDD),53,60 which play important roles in cell protection against oxidative stress initiated by excess ROS and reactive nitrogen species (ROS) and in maintaining a proper thyroid function. GPx enzymes are probably the most important selenoproteins for their function as antioxidants by reducing peroxides such as hydrogen peroxide. The sulfur-containing peptide, glutathione (GSH), is a necessary cofactor in the reduction of peroxides and acts as a reducing substrate; however, sulfur compounds themselves do not exhibit GPx activity.100,101 As known, the presence of ROS can cause the oxidation of LDL, which is associated with the initiation of atherogenesis in heart diseases.102 The cancer preventive trials based on Se supplementation that are currently being undertaken will provide more information on optimal Se intake as well as strategies in the treatment of other potential human diseases associated with low Se status including those related to immunity and thyroid hormones.53 TABLE 30.5

Copper, zinc, and manganese are also involved in the antioxidant cellular mechanism

Polar phenolic compounds The polyphenol content of many foods is documented in nutrient databases such as the Phenol-Explorer and the United States Department of Agriculture (USDA) flavonoid and proanthocyanidin databases.4,61,103,104 Moreover, the levels of the different groups of phenolic compounds present in commonly consumed nuts including pistachios and their corresponding antioxidant

Content and nutritional value of antioxidant minerals in raw and roasted pistachios. Raw (1.5 oz)

Selenium (μg) Copper (μg)

3.0 553

% DRI 5.4 61.4

Roasted (1.5 oz) 4.3 550

% DRI

DRI

7.7

55

61.1

900 11

Zinc (mg)

2.2

8.5

2.34

9.0

Manganese (mg)

0.51

22.2

0.53

23.0

2.3

DRI, Dietary reference intakes (adult males, 19
50 years).18 1.5 oz 5 42.5 g. Data are from Tsantili E, Takidelli C, Christopoulos MV, Lambrinea E, Rouskas D, Roussos PA. Physical, compositional and sensory differences in nuts among pistachio (Pistachia vera L.) varieties. Sci Horticulturae 2010;125(4):562
8; Bullo´ M, Juanola-Falgarona M, Herna´ndez-Alonso P, Salas-Salvado´ J. Nutrition attributes and health effects of pistachio nuts. Br J Nutr 2015;113(2):S79
93; Schulze-Kaysers N, Feuereisen MM, Schieber A. Phenolic compounds in edible species of the Anacardiaceae family
a review. RSC Adv 2015;5:73301
14; USDA. National Nutrient Database for Standard Reference; 2018. Release April 2018.14
17

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30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

activities, together with evidence for the health benefits of nuts have been reviewed and discussed.6,11

Pistachios serve as a good source of polar phenolics Walnuts, pecans, and pistachios show the highest phenolic contents, whereas hazelnut, peanuts, and almonds contain a significantly lower amount.11,17 Total polar phenolics (TPP) in pistachio nuts show a wide variety-dependent content ranging from 1600 mg/kg for the Kastel variety to more than threetimes that (4900 mg/kg) for Larnaka. As known, polar phenolic compounds can be classified into several families. Flavanols are the most abundant phenolics found (about 90%, ranging from 1500 to 4500 mg/kg), where the cultivars Kastel and Larnaka presented the lowest and highest contents respectively.61 Other phenolic groups such as anthocyanins (from 54 to 218 mg/kg), flavonols (from 76 to130 mg/kg), flavanones (from 12 to 71 mg/kg), and gallotannins (from 4 to 46 mg/kg) are also found. These results are similar to those obtained by other authors.6,11,17 As expected due to their relatively high-water solubility only low TPP concentrations are found in VPO (16
58 mg/kg) according to literature data,40,61 but similar to other virgin seed oils, such as soybean, sunflower, rapeseed and corn (10 2 40 mg/kg105). Ojeda-Amador et al.40 described TPP contents in coldpressed VPO between 16 to 23 mg/kg studying eight different pistachio cultivars; whereas higher values (25
54 mg/kg) were reported by other authors32,40,75,93 as well as in commercial VPO.40

Antioxidant and biological activity of phenolics from pistachios It is relevant to remark that the antioxidant activities of different nuts vary widely based on the assay type. This suggests the need to perform more than one type of antioxidant activity method to consider the various mechanisms of antioxidant action and the limitations of each assay.106 Indeed, a number of in vitro chemical methods (e.g., FRAP, ORAC, DPPH) and biological assays (mainly based on lipoprotein oxidation) are used to determine the antioxidant activities of nuts.11 Concerning the antioxidant capacity of the TPP of pistachios, a wide variety-dependent range was observed. Larnaka presents the highest Trolox equivalent antioxidant capacity (TEAC) for both DPPH (35 mmol/kg) and ORAC (330 mmol/kg) assays.61 Mateur and Avdat show a TEAC of 28 and 27 mmol/kg (DPPH) and 282 and 168 mmol/kg (ORAC) respectively, being the cultivars with the second-highest antioxidant activities. Whereas

Kastel and Kerman are the varieties with the smallest TEAC values, with 13 and 10 mmol/kg (DPPH) and 89 and 61 mmol/kg (ORAC) respectively.61 These results are similar to those obtained by Rodrı´guez-Bencomo et al.,62 who studied the DPPH antioxidant activity of a Turkish pistachio cultivar (Uzun). Moreover, Wu and Prior 63 developed a database regarding ORAC in foods, reporting a value of 76 mmol/kg for pistachio nuts, which is close to what was observed for the Kastel and Kerman cultivars.61 These TEAC values together with the commonly known biological activities of pistachio’s phenolics are reported in Table 30.3. Polar extracts rich in phenolic compounds inhibit ROS release and the expression of cytokines associated with acute and chronic inflammation86; whereas Gentile et al.107 evaluated the effects of a hydrophilic extract of P. vera L. on the production of ROS in RAW 264.7 macrophage cells. A hydrophilic extract of ground pistachio kernel was evaluated as a new chemotherapeutic candidate,108 and the results showed a significant decrease in MCF-7 breast cancer cells viability in a dose- and time-dependent manner by induced intracellular ROS generation and that cell death was in an apoptosis-independent manner.108

Effect of roasting on the properties of pistachios Roasting leads to a significant decrease in the antioxidant activity of hazelnut, macadamia, and walnut, whereas in almond and pistachio the antioxidant activity remains stable or is even slightly enhanced.11,109 Furthermore, antioxidant activity is restored in hazelnut, almond, and walnut at the most intense roasting conditions.110,111 This effect can be explained by the loss of polyphenols due to thermal treatment, which is counteracted by the formation of antioxidant-active compounds due to Maillard reactions as discussed by Ac¸ar et al.111 Moreover, it is relevant to remark that most of the antioxidative compounds of nuts such as polyphenols are located in the skin and their removal is accompanied by a loss of antioxidant activity.21,112 Therefore nuts roasted under suitable conditions demonstrated excellent phenolic compositions and antioxidant activities and should be recommended for daily consumption.11

Summary points • Pistachios contain cardioprotective constituents such as oleic acid, phytosterols, phenolics, and tocopherols, with potential high antioxidant and antiinflammatory activities. A remarkable difference in these healthy components is observed among varieties.

II. Antioxidants and Pathology

References

• The fatty acid profile of pistachios is characterized by high contents of oleic acid (from 51% to 81%), similar to olive oil, and linoleic acid (13% 2 31%). • A high phytosterol content (2100 2 7600 mg/kg), as compared to virgin olive oil (1100 2 2100 mg/kg) and other vegetable oils, is found in virgin pistachio oil. • Pistachios are rich in different forms of tocopherols and tocotrienols, possessing a powerful lipid-soluble antioxidant. Pistachios and walnuts contain almost exclusively the γ-tocopherol form ( . 90%). • They contain significant amounts of Zn and Se, both minerals with recognized antioxidant effects that are involved in the prevention of cardiovascular diseases and some types of cancer. • Total polar phenolics in pistachio nuts show a wide variety-dependent content ranging from 1600 mg/ kg for the Kastel cultivar to 4900 mg/kg for Larnaka and corresponding TEAC. Flavanols are the most abundant phenolics found (about 90%). • All these components may contribute to antioxidant defense and counteract oxidative damage and oxidative stress by scavenging and neutralizing free radicals. Indeed, tree nuts have been referred to as a natural functional food.

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45. 80. Moreau RA, Whitaker BD, Hicks KB. Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Prog Lipid Res 2002;41(6):457
500. 81. Awad AB, Fink CS. Phytosterols as anticancer dietary components: evidence and mechanism of action. J Nutr 2000;130 (9):2127
213. 82. Bouic PJ. The role of phytosterols and phytosterolins in immune modulation: a review of the past 10 years. Curr Opin Clin Nutr Metab Care 2001;4(6):471
5. 83. Aparicio R, Luna G. Characterisation of monovarietal virgin olive oils. Eur J Lipid Sci Technol 2002;104 (9
10):614
27. 84. Saber-Tehrani M, Givianrad MH, Aberoomand-Azar P, Waqif-Husain S, Jafari Mohammadi SA. Chemical composition ´ of Irans pistacia atlantica cold-pressed oil. J Chem 2012;2013:1
6.

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85. Gong Y, Pegg RB, Carr EC, Parrish DR, Kellett ME, Kerrihard AL. Chemical and nutritive characteristics of tree nut oils available in the U.S. market. Eur J Lipid Sci Technol 2017;119 (8):1
15. 86. Grace MH, Esposito D, Timmers MA, Xiong J, Yousef G, Komarnytsky S, et al. In vitro lipolytic, antioxidant and anti-inflammatory activities of roasted pistachio kernel and skin constituents. Food Funct 2016;7:4285
98. 87. Stuetz W, Schlo¨rmann, Glei M. B-vitamins, carotenoids and α-/γ-tocopherol in raw and roasted nuts. Food Chem 2017;221:222
7. 88. Eggersdorfer M, Wyss A. Carotenoids in human nutrition and health. Arch Biochem Biophysics 2018;652:18
26. 89. Giordano E, Quadro L. Lutein, zeaxanthin and mammalian development: metabolism, functions and implications for health. Arch Biochem Biophys 2018;647:33
40. 90. Giuffrida D, Saitta M, La Torre L, Bombaci L, Dugo G. Carotenoid, chlorophyll and chlorophyll-derived compounds in pistachio kernels (Pistacia vera L.) from Sicily. Italian J Food Sci 2006;18(3):313
20. 91. Alasalvar C, Shahidi F. Natural antioxidants in tree nuts. Eur J Lipid Sci Technol 2009;111:1056
62. 92. Ballistreri G, Arena E, Fallico B. Influence of ripeness and drying process on the the polyphenols and tocopherols of Pistachia vera L. Molecules 2009;14:4358
69. 93. Ling B, Xuanmin Y, Li R, Wang S. Physicochemical properties, volatile compounds, and oxidative stability of cold pressed kernel oils from raw and roasted pistachio (Pistacia vera L. Var Kerman). Eur J Lipid Sci Technol 2016;118(9):1368
79. 94. Brigelius-Flohe´ R. Bioactivity of vitamin E. Nutr Res Rev 2006;19 (2):174
86. 95. Jiang Q, Christen S, Shigenaga MK, Ames BN. γ-Tocopherol, the major form of vitamin E in the US diet, deserves more attention. Am J Clin Nutr 2001;74(6):714
22. 96. Wagner K, Kamal-Eldin A, Elmadfa I. Gamma-Tocopherol
an underestimated vitamin? Ann Nutr Metab 2004;48(3):169
88. 97. Schaffer S, M€υller WE, Eckert GP. Tocotrienols: constitutional effects in aging and disease. J Nutr 2005;135:151. 98. Huang H-Y, Caballero B, Chang S, et al. The efficacy and safety of multivitamin and mineral supplement use to prevent cancer and chronic disease in adults: a systematic review for a National Institutes of Health state-of-the science conference. Ann Intern Med 2006;145:372
85. 99. Hercberg S, Kesse-Guyot E, Druesne-Pecollo N, et al. Incidence of cancers, ischemic cardiovascular diseases and mortality during 5-year follow-up after stopping antioxidant vitamins and minerals supplements: a postintervention follow-up in the SU. VI.MAX Study. Int J Cancer 2010;127:1875
81. 100. Mugesh G, Singh HB. Synthetic organoselenium compounds as antioxidants: glutathione peroxidase activity. Chem Soc Rev 2000;29:347
57. 101. Battin EE, Brumaghim JL. Antioxidant activity of sulfur and selenium: a review of reactive oxygen species scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms. Cell Biochem Biophys 2009;55:1
23. 102. Zhang H, Luo Y, Zhang W, He Y, Dai S, Zhang R, et al. Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerotic lesions. Am J Pathol 2007;170:1108
20. 103. USDA. USDA database for the proanthocyanidin content of selected foods, release 2. ,http://www.ars.usda.gov/nutrientdata.; 2015 [accessed 05.12.18]. 104. USDA. USDA database for the isoflavones content of selected foods, release 2.1. ,http://www.ars.usda.gov/nutrientdata.; 2015 [accessed 05.12.18].

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30. Pistachio nut, its virgin oil, and their antioxidant and bioactive activities

105. Siger A, Nogala-Kalucka M, Lampart-Szczapa E. The content and antioxidant activity of phenolic compounds in coldpressed plant oils. J Food Lipids 2008;15(2):137
49. 106. Shahidi F, Ambigaipalan P. Phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects
a review. J Funct Foods 2015;18:820
97. 107. Gentile C, Tesoriere L, Butera D, Fazzari M. Antioxidant activity of Sicilian pistachio (Pistachio vera L. var Bronte) nut extract and its bioactive components. J Agric Food Chem 2007;55:643
8. 108. Reboredo-Rodrı´guez P, Gonza´lez-Barreiro C, Cancho-Grande B, Simal-Ga´ndara J, Giampieri F, Forbes-Herna´ndez TY, et al. Effect of pistachio kernel extracts in MCF-7 breast cancer cells: Inhibition of cell proliferation, induction of ROS production, modulation of glycolysis and of mitochondrial respiration. J Funct Foods 2018;45:155
64. 109. Ojeda-Amador RM, Trapani S, Fregapane G, Salvador MD. Phenolics, tocopherols, and volatiles changes during virgin

pistachio oil processing under different technological conditions. Eur J Lipid Sci Technol 2018. 1800221. 110. Schlo¨rmann W, Birringer M, Bo¨hm V, Lo¨ber K, Jahreis G, Lorkowski S, et al. Influence of roasting conditions on healthrelated compounds in different nuts. Food Chem 2015;180:77
85. ¨C 111. Ac¸ar O ¸ , Go¨kmen V, Pellegrini N, Fogliano V. Direct evaluation of the total antioxidant capacity of raw and roasted pulses, nuts, and seeds. Eur Food Res Technol 2009;229:961
9. 112. Arcan I, Yemenicioglu A. Antioxidant activity and phenolic content of fresh and dry nuts with or without the seed coat. J Food Composition Anal. 2009;22:184
8.

Further reading USDA. USDA database for the flavonoid content of selected foods, release 3.2. ,http://www.ars.usda.gov/nutrientdata.; 2015 [accessed 05.12.18].

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C H A P T E R

31 Sambucus ebulus L., antioxidants and potential in disease Aleksandra Cvetanovi´c Department of Biotechnology and Pharmaceutical Engineering, Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

List of abbreviations ACE EC50 IC50 KAE LOD NA SCW WHO

acarbose equivalent effective concentration (concentration of extract that gives half-maximal response). inhibitory concentration (concentration of extracts for achieving inhibitions of 50%) kojic acid equivalent limit of detection not analyzed subcritical water World Health Organization

Introduction In the past few decades, there has been increased interest from the public in natural sources of biologically active molecules and, therefore, an increased number of people use natural herbal drugs, both as prevention and in therapy. The traditional manner of medical treatment that relies on the use of preparations of natural origin, mostly herbal, has been present in many countries for centuries. In some areas, less than 20% of the population have access to generic drugs or other health care products and among these nations traditional medicine has precedence.1,2 On the other hand, countries like China and India bring together conventional medicine and traditional medicine, and for this reason many traditional preparations that have been made for centuries are now recognized as official medicine as well. A significant increase in interest has also been noticed in industrially developed countries, both on account of the insufficient therapeutic outcomes of conventional drugs and because of the negative effects that may be

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00031-7

caused by a prolonged application of strong synthetic drugs.3,4 On the basis of a large number of conducted studies, contemporary researches have confirmed many activities of plants that have been well known in traditional medicine for centuries and, in many cases, the carriers of these biological activities have been determined.5 On the other hand, some favorable effects of many plants that have until recently been considered weeds are recognized now. On account of their specific compositions, many plants may be used in the treatment of some of the most serious illnesses or they can help with curing these. This has all contributed to greater attention being paid to self-seeding plants, although cultivated, edible, and aromatic plants have had precedence until recently. One of the many self-seeding plant species that has been slightly forgotten over the years, but that absolutely deserves its rehabilitation is Sambucus ebulus L. Although it grows all over as a weed with an unpleasant smell, this plant also has healing properties. S. ebulus L., also known as danewort, dane weed, danesblood, dwarf elder, walewort, dwarf elderberry, elderwort, and blood hilder, is a perennial plant from the family Adoxaceae and the order Dipsacales. This plant that has an exceptionally strong, white, crawling root that grows upright and can reach a height of up to 2 m. Its leaves reach up to 15
30 cm long and consist of 5
11 long, pointy leaflets, the smell of which may be repulsive. The white flowers that are located at the top of the stem (gathered into an umbel) have a characteristic smell of bitter almonds. The plant flowers from May to the end of July. The fruit of this plant comes in the form of round, black, juicy berries that have a diameter of 5
10 mm and

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322

31. Sambucus ebulus L., antioxidants and potential in disease

they reach their full maturity between August and September (Figs. 31.1
31.4). In order to grow well, the plant mostly favors sandy soil, but it can also be frequently found in clay soil. It grows on acid, alkaline, and neutral ground, and is well known for its ability to resist high winds and polluted air. On the other hand, S. ebulus L. does not grow in the proximity of seas and sea air.6 S. ebulus L. has a wide distribution along the edges of forests, beside roads, and common grasslands throughout Europe, North America, and West Asia. The use of S. ebulus L. in the traditional medicine of the peoples living in these areas is deeply rooted.

Traditional use

FIGURE 31.2 Sambucus ebulus fruit (berry).

The use of S. ebulus L. in medicine and diets dates back to antiquity. The first data on its therapeutic applications may be found in Pliny’s work, Naturalis Historia, while the famous Greek physician Dioscorides described in detail its multiple therapeutic properties in his book De Materia Medica. Numerous preparations made from this plant have been used for centuries on the territory of present-day France as well as in the whole of West Europe. The plant used to play an exceptionally important role in the traditional medicine of ancient Persia and, therefore, a large number of preparations based on S. ebulus L. are successfully used in Iran even today. Nowadays, it is considered a highly important plant in Romania, Turkey, and almost all of the countries on the Balkan Peninsula, especially Bulgaria.7 These traditionally made healing preparations include all parts of this plant. The root is picked early in spring or in autumn, the leaf is taken when the

FIGURE 31.1 Sambucus ebulus flower.

plant is blossoming, and the fruit are picked when they are fully mature. Each one of these plant parts is used for different purposes. Thus, for instance, the root is used in the case of rheumatism, arthritis, sciatica, and neuralgias. Its use against psoriasis is particularly important and well spread because of its ability to connect and excrete uric acid from the body. The leaf is recommended for the detoxification of the liver and for regulating the function of the kidneys. Also, the healing preparations made of danewort leaf in the traditional manner are used for treating the common cold and a sore throat as well as for lowering the body temperature or for wound healing. The fruit has multiple uses and its roles as a purgative and immunological stimulator are particularly important. The plant has often been used in the case of bee stings or snake bites, and also for edema, eczema, or inflammatory processes. There is a large number of preparations that are based on S. ebulus L. that are traditionally used in the case of some types of cancer. Also, there is a wellknown traditional medicine against Helicobacter pylori.8 In addition to traditional medicines, this plant is used for many other purposes as well. Thus, for instance, its fruit are used for getting a blue colored ink, while its root juice is used for hair coloring. Because of their unpleasant smell, the leaves of this plant are used for repelling rodents, primarily mice and moles. In some parts of Bosnia and Herzegovina, the fruit is used for brewing rakija (traditional local brandy). Still, it is important to take into account that ingesting the fruit of this plant in large quantity may lead to nausea and vomiting. Also, the inadequate use of the leaves may cause dermatitis. Any use of S. ebulus L. requires a thermal treatment of all of its parts in order for some of its components with toxicological properties to be neutralized.

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Chemical composition

FIGURE 31.3 Sambucus ebulus root.

FIGURE 31.4 Sambucus ebulus leaf.

Chemical composition The broad spectrum of the actions of S. ebulus L. was the driving force behind the scientists who carried out detailed analyses of its chemical composition. Also, many biological and pharmacological characteristics

323

have been confirmed on the basis of numerous studies that have helped isolate the main active principles responsible for its given activities. Of the more important bioactive constituents of this plant are polyphenolic compounds, which have been identified in all of its parts. Researches have shown that the yield of the total phenols and flavonoids is highest in the leaves, while the fruit and the root contain smaller quantities of these components. The different distributions of polyphenols in the different parts of S. ebulus L. is caused by numerous factors such as the type, quality, and composition of the soil, the quantity of available light, the temperature, air humidity, and other climatic and atmospheric conditions. All of these factors have a direct impact on the quantity of synthesized polyphenolic compounds in the different parts of the plant material as well as the fact that many polyphenols may be synthesized in different parts of the plant and then accumulated in other parts. In addition, the leaves are the part of the plant in which three essential functions required for the survival of plants take place, namely photosynthesis, transpiration, and gas exchange; therefore, there is a larger quantity of phenols and flavonoids in this part of the plant than in the root or the fruit. The differences in the quantity of these bioactive molecules lie in the fact that in different parts of the plant there are different connections between the plant tissue and the observed components, which has a direct impact on the degree of availability of these compounds. The polarity of the solvent used for the isolation of the polyphenols of S. ebulus L. directly impacts their concentration in extracts, but the selection of the solvent also depends on the part of the plant from which the polyphenols are extracted. Thus, for instance, the polyphenolic compounds present in the fruit and the root are best extracted using ethyl acetate, while in the case of the leaves, the highest degree of efficiency is achieved by applying methanol.9 The same selection of solvents may also be applied in the case of there being an interest in getting a high yield of flavonoid compounds. The polyphenolic compounds of S. ebulus L. belong to the classes of anthocyanins, flavonoids (apigenin, luteolin, naringenin, kaempferol, epicatechin, and quercetin), flavonoid glycosides (rutin, luteolinglycoside, and apigenin-glycoside), and phenolic acids (hydrohybenzoic acid, caffeic acid, vanillic acid, chlorogenic acid, syringic acid, p-coumaric acid, protocatechuic acid, ferulic acid, sinapic acid, gallic acid, cinnamic acid, and rosmarinic acid)10,11 (Table 31.1). In addition to polyphenolic compounds, many other bioactive components have been detected in this valuable plant and its extracts. The flower is rich in the components of the essential oil (0.3%), while a mature fruit contains only 0.01% of the essential oil.12 The

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324 TABLE 31.1

31. Sambucus ebulus L., antioxidants and potential in disease

Phenolic compounds of Sambucus ebulus. Root

Compound

Structure

SCW

Leaf Hexane

SCW

Berry Hexane

SCW

Hexane

1

2

1

2

1

2

NA

2

NA

2

NA

1

Caffeic acid

1

2

1

2

1

2

Vanillic acid

2

2

2

2

2

1

Chlorogenic acid

1

2

1

1

1

2

Syringic acid

2

2

1

1

2

1

p-Coumaric acid

1

2

1

1

1

1

Ferulic acid

1

1

1

1

1

1

Sinapic acid

, LOD

1

, LOD

1

, LOD

1

Cinnamic acid

2

NA

1

NA

2

NA

Gallic acid

1

NA

1

NA

1

NA

NA

2

NA

1

NA

1

Rutin

2

1

1

1

1

1

Quercetin

2

1

1

1

, LOD

1

Protocatechuic acid

p-Hydrohybenzoic acid

Rosmarinic acid

(Continued)

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325

Chemical composition

TABLE 31.1

(Continued) Root

Compound

Structure

SCW

Leaf Hexane

SCW

Berry Hexane

SCW

Hexane

Luteolin

NA

1

NA

1

NA

1

Luteolin-glycoside

NA

2

NA

2

NA

2

Apigenin

NA

1

NA

1

NA

1

Apigenin-glycoside

NA

2

NA

2

NA

2

Naringenin

2

2

2

1

2

1

Naringin

1

NA

1

NA

1

NA

Catechin

1

NA

1

NA

2

NA

Epicatechin

1

NA

1

NA

1

NA

Kaempferol

NA

1

NA

1

NA

1

LOD, Limit of detection; NA, not analyzed; SCW, subcritical water.

dominant components of the S. ebulus L. essential oil are β-bisabolene, germacrene D, geranyl acetate, and α-cubebene. In addition to these, the components that go into the composition of the essential oil with a 2%
5% share are α-bourbonene, β-caryopyllen, β-caryopyllen oxide, trans-verbenol, trans-carvyl acetate, eugenol, δ-elemene, terpinen-4-ol, cis-carveol, and chavicol (Table 31.2). As many as over 50 different compounds go into the composition of the essential oil with an individual share of less than 2%.13 The more important lipophilic components that go into the composition of S. ebulus L. are saturated and unsaturated fatty acids (palmitic acid, oleanic acid,

oleic acid, octadecanoic acid, vaccenic acid, and linoleic acid), triterpenes (α- and β-amyrin, urosolic acid, and maslinic acid), phytosterols (β-sitosterol, stigmasterol, and campesterol), and diterpenoids (dehydroabietic acid). The nutritive value of the fruit is also contributed to by a significant quantity of sugars, fibers, vitamins, and minerals. Cyanogenic glycosides, present in trace amounts, have been detected in all the parts of the plant.12,14,15 Out of the large number of compounds that go into the composition of S. ebulus L. and its extracts, those that have been studied the most by far are lectins. This large group of compounds may be present in high

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326 TABLE 31.2 Compound

31. Sambucus ebulus L., antioxidants and potential in disease

Dominant components in Sambucus ebulus essential oil. Structure

Compound

Structure

β-Bisabolene

β-Caryopyllen oxide

Germacrene D

Terpinen-4-ol

α-Cubebene

Cis-carveol

Geranyl acetate

Chavicol

β-Caryopyllen

Iso-estragol

α-Bourbonene

trans-Carvyl acetate

trans-Verbenol

Eugenol

δ-Elemene

concentrations in all the parts of the plant and they may serve different functions. Lectins that inhibit protein synthesis (ebulins) have been isolated from different parts of S. ebulus L., that is, in the leaves (ebulin 1), fruit (ebulin f), and rhizome (ebulin r1 and r2). The lectins contained in the stems and flowers are structurally similar to those from the fruit and the leaves. The flower contains two types of lectins that are able to bind D-galactose, namely ebulin blo (A-B toxin) and SELblo (B-B lectin). Studies have shown that at an increased concentration ebulin blo has a high degree of toxicity and it is for this reason that some researchers call it a biological weapon.16
19 On the other hand, the toxicity of these proteins may be compromised by exposing them to a high temperature. It is for this reason that most of the preparations that contain this

plant and that are intended for oral use are subjected to thermal processing (most often to boiling for several hours), thus, eliminating the toxicity.

Antioxidant potential Free radicals constitute a group of highly reactive compounds that an organism may use in its fight against pathogens present within the organism. However, when these are present in high concentrations, they may damage cells through different signal paths. An increased concentration of free radicals and/ or a compromised defense system may lead to oxidative stress and different ailments.20,21 As a response to oxidative stress, organisms have developed a series of

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327

Antioxidant potential

complementary antioxidant enzymes and nonenzymatic molecules, which, due to their antioxidant properties, protect biomolecules from oxidative damaging. In addition to such natural systems and molecules, the antioxidants ingested from food also contribute to an organism’s defense system against oxidative damaging. The best known natural exogenous antioxidants (which cells do not synthesize, but are rather ingested with food) are vitamins and polyphenols.21,22 The activity of polyphenols is caused by different factors, which among others also include OH connection dissociation energy, the possibility of the delocalization and resonant stabilization of the unpaired electron of the polyphenol compound’s free radical, and steric factors due to the presence of voluminous groups linked to the aromatic ring.23 The antioxidant efficiency of polyphenolic compounds is determined by the constants of the speeds of their reactions with free radicals.24 Due to their high antioxidant properties, polyphenolic compounds, especially flavonoids, are used instead of synthetic antioxidants.25 S. ebulus L. constitutes an exceptionally important source of these compounds and it is, therefore, a powerful source of natural antioxidants. The antioxidant activity of the extracts of S. ebulus L. has been confirmed in a large number of studies. Just like the quantity of the total polyphenolic compounds, the antioxidant activity of the extracts of S. ebulus L. is caused by the manner of preparation and the polarity of the solvent used. The extracts obtained with petrol ether show the lowest activity in the neutralization of 1.1-diphenyl-2-picrylhydrazyl (DPPH•) radicals in comparison to water, alcohol, acetone, and ethylacetate extracts. Since the quantity of antioxidant molecules, primarily polyphenols, may be drastically different in different parts of the plant, the antioxidant activity will vary in relation to the part of the plant observed.26 Thus, for instance, methanol and petrol ether extracts from the leaves have a higher activity in comparison to the root and the fruit, whereas in the case of acetone extracts, it is the root that shows the highest activity. The highest concentration of molecules with antiradical capabilities from the fruit is extracted using ethyl acetate9 (Table 31.3). The TABLE 31.3

antioxidant activity of these extracts may be increased by applying advanced extraction techniques, thus, increasing the quantity of the antioxidant agents in the obtained product. Extracts prepared with water in subcritical state are marked by a high degree of antioxidant and antiradical activity, in addition to which they are also considered safe and green products. With an extraction of the root using water at a temperature of 140 C and a pressure of 40 bars, it is possible to get an extract, which, when applied at a concentration of 81 μg/mL, may neutralize 50% of the free DPPH radicals in a mixture. In the case of the leaves and the fruit, the inhibitory concentrations are lower than in the case of the root, and these amount to 56 and 69 μg/mL respectively.11 In the case of traditional techniques and when using the same solvent (water), the inhibitory concentration is significantly higher and it amounts to 202.50 mg/mL, while methanol extracts show an activity that is several times lower (723.62 mg/mL).27 The Soxhlet extraction, which is taken as a reference technique, renders extracts, which, at the concentration of 400 mg/mL, may inhibit 49.16% of the DPPH radicals.28 The advantage of extracting S. ebulus L. using subcritical water lies also in the fact that the increased temperature during the extraction leads to the denaturation of ebulin, which means that any possible toxicity may be avoided. Extracts of S. ebulus L. obtained with preheated water are opulent with gallic acid, which is a phenolic acid with a strongly-proven high antioxidant ability.11 Moreover, this antioxidant is used often as a standard in different antioxidant assays. The presence of gallic acid in a high concentration (in root extract 5 208.84 mg/L; in leaf extract 5 141.35 mg/L; in berry extract 5 868.98 mg/L) contributes to the high antioxidant ability of subcritical water S. ebulus L. extracts.11 Apart from gallic acid, the presence of other phenolic acids as well as anthocyanins with strong antioxidant capacity donates the overall antioxidant activity of such extracts. Studies on the antioxidative potential of S. ebulus L. berries have shown that their aqueous extracts modulate antioxidant gene expression, which could be one of the possible mechanism of its antioxidant activity.5 This is mediated with polyphenols in berries, especially anthocyanins.29,30 The

Antioxidant (DPPH scavenging) activity of Sambucus ebulus extracts depending on plant part and solvent used.9,11 IC50 values (μg/mL)

Extract

Subcritical water

Water

Methanol

Acetone

Ethyl acetate

Leaves

0.056

290.46

47.37

167.24

483.43

Roots

0.081

251.62

88.46

52.05

146.11

. 1000

Fruit

0.069

128.23

82.15

171.97

67.38

. 1000

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Petroleum ether 390.32

328

31. Sambucus ebulus L., antioxidants and potential in disease

influence of extraction solvent on the antioxidant activity of different parts of S. ebulus L. is demonstrated in Table 31.3.9,11 In addition to the neutralization of free radicals, the extracts of S. ebulus L. are also marked by a high reduction capability. In a method based on monitoring the reduction of Fe31- Fe21, the subcritical water extracts of the leaf, fruit, and root have proved to be significant donors of electrons and they have achieved 50% reductions with concentrations of 0.15, 0.24, and 0,31 mg/mL respectively.11 The reduction power of S. ebulus L. percolates obtained by water and ethanol has been tested within the scope of concentrations of 25
800 μg/mL, and has shown to be lower in comparison to vitamin C, the reduction capacity of which is used as the reference value in the applied method.27 Although S. ebulus L. extracts show the ability to act as neutralizers of nitric oxide, their activity is much lower than the activity of standard compound quercetin. However, it was noticed that aqueous extracts are more potent than methanolic ones.31 For hydrogen peroxide it has been shown that the cell membrane does not represent a barrier to the diffusion of this specie within the cell or between cells, and can induce oxidative stress virtually anywhere in the cell.32 This molecule can cause cytotoxicity, thus, the ability of natural products to scavenge this specie is important. The methanolic extract of S. ebulus L. is able to scavenge hydrogen peroxide (IC50 5 59.5 μg/ mL) and its activity is similar to the activity of quercetin (IC50 5 52.0 μg/mL), but lower than that of ascorbic acid (IC50 5 21.1 μg/mL).31 Determination of lipid peroxidation inhibition is used to determine the total antioxidant capacity of natural products. Many natural antioxidants inhibit the lipid peroxidation process in vitro. The process of the peroxidation of unsaturated fatty acids is the main cause of oxidative damage to cell membranes, but also of all other biological systems containing lipids. The estimation of the effectivity of extracts to inhibit the process of lipid peroxidation is necessary in order to examine the overall biological activity of the extracts. S. ebulus L. methanolic extracts show weak activity in the inhibition of the lipid peroxidation process in a period of 24 hours (50%). However, exposure to the extracts for a longer interval (48 hours) induces a higher activity (86%). On the other hand, a longer period than this leads to a decrease in activity (at 72 hours activity reaches 50%).31 As part of determining and defining the antioxidant activity of plant extracts, it is important to measure their ability to act as chelators of metal ions. The most commonly used method is based on the complexing of iron (II) ions. It is known that during the Fenton reaction, iron can participate in the formation of highly reactive

FIGURE 31.5 Antioxidant ability of Sambucus ebulus flowers.31

OH radicals, thereby affecting the cell damage process. Minimizing ferro ion can lead to protection against oxidative damage. Control of the level of the iron can be a potential strategy for the prevention or control of Alzheimer’s disease.33 Further, it can reduce iron-related complications and improved overall survival in some diseases such as Thalassemia major.34,35 The measurement of the ability of S. ebulus L. extracts to act as iron chelators is done using the ferrozine method. Ferrozine allows for the quantitative formation of complexes with Fe(II) ions. In the presence of chelating agents, the complex can be decomplexed, or the intensity of the red color that originates from the ferrozine complex may be decreased. It was noticed that extracts of S. ebulus L. flowers obtained by percolation with methanol express weak activity in iron ion chelating (IC50 5 1.3 6 0.07 mg/mL). Also, this activity of S. ebulus L. is dose dependent31 (Fig. 31.5). The ability of the extracts of S. ebulus L. to inhibit free radicals and protect cells against oxidative damaging makes these extracts protective agents in the case of the teratogenicity of albendazole, which has been confirmed by in vivo testing on rats.36

Anticancer activity Cancer chemotherapy involves the use of compounds (most often chemically synthesized) that may slow down or completely stop the growth of cancerogenic cells or they can lead to the death of those cells. The treatments in the fight against tumor diseases may also include the use of radiation. However, this treatment and the one using chemically synthesized compounds often have a lethal impact on normal or rather healthy cells, which causes a side effect. Such treatments often lead to the weakening of the immune system and, thus, patients become susceptible to different infections. Therefore in the past few years the focus of

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Other biological activities of Sambucus ebulus

the world’s scientific community has been on the search for chemotherapeutics of low toxicity and broad spectrum of actions. A large number of anticancerogenic compounds are in fact primary and secondary plant metabolites, both in their basic and in their structurally modified forms (stilbens, lignans, steroids, sesquiterpenes, and diterpenes, etc.). Among these molecules, particular attention has been paid to the polyphenolic compounds of plants, primarily flavonoids.37 Plant extracts and their phenolic compounds may act on numerous targeted molecules of the signal pathways in malignantly transformed cells, simultaneously attacking, in this way, several features of cancer. Different types of cancer use different mechanisms so that the extracts of different plants have specific anticancerogenic effects in line with the different types of cancer. The importance of polyphenolic compounds is reflected partly in their antioxidant activity by which they protect biomolecules (DNA, RNA, proteins, lipids, etc.) against oxidative damaging.38,39 Malignant cells are characterized by a higher level of endogenic oxidative stress and a modified redox status in comparison to normal cells and, therefore, they show increased sensitivity to phenolic compounds in comparison to normal cells.40 Phenolic antioxidants show their anticancerogenic activity by modulating the components of the signal pathways that regulate the cell cycle.38,41,42 In addition, plant polyphenols, headed by flavonoids, are characterized by their ability to lead to the morpho-functional regeneration of organisms while at the same time increasing their defense system. Because of all of the above, nowadays there is major interest, both in the scientific world and in industry, to use plants as a source of leading bioactive anticancerogenic substances as well as for the development of standardized herbal drugs.22 S. ebulus L. has an abundance of bioactive molecules, the anticancerogenic effects of which have been corroborated. Extracts made of the root, the fruit, and the leaves of this plant have shown their cytotoxic activity toward the cell lines of different tumors. In comparison to the root and the fruit, the leaves of this plant have a higher degree of anticancerogenic properties. The ethyl-acetate extract of the fruit of S. ebulus L. has shown a higher degree of cytotoxicity toward tumor cells (HepG2 and CT26) than toward normal ones (CHO and fibroblasts). The inhibitory concentration, that is, the concentration that inhibits 50% of the cell growth, in the case of human hepatocarcinoma, is 97.03 μg/mL, while in the case of colon cancer it amounts to 152.70 μg/mL.43 The values of these inhibitory concentrations are significantly higher in comparison to the those prescribed by the criterion (IC50 , 30 μg/mL) and, therefore, although ethyl-acetate extracts indicate antitumor activity, the high inhibitory concentration cannot classify them into antitumor agents

329

FIGURE 31.6 Cytotoxic activity of subcritical water extracts of the roots, leaves, and fruit of Sambucus ebulus.11

with high potential in the further studies. On the other hand, by applying pressurized water, it is possible to obtain promising results when it comes to the extraction of molecules with antitumor effects from S. ebulus L. (Fig. 31.6). In vitro studies have shown that when it comes to cervical cancer, extracts prepared from the leaves of this plant show an extremely high level of cytotoxicity (IC50 5 0.58 μg/mL). Also, an exceptional level of activity of the leaves has been recorded in the case of lung adenocarcinoma (IC50 5 0.76 μg/mL) and colorectal adenocarcinoma (IC50 5 1.89 μg/mL). With inhibitory concentrations of less than 2 and 9 μg/mL respectively, a high activity has also been observed in the case of the fruit and the root of this plant.11 This high level of activity has opened a way for this plant to be included into further, comprehensive studies. It is important to study in future researches the selectivity of these highly active extracts toward normal cells, establish which compounds from the composition of the extracts play dominant roles, and to clarify the mechanisms of their actions. For the time being, a high degree of correlation has been established between the contents of polyphenolic compounds and the anticancerogenic activity of the extracts of S. ebulus L. However, in addition to polyphenolic compounds, a whole number of other compounds as well as their synergetic effect may also be included in the observed activity. The presence of immunotoxin created by ribosomeinactivating proteins is of particular importance.18,19

Other biological activities of Sambucus ebulus Antidiabetic activity Diabetes (diabetes mellitus) is a chronic, incurable metabolic disorder, which is characterized by hyperglycemia,

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that is, by permanently elevated levels of glucose in the blood. The number of causes that condition the onset of this disease is exceptionally high, but the disease most often starts with a reduced secretion and/or a reduction of the biological action of insulin. Nowadays diabetes is one of the most frequent health problems with its prevalence continuously rising. According to data from the World Health Organization (WHO), the number of people suffering from diabetes in 2015 was 415 million, but the number of individuals with this endocrine ailment is expected to go up, and it is estimated that by 2040 this figure is going to increase to as much as 642 million. It is believed that this tendency in the number of the ill is a consequence of the modern lifestyle and an increase in the number of external etiological factors, with obesity being one of the most pronounced. Due to the importance of the prevention and treatment of this disease, there is increased interest in finding new approaches to the treatment and control of the progress of this disease. In the past few years, one of the most acceptable theories for the control of diabetes has been the inhibition of the action of the key enzymes, that is, of α-amylase and α-glucosidase, the main enzymes in the catabolism of carbohydrates.44 These enzymes catalyze the hydrolysis of the α-(1.4) link in carbohydrate molecules.45 The inhibition of these enzymes is considered an extremely important tool for the control of the level of glucose in the blood of patients suffering from diabetes. In connection with this, several synthetic inhibitors of said enzymes have been synthesized. However, it has been shown that these synthetic molecules have adverse effects on organisms including gastrointestinal difficulties and problems in digestive organs.46 On account of this, there is increased attention being paid to finding natural and safe inhibitors. Natural inhibitors are often constituents of plants or rather of their extracts. The extracts of S. ebulus L. may be considered as an important source of the α-amylase and α-glucosidase inhibitors. Like most natural extracts, the extracts of S. ebulus L. are characterized by a greater ability to inhibit glucosidase in comparison to amylase. Also, different parts of this plant display different affinities toward their inhibition. Thus, for instance, the leaf extracts show a significantly higher capacity to inhibit glucosidase (2.04 mmol ACE/g) in comparison to the fruit extracts (1.40 mmol ACE/g) or the root extracts (0.37 mmol ACE/g). On the other hand, in the case of amylase, the root exhibits the highest degree of inhibition to this enzyme (0.48 mmol ACE/g), while the fruit (0.45 mmol ACE/g) and leaf extracts (0.42 mmol ACE/g) show lower degrees of activity.11 Numerous studies have shown a correlation between the contents of some polyphenolic compounds and antidiabetic activity.47
49 In this regard, the high activity of leaf extracts toward the inhibition of glucosidase may be

closely linked to their high content of chlorogenic and caffeic acids, compounds that are considered to be antidiabetic agents. The high activity of the root toward the inhibition of amylase may be a consequence of the high content of gallic acid in it.50,51

Activity toward the inhibition of tyrosinase Tyrosinase is the key enzyme in the synthesis of melanin and other pigments, and its increased concentration in organisms may lead to the onset of a melanoma. For this reason, the inhibition of this enzyme may be important for the control of different skin disorders, especially of pigmentation disorders.52,53 So far, several inhibitors of this enzyme have been synthesized, however, they exhibit side effects such as a high level of cytotoxicity or they lead to the onset of dermatitis.54 On account of this, there is a need to find and isolate natural inhibitors of this enzyme. S. ebulus L. from the territory of south-east Serbia has proved to be a significant source of molecules capable of inhibiting tyrosinase with a large potential to be applied in the therapy of skin diseases linked to an overproduction of melanin. Extracts of the leaves of S. ebulus L. prepared by heated water exhibit a high degree of inhibition of the activity of tyrosinase (19.67 mg KAE/g). Their activity is higher in comparison to fruit extracts (10.94 mg KAE/g) and root extracts (9.32 mg KAE/g). Regardless of the differences between the parts of the plant material, all three parts of the plant may be considered a good source of antityrosinase agents.11

Antidepressant activity Over the past few decades, continuous efforts have been devoted to developing a mechanism that can moderate and control the course of depression. In vivo studies on mice have shown an antidepressant action of methanol percolates of S. ebulus L. Applied in dosages between 200 and 1200 mg/kg, using forced swimming test (FST) and tail suspension tests (TST) tests, these extracts have shown a high level of activity that was directly related to the applied dosages. Still, even the lowest applied dosage has showed a significant effect. Although imipramine exhibited a higher activity, promising effects have been achieved. The study did not observe any mortality in a 48 hours period when applying a dosage of 3 g/kg.55 Although this plant is considered a significant source of molecules with the potential to be applied as antidepressants, more comprehensive studies are required and they have been intensively conducted over the past few years.

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References

Application in other areas of pathology Numerous studies have proven the benefits of natural products derived from plants. For many of these substances, the mechanisms of action are known, and they are classified as compounds with added values. Some of these belong to the classes of phenols, flavonoids, carotinoids, coumarins, essential oils, etc. On the other hand, for many plants and their constituents only some pathological effects have been noticed and proven. In the case of S. ebulus L., the potential pathological effects have been explored for the extracts obtained by methanol, n-hexane, and ethyl acetate. The results showed that ethyl-acetate extract has hepatotoxic and nephrotoxic effects in mice. In a group of animals which were administered with this extract, significant changes in the tissues of the liver and kidneys were noticed using light microscopy.56
58 Some of the most obvious changes were fatty changes of the renal tubular epithelium. The monitoring of these changes showed that the epithelium was necrotic, but also it was noticed that the epithelial lining of the tubules was preserved. Apart from this, basic alterations in hepatic tissue samples such as apoptotic cells, necrotic hepatic parenchyma cells, central vein dilation, and Kupffer cells hypertrophy were demonstrated. Hepatic disorders were dose dependent. Further, tubular necrosis and interstitial inflammation were seen in nephropathological assays. Additional investigations proved that the intake of vitamins, particularly vitamins C and E, can protect the tissues from hepatotoxicity and nephrotoxicity, which can be caused by the action of ethyl-acetate extracts of S. ebulus L.56
58

Conclusion Because of their exceptional biological potential, the products of the ancient plant S. ebulus L. have broad applications, even in modern times. Its exceptionally rich chemical composition includes numerous compounds of lipophilic and hydrophilic character, among which there are also those with extremely high pharmacological potential. Some of the positive effects of this plant are its high antioxidant, antiradical, antiproliferative, and antidiabetic capacities. On the other hand, like any other herbal material, S. ebulus L. may also have potentially adverse effects, which makes it necessary to have a strictly controlled use and preparation of the products made from this plant before using them in therapy for some diseases. Its high activity and the necessity to ascertain accurate therapeutic dosages as well as the possible side effects impose the

need to have further, strict preclinical and clinical trials, which also constitutes a new platform for the detection and characterization of the pharmacologically active compounds that could, as safe agents, find their application in the contemporary pharmaceutical industry.

Summary points • S. ebulus L. is a rich source of antioxidants. • S. ebulus L. methanolic extract is a good scavenger of hydrogen peroxide. • S. ebulus L. extracts may cause 86% inhibition of the lipid peroxidation process in vitro for 48 hours. • S. ebulus L. extracts show weak ability to act as neutralizers of nitric oxide and weak activity in the iron ion chelating process. • Gallic acid is one of the main compounds responsible for the good antioxidant activity of subcritical water extracts of S. ebulus L. • Nonphenolic constituents of S. ebulus L. are bioactive constituents of this plant. • Pathological effects of S. ebulus L. extracts. • Coherence between antioxidant capacity of S. ebulus L. and its other biological activities.

Acknowledgment The author is grateful to Verica Risti´c for her support in translation.

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C H A P T E R

32 Selenium usage and oxidative stress in Graves’ disease and Graves’ orbitopathy Michele Marino`1, Giulia Lanzolla1, Giovanna Rotondo Dottore1 and Claudio Marcocci2,3 1

Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Pisa, Italy 2Department of Clinical and Experimental Medicine, Endocrinology Unit II, University of Pisa and University Hospital of Pisa, Pisa, Italy 3Endocrinology Unit, University of Pisa and University Hospital of Pisa, Pisa, Italy

List of abbreviations 8-OHdG ATD CAT EUGOGO GAGs GC GO-QOL GPX GSH GSSG H2O2 HA HSP-72 IFN-γ IL1-R IL1-RA IL-1-α IL-β MDA O2
OH OS ROS SeMCys SOD TBARS TNF-β TRAb TRs TSH-R

8-hydroxy-20 -deoxyguanosine antithyroid drugs catalase European Group on Graves’ Orbitopathy glycosaminoglycan glucocorticoid GO
specific quality-of-life questionnaire glutathione peroxidase glutathione glutathione disulfide hydrogen peroxide hyaluronic acid heat shock protein 72 interferon-γ IL1-receptor IL1-receptor antagonist interleukin-1-α interleukin-1-β malondialdehyde superoxide anions hydroxyl radicals oxidative stress reactive oxygen species selenium-methyl-selenocysteine superoxide dismutase thiobarbituric acid
reacting substances tumor necrosis factor-β thyroid stimulating hormone receptor antibodies thioredoxin reductase thyroid-stimulating hormone receptor

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00032-9

Introduction Graves’ disease is an autoimmune disease affecting primarily the thyroid, although several extrathyroidal manifestations can be observed, namely Graves’ orbitopathy (Fig. 32.1), pretibial myxedema (Fig. 32.2), and Graves’ acropachy.1
4 The prevalence of Graves’ disease is B1%, making it one of the most common autoimmune diseases.1 The main pathogenic mechanism is the stimulation of thyrocyte growth and activity by autoantibodies directed against the thyroid stimulating hormone receptor (TSH-R), which bind to TSH-R and mimic the effects of TSH.5 Graves’ orbitopathy is the most common extrathyroidal manifestation of Graves’ disease, being observed in 25%
30% of patients, whereas it is much rarer in patients with hypothyroid autoimmune thyroiditis and in subjects with subclinical thyroid autoimmunity, but without an overt thyroid dysfunction (euthyroid Graves’ orbitopathy).3 The exact pathophysiology of Graves’ orbitopathy is not completely understood, although it is believed to be an autoimmune disease. According to the most popular hypothesis, both cellular and humoral immunity against TSH-R and other autoantigens expressed in the thyrocytes and orbital

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32. Selenium usage and oxidative stress in Graves’ disease and Graves’ orbitopathy

FIGURE 32.1 Right eye of a patient with a severe Graves’ orbitopathy.

FIGURE 32.2 Elephantiasic pretibial myxedema.

fibroblast would initiate the disease. Once autoreactive T lymphocytes infiltrate the orbital tissues, possibly along with autoantibodies, they trigger the secretion of inflammatory cytokines, chemokines, growth factors, and ROS. The resulting proliferation of fibroblasts and their differentiation into preadipocytes and finally adipocytes promote increased synthesis and secretion of glycosaminoglycan (GAGs), especially hyaluronic acid (HA), leading to the remodeling typical of Graves’ orbitopathy, with extraocular muscle enlargement and fat expansion, which are ultimately responsible for the manifestations of the disease.5,6 Several studies support the role of oxidative stress in the pathogenesis of Graves’ disease and Graves’ orbitopathy.7,8 In this context, the use of antioxidant agents, especially selenium, has been proposed in the treatment of these conditions.

Applications to other areas of pathology Oxidative stress acts as a single component of a multifactorial process, together with other elements such as apoptosis, cell-signaling, receptor-mediated responses, and others. Over the past few years, endocrine disrupting compounds have gained interest in human physiopathology and seem to be able to affect

energy metabolic homeostasis and oxidant/antioxidant balance in thyroid tissue and other endocrine systems.9 For example, cadmium (Cd), a ubiquitous toxic metal, has been shown to increase cell proliferation and reduce apoptosis. The role of Cd in autoimmune diseases and in thyroid cancer has been demonstrated and the mitochondrial damage and ROS formation induced by Cd may explain, at least in part, its role in the cellular balance between apoptosis and survival.10 Currently, the crosstalk between thyrocytes metabolism, redox balance, and oncogenic mutations remains poorly characterized, but studies have linked oxidative stress to thyroid cancer.11 The expression of glutathione peroxidase (GPX) and thioredoxin reductase (TRs) in thyroid cancer cells seems to be significantly higher compared to healthy cells.12 Furthermore, a pivotal role of cellular metabolic balance and redox state regulation in papillary thyroid carcinoma has been suggested.13

Oxidative stress in Graves’ hyperthyroidism Oxidative stress plays a role in hyperthyroidism, according to several lines of evidence.7,8 The maintenance of a cell redox state is essential for preserving cellular homeostasis. ROS, namely hydrogen peroxide (H2O2), hydroxyl radicals (OH ), lipid peroxides, and superoxide anions (O2), contain unpaired electrons that make them highly reactive. Thus ROS act as oxidizing agents, thereby perturbing intracellular reactions and damaging cellular components. Under physiological conditions, antioxidant substances including superoxide dismutase (SOD), GPX, catalase (CAT), and glutathione (GSH), defend cells from ROS activity. It is known that thyrotoxicosis is a hypermetabolic state leading to an increased consumption of oxygen with a dysfunction in the mitochondrial respiratory chain and elevated intracellular ATP consumption. This condition is linked with an increase in ROS in peripheral tissues, which saturates antioxidant systems.8 Moreover, the autoimmune reaction may cause ROS release also within the thyroid.8,14
16 These observations support the dual action of oxidative stress in

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Graves’ disease. In peripheral tissues, ROS cause tissue damage, thereby contributing the clinical features of hyperthyroidism. In the thyroid, ROS worsen autoimmunity due to the damage caused to epithelial cells and the exposure of autoantigens to the immune system. Yamada et al. have demonstrated that plasma malondialdehyde (MDA), a marker of lipid peroxidation, is higher in thyrotoxic rats than in euthyroid animals.16 In the same animals, the sustained oxidative stress induced by thyrotoxicosis promotes an increase in erythrocyte antioxidant parameters such as SOD and GPX. Interestingly, plasma MDA as well as erythrocyte SOD and GPX levels are reduced by concomitant administration of vitamin E, a powerful inhibitor of lipid peroxidation and oxidative stress. Vitamin E was also found to be protective against a thyroxineinduced increase of lipid peroxidation in the cardiac and skeletal muscles in rats.16 The majority of studies evaluating the effects of thyroid diseases on the oxidative status in humans have been performed in patients with Graves’ disease, either under hyperthyroid conditions or after the restoration of euthyroidism with antithyroid drugs (ATD) or radioiodine.17
24 Patients with untreated hyperthyroidism have higher levels of oxidative stress parameters including H2O2, lipid peroxides, thiobarbituric acid
reacting substances (TBARS), and MDA in the serum, plasma, and erythrocytes, compared with euthyroid subjects. The serum concentrations of thyroid hormones correlate with levels of lipid peroxidation products in patients with overt hyperthyroidism. These findings do not seem to be specific for Graves’ disease, but rather for thyrotoxicosis as patients with subclinical hyperthyroidism, due to toxic multinodular goiter, have higher values of the mentioned oxidative stress markers.25 The condition of oxidative stress in thyrotoxic patients has been confirmed by the evaluation of the total oxidative status in both Graves’ disease and toxic multinodular goiter with no differences between Graves’ patients with and without Graves’ orbitopathy. Antithyroid medications reduce the levels of oxidative stress markers due to the restoration of euthyroidism and possibly to their antioxidant properties.18,21,26 Conflicting results have been obtained regarding the activity of the antioxidant defense system in thyrotoxic patients. Most studies reported an increase in antioxidant defense enzymes, which remove ROS and ensure a homeostatic response in order to balance thyrotoxicosis-induced increased ROS generation. Komosinska-Vassev et al.27 assessed erythrocyte SOD, CAT, and GPX in patients with Graves’ disease, demonstrating an increase compared with age-matched controls. However, no differences in serum glutathione reductase and total antioxidant

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status were found. The authors suggested that the discrepant findings between erythrocyte and serum antioxidant activities depended on the rapid exhaustion of the antioxidant system. Bednarek et al.23 found a rise in SOD and CAT in the plasma of patients with Graves’ disease of a short duration (1
2 months) compared with healthy controls, but not of GPX and glutathione reductase, which were instead decreased. Abalovich et al.22 evaluated hyperthyroid Graves’ patients showing that erythrocyte SOD and CAT activities were decreased compared to controls, without differences concerning erythrocyte GPX and plasma total reactive antioxidant potential. More recently, Aslan et al.28 reported a decrease in the serum total antioxidant capacity in hyperthyroid patients with an average duration of hyperthyroidism of 2.3 6 1.5 months compared with controls. The duration of hyperthyroidism at the time of the evaluation can explain, at least in part, the conflicting data on antioxidant activity in patients with Graves’ disease. An increment in antioxidant defense mechanisms, thereby balancing the increased oxidative stress, may prevail in patients with hyperthyroidism of a short duration. On the other hand, in patients with hyperthyroidism of a longer duration, the antioxidant defense systems are exhausted, thereby explaining the decreased cellular and serum antioxidant activities.29,30 The restoration of euthyroidism with ATD repairs the oxidative balance, therefore, improving the activity of the intra- and extracellular antioxidant defense systems.31 Although the scavenging effects of ATD may also contribute to these changes, the effects of these drugs on cellular oxidative status are likely due to the restoration of euthyroidism.19
26 An assessment of the oxidant/antioxidant balance in the thyroid tissue of patients undergoing thyroidectomy for Graves’ disease has shown increased levels of free radicals and their scavengers compared with normal thyroid tissue.32

Oxidative stress in Graves’ orbitopathy In Graves’ orbitopathy, tissue hypoxia and oxygen free radical damage play a role in the pathogenesis.5 Inflammation sites are characterized by the presence of ROS and antioxidant mechanisms; oxidative stability is the balance between the formation and elimination of free radicals. Thus oxidative stress is defined as a disruption of this balance due to an increase in ROS production or a decrease in their elimination. A similar condition can cause remarkable damage to cellular components such as membranes, proteins, lipids, and nucleic acids, ultimately resulting in mitochondrial dysfunction and the loss of enzymatic activity.

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The pathogenetic role of ROS in Graves’ orbitopathy is supported by several basic studies performed using cultured orbital fibroblasts as a model.30
39 In 1992, Heufelder et al. demonstrated that H2O2 induces the increased expression of heat shock protein 72 (HSP72), which was involved in antigen recognition and T lymphocyte activation in Graves’ orbitopathy fibroblasts compared with control fibroblasts.33 In a subsequent study, the same authors reported that the enhanced expression of HSP-72 was reduced by antioxidative agents or ATD, which likely promote ROS scavenging activity.31 Burch et al. showed free oxygen damage in vivo in the surgical tissue of Graves’ orbitopathy patients with a greater superoxide-induced proliferation of orbital fibroblasts from Graves’ orbitopathy patients compared with control fibroblasts.34 In addition, H2O2 induced an increase in interleukin-1β (IL-1β) production and GAG accumulation in orbital fibroblasts from normal subjects and Graves’ orbitopathy patients.35 A few studies showing increased oxidative DNA damage, lipid peroxidation, and the release of intracellular ROS in orbital fibroblasts from Graves’ orbitopathy patients compared with control cells also support the role of oxidative stress in Graves’ orbitopathy36
38 Finally, several studies support the hypothesis that oxidative stress enhances cell proliferation and the release of cytokines. Tsai et al.37 and Hondur et al.36 found higher levels of H2O2 and SOD activity as well as decreased GPX activity and reduced glutathione/oxidized glutathione ratio in orbital fibroblasts from Graves’ orbitopathy patients compared with controls. Moreover, the addition of H2O2 is followed by a more-pronounced imbalance of the oxidant/antioxidant ratio in Graves’ orbitopathy fibroblasts than in normal fibroblast.36
38 In addition to basic studies, there is clinical evidence supporting the role of oxidative stress in Graves’ orbitopathy. Cigarette smoking, one important risk factor for Graves’ orbitopathy, enhances in vitro generation of ROS and decreases antioxidant defense.39 Human studies provide further support for the role of oxidative stress. Patients with Graves’ hyperthyroidism of recent onset, either with or without Graves’ orbitopathy, have higher SOD and CAT plasma levels than healthy controls. However, no differences concerning GPX and TRs were observed.22 The restoration of euthyroidism was followed by a normalization of these markers only in patients without Graves’ orbitopathy, suggesting that orbital inflammation contributes markers of oxidative stress.19
21 Tsai et al. found higher urinary levels of 8-hydroxy-20 -deoxyguanosine (8OHdG), a marker of oxidative DNA damage, in patients with active Graves’ orbitopathy compared with healthy controls.37 Furthermore, glucocorticoid (GC) administration reduced 8-OHdG urinary levels in

association with a reduction of Graves’ orbitopathy activity and severity. In line with the knowledge that smoking plays a major role in Graves’ orbitopathy (Fig. 32.3),40 smokers had higher levels of urinary 8OHdG compared with nonsmoker Graves’ orbitopathy patients.37 The relationship between oxidative stress and GC in Graves’ orbitopathy has also been studied. Akarsu et al.41 reported that serum MDA was lower in Graves’ orbitopathy patients compared with healthy controls and patients with Graves’ hyperthyroidism. Furthermore, a positive correlation between the Graves’ orbitopathy clinical activity score and MDA was observed. The study group included 33 Graves’ disease patients with moderately severe and active Graves’ orbitopathy, 20 Graves’ disease patients without Graves’ orbitopathy, and 15 healthy controls. After the random assignment of Graves’ orbitopathy patients to intravenous or oral GC therapy, patients were reevaluated during and after withdrawal of the therapy. Serum MDA was reduced in both treatment groups without differences between Graves’ disease patients and controls. Abalovich et al.22 and Bednarek et al.23 reported similar data. On the other hand, variations in serum GSH during treatment were not significant. These studies suggest that oxidative stress is involved in the pathogenesis of Graves’ orbitopathy, thereby maintaining the active orbital inflammatory process.

Use of antioxidants in the management of Graves’ orbitopathy The potential role of oxidative stress in the pathogenesis of Graves’ orbitopathy is the rationale for the use of antioxidants in the management of mild Graves’ orbitopathy. In the natural history of mild Graves’ orbitopathy, a spontaneous improvement occurs in B20% of patients, whereas 65% present a static eye disease and a small proportion (B15%) progress to moderate-to-severe disease.6 In these patients, the benefits of the “major” treatments are not sufficient to justify the risks that these treatments carry,6,40 and local measures such as artificial tears, ointments, and sunglasses are generally preferred. Nevertheless, similarly to those with moderate-to-severe Graves’ orbitopathy, patients with mild Graves’ orbitopathy also complain about an impairment in their quality of life.42 Thus, some treatment should also be offered to these patients. An ideal treatment for mild Graves’ orbitopathy should be effective, affordable, well tolerated, and widely available. Based on the role of oxidative stress in the complex pathogenetic milieu of Graves’ orbitopathy, an antioxidant approach has been proposed.

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FIGURE 32.3 Risk factors for Graves’ orbitopathy.

Selenium The Swedish chemist Berzelius described selenium as a trace mineral in 1817. Selenium exerts its functions by its incorporation as selenocysteine into selenoproteins, namely GPX, TRs, and iodothyronine deiodinase, all of which have antioxidant enzymatic functions.43 In humans, the dietary sources of selenium guarantee the intake of selenium.43,44 The bioavailability of the mineral is quite variable depending on the type of food. The main reason is the varying selenium contents in the different soils used for growing crops and fodder. Selenium intake from dietary sources is followed by its absorption in the gastrointestinal tract and transportation to the liver, where it binds to several glycoproteins, resulting in the formation of selenoglycoproteins. Selenoglycoproteins reach the bloodstream and finally peripheral tissues, where their concentrations are proportional to the degree of oxidative stress.43
45 Selenium status can be assessed by measuring serum total selenium or selenoproteins including GPX-3 and selenoprotein P. The geographical area influences the selenium status, which is high in North America and relatively low in most European countries. The recommended daily intake of selenium is 53 μg for women and 60 μg for men.45 The average selenium intake is 40 μg/day in Europe, and 93 μg/ day for women and 134 μg/day for men the United States.45 Overdosages of selenium have been associated with increased risk of type 2 diabetes or malignancies, although these observations await confirmation.43,46
48 Because of this reason, and even though selenium intake may be lower than recommended, it is important to measure serum selenium before administering the supplement to avoid overdosage in subjects with baseline serum concentrations higher than 122 μg/L.48 However, studies reported no adverse events for selenium doses not exceeding 200 μg/day, including one in which relatively high levels of selenium were reached (B190 μg/L after 90 days of treatment).47 It is possible that selenium leads to subclinical alterations detectable only by testing, whereas the real clinical impact of these alterations may not be relevant.

Selenium supplements contain selenomethionine or sodium selenite.43 There are no differences between the two types of supplements because selenium is mainly used for the biosynthesis of selenoproteins. However, once selenoproteins are saturated, selenite can no longer be used for selenoproteins synthesis and it is excreted. On the contrary, selenomethionine can further increase serum selenium through its unregulated incorporation into proteins. Consequently, the effect of selenite is strictly linked to the individual state of selenium, whereas with selenomethionine, the concentration of selenium also increases in subjects with a sufficient selenium concentration.

Use of selenium in Graves’ hyperthyroidism The role of ROS in thyrotoxicosis in general and in Graves’ hyperthyroidism in particular should be relevant, especially under conditions of selenium deficiency. The main hypothesis is that selenium deficiency leads to an insufficient counteracting response to ROS activity, thereby worsening the effect of oxidative stress. To address this issue, some studies evaluated the course of the disease in hyperthyroid patients treated with selenium, alone or with other antioxidant agents, in association with ATD drug therapy.8 Vrca et al.49 reported that supplementation with a relatively low dose of selenium (60 μg/day) given within a mixture of antioxidants and concomitantly to methimazole, promotes a better response to treatment in terms of reductions of LDLcholesterol levels compared with patients given methimazole alone. On the other hand, there was no apparent advantage in terms of the control of thyroid function.49 In another study, Guerra et al.14 observed a better biochemical and clinical control of hyperthyroidism in patients treated with methimazole and an antioxidant mixture containing a small amount of selenium (15 g/ day) compared with methimazole alone. Three studies were conducted using “pure” selenium, rather than a mixture of antioxidants.50
52 In the first one, hyperthyroid patients with selenium deficiency (serum selenium B50 ng/mL) were randomized to receive methimazole and levothyroxine (block-and-replace regimen), either

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associated with placebo or selenium (200 g/day), resulting in better biochemical control of hyperthyroidism.51 In another randomized clinical trial, no beneficial effects of selenium were observed in patients with Graves’ hyperthyroidism treated with methimazole regarding short-term, prompt control of thyroid hyperfunction.47 However, in the latter study patients were seleniumsufficient, which may explain the discrepancy. A third, randomized, clinical trial, quite similar to the second one, generated nearly identical results.53

Use of selenium in Graves’ orbitopathy The effects of selenium in orbital fibroblasts were investigated using primary cultures of orbital fibroblasts

from Graves’ orbitopathy patients and control subjects.51,54 Rotondo Dottore et al.54 provided some cellular bases for the beneficial effects of selenium in patients with Graves’ orbitopathy (Figs. 32.4 and 32.5). In a first study, cells were treated with H2O2 to induce oxidative stress, after preincubation with selenium(methyl)-selenocysteine (SeMCys). The measurement of glutathione disulfide (GSSG), GPX activity, cell proliferation, and HA and proinflammatory cytokines showed that the administration of SeMCys to fibroblasts from Graves’ orbitopathy patients or control subjects reduces oxidative stress, cell proliferation, and HA synthesis. Both fibroblasts from Graves’ orbitopathy patients and from the subjects without Graves’ orbitopathy underwent the same modifications after incubation with SeMCys, suggesting that SeMCys protects cells from

FIGURE 32.4 (A) Combined effects of H2O2 (5 μMol) and selenium (10 μMol) or its control methylcysteine (MCys) (10 μMol) on GSSG in

fibroblasts from patients with Graves’ orbitopathy (GO fibroblasts) or from control subjects.  and  P 5 .02 versus H2O2; ‡ and ‡‡ P 5 NS versus H2O2; P 5 NS between Graves’ orbitopathy and control fibroblasts; (B) combined effects of H2O2 (5 μMol), selenium (10 μMol) or MCys (10 μMol) on cell proliferation in Graves’ orbitopathy and control fibroblasts.  P 5 .02 versus untreated cells;  P 5 .02 versus H2O2; P 5 .003 between Graves’ orbitopathy and control fibroblasts; (C) combined effects of H2O2 (5 μMol) and selenium (10 μMol) or its control MCys (10 μMol) on HA release in Graves’ orbitopathy and control fibroblasts.  P 5 .02 versus H2O2; P 5 .02 between Graves’ orbitopathy and control fibroblasts. Source: Reprinted with permission of Dottore, M. Leo, G. Casini, F. Latrofa, L. Cestari, S. Sellari-Franceschini, et al., Anti-oxidant actions of selenium in orbital fibroblasts: a basis for the effects of selenium in Graves’ orbitopathy, Thyroid 2017, Volume 27, pp. 271
278, published by Mary Ann Liebert, Inc., New Rochelle, NY.

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(A) Combined effects of H2O2 (5 μMol) and selenium (10 μMol) or its control MCys (10 μMol) on TNF α in fibroblasts from patients with Graves’ orbitopathy (GO fibroblasts) or from control subjects  and  P 5 .02 versus untreated cells; ‡ and ‡‡ P 5 .02 versus H2O2; (B) combined effects of H2O2 (5 μMol) and selenium (10 μMol) or MCys (10 μMol) on IL-1β in fibroblasts from Graves’ Orbitopathy patients or control subjects.  P 5 .03 and  P 5 .03 versus untreated cells; (C) combined effects of H2O2 (5 μMol) and selenium (10 μMol) or MCys (10 μMol) on IFNγ in fibroblasts from Graves’ orbitopathy patients or control subjects  P 5 .02 and  P 5 .02 versus H2O2; ‡P 5 .023 and ‡‡P 5 .05 versus H2O2; P 5 NS between Graves’ orbitopathy and control fibroblasts for all three panels. Source: Reprinted with permission of Thyroid 2017, Volume 27, pp. 271
278, published by Mary Ann Liebert, Inc., New Rochelle, NY.

FIGURE 32.5

oxidative stress regardless of the underlying condition and/or genetic background.54 In a second study, H2O2 was shown to exert a dual effect on cell proliferation.51 At low concentrations, proliferation increased, whereas high concentrations reduced cell vitality leading to a progressive decrease of cell proliferation. SeMCys counteracted the increased proliferation induced by highdose H2O2 in orbital fibroblasts.51 Although HA release was not affected by H2O2, SeMCys reduced HA synthesis in Graves’ orbitopathy, but not in control fibroblasts, compared with cells treated with H2O2, thereby bringing HA to levels that were even lower than those observed in untreated cells. These findings possibly suggest that selenium acts on HA release, at least in part, regardless of the oxidative stress induced by H2O2, through mechanisms that remain to be investigated. Oxidative stress is followed by the release of proinflammatory cytokines including tumor necrosis factor

α (TNFα), IL-1β, and interferon-γ (IFNγ), not only by immunocompetent cells, but also fibroblasts. As shown in Fig. 32.5, the H2O2-induced release of these cytokines, except for IL1β, was virtually abolished by SeMCys in both Graves’ orbitopathy and control fibroblasts, suggesting that selenium acts on the oxidative stress
induced release of proinflammatory cytokines, which may be responsible for some of the beneficial effects of selenium observed in Graves’ orbitopathy fibroblasts.54 As mentioned previously, H2O2 is cytotoxic at concentrations .5 μM because of high oxidative stress.51 Interestingly, SeMCys seems to be capable of rescuing in part from the cytotoxic actions of H2O2 when used at a 50 μM concentration in orbital fibroblasts, with a reduction of ROS production, and consequently of cell necrosis and apoptosis.51 Although the effects of selenium in orbital fibroblasts were

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largely observed in both Graves’ orbitopathy and control fibroblasts,51,54 this is not a limitation for the clinical use of selenium. Thus normal fibroblasts, unlike Graves’ orbitopathy fibroblasts, are not subjected to oxidative stress. Therefore selenium has only minimal actions under physiological conditions as shown by toxicity experiments on cell vitality, which demonstrated that selenium is poorly cytotoxic even at very high concentrations.51,54 The first promising results on the use of antioxidants in patients with Graves’ orbitopathy were reported by a pilot study conducted in 2000.52 Eleven patients with newly diagnosed Graves’ orbitopathy were treated for 3 months with an antioxidant mixture

containing allopurinol (300 mg/d) and nicotinamide (300 mg/d) or a placebo. The eye disease improved in 9 of the 11 (82%) patients treated with the antioxidant mixture and only in 3 of the 11 (27%) patients treated with the placebo. Overall, soft tissue involvement was the feature that improved mostly in the active treatment group, and no side effects of the antioxidants were reported. In 2011, the European Group on Graves’ Orbitopathy (EUGOGO) performed a randomized, double-blind, placebo-controlled, multicenter, clinical trial to investigate the effect of selenium and pentoxifylline in mild Graves’ orbitopathy.55 The study included 159 patients with mild signs or symptoms of

FIGURE 32.6 (A) Graves’ orbitopathy-specific quality-of-life questionnaire (GO-QOL) at 6 and 12 months in patients with mild Graves’ orbitopathy treated with selenium, placebo or pentoxifylline. (B) Graves’ orbitopathy outcome at 6 and 12 months based on a composite ophthalmological score. Source: Reprinted by Marcocci C, Kahaly GJ, Krassas GE, Bartalena L, Prummel M, Stahl M, et al. Selenium and the course of mild Graves’ orbitopathy. N Engl J Med 2011;364:1920
31.

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References

Graves’ orbitopathy of less than 18 months of duration. The study lasted 1 year, with a period of 6 months for intervention and a period of 6 months for follow-up. The patients were assigned to one of the three treatment arms, namely selenium (sodium selenite 100 mcg twice/day), pentoxifylline (600 mg twice/day), or placebo (twice/day). As shown in Fig. 32.6, the ophthalmological outcome at the end of treatment was significantly better in the group treated with selenium. In particular, Graves’ orbitopathy improved in 61% of the patients in the selenium group compared with the 36% of the placebo group, while it worsened in 7% of the former and in 26% of the latter group (Fig. 32.6). A specific quality-of-life questionnaire was administered to patients and it was also improved to a greater extent in patients treated with selenium.

Summary points • This chapter focuses on the use of selenium in Graves’ disease and Graves’ orbitopathy. • Selenium is a trace mineral with antioxidant actions. • Hyperthyroidism is associated with an increased production of ROS. • Tissue hypoxia and oxygen free radical damage play a role in the orbital tissue remodeling of Graves’ orbitopathy. • Selenium has been proposed for treatment of Graves’ disease and Graves’ orbitopathy.

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24. 42. Terwee CB, Gerding MN, Dekker FW, Prummel MF, Wiersinga WM. Development of a disease specific quality of life questionnaire for patients with Graves’; ophthalmopathy: the GO-QOL. Br J Ophthalmol 1998;82:773
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43. Rayman MP. The importance of selenium to human health. Lancet 2000;356:233
41. 44 Steinbrenner H, Speckmann B, Klotz LO. Selenoproteins: antioxidant selenoenzymes andbeyond. Arch Biochem Biophys 2016;595:113
19. 45. Kipp AP, Strohm D, Brigelius-Flohe´ R, Schomburg L, Bechthold A, Leschik-Bonnet E, et al. German nutrition society (DGE) revised reference values for selenium intake. J Trace Elem Med Biol 2015;32:195
9. 46. Stranges S, Marshall JR, Natarjan R, Donahue RP, Trevisan M, Combs GF, et al. Effects of long term supplementation on the incidence of type 2 diabetes: a randomized trial. Ann Int Med 2007;147:217
22. 47. Leo M, Bartalena L, Rotondo Dottore G, Piantanida E, Premoli P, Ionni I, et al. Effects of selenium on short-term control of hyperthyroidism due to Graves’ disease treated with methimazole: results of a randomized clinical trial. J Endocrinol Invest 2017;40:281
7. 48. Hegedu¨s L, Bonnema SJ, Winther KH. Selenium in the treatment of thyroid diseases: an element in search of the relevant indications? Eur Thyroid J 2016;5:149
51. 49. Vrca VB, Mayer L, Skreb F, Raheli´c D, Maruˇsi´c S. Antioxidant supplementation and serum lipids in patients with Graves’ disease: effect on LDL-cholesterol. Acta Pharm 2012;62:115
22. 50. Calissendorff J, Mikulski E, Larsen EH, Mo¨ller M. A prospective investigation of Graves’ disease and selenium: thyroid hormones, auto-antibodies and self-rated symptoms. Eur Thyroid J 2015;2:93
8. 51. Rotondo Dottore G, Chiarini R, De Gregorio M, Leo M, Casini G, Cestari L, et al. Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in Graves’ orbitopathy. Endocr Nov 2017;58(2):386
9. 52. Bouzas EA, Karadimas P, Mastorakos G, Koutras DA. Antioxidant agents in the treatment of Graves’ ophthalmopathy. Am J Ophthalmol 2000;129:618
22. 53. Kahaly GJ, Riedl M, Ko¨nig J, Diana T, Schomburg L. Double-blind, placebo-controlled, randomized trial of selenium in graves hyperthyroidism. J Clin Endocrinol Metab 2017;102:4333
41. 54. Rotondo Dottore G, Leo M, Casini G, Latrofa F, Cestari L, Sellari-Franceschini S, et al. Anti-oxidant actions of selenium in orbital fibroblasts: a basis for the effects of selenium in Graves’ orbitopathy. Thyroid 2016;27:271
8. 55. Marcocci C, Kahaly GJ, Krassas GE, Bartalena L, Prummel M, Stahl M, et al. Selenium and the course of mild Graves’ orbitopathy. N Engl J Med 2011;364:1920
31.

Further reading Tsai CC, Kao SC, Cheng CY, Kau HC, Hsu WM, Lee CF, et al. Oxidative stress change by systemic corticosteroid treatment among patients having active graves ophthalmopathy. Arch Ophthalmol 2007;125:1652
6.

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C H A P T E R

33 Antioxidant potential of Stevia rebaudiana (Bertoni) Kashif Ameer1,2, Gui-Hun Jiang3, Rai Muhammad Amir1 and Jong-Bang Eun2 1

Institute of Food and Nutritional Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan 2Department of Food Science and Technology and BK 21 Plus Program, College of Agriculture & Life Sciences, Chonnam National University, Gwangju, South Korea 3School of Public Health, Jilin Medical University, Jilin, P.R. China

List of abbreviations AA ABTS ADI BHA BHT CAE CE CgE CTC DPPH DW EAE EC50 ESI-MS EFSA FC FDA FRAP GA GAE GRAS HPLC-DAD HVED IC50 kcal MCF-7 NBT NMR NO OH ORAC PEF QE QTOF

ascorbic acid 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) acceptable daily intake butylated hydroxyanisole butylated hydroxytoluene conventional alcoholic extract catechin equivalent cyanidin-3-glucoside equivalent condescended tannin content 2,2-diphenyl-1-picrylhydrazyl dry weight ethyl acetate extract half maximal effective concentration electrospray ionization/mass spectrometry European food safety authority Folin
Ciocalteu Food and Drug Administration ferric reducing ability of plasma gallic acid gallic acid equivalent generally recognized as safe high-performance liquid chromatography/diodearray detection high voltage electrical discharges half maximal inhibitory concentration kilocalorie Michigan cancer foundation-7 nitroblue tetrazolium nuclear magnetic resonance nitric oxide hydroxyl oxygen radical absorbance capacity pulsed electric field quercetin equivalent quadrupole-time of flight

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00033-0

Reb-A Reb-B RNS ROS RSA SG US TAC TAE TBARS TE TEAC TFC TPC W/V

rebaudioside-A rebaudioside-B reactive nitrogen species reactive oxygen species radical-scavenging activity steviol glycoside ultrasonication total anthocyanin content tannic acid equivalents thiobarbituric acid reactive substances Trolox equivalent Trolox equivalent antioxidant capacity total flavonoid content total phenolic content weight by volume

Introduction The induction of oxidative stress by free radicals may yield several health implications with pathological and degenerative processes including Alzheimer’s disease, cancer, coronary heart disease, and aging. As oxygen consumers, all aerobic organisms possess sophisticated antioxidant mechanisms to ensure protection against damage caused by oxidative stress to biological molecules such as lipids, nucleic acids (DNA or RNA), and proteins. Increased oxidative stress is a strong etiological contributor to the development of atherosclerosis, tumorigenesis, diabetes, ocular disease, and neurodegenerative diseases (Alzheimer’s and Parkinson’s diseases).1,2 Plant matrices and their byproducts such as leaves, shoots, fruit, and peels serve as important sources

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33. Antioxidant potential of Stevia rebaudiana (Bertoni)

of natural bioactive and antioxidant compounds for the replacement of synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).3 They are also well established as members of folklore and traditional medicines in the various pharmacopeias of different countries.2 Stevia is among those plants with pharmaceutical and therapeutic significance that have been reported in the traditional pharmacopeias of Guaranı´ tribes in Paraguay, Brazil, Japan, India, China, and Korea. Stevia is a member of the Asteraceae family (tribe Eupatoricae) and is a perennial shrub native to Paraguay and Brazil. Stevia has a long history of ethnobotanical use as a food and medicine in several countries including Japan, Paraguay, Brazil, and South America.4 In Europe and the United States, stevia has been approved as a natural sweetener and a food additive, with an acceptable daily intake (ADI) of 4 mg/kg body weight declared by the European Food Safety Authority (EFSA) and the US Food and Drug Administration (FDA); furthermore, stevia was granted generally recognized as safe (GRAS) status in 2008 by the FDA.5,6 The leaves of the stevia plant have been employed globally as a natural sweetener, a sugar substitute, and a taste modifier; in addition, it has been acknowledged as “the third glycogen of the world.” Furthermore, extensive usage of stevia leaves has not been shown to result in any adverse effects on the human body.5 Several medicinal and nutritional issues that have arisen from the increased consumption of sucrose- and fructose-sweetened beverages have led to the prevalence of type 2 diabetes mellitus, obesity, and metabolic syndrome in elderly people and children worldwide. The profile of stevia leaves contains the stevia glycosides (SGs) of stevioside (4%
20% DW), rebaudiosideA (Reb-A; 3% DW), dulcoside-A (0.5% DW), and trace amounts of steviolbioside and rebaudioside-B (Reb-B). Of all the diterpene glycosides, stevioside and Reb-A are the most widely studied.6 Most drug entities, natural or synthetic, that exhibit anticancer effects are found to inhibit the synthesis of new genetic materials and cause irreversible damage to DNA or its precursors after interaction.7 Diterpene glycosides including stevioside, Reb-A, isosteviol, and steviol, have been reported to confer therapeutic benefits such as antihypertensive, anti-inflammatory, anticancer, antihyperglycemic, anti-amnesia, antibacterial, immunomodulatory, and diuretic effects.8 The anticancer properties of SGs have been correlated with the mediation of apoptosis by stevioside, which provides a vital contribution to tissue homeostasis, differentiation, and growth regulation; SGs have also been regarded as a significant target for the development of novel anticancer therapies. Apoptotic cell death is also regulated by an important regulator known as reactive oxygen species (ROS) through the

induction of cytochrome C release and the activation of caspases.9 Other than the interest in the sweetening components of stevia, several reports have stressed the pharmaceutical and therapeutic significance of the bioactive constituents from stevia, that is, the polyphenols, which can be employed on a commercial scale for the development of nutraceutical, cosmeceutical, and pharmaceutical products. Crude extracts of stevia have been reported to have a strong antioxidant potential, which can be further utilized in the form of natural extract preparations and derivatives suitable for health-conscious consumers in order to produce a diverse range of healthful objectives with special focus on the cosmetic and food industries. Hence the antioxidant potential and the pertinent bioactive properties of natural stevia are highly desirable for consumers and producers worldwide.6,10

Applications in other areas of pathology In the past few years, stevia has emerged as nutritionally and medicinally important to consumers of various countries such as Brazil and Paraguay. Stevia extracts have been used as a dietary supplement to prevent the onset of various diseases and to alleviate aging-related issues including hypertension, obesity, diabetes, infections, and dental caries. Moreover, the nutritional implications of stevia extract as an oxidative stress reducer in disease prevention has been well described in published literature.11 A brief account of the biological properties of stevia and its health benefits against various oxidative stress
mediated diseases is given here.

Effectiveness against cancer The effectiveness of stevia against tumorigenesis, especially in the case of skin tumors, has been reported by consumers of stevia leaf powder and its derivatives. Moreover, published reports have stressed the anticancer potential of stevia extracts as a chemopreventive agent and necessitated further clinical trials and animal model studies to ascertain its efficacy on other complex types of carcinomas such as liver, lung, adeno, squamous, and renal cell carcinomas. Specifically, stevioside (10 μM) was reported to induce apoptotic cell death in the MCF-7 breast cancer cell line through caspase activation, the release of cytochrome C, and the loss of mitochondrial transmembrane potential.9

Effectiveness against cariogenesis The excessive usage of refined sugars leads to tooth decay and the development of caries (cavities); hence,

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Antioxidant potential against free radicals and stevia potential

sugar-substitutes such as stevia may act as preventive agents with pertinent significance against cariogenesis. Experiments on orally consumed stevia have established its safety owing to its antimicrobial activity and absence of any toxicity.11 One murine study reported a reduction in caries and the inhibition of the microbial activity in stevia-fed rats compared with sucrose-fed mice, which experienced a profound increase in cariogenic activity.12

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of only 2.67 kcal/g, which is negligible; hence, pure stevia extract preparations do not contribute to the caloric intake of consumers. Obese and overweight people may consume stevia as a natural sweetener and a sugar substitute to reduce their caloric intake. Consequently, the incidence of lifestyle-related disorders will be minimized owing to the maintenance of normal blood glucose levels and the subsequent prevention of the onset of type 2 diabetes; these actions can be attributed to the regulation of gluconeogenesis and the antihyperglycemic activity of stevioside.15,16

Effectiveness against atherosclerosis Plaque formation is the main culprit in the development of atherosclerosis and coronary heart diseases. A study on insulin-resistant mice concluded that stevioside had an ameliorative role, rendering improvements in insulin signaling and bolstering the antioxidant defenses. The consumption of stevioside did not lead to any changes in the glycemic response in this murine study.13

Effectiveness against hypertension Coronary heart disease is a major global health issue and high blood pressure is the leading causal agent for the increased incidence of heart disease and renal failure. Hypertension is also termed as a “silent killer” as it can remain for years without any pertinent symptoms and the absence of these symptoms usually leads to increased morbidity from this ailment. The consumption of stevioside has been linked to reductions in hypertension owing to its antihypertensive properties as reported by the findings of murine and animal (anesthetized dogs) studies. Along with antihypertensive drugs, stevioside has been reported as an effective remedy for hypertensive subjects, and several longterm clinical studies have confirmed that systolic and diastolic pressures were reduced significantly owing to the prolonged consumption of stevia extracts rich in stevioside.14

Effectiveness against obesity, diabetes, and in blood glucose homeostasis Obesity is one of the primary causal agents for the development of increased oxidative stress in the human body. The onset of metabolic syndrome has been linked with increases in obesity. An increase in body mass index, an indication of obesity, is followed by an increase in oxidative stress. Stevia is considered to be a noncaloric or zero-calorie natural sweetener and, on a dry weight basis, has a caloric contribution

Antioxidant potential against free radicals and stevia potential Different forms of free radicals moieties have been reported, and these include hydroxyl radical (OH•), sulfur-centered thiyl radicals (RS•), superoxide (O2 2 ), nitric oxide (NO•), and carbon- and oxygen-centered radicals. There is clear evidence that free radicals, specifically O2 in addition to other ROS and reactive 2 nitrogen species (RNS), are generated continuously in vivo, and organisms, including humans, have evolved antioxidant defense mechanisms to ensure protection against ROS and RNS.7 The reactivity of O2 2 and hydrogen peroxide (H2O2) is much lower than that of OH•, but excessive amounts of O2 2 and H2O2 may lead to oxidative injury at the cellular level. Extracts obtained from stevia leaves have been reported to exhibit strong antioxidant activities. This high degree of antioxidant activity was also evident from its inhibitory activity, as stevia leaf extract inhibited the formation of hydroperoxide in sardine oil and was reported to be stronger than green tea extract or DL-α-tocopherol. As a consequence of oxidative stress, lipid peroxidation has been reported to be a significant phenomenon leading to arterial injuries and can act as a major contributory factor to the formation of arteriosclerotic lesions.7,16,17 Hence it has been indicated that the antioxidant activity of stevia leaf extract may decrease the incidence of injury caused by lipid peroxidation and could lead to a significant reduction in the generation of ROS and RNS moieties in the cells owing to its radical-scavenging ability. A detailed description of the damage caused by oxidative stress is presented in Fig. 33.1. The antioxidant effect of stevia leaf extract has been ascribed to its radical-scavenging ability, especially its action against superoxides and free radical (unpaired) electrons.6 According to a published report, the overconsumption of refined sugars in general, and more specifically, of sucrose, has been found to exhibit a contributory role to the etiology of various metabolic disorders such as dental caries, cancer, inflammatory bowel syndrome, candidiasis, and type 2 diabetes. In contrast, stevia extracts were reported by Chatsudthipong and

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33. Antioxidant potential of Stevia rebaudiana (Bertoni)

FIGURE 33.1 Potential of Stevia rebaudiana (Bertoni) against damages caused by oxidative stress. Different levels of oxidative stress with effects and chemical phenomena. Inflammation and carcinomas caused by oxidative stress and antioxidant compounds and phenolic acids in stevia composition.

FIGURE 33.2 Antioxidant profile of Stevia rebaudiana (Bertoni) and radical-scavenging effects against reactive oxygen species to maintain overall health wellness (A). Antioxidant profile of stevia comprising flavonoids, condensed tannins, anthocyanins, phenolic acids, and chlorophyll against reactive oxygen species and for maintaining homeostasis (B).

Muanprasat18 to be effective for the promotion of human health and wellness through their pharmacokinetic behavior and therapeutic benefits. Moreover, the antioxidant potential of stevia has been compared with those of other medicinal plants such as Kwao Krua (Pueraria mirifica), turmeric (Curcuma longa Linn.), Fa Thalai, Andrographis paniculata (Burm.f.) Nees, and candlestick plant (Cassia alata Linn.). In addition, the maximum

antioxidant activity of the acetone and methanolic extracts of the stevia plant have been reported followed by that of the ethanolic extracts. The antioxidant profile of stevia comprises of flavonoids, condensed tannins, anthocyanins, phenolic acids, and chlorophyll (Fig. 33.2) and these play their role in the maintenance of homeostasis with respect to the overall well-being of humans (Table 33.1).

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Antioxidant potential against free radicals and stevia potential

TABLE 33.1

Recovery of antioxidant and bioactive compounds from Stevia rebaudiana (Bertoni) leaves.

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

Radical-scavenging activities Spectrophotometric measurement at 517 nm. DPPH radical inhibition against gallic acid, Trolox, and BHA

% DPPH-inhibition: Water extract (leaves 5 39.86%, callus 5 55.42%) Methanolic extract (leaves 5 33.17%, callus 5 56.82%)

Water extract showed higher percent of inhibition as compared to methanolic extract.

[19]

Water and methanolic FRAP-RSA extracts of stevia leaves and callus

Spectrophotometric determination at 593 nm. Ferrictripyridyl triazine Reduction to ferrous state in presence of antioxidant compounds

Stevia leaf (FRAP-RSA): (1) water extract (38.24 mg TE/g DW), (2) methanolic extract (37.40 mg TE/g DW) Stevia callus (FRAPRSA): (1) water extract (37.36 mg TE/g), (2) methanolic extract (34.37 mg TE/g)

Water extracts of both leaf and callus showed similar activity, while methanolic extract of callus led to decreases in FRAP-RSA as compared to leaf methanolic extract.

[19]

3.

Freeze-dried concentrated extracts

Trolox equivalent antioxidant capacity (TEAC)

TEAC and ability of antioxidant molecules regarding quenching of ABTS molecule was assessed by UV-Vis spectrophotometer at 734 nm

Stevia extracts demonstrated the highest antioxidant capacity (2.41 μmol TE/mg DW)

Highest antioxidant activity was obtained from ethanolic stevia extracts followed by acetone, methanol, distilled water, and acetic acid.

[20]

4.

Methanolic and ethanolic extracts of stevia leaf powder

DPPH

DPPH-RSA evaluation by stable DPPH radical conversion to 1; 1diphenyl-2-picryl hydrazine

Percent inhibition of methanolic stevia extracts was 77.68%, which was about 10 times higher as compared to ethanolic extract (67.07%).

High discoloration was an indicative of the increased RSA.

[21]

5.

Fractionation of stevia extracts with organic solvents

DPPH, ABTS, and hydroxyl-RSAs

All RSA were determined by spectrophotometer against quercetin standard.

Methanolic extracts showed higher DPPHRSA (47.66 μg/mL) than ethyl-acetate stevia extract (9.26 μg/mL). ABTS (28.6 μg/mL) and OH-RSAs (33.9 μg/mL) were also quite high as compared to ethyl acetate extract.

Ethyl acetate fraction showed the highest RSA.

[22]

6.

Ethyl acetate and methanolic crude extracts of stevia leaves obtained by fractionation

Thiobarbituric acid reactive substances (TBARS) assay for assessment of antilipoperoxidant activity of crude stevia extract

Assessment of antilipoperoxidant activity of crude stevia extract by TBARS assay and finally absorbance was measured by spectrophotometer at 532 nm against BHT as standard

Even at 1 mg/mL concentration (IC50 values: 2.1 mg/mL), the methanolic (CAE) extract demonstrated TBARS inhibition up to 32% as compared to BHT (87.7% TBARS inhibition at 0.05 mg/mL)

Concentrationdependent increase in activity was observed in the case of methanolic extract.

[22]

1.

Leaves and callus (water & methanolic extract)

2.

DPPH-RSA

(Continued)

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33. Antioxidant potential of Stevia rebaudiana (Bertoni)

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

Spectrophotometric determination at 517 nm and ascorbic acid (AA) was used as the standard compound

Stevia leaf powder exhibited higher RSA effect in concentrationdependent manner in the range of 20
200 μg/mL. At 200 μg/mL, the IC50 values for both stevia extract and AA were 68.76% and 82.58% inhibition respectively.

The lower IC50 value [23] was corresponded to increased DPPH-radical inhibition activity.

Soxhlet-extracted S. rebaudiana extracts from air-dried leaves

Hydroxyl (OH)-RSA Spectrophotometric determination at 532 nm was observed for the developed pink chromogen against AAOH-RSA was measured in terms of percentage inhibition of deoxyribose degradation

The percent inhibition by stevia extract on OHRSA was in the range of 38.53%
76.61%. The IC50 values for both stevia extract and AA were 81.08 and 71.41 μg/ mL.

The OH-radical quenching ability of stevia extract was associated with the prevention of chain reactions and lipid peroxidation.

[23]

9.

Soxhlet-extracted S. rebaudiana extracts from air-dried leaves

Nitric oxide (NO)RSA

A chromophore was formed due to Greiss reagent that produced chromophore and absorbance was further recoded spectrophotometrically at 546 nm against standard solution of AA.

Strong inhibitory effect of stevia on NO was observed. The percent inhibition at 200 μg/mL was 58.29% and 71.52% for stevia extract and AA (reference compound) respectively. While IC50 values were 132.05 (ethanolic stevia extract) and 66.01 (AA).

NO radicals were significantly scavenged by ethanolic extract of stevia leaves.

[23]

10.

Ethanolic extract of S. rebaudiana leaf

Superoxide (O22)RSA

Superoxide (O22)-RSA of stevia extracts were measured by NBT (nitroblue tetrazolium reagent) method spectrophotometrically at 560 nm against AA (reference compound). The decrease in absorbance was indicative of the enhanced superoxide (O22)-RSA.

The superoxide (O22)RSA of ethanolic stevia extract was recorded in the range of 26.19%
70.84%. IC50 values were 109.01 μg/mL for stevia extract and 36.69 μg/mL for AA.

Superoxide radicals [23] were reported as the most reactive form of ROS. Overall, the authors reported superoxide (O22)-RSA to be statistically significant and low. The lowered extent of superoxide (O22)-RSA was attributed to the presence of other reactive and bioactive substances and nutrients in the stevia extracts.

11.

Hot water extract from the finely ground powder of S. rebaudiana stem waste and leaves

Fish oil oxidation inhibition and measurement of DPPH and oxygen radical absorbance capacity (ORAC)RSA

Microplate reader determination of DPPHRSA at 520 nm against Trolox standard. ORAC assay was performed by microplate fluorometer at excitation and emission wavelengths

About 1000 ppm of stevia stem extract was incorporated in fish oil and peroxide values were used for evaluation. Stevia stem extract was more effective in reducing hydroperoxides even after 2 days. Waste

Extract from the stevia stem showed higher DPPH and ORAC-RSA and efficacy for inhibition of fish oil oxidation

7.

Soxhlet extraction from DPPH-RSA of S. rebaudiana airdried leaves using ethanol as solvent

8.

[5]

(Continued)

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Antioxidant potential against free radicals and stevia potential

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

of 485 nm and 538 nm respectively.

of stevia stem showed significant decreases in TBARS values. Stevia leaf extract showed higher DPPH-RSA as compared to stem extract. ORAC-RSA values were 1.15 μmol TE/mg DW (stem) and 1.88 μmol TE/mg DW (leaves).

Remarks

Reference

[24]

12.

Oven-dried and frozen DPPH and FRAP fresh hydroalcoholic measurement extracts of S. rebaudiana

A 96-well microplate reader was used for determination of DPPHRSA at 515 nm and results were expressed as percentage. FRAP-RSA was assessed by reduction of Ferric to Ferrous by recording absorbance at 690 nm in microplate reader and results were expressed as EC50 values against Trolox standard.

Oven-dried stevia samples resulted in higher DPPH and FRAPRSA as was evident from the lower EC50 values (22.87 and 28.79 μg/mL) as compared to frozen fresh stevia samples (50.66 and 39.73 μg/mL) respectively.

Oven-drying was reported to be a more effective treatment for enhanced antioxidant potential of stevia leaves in comparison with freezing of fresh stevia samples.

13.

S. rebaudiana extract obtained by aqueous maceration from airdried stevia leaves

Spectrophotometric measurements were carried out for all RSAs against AA at wavelengths of: (DPPH) 517 nm, (OH) 532 nm, (NO) 546 nm, and (O22) 560 nm.

At a maximum concentration of 200 μg/ mL, the aqueous stevia extract and AA showed percent DPPH inhibitions of 72.37% and 82.58% respectively. IC50 values (μg/mL) for RSA were:

Contention about stevia [25] as natural source of antioxidants with significant antioxidant potential was confirmed.

Determination of RSA (DPPH, OH, NO, and O22 activities)

DPPH-RSA 5 83.45; AA 5 26.75 OH-RSA 5 100.86; AA 5 71.41 NO-RSA 5 98.73; AA 5 66.01 O2—RSA 5 100.86; AA 5 36.69 14.

Extracts of stevia leaves and callus were obtained by reflux extraction

Assessment of DPPH, OH, and O22-RSA

RSA measurement by spectrophotometer

At a concentration range of 10
100 μg/mL stevia leaves showed DPPHRSA in the range of 3.38%
10.15%. The OH and O22 RSA of stevia leaf extract were found in the ranges of 10.40%
42.85% and 5.62%
38.26% respectively. O22 RSA for callus was 3.35%
25.73%.

Leaf extract showed increasingly higher antioxidant activity as compared to callus and could be used as a natural source of antioxidants in nutraceuticals and health foods.

[26]

(Continued)

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33. Antioxidant potential of Stevia rebaudiana (Bertoni)

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

DPPH and ABTSRSA

DPPH-RSA was determined in terms of inhibition percentage and optical density was recorded at 517 nm against AA. Similarly, ABTS-RSA was determined at a wavelength of 734 nm.

DPPH IC50 5 essential oil extract (2.9 μg/mL), water extract (5 μg/mL) and methanol
water extract (19.26 μg/mL) ABTS-RSA (mg AA equivalent/g DM) 5 essential oil extract (0.22), water extract (0.67) and methanol
water extract (1.17).

The antioxidant activity [27] exhibited by the essential oil was attributed to the presence of phenolic components in the oil such as carvacrol and thymol. Moreover, stevia leaves have been reported to be helpful in the prevention of inflammatory issues mediated by increased NO generation.

Hydrodistillation of Total anthocyanin dried stevia leaves was content (TAC) carried out to get three fractions: essential oils, water extract and methanol
water extract

Spectrophotometric determination of 510 and 700 nm wavelengths and differences of absorbance corresponded to TAC in terms of mg cyanidin-3glucoside equivalents (CgE)

The essential oil fraction did not show any detectable TAC.

Methanol in aqueous phase was a favorable solvent for enhanced recovery of TAC from stevia.

[27]

17.

Blended fruit juice TAC (mango and papaya) with infusions of stevia leaf powder up to 2.5% (W/V)

TAC determination by spectrophotometric method at 520 nm against CgE

Fruit juice blended with stevia infusions showed TAC of 11.8 mg/L. Different intensification techniques did not affect TAC.

Pulsed electric field resulted in higher retention of bioactive compounds including anthocyanins.

[28]

18.

Stevia-added choke berry juice

HPLC determination of anthocyanins at operating conditions of: column temperature of 20 C, detection wavelength of 520 nm, mobile consisted of aqueous phosphoric acid and methanol

Highest anthocyanin compound was cyanidin 3-galactoside (1014.98 mg/L), which accounted for about 46.73% of TAC

Green powder of stevia was considered to be a rich source of anthocyanins after processing.

[29]

Phenolic compounds Phenolic compounds charcaterization were identified and characterized by LC coupled with quadrupole-time of flight (QTOF), accurate mass spectrometer (MS), and dual electrospray ionization.

21 phenolic compounds were identified in polar stevia extracts that were broadly divided into three classes, namely caffiec acids, flavonoids, quinic acids, and derivatives. Other notable phenolic compounds were 5caffeoylquinic acid, 3caffeoylquinic acid, kaempferol rhamnoside, quercetin, rutin, and

Polar extract of stevia leaves demonstarted significanlty higher phenolic compounds.

[30]

15.

Dried stevia leaves were subjected to hydrodistillation using water and methanol
water, and essential oils were obtained from stevia leaf extract

Total anthocyanin content 16.

TAC

• TAC of water extract was 0.35 mg Cg/g DM • TAC of methanol
water extract was 0.67 mg Cg/g DM

Total phenolic content and phenolic compounds 19.

Maceration extract of stevia leaf powder

(Continued)

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Antioxidant potential against free radicals and stevia potential

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

quercetin-3-O-arabinoside. Oxidative stress can be prevented by antiinflammatory effect of one of the main identified compounds (austroinulin). 20.

Stevia leaf and stem extracts

Phenolic compounds identification and determintaion of total phenolic compounds (TPC)

Islotaion and identiication of phenolic compounds were carried out by HPLC followed by NMR spectroscopy. TPC of stevia leaf and stem extracts were determined by Folin
Ciocalteu (FC) colorimetric method againts GA standard.

Main identified phenolic TPC was highest in compounds were vanillic stevia leaf extract using acid, protocatechuic acid, water as solvent caffeic acid, chlorogenic acid, and cryptochlorogenic acid. The TPC of stevia leaf and stem extracts were in the range of 46.14 to 71.46 mg GAE/g.

[5]

21.

Dried stevia leaves

Determintaion of polyphenolic compounds

HPLC determintaion and methanol and formic acid were used as mobile phase.

Ellagic acid, coumarin, hesperidin, chlorogenic acid, rosmarinic acid, eugenol, and vanillin were main phenolic compounds.

Higher contents of phenolic compounds were related with enhanced antioxidant potential of stevia leaves.

[31]

22.

Extracts of finely ground leaves were recovered by solvent extraction using aqueous solutions of organic solvents

TPC determintaion

FC clorimetric method at 725 nm against tannic acid as the standard compound and results were shown as tannic acid equivalents (TAE)/ 100 g

70% methanol resulted in recovery of the highest TPC from stevia leaves, which was 3.52 mg TAE/100 g. TPC of water extract were also equivalent.

High TPC contributed to higher RSA.

[32]

23.

Stir-aided hydroalcoholic extracts of oven-dried stevia leaves

HPLC

Phenolic compounds were quantified by HPLC-DAD-ESI/MS

Total 18 phenolic compounds were identified. Main compounds were kaempferol-3-Oglucoside, quercetin-3-Orutinoside, 3-O-, 4-O-, 5O-caffeoylquinic acid, and quercetin-3-Oglucoside.

Quercetin-O-pentosyldeoxyhexoside and kaempferol-O-hexoside were not detected.

[24]

24.

Maceration extract of TPC air-dried stevia powder

Folin
Ciocalteau reagent method 760 nm against GA standard

TPC of 56.74 mg GAE/g were correlated with higher TPC and TFC

Higher antioxidant compounds may be helpful in quenching free radicals.

[25]

25.

Stevia leaf and callus extract

Phenolic compounds HPLC-DAD (280 nm)

Leaf extract TPC were Pyrogallol (951.3 mg/ 100 g), 4-methoxybenzoic higher than callus. acid (33.81 mg/100 g), 4methylcatechol (25.61 mg/100 g), pcoumaric acid (30.47 mg/100 g), sinapic acid (9.03 mg/100 g), and cinnamic acid (2.42 mg/ 100 g) were main

[26]

(Continued)

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33. Antioxidant potential of Stevia rebaudiana (Bertoni)

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

Stevia extract was rich in antioxidant compounds.

[33]

compounds. TPC was 130.67 μg CE/100 g. 26.

Water extraction assisted by electric discharge (HVED), pulsed electric field (PEF) and sonication (US)

phenolic profile determination

Spectrophotometer and HPLC-based quantification

TPC on DM basis were: US extract 5 9
16 mg GAE/g PEF extract 5 10.28
21 mg GAE/g HVED extract 5 14.23
29.14 mg GAE/g Chlorogenic acid, caffiec acid, ferulic acid, protocatechuic acid, and GA were the main compounds.

27.

Stevia callus and leaf extract

TPC

Colorimetric TPC ranged Higher TPC were determination at 620 nm 25.18
35.86 mg/g DW in correlated with high againts GA standard leaf and callus extract RSA in both extracts. respectively

[19]

Total flavonoid content 28.

Solvent extraction using aqueous solutions of methanol, ethanol, and acetone

TFC measurement

Aluminum chloride (AlCl3) colorimetric method was employed at 430 nm against quercetin standard

Aqueous and acetone fractions showed the maximum TFC (0.14 g QE/100 g)

Water and acetone led to maximum TFC recovery.

[32]

29.

Solvent extracts using water, ethanol, and glycol mixture

TFC determination

Spectrophotometric measurement against quercetin standard

Extract obtained by aqueous
glycol mixture showed the highest flavonoid content (3.6 mg/g DW), which was twice the TFC from distilled water (2.3 mg/g DW) and ethanolic extract (2 mg/g DW).

Due to its significant antioxidant potential, stevia might be useful as a healthful ingredient for food, cosmetics, and dietary supplements.

[10]

30.

Essential oils, water, and methanol
water fractions of hydrodistilled stevia extract

TFC measurement

Colorimetric Aqueous and determination at 510 nm methanol
water as CE on DW basis fractions showed equivalent TFC of 20.68 and 23.46 mg CE/g DM respectively

Stevia leaves may serve [27] as an excellent source of antioxidants.

31.

Leaf and callus extract of stevia

Flavonoid compounds determination

Spectrophotometric measurement in terms of mg QE

TFC of stevia leaf and callus extracts were: Leaf extract TFC 5 15.64 μg QE/mg Callus extract TFC 5 1.57 μg QE/mg

Stevia leaf exhibited higher antioxidant potential as compared to callus.

[26]

32.

Fractioned leaf extract

TFC measurement

Spectrophotometric measurement of TFC at 415 nm against quercetin standard

Ethanolic fraction had the highest TFC (125.64 mg QE/g DW) followed by 1-butanol and dichloromethane

Ethanol was the most suitable solvent for maximizing TFC recovery.

[34]

(Continued)

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References

TABLE 33.1

(Continued)

Sr. No. Sample form

Parameter

Assay/detection technique/separation approach

Recovery of target bioactive compound

Remarks

Reference

Condensed tannins content 33.

Stevia leaf powder

Tannins determination

Extract of stevia was analyzed by standard method

Stevia extract showed higher tannins content (0.01 mg/100 g DW)

High tannins content in stevia extract established its antioxidant potential.

[11]

34.

Solid
liquid extraction from powder and flower using various organic solvents

Phytochemical analysis including condensed tannins content (CTC)

Tannins were determined by mixing 200 mg stevia with distilled water. FeCl3 was used to indicate presence of tannins in extract.

Ethanolic extract Stevia leaf extract demonstrated the highest showed more CTC significant quantities of condensed tannins as compared to flower extract.

[35]

35.

Hydrodistilled stevia CTC determination leaf extract followed by fractionation

HCl-methanol method yielded supernatant, which was analyzed spectrophotometrically at 500 nm.

Methanolic-water fraction showed the maximum CTC (10.20 mg CE/g DM) in comparison with water fraction (8.15 mg CE/g DM).

Essential oil fraction did not show any CTC.

[27]

Wide range of antioxidant compounds of different classes from various matrices of stevia plant and recovery of the antioxidant and bioactive compounds by standards assays/protocols.

Conclusion and future perspective Stevia is a rich source of phytochemicals of which regular consumption may provide beneficial effects against oxidative stress, thereby promoting overall health and wellness, according to the ADI recommended by the FDA and EFSA. In addition to sweeteners, the natural phytoconstituents of stevia could be consumed in the form of extract preparations and ingredients in food formulations to offer therapeutic benefits including diuretic, antidiarrheal, antihyperglycemic, anti-inflammatory, antihypertensive, and immunomodulatory effects. Further studies are needed to achieve greater insight into the potential practical applications in the food, medicinal, and therapeutic sectors. Furthermore, in-depth studies are needed to determine the possible chemical transformations of SGs by human microflora at the microbiome level.

• Stevia rebaudiana (Bertoni) as a shrub of South American origin has long since been known due to the sweet steviol glycosides in its leaves and callus. • Along with its sweetening potential, more than 100 compounds including antioxidants (polyphenols, anthocyanin, flavonoids, and tannins) have been found in stevia exhibiting medicinal and therapeutic significance. • Stevia may be helpful in improving antioxidant intake in human diets by following its ADI (4 mg/kg body weight) according to international approved guidelines. • Stevia extract preparations may be employed as a natural sweetener for the alleviation of health issues caused by oxidative stress such as cancer, liver injury, obesity, diabetes, hypertension, inflammation, and neurodegenerative diseases.

References Summary points • Oxidative stress due to free radicals and reactive oxygen species has been related to the onset of several chronic diseases, aging, and adverse effects on normal tissue functioning.

1. Mecocci P, Boccardi V, Cecchetti R, Bastiani P, Scamosci M, Ruggiero C, et al. A long journey into aging, brain aging, and alzheimer’s disease following the oxidative stress tracks. J Alzheimer’s Dis 2018;62:1319
35. 2. Hassan W, Noreen H, Rehman S, Gul S, Kamal MA, Kamdem JP, et al. Oxidative stress and antioxidant potential of one hundred medicinal plants. Curr Top Med Chem 2017;17:1336
70.

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3. Ameer K, Shahbaz HM, Kwon JH. Green extraction methods for polyphenols from plant matrices and their byproducts: a review. Compr Rev Food Sci Food Saf 2017;16:295
315. 4. Carakostas MC, Curry LL, Boileau AC, Brusick DJ. Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages. Food Chem Toxicol 2008;46:1
10. 5. Yu H, Yang G, Sato M, Yamaguchi T, Nakano T, Xi Y. Antioxidant activities of aqueous extract from Stevia rebaudiana stem waste to inhibit fish oil oxidation and identification of its phenolic compounds. Food Chem 2017;232:379
86. 6. Thomas JE, Glade MJ. Stevia: it’s not just about calories. Open Obes J 2010;2:101
9. 7. Halliwell B. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch Biochem Biophys 2008;476:107
12. 8. Ameer K, Bae SW, Jo Y, Lee HG, Ameer A, Kwon JH. Optimization of microwave-assisted extraction of total extract, stevioside and rebaudioside-A from Stevia rebaudiana (Bertoni) leaves, using response surface methodology (RSM) and artificial neural network (ANN) modelling. Food Chem 2017;229:198
207. 9. Paul S, Sengupta S, Bandyopadhyay TK, Bhattacharyya A. Stevioside induced ROS-mediated apoptosis through mitochondrial pathway in human breast cancer cell line MCF-7. Nutr Cancer 2012;64:1087
94. 10. Gaweł-Be˛ben K, Bujak T, Nizioł-Łukaszewska Z, Antosiewicz B, Jakubczyk A, Kara´s M, et al. Stevia rebaudiana Bert. leaf extracts as a multifunctional source of natural antioxidants. Molecules 2015;20:5468
86. 11. Savita SM, Sheela K, Sunanda S, Shankar AG, Ramakrishna P. Stevia rebaudiana
a functional component for food industry. J Hum Ecol 2017;15:261
4. 12. Ferrazzano GF, Cantile T, Alcidi B, Coda M, Ingenito A, Zarrelli A, et al. Is Stevia rebaudiana Bertoni a non cariogenic sweetener? A review. Molecules 2016;21:1
12. 13. Geeraert B, Crombe´ F, Hulsmans M, Benhabile`s N, Geuns JM, Holvoet P. Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant mice. Int J Obes 2010;34:569
77. 14. Liu JC, Kao PK, Chan P, Hsu YH, Hou CC, Lien GS, et al. Mechanism of the antihypertensive effect of stevioside in anesthetized dogs. Pharmacology 2003;67:14
20. 15. Ruiz-Ruiz JC, Moguel-Ordon˜ez YB, Matus-Basto AJ, SeguraCampos MR. Antidiabetic and antioxidant activity of Stevia rebaudiana extracts (Var. Morita) and their incorporation into a potential functional bread. J Food Sci Technol 2015;52:7894
903. 16. Bender C. Stevia rebaudiana’s antioxidant properties. In: Merillon JM, Ramawat K, editors. Sweeteners. Reference series in phytochemistry. San Diego, Cham: Springer International Publishing; 2016. p. 161
8. 17. Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57:715S
725SS. 18. Chatsudthipong V, Muanprasat C. Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol Ther 2009;121:41
54. 19. Tadhani MB, Patel VH, Subhash R. In vitro antioxidant activities of Stevia rebaudiana leaves and callus. J Food Compos Anal 2007;20:323
9. 20. Phansawan B, Poungbangpho S. Antioxidant capacities of Pueraria mirifica, Stevia rebaudiana Bertoni, Curcuma longa Linn., Andrographis paniculata (Burm.f.) Nees. and Cassia alata Linn. for the development of dietary supplement. Kasetsart J - Nat Sci 2007;41:548
54.

21. Ahmad N, Fazal H, Abbasi BH, Farooq S. Efficient free radical scavenging activity of Ginkgo biloba, Stevia rebaudiana and Parthenium hysterophorous leaves through DPPH (2, 2-diphenyl-1picrylhydrazyl). Int J Phytomed 2010;2:231
9. 22. Ghanta S, Banerjee A, Poddar A, Chattopadhyay S. Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana bertoni, a natural sweetener. J Agric Food Chem 2007;55:10962
7. 23. Shukla S, Mehta A, Bajpai VK, Shukla S. In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert. Food Chem Toxicol 2009;47:2338
43. ˆ , Sousa MJ, Santos-Buelga C, 24. Barroso M, Barros L, Rodrigues MA Ferreira ICFR. Stevia rebaudiana Bertoni cultivated in Portugal: a prospective study of its antioxidant potential in different conservation conditions. Ind Crop Prod 2016;90:49
55. 25. Shukla S, Mehta A, Mehta P, Bajpai VK. Antioxidant ability and total phenolic content of aqueous leaf extract of Stevia rebaudiana Bert. Exp Toxicol Pathol 2012;64:807
11. 26. Kim I, Yang M, Lee O, Kang S. The antioxidant activity and the bioactive compound content of Stevia rebaudiana water extracts. LWT Food Sci Technol 2011;44:1328
32. 27. Muanda FN, Soulimani R, Diop B, Dicko A. Study on chemical composition and biological activities of essential oil and extracts from Stevia rebaudiana Bertoni leaves. LWT Food Sci Technol 2011;44:1865
72. 28. Carbonell-Capella JM, Buniowska M, Barba FJ, Grimi N, Vorobiev E, Esteve MJ, et al. Changes of antioxidant compounds in a fruit juice-Stevia rebaudiana blend processed by pulsed electric technologies and ultrasound. Food Bioprocess Technol 2016;9:1159
68. ˇ Zlabur ˇ 29. Sic J, Dobriˇcevi´c N, Gali´c A, Pliesti´c S, Vo´ca S. The influence of natural sweetener (Stevia rebaudiana Bertoni) on bioactive compounds content in chokeberry juice. J Food Process Pres 2018;42:e13406. 30. Molina-Calle M, Priego-Capote F, Luque, de Castro MD. Characterization of stevia leaves by LC
QTOF MS/MS analysis of polar and non-polar extracts. Food Chem 2017;219:329
38. 31. Najafian S, Moradi M. Polyphenolic compounds (HPLC analysis) and antioxidant activity of Stevia rebaudiana (Asteraceae) by FRAP and DPPH assay in greenhouse and free space condition. Intl J Farm & Alli Sci 2017;6:49
55. 32. Shuvo MMA, Mamun M Al, Absar N. In vitro determination of total phenolics, flavonoids and free radical scavenging activities of Stevia rebaudiana dry leaves powder in different solvents extract. EC Nutr 2018;3:71
8. 33. Barba FJ, Grimi N, Vorobiev E. Evaluating the potential of cell disruption technologies for green selective extraction of antioxidant compounds from Stevia rebaudiana Bertoni leaves. J Food Eng 2015;149:222
8. 34. Preethi D, Sridhar TM, Josthna P, Naidu CV. Studies on antibacterial activity, phytochemical analysis of Stevia rebaudiana (Bert.) An important calorie free biosweetner. J Ecobiotechnol 2011;3:5
10. 35. Joseph D, George J, Mathews MM, Mathew F, Varghese B, Sunny B, A comprehensive exploration on therapeutic options of Stevia rebaudiana with emphasize on anti-diabetic attribute. Res J Pharm Technol 2019;12:4981
4988.

Further reading Jahan IA, Mostafa M, Hossain H, Nimmi I, Sattar A, Alim A, et al. Antioxidant activity of Stevia rebaudiana Bert. leaves from Bangladesh. Bangladesh Pharm J 2010;13:67
75.

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C H A P T E R

34 Tea antioxidants in terms of phenolic and nonphenolic metabolites Protiva Rani Das and Jong-Bang Eun Department of Food Science and Technology and BK 21 Plus Program, Graduate School of Chonnam National University, Gwangju, South Korea

List of abbreviations •

OH O2 ATP C CAT CG DNA EC ECG EGC EGCG GC GSHPx H2O2 HDL iNOS LDL LOOH mtDNA NO O2 O2•2 ONO2 2 Prx RNA RNS RO• ROO• ROS RS SOD 1

hydroxyl radical singlet oxygen adenosine triphosphate catechin catalase catechin gallate deoxyribonucleic acid epicatechin epicatechin gallate epigallocatechin epigallocatechin gallate gallocatechin glutathione peroxidase hydrogen peroxide high-density lipoprotein nitric oxide synthase low-density lipoprotein lipid hydroperoxide mitochondrial DNA nitric oxide oxygen superoxide anion radical peroxynitrite peroxiredoxin ribonucleic acid reactive nitrogen species alkoxyl radical peroxyl radical reactive oxygen species reactive species superoxide dismutase

Introduction Free radicals, reactive species (RS), reactive oxygen species (ROS) and reactive nitrogen species (RNS), and Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00034-2

oxidative stress are widely correlated with several pathogenicities. Free radicals with an unpaired electron were initially described by Moses Gomberg more than a century ago.1 Since 1950, the roles of free radicals in biological systems and their associated pathogenicities have been widely explored.2 Free radicals majorly occur in the mitochondria (90%), cell membranes, and cytoplasm. Electron transfer in biochemical reactions is mediated by RS during metabolic processes. Therefore the electron transport chain coupled with an appropriate amount of RS has been found to be associated with the generation of adenosine triphosphate (ATP).3 RS production can be controlled by the antioxidant defense mechanism to maintain homeostatic levels of RS and antioxidants. Nevertheless, an imbalance between the generation and elimination of RS is associated with oxidative stress. In living organisms, acute or chronic oxidative stress disrupts the cellular metabolism by oxidizing cellular components.3 Consequently, oxidative stress may also cause several health complications such as metabolic disorders, cardiovascular disorders, neurological disorders, cancer, and various infections.4 To maintain low RS levels, biological systems upregulate intracellular antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and peroxiredoxin (Prx).5 Besides endogenous antioxidants, dietary supplementation of natural antioxidants is crucial in the antioxidant mechanism. Among various sources of dietary antioxidant-rich foods and beverages, tea is widely consumed because it is rich in antioxidants and has several health benefits.6 The major tea antioxidants include flavonoids, phenolic acids, amino acids, polysaccharides, organic acids, and other nutrients. In this chapter, the oxidative stress
related

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© 2020 Elsevier Inc. All rights reserved.

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34. Tea antioxidants in terms of phenolic and nonphenolic metabolites

pathophysiologies of several diseases are revised and the role of phenolic and nonphenolic metabolites present as antioxidants in tea is discussed.

Oxidative stress associated pathogenesis A glance at free radicals, reactive species, and oxidative stress Unstable and highly reactive molecular species comprising unpaired electrons in their outer atomic orbital are collectively known as free radicals, which form the byproducts of normal cellular metabolic reactions.4 A reduction in consumed oxygen from molecular oxygen (O2) by a one-electron consecutive pathway liberates superoxide anion radical (O2•2).3 ROS/RNS are the byproducts of molecular O2 metabolism. Commonly generated RS include hydroxyl radical (•OH), singlet oxygen (1O2), alkoxyl radical (RO•), O2•2, hydrogen peroxide (H2O2), peroxyl radical (ROO•), and lipid hydroperoxide (LOOH).5 These radicals are crucial in the synthesis of ATP via the electron transport chain reaction and other important biological processes. The strict balancing of continuously produced RS through the activation of the antioxidant defense system in humans is a natural process.3 Nevertheless, oxidative stress may arise when there is an imbalance between increased RS production and reduced antioxidant levels.4

RS generated by cellular metabolism via environmental factors causes modifications in the amino acids of proteins associated with cellular dysfunctions, protein
protein cross-linkages, and degradation. Nitrotyrosine is an identified biomarker of oxidative protein damage that was found in ROS-mediated oxidative damage to tyrosine.7 Specific amino acids including lysine, methionine, proline, histidine, arginine, and cysteines are more vulnerable to oxidative damage than others. Modified proteins cause several health complications including aging.8 Sulfur reactive radicals generated by carbonyl groups (ketones and aldehydes) and thiol groups lead to alterations in protein structures and loss of their functions.9 The nucleic acids, DNA and RNA, are essential biomolecules for life. Oxidative stress is highly reactive in damaging DNA and RNA molecules. Oxidative damage of DNA majorly occurs due to •OH and peroxynitrite (ONO22).4,5 A biomarker, namely 8-hydroxy-deoxyguanosine, has been identified due to DNA oxidation.10 Oxidative stress
mediated DNA damage alters the DNA bases, damages the double strand, and breaks down the single strand, leading to several genetic disorders. The oxidative damage of cellular polyunsaturated fatty acids leads to lipid peroxidation.5 Lipid peroxidation is associated with cellular dysfunctions, cellular damage, loss of cell membrane flexibility, and tissue injuries that lead to chronic disorders. It can break down aldehydes, some of which are biologically active in increasing cellular toxicity and attacking different cell parts.5

Oxidative stress in human biology Several factors are responsible for imbalances in the steady-state levels of RS and antioxidant levels. These include majorly reduced antioxidant levels, increases in autoxidation due to higher levels of exogenous and endogenous compounds, and reduced generation of oxidative enzymes and antioxidants (low molecular weight).3 Besides exterior oxidants/stimuli, increased levels of external O2 are also responsible for the oxidative stress associated with severe tissue injury.

Oxidative stress
induced cellular damage Oxidative stress leads to the oxidation and dysfunctioning of biomolecules, imbalance in the levels of homeostasis and cellular metabolism, and cellular/tissue damage.4 Cell membranes comprise higher levels of polyunsaturated lipids prone to oxidation by RS. Lipid peroxidation mediated by RS increases their permeability to the cell membrane and causes cell death. Oxygen metabolism generates •OH, O2•2, and H2O2, which are highly responsive to protein, nucleic acid, and lipid damage.

Oxidative stress
induced health hazards Oxidative stress in the human body evokes the pathogenesis of several diseases, in particular, carcinogenesis, metabolic disorders, chronic obstructive pulmonary disease, mutagenesis, neurodegenerative disorders, cardiovascular disorders, genetic disorders, aging, and several others. These pathogeneses majorly occur due to the excessive generation of RS by endogenous and exogenous factors, which are not eliminated because of an impaired antioxidant defense system. Several severe human pathogeneses linked to oxidative damage are summarized here.

Metabolic syndrome Oxidative stress is positively correlated with metabolic syndromes such as hyperglycemia, hypertension, atherosclerosis, hyperlipidemia, insulin resistance, cardiovascular diseases, vascular alternations, obesity, and others.11 Oxidative stress is correlated with reduced high-density lipoprotein (HDL) and raised low-density

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induced pathogenesis

lipoprotein (LDL). Increased LDL is related to the development of metabolic syndromes. Oxidative stress
induced insulin resistances are majorly regulated by the homeostasis equilibrium between the generation of H2O2 level and impaired antioxidant defense.12 O2•2, a major ROS species generated by the angiotensin II type 1 receptor, causes the neutralization of nitric oxide (NO) and the stimulation of ROS production, which may result in increased risk of hypertension.12 Dysglycemia causes diabetic complications by initiating oxidative stress. A mitochondrial electrontransfer chain reaction generates excessive O2•2 due to activated oxidative stress in diabetic patients. Oxidative stress is reported to be significantly correlated with increased fat distribution and adiposity, which result in obesity.13

Aging Oxidative stress is reported to cause cellular dysfunction and death.12 The aging process is closely linked to the dysfunctioning of cellular components, in particular the mitochondria, the main intracellular source of O2•2. Oxidative stress damages mitochondrial constituents such as lipids, proteins, and mitochondrial DNA (mtDNA), and it contributes to the loss of their bioenergetic functions, leads to aging, impairs respiratory chain complexes, and enhances mtDNA mutation.14

Cardiovascular diseases The term cardiovascular disorders collectively refers to various types of heart diseases such as hypertension, atherosclerosis, cardiomyopathy, cardiac hypertrophy, congestive heart failure, ischemic syndrome, and several others. The underlying mechanism behind oxidative stress under various pathophysiological conditions and their relation with inflammation remains unclear; however, reported evidence provides a positive correlation between oxidative stress and inflammation, which is associated with acute coronary artery diseases. The increasing permeability of Ca21 into the vascular myocytes due to oxidative stress stimulates neointimal hyperplasia, resulting in a higher incidence of atherosclerosis and vasoconstrictions, further leading to hypertension. Excessive intracellular Ca21 concentrations are also linked to myocardial cell damage.15

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(HCV). The excessive generation of RS has been determined as the predominant factor for HCV-associated pathogeneses.16 Rotavirus-infected murine ileum stimulates the increased production of NO, nitric oxide synthase (iNOS), and mRNA levels of inducible iNOS as well as decreasing the activity of antioxidant enzymes like SOD and GSHPx.17 Therefore NO production, iNOS induction, RS accumulations, and glutathione content reduction are all mediators of dengue virus infection and cause severe pathogeneses.18 Lung injury due to influenza virus infection leads to the generation of ROS.16

Cancer In human biology, the loss of the antioxidant defense system can generate oxidative stress and damage cellular components such as DNA, proteins, phospholipids, RNA, and carbohydrates. Among several signaling pathways for carcinogenesis, oxidative stress
induced genetic mutations are considered as a major risk factor. ROSstimulated oxidized DNA produces 8-hydroxy-20 -deoxyguanosine, which is potentially capable of DNA mutation by intensifying carcinogenesis and aging.19 Telomere (tumor suppressor gene) and cell cycle
regulated genes are considerably sensitive to oxidative damage to DNA. In contrast, biological mutation is also associated with the initiation of reactive molecules such as aldehydes and epoxides due to the reaction of oxidized lipids and metals.20

Neurodegenerative disorders Oxidative stress is a crucial factor in several neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, epilepsy, tardive dyskinesia, Huntington’s disease, and several other central nervous system disorders. Human brain tissue is vulnerable to oxidative stress and it causes increased levels of oxidized DNA, proteins, and lipids as well as a decreased level of glutathione in patients with Parkinson’s disease.4 Increased levels of oxidized RNA lead to decreased levels of antioxidant enzymes, the deposition of amyloid plaque due to oxidative stress, and the dysregulation of iron and copper homeostasis, which are evident in Alzheimer’s disease.4

Infection Infections caused by bacteria, viruses, or parasites elicit the generation of ROS/RNS. Viral infection
induced ROS generations are associated with damage to various biological constituents. Mitochondrial ROS generation is regulated by the core proteins of the hepatitis C virus

Role of antioxidants in oxidative stress
induced pathogenesis Antioxidants can neutralize reactive radicals by donating an electron and stabilizing them. The modulations of

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34. Tea antioxidants in terms of phenolic and nonphenolic metabolites

free radical reactions involve enzymatic antioxidants or nonenzymatic antioxidant mechanisms. Nonenzymatic antioxidants include natural and synthetic antioxidants. Several studies have reported the potentiality of the antioxidant mechanisms of dietary antioxidants from natural food sources against oxidative stress
induced human pathogeneses.21

Enzymatic antioxidants Biologically, a body can protect itself from the adverse effects of ROS via its enzymatic antioxidant mechanism.5 Antioxidant enzymes mainly include SOD, CAT, GSHPx, and Prx. SOD, CAT, and GSHPx are synergistically crucial in preventing oxidative damage. These enzymes are present in the mitochondria and cytosol. SOD catalyzes O2•2 in the presence of cofactors (copper, manganese, or zinc) to produce O2 and H2O2. Followed by the CAT catalysis of H2O2 to H2O and O2, while the enzyme GSHPx has the potential to catalyze H2O2 and fatty acid hydroperoxides. This enzyme converts H2O2 to H2O. The reduction of H2O2, organic hydroperoxides, and ONO22 is catalyzed by Prx.5

Natural dietary antioxidants Natural dietary compounds with antioxidant activities include phenolic metabolites such as flavonoids, phenolic acids, tannin, lignin, and anthocyanin, etc.,6,22 and nonphenolic metabolites including polysaccharides, amino acids, vitamins, carotenoids, organic acids, and several others.23
27 These bioactive metabolites are widely distributed in different plant-based foods and beverages, and they reveal potential antioxidant activities as well as numerous health benefits. Consequently, the dietary intake of antioxidant-rich food and beverages is increasing with time to fulfill consumer demands.

Phenolic and nonphenolic antioxidant metabolites Phenolic antioxidants Plant secondary metabolism produces phenolic compounds with at least one hydroxyl aromatic ring substituted in their chemical structures.22 These phenolic compounds can be classified into major groups based on their carbon chain.6 The major classes of these compounds with higher antioxidant activities include flavonoids, tannins, phenolic acids, chalcones, and coumarins. The antioxidant activity of phenolic compounds majorly depends on their chemical structures.22 Flavonoids are considered to be the most widely distributed antioxidant compounds

in plant-based food and beverages. The underlying mechanism of phenolic antioxidant compounds to delay or inhibit oxidative degradation reactions involves several pathways, namely (1) scavenging RS by donating electrons and making them unreactive so that they cannot initiate peroxidation, (2) chelating metal ions to inhibit their capacity to generate free radicals, (3) preventing the generation of peroxides by quenching O2•2, (4) terminating oxidative chain reactions, and/or (5) decreasing localized O2 concentrations.28 Nonphenolic antioxidants Besides phenolic compounds, other groups of compounds that have health benefits include carotenoids, alkaloids, amino acids, organic acids, polysaccharides, and others. Carotenoids are isoprenoid pigments, which have been reported as potential antioxidants.23 Natural bioactive polysaccharides act as potent antioxidants.27 The health benefits of amino acids are immense. Moreover, besides its other biological activities, amino acid can prevent oxidative stress.29

Tea as a major source of dietary antioxidant A glance at tea Tea, manufactured from the leaves of Camellia sinensis, is majorly consumed worldwide. The popularity of tea is positively correlated with its high content of bioactive metabolites such as polyphenols, amino acids, organic acids, and polysaccharides, and its pleasing aroma and taste. Based on the processing and manufacturing of tea leaves, four major types of teas are available, namely white tea, green tea, oolong tea, and black tea30 (Fig. 34.1). In terms of the processing of tea and based on the degree of fermentation, white tea is classified as completely unfermented. The exact definition of white tea is not clear; however, it can be defined as “the plucking of selected parts, especially very young leaves and bud of the tea plant.”31,32 Green tea is known as nonfermented tea, oolong tea undergoes a semifermentation process during manufacturing, and black tea is fully fermented tea.33 Among these, white tea is considered as the oldest form of tea due to its simple processing method; however, it is less explored and is preferred over green tea in Europe.32 The worldwide consumption of tea includes a 76%
78% preference for black tea, followed by 20%
22% for green tea, and 2% for oolong tea.34 The chemical composition of tea is basically influenced by the manufacturing process, where tea undergoes oxidative and enzymatic changes.

Phenolic compounds in tea The phenolic compositions of tea can vary according to the different growth stages, varieties, plucking

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361

FIGURE 34.1 Diagram of major types of tea. The manufacturing process of tea leaves categorizes them into different types and affects their chemical compositions.

times, origins, manufacturing processes, seasons, positions on the flushing shoot, fermentation rates, and other factors. The most effective phenolic antioxidants present in tea are flavonoids, in particular, catechins. Tea catechin is altered during processing and can be reduced with the degree of fermentation. Unfermented tea such as white tea or green tea comprises higher catechin than fermented or semifermented tea.31 Table 34.1 represents the list of phenolic antioxidants that are commonly present in different types of tea. Green tea possesses higher contents of polyphenols in comparison with black or oolong tea due to the lack of a fermentation process. The deactivation of polyphenol oxidase by steaming green tea leaves at a high temperature prevents catechin oxidation, thereby retaining the monomeric forms of polyphenols.35 The paramount polyphenols present in green tea leaves are catechins, which make up 25%
35% of its dry weight. Catechins are mainly flavonoids such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), catechin (C), epicatechin (EC), gallocatechin (GC), and catechin gallate (CG).36 Hydroxybenzoic phenolic acids, in particular gallic acid, are also found in green tea, and are present in both free and cell-wallbound form.6 The major flavonols present in tea include kaempferol, quercetin, and myricetin, which remain considerably unaffected during processing.37 Higher contents of proanthocyanidins and lower bisflavanols are also determined in green tea.31 Nevertheless, the proanthocyanidins contents are determined as ten times lower than those of catechins.38

Semifermented oolong tea undergoes a moderate level of oxidation and produces monomeric theaflavins, thearubigins, and catechins. Partial oxidization creates a lower concentration of polymeric polyphenols and a higher concentration of EGCG in oolong tea than in black tea.39 In contrast, black tea contains lesser amounts of monomeric polyphenols (3%
10% of solids) and higher concentrations of polymers (23%
25% of solids) than green tea.37 The entire fermentation process for black tea involves oxidative and enzymatic changes, thereby resulting in the transformation of monomeric polyphenols to polymeric polyphenols, namely theaflavins and thearubigins. Consequently, lower catechins and higher gallic acids are found in black tea during fermentation. Increases in gallic acid are observed due to the ester hydrolysis of 3-galloyl substituted catechins during fermentation.40 Under enzymatic catalysis, epicatechins (EC, ECG, EGC, and EGCG) dimerize to theaflavins. Theaflavins contribute to the yellowish-brown and thearubigins to the reddish-brown color of black tea.37 Theaflavin isomers, namely, neotheaflavin, isotheaflavin, theaflavic acids, theaflavates, and theaflagallins, etc., are present as minor components in comparison to theaflavins.40 To date, the identified thearubigins in black tea include theadibenzotropolones (A, B, and C), dibenzotropolones, tribenzotropolones, and theatribenzotropolones.41 Moreover, black tea also comprises large amounts of bisflavanols. The breakdown of galloylated proanthocyanidins can occur during fermentation.42

II. Antioxidants and Pathology

362 TABLE 34.1

34. Tea antioxidants in terms of phenolic and nonphenolic metabolites

Commonly present phenolic antioxidant metabolites in tea.

Phenolic metabolites

White tea

Green tea

Oolong tea

Black tea

References

Catechins:

ü (Higher)

ü (Higher)

ü (Lower)

ü (Lower)

[36]

3

3

ü (Higher)

ü (Higher)

[39]

Bisflavanols

ü (Lower)

ü (Lower)

ü (Higher)

ü (Higher)

[38]

Flavonols:

ü

ü

ü

ü

[37]

Proanthocyanidins

ü (Higher)

ü (Higher)

ü (Lower)

ü (Lower)

[31]

Phenolic acids: Hydroxybenzoic acids:

ü (Lower)

ü (Lower)

ü (Higher)

ü (Higher)

[6, 40]

• • • • • •

EGCG ECG EGC EC CG GC

Theaflavins Thearubigins

• Quercetin • Kaempferol • Myricetin

• • • • • •

Gallic acid Protocatechuic acid Vanillic acid Galloylquinic acid Coumaryl quinic acid Galloylated glucose derivatives

ü, present; 3 , absent. These phenolic compounds present in tea have antioxidant properties.

Nonphenolic compounds in tea Besides phenols, other compounds present in tea include amino acids, polysaccharides, methylxanthines, organic acids, carotenoids, and carbohydrates.24,26 Table 34.2 represents the list of commonly present nonphenolic antioxidants in different types of tea. Free amino acids are also found in tea, which can be altered via fermentation during the manufacturing process.25 Among all these, nonproteinogenic L-theanine is the predominant amino acid (approximately 2
5 mg/g leaf) found in tea, which accumulates in higher contents in young and fresh tea leaves.26 The highest content of L-theanine is determined in white tea and green tea followed by oolong tea and black tea.43 Polysaccharides in tea are acidic, nonstarch, proteinbound compounds, comprising neutral sugar (44%), uronic acid (43%), and proteins (9%). The molecular weight (MW) distribution of polysaccharides content is affected by the various tea processing methods. Oolong tea contains the highest polysaccharides content (4.6%) followed by black tea (4.2%) and green tea (4.0%) based on their dry weights.44 Two

polysaccharides, namely polysaccharide-A (MW .100 kDa) and polysaccharide-B (MW 10 kDa), were identified in green tea infusions.45 The MW distribution of green tea polysaccharides normally ranges from 9.2 to 251.5 kDa.44 Black tea comprises protein-bound polysaccharide 46 and its MW distribution normally ranges from 3.8 to 32.7 kDa 44; whereas, the MW distribution of polysaccharide in oolong tea ranges from 5.3 to 100.9 kDa.44 Methylxanthines present in tea majorly consist of caffeine, theobromine, and theophylline. In tea leaves, caffeine present as the major alkaloid constitutes approximately 3.5% of the dry weight.35 The degree of fermentation of tea leaves significantly affects the caffeine contents, which can be increased with a high degree of fermentation. Among all tea types, fermented tea contains higher caffeine contents followed by semifermented and nonfermented tea.47 Furthermore, carotenoids are found in tea leaves with a dry weight of 36
73 mg/100 g.48 The major carotenoids in tea include β
carotene, lutein, and zeaxanthins, which can be changed during tea processing and preparation methods.

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363

Conclusions

TABLE 34.2

Commonly present nonphenolic antioxidant metabolites in tea.

Nonphenolic metabolites

White tea

Green tea

Oolong tea

Black tea

References

Methylxanthine alkaloids:

ü (Lower)

ü (Lower)

ü (Higher)

ü (Higher)

[47]

ü ü (Higher) (Higher)

ü (Lower)

ü (Lower)

[43]

ü (Higher, MW distribution ranged from 3.8
32.7 kDa)

[44]

• Caffeine • Theobromine • Theophylline Amino acids: • L-theanine

Polysaccharides ü (Lower)

ü ü (Lower, MW distribution ranged (Higher, MW distribution from 9.2
251.5 kDa) ranged from 5.3
100.9 kDa)

ü, present; 3 , absent; MW, molecular weight. These nonphenolic compounds present in tea have antioxidant properties.

Antioxidant activities of phenolic and nonphenolic metabolites in tea Several studies have reported the antioxidant property of tea polyphenols, in particular, flavonoids.35 The presence of an aromatic ring and hydroxyl groups in the chemical structures of phenolic compounds are majorly associated with the antioxidant properties of tea polyphenols, which can neutralize and scavenge free radicals. Tea catechins like EGCG, EC, and ECG have been reported to exert potential antioxidant effects by donating an electron to free radicals and scavenging ROS such as O2•2, 1O2, and ROO•.49 The hydroxyl and carboxyl groups in tea polyphenols chelate with metals. The phenolic antioxidants in tea can inhibit lipid peroxidation by binding with lipid alkoxyl radicals, depending on the number of hydroxyl groups present in the molecular structures, specifically at the 30 and 40 positions in the B ring.35 Tea phenolic acids like gallic acid, exert numerous health benefits and antioxidant effects.6 The possible antioxidant mechanism of the major catechins in tea is presented in Fig. 34.2. Among the nonphenolic metabolites in tea, polysaccharides have been determined as potential antioxidants against •OH.50 The antioxidant capacity of tea polysaccharides is dependent on their molecular size, monosaccharide composition, and free radical type.51 The antioxidant action of polysaccharides is presented in Fig. 34.3. Caffeine, in appropriate concentrations in tea, exert antioxidant effects by quenching •OH production and other antioxidant mechanisms. The presence of amino acids and organic acids in tea not only contributes to the taste, but also relates potential health benefits. Amino acids have several reported biological

activities. The antioxidant activities of amino acids have been determined as inhibiting oxidative stress
induced interleukin 8 (IL-8) production and inactivating the nuclear factor-kappaB (NF-κB) in Caco-2 intestinal epithelial cells.29 The protective effects of amino acids against tissue oxidative stress are structure dependent.52 Organic acids, particularly ascorbic acid, have been reported to act as potent antioxidants by radical scavenging.5 Carotenoids exhibit antioxidant activities by quenching 1O2 and scavenging other ROS.23 Nevertheless, studies regarding the antioxidant mechanisms of nonphenolic metabolites in tea are scarce.

Conclusions Oxidative stress occurring due to the modulation of intracellular RS levels is considered as a major factor in several human pathogeneses. Natural dietary antioxidants have been reported to maintain intracellular, steady-state levels of RS and antioxidant systems. Antioxidant compounds act by free radical scavenging or by quenching their production and/or chelating activities to prevent oxidative damage. Therefore more research is necessary for understanding the specific underlying mechanisms responsible for oxidative stress
induced health issues. In contrast, the antioxidant reaction mechanism of dietary antioxidants in terms of both phenolic and nonphenolic metabolites also requires immense research to prevent oxidative stress
related pathogeneses. The preventive approaches of natural dietary antioxidants could replace synthetic drugs to prevent oxidative stress and to improve human health and well-being.

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364

34. Tea antioxidants in terms of phenolic and nonphenolic metabolites

FIGURE 34.2 Antioxidant mechanism of major phenolic compounds (catechins) in tea. Tea catechins containing an aromatic ring with hydroxyl groups in their chemical structures are positively correlated with their antioxidant actions.

Application in other areas of pathology In this chapter, the roles of RS in the damage of cellular components and their associated disorders are briefly described. Oxidative stress due to the excessive generation of RS, especially O2•2, •OH, 1O2, and H2O2 are positively correlated with increased risk of metabolic disorders. Actually, these RS have also been linked with other diseases including cancer,19 aging,14

cardiovascular disorders,15 infectious diseases,16 and neurodegenerative disorders.4 Natural dietary antioxidants, especially tea bioactive compounds, have been shown to act as strong antioxidants to prevent oxidative stress in relation to their associated disorders. Tea phenolic antioxidant metabolites have an aromatic ring and hydroxyl groups in their chemical structures to neutralize or scavenge free radicals49 as well as being capable of binding with

II. Antioxidants and Pathology

References

365

FIGURE 34.3 Antioxidant properties of major nonphenolic compounds (polysaccharides) in tea. The antioxidant action of polysaccharides is mainly dependent on their molecular weight, conjugation forms, and synergistic characteristics.

lipid alkoxyl radicals to inhibit lipid peroxidation.35 Besides phenolic metabolites, nonphenolic tea metabolites have also been associated with strong antioxidant activities. The antioxidant activities of major nonphenolic tea metabolites such as polysaccharides are mainly dependent on their molecular size, structure, and free radical type.51

Summary points • This chapter summarized oxidative stress
induced human pathogeneses and the antioxidative roles of natural dietary compounds. • Reactive species induce oxidative stress in a biological system.

• Oxidative stress is positively correlated with several pathophysiological complications. • Oxidative stress impairs cellular components and their functions. • Natural dietary antioxidants have the potential to prevent oxidative stress. • Tea metabolites are predominate dietary antioxidants against oxidative stress. • Tea antioxidants include both phenolic and nonphenolic metabolites. • Tea antioxidants are prospective for human well-being.

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48. 19. Matsui A, Ikeda T, Enomoto K, Hosoda K, Nakashima H, Omae K, et al. Increased formation of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, in human breast cancer tissue and its relationship to GSTP1 and COMT genotypes. Cancer Lett 2000;151:87
95. 20. Noda N, Wakasugi H. Cancer and oxidative stress. J Jpn Med Assoc 2001;44:535
9. 21. Gordon MH. Significance of dietary antioxidants for health. Int J Mol Sci 2012;13:173
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24. Gundogdu M, Ozrenk K, Ercisli S, Kan T, Kodad O, Hegedus A. Organic acids, sugars, vitamin C content and some pomological characteristics of eleven hawthorn species (Crataegus spp.) from Turkey. Biol Res 2014;47:21
6. 25. Keenan EK, Finnie MDA, Jones PS, Rogers PJ, Priestley C. How much theanine in a cup of tea? Effects of tea type and method of preparation. Food Chem 2011;125:588
94. 26. Thippeswamy R, Mallikarjun KG, Gouda M, Rao DH, Martin A, Gowda LR. Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection. J Agric Food Chem 2006;54:7014
19. 27. Wang J, Hu S, Nie S, Yu Q, Xie M. Reviews on mechanisms of in vitro antioxidant activity of polysaccharides. Oxid Med Cell Longev 2016;64:1
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320. 29. Son DO, Satsu H, Shimizu M. Histidine inhibits oxidative stressand TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells. FEBS Lett 2005;579:4671
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C H A P T E R

35 Thymoquinone: the active compound of black seed (Nigella sativa) Hatice Gu¨l Anlar1 and Merve Bacanli2 1

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Zonguldak Bulent Ecevit University, Zonguldak, Turkey 2Department of Pharmaceutical Toxicology, Gu¨lhane Faculty of Pharmacy, University of Health Sciences, Ankara, Turkey

List of abbreviations ALT APAP AST ATP Bax Bcl BHT CAT CdCl4 CPK CYP DEP EAE GABA Gpx GR GSH GSSG GST HbA1c HO-1 IC50 Ig IL LDH LTC4 MAPK MDA MBC MIC MPO MRP MS MTT NA NAC

alanine aminotransferase acetaminophen aspartate aminotransferase adenosine triphosphate Bcl-2-associated X protein B-cell lymphoma butylated hydroxytoluene catalase cadmium tetrachloride creatine phosphokinase cytochrome diesel exhaust particle allergic encephalomyelitis γ-aminobutyric acid glutathione peroxidase glutathione reductase glutathione oxidized glutathione glutathione-S-transferase glycated hemoglobin heme oxygenase-1 the half maximal inhibitory concentration immunoglobulin interleukin lactate dehydrogenase leukotriene C4. mitogen-activated protein kinase malondialdehyde Minimum bactericidal concentration minimum inhibitory concentrations myeloperoxidase multidrug related protein metabolic syndrome 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nicotinamide N-acetylcysteine

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00035-4

NF-κB NO Nrf2 OTA OVA PEFR PGs PI PPAR-γ QR SEM SiHa SOD STAT3 STZ TBARS TNF-α TQ

nuclear factor kappa B nitric oxide NF-E2-related factor-2 organic anion and cation transporters ovalbumin peak expiratory flow rate prostaglandins pulmonary index peroxisome proliferator-activated receptor gamma quinone reductase standard error of the mean squamous carcinoma cells superoxide dismutase signal transducer and activator of transcription 3 streptozotocin thiobarbituric acid reactive substances tumor necrosis factor alpha thymoquinone

Introduction Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4benzoquinone) is found in the oils and extracts obtained from Nigella sativa L. (Ranunculaceae) (Fig. 35.1),

FIGURE 35.1 Nigella sativa plant, its seeds, and the chemical structure of thymoquinone. The figure shows the chemical structure of thymoquinone.

369

© 2020 Elsevier Inc. All rights reserved.

370

35. Thymoquinone: the active compound of black seed (Nigella sativa)

also known as black cumin and black seed, which has been cultivated and used all over the world for many centuries.1,2 It has been used as a folk remedy for the prevention and/or therapy of pulmonary diseases like coughs, bronchitis, asthma, and chest congestion and for gastrointestinal diseases, diabetes, infection, and inflammation.3

proinflammatory cytokines and mediators, nuclear factor kappa B (NF-κB), the signal transducer and activator of transcription 3 (STAT3), peroxisome proliferatoractivated receptor gamma (PPAR-γ), and mitogenactivated protein kinase (MAPK).7

Antioxidant activity Bioavailability and kinetics It is known that TQ has a slower absorption rate when given orally to rabbits compared to intravenous administration. It showed linear kinetics following intravenous administration and it has rapid elimination.4 Numerous studies have shown that TQ and N. sativa interfere with drug metabolizing enzymes and, hence, result in alteration of the blood levels of certain drugs when taken together. TQ is a potent inhibitor of cytochrome (CYP)1A2 and CYP3A4 while it induces the activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR) in rabbits.5 TQ also inhibits various CYP isoenzymes in human liver microsomes such as CYP1A2, CYP3A4, CYP2C9, and CYP2D6.4 Moreover, TQ demonstrated a synergistic effect with glibenclamide on blood glucose levels by reducing the hepatic protein expressions of CYP2C11 and CYP3A2 enzymes.6

Thymoquinone and health TQ has been suggested to have biological properties including antioxidant, antibacterial, antiinflammatory, antidiabetic, anticancer, hepatoprotective, nephroprotective, and analgesic properties. Its antioxidant effects depend on its free radical and superoxide radical scavenging activities and also its preserving of the activities of various antioxidant enzymes (Fig. 35.2). It is also claimed that TQ affects tumor suppressors,

In in vitro and animal studies, N. sativa was claimed to exhibit strong antioxidant activity.8 Black seed powder supplementation antagonized hepatocarcinogeninduced oxidative stress by normalizing glutathione (GSH) and nitric oxide (NO) levels.9 It was demonstrated that TQ pretreatment decreased the levels of conjugated diene and malondialdehyde (MDA) and the activities of GPx, catalase (CAT), and superoxide dismutase (SOD) which increased by 1,2-dimethylhydrazine in the erythrocytes of male Wistar rats.10 TQ also effectively scavenged free radicals in collagen-induced arthritis in Wistar rats. 21 days of TQ (5 mg/kg bw/daily) treatment ameliorated the altered biochemical parameters such as GSH, CAT, SOD, myeloperoxidase (MPO), and inflammatory mediators like tumor necrosis factor alpha (TNF-α), interleukin (IL)1β, IL-6, and IL-10 (Fig. 35.3).11 Hosseinzadeh et al.12 evaluated the antioxidant effects of TQ and black seed oil in ischemiareperfusion injury and they found that pretreatment with TQ and black seed oil decreased the MDA levels, which were previously increased (Fig. 35.4).

Antidiabetic activity TQ treatment (3 mg/mL) reduced the serum glucose and MDA levels while increasing the serum insulin and SOD levels in streptozotocin (STZ)-induced diabetic rats. Similarly, other toxic effects related to FIGURE 35.2 Beneficial effects of thymoquinone on human health. The figure shows the beneficial effects of thymoquinone on human health.

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371

Anticancer activity Control

9

CIA

CIA+TQ

0.9

7

0.8

6 5 #

4 3 2

μ g GSH/g of tissue

8

#

0.7

*

0.6 0.5 0.4 0.3 0.2

1

0.1

0

0 Control

CIA

CIA+TQ

(C)

Control

CIA

CIA+TQ

(D) 12 10

Unit/mg protein

(B)

*

## 8

**

6 4 2 0 Control

CIA

CIA+TQ

μ mole of H2O2 consumed/min/mg protein

nmol of TBARS formed/hr/mg protein

(A)

20 16 ## 12

* 8 4 0 Control

CIA

CIA+TQ

FIGURE 35.3 Antioxidant effects of thymoquinone in collagen-induced arthritis in Wistar rats. The figure shows the antioxidant effects

MDA conc. (nmol/g tissue)

of thymoquinone (5 mg/kg bw) treatment on the level of enzymatic and nonenzymatic antioxidants in the joints of rats.11 (A) Lipid peroxidation, (B) GSH levels, (C) SOD activity, (D) CAT activity in the joints of rats immunized with collagen type II after treatment with thymoquinone. Data are expressed as mean 6 SEM of six rats. TP , .05 versus control, #P , .05, ##P , .01 versus collagen-induced arthritis (CIA) group.

Ischemia–reperfusion Sham Phenytoin (50 mg/kg) TQ 2.5 mg/kg TQ 5 mg/kg TQ 10 mg/kg

350 300 250 200 150 100

***

*** ***

***

50 0

FIGURE 35.4 Effect of thymoquinone on lipid peroxidation following global cerebral ischemia. The figure shows the beneficial effect of thymoquinone on MDA levels measured in 10% homogenates of hippocampus portion from rats subjected to 20 min of ischemia.12 Values are mean 6 SEM (n 5 10).  P , .001 as compared with vehicle (normal saline plus 0.8% Tween 80) treated animals.

diabetes like mitochondrial vacuolization and fragmentation, heterochromatin aggregates, and segregated nucleoli were ameliorated.13 In a similar study by Pari and Sankaranarayanan,14 oral administration of TQ at doses of 20, 40, 80 mg/ kg/bw for 45 days improved the glycemic status by increasing the insulin and hemoglobin and decreasing

the glucose and glycated hemoglobin (HbA1c) levels in STZ-nicotinamide (NA)-induced diabetic rats (Table 35.1). Additionally, TQ treatment restored the altered activities of carbohydrate metabolizing enzymes such as hexokinase, glucose 6-phosphatase, glucose-6-phosphate dehydrogenase, and fructose 1,6bisphosphatase in the liver of diabetic rats. N. sativa seeds (2 g/day) were used as adjuvant therapy in patients and the beneficial effects of the N. sativa seeds on the glycemic control of type 2 diabetes mellitus patients (n 5 94) were studied. It was demonstrated that this supplementation significantly reduced fasting blood glucose, blood glucose level 2 hours postprandially, and glycated hemoglobin (HbA1c) levels without significant changes in body weight. Furthermore, insulin resistance was significantly reduced while β-cell function was increased at 12 weeks of supplementation.15

Anticancer activity There are several studies showing the antiproliferative effects of TQ against colon, pancreatic, and bone cancer cells.16
18

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372 TABLE 35.1

35. Thymoquinone: the active compound of black seed (Nigella sativa)

Effects of thymoquinone on the level of plasma glucose and insulin in diabetic rats.

Groups

Plasma glucose (mg/dL)

Plasma insulin (μU/mL)

Normal control

a

93.36 6 7.15

16.98 6 1.30a

Normal and thymoquinone (80 mg/kg)

94.30 6 7.22a

17.44 6 1.34a

Diabetic control

283.17 6 21.68b

6.46 6 0.49b

Diabetic and thymoquinone (20 mg/kg)

226.18 6 17.31c

7.95 6 0.61c

Diabetic and thymoquinone (40 mg/kg)

163.32 6 12.57d

10.25 6 0.78d

Diabetic and thymoquinone (80 mg/kg)

110.24 6 8.31e,a

14.95 6 1.15e

The table shows the effects of thymoquinone treatment on the level of plasma glucose and insulin in diabetic rats and controls.14 Values are mean 6 SD for 6 rats in each group. a
e In each column, different superscript letters mean significant differences at P , .05.

The cytotoxic effects of TQ in squamous carcinoma (SiHa) cells were examined and compared to cisplatin. As a result of trypan blue dye exclusion and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests, it was observed that TQ has more cytotoxic activity than cisplatin against SiHa cells. Besides, it was less cytotoxic toward normal cells (3T3-L1 and Vero). In a cell cycle analysis, TQ exposure increased apoptosis via downregulation of the B-cell lymphoma (Bcl)-2 protein.19 The antiproliferative effects of TQ against breast cancer cells (BT-474, MCF-7, and MDA-MB-231) were determined by examination of cell viability, migration, and invasion. It was found that TQ significantly inhibited cell proliferation and its antiproliferative effect increased when it was combined with 5-fluorouracil and doxorubicin. Peroxisome proliferator-activated receptor gamma (PPAR-γ) mediates antitumor activity in several cancer types and TQ was able to increase PPAR-γ activity via the downregulation of related genes including survivin, Bcl-2, and Bcl-xL. It also induced apoptosis via the activation of caspase 7, 8, and 9 in a dose-dependent manner.20 In an animal study, 20-methylcholanthrene was used to induce fibrosarcoma in Swiss albino mice 1 week after the administration of TQ (0.01% in drinking water). It was observed that pretreatment with TQ remarkably reduced tumor incidence and tumor burden, delayed the onset of tumors, and reduced the mortality rate. TQ increased GSH, GST, and quinone reductase (QR) activities and decreased lipid peroxidation due to its antioxidant effects. Similarly, it decreased the viability of cancer cells with IC50 values of 15 μm.21 The chemosensitizing effects of TQ were evaluated in gastric cancer cells in in vivo and in vitro conditions. According to the results, TQ significantly increased the apoptotic effects induced by 5-fluorouracil (5-FU) and enhanced the 5-FU-induced killing activity by the activation of caspase-3 and caspase-9, mediating the

upregulation of the proapoptotic protein Bcl-2associated X protein (Bax), and the downregulation of antiapoptotic protein Bcl-2. Moreover, the combination of TQ and 5-FU had more efficacy than either agent alone in a xenograft tumor mouse model.22

Cardiovascular activity TQ may protect the cardiovascular system against diesel exhaust particle (DEP)
induced toxic effects. DEP caused lung inflammation and lung function impairment as well as increases in IL-6 levels, and decreases in systolic blood pressure, platelet numbers, and SOD activity, and pretreatment with TQ at a dose of 6 mg/kg ameliorated these effects.23

Effects on gastrointestinal system Ischemia-reperfusion-induced gastric lesions are known to be linked with free radical formation. ElAbhar et al. studied24 the gastroprotective effects of TQ using different doses ranging from 5 to 100 mg/kg and N. sativa oil at doses of 2.5 and 5 mL/kg in male Wistar rats, and they showed that both treatments possess gastroprotective effects against gastric lesions, and these effects were claimed to be related to the conservation of the gastric mucosal antioxidant/oxidant balance. Similarly, they reported that TQ corrected the altered oxidative stress parameters in a comparable manner to that of the reference drug, omeprazole, in animal pyloric ligation models. Besides its antioxidant properties, TQ might have a protective role against gastrointestinal disorders by its inhibition of acid secretion, proton pump, and neutrophil infiltration while enhancing nitric oxide production and mucin secretion.25 Both N. sativa oil and TQ had gastroprotective effects against alcohol-induced injury, which was revealed by the ulcer index values. They remarkably

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Nephroprotective activity

alleviated most of the biochemical adverse effects relating to oxidative stress of gastric ulcers such as increases in lipid peroxidation and decreases in gastric GSH levels and the enzymatic activities of GST and gastric SOD. But it was reported that TQ has a lesser gastroprotective effect than black seed.26 TQ had ameliorative effects on inflammatory bowel disease induced by dextran sodium sulfate. Colonic MPO activity was altered, MDA and GSH levels were regulated, and body weight loss and the appearance of diarrhea were reduced with TQ application at doses of 5, 10, and 25 mg/kg orally. These result emphasized that it could serve as a therapeutic agent for the treatment of inflammatory bowel disease.27 Helicobacter pylori is an important risk factor for chronic gastritis, peptic ulcer, and gastric carcinoma. ROS is suggested to be a major factor in H. pylori
induced gastric injury. In a study conducted with 88 adult patients having H. pylori infections, TQ exhibited clinically useful anti-H. pylori activity comparable to a triple therapy comprising of amoxicillin, omeprazole, and clarithromycin.28

Hepatoprotective effects In a study using Swiss albino male mice, the effects of TQ (10 μmol/L) against cadmium tetrachloride (CdCl4)
induced hepatotoxicity were evaluated. Oxidative enzymes like SOD and CAT, nonenzymatic antioxidants like GSH and nonprotein thiol, oxidative stress markers like protein carbonyl, and protein levels were determined and when compared with the CdCl4 group, it was found that pretreatment with TQ modulated these altered biomarkers. The results of this study strengthened the hypothesis that the beneficial effects of TQ are depend on its antioxidant activity.29 Acetaminophen (APAP) is frequently used for its analgesic and antipyretic properties, but it causes hepatic necrosis when it is taken in extremely high doses. N-acetylcysteine (NAC) has been used for the treatment of APAP-induced hepatotoxicity, but it has some side effects, especially allergic reactions. Therefore the treatment of APAP-induced hepatoxicity with natural antioxidants has gained attention. The cytoprotective effects of TQ against APAP-induced hepatotoxicity were investigated in Wistar albino rats, where 500 mg/kg APAP was given orally and after that TQ at a total dose of 15 mg/kg was administrated. TQ decreased the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and SOD as well as the levels of MDA and oxidized glutathione (GSSG), which were increased by APAP. In a histopathologic examination of the liver, morphological disorders such as necrotic hepatocytes and

cytoplasmic vacuolization were found less frequently in TQ-treated groups.30 In a similar study, the supplementation of TQ (2 mg/kg/day) for 5 days before APAP administration significantly decreased serum ALT, total nitrate/nitrite, and hepatic lipid peroxides, and increased adenosine triphosphate (ATP) and hepatic reduced GSH. These results confirmed the beneficial effects of TQ on APAP-induced hepatotoxicity possibly via increased resistance to oxidative and nitrosative stresses as well as improved mitochondrial energy production.31

Nephroprotective activity Cisplatin (cis-diamminedichloroplatinum II) and other platinum analogs have been widely used in the treatment of several cancers such as breast, testicular, lung, brain, and prostate. But the toxic effects of these drugs limit their usage and nephrotoxicity is one of their main undesired effects. The protective effects of TQ on cisplatin-induced renal toxicity were evaluated in male rats. TQ was administrated at a dose of 10 mg/kg bw in drinking water for 5 days to rats and it was reported that cisplatin caused increasing levels of the efflux transporter multidrug related protein (MRP) 2, MRP 4, MDA, and 8-isoprostane while decreasing the expression of tubular organic anion transporters (OAT) 1 and OAT 3, and organic cation transporters (OCT) 1 and OCT 2. TQ treatment reversed these changes.32 In another study by the same group, it was demonstrated that NF-κB, serum urea, and creatinine levels, which were increased by cisplatin, were significantly decreased by TQ (10 mg/kg bw). Also, it was found that TQ supplementation significantly ameliorated cisplatin nephrotoxicity via increasing heme oxygenase-1 (HO-1) and NF-E2related factor-2 (Nrf2) levels.33 TQ might have a protective effect against kidney toxicity caused by gentamicin as a result of its antioxidant, antiapoptotic, and antiinflammatory properties. Gentamicin (80 mg/kg ip) increased serum creatinine, blood urea nitrogen, thiobarbituric acid reactive substances (TBARS), and total nitrate/nitrite levels while it decreased the activities of GPx and CAT and the levels of ATP and GSH. TQ administration (50 mg/L) ameliorated these alterations. All of these results emphasize that TQ could have protective effects with antioxidant and free radical scavenging properties as well as preventive effects on energy decline against nephrotoxicity caused by gentamicin.34 Aycan et al.35 revealed that TQ (10 mg/kg) treatment also has a therapeutic effect on APAP-induced nephrotoxicity in rats. It ameliorated the blood urea and creatinine levels, serum NO activity, and tissue

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35. Thymoquinone: the active compound of black seed (Nigella sativa)

FIGURE 35.5 Effects of thymoquinone on the APAP-induced nephrotoxicity. Histopathological view of rat kidney tissue.35 (A) Normal morphology of rat kidney tubules in control group, (B) similar to normal morphology of rat kidney tubules in TQ group (C) grade 3 damage of rat tubules epithelial in APAP group (arrows) (D) grade 1 damage of rat tubules epithelial in APAP 1 TQ group (arrows).

MDA levels. In addition to this, it restored kidney tissue damage (Fig. 35.5).

Effects on pulmonary system Asthma is a chronic respiratory allergic disease with a high prevalence. Current treatments for asthma have some limitations and, therefore, researchers have begun focusing on herbal treatments for the disease. It was suggested that regulatory T cells and immunosuppressive cytokines like IL-10 have a role in the pathogenesis of asthma and the efficacy of immunotherapy. In a study conducted to determine the effects of black cumin powder, which contains TQ, on the pathogenesis and controlling of asthma in mild asthmatic children, it was observed that the powder could increase the efficacy of specific immunotherapy.36 Similarly, beneficial effects of N. sativa oil on the wheeze associated with lower respiratory tract illness were demonstrated in a study with 84 patients via the determination of the pulmonary index (PI) and peak expiratory flow rate (PEFR). TQ is claimed to be responsible for these effects due to its bronchodilatory and antiinflammatory activities and its high content in the plant oil.37

Effects on the nervous system Gilhotra and Dhingra38 explored the effects of TQ on NO and γ-aminobutyric acid (GABA) pathways, which play an important role in anxiety, in male Swiss albino mice. The animals were divided into two groups (unstressed and stressed), TQ at doses of 10 and 20 mg/kg was given, and behavioral testing using an elevated plus maze, light/dark test, and social

interaction test was done. TQ at both doses exhibited significant anxiolytic activity effects without altering nitrite levels, but the higher dose (20 mg/kg) of TQ increased the GABA content in unstressed mice. However, in unstressed mice, only the higher dose (20 mg/kg) produced significant antianxiety activity with a significant decrease in plasma nitrite and reversal of the decreased brain GABA content. Methyleneblue pretreatment enhanced these effects, which strengthened the involvement of NO and GABA pathways in the antianxiety effects of TQ.

Antiinflammatory activity Inflammation is one of the main characteristics of many diseases. Oxidative stress and infections activate the expression of inflammatory genes, which result in the promotion of a cascade of inflammatory mediators such as cytokines, eicosanoids, and lytic enzymes. Therefore the prevention of the inflammatory process could be a promising treatment for many disorders. N. sativa with its main active compound TQ have exerted antiinflammatory effects via the inhibition of inflammatory cytokines in osteoporosis due to its antiinflammatory and antioxidant properties.39 The effects of TQ on leukotriene formation, which are important mediators in asthma and inflammatory processes, were studied in human blood cells. TQ significantly inhibited eicosanoid generation through the inhibition of both lipoxygenase and leukotriene-4 synthase pathways in a concentration and time-dependent manner (Fig. 35.6).40 In animal studies, TQ inhibited the production of 5lipoxygenase products and 5-hydroxyeicosatetraenoic acid with IC50 values of 0.26 6 0.02 and 0.36 6

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Applications in other areas of pathology

FIGURE 35.6 Effect of thymoquinone on leukotriene C4 synthase activity in human platelets. The figure shows time-course of the effect of thymoquinone on leukotriene C4 synthase activity in human platelet cells.40 LTC4; leukotriene C4. Error bars indicate standard error of the mean.  Significantly different from the control, P , .05.

0.02 μg/mL respectively from the polymorphonuclear leukocytes of rats. These effects are considered to be due to the antioxidative action of TQ.41 Likewise, crude fixed oil of N. sativa and TQ inhibited eicosanoid synthesis by interrupting the cyclooxygenase and 5lipoxygenase pathways of the arachidonate metabolism in rat peritoneal leukocytes in a dose-dependent manner.42 These effects were confirmed by another similar study and support the traditional use of N. sativa and its derived products as a treatment for rheumatism and related inflammatory diseases.43 Mezayen et al.44 studied the potential antiinflammatory effects of TQ on allergic airway inflammation. Since prostaglandins (PGs), the proinflammatory mediators derived from arachidonic acid metabolism by COXs, play an important role in modulating inflammatory and allergic immune responses, the effects of TQ on PGs were investigated in a BALB/c mouse model. Ovalbumin (OVA; 20 g) was used for the sensitization and the mice were treated with TQ (3 mg/kg ip) for 5 days after the sensitization. The results of this study demonstrated that TQ mitigated OVA-induced airway inflammation by inhibiting COX-2 expression and PGD2 synthesis. Also, it remarkably decreased lung eosinophilia, goblet cell hyperplasia, Th2 cytokines, and immunoglobulin (Ig) production. Possible alleviating effects of TQ and N. sativa on food allergies were studied in OVA-sensitized BALB/c mice. These animals were pretreated with N. sativa and TQ and subsequently challenged intragastrically with OVA. It was demonstrated that both treatments significantly alleviated the symptoms and immune biomarkers in OVA-induced allergic diarrhea by decreasing plasma mouse mast cell protease-1 and intestinal mast cell numbers.45

375

Inflammation is also a significant factor in the progression of solid tumor malignancies. TQ activated apoptosis and inhibited pancreatic ductal adenocarcinoma cell proliferation by reducing the expression of TNF-α, IL-1β, and Cox-2.46 The role of oxidative stress in the onset and progression of allergic encephalomyelitis (EAE) has been previously described. Mohamed et al.47 investigated the antiinflammatory effects of TQ in EAE, which is an autoimmune demyelinating disease of the central nervous system, and it is widely accepted as an animal model for human multiple sclerosis. They induced EAE using myelin basic protein emulsified with complete Freund’s adjuvant in female Lewis rats. TQ (1 mg/kg) was given to the rats after the EAE was induced and it caused no perivascular inflammation and increases in GSH levels, which were decreased before. These results suggest that TQ may have a role in the treatment of multiple sclerosis due to its antioxidant properties. N. sativa oil was reported as a potent adjuvant for the treatment of allergic diseases such as bronchial asthma, allergic rhinitis, and atopic and hand eczema in randomized, controlled, and double-blinded clinical trials. TQ was mentioned in these studies as a responsible active component of the plant.48,49

Applications in other areas of pathology In a study, N. sativa oil (50
400 mg/kg po) suppressed the nociceptive response in tail-pinch acetic acid
induced writhing, hot-plate and in the early phase of formalin tests in a dose dependent manner. TQ (2.5
10 mg/kg po, 1
6 mg/kg ip, and 1
4 μg/ mouse iv) reduced the nociceptive response both in the early phase and in the late phase of a formalin test. It was claimed that the antinociceptive effects were produced through indirect activation of the κ-opioid receptor and supraspinal μ1 subtypes.50 Al-Rasheed et al.51 examined the ameliorative effects of N. sativa and garlic against fructose-induced metabolic syndrome (MS) in albino male rats. MS was induced by the administration of fructose as a 10% solution in drinking water for 8 weeks and rats were divided into five groups, namely control, MS, MS 1 N. sativa (200 mg/day), MS 1 garlic (250 mg/kg/day), and MS 1 N. sativa (200 mg/day) 1 garlic(250 mg/kg/day). After the 8 weeks of treatment, blood glucose, biochemical parameters in serum, and liver tissues were analyzed. In the MS group, significantly increased body and liver weight, fasting blood glucose, serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels with significantly decreased high-density lipoprotein cholesterol and the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and CAT

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35. Thymoquinone: the active compound of black seed (Nigella sativa)

were observed compared to the control group. The administration of N. sativa or garlic modulated all these altered parameters and might be useful in MS therapy. In a study evaluating the effects of TQ on methotrexate-induced testicular toxicity in male C567BL/ 6 mice, it was reported that methotrexate caused significant oxidative damage. Also, in the histopathological examination, edema, severe disruption in the seminiferous epithelium, interstitial space dilatation, and reduced diameter of seminiferous tubules were observed in the methotrexate group. TQ (10 mg/kg/day ip) treatment for 4 days ameliorated these unwanted changes.52 N. sativa seeds have been used for the treatment of infertility in traditional medicine and these effects were confirmed by investigation in 80 infertile Iranian men. N. sativa oil (5 mL) was given to patients and it was shown that sperm count, motility and morphology, semen volume, pH, and round cells were improved significantly without any adverse effects. It was claimed that the antioxidant compounds of N. sativa such as TQ, vitamin E, selenium, and unsaturated fatty acid are responsible for this effect of the plant.53

Toxic effects of thymoquinone The lethal oral and intraperitoneal doses of TQ were 104.7 and 870.9 mg/kg in mice respectively. These values are much higher than the doses required for its antiinflammatory, antioxidative, and anticarcinogenic effects.54,55 In subchronic toxicity studies, Bamosa et al.19 reported no toxicity after subacute oral administration of TQ (90 mg/kg) in mice for 90 days. Badary et al.56 reported that acute administration of TQ (2 and 3 g/kg) resulted in hypoactivity and difficulty in respiration. 24 hours after, GSH levels were significantly decreased while the plasma urea and creatinine levels, and ALT, lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) enzyme activities were significantly increased. They showed that TQ (0.01%, 0.02%, and 0.03% in drinking water) caused no mortality or signs of toxicity like changes of toxicological significance in body and organ weights, food and water intake, or urine and feces output, tissue GSH, plasma concentrations of urea, creatinine and triglycerides, and enzyme activities of ALT, LDH, and CPK in the subchronic toxicity study. There was no gross or microscopic tissue damage in the histological examination, however, it produced a significant decrease in fasting plasma glucose levels. Based on these results, TQ has been considered relatively less toxic than butylated hydroxytoluene (BHT), a well-known antioxidant; only its hypoglycemic effects must be considered, especially in diabetic patients and pregnant women.

Conclusion The use of herbal medicine is gaining popularity worldwide, therefore, research interest in these compounds has become important. TQ, the most active compound of N. sativa, has remarkable pharmacological properties against a large variety of diseases such as cancer, diabetes, antiinflammatory disorders, and infertility. Although it was found to be relatively safe, it also caused drug interactions and hypoglycemia. Further preclinical and clinical research is needed to confirm the beneficial effects of TQ and to gain insight into the mechanisms underlying the effects of TQ on human health.

Summary points • This chapter focuses on the health-beneficial effects of thymoquinone, which is a phenolic compound found in the extract and oil of N. sativa. • It has been suggested that ROS play an important role in the pathogenesis of a large variety of diseases. • It has been shown that thymoquinone has important pharmacological properties like antioxidant, antiinflammatory, and anticancer activities. • Promising results support its application in the treatment of important diseases. • However, the data show that thymoquinone also causes some side effects and drug interactions. • Therefore further studies are needed to gain insight into its beneficial health effects.

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C H A P T E R

36 Yacon (Smallanthus sonchifolius) use as an antioxidant in diabetes Ana Paula Costa Rodrigues Ferraz1, Je´ssica Leite Garcia1, Mariane Ro´vero Costa1, Carol Cristina Va´gula de Almeida1, Cristina Schimitt Gregolin2, Pedro Henrique Rizzi Alves3, Fabiana Kurokawa Hasimoto3, Carolina B. Berchieri-Ronchi1, Klinsmann Carolo dos Santos4 and Camila Renata Correˆa1 1

2

Department of Pathology, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil, Medical School, Mato Grosso State University (UFMT), Sinop, MT, Brazil, 3Institute of Bioscience, Medical School, Sa˜o Paulo State University (UNESP), Botucatu, SP, Brazil, 4Clinical Research Centre (CRC), Lund University Diabetes Centre, Lund University, Malmo¨, Sweden

List of abbreviations AGE CAT eNOS FOS GSH-Px MRSA PPARS ROS SCFA’s SLs SOD T1DM T2DM

advanced glycation end products catalase endothelial nitric oxide synthase fructooligosaccharides glutathione peroxidase methicillin-resistant Staphylococcus aureus peroxisome proliferator-activated receptors reactive oxygen species short-chain fatty acids sesquiterpene lactones superoxide dismutase type 1 diabetes mellitus type 2 diabetes mellitus

Introduction: from oxidative stress to antioxidants acting on diabetes Diabetes mellitus is a complex chronic disease characterized by elevated levels of blood glucose and deficient production and/or action of insulin, which is a polypeptide hormone produced by pancreatic β-cells (islets of Langerhans) with anabolic effects in various tissues, participating on glycogen, triacylglycerol, and protein metabolism.1 Several factors are involved in

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00036-6

this pathology, although nutritional and autoimmunity are the main predictors of its physiopathogenesis.2 The major metabolic abnormalities that occur in type 1 and 2 diabetes are hyperglycemia, hypertriglyceridemia, and ketoacidosis, which occur during insulin deprivation and affect the liver, and skeletal muscle and adipose tissues.3 Deregulated insulin signaling leads to an excessive utilization of free fatty acids as an energy source in cardiac and skeletal muscle tissues, which could lead to a process called lipotoxicity and, consequently, oxidative stress.4 Dysmetabolism may be linked to increased production of free radicals, which could lead to oxidative damage of DNA, proteins, and lipids as well as the activation of stress-sensitive pathways and the development of stress under the diabetic condition.5 Alternative therapies based on herbal/medicinal plants have been shown to possess some contribution toward reducing diabetes-related oxidative stress damages to underlying pharmacological and molecular pathways based on their phytochemical profile by ameliorating the redox state balance.6 Specifically, several studies have shown the antioxidant activity of natural compounds suggesting strong hydrogen- and electron-donating capacities due to their antioxidant potential in combination with

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36. Yacon (Smallanthus sonchifolius) use as an antioxidant in diabetes

reactive species scavenging, enzymatic activation, or inhibition of diabetic conditions.7 Among these medicinal plants, yacon or Smallanthus sonchifolius is considered a functional food due to the bioactive compounds present in its roots and antioxidants in its leaves.8 Based on this information, this chapter aims to elucidate the effects of S. sonchifolius on diabetic-related complications based on its phytochemical profile.

Antioxidants and diabetes mellitus Scientific evidence shows around 800 medicinal plants and more than 200 bioactive compounds identified as therapeutic alternatives for diabetes treatment.9 Medicinal/herbal plants and diet-based polyphenol-rich foods play a vital role in the production of the antioxidant defense system by providing essential antioxidant compounds such as vitamin E, C, and β-carotene, phenols including flavonoids, and essential minerals that form important antioxidant enzymes. Diet also has an important effect in the oxidation process by affecting the substrates that are subject to oxidation.10 Studies in humans have demonstrated the beneficial effects of various antioxidants in diabetic-related complications. Several antioxidant compounds from herbal medicine are involved in various diabetic pathological processes6 such as hyperglycemia,11 inflammation,12 insulin depletion,13 and vascular damages.14

Antioxidant activity in hyperglycemia and cardiomyopathy Hyperglycemia occurring in diabetes mellitus is caused by increased levels of glucose hepatic production and diminished peripheral glucose utilization by peripheral tissues (muscle and adipose tissue), which in turn lead to increased breakdown of structural proteins and lipolysis, leading to weight loss and higher circulating lipid levels.15 Due to diminished glucose uptake, fatty acids are used as the major energy source. The lipolysis process involves the hydrolysis of triacylglycerol stored in adipose tissue to glycerol and fatty acids. As a result, fatty acids are released into the circulation, thus, increasing their availability as an energy source and resulting in their excessive oxidation in cardiac tissue and skeletal muscle.16 Additionally, the increased oxidation of fatty acid leads to decreased ATP production in the mitochondria since this kind of energy is mainly provided from glucose oxidation. Moreover, this process is an initial unfavorable energetic state associated with the overproduction of reactive oxygen species (ROS). Lipid energy

metabolism is activated by a superfamily of nuclear ligand-activated transcription factors known as peroxisome proliferator-activated receptors (PPARs), which regulate the genes commonly involved in lipid utilization, lipoprotein metabolism, and insulin action.17 Antioxidants can act as PPAR-α and PPAR-γ agonists, providing lower fatty acids oxidation and ROS production.18 Kaviarasan and Pugalendi19 showed that the flavonoid-rich fraction from Spermacoce hispida seed promoted the upregulation of PPARα gene expression, which results in fatty acid catabolism and alleviates liver and kidney damages. Also, Zheng et al.20 demonstrated the antidiabetic activity of flavonoids present in Selaginella tamariscina (Beauv.) through increased PPAR-γ expression in adipose tissue and increased protein expression of IRS-1 in hepatic and skeletal muscle tissues. Oxidative stress, among other mechanisms, is shown as an onset to diabetes complications. It is known that in cardiovascular conditions excessive reactive species such as hydroxyl radicals (•OH), superoxide radicals (•O22 ), and peroxides (H2O2) are produced continuously by NADPH oxidases, xanthine oxidases, mitochondrial enzymes, and dysfunctional endothelial nitric oxide synthase (eNOS).21 To counteract these free radicals and to avoid these reactions, endogenous antioxidant enzymes (e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) are activated, which in diabetic patients is clearly reduced.22 Maritim et al.23 demonstrated that antioxidant compounds such α-lipoic acid and vitamin C and E have the capability to increase intracellular glutathione levels and restore SOD contents in cardiac tissue after 14 days of treatment. ROS production can also mediate several molecular pathways such as NF-κB activation, which increases inflammatory mediators and stimulates other free radicals production.24 In hyperglycemia, free radicals overproduction can activate polyol, hexosamine, protein kinase C, and AGE pathways, which are involved in structural damage.25 In general terms, antioxidants prevent oxidant-induced cell damages by reducing ROS generation via several mechanisms.26

Smallanthus sonchifolius: origins and ethnobotanical characteristics Yacon or S. sonchifolius of the Asteraceae family has some common names in the Andean, Aymara, and Quechua languages (Yacu and Unu meaning “water” as well as Yakku meaning “watery,” Llaqon, Llacum, Llacuma, or Yacumpi, the Quechua words that evolved into “Yacon,” Aricoma, Aricuma, Jicama, Chicama, Shicama, Jiquima, Jiquimilla (Ecuador), Poirre De Terre (French), and Yacon Strawberry (English)). It is an indigean

II. Antioxidants and Pathology

The nutritional characteristics and phytochemical profile of Smallanthus sonchifolius

perennial herbaceous plant native to the Andes mountains27 with botanical expansion in regions of Venezuela to the northwest of Argentina as well as western Europe, New Zealand, Japan, the United States, the Czech Republic, and the State of Sa˜o Paulo.28 In 1845, it was described for the first time as Polymnia sonchifolia Poepp. by Eduard Friedrich Poeppig. Thereafter in 1978, Harold Ernest Robinson established the Smallanthus genre by separating Polymnia into two different species, namely Smallanthus and Polymnia, which presents the name Polymnia edulis Wedd. as its botanical synonym published in 1857.29 This plant is a perennial herb (Fig. 36.1), 1.5
3 m tall that is composed of an aerial system with lower ovate and hastate leaves, upper ovate-lanceolate leaves, and connate and auriculate leaves at the base (Fig. 36.1A). The flower of the yacon plant is yellow to bright orange and inflorescences that are terminal with 1
5 axes, each one with 3 capitula (Fig. 36.1B), and, finally, with tuberous roots and short rhizomes system composed of 4
20 tuberous storage brown, pink, purplish, or cream or ivory white roots (growing 50
100 cm in height) (Fig. 36.1C).28 The organization of the vascular system of the yacon is typical of roots since they are subterranean bodies of caulinar nature, formed by short, thick sympodial rhizomes or “corona.” It adapts to a wide range of soils, although it grows better in rich, moderatelydeep to deep soils that are well-structured and drained, most composed in lateritic soils adjusted by dolomitic limestone and can tolerate a wide pH range from acid to weak alkaline.30

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The S. sonchifolius cultivate has several advantages in that it presents a defense system in its leaves and glands, making insect attacks and access to the plant difficult and contributing to cultivations without the use of pesticides and with lower chemistry contaminations.31

The nutritional characteristics and phytochemical profile of Smallanthus sonchifolius The S. sonchifolius cultivation has nutritional purposes due to its consumption as tea of its leaves, raw and organic extracts, and its in -peeled and fresh in natura or cooked roots, crisp form and juicer32 with lower energy content (619
937 kJ/kg of fresh matter) provided by its 70% water’s composition.33 It has a sweet taste due to the presence of fructose.32 Fructose is derived from fructan, which consists of any carbohydrate with one or more fructosyl-fructose linked with osidic bonds; it could be linear or branched fructose (oligo) polymers such inulins or fructooligosaccharides.34 The higher proportion of nutrients in the tuberous roots of S. sonchifolius regards in the special attention to inulin and fructooligosaccharides (FOS) mentioned previously.35 The main fructooligosaccharides including kestose and nystose present in S. sonchifolius are represented in Fig. 36.2. Inulin-type fructans are found in garlic, leek, bananas,36 and in some Northeast Brazilian species.37 These show around 1
4 g (US) and 3
11 g (Europe)

FIGURE 36.1 Smallanthus sonchifolious. Botanical specimens of yacon. (A) Yacon leaves; (B) yacon flowers; (C) yacon root system.

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36. Yacon (Smallanthus sonchifolius) use as an antioxidant in diabetes

TABLE 36.1

FIGURE 36.2 Fructooligosaccharides. Kestose and nystose.

Yacon roots and its nutritional composition. Fresh weight basis

Dry weight basis

Water (%)

93
70




Ash (%)

0.3
2.0

1.1
6.7

Protein (%)

0.4
2.0

1.3
7.3

Fat (%)

0.1
0.3

0.4
1.0

Fiber (%)

0.3
1.7

1.0
5.7

Calcium (mg/g)

23




Phosphorus (mg/g)

21




Iron (mg/g)

0.3




Retinol (mg/g)

10




Carotene (mg/g)

0.08




Thiamin (mg/g)

0.01




Riboflavin (mg/g)

0.1




Niacin (mg/g)

0.33




Ascorbic acid (mg/g)

13




Nutritional composition of Yacon roots.

FIGURE 36.3 Inulin structure in yacon. Inulin’ structure representation. N 5 B35.

average daily consumptions and are used as sugar substitutes in miscellaneous products.38 The S. sonchifolius roots accumulate around 60% (on dry basis) of insulin type β (2-1) fructans, mainly oligomers (GF2
GF16) (Fig. 36.3).32 The main contents of carbohydrates in S. sonchifolius roots are fructose (350 mg/g DW), glucose

(158 mg/g DW), sucrose (74 mg/g DW), and lowpolymerization degree (DP) oligosaccharides (GF2
9 around 201 mg/g),39 which oscillate during the growing cycle and harvest.40 Additionally, S. sonchifolius holds other types of nutrients such proteins (1.3%
7.3% DW), fat (0.4%
1.0% DW), and fiber (1.0%
5.7% DW), which are represented in Table 36.1. The large dark green leaves and tubers of S. sonchifolius include the polyphenols group demonstrated in Fig. 36.4A
F including chlorogenic acid, caffeic acid, quercetin, protocatechuic acid, p-coumaric acid, and ferulic acid as major antioxidant compounds (Fig. 36.4).41 Other polyphenols from S. sonchifolius leaves that are bioactive compounds are in the form of polyphenol ions of saturated and polyunsaturated fatty acids such as dihexose and derivatives of palmitic, oleic, and linoleic acids34 together with sesquiterpene lactones42 and essential oils such as β-pinene, β-caryophyllene, and γ-cadinene.43 The different techniques as well as types of solvents used in S. sonchifolius extract preparation are divergent in literature. For instance, Dos Santos et al.44 show quercetin as a major compound found in S. sonchifolius leaves using 70% ethanol as a solvent.44 Genta et al.45 formulated a butanolic S. sonchifolius extract that demonstrated the presence of caffeic acid and chlorogenic acid. These report data may explain how the preparation of the extract can affect the concentration and biologically active principle of the polyphenolics profile.

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Smallanthus sonchifolius nutrients and its interaction in diabetes

383

FIGURE 36.4 Polyphenols profile in yacon. Polyphenols in Smallanthus sonchifolious. (A) Chlorogenic acid, (B) caffeic acid, (C) quercetin, (D) protocatechuic acid, (E) p-coumaric acid, and (F) ferulic acid.

Smallanthus sonchifolius nutrients and its interaction in diabetes Several studies have evaluated the effect of the crude extract of S. sonchifolius leaves and roots, obtained in different ways, on the glycemia of diabetic animals. Important data report that a significant decrease in postprandial blood glucose levels can be explained by the stimulation of pancreatic mechanisms, the regeneration or protection of β-cells that were partially destroyed with the use of streptozotocin for the induction of experimental type 1 diabetes mellitus (T1DM), the potentiation of insulin secretion, and the possible increase in peripheral glucose utilization.46
48 Based on these studies, the antioxidant and antidiabetogenic effects of S. sonchifolius on β-pancreatic cells is attributed. In other studies, it was verified that treatment with hydroethanolic extract of S. sonchifolius leaves promoted an improvement in the glycemic and lipid profiles, an increase in the activity of antioxidant enzymes in the skeletal44 and cardiac muscles, and improvement of the alterations related to diabetic cardiomyopathy (fibrosis and cellular disorganization) and of the Langerhans islet architecture and function.49 Additionally, its shows an antioxidant potential in literature.50 Hydroethanolic extract of S. sonchifolius leaves upregulated the glycemic condition, reducing blood glucose levels and, consequently, insulin resistance.51 The hypoglycemic effect may be due to its phytochemical profile, since the caffeic acid is associated with

reducing blood glucose through glycogenesis modulation.52 Whereas chlorogenic acid (CGA) could modulate the antioxidant status and lipid and glucose metabolism.53 It is known that the number of hydroxyl groups (OH) present (trihydroxy phenolic acids, dihydroxy (catechol), and mono-hydroxyl) in phenols structure comprise the antioxidant properties. Based on this knowledge, chlorogenic acid and its metabolites present in S. sonchifolius leaves are possibly associated with catechol groups, which, in turn, can diminish the production of IL-8 though inhibiting the activation of this cytokine and PKD-IKK-NFkB signaling. Additionally, this compound could decrease the mRNA expression of macrophage inflammatory protein 2 (MIP-2, a mouse homolog of IL-8).54 The main mechanism for the rapid glucose regulation in diabetes as well as the lower levels of triglycerides from chlorogenic acid improvement could be associated with the prevention of glucose-6phosphatase translocase 1 and glucose-6-phosphatase (provides glucose starvation) activities in the liver and small intestine. In particular, this compound can enhance phosphorylation, adiponectin (a glucose hormone regulator) and adiponectin receptors of AMP-activated protein kinase (AMPK).55 Therapeutic strategies such as S. sonchifolius use lead to glycemic homeostasis and reduce the metabolic shift generated in the diabetic condition, favoring the use of glucose and decreasing the oxidation of fatty acids and, consequently, improving the recovery of cardiac and muscle tissues.

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36. Yacon (Smallanthus sonchifolius) use as an antioxidant in diabetes

Applications in other areas of pathology S. sonchifolius could be considered a food with multiple functions and effects on the human body as well as a high source of functional ingredients to the food industry. It has others types of applications due to its prebiotic,56 antibacterial,57 and antimicrobial58 effects. The most interesting additional application of S. sonchifolius is that its prebiotic effect from inulintypes fructans and FOS has interesting nutritional and function actions on the gastrointestinal tract as these substances are resistant to the hydrolytic activity in the stomach they go through a fermentation process in the large bowel, thus, increasing bacterial populations and, consequently, fecal mass, thereby reducing disease risk development.34 Campos et al.56 reported that an S. sonchifolius flour diet promoted colon heath benefits improving short-chain fatty acids (SCFAs) in a male guinea pigs model. Specifically, a study by Reina et al.59 showed that S. sonchifolius fermentation improves a progressive drop in pH, enhancing Leuconostoc species production in the intestinal tract.59 In other hand, de Andrade et al.60 isolated sesquiterpene lactones (SLs) from a dichloromethane extract of S. sonchifolius and reported antibacterial proprieties against S. aureus and others Gram-negative populations.60 Corroborating with this study, Joung et al.57 reported various types of S. sonchifolius leaf extracts against six strains of methicillin-resistant S. aureus (MRSA).57 For these reasons, S. sonchifolius seems to be a plant prebiotic through its nondigestible oligosaccharides and against Staphylococcus aureus as an antimicrobial effect, promising great perspectives in biotechnology, clinical trials, and practice.

Summary points • Deregulated metabolism, also referred as dysmetabolism, which occurs in diabetes promotes the overproduction of free radicals as well as the depletion of the endogenous antioxidant system, which, in turn, leads to oxidative damage in different components of cellular and structural compartments. • Therapy approaches aiming to attenuate/alleviate oxidative stress have been shown to be an efficient strategy in promoting the regulation of metabolism under the diabetic condition. • The use of herbal/medicinal plants for the treatment of many chronic diseases such as diabetes and its complications has been recognized by a number of scientists and physicians based on their therapeutic properties.

• Phytochemicals from herbal medicines, specially antioxidants, play an important role in the antioxidant/oxidative balance in the diabetic condition. • Yacon, a perennial plant, rich in phenolic compounds, possess antioxidant and antiinflammatory properties. • Evidences have emerged regarding hypo- and antihyperglycemic effects of different yacon leaf and root extracts. • The potential therapeutic effects of yacon could contribute to the detection of new targets and treatments for diabetes and its complications.

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9. 59. Reina LD, Pe´rez-Dı´az IM, Breidt F, Azcarate-Peril MA, Medina E, Butz N. Characterization of the microbial diversity in yacon spontaneous fermentation at 20 C. Int J Food Microbiol. 2015;16 (203):35
40. 60. de Andrade EF, Carpine´ D, Dagostin JLA, Barison A, Ru¨diger AL, de Mun˜iz GIB. Identification and antimicrobial activity of the sesquiterpene lactone mixture extracted from Smallanthus sonchifolius dried leaves. Eur Food Res Technol. 2017;243(12).

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37 Screening procedures and tests for antioxidants Jan Borlinghaus1,2, Jana Reiter1,2, Michael Ries1,2 and Martin C.H. Gruhlke1 1

Department of Plant Physiology, Worringer Weg 1, Aachen, Germany 2LumiBioSciences, c/o Worringer Weg 1, Aachen, Germany

List of abbreviations ABTS DCPIP DHA DHAR ε MTT OS PES PMS PPP ROS RSS

2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) 2,6-dichlorophenol indophenol dehydroascorbate dehydroascorbate reductase molar extinction coefficient 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide oxidative stress phenazine ethosulfate phenazine methosulfate pentose-phosphate pathway reactive oxygen species reactive sulfur species

Introduction Antioxidants and oxidative stress Oxidative stress (OS) is a major factor related to the development of severe diseases like cancer, neurodegeneration, diabetes, and cardiovascular diseases.1,2 Also, the aging process is closely linked with OS and subsequent cellular redox events.3 OS was initially defined by Sies as a disequilibrium between pro- and antioxidants in favor of prooxidants.4 Both intrinsic factors and extrinsic factors can trigger OS, for example, by the formation of reactive oxygen species (ROS). ROS are a primary source of OS and are generated during mitochondrial oxidative metabolism as well as in response to xenobiotics or the invasion of pathogens.5 However, subsequently (or in some cases independently of ROS), other reactive species like reactive nitrogen species (RNS) or reactive sulfur species (RSS) are produced during OS.6,7 In some cases, RNS, RSS, or other reactive species are “oxidants of the second generation” that are formed due to the reaction of ROS Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00037-8

with other molecules. ROS are chemically seen as a heterogenous group and include both radicals (such as superoxide or hydroxyl radicals) and nonradical compounds (such as hydrogen peroxide). Hence the reactivity of ROS, or in general reactive species, are different. While many ROS or RNS attack a broad spectrum of biomolecules from DNA to lipids of the membrane, RSS, for instance, are fairly specific to thiol groups or sulfenic acids (Gruhlke & Slusarenko, 2012). This makes RSS, in contrast to other reactive species, quite special. However, especially in the case of disease, RSS seem mainly to be “oxidants of the second generation,” which are formed as a consequence of reactions with other reactive species such as ROS. It is not only true for humans, but also for other organisms such as plants, that RSS are important oxidants of the second generation.8 However, since OS has been identified as a dramatic risk factor for health, an important strategy is lowering the OS degree by adding antioxidants that counteract OS.9,10 This is a prominent strategy in pharmacy. A prominent strategy is, hence, the scavenging of reactive species like ROS that either cause or are associated with a certain disease using compounds that easily react with them. This strategy is called antioxidant therapy.11,12 The estimation of antioxidative capacity is hard since there are various in vitro tests, but biological relevance (due to the question of bioavailability) is not necessarily given.13,14 Also, for cosmetics, this approach is of increasing interest since OS is strongly related to aging. Therefore one common strategy in cosmetics is to apply antioxidants to the skin to at least delay the consequences of aging.15,16 Therefore in this chapter, some present tests are presented and a view into the future regarding how the testing of antioxidants should further develop is given.

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Oxidative stress and pathology Many organ systems exhibit pathological processes and develop diseases in cases where the equilibrium between pro- and antioxidants is disturbed. Severe problems with the heart and lungs such as cardiovascular diseases (CVDs) and chronic obstructive pulmonary disease (COPD), neurogenerative diseases, diabetes, and cancer are examples of the plethora of diseases linked to OS.17 In particular, diseases commonly correlated with aging like CVDs or neurodegenerative diseases are associated with inflammatory events and mediated by the secretion of different interleukins and cellular senescence.18 With increasing age, the ability of the cell to defend against oxidative stress (e.g. by glutathione peroxidases or superoxide dismutases) decreases, which in turn leads to an increase in the measurable oxidative stress in cells.19 Theoretically, the concept of applying external antioxidants that are able to scavenge reactive species makes absolute sense. Since OS leads to inflammatory processes (e.g., release of interleukins and enhanced expression of NF-κB),20 a supply of alternative antioxidants might counteract these processes.21,22 Nonetheless, the problem of the bioavailability of these antioxidants remains an important issue. A clear correlation is given between OS and diabetes-related complications. OS promotes prothrombic reactions and cardiovascular problems in type II diabetes.23 OS has also been shown to be a central part in the pathophysiology of dementia.24 The application of natural products with either direct antioxidants or that induce cellular antioxidative defense mechanisms could, therefore, be a strategy to act against these kinds of diseases.25

Classification of antioxidants Due to their chemical behavior, the major concept antioxidants can be classified into different classes. The easiest classification is antioxidants, sensu strictu. That means, these are compounds that counteract directly against oxidants, for example, by the reduction of oxidants. The most important example for such molecules is ascorbic acid (vitamin C), which can react with prooxidants like hydrogen peroxide in a direct manner. An electron is transferred from the reduced vitamin C to hydrogen peroxide under the formation of water and dehydroascorbate (DHA).26 This, however, can be regenerated by an enzymatic system, where electrons (ultimately from the primary metabolism) are transferred to DHA to re-reduce this compound as a recycling step. Antioxidants, strictly seen, are direct antagonists of oxidants and detoxify them by a reaction with them.

Since these direct antioxidants are consumed by their reaction with prooxidants, a recycling step is needed, as already described, for the ascorbate/DHA. An enzyme, DHA reductase (DHAR), reduces DHA to ascorbate. The electrons that are needed for this reaction are supplied by reduced glutathione (GSH). Glutathione, a tripeptide containing cysteine, is one of the major electron donors within the cell. Besides for the reduction of DHA, glutathione provides further electrons for the reduction of oxidized protein cysteine residues by the enzyme glutaredoxin (Grx). A complementary system to re-reduce oxidized cysteine residues of proteins is the thioredoxin (Trx) system; the electrons for this enzyme system are provided by NADPH. NADPH, however, is also an electron donor for the enzyme reducing oxidized glutathione (GSSG) to its reduced form GSH by an enzyme called glutathione reductase. NADPH is an electron donor with a very low reduction potential and a direct link to the primary metabolism [NADPH is, for instance, produced in the pentose-phosphate pathway (PPP)].27,28 Chemically in the same line, but from a biological point of view, different are reductants acting as antioxidants. Reductants are compounds that reduce oxidized targets of the cell. For chemists, these are clearly reducing compounds, glutathione can act as such since it is able to react with disulfides in a thiol
disulfide exchange reaction, while a direct reaction of glutathione and hydrogen peroxide is very slow. Therefore the chemical function of glutathione as an electron donor clearly prevails its role as a direct antioxidant. Besides chemicals that are able to reduce oxidatively damaged biomolecules, many publications also list enzymes that contribute to this repair process (like the already mentioned Trx and Grx) as reductants. A last class of antioxidants are inducers of antioxidative defense. Counterintuitively, these compounds are in many cases prooxidants that react with, for example, transcription factors that contribute to the antioxidant defense system. A prominent example of such transcriptional regulatory systems is the Keap/ Nrf2 system in mammalian cells. Electrophilic compounds, for example, are able to react with cysteine residues of the KEAP1 protein, leading to the detachment from the NRF2 protein and subsequent activation of the transcriptional regulator system29,30.

The problem of screening for antioxidants There are many methods that are used to screen for antioxidants in different manners.31
36 Since antioxidants, as seen, are not a “unique” class of chemical compounds, the problem arises of how to screen for such compounds and to evaluate both the chemical

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and biological activities as antioxidant. The chemical and biological modes of action are so diverse that it is impossible to use a single screening method. Furthermore, screening for antioxidants is strongly correlated with the category that a given compound belongs to. An antioxidant, sensu strictu, can definitely not be screened with the same procedure as an inducer of antioxidative defense. An inducer of oxidative defense is active on the biological level and cannot be screened with chemical in vitro methods. The chemistry involved and the reaction mode are far too different. Therefore screening for antioxidants cannot be seen in one view and needs to be seen in respect of the particular type of antioxidant being screened. Especially for antioxidants, in the strict sense, a couple of different in vitro assays are used. These assays rely on the principle that the potential antioxidant directly interacts with the prooxidant (e.g., a radical), leading to a delayed or lowered reaction with an indicator (e.g., oxidation of fluorescein; oxidized fluorescein has weak fluorescence) for the oxidative activity of the prooxidant. In principle this can give an idea of the antioxidative capacity of a certain compound, but it has no statement regarding the biological relevance of this finding. A compound, for instance, that is, not taken up by the cell or immediately metabolized cannot serve as an antioxidant in vivo, even though the in vitro assay suggests it. Hence such findings need to be examined carefully in view of the physiological situation in the cell or the total organism.13,34 Therefore there is a need for a test system in vivo that also takes physiological parameters into account. Nevertheless, a cell-based system also always has the drawback that the situation in a total, multicellular organism is different to that in a single cell system. If an antioxidant is taken up with nutrients, this might lead to a totally different situation compared to a labbased system, where a compound is directly applied to cells and taken up directly from the medium. Such a cell assay can never reflect the modifications to a particular antioxidant due to digestions. Therefore it is necessary to exercise caution regarding the strength of a statement on the basis of a screening. The finding that a compound has an in vitro antioxidative capacity does not mean that is has the same activity in vivo. And a cell-based in vivo assay does not guarantee that the same is true for the total organism.

Testing for antioxidants One of the best-known antioxidant screening procedures is the oxygen radical absorbance capacity (ORAC) assay. This test is based on the idea that an oxygen radical generating compound (2,20 -azobis(2-

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amidinopropan) dihydrochloride) produces ROS, which are able to oxidize fluorescein. This is a highly fluorescing compound, while its oxidation product has a very weak fluorescence. Due to this fact, the fluorescence of the solution decreases with time since the ROS oxidize the fluorescein. If an antioxidant is added to the test, the antioxidant reacts with the ROS, which, therefore, delays the oxidation of fluorescein until the antioxidant is used. To calibrate the system, mostly Trolox, a synthetic vitamin E analogue, is used as a reference compound with known antioxidative activity in this assay. With this it is possible to express the antioxidative capacity in Trolox equivalents as the unit37 (Fig. 37.1). Another prominent assay used to determine the antioxidative capacity of compounds (mainly from food) is the 2,20 -azino-bis (3-ethylbenzothiazoline6-sulfonic acid) assay, known as the ABTS assay. The basis of this assay is that the radical form that a compound has, in contrast to its nonradical form, shows a strong absorption at 420 nm light (ε 5 3.6 3 104 M21 cm21).38 ABTS is mainly used to follow the enzyme reactions of hydrogen peroxide
consuming enzymes such as laccases or peroxidases. Hydrogen peroxide is in these cases added to, for example,. copper ions, which undergo Fenton chemistry to produce free radicals. Also, radical generators such as persulfate may act as a source of radicals. The radical form of the ABTS absorbs light in the area of blue light and this radical form of the ABTS is created by the radicals in the reaction in the manner of a chain reaction. Similar to ORAC, a reaction of the antioxidant with the free radical delays the formation of the ABTS-radical until the antioxidant is consumed. Again, this assay can also be calibrated with a standard antioxidant (Trolox, for instance)39 (Fig. 37.2).

Testing for electron donors As stated previously, there are various enzyme cascades depending on electron donors. Finally, all electron donors are somehow connected to the primary metabolism of the cell. NADPH is an electron donor with a very low reduction potential. The NADPH/ NADP1 redox couple is linked via thioredoxins to the protein thiol/disulfide redox system and via glutathione reductase to the GSH/GSSG redox couple. Glutathione, however, is linked via the Grx system to protein thiols and via dehydroascorbate reductase (DHR) to the ascorbate/DHA redox couple. To test the activity of antioxidants, in many cases, the redox state (ratio of the amount of reduced to oxidized compound) of these are tested. This is an indirect method to check the effect of a potential antioxidant in the cell.

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FIGURE 37.1 Principle of oxygen radical absorbance capacity (ORAC) assay. AAPH is a radical generator, leading to the formation of ROS. Fluorescein is used as an indicator, since it is a strongly fluorescent compound, whereas its oxidized form has very weak fluorescent properties. If an added compound is active as an antioxidant, the compound reacts with the ROS formed and, therefore, delays the reaction of ROS with fluorescein, as seen in the schematic graph.

Principle of 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Also, here a radical generating system is used that produces ROS oxidizing ABTS to a blue-colored product. Since an antioxidant delays the reaction, the effect of the antioxidant is compared to an untreated (no antioxidant added) control.

FIGURE 37.2

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Introduction

NADPH is, due to its chemical and structural similarity to NADH, not easy to quantify. There are some enzymatic assays that can be used to measure NADPH with quite high sensitivity40 (Fig. 37.3). On the other hand, glutathione is the central hub of the redox network of the cell. Therefore the glutathione redox-buffer system is routinely used to monitor the oxidative status of cells. Assuming an equilibrium between all redox compounds (NADPH, protein thiols, glutathione, etc.,), which is due to kinetic factors not totally given, glutathione is the central hub of the cellular redox environment. Hence there are some routine methods to measure glutathione in cells. Similar to the NADPH cycling method mentioned previously, there is also a glutathione cycling assay using glutathione reductase as the enzyme and NADPH as the electron delivering co-substrate. The basis of this assay is the reduction of the noncolored disulfide, dithionitrobenzoic acid (DTNB), by GSH in a thiol
disulfide exchange reaction. The monomer (thionitrobenzoic acid; TNB) is yellow and absorbs light at a maximum of 412 nm. To measure total glutathione, a cell lysate is used. In the first step, GSH reduces DTNB to TNB, while GSH is oxidized to GSSG. From then on, this GSSG is a substrate of GR and the increase of TNB-mediated absorption is dependent on the concentration of substrate GSSG. Since this can be calibrated with known concentrations of GSSG, this can be used to quantify glutathione. This, however, measures only total glutathione. To distinguish between oxidized and reduced glutathione, GSH is derivatized with 2-vinylpyridine (2-VP). The remaining glutathione is oxidized glutathione and GSH is then calculated by subtracting the GSSG concentration from the total glutathione41 (Fig. 37.4).

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New strategies to measure antioxidants Biological relevance is always a big issue when testing antioxidants. Not only a given antioxidant on its own, but other compounds, for example, in food, can also influence the uptake and, hence, the bioavailability of antioxidants.42 These effects are hard to notice in a cell culture
based assay since they are relevant for total organisms. Furthermore, one has to differentiate between “bioavailability” and “bioeffectivity.” A compound that is taken up by an organism or cell is not necessarily effective as an antioxidant.43 This cannot be reflected in an in vitro test at all and needs, at least, cellular assays. Since many currently available tests are in vitro methods, these assays are not able to include aspects like uptake in the cell or metabolization. Therefore new strategies to measure antioxidants will be necessary. There are many possibilities to quantify OS in cells, from different dyes and reagents to sophisticated genetically-encoded sensors like roGFP. Using these methods in cells having constitutive OS upon exposure to an (potential) antioxidant might be a promising strategy to estimate the activity as an antioxidant in the biological context of the cell. Although many cancer cell lines exhibit constitutively enhanced OS levels, these cells also have other physiological alterations that might influence assay results. Thus other cell lines need to be considered and engineered for a defined level of OS in cells combined with an optimally endogenous detecting system for OS. A further factor that needs to be considered is the increasing automatization of lab technology, which needs to be reflected in newly developed assays.44 If an assay is not usable in automized facilities, it cannot

FIGURE 37.3 Some examples of enzymatic assays to determine the NADPH pool in cells. All of these assays rely on the regeneration of NADP1 to NADPH using 6-phosphoglycerate dehydrogenase. Data according to.40 DCPIP, 2,6-Dichlorophenol indophenol; MTT, phenazine ethosulfate; PES, phenazine ethosulfate; PMS, phenazine methosulfate.

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37. Screening procedures and tests for antioxidants

FIGURE 37.4 Principle of glutathione reductase recycling assay. Total glutathione is measured by the absorbance of thionitrobenzoic acid upon reduction by GSH. GSH is oxidized to GSSG and this again is recycled by glutathione reductase. Since the reaction rate depends on the substrate concentration (GSSG), the increase in yellow color is directly proportional to the concentration of glutathione. Oxidized glutathione is measured by adding 2-vinylpyridine, which reacts with GSH (but not GSSG) in the initial step of the reaction. Thus only GSSG will undergo cycling and be measured.

be used in high-throughput screenings for new antioxidants or for tests of the effectivity of certain products, for example, pharmaceutics or cosmetics. Taken together, there is quite a broad toolkit for in vitro testing.45 This is useful to check for the chemical activity of an antioxidant; however, for biological evaluation, there is a need for new screening strategies that also reflect the aspect of the bioavailability of antioxidants.

Summary points • Antioxidants are a diverse group of compounds counteracting oxidative stress. • Some so-called antioxidants interact directly with prooxidants, while others induce the intrinsic antioxidant defense of the cell. • Different in vitro and in vivo methods exist to quantify antioxidants. • Since the uptake and metabolization of potential antioxidants is not taken into account, in vitro tests should be examined carefully. • New methods for antioxidant measurement are needed.

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38 Recommended resources for pathology: oxidative stress and dietary antioxidants Rajkumar Rajendram1,2, Vinood B. Patel3 and Victor R. Preedy2 1

2

College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Department of Nutrition and Dietetics, School of Life Course Sciences, King’s College London, London, United Kingdom 3School of Life Sciences, University of Westminster, London, United Kingdom

List of abbreviations DNA OS ROS WHO

Deoxyribonucleic acid oxidative stress reactive oxygen species World Health Organisation

Introduction Metabolic energy production generates highly toxic reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hypochlorous acid (HClO), and free radicals (e.g., hydroxyl radical and superoxide anions).1,2 These oxidants initiate toxic chain reactions (e.g., lipid peroxidation) or oxidize nucleic acids and proteins.1,2 Mutations and even cancer may occur if damage to DNA is not repaired.1,2 Damage to proteins results in their degradation and the inhibition of their function.1,2 Oxidative stress (OS) has been implicated in a variety of diseases including perioperative conditions, Alzheimer’s disease, Parkinson’s disease, diabetes mellitus, reflux esophagitis, hypertension, prostatitis, as well as other conditions described elsewhere in this book.1
6 Arguably, antioxidants may ameliorate some TABLE 38.1

of these conditions or cellular pathways and processes contained therein. These antioxidants and therapeutic agents are extensively described in this book. However, it is also important to point out that in many diseases, a reduced appetite associated with disease may also result in OS. This is because, in simple terms, there is a reduced intake of dietary antioxidants. Malabsorption and maldigestion in some conditions affecting the gastrointestinal tract will also cause OS. It is now difficult even for experienced scientists to remain up to date. To assist colleagues who are interested in understanding more about this field, several tables have, therefore, been produced containing upto-date resources in this chapter. The experts who assisted with the compilation of these tables of resources are acknowledged.

Resources Tables 38.1
38.4 list the most up-to-date information on the regulatory bodies and professional societies (Table 38.1), journals (Table 38.2), books (Table 38.3), and online resources (Table 38.4) that are relevant to

Regulatory bodies, professional societies, and organizations.

American Botanical Council (ABC) cms.herbalgram.org/herbstream/library/homePage American Herbalists Guild www.americanherbalistsguild.com American Herbal Products Association www.ahpa.org (Continued)

Pathology. DOI: https://doi.org/10.1016/B978-0-12-815972-9.00038-X

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38. Recommended resources for pathology: oxidative stress and dietary antioxidants

(Continued)

American Society for Investigative Pathology asip.org Antioxidants.org www.antioxidants.org/ginkgo-biloba Arizona Society of Pathologists www.azpath.org Boston University School of Medicine www.bumc.bu.edu/gms/pibs/faculty-research-areas/physiology-pathophysiology/ Chiro.org Chiropractic Resource Organization chiro.org/wordpress/tag/antioxidants/ College of American Pathologists www.cap.org Dietitians in Integrative and Functional Medicine (DIFM) practice group through the Academy of Nutrition integrativerd.org European Society of Pathology www.esp-pathology.org Free Radicals Chemistry Club orgsync.com Institute for Functional Medicine www.functionalmedicine.org International Anesthesia Research Society (IARS) iars.org/about-iars International Society of Antioxidant in Nutrition and Health www.isanh.com Israel Society for Oxygen and Free Radical Research isofrr.net.technion.ac.il/ Japanese Society of Pathology pathology.or.jp/about_jsp/about-jsp.html Johns Hopkins University ehe.jhu.edu/research/research-areas/toxicology-physiology-and-cell-biology.html McGill University Department of Pathology www.mcgill.ca/pathology/about/definition Montana Society of Pathologists - Montana Medical Association mmaoffice.org/societies/montana-society-of-pathologists National Center for Complementary and Integrative Health nccih.nih.gov National Institute of Health www.nih.gov National Institutes of Health (NIH) Office of Dietary Supplements ods.od.nih.gov Natural Medicines Database (formerly Natural Standard and Natural Medicines Comprehensive Database) naturalmedicines. therapeuticresearch.com Natural Products Association www.npainfo.org Nutrition International www.nutritionintl.org Office of Dietary Supplements ods.od.nih.gov/Health_Information/Health_Information.Aspx Oxygen Club of California www.oxyclubcalifornia.org Pathological Society of Great Britain & Ireland www.pathsoc.org Public Pathology
Australia publicpathology.org.au Royal College of Pathologists regulatory landscape for pathology services www.rcpath.org/discover-pathology/news/the-regulatorylandscape-for-pathology-services.html Royal DSM www.dsm.com/corporate/home.html Stanford Medicine med.stanford.edu/mcp.html Science Nordic sciencenordic.com SCS global services www.scsglobalservices.com/services/antioxidants Society for Redox Biology and Medicine sfrbm.org Society for Free Radical Research Asia sfrrj.umin.jp/asia Society for Free Radical Research Australasia SFRR(A) www.sfrr-australasia.org/ (Continued)

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Resources

TABLE 38.1

399

(Continued)

Society for Free Radical Research - Europe (SFRR-E) www.sfrr-europe.org/ Society for Free Radical Research International www.sfrr.org Society for Redox Biology and Medicine sfrbm.org Society of Toxicologic Pathology www.toxpath.org Society of Toxicology www.toxicology.org South west London pathology www.swlpath.nhs.uk University Magna Graecia of Catanzaro, Italy web.unicz.it/it University Tor Vergata of Rome, Italy web.uniroma2.it WHO www.who.int Wings for Life International www.wingsforlife.com/en This table lists the regulatory bodies, professional societies, and organizations involved with pathology, oxidative stress, and dietary antioxidants.

TABLE 38.2

Journals relevant to pathology: oxidative stress and dietary antioxidants.

PLoS One Oxidative Medicine and Cellular Longevity International Journal of Molecular Sciences Free Radical Biology and Medicine Scientific Reports Biomedicine and Pharmacotherapy Nutrients Redox Biology Molecular Medicine Reports Antioxidants and Redox Signaling Biochemical and Biophysical Research Communications Biomed Research International Molecular Neurobiology Oncotarget Life Sciences European Journal of Pharmacology Chemico Biological Interactions Advances in Experimental Medicine and Biology Molecules Current Pharmaceutical Design Food and Chemical Toxicology Cellular Physiology and Biochemistry Free Radical Research Journal of Ethnopharmacology Food and Function International Immunopharmacology Frontiers in Physiology Frontiers in Pharmacology Journal of Biological Chemistry Biological Trace Element Research Journals publishing original research and review articles related to pathology, oxidative stress, and dietary antioxidants. The top 30 journals that have published the highest number of articles over the past 5 years are listed. Although Scopus was used to generate this list, other databases or the use of refined search terms will produce different results.

III. Techniques and Resources

400 TABLE 38.3

38. Recommended resources for pathology: oxidative stress and dietary antioxidants

Relevant books.

Acai: An Extraordinary Antioxidant Rich Palm Fruit from The Amazon. Schauss AG. Biosocial Publications, 2008. Antioxidants in Health and Disease. Zampelas A, Micha R. CRC Press, 2015. Antioxidants in Sport Nutrition. Lamprecht M. CRC Press/Taylor & Francis, 2015. Bioactive Natural Products: Chemistry and Biology. Brahmachari G. John Wiley & Sons, 2015. Bioactive Nutraceuticals and Dietary Supplements in Neurological and Brain Disease Prevention and Therapy. Watson RR, Preedy VR. Academic Press, 2015. Biochemistry of Oxidative Stress. Gelpi RJ, Boveris A, Poderoso JJ. Springer International Publishing, 2016. Cancer and Chemoprevention: An Overview. Rashid S. Springer, 2017. Food Antioxidants. Hudson BJ. Springer, 1990. Free Radicals and Antioxidant Protocols. Uppu RM, Murthy SN, Pryor WA, Parinandi NL. Humana Press, 2010. Free Radicals in Biology. Pryor WA. Academic Press, 1974. Free Radicals in Biology and Medicine. Halliwell B, Gutteridge J. Oxford University Press, 2007. Free Radicals in Human Health and Disease. Rani V, Yadav U. Springer, 2015 ¨ zben T. Springer, 1998. Free Radicals, Oxidative Stress, and Antioxidants. O Free Radicals: The Secret Anarchy of Science. Brooks M, Abrams HN. 2013 Human Diseases: Systemic Approach (8th edition). Zelman M. Prentice Hall, Inc. 2015 Inflammation, Aging, and Oxidative Stress. Bondy SC, Campbell A. Springer International Publishing, 2016. McKee’s Pathology of the Skin (5th Edition). Calonje JE, Brenn T, Lazar AJ, Billings S. Elsevier, 2019. Molecular Pathology and Diagnostics of Cancer. Coppola D. Springer, 2014. Neurodegeneration and Alzheimer’s disease: The role of Diabetes, Genetics, Hormones, and Lifestyle. Martins RN, Brennan CS, Fernando B, Brennan M, Fuller SJ. Wiley, 2019. Nutraceuticals efficacy, safety and toxicity. Gupta RC. Elsevier, 2016. Oxidative Stress and Nanotechnology: Methods and Protocols. Armstrong D, Bharali DJ. Humana Press, 2013. Oxidative Stress in Cancer Biology and Therapy (Oxidative Stress in Applied Basic Research and Clinical Practice). Spitz DR, Dornfeld KJ, Krishnan K, Gius D. Humana Press, 2012. Oxidative Stress in Human Reproduction Shedding Light on a Complicated Phenomenon. Agarwal A, Sharma R, Gupta S, Harlev A, Ahmad G, du Plessis SS, Esteves SC, Wang SW, Durairajanayagam D, Springer International Publishing, 2017. Oxidative Stress and Redox Regulation. Jakob U, Reichmann D. Springer Netherlands, 2013. Oxidative Stress and Cardiorespiratory Function. Pokorski M. Springer International Publishing, 2015. Progress in Brain Research-Natural compounds and retinal ganglion cell neuroprotection. Morrone LA, Rombola` L, Corasaniti MT, Bagetta G, Nucci C, Russo R. Elsevier, 2015. Robbins & Cotran Pathologic Basis of Disease (9th edition). Kumar V, Abbas AK, Aster JC. Elsevier, 2014. Systems Biology of Free Radicals and Antioxidants. Laher I. Springer, 2014. Tropical Fruits: From Cultivation to Consumption and Health Benefits, Fruits from the Amazon. Todorov SD, Pieri FA. Food Science and Technology, 2018. Underwood’s Pathology: a Clinical Approach (7th Edition). Cross S. Elsevier, 2018 This table lists books on pathology, oxidative stress, and dietary antioxidants.

III. Techniques and Resources

References

TABLE 38.4

401

Relevant online resources, information, and emerging technologies.

Abcam www.abcam.com/primary-antibodies/next-generation-ihc-techniques Adolfo Lutz Institute www.ial.sp.gov.br/ial/publicacoes/livros/metodos-fisico-quimicos-para-analise-de-alimentos Bio-Rad www.bio-rad.com/en-id/product/qx200-droplet-digital-pcr-system?ID 5 MPOQQE4VY PREMIER Biosoft www.premierbiosoft.com/tech_notes/tissue-microarray.html Scielo www.scielo.com.br Science Direct www.sciencedirect.com This table lists some internet resources, information, and emerging technologies relevant to pathology, oxidative stress, and dietary antioxidants. Sites listed in Table 38.1 may also have tools or resources within them.

an evidence-based approach to pathology, OS, and dietary antioxidants.

Acknowledgments The authors would like to thank the listed authors for contributing to the development of this resource. Abenavoli L, Al-Azzawi M, Ameer K, Brennan M, De Lorenzo A, De Santis GL, Ekici Gu¨nay N, Gualtieri P, Pala D, Rezazadeh K, Romano L, Sadhukhan P, Sil PC.

Summary points • Oxidative stress is involved in the pathogenesis of disease. • These include, for example, perioperative conditions, Alzheimer’s disease, Parkinson’s disease, diabetes mellitus, reflux esophagitis, hypertension, and prostatitis. • Reduced appetite or malabsorption associated with these diseases may also result in oxidative stress. • Other conditions associated with oxidative stress are also described in this book. • Dietary antioxidants have the putative potential to ameliorate some of the pathways, processes, and conditions associated with oxidative stress.

• This chapter lists resources relating to the regulatory and professional bodies, societies, journals, books, and websites that are relevant to an understanding of oxidative stress and antioxidants.

References 1. Vertuani S, Angusti A, Manfredini S. The antioxidants and proantioxidants network: an overview. Curr Pharm Des 2004;10: 1677
94. 2. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44
84. 3. Stevens JL, Feelisch M, Martin DS. Perioperative oxidative stress: the unseen enemy. Anesth Analg 2019 Oct 9. Available from: https://doi.org/10.1213/ANE.0000000000004455 [Epub ahead of print]. 4. Deng Y, Pan L, Qian W. Associations between the severity of reflux esophagitis in children and changes in oxidative stress, serum inflammation, vasoactive intestinal peptide and motilin. Exp Ther Med 2019 Nov;18(5):3509
13. 5. Mikhael M, Makar C, Wissa A, Le T, Eghbali M, Umar S. Oxidative stress and its implications in the right ventricular remodeling secondary to pulmonary hypertension. Front Physiol 2019 Sep 24;10:1233. 6. Ihsan AU, Khan FU, Khongorzul P, Ahmad KA, Naveed M, Yasmeen S, et al. Role of oxidative stress in pathology of chronic prostatitis/chronic pelvic pain syndrome and male infertility and antioxidants function in ameliorating oxidative stress. Biomed Pharmacother 2018 Oct;106:714
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Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Abdominal aortic constriction (AAC) model of cardiac hypertrophy, 216 Ac¸aı´ (Euterpe oleracea Martius), 127
128, 132t animal model assays, 130 applications, 131 flavonoids, 128
129 human studies, 130
131, 131f polyphenols, 128 in vitro bioassays, 129
130 Acceptable daily intake (ADI), 345
346 Acetaldehyde, 18 Acetaminophen (APAP), 373 Acetaminophen-induced hepatotoxicity, 283 Acetate-induced colitis, 285t Acetate/malonate pathway, 298 Acetate pathway, 290 Acetic acid-induced colitis, 283 Acetyl salicylic acid, 269t Acetylcholinesterase (AChE) inhibitors, 135
136 Acne vulgaris, 219t Activated macrophages, 190
191 Acute cholestasis, 251f Acute exercise, 284 Acute inflammation, 198
199 Acute tubular necrosis, 282
283 Adequate sexual and erectile functionality, 19
20 Adipokines, 297
298 Adipose tissue, 300
301 Advanced glycation end-products (AGEs), 29, 30f, 32
33, 40, 42
43, 43f, 191
192 Advanced lipoxidation end-products (ALE), 40 African pear. See Dacryodes edulis Agaricus bisporus, 290, 291t, 292 Aging, 150t, 153
154 epigallocatechin gallate (EGCG), 220 tea, 359 Albendazole, 328 Alcoholic beverages, 255 Alcoholic liver disease (ALD), 130 definition, 260 epidemiology, 260
261 etiopathogenesis, 260, 261f malnutrition, 262 treatment, 261
262 Alcoholic steatohepatitis (ASH), 260 Alcohol-induced injury, thymoquinone, 372
373 Alkaloids, 206t

Alkoxyl radicals, 5t, 6 Allergic encephalomyelitis (EAE), 375 Allopurinol, 342 Allyl sulfides, 84 α-amylase, 207
208, 329
330 α-glucosidase, 207
208, 330 Alpha-lipoic acid (α-LA), 72t, 215 Alpha-tocopherol, 257, 314, 314t α-tocopherol equivalents (TE), 313
314 Alzheimer’s disease (AD), 123
124, 201, 216
219, 217t, 235, 272 ascorbic acid, 160 bilberry anthocyanins, 184
185 cause of, 135
136 characterization, 135 galanthamine, 138
139, 138f Ginkgo biloba (GB) extract, 241, 242f Nrf2, 80
81 pathogenesis of, 135 pharmacotherapy of, 138
139, 138f Amaryllidaceae alkaloid (AA), 136 antioxidant/neuroprotector effects of, 141t applications, 142 biosynthetic pathway of, 139, 140f in oxidative stress and neuronal cell damage, 139
141 types of, 139, 140f Amino acids, 88, 363 Amoxicillin, 373 Amyloid-β, 201 Amyotrophic lateral sclerosis (ALS) ascorbic acid, 161 Ginkgo biloba (GB) extract, 243 Anaerobic exercise, 284 Angiogenesis, CUR, 201 Animal bone disease models, anthocyanins, 149
155 aging model, 153
154 healthy animals, 154
155 hormone deficiency model, 149
153 inflammatory bone disease model, 153 Animal models Ac¸aı´ (Euterpe oleracea Martius), 130 mushrooms, antioxidants of, 294 PON1, 98
100 Anthocyanin-rich foods, 148t, 153 animal studies, 150t cell studies, 148t human studies, 154t Anthocyanins, 130, 180, 180f, 316 animal bone disease models, 149
155 aging model, 153
154

403

healthy animals, 154
155 hormone deficiency model, 149
153 inflammatory bone disease model, 153 applications, 156 bilberry anticancer activity, 181
182 cell studies, 147
149 anthocyanin compounds, 148t, 149, 150t berry extracts, 147, 148t, 150t black rice extract, 147, 148t dried plum, 147
149, 148t, 150t, 153 Clitoria ternatea, 190 content in consumed foods, 147t daily intake of, 179 dietary as antioxidants, 147 human studies epidemiologic studies, 155
156 randomized controlled trials, 155 humans and animals consume, 181 on inflammation and oxidative stress, 155f oxidative stress bone physiology and pathology, 146
147 and inflammation, 146 plants contain, 181 Antiatherogenic functions, 101t Antibiotics, 92
93 Anticancer activity curcumin (CUR), 200 Sambucus ebulus L., 328
329 thymoquinone (TQ), 371
372 Antidepressant activity, Sambucus ebulus L., 330 Antidiabetic activity of phenolics, 304t Sambucus ebulus L., 329
330 thymoquinone (TQ), 370
371 Antidiabetic drugs, 301 Antiinflammatory activity curcumin (CUR), 198
200, 200t pistachio nut, 312t polyphenols in, 227
228 thymoquinone (TQ), 374
375 Antimicrobial agent, curcumin (CUR) as, 201
202 Antineurodegenerative effect, curcumin (CUR), 201 Antiobesogenic drugs, 301 Antiobesogenic properties of phenolics, 302t Antioxidant, 289 Ac¸aı´ (Euterpe oleracea Martius), 127
128, 132t animal model assays, 130

404 Antioxidant (Continued) applications, 131 flavonoids, 128
129 human studies, 130
131, 131f in vitro bioassays, 129
130 polyphenols, 128 artichoke leaf extract, 174
175 in chronic obstructive pulmonary disease (COPD), 53f classification of, 390 Clitoria ternatea flowers, 190
192 defenses, 171 dietary anthocyanins, 147 electron donors, testing for, 391
393 endogenous, 214, 237 nonprotein antioxidants, 215 protein antioxidants, 215 exogenous, 214
216, 237 extra virgin olive oil, 229
231, 230f hydroxytyrosol, 230
231, 230f oleocanthal, 230f, 231 oleuropein, 230, 230f tyrosol, 230, 230f tyrosol glucuronides and sulfates, 231 Graves’ orbitopathy management, 338 hypothesis, 90
92 lycopene, 249 in male infertility treatment, 23
24 nutritional sources, 23 phytonutrients, 23
24 supplementary antioxidants, 23 measurement strategies, 393
394 methylsulfonylmethane (MSM) antioxidant enzyme production/activity, 279
280 biophysical properties, 279 free radical scavenging, 280
281 redox hub, 280, 280f of mushrooms, 290
292 cell lines model, 292
293 on diseases using animal models, 294 phenolic compounds, 290
292 polysaccharides, 290 vitamins, 292 in vitro and in vivo methods, 294
295 oxidative stress (OS), 299
300, 389 paradox, 24 polyphenols in, 228
229, 228f protective effect antioxidant defense system, improvement of, 208
209 hyperglycemia-induced free radicals, scavenging of, 208
209 redox signaling, 90
92, 92f, 93f screening, problem of, 390
391 Stevia rebaudiana (Bertoni), 347
354, 348f against free radicals and stevia potential, 347
354, 348f and bioactive compounds, 349t testing for, 391 thymoquinone (TQ), 370, 371f Antioxidant minerals, pistachio nut, 314
315 content and nutritional value of, 315t copper, zinc and manganese, 315, 315t selenium proteins, 315, 315t

Index

Antioxidant response elements (AREs), 77, 80
84, 237 Antioxidant vitamins (ACE), 313 ascorbic acid (vitamin C), 314 carotenoids, 313 content and nutritional value of, 314t tocopherols and tocotrienols, 313
314 Antiplatelet activity, 230 Antithyroid drugs (ATD), 337 Antitumoral properties, curcumin (CUR), 200 Anxiety disorders, 163 Apigenin, 324t Apigenin glycosides, 173, 324t Apolipoprotein E (APOE) gene, 160 Apoptosis, 22, 227
228 of retinal cells, 33, 33f Arachidonic acid, 106 Arsenic trioxide, 111
112 Artichoke leaf extract, 169
173 antioxidant effects of, 174
175 applications, 175 bioactive components, 171
173 polyphenolic compounds, 171
173 triterpenes and sesquiterpene lactones, 173 caffeoylquinic acids in, 172f flavonoids in, 172f metabolic syndrome, 173
175, 174t, 175f oxidative stress, 173
175 pharmacokinetics, 173 safety, 173 sesquiterpene lactones, 172f Arylesterase (ARE), 100
102 Ascorbate, 215
216 Ascorbic acid (vitamin C), 69, 257, 314, 363, 390 antidepressant-like effect of, 163f applications, 164 dry form of, 159 neurodegenerative diseases, applications in, 160
161, 164f Alzheimer’s disease, 160 amyotrophic lateral sclerosis, 161 multiple sclerosis, 161 Parkinson’s disease, 160
161 neuroprotective effect of, 161f in neuropsychiatric disorders, 161
163 anxiety disorders, 163 bipolar disorder, 162
163 major depressive disorder, 162 schizophrenia, 161
162 oxidation process, 160f in psychiatric diseases, 164f Asplenic patient, 65 Asthma, 374 Atherogenic process, 100f Atheroma plaques, 42
43, 43f Atherosclerosis, 42
43, 98
100, 217t, 218t, 219t Stevia rebaudiana (Bertoni) against, 347 ATP synthesis process, 40 A-type proanthocyanidin, 181 Autoimmune/inflammatory conditions, 19

Axonal restoration, Ginkgo biloba (GB) extract, 241 Axons, 236 Azoospermic, 272

B Bacterial neurotoxins, 241 Bcl-2-associated X protein (Bax), 372 Benzoic acids, 292 Berries, 179 Berry extracts, 147, 148t, 150t Berry Health Benefits Symposium (BHBS), 179 β-amyloid, 185 β-carotene, 215
216, 247, 257, 313, 314t β-cell regeneration and insulin secretion, 208 Beta-glucan, 290, 292 Beta-glucan extraction methods, 292 Beta-thalassemia intermedia (β-TI), 120
121, 121t, 123t β-thalassemia major (β-TM), 117
121, 119t, 120t, 121t, 122t, 123t Bilberry anthocyanins Alzheimer’s disease (AD), prevention and treatment of, 184
185 cardiovascular disease, prevention and treatment of, 183 chemistry of, 180
181, 181f against chemotherapy and radiation therapy, 182
183 diabetes, prevention and treatment of, 183
184 and macular degeneration, 184 Bile duct ligation (BDL), 250
251 Bilirubin, 215 Binge drinking, 260
261, 261f Bioaccessibility, 295 Bioavailability, 182, 192, 197, 199, 257, 295, 393 extra virgin olive oil (EVOO), 226 lycopene, 248
249 methylsulfonylmethane (MSM), 279f Bioconjugation, 197 Bioeffectivity, 393 Biomarkers, 93
94, 130 Biosynthetic pathway, amaryllidaceae alkaloid, 139, 140f Biotin, 292 Bipolar disorder, 162
163 Bisphenol A, 269t Bisphosphonates, 145
146 Black rice extract, 147, 148t Black tea, 216, 361 Blood glucose homeostasis, 347 Body mass index (BMI), 100
101, 258, 300 Bone health, anthocyanin-rich foods and compounds on, 145
146, 148t, 153 animal studies, 150t cell studies, 148t human studies, 154t Bortezomib, 111
112 Bovine serum albumin (BSA), 191
192 Brain, methylsulfonylmethane, 281
282 Brain injury, 80
81 Breast cancer, bilberry anthocyanin, 182

Index

2-bromopropane, 269t, 271 Bronchoalveolar lavage fluid (BALF), 56
58 Busulfan, 269t Butter fruit tree. See Dacryodes edulis Butylated hydroxyanisole (BHA), 345
346 Butylated hydroxytoluene (BHT), 193, 345
346

C Cadmium (Cd), 269t, 336 Caenorhabditis elegans, 130 Caffeic acid, 138
139, 291t, 292
294, 302t, 324t, 330 Caffeine, 362
363 Caffeoylquinic acids, 172f, 173 Calcium alginate microencapsulation technique, 192 Calprotectin, 89
90 Camellia sinensis, 360 Cancer, 257 chemotherapy, 328
329 epigallocatechin gallate (EGCG), 219
220 formation stages initiation, 219
220 progression stage, 219
220 promotion, 219
220 lycopene, 249
250 Stevia rebaudiana (Bertoni) against, 346 tea, 359 Cancer stem cells (CSCs), 202 Canonical regulatory mechanism, 77 Carbohydrate digestion and absorption, 207
208 Carbohydrates, 259
260 Carbon tetrachloride (CCl4), 282
283 Cardiac hypertrophy, 217t, 218t, 219t Cardiopulmonary system, methylsulfonylmethane, 282 Cardiovascular disease (CVD), 39
40, 97
98, 390 bilberry anthocyanins, 183 epigallocatechin gallate (EGCG), 216 lycopene, 250 mechanisms, 40f mitochondrial production of reactive species, 40
41, 41f oxidative stress in, 41
46 applications, 46 hyperlipidemia and atherosclerosis, 42
43 hypertension, 43
45, 44f inflammation, 45
46, 46f ischemia and reperfusion, 41
42, 42f pathophysiology of, 40 risk factors for, 97 tea, 359 Cariogenesis, Stevia rebaudiana (Bertoni) against, 346
347 Carotenoids, 247, 248f, 313, 360, 362
363 Catalase (CAT), 7, 214
215, 280, 282
283, 285t, 311, 337, 373 Catechins, 304t, 324t, 360
361, 363 Cefixime, 197 Celecoxib, 111
112 Cell lines model, 292
293

Cell studies, anthocyanins, 147
149 anthocyanin compounds, 148t, 149, 150t berry extracts, 147, 148t, 150t black rice extract, 147, 148t dried plum, 147
149, 148t, 150t, 153 Cellular damage, tea, 358 Cellular enzymatic antioxidants, 299 Cellular redox states, 7, 11 Cellular redox imbalance, 159 Central nervous system, ascorbic acid antidepressant-like effect of, 163f applications, 164 dry form of, 159 neurodegenerative diseases, applications in, 160
161, 164f Alzheimer’s disease, 160 amyotrophic lateral sclerosis, 161 multiple sclerosis, 161 Parkinson’s disease, 160
161 neuroprotective effect of, 161f in neuropsychiatric disorders, 161
163 anxiety disorders, 163 bipolar disorder, 162
163 major depressive disorder, 162 schizophrenia, 161
162 oxidation process, 160f in psychiatric diseases, 164f Central nervous system (CNS) disorders, 235
237 Cerebral failure, Ginkgo biloba (GB) extract, 240
241 Cerebral ischemia, 136 Ceruloplasmin, 315 Chemotherapy bilberry anthocyanins, 182
183 cancer, 328
329 Children, Paraoxonase 1 (PON1) in antiatherogenic functions, 101t applications, 102 gene polymorphisms, 98
100 general characteristics, 98
100 multiple activities and substrates, 101f paraoxon, 98
100 in pediatric populations, 100
102 phenylacetate, 98
100 Chitin, 291t Chitosan, 290 Chlorogenic acid (CGA), 173
175, 324t, 383 Chromatography-mass spectrometry (GCMS), 206, 206t Chronic granulomatous disorder, 240 Chronic hemolysis, 67 of sickle RBCs, 72
73 Chronic inflammation, 198
199, 201 Chronic low-grade inflammation, 281, 284 Chronic noncommunicable diseases (NCDs), 225 Chronic obstructive pulmonary disease (COPD), 49
50 antioxidant in, 53f cigarette smoking, 49
50, 56
58, 59f constant airflow restriction, 49 oxidative stress antioxidative defense status, 58f applications, 62

405 cellular reactive nitrogen species, 60
62 cellular reactive oxygen species, 58
59, 60f environmental reactive species, 56
58, 59f in pathogenesis, 50
56, 51f Chronic smoking, 18 Chronic vaso-occlusions, 72
73 Cinnamaldehyde, 84 Cinnamic acid, 291t, 324t Cisplatin, 111
112, 182, 269t, 271, 373 c-Jun N-terminal kinase (JNK) pathways, 20 Clarithromycin, 373 Clitoria ternatea flowers, 189, 190f AAPH-induced morphological changes of erythrocytes, 191f antioxidant activity of, 192t antioxidant properties of, 190
192 applications, 193
194 bioactive components, 190 calcium alginate beads of, 192f, 192t food uses, 193 human study, 193, 194f pharmacological properties of, 189 toxicity, 193 use of, 190 Coenzyme Q (CoQ), 215 Cognitive processing, 281
282 Cold-pressed oils, 310
311 Colorectal cancer, bilberry anthocyanin, 182 Comorbidities, 200
201 Complete Freund’s adjuvant (CFA), 149 Condensed tannins, 299 Copper, pistachio nut, 315, 315t Corynebacterium diphtheriae, 89
90 Coumarins, 128 COX-1. See Cyclooxygenase-1 (COX-1) COX-2. See Cyclooxygenase-2 (COX-2) Cryptorchidism, 19 Cuprizone-mediated demyelination animal model, 161 CUR-loaded polycaprolactone nanoparticles (NPC), 202 Curcumin (CUR), tissue pathology, 83, 111
112, 118, 198f, 199f activity in myocardial diseases, 200
201 anticancer properties of, 200 antiinflammatory activity, 198
200, 200t as antimicrobial agent, 201
202 antineurodegenerative effect, 201 antitumoral properties, 200 applications, 202 bacterial cells treated with, 197
198 bile metabolites, 198 cell activation mechanisms, 199t in gastric cancer, 200 iron chelator, oxidative stress under ironoverloaded conditions, 121
123, 122t metabolic process of, 197 metabolites of, 198 on neuroblastoma tumors, 200 properties of, 198 sirtuins, 198 therapeutic challenges, 202, 202t Curcuminoids, 197

406 Cyanidin, 147t, 149 Cyanidin-3-glucoside, 128, 129f Cyanidin-3-rutinoside, 128, 129f Cyanidin-3-sambubioside, 128, 129f Cyanidin chloride, 153 Cyanogenic glycosides, 325 Cyanoropicrin, 173
175 Cyclic-AMP response element binding protein (CREB), 241 Cyclooxygenase (COX), 207, 239 COX-1, 231 COX-2, 109
110, 190
191, 207, 230
231, 375 Cyclophosphamide, 269t, 271 Cynara scolymus, 169 Cynarin (1,3-Odicaffeoylquinic acid), 173
175 Cysteine, 88 Cytochrome p450 enzymes, 283 Cytokines colony stimulating factor 1 (CSF1), 146 Cytotoxic peroxyl and alkyl radicals, 20
21

D Dacryodes edulis, 209, 210f bioactive compounds of, 206, 206t diabetic pathogenesis, 207 dietary carbohydrate, 207 hyperglycemia-induced oxidative stress and diabetic complications, 207 insulin, 207 preliminary phytochemistry, 205
206, 206t flavonoids, 205
206, 206t saponins, 206, 206t tannins, 206, 206t protective effects, in diabetes pathology, 207
209 antioxidant protective effect, 208
209 β-cell regeneration and insulin secretion, 208 carbohydrate digestion and absorption, 207
208 glucose uptake and utilization, 208 lipid metabolism, 208 Damage-associated molecular pattern (DAMP), 45, 46f Deferasirox (DFX), 118, 119t, 120t, 121t, 122t, 123t Deferiprone (DFP), 118, 119t, 120t, 121t, 122t, 123t Deferoxamine (DFO), 118, 119t, 120t, 121t, 122t, 123t Dehydroascorbate (DHA), 159, 280 Delphinidin, 147t, 149, 153 Dementia defined, 136 pharmacotherapy of, 138
139, 138f Deoxyribonucleic acid (DNA), 9 Detrimental effects, 289 Dexamethasone, 269t Diabetes mellitus (DM), 80, 217t, 379 antioxidants in, 380 bilberry anthocyanins, 183
184 defined, 29, 329
330 hyperglycemia, 380

Index

male reproductive disorders, oxidative stress, 19 Stevia rebaudiana (Bertoni) against, 347 Yacon (Smallanthus sonchifolius), 381f applications, 384 hydroethanolic extract of, 383 inulin structure in, 382f origins and ethnobotanical characteristics, 380
381 polyphenols profile in, 382f roots and its nutritional composition, 382f, 382t Diabetes pathology, Dacryodes edulis on, 207
209 antioxidant protective effect, 208
209 β-cell regeneration and insulin secretion, 208 carbohydrate digestion and absorption, 207
208 diabetic pathogenesis, 207 dietary carbohydrate, 207 hyperglycemia-induced oxidative stress and diabetic complications, 207 insulin, 207 glucose uptake and utilization, 208 lipid metabolism, 208 Diabetic retinopathy (DR) advanced glycation end products, accumulation of, 32 enhanced polyol pathway, 32 epigenetic modifications in, 33
35 DNA methylation, 34 histone modification, 34 microRNAs, 34 sirtuin proteins (SIRTs), 34
35, 35f hexosamine pathway, activation of, 32
33 molecular mechanisms of, 29 nonproliferative diabetic retinopathy, 34f pathogenesis of, 29, 30f protein kinase C, activation of, 32 reactive oxygen species, 30
31 enzymatic production, 31, 31f nonenzymatic production, 30
31 structural and functional alterations in, 33 apoptosis of retinal cells, 33, 33f basement membrane thickening, 33 retinal microvasculature, 33 Diacylglycerol (DAG), 32 Diesel exhaust particle (DEP)
induced toxic effects, 372 Diet-induced obesity model, 150t Dietary antioxidants, resources journals relevant to pathology, 397
401, 399t online resources, information and emerging technologies, 397
401, 401t regulatory bodies, professional societies, and organizations, 397
401, 397t relevant books, 397
401, 400t Dietary carbohydrate, 207 Dietary fibers, 208 Dietary reference intake (DRI), 312 Dihydrocurcumin, 198 Dihydroferulic acid (DHFA), 173 7, 8-dihydro-8-oxo-guanosine (8-oxoG), 9
10

Dimethyl fumarate (DMF), 81 Dimethylsulfide, 277 Dimethylsulfoxide (DMSO), 277, 279 Diterpene glycosides, 346 Dithionitrobenzoic acid (DTNB), 393 Dizocilpine, 137 DNA integrity, 21 DNA methylation, 34 Docetaxel, 111
112 Docosahexaenoic acid (DHA), 83, 201 Doxorubicin (DOX), 111
112, 182, 269t, 372 Dried plum, 147
149, 148t, 150t, 153
155 Drosophila melanogaster model, 130 Drug-loaded mesoporous silica nanoparticles (MSNs), 112 Dysglycemia, 358
359 Dyslipidemia, 97 Dysmetabolism, 379
380

E Eben tree. See Dacryodes edulis Ebulin blo (A-B toxin), 325
326 Edible mushrooms cinnamic acid content of, 291t glucan content and distribution of, 291t phenol contents in, 290
292 phenolic acid compounds in, 293f phenolic acids content of, 291t trehalose, chitin and ergosterol of, 291t Eeratozoospermic, 272 Eicosapentaenoic acid (EPA), 83, 201 Electron donating mechanism, 209 Elephantiasic pretibial myxedema, 336f Ellagic acid, 111 Endogenous antioxidants, 112, 117
118, 237, 300 enzymes, 209, 300 nonprotein antioxidants, 215 alpha-lipoic acid (α-LA), 215 bilirubin, 215 coenzyme Q (CoQ), 215 ferritin, 215 glutathione (GSH), 215 uric acid, 215 protein antioxidants catalase (CAT), 215 glutathione peroxidase (GPx), 215 superoxide dismutases, 215 Endogenous nonenzymatic antioxidants, 300 Endogenous nonprotein antioxidants, 215 alpha-lipoic acid (α-LA), 215 bilirubin, 215 coenzyme Q (CoQ), 215 ferritin, 215 glutathione (GSH), 215 uric acid, 215 Endogenous protein antioxidants catalase (CAT), 215 glutathione peroxidase (GPx), 215 superoxide dismutases, 215 Endogenous reactive oxygen species and antioxidants, 16
18 immature spermatozoa, 18 leukocytes, 16
17

407

Index

Endogenous sources, intracellular ROS, 106
107 Endoplasmic reticulum, free radicals (FRs), 7 Endothelial cells, 137 Endothelial nitric oxide synthase (eNOS), 31
32, 60
61, 106 Endothelin-1, 33 Endotoxin, 190
191, 258 Entoloma lividoalbum, 292 Enzymatic antioxidants, 360 tea, 360 Enzymatic production, 31, 31f Epicatechin, 324t Epidemiologic studies, 155
156 Epidermal growth factor receptor (EGFR), 230 Epigallocatechin gallate (EGCG), 83 and aging, 220 and cancer, 219
220 and cardiovascular diseases, 216 efficacy of, 219t in vitro studies, protective effect of, 218t in vivo studies, protective effect of, 217t and neurodegenerative disease, 216
219 pharmacological actions of, 216
220 protective role in various pathological diseases, 220f, 221 Erectile dysfunction (ED), 19
20 Ergosterol, 291t, 292 Ergothioneine, 292 Erythrocytes, 69, 191, 191f Erythromycin, 201
202 E-selectin, 98 Essential fatty acids (EFAs), 309, 312
313 Essential hypertension, 43 Essential linoleic acid, 312 Etiopathogenesis, 16 European Food Safety Authority (EFSA), 225
226 European Group on Graves’ Orbitopathy (EUGOGO), 342
343 Euthyroidism, 338 Excess residual cytoplasm, 18 Excitotoxicity, 136
137 Exercise-induced oxidative stress, 285t Exhaled nitric oxide (eNO), 60
61 Exogenous antioxidants, 215
216, 237, 300, 326
327 Exogenous reactive oxygen species and antioxidants lifestyle factors, 18 radiation, 18 toxins, 18 Exogenous sources for free radicals (FRs), 8f intracellular ROS, 107 Extra virgin olive oil (EVOO) antioxidant activities, 229
231, 230f hydroxytyrosol, 230
231, 230f oleocanthal, 230f, 231 oleuropein, 230, 230f tyrosol, 230, 230f tyrosol glucuronides and sulfates, 231 applications, 231 bioavailability of, 226

Mediterranean diet (MD), 256, 261
262 olive oil characteristics, 226 extraction procedures, 226, 227f polyphenols in, 225
226, 228t antioxidant activities, 228
229, 228f antioxidant and antiinflammatory properties, 227
228 chemistry, 226
227 mechanisms of action, 229f phase II gene expressions, 229f qualified health claim, 231

F Fasting serum hippuric acid, 183 Fatty acid synthase (FAS), 208 Fatty streak, 97
98 Fenton reaction, 5
6, 40, 88
89, 92
93, 117, 159, 328 Fermented papaya preparation (FPP), 123 Ferric reducing antioxidant power (FRAP), 190, 193, 209 Ferritin, 215 Ferrous ion chelating power (FICP), 190 Ferrozine, 328 Ferulic acid, 293, 324t Fetal Hb (HbF), 67, 72 Filtration, 226 Flammulina velutipes, 290 Flavanones, 316 Flavonoids, 128
129, 170, 173
175, 205
206, 206t, 227, 228t, 239, 265, 290, 298, 299f, 323, 360 artichoke leaf extract, 172f Clitoria ternatea, 190 cyanidin-3-glucoside, 128, 129f cyanidin-3-rutinoside, 128, 129f cyanidin-3-sambubioside, 128, 129f Ginkgo biloba (GB) extract, 238
239 peonidin-3-glucoside, 128, 129f peonidine-3-rutinoside, 128, 129f structure of, 128f Flavonols, 316, 361, 362t 5-fluorouracil, 111
112, 372 Forced exhalation volume at 1 sec (FEV1), 60
61 Formaldehyde, 269t FOXO transcription factors, modulation of, 109 Free amino acids, 362 Free fatty acids (FFAs), 207, 300
301 Free radical scavenging, 184f, 191
192, 255, 266
267, 280
281, 370 Free radicals (FRs), 3
4, 30, 70
72, 127
129, 135
136, 159, 299
300, 357 applications, 11 biomolecules, accumulation on, 106f defined, 4, 358 general characteristics, 4 in health and disease, 10
11 adaptation, 10 cell death, 10
11 tissue injury, 10 initiation, propagation and termination, 299
300

and nonfree radicals, 4
7 alkoxyl and peroxyl radicals, 6 hydrogen peroxide (H2O2), 5 hydroxyl radical, 5
6 hypochlorous acid (HOCl), 6 nitric oxide, 6
7 ozone (O3), 6 reactive nitrogen species, 7 singlet oxygen, 6 superoxide radical, 4
5 oxidative chain reactions, 4 initiation, 4 propagation, 4 termination, 4 oxidative stress, 8
10, 9f deoxyribonucleic acid, 9 lipids, 10 proteins, 9 ribonucleic acid (RNA), 9
10 sources of, 7 endoplasmic reticulum, 7 exogenous, 8f mitochondria, 7 peroxisomes, 7 typical endogenous, 8f Stevia rebaudiana (Bertoni), 345
346 tea, 358 Friedreich’s ataxia, 123
124 Fructooligosaccharides, 381, 382f

G Galanthamine, 138
141, 138f Gallic acid, 290, 291t, 292, 294, 302t, 304t, 324t, 361, 363 Gallotannins, 316 γ-tocopherol, 313
314, 314t Gastric cancer curcumin (CUR), tissue pathology, 200 lycopene, 250 Gastric emptying, 208 Gastrointestinal diseases, lycopene, 251
252 Gastrointestinal tract, methylsulfonylmethane, 283 Gene therapy, 67 Generally recognized as safe (GRAS), 248, 277
278, 345
346 Genitourinary tract infection, male reproductive disorders, 18
19 Genotoxicity, 250 Gentamicin, 269t, 373 Ghosal’s numbering system, 139 Ginkgetin, 239 Ginkgo biloba (GB) extract in nerve regeneration, 235, 238
243 components of, 239f in experimental neuronal models and diseases, 240
243 Alzheimer’s disease, 241, 242f amyotrophic lateral sclerosis, 243 axonal restoration, 241 cerebral failure, 240
241 glaucoma, 243 Huntington’s disease, 242 multiple sclerosis, 243 Parkinson’s disease, 242

408 Ginkgo biloba (GB) extract in nerve regeneration (Continued) pheochromocytoma, 243 flavonoids, 238
239 nerve regenerative mechanisms of action of, 239
240 neuroprotective, 239
240 oxidative stress-related neurodegenerative diseases, 240f pharmacological effects of, 239f terpenoids, 238
239 Ginkgolic acid, 238
239 Ginkgolide B (BN-52021), 239
240 Glaucoma, Ginkgo biloba (GB) extract, 243 Glial cells, 159 Gliotoxicity, 137 Glucocorticoid (GC), 338 Glucolipotoxicity, 207 Glucose oxidation, 30
31 Glucose transporter (GLUT4), 207
208 Glucose uptake and utilization, 208 Glutamate, 136
137 Glutamate-cysteine ligase (GCL), 311 Glutaredoxin (Grx), 390 Glutathione (GSH), 32, 89
90, 112, 215, 280, 283
284, 285t, 315, 370, 390, 393 Glutathione peroxidase (GPx), 7, 40, 130, 171, 215, 280, 311, 315, 337, 373 Glutathione reductase (GR), 311 Glutathione S-transferase (GST), 280 Glycemia, 208 Glycosides, Clitoria ternatea, 190 Glyphosate, 259 Gold nanoparticles (GNPs), 112 Gourmet oils, 310
311 Graves’ disease, 335 Graves’ hyperthyroidism, 338 oxidative stress in, 336
337 selenium in, 339
340 Graves’ orbitopathy, 335
336, 336f, 338 antioxidants in management, 338 oxidative stress in, 337
338 risk factors for, 339f selenium in, 340
343, 340f, 341f Graves’ orbitopathy-specific quality-of-life questionnaire (GO-QOL), 342f Great Oxidation Event, 88
89 Green tea, 360
361 GSK-3β-mediated signaling, 162
163 Gut microbiota, 259, 260f

H Haber-Weiss reactions, 40 Health hazards, 358 Healthy animals, 150t, 154
155 Healthy diet, 225 Healthy oils, 310
311 Heart failure, 217t, 218t Helicobacter pylori, 89
90, 252, 322, 373 Hematoma-associated mechanisms, 80
81 Hematopoietic stem cell transplantation, 67 Heme oxygenase-1, 162 Hemoglobinopathies, 19 Hepatic disorders, 250
252, 331 Hepatic fibrosis, CUR, 202

Index

Hepatitis C virus (HCV), 359 Hepatoprotective effects, 373 Hereditary hemochromatosis, 117 Hesperetin, 302t Hesperidin, 251
252 Hexosamine pathway, 29, 30f, 32
33 High-density lipoprotein (HDL), 98, 99t, 100f, 131, 358
359 High-molecular-weight food polyphenols, 295 High performance liquid chromatography (HPLC), 206, 206t Histone modification, 34 Homocysteines, 19 Homogentisic acid, 290, 291t, 293 Hormone deficiency model, 149
153 Human immunodeficiency virus (HIV), 19 Human immunodeficiency virus type 1 (HIV-1)-Tat, 280, 282, 285t Human studies, anthocyanins epidemiologic studies, 155
156 randomized controlled trials, 155 Huntington’s disease (HD), 237 Ginkgo biloba (GB) extract, 242 Hyaluronic acid (HA), nanomicelles with, 199
200 Hydrogen peroxide (H2O2), 5, 16, 137, 146, 159, 328, 338, 341
342, 341f, 347
348, 360, 391 Hydrolyzable tannins, 299 Hydroperoxyl radical, 5t Hydroxy-isocromans, 228t Hydroxybenzoic acid (HBA), 290, 292, 298 Hydroxybenzoic phenolic acids, 361 Hydroxycinnamic acid (HCA), 290, 292
293, 298 consist of, 298 Hydroxyl radical, 5
6, 5t Hydroxyl radical scavenging activity (HRSA), 190 Hydroxytyrosol, 81
82, 230
231, 230f, 302t Hydroxyurea, 66, 68t, 72, 72t, 123 Hypercholesterolemia, 97
98 Hyperglycemia, 29, 32, 35, 80, 183
184, 205, 297
298, 380 Hyperglycemia-induced free radicals, scavenging of, 208
209 electron donating mechanism, 209 metal ion chelation, 209 proton-donating mechanisms, 208
209 Hyperglycemia-induced oxidative stress and diabetic complications, 207 Hyperhomocysteinaemia, 19 Hyperlipidemia, 42
43 Hypermethylation, 34 Hypertension etiology of, 43 mitochondrial dysfunction in, 45 Nicotinamide adenine dinucleotide phosphate oxidase, 44 reactive species and, 44f Stevia rebaudiana (Bertoni) against, 347 uncoupled endothelial nitric oxide synthase, 45 xanthine oxidase, 44
45

Hyperthermia, 19 Hypertrophy, 201 Hyperuricemia, 45 Hypochlorous acid (HOCl), 6, 52
56 Hypogonadism, 20 Hypothalamic inflammation, 20 Hypothalamic-pituitary-adrenal axes, 281
282 Hypothalamic-pituitary-thyroid axes, 281
282

I Ibuprofen, 231 Idebenone, 121
123, 122t Imipramine, 330 Immature spermatozoa, 18 Immune-mediated inflammatory diseases (IMIDs), 228 Indol, 266 Inducible nitric oxide synthase (iNOS), 6
7, 60
61, 190
191, 282, 359 Infections, tea, 359 Inflammation, 198
199, 219t, 237
238, 375 anthocyanins, 146, 155f cardiovascular disease (CVD), 45
46, 46f methylsulfonylmethane, 281, 281f Inflammation-induced model, 150t Inflammatory bone disease model, 153 Inflammatory bowel disease (IBD), 283 thymoquinone, 373 Inherited hemoglobinopathies, 117 Insulin, 207 Dacryodes edulis, 207 resistance, 301, 371 Insulin growth factor (IGF)-I, 102 Intercellular adhesion molecule-1 (ICAM-1), 31 Interferon gamma (IFN-γ), 146 Interleukin-1, 239 Interleukin (IL)-1β, 283 Interleukin-6 (IL-6), 146, 283 Interleukin 10 (IL-10), 19 International Index of Erectile Function (IIEF) scores, 20 Interorgan pathologies, 284f Intracellular reactive oxygen species, 105
106, 110
111 on cellular homeostasis, 107f regulation of in catabolism, 107 production, 107 sources of, 106
107, 108f endogenous, 106
107 exogenous, 107 upregulation of, 111
112 Intracellular redox homeostasis on cancer progression and therapy applications, 112
113 intracellular ROS, 105
106, 110
111 catabolism, regulation of, 107 on cellular homeostasis, 107f endogenous sources, 106
107 exogenous sources, 107 production, regulation of, 107 sources of, 106
107, 108f

409

Index

upregulation of, 111
112 oxidative stress in carcinogenesis, 107
110 FOXO transcription factors, modulation of, 109 Keap1-Nrf2 pathway, modulation of, 109 mitogen activated protein kinase, modulation of, 108 NF-κB, modulation of, 109
110 p53, modulation of, 109 STAT family proteins, modulation of, 110 targeting oxidative stress in cancer therapeutics, 110
112, 111f antioxidant-mediated therapeutic strategy, 111 intracellular ROS levels, upregulation of, 111
112 Intracellular signaling and angiogenesis in neuronal regeneration, 237
238 Inulin structure in Yacon, 382f Iodothyronine deiodinases (IDD), 315 Ionizing radiation (IR), 182
183 Iron chelation, 328 Iron chelator, oxidative stress under ironoverloaded conditions, 118 applications, 123
124 curcumin, 121
123, 122t idebenone, 121
123, 122t multiantioxidants, 123, 123t N-acetylcysteine (NAC), 119
120, 121t silymarin, 118
119, 120t vitamin C, 118, 119t vitamin E, 120
121, 122t Ischemia-reperfusion-induced gastric lesions, 372 Ischemic reperfusion (IR), 41
42, 42f, 269t Isoflavonoids (3-benzopyrans), 298

K Kaempferol, 302t, 304t, 324t, 362t Kelch-like ECH-associated protein 1 (Keap1), 69, 77, 80, 240, 241f modulation of, 109 protein structure of, 78f ubiquitination regulation of Nrf2, 78f Kidney, methylsulfonylmethane, 282
283 Kotronen index, 258
259 Kupffer cells hypertrophy, 331

L Lactobacillus, 259 Lectins, 325
326 Lentinula edodes (Shiitake), 290, 291t Leukocytes, 16
17 Leukocytospermia, 16
17 Levothyroxine, 339
340 Lewy bodies, 160 Leydig cells, 271
272 L-glutamine, 67, 70, 72, 72t Lifestyle factors, 18 Lignans, 299 Lipid energy metabolism, 380 Lipid hydroperoxides (LOOH), 100f Lipid metabolism, 208

Lipid peroxidation (LPO), 6, 10, 15
16, 20
22, 67
72, 111
112, 137
138, 162, 238, 358, 372
373 in foods, 193 S. ebulus L., 328 thymoquinone (TQ) on, 371f Lipid peroxyl radical, 5t, 10 Lipids, 10 Lipogenesis, 208 Lipolysis process, 380 Lipophilic components, 325 Lipopolysaccharide (LPS), 146, 190
191, 241 Liposoluble vitamin E compounds, 98 Lipotoxicity, 379 Liquid chromatography-mass spectrometry (LC-MS), 206, 206t Listeria monocytogenes, 89
90 Liver damage, 260
262 Liver disease, Mediterranean diet (MD), 255
257 alcoholic liver disease definition, 260 epidemiology, 260
261 etiopathogenesis, 260, 261f malnutrition, 262 treatment, 261
262 applications, 257 extra virgin olive oil, 256 nonalcoholic fatty liver disease (NAFLD), 259f, 260f definition, 258 etiopathogenesis, 258 risk factors for, 258t treatment, 258
260 pyramid, 256f Liver, methylsulfonylmethane, 283 Living fossil. See Ginkgo biloba (GB) extract Low-density lipoprotein cholesterol (LDL-C), 97
98, 309 Low-density lipoprotein (LDL), 42, 98, 100f, 131, 230, 250, 315, 358
359 Low-molecular weight antioxidants, 311 Lutein/zeaxanthin, 314t Luteolin, 173
175, 304t, 324t Luteolin-glycoside, 324t Lycopene, 84 antioxidant effects, 249 applications, 252 bioavailability, 248
249 Blakeslea trispos, 248 cancer, 249
250 cardiovascular diseases, 250 chemical structure of, 247, 248f chemistry and sources, 248 dietary intake of, 248 gastrointestinal diseases, 251
252 hepatic and renal diseases, 250
252 isomers of, 249f natural, 248, 249f neurodegenerative diseases, 252 pharmacokinetics, 248
249 synthetic, 248, 249f tomato products, content of, 249t types of, 249f

M Macronutrients, 261
262, 295 content and nutritional value of, 310t Macrophages, 52
53 Macular degeneration, bilberry anthocyanins, 184 Maillard reactions, 310, 316 Major depressive disorder, 162 Malaria hypothesis, 66
67 Male fecundity, 19 Male hypogonadism, 19
20 Male infertility, 265 antioxidants in treatment, 23
24 nutritional sources, 23 phytonutrients, 23
24 supplementary antioxidants, 23 etiology of, 15 Male reproductive disorders, oxidative stress antioxidant paradox and reductive stress, 24 antioxidants in male infertility treatment, 23
24 nutritional sources, 23 phytonutrients, 23
24 supplementary antioxidants, 23 apoptosis, 22 applications, 24 autoimmune/inflammatory conditions, 19 cryptorchidism and varicocele, 19 diabetes, 19 DNA integrity, 21 erectile dysfunction (ED), 19
20 genitourinary tract infection, 18
19 hypogonadism, 20 lipid peroxidation, 20
21 mitochondrial dysfunction, 21
22 reactive oxygen species in male reproductive tract, 15
16, 17f assessment of, 22 endogenous reactive oxygen species and antioxidants, 16
18 exogenous reactive oxygen species and antioxidants, 18 redox biology in male reproduction, 16, 17f systemic infections, 19 Male reproductive system antioxidant system in, 265
266 melatonin, 267
268 Malnutrition, 261
262 Malondialdehyde (MDA), 20
21, 117
118, 135
136, 171, 199
200, 209, 282
283, 285t, 294, 337, 370, 373
374 Malvidin, 147t, 149 Mammalian target of rapamycin (mTOR) signaling, 89, 201, 237 Manganese, pistachio nut, 315, 315t Manganese (Mn), 130 Mangiferin, 111
112 MD. See Mediterranean diet (MD) Mediterranean adequacy index (MAI), 259
260 Mediterranean diet (MD), 23, 225, 249, 255
257 alcoholic liver disease

410 Mediterranean diet (MD) (Continued) definition, 260 epidemiology, 260
261 etiopathogenesis, 260, 261f malnutrition, 262 treatment, 261
262 applications, 257 extra virgin olive oil, 256 nonalcoholic fatty liver disease (NAFLD), 259f, 260f definition, 258 etiopathogenesis, 258 risk factors for, 258t treatment, 258
260 pyramid, 256f Melatonin, 265 antioxidative properties, 266
272, 269t application, 272 cells, role in, 268f and male reproductive system, 267
268 semen and sperm quality, 272 synthesis in pinealocytes, 267f testicular tissue protection, 268
272 Memantine, 135
136 Metabolic diseases, PON1, 102 Metabolic syndrome (METS), 100
101, 170 antioxidants and, 171 artichoke leaf extract, 173
175, 174t, 175f defined, 169
170 diagnosis of, 170t oxidative stress and, 170
171 tea, 358
359 Metal chelators, 311 Metal ion chelation, 209 Metformin, 100
101 Methimazole, 339
340 20-methylcholanthrene, 372 Methyl-mercury (MeHg), 130 Methylsulfonylmethane (MSM) antioxidant effects antioxidant enzyme production/activity, 279
280 biophysical properties, 279 free radical scavenging, 280
281 redox hub, 280, 280f applications, 284
285 oxidative stress markers, 285t in oxidative stress pathology, 281
285 brain, 281
282 cardiopulmonary system, 282 gastrointestinal tract, 283 inflammation, 281, 281f kidney, 282
283 liver, 283 musculoskeletal system and exercise, 283
284 pharmacokinetic studies in rats, 278
279 tissue distribution and bioavailability of, 279f toxicity, 277
278, 278t Methylxanthines, 362, 363t Microbiome, 131 Microglial activation, 238, 242f Micronutrient deficiencies (MNDs), 23 Micronutrients, 256, 261
262

Index

MicroRNAs (miRNAs), 34 Microwave radiation, 269t Middle cerebral artery occlusion (MCAo), 230 MiOXSYS system, 22 Mitochondria, free radicals (FRs), 7 Mitochondrial dysfunction, 21
22, 170
171 in hypertension, 45 Mitochondrial membrane potential (MMP), 21
22 Mitochondrial transcription factor A (Tfam), 230 Mitogen activated protein kinase (MAPK), 107
108 modulation of, 108 Mobile-detoxifying systems, 69 Monocyte-derived macrophages, 42 Monocytes, 98 Monodehydroascorbate (mDHA), 280 Monounsaturated fatty acids (MUFAs), 225
226 MPO. See Myeloperoxidase (MPO) Multiantioxidants, iron chelator, 123, 123t Multiple sclerosis (MS), 161, 235 Ginkgo biloba (GB) extract, 243 Nrf2, 80
81 Musculoskeletal system and exercise, 283
284 Mushrooms, 289 A. bisporus, 292 antioxidants of, 290
292 cell lines model, 292
293 on diseases using animal models, 294 phenolic compounds, 290
292 polysaccharides, 290 vitamins, 292 in vitro and in vivo methods, functioned food, 294
295 applications, 295 eaten, 290f Myelin, 237 Myeloperoxidase (MPO), 45, 46f, 52
53, 56
58, 100f, 280, 283, 285t Myocardial diseases, curcumin (CUR), 200
201 Myricetin, 362t

N N-acetylcysteine (NAC), 72t, 118
120, 121t, 373 NADPH, 390
391, 393 NAFLD. See Nonalcoholic fatty liver disease (NAFLD) Nanomicelles with hyaluronic acid (HA), 199
200 Naringenin, 302t, 304t, 324t Naringin, 324t Natural bioactive polysaccharides, 360 Natural dietary antioxidants, 360 tea, 360 Natural lycopene, 248, 249f Nelfinavir, 111
112 Neoflavonoids (4-benzopyrans), 298 Neolignans, 299 Neoplastic transformation, 219
220

Neovascularization, 237
238 Nephrotoxicity, 373 Nerve regeneration, Ginkgo biloba (GB) extract in, 235, 238
243 components of, 239f in experimental neuronal models and diseases, 240
243 Alzheimer’s disease, 241, 242f amyotrophic lateral sclerosis, 243 axonal restoration, 241 cerebral failure, 240
241 glaucoma, 243 Huntington’s disease, 242 multiple sclerosis, 243 Parkinson’s disease, 242 pheochromocytoma, 243 flavonoids, 238
239 nerve regenerative mechanisms of action of, 239
240 neuroprotective, 239
240 oxidative stress-related neurodegenerative diseases, 240f pharmacological effects of, 239f terpenoids, 238
239 Neural stem cells (NSCs), 293 Neuroblastoma tumors, curcumin, 200 Neurodegenerative diseases, 201 ascorbic acid, 160
161, 164f Alzheimer’s disease, 160 amyotrophic lateral sclerosis, 161 multiple sclerosis, 161 Parkinson’s disease, 160
161 EGb761, 241f, 242f epigallocatechin gallate (EGCG), 216
219 lycopene, 252 and neuronal cell damage, 136
137 tea, 359 Neurodegenerative processes, 235
237 Neuroinflammatory processes, 237 Neuronal cell damage amaryllidaceae alkaloid in oxidative stress, 139
141 neurodegenerative diseases, 136
137 oxidative stress related with, 137
138 Neuronal nitric oxide synthases (nNOS), 6
7, 60
61 Neuroprotection, 236
237 Neuropsychiatric disorders, ascorbic acid, 161
163 anxiety disorders, 163 bipolar disorder, 162
163 major depressive disorder, 162 schizophrenia, 161
162 Neuroregenerative processes, 235
237 definition of, 236 Neurotoxic oligomer amyloid-β (Aβ), 137 Neurovascular unit (NVU), 136
137 Neutrophils, 52
53 Nicotera, 259
260 Nicotinamide, 342 Nicotinamide adenine dinucleotide (NAD) redox, 72 Nicotinamide adenine dinucleotide phosphate oxidase (Nox), 30
31, 43
44, 58
59, 89, 107, 281
282

Index

Nicotine, 269t Nicotinic acetylcholine receptor (nAChR), 138 Nigella sativa, thymoquinone anticancer activity, 371
372 antidiabetic activity, 370
371 antiinflammatory activity, 374
375 antioxidant activity, 370 antioxidant effects of, 371f on APAP-induced nephrotoxicity, 374f applications, 375
376 bioavailability and kinetics, 370 cardiovascular activity, 372 gastrointestinal system, effects on, 372
373 and health, 370, 370f hepatoprotective effects, 373 on leukotriene C4 synthase, 375f on lipid peroxidation, 371f nephroprotective activity, 373
374 nervous system, effects on, 374 plasma glucose and insulin in diabetic rats, 372t pulmonary system, effects on, 374 toxic effects of, 376 Nitric oxide (NO), 5t, 6
7, 20, 31, 44
45, 50, 60
61, 67
69, 358
359, 370 Nitric oxide synthase (NOS), 6
7, 20 Nitrogen dioxide, 5t Nitrogen monoxide, 6
7 Nitrosative stress (NS), 8
9 Nitrotyrosine, 61
62 NLRP3 inflammasome, 281 N-methyl-D-aspartic acid (NMDA) receptor antagonists, 135
136 No-observed-adverse-eventlevel (NOAEL), 277
278 Nobiletin, 302t Nonalcoholic fatty liver disease (NAFLD), 259f, 260f definition, 258 etiopathogenesis, 258 risk factors for, 258t treatment, 258
260 Nonalcoholic steatohepatitis (NASH), 258 Noncommunicable diseases (NCDs), 228 Nonenzymatic antioxidants, 299, 359
360 Nonenzymatic processes, 106 diabetic retinopathy, reactive oxygen species, 30
31 Nonfermented tea, 360 Nonfree radicals and free radicals (FRs), 4
7 alkoxyl and peroxyl radicals, 6 hydrogen peroxide (H2O2), 5 hydroxyl radical, 5
6 hypochlorous acid (HOCl), 6 nitric oxide, 6
7 ozone (O3), 6 reactive nitrogen species, 7 singlet oxygen, 6 superoxide radical, 4
5 Nonphenolic antioxidant metabolites, tea, 360, 362
363, 363t, 365f Nonproliferative diabetic retinopathy, 34f Nontransferrin-bound iron (NTBI), 117

NSAIDs, 67, 68t, 231 Nuclear factor-erythroid 2-related factor 2 (Nrf2), 69, 77, 128
129, 201, 237, 240, 241f, 267, 279
280, 390 dietary compounds, 81
84 function, 77
80, 79f intranuclear mechanism of, 79f oxidative stress in carcinogenesis, 109 protein structure of, 78f regulatory mechanism of, 80 relation with disease, 80
81 transcription factor, 77
80 ubiquitination regulation of, 78f Nuclear factor kappa B (NF-κB), 41
42, 109
110 Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), 146 Nuclear respiratory factor 1 (Nrf1), 230 NutraNanoSpheres (NNS), 182 Nutrient analysis critical control point (NACCP) process, 257 Nutrition, 127

O Obesity, 149, 219t, 257
258 defined, 300 incidence of, 297
298 oxidative stress and, 300
301 prevalence of, 300 Stevia rebaudiana (Bertoni) against, 347 therapeutic applications of, 301
305 Obstructive jaundice, lycopene, 250
251, 251t Ochratoxin A (OTA), 250, 250f Oleic acid, 312
313 Oleocanthal, 230f, 231 Oleuropein, 83, 230, 230f Olive oil, extra virgin olive oil characteristics, 226 extraction procedures, 226, 227f Omega-3 fatty acid emulsion system, 293 Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), 83 Omeprazole, 372
373 Oncogenesis, 219
220 Oolong tea, 362 O-phenylenediamine (OPD) oxidation, 280
281 Opioids, 68t Orchidectomy (ORX), 149, 153 Organic acids, 363 Orlistat, 301 Osmolality, 282
283 Osmolarity, 282
283 Osteoarthritis, 285t Osteoblasts, 146 Osteoclasts, 146 Osteocytes, 146 Osteoporosis, 149 Osteoprotegerin (OPG), 146 Ovalbumin, 375 Ovariectomy (OVX), 149
153, 150t Oxaliplatin, 111
112 Oxidation of lipids initiation, 128

411 propagation, 128 terminus, 128 Oxidation-reduction potential (ORP), 22 Oxidative chain reactions, 4 initiation, 4 propagation, 4 termination, 4 Oxidative nitrosative stress in peripheral nerve regeneration, 236f Oxidative stress, 258, 282
283, 289, 341 in Graves’ hyperthyroidism, 336
337 in Graves’ orbitopathy, 337
338 and male reproduction, 265 methylsulfonylmethane (MSM), 281
285 brain, 281
282 cardiopulmonary system, 282 gastrointestinal tract, 283 inflammation, 281, 281f kidney, 282
283 liver, 283 musculoskeletal system and exercise, 283
284 neuronal cell damage, 137
138 Oxidative stress markers, MSM, 285t Oxidative stress (OS), 98, 127, 170, 228, 242, 297
298, 397 anthocyanins bone physiology and pathology, 146
147 and inflammation, 146 and antioxidants, 389 artichoke leaf extract, 173
175 bilberry anthocyanins Alzheimer’s disease (AD), prevention and treatment of, 184
185 cardiovascular disease, prevention and treatment of, 183 chemistry of, 180
181, 181f against chemotherapy and radiation therapy, 182
183 diabetes, prevention and treatment of, 183
184 and macular degeneration, 184 in cancer therapeutics, 110
112, 111f antioxidant-mediated therapeutic strategy, 111 intracellular ROS levels, upregulation of, 111
112 in carcinogenesis, 107
110 FOXO transcription factors, modulation of, 109 Keap1-Nrf2 pathway, modulation of, 109 mitogen activated protein kinase, modulation of, 108 NF-κB, modulation of, 109
110 p53, modulation of, 109 STAT family proteins, modulation of, 110 in cardiovascular disease, 41
46 applications, 46 hyperlipidemia and atherosclerosis, 42
43 hypertension, 43
45, 44f inflammation, 45
46, 46f

412 Oxidative stress (OS) (Continued) ischemia and reperfusion, 41
42, 42f chronic obstructive pulmonary disease (COPD) antioxidative defense status, 58f applications, 62 cellular reactive nitrogen species, 60
62 cellular reactive oxygen species, 58
59, 60f environmental reactive species, 56
58, 59f in pathogenesis, 50
56, 51f Clitoria ternatea, 189, 193 Dacryodes edulis, 205, 208
209 defined, 50, 87
88, 213
214, 389 diabetic retinopathy. See Diabetic retinopathy (DR) disease, associated with, 214 free radicals (FRs), 8
10, 9f deoxyribonucleic acid, 9 lipids, 10 proteins, 9 ribonucleic acid (RNA), 9
10 in male reproductive disorders apoptosis, 22 autoimmune/inflammatory conditions, 19 cryptorchidism and varicocele, 19 diabetes, 19 DNA integrity, 21 erectile dysfunction (ED), 19
20 genitourinary tract infection, 18
19 hypogonadism, 20 lipid peroxidation, 20
21 mitochondrial dysfunction, 21
22 redox biology in male reproduction, 16, 17f systemic infections, 19 male reproductive tract, reactive oxygen species in, 15
16, 17f assessment of, 22 endogenous reactive oxygen species and antioxidants, 16
18 exogenous reactive oxygen species and antioxidants, 18 metabolic syndrome, 170
171 in neurodegenerative diseases, 240f in neurodegenerative processes, 238 obesity, 300
301 and pathology, 390 pistachio nut, 311 reactive species and antioxidants, 299
300 redox signaling pathogen elimination in, 89, 90f pathogen thriving in, 89
90, 91f resources journals relevant to pathology, 397
401, 399t online resources, information and emerging technologies, 397
401, 401t regulatory bodies, professional societies, and organizations, 397
401, 397t relevant books, 397
401, 400t sickle cell disease (SCD), 69
72, 71t clinical consequences of, 69
72

Index

red blood cells in coping, physiology of, 69 sickle red blood cells, physiology of, 69, 70f type 2 diabetes (T2D), 301 Oxidative stress
induced insulin resistances, 358
359 Oxidized glutathione (GSSG), 32, 280, 284, 373, 390, 393 Oxidized low-density lipoprotein (oxLDL), 42, 98, 171 Oxidized phospholipids (oxPLs), 98
100 Oxygen-containing free radicals, 105 Oxygen paradox, 3 Oxygen radical absorbance capacity (ORAC), 193, 294
295, 316, 391, 392f OxyR, transcription factor, 88, 88t Ozone (O3), 6

P p53, modulation of, 109 Paclitaxel, 111
112 Paradigm shift, 90 Paraoxon, 98
100 Paraoxonase (PON), 130 Paraoxonase 1 (PON1), in children antiatherogenic functions, 101t applications, 102 gene polymorphisms, 98
100 general characteristics, 98
100 multiple activities and substrates, 101f paraoxon, 98
100 in pediatric populations, 100
102 phenylacetate, 98
100 Parenteral nutrition, 262 Parkinson’s disease (PD), 216
219, 235 ascorbic acid, 160
161 Ginkgo biloba (GB) extract, 242 Nrf2, 80
81 Pathogen-associated molecular pattern (PAMP), 45, 46f p-Coumaric acid, 291t, 292
293, 324t Peak expiratory flow rate (PEFR), 374 Pelargonidin, 147t Pentose-phosphate pathway (PPP), 390 Peonidin, 147t Peonidin-3-glucoside, 128, 129f Peonidine-3-rutinoside, 128, 129f Perception shift, 90, 91f Peripheral nerve injury, 235
236 EGb761, 241f Peripheral nerve regeneration, 236f Peripheral nervous system, 237 Peroxidases, 214 Peroxisome proliferator-activated receptor gamma (PPAR-γ), 146
147, 372 Peroxisome proliferator-activated receptors (PPARs), 380 Peroxisomes, 105
106, 111
112 free radicals (FRs), 7 Peroxyl radicals, 5t, 6 Peroxynitrite, 61
62 Petunidin, 147t P-glycoprotein, 138
139 Pharmacokinetics

artichoke leaf extract, 173 lycopene, 248
249 Methylsulfonylmethane, 278
279 methylsulfonylmethane (MSM), 278
279 Pharmacotherapy, of dementia, 138
139, 138f Phase 2 response, 77 Phenol, 206t Phenolic acids, 128, 227, 228t, 290, 298, 327
328, 362t Phenolic antioxidants, 328
329, 360
361, 362t, 364f Phenolic compounds, 247, 292, 295, 297
298, 299f, 300 phenol contents in edible mushrooms, 290
292 polyphenols, 290 shikimate/phenylpropanoid acid pathway, 298 structures and antioxidant ability, 292 Phenolic hydrogen, 292 Phenolics, 298 antidiabetic properties of, 304t antiobesogenic properties of, 302t antioxidant and biological activity of, 316 classes of, 298 flavonoids, 298, 299f lignans and neolignans, 299 obesity defined, 300 incidence of, 297
298 oxidative stress and, 300
301 prevalence of, 300 therapeutic applications of, 301
305 phenolic acids, 298 structural feature of, 298 tannins, 299 therapeutic applications of, 301
305 type 2 diabetes (T2D), 297
298, 301 oxidative stress and, 301 therapeutic applications of, 301
305 Phenols, 205, 323 Phenylacetate, 98
100 Phenylalanine, 298 Phenyletanoids, 227, 228t Phenylpropanoid pathway, 298 Pheochromocytoma, Ginkgo biloba (GB) extract, 243 Phosphoinositide 3-kinase (PI3K)/AKT pathway, 237 Phospholipids (PL), 313 Photosynthesis, 323 p-Hydrohybenzoic acid, 324t p-Hydroxybenzoic acid, 291t Physical inactivity, 283
284 Phytochemicals, 207
208, 247 carotenoids, 247 Phytocompounds, 257 Phytonutrients, male infertility treatment, 23
24 Phytosterols, 259
260, 313 Clitoria ternatea, 190 Piperlongumine, 112 Pistachio nut, 309
311 antioxidant minerals, 314
315

413

Index

content and nutritional value of, 315t copper, zinc and manganese, 315, 315t selenium proteins, 315, 315t antioxidant vitamins (ACE), 313 ascorbic acid (vitamin C), 314 carotenoids, 313 content and nutritional value of, 314t tocopherols and tocotrienols, 313
314 applications, 311 beneficial lipidic components, 312
313 content and nutritional value of, 310t oleic and essential fatty acids, 312
313 phospholipids (PL), 313 phytosterols, 313 consumption and uses of, 310
311 health-promoting effects and biological activities of, 312t highly nutritious, 309 macronutrients, content and nutritional value of, 310t polar phenolic compounds, 315
316 effect of roasting, 316 phenolics, antioxidant and biological activity of, 316 total polar phenolics (TPP), 316 reactive oxygen species and oxidative stress, 311 Pistachio oil, 311 Pistacia vera L., 309 Plaque formation, 347 Plasma-free fatty acids (FFA), 171 Platelet-activating factor (PAF), 239
240 Pleurotus eryngii (king oyster mushroom), 291t Pleurotus ostreatus (oyster mushroom), 290, 291t Polar phenolic compounds, 315
316 effect of roasting, 316 phenolics, antioxidant and biological activity of, 316 total polar phenolics (TPP), 316 Polymnia edulis Wedd., 381 Polyol pathway, 29, 30f, 32 Polyphenol, 315
316 Polyphenolic compounds, 171
173, 328
330 antioxidant efficiency of, 327
328 of S. ebulus L., 323
325, 324t Polyphenols, 83, 128
129, 131, 146, 181, 192, 216, 225
226, 228t, 255
257, 290, 323, 326
327, 361, 363, 382f antioxidant activities, 228
229, 228f antioxidant and antiinflammatory properties, 227
228 chemistry, 226
227 mechanisms of action, 229f phase II gene expressions, 229f PolyQ proteins, 242 Polysaccharides, 290, 294 in tea, 362, 363t Polyunsaturated fatty acid (PUFA), 10, 15
16, 215, 238, 281
282, 358 PREDIMED study (Prevention with Mediterranean Diet), 311 Proanthocyanidins, 361, 362t Probiotics, 259, 261
262

Procarbazine, 269t Programmed neuronal death, 235
236 Prooxidants, 87
88, 90
92, 92f, 111
112, 127, 390
391 Propidium iodide (PI), 197
198 Prostaglandins (PGs), 375 Prostate cancer epigallocatechin gallate (EGCG), 219
220 lycopene, 249
250 Prostate infection, 18
19 Protein glycation, 191
192 Protein kinase C (PKC), 29, 30f, 32 Proteins, 9 Protocatechuic acid, 291t, 293
294, 324t tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity, 294 Y-maze behavioral test, 294 Proton-donating mechanisms, 208
209 Provitamin A, 313 P-selectin, 98 Psoriasis, Sambucus ebulus L., antioxidants, 322 PUFA. See Polyunsaturated fatty acid (PUFA) Pulmonary index (PI), 374 Pulmonary neutrophilic inflammation, 52
53 Pyridoxine, 292

Q Q192R polymorphism, PON1, 100
101 Quercetin, 83, 111
112, 208, 302t, 304t, 324t, 362t

R Radiation-induced breast cancer, 219t Radiation radiofrequency, 269t Radiation therapy, 18 bilberry anthocyanins, 182
183 Radioiodin, 337 Randomized controlled trials, 155 Reactive nitrogen species (RNS), 4, 5t, 7, 9, 50, 50t, 60
62, 105
106, 135
136, 235
236, 266
267, 289, 299
300, 389 cellular sources, 61t environmental sources, 61t in regulation of cellular function, 11t Reactive oxygen species (ROS), 4, 5t, 9, 50, 50t, 52
53, 58
59, 60f, 77, 90, 98
100, 105
106, 127, 130, 131f, 135
137, 146
147, 159, 170
171, 189, 192, 197, 214, 214t, 227
228, 235
236, 238, 247, 260, 265, 282
284, 285t, 289, 292, 297
298, 300
301, 346, 380, 389 ascorbic acid (vitamin C), 314 in cancer development, 109f cellular sources, 61t diabetic retinopathy (DR), 30
31 enzymatic production, 31, 31f nonenzymatic production, 30
31 environmental sources, 61t excessive, effect on, 21f inflammation, 46f on male fertility, 266f in male reproductive tract, 15
16, 17f

assessment of, 22 endogenous reactive oxygen species and antioxidants, 16
18 exogenous reactive oxygen species and antioxidants, 18 mitochondrial production of, 40
41, 41f oxidative stress and, 30
31 pistachio nut, 311 in regulation of cellular function, 11t Reactive species (RS), 88t, 299
300, 357 antioxidants with putative therapy, redox signaling, 92
93 and hypertension, 44f mitochondrial production of, 40
41, 41f tea, 358 Reactive sulfur species (RSS), 389 Receptor activator of nuclear factor kappa B ligand (RANKL), 146
147 Receptor activator of nuclear factor kappa B (RANK), 146
147 Receptor for advanced glycation end products (RAGE), 32, 42
43 Recurrent pain, 65 Redox biology in male reproduction, 16, 17f Redox hub, 280, 280f, 283 Redox signaling antioxidants, 90
92, 92f, 93f applications, 94 definition, 88 mechanisms, 88, 88t oxidative stress pathogen elimination in, 89, 90f pathogen thriving in, 89
90, 91f paradox, 88
89 perception shift, 90, 91f reactive species and antioxidants with putative therapy, 92
93 Reduced glutathione (GSH), 69 Reductive stress (RS), 22, 24 Renal diseases, lycopene, 250
252 Resveratrol, 83, 89
90, 111
112, 304t Retinol, 314t Retinopathy, 100
101 Rheumatoid arthritis, 149 Riboflavin, 292 Ribonucleic acid (RNA), 9
10 Rofecoxib, 138
139 Rosmarinic acid, 324t Runt-related transcription factor 2 (Runx2), 146 Rutin, 304t, 324t

S Saccharomyces cerevisiae, 292 Safoutier. See Dacryodes edulis Salmonella typhimurium, 89 Sambucus ebulus L., 321
322 anticancer activity, 328
329 antidepressant activity, 330 antidiabetic activity, 329
330 antioxidant potential, 326
328, 327t application, 331 chemical composition, 323
326 dominant components in essential oil, 326t ethyl-acetate extracts, 329, 331

414 Sambucus ebulus L. (Continued) flower, 322f fruit, 322f leaf, 323f polyphenolic compounds, 323
325, 324t root, 323f traditional use, 322 tyrosinase inhibition, 330 Saponifiable fraction, 226 Saponins, 206, 206t Sclerosis diseases, 201 Secoiridoids, 227, 228t Secondary brain injury, 80
81 SELblo (B-B lectin), 325
326 Selenium in Graves’ hyperthyroidism, 339
340 in Graves’ orbitopathy, 340
343, 340f, 341f intake, 339 pistachio nut, 315, 315t Selenium-(methyl)-selenocysteine (SeMCys), 340
341 Selenoglycoproteins, 339 Selenoproteins, 339 Semen and sperm quality, 272 Semifermented oolong tea, 361 Semiquinones, 56
58 Sertoli cells, 268, 271
272 Sesquiterpene lactones (SLs), 173, 384 Shikimate pathway, 290 Shizophrenia, 161
162 Short-chain fatty acids (SCFAs), 384 Sickle cell anemia (SCA), 70
72, 71t Sickle cell disease (SCD), 65
66, 66f agents targeting antioxidant defense in, 72, 72t applications, 73 clinical manifestations, 67, 68t epidemiology and global burden of, 66
67 history of, 65
66, 66f oxidative stress in, 69
72, 71t clinical consequences of, 69
72 red blood cells in coping, physiology of, 69 sickle red blood cells, physiology of, 69, 70f pathophysiology of, 67
69 Sickle Hb (HbS), 65, 67, 72
73 Silent killer. See Hypertension Silymarin, 118
119, 120t Simple steatosis, 258 Sinapic acid, 324t Singlet oxygen, 6 Sirtuin 1 (Sirt1), 42
43, 198 Sirtuin proteins (SIRTs), 33
35, 35f Sistosterol, 208 Sodium-dependent glucose transporter (SGLT-1), 208 Soluble dietary fiber, 295 Soxhlet extraction, 327
328 SoxR, 88 Sperm DNA fragmentation (SDF), 18, 21 Spermatogenesis, 16, 17f, 18, 22, 268
271 Squamous carcinoma (SiHa) cells, thymoquinone, 372 Staphylococcus aureus, 89
90

Index

STAT family proteins, modulation of, 110 Steatosis, 258 Steroidogenic acute regulatory (StAR) protein, 20 Stevia glycosides (SGs), 346 Stevia rebaudiana (Bertoni) antioxidant and bioactive compounds from, 349t antioxidant potential against free radicals and stevia potential, 347
354, 348f applications, 346 effectiveness against atherosclerosis, 347 blood glucose homeostasis, 347 cancer, 346 cariogenesis, 346
347 diabetes, 347 hypertension, 347 obesity, 347 Stilbenes, 128 Streptozotocin, 269t Sulfonylureas, 301 Sulforaphane (1-isothiocyanato-4(methylsulfinyl) butane), 84 Sulfur reactive radicals, 358 Superoxide, 7, 30
31, 31f Superoxide-centric, 4 Superoxide dismutase (SOD), 4, 40, 214
215, 280, 283, 285t, 300, 311, 337, 370
373 Superoxide dismutase-1 (SOD-1) gene, 243 Superoxide radical, 4
5 Superoxide radical scavenging activity (SRSA), 190 Synthetic lycopene, 248, 249f Syringic acid, 324t Systemic infections, 19

T Tail suspension test (TST), 162 Tannins, 128, 206, 206t, 299 Tartrate-resistant acid phosphatase (TRAP), 146 Tea antioxidants in, 359
363 application, 364
365 enzymatic antioxidants, 360 glance at tea, 360 natural dietary antioxidants, 360 nonphenolic antioxidant metabolites, 360, 362
363, 363t, 365f oxidative stress associated pathogenesis, 358
359 aging, 359 cancer, 359 cardiovascular diseases, 359 cellular damage, 358 free radicals, 358 health hazards, 358 in human biology, 358 infections, 359 metabolic syndrome, 358
359 neurodegenerative disorders, 359 reactive species, 358 phenolic antioxidant metabolites, 360
361, 362t, 363, 364f

types of, 361f Ternatin anthocyanins, 190
191 Terpenoids, 238
239 Testicular ischemia, 19 Testicular tissue protection, melatonin, 268
272 Testis, beneficial and antioxidative effects of melatonin on, 269t Tetracyclines, 197 Tetrahydrocurcumin, 121
123, 198 Tetrahydroisoquinoline alkaloids, 139 Thapsigargin, 111
112 Theaflavins, 361, 362t Thearubigins, 361, 362t Thiamine, 292 Thiobarbituric acid reactive substances (TBARs), 130, 193, 373 Thioredoxin/peroxiredoxin antioxidant system (Trx/Prx), 281
282 Thioredoxin reductase (TrxR), 315 Thioredoxin (Trx) system, 390 Thymoquinone (TQ) anticancer activity, 371
372 antidiabetic activity, 370
371 antiinflammatory activity, 374
375 antioxidant activity, 370 antioxidant effects of, 371f on APAP-induced nephrotoxicity, 374f applications, 375
376 bioavailability and kinetics, 370 cardiovascular activity, 372 chemical structure of, 369f gastrointestinal system, effects on, 372
373 and health, 370, 370f hepatoprotective effects, 373 on leukotriene C4 synthase, 375f on lipid peroxidation, 371f nephroprotective activity, 373
374 nervous system, effects on, 374 plasma glucose and insulin in diabetic rats, 372t pulmonary system, effects on, 374 toxic effects of, 376 Thyroid stimulating hormone receptor (TSHR), 335 Thyrotoxicosis, 336
337 TNF-α, 239 Tocopherols, 160
161, 313
314 Tocotrienols, 313
314 Total antioxidant capacity (TAC), 22, 271, 284, 285t Total polar phenolics (TPP), 316 Toxicity Clitoria ternatea flowers, 193 methylsulfonylmethane (MSM), 277
278, 278t Toxoplasma gondii, 89 Transferrin iron-binding capacity (TIBC), 117 Traumatic brain injury (TBI), 80
81 Trehalose, 290, 291t, 292, 294 Triterpenes, 173 Trolox, 129
130 Trolox equivalent antioxidant capacity (TEAC), 190, 193, 284, 285t, 316

415

Index

Trolox equivalents, 391 Tumor necrosis factor-α (TNFα), 146, 197, 282 Type 1 diabetes mellitus (T1D), 100
101 Type 2 diabetes (T2D), 217t, 241, 297
298, 301 bilberry anthocyanins, 183
184 Clitoria ternatea, 193
194 oxidative stress and, 301 selenium, 339 therapeutic applications of, 301
305 Type 2 sodium-dependent transporters (SVCT2), 159 Tyrosinase inhibition, Sambucus ebulus L., 330 Tyrosine nitration, 7 Tyrosol, 81
82, 230, 230f Tyrosol glucuronides and sulfates, 231

U Ubiquinol-10, 98 Ultraviolet (UV) radiation, 192 Uncoupled endothelial nitric oxide synthase, 45 Unfermented tea, 360
361 United States Food and Drug Administration (USFDA), 277
278 Unsaponifiable fraction of oil, 226 Uric acid, 215 Urinary iron excretion (UIE), 118

V Vancomycin, 197 Vanillic acid, 324t

Varicocele, 19 Vascular cell adhesion molecule-1 (VCAM1), 98 Vascular dementia, 136
137 Vascular endothelial growth factor (VEGF), 31
34, 201 bilberry anthocyanin, 182 Vascular failure, 19
20 Vascular smooth muscle cells (VSMC), 171 Vaso-occlusive crises (VOC), 66
69 Virgin nut oils, 310
311 Virgin olive oil, 313 Virgin pistachio oils (VPOs), 313
314, 316 Vitamin A, 23 Vitamin C (ascorbic acid), 69, 160
161, 215
216, 257, 314, 314t, 363, 390 antidepressant-like effect of, 163f applications, 164 dry form of, 159 iron chelator, oxidative stress under ironoverloaded conditions, 118, 119t neurodegenerative diseases, applications in, 160
161, 164f Alzheimer’s disease, 160 amyotrophic lateral sclerosis, 161 multiple sclerosis, 161 Parkinson’s disease, 160
161 neuroprotective effect of, 161f in neuropsychiatric disorders, 161
163 anxiety disorders, 163 bipolar disorder, 162
163 major depressive disorder, 162 schizophrenia, 161
162

oxidation process, 160f in psychiatric diseases, 164f Vitamin D2, 291t, 292 Vitamin D deficiency, 292 Vitamin E, 120
121, 122t, 159, 214
216, 225
226, 313
314, 314t

W Wallerian degeneration, 236 Whiskers-cut model, 185 White tea, 360 Wnt signaling, 146
147

X Xanthine oxidase (XO), 44
45, 128
129

Y Y-maze behavioral test, protocatechuic acid, 294 Yacon (Smallanthus sonchifolius), 381f applications, 384 hydroethanolic extract of, 383 inulin structure in, 382f origins and ethnobotanical characteristics, 380
381 polyphenols profile in, 382f roots and its nutritional composition, 382f, 382t

Z Zinc, pistachio nut, 315, 315t