Recent Frontiers of Phytochemicals: Applications in Food, Pharmacy, Cosmetics and Biotechnology 9780443191435

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Recent Frontiers of Phytochemicals Applications in Food, Pharmacy, Cosmetics, and Biotechnology

Recent Frontiers of Phytochemicals

Applications in Food, Pharmacy, Cosmetics, and Biotechnology

Edited by Siddhartha Pati NatNov Bioscience Private Limited, Balasore, Odisha, India

Tanmay Sarkar Malda Polytechnic, West Bengal State Council of Technical Education, Government of West Bengal, Malda, West Bengal, India

Dibyajit Lahiri Department of Biotechnology, University of Engineering and Management, Kolkata, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2023 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. ISBN: 978-0-443-19143-5 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Candice G. Janco Acquisitions Editor: Gabriela D. Capille Editorial Project Manager: Dan Egan Production Project Manager: R. Vijay Bharath Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Dedication Dr. Siddhartha Pati wants to dedicate the book to his mother, Mrs. Rajeswari Pati; father, Mr. Trilochan Pati; and wife, Mrs. Rosina Rosalin. Dr. Tanmay Sarkar wants to dedicate the book to his mother, Mrs. Arati Sarkar; father, Mr. Bholanath Sarkar; and wife, Mrs. Kohima Kirtonia. Dr. Dibyajit Lahiri wants to dedicate the book to his late grandmother, Mrs. Ila Guha; mother, Mrs. Madhumita Lahiri; father, Mr. Debasish Lahiri; and wife, Mrs. Ankita Lahiri.

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Contents List of contributors Preface Acknowledgment

xix xxv xxvii

1. Extraction, isolation, and characterization of phytochemicals, the bioactive compounds of plants

1

D. Sruthi, M. Dhanalakshmi, H.C. Yashavantha Rao, Ramalingam Parthasarathy and C. Jayabaskaran 1.1 Introduction 1.2 Extraction of phytochemicals 1.2.1 Solvent extraction method 1.2.2 Steam distillation method 1.2.3 Pressing method 1.2.4 Sublimation method 1.3 Isolation and purification of phytoconstituents 1.3.1 Classical isolation methods 1.3.2 Modern separation technologies 1.4 Identification of phytochemicals 1.4.1 Spectral technologies 1.5 Conclusion Acknowledgments References

2. Importance and extraction techniques of functional components

1 1 1 3 3 3 4 4 5 6 6 7 7 8

9

Amreen S. Quadri, Aniket P. Sarkate, Nilesh Prakash Nirmal and Bhagwan K. Sakhale 2.1 Introduction 2.1.1 Phytochemicals and their therapeutic effect 2.1.2 Phytochemicals from different food sources 2.2 Current techniques for extraction of phytochemicals 2.2.1 Conventional methods of extraction 2.2.2 Nonconventional methods for plant extraction 2.3 Characterization of phytochemicals 2.3.1 Determination of total flavonoid content (TFC)

9 9 10 21 21 22 23 23

2.3.2 Determination of total phenolic content (TPC) 2.3.3 Antioxidant activity—DPPH scavenging method (AA) 2.3.4 Antimicrobial activity 2.3.5 Assessment of minimum inhibitory concentration (MIC) 2.3.6 Antioxidant capacity 2.4 Conclusion 2.5 Future considerations for effective extraction of phytochemicals References

3. Novel extraction conditions for phytochemicals

23 23 23 24 24 24 24 24

27

Manas Ranjan Senapati and Prakash Chandra Behera 3.1 Introduction 3.2 Pre-extraction conditions 3.2.1 Collection 3.2.2 Drying 3.2.3 Grinding 3.2.4 Storage 3.3 Selecting a pre-extracting sample preparation 3.3.1 Fresh or dried samples 3.3.2 Ground or powdered samples 3.4 Extraction conditions 3.4.1 Solvent system for extraction 3.4.2 Extraction methods 3.5 Selection approach for a suitable extraction method 3.6 Conclusion References

4. Novel extraction and characterization methods for phytochemicals

27 29 29 31 32 32 32 32 32 33 33 34 50 50 54

63

Ratnnadeep C. Sawant, Subhash R. Somkuwar, Shun-Yuan Luo, Rahul B. Kamble, Deepa Y. Panhekar, Yeshwant R. Bhorge, Rupali R. Chaudhary and S. Abdul Kader 4.1 Introduction

63 vii

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Contents

4.2 Extraction methods 4.2.1 Introduction 4.2.2 Organic solvent extraction 4.2.3 Modified percolation 4.2.4 Extraction with supercritical gases 4.2.5 Direct steam distillation 4.2.6 Microwave-assisted extraction 4.2.7 Other extraction methods 4.2.8 Extraction of essential oil 4.2.9 Accelerated solvent extractor 4.3 Separation techniques 4.3.1 Introduction 4.3.2 Chromatographic techniques 4.4 Applications of chromatography techniques 4.4.1 Non-chromatographic techniques 4.5 Characterization methods 4.5.1 Introduction 4.5.2 Gas chromatogram 4.5.3 UV and visible spectrum 4.5.4 1H-NMR and 13C-NMR spectra 4.5.5 Mass spectrometry 4.5.6 GC
Ms spectrum 4.5.7 Two-dimensional NMR spectrum 4.5.8 X-ray spectroscopy 4.6 Conclusions and future directions References

5. Phytochemicals: recent trends in food, pharmacy, and biotechnology

63 63 64 66 67 67 67 67 68 68 68 68 68 74 75 75 75 77 78 78 79 79 80 81 81 82

85

Ayushman Gadnayak and Budheswar Dehury 5.1 Introduction 5.2 Bioactive phytochemicals 5.3 Antioxidant and antimicrobial properties of phytochemicals 5.4 Phytochemicals from the agri-food by-products 5.4.1 Phenolic compounds 5.4.2 Dietary fiber 5.5 Pharmacological aspects of phytochemicals 5.5.1 Elaeagnus angustifolia 5.5.2 Lawsonia inermis 5.5.3 Holarrhena antidysenterica (L.) 5.6 Nanodrug delivery of the phytochemicals in treating cancer 5.7 Current limitations and future of phytochemicals 5.8 Conclusion and future prospect References

85 85 86 87 88 88 88 88 88 88 89 91 92 92

6. Phytochemicals as bioactive ingredients for functional foods

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R.S. Agrawal, R.C. Ranveer, N.B. Rathod and Nilesh Prakash Nirmal 6.1 Introduction 6.2 Phytonutrients 6.3 Health-promoting ability of phytochemicals 6.4 Biological activities of phytochemicals 6.4.1 Antioxidant 6.4.2 Immunity booster 6.4.3 Anticholesteremic 6.4.4 Antidiabetic 6.4.5 Anticancer 6.4.6 Renoprotective 6.4.7 Neuroprotective 6.4.8 Antiviral (special reference to SARS-CoV-2) 6.5 Phytochemicals-based functional foods 6.5.1 Functional drinks/beverages 6.5.2 Functional bakery and confectionery 6.5.3 Functional dairy products 6.5.4 Meat products 6.6 Future perspective 6.7 Conclusion References

7. Exploring the role of Mahua as a functional food and its future perspectives

95 95 96 98 98 98 100 101 101 102 102 102 103 103 104 104 105 105 105 106

109

Monika Mishra, Subhaswaraj Pattnaik, Harvinder Singh and Pradeep Kumar Naik 7.1 Introduction 7.1.1 Nontimber forest products 7.1.2 Botanical description 7.1.3 Microscopy of mahua 7.1.4 Uses of different parts of mahua 7.2 Traditional uses 7.3 Nutritional and phytochemical profiling 7.3.1 Nutritional analysis of mahua 7.3.2 Comparative nutritional profile 7.3.3 Effect of geographical distribution on the flower composition 7.4 Pharmaceutical uses and pharmacological importance 7.4.1 Industrial uses 7.4.2 Biodiesel 7.4.3 Biological activity 7.5 Mahua as a functional food 7.5.1 Processing of flowers

109 109 109 110 110 110 111 111 112 112 112 112 113 113 116 116

Contents

7.5.2 Value-added food products 7.5.3 Health benefits of mahua 7.6 Current trends and future perspectives Acknowledgment References

117 118 118 118 118

8. Functional beverages: an emerging trend in beverage world 123 Namrata A. Giri, Bhagwan K. Sakhale and Nilesh Prakash Nirmal 8.1 Introduction 8.1.1 Need for functional beverage 8.1.2 Classification of beverage 8.1.3 Types of beverages 8.2 Market of nutraceutical or functional beverages 8.3 Soft drinks 8.4 Nonalcoholic beverages 8.4.1 Cereal-based fermented nonalcoholic beverages 8.4.2 Market of nonalcoholic beverages 8.5 Probiotics beverages 8.6 Fruits-based beverages 8.7 Fermented beverages 8.8 Whey-based beverages 8.9 Micronutrient-fortified beverage 8.10 Beverages rich in antioxidants and herbs 8.11 Prebiotic beverages 8.12 Sports or energy drinks 8.13 Storage study of beverages 8.14 Health safety of drinks 8.15 Consumer demand for beverages References

123 123 124 124 125 125 126 126 128 128 130 132 132 133 134 136 137 138 138 138 138

9. Recent targeted discovery of phytomedicines to manage endocrine disorder develops due to adapting sedentary lifestyle 143 Vijeta Bhattacharya, Namrata Mishra, Radha Sharma, Subodh Kumar Dubey, Balakumar Chandrasekaran and Mohammad F. Bayan 9.1 Introduction 9.1.1 Introduction of endocrine glands and endocrine hormones. What is endocrine system 9.1.2 Introduction of endorinological disorders such as thyroid, diabetes mellitus, polycystic ovarian syndrome

143

143

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9.1.3 Why endocrine disorder occurs due to sedentary lifestyle 9.1.4 Relation between endocrine disorder and sedentary lifestyle 9.1.5 Physiology and mechanism behind endocrine disorder 9.2 Concept of herbal targeted drug delivery 9.3 List of most effective phytochemicals/ phytomedicinal herbs 9.3.1 Used to treat endocrine disorder with the help of targeted drug delivery 9.4 List of novel phytomedicinal formulations in pharmacy to target the endocrine glands and hormone for the treatment of various major endocrine disorders 9.4.1 Types of novel herbal drug delivery systems 9.5 Application of phytomedicine in modern drug development in pharmacy 9.6 Advantages of herbal phytomedicines in modern system 9.6.1 Factors responsible for increased self-medication with herbal medicine 9.7 Conclusion References

10. Current updates on phytopharmaceuticals for cancer treatment

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149 150 151 154 155

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155 157 158 159

159 159 160

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Anshita Gupta Soni, Srushti Mahajan and Pankaj Kumar Singh 10.1 Introduction 10.2 Phytochemicals unexplored 10.3 Molecular mechanism of phytochemicals in preventing cancer 10.3.1 Targeting molecular pathway of cancerous cell 10.3.2 Targeting cell proliferation 10.3.3 Targeting oxidative stress and redox signaling 10.3.4 Genome instability 10.3.5 Modulation of membrane 10.3.6 Targeting immune surveillance and inflammation 10.3.7 Apoptosis and autophagy 10.4 Strategies to improve phytochemical drugability 10.5 Drug delivery approach to improve phytochemical drugability

163 164 169 169 169 169 169 170 170 170 170 172

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Contents

10.6 Phytochemicals in clinical and preclinical stages for preventing cancer 10.7 Insights on phytochemicals as dietary recommendation in cancer 10.8 Conclusion and future perspectives References

11. Phytochemicals in prostate cancer

172 172 175 176

179

Abdel Rahman Al Tawaha, Rose Abukhader, Ali Qaisi, Abhijit Dey, Siddhartha Pati, Abdel Razzaq Al-Tawaha, Iftikhar Ali and Mohamad Shatnawi 11.1 Introduction 11.2 Types of prostate cancer 11.2.1 Small-cell carcinoma 11.2.2 Neuroendocrine prostate tumor 11.2.3 Transitional cell carcinomas of prostate gland 11.2.4 Sarcomas of prostate glands 11.3 Causes of prostate cancer 11.3.1 General causes of prostate cancer 11.3.2 Genetic causes of prostate cancer 11.4 Symptoms of prostate cancer 11.4.1 Advanced symptoms 11.5 Test to identify prostate cancer 11.6 Prostate cancer treatments 11.6.1 Surgery 11.6.2 Radiation 11.6.3 Proton beam radiation 11.6.4 Hormone therapy 11.6.5 Chemotherapy 11.6.6 Immunotherapy 11.6.7 Bisphosphonate therapy 11.6.8 Cryotherapy 11.6.9 High-intensity focused ultrasound 11.6.10 Prostate cancer vaccine 11.7 Prevention of prostate cancer 11.7.1 Physical activity, diet, and body weight 11.7.2 Mineral, vitamins, and supplements 11.7.3 Medicines 11.7.4 5-Alpha-reductase inhibitors 11.7.5 Aspirin 11.8 Phytochemicals in prostate cancer 11.9 Phytochemicals and conventional medical practice 11.10 Effects of specific plant families extracts on human prostate cancer cells 11.10.1 Juglandaceae

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

11.10.2 Crassulaceae 11.10.3 Moraceae 11.11 Prostate cancer risk factors 11.11.1 Age 11.11.2 Race 11.11.3 Diet 11.11.4 Obesity 11.11.5 Environmental exposures 11.11.6 The past of the family 11.12 Conclusion References Further reading

12. Therapeutic phytochemicals from Plumbago auriculata: a drug discovery paradigm

185 185 185 185 185 186 186 186 186 186 187 187

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Khalida Bloch, Vijay Singh Parihar, Minna Kelloma¨ki, Sirikanjana Thongmee and Sougata Ghosh 12.1 12.2 12.3 12.4 12.5

Introduction Traditional uses Phytochemistry Plumbagin Medicinal uses 12.5.1 Antimicrobial activity 12.5.2 Anticancer and cytotoxic activity 12.5.3 Antioxidant activity 12.5.4 Antiobesity 12.5.5 Antiulcer activity 12.6 Nano-biotechnology 12.7 Other properties 12.8 Future perspectives 12.9 Conclusion Acknowledgments References

13. Alkaloids as potential anticancer agent

189 189 190 192 193 193 193 193 194 195 195 198 198 199 199 199

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Mayuri A. Patil, Aniket P. Sarkate, Nilesh Prakash Nirmal and Bhagwan K. Sakhale 13.1 Introduction 13.2 Theoretical relevance 13.2.1 Types of alkaloids 13.2.2 Targeted pathways in cancer treatment 13.3 Biological source, mechanism of action, and applications of indole alkaloids 13.3.1 Vinblastine 13.3.2 Vincristine 13.3.3 Vindesine 13.3.4 Vinflunine 13.3.5 Camptothecin

203 203 203 204 205 205 206 207 208 209

Contents

13.3.6 Montamine 13.4 Biological source, mechanism of action, and applications of isoquinoline alkaloids 13.4.1 Berberine 13.4.2 Noscapine 13.4.3 Liriodenine 13.4.4 Sanguinarine 13.5 Biological source, mechanism of action, and applications of Taxus alkaloid 13.5.1 Taxol 13.6 Aporphinoid alkaloids 13.7 Emetine and related alkaloids 13.8 Biological source, mechanism of action, and applications of Cephalotaxus alkaloids 13.8.1 Cephalotaxine 13.8.2 Homoharringtonine 13.9 Biological source, mechanism of action, and applications of pyrrolizidine alkaloids 13.9.1 Clivorine 13.10 Anticancer alkaloids with future perspective References

14. Potential phytochemicals as microtubule-disrupting agents in cancer prevention

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210 211 212 213 213 214 214 215 216

217 217 218

219 219 219 219

225

Showkat Ahmad Mir, Archana Padhiary, Ashwariya Pati, Sheary Somam Tete, Rajesh Kumar Meher, Iswar Baitharu, Auwal Muhammad and Binata Nayak 14.1 Introduction 14.2 Molecular basis of microtubule dynamics 14.3 Factors affecting microtubule dynamics in cancer cells 14.4 Intracellular stress in cancer 14.5 Targeting microtubules in cancer 14.5.1 Ovarian cancer 14.5.2 Colon cancer 14.5.3 Breast cancer 14.5.4 Oral Squamous Cell Carcinoma 14.6 Alkaloids as microtubulin-disrupting agents 14.6.1 Vinca alkaloids 14.6.2 Vinca alkaloids and their mechanism of action against microtubulin

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14.6.3 Vinca domain 14.6.4 Therapeutic relevance 14.6.5 Side effects of vinca alkaloids 14.7 Taxol as a therapeutic agent disrupting cell polymerization 14.7.1 Mechanism of action of taxol phytochemicals 14.7.2 Interplay of taxanes with microtubule site 14.7.3 Therapeutic relevance of taxol concerning microtubulin dynamics 14.7.4 Side effects of taxanes on treated patients 14.8 Colchicine as a microtubule-disrupting agent 14.8.1 Mechanism of action 14.8.2 Colchicine binding site and their interplay with the microtubule 14.8.3 Therapeutic relevance 14.8.4 Side effects of colchicine 14.9 Curcumin, a phenolic compound, disrupts microtubule function 14.9.1 Mechanism underlying polyphenols as microtubulinbinding target 14.9.2 Binding of curcumin polyphenol with microtubule 14.9.3 Therapeutic relevance of curcumin against microtubule 14.9.4 Side effects of curcumin 14.10 Noscapine therapeutic agents disrupting microtubule dynamics 14.10.1 Mechanism of action 14.10.2 Noscapine binding site 14.10.3 Therapeutic relevance of noscapine against cancer 14.10.4 Toxicity remarks of noscapine on subjected patients 14.11 Coumarin’s background and therapeutic activities 14.11.1 Mechanism and binding site against microtubule 14.11.2 Therapeutic relevance 14.11.3 Toxicity remarks of coumarin and its analogs 14.12 Discussion 14.13 Conclusion Acknowledgment References

xi

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233 233 233 234

234 234 235 235

235 236 236 236 236 237 237 238 238 238 239 239 239 239 240 241 241

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15. Therapeutic effectiveness of phytochemicals targeting specific cancer cells: a review of the evidence

247

Pooja Ravi, Mona Isaq, Yarappa Lakshmikant Ramachandra, Prathap Somu, Padmalatha S. Rai, Chandrappa Chinna Poojari, Kumar Hegde Biliyaru Anand, K. Shilali, Asma Musfira Shabbirahmed and Mohanya Kumaravel 15.1 Introduction 15.2 Strategies for identification of phytochemicals with pharmaceutical potential 15.3 Perceptions of phytochemicals as anticancer agents in the history 15.4 Synthetic analogs for plant-derived compounds: enhancement and application 15.5 Classification of phytochemicals 15.5.1 Alkaloids 15.5.2 Polyphenol 15.5.3 Terpenoid 15.5.4 Thiols 15.6 Plant-derived phytochemicals currently in use for various cancer treatments 15.7 Curcumin 15.8 Quercetin 15.9 Vinca alkaloids 15.10 Camptothecin 15.11 Cervical cancer and phytochemicals 15.12 Current scenario and future perspective Competing interests References

247

248 249

250 250 250 250 251 251

251 251 255 255 255 255 257 257 257

16.3.3 Shortcomings of conventional therapy: hormone therapy (endocrine therapy) 16.4 Role of complementary and alternative medicine (CAM) in breast cancer treatment 16.5 Phytochemicals: traversing a new window in breast cancer therapy 16.5.1 Alkaloids 16.5.2 Terpenoids 16.5.3 Flavonoids 16.5.4 Carotenoids 16.5.5 Phytosterols and phytostanols 16.5.6 Cardiac glycosides 16.6 Phytochemicals and ER(1) breast cancer 16.7 Phytochemicals and HER(2) breast cancer 16.8 Phytochemicals used for triplenegative breast cancer (TNBC) 16.9 Role of phytochemicals in modulating noncoding RNA expression in BC cells 16.10 Phytochemical interventions in healing cancer-associated MDR 16.10.1 Secondary metabolites and ABC transporters: a tale of super cross-opposition 16.11 Diet and dietary phytochemicals in chemosensitization 16.12 Challenges and perspectives: into the future of BC phytochemical interventions 16.13 Conclusion References

17. Phytochemicals and cancer

265

266 267 267 269 271 271 273 274 274 276 276 276 278

281 283

284 285 285

295

Mayuri Iyer, Kavita Pal and Vijay Upadhye

16. Understanding the role of the natural warriors: phytochemicals in breast cancer chemoprevention 261 Prarthana Chatterjee, Suchetana Gupta and Satarupa Banerjee 16.1 Introduction 16.2 Breast cancer: definition, subtypes, and conventional therapies 16.3 Perils of conventional BC therapies 16.3.1 Shortcomings of conventional therapy: chemotherapy 16.3.2 Shortcomings of conventional therapy: radiotherapy

261 262 263 263 265

17.1 Introduction 17.1.1 Terpenes (isoprenoids) and terpenoids 17.1.2 Polyphenols 17.1.3 Alkaloids and other nitrogencontaining constituents 17.2 Role of phytochemicals in various diseases 17.2.1 Diabetes 17.2.2 Hypertension 17.2.3 Cardiovascular disorders 17.2.4 Neurodegenerative disorders 17.2.5 Inflammatory bowel disease (IBD)

295 295 295 296 296 296 297 297 297 298

Contents

17.3 Phytochemicals in cancer 17.3.1 Phytochemicals in chemoprevention 17.3.2 Phytochemicals as chemotherapeutic agents 17.3.3 Phytochemical in alleviation of chemotoxicity 17.3.4 Phytochemical in conjugation with chemotherapy: a synergistic anticancer effect References

300 300 303

Sachin Puri, Namita Hegde, Siddhi Sawant, Ganesh Latambale and Kapil Juvale

303

19.1 Introduction 19.1.1 Importance of phytochemicals 19.1.2 Classification of phytochemicals source and their effectiveness against cancer 19.1.3 Phytochemicals currently in use as cancer therapeutics 19.1.4 Flavonoids—introduction and classification with their chemical structure 19.1.5 Mechanism action of flavonoids 19.1.6 Flavonoid compounds for anticancer activity 19.1.7 Future prospects of phytochemicals in cancer treatment 19.2 Conclusion References

303 304

18. Phytochemicals as a complementary alternative medicine in cancer treatment 309 Kajari Das, M. Dhanalakshmi, Medha Pandya, D. Sruthi and Sushma Dave 18.1 Introduction 18.2 Role of oxidative stress in carcinogenesis 18.2.1 Oxidative stress and antioxidant defense mechanism 18.2.2 ROS-dependent cellular metabolic pathways in cancer cells 18.2.3 Plant-derived antioxidants for the amelioration of oxidative stress 18.3 Mode of action of phytochemicals for cancer prevention by targeting cellular signaling transduction pathways 18.3.1 Anti-inflammatory targets 18.3.2 Growth factor signaling targets 18.3.3 Apoptosis targets 18.3.4 Targets of phytochemicals in cell cycle pathways 18.3.5 Targets in other important pathways 18.4 A historical perspective of plant-derived drugs used popularly in cancer 18.4.1 Important secondary metabolites in cancer treatment 18.4.2 Other important phenolic compounds studied on cancer targets 18.4.3 Phytochemicals in clinical trials 18.4.4 Common dietary phytochemicals 18.5 Phytochemicals induce cancer cell apoptosis and autophagy 18.6 Gut microbiota in gastrointestinal malignancy—a potential target for phytotherapy 18.7 Plant-derived drugs 18.8 Conclusion 18.9 Challenges References

19. Applications of phytochemicals in cancer therapy and anticancer drug development

309 310 310 311 311

313 314 315 315 316 317 318 320

322 325 325 325

326 329 329 329 330

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335 337

338 339

342 343 344 345 345 346

20. Bioactivity, medicinal applications, and chemical compositions of essential oils: detailed perspectives 353 Sonali S. Shinde, Aniket P. Sarkate, Nilesh Prakash Nirmal and Bhagwan K. Sakhale 20.1 Introduction 20.2 Chemistry of essential oils 20.2.1 Terpenes 20.2.2 Phenylpropanoids 20.2.3 Nitrogen- and sulfur-containing compounds in essential oils 20.3 Biological activity of essential oils 20.3.1 Introduction 20.3.2 Antimicrobial activity 20.3.3 Anticancer activity 20.4 Medicinal applications of essential oils 20.5 Conclusion References

21. Biological potential of essential oils in pharmaceutical industries

353 353 354 358 359 360 360 360 361 362 363 363

369

M. Anjaly Shanker, Anandu Chandra Khanashyam, Priyamvada Thorakkattu and Nilesh Prakash Nirmal 21.1 Introduction 21.2 Bioactive components of essential oils 21.3 Biological activities of EO 21.3.1 Antimicrobial properties

369 370 372 372

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21.4 Cancer-preventing function 21.5 Antioxidant and anti-inflammatory properties 21.6 Role in cardiovascular diseases 21.7 Antidiabetic agents 21.8 Other important properties 21.9 Application of EO in pharmaceutical industry 21.10 Future perspective and conclusion References

374 374 375 375 377 377 378 378

22. A review on marine-based phytochemicals and their application in biomedical research 383 Rousan Khatun, Sikha Singh, Navneet Kumar Dubey and Alok Prasad Das 22.1 Introduction 22.2 Phytochemicals from marine resources 22.3 Metabolic process to form marine phytochemicals 22.4 Bioactive potential of marine phytochemical 22.4.1 Antibacterial activity 22.4.2 Antifungal activity 22.4.3 Antiviral agent 22.4.4 Anticancer agents 22.5 Biomedical applications of marine phytochemicals 22.5.1 Pharmaceuticals 22.5.2 Therapeuticals 22.5.3 Nutraceuticals 22.6 Conclusion References

23. Phytochemicals in biofilm inhibition

383 384 387 387 388 388 389 389 390 390 391 391 392 393

397

Anandu Chandra Khanashyam, M. Anjaly Shanker, Pinchu Elizabath Thomas, Karthik Sajith Babu and Nilesh Prakash Nirmal 23.1 23.2 23.3 23.4

Introduction Biofilm formation Inactivation mechanism of biofilm Role of phytochemicals in biofilm inhibition 23.5 Phenolics 23.6 Terpenoids 23.7 Organic acids 23.8 Other phytochemicals 23.8.1 Alkaloids 23.9 Sulfur- and nitrogen-containing phytochemicals 23.10 Future perspective and conclusion References

397 397 399 401 401 405 406 407 407 407 408 408

24. New perspectives and role of phytochemicals in biofilm inhibition

413

Pravin R. Vairagar, Aniket P. Sarkate, Nilesh Prakash Nirmal and Bhagwan K. Sakhale 24.1 Introduction 24.2 Biofilm development and its health hazards 24.2.1 Factors influencing biofilm development 24.2.2 Stages in biofilm development 24.2.3 Microorganisms associated with biofilms and their health hazards 24.3 Occurrence of biofilms 24.3.1 Biofilm on food contact surfaces 24.3.2 Biofilms in food products 24.4 Phytochemicals in biofilm inhibition 24.4.1 Phytochemicals associated with biofilm inhibition 24.4.2 Mode of action of phytochemicals on biofilm 24.4.3 Target areas of phytochemicals 24.5 Conclusion References

413 413 414 415 416 418 418 419 420 420 424 424 425 425

25. Novel perspectives on phytochemicalsbased approaches for mitigation of biofilms in ESKAPE pathogens: recent trends and future avenues 433 Subhaswaraj Pattnaik, Monika Mishra, Harvinder Singh and Pradeep Kumar Naik 25.1 Introduction 25.1.1 An introduction to biofilm and historical perspectives 25.1.2 An insight into the process of biofilm formation 25.1.3 Ultrastructure of biofilm communities 25.1.4 Impact of bacterial biofilm 25.2 Biofilm-mediated drug resistance in ESKAPE pathogens 25.2.1 Regulation of specific virulence genes associated with biofilms 25.3 Mitigation of biofilm architecture: current therapeutic trends 25.3.1 Synthetic and semisynthetic derivatives as biofilm inhibitors 25.3.2 Microbial secondary metabolites for biofilm inhibition 25.4 Phytochemicals-based mitigation strategies against biofilm formation

433 433 434 434 435 435 436 436 437 437 438

Contents

25.4.1 Crude plant extracts against biofilm formation in ESKAPE pathogens 25.4.2 Phytochemicals involved in the inhibition of biofilm formation in ESKAPE pathogens 25.5 Current trends in biofilm inhibition 25.5.1 In silico approaches for phytochemicals-based mitigation of biofilm formation 25.5.2 Nano-based formulation using plant-derived phytochemicals for biofilm inhibition 25.6 Future perspectives Key points Acknowledgment References

26. Phytochemicals in downregulation of quorum sensing

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438 445

445

445 446 446 447 447

455

Ipsita Mohanty, Rojita Mishra, Amrita Kumari Panda, Arabinda Mahanty and Satpal Singh Bisht 26.1 Introduction 455 26.2 Biofilm formation and quorum sensing 455 26.3 Mechanism of quorum sensing in bacteria 456 26.4 Phytochemicals as quorum-sensing inhibitors 457 26.4.1 Grouping of phytochemicals as QS inhibitors 457 26.4.2 Taxa and habitats intersected and interacted with QS inhibition 459 26.4.3 Necessities and low falls in QS inhibition 459 26.5 Clinical studies 459 26.6 Mechanism of phytochemicals involved in quorum-sensing inhibition 459 26.7 Conclusion 459 Acknowledgment 461 References 461

27. Phytoconstituents-based nanoformulations for neurodegenerative disorders

463

Mithun Singh Rajput, Nilesh Prakash Nirmal, Viral Patel, Purnima Dey Sarkar and Manan Raval 27.1 Introduction 27.2 Key issues associated with neurodegenerative diseases 27.3 Significance of nanotechnology in neurodegenerative disorders: incapacitating the blood
brain barrier

27.4 Phytoconstituents and their general mechanism of actions pertaining to neuroprotection 27.5 Phyto-nanomedicine in the management of neurodegenerative disorders 27.6 Nanoformulations in tackling neurodegeneration: preclinical proofs 27.6.1 Phytoconstituents-based nanoformulations for Alzheimer’s disease 27.6.2 Phytoconstituents-based nanoformulations for Parkinson’s disease 27.6.3 Phytoconstituents-based nanoformulations for amyotrophic lateral sclerosis 27.6.4 Phytoconstituents-based nanoformulations for stroke (cerebral ischemia) 27.6.5 Phytoconstituents-based nanoformulations for other neurodegenerative diseases 27.7 Limitations of nanotechnology-based approaches for management of neurodegenerative disorders 27.8 Future outlook and conclusion References

28. Oxidative stress and its management through phytoconstituents

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465

465 466 466

469

471

472

472

473

476 477 477

483

Prakash Chandra Behera and Manas Ranjan Senapati 28.1 Introduction 28.2 Oxidative stress and free radicals 28.2.1 Effect of oxidative stress 28.2.2 Defense of oxidative stress 28.3 Antioxidants 28.3.1 Phytoconstituent as antioxidant 28.4 Antioxidative effect of phytoconstituents 28.4.1 Mechanism of action 28.5 Conclusion References

29. Phytochemicals: an immune booster against the pathogens 463

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483 483 484 485 486 486 488 490 492 493

501

Kena Premshankar Anshuman 29.1 Introduction 29.2 Secondary metabolites 29.2.1 Phenolic compounds 29.2.2 Phytoestrogens

501 502 502 502

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29.2.3 Flavonoids 29.2.4 Alkaloids 29.2.5 Terpenes 29.2.6 Carotenoids 29.2.7 Phytosterols 29.3 Phytotherapy 29.4 Phytomedicine 29.5 SARS-CoV-2 References

30. Phytochemicals: recent trends and future prospective in COVID-19

503 503 503 504 504 504 505 505 505

511

Dhwani Upadhyay, Arti Gaur, Maru Minaxi, Vijay Upadhye and Prasad Andhare 30.1 Introduction 30.1.1 SARS-CoV-2 and COVID-19 30.1.2 Plants’ role in COVID-19 treatment 30.1.3 Phytochemicals and their role in COVID-19 30.1.4 List of various targetable sites in SARS-CoV-2 infection with human cell 30.2 Virus-based targets 30.2.1 Structural-based proteins 30.2.2 Nonstructural proteins 30.3 Host-based targets 30.3.1 Host proteins 30.3.2 Epigentic mechanism 30.3.3 Pathways 30.3.4 Effects of phytochemicals from honey against COVID-19 30.4 Conclusion and future prospective References

511 511 511 512

512 513 513 515 516 516 517 517 521 522 528

31. Phytochemicals—a safe fortification agent in the fermented food industry 535 Renitta Jobby, Sneha P. Nair, Vaishnavi Murugan, Simran Khera and Kanchanlata Tungare 31.1 Introduction 31.2 Types of phytochemicals 31.2.1 Alkaloids 31.2.2 Polyphenols 31.2.3 Terpenoids 31.2.4 Organosulfur compounds 31.2.5 Phytosterols 31.2.6 Carotenoids 31.2.7 Other phytochemicals 31.3 Health benefits of phytochemicals 31.3.1 Oxidative stress amelioration 31.3.2 Reducing inflammation

535 535 535 536 536 536 536 536 536 536 537 537

31.3.3 Cardiovascular protection 31.3.4 Anti-obesity activity 31.3.5 Anti-diabetes activity 31.3.6 Anticancer activity 31.3.7 Antimicrobial activity 31.4 Fortification in the fermentation industry 31.4.1 Vitamin fortification 31.4.2 Iron fortification 31.4.3 Calcium fortification 31.4.4 Fortification with phenolics 31.5 Effect of fermentation on phytochemicals 31.6 Use of phytochemicals as a safe fortifying agent 31.6.1 Cantaloupe (C. melon) incorporated into yogurt 31.6.2 Soy isoflavones used in the fermentation of probiotics and beverages 31.6.3 Whole-bread preparation using cupuassu (Theobroma grandiflorum) peel 31.7 Limitations 31.8 Conclusion References

537 537 537 537 538 538 539 539 539 539 540 541 541

541

541 542 542 542

32. Molecular docking study of bioactive phytochemicals against infectious diseases 545 Sanjeev Kumar Sahu, Thatikayala Mahender, Iqubal Singh, Pankaj Wadhwa, Paranjeet Kaur and Kuldeep Bansal 32.1 Introduction 32.1.1 Molecular docking 32.2 Molecular docking studies of plant products as anti-coronal agents 32.3 Molecular docking studies of plant products as anti-leishmanial agents 32.4 Molecular docking studies of plant products as antitubercular agents 32.5 Conclusion References

33. Phytochemicals in structure-based drug discovery

545 545 546 550 557 566 566

569

Amit Kumar, Jaya Baranwal, Amalia Di Petrillo, Sonia Floris, Brajesh Barse and Antonella Fais 33.1 Introduction 33.1.1 Phytochemicals—medicinal properties

569 570

Contents

33.2 Phytochemicals screening of plant extracts 33.3 Phytochemicals from Phytolacca dioica L. seeds extracts—case study I 33.4 Phytochemicals composition and biological properties of seed extracts from Washingtonia filifera—case study II 33.5 Phytochemicals—opportunities and challenges 33.5.1 Phytochemicals as vegan food ingredients 33.5.2 Plant-based ingredients 33.5.3 Dietary supplements 33.5.4 Effect of COVID-19 on phytochemicals demand 33.5.5 Transfer of phytochemicals into pharmaceuticals—Challenges References

34. Modulation of drug resistance in leukemia using phytochemicals: an in-silico, in-vitro, and in-vivo approach

571 574

575 579 579 579 580 580 580 580

583

Urja Desai, Medha Pandya, Hiram Saiyed and Rakesh Rawal 34.1 Introduction 34.2 Drug resistance: therapeutic failure in leukemia 34.2.1 Proteins/genes responsible for drug-resistance leukemia 34.3 Combination index method and synergism 34.4 Phytochemicals as chemosensitizer and modulators 34.4.1 Computational approach to target multidrug resistance 34.4.2 In vitro analysis of phytochemicals as multidrug resistance reversal 34.4.3 In vivo analysis of phytochemicals as multidrug resistance-reversing agents 34.5 Conclusions and future prospects Acknowledgment References

35. Phytochemical and bioactive potentialities of Melastoma malabathricum

583 585 586 587 588 588 590

593 594 595 595

601

Mansi Tiwari, Mridula Saikia Barooah and Deepjyoti Bhuyan 35.1 Introduction 35.2 Ethno-medicinal practices

601 602

35.3 Phytochemical constituents 35.4 Pharmacological potentialities 35.4.1 Antioxidative potential 35.4.2 Antimicrobial potential 35.4.3 Wound-healing potential 35.4.4 Antidiarrheal property 35.4.5 Anti-ulcer property 35.4.6 Hepatoprotective potential 35.4.7 Antidiabetic potential 35.4.8 Antinociceptive property 35.4.9 Anti-cancerous property 35.5 Conclusion and future perspective References

36. Bioactivity of essential oils and its medicinal applications

xvii

605 607 607 608 608 609 609 610 610 610 611 611 611

617

Abdel Rahman Al Tawaha, Rose Abukhader, Ali Qaisi, Abhijit Dey, Abdel Razzaq Al-Tawaha and Iftikhar Ali 36.1 Introduction 36.2 Chemical structure of flavonoids 36.3 Flavonoids activity against multidrugresistant microbes 36.3.1 Inhibitory activity against cell envelope synthesis 36.3.2 Inhibitory activity against DNA synthesis 36.3.3 Inhibitory activity against ATP synthesis 36.3.4 Inhibitory activity against bacterial toxins 36.3.5 Inhibitory activity against biofilm formation 36.3.6 Membrane-disrupting activities 36.3.7 Inhibitory activity against efflux pumps 36.3.8 Inhibitory activity against bacterial motility 36.4 Conclusion Ethics declarations Ethical approval Consent to participate Consent to publish Authors contributions Funding Competing interests Availability of data and materials References

37. Essential oils as anticancer agents

617 618 619 620 621 622 622 623 624 624 625 625 625 625 626 626 626 626 626 626 626

629

Vilas Jagatap, Iqrar Ahmad, Aakruti Kaikini and Harun Patel 37.1 Introduction

629

xviii

Contents

37.2 Anticancer potential of essential oils 37.3 Conclusion and future perspective Abbreviations References

629 640 640 641

38. Molecular docking study of bioactive phytochemicals against cancer 645 Sandhya Jain and Surya Prakash Gupta 38.1 Introduction

645

38.2 Molecular docking of bioactive phytochemicals with anticancer properties 38.3 Conclusion References Index

645 647 647 649

List of contributors Rose Abukhader Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan R.S. Agrawal Department of Patronage Traditional and Speciality Foods, MIT School of Food Technology, MIT Art, Design, and Technology University, Pune, Maharashtra, India Iqrar Ahmad Division of Computer-Aided Drug Design, Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Dhule, Maharashtra, India Iftikhar Ali Center for Plant Sciences and Biodiversity, University of Swat, Charbagh, Pakistan Abdel Razzaq Al-Tawaha Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, Serdang, Selangor, Malaysia

Jaya Baranwal DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering & Biotechnology, New Delhi, India Mridula Saikia Barooah Department of Food Science and Nutrition, College of Community Science, Assam Agricultural University, Jorhat, Assam, India Brajesh Barse Confederation of Indian Industry (CII), New Delhi, India Mohammad F. Bayan Faculty of Philadelphia University, Amman, Jordan

Pharmacy,

Prakash Chandra Behera Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubanewswar, Odisha, India

Kumar Hegde Biliyaru Anand Department of Botany & Biotechnology, Shri Dharmasthala Manjunatheshwara College, (Autonomous), Ujire, Karnataka, India

Vijeta Bhattacharya School of Pharmacy, ITM University, Gwalior, Madhya Pradesh, India; IPS College of Pharmacy, Shivpuri Link Road, Gwalior, Madhya Pradesh, India

Prasad Andhare Biological Sciences, PDPIAS, Charotar University of Science and Technology, Changa, Gujarat, India

Yeshwant R. Bhorge Department of Chemistry, Marotrao Pantawane Mahavidyalaya, Nagpur, India

M. Anjaly Shanker Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonepat, Haryana, India Kena Premshankar Anshuman Department of Microbiology, Sir P.P. Institute of Science, MK Bhavnagar University, Bhavnagar, Gujarat, India Karthik Sajith Babu Department of Animal Sciences and Industry/Food Science Institute, Kansas State University, Manhattan, KS, United States Iswar Baitharu Department of Environmental Sciences, Sambalpur University, Burla, Odisha, India Satarupa Banerjee School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India Kuldeep Bansal Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Abo Akademi University, Turku, Finland

Deepjyoti Bhuyan Department of Food Science and Nutrition, College of Community Science, Assam Agricultural University, Jorhat, Assam, India Satpal Singh Bisht Department of Zoology, Kumaun University, Nainital, Uttarakhand, India Khalida Bloch Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India Balakumar Chandrasekaran Faculty of Philadelphia University, Amman, Jordan

Pharmacy,

Prarthana Chatterjee School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India Rupali R. Chaudhary Department of Botany, Shri Sant Gadge Maharaj Mahavidyalaya, Nagpur, India Kajari Das Department of Biotechnology, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India

xix

xx

List of contributors

Sushma Dave Department of Applied Sciences, JIET Jodhpur, Jodhpur, Rajasthan, India

Mayuri Iyer Cachar Cancer Hospital and Research Centre (CCHRC), Silchar, Assam, India

Budheswar Dehury Bioinformatics Division, ICMRRegional Medical Research Centre, Bhubaneswar, Odisha, India

Vilas Jagatap Division of Computer-Aided Drug Design, Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Dhule, Maharashtra, India

Urja Desai Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India Abhijit Dey Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

Sandhya Jain Department of Pharmaceutical Sciences & Technology, Faculty of Pharmaceutical Sciences & Technology, AKS University Satna, Satna, Madhya Pradesh, India

M. Dhanalakshmi Research and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India

C. Jayabaskaran Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India

Amalia Di Petrillo Department of Medical Sciences and Public Health, University of Cagliari, Monserrato, Italy

Renitta Jobby Amity Institute of Biotechnology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India; Amity Centre of Excellence in Astrobiology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India

Navneet Kumar Dubey Victory Biotechnology Co. Ltd., Taipei City, Taiwan Subodh Kumar Dubey School of Pharmacy, ITM University, Gwalior, Madhya Pradesh, India Antonella Fais Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy Sonia Floris Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy Ayushman Gadnayak Department of Bioinformatics, Odisha University of Agriculture & Technology, Bhubaneswar, Odisha, India

Kapil Juvale Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India S. Abdul Kader Department of Plant Biology and Plant Biotechnology, Presidency College, Chennai, Tamil Nadu, India Aakruti Kaikini Department of Diabetes and Obesity, King’s College London, London, United Kingdom

Arti Gaur Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India

Rahul B. Kamble Department of Botany, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India

Sougata Ghosh Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India; Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand

Paranjeet Kaur Chitkara of College of Pharmacy, Chitkara University, Punjab, India

Namrata A. Giri ICAR-National Research Centre on Pomegranate, Solapur, Maharashtra, India

Minna Kelloma¨ki Biomaterials and Tissue Engineering Group, BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland

Suchetana Gupta Phil and Penny Knight Campus for Accelerating Scientific Impact, University of Oregon, Eugene, Oregon, United States

Anandu Chandra Khanashyam Department of Food Science and Technology, Kasetsart University, Chatuchak, Bangkok, Thailand

Surya Prakash Gupta Department of Pharmaceutical Sciences & Technology, Faculty of Pharmaceutical Sciences & Technology, AKS University Satna, Satna, Madhya Pradesh, India

Rousan Khatun Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India

Anshita Gupta Soni Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Durg, Chhattisgarh, India Namita Hegde Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India Mona Isaq Department of Biotechnology & Bioinformatics, Kuvempu University, Shivamogga, Karnataka, India

Simran Khera Amity Institute of Biotechnology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India Amit Kumar Department of Electrical and Electronic Engineering, University of Cagliari, Cagliari, Italy Pankaj Kumar Singh Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana, India

List of contributors

Mohanya Kumaravel Department of Biotechnology, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences (Deemed-to-be University), Coimbatore, Tamil Nadu, India Ganesh Latambale Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India

xxi

Binata Nayak School of Life Sciences, Sambalpur University, Burla, Odisha, India Nilesh Prakash Nirmal Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, Thailand Archana Padhiary School of Life Sciences, Sambalpur University, Burla, Odisha, India

Shun-Yuan Luo Department of Chemistry, National Chung Hsing University, Taichung, Taiwan R.O.C.

Kavita Pal Advanced Centre for Treatment, Research, Education in Cancer, Tata Memorial Centre, Mumbai, Maharashtra, India

Srushti Mahajan Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana, India

Amrita Kumari Panda Department of Biotechnology, Sant Gahira Guru University, Ambikapur, Chhattisgarh, India

Arabinda Mahanty Crop Protection Division, National Rice Research Institute, Cuttack, Odisha, India

Medha Pandya Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar, Gujarat, India

Thatikayala Mahender School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India; Avanthi Institute of Pharmaceutical Sciences, Hayathnagar, Hyderabad, India Rajesh Kumar Meher Department of Biotechnology and Bioinformatics, Sambalpur University, Burla, Odisha, India Maru Minaxi Biological Sciences, PDPIAS, Charotar University of Science and Technology, Changa, Gujarat, India Showkat Ahmad Mir School of Life Sciences, Sambalpur University, Burla, Odisha, India Monika Mishra Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar, Sambalpur, Odisha, India Namrata Mishra IPS College of Pharmacy, Shivpuri Link Road, Gwalior, Madhya Pradesh, India Rojita Mishra Department of Botany, Polasara Science College, Polasara, Odisha, India Ipsita Mohanty Departments of Pediatrics, Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, United States Auwal Muhammad Department of Physics, Kano University of Science and Technology, Wudil, Nigeria Vaishnavi Murugan Amity Institute of Biotechnology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India Pradeep Kumar Naik Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar, Sambalpur, Odisha, India Sneha P. Nair Amity Institute of Biotechnology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India

Deepa Y. Panhekar Department of Chemistry, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India Vijay Singh Parihar Biomaterials and Tissue Engineering Group, BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Ramalingam Parthasarathy Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India Harun Patel Division of Computer-Aided Drug Design, Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Dhule, Maharashtra, India Viral Patel Department of Pharmaceutics, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand, Gujarat, India Ashwariya Pati School of Life Sciences, Sambalpur University, Burla, Odisha, India Siddhartha Pati NatNov Bioscience Private Limited, Balasore, Odisha, India Mayuri A. Patil Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Subhaswaraj Pattnaik Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar, Sambalpur, Odisha, India Chandrappa Chinna Poojari Department of Biotechnology, Shridevi Institute of Engineering & Technology, Tumkur, Karnataka, India Alok Prasad Das Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India

xxii

List of contributors

Sachin Puri Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India Ali

Qaisi Department of Pharmaceutical Sciences, School of Pharmacy, University of Jordan, Amman, Jordan

Amreen S. Quadri Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Padmalatha S. Rai Department of Biotechnology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Udupi, Karnataka, India Mithun Singh Rajput Department of Pharmacology, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand, Gujarat, India Yarappa Lakshmikant Ramachandra Department of Biotechnology & Bioinformatics, Kuvempu University, Shivamogga, Karnataka, India R.C. Ranveer Department of Post-Harvest Management of Meat, Poultry and Fish, Post-Graduate Institute of Post-Harvest Management, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Raigad, Maharashtra, India N.B. Rathod Department of Post-Harvest Management of Meat, Poultry and Fish, Post-Graduate Institute of Post-Harvest Management, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Raigad, Maharashtra, India Manan Raval Department of Pharmacognosy, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand, Gujarat, India Pooja Ravi Department of Biotechnology & Bioinformatics, Kuvempu University, Shivamogga, Karnataka, India Rakesh Rawal Department of Life Sciences, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India Sanjeev Kumar Sahu School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India Hiram Saiyed Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India Bhagwan K. Sakhale Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India

Purnima Dey Sarkar Department of Medical Biochemistry, M.G.M. Medical College, Indore, Madhya Pradesh, India Aniket P. Sarkate Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Ratnnadeep C. Sawant Department of Chemistry, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India Siddhi Sawant Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India Manas Ranjan Senapati Agro-Polytechnic Centre (Animal Science), Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India Asma Musfira Shabbirahmed Department of Biotechnology, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences (Deemed-to-be University), Coimbatore, Tamil Nadu, India Radha Sharma Shriram College of Pharmacy, Banmore, Madhya Pradesh, India Mohamad Shatnawi Biotechnology Department, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt, Jordan K.

Shilali Department of Biotechnology & Bioinformatics, Kuvempu University, Shivamogga, Karnataka, India

Sonali S. Shinde Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Harvinder Singh Leaders Institute, Woolloongabba, Queensland, Australia Iqubal Singh School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India Sikha Singh Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India Subhash R. Somkuwar Department of Botany, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India Prathap Somu School of Chemical Engineering, Yeungnam University, Gyeongsan, Gyeongsangbuk, Republic of Korea D. Sruthi Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India Abdel Rahman Al Tawaha Department of Biological Sciences, Al Hussein Bin Talal University, Maan, Jordan Sheary Somam Tete School of Life Sciences, Sambalpur University, Burla, Odisha, India

List of contributors

Pinchu Elizabath Thomas Pinchu Elizabath Thomas, MACFAST (Mar Athanasios College for Advanced Studies Thiruvalla), Kottayam, Kerala, India

xxiii

Dhwani Upadhyay Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India

Sirikanjana Thongmee Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand

Vijay Upadhye Center of Research for Development (CR4D), Department of Microbiology, Parul Institute of Applied Sciences (PIAS), Parul University, Waghodia, Gujarat, India

Priyamvada Thorakkattu Department of Animal Sciences and Industry/Food Science Institute, Kansas State University, Manhattan, KS, United States

Pravin R. Vairagar Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India

Mansi Tiwari Department of Food Science and Nutrition, College of Community Science, Assam Agricultural University, Jorhat, Assam, India

Pankaj Wadhwa School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India

Kanchanlata Tungare School of Biotechnology and Bioinformatics, D. Y. Patil Deemed to be University, Belapur, Maharashtra, India

H.C. Yashavantha Rao Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India

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Preface Medicinal plants (herbs) and tonics have been utilized in traditional medical practices from prehistoric times to cure ailments and provide health benefits. In general, naturally occurring chemicals have complex bioactive effects, and their effectiveness is difficult to ascertain. As a result, comprehensive approaches to phytochemical production and biological activity are required. Carotenoids, phenolic acids, flavonoids, and lignans have been found in medicinal plants and have a wide variety of biological activities, including antioxidant and antibacterial actions, as well as anticancer and antiinflammatory effects and neuroprotection. Phytochemicals have entered the human diet and life since the birth of mankind on the Earth, through the consumption of plant foods and the application of herbal treatments, in a process of coevolution between plants and people. This coevolutionary interaction has resulted in humans’ reliance on food and medicinal plants as the sources of macronutrients, micronutrients, and bioactive phytochemicals. Indeed, substantial data suggest that a plant-rich diet may help to reduce the risk of chronic-degenerative conditions such as cardiovascular and neurodegenerative diseases, several malignancies, and diabetes. Phytochemicals can also be used as dietary supplements to improve the physiological activities of the body in healthy people. Furthermore, because of their multitarget mechanism of action, phytochemicals can be used as adjuvant agents and sensitizers in traditional antibiotic and anticancer therapy, reducing the potential of selecting resistant microbial strains and cancer cells. However, certain preclinical (i.e., in vitro and in vivo) pharmacological properties of phytochemicals must be supported by clinical investigations in healthy and unhealthy individuals, as well as their optimal dosage, administration method, potential side effects, and medication interaction. Similarly, in observational epidemiological investigations, a causal association between nutrition and disease risk has yet to be thoroughly established. This book mainly intends to highlight high-quality chapters presenting and addressing the many processes of potential phytochemical evaluation of known and little-known sources, with a focus on works, including phytochemical and pharmacological evaluations, as well as computational research into the structures and pharmacological mechanisms of natural products and its various application such as medicine, food, and biotech.

Siddhartha Pati Tanmay Sarkar Dibyajit Lahiri

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Acknowledgment The editors would like to express their gratitude to everyone who contributed to this project, but they would especially want to thank the authors and reviewers who took part in the evaluation process. This book would not have been possible without their help. The editors would first like to express their gratitude to all of the authors for their efforts. We sincerely appreciate the authors of the chapters who gave their time and knowledge to this book. Second, the editors are grateful to the reviewers for their contribution in improving the quality, coherence, and content of the chapters. Last but not least, the editors would like to thank the following others for their help and support: Dr. Runu Chakraborty, Professor, Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata, India Dr. Sebak Ranjan Roy, Former Professor, Maulana Abul Kalam Azad University of Technology, Haringhata, India Sri Snehasis Guha, Principal-in-Charge, Malda Polytechnic, Malda, West Bengal, India Dr. Bisnu Prasad Dash, Former Professor, Department of Bioscience and Biotechnology, Fakir Mohan University, India Dr. Debabrata Panda, Director, NATNOV Bioscience Pvt. Ltd., India

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Chapter 1

Extraction, isolation, and characterization of phytochemicals, the bioactive compounds of plants D. Sruthi1, M. Dhanalakshmi2, H.C. Yashavantha Rao1, Ramalingam Parthasarathy1 and C. Jayabaskaran1 1

Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India, 2Research and Development Centre, Bharathiar University,

Coimbatore, Tamil Nadu, India

1.1

Introduction

Plants are rich in phytoconstituents which are broadly classified as primary and secondary metabolites. The primary metabolites like carbohydrates, proteins, and lipids are responsible for the growth and development of the plant. The secondary metabolites mainly include three classes, viz., terpenoids, alkaloids, and phenolics, and are involved in functions like plant survival through defense mechanisms. The health benefits of phytochemicals especially secondary metabolites have been discussed to a significant extent by researchers (Sruthi & Jayabaskaran, 2021). The chemical structures of such high-value constituents are complex, and such compounds should be obtained in purified form for their structural elucidation and bioactivity studies. This can be achieved with the aid of new technologies and methods that emerged in recent years for the extraction, isolation, and characterization of phytochemicals (Feng et al., 2019). Extraction is the initial step in natural product research to isolate a compound of interest. The process of extraction enables to get the target phytochemical to the maximum recovery and is further aimed to reduce/avoid the unwanted chemical constituent (Feng et al., 2019). The next step is the isolation of phytochemicals from the extract, and this can be performed through separation. Through the process of separation, different compounds of extracts can be isolated and purified step by step until we get the purified monomeric compound/s of interest (Feng et al., 2019). The phytochemical isolation and extraction can be performed through a combination strategy including classical methods like solvent extraction and modern separation techniques like chromatography (Sruthi & Jayabaskaran, 2021). The isolated pure phytochemicals further are subjected to the essential part, the identification or structural elucidation. The chemical structure can provide vital information for further studies including bioactivities, structural modifications, synthesis of active compounds, metabolic studies, etc. (Feng et al., 2019). The identification is mainly performed through spectral analysis as it consumes little sample to generate possible structural information regarding the target molecule. As the recovery of pure phytochemicals is often less, the spectral analysis is the frequently used method by natural product researchers (Silverstein & Bassler, 1962). This chapter thus describes different extraction methods for phytochemicals and further discusses the techniques for their isolation and characterization. The general approach in extraction, isolation, and characterization of phytochemicals is schematically summarized in Fig. 1.1.

1.2

Extraction of phytochemicals

1.2.1 Solvent extraction method The extraction of phytochemicals using different solvents is the commonest extraction method. During solvent extraction, initially the solvent is diffused into the cell membrane, and then, it has to dissolve the solutes which results in intracellular and extracellular concentration gradient and enables the diffusion of solute out of the cells (Luo, 2016). The main parameter to standardize in the solvent extraction method is the selection of suitable solvent for extracting the Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00033-5 © 2023 Elsevier Inc. All rights reserved.

1

2

Recent Frontiers of Phytochemicals

FIGURE 1.1 Summarized general approach for extraction, isolation, and characterization of phytochemicals.

target phytochemicals. Hydrophilic or polar phytochemicals like carbohydrates and amino acids can be solubilized and extracted with polar solvents whereas nonpolar constituents like terpenoids can be extracted using nonpolar solvents. The routinely used extraction solvents for phytochemicals and their polarity order from nonpolar to polar are as follows: petroleum ether/hexane . dichloromethane . chloroform . ethyl acetate . acetone . methanol . ethanol . water. Solvent extraction can be performed either as cold extraction or hot extraction depending on whether the plant sample is heating or not. On the other hand, solvent extraction can be explained in different methods including immersion method, percolation method, decoction method, refluxing method, constant refluxing method, supercritical fluid extraction method, ultrasonic extraction method, and microwave-assisted extraction method (Feng et al., 2019). Immersion method is used to extract temperature-sensitive compounds and is usually performed at room temperature or low temperature (,80 C). The compounds like starch, pectin, gum, etc., can be extracted by this method. Briefly, the powdered/crushed plant sample is taken in the container and immersed in suitable organic solvents for a given period. Additionally, stirring or shaking can also give to enhance the extraction rate. Though it is a simple method to perform, this method has disadvantages like the low extraction rate and high chance of contamination in the aqueous extract. In the percolation method, the plant sample is loaded in the percolation apparatus and immersed with suitable organic solvent for 24
48 hours, and the percolates from the bottom of the percolation apparatus are collected. Constant addition of new solvent at the top of the percolation apparatus is required during this process. This method is efficient than the immersion method as it sustains concentration difference throughout the process. However, this method has disadvantages including the complexity of the procedure, more consumption of solvents, and long duration (Feng et al., 2019). Decoction method is a hot extraction method. The powdered or thin pieces of plant sample are taken into a suitable container and heated to boil after adding water to it. With the aid of temperature, the phytoconstituents are extracted. This method is easy to operate, but undesired compounds may also get extracted. Further, this method is not suitable for volatile and thermally sensitive compounds and also does not have the option to extract plants with abundant starch content (Gray et al., 2012). Refluxing method involves the solvent extraction of phytochemicals using heating and refluxing. This method is suitable to extract lipophilic compounds like steroids. The refluxing process

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minimizes the wasting of solvents and also deduces the toxic exposure to operator and environmental pollution. This method is efficient but not suitable for temperature-sensitive compounds as this method requires longtime exposure to heat. In continuation to refluxing method, the constant refluxing method is developed. This is the extraction method using the Soxhlet apparatus which overcomes the disadvantage of consuming large amount of solvents in the previous method. But this method is also not an option to extract thermally sensitive phytochemicals (Feng et al., 2019). Supercritical fluid extraction (SFE) method involves the extraction of phytochemicals using supercritical fluids. If the solvent is at a temperature and pressure above its critical point, a single homogenous fluid is formed which is known as supercritical fluid (SF). Hence, it concurrently shows the properties of both gas and liquid (Feng et al., 2019; Murga et al., 2000). By controlling different parameters, SF in its supercritical state can selectively extract the compounds with different polarities, boiling points, and molecular weights consecutively (Ty´skiewicz et al., 2018). CO2 is the extensively used SF whose critical temperature (31.26 C) is near to room temperature and critical pressure (7.2 MPa) is also not too high. The other advantages which accredited CO2 as the commonly used SF include its nontoxicity, low cost, noninflammable, odorless, chemical stability, and so on. As CO2 is nonpolar in nature, it is mainly used for the extraction of nonpolar compounds. However, by adding entrainers (e.g., ethanol, water, acetone, methanol, ethyl acetate, acetonitrile, etc.), the extractability of SF CO2 for polar compounds can be improved. As this method can be performed at near room temperature, the thermal loss of compounds is negligible, and further, the organic solvent residue is not retained in the extract, and hence, the extract obtained through SFE is of high purity and recovery. Hence, SFE is extensively used for the extraction of alkaloids, phenolic compounds, terpenoids, glycosides, essential oils, etc. (Feng et al., 2019). Ultrasonic extraction (UE) method involves the solvent extraction of phytochemicals assisted by ultrasound. In this method, cavitation bubbles are created near the sample tissue with the aid of ultrasound waves, and further, they break the cell wall which results in the release of the cell content into the solvent (Altemimi et al., 2016). Improved extraction efficiency and less extraction time are the advantages of this method, and recently, ultrasonic extraction attracted natural product research for extracting compounds like isoflavones (Feng et al., 2019; Rostagno et al., 2003). In microwaveassisted extraction (MAE) method, the phytochemicals are extracted from plant samples through their interaction with the microwave. As a result of molecular motion induction, the microwave generates heat which results in the rupturing of cell wall and further release of cellular active constituents (Sookjitsumran et al., 2016). Less degradation of phytochemicals, lower energy consumption, shorter time, and less environmental pollution are the advantages of this method and are widely used in the extraction of compounds like polyphenols (Pan et al., 2003).

1.2.2 Steam distillation method Steam distillation is appropriate for the extraction of volatile components with high boiling point. These compounds can be recovered from the sample through distillation with superheated or saturated steam (Prado et al., 2015). This method is mainly employed for the extraction of essential oil and also some alkaloids and phenolics (Feng et al., 2019). In this method, the volatile compounds are escaped from the raw material and vaporized upon using steam or boiled water. The resulting vapor is cooled and condensed as separate phase immiscible with water. The steam distillation can further be categorized as dry steam distillation, hydrodistillation, and direct steam distillation (Prado et al., 2015).

1.2.3 Pressing method If the active chemicals are present in the juice of the plant in comparatively high amount, we can directly extract the juice from the raw material through the pressing method. Essential oils like orange peel and lemon oils can also be extracted from plant tissues through mechanical pressing. We can perform this at room temperature, and thus, heatsensitive compounds can be saved. However, the presence of large impurities in terms of mucoid substances, water, and cell tissues in the final product and the difficulty to press the entire volatile oil in plants, are some drawbacks of this method (Feng et al., 2019).

1.2.4 Sublimation method Sublimation is the process of direct conversion of solid material into steam without melting after heating. Natural products have sublimation properties (e.g., alkaloids, benzoic acids, coumarins, etc.) and can thus be extracted directly from the raw material through the sublimation method (e.g., caffeine extraction from tea). Because of the low recovery

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rate, this method is not recommended for large-scale production. Due to prolonged heat exposure in this method, natural products can easily carbonize, and thermal breakdown occurs frequently (Feng et al., 2019).

1.3

Isolation and purification of phytoconstituents

Once we get the extract, the next process is to separate the active phytochemicals from the extract one by one through isolation and then purify them into their monomeric structures. There are classical isolation methods and modern separation technologies for the isolation and purification of phytochemicals. This section explains these methods and their specific role and applications in phytochemical isolation and purification.

1.3.1 Classical isolation methods Solvent extraction, crystallization, precipitation, fractional distillation, dialysis, and salting out are the classical methods for the isolation of phytochemicals (Feng et al., 2019; Yubin et al., 2014). Solvent extraction method include acid and basic solvent method and polarity gradient extraction method. In acid and basic solvent method, the compounds in a mixture can be separated based on its acidity or alkalinity. The alkaline compounds like alkaloids can be separated from the mixture by allowing to react with organic acids (0.1%
1% HCl/ H2SO4/CH3COOH/C4H6O6), and the resultant salt can be separated from the non-alkaline components. Same way, acidic nature compounds can be separated by forming salt through the reaction with bases, and the salt can be dissolved in water. Further, natural products with lactone/lactam structures are possible to saponify, and upon dissolving in water, they can be isolated from water-insoluble compounds. Since the acidity and alkalinity of compounds are different, we can also separate the compounds from the total extract through pH gradient extraction (Feng et al., 2019; Yubin et al., 2014). In contrast, polarity gradient extraction method involves the separation of phytochemicals based on the different polarities of each compound. This is normally achieved by selecting a two-phase solvent system based on the polarity of the compounds in the selected extract (e.g., for strong polarity—butanol: water; moderate polarity—ethyl acetate: water; least polarity—chloroform/ether: water). Briefly, the plant extract is dissolved in water and added into separating funnel and extracted sequentially with petroleum ether/cyclohexane, then ethyl acetate/chloroform, and finally with water-saturated n-butanol (Feng et al., 2019) (Fig. 1.2).

FIGURE 1.2 Schematic representation of a common polarity gradient extraction method.

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Crystallization method is an effective way to get pure compounds and involves the precipitation of solute of interest from the mother liquor. Initial crystals are often impure and need recrystallization. If the phytochemical concentration is very high in plant material, we will get pure crystals by slightly concentrating or cooling the extract after extraction with suitable solvents. If we cannot crystallize a compound with single solvent, a mixed solvent system (one has high and one has low solubility to the compound to be crystallized) can be used. Briefly, the sample is heated and dissolved in minimum volume of solvent of high solubility, and further, a turbid solution is prepared by adding the low soluble solvent to the hot solution. Again, the first solvent with high solubility is added, and upon reaching saturation, the compound is crystallized while cooling (Feng et al., 2019). Precipitation is the method of separation which involves the formation of precipitate of phytochemicals either through their reaction with some reagents or their direct precipitation from the solution by adding certain reagents, thereby reducing its solubility. There are solvent precipitation method and exclusive reagent precipitation method, based on the addition of solvents or reagents (Tang et al., 2011). In the solvent precipitation method, the compound can be precipitated from the solution by changing its solubility through the addition of certain solvents. On the other hand, some reagents (e.g., alkaloid precipitation reagent—Reynolds ammonium) can react selectively with a particular compound which will result in reversible precipitation, and this separation method is termed as exclusive reagent precipitation method (Feng et al., 2019). Fractional distillation is the method of separation of compounds from the liquid mixture depending on the difference in their boiling points. It can be categorized as vacuum, atmospheric, and molecular distillation and is mainly used for the separation of liquid alkaloids and essential oils. If the boiling point difference of the compounds is .100 C, we can separate them through repeated distillation, whereas if that difference is ,25 C, the compounds can be separated by fractionation column. If the boiling point difference is small, fine fractionation measures are required for the separation (Hanif et al., 2017). Dialysis involves the separation, concentration, or purification of substances based on their penetration across the semipermeable membrane/dialysis bag under the action of difference in concentration, potential, or pressure (Feng et al., 2019; Tahara et al., 2017). Salting-out method reduces the solubility of certain compounds upon adding inorganic salts (up to saturated state/to a particular concentration) to the plant water extract. The less soluble compound can be thus separated from the other water-soluble constituents. Sodium chloride, magnesium sulfate, sodium sulfate, and ferric sulfate are some of the inorganic salts commonly used for the salting-out method (Azmir et al., 2013; Feng et al., 2019).

1.3.2 Modern separation technologies Chromatography and electrophoresis are explained in this section as these two methods are widely accepted recently in natural product research.

1.3.2.1 Chromatography techniques Chromatography is the widely accepted method of isolation and purification of phytomolecules. It is also an accepted method for the identification and molecular mass determination of phytochemicals. They are mainly partition, adsorption, affinity, ion exchange, and gel filtration chromatographic techniques. Partition chromatography separates the molecules based on the partition coefficients between stationary and mobile phases. There are normal-phase and reverse-phase partition chromatography. The polarity of the stationary phase is stronger than that of the mobile phase in normal phase and vice versa in reverse-phase partition chromatography. The commonly used stationary phase in the normal phase includes silica gel, cellulose powder, etc., whereas that in the reverse phase is mainly octadecyl silylated silica. Normal-phase partition chromatography is widely used for the separation of polar and mid-polar phytochemicals whereas reverse phase is mainly applicable for the separation of nonpolar and moderately polar phytochemicals. In adsorption chromatography, the molecules get separated based on the difference in the adsorptive capacity of the adsorbent (e.g., silica gel, alumina) toward each molecule to be separated. Silica gel is widely used for the separation of almost all classes of phytochemicals whereas alumina is mainly for alkaloids, steroids, and terpenoids (Feng et al., 2019). Affinity chromatography involves the separation of molecules based on their affinity toward the specific ligand that binds with the adsorption medium. This technique can thus selectively and specifically isolate the compound of interest from the complex mixture. Recently, its application has grown along with the development of new technologies (Cao et al., 2019). On the other hand, ion exchange chromatography separates the phytomolecules as per the difference in the dissociation degrees. This method is apt for isolating ionic compounds like

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amino acids, alkaloids, peptides, etc., (Feng et al., 2019). Gel filtration chromatography works with the principle of molecular sieve and separates the compounds based on the gel’s pore size and the molecule’s pore size. Dextran gel, hydroxyl propyl dextran gel, etc., are the gels commonly used in this method (Porath, 1997). Apart from the aforementioned conventional chromatographic techniques, new technologies enabled the development of highly effective and pre´cised chromatographic methods for the separation and purification of biomolecules. These include high-performance liquid chromatography (HPLC), droplet countercurrent chromatography (DCCC), and high-speed countercurrent chromatography (HSCCC), which play crucial roles in the isolation and purification of phytochemicals (Feng et al., 2019). HPLC is a rapid and efficient separation method with the aid of high pressure. It is developed from the conventional column chromatography and operates on the principle of adsorption, partition, ion exchange, gel filtration, or other methods. HPLC is used not only for the separation, but also for the quantitative analysis and qualitative identification of phytochemicals preferably nonvolatile compounds. Preparative HPLC is suitable for the large-scale purification of phytomolecules whereas analytical HPLC enables the identification and purity checking. UV detectors, diode array detectors, and differential refractive index detectors are the commonly used detectors in HPLC. Recently, mass spectrometry detectors (LC-MS) were also established. (Feng et al., 2019; Ji et al., 2018). DCCC is an updated liquid
liquid partition chromatography and works on the basis of countercurrent partition method. When the mobile phase goes through a liquid stationary phase column, droplets will be formed. These droplets of the mobile phase have effective contact with stationary phase and thus constantly form a new surface in thin partition extraction tubes. As a result, the solutes get partitioned between two-phase solvents, and thus, the chemical constituents get separated (Feng et al., 2019). Plant secondary metabolites like indole alkaloids are isolated using DCCC (Cardoso & Wilegas, 1999). HSCCC is another liquid
liquid chromatography based on partition coefficient, and it has no solid support and is encountered only by solvent and Teflon tube. High purity, high speed, and good reproducibility make this technique useful for the isolation and purification of a wide range of phytochemicals like alkaloids, saponins, flavonoids, lignans, proteins, terpenoids, and sugars (Gu et al., 2004).

1.3.2.2 Electrophoresis techniques Electrophoresis is another biochemical method of separation of biomolecules. High-performance capillary electrophoresis (HPCE) is one of these techniques. It is a combination of conventional electrophoresis with modern microcolumn separation techniques. This method effectively separates large and small molecules in a hollow and thin capillary. It includes capillary zone electrophoresis and capillary gel electrophoresis (Feng et al., 2019). Phytochemicals (e.g., components from the tea) are effectively separated by this technique (Horie & Kohata, 2000).

1.4

Identification of phytochemicals

Once we get the purified compound, the next is to identify its chemical structure which can be termed as structural elucidation. The identification is a crucial part for the further characterization of the compound. However, the purity of the isolated compound must be checked prior to the identification. Purity can be checked preliminarily with the physical properties (shape, color, melting point, etc.) of the compound preferably of crystals. Thin-layer chromatography, HPLC, and gas chromatography are widely used for the purity checking of isolated compounds. In TLC, a compound is thought to be pure if it gives only one spot in at least three different solvent systems. GC and HPLC are important techniques for the determination of the purity of phytochemicals. GC is mainly used for volatile and HPLC for nonvolatile constituents. Once the purity is confirmed, the next is to identify the compound. There is no strict strategy for identification, and it purely depends on the experience and skill of the researcher. However, the whole process of structural elucidation runs through a common pattern for both known and unknown phytochemicals and mainly depends on spectral technologies.

1.4.1 Spectral technologies Spectral technologies are the main means to identify the chemical structures of known and unknown compounds. The applications of ultraviolet
visible (UV-vis), infrared (IR), nuclear magnetic resonance (NMR), and mass (MS) spectra in phytochemical identification are introduced briefly in this section. UV
vis spectrum is a type of electron transition spectrum, which is generated after the molecules absorbing the electromagnetic waves with wavelength at the range of 200
800 nm. The valence electrons in the molecules absorb light of certain wavelengths and jump to the excited state from the ground state, and then UV spectra are recorded.

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Compounds with conjugated double bonds, α,β-unsaturated carbonyl groups (aldehydes, ketones, acids, and esters), and aromatic compounds show strong absorption in UV spectra, and hence, this is mainly used to know the presence of conjugated systems in the structures. Since the UV
vis spectra can provide a part of the structural information, it is just an auxiliary method in the identification process (Feng et al., 2019). In contrast, IR is the technique used to determine mainly the functional groups and the aromatic ring substitution types. IR is also helpful for determining the configuration of phytochemicals. IR is based on the vibration
rotational energy level transition of a compound ranging from 4000 to 625 cm21. The region above 1250 cm21 is the functional group region whereas 1250
625 cm21 is the fingerprint region (Feng et al., 2019). In MS, after the molecules are ionized and enter into the collector under the action of electric and magnetic fields, mass and strength information of molecular and fragment ions are recorded. As a result, the information of mass and strength of molecular and fragment ions are recorded as spectrum with mass-to-charge ratio (m/z) versus intensity. In recent era, MS play essential role in natural product research by involving determination of molecular weight and elemental composition, detection of functional groups by cleavage fragments, identification of compound types, and also to determine carbon skeletons. High-resolution mass spectrometry (HR-MS) will give you the information regarding molecular formula and molecular weight. As per the ion source type, commonly employed MS include electron impact mass spectrometry (EI-MS), field desorption mass spectrometry (FD-MS), chemical ionization mass spectrometry (CI-MS), fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption mass spectrometry (MALDI-MS), and tandem mass spectrometry (MS
MS). Nowadays, chromatography coupled with mass spectrometry, viz., liquid chromatography
mass spectrometry (LC
MS) and gas chromatography
mass spectrometry (GC
MS), are widely in practice for the sensitive identification and precise structural characterization (Feng et al., 2019; Kumar et al., 2018). NMR, particularly hydrogen and carbon spectrum, is the most important and widely accepted spectroscopic method to identify the chemical structures of both known and unknown compounds. Briefly, the compound is irradiated by electromagnetic waves in a magnetic field. The transitions in the energy level occur after the atomic nuclei with magnetic distance absorb a certain amount of energy, and then, NMR spectrum is obtained by mapping the absorption strength with the frequencies of the absorption peaks. NMR spectra provide structural information regarding the type and number of hydrogen and carbon atoms in the molecule, the modes they are connected, configuration, the surrounding chemical environment, and conformation (Monakhova et al., 2013). Solid, liquid, and gaseous molecules can be analyzed by NMR. The solvents used to dissolve the sample for NMR analysis should be deuterated, and the commonly used deuterated reagents include CDCl3, CD2Cl2, D2O, DMSO-d6, D6C6, etc. There is proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) (1H-1H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, TOCSY spectrum, and HSQC-TOCSY spectrum) (Feng et al., 2019).

1.5

Conclusion

This chapter has dealt with various methods of extraction of high-value phytochemicals and various techniques for their isolation and characterization. Recently, natural product research preferably phytochemicals received much attention due to their enormous health potential. The plants are extensively researched for its high-value phytochemicals and also for their vast therapeutic potential. However, further clinical trials and well-studied human interventions are required to validate the health benefits of such phytochemicals. Besides, lots of plants are still in “under-explored” category in terms of its chemical assembly and pharmacological potential. Further, there is an increased interest for complementary and alternative medicine that will also intensify the potential functionalities of highly valued phytomolecules in the future. Hence, getting the basic knowledge on how to extract, separate, and characterize such phytochemicals is essential for contributing to natural product research.

Acknowledgments Dr. D. Sruthi acknowledges the Department of Health Research (DHR), Government of India, New Delhi, for her award Young ScientistHRD Scheme (YSS/2019/000035/PRCYSS). She is also grateful to Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi, for her award National Post-Doctoral Fellowship (PDF/2017/000339). Acknowledgment is further extended to Dr. T. John Zachariah, Former Principal Scientist, and Acting Director, ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, for his valuable guidance on plant secondary metabolites during the doctoral period of Dr. D. Sruthi.

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345.

Chapter 2

Importance and extraction techniques of functional components Amreen S. Quadri1, Aniket P. Sarkate1, Nilesh Prakash Nirmal2 and Bhagwan K. Sakhale1 1

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India, 2Institute of Nutrition,

Mahidol University, Salaya, Nakhon Pathom, Thailand

2.1

Introduction

Plants generate a spread of secondary metabolites as a part of their arms during growth. These secondary metabolites or bioactive compounds or functional components have strong inhibitory activity against microorganisms like bacteria and fungi (Anwar et al., 2009). Bacteria like fungal infections pose a giant threat to mankind, and indiscriminate use of antimicrobial drugs has caused resistance in microbes. Because they need the smallest amount of antibiotic-related side effects and better activity against drug-resistant strains, researchers have focused their attention toward phytochemicals (Khan et al., 2009). Bioactive compounds abundant in plants include phenolic acids, flavonoids, tannins, and alkaloids. The antimicrobial characteristics of certain polyphenol classes are investigated to develop novel therapies for the treatment of various microbial infections (Safdar et al., 2017). The term “bioactive” consists of two words: bio- and -active. In etymology, bio-from the Greek (β
ιo-) “bios” [bio-, -bio] refers to life, and -active from the Latin “activus,” means dynamic, filled with energy, with energy, or involves an activity. This activity presents all the phenomena from which it manifests a type of life, a functioning, or a process. As already mentioned, the term bioactive food compound or components (bioactive dietary) is sometimes related to only positive effects on the organism. Such a definition considers that the bioactive dietary influences the state of health and thus has a biological value beyond their calorie content (Guaadaoui et al., 2014). Some food constituents that are typically found in small quantities are vitamins, minerals, and other nutrients. The researchers are studying these substances to see if they have any harmful effects on health. Phenolic compounds, which include flavonoids, are found in both plants and animals and are studied extensively in cereal crops, legumes, nuts, olive oil, vegetables, fruits, tea, and other plant-based foods. Many different functional components appear to have health benefits. Before we can provide science-based dietary recommendations, a lot of research will need to be done. Despite this, there is enough evidence to recommend eating foods rich in functional components. From a practical perspective, a diet that is high in fruits, vegetables, whole grains, legumes, oils, and nuts is typically the best option. (Kris-Etherton et al., 2002). Liu (2013) has defined phytochemicals as bioactive compounds found in plants that are associated with reducing the risk of major noncommunicable chronic diseases.

2.1.1 Phytochemicals and their therapeutic effect Functional components play a central role in high-value development within the industry. Functional components are identified from diverse sources, and their therapeutic benefits, nutritional value, and protective effects in human and animal healthcare have underpinned their application as pharmaceuticals and functional food ingredients. The advantages of those compounds are the result of their proven bioactive properties, including antioxidant, anti-inflammatory, and antimicrobial effects. Most functional components are highly effective at trapping reactive species, which means they help improve endogenous antioxidant defenses. This makes them a great approach for fighting oxidative stress and related diseases (Barba et al., 2014). These functional components/bioactive compounds are found in very small Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00017-7 © 2023 Elsevier Inc. All rights reserved.

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amounts in plant source, and certain food development may be accustomed to improve the physiological state. Lycopene, resveratrol, lignan, and tannins are common functional components. These pure compounds or standardized extracts are reported to own many beneficial activities like antimicrobial, anticancer, analgesic, and wound-healing activity. Bioactive compounds are present in small amounts in food items, and their effect on human health is being continuously investigated. Epidemiological data support that prime intake of natural functional foods, like specific fruits and vegetables, which are rich in bioactive compounds, is related to decreased risk of chronic diseases, like cardiovascular diseases, cancer, metabolic syndrome, diabetes type II, and obesity. Natural bioactive compounds—such as resveratrol, epigallocatechin, curcumin, oleuropein, sulforaphane, quercetin, ellagic acid, anthocyanins, b-glucans, and other biomolecules—are related to the pathophysiology of cardiovascular diseases, diabetes, metabolic syndrome, and cancer. However, more clinical and epidemiological studies are essential so as to confirm their possible effect. Resveratrol (3,5,4-trihydroxystilbene) (RSV) has been detected mainly in grapes, red wine, and pomegranates, and it is the most stilbene present in the human diet. Epigallocatechin-3-gallate (EGCG) is the major polyphenol from tea with high antioxidant and scavenging properties. It is the ester of acid with epigallocatechin and is characterized by two triphenolic groups. Curcumin (CUR) exhibits possible anti-inflammatory, antioxidant, and chemopreventive actions, and it is a polyphenol from herbaceous plant Linnaeus (Zingiberaceae). Quercetin (QR) may be a flavonol found in various foods, like berries and tea; its antioxidant activity is the results of three important groups: the 4-oxo group together with the 2,3-alkene, 3- and 5-hydroxyl groups and therefore the hydroxyl groups of B ring. Ellagic acid (EA) applies high antioxidant and radical scavenging activity, and it is a polyphenol derived from ellagitannins mostly found in nuts and berries. Anthocyanins (ACs) are water-soluble pigments with purple, red, and blue colors and are present in fruits, especially in pomegranates, berries, and red grapes. Anthocyanins have been studied as bio-functional molecules with possible high anti-inflammatory, antioxidant, and chemoprotective effects (Konstantinidi & Koutelidakis, 2019).

2.1.2 Phytochemicals from different food sources Natural products from fruits and vegetables, either as pure compounds or as standardized extracts, provide unlimited opportunities for brand-new development thanks to the unequaled availability of chemical diversity. As extraction is the most vital step in the analysis of constituents present in fruits and vegetables, the strengths and weaknesses of various extraction techniques are discussed. The analysis of bioactive compounds present within the fruits and vegetables involves the applications of common phytochemical screening assays, and chromatographic techniques like HPLC and TLC and nonchromatographic techniques like immunoassay and Fourier transform infrared (FTIR) have to be studied (Sasidharan et al., 2011).

2.1.2.1 Tomato Ali et al. (2020) reviewed nutritional composition and bioactive compounds in tomatoes and their impact on human health and disease. Tomato (Solanum lycopersicum Mill) has been called a nutritious fruit for an extended time, but its antioxidant properties that demonstrate health benefits including alleviation of chronic and cardiovascular diseases are more pronounced in the last decades. The fruit contains numerous functional components with antioxidant properties, which are attributed to carotenoids, total phenolics (TP), and vitamin C. It also has been shown that the antioxidant potential of tomato varies among pulp, skin, and seed of fruits and its ripening stages. Lycopene, a potent antioxidant carotenoid found in tomatoes and other fruits, is believed to protect against prostate and other cancers and inhibit tumor cell growth in animals. Lycopene is the main dietary carotenoid in tomato and tomato-based food products, and lycopene consumption by humans has been reported to guard against cancer, cardiovascular diseases, cognitive function, and osteoporosis (Ali et al., 2020) (Fig. 2.1).

2.1.2.2 Onion Benı´tez et al. (2011) studied bioactive compound content in numerous onions. The study indicates there is a substantial debate over onions (Allium cepa L.) having the precise components accountable for the health-benefiting effects. Two main groups of chemical compounds are proposed: flavonoids and alk(en)yl cysteine sulfoxides (ACSOs) (Mogren et al., 2007). Onion may well be used as a source of food ingredient, since it has been reported that onion may be a potent cardiovascular and anticancer agent, with hypocholesterolemic, antithrombotic and antiplatelet activity, and antioxidant effects, besides the antiasthmatic and antibiotic effects (Moreno et al., 2006). Onion contains assorted phytochemicals, including organosulfur compounds, phenolic mixtures, polysaccharides, and saponins. The phenolic and

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FIGURE 2.1 Chemical structures of (A) lycopene, (B) β-carotene, and (C) vitamin A.

FIGURE 2.2 The substance designs of the fundamental organosulfur and phenolic intensifies in onion.

sulfur-containing compounds, including onionin A, cysteine sulfoxides, quercetin, and quercetin glucosides, are the major bioactive constituents of onion (Zhao et al., 2021) (Fig. 2.2).

2.1.2.3 Garlic Dhwani et al. (2021) reviewed different extraction and quantification methods of allicin from garlic. Garlic (Allium sativum) is widely used as a condiment in food and possesses a good range of therapeutic effects. It contains high medicinal properties and acts as a functional food. Garlic is employed to treat many diseases and is sometimes used as an antibacterial, antifungal, antidiabetic, antioxidant, and antiviral compound (Singh & Singh, 2019). Garlic extracts exhibit high radical scavenging or high antioxidant potential. These health benefits are attributed to the assorted functional components present in garlic. The thiosulfinates present in garlic are essential organosulfur compounds which are a source of the nutritional effects of garlic (Petropoulos et al., 2018). The most important bioactive compound present in garlic is allicin, which is not available naturally but obtained by the destruction of the cytomembrane by chopping, cutting, or grinding the bulbs or clove. This compound allicin is chargeable for the pungent smell of garlic while alliin is liable for its flavor. Garlic is a best natural source of bioactive sulfur-containing compounds and has promising applications in

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FIGURE 2.3 The chemical structures of the main organosulfur compounds in garlic.

the development of functional foods or nutraceuticals for the prevention and management of certain diseases (Shang et al., 2019). Organosulfur intensifies in garlic and onions, isothiocyanates in cruciferous vegetables, and monoterpenes in citrus natural products, cherries, and spices have anticarcinogenic activities in trial models, yet as cardioprotective impacts (Fig. 2.3).

2.1.2.4 Beetroot Salamatullah et al. (2021) studied the bioactive and antimicrobial properties of oven-dried beetroot (pulp and peel) using different solvent processes. Beetroot (Beta vulgaris var. rubra L.; BVr) may be a member of the Chenopodiaceae family and is typically consumed in powdered form, supplemental juice, bread, pickle and further as pureed, boiled, and even processed as jam. BVr is one of the richest vegetables because of the presence of several phenols like catechin, vanillic, caffeic, epicatechin, p-hydroxybenzoic, p-coumaric, protocatechuic, ferulic, and syringic acids and other active compounds like betalains (betacyanins and betaxanthins), folates, flavonoids, and carotenoids (Fig. 2.4).

2.1.2.5 Ginger Srirejeki et al. (2018) conducted a study on the aqueous extract composition of spent ginger (Zingiber officinale var. Amarum) from volatile oil distillation. It has a particular spicy flavor and a nice aroma since many centuries, and it has been extracted by boiling in water for beverage. Ginger compounds act as antioxidants which are phenolic compounds (such as 6-gingerol and 6-shogaol), alanine, and water-soluble vitamin. Antioxidant compounds have a crucial role within the human health and also are widely used as food additives to stop food damage. The extraction of the functional components from spent ginger using an organic solvent is the hottest method recently (Fig. 2.5).

2.1.2.6 Turmeric Turmeric (Curcuma longa L., Zingiberaceae) is particularly popular worldwide thanks to its attractive culinary, cosmetic, and medicinal uses. Specifically, the interest of this tuberous species resides in its exploitation as a coloring and flavoring agent, furthermore as in its numerous pharmacological activities, like antioxidant, anticancer, antiinflammatory, neuro- and dermo-protective, antiasthmatic, or hypoglycaemic, being recently reported that turmeric can even potentially contribute against the life-threatening viral disease COVID-19 by inhibiting the most protease enzyme (Iba´n˜ez & Bla´zquez, 2020) (Fig. 2.6).

2.1.2.7 Kiwi The kiwi is the best-known fruit of the Actinidia genus (Actinidiaceae family), which has become a really popular product throughout the planet because of its nutritional and organoleptic properties and its health benefits. Numerous studies have shown the high content of kiwi in bioactive compounds of interest, like phenolic compounds, vitamins, and carotenoids. These compounds are reported for his or her antioxidant, anti-inflammatory, and antimicrobial activities, among other beneficial properties for health like its use as prebiotics (Chamorro et al., 2021). Kiwi is high in bioactive compounds of interest, such as phenolic compounds, vitamins and carotenoids (Fig. 2.7).

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FIGURE 2.4 Classification of the two main groups of betalains present in beetroot. FIGURE 2.5 Ginger plant, rhizome, and active components (6-gingerol, 6-paradol, and 6-shogaol).

2.1.2.8 Dragon fruit Dragon fruit has been reported as a source of functional components like polyphenols, vitamin C, antioxidant pigments, etc. In red-fleshed dragon fruit, the foremost important antioxidant pigments are betacyanins and betaxanthins. Dragon fruit, as in many other fruits and vegetables, is additionally rich in antioxidants that help to cut back the incidence of degenerative diseases like arthritis, arteriosclerosis, cancer, heart diseases, inflammation, and brain dysfunction (Senadheera & Abeysinghe, 2015).

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FIGURE 2.6 Main compounds found in the rhizomes.

Dragon fruit mainly contains betanin (betanidin-5-Oβ-glucoside), phyllocactin, hylocerenin, and their isomer forms with a minority group of unidentified betacyanin compounds. Phenolic compounds in dragon fruit consist mainly of gallic acid (GA) and ferulic acid with minor amounts of other hydroxycinnamic acids (Kim et al., 2011) (Fig. 2.8).

2.1.2.9 Clove Clove is one of the key vegetal sources of phenolic compounds like flavonoids, hydroxybenzoic acids, hydroxycinnamic acids, and hydroxyphenyl propane. The most bioactive compound of clove is Eugenol, which is found in concentrations starting from 9381.70 to 14,650.00 mg per 100 g of fresh material. The phenolic acids are the compounds found in higher concentrations (Mohammed et al., 2016) (Fig. 2.9).

2.1.2.10 Whole grain The whole grain has been defined to incorporate the intact, ground, cracked, or flaked caryopsis, whose principal anatomical components—the starchy endosperm, germ, and bran—are present within the same relative proportion as they exist within the intact caryopsis. Whole grains are a decent source of dietary fiber, vitamins, minerals, and bioactive compounds, which are suggested to contribute to their protective effects as compared to sophisticated grains. The bulk

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FIGURE 2.7 Bioactive compounds of kiwi.

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FIGURE 2.8 (A) Chemical structure of betacyanin. (B) Structure of phenolic acids.

FIGURE 2.9 Structure of Eugenol.

of bioactive compounds of whole grains are present within the bran/germ fraction of cereal grains (Gani et al., 2012) (Fig. 2.10).

2.1.2.11 Finger millet Finger millet is one of the underutilized small grains. Demand for research in millet is increased as a result of its abundant bioactive compounds. These compounds, including ferulic acid-rich arabinoxylan or ferluxan, ferulic acid, caffeic acid, and quercetin, are associated with certain health-promoting properties and are biologically available in cereals. Ragi has shown potential dietary supplement benefits as a result of the recent focus on self-healing substances over synthetics in the treatment of diet-related disorders. Recent studies on cereals have reported several important health effects such as anti-diabetes, antioxidant, anti-inflammatory, and antibacterial properties (Okwudili et al., 2017) (Fig. 2.11).

2.1.2.12 Fish Fish are sources of various abundant bioactive compounds. It is stated that proteins (amino acids/peptides/hydrolysates) and fatty acids, vitamins, and minerals are among the bioactive compounds in fish. The advantage of animal oil consumption is attributed to Omega-3 polyunsaturated fatty acids (PUFAs) level, specifically thanks to the presence of the omega-3 (EPA) and omega-3 fatty acid (DHA) which may lower pressure and viscosity (Kundam et al., 2018; Yi et al., 2014) (Fig. 2.12).

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FIGURE 2.10 Structures of bioactive phytochemicals present in wheat.

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FIGURE 2.11 Structure of finger millet arabinoxylan.

FIGURE 2.12 The chemical structures of EPA and DHA.

2.1.2.13 Meat Meat and meat products are a decent source of bioactive compounds with a positive effect on human health like vitamins, minerals, peptides, or fatty acids. Growing food consumer awareness and intensified global meat producers’ competition put pressure on creating new healthier meat products. So as to satisfy these expectations, producers use supplements with functional properties for animal diets and as direct additives for meat products. In the food companion aggregate, meat is distributed as a protein food group along with flesh, fish, and eggs (Lachance & Fisher, 2005). Since meat contains a cornucopia of proteins with high natural value, regarded as nutritive, meat is an abecedarian source of essential amino acids. Meat is also an excellent source of some precious minerals and vitamins (Biesalski, 2005; Higgs & Mulvihill, 2002). Some of these nutrients are moreover not present or have inferior bioavailability in other foods. In addition to these introductory nutrients, important attention has lately been paid to meat-grounded bioactive composites, similar to conjugated linoleic acid. Several attractive meats-based bioactive substances have been studied for their physiological properties (Arihara et al., 2004, 2005). Such substances include conjugated linoleic acid (CLA), carnosine, anserine, L-carnitine, glutathione, taurine, coenzyme Q10, and creatine (Arihara & Ohata, 2008) (Fig. 2.13).

2.1.2.14 Flaxseed Flaxseed has become the main focus of many researchers due to its health-promoting and disease-preventing properties, such as flaxseed fiber, protein, lignan, unsaturated carboxylic acid, potassium, etc. Flaxseed is considered as nutraceutical and also as a functional food. It has emerged as an attractive nutritional food because of its exceptionally high content of alpha-linolenic acid (ALA), dietary fiber, high-quality protein, and phytoestrogens. Flaxseeds contain about 55% ALA, 28%
30% protein, and 35% fiber. Owing to its excellent nutritional profile and potential health benefits, it has become an attractive ingredient in diets specially designed for specific health benefits (Gutte et al., 2015) (Fig. 2.14).

2.1.2.15 Pomegranate The health benefits of pomegranate are attributed to its big selection of phytochemicals, which are predominantly polyphenols, including primarily hydrolyzable ellagitannins, anthocyanins, and other polyphenols. Polyphenol-rich fractions derived from the pomegranate fruit have been studied for their potential chemopreventive and/or cancer-therapeutic

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FIGURE 2.13 Representative meat-based bioactive compounds.

FIGURE 2.14 (A) (a) The basic structure of lignans consisting of two interlinked phenylpropanoid molecules, (b) secoisolariciresinol diglucoside, a dibenzylbutane lignan, (c) sesamin, a furanofuran lignin. (B) Chemical structure of the essential fatty acids, alpha-linolenic acid (above) and linoleic acid (below).

effects in several animal models. Although data from in vitro and in vivo studies look convincing, well-designed clinical trials in humans are needed to ascertain whether pomegranate can become part of our armamentarium against cancer (N Syed et al., 2013) (Fig. 2.15). Kris-Etherton et al. (2002) described bioactive compounds in foods with their role in the prevention of ailments and cancer. The study concludes with recommendations for diet rich in fruits, vegetables, whole grains, legumes, oils, and nuts. Guaadaoui et al. (2014) described what is a bioactive compound, a combined definition for a preliminary consensus. This paper tried to seek out a combined definition which might be a platform to develop the “bioactive” concept, and reach a solid consensus on the term “bioactive compound.” We use, for the primary time, the term “biocompounactive” to more simplify the employment of bioactive compounds in relation to their physicochemical and biological properties.

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FIGURE 2.15 Chemical constituents of pomegranate.

2.1.2.16 Factors affecting extraction techniques A number of factors have influenced the extraction efficiency of phytochemicals from plant and animal materials. The factors influencing the extraction efficiency of phytochemicals from materials are shown below. Investigating these factors can help establish the optimal conditions for the extraction of the target compounds from the plant matrix effectively. Raw materials play a vital role in the extraction efficiency of their bioactive compounds due to specific matrix, structure, and phytochemicals; therefore, the extractability of the phytochemicals varies betting on the kind of fabric. Different parts of stuff of the same plant, like leaves, stems, roots, and flowers, have different extractability of the target compounds. In addition, fresh, dried, or ground plant materials with small particle sizes have different extraction efficiencies when the extraction is performed under identical conditions. Although rare specific studies are reported to match the impact of various drying conditions and particle sizes on bioactive compounds, it is worthwhile to contemplate future studies for the higher selection and preparation of starting materials for the extraction of bioactive compounds (Al Ubeed et al., 2022) (Fig. 2.16). Current trends within the food industry and therefore the continuous look for healthy products suggest that consumer’s interest in natural and high-quality foods is increasing. Moreover, the worldwide health crisis created by COVID19 redirected the present consumer attitude, perception, and behavioral patterns to reduction of garbage and also regarding the food products consumed. Hence, attention to natural functional compounds has increased within the past year especially thanks to the consumers’ awareness regarding the evidence that a healthy diet contains a positive impact on health; therefore, consumers worldwide became more health-conscious. Furthermore, agro-food may be a rich source of various bioactive compounds with content looking on the category of the food source, like fruit and vegetables, dairy, meat and fish, cereals and roots, tubers, and oilseed (Vilas-Boas et al., 2021). Several methods are used for the extraction of polyphenol compound from numerous sorts of plants. Extraction through the Soxhlet apparatus with a special polar solvent like water, methanol, ethanol, or ester may be a conventional

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FIGURE 2.16 Factors affecting the extraction efficiency of bioactive compounds.

method. There are some advanced methods of extraction like microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), ultrasound-assisted extraction (UAE), and high hydrostatic pressure extraction (HHPE), which showed different quantitative and qualitative results. However, developing a singular standard method for the extraction of various forms of plants is still challenging. Scientists are attempting to seek out an efficient, cost-effective, and eco-friendly method for polyphenol extraction (Safiullah et al., 2018). The review aims to supply an outline of phytochemicals, conventional (Soxhlet, maceration, and hydrodistillation) and emerging (supercritical fluid, subcritical fluid, microwave-assisted, ultrasonic-assisted, enzyme-assisted, and pulsed electric field-assisted) technologies, identification of functional components with variety of chromatographic and spectrophotometric methods, as well as various analytical methods. This review also highlights the importance of functional components.

2.2

Current techniques for extraction of phytochemicals

2.2.1 Conventional methods of extraction 2.2.1.1 Soxhlet extraction Soxhlet extraction was first used for lipids; however, it is not limited any longer. A dry sample is placed in a thimble, which is further placed in an exceedingly distillation flask containing the solvent of interest. Siphon unloads the answer back within the distillation flask. The method is repeated until the ultimate compound of interest is extracted.

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2.2.1.2 Maceration Maceration is usually employed in homemade tonics. It is popular and accustomed to obtain volatile oil and functional components. The material is ground into smaller particles and mixed with a solvent. Then, the menstruum (solvent) is added to a closed vessel, and therefore the liquid is strained to recover the solid residues. The strained liquid is separated from impurities by filtration.

2.2.1.3 Hydrodistillation Hydrodistillation is often used for essential oils from plants, which might be done via water, steam, or direct steam distillation. During this method, the stuff is boiled in water and direct steam is injected. Indirect cooling condenses the vapor, and also, the separator separates the oil and functional components from the water. This method is not used for thermolabile compounds.

2.2.2 Nonconventional methods for plant extraction There are quite a few modern nonconventional methods which are preferred compared to the standard methods. A number of key modern methods are supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), microwaveassisted extraction (MAE), enzyme-assisted extraction (EAE), pulsed force field extraction (PEF), and ultrasoundassisted extraction (UAE).

2.2.2.1 Enzyme-assisted extraction (EAE) This method is widely accepted and used because it is novel, nontoxic, and non-inflammable. In some plant matrices, phytochemicals are present within the cell cytoplasm and retained by hydrogen or hydrophobic bonding, which do not seem to be accessible by solvent extraction. The addition of specific enzymes like cellulose, amylase, and pectinase as a pretreatment enhances the recovery by breaking the cytomembrane and hydrolyzing the structural polysaccharides and lipids. Extraction of oils from various seeds is achieved using enzyme-assisted aqueous extraction. The extracted oils were found to contain a better amount of free fatty acids and phosphorus content compared to the traditional methods. Enzyme composition, concentration, particle size, and hydrolysis time are considered as key factors of EAE.

2.2.2.2 Supercritical extraction One of the advanced environmental-friendly extraction methods for solid or liquid substances is supercritical fluid extraction (SFE) method, and desirable separation of components is often extracted through this analytical technique. This method produces solvent between vapor and liquid phase, through temperature and pressure conditions. By this example, physical changes accrue inside the solvent like diffusivity, compressibility, gas-like viscosity, liquid-like densities, and reduction in physical phenomenon. However, several extraction solvents are used for SFE methods like greenhouse emission, fluoroform, ammonia, n-pentane, propane, ethane, laughing gas, and water, but the foremost commonly used extraction solvent is greenhouse gas (Safiullah et al., 2018).

2.2.2.3 Microwave-assisted extraction Microwave-assisted extraction (MAE) could be a new extraction technique that mixes microwave and traditional solvent extraction. It is an advantageous technique thanks to shorter extraction time, higher extraction rate, less requirement of solvent, and lower cost over the traditional method of extraction of compounds.

2.2.2.4 Ultrasound-assisted extraction (UAE) Ultrasound passes through a medium by creating cavitation, that is, compression and expansion. Liquid materials will be exploited using this cavitation effect. UAE facilitates the leaching of organic and inorganic compounds from the plant matrix, by intensifying the mass transfer. Ultrasound extraction is found to be suitable for functional components from herbal plants. It is effective for achieving efficient mixing, faster energy transfer, reduced thermal gradients, and selective extraction.

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2.2.2.5 Pulsed electric field extraction (PEF) PEF treatment is reported to be useful for improving the pressing, extraction, and diffusion on the process. PEF mechanism works on the principle of the destruction of semipermeable membrane structure for enhanced extraction and decreased extraction time. PEF treatment is applied to enhance the discharge of intracellular compounds by increasing the semipermeable membrane permeability. Supporting the look of the treatment chamber, PEF process could either be operated in batch or continuous process. PEF treatment parameters like intensity, specific energy, treatment time, temperature, and property of the plant matrix are liable for the effectiveness of the treatment.

2.3

Characterization of phytochemicals

After extraction, bioactive compounds are isolated, purified, and identified to check for the presence of specific amounts of specific compounds. These compounds are classified according to their functional activity as determined by various bioactivity assays. Many analytical methods are used to isolate, purify, and identify bioactive compounds, and screening for these methods is consistent with ease, specificity, and speed (Altemimi et al., 2017; Sasidharan et al., 2011). Chromatography techniques are common for separating the compound of interest from a mixture of extracts. Bioactive compounds are separated and purified according to their adsorption properties, molecular size, ionic strength, boiling point, etc. The chemical assays are demonstrated for the determination of phenolic compounds, tannins, vitamins, flavonoids, pectin, and carboxylic acid content in various analyses within which the quantities of these compounds are calculated using the equivalent of standards, like acid, tannin, catechin, galacturonic acid, monounsaturated fatty acid, and so on. After that, there are several bioactivity assays, like different radical scavenging (DPPH, ABTS), oxygen-reducing power (ORAC), carotene bleaching, iron-chelating (FRAP) assays, microbial inhibition capacity for determination of antioxidant activity, antimicrobial activity, respectively (Ivanovi´c et al., 2020; Trigo et al., 2022). The separation and isolation of bioactive compounds are determined by chromatographic methods, distinguished by their polarity. Gas
liquid chromatography (gas chromatography) is employed, while the extract contains some slight volatile compounds, and liquid
solid chromatography (thin-layer chromatography [TLC]) and high-performance liquid chromatography (HPLC) are used, while the mixture contains high relative molecular mass molecules (Altemimi et al., 2017). The presence of polar compounds in those compounds is separated to the opposite side of the column, leaving the mixture. Finally, after the isolation of bioactive compounds, various spectroscopic methods are used for identification, composition, and bonding inside the molecules. The essential principle of those methods is the absorption of radiation by the molecules, which supplies a spectrum. The spectra are meant for identification as every spectrum is specified for every sort of bonding within the molecule. UV
visible, Fourier transform infrared spectroscopy (FTIR), nuclear resonance (NMR), and spectrographic analysis (MS) are some samples of those methods (Ivanovi´c et al., 2020; Sasidharan et al., 2011).

2.3.1 Determination of total flavonoid content (TFC) The TFC may be measured using the chloride spectrophotometric method as described by Horincar et al. (2019) with slight modifications.

2.3.2 Determination of total phenolic content (TPC) The TPC may be measured spectrophotometrically using the Folin
Ciocalteu (FC) method as described by Horincar et al. (2019).

2.3.3 Antioxidant activity—DPPH scavenging method (AA) The AA is determined using the DPPH free radical scavenging activity as mentioned by Horincar et al. (2019).

2.3.4 Antimicrobial activity Antimicrobial activity of extracts is assessed against Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, and Monilia albicans using agar well diffusion assay.

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2.3.5 Assessment of minimum inhibitory concentration (MIC) MIC of the extracts (methanol and aqueous) will be determined using the microbroth dilution method.

2.3.6 Antioxidant capacity Antioxidant capacity is measured using the oxygen radical absorbance capacity (ORAC) assay and adapted to a 96-well FLUOstar Omega Plate Reader (BMG Labtech Inc., Durham, NC).

2.4

Conclusion

Phytochemicals are naturally present in many foods. They are found in plants, and their consumption generally has beneficial health benefits. Nutrients in food nurture and support our bodies to stay fit. But it has also been proven that certain foods, additionally, provide health values like inhibition or treatment of varied styles of ailments. Recently, plant functional compound has achieved remarkable significance for its food conservative traits, radical scavenging, and containing health benefits for several diseases. Preclinical, clinical, and epidemiological researches suggest that phytochemicals could also with its antioxidant and anti-inflammatory properties are effective in treating various illnesses. Because of public awareness, health benefits, and consumer’s high demand, the international market for functional compound is remarkably increasing. The high consumption rate of plant-rich functional compound by patients and elder people also had a vital role within the global market growth. Currently, scientists are engrossed with the health advantages derived from foods, and there are abundant substantiation of the promotion of human health by foods and their constituents. The rise in ailments, like cancer, diabetes, hypertension, obesity etc., related to foods has prompted people to prefer foods and food products that also provide functional and health benefits. This review describes both traditional and new techniques used to extract bioactive compounds from different food sources. In addition, bioactive compounds extracted from extraction techniques can be characterized using chromatography and spectrophotometry. Improving extraction techniques with little or no solvent can have a significant impact on sustainable bioprocesses. In addition, the characterization of bioactive compounds by fast, sensitive, and inexpensive methods for use and incorporation in a variety of fields is useful. Extracted bioactive compounds are often incorporated into a variety of products in the pharmaceutical and food industries.

2.5

Future considerations for effective extraction of phytochemicals

Plant material may be a rich and diversified source of phytochemicals, but it would be challenging to possess an optimal extraction technique or condition to recover all bioactive molecules from it. Although numerous conventional and advanced techniques are employed to extract phytochemicals, there are still gaps for future studies to know more about the materials, the impact of various extraction parameters, and therefore the stability of derived phytochemicals. Rare comprehensive studies have compared the phytochemicals, between different species and their different parts, like leaves, stems, flowers, and roots. Additionally, the influence of geographical locations, harvesting season, harvesting time, and cultivation methods on the phytochemicals must be considered to possess high-quality starting materials for extraction. Systematic investigations are necessary to grasp the impact of various transportation, storage, and drying conditions and methods on the retention of phytochemicals from different sources. As these phytochemicals are sensitive, storage and transportation under appropriate conditions are crucial. Additionally, drying can affect phytochemicals, and further studies on different drying conditions and methods are needed to spot the simplest drying conditions and methods before the extraction process.

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315. Dhwani, S., Poojitha, P., & Gurumoorthi, P. (2021). A review on different extraction and quantification methods of allicin from garlic. Journal of Xidian University, 15(6), 183
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53. Iba´n˜ez, M. D., & Bla´zquez, M. A. (2020). Curcuma longa L. rhizome essential oil from extraction to its agri-food applications. A review. Plants, 10 (1), 44. Ivanovi´c, M., Islamˇcevi´c Razborˇsek, M., & Kolar, M. (2020). Innovative extraction techniques using deep eutectic solvents and analytical methods for the isolation and characterization of natural bioactive compounds from plant material. Plants, 9(11), 1428. Khan, R., Islam, B., Akram, M., Shakil, S., Ahmad, A., Ali, S. M., & Khan, A. U. (2009). Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules (Basel, Switzerland), 14(2), 586
597. Kim, H., Choi, H. K., Moon, J. Y., Kim, Y. S., Mosaddik, A., & Cho, S. K. (2011). Comparative antioxidant and antiproliferative activities of red and white pitayas and their correlation with flavonoid and polyphenol content. Journal of Food Science, 76(1), C38
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88. Kundam, D. N., Acham, I. O., & Girgih, A. T. (2018). Bioactive compounds in fish and their health benefits. Asian Food Science Journal, 4(4), 1
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392S. Available from https://doi. org/10.3945/an.112.003517. Mogren, L. M., Olsson, M. E., & Gertsson, U. E. (2007). Effects of cultivar, lifting time and nitrogen fertiliser level on quercetin content in onion (Allium cepa L.) at lifting. Journal of the Science of Food and Agriculture, 87(3), 470
476. Mohammed, K. A. K., Abdulkadhim, H. M., & Noori, S. I. (2016). Chemical composition and anti-bacterial effects of clove (Syzygium aromaticum) flowers. International Journal of Current Microbiology and Applied Sciences, 5(2), 483
489. Moreno, F. J., Corzo-Martı, M., Del Castillo, M. D., & Villamiel, M. (2006). Changes in antioxidant activity of dehydrated onion and garlic during storage. Food Research International, 39(8), 891
897. N Syed, D., Chamcheu, J. C., M Adhami, V., & Mukhtar, H. (2013). Pomegranate extracts and cancer prevention: Molecular and cellular activities. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 13(8), 1149
1161. Okwudili, U. H., Gyebi, D. K., & Obiefuna, J. A. I. (2017). Finger millet bioactive compounds, bioaccessibility, and potential health effects
A review. Czech Journal of Food Sciences, 35(1), 7
17. ˆ ., Ntatsi, G., Petrotos, K., Barros, L., & Ferreira, I. C. (2018). Nutritional value, chemical characterization and bulb Petropoulos, S. A., Fernandes, A morphology of Greek garlic landraces. Molecules (Basel, Switzerland), 23(2), 319.

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Safdar, M. N., Kausar, T., Jabbar, S., Mumtaz, A., Ahad, K., & Saddozai, A. A. (2017). Extraction and quantification of polyphenols from kinnow (Citrus reticulate L.) peel using ultrasound and maceration techniques. Journal of Food and Drug Analysis, 25(3), 488
500. Safiullah, Jauhar, Mohammad Rashedi, Ismail-Fitry, Gun Hean, Chong, Mahmud, Ab. Rashid Nor-Khaizura, & Wan Ibadullah, Wan Zunairah (2018). Polyphenol compounds from pomegranate (Punica granatum) extracted via various methods and its application on meat and meat products: A review. Journal of Advanced Research in Applied Sciences and Engineering Technology, 12(1), 1
12. Salamatullah, A. M., Hayat, K., Alkaltham, M. S., Ahmed, M. A., Arzoo, S., Husain, F. M., & Al-Harbi, L. N. (2021). Bioactive and antimicrobial properties of oven-dried beetroot (pulp and peel) using different solvents. Processes, 9(4), 588. Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K. M., & Latha, L. Y. (2011). Extraction, isolation and characterization of bioactive compounds from plants’ extracts. African Journal of Traditional, Complementary and Alternative Medicines, 8(1). Senadheera, S. P. N. M. K., & Abeysinghe, D. C. (2015). Functional components and total antioxidant capacity of different tissues of two pitaya (dragon fruit) species grown in Sri Lanka. Journal of Food and Agriculture, 8(1 & 2), 33
40. Shang, A., Cao, S. Y., Xu, X. Y., Gan, R. Y., Tang, G. Y., Corke, H., & Li, H. B. (2019). Bioactive compounds and biological functions of garlic (Allium sativum L.). Foods, 8(7), 246. Singh, R., & Singh, K. (2019). Garlic: A spice with wide medicinal actions. Journal of Pharmacognosy and Phytochemistry, 8(1), 1349
1355. Srirejeki, S., Manuhara, G. J., Amanto, B. S., Atmaka, W., & Laksono, P. W. (2018). The effect of water volume and mixing time on physical properties of bread made from modified cassava starch-wheat composite flour. IOP conference series: Materials science and engineering, 333(1), 012072, IOP Publishing. Trigo, J. P., Alexandre, E. M., Saraiva, J. A., & Pintado, M. E. (2022). High value-added compounds from fruit and vegetable byproducts
Characterization, bioactivities, and application in the development of novel food products. Critical Reviews in Food Science and Nutrition, 60(8), 1388
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6. Zhao, X. X., Lin, F. J., Li, H., Li, H. B., Wu, D. T., Geng, F., & Gan, R. Y. (2021). Recent advances in bioactive compounds, health functions, and safety concerns of onion (Allium cepa L.). Frontiers in Nutrition, 8.

Chapter 3

Novel extraction conditions for phytochemicals Manas Ranjan Senapati1 and Prakash Chandra Behera2 1

Agro-Polytechnic Centre (Animal Science), Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India, 2Department of Veterinary

Biochemistry, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubanewswar, Odisha, India

3.1

Introduction

In consonance with the World Health Organization (WHO), more than 80% of the world’s population relies on indigenous traditional knowledge/medicine for their primary healthcare needs. Traditional medicine is the natural plant either as standardized extracts or as pure compounds (Patel et al., 2021). Medicinal plants are the richest bioresource of drugs of traditional systems of medicine, nutraceuticals, food supplements, pharmaceutical intermediates, and chemical entities for crude and synthetic drugs (Rasul, 2018). Traditional knowledge of medicinal plants is a guide for searching and developing new cures. Despite inadequate knowledge of the active chemical constituents, mechanisms of action, and efficacy of herbal medicines, it has been used as the option of choice in the treatment of human beings and animals from time immemorial. The phytopreparations are beneficial due to cheaper cost, local availability, easy acceptability, and involvement of simple techniques. Plants are contemplated as autogenetic factories for the production of various phytochemicals. A large number of secondary metabolites like alkaloids, phenolics, steroids, terpenoids, flavonoids, etc., are amalgamated by plants in addition to compounds that are needed for their growth and reproduction. These plant metabolites, by virtue of their potent radical scavenging/transition metal ion chelating activities to inhibit the oxidizing chain reactions, significantly reduce the risks of cancer, cardiovascular disease, diabetes, inflammation, infectious, and geriatric diseases (Table 3.1). Therefore floral medicinal TABLE 3.1 Pharmaceutical importance of phytoconstituents. Sl. no.

Phytoconstituent

Pharmaceutical importance

References

1

Alkaloids

Anticancer, preanesthetic in surgery, child birth, ophthalmology, in delirium, tremor, menia, and Parkinsonism

Soni et al. (2012)

2

Beta-carboline alkaloids

Antidepressant, antitumor, antibacterial, anti-inflammatory, assisting detoxifying enzymes, cardioprotective

Patel et al. (2012)

3

Flavonoids

Lowers blood cholesterol, antioxidants, antihypertensive, antimicrobleeding, hepatoprotective, antibacterial, anti-HIV-I reverse transcriptase, antidengue, inhabitants of viral polymerase

Kumar and Abhay (2013)

4

Phenolics

Anticancer

Ozcan et al. (2014)

5

Steroid

Anticancer, antiasthmatic, antidiabetic

Sheikh et al. (2013)

6

Tannins

Immunomodulatory, antibacterial, antifungal, hypolipidemic, cardioprotective

Tariq and Reyaz (2013)

7

Terpenes

Anticancer, antioxidant, antiviral, antiatherosclerotic

Chemat et al. (2005)

8

Anthocyanin

Immunomodulatory, anti-inflammatory, antioxidant, colorants of pharmaceutical and food products

Yang and Zhai (2010)

9

Saponins

Inhibits dental caries, antihypercalciuria, antidote for lead poisoning, anticancer, hepatoprotective

Kwon et al. (2003)

Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00019-0 © 2023 Elsevier Inc. All rights reserved.

27

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Recent Frontiers of Phytochemicals

formulations find a place in alternative medicines as a result of which exploration of those plant metabolites so-called phytochemicals led the researchers toward its identification and isolation (Akhtar et al., 2019). The isolated active phytomolecules serve as starting material for laboratory synthesis and development of drugs with promising potency. The efficacy of developed drugs is validated by the effect study of the phytoconstituents either in vivo or in vitro. Effect study is possible after extraction of phytoconstituents under suitable conditions for better and effective harvest. The physical property, chemical nature, solubility, and sensitivity to pH and temperature of these diverged phytoconstituents vary within and between the plant species. In order to harvest more phytoconstituents both qualitatively and quantitatively, advanced extraction tool with respect to the use of the solvent, temperature, pressure, and time for different components independently or in combination is very much precious. In view of the growing demand for herbal therapeutic molecules, the development of innovative extraction techniques to recover phytoconstituents in a sustainable and eco-friendly manner is the need of the hour. Therefore the isolation of such compounds needs a specific extraction technique involving the most appropriate solvents. For attaining the desired analytes from the plant extract for experimental purposes involves the proper and timely collection of the plant sample, authentication by an expert, adequate drying and grinding followed by extraction, fractionation, and isolation of the bioactive compound where applicable (Abubakar & Haque, 2020). Extraction, screening, and detection of phytochemicals with different extraction techniques and the nature/volume of solvent advocate better for the effect study. Depending on the physical nature and the chemical properties of phytoconstituents, various conventional extraction methods including infusion, digestion, decoction, percolation, and maceration are commonly practiced in the herbal industry for crude extraction (Huda et al., 2016). However, traditional methods of extraction are time-consuming, at least 2
7 h with the consumption of a large amount of solvents, and also, the targeted molecules might be decomposed due to high temperatures in Soxhlet extraction (Afoakwah et al., 2012). Therefore, there was a space for the invention of new techniques owing to the development of alternative extraction techniques like accelerated solvent extraction (ASE), microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), and supercritical fluid extraction (SFE) that have gained increasing interest during the last three decades (Co et al., 2012). These techniques reduced the consumption of organic solvent, shortened extraction time, alteration in extraction temperature and pressure, and enabled automation concerning to compensate for the increasing demand for natural products (Table 3.2).

TABLE 3.2 A brief summary of various extraction methods for natural products (Arora & Itankar, 2018). Method

Solvent

Temperature

Pressure

Time

Volume of organic

Polarity of natural

solvent consumed

products extracts

Maceration

Water, aqueous and nonaqueous solvents

Room temperature

Atmospheric

Long

Large

Dependent on extracting solvent

Percolation

Water, aqueous and nonaqueous solvents

Room temperature occasionally under heat

Atmospheric

Long

Large

Dependent on extracting solvent

Decoction

Water

Under heat

Atmospheric

Moderate

None

Polar compounds

Reflux extraction

Aqueous and nonaqueous solvents

Under heat

Atmospheric

Moderate

Moderate

Dependent on extracting solvent

Soxhlet extraction

Organic solvents

Under heat

Atmospheric

Long

Moderate

Dependent on extracting solvent

Pressurized liquid extraction

Water, aqueous and nonaqueous solvents

Under heat

High

Short

Small

Dependent on extracting solvent

Supercritical fluid extraction

Supercritical fluid (usually S-CO2), sometimes with modifier

Near room temperature

High

Short

None or small

Nonpolar to moderate polar compounds

Ultrasound-assisted extraction

Water, aqueous and nonaqueous solvents

Room temperature or under heat

Atmospheric

Short

Moderate

Dependent on extracting solvent

Microwave-assisted extraction

Water, aqueous and nonaqueous solvents

Room temperature

Atmospheric

Short

None or moderate

Dependent on extracting solvent

Pulsed electric field extraction

Water, aqueous and nonaqueous solvents

Room temperature or under heat

Atmospheric

Short

Moderate

Dependent on extracting solvent

Enzyme-assisted extraction

Water, aqueous and nonaqueous solvents

Room temperature or heated after enzyme treatment

Atmospheric

Moderate

Moderate

Dependent on extracting solvent

Hydrodistillation and steam distillation

Water

Under heat

Atmospheric

Long

None

Essential oil (usually nonpolar)

Novel extraction conditions for phytochemicals Chapter | 3

29

The biological activities of the phytoconstituents extracted in these techniques using different solvents exhibited variations during the effect study. Based on varied chemical characteristics, polarities and uneven distribution in the plant matrix, recovery of phytophenols, the antioxidant compounds are typically accomplished through different extraction techniques and the nature of the solvent (Chan et al., 2011; Devgun et al., 2009). The phytochemicals though having a very significant vital role in life science, the quantity of these active ingredients in the plant is always fairly low. Again, intensive laboratory technology and time-consuming extraction and isolation process have been the bottleneck of the application of phytochemicals in drug development. Therefore there is an urgent need to develop effective and selective methods for the extraction of these natural bioactive compounds. Hence, this review intends to provide a comprehensive view of novel extraction conditions for phytochemicals.

3.2

Pre-extraction conditions

Plants are rich sources of unique chemical substances such as alkaloids, glycosides, tannins, steroids, volatile oils, resins, phenols, and flavonoids deposited in leaves, flowers, bark, seeds, fruits, and roots (Duraipandiyan et al., 2006). The recovery of these phytocomponents depends upon the extraction technique. Based on their physical and chemical properties, four different pre-extraction conditions are followed strictly in the process of isolation and identification of the bioactive compounds from the plant materials.

3.2.1 Collection The process of collection is the gateway to the recovery of bioactive phytocompound of our interest considering their qualitative and quantitative characteristics. The proportion of active principles in the plant is influenced by environmental factors, species, place of collection, cultural factors, the biological value of the cultivar, processing methods, etc. Unambiguous identification of plant species appears often difficult due to changes in plant taxonomy (Bucar et al., 2013). In this context, the focus must be given to environmental, ecological, and social factors during collection to ensure quality (Patel et al., 2021).

3.2.1.1 Quality consideration 3.2.1.1.1

Taxonomical authenticity of species

It is mandatory to perceive the taxonomical identity of the plant species before its collection by searching the literature. Genus, species, subspecies (if any), and citation of author must be included during recognition. In case of reporting a

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Recent Frontiers of Phytochemicals

new medicinal plant species, which has no entry in any literature, the Botanical Survey Institute, Forest Research Institute, or any authorized national or local herbaria must be communicated to substantiate its identity (Bucar et al., 2013; Upton et al., 2020). 3.2.1.1.2

Collection of healthy plant at its unerring phenological phase

The subject of plant samples to be collected should be healthy ones without any infestation (insect, pest, and fungi) and infection (bacteria and virus) (Oyewole et al., 2018). Since the concentration of active principles differs according to the various parts and stages of plant growth, collection should be done at the proper developmental stage to accomplish the maximum amount of bioactive compounds. The harvesting period for the plant samples must be documented with respect to its phenological stage, dates, and months as well (Mendes et al., 2017). 3.2.1.1.3

Climatic condition and area of collection

Collection of samples should be avoided during rain, moist, or at high humid conditions in order to give a wide berth to insect, pest, or fungal assault. In case of collection in wet weather, the sample should be processed to remove the maximum amount of water as soon as possible. Plant samples should be collected from the area denude of dusts, insects, chemicals, harmful fumes, etc. The places like industrial and mining areas, public utilities, crematoria, sewage lines, hospitals and dispensaries, garages, anthills, roadside areas with vehicle pollution, or the area near these places should be avoided during sample collection (National Medicinal Plant Board, 2009). 3.2.1.1.4 At liberty from undesirable stuffs during collection Collected samples should be unchained from immature or decadent parts of plants, foreign stuffs, and harmful weeds that could degrade the quality and quantity of extract (Patel et al., 2021).

3.2.1.2 Ecological consideration 3.2.1.2.1

Significance of preservation and restoration of species

The collector should be aware of endemic plant species found in his collection site and follow prevailing ecological and legal guidelines to eliminate probable threat to the species (Cragg et al., 1993). So, the contemporary protection status of desired plant species must be known to forest, wildlife officials, and collectors as well (Rai et al., 2000). The collector should remember that some species of therapeutic plants are terrorized by overharvesting in several parts of the world leading to its inclusion in the Law of Biodiversity, and its harvesting is now forbidden (Coors et al., 2019; Luo et al., 2019). Therapeutic plant material should only be collected to the extent that they can regenerate without making injury to the parts like roots, stems, leaves, flowers, fruits, and seeds during collection. 3.2.1.2.2

Area of sustenance and habitat management

The amount of collected plant samples should be proportional to the allocation of the species in its sustainable geographical area. Damage to the habitat of the plant should be minimized to ensure its long-term viability and care to be taken to avoid uprooting the linked species during the collection of roots or secretive parts of research. In the case of the collection of plant samples in the wild, extreme vigilance should be exercised to avoid destroying its specific ecosystems (Chemat et al., 2012). 3.2.1.2.3

Equipment for collection

Pertinent equipment is to be used for digging, cutting, peeling, sorting, and other activities to prevent unfortunate injury to the plant of interest. Apparatus made of nontoxic material, cleaned and devoid of any contaminants like chemicals, paints, and lubricants, are to be used for purity of the harvesting molecules.

3.2.1.3 Social consideration 3.2.1.3.1

Availability for local use

The accessibility of desired species for local use should not be abused by collocated assembly of therapeutic plant products from the wild.

Novel extraction conditions for phytochemicals Chapter | 3

3.2.1.3.2

31

Reasonable pricing and sharing of benefits

Collectors of therapeutic parts of the various plant species sometimes do not obtain revenues as compared to their efforts within India as per the recent supply chain arrangement. There is a need for rational pricing mechanism for all harvested species. In this context, the Biological Diversity Act, 2002, has included benefit sharing option for medicinal plant properties and related information in India. Therefore a fair and equitable profit-sharing strategy should be created and followed by all partners in medicinal plant products. 3.2.1.3.3 Health stature of collectors Individuals with open wounds or inflammations or infections on their skin should stay away from the primary processing system. Natural elements like plant exudates, pollen, and aromas should be avoided by individuals with allergies. Collectors should wear relevant personal protective equipment such as safety gloves, shoes, eye, and nose protection when obtaining the product from wild habitats. 3.2.1.3.4 Cultural ethics In India, certain plant species are correlated with social and religious values. In some tribes, some plants are revered as sacred, and only a small number of populations of those plants may be harvested, as a consequence of which harvesting and postharvest management of medicinal plant products must follow and respect the ethical essence and norms of the local community and the realm as well (WHO, 2003).

3.2.2 Drying Collected plant samples are to be dehydrated properly before packing and further processing. Innumerable plant samples like rhizomes, woody parts, pulpy fruits, fleshy roots, stems, leaves, and petals and those containing polysaccharides require additional attention for adequate drying. Morphologically thick, meaty, or large samples should be cut or sliced into small and thin pieces to ensure requisite drying. Based on the physical and chemical nature of samples and bioactive compounds to be extracted, the following drying steps are to be undertaken (Abdullah, Razak Shaari et al., 2012).

3.2.2.1 Air-drying The period of air-drying varies from 3
7 days to 1 month and even up to 1 year depending on the types of samples to be dried (e.g., leaves or seeds). Although the leaves and stems can be air-dried together at room temperature, there is a risk of predisposed contamination at teetering temperature conditions (Fokunang, Ronel et al., 2017; Pougoue et al., 2020). Since the drying method is of long duration without the involvement of any lab equipment, heat-labile compounds are preserved at room temperature. Therefore shade-drying is preferred by many researchers (Sharma & Janmeda, 2017; Temidayo, 2013; Tzanova et al., 2018).

3.2.2.2 Oven-drying It is one of the pre-extraction practices to remove or reduce the moisture content of the samples by employing thermal energy. It is the easiest and the most rapid thermal process of sample preparation within a short duration for extraction and preservation of phytochemicals (Abdullah, Shaari et al., 2012; Mediani et al., 2013). The thermal procedure needs to be standardized and validated with respect to the range of temperature and holding time for an effective extraction before use. Oven-drying at 40 C
45 C is easy and time-consuming. Some thermolabile compounds are destroyed on exposure to high temperatures for a short period (Fokunang et al., 2018; Tembe Fokunang, Pougoue et al., 2019; Venugopal & Liu, 2012). In contrast, oven-drying at 40 C can increase the total polyphenol content and biological activity of phytochemicals (Yi & Wetzstein, 2011).

3.2.2.3 Freeze-drying (lyophilization) Sublimation is a method of converting a solid directly into gas phase surpassing the liquid phase. Before lyophilization, the herbal sample is frozen at 280 C to 220 C to solidify the remaining liquid accommodated in the samples for 12 h or overnight to lyophilize the sample quickly by avoiding the frozen liquid in the sample from melting. The procedure includes wrapping the mouth of the container with needle-poked parafilm to prevent the sample loss during the process (Venugopal & Liu, 2012). Freeze-drying method preserves and yields a greater quantity of phenolic compounds as the bioactive metabolites as compared to air-drying (Jiofack et al., 2011; Wilcox et al., 2007). On a triparty comparison between air, freeze, and microwave-drying, freeze-drying appears complex and expensive for which its use is restricted to delicate, thermal-sensitive compounds of high value (Azwanida, 2015).

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Recent Frontiers of Phytochemicals

3.2.2.4 Microwave-drying The process involves the combined effect of both electric and magnetic fields by using electromagnetic radiation. Here, the alignment of molecules on the electric field with a sturdy or instigated dipole moment and ionic induction of the electric field prompts simultaneous heating and oscillation of the molecules by dipolar rotation (Tembe Fokunang, Fokunang et al., 2019). At the same time, oscillation engenders molecules for collision resulting in rapid heating of the samples. This approach can extricate drying time with the drawback of causing phytochemical deterioration (Kaufmann & Christen, 2002).

3.2.3 Grinding The dried plant samples are ground for leaching out the bioactive compounds from disrupted cells. Grinding facilitates the samples into homogenized small-sized particles in coarse or powdered form and greater surface contact with extraction solvents required for optimum extraction (Arnold & Schepers, 2004; Borhan et al., 2013; Methods Optimization in Accelerated Solvent Extraction, 2013). Traditional mortar and pestle and electric blenders or mills are typically used to reduce the particle size of a sample. Particle size of less than 0.5 mm enhances penetration of solvents with the target analytes and diffusion of solutes for which it is considered the optimum size for efficient extraction (Borhan et al., 2013; Ngono et al., 2018). Difficulty in filtration is observed with too fine particle size due to excessive absorption of solvent. Generally, powdered plant materials are ideal for the effective extraction of phytochemicals (Arora & Itankar, 2018; Hossain & Rahman, 2015; Pk et al., 2019; Zhang et al., 2016, 2018).

3.2.4 Storage Preservation of plant samples avoids the adverse effects of temperature, humidity, and contamination and facilitates storage for future use. The conditions of storage vary with the nature of the samples. Separate climate (temperature and humidity) regulated storage facilities for hygroscopic and volatile materials, and closed containers in a safe location for storage of oils, resins, gums, and other flammable products are recommended. The containers are specific to the samples and are labeled properly with all the pertinent information about the samples. Bulk amounts of samples are compressed manually/mechanically to take the edge off storage space requirements and to clear the way for transportation. Containers especially gunny bags, jute bags, and corrugated boxes are not stacked directly on the floor, and containers of different samples are not towered together. The samples are used within its legitimate shelf life period as tagged on the container (Patel et al., 2021; Salminen, 2003).

3.3

Selecting a pre-extracting sample preparation

3.3.1 Fresh or dried samples In medicinal plant research, both fresh and dried samples are used. Dried samples are selected concerning the time required for experimental design (Amid & Adenan, 2010; Azame et al., 2020; Bezejea et al., 2019; Fokunang, TembeFokunang et al., 2017). Since fresh samples are fragile and deteriorate faster than dried samples, a maximum of 3 h gap between harvest and experiment is advisable to retain its freshness. Although no significant effect is reported in the total phenolic contents of fresh and dried samples, dried sample yields more flavonoids (Fokunang, 2016; Fokunang, TembeFokunang et al., 2017).

3.3.2 Ground or powdered samples The curtailment of particle size increases the contact surface area between samples and extraction solvents. Conventionally, grinding can result in smaller coarse samples whereas powdered samples have a more homogenized and even smaller particle size, thus providing a better contact surface with the extraction solvents (Azame et al., 2020; Mukherjee et al., 2011; San et al., 2013). Based on the type of samples (ground/powdered), different extraction techniques like infusions, percolation, decoctions, etc., are developed to yield tinctures, fluid, semisolid, and powdered extracts. Such preparations popularly are called galenicals, named after Galen, the second-century Greek physician. The use of pectinolytic and a cell wall polysaccharide-degrading enzyme in sample preparation facilitates small-sized particles and enhances enzyme action in enzyme-assisted extraction process (TEMBE et al., 2019; Tembe-Fokunang et al., 2018; Vongsak et al., 2013).

Novel extraction conditions for phytochemicals Chapter | 3

3.4

33

Extraction conditions

Extraction is the first and an important step in the itinerary of phytochemical processing for the discovery of bioactive constituents from the plant materials. The components of the extraction conditions affecting the quality and quantity of phytochemicals of an extract are temperature, duration, particle size, holding time, solvent (type, nature and polarity, pH, concentration, and solvent-to-sample ratio), and type of analytes to be extracted. Therefore the selection and optimization of suitable extraction process are critical for upscaling purposes, that is, from bench scale to pilot plant level (Dhanani et al., 2017; Patel et al., 2021; Roby et al., 2013). Though high temperature increases diffusion and solubility, too high temperature causes loss of solvents, extraction of undesirable impurities, and the decaying of thermolabile components. The efficiency of extraction is directly proportional to extraction duration up to a certain time range. Increasing the time does not affect the extraction after attaining the equilibrium of the solute inside and outside the solid material. The extraction yield is higher owing to a higher solvent-to-solid ratio; however, too high solvent-to-solid ratio requires more volume of extraction solvent and a long time for concentration as well.

3.4.1 Solvent system for extraction Solvent extraction is the most extensively used method for the extraction of bioactive compounds from the plant samples. The method progress with the stages of (1) the solvent penetrates the solid matrix; (2) the solute dissolves in the solvents; (3) the solute is diffused out of the solid matrix; and (4) the extracted solutes are finally collected. Any factor augmenting the diffusivity and solubility in the above steps facilitates the extraction. The following characteristics are considered during the selection of a solvent system in the extraction process (Barwick, 1997; Oancea et al., 2012; Zhang et al., 2018). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Safe to use, nonhazardous, and nonflammable; Low cost as viable; Low toxicity; Capable to extract the effective ingredient leaving the inert stuff behind; Low viscosity to permit ease of penetration; Promotion of rapid physiologic absorption of the extract; Preservative action; Rapid recovery and separation from the extract; Ease of evaporation at low heat; Low boiling temperature to avoid degradation by heat; Incompetence to cause the extract to dissociate; Never react with the extract.

Moreover, solvent system selection highly depends on the specific chemical nature of the phytochemical being targeted for extraction. Different solvent systems are required to extract bioactive compounds from natural products as tabulated in Table 3.3. Polar solvents such as methanol, ethanol, or ethyl acetate are used for extraction of hydrophilic compounds, whereas for extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/ methanol in ratio of 1:1 is used. In some cases, extraction with hexane is used to produce chlorophyll-free extract (Kaufmann & Christen, 2002; Mediani et al., 2013). TABLE 3.3 Solvents used for active component extraction (Paulucci et al., 2013; Sasidharan et al., 2011). Solvent name

Name of the bioactive metabolites

Water

Anthocyanins, lectins, polyphenols, saponins, starches, tanins, terpenoids

Ethanol

Alkaloids, flavonol, polyacetylenes, polyphenols, sterols, tannins, terpenoids

Methanol

Anthocyanins, flavones, lactones, phenones, polyphenols, quassinoids, saponins, tanins, totarol, terpenoids, xanthoxylin

Chloroform

Flavonoids, terpenoids

Ether

Alkaloids, coumarins, fatty acids, terpenoids

Acetone

Flavonols, phenol

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3.4.1.1 Selection of solvents (on the basis of polarity) The solvent choice is influenced by the nature of the plant, the portion of the plant to be extracted, the nature of the bioactive components, types of extraction techniques, and the availability of solvent. Polar solvents like water, methanol, ethanol, butanol, and acetone are used in polar compound extraction, while nonpolar solvents like dichloromethane, ether, and hexane are used in nonpolar chemical extraction along with its advantages and disadvantages as tabulated in Table 3.4. The traditional method for liquid
liquid extraction involves the use of two miscible solvents such as water
dichloromethane, water
ether, or water
hexane. Water is present in all of the mixtures due to its high polarity and miscibility with organic solvents. During fractionation, the selected solvent is added in order of increasing polarity, starting with the least polar n-hexane and ending with the most polar water. Five solvents can be used during fractionation by mixing two low-polarity solvents (n-hexane, chloroform), two medium-polarity solvents (dichloromethane, n-butanol), and one highpolarity solvent (water) (Nawaz et al., 2020). Various solvents (methanol, hexane, and ethyl alcohol) of differing polarities are used for the extraction of different phenolic compounds from plant parts (leaves and seeds). Multiple solvents are used sequentially to limit the amount of analogous compounds in the desired yield. The polarity of common solvents can be cited as hexane , chloroform , ethyl acetate , acetone , methanol , water (Wong & Kitts, 2006). Acetone and N,N dimethyl-formamide (DMF) are highly effective at extracting antioxidants, and methanol is more effective in extracting a large amount of phenolic contents from plant samples as compared to ethanol. Ethanol extracts higher concentrations of phenolics as compared to acetone, water, and methanol (Anokwuru et al., 2011; Koffi et al., 2010; Ruan et al., 2008). The extraction of flavonoids is based on similar principles of polyphenols extraction. The process involves methanol, ethanol, acetonitrile, acetone, or their mixtures with water depending on the required solvent polarity and the type of flavonoid (Stobiecki & Kachlicki, 2006; Tsao, 2010). The absolute choices of solvents for less polar flavonoids (isoflavons, flavonones, and flavones) are acetone, chloroform, methylene chloride, and diethyl ether and those for more polar flavonoid fractions are alcohol and alcohol
water mixture. However, the extraction of flavonones and anthocyanins depends on the pH of the solvent (Marston & Hostettmann, 2006). Since the presence of lipophilic substances affect the composition profile of flavonoids and their derivatives in the extracts, an additional cleaning procedure (solid-phase extraction) is advisable. Lipophilic compounds from the plant material are removed by using n-hexane (Cosa et al., 2006; Musa, 2015) or petroleum ether (Zhang et al., 2016).

3.4.2 Extraction methods Extraction is followed either by: (1) fresh plant samples can be homogenized or macerated with alcohol, (2) powdered dried material is directly extracted, or (3) first defeat the material and then extraction of desired component TABLE 3.4 Properties of solvent for extraction. Solvent

Nature

Advantage

Disadvantage

References

Polar

Water

Dissolves a wide range of polar compounds, inexpensive, nontoxic, and nonflammable

Stimulates the growth of germs and mold, causes hydrolysis, and requires a lot of heat

Castro-Puyana et al. (2017); Das et al. (2010)

Alcohol

Self-preservative, harmless at low concentration, requires modest of heat for extraction

Combustible, volatile, unable to dissolve fats, gums, or wax

Sun, Wu et al. (2015)

Ionic liquid (green solvent)

Extremely heat stable, intense miscibility with water and other solvent, attracts and transmits microwave, ideal for liquid
liquid extraction, nonflammable

Not suitable for the formulation of extracts

Vishwakarma (2014); Xiao, Chen et al. (2018)

Chloroform

Colorless with sweet odor, soluble in alcohols, extract lipids terpenoids, flavonoids, and oils, easily absorbed and processed by the body

Contains sedative and carcinogenic properties

Torres et al. (2014)

Ether

Low boiling point, miscible with water, no taste, unaffected by acids, bases, or metals, extract alkaloids, terpenoids, coumarins, and fatty acids

Extremely flammable and volatile in nature

Torres et al. (2014)

Nonpolar

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(Charishma & Thiruvengadarajan, 2013). Various extraction techniques, including conventional and recent advanced methods, have gained increasing interest during the last three decades across the globe for phytochemical processing of medicinal plants (Co et al., 2012). Some newer methods are still unfolding, whereas the existing ones are experiencing moderations. One disadvantage of the classical solid
liquid techniques is the large amount of plant material remaining after the process, which is still rich in bioactive compounds besides the enhanced weight of raw plant material, the large amount of solvent used, energy, and time consumption. Also, some methods are not suitable for phytoconstituents with the risk of thermal degradation. That being the case, selecting a pertinent method of extraction is a very vital issue which depends on innumerable factors (Ahmad et al., 2019).

3.4.2.1 Factors to be considered for selecting a method of extraction The selection of the extraction method depends on (1) Nature of material and components to be extracted: Heat-stable plant material is extracted using Soxhlet extraction or microwave-assisted extraction, whereas plant materials those are not heat stable are extracted using maceration or percolation. (2) Available solvent system: Maceration is a suitable method, if the solvent of extraction is water, but for volatile solvent, percolation and Soxhlet extraction are more appropriate. (3) Cost of the drug: Cheap drugs are extracted using classical conventional methods, whereas costly drugs are preferably extracted using advanced techniques. (4) Duration of extraction: Maceration is suitable for plant material requiring long exposure to the solvent systems, whereas techniques such as microwave- or ultrasound-assisted extraction are used for a shorter duration. (5) Required extract volume: Large volume products such as tinctures are prepared by maceration, whereas concentrated products are produced by percolation or Soxhlet extraction. (6) Intended use: Extracts intended for consumption by human are usually prepared by maceration, whereas products intended for experimental testing are prepared using other methods in addition to maceration. (7) Influence of solvent: Influence of solvent on extraction method can only be achieved either by altering the pH or polarity of the solvent. A component may behave like strong electrolyte or nonelectrolyte, depending on the pH of the solution. Precipitation of components occurs, when the pH of the solution is adjusted to such a value at which unionized molecules are produced in sufficient concentration to surpass its solubility (Azwanida, 2015; Majekodunmi, 2015; Pandey & Tripathi, 2014). It is always sensible to consult specific literature in order to intercept the avoidable loss of desired bioactive metabolites caused by the use of an ill-suited extraction technique. The simplest extraction processes employed may be classified as (1) extraction with organic solvents by percolation, maceration, reflux, and Soxhlet extraction and (2) extraction with water by infusion, decoction, and steam distillation. The most popular method of extraction is to use a liquid solvent at atmospheric pressure, possibly with the application of heat. Other methods include steam distillation, supercritical fluid extraction, and the use of liquefied gases under moderate pressure. The choice of method depends on the factors listed above as well as the intrinsic advantages and disadvantages of the procedures (Kar, 2007).

3.4.2.2 Classification of extraction method 3.4.2.2.1

Maceration

Maceration is a technique now generally adopted in usage for natural products/medicinal plant research. This is an extraction procedure in which coarsely or powdered plant samples like leaves, bark, or root are placed inside a closed container along with a defined solvent. This setup is left to stand at room temperature for a minimum of 72 h with stirring in a regular interval. The process is conditioned to soften and break up the plant’s cell wall in order to liberate the soluble bioactive metabolites. Extraction is aided by frequent agitation during maceration by two processes: (1) promote diffusion and (2) separation of concentrated solution from the sample surface by introducing additional solvent to the system to increase extraction yield. After 3 days, the whole mixture is then sieved out, and the marc is pressed to recover the maximum amount of occluded solution. The acquired pressed-out liquid and the strained solvent are combined to face for clarification either by filtration using Whatman no 1 filter paper or through decantation after standing. In this technique, heat is transferred by convection and conduction, and the choice of the solvent is basically playing the most critical role to determine the type of phytochemical extracted from the samples (Handa et al., 2008; Hossain et al., 2014; Ingle et al., 2017; Morata et al., 2018; Selvamuthukumaran & Shi, 2017; Trusheva et al., 2007). When the solvent is water and the period of maceration is long, a small quantity of alcohol may be added to prevent microbial growth. Seventy percent of methanol is an efficient solvent for extracting flavonoids by maceration (Damia´n-Reyna et al., 2016; Laghari et al., 2011; Sulaiman et al., 2011). The extracts obtained by maceration are complex mixtures, and it is necessary to carry out further purification by re-extrication (Lin et al., 2016), column chromatography (Jovanovi´c et al., 2017), or a combination of both methods (Ma et al., 2013). This technique is a simple and effective

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method for the extraction of phenolic compounds and can be translated into economic benefits with nearly identical extraction yields as compared to other routine methods (Albuquerque et al., 2017; Jovanovi´c et al., 2017). Strength: Though being the most traditional and conventional method of extraction, this technique is the easiest and simple. This method is convenient and very suitable for thermolabile plant material. Maceration has long been a popular and cost-effective home method for tincture or tonic preparation. In addition, this method is utilized to extract essential oils and active chemicals from plant matter. In most cases, the maceration method entails many extraction phases. Alteration in the temperature and the choice of extraction solvents can enhance the extraction process. Thus it also proves to be a flexible method as per our convenience. The technique of maceration is the easiest, and no special lab equipment is required (Abubakar & Haque, 2020; Patel et al., 2021). Limitations: Being the simplest form of extraction technique, no major limitations in this method are persisting. But the limitation lies in the choice of the use of organic extraction solvent. The more complex the extraction solvent is, the more purification techniques need to be employed to obtain the pure form of the active constituent. The main disadvantages of this method are the large volume of solvents, the long processing time, the long extraction time, and the need for future purification. In case purity is an issue, advanced extraction technology should be considered. 3.4.2.2.2 Infusion This is an extraction process that uses the same principle as maceration having only one difference of shorter keeping time. The sample is ground into a fine powder and then placed inside a clean close container along with hot or cold extraction solvents. This method is suitable for the extraction of bioactive constituents that are readily soluble. In addition, it is an appropriate method for the preparation of fresh extract before use. The solvent-to-sample ratio is usually 4:1 or 16:1 depending on the intended use (Handa et al., 2008; Hossain et al., 2014; Ingle et al., 2017). 3.4.2.2.3 Percolation (exhaustive extraction) The apparatus used in this process is called percolator. It is a narrow cone-shaped glass vessel with opening at both ends. A dried, ground, and finely powdered plant material is moistened with the more quantity solvent of extraction in a clean container and kept for 4 h. Subsequently, the content is then transferred into percolator with the lower end closed and allowed to stand for 24 h at room temperature (Hossain et al., 2014). The solvent of extraction is then poured from the top until the sample is completely saturated. The lower part of the percolator is then opened, and the liquid is allowed to drip slowly. Some quantity of solvent was added continuously, and the extraction took place by gravitational force, pushing the solvent through the drug material downward. The addition of solvent stopped when the volume of solvent added reached 75% of the intended quantity of the entire preparations. The marc is then pressed, and the final amount of solvent is added to get the required volume. The final volume of the extract is clarified by filtration or by standing followed by decanting. It is more efficient than maceration because it is a continuous process in which the saturated solvent is constantly being replaced by fresh solvent. This is the most frequently used method to extract phytochemicals for the preparation of tinctures and fluid extracts since it does not require much manipulation or time (Gao et al., 2009; Zhang, Wang et al., 2014). It is a continuous process in which the saturated solvent is constantly being displaced by a fresh one. Normally, percolation is not used as a continuous method because the sample is steeled in solvent in the percolator for 24 h (up to three times), and then, the extracted materials are collected and pooled. The percolation process is generally conducted at moderate rates such as using 6 drops/min until the extraction is completed before evaporation to obtain a concentrated extract (Nn, 2015; Rathi et al., 2012). In general, in the process of percolation, particularly in the manufacturing of concentrated preparations like liquid extracts, the following problems may arise: 1. If the active substances are thermolabile, evaporation of large volume of dilute percolate may result in partial loss of the active constituents. 2. In the case of alcohol
water mixture, evaporation results in preferential vaporization of alcohol leaving behind an almost aqueous concentrate which may not be able to retain the extracted matter in solution and hence gets precipitated. In such cases, the modification in the general process of percolation is required as given below. Reserved percolation In this case, the extraction is done through the general percolation procedure. At last, the evaporation is done under reduced pressure in equipment like a climbing evaporator to the consistency of a soft extract (semisolid) such that all the water is removed. This is then dissolved in the reserved portion which is strongly alcoholic and easily dissolves the evaporated portion with any risk of precipitation (Madziga et al., 2010).

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Decoction

The working principle of decoctions is the same as that of infusions, but the samples are boiled with that of solvent for a specified period or till a definite volume is attained. This technique involves continuous hot extraction using specified volume of water as a solvent. It is usually the method of choice when working with tougher and more fibrous plant or its parts like barks, roots, and woods in its dried, grinded, or powdered form which have water-soluble bioactive compounds. Depending on the type of plant material used, strong decoctions are prepared in two general ways. The first involves boiling the mixture longer. This is usually indicated when working with larger woody pieces of bark. Longer boiling time, up to 2 h or more, is sometimes necessary to break down, soften, and extract the larger pieces. Alternatively, when smaller woody pieces are used yet a stronger remedy is wanted, the decoction is prepared as above with shorter boiling time of up to 20 min, and then, it is allowed to sit/soak overnight before straining out the herb. When straining, again, make sure to press on the cut herb pieces in the strainer to get as much moisture/decoction out of the herb pieces. The extract from this process contains a large amount of water-soluble impurities. It cannot be used for the extraction of thermolabile or volatile components as it encounters hydrolysis, dehydration, decarboxylation, and addition reactions. The hydrolysis efficiency was strongly affected by pH, temperature, and the amount of samples. This process might enhance the dissolution of some bioactive compounds compared with the maceration process. The high temperature in this process deactivated the activity of the β-glucuronidase and prevented the transformation of glycosides to their aglycones (Kamboj, 2010; Li, Lai et al., 2010; Zhang et al., 2013; Zhang, Chen et al., 2014). This process is typically used in the preparation of ayurvedic extracts called quath or kawath. The starting ratio of the crude sample to water is fixed, for example, 1:4 or 1:16; the volume is then brought down to one-fourth of its original volume by boiling during the extraction procedure. Then, the concentrated extract is filtered and used as such or processed further (Pandey & Tripathi, 2014; Tembe-Fokunang et al., 2018). 3.4.2.2.5

Expression

Expression, referred to as cold pressing, is a method of extraction specific to citrus essential oils. In older times, expression was done in the form of sponge pressing, which was literally accomplished by hand. The oil released in this process is absorbed by the sponge, and it was recovered back by squeezing the sponge. It is reported that oil produced through this way contains more of the fruit odor than oil produced by any other method. Thermosensitive essential oils are extracted by this method usually achieved by a tincture press. In general, expression involves squeezing any plant material at great pressure to press out oils or other liquids. The process is carried out by hand-operated presses or crushes in isolated rural areas or by gigantic mechanical presses in industrial centers. This involves disruption of the cellular structure by the application of pressure to the material and allows oil to flow out of it. It is frequently used for the extraction of soya oil, sunflower oil, and olive oil. The rupture in the cell kernel causes the elution of fixed oils from plant material through this method (Adekunle & Adekunle, 2009). 3.4.2.2.6

Distillation

Distillation is a process of separation of components of a mixture of two or more liquids by virtue of their vapor pressure difference. There are three systems of distillation described as follows. Hydrodistillation It is the oldest method being used for the separation of essential oil. In a crude metallic distillation unit, plant material is kept along with boiling water. This process obeys the principle of osmotic pressure to diffuse oil from the oil glands. The essential oil of a plant consists of many compounds which generally boil between 150 C and 300 C. The vapor passes through a coiled tube contained in a water bath, and the condensate is obtained at the bottom of the condenser tube. The disadvantages are that the heat is difficult to control, and hence, the rate of distillation is variable. Also, the possibility exists for local overheating and burning of the sample which can lead to extract of lowquality oil. Steam distillation A process of extracting essential oils from plant products through heating and evaporation process is known as steam distillation. Steam distillation is a popular method for the extraction of volatile oils (essential oils) from plant material. This can be carried out in a number of ways. One method is to mix the plant material with water and heat it to boiling (distillation with water). The vapors are collected and allowed to condense, and the oil is separated from the water. It resembles hydrosteam distillation except that no water is kept in the bottom of the still. This method is efficient and gives higher yields. However, it is not generally employed for delicate flowers. To maximize the yields of the oils, precautions must be taken to ensure efficient condensation of the steam and vaporized oil and collection of

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the condensate in such a way as to prevent loss of the volatile material. However, to avoid the risk of explosion, a completely closed system must not be used. The advantages of this type of dry steam distillation are that it is relatively rapid; therefore, charging and emptying the still are much faster, and the consumption of energy is lower. The rapid distillation is also less likely to damage those oils which contain reactive compounds, for example, esters. However, it cannot be used where the oil contains hydrolyzable components such as esters or those that are easily degradable (Doughari et al., 2009; Vlietinck, 2010). Hydrosteam distillation To overcome the drawback of hydrodistillation, modifications in techniques were developed. In this technique, plant material is supported on a perforated grid or screen inserted at some distance above the bottom of the still. Water filled below the grid is heated which produces saturated and wet steam, and thus produced steam pass through plant material and vaporized essential oil. 3.4.2.2.7

Soxhlet extraction

Named after Franz Ritter von Soxhlet, a German agricultural chemist, it is the best method for the continuous extraction of a solid by a hot solvent. It is a standard technique that has been used for a long time and is the main reference for evaluating the performance of other solid
liquid extraction methods. In this method, fat and oil from solid material are extracted by repeated washing with organic solvent under reflux. Organic solvents commonly used are hexane and petroleum ether. It is an apparatus of a specialized glass refluxing unit mainly used for organic solvent extractions. The powdered solid material is placed in a thimble made up of sharp filter paper or cellulose and is placed inside the Soxhlet apparatus. The apparatus is fitted to a round-bottomed (RB) flask containing the solvent and to a reflex condenser. The solvent in the RB flask is boiled gently, and the vapor passes up through the side tube, is condensed by the condenser, falls into the thimble containing the material, and slowly fills the Soxhlet. When the solvent reaches the top of the attached tube, it siphons over into the flask, thus removing the portion of the extracted substance, and this procedure is repeated and continued until a drop of solvent from the siphon tube is unable to leave residue when evaporated. On a small scale, it is used mainly as a batch process; it can become much more economical and viable when converted into a continuous extraction procedure on a medium- or large-scale extraction. The optimum sample for this extraction is a dry, finely divided solid, and numerous variables such as temperature, solvent sample ratio, and solvent type must be taken into account (Zygler et al., 2012). Soxhlet extraction is usually a well-established technique with wide industrial applications, better reproducibility, and efficiency along with the least extract manipulation over other novel extraction methods such as ultrasound-assisted, microwave-assisted, supercritical fluid, or accelerated solvent extractions. However, compared with the novel fast extraction techniques, it is an old-fashioned, time- and solvent-consuming extraction technique. Some solvents used in the conventional Soxhlet have recently been questioned because of their toxicity. When compared to advanced extraction methods such as supercritical fluid extraction (SFE), this approach is deemed unfriendly to the environment and may lead to pollution. The researcher used solvents with different polarities (hexane, dichloromethane, ethyl acetate, and methanol) starting with the low polar solvent and ending with the high polar one. Thus the selectivity of some thermally stable flavonoids can be attained by Soxhlet extraction. However, different solvents have to be used for several successive extractions. The researchers explained this with its oxidation and degradation during the extraction process (Anuradha et al., 2010). The extracts obtained by Soxhlet extraction have a complex composition, and the flavonoid fraction must be re-extracted to be purified or could be purified by liquid chromatography (Zhang et al., 2016). Compared to percolation, the advantages of Soxhlet extraction are the shorter processing time and the possibility for automation. After quantitative extraction by classical solid
liquid methods like Soxhlet or maceration, the purification in one step can lead to the preparation of extract rich in flavonoids. The Soxhlet extraction method integrates the advantages of the reflux extraction and percolation, which utilizes the principle of reflux and siphoning to continuously extract the herb with fresh solvent. It is an automatic continuous extraction method with high extraction efficiency that requires less time and solvent consumption than maceration or percolation (Azwanida, 2015). The high temperature and long extraction time in the Soxhlet extraction will increase the possibilities of thermal degradation. The longtime requirement and the requirement of large amounts of solvent lead to wide criticism of the conventional Soxhlet extraction method. The concentrations of both total polyphenols and total alkaloids from the Soxhlet extraction method at 70 C decreased as compared to those from the maceration method applied under 40 C (Chin et al., 2013; Wei et al., 2013). Practical issues for Soxhlet extraction Solvent choice: A suitable extracting solvent should be selected for the extraction of the targeted phytoconstituent using this extraction method. Different solvents will yield different extracts and

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compositions. The most widely used solvent to extract edible oils from plant samples is hexane as it has a fairly narrow boiling point range of approximately 63 C
69 C. It is also an excellent oil solvent in terms of oil solubility and ease of recovery. However, n-hexane, the main component of commercial hexane, is listed as No. 1 on the list of 189 hazardous air pollutants by the US Environmental Protection Agency. The use of alternative solvents such as isopropanol, ethanol, hydrocarbons, and even water has increased due to environmental, health, and safety concerns. Alternative solvents often result in less recovery due to a decreased molecular affinity between solvent and solute. The costs of alternative solvents could be higher. A cosolvent is sometimes added in order to increase the polarity of the liquid phase. A mixture of solvents such as isopropanol and hexane has been reported to increase the yield and kinetics of extraction (Mamidipally & Liu, 2004; Zarnowski & Suzuki, 2004). Matrix characteristics: Soxhlet extraction strongly depends on matrix characteristics and particle size as the internal diffusion may be the limiting step during extraction (Luque-Garcia & Luque de Castro, 2004). Operating conditions: During Soxhlet extraction, the solvent is usually recovered by evaporation. The extraction and evaporation temperatures have a significant effect on the quality of final products. The high boiling temperature for solvent recovery can be decreased by using vacuum or membrane separation to recover the solvent. Advantages and disadvantages of Soxhlet extraction Advantages: (1) The displacement of transfer equilibrium by repeatedly bringing fresh solvent into contact with the solid matrix, (2) maintaining a relatively high extraction temperature with heat from the distillation flask, (3) no filtration requirement after leaching, and (4) large amounts of bioactive metabolites can be extracted with a very small amount of solvent, saving time, energy, and financial costs. Disadvantages: (1) The extraction time is long, (2) a large volume of solvent is used, (3) agitation cannot be provided to accelerate the process, (4) the large amount of solvent used requires an evaporation/concentration procedure, (5) the possibility of thermal decomposition of the target compounds, (6) hazardous and flammable liquid organic solvents, and (7) if required, high-purity solvents may increase the cost (Kar, 2007; Tembe-Fokunang et al., 2018). 3.4.2.2.8 Microwave-assisted extraction MAE has attracted the attention of researchers as a technique to extract bioactive compounds from a wide variety of plants and natural residues. Microwave-assisted extraction is one of the advanced techniques under thought nowadays. In MAE, microwave vitality is utilized to concentrate plant metabolites with the solvents. This system has demonstrated its wellbeing for the vast majority of the specimens because of the ease to handle and to understand steadiness. Exploration is continuing for the functional use of microwaves for business creation of phytoconstituents, yet at the same time in the early stages. Recently, advanced techniques have become available to reduce the loss of bioactive compound without increasing the extraction time. Therefore MAE is demonstrated to be a good technique in multiple fields, especially in the medicinal plant area. Moreover, this technique reduced the losses of the biochemical compounds being extracted. It has been used as an alternative to conventional techniques for the extraction of antioxidants because of its ability to reduce both time and extraction solvent volume. In fact, the main objective of using MAE is to heat the solvent and extract antioxidants from plants with a lesser amount of these solvents. Heating the solvents and plant tissue using microwave increases the kinetic of extraction to facilitate the partition of analytes from the sample matrix into the solvent. Microwave radiation interacts with dipoles of polar, polarizable materials cause heating near the surface of the materials, and heat is transferred by conduction. Dipole rotation of the molecules induced by microwave electromagnetic disrupts hydrogen bonding, enhancing the migration of dissolved ions and promoting solvent penetration into the matrix. In nonpolar solvents, poor heating occurs as the energy is transferred by dielectric absorption only (Ballard et al., 2010; Devgun et al., 2009; Kingston & Jessie, 1998; Li et al., 2012). Principles and mechanisms Microwaves are part of electromagnetic spectrum of light with a frequency of 300 MHz to 300 GHz, and the wavelengths of these waves range from 1 cm to 1 m. These waves are made up of two perpendicular oscillating fields which are used as energy and information carriers. The first application of microwaves includes its interaction with specific materials which can absorb a part of its electromagnetic energy and can convert it into heat. Commercial microwaves use 2450 MHz of energy for this purpose which is almost equivalent to 600
700 W (Afoakwah et al., 2012; Mandal et al., 2007). Microwaves are transmitted as waves, which can penetrate biomaterials and interact with polar molecules such as water in the biomaterials to create heat. Consequently, microwaves can heat a whole material to penetration depth simultaneously. The mechanism of MAE is different from other types of extraction methods because the extraction occurs as a result of changes in the cell structure caused by electromagnetic waves (Azwanida, 2015). This process of extraction involves a synergistic combination of mass and heat transfers working in

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the same direction whereas the mass transfer in conventional methods occurs from inside to outside of the substrates, and heat transfer occurs from the outside to inside of the substrate. The series of phenomenological steps that occur during the MAE are: (1) the irradiation heat from a microwave is transferred to the solid through the microwavetransparent solvent without absorption, (2) the intense heating of the above results in residual microwave-absorbing in the solid being heated up, (3) the heated moisture evaporates and creates a high vapor pressure, (4) the high vapor pressure breaks the cell of the substrate, and (5) cell wall breakage enhances the releases of the extract from the samples. MAE offers rapid delivery of energy to a total volume of solvent and solid plant matrix with subsequent heating of the solvent and solid matrix, efficiently and homogeneously. Because water within the plant matrix absorbs microwave energy, cell disruption is promoted by internal superheating, which facilitates the desorption of phytochemicals from the matrix, improving the recovery of bioactive compounds (Chemat & Cravotto, 2013; Raut et al., 2015). Furthermore, the migration of dissolved ions increased solvent penetration into the matrix resulting in a heating effect due to increased kinetic energies of ions as well as friction between ions due to their continuous movements and change in directions and thus facilitated the release of the chemicals. Destruction of hydrogen bonding also increases the penetrating efficiency of the solvents into the plant matrix. The effect of microwave energy is thus strongly dependent on the dielectric susceptibility of both the solvent and the solid plant matrix. There are two types of commercially available MAE systems: closed vessels system under controlled pressure and temperature and open vessels system, working at atmospheric pressure. The closed vessels MAE system, having Teflon cells, is generally used for extraction under drastic conditions such as high extraction temperature. The pressure in the vessel essentially depends on the vapor pressure, volume, and boiling point of the selected solvents. The advantages of closed vessel systems are: (1) higher temperatures are attained rapidly due to increased pressure inside the vessel, (2) volatile compounds are not wasted in the environment but remain as a part of the extract, (3) there is no or lesser risk of contamination due to closed systems, (4) as solvent is not evaporated during heating, a very little amount of solvent is required in this procedure, and (5) the procedures like acid digestions become safer as fumes do not come out, so it becomes easy to handle such procedures. In open vessel systems, quartz cells are used, work under atmospheric pressures, and depend solely on boiling points of solvents for heating purposes to attain maximum temperature. Solvent is heated and refluxed back through condenser, like Soxhlet extraction, and cellulose cartridge can also be used to carry plant sample as it helps to avoid an extra step of filtration. Open vessel system can be used to extract larger quantities of plant sample. Open vessel systems are also considered safer in solvent handling (Tatke & Jaiswal, 2011). Several forces that include physicochemical relations and interactions like chemical interactions, driving forces, interstitial diffusion, and dispersion forces can be seen during the process, and the strength and persistence of properties can be related to the characteristics of the extraction solvent such as polarity, solubility in water, purity, solubilization (Shams et al., 2015). Practical issues for microwave assisted extraction Several studies had been done on optimizing the factors for MAE to achieve optimal yields from the plant samples. The operative parameters that determine the efficiency of MAE include matrix characteristics, solvent-to-sample ratio, solvent choice and operating conditions like microwave power and extraction temperature, irradiation time, stirring effect, microwave energy density, etc. Hence, understanding the influences and interactions of these parameters on the extraction process is highly essential (Veggi et al., 2013). Matrix characteristics: As MAE depends on the dielectric susceptibility of solvent and matrix, better recoveries can be obtained by moistening samples with a substance that possesses a relatively high dielectric constant such as water. If a dry sample is rehydrated before extraction, the matrix itself can thus interact with microwaves and hence facilitate the heating process. The microwave heating leads to the expansion and ruptures of cell walls and is followed by the liberation of phytochemicals into the solvent resulting in improving the yield. In this case, the surrounding solvent can have a low dielectric constant and thus remains cold during extraction. This method can be used to extract thermosensitive compounds such as essential oils. However, it was found that it was impossible to perform a good MAE for completely dry as well as for very wet samples when a nonpolar solvent such as hexane was used as the extraction solvent (Brachet et al., 2002). Plant particle size usually has a significant influence on the efficiency of MAE. The particle sizes of the extracted materials are usually in the range of 100 μm
2 mm. Fine powders can enhance the extraction because the limiting step of the extraction is often the diffusion of chemicals out of the plant matrix, and the larger surface area of a fine powder provides contact between the plant matrix and the solvent. Nevertheless, too much finest of the plant sample may generate some technical difficulties. Hence, filtration or centrifugation is employed in the preparation of the plant samples (Mandal et al., 2007; Spar Eskilsson & Bjorklund, 2000). Solvent-to-sample ratio: During extraction, the solvent volume must be sufficient to ensure that the solid matrix is entirely immersed. Generally, a higher ratio of solvent volume to solid matrix mass in conventional extraction techniques can increase the recovery. However, in the MAE, a higher ratio may give lower recoveries. This is probably due

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to inadequate stirring of the solvent by microwaves. Some literature had reported that the extraction solution must not exceed 30%
34% (w/v). In the previous studies, the solvent-to-feed ratio between 10:1 (mL/g) and 20:1 (mL/g) had been reported to give optimal yields. The volume of extracting solvent is another important factor. A large volume of solvent requires more energy and time to condense the extraction solution in the purification process. Also, high volume of solvent may give lower recoveries because of nonuniform distribution and exposure to microwave (Chemat et al., 2005; Veggi et al., 2013). Solvent choice: Solvent choice for MAE is dictated by the solubility of the extracts of interest, by the interaction between solvent and plant matrix, and finally by the microwave-absorbing properties of the solvent determined by its dielectric constant (Chan et al., 2011). The dielectric constant of some commonly used solvents is illustrated in Table 3.5. The results of the researchers showed that both efficacy and selectivity of MAE depend significantly on the dielectric constant of the extraction solvent mixture. Usually, the chosen solvent should possess a high dielectric constant and strongly absorb microwave energy. Solvents such as ethanol, methanol, and water are sufficiently polar to be heated by microwave energy. Nonpolar solvents with low dielectric constants such as hexane and toluene are not potential solvents for MAE. Studies had shown that the addition of a small quantity of water to polar solvent resulted in higher diffusion of water into the cells of the matrix, leading to effective heating and thus facilitating the transport of compounds into the solvent at higher mass transfer rates. The extracting selectivity and the ability of the solvent to interact with microwaves can be modulated by using mixtures of solvents. One of the most commonly used mixtures is hexane
acetone. A small amount of water (e.g., 10%) can also be incorporated in nonpolar solvents such as hexane, xylene, or toluene to improve the heating rate (Brachet et al., 2002; Spar Eskilsson & Bjorklund, 2000). Operating conditions: Microwave power and extraction temperature: In MAE, microwave power and temperature of extraction are important factors that affect the extraction yield. The higher microwave power can lead to an increase in the temperature of the system resulting in the increase of the extraction yield until it becomes insignificant or declines. An increase in temperature can result in increased solvent efficacy because of drop in surface tension and viscosity, enhancing the solvent to solubilize solutes and improving matrix wetting and penetration. However, the efficiency of MAE increases with the increase in temperature until an optimum temperature is reached. Microwave power is also related to the quantity of sample and the extraction time required. The power provides localized heating in the plant matrix and acts as a driving force for MAE to destroy the plant matrix so that the solute can diffuse and dissolve in the solvent. Therefore increasing the microwave power will generally improve the extraction yield and result in a shorter extraction time. On the other hand, if microwave power is too high, leading to the degradation of thermolabile compounds in the plant matrix, then extraction yield will be poor (Terigar et al., 2010; Veggi et al., 2013). It is then important to select the appropriate microwave power to reduce the extraction time required to reach the set temperature and avoid a “bumping” phenomenon. Irradiation time: One of the importances of MAE over conventional methods is that the irradiation/extraction time is very short. The usual time ranges from a few minutes to half an hour depending on the plant sample/matrix so as to avoid possible oxidation and thermal degradation. The irradiation time is affected by the dielectric property of the

TABLE 3.5 Dielectric constant of some commonly used solvents (Chan et al., 2011; Tatke & Jaiswal, 2011). Sl. no.

Solvent

Dielectric constant at 20 C

1

Hexane

1.89

2

Toluene

2.40

3

Chloroform

4.80

4

Dichloromethane

8.90

5

Acetone

20.70

6

Ethanol

24.30

7

Methanol

32.60

8

Dimethylformamide

37.70

9

Dimethyl sulfoxide

45.00

10

Water

78.50

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solvent used. Solvents such as ethanol, water, and methanol may heat up rapidly on longer exposure which can result in the degradation of thermolabile compounds in the extracts. Increased time of irradiation can improve the recovery yield; nevertheless, the increased yield can decline at prolonged irradiation time. Sometimes, if the extraction will take a longer time, the plant materials are extracted through multiple stages by utilizing consecutive extraction cycles. Here, a new solvent is introduced to the residues; the procedure is then repeated to ensure the exhaustion of the plant sample. The use of this process helps higher recovery yield with no excessive heating. The nature of the plant sample and solute determines the number of extraction cycles and time as well (Chan et al., 2011; Raut et al., 2015). Stirring effect: Mass transfer processes in the solvent phase are usually enhanced by stirring. The equilibrium between the vapor and aqueous phases is achieved more rapidly. The use of a stirrer in MAE accelerates the extraction process by increasing the dissolution and desorption of bioactive compounds in the sample matrix. Thorough stirring can reduce the drawbacks possessed when using a low solvent-to-solid ratio and minimized the mass transfer barrier (Chan et al., 2011; Ruan & Li, 2007). Microwave energy density: There are three heating operational modes employed in the performance evaluation of MAE. These include the constant-power heating mode, intermittent heating mode, and constant temperature heating mode. The study reported that the constant-power heating mode presents the standard practice in the extraction of thermally sensitive active constituents of the plant matrix. It is worth to note that microwave power alone does not provide an adequate explanation as to how energy is being absorbed in the extraction of the biological medium. Therefore some study was conducted in order to establish the interrelationship between the microwave energy density and the extraction yield. From the research, it was concluded that for a unit of extracting solvent, microwave energy density is the most important factor affecting the extraction efficiency in MAE due to an accelerated effect on the ionic conduction and dipole rotation which in turn leads to an increase in the extraction yield. Polar solvent rates of absorption improve with increasing power and ultimately result in higher heating and extraction rate (Chan et al., 2015; Desai et al., 2010). Potential applications of microwave assisted extraction MAE is a reliable source of extraction of phytoconstituents. A lot of literature has been available for the extraction of phenolics (Gallo, Rasalia et al., 2010), flavonoids (Xiao, Lujia et al., 2008), essential oils (Wang et al., 2010), and glycosides (Javad et al., 2014). This is also applied for the analysis of heavy metals and other pollutants present in the soil samples with decreased test time, lesser power, and solvent consumption. It is also in use for the synthesis of pharmaceutical products. These results are guiding toward the wide range of commercial applications of MAE in the field of pharmaceuticals, food products, dentistry, etc., (Desrosiers et al., 2009; Sadeghi et al., 2017). The potential applications of MAE over conventional extraction are associated with a drastic reduction in organic solvent consumption and extraction time. It was also found that the presence of water in the solvent of methanol had a beneficial effect and allowed faster extractions than with organic solvent alone. A higher extraction yield can be achieved using MAE in a shorter extraction time. A study revealed that MAE recovered 92.1% of desired analytes within 12 min of extraction time, while several-hour Soxhlet extraction only achieved about 60% recovery (Kaufmann et al., 2001a; Pan et al., 2002). Advantages and disadvantages of microwave assisted extraction MAE has been considered a potential alternative to traditional solid
liquid extraction for the extraction of metabolites from plants. By considering economical and practical aspects, it is a strong novel extraction technique for the extraction of phytochemicals. Improved recoveries of the active constituent from the sample can be achieved with constant reproducibility. It has been used for extraction for several reasons like (1) remarkably required less extraction time, ranging from a few seconds to a few minutes (15 s to 20 min), (2) less amount of solvent, only a few milliliters of solvent is required, (3) extraction yield is improved, (4) better precision and accuracy as provided by the automation of the instrument, (5) favorable for thermolabile constituents, (6) heavy metals and pesticide residue which are present in minute traces can be extracted from a few milligrams of plant sample, and (7) during extraction, it provides agitation, by which the mass transfer phenomenon is improved (Mandal et al., 2007). However, compared to SFE, additional filtration or centrifugation is necessary to remove the solid residue during MAE. It is done with caution of using proper conditions in order to avoid thermal degradation. This method is limited to small molecule phenolic compounds such as phenolic acids (gallic acid and ellagic acid), quercetin, isoflavin, and transresveratrol as they are stable under microwave heating conditions up to 100 C for 20 min. Excessive exposure to microwave radiations results in a drastic decrease in the yield of phenolics and flavanones, mainly caused due to oxidation of compounds. Tannins and anthocyanins are not likely suitable for MAE as they are easily subjected to degradation at high temperature. Furthermore, the efficiency of microwaves can be very poor when either the target compounds or the solvents are nonpolar or when they are volatile (Kaufmann et al., 2001a; Spar Eskilsson & Bjorklund, 2000).

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Accelerated solvent extraction

Principles and working mechanisms ASE is a solid
liquid extraction process operated at elevated temperature, usually between 50 C and 200 C and at pressures between 10 and 15 MPa. Therefore ASE is known as pressurized liquid extraction (PLE) or enhanced solvent extraction system (ESE) or pressurized fluid extraction (PFE) or high-pressure solvent extraction (HPSE) by different research groups. Increased temperature accelerates the extraction process by increasing the extraction kinetics, and elevated pressure keeps the solvent in the liquid state above its boiling point resulting in a high solubility and diffusion rate of lipid solutes in the solvent and enforces the solvent to the matrix with a high penetration power, thus achieving safe and rapid extraction (Lv et al., 2010; Nieto et al., 2010). With the use of a small amount of solvent, it is an efficient form of liquid solvent extraction as compared to maceration and Soxhlet extraction. The solvent is still below its critical condition during ASE. But pressure allows the extraction cell to be filled faster and helps to force liquid into the solid matrix. Elevated temperatures enhance the diffusivity of the solvent resulting in increased extraction. Considering the facts and findings, the most important parameters for this technique are the polarity of the extraction solvent, temperature, pressure, and the number of extraction cycles which control the process mechanisms (Zgorka, 2009). The sample is packed with inert material such as sand in the stainless steel extraction cell to prevent the sample from aggregating and further blocking the tubing system of the apparatus. A packed ASE cell includes layers of sand
sample mixture in between cellulose filter paper and sand layers. This automated extraction technology is well adapted to control the temperature and pressure for each sample and takes less than an hour for extraction. Compared to other solvent techniques, ASE also critically depends on the solvent types. Cyclohexane and acetone solution at the ratio of 6:4 v/v with 5-min heating at 50 C has been shown to yield the highest amount of phytochemicals with 68.16% purity. Although the solvent used in ASE is usually organic solvents, pressurized hot water or subcritical water can also be used in ASE apparatus, which is usually called pressurized hot water extraction (PHWE) or subcritical water extraction (SWE) (Eskilsson et al., 2004; Sulaiman et al., 2011). Advantages and disadvantages of accelerated solvent extraction The use of carbon dioxide and water, nontoxic extracting solvents, has economic and environmental benefits. ASE is considered to be a potential alternative technique to SFE for the extraction of polar compounds. It dramatically decreased the consumption of extraction time and solvent as well and had better repeatability compared to other methods. Also, it offers the possibility to manipulate extraction temperature and steps to be followed during the purification of extracts (Brachet et al., 2001; Kaufmann et al., 2001b). Particular attention should be paid to the ASE performed with high extraction temperature, which may lead to the degradation of thermolabile compounds. Potential applications of accelerated solvent extraction ASE has been successfully used by the researchers at various investigations, researches, and development laboratories for extracting many types of natural bioactive analytes including saponins, flavonoids, essential oils, etc. Some researchers believed it could not be used to extract thermolabile compounds due to its high extraction temperature, while others were in belief that it could be used for the extraction of the same because of its shorter extraction time. Some bioactive compound like anthocyanin is thermolabile, but its degradation rate is timedependent. Hence, the high-temperature, short-duration PLE extraction conditions could overcome the disadvantage of high temperature employed in the extraction. PHWE has industrial applications; however, the degradation of glucoside and malonyl forms was detected at higher temperature levels during PHWE (Moras et al., 2017; Vergara-Salinas et al., 2013). 3.4.2.2.10 Ultrasound-assisted extraction/sonication Principles and mechanisms Ultrasonic-assisted extraction (UAE), also called ultrasonic extraction or sonication, uses ultrasonic wave energy in the extraction. Ultrasound in the solvent-producing cavitation accelerates the dissolution and diffusion of the solute as well as the heat transfer, which improves the extraction efficiency. Sound waves, which have frequencies higher than 20 kHz, are mechanical vibrations in solid, liquid, and gas. Unlike electromagnetic waves, sound waves must travel in a matter, and they involve expansion and compression cycles during travel in the medium. Expansion pulls molecules apart, and compression pushes them together. The expansion can create bubbles in a liquid, grow, and finally collapse to produce negative pressure. Close to a solid boundary, cavity collapse is asymmetric and produces high-speed jets of liquid. The liquid jets have a strong impact on the solid surface. Two general designs of ultrasound-assisted extractors are ultrasonic baths or closed extractors fitted with an ultrasonic horn transducer. The mechanical effects of ultrasound induce a greater penetration of solvent into cellular materials and improve mass transfer. Ultrasound in extraction can also disrupt biological cell walls, facilitating the release of contents. Therefore efficient cell disruption and effective mass transfer are cited as two major factors leading to the enhancement of extraction

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with ultrasonic power. In contrast to conventional extractions, the mechanical effects of ultrasound cause the rupture of cell walls and release the cell contents over a shorter period as evident from the scanning electron micrographs (Chemat et al., 2017; Vinatoru et al., 2017). Practical issues for ultrasonic assisted extraction It is necessary to take into account sample characteristics such as moisture content and particle size and solvent used for the extraction in order to obtain an efficient and effective UAE. Furthermore, many factors govern the action of ultrasound including frequency, pressure, temperature, and time of extraction/sonication time. Ultrasound frequency has great effects on extraction yield and kinetics. However, the effects of ultrasound on extraction yield and kinetics differ depending on the nature of the plant material to be extracted. A small change in frequency can increase the yield of extract; however, ultrasound has weak effects on both yield and kinetics for the extraction of oil from plant samples. The ultrasonic wave distribution inside an extractor is also a key parameter in the design of an ultrasonic extractor. The maximum ultrasound power is observed in the vicinity of the radiating surface of the ultrasonic horn. Ultrasonic intensity decreases rather abruptly as the distance from the radiating surface increases. Also, ultrasound intensity is attenuated with the increase in the presence of solid particles. In order to avoid standing waves or the formation of solid-free regions for the preferential passage of the ultrasonic waves, additional agitation or shaking is usually used (Romdhane & Gourdon, 2002). Operating conditions The use of ultrasound allows changes in the processing condition such as alteration in temperature and pressure from those used in extractions without ultrasound. As per the findings of the research, for solid
hexane extraction of plant sample without ultrasound, extraction yield increases with an increase in the extraction temperature, and maximum yield is achieved at 66 C. With ultrasound, the effect of temperature in the range of 40 C
66 C on the yield is negligible, such that optimal extraction occurs across the range of temperature from 40 C to 66 C. Therefore the use of ultrasound-assisted extraction is advisable for thermolabile compounds, which may be altered under Soxhlet operating conditions due to the high extraction temperature. However, it should be noted that since ultrasound generates heat, it is important to accurately control the extraction temperature. The sonication time should also be considered carefully as excess sonication can damage the quality of extracts. To increase mechanical stressing of the cells so-called interface friction, treatment with the ultrasound plays a major role owing to the decomposition of the alkaloids in samples which is observed after ultrasound treatment on the laboratory scale at 20 kHz, whereas the content of digitalis glycosides decreases when an ultrasound output represents the optimum formation of hydrogen peroxide during the extraction (Kar, 2007; Romdhane & Gourdon, 2002). Advantages and disadvantages of ultrasonic assisted extraction UAE is an inexpensive, simple, and efficient alternative to conventional extraction techniques. The main benefits of the use of ultrasound in solid
liquid extraction include the increase in extraction yield and faster kinetics. Ultrasound can also reduce the operating temperature allowing the extraction of thermolabile compounds such as anthocyanin from flower parts, in order to reduce extraction time and avoid high-temperature exposure. UAE includes low solvent and energy consumption and reduction of extraction temperature and time, which make it a simple and relatively low-cost technology. Compared with other novel extraction techniques such as microwave-assisted extraction, the ultrasound apparatus is cheaper, and its operation is easier and confers it to be a simple and low-cost technology that can be utilized to extract phytochemicals on a small or large scale. Furthermore, the UAE can be used with any solvent for extracting a wide variety of natural compounds. This makes UAE an alternative to maceration and Soxhlet extraction. However, the effects of ultrasound on extraction yield and kinetics may be linked to the nature of the plant matrix. The presence of a dispersed phase contributes to the ultrasound wave attenuation, and the active part of ultrasound inside the extractor is restricted to a zone located in the vicinity of the ultrasonic emitter. Therefore those two factors must be considered carefully in the design of the UAE. Conversely, the generation of free radicals by ultrasonic energy greater than 20 kHz may have an unacceptable effect on active phytochemicals. Therefore all UAE parameters like temperature, extraction time, polarity and amount of solvent, amount and type of sample, ultrasound frequency and intensity, and number of pulses must be carefully optimized in order to avoid thermal degradation of phenolic compounds (Chemat et al., 2004; Corrales et al., 2008; Vernes et al., 2019). Potential applications of ultrasonic assisted extraction UAE has been used to extract desired analytes from plants such as essential oils, lipids, and dietary supplements. Extraction rates of bioactive compound by UAE with hexane were 1.3
2 times more rapid than those by conventional extraction depending on temperature. The ultrasound was also

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applied to the cartridge of a Soxhlet extraction for the extraction of total fat from seeds such as sunflower, rape, and soybean seeds. The use of ultrasound reduced the extraction by at least half of the time needed by conventional extraction methods without any change in the composition of extracted oils. UAE was considered as an efficient method for extracting phenolics, antioxidants, steroids, triterpenoids from desired plant samples (Chemat et al., 2004, 2017; Vinatoru et al., 2017). 3.4.2.2.11 Supercritical fluid extraction SFE is a green method that can be used as an alternative to traditional solvent extraction. It has a lower environmental impact and satisfies consumers’ desire for safe, natural, and high-quality components. This technology is being used by an increasing number of businesses, and it is said to provide a competitive advantage. SFE is a method in which a gas is delivered to an extraction vessel at a temperature and pressure above its critical limit in order to extract the desired component or mixture of compounds from a product matrix with a general objective of reducing the use of organic solvents and increased sample throughput (Gallego et al., 2019). Principles and mechanisms Supercritical state is achieved when the temperature and the pressure of a substance are raised over its critical value. The supercritical fluid has characteristics of both gases and liquids. Compared with liquid solvents, supercritical fluids have several major advantages: (1) the dissolving power of a supercritical fluid solvent depends on its density, which is highly adjustable by changing the pressure or/and temperature; (2) the supercritical fluid has a higher diffusion coefficient and lower viscosity and surface tension than a liquid solvent, leading to more favorable mass transfer. During SFE, raw plant material is loaded into an extraction vessel, which is equipped with temperature controllers and pressure valves at both inlet and outlet to keep desired extraction conditions. The extraction vessel is pressurized with the fluid by a pump. The fluid and the dissolved compounds are transported to separators, where the salvation power of the fluid is decreased by decreasing the pressure or increasing the temperature of the fluid. The product is then collected via a valve located in the lower part of the separators, and then, the fluid is further regenerated and recycled (Gallego et al., 2019; Sihvonen et al., 1999). Practical issues for supercritical fluid extraction To develop a successful SFE, several factors must be taken into consideration. These factors include the selection of supercritical fluids, plant material preparation, modifiers, solubility of analytes, and extraction time. Solvent choice: Selection of supercritical fluids is critical for the development of an SFE process. With a reduction in the price of carbon dioxide and restrictions on the use of other organic solvents, carbon dioxide has begun to move from some marginal applications to being the major solvent for supercritical fluid extraction. The critical state of carbon dioxide fluid is realized at temperature of only 31 C and pressure of 7.3 MPa. Also, carbon dioxide is nonflammable and nontoxic. Supercritical CO2 is a good solvent for the extraction of nonpolar compounds such as hydrocarbons. Recently, argon has been used as a substitute for carbon dioxide, because it is inert and less expensive. The component recovery rates generally increase with increasing pressure or temperature, and the highest recovery rates for argon are obtained at 500 atmospheric pressure and 150 C. To extract polar compounds, some polar supercritical fluids such as Freon-22, nitrous oxide, and hexane have been considered. However, their applications are limited due to their unfavorable properties with respect to safety and environmental considerations. Although supercritical water has certain advantages such as higher extraction ability for polar compounds, it is not suitable for thermally labile compounds. Many phytochemicals such as phenolics, alkaloids, and glycosidic compounds are poorly soluble in carbon dioxide and hence not extractable (Lang & Wai, 2001; Patil et al., 2013). Role of modifiers: Techniques aimed at overcoming the limited solubility of polar substances in supercritical CO2 have been sought. The addition of polar cosolvents (modifiers) to the supercritical CO2 is known to significantly increase the solubility of polar compounds. Among all the modifiers including methanol, ethanol, acetonitrile, acetone, water, dichloromethane, and ethyl ether, methanol is the most commonly used because it is an effective polar modifier and is miscible up to 20% with CO2. However, ethanol may be a better choice in SFE of phytochemicals because of its lower toxicity. Furthermore, the use of methanol as a modifier requires a slightly higher temperature to reach the supercritical state, and this could be detrimental for thermolabile compounds. The best modifier usually can be determined based on preliminary experiments. A mixture of modifiers can also be used in SFE with one disadvantage of using a modifier is that it can cause poor selectivity (Hamburger et al., 2004; Lang & Wai, 2001). Preparation of plant materials: Preparation of plant materials is another critical factor for the SFE of plant bioactive compounds. Frequently, fresh plant materials are used in the SFE. However, when fresh plant materials are extracted, the

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high moisture content can cause mechanical difficulties such as restrictor clogging due to ice formation. Although water is only about 0.3% soluble in supercritical CO2, highly water-soluble solutes would prefer to partition into the aqueous phase, resulting in low efficiency of SFE. In order to minimize the above constraints, some chemicals such as Na2SO4 and silica gel are thus mixed with the plant materials to retain the moisture for SFE of fresh materials (Lang & Wai, 2001). Plant particle size is also an important criterion for a good SFE process. Large particles may result in a long extraction process because the process may be controlled by internal diffusion. However, fine powder can speed up the extraction but may also cause difficulty in maintaining a proper flow rate owing to a small decrease in the total yield of the desired compounds. Therefore in this case, some rigid inert materials such as glass beads or sea sand are packed with the fine plant powder to maintain the desired permissibility of the particle bed (Sihvonen et al., 1999). Solubility of analytes: The solubility of a target compound in a supercritical fluid is a major factor in determining its extraction efficiency. The temperature and density of the fluid control the solubility. The choice of a proper density of a supercritical fluid such as CO2 is the crucial point influencing solvent power and selectivity and the main factor determining the extract composition. It is often desirable to extract the compound right above the point where the desired compounds become soluble in the fluid so that the extraction of other compounds can be minimized. On that account, SFE could selectively extract substances from the plant materials by controlling the conditions such as temperature and pressure (fluid density) (Cherchi et al., 2001). Extraction time: The extraction time has been proven to be another parameter that determines extract composition. Lower molecular weight and less polar compounds are more readily extracted during supercritical CO2 extraction since the extraction mechanism is usually controlled by internal diffusion. Therefore the extract composition varies with the extraction time. However, some literature reported that the increase in CO2 flow rate did not seem to influence the extract composition rather it increased the extraction rate (Cherchi et al., 2001; Coelho et al., 2003). Potential applications of supercritical fluid extraction SFE is a potential alternative to conventional extraction methods using organic solvents for extracting biologically active components from plants. It has been used to extract plant materials, especially lipids, essential oils, and flavons. SFE can prevent the oxidation of lipids as the extracted oil by SFE was more protected against oxidation of the polyunsaturated fatty acids than the n-hexane-extracted oil. Oil extracted with supercritical CO2 was clearer than the one extracted by n-hexane. SFE can achieve higher yield and quality of essential oils, flavors, and natural aromas than conventional steam distillation (Cherchi et al., 2001; Coelho et al., 2003). Many active substances in plants such as phenolics, alkaloids, and glycosidic compounds are poorly soluble in CO2 and hence not extractable. Modifiers such as methanol and ethanol are widely used in the supercritical CO2 extraction of polar substances. Supercritical CO2 modified with 15% ethanol gave higher yields than pure supercritical CO2. Similarly, supercritical CO2 extraction with methanol in the range of 3%
7% as a modifier was proven to be a very efficient and fast method to recover a higher percentage of analytes from plant samples (Ellington et al., 2003; Hamburger et al., 2004). Advantages and disadvantages of supercritical fluid extraction SFE offers unusual possibilities for selective extractions and fractionations because the solubility of a supercritical fluid can be manipulated by changing the pressure and/ or temperature of the fluid. Furthermore, supercritical fluids have a density of a liquid and can solubilize a solid like a liquid solvent. The solubility of a solid in a supercritical fluid increases with the density of the fluid which can be achieved at high pressure. The dissolved phytochemical compounds can be recovered from the fluid through the reduction of the density of the supercritical fluid by decreasing its pressure. Therefore, SFE can eliminate the concentration process, which usually is time-consuming. Furthermore, the solutes can be separated from a supercritical solvent without loss of volatiles due to the extreme volatility of the supercritical fluid. Additionally, the diffusivity of a supercritical fluid is one to two orders of magnitude higher than that of other liquids, which permits rapid mass transfer, resulting in a larger extraction rate than that obtained by conventional solvent extractions. The low supercritical temperature of CO2 makes it attractive for the extraction of heat sensible compounds. As SFE uses no or only minimal organic solvent (organic modifiers) in extraction, it is a more environmentally friendly extraction process than conventional solventsolid extraction owing to its nontoxic, nonflammable, inexpensive, lowering running costs, and reducing CO2 emissions into the atmosphere. SFE can be directly coupled with a chromatographic method for simultaneously extracting and quantifying highly volatile extracted compounds (Murga et al., 2000; Patiland & Shettigar, 2010). The huge disadvantage of this extracting technique was the use of an expensive installation due to its initial cost of equipment being very high. The efficiency of supercritical CO2 regarding polar analytes may be increased by adding a modifier. The solvating properties of the fluid could be adjusted by optimization of pressure and/or temperature, which led to relatively high selectivity. The most important factor for the efficiency of the extraction method was the high solubility of the target substances in the extract. Therefore many parameters that affect the solubility and the resulting yield had to be taken into

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account. Acidification of the medium is required for higher anthocyanin content in the extracts. However, the degradation of the anthocyanins during storage was higher, which led to the loss of the intensive color. However, the economics and burdensome operating conditions of the SFE processes have restricted the applications to some very specialized fields such as essential oils, anthocyanins, coffee extraction, etc., and university research (Ghafoor et al., 2010; Murga et al., 2000). 3.4.2.2.12

Enzyme-assisted extraction

The cell membrane, cell wall, and micelles are composed of macromolecules like polysaccharides and proteins. During extraction, the coagulation and denaturation of proteins at high temperatures are the main barriers for the extraction of natural products. Hence, EAE is an enzymatic pretreatment which is carried out by the addition of specific hydrolyzing enzymes during the extraction step. Extraction efficiency is enhanced by EAE due to the hydrolytic action of the enzymes on the components of the cell wall, cell membrane, and micelles resulting in the disruption of macromolecules inside the cell owing to facilitate the release of the natural product. Cellulose, α-amylase, and pectinase are generally employed in EAE. This approach is considered a novel and effective technique for the extraction of a large group of secondary plant metabolites with antioxidant properties (Liu et al., 2016; Maier et al., 2008). In this regard, several parameters must be taken into account in order for the extraction process to be effective such as reaction temperature, extraction time, pH of the system, enzyme concentration, and the particle size of the substrate. The polysaccharide yield under the optimized EAE condition using glucose oxidase increased by more than 250% compared with that from nonenzyme treated methods. The EAE is suitable for the extraction of various bioactive substances from plant matrices, but the fraction obtained after filtration is rich in water-soluble small molecule compounds, including flavonoids. After this procedure, it is possible to isolate the flavonoids step by step by changing the pH and adding different solvents. However, this makes EAE a nonselective method regarding flavonoid extraction from plant materials (Chen et al., 2014; Liu et al., 2016). 3.4.2.2.13

Solid-phase microextraction

It is a sample preparation process that extracts target analytes from the sample matrices using small amounts of extraction phases. SPME, like solid-phase extraction (SPE), includes the partitioning of analytes from sample matrices to extraction phases, which is determined by chemical potential gradients between the sample matrices and extraction phases. The amounts of analytes extracted by the extraction phases are at their peak when the analytes attain partition equilibrium between the sample matrices and the extraction phases. When the extracted analytes are close enough to its theoretical equilibrium extraction quantity, the equilibrium state is frequently deemed and reached in practice (e.g., no less than 95% of the theoretical equilibrium extraction amount). Before that, SPME is thought to be in a state of preequilibrium. SPME can be halted during the pre-equilibrium stage, especially if sufficient sensitivity can be guaranteed, but the time efficiency for the sample preparation step is the primary issue (Zwir-Ferenc & Biziuk, 2006). The extraction stages in SPME are often mounted on supporting substrates or manufactured as monolithic fibers or thin films. Since SPME commonly uses relatively small amounts of extraction phases, absolute recoveries are typically low. As a result, SPME is considered a nonexhaustive extraction technique. It can be used to determine the free concentrations of analytes in biological and environmental samples when the extracted amount is small in comparison to the total amount of the analytes in the sample matrix and does not significantly alter the distribution of the analytes in the sample matrix. Since large portions of the extracted analytes can be successfully transferred to the analytical instruments for analysis, even though the absolute recoveries of SPME are often extremely low, very satisfactory sensitivities can still be reached when coupling it with chromatography or directly with mass spectrometry. In comparison to SPE, another advantage of SPME is that it requires no or very little solvent. This makes SPME intriguing in light of the current push to develop green sample preparation approaches. In combination with gas chromatography (GC), it has evolved as one of the best efficient and practical technologies for analyzing volatiles and semivolatiles from a variety of sources. It has also been used to extract polar analytes and even to extract a wide range of analytes with different polarities simultaneously by designing extraction phases with appropriate polarities and functional groups. It is also an exciting in vivo sampling method for studying the status and processes in living systems due to its low invasiveness to living animals and plants. Sulfur compounds in air samples are measured using SPME, a solvent-free sample preparation method that allows sampling, isolation, and enrichment in a single step (Xu & Ouyang, 2019). 3.4.2.2.14

Reflux extraction

Reflux extraction is more efficient than percolation or maceration or sonication or Soxhlet extraction and requires less extraction time and solvent. It cannot be used for the extraction of thermolabile natural products. Refluxing with 70% ethanol

48

Recent Frontiers of Phytochemicals

provided the highest yield of the natural biometabolites among the extracts prepared by different extraction methods. In this hot extraction process, the material is treated with boiling solvent. The solvent vapor is recycled by a condenser fitted on top of the container. This method was found to be better than the decoction method, and the highest yields were obtained with 60% ethanol as the extraction solvent (Kongkiatpaiboon & Gritsanapan, 2013; Zhang et al., 2018). 3.4.2.2.15

Countercurrent extraction

In CCE, the wet raw material is pulverized to turn into fine slurry using toothed disk disintegrators. In this process, the material for extraction in the form of a fine slurry is moved in one direction within a cylindrical extractor as it comes in contact with the extraction solvent. Then, the further movement of the starting material leads to the extraction of more concentrated extract. Complete extraction is achieved when the quantities of solvent, crude material, and their flow rates are optimized. The process has minimal risk from high temperatures, is very efficient, and requires little time. At the end of the process, sufficient concentrated extract is produced at one end of the extractor while the waste that is free of visible solvent comes out from the other end (Xia et al., 2016; Zhang et al., 2018). Advantages of the countercurrent extraction process 1. A handsome quantity of the plant material can be extracted with a much smaller volume of solvent as compared to other methods like maceration, decoction, and percolation. 2. CCE is commonly done at room temperature, which spares the thermolabile constituents from exposure to heat and is employed in most other techniques. 3. As the pulverization of the crude product is done under wet conditions, the heat generated during combustion is neutralized by water. This reduces the thermolabile constituents from exposure to heat. 4. This extraction procedure has been rated to be more efficient and effective than continuous hot/Soxhlet extraction (Fonmboh et al., 2020). 3.4.2.2.16 Pulsed electric field extraction Pulsed electric field (PEF) uses short pulses of electricity (from μs to ms) under high-intensity electric fields (kV/cm), which leads to the formation of pores on the cell membranes by improving the extraction through diffusion processes, causing the permeabilization of the cell membrane. This method ensures nonthermal permeabilization of cellular membranes and prevents the cell walls from undergoing thermal alteration. Using this technique as an extraction or as a pretreatment method, it not only enhanced yields of bioactive compounds, but also improved antimicrobial effectiveness for different extracts (Fincan et al., 2004; Pina-Pe´rez et al., 2018). PEF extraction significantly increases the extraction yield and decreases the extraction time because it can increase mass transfer during extraction by destroying membrane structures. The effectiveness of PEF treatment depends on several parameters including field strength, specific energy input, pulse number, and treatment temperature. PEF extraction is a nonthermal method and minimizes the degradation of thermolabile compounds. The yield of the PEF extraction method is higher than those of MAE, heat reflux extraction, UAE, and PLE. The entire PEF extraction process took much less time than the other tested methods. Also, the extraction yield of phenolic content (eight times) and antioxidant activity (30 times) were achieved after the PEF treatment compared to untreated samples (Guderjan et al., 2005; Hou et al., 2010). 3.4.2.2.17 Phytonic process A new technology involves a new solvent, based on hydrofluorocarbon used to optimize its remarkable properties in the extraction of plant materials to offer great environmental advantages as well as health and safety benefits over traditional processes. It is used for the production of high-quality biological extracts. Advanced photonics limited in Manchester, United Kingdom, has successfully developed this patented technology called “the phytonic process.” The products extracted by this process are phytochemicals and fragrant compounds of essential oils which can be used directly without further physical or chemical treatment. The properties of the new generation of fluorocarbon solvents have been applied to the extraction of plant materials. The chemical name of the solvent, used in this process is 1, 1, 2, 2-tetra-fluoro-ethane, well known as hydrofluorocarbon-134a (HFC-134a) that was developed as a substitute for chlorofluorocarbons. The boiling point of this solvent is 225 C, and it is neither flammable nor toxic. Unlike chlorofluorocarbons, it does not deplete the ozone layer and has a vapor pressure of 5.6 bar at room temperature. By all standards, this is a poor solvent as it does not mix with mineral oils or triglycerides and does not dissolve plant waste. In this process, the solvents can be adopted and customized by using modified solvents with HFC-134a. The process can also be made highly selective in extracting a specific class of phytochemicals, and other modified solvents can be used to extract a

Novel extraction conditions for phytochemicals Chapter | 3

49

broader spectrum of components. The biological products made by this process have an extremely low residual solvent. The residuals are known to be below undetectable levels. These solvents are neither acidic nor alkaline in nature and, therefore have only minimal potential reaction effects on the herbal materials. The crude plant sample is sealed so that the solvents can be constantly recycled and well recovered at the end of each reaction cycle. The only utility needed to operate these systems is electricity with very low consumption of energy. There is no scope for loss of the solvents even if some solvents do escape, and they contain no chlorine and thus pose no threat to the ozone layer. The waste biomass from the herbal plant product is dry, environmentally friendly, and easier to manage (Onwukaeme et al., 2007; Parekh & Chanda, 2007). Advantages of the phytonic process This process is cool and gentle compared to others which require high temperatures. Its products are sustained beyond the room temperature. No vacuum stripping is required as in other processes which can lead to the loss of precious volatiles. The process is carried out at neutral pH. So, in the absence of oxygen, the products are never predisposed to damage due to acid hydrolysis or oxidation. The technique is very selective, so it offers a choice of operating conditions and a choice of end products. It is an eco-friendly process, requires a minimum amount of electrical energy, and releases very little harmful emissions into the atmosphere. The resulting waste product is innocuous and poses no problem with waste disposal. The solvents used in the technique are not flammable, toxic, or ozone-depleting, and these are completing the recycling process smoothly within the system (Rahalison et al., 1991). Use of the phytonic process This process can be used wildly in the field of biotechnology such as in the food and herbal drug industry. In most cases, it is used in the production of pharmacologically active intermediates and topquality pharmaceutical-grade antibiotic extracts. However, the fact that it is used in all these areas in no way concerns its use in other areas. The technique is also used in the extraction of high-quality essential oils, oleoresins, natural food, colors, flavors, and aromatic oils from different herbal plant materials. The technique is well adopted for refining crude products obtained from other extraction processes. It provides extraction without waxes or other contaminants thus helping in the removal of many biocides from contaminated biomass (Parekh & Chanda, 2007; Rahalison et al., 1991). 3.4.2.2.18 Negative pressure cavitation extraction NPCE is a new type of cavitation, suitable for the isolation of thermo-unstable plant byproducts. The main working principle behind this process is a continuous introduction of nitrogen stream into the extraction system. Because of the negative pressure, N2 bubbles arise in the liquid
solid system with the formation of a highly unstable gas
liquid
solid phase. These exercises promote the turbulence, collision, and mass transfer between the solvent and the plant matrix, which leads to an effective extraction of the target analytes. A constant low temperature is maintained during this operation, thus preventing the degradation of thermosensitive compounds and making it effective, simple, low cost, and eco-friendly as well. NPCE shows potential for industrial application. Another advantage of this method is thermosensitive compounds such as isoflavonoids which can be quantitatively extracted at room temperature. The researchers concluded that NPCE has a good extraction efficiency compared to the other conventional extraction methods. Its efficiency increased significantly in combination with ultrasonication (Liu et al., 2009; Wang et al., 2020; Zhang et al., 2011). 3.4.2.2.19

Matrix solid-phase dispersion

MSPD is a popular alternative to the solid
liquid extraction method. This technique facilitates the extraction, fractionation, and preparation of the target compounds. The process involves simultaneous homogenization, extraction, and purification, during which most of the constraints are eliminated, associated with the classical methods. Dried leaf samples were blended with hydrophobic C18-resin and placed in small columns, and isoflavones were extracted with a mixture of dichloromethane and methanol. Contrary to the standard extraction techniques, MSPD needs no specific labor or expensive equipment rather it is fast, requires less solvent, and more environmentally friendly. The selectivity depends on the eluent choice. Graphene-encapsulated silica experienced better extraction efficiency regarding the target analytes as compared to other five sorbents such as graphene, silica gel, C18-resins, diatomaceous earth, and neutral alumina (Hong et al., 2015; Sun, Wu et al., 2015; Visnevschi-Necrasov et al., 2009). 3.4.2.2.20

Enfleurage (extraction with cold fat)

Enfleurage is the process of extraction of delicate fragrance by absorbing it from samples (e.g., flowers) in contact with cold fats. This process is adopted for fragrant flowers of jasmine and tuberose, which continue to manifest their characteristic fragrance even in plucked condition. The flower petals are spread over a layer of refined fat that picks up the

50

Recent Frontiers of Phytochemicals

odor of the flowers. Then, the saturated fat is treated with a solvent, usually alcohol, in which the fragrant components are soluble. The residual fat dissolved in alcohol may be removed by cooling the alcohol extract to 20 C, when fat separates out. The alcohol is evaporated under reduced pressure, and pure oils are obtained. Refined lard or beef suet is preferred for this method of extraction. Fat is thinly layered on both sides of a glass plate supported on a rectangular wooden frame. Fresh fragrant flowers are lightly layered on fat-coated chassis. It gives a much greater yield of flower oil than other methods. Despite this advantage, it has lately been replaced by extraction with volatile solvents because enfleurage is a very delicate and lengthy process requiring much experience and labor (Doughari et al., 2009).

3.5

Selection approach for a suitable extraction method

Since bioactive compounds occurring in plant samples consist of multicomponent mixtures, so their extraction, separation, and determination still create problems in ethnomedicine development process. Several novel extraction techniques have been developed as an alternative to conventional extraction methods, offering advantages with respect to extraction time, extraction temperature, nature and volume of solvent, solvent-to-sample ratio, extraction yields, and reproducibility. A comparison of different extraction methods is projected in Table 3.6. Irrespective of above terms and conditions, several other factors like collection, identification, authentication, sample preparation, etc., are also equally important for selection of novel extraction methods. So, selection approach for a suitable extraction method is described as follows (Fonmboh et al., 2020). 1. The binomial authentication and validation of collected plant samples need to be done prior to onset of any preextraction process. All foreign materials need to be removed from the sample as well. 2. The suitable part of the plant must be used, and the age, time, place, time of collection, and season of harvest should be recorded. 3. The pre-extraction conditions are to be followed strictly which generally depend on the nature of its desired phytochemicals. A suitable weight correction is to be needed when the crude herbal product with high moisture content has to be used for extraction. 4. The drying methods need to be specified. Any technique that produces heat should be avoided. The coarse plant material should be passed through sieves in order to obtain the required particles of homogenous size. 5. Nature of desired phytochemicals requires a healthy discussion. (1) It is always advisable to use a suitable solvent system as per polarity character of analytes to be extracted. (2) If the bioactive compounds are thermolabile, then the extraction methods such as cold percolation, maceration and countercurrent extraction (CCE) are preferred. For thermostable compounds, it is suitable to use the Soxhlet extraction method when there is a nonaqueous solvent and the decoction extraction when water is the main solvent. (3) Precautionary measures to be taken during dealing with phytochemicals such flavonoids and phenyl propanoids those are easily degraded in organic solvents. (4) The extraction technique where involvement of heat is needed, it is advisable to avoid temperatures that are higher than normal; for example, some glycosides may likely break when exposed to higher temperature. (5) There is need for the standardization of extraction time as insufficient time means incomplete extraction. Unwanted bioactive constituents may also be extracted with longer extraction time. Generally, if tea is boiled for a long duration, tannins can be extracted which gives astringency to the final product preparation. (6) The number of cycles of extractions required for complete extraction is as important as the duration of each extraction. 6. The quality and quantity of solvents used for extraction must be specified and well controlled. 7. The concentration and drying procedures of products should ensure that there is safety and stability of the bioactive metabolites. Drying under reduced pressure by using a rotavapor and lyophilization although costly is commonly used. 8. Consideration should be taken on designing and standardization of the extraction method keeping in view of the characteristics of the extractor.

3.6

Conclusion

Plants are an inexhaustible source of phytochemicals, those are playing a pivotal role for drug development over the past few decades, and progress is still on. Several works have been done on medicinal plant either to investigate or prove a reported claim of biological activity or to mimic its traditional medicinal use based on ethno-medicinal survey. A lot of research teams compared classical and modern methods for the extraction of those phytochemicals from plant materials and discussed their pros and cons. The need to extract these phytochemicals from plant materials prompts

TABLE 3.6 Comparison of different extraction methods. Sl. no.

1

2

Plant/compound

Artemisia annua L./ artemisinin

Sweet grass leaves/ dihydroxycoumarin

Method of extraction/yield MAE (92.1%)

Extraction condition P/T

Solvent

650 W 

SFE (33.2%)

35 C

Soxhelt (60.4%)

35 C

CO2



MAE (0.42%)

200 W/80 C

Acetone

SFE (0.49%)

40 C

20% Ethanol

Soxhlet (0.46%) 3

Licorice roots/ glycyrrhizic acid

MAE (2.26%)

Acetone 



700 W/85 C
90 C

UAE (2.26%)

Ethanol

Soxhlet (2.5%) 4

5

6

Green tea leaves/tea polyphenols, tea caffeine

Grape fruit/pectin

Ganoderma atrum/ polyphenol

MAE (30%) UAE (28%)

700 W/20 C 



20 C
40 C

t

15

12 min

6

2.5 h

12

6h

10

15 min

50

6h

10

4 min

10

20.5 h

10

10 h

20

4 min

20

90 min

85 C

20

45 min

900 W/20 C

30

6 min

30

25 min

30

90 min

25

5 min



UAE (17.92%)

70 C

Heat Batch (19.16%)

90 C

MAE (5.11%)

Water



90 C

10% Ethanol

55 C

CO2 1 Ethanol

3h

10% Ethanol

3h

UAE (1.72%)

25



HRE (2.22%)

95 C

MAE (90.47%)

60 W/50 C

UAE (71.42%)



Acetone

150 W/21 C

Soxhlet (20.10%) SFE (69.36%)



50 C

CO2 1 Ethanol

Hao et al. (2002)

Grigonis et al. (2005)

2h

MAE (27.81%)

Shaking (2.58%)

Turmeric plant/ curcumim

R

HRE (28%)

SFE (1.52%)

7

50% Ethanol



References

Pan et al. (2000)

Pan et al. (2003)

Bagherian et al. (2011)

Chen et al. (2007)

30 min

25

1h

3

5 min

3

5 min

5

8h

Wakte et al. (2011)

240 min

(Continued )

TABLE 3.6 (Continued) Sl. no.

8

Plant/compound

Silybum marianum (L.) (milk thistle)/Silybinin

Method of extraction/yield MAE (1.37%)

Extraction condition P/T

Solvent

R

t

600 W

80% Ethanol

25

12 min

Soxhlet (1.09%)

100

Stirring (0.48%)

10

11

12

13

Coriandrum sativium/ phenolics content

MAE (0.082%)

MAE (1.679%)

Cuminum cyminum/ phenolics content

MAE (1.159%)

Crocus satious/ phenolics content

MAE (2.939%)

Morinda citriflora (roots)/total phenolics content

MAE (95.91%)

50% Ethanol



200 W/50 C

50% Ethanol

UAE (0.506%) 200 W/50 C

50% Ethanol

UAE (0.290%) 200 W/50 C

50% Ethanol

720 W/60 C 

60 C

15

16

17

18

18 min

10

30 min

20

18 min

10

30 min

20

18 min

10

30 min

20

18 min 30 min

80% Ethanol

100

15 min

Ethanol

100

60 min

100

3 days

100

4h

Maceration (63.33%)

14

20

10

UAE (0.500%)

UAE (62.23%)

12 h

24 h 200 W/50 C

UAE (0.041%)

Cinnamomum zeylanicum/phenolics content



Soxhlet (97.74%)

100 C

Tomato/total phenolics content

MAE (0.646%)

100 W

Methanol

50

45 min

Shaker (0.603%)

45 C

60% Ethanol

50

15 h

Foeniculum vulgare Miler (seeds)/total phenolics content

MWHD (1.14%)

300 W/100 C

Water

Iochroma gesnerioides (leaves)/iochromolide

MAE (0.85%)

Xanthoceras sorbifolia/ triterpene saponins

MAE (11.62%)

Gymnuema sylvestre/ gymnemagenin

HD (0.265%) 25 W

Methanol

Soxhlet (0.81%) 900 W/50 C

40% Ethanol



2

200 s

8

319 s

50

40 s

100

6h

30

21 min

UAE (6.78%)

250 W/50 C

30

180 min

Reflux (10.82%)

800 W/50 C

30

270 min

MAE (4.3%)

280 W

25

6 min

Reflux (3.3%)

Dhobi et al. (2009)

24 h

Maceration (0.36%) 9

References



95 C

85% Methanol

100

6h

Maceration (1.7%)

100

24 h

Stirring (2.2%)

100

24 h

Gallo, Ferracane et al. (2010) Gallo, Ferracane et al. (2010) Gallo, Ferracane et al. (2010) Gallo, Ferracane et al. (2010) Hemwimon et al. (2007)

Hongyan et al. (2012)

Kapa´s et al. (2011)

Kaufmann et al. (2001)

Li, Zu et al. (2010)

Mandal et al. (2009)

19

Melilotus officinalis (yellow sweet clover)/ coumarin

MAE (0.398%)

100 W/50 C

UAE (0.357%)

50 C

Soxhlet (0.216%) 20

21

Salvia miltiorrvhiza (dried root)/ cryptotanshinone

Radix astragali (dried root)/astragalosides

MAE (0.23%)



80 C

50% Ethanol

20

10 min

20

60 min

95% Ethanol

17

8h

95% Ethanol

10

2 min

Reflux (0.24%)

10

45 min

UAE (0.25%)

10

75 min

Soxhlet (0.25%)

10

95 min



25

15 min

Soxhlet (0.770%)



90 C

20

4h

Reflux (0.761%)

90 C

20

1h

UAE (0.519%)

100 W

20

40 min

20

12 h

25

2 min

MAE (0.788%)

700 W/70 C

80% Ethanol

Maceration (0.411%) 22

Ipomoea batatas (sweet potato)/total phenolics

MAE (6.115%)

23

Tobacco leaves/ solanesol

MAE (0.91%)

700 W

HRE (0.87%)



24

Radix asragali (root of astragalus)/flavonoids

123 W

CSE (5.969%)

60 C 

53% Ethanol 60% Ethanol

30

120 min

Hexane: Ethanol (1:3 v/v) 1 NaOH

10

40 min

10

180 min

MAE (0.129%)

110 C

95% Ethanol

25

50 min

Soxhlet (0.119%)

85 C

Methanol

25

4h

UAE (0.074%)

60 C

20

60 min

HRE (0.093%)

75 C

25

4h

90% Ethanol

Martino et al. (2006)

Pan et al. (2001)

Yan et al. (2010)

Song et al. (2011)

Zhou and Liu (2006)

Xiao, Han et al. (2008)

NB, CSE, conventional solvent extraction; HD, hydrodistillation; HRE, heat reflux extraction; MAE, Microwave-assisted extraction; MWHD, microwave-assisted hydrodistillation; P, power, R, solvent/sample (mL/g); SFE, supercritical fluid extraction; T, extraction temperature; t, extraction time; UAE, ultrasound-assisted extraction.

54

Recent Frontiers of Phytochemicals

continued searching for economically and ecologically feasible extraction technologies. But intensive laboratory technology and time-consuming extraction and isolation process, however, hindered the application of phytochemicals in drug development. Growing body technology continues to develop more new automated and rapid techniques for extraction, isolation, and characterization of phytochemicals which might reach the requirement of high-throughput screening. The focus of our study in this chapter is the selectivity of the reviewed methods with increased interest in the natural compounds obtained from plants which will lead to the development of new methods and techniques or will improve the existing ones. Nonetheless, the rate of success and the authenticity of these findings depend on the accuracy in selection of solvents, selection and proper execution of extraction methods, phytochemical screening, fractionation, and identification techniques. The sample preparation and the extraction steps are equally important in the development of a selective extraction method. The modern extraction methods, known as green extraction methods, including UAE, MAE, SFE, and PLE, have also been the subject of increased attention in recent years due to their high extraction yields, selectivity, stability of the target extracts and possess safety merits. Some of those green methods have become routine sample preparation methods for analytical purposes. Traditional solid
liquid extraction methods require a large quantity of solvent- and are time-consuming. The large amount of solvent used not only increases operating costs but also causes additional environmental problems. However, novel extraction techniques have only been found in a limited field of applications. So, more research is needed to improve the understanding of extraction mechanism, remove technical barriers, improve the design, and scale up of the novel extraction systems for their industrial applications. In conclusion, there is no unique universal extraction method, and each extraction procedure is specific to certain plants. The design of sample preparation and extraction methods must be consistent with the study objectives, samples, and target compounds. Last but not least, its optimization is important for a variety of applications, for example, preparation of extracts for pharmaceutical usage, with a minimum level of impurities. In addition, when the method selected as the most appropriate one, it needs to be standardized and must achieve an acceptable degree of repeatability and reproducibility.

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Chapter 4

Novel extraction and characterization methods for phytochemicals Ratnnadeep C. Sawant1, Subhash R. Somkuwar2, Shun-Yuan Luo3, Rahul B. Kamble2, Deepa Y. Panhekar1, Yeshwant R. Bhorge4, Rupali R. Chaudhary5 and S. Abdul Kader6 1

Department of Chemistry, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India, 2Department of Botany, Dr. Ambedkar College, Deekshabhoomi,

Nagpur, India, 3Department of Chemistry, National Chung Hsing University, Taichung, Taiwan R.O.C., 4Department of Chemistry, Marotrao Pantawane Mahavidyalaya, Nagpur, India, 5Department of Botany, Shri Sant Gadge Maharaj Mahavidyalaya, Nagpur, India, 6Department of Plant Biology and Plant Biotechnology, Presidency College, Chennai, Tamil Nadu, India

4.1

Introduction

Natural products, such as plant extracts, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Cosa et al., 2006). According to the World Health Organization (WHO), more than 80% of the world’s population relies on traditional medicine for their primary healthcare needs. The use of herbal medicines in Asia represents a long history of human interactions with the environment. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases (Duraipandiyan et al., 2006). Due to the development of adverse effects and microbial resistance to the chemically synthesized drugs, men turned to ethnopharmacognosy. They found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effects. Many beneficial biological activities such as anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic, and wound healing activity were reported. In many cases, people claim the benefit of certain natural or herbal products. However, clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim. Clinical trials directed toward understanding the pharmacokinetics, bioavailability, efficacy, safety, and drug interactions of newly developed bioactive compounds and their formulations (extracts) require careful evaluation. Clinical trials are carefully planned to safeguard the health of the participants and answer specific research questions by evaluating for both immediate and long-term side effects, and their outcomes are measured before the drug is widely applied to patients. Phytochemically, the extraction of bioactive compounds could be defined as a separation procedure employed for the recovery and purification of plant materials, rendering them useful in a wide range of applications (Gil-ch et al., 2013). Conventional techniques of phytochemical extraction with biological activities include maceration and Soxhlet extraction; these methods have been associated with a high consumption of organic solvents that limits the application of bioactive extracts due to solvent toxicity (Da Porto et al., 2013). In addition, longtime extraction is required, which involves high energetic consumption causing an incremental cost. Thus far, the implementation of novel extraction technologies, using different mechanisms such as ultrasound, microwave energy, supercritical fluids, and accelerated solvent extraction (ASE), has been promoted (Bendicho et al., 2012); the main objective of these methods is to reduce extraction time and energy consumption which is reflected in the lowering of the final cost. A common aspect of these technologies is that they are sustainable because they protect both the environment and consumers’ health and enhance the economic and innovative competitiveness of industries (Armenta et al., 2015). A brief summary of the general approaches in extraction, separation, and characterization of bioactive compounds from plant extract is shown in Fig. 4.1.

4.2

Extraction methods

4.2.1 Introduction Extraction is the crucial first step in the analysis of medicinal plants because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization. The basic operation included steps, Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00035-9 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 4.1 A brief summary of the general approaches in extraction, separation, and characterization of bioactive compounds from plant extract.

such as prewashing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted, or destroyed during the preparation of the extract from plant samples. If the plant was selected on the basis of traditional uses (Fabricant & Farnsworth, 2001), then it is needed to prepare the extract as described by the traditional healer in order to mimic as closely as possible the traditional “herbal” drug. The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol, or ethyl acetate. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 is used. In some instances, extraction with hexane is used to remove chlorophyll (Cosa et al., 2006). As the target compounds may be nonpolar to polar and thermally labile, the suitability of the methods of extraction must be considered. Various methods, such as sonification, heating under reflux, Soxhlet extraction shown in Fig. 4.2 and others, are commonly used (The Japanese Pharmacopeia, 2001; United States Pharmacopeia & National Formulary, 2002; Pharmacopoeia of the People’s Republic of China, 2000) for the plant samples extraction. In addition, plant extracts are also prepared by maceration or percolation of fresh green plants or dried powdered plant material in water and/or organic solvent systems. The other modern extraction techniques include solid-phase micro-extraction, supercritical fluid extraction (SFE), pressurized liquid extraction, microwave-assisted extraction (MAE), solid-phase extraction, and surfactant-mediated techniques, which possess certain advantages. These are the reduction in organic solvent consumption and sample degradation, elimination of additional sample cleanup and concentration steps before chromatographic analysis, improvement in extraction efficiency, selectivity, and kinetics of extraction. The ease of automation for these techniques also favors their usage for the extraction of plant materials (Huie, 2002).

4.2.2 Organic solvent extraction The demand for bioactive compounds identified from natural plant materials and its products is increasing day by day and used in various sectors such as food, beverage, cosmetics, and pharmaceutical industries. Many valuable natural materials have traditionally been extracted with organic solvents. However, some of the organic solvents are believed to be toxic, and the extraction conditions are often harsh. A simple method using ethanol (a food-grade solvent) instead of methanol was applied for the extraction of phenolic compounds from various citrus peels. The effects of the following parameters, the conditions of the peel samples, effect of repeated extraction, different types of organic solvents, the concentration of the solvent, and temperature of extraction, were examined. Maceration, percolation, cold (Fig. 4.3) and countercurrent extraction (CCE), etc., are the most conventional extraction techniques. Despite the emergence of alternative extraction techniques, conventional extraction techniques are widely applied mainly due to its low cost and easy installation (Dru˙zy´nska et al., 2007). The extraction methods mostly used have been discussed below.

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FIGURE 4.2 Soxhlet extraction setup.

FIGURE 4.3 Different extraction methods: (A) cold percolation, (B) solvent extraction, (C) supercritical fluid extraction, (D) microwave-assisted extraction.

4.2.2.1 Maceration The most widely used conventional extraction procedure is maceration, which is considered as a steady-state extraction technique. In this process, the whole or powdered medicinal or aromatic plant sample is placed in a closed vessel with the solvent, and it is allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The closed vessel is used so that the evaporation of the extracting solvent could be prevented, and no variation between different batches of the same plant can occur. The mixture is then strained, the marc that is the undissolved part after extraction is pressed to recover a large number of occluded solutions, and the two fractions of the liquids are combined. Finally, when equilibrium is achieved the combined liquids are clarified by

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filtration or decantation after standing, and the marc may be strained through a special press. As the system is static, except for occasional shaking, molecular diffusion is considered to be the main process, which is a very slow procedure. Occasional shaking can assist diffusion and can also ensure dispersal of the concentrated solution accumulating around the surface of the particles, thereby bringing fresh extract to the particle surface for further extraction. Generally, plant material in fine powder form is never used, as it makes subsequent clarification of the extract difficult. Although for small amounts of solvent, agitation is not a serious problem; when the technique is applied on an industrial scale with big sample vessels, occasional shaking is difficult. For this reason, techniques such as circulatory extraction and multistage extraction appropriate equipment have been developed. In order to increase the extraction yield, repeated maceration can be implemented. Double or triple maceration can be extremely useful, especially when the target components are of high value and in cases where the concentrated infusion contains oil that is considered volatile. Usually, evaporation of the second and the third extract is required before combination with the first extract, in order to decrease the total volume of solvent (Handa et al., 2008).

4.2.2.2 Percolation Percolation is an extraction technique used for drugs of high cost and preferably for thermolabile active components. Percolation is a technique based on continuous flow of the solvent, which passes through a nonmoving bed that contains the crude plant material, in order to extract the soluble active components from it. The device used here is called a percolator, and it is a narrow, cone-shaped vessel open at its both ends. In this process, the solid plant sample is moisturized with an appropriate amount of the specified solvent and allowed to stand for approximately 4 h in a container that is well closed. Then, the mass is packed, the top of the percolator is closed, more solvent is added, and a shallow layer is formed above the mass. As the next step, the mixture is allowed to macerate in the closed percolator for 24 h. The outlet of the percolator is then opened, and the liquid contained therein drips slowly. More solvent is added as required, until the percolate measures about three-quarters of the required volume of the final extract. The solid then is pressed, and the liquid is added to the percolate. Sufficient solvent is added to produce the required volume, and the combined liquid can be clarified by filtration or by standing followed by decanting. Due to the fact that there is minimum content of fine solid particles in the extract, filtration can also take place. Moreover, the solid material requires little mechanical treatment, because it does not need to move in the extraction device. In order to increase the extraction efficiency in percolation, agitation could be provided with the use of a mechanical stirrer. Alternatively, repeated circulation of the extract back to the percolator can be implemented. In addition, if the active component is thermostable, the solvent temperature can increase, which will lead to higher solubility of the bioactive compounds of the plant sample and to higher extraction efficiency (Azmir et al., 2013; Handa et al., 2008).

4.2.3 Modified percolation When a more concentrated product is needed, modified percolation can be implemented in order to extract the target phytochemical components of the medicinal or aromatic plant. This technique includes also an evaporation step, especially when the solvent is dilute alcohol. The process starts with the imbibition of the crude drug, followed by maceration, percolation, and collection of 1000 mL of percolate. The percolation and collection steps are continued until the drug is completely exhausted, and the extraction procedure comes to its end (Handa et al., 2008).

4.2.3.1 Cold percolation The different plant parts were dried in an artificial environment at low temperatures (50 C
60 C), and dried powder was then further used for extraction purposes using various solvents. Weigh the dried powder, add into conical flask with respective solvents, and allow keeping at room temperature for 30 min shaking after every 24 h for 7 days. Finally, filter the extract using Whatman filter paper under vacuum, and dry it at room temperature in watch glass dish. Note down the weight of each dish prior to drying the extracts and after drying too. Calculate the weight of the extract from the difference (Harborne, 1973).

4.2.3.2 Countercurrent extraction In CCE, the wet raw plant material is pulverized using toothed disk disintegrators in order to produce a fine slurry. In this extraction technique, the plant sample is moved in one direction inside a cylindrical extractor where it comes in direct contact with the extraction solvent. The further the starting material moves, the more concentrated the obtained

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extract becomes. With optimization of the quantities of solvent and plant sample and their flow rate, complete extraction can also be achieved. The main benefits of this procedure are its high efficiency, the low extraction time, and the lack of risks due to high temperature since it is normally done at room temperature. Also, compared to other extraction techniques the unit quantity of the medicinal or aromatic plant material can be extracted with the use of a significantly smaller solvent volume (Handa et al., 2008).

4.2.4 Extraction with supercritical gases SFE involves the use of gases, usually CO2, and compressing them into a dense liquid. This liquid is then pumped through a cylinder containing the material to be extracted. From there, the extract-laden liquid is pumped into a separation chamber where the extract is separated from the gas, and the gas is recovered for reuse. Solvent properties of CO2 can be manipulated and adjusted by varying the pressure and temperature. The advantages of SFE are no solvent residues are left in it as CO2 evaporates completely (Patil & Shettigar, 2010).

4.2.5 Direct steam distillation With direct steam distillation, extraction occurs with the use of steam generated outside the tank in a steam generator or a boiler. The plant is again supported above the steam inlet, as in the previous case of water and steam distillation. By using direct steam distillation, the extraction time can be significantly reduced, and this is the reason why this extraction modification is recommended for distillation of high-boiling oils and hard materials such as roots and woods (Azmir et al., 2013; Handa et al., 2008).

4.2.6 Microwave-assisted extraction It is simply termed as microwave extraction, which combines microwave and traditional solvent extraction. Heating the solvents and plant tissue using microwave increases the kinetic of extraction, which is called MAE (Delazar et al., 2012). The target for heating in dried plant material is the minute microscopic traces of moisture that occurs in plant cells. The heating up of this moisture inside the plant cell due to microwave effect results in evaporation and generates tremendous pressure on the cell wall. The cell wall is pushed from inside due to the pressure, and the cell wall ruptures. Thus, the exudation of active constituents from the ruptured cells occurs, hence increasing the yield of phytoconstituents (Gordy & Smith, 1953; Goldman, 1962).

4.2.7 Other extraction methods 4.2.7.1 Enzymatic extraction The enzymatic process is claimed to offer a number of advantages over mechanical
thermal comminution of several fruit pulps. In particular, the use of cellulases and pectinases has been an integral part of the modern fruit processing technology involving the treatment of fruit masses. The enzyme treatment not only facilitates easy pressing and an increase in juice recovery but also ensures the highest possible quality of the end products (Kilara, 1982; Roumbouts & Pilnik, 1978). These enzymes not only help in softening the plant tissue but also lead to the release of cell contents that may be recovered with high yield (Sreenath et al., 1984).

4.2.7.2 Ultrasonic extraction The development of modern techniques, such as extraction assisted by microwave or ultrasound, is intended to overcome these difficulties by increasing extraction efficiency, selectivity, and kinetics. In this context, the use of ultrasound or sonication to break the cell membranes has the advantage of reducing considerably the extraction time and increasing the extract yield. Ultrasonic extraction uses ultrasonic vibrations to extract samples with polar solvents in an ultrasonic bath. This is often used for chemical extraction from solid samples because it is simple. Ultrasound waves were employed to extract active compounds, such as saponins, steroids, and triterpenoids from Chresta spp, about three times faster than with the traditional extraction methods (Schinor et al., 2004). The ultrasonic field enables the generation, locally, of microcavitations in the liquid surrounding the plant material. The effects are twofold: mechanical disruption of the cell’s wall, releasing its content and local heating of the liquid, increasing the extract diffusion. The kinetic energy is introduced in the whole volume following the collapse of cavitation bubbles at or near walls or interfaces, thus improving the mass transfer across the solid
liquid interface. The mechanical effects

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of ultrasounds induce a greater penetration of solvent into cellular membrane walls, facilitating the release of contents of the cells and improving mass transfer (Alupului et al., 2009; Keil, 2007).

4.2.8 Extraction of essential oil In 1980, SFE was first carried out on a commercial scale, while its industrial-scale application includes processes such as decaffeination of coffee beans and tea, extraction of essential oils, oleoresins, and flavoring compounds from herbs and spices as well as extraction of high-valued bioactive compounds from a great variety of natural matrices (Zekovi´c et al., 2017).

4.2.9 Accelerated solvent extractor The analysis of contaminants in soil or sediments (pesticides, polychlorinated biphenyls—PCBs, polycyclic aromatic hydrocarbons—PAHs) involves an extraction step, which can be performed in different ways (Lindhardt et al., 1994). Most common and simple techniques, such as shake flask extraction, Soxhlet extraction, or sonication, use large volumes of solvent, and they are time-consuming and tedious. There are several new techniques, including MAE, SFE, and ASE, which are faster, use less extraction fluids than the “classic” extraction techniques, and can be readily automated. The use of these techniques requires the development of appropriate operation parameters. The better this optimization step is performed, the less complicated the sample cleanup method can be used. This article reviews some aspects of the theory, practice, and applications of ASE (also known as pressurized fluid extraction—PFE, or pressurized liquid extraction—PLE)—the method that was first described in 1995 (Richter et al., 1995; Ezzell et al., 1995).

4.3

Separation techniques

4.3.1 Introduction Natural products (i.e., plant-derived chemicals) are important sources for drug development. Since the chemical constituents of plants are complicated, pure compounds must be obtained from them via extraction and isolation before structure identification, bioactivity screening, and so on. In recent years, new technologies and methods of extraction, isolation, and structural identification have come forth, which promote the speed of extraction and analysis of phytochemicals. Extraction is the first step to separate the desired natural products from the raw materials in phytochemistry research, and the purpose of extraction is to get the objective chemical constituents to the utmost extent and avoid or reduce the solution of unwanted constituents. The components in the extract are complex and contain a variety of natural products that require further separation and purification to obtain the active fraction or pure natural products. The separation depends on the physical or chemical difference of the individual natural product. The separation of phytochemicals is a process of isolating the constituents of plant extracts or effective parts one by one and purifying them into monomer compounds by physical and chemical methods. Classical isolation methods such as solvent extraction, precipitation, crystallization, fractional distillation, salting out, and dialysis are still used commonly. But the modern separation techniques such as column chromatography, high-performance liquid chromatography (HPLC), ultrafiltration, and high-performance liquid drop countercurrent chromatography also play an important role in the separation of phytochemicals (Alberti et al., 2018; Brusotti et al., 2014; Hosler & Mikita, 1987). Since bioactive compounds occurring in plant material consist of multicomponent mixtures, their separation and determination still create problems. Practically, most of them have to be purified by the combination of several chromatographic techniques and various other purification methods to isolate bioactive compounds.

4.3.2 Chromatographic techniques Chromatography is the most commonly used technique for the separation and identification of chemical compounds present in natural products because of its high separation efficiency, rapidity, and simplicity. Chromatography was first developed by a botanist M. Tswett in 1906 for the separation of colored substances into their components. Chromatography is based on the general principle of distribution of components of a mixture of organic compounds between two phases: 1. the stationary phase and 2. the mobile phase. The material on which the various components are adsorbed is called the stationary phase, while the mixture to be separated is dissolved in a suitable medium either liquid

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or gas which constitutes the mobile phase. The moving phase is made to run on a stationary phase, and the separation is based on the principle that the components of a mixture present in the moving phase move at a different rate through the stationary phase. The chromatography is based on two general phenomena, namely, adsorption and partition. When the stationary phase is solid, the basis of separation is adsorption. But when the stationary phase is liquid, the basis is partition. Chromatographic separations take advantage of the fact that different substances are partitioned differently between these two phases. It is used to separate compound present in fractionized extract and also from callus extract. The chemical compounds are separated based on their size, shape, and charge. Also, the separation of the compound depends on the usage of the solvent. During the separation process, the analyte in solvent moves through solid phase that acts as a sieving material, and as the molecule proceeds further through the molecular sieve, it gets separated.

4.3.2.1 Paper chromatography Paper chromatography was introduced by Schonbein (1865) under the name capillary analysis. In paper chromatography, a sheet of filter paper is used as a solid phase which acts as the inert phase, and a pure solvent or a mixture of solvent is used to act as the mobile phase. One of the advantages of paper chromatography is that separations are carried out simply on sheets of filter paper, which acts as both support and medium for separation (Harborne, 1998). Another advantage is the considerable reproducibility of retention factor (Rf) values. During the experiment, the sample is placed near the bottom of the filter paper, and then, the filter paper is placed in chromatographic chamber with solvent. The solvent moves forward by capillary action carrying soluble molecules along with it. Low-porosity filter paper will produce a slow rate of movement of the solvent, and thick filter papers have increased sample capacity. The mixture undergoes partly adsorption on the filter paper and partly partition between the water molecule attached to the cellulose fiber and the solvent. Based on the amount of adsorption and partition, the components in the mixture travel at different rates on the filter paper and get separated from one another. After some time, the paper strip is taken out, dried, and the position of the different components is noted. If the position of different components is not visible on the filter paper, then it can be located with the help of a suitable reagent like ninhydrin that is used to identify different types of α-amino acids present in a mixture. The various components present in a mixture are identified by comparing the calculated Rf value with the reference substance or with the standard values given in the data book.

4.3.2.2 Thin-layer chromatography Thin-layer chromatography (TLC) (Fig. 4.4) is a form of adsorption chromatography (Hahn-Deinstrop, 2000) where samples are separated based on the interaction between a thin layer of adsorbent attached to the plate. TLC is a simple, quick, and inexpensive procedure that gives the researcher a quick answer about the presence of several chemical compounds in a mixture. Compared to paper chromatography, the special advantage of TLC is the versatility, speed, and sensitivity. It is mostly employed for the separation of low-molecular weight compounds. It is also used to support the identity of a compound in a mixture when the Rf value of a compound is compared with the Rf value of a known compound, along with other tests like the spraying of phytochemical screening reagents (which cause color changes watch glass

thin layer chromatography plate

beaker

pencil line M

spot of mixture

FIGURE 4.4 Thin-layer chromatography setup.

solvent

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TABLE 4.1 Different adsorbent used to separate various compounds. Sr. no.

Adsorbent

Use to separate

1

Silica gel

Amino acids, alkaloid, sugars, fatty acids, lipid, etc.

2

Aluminum

Alkaloids, phenols, steroids, vitamins, and carotenes

3

Celite

Steroids and inorganic cations

4

Cellulose powder

Amino acids, food dyes, alkaloids

5

Starch

Amino acids

6

Sephadex

Amino acids, proteins

according to the phytochemicals present in the plant extract) or by viewing the plate under the UV light. The first practical application of TLC was given by Stahl. TLC has also been used for confirmation of purity and identity of isolated compounds. The standardized thin-layer chromatographic procedure can be used effectively for the screening, analysis, as well as quality evaluation of the plant or its derived herbal products. Newer approaches in TLC enable the analyst to separate and determine the useful natural products in complex mixtures of plant products. With the use of an applicator, a thin layer of an adsorbent, such as silica gel formed from a slurry in a suitable solvent, is spread across a glass plate of size 5 3 2 cm. Different adsorbents used to separate various compounds are enlisted in Table 4.1. The sample extract is spotted on the TLC plates. The separated mixture is dissolved in a suitable solvent and placed on a glass plate using a fine capillary at a distance of about 2 cm from the bottom. The dry plate is then placed in a development chamber containing a suitable solvent or a mixture of solvents in a vertical position. It should be noted that the spot marked on it should not be dipped in the solvent during the operation. The solvent will progressively increase due to capillary action once the compartment is closed. A number of fine spots form when the mixture separates. Finally, the plate is removed and dried. Colored components can be found using the naked eye, while colorless components can be found using UV light, iodine, and sulfuric acid mixed with an oxidizing agent such as potassium permanganate, nitric acid, and other methods. The components can be identified using the Rf value or by using a suitable procedure to elute each component separately. The relative adsorption of each component of a mixture is expressed by a retention factor known as the Rf value. The Rf value is calculated as the distance traveled by the solute to the distance traveled by the solvent. The compounds from the spots are scrubbed and used for further screening. Rf 5 Distance traveled by the solute/distance traveled by the solvent.

4.3.2.3 Gas
liquid chromatography Gas
liquid chromatography (GLC, also called gas chromatography; Fig. 4.5), is a method for the separation of volatile compounds (Littlewood, 1962). It is well known that many pharmacologically active constituents in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by GLC is very important in the analysis of herbal medicines. GLC provides both qualitative and quantitative data on plant substances since measurements of the area under the peaks shown on the GLC trace are directly related to the concentrations of the different components of the original mixture. This technique depends on the redistribution of the components across a stationary phase or support material in the form of a liquid, solid, or combination of both and a gaseous mobile phase. The rate of migration for the chemical species is determined through its distribution in the gas phase. For example, a species that distributes itself 100% into the gas phase will migrate at the same rate as the flowing gas, whereas a species that distributes itself 100% into the stationary phase will not migrate at all. Species that distribute themselves partly in both phases will migrate at an intermediate rate (Burchfield & Storrs, 1962). Gas chromatography involves a sample being vaporized and injected into the head of the chromatographic column. The sample is then transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. The GLC apparatus can be set up in such a way that the separated components are further subjected to spectral or other analysis. Most frequently, GLC is automatically linked to mass spectroscopy (Ms), and the combined GC
Ms apparatus has emerged in recent years as one of the most important of all techniques for phytochemical analysis.

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FIGURE 4.5 Schematic representation of a standard GLC.

Column oven heat control Detector oven Column oven

Detector reference D

Vaporizer heat control

Detector oven heat control

R

Recorder of signal current

Column

Heat exchange coils

Vaporizer Exhaust

Flow meter Regulator

Gas-inlet Panel valve connection

Carrier gas tank

Pressure gauze Sample

sample HPLC pump

Detector

Injector

HPLC column Data aquision

HPLC solvent

waste FIGURE 4.6 Schematic representation of a standard HPLC.

4.3.2.4 High-performance liquid chromatography High-performance liquid chromatography (Fig. 4.6) also known as high-pressure liquid chromatography (HPLC) is an analytical technique used for the separation and determination of organic and inorganic solutes in any samples especially biological, pharmaceutical, food, environmental, industrial, etc., (Hancock, 1990). HPLC is useful for a compound that cannot be vaporized or that decomposes under high temperature, and it provides a good complement to gas chromatography for the detection of compounds (Katz, 1995). Further, HPLC is a flexible, stable, and commonly used process for natural product isolation (Cannell, 1998), especially suitable for the separation and characterization of polyphenolic compounds (Roblova´ Mgr, 2016). It is also used for the detection, chemical separation, purification, and quantification of drugs either in their active pharmaceutical ingredient or in their formulations during the three main phases of drug discovery, drug production, and manufacturing. HPLC separates compounds on the basis of their interactions with solid particles of tightly packed column and the solvent of the mobile phase. Modern HPLC uses a nonpolar solid phase, like C18, and a polar liquid phase, generally a mixture of water and another solvent. High pressure of up to 400 bars is required to elute the analyte through column

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before they pass through a diode array detector (DAD). This DAD measures the absorption spectra of the analytes to aid in their identification. Currently, this technique is gaining popularity among various analytical techniques as the main choice for fingerprinting study for the quality control of herbal plants (Fan et al., 2006). Natural products are frequently isolated following the evaluation of a relatively crude extract in a biological assay in order to fully characterize the active entity. The biologically active entity is often present only as minor component in the extract, and the resolving power of HPLC is ideally suited to the rapid processing of such multicomponent samples on both an analytical and preparative scale. Many benchtop HPLC instruments now are modular in design and comprise a solvent delivery pump, a sample introduction device such as an auto-sampler or manual injection valve, an analytical column, a guard column, a detector, and a recorder or a printer. Purification of the compound of interest using HPLC is the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, flow rate, suitable detectors and columns to get an optimum separation. Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC, a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels at which the assay will be performed. UV detectors are popular among all detectors because they offer high sensitivity (Li et al., 2004) and also because majority of naturally occurring compounds encountered have some UV absorbance at low wavelengths (190
210 nm) (Cannell, 1998). Besides UV, other detection methods are also being employed to detect phytochemicals among which is the DAD coupled with mass spectrometer (Ms) (Tsao & Deng, 2004). Liquid chromatography coupled with mass spectrometry (LC/Ms) is also a powerful technique for the analysis of complex botanical extracts (Cai et al., 2002; He, 2000). It provides abundant information for the structural elucidation of the compounds when tandem mass spectrometry (MSn) is applied. Therefore, the combination of HPLC and Ms facilitates rapid and accurate identification of chemical compounds in medicinal herbs, especially when a pure standard is unavailable (Ye et al., 2007). The processing of a crude source material to provide a sample suitable for HPLC analysis, and the choice of solvent can have a significant bearing on the overall success of natural product isolation. The source material, for example, dried powdered plant, will initially need to be treated in such a way as to ensure that the compound of interest is efficiently liberated into solution. In the case of dried plant material, an organic solvent (e.g., methanol, chloroform) may be used as the initial extractant, and following a period of maceration, solid material is then removed by decanting off the extract by filtration. The filtrate is then concentrated and injected into HPLC for separation. The usage of guard columns is necessary for the analysis of crude extract as many natural product materials contain a significant level of strongly binding components, such as chlorophyll and other endogenous materials that may in the long-term compromise the performance of analytical columns.

4.3.2.5 High-performance thin-layer chromatography High-performance thin-layer chromatography (HPTLC) is a planar chromatography where the separation of sample components is achieved on high-performance layers with detection and data acquisition. These high-performance layers are plates pre-coated with a sorbent of particle size 5
7 μm and a layer thickness of 150
200 μm. The reduction in thickness of layer and particle size results in increasing the plate efficiency as well as nature of separation. HPTLC gives chromatogram, that is, separated samples after chromatography can be inspected by the eyes only in the case of HPTLC. The main difference between TLC and HPTLC is the particle and pore size of sorbents illustrated in Table 4.2.

4.3.2.6 Capillary electrophoresis It is an instrumental analysis method developed by combining classical electrophoresis with modern microcolumn separation technologies in the late 1980s. In pharmaceutical analysis, the most commonly used separation modes are capillary zone electrophoresis, micellar electrokinetic capillary chromatography, and capillary gel electrophoresis. Capillary electrophoresis (Fig. 4.7) is an efficient separation technology of large and small molecules in a hollow and thin inner diameter capillary (10
200 μm). The two ends of the capillary are immersed in a buffer solution, and electrodes

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TABLE 4.2 Differences between HPTLC and TLC. Criteria

HPTLC

TLC

Layer of sorbent

100 μm

250 μm

Efficiency

High due to smaller particle size generated

Less

Separations

3
5 cm

10
15 cm

Analysis time

Shorter migration distance and the analysis

Less

Solid support

Silica gel for normal phase and C8, C18 for reverse phase

Silica gel, alumina

Sample spotting

Auto-sampler

Manual spotting

Scanning

UV/visible/fluorescence scanner scans the entire chromatogram qualitatively and quantitatively, and the scanner is an advanced type of densitometer

Not possible

HPTLC, High-performance thin-layer chromatography; TLC, thin-layer chromatography.

Fused Silica Capillary Detector

Signal

Complex

FIGURE 4.7 Schematic representation of a capillary electrophoresis setup.

Ligand

Migration Time

Anode

Cathode Analyte Sample Plug

Laser

Inlet Electrolyte Reservoir

Outlet Electrolyte Reservoir

connected with a high-voltage power supply are inserted separately. The voltage makes samples migrate along the capillary. According to the charge and volume of the separated substances, various molecules are separated under high voltage. Sample injection could be accomplished by pressing the sample into a capillary tube by atmospheric pressure or voltage. In zone capillary electrophoresis, separation could be achieved by the movement of electrophoresis and electroosmotic flow. The strength of electroosmotic flow depends on the strength of electric field, pH value of electrolyte, composition of buffer solution, ionic strength, internal friction, and so on. HPCE has the advantages of high efficiency, microamount, economy, high automation, and wide application. However, it has the disadvantages of poor preparation ability, low sensitivity, and poor separation reproducibility.

4.3.2.7 Countercurrent chromatography Countercurrent chromatography (CCC) (Fig. 4.8) is a form of liquid
liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force (Berthod et al., 2009; Ito & Bowman, 1970). The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube, or a series of chambers connected with channels, which contain both phases. The resulting dynamic mixing and settling action allow the components to be separated by their respective solubility in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation (Liu et al., 2015). CCC is used to separate, identify, and quantify the chemical components of a mixture. CCC and related

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FIGURE 4.8 Schematic representation of countercurrent chromatography.

pump

injecon valve detector

staonary phase

mobile phase

CCC centrifuge with three mullayer coils

fracon collector

liquid
liquid separation techniques have been used on both industrial and laboratory scales to purify a wide variety of chemical substances (Friesen et al., 2015). 4.3.2.7.1

Droplet countercurrent chromatography

Droplet countercurrent chromatography (DCCC) is an improved liquid
liquid partition chromatography based on the countercurrent partition method. It was introduced by Tanimura, Pisano, Ito, and Bowman in 1970. DCCC uses only gravity to move the mobile phase through the stationary phase which is held in long vertical tubes connected in series. In the descending mode, droplets of the denser mobile phase and sample are allowed to fall through the columns of the lighter stationary phase using only gravity. The formation of droplets is required when the mobile phase passes through a liquid stationary phase column. Droplets of mobile phase contact with stationary phase effectively and form new surfaces in thin partition extraction tubes constantly, which promotes the partition of solutes in two-phase solvents, and the chemical components of mixtures are isolated in immiscible two-phase droplets due to different partition coefficients. This method is suitable for the separation of phytochemicals with strong polarity. The separation effect is usually better than countercurrent partition chromatography, and there is no emulsification phenomenon. Furthermore, nitrogen is used to drive the mobile phase so the separated substance will not be oxidized by oxygen in the atmosphere. However, the solvent system which can generate droplets must be selected in this method, the amount of sample treated is small, and special equipment is needed. DCCC possesses good reproducibility and can handle crude extract samples of milligram to gram grade. It can be used in either acidic or basic conditions. Because no solid separation carriers are used, the phenomenon of irreversible adsorption and band broadening of chromatographic peaks can be avoided. Compared with preparative HPLC, DCCC consumes less solvent, but the separation time is longer and the resolution is lower. With the rapid development of high-speed countercurrent chromatography (HSCCC), the use of DCCC for natural product separations was less preferred (Hostettmann et al., 1979). The main limitation of DCCC is that flow rates are low, and poor mixing is achieved for most binary solvent systems. 4.3.2.7.2

High-speed countercurrent chromatography

HSCCC is also a liquid
liquid partition chromatography. It is another mild form of chromatography with no solid support and hence no chance of loss of substrate by binding to the column. The only media encountered by the sample are solvent and Teflon tubing. The former is common to all forms of chromatography and the latter to most. The chemical constituents with higher partition coefficient in mobile phase are eluted first, whereas those with higher partition coefficient in stationary phase are eluted later. HSCCC could avoid the shortcomings of irreversible adsorption and abnormal tailing of chromatographic peaks caused by solid carriers in liquid chromatography because it does not need solid carriers. The sample recovery is near 100% from chromatography. It also has the advantages of good reproducibility, high purity of separated compounds, and fast speed. It is suitable for the isolation and purification of wide kinds of phytochemicals such as saponins, alkaloids, flavonoids, anthroquinoids, lignans, triterpenes, proteins, and carbohydrates (Yoichiro & Walter, 1996).

4.4

Applications of chromatography techniques

1. It is used for characterizing and isolating organic compounds such as amino acids, alcohol, amines, acids, antibiotics, etc. 2. It is used to find the amount of chemicals in each pharmaceutical product.

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3. 4. 5. 6. 7.

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It is also used in hospitals to detect the alcohol level in the patient’s bloodstream. It is used in clinical tests like urine analysis, antibiotic analysis, etc. The level of pollutants in water can be identified. It is used in forensic labs to identify components in the samples collected from the suspects. It is also used in the food industry to identify the nutritive value of food additives and their components in the food.

4.4.1 Non-chromatographic techniques 4.4.1.1 Immunoassay An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. The molecule detected by the immunoassay is often referred to as an “analyte,” and hence, immunoassays are becoming important tools in bioactive compound analyses. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of an unknown sample. Immunoassays show high specificity and sensitivity for receptor binding analyses, enzyme assays, and qualitative as well as quantitative analytical techniques. Enzyme-linked immunosorbent assay (ELISA) based on MAbs are in many cases more sensitive than conventional HPLC methods. Monoclonal antibodies (MAbs) can be produced in specialized cells through a technique known as hybridoma technology. Such MAbs are used to determine similar drugs in the plant extract mixture through ELISA.

4.4.1.2 Phytochemical screening assay “Phytochemicals” are chemical compounds found in plants, and the term is often used to describe a large number of secondary metabolites derived from the plants. Phytochemical screening assay is a simple, quick, and inexpensive procedure that gives a quick answer to the researcher about the presence or absence of various types of phytochemicals as mentioned above in the given sample. It is an important tool in bioactive compound analyses. After obtaining the crude extract or active fraction from the plant material, phytochemical screening test can be performed using the experimental procedures given in Table 4.3 (Gul et al., 2017) to get an idea regarding the type of phytochemicals present in the sample extract mixture or fraction.

4.4.1.3 Fourier transform infrared spectrum Fourier transform infrared spectroscopy (FTIR; Fig. 4.9) is a valuable tool for the characterization and identification of compounds or functional groups (chemical bonds) present in an unknown mixture of plant extract (Eberhardt et al., 2007; Hazra et al., 2007). In addition, FTIR spectra of pure compounds are usually so unique that they are like a molecular “fingerprint.” For most common plant compounds, the spectrum of an unknown compound can be identified by comparison to a library of known compounds. Samples for FTIR can be prepared in a number of ways. For liquid samples, the easiest way is to place one drop of sample between two plates of sodium chloride. The drop forms a thin film between the plates. Solid samples can be milled with potassium bromide (KBr) and then compressed into a thin pellet which can be analyzed. Otherwise, solid samples can be dissolved in a solvent such as methylene chloride, and a few drops of the solution are then placed onto a single-salt high-attenuated total reflectance (HATR) plate. The solvent is then evaporated off, leaving a thin film of the original material on the plate. The spectrum was recorded in terms of percentage transmittance. The peaks at specific wave number were assigned by bonding and functional group as per the reference given in Varian FTIR instrument manual.

4.5

Characterization methods

4.5.1 Introduction In identifying a plant constituent, once it has been isolated and purified, it is necessary first to determine the class of compound and then to find out which particular substance it is within that class. Its homogeneity must be checked carefully beforehand; that is, it should travel as a single spot in several TLC or PC systems. The class of compound is usually clear from its response to color tests, its solubility and R, properties, and its UV spectral characteristics. Biochemical tests may also be invaluable: the presence of a glucoside can be confirmed by hydrolysis with

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TABLE 4.3 Preliminary phytochemical screening tests. S. no.

Phytochemicals

Procedure

Results

1

Anthraquinones

Take 6 g of the crude drug powder sample in a conical flask. Add 10 mL of benzene, and filter it after 10 min. Again, add 10 mL of 10% ammonia solution to the filtrate, and shake vigorously for 30 s.

Appearance of pink, violet, or red color indicates the presence of anthraquinones.

2

Tannins

Add 10 mL of bromine water to the 0.5 g aqueous extract.

Decoloration of bromine water indicates the presence of tannins.

3

Saponins

Take a small quantity of aqueous crude extract in a test tube. Add 5 mL of distilled water and mix thoroughly. Again add a few drops of olive oil and again mix vigorously.

Formation of foam indicates the presence of saponins.

4

Flavonoids

Shinoda test: Mix pieces of magnesium ribbon and conc. HCl with aqueous crude extract. Alkaline reagent test: To the aqueous crude extract, add 2 mL of 2.0% NaOH. When concentrated yellow color appears, add a few drops of diluted acid.

Appearance of pink color after few minutes indicates the presence of flavonoid. Disappearance of yellow color indicates the presence of flavonoids.

5

Glycosides

Liebermann’s test: Add 2 mL of acetic acid and 2 mL of chloroform to the aqueous crude extract, and allow the mixture to cool. Then add conc. H2SO4. Keller
Kiliani test: Add 4 mL of glacial acetic acid and 1 drop of 2% FeCl3 to the 10 mL aqueous crude extract. Then add 1 mL of conc. H2SO4. Salkowski’s test: Add 2 mL conc. H2SO4 to the aqueous crude extract.

Appearance of green color indicates the presence of glycosides. Formation of a brown ring between the layers indicates the presence of cardiac glycosides. Formation of a reddishbrown color indicates the presence of glycoside.

6

Terpenoids

Add 2 mL of chloroform to 5 mL aqueous crude extract. Evaporate the mixture using water bath and then boil with 3 mL conc. H2SO4.

Appearance of gray color indicates the presence of terpenoids.

7

Steroids

Add 2 mL of chloroform and conc. H2SO4 to the 5 mL aqueous crude extract.

Appearance of red color in the lower chloroform layer indicates the presence of steroids.

8

Phenols

Take 20 mL of distilled water in a test tube, add few powdered samples, boil it, and then filter the mixture. To the filtrate add 3
4 drops of 0.1% ferric chloride.

Appearance of brownish green or blue color indicates the presence of phenols.

9

Alkaloids

Add 1% HCl to the crude extract and warm it. After filtering, add Mayer’s reagent (mercuric chloride 1 potassium iodide in water) to the filtrate.

Formation of yellow colored precipitate indicates the presence of alkaloids.

10

Carbohydrates

Molisch’s test: Dissolve the crude sample powder in 5 mL of distilled water and filter. To this filtrate, add two drops of alcoholic α
-naphthol solution in a test tube. Then using a dropper along with side of test tube, disposed tubes and pour drop wise conc. sulfuric acid carefully.

Formation of violet color at the junction or interface of two liquids indicates the presence of carbohydrates.

8-glucosidase, of a mustard oil glycoside by hydrolysis with myrosinase, and so on. For growth regulators, a bioassay is an essential part of identification. Complete identification within the class depends on measuring other properties and then comparing these data with those in the literature. These properties include melting point (for solids), boiling point (for liquids), optical rotation

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FIGURE 4.9 Fourier transform infrared spectroscopy.

(for optically active compounds) and R, or RRt (under standard conditions). However, equally informative data on a plant substance are its spectral characteristics: these include ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), and mass spectral (Ms) measurements. A known plant compound can usually be identified on the above basis. Direct comparison with authentic material (if available) should be carried out as final confirmation. If authentic material is not available, careful comparison with literature data may suffice for its identification. If a new compound is present, all the above data should be sufficient to characterize it. With new compounds, however, it is preferable to confirm the identification through chemical degradation or by preparing the compound by laboratory synthesis. Spectroscopic techniques such as IR, UV
visible, NMR, and Ms provide sufficient information for quantitative and qualitative analysis of phytochemicals. Basically, the organic molecule, when allowed to interact with electromagnetic radiations, absorbs in certain region depending upon its structure and produces a spectrum. The spectrum obtained is specific to certain functional groups, which helps in the identification of the molecule. Due to the fact that plant extracts usually occur as a combination of various types of bioactive compounds or phytochemicals with different polarities, their separation still remains a big challenge for the process of identification and characterization of bioactive compounds. It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex chromatography, and HPLC should be used to obtain pure compounds. The pure compounds are then used for the determination of structural and biological activity. Besides that, non-chromatographic techniques such as immunoassay, which use MAbs, phytochemical screening assay, FTIR, can also be used to obtain and facilitate the identification of the bioactive compounds.

4.5.2 Gas chromatogram The first gas chromatograms were generated by an automated titration system. But, in 1954, Ray used the temperature (and hence electrical resistance) change of a filament of a thermal conductivity-measuring device—a katharometer as a means of detection (Ray & Appl, 1954). The katharometer remained popular for packed-column work because of its response to most analytes, but the requirement of trace analysis and the development of the capillary column quickly resulted in a new emphasis. Rather than using bulk properties (based, e.g., on gas density, flow impedance, and gravimetry—a sensitive version of the latter was proposed by Martin (Burchfield & Storrs, 1962) in 1962 as potentially the “ideal detector for GC”), more sensitive ionization-based detectors were investigated. GC expanded with great rapidity over the two decades following its invention in 1952, and much of current practice has its roots in that period. The introduction of robust, efficient, and reproducible fused-silica capillary columns and the provision of relatively

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Syringe

PG

Detector Electrometer

Recorder

FIGURE 4.10 Schematic diagram of a basic gas chromatograph.

Detector x

Injector

TRAP PR2

PRI

G

Fuel Gas(es)

ADC

Column Data System NV

Make up Gas Column Oven

inexpensive but reliable equipment for GC-Ms provided a crucial new impetus in the 1980s. High-speed GC, time-offlight Ms, AED, and comprehensive GC x GC now promise further expansion. The versatility of modern GC will expand its application areas. A schematic diagram of a basic gas chromatograph is shown in Fig. 4.10.

4.5.3 UV and visible spectrum UV
visible spectroscopy can be utilized for the identification of bioactive compounds in both isolated and mixture forms. It can be used to identify various phytochemicals with the help of maximum absorption (λ max) values corresponding to their structural features, for example, total phenolic extract (280 nm), flavones (320 nm), phenolic acids (360 nm), etc., (Altemimi et al., 2017). This spectroscopy technique requires less time for observation and is available at low cost (Rasul, 2018). The value of UV and visible spectra in identifying unknown constituents is related to the relative complexity of the spectrum and the general position of the wavelength maxima. If a substance shows a single absorption band between 250 and 260 nm, it could be anyone of a considerable number of compounds (e.g., a simple phenol, a purine or pyrimidine, an aromatic amino acid, and so on). If, however, it shows three distinct peaks in the 400
500 nm region, with little absorption elsewhere, it is almost certainly a carotenoid. Furthermore, spectral measurements in two or three other solvents and comparison with literature data might even indicate which particular carotenoid it is. The above statements suggest that absorption spectra are of especial value in plant pigment studies, and this is certainly true for both water- and lipid-soluble plant coloring matters. Other classes which show characteristic absorption properties include unsaturated compounds (particularly polyacetylenes), aromatic compounds in general (e.g., hydroxycinnamic acids), and ketones. The complete absence of UV absorption also provides some useful structural information. It is indicative of the presence of saturated lipids or alkanes in lipid fractions of plant extracts, or of organic acids, aliphatic amino acids, or sugars in the water-soluble fractions. Because of space limitations, the spectral properties of only a very limited number of plant constituents can be given in this book. These are mainly recorded in the form of tables of spectral maxima, but a few illustrations of spectral curves are included in later pages.

4.5.4

1

H-NMR and 13C-NMR spectra

NMR spectroscopy gives physical, chemical, and biological properties of matter. One-dimensional technique is routinely used, but the complicated structure of the molecules could be achieved through two-dimensional NMR (2D NMR) techniques. Solid-state NMR spectroscopy is used for the determination of molecular structure of solids. Radiolabeled 13C NMR is used to identify the types of carbon present in the compound. 1H-NMR is used to find out the types of hydrogen present in the compound and to find out how the hydrogen atoms are connected. NMR spectroscopy is used to determine the magnetic properties of certain nuclei such as 1H, 13C, 19F, and 31P (Rasul, 2018). When magnetically active nuclei interact with the radiofrequency region of electromagnetic radiations, it produces a signal with a frequency characteristic of the external magnetic field applied and resonates when its oscillation frequency becomes equivalent to the intrinsic frequency. It is usually measured in terms of chemical shift which depends upon the strength of the applied magnetic field, the chemical environment, and the magnetic properties of the nuclei involved (Altemimi et al., 2017). NMR spectroscopy has enabled us to predict the structure of the bioactive molecules by analyzing the frequencies (or chemical shift values) of signals and determining the positions of different nuclei.

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FIGURE 4.11 Mass spectrometer.

4.5.5 Mass spectrometry Mass spectrometry (Fig. 4.11) is a powerful analytical technique for the identification of unknown compounds, quantification of known compounds, and elucidating the structure and chemical properties of molecules. Through the Ms spectrum the molecular weight of sample can be determined. This method is mostly employed for the structural elucidation of organic compounds, for peptide or oligonucleotide sequencing, and for monitoring the existence of previously characterized compounds in complex mixtures with high specificity by defining both the molecular weight and a diagnostic fragment of the molecule simultaneously. Mass spectroscopy involves the bombardment of organic molecules either with electrons or lasers, thereby converting them to highly energetic charged species. Using this spectroscopic technique, the relative molecular mass of fragmented ions can be determined along with the information about places of fragmentations that can be subsequently used to predict the molecular formula of the bioactive compound (Ingle et al., 2017). Thus, Ms provides abundant information required for structural determination. For example, for structural elucidation of phenolic compounds, Ms has been found a highly efficient analytic tool with electrospray ionization (ESI), a preferred way of generating charged species from macromolecules (phenolic compounds) (Altemimi et al., 2017).

4.5.6 GC
Ms spectrum Gas chromatography
mass spectrometry (GC
Ms, Fig. 4.12) analysis is an effective testing and troubleshooting tool to identify and quantify chemicals in a complex mixture (Al-Rubaye et al., 2017). Some of the technical fields using GC
Ms analysis are perfume industry (Van Asten A, 2002), food (Chiu & Kuo, 2020; Vene et al., 2013), hydrocarbon fuels research pharmacy research (He et al., 2016), forensic (Bridge et al., 2018), and sometimes it becomes more powerful to detect chemical warfare agent (Li et al., 2020). GC and Ms provide distinct but complementary results; while GC separates components of a mixture, Ms can analyze and identify these components. These methods were first used in tandem in the 1950s and are still widely applied in clinics and laboratories worldwide (Simon-Manso et al., 2013). Additionally, the separation by gas chromatography also provides information and contributes to unambiguous identification of organic substances. The process of identification is nowadays supported by automated search algorithms; however, a profound identification needs an in-depth knowledge of interpretation of both mass spectrometric and gas chromatographic data. Therefore, this study is addressed to collect and summarize the way to read and interpret GC
Ms output in a practical and systematic manner. The principal aspects of GC
Ms data interpretation are given in advance by a brief description of up-to-date processes of identification and, finally, by some examples from laboratory research background and industrial uses as technical matter analyses.

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FIGURE 4.12 GC
Ms spectrometer.

FIGURE 4.13 Bruker 700 MHz nuclear magnetic resonance (NMR) spectrometer.

4.5.7 Two-dimensional NMR spectrum NMR (Fig. 4.13) is a highly versatile spectroscopic technique with applications in a large range of disciplines, from chemistry and physics to biology and medicine. It is widely used as an analytical tool in all chemistry laboratories and industries, where it provides a superior way to extract structural and quantitative information at site-resolved level, and hence enables the unambiguous elucidation of inorganic and organic molecular structures (Batta et al., 1997; Simpson, 2008). NMR is also very well recognized in biochemistry for its ability to deliver the folding and geometry of large macromolecules, alone or in complexes with drugs or with other biomacromolecules, under nearly physiological conditions (Cavanagh et al., 2006; Wu¨thrich, 1986). The capabilities of NMR to shed light on dynamic chemical, biophysical, and biological processes over a wide range of timescales by a range of complementary methods are also well known (Tycko, 2003). Enabling many—in fact most—of these applications is a revolutionary proposition by J. Jeener, dating back to 1971 and involving the concept of multidimensional NMR spectroscopy acquisitions. By providing a way of spreading the spectral peaks over a 2D frequency plane rather than along a 1D frequency axis, Jeener’s idea literally changed the face of magnetic resonance as it offered: (1) a much better way of discriminating resonances than what could be offered by 1D traces—even at much higher fields—and (2) a much richer and wider range of experiments for extracting structural and dynamic information maps, of a kind that could only be dreamed of by other spectroscopic methods relying on one-dimensional acquisitions (Jeener, 1971; Aue et al., 1976). As a result of this, 2D NMR, as well

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81

X-Ray Spectroscopy Data output device Amplifier Detector Computer Electron beam

Display

X-ray signal Sample

FIGURE 4.14 X-ray spectrometer.

as its higher-dimensional variants, has now become a routine analytical tool for the elucidation of small organic molecules, and a method of choice for the study of solution- and solid-state macromolecular structures.

4.5.8 X-ray spectroscopy X-ray spectroscopy is an excellent method to determine the structure of a compound. However, this technique requires the availability of a compound as a single crystal. Most chemists find this process very tedious, time-consuming, and it requires a skillful hand. In the event when other spectral methods fail to reveal a compound’s identity, X-ray spectroscopy (Fig. 4.14) is the method of choice for structural determination where other parameters such as bond lengths and bond angles are also determined. However, advances in technology have made it possible to have an NMR spectrum complete in as little as 1 min. X-ray spectrum analysis is an element analysis of the material on the basis of its X-ray spectrum. A qualitative X-ray spectrum analysis is performed using Moseley’s law. Quantitative X-ray spectrum analysis is performed by the intensity of the lines employing a crystal analyzer, scintillation and ionization counter, and coordinate plotter. All elements with atomic number Z9 (sometimes lighter elements) may be determined by X-ray spectrum analysis methods (Kljuev, 2001).

4.6

Conclusions and future directions

Phytochemicals have long been used as interesting biological and medicinal sources which are considered to be natural, active, safe, and sustainable sources for human and veterinary health benefits. Plants with medicinal properties play an increasingly important role in the food and pharmaceutical industries for their functions in disease prevention and treatment. Nature is an exceptional source of high variety of phytochemicals, many of these have been identified, and still n number remains unknown to the scientific world. In recent years, there is an increasing interest in phytochemicals throughout the world due to their bioaccessibility and bioavailability in humans with some proven health benefits, including antioxidant, protective action for cardiovascular diseases, cytotoxicity, anti-inflammatory, anticancer, obesity, immune-modulatory, antimicrobial, diabetes mellitus, hypoglycemic action, other degenerative diseases, putative properties to use in foods, functional foods, nutraceutical products, and as a therapeutic source for managing several biological interventions. Natural products have contributed for searching of novel phytochemicals and natural products for the drug development process in recent years and continue to do so. The lab-intensive and time-consuming extraction and isolation processes, however, have hindered the application of natural products in drug development. Since bioactive compounds occurring in plant material consist of

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multicomponent mixtures, their separation and determination still create problems. Practically, most of them have to be purified by the combination of several chromatographic techniques and various other purification methods to isolate bioactive compound/s. The ever-growing demand to extract plant bioactive compounds encourages continuous search for convenient extraction methods. Conventional extraction techniques such as liquid
liquid extraction, maceration, percolation, reflux, and Soxhlet extraction techniques are time- and energy-consuming, have low extraction efficiency, and generate organic pollution harmful to human health and the environment. In order to bypass these drawbacks, numerous efforts have recently been made to streamline green and efficient extraction procedures such as microwave and SFE as well as PEF, MAE, and UAE. These techniques have several merits such as increasing the extract yield, simplicity of operation, low cost, decreasing the thermal degradation, selective heating of vegetal material, reducing the usage of organic solvent, environmental safety, etc. The chromatography advancement and awareness about the environment are two important factors for the development of most nonconventional extraction processes. However, understanding every aspect of nonconventional extraction process is vital as most of these methods are based on different mechanisms, and extraction enhancement has resulted from different processes. Regarding extraction, reflux extraction is the most commonly employed technique for preparative separation. The novel extraction and characterization of phytochemicals method play an important role in phytochemistry research because it is necessary to extract the specific phytochemical from the plant material without altering its nature and properties. As technology continues to develop, more and more new automatic and rapid techniques have been created to extract and separate natural products, which might reach the requirement of high-throughput screening. The modern extraction methods, also regarded as green extraction methods, including UAE, MAE, SFE, and PLE, have also been the subject of increased attention in recent years due to their high extraction yields, selectivity, stability of the target extracts, and process safety merits. Nonetheless, the rate of success and the authenticity of these findings depend on the accuracy in the selection of solvents, selection and proper execution of extraction methods, phytochemical screening, fractionation, and identification techniques. Some of those green methods have become routine sample preparation methods for analytical purposes. Proper choice of standard methods also influences the measurement of extraction efficiency. Novel extraction methods provide high diversity of phytochemical compounds, giving high biological activity. In the view of increasing economic significance of bioactive compounds and commodities rich in these bioactive compounds, efforts should be made to develop more sophisticated extraction processes with some novel, consistent, and standardized methods in the future.

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95

Chapter 5

Phytochemicals: recent trends in food, pharmacy, and biotechnology Ayushman Gadnayak1 and Budheswar Dehury2 1

Department of Bioinformatics, Odisha University of Agriculture & Technology, Bhubaneswar, Odisha, India, 2Bioinformatics Division, ICMR-

Regional Medical Research Centre, Bhubaneswar, Odisha, India

5.1

Introduction

Phytochemicals, often considered as phytonutrients, are bioactive and natural compounds rich in various foods like fruits, nuts, whole grain products as well as legumes, and dark chocolates. There are numerous phytochemicals in which only a small amount is being isolated and identified from the plants (Go´mez-Garcı´a et al., 2020). The most common phytochemicals found in food are carotenoids, flavonoids, ligands, saponins, cyanidin, isoflavones, polyphenols, and indole. The biodiversity in the resources of the phytochemicals has given a renewable and unique resource to discover the potential of using such agents in the discovering novel drugs, development of therapeutics against cancer, and supplementary foods. Much of the present evidence on the advantages of the phytochemicals have come from previous researches and their clinical applications. After intake of phytochemicals in their diet, individuals have shown significantly lower risk of certain types of cancers as well as cardiovascular diseases. However, there is no accurate evidence of using any specific phytochemical to eliminate the chances of cancer. However, there are promising evidences that indicates the potential use of phytochemicals to: G G G G G G

Protect the cells and DNA from damage that in turn leads to lower the risk of cancer, Enhance the immune system, Reduce inflammations, Slow rate of growth for some of the cancer cells, Regulate the hormones, and Lowers the risks of chronic diseases such as cardiovascular diseases and obesity.

Phytochemicals including carotenoid, flavonoids, anthocyanin, isothiocyanates, and zeaxanthin helps in inhibiting the growth of cancer cells and lowers the occurrence of cardiovascular diseases. Carotenoid can be found in cooked tomatoes, carrots, orange, and sweet potatoes. On the other hand, flavonoids that are found in berries, apples, soybeans, coffee, walnuts, and whole grains have the capability to fight inflammation, reduce tumor growth, and decrease the chances of DNA damage. Anthocyanins, found in berries help in lowering blood pressure, therefore, they can be used as a daily remedy for maintaining hypertension. Lastly, zeaxanthin is found in leafy green lie chard, and spinach is demonstrated to promote eye health (Roswellparki, 2019).

5.2

Bioactive phytochemicals

The traditional Mediterranean diet can be considered as a well-known pattern that is directly interlinked with longevity as well as the improvement of the quality of life as it decreases the risk of certain chronic diseases (Go´mez-Garcı´a et al., 2020). For instance, studies have shown promising roles of certain phytochemicals present in honey and berries (Roswellparki, 2019). Such studies have demonstrated the potential role of phytochemicals in inactivation of carcinogens, thereby reducing the proliferation of cells including apoptosis and cell arrest as well as preventing angiogenesis in certain types of tumors as illustrated in Fig. 5.1. Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00031-1 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 5.1 Diverse spectrum of applications of bioactive phytochemicals derived from edible flowers

Edible flowers have been studied to be rich in bioactive phytochemicals. Some of these flowers exhibit a broad spectrum of anti-inflammatory and antioxidant properties, while, some are studied to possess anticancer and antidiabetic properties. For example, Arbutus species have shown promising antioxidant and antimicrobial activities, thus being used as agents for drug discovery. Metabolic syndromes observed in certain chronic diseases like cardiovascular diseases, obesity, hypertension (HTN), type 2 diabetes (T2D), and nonalcoholic fatty liver disease (NAFLD). In this context, dietary and lifestyle modifications are demonstrated to be efficient approaches to control metabolic syndromes. A recent review by Xiao and Bai highlights the favourable effects of phytochemicals in regulating glucose metabolism and modulation of the host glucose metabolism (Xiao & Bai, 2019). Circadian rhythm can be considered as a physiological activity rhythm that is driven by the “internal circadian clock” and other external stimuli. The disruption of the same can cause several diseases like metabolic syndrome, heart disease, and obesity. Hence, promising therapeutics obtained from phytochemicals, which have the potential to treat or prevent the occurrence of these complex disorders. However, micronutrient supplementations can affect human health, but the results are not conclusive due to lack of evidence (Xiao & Bai, 2019). Furthermore, it has also been demonstrated that micronutrients can improve the conditions of heart and enhance the quality of life of the patients with heart-related ailments. The dynamic interaction between host immune system and carcinogenesis and nutritional immunology, i.e., the use of natural compounds as immunomodulators in cancer patients, is beginning to develop. Although the interactions between the immune system and the tumor microenvironment are intricate, modulation of specific immune cells and cytokines by certain phytochemicals, viz. carotenoids, curcumin, and beta-glucans, have been studied to show promising immune-modulating effects in patients with cancer (Pan et al., 2019).

5.3

Antioxidant and antimicrobial properties of phytochemicals

The adverse effects of the synthetic microbial agents as well as the global demands for safe food that possesses minimal chemical preservatives have enhanced the demands for the antimicrobial agents that are mainly plant-based (Pimentel-Moral et al., 2020). The plans have shown to harbor various types of phytochemicals that have evolved

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more likely as a defense against adverse environmental conditions, that is, pathogenic microbes as well as insects and pests. The unique chemical structure of the phytochemicals is generally classified into several groups like polyphenols, sulfur-containing products, carotenoids, alkaloids, and terpenes. Polyphenols are considered as secondary compounds that possesses several antimicrobial, antioxidant, anticancer, and antihypertensive activities (Pimentel-Moral et al., 2020). Depending on the chemical structure of polyphenols, they can be subgrouped into two classes, that is, flavonoids and nonflavonoids. Alkaloids are the organic bases of nitrogen which are derived from plant and have been widely used as antimicrobial agents from ancient times. Sanguinarine, as well as piperine, possesses a wide spectrum of antimicrobial activity against several microbes like Staphylococcus aureus, Escherichia coli, and Bacillus subtilis (O’Shea et al., 2012). A range of alkaloids like xinghaiamine, ascididemin, clausenol, chelerythrine, and agelasine have been demonstrated to possess strong antimicrobial effects against gram-negative and grampositive bacteria along with some naturally available alkaloids such as squalamine, tetrandrine, and lysergol (Kovaˇcevi´c et al., 2018). Free radicals are considered as unstable and highly reactive compounds that damage the overall food quality and confer adverse effects on the nutritional food composition. Singlet oxygen, hydroxyl free radicals, superoxide anion, and nitric oxide are some of the important free radicals (Go´mez-Garcı´a et al., 2020). Certain antioxidants from phytochemicals can communicate with free radicals, which in turn can prevent the damage to cellular organs (Choudhari et al., 2020). Black and green teas are proven to be useful sources of antioxidants in a palatable form that is available to an important number of the population of the world. A recent study has demonstrated that the solvent extracts, such as hexane, ethyl acetate; methanol, and ethanol of Sargassum serratifolium, exhibits a broad spectrum of antioxidant activity (Jamali et al., 2020).

5.4

Phytochemicals from the agri-food by-products

Epidemiological studies have demonstrated that the consumption of vegetables and fruits can add health benefits that generally contribute to organic micronutrients such as polyphenols, fiber, vitamin C, carotenoids, and others (Zhao et al., 2019). Among the peels, fruit pomace, and plant leaves, husk seed oil, as well as crop, are of greater importance nowadays as they are rich in hydrophilic and lipophilic constituents that can be considered as the precious natural antioxidants. The three-dimensional representation of some of the important bioactive phytochemicals are shown in Fig. 5.2. These compounds are considered as beneficial organic pigments that are mainly synthesized by fungi, bacteria, and plants. They are present in considerable amounts in certain fruit tissues and play a crucial role in human health by protecting tissues and cells from the destructive effects of the singlet oxygen. Some of these compounds are also used as natural colorants in case of the food industry (Kis et al., 2020). The processing of tomato industries can be demonstrated as an example that generally generates a large number of by-products and peels. Seeds represented in the 10%
40% of the total tomatoes are processed. The by-products that are generated are combined with the important characteristics of its components (Wagh et al., 2009). FIGURE 5.2 The two-dimensional representation of the bioactive phytochemicals.

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5.4.1 Phenolic compounds Phenolic compounds are considered as a much-diversified category of the secondary plant metabolites that include phenolic acids, flavonoids, lignins, lignans, stilbenes, and tannin. Phenolics are seen to be present in all types of plant origin, beverages, fruits, and the processing of the plants those results in the generation of by-products that contain bioactive compounds. For example, grapes are considered as the world’s largest crop that is produced more than 60 tonnes annually and has phenolic compounds that are used as by-products (Wagh et al., 2009). Several promising usages of these by-products that are rich in the phenolic compounds can be considered as one of the crucial health promoters, and can be used in food additives as well as cosmetics (Mani et al., 2020).

5.4.2 Dietary fiber Since ancient times dietary fibers are used in nutrition as well as health and have received wide attention in research for health benefits. Fibers are referred to as the mixture of non-starch polysaccharides that control digestion by the action of enzymes in the case of the gastrointestinal canal (Chaudhary et al., 2018). The total dietary fiber in mango byproducts ranged from 40% to 72%. The mango peels contain dietary fiber that is useful in cosmetic, food, pharmaceutical, and nutraceutical applications (Zhang et al., 2019).

5.5

Pharmacological aspects of phytochemicals

There is a growing amount of interest in the pharmacological aspects of phytochemicals due to their effectiveness, and minimal side effects. Below are a list of compounds with their pharmacological aspects from diverse plant species.

5.5.1 Elaeagnus angustifolia Elaeagnus angustifolia exhibits a wide variety of biological and pharmacological activities. This species is also known as Russian olive, which is used worldwide as a remedy and nutritional agent in the management of certain illnesses (Forni et al., 2019). Previous studies have demonstrated that the plant contains a wide spectrum of therapeutic and pharmacological effects that include insecticidal, antimicrobial, wound healing, antioxidant, cardioprotective, insecticidal, hypolipidemic, gastroprotective, antitumor, antimutagenic, and antinociceptive activities (Lillehoj et al., 2018). It has also been reported to contain steroids, polysaccharides, beta-carboline, phenols, phenolic acids, as well as ketones, pyrimidines, esters, and certain nutritive compounds.

5.5.2 Lawsonia inermis The use of Lawsonia inermis for cosmetic and medicinal purposes is linked to modern and ancient cultures in Asia and North Africa. The literature works in this area reveal that L. inermis plays a crucial role in regular lives giving medicinal and psychological benefits. From the extracts of these compounds, around 70 types of phenolic compounds have been isolated, which have pharmacological effects (Khatoon et al., 2020). Extensive research into the pharmacological as well as phytochemistry of the stems of henna forms the use as a cure for several medical conditions. Around 100 secondary metabolites have been isolated from the subterranean as well as the aerial parts. On the other hand, the safety assessment of the compound has revealed that the compound does not have any acute or chronic toxicity.

5.5.3 Holarrhena antidysenterica (L.) Holarrhena antidysenterica (L.) belongs to the Apocynaceae family and is widely used in the Indian ayurvedic medicine system in order to treat diarrhea and dysentery. A methanolic bark extract has been shown to lower malondialdehyde levels as well as nitric oxide and increase the concentration of superoxide dismutase as well as glutathione in colitis in male albino rats. Recent studies based on the leaf extract isolated from this plant have also revealed its antioxidant properties. A “CNS stimulating activity” has also been found in the extracts of this species. Furthermore, it also has anthelminthic, antimicrobial, and antimutagenic activity (Khatoon et al., 2020).

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5.6

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Nanodrug delivery of the phytochemicals in treating cancer

In spite of the greatest advantages in the treatment choices available for cancer patients, there is no accurate method that can cure cancer. There are a wide variety of phytochemicals that are known to express anticancer properties. In order to increase the minute backlogs, scientists have employed advanced nano-formulation technology. A particular nanomaterial is being used in the system that varies in size and is used to deliver or carry any substance or drug. In lung cancer, a small molecule called polyphenol honokiol can be found effective. The compound is loaded into the micelles depending on the poly(ἐ-caprolactone)-poly(ethylene glycol)-poly(ἐ-caprolactone) copolymer or PCEC in which the size is 51 nm (Ghildiyal et al., 2020). The recent development in biotechnical advancement based on the phytochemicals for drug delivery is summarized in Fig. 5.3. Phytochemical in polymeric nanoparticle, liposome, micelle, and metallic nanoparticle can be seen with adjacent drug particles. Triptolide can be considered as a terpenoid tri-epoxide that is purified from the Tripterygium wilfordii, a Chinese herb, where the compound is loaded onto the polymeric micelles and is synthesized using the MePEG-PLA through a solvent evaporation method. The wide spectrum of anticancer properties, phytochemicals, and nanocarriers in cell lines is summarized in Table 5.1. The effects of the flavone apigenin against the melanoma while delivering in combination with the poly(lactic-co-glycolide) nanoparticles are demonstrated to be significant. FIGURE 5.3 Recent advances in drug delivery with phytochemicals.

TABLE 5.1 Anticancer properties of diverse phytochemicals. Type of cancer

Cell line tested

Phytochemical

Nanocarrier

Lung

AS49 human lung adenocarcinomic cells

Honokiol

Poly(i-caprolactone)-poly (ethylene glycol)-poly(icaprolactone) copolymer micelle

NCI HA60 nonsmall cell lung carcinoma cells

Ferulic acid

Poly(lactide-co-glycolide) (PLGA) nanoparticles

AS49 human lung adenocarcinomic cells

B-Lapachone

Poly(ethylene glycol)-co-poly (D,L-lactic add) (PEG-PLA) polymer micelles

AS49 human lung adenocarcinomic cells

6-Lapachone and paclitaxel

Poly(ethylene glycol)-co-poly (D,L-lactic add) (PEG-PLA) polymer micelles

H292 lung cancer cells

Luteolin

Polylactic acid and polyethylene glycol (PLA-PEG) nanoparticle

MCF-human breast adenocarcinoma cell line and MDA-MB-453

Curcumin

Silk fibroin and chitosan (SFCS) polymers nanoparticles

SK BR-3 human breast cancer cells

Noscapine

Human serum albumin (HSA) nanoparticle

MDA-MB-231 breast cancer cells

Ursolic acid

pH-sensitive liposomes

Breast

(Continued )

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TABLE 5.1 (Continued) Type of cancer

Cell line tested

Phytochemical

Nanocarrier

MCF-7 human breast adenocarcinoma cell line

Thymoquinone

Liposomes modified with Triton X-100

MDA-MB-231 breast cancer cells

Silibinin

Lipid nanoparticles containing D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and phosphatidylcholine

MCF-7 human breast adenocarcinoma cell line

Gallic acid

PAMAM dendrimers

Colorectal

HT29 human adenocarcinoma cells

Triptolide

MePEG-PLA copolymer micelle

CT26 murine colon carcinoma cells

Honokiol

Monomethoxy poly(ethylene glycol) (MPEG) and poly(icaprolactone) (PCL) starshaped micelle

HCT 116 human colon cancer cells

Thymoquinone

Poly(lactide-co-glycolide) (PLGA) nanoparticles

A375 skin melanoma and HaCaT keratinocytes

Apigenin

Poly(lactic-co-glycolide) nanoparticles

B16 and B16F: 10 melanoma cells

Combretastatin A-4 and Doxorubicin

RGD (arginylglycylaspartic acid)-modified liposomes

Mel 928 melanoma cells

Epigallocatechin 3-gallate (EGCG)

Polylactic acid, polyethylene glycol nanoparticles

A2780 human ovarian cancer cells

Honokiol

Monomethoxy poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) nanocarrier

A2780CP ovarian cancer cells

Curcumin

Poly(lactic-co-glycolide) (PLGA) nanoparticle

SKOV ovarian cancer cells

Resveratrol

Bovine serum albumin nanoparticles

SKOV-3 ovarian cancer cells

Curcumin

Poly(2-hydroxyethyl methacrylate) (PHEMA) nanoparticles

Melanoma

Ovarian

Prostate

LNCaP human prostate adenocarcinoma cells and PCa prostate cancer cells PC3 human prostate cancer cells

Polylactic acid, polyethylene glycol, prostate-specific membrane antigen (PSMA) ligands Epigallocatechin 3-gallate (EGCG)

PCa prostate cancer cells

Bovine serum albumin nanoparticles Polylactic acid-polyethylene glycol (PLA-PEG) nanocarrier

LNCaP human prostate adenocarcinoma cells, PC3 human prostate cancer cells, and DU-145 PC3 human prostate cancer cells

Curcumin

Poly(lactic-co-glycolic acid) (PLGA) nanospheres

PTEN CaP8 mouse prostate epithelium cancer cells

Curcumin and resveratrol

Curcumin and resveratrol nanoemulsions (Continued )

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TABLE 5.1 (Continued) Type of cancer

Cell line tested

Phytochemical

Nanocarrier

Cervical

HeLa cervical cancer cells

Curcumin

Bovine casein micelles Alginate
chitosan
pluronic composite nanoparticles

HepG2 hepatocellular carcinoma cells

Berberine

Berberine polyethenyl glycol (PEG) liposome

Berberine

Berberine and D-a tocopheryl polyethylene glycol 1000 succinate (TPGS) nanosuspension

Gambogic acid

Lactoferrine nanoparticles

FIGURE 5.4 Uses of bioactive phytochemicals.

5.7

Current limitations and future of phytochemicals

Though the plant products are used as antimicrobial as well as antioxidant agents, bioavailability of their raw materials, seasonal variation, and their high cost in comparison with synthetic perspectives restrict their commercial usage. Most of the available information based on in vitro tests are limited and insufficient, thereby limiting the understanding of the functional utility as well as the perspective of the phytochemicals (Ashfaq et al., 2021). Furthermore, the study of the distribution, metabolism, absorption bioavailability, as well as toxicity in case of in vivo application, is limited in the literature. Therefore, phytochemicals frequently cannot express their full effects in terms of food and pharmaceutical industries. Hence, it is of immense importance to undertake research on multidisciplinary aspects of the use of phytochemicals (Ranjan et al., 2019). Furthermore, there is also a strict need for stability evaluation, sensory acceptance, and regulatory aspects of phytochemicals either exclusively or in combination with the food system. Different types of activities of bioactive phytochemical such as promotion, inhibition, and regulation are illustrated in Fig. 5.4. Fig. 5.4 shows phytochemicals that can promote apoptosis and may inhibit reception,

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metastasis, and angiogenesis. Epigenetic alterations coupled with certain state-of-the-art modern techniques can help in commercial production and increase the utility of such materials in drug delivery. These approaches include the use of waste material, biotechnological approach, nanotechnological approach, and bioinformatics approaches.

5.8

Conclusion and future prospect

Considering the above facts, it can be concluded that phytochemicals are biologically active compounds including carotenoids, flavonoids, terpenes, polyphenols, etc., that possess a wide spectrum of biological activities, with multifaceted uses. Thus, the phytochemicals can be used in various aspects that are associated with lowering the chances of cancer, DNA damage, and certain chronic diseases like cardiovascular disease hypertension and obesity. Phytochemicals can be obtained from various sources including the agri-food products and from certain edible flowers like silk cotton tree and dog flower. They can also be useful in drug delivery and development. Several studies over the past decade have shown promising applications of phytochemicals in drug discovery and treatment. Therefore, we observe a increasing trend of applications of phytochemicals in clinical science. Studies have also shown the promising application of phytochemical compounds as nanoparticles in drug delivery, cancer research, cosmetics, and food supplements. The limitations in the studies of phytochemicals have to be addressed in order to scale-up the availability of the phytochemicals for drugdevelopment related activities. As mentioned earlier, the clinical applications of phytochemical are broad, here we have provided some insights into the usefulness of phytochemicals and their synthetic derivatives for the treatment of cancer. Understanding their mode of action and molecular mechanism is important to strategize the current increasing usage of phytochemicals as antioxidants. In future, certain explorative studies need to be conducted based on the current knowledge in order to provide a holistic review of the use of phytochemicals as neuroprotective, and anticancer drugs. Thus, an in-depth analysis of the mechanism of actions of these phytochemicals can broaden their future usage in treatment of various diseases and making lives better.

References Ashfaq, F., Ali, Q., Haider, M. A., Hafeez, M. M., & Malik, A. (2021). Therapeutic activities of garlic constituent phytochemicals. Biological and Clinical Sciences Research Journal, 2021(1). Chaudhary, P., Sharma, A., Singh, B., & Nagpal, A. K. (2018). Bioactivities of phytochemicals present in tomato. Journal of Food Science and Technology, 55(8), 2833
2849. Choudhari, A. S., Mandave, P. C., Deshpande, M., Ranjekar, P., & Prakash, O. (2020). Phytochemicals in cancer treatment: From preclinical studies to clinical practice. Frontiers in Pharmacology, 1614. Forni, C., Facchiano, F., Bartoli, M., Pieretti, S., Facchiano, A., D’Arcangelo, D., Norelli, S., Valle, G., Nisini, R., Beninati, S., & Tabolacci, C. (2019). Beneficial role of phytochemicals on oxidative stress and age-related diseases. BioMed Research International, 2019. Ghildiyal, R., Prakash, V., Chaudhary, V. K., Gupta, V., & Gabrani, R. (2020). Phytochemicals as antiviral agents: Recent updates. Plant-derived bioactives (pp. 279
295). Singapore: Springer. Go´mez-Garcı´a, R., Campos, D. A., Aguilar, C. N., Madureira, A. R., & Pintado, M. (2020). Valorization of melon fruit (Cucumis melo L.) by-products: Phytochemical and biofunctional properties with emphasis on recent trends and advances. Trends in Food Science & Technology, 99, 507
519. Jamali, S. N., Assadpour, E., & Jafari, S. M. (2020). Formulation and application of nanoemulsions for nutraceuticals and phytochemicals. Current Medicinal Chemistry, 27(18), 3079
3095. Khatoon, E., Banik, K., Harsha, C., Sailo, B. L., Thakur, K. K., Khwairakpam, A. D., Vikkurthi, R., Devi, T. B., Gupta, S. C., & Kunnumakkara, A. B. (2020). Phytochemicals in cancer cell chemosensitization: Current knowledge and future perspectives. Seminars in cancer biology. Academic Press, June. Kis, B., Avram, S., Pavel, I. Z., Lombrea, A., Buda, V., Dehelean, C. A., Soica, C., Yerer, M. B., Bojin, F., Folescu, R., & Danciu, C. (2020). Recent advances regarding the phytochemical and therapeutic uses of Populus nigra L. buds. Plants, 9(11), 1464. Kovaˇcevi´c, D. B., Maras, M., Barba, F. J., Granato, D., Roohinejad, S., Mallikarjunan, K., Montesano, D., Lorenzo, J. M., & Putnik, P. (2018). Innovative technologies for the recovery of phytochemicals from Stevia rebaudiana Bertoni leaves: A review. Food Chemistry, 268, 513
521. Lillehoj, H., Liu, Y., Calsamiglia, S., Fernandez-Miyakawa, M. E., Chi, F., Cravens, R. L., Oh, S., & Gay, C. G. (2018). Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Veterinary Research, 49(1), 1
18. Mani, J. S., Johnson, J. B., Steel, J. C., Broszczak, D. A., Neilsen, P. M., Walsh, K. B., & Naiker, M. (2020). Natural product-derived phytochemicals as potential agents against coronaviruses: A review. Virus Research, 284, 197989. O’Shea, N., Arendt, E. K., & Gallagher, E. (2012). Dietary fibre and phytochemical characteristics of fruit and vegetable by-products and their recent applications as novel ingredients in food products. Innovative Food Science & Emerging Technologies, 16, 1
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Pan, P., Huang, Y. W., Oshima, K., et al. (2019). The immunomodulatory potential of natural compounds in tumor-bearing mice and humans. Critical Reviews in Food Science and Nutrition, 59(6), 992
1007. Available from https://doi.org/10.1080/10408398.2018.1537237. Pimentel-Moral, S., Ca´diz-Gurrea, M. de. l. L., Rodrı´guez-Pe´rez, C., & Segura-Carretero, A. (2020). Recent advances in extraction technologies of phytochemicals applied for the revaluation of agri-food by-products. Functional and Preservative Properties of Phytochemicals, 209
239. Ranjan, A., Ramachandran, S., Gupta, N., Kaushik, I., Wright, S., Srivastava, S., Das, H., Srivastava, S., Prasad, S., & Srivastava, S. K. (2019). Role of phytochemicals in cancer prevention. International Journal of Molecular Sciences, 20(20), 4981. Roswellparki, 2019. For the health benefits of phytochemicals, “Eat a Rainbow.” Retrieved 4.06.2022 from https://www.roswellpark.org/cancertalk/ 201912/health-benefits-phytochemicals-eat-rainbow. Wagh, V. D., Wagh, K. V., Tandale, Y. N., & Salve, S. A. (2009). Phytochemical, pharmacological and phytopharmaceutics aspects of Sesbania grandiflora (Hadga): A review. Journal of Pharmacy Research, 2(5), 889
892. Xiao, J., & Bai, W. (2019). Bioactive phytochemicals. Critical Reviews in Food Science and Nutrition, 59(6), 827
829. Zhang, L., Virgous, C., & Si, H. (2019). Synergistic anti-inflammatory effects and mechanisms of combined phytochemicals. The Journal of Nutritional Biochemistry, 69, 19
30. Zhao, C., Liu, Y., Lai, S., Cao, H., Guan, Y., San Cheang, W., Liu, B., Zhao, K., Miao, S., Riviere, C., & Capanoglu, E. (2019). Effects of domestic cooking process on the chemical and biological properties of dietary phytochemicals. Trends in Food Science & Technology, 85, 55
66.

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Chapter 6

Phytochemicals as bioactive ingredients for functional foods R.S. Agrawal1, R.C. Ranveer2, N.B. Rathod2 and Nilesh Prakash Nirmal3 1

Department of Patronage Traditional and Speciality Foods, MIT School of Food Technology, MIT Art, Design, and Technology University, Pune,

Maharashtra, India, 2Department of Post-Harvest Management of Meat, Poultry and Fish, Post-Graduate Institute of Post-Harvest Management, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Raigad, Maharashtra, India, 3Institute of Nutrition, Mahidol University, Salaya, NakhonPathom, Thailand

6.1

Introduction

Plant-derived medicinal products are gaining renowned consumer interest in scientific communities around the world due to their positive effects on human health and minimum/no side effects. Plants are linked to biologically important novel chemical compounds making them a potential source for the development of medicinal compounds whose characterization has led to the development of novel and low-cost medications with high therapeutic potential (Ukwuani et al., 2013). The secondary products formed from plant materials, generally referred to as phytochemicals, are responsible for beneficial medicinal effects. Phytochemicals are biologically active, naturally occurring chemical compounds that provide health benefits to humans as macronutrients and micronutrients. These compounds play a significant role in their growth and development but also protect plants from environmental hazards like UV exposure, pollution, stress, drought, extreme temperatures, and pathogenic attack. Phytochemicals accumulate in various parts of plants, including the roots, stems, leaves, flowers, fruits, and seeds, and are responsible for the variety of colors and flavors found in fruits and vegetables. Phytochemical concentration ranges from plant to plant depending on variety, processing, cooking, and growth conditions. They are thought to act as synergistic agents allowing nutrients to be used more effectively by the body (Nyamai et al., 2016). Plant-based foods like fruits, vegetables, and grains contain several bioactive phytochemicals which are associated with a decrease in the risk of chronic diseases due to their part in antioxidant and free radical scavenging effects (Zhang, Gan, et al., 2015). Phytochemicals have great antioxidant potential and are of great interest due to their beneficial effects on the health of human beings, and they give immense health benefits to the consumers. Phytochemicals extracted from medicinal plants and dietary sources are often biologically active and have attracted the attention of researchers and pharmaceutical industries around the world. Consumers’ attitudes toward a healthy and well-balanced diet are becoming more favorable all over the world. As a result, there has been an increase in demand for foods that improve health and well-being, such as functional foods containing phytochemicals and probiotics. The concept of functional foods raises concerns about food security and appropriate selection of an adequate diet, beyond classical adequacy in energy, protein, essential fats, vitamins, and minerals. Today, it is recognized that foods not only provide basic nutrition, but can also prevent diseases and ensure good health and longevity. Functional foods may contain significant levels of biologically active components that impart health benefits. Several factors like scientific advances, consumer demand, increasing healthcare costs, an aging population, technical advances in the food industry, and changing regulatory environment have stimulated the field of functional foods (Hasler, 1996). This chapter will provide a brief overview of the role of phytonutrients biological activity in human health promotion and its potential application in functional food product development.

6.2

Phytonutrients

Plant sources contain many bioactive compounds in addition to those which are traditionally considered as nutrients, such as vitamins and minerals. Phytochemicals, also referred to as phytonutrients, refer to a wide range of potentially Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00028-1 © 2023 Elsevier Inc. All rights reserved.

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helpful chemicals generated by plants. These are naturally occurring compounds found in plant-based foods like fruits, vegetables, whole grains, legumes, beans, herbs, spices, nuts, and seeds. Their widespread presence in the diet and apparent low toxicity suggest that phytochemicals have enormous potential to improve human health and prevent the risk of diseases. Thus they can also be referred to as bioactive food components (Nyamai et al., 2016). Phytonutrients that are precious for human nutrition are identified and isolated from plants and related foods. Phytonutrients have been classified into major groups according to their chemical structures and functional characteristics. Some groups of phytochemicals which have or appear to have significant health potentials are carotenoids, phenolic compounds (flavonoids, phytoestrogens, phenolic acids), phytosterols and phytostanols, tocotrienols, organosulfur compounds (allium compounds and glucosinolates), and nondigestible carbohydrates (dietary fiber and prebiotics). Fruits and vegetables with deeper and brighter colors are prevalent sources of phytochemicals. The best way to ensure a variety of phytochemicals and essential nutrients in a diet is to consume a rainbow of plant-based foods. The main classes of phytonutrients are shown in Fig. 6.1.

6.3

Health-promoting ability of phytochemicals

Currently, plant-derived phytochemicals are emerging as promising drug candidates in the prevention and treatment of various metabolic disorders. Phytotherapy is gaining more consumer attention that involves the use of natural origin extracts as medicine or health-promoting agents (Berger & Shenkin, 2006). Phytochemicals are traditionally known to act as an effective barrier for the occurrence of several morbid health conditions by maintaining physiological systems. New experimental research is emerging that shows multiple effects of fruits, vegetables, and their phytonutrients that demonstrate positive concern toward human health. These chemical compounds play multiple preventive activities mainly antidiabetic, anti-inflammatory, antiaging, antimicrobial, antiparasitic, antidepressant, anticancer, antioxidant, and wound healing. The role of phytochemicals as health-promoting and disease-preventing constituent has gained increased interest worldwide particularly because of their availability, low cost, and minimal side effects. The potential health effects of phytochemicals are associated with numerous mechanisms, including prevention of oxidant formation, scavenging of activated oxidants, reduction of reactive intermediates, induction of repair systems, and promotion of apoptosis. As most of the developing countries are facing dietary and lifestyle-related diseases and an increasing aging population, this leads to increased interest and demand for food which is fortified with essential nutrients and bioactive compounds that fight against many health-related problems. Phytochemicals with increased bioactivity and little toxicity may be the most efficient alternative way for treating most diseases. Dietary intake of phytochemicals promotes health benefits and protects against chronic-degenerative disorders, such as cancer, cardiovascular and neurodegenerative diseases, diabetes, high blood pressure, inflammation, and microbial infections. The major phytonutrients of nutraceutical importance, their source, and health benefits are summarized in Table 6.1 (Charu & Dhan, 2014).

Phytosterols

Phytonutrients

Beta sitosterol, Campesterols Diosgenin

Polyphenols

Flavonoids, Phenolic acids, Tannins, Coumarins

Alkaloids

Berberine, Theobromine, Camptothecin

Organosulfur compounds

Indoles, Isothiocyanates Allyl suphur compounds

Terpenoids

D-limonene, Acetic acid, Glycyrrhizic acis

FIGURE 6.1 Classification of phytonutrients.

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TABLE 6.1 Major phytonutrients of nutraceutical importance, their sources, and health benefits. Phytonutrient

Food sources

Health benefits

Reference

Anthocyanins

Blackberry, cherry, orange, purple corn, raspberry, and red grape

Antioxidants, antiinflammatory, antiallergic, and lowers blood pressure

Choung et al. (2001)

Carotenoids

Carrots, leafy greens, red, oranges, yellow vegetables, pumpkin

Cell growth regulation, modulating gene expression, immune response, antioxidants

Paiva and Russell (1999)

Lycopene

Tomato, apricots, papaya, watermelon

Lowers risk of atherosclerosis and prostate cancer

Merve et al. (2017)

Resveratrol

Blueberry, peanuts, red grapes, red wine

Antioxidant, anticancer, antiaging, antidiabetic

Valenzano et al. (2006)

Phytosterol

Vegetables, nuts, fruits, seeds

Anticancer, hypolipidemic, reduces atherosclerosis

Charu and Dhan (2014)

Flavonoids

Berries, coffee, legumes, tea, cocoa, peanuts, spices, onion, apple, green vegetables, olive oil, walnuts

Antioxidant, antiviral, inhibition of hydrolytic and oxidative enzymes, and fights inflammation

Tanwar (2012)

Quercetin

Red onions, buckwheat, red grapes, green tea, and apple skin

Strong antioxidant, reduces LDL oxidation, blood thinner

Kumar and Andy (2012)

Isoflavonoids

Soybeans and soy-based products

Antioxidant, inhibits tumor growth, prevention of osteoporosis

Giles and Wei (1997)

Limonoids

Citrus fruits

Anticancer, antibacterial, antiviral, insecticidal

Ozaki et al. (1995)

Polyphenols

Cereals, legumes, oilseeds, fruits, vegetables, beverages

Antioxidant, anticarcinogenic, anti-inflammatory, antidiabetic, antiviral, antiallergic, antiaging

Mildner-Szkudlarz et al. (2013)

Omega-3 fatty acids

Fishes, soybean, flax oil

Lowers cholesterol, reduces high blood pressure, protects from heart attack, improves memory

Pawlosky et al. (2001)

Phytoestrogens

Soybeans, wheat, barley, corn, oats

Anticancer, antioxidant

Prakash and Gupta (2011)

Terpenoids

Plants, marine organism, algae

Slows cancer cell growth, strengthens immune function, limits production of cancerrelated hormones, fights viruses, works as antioxidant

Tholl (2006)

Probiotics

Fermented foods like milk, curd, fermented vegetables

Antimicrobial, lactose intolerance, immune enhancement

Pathan et al. (2017)

Isothiocyanates

Cruciferous vegetables like broccoli, cabbage, cauliflower

Antioxidants, protection against cancer, and cardiovascular disease

Charu and Dhan (2014)

Lutein and zeaxanthin

Dark, leafy greens, such as spinach

Promotes eye health

Simran and Suvartan (2019)

Fibers

Green leafy vegetables, oats

Reduces blood cholesterol, cardiovascular diseases

Slavin (2013)

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Biological activities of phytochemicals

Food intake plays a vital role in the modulation of the risk of cardiometabolic diseases. Plants are always considered as one of the most reliable sources for the management of diseases, and different synthetic drugs are derived from them directly or indirectly. Plant sources are very much popular since the ancient time as they are relatively safer and much cheaper alternatives than synthetic drugs. Plants and plant products can exert promising health-promoting and diseasepreventing abilities like antioxidant, immunity boosting, anticholesteremic, antidiabetic, anticancerous, renoprotective, neuroprotective, antiviral, etc., (Table 6.2). The presence of phytonutrients like flavonoids, alkaloids, and tannins render these life-saving therapeutic activities.

6.4.1 Antioxidant During aerobic metabolism, reactive oxygen species are produced in the human body leading to diseases like cancer, cardiovascular disease, etc. Antioxidants are substances acting as health-protecting factors that prevent damage to the cells from free radicals and are also used as natural food preservatives. Natural antioxidants being secondary metabolites of phytochemicals are more commonly preferred over synthetic antioxidants, which are found to impose side effects (Duduku et al., 2007). Plants produce a very impressive array of antioxidant compounds that include carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, and tocotrienols to prevent oxidation of the susceptible substrate (Hollman, 2001). Some of the commonly known antioxidants include beta-carotene, ascorbic acid, and alpha-tocopherol that block free radicals directly within the human body (Hayek, 2000). Natural antioxidants from plant sources are safer for health and have better antioxidant activity. Phytochemicals are non-nutrient bioactive components that are primarily responsible for neutralizing free radicals after oxidative stress and removing their power to create damage. Fruits like berries, grapes, Chinese dates, pomegranate, guava, sweetshop, persimmon, Chinese wampee, and plum are rich in antioxidant phytochemicals. Besides, fruit wastes (peel and seed) also contain high contents of antioxidant phytochemicals. Some vegetables like cowpea, caraway, green soybean, ginseng leaf, and broccoli are found to have high antioxidant capacities. Among cereal grains, pigmented rice, such as black rice, red rice, and purple rice, possesses high contents of antioxidant phytochemicals (Yu-Jie Zhang, 2015). Lycopene is a potent dietary antioxidant reported to have the highest capacity of capturing the singlet oxygen and scavenging free radicals (Merve et al., 2017). Polyphenolic compounds like catechins exhibit potent antioxidant properties, although in some cases they may act in the cell as pro-oxidants. Tea catechin is effective scavenger of reactive oxygen species and act as a powerful antioxidant agent (Charu & Dhan, 2014). Various processes of extraction such as Soxhlet extraction, subcritical water extraction (SWE), pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE) are used to obtain antioxidants from plant matrices (Duduku et al., 2007).

6.4.2 Immunity booster A well-functioning immune system plays a significant role in the maintenance of ordinary physiological and immunological functions. The balanced immune system enhances the body’s defense mechanism against diseases and infections. Phytochemicals provide an exciting opportunity to maintain the best health conditions through balanced and properly administered daily nutrition. These are considered as powerful and valuable resources of active compounds that play an imperative role to stimulate the immune system. The use of natural food sources such as fruits and vegetables in diet could support the body’s natural defense by strengthening our immune response. The application of selective nutrients to modulate the activity of the immune system is referred to as immunonutrition. The nutrients that have a specific function in immunomodulation are known as “immunonutrients” or “immunity regulators” which include vitamins, minerals, amino acids (arginine and glutamine), nucleotides, probiotics, and omega3 fatty acids (Robert, 2009). These natural bioactive compounds derived from foods can be taken as supplements at a much higher concentration than diet that could provide the basic foundation of functional foods that affect the immune system. These include antioxidants from fruits and berries, disease-fighting compounds from spices and fatty acids in fish (Jim & Edward, 2010). The importance of fruits and vegetables as modulators of the immune system is well established. Researchers have proven that fruits are abundant in bioactive compounds like vitamins, minerals, and phytochemicals like β-carotene, flavonoids, tannins, and phenolics. Natural antioxidants including vitamin A, C, E, polyphenols, and minerals like selenium improve our immune system by quenching the free radicals. These components have shown their potential to enhance our immunity by supporting the proliferation of lymphocytes, reducing oxidative

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TABLE 6.2 Overview of the mechanism of action of phytonutrients in the prevention of disorders. Phytonutrient

Activity

Mechanism of action

Reference

Alkaloids

Antioxidant

Free radical scavenging activity

Nyamai et al. (2016)

Antidiabetic

Increases insulin secretion, inhibition of digestive enzymes, increases glucose uptake

Mengjie et al. (2021)

Antiviral

Decreases viral entry and replication

Mohammad et al. (2021)

Antioxidant

Effectively scavenges free radicles

Venugopal and Adluri (2007)

Antidiabetic

Inhibits hyperglycemia and oxidative stress, improves β-cell functions

Switi et al. (2014)

Anti-inflammatory

Inhibition of enzymes like cyclooxygenase 2

Venugopal and Adluri (2007)

Anticancer

Inhibits invasion, enhances efficacy of radiotherapy, suppresses ROS production

Merve et al. (2017)

Capsaicin

Pancreatic cancer, liver cancer, lung cancer

Decreases cell proliferation, increases apoptosis

Jim and Edward (2010)

Flavonoids

Antimicrobial

Complex with cell wall, cytoplasmic membrane damage by perforation, inhibits DNA synthesis

Panche et al. (2016)

Hyperlipidemia

Reduces LDL cholesterol, increases HDL cholesterol

Tanwar (2012)

Hypertension

Improves vascular function by increasing the bioavailability of nitric oxide

Jaime et al. (2015)

Isoflavones

Anticancer

Inhibits cell proliferation, decreases invasion, enhances apoptosis

Hye-Ji et al. (2019)

Lycopene and catechins

Antioxidant

Radical scavenger, prevention of lipid peroxidation and DNA damage, regeneration of nonenzymatic antioxidants like vitamin C/E

Merve et al. (2017)

Anticancer

Increases antioxidant potential, decreases oxidative damage to lipids, proteins, and DNA, antiangiogenic activity, protection against oxidative stress

Jim and Edward (2010)

Antioxidant

Activation of antioxidant enzymes, scavenging of ROS

Jim and Edward (2010)

Antiviral

Signaling pathways for cell survival and proliferation, binding to cell surface

Antiaging

Improves lipid metabolism, suppresses inflammation and ROS production

Anticancer

Induces apoptosis, improves RA sensitivity, enhances the efficacy of radiotherapy

Curcumin

Polyphenols

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damage to cells, scavenging free radical species, and improving anti-inflammatory as well as immunomodulatory mechanisms (Shruti et al., 2022). Plant-derived nutraceuticals like alkaloids, phenolics, saponins, and glycoproteins have been reported to possess anti-inflammatory and immunomodulating properties. Moreover, terpenoids, polysaccharides, and fatty acids have also received considerable attention as possible immune-modulating agents for further application in immune disorders. The research study has shown immune-boosting effects of vitamin C that increase the production of infectionfighting white blood cells and antibodies and also help to prevent the entry of viruses. Fruit juices like orange, guava, and kiwi juice have been recognized as excellent sources of vitamin C whereas vegetables like red capsicum, broccoli, colored berries, and tomatoes are rich sources of vitamin C (Rose, 2007). Vitamin E also got attention as an immune booster having food sources like whole oats, wheat germ, avocado, raw nuts, seeds, fish, poultry, meat, and eggs. Seeds (pumpkin seeds) and nuts (walnuts, Brazil nuts, and almonds) are considered as natural sources of essential amino acids and essential fatty acids that have a positive effect on the immune system (Turner, 2007). Mushrooms can also help to boost the immune system, and the best-known example of an immune-active mushroom is Shiitake mushroom that contains lentinan as an immune-enhancing ingredient (Jim & Edward, 2010). The major phytochemicals present in honey, ginger, turmeric, garlic, black pepper, garlic, clove, onion, basil, and ginseng have shown an active role in enhancing immunity and also possess antiviral properties to fight against COVID-19. A number of amino acids like arginine are currently used for food fortification owing to their beneficial effect on the immune system. Omega-3 fatty acids from fish, flaxseed, nuts, and seeds have shown their potential to boost the immune system (Kolakowski et al., 2006). β-Glucan has an exciting opportunity to become a primary immunestimulating functional component to improve animal health status, due to its ability to enhance the host resistance to viral, bacterial, fungal, and parasitic infections (London, 2008). Consumers’ interest in probiotic-enhanced immunity is also increasing day by day. The present pandemic situation has increased the demand for plant-based functional foods for enhancing the immunity of all aged groups against COVID-19. The well-maintained innate immune system is always the best defense against any infections. The famous quote, “Prevention is always better than cure,” is considerably suitable to summarize the significant potential of functional foods to enhance the immune system. Immunomodulation by phytochemicals represents an interesting tool to be exploited for health-promoting and disease-preventing purposes owing to their multiple bioactivities, well tolerability, and good patient compliance (Antonella et al., 2020).

6.4.3 Anticholesteremic With changes in eating habits and lifestyle, the incidence of hypercholesterolemia has increased significantly. Hypercholesterolemia is characterized by elevated levels of total cholesterol or low-density lipoprotein cholesterol (LDL) or lower levels of high-density lipoprotein cholesterol (HDL). Hypercholesterolemia can be diagnosed with lifestyle modifications and dietary alterations. Hypercholesterolemia is an important risk factor for atherosclerosis, and cardiovascular diseases are considered as a silent health problem. The use of synthetic drugs like statins, niacin, and ezetimibe is increased to reduce or maintain acceptable blood cholesterol levels (Scott et al., 2022). Natural products have high lipid-lowering potential with minimal or no side effects and are thus considered as viable alternatives to synthetic drugs. The growing global interest in phytochemicals capable of lowering cholesterol level is rapidly increasing the consumers’ interest and demand. Recently, phytochemicals are considered as potential hypocholesterolemic agents. The phytochemical compounds like phytosterols, phenolics, saponins, alkaloids, dietary fibers, and lectins are known as natural cholesterol busters due to their cholesterol-lowering effects (Zhaohui et al., 2017). These busters not only decrease cholesterol oxidation and absorption but also increase cholesterol catabolism and elimination. Most of these busters are found in cereals, oatmeal, fruits, vegetables, legumes, and fermented foods. Phytosterol consumption in human diets represents an effective means of improving lipid profiles by suppressing intestinal cholesterol absorption. These can significantly reduce low-density lipoprotein cholesterol concentrations and reduce the risk of coronary heart diseases (Peter et al., 1997). Research studies emphasize that phytosterols should be a part of heart-healthy eating diets due to its anticholesteremic effects. Phenolic compounds are well documented with beneficial roles for human health due to their potential antioxidant as well as cholesterol-lowering properties (Zhaohui et al., 2017). Saponins are naturally occurring compounds in many plants and are characterized by surface-active foaming properties (Ozaifa et al., 2022). This helps to inhibit cholesterol absorption and thus decreases serum and liver cholesterol. Phytochemical saponins have a wide spectrum of activities like antifungal, antibacterial, antihypercholesterolemia, and inhibition of cancer cell growth. The saponins extracted from fenugreek and asparagus exhibited great protective effects against hypercholesterolemia. This is due to the inhibition of cholesterol absorption

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from the intestine due to the formation of insoluble complexes between cholesterol and saponin (Amany Ali et al., 2019). Plant lectins are carbohydrate-binding proteins. Dietary lectins are able to bind with intestinal brush border membranes which stimulate the release of anorectic neuropeptides that produce satiety and reduce food intake (RamirezJime´nez et al., 2015). Dietary fibers form complexes with dietary fats, cholesterol, and bile acids. Thus digestion of fat by pancreatic lipases is inhibited, while hepatic bile synthesis and cholesterol excretion are enhanced (Giovane & Napoli, 2010). Dietary fibers promote the growth of intestinal microflora such as Lactobacillus acidophilus. Therefore dietary fibers that selectively stimulate the growth and activity of beneficial microflora are known as “prebiotics” (Slavin, 2013).

6.4.4 Antidiabetic Diabetes mellitus (DM), the most prevalent health problem worldwide is characterized by increased blood glucose levels and insufficiency in insulin activity and/or insulin secretion Currently, phytochemicals identified from medicinal plants are emerging as promising drug candidates in the prevention and treatment of diabetes. Phytonutrients like alkaloids, glycosides, dietary fibers, polysaccharides, and phenolics such as flavonoids, terpenoids, and steroids have been reported as the most potent antidiabetic compounds (Firdous, 2014). Alkaloids like berberine isolated from the barberry tree exert a wide range of antidiabetic activities by improving the action of insulin. Resveratrol present in many plants including grapes, plums, and nuts has been shown to be effective in modulating blood glucose levels and decreasing insulin resistance (Mengjie et al., 2021). The jamun seeds contain important glycoside “Jambolin” which possesses hypoglycemic action by either preventing conversion of starch into sugar or increasing insulin secretion from β-cells. Flavonoids (anthocyanins, catechins, flavanols, flavones, flavanones) represent beneficial groups of plant metabolites with hypoglycemic potential. Fruits, vegetables, beverages, chocolates, herbs, and plants are natural sources of flavonoids that exert antidiabetic properties partly due to their antioxidant potential and partly due to their capability to modulate some cell signaling. Hesperidin, predominantly abundant in citrus fruits, plays an important role in preventing the progression of hyperglycemia. Naringenin, rutin, kaempferol, quercetin, genistein, and daidzein have been known to possess an enormous array of pharmacological activities with antidiabetic potential (Patrick & Isaac, 2018). Epigallocatechin gallate, a natural phytochemical predominantly found in green tea, has also emerged as a potential antioxidant and antidiabetic agent (Mengjie et al., 2021). A massive range of cohort studies has proven the role of dietary fiber in the management of diabetes. High-fiber diets especially with soluble fibers offer some improvements in carbohydrate metabolism, regulate blood sugar, reduce total cholesterol, LDL cholesterol and are associated with other beneficial effects in patients with diabetes. The oral administration of β-glucans, dietary fiber present in barley in type 2 diabetic, and high-fat diet-induced obese mice resulted in a significant reduction in blood glucose level (Cao et al., 2017). Curcumin, a bioactive molecule predominantly present in the Curcuma longa plant, has been associated with various pharmacological and biological effects. The metabolic effects of curcumin were accompanied by delay in diabetes development, improvement in β-cell functions, prevention of β-cell death, and decreased insulin resistance (Switi et al., 2014). Massive research showed the potential use of fenugreek seeds as an adjuvant in the management of diabetes due to improved glycemic control and reduced insulin resistance. The phytochemicals associated with antidiabetic activity of fenugreek seeds are trigonelline, nicotinic acid, and coumarin. Research investigation revealed that fenugreek seed powder is a potent natural food source that has the capacity to control diabetes (Genet et al., 2019). The benefit of large array of these bioactive phytochemicals and their therapeutic potential lies principally in their anti-inflammatory properties.

6.4.5 Anticancer Phytochemicals exhibit pharmacological effects for the management of chronic-degenerative diseases like diabetes and cancer. Phytonutrients are considered as most promising options with better treatment effectiveness in cancer patients and decreased adverse conditions. Capsaicin, a major alkaloid and active component of chili peppers, acts as a cancerpreventing agent. It inhibits the carcinogens activity through numerous pathways and can improve patients’ sensitivity to chemoradiotherapy, reduces treatment dosages, and improves patients’ tolerance. The natural polyphenol and dietary phytochemicals present in green tea are catechins that exert a synergistic cancer cell growth inhibitory effect and antioxidant potential. EGCG is the most abundant and powerful antioxidant in green tea and is associated with cancer chemoprevention. It is the active form of catechin that can strongly engender apoptosis and inhibit growth in several types of cancers like colon, kidney, breast, and brain cancers (Charu & Dhan, 2014). Lower incidence of cancer is associated

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with dietary consumption of lycopene, an antioxidant carotenoid mainly found in tomatoes. It plays an important role in the prevention and treatment of cancer typically prostate cancer by multiple mechanisms like scavenging of ROS, upregulation of detoxification systems, and interference with cell proliferation (Merve et al., 2017). The phytochemical curcumin has been recognized for its activity against cancer cells in several different types of cancer such as prostate, colorectal, breast, pancreatic, brain, head, and neck cancer. Plant metabolites with antitumor activity are lupeol, asiatic acid, celastrol, auraptene, ursolic acid, saidmanetin, indole-3-carbinol, and hypericin. These substances have been shown to affect signaling to control cell growth and apoptosis, immune response, and stromal microenvironment. (Irina et al., 2019). Isothiocyanates are major biologically active compounds present in cruciferous vegetables such as broccoli, cabbage, cauliflower and have been identified as promising anticarcinogenic agents. Benzyl isothiocyanate demonstrated anticancer activity by suppressing various critical hallmarks of cancer like tumor growth, cellular proliferation, angiogenesis, apoptosis, etc., (Alok Ranjan et al., 2019). Other phytochemicals like resveratrol, quercetin, berberine, and isoflavones have also shown chemopreventive properties and can be potentially used for cancer prevention and may mitigate associated toxicity of synthetic drugs.

6.4.6 Renoprotective Chronic renal disease is a condition characterized by a gradual loss of kidney function over time. Its prevalence rate is increasing with the increased diabetes and hypertension that has a profound impact on human health. As the management of chronic renal failure is exceedingly expensive, much attention has been given to the use of phytochemicals in providing renoprotective effects. Potentially active phytochemicals can be identified and isolated from medicinal plants for effective therapy in renal disorders. Numerous plant-derived compounds are being screened worldwide to validate their application in the management of renal disorders (Parakh et al., 2022). Medicinal plants are associated with the modulation of chronic kidney disease with various phytochemicals, including curcumin, resveratrol, capsaicin, quercetin, and genistein. Flavonoids derived from plants can become pharmacological agents as they play an essential role in renoprotective action. The antitumor activity of flavonoids helps to suppress renal carcinoma cell proliferation and is reported as a cost-effective alternative (Mario et al., 2022).

6.4.7 Neuroprotective The use of plants as a supplementary treatment for neurodegenerative disorders such as Alzheimer’s and Parkinson’s is supported by the reviewed studies. Researchers have suggested that phytochemicals such as capsaicin (found in red pepper), curcumin (found in the spice turmeric), epigallocatechin gallate (catechin in tea known as EGCG), and resveratrol (found in grapes, wine, and peanuts) may have neuroprotective effects (Lee et al., 2012). Phytochemicals help to maintain the brain’s chemical balance by influencing the function of receptors for the major inhibitory neurotransmitters. Neuroprotection refers to the strategies and relative mechanisms able to defend the central nervous system (CNS) against neuronal injury. Recently, there has been intense interest in the potential of alkaloids, phenols, flavonoids, terpenoids, and saponins to modulate neuronal function and prevent age-related neurodegeneration (Kumar & Khanum, 2012).

6.4.8 Antiviral (special reference to SARS-CoV-2) Due to the outbreak of the recent surpassing COVID-19 pandemic, several researchers have focused on the use of natural compounds as an effective alternative for discovering drug molecules to treat viral diseases and their complications. Most in vitro and in vivo studies are in progress for screening the effectiveness of phytochemicals against coronaviruses (especially SARS-CoV-2), computer docking models studies on predicting the anti-CoVs effects of these compounds against the coronavirus family members such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Research showed that natural polyphenol compounds like quercetin (Chiow et al., 2016), kaempferol (Schwarz et al., 2014), myricetin (Yu et al., 2012), apigenin (Ryu et al., 2010a), and resveratrol (Wahedi et al., 2020) hold prominent activities against coronaviruses. Lung injury is the main COVID-19 complication that happens with inflammatory cascades by SARS-CoV-2 (Fakhri et al., 2020). Alkaloids are nitrogen-containing phytochemicals that interact with the coronavirus structural proteins, including spike (S) glycoprotein and nucleocapsid (N) on the virus surface, as well as nonstructural angiotensin-converting enzyme 2 (ACE2) in the cell membrane and, in turn, inhibit the enzymes involved in coronavirus replication. Marine/ plant-derived alkaloids like berberine, tetrandrine, cepharanthine, lycorine, ergotamine, palmatine, noscapine, and

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quinine with prominent anti-SARS-CoV-2 effects along with antipyretic, anti-inflammatory, antitussive and lung injury, immunomodulatory, and protective effects against neurotoxicity, cardiotoxicity, nephrotoxicity, and hepatotoxicity could be promising candidates for COVID-19 treatment (Mohammad et al., 2021). Hesperidin, which is abundant in citrus, has shown a potential inhibitory effect on ACE2; thereby, it could be a good candidate for clinical trials on SARS-CoV-2 (Haggag et al., 2020). The phytochemicals in essential oils are among the other anti-HCoV natural compounds (Nadjib, 2020). The essential oil component of garlic showed a potent inhibitory effect against the SARS-CoV-2 receptor ACE2. The major constituents of the essential oil of garlic are organosulfur compounds having many pharmacological activities (Thuy et al., 2020). Ayurvedic system of medicine and the Ministry of Ayush recommend the rational use of herbal drugs to combat COVID-19 (Remya & Minnie, 2020). Therefore, plant extracts and phytochemicals have attracted the attention of many researchers as potential sources for viral inhibitors to discover new anti-CoV agents regarding controlling-related complications. This COVID pandemic situation has witnessed a tremendous resurgence in the interest and use of medicinal plants.

6.5

Phytochemicals-based functional foods

The growing demand for nutritious, healthy, and sustainable but at the same time attractive food products acts as driving forces for the future of food processing. The growing understanding of the relationship between diet and health has led to the development of functional foods. The surge of interest in phytochemicals as associated with health promotion effects beyond their nutritional value has been increasingly accepted in recent years, and the specific effects of nutrition prevention on disease have led to the development of functional foods (Monica & Ioan, 2019). Functional foods are sometimes also called nutraceuticals, pharmafoods, or designer foods. Functional foods may be defined as any food that has a positive impact on an individual’s health, physical performance, or state of mind in addition to its nutrient value. Examples may include conventional foods; fortified, enriched, or enhanced foods; and dietary supplements. Functional foods provide essential nutrients beyond quantities necessary for normal maintenance, growth, and development and/or provide other biologically active components that impact health benefits or desirable physiological effects. As the role of diet in the prevention of human ailments such as cancer, cardiovascular diseases, and obesity has become more evident, many consumers are increasingly seeking functional foods to improve their diets. Consequently, there is a trend to search for natural raw materials rich in dietary fiber and high in antioxidant capacity as functional ingredients for the food industry (Mildner-Szkudlarz et al., 2013). Due to the popularity and increasing interest in the functional food concept, the relationship between food and health has an increasing impact on food innovation. The main markets for functional foods are beverages, dairy products, bakery and confectionery products, and breakfast cereals.

6.5.1 Functional drinks/beverages Beverages are considered as the most active functional food category that helps to hydrate the body and to regain energy rapidly. A functional beverage is a nonalcoholic drink product that is formulated with ingredients, such as raw fruits, herbs, vitamins, minerals, amino acids, and other bioactive compounds that provide specific health benefits (Niharika et al., 2014). These are considered as an excellent delivering medium for nutrients and bioactive compounds like vitamins, minerals, antioxidants, ω-3 fatty acids, plant extracts, fiber, prebiotics, and probiotics (Maria et al., 2014). Functional beverages can be useful to enhance the immune system, improves gut or cardiovascular health, and helps with indigestion and weight management. Functional beverage product category includes dairy-based beverages (probiotics, fortified drinks), functional milk (extra calcium, omega-3, and vitamin fortified drinks), juices (vitamins and omega-3 fortified drinks), functional waters (vitamin and mineral fortified drinks, sports and energy drinks, herbal drinks, and health and wellness drinks) (Irene, 2019). Probiotics are defined as foods containing live microorganisms (mainly bacteria) which, when consumed in adequate amounts, can confer health benefits to the host. Food professionals are searching for new vehicles to deliver the health benefits of probiotics. The growth of functional drinks can be attributed to the increasing demand for high-performance drinks in sports. Functional sports drinks help in rehydration, energy replenishment, improvement of mental focus and athletic performance, and/or to prevent joint pain. Sports drinks are developed with essential electrolytes like sodium, potassium, chloride, calcium, phosphate, and magnesium, which are lost by sweating during training and/or competition (Evans et al., 2017). The inclusion of salt in sports drinks is known to perform physiological functions like fluid retention and supply energy.

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Amino acids are used to slow fatigue and improve muscle function, while vitamin B are used to boost metabolism and generate energy. Simple CHO can be used for a quick energy burst, whereas complex CHO provides sustained energy (Stefania et al., 2018). Plant-based beverages are usually made from fruits, vegetables, and herbs, alone or combined with other ingredients like fiber, vitamins, and minerals. The growing global interest in herbal teas/beverages among health-conscious consumers is due to the major source of bioactive compounds such as carotenoids, phenolic acids, flavonoids, coumarins, alkaloids, polyacetylenes, saponins, and terpenoids. Scientific evidence shows that these bioactive compounds render a myriad of biological effects of tea (Chandrasekara & Shahidi, 2018). Plant-based beverages have the efficient potential for maintaining health due to antioxidant properties that combat free radicals.

6.5.2 Functional bakery and confectionery Increased consumption of bakery and confectionery food products among all age group consumers is expected to propel the overall functional confectionery market, in the upcoming years. The bakery sector is likely to flourish due to the growing number of cereal-based products. Flour-based confectionery products are considered as an ideal medium for fortification and nutritional improvement by which functional foods can be delivered to consumers in an acceptable food. The most significant impact on increasing the functioning and nutritional content of flour-based confectionery is probably the substitution of flour in recipes with other types of flours, such as wholemeal wheat flour and flours from other cereals and noncereals like oat, barley, rice, soy, buckwheat, flaxseed, etc. Whole grains are high in dietary fiber, trace minerals, antioxidants, and phenolic compounds which play a beneficial role in human health by lowering the risk of cancer, diabetes, obesity, and cardiovascular disease (Sanja & Asima, 2021). Pseudo-cereals like buckwheat, amaranth, and quinoa are of great interest not just because of their excellent nutritional profile but also because they are gluten-free. Furthermore, protein fortification of biscuits is of current interest, and they can be made from composite flours, such as wheat flour fortified with soy, peanut, corn germ flour and with supplementation of health-promoting ingredients like whey protein concentrate and skimmed milk powder (Aggarwal et al., 2016). Different cereals, legumes, and fruits have been widely recognized as important sources for the improvement of flour-based confectionery and the provision of functional properties. Fruits have recently attracted a lot of attention as a source of bioactive compounds due to their antioxidant, anticarcinogenic, antimutagenic properties, and they had significant relevance to bakery and confectionery industry, especially for biscuits, cakes, and other bakery products. Confectionery market is traditionally based on the use of sugar. Candies, chewing gum, and chocolate are wellknown products in the confectionery market. Although sugar confectionery has an established place in the market, there is additional interest in sugar reduction in the confectionery sector. Sugar alcohols (polyols) are considered as building blocks of sugar-free confectionery. The utilization of polyols offers functional benefits to consumers like reduced energy products, reduced carcinogenicity, and other health benefits such as possible antineoplastic and prebiotic effects and reduced hyperlipidemia (Albert Zumbe et al., 2001). Confectionery sector has focused on the production of lowcalorie, high-fiber foods in response to the public interest for low-calorie and functional products. A surge in demand for sugar-free products due to increased health awareness among consumers could help in the growth of the functional confectionery market in the coming years. Reducing or substituting sugar for other sweeteners, reducing fat in recipes, and complete elimination of fat could decrease the caloric value of flour-based confectionery.

6.5.3 Functional dairy products Dairy products are natural healthy products having a major contribution to the functional food market. The fortification of bioactive components in dairy-based beverages makes them functional beverages as they provide immense health benefits. Functional dairy products include fortified dairy beverages like probiotics, prebiotics, vitamins, minerals fortified, and whey-based beverages. Vitamin and mineral fortification of dairy-based beverages has been carried out to compensate for the loss of minerals and vitamins during processing. Whey, a by-product of the cheese industry, has also been utilized for the development of whey-based functional beverages (Deepak & Sheweta, 2019). Fermented dairy products have been considered to have enormous health benefits and thus broadening the product range to other types of health-promoting products. Probiotics represent the novel buzzword in human dietary selection and are outstanding components of functional foods. The probiotic market is growing directly in accordance with the increased awareness of functional foods for health in humans. It has been predicted that probiotic foods comprise about 60%
70% of the total functional food market (Pathan et al., 2017). The use of probiotics in dairy and nondairy

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beverages has been extensively investigated worldwide. Dairy products like yogurt, lassi, ice cream, cheese, etc., have long been considered as important vehicles for the delivery of probiotics. Probiotics have three possible modes of action: generating immunostimulants, producing antimicrobial compounds, and competitively excluding other bacteria (Jim & Edward, 2010). The health benefits of probiotic bacteria depend on their viability. According to the International Federation for Dairy (IDF), at least 107 probiotic bacterial cells should be alive at the time of consumption per gram of product. Consumers’ growing health concerns have increased interest in nondairy probiotic beverages rather than dairy-based products due to cholesterol in dairy-related products (Behera & Panda, 2020). This has led to the development of diverse nondairy probiotics with some alternative sources like fruit, vegetable, grains, and legumes. The cultured dairy products can be added with beta-glucan of oats, isoflavones of soy protein, carotenoids, and flavonoids of certain fruits to produce functional dairy products having dynamic and promissory biological effects. Beneficial effects attributed to nondairy probiotic beverages include high phytochemical content, lower cholesterol and high blood pressure, and improve immune system (Daniela et al., 2018). Nondairy probiotic beverages can also help as a healthy substitute for dairy probiotics and are of great interest to lactose-intolerant consumers (Amin, 2018). Different extraction and encapsulation technologies have been used to obtain target food bioactive ingredients and to ensure an effective functionalization of dairy products (Amin, 2018). The phytochemicals are used as functional food, soft drinks, and many other food items, which are having good nutrient value and significant importance, economically.

6.5.4 Meat products With the advancement of the functional food concept, manufacturers of meat products have modified their formulas with the novel addition of phytonutrients to add functional properties to meat products. Meat products can be modified by adding ingredients considered beneficial for health or by eliminating or reducing components that are considered harmful (Bhat & Bhat, 2011). In the functional food market, meat products are also considered as excellent tools for introducing various bioactive compounds, to obtain functional meat products. Functional meat products are developed with the aim of minimizing the pro-oxidant effects associated with high meat consumption. Meat products can be functionally modified by the addition of fibers to impart more health benefits. The comminuted meat products can be developed with nonmeat proteins from plant sources like soy, Bengal gram, and buckwheat proteins. Texturized properties of soy protein offered the opportunity to develop meat analogs. Research studies have been conducted for the development of reformulated meat-based functional foods with the reduction in animal fat and the addition of bioactive compounds to achieve a functional effect. Therefore meat-based functional foods are being considered as an excellent opportunity to improve the nutritional status of meat products and address the changing demands of the marketplace.

6.6

Future perspective

The search for new plant sources for pharmaceutical and agrochemical industries is an ongoing process that requires continual optimization. The development of functional foods appears as an interesting trend with large market potential; the increasing demand and interest in sustainable food ingredients seem also promising. The “new” nutraceuticals of plant origin may evolve to be considered a vital aspect of dietary disease-preventive food components. Phytochemical functional foods will establish itself as a standard reference in one of the most important sectors in the functional foods market.

6.7

Conclusion

Consumers’ demand for healthier food products that prevent nutrition-related diseases and improve physical and mental well-being has led to the accelerated growth of the functional foods market. Colorful fruits and vegetables can paint a beautiful picture of health owing to the presence of phytonutrients. Phytochemicals are a natural source of healthpromoting compounds and can be a greater opportunity for developing fortified food with these functional ingredients. But extensive research and epidemiological study are required for the manufacturing of food products with bioactive compounds which can improve and maintain health.

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Chapter 7

Exploring the role of Mahua as a functional food and its future perspectives Monika Mishra1, Subhaswaraj Pattnaik1, Harvinder Singh2 and Pradeep Kumar Naik1 1

Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar,

Sambalpur, Odisha, India, 2Leaders Institute, Woolloongabba, Queensland, Australia

7.1

Introduction

7.1.1 Nontimber forest products In a variety of ways, forests and trees contribute to the livelihood of rural families. Forests are the main source of nontimber forest products (NTFPs) for well-being, domestic consumption, and earnings for people living in rural areas. Though NTFPs play a significant role in employment for rural people, they habitually put their hopes on potential gains generated from the marketing of NTFPs for poverty mitigation and maintenance of the natural resource base. Moreover, the NTFPs have directly influenced the economic status of the rural people (Kumar & Meena, 2018). Harvesting NTFPs can provide a safety net or green social security to billions of poor in the form of low-cost building materials, income, fuel, food supplements, and traditional medicines. Out of 20% of NTFPs, only 0.8% have been commercially utilized (Maithani, 1994). NTFP deeds hold expectations for integrated forms of growth that yield higher rural income and conserve biodiversity without competing with agriculture (Sharma, 1992). Madhuca indica commonly known as Mahua is an important nontimber forest product (NTFP) that is mostly found in Central and Eastern India, and it is directly linked with the tribal economic status in different ways. Although all parts of the mahua tree are used, mainly mahua seeds and flowers are collected by the tribes for self-consumption and for sale to raise money to maintain their daily life. Mahua is contributing to the economic status of the tribal society. There is a clear linkage between various socioeconomic factors that affect the level of dependency of mahua in the tribal society (Nayak & Sahoo, 2020).

7.1.2 Botanical description Taxonomical classification: Kingdom Plantae, Division Magnoliophyta, Class Magnoliopsida, Order Ericales, Family Sapotaceae, Genus Madhuca, Species Longifolia. Mahua trees are distributed in different parts of India as well as Asian countries like Philippines, Pakistan, Sri Lanka to Australia. In central India, it is found near the river banks and in the semi-evergreen forests. Mahua trees are abundantly found in different states of India like Uttar Pradesh, Madhya Pradesh, Odisha, Jharkhand, Chhattisgarh, Andhra Pradesh, Maharashtra, Bihar, West Bengal, Karnataka, Gujarat, Rajasthan. The annual production of mahua Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00030-X © 2023 Elsevier Inc. All rights reserved.

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flowers is 45,000 million tonnes. The yield of every tree varies from 83 to 320 kg every year (Pinakin et al., 2018). Patel et al. (2012) studied that mahua is a medium-sized to large deciduous tree, usually with a short and large rounded crown found all over the green forest part of India up to an altitude of 1200 m, and the height of the tree is 12
15 m. The bark is a dark brown color and is slightly cracked. The size of the leaves is 10
30 cm and is thick and leathery. According to Bisht et al. (2018), most of the leaves are pointed at the tip, clustescent glabrred near the end of branches, epileptic or elliptic-oblong 7.5
23 cm. Flowers are small and fleshy, dull or pale white and define fascicles near the end of branches. The corolla of the flowers are tubular, freshly pale, yellow aromatic and caduceus. Fruits are 2
6 cm long, fleshy, and greenish (Jha & Mazumder, 2018).

7.1.3 Microscopy of mahua The microscopic study was observed by Yadav et al. (2011) which shows that the seeds are dark brown, and the number varies from 1 to 4. The stem is cylindrical, decumbent, and branched. The fruits are fleshy, green, and ovoid type. The flowers are abundant in number and present at the ends of the branches, drooping on the pedicles. Corollas are yellowish-white whereas the tubes are fleshy. Stamens vary from 20 to 30 in number, and the anthers are hispid at the back with stiff hair. The petiole is short. The leaves are clustered at the end of the branches. Leaves and petiole are pubescent or tomentose. Leaves are coriaceous, elliptic or oblong-elliptic, shortly acuminate, and base cuneate.

7.1.4 Uses of different parts of mahua 7.1.4.1 Flowers Liquor is produced from corolla as it is a rich source of fermentable sugar. Per tonne of dry mahua flower gives 80
217 L of ethyl alcohol (Vimal & Tyagi, 1984). Anonymous (1962) stated that flowers can be used for the distillation of country liquor.

7.1.4.2 Fruits They can be either eaten raw or cooked. At the ripening stage, fruits are rich in starch which converts to sugar after 2
3 days of plucking (Anonymous, 1962).

7.1.4.3 Seeds An average mahua tree yields 100 kg of seeds per year. The seed kernels contain aspartic, glutamic, glycine, serine, proline, and alanine. The seed coat contains 2% of quercetin and dihydroquercetin with some tannins and thioglucosides (Banerji & Mitra, 1996).

7.1.4.4 Mahua oil It can be used washing soaps, manufacturing candles, lubricants, bathing oil, production of steric acid, etc. (Douli et al. 1968a, 1968b).

7.1.4.5 Cake Mahua-deoiled cake contains carbohydrates, proteins, and ash. Due to its toxic effects, it cannot be used as cattle feed, but when it is detoxified it can be given to cattle (Mitra & Misra, 1967). They are also used as wormicide and fish poison but do not decompose and nitrify in soil (Misra & Mitra, 1968). The consumption of mahua seed cake causes hemolysis due to the presence of saponin (Birk, 1969). Saponin was removed by the use of different solvents like water, ethanol, isopropanol. Detoxification was done with minimal loss of protein and carbohydrates (Sen Gupta, 1980).

7.2

Traditional uses

For many years, there are a lot of traditional uses for each and every part of the mahua tree. Mahua plays a vital role in the treatment and prevention of many diseases. Different parts of the tree are used to treat different types of diseases. According to Patel et al. (2012), in Ayurveda mahua flower is used as a cooling agent, acute chronic tonsillitis, pharyngitis, ulcer, and bronchitis. The flowers are used as a tonic, analgesic, and diuretic. Jha and Mazumder (2018)

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clearly mentioned that the flowers have shown some activity against swelling, itching, and fractures. According to Bisht et al. (2018), bark juice is orally taken for the treatment of diarrhea, and it is also used in the treatment of chronic tonsillitis, leprosy, and fever. Sometimes the bark is used to cure rheumatism, bronchitis, and diabetes. Patel et al. (2012) cited that besides all these properties bark is a good therapy used to cure bleeding and spongy gum. Verma et al. (2014) discussed that the leaves of mahua play a significant role in the treatment of chronic bronchitis and Cushing’s disease. The leaves also show several activities like wound healing, hepatoprotective, antioxidant, antimicrobial, verminosis, gastropathy, dipsia, bronchitis, dermatopathy, rheumatism, cephalgia, and hemorrhoids. Pinakin et al. (2018) quote that the fat gained from mahua seeds has many therapeutic claims such as emuluscent property, used in skin infection, rheumatism, headache, laxative, piles, and sometimes as galactagogue. According to Bisht et al. (2018), traditionally mahua is utilized in food production. G G G

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Due to the presence of a high amount of sugar, mahua is used as a sweetener in the preparation of many food items. Sundried flowers are boiled with tamarind, and sal seeds and are consumed by the poor tribal people. Flowers that are left after fermentation and distillation are used as cattle fed to improve the health status of the cattle. Fermented flowers are used for the preparation of country liquor having an alcohol amount of 30%
40%. The seed oil is used as cooking oil.

7.3

Nutritional and phytochemical profiling

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Sugars: The sugar contents of mahua flowers are dextrose, levulose, maltose, sucrose, etc. According to Fowler et al. (1920), when the flower is ready to fall it has high sugar content. In the flower, the amount of levulose is greater than dextrose. In the storage period, the amount of sucrose decreases, but in the shedding stage of corolla the amount of sucrose is high. Sutaria & Magar (1955a, 1955b, 1955c) have done a paper chromatographic experiment of unhydrolyzed mahua extract which revealed the presence of maltose, sucrose, arabinose, fructose, and rhamnose, but the hydrolyzed extract revealed the presence of galacturonic acid. Polysaccharide: The water-soluble polysaccharides were extracted and gel-filtered to obtain a homogeneous fraction. D-galactose, L-arabinose, L-rhamnose, D-xylose, and D-glutaric acid were found in the first fractionation. The second fractionation was done by Sarkar & Chaterjee (1984), and they reported the presence of D-glucose, L-arabinose, and D-glucuronic acid. Protein and amino acid: The nitrogen content of the underdeveloped flower is higher than the fully developed flower, that is, 0.65%
1.1%. The protein content of the flower fluctuates from 4.4% to 7% (Belavady & Balasubramanian, 1959; Jayasree et al., 1998). Sutaria & Magar (1955a) reported the presence of eleven amino acids like lysine, arginine, aspartic acid, glutamic acid, threonine, valine, tryptophan, phenylalanine, isoleucine, leucine, and proline. Jayasree et al. (1998) discussed that the protein content of mahua flower was superior when compared with groundnut protein. Fat and fatty acid: The fat content of mahua flower is very low. As reported by Jayasree et al. (1998) and Sutaria & Magar (1955a, 1955b, 1955c) the fat content differs from 0.09% to 1.3%. Sutaria & Magar (1955a) stated that mahua flowers contain linoleic, oleic, palmitic, and stearic acid. Vitamins: Different researchers have evaluated different vitamin contents of fresh mahua flowers. Ascorbic acid content varies from 36.99 to 7 mg. Thiamine content is 140.3 μg (Sutaria & Magar, 1955b). Riboflavin is found to be 878 μg (Belavady & Balasubramanian, 1959). Niacin was found to be 5.2 mg (Belavady & Balasubramanian, 1959), and folic acid content is 214 μg (Sutaria & Magar, 1955b). Minerals: Different minerals have been reported in mahua flowers such as Ca, P, Fe, Mg, Na, and K. Out of these, Na is 25.27 mg/100 g, K is 1.2 mg/100 g, and Zn is 0.926 mg/100 g (Sutaria & Magar, 1955a). Enzymes: Fowler et al. (1920) detected enzymes in different developmental stages of the flowers such as amylase, maltase, invertase, catalase, and oxidase. Saponins: It can be termed as an anti-nutritional factor, but it has both positive and negative consequences. In the positive aspects, it helps in lowering the serum cholesterol level in humans. On the negative side, it hinders the productive performance of nonruminants. They also show the toxic effect that leads to death in the rats (Goodhart & Sahil, 1980; Mulky & Gandhi, 1977).

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TABLE 7.1 Phytochemical screening of mahua flower extract. Sl. no

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Ethanolic extract

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Proteins

Biuret test

1ve

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Carbohydrates

Molisch’s test

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1ve

Mishra & Usha (2019), Patel et al. (2019)

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Ninhydrin test

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Sudan test

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Tannic acid test

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Alkaline reagent test

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Lactic acid test

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7.3.2 Comparative nutritional profile As per the report of Gopalan et al. (2004) and Jayasree et al. (1998), the amount of protein content of dry mahua flower was 6.67% which is much higher when compared with other common fruits like apple, banana, mango, and raisins. Likewise, it has a lower amount of fat, that is, 0.09%. Carbohydrate content of fresh mahua flower is 68%. Calcium content is found to be 139%. From the different reviewers, it can be said that fresh and dry mahua flowers are having all the essential nutritional compounds.

7.3.3 Effect of geographical distribution on the flower composition According to Elworthy (1887), there is an effect of climatic changes on the sugar composition of mahua flowers. The flowers were collected from Hyderabad, Jabalpur, Gujarat, and Mirzapur. The cane sugar % was found to be maximum, that is, 17.1% in the flower collected from Hyderabad. The inverted sugar % of the flowers collected from Gujarat was 45.3%. Dextrose % of the flowers collected from Mirzapur was 43.6%, whereas the total sugar % of the flowers from Hyderabad was 57.1%. According to Roy & Rao (1959), flowers collected from the Mandla district of Madhya Pradesh were found to contain reducing sugar (57.3%), nonreducing sugar (9.4%), starch (3.6%), protein (6.8%), ash (4.5%), and other components (18.4%), respectively. Phytochemical screening of mahua plant extract was studied by different researchers who have studied the presence or absence of different phytoconstituents. The detailed study of phytochemical screening of mahua flower extract is depicted in Table 7.1. Phytochemical profiling of different parts of mahua plant was done. In Table 7.2, reviewers have found the presence of various chemical constituents which were also responsible for different biological activities. GC-Ms analysis of seed, leaf, bark, and flower was done by Ranjana et al. (2018). Certain important phytochemicals were found such as hexylcyclohexane, octylcyclohexane, E-14-hexdecenal, pentadecan-8-one, 8-octadecanone, dibutyl phthalate. Annalakshmi et al. (2013) did the GC-Ms and HPTLC analysis of leaf extract of mahua, and they found that there were several known and unknown bioactive compounds present which were mainly responsible for the treatment of different diseases.

7.4

Pharmaceutical uses and pharmacological importance

7.4.1 Industrial uses Patel et al. (2019) observed a lot of traditional utilization of mahua tree. Mahua can be used industrially which can upgrade the economic status of the country. Flowers that are used for the preparation of country liquor can be fermented to obtain spirits, ethanol, acetone, lactic acids, vinegar, etc. Soon after the alcohol production, the remaining pulp and other ingredients are used for the production of biofertilizer. According to Mishra & Poonia (2019), mahua biofertilizer can be said as a good fertilizer as there is a presence of nitrogen, phosphorus, and potassium along with calcium. After the removal of oil from the seed, the seed cake may be used for cattle feed (Ramadan et al., 2016). Many researchers

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TABLE 7.2 Phytochemical profiling of mahua. Sl. no

Plant part

Chemical constituents

References

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Leaves

D-glucoside,

stigmasterol, β-carotene, xanthophylls, erythrodiol, palmitic acid, myricetin, 3-O-arabinoside, 3-O-L-rhamnoside, quercetin, 3galactoside; 3β-caproxy and 3β-palmitoxy-olean-12-en-28-ol, oleanolic acid, β-sitosterol, 3-O-β-D-glucoside, 3β-caproxyolcan-12-en-28-ol, β-carotene, n-octacosanol, sitosterol, quercetin, β-sitosterol-β-D-glucoside, n-hexacosanol. 3-O-arabinoside, xanthophylls, 3-O-L-rhamnoside, n-octacosanol, 3β-caproxyolcan-12-en-28-ol, β-sitosterol and 3-O-β-D-glucosideand sitosterol

Verma et al. (2014), Khare et al. (2018), Mishra & Pradhan (2013)

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Myristic, palmitic, and stearic acids, α-alanine, aspartic acid, cystine, glycine, isoleucine and leucine, lysine, methionine, proline, serine, threonine, myricetin, quercetin, Mi-saponin A, saponin B, arachidic, linolenic, oleic, quercetin

Verma et al. (2014), Khare et al. (2018), Mishra & Pradhan (2013)

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Fruits

n-Hexacosanol quercetin and dihydroquercetin, β-sitosterol and its 3β-D-glucoside, α- and β-amyrin acetates

Verma et al. (2014), Mishra & Pradhan (2013)

04

Flower

Vitamins A and C

Verma et al. (2014)

05

Bark

α- and β-Amyrin acetates, 3β-monocaprylic ester of erythrodiol and 3β-capryloxy oleanolic acid, ethylcinnamate, α-terpineol, and sesquiterpene alcohol

Khare et al. (2018), Mishra & Pradhan (2013)

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Nut-shell

n-Hexacosanol quercetin and dihydroquercetin, β-sitosterol and its 3β-D-glucoside

Mishra & Pradhan (2013)

and reviews suggest that mahua flowers and mahua-deoiled cake can be used as cattle feed to provide proper nutrition to the cattle at an affordable rate. A cream formulated from mahua oil is nontoxic, less expensive, and biodegradable. So it could be used as an effective emulsifier for formulating cream (Mahajan et al., 2017). Because of lots of uses, mahua is giving employment and opportunity to the tribal people. They collect every part of the mahua tree and sell it in the local market, and in return they get money. But the amount is not sufficient for survival. Some government schemes should be provided for all such people who are completely dependent on mahua for their livelihood.

7.4.2 Biodiesel Ghadge & Raheman (2006) optimized the biodiesel production using a surface methodology. The surface methodology process gave a yield of 98% mahua biodiesel that satisfies the properties of both American and European standards of biodiesel. Puhan et al. (2005) stated that mahua can be used as a source of renewable energy in India. They observed that in mahua oil methyl ester has lower emission compared with other esters. Raheman & Ghadge (2007) observed that the blend of mahua oil with high-speed diesel can be used as an alternative fuel that causes low pollution. Due to the increase in the price of oil, mahua oil can be used as a lubricant for maintenance purposes. Apart from the environmental benefits, Suhane et al. (2013) experimentally revealed that the addition of mahua oil with 90T oil showed good wear-riding traits. Goud et al. (2006) developed epoxides by using hydrogen peroxides using mahua oil. (Kapilan & Reddy, 2008) concluded that an engine ran effortlessly with methyl ester of mahua oil and B20, but there was a heavy emission when only mahua oil was used as a fuel.

7.4.3 Biological activity G

Therapeutic property: Mishra & Usha (2019) reported that from ages mahua has a lot of therapeutic potentials. Different parts of the plants have different therapeutic and pharmacological uses. Decoction of mahua flowers is used to quench the thirst. It is used as a cooling agent, and it is also used to treat tonsillitis, bronchitis, and inflammation. It has astringent properties so sometimes it is used in the treatment of piles, diarrhea, and colitis. The flowers are used to treat eczema, skin diseases, eye diseases, and arrest bleeding. It is also used to treat burning sensations, heart diseases, and ear complaints. The flower extract is used to increase the quantity of seminal fluids.

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Anti-inflammatory activity: The inflammation is caused due to the release of several chemicals like histamine and serotonin from the damaged cell. Inflammation is a self-protective mechanism of the body (Yadav et al., 2012). The vital mechanism of the anti-inflammatory drugs is reflected due to the inhibition of PG synthesis at the site of injury. According to Gaikwad et al. (2009), the potential of anti-inflammatory drugs is measured by their effectiveness to prevent COX. The anti-inflammatory activity is directly proportional to the inhibition of COX. The aerial parts of the M. indica are used in the action of inflammation. The aerial part was extracted by using Soxhlet apparatus. The extract was concentrated under vacuum-sounding apparatus for 30 minutes. The result was satisfactory when the solution was given to male Wistar rat. Analgesic activity: Pain killer can also be used for the word analgesic. Analgesic are the group of drugs that are used to give relief from pain without disturbing the consciousness. Madhuca longifolia exhibited the analgesic effect from both methanolic and ethanolic extracts. The activity was assessed on acetic acid writhing. Six animals were orally given the methanolic extract of M. longifolia. The number of writhing during the subsequent 30 minutes was witnessed after acetic acid injection (Verma et al., 2014). Anti-analgesia is expressed as the decrease in the number of abdominal constrictions between control animal and mice pretreated with the extract. The analgesic effect was also screened through the tail flick, hot plate, and chemical graded doses of both aqueous and alcoholic extract of M. longifolia (4.0
64.0 mg/kg, i.m. for 3 days) produced a dose-dependent analgesic effect in all the three nociceptive methods carried out either in rats or mice (Patel et al., 2012). Antipyretic activity: Antipyretic can be defined as a term that works against fever. The prostaglandin is responsible for fever, and the antipyretics causes the hypothalamus to override the prostaglandins which results in a reduction in fever (Khare et al., 2018). In animal experiments, the mahua extract was found to reduce fever (Patel et al., 2019). Antihyperglycemic activity: The important hypoglycemic effects of Mahua bark in diabetic rats specify that this effect can be facilitated by stimulation of glucose consumption by peripheral tissues. The consequences of the study specified the ethanolic extract of Mahua bark to have a hypoglycemic effect on STZ-induced diabetic rats. In all groups except for glibenclamide, at 30 minutes of initiating the glucose tolerance test, blood glucose concentration was higher than at zero time but decreased significantly from 30 to 120 minutes (Yadav et al., 2012). Methanolic extracts were improving glucose operation; thus the blood glucose level was expressively reduced in glucose-loaded rats. Methanolic extract of Madhuca has considerably reduced the serum glucose level in streptozotocin and STZNIC-induced diabetic rats. According to Patel et al. (2012), the crude methanolic extract of Mahua leaves confirmed dose-dependent reductions in serum glucose level succeeding administration in glucose-loaded mice. The serum glucose levels were found to be considerably reduced at doses of 100, 250, and 500 mg extract per kg body weight. Antiulcer activity: Ulcer is considered as a common complaint of gastrointestinal tract (Patel et al., 2019). Due to the imbalance in the defensive and attacking factor of GIT, there is a result in ulcer. The alcoholic extract of mahua flower is used to study the antiulcer activity. The experiment was conducted with the help of Wistar rats by pylorus ligation method, and the standard drug used was ranitidine. The alcoholic extract of mahua and the standard drug were given simultaneously 2 days before pylorus ligation, and the gastric juice was measured. The increase in the amount of gastric juice could be due to the inhibition of histamine which exaggerates acid release (Mohod & Bodhankar, 2013). Antioxidant activity: Free radicals are the cause of various diseases such as aging, cancer, heart disease, etc. Generally, antioxidants protect our cells from free radicals (Verma et al., 2013). Higher the phenolic content, more the antioxidant activity because the phenol groups donate the hydrogen atom. The antioxidant power of a drug depends upon two mechanisms, first to prevent oxidation by oxidizing itself or second by generating a layer of protection over the material. The methanolic bark extract of mahua showed antioxidant activity, that is, capable of donating hydrogen atoms (Khare et al., 2018). Antifertility activity: Some studies revealed that mahua has antifertility activity. The term antifertility means a substance that inhibits the ability to produce offspring (Verma et al., 2014). Male and female mice were taken for the study, and the study revealed that the percentage of fertility in the case of males and females has decreased in atropine-induced mice. It decreases the sperm count and reduced the spermatozoa density (Patel et al., 2012). Dermatological activity: As the condition of the environment is changing, there are a lot of skin-related problems people are facing in their daily life (Verma et al., 2014). There are several marketed cosmetic products which sometimes cause skin irritation, itching, etc. Mahua seed oil could be used as an alternative for the treatment of various skin-related problems. Traditionally, the tribes of many states use the mahua oil as a lotion (Patel et al., 2019). Hepatoprotective activity: To study the hepatoprotective activity, the albino rats were injected with CCl4 or carbon tetrachloride. Mahua methanolic extract has shown hepatoprotective activity (dose-dependent 300 mg/kg body weight) (Patel et al., 2019). The methanolic extract was proved to be effective against lowering the serum level of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphate (ALKP), and total bilirubin. It has also increased the level of total proteins and albumin (Yadav et al., 2012).

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Antibacterial activity: According to Khare et al. (2018), the flower extract shows antibacterial activity against Escherichia coli and has the ability to resist rice pest disease. By disk diffusion method, the methanolic extract of dried bark shows a significant effect against Bacillus subtilis, Staphylococcus aureus, Staphylococcus, E. coli. Patel et al. (2019) also informed that the ethanolic extract showed to be having good antibacterial activity. For the treatment of diarrhea, the methanolic extract of mahua flowers showed effective results against S. aureus, Salmonella typhi, Bacillus pumilus, Proteus vulgaris, E. coli, B. subtilis, Candida albicans, Pseudomonas aeruginosa, and Micrococcus luteus. The methanolic extract of mahua was used to study the antimicrobial study by disk diffusion method in which three fungal strains and three bacterial strains were taken, that is, E. coli, Pseudomonas, S. aureus, Aspergillus niger, Penicillium spp., Scytalidium spp. All of them showed positive results (Patel et al., 2012). Antiepileptic activity: Verma et al. (2014) and Patel et al. (2012) revealed that M. longifolia retains significant antiepileptic activity as it amplified the onset time of seizure and reduces the period of seizures. The methanolic extract of mahua has shown antiepileptic activity. The mouse was induced with pentylenetetrazol-induced seizures (PTZ), and diazepam has been used as a standard drug. The researchers have successfully studied the involvement of benzodiazepine and opioid receptor. Antinociceptive and antidiarrheal activity: Khare et al. (2018) and Patel et al. (2019) studied that diclofenac sodium was used as a standard drug for the comparison of the response. The mice showed antinociceptive and antidiarrheal activity in the alcoholic extract of the bark. Mahua bark extract showed to be effective in monitoring the writhing reflex induced by acetic acid at a dose of 250 and 500 mg/kg of body weight by oral route. Antihelminthic activity: Mahua plant has numerous phytochemical constituents in it. It contains protein, starch, phenols, flavonoids, tannins, terpenoids, and many more (Khare et al., 2018). The study was done by in vivo method, and Pheretima posthuma (adult Indian earthworm) was used in the study. Metronidazole was used as a standard drug. The alcoholic and hydroalcoholic extract has shown antihelminthic activity due to the presence of tannins and other phytochemical constituents (Patel et al., 2019). Immunomodulatory activity: The alcoholic extract of mahua showed significantly good immunomodulatory activity. Cyclophosphamide-induced myelosuppression in mice was tested. The mahua extract improved the DTH response and antibody titer value and also triggered the renovation of total leukocyte count (TLC) and differential leukocyte count (DLC) (Khare et al., 2018). Larvicidal and ovicidal activity: Not only the mahua oil but also the mahua seed cake have shown larvicidal and ovicidal activity against Meloidogyne incognita, and they were also effective against larval growth from the egg sacs of cyst nematodes (Khare et al., 2018). Antidiabetic activity: Yadav et al. (2012) reviewed that the alcoholic extract of mahua leaf and bark was used for the study of the antidiabetic property. The albino Wistar rat was induced by streptozotocin, and after 30 days, it was compared with insulin-treated rat. The experiment showed a decrease in blood sugar level. Spasmolytic activity: Saponin is responsible for the spasmolytic activity in the alcoholic extract of mahua. The saponins extracted from mahua leaves influenced a major spasmolytic activity. The saponins existing in the leaves, and seeds of M. longifolia possessed spasmolytic property on isolated guinea pig ileum (Patel et al., 2019). Spermicidal activity: Khare et al. (2018) studied the steroid and triterpenoid saponins existing in M. longifolia seeds which influenced clear spermicidal activity. Insecticidal and pesticidal activity: Mahua cake influenced an important insecticidal and pesticidal action against phytonematode. Mahua has shown pesticidal activity against Tetranychus urticae (Khare et al., 2018). Wound-healing activity: According to Yadav et al. (2012) and Jha and Mazumder (2018), methanolic extract of mahua bark was used to treat the wound of the mice. Betadine was used as a standard drug. Mahua extract-treated animals showed a substantial decrease in wound area and period of epithelization. Mahua extract showed a faster wound-healing activity than the standard drug. Nephroprotective activity: Against acetaminophen-induced nephrotoxicity, the alcoholic extract of mahua possesses nephroprotective activity. There was an increase in the levels of serum urea, hemoglobin (Hb), total leukocyte count, creatinine, filled cell volume, DLC, mean corpuscular volume, and upraised body weight sideways with condensed levels of neutrophils, mean corpuscular Hb content, mean corpuscular hematocrit, granulocytes, uric acid, and platelet concentrations (Khare et al., 2018; Patel et al., 2019). Neuropharmacological activity: For the study of neuropharmacological activity, tramadol hydrochloride, diazepam, and chlorpromazine were used as a standard drug against Swiss albino male mice. The activity was measured via phenobarbitone sodium-induced sleep and their antagonistic regaining righting effects by mahua extract. Actophotometer was used for spontaneous motor activity. There was a decrease in sleeping time and a reduction in spontaneous motor activity with the use of actophotometer (Patel et al., 2019).

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Rheumatic arthritis: Patel et al. (2019) considered that arthritis can also be called as an inflammatory disorder which is usually caused by a lack of synovial fluid. Lack of synovial fluid can cause loss of lubrication which in turn may cause severe pain in the joints. Mahua oil was used for the action of rheumatic arthritis. Anticancer activity: According to Yadav et al. (2012), the cytotoxic activity against Ehrlich ascites carcinoma cell lines using different in vitro cytotoxic assays at 200 μg/mL was shown by the alcoholic extract of mahua leaves. Patel et al. (2012) found that both extracts revealed important cytotoxic action, but greater cytotoxic activity was found in ethanol extract. Toxicity: A combination of saponins from M. longifolia seed did not disclose any cholinergic action, though it was shaped at an advanced concentration (Patel et al., 2019). The saponin is tremendously toxic after it has been induced parentally, and LD50 by the IV route was found to be 50
70 times higher than the oral route. In the root of M. indica, the extreme quantity of phenol was detected, that is, 46.0 mg/g dry weight. These complexes showed an important part in the precursor of toxic constituents. Patel et al. (2012) also described having toxic biochemical such as aflatoxine in M. indica seed oil. Rajgor et al. (1986) took 24 albino rats and fed them with ordinary boiled and pressure-cooked mahua flowers which showed that there was no change in food intake. But the organ weight was significantly lower in the ordinary-fed group compared to the pressure cooker fed. Bora & Singh (1994) observed the feeding of dried mahua as a substitute for maize. They concluded that dried flowers can be incorporated into the gill ration as a substitute for maize. Kotwal (2000) studied the feeding diet of processed (without saponin) and unprocessed (with saponin) mahua syrup on the biochemical and histopathological status of albino rats. According to the findings, the unprocessed syrup showed some desirable changes in the parameters, but the processed syrup does not show any changes.

7.5

Mahua as a functional food

7.5.1 Processing of flowers 7.5.1.1 Collection of flowers The primary collectors or usually the tribal families collect the fresh flowers in the early morning (Mishra & Poonia, 2019). A bamboo stick is used to pluck the flowers from the trees. Before the collection of flowers, the ground is cleaned, and the floor is cleared from leaves and grass (Bakhara et al., 2016; Chandel et al., 2018). The time required for the collection of flowers may vary because the mahua flowers are collected manually from the ground.

7.5.1.2 Preprocessing The flowers are preprocessed to increase their shelf life (Mishra & Poonia, 2019). The preprocessing methods include washing, stamen removal, and blanching with preservatives. Sometimes, the stamen is removed manually or mechanically. To reduce the rate of spoilage, the flowers are free from moisture which includes shed drying, sun drying, and tray drying (Kumari et al., 2018).

7.5.1.3 Drying Before drying pretreatment was given to the mahua flower with 4.1 minutes of blanching, 1285 ppm KMS, and 0.77% citric acid concentration (Pinakin et al., 2018). The tribals used to dry the flowers by spreading them in a clean dry place for 3
4 days under the sun (Chandel et al., 2018). After the flowers are completely dried, the stamen is detached from the flowers manually. Stamen removal is necessary because it gives a bitter taste which is mostly disliked by the customers (Bakhara et al., 2016). Storage: The dried flowers are stored in dark rooms packed in gunny bags. But the flowers absorb moisture easily from the earthen floor or roof which is more prone to bacterial and fungal spoilage (Bakhara et al., 2016; Mishra & Poonia, 2019).

7.5.1.4 Postharvest spoilage of flowers Due to the lack of storage facility, about 30% of the flowers get spoiled. Fresh flowers are highly nutritious and have high moisture content which leads to bacterial and fungal spoilage (Behera & Swain, 2013). The bacteria that involve in the spoilage are Bacillus, Micrococcus, Siderococcus, Nocardia, and Pseudomonas, and the fungi that are involved in the spoilage include A. niger, Aspergillus flavus, Penicillium, and Rhizopus (Mishra & Poonia, 2019). Due to bacterial and fungal spoilage, the amount of sugar and ascorbic acid decreases. Certain insects and larvae are also involved

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in the spoilage of the flowers which belong to the family of Noctuidae, Anthocoridae, Cucujidae, Bostrychidae, Tephritidae, and Formicidae (Behera & Swain, 2013).

7.5.1.5 Methods of preservation Since mahua is a multipurpose tree having high moisture content and is rich in nutritional compounds, it is more likely to have spoilage. One of the spoilage is caused by Fusarium solani (Verma et al., 2014). The spoilage of the flowers is mainly due to its high moisture content, lack of storage facilities, and inadequate knowledge of further processing. G

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Moisture content of the flowers can be reduced by the use of modern machinery such as oven drying, hot air drying, tray drying, solar drying, cabinet drying, etc. Reduction in moisture content is an unfavorable environment for the growth of microorganisms. Humectants are used to keep moisture away from the flowers. To check the spoilage, the flowers are treated with UV and certain antibiotics. The flowers should be kept in a controlled atmosphere. Government warehouses should be built-in tribal areas so that the poor can avail the facilities and can keep their raw materials in a good condition. Das et al. (2010) found that the proper use of liquid nitrogen to powder the flowers can lead to a decrease in spoilage, and deep-freeze flowers are found to be less prone to microbial growth.

7.5.2 Value-added food products Sugar syrup is the oldest food product that is prepared by researchers. Sugar syrup was prepared by Abhyankar & Narayana (1942) in which they found that there is 75%
87% of reducing sugar present in the flowers. In the process of preparing the sugar syrup, they extracted 90% of the sugar with hot water. The syrup was clarified with the addition of slacked lime followed by superphosphate. The syrup was passed through activated charcoal and evaporated to a consistency of 70
75 Brix. Shrivastava et al. (1970) extracted the sugar syrup in two different ways. The first method includes the extraction process directly from fresh water, and the second method was the extraction done from the previous extraction. The second method was more effective and economical that takes less time. Chand & Mahaptra (1983) prepared the sugar syrup from dried mahua flowers. The syrup was first clarified using lime, and pH was maintained by adding citric acid solution. The addition of 50 ppm of metabisulphite to the syrup has increased the shelf life. Since mahua is rich in sugar, it has the potential for making good-quality fermented products. Soni & Dey (2013) observed that the fermented mahua when added with guava improved the flavor of the wine. The addition of guava has increased the antioxidant activity. Singh et al. (2013) investigated that mahua flower juice has a maximum yield of ethanol at 25 C with a pH 4.5 after 14 days of fermentation. The alcoholic beverage developed by Singh et al. (2013) has the characteristic flavor and aroma of mahua flowers. Dushing & Surve (2019) developed wine from mahua flower extract and pomegranate fruit juice. The result showed that S. cerevisiae (NCIM-3215) produced better results after 7 days of fermentation, and the wine was acceptable at a proportion of (20:80). Yadav et al. (2009) standardized a pretreatment condition for mahua wine. They have optimized that a dip of KMS for 10 minutes leads to a decrease in microbial population, and heating at 100 C and 500 ppm KMS sulphitation are required for self-stable mahua flower juice. Patel et al. (2016) introduced an antioxidant-rich beverage from mahua flower and amla. Several blends were prepared, but a blend of 40 Brix mahua having 50% amla was best of all. Singh et al. (2018) developed cupcakes from mahua flower syrup which has 100% replaced sugar, and the product was also acceptable by the panelist. Likewise, Ravat & Dixit (2017) developed gluten-free biscuits incorporated with mahua powder as natural sugar. The gluten-free biscuits had a shelf life of 60 days. According to Pinakin et al. (2018), several researchers have developed value-added products from mahua flowers such as puree and sauces, juice, mahua jam, jelly, marmalade, mahua bar, mahua candy, mahua toffee, mahua cake, mahua squash, mahua ladoo, mahua RTS. The seed oil can be used as food supplements having proper health benefits (Yadav et al., 2011). The chemical composition of mahua oil is the same as other edible oils. Rukmini (1990) compared mahua oil with groundnut oil which showed maximum similarities in the nutritional properties. Other uses: G

Mahua yeast was extracted from the alcohol factory that contains some vitamins in it. The yeast extracted from mahua was far better than distillery yeast (Daver & Ahmed, 1944).

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A new type of yeast Zygosaccharomyces mahwae was isolated by Lender (1992). Organic manure was prepared by adding mahua to the waste organic matter (Fowler & Gilber, 1930). The role of molasses, mahua flower, and mannitol in nitrogen fixation was reported by Patel & Kibe (1951). Hasan et al. (1928) reported that when the cattle were given mahua there was an increase in the health status and quality of the milk. A new agar medium for the fungal culture was developed by Saha & Singh (1991).

7.5.3 Health benefits of mahua According to Mishra & Pradhan (2013), there are several health benefits of mahua. It can be used to clear chronic bronchitis problems and can be used as a remedy for cough and tonsillitis. For testis inflammation, mahua leaves are used. A decoction of bark in water is taken orally for the relief of rheumatism. Sometimes oil is also used in the affected area to get relief. It can be used for the treatment of diabetes. Mahua seed oil has laxative properties so it can be used for constipation and piles. Mahua leaves are effective against eczema and bleeding gums. Mahua leaf ash is used to cure itching. The feeding mothers consume mahua flowers to increase lactation.

7.6

Current trends and future perspectives

After the study of all the information available, we can conclude that the mahua tree is a boon to our environment. Nature has gifted us with this tree which is a rich source of natural sugar and different phytochemical constituents. Mahua is a perfectly balanced and fully synchronized plant that gives traditional, pharmacological, and economic benefits. Besides its utilization as food, fodder, and fuel, it has antibacterial, anticancer, hepatoprotective, antihyperglycemic, analgesic activities, etc., with certain health importance which helps to treat bronchitis, cough, piles, eczema, bleeding gums. It has also certain important chemical constituents which include flavonoids, glycosides, alkaloids, tannins, and terpenoids. For a long time, mahua is being used in the production of country liquor, but if the tribes are trained with available food technologies, they can market the mahua food products which in turn can raise their standard of living. To increase the commercial utilization of the flowers, there should be an implementation of advanced technologies by which they can make different value-added food products. Government and NGOs should aware people of the utilization of mahua flowers by conducting different entrepreneurship programs by which there will be an enhancement of income sources among the poor people.

Acknowledgment We would like to acknowledge OHEPEE, Govt. of Odisha, for providing financial support.

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1070. Lender, A. (1992). Indian Mahwa. Schweizerische Apotheker-Zeitung, 60, 713
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97. Maithani, G. P. (1994). Management Perspectives of Minor Forest Produce. MFP News. Mishra, A., & Poonia, A. (2019). Mahua (Madhuca longifolia) flowers: review on processing and biological properties. Nutrition and Food Science, 49(6), 1153
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2207. Patel, M., Jaiswal, S., Naik, B., Naik, M., Saxena, D., & Naik, S. N. (2016). Development of antioxidant rich beverage from Mahua (Madhuca indica) and Amla (Emblica officinalis). Journal of Scientific and Industrial Research, 75(1), 35
39. Patel, P. K., Janghel, V., Chandel, S. S., & Sahu, J. (2019). Madhuca indica (Mahua)-pharmaceutical, nutraceutical and economical importance for tribal people of Chhattisgarh state. International Journal of Pharmaceutical and Phytopharmacological Research, 9(3), 16
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326. Patel, P. K., Prajapati, N. K., & Dubey, B. K. (2012). Madhuca indica: A review of its medicinal property. International Journal of Pharmaceutical Sciences and Research, 3(5), 1285
1293. Pinakin, D. J., Kumar, V., Kumar, A., Gat, Y., Suri, S., & Sharma, K. (2018). Mahua: A boon for pharmacy and food industry. Current Research in Nutrition and Food Science, 6(2), 371
381. Available from https://doi.org/10.12944/CRNFSJ.6.2.12. Puhan, S., Vedaraman, N., Ram, B. V., Sankarnarayanan, G., & Jeychandran, K. (2005). Mahua oil (Madhuca Indica seed oil) methyl ester as biodiesel-preparation and emission characterstics. Biomass and Bioenergy, 28(1), 87
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2573. Rajgor, N., Gujral, S., Gopaldas, T., et al. (1986). Preliminary trials to investigate the feasibility of using Mahuda flowers (Mahuda indica or Bassia latifolia) as energy source in an experimental model. Indian Journal of Nutrition and Dietics, 23(1). Ramadan, M. F., Mohdaly, A. A. A., Assiri, A., Tadrod, M., & Niemeyer, B. (2016). Functional characteristics, nutritional value and industrial applications of Madhuca longifolia seeds: an overview. Journal of Food Science and Technology, 53, 2149
2157. Ranjana, K., Amit, P., & Kumar, S. A. (2018). GC-MS analysis of methanol extract from bark, flower, leaf and seed of Madhuca indica JF gmel. Journal of Pharmacognosy and Phytochemistry, 7(2), 3259
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Chapter 8

Functional beverages: an emerging trend in beverage world Namrata A. Giri1, Bhagwan K. Sakhale2 and Nilesh Prakash Nirmal3 1

ICAR-National Research Centre on Pomegranate, Solapur, Maharashtra, India, 2Department of Chemical Technology, Dr. Babasaheb Ambedkar

Marathwada University, Aurangabad, Maharashtra, India, 3Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, Thailand

8.1

Introduction

Fruits and vegetables are nutritious ingredients in the beverage as it provides vitamins, minerals, and antioxidants. Fruit juices and beverages are one of the best value-added, processed products which are convenient to use and also fulfill the daily requirements of fruits and vegetables in diet. New product introductions in the health drink and fruit juice categories were found to reach over 700 new offerings in the year 2003, up 40% over 2002 (Anon, 2003). The functional beverage sector has been reported to be the fastest-growing segment (Roberts, 2009). Fruit-based functional beverages enriched with nutraceuticals with refreshing flavors and tastes are being preferred over aerated drinks by healthconscious consumers, in particular. The addition of nutraceuticals in the drink provides great scope to offer health benefits such as improved antioxidant profile and protection against various chronic metabolic diseases. Nutraceutical beverages are the fastest annual increasing market in the world with annual growth rate of 13.6% between 2002 and 2007 (Heckman et al., 2010). Nowadays, consumer is having great demand for this kind of beverage due to the taste and acceptability and health-promoting ingredients available in the drink. With these requirements, it is necessary to formulate beverage of high quality with good taste, which is needed for health promotion and disease prevention. The establishment of functional beverages containing health-promoting ingredients is a top priority of modern technology. The functional beverage contains different health-promoting ingredients like ascorbic acid, tocopherol, β-carotene, etc., and offers benefits of dietary phytochemicals (Schnitter, 2001). At present, the functional beverage is the most significant category of new product development (NPD) in recent years (Sorenson & Bogue, 2005) as consumer behavior is 13.79% influenced by healthy factors (Jaisam & Utama-ang, 2008). The main components of soft drinks are water, acidulants, flavorings, food colors, preservatives, and other functional ingredients (Ashurst, 1998). The use of natural ingredients makes the drinks more acceptable (Sharma, 2005). The product acceptability is different among consumers as taste blindness occurs in 22.2% of women and 25.9% of men (Meyer, 2007). However, there is also difference in beverage consumption between men and women. So the functional beverages need to be promoted as convenient, nutritious, and tasty formulations with specific health benefits for the target population (Sharma, 2005).

8.1.1 Need for functional beverage The present market has great potential for new product development, among that the functional beverage category is one of the most significant (Sorenson & Bogue, 2005) because the consumer behavior is 13.79% influenced by healthy factor (Jaisam & Utama-ang, 2008). The market for functional drinks is expected to expand further because the change in lifestyle leads to lifestyle diseases such as diabetes, hypertension, etc. (McCoy, 2005). The functional nutrients are present in functional drinks such as ascorbic acid, tocopherol, β-carotene, etc., and offer benefits of dietary phytochemicals (Schnitter, 2001). Consumption of food containing functional ingredients would help to deliver good health and wellness to consumers (Sharma, 2005). Chen et al. (2009) reported that the functional beverages have no cytotoxicity Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00002-5 © 2023 Elsevier Inc. All rights reserved.

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and mutagenicity effects. This trend, however, is changing, as interest in immunity, cancer, and heart health grows. Also, the market for functional foods is in its infancy in many countries; however, product innovation throughout a number of sectors, such as drinks, bakery, and probiotics, is evident, with trends generally following those of the United States and United Kingdom (Luckow & Delahunty, 2004).

8.1.2 Classification of beverage Beverages can be classified majorly into three groups based on their content in the form of fruit juice, synthetic flavor, addition of CO2, and health benefits of beverage. The classification of soft drinks is mentioned as follows: 1. Ready-to-drink essence-flavored beverages; 2. Ready-to-drink beverages containing fruits or fruit juice; and 3. Ready-to-drink beverages after dilution. The classification of beverages is presented in Table 8.1 (Kregiel, 2015). Functional drinks are rapidly growing and demanding subsector of the market and include drinks promoting health benefits along with the prevention of disease, improving immunity and digestion, and helping to boost energy (Tenge & Geiger, 2001). The beverages are known as functional because it contains health-providing ingredients such as microelements (vitamins, minerals) and nutraceuticals. The market for functional beverage is vast and can be targeted based on age, gender, and nutritional requirement.

8.1.3 Types of beverages Functional beverages are becoming more popular in the market, and it is valued at US $25 bn in 2005 (Datamonitor, 2006). Based on the Mintel Business Market Research report (Mintel International Group, 2009), the functional drinks and natural RTD beverages market has grown to $23 bn. It was reported that the sales of functional beverages and energy drinks in 2009 were 9 billion and 1.03 million, respectively. The sales of diet drinks, water, and fruit juices showed considerable growth. As the different types of beverages are there in the market, globally it can be divided into four segments: drinks, milk-based drinks, soft drinks, and alcoholic drinks. Hot drinks can be further classified into tea, coffee, and hot malt-based products; the second segment of milk-based drinks consists of segments like white drinking milk and flavored milk products. The third segment of soft drinks can be further categorized into subcategories which

TABLE 8.1 Classification of beverages (Kregiel, 2015). Type of soft drink

Description

Bottled water

Potable water, water with flavorings, and minerals/vitamins. 1. Still water: noncarbonated, mineral, spring, or table water, with or without added flavorings and vitamins/ minerals. 2. Carbonated water: mineral, spring, or table water, low carbonated waters, naturally sparkling or sparkling by CO2 injection. 3. Flavored water: unsweetened water, with essences and/or aromatic substances. Potable water sold in packs of over 10 L for use in dispensers. Sweetened, beverages with carbon dioxide, syrups for home dilution, and out-of-home carbonated soft drinks. 100% pure fruit or vegetable juice without ingredients, except permitted minerals and vitamins, with sweetening agents (less than 2%). Diluted fruit/vegetable juice and pulp, with sweetening agents, minerals, and vitamins. Flavored ready-to-drink, noncarbonated beverages, containing fruit or non-fruit flavors or juice content (to 25%). Non-ready-to-drink products, marketed as concentrates for home consumption including fruit and non-fruitbased products and flavors. Non-ready-to-drink products in powder form. Tea-based or coffee-based drinks and non-ready-to-drink powders and liquid concentrates for dilution.

Bulk/hot water Carbonates Juice Nectars Still drinks Squash/syrups Fruit powders Iced/ready-to-drink tea/coffee drinks Sports drinks Energy drinks

Products described as “isotonic,” “hypertonic,” or “hypotonic,” still or carbonated, ready-to-drink, or non-readyto-drink powders and concentrates; also fruit and non-fruit flavored drinks. Energy-enhancing drinks, mainly carbonated and containing taurine, guarana, glucose, caffeine, exotic herbs and substances, minerals, and vitamins.

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include bottled water; carbonated soft drinks; dilutables, also known as squash and including powders, cordials, and syrups; 100% fruit juice and nectars with 25%
99% juice content; still drinks, including ready-to-drink (RTD) teas, sports drinks, and other noncarbonated products with less than 25% fruit juice. Alcoholic drinks which form the fourth segment are comprised of products like beer, wine, spirits, cider, sake, and flavored alcoholic beverages (premixed spirits) (Mulvihill, 1992).

8.2

Market of nutraceutical or functional beverages

Functional foods or nutraceuticals are the best treatment regime for curing or managing various lifestyle diseases like diabetes, obesity, cancer, arthritis, hypertension, etc. . Nutraceuticals are gaining an important position in the growing health market of India as well as in the world. By giving complementary benefits, it will play an important role in the 21st therapeutic scenario (Singh et al., 2012). The Indian nutraceutical market was valued at $1480 million in 2011 and grew to $2731 million in 2016 at Compound Annual Growth Rate (CAGR) of 13%. Factors supporting the growth of nutraceuticals in India are increasing obesity in the population and rising instances of diabetes and cardiovascular diseases as government funding in vitamin fortification (Gupta et al., 2013). It was reported that the total global beverage industry had a business of around $1755.4 billion in 2010, which depicted a CAGR of 2.3% for the period spanning 2007
16. The beverage industry increased, with an anticipated CAGR of 3.4% for the 5 years 2011
16, which was expected to increase the value of the industry to about $2072.9 billion by the end of 2016. In the present era, consumers are conscious about their health and demand food which is nutritious and healthy. Among that, regular consumption of functional beverage can also fulfill the nutritional requirement. Beverages are more preferred over other food because of their convenience, thirst-quenching ability, good taste, etc. It was reported that the global market for functional foods with specific health claims was USD $43.27 billion (Surrey, 2014) in 2013. It grew by 25% in 2017 (Bonar, 2017). Functional beverages occupied over half (US $99 billion) (Euromonitor International, 2019) of the total market value (US $168 billion) of functional foods in 2019 (Euromonitor International, 2019), with approximately 1/3 of the market value (US $36 billion) being contributed by the Asia Pacific region (Euromonitor International, 2019).

8.3

Soft drinks

The consumption of soft drinks was found to have increased dramatically over the past several decades with the greatest increase among children and adolescents. Excessive intake of soft drinks with high sugar and acid content both in regular and diet could cause detrimental impacts on dental and general health including dental caries, dental erosion, overweight, obesity, and increased risk of type 2 diabetes. The sugar tax has raised the level of awareness; however, it is necessary to educate patients about the harmful effects of different types of soft drinks as it is not always easy for individuals to know from drink labeling what they actually contain (Tahmassebi & BaniHani, 2020). It is estimated that soft drinks worth nearly 2 crores of rupees are marketed annually in India. In addition to a number of large units, manufacturing RTS of standard quality, small units both in rural and semi-urban is also manufacturing soft drinks. However, very little data are available regarding the exact nature of such drinks (Phillips, 1992). Garg and Ahuja (2015) reported that the high consumption of soft drinks in the past few years has attracted negative attention worldwide due to its possible adverse effects, leading the health-conscious people to find alternative nutraceutical or herbal health drinks. The nutraceutical beverages were developed by the utilization of some easily available and well-known traditional herbs having nutritional potential. The key ingredients were selected as bael, amla, lemon juice, ashwagandha, and poppy seeds based on their household routine use in the summer with proven refreshing, cooling, and energetic feeling for ages. The physicochemical analysis of the prepared drink was found to contain optimum level of titratable acidity, total soluble solids (TSSs), and pH which were in accordance with the commercial recommendations. There were no bacterial colonies found in the product and therefore were found within limits. The formulation was found to contain flavonoids (80 mg/100 mL), phenolics (103 mg/100 mL), vitamin C (250 mg/100 mL) and has antioxidant potential (75.52%) apart from providing several other essential vitamins, minerals, and healthy components. The developed nutraceutical drink provides an economical and feasible option for consumers with very good taste combined with potential health benefits. The prepared drink is potentially capable to replace the synthetic soft drinks available in the market.

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Nonalcoholic beverages

Nonalcoholic beverages are also known as virgin drinks which do not contain alcohol as yeast is not added to convert sugar into alcohol during the fermentation process. In the United States, this term is defined as a beverage that contains less than 0.5% alcohol by volume. Most of the drinks are classified under this category as tea, coffee, fermented and non-fermented milk, fruit and vegetable beverages, herbal juices like parsley, holy basil, aloe vera, mint, dill, rosemary, etc., juices from oil seeds like coconut, sesame seed, badam, carbonated beverages, etc. (Anilakumar et al., 2017). There is constant increase in the growth of nonalcoholic beverage industries especially in developing countries, and the major players for beverage in today’s market are Coca Cola, Nestle, Dabur, Pepsi, Parle Agro, etc. Nonalcoholic beverages include drinks with carbonation, without carbonation, and hot beverages. According to a report stated by Sowmiya (2016), the market for carbonated drink increased by 10%
12% annually and for fruit-based drink by 35%
40% annually. Moreover, the functional drink was sold as an energy drink, its market size has grown by 25% in India, and the major contributors are Parle Agro, Dabur, and HUL. There are different types of beverages available in the market such as alcoholic and nonalcoholic beverages. These drinks are produced using cereals by fermentation especially in some African countries and are very popular because of the social, religious, and therapeutic values associated with them (Aka et al., 2008; Nwachukwu et al., 2010). The overall nonalcoholic beverage market in India witnessed aggregate sales of 20 billion liters, worth USD 10 billion in the year 2015
16. Out of the total volume sales of 20 billion liters of nonalcoholic beverage in India in 2015
16, off-trade sales are estimated at 13.75 billion liters (about 69%), whereas on-trade sales are estimated at 6.25 billion liters (about 31%). The import segment in nonalcoholic beverage is largely dominated by fruit pulp and juicebased drinks. The Indian consumer is increasingly becoming health-conscious, and this has resulted in the growth of natural and healthier beverage products. Increasingly, many established players are moving into 100% juice and nectar categories. Also, cold-pressed juices that preserve better nutrition are catching the attention of the healthier consumer bracket in India. Over the past 2 years, several gourmet players in this category are gaining popularity in the metropolitan cities in India (http://www.insideindiatrade.com/ Non Alcoholic Beverage Sectoral Report/2017, 2017). Nonalcoholic beverages are preferred and liked by almost all age group consumers especially by children, pregnant women, sick, and old people whereas alcoholic beverages are more preferred by men. Their production varies from one region to another, but essentially includes malting, brewing, and fermentation feedstock for millet, maize, and mainly sorghum. Moreover, the alcoholic fermentation is initiated by pitching sweet wort with a portion of the previous brew or dried yeast harvested from previous beverage (Aka, 2009; Maoura et al., 2005; N’guessan et al., 2011). A wide range of cereals and raw materials were used for fermented foods and beverages. Cereal grains such as sorghum (Sorghum bicolour (L.) Moench), millets (pearl and finger millets) (Pennisetumglaucum (L.) and Eleusinecoracana), and maize (Zea mays (L.)) were common substrates usually used in Africa to produce a wide variety of beverages .

8.4.1 Cereal-based fermented nonalcoholic beverages In recent years, cereals and its ingredients are accepted as functional foods because they provide dietary fiber, proteins, energy, minerals, vitamins, and antioxidants required for human health. Arabinoxylan and β-glucan are examples of such dietary fibers. β
glucan is a soluble fiber that has the ability to increase solution viscosity and possible rapid fermentation in the small intestine. β-glucan can delay gastric emptying and increases gastrointestinal transit time and luminal viscosity (Saikia & Deka, 2011). Cereals also contain resistant starch carbohydrates, galacto- and fructooligosaccharides. Charalampopoulos et al. (2002) showed that cereals are good fermentable substrates for the growth of probiotic microorganisms. Cereals have been acclaimed to prevent cancer and cardiovascular diseases, reduce tumor, lower blood pressure, limit the incidents of heart diseases, control cholesterol level and rate of fat absorption, delay gastric emptying, and supply gastrointestinal health. The potential of cereal nutrients in the control of coronary heart disease is well known. These nutrients include fiber, vitamin E, selenium, folate, linoleic acid as well as phenolic acids with antioxidants properties. It was reported that the natural lactic fermentation of cereal results in a decrease in carbohydrate content together with increase in some amino acids and B-group vitamins. Further, fermentation results in reduction of antinutritional factors, leading to an increment in mineral availability and protein and starch digestibility. In recent times, cereals are gaining popularity as a probiotic delivery vehicle due to their inherent attributes. Cereal grains can serve as a suitable carrier matrix of probiotic cultures by supporting the growth and survival of organisms (Ganguly et al., 2021). The technology used for obtaining cereal or pseudo-cereal-based milk substitutes primarily involves the extraction of selected plant material, and the obtained beverages differ in their chemical composition and nutritional value (the

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Dehulled millet grains

Clean

Steep

Wet mill with the addition of spices

Wet-sieve

Allow to settle

Decant

Slurry in cold water + slurry in boiling water Add sweetener and mix

Bottle

Kunu-zaki FIGURE 8.1 Process flowchart for manufacturing of Kunun-zaki (Achi & Asamudo, 2019).

content of proteins, lipids, and carbohydrates, glycemic index, etc.) due to the chemical diversity of the cereal and pseudo-cereal raw materials and the operations used for their production. Beverages made from cereals or pseudocereals are an excellent matrix for the growth of lactic acid bacteria, and the lactic acid fermentation not only produces desirable changes in the flavor of fermented beverages and the biological availability of nutrients but also contributes to the formation of functional compounds (e.g., vitamin B) (Ziarno & Cicho´nska, 2021). Kunun-zaki is a millet-based nonalcoholic fermented beverage widely consumed in the northern parts of Nigeria. This beverage is however becoming more widely consumed in southern Nigeria, owing to its refreshing qualities. Adeyemi and Umar (1994) described the traditional process for the manufacture of Kunun-zaki. The process involves the steeping of millet with spices (ginger, cloves, and pepper), wet sieving, and partial gelatinization of the slurry, followed by the addition of sugar and bottling. The fermentation which occurs briefly during the steeping of the grains in water over 8
48 hours period is known to involve mainly lactic acid bacteria and yeasts. The process flowchart is given in Fig. 8.1. Kvass is a cereal-based beverage traditionally produced from fermented rye and barley malt, rye flour, and stale rye bread in Lithuania and other eastern European countries. It is a nonalcoholic beverage, so the ethanol content should be negligible, and it is considered to be spoiled if alcohol accumulates to higher levels. Kvass undergoes nonthermal processing after fermentation and thus contains high cell counts of viable yeast and lactic acid bacteria (LAB). The application of such LAB for nonalcoholic cereal-based beverage production provides new opportunities for the development of functional fermented products (Basinskiene et al., 2016). They developed a new technology for making traditional Lithuanian nonalcoholic beverage Kvass from fermented cereals by extending the spectrum of raw materials (extruded rye) and applying new biotechnological resources (xylanolytic enzymes and LAB) to improve its functional properties. Beverages fermented by LAB had lower pH values and ethanol volume fraction compared to the yeast-fermented beverage. The acceptability of the beverage fermented by Lactobacillus sakei was higher than that of Pediococcus pentosaceus or yeast-fermented beverages and similar to the acceptability of commercial Kvass made from malt extract. The results showed that extruded rye, xylanolytic enzymes, and LAB can be used for the production of novel and safe highvalue nonalcoholic beverages.

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8.4.2 Market of nonalcoholic beverages The beverage industry in India constitutes around USD 230 million among the USD 65 billion food processing industry. The major sectors in the beverage industry are tea and coffee which are not only sold heavily in the domestic market but are also exported to a range of leading overseas markets. Among the hot beverages manufactured at large scale, tea is the most dominant beverage that is ruling both the domestic and international markets even today. Worldwide, India ranks fifth in coffee production and accounted for 4% of the world’s production. The turnover of tea is also reported to grow annually by 1%
2% in India (Sowmiya, 2016).

8.5

Probiotics beverages

Probiotics are living microorganisms (microscopic organisms) that, when taken by mouth, benefit your health by improving the balance of bacteria in the intestines. These microorganisms are most often bacteria, but also include other kinds of organisms such as yeast. In the market, most probiotic drinks are available but these are milk-based. The group of consumers with lactose intolerance avoids milk-based foods. Probiotic microflora displays numerous health benefits beyond providing basic nutritional value. The health benefit reported by probiotics is the improvement in gut health and the prevention of intestinal infections and stimulation of the immune system (Kailasapathy & Chin, 2000). Infection prevention is increasingly preferred over using the traditional action of chemotherapy with antibiotics that rise the concern over the development of antibiotic resistance and has placed probiotics at the fore. Probiotics are “live microorganisms which, when administered in adequate amounts, confer a health benefit to the host.” The most common types of probiotics are lactic acid bacteria (LAB) and include species from the Lactobacillus, Pediococcus, and Bifidobacterium genera. Probiotic bacteria are beneficial bacteria that provide therapeutic effects on their host when ingested, and probiotic food products should contain at least 106 CFU/100 g to transfer beneficial effects to the host (Hekmat & Reid, 2006; Rybka & Kailasapathy, 1995). The increasing consumption of probiotics parallels the growing trend in vegetarianism and diets that promote health and wellness (Martins et al., 2013). Therefore, the growing number of vegetarian individuals reinforces the importance of the need to develop nondairy probiotic alternatives. Gardiner et al. (2002) stated that Lactobacillus rhamnosus GR-1 has been proven to help maintain a favorable microbial balance in the intestine and can survive in the intestinal tract without induction of systemic immune or inflammatory responses. The diet of Asian people lacks meat and dairy foods and takes more plant-based foods. Asian consumers have more choices of demanding plant-based beverages as compared to dairy. The average per capita dairy consumption (including fluid milk, butter, cheese, nonfat dry milk powder, and whole milk powder) in milk equivalent in the last decade for the major dairy markets was 10.2 kg in China, 71.8 kg in India, 7.8 kg in Indonesia, 97.6 kg in Japan, 67.8 kg in Malaysia, 24 kg in the Philippines, 80 kg in South Korea, 28.7 in Thailand, and 8.6 kg in Vietnam. This contrasts with per capita consumption of 330 kg in the European Union, 310 kg per capita in Australia, and 251 kg per capita in the United States (Dong, 2006). With the fact of more demand for nondairy probiotics, cereals, fruits, and vegetables may be potential substrates, where the healthy probiotic bacteria will make their mark, both in developing and developed countries. Morya et al. (2017) developed low-fat probiotic beverage using whey and sorghum. Skim milk and whey prepared from skim milk were used as the base material for low-fat probiotic beverage. For base material, cereal was added at different levels for each sample combination. The addition of stabilizer, mixing, and mixture were pasteurized at 63 C for 30 minutes and cooled at below 40 C. Then, 2% of the prepared mixture and culture was added to it. It was incubated at 37 C
42 C/6 hours, and 12% sugar syrup was added into it and agitated. Then, homogenization at 250
300 psi was done, and the prepared beverage was filled into presterilized bottles and corked tightly and stored at (4 C 6 1). The process flowchart for preparation of low-fat probiotic (Lactobacillus acidophilus) beverage from whey and sorghum is given in Fig. 8.2 (Morya et al., 2017). Probiotics products are available in plenty in the market today, but in the form of fermented milk and yogurts which cannot be consumed by lactose-intolerant people. Based on that, there is an increase in demand for the cereal-based probiotic products. Owing to health considerations, from the perspective of cholesterol in dairy products for developed countries, and economic reasons for developing countries, alternative raw materials for probiotics need to be searched (Deshpande et al., 2015). Cereals could be alternative ingredients for the milk-based probiotic products. Cereals are a major source of fibers in the diet, and the main active component of cereal fibers is β-glucan. Numerous scientific studies demonstrated the hypocholesterolemic effect of this compound, bringing a 20%
30% reduction of LDL cholesterol. The overall effect is the reduction of cardiovascular disease risk (Gallaher, 2000; Wrick, 1994). Beta-glucan is also considered a prebiotic as it can support the growth of some beneficial bacteria in the colon (Stark & Madar, 1994).

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Whey and skim milk Stabilizer

Sorghum Mixing

Pasteurization (63°C / 30 min)

Cooling (40°C) Addition of probiotic culture Incubation (37-42°C / 6 hrs)

Dahi

Addition of sugar syrup (12%)

Agitation

Homogenization (250psi and 500 psi)

Filling

Packaging

Storage (4°C±1) FIGURE 8.2 Preparation of low-fat probiotic (Lactobacillus acidophilus) beverage from whey and sorghum (Morya et al., 2017).

The cereals with the highest β-glucan content are oats and barley (Manthey et al., 1999; Wood & Beer, 1998). The beneficial effects of food with added live microbes (probiotics) on human health are being increasingly promoted by health professionals. Garg et al. (2015) determined the suitability of orange and carrot as raw material for developing probiotic orange and carrot-blended RTS beverage using L. acidophilus NCDC14. The standardized orange and carrot-blended juice (50:50 ratio) was fortified with L. acidophilus at 1012 cfu/mL and stored at 4 C. The viability of L. acidophilus was studied by plate count method using MRS agar for 40 days. The titratable acidity got increased significantly (1.344%) in the RTS after 40 days of storage at 4 C. pH and vitamin C got decreased whereas β-carotene got increased from 560 to 672 μg/100 g. The initial probiotic count was recorded as 25.2 3 1012 cfu/mL, and the viable cell count started decreasing gradually to 10.1 3 1010 cfu/mL after 40 days of storage. This study revealed that fruit and vegetableblended juice fortified with probiotics can serve as a better alternative for dairy-based probiotic products with additional nutritional profile. Similarly, Profir and Vizireanu (2013) investigated the consumer acceptance of fermented juice made from beetroot, carrot, and celery. This juice has been inoculated with three probiotic strains: L. acidophilus, Lactobacillus casei, and Saccharomyces boulardii. After lactic fermentation, the functional beverage has been evaluated by trained panelists. He and Hekmat (2015) measured the survival of L. rhamnosus GR-1 in nondairy probiotic beverages over a 28-day storage period and determined which sample was most preferred on measures of appearance, consistency, flavor, texture, and overall acceptability. Three nondairy samples and one control milk sample were prepared. The nondairy samples were soy (1:3 product-to-water ratio), almond (1:3), and peanut (1:5). L. rhamnosus GR-1 remained viable (107 CFU/mL) in all samples over 28 days storage period, and changes over time were dependent on the sample (P 5 .03). The results of the sensory evaluation (n 5 90) showed that the soy and peanut samples were significantly

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different (P , .03) from the control milk sample in appearance, consistency, flavor, texture, and overall acceptability. The almond sample was not rated significantly different (P..05) from the milk control in all categories. The results of the study suggested that probiotic almond milk may be a feasible substitute for conventional probiotic milk beverages, particularly for vegetarians who cannot consume dairy products and individuals with lactose intolerance. A probiotic drink from carrot juice and beetroot juice with L. acidophilus, L. casei, and Bifidum longum was investigated by Chauhan et al. (2021). The proximate composition of probiotic juice revealed 26% increase in protein content and reduction of 17% in the level of carbohydrates as compared to that in the fresh carrot and beetroot juice. Sensory analysis of fresh and probiotic carrot beetroot juice revealed a 20% elevation in the overall acceptability of probiotic carrot beetroot juice over fresh juice. The data obtained from this study provide new insights into projection of fermented carrot beetroot juice as an appropriate media for the growth of probiotics. Consumers are demanding healthier foods, and the increasing drawbacks associated with dairy-based products have driven efforts to find plant-based probiotic alternatives. Consequently, this study aimed to evaluate the suitability of a teff-based substrate for delivering the potential probiotics, L. rhamnosus GG (LGG) and Lactobacillus plantarum A6 (LA6), with a view to developing probiotic functional beverages. Single-strain and mixed-strain fermentations were performed without any pH control. A combination of the two evaluated lactobacilli strains reduced fermentation time. In conclusion, a substrate made of whole-grain teff flour without any supplement could be used as a substrate to produce functional probiotic beverages (Alemneh et al., 2021).

8.6

Fruits-based beverages

The natural extract obtained by pressing the fruit or vegetable tissue is referred to as juice. Juice is commonly consumed as a beverage or used as an ingredient or flavoring in foods or other beverages, such as smoothies. Fruit juicebased beverages are the blend of juice, sugar, and artificial flavor in the correct proportion and processed using pasteurization, canning, or concentration. The health-benefiting components present in fruit juice are the presence of vitamins, phytochemicals, minerals, etc. The fruit is rich in prebiotic dietary fiber, vitamins, minerals, and polyphenolic flavonoid antioxidant compounds. According to a new research study, mango fruit has been found to protect against colon, breast, leukemia, and prostate cancers. Several trial studies suggest that polyphenolic antioxidant compounds in mango are known to offer protection against breast and colon cancers. Mango fruit is an excellent source of vitamin A and flavonoids like beta-carotene, α-carotene, and β-cryptoxanthin. In total, 100 g of fresh fruit provides 765 mg or 25% of recommended daily levels of vitamin A. Together, these compounds are known to have antioxidant properties and are essential for vision. Vitamin A is also required for maintaining healthy mucus membranes and skin. Consumption of natural fruits rich in carotenes is known to protect the body from lung and oral cavity cancers. The extracted pulp was used for the preparation of mango RTS beverage. The required quantity of pulp was added to the measured quality of water, and ground sugar was also added to it. In all 14 treatments, similar method was used. The pulp and sugar were mixed thoroughly and heated up to 65 C to dissolve them properly. It was homogenized with juicer cum mixture and then strained with a muslin cloth to remove impurities if any. The sodium benzoate at 700 ppm was used as a preservative for the prepared RTS beverage. The RTS was then filled in sterilized glass bottles (200 mL capacity) and sealed with a crown cork. The guava-mango ready-to-serve (RTS) drink and squash were developed by Yadav et al. (2015) using guavamango blends. Total sugars and acidity increased, while ascorbic acid decreased in both the beverage blends with the increase in storage duration. The color and appearance, flavor, taste, mouth feel, and overall acceptability of guava mango beverages decreased significantly with the advancement in storage period; however, their overall rating remained above the acceptable level even after 3 months of storage. Overall acceptability also increased with the increase in the proportion of pulp (15%
20%) in RTS drink and (30%
40%) in squash. RTS drink prepared with 20% pulp (20 guava:80 mango), 14% total soluble solids (TSSs), and 0.26% acidity was found most acceptable, while squash prepared with 40% pulp (20 guava:80 mango), 50% TSS and 1.0% acidity was found most acceptable. The soymilk fortified with mango pulp helps to improve the nutritional as well as therapeutic value of beverages. It would offer more health benefits as compared to plain fruit beverage to the consumer. Mango pulp is added to soymilk to enhance its vitamin A, C, and mineral contents. It also provides sweetness and masks the beany flavor of soymilk to some extent (Lee et al., 1990). Sakhale et al. (2012a, 2012b) developed the soymilk-based mango RTS beverage. The ready-to-serve (RTS) beverage was prepared by blending soymilk with mango pulp in different combinations such as 80:20, 70:30, 60:40, and 50:50 and analyzed for various physicochemical and sensory characteristics for its overall acceptability. The study revealed that the RTS beverage prepared by blending the soymilk and mango pulp in an equal

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proportion (50:50) was found better in almost all physicochemical and sensory quality parameters as compared to the other combinations. The blend of fruit juices with herbs, milk, spice extract, etc., can be utilized for the development of beverages having health benefits. Several attempts were made by researchers to develop fruits-based beverages having superior quality and longer shelf life. Pushkala and Srividya (2014) formulated a spiced functional RTS beverage blend using aloe gel (AG) and papaya. Aloe gel (30%), papaya pulp (15%), spice extract (5%), and citric acid (0.1%) were mixed in a given proportion to prepare the blend with TSS of 15  Brix. The SAGPB exhibited superior quality characteristics compared to SPB both in fresh and stored samples. The SPB was acceptable for up to 4 months and SAGPB for 5 months. The results indicate that nutraceutical-rich AG could be successfully utilized to develop functional fruit beverages with improved quality and shelf life. Aloe-fruit beverage blends could also offer an attractive means of increasing the consumption of unpalatable aloe gel/juice. The developed aloe-gel-fruit-based spiced functional beverage blend could be promoted as a nutraceutical product with multiple benefits to the consumers. Similar type of beverages rich in protein was prepared by Bansode et al. (2009) by using plum juice, sugar, and whey protein concentrate (WPC). The RTS plum beverage prepared by using 30% WPC and stored in refrigerator was found better with respect to chemical composition and sensorial quality. Beverages containing fruit juice along with separated milk and reconstituted skim milk were developed by Shukla et al. (2003) by blending juice/pulp from apples, bananas, guavas, litchis, and mangos at four different concentrations (100, 200, 300, and 400 g/L). Organoleptic evaluation of the beverages showed that apple juice and guava pulp could be blended at up to 300 and 100 g/L in milk products, respectively. Banana and mango pulp could also successfully be used at up to 200 g/L in separated milk and reconstituted skim milk. Litchi juice could be blended up to 300 g/L in separated milk and 200 g/L in reconstituted skim milk. Vadakkan et al. (2010) studied the effects of total soluble solids (TSSs), acidity, and carbonation levels on the quality of sweet orange beverage. The sweet orange ready-to-serve (RTS) carbonated beverage was prepared by extracting and clarifying the juice and adjusting the total TSS to 56, 60, 64, and 68 Brix with sugar and acidity to 0.5%, 1.0%, and 1.5% with citric acid. The beverage carbonated at 120 psi pressure scored highest for sensory quality than those at 75 and 120 psi. Aonla RTS beverage rich in vitamin C was prepared from the aonla fruits blanched for 3 minutes, and blending the juice with 15% sugar, 3% ginger, and 2% cumin seed extract was found significantly better in almost all sensory quality characteristics (Kanakdande et al., 2007). Jain and Khurdiya (2004) developed vitamin C-enriched fruit juice-based ready-to-serve beverages through blending of Indian gooseberry juice. Physical and nutritional qualities of fruits, viz., apple, lime, pomegranate, Perlette grape, and Pusa Navrang grape were analyzed and compared with those of Indian gooseberry (Emblica officinalis Gaertn.). Indian gooseberry juice contained the highest vitamin C (478.56 mg/100 mL). Hence, when gooseberry juice was blended with other fruits juice for the preparation of ready-to-serve (RTS) beverages, it boosted their nutritional quality in terms of vitamin C content. On the basis of overall sensory quality and vitamin C content, RTS beverage prepared by blending gooseberry and Pusa Navrang grape juice in 20:80 ratio was found to be the best. Pomegranate juice is useful for the patients suffering from leprosy, high cholesterol levels, heart patients, and kidney problems. Pomegranate juice contains about 8.0 mg ascorbic acid/100 mL of juice and is a good source of vitamin B (pantothenic acid), minerals, Na, K, Fe, Cr, and Cu, and polyphenols such as tannins and flavonoids (Heyn, 1990). Pomegranates have high-fiber contents; however, fiber is entirely contained in the edible seeds which also supply unsaturated oils. The most abundant polyphenols in pomegranate juice are the hydrolysate tannins called ellagitannins (Heber, 2008) formed when ellagic acid binds with a carbohydrate. Punicalagins are unique pomegranate tannins with free radical scavenging properties. Punicalagins are absorbed into the human body and may have dietary values as an antioxidant (Schubert et al., 1999). Dhumal et al. (2013) studied the preparation of pomegranate juice concentrate by different heating methods. Clarified pomegranate juice of cv. Bhagwa was concentrated by various heating methods. The final juice concentration of 65 Brix was achieved in 37, 78, and 106 minutes by using atmospheric heating, microwave, and rotary vacuum evaporation techniques, respectively. Maximum concentrate recovery on weight (24.35%) and volume (20.44%) basis was recorded in the rotary vacuum heating process. It also recorded maximum moisture (24.46%), polyphenols (833.92 mg/100 g fruit), anthocyanins (321.97 mg/100 g fruit), antioxidant activity (76.41%) with maximum overall acceptability score (8.607). The maximum extent of color loss on Hunter parameters was recorded in the microwave heating method while the minimum was in rotary vacuum evaporation. Highest viscosity (120.20 mPa.s) was noted in juice concentrate prepared by microwave heating. Gupta et al. (2017) studied the effect of keeping the nutritional and functional attributes of papaya pulp potential of whey to be used in nutritious and health-promoting beverage. Paneer whey is used as raw material, and three levels of

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Soy extract or/and Quinoa extract Addition of Soybean Oil, Lactose and stabilizer Homogenization (5 min.) at room temperature

FIGURE 8.3 Process flowchart for the production of fermented beverages (Bianchi et al., 2015).

Thermal treatment (under stirring) until 50°C

Addition of sugar (50°C)

Thermal treatment (under stirring) until 80°C Milk powder addition (80°C) Homogenization / 5 min

Pasteurization (95°C/ 5 min.) Fructo-oligosaccharides addition Cooling at 37 °C (in ice bath)

Inoculation with L. casei

Incubation (pH 4.8)

Cooling and storage (5 °C)

sugar (8%, 10%, and 12%) and three levels of papaya pulp (10%, 20%, and 30%) are used for the preparation of papaya pulp-based whey beverage. This product is stored at 5 C at a refrigerated temperature. The product sample contains 10% sugar and 20% papaya pulp having maximum acceptability and optimum level of chemical percentage.

8.7

Fermented beverages

Fermented dairy products are generally good food matrices for the development of functional foods, but the consumption of these products is limited due to growing vegetarianism and a large number of individuals who are lactoseintolerant or on cholesterol-restricted diets. Probiotic bacteria are beneficial bacteria that provide therapeutic effects on their host when ingested, and probiotic food products should contain at least 106 CFU/100 g to transfer beneficial effects to the host (Hekmat & Reid, 2006; Rybka & Kailasapathy, 1995). Fermented dairy products are the generic carrier matrices of probiotic microorganisms, with yogurt and fermented milk as the most commonly marketed products (Martins et al., 2013). The fermented beverages were produced with various proportions of aqueous extracts of soy and quinoa, 2% lactic culture (L. casei LC-1), 6% sucrose, 0.8% soybean oil, 1% food grade lactose, 0.14% Recodan TM RS-B stabilizer, 2.5% milk powder, and 3% fructo-oligosaccharides. The process flowchart for the production of fermented beverage is shown in Fig. 8.3 (Bianchi et al., 2015).

8.8

Whey-based beverages

Whey and whey-derived products besides being excellent nutritional ingredients have wide-ranging and excellent functional characteristics supplying flavor, consistency, color, and overall appearance in variety of foods. An attempt is therefore made to produce the nutritionally improved fermented beverage using dairy by-product, whey in conjunction with cereals and beneficial organisms that may provide health benefit to the consumer of all age groups.

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The whey-based beverages are the best alternative to fruit beverages added with artificial color and flavor. These are more nutritious and with appealing taste and flavor. Alternately, the growing demand for whey-based beverage is also beneficial for dairy industries and beverage manufacturers. Such type of drinks is more suitable for the consumers where there are deficiencies of certain nutrients, and whey is considered as a dairy by-product, rich in water-soluble vitamins, whey proteins, etc. (Naik et al., 2009) and could be used as a preventive measure for the diseases such as anemia, liver, and bone-related complaints (Cruz et al., 2009). The whey from dairy industries can be used for product diversification into fermented and non-fermented beverages and can substitute water for the preparation of the nutritious drink. The whey beverage with natural fruit pulp can be prepared by blending it with pulp or juice from different pulp which will not only enhance the organoleptic properties but also nutritional value. Mango pulp fortified whey beverage was developed and evaluated by Sakhale et al. (2012a, 2012b). Protein-rich beverage can be prepared using whey which is easy to digest, having higher nutritional values and health-benefiting properties. Five general trends have influenced food and beverage innovations since 1985: convenience, pleasure, ethnic fusion, tradition, and, importantly, health and wellness. Sales of energy drinks and ready-todrinks have grown to $23 billion. Whey beverages are generally classified into four basic types: mixtures of whey (processed or unprocessed, including UF permeates) with fruit or (rarely) vegetable juices; dairy-type, “thick” beverages (fermented or unfermented); thirst-quenching carbonated beverages (the “Rivella-type”); and alcoholic beverages (beer, wine, or liqueurs) (Chavan et al., 2015). The whey-based beverage containing fruit pulp such as apple, orange, peach, and pear added with refined sugar and citric acid was studied by Djuri´c et al. (2004). The blend of whey along with orange and pear juice was sensorial depending on the quantity of sugar added whereas blend of peach and whey with 2% of refined sugar was more acceptable with reference to organoleptic properties. Fortification of mango pulp in whey improves the nutritional as well as therapeutic value of beverage. Whey-based fruit juice beverage would offer several distinct nutritional advantages over the plain fruit beverage to the consumer. Mango pulp is added to whey to enhance its vitamin A, C, and mineral contents. Whey-based beverage added with mango pulp Cv. Kesar (at 20%, 25%, and 30%) was developed and evaluated by Sakhale et al. (2012a, 2012b). It was reported that beverage with 70% whey and 30% mango pulp offers a higher overall acceptability score, and a solo provides 9.80 mg/100 g of ascorbic acid. Storage stability and sensory analysis of UHT-processed whey-banana beverages were studied by Koffi et al. (2005). Selected characteristics of whey-fortified banana beverages stored at 4 C, 20 C, 30 C, and 40 C were monitored at specific time intervals over a 60-day storage period. A sensory descriptive analysis panel generated terms to describe and quantify the sensory characteristics of the whey-banana beverage stored at 4 C. The product was a sour, sweet, smooth beverage, with distinctive banana flavor and minimum off-flavor. A consumer panel indicated that sourness and acidity were critical quality factors. The main differences detected were for sedimentation (greater at 40 C) and serum separation (min. at 4 C). Kanchana et al. (2021) developed and evaluated whey-based herbal beverages using extract from brahmi and mint along with jaljeera powder. The pH, titrable acidity (% lactic acid), and total soluble solids (0Brix) of whey used for the development of whey-based herbal drinks were found to be 5.21, 0.23, and 6.4, respectively. The proximate composition of whey such as lactose (g/100 g), protein (g/100 g), and fat (g/100 g) were 4.28, 0.28, and 0.13, respectively. The whey (65%), sugar (11%), brahmi extract (3%), mint extract (2%), and jaljeera powder (0.15%) were standardized for the formulation of whey-based herbal drinks. Among the three variations, whey-based jaljeera drink scored the highest sensory acceptability. The pH, titrable acidity (% citric acid), total soluble solids (0Brix), lactose (g/100 g), protein (g/ 100 g), fat (g/100 g), total reducing sugar (g/100 g), total sugar (g/100 g), calcium (mg/100 g), ascorbic acid (mg/ 100 g), and total phenol (mgGAE/100 g) of whey-jaljeera drink (H3) were 5.24, 0.21, 12.0, 2.78, 0.46, 0.30, 3.43, 14.43, 55.27, 0.12, and 14.26, respectively.

8.9

Micronutrient-fortified beverage

Micronutrient malnutrition has been recognized not only to be widespread, but also to cause serious health, developmental, and economic problems. More than 2 billion people worldwide are affected by micronutrient malnutrition. In addition, children in developing countries are prone to infection, including chronic infection with a variety of parasites. The combined effects of undernourishment and chronic helminth infections can be serious. In areas where both are common, much of the student population may carry a significant health burden, resulting in delays in physical, emotional, and intellectual growth that have the potential to prevent the children from reaching their full potential in both physical and mental development.

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The food category of beverages encompasses a wide range of products, including fruit juices and drinks, milks and milk drinks, chocolate (malt) beverages, instant flavored drinks, nectars, meal replacers, supplements for pregnancy and lactation, sports drinks, and others. Micronutrient-fortified foods, including beverages, are becoming increasingly popular in many countries. In a recent survey in the United States, more than half of the respondents reported consuming micronutrient-fortified fruit juices or drinks several times weekly. The contribution to micronutrient intakes from fortified foods in the United States ranged from 6% for vitamin B6 and folk acid up to 24% for iron and vitamin B1. Ziauddin Hyder et al. (2007) studied the effect of a multiple-micronutrient-fortified beverage on hemoglobin, iron, and vitamin A status and growth in adolescent girls in rural Bangladesh as the adolescent girls have high nutrient needs and are susceptible to micronutrient deficiencies. A total of 1125 girls (Hb $70 g/L) enrolled in a randomized, doubleblind, placebo-controlled trial and were allocated to either a fortified or non-fortified beverage of similar taste and appearance. The beverage was provided at schools 6 days/week for 12 months. The fortified beverage increased the Hb and sFt and retinol concentrations at 6 months (P, 0.01). Adolescent girls in the non-fortified beverage group were more likely to suffer from anemia (Hb, 120 g/L), iron deficiency (sFt, 12 mg/L), and low serum retinol concentrations (serum retinol, 0.70 mmol/L) (OR 1/4 2.04, 5.38, and 5.47, respectively; P, 0.01). The fortified beverage group had greater increases in weight, MUAC, and BMI over 6 months (P, 0.01). Consuming the beverage for an additional 6 months did not further improve the Hb concentration, but the sFt level continued to increase (P 1/4 0.01). The use of multiple-micronutrient-fortified beverage can contribute to the reduction of anemia and the improvement of micronutrient status and growth in adolescent girls in rural Bangladesh. Grant et al. (2015) studied the intervention of multiple-micronutrient-fortified nondairy beverage to reduce the risk of anemia and iron deficiency in school-aged children in low-middle income countries. Multiple-micronutrient (MMN) fortification of beverages was found an effective option to deliver micronutrients to vulnerable populations. Results of school-aged children were included in the meta-analysis. Compared to isocaloric controls, children who received MMN-fortified beverages for 8 weeks to 6 months showed significant improvements in hemoglobin. MMN-fortified beverage interventions could have major programmatic implications for reducing the burden of anemia and iron deficiency in school-aged children in low-middle income countries.

8.10

Beverages rich in antioxidants and herbs

The medicinal value of fruit beverages can be enhanced by the incorporation of herbal extracts. Fruits juice could be enriched by the addition of herbal extract for the preparation of beverages which improves taste, aroma, and nutrition and also contributes to medicinal values. There is always a demand from the consumers all over the world for new food products which are nutritious with a delicate flavor. Herbal beverages in the form of RTS, squashes, appetizers, and health drinks are important from the nutritional point of view. Some herbs like tulsi, aswagandha, aloe vera, shatavari, etc., can be utilized in order to enhance the medicinal value of functional beverages. Tulsi (Ocimum sanctum) is believed to have health benefits due to their anthelmintic activity and polyphenols (Gangrade et al., 2000). Juice or infusion of the tulsi leaves is used in the treatment of bronchitis, catarrh, digestive complaints, arthritis, ringworms, hypertension, heart attack, cancer, viral hepatitis, and diabetes. The leaves and seeds of tulsi are reported to have diuretic and laxative properties. Arugampul (Cynodon dactylon) is one of the most commonly occurring weeds in India. It is a bitter plant having antimutagenic, antitumuorigenic, and antigenotoxic activity and can be used as the best chemopreventive agent (Annapurani & Bhagavathy, 2000). Thamilselvi et al. (2015) prepared lime-based herbal-blended RTS beverage using tulsi and arugampul. The manufacturing process is shown in Fig. 8.4. There are different categories of functional beverage which may include energy beverage, sports drink, vitaminfortified beverage, antioxidant-enriched beverage, etc. (Wootton-Beard & Ryan, 2011). The beverage enriched with natural antioxidant as a functional ingredient can be consumed as a dietary requirement and helps to fight against free radicals and protect the body against oxidative stress. Consumers are increasingly better informed about the major role of beverages and foods in diet and health and hence desire functional beverages that contribute to preventing or inhibiting the progression of degenerative diseases caused by oxidative stress (Ozen et al., 2012). Functional beverages are often widely valued (Kausar et al., 2012) with vegetable and fruit beverages also enjoying wide commercial acceptance along with dairy beverages (Davoodi et al., 2013). Awe et al. (2013) documented the antioxidant benefits of Hibiscus sabdariffa extract “HSE,” cocoa and ginger beverage blends as novel functional beverage. Pruthi and Singh (2014) developed and evaluated antioxidant-rich, wheatgrass-based nourishing beverage. Wheatgrass juice serves as a highly beneficial source of therapeutic and nutritional value. The effect of vitamin E in wheatgrass juice can be utilized along with vitamin A from basil and vitamin C from lemon juice in improving its

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Extraction of Lime juice

Addition of herbal extracts (Tulsi and Arugampul)

Mixing with filtered 10-15°Brix sugar syrup

Heating at 80°C for 15 minutes

Bottling

Crown corking

Pasteurization at 90°C for 25 minutes

Cooling

Labelling FIGURE 8.4 Flowchart for the preparation of herbal-blended lime-based ready-to-serve beverage (Thamilselvi et al., 2015).

antioxidant activity as the combination of the three helps in reducing the blood LDL and blood glucose levels more significantly. This study involved the preparation of four samples of wheatgrass-based beverage using lemon and basil juice in different proportions so as to add more antioxidants to the beverage. Sample a (having combination of juices such as wheatgrass 85%, lemon juice 10%, basil juice 5%) was best accepted by panel members. It was concluded that a nutritively enriched low-cost wheatgrass-based beverage could be developed with more acceptability and beneficial effect. The demand for functional foods and drinks with health benefit is on the increase. The author carried out an experiment to formulate and investigate the effects of blends of two or more pineapple, orange juices, carrot, and Hibiscus sabdariffa extracts (HSEs) on the antioxidant properties of the juice formulations in order to obtain a combination with optimal antioxidant properties. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20 -azino-bis(3-ethylbenzothiazoline6-sulfonic acid) (ABTS) radical scavenging abilities, ferric-reducing antioxidant potential (FRAP), vitamin C, total phenolics, and total carotenoids contents of the formulations were evaluated as a test of antioxidant property. In all the mixtures, formulations having HSE as part of the mixture showed the highest antioxidant potential (Oluwatoyin et al., 2016). Herbal beverage was developed by Isabela et al. (2012) by using herbal mate extract which is having high antioxidant activity and antimicrobial property due to the presence of polyphenols. This was a fermented beverage, and the microbial strain used was L. acidophilus. The sensorial properties of fermented herbal beverage were studied and reported that it could be a nondairy-based fermented probiotic herbal beverage enriched in antioxidant best suited for lactose-intolerant consumer. Such type of products offers good functionality with respect to gut health and other health benefits. Chauhan et al. (2012) designed a functional herbal RTS beverage using herbal plants like Tinospora cordifolia and Ocimum (basil). Nutraceutical-rich extracts of these herbs were added to the fresh juice of sweet orange for the preparation of refreshing, thirst-quenching, and energizing ready-to-serve drink that not only improves the health but also fulfills the nutritional requirements. Herb-mixed beverage having formulation 6% basil and 1.5% T. cordifolia was found to be optimum among the other formulations. The above-optimized beverage can be stored effectively for 2 months. The extracts of these herbs could be used as a valuable ingredient for the production of herbal beverage with all the important properties and medicinal characteristics of tinospora and basil herbs. This can be a good health drink with phenolics and vitamin C as antioxidants. The functional beverages rich in antioxidants were developed by using high antioxidant capacity mixture of extruded whole maize (EMF) and chickpea (ECF) flours suitable to elaborate a nutraceutical beverage. Extruder operation conditions were extrusion temperature (ET, 120 C
170 C) and screw speed (SS, 120
200 rpm). The desirability method was applied to obtain optimum maximum values for the two response variables (AoxA 5 antioxidant activity, A 5 acceptability). The nutraceutical beverage could be used for health promotion and disease prevention (Jesus et al., 2012).

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Herbal beverage with functional characteristics was prepared using herbs such as Phyllanthus emblica, Terminalia chebula, Kaempferia parviflora, Centella asiatica, Nelumbo nucifera, Rauvolfia serpentina, Ginkgo biloba, Crocus sativus, and Clitoria ternatea which possess high antioxidant activity and anti-acetylcholinesterase activities (Suree et al., 2015). The herbal beverage was converted into alcoholic beverage by blending with grapes and in the ratio of 40:60. The fermented beverage was reported high in phytonutrients and antioxidant activity (95.99%) due to the addition of herbal beverage.

8.11

Prebiotic beverages

Prebiotics are nondigestible food ingredients that affect the host by selectively targeting the growth and/or activity of one or a limited number of beneficial bacteria in the colon, and thus have the potential to improve health (Gibson et al., 2004 ). Prebiotics are a very specific type of food. While many of the food ingredients we consume are digested immediately, prebiotics are a healthy nondigestible food ingredient. Furthermore, prebiotics are heat-resistant, which keep them intact during the baking process and allow them to be incorporated into everyday food choices. By consuming a nondigestible ingredient, it allows for the growth of bio-cultures by reaching the intestine unaffected by the digestion process. This can provide good digestive health. The positive effects prebiotics have by reaching the intestine in an unaltered form are known as the prebiotic effect. Prebiotics are dietary substances (mostly consisting of non-starch polysaccharides and oligosaccharides poorly digested by human enzymes) that nurture a selected group of microorganisms living in the gut. They favor the growth of beneficial bacteria over that of harmful ones. Buttermilk-based fermented drink using barley and fructo-oligosaccharide as functional ingredients were developed by Sheth and Hirdyani (2016). Barley was weighed (150 gm), washed, soaked in water for 30 minutes, and then cooked for 25 minutes. The cooked barley was then cooled at room temperature. Buttermilk (600 mL) was then added and blended in an electric mixer for 2 minutes to a smooth consistency, without lumps. The mixture was then sieved through 1000 μ sieve and kept for incubation at 35 C for 8 hours for fermentation. The process flowchart for the preparation of functional beverages from barley is given in Fig. 8.5 (Sheth & Hirdyani, 2016). Barley (150gms)

Soak (30 minutes)

Cook with 1000 ml of water (25 minutes)

Grind to a paste

Mix the ground millets with 600 ml buttermilk, stir continuously (10 minutes), and sieve (1000 Psieve)

Fermentation (8 hours at 35°C)

Addition of FOS (5%)

Addition of colors (0.3%) and flavours (0.3%) / Salt (0.4%) and jeera (0.6%)

Functional beverage (850 ml) Cool to 5°C and store in refrigerator (5-7°C) FIGURE 8.5 Process flowchart for preparation of functional beverage (Sheth & Hirdyani, 2016).

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Marina et al. (2016) formulated a cashew nut beverage with added mango juice and prebiotic substances by means of evaluating its sensory characteristics and physicochemical optimization. The means comparison test showed that the formulation that allows for the beverage’s greatest acceptance must contain 40% mango juice and 8% sugar. The beverage’s acidity was influenced only by the juice content, which, besides making formulations significantly more acid, did not affect their acceptance. Inulin is also used as a functional ingredient for the preparation of prebiotic beverages. It is a compound extracted from the chicory root and is a fructan of great importance commercially, with a fully proven prebiotic function. Among the health benefits for the host is the fact that fermentation by the bacteria produces short-chain organic acids, which are attributed the reduction effect of lipids and cholesterol and, therefore, a possible reduction in the risk of hypertension. A fermented drink by diversifying the quantities of L. acidophilus inoculums and prebiotic fiber in the form of inulin was developed by William et al. (2014) and used the total dry extract of whey and sucrose. After 28 days of storage period, the viable L. acidophilus cell counts had decreased for all treatments but were still above the minimum count of 7 log CFU/mL recommended by the Brazilian legislation. Soluble dietary fiber can be applied in different types of beverages such as energy drinks, sports drinks, carbonated beverages, and protein-based beverages in order to achieve enhanced functional properties (Beristain et al., 2006). Ausra et al. (2014) evaluated the rheology, technological, and sensory characteristics of fortified drink products with fibers. Yogurt was prepared with more benefits to health and to replace conventional stabilizers with newer, healthier ones. Orange pulp, pectin, bamboo, and cane fibers were utilized. This study evaluated the effect of the supplementation of the same dietary fiber on the syneresis, stability, pH, acidity, dry matter content, viscosity, and sensory evaluation of yogurt, juices, and juice drinks. Results indicate that the Citri-Fi fiber is an almost applicable ingredient for the design of new high value-added yogurt, juices, and juice drinks. Most probiotic foods at the markets worldwide are milk-based, and very few attempts are made to the development of probiotic foods using other fermentation substrates such as cereals like soybean and its derivatives. It has good potential for application in the functional food industry, because they contain a large quantity of components that are beneficial to health, such as proteins, isoflavones, fiber, essential fatty acids, oligosaccharides, etc. Tahis et al. (2014) evaluated chemical, sensory properties, and stability of a functional soy product with soy fiber and fermented with probiotic kefir culture. The product was characterized by chemical composition, color, and sensory analysis. The functional soy product presented better chemical composition and difference in color compared to the fermented product without fiber. Sensory analysis showed that the functional soy product had good acceptance and had better firmness and reduced syneresis compared to fermented product without fiber.

8.12

Sports or energy drinks

The sports nutrition market—currently worth an estimated $20 billion globally (Transparency Market Research Report, 2013
2019)—is in a phase of significant growth as it increasingly moves from a niche market into the mainstream. In Asia, the sports and energy drinks segment has expanded particularly quickly, with India and China recording average growth rates of almost 39% and 21%, respectively, between 2007 and 2012 (Ken Research, 2013). It was predicted that there will be a continuous strong increase in performance which is expected across the region, with predictions of a further 11.5% growth from 2012 to 2017 (Ken Research, 2013). As the segment grows more competitive and widespread, manufacturers are launching highly targeted beverages which offer a range of nutritional benefits to meet a variety of performance requirements. With proven benefits across a range of key areas of sports performance, collagen peptides are increasingly seen as the ideal ingredient by many manufacturers. The interest in fitness is continuously increasing in Indian and across Asia among the sports person. They are looking for effective protein-rich ingredients in sports drink to fulfill their requirements. To meet the levels of protein required to achieve their goals and ensure muscle regeneration, athletes commonly rely on sports nutrition beverages and bars. To ensure sufficient uptake, the protein used in these foods needs to be easily digestible and highly bioavailable. Collagen peptides are particularly attractive to integrate into high-protein formulations, combining the effect of different protein sources, to create optimal sports nutrition recovery products (Mai Nygaard, Global Product Manager, 2015). Date palm and watermelon fruit with the addition of sucrose, glucose, sodium chloride, ascorbic acid, sodium metabisulfite, and water were used in the production of isotonic sports drink, and a commercial sports drink was used as the reference sample by Oluwole et al. (2019). The energy, carbohydrate, osmolality, and pH of the drink were analyzed using standard methods. A significant difference was observed in the energy, pH, and osmolality content of the drinks, but no significant difference was observed between the carbohydrate content of the drinks. The developed drink had carbohydrate

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content of 7.3%, energy of 30.1 Kcal, pH of 3.7, and osmolality of 323 mOsm/kg H2O while the commercial sports drink had carbohydrate content of 6.7%, energy of 26.6 Kcal, pH of 3.1, and osmolality of 290 mOsm/kg H2O.

8.13

Storage study of beverages

The functional beverages should have a long shelf life with good overall acceptability in terms of taste and quality. The shelf life of functional beverages depends on the combination and level of ingredients added with the types of processing done. Bhuiyan et al. (2012) conducted the experiment to develop functional beverage based on taste preference and studied its storage life. The study showed that sweetness, sourness, and their interaction have significant effect on overall acceptability in the development of functional beverage. Organoleptic test indicated that men and women preferred sweet and sour beverage, respectively. TSS and pH showed linear whereas acidity and vitamin C concentration showed an inverse relation with storage period. The types of packaging materials used for packing and the addition of preservatives in beverages also decided the storage stability of functional beverages. The study was carried out to find out optimum levels of juice and other ingredients for the preparation of RTS beverages and to evaluate shelf life. The RTS was stored with six treatments in three different packaging materials, glass bottles, standing pouch, and PET bottles. The RTS beverages were stored at ambient temperature (27 C 6 5 C) and cold temperature (5 C 6 2 C). The RTS with pasteurization 1 100 ppm sodium benzoate stored at a cold temperature was found to be more acceptable as compared to other samples stored at ambient temperature after 90 days of storage (Khade, 2015). Khasanov and Matveeva (2020) determined the shelf life of a functional beverage based on plant raw materials by accelerated testing based on the Arrhenius model used. As a controlled indicator, the concentration of anthocyanins is selected, which determines the functionality of beverage. The test was carried out at a temperature of 50 C. The control sample was stored at a temperature of 20 C. Fixing the value of anthocyanins was performed every 3
4 days. As a result, the critical value was the value of anthocyanins on day 14. It fell by 55% from the original value. Thus, according to the calculations and the law of Arrhenius, the shelf life of the drink was 3.7 months.

8.14

Health safety of drinks

The health safety of any food product is prime consideration for the consumer, and in the case of beverage, it mostly depends on the content of beverage, the microorganism involved, and the packaging material used. It is essential to mention the ingredients, level, and date of manufacture on the level of beverage. The beverage manufacturer should also follow national and international standards for the preparation of beverages (Dorota, 2015).

8.15

Consumer demand for beverages

Nowadays, consumers are demanding the products which are innovative and enhance health benefits. Drinks like flavored waters, isotonics, yogurt drinks, energy drinks, functional RTD teas, and products similar to the segment will be shaping the future market of the beverage industry in comparison to the traditional products like 100% juice, colas, milk, teas, and others. In the market, different types of beverages are available which are more attractive and expensive but less effective as synthetic beverage. At present time, there is demand for low-cost health beverage as people always avoid the synthetic beverage from the standpoint of hypertension, diabetes, and obesity. So, a beverage that satisfies need-based demand of the majority of the population including those suffering from obesity, hypertension, and diabetes should be developed.

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2153.

Chapter 9

Recent targeted discovery of phytomedicines to manage endocrine disorder develops due to adapting sedentary lifestyle Vijeta Bhattacharya1,2, Namrata Mishra2, Radha Sharma3, Subodh Kumar Dubey1, Balakumar Chandrasekaran4 and Mohammad F. Bayan4 1

School of Pharmacy, ITM University, Gwalior, Madhya Pradesh, India, 2IPS College of Pharmacy, Shivpuri Link Road, Gwalior, Madhya Pradesh,

India, 3Shriram College of Pharmacy, Banmore, Madhya Pradesh, India, 4Faculty of Pharmacy, Philadelphia University, Amman, Jordan

9.1

Introduction

9.1.1 Introduction of endocrine glands and endocrine hormones. What is endocrine system 9.1.1.1 The endocrine system and disorders 9.1.1.1.1

The endocrine system

The endocrine system is a series of glands that produce and secrete hormones that the body uses for a wide range of functions. These control many different bodily functions, including: 1. 2. 3. 4. 5. 6. 7.

respiration, metabolism, reproduction, sensory perception, movement, sexual development, and growth.

Hormones are produced by glands and sent into the bloodstream to the various tissues in the body. They send signals to those tissues to tell them what they are supposed to do. When the glands do not produce the right amount of hormones, diseases develop that can affect many aspects of life (Sinha et al., 2013).

9.1.1.2 The main hormone-producing glands 1. Hypothalamus: The hypothalamus is responsible for body temperature, hunger, moods, and the release of hormones from other glands and also controls thirst, sleep, and sex drive. 2. Pituitary: Considered the "master control gland," the pituitary gland controls other glands and makes the hormones that trigger growth. 3. Parathyroid: This gland controls the amount of calcium in the body. 4. Pancreas: This gland produces the insulin that helps control blood sugar levels. 5. Thyroid: The thyroid produces hormones associated with calorie burning and heart rate. 6. Adrenal: Adrenal glands produce the hormones that control sex drive and cortisol, the stress hormone. 7. Pineal: This gland produces melatonin which affects sleep. Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00021-9 © 2023 Elsevier Inc. All rights reserved.

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8. Ovaries: Only in women, the ovaries secrete estrogen, testosterone, and progesterone, the female sex hormones. 9. Testes: Only in men, the testes produce the male sex hormone, testosterone, and produce sperm. Some of the factors that affect endocrine organs include aging, certain diseases and conditions, stress, the environment, and genetics.

9.1.1.3 Factors which affect endocrine disorders Everyone’s body undergoes changes, some natural and some not, that can affect the way the endocrine system works (Sicree et al., 2006). Some of the factors that affect endocrine organs include puberty, aging, pregnancy, the environment, genetics, and certain diseases and medications, including naturopathic medicine, herbal supplements, and prescription medicines such as opioids or steroids. 9.1.1.3.1 Aging Despite age-related changes, the endocrine system functions well in most older people. However, some changes occur because of either damage to cells during the aging process or medical issues that the aging body accumulates, or genetically programmed cellular changes. These changes may alter the following: 1. 2. 3. 4. 5.

hormone production and secretion, hormone metabolism (how quickly hormones are broken down and leave the body), hormone levels circulating in blood, target cell or target tissue response to hormones, rhythms in the body, such as the menstrual cycle.

For example, increasing age is thought to be related to the development of type 2 diabetes, especially in people who might be at risk for this disorder. The aging process affects nearly every gland. With increasing age, the pituitary gland (located in the brain) can become smaller and may not work as well, which although may provide sufficient hormonal signaling for the continuity of life. For example, the production of growth hormone might decrease, which is likely not a priority in an aging individual; this is also an example of genetic programming that we have evolved as species to adapt to. Decreased growth hormone levels in older people might lead to problems such as decreased lean muscle, decreased heart function, and osteoporosis. Aging affects a woman’s ovaries and results in menopause, usually between 50 and 55 years of age. In menopause, the ovaries stop making estrogen and progesterone and no longer have a store of eggs. When this happens, menstrual periods stop. 9.1.1.3.2 Diseases and conditions Chronic diseases and other conditions may affect endocrine system function in several ways. After hormones produce their effects on their target organs, they are broken down (metabolized) into inactive molecules. The liver and kidneys are the main organs that break down hormones. The ability of the body to break down hormones may be decreased in people who have chronic heart, liver, or kidney disease. Abnormal endocrine function can result from: 1. 2. 3. 4. 5. 6. 7.

congenital (birth) or genetic defects (see section on genetics below), surgery, radiation, or some cancer treatments, traumatic injuries, cancerous and noncancerous tumors, infection, autoimmune destruction (when the immune system turns against the body’s organs and causes damage), medications or supplements.

In general, the abnormal endocrine function creates a hormone imbalance typified by too much or too little of a hormone. The underlying problem might be due to an endocrine gland making too much or too little of the hormone, or to a problem breaking down the hormone. 9.1.1.3.3

Stress

Physical or mental stressors can trigger a stress response. The stress response is complex and can influence heart, kidney, liver, and endocrine system function. Many factors can start the stress response, but physical stressors are the most

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important. For the body to respond to, and cope with physical stress, the adrenal glands make more cortisol. If the adrenal glands do not respond, this can be a life-threatening problem. Some medically important factors causing a stress response are: 1. 2. 3. 4. 5. 6.

trauma (severe injury) of any type, severe illness or infection, intense heat or cold, surgical procedures, serious diseases, allergic reactions.

Other types of stress include emotional, social, or economic, but these usually do not require the body to produce high levels of cortisol to survive the stress. 9.1.1.3.4

Environmental factors

An environmental endocrine-disrupting chemical (EDC) is a substance outside of the body that may interfere with the normal function of the endocrine system. Some EDCs mimic natural hormone binding at the target cell receptor. (Binding occurs when a hormone attaches to a cell receptor, a part of the cell designed to respond to that particular hormone.) EDCs can start the same processes that the natural hormone would start. Other EDCs block normal hormone binding and thereby prevent the effects of the natural hormones. Still, other EDCs can directly interfere with the production, storage, release, transport, or elimination of natural hormones in the body. This can greatly affect the function of certain body systems. EDCs can affect people in many ways: 1. 2. 3. 4. 5.

disrupted sexual development, decreased fertility, birth defects, reduced immune response, neurological and behavioral changes, including reduced ability to handle stress.

9.1.1.3.5 Genetics Your endocrine system can be affected by genes. Genes are units of hereditary information passed from parent to child. Genes are contained in chromosomes. The normal number of chromosomes is 46 (23 pairs). Sometimes extra, missing, or damaged chromosomes can result in diseases or conditions that affect hormone production or function. The 23rd pair, for example, is the sex chromosome pair. A mother and father each contribute a sex chromosome to the child. Girls usually have two X chromosomes while boys have one X and one Y chromosome. Sometimes, however, a chromosome or piece of a chromosome may be missing. In Turner syndrome, only one normal X chromosome is present, and this can cause poor growth and a problem with how the ovaries function. In another example, a child with Prader
Willi syndrome may be missing all or part of chromosome 15, which affects growth, metabolism, and puberty. Your genes also may place you at increased risk for certain diseases, such as breast cancer. Women who have inherited mutations in the BRCA1 or BRCA2 gene face a much higher risk of developing breast cancer and ovarian cancer compared with the general population (Table 9.1).

9.1.2 Introduction of endorinological disorders such as thyroid, diabetes mellitus, polycystic ovarian syndrome 1. Thyroid and polycystic ovarian syndrome Thyroid disorders and polycystic ovary syndrome (PCOS) are two of the most common endocrine disorders in the general population. Although the etiopathogenesis of hypothyroidism and PCOS is completely different, these two entities have many features in common. An increase in ovarian volume and cystic changes in ovaries have been reported in primary hypothyroidism. On the other direction, it is increasingly realized that thyroid disorders are more common in women with PCOS as compared to the normal population (Sirmans and Pate, Epidemiology; Tehrani et al., 2011). Whether this is due to some common factors predisposing an individual to both disorders or due to a pathophysiological

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TABLE 9.1 Hormones synthesized and secreted by dedicated endocrine glands. S. no

Glands

Hormones

1

Pituitary gland

Growth hormones Prolactin Adrenocorticotropic hormone Thyroid-stimulating hormone Follicle-stimulating hormone Luteinizing hormone

2

Thyroid gland

Tetraiodothyronine (T4; thyroxine) Triiodothyronine (T3) Calcitonin

3

Parathyroid gland

Parathyroid hormone

4

Islets of Langerhans (endocrine pancreas)

Insulin Glucagon Somatostatin

5

Adrenal gland

Epinephrine Norepinephrine Cortisol Aldosterone Dehyroepiandrosterone sulfate

6

Hormones synthesized by gonadsa. Ovariesb. Testes

Estradioal-17beta Progesterone Inhibin Testosterone Anti-Mullerian hormone Inhibin

connection between the two disorders has not been established until now. The purpose of descriptive and exploratory reviews is to explore the relationship between these two disorders. To generate a hypothesis linking these disorders, the terms “PCOS,” “autoimmunity,” “subclinical hypothyroidism,” “thyroid autoimmunity,” “thyroid autoantibodies,” “leptin,” obesity,” and “thyroiditis” were searched in various combinations in PubMed, Google Scholar, and Embase. Relevant articles from this search as well as from cross-references were retrieved and included. Elucidation of this relationship between thyroid disorders and PCOS requires answers to two questions: What happens to ovaries in thyroid disorders? (2) What happens to thyroid in PCOS?

9.1.2.1 What happens to ovaries in thyroid disorders? This question is relatively easier to answer, and the pathways leading to change in ovarian morphology in hypothyroidism are well-known. In the presence of hypothyroidism, ovarian morphology becomes polycystic. Hence, thyroid disorders are one of the exclusion criteria before making a diagnosis of PCOS in any woman. The underlying pathophysiology has been outlined in Fig. 9.1. Rise in thyrotropin
thyrotropin hormone (TRH) in primary hypothyroidism leads to increased prolactin and thyroid-stimulating hormone (TSH). Prolactin contributes toward polycystic ovarian morphology by inhibiting ovulation as a result of the change in the ratio of follicle-stimulating hormone (FSH) and luteinizing hormone and increased dehydroepiandrosterone from the adrenal gland. Increased TSH also contributes due to its spill-over effect on FSH receptors. Increased collagen deposition in ovaries as a result of hypothyroidism has also been suggested. The severity of ovarian morphology also depends on the duration and severity of underlying primary hypothyroidism. In most severe cases like long-standing untreated cases of congenital hypothyroidism, ovarian morphology can be very striking and can even be mistaken for ovarian malignancies. These cases have been given an eponym Van Wyk and Grumbach syndrome, after the scientist who first described the case (Aurora & Punjabi, 2013). In a study, on somewhat less severe primary hypothyroidism, by treatment naı¨ve females with primary hypothyroidism, with mean TSH 57.1 mcg/dl, underwent evaluation of ovarian volume before and after replacement with thyroxine

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FIGURE 9.1 Pathophysiology of thyroid disorder.

(Low, 2011). Twenty-six healthy normal controls were also recruited. Ten of 26 hypothyroid females had polycysticappearing ovaries on ultrasound sonography test at baseline. All women with primary hypothyroidism had significantly higher ovarian volumes than controls. Even the subgroup without polycystic-appearing ovaries had significantly higher ovarian volumes. However, there was no correlation of TSH levels with cyst formation. The most remarkable finding of this study was the normalization of ovarian volume in all patients, with or without polycystic-appearing ovaries, after the replacement of thyroxine (Low, 2011; Taylor & Ashwal, 2012). These girls also had higher hirsutism score, the lower number of annual menstrual cycles, and higher insulin resistance score. However, two factors need to be considered before drawing any conclusion from this study. First, there seems to be a cosegregation of PCOS and CLT cases, leading to the classic “which comes first, the chicken or the egg?” dilemma: whether autoimmune thyroiditis predisposes subjects to develop characteristics suggestive of PCOS or whether PCOS is a forerunner of autoimmune thyroiditis remains speculation. Second, this study recruited adolescents with a mean age of 14.7 years (Low, 2011). In this age group, the diagnosis of PCOS is not always tenacious, as normal girls of this age may have polycystic-appearing ovaries, acne, and menstrual abnormalities. In summary, therefore hypothyroidism can lead to polycystic morphology of the ovaries. While this morphology can vary with the severity and duration of hypothyroidism, there is no evidence to suggest that primary hypothyroidism can lead to PCOS.

9.1.2.2 What happens to thyroid in polycystic ovary syndrome? The prevalence of subclinical thyroid function in the general population has been estimated at around 10%, but in reproductive years this prevalence is considerably low at 4%
6% (Nammi, The; Singh et al., Effect). In recent years, a number of publications have reported an increased incidence of thyroid disorders in females with PCOS. Sinha et al. compared 80 PCOS females with 80 controls and found a significant higher prevalence of goiter (27.5% vs 7.5%) and subclinical hypothyroidism (22.5% vs 8.75%) in PCOS patients as compared to controls. Another study conducted in young women with PCOS found prevalence of subclinical hypothyroidism (defined as TSH . 4.5 IU/mL) to be 11.3% (mean TSH level leads to PCOS of 6.1 6 1.2 mIU/L). There was no difference in two groups (with or without subclinical hypothyroidism) with respect to BMI, waist circumference, or Ferriman
Gallwey score (Singh et al., Effect). Lowdensity lipoprotein cholesterol (LDL-C) was found to be significantly higher in the cohort with subclinical hypothyroidism. The pathophysiological pathway connecting these two disorders has not been clearly delineated as of now. The most obvious connection, perhaps, is the increased BMI and insulin resistance common to both conditions. Increase in BMI is an integral part of PCOS and is seen in a large majority (54%
68%) of these cases (Bisht and Sisodia, Anti). The link between thyroid functions and obesity is again an interesting one, with unclear pathophysiological mechanisms; there is, however, enough evidence to say that TSH is higher in people with high BMI (Asgary et al., 2012; Khan et al., Cinnamon). The proposed link is depicted in Fig. 9.2. Obesity is associated with an altered milieu with increase in proinflammatory markers and increase in insulin resistance. This, through undefined mechanisms, leads to decreased deiodinase-2 activity at the pituitary level resulting in relative T3 deficiency and increase in TSH levels (Asgary et al., 2012). Another pathway, based on leptin, has been hypothesized to explain this observation. Increased leptin in obesity has been proposed to act directly on the hypothalamus resulting in increased TRH secretion (Eddouks et al., 2005). Raised TSH levels, with any of these two pathways, act on adipocytes to increase their proliferation. In culture studies, TSH has been shown to increase the proliferation of adipocytes as well as increase in production of proinflammatory markers from adipocytes, acting on TSH receptors present in adipocytes. Muscogiuri et al. recently studied 60 euthyroid subjects to find a correlation of TSH (within normal range) to either adipose tissue or insulin resistance. In univariate analysis, both adiposity and insulin resistance were significantly associated with raised TSH, but after multivariate regression, visceral adipose tissue volume was found to be the only predictor of TSH (P 5 0.01) (Asgary et al., 2012). Another interesting observation about TSH-lowering effect of metformin has been reported in both PCOS and non-

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FIGURE 9.2 Pathophysiology of polycystic ovaries in patients with primary hypothyroidism.

FIGURE 9.3 Hypothesis linking adiposity and raised thyroidstimulating hormone.

PCOS populations (Samarghandian et al., 2012; American Thyroid Association, 2013). Metformin has been shown to lower TSH in persons with clinical and subclinical hypothyroidism, but not in euthyroid people (Samarghandian et al., 2012). However, there is not enough evidence to suggest that this TSH-lowering effect of metformin is mediated by lowering insulin resistance. A change in the affinity or in the number of TSH receptors, an increase in the central dopaminergic tone, and the direct effect of metformin on TSH regulation have been proposed as potential explanations (American Thyroid Association, 2013). The pitfall of this pathway is that it fails to explain the increased incidence of thyroid autoimmunity in patients with PCOS. Thyroid autoimmunity is increased in patients with PCOS. Females with PCOS have higher thyroid antibody levels and larger thyroid volumes, and their thyroids are more hypoechogenic (compatible with thyroiditis) (Fig. 9.3). 2. Diabetes mellitus Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion or action, or both. Diabetes mellitus, commonly referred to as diabetes (as it will be in this article), was first identified as a disease associated with "sweet urine," and excessive muscle loss in the ancient world. Elevated levels of blood glucose (hyperglycemia) lead to spillage of glucose into the urine, hence the term sweet urine. Normally, blood glucose levels are tightly controlled by insulin, a hormone produced by the pancreas. Insulin lowers the blood glucose level. When the blood glucose elevates (e.g., after eating food), insulin is released from the pancreas to normalize the glucose level. In patients with diabetes, the absence or insufficient production of insulin causes hyperglycemia.

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Blood sugar level in normal and diabetic patients. Category of a person

Fasting value

Normal Early diabetes Established diabetes

Minimum value 70 101 More than 126

Postprandial Maximum value 100 126


Value 2 h after consuming glucose Less than 140 140 to 200 More than 200

*All values are in mg/100 mL.

9.1.2.3 Causes of diabetes Insufficient production of insulin (either absolutely or relative to the body’s needs), production of defective insulin (which is uncommon), or the inability of cells to use insulin properly and efficiently lead to hyperglycemia and diabetes. This latter condition affects mostly the cells of muscle and fat tissues and results in a condition known as "insulin resistance." This is the primary problem in type 2 diabetes (Inzuchi et al., 1998; Khanfar et al., 2003). The absolute lack of insulin, usually secondary to a destructive process affecting the insulin-producing beta cells in the pancreas, is the main disorder in type 1 diabetes. In type 2 diabetes, there also is a steady decline of beta cells that adds to the process of elevated blood sugars. Essentially, if someone is resistant to insulin, the body can, to some degree, increase the production of insulin and overcome the level of resistance. After time, if production decreases and insulin cannot be released as vigorously, hyperglycemia develops. Glucose is a simple sugar found in food. Glucose is an essential nutrient that provides energy for the proper functioning of the body cells. Carbohydrates are broken down in the small intestine, and the glucose in digested food is then absorbed by the intestinal cells into the bloodstream and is carried by the bloodstream to all the cells in the body where it is utilized. However, glucose cannot enter the cells alone and needs insulin to aid in its transport into the cells. Without insulin, the cells become starved of glucose energy despite the presence of abundant glucose in the bloodstream. In certain types of diabetes, the cells’ inability to utilize glucose gives rise to the ironic situation of "starvation in the midst of plenty." The abundant, unutilized glucose is wastefully excreted in the urine. Insulin is a hormone that is produced by specialized cells (beta cells) of the pancreas. (The pancreas is a deep-seated organ in the abdomen located behind the stomach.) In addition to helping glucose enter the cells, insulin is also important in tightly regulating the level of glucose in the blood. After a meal, the blood glucose level rises. In response to the increased glucose level, the pancreas normally releases more insulin into the bloodstream to help glucose enter the cells and lower blood glucose levels after a meal. When the blood glucose levels are lowered, the insulin release from the pancreas is turned down (Matthaus & Ozcan, 2005). It is important to note that even in the fasting state there is a low steady release of insulin that fluctuates a bit and helps to maintain a steady blood sugar level during fasting. In normal individuals, such a regulatory system helps to keep blood glucose levels in a tightly controlled range. As outlined above, in patients with diabetes, the insulin is either absent, relatively insufficient for the body’s needs, or not used properly by the body. All of these factors cause elevated levels of blood glucose (hyperglycemia)

9.1.3 Why endocrine disorder occurs due to sedentary lifestyle 1. Leading a sedentary lifestyle is becoming a significant health issue nowadays. Sedentary lifestyles become visible to be increasingly widespread in many nations which can lead to a chronic health situation. Most importantly, leading a sedentary lifestyle may lead to endocrinological disorders. The endocrine system is a network of glands and organs located throughout the body. The main function of the endocrine system is to regulate the range of bodily functions through the release of hormones. When the function of the endocrine system is disrupted, then it may lead to hormonal imbalance. For the management of major endocrinological disorders such as diabetes, thyroid, PCOS (female hormonal imbalance), many ethnomedicinal herbs are used. This present work aimed to focus on ethnomedicinal herbs to target the endocrine glands for major endocrinological disease management. Ethnomedicinal herbs are the traditional medicinal herbs or we can say ethnic medicinal herbs which are used for the treatment of many wide varieties of diseases.

9.1.3.1 Rationale 1. Endocrine disorder is a noncommunicable disease that can occur due to hormonal imbalance. 2. Endocrine system consists of different types of glands which are situated in different parts of the body. The main function of these glands is to secrete hormones.

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3. When there is overproduction and less production of hormones by the endocrine glands, this may lead to endocrine disorder. 4. The endocrine disruption mainly occurs due to having sedentary lifestyle. 5. Nowadays, due to leading a sedentary lifestyle most people worldwide are facing endocrine disorders due to hormonal imbalances. 6. Different phytomedicinal herbs are there which can easily manage the major endocrinological disorder which is very common nowadays. 7. In today’s world, people are facing problems due to endocrine disorders. If endocrine disruption occurs in our body, it may lead to hormonal imbalance, and hormonal imbalance in our body may lead to major lifestyle diseases such as 1. Type-2 diabetes, 2. Hypothyroidism and hyperthyroidism, 3. PCOS (female hormonal imbalance). This above disease is very much spreading nowadays only because of sedentary lifestyle which is the major cause of the endocrinological disorder. 9.1.3.1.1 Endocrine glands and endocrine hormones The endocrine system is a series of glands that produce and secrete hormones that the body uses for a wide range of functions. These control many different bodily functions, including: 1. 2. 3. 4. 5. 6. 7.

respiration, metabolism, reproduction, sensory perception, movement, sexual development, and growth.

Hormones are produced by glands and sent into the bloodstream to the various tissues in the body. They send signals to those tissues to tell them what they are supposed to do. When the glands do not produce the right amount of hormones, diseases develop that can affect many aspects of life.

9.1.4 Relation between endocrine disorder and sedentary lifestyle The country has been undergoing a rapid transition in health over the past several decades—a shift from infectious diseases to noncommunicable diseases (NCDs). This burden of NCDs had to be matched with appropriate responses in research, and ICMR decided to set up National Centre for Disease Informatics and Research (NCDIR), Bengaluru, in 2011 with the purpose of collection, analysis, and reporting of etiological, clinical, diabetes, cardiovascular diseases, stroke, and other determinants. The ICMR National Cancer Registry Program (NCRP), since 1982, collects and reports data on cancer incidence, burden, and trends. Additionally, registries on stroke and heart failure and endocrine disorders have also been initiated in 2017 along with a national survey on NCD risk factors. NCDIR functions in a disease informatics hub for noncommunicable diseases to inform policy, program, and decision-making (Damsch et al., 2011b; Dunaif, 1997; Mueller et al., 2009; Speroff, 2011). ICMR along with the Public Health Foundation of India (PHFI) and Institute for Health Metrics and Evaluation (IHME) published state-level disease burden reports mapping the pattern of diseases burden from 1990 to 2016 which showed the rising burden due to noncommunicable diseases. Over the past century, India has transitioned from an era dominated by disease burden attributed to infectious diseases, childhood and maternal deaths to an era of lifestyle diseases-related chronic diseases. Noncommunicable diseases (NCDs): In 2017, India witnessed 61.8% death due to noncommunicable diseases. These are a group of chronic diseases that begin in an early phase of life and continue to progress if not appropriately intervened over the span of life leading to sickness and untimely death. The rise in obesity among adults and children in the country is propelled by over-consumption of food high in fats, carbohydrates, and salt and thus seeks our immediate attention to limit its damages due to diabetes, heart diseases, and endocrine disease. It is estimated that there are 19% overweight men and 21% women and 2% overweight and obese children in the country (Huang & Matthew Peterson, 2007; Nada, 2013).

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9.1.5 Physiology and mechanism behind endocrine disorder Thyroid-stimulating hormone (thyrotropin) (TSH)—TSH is synthesized and released from cells within the anterior pituitary gland, known as thyrotrophs. This hormone is composed of two subunits: one alpha and one beta. TSH is one of the four hormones which share the same alpha unit. The beta unit is what makes TSH unique and determines its specificity within the human body. Due to the physiologic effects of thyroxine (T4) and triiodothyronine (T3), these two hormones will help tightly control the levels of TSH released into the body. Minute increases in serum T3 and T4 will result in TSH inhibition. Conversely, small decreases in serum T3 and T4 result in increased TSH. T3 and T4 levels will also work to increase/decrease TRH through a negative feedback look, another mechanism for modulating TSH levels. TSH levels will slowly change depending on several factors, such as the initial TSH level, the hormone given (T3 or T4), and the dose of the hormone given. A higher TSH level will take longer to decrease and will gradually decline over several days. TSH levels will respond faster to T3 than T4 (Ganie et al., 2011; Gasbarrone & Franzese, 2005; Roos et al., 2007; Uzunlulu et al., 2007). Additionally, when given a higher dose, TSH will respond more rapidly. If high doses of T3 are administered, TSH levels will begin to decline over the course of several hours in hypothyroid patients. Other inhibitors of TSH include somatostatin, dopamine, and glucocorticoids. Dopamine can cause a rapid decrease in TSH levels, and accordingly, dopamine antagonists can acutely raise TSH levels. Patients in the intensive care unit (ICU) often have altered TSH levels when receiving dopamine or dopamine antagonists. TSH is one of the four endocrine hormones (hCG, TSH, LH, FSH) that all share the same alpha unit. There are pathologic states, such as choriocarcinoma, where the severe elevation of HCG leads to hyperthyroidism symptoms because TSH receptors bind HCG due to the shared alpha unit. It is discussed in detail here. TSH is an essential hormone for the thyroid. It stimulates each step in hormone synthesis within the thyroid, affects the expression of multiple genes, and can cause thyroid hyperplasia/hypertrophy. The action begins when TSH binds to a plasma membrane receptor, activating adenylyl cyclase, which increases cyclic adenosine monophosphate (cAMP), resulting in the activation of several protein kinases. Via the same receptor, TSH stimulates phospholipase C, increasing phosphoinositide turnover, protein kinase C activity, and intracellular calcium concentration. How the above steps specifically link to T3 and T4 synthesis, release, and other thyroid metabolic processes are not fully understood.

9.1.5.1 Thyroid gland General T3/4 actions—thyroid hormones are crucial throughout the entire life of an individual. In childhood development, thyroid hormones help develop several body systems, particularly the brain. In adulthood, the thyroid hormones help drive metabolic activity and function of nearly all organs. Since it is necessary for so many different systems, a constant thyroid hormone supply is required, yet the total serum levels are always tightly controlled; if not, pathology will occur. Two mechanisms control the production of thyroid hormones. The first is through hormonal pathways and negative feedback loops. Levels of TRH, TSH, T4, and T3 will signal the thyroid whether to increase or decrease thyroid hormone levels. The second is via hormone consumption by extrathyroidal tissues based on nutritional, hormonal, and illness-related factors—the effect varies depending on the tissue. The first mechanism helps protect the thyroid from hyper/hypo-secreting, and the second mechanism helps respond to rapid changes within the tissue. As previously mentioned in the TSH section, there are two thyroid hormones, T3 and T4. However, before either hormone can begin synthesis, iodide must undergo oxidation to iodine and become incorporated into tyrosine residues within the colloid. Iodide is an essential ion in thyroid physiology and will be discussed again in pathophysiology. The remaining steps of hormone synthesis include combining two diiodotyrosine (DIT) molecules to make T4 or combining one monoiodotyrosine and one DIT to create T3. Thyro-globin is a glycoprotein that incorporates into exocytotic vesicles which fuse to the apical cell membrane—only when these steps have occurred can iodination and coupling of T4 and T3 happen. To release these hormones into the extracellular fluid and eventually circulation, thyroglobulin must be resorbed into the thyroid follicular cells to create colloid droplets. These colloid droplets fuse with lysosomes to create phagolysosomes, allowing hydrolyzation of the thyroglobulin allowing hormone secretion. T4 (99.95%) and T3 (99.5%) are mostly bound within the bloodstream, preventing it from being metabolically active. The proteins that bind T4 and T3 listed in most common to least common are as follows: thyroxine-binding globulin (TBG), transthyretin (TTR), albumin, and lipoproteins. The remaining 0.02% of free T4 leaves only 2 ng/dL within the body. Similarly, 0.05% of T3 leaves only 0.4 ng/dL. Since most T4 and T3 are bound within the serum, changes in serum concentrations of binding proteins result in drastic effects on serum total T4 and T3. However, changes in binding proteins do not affect the free hormone concentrations or the rate T4, and T3 gets metabolized.

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9.1.5.2 Thyroxine (T4) T4 is less metabolically active and produced exclusively within the thyroid. The daily production rate is 80 to 100 mcg and degraded at roughly 10% per day. Approximately, 80% is de-iodinated—of this, 40% converts to T3, and the other 40% converts to reverse T3 (rT3). The final 20% conjugates to tetraiodothyroacetic. The conversion of the periphery of T4 to T3 is mediated via the enzyme 5’-deiodinase. T3 is the primary metabolite of T4, which has physiologic activity; other T4 metabolites are inactive. This conversion process is regulated in extrathyroidal tissue. Thus, T3 production may change independently of the pituitary-thyroid state.

9.1.5.3 Triiodothyronine (T3) T3 is the primary metabolic hormone from the thyroid and is the driver behind metabolic and organ processes. About 80% of T3 production is in extra thyroid tissue from the deiodination of T4. The remaining 20% gets synthesized within the thyroid. Daily production is 30 to 40 mcg, but the extrathyroidal reserve of T3 is roughly 50 mcg. The fraction of T3 produced throughout the body from T4 varies considerably from tissue to tissue. Certain tissues like the anterior pituitary and liver contain high levels of T3 nuclear receptors, making them more responsive to serum T3. T3 acts by modifying gene transcription. Due to the wide-reaching effects of T3, it affects nearly all tissues and ability to synthesize protein and turnover substrate. The nuclear actions of T3 will depend on four factors: availability of hormone, thyroid hormone nuclear receptors (TRs), availability of receptor cofactors, and DNA regulatory elements. Within most tissue, T3 enters by simple diffusion. However, in the brain and thyroid, T3 is actively transported into cells. Depending on the tissue, T3 will have different actions, which are determined by the local production of T3 and the quantity and distribution of TR isoforms. The isoforms consist of TR-alpha-1 and 2 and TR-beta-1, 2, and 3. There are insufficient studies on the TR isoforms, but due to regional or cell-specific distributions of the TRs, it is suggestive of different functions even within the same tissue. For example, TR-alpha is the dominant isoform in the brain, but TR-beta-2 is present at very high levels within the hypothalamus and pituitary. The data that do exist for TR isoforms come primarily from knockout mice with TR gene point mutations. Mice with TR-alpha deletion show poor feeding and growth, slowed heart rate, low basal body temperature, and reduced bone mineralization. Mice with inactivation of the TR-beta gene showed indications of inappropriately normal serum TSH levels, high serum T4 concentrations, and thyroid gland hyperplasia. Finally, knockout mice without both TR-alpha and beta genes showed thyroid hyperplasia and markedly high serum concentrations of T4, T3, and TSH—which were 11 times, 30 times, and 160 times greater than normal, respectively. As previously mentioned, T3 binds to TR on the nucleus resulting in the modulation of gene expression. All genes affected have specific DNA sequences that bind TR with high affinity. Ultimately, the human genome project provided data that allows specific DNA sequences to be identified. Without these specific DNA sequences, T3-dependent gene activation will be minimal or absent completely. Different tissues have one of three deiodinases within the periphery that convert the prohormone T4 to active T3 of which three enzymes will be expressed depending on a specific pattern of development and tissue type [68,71]. 1. Type 1 5’-deiodinase (Dio1) is found primarily in the liver, kidneys, and muscle. Dio1 was found to have reduced activity in hypothyroid subjects. 2. Type 2 5’-deiodinase (Dio2) was studied in rodents and displayed a higher prevalence in the cerebral cortex, brown fat, and the pituitary. In humans, Dio2 also expresses in skeletal muscle, heart, and thyroid. Within humans, Dio2 produces the majority of circulating T3. Dio2 was found to increase in hypothyroid and iodine-deficient subjects. 3. Type 3 5’-deiodinase (Dio3) actually inactivates T4 and is found primarily in the placenta, skin, skeletal muscle, and the developing brain. It is essential for sensory development, particularly within the inner ear. During human development, Dio3 is expressed first; as Dio1 and Dio2 increase, Dio3 expression will decrease. 4. It is well known that T4 and T3 have wide-reaching effects and can influence nearly every organ system within the body, specifically, three major areas include bones, heart, and metabolism regulation (Hosseinpanah et al., 2011; Longe et al., 2005; Patwardhan et al., 2004; Rates, 2001; Robinson & Zhang, 2011; Sheng-Ji, 2001; Tuzcu et al., 2005). 5. Bones—Patients in infancy who were born with congenital hypothyroidism and are not treated with hormone replacement will have delayed epiphyseal development and poor growth. This same finding was present in patients with thyroid hormone resistance. All TR isoforms are expressed in bones. However, TR-alpha and alpha/beta knockout mice showed abnormal bone development. 6. Heart—Patients who have T3 resistance will demonstrate elevated T3 levels, resulting in tachycardia. This likely demonstrates that patients who are resistant to T3 will not have cardiac resistance. Patients who have TR-beta

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8. 9.

10.

11.

12.

13.

14.

15.

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mutations will phenotypically present this way, a concept supported by T3-beta knockout mice that also do not have cardiac resistance to T3. Metabolism regulation—T3 regulates the metabolic rate and can influence modest body weight changes. Humans with TR-beta mutations and T3 resistance demonstrate increased T3-alpha activity—this stimulation results in increased feeding and increased fatty acid oxidation. T3 also influences glucose metabolism, increasing its uptake. When Dio2 is not functioning properly, an association with glucose intolerance is evident. It is not yet well studied, but patients with impaired mitochondrial oxidative metabolism (seen in metabolic syndrome and type 2 diabetics) may have reduced T3 hormone action. The complications of improper metabolism will be discussed in thyroid hormone pathology. Pancreas Insulin—Directly and indirectly, insulin affects several tissues; however, this article focuses on adipose tissue, muscle, and the liver. Insulin is a 51-amino acid peptide that is synthesized and secreted by the beta cells of the pancreas. Its action begins when it binds a cell membrane heterotetrameric receptor. The receptor has two alpha subunits which function to bind insulin, two beta subunits which transduce the signal. Through a cascade of cell signaling, insulin is a powerful regulator of metabolic action. When there is a breakdown in cell signaling, resistance, or decrease in insulin, many different pathologies can occur—see pathology section for more information. Several factors may act to either further stimulate insulin release or inhibit it. Glucose, mannose, leucine, and vagal stimulation will all increase insulin secretion. Alpha-adrenergic effects, somatostatin, and several drugs can inhibit insulin secretion. One of the primary functions of insulin is to control glucose levels. Glucose can be attained from three sources: gluconeogenesis, oral intake, and glycogenolysis. Once glucose is inside cells, one of the two things will occur—it can be stored as glycogen or undergo glycolysis and convert to pyruvate. Insulin modulates what happens to glucose in a few different ways, such as stimulating glycogen synthesis, increasing glucose transport into muscle and adipose, inhibiting glycogenolysis and gluconeogenesis, and increasing glycolysis in muscle and adipose. While most tissues can produce glucose within its cells, only the kidney and liver possess glucose-6-phosphatase, which is needed to release glucose into the blood. The liver produces 80%
90% of glucose in patients without glucoserelated pathology, making the liver the primary target for insulin. Through different pathways, insulin acts upon the liver, both directly and indirectly. Directly, insulin inhibits hepatic glycogen phosphorylase, the glycogenolytic enzyme, thereby inhibiting glucose output. Indirectly, insulin decreases the flow of glucose precursors, along with decreased glucagon secretion. A study of insulin infusion in dogs demonstrated the primary effects of insulin on hepatic glucose due to the direct insulin pathway. However, with the infusion of substantial amounts of insulin, the indirect effect became more predominant. The utilization of glucose is possible through cellular uptake, made possible by glucose transporters, GLUT1,2,3,4, and 5. GLUT-4 is the primary transporter in muscle and adipose; it resides within the cytoplasm until an insulin signal causes translocation to the cell membrane. When the body is in a euglycemic state, most glucose uptake, which is mediated by insulin, will occur in the muscle. Less than 10% of glucose is taken up by adipose tissue, primarily due to insulin-inhibiting lipolysis. Muscle will get most of the glucose because when free fatty acids are not available, increased glucose uptake is required to supply muscle tissue. Insulin optimizes glycolysis in muscle by catalyzing the glycolytic pathway by increasing hexokinase and 6-phosphofructokinase activity. As previously mentioned, in euglycemic states, insulin inhibits lipolysis. After a meal, insulin will promote triglyceride storage in adipose cells. This is mediated via three primary mechanisms. First, insulin increases the clearance of chylomicrons rich in triglycerides by increasing lipoprotein lipase. However, insulin only stimulates lipoprotein lipase expression in adipose tissue; in muscle, insulin actually inhibits lipoprotein lipase, resulting in triglyceride storage. The second mechanism is via insulin-stimulated re-esterification of fatty acids into triglycerides in adipose cells. Finally, the third mechanism is by insulin-inhibiting lipolysis. The overall effect of fat metabolism by insulin is to potently reduce hepatic gluconeogenesis and hepatic glucose release by blocking the supply of fatty acids to the liver (Longe et al., 2005; Mendonca-Filho, 2006). Ketone and insulin dynamics—Under physiologic states which result in deficient insulin levels, such as prolonged fasting or uncontrolled diabetes mellitus, fat is mobilized to meet metabolic demands. The liver is unable to handle all the fatty acids being shuttled its way, resulting in ketone body production. This is a result of incomplete betaoxidation of the long-chain fatty acids which are oversupplied to the liver. Ketoacids can be employed as fuel in extrahepatic tissue, such as skeletal muscle and the heart. However, under very prolonged periods of fasting, the brain will also use ketoacids for energy. Insulin acts to keep the levels of ketone bodies low; it potently drops

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16. 17. 18. 19.

20.

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circulating levels via three mechanisms. First, insulin inhibits lipolysis, so the fatty acids needed to make ketone bodies are not available. Second, insulin will act within the liver to directly inhibit ketogenesis. Third, insulin helps increase the peripheral clearance of ketone bodies. Protein metabolism and paracrine effects—As previously mentioned, insulin inhibits gluconeogenesis, and this keeps amino acids readily available for protein synthesis. Insulin expedites the transportation of amino acids into the liver and skeletal muscle. Also, insulin escalates the amount and efficiency of ribosomes. Lastly, insulin inhibits protein breakdown; roughly, 40% of proteolysis is influenced by insulin. The net result is increased protein synthesis. Insulin has many influences on other hormones within the body. The pancreatic islet (Ernst, 2007; Schulz et al., 2001) cells have alpha, beta, and delta cells. Alpha secretes glucagon, beta secretes insulin, and delta secretes somatostatin. When these hormones get secreted, they have paracrine effects on the surrounding cells. Insulin specifically will reach alpha cells first and inhibit the release of glucagon, which causes an increased effect of its metabolic actions. In hyperglycemic states, somatostatin will also be secreted, inhibiting alpha cells from releasing glucagon to reduce glucose levels. Insulin has other functions outside of energy metabolism, which are important for the clinical setting, as abnormal responses to insulin can lead to several different pathologies. Insulin impacts steroidogenesis, fibrinolysis, vascular function, and growth.

Concept of herbal targeted drug delivery

Various drug delivery and drug targeting systems are currently under development to minimize drug degradation and loss, to prevent harmful side effects, and to increase drug bioavailability and the fraction of the drug accumulated in the required zone. Among drug carriers, one can name soluble polymers, microparticles made of insoluble or biodegradable natural and synthetic polymers, microcapsules, cells, cell ghosts, lipoproteins, liposomes, and micelles. Targeting is the ability to direct the drug-loaded system to the site of interest. Two major mechanisms can be distinguished for addressing the desired sites for drug release: (1) passive and (2) active targeting. An example of passive targeting is the preferential accumulation of chemotherapeutic agents in solid tumors as a result of the enhanced vascular permeability of tumor tissues compared with healthy tissue. A strategy that could allow active targeting involves the surface functionalization of drug carriers with ligands that are selectively recognized by receptors on the surface of the cells of interest. Since ligand
receptor interactions can be highly selective, this could allow more precise targeting of the site of interest. For over 20 years, researchers have appreciated the potential benefits of nanotechnology in providing vast improvements in drug delivery and drug targeting. Improving delivery techniques that minimize toxicity and improve efficacy offers great potential benefits to patients and opens up new markets for pharmaceutical and drug delivery companies. Presently, novel drug delivery systems have been widely utilized only for allopathic drugs, but they have their limitations; hence, turning to safe, effective, and time-tested ayurvedic herbal drug formulation would be a preferable option. In the case of herbal extracts, there is a great possibility that many compounds will be destroyed in the highly acidic pH of the stomach. Other components might be metabolized by the liver before reaching the blood. As a result, the required amount of the drug may not reach the blood. If the drug does not reach the blood at a minimum level, which is known as “minimum effective level,” then there will be no therapeutic effect. Phytopharmaceuticals are pharmaceuticals using traditional compounds derived from botanicals instead of chemicals. Natural ingredients are more easily and more readily metabolized by the body. Therefore they produce fewer, if any, side effects and provide increased absorption in the bloodstream resulting in more thorough and effective treatments. Pharmaceuticals made from chemical compounds are prone to adverse side effects. The human body will have a tendency to reject certain chemical compounds which do not occur naturally (Ernst et al., 2006). These rejections occur in the form of side effects, some as mild as minor headaches and others as severe as to be potentially lethal. It is important to note while phytopharmaceuticals produce fewer to no side effects, chemical interactions with other prescription drugs can occur. Furthermore, as they are single and purified compounds, they can be easily standardized making it easier to incorporate them in modern drug delivery systems compared to herbs. As herbal novel drug delivery systems have a lot of potential, several researchers are working toward developing novel drug delivery systems like mouth-dissolving tablets, sustained and extended-release formulations, mucoadhesive systems, transdermal dosage forms, microparticles, microcapsules, nanoparticles, implants, etc. Herbal medicines have been widely used all over the world since ancient times and have been recognized by physicians and patients for their better therapeutic value as they have fewer adverse effects as compared with modern medicines. The drugs of ayurvedic origin can be utilized in a better form with enhanced efficacy by incorporating modern dosage forms. However, phytotherapeutics needs a scientific approach to deliver the components

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in a novel manner to increase patient compliance and avoid repeated administration. This can be achieved by designing novel drug delivery systems for herbal constituents (Marcus & Grollman, 2002). Novel drug delivery systems not only reduce the repeated administration to overcome noncompliance, but also help to increase the therapeutic value by reducing toxicity and increasing bioavailability, and so on. Recently, pharmaceutical scientists have shifted their focus to designing a drug delivery system for herbal medicines using a scientific approach. The novel research can also aid in capturing as well as to remain in the market. But there are many challenges with herbal drugs which need to be overcome like the difficulty of conducting clinical research in herbal drugs, development of simple bioassays for biological standardization, pharmacological and toxicological evaluation methods’ development, investigation of their sites of absorption, toxic herbal drugs in use, discovering various animal models for toxicity and safety evaluation, legal and regulatory aspects of herbal drugs, and so on.

9.3

List of most effective phytochemicals/phytomedicinal herbs

9.3.1 Used to treat endocrine disorder with the help of targeted drug delivery 9.3.1.1 Major endocrinological disorder and natural products/herbs used in the treatment of endocrinological disorder 9.3.1.1.1 Diabetes Diabetes is an endocrine disease that causes when your blood glucose is too high, and insulin is a hormone made by the pancreas and helps glucose from food get into your cell to be used for energy. 9.3.1.1.2

Herbs used in diabetes

1. Capparis spinosa; family-Capparaceae, 2. Urtica dioica; family-Urticaceae, 3. Ginseng; family-Araliaceae. 9.3.1.1.3

Thyroid

Thyroid disorders are very common endocrine disorders and tend mainly to occur in women. When hormonal imbalance occurs, then this disorder may happen. 9.3.1.1.4 Herbs used in thyroid 1. Bladder wark; family-Fucaceae, 2. Withania somnifera; family-Solanaceae, 3. Indian gooseberry; family-Phyllanthaceae. 9.3.1.1.5

Polycystic ovary syndrome

Polycystic ovary syndrome, or PCOS, is the most common endocrine disorder in women of reproductive age. The syndrome is named after the characteristic cysts which may form on the ovaries, though it is important to note that this is a sign and not the underlying cause of the disorder (Table 9.2). 9.3.1.1.6 Herbs used in polycystic ovary syndrome 1) Nigella sativa, family-Ranunculaceae, 2) Chasteberry, family-Lamiaceae, 3) Marjoram, family-Lamiaceae; 4) Gymnema, family-Apocynaceae;

9.4 List of novel phytomedicinal formulations in pharmacy to target the endocrine glands and hormone for the treatment of various major endocrine disorders 1. In the past few decades, considerable attention has been concentrated on the evolution of a novel drug delivery system (NDDS) for herbal drugs.

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TABLE 9.2 Herbs and phytoconstituents used in the treatment of endocrine disorders. S. no

Endocrine disorder

Phytomedicinal herbs

Phytochemical constituents

1.

Diabetes mellitus

1. Capparis spinosa (root extract) 2. Urtica dioica (leaf extract) 3. Ginseng

1. Glycosinolates, glycosides 2. Eriocitrin, rosmarinic acid 3. Ginsenosides, saponin

2.

Thyroid

1. Bladder wark 2. Withania somnifera (root extract) 3. Indian Gooseberry

1. Iodine 2. N-methyl-alanine, tropine, 6-hydroxy.4-methyl, 2-benzopyrone, caffeic acid, epinephrine 3. Punicafolin, phyllanemblinin A, kaempferol, ellagic acid, gallic acid

3.

Polycystic ovarian syndrome

1. 2. 3. 4.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Nigella sativa (seed extract) Chasteberry Marjoram Gymnema

Thymoquinone Carvacrol Limonene Palmitic acid Casticin Sabinene Agnuside Aucubin Sabinene Terpinene Terpinen-4-ol Terpineol Gymnemic acid, tartaric acid, gurmarin, stigmasterol, betaine

2. Conventional dosage forms including prolonged-release dosage forms are unable to satisfy both holding the drug component at a distinct rate as per directed by the requirements of the body all through the period of treatment as well as directing the phytoconstituents to their desired target site to obtain almost therapeutic response. 3. In phytoformulation research, developing nanosized dosage forms (polymeric nanocapsules, liposomes, solid lipid nanoparticles, phytosomes, and nanoemulsion) has a number of advantages for herbal drugs including enhancement of solubility and bioavailability, protection from toxicity, enhancement of pharmacological activity, enhancement of stability (Casey et al., 2007). 4. Thus the nanosized NDDS of herbal drugs has a potential future for enhancing the activity and overcoming problems associated with the plant medicines. 5. Herbal drugs are getting more popular in the modern world for their diligence to cure a variety of diseases with less toxic effects and better therapeutic effects. 6. Incorporation of novel drug delivery technology to herbal plant actives minimizes the drug degradation or presystemic metabolism and serious side effects by the accumulation of drugs in the nontargeted areas and improves the case of administration in pediatric and geriatric patients. 7. Conventional dosage forms, including prolonged-release dosage forms, are unable to fulfill the ideal requirements of novel carriers such as the ability to deliver the drug at targeted site. 8. For good bioavailability, natural products must have a sound balance between hydrophilicity (for dissolving into the gastrointestinal fluids) and lipophilicity (to cross lipidic biomembranes). 9. Many phytoconstituents such as polyphenolics have good water solubility, but poorly absorbed. 10. Thus the nanosized NDDS of herbal drugs has a potential future for enhancing the natural process and overwhelming problems related to plant medicines. 11. Novel herbal drug carriers cure particular disease by targeting just the affected zone inside a patient’s body and transporting the drug to that region. 12. NDDS is advantageous in giving up the herbal drug at a predetermined rate and delivering of the drug at the site of action which minimizes the toxic effects or increases in bioavailability of drugs. 13. In the present study, an effort has been induced to touch on various aspects and applications related to novel herbal drug preparations.

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9.4.1 Types of novel herbal drug delivery systems Several approaches in the case of new herbal drug delivery system include different types of expressions such as liposomes, phytosomes, pharmacosomes, nanoparticles, niosomes, microspheres, transferosomes, ethosomes, transdermal drug delivery system (TDDS), and proniosomes which are discussed. Liposomes 1. These are microparticulate or colloidal carriers, usually 0.05
5.0 mm in diameter which form spontaneously; when certain lipids are hydrated in aqueous media, the liposomes are spherical particles that encapsulate a fraction of the solvent in which they freely pass around or float into their interior. 2. They can carry one, several, or multiple concentric membranes. Liposomes are constructed of polar lipids which are characterized by having a lipophilic and hydrophilic group of the same molecules. 3. The primary advantages of using liposomes include a. The high biocompatibility, b. The easiness of preparation, c. The chemical versatility that allows the loading of hydrophilic, amphiphilic, and lipophilic compounds, d. The simple introduction of their pharmacokinetics proprieties by varying the chemical composition (Guo et al., 2007). Phytosomes 1. Most of the bioactive constituents of phytomedicines are flavonoids, which are poorly bioavailable when taken orally. 2. Water-soluble phytoconstituent molecules (mainly polyphenols) can be converted into lipid-compatible molecule complexes which are called phytosomes. 3. Phytosomes are more bioavailable as compared to simple herbal extracts owing to their enhanced mental ability to skip through the lipid-rich biomembranes and finally arrive at their origin. 4. The lipid-phase substances employed to make phytoconstituents lipid-compatible are phospholipids from soy, mainly phosphatidyl chloride. 5. Phytosomes complexes show better pharmacokinetic and therapeutic profiles than their noncomplexed herbal extract. 6. The phytosomes technology has markedly enhanced selected phytochemicals. Nanoparticles 1. Nanoparticles are efficient delivery systems for the delivery of both hydrophilic and hydrophobic drugs. 2. Nanoparticles are the sub-micron-sized particles ranging 10
1000 nm. 3. The major goal behind designing nanoparticles as a delivery arrangement is to control particle size, surface properties, and release of pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically optimal rate and dose regimen. 4. Nanosphere has a matrix-type structure in which the active ingredient is dispersed throughout whereas the nanocapsules have a polymeric membrane and active ingredient care. 5. Nanoparticles possess many advantages such as increasing compound solubility, reducing medicinal doses, and improving the absorbency of herbal medicines compared with the respective crude drug preparations. Niosomes 1. Niosomes are multilamellar vesicles formed from nonionic surfactants of the alkyl or dialkyl polyglycerol ether class and cholesterol. 2. Niosomes are different from liposomes face in that they offer certain advantages over liposomes. Proniosomes 1. Proniosomes gel system is a step forward to noisome, which can be utilized for various applications in the delivery of actives at desired sites. 2. Proniosomal gels are water-soluble carrier particles that are coated with surfactant and can be hydrated to form niosomes dispersion immediately before use. 3. Proniosomal gels are the formulations which on in situ hydration with water from the skin are converted into niosomes.

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Ethosomes 1. Newer advancements in patch technology have led to the development of ethosomal patch, which consists of drug in ethosomes. 2. Ethosomal systems are made up of soya phosphatidylcholine, ethanol, and water. They may form multilamellar vesicles and have high entrapment capacity for particles of various lipophilicity. 3. The elastic vesicles and transferosomes have also been used as drug carrier for a range of small molecules, peptides, proteins, and vaccines. 4. Ethosomes have a high deformability and entrapment efficiency and can penetrate through the skin completely and improve drug delivery through the skin. 5. The ethosomes carrier also can provide an efficient intracellular delivery for both hydrophilic and lipophilic drugs. Transferosomes 1. Transferosomes are specially optimized particles or vesicles that can respond to external stress by rapid and energetically inexpensive, shape transformations. 2. The development of novel approach such as transferosomes has immensely contributed to overcoming problem faced by transdermal drug delivery such as being unable to transport larger molecules, penetration through the stratum corneum is the rate-limiting step, and physicochemical property of drugs hinders their transport through skin. These elastic vesicles can squeeze themselves through skin pores many times smaller than their size and can transport larger molecules.

9.5

Application of phytomedicine in modern drug development in pharmacy

1. Phytomedicine or the use of herbal medicine with therapeutics properties has been around since the dawn of human civilization. 2. A phytopharmaceutical preparation or herbal medicine is any manufactured medicine obtained exclusively from plants either in the crude state or as pharmaceutical formulation. 3. Although the industrial revolution and the development of organic chemistry resulted in a preference for synthetic products, the World Health Organization (WHO) reports between 70% and 95% of citizens in a majority of developing countries still rely on traditional medicine as their primary source of medication. 4. In the last decades, herbal medicine has seen some form of revival advancing at a greater place in community acceptance of their therapeutic effects. This field is bringing forward new lead drug discoveries as well as safe and efficacious plant-based medicines. In turn, this leads to growing number of sales of commercialized medicinal herbs and most importantly growing number of pharmaceutical companies that involve in the research and development of plants as a source of modern medicine. 5. What chemists have been desperately seeking, mother nature has already plenty of stock. Current categories of herbal medicines: 1. The term “herbal medicine” is fraught with misconception that stems from the diversity of its approaches. 2. Herbal medicine can be categorized into three general groupings, namely, phytotherapy, over-the-counter (OTC) herbal medicine, and traditional herbalism. 3. Among these, phytotherapy is the one that adheres to scientific methodology and generates reasonably sound data. Based on the principles of phytotherapy, a herb contains a number of pharmacologically active compounds that should be seen as a single unit. 4. The whole extract can be standardized and clinically tested for a distinct clinical condition. This prominent feature differentiates phytotherapy from conventional pharmacotherapy, which generally favors the more reductionist approach. 5. The majority population of the developing world relies on traditional herbal medicine as the primary source of treatment for illness. 6. Approximately, 25% of drugs prescribed worldwide came from plants, with 121 such active compounds being in current use. There are 252 drugs considered basic and essential by the WHO of which 11% are exclusive of plant origin. 7. Plants can be used as therapeutic resources in several ways—herbal teas or other home remedies, crude extracts, and extraction with purification to isolate an active compound. 8. Among the most important rate of herbal medicine in modern drug development is the identification of plants with useful therapeutic compounds. 9. This is where the modern field of phytosciences comes in this field attempts to verify the health benefits of plants commonly used in traditional medicines and their mechanism of action.

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Advantages of herbal phytomedicines in modern system

The use of herbal medicines and phytonutrients or nutraceuticals continues to expand rapidly across the world with many people now resorting to these products for the treatment of various health challenges in different national healthcare settings (WHO, 2004). This past decade has obviously witnessed a tremendous surge in acceptance and public interest in natural therapies both in developing and developed countries, with these herbal remedies being available not only in drug stores, but now also in food stores and supermarkets. It is estimated that up to four billion people (representing 80% of the world’s population) living in the developing world rely on herbal medicinal products as a primary source of healthcare and traditional medical practice which involves the use of herbs viewed as an integral part of the culture in those communities. The use of herbal remedies has also been widely embraced in many developed countries with complementary and alternative medicines (CAMs) now becoming mainstream in the United Kingdom and the rest of Europe, as well as in North America and Australia. In fact, while places like the United Kingdom have a historical tradition of using herbal medicines the use is also widespread and well established in some other European countries. In these developed countries, the most important among many other reasons for seeking herbal therapy is the belief that it will promote healthier living. Herbal medicines are, therefore often viewed as a balanced and moderate approach to healing, and individuals who use them as home remedies and over-the-counter drugs spend a huge amount of money (in excess of billions of dollars) on herbal products. This explains in part the reason sales of herbal medicines are booming and represent a substantial proportion of the global drug market. As the global use of herbal medicinal products continues to grow and many more new products are introduced into the market, public health issues and concerns surrounding their safety are also increasingly recognized. Although some herbal medicines have promising potential and are widely used, many of them remain untested, and their use is also not monitored. This makes knowledge of their potential adverse effects very limited and identification of the safest and most effective therapies as well as the promotion of their rational use more difficult. It is also common knowledge that the safety of most herbal products is further compromised by lack of suitable quality controls, inadequate labeling, and the absence of appropriate patient information. It has become essential, therefore to furnish the general public including healthcare professionals with adequate information to facilitate a better understanding of the risks associated with the use of these products and to ensure that all medicines are safe and of suitable quality. Discussion in this review is limited to toxicity-related issues and major safety concerns arising from the use of herbal medicines as well as factors promoting them. Some important challenges associated with the effective monitoring of the safety of these herbal remedies are also highlighted with a view to helping refocus relevant regulatory agencies on the need for effectiveness and ensuring adequate protection of public health and promoting safety.

9.6.1 Factors responsible for increased self-medication with herbal medicine Essentially, herbal remedies consist of portions of plants or unpurified plant extracts containing several constituents which are often generally believed to work together synergistically. The recent resurgence of public interest in herbal remedies has been attributed to several factors some of which include (1) various claims on the efficacy or effectiveness of plant medicines, (2) preference of consumers for natural therapies and a greater interest in alternative medicines, (3) erroneous belief that herbal products are superior to manufactured products, (4) dissatisfaction with the results from orthodox pharmaceuticals and the belief that herbal medicines might be effective in the treatment of certain diseases where conventional therapies and medicines have proven to be ineffective or inadequate, (5) high cost and side effects of most modern drugs, and (6) improvements in the quality, efficacy, and safety of herbal medicines with the development of science and technology.

9.7

Conclusion

This study reported that herbal medicine may have beneficial effects on different endocrine disorders that occurs due to lifestyle changes. Lifestyle diseases are becoming more common these days and affecting the majority of the population. Lack of physical activity unhealthy lifestyle, poor diet, poor sleeping pattern, alcohol, smoking, and stress are some of the causes of lifestyle disease.

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Lifestyle diseases are linked with the way people live their lives. Diabetes, thyroid, and hormonal imbalance are the most common endocrine disorder nowadays which are related to each other. This review aimed to investigate the role of herbal phytomedicinal substances in the treatment of major endocrine disorders.

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20. Damsch, S., Eichenbaum, G., Tonelli, A., Lammens, L., Van den Bulck, K., Feyen, B., et al. (2011b). Gavage-related reflux in rats: Identification, patho- genesis, and toxicological implications (review). Toxicologic Pathology, 39, 348
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616. Ganie, M. A., Laway, B. A., Wani, T. A., Zargar, M. A., Nisar, S., Ahamed, F., et al. (2011). Association of subclinical hypothyroidism and phenotype, insulin resistance, and lipid parameters in young women with polycystic ovary syndrome. Fertility and Sterility, 95, 2039
2043. Gasbarrone, A., & Franzese, I. T. (2005). Relationship between insulin secretion, and thyroid and ovary function in patients suffering from polyystic ovary. Minerva Endocrinology, 30, 193
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872. Khan A., Safdar M., Ali Khan M.M., Khattak K.N., Anderson R.A. (2003). Cinnamon improves glucose and lipids of people with type 2 diabetes. Khanfar, M. A., Sabri, S. S., Zarga, M. H., & Zeller, K. P. (2003). The chemical constituents of Capparis spinosa of Jordanian origin. Natural Product Research, 17, 9
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79. Sicree, R., Shaw, J., & Zimmet, P. (2006). (3rd ed.). Diabetes and impaired glucose tolerance. Delice Gan. Diabetes Atlas, (51). International DiabetesFederation. Singh S.N., P. Vats, S. Suri, R. Shyam, Kumria M.M.L., Ranganathan S., Sridharan K. (2001). Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in streptozotocin induced diabetic rats. Sinha, U., Sinharay, K., Saha, S., Longkumer, T. A., Baul, S. N., & Pal, S. K. (2013). Thyroid disorders in polycystic ovarian syndrome subjects: A tertiary hospital based cross-sectional study from Eastern India. Indian Journal of Endocrinology and Metabolism, 17, 304
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531. Taylor, D. A., & Ashwal, S. (2012). Impairment of consciousness and coma. In K. F. Swaiman, S. Ashwal, D. M. Ferriero, & N. F. Schor (Eds.), Swaiman’s pediatric neurology (5th ed., pp. 1063
1067). [Edinburgh]: Elsevier Saunders. Tehrani, F. R., Simbar, M., Tohidi, M., Hosseinpanah, F., & Azizi, F. (2011). The prevalence of polycystic ovary syndrome in a community sample of Iranian population: Iranian PCOS prevalence study. Reproductive Biology and Endocrinology, 9(1), 39. Tuzcu, A. B. M., Gokalp, D., Tuzun, Y., & Gunes, K. (2005). Subclinical hypothyroidism is associated with early elevated high sensitive C-reactive protein (low grade inflammation) and fasting hyperinsulinemia. Endocrine Journal, 52, 89
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76.

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Chapter 10

Current updates on phytopharmaceuticals for cancer treatment Anshita Gupta Soni1, Srushti Mahajan2 and Pankaj Kumar Singh2 1

Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Durg, Chhattisgarh, India, 2Department of Pharmaceutics, National Institute of

Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana, India

10.1

Introduction

Phytochemicals are those compounds which are contributing at a wider range as therapeutics, cosmetic, nutraceuticals as well as dietary supplements These plant-derived bioactives have proven themselves as potent therapeutic agents in various disorders. Different studies have postulated the promising role of phytochemicals as anticancer, antiviral, antiinflammatory, antioxidant, antimicrobial, and antinociceptive drugs (Reddy & Rao, 2004). In an era of tremendous revolution in medicines, the search for potent anticancer drugs is still under research. Cancer is a devastating disorder crucially invading major of the world’s population, and an estimation of around 9.9 million people died by 2020
21 due to this dreadful disease. Cancer and its treatment have become a pivotal issue in public health globally. In the current scenario, the rate of occurrence of neoplastic disease has increased tremendously (Baur & Sinclair, 2006). The most significant of them remain oral cancer and breast cancer, next to lung cancer. The occurrence of these types of cancer is equally common in men and women both, while the ratio of inception of blood cancer, lymph, and brain cancer was found common in children. There are several reasons due to which the death rates increase in cancer. It is a disease that develops its foundation inside the cell long before its symptoms arrive. Some of the contributory factors include: 1. 2. 3. 4. 5. 6.

Abnormal lifestyle Excessive tobacco use Physical and chemical carcinogens Alcohol use Unhealthy diet Interaction with biological carcinogens

Another accelerating death rate factor is a lack of awareness regarding cancer, its detection, and treatment, which may lead to the turmoil in the life of the cancer patient. The treatment options for cancer also involve a range of therapies depending upon the type of cancer like: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Immunotherapy Chemotherapy Radiation therapy Hormonal therapy Gene-mediated therapy Photodynamic therapy Targeted therapy including monoclonal antibodies treatment Surgical removal of cancerous cells or tumors Combinational therapy

Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00023-2 © 2023 Elsevier Inc. All rights reserved.

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The main objective behind all these therapies is to eradicate cancer cell and restore normal cell. The main issue that remains with such therapies is their side effects or adverse effects. Chemotherapy is the mainstay of cancer treatment (Saiko et al., 2008). It is used in various types of cancers inducing tumors where it shrinks the size of the tumors and finally kills it. But during the process of treatment through chemotherapy, several hurdles are being faced by the patients which lead to failure of therapy and ultimately patient dies (Fig. 10.1). Out of all these contributing factors, autophagy and apoptosis are two crucially involved processes which act as a double edged sword in treating cancer as well as in developing chemoresistance (Harikumar & Aggarwal, 2008). Autophagy means self-engulfing, and apoptosis means self-killing or programmed cell death. Both processes are responsible to maintain metabolic homeostasis by the removal of abnormal proteins and dead or irreparable cell organelles. While the role of both these processes has become contradictory in cancer, the role of autophagy is to act as a tumor suppressor by regulating reactive oxygen species inside the cell. But studies suggest that cancer stem cells lead to faulty autophagy leading to chemotherapy-induced cytotoxicity. Similarly, cancer cell shows chemoresistant by escaping from pro-apoptotic signals, faulty apoptosis initiation, and regulation. Hence, it can be considered that to develop a better chemotherapeutic agent, these triggering factors should be kept in context to avoid therapeutic failure (Bishayee, 2009; Zhu et al., 2013). Moreover, in a recent study by Deng and co-workers, it is clear that phytochemicals have the potential to interfere with the process of autophagy and apoptosis, but detailed scientific findings are required to learn the consequence of phytoactives in modulating the chemoresistance of cancer cells (He et al., 2015; Salmani et al., 2017).

10.2

Phytochemicals unexplored

An era has been passed accounting for the role of phytochemicals in treating cancer. Various phytochemicals from past to present have shown remarkable results in either suppressing the cancer cell growth or inhibiting its proliferation. Potent anticancer drugs like vincristine or vinblastine to taxol or curcumin are of phyto-origin. Phytochemicals have not only proven their potential when administered solely but also when given in combination with existing drugs. They have demonstrated their action in downregulating the drug-resistance gene (Gupte & Mumper, 2009; Yang et al., 2018) (curcumin when administered with docetaxel in human breast cancer cell lines) (Fig. 10.2; Table 10.1). In spite of all these phytochemicals, there are some other phytochemicals which have shown promising anticancer effects in recent studies. Recent findings showed the promising anticancer potential of leaf extract of Leea indica on DU-145 and PC-3 human prostate cancer cell lines (Ghagane et al., 2017). L. indica Meer is an originally Malaysianorigin plant belonging to the family Leeaceae and is claimed to have a cascade of pharmacological activities. Their study confirmed that the plant possesses variety of secondary metabolites, has strong antioxidant activity, and has significant anticancer effect on DU-145 and PC-3 cell lines with a IC50 value of 529.44 6 42.07 and 547.55 6 33.52 μg/mL, respectively. Similarly in a study conducted by researchers, interesting results of Eclipta alba plant extract on HCT-116 colon cancer cell lines were found (Nelson et al., 2020). Scientists tested the plant extract in fine different cell lines, such FIGURE 10.1 Chemotherapy resistance and its contributing factors.

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FIGURE 10.2 Different methods of suppression of cancer progression by phytochemicals.

as, human colorectal carcinoma (HCL-116), human prostate cancer (PC-3), Michigan Cancer Foundation breast cell lines (MCF-7), and renal cell carcinoma (RCC-45) along with human fibroblast cell line (WI-38) using MTT assay. Out of all four cell lines, the extract showed significant (P , 0.005) specificity against human colorectal cell lines (HCT-116) while nontoxicity toward WI-38 cells which confirms its anticancer effect on HCT-116 cell lines. In a recent study, a novel plant extract of Adenoma bracteosum (Bonati) was studied on human lungs and liver cancer cells. The in vitro cytotoxicity assay was done with different fractions of A. bracteosum using SRB assay on NCIH460 and HEPG 2 cancer cell lines. The result of the study showed that the ethanolic and chloroform extracts of the plant exhibited significant activity on both cell lines. The extract further taken for bioactive compound isolation by NMR spectra revealed the presence of xanthomicrol, 5,40 dihydroxy-6,7,8,30 -tetramithoxy flavone and ursolic acid (Kumar et al., 2019). In an another study, anticancer activities of five plants, namely, Calotropis procera, Moringa oleifera, Millettia pinnata, Basella alba, and Euphorbia nerrifolia, collected from tribal areas of Jharkhand were studied. All five plant extracts were assessed for their toxicity in A549 cell lines. In the said study, out of all five extracts M. pinnata, ethyl acetate extract showed significant potentiality against A549 cell lines (Purnamasari et al., 2019). The metabolic extract of Fiscus carica leaves and fruits against Huh7 liver cancer cells for proliferation, apoptosis, and necrosis activity was also evaluated in one research study (Mykhailenko et al., 2020). The MTT assay was used to evaluate the anticancer potential of the extract, while using Annexing biomarker V-PI and flow cytometry to study apoptosis and necrosis. The study showed that the leaf extract of the plant F. carica was more anticancer than fruit extracts of the same plant. Similarly, in an another elaborated study, Ukraine origin plants chiefly Crocus sativus, Jurinea bucharica, and Gherkin hybrid zefir were evaluated against several human tumor cell lines (MCF-7, Hepe2, HCT-116, Hela, Hl-60, and HaCaT). The extracts showed an exponential activity spectrum against cancer cell lines out of which gladiolus G. hybrid Zefir showed maximum anticancer potential against all cell lines (Al-Saraireh et al., 2021). The anticancer potential of Euphorbia hierosolymitana a traditionally restricted medicinal plant of eastern Mediterranean countries was exploited. The phytochemical investigation clearly indicated a wide range of secondary metabolites in two different extracts (ethyl acetate and n-butane) of the plant. The GC-MS studies of the extracts showed the occurrence of pyridine derivative along with desulphosinigrin along with other primary chemical constituents. The anticancer activity of ethyl acetate extract was found to be maximum (Majoumouo et al., 2020). The potential of metabolic and aqueous extracts of Bersama engleriana against four cell lines in order to find new therapeutic moiety was also explored in one study. Preliminary phytochemical analysis of the plant confirmed the presence of polyphenols (Mahmod & Talib, 2021). Methanolic extract of B. engleriana exhibited significant antiproliferative activities with an IC50 value of 60 6 0.91 on U-87MG, 53.73 6 0.77 on MG-63, and 50.91 6 0.46 μg/mL on MIA PaCa-2 cell lines. The results showed that B. engleriana could be seen as a novel therapeutic agent against cancer. In 2021, Asama et al. investigated the anticancer activity of Mandragora autumnalis by studying its activity in vitro and in vivo. The ethanolic extract of the plant was studied on human breast cancer cell lines and human colon cancer cell lines (HCT-116) along with human lung adenocarcinoma (A549) cell lines. The study showed that ethanol extract

TABLE 10.1 List of some important medicinal plants and their phytochemicals and their role in cancer treatment. S.No.

Phytochemical

Biological source/class of compound

Mechanism of action

Therapeutic use

Types of cancer

References

1.

Curcumin

Curcuma longa/polyphenol

Curcumin shows apoptosis induction, It inhibits the molecular signals, acts as free radical scavenger, Its shows anti-inflammatory response on tumor microenvironment

Anti-inflammatory, anticancer, antibacterial.

Breast cancer Skin cancer and tumor

Reddy and Rao (2004)

2.

Resveratrol

Grapes, berries/ polyphenolic

Resveratrol overcomes the neoplast cell development and progression, activating automatic cell dying It shows anti-inflammatory properties and angiogenesis as well as stop the metastatic extend of neoplast

Anti-inflammatory, anticancer Analgesic Antidiabetic

Breast, colon, prostate, liver, and lung cancer

Baur and Sinclair (2006), Bishayee (2009), Harikumar and Aggarwal (2008), Saiko et al. (2008)

3.

Apigenin

Oregano/flavone

Apigenin shows antiangiogenic properties which are connected with the regulation of various pathways, Its shows induction of apoptosis, suppression of neoplast cell alteration, These compounds show inhibitory action on nicotine smoke-related carcinoma

Antioxidant Anti-inflammatory Antihypertensive Antibacterial Antiviral Anticancer

Colorectal and colon cancer Pulmonary cancer, Melanoma, Prostatic cancer Osteosarcoma Breast cancer

He et al. (2015), Salmani et al. (2017), Yang et al. (2018), Zhu et al. (2013)

4.

Epigallocatechin gallate

Green tea/flavone-3-ol

Tea catechins along with EGCG show redox properties and interact with ROS species, Metal chelating properties of EGCG which assist in the anticipation of ROS species production

Anticancer Heart disease Antidiabetic

Breast cancer

Gupte and Mumper (2009), Khan et al. (2011)

5.

Genistein

Genista tinctoria/isoflavones

Estrogen binding Genistein phytochemical competes with 17β-estradiol, The result says substance inhibits the production and multiplication of estrogen and androgen receptorpositive in vitro, These substances block protein tyrosine kinase signaling mechanism using protein tyrosine kinase (PTK) inhibition

Anticancer

Prostate cancer Bladder cancer Breast cancer

Evans et al. (1995), Huang et al. (1992), Okura et al. (1988), Peterson and Barnes (1991), Ullrich and Schlessinger (1990)

6.

Piperlongumine

Piper longum L./alkaloidal

These compounds activate autophagy-mediated cell apoptosis via inhibition of protein kinase B in pulmonary cancer. Piperlongumine as well shows anti-inflammatory response through transcription factors nuclear factor of kappa B

Anticancer

Melanoma, pancreatic cancer, colon cancer, orosquamous cell, nonsmall cell pulmonary cancer, GI cancer, bile duct cancer, prostatic cancer

Fofaria and Srivastava (2014), Fofaria and Srivastava (2015), Han et al. (2014), Wang et al. (2015)

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and n-hexane extract have the potential to reduce the expression of VEGF in MCF-7 cell lines (Wambua Mukavi et al., 2020). Justus and his colleagues studied the anticancer profile of Kigelia africana (Lam), a native plant from Africa. Methanolic extract of the plant was studied against SRB assay on human breast cancer cell lines (HCC1937). The study showed that the plant exhibited significant anticancer activity with an IC50 value of 26.02 6 0.1 μg/mL as compared to the standard (Ndlovu et al., 2021). Various drugs have also shown potential activity against cervical cancer. In one such study by Mxloisi and coworkers, anti-HELA cell activity of the acetone extract of Seriphium plumosum, Toona ciliata, and Schkuhria pinnata was evaluated. In preliminary phytochemical analysis, S. plumosum and T. ciliata showed exceptionally higher flavonoidal content. Based on cell proliferation inhibition test, all three extracts showed anticancer, antioxidant, and free radical scavenging activity in T. ciliata . S. plumosum . S. pinnata order (Vaksman et al., 2014). Other than these scientific findings, there are many more phytoconstituents which are still unexplored and undocumented scientifically. Many of the studies on these plants are at a primary stage and lack proper scientific validation. Several studies on phytochemicals are done every year, but they do not undergo further assessment either for lead identification or for analyzing their synergistic mechanism of action due to improper research findings (Table 10.2).

TABLE 10.2 List of phytochemicals used in types of cancer (Banerjee et al., 2002; Bhattacharya et al., 2018; De La Chapa et al., 2018; de Lima et al., 2018; Dou et al., 2018; Garnier, 1991; Huang et al., 2017; Islam et al., 2018; Kuppusamy et al., 2017; Lin et al., 2016; Wu et al., 2017, 2018; Zhang et al., 2017; Zhao et al., 2016). S.No.

Phytochemical

Types of cancer

1.

6-Shogaol

Lung cancer and prostate cancer

2.

Allicin

Liver bile duct carcinoma and lung adenocarcinoma

3.

Alpinumisoflavone (AIF)

Renal cell carcinoma and esophageal squamous cell carcinoma

4.

Apigenin (APG)

Tumor

5.

Andrographolide

Tumor

6.

Baicalein

Tumor

7.

Curcumin

Skin cancer and tumor

8.

Decursin

Prostate cancer

9.

Epigallocatechin (EGCG)

Breast cancer

10

Emodin

Lung epithelial (A549) cells

11.

Gingerol

Breast cancer

12.

Glycyrrhizin (GA)

Nonsmall cell lung cancer

13.

Thymol

Tumor

14.

Withaferin A (WA)

Tumor

15.

Genistein

Prostatic carcinoma, bladder cancer, breast cancer

16.

Piperlongumine

Carcinoma, colon cancer, orosquamous cell carcinoma, small cell lung cancer, GI tract cancer, bile duct cancer, prostate cancer, pancreatic cancer.

17.

Berberine

Colorectal cancer

18.

Quercetin

Prostate cancer

19.

Resveratrol

Low-grade GI neuroendocrine tumor cancer

20.

Sulforaphane

Lung cancer in former smokers

21.

Ellagic acid

Lung cancer, prostatic carcinoma, colon cancer, breast cancer

22.

Lycopene

Metastatic colorectal cancer

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169

Molecular mechanism of phytochemicals in preventing cancer

Phytochemicals can be considered as pleiotropic by nature. They are responsible for striking different molecular pathways associated with the pathophysiology of a disease. Phytochemicals act synergistically through cellular targets leading to modulation in signaling mechanism of cancer cells shielding normal cells (de Lima et al., 2018; Lin et al., 2016; Wu et al., 2018). The molecular mechanism of action of phytochemicals is a multistep process that can be discussed as follows.

10.3.1 Targeting molecular pathway of cancerous cell Phytochemicals interfere with the intracellular target and cause direct effect on molecular pathways of the cancerous cells. In the process of modulating molecular pathways, phytochemicals show affinity toward specific molecular target through which they inhibit carcinogenesis and also block mutagenic activity (Banerjee et al., 2002; De La Chapa et al., 2018). Numerous researches have suggested that certain phytochemicals like curcumin, resveratrol, green tea polyphenols, erucin, vitamin E, dioscin, and kaempferol have proven evidence of targeting molecular pathways.

10.3.2 Targeting cell proliferation Phytochemicals crucially act on the stage of cell proliferation leading to apoptosis. Chiefly, phytosterols are those chemical entities which specifically act on the transduction of signals causing improper cell proliferation and ultimately apoptosis (Kuppusamy et al., 2017). Phytosterols are chemically the member of 4-desmethylsterol group and include β-sitosterol, stigmasterol, campesterol, etc., commonly occurring in whole grains, nuts, and seeds. β-sitosterol is chemically analogous to cholesterol and exhibits antiproliferative activity against various types of cancer (Adnyana et al., 2012) (Fig. 10.3).

10.3.3 Targeting oxidative stress and redox signaling Phytochemicals have a profound effect in suppressing the entire process of carcinogenesis by acting on the redox signaling mechanism, which has been proved both in vitro and in vivo. Phytochemicals directly act on these cancerous biomarkers which play important role in activating successive steps of carcinogenesis (Kashyap et al., 2019). These biomarkers could be enzymes like glutathione S-transferase, NADPH, lipase or MMPs, and quinine oxidoreductase (NQOI). These phytochemicals lead to the activation of cytoprotection enzymes like NF-E2-related factor 2 (Nrf2) which gets initiated not only in the presence of oxidative stress but also by the presence of phytochemicals. This Nrf2 is responsible for maintaining genomic integrity and minimizing the effect of free ion chemicals and DNA-related oxidative stress. Quercetin, rutin green tea, and various phytochemicals belonging to the family of Cruciferae act by reducing reactive oxygen species inside the cell (Akram et al., 2017). They not only inhibit the conversion of procarcinogens into DNA-damaging species but also prevent DNA mutation.

10.3.4 Genome instability Phytochemicals are the only chemical entities which can alter or stabilize genome instability. Genome instability can be defined as the tendency of altering the genome frequently in the life cycle of a cell. Genome instability is the most contributing factor to tumorigenesis. Curcuminoids are those phytochemicals which have the capability to inhibit genome FIGURE 10.3 Demonstrates an example of β-sitosterol mechanism of targeting cell proliferation.

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instability and tumorigenesis. Resveratrol, quercetin, and genistein also carry out DNA repair by preventing genome instability (Al Dhaheri et al., 2014).

10.3.5 Modulation of membrane Phytochemicals like curcumin have the property to adhere to the tumor cell membrane which causes alteration in membrane properties (thickness, fluidity, and elasticity) causing disturbance in the structure and function of membrane proteins like epidermal growth factor receptor (EGFR), CFTR, and potassium channels (Alers et al., 2012). Curcumin acts on the coupling between membrane protein and lipid junction. Capsaicin and geinstein also act by modulating cell membrane.

10.3.6 Targeting immune surveillance and inflammation Various phytochemicals have also been found which increase immune surveillance by enhancing T-cells effectors against cancer cells. They also play a critical role in inhibiting inflammation which is a key component of tumor progress (Alfarouk et al., 2015). During cancer growth, innate immune system causes cancer invasion by migration and metastasis and involves chemokines and their receptors. Phytochemicals decrease the protein expression of inflammatory biomarkers like COX-2 and PGE-2 and can be used in novel therapy of cancer treatment. Examples include luteolin which exert a pro-oxidant activity in tumor cells, and due to high lipophilicity, it is a good drug candidate in treating skin and brain cancer, as it can penetrate human skin and could also cross blood
brain barrier (Andualem et al., 2014; Aung et al., 2017).

10.3.7 Apoptosis and autophagy In cancer, faulty apoptosis and autophagy are caused by oncogenes which are itself produced as a result of genomic instability ´ valos et al., 2014; Baliga et al., 2011). These faulty autophagy and apoptosis regulators suppress the selective normal autop(A hagy, cause excessive ROH production, and enhance tumor generation. Phytochemicals like apigenin serve as a key agent in suppressing autophagy by inhibiting PI3K/AKT/mTOR pathway. Similarly, allicin, berberine, celastrol, curcumin, fisetin, and many more phytochemicals have been reported to inhibit autophagy through mTOR pathway whereas certain phytochemicals like angelicin, luteolin, kaempferol, etc., cause an increase in cellular toxicity and mediate apoptosis by decreasing the expression of antiapoptotic proteins (Bcl-XL, Bcl-2) (Balli et al., 2016; Barrajo´n-Catala´n et al., 2010) (Fig. 10.4).

10.4

Strategies to improve phytochemical drugability

Phytochemicals have proven themselves as significant competitors to existing anticancer drugs; the issues of poor bioavailability and solubility hinder their path as drug of choice in cancer chemoprevention (Battu & Kumar, 2012). Phytochemicals hold ground where synthetic or conventional drug founds limitations. The hurdles faced by conventional anticancer regimens are: 1. 2. 3. 4. 5.

Low solubility Unsuitability for oral administration Short half-life Poor specificity with associated side effects Multidrug resistance

Similarly, phytochemicals also face certain issues related to drugability. The major concern while delivering a phytochemical in vivo remains in their bioavailability and stability issues. Various studies have shown that many phytochemical compounds have a lower bioavailability and unsuitability in the gut. These issues cannot be overcome by adjusting the dose or duration of dosing. It has been observed that the issue of the bioavailability of anticancer drugs is the lowest priority while discovering novel anticancer drug (Battu & Kumar, 2012). Simply assessing the plasma level of an anticancer drug does not signify its bioavailability as its concentration at the target site is the key to efficacy; many phytochemicals when tested in vitro give promising results but when evaluated they fail to achieve optimum effective concentration at the target site which can trigger or modulate the metabolic pathway responsible for unveiling tumorigenesis/cancer genesis (Fig. 10.5). Bioavailability of entire phytochemicals having anticancer nature cannot be accurately predicted because not all chemical/phytochemical compounds follow “Lipinski’s rule of five” which states that any compound will have better

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FIGURE 10.4 An overview of promising aspects of phytochemicals as potent anticancer drug. Pic Courtsey: The Hong Phong Nguyen, V. Bharath Kumar, Vinoth Kumar Ponnusamy, Thi Thu Thao Mai, Phuong Tran Nhat, Kathirvel Brindhadevi, Arivalagan Pugazhendhi,; Phytochemicals intended for anticancer effects at preclinical levels to clinical practice: Assessment of formulations at nanoscale for non-small cell lung cancer (NSCLC) therapy. Process Biochemistry, 104, 2021, pp. 55
75, downloaded from science direct on 19th july 2022.

FIGURE 10.5 Factors which contribute to limiting bioavailability of the phytochemicals.

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bioavailability if it contains not more than 5 hydrogen bond donors, not more than 10 hydrogen acceptor. Also, its molecular mass should not be more than 500 daltons and log P value less than 5 and must have less than 10 rotatable bonds. Phytochemicals like curcumin polyphenols do not follow this rule and hence possess poor bioavailability. Similarly, phytochemicals like genistein and biochanin A follow the “rule” and must have good absorption properties but are excreted out fast, causing limitations to their bioavailability (Bezerra et al., 2008). Hence, to improve the drugability of phytochemicals entire bioavailability approach of every single entity should be taken into account to overcome their limitations. For example, many phytochemicals when administered orally undergo rapid conjugation by glucuronidation in the intestine and liver leading to poor bioavailability of the compound. In such cases, any phytochemicals which can prevent or inhibit glucuronidation can be coadministered. As such case, when curcumin and piperine (compound of black pepper) were administered together increased the circulating curcumin plasma level and subsequently increased bioaccumulation of the drug at the target site by suppressing glucuronidation (Bhatia & Das, 2020). Similarly, when resveratrol was coadministered with quercetin and myricetin, the bioavailability of resveratrol increased due to the inhibition of glucuronidation and sulfation by quercetin and myricetin.

10.5

Drug delivery approach to improve phytochemical drugability

A smart yet promising way to subside the limitation of phytochemicals drugability is the development of drug-loaded carrier system. They have the characteristics: G G G

Modulate pharmacokinetics of existing drugs Can target drug to desired site of action Lower systemic toxicity

10.6

Phytochemicals in clinical and preclinical stages for preventing cancer

Evidence-based studies have postulated several phytoconstituents as promising lead molecules for the treatment of cancer. Some of the phytoconstituents are summarized below which are under clinical and preclinical trial phases and have shown remarkable anticancer effect in different cell lines (Tables 10.3 and 10.4).

10.7

Insights on phytochemicals as dietary recommendation in cancer

Dietary supplements are a class of compounds which are made from food or food-like substances and have the appearance of drug (Hema et al., 2018; Kar, 2016). Nutraceuticals belong to a category of dietary supplements which are totally made up of food and contribute to the prevention as well as protection from diseases. Phytochemicals coming under the category of nutraceuticals belonging to the dietary supplement regimen play a crucial role in treating cancer. The phytochemicals which exert an effect on cancer cells can be truly termed as “nutraceuticals.” Nutraceuticals are a category of dietary supplements (Vaiyapuri et al., 2020). These nutraceuticals can be acquired from different sources like: 1. 2. 3. 4.

Natural resource From food industry Newly bioengineered microorganism, agroproducts, or active molecules Herbal supplements

Nowadays, these dietary supplements have taken the form of genetically engineered “customized and processed products” which very soon would be called as “personalized processed products” to treat different diseases. These chemical compounds are of different types: 1. 2. 3. 4. 5. 6.

Isoprenoid derivatives Phenolic compounds Carbohydrate derivatives Fatty acid and structural lipids Amino acid devotions Minerals

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TABLE 10.3 List of phytoconstituents used in preclinical trial on different types of cancer. S.No.

Phytoconstitents

Biological source

Cancer

Result

References

1.

6-Shogaol

Zingiber officinale, (Zingiberaceae)

Lung cancer and prostate cancer

6-Shogaol inhibited the expansion of NCI-H1650 pulmonary cancer

Huang et al. (2017)

2.

Allicin

Allium sativum (Amaryllidaceae)

Liver bile duct carcinoma and lung adenocarcinoma

Allicin overcomes the extent of MMP-2 also with lowering the action of the STAT3

Zhang et al. (2017)

3.

Alpinumisoflavone (AIF)

Derris eriocarpa (Leguminosae)

Renal cell carcinoma and esophageal squamous cell carcinoma

Alpinumisoflavone stop expansion and metastasis of 786-O human kidney cells

Bhattacharya et al. (2018)

4.

Apigenin (APG)

Colon and colorectal cancer, pulmonary cancer, Melanoma, Prostatic cancer Osteosarcoma Breast cancer

Apigenin prevents tumor expansion which can be allied by lowering Ki67 expression and induction of apoptosis

Islam et al. (2018)

5.

Andrographolide

Andrographis paniculata (Acanthaceae)

Tumor

Andrographolide inhibited tumor enlargement via blocking tumor alteration to hypoxic condition

Dou et al. (2018)

6.

Baicalein

Scutellaria baicalensis (Lamiaceae)

Tumor

Baicalein stops tumor expansion and activates apoptosis

Zhao et al. (2016)

7.

Curcumin

Curcuma longa (Zingiberaceae)

Skin cancer and tumor

Curcumin stops the development of tumor cells via mechanisms with cell cycle arrest, autophagy, and reduces the protein kinase B

Wu et al. (2017)

8.

Decursin

Angelica gigas Nakai

Prostate cancer

Decursin decreases tumor development and pulmonary metastasis

Garnier (1991)

9.

Epigallocatechin (EGCG)

Green tea

Breast cancer

EGCG reduced tumor via inducing apoptosis and inhibiting proliferation

Garnier (1991)

10.

Emodin

Rheum palmatum L. (Polygonaceae)

Lung epithelial (A549) cells

Emodin prevents the expansion of pulmonary epithelial (A549) cells with activating ER stressdependent apoptosis

Lin et al. (2016)

11.

Gingerol

Z. officinale Roscoe

Breast cancer

Gingerol suppressed the orthotopic tumor development and metastasis of mouse

de Lima et al. (2018)

12.

Glycyrrhizin (GA)

Glycyrrhiza glabra L.

Nonsmall cell pulmonary cancer

Glycyrrhizin overcomes the TxAS and PCNA expression

Wu et al. (2018)

(Continued )

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Recent Frontiers of Phytochemicals

TABLE 10.3 (Continued) S.No.

Phytoconstitents

Biological source

Cancer

Result

References

13.

Resveratrol

Grapes, berries

Breast cancer, colon cancer, prostatic, hepatic, and pulmonary

Resveratrol overcomes tumor development and metastasis to the pulmonary in mice

Banerjee et al. (2002)

14.

Thymol

Thymus vulgaris, Origanum vulgare

Tumor

Thymol overcomes the tumor size with lowering cell proliferation and activating apoptosis

De La Chapa et al. (2018)

15.

Withaferin A (WA)

Withania somnifera (Solanaceae)

Tumor

Withaferin suppressed the neoplast expansion of colorectal (HCT-116) cells overexpressing protein kinase and also formed microvessel

Kuppusamy et al. (2017)

TABLE 10.4 List of phytoconstituents used in clinical trial on different types of cancers. S.No.

Phytoconstituents

Biological name

Cancer types

Result

1.

Berberine

Berberis sp. Family—Berberidaceae

Colon or colorectal cancer

Berberine prevents the repetition of cancer

2.

Curcumin

Curcuma longa Linn Family—Zingiberaceae

Metastatic breast cancer

Curcumin helps in superiority of life.

3.

Epigallocatechin

Camellia sinensis, Family— Theaceae

Colon cancer

Epigallocatechin modifies methylation pattern contrast to baseline

4.

Lycopin

Solanum lycopersicum Family— Solanaceae

Metastatic colorectal cancer

Treat skin toxicity alone or in grouping with panitumumab

5.

Quercetin

Prostatic sarcoma

These substances and their methylated metabolites in prostatic tissue

6.

Resveratrol

Arachis hypogaea Family—Ericaceae

Low-grade gastrointestinal neuroendocrine carcinoma

Single-pass transmembrane receptor activation, toxicity

7.

Sulforaphane

Brassica oleracea Family— Brassicaceae

Pulmonary cancer

Sulforaphane shows cell buildup marker Ki-67

Dietary supplements or nutritional supplements have proven themselves as a million-dollar industry. As per the study conducted by Global Market Insights (Report ID: GMI4179), Onco-Nutrition and Supplements Market Size is expected to record a healthy CAGR between 2022 and 2028 driven by the rising need for proper nutrition for living well during and after cancer treatment. Major companies in the onco-nutrition and supplements market comprise Danone, Abbott, B. Braun Melsungen AG, Global Health Products, Hormel Foods, Nestle S.A., and others. These players are focused on the launch of mergers, acquisitions, partnerships, collaborations, and the development of novel onco-nutrition products for maintaining their position in the market (Pandey & Madhuri, 2009; Van der et al., 2017). The marketing and promotional concept fueling the role of dietary supplement as anticancer supplements had gimmicked the market value and customer’s confidence in such products. But the term anticancer supplement is truly misleading. There are certain dietary supplements that could help in reducing the risk of cancer or could help the body to

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TABLE 10.5 List of dietary supplements and their probable role in preventing cancer. S.No.

Phytochemical

Target pathway

1.

Ellagic acid (EA)

EA targets cell apoptosis, cell cycle, insulin-like growth factor signaling, nuclear factor kappa B signaling, protein 53 signaling, telomerase enzyme, hormone signaling

2.

EGCG

EGCG targets, cell cycle, cytokine signaling, lipid metabolism, nuclear factor kappa B signaling, Hedgehog signaling protein kinase signaling, telomerase enzyme, detoxification enzymes and antioxidants, cell apoptosis, vascular endothelial development factor signaling, multidrug resistance, hormone signaling, folate biosynthesis pathway, JAK signaling

3.

Genistein

Genistein targets protein kinase B signaling pathway, wingless-related integration site signaling, endothelial
mesenchymal transition signaling, nexus communicans, MAPK signaling, nuclear factor kappa B signaling, protein 53 signaling, telomerase enzyme, vascular endothelial growth factor signaling, detoxification enzymes and antioxidants, hormone signaling, Hedgehog signaling, cell apoptosis

4.

Quercetin

Quercetin targets, cell apoptosis, cell cycle, nexus communicans, insulin-like growth factor signaling, lipid metabolism, nuclear factor kappa B signaling, Hedgehog signaling, protein kinase B signaling, ErbB2 signaling, antioxidant/detoxification enzymes

5.

Resveratrol

Protein kinase B signaling, cell apoptosis, cell cycle, hormone signaling, nuclear factor kappa B signaling, Notch signaling, protein 53 signaling, protein kinase B signaling, telomerase enzyme, ErbB2 signaling, lipid metabolism

6.

Curcumin

Curcumin targets Akt/mTOR signaling, cell apoptosis, cell cycle, cytokine signaling, insulin-like growth factor signaling, MAPK signaling, multidrug resistance, nuclear factor kappa B signaling, notch signaling, protein 53 signaling, wingless-related integration site signaling, vascular endothelial growth factor signaling, Hedgehog signaling, antioxidant/detoxification enzymes

7.

Lycopene

Lycopene targets protein kinase B signaling, antioxidant/detoxification enzymes, cell cycle, nexus communicans, insulin-like growth factor signaling, lipid metabolism, rat sarcoma signaling

recover from the disease, but they cannot replace the mainstay of cancer therapies (Park et al., 2016; Perumal Samy & Gopalakrishnakone, 2010; Pistritto et al., 2016; Priya et al., 2012; Rosangkima & Prasad, 2004). American Cancer Society also suggests to take dietary supplements for the patients recovering from cancer which include vitamins, minerals, herbs, botanicals, prebiotics, probiotics, enzymes, amino acids, etc., in order to speed up the recovery process of the patients. But the first and foremost thing while administrating nutritional or dietary supplements in cancer patients is to monitor their drug
herb or herb
herb interaction and associated side effects with the anticancer drugs. Various health agencies are trying to aware people regarding the safe and healthy use of dietary supplements. In fact the use of tomato, soy, phytoestrogens, and cruciferous vegetables has been also recommended by FDA to avoid risk and chances of occurrence of cancer. Few diets have been chiefly recommended for cancer prevention for the patients by National Cancer Institute like NTP-2000 and NIH-07 to overcome the deficiencies of vitamins C, D, E and loss of cellular immunity (Rallabandi et al., 2020; Seca & Pinto, 2018; Srivastava, 2014) (Table 10.5).

10.8

Conclusion and future perspectives

Due to the tremendous rise in the cases of this fatal disease, there is an urgent need to develop novel therapeutic strategies as cure. The most challenging feature of drug designing for anticancer drugs is the complicated pathophysiology and intrinsic nature of the disease which vary from person to person. Various types of cancers have been reported which have become resistant to certain chemotherapeutics due to variation of intrinsic cofactors, faulty signaling mechanisms, and initiation of defective autophagy of cancerous cells. There is a great need to explore not only the signaling mechanism involved in the process of uncontrollable cell growth but also to mark the role of several intermediate factors which acts as connecting circuits between autophagy and apoptosis. Targeting these connecting links would also render us to design more site-specific drug delivery systems for different types of cancers. Moreover, there is also a concept to design personalized targeted drug delivery systems for cancer patients because the physiology of a patient, the nature of the drug and its extent of therapeutic activity, pharmacokinetic and pharmacodynamic effect and its adverse reactions vary from person to person. In this regard, scientists are of the view that plant products and their bioactives could be a

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potential source of anticancer drugs. Plant bioactives are synergistic in nature and action. They act at multiple molecular levels and provide an overall therapeutic effect. These phytoconstituents not only interfere with cellular signaling pathways but also reduce the level of reactive oxygen species, suppress different biomarkers of cancers, and competitively fight with tumor-associated enzymes for inhibiting the growth and progress of cancerous cells. The basic hurdle of delivery of phytoconstituents in vivo remains in their poor solubility and bioavailability which could be removed through the use of nanovesicular systems. Nanotechnology-based drug delivery system has facilitated the drug delivery of phytoactives to a greater extent making them clinically effective and promising for future use. But still there is a need for more evidence-based studies of phytoactives which could prove them as potent candidates in treating cancer.

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7, [CrossRef] [PubMed].

Chapter 11

Phytochemicals in prostate cancer Abdel Rahman Al Tawaha1, Rose Abukhader2, Ali Qaisi3, Abhijit Dey4, Siddhartha Pati5, Abdel Razzaq Al-Tawaha6, Iftikhar Ali7 and Mohamad Shatnawi8 1

Department of Biological Sciences, Al Hussein Bin Talal University, Maan, Jordan, 2Faculty of Medicine, Jordan University of Science and

Technology, Irbid, Jordan, 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Jordan Amman, Amman, Jordan, 4

Department of Life Sciences, Presidency University, Kolkata, West Bengal, India, 5NatNov Bioscience Private Limited, Balasore, Odisha, India,

6

Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, Serdang, Selangor, Malaysia, 7Center for Plant Sciences and

Biodiversity, University of Swat, Charbagh, Pakistan, 8Biotechnology Department, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt, Jordan

11.1

Introduction

The word cancer originated from a Latin word that means crab. The ancients used the word to mean a malignancy. Abnormal and uncontrolled division of cells is known as cancer. Cancer is not a single disease. It is a cluster of more than 100 various and distinctive diseases. Cancer may harm any cell, tissue, and organ of the body. All cancers are named for the type of tissue or organ in which they start (Blank et al., 2016). Cancer is well known with different names and various categories such as carcinoma, sarcoma (osteosarcoma, Kaposi sarcoma, and leiomyosarcoma), leukemia, lymphoma (Hodgkin and non-Hodgkin), melanoma, multiple myeloma, brain and spinal cord tumors. Furthermore, tumors include neuroendocrine tumors, germ cell tumors, and carcinoid tumors (Wang et al., 2018). An individual may inherit the specific type mutation in a gene from parents, and there would be an increased chance to get affected by cancer. Genetic cancer includes prostate cancer, colon cancer, ovarian cancer, breast cancer, uterine cancer, pancreatic cancer, and skin cancer. The type of cancer found in the prostate gland is known as prostate cancer. It is a general type of cancer. Most prostate cancer develops steadily and remains in the prostate glands, where it may not produce critical infection. However, some types of cancer need minimal or maybe recovery without any treatment, and other kinds of prostate cancer are aggressive and proliferate quickly. Cancer cells are formed in the tissue of the prostate in prostate cancer. Cell alteration may start 10, 20, or even 30 years before a cancer tumor becomes big enough to cause symptoms. Prostate cancer may expand by the time symptoms appears; cancer may already be advanced (Lee & Shen, 2015). The prostate is an important and small gland found in men. It is a segment of the male reproductive system. The size and shape of the prostate are similar to walnut. Its site is below the bladder, lower in the pelvis, and just in front of the rectum. Prostate plays role in semen makeup, the milky liquid that takes sperm from the testicles through the penis when a man ejaculates. Parts of the urethra are surrounded by the prostate, a tube that carries urine out of the bladder and through the penis. As man’s age increases, the size of the prostate is also expanding. As a result, the urethra becomes narrow and urine flow is decreased. This is known as benign prostatic hyperplasia, and it is not acute like prostate cancer (Rawla, 2019).

11.2

Types of prostate cancer

11.2.1 Small-cell carcinoma Small-cell prostate cancer is an advanced type of cancer. Less than 2 in every 100 prostate cancer (less than 2%) are small-cell prostate cancer (Litwin & Tan, 2017). They can also be classed as a type of prostate cancer and have advanced cancer by the time they are diagnosed. This means that cancer may infect other parts of the body like tissues and bones. Specific symptoms of small-cell carcinoma are difficulty passing, pain, and confusion, but some cases may also have symptoms like pricking, tingling, or numbness in arms, hands, legs, and feet, muscle cramps, difficulty Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00022-0 © 2023 Elsevier Inc. All rights reserved.

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walking, and a problem with your memory. The main treatments for small-cell prostate cancer are chemotherapy, radiotherapy, and surgery (Litwin & Tan, 2017).

11.2.2 Neuroendocrine prostate tumor The type of cancer that is called the lethal subtype of prostate cancer. The tumor predominantly developed from the adenocarcinoma in action to drug-induced androgen receptor signaling inhabitation, although the whole procedure behind this transdifferentiation is a subject of discussion. Few effective treatment options are available for its proper cure. The survival of the men with neuroendocrine prostate tumor (NEPC) is very poor. To enhance clinical results, an appreciation of the molecular mechanism maintaining NEPC growth is crucial. The only treatment presently available for NEPC is platinum-based chemotherapy (Ilic et al., 2013).

11.2.3 Transitional cell carcinomas of prostate gland A rare form of prostate cancer is called transitional cell carcinoma of the prostate, or urothelial carcinoma of the prostate. It is very dangerous, but it is curable if it is noticed earlier. In this type of prostate cancer, urothelial cells make up the lining inside the parts of the urinary tract. In men, a part of the urethra passes from the prostate gland. Transitional cell carcinoma of the prostate is initiated in the section of the urethra, or prostate ducts. It spreads easily to other parts of the prostate gland, the bladder, and the seminal vesicles. It can get into nearby lymph nodes and even spread far throughout the body to the bones and organs like the liver and lungs. Men in middle age are the ones most likely to get transitional cell carcinoma of the prostate. The ratio of age at the time of diagnosis is 55 years. If it is only founded at the spot where it started, the doctors may recommend surgery to eliminate the tumor and possibly some attached tissue. Immunotherapy, chemotherapy, and radiation treatment are also applied to cure transition cell carcinoma of the prostate (Wang et al., 2018).

11.2.4 Sarcomas of prostate glands Young prostatic stromal sarcoma is an advanced malignant tumor. The key symptoms are urinary retention secondary to bladder outlet obstruction. The level of the prostatic-specific antigen is normal. Image films show a mass of prostate with or without pelvic organ invasion based on the tumor aggressiveness. A man with prostatic stromal sarcoma is attached with leukocytosis and neutrophilia, urinary obstruction, hypodense prostate areas, and prostate enlargement on CT images, simulating prostatitis with abscess formation. Antibiotic and analgesic treatments were recommended. Instead of neutrophilia and leukocytosis, the person did not express other clinical features of either prostatic abscesses, since blood and urinary culture were negative (Giunchi et al., 2019).

11.3

Causes of prostate cancer

11.3.1 General causes of prostate cancer During this era, work with infectious particles generates only meager results which seemed not relevant to human beings. It is confirmed that a variety of viruses cause prostate cancer in males, and this groundbreaking evidence began to emerge in the 1980s (Gandaglia et al., 2021). There is now enough proof of carcinogenicity in humans for the human T-cell lymphotropic virus, human papillomavirus, hepatitis B virus, HIV, hepatitis C virus, Epstein
Barr virus, and human herpes virus 8. Many other causes of prostate cancer have also been confirmed which include sunlight, hormones, parasites, pharmaceuticals, alcohol, bacteria, salted fish, fungi, herbs, and wood dust. Some additional causes of cancer were determined by the World Cancer Research Fund, which include red meat, obesity, beta carotene, low fiber diets, not breastfeeding, processed meat enhanced high, and a sedentary lifestyle (Danaei et al., 2005).

11.3.2 Genetic causes of prostate cancer A genetic disease causes the gene to change the function of cell growth and division. Genetic changes alter from person to person because of their specific combination. Even these changes differ within various cells of the same tumor. This alteration may occur due to the errors in the process of cell division, maybe inherited, or due to environmental factors like UV rays.

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Main three types of a gene that tend to be the most affected gene due to cancer are listed below: 1. DNA repair gene, 2. Suppressor gene, 3. Proto-oncogenes. Prostate cancer is a result of the change in the DNA of the normal prostate. Gene is a smaller subunit of DNA that controls the proper functioning of our cells. Various genes control when our cells divide to make new ones, grow, and die. Some genes that play role in cell growth, division, and staying alive are called oncogenes. Certain tumor suppressor genes are important for the normal functioning of cells. About 10% of prostate cancer is caused by inherited genes and is called hereditary cancer. Several mutations in genes produce hereditary cancer. Mutations in genes like CHEK2, RAD51D, ATM, PALB2, MSH2, MSH6, MLH1, BRCA1, BRCA2, PMS2, HPC1, and HOB13 are responsible for prostate cancers (Blackadar, 2016).

11.4 G G G G G G G

Symptoms of prostate cancer

Frequent urge to pass urine, especially at night, Nagging pain in the back, hips, or pelvis, Weak or interrupted urine stream, Painful ejaculation, Pain or burning when passing urine, Blood in the urine or semen, Trouble passing urine.

Lymph nodes of the pelvis are mainly infected by prostate cancer, or it may spread all over the body. It may attack the bones. So, pain in the bones, and importantly in the back, can be a sign of advanced prostate cancer. No symptoms are expressed in the early stages of prostate cancer, but screening can detect alterations that may indicate cancer. Measurement of the level of PSA in the blood is done by screening a trusted source. Cancer is present if the level of PSA is high in the blood (Yennurajalingam et al., 2012).

11.4.1 Advanced symptoms Man, with advanced prostate cancer, may also express no signs or symptoms. Potential symptoms will depend on the spreading rate in the body as well as the size of the tumor (Newby et al., 2015). In addition, the mentioned prostate tumor can involve the following signs and symptoms: (1) tiredness, (2) bone pain, and (3) unexpected weight loss.

11.5

Test to identify prostate cancer

After the prostate cancer physical symptoms and observation, the doctor will do the test to check how far the tumor has been expressed. All men are not recommended for each and every test. It is based on the observation of patient biopsy, a method that notices tissue from an infected person’s prostate gland for a tumor (Gandaglia et al., 2021). Tests that support the doctors checking out the condition of your prostate cancer include: (1) MRI of the prostate, (2) digital rectal exam (DRE), (3) prostate-specific antigen test (PSA), (4) surgery to check the lymph nodes in your pelvis for prostate cancer spread, (5) CT scan of the pelvis and abdomen to see if cancer has spread, and (6) nuclear medicine bone scan to check if cancer has spread to the bones (Hayes & Barry, 2014).

11.6

Prostate cancer treatments

Not one prostate cancer treatment is good for every male, but there are a lot of options. The doctor will note many things when they recommend one of you, including: (1) tumor size and how far it is spread, and this is known as the stage of disease; (2) speed of tumor growth; (3) age and health condition of the patient; and (4) patient’s personal preference. With active surveillance, patients also get regular tests to check on their cancer (Thorn et al., 2011). Treatments recommended by the doctors based on the type, stage, and growth of cancer are as follows.

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11.6.1 Surgery The doctor may recommend surgery after a diagnosis of cancer. They may recommend the removal of your prostate gland and sometimes surrounding tissue also. The critical side effect of an operation is problems controlling the patient urine and problem getting and keeping an erection. Nerves around the prostate are also protected to prevent their side effects (Litwin & Tan, 2017).

11.6.2 Radiation High-energy beams (similar to X-rays) are used in this treatment to kill cancer. This treatment is applied when cancer is low grade or confined to the patient’s prostate only. This is recommended when some cancer cells are left behind after surgery. It is helpful to eradicate cancer that has spread in the bones. External and internal radiotherapy works best for prostate cancer (Daniyal et al., 2014).

11.6.3 Proton beam radiation In this special type of radiation therapy, very small particles attack and destroy cancer cells that have not spread still (Thorn et al., 2011).

11.6.4 Hormone therapy Prostate cancer cells require male sex hormones, like testosterone, to keep growing. It is known as androgen deprivation therapy in a doctor’s language. Levels of testosterone and other male hormones are decreased by hormonal treatment. Other types of treatments cannot allow that hormone to work (Litwin & Tan, 2017).

11.6.5 Chemotherapy Drugs or medicines that are given to patients via mouth or IV pass through your body, killing and attacking cancer cells and shrinking tumors. Doctors recommend chemo when the disease has encircled outside the prostate, where hormone therapy is not working for the patient for patient recovery (Wirth et al., 2007).

11.6.6 Immunotherapy This treatment works with your immunity to fight cancer. Advanced prostate cancer is treated by this therapy (Hayes & Barry, 2014).

11.6.7 Bisphosphonate therapy When the disease reaches the bone, these drugs can kill the pain and prevent fractures (Litwin & Tan, 2017).

11.6.8 Cryotherapy For patients having early stage of prostate cancer, the doctor may finalize to destroy the cancer cells by freezing them. They put probes or small needles in the patient prostate to deliver the very cold gas that kills the cells. It is usually the least recommended treatment by doctors (Thorn et al., 2011).

11.6.9 High-intensity focused ultrasound Sound waves are generated by this device. These waves deliver heat energy to kill cancer cells. It is undefined how good it works (Litwin & Tan, 2017).

11.6.10 Prostate cancer vaccine Body defense is boosted by vaccines so it can fight an infection or disease. Vaccines encourage patient immunity to attack cancer cells. When hormone therapy fails to control prostate cancer, this treatment works best. Research could

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not confirm whether it stops or decreases cancer growth, but it is noted that men live longer with prostate cancer (Gulley et al., 2014).

11.7

Prevention of prostate cancer

11.7.1 Physical activity, diet, and body weight Some researchers stated that men who are obese or overweight have a greater risk of developing advanced prostate cancer that may be fatal in the future. Men whose diets are rich in calcium and dairy products have a greater risk of prostate cancer (Cuzick et al., 2014). To reduce the risk of prostate cancer, best advices about activity and diet are: G G G

Remain active physically, Keep maintaining your body weight, Use a healthy eating pattern, and eat a variety of colorful vegetables, fruits, and grains. Avoid processed and red meats and beverages (Thompson et al., 2014).

11.7.2 Mineral, vitamins, and supplements Some studies revealed that selenium or vitamin E may decrease the pattern of prostate cancer. Isoflavones and soy play important role in the eradication of prostate cancer (Barry & Simmons, 2017).

11.7.3 Medicines Prostate tumors may be controlled by using some drugs.

11.7.4 5-Alpha-reductase inhibitors Testosterone is changed into dihydrotestosterone (DHT) by the action of the 5-alpha-reductase enzyme, and DHT is the key enzyme that causes the prostate to grow. This drug blocks the enzyme from forming DHT. These drugs are given to reduce the non-cancerous growth of the prostate (Thompson et al., 2014).

11.7.5 Aspirin Some reports explain that for men who take daily aspirin, the risk of dying from prostate cancer is reduced in them. But much usage of aspirin can also have side effects, including a higher risk of bleeding in the digestive system. It is not recommended by most doctors to cure prostate cancer only (Gandaglia et al., 2021).

11.8

Phytochemicals in prostate cancer

On a global scale, prostate cancer is the second most common type of cancer to be diagnosed in men and ranks as the fifth leading cause of death in men caused by cancer. In 2018, there were 1.276 million cases, and 0.359 million people lost their lives as a result. It is the eighth leading cause of death in both men and women combined and the fourth most common type of cancer to be diagnosed in the United States after lung, breast, and colon cancers in terms of incidence (Bray et al., 2018). If the current trend keeps going, the number of men diagnosed with prostate cancer around the world will reach 2.2938 million in the year 2040. Although prostate cancer is the most commonly diagnosed form of cancer in more than half of the world’s countries (105 out of 185), the death rate due to prostate cancer is highest in Southern Africa (26.4%), followed by the Caribbean (25.4%), and then middle Africa (22.4%). Cancer of the prostate is the most common form of malignancy found in men in both the United Kingdom and the United States (Bray et al., 2018; Ferlay et al., 2019). Cancer of the prostate is a diverse disease that ranks as the second deadliest form of malignancy in men and is the most common type of cancer to be diagnosed in men. Traditional medicinal plants have been used both to treat a wide range of diseases and to develop new pharmaceuticals. Plants used for medical purposes have the potential to be sources of naturally occurring bioactive compounds such as alkaloids, phenolic compounds, terpenes, and steroids. Over the course of the last century, there has been an alarming rise in the prevalence of chronic diseases such as cancer, which has become one of the most challenging situations for the public health systems of underdeveloped and developing countries. Because of its increasing incidence, mortality rate, and high treatment cost, cancer is

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one of the most pressing public health concerns in every nation. This is true for people of both sexes and all ages. In general, cancer continues to be not only a factor in the enormous toll it takes on people’s health but also the world’s second leading cause of death from disease. Cancer is caused by the growth of cells out of control. This can happen in different tissues and spread to tissues nearby and far away. Even though there has been a lot of progress in cancer biology, it is still one of the leading causes of death, and those who do survive can have permanent problems (such as physical, cognitive, psychosocial, and treatment side effects). It is very worrying that cancer is a common disease and that the number of diagnoses is rising sharply around the world. There are a lot of possible reasons for this rise, but the most important one is that people are changing the way they live. Because prostate cancer is a diverse disease, determining the characteristics that put a patient at risk is essential to determine how the disease will behave. In epidemiological research, it has been repeatedly stressed that naturally occurring dietary substances exhibit chemopreventive qualities and might easily suppress a variety of cancers, including that of the prostate. This is one of the malignancies that has been the focus of these investigations. However, there has been a lack of consensus regarding the optimal plant-based diet, the minerals and phytochemicals that go along with it, and how they affect prostate health. In this sense, the purpose of the present review is to provide a detailed overview of the physiopathology of prostate cancer, including the primary risk factors and current therapeutic strategies, and of the role of plant-derived phytochemicals, including plant extracts and its corresponding isolated compounds, in prostate cancer. Specifically, the review will focus on the role of plant extracts and its corresponding isolated compounds.

11.9

Phytochemicals and conventional medical practice

The consumption of meals produced from plants is linked to a multitude of health benefits, including the reduction or elimination of one’s risk of developing chronic diseases. Consumption of these foods results in the delivery of a diverse range of nutrients and phytochemicals, all of which contribute to the maintenance of normal growth and the prevention of the onset of a wide variety of chronic diseases. Phenolics, alkaloids, nitrogen-containing compounds, organosulfur compounds, phytosterols, and carotenoids are the six major categories that are used to organize identified phytochemicals. Secondary plant metabolites known as phenolics play an important function in plant defense and are produced by plants. It is interesting to note that some of these responsibilities include fighting cancer, reducing inflammation, and killing microorganisms. To this day, several thousand different phenolic compounds have been extracted from a wide range of plant sources, including fruits, grains, and legumes. In reaction to environmental stress, plants develop the secondary metabolite known as alkaloids. These chemicals have been shown to exhibit a variety of features, including cytotoxicity and efficacy against inflammation. Isothiocyanates are among the organosulfur compounds generated from plants that have been subjected to the greatest research. It is essential to note that this class of chemicals possesses chemoprotective action. Carotenoids and polyphenols are two types of compounds that are known to possess antiinflammatory and antioxidant properties. In addition, the phytochemicals in question offer protection against the oxidative stress that is linked to a variety of persistent disorders, such as diabetic retinopathy and macular degeneration. Phytochemicals are a fascinating class of potential new therapeutic reagents for the prevention and treatment of cancer, particularly in light of the fact that they have a long history of usage in traditional medicine and have the potential to inhibit the growth of cancer cells.

11.10 Effects of specific plant families extracts on human prostate cancer cells Important plant families have been studied in recent years for their potential to be used as anticancer treatments. These plants’ anticancer properties are directly related to the bioactive compounds found in their extracts. While many of these plants’ bioactive compounds are similar, recent research has identified unique components that may be useful as chemotherapeutic agents.

11.10.1 Juglandaceae Juglandaceae are monoecious or dioecious trees or shrubs with pinnate or trifoliolate leaves, flowers small, corolla absent, male flowers in catkins, female flowers at shoot tips, the ovary inferior with 2
3 carpels and locules (1 loculate above), the subtending bracts fusing and forming an outer husk in many taxa, and the fruit a 1-seeded nut, samara. Members of this family are mostly found in temperate zones. They are made up of trees and shrubs that produce nuts such as walnut. One of the most common phytochemicals produced by members of this family is juglone. Juglone has been shown to inhibit EMT, migration, and invasion of LNCaP and LNCap-A1 cells via the glycogen synthase kinase-

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3/snail-dependent pathway. EMT is especially important in cancer progression because epithelial cancer cells that transform to a mesenchymal phenotype are more invasive and thus more likely to metastasize. Furthermore, because bone metastases significantly reduce patient survival, inhibiting this process represents a potent therapeutic target for slowing the progression of prostate and other cancers.

11.10.2 Crassulaceae Crassulaceae are a family of dicotyledonous perennials that can take the form of herbaceous, shrubby, or even tree-like plants. The warm, arid regions of the planet are the natural habitat for these plants. Uronic acid can be found in an extract made with methanol from the roots of Kalanchoe gastonis-bonnieri. The results of this study showed that DU145, LNCaP, and PC-3 cells that were exposed to this extract underwent apoptosis that was mediated by caspase-8. One of the defining characteristics of cancer cells is their ability to thwart the cell death process known as apoptosis. As a result, substances that are capable of inducing apoptosis in cancer cells are necessary for inhibiting a significant characteristic of the cancer phenotype. Caspases 3 and 8 are both considered to be regulators of the programmed cell death process. Caspase-3 expression has been demonstrated in the past to be connected with a higher likelihood of survival for breast cancer patients, which is an intriguing finding. These observations strongly suggest that inducing the production of caspase-8 could potentially boost the survival rate of prostate cancer patients.

11.10.3 Moraceae The trees and shrubs that belong to the family Moraceae are either deciduous or evergreen, depending on the species, and they are found naturally in tropical and subtropical locations. While several genera are prized for their edible fruits, others are known for the waxy or latex-like compounds they generate. Flavonoids, triterpenoids, ascorbic acid, and chlorogenic acid are only some of the phytochemicals that have been extracted from members of the Moraceae family. Other phytochemicals include phenolic acids. Apoptosis was triggered in LNCaP and PC-3 cells by extracts from the angustifolia and deltoidea kinds of the fig tree (Ficus deltoidea). These extracts also hindered the cells’ ability to invade and migrate. In addition, the expression of vascular endothelial growth factor-A in PC-3 cells was regulated by these plant extracts, which led to angiogenesis being suppressed. Angiogenesis is a process that contributes to the development of tumors. Extracts from the fruit of Morus nigra, which contain high quantities of ascorbic and chlorogenic acids, had a similar effect on PC-3 cells, causing them to enter an arrested state in the Gap 1 phase of the cell cycle and inducing them to undergo apoptosis. Cancer cells are characterized by fundamental traits like angiogenesis and uncontrolled proliferation. In this way, the inhibition of either mechanism has the ability to restrain the expansion of cancer cells and the creation of tumors.

11.11 Prostate cancer risk factors Over the course of their lifetimes, the majority of men will develop prostate cancer at some point. On the other hand, there are certain risk factors that, when combined, increase the probability of developing the disease.

11.11.1 Age Men under the age of 40 have an extremely low risk of developing aggressive prostate cancer, and the disease affects six out of ten men aged 65 and older. However, the likelihood of developing prostate cancer increases with advancing age. The American Cancer Society estimates that approximately 60% of all prostate cancers are diagnosed in men over the age of 65 (Rebbeck, 2017).

11.11.2 Race According to the Centers for Disease Control and Prevention (CDC), men of African-American descent have the highest rate of prostate cancer, followed by men of Hispanic origin, men of American Indian or Alaska Native descent, men of white origin, and men of Asian or Pacific Islander descent. According to the findings of the American Cancer Society, African-American men have a mortality rate from prostate cancer that is two times higher than that of white men. It is possible that men from Scandinavia also have a higher risk of prostate cancer. Throughout recorded history, the incidence rate in East Asia (including China and Japan) has been relatively low. However, when men of Chinese and Japanese descent immigrate to the United States, they are at an increased risk of developing prostate cancer in comparison to the populations from which they originally came.

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11.11.3 Diet According to the findings of some medical studies, the typical diet of people living in industrialized Western nations may be a contributing factor in the development of prostate cancer. Take into consideration the following information regarding diet and its influence on the risk of developing prostate cancer. Studies have shown that men who consume a diet that is high in fat, particularly one that is high in red meat or high-fat dairy products, may have an increased risk of developing prostate cancer. Fruits and vegetables: It has been suggested that diets that are high in fruits and vegetables may reduce the risk of prostate cancer; however, it is not known which nutrient or nutrients may be responsible for this effect. Carotenoids: It has been demonstrated that carotenoids, such as lycopene, can inhibit the growth of human prostate cancer cells that have been grown in the laboratory. Tomatoes that have been processed are the most important source of lycopene. However, it is unclear whether or not lycopene influences the risk of prostate cancer in men.

11.11.4 Obesity According to the findings of the vast majority of studies, being obese does not influence the overall risk of developing prostate cancer. On the other hand, men who are obese may have an increased risk of developing more aggressive forms of prostate cancer.

11.11.5 Environmental exposures According to the findings of some studies, men who are exposed to particular kinds of chemicals have an increased likelihood of developing prostate cancer. As an illustration, firefighters are routinely exposed to the byproducts of combustion, whereas farmers are regularly exposed to agricultural chemicals. Because of the possible exposure to chemicals, men who work in these occupations may have an increased risk of developing prostate cancer.

11.11.6 The past of the family The presence of a positive family history is also an important risk factor. According to the American Cancer Society, if either your father or your brother had prostate cancer, your risk of developing the disease is more than doubled. If either of your parents had breast cancer, your risk of developing breast cancer is lower. Men who have several affected relatives, particularly if those relatives were young when they were diagnosed with prostate cancer, have a significantly increased risk of developing prostate cancer themselves.

11.12 Conclusion Cancer is a significant public health problem that continues to be the leading cause of death on a global scale. Scientists have been able to develop a wide variety of cancer-fighting medications as they have gained a deeper understanding of the molecular processes that drive the disease’s progression. However, the use of pharmaceuticals that are derived from chemicals has not resulted in a significant improvement in the overall survival rate over the course of the past few decades. As a direct result of this, novel strategies and groundbreaking chemoprevention drugs are required to supplement the currently available prostate cancer treatments in order to boost the efficacy of these treatments. Phytochemicals, which are molecules that occur naturally in plants and which are also sources for cancer therapy, are important resources for the development of novel medications and are also sources for cancer therapy. Phytochemicals can be found in a wide variety of plant species. Chemoprevention is the practice of lowering the risk of developing cancer in otherwise healthy people through the administration of man-made, natural, or biological substances. Chemopreventive drugs inhibit the development of cancer either by preventing the DNA damage that results in malignancy or by reversing or inhibiting the division of premalignant cells that have DNA damage. Either way, this results in the suppression of cancer formation. In clinical trials involving patients with breast, prostate, and colon cancer, this strategy has been demonstrated to be beneficial. There has been an ongoing increase in the number of cancer cases, conventional chemotherapies have not been successful in controlling prostate cancer, and there is excessive toxicity associated with chemotherapies. These three factors call for a new approach.

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References Barry, M. J., & Simmons, L. H. (2017). Prevention of prostate cancer morbidity and mortality: Primary prevention and early detection. Medical Clinics, 101(4), 787
806. Blackadar, C. B. (2016). Historical review of the causes of cancer. World Journal of Clinical Oncology, 7(1), 54. Blank, C. U., Haanen, J. B., Ribas, A., & Schumacher, T. N. (2016). The “cancer immunogram.”. Science (New York, N.Y.), 352(6286), 658
660. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6), 394
424. Cuzick, J., Thorat, M. A., Andriole, G., Brawley, O. W., Brown, P. H., Culig, Z., . . . Wolk, A. (2014). Prevention and early detection of prostate cancer. The Lancet Oncology, 15(11), e484
e492. Danaei, G., Vander Hoorn, S., Lopez, A. D., Murray, C. J., & Ezzati, M. (2005). Comparative Risk Assessment collaborating group (Cancers, 2005). Causes of cancer in the world: Comparative risk assessment of nine behavioural and environmental risk factors. The Lancet, 366(9499), 1784
1793. Daniyal, M., Siddiqui, Z. A., Akram, M., Asif, H. M., Sultana, S., & Khan, A. (2014). Epidemiology, etiology, diagnosis and treatment of prostate cancer. Asian Pacific Journal of Cancer Prevention, 15(22), 9575
9578. Ferlay, J., Colombet, M., Soerjomataram, I., Mathers, C., Parkin, D. M., Pin˜eros, M., & Bray, F. (2019). Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. International Journal of Cancer, 144(8), 1941
1953. Gandaglia, G., Leni, R., Bray, F., Fleshner, N., Freedland, S. J., Kibel, A., . . . La Vecchia, C. (2021). Epidemiology and prevention of prostate cancer. European Urology Oncology. Giunchi, F., Fiorentino, M., & Loda, M. (2019). The metabolic landscape of prostate cancer. European Urology Oncology, 2(1), 28
36. Gulley, J. L., Madan, R. A., Tsang, K. Y., Jochems, C., Marte´, J. L., Farsaci, B., . . . Schlom, J. (2014). Immune impact induced by PROSTVAC (PSA-TRICOM), a therapeutic vaccine for prostate cancer. Cancer Immunology Research, 2(2), 133
141. Hayes, J. H., & Barry, M. J. (2014). Screening for prostate cancer with the prostate-specific antigen test: A review of current evidence. JAMA: The Journal of the American Medical Association, 311(11), 1143
1149. Ilic, D., Neuberger, M. M., Djulbegovic, M., & Dahm, P. (2013). Screening for prostate cancer. Cochrane Database of Systematic Reviews (1). Lee, S. H., & Shen, M. M. (2015). Cell types of origin for prostate cancer. Current Opinion in Cell Biology, 37, 35
41. Litwin, M. S., & Tan, H. J. (2017). The diagnosis and treatment of prostate cancer: A review. JAMA: The Journal of the American Medical Association, 317(24), 2532
2542. Newby, T. A., Graff, J. N., Ganzini, L. K., & McDonagh, M. S. (2015). Interventions that may reduce depressive symptoms among prostate cancer patients: A systematic review and meta-analysis. Psycho-Oncology, 24(12), 1686
1693. Rawla, P. (2019). Epidemiology of prostate cancer. World Journal of Oncology, 10(2), 63. Rebbeck, T. R. (2017). Prostate cancer genetics: Variation by race, ethnicity, and geography. Seminars in radiation oncology (Vol. 27, pp. 3
10). WB Saunders. Thompson, I. M., Cabang, A. B., & Wargovich, M. J. (2014). Future directions in the prevention of prostate cancer. Nature Reviews Clinical Oncology, 11(1), 49
60. Thorn, C. F., Marsh, S., Carrillo, M. W., McLeod, H. L., Klein, T. E., & Altman, R. B. (2011). PharmGKB summary: Fluoropyrimidine pathways. Pharmacogenetics and Genomics, 21(4), 237. Wang, G., Zhao, D., Spring, D. J., & DePinho, R. A. (2018). Genetics and biology of prostate cancer. Genes & Development, 32(17
18), 1105
1140. Wirth, M. P., Hakenberg, O. W., & Froehner, M. (2007). Antiandrogens in the treatment of prostate cancer. European Urology, 51(2), 306
314. Yennurajalingam, S., Atkinson, B., Masterson, J., Hui, D., Urbauer, D., Tu, S. M., & Bruera, E. (2012). The impact of an outpatient palliative care consultation on symptom burden in advanced prostate cancer patients. Journal of Palliative Medicine, 15(1), 20
24.

Further reading Abate-Shen, C., & Shen, M. M. (2000). Molecular genetics of prostate cancer. Genes & Development, 14(19), 2410
2434. Cao, Y., & Giovannucci, E. (2016). Obesity and prostate cancer. Obesity and Cancer, 137
153. Denmeade, S. R., & Isaacs, J. T. (2002). A history of prostate cancer treatment. Nature Reviews. Cancer, 2(5), 389
396. Khan, H. M., & Cheng, H. H. (2022). Germline genetics of prostate cancer. The Prostate, 82, S3
S12. Pienta, K. J., & Esper, P. S. (1993). Risk factors for prostate cancer. Annals of Internal Medicine, 118(10), 793
803.

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Chapter 12

Therapeutic phytochemicals from Plumbago auriculata: a drug discovery paradigm Khalida Bloch1, Vijay Singh Parihar2, Minna Kelloma¨ki2, Sirikanjana Thongmee3 and Sougata Ghosh1,3 1

Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India, 2Biomaterials and Tissue Engineering Group, BioMediTech,

Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland, 3Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand

12.1

Introduction

Conventional medicine to combat the human health problems and life-threatening diseases is not only expensive but also has several side effects. Medicinal plants that are used since ancient times to treat and cure deadly diseases can serve as potential complementary and alternative medicine. Formulations developed from plant-derived secondary metabolites have broad-spectrum therapeutic applications with negligible or no adverse effects. Plants can either produce and secrete or store these bioactive principles in their tissues. Plumbago auriculata is a medicinal plant from the family Plumbaginaceae (Leadwort family) that was first described by Antoine Laurent De Jussieu in 1789. Plumbaginaceae with 24 genera and 400 species includes herbs, lianas, and shrubs which occur even in saline habitats. P. auriculata is a perennial, evergreen shrub that is mostly native to South Africa. It is also distributed in the tropical and subtropical regions and is commonly known as Cape Plumbago or Cape Leadwort as it is widely located in the Cape regions of South Africa (Singh, Naidoo, & Bajinath, 2018; Singh, Naidoo, Mocktar, et al., 2018). P. auriculata can tolerate high humidity and elevated temperature. The plant can grow up to 3 m in height with erect and climbing stems. The leaves are thin in texture and have miniscule glandular dots, elliptic, and greyish green beneath with a whitish scale. Salt glands are present on both surfaces of the leaves. The flower colors of P. auriculata can vary from sky blue, deep blue to white. The flowers are salver-shaped with 2.5
3 cm long, and flowering occurs throughout the year (Ferrero et al., 2009). The corolla is blue, and the calyx possesses glandular and nonglandular hair known as trichomes. The fruit is long-beaked, and the seed is dark brown with a length of 7 mm (Luteyn, 1990). P. auriculata produces a major compound known as Plumbagin. The plant is rich in tannins, phenols, alkaloids saponins, proteins, and sugars. This chapter emphasizes the detailed traditional uses, phytochemistry, medicinal, and other properties of P. auriculata. Fig. 12.1 gives a schematic representation of promises of P. auriculata in drug development.

12.2

Traditional uses

The stems, roots, and leaves of P. auriculata possess neuroprotective, hepatoprotective, cardiotoxic, and antiatherogenic properties (Deshpande et al., 2014). The roots and leaves are used for the treatment of edema, headache, lesions on the skin, piles, diarrhea, rheumatism, warts, and fractures (Elgorashi et al., 2003). Aerials parts of the plant are also used in the treatment of malaria, mostly endemic in Mozambique (Ramalhete et al., 2008). People of Manchale Village located in India use a paste made from the root prepared in water for the treatment of piles (Poornima et al., 2012). The fresh root extract prepared in rainwater is used by Indian tribes to combat acidity and also for treating diarrhea in cows (Dold & Cocks, 2001; Jain et al., 2010). Hence, P. auriculata is not only used to treat diseases in humans but also livestock (McGaw & Eloff, 2008). Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00027-X © 2023 Elsevier Inc. All rights reserved.

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FIGURE 12.1 Schematic representation for the scope of drug discovery from P. auriculata. The diagram shows the collection of the P. auriculata plant samples followed by their size reduction for increasing the surface area for efficient extraction. The sequential extraction of diverse phytochemicals can be achieved using the Soxhlet apparatus using both nonpolar and polar solvents. Bioassay-guided fractionation results in the isolation of the bioactive principles that can be further purified using chromatographic techniques. The pure compounds can be checked for therapeutic activity using in vitro and in vivo experimental models. The bioactive compounds with confirmed therapeutic potential can be further taken ahead for drug development.

12.3

Phytochemistry

P. auriculata is of immense medical interest due to the predominance of diverse phytochemical constituents with promising therapeutic advantages. Secondary metabolites from the plant with healing properties include saponins, tannins, alkaloids, glycosides, flavonoids, phenols, and naphthoquinones, apart from carbohydrates, proteins, oils, and fats which are present in aerials parts of the plant as listed in Table 12.1. The phytochemical constituents can be extracted sequentially in different solvents such as methanol, hexane, acetone, chloroform, petroleum ether, ethanol, toluene, and ethyl acetate and analyzed using gas chromatography
mass spectrometry (GC-Ms). The flowers of the plant contain azalein, capensinidin, and capensindin-3-rhamnoside while leaves show the presence of luteolin and glycosides as shown in Fig. 12.2. The major secondary metabolite present in P. auriculata is hydroxyl naphthoquinone known as plumbagin. The roots of the plant possess several chemical constituents such as α-amyrin, α-amyrin acetate, glucopyranoside, and isoshinanolone. A new natural compound known as capensisone is found in P. auriculata (Saji & Antony, 2015). More than 20 phytochemical compounds were screened from the hexane extract of P. auriculata leaves among which 13-docosenamide was the major compound. This compound was identified the first time in the cerebrospinal fluid of sleep-deprived cats, rats, and humans. 13-Docosenamide also reduces mobility in rats (Ben-Shabat et al., 1998). The fatty acid ester, 2-plamitoylglycerol (hexadecanoic acid 2-hydroxyl-1-(hydroxymethyl) ethyl ester), present in the hexane leaf extract increased the inhibitory activity of 2-arachidonylglycerol on adenylyl cyclase (Gallily et al., 2000). 9-Octadecenamide was also obtained in the calyx extract of P. auriculata prepared in hexane which exhibited anticancer activity (Abubakar & Majinda, 2016). A linolenic ester obtained in the plant extract known as 9,12-octadecadienoic acid methyl ester with anti-inflammatory, antiarthritic, antihistamine, anticancer, antieczemic, anticoronary, antiacne, insecticidal, and nematicidal activity was also reported (Sharmila et al., 2016). An alcoholic compound known as nanodecanol exhibited antimicrobial and cytotoxic activities (Kuppuswamy et al., 2013).

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TABLE 12.1 Phytochemical constituents present in P. auriculata. Plant parts

Phytochemical

References

Aerial parts

Palmitic acid

Ribeiro de paiva et al. (2005)

Epi-isoshinanolone Plumbagic acid Sitosterol 3-O-glucosylsitosterol Gallic acid

Melk et al. (2021)

Chlorogenic acid Catechin Methyl gallate Caffeic acid Syringic acid Pyro catechol Rutin Ellagic acid Coumaric acid Vanillin Ferulic acid Naringenin Taxifolin Cinnamic acid Kaempferol Leaves

Apigenin

Saji and Antony (2015)

Luteolin Flower

Azalein Capensinidin Capensinidin-3-rhamnoside

Leaves and flowers

2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran4-one

Rios-Chavez et al. (2019)

5-Hydroxymethyl-2-furancarboxaldehyde Ethyl α-D-glucopyranoside Caffeine Theobromine n-Hexadecanoic acid Phytol γ-Sitosterol Lupenone Lupeol (Continued )

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TABLE 12.1 (Continued) Plant parts

Phytochemical

References

Roots

α-Amyrin acetate

Singh, Naidoo, and Bajinath (2018), Singh, Naidoo, Mocktar, et al. (2018)

Plumbagin

Taneja et al. (2021)

α-Amyrin

Singh, Naidoo, and Bajinath (2018), Singh, Naidoo, Mocktar, et al. (2018)

β-Sitosterol Diomuscinone Capensisone Isoshinanolone β-sitosterol-β-glucopyranoside Isoshinanolone Z-11-Octadeen-1-yl-acetate

Lakshmanan et al. (2016)

Heptadecanoic acid 4,5,7-Trihydroxy isoflavone E,E,Z-1,3,12-nonadecatrine-5, 14-diol Ethanol, 2-(9-octadecenyloxyl)-, (E) 13-Docosenoic acid, methyl ester (Z) Bis(2-ethyl hexyl) phthalate 10,12,14-Nonacosatriynoic acid

12.4

Plumbagin

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, C11H8O3) is a broad-spectrum potent bioactive compound present in roots, stems, and leaves of the genus Plumbago. The roots of the plant contain a high concentration of plumbagin (Padhye et al., 2012). Plumbagin was isolated for the first time in the year 1829 using solvent extraction of plant powder (Tyagi & Menghani, 2014). Plumbagin is a naturally occurring organic yellow-colored compound soluble in a variety of solvents such as acetone, benzene, ethanol, methanol, acetic acid, and chloroform and slightly soluble in hot water with a melting point of 72 C
79 C. The name plumbagin is derived from its plant source Plumbago (Taneja et al., 2021). This compound shows a broad range of activities such as anticancer, cardiotonic, radio-modifying, leishmanicidal, anti-inflammatory, antimicrobial, antifertility, antidiabetic, antioxidant, and antimalarial activities. Plumbagin is also used in the treatment of intestinal, neural, cardiac, and rheumatic pain relief (Galal et al., 2013). The leaves and stems of P. auriculata have considerably more amount of plumbagin as compared to other Plumbago species (Singh, Naidoo, & Bajinath, 2018; Singh, Naidoo, Mocktar, et al., 2018). It shows potential abilities such as the generation of reactive oxygen species (ROS), chelating of metal, and capacity to tolerate redox cycling. It also exhibits plasmid curing ability which refers to the removal of conjugable multidrug-resistant plasmids from various bacterial strains. Additionally, several drug molecules are conjugated with plumbagin for the formulation of anticancer agents. It has been reported that human serum albumin combined with plumbagin enhances cell apoptosis, cell cycle arrest, and cytotoxicity in cancer cells. It also exhibits antiproliferative activity in human breast carcinoma cells. It can inhibit the epithelial
mesenchymal transition (EMT) and cell proliferation which induces autophagy, DNA damage, angiogenesis, metastasis, reduction of cell viability, and expression of the p53 gene as has anticancer activity against prostate cancer, renal cancer, lung cancer, breast cancer, oral cancer, ovarian cancer, pancreatic cancer, liver cancer, skin cancer, brain cancer, cervical cancer, esophageal cancer, osteosarcoma, glioblastoma, cholangiocarcinoma, and leukemia (Taneja et al., 2021). Higher concentration of plumbagin delays the germination of fungus. It also inhibits the topoisomerase-II, an enzyme involved in rheumatoid arthritis, and suppresses the activation of NF-kB, a ubiquitous transcription factor in tumor cells that affects the functions of leukocytes in immune responses. A wide range of bacterial strains such as Staphylococcus aureus, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Escherichia coli are inhibited by plumbagin (Padhye et al., 2012). It can act as an irritant to smooth the muscle of the uterus when taken orally at

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a higher concentration while it shows an antifertility effect at a concentration of 1 mg/100 g of body weight in female rats. Efficient in vitro and in vivo antimalarial activity was shown by plumbagin against K1 and 3D7 Plasmodium falciparum and Plasmodium berghei infected mouse models, respectively (Sumsakul et al., 2014). It also reduces the blood glucose level significantly by increasing the activity of hexokinase and decreasing the activity of glucose-6-phosphate and fructose-1,6-bisphosphate (Sunil et al., 2012). Further, it acts as a promising antioxidant that can reduce catechol-induced DNA damage and inhibits ascorbate and NADPH-dependent lipid peroxidation in the lymphoma cells of the mouse (Demma et al., 2009).

12.5

Medicinal uses

12.5.1 Antimicrobial activity Antibacterial activity of root extracts of P. auriculata prepared in water, chloroform, methanol, and ethanol was tested against 150 different strains of E. coli isolated from water and fecal samples of patients collected from the Mthata region, Eastern Cape, South Africa, using the Kirby
Bauer disk diffusion method. The ethanolic extract showed the highest antibacterial activity against all isolated E. coli strains (Muringani & Makwikwi, 2017). In another study, the aerial parts of the plant were used to evaluate the antibacterial activity. The aerial parts were dried, and extracts were prepared in solvents such as ethanol, petroleum ether, and chloroform. Antibacterial activity was checked using the agar well diffusion method against Streptococcus pyogenes, Pseudomonas aeruginosa, S. aureus, Bacillus subtilis, and Morganella morganii. Amikacin antibiotic (30 μg/disk) was used as a control. Ethanolic extract of P. auriculata showed the highest antibacterial activity (70%) with a 23 6 0.3 mm zone of inhibition. P. auriculata showed more than 50% activity against gram-positive bacteria and 26% against gram-negative bacteria. It was further concluded that the presence of alkaloids and phenolic compounds present in the plant might be responsible for the antibacterial activity (Tharmaraj & Antonysamy, 2015). Patwardhan et al. (2015) showed that the root extract of P. auriculata exhibits plasmid curing potential. The root extracts were prepared using the Soxhlet apparatus with petroleum ether, chloroform, acetone, and ethanol. After 24 hours, the extracts were filtered and concentrated to dryness using a rotary evaporator. Plasmid curing was performed in E. coli, Proteus vulgaris, and Klebsiella pneumoniae. The ethanolic extract exhibited strong antibacterial activity against P. vulgaris followed by K. pneumoniae, E. coli, and P. aeruginosa. Among all extracts, the root extract prepared in ethanol showed maximum plasmid curing activity as compared to petroleum ether, chloroform, and acetone. The ethanolic root extract of P. auriculata cured plasmids with 32%, 30%, 15%, and 13% curing efficiency in P. vulgaris, K. pneumoniae, E. coli, and P. aeruginosa, respectively (Patwardhan et al., 2015). The ethanolic root extract of P. auriculata showed significant antibacterial activity against Helicobacter pylori. The roots were collected and dried. The dry roots were converted to powder and soaked in lime water. The lime water was changed until the red color disappeared. Further, they were dried, the extract was prepared using the Soxhlet apparatus for 6 hours, and ethanol was used as solvent. The antibacterial activity using 100 μg/mL of extract was performed on a Columbia agar plate where a zone of inhibition of 1.73 cm was observed against H. pylori (Paul et al., 2013).

12.5.2 Anticancer and cytotoxic activity Medicinal plants have promising anticancer activity that is exploited substantially for drug development to control and inhibit cancer and its associated clinical complications. The dose-dependent cytotoxic activity in HGE-17 cell lines was tested using 50
250 μg/mL of ethanolic root extract of P. auriculata using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. IC50 value of P. auriculata was found to be 278.59 μg/mL (Paul et al., 2013). Anticancer activity of methanolic root extract of P. auriculata was analyzed against human lung adenocarcinoma (A549) and ovarian teratocarcinoma cell line (PA1) using MTT assay. The cells were treated with different concentrations of drug (5
60 μg/mL) and incubated for 24 and 48 hours. A significant reduction in cell viability was observed in both cell lines. The IC50 values of methanolic extract were 45 and 10 μg/mL for the A549 cell line whereas they were 10 and 60 μg/mL for the PA1 cell line at 24 and 48 hours, respectively. Beta-sitosterol in the plant extract was speculated to be involved in the anticancer activity (Lakshmanan et al., 2016).

12.5.3 Antioxidant activity Oxidative stress due to high levels of free radicals is associated with several diseases. Hence, antioxidants of herbal origin can help to scavenge free radicals and control the level of reactive oxygen species (ROS). Leaf extract of P. auriculata was used to check the antioxidant activity. The leaves were collected, dried, and converted into fine powder. About 10 g of leaves

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were extracted in methanol, acetone, and water separately. Ferric reducing assay was performed using methanolic extract where Perl’s Prussian blue color obtained by the formation of iron (II)-ferrocyanide complex was measured at 700 nm. The presence of antioxidant can be determined by the reduction of Fe31 to Fe21 by donating electron. In the case of ferrix reducing power assay, the IC50 value was 70.79 μg/mL while for trolox it was 50.12 μg/mL. Additionally, the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity was performed using different concentrations of methanolic plant extracts. The IC50 value for P. auriculata was 21.38 μg/mL for DPPH radical scavenging activity while for trolox it was 1.85 μg/mL (Jaradat et al., 2016). The antioxidant activity of the methanolic root extract of P. auriculata was determined using DPPH and FRAP assays. The presence of sitosterol in the plant may be the major reason for the antioxidant activity. For DPPH free radical scavenging activity, P. auriculata showed IC50 value at 260 μg/mL. For ferric reducing power assay, maximum absorbance at 0.321 nm was observed in the case of methanolic root extract of P. auriculata (Lakshmanan et al., 2016).

12.5.4 Antiobesity Sedentary lifestyle associated with weight gain and obesity enhances the risk of diabetes, hypertension, cancer, and sleep disorders. Hence, developing an herbal remedy for controlling obesity is one of the key areas of present researches. Porcine pancreatic lipase inhibitory assay was employed to check the antiobesity effect of P. auriculata leaf extract. Pancreatic lipase enzyme (1 mg/mL) was treated with various concentrations of the plant extract while p-

FIGURE 12.2 Phytochemical diversity of P. auriculata.

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FIGURE 12.2 (Continued).

nitrophenyl butyrate (PNPB) was used as a substrate. The hydrolysis of p-nitrophenolate to p-nitrophenol was determined spectrophotometrically at 405 nm. The methanolic extract showed IC50 value of 130.32 μg/mL indicating lipase inhibitory activity of P. auriculata (Jaradat et al., 2016).

12.5.5 Antiulcer activity Roots of P. auriculata were used to check the antiulcer activity. The roots were collected, dried, and converted into a fine powder and extracted using the Soxhlet apparatus for 6 hours with ethanol as a solvent. Evaluation of antiulcer activity was carried out using 80% ethanol-induced ulcer model in goat intestine. Additionally, the in vivo antiulcer potential was evaluated in aspirin-induced model and ethanol-induced model in rats. P. auriculata showed 96.04% protection from ulcers in in vitro analysis. In the aspirin-induced model, P. auriculata (300 mg/kg) showed significant ulcer reduction with 0.83 6 0.40, 1.92 6 0.55, and 5.13 6 0.05 for ulcer number, ulcer score, and ulcer index, respectively. The extract showed 40.45% antiulcer activity. In the case of the ethanol-induced model, the plant extract (300 mg/kg) showed a significant ulcer reduction with 1.17 6 0.40, 5.25 6 0.81, and 6.92 6 0.05 for ulcer number, ulcer score, and ulcer index, respectively. The protection against ulcer in both models indicates that the extract possesses cytoprotective activity (Ittiyavirah & Paul, 2016).

12.6

Nano-biotechnology

Biogenic nanoparticles are advantageous as their synthesis does not involve hazardous chemicals for reduction and stabilization unlike chemical and physical methods for nanoparticle synthesis (Bloch et al., 2021; Ghosh & Webster, 2021a, 2021b, 2021c). Various bacteria, cyanobacteria, fungi, algae, and plants are reported for the synthesis of

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nanoparticles which is considered as a rapid, efficient, and environmentally benign approach (Ghosh, Bhagwat, et al., 2022; Ghosh, Sarkar, et al., 2022; Nitnavare et al., 2022). The rich phytochemistry of medicinal plants has led to the exploration of their promising nano-biotechnological potential (Ranpariya et al., 2021). Polyphenols, flavonoids, ascorbic acid, citric acid, tannins, starch, saponin, alkaloids, and others can play significant roles as reducing and stabilizing agents. Hence, P. auriculata with potential phytochemical diversity is reported to synthesize various metal and metal oxide nanoparticles. Zinc oxide nanoparticles (ZnONPs) were synthesized using aerial parts of P. auriculata. The plant powder (100 g) was extracted using 90% ethanol which was allowed to react with zinc acetate for 20 minutes at pH 12 that resulted in the precipitation of ZnONPs. The nanoparticles were recovered by centrifugation followed by washing and freezedrying. The average particle size was 10.58 6 3.350 nm with a zeta potential of 219.6 mV. Transmission electron microscope (TEM) images revealed that the size of the particle was in the range of 5.08 6 6.56 nm while some bigger particles of size 38.29 6 6.88 nm were also observed in scanning electron microscope (SEM) images as shown in Fig. 12.3. Fourier transform infrared (FTIR) spectroscopic analysis revealed the involvement of active groups for alcohols, ethers, carboxylic acid esters, and phenolic compounds in the synthesis and stabilization of the phytogenic ZnONPs. The antiviral activity of ZnONPs was checked against avian metapneumovirus (aMPV) subtype B. Significant antiviral activity of uncalcinated ZnONPs was 52.48 6 1.57 and 42.67 6 4.08 μg/mL with 50% cytotoxic concentration (CC50) and 50% inhibition concentration (IC50), respectively. The inhibition percentage was 99%, and the selectivity index was 1.23 (Melk et al., 2021). In another study, an aqueous extract of P. auriculata was used for the synthesis of silver nanoparticles (AgNPs), and its antibacterial and antilarvicidal activities were evaluated. The leaf extract was reacted with 1 mM silver nitrate (AgNO3) in darkness for 24 hours at 25 C 6 2 C. The resulting AgNPs, as indicated by the appearance of the brown color in the reaction mixture, were mostly spherical and hexagonal with , 50 nm size. The average hydrodynamic particle size was in the range of 20
500 nm with a zeta potential of 217.1 mV. FTIR analysis showed that the AgNPs were associated with phytochemicals as revealed from the strong peaks at 3381 cm21 (primary amines), 2916 cm21 (alkanes), 2848 cm21 (aldehyde), 1607 cm21 (amines), and 1207 cm21 (aliphatic amines). Hence, it can be concluded that secondary metabolites present in plants acted as stabilizing agents in the synthesis of AgNPs. X-ray diffraction showed a face-centered cubic structure indexed by Bragg’s reflection to the (111, 200, 220, 311) plane. EDX analysis confirmed the presence of elemental silver. The antibacterial activity was checked against gram-positive B. subtilis and S. aureus and gram-negative E. coli and K. pneumonia as shown in Fig. 12.4. The maximum zone of inhibition was obtained against S. aureus followed by E. coli, B. subtilis, and K. pneumoniae which were 10 6 1.5, 12 6 2.5, 8 6 1.0, and 14 6 1.7 nm, respectively, at an AgNPs concentration of 20 μg/mL. The antilarvicidal activity was checked against Aedes aegypti and Culex quinquefasciatus. The maximum mortality was observed in the fourth instars of A. aegypti with LC50 values of 45.1 μg/mL, while C. quinquefasciatus showed LC50 value of 41.1 μg/mL. With the increase in the concentration of AgNPs, the rate of mortality also increased. The molecular docking of plumbagin with the mosquito salivary proteins was also carried out, as it was observed in previous studies that plumbagin was responsible for larvicidal activity against Anopheles gambiae. Molecular docking was used to examine the interaction and

FIGURE 12.3 Morphological analysis of ZnONPs. (A) TEM analysis of ZnONPs; (B) SEM analysis of ZnONPs. Reprinted from Melk, M.M., ElHawary, S.S., Melek, F.R., Saleh, D.O., Ali, O.M., El Raey, M.A., & Selim, N.M. (2021). Nano zinc oxide green synthesized from Plumbago auriculata Lam. alcoholic extract. Plants. 10(2447), 1
14. (Open access).

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FIGURE 12.4 Antibacterial activity of green synthesized AgNPs. (A) Bacillus subtilis; (B) Staphylococcus aureus; (C) Escherichia coli; (D) Klebsiella pneumoniae. Reprinted from Govindan, L., Anbazhagan, S., Altemimi, A.B., Lakshminarayanan, K., Kuppan, S., Pratap-Singh, A., & Kandasamy, M. (2020). Efficacy of antimicrobial and larvicidal activities of green synthesized silver nanoparticles using leaf extract of Plumbago auriculata Lam. Plants. 9 (1577), 1
13.(Open access).

binding of plumbagin with D7 salivary protein present in A. aegypti and odorant-binding protein (OBP) on C. quinquefasciatus. Plumbagin showed a docking score of 26.71 (kcal/mol) and a 12.1 μM inhibitory constant against D7 protein in A. aegypti, whereas the docking score of plumbagin with OBP of C. quinquefasciatus was 27.48 (kcal/mol) with 3.31 μM of inhibitory constant. This indicated that plumbagin showed a higher binding affinity toward D7 salivary protein and OBP protein. Hence, it may be used for antimalarial and antifilarial drug development (Govindan et al., 2020). Yet in another study, the antioxidant, antituberculosis, and dye degradation potential of AgNPs synthesized from ethanolic leaf extract of P. auriculata were evaluated. The UV
Vis spectra showed the absorbance maxima of AgNPs at 428 nm. The XRD peaks confirmed the face-centered cubic crystal structure of silver. The spherical-shaped AgNPs were in size ranging from 15 to 45 nm. FTIR analysis showed that phenols, flavonoids, and sugars present in the extract may serve as reducing agents in the formation of AgNPs. The antituberculosis activity was carried out using microplate Alamar blue assay (MABA) against M. tuberculosis. Streptomycin and pyrazinamide were used as standard. The phytogenic AgNPs showed a minimum inhibitory concentration (MIC) of 1.6 μg/mL while the plant extract showed MIC value of 25 μg/mL. Antioxidant activity with 93% and 65% DPPH radical scavenging was observed at 40 μg/mL of plant extract and AgNPs with IC50 values of 15.9 and 28.5 μg/mL, respectively. A high DPPH radical scavenging activity was exhibited by ethanol extract as compared to AgNPs. The degradation of two azo dyes, Congo red and malachite green, by AgNPs was accomplished within 2 hours which was marked by a decrease in the absorbance due to the breaking of the azo bonds as shown in Fig. 12.5. Similarly, AgNPs may transfer hydrogen to the central carbon present in malachite green loss of conjugation in rings which leads to a reduction in absorbance (Jaryal & Kaur, 2017). Another study reported the synthesis of AgNPs from leaf and calyx extract of P. auriculata. The phytogenic AgNPs showed a surface plasmon resonance-associated peak between 420 and 460 nm in the UV
visible spectra. TEM images showed spherical- and oblong-shaped AgNPs with sizes ranging from 15 to 30 nm. It is important to note that the AgNPs synthesized at 24 C were 15.22
26.5 nm in size while those synthesized at 60 C were aggregated with a size ranging from 18.33 to 29.48 nm. FTIR analysis showed the involvement of
C 5 O and
OH functional groups in the reduction of the metal ions to the nanoparticles and their further stabilization. The antibacterial activity of AgNPs against E. coli, S. aureus, K. pneumoniae, and Salmonella typhimurium was confirmed with the MIC equivalent to 1.25, 62.5, and 250 μg/mL against E. coli, S. aureus, and K. pneumoniae, respectively (Singh, Naidoo, & Bajinath, 2018; Singh, Naidoo, Mocktar, et al., 2018).

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FIGURE 12.5 Mechanism of degradation of Congo red and malachite green. Reprinted with permission from Jaryal, N., & Kaur, H. (2017). Plumbago auriculata leaf extract mediated AgNPs and its activities as antioxidant, anti-TB and dye degrading agents. J. Biomater Sci. Polym. Ed. 28 (16), 1847
1858. Copyright r 2017 Informa UK Limited, trading as Taylor & Francis Group.

12.7

Other properties

Cold tolerance in P. auriculata was reported as the differentiation of flower buds halts at 15 C in addition to the yellowing of leaves with decreasing temperature. To understand the mechanism behind the response of plants toward low temperatures, chlorophyll fluorescence parameters in combination with transcriptome sequencing and real-time PCR (RT-qPCR) were conducted. It was speculated that the cold response of P. auriculata can be due to the damage to the photosynthetic system. The cold stress affects the thylakoid membrane, hence reducing photosynthesis. It was observed that P. auriculata regulates several metabolic pathways to combat cold stress. The increase in sugar metabolism, enhancement of ROS clearance mechanism, and reduction in the sites of enzymes for lipid metabolism reducing the biosynthesis of cutin, suberin, and wax were established as the underlying mechanisms for self-protection. Such an alteration of the metabolic pathways aids the plant to survive in winter in cold tropical regions. Critical damage to the photosynthetic system was confirmed as the major reason for the inhibition of blossom in low temperatures (Li et al., 2020). Some studies have also reported the toxic effects of P. auriculata apart from its high medicinal values. As stated earlier, plumbagin is the major compound found in the genus Plumbago, which can exhibit toxicity as it generates the reactive oxygen species (Jose et al., 2014). At a low dose, it acts as an irritant inhibiting mitosis. Plumbagin at higher doses can cause respiratory failure and paralysis. It also shows nucleotoxic and cytotoxic effects. The common side effects of plumbagin include skin rashes, diarrhea, an increase in white blood cells and neutrophil counts, toxicity to hepatic cells, and an increase in the levels of acid phosphatase and serum phosphatase. The thorns and juice sap of the plant result in dermatitis. In the tribal regions, the roots of P. auriculata are used to heal the wound at a very low dose for a limited period as more exposure and high concentration can be lethal due to severe irritation (Jain et al., 2010).

12.8

Future perspectives

Medicinal plants with a plethora of the phytochemicals can serve as potential complementary and alternative medicine. The rich phytochemistry of P. auriculata imparts therapeutic properties due to their bioactive nature. However, there is

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a huge scope to explore the effect of these phytochemicals on bacterial biofilm which is generally associated with critical nosocomial infections. The isolated bioactive principles can also be checked for the plasmid curing activity as the transfer of multidrug resistance among bacteria is plasmid-mediated. There is a lacuna in the area for evaluation of the nano-biotechnological potential of P. auriculata. Hence, more researches are needed in this area for the synthesis of gold, silver, platinum, palladium, copper, iron, and other nanoparticles with biomedical, environmental, and agricultural applications (Ghosh et al., 2015; Ghosh, Chacko, et al., 2016; Ghosh, Gurav, et al., 2016). Hazardous dyes are potential threats to the environment and hence should be removed effectively from the industrial effluents before they are released into the environment. Hence, nanoparticles synthesized from these medicinal plants can be checked for the photocatalytic dye degradation (Ghosh, 2018; Shende et al., 2017, 2018). Similarly, these nanoparticles can be explored for the targeted drug delivery against cancer, diabetes, and infections (Bhagwat et al., 2018; Ghosh, 2019). Nanotechnology has also recently been used for tissue regeneration and fabrication of 3D-printed scaffolds. However, the chemically and physically synthesized nanomaterials are often associated with hazardous chemicals that are used during their synthesis and stabilization (Ghosh et al., 2018; Ghosh & Webster, 2021b, 2021c; Ghosh, Bhagwat, et al., 2022). Further, P. auriculata can be used for developing biochar for enhanced removal of heavy metals, organic wastes, and toxic dyes (Luikham et al., 2018). The endophytic diversity of the plant can also be explored which will provide an opportunity to exploit the untapped microbial endophytes for screening and synthesis of bioactive compounds. Thus, this remarkable medicinal plant provides numerous applications and scope for future research.

12.9

Conclusion

P. auriculata is a traditional medicinal plant that is used in folk medicine globally. The plant possesses a diverse group of phytochemicals such as phenols, alkaloids, tannins, proteins, and carbohydrates. The bioactive compounds act as potent anticancer, antimicrobial, antioxidant, and antidiabetic agents. The lack of exploration of the phytochemical diversity of this plant for therapeutic, agricultural, and environmental applications provides an opportunity for research in the formulation of novel drugs, phytoremediation, and water treatment processes. The promising medicinal application expands the new horizons to overcome multidrug-resistant (MDR) organism-associated infections. This chapter also provides the insights for the phytogenic synthesis of nanoparticles from P. auriculata to combat MDRs and increase the scope for finding new nanomedicine and drug development.

Acknowledgments Ms. Khalida Bloch is thankful to the Student Startup & Innovation Policy (SSIP) of the Government of Gujarat and RK University for funding (U-0647/SSIP/RKU/SOS/2021-22/13). Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok, Thailand, for Post-doctoral Fellowship and funding under Reinventing University Program (Ref. No. 6501.0207/10870 dated November 9, 2021 and Ref. No. 6501.0207/9219 dated September 14, 2022).

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17. Ramalhete, C., Lopez, D., Mulhovo, S., Rosario, V. E., & Ferreira, M. J. U. (2008). Antimalarial activity of some plants traditionally used in Mozambique. Workshop Plantas Medicinais e Fitoterapeuticasnos Tropicos, 31, 1
9. Ranpariya, B., Salunke, G., Karmakar, S., Babiya, K., Sutar, S., Kadoo, N., Kumbhakar, P., & Ghosh, S. (2021). Antimicrobial synergy of silverplatinum nanohybrids with antibiotics. Frontiers in Microbiology, 11610968. Ribeiro de paiva, S., Figueiredo, M. R., & Kaplan, M. A. C. (2005). Isolation of secondary metabolites from roots of Plumbago auriculata Lam. by countercurrent chromatography. Phytochemical Analysis: PCA, 16(4), 278
281. Rios-Chavez, P., Perez-Gonzalez, J., Salgado-Garciglia, R., Ramirez-Chavez, E., Molina-Torres, J., Martinez-Trujillo, M., & Carreon-Abud, Y. (2019). Antibacterial and cytotoxicity activities and phytochemical analysis of three ornamental plants grown in Mexico. Anais da Academia Brasileira de Ciencias, 91(2), e20180468. Saji, A., & Antony, T. (2015). Plumbago auriculata Lam. International Journal of Pharmaceutical Sciences Review and Research, 3(2), 281
284. Sharmila, M., Rajeswari, M., Indhiramuthu, J., & Geetha, D. H. (2016). GC-MS analysis of bioactive compounds of Amaranthus polygonoides Linn. (Amaranthaceae). International Journal of Advances in Applied Sciences, 1(1), 174
180. Shende, S., Joshi, K. A., Kulkarni, A. S., Charolkar, C., Shinde, V. S., Parihar, V. S., Kitture, R., Banerjee, K., Kamble, N., Bellare, J., & Ghosh, S. (2018). Platanus orientalis leaf mediated rapid synthesis of catalytic gold and silver nanoparticles. Journal of Nanomedicine & Nanotechnology, 9(2)1000494. Shende, S., Joshi, K. A., Kulkarni, A. S., Shinde, V. S., Parihar, V. S., Kitture, R., Banerjee, K., Kamble, N., Bellare, J., & Ghosh, S. (2017). Litchi chinensis peel: A novel source for synthesis of gold and silver nanocatalysts. Global Journal of Nanomedicine, 3(1)555603. Singh, K., Naidoo, Y., & Bajinath, H. (2018). A comprehensive review on the genus Plumbago with focus on Plumbago auriculata (Plumbaginaceae). African Journal of Traditional, Complementary and Alternative Medicines, 15(1), 199
215. Singh, K., Naidoo, Y., Mocktar, C., & Bajinath, H. (2018). Biosynthesis of silver nanoparticles using Plumbago auriculata leaf and calyx extracts and evaluation of their antimicrobial activities. Advances in Natural Sciences: Nanoscience and Nanotechnology, 9(3)035004. Sumsakul, W., Plengsuriyakarn, T., Chaijaroenkul, W., Viyanant, V., Karbwang, J., & Na-Bangchang, K. (2014). Antimalarial activity of plumbagin in vitro and in animal models. BMC Complementary and Alternative Medicine, 14, 15. Sunil, C., Duraipandiyan, V., Agastian, P., & Ignacimuthu, S. (2012). Antidiabetic effect of plumbagin isolated from Plumbago zeylanica L. root and its effect on GLUT4 translocation in streptozotocin-induced diabetic rats. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 50(12), 4356
4363. Taneja, N., Alam, A., Patnaik, R., & Taneja, T. (2021). Unmasking the potential role of plant based medicine “plumbagin” in oral cancer-A novel paradigm. Oral Science International, 19(1), 3
18. Tharmaraj, R. J. J. M., & Antonysamy, J. M. (2015). Screening of bacterial activity of selected Plumbago species against bacterial pathogens. Journal of Microbiology & Experimentation, 2(6), 00070. Tyagi, R., & Menghani, E. (2014). A review on Plumbago zeylanica: A compelling herb. International Journal of Pharmaceutical Sciences and Research, 5(4), 119
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Chapter 13

Alkaloids as potential anticancer agent Mayuri A. Patil1, Aniket P. Sarkate1, Nilesh Prakash Nirmal2 and Bhagwan K. Sakhale1 1

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India, 2Institute of Nutrition,

Mahidol University, Salaya, Nakhon Pathom, Thailand

13.1

Introduction

Cancer has recently achieved a high level in terms of morbidity and mortality, and it has become a serious public health issue around the world. Numerous studies are focused on finding new chemicals for the treatment of cancer, with many focusing on plant-derived compounds that have therapeutic capability and are widely utilized in traditional treatments (Shi et al., 2006). Cancer was originally linked to defective genes around 1980, and since then, cancer research has grown into a prominent field of study, bolstering the foundations of modern biology to a large extent. The effort of deciphering the human genome sequence was one of those out-of-the-ordinary activities that have been fueled in large part by cancer research, and many of the exciting insights into the genetic circuits that drive developmental processes have also originated from cancer research (Nahata, 2017). Plant-derived chemical substances have been employed to cure human ailments since the dawn of civilization. Natural products have gotten a lot of press in recent years because of their promise as innovative cancer-preventative and therapeutic agents (Newman, 2008). There is growing evidence that plant-derived chemicals can suppress several stages of tumor cell growth and associated inflammatory processes, implying that these products are important in cancer prevention and treatment (Haque et al., 2016). With the invention and progress in the development of the vinca alkaloids, vinblastine and vincristine, as well as the isolation of cytotoxic podophyllotoxins, the quest for anticancer medicines from plant sources began in earnest in the 1950s. The accomplishments were made feasible by the screening and identification of several anticancer alkaloids from tree species, modern technology, and advancements in instrumentation techniques. Taxol and camptothecin, two lead alkaloids extracted from trees that have anticancer characteristics and have been developed as medications or are in the early phases of research, were discovered in the 1960s (Chabner & Roberts, 2005). In the mechanism of action on tumor cells, alkaloids and their congeners target DNA replication or protein synthesis, resulting in neoplastic cell death (Faddeeva & Beliaeva, 1997).

13.2

Theoretical relevance

Although numerous chemotherapeutic treatments are utilized to treat cancer, cancer recurrence and drug resistance are the root causes of high mortality. Phytochemicals, or natural chemicals derived from plants, have been shown to have anticancer properties and to be beneficial in treating a variety of disorders (Chandrasekar et al., 2018). Alkaloids, which are usually colorless and odorless crystalline solids, are one of these phytochemicals. They can, however, look like yellowish liquids (Ruano et al., 2016). Various research on animal and human models demonstrated the potential value of alkaloids and their powerful biological effects at low doses. Alkaloids were well known and used in traditional medicine by people of all ages for their various activities. They lacked direct methods for isolating pure chemicals though (Santhi et al., 2022).

13.2.1 Types of alkaloids Alkaloids are organic nitrogen-containing bases that exist naturally and have a wide range of physiological effects on humans and other animals (Bhattacharya & Naitam, 2019). Alkaloids can be classified into three kinds based on their biosynthesis pathway, molecular structure, and molecular precursor: Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00034-7 © 2023 Elsevier Inc. All rights reserved.

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1. Heterocyclic alkaloids (true alkaloids), 2. Non-heterocyclic alkaloids (protoalkaloids), 3. Pseudo-alkaloids. True alkaloids and protoalkaloids are made from amino acids, whereas pseudo-alkaloids are not. 1. Heterocyclic alkaloids (true alkaloids): They are cyclic amino acid derivatives that are chemically complicated and physiologically active. These extremely reactive heterocyclic alkaloids obtained from nature can produce salts that are water-soluble in nature with organic acids like tartaric, acetic, oxalic, lactic, malic, and citric acids due to the presence of intra-cyclic nitrogen atoms and significant biological activity. Nicotine, cocaine, quinine, dopamine, morphine, geissospermine, piperine, berberine, and gasoline are some of the alkaloids discovered in these groups, with cocaine, morphine, and quinine being the most common true alkaloids found in nature (Dey et al., 2020). 2. Non-heterocyclic alkaloids (protoalkaloids): Protoalkaloids are produced from amino acids or biogenic amines and have a nitrogen atom outside the ring that remains as a side chain rather than being part of the heterocyclic system. Examples include mescaline, colchicine, cathinone, and other protoalkaloids, which are rare in nature. Mescaline, often known as peyote, is a phenylethylamine alkaloid obtained from the plant Lophophora williamsii (Birajdar et al., 2013). 3. Pseudo-alkaloids: Pseudo-alkaloids are substances with carbon skeletons that are not made up of amino acids. Amination or transamination of amino acid precursors or post-cursors produces pseudo-alkaloids in reality. Acetate and phenylalanine-derived pseudo-alkaloids, as well as steroidal alkaloids, might be found. Coniine, capsaicin, ephedrine, solanidine, caffeine, theobromine, and pinidine are examples of pseudo-alkaloids (Dey et al., 2020).

13.2.2 Targeted pathways in cancer treatment Cancer is a multifactoral heterogeneous metabolic disorder that disrupts cellar homeostasis irreversibly. The following six key hallmarks of tumor growth are caused by this impairment: (1) uncontrolled cell division and differentiation, (2) activated proliferative signaling, (3) activated angiogenesis, (4) replicative immortality, (5) metastatic development, and (6) cell death resistant (Robinson, 1974). It is a pemetrexed-induced apoptosis regulator that is modulated by vinca alkaloids. Another frequent and influential class of anticancer drugs is tubulin-binding compounds, which disrupt microtubule assembly and trigger mitotic arrest (Chikati et al., 2018). Alkaloids have been associated with cancer management and prevention, including the prevention and regulation of oxidative stress and inflammation, or both. Vincristine, vinblastine, vinorelbine, and vindesine are vinca alkaloids that have exhibited anticancer action in MCF-7 and MDA-MB-231 breast cancer cells. The development of resistance is a common occurrence during treatment. Some chemotherapeutic-resistant cell lines, such as MCF-7, a docetaxel-resistant cell line that was addressed with the alkaloid colchicine in a study, have shown sensitivity to alkaloids with the advancement of cancer therapy development, and a tendency toward novel “targeted” therapies has emerged to reduce the severity and toxicity of “cytotoxic chemotherapies” such as vinca alkaloids. Phenanthrene alkaloid, berberine, is found in the roots and bark of Berberis, Hydrastis canadensis, and Coptis chinensis, among other plants. In one experiment, anoikis-resistant MCF-7 and MDA-MB-231 cells were found to efficiently limit growth by producing cell cycle arrest (Gahlot et al., 2013). In vivo experiments with vinblastine and in vitro research with vincristine, vinca alkaloids, successfully suppressed the development of pemetrexed-resistant tumors, according to another study. Vinblastine, vincristine, vindesine, and vinorelbine are well-known antimitotic drugs, and structural differences between these molecules have been shown to alter anticancer activity and toxicity. High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that plays a role in inflammation, cell migration, death of cells, and tumor metastasis. It binds to receptors for advanced glycation end products, which are involved in inflammation, tumor cell proliferation, migration, and penetration (Kaur, 2013). Cancer (2019) studied temozolomide-sensitive U87MG and TMZ-resistant T98G and TMZ-resistant T98G from human glioblastoma, and the anticancer potential of the alkaloid papaverine was also studied (GGB). Papaverine reduced the high mobility group box 1 protein in temozolomide-sensitive U87MG and TMZ-resistant T98G human glioblastoma cells, limiting tumor growth promotion (Lakhanpal et al., 2014). During tumor malignancy, the antioxidative transcription nuclear factor Nrf2 is observed to increase in cancer cells such as colonic, thyroid, endometrial, lung, breast, and pancreatic cancer cells. Modifications in the gene may come about as a result of this. The alkaloid trigonelline decreased nuclear levels of the Nrf2 protein but not total expression. Surprisingly, no side effects have been recorded in human tests with trigonelline (Santhi et al., 2022).

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Biological source, mechanism of action, and applications of indole alkaloids

Vinca alkaloids (VAs) were the very first antimitotic drugs from the plant kingdom to enter the pharmacological market, and they were quickly recognized as efficient and useful treatments that were desperately required in the healthcare system. VAs are frequently utilized as anticancer medications, either alone or in combination with other treatments, to treat breast cancer, osteosarcoma, and acute lymphocytic leukemia. They were first used to treat juvenile hematologic malignancies and subsequently expanded to include solid and adult hematologic malignancies (Johnson et al., 1960; Moudi et al., 2013; Silvestri, 2013).

13.3.1 Vinblastine (Fig. 13.1) FIGURE 13.1 Structure of vinblastin.

Vinca alkaloid, obtained from Catharanthus roseus, is among the most studied classes of anticancer drugs. Solid tumors have long been treated using dimeric alkaloids from C. roseus in combination with chemotherapy. Vinca alkaloids, including vinblastine, vincristine, vindesine, and vinorelbine, are now used in therapeutic practice (Noble et al., 1958). Vinblastine (VLB) is an active chemical found in nature. The salt of an alkaloid derived from Vinca rosea Linn., a popular flowering herb known as the periwinkle, is vinblastine sulfate (more appropriately known as C. roseus G. Don). Vinca leukoblastine, or VLB, was the previous generic name (Haque et al., 2016). Particularly, vindesine and vinorelbine, semisynthetic equivalents of vinblastine, are in therapeutic use among the many derivatives produced. More recently, vinflunine, a novel Vinca alkaloid, has been produced (Fahy et al., 2008). It is an oncolytic agent with a stathmo kinetic effect. Developing cells are halted in metaphase when treated in vitro with this mixture. Vinblastine is not to be administered intramuscularly, subcutaneously, or intrathecally. Drugs that disrupt microtubules, such as vinblastine, colcemid, and nocodazole, have been reported to work in two ways. They decrease microtubule dynamics at low doses and lower microtubule polymer mass at higher values (Haque et al., 2016). Vinblastine, an autophagy maturation inhibitor, when combined with nanoliposomal C6-ceramide (an autophagy inducer), increased cell apoptosis in HepG2 (human hepatocarcinoma) and LS174T (human colon) cell lines by increasing autophagic vacuole accumulation and reducing autophagy maturation. The deletion of Beclin-1, a protein related to autophagy, was inhibited by this combined therapy. Nausea and vomiting that can last just under 24 hours, stomach ache, constipation, diarrhea, jaw pain, headache, or other soreness, thinned or brittle hair, and easily burnt exposed regions of the skin are all side effects of vinblastine. Vinblastine is an anticancer drug used to treat cancers including Hodgkin’s lymphoma, non-lymphoma, Hodgkin’s breast cancer, testicular cancer, mycosis fungoides, Kaposi’s sarcoma, and Letterer
Siwe disease. Non-small cell lung cancer, bladder cancer, head and neck cancer, cervical cancer, idiopathic thrombocytopenia purpura, and autoimmune hemolytic anemia are all treated with vinblastine. In patients with leucopenia, vinblastine sulfate is not recommended. It should not be taken if a bacterial infection is present. Infections like these should be dealt with as soon as possible (Haque et al., 2016).

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13.3.2 Vincristine (Fig. 13.2) FIGURE 13.2 Structure of vincristine.

The vindoline component and the catharanthine-type section of vincristine have a nonsymmetrical dimeric structure, with two indole-type nuclei linked by a carbon
carbon bond. The Food and Drug Administration (FDA) authorized its clinical usage for cancer treatment in 1963. It was one of the first plant-based anticancer medicines to be approved by the FDA. It is a naturally-occurring alkaloid isolated from the leaves of C. roseus (L.) G. Don (previously V. rosea L.) that has been used in adult and pediatric oncology to treat acute lymphoblastic leukemia. Vincristine and related vinca alkaloids are mitotic poisons Skubnı´k et al. (2020), notably tubulin-binding chemicals, whose biological effects are derived from interrupting microtubule activity. Tubulin heterodimers form microtubules, which are polymeric fibers. The subunits of the protein tubulin form dimers, and the binding site for vincristine is found on the subunit at the heterodimer’s boundary. As a result, vincristine and other vinca alkaloids are the only tubulinbinding drugs found thus far which do not absolutely bind one tubulin heterodimer (Field et al., 2014). This critical characteristic is essential to the vinca alkaloids’ unique mechanism of action. The chemicals have the ability to break microtubule fibers at large dosages, in particular. The fibers then unite with one another, always bound by the vinca alkaloid. In the mitotic spindle, which is responsible for the separation of chromatids during mitosis, such unevenly organized, often spiraling fibers are unable to fulfill their purpose (Himes, 1991). Low concentrations of vinca alkaloids, which bind the terminals of microtubule fibers and maintain microtubule dynamics, likewise impede this function (Jordan et al., 1991). Nevertheless, neurotoxicity is the most serious adverse effect of vincristine therapy, as seen by vincristine-induced peripheral neuropathy (VIPN). This consequence has a significant influence on patients’ well-being; it is a burdensome and unpleasant condition characterized by numbness, tingling, or severe sensations in the hands and feet, as well as muscle weakness. Unfortunately, because the molecular basis for this impact of vincristine was unknown for a long time, it was impossible to estimate the likelihood of a VIPN breakout in a specific patient. Some of the markers have, however, been identified in recent years, albeit the significance of the published data must still be confirmed in practice (Verma et al., 2020). Sphingomyelin/cholesterol (SM/Chol) liposomal vincristine (Marqibo) was licensed by the FDA in 2012 to treat people with relapsed acute lymphoblastic leukemia (New Drug Application: 202497). Vincristine can be loaded into conventional liposomes such as SM/Chol liposomes, but some other types of liposomes, such as PEGylated liposomes, have already been tested, despite the fact that SM/Chol liposomal vincristine has a longer circulation time, lower liposome leakage, and a better antitumor effect than PEGylated liposomal vincristine. Marqibo is now being tested in pediatric patients with recurrent or chemotherapy-resistant solid tumors and leukemia (ClinicalTrials.gov Identifier: NCT01222780). Other vincristine encapsulated formulations are also being tested in clinical trials for cancers like small-cell lung cancer (ClinicalTrials.gov Identifier: NCT02566993), advanced cervical cancer (ClinicalTrials.gov Identifier: NCT02471027), and liver cancer (ClinicalTrials.gov Identifier: NCT00980460) (Ana, 2018).

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13.3.3 Vindesine (Fig. 13.3) FIGURE 13.3 Structure of vindesine.

C. roseus is the most well-known medicinal plant, and it has been used as medicine since antiquity. Out of 345 bioactive phytochemicals, the species contains more than 150 indole alkaloids including vindesine, making it a wellknown herbal medicine with anticancer properties (Ferreres et al., 2008; Kumar et al., 2021). Vindesine, a vinca alkaloid derived from vinblastine, is used to treat a number of malignancies, the most frequent of which is acute lymphocytic leukemia. It operates by blocking tubulin mitotic function, which prevents cells from beginning metaphase mitosis. Vindesine inhibits the entry of cells into metaphase mitosis. At dosages aimed to prevent 10%
15% of cells from initiating mitosis, it is 3 times more effective than vincristine and nearly 10 times more effective than vinblastine in vitro tests. Vincristine and vindesine are approximately similar at doses that stop 40%
50% of cells in mitosis. Vinblastine creates a large number of post-metaphase cells, whereas vindesine produces a modest amount. Vindesine is a second-generation semisynthetic vinca alkaloid with broad-spectrum anticancer efficacy and little neurotoxicity in vitro. In preclinical studies, vindesine inhibits Ridgeway osteogenic sarcoma, Gardner lymphosarcoma, P154 leukemia, and P388 leukemia in mice, as well as murine B16 melanoma and S180 ascites tumor. When delivered peritoneally to L1210 leukemia cells, vindesine plus methotrexate resulted in a 200% increase in the life span of mice. In a randomized phase III trial, vindesine in combination with epirubicin had no effect on the drug’s efficacy (Joel, 1995; Nielsen et al., 1990). In another trial, patients with advanced breast cancer were randomized to receive vindesine (3 mg/m2) with adriamycin, which resulted in a 63% overall response and a 43-week survival rate. In 63 patients having incurable non-small cell lung cancer, single-agent vindesine (3 mg/m2) resulted in a 14% response rate, compared to 0% for vincristine (Smith et al., 1980). Vindesine is also used to cure nonsmall cell lung cancer and vincristine-resistant juvenile chronic lymphocytic leukemia. Patients who reverted after having vincristine as part of a multi-agent treatment program have shown promise with vindesine. Vindesine is used to treat leukemia, non-small cell lung cancer, and lymphoma as part of combination therapy (Dhyani et al., 2022).

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13.3.4 Vinflunine (Fig. 13.4) FIGURE 13.4 Structure of vinflunine.

Vinflunine, a new generation of vinca alkaloids, was synthesized using a semisynthetic technique including superacidic chemistry. In mouse tumors and human tumor xenografts, the fifth novel vinca alkaloid outperforms the others (Hill et al., 1999; Kruczynski et al., 1998). Vinflunine is now being explored in clinical settings after preclinical discoveries of favorable antitumor actions such as microtubule dynamics disruption, antiangiogenesis, and extended multidrug resistance development (Barret et al., 2000; Pourroy et al., 2006; Kruczynski et al., 2006). Vinflunine’s main effects include a decrease in microtubule growth rate, a rise in growth duration, and a drop in shortening duration. They did not diminish the rate of shortening or increase the proportion of time the microtubules spent in an attenuated state, in contrast to vinblastine, and neither growth nor shortening was detected. Treadmilling was also inhibited less significantly by vinflunine and vinorelbine than by vinblastine. The various effects of these medications on microtubule dynamics are anticipated to have different effects on mitotic spindle function, resulting in different impacts on cell cycle progression and cell death (Alain et al., 2002). Vinflunine prevents cells from entering mitosis and causes them to die via inducing apoptosis. Vinorelbine, vinblastine, and vincristine have comparable interactions with tubulin’s vinca alkaloid-binding domain. Vinflunine attaches to the domain more slowly compared to other vinca alkaloids, a property that indicated decreased neurotoxicity, which was validated in clinical tests (Kruczynski et al., 1998). Normal cells have “normal” checkpoint mechanisms, which make them more resistant to vinflunine and vinorelbine because they suppress microtubule dynamics fewer effectively, but cancer cells, which usually have deficient checkpoint mechanisms, are much more vulnerable to vinflunine and vinorelbine. Owing to its unique constellation of effects on microtubule dynamics and tubulin treadmilling, this explains their better antitumor activities in in vitro investigations. In general, vinflunine’s diversified action on microtubules is anticipated to have different effects on the mitotic spindle’s functions and differing effects on cell cycle advancement and cell death (Hill, 2001). Vinflunine was found to be successful in the management of human epidermal growth factor receptor-2 (EGFR-2)positive metastatic breast cancer in phase II clinical trial (Yardley et al., 2010). Vinflunine had an excellent prognosis in phase III trial, but it did not outperform other regularly used alkylating drugs in terms of overall survival (Cortes et al., 2018).

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13.3.5 Camptothecin (Fig. 13.5)

FIGURE 13.5 Structure of campothecin.

Camptothecin was first extracted from Camptotheca acuminata Decne (Nyssaceae), a Chinese esthetic tree known as the “tree of joy” in China. Out of 1000 different plant extracts examined for antitumor activity, the extract of C. acuminata was the only one that exhibited efficiency, and the active ingredient extracted was identified as camptothecin (Rahier et al., 2005). DNA is generally found in the form of a supercoiled double helix. It unwinds during replication, with single strands acting as templates for the creation of new strands. Transient cleavage of one or both strands of DNA is required to relieve the torsional tension that accumulates ahead of the replication fork. This is made easier by topoisomerases (Topo). Breaks are caused by Topo-II, whereas single-strand breaks are caused by Topo-I. This movement permits the broken strand to rotate around the undamaged strand. The broken strand is then relegated by Topo-I to restore the integrity of the double-stranded DNA. Single-strand breaks are induced by camptothecin in a reversible manner, reducing the cell’s ability to replicate. It keeps the cleavable combination between Topo-I and the DNA stable. These nonlethal stabilized breaks are totally recoverable (Schneider et al., 1990). Single-strand breaks are transformed into irreversible double-strand breaks when a DNA replication fork collides with the cleavable complex (Hsiang & Liu, 1988). Caspase activation is thus responsible for apoptosis cell death. When caspase activity is inhibited, cells transit from apoptosis to a temporary G1 arrest, which is followed by cell necrosis (Sane & Bertran, 1999). In 1966, Wall and Wani extracted 20-(S)-camptothecin (CPT) from the bark of C. acuminata, but they rapidly discovered that CPT has a number of drawbacks, including low stability and solubility (Wall et al., 1966). A year after CPT was developed, Wall and Wani discovered paclitaxel, an anticancer medication with a similar promise (Wani et al., 1971). Despite the fact that both medicines had potent anticancer properties (Slichenmyer & Von Hoff, 1990), CPT’s poor solubility and unexpected adverse medication interactions favored the advancement of paclitaxel as a broad-spectrum chemotherapy (Wall, 1998). The CPTs, on the other hand, drew a lot of attention in the late 1980s when the molecular target was discovered: DNA topoisomerase I (TOP I), which is thought to be the solitary point of biological activity (Pommier et al., 1987; Thomsen et al., 1987; Jaxel et al., 1988; Kjeldsen et al., 1988). Crystal structures of CPT and a number of certain other compounds eventually validated the binding pocket (Redinbo et al., 1998; Staker et al., 2002). TOP I is a necessary enzyme that relieves supercoiled DNA by forming single-strand breaks and religation prior to transcription. CPT hinders religation and causes apoptosis when it binds to TOP I (Pommier, 2006). Various compounds have been created to alleviate the solubility and stability concerns associated with camptothecin. Only two CPT derivatives, irinotecan and topotecan, are licensed for clinical usage, despite the fact that a variety of larger and smaller molecule medicines are now in clinical development. Irinotecan is a drug that is now being used to treat metastatic colorectal cancer. Topotecan is a treatment for ovarian cancer, cervical cancer, and small cell lung cancer that has been FDA-approved. Tertiary amine cations are used in these derivatives to increase solubility and, as a result, lactone stability (Sharma et al., 2010).

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13.3.6 Montamine (Fig. 13.6) FIGURE 13.6 Structure of montamine.

Centaurea montana (family: Asteraceae alt. Compositae) is a tall plant with enormous, reddish, blue center flower heads that is native to Australia, Belgium, and Italy, as well as being cultivated in many other countries, while a number of flavonoids, acetylenes, and a lignan, arctigenin, had previously been described from the aerial parts of C. montana. A dimeric indole alkaloid, namely, montamine, was screened and quantified from the seeds (family: Asteraceae). These chemicals were found to suppress the growth of the CaCo-2 colon cancer cell line (Shoeb et al., 2006). MTT was used to test the in vitro cytotoxicities of most of the chemicals identified and defined against colon cancer cell lines, CaCo2. Montamine, a dimeric indole alkaloid with an IC50 of 43.9 μm, showed substantial anticancer activity in vitro. The production of montamine from the dimerization of moschamine enhanced the cytotoxicity by twice. The distinctive structural properties will almost probably be used as a template for developing anticancer drugs (Shoeb et al., 2006).

13.4 Biological source, mechanism of action, and applications of isoquinoline alkaloids One of the most diverse categories of natural chemicals is alkaloids with an isoquinoline component. Isoquinoline is a heterocyclic molecule made up of a benzene ring bonded to a pyridine ring at C3/C4 (Satyajit & Lutfun, 2007). Isoquinoline alkaloids are divided into subgroups depending on structural variability: benzylisoquinoline, aporphine, protoberberine, benzo[c]phenanthridine, protopine, phthalide isoquinoline, morphine, emetine, and pavine (Hostalkova et al., 2019). The protoberberine class includes isoquinoline alkaloids such as berberine, palmatine, coralyne, and coptisine, whereas the benzo [c] phenanthridine class includes sanguinarine, chelerythrine, and chelidonine. The benzylisoquinoline alkaloid class includes noscapine and scoulerine. The phytoceutical action of the most common isoquinoline alkaloids has been extensively studied (Yun et al., 2021). Isoquinoline alkaloids have anticancer properties that are remarkable. Novel chemotherapy regimens involving isoquinoline alkaloids and/or isoquinoline-rich plants have been researched. In numerous cancer cell lines, they effectively trigger apoptosis (Choi et al., 2007; Havelek et al., 2016). Isoquinoline alkaloids show significant anticancer effects through cell cycle arrest, apoptosis, and autophagy, leading to cell death, according to research based on in vivo and in vitro models (Al-ghazzawi, 2019).

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13.4.1 Berberine (Fig. 13.7)

FIGURE 13.7 Structure of berberine.

Rhizoma coptidis was used to extract berberine. By suppressing the enzyme cyclooxygenase-2 (COX2), which is highly generated in colon cancer cells and plays a critical role in colon carcinogenesis, it may have potential chemopreventive properties against colon tumor growth. Cancer develops as a result of changes in the cell cycle (Jahagirdar et al., 2018; Matera & Saif, 2017). BBR was found to control cell cycle and reduce cell growth in a variety of malignancies in studies (Wang, Song, et al., 2016; Wang, Wang, et al., 2016; Xiao et al., 2018). BBR inhibited the expression of cyclin D1 and cyclin E1 in A549 lung cancer cells, causing them to enter the G1 phase of the cell cycle (Xiao et al., 2018). In colorectal cancer cells, a mixture of an Hsp90 inhibitor and BBR decreased cell development via inhibiting CDK4 expression and modulating cyclin D1 (Su et al., 2015). BBR decreased cyclin D1 expression in HepG2 human hepatoma cells in vitro and in vivo. Furthermore, in several cancer cells, BBR stopped the cell cycle at G1 by reducing cyclin B1 levels and inhibiting CDC2 kinase indirectly (Li et al., 2000). BBR enhanced the production of p53 and p21 in HBT-94 chondrosarcoma cells by regulating the activation of the PI3K/Akt and p38 signaling pathways, resulting in G2/M phase arrest (Eo et al., 2014). BBR halted MDA-MB-231 breast cancer cells in the S phase, which led to the cancer cells’ high chemosensitivity (Gao et al., 2019). BBR has also been demonstrated to have an effect on the cell cycle by regulating Rb. BBR operated on the 30 UTR of Rb, inhibiting Rb mRNA breakdown, stabilizing Rb translation, and stopping cell cycle progression (Chai et al., 2014). BBR also hindered Rb protein phosphorylation, which inhibited the transcriptional activator E2F from dissociating from Rb and slowed the transition from G1 to S phase (Wu et al., 2015). BBR suppresses cancer cell growth by interfering with the cell cycle. BBR has been shown in current research to lower cholesterol levels and glycemic index while also having antitumor properties (Gu et al., 2011; Yao et al., 2015). BBR reduced cholesterol levels by inhibiting HMG-CoA reductase and improving the stability of LDLR mRNA by associating with the 3-UTR of the LDL receptor (LDLR) (Kong et al., 2004). In vivo studies revealed that BBR reduced nonalcoholic fatty liver disease by stimulating SIRT3 (Xu et al., 2019). BBR increased ROS generation in foam cells, which enhanced cholesterol permeability, and inhibited mTOR and Akt activation, which caused autophagy (Kou et al., 2017). The mechanisms of BBR’s hypoglycemic effects have also been thoroughly researched. BBR increased insulin action by inhibiting mitochondria and activating AMPK, according to studies (Lee et al., 2006; Turner et al., 2008). BBR improved sensitivity to insulin in liver and muscle cells by increasing InsR expression (Chen et al., 2010; Zhao et al., 2012). BBR has been demonstrated to have antitumor properties in investigations on lung cancer, cervical cancer, liver cancer, leukemia, and other cancers (Li et al., 2008; Qi et al., 2014).

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13.4.2 Noscapine (Fig. 13.8)

FIGURE 13.8 Structure of noscapine.

Noscapine, originally named as narcotine (renamed noscapine by the A.M.A. Council on Drugs in 1958), is a phthalide isoquinoline alkaloid found in opium. It is the second most abundant alkaloid after morphine. One of the first alkaloids identified from Papaver somniferum was noscapine. Despite the fact that it is located in a plant with a high alkaloid content, it chemically and pharmacologically has no similarities to the narcotic alkaloids found in P. somniferum (Segal et al., 1957). Earlier toxicity testing revealed that it was the least hazardous of the alkaloid opiates (Krueger et al., 1941; Winter & Flataker, 1961). The phenanthrene nucleus underpins the structures of codeine, morphine, thebaine, and other opiate chemicals, while it is a tetrahydroisoquinoline derivative. Because noscapine has two stereogenic centers, it can be divided into four stereoisomers. Noscapine promotes apoptosis in several cell lines and stops dividing cells from entering metaphase (Ye et al., 1998). Microtubule-binding medicines like paclitaxel, docetaxel, and the vinca alkaloids are now used in cancer treatment trials. Consequently, the high toxicity and poor water solubility of these medicines have restricted their use in cancer chemotherapy. Furthermore, their usage has been impeded by the emergence of drug tolerance caused by a variety of mechanisms, including P-glycoprotein overexpression (Gottesman & Pastan, 1993), tubulin isotype expression changes (Kavallaris et al., 1999), as well as the occurrence of tubulin mutations (Giannakakou et al., 1997). As a result, microtubule-based drugs like noscapine (Verma et al., 2006) are in high request for development and/or research. In both paclitaxel-sensitive and paclitaxel-resistant human ovarian cancer cells, noscapine has been shown to efficiently suppress proliferation and promote apoptosis. This is based on the fact that noscapine binds to tubulin at a different position than paclitaxel, as evidenced by the fact that noscapine has no effect on paclitaxel affinity to tubulin. The JNK pathway, which is activated by the c-Jun NH2 terminal kinase (JNK), has been shown to have a role in noscapineinduced apoptosis (Zhihu et al., 2002). While noscapine is an old antitussive medicine, it has been proven to have novel clinical applications, such as anticancer. This medicine and its analogs have a lot of potential as anticancer agents because they are water-soluble and may be taken orally (Mahmoudian & Rahimi-Moghaddam, 2009). Even though many chemotherapeutic drugs are resistant to colorectal cancer, noscapine promotes apoptosis in a p53-dependent way that requires p21 activation. As a result, in colon cancer cells that express both p53 and p21, noscapine can trigger apoptosis (Aneja et al., 2007). It is also used in human non-small cell lung cancer, glioblastoma, ovarian carcinoma, melanoma, breast cancer, and lymphoma (Mahmoudian & Rahimi-Moghaddam, 2009).

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13.4.3 Liriodenine (Fig. 13.9)

FIGURE 13.9 Structure of liriodenine.

On human lung cancer cells and various types of human cancer cells, the liriodenine isoquinoline alkaloid extracted from Cananga odorata (Annonaceae) has effective cytotoxic, antiproliferative, and apoptosis-inducing effects (Chang et al., 2004). Both in vivo and in vitro, it was discovered to be a strong inhibitor of topoisomerase II (EC 5.99.1.3) (Wooa et al., 1997). Liriodenine suppressed the growth of CAOV-3 human ovarian cancer cells by inducing apoptosis by the mitochondrial signaling system and stopping cell cycle development in the S phase. Nordin et al. (2015) found that it produced overexpression of Bax and downregulation of Bcl-2 and survivin proteins. Despite its anticancer properties, chelidonine’s use is restricted due to multidrug resistance. Encapsulated chelidonine outperformed free chelidonine in preventing cell cycle progression at the G2/M phase in HepG2 cells (Paul et al., 2013). Liriodenine dramatically lowered cellular vitality, caused apoptosis, enhanced the development of apoptotic nucleoli, and enhanced caspase-3 activity in MCF-7 cells, according to the findings. On human lung cancer cells (Chang et al., 2004) and human ovarian cancer cells, liriodenine has anticancer properties (Nordin et al., 2015). According to studies on breast cancer, the folliclestimulating hormone stimulates the synthesis of VEGF and facilitates the expression of hypoxia-inducible factor (Groves et al., 2009; Liu & Qian, 2012). Growth, invasion, motility, and lumen creation in cancer cells can all be aided by VEGF (Liu & Qian, 2012). The present investigation discovered that 10 μM liriodenine dramatically reduced the production of VEGF protein in MCF-7 cells. Li et al. (2000) proposed that liriodenine promotes apoptosis in human laryngo carcinoma cells by inhibiting VEGF expression. As a result, VEGF expression may play a key role in MCF-7 cell death induced by liriodenine.

13.4.4 Sanguinarine (Fig. 13.10) FIGURE 13.10 Structure of sanguinarine.

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Isoquinoline alkaloids produced from greater celandine include sanguinarine. Haseeb et al. (2007) found that it promotes apoptosis in A549 human lung cancer cells, pancreatic carcinoma Aspc-1 and Bxpc-3 cells, and is efficient toward melanoma skin cancer. In human cervical cells, it is also efficient against multidrug resistance (Byeong-Churl et al., 2009; Ilaria et al., 2009; Zhihu et al., 2002). Sanguinarine caused apoptosis in pancreatic carcinoma cells Aspc-1 and Bxpc-3, as well as human lung cancer cells A549, was proven to be effective against melanoma (Ahsan et al., 2007; Jang et al., 2009; Stefano et al., 2009), and was discovered that it was efficient toward multidrug resistant human cervical cancer cell lines. Sanguinarine connects to the G-quadruplex of the KRAS promoter, which is linked directly to the KRAS mutation for cancer treatment, and it is beneficial against SW620 cells. Sanguinarine was discovered to be a potent cytotoxic agent by inducing autophagy, particularly in malignant glioma cells that are resistant to drug-induced apoptosis. It promoted the development of acidic vesicular organelles and GFP-LC3 punctate after treatment, as well as the conversion of LC3-II. It also enhanced the levels of Atg5 and Beclin-1 expression (Pallichankandy et al., 2015). Sanguinarine, in combination with digitonin and doxorubicin, restored Caco-2 and CEM/ADR5000 cancer cells’ multidrug resistance to the medication doxorubicin (Eid et al., 2012). In two-drug combos (sanguinarine 1 doxorubicin), the IC50 value of doxorubicin was lowered by 17 sixfold, and in three-drug combinations (sanguinarine 1 digitonin 1 doxorubicin), it was decreased by 35-fold.

13.5

Biological source, mechanism of action, and applications of Taxus alkaloid

The yew trees Taxus baccata and Taxus brevifolia are members of the Taxus genus (Taxaceae). The English yew (T. baccata) is a common name for T. baccata. The Pacific yew is the more frequent name for T. brevifolia, but it is also known as the Western or American yew. The FDA classified it as the only authentic source of paclitaxel (Taxol), a highly publicized anticancer medication. Arils are the vivid red fruits of yew trees, and each one contains a single seed. The only portions of the plant that do not contain deadly alkaloids called taxines are the arils, which are fairly pleasant (Nahata, 2017).

13.5.1 Taxol Paclitaxel is biosynthesized and found in the leaves of a variety of Taxus species, and the easy semisynthetic transformation of baccatins to paclitaxel, as well as active paclitaxel analogs like docetaxel (Taxotere), has offered a substantial, renewable natural supply of this essential family of medications. (Fig. 13.11) FIGURE 13.11 Structure of taxol.

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Paclitaxel conflicts with microtubule growth’s natural function. Paclitaxel attaches to the tubulin subunit. Tubulin is the “building block” of microtubules, and paclitaxel binds to these construction blocks, locking them in place. The microtubule/paclitaxel complex that results is unable to disintegrate. Since the shortening and lengthening of microtubules (known as dynamic instability) are required for their function as a cell’s transportation highway, this has a negative impact on cell function. According to additional studies, paclitaxel induces programmed cell death (apoptosis) in cancer cells by attaching to the apoptosis-inhibiting protein Bcl-2 (B-cell leukemia 2) and therefore inhibiting its action. Nausea and vomiting, appetite loss, alterations in taste, thinning or brittle hair, soreness in the arms or legs that lasts 2
3 days, changes in the color of the nails, and tingling in the hands or toes are all typical adverse effects. Unusual bruising or bleeding, pain/redness/swelling at the injection site, change in regular bowel habits for more than 2 days, fever, chills, cough, sore throat, difficulty swallowing, lightheadedness, shortness of breath, severe tiredness, skin rash, facial flushing, female infertility due to ovarian damage, and chest pain are all possible side effects (Haque et al., 2016). Paclitaxel is a drug that is utilized to treat a range of malignancies, including breast, ovarian, and non-small cell lung cancer, as well as Kaposi sarcoma. It has also gotten a lot of interest because of its potential for treating psoriasis, MS, and rheumatoid arthritis. The semisynthetic derivative docetaxel is mostly used to cure breast cancer. The fact that more than a dozen taxanes analogs are in clinical or preclinical research demonstrates the relevance of this class of anticancer drugs.

13.6

Aporphinoid alkaloids

Aporphinoids are a large subgroup of benzylisoquinoline molecules among alkaloids, with over 500 alkaloids discovered to date. Annonaceae, Lauraceae, Monimiaceae, Menispermaceae, Hernandiaceae, and Ranunculaceae are just a handful of the plant families where they can be found. Two aporphines are commercially accessible as pharmaceuticals. One seems to be boldine, which has been isolated from the leaves and bark of the South American tree Peumus boldus, and has been shown to have free radical scavenging (antioxidant) capabilities along with increased bile secretion (choleretic) (Bruneton, 1999). (Fig. 13.12)

FIGURE 13.12 Structure of aporphine.

Although the modes of action of these benzylisoquinoline compounds are unknown, DNA-manipulating enzymes such as polymerases and topoisomerases are commonly mentioned as potential targets. Aporphinoids have a diverse set of biological characteristics (Rios et al., 1989, 2000). Several of them are effective dopaminergic drugs with adrenergic and serotonergic transmission actions. Some aporphinoids also have antiplatelet and vasodilator properties (by inhibiting extracellular calcium entrance and sometimes tissue-specific changes in intracellular calcium movements). Antioxidant capabilities of phenolic and non-phenolic aporphines, and also antibacterial, antiviral, and cytotoxic activities, have also been identified (Stevigny et al., 2005).

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Emetine and related alkaloids

The roots of Cephaelis ipecacuanha have long been used by Brazilian Indians to treat diarrhea. It was first introduced to Europe in the year. Scientists isolated a primitive version of emetine in 1817, but it was later shown to contain a mixture of emetine, cephaeline, and psychotrine. Various additional alkaloids have now been identified and their chemistry investigated. The studies of emetine’s therapeutic usage were limited to amebiasis and a few additional protozoal illnesses till recently. Initial reports of tumor regression after emetine therapy were mostly dismissed. However, after the revelation of emetine’s efficacy against certain nonspecific granulomatous lesions, interest has grown again. (Fig. 13.13)

FIGURE 13.13 Structure of emetine.

Lewisohn (1918) described the anticancer action of EMT on malignant human tumors for the very first time in 1918, but because he was unable to replicate the result in experimental animals, he concluded that the medicine had no antitumor qualities and that the tumor regression had to be random. However, Van Hoose (1919) documented the remission of various cancers in a number of patients by EMT in the subsequent year. Further reports of EMT’s efficacy in rat Yoshida sarcoma (Isaka, 1950), intra-abdominal and retroperitoneal nonspecific granulomas (Grollman, 1965), and murine leukemia (Isaka, 1950) followed (Jondorf et al., 1970). Furthermore, the efficacy of dehydroemetine, an analog of EMT, has been demonstrated in chronic granulocytic leukemia (Abd-Rabbo, 1966), different malignancies (Abd-Rabbo, 1969), Hodgkin’s disease, and rectal adenocarcinoma (Wyburn-Mason, 1966). Phase I and II clinical studies using EMT were conducted in the early 1970s based on these reports (Kane et al., 1975; Mastrangelo et al., 1973; Moertel et al., 1974; Panettiere & Coltman, 1971; Siddiqui et al., 1973; Street, 1972). Nevertheless, the medicine was pulled from human trials (Von Hoff et al., 1977) due to its relatively limited therapeutic index, heart toxicity, and other side effects that were also seen in amebic patients (Knight, 1980). Until then, the drug has been employed in in vitro experiments demanding protein biosynthesis inhibition (Akinboye et al., 2012). EMT has also been identified as a regulator of various cancer-related biochemical pathways, according to the findings of these current investigations. Indeed, Akinboye and Bakare (2011) demonstrated that EMT exerts its antitumor effect via apoptosis via processes such as protein biosynthesis inhibition, DNA interaction, and modulation of proapoptotic molecules. Several research has also looked into the role of EMT in cancer growth inhibition and its biological targets utilizing a range of human carcinoma cell lines in current decades. New compounds have also been developed, which have been found to be effective while being less harmful to normal cells. In addition, the medicine has been studied in conjunction with other drugs to see if there is a synergistic antitumor impact that would justify a lower dose. EMT for prostate, breast, and normal human prostatic epithelial cell line cancer: EMT was derivatized at its N-20 position so that it could be given as a prodrug to cancer cells, where it would be triggered by an enzyme called fibroblast activation protein (FAP), which is abundantly expressed in metastatic tumors. FAP activation of 11 peptidyl EMT prodrug analogs was

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investigated in vitro. One of the prodrugs, a dipeptidyl peptidase-4 (DPPIV) activatable derivative, was demonstrated to activate EMT (70% in 24 hours), and cytotoxicity experiments revealed its equipotence to EMT when FAP and DPPIV were present. In the normal cell line, PrEC, the prodrug, was nearly 200 times fewer cytotoxic than EMT (Akinboye & Bakare, 2011). Bladder cancer: Low nanomolar doses of EMT were shown to effectively suppress the expression of HIF1 and HIF2, but not HIF1. Protein synthesis suppression and proteasomal breakdown both contributed to the decrease in HIF expression. Given the crucial function of HIF proteins and hypoxia signaling in driving tumor development and progression, it was postulated that cancer patients would benefit from treatment with an HIF inhibitor as EMT (Foreman et al., 2014). Ovarian cancer: The combination of cisplatin and EMT not just significantly increased apoptosis but also inhibited tumor cell colony development. The activation of caspases 3, 7, and 8 and the downregulation of bcl-xL by EMT were required for apoptosis (Sun et al., 2015). Lung cancer: Human non-small cell lung cancer (NSCLC) cells are inhibited in their motility and invasion by EMT. The drug selectively inhibits two (p38 and ERK) of the three main mitogen-activated protein kinases (MAPKs), p38, ERK, and JNK, resulting in the selective downregulation of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), two major gelatinases that can deteriorate extracellular matrix components and enable cancer cells to disperse out from their origin (Kim et al., 2015). Pancreatic cancer cell: EMT was discovered to sensitize pancreatic tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Myeloid cell leukemia sequence-1 (Mcl-1) is thought to play a role in pancreatic cancer cell sensitivity to TRAIL, and EMT promotes apoptosis in TRAIL-resistant pancreatic cancer cells by reducing Mcl-1 protein function (Han et al., 2014).

13.8

Biological source, mechanism of action, and applications of Cephalotaxus alkaloids

Cephalotaxus alkaloids are a group of plant secondary metabolites that have been studied for over 60 years. Cephalotaxus extracts were found to have considerable antileukemia efficacy in mice. Following the identification of potential anticancer activity in novel Cephalotaxus derivatives by Chinese, Japanese, and American researchers, the family underwent comprehensive structure elucidation and biological studies. The biological activities of Cephalotaxus alkaloids are primarily focused on the antileukemic activity of homoharringtonine (HHT), which has shown significant perks in the treatment of orphan myeloid leukemia and was authorized by the European Medicine Agency in 2009 and the United States Food and Drug Administration in 2012.

13.8.1 Cephalotaxine Cephalotaxine is a secondary metabolite of Cephalotaxus harringtonia, a coniferous plant in the Taxaceae family, along with a variety of related chemicals. Secondary metabolites are nonessential substances in the body that are divided into three categories: phenolics, which comprise hydroxylated aromatic rings; terpenoids that are derivatives of polymeric isoprenes and are synthesized innately through the mevalonic acid pathway; and alkaloids, which are nitrogencontaining nonprotein compounds (Seca, 2018). These metabolites have shown to be promising anticancer drugs, and plant metabolites, such as cephalotaxine, have the perfect composition for clinically tested molecules. (Fig. 13.14)

FIGURE 13.14 Structure of cephalotaxine.

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Because it is resistant to tyrosine kinase receptor antagonists, CET is a potential parent of omacetaxine, a protein translation blocker that has been utilized to cure chronic myeloid leukemia (Gandhi et al., 2014). On ribosomes, normal protein translation takes three steps. The A, P, and E sites are the three sites in the ribosome. When the amino acidlinked transfer RNA (tRNA) enters the translation process, it first attaches to the corresponding mRNA at the acceptor (A site). Then, it goes to the peptidyl-tRNA (P site) and transfers the amino acid to the incoming amino acid, enabling the next tRNA to take in the next amino acid and resume protein translation/production. The ribosome is detached from the deacylated tRNA by moving it to the E site. The usage of omacetaxine, a competing inhibitor that competes with tRNA for binding to the A site, inhibits translation (protein synthesis). No additional tRNAs can attach to the binding domain until omacetaxine has covalently bound to it, inhibiting the synthesis of proteins, especially those with short half-lives. Apoptosis occurs in cells that rely on the proteins produced by translation, halting the proliferation of chronic myeloid leukemia cells and facilitating tumor regression (Winer & DeAngelo, 2018). CET and its derivatives are effective over a variety of cancers and viruses, and they have the ability to cure different types of leukemia, CRC, HIV, and a variety of other disorders.

13.8.2 Homoharringtonine Homoharringtonine is another plant-derived substance in therapeutic usage. Homoharringtonine was discovered in the C. harringtonia var. drupacea tree in China (Cephalotaxaceae). Elliptinium has been extracted from a variety of Apocynaceae species, including Bleekeria vitiensis, a Fijian medicinal plant reported to have anticancer effects. In China, a racemic mixture of harringtonine and homoharringtonine (HHT) is being utilized to treat acute and chronic myelogenous leukemia with great effectiveness (Itokawa et al., 1993). (Fig. 13.15)

FIGURE 13.15 Structure of homoharringtonine.

The remarkable antiproliferative action of homoharringtonine on murine P-388 leukemia cells with IC50 values of 17 nM sparked curiosity (Chang et al., 2017). In fact actually, homoharringtonine or a combination of cephalotaxine esters has been utilized to treat hematological malignancies in China since the 1970s (Lu & Wang, 2014). Yet, it was not until the discovery of the above-mentioned semisynthetic process that homoharringtonine became popular in Western medicine. Homoharringtonine is a first-in-class protein translation inhibitor, meaning it stops protein synthesis at the extension stage. In reality, homoharringtonine binds to the A site of the large ribosomal subunit, preventing the charged tRNA from accessing the A site and, as a result, the generation of peptide bonds (Gandhi et al., 2014). Because this medicine does not specifically target proteins, it works by disrupting proteins with a high turnover rate, such as the leukemic cells’ increased short-lived oncoproteins BCR-ABL1 and antiapoptotic proteins (Mcl-1, Myc), causing cells to apoptose (Gandhi et al., 2014). Other techniques have previously proposed that it can alter signaling pathways such as the JAK-STAT5 pathway by regulating protein tyrosine kinase phosphorylation (Li et al., 2017) and stimulating the TGF pathway by phosphorylating Smad3 (Chen et al., 2017). The market authorization of homoharringtonine, as well

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as ongoing preclinical and clinical research, suggests that it could be used to treat additional hematological cancers. For example, the generated long-lasting hematologic and cytogenetic effects irrespective of mutational status (Cortes et al., 2015; Damlaj et al., 2016) have the potential to destroy stem/progenitor cells effectively (Allan et al., 2011; Damlaj et al., 2016) and may have a contribution in acute myeloid leukemia (Lam et al., 2016). Refined homoharringtonine has already been observed to elicit full hematologic relapse in patients with late chronic-phase chronic myelogenous leukemia, such as those that are unresponsive to standard treatment. Elliptinium is a drug that is used to manage breast cancer in France (Itokawa et al., 1993).

13.9

Biological source, mechanism of action, and applications of pyrrolizidine alkaloids

13.9.1 Clivorine In human liver L-02 cells, pyrrolizidine alkaloid derived from the Chinese plant Ligularia hodgsonii Hook displayed antiproliferative action (Ji et al., 2005). It also activated autophagy in Huh-7.5 cells by accumulating autophagosomes. Because chloroquine (a lysosomal inhibitor) was present, it produced a rise in LC3B expression as well as an increase in LC3B-I to LC3B-II conversion. Despite this, hepatotoxicity resulted in numerous cell deaths (Liu et al., 2017). Clivorine inhibited cell proliferation and induced mitochondria-mediated apoptosis in human liver L-02 cells, according to Ji et al. (2008). This endeavor included degrading Bcl-xL protein, releasing cytochrome c from mitochondria, and activating caspases-3/9 (Ji et al., 2002). A current research discovered that after clivorine exposure, PC12 cells experienced apoptosis at concentrations more than 50 μM, whereas lower dosages reduced neuronal development via the TrkA/Akt signaling pathway (Xiong et al., 2016). Liu et al. (2017) studied that in Huh-7 cells, clivorine modulated the mRNA expression of the autophagy-associated gene LC3 (microtubule-associated protein 1 light chain 3). Autophagy appears to have an effect on PA toxicity, according to the findings by using three retronecine-type PAs (senecionine, seneciphylline, monocrotaline) and one otonecine-type PA clivorine at varied dosages to investigate the harmful effects of PAs on human hepatoma Huh-7.5 cells. Their impacts on cell proliferation were studied, as well as the underlying mechanisms, which included autophagy. Data show that all PAs are cytotoxic, with clivorine being the most unique among them. Their toxicities may be caused by the same apoptotic pathway, but autophagy may defend against toxic stimuli in the initial stages.

13.10 Anticancer alkaloids with future perspective Identifying naturally produced chemicals effective for slowing, delaying, or rectifying the multistage carcinogenesis process has received a lot of attention in recent decades (Sharma et al., 2010). Medications of choice are anticancer drugs with few adverse effects, induce apoptosis, and target specific cytotoxicity to cancer cells (Nahata, 2017). Alkaloids look promising as anticancer treatments because they inhibit the enzyme topoisomerase, which is involved in DNA replication, induce apoptosis, and alter a variety of intracellular targets and signaling cascades. These alkaloids, which have a variety of chemical structures and exhibit varying cytotoxicity against different cancer cell lines, may only be fully understood by further molecular research and molecular docking analysis. Anticancer alkaloids were found to be quite diverse. Alkaloids with anticancer activity can be found in a variety of places. The majority of the above-mentioned alkaloids are divided into different families, and their biosynthesis differs as well. It is a difficult effort to identify similar modes of action for alkaloids, because within every structural class, compounds have been demonstrated to have distinct cellular and molecular processes. Restriction of cellular proliferation, change of DNA replication and cell cycle progression, induction of apoptosis, autophagy, and necroptosis, suppression of angiogenesis, and manipulation of numerous signaling molecules and pathways are the key and yet most investigated methods. The majority of anticancer researches has so far been conducted in vitro. As a result, more in vivo and randomized clinical trials are required for medicines that show promise in vitro.

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473. Wu, H. L., Chuang, T. Y., Al-Hendy, A., et al. (2015). Berberine inhibits the proliferation of human uterine leiomyoma cells. Fertility and Sterility, 103(4), 1098
1106. Wyburn-Mason, R. (1966). Dehydroemetine in chronic leukaemia. Lancet, 1, 1266
1267. Xiao, Y., Tian, C., Huang, T., et al. (2018). 8-cetylberberine inhibits growth of lung cancer in vitro and in vivo. Life Sciences, 192, 259
269. Xiong, A., Yan, A. L., Bi, C. W. C., Lam, K. Y. C., Chan, G. K. L., Lau, K. K. M., et al. (2016). Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 23, 931
938. Xu, X., Zhu, X. P., Bai, J. Y., et al. (2019). Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3. The FASEB Journal, 33(6), 7289
7300. Yao, J., Kong, W., & Jiang, J. (2015). Learning from berberine: Treating chronic diseases through multiple targets. Science China Life Sciences, 58 (9), 854
859. Yardley, D. A., McCleod, M., Schreiber, F., Murphy, P., Patton, J., Thompson, D. S., Shastry, M., Rubin, M., Melnik, M., Burris, H. A., & Hainsworth, J. D. (2010). A phase II trial of vinflunine as monotherapy or in combination with trastuzumab as first-line treatment of metastatic breast cancer. Cancer Investigation, 28, 925
931. Ye, K., Ke, Y., Keshava, N., et al. (1998). Opium alkaloid noscapine is an antitumor agent that arrest metaphase and induces apoptosis in dividing cells. Proceedings of the National Academy of Sciences of the USA, 95, 1601
1606. Yun, D., Yoon, S. Y., Park, S. J., & Park, Y. J. (2021). The anticancer effect of natural plant alkaloid isoquinolines. International Journal of Molecular Sciences, 22, 1653. Zhao, H. L., Sui, Y., Qiao, C. F., et al. (2012). Sustained antidiabetic effects of a berberine-containing Chinese herbal medicine through regulation of hepatic gene expression. Diabetes, 61(4), 933
943. Zhihu, D., Shou-Ching, T., Priya, W., Xiaolong, Y., Alan, P., & Andrejs, L. (2002). The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death. Biochemical Pharmacology, 63(8), 1415
1421.

Chapter 14

Potential phytochemicals as microtubule-disrupting agents in cancer prevention$ Showkat Ahmad Mir1, Archana Padhiary1, Ashwariya Pati1, Sheary Somam Tete1, Rajesh Kumar Meher2, Iswar Baitharu3, Auwal Muhammad4 and Binata Nayak1 1

School of Life Sciences, Sambalpur University, Burla, Odisha, India, 2Department of Biotechnology and Bioinformatics, Sambalpur University,

Burla, Odisha, India, 3Department of Environmental Sciences, Sambalpur University, Burla, Odisha, India, 4Department of Physics, Kano University of Science and Technology, Wudil, Nigeria

14.1

Introduction

Microtubules (MTs) are extremely strong tube-shaped cytoskeleton filaments that are mostly responsible for intracellular transport and maintaining cellular morphology. The rigidness of the MTs is very much important for all the biological functions that occur within a cell. The transformation of MTs has an important role in the division of cells and also intracellular transportation by clearing the way for intracellular vesicular traffic (Anand et al., 2011). The constituent protein of the microtubule, tubulin, shapes a heterodimer that is arranged in the form of two homologous polypeptides, those are designated as α- and β-tubulin. The length of each of the tubulin heterodimers is about 8 nm, and each interacts longitudinally and laterally first to form the protofilaments and then to MTs. One of the fascinating features of tubulin protein is that it can be present in multiple isoforms in eukaryotic organisms and its various posttranslational modifications such as polyglutamylation, acetylation, detyrosination, and phosphorylation (Banerjee et al., 2010). The foremost characteristic of MTs is their dynamics; they can expand and shrink continuously by reversible attachment and detachment of α- and β-tubulin heterodimers. In humans, MTs are formed of numerous isoforms of tubulin which are α, β, γ, o¯ , h, and z tubulins. The various tubulin isomers are different from each other in the C-terminal end sequence which functions as one of the binding domains in the microtubule-associated proteins (MAPs). The linear tubulin heterodimers are arranged linearly to form the protofilaments that later get joined to form a hollow cylindrical structure carrying out cell migration and mitosis (Jordan & Wilson, 2004). These microtubular structures undergo frequent polymerization and depolymerization phenomenon to maintain their dynamic equilibrium (Desai & Mitchison, 1997) which gets targeted by several anti-microtubule agents often leading to microtubule disruption, cell cycle arrest, and apoptosis (Ballatore et al., 2012), and considering this, microtubule can be targeted for the development of potential anticancer drugs. Currently, phytochemicals obtained from plants have been authentic in treating various health-related ailments, and they are screened based on their less toxicity and potential for overcoming drug resistance diseases. Phytochemicals are biologically active compounds obtained naturally from different plant sources and classified based on their chemical structures. These include alkaloids, phenolic, and nitrogen-containing compounds. Between 1981 and 2010, around 70% of new natural compounds and their analogs have been reported. Approximately, 75% of natural phytochemicals have been authorized for the treatment of cancer-related disorders (Craig, 1997, 1999) that originate from plants, marine organisms, and microorganisms. There are various natural phytochemicals agents, such as vinca alkaloids (VAs), paclitaxel (PTX), colchicine, combretastatins, epithilones, etc., that can target the microtubules either by stabilizing or destabilizing the polymerization activity eventually disturbing the microtubule’s dynamic equilibrium which ultimately results in the cell cycle (G2/M phase) arrest, abnormal cell division, apoptosis, and cell death (Kamal et al., 2014).

$

All authors contributed equally.

Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00020-7 © 2023 Elsevier Inc. All rights reserved.

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For survival and growth and to regulate all the cellular processes in both the normal and the cancer cells, cytoskeletal proteins are needed which contain many different protein subfamilies including microtubules, intermediate filaments, and actin. Defects in the structure or properties of microtubules can cause various severe diseases including cancer. During the transformation from normal cells to cancerous cells, the dynamic network of interlinking protein filaments can be rejuvenated to support or help the development of cancer cells through the advancement of their growth, survival, and invasion which results in the acquiring of tumor cells with a different sign of cancer. The agents like vinblastine (VBL), taxol, etc., bind with the microtubules and are mostly used for the treatment of various cancer types such as the neck, breast, prostate, lung, head, ovary esophagus, stomach, and bladder. In the newer studies, microtubules are day by day fetching higher attention and are better understood as one of the key mediators with the other cytoskeletal proteins such as intermediate filaments, actin, and proteins in the cell
cell junction also including other cellular regulators like Rho-GTPases and myosin.

14.2

Molecular basis of microtubule dynamics

From various studies, it was found that both the α- and β-tubulin have high similarities in structure, and both of these are composed of many large secondary structures which include 10β strands, namely, S1
S10 and 12α helices, namely, H1
H12; both of these are linked by loops. With these larger structures, six helical structures are also present which are named H1’, H2’, H2’’, H3’, H9’, and H11’ (Lo¨we et al., 2001). These entire combinations make the protein body, this protein body can be divided into N-terminal, intermediate, and C-terminal domain, and N-terminal is also named the nucleotide-binding domain. The N-terminal constitutes amino acids from 1 to 205, and it comprises about 6 parallel β strands which are S1
S6 structurally and alternates with the helices H1
H7 which are joined by the loops; likewise, the intermediate domain also contains amino acids from 206 to 381, and these are formed by the three α helices H8
H10, also structurally alternating with four of the β strands S7
S10. The last part, the C-terminal domain, consists of amino acids from 382 to 41, and it is composed of only two α helices named H11 and H12 which is followed by various numbers of unorganized amino acids which extend behind the globular part which is also named tubulin tail (Lo¨we et al., 2001). The intermediate domains and the N-terminal, also known as nucleotide-binding domains, are the major part of globular protein which has four detectable functional surfaces named H3 surface, ML surface plus end, and minus end. From the adjacent tubulins, both the plus and minus interact longitudinally, and it is very important for the development of both protofilaments and heterodimers. Nucleotide-binding pocket (NBP) is included in the plus-end surface where the GTP or GDP is shattered. The plus end of the α-tubulin is highly associated with the minus end of the β-tubulin in the heterodimer, so the NBP is permanently attached to GTP. But the plus end of the β-tubulin including NBP when it is exposed allows the exchange of GTP to GDP. The conversion of GTP to GDP is found to be one of the most important factors as it helps in regulating the dynamics of the microtubule by bringing out some of the conformational alterations in the tubulin dimer, and the other tubular regions like C-terminal domains and intermediate domains are found to be involved in this process. The helices H11 and H12 of the C-terminal domain are arranged on the outside of the MT which is continually exposed to the cytoplasmic part, and from these two helices, a surface was formed where the motor or MAPs can bind (Lo¨we et al., 2001). Both the isoforms of α- and β-tubulin go through various posttranslational modifications which change the structure of microtubules and heterodimers; the posttranslational modification in tubulin can bring changes in microtubule dynamics by determining the relationship between microtubule-interacting protein and microtubules which can bring about changes in microtubules like its stability, destability and also can cut microtubules.

14.3

Factors affecting microtubule dynamics in cancer cells

A small change or alteration of structure in the microtubules can cause cancer. From several studies, it was found that in many types of cancer, the expression of β-tubulin is very high, and it was found to be correlated with chemotherapy drug resistance, clinical behavior, and poor outcomes for the patient. The increase in expression of β1 was seen in various types of cancers including kidney, breast, and colon cancer, but the expression of β1-tubulin was found to be decreased in prostate cancer. Experimentally, lowering the level of β1tubulin by mir-195 or microRNA or siRNA stimulates the adenocarcinoma cell lines to eribuline and PTX, which indicates a direct relationship between the β1-tubulin level and MTA resistance in non-small cell lung adenocarcinomas.

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In several cancer types, primarily cancers of epithelial origin, the βII tubulin was found inside the nucleus of cancer cells as well as the non-transformed cells that are present adjoining the cancer cells. The isoform of tubulin βIII is associated with tumorigenesis, and it was fully confirmed in a model of the pancreatic cell line. Suppressing the expression of βIII by mir-200c microRNA and shRNA reduces the growth of cancer cells and the potential of tumorigenicity both in vivo and in vitro in xenographic and orthotopic pancreatic cancer, and it was confirmed by studies in mouse models.

14.4

Intracellular stress in cancer

For the growth and proper functioning of cells, the cytoskeletal molecule is very much important, but when a normal cell gets transformed into a cancer cell at that time mutations in the cytoskeletal molecules are found. Some examples of these are mutations of β-catenin which are seen in the prostate, liver, and colon cancer which are found to develop carcinogenicity through a Wnt-dependent pathway. On the other hand, the mutations also can be seen in cytoskeletal protein-related coding (CPCR) with the cytoskeletal-related proteins and with the genome which have been marked in various types of cancers like ovarian, breast, and melanoma. Several isotypes of tubulin like βIII, βV, and βVI can function as oxidative stress sensors and as redox switches. Sometimes oxidative stress can be carcinogenic as the reactive oxygen species (ROS) can damage the molecules like proteins, lipids, and DNA in the cells. It also encourages cancer growth by performing cell invasion and migration through the epithelial
mesenchymal transition (EMT) thereby regulating tumor metastasis.

14.5

Targeting microtubules in cancer

With the advancement of technology recently microtubule-determined chemotherapeutic agents are introduced for the treatment of cancer. VBL and PTX are the two major classes of microtubule specific and are used as chemotherapeutic agents. The recognition of inhibitors like IMB5046 has antitumor properties in many cell lines. Taxanes were used for the treatment of various forms of cancer. The first clinically approved drug PTX was used for the treatment of cancer. Since 1992 PTX has been used for the treatment of ovarian cancer; then, in 1994 it was used for the treatment of breast cancer (Engels et al., 2005). Nowadays, one of the most promising derivatives was found to be cabazitaxel which was clinically approved for the treatment of hormone-resistant prostate cancer. The MT structure along with GTP at the positive end and GDP at the α and β interfaces are represented in Fig. 14.1.

14.5.1 Ovarian cancer Many types of proteins are found in the extracellular matrix (ECM), but there are some proteins like fibronectin that govern and produce resistance toward chemotherapy by activating the intracellular pathways. Due to the loss of protein in the ECM, transforming growth factor beta-induced (TGFBI) occurs in resistance to mitotic spindle and PTX abnormalities in ovarian cancer cells. But when the PTX-resistant cells get treated with the recombinant form of TGFBI

FIGURE 14.1 The α and β dimers are represented as green and blue, whereas the GTP and GDP are represented as ball-shaped models in yellow and red.

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proteins, it shows integrin-dependent reestablishment of PTX sensitivity; then, the microtubules get stabilized by Rho and FAK-dependent pathway. After performing immunohistochemical staining for the protein TGFBI in the ovarian cancer cells treated with PTX, from a clinical trial it was found that the morphological changes of PTX created cytotoxicity, and these were regulated to only those areas where there is a strong expression of the protein TGFBI. Taxanes are a type of drug that can stabilize the microtubules and are tremendously used as one of the influential chemotherapeutics means for treating solid tumors. It was found that the ECM can bring out physiological changes in taxane sensitivity by stabilizing the microtubules.

14.5.2 Colon cancer Colon cancer is one of the leading types of cancer among all cancer types. Endothelial cell trans-differentiation also named an endothelial-to-mesenchymal transition (EndMT) plays a pivotal role in the progression of cancer. It is also functionally associated with different cellular morphology which has the ability to migration and polarity resulting in the reorganization of the microtubule cytoskeleton. It was found that the cells of colon cancer can regulate EndMT in endothelium by the upregulation and phosphorylation of tubulin β3. Advanced stages of colon cancer were treated with therapies that inhibit the expression of β3 and its phosphorylation by blocking the recruitment of β3’ to microtubules. Anti-inflammatory chemotherapeutics can be also a promising tool in the future course of time.

14.5.3 Breast cancer The different isoforms of β tubulins are having different functions in the cancer cells’ response to extrinsic factors also including MT dynamics. In the patients suffering from breast cancer, it was found that both the isoforms of tubulin βI and βIII were upregulated, and their response to taxane-based therapy was very poor, but when the level of both the isoforms was low then the maximum number of patients had responded well to the therapy. In the group of patients, where the level of one of the β-tubulin isoforms was low and another is high, then the patient’s response was intermediary.

14.5.4 Oral Squamous Cell Carcinoma In the treatment of various types of cancer, the drugs like microtubule inhibitors such as microtubule stabilizers and destabilizers are used. When the normal cells get converted to cancer cells, then the JAK/STAT pathway is abnormally regulated in the transformed cell; it plays several roles including apoptosis, invasion, proliferation, migration, angiogenesis, and drug resistance. The inhibitors of microtubules have been found to inhibit the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in several types of cancer cell types. A microtubule inhibitor MPT0B098, when treated with oral squamous cell carcinoma (OSCC) cells, the protein level of suppressor of cytokine signaling 3 (SOCS3) increases thereby promoting apoptosis, cell cycle arrest, and growth inhibitor. The gathering of protein SOCS3 can bind to TYK2 and JAK2 and inhibit its activity which results in a loss of STATs regulation and phosphorylation. When the STATs activity was inhibited, it stimulates OSCC cells to MPT0B098 cytotoxicity which indicates that STAT3 is a medium of drug resistance in OSCC. Likewise, it was also observed that there is another microtubule-depolymerizing drug named nocodazole which is also capable of affecting the stability of SOCS1 proteins.

14.6

Alkaloids as microtubulin-disrupting agents

Alkaloids consist of nitrogen atoms either associated with the heterocyclic ring structure or reside inside it. These alkaloids containing nitrogen moiety exhibiting active components that have already been implemented as chemotherapeutic drugs like camptothecin and vinblasine (Huang et al., 2007). They are classified based on their biosynthetic pathways. They are diverse and are distributed in higher plants belonging to Papaveraceace, Leguminosae, Loganiaceae, Ranunculaceae, and Menispermaceae.

14.6.1 Vinca alkaloids The VAs are the oldest groups of naturally occurring or semisynthetic nitrogenous compounds extracted from the Catharanthus roseus (Fig. 14.8) (Madagascar pink periwinkle plant) used for the treatment of cancer (Brogan, 2010; Kufe et al., 2003) though this plant produces innumerable monoterpenoid indole alkaloids (TIA) in different

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Structure of Vinca alkaloid showing catharanthine nucleus and vindoline nucleus

F F

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CONH2 H3CO

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H

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CO2CH3 H3CO

N

H OH

CH3

CH3

Vinflunine

Vindensine Vinca Alkaloids FIGURE 14.2 The 2D structures of vinca alkaloids represent the catharanthine and vindoline nucleus.

organs (Barnett et al., 1978). The VAs present in Catharanthus are bulky amounts consisting of an indole nucleus (catharanthine portion) and a dihydro indole nucleus (vindoline portion) linked by a carbon
carbon ring (Kameswrarao et al., 1997). Over time, the VAs have prevalent applications in cancer therapy, especially in hematological, lymphatic neoplasm, osteosarcoma, breast cancer, and solid tumors either as single or in combination dosage (Johnson et al., 1960,1963; Johnson, 1968). They get associated with the microtubules causing mitotic dysfunction and inhibiting the proliferation of the cells leading to apoptotic death. Vincristine (VCR) and VBL occur naturally from the leaves, whereas vindesine (VDS) and vinorelbine (VRL) are semisynthetic drugs that are used as tubulin-disrupting agents (Em Sutrisna, 2015) Fig. 14.2.

14.6.2 Vinca alkaloids and their mechanism of action against microtubulin Vinblastine crosslinks with the alpha-tubulin and beta-tubulin resulting in the formation of crystalline tubulin-vinca alkaloid structures via the polymerization of microtubulin molecules (Haskins et al., 1981). VAs act in a concentrationdependent manner for tubulin polymerization fluctuations. VAs in their lower concentration directly inhibit the addition of tubulins at the positive end thereby restricting the elongation of microtubules.

14.6.3 Vinca domain The vinca binding site was under wrap till the discovery of the X-ray structure of the vinblasin-RB3 protein stathmin-like ˚ . Vinblastine binds to the interface of alpha/beta (alpha-2/beta-1)-tubulin domain (RB3-SLD) complex at a resolution of 4.1 A heterodimer in a head-to-tail fashion such that 80% of its surface remain buried within the tubulin heterodimeric complex (Gigant et al., 2005). In detail, it was found that vinca site is exactly located in the beta-tubulins plus-end surface as H6 residues and H6
H7, T5 loops. The ligands of the vinca site also interact with the subsequent alpha-tubulin’s minus end structure along with loops H10, S9, and T7 (Doodhi et al., 2016; Gigant et al., 2005) (Fig. 14.3). The association of ligands at the beta-tubulins plus-end site alters its surface forming a curved structure that hinders the addition of tubulins at the microtubules plus end inhibiting and destabilizing the microtubule wall (Gigant et al., 2005). The polymerization of free tubulins gets obstructed as the vinca site ligands form tubulin oligomeric rings (Alday & Correia, 2009; Gigant et al., 2005). Approximately, 16
17 VAs binding sites have been identified in the microtubules which are located at each microtubule end. The binding of VAs to these sites is rapid and reversible. It often leads to the inhibition of the microtubule’s assemblage

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O O O

HN

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O

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CH3

H

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Cryptophycine A (R1=Me, R2 = Cl) Cryptophycine B (R1=Me, R2=H)

Dolastatin O

O

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H O

O

O OH

O

H O

H

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O O

O

O

N

H O

N

O Cl

O

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Rizoxine Vinca domain interacting compounds

Maytansine

FIGURE 14.3 The 2D structure of phytochemicals binds with the vinca domain of the MT.

termed kinetic capping (Cormier et al., 2010; Jordan et al., 1992). Vinca domain is also the binding site for other naturally occurring compounds like cryptophycin, dolastatin, rhizoxin, or maytansine (Gordon et al., 2011).

14.6.4 Therapeutic relevance The application of VAs is generally used as a combination of medicinal chemotherapy (Kufe et al., 2003). It has already been reported that VBL possesses maximum potential in treating malignant angiogenesis, testicular carcinomas, lymphomas (Hodgkin and non-Hodgkin), breast cancers, and germ cell tumors. Blockage in the major steps in angiogenesis was observed when malignant tumor cells were treated with 0.1
1.0 pmol/L concentration of VBL. This low dosage did not have any effect on the normal fibroblast cells as well as lymphoid tumors; rather, combined use of low-concentration VBL and antibodies against vascular endothelial growth factor (VEGF) resulted in the elevation of antitumor activity. The most considered VAs for clinical trials as anticancer agents were VCR, VBL, VRL, and VDS. Out of these, VBL and VCR are FDAapproved in 1961 and 1963, respectively. VBL, VCR, and VRL were approved for usage in the United States (Kufe et al., 2003), and recently, vinflunine (VFL), a new synthetic VA, has been approved for medicinal usage in Europe (Bennouna et al., 2008). Both VRL and vindesine (VDS) show similar consequences as VBL. VRL has been checked to possess antitumor potential in patients suffering from breast cancer and osteosarcoma. Gregory and Smith (2000) have been FDAapproved in 1994 (Kufe et al., 2003). VDS shows antineoplastic property that has been reported in several carcinomas, lymphocytic and myeloid leukemia, melanoma, and solid tumors (Joel, 1995). VCR has been approved for treating several nonmalignant hematological diseases, including leukemia, lymphomas, neuroblastoma, Hodgkin’s disease, and rhabdomyosarcoma (Kufe et al., 2003). Other than these natural and semisynthetic VAs, VFL is a synthetic fluorinated VA that falls under the category of a third-generation drug (Bonfil et al., 2002) and has been used as first-line treatment for breast cancer and as second-line treatment for transitional cell carcinoma of the urothelium (TCCU) in Europe. Clinically, this compound has also been implemented on solid tumors, lung cancers, breast cancers, and several malignancies. It was approved by European Medicines Agency in the year 2012 (Bennouna et al., 2008).

14.6.5 Side effects of vinca alkaloids VAs have been extensively implemented for various clinical therapies, yet their use is getting limited due to their negative effects on health conditions. VCR shows severe neurological toxicity, constipation, bone marrow suppression, and

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vomiting. VBL often leads to white blood cell toxicity, dyspnea, chest pain, and other gastrointestinal disorders (Kufe et al., 2003). VDS has been approved only by certain countries pertaining to its negative effects (Dyke et al., 1979). VRL falls under second-generation VAs endowed with vast antiproliferative activity. It showed high potency in treating advanced breast cancer and metastatic non-small lung cancers; along with that, they are less neurotoxic in comparison with VBL and VCR (Budman, 1997; Gregory & Smith, 2000; Joel, 1995). Besides these, the major dose-limiting drawback reported by the use of VAs is neutropenia. Health issues related to gastronomal toxicity are common in VCR and other VAs when used in high doses. Mucositis is most common in VBL in comparison to VDS or VBL and is less frequent in VCR. Apart from these, synthetic VA, VRL when injected intravenously leads to venous irritation and inflammation at the administered site (Budman, 1997); other side effects include anemia, peripheral neuropathy, hair loss, etc., (Kufe et al., 2003). Among all VAs, VFN has been reported as the superior compound being the least neurotoxic and possessing high anti-cancerous activity (Bonfil et al., 2002). The most probable issue that limits the success rate of VAs in a clinical trial is due to the initiation of certain cellular mechanisms resisting these compounds which includes a variation of beta-tubulin structure, defects in apoptotic responses, multidrug resistance mechanisms reducing the anticancer, antiproliferative activities of VAs. To suppress the toxicological aspects of VAs, they are often administered as combined clinical therapies such that the other drugs can counteract the mechanism of action of VAs. Combination trials have reduced the toxicological disadvantages of VAs like peripheral neurotoxicity and impairment of tendon reflexes that leads to paralysis due to motor neuron dysfunction. Drug-induced perturbation of microtubules may cause sensory defects and paresthesia as a result of decreased axonal transport.

14.7

Taxol as a therapeutic agent disrupting cell polymerization

Taxols, having the generic name paclitaxel (PTX), are naturally derived secondary metabolites derived from plants belonging to Taxus species and a certain extent from Coniferales and fungus Taxus chinensis. Taxus brevifolia (Pacific yew tree) was the first plant from which this active compound was first isolated by M. Wall and M. Wani; Wani in 1971 gave the detailed structure of taxol being a diterpene and possessing a distinct taxane ring along with tetramembered octane ring and an ester chain at C-13 (Fig. 14.4). Due to the slow growth of T. brevifolia, it could not keep up with the pace of this high demanding drug. Thus, mass production of PTX was carried from the endophytic fungus Taxomyces andreanae which was isolated from T. brevifolia phloem. Along with PTX and other taxane compounds docetaxel (semisynthetic, second-generation) and cabazitaxel have also been proven to be excellent first-line chemotherapeutic approaches in early breast cancer patients, and recently, PTX is manufactured semisynthetically by altering 10-diacetyl baccatins III isolated from Taxus baccata O

O O O

O NH

O

O

HO

O

O

O

OH

OH O

NH

O

O

O

O

O

NH

H

O

H O

OH

O O

H O

OH

Paclitaxel

OH O

O

O

O

O

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Larotaxel

Docetaxel

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H O

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O O

O O

O

OH O

O

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O H O

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OH

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H

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O

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Cabazitaxel O

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S

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O

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N

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NH

O

H O

O

OH

O OH

O

OH

O

Epothilone A (R=H) Epothilone B (R= CH3)

O

OH

O

O

Ixabepilone

Zampanolide

Taxane site interacting compounds FIGURE 14.4 Taxane-based phytochemicals interact with the MT and disrupt cell cycle polymerization.

Taccanolide

O

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(European yew). The structure of docetaxel differs from PTX only at two positions, and a hydroxyl group is substituted at C-10 in place of acetate ester making this compound more water-soluble as compared to PTX (Clarke & Rivory, 1999). As PTX is an antineoplastic, antimitotic, microtubule drug stabilizer (Kellokumpu-Lehtinen et al., 2013), several clinical trials of PTX as taxol and their alternatives had shown positive results in treating different types of cancers including ovarian, Kaposi’s, non-small cell lung sarcoma, leukopenia, and mostly breast cancer. The beneficial therapeutic relevance of coadministration of PTX with other drugs has also been documented (Kocher et al., 2020; Kumar et al., 2016).

14.7.1 Mechanism of action of taxol phytochemicals PTX shows a distinct mechanism by binding directly to the microtubule filament and stabilizing it unlike other anticancer drugs like VCR or VBL that function antagonistically destabilizing the microtubule polymerization. PTX helps in the aggregation of tubulin into stable tubulin polymers by reducing its critical concentration. The stability of these polymerized microtubules hinders the cell division process, due to ineffective chromosomal segregation leading to mitotic checkpoint arrest at the G2/M phase and then apoptosis. They also become insensitive toward cold temperatures or calcium concentration (Collins & Vallee, 1987). One of the properties of PTX that makes it unique from other chemotherapeutic agents is that it can polymerize the tubulins even in the absence of GTP and other MAPs (Priyadarshini & Keerthi, 2012). As suggested earlier, the action of taxol is concentration-dependent; that is, a concentration . 10 nM results in mitotic inhibition, whereas a concentration less than that led to cell death due to anomalous mitosis (Chen & Horwitz, 2002), and repressed microtubule dynamics was also observed (Jordan et al., 1993,1996).

14.7.2 Interplay of taxanes with microtubule site Taxane targets the beta subunit of the microtubule heterodimer (Fig. 14.5). The binding site is especially located in the hollow microtubule lumen (Freedman et al., 2009). As reported, the microtubule is arranged as repetitive globular proteins creating small nanopores in between each adjacent protofilament. Taxane enters through these nanopores to gain access to the binding site present on the luminal side (Buey et al., 2007). In detail, the taxane site is present near the ML surface of the microtubule’s inner side (lumen) constituting hydrophobic components H7, S7 and loops H6
H7, S9
S10, and M-loop S7
H9 (Kellogg et al., 2017; Lo¨we et al., 2001). The binding stability of the taxane site varies

FIGURE 14.5 Phytochemicals that interact with the colchicine binding site were isolated from http://www.rcbs.com and represent the respective phytochemical with space and ball model.

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from compound to compound. Generally, the compounds show hydrophobic and polar interaction with the amino acids in the binding site that stabilize the adjacent heterodimers resulting in the overall stability of microtubules (Alushin et al., 2014; Kellogg et al., 2017). The binding of taxane with the heterodimer occurs in 1:1 ratio (Jordan & Wilson, 2004) along with the microtubular length, and only a single taxane can stabilize innumerable tubulin heterodimers (Derry et al., 1995; Jordan & Wilson, 2004). PTX arrests the compaction of heterodimers in their GDP form and stabilizes the microtubule allosterically (Alushin et al., 2014; Kellogg et al., 2017). PTX is especially used as a chemotherapeutic agent against breast and ovarian cancer. The other compounds that can interact with this taxane site stabilizing the microtubules and acting as anti-cancerous agents are docetaxel (breast, lung), larotaxel (lung, bladder), ixabepilone (breast), epothilone B (lung), cabazitaxel (prostrate), and others (Choy, 2001; Die´ras et al., 2008; Engels et al., 2005; Larkin & Kaye, 2007). Zampanolide and taccalonolide A showed covalent interaction with the taxane site amino acids ASN 228/HIS 229 and ASP 226 (Field et al., 2017).

14.7.3 Therapeutic relevance of taxol concerning microtubulin dynamics Taxol possesses high therapeutic relevance in treating breast, ovarian, and lung cancers along with Kaposi’s sarcoma and has been approved by the FDA and EMA. FDA approved the use of taxol in ovarian and breast cancer in the years 1992 and 1994, respectively. Apart from these, semisynthetic taxol derivatives, that is, Taxotere and cabazitaxel, have also been approved by FDA for the treatment of breast cancer and prostate cancers, respectively. The estimated response rate of taxol for ovarian cancer is 30% and for metastatic breast cancer is 56% (Barkat et al., 2019). Besides the use of conventional PTX, other technologies like nanocarrier systems use nab-PTX (Efferth et al., 2017; Jadhav et al., 2017) and glutathione/naringin-encapsulated PTX micelles (Jabri et al., 2019). Folate-coated PTX liposomes, tannic acid nanoparticles loaded with PTX (Chowdhury et al., 2019), combined use of folate
curcumin conjugate, and lipid nanoparticles-packed PTX (Baek & Cho, 2017) enhanced the efficacy of PTX against breast cancer and other related sarcomas, made PTX more biocompatible (in vivo and in vitro), and reduced the side effects associated with PTX.

14.7.4 Side effects of taxanes on treated patients The major toxic side effects caused due to PTX usage were neuropathic and hypersensitive responses. Due to the poor solubility status of PTX, it needs to be administered along with a lipid-based solvent; these solvents generally limit the effectiveness of the drug delivery system that led to histamine hypersensitivity, cardiotoxicity, and neuropathic disorders of the sense organs. Hypersensitivity reaction is observed within the first 10 minutes of PTX administration that includes pulmonary-related disorders such as shortness of breath (dyspnea), tightening of bronchi muscles (bronchospasm), dermatological disorders (urticaria, erythematous rash, angioedema), low blood pressure, fever, chest/abdominal pain, etc. To reduce the hypersensitivity caused by PTX, anti-inflammatory (dexamethasone) or anti-histamine (cimetidine, diphenhydramine) drugs are administered as prior medications. The onset of neuropathy-related disorders is directly proportional to the frequency of the PTX dose-administered side effects like neutropenia, severe infections, and fever which have also been observed by Sparano. Besides neuropathic, pulmonary, and dermatology-related disorders, cardiotoxic effects like irregular heartbeat (bradyarrhythmias, tachyarrhythmias), weakening of the heart muscles (cardiac ischemia), and atrioventricular blockage have also been reported by Rowinsky et al. To reduce cardiotoxicity, the combined use of doxorubicin led to severe heart failure (Gianni et al., 1995).

14.8

Colchicine as a microtubule-disrupting agent

Colchicine is a naturally derived lipophilic alkaloid extracted from Colchicum autumnale’s (meadow saffron) seeds, flowers, and corn, and it belongs to the family Liliaceae. The precursors for colchicine are aromatic amino acids, tyrosine and phenylalanine (Bennouna et al., 2008; Dasgeb et al., 2018). The X-ray crystallographic structure of colchicine includes trimers of A, B, and C composed of trimethoxy benzene ring, methoxy tropone ring, and heptad membered ring having acetamido group at C7, respectively, (Bhattacharyya et al., 2008). Rings A and C bind to the beta-tubulin subunit, whereas ring B binds to the alpha-tubulin subunit of the microtubule; however, the addition of any bulky substituent in ring A results in the reduction of binding affinity to the beta-tubulin (Bhattacharyya & Wolff, 1974). Colchicine served as an unapproved therapeutic drug against gout, pericarditis, Paget’s disease, familial Mediterranean fever, biliary, hepatic cirrhosis, amyloidosis, and Behcet’s disease. In 2009, colchicine was approved by US Food and Drug Administration (FDA) to be used as a mono-therapeutic agent for acute gout flares and Mediterranean fever.

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Later, it was discovered that colchicine has been more effective in killing cancer cells rather than normal cells due to its microtubule inhibition property (Fig. 14.5). But pharmaceutically its role as an anti-cancerous drug is limited due to its high toxicity and low therapeutic index (Kumar et al., 2017; Lin et al., 2013).

14.8.1 Mechanism of action Colchicine acts as a microtubule-disrupting agent by inhibiting the assembly and polymerization of the tubulin subunits. The microtubule disruption results in metaphase arrest and then apoptosis like any other anti-microtubule drug (Bhattacharyya et al., 2008). Colchicine specifically binds at the interface of alpha- and beta-tubulin (CYS 241) by hydrogen/hydrophobic interactions to form a tubulin
colchicine complex (Gigant et al., 2005). Colchicine gets copolymerized into microtubules due to its high binding affinity toward soluble tubulin; the first reversible colchicine betatubulin complex formed generates a curved structure of the tubulin dimer due to the steric hindrance between colchicine alpha-tubulins (alpha-101, alpha-181) and GTP that leads to the formation of the poorly reversible colchicine
tubulin complex (Bhattacharyya et al., 2008; Hastie, 1991). Colchicine can serve its purpose as a microtubule disruptor only after the formation of colchicine
tubulin complex, which gets bound to the end of the microtubule inhibiting elongation by “end-conserving mechanism” which suggests that this complex reduces the rate of tubulin addition rather completely preventing it (Chae et al., 2008; Cronstein et al., 1995). Colchicine acts in a dose-dependent manner, that is, at a low dosage results in microtubule arrest, whereas at a high dosage, it leads to microtubule degradation/depolymerization (Bhattacharyya et al., 2008; Leung et al., 2015), but the treatment of colchicine at 50 nM concentration blocks all the cells undergoing mitosis. Similarly, cells at prophase are more sensitive toward low colchicine concentration, and at high doses, all the metaphasic cells get arrested. It acts as an antimitotic drug hampering the normal mitosis of the cell redirecting it toward an abnormal cell cycle (Bhattacharyya et al., 2008; Leung et al., 2015) termed “colchicine mitosis” (c-mitosis) specified by the absence of mitotic spindle apparatus disintegrating the nuclear membrane, condensation of chromosomes, and fixed centromere (Frankhauser & Humphrey, 1952); along with that, it also destabilizes the tubulins and mutilates the pre-existing blood vessels formed within the tumor vasculature. The anti-inflammatory effects of colchicine on leukocytes are also associated with microtubule and downstream cellular response disruption (Leung et al., 2015).

14.8.2 Colchicine binding site and their interplay with the microtubule The colchicine binding site is situated toward the positive end of beta-tubulin within the interface of alpha- and betatubulin heterodimer. This binding site constitutes of both polar and nonpolar residues H7, H8, S7, S8, and T7 loop (H7
H8). The detailed colchicine binding site came to the limelight after exploring the tubulin complex with DAMA˚ . In tubulin DAMA-colchicine complex, A and C rings intercolchicine and RB3-SLD complex at a resolution of 3.5 A act with the beta-tubulin, whereas the B ring interacts with the alpha-tubulin. This complex interacted with the beta subunit by (S8 and S9 strands, the T7 loop, and H7
H8 helices) and the alpha subunit by (the T5 loop), respectively. The residues S7
S9 termed the microtubule loop (M-loop) are the primary element for microtubule interaction (Bhattacharyya et al., 2008). As reported earlier, the polymerization of tubulin heterodimers at the microtubule polymerization end results in a conformational change in beta-tubulin (S8
S9) bringing them to closer proximity subsequently, leading to the transition from curved to straight conformation; thus, colchicine site gets contracted (Dorle´ans et al., 2009), but binding of colchicine hinders the conformational change mechanism, and binding pocket is stabilized in its curved structure inhibiting the microtubule polymerization. This site is not only restricted to the binding of colchicine, but certain less toxic synthesized analogs and naturally obtained molecules including combretastatin A-1 phosphate, combretastatin A-4 (combretastatin derivative), indibulin (indole derivative), podophyllotoxin estradiol 2-ME (natural compound) also interact with this site. These compounds were also effective in cell cycle arrest, tumor inhibitors, microtubule disruption, and apoptosis serving the same purpose as colchicine (Checchi et al., 2003; Lu et al., 2012).

14.8.3 Therapeutic relevance Colchicine has been approved by FDA in 2009 for the treatment of gout and Mediterranean fever. Besides, other therapeutic applications of colchicine include the inhibition of migrant malignant cells (Charpentier et al., 2014), cell blebbing by (Rho effector kinase/myosin light chain pathway), inhibiting new blood vessel formation from the pre-existing ones within the tumor vasculature (angiogenesis inhibition) (Ganguly et al., 2013), activation of caspases due to

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mitochondrial efflux of cytochrome c (Cyt-c) leading to apoptosis (Bhattacharyya et al., 2008), anti-inflammatory response on leukocytes also being reported (Leung et al., 2015). Though colchicine or colchicine site-binding compounds have not been approved as anticancer drugs, yet several are under phase I/III clinical trials (Kapoor et al., 2018). Previous studies corroborate that 2
6 ng/mL of colchicine is the clinically acceptable concentration that is used for the treatment of hepatic carcinoma (Lin et al., 2013) and bile duct carcinoma (cholangiocarcinoma). In total, 6 ng/ mL concentration of colchicine showed similar antitumor activity as 1 mg/mL epirubicin (anthracycline drug), which is used as a chemotherapeutic drug against various cancers (Lunardi et al., 2003).

14.8.4 Side effects of colchicine Though colchicine is known to be an effective antimitotic, anti-inflammatory, and anti-fibrotic agent, due to its highly toxic effects on normal cells its usage on cancer cells is limited (Bhattacharyya et al., 2008). The central pathway for colchicine metabolism occurs by hepatobiliary excretion which accounts for 5%
10% of renal excretion. The potent dosage of this drug is reported to be 0.015 mg/kg, whereas a dosage exceeding 0.1 mg/kg results in gastrointestinal ataxia, bone marrow aplasia, and several other disorders. Ingestion of colchicine greater than 0.8 mg/kg showed a higher fatality rate (Finkelstein et al., 2010). Upon administration of the prescribed dosage for treating familial Mediterranean fever and acute gout flares, the most frequent adverse effects observed are gastrointestinal disorders (abdominal pain, dysentery, vomiting) and pharyngolaryngeal soreness, which later subsided upon lowering the dosage. Besides that, blood abnormalities lead to neutropenia, leukopenia, thrombocytopenia, granulocytopenia, pancytopenia, and bone marrow aplasia (US FDA; 2013). The coadministration of colchicine with statins, cyclosporin, or digoxin results in muscular disorders (myopathy/rhabdomyolysis), and fatal consequences were also reported in patients coadministered with Pgp inhibitors and CYP3A4 inhibitors.

14.9

Curcumin, a phenolic compound, disrupts microtubule function

Curcumin is a polyphenolic compound naturally obtained from the rhizomes of Curcuma longa Linn. that belongs to the family Zingiberaceae (Aggarwal et al., 2003) (Fig. 14.8). Curcumin is an asymmetrical ligand consisting of alpha
beta unsaturated diketone structures adjoined by 4-hydroxy-3-methoxyphenyl moiety. It is known to possess a wide range of biological activities (Aggarwal et al., 2007) including anti-inflammatory (Huang et al., 1991), antioxidant, antiangiogenic (Arbiser et al., 1998), neuroprotectant (Bala et al., 2006), antifungal, antiviral (Kim et al., 2003), and wound healing effects (Biswas & Mukherjee, 2003). Recent studies have revealed curcumin as an anticancer agent that can prevent initiation, progression, metastasis, and angiogenesis in several tumor cells (Aggarwal & Sung, 2009; Goel et al., 2008; Kawamori et al., 1999) by interacting with microtubules in their specific binding site (Fig. 14.5) to suppress the microtubule’s dynamic instability (Banerjee et al., 2010; Chakraborti et al., 2011; Gupta et al., 2006), suppressing the regulation of antiapoptotic genes, activating the caspases and upregulating anticancer (P53) genes (Fujiwara et al., 2008). As per previous reports, the types of cancers that have been inhibited by curcumin are breast cancer, colorectal cancer, prostate cancer, and basal cell cancer (Chauhan, 2002; Dorai et al., 2001; Jee et al., 1998; Kawamori et al., 1999; Li & Lin-Shia, 2001).

14.9.1 Mechanism underlying polyphenols as microtubulin-binding target The mechanism by which curcumin exactly acts is still unclear. As suggested by Jordan (2002), curcumin completely inhibits the polymerization of microtubules leading to the accumulation of early mitotic cells without affecting tubulin’s function. Curcumin can directly bind to the purified tubulin subunits even at 1 μM concentration (Cheng et al., 2001) in a concentration-dependent manner. The binding of the curcumin
tubulin complex to the microtubule leads to conformational changes in tubulin along with a decrease in GTPase activity. These alterations bring about instability in the microtubule’s dynamic equilibrium marked by abnormal localization of the Eg5 mitotic kinesin in breast carcinoma cells (Gupta et al., 2006). Curcumin has shown the effects like mitotic spindle disruption and anaphase movement inhibition that in turn led to the formation of micro-nucleation in MCF-7 breast cancer cells (Holy, 2002). From several studies, it has been found that curcumin causes the death of cancer cells by inhibiting their proliferation, metastasis, and inducing apoptosis (Bharti et al., 2003; Bush et al., 2001; Chen et al., 1999; Choudhuri et al., 2005; Jaiswal et al., 2002) that could be either by caspase-dependent or caspase-independent (Aggarwal et al., 2003; Anto et al., 2002), but attaining apoptosis is rather a late process; the main cause for cell death is due to G2/M phase arrest, changes in cell and nucleus, and majorly due to mitotic catastrophe leading to cell death (Castedo et al., 2004). The cells treated with

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curcumin are frequently observed with defects like monopolar spindles and abnormal chromatin (Banerjee et al., 2010; Basile et al., 2009; Holy, 2002). From a detailed study, it has been revealed that downregulation of Aurora-A kinase and mislocalization of Aurora-B were observed (Liu et al., 2011). In curcumin-induced mitotic inhibition, increased Mad2 and BubR1 resulted in the improper attachment of spindles to the kinetochore (spindle assembly checkpoint). Curcumin directly binds to the anaphase-promoting complex (APC) reducing its activity; thereby, the metaphase to anaphase cell cycle progression gets hampered (Lee et al., 2012). The accurate mechanism is still unclear. However, curcumin is related to ROS production, topoisomerase inhibition, and chromosomal modifications (Giri et al., 1990; Jiang et al., 2010; Lopez-Lazaro et al., 2007).

14.9.2 Binding of curcumin polyphenol with microtubule Though the exact binding location of curcumin is still under study, Gupta et al. reported that the binding of curcumin to the tubulin was partially repressed by colchicine and podophyllotoxin, whereas no effect was observed by VBL to the curcumin
tubulin binding site (Gupta et al., 2006). This indicates curcumin shares a part of the colchicine and podophyllotoxin binding site and is distinct from that of the VBL binding site. S. Chakraborti et al. (2011) investigated the ˚ far from the colchicine binding site and more closure to the VBL domain located at curcumin binding site to be 32 A the interface of alpha- and beta-tubulin heterodimer. According to the studies conducted by Gutie´rrez-Gutie´rrez et al. (2017), on Giardia, the binding of curcumin was at the interface adjacent to the VBL domain, and the predicted hydrogen bonding interaction with the amino acid residues was ARG 2, SER 165, THR 253, GLN 256 (alpha-tubulin), and LYS 103 (beta-tubulin); van der Waals and hydrophobic interaction were also observed between the phenyl ring of curcumin and TRP 407 residue of beta-tubulin (Fig. 14.5).

14.9.3 Therapeutic relevance of curcumin against microtubule Curcumin has been widely used as an anti-inflammatory, anticarcinogenic, antioxidant, and antimicrobial drug. It is currently under phase II clinical trial in patients suffering from pancreatic cancer (Dhillon et al., 2008). Curcumin concentration at 5
50 μM has been reported to inhibit the cell cycle along with the proliferation of different cancer cell lines (Anand et al., 2011; Lee et al., 2009). One relevant cause of curcumin that makes it a suitable therapeutic agent is its low toxicity; that is, consuming 10 g of curcumin per day does not show any side effects (Anand et al., 2008) rather prevents the cancer cells from proliferating without affecting the normal healthy cells (Boon & Wong, 2004). Several studies have revealed that curcumin modulates cancer cell signaling pathways and increases the potential of antitumor drugs as well as increases the sensitivity of the chemotherapy-resistant cancer cells, which makes it a potent anticancer compound. Furthermore, it can induce apoptosis and inhibit cell proliferation and cell cycle progression. It is also known to enhance the radiotherapy treatment of lung cancer cells by regulating various signaling pathways (KeyvaniGhamsari et al., 2020). Despite all these findings, due to limited clinical trials on human subjects, further studies need to be conducted to get authenticated results regarding curcumin. Furthermore, curcumin can enhance the efficacy of radiotherapy treatment of lung carcinoma by targeting numerous signaling pathways (Ashrafizadeh et al., 2020).

14.9.4 Side effects of curcumin Though no such toxicity of curcumin usage has still been reported, yet side effects like dizziness, urticaria, diarrhea, or nausea can be observed. High consumption of curcumin can be risky for pregnant women (Hsu & Cheng, 2007). The major limitations of curcumin are its low bioavailability (Anand et al., 2007). On oral administration, curcumin gets metabolized into conjugants (curcumin glucuronide and curcumin sulfates) in the intestine, liver, and intraperitoneal administration results in its reduction into tetrahydro-, hexahydro-, or octahydro-curcumin resulting in low level or a negligible amount of curcumin metabolites in the serum or tissue of an animal or human subjects. The curcumin conjugates or the reduced products are biologically inactive and, thus, possess no functional activity.

14.10 Noscapine therapeutic agents disrupting microtubule dynamics Noscapine is a phthalideisoquinoline alkaloid isolated from the Papaveraceae family including Papaver somniferum (Fig. 14.8). It is known as a nonnarcotic opium derivative which constitutes 1%
10% of opium’s alkaloid content and lacks in having any kind of sedative, analgesic, excitatory, or respiratory depressant properties (Altinoz et al., 2019). For a long time, noscapine has been clinically used for treating cough (Ebrahini et al., 2003). Extensive studies on

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noscapine were conducted based on the presence of lactone ring and its structural resemblance with many other antimicrotubular agents (Karlsson & Dahlstrom, 1990) (Fig. 14.6). As reported, noscapine has the potential to induce mitotic arrest and apoptosis in various cancer cells including thymic epithelial tumor, lymphoma, and breast carcinoma.

14.10.1 Mechanism of action The accurate mechanism by which noscapine act is still unknown, but from several investigations it has been reported that noscapine even at 2 μM concentration can act as an antimicrotubule-targeting agent, arresting the G2/M phase cell cycle leading to apoptosis. In HeLa cells, treatment of noscapine resulted in the formation of abnormal multipolar spindles leading to a mitotic arrest by modifying the microtubular instability, predominately by increasing the attenuated state leading to apoptosis. Ye et al. confirmed that the binding of noscapine to tubulin increases the polymerization frequency of the microtubule causing mitotic arrest. Noscapine conducts the cell cycle arrest and apoptosis in glioma cells via the C-Jun-N terminal kinase pathway and colon cancer via p53- and p21-dependent mechanism (Aneja, Dhiman, et al., 2007; Aneja, Ghaleb, et al., 2007). It was also noted that noscapine was able to bring about a similar fate of cell death in both apoptosis-resistant/prone cells, and it also inhibited xenograft tumor cells in mice (Heidari et al., 2007; Jackson et al., 2008). Noscapine mainly targets the kinetic stabilization of the microtubule structure and not on the microtubules, polymerization, or depolymerization which is a unique property of noscapine.

14.10.2 Noscapine binding site To predict the binding site of noscapine, Ye et al. carried out a study on the binding interaction of colchicine and noscapine to specific sites of the microtubule. Although noscapine and colchicine share similar structural moieties, they differ in their stereo structure thus binding to different sites of the microtubule. Naik et al. conducted a blind docking approach to find the specific location of noscapine binding. Noscapinoid showed a better binding score at the colchicine binding site, which suggests both colchicine and noscapine binding sites overlap with each other or the site is very near to it, and its mode of action on microtubule assembly is different from that of colchicine. The computationally predicted noscapinoid binding site is hydrophobic in nature (Naik et al., 2011). Other noscapine derivative such as 7Aaminonoscapine binds to the microtubule at the colchicine binding site and blocks the curve to the straight transition process of tubulin blocking the tubulin assembly. It has been reported that noscapine can induce cell arrest and cell death in both PTX-sensitive and -resistant ovarian cancer cells, which indicates noscapine possesses a different binding site than PTX.

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14.10.3 Therapeutic relevance of noscapine against cancer Noscapine showed positive results in both its lower concentration (20 mg/kg) and higher concentration (120 mg/kg), but it showed an antitumor effect at the higher dose. Though the binding affinity of noscapine to tubulin is weak as compared to colchicine, yet it is sufficient for a mitotic arrest. As a result, it acts as an anticancer agent. It can effectively block both the PTX-sensitive and -resistant human ovarian cancer cell lines. Clinically, it can act as a tranquilizer, anti-stroke, and antitumor agent. When noscapine was coadministered with VCR and doxorubicin, it altered the tumoral resistance and increased their efficacy in the OVCAR-3 cell line which suggests noscapine could be a potential adjuvant chemotherapeutic drug candidate. Current studies indicate noscapine increases the sensitivity of glioma cancer cells toward radiation resulting in tumor suppression such that it can be used in combination with radiotherapy. It is under phase I/II clinical trial for the treatment of non-Hodgkin’s lymphoma (Aneja, Dhiman, et al., 2007; Aneja, Ghaleb, et al., 2007).

14.10.4 Toxicity remarks of noscapine on subjected patients Clinically, noscapine fulfills all the criteria of being nontoxic as reported via ADMET pharmacokinetic profiling and showing no cytotoxic effects on normal cells (Aneja, Dhiman, et al., 2007; Aneja, Ghaleb, et al., 2007) Application of a high dose (3 g) of noscapine on humans and 30 times the therapeutic dosage in rats and dogs showed no elevated toxicities, except headache, nausea, and vomiting in human subjects and some histologic changes in the liver of rats and dogs. Administration of noscapine by parenteral and intravenous infusion can lead to anaphylaxis reaction and infection at the injected site instigating pain, and a blood clot in the vessels and arteries (Ke et al., 2000, Landen et al., 2002, 2004).

14.11 Coumarin’s background and therapeutic activities Coumarins are a group of naturally occurring heterocyclic compounds which are found in a large group of plant families including Rutaceae, Compositae, mulberry, Umbelliferae, etc., (Hassanein et al., 2020). Tonka bean (seed, stem, leaves, flowers, fruits), Dipteryx odorata (seeds), and Cinnamon cassia are known to be rich in coumarin (Baker, 2015; Bayoumi, 2019; Bovell-Benjamin & Roberts, 2016) (Fig. 14.8). This compound and its derivatives have been found to have a broad range of therapeutic activities including antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and many more. Coumarins can be distinguished into five classes based on their side chains: simple coumarins, dicoumarin, isocoumarin, pyranocoumarins, and furocoumarin (Fig. 14.7). In recent years, both natural and synthetic O O

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occurring coumarin compounds have been found to show one of the most potent anticancer activities (Emani & Dadashpour, 2015) along with the brain and cardiovascular disorders (Borges et al., 2013).

14.11.1 Mechanism and binding site against microtubule Coumarin is known to be an ideal chemotherapeutic agent against innumerable cancer cell lines. But only a few studies have been conducted on coumarin being an anti-microtubule compound. Cao et al. synthesized four new substituted coumarins and tested them against various types of cancers. Among them, a compound named 5-chloro-n-(2-methoxy5-(Methyl (2-oxo-2HChromen-4-yl) amino) pentanamide (compound 65) exhibited the ability to disrupt the network of microtubules inhibiting its polymerization hepatocellular carcinoma cells. It showed a similar mechanism to that of colchicine and also interacted at the colchicine binding site of the microtubule. It also led to the inhibition of human ovarian cancer cells in the G2/M phase resulting in apoptosis (Cao et al., 2016). Hydroxy-coumarin and esculetin arrest cell growth and proliferation of the G1 phase cell cycle in leukemia and prostate cancer (Kennedy & Lacy, 2004; Leung et al., 2005). In vitro and in vivo studies of Ferulin C against breast cancer cells also suggested its contact with the beta-tubulin subunits via the colchicine site. After binding to the beta-tubulin subunit, Ferulin C inhibits its aggregation inducing microtubule instability. This resulted in G1/S cell cycle arrest through the p21
Cip1/Waf1
CDK2 signaling pathway.

14.11.2 Therapeutic relevance There are several reports regarding coumarin playing a pivotal role as an anticancer agent including prostate cancer, skin cancer, and kidney cancer. Phase 1 clinical trial of coumate (sulfatase inhibitor) has been reported in postmenopausal breast cancer women. Coumarin is known to be effective against breast cancer as it can regulate the estrogen receptor by forming estrogen
coumarin conjugates (Jime´nez-Orozco et al., 2011). Both coumarin and umbelliferone were reported to inhibit the G1 phase of the cell cycle in lung cancer cells (Lopez-Gonzalez et al., 2004). Warfarin has also been used for the treatment of lung cell carcinoma. Coadministration of coumarin with the standard chemotherapeutic drugs showed better results for the treatment of lung cancer and inhibition of tumor growth (Kostova, 2014). As reported, coumarin and its derivatives undergo different mechanisms of action for different types of cancer cells, and it helps reduce the side effects caused during radiotherapy (Klenkar & Molnar, 2015). Other than its anticancer effects, this compound and its derivatives have a wide range of antibacterial, antiviral, antifungal, anti-inflammatory, and analgesic properties.

14.11.3 Toxicity remarks of coumarin and its analogs Coumarin obeys the Lipinski’s rule of five which is the most common criteria that make a compound an ideal drug candidate (Galkin et al., 2009). The use of coumarin has been banned by Food and Drug Administration (FDA) in 1952 after the reports of hepatotoxicity in animals (Egan et al., 1990). Though the administration of coumarin in high doses leads to hepatotoxic effects, yet they expressed positive results in reducing the diseases pertaining to brain, heart, and cancer (Bovell-Benjamin & Roberts, 2016). The metabolism pathway of coumarin via the 3,4-epoxidation pathway is the major cause of the formation of toxic metabolites; however, the 7-hydroxylation pathway is most frequent and nontoxic to humans (Bovell-Benjamin & Roberts, 2016). Psoralen is known to cause phototoxicity; however, its toxicity is beneficial in treating psoriasis (Asif, 2015; Chaudhary et al., 1986), whereas umbelliferone has been reported as nontoxic (Chaudhary et al., 1986) (Fig. 14.8).

14.12 Discussion The arrangement of microtubules has a very important function in cell division. So, it was found that there are a large number of microtubule-targeting agents (MTAs) that were found to be applicable as therapeutic drugs in treating several cancer types. Understanding the effect of anti-tubulin or anti-microtubule compounds on the stability, polymer mass, and dynamics in microtubules is very difficult. Primarily, MTAs have been found to be divided mainly into two categories. The first category of compounds was microtubule-stabilizing agents (MSAs); these compounds when bound with the tubulin heterodimer expand the lateral interaction within the heterodimers, but at higher concentration, it leads to the stabilization and polymerization of MTs, which results in an increased mass of polymer in the cell. The second category of compounds was microtubule-destabilizing agents (MDAs) when its longitudinal interaction was decreased or

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FIGURE 14.8 Origin of the phytochemicals isolated from different sources against MT.

inhibited within the heterodimers at a very high concentration; then, it results in the microtubule depolymerization also decreasing polymer mass. Especially in the lower concentration when it was used in the therapeutic applications, both the MTA types show no effect on the mass of polymer as well as in cytoskeletal features. Up to date, six binding sites were recognized on the surface of tubulin, and several small molecules were identified that can alter the inter- and intratubulin molecules. In the usual physiological state, the network of the cytoskeleton in the cell is deformation resistance, but when it was seen on the malignant cells then the rearrangement of the cytoskeleton can take place. The alteration in structure and arrangement of cytoskeletal molecules during the transformation from normal cells to malignant cells involves the cytoskeleton and their related molecules including MAPs, microtubules, actin stress fiber, and microfilaments. Chemotherapies related to microtubules have been found to be available for clinical use. At first, it was proposed that the presence of tubulin β3 is linked with chemoresistance in malignant cells. The different subtypes have been found to be a marker of VAs or taxane-based to different drugs of various cancer types breast, pancreas (Lee et al., 2009), stomach, lung, ovary. From the recent studies, it was found that β3-tubulin was recognized as a tumor intruding marker related to EMT. Other than the chemical therapeutic agents, phytochemicals can be screened according to their nontoxic ADMET profiles for the treatment of different types of cancer, inhibition of tumor outgrowth, and metastasis. The phytochemicals belonging to alkaloids, nitrogen-containing groups, and polyphenolic compounds possess a wide range of pharmacological benefits. The compounds belonging to these groups include Vas (VBL, VCR, VRL, VDS, VFL), taxol, curcumin, noscapine, coumarin that have been reported to be anti-microtubule agents. They target the microtubules either by polymerization or depolymerization, subsequently leading to the disruption of the cell cycle, mitotic arrest, and apoptosis of the abnormal cells. The phytochemicals bind to specific binding sites of the microtubules to carry out these processes. Many of these drugs have been clinically approved by the FDA and other medicinal agencies for treatment purposes. There are many potent phytochemicals that are under wrap that require extensive studies and clinical trials to be proved as curative agents for various diseases.

14.13 Conclusion MTAs are one of the most important categories of anticancer drugs in various cancer types. Thus, it is very important to find out novel phytochemicals for the treatment of various cancer types by targeting microtubules which disrupt the cell polymerization and regulate cancer cell death. It is also worthy to mention that toxicity is the major concern among drugs reported which corroborates the novel nontoxic efficient phytochemicals that could meet the present motive and serve as better anticancer drugs.

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Acknowledgment We are very thankful to Professor, Pradeep Kumar Naik, Department of Biotechnology and Bioinformatics, Sambalpur University, for the necessary suggestions and discussion of the efficacy of novel phytochemicals as anti-tubulin agents. Showkat Ahmad Mir wishes to acknowledge the “DBT-BUILDER,” Sambalpur University, Interdisciplinary Life Science Programme for Advance Research and Education for providing the fellowship.

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˚ resolution. Journal of Molecular Biology, 313, Lo¨we, J., Li, H., Downing, K. H., & Nogales, E. (2001). Refined structure of A_-tubulin at 3.5 A 1045
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141.

Chapter 15

Therapeutic effectiveness of phytochemicals targeting specific cancer cells: a review of the evidence Pooja Ravi1, Mona Isaq1, Yarappa Lakshmikant Ramachandra1, Prathap Somu2, Padmalatha S. Rai3, Chandrappa Chinna Poojari4, Kumar Hegde Biliyaru Anand5, K. Shilali1, Asma Musfira Shabbirahmed6 and Mohanya Kumaravel6 1

Department of Biotechnology & Bioinformatics, Kuvempu University, Shivamogga, Karnataka, India, 2School of Chemical Engineering, Yeungnam

University, Gyeongsan, Gyeongsangbuk, Republic of Korea, 3Department of Biotechnology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Udupi, Karnataka, India, 4Department of Biotechnology, Shridevi Institute of Engineering & Technology, Tumkur, Karnataka, India, 5Department of Botany & Biotechnology, Shri Dharmasthala Manjunatheshwara College, (Autonomous), Ujire, Karnataka, India, 6Department of Biotechnology, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences (Deemed-to-be University), Coimbatore, Tamil Nadu, India

15.1

Introduction

Cancer is regarded as one of the leading causes of death worldwide with 7.9 million deaths in 2007 according to WHO 2011. Also, it is one of the extremely outrageous diseases in developing and underdeveloped nations due to the deficit of advanced therapies and care (George et al., 2021). Each year, approximately 11 million individuals are confirmed with cancer, causing millions of deaths. Several studies specify that different types of cancers (stomach, lungs, cervix uteri, liver, breasts, and colorectal) cause around 13% of death annually (Omara et al., 2020). Considerable efforts were successively made for distinguishing the epitome approach for a cancer drug investigation, which later developed as the leading thrust of study for the following several eras (Goldin, 1979). There are several reports that state the mortality rate due to cancer is higher in women than men. Men owed a higher occurrence rate for tumors of the prostate (15.0%), lung (16.7%), stomach (8.5%), colorectum (10.0%), and liver (7.5%), whereas women showed increased occurrence rates around breast (25.2%), lung (8.7%), colorectum (9.2%), stomach (4.8%), and cervix (7.9%) cancers (Alves-Silva et al., 2017). Malignancy is the uncontrolled growth of abnormal cells wherever in the human body. The developed aberrant cells are named malignant cells or tumor cells. The malignant cells can permeate into healthy tissues (Anitha et al., 2014; Chacko et al., 2015) Several cancer and the irregular units that comprise the malignant tissue are subsequently recognized through the particular tissue where these irregular cells are created (for instance, cervical cancer, breast cancer, and colorectal cancer). Often, these cells can segregate from the primary source of mass cells, propagate through various routes (blood and lymph), and reside in new organs from which these cells can once again imitate the same cycle. This development departing from one part and multiplying in another part of the body is called metastatic condition or metastasis (Sreejaya & Santhy, 2013). The conventional therapeutic interventions used for cancer mainly were chemotherapeutic surgery, delivery, or therapy using radiation. These aberrant cells divide quickly, expand, and influence adjoining cells. The therapy using radiation promptly damages the malignant cells even though the chemotherapeutic procedure affects the cancer cell surroundings. Presently, cancer treatment is achieved by malignant surgery, (in the instance of malignancies that have spread) although curative efficacy is comparatively lesser. Cytotoxic treatment is used for chemotherapy which is frequently utilized for the management of several types of cancers (Skeel & Khleif, 2011). These cytotoxic drugs stereotypically sustain abundant dose-confining normal tissue complexities (Tack et al., 2004). Therapeutic chemicals are delivered in a synchronized manner to deliver a combined effect in two or more Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00039-6 © 2023 Elsevier Inc. All rights reserved.

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distinct pathways. There are several biological activities such as chemotherapeutic agents, and due to permeability limitations, they do not have the highest output in terms of therapeutic response, side effects, dosage, and solubility. Developing therapeutic efficiency of chemotherapy is difficult as they search for an alternative method that may enhance the accumulation, and cytotoxic medicines are persistently retained in drug-resistant cancer cells without causing any negative effects (Longley & Johnston, 2005). The existing therapeutic approaches and the medical applications of anticancer agents are eternally accompanied by various acute and unfavorable adverse effects such as nausea, hair loss, medication resistance, nausea, fatigue, anemia, bone marrow suppression, peripheral neuropathy, neurological dysfunctions, numbness, gastrointestinal, and cardiac complications and illnesses (Tipton, 2003). Various kinds of treatments like immunotherapy, radiation therapy, chemotherapy, or surgical procedures are accessible to cure different types of cancer. Nowadays, chemotherapy is considered a major source of treatment to control extensive sorts of malignancies (Gottesman et al., 2002). A major drawback in existence is multidrug resistance (MDR), a key mechanism in the intrinsic overexpression of certain proteins following recurrent treatment. Protein expression hinders penetration through resistant cells in a specific way. The absorptivity possibilities of chemotherapeutics, nanocarriers, and a multidentate strategy for MDR therapy are being investigated. Nanocarriers have made significant progress in the delivery of chemotherapeutic drugs due to their physicochemical features and high drug-loading capacity. In several types of tumors, MDR proteins limit treatment efficacy, resulting in a varied drug penetration rate. The detection of biomarkers before or during therapy was an alternative therapeutic technique. The acuteness and extent of the usual side effects differ with the drugs used, the dosage, and the schedule of administration. Although, for cancer patients, the risen levels of lethargy during treatment persist as a concern it affects the majority of patients during radiotherapy. Late effects involve radiation necrosis, dementia, and effects on complex cognitive functioning (Parnell & Woll, 2003). Patients with malignant glioma are regularly treated with postoperative peripheral beam radiotherapy. The larger dose amount should consolidate the enriching tumor along with a restricted margin (Leibel & Sheline, 1987). Radiation dose strengthening and radiation sensitization techniques are not advised as standard therapy for patients older than 70 years. The molecular and genetic pathways behind cancer have typically been investigated in 2D models employing monolayers of cultured cancer cells. For instance, several reports have revealed particular phases of variations of oncogenes and tumor suppressor genes that influence oncogenesis. Targeted therapy is considered a rising field in cancer research. Tumor DNA sequencing and other approaches are now used to identify new targets, corresponding to the specific tumor, and to suppress them, new medications can be developed. In the upcoming years, the idea of personalized medicine will revolutionize cancer therapy because of its efficacy and cost-effectiveness. Although 3D culturing models range from simple cancer cell spheroids to models with several cell lines, these models are being used to imitate the tumor microenvironment and provide a balance between the reductionist method of isolating cancer cells as a 2D monolayer and emerging human tumors in xenogeneic hosts (Nyga et al.).

15.2

Strategies for identification of phytochemicals with pharmaceutical potential

Plants with medicinal properties play a significant role especially in the field of the pharmaceutical industry for the treatment of various diseases. They might vary with longitude, latitude, altitude, climate, age, and seasonal diversity from species to species. Each plant parts contain many numbers of pharmacological functions. These bioactive phytochemicals are majorly used as anticancer drugs. Extraction assay, combinatorial chemistry, and bioassay-guided fractionation are all phases of phytochemical separation. For the separation of bioactive compounds, various analytical techniques have been used. The process starts with natural extract analysis by using dry or wet plant material by confirming the biological activity. Then, active plant extracts were fractionated and analyzed for biological activity using GCMS, TLC, UV-vis, LCMS, NMR, HPLC, and FTIR. Different solvents are utilized for the separation of different active chemicals based on their polarity groups. For distillation, Sephadex, Superdex, Silica, or any other appropriate matrix can be employed. Some of the coloring agents are also employed to detect natural chemicals in endangered species or therapeutic plants (e.g., vanillin-sulfuric acid). These protocols may vary from species to species. Moreover, the bioactive chemicals’ concentration, safety, and volume must all be very high, which can be achieved by utilizing highly pure solvents and matrices, as well as careful handling. Once the purification is done for the phytochemicals, they should have antitumor effects in vitro or in vivo. If the compound showed anticancer activity, then it has to undergo various tests such as pharmacodynamics, metabolic outcome, pharmacokinetics, biosafety, side effects, drug interactions, immunogenicity, dosage concentration, and other terms used for future drug design. A detailed schematic diagram is shown in Fig. 15.1 that depicts the biosynthesis, optimization, characterization, evaluation, and prospective application of bioactive compounds as cancer treatment agents.

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FIGURE 15.1 Anticancer phytochemical synthesis, optimization, characterization, and potential application as a cancer treatment agent are described.

15.3

Perceptions of phytochemicals as anticancer agents in the history

Biological products are an important source of cancer treatment. Particularly, secondary metabolites are significant for the natural mechanism in the human body to function properly. There are various reports suggesting that phytochemicals suppress highly expressed proteins, amino acids, hormones, and enzymes in diverse ways. Plant-derived compounds are used to suppress malignant tumors and minimize treatment resistance. These can modulate the expression of defective genes, modify the genetic expression of certain genes like p21 and p53 during mitosis or meiosis, or even execute a DNA repair mechanism. Considering the awareness of biological molecules to cure malignancies, chemical leads molecules, as well as medicinal plants, and other natural resources. Semisynthetic analogs with enhanced pharmacological properties are being developed by scientists (Efferth, 2006). Several phytotherapeutic techniques have been documented as a result of extensive research done by chemists and pharmacologists (Jiang et al., 2005). These phytochemicals aid in modifying the rate at which protective enzymes are synthesized, and they have also exhibited antioxidant and relative oxygen-producing capabilities by modulating various pathways (Nasri, 2013). Some of the cytotoxic drugs hinder angiogenesis and show minimal toxicity. Surveys on new plants will lead to the invention of novel anticancerous medications whose success has phenomenal importance (Bahmani et al., 2017). Meanwhile, specialized target drugs have been designed to attack tumor-related proteins, they are the foundation of accurate medicine, biological molecules procured from plant resources signify a valuable source for specific remedies because they affect many targets at the same time, and pharmacological combinations behave in a multi-specific manner. Using network pharmacology, the complexity of pharmacologic networks and new signaling networks that are visible in malignancies can be found (Efferth et al., 2017). Plant-derived anticancer medicines such as vincristine, etoposide, paclitaxel, and camptothecin are some examples. Phytochemicals are also useful in targeted cancer therapy; according to a large body of research, this chapter presents a review of a highly effective combination of standard chemotherapy medicines with plant-derived bioactive chemicals, such as cisplatin, irinotecan, docetaxel, topotecan, paclitaxel, mitomycin-C, 5fluorouracil, vinblastine, and doxorubicin. A synergistic combination necessitates the usage of two components at the same time, i.e., mixing a synthetic anticancerous agent with a plant compound in the same experiment, rather than just comparing or using substances in parallel (Pezzani et al., 2019). Indeed, the preclinical evidence presented in various studies has been validated by rigorous clinical trials (randomized double-blind), and a vast number of clinical trials have been published.

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Synthetic analogs for plant-derived compounds: enhancement and application

One of the major limiting factors of secondary metabolites isolated from plants is their low solubility or inadequate bioavailability which obstructs their use in prerequisites for clinical purposes. The ultimate solution for this problem is the use of semisynthetic or synthetic analogs for distinguishing plant-derived compounds which has been implemented (Fridlender et al., 2015). One of the major examples is to improve therapeutic characteristics, and morphine has been converted to morphine-6-glucuronide. Paclitaxel (Taxol) and the correspondent’s docetaxel (Taxotere) and cabazitaxel (Jevtana) are some examples of synthetic anticancer analogs produced commerically. Camptothecin and analogs belotecan (Camptobell), irinotecan (Camptosar); vinblastine (Vumon), topotecan (Hycamtin), vincristine (Oncovin), and vinorelbine (Navelbine); and their analogs vindesine (Eldisine) and podophyllotoxin and teniposide (Vumon) and analogs etoposide were few other examples for plant based anticancer agents (Etopophos) (Sharifi-Rad et al., 2019). Most of the analogs have similar pharmaceutical characteristics in different diseases. Several reports show that the production of novel advanced pharmaceuticals is efficiently exploited for 64% of drugs produced from natural sources (Newman & Cragg, 2016). It is clear that effective drug development and discovery necessitate robust interdisciplinary collaboration association involving biological products, optimization can be accomplished by combination, total synthesis, medicinal chemistry, and combinatorial biochemistry, and all of these must be integrated into good biology. Also with the advent of high-throughput screening, a large number of potential precursor materials can be quickly evaluated, allowing for accurate determinations of which prototype ligand should be developed further as therapeutic drugs (Cragg & Pezzuto, 2016).

15.5

Classification of phytochemicals

In pharmaceutical treatment, natural compounds play a key role, particularly in the case of antitumor drugs. Beneficial component classes such as steroids, fatty acids, glycosides, terpenes, flavonoids, alkaloids, tannins, and phenolics have been identified in ethnomedicine plant analysis (Rex et al., 2018). They are responsible for enhancing DNA repair pathways and directly affect the central hallmark of cancer progression and metastasis. The pure, chemically well-defined drugs obtained naturally have been used before being used as a drug, and advanced medicines are either transformed directly or through chemical processes.

15.5.1 Alkaloids Alkaloids are essential chemical substances that can be used to develop novel drugs (Mondal et al., 2019). Alkaloids represent an extremely diverse chemical group with a ring structure having nitrogen atoms. Basic nitrogen is present in the heterocyclic ring (Khan, 2015). However, alkaloids are the group of natural herbs with the most biological activity, and some of these compounds, camptothecin and vinblastine, have already been turned into chemotherapy medicines (Lu et al., 2012). The primary mechanism of action for numerous alkaloids investigation indicated several active pathways that are not confined to one or two treatments (Khattak & Khan, 2016). In cancer cells, molecular mechanisms of action involve cell cycle disruption, phagocytosis, angiogenesis, intrinsic and extrinsic apoptosis, glycolysis inhibition, stress and modulation of immune function, anti-inflammatory reactions, cellular metabolism, and invasion and metastasis suppression. Plant alkaloids have an impact on a variety of underlying signaling mechanisms (Efferth & Oesch, 2021). Some alkaloids (capsaicin, piperine) appear to enhance tumor growth and metastasis or to act as co-carcinogens, whereas others appear to be genotoxic (caffeine, sanguinarine, harmine). Consequently, cancer-curing medications must not be genotoxic or carcinogenic. Caffeine has been known to help the development of mammary glands in addition to DNA-damaging chemicals that induce cancer. This finding could be regarded as a proliferative impact, which is undesirable in anticancer medicines as well (Youns et al., 2010).

15.5.2 Polyphenol Polyphenols are secondary metabolites found in plants that help them defend themselves against various stresses. Flavonoids and non-flavonoids are the two major classifications (Asensi et al., 2011). These are natural chemicals that all have a ring structure with more than one hydroxyl group and are generated from phenylalanine. Phenols, flavonoids, their derivatives, stilbenes, and lignans are among the many antioxidants that fall under this category. Polyphenols have various targets in carcinogenesis, drug and radiation resistance mechanisms, tumor cell proliferation and apoptosis, inflammation, invasive dissemination, angiogenesis, and drug and radiation resistance and processes (Damianaki et al., 2000). Polyphenols inhibit platelet activation, capillary permeability, lipid peroxidation, and enzyme systems like

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lipoxygenase, among other biological activities. The following phytochemicals are useful in the chemoprevention of cervical cancer in experimental investigations. Anticarcinogenic, antimutagenic, and anti-inflammatory characteristics are all present in them (Briguglio et al., 2020). Combining conventional medications with polyphenols has several benefits, including the development of more effective anticancer therapies with fewer adverse effects on patients’ health (Sharma et al., 2018). Carcinogenesis is influenced by phenolic compounds that activate cell immune defenses such as hindering anti-inflammatory and anticellular growth signaling pathways, as well as detoxification and antioxidant enzyme complexes, all of which lead to cell cycle arrest and/or cellular death (Sa´nchez-Tapia et al., 2019).

15.5.3 Terpenoid Terpenoids, which are made up of “isoprenoid” units, are the most diverse categories of bioactive chemicals, with over 40,000 different molecules, and each year, numerous new compounds are identified (Sacchettini & Poulter, 1997). Terpenoids are categorized into various groups, depending on the amount of building units, such as monoterpenes (e.g., carvone, perillyl alcohol, limonene-d, and geraniol), diterpenes (e.g., trans-retinoic acid and retinol), triterpenes (e.g., lupeol, ursolic acid, oleanolic acid, and betulinic acid), and tetraterpenes (e.g., lutein, α-carotene, and β-carotene lycopene) (Rabi & Bishayee, 2009). Many triterpenoids have been found to inhibit the growth of a wide variety of cell types while causing no harm to healthy cells (Setzer & Setzer, 2003). Through the transcription and growth factor regulation, as well as intracellular signaling processes, these triterpenoids and their derivative products act at multiple levels of tumor development, inhibiting the development of carcinogenesis, inducing tumor cell differentiation and apoptosis, and suppressing the tumor angiogenesis, invasion, and metastasis (Liby et al., 2007). Terpenoids individually illustrate how complicated signaling pathways like NF-kB, PI3K/AKT/mTOR, AMPK, MAPK/ERK/JNK, and reactive oxygen species can induce phagocytosis by various secondary metabolites. Furthermore, autophagy induction in tumor cells can be either damaging or beneficial (El-Baba et al., 2021). By triggering cell cycle arrest and death, many terpenoids have anti-colon cancer activities (Sharma et al., 2017).

15.5.4 Thiols Thiols are a type of secondary metabolite which has sulfur atoms in its constitution and can be found in plants in small quantities. Several aspects of cell metabolism are dependent on thiol
disulfide exchange processes. A nucleophilic thiolate binds to one of the two sulfur atoms of the target disulfide bond in these reactions. Thiols are a group of highly reactive chemicals that play an important role in maintaining cellular redox equilibrium (Pivato et al., 2014). The thiol moiety is one of the cell’s most powerful nucleophilic groups. It has taken part in a variety of biochemical processes, where thiol-containing compounds have an important function in maintaining intracellular redox balance, commonly known as oxidative stress. Apoptosis is another well-known role of phytochemicals (Zhang et al., 2004).

15.6

Plant-derived phytochemicals currently in use for various cancer treatments

Phytochemicals, or naturally occurring plant substances, are used to develop new medications and are also used to treat cancer. It can be categorized based on its chemical structure (Kapinova et al., 2017). Taxol analogs and vinca alkaloids such as podophyllotoxin, vinblastine, vincristine, and analogs are some of the examples. Typically, phytochemicals interact by modulating the molecular mechanisms in cancer growth and progress. Enhanced antioxidant level, inhibition of proliferation, stimulation of cell cycle arrest and apoptosis, carcinogen inactivation, and immune system control are some of the individual approaches (Choudhari et al., 2020). They have a wide and multifaceted set of activities on a variety of molecular targets and signaling pathways, including membrane receptors (Deng et al., 2017). Anticancer activities of phytochemicals have been investigated in vitro and in vivo. They have an independent and overlapped process that scavenges free radicals to slow down carcinogenic activity (Lee et al., 2013). This report describes several compounds derived from plants that are used to treat cancer, and their chemical structures are shown in Fig. 15.2.

15.7

Curcumin

Curcumin is a phenolic extract from the Curcuma longa plant found to have therapeutic activities (Giordano & Tommonaro, 2019). Curcumin has epitome characteristics for a chemopreventive agent with its low toxicity to healthy cells, easy accessibility, and affordability. Curcumin’s anticancer properties are primarily due to its major role in several biochemical mechanisms engaged in programmed cell death regulation (Park et al., 2013). Curcumin stimulates

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cisplatin-treated cervical cancer cells, lowering the drug’s chemotherapeutic dosage. The NFB, p53-caspase-3 pathway has been demonstrated to increase paclitaxel-induced apoptosis more actively in cervical cancer cell lines. Transcription factors, receptors, inflammatory markers, growth regulators, and enzymes are among the signaling pathways targeted by the compound. Curcumin possesses anticancer properties through various mechanisms, including proapoptotic stimulation, survival signal reduction, and reactive oxygen stress (ROS) scavenging to various extents. In a hepatocarcinoma cell line, it primarily disrupts the cell cycle which has cytotoxic effects and manages antiproliferation and apoptosis activity. Curcumin is recognized as an effective agent to be used in combination with chemotherapy for uterine cancer, boosting apoptosis and inhibiting cancer by significantly lowering the expression of cell proliferation signals, based on its potential to activate multiple targets (Devassy et al., 2015).

FIGURE 15.2 Structures of the plant-derived compounds reported having anticancer potential.

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FIGURE 15.2 (Continued).

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FIGURE 15.2 (Continued).

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Quercetin

Quercetin, a flavonoid derived from plants, is known to have anticancer properties. Numerous studies demonstrate quercetin’s proapoptotic effect in cancer cells, which is produced in plants via phenylpropanoid cascade, starting with phenylalanine (Chirumbolo, 2013). The nearest metabolites for quercetin are naringenin and eriodictyol, which lead to its production via dihydro-quercetin intermediates. It promotes cell cycle arrest and enhances apoptotic activation. Apoptosis induction is the ultimate objective of cancer therapy, and quercetin’s intrinsic capacity to target apoptotic sites makes it a possibility for cancer investigations (Choi et al., 2008). It has the potential to be used as an antitumor agent because it inhibits the growth of tumor-affected blood vessels. Quercetin inhibits tumor growth by inhibiting the angiogenesis pathway controlled by VEGFR-2, repressing the expression of the downstream regulatory factor AKT, and restraining tumor growth in prostate and breast malignancies. Apart from its many advantages, quercetin has many drawbacks, including low bioavailability, poor absorption, quick metabolism, chemical inertness, and rapid systemic clearance. As a result, the use of quercetin analogs and nanotechnology-based techniques to target quercetin may be able to overcome these restrictions. As a result, it seems like these frameworks have the potential to expose new vistas in the use of quercetin as a potential therapeutic approach, either alone or in combination with other medications, in the treatment of many malignancies, including ovarian cancer.

15.9

Vinca alkaloids

Robert Noble and Charles Thomas Beer, two Canadian scientists, discovered vinca alkaloids in the Madagascar periwinkle plant in the 1950s. The Apocynaceae family includes Catharanthus roseus (basionym Vinca rosea). They originated from the pink periwinkle plant C. roseus G. and are either occurring naturally or with semisynthetic nitrogenous bases (Moudi et al., 2013). In vitro, vinca alkaloids, and other microtubule destabilizing drugs can stop neoplastic angiogenesis. Even though structural similarities between the vinca alkaloids are striking, their toxicologic characteristics are vastly diverse. The principal mechanisms of vinca alkaloid cytotoxicity include associations with tubulin and disturbance of microtubule function, notably of the microtubules that constitute the mitotic spindle mechanism, resulting in metaphase arrest (Himes, 1991). Vinca alkaloids suppress cell proliferation largely through their impact on mitotic spindle microtubules, but they can also decrease cell proliferation through additional pathways or a combination of processes. In vitro interactions of vinca alkaloids with tubulin and microtubules have been intensively investigated. They attach to tubulin and can prevent tubulin polymerization into microtubules in vitro and in vivo at higher concentrations. Tubulin self-associates as a result of the vinca alkaloids induction. This binding has the outcome of preventing tubulin polymerization and microtubule assembly, cell cycle metaphase arrest, and inducing apoptosis. Many cancers, such as Hodgkin’s lymphoma, leukemia, NSCLC, breast cancer, brain cancer, melanoma, bladder cancer, and testicular cancer, are treated with them in association with other chemotherapeutic drugs. At the molecular level, the antiproliferative cascade generated by VAs is still unclear, while some clinical research demonstrates that p53, Bcl-2, Bcl-x, and other gene products are directly linked to the regulation of the balance between cell proliferation and apoptosis involved (Howard et al., 1980).

15.10 Camptothecin In the late 1950s, camptothecin (CPT), a plant alkaloid derived from Camptotheca acuminata and reported to inhibit a DNA-replicating enzyme topoisomerase-I. Camptothecin stabilizes by blocking topoisomerase and causing a doublestrand DNA break during replication leading to cell death according to the theory of single-strand break in DNA’s phosphodiester backbone (Berrada et al., 2005).

15.11 Cervical cancer and phytochemicals Cervical cancer has been the most lethal cancer in women, and it affects the majority of individuals. Every year, it causes more than five million patients with cervical cancer (CC) having a 90% mortality rate (Bray et al., 2018). Out of every 1 million, 30%
40% of women are affected by cervical cancer. The main cause of this disease is the persistent infection with sexually transmitted human papillomavirus (HPV). Prevention and treatment of cervical cancer are evolving in terms of knowledge, innovation, and execution, with effective and scalable treatments on the horizon. The major quality test for screening is to determine the lesion and also to facilitate the important variables which are used to reduce morbidity for effective treatment. Diagnosis of cervical cancer is exceedingly low, especially in developing and undeveloped countries, due to the lack of qualified health workers. Further, we have tabulated phytochemicals reported to have anticervical cancer activity and their mechanism of action (Table 15.1).

TABLE 15.1 Anticervical cancer phytochemicals and mechanism of action. Phytochemicals

Sort

Type of cell

Observation

Activity

Mechanism of action

References

Quercetin

Flavonoids

HeLa

In vitro

Induction of apoptosis as an antiproliferative strategy

Increased expression of proapoptotic cytochrome c, Bcl-2 family proteins, Apaf-1, and caspases with downregulation of antiapoptotic Bcl-2 proteins and survivin, G2/M phase cell cycle arrest

Priyadarsini et al. (2010)

Isoliquiritigenin

Flavonoids

HeLa

In vitro

Antiproliferation

Enhanced p21 expression in a p53-dependent manner with downregulation of cdc2, cdc25C, and cyclin B expression, regulation of the Bcl-2 family protein expression, phosphorylates Chk2, and promotes the aggregation of inactive cdc25C and cdc2. Induction of G2/M phase cell cycle arrest

Shafi et al. (2009)

Naringin

Flavonoids

SiHa

In vitro

Antiproliferation

Naringin activated both death receptor and mitochondrial mechanisms involved in apoptosis induction

Ying et al. (2012)

Luteolin

Flavonoids

HeLa

In vitro

Apoptosis induction and inhibition of tumor growth

Sensitization of HeLa cells caused by luteolin by activating TRAIL-induced apoptosis through both extrinsic and intrinsic apoptotic mechanisms

Szliszka et al. (2011)

Isoflavone

Flavonoids

HeLa

In vitro

Antiproliferation and apoptosis induction

Apoptosis induction via the mitochondrial mechanism

Kim et al. (2010)

Fisetin

Flavonoids

HeLa

In vitro

Antiproliferation, apoptosis induction, significantly decreased tumor growth

ERK1/2 inhibition by PD98059 and ERK1/2 phosphorylation lead to the caspase-8/3 pathway activation

Li et al. (2010)

Cannabidiol

Cannabinoids

HeLa

In vitro

Anti-invasion

TIMP-1 upregulation leads to decreased invasion. Cannabidiol-induced TIMP-1 expression knockdown by siRNA results in a reversal of the cannabidiolelicited decrease in tumor cell invasiveness

Tayarani-Najaran et al. (2010)

Gallic acid

Phenols

HeLa

In vitro

Apoptosis induction

Cell death is induced by apoptosis and/or necrosis as a result of enhanced ROS production with the reduction in GSH

Jiang et al. (2021)

Resveratrol

Polyphenols

HeLa

In vitro

Antiproliferation

Induction of cell apoptosis

Tavakkol-Afshari et al. (2008)

Catechin hydrate

Polyphenols

CaSki, SiHa, HeLa, C-33A

In vitro

Apoptotic induction

Regulates p53 and caspase-3, 8, and 9 expression

Rejiya et al. (2009)

Methyl jasmonate

Stress hormones in plants

SiHa, HeLa, C-33A

In vitro

Antiproliferation

Enhances the expression of Bax with the decreased expression of p53 and p21

Kwon et al. (2006)

Oxidized lutein

Carotenoids

HeLa

In vitro

Antiproliferation and apoptosis induction

The free radical scavenging activity of oxidized lutein induces apoptosis

Wu et al. (2011)

EGCG and RA

Polyphenols, retinoids

HeLa

In vitro

Antiproliferation

EGCG and RA together cause the inhibition of telomerase activity, thereby inducing apoptosis

Ramer et al. (2010)

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15.12 Current scenario and future perspective The most common cause of mortality in the world is cancer. The development of novel therapeutics for cancer prevention is critical. Around 60% of the current anticancer drugs are derived from natural sources. Nature continues to be an abundant source of biologically active and diverse chemotypes, while relatively only a few of the actual isolated natural products are developed into clinically effective drugs in their own right. Several bioactive compounds are recognized as chemotherapeutic and chemopreventive agents in cancer therapy. These molecules are frequently used as models for the development of more effective analogs and prodrugs using chemical methods such as total or combinatorial (parallel) synthesis or biosynthetic pathway alteration. Ethnopharmacological knowledge is used in several ways for drug discovery, and it is backed up by a broad range of disciplines including pharmacology, biochemistry, medicinal chemistry, molecular and cellular biology, as well as natural product chemistry, which is crucial for harvesting phytochemical potential. Furthermore, improvements in the formulation could lead to more effective drug administration to patients, or the conjugation of toxic natural molecules to monoclonal antibodies or polymeric carriers that specifically target epitopes on tumors of interest could lead to the development of effective targeted therapies. The importance of a multidisciplinary approach in the optimization of potential biological drugs from natural product sources has been thoroughly examined, as has the critical role of natural products in the discovery and development of novel anticancer agents.

Competing interests The authors declare that they have no competing interests.

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Chapter 16

Understanding the role of the natural warriors: phytochemicals in breast cancer chemoprevention Prarthana Chatterjee1, Suchetana Gupta2 and Satarupa Banerjee1 1

School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India, 2Phil and Penny Knight Campus for Accelerating

Scientific Impact, University of Oregon, Eugene, Oregon, United States

16.1

Introduction

Breast cancer (BC) is the leading cause of malignancy-related death in women worldwide. Globally, BC is accounted for one out of every four cases of cancer diagnosed in women. In 2020 suppressing lung cancer, BC has emerged as the most common cancer diagnosed globally (Breast Cancer Statistics and Resources, 2022). According to an estimate provided by the American Cancer Society (ACS), about 287,850 BC-related deaths will be recorded by the end of 2022 worldwide. Presently, one out of every eight women in the United States is prone to BC-related vulnerabilities (Breast Cancer Statistics, 2022). Despite many advances in the field of oncobiology and molecular research, the development of multidrug resistance (MDR), tumor relapse, and lack of novel biomarkers, assisting in early tumor screening, are some of the major therapeutic drawbacks that contribute to the steady exfoliating mortality caused by this neoplasty. The current BC therapeutic regimen is mainly built on classical convictions of chemotherapy, radiation therapy, surgical interventions, and endocrinal targeted approaches, which initially though effective seems to become insensitive and resistant, as the tumors proliferate with time, often leading to widespread malignant invasion and distant metastasis (Singh et al., 2021). Alternatively, though the main aim of every antineoplastic strategy is to prolong survival without disturbing the standard quality of life, several synthetic drugs used in clinical practices are often accompanied by many adverse side effects, adding to the distress of the already ill patients. The excessive social and economical burden, imposed by BC, is thus a major health crisis, faced by several families of cancer-surviving patients worldwide, affecting the global healthcare economy (Fridlender et al., 2015). Hence, scientists all over the world are striving relentlessly, to find an alternative, least toxic, inexpensive, and target-driven therapeutic strategy to combat the surge of BC tumorigenesis. Several plant-derived bioactive secondary metabolites (phytochemicals) obtained from different natural sources have been used from time immemorial to fight several human diseases and pathologies. The different classes of phytochemicals such as alkaloids, flavonoids, terpenoids, and carotenoids have multiple pharmacological activities, which validate their indigenous use against cancer, diabetes, microbial infections, cardiological, neurological, and other inflammatory disorders of human bodies (Mechanistic evaluation of phytochemicals in breast cancer remedy: Current understanding and future perspectives, 2022). Epidemiological evidence suggests that the antiproliferative role of phytochemicals, which is exploited in tumor biology, is generally attributed to their antioxidant, anti-inflammatory, cytotoxic, apoptosis-mediating chemosensitizing abilities. These phytochemicals generally modulate various tumor-inducing signaling pathways, through their interaction with several molecular targets, which include genetic and epigenetic cofactors, transcription kinases, cell cycle checkpoint inhibitors, and metabolic enzyme modulators (Losada-Echeberrı´a et al., 2017). Thus, these bioactive compounds restore the genetic stability of the cells, by suppressing mutations, reversing epithelial
mesenchymal transitions (EMTs), inducing apoptosis, as well as preventing angiogenesis and proliferation of the malignant entities. Preclinical studies reveal promising outcomes of using these plant-derived compounds in cancer treatment and diagnosis, when used along with conventional chemotherapeutic derivatives, to obtain a combined efficacy greater than the sum of their additive individual effects (Rizeq et al., 2020). This chapter thus discusses the Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00004-9 © 2023 Elsevier Inc. All rights reserved.

261

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Recent Frontiers of Phytochemicals

fundamentals of BC and the perils of conventional therapeutic approaches, the problems of MDR and chemoresistance, and, finally, the role of complementary and alternative medicine (CAM)-based approaches in restoring chemosensitivity and improvising tumor prognosis and therapy outcomes in different subclasses of BC.

16.2

Breast cancer: definition, subtypes, and conventional therapies

Breast cancer (BC), the most frequently diagnosed cancer in women worldwide, accounts for 11.7% of all types of cancers reported annually (Sung et al., 2021). It refers to the uncontrolled division and unrestricted growth of the cells in the mammary tissues of the female body. The mass of abnormal cells, identified as tumors formed as a result of the malignant invasion of the cancer cells, often spreads to different distant primary physiological organs of the body, including lymph nodes, spleen, bones, brain, lungs, liver, and kidney, by a process known as metastasis, through the different blood and lymph vessels in the body (Breast cancer: Types of treatment, 2022). The female mammary tissue is generally composed of three primary parts, the connective tissues, ducts, and lobules. The milk-producing glands are known as lobules, which are carried to the nipples through the ducts, and the surrounding adipose tissue holding it all together comprises the connective tissues. Most breast tumors are reported to initiate either from the ducts or the milk-producing lobules (CDCBreastCancer, 2022). Being the second most commonly diagnosed cancer globally, the incidence of BC is associated with several genetic, epigenetic, lifestyle, and dietary-induced risk factors, which are expanding exponentially. It is reported that only 10% of BC cases are hereditary, pushing the female age group ranging between 20 and 50, to enhanced malignant vulnerabilities. Multiple endogenous and exogenous factors synergistically govern the pathology of the disease, accounting for its very poor prognosis (Breast cancer - Risk factors and prevention, 2012). Owing to the various histopathological and molecular disparities between the different subtypes of BC, neoplasia is generally considered to be genetically ambiguous and heterogeneous, producing different responses to diagnostic strategies and prognoses (Giani et al., 2021). The BC etiology and the proliferation of the neoplastic cells are generally mediated by multiple biomarker-based signaling pathways and mechanisms (Cheang et al., 2008). Hence, identification of the key molecular markers plays an important role in assessing the prognosis and severity of the disease. The four major biomarkers dominating BC etiology and prognosis include the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cellular protein Ki67 of which ER and PR confer the hormonal response of the BC, being the major target in hormone-mediated endocrine therapy in BC therapeutics whereas the expression of HER2 is known to enhance tumor aggression in about 10%
15% of BC cases, being sensitive to antibody-mediated monoclonal immunotherapeutics. Lastly, Ki-67 predicts the severity of tumors, assesses the degree of proliferation, and predicts the prognosis of the disease. High expression of this marker protein generally corresponds to a poor prognosis (Losada-Echeberrı´a et al., 2017). Based on the expression of these molecular biomarkers, immunohistochemical methods and genome profiling classify BC into four distinct molecular subtypes: 1. Luminal A: They constitute about 50%
70% of BC cases, expressing both the hormone receptors ER and PR, with HER2 negative and report the best prognosis, least instances of relapse, along with reduced rates of proliferation, corresponding to only level 1 or 2 in histological tumor grading (Gao & Swain, 2018). 2. Luminal B: This category of BC also expresses both ER and PR, but HER2 overexpression may be present or absent. They are comparatively more aggressive with a higher rate of tumor progression and proliferation than Luminal A. The size of the tumor in Luminal B BCs is larger, diagnosed at a much-advanced stage, with a higher histological tumor grade and positive dissemination of lymph nodes than Luminal A. Although they too have high rates of overall survival (OS), it is not as high as compared to Luminal A, with a poorer prognosis and a higher expression of biomarker Ki67 (Losada-Echeberrı´a et al., 2017; Wu et al., 2016). Table 16.1 highlights the major BC subtypes along with their receptor expressions and representative cell lines. TABLE 16.1 The major BC subtypes along with their receptor expressions and representative cell lines. BC subtype

ER expression

PR expression

HER2 expression

Ki67 expression (%)

Major cell lines

Luminal A

1

1

2

,15

MCF-7, T47D, SUM185

Luminal B

1

1

1/2

.15

BT 474, ZR-75

HER2

2

2

1

.15

MDA-MB-435, AU565, and SKBR3

TNBC

2

2

2

.15

MDA-MB-231, MDA-MB-468, MDA-MB-453 and BT549

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The majority of the BC cases belong to the luminal category, with significant sensitivity to conventional endocrine therapy, as they are positive to both the hormone receptors, ER and PR, respectively. Thus, the use of aromatase inhibitors (AIs), such as letrozole, anastrozole, and selective estrogen receptor modulators (SERMs), such as tamoxifen, forms the primary treatment regimen in patients with luminal BC (Tang et al., 2016). 3. HER2 positive: Patients with HER2 amplification lack the ER and PR expressions, with a higher histological tumor grade, virulent malignant invasion, and positive lymph node amplification, compared to the luminal (both A and B) category of BC. They generally constitute 5%
10% of all the BC cases, diagnosed at a stage much earlier than that diagnosed in luminal BCs. The prognosis of HER2-positive BCs has currently improved owing to the administration of HER2 inhibitory drugs such as lapatinib and monoclonal anti-HER2 derivatives such as pertuzumab and trastuzumab, indicating their sensitivity to targeted conventional therapy though the adverse drug side effects are still a lingering cause of concern in HER2-positive BC (Targeted therapies in HER2-positive breast cancer, 2015). 4. Triple-negative breast cancer (TNBC): This is a hormone-refractory most virulent subtype of BC, which lacks all three hormone receptors, ER, PR, and HER2. It is the most lethal molecular type of BC, with a high degree of tumor proliferation, angiogenesis, elevated histological tumor grade, enhanced degree of distant metastasis, and malignant invasion (Mechanistic evaluation of phytochemicals in breast cancer remedy: Current understanding and future perspectives, 2022). TNBC tumors are generally characterized by a reduced OS and disease-free survival (DFS), and higher chances of cancer relapse, characterized by the worst prognosis, among all the subcategories of BC. Being insensitive to conventional approaches of hormonal or endocrine therapy, the treatment options in TNBC are largely constricted, to only narrow regiments of classical chemo and radiation therapeutic paradigms (Giani et al., 2021). The frequently used chemotherapeutic drugs in TNBC include poly(ADP ribose) polymerase inhibitors (PARP), taxanes, anthracyclines, and other platinum-based agents (Atcı et al., 2021). In general, currently, chemotherapy, radiation therapy, endocrine, and targeted therapies are the major therapeutic interventions employed in BC patients, which are discussed in detail in the later section of the chapter. But the several adverse side effects of this conventional anticancer approach, including MDR and loss of sensitivity to chemotherapeutic drugs, often fail to produce the desired and optimistic antineoplastic effects. These complications are often lifethreatening and thus compel the researchers to look forward to other better alternatives, aiding the progress in the field of oncotherapeutic research. Table 16.2 illustrates the commonly used chemotherapeutic drugs for BC treatment.

16.3

Perils of conventional BC therapies

The initiation of cancer in the mammary tissue is marked by the sudden uncontrolled growth and division of the normal cells, giving birth to an atrocious mass of fibrous cells, called tumors. The behavior, stage, and biology of these mass of malignant cells generally direct the treatment paradigm of BC patients. The deciding factors that generally contribute to designing a personalized patient-specific treatment strategy include the prediction of the tumor subtype and status of the endocrine receptors (ER, PR, and HER2), several general and genomic tests to determine the hereditary history, and some fundamental treatment strategies used as neoadjuvant therapy, during the initiation of cancer treatment in its early stage and also for locally advanced BC. Thus, chemotherapy, radiation therapy, endocrinal therapy (in the case of receptor-positive BCs), immunotherapy, palliative therapy, biomarker-based targeted therapy, and surgical methods are the primary conventional protocols dominating BC diagnosis (Breast cancer: Types of treatment, 2022). Chemotherapy and radiation therapy remain the principal treatment regiments in BC patients, owing to their ability to reduce the tumor size and prevent their proliferation at their primary or detect any mutations in inherited genes such as BRCA1 and/or BRCA2, the general and gynecological health of the patient, and menstrual rhythms. Though the medical practitioners and oncologists tailor a specific therapeutic strategy depending on the physiological needs of each patient, there are metastasizing sites; still, the outcome of the treatment protocol differs from patient to patient of BC, even among the ones with the same subtype, stage, and grade of the tumor (Assessment of the evolution of cancer treatment therapies - PubMed, 2022).

16.3.1 Shortcomings of conventional therapy: chemotherapy Chemotherapy generally deals with the intravenous or oral administrations of certain synthetic drugs, to prevent the growth, proliferation, invasion, and metastasis of tumor cells, keeping the healthy cells intact. The chemotherapy that is undertaken before surgery is known as neoadjuvant chemotherapy, whereas the sessions of chemotherapy post-surgery are known as adjuvant chemotherapy. The entire chemotherapy regimen of a BC patient generally comprises selective rounds of chemo-sessions spread over a specific period, given in fixed intervals, to recover from the effects of the

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TABLE 16.2 The commonly used chemotherapeutic drugs for BC treatment (Breast cancer: Types of treatment, 2022; Chemotherapy for breast cancer, 2022; Treatments used for triple-negative breast cancer, 2022; New metastatic breast cancer treatments). Common chemotherapeutic drugs

Chemotherapeutic drug combination used

Drugs for HER-2-positive breast cancer

Drugs for metastatic breast cancer

Chemo-drugs for TNBC (used in combination or singly)

Paclitaxel

Docetaxel and cyclophosphamide (TC)

Paclitaxel and trastuzumab (TH)

Docetaxel, paclitaxel, and abraxane (albumin-bound paclitaxel)

Olaparib and talazoparib (PARP inhibitor)

Epirubicin

Cyclophosphamide, epirubicin, and 5-FU (CEF)

Carboplatin and trastuzumab (TCH)

Ixabepilone

Pembrolizumab (immunotherapeutic conjugate)

Fluorouracil

Cyclophosphamide, doxorubicin, and 5-FU (CAF)

Paclitaxel or docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP)

Eribulin

Gemcitabine and capecitabine (antimetabolites)

Capecitabine

Epirubicin and cyclophosphamide (EC)

Doxorubicin, cyclophosphamide, paclitaxel or docetaxel, and trastuzumab (ACTH)

Gemcitabine, vinorelbine

Sacituzumab govitecan (antibody drug conjugate)

Docetaxel

Doxorubicin and cyclophosphamide (AC)

Doxorubicin, cyclophosphamide, paclitaxel or docetaxel and trastuzumab and pertuzumab (ACTHP)

Capecitabine

Adriamycin and epirubicin (anthracyclines)

Doxorubicin

Cyclophosphamide, methotrexate, and 5-FU (CMF)

Tamoxifen

Cisplatin and carboplatin

Carboplatin and cisplatin (platinum agents/alkylators)

Carboplatin

Capecitabine (Xeloda)

fam-trastuzumab and deruxtecan (Enhertu)

Doxorubicin, Doxil (liposomal doxorubicin), and epirubicin

Docetaxel and cyclophosphamide (TC)

Abraxane

Docetaxel, doxorubicin, and cyclophosphamide (TAC)

Pertuzumab, trastuzumab, and hyaluronidase-zzxf (Phesgo)

Atezolizumab

Epirubicin and cyclophosphamide (EC)

Methotrexate

Neratinib and capecitabine combinations

Atezolizumab

Alpelisib

Docetaxel, doxorubicin, and cyclophosphamide (TAC)

Cyclophosphamide

Trastuzumab and docetaxel (Hylecta)

Alpelisib

Talazoparib

Doxorubicin and cyclophosphamide followed by paclitaxel or docetaxel (AC - T)

chemotherapeutic drug. The interval between the administrations of two chemotherapeutic sessions is generally maintained to be twice a week, once a week, once a fortnight, or 3 weeks, depending upon the drug used and the specificity of the regiment. The entire cycle of chemotherapy generally ranges from 3 to 6 months, and each time the cycle is repeated to mark the start of a new chemo schedule. Clinical reports reveal that in certain cases, a combination of two or more drugs is used during adjuvant therapies, as multiple combinations of drug doses are found to be therapeutically more effective than a monotypic dose administered (Chemotherapy for breast cancer, 2022). The major chemotherapeutic drugs include alkylating agents, antimetabolites, anthracyclines, platinum-based agents, PARP, and microtubule inhibitors (Kreidieh et al., 2016). However, most chemotherapeutic drugs cause some adverse ill effects leading to the physical distress of the patients. More than often, patients develop intrinsic and/or acquired resistance to these synthetic drug doses, ultimately leading to drug ineffectiveness and MDR. For instance, dermal hyperpigmentation is frequently observed in patients when treated with alkylating agents such as cisplatin, carboplatin, and cyclophosphamide. Similarly, the administration of 5-FU and Doxil (liposomal doxorubicin) causes “hand and foot” in patients of BC (Rizeq et al., 2020). Researches reveal women surviving BC are also prone to a higher risk of developing unrelated cancer, known as “second cancer” as a fatal side effect of chemotherapy. It might have its origin in the same breast in the case of BC survivors who underwent a lumpectomy, or in the opposite breast for those who have undergone mastectomy. The frequently developed second cancer, as a result of chemotherapeutic stress in BC patients, is acute myelogenous leukemia (AML). Alarmingly,

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FIGURE 16.1 The various adverse effects encountered in BC patients undergoing neoadjuvant and adjuvant chemotherapy.

chemotherapy-induced myeloid leukemia has a cure rate of only 10%, being highly resistant to subsequent chemotherapies (Rheingold et al., 2003). Another major disadvantage of conventional therapeutic approaches is the cancer relapse, which is more frequently observed in TNBC. Chemotherapeutic approaches alone are not enough to evict all the CSC existing in the TME, stagnating as a minimal residual disease (MRD) (Doll & Peto, 1981). The financial burden encompassing BC treatment through chemotherapy also plays a major role in contributing to the annual cancer-related mortality, especially in the second- and third-world countries. Fig. 16.1 depicts the various adverse effects encountered in BC patients undergoing neoadjuvant and adjuvant chemotherapy.

16.3.2 Shortcomings of conventional therapy: radiotherapy Radiation therapy is one of the most utilized treatment paradigms in BC conventional therapeutics. The advent of radiotherapy in the field of modern oncology took place as early as 1900, immediately following chemotherapy. It represents a trustworthy multidisciplinary cancer therapeutic paradigm, which involves using high-energy radiation particles for killing aggressive malignant cells, with minimal disturbance to the neighboring healthy cells in the surrounding (Provision and use of radiotherapy in Europe, 2022). Radiation therapy aids in preventing cancer metastasis by reducing the tumor size. Often used in combination with chemotherapy in managing BC, radiation therapy helps to control distant metastasis in BC patients, limiting the migration of the tumor cells, to the surrounding lymph nodes, brain, lungs, and bones. It is usually given to the BC patients either after mastectomy or breast-conserving surgery (BCS), as adjuvant therapy, over a specific period post-surgery, depending on patient-specific physiological needs. Of the two types of radiation therapy, used in BC patients, external beam radiation therapy (EBRT) and brachytherapy, the former is the most commonly used. It involves directing a radiation beam at the site of surgery, to permanently evict any remnants of malignancy. But this radiation-based therapeutic paradigm is often accompanied by a plethora of side effects, including fatigue, skin darkening, localized swelling in the breast, development of lymphedema and seroma, breast pain, damage to the adipose tissue of the breast, and weakening of ribs (Radiation for breast cancer, 2022). Moreover, often the tumor cells acquire resistance and become insensitive to radiation therapy. The principal causes of MDR developed due to radiotherapeutic approaches involve the development of a hypoxic environment in the intratumoral TME, and aberration of the major glycolytic and mitochondrial signaling pathway, which leads to reduced formation of reactive oxygen species (ROS) (Liskova et al., 2021). This restores the longevity of the damaged DNA of the malignant cells, ensuring their renewal of stem-cell-like properties. Hence, there lies a pressing urgency, to discover novel inexpensive therapeutic avenues to overcome the toxic side effects and MDR that frequently move hand-in-hand with conventional tumor treatment procedures. Fortunately, the majority of these CAM-based chemotherapeutics and chemopreventive strategies are inspired by the natural phytochemicals, obtained from the plants (Rizeq et al., 2020).

16.3.3 Shortcomings of conventional therapy: hormone therapy (endocrine therapy) In addition to chemo and radiation therapy, another targeted adjuvant therapy widely used in BC treatment for hormone-sensitive patients is hormone therapy or endocrine therapy. It is generally rendered to patients before or post

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the completion of the chemotherapeutic courses. Currently, the endocrinal drugs commonly used in BC therapeutics are subdivided into three categories: (1) aromatase inhibitors (AIs), which reduce estrogen secretion, by inhibiting the activity of aromatase, (2) selective estrogen receptor modulators (SERMs), which inhibit estrogen activity, and (3) selective estrogen receptor downregulators (SERDs) which bring about ubiquitination and destabilization of ER. Despite the therapeutic success of endocrinal therapy, in preventing cancer relapse, and prolonging life expectancy, hormone-positive BC often develops resistance to this endocrine therapy (Fan et al., 2015). The major cause of the development of MDR in ER-driven antiestrogen endocrine therapy is attributed to the loss of expression of ER or mutations in the hormone receptor. Loss of expression of the receptor protein is mainly due to some methylation changes occurring in the epigenome of ER, being reported only in a minor group of ER-positive BC patients. Mutations affecting ER expression pattern are mainly due to point mutations and deletion, bringing about a random inherited change in the primary transcriptional signaling pathways governing ER expression (Estrogen receptor expression in benign breast epithelium and breast cancer risk - PubMed, 2022). Moreover, half of the patients with ER-positive breast cancer lack the progesterone receptor (PR). The absence of PR often contributes to the metastatic spread of the disease along with the enhanced level of HER2 expression via upregulation of the PI3k/Akt/mTOR pathway, with subsequent loss of ER expression and function. Since ER 1 /PR 1 BC was found to be more therapy sensitized than ER 1 /PR-BC, loss of PR in Luminal A BCs is considered to be a major prognostic indicator in assessing the outcome of the endocrine therapy (Tovey et al., 2005). Thus, addressing the insensitivity of the PR tumors in Luminal A BCs and their enhanced correlation with HER2 activity will pave a novel avenue in addressing therapy-induced resistance in patients with BC. Development of MDR, loss of sensitization of cancer cells to endocrine therapy, and limited applicability in hormone-refractory and metastatic HER2-positive BC are the major blockages encountered in endocrine therapy (Fan et al., 2015). Such limitations encountered in all the conventional therapeutic approaches of BC thus indeed call for the mining of a novel, less toxic branch of cancer therapeutics.

16.4

Role of complementary and alternative medicine (CAM) in breast cancer treatment

Owing to the numerous physical, psychological, and physiological stresses induced by conventional cancer therapeutic approaches, “complementary and alternative medicine” (CAM) is emerging as a consolidated healthcare alternative in treating BC patients (Wang et al., 2018; Buzyn, 2014). According to the National Center for Complementary and Alternative Medicine, CAM constitutes any practices, products, or systems of the healthcare fraternity which does not belong to the conventional or preliminary line of medical diagnosis (Molassiotis et al., 2006). CAM includes a broad spectrum of treatment approaches, including the use of aromatherapy, herbal medicines, homeopathy, spiritual and relaxational retreats, dietary phytochemicals, chiropractic therapy, and reflexology (Rizeq et al., 2020; Trends in the use of complementary health approaches among A, 2022). Moreover, CAM has been subdivided into two categories, by the National Center for the Complementary and Integrative Health (NCCIH): (1) the branch of CAM dealing with “mind and body practices” which includes meditation and yoga, aromatherapy, use of essential oils, chiropractic and reflexology therapies and (2) the use of plant-derived natural metabolites—such as alkaloids, carotenoids, polyphenols, flavonoids, herbs, and probiotics (Paepke et al., 2020). Dietary and behavioral elements have been identified by several clinical and epidemiological trials as the key contributors to regulating the prevalence and risk of many cancers, including BC. This has led to enhanced curiosity in exploring numerous plant-derived secondary metabolites and phytochemicals as an established enriched resource of CAM (Rizeq et al., 2020). CAM is not predicted to replace or substitute the primary line of cancer adjuvant diagnosis completely. But statistics show an inevitable use of CAM approaches among cancer patients, owing to its ability in rendering a post-cancer diagnostic disease-free life, reducing the perils of post-surgery complications, and benefitting the overall well-being of the individual (Trends in the use of complementary health approaches among A, 2022; Calcagni et al., 2019). Circumstantially, BC contributes to more than half of the cancers in the entire population of Western countries. Reports suggest that patients with BC utilize CAM more than patients suffering from any other cancers such as prostate cancer, colorectal cancer, etc. About 50% of women confirmed the use of CAM when diagnosed with BC, as an alternative healthcare well-being strategy (Wanchai et al., 2010). It has been surveyed that the rate of CAM use among women diagnosed with BC is presently as high as 90%. In a population of women undergoing radiotherapy, about 90% of them have succumbed to CAM, as a means of overcoming radiotherapeutic ills (Micke et al., 2009). CAM thus a sophisticated, mostly post-surgery treatment approach is found to be a choice more prevalent among the younger, educated population of financially affluent BC patients, undergoing comparatively less mainstream medical consultation postadjuvant therapy (Paepke et al., 2020).

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Phytochemicals present in recommended combinations in diet or as medicines have been observed to prevent the risks of an onset of BC, as well as help in better recovery in patients, post-chemo and radiation therapy. Cancer, in general, is attributed to uncontrolled cell division, which results from aberrant mutations in the cells as well as changes in the epigenome, which brings about a collapse in the general transcriptional mechanisms of the major tumor biomarker pathways (Hardy & Tollefsbol, 2011). Substantial reports suggest that bioactive phytochemicals, when included in the dietary regimen of a BC patient, bring about positive changes in the epigenome, altering the dysregulated state of the epigenetic targets and their molecular pathways. Hence, an epigenetic diet delineates the chemotherapeutic effects of the phytochemicals in combating tumor proliferation and spread. Interestingly, discovered in the post-genomic era of medicine, nutrigenomics is an emerging new branch in the field of oncogenic chemoprevention, which arose from an amalgamation of genomics and nutritional dietary alternatives (Sellami & Bragazzi, 2020). Advancements in the field of nutrigenomics, therefore, represent a promising opportunity, to design patient-specific personalized dietary regimens for women with a higher risk of BC or diagnosed with the disease, by decoding the different molecular mechanisms associated with every stage of BC tumorigenesis and its subtypes, in the light of epigenetic interactomics (Burdge et al., 2012).

16.5

Phytochemicals: traversing a new window in breast cancer therapy

Researches reveal that around 10% of cancers are only caused by predispository hereditary factors and the remaining 90% being attributed to several acquired and epigenetic risks. Free radicals and ROS are potent carcinogens present in abundance in our food and surroundings. Hence, it is of primary importance to maintain an equilibrium between the detoxification pathway of our bodies and the number of dietary toxins we are daily intaking. A conscious change in diet might thus reduce one significant element of the acquired risk factor of carcinogenesis (Anand et al., 2008). Thus, the idea of chemoprevention in the light of phytochemical-based therapeutics is emerging as a breakthrough in the field of gynecological oncology. The success of these natural dietary phytochemicals is owed to their ability in suppressing the oncogenic expression of several molecular targets and restoring the latter’s normal cell signaling abilities, validated through many ongoing preclinical analyses (Critical reviews in food science and nutrition, 2022). The expenses of cancer treatment are often unaffordable and financially inaccessible to several low-income and mediocre section of the society, who ultimately succumbs to the satanic grip of tumorigenesis. On the contrary, compared to the conventional oncogenic therapeutic approaches, the plant-derived secondary metabolites offer a novel treatment regimen, which is inexpensive and less toxic in its mode of activity (Prostate cancer management: long-term beliefs epidemic developments in the early twenty-first century and 3PM dimensional solutions - PubMed, 2022). This noninvasive method of anticancer paradigm thus appears to be promising in preventing the progress of carcinogenesis and also relaxes the major economic concern in the global healthcare society (Jain et al., 2021). Owing to the lack of targeted therapy and absence of the endocrine receptors, TNBC is the most aggressive molecular subtype of BC, accounting for a higher rate of recurrence and mortality. So researchers are actively searching for some natural compound-based treatment alternatives specifically for TNBC. Among thousands of phytochemicals screened for chemotherapeutic efficacy in TNBC cell line, commonly used MDA-MB-231 and BT549, some showed promising results in preliminary preclinical and clinical studies (Mitra & Dash, 2018). Among the commonly available compounds already reported to be effective against TNBC, resveratrol and curcumin showed potent tumor inhibitory properties. The catechin compound epigallocatechin-3-gallate (EGCG) suppresses the malignant invasion and selfrenewal property of the cancer cells within the TME. Carnosol, a diterpene on the other hand, controls tumor growth and metastasis by promoting ROS-mediated apoptosis (Carnosol induces ROS-mediated beclin1-independent autophagy and apoptosis in triple negative breast cancer, 2022). Hence, the discovery of these antineoplastic properties in these natural compounds raises the hope of developing a parallel less-toxic therapeutic armamentarium to combat TNBC. In this literature, several plant-derived dietary compounds, along with their mode of action and potential in different subcategories of BC therapeutics, are discussed, to enable further futuristic studies.

16.5.1 Alkaloids Alkaloids represent a diverse group of plant-derived secondary metabolites, acting as a major mine of drug discovery. They are the nitrogen-containing cyclic phytocompounds, mainly retrieved from green plants, predominantly found in the members of Leguminosae, Ranunculaceae, Papaveraceae, Solanaceae, Menispermaceae, Amaryllidaceae, and Loganiaceae families. The most basic forms of alkaloids are derived from amino acids, whereas several others are also formed from the modifications of various other classes of secondary metabolites, including flavonoids, steroids, terpenoids, and carotenes. They possess a plethora of pharmaceutical activities such as anticancer, anti-inflammatory,

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antioxidant, and antinociceptive properties, making them excellent candidates for small-molecule inhibitors of several diseases (Mondal et al., 2019). Although alkaloids have no strict system of classification, they can further be subcategorized into protoalkaloids, true alkaloids, and pseudoalkaloids. Protoalkaloids are the ones, which are derived from the amino acids but lack a nitrogen-containing heterocyclic ring, and examples include hordenine, colchicine, ephedrine, jurubine, taxol, mescaline, and erythromycin, whereas the alkaloids directly derived from the amino acids, with a basic nitrogen atom and a heterocyclic ring, are known as true alkaloids. Based on variation in the structure of the heterocyclic ring, true alkaloids are further classified into 14 subgroups—quinolone, isoquinoline, quinolizidine, pyrrolizidine, pyrrolidine, piperidine, acridine, tropone, indolizidine, oxazole, benzopyrrole, piperidine, imidazole and pseudoalkaloids (Therapeutic value of steroidal alkaloids in cancer, 2022). Lastly, the pseudoalkaloids are the ones that are derived from the structural modification of the groups of secondary metabolites, and not directly from the amino acids, but they contain the nitrogen-containing heterocyclic ring, for example, solanidine and delphidine (Shin et al., 2018). Among the three subclasses of the alkaloids, true alkaloids are abundantly available in nature. Alkaloids are the most prominent components present in herbaceous plants, many of which have already been implemented as potent chemotherapeutic drugs (Lu et al., 2012). Vinca alkaloids comprising vinblastine, vincristine, vindesine, vinflunine, and vinorelbine, extracted from the roots of Catharanthus roseus, belonging to the family Apocynaceae represent a significant subvariety of alkaloids in cancer treatment. Of the vinca alkaloids mentioned, vincristine and vinblastine are of primary importance in oncological pharmaceutics. Their preliminary role as an antimitotic microtubule inhibitor contributes to their antiproliferative efficacy in BC therapeutics (Mechanistic evaluation of phytochemicals in breast cancer remedy: current understanding and future perspectives, 2022). Table 16.3 enlists the different members of the alkaloids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. TABLE 16.3 The different members of the alkaloids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Phytochemical name

BC cell line

Mechanisms of action

References

Berberine

MCF-7 and MDA-MB-231

Promotes apoptosis by downregulating the level of metadherin and Bcl-2 levels.

Sun et al. (2019)

Evodiamine

MCF-7 and MDA-MB-231

Inhibits tumor proliferation by promoting Bax/Bcl-2 and caspase-7 regulated apoptosis and cell cycle arrest at G0/G1 phase.

Takeshima et al. (2014)

Piperine

MCF-7, T47D, and MDA-MB-231

Prevents the growth and renewal of CSCs. Promotes apoptosis by G2/M phase cell cycle arrest, and Bax/Bcl-2 modulation inhibits metastasis by downregulating MMP2 and MMP-9 expression.

Cancer chemoprevention and piperine: molecular mechanisms and therapeutic opportunities - PMC (2022)

Tetrandrine

MDA-MB-231

Prevents tumor growth by promoting apoptosis along with downregulation of the mTOR/PI3k/AKT signaling pathway.

Luan et al. (2020)

Sanguinarine

MDA-MB-231

Prevents metastasis and EMT in BC cancer cells, by downregulating the MMP-2/9 expression and HIF-1α/TGF-β signaling pathway.

Ghauri et al. (2021)

Lycorine

MDA-MB-231

Inhibits tumor growth by targeting the STAT-3 signaling pathway, inhibits metastasis by downregulation of MMP2 and MMP-9 expression, and promotes cellular apoptosis through upregulation of Bax.

Wang et al. (2017)

α-Solanine

On mice mammary model

Prevents tumor proliferation, metastasis, and angiogenesis by downregulating the VEGF and mTOR/ AKT signaling pathway, reducing expression of MMP-2 and MMP-9, respectively.

Mohsenikia et al. (2016)

Piplartine (piperlongumine)

MDA-MB-231

Promotes apoptosis by inhibiting PI3K-Akt/mTOR and JAK2-STAT3 signaling pathways.

Chen et al. (2019)

Tryptanthrin

MCF-7

Inhibits doxorubicin-induced multidrug resistance in BC by downregulating the ABC transporter gene MDR1.

Yu et al. (2007)

Quinoline

MCF-7

Promotes apoptosis by S phase cell cycle arrest through DNA intercalation.

Iqbal et al. (2019)

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The antitumor efficacy of vincristine is exhibited in the TNBC cell line MDA-MB-231 and Luminal A cell line MCF-7, where a mixture of Thymus caramanicus and vincristine induces apoptosis of the malignant cells by promoting the expression of caspase-3 with subsequent downregulation of the cyclin D molecule. Similar results were obtained when a combined mixture of vincristine and Satureja khuzestanica was administered in the MCF-7 cell line which showed caspase-mediated induction of apoptosis (Molecular docking prediction and in vitro studies elucidate anticancer activity of phytoestrogens, 2022). Moreover, in an in vivo administration of a liposomal combination of vincristine and dasatinib in the TNBC cell line, MDA-MB-231, inhibition of stem cell renewal was noted. The liposomal combination of the drugs also induces caspase-dependent induction of apoptosis, through caspases 3, 8, and 9, accompanied by subsequent elevation of ROS synthesis, and proapoptotic factors, Bax and Mcl-1 (Zeng et al., 2015). Vinblastine, on the other hand being a potent microtubule inhibitor, prevents tubulin-induced microtubule polymerization and angiogenesis, in the luminal BC cell line MCF-7. The compound is reported to promote apoptosis in the docetaxel-resistant BC cell line of MCF-7, by enhancing PARP cleavage and interrupting tubulin polymerization. Additionally, the apoptosis-inducing effect of vinblastine was also observed in MDA-MB-231, where a combination mixture of docetaxel and vinblastine is observed to induce apoptosis through downregulation of BIRC5-encoded survivin, a potent antiapoptotic factor (Sensitivity of docetaxel-resistant MCF-7 breast cancer cells to microtubuledestabilizing agents including vinca alkaloids and colchicine-site binding agents, 2022). A similar result of synergistic promotion of apoptosis is also revealed in the administration of deguelin, vinblastine, and docetaxel, in TNBC. Therefore findings reveal that survivin downregulation may be an optimistic mechanism of alkaloid-induced apoptosis in BC therapeutics. Thus, we see alkaloids play an essential role in BC chemoprevention and therapeutics via modulating the apoptosis pathway in tumor carcinogenesis (Choudhari et al., 2019).

16.5.2 Terpenoids Terpenoids, commonly known as terpenes, are the most abundant phytochemicals, produced by different living organisms such as plants, bacteria, yeast, and fungi. Composed of isoprene units (C5H8)n, terpenoids are known to play pioneer roles in regulating several molecular and biochemical mechanisms in various cellular entities (Siraj et al., 2021). Although produced from different living sources, green flowering plants are the major reservoirs of terpenoid production. Yet 80,000 terpenoids are reported from the green plants alone, with new compounds being added every year annually (Pichersky & Raguso, 2018). The chemical structure of terpenoids is composed of two 5-C building blocks of isoprenes (C5H8)n, where n denotes the number of isoprene units present. Based on the number of isoprene building blocks, terpenoids are classified into monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids (C25), triterpenoids (C30), tetraterpenoids (C40), and polyterpenoids (Rabi & Bishayee, 2009). The number of isoprene units present in mono-, di-, tri-, and tetraterpenoids is found to be 2, 4, 6, and 8, respectively (Siraj et al., 2021). These organic hydrocarbons are important sources of secondary metabolites and possess significant pharmacological properties, including potent anticancer, antioxidant, anti-inflammatory, antiparasitic, antimicrobial, and anti-immunomodulatory activities. Hence, epidemiological reports suggest that intake of a terpenoid-enriched diet of fruits, pulses, whole grains, and green leafy vegetables reduces the risk of several life-threatening diseases including tumor malignancy (Rabi & Bishayee, 2009). Among the different groups of isoprene compounds reported from plants, monoterpenoids and triterpenoids have the most validated chemotherapeutic and chemopreventive properties, as observed in several preclinical studies. The chemopreventive activities of the monoterpenoids are greatly elucidated in several cancers including the breast, lungs, pancreas, colon, prostate, and liver carcinomas (Kris-Etherton et al., 2002). Derivatives of the isopentyl phosphate oligomers, the triterpenoids comprise the biggest group of plant secondary metabolites, consisting of about 20,000 reported natural compounds (Liby et al., 2007). The chemopreventive activities of the triterpenoids are exhibited by their ability to regulate the different stages of carcinogenesis in breast, colon, skin, and prostate cancers. Ongoing phase I and II clinical trials reveal the inhibitory effect of triterpenoids on tumor growth, proliferation, and the onset of metastasis. Triterpenoids promote apoptosis of the malignant cells, by regulating the expression of a number of epigenetic and transcriptional signaling factors, without exerting any harmful effect on the healthy cells in the tumor surroundings (Burke et al., 2002). The plethora of chemotherapeutic activities of terpenoids thus has ignited much interest in their commercial and economic availability. Table 16.4 enlists the different members of the terpenoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Terpenoids thus exhibit various biological chemopreventive activities, which are far better with little or no atrocious side effects, as compared to any single synthetic chemotherapeutic drug. The chemotherapeutic property of terpenoids is attributed to their wide array of action mechanisms, such as the promotion of apoptosis, reduction of oxidative stress, inducing epigenetic silencing of genes, bringing about the cell cycle arrest, inhibiting malignant cell proliferation and

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TABLE 16.4 The different members of the terpenoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Phytochemical name

Class of terpenes

BC cell line

Mode of action

References

Withaferin-A

Triterpene

MCF-7

Promotes apoptosis by downregulating the concentration of cyclin D1 and NF-κB signaling.

Mohan et al. (2004)

Farnesol

Sesquiterpenes

MCF-7

Inhibits tumor growth by estrogen receptor downregulation via modulating farnesoid-X receptor-mediated signaling pathway.

Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptormediated estrogen receptor activation (2022)

Asiatic acid

Triterpene

MCF-7 and MDAMB-231

Promotes apoptosis by cell cycle S/G2 checkpoint arrest.

Rabi and Bishayee (2009)

Perillyl alcohol and d-limonene

Monoterpene

MCF-7, T47D, and MDA-MB-231

Promotes apoptosis and tumor proliferation by G0/ G1 transition and cyclin D1 downregulation.

Bardon et al. (1998)

Betulinic acid

Triterpenes

MCF-7

Promotes apoptosis by upregulating pro-apoptosis inducing Bax and downregulating cyclin D1 and Bcl-2.

Rabi and Bishayee (2009)

Ginsenoside Rk1

Triterpene

MDA-MB-231

Inhibits tumor invasion by MMP-2 downregulation, induces cell cycle arrest at M0/ M1 phase, enhances p53 and p21 expressions, decreases cyclin D1 and CDK4 regulation, and increases the production of caspases 3, 8, and 9 and Bax regulation, with a subsequent decrease in Bcl-2 expression.

Koh et al. (2020)

Pristimerin

Triterpene

MDA-MB-231

Promotes apoptosis by Bax/Bcl-2 modulation.

Rabi and Bishayee (2009)

Oridonin

Diterpene

MCF-7 and MDAMB-231

Promotes autophagy and apoptosis by upregulating Bax expression and ROS-regulated Nrf2 signaling pathway.

Cui et al. (2007a)

Actein

Triterpene

MDA-MB-231

Inhibits tumor metastasis by downregulating the EGFR, NF-κB, and AKT signaling pathways.

Wu et al. (2018)

Vitamin E succinate

Tetraterpene

MCF-7 and MDAMB-435

Inhibits tumor growth and metastasis by promoting apoptosis.

Rabi and Bishayee (2009)

Ganoderic acid

Triterpene

MDA-MB-231

Inhibits tumor invasion by downregulating CDK4 expression and altering the NF-κB signaling pathway.

Jiang et al. (2008)

Ursolic acid

Triterpene

MCF-7 and MDAMB-231

Promotes apoptosis by G0/G1 cell cycle arrest.

Yin et al. (2018)

Geraniol

Monoterpene

MCF-7

Inhibits tumor growth by G1 phase cell cycle arrest.

Bardon et al. (1998)

Gypenosides

Triterpene

MCF-7 and MDAMB-231

Suppresses malignant invasion by G0/G1 cell cycle arrest by downregulating the transcription factor E2F1.

Zu et al. (2021)

Excisanin A

Diterpene

MDA-MB-468 and MDA-MB-231

Inhibits metastasis by downregulating MMP-9 and MMP-2 expression and prevents tumor migration by deregulating the AKT signaling pathway.

Excisanin A et al. (2022)

Andrographolide

Diterpenoid

MCF-7, T47D, MDA-MB-231, and BT549

Prevents angiogenesis and metastatic invasion by downregulating COX2 expression and VEGF signaling.

Peng et al. (2018)

Cucurbitacin-B

Tetracyclic triterpene

MCF-7 and MDAMB-231

Inhibits tumor cell proliferation by induction of apoptosis, G2-M phase cell cycle arrest, and degradation of the microtubule polymerization.

Duangmano et al. (2012)

Lupeol

Pentacyclic triterpene

MDA-MB-231

Inhibits tumor proliferation and metastasis by downregulation of MMP-2 and AKT signaling.

Screening of phytochemicals as potential inhibitors of breast cancer using structure based multitargeted molecular docking analysis (2022)

Carnosol

Diterpene

MDA-MB-231

Promotes ROS-mediated apoptosis along with downregulation of Bcl-2 expression and inhibits malignant cell migration by G2 phase cell cycle arrest.

Carnosol induces ROS-mediated beclin1independent autophagy and apoptosis in triple negative breast cancer (2022)

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inflammation, and modulation of several cellular signaling pathways (Thoppil & Bishayee, 2011). Since the incidence of BC has always been on the rise, terpenoid-containing dietary regimens appear to be a promising strategy to prevent the incidence, relapse, and spread of the disease. Asserting the results obtained from preclinical and phase I/II clinical trials, the chemopreventive role of terpenoids should be further validated in (Thoppil & Bishayee, 2011) in vivo animal and human tumor models, to assess the chemotherapeutic performance of these dietary elements on a more practical and translational basis.

16.5.3 Flavonoids Flavonoids are the largest diverse subgroup of polyphenolic secondary metabolites produced by green plants, found abundantly in fruits, leafy vegetables, medicinal plants, and nuts. The basic 15-C structural framework of flavonoids consists of two benzene rings and one heterocyclic ring connected by a pyrane carbon bridge (Liskova et al., 2021). Based on the structural differences, flavonoids are further subcategorized into different subclasses based on their structural disparity (Fig. 16.2) (Shin et al., 2018). The antioxidant, anti-inflammatory, antimetastatic, proapoptotic, and antiangiogenic properties of flavonoids enable their efficacy as potential antineoplastic agents. The majority of flavonoids execute their anticancer activity through the induction of tumor cell death by apoptosis and inhibition of malignant invasion and metastasis of the tumor cells (Ranjan et al., 2019). Table 16.4 summarizes the activities of different classes of flavonoids in tumor suppression in BC (Table 16.5). Despite the preclinical excellency of flavonoids as antiproliferative agents, their success in clinical trials is still undergoing critical evaluation and research. Increased clinical trials should be executed in humans using the flavonoid compounds in sole composition or with chemotherapeutic adjuvants in larger samples, to ligate the gaps in flavonoid research, and their application as potential cancer therapeutic agents (Jain et al., 2021). Table 16.4 enlists the different members of flavonoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC, while Fig. 16.2 depicts different subclasses of flavonoids and the primary members representing each of them.

16.5.4 Carotenoids Carotenoids are tetraterpenoid lipophilic photochromatic pigments found to be present in several groups of green plants, cyanobacteria, and fungi (Carotenoids modulate the hallmarks of cancer cells
ScienceDirect, 2022). The red, orange, and yellow color of various fruits, flowers, and vegetables is attributed to the presence of the carotene pigments in their chromoplast. To date, approximately, 600 such lipophilic pigments have been identified, of which 20 are present in human tissues alone. As carotenoids are not synthesized by human and animal sources, the main source of carotenoids in the human body is through the intake of carotene containing leafy vegetables and fruits. Since carotenoids are lipophilic, they have the same absorption mechanism as that of diet fat (Cazzonelli, 2011). FIGURE 16.2 The different subclasses of flavonoids and the primary members representing each of them.

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TABLE 16.5 The different members of flavonoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Name of the flavonoid

BC cell line

Mode of action

References

Quercetin

MCF-7

Prevents the proliferation of tumor cells by inhibiting the expression of the TF twist, downregulating the MAPK pathway, and induces cytochrome-mediated apoptosis by enhancing the expression of Bax and caspase-3. It also prevents CSC renewal by inhibiting the Notch, Sonic hedgehog, and Wnt/β catenin pathway.

Ezzati et al. (2020)

Rutin

MDA-MB-231

Induces apoptosis in TNBC cells, by enhancing the expression of caspases 3, 8, and 9.

Perk et al. (2014)

Phloretin

MCF-7 and MDA-MB-231

Inhibits tumor proliferation and improves chemosensitization of TNBC cells by preventing cytoprotective autophagy.

Chen et al. (2021)

Kaempferol

Luminal A MCF-7

Prevents angiogenesis by downregulation of the VEGF signaling pathway and inhibits tumor proliferation by arresting G2/M phase cell cycle transition.

Kim and Choi (2013)

Nobiletin

MCF-7, MDA-MB-435, and MDAMB-231

Induction of apoptosis by upregulation of TSG P-53, caspase-3, and Bax/Bcl-2 and reduces the migration of tumor cells by NF-κB and Nrf2 pathways.

Liu et al. (2018)

Fisetin

MDA-MB-231

Inhibits metastasis and reverses EMT through downregulation of the PTEN/Akt/GSK3β pathway.

Li et al. (2018a)

Procyanidin

MCF-7 and MDA-MB-231

Induces apoptosis by upregulation of Bax and downregulation of Bcl-2 expression.

Gao and Tollefsbol (2018)

Glycitein

SKBR3 and MDA-MB-231

Prevents tumor migration and invasion through downregulation of MMP-8 and MMP-13 expression.

Zhang et al. (2015)

Genistein

MCF-7 and MDA-MB-231

Promotes tumor suppression through downregulating Akt and NF-κB signaling pathways and cell cycle arrest at G0/G1 and G2/M transition phase.

Rizeq et al. (2020)

Wogonin

MDA-MB-231

Prevents metastasis and invasiveness by downregulating the expressions of MMP-9 and IL-8.

Go et al. (2018)

Delphinidin

SKBR3

Induces autophagy and apoptosis by downregulating the mTOR and activation of the AMPK signaling pathway.

Chen et al. (2018)

Morusin

MCF-7 and MDA-MB-231

Induces apoptosis by decreasing the secretion of survivin, upregulating the expression of Bax protein, inhibits tumor proliferation by downregulating STAT3 signaling.

Kang et al. (2017)

Luteolin

MDA-MB-231

Induces apoptosis by inhibiting STAT3 expression, and prevents migration of malignant cells by downregulating Akt and ERK signaling pathways.

Luteolin inhibits breast cancer development and progression in vitro and in vivo by suppressing notch signaling and regulating MiRNAs
PubMed (2022)

Hispidulin

MCF-7

Reverses EMT by downregulating Smad 2/3 signaling pathway.

Kim and Lee (2021)

Chrysin

MCF-7 and T47D

Induces apoptosis by promoting telomerase shortening through inhibition of the activity of telomerase and inhibits malignant invasion by promoting downregulation of cyclin D1, CDK4, and CDK6 expressions.

Rasouli and Zarghami (2018)

Generally, a molecule of C40 isoprenoid forms the structural backbone of the carotenoid molecule, present in a tail
tail linkage comprising two C-20 units, which might contain an acyclic ring or a ring with diverse terminal modifications. Additionally, some lower group of organisms like bacteria also synthesizes partially degraded carotenoids, known as norcarotenoids or apocarotenoids, composed of C45 or C50 backbone moieties, formed by the addition of C40 (Carotenoids modulate the hallmarks of cancer cells
ScienceDirect, 2022). Carotenoids are further subcategorized into oxygendevoid carotenes, such as lycopene, α-carotene, β-carotene, and oxygen-rich xanthophylls, containing a minimum of one oxygen atom in their hydrocarbon chain, which includes fucoxanthin, zeaxanthin, and neoxanthin. The anti-inflammatory, antiproliferative, anti-immunomodulatory, and proapoptotic activities constitute the prime pharmacological functions of

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TABLE 16.6 The different members of the carotenoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Carotenoid name

BC cell line

Mode of action

References

Luteolin

1. MDA-MB-231

1. Inhibits tumor cell inhibition and angiogenesis through downregulating the expression of cyclin D1 and inhibiting the Notch and VEGF signaling pathway.

1. Sun et al. (2015)

2. MCF-7 3. BT-474 and T47D

2. Inhibits proliferation of the malignant cells, by downregulation expression of STAT3 protein and EGFR signaling pathway.

2. Luteolin promotes cell apoptosis by inducing autophagy in hepatocellular carcinoma (2017) 3. Sun et al. (2015)

3. Promotes apoptosis and prevents proliferation and angiogenesis of malignant cells by downregulating VEGF signaling and decreasing the expression of ALDH and CD44. Lycopene

1. MCF-7 2. MDA-MB-468

1. Inhibits malignant growth by cell cycle arrest at G1/S and G0/G1 stage and cyclin D downregulation.

1. Nahum et al. (2001) 2. Takeshima et al. (2014)

2. Arrests cell cycle regulation by inhibiting β-tubulin polymerization and CK19 and CK8/1 expression and induces apoptosis by enhancing PARP cleavage and Bax upregulation. Astaxanthin

MCF-7 and MDA-MB-231

Inhibits malignant cell migration and metastasis by downregulating MMP-2 and MMP-9 expressions, and inhibits neuronal death by modulating the Wnt/β catenin signaling pathway.

Antioxidants (2022)

β-carotene

MCF-7

Induces apoptosis by enhancing ROS generation and PPARγ activation

Cui et al. (2007b)

Violaxanthin

MCF-7

Induction of caspase-dependent early metastasis.

Pasquet et al. (2011)

Halocynthiaxanthin

MCF-7

Promotes apoptosis by enhanced DNA fragmentation and increased regulation of Bax, with subsequent downregulation of Bcl-2 expression.

Konishi et al. (2006)

Crocetin

MDA-MB-231

Inhibits malignant inhibition and metastasis, by downregulation of MMP-2 and MMP-9 expression, prevents tubulin polymerization, promotes upregulation of caspases 3, 8, and 9 expressions, and induces apoptosis by upregulating Bax/Bcl-2 ratio.

Hoshyar and Mollaei (2017)

carotenoids (Koklesova et al., 2020). Epidemiological studies reveal anticancer properties reported in various carotenoids like lycopene, lutein, β-carotene, α-carotene, neoxanthin, and violaxanthin in BC cells lines such as MDA-MB-231, T47D, BT-474, and MCF-7. The major mechanisms by which carotenoids mediate their antineoplastic role are through cell cycle arrest, immunomodulation, induction of apoptosis, inhibition of metastasis, and alteration of several growth and biochemical signaling pathways (Giani et al., 2021). Among the several lifestyle and epigenetic factors responsible for the incidence of BC, the dietary regimen of an individual plays a primary role in the prevention and control of the disease. Intake of antioxidants in the form of carotenoids in the diet is thus a significant step in embracing phytochemical-based chemotherapeutic prevention of BC (Sheu et al., 2010). Preclinical studies support the role of carotenoids in combating tumor-mediated cellular stress by alteration of several pro-tumorigenic pathways. Moreover, the carotenoids stabilize the oxidative unrest in the BC cells caused by several alkylating and platinum-based chemotherapeutic drugs (Giani et al., 2021). It quenches the acute oxidative thirst of the malignant cells by dWXZ ROS generation by the virtue of their strong anti-oxidation property (Lamson & Brignall, 1999). Hence, a combination of carotenoids along with the chemotherapeutic agents, promises a strong phytochemical-based CAM alternative in BC therapeutics. Table 16.6 enlists the different members of the carotenoids, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC.

16.5.5 Phytosterols and phytostanols Plant sterols are C28 and C29 derivatives of steroid alcohol, constituting the integral component of the cellular and plasma membrane of plants. They being unable to be synthesized within the human body is uptaken in the diet through lipid-rich plant food, particularly nuts and vegetable oil. The plant sterols generally exist in both sterol and stanol forms, where the bioavailability and the dietary significance of sterols exceed much higher than that of stanols (Jiang et al., 2019).

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The dietary absorption of phytosterols is 0.4%
3.5%, generally exceeding that of phytostanols which ranges from 0.02% to 0.3%. Globally, the daily intake of plant sterols in the diet is estimated to range between 200 and 400 mg, a figure that is almost equivalent to daily cholesterol uptake in humans. Although the consumption level of cholesterol and plant dietary sterols collide, there is a noteworthy difference in the level of absorption of these two categories of sterol compounds (Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet - PubMed, 2022). The poor absorption of the phytosterols in the human body is majorly attributed to their ABC transporter-mediated efflux from the cellular enterocytes, resulting in plasma sterol level of less than 1 , mg/dL in blood. But despite the reduced plasma-level concentration of plant sterols, they play significant roles in controlling several biological functions in the human body (Anticancer effects of phytosterols, 2022). Their ability to attenuate several biological and biochemical pathways establishes themselves as novel therapeutic alternatives for cancer treatment. The most commonly available dietary phytosterols include stigmasterol, campesterol, β-sitosterol, and campestanol, of which the presence of β-sitosterol is most significant. A number of phytosterols are reviewed to exhibit antineoplastic properties in BC, by modulating various cellular mechanisms, mainly by promoting apoptosis, inhibiting metastasis, and limiting the migration of malignant cells. β-Sitosterol induces apoptosis in hormone-insensitive TNBC cell line MDAMB-231, by upregulation of caspases 3, 8, and 9. The sterol is also reported to prevent metastasis in TNBC by downregulating the expression of proteins MMP-2 and MMP-9 (Jiang et al., 2019). Additionally, it prevents tumor proliferation by upregulating the expression of the TSG MiR-10A via downregulating the PI3k-Akt pathway (Xu et al., 2018). In MCF-7 and HER2-positive cell line T47D, stigmasterol is known to promote apoptosis by induction of oxidative stress (Sianipar et al., 2021). Additionally, another plant sterol, campesterol, is also known to reduce tumor cell proliferation and expansion by inhibiting cell cycle transition at the G2/M phase (Anticancer effects of phytosterols, 2022). The apoptotic and cell cycle arrest activity of phytosterols are mainly mediated through their ability to induce sphingolipid metabolism along with ceramide formation. Researches suggest that endocrinal and immunological responses mediated by plant sterols are also assumed to have antineoplastic effects on breast tumor growth and survival (Bradford & Awad, 2007).

16.5.6 Cardiac glycosides Cardiac glycosides are cardiotonic phytosterols, mainly obtained from green plants and other amphibian sources as well, having wide application in cancer and cardio remediations. The sugar component, glycone, and its steroid counterpart, aglycone, comprise the core structure of CGs. CGs have an α-pyrone or a butyrolactone ring and a sugar moiety, positioned at the β-17 and β-3, respectively (Prospects and therapeutic applications of cardiac glycosides in cancer remediation, 2022). The steroidal nucleus constitutes the aglycone subunit of the CGs, whereas the functional activity of CGs is attributed to the compound’s lactone ring. Fructose, glucose, mannose, galactose, and digitalose are the main monosaccharides found to be present mainly in the sugar moieties of CGs. The overall therapeutic potency of the CG is associated with the type of monosaccharide present in the sugar moiety (Liu et al., 2021). Based on the functional group present in β-17, CGs are classified into (1) bufadienolides and (2) cardenolides. They can be differentiated based on the six-membered α-pyrone ring and the five-membered butyrolactone ring present in bufadienolides and cardenolides, respectively (Plant-derived cardiac glycosides: Role in heart ailments and cancer management
PubMed, 2022). The CGs are mainly confined in the angiosperm families, Crassulaceae, Fabaceae, Brassicaceae, Asclepiadaceae, Ranunculaceae, and Apocynaceae, with roots, barks, leaves, and stems being the plant parts having the most therapeutic values. Some of the widely reported CGs used in disease prevention and therapeutics include calotropin, lanatoside, thevetin, digitoxin, digoxin, bufalin, oleandrin, and ouabain (Kumavath et al., 2021). Despite the several therapeutic applications of glycosides, reported in cancer and other pathological treatments, the administration of this steroidal secondary metabolite is often restricted practically. Hormonal fluctuations with a consequent reduction in sexual libido, low bioavailability, and enhanced mortality due to toxic electrophysiological effects are some of the factors, limiting their practical clinical applications. Thus, bioactivity profiling of the CGs is suggested to improvise their safe therapeutic use, by quilting these applicatory loopholes of glycosides, in modern-day cancer treatment (Mladˇenka et al., 2018). Table 16.7 enlists different cardiac glycosides, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC.

16.6

Phytochemicals and ER(1) breast cancer

Luminal A or ER(1) BC constitutes about 80% of BC cases reported annually of which 65% are PR(1) (Lumachi et al., 2015). Endocrine therapy involving the use of aromatase inhibitors and selective estrogen receptor modulators (SERMs), such as tamoxifen, remains the main exploited option in ER(1) BC (Israel et al., 2018). The aromatase inhibitors such as

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TABLE 16.7 Different cardiac glycosides, which possess validated chemotherapeutic activities in the different malignant subtypes and cell lines of BC. Name of glycoside

BC cell line

Mechanism of action

References

Bufalin

MDA-MB-231

Inhibits tumorigenesis and promotes apoptosis by enhancing ROS generation through modulating the RIP1/ RIP3/PARP-1 pathways.

Li et al. (2018b)

Oleandrin

MCF-7, MDA-MB-231, and SKBR3

Induces mitochondrial-derived apoptosis, by promoting endoplasmic reticulum stress.

Li et al. (2020)

Ouabain

MCF-7 and MDA-MB-231

Antiproliferative action of malignant cells by downregulation of STAT3 expression and induction of apoptosis via enhanced production of caspases.

Du et al. (2021)

Proscillaridin

MCF-7 and MDA-MB-231

Prevents the growth of tumor cells by inhibiting DNA topoisomerase I and II activity.

Winnicka et al. (2008)

Lanatoside C

MCF-7, MDA-MB-231, and MDA-MB-435

Inhibits malignant cell growth by G2/M phase cell cycle arrest and downregulating Wnt/B, MAPK, and PI3k/AKT/ mTOR signaling pathway.

Reddy et al. (2019a)

Peruvoside

MCF-7, MDA-MB-231, and MDA-MB-435

Promotes autophagy and apoptosis through downregulating MAPK and PI3k/AKT/mTOR signaling pathways.

Reddy et al. (2020)

Strophanthidin

MCF-7

Induces apoptosis by G2/M phase cell cycle arrest and downregulating Wnt/B and PI3k/AKT/mTOR signaling pathways.

Reddy et al. (2019b)

Convallatoxin

MCF-7, MDA-MB-231, and MDA-MB-468

Induces apoptosis by G0/G1 cell cycle arrest, upregulation of Bax, and downregulation of Bcl-2.

Kaushik et al. (2017)

Digitoxin

MCF-7 and MDA-MB-468

Reduces tumor cell migration by cell cycle arrest at G0/G1 stage, enhances superoxide production, and induces apoptosis by downregulating the nuclear kappa-β pathway.

Kulkarni et al. (2017)

Digoxin

MDA-MB-231

Induces apoptosis by enhancing Bax expression and downregulating Bcl-2 expression level.

Anti-proliferative effect of digoxin on breast cancer cells via inducing apoptosis (2017)

letrozole, anastrozole, and exemestane prevent ER carcinogenesis by inhibiting estrogen synthesis. On the other hand, the SERM, tamoxifen, prevents tumor invasion and metastasis by inhibiting the binding of the endocrinal entities at the sites of the hormone receptor. Despite the therapeutic efficacy of endocrinal therapy, in enhancing the 5-year survival rate of ER(1) patients, cancer cells frequently develop MDR, which makes them insensitive to hormonal treatment. Hence, phytochemicals in combination with endocrinal and chemotherapeutic drugs are often implemented as promising antitumor therapeutic alternatives (Hormone therapy for breast cancer fact sheet
NCI, 2021). Several plant-derived bioactive molecules have shown a beneficial role in suppressing tumor growth in ER(1) cases. They mainly mediate their antineoplastic roles by blocking estrogen synthesis, thus acting as potent natural SERBs. Isoflavones, such as genistein and daidzein, inhibit metastasis by downregulating MMP-2 and MMP-9 and induce metastasis by G0/G1 cell cycle arrest. Additionally, genistein together with ipriflavone inhibits distant metastasis in bones, in the advanced stage of ER(1) BC (Lumachi et al., 2015). The alkaloid berberine also promotes apoptosis in ER(1) cells by upregulating the level of Bax and retarding the Bcl-2 expressions in the malignant cells. EGCG, a polyphenol extracted from green tea, also prevents angiogenesis in ER(1) BC cells, by downregulating the VEGF signaling pathway in the tumor cells. EGCG also promotes apoptosis in the MCF-7 cell line of BC, together with tamoxifen and other inhibitors of histone deacetylases. On the other hand, flavonoids such as kaempferol and quercetin also exhibit anticancer activities in a concentration-dependent manner. Kaempferol exhibits antiproliferative activity when administered at a concentration of 50
100 μM in MCF-7 cells, whereas induces tumor growth at a concentration lower than the optimum of 5
10 μM (Tao et al., 2015). On the other hand, carotenoids such as β-carotene and violaxanthin are also reported to induce caspase-dependent apoptosis and prevent early-stage metastasis through caspase upregulation of 3, 8, and 9, enhancing ROS generation and PARP cleavage. Secondary metabolites, such as curcumin, also show potent efficacy as antiproliferative agents by downregulating several cellular signaling mechanisms such as Wnt-β catenin, NF-κB, and mTOR-AKT signaling pathways (Israel et al., 2018). Along with the ones mentioned here, many other phytochemicals acting against the Luminal A cell line, MCF-7, are already discussed in the tables belonging to specific classes of bioactive molecules, in the previous sections of the chapter in which the majority of them are undergoing present clinical trials and bloom as potent therapeutic weapons, leading the future of the upcoming ER(1) BC therapeutic regiment.

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Phytochemicals and HER(2) breast cancer

HER2-positive BC is one of the aggressive molecular BC subtypes, which contributes to about 20% of all the BC cases reported annually, showing an enhanced expression of the human epidermal growth factor protein 2, as suggested by its name. Overexpression of HER2, a growth protein belonging to the epidermal growth factor receptor (EGFR) family, drives the massive proliferation of the tumor cells (Siddiqui et al.). Despite the scope of implementing targeted therapy, along with the conventional chemo and radiotherapy in the diagnosis of this disease, the mortality rate remains still alarming owing to the highly metastatic nature of these malignant BC cells. Although trastuzumab-based chemotherapy is the primary line of treatment in HER2-positive patients, owing to several drawbacks and ill effects of the chemotherapeutic regimen, a growing inclination toward administering phytochemicals as an axillary branch of HER2 is observed lately (Israel et al., 2018). Preclinical studies reveal that various phytochemicals act as natural tyrosine kinase inhibitors in metastatic HER2-positive patients. Grape polyphenol inhibits tumor growth in HER2-positive cells by downregulating the PI3K/Akt/mTOR pathway and elevating the expression of the protein AMP kinase (Castillo-Pichardo et al., 2009). Resveratrol, a phytoalexin, and a stilbenoid induce apoptosis in metastatic MDA-MB-453 cell lines by downregulating the NF-κB along with subsequent elevating of the TGFβ1 signaling pathway. On the other hand, kaempferol reduces metastasis in the HER2-positive cell line SKBR3, by suppressing MMP-2 and MMP-9 activity by modulating the MAPK pathway (Choi & Ahn, 2008). Zerumbone, a sesquiterpene, is reported to induce apoptosis in the metastatic cell line, through upregulation of Bax, with a significant decrease of Bcl-2 expression in the tumor cells (Siraj et al., 2021). The quassinoids, brusatol, is known to enhance the antiproliferative effect of trastuzumab, the primary drug in HER2-positive treatment in BT474 and SKVO3 by the inhibition of HER2-AKT/ERK1/2 and Nrf2/HO-1 signaling pathways (Nrf2 Inhibitor, 2022). The triterpene betulin deregulates the NF-κB and MAPK signaling pathway, thereby preventing the secretions of oncogenic chemokines TNF-α and IL-1 in the metastatic HER2 cells (Siraj et al., 2021). Thus, impressive inferences of the plant-derived compounds on the HER2 cell line indicate a better future in the prognosis and control of the metastatic HER2-positive BC, in combination with the synthetic chemotherapeutic derivatives.

16.8

Phytochemicals used for triple-negative breast cancer (TNBC)

The extreme invasiveness and highly metastatic nature of the TNBC cells are attributed to their molecular heterogeneity and basal phenotype, occurring as a result of the tumor
stromal interaction. Due to the absence of the three major hormone receptors in the TNBC cells, ER, PR, and HER2, conventional endocrinal and targeted therapy are inapplicable for TNBC diagnosis (Screening of phytochemicals as potential inhibitors of breast cancer using structure based multitargeted molecular docking analysis - ScienceDirect, 2022). The exclusive molecular heterogeneity of the TNBC subtype contributes to their distinct clinical characteristics, which often correspond to the molecular grade of the tumor. The commonly implemented therapeutic approaches in TNBC treatment include the use of chemotherapeutic drugs, such as anthracyclines, taxanes, and platinum-based agents, like cisplatin and carboplatin. Additionally, molecular signaling inhibitors such as angiogenesis inhibitors, EGFR inhibitors, PARP inhibitors, and tyrosine kinase inhibitors are some of the means of vague combinational therapy used to combat tumorigenesis in TNBC (Israel et al., 2018). But prolonged usage of these methods as antineoplastic alternatives in TNBC treatment corresponds to tumor relapse, loss of chemosensitization, development of MDR, and distant metastasis, in major organs like CNS, bones, lungs, and kidneys (Liu et al., 2019). Researches reveal several plant-derived bioactive compounds, to modulate the signaling pathways, mediate epigenetic alteration, amend miRNA expressions, reduce hypoxic stress, and induce apoptotic cell death in MDA-MB-231, the most commonly used TNBC cell line, which contributes to tumor growth, migration, and invasion in the humble cells (Anders & Carey, 2009). The use of these phytochemicals along with their adjuvant chemo and radiation therapeutic counterparts may lead to the improvisation of the worst prognosis in TNBC, to a degree better than what it is at present. Further investigations and translational research are thereby needed to gain a better insight into the biomechanism of these compounds as novel antineoplastic agents for this hormone-resistant BC subtype. Table 16.8 shows some plant metabolites that possess validated chemotherapeutic activities in MDA-MB-231 and MDA-MB-468 TNBC cell lines.

16.9

Role of phytochemicals in modulating noncoding RNA expression in BC cells

MicroRNAs (miRNAs) and the long noncoding RNA (lncRNAs) are two major subcategories of noncoding RNAs (ncRNA), which plays an important role in maintaining the physiological homeostasis of the body. Therefore expressional dysregulation of the ncRNA leads to the pathogenesis of several human diseases. Evidence suggests that the differential expression of the ncRNAs, especially the miRNAs, plays a major role in initiating tumorigenesis in the BC

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TABLE 16.8 Some plant metabolites that possess validated chemotherapeutic activities in MDA-MB-231 and MDAMB-468 TNBC cell lines. Phytochemical name

Phytochemical class

Mechanism of action

References

Berberine

Alkaloid

Promotes apoptosis by downregulating the level of metadherin and Bcl-2 levels.

Treatments used for triplenegative breast cancer (2022)

Piperine

Alkaloid

Prevents the growth and renewal of CSCs. Promotes apoptosis by G2/M phase cell cycle arrest and Bax/Bcl-2 modulation inhibits metastasis by downregulating MMP-2 and MMP-9 expression.

Li et al. (2011)

Tetradine

Alkaloid

Prevents tumor growth by promoting apoptosis along with downregulation of the mTOR/PI3k/AKT signaling pathway.

Luan et al. (2020)

Evodiamine

Alkaloid

Inhibits tumor proliferation by promoting Bax/Bcl-2 and caspase-7 regulated apoptosis and cell cyle arrest at G0/G1 phase

Takeshima et al. (2014)

Wogonin

Flavonoid

Prevents metastasis and invasiveness by downregulating the expressions of MMP-9 and IL-8.

Go et al. (2018)

Fisetin

Flavonoid

Inhibits metastasis and reverses EMT through downregulation of PTEN/Akt/GSK3β pathway

Li et al. (2018a)

Genistein

Flavonoid

Promotes tumor suppression through downregulating Akt and NFκB signaling pathways and cell cycle arrest at G0/G1 and G2/M transition phase.

Rizeq et al. (2020)

Phloretin

Flavonoid

Inhibits tumor proliferation and improves chemosensitization of TNBC cells by preventing cytoprotective autophagy.

Chen et al. (2021)

Crocetin

Carotenoid

Inhibits malignant inhibition and metastasis, by downregulation of MMP-2 and MMP-9 expression, prevents tubulin polymerization, promotes upregulation of caspases 3, 8, and 9 expressions, and induces apoptosis by upregulating Bax/Bcl-2 ratio.

Hoshyar and Mollaei (2017)

Lycopene

Carotenoid

Arrests cell cycle regulation by inhibiting β-tubulin polymerization and CK19 and CK8/1 expression induces apoptosis by enhancing PARP cleavage and Bax upregulation.

Aggarwal et al. (2009)

Astaxanthin

Carotenoid

Inhibits malignant cell migration and metastasis by downregulating MMP-2 and MMP-9 expressions and inhibits neuronal death by modulating the Wnt/β catenin signaling pathway.

Go et al. (2018)

Luteolin

Carotenoid

Inhibits tumor cell inhibition and angiogenesis by downregulating the expression of cyclin D1 and inhibiting the Notch and VEGF signaling pathway.

Li et al. (2018a)

Bufalin

Cardiac glycosides

Inhibits tumorigenesis and promotes apoptosis by enhancing ROS generation through modulating the RIP1/RIP3/PARP-1 pathways.

Li et al. (2018b)

Digoxin

Cardiac glycosides

Induces apoptosis by enhancing Bax expression and downregulating Bcl-2 expression level

Winnicka et al. (2008)

Digitoxin

Cardiac glycosides

Reduces tumor cell migration by cell cycle arrest at G0/G1 stage, enhances superoxide production, and induces apoptosis by downregulating the nuclear kappa-β pathway.

Kulkarni et al. (2017)

Carnosol

Terpenoid

Promotes ROS-mediated apoptosis along with downregulation of Bcl-2 expression and inhibits malignant cell migration by G2 phase cell cycle arrest.

Carnosol induces ROSmediated beclin1-independent autophagy and apoptosis in triple negative breast cancer (2022)

Lupeol

Terpenoid

Inhibits tumor proliferation and metastasis by downregulation of MMP-2 and AKT signaling.

Screening of phytochemicals as potential inhibitors of breast cancer using structure based multitargeted molecular docking analysis ScienceDirect (2022)

Andrographolide

Terpenoid

Prevents angiogenesis and metastatic invasion by downregulating COX2 expression and VEGF signaling

Peng et al. (2018)

Ganoderic acid

Terpenoid

Inhibits tumor invasion by downregulating CDK4 expression and altering the NF-κB signaling pathway.

Jiang et al. (2008)

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cells. The ncRNAs take part in tumor growth, proliferation, angiogenesis, EMT, and metastasis of the cancer cells. Hence, miRNAs and lncRNAs are considered strong oncogenic targets. The oncogenic phenotype of the tumor cells can be reversed by restoring the regulation of the miRNAs and the lncRNAs (Ruiz-Manriquez et al., 2022). The miRNAs which take part in oncogenesis are collectively known as onco-miRs. The level of onco-miRs is generally upregulated in the malignant cells, which targets the TSGs, bringing their downregulation in the tumor cells. The oncomiRs exert their tumor-promoting activity either by targeting a plethora of genes or by a single gene that can be dysregulated by numerous miRNAs (Key, 2011). Studies revealed several secondary metabolites derived from various plant sources which modulate the expression of these ncRNAs, thereby diluting cancer invasion and development (Saghafi et al., 2019). The major phytochemicals that are reported in cancer chemotherapeutics and modulating ncRNA expressions include flavonoids, terpenoids, polyphenols, and curcumins (Varghese et al., 2020). The natural compounds exert their antineoplastic effect by regulating a number of molecular mechanisms, of which epigenetic silencing of the differentially overexpressed oncogenes is gaining considerable importance. The plant-derived compounds alter the cancerpromoting epigenetic configuration by upregulating the miRNA of cancer-specific tumor-suppressor genes (TSGs), thereby restoring the nonmalignant phenotype (Chuang & Jones, 2007; Flabouraris & Karikas, 2016). Table 16.9 shows the phytochemicals derived from different classes of plant compounds that exert chemotherapeutic activity by restoring BC miRNA regulation. Studies concerning miRNA profiling in different molecular subtypes of BC reveal an uptight association of these ncRNAs with the different stages of progression of tumorigenesis. It helps in better predicting the therapy outcomes, rate of relapse, and the probable risks of developing chemoresistance in the targeted cancer cells (Varghese et al., 2020). For instance, an enhanced expression of the miRNA-155 indicates advanced stages of TNBC, whereas an upregulated expression of miRNA-93 echoes lymph node metastasis in the basal subtype of BC. On the other hand, the downregulation of the TSG miRNA 153 is associated with advanced stages of weak tumor prognosis (Litchfield et al., 2015). Hence, it is worth noting that miRNA-based studies are gaining traction, as potential therapeutic biomarkers in assessing BC outcomes and prognosis (Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer - PubMed, 2022). Advances in the field of oncology therapeutics identify the various plant-derived metabolites, to regulate the expression of the BC-associated miRNAs, aiding in the prevention of BC tumor proliferation, growth, and metastasis (Abbasi et al., 2018). The phytochemicals can bind directly to the target miRNAs or interact with the major effector proteins associated with the candidate miRNA. They may also modulate the expression of the miRNAs by targeting or modulating the expression of their host genes (Abbasi et al., 2018). Because a single miRNA can target multiple mRNAs, hence these dietary metabolites can exert their regulatory control over the tumor cells in various ways by dominating the host miRNAs (Kang, 2019). Additionally, it has also been accounted that the combined use of the miRNA targeting phytochemicals along with the conventional synthetic drugs enhances the latter’s cytotoxic tumor-killing therapeutic activity. But until lately, researches reveal that, due to their poor bioavailability and success in in vivo studies, the therapeutic efficacy of the dietary phytochemicals has been established, only on a constricted number of BC miRNAs, hinting that much work is yet to be done to investigate more such phytochemical-targeting miRNAs and decode their molecular mechanisms to aid in both BC CAM and in vivo clinical studies (Abbasi et al., 2018).

16.10 Phytochemical interventions in healing cancer-associated MDR In modern clinical practice, the most exploited diagnostic modality for breast cancer treatment is chemotherapy. But the antineoplastic effect of this widely used paradigm is often accompanied by some severe side effects. One of the major drawbacks of chemotherapeutic strategies encountered in BC diagnosis is the acquisition of MDR by the cancer cells, acquired during and/or after the course of the preliminary adjuvant therapy. MDR is a principal defense mechanism exhibited by the tumor cells in response to the different therapeutic strategies employed to curb their invasion and spread. The development of MDR ultimately leads to cancer relapse and widespread metastasis, proving to be fatal in about 90% of patients (Hamed et al., 2019). Resisting MDR is therefore an impending challenge in the field of oncogenic therapeutics (Tinoush et al., 2020). Attempts are thus being made to design strategies to nullify the cytotoxic effects of conventional synthetic drugs of chemotherapy and replaced them with substitutes possessing the least cellular toxicity and highest antineoplastic potency (Guo et al., 2016). Malignant cells exhibit many innate and acquired mechanisms of broad-spectrum cross-resistance to curb the therapeutic potency of the several antineoplastic methods predominating breast cancer treatment (Hamed et al., 2019). In tumor cells, acquired resistance develops post the incorporation of adjuvant chemotherapy, whereas innate resistance is inherent and retains even prior to the administration of the chemotherapeutic agents (Wang et al., 2019). Intrinsic or innate modes of resistance in MDR include (1) reduced

TABLE 16.9 Enlists the roles of different dietary phytochemicals in miRNA regulation in BC. Phytochemical name

Phytochemical class

Target miRNA

Mechanism of action

References

Quercetin

Flavonoid

Upregulates miR-183, miR-146a, miR-125a, miR-381, miR-19b, miR98, miR-106a. Downregulates miRNA27a

Promotes apoptosis, by upregulation of apoptosis-promoting factors Bax and caspase3 and downregulation of EGFR signaling pathway.

Tao et al. (2015)

Curcumin

Polyphenol

Upregulates miR-19a, miR-19b, miR-181b

Promotes apoptosis by downregulating the expression of the apoptosis blocker Bcl-2 and Bmi-1, which promotes self-renewal of CSCs.

Aggarwal et al. (2005)

Soy

Isoflavones

Downregulates miR-223, miR-155, and miR27a

Inhibits EMT, malignant cell growth, proliferation, and invasion and restores the aberrant Wnt signaling pathway.

Soy isoflavone genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein - PMC (2022)

Resveratrol

Polyphenol

Upregulates miR-125b-5p, miR-122-5p, miR409-3p, miR-200c-3p, miR-542-3p, miR-663, miR-744

Promotes apoptosis and repairs defects of the cell cycle mechanisms.

Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death (2022)

EGCG

Catechin

Upregulates miR-16 and miR-126

Inhibits tumor growth and promotes apoptosis. Prevents M2 polarization and deregulates TGF-β and IL-6-mediated protumorigenic signaling in malignant cells.

Jang et al. (2013)

A mixture of emodin and curcumin

Polyphenol

Upregulates miR-34a

Downregulation of apoptosis inhibiting Bcl-2 and Bmi-1.

Jobin et al. (1999)

Garcinol

Polyisoprenylated benzophenone

Upregulates miR-200 and let7

Reversal and inhibition of EMT. Also alters dysregulated Wnt signaling pathway.

Garcinol regulates EMT and Wnt signaling pathways in vitro and in vivo leading to anticancer activity against breast cancer cells - PubMed (2022)

Ellagic acid

Polyphenol

Upregulates miR-34c, miR-182, miR183, miR375, miR429, and miR196c. Downregulates miR335, miR127, miR122, miR206, and miR205.

Inhibits tumor growth by downregulating BCpromoting genes fox01, fox03a, and Bcl-2.

Munagala et al. (2013)

Diallyl disulfide

Organosulfur compound

Upregulates miR-34a

Inhibits tumor growth, invasion, and metastasis by Ras/ERK and SRC expression.

Xiao et al. (2014)

Genistein

Organosulfur compound

Downregulates miR-155

Inhibits malignant cell proliferation by modulating the expression of β catenin, fox03, and PTEN.

Soy isoflavone genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein - PMC (2022)

Sulforaphane

Organosulfur compound

Upregulated miR140 Downregulated miR21 and miR-29a

Prevents self-renewal of CSCs by ALDH1 and sox9 expression modulation.

Li et al. (2014)

3,30 Diindolylmethane

Indole compound

Upregulates miR-200b, miR-212, miR-200b, and miR-132

Suppresses malignant growth and metastasis by downregulation of sox4 and foxM1 genes along with modulating the Akt signaling pathway.

Holzapfel et al. (2017)

(Continued )

TABLE 16.9 (Continued) Phytochemical name

Phytochemical class

Target miRNA

Mechanism of action

References

Indole-3-carbinol

Indole compound

Upregulates miR-34a

Promotes apoptosis and inhibits tumor cell growth by upregulating p21 and p53 expression.

Hargraves et al. (2016)

Pomegranate polyphenols

Polyphenol

Downregulates miR-155 and miR-27a

Inhibits tumor cell invasion and proliferation by ZBTB10, PI3k, and VEGF expression modulation.

Banerjee et al. (2012)

Luteolin

Flavonoid

Upregulates miRNA-181a, miR-34a, miRNA-203, miRNA-224, miRNA139-5p, and miRNA-246. Downregulates miRNA-21 and miRNA-155.

Inhibits tumor growth and angiogenesis by downregulating Notch-1, PI3k/Akt, and VEGF signaling pathways, and decreasing cyclin D1 and MMP-2/9 concentration.

Luteolin Inhibits Breast Cancer Development and Progression In Vitro and In Vivo by Suppressing Notch Signaling and Regulating MiRNAs - PubMed (2022)

Silibinin

Flavonolignan

Downregulates miRNA-21 and miR-17-92

Promotes apoptosis by upregulating apoptosis-promoting proteins APAF-1 and CASP-9. Inhibits tumor angiogenesis by downregulating VEGF signaling.

Zadeh et al. (2015)

Sinomenine

Alkaloid

Upregulates miRNA-29

Inhibits migration and invasion of malignant cells by upregulating the PDCD4-1 axis.

Gao et al. (2019)

Betulinic acid

Terpenoid

Downregulates miRNA-27a

Inhibits tumor growth by downregulating Myt-1 and ZBTB10 expression.

The Effects of betulinic acid on microRNA27a regulated target genes in MDA-MB-231 breast cancer cells - Talcott - and 2008 (2022)

Triptolide

Terpenoid

Upregulates miRNA-146a

Prevents tumor cell metastasis and angiogenesis by downregulating the RhoGTPase signaling pathway.

Liu et al. (2019)

Cardamonin

Chalcone

Downregulates miRNA-21

Inhibits EMT and VEGF-mediated angiogenesis.

Jiang et al. (2015)

β-Sitosterol-D-glucoside

Sterol

Upregulates miR10a

Promotes apoptosis by downregulating the PI3K/Akt pathways and the antiapoptotic factors Bcl-xl and Bcl-2.

Xu et al. (2018)

Vulpinic acid

Butenolide

Upregulates miR-769-5p, miR-197-3p, miR1268a, miR-6740-5p, miR-16-1-3p, miR-4235p, miR-2861, miR-132-3p, and miR-196b-5p

Promotes apoptosis by upregulation of proapoptotic factors Bax and downregulation of pro-caspases 3/9.

Cansaran-Duman et al. (2021)

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susceptibility to anticancer drugs, as seen in the cells of TNBC, due to an underlying inherent genetic mutation, (2) reduced disease-free survival (DFS) and relapse due to tumor heterogeneity of insensitive cancer stem cells (CSCs), and (3) epigenetic regulation of intrinsic molecular pathways, conferring neoplastic resistance to antiproliferative drugs. The innate resistance contributed by the CSCs is often assumed to be “acquired” initially, since most of the tumor cells are stalled and deceased during the initial stages of the chemotherapy paradigm, as they later become resistant during the final stages of the adjuvant treatment, making MDR seem apparently “acquired.” But it is the inherent self-renewal proliferative characteristic of the innate CSCS that mediates the malignancy relapse and tumor stemness (Frank et al., 2010). On the other hand, the development of MDR by acquired resistance may often be a result of (1) accidental activation of a secondary proto-oncogene during diagnostic paradigm, (2) altered tumor microenvironment, (3) change in differential expression of drug target due to secondary mutation acquired during the treatment, and (4) acquisition of EMT and avoidance of cellular senescence (Huang et al., 2016). The cancers exhibiting high intensity of chemotherapy detoxification, being extremely resistant to neoadjuvant courses of chemotherapy, as seen in cases of renal and gastrointestinal cancers, are perceived to possess the innate origin of MDR. But gynecological cancers of the breast and ovary generally develop resistance during the treatment and are known to have achieved an acquired type of multidrug (MD) resistance (Hamed et al., 2019). The major attributes characterizing MD resistance in breast cancer cells include overexpression of the membrane-bound ATP-binding cassette (ABC) transporter proteins resulting in enhanced cytotoxic drug efflux. The pharmacological impairment of adjuvant treatment involving the insufficient and suboptimal concentration of intracellular drug also contributes to the enhanced proliferation of invasive cells strengthening chemotherapeutic resistance. MDR-amplified angiogenesis of malignant cells coupled with poor vasculature of the latter further builds up the physiological aspect of chemotherapeutic failure resulting in poor drug distribution in the malignant proliferative cells (Breast cancer statistics and resources, 2022; Rizeq et al., 2020). It has been reported that with the incidence of MDR during the ongoing treatment, the prescribed drug doses are enhanced manifold times and then the dose that was previously administered during the initial stage of the adjuvant therapy, to control the sporadic spread and metastasis of the disease, exposing the healthy cells to greater toxic vulnerabilities (Cancer therapy resistance: chasing epigenetics, 2014). In addition to the MDR that develops from the multiple administration of the doses of these commercially available chemotherapeutic drugs, a high rate of histological toxicity is also encountered in the healthy tissues, even at dosages undertaken below the prescribed minimum owing to their very restricted window of therapeutic index. The resulting cellular and histopathological toxicity indeed greatly compromise the positive effects of the chemotherapy, the toxicity often overweighing the anticancer beneficial effects of chemo-based adjuvants, for which drugs were employed in the very first place naturally (Guo et al., 2016). Thus, sole reliance on synthetic forms of convectional chemo-based agents is questionable, as far as overcoming resistance in sporadic cancers, like that of the breast is concerned. BC, along with its most lethal aggressive subtype, TNBC, often fails to address such concordant adverse ill effects of MDR and falls prey to its lethal conquest. Chemosensitization of the tumor cells thereby appears as the only possible alternative for negating the ever-progressing aggression of the malignant cells. Hence, screening for other chemoindependent cytotoxic drug alternatives, as singular doses or in combination with adjuvants, has become a more pronounced requirement. Natural compounds with the least or no cytotoxicity, exhibiting antitumor properties at low concentration with enhanced anticarcinogenic effects and dosage efficacy, are therefore subjected to several ongoing preclinical and clinical trials, for widening their scope in translational cancer diagnosis (Guestini et al., 2017). Phytochemicals flavonoids, alkaloids, and polyphenols owing to their ability to accumulate within the cancer tissues serve as potent contenders for chemosensitization by bypassing their synthetic systemic convectional chemotherapeutic counterparts (Guestini et al., 2017).

16.10.1 Secondary metabolites and ABC transporters: a tale of super cross-opposition Cancer cells exhibit MDR mainly by decreasing the intracellular concentration of the antiproliferative drugs within the tumor tissues, by the overexpression of aldehyde dehydrogenase (ALDH), and a special category of membrane-bound ATP-binding cassette protein, known as ABC transporters (Liu et al., 2020). In healthy tissues, they generally participate in the exchange and transportation of several intrinsic and foreign biochemical substances including small molecules of lipid, amino acids, sugar, and cholesterol across the biological membranes of the host cells (Chen et al., 2016). The ABC transporter proteins are generally encoded by 49 genes and are divided into 7 subfamilies, namely, ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, and ABCG (Chen et al., 2016; Huang, 2007). But despite the physiological diversities among the members of the different subgroups of the transmembrane protein family, their fundamental structure remains the same. It is composed of two major subunits—alpha-helix containing transmembrane domain (TMD)

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and a nucleotide-binding (NBD) domain. The ATP transporters generally induce MDR either by decreasing the drug uptake from the target organelle or by accelerating their extracellular efflux (Karthikeyan & Hoti, 2015). The ATPase activity of the transporter protein remains confined in the NBD domain, which provides the necessary energy currency required for the ATP hydrolysis, which in turn promotes the enhanced chemotherapeutic drug efflux in the tumor cells (Yu et al., 2016; Durmus et al., 2015). The cancer cells when subjected to the administration of the chemotherapeutic drugs enhance the expression of these transporter proteins. These ATP cassette-binding proteins thus translocate the adjuvant drugs into the extracellular matrix of the cells, using the former as the foreign substrate (Karthikeyan & Hoti, 2015). Although all the members of the transporter protein subfamilies take part in the establishment of MDR in the neoplastic cells, the role of P-glycoprotein (p-gp, or ABCB1), BCRP (breast cancer resistance protein, or MXR, mitoxantrone resistance protein, or ABCG2), and MRP-1 (MDR- associated protein-1, or ABCC1) is reported to be the most significant. A thorough investigation of the mechanism of MDR thus hints at the urgency of mining novel natural modulators and inhibitors of ABC transporters to restore the sensitivity of malignant cells to chemotherapy and relieve the stress of insensitive chemoresistance. The widespread structural, pharmacological, and toxicological versatility of the plant secondary metabolites coupled with their minimal innate toxicity and significantly favorable oral bioavailability supports their candidature to serve as potent natural metabolites in combating chemotherapy-mediated resistance (Frontiers & secondary metabolites, 2022). The natural metabolites thus serve as the latest and the fourth generation of chemosensitizers (Limtrakul et al., 2005). The diverse toxicological and pharmacological attributes of alkaloids account for their immense application in overcoming adjuvant-derived MDR in BC cells. Based on similarities in functional groups, and skeletal and biochemical structures of these nitrogen-containing organic compounds, they are employed in executing anti-MDR roles in the various neoplastic cell lines (Wink, 2012). An isoquinoline alkaloid, glaucine, when applied in combination with the adjuvant chemotherapy paradigm, is reported to overcome ABCB1 (also known as p-gp) transporter-induced MDR, in the most common hormone receptor-positive metastatic breast adenocarcinoma cell line MCF7/ADR thereby restoring the cell’s chemoreceptiveness to doses of mitoxantrone and adriamycin (Lei et al., 2013). The resistance reversal property of the quinolone derivatives is attributed primarily to the presence of the quinolyl functional group in its ring, as the replacement of the latter by a naphthyl or a phenyl ring, deteriorated the compound’s chemosensitive stability (Suzuki et al., 1997). Quinidine and galantamine homodimers inhibit the efflux of intracellular drugs doxorubicin (DOX), rhodamine-123 (Rh-123), paclitaxel (also known as taxol), and mitoxantrone (MTX) in MDR breast cancer cell line MC-7/DX1 (Tinoush et al., 2020; Pires et al., 2009; Namanja et al., 2009). The BCRP-enriched TNBC cell line MDAMB-231 is known to show enhanced susceptibility to antiproliferative drugs MTX and camptothecin (CPT) treatment when administered in combination with a beta-carboline-based alkaloid hormone (Ma & Wink, 2010). An essential dietary alkaloid, member of the Piperaceae family, piperine isolated from the roots of Piper longum and Piper nigrum also exhibits significant MDR reversing activity. They restore the DOX concentration in the MC-7/DOX cells, along with a subsequent decrease in the gene expression levels of the ABC transporter proteins, ABCG2 and ABCB1, thereby maintaining the optimum intracellular drug concentration level within the tumor cell (Li et al., 2011). Piperine is the first reported and the most commonly used bioavailability enhancer for anticancer drugs, as it shows profuse P-glycoprotein (p-gp, ABCB1) and CYP3A4 (a cytochrome P450) inhibitory activity that prevents the efflux of several intracellular antiproliferative drugs (Atal & Bedi, 2010). The alkaloid is reported to inhibit several cytochrome P450 enzyme systems, majorly by glucuronidation, which brings about the metabolism of more than 50% of FDA-approved chemotherapeutic drugs. So the alkaloid when added in combination with the resveratrol, an anticarcinogenic agent, shows improved chemotherapeutic effect with enhanced intracellular plasma concentration, compared to when the drug was administered in sole composition (Johnson et al., 2011) Several polyphenolic flavonoid compounds are also reported to have potent MDR inhibitory activity, acting as a natural reservoir of phytoestrogens. The p-gp or ABCB1 glycoprotein inhibitory effect of the flavonoids is generally accomplished by the hydroxyl groups present in the carbon atoms numbered C5 and C7 (Sheu et al., 2010). Epigallocatechin gallate enhances MTX concentration in the breast cancer cell line MCF-7/TAM with a subsequent decrease in the level of the transporter proteins p-gp and BCRP or MXR, thereby reducing tumor invasion and angiogenesis. The flavonoid is also reported to make the cell chemosensitive to Rh-123 toxicity, thereby decreasing tumor cell viability (Epigallocatechin-3, 2022). MTT assay results recorded, honokiol, a neo-lignan biphenolic flavonoid to enhance the Rh-123 intracellular concentration and cytotoxicity along with deterioration of the MRP-1 and p-gp gene expression level in the MCF-7/ADR cell line of BC (Xu et al., 2006). Quercetin inhibits MDR in the BC cell lines MCF-7 and MCF/DOX by enhancing the chemosensitivity of tumor cells to drugs such as vincristine, DOX, and paclitaxel, by eradication of CSCs through nuclear translocation activity of YB-1 and reduction in the gene expression level of p-gp (Quercetin reversed MDR in breast cancer cells through down-regulating P-gp expression and eliminating cancer stem cells mediated by YB-1 nuclear translocation - PubMed, 2022).

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As the treatment paradigm is already very constricted in TNBC owing to the absence of all the three major hormone receptors (ER, PR, and HER2), chemotherapy remains to be the most viable treatment option for patients. So the development of MDR in TNBC patients is a real fatal challenge. A few natural compounds in combination with the conventional adjuvants reported overcoming MDR are thus being subjected to clinical and preclinical trials. The polyphenol baicalein shows MDR reversal activity in the TNBC cell line MDA-MB-231 by modulating the formation of the mammosphere through downregulation of ABC transporter ABCG2 and also inhibits CSC growth by altering the expression of the pluripotent factors OCT3/OCT4. It is also recorded to target frequently occurring metastasis in TNBC cells through apoptosis induction by downregulating interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), as the latter is also known to contribute to the weaker prognosis and rapid relapse in patients with TNBC (Koh et al., 2019). The flavonoid is reported to make the chemo-insensitive TNBC cells sensitized to both chemo and radiation therapy. Aberrant molecular functioning of the tyrosine kinase pathway, c-Met, promotes tumor aggressiveness and metastasis in TNBC. Rutin (quercetin 3-O-β-D-glucoside) enhances chemosensitivity in the MDA-MB-231 cell line and reverses MDR, by altering the c-Met kinase pathway (Rutin as a novel c-Met inhibitory lead for the control of triple negative breast malignancies - PMC, 2022). NF-κB is a primary signaling molecule promoting cancer cell growth and proliferation. Polyphenolic curcumin makes the TNBC cell line sensitive to antiproliferative agents like paclitaxel by dysregulating the NF-κB signaling pathway (Curcumin inhibits cell proliferation of MDA-MB-231 and BT-483 breast cancer cells mediated by down-regulation of NFκB cyclinD and MMP-1 transcription - ScienceDirect, 2022). Alternatively, reports suggest a combination of paclitaxel and curcumin in the MDA-MB-231 cell line, affect the tumor size, and promote apoptosis by reducing the MM9 expression level (Aggarwal et al., 2005). Tetrahydrocurcumin, a derivative of the polyphenolic flavonoid, inhibits the overexpression of the ABC transporter proteins, including p-gp, MRP-1, or ABCC1, and MXR thereby enhancing the chemosensitivity of the MDA-MB-231 cell line (Mitra & Dash, 2018). In addition to these several phytochemicals reported in BC, including its most invasive subtype TNBC, associated with MDR reversal, nanoparticles-coated phytochemical drug combinations targeting resistance-related biomarkers in the treatment of BC are an optimistic therapeutic alternative in combating metastasis and relapse in patients undergoing BC therapy (Guestini et al., 2017). The natural compounds, in combination with conventional medicines, or as a single “stand-alone” therapeutic system tend to amplify chemosensitivity and accelerate drug uptake in malignant cells by modulating several molecular pathways, without necessarily reproducing any toxic side effect (Mitra & Dash, 2018).

16.11 Diet and dietary phytochemicals in chemosensitization BC is well interpreted to be prevented by avoiding several lifestyles, dietary, and endocrinal risks (Aggarwal et al., 2009; Thomson, 2012). Although a rigid interassociation between a balanced diet and chemoprevention is yet to be drawn, the dietary regimen of an individual plays an important role in cancer therapeutics and the probability of its incidence (Choudhari et al., 2019). The plant-derived dietary compounds comprise a diverse group of several bioactive complexes classified based on their fundamental biochemical structure—alkaloids, polyphenol flavonoids, carotenoids, terpenoids, and likewise. Recently, a total of 5000 dietary compounds have been recognized to possess anticancer properties, though a large number is yet to be discovered and reported to conclude their health benefit in cancer prevention when kept in the diet as a whole (Ames & Wakimoto, 2002). They are found to occur in the food products commonly available such as vegetables, fruits, spices, condiments, and different plant extracts (Key, 2011). They have attracted the widespread attention of the scientific community because of their pleiotropic ability to exert their control in almost every stage of the altered cell cycle. The reported chemosensitizing and chemopreventive properties of these compounds are generally attributed to their cell cycle arresting ability, excellent bioavailability, antineoplastic, antiproliferative, and apoptosis-promoting characteristics (Braicu et al., 2017). These phytochemicals exert their antineoplastic potential by targeting specific aberrated molecular and biochemical pathways within the tumor cells (Sharma et al., 2018). Among all the phytochemicals reported, flavonoids are the most profusely available natural antioxidants found to be present in our daily diet regimen (Choi & Ahn, 2008). Fisetin, a polyphenolic compound, is reported to possess appreciable oral bioavailability, modulates autophagy, and brings about apoptosis in the BC cell line MC-7. Apigenin, a member of trihydroxyflavone, elevates the formation of cellular autophagosomes and enhances LC3 lipidation through L3 II/ I ratio upregulation, thereby promoting an autophagy flux in the metastatic MDA-MB-231 TNBC cell line (Lu et al., 2020). The dietary polyphenol, kaempferol, arrests the cell cycle transition of the malignant cells at the G2/M stage, thus acting as a potent antiproliferative agent in the BC cell lines MC-7 and MDA-MB-231. Kaempferol also induces apoptosis in metastatic breast carcinoma cell line MDA-MB-453 through the fragmentation of the misaligned DNA and by altering the expression level of the series of caspases 3, 7, and 9 along with induction of phosphorylation in the tumor-suppressor P53 assay (Choi & Ahn, 2008). Resveratrol, an important phytoalexin-based polyphenol, and a major

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antioxidant, abundantly occurring in berries and grapes shows potent anticarcinogenic activity in the BC cells. In both Luminal A BC cell line MC-7 and the characteristic TNBC cell line MDA-MB-231, resveratrol induces chemosensitization through apoptosis induction in the tumor cells by promoting TRAIL sensitivity and G1 cell cycle arrest of the malignant cells (Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol - PubMed, 2022). Epigallo-catechin-3-gallate (EGCG), the principal catechin extracted from green tea leaves, contributes to the significant reversal of MDR in the BC cell lines of Luminal A (MCF-7) and TNBC (MDA-MB-231), respectively. The phytochemical despite its poor bioavailability and intestinal absorption, when used in combination with other chemotherapeutic phytochemicals, exerts a synergistic role in inhibiting cell growth and viability of the MCF-7 and MDA-MB-231 cells by 50% when applied in a dosage of 100 μm (Sheng et al., 2019). The catechins revive chemosensitivity in the BC cells, by mostly repairing the impaired Wnt/β catenin pathway. The level of GSK3β present in the tumor cell is inversely proportional to the level of βcatenin accumulated within it. EGCG exerts its anti-tumorigenic effect by inhibiting the degradation of GSK3β, thereby subsequently decreasing the concentration of the βcatenin within the cancer cell. The subsequent deregulation of the βcatenin concentration, followed by EGCG-mediated ubiquitination of the molecule, prevents the oncogenic transition of the proto-oncogene cMyc along with cyclin D1, thereby preventing malignant growth and proliferation of BC cells (Jain et al., 2021). Interestingly, in addition to its chemotherapeutic properties, the radiotherapeutic property has also been observed in the case of breast cancer patient, as EGCG prevents radiotherapy-induced dermatological fatigues in patients of BC (Mechanistic evaluation of phytochemicals in breast cancer remedy: current understanding and future perspectives, 2022). Dietary folic acid derivatives restore chemosensitization in resistant TNBC tumor cells by repairing the S-adenosyl-L-methionine (AdoMet)-derived DNA repair mechanism (Tio et al., 2014). Alternatively, it has also been reported that AdoMet enhances the expression of miR-449a and miR34C in the TNBC cell lines MDA-MB-468 and MDA-MB231 promoting the apoptosis effect of chemotherapeutic sulfonium compounds by modulating several driver genes of the underlying oncogenic pathways (Coppola et al., 2020). Curcumin obtained from the rhizomes of Curcuma longa also brings about chemosensitization of BC cell lines by modulating several oncogenic targets. It sensitizes the TNBC cell lines MDA-MB-231 and MDA-MB-468 to cisplatin by downregulating the lncRNA CCTA1. The latter acts as an antagonistic prognostic biomarker in TNBC treatment, since high expression of CCTA1 is correlated with poor prognosis in TNBC. Cisplatin mediates the downregulation of the lncRNA modulating the P13k/AKT/mTOR pathway. Curcumin also mediates chemosensitization of the chemoresistant TNBC cells to 5-fluorouracil (5-Fu), the drug enhancing apoptosis in the tumor cells. The chemosensitization of 5-FU is essentially brought about in two ways: enhancement of chemosensitization through the dysregulation of the TNF-a¨-mediated NF-κB pathway by the inhibition of proteasomal activities and secondly through the inhibition of thymidylate synthase (TS), which is a major precursor in the biosynthesis of DNA, and hence a primary tumor target (Rose et al., 2002; Ponce-Cusi & Calaf, 2016). Moreover, chemosensitization property by regulating the level of intracellular reactive oxygen species (ROS) has also been reported in the dietary carotenoid lycopene, a phytochemical abundantly found to be present in tomato, a member of the taxonomic family Solanaceae. Reduced risk in TNBC stem cell proliferation and renewal is achieved by the enhanced production of the antioxidants, superoxide dismutase-1 (SOD-1) and glutathione-S-transferase-omega-1 by lycopene. The phosphorylation inhibition of the molecules of the Akt/mTOR pathway, followed by subsequent upregulation of the proapoptotic protein Bax, confirms the apoptosis-inducing activity of the carotenoid (Takeshima et al., 2014; Holzapfel et al., 2017). Among the many, these are some of the compounds, whose chemosensitization ability is reviewed based on their respective disease-specific importance. Thus supported by several preclinical and clinical epidemiological reports, chemosensitization and chemoprevention by plant-derived dietary compounds are conveniently available, cost-effective, least side effect, and therapeutically approved treatment alternatives for controlling the onset and management of BC. Though much advancement has been done in the field of molecular oncology, the critical mode of action of these phytochemicals is yet to be deciphered completely (Mechanistic evaluation of phytochemicals in breast cancer remedy: current understanding and future perspectives, 2022). The therapeutic effects of these secondary metabolites are thus attributed to being a combined success tale of many plant compounds taken together, then a single estimated individually. Hence, a multiplex combination of several phytochemicals amalgamated with their possible prospective oncotargets plays a retroactive and synergistic role in combating the different stages of tumorigenesis.

16.12 Challenges and perspectives: into the future of BC phytochemical interventions Both in developing and in already developed countries, BC is the leading cause of malignancy-related female mortality worldwide. As reported by several epidemiological analyses, there exists an inversely proportional relationship between

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the dietary reliance on phytochemicals and the incidence of BC. Thus, the significant contribution of the plant-derived secondary metabolites in cancer therapeutics is evident and cannot be overlooked undoubtedly (Abbasi et al., 2018). To combat drug resistance, an outcome-oriented therapeutic approach involving phytochemicals in combination with chemotherapeutic agents is yet to be developed. Despite the several advantages of phytochemical administration in cancer patients, as an alternative therapeutic paradigm, the standardized long-term protocol and the cellular specificity of these dietary elements in BC therapeutics are major concerns for scientists worldwide. Moreover, the stability of these secondary metabolites during large-scale commercial manufacturing is uncertain as well (Mitra & Dash, 2018). The antiproliferative effects mediated by the phytochemicals are generally thought to be an amalgamation of several singular mechanisms, which often when tested in in vivo studies stand out to be ineffective. So identification of the specific molecular targets, of the phytochemicals, is an acute necessity, as often pathway deregulation by them exposes the host cells to extreme malignancy. In most of the phytochemicals reported to inhibit malignant growth in BC, they have exhibited a dosage-specific inhibitory ability. Hence, a proper insight into the bioavailability and the exact optimum concentration which corresponds to the inhibitory activity of the phytochemicals in the BC cells should be prescribed as well. It has been reported that a dosage lower than the optimum often promotes tumor growth in the host cell, whereas a dosage higher than the prescribed causes additional ill effects (More et al., 2021). Poor bioavailability of the phytochemical conjugates following absorption should also be taken into serious consideration, by pharmaceutical companies, as it largely compromises the drug performance and clarity (Koh et al., 2020). Strategies such as emulsion formation, nanoformulations, and encapsulations of the drug molecule opted in the future might aid in evading such drawbacks. Moreover, in several preclinical studies performed, small sample size, lack of control or placebo group, and shortrun trials are some of the major flaws encountered. So anticancer drug validations by these studies, in many cases, appear to be inconclusive and faulty (Mitra & Dash, 2018). Executing control trials, coupled with highly standardized protocols, administered in larger samples is thus recommended for proper validation of these small-molecule natural inhibitors. However, researchers are still optimistic, regarding the efficacy of these phytochemicals to dominate the major line of cancer therapeutic paradigm in the upcoming era, with the minor alterations of the prevailing drawbacks, impressed by their minimal intrinsic toxicity in the healthy cell, and extremely notorious delivery in the TME.

16.13 Conclusion In closing, since time immemorial, phytochemicals have been a very effective companion in combating several human diseases including cancer. BC being a major reason for death in women of all age groups worldwide, combating the disease is a major challenge, with the best effective and least toxic alternative. Until today, the majority of the chemotherapeutic drugs used in BC treatment are either a mimic or a derivative of the diverse variety of natural compounds available, the most prominent of them being taxanes. Studies reveal that the synergistic role of phytochemicals, amalgamated with conventional chemotherapeutics, enhances the probability of overcoming tumor toxicity. Phytochemicals, a potential CAM-based tool for cancer prevention, are known to retard the different stages of cancer progression and metastasis through a plethora of molecular mechanisms, such as immunomodulation of cytokines, induction of apoptosis, inhibition of EMT, prevention of metastasis and angiogenesis, by a number of genetic and epigenetic pathways. The plant-derived secondary metabolites thus exert their antineoplastic activities on the tumor cell by dominating the major signaling pathways such as NF-κB, Wnt-β catenin, Notch, EGFR, VEGF signaling, through expressional alteration of either the oncogenes, TSG, or by the regulation of the pro-tumorigenic miRNAs. The work thus gives a comprehensive update on the chemotherapeutic mechanisms of the different classes of bioactive compounds and their dietary privileges and addresses the potency and efficacy of these phytochemicals as an optimistic derivative of CAM, elucidating their pivotal role in overcoming therapy-induced MDR, accelerating chemosensitization and in achieving therapeutic excellence.

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280. Available from https://doi.org/10.1016/j.canlet.2005.11.031. Xu, H., et al. (2018). Anti-breast-cancer activity exerted by β-sitosterol-d-glucoside from sweet potato via upregulation of microRNA-10a and via the PI3K-Akt signaling pathway. Journal of Agricultural and Food Chemistry, 66(37), 9704
9718. Available from https://doi.org/10.1021/acs. jafc.8b03305. Yin, R., Li, T., Tian, J. X., Xi, P., & Liu, R. H. (2018). Ursolic acid, a potential anticancer compound for breast cancer therapy. Critical Reviews in Food Science and Nutrition, 58(4), 568
574. Available from https://doi.org/10.1080/10408398.2016.1203755. Yu, J., Zhou, P., Asenso, J., Yang, X.-D., Wang, C., & Wei, W. (2016). Advances in plant-based inhibitors of P-glycoprotein. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(6), 867
881. Available from https://doi.org/10.3109/14756366.2016.1149476. Yu, S.-T., Chen, T.-M., Tseng, S.-Y., & Chen, Y.-H. (2007). Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells. Biochemical and Biophysical Research Communications, 358(1), 79
84. Available from https://doi.org/10.1016/j.bbrc.2007.04.107. Zadeh, M. M., Ranji, N., & Motamed, N. (2015). Deregulation of miR-21 and miR-155 and their putative targets after silibinin treatment in T47D breast cancer cells. Iranian Journal of Basic Medical Sciences, 18(12), 1209
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7817. Zu, M.-L., et al. (2021). Gypenoside LI arrests the cell cycle of breast cancer in G0/G1 phase by down-regulating E2F1. Journal of Ethnopharmacology, 273, 114017. Available from https://doi.org/10.1016/j.jep.2021.114017, Jun.

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Chapter 17

Phytochemicals and cancer Mayuri Iyer1, Kavita Pal2 and Vijay Upadhye3 1

Cachar Cancer Hospital and Research Centre (CCHRC), Silchar, Assam, India, 2Advanced Centre for Treatment, Research, Education in Cancer,

Tata Memorial Centre, Mumbai, Maharashtra, India, 3Center of Research for Development (CR4D), Department of Microbiology, Parul Institute of Applied Sciences (PIAS), Parul University, Waghodia, Gujarat, India

17.1

Introduction

Phytochemicals commonly consist of secondary metabolites that are synthesized as by-products of primary metabolism in plants, algae, and fungi, which are formed under environmental pressure playing a crucial role in protecting plants against environmental stresses. Phytochemicals are naturally occurring, biologically active chemical compounds associated with numerous health benefits and nutrition in humans. There is no systematic categorization of these phytochemicals. However, it can be broadly classified based on the presence of functional groups. According to Harborne and Baxter, phytochemicals are classified into six major categories based on their chemical structures and characteristics, namely, alkaloids and other nitrogen-containing compounds, carbohydrates, lipids, phenolic, and terpenoids. Some phytochemicals may be toxic, while many others have a wide range of benefits. Phytochemicals that are lethal act as defensive agents for plants against insects (D. Singh, 2014), various types of pathogens and pollutants (Iriti & Faoro, 2009), and herbivores.

17.1.1 Terpenes (isoprenoids) and terpenoids Terpenes, also known as isoprenoids, are a diverse group of naturally occurring phytochemicals. Terpenes and terpenoids are considered to be the same, but have slight chemical differences. Terpenes are volatile, unsaturated 5-carbon cyclic compounds, which are built up of isoprene monomeric units, whereas terpenoids are a modified class of terpenes with different functional groups at different positions. Terpenes are majorly classified based on the number of these isoprene units and their organization, namely, monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and triterpenes (C30), and terpenoids include iridoids, sesquiterpenoids, sesquiterpene lactones, diterpenoids, triterpenoid saponins, steroid saponins, cardenolides and bufadienolides, phytosterols, cucurbitacins, nortriterpenoids, other triterpenoids, and carotenoids. Terpenes are widespread in nature, mainly in plants as constituents of essential oils. Terpenes are found in plant species such as Pinus ponderosa (Pinaceae), spices (sage, rosemary, caraway, cumin, clove, and thyme), Cretan propolis, Helichrysum italicum, and Rosmarinus officinalis. Terpenes have characteristics such as fragrance, taste, and color that primarily act as pesticides and repel parasites. They also have various antimicrobial properties to help the plant build immunity against bacteria, plasmodium, fungi, and viruses (Cox-Georgian et al., 2019).

17.1.2 Polyphenols The largest range of phytochemicals is the class of polyphenols that are widely distributed in higher plants. Phenols are made of hydroxyl group (2OH) that are directly bonded to an aromatic hydrocarbon group. Polyphenols broadly comprise phenols, flavanoids, stilbenes, tannins, lignans, xanthones, quinines, coumarins, phenylpropanoids, and benzofuranoids. Polyphenols are rich in fruits, whole grains, nuts, seeds, beverages, spices, and seasonings (Pe´rez-Jime´nez et al., 2010). In the nutraceutical industry, polyphenols are commonly advertised and linked with tea, cinnamon, and several fruits, especially berries. Polyphenols have immense applications other than in the field of health and nutrition. Due to Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00007-4 © 2023 Elsevier Inc. All rights reserved.

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characteristic colors imparted by some polyphenols, they are used as natural dyes and coloring agents in the food industry. Additionally, they are also widely exploited in cosmetics due to a combination of antioxidative, anti-inflammatory, antiaging, antimicrobial, and solar photoprotective characteristics (Zillich et al., 2015; de Lima Cherubim et al., 2020). Some commonly known plants rich in polyphenols are Mentha balsamea (mint), Castanea sp. (chestnuts), Syzygium aromaticum (cloves), Camellia sinensis (tea), Vitis vinifera (grape), and Theobroma cacao (chocolate).

17.1.3 Alkaloids and other nitrogen-containing constituents The name alkaloids are derived from “alkaline” that constitute nitrogen-containing bases. They are usually organic bases and form salts with acids (Roy, 2017). The alkaloids include Amaryllidaceae, betalain, diterpenoid, indole, isoquinoline, lycopodium, monoterpene, sesquiterpene, peptide, pyrrolidine and piperidine, pyrrolizidine, quinoline, quinolizidine, steroidal, and tropane compounds (Izzah Ahmad et al., 2019) (Fig. 17.1). Other nitrogen-containing constituents include nonprotein amino acids, amines, cyanogenic glycosides, glucosinolates, purines, and pyrimidines (Izzah Ahmad et al., 2019).

17.2

Role of phytochemicals in various diseases

Two major streams of medicine, Ayurveda and Unani, use treatment regimens based on a combination of various phytochemicals. Both Ayurveda and Unani have stood the test of time and have been practiced since ancient times. They are effective treatment methods in today’s time, especially because of their minimal side effects. Indians generally have a saying with the following implied meaning, “the diet you consume is your medicine.” Consuming a healthy and wholesome diet ensures a holistic supply of naturally occurring phytochemicals with health-protecting benefits. In fact, numerous allopathic drugs have been derived from various phytochemicals such as aspirin, quinine, digoxin, etc.

17.2.1 Diabetes Hyperglycemia is a metabolic disorder that gives rise to a plethora of complications. The major complications include diabetic nephropathy, retinopathy, neuropathy, delayed wound healing, heart attack, peripheral vascular disturbances, and diabetic ketoacidosis. Type-1 or insulin-dependent diabetes mellitus is an autoimmune disorder characterized by FIGURE 17.1 Phytochemical - an overview.

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the destruction of pancreatic beta cells, whereas type-2 or insulin-independent diabetes mellitus patients present with insulin resistance compensated by hypersecretion of insulin. Teoh and Das reported approximately 200 pure bioactive compounds possessing potent hypoglycemic properties (Teoh & Das, 2018). Some of the plants commonly known to reduce blood glucose levels are Phyllanthus amarus (Adedapo & Ofuegbe, 2014; Modak et al., 2007; Patel et al., 2011), Mangifera indica (Ganogpichayagrai et al., 2017; Kumar et al., 2021), Azadirachta indica (Atangwho et al., 2012; Saleem et al., 2018; Yarmohammadi et al., 2021; Yarmohammadi et al., 2021), Allium sativum (Batiha et al., 2020; Modak et al., 2007; Ryu & Kang, 2017; Shang et al., 2019), Aloe barbadensis (Pothuraju et al., 2016; Shakib et al., 2019), Salacia reticulate (Y. Li et al., 2008; Medagama, 2015; Shivaprasad et al., 2013), Murraya koenigii (Samanta et al., 2018; Balakrishnan et al., 2020), Punica granatum (Salwe et al., 2015), ˇ ´ , 2020). and Nigella sativa (Hassan & Sudomova

17.2.2 Hypertension Systemic arterial hypertension is commonly characterized by persistent high blood pressure (BP) in the systemic arteries. Blood pressure (BP) is commonly expressed as the ratio of systolic BP to diastolic BP. The most common preventable risk factor for a lieu of health conditions is hypertension such as heart failure, coronary heart disease, stroke, myocardial infarction, atrial fibrillation, peripheral artery disease, chronic kidney disease (CKD), and cognitive impairment, being the single leading contributor to many causes of death and disability worldwide. The most effective interventions are lessening body weight, reduced sodium intake, increased potassium intake, increased physical activity, lowered consumption of alcohol and diets like the Dietary Approaches to Stop Hypertension (DASH) diet that combines several elements which favorably affect BP and physical exercise. First-line antihypertensive medications include ACE inhibitors, angiotensin II receptor blockers (also known as sartans), beta blockers, dihydropyridine calcium channel blockers, and thiazide diuretics (Oparil et al., 2019). Plants with antihypertensive phytochemicals are Allium sativum (Batiha et al., 2020; Modak et al., 2007; Ryu & Kang, 2017; Shang et al., 2019), Withania somnifera (Kalra & Kaushik, 2017; Sandhu et al., 2010), Moringa oleifera (Saini et al., 2016; Vergara-Jimenez et al., 2017), Terminalia arjuna (Amalraj & Gopi, 2017; Dwivedi & Chopra, 2014; Sandhu et al., 2010), and Loranthus micranthus (Zorofchian Moghadamtousi et al., 2013; Iwalokun et al., 2011).

17.2.3 Cardiovascular disorders Cardiovascular disease (CVD) is another potential cause of death and disability in developed countries. Nowadays, news reports heart failures in even normal young adults. The etiology of CVD is very complex. However, the overproduction of oxidants is one of the main pathogenic factors. Oxidative damage can cause endothelial cell injuries and deleterious vasodilator effects. However, antioxidant phytochemicals such as the class of polyphenols scavenge reactive oxygen radicals (ROS) inhibiting cellular damage. The cardioprotective effects are mainly due to their antioxidative, antihypercholesterolemic, antiangiogenic, anti-ischemic, inhibition of platelet aggregation, and antiinflammatory activities that result in reduction of the risk of cardiovascular disorders. Some of the commonly known phytochemicals known to cure CVDs are resveratrol (Hung et al., 2000; Kazemirad & Kazerani, 2020; Pagliaro et al., 2015; Riba et al., 2017), anthocyanins, dehydroglyasperin C, β-carotene, lycopene, phlorizin, and berberine. Some plants with the ability to treat heart diseases are Allium sativum, Curcuma longa (Pagliaro et al., 2015; Pourbagher-Shahri et al., 2021; Qin et al., 2017; Wang et al., 2012), Hibiscus sabdariffa (Abubakar et al., 2019; Micucci et al., 2015; Najafpour Boushehri et al., 2020; Riaz & Chopra, 2018), Ocimum sanctum, and Allium cepa (Chakraborty et al., 2022; W. Li et al., 2021).

17.2.4 Neurodegenerative disorders Neurodegenerative diseases are a heterogeneous, group of chronic and untreatable conditions, characterized by progressive functional impairment of the nervous system, induced by the deterioration of neurons, myelin sheath, neurotransmission, and movement control. Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, and multiple sclerosis are some of the commonly known neurodegenerative disorders (Forni et al., 2019). Neurodegenerative diseases are affected by factors such as stimulating nuclear factor (elytroid-derived 2)-like 2 (Nrf2) in the antioxidant system, sirtuin and forkhead box O (FOXO) transcription factors, and chaperones and neurotropic factors, and by inhibiting acetylcholinesterase (AChE) activity (Venkatesan et al., 2015). Compounds with antioxidant and anti-inflammatory activities have the potential to treat neurodegenerative diseases. For example, ladostigil

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acts as a neuroprotective agent and has been suggested to be effective in treating AD and PD. According to the Indian traditional medicine, Ayurveda, Mucuna pruriens (velvet bean) extract effectively helps in managing memory impairment in PD by reducing GSH, DPPH radicals, and ROS content. Evidently, curcuminoid usage in routine Indian diet lowers AD prevalence in India by attenuating inflammatory damage through cytokine production inhibition and microglia activation (Venkatesan et al., 2015).

17.2.5 Inflammatory bowel disease (IBD) Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the GI tract, which mainly includes Crohn’s disease and ulcerative colitis in the clinical context (Prideaux et al., 2012). Intestinal permeability is increased by mucosal damage which leads to bacterial translocation and inflammation (Hossen et al., 2020). IBD symptoms include abdominal pain, diarrhea, bloody stools, weight loss, and the influx of neutrophils and macrophages that produce cytokines, proteolytic enzymes, and free radicals that result in inflammation and ulceration (Guan, 2019). Phytochemicals such as mangiferin, norisoboldine, and curcumin suppress the pro-inflammatory cytokines and stimulate anti-inflammatory cytokine production. Under stress, ROS hinders crucial intestinal enzymes and signaling molecules. The loss of regulation of these enzymes and free radicals is a marker of colonic tissue damage and leukocyte infiltration (Hossen et al., 2020). Several phytochemicals act as antioxidants and ameliorate the deleterious effects posed by ROS production, especially the class of phenols. Phytochemicals also are strong gut microbiome modulators. Many phytochemicals such as polyphenols, carotenoids, phytosterols/phytostanols, lignans, alkaloids, glucosinolates, and terpenes possess antioxidant and anti-inflammatory properties altering gut microbial composition positively. Some polyphenols act as prebiotics and can be metabolized in the gut into bioactive constituents utilized by the gut commensals (Dingeo et al., 2020). A few polyphenols show to influence bacterial communication, interacting with quorum sensing (Sudheer et al., 2022) (Table 17.1).

TABLE 17.1 Phytochemicals from different sources being used in the treatment of different diseases. Disease/disorder

Plant

Active phytochemicals

Reference

Diabetes

Phyllanthus amarus

Amariin, epibubbialine, furosin, gallic acid, phyllanthenone, phyllanthusiin, kaempferol, phyllantheol, geraniin, linalool, phyllanthin D, phyllanthin, hypophyllanthin, nirurin niranthin, phytol, quercetin, securinine

Adedapo and Ofuegbe (2014), Modak et al. (2007), J. R. Patel et al. (2011)

Mangifera indica

Mangniferin, catechin, isoquercitin, hyperin, kainic acid, gallic acid, norathyriol, quercetin, ellagic acid

Ganogpichayagrai et al. (2017), M. Kumar et al. (2021)

Azadirachta indica

Azadirachtin, nimbilin, nimbolinin, mahmoodin, azadirachtol, nimonol, salimuzzalin, limocinone, salannin, α-linolenic acid

Atangwho et al. (2012), Saleem et al. (2018), Yarmohammadi et al. (2021)

Allium sativum

Allicin, diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), ajoene, S-allyl-cysteine (SAC), alliin, desgalactotigonin-rhamnose, protodesgalactotigonin, proto-desgalactotigonin-rhamnose, voghieroside D1, sativoside B1-rhamnose, and sativoside R1, β-resorcylic acid, pyrogallol, gallic acid, rutin, protocatechuic acid, quercetin

Batiha et al. (2020), Modak et al. (2007), Ryu and Kang (2017), Shang et al. (2019)

Murraya koenigii

Mahanine, mahanimbine, koenimbine, murrayafoline, girinimbine, murrayazoline, koenoline

Samanta et al. (2018), Balakrishnan et al. (2020)

Phaseolus vulgaris L.

Chlorogenic acid, syringic acid, caffeic acid, kaempferol, pelargonidin, cyanidin, delphinidin, sugars, fatty acids, and tocopherols

Jawaid et al. (2017)

Hypertension

(Continued )

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TABLE 17.1 (Continued) Disease/disorder

Cardiovascular disorders

Neurodegenerative disorder

Inflammatory bowel disease (IBD)

Plant

Active phytochemicals

Reference

Withania somnifera

Withaniol, somnirol, somnitol, withanic acid, phytosterol, ipuranol, stearic, cerotic, palmitic, oleic acid, withanine, somniferine, somnine, linoleic acids, withaferin A, withanolide D

Kalra and Kaushik (2017), Sandhu et al. (2010)

Moringa oleifera

Glucomoringin, quercetin, apigenin, kaempferol, isothiocyanates, nitriles, thiocarbamates, glucosides, rutinosides, malonyl glucosides, quercetin, kaempferol, isorhamnetin, glucosinolates, glucotropaeolin

Saini et al. (2016), Vergara-Jimenez et al. (2017)

Terminalia arjuna

Arjunin, arjunolone, arjunetin, terminic acid, luteolin, gallic acid, kaempferol, pelargonidin, pyrocatechols, punicallin, castalagin, casuariin, casuarinin

Amalraj and Gopi (2017), Dwivedi and Chopra (2014), Sandhu et al. (2010)

Loranthus micranthus

Linamarin gallate, walsuraside B, catechin, epicatechin, epicatechin 3-O-gallate, rutin, peltatoside, lupeol, lupinine, loranthoic acid

Zorofchian Moghadamtousi et al. (2013), Iwalokun et al. (2011)

Curcuma longa

Curcumin, turmerol, D-α-phellandrene, veleric acid, phellandrene, borneol, zingiberene, sesquiterpene, ferulic acid, vanilic acid, β-phellandrene, turmerone, and α-turmerone

Pagliaro et al. (2015), Pourbagher-Shahri et al. (2021), Qin et al. (2017), Wang et al. (2012)

Hibiscus sabdariffa

Delphinidin-3-glucoside, sambubioside, and cyanidine3-sambubioside, gossypetin, hibiscetin, protocatechuic acid, eugenol, β-sitoesterol and ergoesterol, flavylium, delphinidine-3-sambubioside, cyanidine-3sambubioside

Abubakar et al. (2019), Micucci et al. (2015), Najafpour Boushehri et al. (2020), Riaz and Chopra (2018)

Allium cepa

Pyruvic acid, iosulfinates, isoalliin, alliin, deoxyalliin, cycloalliin, ceposide A and ceposide B, glucose, fructose, and sucrose, fructooligosaccharides (FOS)

Chakraborty et al. (2022), Li et al. (2021)

Curcuma longa

Curcumin, turmerol, D-α-phellandrene, veleric acid, phellandrene, borneol, zingiberene, sesquiterpene, ferulic acid, vanilic acid, β-phellandrene, turmerone, and α-turmerone

Berry et al. (2021)

Camellia sinensis

Epigallocatechin-3-galate

Malar et al. (2020), Nunes et al. (2015)

Zingiber officinale

6-Gingerol, 6-shogaol, 10-gingerol, gingerdiones, gingerdiols, paradols, 6-dehydrogingerols, 5-acetoxy-6gingerol, 3,5-diacetoxy-6-gingerdiol, and 12-gingerol

Arcusa et al. (2022), Talebi et al. (2021)

Vitis vinifera

Resveratrol, pterostilbene, gallic acid, ferulic acid, caffeic acid, caftaric acid, syringic acid, quercetin, kaempferol, (1) catechin, epicatechin, anthocyanin, proanthocyanidin, ampelopsin A, vitisin A, vitisin B.

Insanu et al. (2021)

Piper longum

Piperine, pipernonaline, piperettine, asarinine, pellitorine, piperundecalidine, piperlongumine, piperlonguminine, retrofractamide A, pergumidiene, brachystamide-B, a dimer of desmethoxypiplartine, Nisobutyl decadienamide, brachyamide-A, brachystine, pipercide, piperderidine, longamide, dehydropipernonaline piperidine, and tetrahydro piperine

Haq et al. (2021), Huang et al. (2020), Huang et al. (2020), S. Kumar et al. (2011)

Citrus paradisi

Quercetin, rutin, isoquercitrin, kaempferol, myricetin, naringin, naringenin, hesperidin, hesperetin, poncirin, apigenin, diosmin, luteolin, nobiletin, and tangeretin

Musumeci et al. (2020)

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Phytochemicals in cancer

Nearly 15 million new cases of cancer are detected worldwide every year (https://www.cancer.gov/about-cancer/understanding/statistics). In order to combat cancer, various kinds of medical treatments such as chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, photodynamic therapy, targeted therapy, surgery, palliative care, and a combination of these are increasing. However, chemotherapy is yet a widely accepted first line of treatment for a wide range of cancers remaining a principal mode of treatment. The role of chemotherapy was limited to treating epithelial malignancies and symptomatic metastatic disease for almost 30 years. Nevertheless, nowadays, cancer chemotherapy has found three main applications: (1) it can cure a few types of malignancies such as childhood leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, and germ cell malignancies; (2) it has a palliative role for most metastatic malignancies; and (3) it has been used as an adjuvant in several types of resected epithelial malignancies. Chemotherapeutic agents are majorly classified into alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan, busulfan), antimetabolites (e.g., 5-fluorouracil, capecitabine, methotrexate, gemcitabine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin), topoisomerase inhibitors (e.g., topotecan, irinotecan, etoposide, teniposide), and mitotic inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine) (Sak, 2012). Phytochemicals play crucial roles during chemotherapy regimens, given the fact that chemotherapy is a very aggressive form of anticancer treatment. Phytochemical-based intervention during chemotherapy is tremendously beneficial. It increases the efficacy of treatment, decreases debilitating chemotherapy-induced toxicity and severity of comorbid conditions, reduces drug resistance, and improves the quality of life. Fig. 17.2 depicts, in brief, the role phytochemicals played in chemotherapy.

17.3.1 Phytochemicals in chemoprevention Chemoprevention means using a strategy to prevent, suppress, or reverse the initial phase of tumorigenesis/oncogenesis or to inhibit the invading potential of premalignant cells using natural, synthetic, or biological agents. Clinically, chemoprevention is categorized into three types, primary, secondary, and tertiary. Primary chemoprevention is suitable for the general population with no cancer and populations at high risk of developing cancer in their lifetime. Secondary chemoprevention is intended for patients with premalignant lesions, which may progress to invasive cancer. Tertiary chemoprevention is to prevent the recurrence of cancer (Ranjan et al., 2019). Many dietary phytochemicals are studied to possess a significant chemopreventive potential and are an emerging field of interest to combat cancer. Several epidemiological studies report regular fruit and vegetable intake significantly reduces cancer incidence. Plant-derived bioactive compounds play a significant role in cancer chemoprevention. Common chemopreventive phytochemicals are curcumin, resveratrol, quercetin, folate and folic acid, indole-3-carboinol, genistein, apigenin, and epigallocatechin gallate. Phytochemical-based chemoprevention is potent and cost-effective which can lead to the development of new and alternative methods of cancer prevention and therapy (Hosseini & Ghorbani, 2015) (Table 17.2).

FIGURE 17.2 Role of phytochemicals in chemotherapy.

TABLE 17.2 Molecular structure of curcumin. Phytochemical

Common dietary sources

Against cancer

Curcumin

Turmeric

G G G G G G

1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione

G G G G G

Resveratrol

Red grapes, peanuts, soybeans, and pomegranates

G G G G G G G

G

Breast cancer Colon cancer Gastric cancer Hepatocellular carcinoma Lung cancer Prostate cancer Skin cancer

G Apoptosis induction via Bax, Bak, PUMA, Noxa, Bim, p53, TRAIL, TRAIL-R1/Dr4, and TRAIL-R2/ Dr5 upregulationBcl-2, Bcl-XL, and Mcl-1 downregulation G Growth arrest at G1 and G1/S phases by inducing the expression of CDK inhibitors p21/WAF1/CIP1 and p27/KIP1 G Reduction of inflammation via inhibition of prostaglandin production, COX-2 activity, and NFκB activity G Modulation of cell signaling pathway

Shankar et al. (2007) Ashrafizadeh et al. (2021) Alavi et al. (2021) Xiao et al. (2019)

Onions, apples, broccoli, tomatoes, and citrus fruit

G

ROS signaling inhibitor: Diethylnitrosamine (DEN) inhibition G Superoxide dismutase, glutathione peroxidase, catalase, GSH, and total glutathione upregulation G Wnt/β-Catenin/T cell factor signaling inhibition G p53 activity induction G Ras proteins inhibition G PI3K inhibition

Rather and Bhagat (2020), Vafadar et al. (2020), Gibellini Lara et al. (2011)

G G G G G

Acute monocytic leukemia cells Breast cancer Epidermoid carcinoma Hepatocellular carcinoma Lung adenocarcinoma Skin cancer

G G G G G

G

Induction of phase II enzymes via activation of Nrf 2 Apoptosis induction Modulation of cell cycle regulators NF-κB inhibition Angiogenesis inhibition Microtubule polymerization inhibition

References

Bladder cancer Brain cancer Colon cancer Kidney cancer Leukemia Multiple myeloma Esophageal cancer Oral cancer Prostate cancer Skin cancer Stomach cancer

trans-3,4,5,-trihydroxystilbene

Quercetin

Chemopreventive mode of action

G. Kumar et al. (2016) Guangyang et al. (2015) Belcaro et al. (2014) van’t Land et al. (2004) Rahmani et al. (2014) Mansouri et al. (2020) Punatar et al. (2022)

(Continued )

TABLE 17.2 (Continued) Phytochemical

Common dietary sources

Against cancer

Apigenin

Basil, Biloba, Celery, Tea, Chamomile, Cilantro, Flax, Licorice, Mint, Oregano

G G G G G G G G

40 ,5,7,-trihydroxy-flavone

G G G G G G

Adrenocortical cancer Breast cancer Cervical cancer Colon cancer Endometrial cancer Gastric cancer Hepatocellular cancer Leukemia Lung cancer Neuroblastoma Ovarian cancer Prostate cancer Skin cancer Thyroid cancer

Chemopreventive mode of action G G

G

G

G

G

G

G

G

G

Epigallocatechin Gallate

Green Tea

G G G G G

Breast cancer Esophageal cancer Colorectal cancer Gastric cancer Oral cancer

G

G

G G

G G G

Anthocyanins

Black rice

Breast cancer Human colorectal carcinoma

References

Promotes metal chelation Promotes free radicals scavenging stimulates phase II detoxification enzymes Increases glutathione (GSH) concentration Suppresses COX-2 and iNOS expression in macrophage Suppresses TNF-induced (NF)-κB activation in HUVEC Inhibits PKC, MAPK, ornithine decarboxylase, tyrosine kinases, and casein kinase 2 activity Downregulates Na1/Ca212 exchanger expression Induces a reversible G2/M and G0/G1 arrest via p34 (cdc2) kinase inhibition and p53 protein stability Downregulates cyclin D1, D3 and cdk4 expression Suppresses VEGF expression

D. Patel et al. (2011) Nozhat et al. (2021) Javed et al. (2021) Mahbub et al. (2022) Moein et al. (2022) Hnit et al. (2022)

Suppression of VEGF, NFkB, c-fos, and cyclin D1 expression Capillary endothelial cell proliferation inhibition Induction of TIMP-1 and TIMP-2 Suppresses MMP-2 activity and focal adhesion kinase (FAK), membrane type1-MMP (MT1-MMP), and Bcl-XL p53 stabilization Inhibits IDO expression JAK/STAT3 inhibition

Hanxi et al. (2015) Li et al. (2020) Amin et al. (2021) Alserihi et al. (2022) Bimonte et al. (2019)

Suppresses metastasis by targeting RAS/ RAF/MAPK pathway Reduces proliferation by inducing caspase3 activation and p21 upregulation

Xiang-Yan et al. (2015) Anwar et al. (2016) Medic et al. (2019) Shi et al. (2021)

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17.3.2 Phytochemicals as chemotherapeutic agents Approximately, 50% of approved anticancer drugs from 1940 to 2014 originate from natural products. Phytochemicals reduce the rate of carcinogenic process by scavenging free radicals, suppressing survival and proliferation of malignant cells, and diminishing invasiveness and angiogenesis of tumors. According to a review article by Choudhari et al., there are four major classes of clinically used plant-derived anti-cancerous compounds including vinca alkaloids, taxane diterpenoids, camptothecin derivatives, and epipodophyllotoxin. Vinca alkaloids are obtained from the pink periwinkle plant Catharanthus roseus (Apocynaceae). The vinca alkaloids achieve cytotoxic effects by binding to β-tubulin affecting cellular functions such as maintenance of cell shape, motility, and transport between organelles. Vinblastine and vincristine are the two naturally isolated alkaloids that have been used in clinical oncology for almost 50 years treating a variety of cancers, including leukemia, Hodgkin and non-Hodgkin lymphomas, advanced testicular carcinoma, breast and lung cancers, and Kaposi’s sarcoma. Taxanes were first isolated from the bark of Yew tree. Taxanes exert an anticancer effect by stabilization of microtubules, resulting in cell cycle arrest and aberrant mitosis. Paclitaxel, a semisynthetic derivative, is primarily used against breast, ovarian, pancreas, prostate, and lung cancers. Camptothecin is a quinolone alkaloid obtained from Camptotheca acuminata. Camptothecin complexes with type-I DNA topoisomerase prevent cleavage and religation of DNA leading to DNA double-strand breaks. At present, irinotecan and topotecan are the two FDA-approved semisynthetic camptothecin derivatives for the treatment of advanced cancers of the large intestine and rectum, and recurring ovarian, small cell lung cancer, and cervical cancer, respectively. Podophyllotoxin is a natural product isolated from Podophyllum peltatum and Podophyllum emodi (Berberidaceae). Podophyllotoxin reversibly binds to tubulin, whereas its key derivatives etoposide and teniposide inhibit topoisomerase II, inducing topoisomerase II-mediated DNA cleavage. Moreover, podophyllotoxin also exhibits potential anti-multidrug-resistant (MDR) activity against diverse drug-resistant tumor cells (Choudhari et al., 2020). Some studies have demonstrated that certain phytochemicals have better selectivity than chemically derived drugs. For example, the ethanolic extract of Senecio graveolens causes selective cytotoxicity in breast cancer cells via hypoxia-mediated mitochondrial dysfunction and oxidative stress. Ashwagandha leaf extract and its purified component withanone cause selective killing of cancer cells by inducing ROS-mediated damage. Solamargine, a steroidal glycoalkaloid found in plants of the Solanaceae family, induces cell death selectively in human melanoma cancer cells via the extrinsic lysosomal
mitochondrial death pathway. Rhodiola rosea extracts selectively inhibit the growth of urinary bladder cancer cells while having minimal effect on nonmalignant bladder epithelial cells (V. K. Singh et al., 2019).

17.3.3 Phytochemical in alleviation of chemotoxicity Chemotherapy, the first line of anticancer treatment, has been shown to induce an overall prevalence of 75%
100% [Ma1] of chemotherapy-induced toxicity. Some of the well-known implications of chemotherapy are cardiotoxicity, nephrotoxicity, neuropsychotic conditions, fertility-related issues, and gastrointestinal toxicity. The chief complaint related to chemotoxicity is regarding compromising the quality of patient life and further compromising the treatment regimen. Mittra et al. reported that a combination of resveratrol-copper successfully suppresses toxic cytological effects, such as DNA damage, apoptosis, and inflammation, induced by adriamycin, cyclophosphamide, cisplatin, methotrexate, and paclitaxel (Mittra et al., 2017). Another group of scientists has reviewed dietary phytochemicals to treat chemobrain or chemofog where cancer survivors treated with chemotherapy develop long-term cognitive impairments, affecting their quality of life. They reviewed the effects of astaxanthin, omega-3 fatty acids, ginsenoside, cotinine, resveratrol, polydatin, catechin, rutin, naringin, curcumin, dehydrozingerone, berberine, C-phycocyanin, the higher fungi Cordyceps militaris, thyme (Thymus vulgaris), and polyherbal formulation. Studies have reported that Chinese ginseng-derived saponins are able to protect against cyclophosphamide-induced genotoxicity and apoptosis in bone marrow cells and peripheral lymphocytes and American ginseng can attenuate nausea and vomiting induced by cisplatin. Polyphenol quercetin is able to potentiate the cytotoxic effect of cisplatin while protecting normal renal cells from cisplatin toxicity (V. K. Singh et al., 2019). However, it is found that the field of chemotherapy-induced toxicity prevention has not found prominence to a large extent although it is a serious issue to be addressed.

17.3.4 Phytochemical in conjugation with chemotherapy: a synergistic anticancer effect Phytochemicals combined with chemotherapy as an anticancer regimen have proven to be highly effective and beneficial for cancer victims. Due to the increase in phytochemical-induced anticancer efficacy of chemotherapy, the dose required to treat malignancies by default reduces and thereby reduces the deleterious effects of chemotoxicity.

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Quercetin synergistically with busulfan acts against human leukemia cells whereas with doxorubicin in cultured multidrug-resistant human breast cancer cells. It increases the cytotoxic effect of cyclophosphamide and decreases resistance to gemcitabine, topotecan, vincristine, tamoxifen, paclitaxel, and doxorubicin. Catechins from green tea increase the therapeutic effect of doxorubicin in drug-resistant tumors in animal studies. Green tea helps in the selective accumulation of chemotherapeutic agents in neoplastic cells and not in the normal tissue thereby enhancing its antitumor activity. Several phytochemicals such as flavonoids can enhance the drug bioavailability by inhibiting ATP transportersmediated drug efflux in vitro. It is recommended therefore that the diet of cancer patients treated with chemotherapy should be rich in herbal constituents (including catechins, kaempferol, naringenin, quercetin, silymarin), fruits and berries, and spices. To ensure the removal of toxic waste products, stimulation of phase II detoxification enzymes is crucial. For this purpose, cancer patients should increase the intake of isothiocyanates, found in various cruciferous vegetables, particularly Brussels sprouts and red cabbage, naringin, in grapefruit, and parsley, and spice their food with curcumin (Sak, 2012).

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Chapter 18

Phytochemicals as a complementary alternative medicine in cancer treatment Kajari Das1, M. Dhanalakshmi2, Medha Pandya3, D. Sruthi4 and Sushma Dave5 1

Department of Biotechnology, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha,

India, 2Research and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India, 3Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar, Gujarat, India, 4Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India, 5Department of Applied Sciences, JIET Jodhpur, Jodhpur, Rajasthan, India

18.1

Introduction

Cancer, also known as a malignant neoplasm in medical terms, is a complicated illness that develops over time as a result of a gradual accumulation of genetic and epigenetic alterations that allow cells to escape normal cellular and environmental restrictions. Hippocrates (460
370 BCE), a Greek physician, is known as the “Father of Medicine” because of his humoral theory based on observation of clinical signs and rational conclusions. He also coined the term “cancer” as carcinos and carcinoma to designate non-ulcer-forming and ulcer-producing cancers, respectively. Celsus, a Roman physician, subsequently translated this Greek phrase into English, cancer (28
50 BCE). Another Roman physician, Galen, introduced the term oncos to characterize tumors, which is now often used as part of the name of oncologists (Hajdu, 2012). Cancer is presently thought to be a multigene, multistep illness that begins with a single aberrant cell with an altered DNA sequence. When the unrestrained multiplication of these aberrant cells is followed by a second mutation, the stage becomes somewhat irregular. A tumor develops as a result of repeated rounds of mutation and the selective growth of these cells. A typical and healthy body has 30 trillion cells. Normal cells only multiply when they are told to by other cells in their environment. This teamwork is necessary to ensure that each tissue retains the proper size and shape to the body’s requirements (Bianconi et al., 2013). Cancer cells, on the other hand, do not cooperate in this way. They have the potential to move from the spot where they originated as they follow their own intrinsic instructions for reproduction. They invade adjacent tissues, producing new ones. Tumors’ malignant cells allow them to become more aggressive over time. They also disturb tissues and organs that are necessary for the organism’s overall existence (Warrell et al., 2005). Several genetic alterations in normal cells are responsible for carcinogenesis. The whole process of carcinogenesis involves several stepwise changes in chemical, physical, biological, or genomic processes in the cells. There are three steps such as initiation, promotion, and progression. The first step starts with a gene mutation in a normal cell that finally progresses into malignant tumors from benign tumors (Dawood et al., 2014). Gene mutation may be associated with chromosomal translocation or deletion and dysregulated expression or activity of signaling pathways. Genetic alteration in the first stage is basically the activation of oncogenes and/or the deactivation of tumor suppressor genes (Daga et al., 2018). The first stage is also associated with uncontrolled cell growth and inactivation of apoptotic mechanisms that subsequently lead to the acquisition of metastatic properties. The ability to self-renew and differentiate into many specialized cell types are two properties that define stem cells. This approach has been expanded beyond adult and embryonic stem cells to include cancer stem cells (CSCs) and induced pluripotent stem (IPS) cells (Barati et al., 2021). More stem cells that are undifferentiated are produced by selfrenewal. Stem cells create adult cell types via differentiation. Cancer stem cells (CSCs) are a tiny fraction of tumor cells that may self-renew, differentiate, and become tumor-producing when introduced into an animal host. To recognize and enrich CSCs, a range of cell surface markers, including CD44, CD24, and CD133, are often utilized (Huang et al., 2021). The characteristics of CSCs are regulated by a network of microRNAs, Wnt/catenin, Notch, and Hedgehog Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00006-2 © 2023 Elsevier Inc. All rights reserved.

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signaling pathways (Yoshida et al., 2021). Emerging research showing that CSCs are resistant to traditional chemotherapy and radiation therapy and that CSCs are very likely the cause of cancer metastasis has reinforced the therapeutic importance of CSCs. For the development of new anticancer drugs, CSCs are thought to be a crucial target. Cancer is caused by internal factors that account for about 5%
10% and by environmental/acquired factors that account for about 90%
95%. The factors responsible for cancer are hereditary factors, environmental factors, tobacco, alcohol, obesity, environmental pollution, radiation, infectious agents, and diet. Hanahan and Weinberg suggested ten hallmarks of cancer which are endowed with a firm basis for understanding the biology of cancer (Hanahan & Weinberg, 2011): (1) sustaining proliferative signaling, (2) insensitivity to anti-growth signals, (3) evading apoptosis, (4) limitless reproductive potential, (5) inducing angiogenesis, (6) activating invasion and metastasis, (7) genome instability and mutation, (8) characteristic tumor-promoting inflammation, (9) reprogramming energy metabolism, and (10) evading immune destruction. Cancers can be categorized according to their primary site of origin or by histology or tissue type (i.e., carcinoma, sarcoma, leukemia, lymphoma, and myeloma). Individual cancers are also categorized based on their grade and stage (e.g., stage IV). The most traditional forms of cancer treatment are surgery, radiation, and chemotherapy. These treatments can be applied either independently or in conjunction with other treatments. Treatment must be tailored to the specific needs of each patient because every malignancy is unique. Various factors, including the patient, the disease, the stage of the disease, performance level, and other disorders, will determine the precise treatment option or combination of alternatives. Targeted therapy, immunotherapy, hormone therapy, and bone marrow transplantation are further cancer treatment approaches (Emens, 2010). Traditional chemotherapy has a lot of negative side effects despite being successful. For instance, individuals receiving treatments based on platinum-alkylating drugs, topoisomerase inhibitors, and mitotic inhibitors experience cutaneous hypersensitivity reactions; hyperpigmentation is common when alkylating medicines such as ifosfamide, cyclophosphamide, and thiotepa are used (Diehl, 2018). A lot of liposomal doxorubicin, daunorubicin, and 50 -fluorouracil patients have hand and foot syndrome. Recurrence is one of the main drawbacks of traditional therapy since not all cancer stem cells are completely eliminated from the body. Additionally, multidrug resistance (MDR) development is a critical therapeutic problem. Therefore, it is essential to discover new methods and treatments that could provide comparatively affordable regimens with fewer undesirable side effects (Ye et al., 2019). Phytochemicals from plants, in particular, are the main inspiration for many of these complementary and alternative therapies that are either chemopreventive or chemotherapeutic. Complementary and alternative medicine (CAM) includes approaches and chemicals that are not often thought of as conventional treatments. In fact, CAM refers to the utilization of dietary phytochemicals or their naturally occurring or synthesized derivatives for the treatment and prevention of cancer. CAM includes a variety of therapeutic modalities, including nutritional supplements, herbal therapies, and homeopathic treatments (Mainardi et al., 2009). Epidemiological and preclinical research has revealed that nutritional and behavioral factors may have a major impact on the occurrence of some cancers. As a result, there is a greater interest in dietary phytochemicals. Dietary phytochemicals are naturally occurring bioactives that are present in a variety of foods, including fruits, vegetables, plants, and spices. They have qualities that are anti-inflammatory, antioxidative, proapoptotic, and antiproliferative, and they are especially extensively known for preventing the growth of certain cancer cells (Choudhari et al., 2020). Numerous reports suggested that phytochemicals and nutraceuticals have promising inhibitory effects on inflammatory and oxidative pathways/mediators implicated in the pathophysiology of lungs damage after COVID-19 infection throughout the pandemic period (Dhanalakshmi et al., 2022; Manjunathan et al., 2022; Pandya et al., 2022). Historically, plants are the most essential and valuable resource in human life that provides all of the basic needs such as food, clothing, shelter, and medicines (Mahady, 2001; Talalay & Talalay, 2001). Since ancient times, plants and their formulations have been used as therapeutics against various diseases. Various folk medicine practitioners from different philosophies and cultures are successfully applying their herbal preparations to heal a large number of diseases. Ayurveda, the ancient Vedic literature of India, is extensively being used in modern drug development programs (Behere et al., 2013) and has acquired wide acceptance in the present healthcare system.

18.2

Role of oxidative stress in carcinogenesis

18.2.1 Oxidative stress and antioxidant defense mechanism Aerobic organisms are continuously under a grave threat of oxidation hazard. Reactive oxygen species (ROS) are a group of oxygen-derived free radicals that are the bullets that trigger the ignition. ROS are produced in living organisms

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during normal cellular metabolism and exposure to environmental pollutants. ROS levels are maintained below the threshold level by the radical scavenging enzymes. A disbalance between ROS and ROS-neutralizing agents leads to oxidative stress during which increased levels of ROS impair cellular function. The study of oxidative stress is nowadays becoming a very interesting topic among researchers. A little imbalance between ROS and antioxidants can result in huge impairment due to oxidative damage of all the macromolecules (protein, fat, nucleic acid, and carbohydrate) (Azab et al., 2017; Prior & Cao, 1999; Sruthi & Zachariah, 2017) consequently resulting in various diseases such as atherosclerosis, ischemic heart diseases, diabetes, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, and initiation of carcinogenesis or liver diseases. ROS, when properly regulated, can facilitate the progression of various cellular activities (viability, activation, proliferation, and organ function). Antioxidants are enzymes and small molecules that are essential for normal cellular maintenance protecting our body from the scavenging effect of free radicals (Azab et al., 2017; Robinson et al., 1997). During oxidative stress, free radicals are released and start a chain reaction of all macromolecules in their vicinity, which if not terminated can lead to lethality. The chain termination is accomplished by the antioxidant molecules which can safely inactivate free radicals before any vital molecules of cells are damaged. Impaired endogenous antioxidant defense systems can be repaired by the dietary natural antioxidants that not only strengthen the cellular antioxidant system but also maintain the optimal balance by removing the ROS from the system (Albasha & Azab, 2014; Al-Mamary et al., 2002; Azab et al., 2017; Azab & Albasha, 2018; Fetouh & Azab, 2014).

18.2.2 ROS-dependent cellular metabolic pathways in cancer cells The rise in ROS level has a positive effect on cancer initiation where cancer stem cells are also activated. However, the toxic effect of excess ROS has a scavenging effect on the cells (Gorrini et al., 2013). To ameliorate the toxicity, some nuclear transcription factors and oncogenes are activated and diminish the ROS level. Nuclear factor erythroid 2-related factor 2 (Nrf2) is one among them that can defend cancer cells even from DNA damage (Hayes & McMahon, 2006; Hu et al., 2006; Iida et al., 2004; Ma, 2013; Ramos-Gomez et al., 2001; Satoh et al., 2013; Xu et al., 2006). At low levels, it is suggested that ROS may aid cancer progression and survival (Martindale & Holbrook, 2002; Ranjan et al., 2006; Shi et al., 2014; Shimura et al., 2016; Wang et al., 2014; Zhao et al., 2015). There are various important molecules that are activated in presence of certain levels of ROS such as expression of cyclin D1, phosphorylation of extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK), and activation of mitogenactivated protein kinase (MAPK), all of which are connected to cancer apart from stimulating alterations in genomic DNA or DNA damage.

18.2.3 Plant-derived antioxidants for the amelioration of oxidative stress Both plants and animals have intracellular antioxidants to maintain complex systems of redox regulation. Cells are damaged and killed due to oxidative stress created by insufficient levels of antioxidants or inhibition of the antioxidant enzymes and consequently increased levels of ROS (Halliwell, 1999). Plant-derived antioxidants are the exogenous source of ROS scavenging agents such as glutathione, vitamin C, vitamin A, and vitamin E as well as enzymes such as catalase, superoxide dismutase, and various peroxidases available for dietary supplements for the prevention of diseases. Researchers are investigating these compounds for their potential to cure cancer, coronary heart disease, and even altitude sickness. Before formulating any phytochemicals, the tumor-promoting (tumorigenesis, angiogenesis, invasion and metastasis, and chemoresistance) and the tumor-suppressive (apoptosis, autophagy, and necroptosis) functions of ROS have to be properly understood (Galadari et al., 2017). A number of transcription factors involved in ROS-mediated tumorigenic activities are activator protein 1 (AP-1), nuclear factor-κB (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), hypoxia-inducible factor-1α (HIF-1α), and p53 (Klaunig et al., 2010). Intracellular ROS-mediated tumorigenesis and progression can be reduced by using antioxidants alone or a combinatorial dose with conventional anticancer agents. Supporting this data, overexpression of SOD3 resulted due to the treatment in two models (experimental lung metastasis model and syngeneic mouse model) where inhibition of breast cancer metastasis occurred as reported by Teoh-Fitzgerald et al. (2014). On the contrary, for ROS-mediated cancer cell death some anticancer agents have to increase the ROS to a toxic level, thereby activating cell death pathways. The effect of these agents could be explained as either due to increased ROS generation, inhibition of antioxidant defense, or a combination of both. Several such agents are currently being used for the treatment of cancer or are under clinical trials (Tables 18.1 and 18.2). Considering the fact that cancer cells already have increased ROS levels, therapeutic strategies that further

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TABLE 18.1 ROS-dependent anticancer mechanism influenced by phytocompounds in cancer cells (Khan et al., 2022). Compounds/class/plant origin

Mechanism

Andrographolide, Diterpene Andrographis paniculata

Colon cancer in vitro research reveals an increase in intracellular ROS levels

Corosolic acid, Triterpene Lagerstroemia speciosa

Lipid ROS induction and an in vitro kidney cancer investigationBreast cancer in vitro study: rise in ROS generation and fall in VEGF concentration

D-limonene, Monoterpene Citrus aurantium

Caspase-3 and caspase-9 activation and ROS production during an in vitro study of prostate cancer

Glycyrrhizin, Triterpenes Glycyrrhiza glabra

Causes breast cancer to undergo ROS-mediated apoptosis both in vitro and in vivo.

Lycopene, Carotenoid Solanum lycopercicum

Inhibition of ROS-mediated lNF-KB signaling, in vitro study on pancreatic cancer Increase in SOD, and CAT, while decrease in MDA levels, in vivo study on gastric cancer

Sugiol, Diterpene Salvia prionitis

Pancreatic cancer in vitro study: ROS-mediated lNF-KB signaling inhibition In vivo study on gastric cancer: SOD and CAT levels rise as MDA levels fall

Ursolic acid, Triterpene Oldenlandia diffusa

Downregulation of Nrf2 expression, in vitro study on breast cancer

Aloperine, Alkaloid Sophora alopecuroides

ROS activation, in vitro study on ovarian cancer

Artemisinin, Sesquiterpene Artemisia annua

Increased ROS generation in studies on colon cancer conducted both in vitro and in vivo

Harmine, Alkaloid Peganum harmala

Lung cancer in vitro research: suppression of AKT phosphorylation and increased ROS production

Plumbagin, Phenolics Plumbago zeylanica

Colon cancer research using in vitro ROS induction Prostate cancer research including the induction of ROS generation and the activation of ER stress Caspase-9 activation and ROS generation in an in vitro lung cancer investigation

Hesperidin, Flavanoids Citrus limon

Increased ROS levels, controlled MAPK signaling, and in vitro investigation of gastric cancer triggers apoptosis by generating ROS, study of prostate cancer in vitro

TABLE 18.2 Phytochemicals in clinical trials. Candidates in clinical trial phase I/II Compound name and plant

Cancer system

Trial phase

Curcumin Curcuma longa

Leukemia, lung cancer, prostate cancer

Phase I

Pomiferin Maclura pomifera

Breast cancer

Phase I

Flavopiridol Amoora rohituka

Chronic lymphocytic, leukemia, lung cancer, prostate cancer

Phase 1/II

Sulforaphane Brassica oleracea

Prostate cancer, osteosarcoma

Phase 1/II

Omacetaxine Cephalotaxus harringtonia

Hematological cancer

Phase II

Combretastatin Maclura pomifera

Esophageal, small-cell lung, melanoma ovarian cancer

Phase II

Irinotecan Maclura pomifera

Lung, colorectal cancer

Phase II/III

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increase ROS generation can specifically trigger cell death in cancers compared to their normal counterparts. Chemicals like beta-phenylethyl isothiocyanate and 2-methoxyestradiol were shown to selectively induce death in human leukemia cells over normal lymphocytes by causing a further increase in ROS (Trachootham et al., 2006). Mannose, a potential saccharide candidate in disease management, which is present in fenugreek, ivory nuts, guar gums, and even coffee has been found to have anti-tumour effect as it impairs tumour growth and enhances chemotherapy (Gonzalez et al., 2018; Dhanalakshmi, 2023).

18.3 Mode of action of phytochemicals for cancer prevention by targeting cellular signaling transduction pathways The ability of anticancer phytochemicals to control adaptive cellular stress response pathways is one of their usual modes of action. The full picture of how molecules interact to determine the fate of cancer cells is still not entirely apparent. Nevertheless, cellular regulation is carried out by signal transduction, which typically begins with interactions between extracellular substances and cell membrane receptors, followed by cascades consisting of regulatory proteins and/or transcription factors, which affect the expression of oncogenes and defensive proteins and lead to various cell fates (such as apoptosis, cell cycle arrest, proliferation, etc.). Disruption in equilibrium between proliferation and death of cells leads to tumor formation. A number of signal transduction pathways are involved in cell cycle and programmed cell death (apoptosis). Cell injury and DNA damage direct toward apoptosis. According to a study, of 121 drugs being used as cancer therapeutics, 90 were found to be plant-derived, among which 74% were formulated primarily based on traditional practices and faith (Winston & Beck, 1999). Anticancer activity of phytochemicals through apoptosis induction in the intrinsic pathway is believed to start with the release of cytochrome C from the loss of mitochondrial membrane potential. The final targets of cell signaling pathways primarily consist of apoptosis regulators, such as Bax, Bad, Bcl-2 family proteins, and caspases; cell cycle regulators, such as myc and checkpoint kinase (Chks); and proliferation regulators, such as cyclins, cell division cycle proteins (Cdcs), and cyclin-dependent kinases (CDKs). Activation and expression of proapoptotic molecules like caspase, Fas, FADD, p53, and c-Jun lead to apoptotic pathways. Survival pathways like Akt signaling and other pathways are inhibited which involve phosphorylation of ERK, P13K, Raf, surviving gene, STAT 3, and nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB) inhibition and downregulation. In Fig. 18.1, the main signaling pathways are illustrated. The exact mechanism of cancer prevention and cure by phytochemicals is still a topic of research. They can exert a wide and complex range of actions on the nuclear and cytosolic factors of a cancer cell. A biomolecule can suppress the malignant transformation of an initiated pre-neoplastic cell or it can block the metabolic conversion of the pro-carcinogen. The Plant-based Anticancer Compound-Activity-Target database (NPACT) gathers the information related to plant-derived natural compounds proven by several experiments which exhibited the anticancer activity both in vitro and in vivo. It contained more than 1500 compound entries, and every record provided FIGURE 18.1 Significant signaling pathways that promote and progress carcinogenesis.

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information on the structure, already published data from both in vitro and in vivo experiments along with references and values like IC(50)/ED(50)/EC(50)/GI(50), properties like physical, elemental, and topological and several other properties (Mangal et al., 2013).

18.3.1 Anti-inflammatory targets Persistent inflammation caused due to various tissue disturbances raises the risk of cancer. Anti-inflammatory therapies have the potential to enhance the effectiveness of both conventional treatments and next-generation immunotherapies in addition to preventing or delaying the beginning of cancer (Greten & Grivennikov, 2019). Nonsteroidal antiinflammatory medicines (NSAIDs) target cyclooxygenase 1 (COX1) and COX2, which can activate AKT, mTOR, and NF-B to boost cancer cell survival and proliferation either directly or by producing prostaglandin E2 (PGE2) (Pannunzio & Coluccia, 2018). PGE2 can bind to the PGE2 receptor EP4 and trigger intracellular signal transduction when it is generated in a COX2-dependent way. While COX2 works to upregulate the expression of DNA (cytosine 5)methyltransferase 1 and/or 3B (DNMT1/3B), EP4 signaling operates to downregulate the expression of DNA demethylase TET1. Tumor suppressor genes are silenced as a result of the altered expression of both of these epigenetic regulators, which encourages the development of cancer (Hou et al., 2021). Fig. 18.2 demonstrates important phytochemicals used as an alternative medicine to target such inflammatory pathways. The Cox-1 is potential target of apigenin, naringenin, pelargonidin, (2)-epicatechin, peonidin, malvidin, etc. The Cox-2 pathway can be inhibited by curcumin, natsudaidai, brucin, auraptene, etc. More than 30 phytochemicals were identified as potential inhibitors of NF-kB (see Fig. 18.3).

FIGURE 18.2 Anti-inflammatory targets of phytochemicals for cancer prevention.

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FIGURE 18.3 Phytochemicals targeting major growth signaling pathways.

18.3.2 Growth factor signaling targets Growth factor receptor (GFR) signaling responds to various endogenous or exogenous stimuli and activates downstream signaling pathways, thus controlling epithelial cell growth (Tiash & Chowdhury, 2015). They are categorized into various families as per their molecular evolution, target cells, functions, and vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) families, vascular endothelial growth factor (VEGF) family, Wnt family, epidermal growth factor (EGF) family, platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF) family (Tiash & Chowdhury, 2015). PI3K/Akt/mTOR, MAPK, and c-Src are some of the key pathways involved in cell proliferation and differentiation. AKT1 is the most intriguing target out of the roughly 20 targets investigated within the growth factor signaling pathway, with over 25 phytochemicals specifically targeting this protein (Fig. 18.3), including those phytochemicals that are present in our regular diet apple, fisetin, genistein, and gallic acid from green tea, quercetin (onion, apple, broccoli), luteolin (parsley, celery, pepper, dandelion), myricetin (grapes, onions, tea), etc.

18.3.3 Apoptosis targets The natural process of cell death referred to as apoptosis represents a potential target for the treatment of cancer (Garcı´a et al., 2012). Caspases are used by both the intrinsic and extrinsic pathways to carry out apoptosis by cleaving a large number of proteins. The overexpression of antiapoptotic proteins and the underexpression of proapoptotic proteins are two common ways that the apoptotic pathway is inhibited in cancer (McIlwain et al., 2013). Chemotherapy, the most widely used anticancer treatment, is intrinsically resistant to many of these changes. Plant-derived substances that exhibit anticancer activity by triggering the apoptotic pathway are promising new cancer treatments (Ahmed et al., 2019). Numerous phytochemicals, as shown in Fig. 18.4A, target crucial apoptotic pathways such as caspase 3, caspase 8, and caspase 9. There are more than 20 phytochemicals which are acting on all the three targets including allicin from garlic which is part of our daily diet, nimbolide, a neem limonoid, and butein, a chalcone found in Toxicodendron

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FIGURE 18.4 (A) Role of different phytochemicals in the regulation of caspase in apoptosis pathway. (B) Role of different phytochemicals in regulation of BCL2 and BAX apoptosis pathway.

vernicifluum (or formerly Rhus verniciflua), Dahlia, Butea (Butea monosperma). Fig. 18.4B illustrates that more than 40 phytochemicals are effective on BAX and BCl2 pathways.

18.3.4 Targets of phytochemicals in cell cycle pathways The mammalian cell cycle is a highly structured and controlled process that makes sure that genetic material is duplicated and cells are divided. Growth regulatory signals are involved in this regulation, as well as signals sent by proteins

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FIGURE 18.4 (Continued).

that check the integrity of the genome for signs of genetic damage (Otto & Sicinski, 2017). The progression through the four distinct cell cycle phases (G0/G1, S, G2, and M), which are controlled by a number of cyclin-dependent kinases (CDKs) that work in conjunction with their cyclin partners, is necessary for proliferation (Visconti et al., 2016). The activation of cell cycle checkpoints in response to DNA damage can inhibit the activity of CDKs involved in cell cycle regulation, which is tightly regulated and induced by mitogenic signals (Johnson & Shapiro, 2010). There has been a lot of interest in the development of cyclin-dependent kinase inhibitors during the last two decades. This interest arose originally from discoveries that various CDK isoforms play important roles in cancer cell proliferation via loss of cell cycle control, a characteristic trait of cancer (Whittaker et al., 2017). CDKs have now been demonstrated to influence other activities, most notably transcription. Early nonselective CDK inhibitors demonstrated significant toxicity and were found to be inadequately active in most malignancies. Fisetin (found in strawberries, apples, mangoes, persimmons, kiwis, and grapes), hesperidin (from oranges, grapefruit, lemon, and tangerines), tricin (whole cereal grains, such as rice, barley, oats, and wheat), evodiamine, and (1)-gallocatechin are the major inhibitory agents of cyclin B1. Cell cycle target inhibitors from various plant sources are depicted in Fig. 18.5.

18.3.5 Targets in other important pathways Advanced cancer is a complex illness that necessitates therapies that target diverse biological pathways. Several phytochemicals target many dysregulated pathways in cancer cells and so provide an alternative/complementary method of cancer treatment. The sequence of events that leads to tumor cell invasion and metastasis is critical in the prognosis of cancer patients. Tumor metastasis is also a drug target that involves a sequence of phases that result in the establishment

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FIGURE 18.5 Phytochemicals accountable for cell cycle regulation.

of secondary tumors in distant organs and is mostly responsible for cancer mortality and morbidity (Martin et al., 2013). Invasion, metastasis, angiogenesis, and stemness are distinct pathways that are targets of various phytochemicals, as shown in Fig. 18.6. The matrix metalloproteinases (MMPs) play a role in colorectal cancer invasion and metastasis. Curcumin, gingerols, scopoletin, morin, and gallic acid have inhibitory effects on MMP2. Luteolin and quercetin also demonstrated inhibitory effects on the transcription factor TWIST-1. Fig. 18.7 depicts a few unclassified phytochemical targets such as genome integrity, drug resistance, growth factor and hormone signaling, drug metabolism and carcinogen metabolism, and immune evasion.

18.4

A historical perspective of plant-derived drugs used popularly in cancer

The potentials of natural products as therapeutics have been recognized that can fight against various diseases including cancer for over 50 years (Marı´a Elena Martı´nez). In the 1960s, National Cancer Institute (NCI) introduced an anticancer drug screening program intending to find natural products with an anticancer activity that led to the discovery of taxanes (taxol, baccatin III, and 10deacetylbaccatin III) (1964) and camptothecins (1966) (Chabner & Roberts, 2005). Since then, several types of anticancer drugs like apigenin, colchicine, and resveratrol are in clinical use, and most of them belong to the class of small molecule natural products. Functionally, the phytochemicals are of two classes: primary and secondary metabolites (Hussein & El-Anssary, 2019). For normal physiological growth and energy requirements, the plants solely depend on the primary metabolites that are distributed widely in all plants (Hussain et al., 2012; Wink, 2016), while secondary metabolites are required in plants basically for protection against various negative effects of environmental factors (Iriti & Faoro, 2009). Different taxonomic groups of plants possess different types and levels of these phytochemicals (Adeyemi & Mohammed, 2014;

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FIGURE 18.6 Role of phytochemicals in suppression of invasion, metastasis, angiogenesis, and stemness-related pathways in cancer.

FIGURE 18.7 Role of phytochemicals in suppression of genome stability, drug resistance, growth factor and hormone signaling, drug metabolism and carcinogen metabolism, as well as a few unclassified targets, immune evasion.

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Tiwari & Rana, 2015). After providing protection to plants, these chemicals are well known to provide protection to animals and humans as various pharmacological and medicinal sources for many drugs of natural origin to cure diseases from migraine to cancer (Hussein & El-Anssary, 2019; Jain et al., 2019; Velu et al., 2018). A very low-level production of these secondary metabolites in plants is a great concern; hence, a large amount of plant material is used to extract the drug molecules (Dar et al., 2017).

18.4.1 Important secondary metabolites in cancer treatment Plant defense metabolites arise from the isoprenoid, the alkaloid, and the phenylpropanoid pathways which form three major secondary metabolites (Sruthi & Jayabaskaran, 2021; Iriti & Faoro, 2009). Secondary metabolites are categorized primarily as terpenoids, alkaloids, and phenolics based on their chemical nature (Shamina & Sarma, 2001). We are focusing on the selected class of compounds such as terpenoids, alkaloids, and flavonoids, the major contributors from the phenolic category.

18.4.1.1 Terpenes (terpenoids) Terpene class has the largest number of compounds (513) in the Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT) (Mangal et al., 2013) and is ubiquitous in plants with more than 22,000 compounds (Freeman & Gwyn, 2008) basically secondary metabolite products. All terpenes are derived from branched, basic, five-carbon unit isoprene (C5H8) (Bramley, 1997; Goodwin & Mercer, 2003). Fig. 18.8 shows multiple terpenoids that have inhibitory effects on different types of cancers. Andrographolide, lycopene, corosolic acid, and ursolic acid are found to be effective on more than half-a-dozen cancer types. Table 18.2 gives the mechanism of action of these phytochemicals along with the source plant.

18.4.1.2 Alkaloids Alkaloids symbolize another larger group of heterocyclic, physiologically active secondary metabolites. Over 2000 alkaloids are known from plants and are distributed in all parts of the plant. Dicotyledonous plants are richer in alkaloids than monocotyledonous plants, but their distribution to lower plants is limited (Goodwin & Mercer, 2003). Plant parts from Apocynaceae, Rubiaceae, Solanaceae, Papaveraceae, Leguminosae, and Fumariaceae are rich in alkaloids and from Rosaceae and Labiatae are low in alkaloids. Alkaloids have many ecological, biochemical, and physiological functions in plants. They protect plants from insects and predators, act as nitrogen excreta such as uric acid and urea in animals, and also act as nitrogen storage and growth regulators. It also includes maintaining ion balance under chelating power (Goodwin & Mercer, 2003). The most common cancer treatments are chemotherapy and radiation therapy. On the other hand, the emergence of adverse effects from chemotherapy and radiation therapy limits its therapeutic use.

FIGURE 18.8 Terpenoids having anticancer activities in different types of cancers.

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FIGURE 18.9 Alkaloids as anticancer agents against varieties of cancers.

Natural dietary supplements containing nutrients such as grape seed extract, ginseng extract, and curcumin can help people with serious illnesses, oral mucositis, gastrointestinal toxicity, hepatotoxicity, renal toxicity, and damage to the blood system and help recover from the side effects of conventional therapy and radiation therapy. The cardiotoxicity and hepatotoxicity caused by chemotherapy and radiation therapy (Chandrasekar et al., 2018a,b; Patil et al., 2018; Thakur et al., 2020) are also ameliorated. Many chemotherapeutic regimens are used to treat cancer, but cancer recurrence and drug resistance are very frequent. It is the root cause of high mortality. Studies of various phytochemicals, which are natural chemicals derived from plants, show anticancer activity and are effective against other diseases. These phytochemicals contain alkaloids, which are almost colorless and odorless crystalline solids. However, it may also appear as a yellowish liquid (Aniszewski, 2015). Various studies have demonstrated the potential of alkaloids and their strong biological effects in animal and human models at minimum doses including different malignancies. Fig. 18.9 shows alkaloids that are studied in different cancer types, and the mechanisms inferred are tabulated in Table 18.2. In traditional medicine, alkaloids are well known and have been used by people of all ages due to their diverse effects. However, there were no direct means of separating pure compounds. The potential effects of these herbal anticancer drugs have been previously reported. Vinblastine and vincristine anticancer properties are well documented. It acts as an anion scavenger (Zhang et al., 2018). Taxol, originally isolated from Taxus species plants, is the most effective drug against breast and ovarian cancer, leading to mitotic arrest by stabilizing tubulin polymerization (Torres & Horwitz, 1998). Vinca alkaloids are one of the important anticancer agents isolated from Catharanthus roseus, bind to tubulin, and avert the polymerization of tubulin, leading to the disturbance in mitotic spindle assembly (Moreno et al., 1995). Several other compounds such as epothilones, discodermolide, eleutherobin, laulimalide, and isolaulimalide were found to be antimitotic to cells by inhibiting tubulin depolymerization taxanes. Plant-derived anticancer drug camptothecins (irinotecan, topotecan) are found to be an inhibitor of topoisomerase-I (Fronza et al., 2012).

18.4.1.3 Flavonoids Flavonoids are one of the biggest class of polyphenols. Plants with higher flavonoids content are considered to be used to extract chemopreventive agents (Sak, 2014) as several flavonoids possess anticancer and cytotoxic activity (George et al., 2017) (Fig. 18.10). Flavonoids are one of the common components in the human diet found in vegetables, fruits, and some herbs. As per the report in various countries like Finland, Japan, United States of America, Italy, and the Netherlands, the average consumption of important flavonoids as composite foods comprises quercetin (6 mg/day), kaempferol (64 mg/day), myricetin (13 mg/day), luteolin (27 mg/day), and apigenin (33 mg/day) (Shukla & Gupta, 2010). According to the database (Haytowitz et al., 2018) of the United States Department of Agriculture (USDA), different vegetables, fruits, cereals, and beverages are rich sources of different types of flavonoids. There are six subclasses of

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FIGURE 18.10 Flavonoids as therapeutics for different cancers.

flavonoids mainly found in food materials. (1) The predominant flavonols that occur in nutrients and possess protective activity are quercetin, kaempferol, myricetin, and isorhamnetin. (2) Dietary flavones that extensively occurred and are used as nutraceuticals are luteolin and apigenin. (3) Bioavailable flavanones which exhibit several pharmacological actions are hesperetin and naringenin. (4) Flavan-3-ols presence in food maintains its quality such as taste, aroma, and microorganism stability. Several flavan-3-ols like catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin 3gallate, and epigallocatechin 3-gallate provide numerous health benefits. (5) Anthocyanidins are natural food colorants. The major anthocyanins present in plants are cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. (6) Isoflavones are found in a few plants. They are known as phytoestrogens. Isoflavones like genistein, daidzein, and glycitein are present in nutrient supplements. Flavonoids are water-soluble. It possess hydroxyl groups at different positions.

18.4.2 Other important phenolic compounds studied on cancer targets Almost around 400 (B393) phytochemicals are known to interact with the above-mentioned targets of cancer, and discussing each one will be beyond the scope of this chapter. We were discussing the important ones taking the representative compounds from the plant secondary metabolites classes, namely, terpenoids, alkaloids, and phenolics. Here, we discuss a few important groups under phenolics which are getting more attention in antitumor research.

18.4.2.1 Chalcones The tumor necrosis factor
related apoptosis-inducing ligand (TRAIL) is a protein that exists in soluble form or is expressed on the surface of immune effector cells. This ligand induces apoptosis in cancer cells while showing no toxicity for normal cells. Preclinical studies have shown that the resistance to TRAIL can be overcome by chalcones. Butein represented increased sensitivity to TRAIL-mediated cell death and in combination with TRAIL, as seen in TRAILresistant leukemia U937 cells (increased caspase-3 and caspase-8 activation). Upregulation of TRAIL-R2 enhances such mechanisms in hepatoma cancer cells, colon cancer cells, and prostate cancer cells (Kim, 2008; Moon et al., 2010). A combination of butein and TRAIL leads to cleavage of caspase-3, -8, -9, Bid, and poly(ADP-ribose) polymerase (PARP) as well as the release of cytochrome C from the mitochondria into the cytosol in hepatoma HepG2 cancer cells. The combined treatment decreased the expression of Bcl-2, XIAP, IAP-1, and IAP-2 in hepatoma cancer cells (Moon et al., 2010).

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FIGURE 18.11 The molecular targets for chalcones in cancer.

Chalcones in combination with TRAIL can sensitize TRAIL-resistant cancer cells to apoptosis. Chalcones such as butein, xanthohumol, isoliquiritigenin, and licochalcone A augment anticancer activities through different mechanisms in the apoptotic pathway (Fig. 18.11). Preclinical and epidemiological studies have shown that chalcones can inhibit cancerogenesis at very early stages (act of chemoprevention). Xanthohumol and other phenols may contribute to the improved health of beer drinkers. As interest in craft beer rises, the phenolic content of beer is being more widely understood, leading to beer emerging as a functional beverage. Beer polyphenols derive not only from hops and barley, the primary ingredients of beer, but from various other ingredients added to impart additional flavors. While the phenolic profile of beer is thus extensive and varied, xanthohumol is a beer-specific polyphenol, being found exclusively in hops. A valuable flavonoid called licochalcone A (LA) is extracted from the Chinese herb Glycyrrhiza uralensis Fisch. The data gathered show that LA has a wide range of pharmacological activities, such as anticancer, anti-inflammatory,

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antioxidant, antibacterial, antiparasitic, bone protection, blood glucose and lipid management, neuroprotection, and skin protection. Multiple signaling pathways, including PI3K/Akt/mTOR, P53, NF-B, and P38, mediate LA activity. Numerous therapeutic targets, including TNF, VEGF, Fas, FasL, PI3K, AKT, and caspases, are also influenced by the apoptosis-related caspase-3, inflammatory-related MAPK, and oxidative stress-related Nrf2 signaling pathways. Isoliquiritigenin (ISL), a chalcone-structured natural bioactive molecule, exhibits strong anticancer activity in a variety of malignancies both in vivo and in vitro. The authors have provided an overview of the function of ISL and a complete account of its usage to date in various therapeutic plans for the treatment of various tumors, either alone or in combination with other medications (Elrod, 2018; Kło´sek et al., 2017; Li et al., 2019).

18.4.2.2 Flavonols Flavonols are a class of flavonoids that have the 3-hydroxyflavone backbone that has many targets (Fig. 18.12). Fisetin (3,30 ,40 ,7-tetrahydroxyflavone), one of the unique dietary compounds, is found in a variety of fruits and vegetables, including apple, persimmon, grape, strawberry, cucumber, and onion. Fisetin is a potent anticancer agent that has been used to modulate the expressions of Bcl-2 family proteins in various cancer cell lines, including HT-29, U266, MDAMB-231, BT549, and PC-3M-luc-6, as well as to inhibit stages in cancer cells (proliferation, invasion), prevent cell cycle progression, inhibit cell growth, induce apoptosis, and cause polymerase (PARP) cleavage. Fisetin also lowers the activation of NF-B, the level of the oncoprotein securin, and the activation of the PKC/ROS/ERK1/2 and p38 MAPK signaling pathways. Fisetin decreased the levels of TET1 expression while also preventing cell proliferation, invasion, and division (Imran et al., 2021). Particularly, the consumption of foods high in kaempferol has been associated with a lower risk of acquiring various malignancies, such as skin, liver, and colon. Apoptosis, cell cycle arrest in the G2/M phase, downregulation of markers associated with the epithelial
mesenchymal transition (EMT), and signaling pathways involving phosphoinositide 3kinase and protein kinase B are some of the mechanisms of action. In this regard, the results from experimental investigations that looked into the associations between kaempferol intake and cancer prevention are reviewed in this article. Even though there is emerging evidence that kaempferol can be used to prevent cancer, more preclinical and clinical FIGURE 18.12 The molecular targets of flavonols in cancer.

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studies employing kaempferol or foods high in kaempferol are crucial before any public health recommendations or formulations using kaempferol (Imran et al., 2019). As a novel source of therapeutics, medicinal plants have the potential to be utilized alone or in conjunction with other medications to treat a variety of malignancies, including ovarian cancer. Among different natural substances, quercetin has demonstrated excellent anti-inflammatory and anticancer capabilities. Quercetin has a cytotoxic effect on ovarian cancer cells, according to in vitro and in vivo tests. Few clinical researches examined the anticancer properties of quercetin, notably in the case of ovarian cancer, despite the compound showing promising outcomes both in vitro and in vivo. It appears that additional clinical research could allow for the introduction of quercetin as a therapeutic agent either by itself or in combination with other chemotherapeutic medicines in the clinical context (Vafadar et al., 2020). A natural flavonol substance called myricetin (3, 5, 7, 30 , 40 , and 50 -hexahydroxyflavone) is present in a wide range of plants, including berries, oranges, grapes, herbs, teas, and wine. Myricetin has been shown to have good biological activity as an antitumor, anti-inflammatory, and antioxidant agent in the last 10 years, according to a converging body of research. Myricetin has been demonstrated to limit cancer cell invasion and metastasis, cause cancer cell cycle arrest and death, and inhibit cancer cell proliferation in investigations involving numerous types of cancer cells. Myricetin has gained more attention as a possible tumor inhibitor in human patients as a result of these discoveries (Jiang et al., 2019).

18.4.2.3 Flavones, flavanones, isoflavones, and flavanols Flavones are mostly found in leaves, flowers, and fruits. Celery, chamomile, mint, parsley, and red peppers are high in flavones. It has a similar structure to flavonols. A double bond is present in the C2
C3 position of the structural ring, while a keto group is present in the C4 position. The OH group is missing at the C3 position (Kumar & Pandey, 2013). It contains flavones such as apigenin, chrysin, luteolin, baicalein, wogonin, and eupatorin. Flavanones are often referred to as dihydroflavones. It belongs to the flavonoid family. It is made up of a saturated carbon ring. Citrus fruits including lemon, oranges, and grapes contain flavanones. It consists of hesperetin, naringenin, and eriodictyon. Isoflavonoids are a distinct subclass of flavonoids. Isoflavonoids like daidzein and genistein are found mostly in leguminous plants such as soybeans (Iwashina, 2013). Some flavones, flavanones, isoflavones, and flavanols and their molecular targets in cancer are depicted in Fig. 18.13A
D.

18.4.3 Phytochemicals in clinical trials To date, a significant number of phytochemicals have been investigated at the preclinical and clinical stages, either as a single agent or in combination with known anticancer medicines. Many clinical studies have been conducted in recent years to examine the toxicity, pharmacokinetics, and biologically beneficial dosage of phytochemicals against cancer. Table 18.2 explains the extensive research on various phytochemicals which are under clinical trial at different stages (Khan et al., 2022).

18.4.4 Common dietary phytochemicals Fig. 18.14 shows the three most widely used dietary phytochemicals with some of their respective cellular targets: curcumin (Tomeh et al., 2019), gallic acid (Jiang et al., 2021), and geraniol (Cho et al., 2016).

18.5

Phytochemicals induce cancer cell apoptosis and autophagy

During pathophysiological stimuli, apoptosis plays a pivotal role as a cell death mechanism to sustain cellular homeostasis (Chen & Yu, 2013). Malignant cells generally undergo successive genetic mutations that are essential for their survival under pathological stimuli avoiding apoptosis. Cancer cells generally evade apoptosis by disrupting the balance between proapoptotic and antiapoptotic proteins. Reduced caspase activity and impaired death receptor (DR) signaling also help the cancer cells evade apoptosis. Autophagy is a conserved intracellular catabolic degradation mechanism that removes intracellular cargos such as misfolded proteins, foreign particles, and dysfunctional cellular organelles to maintain cellular homeostasis under external stimuli, oxidative stress, and different pathological conditions such as cancer (Kaur & Debnath, 2015; He & Klionsky, 2009). In malignant cells, autophagy can act in two contradictory processes, associated with tumor suppression and tumor progression (Maiuri et al., 2009). As it is believed, in normal cells autophagy is engaged in the removal

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FIGURE 18.13 (A) Molecular targets of flavones. (B) Molecular targets of flavanones. (C) Molecular targets of isoflavones. (D) Molecular targets of flavanols.

of stress products that trigger tumorigenesis, and blocking autophagy with various therapeutics in established tumors may suppress tumorigenesis (Mathew & White, 2011). Targeting autophagy can uncover an alternative cell death mechanism in cancer cells where apoptosis is invaded. The autophagy-modulating phytochemicals might serve as magic bullets against apoptosis-deficient/resistant, multidrug-resistant cancer cells. Dietary phytochemicals are highly potent in controlling cancer being associated with various cell death pathways such as apoptosis, autophagy, cell cycle, inhibition in cellular proliferation, invasion and migration, which are indispensable in enhanced anticancer activity. A number of dietary phytochemicals such as kaempferol, noscapine, codeine, sulforaphane, epigallocatechin-3-gallate (EGCG), curcumin, β-carotene, resveratrol, quercetin, and lycopene are evidenced to induce cell death through modification of apoptosis and autophagy (Patra et al., 2021).

18.6 Gut microbiota in gastrointestinal malignancy—a potential target for phytotherapy Human gastrointestinal tract harbors a huge number of microorganisms, comprising bacteria, viruses, fungi, and archaea combined known as gut microbiota. The entire genome of the gut microbiota is known as the gut microbiome (Sidhu & van der Poorten, 2017; Vivarelli et al., 2019). During the first few years of life, the quality and quantity of the microbiome remain unstable while it stabilizes, maintaining a proper symbiotic balance (Shreiner et al., 2015).

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FIGURE 18.13 (Continued).

The gut and its microbiota can function together in one of two ways: in peaceful coexistence (symbiosis) or in a harmful state (dysbiosis) (Beliza´rio & Faintuch, 2018). Under healthy symbiotic conditions, the gut microbiota performs various beneficiary activities, including the production of vitamins, metabolism of dietary compounds and/or drugs, and protection against gut pathogens (Shreiner et al., 2015; Sidhu & van der Poorten, 2017) whereas under unfavorable

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FIGURE 18.14 Molecular targets of common dietary phytochemicals.

conditions an imbalanced dysbiotic condition develops when the gut is housed with a greater proportion of diseasecausing microbes, which can lead to gastrointestinal tract malignancies (Vivarelli et al., 2019). Certain gut microbes are associated with specific gastrointestinal malignancies, such as Helicobacter pylori and Epstein
Barr virus (EBV) with gastric adenocarcinoma (GC) (Cheng et al., 2016); H. pylori with gastric mucosa-associated lymphoid tissue (MALT) lymphoma (Floch et al., 2017); H. pylori, Escherichia coli, Mycoplasma species, and Streptococcus bovis with colon cancer (Abdulamir et al., 2011; Butt & Epplein, 2019; Tsai et al., 1995); and human papillomavirus (HPV) with squamous cell carcinoma (SCC) of the anal canal (Armstrong et al., 2018; Juneja et al., 2021). Gut microorganisms play a key role in immunomodulation and establish the mucosal innate and adaptive immune system (Jandhyala et al., 2015; Shreiner et al., 2015). Gut microbiota also communicates with the central nervous system through the vagus nerve and hypothalamic-pituitary-adrenal axis by the production of neurotransmitters and their precursor (Shreiner et al., 2015; Sidhu & van der Poorten, 2017). Infections, antibiotic use, specific hygienic practices, dietary changes, and even vacation travel can result in temporarily unstable gut microbiota. However, in most cases, complete recovery is eventually restored (Shreiner et al., 2015).

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The speculated pathogenesis of microbiota-induced gastrointestinal malignancies (GMs) falls into one of the three categories: (1) host cell DNA damage and cytotoxicity, (2) regulation of proinflammatory signals to induce inflammation, and (3) controlling tumorigenic/tumor-suppressing pathways. These three mechanisms may injure the gastrointestinal epithelium, and poor healing can result in neoplastic transformation. For example, cytotoxin-associated gene A (CagA) protein from H. pylori can degrade tumor protein p53 in gastric epithelial cells and activate the β-catenin pathway, which predisposes to gastric cancer and MALT lymphoma (Cheng et al., 2016; Floch et al., 2017). Research studies on phytochemical intervention in gut microbiota influenced cancer development and are at its infant stage. However, the recomposition of microbiota using phytochemicals could be considered as a ray of hope for cancer mystery solution, and scientists could offer coherent answers to many questions related to the cause and cure of various cancers.

18.7

Plant-derived drugs

The Plant-based Anticancer Compound-Activity-Target database (NPACT) gathers the information related to plantderived natural compounds proven by several experiments which exhibited the anticancer activity both in vitro and in vivo. It contained more than 1500 compound entries, and every record provided information on the structure, already published data from both in vitro and in vivo experiments along with references and values like IC (50)/ED (50)/EC (50)/GI (50), properties like physical, elemental, and topological and several other properties (Mangal et al., 2013).

18.8

Conclusion

Ethnopharmacological potential in plants due to the presence of bioactive phytoconstituents is now known to all. Since ancient times, plants have been employed as therapeutic agents in naturopathy. People are inclined toward this alternative therapy to avoid the high expense and severe side effects of conventional chemotherapy as phytochemicals are more widely available and less expensive. Hence, the goal of this study is to provide an overview of the development of natural, safe, effective, and economical therapeutic agents/drugs against various cancers commonly found as plantderived anticancer chemicals. Moreover, the plants considered to have anticancer properties are mostly in crude form and still need advanced research for the isolation of phytochemicals and establishing their cellular and molecular role in treating cancer. In the present article, the authors have attempted to simplify the job of researchers by developing interaction graphs of active components in plants against their appropriate targets.

18.9

Challenges

In this unique situation, the academic environment has begun to look for imaginative sources of information. Anticancer drug blends in regular sauces containing traditional plants. Various investigations are currently underway for in vitro assessment of anticancer properties of a normal mixture of plants also in vivo. In the preclinical stage, you can see some promising mixtures such as sulforaphane and various phenolic compounds. On the other hand, some phytochemicals such as vinca alkaloids and paclitaxel showed positive clinical results and were supported in addition to the treatment of malignant tumors. These mixtures are gradually exempt from restrictions: poor solvency, limited impact, negative side effects, etc. This audit expects to accumulate data about the ongoing phytochemicals utilized for disease treatment and encouraging up-and-comers, fundamental activity components, and furthermore revealed impediments. In this sense, a few systems to face the limits have been thought of, for example, nano-based definitions to further develop solvency or synthetic change to lessen harmfulness. All in all, albeit more exploration is as yet important to foster more proficient and safe phytochemical drugs, and a greater amount of these mixtures may be utilized in future malignant growth therapies. Traditional plants have been generally viewed as a perpetual wellspring of new compounds for the advancement of new drugs and medications. Subsequently, these days specialists have at their total removal a lot of ethnomedicinal and ethnopharmaco-consistent data of totally different plant species which is a device for choosing up-andcomers and also leads the examination of those plants really encouraging. Because of this information, various instances of phytochemicals with restorative properties against various illnesses can be found all through the literature. Cancer is an intricate sickness that consistently costs a few huge numbers of living souls. The uncontrolled multiplication of cells causes the mistaken working of the body, with a considerable rundown of side effects lastly, passing. Considering not only the health and social importance of this disease, but also its economic impact on the health framework, new and useful other options are born.

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It will be checked consistently. For this reason, some basic factors such as medical history, survival, induced tumorigenic changes, possible side effects, and drug harm need to be taken into account. Under these special circumstances, various plant-derived phytochemicals have been discovered and are now used in the treatment of diseases. Current reviews are collecting models that have been shown to apply anticancer effects to preliminary clinical studies and are approved for clinical use, such as vinca alkaloids, paclitaxel, and irinotecan. By the way, these mixtures are not exempt from restrictions such as low solubility, limited effect, and harmful side effects. This article also contains several blends being considered in preclinical and clinical studies. However, further preliminary clinical work is required before further protected use. Finally, after a brief description of the compositional data collected, attempts were made to identify the underlying difficulties in the fight against cancer.

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Chapter 19

Applications of phytochemicals in cancer therapy and anticancer drug development Sachin Puri, Namita Hegde, Siddhi Sawant, Ganesh Latambale and Kapil Juvale Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (W), Mumbai, Maharashtra, India

19.1

Introduction

Cancer is the most common genetic disorder, and it remains a leading cause of mortality and morbidity worldwide, with a significant global impact on both economically developed and developing countries (Frontiers et al., 2022). According to the National Centre for Health Statistics, approximately 19.8 million cancer patients were reported in 2022, with 0.6 million deaths registered worldwide (Siegel et al., 2022). The Cancer Statistics Report of 2020 demonstrated that incidence among men and women was found to be 94.1 per 100,000 persons and 103.6 per 100,000 persons, respectively. From 2010 to 2019, new cases of cancer increased from 18.7 million to over 23 million, and deaths regarding cancer reached 10 million from 8.29 million, respectively, (India’s cancer burden: cases, & deaths increased last decade, COVID-19 widens screening gap, 2022). Chemotherapy and radiotherapy are the only effective treatments available currently, but the toxicity of currently available drugs, climate changes, and lifestyle as well as the development of resistance to existing drugs highlights the need for the development of newer anticancer agents with low toxicity and high efficacy. As a result, many scientists are working hard to develop new and safer alternative strategies that can solve the problem caused by conventional methods (Martı´n Ortega & Segura Campos, 2021). Carcinogenesis is a multistep process that involves multiple signaling pathways. Because of their pleiotropic effects on target events in multiple ways, phytochemicals are considered promising candidates for anticancer drug development. These phytochemicals are currently being studied to determine whether they can be used as potential candidates (those that can inhibit the growth of tumor cells without causing any undesired side effects). Phytochemicals and their analogs have been recognized as anticancer therapy candidates. Herbal medicines have long been and continue to be used as the primary source of medical treatment in developing countries. The natural antiseptic properties of plants have been used in medicine for centuries. As a result, research has focused on the properties and applications of terrestrial plant extracts in the preparation of potential nanomaterial-based drugs for diseases such as cancer (Sivaraj et al., 2014). Higher plants have an advantage in drug discovery over synthetic drugs because they have a long history of human use as food and spices. Active components isolated from secondary metabolites derived from plants are likely to be superior and safer than those derived from synthetic chemicals, and their anticancer activities are being investigated, leading to the development of new clinical trials (Greenwell & Rahman, 2015; Katiyar et al., 2012). Many plant-derived drugs are currently being used to treat cancers such as prostate, breast, and colorectal cancer. Several researchers have identified plant species with anticancer properties, with a focus on those used in herbal medicine in developing countries (Cai et al., 2006; Costa-Lotufo et al., 2005; Fouche et al., 2008; Freiburghaus et al., 1996; Ochwang’i et al., 2014). Phytochemicals, belonging to plants’ secondary metabolites, are a group of powerful compounds that includes a wide range of chemical substances such as flavonoids, polyphenols, steroidal saponins, vitamins, and organosulfur compounds. They mainly focus on plant development, playing a role in the appropriate physiological processes, that is, symbiotic association, reproduction, and interactions with the environment and other organisms (Forni et al., 2019). However, most of the compounds are constitutively produced, and their production can be increased under stressful Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00026-8 © 2023 Elsevier Inc. All rights reserved.

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conditions, in a dependent manner on stressor and growth conditions (Akula & Ravishankar, 2011). Constituents of phytochemicals are important for the proper functioning and survival of plants. These agents contribute protection from herbivores, competitors, and microorganisms by regulating growth (e.g., by delaying the time of seed germination till the arrival of the appropriate time) and pollination control, rhizosphere environment, and fertilization. Some phytochemicals can produce inherent biological properties which include toxic or poisonous effects. Careful monitoring can mostly identify the effect and cause with considerable level of biological effects produced, enveloping the most severe such as organ damage, death, birth defects, and reproductive failure, to more favorable but however economically essential factors which include addiction/ habituation, failure to thrive, or malnutrition (Molyneux et al., 2007). Plant active constituents that are required for plant survival and “housekeeping” of the organism are being studied for their ability to prevent cancerous cells from growing and initiating apoptosis. Table 19.1 discusses the important

TABLE 19.1 List of some important natural products and their phytochemicals against cancer. Sr. no

Cancer type

Phytochemicals

Plant name

Refs.

1

Breast cancer

Harmine

Peganum harmala

Ayoob et al. (2017)

2

Glioblastoma, colon cancer, leukemia

Curcumin, ascorbic acid

Curcuma longa

Ooko et al. (2017)

3

Breast, prostate, and cervical cancer

Steroids, terpenoids, flavonoids, glycosides

Allium wallichii

Bhandari et al. (2017)

4

Breast, liver, and pancreatic cancer

Artemisinin

Artemisia annua

Efferth (2017)

5

Internal tumor

Tannins

Debregeasia saeneb

Tariq et al. (2017)

6

Liver, breast, fibrosarcoma

Eugenol, orientin

Ocimum sanctum

Preethi and Padma (2016)

7

Hepatocarcinoma, prostate, ovary, liver, and colon cancer

Ginkgetin, ginkgolide A and B

Ginkgo biloba

Xiong et al. (2016)

8

Breast and lung cancer

Doxorubicin, spinanine A, rutin, quercetin

Ziziphus spinachristi

Jaradat et al. (2016)

9

Stomach, kidney, prostate, and breast cancer

Licochalcone A, licoagrachalcone

Glycyrrhiza glabra

Zhang et al. (2016)

10

Abdominal cancer

Moringa oleifera protease inhibitor (MoPI)

Moringa oleifera

Srikanth and Chen (2016)

11

Leukemia, melanoma, breast, and lymphoma cancer

Lupeol

Aegle marmelos

Wal et al. (2015)

12

Cervix, ovary, liver, and urinary cancer

Gingerol

Zingiber officinale

Rastogi et al. (2015)

13

Colon cancer

Andrographolide

Andrographis paniculata

Osman et al. (2015)

14

Non-small cell lung cancer

Podophyllotoxin

Podophyllum peltatum

Choi et al. (2015)

15

Lymphoid, glioma, liver, lung, ovary

Sinapic and ferulic acid

Annona coriacea

Formagio et al. (2015)

16

Rectum, testis, lung, cervix, ovary, and breast

Vinblastine, vincristine

Catharanthus roseus

Keglevich et al. (2012)

17

Nasopharyngeal cancer

Alkaloids, saponins, flavonoids, tannins

Carissa spinarum

Sahreen et al. (2013)

18

Breast and ovarian cancer

Paclitaxel

Taxus brevifolia

Cragg and Newman (2005)

19

Prostate, breast, and liver cancer

Berberine, cannabisin

Berberis vulgaris

Pierpaoli et al. (2015)

20

Cervical cancer

Bryophyllin A

Bryophyllum pinnatum

Mahata et al. (2012)

21

Colorectal cancer, colon cancer

Silymarin

Silybum marianum

Ramasamy and Agarwal (2008)

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natural products with their active phytochemicals against specific types of cancer. Among the studied natural products, flavonoids are one of the most important classes of phytochemicals for their effectiveness against different cancers and management of drug resistance compounds. This chapter gives detailed overview of the active principles in plantderived compounds that have antiproliferative properties with an additional focus on the flavonoid class of compounds. Further importance of the phytochemicals in anticancer drug discovery is also discussed to aid in the future development of phytochemicals-based anticancer agents.

19.1.1 Importance of phytochemicals Phytochemicals consist of heterogeneous set of bioactive materials which includes alkaloids, saponins, lignans, terpenes, glycosides, gums, oils, polyphenols, flavonoids, carotenoids, Taxol, podophyllotoxins, and all other secondary metabolites play important role in cancer by preventing tumor initiation, promotion, and progression during the multistage carcinogenesis process and act as a chemopreventive agent (Lee et al., 2011; Murakami, 2009; Priyadarsini & Nagini, 2012). These compounds are present in all fruits, vegetables, grains, and herbs. Marine organisms such as algae, seaweeds, and sponges also contain many bioactive compounds. They have various health benefits including antioxidant, anticancer, antimicrobial, anti-inflammatory antidiabetic, and antibiotic properties (Crozier et al., 2008; Kotecha et al., 2016). Phytochemicals have been seen to be utilized in pharmaceutical products and nutrition, cosmetics, and dietary/nutritional supplements. Secondary metabolism of plants involves hundreds of various enzymes, some of which catalyze particular reactions, providing one product from specific substrate, whereas others may provide more products from a single substrate. This helps the biofactories to provide newer applications by the phytochemicals bioconversion or the manufacture of newer bioactive agents (Watson et al., 2013). The term nutraceutical is derived from “pharmaceutical,” and “nutrition” is a food or dietary product that gives medical and health benefits, such as treatment and prevention of disease. Such products range from dietary supplements, isolated nutrients, and particular diets to herbal products, foods that are engineered genetically, and processed foods including cereals, beverages, and soups (Acharya et al., 2010). A nutraceutical is explained to contain physiological advantages or produce protection from chronic disease. The phytochemicals which include the bioactive substances promote or sustain health and are produced by food intersection and pharmaceutical companies. The most promising molecules for further health-promoting studies are phenolic compounds. These phytochemicals comprise a vast range of almost 8000 unique molecules, that play important roles in the life of plants, where they are widely distributed. These compounds can be divided into phenolic acids, lignans, lignins, stilbenes, tannins, and flavonoids. Even though they are constitutively present, stressful growth conditions and/or changes in growth medium components may further induce their synthesis and can be used to enhance their production by in vitro plant cultures (Forni, 2019; Forni et al., 2016; Lucioli et al., 2017). In plants, phenolics are involved in H2O2 detoxification, providing protection against UV radiation and also acting as enzyme modulators and feeding deterrents for herbivores (Bennett & Wallsgrove, 1994). The broad spectrum of biological activities of phenolics, among which antioxidant (i.e., reducing agents, free radical scavengers, and quenchers of single oxygen formation) and antitumor properties, is widely acknowledged in several studies (Kasote et al., 2015; Pandey & Rizvi, 2009). The presence of at least one phenol ring is important for such activity, with hydroxyl, methyl, or acetyl groups replacing the hydrogen. An increased antioxidant activity has been related to the enhanced number of free hydroxyls and the conjugation of side chains to aromatic rings (Moran et al., 1997). Flavonoids contain the following subclasses: flavonols, flavones, flavanones, flavan-3-ols, isoflavones, and anthocyanidins. They have attracted the attention of the researchers because of their positive effects on a number of diseases as reported in this review. For instance, quercetin and anthocyanins have been reported to be effective in reducing the growth rate of malignant cells, influencing carcinogen metabolism, reducing parameters of tissue inflammation, and inhibiting angiogenesis (Bunea et al., 2013; Forni et al., 2014). According to some authors, the antitumor activities of phenolic compounds may be related to apoptosis, scavenging of radicals, antioxidant, and prooxidant characteristics (Nandi et al., 2007). Terpenoids represent another very large family of plant secondary metabolites (Aharoni et al., 2005). In vitro assays showed that monoterpenes, sesquiterpenes, and diterpenes extracted from aromatic plants have notable antioxidant activity (Yerlikaya, 2013). The hypoglycemic and antioxidant activities of the alkaloid vindoline, vindolidine, vindolicine, and vindolinine, obtained from Catharanthus roseus leaves, have been reported (Tiong et al., 2013). Moreover, vindolicine shows the highest antioxidant effects and also decreases H2O2-induced oxidative damage to pancreatic cells, suggesting it as a potential antidiabetic agent.

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19.1.2 Classification of phytochemicals source and their effectiveness against cancer Several physiologically active phytochemicals have been found as having the ability to inhibit carcinogenesis at various stages, as detailed below.

19.1.2.1 Phenolics Fruits have a high concentration of the secondary metabolite phenolics (Crozier et al., 2008). These compounds include one or more aromatic rings as well as one or more hydroxyl groups (Watson et al., 2013). The primary phenolic chemicals present in food may be divided into three categories: simple phenols and phenolic acids, hydroxycinnamic acid derivatives, and flavonoids. They are necessary for plant development and reproduction, as well as serving as defensive mechanisms against diseases, parasites, and predators (Acharya et al., 2010; Levin, 1971). The increased interest in these chemicals is mostly due to their antioxidant activity and potential to prevent certain illnesses. It is worth noting that the health advantages of phytochemicals will be realized with frequent use and bioavailability (Scalbert & Williamson, 2000). Simple phenols and phenolic acids serve as precursors for the creation of more complex molecules like flavonoids and tannins (Chirumbolo, 2012; Islam et al., 2013). They work in plants’ natural defensive mechanisms to protect them from infectious illnesses and inhibit the growth of dangerous bacteria, viruses, and fungi. Monophenols, 3-ethylphenol, 3, 4-dimethylphenol, and diphenols are among them, and they are likely the most common simple phenol (Chirumbolo, 2012). Hydroxycinnamic acids and their esterified derivatives are virtually entirely derived from p-coumaric acid (PCA), caffeic acid (CA), and ferulic acid (FA) (Mateos et al., 2006). They are commonly found conjugated, typically as esters rather than glycosides (Razzaghi-Asl et al., 2013). The most common phytochemicals in plant food powders are hydroxycinnamic acid derivatives such as chlorogenic acid, coumaric acid, caffeic acid, and sinapic acid (Chaturvedi et al., 2008). As a result, the incorporation of these food plant powders strong in bioactive phytochemicals would benefit food reformulations capable of improving foods in terms of both quality and health endorsement characteristics. This technique will immediately enhance the intake of plant-based foods, which has been shown to be good for health. Flavonoids, a key type of phenolic chemical, were found to have considerable antioxidant activity (Gulcin, 2012). These molecules have been linked to a lower risk of major chronic illnesses and are abundant in fruits, vegetables, and other plant foods (Forster et al., 2013). There are around 4000 different flavonoids known. They usually have two aromatic rings (A and B rings) joined by three carbons, which are usually in an oxygenated heterocycle ring or C ring. Flavonols, flavones, flavanols (catechins), flavanones, anthocyanidins, and isoflavonoids are many forms of flavonoids based on the general structure of the heterocyclic C ring (Murakami, 2013).

19.1.2.2 Organosulfur compounds Organosulfur compounds are organic molecules distinguished by the presence of sulfur-containing functional groups (Giardi et al., 2013). Regular consumption of organosulfur compounds has bioactive qualities, particularly in terms of cardiovascular health. Wattenberg and colleagues evaluated many organosulfur compounds for their potential to inhibit carcinogenesis induced by N-nitrosodiethylamine, with diallyl disulfide emerging as the most effective (Rajkapoor et al., 2005). Several studies have been conducted to evaluate the capacity of natural compounds produced from cruciferous plants and members of the Allium genus to prevent cancer. Some vegetables high in organosulfur compounds include watercress, Chinese cabbage, and broccoli (OSCs).

19.1.2.3 Carotenoids Carotenoids, the most abundant natural pigment, have received a lot of attention due to their provitamin and antioxidant qualities (Stahl & Sies, 2003, 2005). In nature, about 600 distinct carotenoids have been identified. They might be plants, microbes, or animals. They have a skeleton made up of 40-carbon isoprene units (Russo, 2007). Their structure can be cyclized at one or both ends, with varying degrees of hydrogenation and oxygen-containing functional groups. In nature, carotenoid complexes are generally found in the all-trans form. Carotenoids are distinguished by their extensive sequences of conjugated double bonds produced in the molecule’s core. Their form, chemical reactivity, and lightabsorbing characteristics are all outstanding. Carotenoids can react with free radicals and generate new radicals (Valko et al., 2007). An adequate amount of carotenoids can prevent lipid oxidation and accompanying oxidative stress.

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TABLE 19.2 Anticancer properties of phytochemicals (Mollakhalili Meybodi et al., 2017). Phytochemicals

Mechanisms involved in anticancer effect

Phenolic compound

Reduction in incidence of neoplasia induced by chemical carcinogens Prevention of nitrosation of susceptible secondary amines and amides to form highly potent carcinogenic nitrosamines and nitrosamides in different foods Inhibition of promotion of processes involved in cancer Reduction of hyperproliferation of epithelial cells by inhibition of kinases

Organosulfur compounds

Induction of carcinogen detoxification Inhibition of tumor cell proliferation and progression Antimicrobial effect against microorganisms causing cancer Free radical scavenging Inhibition of formation of DNA adduct Induction of cell cycle arrest and apoptosis

Alkaloids

Alteration of metabolism pathways resulting in carcinogens Alteration of tumor metabolism Inhibition of tumor cell growth

Carotenoids

Induction of cell differentiation

Nitrogen-containing compounds

Inhibition of the metabolic activation responsible for formation of carcinogens

19.1.2.4 Alkaloids Alkaloids are a class of chemical molecules with a ring structure that include nitrogen and have a wide spectrum of anticancer activities (Deiters & Martin, 2004). These drugs reduce cancer by inhibiting enzyme topoisomerase activity, which is involved in DNA replication, triggering apoptosis, and increasing p53 gene expression (Chikamori et al., 2010; Su et al., 2015). However, alkaloids have existed long before humans; some of them are structurally identical to neurotransmitters found in the human central nervous system. Given the medical relevance of alkaloids and studies demonstrating their involvement in cell multiplication, they would be exploited as an effective chemopreventive agent in the current drug discovery period (Bhandari, 2015; Ouyang et al., 2014). Alkaloids are classified into the following groups: Amaryllidaceae alkaloids, betalain alkaloids, diterpenoid alkaloids, indole alkaloids, isoquinoline alkaloids, lycopodium alkaloids, monoterpene and sesquiterpene alkaloids, peptide alkaloids, pyrrolidine and piperidine alkaloids, pyrrolizidine alkaloids, quinoline alkaloids. Table 19.2 indicates the mechanisms involved in the anticancer effects of phytochemicals.

19.1.3 Phytochemicals currently in use as cancer therapeutics Vinca alkaloids, taxane diterpenoids, camptothecin derivatives, and epipodophyllotoxin are the four principal types of therapeutically employed plant-derived anticancer agents. Other plant-derived anticancer medicines utilized in addition to these phytochemical families include combretastatins, homoharringtonine (omacetaxine mepesuccinate, cephalotaxine alkaloid), and ingenol mebutate. Poor water solubility and considerable hazardous side effects continue to be a major problem; hence, the present emphasis of the research is on reducing the impact of these variables. Several analogs and prodrugs have been produced in this regard, and techniques to improve water solubility and tumor selectivity have been developed. A brief explanation of a few phytochemicals utilized in cancer treatment is provided below.

19.1.3.1 Vinca alkaloids Vinca alkaloids are a class of medications derived from C. roseus, a pink periwinkle plant (Apocynaceae). Vinca alkaloids cause cytotoxicity by binding to tubulin at a different place than taxanes, preventing polymerization and assembly of microtubules, resulting in metaphase arrest and cell death. Because microtubules are involved in various other

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cellular activities, including cell shape preservation, motility, and organelle transport, vinca alkaloids influence both malignant and nonmalignant cells in the non-mitotic cell cycle. Vinblastine and vincristine are two naturally isolated alkaloids that have been utilized for almost 50 years in clinical oncology (Fig. 19.1). FIGURE 19.1 Vinca alkaloids used as anticancer agents.

These two alkaloids’ semisynthetic equivalents have been synthesized. Vinorelbine and vindesine are the only two clinically authorized semisynthetic equivalents. These medicines are commonly used in combination with chemotherapy to treat malignancies such as leukemia, Hodgkin and non-Hodgkin lymphomas, advanced testicular carcinoma, breast and lung cancers, and Kaposi’s sarcoma. Vinflunine, a gem-difluoromethylenated second-generation vinorelbine derivative, was recently licensed for the treatment of second-line transitional cell carcinoma of the urothelium (TCCU) (Martino et al., 2018).

19.1.3.2 Taxanes Taxanes, which were discovered in the bark of the yew tree, are promising anticancer medications. Taxanes inhibit cancer growth by stabilizing microtubules, causing cell cycle arrest and abnormal mitosis (Fig. 19.2). Paclitaxel, a natural substance extracted from the bark and leaves of Taxus brevifolia, and docetaxel, a semisynthetic derivative, are largely used in the treatment of breast, ovarian, pancreatic, prostate, and lung cancers. A variety of semisynthetic derivatives with better cytotoxicity in resistant tumors, lower toxicity, and improved solubility have been produced FIGURE 19.2 Taxanes used as anticancer agents.

Cabazitaxel, a second-generation docetaxel derivative, for example, has cytotoxic effectiveness against different docetaxel-resistant cancers while causing reduced overall toxicity (Kotsakis et al., 2016; Oudard et al., 2017). Another distinguishing feature of cabazitaxel is its capacity to cross the blood
brain barrier in vivo, which is not possible with other taxanes. Some paclitaxel analogs, including larotaxel, milataxel, ortataxel, and tesetaxel, are now in clinical trials.

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19.1.3.3 Camptothecins Camptothecin is a quinolone alkaloid isolated from Camptotheca acuminata, a Chinese tree. Camptothecin forms bind with type I DNA topoisomerase, blocking DNA cleavage and religation, resulting in a double-strand break and cytotoxicity (Hertzberg et al., 1989). Fig. 19.3 depicts the camptothecin derivatives as anticancer agents.

FIGURE 19.3 Camptothecines as anticancer agents.

At the moment, the two FDA-approved semisynthetic camptothecin derivatives that are clinically active and less toxic than the parent molecule are irinotecan and topotecan. Irinotecan is used to treat advanced malignancies of the large intestine and the rectum. Topotecan, on the other hand, is licensed for the treatment of recurrent ovarian, smallcell lung, and cervical cancer.

19.1.3.4 Podophyllotoxins Podophyllotoxin is a naturally occurring toxin isolated from the plants Podophyllum peltatum and Podophyllum emodi (Berberidaceae). Podophyllotoxin binds tubulin reversibly, but its main derivatives etoposide and teniposide block topoisomerase II, causing topoisomerase II-mediated DNA breakage. Furthermore, podophyllotoxin may have antimultidrug resistance (MDR) efficacy against a variety of drug-resistant tumor cells. Fig. 19.4 depicts prominent podophyllotoxins as anticancer agents. FIGURE 19.4 Podophyllotoxins as anticancer agents.

CIP-36, a podophyllotoxin derivative, has been found to overcome the MDR of the adriamycin-resistant human leukemic cell line K562/ADR via modulating topoisomerase-IIa activity (Cao et al., 2015). CIP-36, on the other hand, failed in clinical trials due to a lack of effectiveness and significant toxicity.

19.1.3.5 Other plant-derived anticancer agents Ingenol mebutate (IM) is a hydrophobic ester of the diterpene ingenol derived from Euphorbia peplus, a common Australian shrub (Euphorbiaceae). IM is licensed for the topical treatment of actinic keratosis, a common skin disease caused by persistent UV radiation exposure that can progress to squamous cell carcinoma if not treated. At high concentrations (200
300 M), IM produces fast induction of cell death in the treated region; at low doses (0.1 M), it starts an inflammatory response capable of removing leftover cells (Skroza et al., 2018).

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Homoharringtonine (HHT) is a naturally occurring ester of the alkaloid cephalotaxine isolated from several Cephalotaxus genus (Cephalotaxaceae) trees that have been licensed for the treatment of chronic myeloid leukemia. HHT binds to the A-site cleft of the large ribosomal subunit, affecting chain elongation and inhibiting protein synthesis. Itokawa et al. (2005) provided a detailed account of the discovery and development of HHT and related chemicals. A semisynthetic form of HHT, omacetaxine mepesuccinate, has been found to be an effective therapy for myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) in individuals who are resistant or intolerable to hypomethylating drugs such as azacitidine and decitabine (Short et al., 2019). The combretastatins are a group of cis-stilbenes derived from Cape bushwillow (Combretum caffrum, Combretaceae), a South African plant. Compounds of the combretastatin family operate on cancer cells indirectly by blocking tubulin polymerization, resulting in the disruption of tumor endothelial cells lining the tumor vasculature and fast vascular collapse in solid tumors (Tozer et al., 2002). The two naturally isolated chemicals are combretastatin A1 and combretastatin A4. CA4P is a phosphate prodrug of combretastatin A4, which has been recognized as an orphan drug by the US Food and Medication Administration (FDA) and is licensed for the treatment of a variety of thyroid and ovarian cancers. Structures of the phytochemicals described above are given in Fig. 19.5.

FIGURE 19.5 Miscellaneous phytochemicals and their derivatives as anticancer agents.

19.1.4 Flavonoids—introduction and classification with their chemical structure The fundamental flavan skeleton is shared by all flavonoids: a 15-carbon phenylpropanoid chain (C6
C3
C6) that comprises two aromatic rings (A and B) connected by a heterocyclic pyran ring (C). Flavonoids are categorized into six primary classes based on their chemical structure, degree of oxidation, and connecting chain unsaturation: flavonoids, flavanones, flavanols, flavonols, flavones, and anthocyanidins are all types of flavonoids (Abotaleb et al., 2018; Durazzo et al., 2019; Panche et al., 2016) (Fig. 19.6).

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FIGURE 19.6 Classes of naturally occuring flavonoids.

19.1.5 Mechanism action of flavonoids When the balance between prooxidant activities and antioxidant defense is disrupted, ROS generation rises and free radicals accumulate. ROS are primarily produced in the electron transport chain of mitochondria as byproducts of cellular oxidative phosphorylation (Murphy, 2009). The amount of ROS created creates oxidative stress, which is involved in the development of inflammatory processes, which leads to the development of many degenerative illnesses and cancer. Flavonoids have a dual role in ROS homeostasis, acting as antioxidants under normal conditions and as powerful prooxidants in cancer cells, activating apoptotic pathways (Hadi et al., 2000; Link et al., 2010). Because of its propensity to stabilize free radicals due to the presence of phenolic hydroxyl groups, flavonoids can directly scavenge ROS and chelate metal ions (Youn et al., 2006). The indirect antioxidant effects of flavonoids are connected to the activation of antioxidant enzymes, the repression of prooxidant enzymes, and the stimulation of antioxidant enzyme and phase II detoxification enzyme synthesis (Fraga et al., 2010). Flavonoid anticancer effects entail both antioxidant and prooxidant actions (Oliveira-Marques et al., 2009; Valko et al., 2007). Isoflavone genistein induced G2/M cell arrest and subsequent ROS-dependent apoptosis in breast cancer cells (Kaushik et al., 2019). Daidzein induced apoptosis in breast cancer MCF-7 cells via ROS production (Jin et al., 2010). Flavanone hesperetin activated the mitochondrial apoptotic pathway, causing apoptosis in gall bladder carcinoma (Pandey et al., 2019), esophageal cancer (Wu et al., 2016), hepatocellular carcinoma (Zhang et al., 2015), and human breast carcinoma MCF-7 cells (Palit et al., 2015). Flavanone naringenin inhibited cancer growth in choriocarcinoma JAR and JEG 3 cell lines by increasing ROS production and activating signaling pathways (Park, Lim et al., 2018). In human epidermoid carcinoma A431 cells, it also triggered an apoptotic cascade (Ahamad et al., 2014). Naringenin inhibited proliferation and migration while inducing apoptosis and ROS production in prostate cancer PC3 and LNCaP cell lines (Lim et al., 2017). Furthermore, in chronic illnesses including cancer, naringenin decreased ROS production and increased the activities of superoxide dismutase, catalase, and glutathione (Zaidun et al., 2018). Because of their prooxidant qualities, cocoa catechins and procyanidins have been demonstrated to produce apoptotic morphological alterations, DNA damage, and apoptosis in epithelial ovarian cancer cells (Taparia & Khanna, 2016). In HepG2 cells, cocoa polyphenolic extract activated the ERK1/2 pathway, boosting the activities of glutathione peroxidase and reductase (Martin et al., 2010). Cocoa catechins and procyanidins also protected Caco2 cells from oxidative stress and cellular death by lowering ROS generation (Rodriguez-Ramiro et al., 2011). Cocoa flavanols have been shown to protect against colon cancer due to their antioxidant characteristics (Martin et al., 2013, 2016). The flavonol quercetin has powerful cancer chemopreventive activities (Rather & Bhagat, 2019; Tang et al., 2019). Recent research found that quercetin inhibited the growth of hepatocellular carcinoma HepG2 cells via lowering intracellular ROS levels (Jeon et al., 2019). It boosted ROS generation and the amount of apoptotic cells in human gastric cancer AGS and human breast cancer MCF-7 cells (Shang et al., 2018; Wu, Kroon et al., 2018). Flavonol kaempferol reduced the development of malignant bladder cells by inducing apoptosis and S-phase arrest via ROS level regulation (Wu, Meng et al., 2018).

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Caspases were activated, and apoptosis was promoted in colorectal cancer HCT116, HCT15, and SW480 cell lines (Choi et al., 2018). Furthermore, kaempferol was found to be lethal to rat hepatocellular carcinoma cells via ROSmediated mitochondrial targeting (Seydi et al., 2018). The anticancer actions of the flavones, apigenin and luteolin, in ovarian cancer cell lines (A2780, OVCAR-3, and SKOV-3) were also linked to alterations in ROS signaling and apoptosis promotion (Salmani et al., 2017; Tavsan & Kayali, 2019). Furthermore, apigenin induced apoptosis in human cervical cancer-derived cell lines such as HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16- and HPV 18-positive), and C33A (HPV-negative) cells via increased ROS generation and the activation of mitochondrial apoptotic pathways (Souza et al., 2017). Flavone chrysin has been shown to increase ROS and lipid peroxidation levels, causing choriocarcinoma (JAR and JEG3) (Park, Park et al., 2018), bladder cancer (Xu et al., 2018), and ovarian cancer (ES2 and OV90) cells to die (Lim et al., 2018). Flavonoids’ antioxidant activity in humans has also been studied. It was discovered that anthocyanin intake in the diet correlated with serum total antioxidant capacity (Alipour et al., 2016). Furthermore, cyanidin caused cell death in DU145 and LnCap human prostate cancer cells via ROS modulation (Sorrenti et al., 2015). Cyanidin and delphinidin increased cellular ROS levels, inhibited glutathione reductase, and depleted glutathione, resulting in cytotoxicity in metastatic (LoVo and LoVo/ADR) colorectal cancer cells (Cvorovic et al., 2010) (Fig. 19.7). FIGURE 19.7 Naturally occurring flavonoids reported for their anticancer activity.

19.1.6 Flavonoid compounds for anticancer activity Numerous studies have shown that flavonoids may scavenge free radicals, regulate cellular metabolism, and prevent oxidative stress
related illnesses (Gorlach et al., 2015; Perez-Vizcaino & Fraga, 2018). There is mounting evidence

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that several flavonoids have anticancer action; nevertheless, the molecular processes behind this effect have yet to be fully understood. Cancer is a diverse illness defined by uncontrolled cell proliferation and a disrupted cell cycle, which results in the formation of aberrant cells that infiltrate and spread to other regions of the body (Kroemer & Pouyssegur, 2008; Neagu et al., 2019). Internal causes of cancer include oxidative stress, hypoxia, genetic alterations, and a loss of apoptotic function, whereas external causes include increased exposure to stress, pollution, smoking, radiation, and UV rays (Blackadar, 2016). The key features of cancer cells include altered metabolism, disrupted cell cycles, frequent mutations, resistance to immune response, chronic inflammation, metastasis development, and angiogenesis promotion (Neagu et al., 2019). Cancer seems to be a metabolic illness characterized by varying degrees of mitochondrial dysfunction and metabolic changes (Bock & Tait, 2019; Kroemer & Pouyssegur, 2008). Mitochondria are vital for cellular energy supply, metabolic control, cell death signaling, and the formation of reactive oxygen species (ROS). Tumor cells’ key metabolic changes include enhanced aerobic glycolysis (Lebelo et al., 2019), unregulated pH (Chiche et al., 2010), decreased lipid metabolism (Zaidi et al., 2013), increased ROS production (Weinberg et al., 2019), and reduced enzyme activity (Lu & Wang, 2018). As a result, the extracellular environment becomes acidic and more inflammatory (Lee & Kim, 2016), glutamine-driven lipid biosynthesis increases and upregulates the pathways involved in tumorigenesis initiation and metastasis (Vegliante et al., 2018), cardiolipin levels decrease in membranes, causing impaired enzyme activities (Kiebish et al., 2008; Seyfried et al., 2014; Zhong et al., 2017), mitochondria are hyperpolarized (Neagu et al., 2019), and this effect correlates with cancer cell malignancy and invasiveness (Neagu et al., 2019).

19.1.7 Future prospects of phytochemicals in cancer treatment Medicinal plants continue to be an important source of novel pharmacological leads. One significant advantage of medicinal plant-based drug development is the availability of ethnopharmacological knowledge, which provides a great opportunity to narrow the vast array of potential leads to more promising ones. To realize the full potential of phytochemicals, a novel approach of integrated drug discovery is required, in which ethnopharmacological knowledge is supported by broad interdisciplinary forces involving medicinal chemistry, pharmacology, biochemistry, molecular and cellular biology, and natural product chemistry. Furthermore, advancements in analytical technology and computational approaches, as well as the development of self-teaching artificial intelligence systems, will make it easier to identify new phytochemical lead entities for pharmacological investigation. The findings obtained in different rounds of clinical trials, as well as intriguing preclinical results, indicate that ways and means to transfer phytochemicals “from bench to real-life circumstances” are on the horizon, according to the current study. Despite the potential of phytochemicals as cancer therapeutic agents, there are several limitations that must be addressed. For example, most phytochemicals investigated at the preclinical stage lack knowledge of their molecular interactions with various signaling molecules. To address difficulties with molecular targets and pathways, in silico methodologies such as molecular docking must be used to understand the interaction of phytochemicals in various signaling pathways, which can then be confirmed using various in vitro and in vivo models. Most associated clinical studies include methodological faults, such as a lack of a control or placebo group, limited sample numbers, and a short study duration. As a result, it is too early to infer that many phytochemicals have anticancer properties, and large-scale and well-controlled clinical trials are required to evaluate their efficacies, side effects, and safety before they can be used to treat cancer. Furthermore, considerable standardization of methodologies for evaluating their bioavailability, effectiveness, safety, quality, composition, manufacturing processes, and regulatory and approval standards on promising phytochemicals is required to match the worldwide standard. The pharmaceutical sector, paradoxically, has a wealth of information and experience in medication creation. As a result, integrating the benefits of traditional and contemporary medicine has previously been proposed as a viable way to discovering and bringing novel plant-derived drugs to market. Combinations of chemotherapeutic drugs with phytochemical substances have been shown to produce synergistic or additive effects in cancer cells with tolerable side effects (Li et al., 2013; Pezzani et al., 2019). Thus, due to their low intrinsic toxicity in normal cells but dramatic effects in malignant cells, phytochemicals’ anticancer and chemopreventive qualities have piqued the interest of oncology researchers in recent years (Li et al., 2013).

19.2

Conclusion

Carcinogenesis is a complicated and heterogeneous process involving multiple combinations of genetic and epigenetic processes that occur in a single cell to produce a neoplastic deformity. Taking into account the various stages of cancer,

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initiation, development, and advancement, the second step is the most important to consider for cancer chemoprevention. It is expected that correct lifestyle changes might prevent more than two-thirds of all human malignancies. Phytochemicals are mostly found in fruits and vegetables, and their consumption might help in the prevention of the cancer. Natural products have been source of inspiration for the drug development, especially in cancer; many modern drugs have been developed by chemical modification of phytochemicals. As the currently available anticancer drugs are failing due to drug resistance, the identification of new cancer therapeutics is most urgently needed. The need of finding new therapeutics can be fulfilled to a good extent by studying new phytochemicals with anticancer effectiveness.

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Chapter 20

Bioactivity, medicinal applications, and chemical compositions of essential oils: detailed perspectives Sonali S. Shinde1, Aniket P. Sarkate1, Nilesh Prakash Nirmal2 and Bhagwan K. Sakhale1 1

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India, 2Institute of Nutrition,

Mahidol University, Nakhon Pathom, Thailand

20.1

Introduction

Essential oils are oily or lipid-like plant compounds that have a strong aroma (Bakkali et al., 2008; Burt, 2004). They are natural, complex, volatile plant components that are oily or lipid-like in nature. They are soluble in some alcohol, lipids, and hydrophobic substances, and these essential oils are mostly in liquid form at room temperature (Carson & Hammer, 2011; Giannakopoulos et al., 2017). Flowers (orange, lavender, pink, flower bud in case of clove), leaves (eucalyptus, bay leaf, thyme, mint, savory, pine needles, sage), rhizomes (ginger and sweet flag), seeds (coriander and carvi), wood and bark (sandalwood), roots (vetiver and citrus epicarps), fruits (anise, fennel) (Ahmed & Eapen, 1986). Solvent and supercritical fluid extraction, fermentation, enfleurage, or expression under pressure are some of the processes employed in commercial production, but low- or high-pressure steam or hydrodistillation is the most common (Lahlou, 2004). Many factors, including environment, plant nutrition, and stress, can alter the presence, yield, and content of essential oils (Croteau, 1986). Essential oils are also known as volatile oils, plant oils, ethereal oils, or aetheroleum; however, for the purposes of this chapter, the term “essential oil” will be used. Essential oils (EOs) are secondary metabolites that plants produce in response to stressful circumstances or to battle infectious or parasitic organisms (Rauha et al., 2000). Secondary plant metabolites have traditionally been described as all plant-produced chemicals that do not appear to be needed for plant growth. Secondary plant metabolites have traditionally been defined as all plantproduced chemicals that do not appear to be needed for the growth and development of plants and/or have no evident purpose (Croteau et al., 2000). Volatile oils are extremely complicated chemical combinations. Sesquiterpenes and monoterpenes, which are hydrocarbons with the molecular formula (C5H8)n, are the major constituents of the oils. Esters, ethers, alcohols, ketones, aldehydes, phenols, and oxides are oxygenated chemicals generated from these hydrocarbons. There are over 3000 sesquiterpene and 1000 monoterpene structures, according to estimates. Particular sulfuror nitrogen-containing compounds and phenylpropenes are examples of other chemicals. Every year, hundreds of novel natural compounds are separated and identified, but only a few have data on their biological functions (Lal et al., 2020). In the perfumery, cosmetics industries, and aromatherapy, these essential oils are widely employed. Aromatherapy is a therapeutic approach that uses essential oils to provide inhalations, massages, and baths (volatile oils). Essential oils (EOs) also function as chemical messages that help the plant govern and regulate its surroundings (ecological role) by repelling predators, attracting pollinators, inhibiting seed germination, and coordinating with other plants. In addition, EOs have insecticidal, deterring, and antifungal properties (Masango, 2005).

20.2

Chemistry of essential oils

Essential oils are made up of a complex blend of polar and nonpolar molecules (Dima & Dima, 2015). The essential oil content is determined by the extracted plant’s species, geographic region, harvest time, and extraction method (Bohra Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00010-4 © 2023 Elsevier Inc. All rights reserved.

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et al., 1994). Essential oils contain a range of chemical classes, primarily terpenes, but also phenylpropanoids, and they may contain nitrogen or sulfur, though in reduced amounts and often, but not always, in minor proportions (Filly et al., 2014). They are usually low-molecular-weight molecules with poor water solubility (Morsy, 2017). The following are the major main groups of EOs (Alam et al., 2010): 1. Terpenes, which are connected to isoprene, are one type of terpene and straight-chain compounds not containing any side chain; 2. Phenylpropanoids (benzene derivatives); 3. Miscellaneous group or sulfur and nitrogen compounds of essential oils (Sharopov et al., 2015).

20.2.1 Terpenes Terpenes are the largest group of phytochemicals, and a study of more than 30,000 structures in essential oils is done (Little & Croteau, 1999). Terpenes are photosynthesized through the mevalonic acid pathway in the cytoplasm of plant cells (Dung et al., 2008). The word “terpene” originates from the isolation of some major turpentine compounds, isolation of monoterpenes from the resin extract of many Pinus spp. Terpenes are also called as chain of polymers of isoprene (C5H8) interlinked in a repetitive head-to-tail manner (Chinou, 2005) as shown in Fig. 20.1.

20.2.1.1 Biosynthesis of terpenes Plants do synthesis of terpenes via two different biological pathways (Cairo et al., 2010). The mevalonic acid pathway or mevalonate pathway was the first process, which produces sterols, ubiquinones, and sesquiterpenes, and occurs mostly in the cytoplasm, endoplasmic reticulum, and mitochondria (Hosfield et al., 2004; Liu et al., 2008). The mevalonate pathway is used by archaebacteria as well as most eukaryotes including humans (Oldfield & Lin, 2012). Prominently, the occurrence and function of the second pathway were confirmed and described, and it is recognized as the non-mevalonic acid or MEP pathway. It is also called as methyl
erythritol phosphate pathway. The location of this pathway is in phyto-cell plastids, and this is an important pathway for the formation of diterpenes, hemiterpenes, monoterpenes, and other higher terpenes not found in essential oils, like carotenoids and chlorophyll phytol (Bohlmann & Keeling, 2008; Dubey et al., 2003). There is no such typical isolation method for both pathways for the synthesis of terpene and secondary metabolites. These are generally produced in one pathway in any plant cell area which may forward to the next pathway or other compartments of plant cell. These pathways produce isopentenyl diphosphate (IPP) and its isomer molecule such as dimethylallyl diphosphate (DMAPP) (Croteau et al., 2000a,b; Keszei et al., 2008). This is the basic unit of terpene molecules. Various combinations of these precursors produce geranyl diphosphate, i.e., (DMAPP 1 IPP), farnesyl diphosphate is nothing but (DMAPP 1 2 IPP), and geranylgeranyl diphosphate is (DMAPP 1 3 IPP), so all these are main parent compound for monoterpenes, triterpenes, sesquiterpenes, diterpenes, tetraterpenes (Bouvier et al., 2005). Though there are some exceptions from Lamiaceae, Asteraceae, and Apiaceae families, where irregularity in the production of sesquiterpenes, monoterpenes, and diterpenes by the head-to-head linkage of either 1DMAPP or GPP or 2 DMAPP or 2 GPPs, respectively. Specifically, precursor is from iso-pentenyl diphosphate (IPP, 2), the C5 substrates dimethylallyl diphosphate (DMAPP, 1) and mainly by condensing DMAPP with one or more IPP moieties in a “head-to-tail” or 10
4 fashion to produce (C10) geranyl diphosphate (GPP, 3), (C15) farnesyl diphosphate (FPP, 4), or (C20) geranylgeranyl diphosphate (GGPP, 5) as shown in Fig. 20.2.

FIGURE 20.1 Chain of polymers of isoprene (C5H8) interlinked in a repetitive head-to-tail manner.

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FIGURE 20.2 Hemi-, mono-, sesqui-, di-, tri-, and tetraterpene natural products.

FIGURE 20.3 Acyclic monoterpenes.

20.2.1.2 Monoterpenes The combinations of isoprene units are called terpene. Monoterpenes (C10H16) are formed by the linkage of two isoprene units (minimum one double bond). These terpenes have a hydrocarbon backbone that can be reconstructed into acyclic, cyclic, or aromatic (Sell, 2006). Acyclic monoterpenes present in essential oils may have either linear or regular structures with head-to-tail isoprene unit linkage, like the (E) and (Z) isomers of β-ocimene or the hydrocarbons myrcene. The trans
cis nomenclature for stereoisomers has been replaced by the (E) and (Z) notation for stereoisomers (McNaught & Wilkinson, 1997). Linalool, citronellol, and geraniol are instances of acyclic monoterpenes present in essential oils, as shown in Fig. 20.3. Depending on ring size, cyclic monoterpenes can be classified as monocyclic monoterpenes, bicyclic monoterpenes, and tricyclic monoterpenes (George et al., 2015) as shown in Fig. 20.4. These compounds undergo oxidization easily as they react rapidly with air and heat sources (Hunter, 2010). Monocyclic monoterpenes, which are formed from the p-menthane skeleton by cyclization of noncyclic monoterpenes, are the most common monoterpenes found in nature. Terpinene, limonene, r-terpinene, and terpinolene are major monoterpenes from this group, also aromatic hydrocarbon like p-cymene and its hydroxylated derivatives such as thymol and carvone both of which have antibacterial activity (Hu¨snu¨ et al., 2007) as shown in Fig. 20.4. The biosynthesis of bicyclic monoterpenes begins with the cyclization of monocyclic monoterpenes. They can be further divided into bornane, fenchane, camphane, thujane, sabinene pinane, and carane depending on the base from which they are formed (Taiz & Zeiger, 2010). In pine oils, main components are α-pinene and β-pinene, specifically pine oils, and these are bicyclic monoterpenes produced by intramolecular arrangement of the universal intermediate, that is, α-terpinyl cation, producing the bicyclic structure as shown in Fig. 20.5. In comparison to bicyclic and monocyclic monoterpenes, tricyclic monoterpenes are uncommon in essential oils. Two main examples found in essential oils are pinene oxide and tricyclene. One tricyclic monoterpene structure is given in Fig. 20.6.

20.2.1.3 Straight-chain components not containing any side chain Acyclic non-terpenoid oxygen derivatives like aldehydes, ketones, alcohols, ethers, esters, and acid and straight-chain hydrocarbons, starting range from n-heptane till compounds with 35 carbon atoms components (Adams & Wright, 2012). The leaf alcohol, that is, 3(Z)-hexen-1-ol, as shown in Fig. 20.7, and its esters derivatives give intense grassy green smell of freshly cut green leaves or grass via the pathway known as octadecenoic pathway (Hatanaka et al., 1995).

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FIGURE 20.4 Monocyclic monoterpenes.

FIGURE 20.5 Bicyclic monoterpenes.

FIGURE 20.6 Tricyclic monoterpenes.

FIGURE 20.7 Chemical stucture of leaf alcohol.

20.2.1.4 Sesquiterpenes Sesquiterpenes (C15H24) are the second largest group of monoterpenes (Gigot et al., 2010; Vasiliev et al., 2003). Three units of isoprene monomers form one sesquiterpene (Chooi et al., 2013). Sesquiterpenes are unsaturated compounds. There are linear, branched, or cyclic sesquiterpenes. Sesquiterpenes are unsaturated compounds. Cyclic sesquiterpenes can be classified into monocyclic, bicyclic, or tricyclic (George et al., 2015). Diterpenes are produced by the head-totail interlinkage of four isoprene monomers followed by other substitutions. Many essential oils contain acyclic sesquiterpenes, including the isomers nerolidol and farnesol, and even the αand β-structural isomers of farnesene. In nature, (E) isomers are more prevalent than (Z) isomers, and (E)-nerolidol can be found in a variety of economically significant essential oils, such as neroli oil from Citrus aurantium blossoms

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(Fugh-Berman & Myers, 2004). There have been identified essential oils that contain over 90% (E)-nerolidol (Limberger et al., 2005). Farnesol is a main constituent of essential oil extracted from rose flower and aromatic oils from Santalum spicatum, Australian sandalwood (Piggott et al., 1997). Santolina spp. has been found to have irregular acyclic sesquiterpenes (Asteraceae) (Ferrari et al., 2005; Liu et al., 2007) as shown in Fig. 20.8. Monocyclic, bicyclic, and tricyclic sesquiterpenes are all types of cyclic sesquiterpenes. Abscisic acid, -bisabolene, and its oxygenated derivatives, -bisabolol, are all monocyclic sesquiterpenes found in large amounts in chamomile (Matricaria chamomilla) oils (Ganzera et al., 2006). Eudesmol, widdrol, guaiol, and azulenes are examples of bicyclic sesquiterpenes. Azulenes, for example, chamomile oil from chamazulene and oil from Artemisia arborescens, are responsible for the blue color of several essential oils (Sinico et al., 2005). Many essential oils contain bicyclic caryophyllene, the most prevalent of which is -caryophyllene, which can also be a considerable component (Henriques et al., 1993; Sabulal et al., 2006). Tricyclic sesquiterpenes include cedrene and santalol. Many essential oils contain cedrene, including cedarwood oils obtained from Cedrus and Juniperus species (Adams, 1991; Lis-Balchin et al., 1998), and sandalwood oil from Santalum album contains santalols (Sangwan et al., 2001).

20.2.1.5 Diterpenes Head-to-tail linkages of four isoprene subunits are proceeded by replacements and/or rearrangement to form diterpenes. They are the most vital components of various plant resins (Langenheim, 2003). The content of diterpenes, triterpenes, and tetraterpenes is at a very low level in essential oils (Ding & Lee, 2019). They can recover by increasing steam distillation periods (Berger, 2007) and mainly recover by the extraction method. Like monoterpenes and sesquiterpenes, these diterpenes may be acyclic or cyclic as shown in Fig. 20.9. Example of acyclic diterpenes is phytol. It forms the hydrophobic branch linkage of chlorophyll, and it is observed in the green leaves of all plants (Croteau et al., 2000a,b; Obst, 1998). It is one common ingredient of many essential oils (Hoet et al., 2006; Wu et al., 2004). Plaunotol is the second most important element of Croton stellatopilosus (family: Euphorbiaceae; previously known as Croton sublyratus) also called as a Thai medicinal plant (Jahangir et al., 2009; Kose et al., 2007). The monocyclic camphorene (also known as dimyrcene), a constituent of camphor oil produced from the plant Cinnamomum camphora (family: Lauraceae), often known as the camphor laurel, is a popular cyclic diterpene in essential oils (Breitmaier, 2006). The essential oils of Pistacia lentiscus from leaves, also from mastic gum extracted from a similar plant, are utilized to distinguish different isomers of camphorene (Wungsintaweekul et al., 2008). Essential oils also include bicyclic and tricyclic diterpenes. The clerodanes and labdanes are two structural categories of bicyclic diterpenes found in essential oils (Boelens & Jimenez, 1991).

20.2.1.6 Norterpenes Norterpenes have eight isoprene units, which are important in plants for a variety of reasons, including their role in photosynthesis (Hirschberg, 2001). They are not found in the compounds of essential oils. They are, nevertheless, comparable to oils in that when their carbon core is broken, primarily through oxidation, apocarotenoids are formed (Auldridge et al., 2006; Kloer & Schulz, 2006). Norterpenoids or norisoprenoids are the main types of apocarotenoids which are obtained by cleavage at the 9
10 and responsible for flavor and aroma (Rodrı´guez-Bustamante & Sanchez, 2007), for example, natural aroma obtained from Boronia megastigma and Rosa damascena as shown in Fig. 20.10.

FIGURE 20.8 Structures of acyclic sesquiterpenes.

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FIGURE 20.9 Structure of diterpenes.

FIGURE 20.10 Structure of damascone.

20.2.2 Phenylpropanoids 20.2.2.1 Biosynthesis of phenylpropanoids Biosynthesis of phenylpropanoids takes place by the plant shikimate pathway. Location of this pathway is intracellular and plastids of plant cells, and this complex path of synthesis has been found for more than 10 years (Schmid & Amrhein, 1995; Vogt, 2010). The synthesis of various phenolic constituents in plants is produced by shikimic acid pathway, and it begins with glucose and produces aromatic amino acids such as tyrosine, tryptophan, and phenylalanine

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(Herrmann & Weaver, 1999). Phenylpropanoid synthesis is from aromatic amino acids like phenylalanine and a small amount of tyrosine (Sangwan et al., 2001). They have a C6C3 chemical structure of a benzene aromatic ring and a three-carbon side chain (Ferrer et al., 2008). When the three-carbon side chain gets connected to aromatic ring and is deducted by two carbons, thus benzenoids are formed. These compounds are named phenylpropanoids (Chappell, 2008; Jirovetz et al., 2003).

20.2.2.2 Phenylpropanoids occurrence in essential oils These phenylpropanoids are aromatic metabolites of plants which give flavors and perfumes to industry, but it contains a comparatively small amount of the essential oils. This non-terpenoid group consists of constituents obtained from npropyl benzene. The aromatic ring may contain methoxy, methylene dioxy groups, and hydroxy groups. The propyl side chain may contain carboxyl group or hydroxyl group. Phenylpropenes contain subfamily of phenylpropanoids. Examples of such phenylpropanoids are methyl chavicol, trans-anethole, isoeugenol, eugenol, vanillin, myristicin, cinnamaldehyde, and safrole as shown in Fig. 20.11.

20.2.3 Nitrogen- and sulfur-containing compounds in essential oils Glucosinolates, aglycones, and their metabolic products, such as isothiocyanates, are nitrogen- and sulfur-containing phytoconstituents (Halkier & Gershenzon, 2006). Aglycones are the nonglycosidic part of a glycoside, a compound formed using a sugar group, called as the glycone which is linked to another group. Glucosinolates are found as mustard oil glucosides, and these are sulfur-containing or nitrogen-containing phytoconstituents obtained from one of eight amino acids and glucose (Fahey et al., 2001). When phyto-enzymes such as myrosinases work on glucosinolates to break the glucose group, an unstable aglycone is released, which undergoes rearrangements to generate thiocyanates, nitriles, and isothiocyanates (Masteli´c et al., 2006). Onion, garlic, shallots, and leek include volatile sulfur components

FIGURE 20.11 Chemical structures of phenylpropenes.

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such as dimethyl sulfide, allyl sulfide, dimethylthiophene, and diallyl disulfide (Iranshahi, 2012). These components are ˇ mainly responsible for the characteristic taste and aroma (Stajner et al., 2006). The chemical structures of sulfurcontaining compounds in essential oils are shown in Fig. 20.12.

20.3

Biological activity of essential oils

20.3.1 Introduction Various biological activities of essential oils lead to more focus on their applications in agriculture, cosmetic, and health and also in food industries (Ballabeni et al., 2004; Tognolini et al., 2007). Biological properties such as antimicrobial, antioxidant, analgesic, anticancer, anti-inflammatory, antiplatelet, and other immunomodulatory and antithrombotic activities of essential oils are explained as follows (Tognolini et al., 2006; Huang et al., 2007).

20.3.2 Antimicrobial activity The antimicrobial activity of aromatic oils extracted from medicinal herbs shows various uses in many revenue sections like perfumes, cosmetics (Liao et al., 2008), sanitary pharmaceuticals, nutraceuticals, pharma, and food industries (Sudipta et al., 2012; Swamy & Sinniah, 2015). In the next part, we have explained the biological activities of various essential oils obtained from both medicinal and aromatic plants (Akthar et al., 2014).

20.3.2.1 Antibacterial activity Essential oils show broad-spectrum inhibition against many bacterial pathogens (Teixeira et al., 2013). Essential oil is permeable through the cell membrane and cell wall as it is lipophilic in nature (Oussalah et al., 2006). Essential oil constituents when coming in contact with fatty acids, phospholipids, and polysaccharides, there is leakage of the cellular contents, loss of membrane integrity, disturbance in proton pump activity (Di Pasqua et al., 2007), and at the end cell death (Ding & Lee, 2019; Saad et al., 2013). Denaturation of cell is another mechanism of action of these essential oils (Gustafson et al., 1998). Carvone acts by breaking the phospholipid bilayer membrane, while terpinen-4-ol disturbs cell respiration and changes the cell membrane permeability (Cox et al., 2000). Carvacrol and p-cymene cause ion leakage by accumulating in the phospholipid part of the cell membrane. Sage essential oil shows major antimicrobial action (Jassbi et al., 2012). Staphylococcus aureus bacteria have a single layer of lipid in membrane which is highly sensitive to essential oils with a high amount of p-cymene. Oregano oil extracted from Origanum vulgare by cold press method shows antimicrobial activity against S. aureus, Salmonella enteritidis, Listeria monocytogenes, Escherichia coli. The inhibition zone area is from 20 to 70 mm, and it depends on the type of target bacteria, for example, Chinese FIGURE 20.12 Chemical structures of sulfur- and nitrogen-containing compounds.

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cinnamon-inhibited E. coli and S. aureus at MIC 470 ppm in microbroth dilution assay. Red bergamot oil shows antibacterial activity due to p-cymene and carvacrol; likewise, trans-cinnamaldehyde shows this effect in Chinese cinnamon essential oil. Radaelli et al. (2016) studied the antimicrobial action of rosemary, basil, peppermint, marjoram, anise, and thyme essential oils against Clostridium perfringens strain A.

20.3.2.2 Antifungal activity Most fungi are responsible for various human infections (Cavaleiro et al., 2006). Crop fields get fungal attack in the field or while storing (Reyes-Jurado et al., 2015). Fungus growth is the major problem in the food industry (Will & Kru¨ger, 1999). Mycotoxin accumulation can be avoided by preventing fungal growth (Varga et al., 2010). Essential oils can disturb the cell life cycle of molds (Sridhar et al., 2003). Because of the low concentration of active constituents in essential oils and the high cost of their production, there are limitations for use as antifungal agents. Thus, the investigation of antifungal constituents in the essential oils is important because of the requirement of synthesizing all these compounds as antifungal agents (Rahman et al., 2011). Serrano et al. (2005) found that the major essential oil constituents of Thymus glandulosus, Origanum compactum, and Moroccan Labiatae are carvacrol, linalyl acetate, and thymol. These oils totally inhibit the growth of the mycelium of Botrytis cinerea at a concentration of 100 ppm. Serrano et al. developed active packaging materials incorporating menthol, eugenol, eucalyptol, and thymol to prevent yeast and mold growth in preserved cherries (Lo´pez et al., 2007). Cinnamon essential oil demonstrated strong antifungal activity against Aspergillus flavus (extremely low minimum inhibitory concentration) (Nasir et al., 2015). Cinnamomum zeylanicum bark essential oil contains cinnamaldehyde, which has antimycotic properties (Rota et al., 2008). At 0.01%, carvacrol, the main active component in oregano oil, was found to completely inhibit Saccharomyces cerevisiae growth. It had 1500 times the strength of oregano oil. Terpinene, the biological precursor of carvacrol, on the other hand, was unsuccessful as a fungicide. Carvacrol disrupted the purpose of rapamycin signaling pathway, causing the cells to die.

20.3.2.3 Antiviral activity The rise of viral resistance to antiviral medications increases the need for novel compounds that are effective in viral infections, and natural products could provide a source of antiviral drugs (Koroch et al., 2007). In a viral suspension test, essential oils of Melaleuca alternifolia and eucalyptus displayed strong antiviral activity against herpes simplex virus type 2 (HSV-2) and herpes simplex virus type 1 (HSV-1) (Valenti et al., 2001). Santolina insularis essential oils also demonstrated direct antiviral actions on both HSV-1 and HSV-2, inhibiting both herpes types’ cell-to-cell transmission (Sinico et al., 2005). Furthermore, it was shown that essential oils in multilamellar liposomes significantly increased antiviral effectiveness against intracellular HSV-1 (Duschatzky et al., 2005; Garcia et al., 2003). Essential oils from Argentine aromatic plants were found to be virucidal against HSV-1 and Junin virus, and the activity was time-dependent and temperature-dependent (Minami et al., 2003; Schuhmacher et al., 2003). The scientists were unable to determine the nature of the active components of the oils that were responsible for the antiviral action. Mentha piperita (Marongiu et al., 2003) and lemon grass (Burke et al., 2004) essential oils demonstrated a direct virucidal effect on HSV-1. Essential oils have been shown to have antiviral activity against poliovirus-1 (Chiang et al., 2005); specificity of the inhibition of viral isoborneol, polypeptide glycosylation, has been reported to be an attractive chemical for suppressing the HSV life cycle (Wang et al., 2006). Linalool was also the most effective against adenoviruses; however, -caryophyllene, carvone, farnesol, -myrcene, cineole, geraniol, fenchone, and -thujone had no effect (Armaka et al., 1999). A study of essential oils from different Melaleuca species found that those with higher levels of 1,8-cineole and terpinene-4ol had more antiviral activity than those with higher levels of methyl eugenol or 1,8-cineole (Farag et al., 2004).

20.3.3 Anticancer activity Cancer is a complicated disorder described by the uncontrolled proliferation of aberrant cells, which results in the development of a tumor (Pavet et al., 2011). Carcinogenesis is a multiphase process involving oxidative damage and the development of malignancies. Secondary metabolites from several plants have been shown to slow the progression of cancer (Rajput & Mandal, 2012). Cytotoxic and anticancer properties are found in essential oil components. They are crucial in the prevention and treatment of cancer (Magalha˜es & de Sousa, 2015). Essential oils can be used in conjunction with cancer treatment to reduce pharmacological side effects (Hadfield, 2001). Essential oils cause cytotoxicity by disrupting cellular integrity, resulting in necrosis and apoptosis, cell cycle arrest, and the loss of crucial organelle

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function (Bhardwaj et al., 2013; Russo et al., 2015). As a result, determining the antitumor activity of the extract oils as well as their safety in normal cell lines is critical (Sieniawska et al., 2016). The essential oil from Ricinus communis leaves has a moderate antiproliferative effect on a cervical cancer cell line. Thujone and 1,8-cineole are made up the majority of the oil’s makeup (Zarai et al., 2012). HT29, A549, and Hep2 cancer cell lines were shown to be significantly cytotoxic by Eugenia caryophyllata essential oil. The high amounts of phenolic chemicals, particularly eugenol, are most likely to blame for the cytotoxicity (Kouidhi et al., 2010). Eugenol was found to be cytotoxic to cytotoxic effect cells HepG2 and colonic cells Caco2 (Yoo et al., 2005), HL60 leukemia cells, (Atsumi et al., 2005), and bone formation cell line U2OS (Ho et al., 2006) in a dose-dependent manner. Essential oil terpenoids inhibit tumor cell growth. The primary components in lavender oil were linalyl and linalyl acetate. They were more harmful than the total essential oil against the cancer cell lines 153BR, HNDF, and HMEC1 (Prashar et al., 2004). MCF-7 mammary cancer cells (Duncan et al., 2004) and PC3 prostatic cancer cells (Kim et al., 2012) were both inhibited by geraniol. It was found to disrupt membrane processes and ion transport, limit DNA synthesis, and shrink colon cancers (Carnesecchi et al., 2004). The proliferation of MCF-7 tumor cell lines was not inhibited by caryophyllene, whereas humulene was cytotoxic. Caryophyllene, on the other hand, increased the cytotoxicity of humulene (Legault & Pichette, 2007). Neuroblastoma cells were unaffected by limonene or linalyl acetate. Their combination, on the other hand, caused apoptosis (Russo et al., 2013). The anticancer characteristics of this synergic effect were consistent in various investigations (El-Shemy et al., 2013). Herb and basil essential oils contain a lot of carvacrol. In colon cancer cell lines HCT116 and LoVo (Fan et al., 2015), as well as human oral carcinoma (Dai et al., 2016), it reduces tumor cell proliferation and promotes apoptosis. HCT116 cells grow slower when exposed to perillyl alcohol (Ma et al., 2016).

20.4

Medicinal applications of essential oils

The bioactivities of essential oils were covered in the preceding section, and several investigations confirmed the uses readily available of essential oils derived from various plants. Due to their high volatility, surface charge, and ease of oxidation, their use is frequently restricted. The usage of essential oils has been expanded to the medical sectors thanks to number of factors that contribute to intervention and the emergence of new innovations such as microscopy and nanotechnology. As a result, the applicability of plant oils in biomedicine is recognized in this part. Essential oil-based microencapsulation: Microencapsulation technology encapsulates chemicals in natural polymer materials to provide a nanoparticle product embedding technology. Microencapsulation mitigates some of the drawbacks of using essential oils, such as high volatility and quick dissipation, and the delayed release of microcapsules can substantially slow down the evaporation of the active ingredients. Furthermore, interaction among plant oils and polymers is a concern for microcapsule products containing essential oils. Spray drying, molecular encapsulation, water phase separation, and refitting are the most common ways for preparing microcapsules; however, the heating phases in the production process might result in a partial loss of essential oils, which is also a major problem. In this case, optimizing and protecting the content of essential oils are critical. To enhance the functional qualities of active ingredients in essential oils, it is critical to use the right sealing materials and packaging technologies. Essential oil-based nano-emulsion: Apart from the two types of active packaging stated above, nano-emulsion has been widely employed in the food sector because of its capacity to solve issues such as essential oil stability, strong odor, and unfavorable impact on food organoleptic features. Because of its nonmetered size and greater diffusion, micro as a delivery technology can help to increase the durability of plant oils, retain the action of bioactive substances, and decrease their different types of samples on food. It is a promising method that can be found in essential oil emulsions made with food-grade materials. Furthermore, as compared to ordinary emulsions, nano-emulsions exhibit several unique physiochemical properties, such as very smallest particle size, excellent transparency, and strong physical stability. The use of aromatic oils nano-emulsion as great packaged foods in the food chain is currently gaining traction, but its commercial potential needs to be investigated further (Garavand et al., 2019). Biomedicine application: Due to their high antioxidant activities and spectral antibacterial characteristics, EOs are widely employed as antibacterial agents and antioxidants. EOs also have powerful therapeutic qualities, including antiinflammatory and antispasmodic characteristics, and can be utilized as local anesthetics. The study of essential oils’ therapeutic effects has grown in popularity in recent years. The nano-drug delivery system, for example, gives a lot of standard value for EOs as medicine, including continuous drug release control, deep tissue penetration of pharmaceuticals with nanoparticle, cellular absorption, and drug therapy protection at the intracellular and extracellular levels (Mehdizadeh et al., 2018). Herbal items for ingestion, containing essential oils, are classed as dietary supplements in the United States. As a result, they are frequently exempt from the more stringent regulations that apply to pharmaceuticals and foods.

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Pharmaceutical products are considered medicinal products in Australia if they contain essential oils and make therapeutic claims (Ghosh et al., 2006). They can be categorized as registered or listed products in Australia’s pharmaceutical regulatory framework. Listed items must meet less strict requirements than registered products and can make smaller therapeutic claims as a result (Forte & Raman, 2000). Different regulatory measures are taken in distinct member states of the European Union, notwithstanding the goal of harmonization among member states (Barnes, 2003). Essential oils synthesized into therapeutic goods may be licensed for distribution under a simplified approach once harmonized, rather than having to meet criteria for an entire product license (Beerling et al., 2002). In most Western countries, over-the-counter (OTC) drugs are intended to treat minor, self-limiting diseases and their symptoms. Many OTC medicines contain essential oils as active components or excipients. For example, eucalyptus oil is present in over 100 over-the-counter (OTC) medicines, designated primarily for the treatment of respiratory infections. Any active pharmaceutical drug with essential oils as the main remedy has been completely licensed as medication by meeting the safety and effectiveness data requirements to be met by new, traditional pharmaceuticals around the world. There are a variety of reasons for this, including a lack of resources to thoroughly investigate and maintain the efficacy and safety of such items. In most circumstances, the intellectual property created as a result of such efforts would not be properly safeguarded to justify the private investment.

20.5

Conclusion

After the study of major varieties of essential oils and their chemistry, it is concluded that various complex and simple chemical constituents are present in various essential oils. There are various biological activities of essential oils. Thus, further task is the use and application of these effective essential oils in various pharmaceutical formulations for better therapeutic effects with minimum side effects.

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Chapter 21

Biological potential of essential oils in pharmaceutical industries M. Anjaly Shanker1, Anandu Chandra Khanashyam2, Priyamvada Thorakkattu3 and Nilesh Prakash Nirmal4 1

Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM),

Sonepat, Haryana, India, 2Department of Food Science and Technology, Kasetsart University, Chatuchak, Bangkok, Thailand, 3Department of Animal Sciences and Industry/Food Science Institute, Kansas State University, Manhattan, KS, United States, 4Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, Thailand

21.1

Introduction

Essential oils (EOs) satiate the class of important natural products obtained from plants, distinguished by strong odor and multicomponent profile with a variety of applications including cosmetics, food, perfume, medicinal, and therapeutic purposes. Defined as secondary metabolites isolated from aromatic plants, they played a significant role in ancient pharmacopoeia. For 5000 years, the use of essential oils has been a key aspect of Indian medicinal practice. Ayurveda is an ancient system of medicine that outlines the medicinal and religious applications of over 700 distinct species of plants and remains as the foundation of Indian healthcare (Gurib-Fakim, 2006). A document from the Ottoman Empire (1571
1878) that was discovered on the island of Cyprus lists 494 herbal remedies and 231 plant species that were utilized by Orthodox monks in their monasteries (Lardos, 2006). Moerman (1996) reported that the Native American communities used around 2564 plant species as medications and about 1625 species as food. In the 16th century, Paracelsus von Hohenheim first used the word “essential oil,” referring to the active ingredient in a medication “Quinta essential” (Guenther, 1950). Hippocrates, regarded as the founder of modern medicine, recorded the therapeutic value of more than 300 plants (Thomson & Schultes, 1978). Assimilation of their chemical constituent profile and their bioactivities, the application of these compounds started compounding with time. Attributing to diverse techniques of extraction of these EOs, the compositional value and quantity are dependent on various factors including the extraction method, soil composition, plant part, age, climate, etc. (Edris, 2007). Several techniques including solvent extraction, water or steam distillation, supercritical fluid extraction, etc., are commonly used to extract EOs and their constituents from different parts of aromatic plants. Latest studies on these extraction techniques intend to the inclusion of greener technologies evading solvent treatment aiming at superior quality products at reasonable expenses (Selvamuthukumaran & Shi, 2017). Essential oil composition is highly variable within and across different plant species, instituting every unveiled functional property. Suitability and significance of these compounds in medical or pharmaceutical functions are due to the presence of these constituents which exhibits different antiseptic and medicinal properties like antimicrobial, anti-inflammatory, wound healing, anticancer, antidiabetic, and other miscellaneous properties (Fig. 21.1). Major advantage of EO application is the synergy of the specific activity of these constituents’ offering superiority than applied discretely as individual modules. The potentiality of these major components is found to be favored by the presence of other minor components in the composition of essential oils (Bakkali et al., 2008). Therapeutic potential of essential oils in biopharmaceuticals is dependent on their quality and safety; hence, it is important to ensure the stability of these components until the expiry period. Ancient medicine has always served as the foundation for modern allopathic medicine, and it is possible that a great number of significant new treatments will be found and made available for purchase in the future, just as they have been up to this point, by following the cues offered by conventional wisdom and experiences. The current chapter provides information on the chemical makeup of certain significant EOs and their primary bioactive single constituents, as well as some features of plant EOs medicinal Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00036-0 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 21.1 Various therapeutic properties of essential oils.

benefits for human health and biological characterization, such as pharmacological characteristics (anti-inflammatory, antioxidant, anticarcinogenic properties, etc.), and antimicrobial effects.

21.2

Bioactive components of essential oils

Essential oils contain complex mixes of volatile molecules and are available as concentrated liquids. Generally, essential oils are characterized based on four criteria: the size or number of carbon atoms, primary biosynthetic origin, the parent backbone, and the character of oxidation by electronegative atoms, which are atoms larger than carbon like oxygen or nitrogen (Sadgrove et al., 2022). Three biosynthetic classes of essential oil constituents—terpenes, phenylpropanoids, or isothiocyanates—are produced by four primary biosynthetic processes. They come from the metabolic routes for mevalonate and methylerythritol phosphate (Zhao et al., 2013), shikimate (Santos-Sa´nchez et al., 2019), and glucosinolate (Malka & Cheng, 2017). It could contain volatile compounds from both non-terpenoid or terpenoid origin. In plants, essential oils are generally found in low concentrations, ranging from 0.1% to 1% (Noriega, 2020). Korkina (2007) reported that both plants and microorganisms have two major groups of metabolites: primary metabolites (essential for survival) and secondary metabolites (released as a defense against predators and pathogens and also protects against other stressful environments). Primary metabolites are precursors of secondary metabolites through various biochemical pathways such as methylation, glycosylation, and hydroxylation. Secondary metabolites play a crucial role in acclimatizing to environmental changes and also serve as signaling molecules and hormones to create a competitive advantage by poisoning enemies. The largest of several classes of secondary metabolites can be divided into (1) terpenes, (2) phenylpropanoids and their derivatives, and (3) nitrogen-containing compounds. Essential oils include components that perform almost all of the chemical processes, and the most basic are hydrocarbons from the terpene family, such as p-cymene, which is comparable to pinene, a component of turpentine oil. The remaining elements are virtually all terpene stages at varying stages of development. Essential oils may be obtained from a variety of plant components (bark, buds, flowers, fruits, leaves, peels, etc.) or the whole plant from a single source. They have several commercial applications as flavors, scents, high-grade lubricants, and pharmaceuticals as a result of their chemical diversity. Terpenes are regarded as the largest group of components in essential oils (De Groot & Schmidt, 2016). Terpenes are also called as terpenoids and can be classified based on the number of isoprene units as mono, di, tri, tetra, and sesquiterpenes (Berger, 2007). Isoprene is a hydrocarbon with the molecular formula C5H8

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and is the building unit of terpenes. The polymerization capacity of isoprene determines the chemical diversity of terpenes (Noriega, 2020). All terpenes have a hydrocarbon backbone that is created from prenyl diphosphates of different lengths. The remaining allylic carbocation intermediates can be encouraged to proceed through intricate chemical cascades after the diphosphate group is ionized, creating a variety of linear and cyclized hydrocarbon backbones that can then be further modified with a wide range of functional groups (Jiang et al., 2016). Although the terms terpenes and terpenoids are frequently used interchangeably, they do have some differences. Terpenes are a combination of isoprene units that are naturally occurring, volatile, unsaturated 5-carbon cyclic compounds that has a scent or a taste to defend themselves against organisms that feed off certain types of plants (Cox-Georgian et al., 2019). Terpenes and isoprenoids, in general, have garnered much attention because of their important physiological/ecological roles (Kempinski et al., 2015) and their broad uses in pharmaceutical applications (Batool et al., 2021). The biosynthesis of all isoprenoids starts from the two universal fivecarbon precursors: isopentenyl diphosphate and dimethylallyl diphosphate. Thin-layer chromatography, gas chromatography, or liquid chromatography are the analytical techniques usually employed for the analysis of terpenes (Jiang et al., 2016). Notably, numerous chemical processes and/or experiments have resulted in the discovery, characterization, and extraction of key elements that are of great interest, particularly the recovery of selected terpenes for industrial use. Monoterpenes (one terpene unit or two isoprene units; examples include linalool, geraniol, and limonene) are the smallest terpenes. They are recognized as the primary ingredient of essential oils, perfumes, and numerous structural isomers since they include the composition C10H16 and are derived from various flowers, fruits, and leaves. Among all the terpene classes, monoterpenes have the strongest aroma. Sesquiterpenes (three isoprene units; examples include caryophyllene, humulene, etc.). Sesquiterpenes, which have the molecular formula C15H24, are much more stable than monoterpenes. They are separated by steam distillation or extraction and then refined using techniques like vacuum fractional distillation or gas chromatography. Interestingly, about a quarter of the terpene fractions in essential oils are monoterpenes and sesquiterpenes (De Groot & Schmidt, 2016). Sesquiterpenoids are created by oxidizing or rearranging isoprene units after they have been converted to sesquiterpenes. Diterpenes are chemical substances that are created naturally and have the molecular formula C20H32. Squalene is the biological precursor of all triterpenes. Triterpenes are made up of three or six isoprene units and contain the chemical formula C30H48, which also includes steroids and sterols. The tetraterpenes (eight isoprene units; examples include carotenoids) are classified according to the number of isoprene units. Tetraterpenes, commonly referred to as carotenoids, are substances that are derived from isoprene units and have the chemical formula C40H56. Since most carotenoids are very unsaturated, isolating and purifying them may be quite challenging. Notably, the varieties of chemical components found in essential oils determine the pathways involved in producing a certain impact. For instance, the application of caryophyllene present in numerous herbs and spices was recently reported to enhance reepithelialization of cutaneous wounds, and linalool found in herbs, spices, and fruits has an anxiolytic effect through the olfactory system (Harada et al., 2018). Lavender essential oils, for example, contain both caryophyllene and linalool. These examples, as well as the fact that they are both found in the same essential oil, demonstrate the importance of understanding what impact each chemical constituent and how it produces those effects. This also means that the way essential oils are used should be altered based on the chemical components that are anticipated to work. There are several techniques available to extract essential oils. Essential oils may be extracted from plant matrices using a variety of approaches that are classed as conventional (using heat to extract volatile material) and innovative extraction approaches. Indeed, by minimizing extraction time, reducing energy utilization, curtailing solvent use, enhancing selectivity in extraction efficiency, etc., the extraction can be more efficient (El Asbahani et al., 2015). The traditional extraction procedures are normally time-consuming and need huge amounts of organic solvent. The use of significant quantities of solvent results in increased expenses, owing to additional costs involved with the acquisition and disposal of hazardous solvents, and the subsequent environmental risks. Alternative extraction methods that use less solvent and take less time have been taken into consideration during the last 10 years. The more recent methods include pressured liquid extraction, supercritical fluid extraction, ultrasound-assisted extraction, and microwave extraction. Terpenes can have a huge variety of structural variations, so the methods used to purify them will vary case-by-case depending on the chemical characteristics of the target or suspected terpene, the physical characteristics, quantity of the starting plant material, and the accessibility of equipment and reagents. Previously, Jiang et al. (2016) discussed protocols for extracting terpenes and terpenoids from different plant sources. The procedures typically include the following steps: disrupting the plant cells to release their chemical components; extracting the sample with a suitable solvent; distilling/trapping compounds; separating the desired terpene from other unwanted contents of the extracts that confound analysis and quantification and using the proper analytical technique. Phenylpropanoids are a large class of organic compounds synthesized from amino acids, phenylalanine and tyrosine, produced through the shikimic pathway by plants. Phenylalanine is converted to cinnamic acid in the presence of the

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enzyme phenylalanine ammonia lyase. Cinnamaldehyde is formed by the reduction of carboxylic acid functional group in the cinnamic acid. Trans-cinnamate 4-monooxygenase hydroxylates cinnamic acid in the 4-position to produce pcoumaric acid, which could be further converted to produce hydroxylated derivatives like umbelliferone (Neelam et al., 2020). Phenylpropanoids could be obtained from several plant-based sources such as fruits, coffees, herbs, vegetables like potatoes, mushrooms, cereals such as barley, wheat, oats, rye, rice bran, and red wine. Cinnamic acid is an active component in cinnamon, clove, black pepper, coriander, and turmeric (Neelam et al., 2020 ). P-coumaric acid can be found in Chinese cabbage, blood orange, and cranberry. Caffeic acid is a very important constituent in chicory and green coffee. Another important phenylpropanoid, ferulic acid, is found in coffee, corn kernel, rice bran, and spinach. Phenylpropanoids are usually found in conjugated forms in plants. They provide a wide range of antimicrobial activity, provide quenching free radicals by acting as strong antioxidants, help in fighting against diabetes, cancer, and cardiotoxicity, provide neuroprotective effects, and also curb inflammations (Neelam et al., 2020).

21.3

Biological activities of EO

21.3.1 Antimicrobial properties Essential oils are volatile chemicals extracted from medicinal and aromatic plants, mostly by steam distillation, from plant components such as flowers, stems, leaves, seeds, roots, and wood. They are a natural alternative to synthetic food additives and have recently gained interest as a prospective source for natural food preservatives due to the increased interest in developing safe, efficient, and natural food preservatives. They are secondary metabolites generated by plants and have antibacterial, antifungal, antiviral, and insecticidal properties. Terpenes, phenolics, aldehydes, alcohols, ketones, etc., are some of the major components present in essential oils which are responsible for their distinct biological activity. Recently, due to the increased incidence of antibiotic resistance and the unpleasant side effects of synthetic drugs, essential oils are gaining more research interest and have encouraged researchers to develop new antibacterial lead compounds for treating a variety of human infections. Moreover, many advantages of utilizing EO as a food preservative have been reported, including increased shelf life, hypoallergenicity, flavor enhancement, and potential health benefits to consumers because of its antioxidant and anticancer properties (Blowman et al., 2018). Even though there are several hypotheses that explain the antimicrobial action of EO, the primary mechanism of bacterial inactivation is by destabilizing the membrane integrity and by disrupting the proton pump (Nieto, 2017). Due to their high lipophilic nature, EO penetrates easily into the bacterial cell membranes and thereby alters the cell membrane permeability. This will lead to leakage of the intercellular components, leading to cell lysis. The penetration of EO into the cell cytoplasm can also affect the metabolic functioning of the cells such as membrane transport, nutrient processing, production of macromolecules, growth regulators, ATP production, etc. (Oussalah et al., 2006). The changes in cell structure could result in a cascade effect, leading to a breakdown of intercellular components (Carson et al., 2002). The antimicrobial activity of essential oils is also due to the decrease in cell membrane potentials, inhibition of proton pumps, and ATP depletion (Turina et al., 2006). Cox et al. (1998) studied the inactivation mechanism of tea tree oil on Escherichia coli and reported that tea tree oil can alter the permeability of cell membrane and thereby stimulate the leakage of K1 ions. This disrupted the glucose depending on respiration cycle in E. coli leading to cell death. The antibacterial action of three major monoterpenes found in essential oils, namely, menthol, thymol, and linalyl acetate is reported due to a change in the lipid fraction of bacterial plasma membranes (Trombetta et al., 2005). A significant depletion of the internal ATP pool in the cell cytoplasm of Bacillus cereus was reported by Ultee et al. (1999) when carvacrol was used to inactivate the cells. EO’s antifungal mechanism is also reported to be similar to that of the antibacterial inactivation mechanism. In general, essential oil exposure causes cellular components to coagulate due to permanent cell membrane damage. EO creates a membrane potential across the cell membrane in yeast cells, disrupting ATP synthesis and causing cell membrane damage (Aleksic & Knezevic, 2014). The EO also infiltrates and damages the fungal cytoplasm via a permeabilization process, resulting in mitochondrial membrane breakdown. Essential oils are also potential antiviral agents . However, further study is necessary to have a thorough understanding of the antiviral activity of EO. EO can inactivate viruses either by interfering with virion envelopment, which is necessary for virion entrance into host cells, or by suppressing viral replication via cellular DNA polymerase inhibition and changes in phenylpropanoid pathways. Isoborneol is a compound that belongs to the class of monoterpene and is abundant in several plant EO. Studies by Armaka et al. (1999) reported that isoborneol affects the glycosylation process of viral proteins, inhibiting the growth of HSV-1. The multiplication ability of HSV-2 viruses has been reported to be inhibited by citronella and citral which are the main active components found in Melissa officinalis EO (Allahverdiyev et al., 2004). The antimicrobial properties of various EO and their active components are summarized in Table 21.1.

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TABLE 21.1 Summary of antimicrobial properties of EOs. Compound/EO

Targeted organism

Comments

References

Artemisia essential oils

E. coli, S. aureus, Staphylococcus epidermidis, C. albicans, C. neoformans, T. rubrum, M. canis, M. gypseum, F. pedrosoi and A. niger

Study identified a total of 110 components which accounted for 71.0%
98.8% of the oil composition. Cineole, Santolina triene, camphene, and α pinene were some of the major active components identified.

Lopes-Lutz et al. (2008)

Achillea clavennae essential oil

S. aureus, S. pyogenes, H. influenzae, K. pneumoniae, and S. pheumoniae (penicillinsusceptible and penicillin-resistant), and P. aeruginosa

The highest antimicrobial activity was reported for K. pneumoniae and S. pheumoniae (penicillin-susceptible and penicillin-resistant). The essential oil was effective in inactivating both gram-positive and gram-negative bacteria.

Skocibusic et al. (2004)

Cuminum cyminum and Foeniculum vulgare Mill.

E. coli and S. typhimurium

F. vulgare EO were more effective than C. cyminum in inactivating the pathogens, and S. typhimurium was reported to be more susceptible to EO as compared to E. coli.

Bisht et al. (2014)

Cumin Seeds (Nigella sativa L.)

S. aureus, B. cereus, B. subtilis, and F. moniliforme

EO showed about 90% zone inhibition against F. moniliforme.

Singh et al. (2014)

Ocimum basilicum

Bacterial strains: B. cereus, M. flavus, S. aureus, E. faecalis, E. coli, P. aeruginosa, S. typhimurium, and L. monocytogenes. Fungal strains: A. fumigatus, A. niger, A. versicolor, A. ochraceus, P. funiculosum, P. ochrochloron, and T. viride

Ocimum EO were most active against the bacterium Micrococcus flavus and showed 10
100 times more antifungal activity compared to the commercial antifungal agents for every strain tested

Beatovic et al. (2015)

Origanum

E. coli, S. enteritidis, and S. essen

In comparison to oils containing the monoterpenic alcohol linalool, oils having a larger percentage of phenolic components (carvacrol and thymol) had a better inhibitory activity.

Penalver et al. (2005)

Clove and Rosemary Essential Oils

S. epidermidis, S. aureus, B. subtilis, P. vulgaris, P. aeruginosa, E. coli, A. niger, and C. albicans

All of the studied bacteria were significantly inhibited by both the essential oils. In experiments using individual microorganisms, the antimicrobial activity of mixtures of the two essential oils revealed their additive, synergistic, or antagonistic activities.

Fu et al. (2007)

Ajwain (Trachyspermum ammi: Umbellifereae) essential oil

Japanese encephalitis virus (JEV)

Postexposure treatment showed a 40% virus inhibition when treated with 0.5 mg/ mL of ajwain oil by PRNT method.

Roy et al. (2015)

Achillea fragrantissima EO

ORF virus

The ORF titer reduced from 5.9 to 1 after 60 min exposure to EO.

Zeedan (2014)

Fortunella margarita

Avian influenza (H5N1) virus

The synergistic interaction of a combination of volatile molecules or their main components is what gives these essential oils their biological activity.

Ibrahim et al. (2015)

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Cancer-preventing function

Cancer is a complicated genetic condition that causes the body’s aberrant cells to develop and spread out of control, posing a threat to human life. Major signaling elements that activate cancer in humans include aging, alterations in lifestyle, hormone changes, and carcinogen exposures. Genomic instability brought on by DNA damage is the primary cause of cancer. Even while certain traditional cancer treatment techniques, such as chemotherapy, radiation therapy, and surgery, are successful, they can have significant adverse effects and toxicities. In general, there are three phases in the development of cancer: (1) initiation, when exposure to a carcinogen causes DNA damage and mutations occur at the cellular level owing to a malfunction in DNA repair processes; (2) promotion, when cell growth causes hyperproliferation, alteration, and inflammation of the cells and tissues; and (3) progression, when tumor formation from preneoplastic cells occurs through clonal expansion. EOs cause cancer cell death by inducing necrosis, apoptosis, causing organelle dysfunction, and cell cycle arrest. This is accompanied by an increase in the cell membrane fluidity, decreased ATP synthesis, change in pH gradient, and a loss of mitochondrial potential, all of which are precursors to cell death (Sharifi-Rad et al., 2017). The anticarcinogenic effects of EO are mainly due to its antioxidant, antimutagenic, and anti-proliferic properties (Bhalla et al., 2013). In recent years, a lot of researchers have been looking into the antioxidant properties of various essential oils in an effort to find safer natural antioxidants. As a result, several studies have identified essential oils as the ideal source of natural antioxidants. Superoxide anions and hydrogen peroxide in eukaryotes form hydroxyl radicals, which are extremely harmful to mitochondrial DNA. An RoS accumulation is caused by mitochondrial DNA damage as it prevents the expression of the electron transport protein. When combined with essential oil, the free radicals produced by the damaged mitochondrial membrane form reactive phenoxy radicals, which then join with ROS to stop further damage. The antimutagenic activity of EO is primarily achieved by: (1) preventing mutagens from penetrating the cell membrane, (2) inactivating the mutagens by direct scavenging, (3) trapping the free radicals created by mutagens, and (4) blocking the enzymes such as cytochrome P450 which is involved in converting pro-mutagens to mutagens, and by efficient error-free DNA repair (Sharma et al., 2022). There are also multiple mechanisms through which EO could exhibit antiproliferative activity such as membrane disruption apoptosis induction (Russo et al., 2015). Bayala et al. (2014) studied the antiproliferative activity of essential oils extracted from seven different plants, namely, Ocimum basilicum, Ocimum americanum, Ageratum conyzoides, Hyptis spicigera, Lippia multiflora, Eucalyptus camaldulensis, and Zingiber officinale. The antiproliferative activity was tested against androgen-resistive and androgen-sensitive prostate cancer cell lines, PC3 and LNCaP, respectively. The study reported that four out of the seven tested plant EOs, O. basilicum, L. multiflora, Z. officinale, and A. conyzoides, showed significant activity against both PC3 and LNCaP cancer cell lines. The anticancer property of garlic essential oils along with other organosulfur compounds such as diallyl sulfide, diallyl disulfide, diallyl trisulfide on HL-60 leukemia cells via apoptosis due to the action of intracellular RoS has been demonstrated by Agassi et al. (2020). Antiproliferative activity of Citrus medica essential oil against human hepatocellular carcinoma (HepG2, EC50 5 0.091 mg/mL), breast adenocarcinoma (MCF-7, EC50 5 0.16 mg/mL), colon adenocarcinoma (Caco2, EC50 5 0.013 mg/mL), leukemic monocytic (THP-1, EC50 5 0.074 mg/mL), malignant melanoma (A375, 0.0057 mg/mL) was reported (Mitropoulou et al., 2017). Furthermore, when tested against skin melanoma A375 cells and healthy skin cells (HaCat), C. medica oil was found to have cancer-specific action (Mitropoulou et al., 2017). A significant reduction in phenobarbitone-induced cytochrome p450 enzyme activity, along with the anticarcinogenic activity of turmeric EO against Dalton’s lymphoma ascites cells (DLA) and Ehrlich ascites carcinoma (EAC) cancer cell lines, was demonstrated in vivo and in vitro by Liju et al. (2014). Another in vitro study on Eucalyptus globulus EO against A549 lung cancer cells showed a significant reduction in number of surviving cells with an increase in concentrations of EO (Adnan, 2019). Saab et al. (2012) reported cytotoxicity of Juniperus oxycedrus EO against acute lymphoblastic leukemia cells CCRF-CEM (drug-sensitive) and CEM/ADR5000 (multidrug-resistant P-glycoprotein-expressing).

21.5

Antioxidant and anti-inflammatory properties

Antioxidants are substances that can prevent or delay the oxidation of free radicals. One of the essential biological characteristics of EOs to combat oxidative stress is their antioxidant capacity. Reactive oxygen species (ROS), such as hydroxyl radicals, hydrogen peroxide, and superoxide anions, cause mitochondrial DNA damage in eukaryotes under oxidation-prone circumstances by inhibiting the electron transport chain, which results in a buildup of ROS. Reactive phenoxy radicals are created when ROS and EO interact, setting off a series of events that prevents additional oxidative damage by combining with ROS once again. Upregulation of antioxidant enzymes (such as superoxide dismutase, catalase, etc.) and non-antioxidants like glutathione is another antioxidant mechanism triggered by EOs. Since an oxidative burst is one of the inflammatory responses, essential oils that can eliminate certain free radicals may also have antiinflammatory effects. Inflammation-related phagocytosis of microorganisms is followed by a sharp rise in oxygen

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consumption. This leads to the production of superoxide anion radicals (O2 2), which are then swiftly converted to hydrogen peroxide (H2O2) either spontaneously or by the enzymatic action of superoxide dismutase. This H2O2 can later react with the polyunsaturated fatty acids, leading to the production of peroxyl radical. Another important type of free radicals that are generated during the process is reactive nitrogen species (RNS) such as nitric oxide and peroxynitrate anion which are produced as a result of immunological reaction by neutrophils and macrophages. ROS and RNS are produced in phagocytes in an effort to neutralize invasive pathogens and play a crucial role in the host defense system. However, their overproduction may result in damage to the inflammatory areas. Furthermore, these reactive species play crucial roles in inflammation by acting as trigger elements or signaling messenger molecules. The inflammatory responses are characterized by an increased endothelial permeability followed by an increased influx of bold leukocytes and an oxidative burst. This will also induce the production of arachidonic acid along with certain enzymes (peroxidase, oxygenase, nitric oxide synthases, etc.) and cytokines (interleukins and tumor necrosis factor-α [TNF-α]) (Gomes et al., 2008). Polyunsaturated fatty acids, such as arachidonic acid, are released from cell membranes by the enzyme phospholipase A2 in response to various inflammatory stimuli. The lipoxygenase (LOX) and cyclooxygenase (COX) pathways metabolize the same fatty acid in various eicosanoids, including prostaglandins (PGs), leukotrienes (LTs), and thromboxane A2. The COX-2 and TNF-α inhibitory activity of cinnamaldehyde along with luciferase inactivation on RAW 264.7 macrophages was reported by Lee et al. (2005). Cinnamon EOs are also reported to inhibit nitric oxide (NO) and prostaglandin E2 (PGE2) synthesis in macrophages (Tung et al., 2008). Inhibition of cytokines, interleukin-1beta (IL-1β), and TNF-α secretion within lipoteichoic acid (LTA) or lipopolysaccharide (LPS)-stimulated murine J774A.1 macrophages by cinnamaldehyde through in vitro studies were reported by Chao et al. (2008). Ballabeni et al. (2010) studied the anti-inflammatory properties of Ocotea quixos essential oil both in vivo and in vitro. The study reported a decrease in LPS-induced COX-2 expression and NO release. Moreover, in feeding of rats with carrageenaninduced rat paw edema, a significant reduction in swelling in a dose-dependent manner was observed. Similar inhibitory effects on carrageenan-induced rat paw edema were also observed in the case of dillapiole, an active component extracted from Piper aduncum L. essential oil (Parise-Filho et al., 2011). Chamazulene and bisabolol are two additional compounds found in chamomile essential oil that have anti-inflammatory properties, likely due to the inhibition of 5-lipoxygenase by suppressing leukotriene production (Kamatou & Viljoen, 2009). Studies have also confirmed that terpinen-4-ol, a major component of tea tree oil is capable of suppressing of TNF-α, IL-1β, and PGE2 in human monocytes (Hart et al., 2000). G

21.6

Role in cardiovascular diseases

Important among the noncommunicable diseases in the world, cardiovascular problems are befalling and recurrent in humans leading to major health issues, even death. Cardiovascular diseases are correlated with certain risk factors including age, gender, race, gene expression which is non-modifiable and also includes other modifiable factors like unhealthy diet, physical inactivity, obesity, hypertension, diabetes, smoking, alcohol misuse, and psychological problems. Minimal incidence of the disease intends toward the better management leading to the detection and modification of these risk factors. An intention to recommend new techniques with minimal side effects indorses the likelihood of using medicinal and aromatic plants and their constituents in managing cardiovascular diseases and the risk factors associated with it. Ribeiro et al. (2010) investigated the efficiency and mechanism triggering the cardiovascular changes stimulated by alpha-terpineol, a vital constituent of essential oils of major aromatic plants. The author validates the presence of terpineol leading to EO-induced hypotension facilitating vasorelaxation. Concentration-dependent release of nitric oxide (NO) and NO/cyclic GMP pathway activation are speculated to be mechanisms behind the induced changes in the system. Intravenous intrusions by EO obtained from Ocimum gratissimum in rats exhibited constant and dosage- or concentration-dependent reductions in mean aortic pressure and heart rate (Lahlou et al., 2004). The hypotensive activity related to the presence of EO can be attributed to eugenol, the major and important volatile constituent of this essential oil. Cymbopogon winterianus essential oil was found to exhibit cardiovascular effects by inducing hypotension and vasorelaxation in male Wistar rats under study (De Menezes et al., 2010). These dose-dependent changes are mainly attributed to the Ca21 channel blocking happening under the influence of constituents of EO. Menezes et al. (2010) evaluated the cardiovascular potential of four monoterpenes and one sesquiterpene in normotensive rats. With the former inducing hypotension with tachycardia, the latter was found to be causing hypotensive effect with bradycardia. The major difference attributed between these compounds is the mechanism followed for achieving cardiovascular changes.

21.7

Antidiabetic agents

Subsisting as a public health problem concerning many populations, diabetic issues are exemplified by hyper- or hypoglycemic conditions subsequent to the metabolism of glucose and abnormalities in insulin secretion and activity. Recent

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studies in controlling diabetes are mainly majored by the utilization of plants species, thereby controlling diabetic issues and other health hazards related to this. Compared to the other aspects, the potential of EO in maintaining this condition is less evaluated and has a potential to be explored. Prevention of diabetic issues follows the inhibition or advancement of different metabolic mechanisms, and the selection is based on the composition of EOs involved in the process. Possessing large amounts of volatile components like geranial (65.4%), neral (24.7%), citrals, etc., medicinal plant species lemon balm (M. officinalis) essential oil is found to have significant antidiabetic potential (Chung et al., 2010). In vivo studies in mice for a period of 6 weeks have exhibited a substantial reduction in glucose levels and improved glucose tolerance which is facilitated by the inhibition of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) enzyme in the liver and glucokinase (GCK) activation. In addition, improved GLUT4, sterol regulatory element-binding protein SREBP-1c, peroxisome proliferator-activated receptors PPAR-g and PPAR-a expression in adipose tissue and liver have also contributed toward the additional anti-hyperglycemic potential of lemon balm EO. Similarly, Yen et al. (2015) reported the efficacy of EOs of M. officinalis assessed on their glucose consumption activity. In vitro antidiabetic screening model grounded on gauging consumption of glucose after 24 hours in a culture medium of 3T3-L1 adipocytes showed that the presence of EO increased the glucose consumption by 63.64% proposing M. officinalis as a potential candidate in managing diabetic issues. The author speculates the possibility of activation of ACC/AMPK pathway boosting the glucose uptake and regulating acetyl CoA carboxylase (ACC) activity leading to the inhibition of lipid accumulation in adipose tissues. Consolidated data on the antidiabetic potential of essential oils from different plant sources are given in Table 21.2. The significance of dosage in amending antidiabetic activity of EO is

TABLE 21.2 Consolidated data on antidiabetic potential of essential oils. Compound/EO

Conditions

Observations

References

Pelargonium graveolens EO

Antidiabetic activity via α-glucosidase inhibition assay at concentration range

Inhibition of enzyme ranging from 28.13 to 74.24 μg/mL for concentration ranging from 31.25 to 1000 μg/mL

Ahamad and Uthirapathy (2021)

Syzygium aromaticum EO

Dose dependency of the activity was tested at concentration from 1 to 100 μg/mL

Increase in activity at increase in concentration. Highest activity of 95.30% at higher concentrations

Tahir et al. (2016)

Lemon balm essential oil

EO at concentration of 0  015 mg/d for 6 weeks in mice

Reduction in glucose levels, improved glucose tolerance and metabolism in liver and adipose tissue makes EO at low concentration as an anti-hypoglycemic agent

Chung et al. (2010)

EO of fenugreek, cinnamon, cumin, oregano

Combination of EOs to enhance insulin sensitivity in Zucker fatty rats (ZFRs) and hypertensive rats (SHRs)

Essential oils helped in lowering the circulating glucose levels and enhancing insulin sensitivity

Talpur et al. (2005)

Lemon balm essential oil

Assayed on glucose consumption activity

Increased glucose consumption by 64% by the presence of EO

Yen et al. (2015)

Cuminum cyminum EO

Dose dependency of the activity was tested at concentration from 1 to 100 μg/mL

Increase in activity at increase in concentration. Highest activity of 83.09% at higher concentration

Tahir et al. (2016)

Safflower essential oil

Comparison of different extracts of safflower EO

EOs showed best in vitro activity against protein tyrosine phosphatase (PTP1B) indicating potential for treating against diabetes

Li et al. (2012)

Origanum vulgare

Combination of EO and rosmarinic acid in regulating amylase activity

Combination of EO and rosmarinic acid was better than operating discretely

Alef et al. (2013)

Eucalyptus camaldulensis essential oil

In vitro antidiabetic activities of major components

Antidiabetic activities of EO exhibited by the α-amylase and α-glucosidase inhibition by noncompetitive mechanism

Sahin Basak and Candan (2010)

Eruca vesicaria EO

Inhibitory potential against α-amylase and α-glucosidase

EO extracted showed potential against α-amylase and α-glucosidase inhibition showing antidiabetic potential

Hichri et al. (2019)

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fairly imperative sphere that needs to be covered in research investigations to reciprocate the actual implication of EO in maintaining glucose levels. Tahir et al. (2016) proposed such a correlation of dosage on the potential of EOs of Syzygium aromaticum and Cuminum cyminum in antidiabetic activity. With major composition of eugenol and α-pinene, the maximum activity was exhibited at higher concentrations of 100 μg/mL and minimum activity at lower concentrations in the case of EOs from both plants. However, general inferences on this aspect entail more revisions on a variety of essential oils from different plant sources. The efficacy of combination of different essential oils in controlling blood glucose levels was evaluated by Talpur et al. (2005). This study investigated the possibility of combinations of essential oils such as oregano, fenugreek, cumin, cinnamon, and myrtle oils in enhancing the insulin sensitivity of Zucker fatty rats (ZFRs) and spontaneously hypertensive rats (SHRs). Combined essential oil proportions were found to be effective in lowering the circulating blood glucose levels and systolic blood pressure enhancing insulin sensitivity. Furthermore, conclusions on the proportions of these combinations require more extended research. Alef et al. (2013) suggested the combination of EOs with other phenolic substances in regulating the amylase activity. Combination of O. vulgare and rosmarinic acid exhibited higher inhibition rates than operating disjointedly. The outcomes propose the necessity of investigations on these combinations along with studies on individual characteristic properties of EOs in regulating the diabetic issues.

21.8

Other important properties

The diversity and therapeutic potentials of essential oils and their volatile components are not only constrained to above-discussed boundaries. Noteworthy outcomes on antimutagenic properties of essential oils were reported by several authors. Ginger essential oil was found to be effective in inhibiting mutagenicity stimulated by different mutagens (Jeena et al., 2014). The inhibition rates were dependent on concentration and the kind of mutagenic constituents encompassed. Results expressed an increase in inhibition percentage with an increase in concentration up to 1 mg/plate. Evandri et al. (2005) proposed the antimutagenic properties of EO of Lavandula angustifolia against the mutagens induced in Salmonella typhimurium strains. Antimutagenic activity was found to be concentration-dependent, with the highest inhibition of 66.4% observed at higher concentrations of 0.80 mg/plate. EOs and their terpene components distinguished by their affordability and high efficacy were reported to be agreeable natural substitutes for synthetic skin penetration enhancers. Amnuaikit et al. (2005) contemplated the possibility and efficacy of employing terpenes like cineole, menthol, and propylene glycol as enhancers to improve the skin penetrations of propranolol hydrochloride. Antistress and neuroprotective properties of eugenol obtained from clove, nutmeg, cinnamon, basil, etc., were reported by different authors. Garabadu et al. (2015) reported the usefulness of eugenol in stress-related and neuroprotective activities during in vivo studies in rats. Reports illustrate that the compound safeguarded restraint stress-induced gastrointestinal dysfunction and also had a prominent involvement in the brain monoaminergic pathways. Inclusion and use of essential oils as an alternative to the synthetic chemicals used to control insects will be accommodating concerns related to health issues in humans. Concentration contingent effectiveness of the EOs of Ipomoea cairica, Centella asiatica, Psidium guajava, Momordica charantia, and Tridax procumbens against malarial vector Anopheles stephensi was reported by Rajkumar and Jebanesan (2007). Dose-related efficacy shows the highest repellence at a concentration of 6%. Among the tested EOs, the highest inhibition was shown by I. cairica, T. procumbens, and M. charantia followed by the other two extracts. Potential of Salvia lavandulaefolia Vahl. extracts and constituents in the treatment of Alzheimer’s disease in a pilot clinical trial was reported by Perry et al. (2003). The study elucidates the improvements in the accuracy of detection of target digits and the increase in false alarm responses in vigilance test (VIGFA) after 6 weeks of treatment.

21.9

Application of EO in pharmaceutical industry

Owning and unveiling functional properties like antioxidant, antibacterial properties, etc., have increased the amount of research on EO with the idea of featuring them in diverse fields of application. Multifarious healing properties of these products have made their application belligerent in the medical field. Essential oils isolated mainly from Pogostemon cablin which had elevated alcohol content were found to be highly effective against ear edema formation (Luo et al., 2019). The study also elucidates the possibility of involving the EOs extracted from other Lamiaceae species in certain pharmacological applications owing to their anti-inflammatory and oxidation potential. Elated antimicrobial properties of these compounds make them a reliable alternative to control the growth of multidrug-resistant microorganisms and regulate infectious diseases related to that (Tariq et al., 2019). Functionalization of these EO with antibiotics and nanoparticles has known to increase the potentiality of their antimicrobial properties (Bilia et al., 2014; Moon et al., 2011;

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Reichling, 2010). Encapsulation of EO has been observed to be enhancing the drug release rates and volatility regulation and influencing their bioactivity. Enhanced rates of microbial activity were detected against E. coli and Salmonella spp. with encapsulation of thymol EO with zein-sodium caseinate nanoparticles (Li et al., 2012). Similarly, amalgamation of silver nanoparticles and cinnamaldehyde EO was exhibiting a faster bactericidal action in bacterial kill curve analysis, and synergy was exemplified to be the justification for the superior outcomes (Ghosh et al., 2013). The possibility of healing of skin wounds studied by Saporito et al. (2018) shows the efficacy of the synergy of eucalyptus EO and lipid nanoparticles based on coco butter, olive oil, and sesame oil in wound repairing and antimicrobial action. Fungal and bacterial infections are an ongoing problem in the case of biomedical devices and implants that are introduced to the human system. In this context, the study by Grumezescu et al. (2012) becomes important, as the study involves the efficacy of hybrid nanomaterial used for Eugenia caryophyllata EO stabilization. Results depict the usefulness of this nanomaterial-EO collaboration in the inhibition of biofilm development emphasizing the scope of these amalgamations as upgraded anti-biofilm layers in biomedical applications. Comparable research by Chifiriuc et al. (2012) also illustrates the expediency in grouping of Rosmarinus officinalis EO with nanoparticles in inhibiting the biofilm formation of Candida albicans and Candida tropicalis accentuating their practicality. A compelling effect on cancerous cells was also observed by the amalgamation of essential oils with different chemotherapeutic agents in nano-based systems. Nano-emulsion-based camphor oil combined with ifosfamide, a chemotherapeutic agent, was operative in controlling MCF-7 breast and HeLa cervical cancer cell lines exhibiting its potent anticancer activities (AlMotwaa et al., 2019). A similar study conducted by AlMotwaa (2021), investigated the possibility of ifosfamide incorporation into nano-emulsion-based clove oil against cervical and breast cancer cells. Amalgamation showed higher toxicity against these cancerous cells in dose-dependent manner, unveiling the undeniable therapeutic properties of the components. Al-Otaibi et al. (2018) also reported the usefulness of the unification of emulsions of EO from ginger and frankincense and antineoplastic agent mitomycin on cervical and breast cancer cells. Cytotoxicity of individual EO and combination treatment was effective, dosage-dependent, and markedly greater than the effectiveness of free or individual mitomycin treatments.

21.10 Future perspective and conclusion With varied pertinence attributing to the presence of diverse bioactive components, there is exceptional desirability pertaining to the areas of applications of these essential oils. Beneficial bioactive properties including antibacterial, anticancerous, antioxidant activities, etc., can be expended as a reliable answer for human concerns associated with the side effects of synthetic medicines. Pronounced association between the constituents of EO and their bioactivity emphasize the importance of the origin and extraction methods on the effectiveness of the whole system. Amid these parameters, the efficacy of these compounds has exclusively paved a way for these EOs from traditional medicine to modern pharmacological applications. The effectiveness of different EOs and their components against various ailments particularly against cancerous cells and infections and other healing properties aided in impelling further studies and research. A better understanding of their chemistry and mode of action has helped in diversifying the field of application of these EOs. Yet accounting for the wide variation of the chemical constituents, their crisscross activity and mechanism of action, a detailed array of evidences, materials and information becomes obligatory. This necessitates further studies on the possibilities and performances of different EOs and their components in the healing of various alignments. In addition to that, the efficacy of amalgamations of nano-based technologies with EO potential aiming at manageable drug delivery and higher bioactivity has anticipated more diversified industrial applications of these products.

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129. Zhao, L., Chang, W. C., Xiao, Y., Liu, H. W., & Liu, P. (2013). Methylerythritol phosphate pathway of isoprenoid biosynthesis. Annual Review of Biochemistry, 82, 497.

Chapter 22

A review on marine-based phytochemicals and their application in biomedical research Rousan Khatun1, Sikha Singh1, Navneet Kumar Dubey2 and Alok Prasad Das1 1

Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India, 2Victory Biotechnology Co. Ltd., Taipei City, Taiwan

22.1

Introduction

In antiquity, plants have consistently been considered for health and inception, whereas the communicable experiential approach gives apprehension of numerous relieving features of plants. Howbeit, slowly but surely, humans have been fascinated to gain knowledge about the characteristics of plants. In the exercise of gaining and utilizing the knowledge about bioactive substances secreted by the plants, humans have discovered varying techniques to study the phytonutrients (plant foods) and their derivatives (i.e., phytochemicals) which depend on relieving nature of plants themselves. This is the origin of phytochemicals that are termed by the study done in the science which is known that plant itself contains natural substances. Several methodologies have evolved to study the phytochemicals finely, fluctuating from the preliminary sample of plant tissue to experienced methods for the illumination of organic structures. Exploration of the latest substances or goods for biomedicals is in the progress of action that is indispensable in frequent increments (Mendoza & Silva, 2019). Formerly, the shade of 10,000 natural substances yields collectively one commercial substance. In the appearance of chemical biology, association altered. At present, the shade of 100,000 structures from first day in chemical biology along with the natural substances shaded in a year gives rise to ,1 commercial substance. Its progress acquires around one decade and 2 years and expenses approximately 350 million dollars or 26,933,585,000.00 (INR). In the time of 1940s, revelation occurred of marine plants as the potential medicinal source for biomedicals. Along with marine plants, so many other marine organisms are also considered the major source of marine-based natural products which are effective in treating fatal diseases or infections or syndromes (i.e., mainly considerable death diseases like cancer and AIDS). Despite the earth being covered with 79% of the aquatic region, most marine organisms are yet to be discovered as a generator of natural products. Bioactive substances originated in marine plants that are beneficial to themselves defending from the fatal outcome of extremely high concentrations of sunlight and O2. Various phytoplanktons, vertebrates, invertebrates, and other microbes which are marine-based are capable of secreting a notable quantity of phytochemicals that retain huge biomedical affairs. Marine natural substances contain an extensive diversity of phytochemicals, tetraterpenoids, and other obtained by-products of keratin, flavonoids, and COS. Phytochemicals, i.e., 1,3,5-trihydroxy benzene, found in Phaeophyta, are derived from the decarboxylation of benzene-1,3,5-triol. These are substantially existing in marine Phaeophyta which reserve various biotic venture which contains biomedical affairs like antifungal, antibacterial, anti-hyper, and hypotensive. Bioactive endorphins are secreted as an outcome of the enzymic hydrolysis of aquatic substances (Barelli et al., 2016) and emerge as derivatives through aquatic garbling (Gouda et al., 2016). Acquired sulfated polysaccharides are of various kinds for seaweed and exhibit a noticeable affair as opposed to cancer and AIDS. Plants and microbes integrate coloring pigments which are studied as tetraterpenoids and have fortunate outcomes in precluding diseases and syndromes. Plasma membranes of eukaryotic animals mostly contain zoosterols and are identified as lowering agents (i.e., cholesterol or fat or lipid). All the aquatic natural substances obtain vast crucial biomedical affairs as Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00013-X © 2023 Elsevier Inc. All rights reserved.

383

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their effective activity as opposed to cancer and other fatal viral diseases, and they too have vital anticonvulsant and antioxidant affairs. Phytonutrients and their derivatives like phytochemicals available in marine plants are encouraging choices to boost the therapeutic effect in patients who are having cancer and decline unfavorable responses. Various phytochemicals are biologically arising natural bioactive or secondary metabolites with antitumor potential impotence. The expansion of the effectiveness and complexity of phytochemicals for anticancer remedies start with examining the extraction of bioactive substances for the natural potentiality of anticancer expertise chased by disinfected or sterile phytochemicals examining the effectiveness of in vitro and in vivo. In some considered reviews, there are various strives that have been created to collect the direction mainly about phytochemicals used as anticancer that are analyzed at the stage of biomedical together with their marketing value and availability. In the case of biomedicals, we reviewed the in vivo activity of phytochemicals. This review’s further focal points of phytonutrients and their derivatives also mention some phytochemicals which are estimated for biomedical applications along with succinct details on the currently used marinebased phytochemicals for anticancer medication. Fungi have the potential to yield the latest substances, and mostly, 7 out of 21 prescribed medications depend upon fungal strains, whereas .90% of fungi are yet to be discovered. Generally, mycorrhiza and sac fungi are outlined by their event within subclinical tissues of plants and were established on living substrates (i.e., ranging from extreme cold to extreme hot and from desert to diversified forest) like hornworts, mosses, lycophytes, ferns, and seed plants. Their enigmatic way of living, universality, and abundance in each herb, combined with the appearing attestation of their constantly giving over ecological consequence, has provoked multiplying eagerness concerning these well-known fungi over few decades. Plants interact with microorganisms varyingly. Esteemed as an untrodden chemically diversifying place, mycorrhiza has the capability to secret bioactive substances which play a vast role in protecting plants themselves from varying pathogens (e.g., microbes and pests), influencing resistance, increasing vegetation, and enhancing the crop-yielding properties (Nicoletti & Fiorentino, 2015). Marine algal (i.e., seaweed) natural bioactive substances or secondary metabolites (i.e., phytochemicals) are varyingly used in biomedicals as active compounds for drugs, and several phytochemicals are used for diseases which are generally hard to cure or nearly non-treatable. Although marine-based phytochemicals have significantly high marketing value, these substances are very fascinating and are increasingly used for biomedical purposes (Barelli et al., 2016). Plant endophytes (“microbes and fungi that lived within plant tissues) are generally not capable of causing disease to plants or their host, and they secret phytochemicals that empower them to live in the competition of various plant endo-tissues without causing any harm to their host. Microbes are very specific to their habitat to compete with other fellow microbes. Those phytochemicals have many significant uses for biomedical to date. Phytochemicals like griseofulvin are antifungal agents, streptomycin is an antibacterial agent, and calicheamicin is an anticancer agent, respectively, produced by fungi and bacteria. Phytochemicals come under the study of interest and use after the discovery of the production of Taxol from plant endophytes like fungus in Northwest Pacific yew tree’s bark and needle. The study of produced phytochemicals for biomedical applications and applying technologies to yield phytochemicals by endophytic microbes. The main focus of the chapter from this book is to discuss the case of the biomedical application of phytochemicals.

22.2

Phytochemicals from marine resources

Marine plants have been utilized to medicate several diseases since ancient times. Phytochemicals from plants as medicine (4500 BCE) still represent live traditions. From antiquity, the recognition of choosing perfect plants, to a specific period of collection, and the process of making medicine with its specific utilization were orally passed down from one generation to the next. These medicines were formulated in the form of tinctures and beverages, and in biochemistry, a systematic study of the active ingredients of marine plants with medicinal properties was conducted varyingly, which opened the door to know about the biomedical importance of those actively present marine plants. This has accelerated the discovery of drugs and led to a miraculous invention in medicine. The first breakthrough that introduced initial drugs came from painkiller morphine isolation from the Papaver somniferum plant. Later, medicinal plants of the 20th century emerged, along with ortho-hydroxybenzoic acid, a predecessor of Salix sp., Erythroxylum coca, and Cinchona officinalis which produce aspirin, cocaine, quinine, respectively, digital spur, and several others with biomedical potential. For several decades, micromolecule-approved drugs which were originated from natural products were templates for artificial change and other biomedical inquiries. This demonstrates the huge potential of the medicinal character of plants, which has been studied in traditional medicine for several decades. Some of the compounds obtained from commercially available phytochemicals dealing with various diseases are listed in Table 22.1.

TABLE 22.1 List of marine phytochemicals along with their biomedical use and sources. Sl. No

Phytochemicals

Sources

Health benefits

References

1

Terpenoids

Tea, thyme, cannabis, Spanish sage, citrus fruits, fungi

Effective against various pathogenic diseases including cancer

Joshee et al. (2019)

2

Fibers

Seed—Kapok, milkweed; stem—nettle, ramie, kenaf; leaves—abaca, manila; fruit—coir

Reduce the risk of chronic, benefits digestion, anticholesterol properties

Sfiligoj Smole et al. (2012)

3

Polysaccharide

Seaweeds

Immunomodulatory, antitumor, antimicrobial, anticoagulant, antithrombotic, antimutagenic, anti-HIV infection, herpes, and HV (HBV, HCV)

Mohammed et al. (2021)

4

Flavonoids

Seagrasses, halophytes

Anti-inflammatory and protect cells from oxidative damage which is supposed to lead to disease, cardiovascular, cancer prevention, antihistamine, antimicrobial

Ballard et al. (2019)

5

Isoflavonoids

Seagrass, algae

Anticancer, antimicrobial

6

Anthocyanidins

Cereal, white-water lily

Antioxidant, effective against angiogenesis, antidiabetic, antitumor, neuroprotection

Jaiswal et al. (2019)

7

Limonoids

Seaweed

Antimalarial, bactericidal, fungicidal, virucidal

Akihisa and Tokuda (2016)

8

Saponins

Sea cucumber

Fungicidal, shark repellent, antitumor, antiinflammatory

Xiao et al. (2018)

9

Lutein

Euglenophyta, cryptophyte, rhodophyta algal species

Prevention of macular degenerative disease, reducing the risk of heart attack and stroke

Saha et al. (2020)

10

Phlorotannins

Seaweed brown algae

Antimicrobial activity

Mogosanu and Bejenaru (2016)

11

Fucosterol

Cystoseira sp., Pelvetia sp.

Protects against neuroinflammation, oxidative stress

Rahman et al. (2021)

12

Glycosides

Cyanobacteria

Anticancer, antitumor

Li et al. (2020)

13

Mycosporine

Red algae

Helps in protecting the skin against UV radiation

Figueroa (2021)

14

Pseudopterosin

Caribbean Sea whip

Anti-inflammatory, analgesic properties

Kohl and Kerr (2003)

15

Cholesterol

Red algae

Antioxidant activity

Hamed et al. (2015)

16

Ergosterol

Chlamydomonas sp.

Antioxidant activity

Hamed et al. (2015)

17

Agar

Gelidium seaweed

Antioxidant activity

Hamed et al. (2015)

18

Alginate

Brown algae

Moderate human appetite and energy intake

Peter et al. (2011)

19

Laminaran

Laminaria algae sp.

Hypotensive agent, antioxidant protection

Hamed et al. (2015)

20

Carrageenan

Tribonema seaweed

Anticancer activity

El-Beltagi et al. (2019) (Continued )

TABLE 22.1 (Continued) Sl. No

Phytochemicals

Sources

Health benefits

References

21

Porphyrin

Chondrus seaweed

Anticancer activity

El-Beltagi et al. (2019)

22

Ulvan

Ulva seaweed

Antioxidant activity

El-Beltagi et al. (2019)

23

Fucoidans

Fucus and cladosiphon seaweed

Antioxidant activity

El-Beltagi et al. (2019)

24

Zeaxanthin

Pyropia seaweed

ADM prevention

El-Beltagi et al. (2019)

25

β-carotene

Kappaphycus seaweed

Cure of erythema, antiproliferative

El-Beltagi et al. (2019)

26

Fucoxanthinol

Corbicula algae sp.

Anti-obesity

El-Beltagi et al. (2019)

27

Fucoxanthins

Laminaria seaweed

Antitumoral activity on lung cancer cells

Lomartire and Gonc¸alves (2022)

28

Phloroglucinol

Ecklonia seaweed

Anti-inflammatory

Lomartire and Gonc¸alves (2022)

29

Sulfate polysaccharide

Gracilaria seaweed

Antidiabetic, anti-obesity

Lomartire and Gonc¸alves (2022)

30

MAA

Nostoc sp.

Antioxidant

Lomartire and Gonc¸alves (2022)

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Current medicinal inventions are depending upon natural products, mainly plant products (i.e., phytochemicals) because they are the most effective and cheaper alternatives (Ghosh et al., 2016; Ghosh & Das, 2015, 2017, 2018). Essential phytochemicals present in marine plant resources act as precursor syntheses of effective drug substances for biomedical applications. Marine-based phytochemicals are of medicinal value i.e., alkalis, alkaloids, tannins, and phenolic substances. More than 180 marine-based phytochemicals are known for antimicrobial properties (Mendoza & Silva, 2019). They contain large amounts of phytochemicals that are effective for the health of humans and act as antioxidants that protect against damage caused by free radicals. Damage is reduced and controlled by forbidding the degeneration of reactive oxygen species (ROS, i.e., crucial to physiological processes in humans) as an antioxidant (Ghosh et al., 2020; Ghosh & Das, 2020). Qualitative and quantitative phytochemical researches need a proper extraction technique.

22.3

Metabolic process to form marine phytochemicals

With the increased use of plastics and plastic products, marine source gets rich in plastics which also affects marine organisms along with their secreted natural products (i.e., phytochemicals). Owing to the increasing plastic pollution and its impact on marine-based organisms, several studies and research must be done to cure and protect marine resources (Bal & Das, 2020; Bhattacharjee et al., 2021; Biswal et al., 2021). Some studies show that microplastics spread by the food chain from the sediments through the plankton to other invertebrates (i.e., marine animals like crustaceans to fish) to terrestrial vertebrates, and this impact of microplastics occurs as aquatic organisms are exposed to contamination of microplastics and act as the carrier of microplastics or plastics (Das & Ghosh, 2018, 2022). Metabolism generally occurs in the living cell as a set of chemical reactions, and it is the process of degrading complex substances into simpler ones. All plants along with marine-based plants are generally autotrophic organisms, and they have types of metabolites, like the primary metabolism that is present in all organisms and contingent digestion that grants them to form and acquire substances of different synthetic natures. Several macromolecules are at the end winding up as the ordinary molecules of all cells, which are essential for their working and the organism they reside in (Das et al., 2012, 2015, 2015a). These are macromolecules, which are in entire plant species and do clone things, i.e., termed as early metabolism. Plants designate compelling amounts of integrated several macromolecules in the fusion of organic molecules array, which does not appear to be directly involved in the physiology of plants (i.e., photosynthesis, respiration, assimilation of nutrients, soluble macromolecules), and these natural products are termed as phytochemicals (sec´ valos & Elena, 2009; Stewart et al., 2014). Phytochemicals are characteristic of higher plants. A ondary metabolite) (A necessary feature of higher plants is that they contain flowers, seeds, and fruits. Its replicative process is distinct from one of the secondaries. They are divided into gymnosperms and angiosperms as their replicative organs are ocular and that is termed spermatophytes. Phytochemicals have organic features and are expressed by their various benefits and appliances in drugs, pesticides, and herbs. The biosynthesis of phytochemicals is generally limited to the definite ´ valos & Elena, 2009). Some marine plant species yield crucial degrees of plant growth and the period of significance (A contingent metabolism of plant synergies with diversity like protecting from environmental stress, relating with reproductivity of plants, to propagating the pollination process of attracting insects.

22.4

Bioactive potential of marine phytochemical

Being uniquely diversifying, marine breeds a wide range of natural substances. Most of the reports show the presence of marine phytochemicals like tannins, flavonoids (i.e., isoflavonoids, anthocyanidins), sterols, glycosides, natural acids, saponins, limonoids, lutein, fibers, and terpenoids as shown in Fig. 22.1. Avicennia marina methanolic concentrations include pentanoic acid, decyl hydroxylamine, pyrrolidine, 4-chlorophenyl, octadecyl isocyanate, thiazolidinediones, and arabinopyranoside. Methyl ester, acyclic aliphatic, and the monosubstituted aliphatic group are the compounds of fatty acid that have been found in Excoecaria agallocha as their constituents. Tissues of marine species (bark, root, and leaf) were scanned as qualitative, semi-qualitative, and quantitative for the exact analysis of phytochemicals. The existence of phytochemicals (like alkaloids, saponins, tannins, flavonoids, and sugar reduction) has been outlined. In the case of quality inspection, tests like Meyers, Dragendorff, Salkowski, and Nothing were executed for alkaloids, glycosides, and saponins, respectively. Other tests like ferric chloride, aluminum chloride, Fehling, and potassium ferrocyanide were executed, respectively, to reduce several other phytochemicals. Flavonoids (i.e., found in leaves) and tannins (i.e., found in stems) in high concentration are found in Rhizophora racemosa, whereas sugar reduction in high concentration is found in Nypa fruticans (Mitra et al., 2021).

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FIGURE 22.1 Shows the list of marine-based phytochemicals use for biomedical.

22.4.1 Antibacterial activity Worldwide, marine resources are under research for their antibacterial activities, as they have highly effective bactericidal properties as opposed to various diseases. Since marine-originated plants are filled with phytochemicals, they are helpful against numerous diseases in vitro as well as in some cases in vivo. It has been suggested that the antimicrobial affairs of Xylocarpus granatum may be owing to the occurrence of triterpene in the methyl alcohol extract. Average bactericidal affairs have occurred owing to the presence of phenolic substances such as tannins. The presence of phytochemicals like glycosides, saponins, tannins, flavonoids, alkaloids, and terpenoids may affect the bactericidal properties of Avicenna officinalis (Mogosanu & Bejenaru, 2016). The existence of flavonoids as the phytochemical in Lumnitzera racemosa extract is also responsible for bactericidal affairs (Mitra et al., 2021). In some reports, it has been studied as a promising bactericidal affair owing to the existence of terpenoids, phenolics, and alkaloids. Hence, biomedical screening of phytochemicals becomes the source for many therapeutic, pharmaceutical, and nutraceutical agents.

22.4.2 Antifungal activity There have been identified seven marine species of fungi for phytochemicals such as Avicennia marina, E. agallocha, L. racemosa, Derris trifoliata, Bruguiera gymnorrhiza, and Acanthus ilicifolius. The trichloromethane (CHCl3) and methyl alcohol (CH3OH) extracts of the E. agallocha test give fungicidal affairs as opposed to fungus. A. mariner chloroform extract (i.e., C. albicans) shows antifungal activity against Niger sp. Trichloromethane extract of D. trifoliata works as opposed to fungal species like Aspergillus niger and Saccharomyces cerevisiae. Trichloromethane extracts from A. ilicifolius result in a reduction in mortality rate, the severity of symptoms, and the wound score of diseased rats caused by a fungus. Several studies have shown the occurrence of a few phytochemicals in trichloromethane extract which are absorbed from the GI tract by intaking orally and producing fungicidal effects. Rhizophora mucronata and Avicennia marina are ethanolic extracts that show antifungal properties as opposed to various penicillium species like Penicillium purpurogenum, Penicillium chrysogenum, Penicillium notatum, Penicillium italicum, A. niger, and Alternaria alternata.

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22.4.3 Antiviral agent In addition to other antimicrobial researches, some eye-catching virucidal research has been also outlined. Some notable viral diseases have successfully been effective in marine-based phytochemicals. According to several reports, the marine-based plant extract was tested for HIV in MT-4 cells. The active extract completely inhibits the absorption of the virus in cells belonging to the family Rhizophoraceae. Rhododendron, a marine associated with virucidal affairs as opposed to HBV, has antimalarial, antioxidant, anti-hepatoprotective, anti-inflammatory, antimicrobial, and other properties. Pongamia pinnata is a marine-based plant whose extract shows promising virucidal affairs as opposed to humans and SIV. Extracts of Acanthus ebracteate and A. ilicifolius are proven to have virucidal properties. Avicennia marina, Avicennia officinalis, Ceriops decandra, Bruguiera cylindrica, E. agallocha, L. racemosa, Rhizophora apiculata, Rhizophora lamarckii, Sonneratia apetala extracts which are dealing with Xanthone derivative from Calophyllum inophyllum (Guttiferae) show anti-HIV properties, and they work against HIV-1 (gp-41 and gp-120) in cell culture.

22.4.4 Anticancer agents Cancer is a process where cells are amplified atypically, and it is an unmanageable mode that overcomes the various tissues. Cancer is considered a fatal disease for decades. Broad case studies have been conveyed concerning and exploring the latest secure medicines that will have under most hostile shock on the human body. Marine sources invariably sanctioned inception for medicines as they are the most dominant aspirants to heal delicate (i.e., chronic and acute) illnesses owing to their NSAIDs, anticarcinogenic, and germicide consequences. It established numerous phytochemicals which got into a trial for their biomedical effectiveness; among those, several phytochemicals possess the capability to take measures for vigorous medicines. Derivatives of phytonutrients have been broadly utilized as anticarcinogenic representatives. Marine resources especially the marine plant species are achieving more interest in the discovery of medicines for cancer treatment. Given the activity of marine-based phytochemicals against cancer, the data of species names along with their respective extracted phytochemicals are described in Table 22.2. Several nanoparticles of phytochemicals (i.e., selenium) are used against cancer as effective chemoprotective compounds. Mainly, the combination of selenium with doxorubicin (i.e., nanoparticles of phytochemicals) caused cell death in cancer cells and effective against cancer treatment. TABLE 22.2 List of marine-based phytochemicals effective against cancer treatment. Sl no

Name of extracted phytochemicals

Origin of phytochemical extraction (species)

References

1

Benzoxazole

A. ilicifolius

Mun˜oz-Ochoa et al. (2010)

2

Betulinic acid

A. officinalis

3

Avicequinone

A. marina and A. alba

Thomas and Kim (2013)

4

Brugin

B. sexangular and B. gymnorrhiza

Mun˜oz-Ochoa et al. (2010)

5

Quinine

C. decandra

Azam et al. (2017), Sappati et al. (2019)

6

Exocoecaria

E. agallocha

7

Eleganonal

B. bifurcate

Silva et al. (2019)

8

Algae extract

D. dichotoma

Kosanı´c et al. (2019)

9

Ethanol extract

C. sinuosa, H. scoparia, P. concrescens

Mun˜oz-Ochoa et al. (2010)

10

Phenol

S. latissima, S. dubyi

Azam et al. (2017), Sappati et al. (2019)

11

Sesquiterpenes

L. luzonensis

Thomas and Kim (2013)

12

Glycosaminoglycan

J. rubens

SpecialChem—The Universal Selection Source: Cosmetics Ingredients (2020)

13

Fucoxanthin

G. oxysperma, U. fasciata

Premalatha et al. (2011), Kelman et al. (2012)

14

Lycopene

A. vaginicola

Mourelle et al. (2017)

15

β-Cryptoxanthin

D. salina

390

22.5

Recent Frontiers of Phytochemicals

Biomedical applications of marine phytochemicals

22.5.1 Pharmaceuticals By nature, marine plant-derived chemicals or phytochemicals are essential compounds for the discovery of new medicine for several decades. Various chemicals are derived from plant species (i.e., phytochemicals), and they are distinctly utilized in pharmaceuticals (Fig. 22.2). In recent years, owing to the increase in the use of phytochemicals in pharmaceutics and turning attention through surveys toward green marine resources are still an underexploited field of interest for research (Mohanty et al., 2016, 2017, 2018; Mohanty & Das, 2022). So, proper planning is prepared to select the high prospects of different marine-based phytochemicals; therefore they can be utilized in research (Mishra et al., 2019a, 2019b, 2021, 2022; Mishra & Das, 2008). Several researches have been conducted which give report on the antibacterial, antiviral, and antifungal activities of marine-based phytochemicals against various organisms. Aside from antifungal, antibacterial, and antiviral properties, another considerable property is antioxidant properties. Exploring the other marine drugs for utilization in pharmaceuticals must deal with scientific research, which will build a way of success in pharmaceutics. Numerous researchers studied the overall process from extraction to the characterization of many marine-based phytochemicals by different techniques (Das et al., 2011; Das & Mishra, 2008; Das & Singh, 2011; Prabhakar et al., 2019; Sanket et al., 2017; Das et al., 2003). Avicenniaceae marina, Avicenniaceae alba, and Avicenniaceae officinalis

FIGURE 22.2 Shows one of the marine sourced phytochemical fucoxanthins’ potential activity for pharmacology.

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391

species owing to their abundance subsequently as species of Bruguiera, Excoecaria, and Rhizophora have become interesting points for various researchers. In general, species like Avicennia, Bruguiera, Acanthus, Rhizophora, and Xylocarpus are especially considered the main source of marine-based phytochemicals. Avicennia marina species provide various phytochemical constituents. The selection of solvents according to their working significance is necessary for the extraction process. Some studies give the idea about commonly used solvents like methanol concerning ethyl alcohol, ethyl ethanoate, trichloromethane, and water. In the separation process for phytochemicals from the species techniques in stationary phage like traditional thin layer chromatography and column chromatography (HPLC-Ms, GC-Ms, LC-Ms) along with silica gel are used. Further process of identification of the extracted phytochemicals is done by the UV, FTIR, NMR, and Ms (Das et al., 2011, 2014, 2015b; Das & Mishra, 2010). Phytochemicals like pseudopterosin, mycosporine, alkaloids, saponins, terpenoids, flavonoids, isoflavonoids, anthocyanidins, limonoids, lutein, phlorotannins, fibers, sterol, polysaccharide, glycosides, and polycyclic musk are qualitatively extracted from marine plant species. Species like Acanthus ilicifolius, Avicennia marina, and Rhizophora stylosa abundantly show bactericidal and fungicidal activity along with other activities like anti-inflammatory, virucidal, anticarcinogenic, and cytotoxic. In short, marine diversity has a lot of pharmaceutical significance as well as that is a productive resource. Marine-based phytochemicals need more specific research and attention to explore future appliances and approaches.

22.5.2 Therapeuticals The study of chemistry or all the chemical properties of marine-based secondary metabolites is the basic and main source for researchers to exploit new research areas as phytochemicals are essential compounds for the field of therapeutics. Marine and other marine derivatives can yield phytochemicals on a large scale as they are capable to survive distinctly. Phytochemicals like alkaloids, terpenoids, and rotenoids are the source of toxins which are primarily secreted by marine plant species. The extraction of natural products from marine plant species is the initial step toward the preparation of phytochemical-based medicines or drugs for therapeutic use. For the extraction of those natural products, several techniques like sequential solvent extraction and other chromatographic separation techniques are used along with an active fraction of those natural products. After isolation and identification, these obtained phytochemicals are applied to test the effectiveness and viability of animal models and then that would be conducted for testing on humans in three phases of a clinical trial which are the essential steps for discovery in the field of therapeutics. In the area of therapeutics, the marine-based plant species’ extract and their effectiveness in activities like antimicrobial, anti-inflammatory, and anticancer are studied by researchers in a wide range. Despite a lot of the latest chemical compounds being isolated from marine-based plants, only a small amount of them have been used in the field of therapeutics with selective and essential biomedical appliances, whereas extracted phytochemical from species like Rhizophora mangle is therapeutically effective in controlling diabetes (Saha et al., 2020). E. agallocha, Bruguiera sexangular, and Avicennia africana are the sources of phytochemical and are effective as opposed to fungicidal, bactericidal, virucidal, anticarcinogenic, anti-HIV, and antitumor affairs (Dettmar et al., 2011; Hamed et al., 2015).

22.5.3 Nutraceuticals Nanoencapsulation is a productive proposal to yield ingredients (i.e., nourishing, dietary additive, and redox-active) for nutraceuticals. These are put in several phytochemical composite substances. Anti-inflammatories, a secondary metabolite remoted from marine plant species, seem efficacious in decreasing tissue plasminogen activator (tPA), and free curcumin induced swelling in rat ears as differentiated. Making ROS is the main briber of several disorders inclusive of cancer and age-related diseases period (with respect to ROS utilizing marine phytochemicals as listed in Fig. 22.3). Other composition uses antioxidants through the integration of the ,microparticles as abating elements. Gold nanoparticles correlated with 3,4,5-trihydroxybenzoic acid and 3,4-dihydroxybenzoic acid exhibited magnificent phytochemical qualities. Changes in high-level and cytoprotective outcomes, as opposed to H2O2 by these gold nanoparticles, were noticed too like chondrocytes, keratinocytes, bfgf, astrocytes, etc. These nanoparticles removed 40% H2O2 at a congregation of 50 microgram mL21, just about similar to carried off by 3000 units/mg of catalase enzyme. Some studies outlined that AuNPs produce and accommodate phytochemicals, polyphenols, and gum arabic was initiated to be attributed by breast cancer cells (i.e., MCF-7) and prostate cancer cells (i.e., PC-3) and displayed magnificent viability of those cells, thus appearing a nonpoisonous choice to utilize for gold nanoparticles as a curative and symptomatic factor. Resveratrol, a phytochemical, gets going in many berries and nuts and is utilized as a dietary additive for sickness related to cardiovascular and cancer that are having vital complications. Generally, UV display, molecular-encapsulated makes of the secondary metabolites in liposomes, solid lipid molecular particles, and polymeric lipid-core molecular

392

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FIGURE 22.3 Portrays ROS with assorted biological molecules. Aquaphobic α-amino carboxylic acids in a polypeptide put up to their attainable consequence of antioxidant. Macroalgae having an activity like antioxidants contains biomedical chemical compounds (i.e., pharmaceutic, nutraceutical and therapeutic chemical compounds) such as phenol, sulphated polysaccharide, methanol extract and ethanol extract from various marine algae species (Halliwell, 2006; Al-Amoudi et al., 2009; Cornish & Garbary, 2010; El-Beltagi et al., 2019).

capsules accelerated the light steadiness of trans-resveratrol, in inclusion to growing it as an aqueous dissolved. One more fascinating encounter in nutraceutical delivery is the smaller than micro-enclose Bifidus-like LAB pointing particular range of the GI tract. Emulsions of particles that were smaller than the microparticles of Lactobacillus delbrueckii sp. bulgaricus in sesame oil consulted preventive impacts on the tissue when unprotected from unnatural peak acid gastric or bile salt situations. Thus, nanoformulations have detected broad implementation and the ability to produce a better result in nutraceutical conveyance.

22.6

Conclusion

Marine regions have antagonistic, majorly in the habitat with varying streams and salinity. As reported earlier, their biochemical peculiarity is located in reality by which they are capable to survive adequately in a halophytic habitat to generate several special phytochemicals that would be effective in the discoveries of numerous herbal medicines. Marinebased phytochemicals show worldwide large-scale resources and give a diversifying service to several biomedical applications. The marine diversity is full of an untapped pool of novel drugs, and related research is based on inventive and investigation stairs. Globally, a huge variety of marine plants have been utilized for biomedical applications. As described in several reports, phytochemical extraction from marine plants is effective against antibacterial, antifungal, and antiviral activities that are relevant to biomedical. Synergistic effects of the fusion of biomedical and phytochemical substances in drug discovery with adequate reaction are essential to reporting. Currently, the anticarcinogenic and antimicrobial attributes of secondary metabolites are fascinating and accelerating importance for research on their depressed lethal intrinsic

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FIGURE 22.4 Shows the overall scenario for use of marine phytochemicals along with their properties in biomedical.

cells but remarkable execution to study on oncology. In this review, an endeavor has been produced to supply a list of the utility of secondary metabolites, i.e., utilized for biomedical examinations. This data will be excessively advantageous to analyze a sequence of supplementary plant extract (i.e., phytochemical-based) medicines to medicate cancer with the lowest negative consequences or reactions. Thus, it may be windup that a prominent concern of attempt and scientific knowledge is needed to reveal the capability of marine plant extract phytochemicals. Such investigation discovery will assist the biomedicals with ideal pharmaceutics, therapeutic, and nutraceutical applications (cited in Fig. 22.4).

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11. Available from https://doi.org/10.1080/01490451.2021.1983670. Mitra, S., Naskar, N., & Chaudhuri, P. (2021). A review of potential bioactive phytochemicals for novel therapeutic applications with special emphasis on mangrove species. Phytomedicine Plus, Singh, R. P., Mishra, S., & Das, A. P. (2020). Synthetic microfibers: Pollution toxicity and remediation, Chemosphere. Mogosanu, G. D., & Bejenaru, C. (2016). Marine Natural Products in Fighting Microbial Infections. Available from https://doi.org/10.1016/B978-012-803642-6.00017-4. Mohammed, A. S. A., Naveed, M., & Jost, N. (2021). Polysaccharides; Classification, chemical properties, and future perspective applications in fields of pharmacology and biological medicine (a review of current applications and upcoming potentialities). Journal of Polymers and the Environment, 29, 2359
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316. Mourelle, M. L., Go´mez, C. P., & Legido, J. L. (2017). The potential use of marine microalgae and cyanobacteria in cosmetics and thalassotherapy. Cosmetics, 4, 46. ´ lvarez, J. I., Zermen˜o-Cervantes, L. A., Martı´nez-Dı´az, S., & Rodrı´guez-Riosmena, R. (2010). Screening of extracts of Mun˜oz-Ochoa, M., Murillo-A algae from Baja California Sur, Mexico as reversers of the antibiotic resistance of some pathogenic bacteria. European Review for Medical and Pharmacological Sciences, 14, 739
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104. Thomas, N. V., & Kim, S. K. (2013). Beneficial effects of marine algal compounds in cosmeceuticals. Marine Drugs, 11, 146
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Chapter 23

Phytochemicals in biofilm inhibition Anandu Chandra Khanashyam1, M. Anjaly Shanker2, Pinchu Elizabath Thomas3, Karthik Sajith Babu4 and Nilesh Prakash Nirmal5 1

Department of Food Science and Technology, Kasetsart University, Chatuchak, Bangkok, Thailand, 2Department of Agriculture and Environmental

Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonepat, Haryana, India, 3Pinchu Elizabath Thomas, MACFAST (Mar Athanasios College for Advanced Studies Thiruvalla), Kottayam, Kerala, India, 4Department of Animal Sciences and Industry/Food Science Institute, Kansas State University, Manhattan, KS, United States, 5Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, Thailand

23.1

Introduction

Microorganisms are not only capable of living as pure cultures of scattered single cells called planktonic cells, but also are able to aggregate at surfaces to form film, flocs, sludge, mars, or biofilm (Flemming & Wingender, 2010). The most common microbial lifestyle in most natural habitats is the one attached to a surface in the form of a biofilm. The surface association is an effective way to stay in a good microenvironment instead of being washed away by currents. Microorganisms make up less than 10% of the dry mass in most biofilms, whereas the matrix makes up more than 90%. The matrix is the extracellular substance that the biofilm cells are embedded in, which is largely created by the organisms themselves. It is made up of a mixture of different types of biopolymers called extracellular polymeric substances (EPSs), which serves as a scaffold for the biofilm’s three-dimensional structure. Although the exact molecular mechanism and the functions of these EPSs are not precisely understood, it is reported that EPS is responsible for aiding surface adhesion and biofilm cohesiveness. Moreover, it can also help in water retention and sorption of organic and inorganic compounds, facilitate the transfer of genetic material, and act as a protective barrier and a nutrient source (Flemming & Wingender, 2010). The characteristics of the microbial cell change dramatically after it gets incorporated with the biofilm matrix. When compared to bacteria in the planktonic stage, bacteria in biofilms have a far higher pattern of adaptive resistance to antibiotics and other disinfectants (Vestby et al., 2020). An increase in resistance toward chemical disinfects for Listeria monocytogenes biofilm in fish and Pseudomonas fluorescens biofilm in meat processing plants has been reported by Papaioannou et al. (2018) and Wang et al. (2018). This can jeopardize the cleaning efficacy of sanitizers and other disinfectants in processing plants and thereby raises the risk of foodborne outbreaks and diseases. Due to their bio-transfer capability, biofilms generated on food contact surfaces act as a continuous reservoir for microbial contamination, posing a danger to the microbiological quality and safety. It is estimated that more than 60% of foodborne outbreaks and 80% of bacterial infections are associated with biofilm (Bridier et al., 2015; Wolcott & Ehrlich, 2008). Cross-contamination from biofilm can also lower the shelf life of the processed products. In addition, biofilms growth in locations like fluid handling systems and plate heat exchangers can cause mechanical blockage and reduce heat transfer efficiency. Moreover, some bacteria found in biofilms can stimulate chemical and biological processes that cause metal corrosion in pipes and tanks. Furthermore, improper chemical usage can lead to the proliferation of disinfectant-resistant bacteria and the emergence of antibiotic cross-resistance (Techaruvichit et al., 2016). Phytochemicals are natural substances that have the potential to be used as alternative natural disinfectants for biofilm reduction (Ta & Arnason, 2015). Studies have reported increased efficacy in biofilm inactivation by industrial disinfectants when used in combination with various essential oils (Vazquez-Sanchez et al., 2018; Va´zquez-Sa´nchez et al., 2018). This chapter deals with the biofilm formation and its inactivation by phytochemicals along with its mode and mechanism of action.

23.2

Biofilm formation

Biofilms are formed by one or more species of microorganisms immersed in an extracellular matrix of different compositions depending on the type of food manufacturing environment and the colonizing species. Most bacteria are now Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00018-9 © 2023 Elsevier Inc. All rights reserved.

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assumed to produce biofilms in nature, and the biofilm formation is highly influenced by the environment and the processes by which individual cell gene expression influences their growth and has attracted researchers’ curiosity worldwide. It is now commonly known that almost all microbes adhere to a surface and develop as biofilms, which is a universal microbial survival strategy (McCarty et al., 2014). Biofilm communities may be difficult to remove once established; therefore, research into their development and mechanisms has a wide range of applications in biomedicine, agro-food, and other industries. Cell densities in biofilms range from 108 to 1011 cells per gram of wet weight (JolivetGougeon & Bonnaure-Mallet, 2014). Various research has been done in order to obtain a better knowledge of biofilms and to find a way to prevent food contamination (Carrascosa et al., 2021). Biofilms have become more tolerant to disinfections in many wide-ranging agro-food sectors. Indeed, biofilm production is influenced by the attachment surface features such as hydrophobicity, electrostatic charge, surface roughness, topography, etc., (Arau´jo et al., 2010; Tang et al., 2011). From a cell properties standpoint of the microbes, the cellular membrane components, appendages, and bacteria-produced extracellular polymeric substances play a crucial role in biofilm formation (Tang et al., 2011). Fig. 23.1 shows a schematic of biofilm formation. Attachment, maturation, and dispersal are the typical steps of biofilm development. Surface attachment is one of the critical steps in biofilm formation as this is the stage when it changes from planktonic life to the biofilm phase. The microorganisms could use a variety of cell appendages for this attachment process which includes fimbriae, pili, flagella, curli fibers, and other outer membrane proteins (Renner & Weibel, 2011). The microorganism could leave the surface and return to the planktonic life or still continue to participate in the biofilm process at this stage (Toyofuku et al., 2016). The initial surface attachment strongly depends on the physical, chemical, and biological interactions. The surface properties also play a crucial role in the initial attachment process. Any type of surface, including metal, glass, plastic, wood, and food products, is susceptible or prone to biofilm formation. However, the initial attachment for biofilm formation also could be dependent on various properties such as texture, surface roughness, surface charge, pH, temperature, and hydrophobicity of the surface. The initial attachment between microbial cell wall and surfaces is characterized by electrostatic and van der Waals forces (Renner & Weibel, 2011). The Derjaguin
Landau
Verwey
Overbeek theory and the thermodynamic methods are also generally used to explain the theories of bacterial adhesion (Hori & Matsumoto, 2010). Hydrophobic interactions influence the attachment of cells to the surface once reversible attachment is established. Cells undergo irreversible attachment after reversible attachment (Petrova & Sauer, 2012; Toyofuku et al., 2016). The reversible attachment becomes irreversible when there is a change from weak interactions of the microorganism to the surface to a permanent bonding involving extracellular polymeric substances (Srey et al., 2013; Stoodley et al., 2002). Bacterial cells begin to form microcolonies after irreversible adhesion. Microcolony development is caused by the simultaneous accumulation and proliferation of microbes and is linked to the creation of extracellular polymeric substances, which aid in the strengthening of the bacteria-substratum relationship and protects the colony from environmental-related stress. Extracellular polymeric substances account for . 90% of the dry mass in most mature biofilms. The extracellular polymeric substances are thought to “glue” cells in an established biofilm together, resisting mechanical stresses and separation of the community from the substrate’s surface (Renner & Weibel, 2011). Proteins, polysaccharides, nucleic acids, lipids, and other biopolymers are all components of

FIGURE 23.1 Schematic diagram showing the stages in the biofilm formation.

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the extracellular polymeric substances and have specific roles in assisting with biofilm maturation. Microcolonies formed are usually defined by their physical size of tens or hundreds of microns. Very recently, Huang et al. (2020) reviewed various methods to probe the formation of biofilms. Indeed, these bacteria release extracellular polymeric substances, which encapsulate them in a layer of entity, forming a physical barrier between the community and the extracellular environment. The chemical communication between cells in the community encourages the synthesis and secretion of extracellular polymeric substances, which varies by species and growth circumstances. It is interesting to note that a number of biofilm structures have been proven to evolve dynamically in response to external factors/conditions. Pseudomonas aeruginosa, for example, takes the shape of a mushroom structure with networks between colonies comprising rod-shaped cells. The recruitment of planktonic cells from the surrounding media as a consequence of cellto-cell communication has been shown in studies of bacterial species in natural environments. In bacteria, quorum sensing is the best-known example of chemical communication. It is a critical step in biofilm development and a way for cells to get information about their surroundings. Pathogenesis, nutrition acquisition, and secondary metabolite synthesis are just a few of the cellular activities that quorum sensing influences. However, it is critical to note that, unlike simple colloidal systems, a bacterial surface is structurally and chemically heterogeneous, thereby increasing the complexity of actual bacterial adhesion. Moreover, microbial adherence to solid surfaces is influenced by cell surface features. The roles of extracellular polymeric substances and cell appendages are to bridge the disparity between the cell body and the substratum. While extracellular polymeric substances are more crucial for biofilm construction than cell adhesion, cell appendages are often required for the first connection between cells and a substratum and serve as adhesins. The final step in biofilm formation is dispersion which is characterized by the transition of some of the cells back to the planktonic stage from a mature biofilm. However, those reverting to planktonic mode could attach all over again to new surfaces. As a result, dispersal represents not only the end of one lifecycle but also the beginning of another (Toyofuku et al., 2016). Biofilm detachment could be caused by either active dispersals, such as increased fluid shear, internal biofilm processes, and by passive dispersals, such as endogenous enzymatic breakdown, or the release of extracellular polymeric substances or surface-binding protein (Hall-Stoodley et al., 2004; McDougald et al., 2012; Toyofuku et al., 2016). Furthermore, biofilm detachment could also be caused by starvation which forces the cells to search for a more nutrient-dense environment (O’Toole et al., 2000).

23.3

Inactivation mechanism of biofilm

Contaminants in food manufacturing facilities often arise from the air, machinery, or food surfaces. The major foodborne pathogens that have been reported to form biofilm in food industries include L. monocytogenes (Mazaheri et al., 2021), Salmonella spp. (Webber et al., 2019), Escherichia coli (Aijuka & Buys, 2019), Pseudomonas spp. (Radovanovic et al., 2020), Vibrio parahaemolyticus (Roy et al., 2021), Clostridium perfringens (Alzubeidi et al., 2018; Charlebois et al., 2020), Campylobacter jejuni (Klanˇcnik et al., 2020), Bacillus spp. (Akbas and Cag, 2016), and Staphylococcus aureus (Miao et al., 2019). Due to its significant adverse effect of biofilm on several human activities, various techniques are used to prevent and remove a biofilm. The high disinfectant resistance of biofilm cells may raise the chance of disinfection failure, resulting in serious health issues and financial losses. Numerous studies have concluded that biofilm resistance to cleaning agents is a highly complex process involving a variety of mechanisms, including: 1. Reduced disinfectant penetration into the biofilm: The concentration of disinfectants required to inactivate a bacterium at a target site is determined by the three-dimensional structure of the cell that is attached to the EPS and biofilm. This compact structure can prevent the penetration of the compounds into deeper layers of the cell, which can protect the organisms against their harmful effects (Flemming & Wingender, 2010). The penetration of antimicrobials into biofilm matrix can also be affected by hydrophobic or electrostatic interactions. These interactions prevent the molecules from penetrating the deeper layers of the biofilm (Zhang, Nadezhina, & Wilkinson, 2011). 2. Altered biofilm cell physiology: Previous research has revealed the physiological differences between planktonic cell and bacteria attached to a biofilm, suggesting that the attachment of a bacterial cell to a surface causes upregulation of the genes involved in EPS synthesis leading to modifications in bacterial membranes (Sauer & Camper, 2001). As the proteins and phospholipids present in the bacterial cell wall are the initial line of bacterial defense against antibacterial agent, this increase in membrane stiffness might prevent disinfectants from accessing the lipid bilayers and boost biofilm cell resistance to disinfectants at the cellular level. Another plausible mechanism of biocide resistance is supported by the finding that some biofilm cells are able to detect the biocide threat and respond more effectively than planktonic cells by deploying protective stress responses (Yuan et al., 2021).

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3. Mutual protection arising from mixed-species biofilms: Surface-associated bacterial communities in the food processing environment often have intricate connections of diverse bacterial species that interact in a variety of ways to form a complex and dynamic network. Such interactions are important in building biofilm matrix and are responsible for a variety of activities, including antimicrobial resistance. In general, as compared to mono-species biofilms, mixed-species biofilms are less responsive to disinfectants (Yuan et al., 2020). 4. Presence of persister cells: Persister cells are a bacterial phenotype found in biofilms in which a tiny percentage of bacterial cells (about 0.1%
10%) are antimicrobial-resistant. Recently, persister cells have been suggested as another explanation for biofilms’ reduced susceptibility to disinfectants (Lewis, 2005). Biofilms are controlled and removed using traditional, physical, and chemical procedures like chlorination, flushing, and UV disinfection. However, concerns about the efficacy and safety of these treatments have remained, promoting the discovery, development, and use of innovative ways for dispersing and/or preventing the formation of biofilms. Recent breakthroughs in biofilm research have unveiled new details about the process of biofilm formation, paving the way for the creation of innovative biofilm prevention and inhibition strategies. Many innovative strategies, including suppression of quorum sensing (QS), bactericidal coating, enzymatic disruption, nanotechnology, bacteriophages, and bioelectric approach, have been successfully researched in the quest for effective biofilm prevention and control options (Sadekuzzaman et al., 2015). The effective mechanisms involved in biofilm inactivation include (Ali & Neelakantan, 2022): (1) inhibition of cell attachment to surfaces (oleanolic acid, curcumin, quercetin, etc.), (2) quorum quenching, (3) disrupting the biofilm matrix (curcumin and berberine), and by inhibiting biofilm recovery (cinnamaldehyde and carvacrol). The schematic representation of biofilm inactivation is shown in Fig. 23.2. Inhibiting cell attachment is an excellent way to avoid biofilm development in the early stages. Therefore, redesigning the surface or covering it with compounds that discourage bacterial adherence would potentially prevent the formation of bacterial biofilm (Muhammad et al., 2020). Coating of surfaces with hydrophobic materials like silicon-based materials such as polyethylene glycol (PEG) is one of the most extensively used antifouling coating in maritime and biomedical industries because of their hydrophilic surface properties, and PEG-coated surfaces have been found to prevent bacterial adherence. Several bacterial species, including Staphylococcus epidermidis, S. aureus, Streptococcus salivarius, E. coli, and P. aeruginosa adhesion, were found to reduce on surfaces with PEG coatings (Roosjen et al., 2003).

FIGURE 23.2 Biofilm inactivation mechanism by phytochemicals.

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Because of their antibacterial and anti-biofilm capabilities, phytochemicals have shown substantial potential to combat microbial pathogens. Pourhajibagher et al. (2018) reported a reduction in the Enterococcus faecalis biofilm formation by 23% on polystyrene microplates after exposure of planktonic cells to curcumin concentrations of 5 g/mL. Similarly, disruption and reduced thickness of E. faecalis biofilm were reported by L. Chen et al. (2016) when berberine was used as an antimicrobial agent. Carvacrol is a phenolic monoterpene that has antibacterial properties. Carvacrol was reported to be capable of efficiently inactivating E. faecalis biofilms as it was able to give a 5 log cfu/mL reduction in 4-day-old E. faecalis biofilms after 15 minutes treatment (Campana & Baffone, 2018). Moreover, only 8% of wounded E. faecalis biofilm cells regrew following 15 minutes of treatment with 1% carvacrol, demonstrating carvacrol’s potent lethal effect. These findings suggest that carvacrol has the ability to prevent E. faecalis biofilms from regrowing. Carvacrol’s antimicrobial activity is attributed to the presence of a functional hydroxyl group (
OH), which ensures high binding affinity of these compounds as microbial enzymes and cell receptors, resulting in hydrogen bond formation and inactivation. The delocalized electron system also contributes to carvacrol’s antimicrobial activity by allowing protons to escape and lowering the pH gradient across the bacterial cell membrane. Furthermore, carvacrol’s strong hydrophobicity allows it to penetrate deeply into bacterial membrane phospholipid bilayers, causing changed conformation of the membrane phospholipid bilayers and culminating in membrane damage and cell lysis (Ali & Neelakantan, 2022). Another mechanism by which biofilm can be inactivated is by interfering with bacterial quorum sensing (QS) which is termed as quorum quenching (QQ). Quorum sensing (QS) is a biochemical process that bacteria use to communicate and modify their behavior in response to cell density and the environment. Bacteria can use this communication system to accomplish processes including virulence factor synthesis, biofilm development, etc., (Remy et al., 2018). QS is characterized by the generation and detection of tiny extracellular molecules called autoinducers (AIs), which are produced in proportion to cell density (Papenfort & Bassler, 2016). Autoinducing peptides (AIPs) have been intensively tested and found to induce QS in gram-positive bacteria, although many gram-negative bacteria have been reported to employ a distinct class of autoinducers termed acyl homoserine lactones (AHLs) (Schuster et al., 2013). The quorum quenching technique uses quorum quenching enzymes such as lactonase, oxidoreductase, acylase, paraoxonase, etc., to inactivate quorum-sensing signals (Chen et al., 2013). The primary mechanisms of QS inhibition are as follows (Paluch et al., 2020): (1) signal molecule production is inhibited; (2) signal molecules are inactivated or degraded by enzymes; (3) act as analogs at receptor site and compete with signal molecules; and (4) signal transduction cascades are blocked. Several plant extracts and active chemicals have been thoroughly examined against certain bacterial biofilms on QS inactivation properties (Ta & Arnason, 2015). Dwivedi and Singh (2016) demonstrated the ability of plant phytochemicals, namely, embelin and piperine, on inhibiting the receptors and molecules involved in QS and thereby inactivating the Streptococcus mutans biofilm formation. Peppermint oil and menthol have been shown to reduce QS by inhibiting the violacein generated by Chromobacterium violaceum, which is controlled by AHL (Husain et al., 2015). Centella asiatica flavonoids inhibited swarming and twitching motility, as well as pyocyanin and biofilm development in P. aeruginosa (Vasavi et al., 2016). Norspermidine, a kind of polyamine, inhibited the expression of genes associated with the QS system in P. aeruginosa, including lasR/I, rhlR/I, and mvfR. As a result, there was less adhesion to the surface. Biofilm generation was restricted, and eradication was quicker with a reduced number of cells adhering to the surface (Qu et al., 2016).

23.4

Role of phytochemicals in biofilm inhibition

Antimicrobial resistance is one of the world’s most serious health threats, and new approaches are required to address it. In the current scenario, the development of innovative antimicrobial agents, especially biofilms, gained importance due to its acceptability and its effectiveness against microorganisms. The major phytochemicals which are used to develop antimicrobial biofilms include phenolics, terpenoids, organic acids, alkaloids, nitrogen, and sulfur-containing compounds (Table 23.1).

23.5

Phenolics

Considered as secondary metabolites, phenolics fall into the class of important natural molecules possessing different bioactive properties and with potential to prevention and treatment of many chronic diseases in human. These compounds can be obtained from many kinds of plant materials by the normal process of physical and chemical extraction methods. Phenolics are known to be contributors as metal chelating agents, free radical eliminators, lipid peroxidation inhibitors, antioxidants, and specifically as excellent inhibitors of many foodborne spoilage and pathogenic bacteria that causes deterioration or poisoning (Lattanzio, 2013). These food-related bacteria have the power to form biofilms by

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TABLE 23.1 The anti-biofilm potential of phytochemicals. Type of compound

Phytochemical specification

Observation

References

Apple extracts

Rutin, caffeic acid, epicatechin, dicaffeoyl quinic acid

Anti-QS activity was due to the synergistic effect of all the phenolic compounds

Fratianni et al. (2011)

Berberine

Quaternary protoberberine alkaloids

Potent biofilm inhibitor effective even at concentrations of 0.0635 mg/mL against the biofilm formation of Klebsiella pneumoniae

Magesh et al. (2013)

Bergamottin

Furocoumarins

Effective in hindering the biofilms of Escherichia coli by an inhibition percentage of 71.9%

Girennavar et al. (2008)

Betulin

Terpenoid

Effective as Chromobacterium violaceum AHL signaling inhibitors and had a negative influence on the biofilm formation of Obesumbacterium proteus strain

Priha et al. (2014)

Canthospermolides

Sesquiterpenoids

Biofilm inhibitory property against Pseudomonas aeruginosa biofilms by around 70% with an application of 2.5 μg/mL

Cartagena et al. (2007)

Carveol and carvone

Terpenoids

Reduction in biofilm by application of 250 μmol and decline in cell aggregation from 90% to 10% (48 mM)

De Carvalho and Da Fonseca (2007)

Cassipourol, β-sitosterol, and α-amyrin

Terpenoids

Able to interrupt the development of biofilms and reported a wide range of inhibition against P. aeruginosa

Rasamiravaka et al. (2017)

Catechin

Flavonoid

Effective against the biofilm formation of P. aeruginosa

Vandeputte et al. (2010)

Cinnamic aldehyde

Phenyl propene

Reduced virulence and increased susceptibility to stress

Brackman et al. (2008)

Citric, malic, and gallic acid

Organic acid

Inhibit Bacillus subtilis biofilms

Akbas and Cag (2016)

Cloudberry extracts

Phenolic compounds

Hindrance on the biofilm formation of brewery bacterial contaminant strain O. proteus

Priha et al. (2014)

Curcumin

Principal curcuminoid

Effective against the biofilm formation of Klebsiella pneumoniae at a concentration of 0.25 mg/mL

Magesh et al. (2013)

Ellagitannins and proanthocyanidins

Phenolic compound

Reported biofilm and quorumsensing inhibitory activities

Lin et al. (2011)

Epigallocatechin-3-gallate

Form of catechin

Effective against young mature biofilms of Stenotrophomonas maltophilia and also contributes to the reduction of viable cells in this film

Vidigal et al. (2014)

(Continued )

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TABLE 23.1 (Continued) Type of compound

Phytochemical specification

Observation

References

Fruit, herb, and spice extracts

Different phytochemicals

Inhibits the QS pathway by interfering the activity and synthesis of AHL

Vattem et al. (2007)

Geraniol, carvacrol, and thymol

Terpenoids

Inhibition rates of more than 75% were observed with carvacrol used at concentrations of 0.03% in the case of Candida albicans and at 0.125% of Candida glabrata and Candida parapsilosis

Dalleau et al. (2008)

Grape (Vitis coignetiae) extracts

Phenolic compounds

Biofilm inhibition percentage of the phenolics from pomace is around 69.5% and that from juice was found to be about 5%

Yano et al. (2012)

Gymnemic acids

Terpenoids

Enhanced the survival rate of C. albicans infected Caenorhabditis elegans (40 μg/mL)

Vediyappan et al. (2013)

Lactic acid

Organic acid

Use of 2% lactic acid on cucumber and reported a decline in the microbial count and a further extension in shelf life

Amrutha et al. (2017)

Limonoids

Terpenoids

Limonoids such as isolimonic acid and ichangin reported bioluminescence inhibition in Vibrio harveyi (6.25 μg/mL)

Vikram et al. (2011)

Malic acid

Organic acid

Synergistic effect of malic acid and sodium hypochlorite showed anti-biofilm activity at concentrations nearly four times lower than their individual effective concentrations

Chauhan et al. (2022)

Nootkatone

Sesquiterpenoid

Inhibition of biofilm mass by 50% and thereby reducing the chance of film formation

Farha et al. (2020)

Organic acid water

Organic acid

Examined the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and their sensitivity to Salmonella typhimurium biofilms. The study found that the age of the biofilm was not connected with its resistance to organic acids

Pande et al. (2018)

p-Coumaric acid, ellagic acid, and kaempferol

Phenolic compound

Biofilm inhibition and reported antimicrobial activity, with diameters ranging from 6.6 to 21.3 mm

Nassima et al. (2019)

Peony flower extracts

Gallic acid, kaempferol-7-Oglucoside, and apigenin-7-Oglucoside

Phenolic extracts show the inhibition rates of 77.93% and 87.03% on biofilms of E. coli and Staphylococcus aureus

Li et al. (2020)

(Continued )

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TABLE 23.1 (Continued) Type of compound

Phytochemical specification

Observation

References

Phloretin

Flavonoid

Effective against the biofilm of E. coli

Lee et al. (2011)

Quercetin

Flavonoid group

Concentration of 80 μg/mL was found to have inhibitory effects toward biofilm formation

Gopu et al. (2015)

Resveratrol

Terpenoids

Exhibited biofilm inhibitory effect against Propionibacterium acnes at 0.32% concentration

Coenye et al. (2012)

Thymol, carvacrol, and eugenol

Terpenoids

inhibit the activity of 24-h-old mature biofilms at a concentration of 1 mg/mL

Raut et al. (2013)

Thymol, citral, and carvacrol

Phenolic and aldehydic terpenes

Effective in inhibiting the biofilm formation of Cryptococcus neoformans

Kumari et al. (2019)

Vanillin

Phenolic compound

A concentration of 0.18 mg/mL reduced the development of biofilm by Aeromonas hydrophila on membrane filter by 90%

Kappachery et al. (2010)

Viridiflorol

Sesquiterpenoid

Inhibition of biofilms of P. aeruginosa and S. aureus at rates of 60% and 40%

Gilabert et al. (2015)

mode of bacterial cell-to-cell communication termed as quorum sensing (QS), thereby causing serious issues of contamination. Phenolics have the potential to control or inhibit the formation of autoinducers like N-acylhomoserine lactone (AHL) molecules, peptide molecules, etc., enzymatic degradation of signaling components and influence the receptor aversion reducing the bacterial mobility and pathogenic behavioral traits (Tako´ et al., 2020). The possibility of different phenolics as anti-biofilm agents was studied in detail by several authors. Plant flavanol quercetin at a concentration of 80 μg/mL was found to be effective against the pathogenic effect of two common gram-negative pathogens, P. aeruginosa and Klebsiella pneumoniae. QS inhibitory activity of quercetin is closely related to the conformational changes happening with the receptor molecules which hindered the biofilm formation (Gopu et al., 2015). Similarly, the virulence factors of P. aeruginosa were found to be inhibited by the presence of catechin which influences the QS hindering the biofilm formation (Vandeputte et al., 2010). Analogous to this, Hentzer et al. (2002) reported the efficacy of a halogenated furanone compound in inhibiting the biofilm formation of P. aeruginosa. The likelihood of penetration into the microcolonies of the strain inhibiting the signaling pathways impacts the QS activity thereby controlling the biofilm formation. The efficacy of utilization of natural components in biofilm inhibition is not limited to these individual components but also extends effectively toward the potential of phenolic extracts from plants, fruits, herbs, etc. Phenolic extracts from berries exhibited AHL inhibitory properties in the case of C. violaceum more specifically the raspberries and cloudberry extracts. Apart from the QS inhibition, extracts unveiled a minor influence on the growth at discrete concentrations (Priha et al., 2014). Positive influence on limiting the biofilm formation of brewery bacterial contaminant strain Obesumbacterium proteus by the presence of cloudberry extracts was also mentioned in the study. Phloretin, major flavonoid in apple extracts, exhibited efficacy against the biofilms of E. coli without altering the growth of planktonic cells (Lee et al., 2011). The potential of different types of these fruits, herbs, and plant extracts in inhibiting the biofilm formation depends on the part of the plant from which extracts are developed. Comparison studies on phenolic-rich Japanese wild grape Vitis coignetiae showed that the inhibitory activity of biofilm formation by S. mutans was higher in the case of pomace phenolics than that of fruit juice. Biofilm inhibition percentage of the phenolics from pomace is around 69.5% and that from juice was found to be about 5% (Yano et al., 2012). The mechanism followed in the inhibition of biofilm is not well explained and can be included in the future scope of the study. The author also explains the possibility of difference in the activity of phenolic extracts of different grape varieties against the inhibitory performance. With varieties including V. coignetiae and

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Campbell early showing the highest inhibitory activity, the least inhibitory activity was exhibited by the white variety Niagara possessing low phenolic percentage. Furthermore, studies also revealed that anti-QS activity of any fruit or plant extract cannot be correlated explicitly with a single phenolic compound and can be attributed to a group of phenolic compounds that are present. The QS inhibition activity of phenolic apple extracts is attributed to the synergistic effect of major phenolic compounds that are present in the system including rutin, epicatechin, dicaffeoylquinic acid, and caffeic acid (Fratianni et al., 2011). In addition to this is the study concentrating on the effectiveness of the phenolic extracts from peony flowers in QS activity inhibition. Phenolic extracts show the inhibition rates of 77.93% and 87.03% on biofilms of E. coli and S. aureus (Li et al., 2020). The author speculates the relation between the concentration and chemical composition of extracts comprising gallic acid, kaempferol-7-O-glucoside, and apigenin-7-O-glucoside and the inhibition levels. These studies put forward the possibility of incorporating natural quorum sensing inhibitors which contributes toward inhibition of biofilm formation thereby maintaining safety and quality. Phenolic compounds extracted from Populus nigra and Populus alba buds exhibited antimicrobial activity, with diameters ranging from 6.6 to 21.3 mm. The three major phenolic compounds present in the extract were p-coumaric acid, ellagic acid, and kaempferol. Also, extracts from P. nigra reported anti-biofilm properties of more than 70% (Nassima et al., 2019). In addition, phenolic compounds from Rubus rosaefolius were evaluated for its antioxidant, antimicrobial, and anti-quorum sensing activities. The phenolic extract inhibited bacteria in the minimum inhibitory concentration range of 491.90
1475.74 mg GAE/L (Oliveira et al., 2016). Cinnamic aldehyde is a phenyl propene which can interfere with AI-2-based quorum sensing in Vibrio spp. by diminishing the DNA-binding ability of LuxR. It was concluded that the application of these kinds of compounds may result in several marked phenotypic changes such as reduced virulence and increased susceptibility to stress (Brackman et al., 2008). Furthermore, the anti-biofilm effect of vanillin was proved under various abiotic stress by Kappachery et al. It was found that pretreatment with vanillin, at a concentration of 0.18 mg/mL, can reduce the development of biofilm by Aeromonas hydrophila on membrane filter by 90% (Kappachery et al., 2010). Ellagitannins and proanthocyanidins are phenolics that have shown biofilm and quorum-sensing inhibitory activities. A 50% inhibition of biofilm in S. aureus was induced by 1,2,3,4,6-penta-Ogalloyl-β-D-glucopyranose, which is a precursor of gallotannins (Lin et al., 2011). Another important group of phenolics includes flavonoids that exhibited an inhibition effect in quorum sensing and biofilm formation. For example, quercetin at a concentration of 6.25 μg/mL showed bioluminescence inhibiting activity in Vibrio harveyi strains. Also, the effect of quercetin against E. coli O157:H7 and V. harveyi BB120 and the inhibition of S. aureus biofilms was explained by Lee et al. (2013).

23.6

Terpenoids

Identified also as isoprenoids, terpenoids fall into a class of abundant and diversified natural products occurring widely in plants. Being divided into different subclasses of monoterpenes, diterpenes, triterpenes, and tetraterpenes, the possibilities of these groups of compounds are difficult to overstate (Ludwiczuk et al., 2017). With the known antimicrobial properties of these compounds, studies on the potential of different subclasses were studied. A consolidated summary of the potential of phenolics and terpenoids is given in Table 23.1. The likelihood of the involvement of different classes of terpenoids against the biofilms of P. aeruginosa and S. aureus was studied in detail by (Gilabert et al., 2015). Viridiflorol which is a sesquiterpenoid was found to be the most potent biofilm inhibitor among the group with 40% inhibition rates in the case of S. aureus and 60% in the case of the other strain. The author also explains the positive effect of other terpenes on controlling the biofilm formation. Equivalently, anti-biofilm efficacy of nootkatone (sesquiterpenoid) extracted from grapefruit against S. aureus was depicted by Farha et al. (2020). Reduction of biofilm mass by 50% and attaining a bacterial cell death of around 79%, this particular terpenoid can be expended as a favorable phytochemical contributing to anti-biofilm formation. The potential of terpenes in inhibiting the biofilm formation of Candida species studied in detail depicts the leeway of using these phytochemicals as effective antifilm agents (Dalleau et al., 2008). Among the terpene derivatives involved in the study, the most effectual in falling the biofilm formation of Candida albicans were geraniol, carvacrol, and thymol. Inhibition rates of more than 75% were observed with carvacrol used at concentrations of 0.03% in the case of C. albicans and at 0.125% of Candida glabrata and Candida parapsilosis, whereas in the case of geraniol and thymol inhibition of about 75% was achievable at concentrations of greater than 0.125% for the former and 0.06% or 0.125% in the case of the latter. Alike, Raut et al. (2013) also illustrated the promising anti-biofilm activity and inhibition of virulence factors of C. albicans underlining the potential of terpene classes. Along with the distinct upshot of these terpenoid compounds on the anti-biofilm potential, these compounds also display synergistic effects elevating their potential. Touil et al. (2020) described such a study which concentrates the potential of plant terpenoids, carvacrol, and

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cuminaldehyde discretely and in combination. With terpenoid combinations showing better results than the individual fractions, the synergistic effect also showed higher biofilm penetration capability. With effective reports on the anti-biofilm formation of terpenes, the mechanism of action remains unmapped. Correlated to this is the work reported by Kumari et al. (2019) which focused on explaining the mechanism and effectiveness of inhibiting Cryptococcus neoformans biofilm with thymol, citral, and carvacrol terpenes. The author speculates the possibility of mechanisms including ROS generation, ergosterol biosynthesis inhibition, and lipid peroxidation in unsettling operative integrity of biofilm. This analysis offers a set of data for the studies focusing on the antibiofilm potential of terpenes classes on different pathogenic bacteria. It was observed that compounds such as thymol, carvacrol, and eugenol were able to inhibit the activity of 24-hourold mature C. albicans biofilms at a concentration of 1 mg/mL (Raut et al., 2013). Terpenoids such as cassipourol, β-sitosterol, and α-amyrin extracted from Platostoma rotundifolium were analyzed for their anti-virulence activities. These terpenoids were able to interrupt the development of biofilms at amounts down to 12.5, 50, and 50 μM for cassipourol, β-sitosterol, and α-amyrin, respectively. Moreover, they reported a wide range of inhibition against P. aeruginosa and confirmed their effectiveness as a therapeutic agent (Rasamiravaka et al., 2017). Additionally, terpenes such as monoterpenes, limonoids, and triterpenes have reported biofilm inhibition and antiquorum sensing properties. Thymol and carvacrol showed effectiveness against biofilms formed by gram-positive and gram-negative bacteria. Moreover, previous studies confirmed that thymol affected the development of L. monocytogenes biofilms and inactivated them at 0.5 mM and 5 mM concentrations, respectively, (Upadhyay et al., 2013). Resveratrol is another bioactive compound that processes antioxidant property and can effectively act against biofilms. It showed biofilm inhibitory effect against Propionibacterium acnes at 0.32% concentration as well (Coenye et al., 2012). Canthospermolides are sesquiterpenoids which aids in reducing the biofilm development. Acanthospermolides obtained from Acanthospermum hispidum DC. reported biofilm inhibitory property against P. aeruginosa biofilms by around 70% with an application of 2.5 μg/mL (Cartagena et al., 2007). Additionally, limonoids such as isolimonic acid and ichangin reported bioluminescence inhibition in V. harveyi at a level of 6.25 μg/mL (Vikram et al., 2011). Besides, the survival rate of C. albicans infected Caenorhabditis elegans was significantly enhanced by treatment with gymnemic acids at a concentration of 40 μg/mL (Vediyappan et al., 2013). A one-third reduction in the amount of biofilm was noted by the application of 250 μmol of carveol and carvone in comparison with the control. Further, the degree of cell aggregation dropped from 90% to 10% which was observed when 48 mM concentration of carvone was used (De Carvalho & Da Fonseca, 2007).

23.7

Organic acids

Organic acids are extensively used as food preservatives due to their antibacterial properties, and they have been declared as generally recognized as safe (GRAS). They are volatile or weak acids relatively stable, metabolized, unabsorbed, and does not leave any chemical residue. Some of the most analyzed organic acids include acetic acid, citric acid, malic acid, formic acid, lactic acid, and maleic acid. As a result, the impact of organic acids such as acetic acid, citric acid, and lactic acid on quorum signaling and their potential in E. coli and Salmonella spp. biofilms were evaluated. About 2% lactic acid exhibited more antimicrobial activity than acetic and citric acids. In addition, a considerable decrease in biofilm formation and motility was reported. Besides, lactic acid and acetic acid showed more anti-quorum activity than citric acid. Summarily, the use of 2% lactic acid on cucumber reported decline in microbial count and further extension in shelf life (Amrutha et al., 2017). Additionally, citric, malic, and gallic acids (1% and 2%) can also be used to inhibit Bacillus subtilis biofilms. In comparison with citric and gallic acids, malic acid showed higher anti-biofilm activity. Besides, citric acid reported more inhibition activity against biofilms than chlorine treatment (Akbas and Cag, 2016). Additionally, the synergistic effect of malic acid and sodium hypochlorite against Cronobacter sakazakii biofilm was studied. Malic acid at a dose of 0.0625 mol/L in grouping with sodium hypochlorite at a level of 0.00004 mol/L was found to be the most efficient combination with fractional inhibitory concentration index of 0.38 against C. sakazakii biofilm. These findings revealed that combining malic acid and sodium hypochlorite has anti-biofilm activity at concentrations nearly four times lower than their individual effective concentrations (Chauhan et al., 2022). Another study examined the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and their sensitivity to Salmonella typhimurium biofilms. The study found that the age of biofilm was not connected with its resistance to organic acids. But the type of product, concentration, and period of exposure were the key determinants for reduction in the number of viable biofilm cells. These findings suggest the future application of the use of organic acids in biofilm control in the poultry sector (Pande et al., 2018). Overall, these results will pave the way for further research in the area of biofilms as potential antimicrobial agents.

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407

Other phytochemicals

23.8.1 Alkaloids Conferred with diverse possibilities of pharmaceutical applications, alkaloids refer to one of the most prevalent and abundant classes of secondary metabolites in plants. Incorporation of these classes of products in inhibition of biofilm activity is still in a nascent stage and entails succor. Oxindole a bicyclic monoterpene alkaloid which is inherently a part of the existing biologically active compounds was found to be operative in inhibiting the biofilm of n P. aeruginosa (El-Hawary et al., 2019). Two different classes of brominated oxindole alkaloids obtained from Callyspongia siphonella crude extracts displayed an inhibition percentage of 49.32% and 41.76%, respectively. Bromopyrrole alkaloids representing a large variety of bioactive components shaped by marine sponges display promising anti-biofilm properties accentuating the potential of the obtainable band (Rane, Sahu, Shah, & Karpoormath, 2014). Sun et al. (2018) studied the marine alkaloid fractions obtained from the marine sponge Stylissa massa which depicted the likelihood of the presence of 32 alkaloids exhibiting inhibitory upshots against a panel of bacteria. Among the identified fractions, phakellin-based alkaloids, especially dibromoisophakellin and dibromophakellin, showed positive influence toward anti-biofilm properties of E. coli promoting the practice of these components for biofilm inhibition. Similarly, Hertiani et al. (2010) also elucidated the efficacy of these bromopyrrole alkaloid derivatives from Indonesian marine sponges Agelas linnaei against the biofilm formation capacity of S. epidermidis. Shifting to other major alkaloid fractions, aporphine alkaloids constitute a subsection of alkaloids descended from isoquinoline with quite a few biologically important properties. Di Marco, Lucero-Estrada, and R. Pungitore (2019) have extensively studied the ability of these 17 aporphinoids fractions in inhibiting the biofilm formation by altering the quorum-sensing capacity of Yersinia enterocolitica. Fractions including oliverine, oliceridine, pachypodanthine, guatterine, and liriodenine exhibited inhibition values of more than 87% with pachypodanthine being the most operative element with minimum biofilm inhibitory concentration of around 125 μmol/L at subinhibitory concentration. The antibacterial activity of these fractions, specifically pachypodanthine and oliceridine, was reported beforehand by Di marco et al. (2019) with minimum inhibitory concentration of around 25 and 100 μmol/L analogs to the reported study. Other fractions like (R)-Bgugaine a natural pyrrolidine alkaloid possessing antibacterial activity have reported competences in altering the quorum-sensing activity and reducing the biofilm formation of P. aeruginosa by 83% (Majik et al., 2013). A clinical study by Dusane, Hosseinidoust, Asadishad, and Tufenkji (2014) is also comparable to the former results which investigated the capability of piperine and reserpine in controlling the colonies of E. coli. These compounds were found to be negatively affecting the mobility of the bacterial populations, increasing the penetration of antibiotics and thereby establishing control over biofilm formation. Comparable to these results is the effectiveness of berberine in controlling the biofilm concentrations in clinical sequesters of K. pneumoniae at an inhibitory concentration of 63.5 μg/mL (Magesh et al., 2013). Even though these inhibitory concentrations need to be evaluated in detail underlining the values of alkaloid composition obligatory for inhibition of particular strain, it offers a comprehensive idea about the progression and is beneficial toward future detailing.

23.9

Sulfur- and nitrogen-containing phytochemicals

A distinctive facet of allium vegetables such as onion, garlic, and cruciferous groups such as cabbages, broccoli, kale, etc., is the presence of specialized sulfur-rich active components beneficial in countless means. It is found that these organosulfur compounds including allicin, ajoene, and isothiocyanates detected in allium and cruciferous vegetable extracts contribute positively toward the alteration of quorum-sensing activity of different strains. With underlying processes remaining imprecise, extracts from garlic oil were found to be effective against quorum-sensing property of P. aeruginosa. Diallyl sulfide, the most profuse sulfur-containing composite in garlic oil at a concentration of 1.28 mg/mL reduced the biofilm-forming potential of the strain (Li et al., 2018). Similarly, Xu et al. (2019) also reported the efficacy of allicin in controlling the quorum-sensing activity and biofilm formation of P. aeruginosa. Two natural isothiocyanates which are abundant in cruciferous vegetables like broccoli, sulforaphane and erucin, were found to have influence on the virulence factors of P. aeruginosa. Sulforaphane at concentrations of 12 μm and above exhibits a strong anti-biofilm activity of P. aeruginosa. Comparable reports elucidate that the anti-biofilm activity of erucin is markedly lower than the former fraction, and the difference can be attributed to the difference in hydrophobicity and oxidation rate of erucin. Added to these, authors also report the effectiveness of thiocyanates like allyl isothiocyanate, sulforaphane in inhibiting the quorum sensing and virulence factors of different strains including C. violaceum, L. monocytogenes, E. coli, and P. aeruginosa (Borges et al., 2014; Borges, Simoes, Serra, Saavedra, & Simo˜es, 2013; Ganin et al., 2013). With underlying mechanisms remaining unclear in these operative studies, the future orbit of the research should be readdressed in that course to have lucidity of the progression. Moving with the latter group of

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nitrogen-containing phytochemicals, Monte, Abreu, Borges, Simo˜es, and Simo˜es (2014) explain the likelihood and efficacy of indole-3-carbinol (I3C) in altering the motility and quorum sensing thereby preventing cell interactions and biofilm formation. I3C was effective against the biofilms of E. coli and S. aureus with minimum inhibitory concentrations of 800 and 400 μg/mL, respectively. The author also elucidates the possibility of combination of the indole-3-carbinol with other antibiotics which increased the inhibitory properties in S. aureus strains. The effectiveness of betacyanin fractions isolated from red spinach and red pitahaya in inhibiting the biofilm activity of S. aureus and P. aeruginosa strains was studied by Yong, Dykes, Lee, and Choo (2019). Comparative studies reported that the betacyanin fractions from red spinach exhibited better inhibitory values against strains of S. aureus and the latter was outshining against the P. aeruginosa populations. The minimum inhibitory concentrations of betacyanin fractions from red spinach were around 0  313
1  25 mg/mL and that of pitahaya were about 0  313
0  625 mg/mL. With underlying mechanisms remaining unclear in these operative studies, the future orbit of the research should be readdressed in that course to have lucidity of the progression.

23.10 Future perspective and conclusion Bacterial biofilm development is ubiquitous and takes place in a series of well-ordered steps. It is the most common bacterial life-form in both natural and artificial environments. The capacity of bacteria to colonize surfaces and form biofilms is seen as a severe threat and can lead to adverse consequences in a variety of fields including food, pharmacy, and healthcare sector. Various strategies and procedures have been developed in an effort to eliminate harmful biofilms, with the majority of them focusing on interference with bacterial attachment and QS, as well as biofilm matrix degradation. Natural compounds, bacteriophages, nanotechnology, bacteriocin, quorum quenching, and enzymes are some of the developing innovative techniques that are promising and may assist to establish anti-biofilm tactics that are superior to the current ones. Furthermore, the “biofilm problem” is predicted to be solved in the near future using a mix of these innovative approaches and traditional procedures (disinfectants, antibiotics, and physical methods). Many phytochemicals found in plants can interfere with bacteria quorum sensing and biofilm development. There are many biosynthetic classes of chemicals that have the potential to inactivate biofilm, but they have not been well investigated. The discovered inhibitors might be a gold mine of lead chemicals for developing and combating antibiotic resistance of bacterial biofilms. The chemistry and mechanism of these chemicals as natural defenses are still unknown and are a fascinating subject for further research.

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cell signalling and biofilm formation by modulating the response regulator LuxO. Microbiology (Reading, England), 157(1), 99
110. Wang, H., Cai, L., Li, Y., Xu, X., & Zhou, G. (2018). Biofilm formation by meat-borne Pseudomonas fluorescens on stainless steel and its resistance to disinfectants. Food Control, 91, 397
403. Available from https://doi.org/10.1016/j.foodcont.2018.04.035. Webber, B., Oliveira, A. P. d, Pottker, E. S., Daroit, L., Levandowski, R., Santos, L. R. d, . . . Rodrigues, L. B. (2019). Salmonella Enteritidis forms biofilm under low temperatures on different food industry surfaces. Cieˆncia Rural, 49(7). Available from https://doi.org/10.1590/01038478cr20181022. Wolcott, R. D., & Ehrlich, G. D. (2008). Biofilms and chronic infections. JAMA: Journal of the American Medical Association, 299(22), 2682
2684. Xu, Z., Zhang, H., Yu, H., Dai, Q., Xiong, J., Sheng, H., . . . He, X. (2019). Allicin inhibits Pseudomonas aeruginosa virulence by suppressing the rhl and pqs quorum-sensing systems. Canadian Journal of Microbiology, 65(8), 563
574. Yano, A., Kikuchi, S., Takahashi, T., Kohama, K., & Yoshida, Y. (2012). Inhibitory effects of the phenolic fraction from the pomace of Vitis coignetiae on biofilm formation by Streptococcus mutans. Archives of Oral Biology, 57(6), 711
719. Yong, Y. Y., Dykes, G., Lee, S. M., & Choo, W. S. (2019). Biofilm inhibiting activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms, 126(1), 68
78. Yuan, L., Hansen, M. F., Roder, H. L., Wang, N., Burmolle, M., & He, G. (2020). Mixed-species biofilms in the food industry: Current knowledge and novel control strategies. Critical Reviews in Food Science and Nutrition, 60(13), 2277
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3891. Available from https://doi.org/10.1080/ 10408398.2020.1809345. Zhang, Z., Nadezhina, E., & Wilkinson, K. J. (2011). Quantifying diffusion in a biofilm of Streptococcus mutans. Antimicrobial Agents & Chemotherapy, 55(3), 1075
1081. Available from https://doi.org/10.1128/AAC.01329-10.

Chapter 24

New perspectives and role of phytochemicals in biofilm inhibition Pravin R. Vairagar1, Aniket P. Sarkate1, Nilesh Prakash Nirmal2 and Bhagwan K. Sakhale1 1

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India, 2Institute of Nutrition,

Mahidol University, Salaya, Nakhon Pathom, Thailand

24.1

Introduction

Biofilms are complex microbial ecosystems formed by one or more species immersed in an extracellular matrix of different compositions depending on the type of food manufacturing environment and the colonizing species. Examples of microorganisms that can comprise these biofilms include bacteria and fungi. The presence of more than one bacterial species in a biofilm has important ecological advantages because it can facilitate the biofilm’s attachment to a surface. For some species, this can even occur in the absence of specialized fimbriae. Mixed biofilms show higher resistance to disinfectants such as quaternary ammonium compounds and other biocides (Meyer, 2015). Biofilms imply major challenges for the food industry because they allow bacteria to bind to a range of surfaces, including rubber, polypropylene, plastic, glass, stainless steel, and even food products, within just a few minutes, which is followed by mature biofilms developing within a few days (or even hours) (Hall-Stoodley et al., 2004). Biofilms have become a major environmental microbiology concern in the food industry over the last 30 years. This topic is prominent due to the potential for contamination of food from biofilms; they are responsible for more than 20% of food poisoning cases and for being up to 1000-fold more tolerant to antibiotics than their planktonic counterparts (Carrascosa et al., 2021). In recent years, drug resistance of human pathogenic bacteria has been extensively reported. Moreover, persistent infections were also observed due to improved resistance of bacteria in biofilm (Davies & Davies, 2010; Davies, 1994). This creates a tremendous economic loss and pressure on the medical community to find alternative approaches for the treatment of diseases related to biofilms. Therefore, efforts have been applied to discover efficient antimicrobial molecules not so vulnerable as current drugs to bacterial resistance mechanisms, including those in biofilms (Li et al., 2011; Saleem et al., 2010). Some natural products have distinctive properties that make them perfect candidates for these much-needed therapeutics (Simoes et al., 2009). Plants produce an enormous array of secondary metabolites (phytochemicals) that are not essential for their normal physiological functions. However, these bioactive compounds are used to protect plants against attacks from microorganisms, herbivores, insects, nematodes, and even other plants (Dixon, 2001; Hemaiswarya et al., 2008). The importance of diverse natural products has been recognized by humans due to their beneficial properties for health. Inclusively, many classes of plant secondary metabolites have demonstrated their potential as antimicrobials or synergists of other products (Prior & Cao, 2000; Saavedra et al., 2010). So, nowadays phytochemicals are a fundamental source of chemical diversity and important components of the current pharmaceutical products (Dixon, 2001; Kubo et al., 2006; Saleem et al., 2010). Therefore, the study of biofilm and the strategies to eliminate them is one of the most important fields of research in the present days. Many reviews on biofilm inhibition strategies have already been done, but this chapter focuses especially on new perspectives of phytochemicals in biofilm inhibition.

24.2

Biofilm development and its health hazards

It is now understood that about 40%
80% of bacterial cells on earth can form biofilms (Flemming & Wuertz, 2019). The formation of biofilms was detrimental in several situations (Coughlan et al., 2016; Dobretsov et al., 2006; Donlan Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00012-8 © 2023 Elsevier Inc. All rights reserved.

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& Costerton, 2002). For example, in food industries, pathogenic bacteria are able to form biofilms inside processing facilities, leading to food spoilage. In hospital settings, biofilms have also been shown to persist on medical device surfaces and on patient’s tissues causing persistent infections (Dongari-Bagtzoglou, 2008; Percival et al., 2015). In view of the serious impact of biofilms on human health and other aspects, researchers and the public have long focused on the prevention and control of harmful biofilms. Generally, bacterial biofilm formation relies on the interaction between the bacterial cells, the substrates, and the surrounding media (Van Houdt & Michiels, 2010). The formation of bacterial biofilms is a multistep process starting with reversible attachment to surfaces aided by intermolecular forces and hydrophobicity and then progressing to extracellular polymeric substances (EPSs) production which enables the cells to permanently adhere to a surface (Bogino et al., 2013; Dunne, 2002). More specially, there are five main phases involved in the biofilm formation process: reversible attachment, irreversible attachment, EPS production, maturation of biofilm, and dispersal/detachment (Stoodley et al., 2002; Toyofuku et al., 2016). In the healthcare settings, biofilms have been shown to develop on medical device surfaces, dead tissues (e.g., sequestra of bones), and inside living tissues (e.g., lung tissue, teeth surfaces) (Alav et al., 2018). They may develop on the surface of biomedical devices such as catheters, prosthetic heart valves, pacemakers, breast implants, contact lenses, and cerebrospinal fluid shunts (Hall-Stoodley et al., 2004; Wu et al., 2015). Within the food industry, biofilms can occur on surfaces contacting with or without foods (Kumar & Anand, 1998; Zottola & Sasahara, 1994). Biofilms are responsible for about 60% of foodborne outbreaks (Han et al., 2017). Therefore, the presence of biofilms in food processing environments poses a significant risk to food safety and the food industry (Galie et al., 2018). In the food processing environments, contaminants mostly come from the surrounding air, equipment, or food surfaces (Kumar & Anand, 1998). Then, biofilms growing in food processing environments may lead to spoilage of food, which in turn can cause serious public health risks to consumers and serious economic consequences (Coughlan et al., 2016; Galie et al., 2018).

24.2.1 Factors influencing biofilm development The formation of biofilm is a dynamic and complex process which includes the initial attachment of bacterial cells to the substratum, physiological changes within the microbe, multiplication of adhered cells to form microcolonies, and finally biofilm maturation (Puttamreddy et al., 2010). Biofilm-associated bacteria demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to the immune system and antibiotics that render biofilm a source of chronic and persistent infections (Wei & Ma Luyan, 2013). It is known that the change of phenotype from planktonic to the sessile form occurs in response to changes in environmental conditions (Chiara et al., 2016). These environmental factors, such as nutrient level, temperature, pH, and ionic strength, can influence biofilm formation (Agarwal et al., 2011). Various factors can influence bacterial adhesion: cell surface properties, such as hydrophobicity, flagellation, and motility, surface properties, such as hydrophobicity and roughness, and environmental factors, such as temperature, pH, availability of nutrients, and hydrodynamic conditions (Table 24.1) (Alotaibi & Bukhari, 2021). Cell surface properties, specifically the presence of extracellular appendages, such as fimbriae and flagella, the interactions involved in cell-tocell communication, and EPS production, such as surface-associated polysaccharides or proteins possibly provide a competitive advantage for one organism in a mixed microbial community (Donlan, 2002; Simo˜es et al., 2010).

TABLE 24.1 Important variables in bacterial cell attachment and biofilm formation (Alotaibi & Bukhari, 2021). Properties of the substratum

Properties of the bulk fluid

Properties of the cell

Hydrophobicity

Temperature

Cell surface hydrophobicity

Conditioning film

pH

Extracellular appendages, such as fimbriae and flagella

Texture or roughness

Flow velocity and nutrient availability

Extracellular polymeric substances

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24.2.2 Stages in biofilm development There are four main stages involved in the development of biofilm: (1) cellular attachment, (2) formation of microcolonies, (3) biofilm maturation, and (4) detachment (Jamal et al., 2018).

24.2.2.1 Cellular attachment The first step of biofilm formation is attachment of bacteria onto a surface (Crouzet et al., 2014). An organism has to overcome the repulsive generated due to negatively charged bacterial membrane and the surface for the purpose to bind on the surface (Palmer et al., 2007). A hydrophilic surface such as glass and metal has more force of repulsion as compared to a hydrophobic surface, for example, plastic. The reduction in repulsive forces corresponds to the increased strength of attachment (Jamal et al., 2018). Attachment is achieved by the presence of flagella and pili on the bacterial membrane (Jamal et al., 2018; Palmer et al., 2007). In the reversible attachment phase, structural support is involved. In reversible binding, bacteria are poorly bound to the surface, and at this stage, they are able to leave the surface. The bacteria returned to its planktonic lifestyle once leaving a surface (Petrova & Sauer, 2012). The important step in the transition from a planktonic to biofilm lifestyle of bacteria is observed when bacteria stay on the surface and undergo irreversible attachment. The surface proteins of bacterial cells aid in the adhesion of cells to a surface resulting in irreversible binding (Caiazza & O’Toole, 2004). The stronger chemical and physical shear forces can be sustained by biofilm as a result of irreversible binding (Rabin et al., 2015).

24.2.2.2 Formation of microcolonies The bacteria start to divide and produce EPS after being attached irreversibly to a surface (Armbruster & Parsek, 2018). A biofilm matrix is formed after the production of EPS which acts as “shelter” for attached cells living in (Flemming et al., 2007). The adhesion of cells to surfaces is due to EPS resulting in permanent attachment (Caiazza & O’Toole, 2004). It was observed that biofilm matrix proteins of biofilm possess adhesion-like properties that mediate cellular attachment. A study showed that RbmA acts as a mediator in this process which is one of the matrix proteins found in the Vibrio cholerae biofilm (Drescher et al., 2016). As a result of EPS-mediated cellular cohesion, bacterial cells are brought together and microcolonies are formed, which is the second step of biofilm formation (Bowen et al., 2018). The coordination between one microcommunity and another is important for substrate exchange, the distribution of essential metabolic products, and also the excretion of harmful substances (Jamal et al., 2018).

24.2.2.3 Biofilm maturation In the biofilm maturation process, a three-dimensional (3D) structure was formed after the maturation of an early biofilm which is developed due to cellular division with continuous production of EPS. The above 3D structure is contributed to by the EPS (produced by the embedded cells), and they are also responsible for maintaining this architecture (Hobley et al., 2015). The facilitation of quorum sensing is observed in the maturation process due to cell-to-cell communication which leads to the release of signaling molecules called autoinducers by cells embedded in the biofilms (Jamal et al., 2018; Preda & S˘andulescu, 2019). Cells express their genes coding for EPS which receives the signals. The production of EPS increased, and the biofilms acquire the previously discussed 3D structure (Jamal et al., 2018; Saxena et al., 2019). The mature biofilm also contains water channels in the matrix acting as a circulatory system as well as functions channel as nutrient distribution and the removal of waste products (Parsek & Singh, 2003; Wilking et al., 2013).

24.2.2.4 Detachment of biofilm After maturation, biofilms undergo a process known as dispersion. In this stage, some of the cells leave the biofilm and return to their planktonic lifestyle (Chua et al., 2014). Once, these cells return to their original free-floating form and are capable to attach onto a new surface, and the cycle starts all over again (Rumbaugh & Sauer, 2020). Active or passive detachment of cells is found from the biofilm. Mechanical forces or external forces such as abrasion, fluid shear, and solid shear are responsible for the passive dispersal of a biofilm (Fleming & Rumbaugh, 2017; Kaplan, 2010), while upregulation and downregulation of genes are involved in active dispersal. Nutrient starvation, insufficient oxygen supply, and changes in temperature are observed as environmental triggers related to the dispersal of biofilms (Kostakioti et al., 2013). Under these conditions, the upregulation of genes responsible for flagella synthesis in bacterial cells provides them the ability to leave the biofilm (Guilhen et al., 2017). Moreover, the production of dispersin B is also

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increased. This enzyme is present in the extracellular matrix and helps to hydrolyze polysaccharides which cause EPS degradation. There is negative impact of increased secretion of dispersin B into the matrix in biofilm formation permitting adherent cells to leave the biofilm easily (Guilhen et al., 2017; Pirrone et al., 2016). Fig. 24.1, as shown, summarizes the various stages of biofilm development.

24.2.3 Microorganisms associated with biofilms and their health hazards Biofilm formation is associated with bacteria, fungi, and protists (Costa-Orlandi et al., 2017; Tasneem et al., 2018). Some examples of biofilm-forming bacteria, fungi, and protists are shown in Fig. 24.2. Foodborne diseases associated with bacterial biofilms on food matrixes or factory equipment may arise via intoxications or infections. Toxins, for example, can be secreted by biofilm found within food processing plants. From there, they can contaminate a food matrix, causing individual or multiple (in the case of an outbreak) intoxications. In either case, the presence of biofilms in a food factory puts human health at risk. The amount of risk is dependent on the bacterial species forming this tridimensional living structure. The main locations for biofilm development depend on the factory type, but may include water, milk, and other liquid pipelines, pasteurizer plates, reverse osmosis membranes, tables, employee gloves, animal carcasses, contact surfaces, storage silos for raw materials and additives, dispensing tubing, packing material, etc. (Camargo et al., 2017). FIGURE 24.1 Steps in biofilm formation (Samrot et al., 2021). Adapted from Samrot, A. V., Abubakar Mohamed, A., Faradjeva, E., Si Jie, L., Hooi Sze, C., Arif, A., . . . Kumar, S. S. (2021). Mechanisms and impact of biofilms and targeting of biofilms using bioactive compounds-A review. Medicina, 57(8), 839.

Biofilm-forming Microorganisms

Bacteria Acinetobacter baumannii Bacillus subtilis Campylobacter jejuni Clostridium difficile Clostridium perfringens Enterococcus faecalis Escherichia coli Fusobacterium nucleatum Klebsiella pneumoniae Listeria monocytogenes Proteus mirabilis Pseudomonas aerobicus Pseudomonas aeruginosa Salmonella spp. Staphylococcus aureus Staphylococcus epidermidis Streptococcus agalactiae Vibrio cholerae

Fungi Apophysomyces elegans Aspergillus fumigatus Blastoschizomyces capitatus Candida albicans Candida parapsilosis Coccidioides immitis Cryptococcus laurentii Cryptococcus neoformans Exophiala dermatitidis Histoplasma capsulatum Lichtheimia corymbifera Malassezia pachydermatis Trichosporon asahii Trichophyton rubrum Trichophyton mentagrophytes Paracoccidioides brasiliensis Pneumocystis carinii Rhizomucor pusillus Rhizopus oryzae

Protists Cryptosporidium spp. Cyclospora cayetanensis Toxoplasma gonidii

FIGURE 24.2 Types of biofilmsforming organisms. Adapted from Samrot, A. V., Abubakar Mohamed, A., Faradjeva, E., Si Jie, L., Hooi Sze, C., Arif, A., . . . & Kumar, S. S. (2021). Mechanisms and impact of biofilms and targeting of biofilms using bioactive compounds-A review. Medicina, 57(8), 839.

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In the food industry, biofilm-forming species appear in factory environments and can be pathogenic to humans because they develop biofilm structures. The processing environments of the food industry, for example, wood, glass, stainless steel, polyethylene, rubber, polypropylene, etc., act as artificial substrates for these pathogens (Abdallah et al., 2015; Colagiorgi et al., 2017). The characteristics of the bacterial growth form on food in a processing environment involve different behaviors when considering cleaning and disinfection processes. Controlling biofilm formations in the food industry can prove difficult when having to decide the right strategy. Examples of these relevant biofilm-forming pathogens for the food industry are briefly described in Table 24.2. Biofilms are responsible for chronic illness and nosocomial infections, industrial pipe fouling, spoilage of foods, contamination of seafood, and dairy products as well as ship hull fouling (Abdallah et al., 2014; Khatoon et al., 2018; Schultz et al., 2011; Zottola & Sasahara, 1994). Therefore, the harmful effects of biofilms on human society are manifold (Muhammad et al., 2020).

TABLE 24.2 Biofilm-forming pathogens in the food industry (Carrascosa et al., 2021). Pathogen

Characteristics

Contaminated food

Example of harmful spoilage effects

Bacillus cereus

Gram-positive, spore-forming, anaerobic, facultative anaerobic

Dairy products, rice, vegetables, meat

Diarrhea and vomiting symptoms

Campylobacter jejuni

Gram-negative, aerobic, and anaerobic

Animals, poultry, unpasteurized milk

Bloody diarrhea, fever, stomach cramp, nausea, and vomiting

Escherichia coli

Gram-negative, rod-shaped

Raw milk, fresh meat, fruits, and vegetables

Diarrhea outbreaks and hemolytic uremic syndrome

Listeria monocytogenes

Gram-negative, rod-shaped, facultative anaerobic

Dairy products, meat, ready-toeat products, fruit, soft cheeses, ice cream, unpasteurized milk, candied apples, frozen vegetables, poultry

Listeriosis in the elderly, pregnant women, and immunecompromised patients

Salmonella enterica

Gram-negative, rod-shaped, flagellate, facultative aerobic

Poultry meat, bovine ovine, porcine, fish

Can cause gastroenteritis or septicemia

Staphylococcus aureus

Gram-positive, non-spore forming, non-motile, facultative anaerobic

Meat products, poultry, egg products, dairy products, salads, bakery products, especially cream-filled pastries and cakes, and sandwich fillings

Methicillin resistance can cause vomiting and diarrhea

Pseudomonas spp.

Psychrotrophic, motile, gramnegative rod-shaped

Fruits, vegetables, meat surfaces, and low-acid dairy products

Produces blue discoloration on fresh cheese.

Geobacillus stearothermophilus

Thermophilic, Gram-positive, spore-forming, aerobic or facultative anaerobic

Dried dairy products

Production of acids or enzymes leading to off-flavors

Anoxybacillus flavithermus

Thermophilic organism, grampositive, spore-forming, facultatively anaerobic, nonpathogenic

Dried milk powder

An indicator of poor hygiene

Pectinatus spp.

Gram-negative, non-spore forming, anaerobic

Beer and brewery environment

Rapid cell growth makes beer turbid and smells like rotten eggs due to the production of sulfur compounds

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Biofilm-forming bacteria contribute to a lot of life-threatening infections and diseases in humans such as cystic fibrosis (CF), otitis media, periodontitis, infective endocarditis (IE), chronic wounds, and osteomyelitis (Akyıldız et al., 2013; Southey-Pillig et al., 2005; Masters et al., 2019). Bacterial biofilms are responsible for approximately 80% of chronic and recurrent microbial infections in the human body. Antibiotic resistance of microbial cells within biofilms has shown 10
1000 times more than their planktonic form (Mah, 2012). The biofilms initially formed on medical implants, such as heart valves, catheters, contact lenses, joint prostheses, intrauterine devices, and dental unit, are responsible for urinary tract and bloodstream infections. These infections can only be treated by removal of the implants which not only increases the cost of the treatment but also becomes problematic for patients (Costerton et al., 2005). Biofilm infections related to host tissue are often chronic, including chronic osteomyelitis, chronic prostatitis, chronic rhinosinusitis, chronic lung infections of cystic fibrosis patients, chronic otitis media, recurrent urinary tract infection, endocarditis, chronic wounds, periodontitis, and dental caries (Burmølle et al., 2010).

24.3

Occurrence of biofilms

Biofilms may form on a wide variety of surfaces, including natural aquatic systems living tissues, indwelling medical devices, and industrial/potable water system piping. The vast majority of microbes grow as biofilms in aqueous environments (Donlan, 2002). These biofilms can be benign or pathogenic, releasing harmful products and toxins, which become encased within the biofilm matrix. Biofilm formation is a phenomenon that occurs in both natural and man-made environments under diverse conditions, occurring on most moist surfaces, plant roots, and nearly every living animal. Biofilms may exist as beneficial epithilic communities in rivers and streams, wastewater treatment plant trickling beds, or in the alimentary canal of mammals (Costerton et al., 1981). Biofilms are not, however, confined to solid/liquid interfaces and can also be found at solid/air or liquid/liquid interfaces. Airborne pathogens and deteriogens have been shown to be important factors in the biodeterioration of surface coatings (Lloyd, 1987). Biofilms at liquid/liquid interfaces have been implicated in hydrocarbon degradation, including fuels, oils, and industrial coolants (Percival et al., 2011). Within the food industry, biofilms can occur on surfaces contacting with or without foods (Kumar & Anand, 1998; Zottola & Sasahara, 1994). Food and food processing environments are the best sites for microbial attachment and biofilm formation. Pathogenic microorganisms can attach to food surfaces, grow on them, and form a biofilm that causes an increase in food safety risk (Hoveida et al., 2019). Biofilms are responsible for about 60% of foodborne outbreaks (Han et al., 2017). Therefore, the presence of biofilms in food processing environments poses a significant risk to food safety and the food industry (Galie et al., 2018). In the food processing environments, contaminants mostly come from the surrounding air, equipment, or food surfaces (Kumar & Anand, 1998). Then, biofilms growing in food processing environments may lead to spoilage of food, which in turn can cause serious public health risks to consumers and serious economic consequences (Coughlan et al., 2016; Galie et al., 2018). In this section, we discussed the occurrence of biofilms in the context of food contact surfaces and within food products.

24.3.1 Biofilm on food contact surfaces In research conducted on Cronobacter sakazakii, it has been reported that this bacterium is able to adhere to different surfaces such as silicon, latex, polycarbonate, stainless steel, glass, and polyvinyl chloride (PVC). Biofilm formation on stainless steel surfaces of food processing plants, leading to foodborne illness outbreaks, is enabled by the attachment and confinement of pathogens within microscale cavities of surface roughness (grooves, scratches) (Awad et al., 2018). The attachment of microorganisms on the food preparation surface could enable microorganisms to form biofilm and become a source of contamination (Iversen et al., 2004). Poor sanitation of food contact surfaces, equipment, and processing environments has been a contributing factor in foodborne disease outbreaks, especially those involving Listeria monocytogenes and Salmonella (Lindsay & Von Holy, 2006). Insufficient and ineffective cleaning practices can cause food residues to remain in food processing and can facilitate bacterial attachment and biofilm formation (Flemming et al., 2016). These surfaces with adherent microbial communities are difficult to sanitize properly since cells within a biofilm are persistent or tolerant to hygienic conditions (Wang, 2019). The production of biofilm and its persistence on different surfaces related to food, medical, and other sectors would be reservoirs for many pathogens that are infectious (Galie et al., 2018). Bacillus cereus is responsible for biofilm formation on food contact surfaces, such as stainless steel pipes, conveyor belts, and storage tanks (Grigore-Gurgu et al., 2019). There are reports indicating that although EHEC can form biofilms on different food industry surfaces, neither an effective means to prevent EHEC biofilm formation nor an

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effective treatment for its infections exists because antibiotic treatment tends to increase the risk of hemolytic uremic syndrome and kidney failure (Lee et al., 2007). Pseudomonas is a heterotrophic, motile, gram-negative rod-shaped bacterium. Pseudomonads are generally ubiquitous psychrotrophic spoilage organisms that are often found in food processing environments, including floors and drains, and also on fruit, vegetables, and meat surfaces, and in low-acid dairy products (Chmielewski & Frank, 2003; Gonza´lez-Rivas et al., 2018). Pseudomonas spp. produce huge amounts of EPS and are known to attach and form biofilms on stainless steel surfaces. Anoxybacillus flavithermus is another gram-positive, thermophilic, and spore-forming organism that is facultatively anaerobic and nonpathogenic (Strejc et al., 2020). A. flavithermus is a potential contaminant of dairy products and poses a problem for the milk powder processing industry, as high levels will reduce milk powder acceptability for both local and international markets (Murphy et al., 1999). A. flavithermus spores are very heat-resistant, and their vegetative cells can grow at temperatures up to 65 C with a significant increase in bacterial adhesion on stainless steel surfaces in the presence of skimmed milk. This indicates that milk positively influences these species’ biofilm formation (Sadiq et al., 2017). A combination of several pathogens can synergistically interact to form biofilms in the food industry. In food processing environments, bacteria are able to exist as multispecies biofilms, from where both spoilage and pathogenic bacteria can contaminate food (Sterniˇsa et al., 2019). In food industries, biofilm-related effects (pathogenicity, corrosion of metal surfaces, and alteration to organoleptic properties due to the secretion of proteases or lipases) are critically important. For example, in the dairy industry several processes and structures (pipelines, raw milk tanks, butter centrifuges, pasteurizers, cheese tanks, packing tools) can act as surface substrates for biofilm formation at different temperatures and involve several mixed colonizing species (Galie et al., 2018).

24.3.2 Biofilms in food products Food is rich in nutrients and suitable for the growth and reproduction of pathogens. Bacteria exist in food in two ways: bacteria can be suspended in liquid food, usually living planktonically; in solid or viscous food, bacteria can easily adhere to the surface of food materials, food processing equipment, and the surface of pipelines and can eventually form a bacterial biofilm (Nitschke et al., 2009). Generally, the growth of pathogenic bacteria such as Escherichia coli O157:H7 and Salmonella enterica can result in cross-contamination from food processing surfaces to food products (Jun et al., 2010). E. coli is a gram-negative and rod-shaped bacterium. Most E. coli strains form part of human intestinal microbiota and pose no health problem. However, the virulence types of E. coli include enterotoxigenic (ETEC), enteroinvasive (EIEC), enteropathogenic (EPEC), and Vero cytotoxigenic (VTEC). O157:H7 EHEC is the most frequent serotype associated with EHEC infections in humans in the United States (Gould et al., 2013). Widespread E. coli dissemination in natural environments is, to a great extent, due to its ability to grow as a biofilm. It is worth considering that several E. coli strains may cause disease in humans and that Enterohemorrhagic E. coli (EHEC) strains are the most relevant for the food industry. EHEC serotype O157:H7 is the human pathogen responsible for bloody diarrhea outbreaks and hemolytic uremic syndrome (HUS) worldwide. They can be transmitted by raw milk, drinking water or fresh meat, fruit, and vegetables, for example, melons, tomatoes, parsley, coriander, spinach, lettuce, etc. (Galie et al., 2018). L. monocytogenes is a gram-positive bacterium and a ubiquitous foodborne pathogen that can appear in soil, food, and water. Its ingestion can result in abortions in pregnant women and other serious complications in the elderly and children. The pathogen can be transmitted to several food types, such as dairy products, seafood, meat, fruit, ready-toeat meals, ice cream, soft cheeses, unpasteurized milk, frozen vegetables, candied apples, and poultry (Rothrock et al., 2017), but it is not known to be resistant to pasteurization treatments. The pathogen proliferates at low temperature and is able to form pure culture biofilms or grow in multispecies biofilms (Milillo et al., 2012). L. monocytogenes can survive under acidic conditions for lengthy periods and can form biofilms that grow without oxygen. Its numbers are likely to rise or lower in biofilms depending on the competing microbes present (Chmielewski & Frank, 2003). Poultry meat is a frequent reservoir for these bacteria in processed food, whose importance as a food pathogen has been demonstrated by the fact that S. enterica biofilm formation on food surfaces was the first reported case in 1966 to possess complex multicellular structures (Duguid et al., 1966). Pseudomonas is a heterotrophic, motile, gram-negative rod-shaped bacterium. Pseudomonads are generally ubiquitous psychrotrophic spoilage organisms that are often found in food processing environments, including floors and

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drains, and also on fruit, vegetables, and meat surfaces, and in low-acid dairy products (Chmielewski & Frank, 2003; Gonza´lez-Rivas et al., 2018).

24.4

Phytochemicals in biofilm inhibition

The majority of current infectious diseases are almost untreatable by conventional antibiotic therapy given the advent of multidrug-resistant bacteria. The degree of severity and the persistence of infections are worsened when microorganisms form biofilms. Therefore, efforts are being applied to develop new drugs not as vulnerable as the current ones to bacterial resistance mechanisms and are also able to target bacteria in biofilms. Natural products, especially those obtained from plants, have proven to be outstanding compounds with unique properties, making them perfect candidates for these much-needed therapeutics (Borges et al., 2016). The conventional physical and chemical methods used for treating biofilms are not performed as expected and also create environmental pollution. The increased resistance of biofilm against antimicrobials and the host immune system imposed the need for new strategies (Rabin et al., 2015). Biological control of biofilms uses certain mechanisms (from living matter, microorganisms, or microbes within the biofilm itself) in order to interfere with their existence (Gule et al., 2016). This is mainly used to target the QS, degrade the extracellular matrix, inhibit microbial adherence, and eliminate persister cells. Various natural compounds derived from plants, fungi, bacteria, and other animals are of great interest in drug discovery due to their enormous advantages (Borges et al., 2015). The misuse of antibiotics also contributed to the development of drug resistance, which might aggravate the bacteria-infected disease. Thus, novel strategies other than antibiotics should be developed to combat the bacterial and biofilm formation. In the last two decades, novel approaches in preventing biofilm formation and QS have been widely developed and reported including natural products from plants. Many plant natural products have demonstrated antimicrobial and chemopreventive properties (Tan & Vanitha, 2004). It is well known that herbal remedies are employed by different human cultures for centuries, and some of those natural products are essential for prevention and treatment (Lau & Plotkin, 2013).

24.4.1 Phytochemicals associated with biofilm inhibition As mentioned, many of the antimicrobial drugs used to effectively treat human diseases have been derived from nature (Newman & Cragg, 2007). The interest in antimicrobials derived from natural sources has increased in the last years due to the fact that many antibiotics had become ineffective in the treatment of microbial infections. The accepted safe status of some of these compounds, associated with lower adverse effects and reduced cost compared with folk pharmaceuticals, was also an encouraging factor (Cowan, 1999; Lin et al., 2000). However, the attention to natural products is not new. On the contrary, this interest remains for centuries with the use of plant extract for the treatment of some diseases, in traditional medicine. In this context, it is known that some dietary phytochemicals, such as essential oils (EO), phenolics, glucosinolates (GLS), and their hydrolysis products, have a wide range of effects on health, preventing the risk of some diseases (Holst & Williamson, 2004; Prior & Cao, 2000; Teixeira et al., 2013). These properties include antibacterial, antiviral, antioxidant, anti-inflammatory, antiallergic, and anticarcinogenic activities, hepatoprotective and antithrombotic effects, and vasodilatory action (Bakkali et al., 2008; Saavedra et al., 2010; Shin et al., 2004; Soobrattee et al., 2005; Srinivasan et al., 2007). Currently, several researchers were able to identify improved strategies for biofilm control. Thus, considering the numerous therapeutic properties of dietary phytochemicals, and the fact that these compounds are thought to be an integral part of both human and animal diets, it is important to study their activity against bacterial biofilms. In fact, as previously demonstrated by some authors, phytochemicals could represent a natural antimicrobial strategy with a significant impact not only against planktonic bacteria but also on bacterial biofilm formation and development. A few phytochemical studies with the potential for anti-biofilm activity along with their mechanism of action in the prevention and control of biofilm are summarized in Table 24.3.

24.4.1.1 Essential oils Essential oils (EOs) are complex mixtures of a large and diverse class of terpenoid and phenolic compounds with strong odor that are synthesized in several plant organs and exhibit broad antibacterial, antiparasitic, antifungal, and antiviral properties (Pandey et al., 2017; Tariq et al., 2019). These volatile compounds play an important role in the protection of plants, against microorganisms (Bakkali et al., 2008). The biological activity of herbs and spices, and particularly their

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TABLE 24.3 Phytochemicals for biofilm prevention and control with mechanism of action.

Essential oils (EO)

Phenolics

Phytochemical/extract

Biofilm prevention and control mechanism

References

Oregano oil (carvacrol), Thyme oil (thymol)

The absence of extracellular polymeric matrix (EPM), reduction in cellular density, and alteration in the surface morphology of Cryptococcus biofilms cells; Inhibits planktonic and sessile cells of S. aureus isolated from food contact surfaces

dos Santos Rodrigues et al. (2017), Kumari et al. (2017)

Mentha suaveolens ssp. Insularis (cis-cis-pmenthenolide)

Inhibition of violacein Production and biofilm formation related to a disruption in the QS mechanism.

Poli et al. (2018)

Cinnamomum and its derivatives (cinnamaldehyde)

Inhibiting flagella protein synthesis and swarming motility, suppressing bacterial attachment, colonization, and biofilm formation in an early stage

Didehdar et al. (2022)

Eugenol

Inhibits biofilm formation, disrupted the cell-to-cell connections, detachment of existing biofilms, and killed the bacteria in biofilms of both methicillin-resistant and methicillin-sensitive strains of S. aureus

Yadav et al. (2015)

Cinnamon oil, coriander essential oil, and its major compound linalool

Inhibits Pseudomonas aeruginosa PAO1 biofilms by hindering the QS process; Antibacterial, anti-biofilm, antiQS, and antioxidant potentials

Duarte et al. (2016), Kalia et al. (2015)

Adiantum philippense crude extract

Effective against inhibition of biofilms of bacteria via hampering the production of EPS

Adnan et al. (2020)

(2)-Epigallocatechin gallate

Inhibited S. aureus biofilm formation

Slobodnı´kova´ et al. (2016)

Ellagic acid and tannic acid

Inhibited biofilm formation of E. coli

Curcumin

Attenuation of virulence in P. aeruginosa

Rudrappa and Bais (2008)

Piper betle ethyl acetate extract and its active metabolite phytol

Inhibits the swarming Motility and hydrophobicity Downregulates QS genes to inhibit biofilm of Serratia marcescens

Srinivasan et al. (2016)

Naringenin

Decrease in cell surface hydrophobicity and exopolysaccharide production in S. aureus biofilm

Wen et al. (2021)

(Continued )

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Recent Frontiers of Phytochemicals

TABLE 24.3 (Continued)

Isothiocyanates (ITCs)

Phytochemical/extract

Biofilm prevention and control mechanism

References

Allyl isothiocyanate, benzyl isothiocyanate, and phenylethyl isothiocyanate extracted from nasturtium and horseradish

Inhibited biofilm formation and metabolic activity in mature biofilms of P. aeruginosa

Kaiser et al. (2017)

Sulforaphane, erucin

Antagonists of transcriptional activator of LasR and inhibition of biofilm formation in P. aeruginosa

Tan et al. (2020)

Iberin

Interference of rhamnolipid production and gene expression of lasB and rhlA in P. aeruginosa

Allicin and ajoene from garlic

Decreases the bacterial adhesion in the initial stages of biofilm formation of P. aeruginosa as it reduces EPS formation and interferes with the QS system; Downregulates rhamnolipid production

Mishra et al. (2020)

medicinal and antimicrobial properties, is due for the most part to EO (Gershenzon, 1991). Food and Drug Administration (FDA) generally considers that many individual components of the EO are safe, which has allowed its use in multiple applications in medical, pharmaceutical, food, cosmetic, and health industry (Bakkali et al., 2008; Swamy et al., 2016). Essential oil and their active components (EO-ACs) have shown to possess a variety of biological and pharmacological properties. Kumari et al. evaluated the effect of six EO-ACs sourced from oregano oil (carvacrol), cinnamon oil (cinnamaldehyde), lemongrass oil (citral), clove oil (eugenol), peppermint oil (menthol), and thyme oil (thymol) against three infectious forms: planktonic cells, biofilm formation, and preformed biofilm of Cryptococcus neoformans and Cryptococcus laurentii. The three most potent EO-ACs, thymol, carvacrol, and citral, showed excellent anti-biofilm activity at a much lower concentration against C. laurentii in comparison to C. neoformans. The effect revealed the absence of extracellular polymeric matrix (EPM), reduction in cellular density, and alteration in the surface morphology of biofilm cells (Kumari et al., 2017). In another study, essential oils from Origanum vulgare L. oregano (OVEO) and carvacrol (CAR) showed efficacy to inhibit the growth of planktonic as well as sessile cells of selected Staphylococcus aureus isolates at higher amounts for later. Also, these are effective at inhibiting biofilm formation by S. aureus on stainless steel surfaces (dos Santos Rodrigues et al., 2017). The anti-quorum sensing activity of 12 essential oils on Chromobacterium violaceum was investigated with special emphasis on cis-cis-p-menthenolide extracted and isolated from a plant endemic to the occidental Mediterranean Sea islands, Mentha suaveolens ssp. insularis, found to act as an inhibitor of violacein production and biofilm formation which might be to a disruption in the QS mechanism (Poli et al., 2018). The biological activities of EO are related to their chemical composition and functional groups. Moreover, the power of essential oils is connected with their main constituents. The oils containing phenols, such as thymol, carvacrol, and eugenol, exhibit the most pronounced activity against diverse microorganisms (Kalemba and Kunicka, 2003). In a study performed by Yadav et al., eugenol exhibits notable activity against methicillin-resistant and methicillin-sensitive S. aureus (MRSA and MSSA) clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may be used to control or eradicate S. aureus biofilm-related infections (Yadav et al., 2015). Furthermore, the activity of cinnamon oil was also tested against Pseudomonas aeruginosa PAO1 and revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric

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substances (Kalia et al., 2015). Similarly, coriander essential oil and its major compound linalool showed antibacterial, anti-biofilm, anti-QS, and antioxidant potentials (Duarte et al., 2016) and Cinnamomum along with its derivatives (cinnamaldehyde) inhibiting flagella protein synthesis and swarming motility suppresses bacterial attachment, colonization, and biofilm formation in an early stage (Didehdar et al., 2022).

24.4.1.2 Phenolics Plant polyphenols represent a large class of biologically active secondary metabolites of plants. They include flavonoids, tannins, anthocyanins, phenolic acids, stilbenes, coumarins, lignans, and lignins (Pereira et al., 2009). These substances play an important role in resistance against various microbial pathogens and protect against free radicals and toxins (Daglia, 2012; Quideau et al., 2011). Phenolics can be found in diverse dietary products, such as vegetables, fruits, chocolates, and beverages (Soobrattee et al., 2005; Rodriguez et al., 2011). Phenolics are considered potential therapeutic agents against a wide range of ailments including neurodegenerative diseases, cancer, diabetes, cardiovascular dysfunctions, inflammatory diseases, and also against aging (Soobrattee et al., 2005; Srinivasan et al., 2007; Jayaraman et al., 2010). Therefore, foods containing phenolics are becoming an important part of diets due to their biological effects, mainly antioxidant potential and also antimicrobial properties. Adiantum philippense crude extract was screened for its phytochemical constituents, antagonistic potential, and effect on bacterial adhesion and biofilm formation against common food pathogens, viz., E. coli, S. aureus, Shigella flexneri, and P. aeruginosa by Adnan et al. The findings represent the bioactivity and potency of A. philippense crude extract against food pathogens not only in their planktonic forms but also against/in biofilms and also correlated these findings with the possible mechanism of biofilm inhibition via targeting adhesin proteins (Adnan et al., 2020). Slobodnı´kova´ et al. reviewed plant polyphenols from which (2)-epigallocatechin gallate and gallic acid have shown inhibition of biofilm formation of S. aureus and E. coli, respectively. The major constituent of turmeric (Curcuma longa L.) roots/rhizomes is the curcumin-inhibited diverse virulence factors in P. aeruginosa, specifically the elastase, protease, and pyocyanin production without affecting bacterial growth in a dose-dependent manner (Rudrappa & Bais, 2008). The anti-QS efficacy of the Piper betle ethyl acetate extract (PBE) and its active metabolite phytol was evaluated against Serratia marcescens and demonstrates the promising anti-QS and anti-biofilm activities. In addition, PBE effectively inhibited the hydrophobicity and swarming motility (Srinivasan et al., 2016). In another study, Wen et al. investigated the anti-biofilm formation properties of the citrus flavonoid naringenin on the S. aureus ATCC 6538 (S. aureus) using subminimum inhibitory concentrations (sub-MICs) of 5B60 mg/L. The study revealed that the thick coating of S. aureus biofilms became thinner and finally separated into individual colonies when exposed to naringenin. The decreased biofilm formation of S. aureus cells may be due to a decrease in cell surface hydrophobicity and exopolysaccharide production, which is involved in the adherence or maturation of biofilms (Wen et al., 2021).

24.4.1.3 Isothiocyanates Isothiocyanates (ITCs) are bioactive products resulting from enzymatic hydrolysis of glucosinolates (GLs), the most abundant secondary metabolites in the botanical order Brassicales, and have the antimicrobial activity against foodborne and plant pathogens (Romeo et al., 2018). Cruciferous plants (family Cruciferae), including cabbage, broccoli, horseradish, brussel sprouts, and mustard, exhibit excellent chemotherapeutic properties (Wilson et al., 2013; Wu et al., 2009) due to the presence of isothiocyanates (ITCs). ITCs are the compounds synthesized from glucosinolates through the hydrolytic action of the enzyme myrosinase present in the plants (Davaatseren et al., 2014). ITCs are known to prevent the biofilm formation by pathogenic bacteria like E. coli, P. aeruginosa, S. aureus, and L. monocytogenes (Abreu et al., 2014). The antimicrobial target compounds, for example, allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), or phenylethyl isothiocyanate (PEITC), are released mostly by enzyme-mediated hydrolysis of the plant-derived glucosinolates (GLs) furnishing ITCs with the substitution pattern of amino acids in their side chain (Halkier & Gershenzon, 2006). Similar compounds found in plants such as nasturtium (Tropaeolum majus) and horseradish (Armoracia rusticana) inhibited biofilm formation and metabolic activity in mature biofilms of P. aeruginosa (Kaiser et al., 2017). Some isothiocyanates, that is, sulforaphane, erucin, and iberin, are reviewed by Tan et al. which show quorum-sensing (QS) activities like antagonists of transcriptional activator of LasR and inhibition of biofilm formation in P. aeruginosa and interference of rhamnolipid production and gene expression of lasB and rhlA in P. aeruginosa, respectively, (Tan et al., 2020). Furthermore, in another review Mishra et al. summarized plant-based antimicrobials with the potential of antibiofilm activity including allicin and ajoene from garlic which showed a decrease in the bacterial adhesion at the initial stages of biofilm formation of P. aeruginosa as it reduces EPS formation and downregulates rhamnolipid production, respectively, (Mishra et al., 2020).

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24.4.2 Mode of action of phytochemicals on biofilm 24.4.2.1 Phytochemicals as quorum-sensing inhibitors An intercellular communication which increases the virulence/pathogenicity of the bacteria is quorum sensing (QS). Empowerment and pathogenicity of bacteria against antibacterial compounds to coordinate phenotype, genotype, and direct physiological activities are by QS in bacteria. They cover, among others, the establishment of structured microbial communities like biofilms, the production of virulence factors, and also secondary metabolites. Certainly, QS systems are synchronized into certain actions essential for biofilm development and differentiation (Jakobsen et al., 2012; Borges et al., 2014, 2015). QS inhibitors are moreover a potential component to surpass the restraints of antibiotics usage to treat biofilm infections. The benefit of not suppressing the cells in their course makes it amicable for disease control and therefore will not apply selective pressure to develop resistance (Qian et al., 2013). The pathways of QS signaling interference can avoid initial biofilm development by modifying its progress by inhibiting the secretion of cellular appendages and adhesins, which affect the development of microcolonies, surface adhesion, bacterial motility, cell auto, and coaggregation and restrict the EPS production. QS inhibition has been found in natural products mainly phytochemicals in the screening tests (Borges et al., 2015). Phytochemicals offer enormous biological activity and chemical diversity with structural complexity and have been perceived as a huge and appealing vault of QS inhibition (Borges et al., 2015; Vattem et al., 2007). In fact, they are similar to what is considered an “ideal” QS inhibition, which has been profoundly effective, chemically stable, molecules of low molecular weight, and being safe to health. Hence, in an era where the effectiveness of the antibiotics is no longer definite, phytochemicals with QS inhibition activity are often gifted measures to support the treatment of bacterial infections (Kalia, 2013; Rasmussen & Givskov, 2006).

24.4.2.2 Phytochemicals as biofilm metal chelators Metal chelator ions like iron, zinc, calcium, copper, magnesium, and manganese are engaged with some natural procedures vital for the development and existence of the microorganisms in their personal atmosphere. All the metals and iron precisely play in pathogenesis and virulence (Porcheron et al., 2013), and as they function as signaling factors, they have also been related to the formation of biofilm (Banin et al., 2005). The primitive phases of biofilm formation like attachment of the cells and microcolony formation constantly require iron sensing which was evidenced by many investigators, as they are essential for the growth, development, and adherence of bacterial cell (O’Toole & Kolter, 1998; Singh et al., 2002). Among plant-derived compounds such as phenolic acids, polyphenols, and flavonoids, the 6, 7-dihydroxy iron having chelating properties and chelation sites are exceptionally compelling in flavonoids. Despite the extraordinary capability of phytochemicals to take up metals, the investigation of their efficacy to treat biofilm-based infections remains to be scarce (Mladˇenka et al., 2011; Jayasinghe et al., 2015; Hatcher et al., 2009). Metal chelating is one among the quinolones testing, as required an equivalent structure to nitroxoline, which is an antibiotic with known antimicrobial and anti-biofilm activities, and known to be associated to chelate several metals (Abouelhassan et al., 2014).

24.4.2.3 Phytochemicals as biofilm efflux pump inhibitors At present day, increasing proof has simplified those systems of efflux pumps which are not just pumps for the transportation of drugs or additional toxic substances out of the cells but have been furthermore required for QS regulation and the ensuing articulation of genes liable for virulency and biofilm formation (Chan & Chua, 2005). Certainly, biofilm formation studies showed this linkage, citing the presence of the upregulation of genes encoding efflux pumps (EPs) (Sun et al., 2014; Soto, 2013; Van Acker & Coenye, 2016; Gupta et al., 2014). Application of EPs is often a vital measure to manage biofilm formation and to diminish biofilm tolerance to antibiotics due to their remarkably dynamic nature (Kvist et al., 2008; Baugh et al., 2014).

24.4.3 Target areas of phytochemicals 24.4.3.1 Preventing microbial adhesion Various factors like pH, ionic strength, temperature, nutrients, genotype, and phenotype of microorganism influence the method of adhesion. The bacterial adhesion mainly depends on the charge, hydrophobicity, presence of adhesion components like flagella, fimbriae, and pili and therefore the EPS structure of microorganism (Donlan & Costerton, 2002).

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The surface property of the fabric on which biofilm is made also plays a vital role in its formation (Gro¨ßner-Schreiber et al., 2009).

24.4.3.2 Control of cellular motility Bacteria show various styles of movements like swimming, swarming, gliding, etc., and these movements play a vital role in biofilm formations. Varying results were observed by different phytochemicals on cellular motility during the different duration of your time. The swarming and swimming motility of P. aeruginosa, Proteus mirabilis, and bacteria species was decreased by methanolic extracts of herb Cuminum cyminum-48. However, cinnamaldehyde and eugenol from laurel decreased the swimming motility of E. coli (Niu & Gilbert, 2004).

24.4.3.3 Change in bacterial static properties The bacterial static property against phytochemicals proves to be helpful in controlling their effects when bacteria were found successful in forming a biofilm. The MIC and MBI values of phytochemicals were needed to be established. The MIC and MBI values for gram-positive bacteria are usually lesser than for gram-negative bacteria (Ta & Arnason, 2015). The morphology of the S. aureus and E. coli cells in biofilm changed when observed after treatment with phytochemicals (essential oils). Reduction in cell size, length, and diameter was observed, the peptidoglycan structure of cell wall gets disrupted, and cell contents leak out and eventually lead to cell death (Simo˜es et al., 2008). Gallic (hydroxybenzoic acid) and ferulic acids (hydroxycinnamic acid) were also tested for their antimicrobial activities against S. aureus and E. coli (Ta & Arnason, 2015).

24.5

Conclusion

Biofilm development in the food industry environment is a major concern because of its potential for food contamination from biofilms. The data available show that microbial species have the ability to form biofilms on various biotic as well as abiotic surfaces in varying environmental conditions. The strategies must be used to inhibit the biofilm formation instead of eradicating once it is formed to avoid the resulting cost and risk to public health. The potential of phytochemicals as effective, inexpensive, and safe natural agents for inhibition of biofilm formation opens up new prospects for controlling biofilm development in various sectors. The efficacy of its use was found superior to other chemicals without increasing environmental pollution and antibiotic resistance of microorganisms. In the future, the active ingredients of more plants or waste by-products of agro-industry should be identified and purified, and these should be used synergistically with other safe methods for the betterment of public health.

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Chapter 25

Novel perspectives on phytochemicalsbased approaches for mitigation of biofilms in ESKAPE pathogens: recent trends and future avenues Subhaswaraj Pattnaik1, Monika Mishra1, Harvinder Singh2 and Pradeep Kumar Naik1 1

Centre of Excellence in Natural Products and Therapeutics, Department of Biotechnology and Bioinformatics, Sambalpur University, Jyoti Vihar,

Sambalpur, Odisha, India, 2Leaders Institute, Woolloongabba, Queensland, Australia

25.1

Introduction

No doubt, the recent development in pharmaceutical sectors has revolutionized our efforts to design and synthesize potential drug molecules from different resources against several health ailments including microbial infections. On contrary, pathogenic microorganisms also undergo gradual physiological, phenotypic, and genotypic transformations to tackle the relative environmental stress associated with the regular and irrational use of antibiotics. Among the several adaptations followed by the pathogenic microorganisms in response to stress conditions, the formation of an extracellular biofilm matrix is considered as a viable opportunity for the residing microflora to explore several heterogeneous microenvironments within the matrix and could bypass the adverse environmental conditions including antibiotic stress (Schlafer & Meyer, 2017). The biofilm formation and development in pathogenic microorganisms are considered as a protective barrier not only against several environmental stresses such as altered pH, salinity, osmolarity, temperature, nutritional availability, and mechanical forces but also provide a protective response against antibiotic treatment and host cell immunity. Hence, understanding the biofilm architecture and its efficient management is considered therapeutically relevant in drug discovery pipelines (Sharma et al., 2019; Zhang, Chen, et al., 2019).

25.1.1 An introduction to biofilm and historical perspectives Microbial biofilms, as discussed earlier, are an adaptation of microbial community toward stress environments by aggregation of microbial communities where the microorganisms tend to produce a matrix of extracellular polymeric substances (EPS) and encase themselves within the self-produced matrix (Flemming et al., 2016). The formed polymeric matrix not only functions as a protective barrier against stress conditions but also allows the attachment of sessile microbiota to several biotic and abiotic surfaces. Within the biofilm matrix, the sessile microflora resides in heterogeneous microenvironments and undergoes several adaptations including modified phenotype, altered physiological responses, and transcriptional regulations (Donlan & Costerton, 2002; Lazar, 2011). From a historical perspective, the concept of biofilm was described for the very first time in 1683. Meanwhile, the etiological role of biofilms in persistent infections was studied by Nils Hoiby way back in the 1970s. The term “biofilm” was first coined by Bill Costerton in 1978 whereas the descriptive report pertaining to empirical features of bacterial biofilms was reported in the early phases of the 2000s (Chandki et al., 2011). As per recent studies, it was evident that approximately 80% of bacteria in the natural environment have the ability to form biofilms (Penesyan et al., 2021). Interestingly, biofilm formation not only occurs under a natural suitable environment but also under several harsh environmental conditions in terms of extensive variations in temperature, pH, salinity, pressure, nutrient availability, and other topographic factors (Shakibaie, 2018). Biofilm mode of lifestyle is considered as one of the Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00005-0 © 2023 Elsevier Inc. All rights reserved.

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most predominant and successful modes of microbial life owing to their incidences in fossilized form for several billion years (Yin et al., 2019). Owing to the importance of biofilms in clinical and healthcare settings, biofilms are categorized as a potential biomarker in understanding the etiology of bacterial infections and their ability to abolish the role of conventional antibiotics in the management of chronic microbial infections (Vestby et al., 2020). The sessile microbial community tends to form biofilms on several biotic (e.g., tissues of the human host) and abiotic (e.g., glass, polystyrene plates, biomedical instruments) surfaces and form the foundations of chronic microbial infections and several other hospital-acquired health ailments thereby creating an arduous challenge to the current antimicrobial therapy (Mu¨sken et al., 2010). In particular, opportunistic nosocomial pathogens with special reference to the ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) have the ability to develop persistent infections by virtue of their ability to form biofilms. It is evident from earlier research works that the nosocomial pathogens with the ability to form biofilms have a higher tendency to exhibit resistance against antibiotics as compared to their planktonic counterparts (Mah et al., 2003; Whiteley et al., 2001). The formation of recalcitrant biofilm communities involves a series of dynamic events where the EPSs forming the biofilm matrix play a pivotal role in emerging chronic infections by providing mechanical stability as well as enhancing the tolerance towards antimicrobial agents. The EPS is mainly composed of exopolysaccharides, proteins, nucleic acids (i.e., extracellular DNA), lipids, etc. (Karygianni et al., 2020). An integral part of any biofilm community is the presence of water channels which forms the circulatory system within the biofilm matrix for the efficient distribution of nutrients to the encased microcolonies and thus facilitates the rapid growth of microorganisms within the matrix (Choudhury et al., n.d.).

25.1.2 An insight into the process of biofilm formation The formation of biofilm is no doubt a rapid but complex process with the involvement of several genetical deviations, physiological processes, metabolic alterations, phenotypic variations, and physicochemical factors. However, the process involves a simple cascade of sequential events starting from (1) initial attachment to the biotic/abiotic surfaces, (2) microcolony formation, (3) establishment of biofilm matrix, (4) maturation of biofilm matrix, and (5) dispersion of biofilms (Jamal et al., 2018; Masa´k et al., 2014). In the first step, microbial cells tend to attach to several biotic or abiotic surfaces using their surface appendages (i.e., flagella and pili) and other physical forces such as electrostatic interactions and Van der Waals forces. Several other factors such as adhesion-associated proteins and hydrophobicity profiles greatly enhance the attachment of microbes to different surfaces. In the subsequent step, the microflora forms an irreversible attachment to the surface by forming microcolonies within the EPS. Several microcolonies correlate with each other and in return gained positive responses in terms of substrate exchange, metabolic product distribution, and other physiological events. In the next step, called the proliferation stage, bacterial cells tend to secrete EPS, which provides a suitable environment for the bacterial cells to communicate with each other through a specific chemical signaling network for the production of several virulence factors, cytotoxic elements, and other important elements responsible for resistance against antibiotics. In the final stage, under a nutrient depletion state, the sessile bacterial cells transformed into motile planktonic forms which further initiate the process of attachment and colonize other surfaces for further initiating the biofilm infection cycle (Jamal et al., 2018; Muhammad et al., 2020). The process of biofilm dispersion is inherently associated with low levels of c-di-GMP, modification of surface adhesion properties, enzymatic degradation of polysaccharides, and disassembling of polymeric matrix (Rumbaugh & Sauer, 2020).

25.1.3 Ultrastructure of biofilm communities The ultrastructure of bacterial biofilms is mainly composed of secreted polymeric matrix called EPS which is made up of exopolysaccharides, proteins, and lipids. In the biofilm matrix, exopolysaccharides form an extensive structural network with carbohydrates like glucose, galactose, and mannose which are the most abundant carbohydrates. The diversity of carbohydrates in biofilm matrix and their production are highly dependent upon several environmental stress conditions. The extracellular proteins and their association with exopolysaccharides are important in the maintenance of biofilm architecture and its stabilization. Apart from these components, the secretion of extracellular DNA (eDNA) also greatly facilitates the formation and development of a biofilm matrix (Rabin et al., 2015). The contribution of each of the components of EPS in biofilm formation and development is spatially different among several species of the same bacterial genus as well as among the different bacterial strains (Okuda et al., 2018). The biofilm architecture forms the basis for highly dynamic and complex biological processes of the embedded heterogenic bacterial population which ultimately control the several pathophysiological events in bacterial pathogenicity. For example, prior to biofilm maturation, a highly specific and cell density-dependent chemical signaling network termed “quorum sensing” (QS)

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functions to communicate among the embedded bacterial population (both intraspecies as well as interspecies communication) for survival during stress and production of several virulence phenotypes responsible for chronic infections (Schilcher & Horswill, 2020).

25.1.4 Impact of bacterial biofilm The biofilm mode of lifestyle has enabled the residing bacterial community to behave socially and function cordially similar to that of differentiated cells in multicellular organisms. Thus, biofilm formation positively controls the infection modulatory behaviors of the embedded bacterial community (Penesyan et al., 2021). The social and collective behavior within the biofilm matrix, however, allows the tendency of nosocomial pathogens to form recalcitrant biofilms resulting in the occurrence of several chronic infections, including cystic fibrosis, chronic pneumonia, periodontitis, and other hospitalacquired infections. As per recent reports, biofilms are generally associated with several life-threatening diseases associated with different organs of the human body. For example, otitis media (auditory organs), atherosclerosis (cardiovascular system), wound infections (skin, integumentary system), cystic fibrosis (respiratory system), bacterial vaginosis (reproductive system), urinary tract infections (urinary system), etc., are associated with biofilm communities and create a global health issue (Vestby et al., 2020). Several bacterial species also harbor the oral environment and also have the tendency to form biofilms on the oral microenvironment (i.e., dental plaques by Streptococcus mutans) and critically affect oral health by producing dental caries and periodontal diseases (Abebe, 2021). As per estimates, biofilm-associated chronic infections result in more than 0.5 million deaths annually across the globe which ultimately results in a decrease in the global economy (Banerjee et al., 2020). Moreover, the biofilms also result in varied infections associated with indwelling biomedical devices such as catheters, heart valves, orthopedic devices, dental implants, peritoneal dialysis catheters, surgical soft tissue prostheses, and contact lenses thus inferring several consequences in terms of health as well as the economy (Borges et al., 2016; Stoica et al., 2017). Thus, biofilms on implanted medical devices are the root cause of the majority of hospital-acquired infections leading to several life-threatening conditions (Dufour et al., 2010). Food industries and food processing sectors are highly prone to the growth and attachment of pathogenic microorganisms and initiate the process of biofilms. Hence, the incidence of biofilm communities on food surfaces and the devices associated with food processing units result in an increased incidence of food safety risks, health ailments, and severe economic loss. Since biofilm modes of lifestyle are difficult to handle using conventional therapeutics and disruption strategies; the development of biofilms in food-based industries has resulted in severe disease outbreaks associated with foodborne pathogenic bacteria (Abebe, 2020). For example, biofilm-forming Bacillus cereus has the ability to withstand the industrial pasteurization process by forming endospores, and thus, it is difficult to remove this bacteria using conventional microbial cleaning approaches. In addition, some bacterial strains under severe stress have the ability to produce several types of diarrheal enterotoxins which ultimately result in the occurrence of diarrhea and food poisoning (Galie et al., 2018).

25.2

Biofilm-mediated drug resistance in ESKAPE pathogens

In the last few decades, antimicrobial resistance (AMR) became a global health issue with an annual death of 0.7 million people across the globe. As per the estimation of the WHO, if we fail to tackle the AMR phenomena, it is expected to cause 10 million deaths by 2050 with a global burden of USD 100 trillion. Among the several factors responsible for the occurrence of AMR, the ability of pathogenic microorganisms to form protective measures in the form of biofilms gained recent recognition. The biofilm formation provides an aided advantage to the bacterial pathogens to exhibit collective recalcitrance for withstanding the wrath of high concentrations of antibiotics. For example, about 80% of chronic and persistent microbial infections are directly associated with biofilm dynamics, which ultimately bypass the antibiotic treatment regimens (Urue´n et al., 2021). Within the biofilm matrix, the gradients in the distribution of nutrients, osmotic pressure, salinity, and oxygen result in the spatial heterogeneity of the bacterial population which strongly influences the relative fitness benefits of the cooperative and competitive phenotypic bacterial population. This spatial arrangement within a biofilm is observed in both monospecies as well as multispecies biofilms (Nadell et al., 2016). The physiological stratification and phenotypic heterogeneity observed within the biofilm matrix are responsible for the generation of a gradient of the bacterial population exhibiting genetic and heritable variations (termed as “resistance”), non-inheritable behavior (termed as “tolerance”), and altered physiological behaviors in response to stress (termed as “persistence”). The bacterial population exhibiting “resistance” could grow even at the exposure to high doses of antimicrobial treatment irrespective of the duration of the treatment regimens. Meanwhile, the bacterial population showing “tolerance” infers a transient ability to sustain antimicrobial therapy. In addition, a subpopulation within the clonal bacterial population exhibits “persistence,” where the bacterial community becomes dormant on exposure to antibiotics and persists for a longer time (Brauner et al., 2016; Flemming et al., 2016).

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The biofilm-mediated resistance to antibiotics in ESKAPE pathogens occurs through several mechanisms including extracellular matrix-mediated restriction of antibiotic penetration, increased production of hydrolyzing enzymes specific to antibiotics, alteration in pathophysiological targets, increased subpopulation of persister cells within the matrix, physiological dormancy with differential metabolic activities, biofilm-mediated upregulation of bacterial efflux machinery, enhanced horizontal gene transfer among the embedded bacterial population, and matrix-mediated binding retardation of antibiotics (De Oliveira et al., 2020; Høiby et al., 2011). The extracellular matrix composition in biofilm architecture also greatly establishes the cross-talk between embedded bacterial communities with an increased rate of genetic material exchange. In this cross-talk, antibiotic resistance gene clusters also tend to disperse among the bacterial population which concomitantly facilitates survival strategies against exposure to antibiotics (Urue´n et al., 2021). For instance, the presence of polyanionic alginate in EPS of P. aeruginosa biofilm provides a protective environment to the embedded bacterial population against the aminoglycoside group of antibiotics (Urue´n et al., 2021). The increased production of extended-spectrum β-lactamase (ESBL) and carbapenemase like metallo-β-lactamase (MBL) in ESKAPE pathogens has critically enabled the resistance to next-generation antibiotics including carbapenems. Similarly, the production of aminoglycoside-modifying enzymes highly contributed to resistance to aminoglycoside antibiotics in A. baumannii (Gedefie et al., 2021; Pandey et al., 2021). Since the majority of antibiotics specifically target actively dividing microbial cells, the occurrence of persister cells with phenotypic heterogeneity and metabolic dormancy within biofilms becomes less susceptible to antibiotics. Similarly, the occurrence of anoxic conditions within the biofilm matrix also critically affects the functional attributes of certain antibiotics which require aerobic metabolic activities as their target sites (Varadarajan et al., 2020).

25.2.1 Regulation of specific virulence genes associated with biofilms As mentioned earlier, approximately 40%
80% of the bacterial population inherently produce biofilms. In particular, ESKAPE pathogens gained considerable attention owing to their widespread virulence attributes and ability to induce chronic infections through biofilm formation. The transcriptomic studies have revealed that regulatory gene expression in the planktonic state resembles that of the biofilm state. The only difference observed in biofilm mode is the regulatory adaptations in terms of physiological and metabolic processes. However, the regulatory genes which are generally associated with the adaptive conditions for anaerobic growth conditions are observed to be comparatively upregulated when the planktonic form transforms into a sessile state during biofilm development (Høiby et al., 2011). Quorum sensing (QS), the highly synchronized and complex cell-to-cell communication, is considered the control center for coordinating several socialistic behavior and pathophysiological processes in response to regulated synthesis, sensing, and response to signaling molecules. It is evident from earlier studies that QS regulatory network plays a pivotal role in biofilm formation and development by modulating the production of several virulence phenotypes such as EPS, alginates, rhamnolipids, cytotoxic elements, hydrophobicity factors, surface motility accessories (i.e., pili, flagella, and fimbriae), pellicle formation, eDNA, and adhesion proteins (Chang, 2018; Lazar et al., 2021; Rutherford & Bassler, 2012; Schiessl et al., 2019; Tahrioui et al., 2019). The presence of psl gene (encoding mannose-rich exopolysaccharides) in the extracellular matrix of multidrugresistant ESKAPE pathogen, P. aeruginosa, acts as first line of defense against exposure to antibiotics during the early stages of biofilm formation (Billings et al., 2013; Joo & Otto, 2012). Further, Psl polysaccharides induce the production of c-di-GMP (key secondary messenger), which drives the phenotypic switching of bacterial cells from planktonic to biofilm state. Hence, c-di-GMP is considered an important phenotype in biofilm development (Dragoˇs & Kova´cs, 2017). The key regulatory signal molecules including acyl homoserine lactones (AHLs) in gram-negative bacteria, autoinducer peptides (AIPs) in gram-positive bacteria, and interspecies signal analogues (e.g., diffusible signal factors (DSFs)) are also involved in biofilm maturation and dispersion (An et al., 2019; McDougald et al., 2012). Since biofilm regulatory signals are responsible for inducing bacterial virulence in chronic microbial infections, it is imperative to develop therapeutic modules with an aim to disrupt the biofilm formation and eradication of the formed biofilm matrix.

25.3

Mitigation of biofilm architecture: current therapeutic trends

During the dispersion phase, biofilm-encased bacterial communities egress from the matrix and enter planktonic mode which is considered as most vulnerable to therapeutic regimes; the post-dispersion phase is considered a therapeutic target for biofilm control approaches (Rumbaugh & Sauer, 2020). Presently, several conventional and alternative therapeutic regimens are considered as control measures against biofilms-associated infections. In particular, bacteriophage

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therapy, mechanical eradication of biofilm by physical techniques, biophysical approaches for improved drug penetration into the biofilm matrix, improved drug delivery systems with localized drug delivery, development of QS inhibitors (QSIs) and anti-biofilm molecules, modified vaccine development targeting biofilm adhesive phenotypes, development of matrix destabilizing agents, mechanistic development of anti-persister strategies, and development of next-generation medical devices with decreased susceptibility to microbial biofilms are considered as promising biofilm control measures (Lazar et al., 2021). Owing to the infection severity of multispecies biofilms, putative drug candidates (i.e., QSIs and anti-biofilm agents) along with a combination of enzymatic treatment regimens could be inevitable in the inhibition and eradication of biofilms (Willems et al., 2016). The recent development of electroceutical dressings provides novel avenues for the remediation of formed biofilm matrix by using electric fields and concomitantly improving the wound healing process (Dusane et al., 2008). The emergence of novel nano-scaled drug delivery systems is also being spatially designed and developed for the mitigation and eradication of microbial biofilms. Among the spatially designed drug delivery nanoplatforms, polymeric nanoparticles, inorganic nanoparticles, dendrimers, and lipid-based vesicular nanoparticles (i.e., liposomes) are considered as influential in escaping biofilm-mediated drug resistance by critically improving the localized delivery of loaded drug molecules at the target sites (Rukavina & Vani´c, 2016). As per the recent trends, pharmacologically relevant natural products and their derivatives gained considerable attention in our efforts in the management of QS-mediated biofilm mechanics in ESKAPE pathogens.

25.3.1 Synthetic and semisynthetic derivatives as biofilm inhibitors The semisynthetic derivatives (amide derivatization) of di-rhamnolipids (derived from Lysinibacillus sp. BV152.1) significantly improved the biofilm inhibition against ESKAPE pathogens, P. aeruginosa PAO1 and S. aureus. Among the amide derivatives, the morpholine derivative exhibited the highest effect on biofilm disruption (Aleksic et al., 2017). Due to the pharmacological importance of antimicrobial peptides in biofilm mitigation, semisynthetic peptide lin-SB056
1 and its dendrimeric derivative (lin-SB056
1)2-K were evaluated for their anti-biofilm properties. As compared to the parent molecule, the dendrimeric derivative exhibited promising anti-biofilm effects against both reference strain, P. aeruginosa PAO1 as well as cystic fibrosis lung isolates of P. aeruginosa (Grassi et al., 2019). In an earlier study, chalcone-linked amine derivative ((E)-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-3-(piperidin-1-yl)propanamide) showed potent anti-biofilm activities against S. aureus IFO 3060 and P. aeruginosa IFO 3448 by modulating the c-di-GMP signaling (El-Messery et al., 2018). Recently, synthetic derivatives of hydroxynaphthoquinone (2-hydroxy-1,4-naphthoquinone) exhibited promising biofilm inhibition potential against methicillin-resistant S. aureus (MRSA) and thus could be considered for clearance of antibiotic resistance phenomenon (Song et al., 2020). In a recent study, a synthetic benzoyl ester derivative of β-amyrin (e.g., β-amyrin 3,4,5-trimethoxybenzoyl ester) exhibited a significant inhibitory effect on biofilm formation in S. aureus (Tamfu et al., 2022). No doubt, synthetic and semisynthetic compounds exhibit promising avenues in the management of biofilm dynamics; their probable toxicity profile, bioavailability, and biodegradability issues limit their widespread potential as antibiofilm agents. In this context, scientific interventions have shifted toward more reliable natural resources as potential alternatives in the regulation of biofilm mechanics and drug resistance phenomena. Natural resources, particularly plant-derived phytochemicals and microbes-derived secondary metabolites, received worldwide scientific attention for their pharmacological relevance and could be considered as reliable and effective therapeutic regimens in the fight against chronic biofilm infections and drug resistance-associated health hazards.

25.3.2 Microbial secondary metabolites for biofilm inhibition Microbial-derived bioactive molecules are considered pharmacologically important candidates in the management of biofilm formation in ESKAPE pathogens. Azithromycin (AZM), an important macrolide antibiotic derived from Saccharopolyspora erythraea, transcriptionally not only downregulated the expression of QS regulatory system in P. aeruginosa, but also critically reduced the expression of multiple flagellar biosynthesis proteins required for biofilm surface attachment (Townsley & Shank, 2017). The presence of bioactive secondary metabolites such as Fenaclon, 2,4di-tert-butylphenol, 1,4-phenylenediacetic acid, 4-ethoxybenzaldehyde, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4dione present in fungal crude extracts of Diaporthe phaseolorum SSP12, Aspergillus ochraceopetaliformis SSP13, and Phomopsis tersa have significantly contributed in attenuation of the QS regulatory virulence phenotypes production and biofilm formation in P. aeruginosa PAO1 (Meena et al., 2020; Pattnaik, Ahmed, et al., 2018; Pattnaik, Ranganathan, et al., 2018).

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Phytochemicals-based mitigation strategies against biofilm formation

Among the alternative therapeutic drug candidates from natural resources, plants and plant-derived phytochemicals are considered to be the most explored resources due to their easy availability, comparatively less complex extraction procedures, and absence of biological threats unlike that of microbial resources. The rich lineage of biologically active, structurally diverse phytochemicals identified from plant sources is inherently reported for widespread pharmacological potential such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, antioxidant, neuroprotective, hepatoprotective, and cardioprotective properties (Fatima et al., 2021; Khare et al., 2021; Mitra et al., 2022; Song et al., 2022). Owing to their extensive pharmacological and pharmaceutical importance, phytochemicals are considered effective avenues for disease prevention and treatment against chronic microbial infections (Yu et al., 2021).

25.4.1 Crude plant extracts against biofilm formation in ESKAPE pathogens In the fight against chronic microbial infections, no doubt antibiotics serve as potential therapeutic agents, but the emergence of resistance to these antimicrobial drugs has urged the scientific community to look for alternative yet effective therapeutic measures to cope with the microbial infections and drug resistance. In this context, pharmacologically relevant plant resources and the derivatized products could be instrumental in providing a rational strategy to counteract drug resistance by targeting the bacterial communication system (Ouedraogo & Kiendrebeogo, 2016). Medicinal plants are a rich source of bioactive secondary metabolites which significantly contribute toward the use of the plant materials as folkloric medicines for several diseases and disorders. Due to the comparatively low toxicity profile and absence of bioavailability issues, plantderived products are considered for therapeutic applications in the fight against microbial infections and biofilm-mediated drug resistance. Since the beginning of the 21st century, a large number of medicinal plants and rare medicinal plants are being actively screened for their ability to attenuate bacterial signaling networks which are associated with bacterial pathogenesis and drug resistance (Banerjee et al., 2017). Table 25.1 depicts the list of crude plant extracts and their bioactive fractions reported for inhibition of QS-regulated virulence and biofilm formation in ESKAPE pathogens (Table 25.1).

25.4.2 Phytochemicals involved in the inhibition of biofilm formation in ESKAPE pathogens Since ancient times, ethnopharmacologically important medicinal plants and their bioactive phytochemicals have been recognized as rich sources of folkloric medicines against several pathogenic microorganisms (Elhawary et al., 2018). The presence of structurally diverse phytochemicals such as alkaloids, flavonoids, anthocyanins, anthraquinone, polyphenols, organosulfur compounds, tannins, terpenes, terpenoids, phenolic acids, etc., in the ethnobotanically important plants has characteristically modulate widespread pharmacological properties (Haripriyan et al., 2018; Qais et al., 2019). Plant-derived phytochemicals are considered as preferred alternatives in the development of potential QSIs and anti-biofilm agents due to their relatively nontoxic nature, biocompatibility, easy availability, biodegradability, and eco-friendly nature (Ghosh et al., 2022; Li et al., 2018). For example, bioactive phytochemicals such as caffeine, estragole, squalene, and neophytadiene identified from ethyl acetate fraction of Camellia sinensis potentially modulate the QS transcriptional regulators in P. aeruginosa. Thus, these compounds could be considered as potential inhibitors of QS-regulated virulence and biofilm formation (Qais et al., 2019). Similarly, flavonoid-rich Citrus sp. reported for the presence of naringin, naringenin, quercetin, kaempferol, apigenin, baicalein, etc., evidently exhibited promising inhibitory effect on QS-mediated production of virulence phenotypes and biofilm (Liu et al., 2017; Peng et al., 2019). The promising antibacterial properties of simple phenols, polyphenols, phenolic acids, and coumarin derivatives are considered in the development of phenolic acids and polyphenols as potential regulators of QS-associated biofilm mechanics (Lemos et al., 2014; Mombeshora et al., 2021). Similarly, the presence of bioactive quinone derivatives as well as terpenes in Nigella sativa extracts resulted in the inhibition of biofilm formation in ESKAPE pathogens (P. aeruginosa, K. pneumoniae, and S. aureus) and hence could be considered in the treatment of drug-resistant infections (Rahman & Roy, 2021). The plant-derived essential oil compounds such as thymol, carvacrol, and geraniol also possess promising antimicrobial and anti-biofilm activities against ESKAPE uropathogen, K. pneumoniae. This result suggested the use of essential oil compounds as a putative source of anti-biofilm agents in the fight against chronic microbial infections (Kwiatkowski et al., 2022). Plant-derived essential oil exhibited promising aspects in targeting several pathophysiological mechanisms associated with drug resistance. For example, interference in a cell density-dependent QS regulatory network, cell membrane permeability, drug efflux pumps machinery, transfer of mobile genetic elements, and biofilm mechanics are possible therapeutic targets in the fight against persistent microbial infections and drug resistance (Khare et al., 2021). The list of phytochemicals reported for QS attenuation and biofilm inhibition in ESKAPE pathogens is listed in Table 25.2.

TABLE 25.1 List of pharmacologically relevant medicinal plants reported for quorum sensing inhibition and mitigation of biofilm mechanics against ESKAPE pathogens. SI. No.

Plant species

Plant family

Target microorganism

Minimum inhibitory concentrations (MIC)

Mechanism of action

References

1.

Amomum tsao-ko

Zingiberaceae

Pseudomonas aeruginosa and Staphylococcus aureus

2, 1 mg/mL, respectively

Inhibition of biofilm formation by modulating QS response

Rahman et al. (2017)

2.

Andrographis paniculata

Acanthaceae

P. aeruginosa

5 mg/mL

Modulation of QS-regulated virulence phenotypes production, biofilm inhibition, and interference in pathogeninduced activation of MAPK pathway

Banerjee et al. (2017)

3.

Anogeissus leiocarpus (DC) Guill. et Perr.

Combretaceae

P. aeruginosa PAO1

1.25 mg/mL

Suppression of QS regulatory genes

(Ouedraogo and Kiendrebeogo, 2016)

4.

Cornus controversa

Cornaceae

P. aeruginosa PAO1

.2%

Anti-biofilm and QS inhibitory properties

Choi et al. (2018)

5.

Cassia alata L.

Caesalpiniaceae

P. aeruginosa




Interference in the production of QSassociated pathogenic factors and inhibition of bacterial motility

Rekha et al. (2017)

6.

Centella asiatica

Apiaceae

P. aeruginosa PAO1

.400 μg/mL

Inhibition of QS-regulated pathogenic factors production, swarming motility, and biofilm formation

Vasavi et al. (2016)

7.

Malva sylvestris

Malvaceae

ESKAPE pathogens (S. aureus, Klebsiella pneumoniae, and Enterococcus faecalis)

BIC50 (minimum biofilm inhibition): 40 mg/mL

Inhibition of biofilm formation

Fathi et al. (2022)

8.

Mangifera indica

Anacardiaceae

P. aeruginosa PAO1

.0.8 mg/mL

Concentration-dependent inhibition of QS-mediated virulence factors production and biofilm formation

Husain et al. (2017)

9.

Laserpitium ochridanum

Apiaceae

P. aeruginosa and S. aureus

0.5 and 0.4 mg/mL respectively

Inhibition of biofilm formation and decreased production of QS-mediated virulence factors

Mileski et al. (2017)

10.

Carum copticum

Apiaceae

Acinetobacter baumannii, K. pneumoniae

25 mg/mL

Disruption of biofilm formation with the highest effect was observed against A. baumannii

Mohammadi et al. (2019)

11.

Syzygium jambos (L.) Alston

Myrtaceae

P. aeruginosa PAO1

1 mg/mL

QS inhibition and biofilm disruption

Rajkumari et al. (2018) (Continued )

TABLE 25.1 (Continued) SI. No.

Plant species

Plant family

Target microorganism

Minimum inhibitory concentrations (MIC)

Mechanism of action

References

12.

Syzygium cumini (L.) ethyl acetate fraction

Myrtaceae

P. aeruginosa and S. aureus




Inhibition of QS regulatory virulence and biofilm formation

Gupta et al. (2019)

13.

Parkia javanica

Fabaceae

P. aeruginosa

180 μg/mL

Attenuation of QS-mediated swarming motility, secretion of virulence factors, and biofilm formation

Das et al. (2017)

14.

Pistacia atlantica

Anacardiaceae

P. aeruginosa

0.5 mg/mL

Inhibition of QS-regulated virulence by targeting transcription regulator, LasR

Kordbacheh et al. (2017)

15.

Solanum torvum

Solanaceae

P. aeruginosa




Disturbance in bacterial virulence at sublethal concentration

Vadakkan et al. (2019)

16.

Psoralea corylifolia

Fabaceae

P. aeruginosa

.1 mg/mL

Inhibition of QS-regulated virulence factors by modulating stable interaction with transcriptional regulators, LasR and RhlR

Husain et al. (2018)

17.

Terminalia bellirica

Combretaceae

P. aeruginosa

.0.5 mg/mL

Reduction of biofilm formation by interfering the QS regulatory behavior

Sankar Ganesh and Ravishankar Rai (2018)

18.

Salvadora persica

Salvadoraceae

Staphylococcus sp.

6.25 mg/mL

Biofilm inhibition

Noumi et al. (2017)

19.

Herba patriniae

Caprifoliaceae

P. aeruginosa




Downregulation of biofilm-associated genes

Fu et al. (2017)

20.

Galla chinensis

Anacardiaceae

P. aeruginosa PAO1

2 mg/mL

Inhibition of QS-mediated swarming motility

Zhang, Djakpo, et al. (2019)

21.

Hypericum perforatum

Hypericaceae

P. aeruginosa PAO1




Downregulation of QS-regulated virulence phenotypes by modulating QS signaling pathway

Do˘gan et al. (2019)

TABLE 25.2 List of plant-derived phytochemicals reported for attenuation of quorum sensing-regulated virulence and biofilm inhibition against ESKAPE pathogens. SI. No.

Chemical class

Phytochemicals

Plant source

Target microorganisms

Minimum inhibitory concentration (MIC)

Mechanism of action

References

1.

Alkaloids

Berberine

Berberis sp.

Pseudomonas aeruginosa PAO1

1.25 mg/mL

Targets RhlR of QS circuit and modulates biofilm mechanics

Aswathanarayan and Vittal (2018)

2.

Hordenine

Hordeum vulgare

P. aeruginosa PAO1

2.5 mg/mL

Suppression of QS regulatory genes and biofilm formation

Zhou et al. (2018)

3.

Reserpine

Rauwolfia serpentina

P. aeruginosa PAO1

0.8 mg/mL

Inhibition of QS-mediated virulence phenotypes, biofilm disruption, and motility

Parai et al. (2018)

4.

Caffeine

Coffea arabica

P. aeruginosa (MTCC 424)

200 μg/mL

Biofilm disruption by interfering QS machinery without affecting the cell viability

Chakraborty et al. (2020)

Proanthocyanidins

Vaccinium macrocarpon L.

P. aeruginosa PA14




Inhibition of QS-controlled virulence determinants

Maisuria et al. (2016)

Anthocyanidins (delphinidin and pelargonidin)




P. aeruginosa PAO1

0.45 mg/mL

Biofilm disruption

Pejin et al. (2017)

Aloe-emodin

Rheum officinale Baill.

Staphylococcus aureus

.1.024 mg/mL

Biofilm inhibition by decreasing the production of extracellular proteins and polysaccharide intercellular adhesion (PIA)

Xiang et al. (2017)

8.

Alizarin (1,2dihydroxyanthraquinone)




Methicillin-sensitive S. aureus (MSSA 6538)

.1 mg/mL

Promotion of anti-biofilm activity by inhibition of hemolytic activity

Lee et al. (2016)

9.

Symploquinone A,C

Symplocos racemosa Roxb.

Methicillin-resistant S. aureus (MRSA)

83
160 μg/mL

Inhibition of biofilm formation at sub-MICs

Farooq et al. (2017)

5.

Anthocyanins

6.

7.

Anthraquinones

(Continued )

TABLE 25.2 (Continued) SI. No.

Chemical class

Phytochemicals

Plant source

Target microorganisms

Minimum inhibitory concentration (MIC)

Mechanism of action

References

10.

Flavonoids and polyphenols

Baicalein

Scutellaria baicalensis

P. aeruginosa, S. aureus 17546

0.256 mg/mL,

Attenuation of QS-mediated virulence phenotypes and biofilm disruption

Chen et al. (2016), Luo et al. (2016)

11.

Quercetin glucopyranoside

Allium cepa

P. aeruginosa

0.4 mg/mL

Modulation of QS-regulated response

Al-Yousef et al. (2017)

12.

Trihydroxyflavone

Alstonia scholaris

P. aeruginosa

0.2 mg/mL

Disruption of biofilm matrix by targeting the QS circuit, LasR

Abinaya and Gayathri (2019)

13.

Calycopterin

Marcetia latifolia

P. aeruginosa (ATCC 27853)

EC50: 34 μM

Inhibition of QS-mediated pyocyanin production and swarming motility

Froes et al. (2020)

14.

Baicalin

Scutellaria baicalensis

P. aeruginosa

.1.024 mg/mL

Biofilm inhibition

Luo et al. (2017)

15.

Curcumin

Curcuma longa

Acinetobacter baumannii

.500 μg/mL

Disruption of biofilm formation by inhibition of pellicle formation and surface motility properties

Raorane et al. (2019)

16.

Pulverulentone A

Callistemon citrinus

Methicillin-resistant S. aureus

125 μg/mL

Attenuation of QS machinery and biofilm inhibition

Shehabeldine et al. (2020)

17.

Fisetin, phloretin




Acinetobacter baumannii

.500 μg/mL

Inhibition of pellicle formation and biofilm formation

Raorane et al. (2019)

18.

Resveratrol




P. aeruginosa

.400 μM

Inhibition of QS-regulated phenotypes by minimizing oxidative stress

Chen et al. (2017)

19.

Naringenin




P. aeruginosa




Competitively inhibited the binding of C12-HSL for binding to its cognate LasR receptor and thus disrupted the expression of QSregulated virulence genes

Hernando-Amado et al. (2020)

20.

Scutellarein

Scutellaria lateriflora

S. aureus

500 μg/mL

Inhibition of biofilmassociated protein (BAP)mediated biofilm formation

Matilla-Cuenca et al. (2020)

21.

Cinnamic acid

Averrhoa carambola

P. aeruginosa

0.5 mg/mL

Impairment of QS-controlled pathogenesis

Rajkumari et al. (2018b)

22.

Cinnamaldehyde

Cinnamomum sp.

P. aeruginosa

.11.8 mM

Modulates c-di-GMP signaling and promotes biofilm disruption

Topa et al. (2018)

23.

Chlorogenic acid

Camellia sinensis

P. aeruginosa, S. aureus

.5 mg/mL

Inhibits QS-regulated pathogenic factors and biofilm dynamics

Wang et al. (2019)

24.

Eugenol

Syzygium aromaticum

P. aeruginosa PAO1

275 μg/mL

Controls production of QSregulated pathogenic factors

Rathinam et al. (2017)

25.

6-Gingerol

Zingiber officinale

P. aeruginosa




Reduces the QS regulatory response

Kim et al. (2015)

26.

Ferulic acid

-

P. aeruginosa PAO1

1 mg/mL

Inhibition of biofilm formation by modulating the production of exopolysaccharides

Pattnaik, Barik, et al. (2018)

Ajoene

Allium sativum

P. aeruginosa




Alters biofilm architecture, rhamnolipid inhibition

Jakobsen et al. (2012)

Allicin

A. sativum

P. aeruginosa




Disruption of the bacterial adhesion in the initial stages of biofilm formation by inhibition of EPS production

Xu et al. (2019)

27.

Phenylpropanoids

Organosulfur compounds

28.

(1)-Dehydroabietic Acid

Coniferous plants

S. aureus

70 μM

Inhibition of biofilm matrix

Fallarero et al. (2013)

Tormentic acid congener

Callistemon viminalis

P. aeruginosa, S. aureus

25 and 12.5 μg/mL

Biofilm disruption by decreasing the release of eDNA and capsular polysaccharides from biofilms

Chipenzi et al. (2020)

31.

Betulin and betulinic acid

Syzygium jambos

P. aeruginosa

250 μg/mL

Interferes with QS-mediated virulence and biofilm architecture

Rajkumari et al. (2018a)

32.

Carvacrol

Origanum vulgare

P. aeruginosa

.3.9 mM

Targets LasI and modulates QS-controlled behavior

Tapia-Rodriguez et al. (2019)

33.

Phytol

Plant chlorophyll

P. aeruginosa

19 μg/mL

Alters bacterial motility profile

Pejin et al. (2015)

29. 30.

Terpenes and terpenoids

(Continued )

TABLE 25.2 (Continued) SI. No.

Chemical class

Phytochemicals

Plant source

Target microorganisms

Minimum inhibitory concentration (MIC)

Mechanism of action

References

34.

Terpenes and terpenoids

Phytol




Klebsiella pneumonia (ATCC BAA-1705; ATCC 700603)

0.125 mg/mL

Reduction of adhesion capabilities of hypervirulent and drug-resistant K. pneumoniae by disruption of biofilm architecture

Adeosun et al. (2022)

35.

Terpinen-4-ol

Pandanus odorifer essential oil

P. aeruginosa

0.5% (v/v)

Mitigation of QS regulatory behaviors in P. aeruginosa by targeting transcriptional regulators, LasR, RhlR, and PqsR; downregulation of QSregulated genes; biofilm inhibition

Bose et al. (2020)

36.

Glycyrrhetinic acid

Glycyrrhiza glabra

P. aeruginosa

160 μg/mL

Inhibits the secretion of pathogenic determinants and biofilm formation

Kannan et al. (2019)

Phytochemicals-based approaches Chapter | 25

25.5

445

Current trends in biofilm inhibition

25.5.1 In silico approaches for phytochemicals-based mitigation of biofilm formation The interdisciplinary aspects of computational tools in determining the effectiveness of putative drug candidates from natural resources have revolutionized the current drug discovery and development pipelines. Particularly, computeraided drug design (CADD) using the molecular docking and molecular dynamics simulation (MDS) tools provide a fast and cost-effective screening of thousands of putative drug candidates against either predefined pathophysiological targets or novel undefined targets. As a result, potential drug-like molecules could be easily filtered and evaluated for their possible role in the mitigation of pathophysiological responses (Abelyan et al., 2020; Wei et al., 2016; Zeng et al., 2018). In the development of potent QSIs and anti-biofilm agents from natural resources against ESKAPE pathogens, the CADD-based in silico approaches provide an insight into the possible mechanism of action. Based on the computational trends, further therapeutic regimens in vitro and in vivo could be decided for the establishment of their candidature as potential inhibitors of QS-mediated virulence and biofilm formation (Adnan et al., 2020; Awadelkareem et al., 2022). Since QS regulatory network is highly dependent on the activation of specific transcriptional regulators (i.e., LasR, RhlR), targeting these regulators could be instrumental in disrupting the QS signaling network. In this regard, CADD approaches provide a virtual platform to define the interactions of the putative drug candidates with the therapeutic regulators. Based on the stability of the interaction, novel candidates could be selected for their efficacy in minimizing the QS transcriptional regulator-mediated virulence and biofilm formation. For example, the stable interactions of bioactive phytochemicals such as mosloflavone, 5-hydroxymethyl furfural (5-HMF), betulin, and cinnamic acid toward QS transcriptional regulators, LasR and RhlR in P. aeruginosa PAO1, were evident from molecular docking and molecular dynamic simulation. The in silico results were further corroborated through in vitro studies which revealed the significant alteration in the production of virulence phenotypes linked to QS circuit and biofilm formation (Hnamte et al., 2019; Rajkumari et al., 2018a, 2018b, 2019). Since bacterial biofilms are highly resistant to antibiotics and the limitations associated with available antibiotics, it is highly important to optimize the doses of conventional antibiotics. In this context, pharmacokinetic (PK) and pharmacodynamic (PD) prediction of antimicrobial agents through computational tools could provide cues to improve the biological effect on drug-resistant pathogens. In recent studies, comparative PK and PD profiles of antimicrobials against both planktonic and biofilm-embedded sessile bacteria were predicted through computational tools, which could facilitate upliftment in the scientific efforts in drug optimization before the treatment regimens (Wu et al., 2015). Since PK/ PD profile is an essential norm for putative drug candidates before being considered for market approval, advanced computational tools such as SwissADME, admetSAR, TOPKAT module of Discovery Studio, etc., are actively engaged in predicting several parameters associated with PK/PD properties. The drug candidates after being passed through the PK/PD filters could be considered for further evaluation (Alam & Khan, 2018; Roman et al., 2018).

25.5.2 Nano-based formulation using plant-derived phytochemicals for biofilm inhibition In scientific efforts to develop alternative therapeutic regimens against microbial infections and biofilms associated with drug resistance, we have received positive feedback to use phytochemicals in the fight against drug resistance patterns in ESKAPE pathogens. However, certain limitations such as lack of target-specific actions, poor solubility issues, and absence of long-term therapeutic effect hinder their applicability in clinical settings. Hence, it is imperative to use species-specific drug delivery platforms for improving drug therapeutics against microbial infections (Li et al., 2019; Subhaswaraj et al., 2020). In this regard, nanotechnological interventions owing to their unique physicochemical properties and biomedical applications could provide a new paradigm for target-specific delivery of putative phytochemical drug candidates for their improved therapeutic efficacy (Zhu et al., 2014). The nanotechnological interventions in drug development pipelines provide several advantages including the slow and sustained release of encapsulated drug moieties/phytochemicals, improved drug stability, increased therapeutic index, and more importantly prolonged therapeutic effect (Zaidi et al., 2017). The presence of phytochemicals such as alkaloids, phenolics, saponins, tannins, terpenoids, etc., in plant extracts is considered as promising reducing and stabilizing agents in the green synthesis of nanoparticles. In this context, N. sativa seed extract was used as stabilizing and reducing agents for the synthesis of zinc oxide nanoparticles (ZnO NPs). The green synthesized ZnO NPs significantly attenuated the QS-mediated biofilm mechanics in P. aeruginosa and the foodborne pathogen, Escherichia coli. Thus, the developed ZnO NPs could be instrumental in the food packaging and food processing industries (Al-Shabib et al., 2016). The reducing ability of medicinal plant extracts was explored for

446

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the synthesis of silver nanoparticles (AgNPs), which characteristically improved biofilm inhibition in pathogenic microorganisms, P. aeruginosa and S. aureus (Mohanta et al., 2020). The biocompatible chitosan nanoparticles also served as encapsulating agents for several phytochemicals and drug moieties. In this regard, bioactive phytochemicals, ferulic acid, and cinnamaldehyde were encapsulated onto chitosan nanoparticles. The encapsulated nanoparticles exhibited a promising response in mitigating QS-mediated virulence phenotypes production and also inhibited biofilm formation. It was also evident from the studies that phytochemicals-encapsulated nanoparticles have shown comparatively improved biological activities than that of nascent ferulic acid and cinnamaldehyde (Pattnaik, Barik, et al., 2018; Subhaswaraj et al., 2018). Later on, chrysin-loaded chitosan nanoparticles also critically improved the anti-biofilm potential of chrysin by concomitant decrease in the production of exopolysaccharides and biofilm formation in S. aureus (Siddhardha et al., 2020). Earlier, baicalein-fabricated gold nanoparticles (BCL-AuNPs) significantly inhibited the QS-regulated virulence phenotypes (e.g., exopolysaccharides, swarming motility) in P. aeruginosa PAO1. The bioactive flavone, baicalein, thus served as a reducing and capping agent for the synthesis of gold nanoparticles. The results thus emphasized the use of phytochemicals-based nanomaterials for the treatment of biofilm-mediated chronic infections (Rajkumari et al., 2017). Using the bioactive components of different plant parts as reducing and capping agents, silver nanoparticles (AgNPs) were synthesized. The synthesized AgNPs exhibited promising biofilm inhibitory potential against MDR P. aeruginosa (Feizi et al., 2018; Habibipour et al., 2019; Singh et al., 2018). Recently, a polymer-stabilized carvacrol-in-water nanocomposite (NC) was designed and developed for effective therapeutic agents against P. aeruginosa biofilm. It was evident from earlier studies that the therapeutic index of phytochemicals critically improved after being used in a combination with nanocomposites by minimizing the MICs against pathogenic microorganisms (Landis et al., 2018; Li et al., 2019). Hence, the bioactive phytochemicals could be used in combination with suitable nanoplatforms not only to improve their therapeutic index but also to improve the localized and sustained release at the target sites for improved and long-term therapeutic effects.

25.6

Future perspectives

As discussed earlier, biofilm is the most predominant mode of ESKAPE pathogens to allow the growth of embedded bacterial communities from environmental stress including the stress of antimicrobial treatment. ESKAPE pathogens being active biofilm producers possess a significant challenge to our healthcare settings and current R&D sectors for antimicrobial therapeutics. In this context, it is imperative to quest for bioactive phytochemicals as promising alternatives to tackle the highly resilient biofilm dynamics. In recent years, phytochemicals are considered as potential agents to quench the pathophysiological factors responsible for biofilm development and drug resistance. Phytochemicals are being actively explored as QSIs, anti-biofilm agents. However, the majority of research works are still in preclinical settings, and more evidence-based scientific investigations need to be carried out for their ability to pass the clinical trials (Das et al., 2021). The emergence of advanced computational tools could provide novel avenues to develop suitable drug candidates from plant origin for their role in QS inhibition and mitigation of recalcitrant biofilm dynamics in ESKAPE pathogens (An et al., 2021; Chaieb et al., 2022). No doubt, computational approaches characteristically improved the screening of putative drug candidates for specific therapeutic applications, but the rate of molecules identified from in silico tools to introduction into the market remains very less. In this context, more sophisticated and advanced tools should be designed to improve the efficacy of such candidates in clinical settings.

Key points Similarly, nanotechnological interventions also revolutionized the therapeutic index of nascent phytochemicals in the fight against chronic microbial infections. In addition, nanomaterial impregnations of anti-biofilm devices also critically improved our sincere efforts to challenge biofilm-associated infections. However, the use of nanotechnology-based efficacy is short term and environmentally toxic. Hence, it is imperative to develop regulatory bodies to address the issues associated with nanotechnological interventions in drug development pipelines (Ramasamy & Lee, 2016; Subhaswaraj et al., 2020). One more interesting aspect is to tackle biofilm-associated infections by using putative phytochemicals in combination with conventional antimicrobials for improving therapeutic efficacy. In addition, drug repurposing could be a viable alternative for the use of phytochemical drugs which were initially used for other therapeutics and could be intertwined with biofilm infections and drug resistance. In this context, more extensive research should be carried out with advanced computational and molecular tools for effective treatment regimens against chronic microbial infections and biofilm-mediated drug resistance in ESKAPE pathogens.

Phytochemicals-based approaches Chapter | 25

447

Acknowledgment We would like to acknowledge OHEPEE, Govt. of Odisha, for providing financial support. The author, Subhaswaraj Pattnaik also acknowledges the Science and Engineering Board (SERB), Department of Science and Technology (DST), Govt. of India, for the award of National Post-Doctoral Fellowship (NPDF) (File Number: PDF/2021/001260).

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Chapter 26

Phytochemicals in downregulation of quorum sensing Ipsita Mohanty1, Rojita Mishra2, Amrita Kumari Panda3, Arabinda Mahanty4 and Satpal Singh Bisht5 1

Departments of Pediatrics, Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, United States, 2Department of Botany, Polasara

Science College, Polasara, Odisha, India, 3Department of Biotechnology, Sant Gahira Guru University, Ambikapur, Chhattisgarh, India, 4Crop Protection Division, National Rice Research Institute, Cuttack, Odisha, India, 5Department of Zoology, Kumaun University, Nainital, Uttarakhand, India

26.1

Introduction

Biofilms are a microbial persistence phenomenon that confers antibacterial resistance and defeats host defense systems and ability to endure physically hostile environment. A protective sheath composed of polysaccharide matrix generated by bacteria levies antimicrobial resistance and persistent chronic infections leading to major clinical problems (Marsh et al., 2022). These surface-adhered bacterial aggregates are present ubiquitously and exert a significant economic burden on almost all sectors including human healthcare, pose food safety challenges, interrupt yield from oil production, and even contaminate household water supplies in the pipelines. A recent study from 2019 has estimated the financial losses to be as high as $5000 billion a year and involves almost all sectors ranging from medical and healthcare, personal care, food and agriculture, homecare products, and finally in marine, oil, and gas sectors (Ca´mara et al., 2022; Mishra et al., 2020). National Institutes of Health in United States have estimated around 80% of infections from medicare facilities are due to biofilms from nosocomial infections and secondary infections to existing diseases (Nourbakhsh et al., 2022). This calls for improved interventions to explore potential anti-biofilm candidates to prevent the progression of the disease and ameliorate the financial burden. Quorum sensing is of focal interest for researchers and clinicians throughout the world because of their significance in the fields of medicine, industry, and agriculture, as inhibition of quorum sensing-mediated mechanisms hinders the pathogenicity and virulence to these microbes (Mahanty et al., 2013; Peter et al., 2019). Hence, the identification and characterization of anti-QS compounds as new antipathogenic and antibacterial candidates play an imperative role in reducing the virulence and pathogenicity. Here comes the potential role of phytochemicals in inhibiting QS-related processes. The current chapter discusses the potential phytochemicals harnessed for QS inhibition and some of the phytochemical sources which are currently under clinical trial. The phenomenon of quorum sensing sheds a whole new light on the process of mitigation of microbial infection, where the plant-derived compounds block essential pathways (like virulence factor expression and biofilm formation) controlled by quorum sensing (Peter et al., 2019).

26.2

Biofilm formation and quorum sensing

Biofilm formation is a complicated multistep process involving cell attachment to solid surface, cell-to-cell adhesion, maturation, and dispersal. During the initial formative stages, when bacteria attach to the substrate, they transform from planktonic cells to sessile growing ones, thereby transitioning from actively dividing and floating phenotype to secretory and immobile phenotype which produces a network of soluble microbial products (SMPs) and extracellular polymeric substance (EPS) enclosing multilayered bacterial clusters (Muhammad et al., 2020). This is followed by biofilm maturation, which often confers a level of protection against a hostile environment. In the final step, the bacterial cells detach and disperse from the clustered colonies, leading to the formation of a potential new biofilm colony in a new location. Within biofilms, bacteria often interact and communicate among themselves via quorum sensing (QS) to coordinate communal behaviors. QS is a vital step in regulating the biofilm formation. Essentially, this is a process by which Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00038-4 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 26.1 Economic impact of biofilms on different sectors of humankind including healthcare, agriculture, sewage management, food safety, oil production, and household water supplies in the pipelines.

FIGURE 26.2 Steps involved in the process quorum sensing in bacteria.

microbes produce, secrete, and subsequently detect small signaling molecules called autoinducers (AIs) to assess their local population density and regulate common physiologic processes such as symbiosis, apoptosis, genetic competence, bioluminescence, sporulation, and virulence (Subramani & Jayaprakashvel, 2019). In nutshell, quorum sensing is an intercellular communication that regulates the formation of biofilms and exerts increased antibiotic resistance. Hence, inhibition of QS and biofilm formation is considered as a new target for antimicrobial chemotherapy against drugresistant bacterial infections (Fig. 26.1).

26.3

Mechanism of quorum sensing in bacteria

Quorum sensing (QS) is the communication between the microbial cells. Similar to any other metabolic process, quorum sensing is also controlled by a network of genes, proteins, and metabolites. This means the communication begins with the production of signaling molecules which gets secreted out of the microbial cell and gets detected by other microbial cells which in turn not only activates the expression of genes necessary for cooperative behaviors but also further production of signaling molecules (Fig. 26.2). The signaling molecules are referred to as autoinducers (AIs). The gram-positive and gram-negative bacteria use different types of quorum sensing approaches. Different types of quorum-sensing systems utilized by different criteria are listed in Table 26.1. Gram-positive bacteria use peptides, called autoinducing peptides (AIPs), as signaling molecules, whereas the gram-negative bacteria produce small molecules known as autoinducers (AIs). Different bacteria use different quorum-sensing mechanisms some of which are described in Table 26.3. The cell wall of gram-positive bacteria is different from those of the gram-negative bacteria. The AIPs in grampositive bacteria are synthesized as pro-AIPs which get transformed into AIPs through cleavage. As the cell walls of gram-positive bacteria are impervious to peptides, specialized transporters are required for extracellular transport of the AIPs. After secretion of the AIPs, it is detected via membrane-bound two-component sensor kinases (Rutherford & Bassler, 2012; Simon et al., 2007). The sensor kinases autophosphorylate at conserved histidines when bound by the AIP. The phosphoryl group is passed from the histidine to a conserved aspartate on a cognate cytoplasmic response regulator protein, and the phosphorylated response regulator controls the expression of QS-target genes (Fig. 26.3).

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TABLE 26.1 Quorum-sensing systems in different bacteria. Gram-negative bacteria Escherichia coli

SdiA

Pseudomonas aeruginosa

Las, Rhl, PQS, IQS

Vibrio fischeri

LuxI, Ain, LuxS

Acinetobacter baumannii

Lux

Gram-positive bacteria Bacillus subtilis

ComQXPA

Clostridium botulinum

Agr

Staphylococcus aureus

Agr

Streptococcus pneumoniae

Lux

FIGURE 26.3 Molecular mechanism of quorum sensing in gram-positive bacteria.

26.4

Phytochemicals as quorum-sensing inhibitors

Botanicals from plants have great potential in controlling the microbial disease. Compounds popularly known as antiquorum sensing compound have a great impact on declining the pathogenicity of bacteria. Mainly, the large spectrum of secondary metabolites like phenols, terpenoids, alkaloids, polyacetylenes, etc., provides a wide range of phytochemicals which act as quorum quenchers (Asfour, 2018). List of bioactive phytochemicals that has been found to be effective in biofilm inhibition are listed in Table 26.2.

26.4.1 Grouping of phytochemicals as QS inhibitors Plants are the goldmines of various metabolites mainly the secondary metabolites like nonpolar hydrocarbons and their derived products. Secondary metabolites like terpenes, aromatic phenolics, and alkaloids which are having nitrogen are bioactive compounds. Many of these compounds have antimicrobial activity. They can be served as potential candidates in the search for future infection therapeutics. Some common phenolic compounds that are very much used in and reported to have antimicrobial potential are catechol, phloretin, warfarin, caffeic acid, eugenol, etc. Similarly, monoterpenes popularly known as essential oils from Santalum album, Cinnamomum verum, Valeriana officinalis, Rosmarinus

TABLE 26.2 Bioactive phytochemicals in biofilm inhibition with evidence from relevant studies. Compound

Source

Experimental details

2-(3, 4-dihydroxyphenyl)1,3-benzodioxole-5carboxaldehyde

Plumula nelumbinis

Brugierol

Bruguiera gymnorhiza

Anti-QS mechanism

Inhibitory concentration

References

Inhibited the production of elastase, rhamnolipids, and pyocyanin

MIC 5 100 μM

Chen et al. (2022)

Reduced virulence factor production, biofilm formation, quorum-sensing molecules, and expression of QS-related genes

MIC 5 32 μg/mL

Dahibhate et al. (2022)

Rhein

Cassia fistula

Inhibited QS-mediated extracellular virulence factors, viz., protease, elastase, pyocyanin, and rhamnolipid

MIC 5 0.15 mg/mL

Peerzada et al. (2022)

Carvacrol

Origanum vulgare

In vitro colorimetric assays, microscopy, cell surface hydrophobicity, gene expression analysis

Streptococcus pyogenes

Downregulation of speB, srtB, luxS, covS, dltA, ciaH, and hasA genes

MIC 5 125 μg/mL

Wijesundara et al. (2022)

Apigenin and luteolin

Gnaphalium hypoleucum DC.

In vitro (crystal violet biofilm assay and SEM analysis)

Chromobacterium violaceum ATCC 12472

1. Downregulating the vioB, vioC, and vioD genes. 2. Reduced swarming activity. 3. Inhibits production of violacein

MIC 5 500 μg/mL

Li et al. (2022)

D-limonene

Citrus paradisi and Citrus reticulata

In vitro (crystal violet biofilm assay). In vivo C. elegans infection model

Escherichia coli, P. aeruginosa PAO1 and PA14

Inhibits swimming motility and formation and structure of biofilms.

MIC 5 5% (v/v)MIC 5 0.125% (v/v)

Wang et al. (2018) D’Almeida et al. (2022)

Eugenol

Pimenta dioica L.

In vitro (crystal violet biofilm assay)

Listeria monocytogenes CECT 933

Inhibits production of violacein by 69.30% at 100 μg/mL

MIC 5 0.048 mg/mL

ALrashidi et al. (2022)

Naringin

Citrus wilsonii Tanaka

In vitro (SEM analysis, confocal laser scanning microscopy). In vivo (Zebrafish infection model)

Pseudomonas spp., Aeromonas hydrophila

Inhibition of alginate and EPS production. Downregulation of ahh1, aerA, lip, and ahyB virulence gene expression

MIC 5 128
512 μg/ mLMBC 5 128
1024 μg/mL MBIC 5 750 μg/mL

Husain et al. (2021) Srinivasan et al. (2020)

Quercetin

Allium cepa

In vitro (field emission SEM analysis)

Salmonella typhimurium

Disturbing cell-to-cell connections and inducing cell lysis

MIC 5 250 μg/mL

Roy et al. (2022)

Morin

Morus alba L.

In vitro (crystal violet biofilm assay and SEM analysis)

Staphylococcus aureus

Downregulation of biofilmforming genes

MIC 5 281.83 μg/mL

Chemmugil et al. (2019)

Ginkgolic acid

G. biloba

In vitro (DNA microarray and qRT-PCR)

Escherichia coli O157:H7

Repressed curli genes, reduced fimbriae production, and causes biofilm reductions

MIC 5 5 μg/mL

Lee et al. (2014)

Phloretin

Apple extracts

In vitro (microdilution and crystal violet biofilm assay)

S. aureus RN4220

Overexpress efflux protein genes

MIC .1024 μg/mL

Lopes et al. (2017)

In vitro quantitative real-time PCR

Pathogenic species

Pseudomonas aeruginosa

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officinalis, Ocimum sanctum, Ocimum aromaticus, Matricaria chamomilla, etc., have numerous potential antimicrobial compounds (Cowan,1999; Stanojevi´c et al., 2016; Pattanayak et al.2010, Mishra et al., 2020). Phytocompounds mainly involved in inhibiting quorum-sensing activity are alkaloids, organosulfur compounds, polyphenols, coumarins, terpenes, flavonoids, and essential oils (Mishra et al., 2020).

26.4.2 Taxa and habitats intersected and interacted with QS inhibition The literature study reveals that a large variety of plant families have bioactive compounds which can be used as QSIs. A wide variety of plant families are represented in the literature from which active compounds were isolated. Mainly, angiosperms more specifically monocots and eudicots are used as QS inhibitors. But cryptogams like gymnosperm, pteridophytes, bryophytes, fungi, and algae are unrepresented groups, containing botanicals which can be utilized in controlling the biofilm by quorum sensing (Ta and Arnason (2015)). Zingiberaceae, Rubiaceae, Lauraceae, and Theaceae families of the tropical and subtropical region contain plants which have active compounds that can be used as QQ. Asteraceae, Lamiaceae, Ericaceae, Berberidaceae, and Apiaceae families of the temperate region also contain bioactive QQ.

26.4.3 Necessities and low falls in QS inhibition Antibiotics were a miracle discovery for controlling the bacterial infection. This boon became a curse with the advent of time because of its improper use and consumption. This leads to the development of multidrug resistance species (Chadha et al., 2021). Recent past it became a great concern for the scientist to ponder and search for alternative strategies like anti-virulence factors which are natural. Botanicals come to the great rescue while using them as antivirulence factor without destroying bacteria. These are mainly the QS inhibitors (Chadha et al., 2021; Kalia et al., 2007). Recent reports also suggest that quorum quenchers also provide less selection pressure, but it is still in research (Kalia et al., 2019; Koul et al., 2016). Quorum quenchers should be chosen very carefully considering some critical factors like molecular weight, specificity, stability, bioavailability, cytotoxicity, and effect on useful gut microbiome. Natural QSIs demand more rigorous and scientific studies before entering clinical trials. These QSIs can be used by formulating a suitable drug delivery system and proper evaluation of its pharmacokinetic parameters. The therapeutic index of QSI will be increased if we consider every parameter into account (Chadha et al., 2021).

26.5

Clinical studies

Even though there are no anti-biofilm agents from plants which have been approved by US Food and Drug Administration yet, few phytochemicals have shown promising results in clinical trials mostly in dentistry and urinary tract infections (Lu et al., 2019) [7]. Table 26.3 enlists some of the phytochemicals with potential anti-biofilm property involved in clinical trials. Camellia sinensis (L.) Kuntze, Punica granatum L., and Lippia sidoides Cham are currently used clinically in the formulation of antiplaque agents (Cardoso et al., 2021).

26.6

Mechanism of phytochemicals involved in quorum-sensing inhibition

The anti-quorum sensing activity of these secondary metabolites is remained as frontiers in today’s research. They are very safe and have fewer side effects. The exact mechanism is still to be understood, but it was found in many studies that their actions are mainly to control cell attachment, adhesion, suppression of polymer formation, interruption in the biosynthesis of extracellular matrix, controlling the pathogenic factor production, etc., (Ghosh et al., 2022). In some recent reviews, it was concluded that phytocompounds mainly act by two general mechanisms (Bouyahya et al., 2022). These are as follows: first through inhibition of the pathway that helps in the generation of QS mediators and second through inhibition of signals that help in QS mediator’s reception.

26.7

Conclusion

The emergence of antimicrobial resistance is among the greatest challenges for the healthcare system. In order to protect human lives from falling prey to challenges, combinations of therapies including phytochemicals, synthetic antimicrobials, and phase therapy would be required. Phytochemicals would thus play an important role in the next generation of treatment of microbial infections. Quorum sensing plays a crucial role in the development and growth of the biofilm. Quorum quenchers are mainly targeted for the AHLS production. Combination therapy and synthetic and natural antimicrobials are the best strategy for anti-quorum sensing agents.

TABLE 26.3 Potential anti-biofilm agents derived from plants under clinical evaluation. Sector

Phytochemicals

Outcome

Reference

Dentistry

Ricinus communis

10% R. communis solutions showed antibacterial efficacy against S. mutans and Candida spp. and in treatment of denture wearers with stomatitis.

Salles et al. (2015); Arruda et al. (2017)

Melaleuca alternifolia

Exerts anti-biofilm effect and inhibits plaque formation and gingivitis in patients with orthodontic treatment

Cardoso et al. (2021)

Azadirachta indica

Exerts anti-biofilm effect and inhibits plaque formation and gingivitis

Cardoso et al. (2021)

Salvadora persica L. (branded as Miswak)

Exerts antiplaque, anti-gingivitis, anticariogenic, promotion of gingival wound healing, whitening properties, orthodontic chain preservation, reduces cariogenic bacterial counts, that is, Streptococcus mutans and Lactobacillus counts, inhibits dental biofilm

Jassoma et al. (2019); Niazi et al. (2018); Nordin et al. (2020)

Camellia sinensis

Controls dental biofilm and used as an adjunct in gingivitis management

Cardoso et al. (2021); Martins et al. (2020); Abdulkareem et al. (2021)

Lemongrass

Decreased oral malodour under 8-day treatment due to its antimicrobial effect

Satthanakul et al. (2015)

Cymbopogon flexuosus, Thymus zygis, and Rosmarinus officinalis

Anti-biofilm potential in the subgingiva

Azad et al. (2016)

Ear infection

Acacia arabica

Inhibited biofilm formation in ear swabs from otitis media-infected patients

Rehman et al. (2016)

Urinary tract infection

Cranberry extract (proanthocyanidin-A, PAC-A)

Exhibits antibacterial effect and is marketed as CISTIMEV PLUS which decreases bacterial load in indwelling urinary catheters in clinical settings

Mantzorou and Giaginis (2018); Singh et al. (2016)

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Acknowledgment Figures in this chapter were created with BioRender.com (https://biorender.com/).

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Chapter 27

Phytoconstituents-based nanoformulations for neurodegenerative disorders Mithun Singh Rajput1, Nilesh Prakash Nirmal2, Viral Patel3, Purnima Dey Sarkar4 and Manan Raval5 1

Department of Pharmacology, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand,

Gujarat, India, 2Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, Thailand, 3Department of Pharmaceutics, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand, Gujarat, India, 4Department of Medical Biochemistry, M.G.M. Medical College, Indore, Madhya Pradesh, India, 5Department of Pharmacognosy, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Anand, Gujarat, India

27.1

Introduction

Neurodegenerative diseases are assorted clusters of ailments marked by the developing degeneration of the composition and activity of the central or peripheral nervous system (Khan et al., 2021). Such lasting progressive damages may cause disability in thinking, movement, cognition, and memory (Moradi et al., 2020). In our aging society, the increase of patients with age-associated NDs is an oppressive issue for medical care, society, and economy. Genetic susceptibility, aging, lifestyle, nutrition, chemicals, specific viruses, and exposure to some environmental toxins (Hodjat et al., 2017) are supposed to be predominant risk factors of NDs. As the worldwide average life expectancy has increased, the prevalence of age-related NDs has risen dramatically. NDs can cause problems related to movement (ataxias) or mental functioning (dementia) and can lead to death, having profound social and economic implications (Wynford-Thomas & Robertson, 2017). The major ND, Alzheimer’s disease (AD), alone represents 60%
70% of dementia cases all over the world (Qiu et al., 2009). Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Huntington’s disease (HD), motor neuron disease, prion disease, spinocerebellar ataxia, multiple sclerosis (Ms), and spinal muscular atrophy also belong to the similar menage that include cognitive and behavioral disturbances (Qiu et al., 2009). According to the WHO study on the top 10 causes of worldwide death, the rate of dementia and age-related NDs-related mortality has increased more than thrice since the year 2000, and dementia was the fifth cause of death in 2016. (WHO, 2018) Thereby, it is predicted that mental and emotional defects will cause emotional, social, and financial burdens on the healthcare system in the future. Neuroprotection is aimed to prevent neuronal death, regenerate neuronal networks, and ameliorate the brain dysfunction as “disease-modifying” therapy (Naoi et al., 2019). Symptomatic relief is the only intervention for the management of such ailments; for example, cholinesterase inhibitors are prescribed to enhance cognition, dopaminergic balance for movement disorders and Parkinsonism, antipsychotic drugs for dementia, analgesic and anti-inflammatory agents for neuronal pain and infections, etc., (Rajput & Sarkar, 2017). Advancement in therapeutic management is required to manage numerous other progressive and grave symptoms of the diseases. Integrative treatments along with potential phytochemicals-based therapies are also on the frontline of research to improve etiology-related therapeutic strategies. Herbal preparation and phytochemicals isolated from plants have been proposed as “herbal medicine” for the treatment of various disorders. A large number of pharmacological or biological activities of the phytochemicals have made them appropriate candidates for the treatment of NDs (Khan et al., 2021). Phytochemicals and herbal nutraceuticals are being used to combat disease symptoms, and various trials employing phytochemicals to prevent NDs have been published (Venkatesan et al., 2015). Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00003-7 © 2023 Elsevier Inc. All rights reserved.

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Etiology of neurodegeneration and neuronal functional impairment may be associated with mitochondrial dysfunction, accumulation of misfolded protein, lack of neurotrophic factor production, endogenous antioxidant enzyme activity depletion, neurotrophin deficiency, and sometimes defects at the genetic and molecular levels (Eltanameli et al., 2022). Due to the different mechanisms involved in the central nervous system (CNS) conditions, it is challenging to have a single treatment strategy to treat these conditions. Besides, the blood
brain barrier (BBB) is another obstacle that hinders potential drugs from crossing into the brain. One of the major hindrances that constrain the progress of effective medications for the management of neurodegenerative disorders is the selective property of the BBB, which averts infiltration of a large number of CNS drugs through brain (Mechan et al., 2006). The brain capillary endothelial cells, which limit trans-cellular transit, and the tight and adherens junctions between the cells, which limit para-cellular flow, are primarily responsible for the BBB’s functional complexity. Consequently, only less than 5% of active ingredients are able to enter the brain (Mechan et al., 2006). Moreover, in order to reach satisfactory therapeutic efficacy in brain, the drugs have to be administered in high doses resulting in severe peripheral side effects. To overcome such limitations, advancement in research on colloidal delivery systems is evident that attain the benefits of particle size reduction, such as liposomes, polymeric nanoparticles, solid lipid NPs, metal nanoparticles-based carriers, cubosomes and emulsions, and, very recently, magnetic nanoparticles (Fakhri et al., 2022; Moradi et al., 2020). Ascertaining the therapeutic efficacy of plant-based bioactive compounds with amalgamation of nanotechnology-based approach is trending, which also exhibits the benefit of lessening side effects (Fakhri et al., 2022). The purpose of this chapter is to review most of the current advancements in the field of neuroprotection by herbal products on various NDs and to elicit their putative mechanisms of action, with a focus on phytochemical nanoformulations.

27.2

Key issues associated with neurodegenerative diseases

Numerous neurological diseases, such as AD, PD, HD, and ALS, are linked to aging. As the population ages, neurodegenerative contagions are becoming a major socioeconomic burden in many countries. In these disorders, a collection of misfolded and totaled proteins in the mind are a common neurotic aggregation. A developing assortment of proof demonstrates that toxic protein conglomeration and neurodegenerative infections are connected. Because of a better understanding of the etiology of these illnesses (Fig. 27.1), groundbreaking efforts for disease-changing therapies have recently greatly increased (Kim & Mook-Jung, 2015).

FIGURE 27.1 Etiology of various neurodegenerative disorders.

Phytoconstituents-based nanoformulations for neurodegenerative disorders Chapter | 27

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27.3 Significance of nanotechnology in neurodegenerative disorders: incapacitating the blood
brain barrier The inability of most medicinal substances to cross the BBB effectively limits the therapy options for NDs. As a result, the BBB is one of the most significant roadblocks to advancement in the treatment of NDs (Cen˜a & Ja´tiva, 2018). In most vertebrate species, the BBB is made up of an evolved form of the brain microvascular system membrane barrier that separates circulating blood from the extracellular fluid in the brain (Ding et al., 2020). The BBB is made up of the basal lamina, which surrounds pericytes and microvessel endothelial cells and contains extracellular matrix proteins such as collagen, heparan sulfate, and laminin (Fakhri et al., 2022; Saraiva et al., 2018). Other structures that make up the BBB include astrocyte endfeet and interneurons. Gap junctions, tight junctions, and adherens junctions already exist in the BBB. Tight junctions, on the other hand, appear to be more significant than other types of BBB connections because they create large amounts of electrical resistance between endothelial cells (Ding et al., 2020). The most important function of BBB is to protect the brain from all outside agents. BBB also protected the brain from changes in ionic composition in the cerebrospinal fluid, as well as facilitating the metabolization of various chemical compounds and the elimination of waste materials (Fakhri et al., 2022; Saraiva et al., 2018). Disruption of extracellular matrix proteins can increase BBB permeability, assisting and improving drug delivery (Fakhri et al., 2022). Nanotechnology, on the other hand, has grown rapidly in recent years and has the potential to provide considerable advances in the identification and treatment of CNS diseases. The term “nanotechnology” refers to the control or manipulation of nanometer-scale engineered materials or devices (Silva, 2006). Because of changes in the arrangement and spacing of surface atoms and molecules, nanomaterials differ dramatically from their macroscale counterparts (Moghimi et al., 2005). Nanomaterial-based technologies have several uses in biomarker identification, treatment, and theranostics. Modified nanomaterials could be utilized to identify and cure damaged cells and tissues on a molecular level, for starters. When a surface has been modified with unique molecules, nanoengineered materials may also preserve medicine release, boost bioavailability, distribute multiple agents, and prevent compounds from degradation (Bhattacharya et al., 2022). Nanomaterials with surface functionalization can be utilized to target and infiltrate the BBB while also enhancing the nanomaterial’s blood half-life (Pardridge, 2016). Nanomaterials are at the top of the list for diagnosing and treating CNS illnesses due to their unique properties. There are a number of promising advantages to using nanoparticles in medicine, including high drug-loading capacity, which reduces the risk of chemical interactions or toxicity; a high surface area-tovolume ratio, which makes parenteral administration easier; the ability to use active and passive drug-targeting strategies; and sustained and continuous dosing options (Bhattacharya et al., 2022; Goldsmith et al., 2014). Nanoparticle size, targeting properties, lipid or water solubility and their respective hydrophobicity or hydrophilicity, chemical and physical stability, surface charge, permeability, biodegradability, biocompatibility, cytotoxicity, drug release profile, and antigenicity of the final product all play a role in the nanoparticle manufacturing materials selection process (Bhattacharya et al., 2022; Goldsmith et al., 2014). Passive and active transfer channels are the most common ways for nanoparticles to penetrate the BBB. In a passive transfer method, gold nanoparticles and tiny lipophilic compounds (400 Da) might flow through the BBB (Pardridge, 2005). In addition, receptor, carrier, and adsorption techniques all play a role in active endocytosis. The entry of different nanostructures such as liposomes and PLGA-based nanoparticles through the BBB is facilitated via receptor-mediated endocytosis (Fornaguera et al., 2015). Low-density lipoproteins, transferrin, lactoferrin, insulin receptors, and their ligands are all active influence receptors (Ding et al., 2020). The surface characteristics of nanoparticles mediate the process of nanoparticle transportation to endothelial cells via adsorption. Because endothelial cells’ plasma membranes are negatively charged, cationic nanoparticles are more likely to undergo this process than negatively charged or neutral nanoparticles. Using this approach, liposomes and gold nanoparticles can penetrate the BBB (Zhou et al., 2018). Carrier-mediated transport routes, such as the amino acid transporter (ASCT2) protein and the glucose transporter 1 (GLUT1) protein, are another method of nanoparticles for improving drug delivery across the BBB (Moradi et al., 2020).

27.4 Phytoconstituents and their general mechanism of actions pertaining to neuroprotection Chemical exposure, lifestyle, low levels of nerve growth factors such as BDNF, NGF, and others, as well as aging, are all factors that contribute to NDs. The most common cause of NDs is advancing age. All physiological and metabolic processes slow down as people age, which affects brain function, growth factors, and neuron damage, leading to NDs. The NDs eventually turn into life-threatening illnesses (Schulz & Deuschl, 2015). It is suggested that natural therapy is used to treat aging persons with the purpose of minimizing drug adverse effects (Albarracin et al., 2012). Vegetables,

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fruits, and herbs should be consumed on a daily basis to maintain a healthy lifestyle. Fruits, herbs, and vegetables are well known as the best sources of phytochemicals (Davinelli et al., 2016). Phytochemicals (phyto 1 chemicals 5 natural chemicals produced from plants) are naturally obtained from plants and have a wide range of medicinal properties (Wang et al., 2018a, 2018b). Apart from their beneficial effects, phytochemicals are viewed as a promising treatment option for a variety of ailments. Fruits- and vegetables-based diet was an integral part of the food component for a healthy lifestyle in ancient times because of its ability to improve immunity and antioxidative activity. This type of food gives you the strength to battle diseases like cancer, tumors, neurodegeneration, diabetes, and so on (Jahan et al., 2017). Polyphenols from vegetables and fruits have long been studied for their possible antioxidative, anti-inflammatory, and neuroprotective properties. As a result, researchers are becoming interested in herbal medications in order to develop polyphenols as a prospective and promising therapy for a variety of diseases (Azam et al., 2019). Our bodies produce free radicals as a result of several metabolic activities, but they also have routes for scavenging them. When the body is unable to balance free radicals, oxidative stress, mitochondrial malfunction, and inflammation develop (Huang et al., 2013). Oxidative stress, mitochondrial dysfunction, and inflammation are all important factors in the development of NDs. We employ a variety of readily available phytochemicals on a daily basis, including resveratrol, quercetin, curcumin, and many others (Khan et al., 2021). Researchers have also explored the importance of phytochemicals found in olive oil, avocado, kiwi, grapes, and a variety of other foods. It is claimed that eating a Mediterranean diet rich in olive oil reduced overall mortality among Parkinson’s and Alzheimer’s patients, indicating a considerable improvement in health condition (Kahkeshani et al., 2015). There is growing evidence that phytochemicals can operate as nutraceuticals, alleviating the symptoms of NDs, although the mechanism of action of these phytochemicals is unknown (Zhang et al., 2015). According to another study, phytochemicals can be a promising therapeutic agent due to their antioxidative and radical scavenging properties. However, more research into mechanistic activity is required. Many hypotheses have been proposed that their function is mediated by modulating enzyme metabolism, gene expression, and the regulation of numerous small cascade molecules that are activated in response to healing and stress (Paredes-Gonzalez et al., 2015). In terms of the key lock idea, another concept emphasized the significance of phytochemicals. Phytochemicals are said to work as ligands, binding to certain receptors on cells and then indirectly participating in a cascade mechanism that results in possible therapeutic efficacy (Krajka-Ku´zniak et al., 2015). Many phytoconstituents have been reported to be neuroprotective by stimulating various signaling pathways, such as resveratrol’s PKA-mediated activation of GSK3, catenin, CREB, and ERK1/2 (Jahan et al., 2018).

27.5

Phyto-nanomedicine in the management of neurodegenerative disorders

A number of synthetic medications have shown promise in the treatment of NDs such as autism, PD, AD, and other chronic disorders. Synthetic medications come with a slew of negative side effects, making them unsuitable for long-term use (Xu et al., 2018). Because of the negative effects of these synthetic medications, scientists have shifted their focus to the use of phytochemicals, which have fewer side effects. Phytochemicals have antioxidative, anticholinesterase, antiinflammatory, and anti-amyloid characteristics, making them a prospective therapeutic agent (Hajialyani et al., 2019). Given that present therapies appear to be insufficient for the aforementioned illnesses, scientists are experimenting with plant-based medications and nanotechnological techniques to see what they can come up with. Nanotheranostics is one such method that is getting widespread interest in the scientific community for the treatment of ND on a global scale. It uses nanoparticles for both diagnostic and treatment at the same time. Tripathy et al. (2018) discovered that this treatment has gained traction in the medical community since it is quite aggressive and directly targets the problem area (Tripathy et al., 2018). Furthermore, variations in terms of disease kind and tailoring based on the patient’s demands can be accommodated, extending the approach’s usefulness (Bar-Zeev et al., 2017). Chemical engineers have devised a revolutionary nanotheranostic device that uses tunable light to activate nanoparticles and opens up new possibilities in the sector (Kim et al., 2013a, 2013b; Vuilleumier et al., 2019). Table 27.1 summarizes many types of plantbased medications used in ND therapies, as well as the traditional methodologies and contemporary advances in plantderived nanomedicine discovered thus far (Table 27.1).

27.6

Nanoformulations in tackling neurodegeneration: preclinical proofs

Recently, pharmaceutical research has focused on developing nanotechnology approaches that are applicable to a variety of medical fields, including medication delivery (Calzoni et al., 2019). As a result, fresh approaches to improving the efficacy, transport across the BBB, bioavailability, and, ultimately, the negative impact of pharmacological drugs

TABLE 27.1 Phyto-nanomedicine in the management of neurodegenerative disorders. Plant

Common name

Family

Major neuroprotective chemical constituent

Mechanism of neuroprotection

References

Acorus calamus

Sweet flag

Acoraceae

α- and β-asarone

Suppression of Aβ-induced neuronal apoptosis, inhibitory function on AChE, improvement in dopaminergic nerve function

Bandelow et al. (2017)

Allium sativum

Garlic

Amaryllidaceae

S-allyl cysteine

Antioxidant, decrease in lipid peroxidation and DNA fragmentation, protection of dopamine levels

Powell et al. (2018)

Bacopa monnieri

Brahmi

Scrophulariaceae

Steroidal bacosides A and B, bacopa saponins F, E, and D

Upregulation of glutathione activity, inhibition of lipid peroxidation, regulation of Hsp 70 and cytochrome P450, and reduction in aggregation of alpha-synuclein in brain

Shang et al. (2019)

Centella asiatica

Spade leaf

Umbelliferae

Asiaticoside, brahminoside, centella saponin, naringin, rutin, sitosterol, quercetin

Decreases Aβ deposition, exhibits antioxidant activity, restores GSH levels, amelioration of decrease in AChE activity in the brain

Chen and Pan (2014), Roy and Awasthi (2018)

Curcuma longa

Turmeric

Zingiberaceae

Curcumin, desmethoxycurcumin, and Bisdesmethoxycurcumin

Prevents Aβ aggregate formation, inhibition of Aβ oligomerization and formation of fibril, causes a macrophage enhancement of Aβ uptake, and inhibits the Aβ-heme complex peroxidase activity

Roy and Awasthi (2018), Chopra et al. (2021), Soares et al. (2021)

Celastrus paniculatus

Jyotishmati

Celastraceae

Sesquiterpene, evoninoate, paniculatine A and B, ofornine, celapagine, malkanginnol, malkanguniol, β-amyrin, and β-sitosterol

Decreases oxidative stress, increases glutathione and catalase levels, decreases AChE levels

Roy and Awasthi (2018), Jakka (2016)

Coriandrum sativum

Dhaniya

Apiaceae

Quercetin 3-glucoronide, protocatechuic acid, glycitin, and caffeic acid

Reduction in size of cerebral infarct, calcium levels, and lipid peroxidation in brain

Amadi et al. (2019), Ashraf et al. (2019), Owoeye et al. (2019)

Galanthus nivalis

Snowdrop

Amaryllidaceae

Galantamine

Inhibiting AChE activity and stimulation of nicotinic receptors

Yang and Chang (2019)

Gingko biloba

Maidenhair tree

Ginkgoaceae

Isorhamnetin, quercetin, kaempferol, ginkgolides A, B, C, J, and M

Inhibition of Aβ aggregation, antioxidative effects, decrease in damage caused due to excitotoxicity and global brain ischemia, inhibits AChE activity

Yuan et al. (2017)

Glycyrrhiza glabra

Yashtimadhuh or liquorice

Leguminosae

Glabridin

Decreases MDA level and increases the superoxide dismutase level while reducing glutathione levels, restored the decreased concentration of dopamine and glutamate in the brain and decreased activity of AChE

Abdolmaleki et al. (2020)

Hypericum perforatum

Millepertuis or hypericum

Hypericaceae

Hyperoside, hypericin, kaempferol, biapigenin, and quercetin

Protects brain cells from cytotoxicity through the reduction of glutathione loss, overload of calcium and cell death mediated through ROS, improvement of microglial viability through the reduction of toxicity by amyloid beta in AD, inhibits AChE and MDA formation in the brain

Bridi et al. (2018)

(Continued )

TABLE 27.1 (Continued) Plant

Common name

Family

Major neuroprotective chemical constituent

Mechanism of neuroprotection

References

Lycopodium serratum

Ground pines or creeping cedar

Lycopodiaceae

Huperzine A

Inhibitor of AChE, protective effects such as amyloid precursor protein metabolism regulation, oxidative stress protection mediated by Aβ, apoptosis, dysfunction of mitochondria, and antiinflammation

Agatonovic-Kustrin et al. (2018)

Melissa officinalis

Lemon balm

Lamiaceae

Quercetin, apigenin, luteolin

Inhibits monoamine oxidases and AChE, protects neurons from oxidative stress

Eudes et al. (2017)

Ocimum sanctum

Tulsi or holy basil

Labiatae

Rosmarinic acid and ursolic acid

Restores choline acetyltransferase expression, increased production of Ach, inhibits peroxidation of lipids, ROS generation, damage to DNA, and depolarization of membranes

Kusindarta et al. (2018), Antonescu Mintas et al. (2021)

Panax ginseng

Ginseng

Araliaceae

Gensenosides

Reduction in the Aβ deposition, preventing apoptosis and neuronal death, suppresses cellular AChE activity and enhances cholinergic metabolism

Ahuja et al. (2018)

Rosmarinus officinalis

Rosemary or Satapatrika

Lamiaceae

Eugenol, carvacrol, oleanolic acid, and ursolic acid

Possesses cytoprotective, antiapoptotic, and antiinflammatory activities

Nieto et al. (2018)

Salvia officinalis

Garden sage

Lamiaceae

Carnosic acid and rosmarinic acid

Low AChE inhibitory effect, inhibits ROS formation, peroxidation of lipids, fragmentation of DNA, activation of caspase-3, and hyperphosphorylation of tau protein

Lopresti (2017)

Terminalia chebula

King of medicines in Tibet

Combretaceae

Arjungenin, sarjunglucoside 1, chebulosides 1 and 2, chebulic acid, chebulinic acid, tannic acid, ellagic acid, punicalagin, terflavin A, rutin, luteolin, and quercetin

Protective effect on neuronal cells against ischemia, stimulates microglia cells death rate

Lee et al. (2017)

Tinospora cordifolia

Giloe

Menispermaceae

Alkaloids, diterpenoid lactones, steroids, glycosides, and aliphatic acids

Increases concentration of glutathione and the expression of the gamma-glutamyl-cysteine ligase and superoxide dismutase, decreases the mRNA expressions of iNOS, increases the dopamine level of the brain

Tiwari et al. (2018)

Withania somnifera

Ashwagandha or Indian ginseng

Solanaceae

Withaferin A, withanolide D, and withanolides

Reduces dopaminergic neurodegeneration

Roy and Awasthi (2018), Raja Sankar et al. (2009)

Zizyphus jujube

Jujube

Rhamnaceae

Oleamide

AChE inhibitory activity, antioxidant properties, upregulation of acetylcholine transferase, increase in acetylcholine level

Roy and Awasthi (2018), Olasehinde et al. (2017)

Aβ, Amyloid beta; AChE, acetylcholine esterase; AD, Alzheimer’s disease; GSH, glutathione; Hsp, heat shock protein; MDA, malondialdehyde; ROS, reactive oxygen species.

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FIGURE 27.2 Phytochemicals-based nanoformulations in tackling neurodegeneration.

used to treat NDs must be developed (Sahni et al., 2018). Nanoparticles used to transport therapeutic chemicals improve biodistribution and pharmacokinetics, allow for multiple medications to be delivered simultaneously, enable focused intracellular drug delivery, and reduce systemic toxicity and adverse effects (Cerqueira et al., 2020) (Fig. 27.2).

27.6.1 Phytoconstituents-based nanoformulations for Alzheimer’s disease The most common neurodegenerative condition, Alzheimer’s disease (AD), is the primary cause of dementia, a clinical condition marked by a gradual decline in two or more general cognitive abilities, such as debilitated cognition, language, executive/visuospatial action, personality, and behavior, resulting in a loss of ability to execute instrumental and/ or fundamental activities (Rajput et al., 2020a). After 3
6 years of amnestic mild cognitive impairments, it is usually detected. Extracellular A plaques and intraneuronal tau-containing neurofibrillary tangles have been seen in the brains of Alzheimer’s patients, which are linked to inflammatory/oxidative stress/apoptotic pathways (Rajput et al., 2020b). The presence of BBB is important in the treatment of neurological illnesses because it prevents many medications from reaching the brain in sufficient concentration (Mechan et al., 2006). So, there is a requirement for nanomedicine therapy to solve such problems. Plants and their phytochemicals have proved to have anti-AD properties (Fakhri et al., 2022). Several studies have revealed resveratrol’s beneficial potential against AD. Because resveratrol leaves the bloodstream quickly, solid lipid nanoparticles (SLNs) were created to encapsulate it and transport it to the brain, where amyloid fibril formation occurs. By suppressing the creation of Aβ1
42 aggregates, SLNs offer a potential, dynamic mechanism for delivering resveratrol to the brain in a specific location and preventing/slowing AD progression (Loureiro et al., 2017). In addition, when compared to an orally administered resveratrol solution, in vivo testing revealed that developed nanostructured lipid carriers (NLCs) injected nasally can effectively prevent AD (Rajput et al., 2018). Curcumin, a phenolic substance obtained from Curcuma longa, has positive effects in the treatment of NDs; however, curcumin’s low bioavailability, fast metabolism, and quick elimination hampered its usefulness (Anand et al., 2007). Even at low dosages, nanocurcumin with increased bioavailability inhibited the development and neurotoxicity of two AD indicators, hyperphosphorylated tau and amyloid misfolding (Cheng et al., 2013). It also regulates various facets of AD, including insulin signaling, cholesterol levels, acetylcholinesterase inhibition, microglial function regulation, and antioxidant activity (Mandal et al., 2020). Curcumin encapsulated in PLGA nanoparticles with a ligand for BBB bridging has low toxicity and reduces Aβ aggregates significantly (Doggui et al., 2012). In vitro and in the hippocampus and subventricular zone in vivo, nanocurcumin-encapsulated PLGA nanoparticles increased the proliferation of endogenous neural stem cells and neuronal development, especially when compared to bulk curcumin (Barbara et al., 2017). Cur-PLGA nanoparticles activated the canonical Wnt/β-catenin pathway in an AD rat model, restoring mediated

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inhibition on hippocampal neurogenesis, cognition, and memory (Tiwari et al., 2014). In vitro, PLGA-poly(ethylene glycol) (PLGA-PEG) nanoparticles coupled with B6 peptide-loaded with curcumin (PLGA-PEGB6/curcumin) may be a viable solution for the treatment of AD by lowering curcumin size, increasing cellular absorption, and improving blood compatibility. The results demonstrated that lowering hippocampal Aβ synthesis and deposit, as well as tau hyperphosphorylation, improved spatial learning and memory function in APP/PS1 mice as compared to native curcumin (Fan et al., 2018). Nanoliposomes, another innovative curcumin formulation, were shown to be stable and monodispersed. These formulations were found to be safe in vitro, suppressed the synthesis of amyloid peptides, and reduced Aβ-induced toxicity to a degree (Lazar et al., 2013). Naringenin is a flavonoid that protects neurons from free radicals and inflammation; however, it does not penetrate biological membranes well. The naringenin-loaded nanoemulsion greatly reduced Aβ’s direct toxic effect on SH-SY5Y cells, which was connected to a decrease in APP and β-secretase expression as well as reduced amyloidogenesis. The amount of phosphorylated tau in SH-SY5Y cells exposed to Aβ was likewise lowered. These findings suggested that a naringenin-loaded nanoemulsion could be used to treat AD (Md et al., 2018). Quercetin, another phytochemical, has shown promise in the treatment of NDs. Quercetin’s therapeutic application was limited due to its low oral absorption. Nanoencapsulated quercetin in zinc nanoparticles greatly enhanced oral absorption and bioavailability of the flavonoid as a potential treatment for AD. Treatment with this orally administered flavonoid improved SAMP8 mice’s cognitive and memory deficiencies (Puerta et al., 2017). When quercetin was integrated into cyclodextrin-dodecyl carbonate nanoparticles, its anti-inflammatory effects on SH-SY5Y cells were enhanced via inhibiting the toll-like receptor 4 (TLR4) and COX-2 signaling pathways, compared to cells treated with free quercetin (Testa et al., 2014). Pretreating rats with quercetin nanoparticles reduced scopolamineinduced behavioral changes, implying that because of its increased bioavailability, it could be employed as a prophylactic strategy against the progression of AD (Palle & Neerati, 2017). In vitro studies revealed that PLGAfunctionalized quercetin nanoparticles have low cytotoxicity, indicating that they can prevent Zn21-Aβ1
42 system neurotoxicity while simultaneously boosting neuron cell survival by inhibiting the system. According to evidence from in vivo studies, giving PLGA-functionalized quercetin nanoparticles to APP/PS1 mice improved cognitive skills and memory (Sun et al., 2016). Epigallocatechin-3-gallate (EGCG), the major catechin present in tea, has been shown to have antioxidant properties. It can increase the production of α-secretase, which can improve the non-amyloidogenic processing of APP, lowering the formation of brain Aβ plaques, which is a hallmark of AD pathogenesis. In vitro, EGCG nanolipidic particles increased neuronal secretase activity by up to 91%, while oral bioavailability in vivo was increased by more than double over free EGCG (Smith et al., 2010). EGCG has been shown to have anti-amyloidogenic, metal chelation, and antioxidant properties. NanoEGCG’s antioxidant and metal chelation properties surpassed those of its free form, lowering cellular toxicity substantially. In addition, an in vitro investigation found that EGCG nanoparticles could protect against Al31-induced Aβ42 fibrillation and neurotoxicity (Singh et al., 2018). EGCG was bound to the surface of selenium nanoparticles in another investigation to reduce EGCG’s cytotoxicity at high concentrations. Due to the poor transport efficiency of EGCG-selenium nanoparticles to the targeted cells, EGCG-selenium nanoparticles were produced, given the peptide’s affinity for neurons. In order to prevent the onset and progression of AD, selenoprotein’s role in antioxidation and neuroprotection is critical. Aβ fibrillation was suppressed, and Aβ fibrils were efficiently disaggregated into safe aggregates using EGCG-stabilized selenium nanoparticles coated with peptides. EGCG-selenium nanoparticles also had a considerable propensity for tagging Aβ fibrils (Zhang et al., 2014). The major component of sesame seed oil is sesamol, a polyphenolic compound. When the pure sesamol and sesamol-SLN groups were compared, the latter was significantly more effective than the pure sesamol group at a dosage of 16 mg/kg, which was roughly equivalent to the rivastigmine effects. By lowering oxidative stress, it was found that placing sesamol in the SLNs is a great way to reduce intracerebroventricular streptozotocin-induced neuronal dysfunction and memory deficits (Sachdeva et al., 2015). Ferulic acid, another phenolic molecule, has been shown to be an effective antioxidant in the treatment of AD. At the levels examined, pure SLN showed no toxicity and the ability to enter human neuroblastoma cells (LAN 5) with zero toxicity. Furthermore, cells treated with ferulic acid-loaded SLN produced fewer reactive oxygen species (ROS) than cells treated with free ferulic acid (Meng et al., 2018). Alkaloids, in addition to phenolic compounds, are another class of phytochemicals that have shown promising neuroprotective benefits in nanoformulations. Berberine is an isoquinoline alkaloid that has been used for millennia to treat NDs, including dementia (Cai et al., 2016). Berberine-loaded multiwalled carbon nanotubes are a fantastic nanostructured construct for getting berberine into the BBB. In addition, the phospholipid-coated and polysorbate-coated multiwalled carbon nanotubes showed good memory function recovery, which corresponded to their ability to transmit neuropharmaceutical substances to brain microglial cells. Because these nanotubes were able to maintain normal biochemical levels in brain

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tissue, they showed promise in reducing Aβ-induced AD (Lohan et al., 2017). Ginsenosides, which are triterpenoids, are another phytochemical with neuroprotective properties. A new nanotherapeutic method that boosts BBB permeability to promote ginsenoside distribution to the brain might aid neuroprotective effects and reduce the formation of Aβ plaques and eventual dementia. PLGA-ginsenoside Rg3 nanoparticles revealed an intriguing new theranostic material ideal for encapsulating natural nutraceuticals for the detection and therapy of Alzheimer’s disease (Aalinkeel et al., 2018). Hence, nanoformulations of phytochemicals (e.g., berberine, curcumin, EGCG, ferulic acid, ginsenoside, huperzine, naringenin, quercetin, rosmarinic acid, resveratrol, and sesamol) from various classes (e.g., alkaloids, phenolic compounds, and terpenoids) have demonstrated therapeutic effects on AD by modulating various pathways like suppression of protein misfolding and aggregation, as well as modifying neuroinflammatory, neuroapoptosis, and neuronal oxidative stress. Nanophytochemicals also alter the pathways outlined above, which improve the bioavailability of secondary metabolites and reduce cellular toxicity.

27.6.2 Phytoconstituents-based nanoformulations for Parkinson’s disease The progressive loss of dopaminergic neuronal cells in the substantia nigra pars compacta portion of the basal ganglia causes PD. The primary pathophysiological aspects of PD include oxidative stress, inflammation, and apoptosis (Mandal et al., 2020). Mutations in the gene α-synuclein, which encodes for a protein that has been identified as one of the contributing factors for the onset of PD, are thought to have a strong hereditary relationship (Sim et al., 2020). There are numerous phytochemicals that have been identified as effective in the treatment of PD (Fakhri et al., 2022). Resveratrol nanoparticles can maintain blood levels for longer periods of time, enhancing bioavailability and, as a result, improving pharmacological efficacy. Consequently, resveratrol nanoparticles proved to be more efficient than naive resveratrol in reducing rotenone-induced PD-like behavioral impairments in rats (Palle & Neerati, 2018). In vitro, Fe3O4-modified resveratrol liposomes, a magnetic targeting drug nanocarrier, displayed sustained and delayed drug release. In the presence of an external magnetic field, in vivo tests indicated that such nanoparticles could easily penetrate the BBB and increase medication concentration at the targeted location (Wang et al., 2018a, 2018b). The nasal mucosa penetration of resveratrol-loaded nanoemulsions for the treatment of PD was rather high in vitro and ex vivo. According to histological and biochemical examinations, the brain tissues of the group given resveratrol nanoemulsions exhibited lower levels of degenerative changes, oxidative stress indicators, and fewer eosinophilic lesions than the positive control group (Pangeni et al., 2014). Resveratrol in polysorbate 80-coated poly(lactide) (PLA) nanoparticles can protect neurons from behavioral and metabolic changes. These findings indicated that resveratrol-loaded PLA nanoparticles coated with polysorbate 80 boosted resveratrol concentration in the brain, suggesting that it could be a promising nanomedical device and adjuvant therapy for NDs like PD (daRocha Lindner et al., 2015). Curcumin, in the form of nanoformulation bound to polybutyl cyanoacrylate, has neuroprotective benefits in PD, passes the blood
brain barrier, and reduced PD symptoms (Mandal et al., 2020). Piperine and curcumin co-encapsulated glyceryl monooleate (GMO) nanoparticles suppressed S protein oligomerization and fibril formation, decreased rotenone-induced toxicity, oxidative stress by reducing GSH depletion induced by rotenone, apoptosis by reducing the ratio of Bcl-2 to Bax, and initiation of the autophagic pathway in vitro. Furthermore, in a PD animal model, in vivo studies revealed that such nanoparticles could cross the BBB, reverse rotenone-induced motor coordination impairment, and prevent dopaminergic neuronal degeneration (Kundu et al., 2016). It was discovered that combining a vitamin E-loaded naringenin nanoemulsion with standard medication (levodopa) efficiently restored the effects of 6-hydroxydopamine (6-OHDA), including increased muscle coordination, grip strength, and swimming activity. In a rat model, it also enhanced naringenin levels in the brain and increased cerebral bioavailability. Although GSH and SOD levels were significantly higher in the group given naringenin nanoemulsion intranasally with levodopa, malondialdehyde (MDA) levels were significantly lower (Gaba et al., 2019). Quercetin is a bioflavonoid found in a number of fruits and vegetables that have been shown to have neuroprotective qualities by mending mitochondrial electron transport chain defects and enhancing mitochondrial quality control (Burgos et al., 2020). Furthermore, quercetin has a great capability for removing ROS. Low solubility and bioavailability have hampered quercetin’s medicinal applications; hence, the bioavailability and efficacy of quercetin nanocrystals were found to be higher in PD-like animals than in naive quercetin. In the hippocampus, antioxidant enzyme activities and total GSH levels increased significantly, while malondialdehyde levels decreased. As a result, nanoformulations of resveratrol, curcumin, piperine, naringenin, quercetin, and gallic acid have demonstrated a variety of therapeutic effects on PD by increasing bioavailability and lowering rotenone’s adverse effects. Nanophytochemicals enhance neurogenesis, block apoptotic pathways, and reduce oxidative stress/inflammation from a molecular standpoint.

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27.6.3 Phytoconstituents-based nanoformulations for amyotrophic lateral sclerosis ALS, also known as motor neuron disease or Lou Gehrig’s disease, is a rapidly progressive ND that causes the failure of muscular movement neurons. ALS attacks motor neurons in the brain, brainstem, and spinal cord, causing progressive motor neuron degeneration and muscle atrophy, paralysis, and death due to respiratory failure. ALS is caused by gene mutations that disrupt regular protein functioning, oxidative stress, protein misfolding and aggregation, mitochondrial dysfunction, and neuroinflammation (Wang et al., 2020). Pathological inclusions, such as SOD1 in ALS, have been found to be equivalent to amyloid fibrils formed in vitro, according to certain studies. In mice models with human SOD1 mutations, fibrillar inclusions characterize the disease of brain tissues (Wang et al., 2002). Inhibiting the formation of SOD1 amyloid could be a useful strategy for fighting ALS. Curcumin inhibited SOD1 fibrillation and favored the formation of smaller, disordered aggregates as a result. Curcumin nanoparticles with a higher water solubility managed aggregation in the same way as curcumin bulk (Bhatia et al., 2015). Ahmadi et al. (2018) developed and executed a randomized clinical trial in patients with ALS to determine the efficacy and safety of oral capsules containing 80 mg nanomicelles curcuminoids coupled with riluzole. The findings demonstrated that nanocurcumin was safe, with no major side effects, increasing the likelihood of survival in treated individuals, particularly those with irritable bladder (Ahmadi et al., 2018). Tripodo et al. also proposed a novel drug delivery system for the treatment of NDs, including ALS. Curcumin and mesenchymal stromal cells were loaded into micelles to create a carried-in-carrier system. According to the findings, these nanoparticles loaded maximally in micelle-laden mesenchymal stromal cells in minutes, and the loading was concentration-dependent. Mesenchymal stromal cells demonstrated cytotoxicity when exposed to bare curcumin; however, naive curcumin micelles protected them from this effect (Tripodo et al., 2015).

27.6.4 Phytoconstituents-based nanoformulations for stroke (cerebral ischemia) Cerebrovascular diseases are the first reason behind physical impairment, the second most vital cause of death, and also the main reason for hospital inmate hospitalization for several patients. Stroke is the most frequent vessel malaise and is the third-largest cause of mortality within the developed world, inflicting 15 million injuries and 5 million fatalities every year. Cerebral ischemia accounts for 80% of all stroke injuries caused by hypoperfusion, thrombosis, or embolism (Gao et al., 2013). Many variables, notably aerophilous stress, apoptosis, and dropsy caused by ionic imbalance and inflammation, are thought to own a task in the pathophysiology of ischemic stroke (Nouri et al., 2019). Clinical trials have indisputable that the biological activities of many seasoner formulations made from natural merchandise are related to their inhibitor properties that is taken into account together with the treatment mechanisms of anemia stroke. The most issue with the mistreatment of phytochemicals in stroke treatment is their poor bioavailability, limiting their effectualness in clinical studies (Mutoh et al., 2016). In rats with transient middle arterial blood vessel occlusion, the intra-arterial injection of resveratrol nanoparticles exhibited extra safety from cerebral ischemia/reperfusion (I/R) damage. Resveratrol nanoparticles reduced aerophilous stress caused by I/R via decreasing MDA, avoided brain edema, saved neurons from apoptosis through decreasing Bax and cleaved caspase-3, and promoted ontogeny by increasing brain-derived neurotrophic issue (BDNF) (Lu et al., 2020). Curcumin therapy protects the arteries in those who are at risk of stroke. Curcumin incorporated into a solid lipid matrix of SLN resists enzymatic degradation during absorption and has long-lasting circulation and lower excretion in vivo after absorption. After treatment with SLN curcumin, the enzyme activities of SOD, CAT, GSH, and the mitochondrial complex improved, but lipid peroxidation, nitrite, and acetylcholinesterase rates decreased (Kakkar et al., 2013). In another study, oral therapy with nanoencapsulated quercetin reduced the loss of pyramidal neurons in the hippocampus CA1 and CA3 subfields and decreased the expression of iNOS and caspase-3 activities, protecting against oxidative damage in young and old rats subjected to I/R. Early therapy with nanomedicines improves the chances of survival due to less neuronal loss (Ghosh et al., 2013). After brain I/R, releasable quercetin-loaded polymer nanocapsules showed increased brain uptake and impressive mitochondrial localization. In both young and old rats, this novel upregulated mitochondrial quercetin supply alleviated histopathological intensity by reducing structural and functional integrity of mitochondria by ROS-protected sequestration by increasing GSH levels and SOD and CAT activities, regulating mitochondrial ROS-driven apoptotic cell death (Ghosh et al., 2017). The main active ingredient in Panax notoginseng is Panax notoginsenoside. When administered orally, Panax’s unique side-loading nuclear envelope notoginseng hybrid liposome vesicle (HLV) delivery method enhanced the bioactivity of the free drug. Furthermore, cerebral water content and infarct volume, which detect cerebral I/R, decreased in the acute myocardial ischemia group compared to the control group, serum lactate dehydrogenase (LDH), and H2O2 and MDA increased significantly, while

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SOD decreased significantly. Compared to the model group, significant restores in enzyme levels occurred in the Panax notoginsenoside-treated group, especially in the Panax notoginsenoside HLV-treated group with improved physicochemical properties (Zhang et al., 2012). With stem cell treatment, the potential to overcome the damage caused by brain ischemia seemed promising. The entrapped active ingredient naringenin has a prolonged release pattern in a nanoformulation of naringenin. The results suggest that naringen-loaded, gelatin-coated polycaprolactone nanoparticles have protective effects on human mesenchymal stem cells by reducing inflammatory responses (TNFα, IL1β, COX2, and iNOS) via NFκB pathways induced by oxygen starvation and glucose (Ahmad et al., 2018). Like another phytochemical, retinoic acid nanoparticles decreased microglial activation in N9 microglial cells; hippocampal organotypic slice cultures effectively inhibited LPS-induced nitric oxide (NO) release and iNOS expression and promoted arginase-1 and IL-4 production (Bernardo-Castro et al., 2021). In summary, nanoformulations of resveratrol, curcumin, quercetin, ginsenoside, naringenin, and retinoic acid have diverse effects on stroke. These therapeutic responses are applied by reducing oxidative stress by enhancing the activation of SOD, CAT, GSH, and complex mitochondrial enzymes. The nanophytochemicals also decrease Bax and cleaved caspase-3, increase BDNF expression, and reduce CA1 and CA3 hippocampal pyramidal neuron loss. The nanophytocompounds also reduce inflammatory mediators (e.g., TNFα, IL1β, COX2, and iNOS) via NFκB signaling pathways and inhibit LPS-induced nitric oxide (NO)-promoted arginas-e1 and IL4 production.

27.6.5 Phytoconstituents-based nanoformulations for other neurodegenerative diseases Nanoformulations have also shown promise in the fight against other NDs, such as HD and Ms. HD is a neurological disorder that is passed down through the family in an autosomal dominant pattern. HD occurs in middle age and continues to death 15
20 years later, with uncontrollable movement issues, cognitive deficiencies, and mental elements as signs and symptoms. An irregular repetition of the huntingtin gene’s triplet cytosine-adenine-guanine (CAG) is defined by a polyglutamine increase at the NH2 terminus of the protein huntingtin (HTT), which is translated at the protein level by a polyglutamine increase at the NH2-terminus of the protein huntingtin (HTT) (Andre et al., 2016). Neuronal death in HD has an origin that is unknown. Glutamate excitotoxicity, mitochondrial dysfunction, inefficient protein degradation, protein misfolding, caspase activation, transcriptional pathway dysregulation, reduced proteasome activity, and proteolysis are the key causes of the ailment (Maiti & Dunbar, 2018). Curcumin SLNs were given to rats who had been administered 3-nitropropionic acid (3-NP). In rodents, this toxin causes HD-like neuropathology, with a decrease in HD-like neurodegeneration and a significant increase in mitochondrial complex function and cytochrome rates. The results showed that curcumin encapsulated with SLN has a distinct advantage over curcumin alone in terms of bioavailability. GSH levels and SOD activity were both restored by curcumin SLNs. Curcumin-SLN-treated rats showed a significant difference in neuromotor coordination when compared to 3-NP-treated rats (Sandhir et al., 2014). Moreover, curcumin-loaded nanoparticles based on amphiphilic hyaluronan-conjugate revealed neuroprotective properties in an in vitro model of HD, according to a recent study (Pepe et al., 2020). In another investigation, treatment with rosmarinic acid-loaded SLNs significantly improves deficits in body weight, beam walk, locomotor, and motor coordination, as well as 3-NP-induced striatal oxidative stress (CAT and GSH). The optimal brain drug concentration demonstrates the relevance of choosing the nasal route over the i.v. route (Bhatt et al., 2015). Ms is a chronic immune-mediated demyelinating disease of the CNS that can be relapsing-remitting or progressive, resulting in axon loss and paralysis. Ms usually strikes individuals between the ages of 20 and 50, and women are more likely than men to develop the disease. Muscle weakness, weak reflexes, muscle spasms, difficulty moving, poor coordination, and imbalance are some of the physical, neurological, and psychiatric symptoms of Ms. Nanotechnology is a promising technique that has made important advances to the diagnosis and treatment of CNS illnesses such as Ms (Dolati et al., 2017). Dimethyl fumarate, like another medicine, is a viable Ms treatment; nevertheless, it has been related to multiple dosages, insufficient brain penetration, and gastrointestinal flushing. On crucial metrics like motor coordination, grip strength, weight, and locomotor activity, studies showed that nanocarriers containing dimethyl fumarate in a once-daily dosage regimen outperformed thrice-daily plain dimethyl fumarate administration (Kumar et al., 2018). According to the same study, SLN-dimethyl fumarate enhances bioavailability, biological residence, and brain absorption, hinting that lipid nanocarrier formulations could be useful in the treatment of Ms (Kumar et al., 2017). In addition, an in vitro study using a mouse brain microvascular endothelial cell model found that cationic SLN had higher permeability values than SLN treated with polysorbate 80. In vivo imaging revealed that polysorbate 80-treated SLN could reach the brain, despite the fact that they predominantly gathered in the liver and spleen (Esposito et al., 2017). Exosomes, or tiny nanovesicles produced by dendritic cells, for example, include proteins and RNAs that aid in the repair of myelin sheaths. Exosomes have been considered as a possible substitute for liposomes in the delivery of pharmaceuticals. Exosomes have negligible toxicity and can cross the BBB fast, making them ideal for therapeutic purposes.

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Dendritic-derived exosomes greatly boosted myelination and accelerated remyelination following injury by inducing preoligodendrocytes to develop into myelin-producing cells. In hippocampal slice cultures, serum exosomes delivered nasally to naive rats increased myelin content, oligodendrocyte precursor cell, and neural stem cell levels while reducing oxidative damage. Inflammation was decreased, and effector T cell activity was regulated by PLGA nanoparticles covalently cross-linked to myelin peptides. After IV therapy, CeO nanoparticles stabilized with citrate/EDTA and acted as an antioxidant in CNS ROS levels in C57/Bl6 MOG EAE mice. This treatment improved clinical symptoms and motor impairments while lowering free radical damage to the CNS (Dolati et al., 2017). Artificial neurotrophins are identified as possible agents against neurodegeneration in another study (Yang et al., 2021). As a result, phytochemical and synthetic medication nanoformulations successfully controlled dysregulated mediators in NDs, preventing neurodegeneration. Preclinical evidence for the use of phytochemical-based nanoformulations in the treatment of various NDs is shown in Table 27.2. TABLE 27.2 Phytochemicals-based nanoformulations for the management of various neurodegenerative disorders. Disease

Phytoconstituent

Nanoformulations

Mechanism of action

References

Alzheimer’s disease

Resveratrol

Solid lipid nanoparticles

Inhibition of the production of Aβ1
42 aggregates

Loureiro et al. (2017)

Nanostructured lipid carriers

Behavioral parameter change (learning and memory)

Rajput et al. (2018)

Nanocurcumin

Prevention of tau hyperphosphorylation and amyloid misfolding

Cheng et al. (2013)

Nanocurcumin

Inhibition of acetylcholinesterase, regulating microglial function and acting as an antioxidant

Mandal et al. (2020)

PLGA nanoparticles

Reduction of Aβ aggregates

Doggui et al. (2012)

PLGA nanoparticles

Increased proliferation of endogenous neural stem cells and neuronal differentiation

Barbara et al. (2017)

PLGA nanoparticles

Promoted neurogenesis, cognition, and memory via activating the canonical Wnt/β-catenin pathway

Tiwari et al. (2014)

PLGA-PEG conjugated nanoparticles

Decreased hippocampus Aβ production and deposit, as well as tau hyperphosphorylation

Fan et al. (2018)

Nanoliposomes

Inhibited amyloid peptide production and reduced Aβinduced toxicity

Lazar et al. (2013)

Naringenin

Nanoemulsion

Decreased expression of APP, β-secretase, and attenuating amyloidogenesis

Md et al. (2018)

Quercetin

Zinc nanoparticles

Behavioral parameter change (learning and memory)

Puerta et al. (2017)

Cyclodextrin-dodecyl carbonate nanoparticles,

Exert anti-inflammatory actions via diminishing the toll-like receptor 4 (TLR4) and COX-2 signaling pathway on neuronal cell lines

Testa et al. (2014)

Nanoparticles

Behavioral parameter change (learning and memory)

Palle and Neerati (2017)

PLGA nanoparticles

Decreasing neurotoxicity of Aβ42 and increasing neuron cell survival

Sun et al. (2016)

Curcumin

(Continued )

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TABLE 27.2 (Continued) Disease

Parkinson’s disease

Amyotrophic lateral sclerosis

Cerebral ischemia

Phytoconstituent

Nanoformulations

Mechanism of action

References

Epigallocatechin-3gallate

Nanolipidic particle

Increased expression of α-secretase, reducing the development of brain Aβ plaques

Smith et al. (2010)

Nanolipidic particle

Incorporating anti-amyloidogenic, metal chelation, and antioxidant activities

Singh et al. (2018)

Selenium nanoparticles

Disaggregation of Aβ fibrils

Zhang et al. (2014)

Sesamol

Solid lipid nanoparticles

Behavioral parameter change (learning and memory) and reducing oxidative stress

Sachdeva et al. (2015)

Ferulic acid

Solid lipid nanoparticles

Behavioral parameter change (learning and memory) and reducing oxidative stress

Meng et al. (2018)

Berberine

Carbon nanotubes

Maintained normal biochemical concentration in Aβ treated brain

Lohan et al. (2017)

Resveratrol

Nanoparticles

Improving behavioral abnormality

Palle and Neerati (2018)

Fe3O4 liposomes

Increased BBB permeability of drug

Wang et al. (2018a, b)

Nanoemulsion

Exerting antioxidant effect and reducing eosinophilic lesions

Pangeni et al. (2014)

PLA nanoparticles

Increased BBB permeability of drug

daRocha Lindner et al. (2015)

Polybutyl cyanoacrylate nanoparticles

Increased BBB permeability of drug and improving behavioral abnormality

Mandal et al. (2020)

Piperine

Glyceryl monooleate nanoparticles

Increased suppression of S protein oligomerization and fibril formation

Kundu et al. (2016)

Naringenin

Vitamin E-loaded naringenin nanoemulsion

Increased BBB permeability of drug, improved behavioral abnormality, and reduced oxidative stress

Gaba et al. (2019)

Quercetin

Nanocrystals

Increased BBB permeability of drug, improved behavioral abnormality, and reduced oxidative stress

Burgos et al. (2020)

Curcumin

Nanomicelles

Increased safety and probability of survival

Ahmadi et al. (2018)

Nanoparticles

Prevented SOD1 fibrillation and promoted the formation of smaller, disordered aggregates

Bhatia et al. (2015)

Micelles

Decreased cerebral toxicity

Tripodo et al. (2015)

Nanoparticles

Reduced oxidative stress caused by ischemia-reperfusion via decreasing MDA, avoided brain edema, rescued neurons from apoptosis and promoted neurogenesis

Lu et al. (2020)

Resveratrol

(Continued )

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Recent Frontiers of Phytochemicals

TABLE 27.2 (Continued) Disease

Huntington’s disease

Multiple sclerosis

Phytoconstituent

Nanoformulations

Mechanism of action

References

Curcumin

Solid lipid nanoparticles

Enhanced prooxidants activity, lowered lipid peroxidation, nitrite, and acetylcholinesterase levels

Kakkar et al. (2013)

Quercetin

Nanoencapsulation

Decreased oxidative stress, reduced loss of pyramidal neurons

Ghosh et al. (2013)

Nanocapsules

Minimization of oxidative stress and regulation of apoptosis

Ghosh et al. (2017)

Panax notoginsenoside

Core
shell hybrid liposomal vesicle

Improved physiochemical parameters in ischemic condition

Zhang et al. (2012)

Naringenin

Gelatin-coated polycaprolactone nanoparticles

Protected mesenchymal stem cells from inflammation

Ahmad et al. (2018)

Retinoic acid

Nanoparticles

Decreased microglial activation and effectively inhibited LPSinduced release of nitric oxide and iNOS production

Bernardo-Castro et al. (2021)

Curcumin

Solid lipid nanoparticles

Increased bioavailability, reduced oxidative stress, and improved neuromotor coordination

Sandhir et al. (2014); Pepe et al. (2020)

Rosmarinic acid

Solid lipid nanoparticles

Improved locomotor activity and motor coordination, reduced oxidative stress

Bhatt et al. (2015)

Dimethyl fumarate

Nanocarriers

Improved locomotor activity and motor coordination

Kumar et al. (2018)

Solid lipid nanoparticles

Enhanced bioavailability, improved locomotor activity and motor coordination

Kumar et al. (2017)

27.7 Limitations of nanotechnology-based approaches for management of neurodegenerative disorders Since each patient’s DNA and neurological functioning and traits are unique, no single strategy can be used to treat all of them, as discovered by Indrasekara et al. (2014). Every person must be identified and treated for these neurodegenerative illnesses on an individual basis, which is a challenging task that does not help in the development of a universal approach or treatment technique (de Lau et al., 2006). Furthermore, Kim et al. (2013a, 2013b) discovered that if the injection is not done correctly, the NPs used in this method may be absorbed by the blood or other body parts rather than the desired target region (Kim et al., 2013a, 2013b). The researchers have called attention to this challenge or limitation. If the medicine is not properly injected, it will not be absorbed or used to treat the target area for neurodegenerative diseases including AD and PD. Some have argued that because just a small amount of the medication really gets to where it is needed, the therapy has yet to be proven to be cent percent effective (Rahman et al., 2015). Safety concerns must be solved in order for this study to progress into viable medicinal drugs. As a reminder, even the most effective NP formulations for brain delivery accumulate widely in other regions of the body, including the spleen, liver, and kidney. As a result, nanoformulations that only activate when they reach the brain (Li et al., 2015) rather than when they reach other sections of the body are crucial. Advances in triggerable nanoformulation technology may soon benefit nanoparticles in regenerative medicine. The development of nanoparticles that target specific brain cells is a critical issue that requires further investigation. Specific brain cells, such as dopaminergic neurons (the principal target in PD), neural stem cells (neuronal repair), or microglia (neuroinflammation), are being targeted, which could advance its potential therapeutic usefulness in the context of NDs.

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The BBB’s protective effect makes it difficult to target molecules to the brain parenchyma; hence, multifunctionality is necessary for brain-targeting NP. In fact, the BBB’s principal function is to safeguard brain tissue from potentially harmful substances. The neurotoxicity of nanoparticles must be investigated for the same reasons that the neurotoxicity of a more traditional therapeutic system must be investigated. Because it plays a role in the neurodegenerative pathogenic process in the vast majority of CNS disorders, microglial activation is an important factor in NP neurotoxicity.

27.8

Future outlook and conclusion

NDs are regarded as the advancing damage of neuronal construction or functioning, further which is typically associated with neuronal death. The most widespread NDs are AD, PD, ALS, stroke, Huntington’s disease, and multiple sclerosis. The pathogenesis of various NDs is associated with ample attributes such as neuronal oxidative stress, neuroinflammation, protein misfolding, and neuroapoptosis (Fig. 27.1). The efficacy of conventional therapies is limited by such complex pathophysiological mechanisms and pharmacokinetic confines, which are attributed to enhanced side effects. These divert the therapeutic strategy toward novel treatments and more advanced alternative formulations. In recent years, phytochemicals have gained much hope as a potential therapeutic agent in the management of various disorders including NDs. The plant secondary metabolites possess proven effectiveness against many ailments; however, their certain properties like less solubility, instability, decreased absorption, low selectivity, and poor bioavailability limit their therapeutic applications. Moreover, chemical degradation, fast metabolism, and rapid clearance are certain properties that contribute to their low plasma levels. Articulating nanoformulations of phytoconstituents (micelle, nanoparticles, solid lipid nanoparticles, liposomes, etc.) controls these pharmacokinetic confines by increasing bioavailability, specificity, cellular uptake, and efficacy (Min et al., 2020). Consequently, nanoformulations are found to be useful in contending various NDs through their multiple beneficial properties like enhanced pharmacokinetics, increased biocompatibility, facilitated permeation through BBB, and intrinsic neuroprotective effects. In recent years, application of various nanoformulations of plant secondary metabolites has shown a bright future against AD, PD, ALS, cerebral ischemic stroke, Huntington’s disease, and Ms. Recent advancements in the new delivery system for plant-based natural drugs have been summarized in this chapter, which may reshape the therapeutic tactics in the management of NDs although additional studies are required for some concerns pertaining to the neurotoxicity of novel delivery systems (Chang et al., 2021). Further preclinical studies are required to explicate the specific tools and validate additional suitable novel systems (e.g., nanoenzymes, peptide engineering techniques, DNA nanocages). Substantiation of the in vitro and in vivo protocols is crucial to extend these nanoformulations from the lab to clinical trials. It is also essential to set precise guidelines for the quality, efficacy, and safety evaluation of these products and accomplish proper clinical trials to ascertain the occurrence of minimal side effects to ease the marketing of these nanoformulations effectively. Moreover, cost-effectiveness in developing such nanoformulations must be taken into consideration for commercial success in the market.

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303.

Chapter 28

Oxidative stress and its management through phytoconstituents Prakash Chandra Behera1 and Manas Ranjan Senapati2 1

Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology,

Bhubanewswar, Odisha, India, 2Agro-Polytechnic Centre (Animal Science), Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India

28.1

Introduction

Living systems constantly struggle against the changing environmental conditions to maintain optimum health and vigor throughout the life through continuous interaction between internal and external factors. When this interaction fails, the balance is disturbed leading to disharmony and disease. A majority of the diseases/disorders are mainly due to the imbalance between prooxidant and antioxidant homeostatic phenomena in the body. Body can protect itself against the damage with the help of RBC and liver cells through reduced glutathione (GSH) together with glutathione-dependent system, glutathione peroxidase, glutathione transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase by scavenging the causative harmful free radicals. Free radicals are fundamental to aerobic life and metabolism. Their endogenous generation is stimulated by insecticides/pesticides/automobile exhaust fumes/industrial waste, etc. Even though the dynamic balance between the generation and scavenging of free radicals is maintained by the body’s oxidative defense mechanisms, the additional burden leads to oxidative stress, tissue injury, and diseases. A diverged variety of disease syndromes in humans and animals like age-related molecular degenerations, angina, arthritis, ascites, atherosclerosis, some type of cancer, diabetes mellitus, fatty liver, Alzheimer’s disease, etc., are attributed to oxidative stress (Hartman et al., 2006). Besides, it has been implicated in the pathogenesis of several viral diseases including hepatitis, influenza, and AIDS through cellular inflammation and transformation of benign viral RNA to a virulent one. Medicinal herbs have been in use in one former another, under indigenous systems of medicine like Ayurveda, Sidha, and Unani (Dubey et al., 2004). More than 3000 plants are recognized for their medicinal value for which more than 80% of the world population depend on phytomedication traditionally (Duraipandiyan et al., 2006). The medicinal value of plants is attributed to bioactive chemical substances where polyphenols and flavonoids dominate over other constituents (Edeoga et al., 2005). Phyto-polyphenols are secondary metabolites/their derivatives/isomers of flavones, isoflavones, flavonols, catechins, and phenolic acids. More than 8000 structural variants of polyphenols and 3000 flavonoids are distributed in different parts of plants to exhibit antioxidative, antibacterial, antifungal, antimutagenic, anticancer, hypolipidemic, and hepatoprotective effects. The action is brought about by scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS), chelating metal ions, interacting with enzymes, adenosine receptors, and biomarkers (Lotito & Frei, 2004; Prior et al., 2003; Rajendran et al., 2004; Silva et al., 2002). Since oxidative stress is considered the basic etiology in the pathogenesis of many diseases/disorders and phytoconstituents are able to reduce such stress and maintain normal health on consumption, this manuscript aims to illustrate the mechanism of oxidative stress development and its management by phyto-polyphenols/constituents.

28.2

Oxidative stress and free radicals

The oxidative stress refers to a serious imbalance between the production of reactive species and antioxidant defense or the disturbance in the prooxidant
antioxidant balance leading to potential damage (Sies, 1991). Oxidative stress can result from the following reasons. Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00014-1 © 2023 Elsevier Inc. All rights reserved.

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1. Diminished levels of antioxidants, for example, mutations affecting the activities of antioxidant defense enzymes such as Cu, Zn, SOD, or glutathione peroxidase, toxins that deplete antioxidant defenses. Many xenobiotics are metabolized by conjugation with GSH; high doses can deplete GSH and cause oxidative stress even if the xenobiotic is not itself a generator of reactive species. Deficiencies in dietary minerals (Zn21, Mg21, Fe21, Cu21, and Se) and/ or antioxidants can also cause oxidative stress. 2. Increased production of reactive species, by exposure of cells to elevated levels of O2 or to other toxins that are themselves reactive species (e.g., NO22) and are metabolized to generate reactive species (parquet) or excessive activation of “natural” systems producing such species. Oxidation is an essential part of aerobic life, and it produces energy in the form of ATP through the process of metabolism. Along the metabolic process, uncoupled electron flow generates free radicals known as ROS which include superoxide (O22), peroxyl (ROO2), alkoxyl (RO2), hydroxyl (HO2), singlet oxygen, and nitric oxide (NO). Unpaired electron-bearing free radicals of oxygen/nitrogen-based molecules are generated during vigorous exercise, inflammation, exposure to certain chemicals, cigarette smoke, alcohol, air pollutants, and high-fat diets (Gutteridge & Halliwell, 1994). The electrons are added to oxygen for the production of superoxide anion, hydroxyl radical, perhydroxyl radical, peroxyl radical, alkoxyl radical, H2O2, and singlet oxygen among which the most common are superoxide, hydroxyl, and hydrogen peroxide radicals those are extremely reactive to cause sequence of damaging effects even though they have a short life. Superoxide radicals are the first step product in the reduction of oxygen to form H2O2 and lead to the formation of hydroxyl free radical and the perhydroxyl radical. Hydroxyl radicals are the most highly reactive species to interact with a variety of molecules by removal/addition of hydrogen to saturated or unsaturated bonds which is relevant with organic lipids and some types of metal ions (Arouma & Halliwell, 1987; Yu, 1994). The hydroxyl radical formation is greatly accelerated by the action of biologically available metal ion Fe21 as a catalyst to reduce oxygen to form superoxide radical which is quickly converted to H2O2 in the Haber
Weiss reaction (Knekt et al., 1994). H2O2 reacts with Fe21 in a Fenton reaction producing extremely reactive hydroxyl radicals (Gutteridge & Halliwell, 1994). Source of free radicals: Sources Free radicals

Non-radicals

Internal

External

Hydroxyl radical (OH )

Hydrogen peroxide (H2O2)

Mitochondria and phagocytes

Superoxide radical (O22)

Singlet oxygen (1O2)

Nitric oxide radical (NO2)

Hypochlorous acid (HOCl)

Lipid peroxyl radical (LOO2)

Ozone (O3)

Xanthine oxidase and peroxisomes Arachidonate pathways and reactions of transition metals Exercise, ischemia, and inflammation

Environmental and industrial pollutants, drugs, pesticides Radiation, ultraviolet light

2

Cigarette smoke Ozone

28.2.1 Effect of oxidative stress Oxidative stress affects different cellular and subcellular organic biomolecules through various mechanisms. DNA on attack by hydroxyl radical generates a huge range of base and sugar-modified products (Dizdaroglu et al., 2002). Initial free radical products attack upon purines, pyrimidines, and deoxyribose and transform into stable end products, whose relative amounts depend on reaction conditions (Alam et al., 1997; Halliwell, 1999). DNA can also be damaged by reactive nitrogen species mainly by nitration and deamination of purines (Lee et al., 2002; Spencer et al., 2003; Yermilov et al., 1995; Zhao et al., 2001b). Oxidative damage to proteins affects the function of receptors, enzymes, and transport proteins, generates new antigens that can provoke immune response and contribute to secondary damage to other biomolecules, and inactivates the DNA repair enzymes and loss of fidelity of damaged DNA polymerases in DNA replication (Casciola-rosen et al., 1997; Halliwell, 1978; Wiseman & Halliwell, 1996). Free radicals attack proteins to generate amino acid radicals which may cross-link/react with O2 to give peroxyl radicals. Individual amino acid oxidation produces kynurenines (from tryptophan), bi-tyrosine, valine and leucine hydroxides, L-di-hydroxy-phenylalanine (L-DOPA), ortho-tyrosine, 2-oxo-histidine, glutamate semialdehyde, and adipic semialdehyde (Giulivi & Davies, 2001; Harth et al., 2001; Headlam & Davies, 2003). Lipids can be oxidized, chlorinated, and nitrated by a range of ROS excluding H2O2, NO2, or O22which are essentially un-reactive with lipids (Halliwell & Gutteridge, 1999). Techniques for the measurement of these products like

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nitrated linoleate (Lim et al., 2002; Lima et al., 2002; Thukkani et al., 2003), lipid oxidation products like diene and thiobarbituric acid (TBA) (Halliwell & Gutteridge, 1999), unsaturated aldehydes like 4-hydroxynonenal and acrolein act as the biomarker of such peroxidation (Fam & Morrow, 2003; Roberts & Morrow, 2002; Uchida, 2003). Peroxidation of PUFAs is a self-propagating reaction of initiation, propagation, and termination that causes membrane alterations and damage to cells through cytotoxic aldehyde byproducts, malondialdehyde (MDA), the C3
C10 straight chain aldehydes, and 4-hydroxynonenal (4-HNE) (Shewfelt & Purvis, 1995). MDA and 4-HNE are strong electrophilic compounds which form covalent adducts with proteins, nucleic acids, and phospholipids (Sodum & Chung, 1988). Lipid hydroperoxides and oxygenated products participate in the signal transduction cascade in cell proliferation, differentiation, maturation, and apoptosis (Barrera et al., 2004; Blokhina et al., 2003; Cejas et al., 2004). ROS and LPO products are cytotoxic and involved in the intracellular signaling mechanisms (Salganik, 2001). Although some of these ROS play positive roles in cell physiology, most of them cause great damage to cell membranes and DNA, inducing oxidation that causes membrane lipid peroxidation, decreased membrane fluidity, and DNA mutations leading to cancer, degenerative, and other diseases (Finkel & Holbrook, 2000). The involvement of radical attacks is also attributed to some pathologies leading to accelerated aging, atherosclerosis, diabetes, weak immunity, cancerogenesis, and weak heart because they impair the K
Na cellular pump, resulting in change of intracellular osmotic pressure by K outflow and Na inflow (Ames, 1998). That is why recent research has been focused on the role of free radicals in the mechanisms of these diseases. The hydroxyl (half-life of 1029 s) and the alkoxyl (half-life of seconds) free radicals are very reactive and rapidly attack the molecules in nearby cells, and probably, the damage caused by them is unavoidable and is dealt with by repair processes. The superoxide anion, lipid hydroperoxides, and nitric oxide are less reactive (Ames et al., 1993). ROS may be very damaging since they can attack lipids in cell membranes, proteins in tissues or enzymes, carbohydrates, and DNA, to induce oxidations, which cause membrane damage, protein modification (including enzymes), and DNA damage. The increased lipid peroxidation activity is mainly due to the formation of highly reactive cytotoxic compounds like oxidative free radicals. ROS plays an effective role in the pathogenesis of different pathological diseases through loss of cell homeostasis by modifying the structure and functions of cell membrane. The most important characteristic of lipid peroxidation is to cause a considerable DNA-MDA adduct by interacting with cellular DNA. However, cells possess elaborate antioxidant defense mechanisms to neutralize the deleterious effects of free radical-induced lipid peroxidation. Enzymatic antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and nonenzymatic antioxidants such as vitamin E and reduced glutathione (GSH) act synergistically with one another to detoxify the effects of lipid peroxidation.

28.2.2 Defense of oxidative stress A crucial defense is provided by antioxidant with detoxification of ROS in the cell by both enzymatic and nonenzymatic systems. The enzymes involved are SOD for catalytic decomposition of the superoxide radical to hydrogen peroxide and oxygen, CAT for decomposition of hydrogen peroxide, GPx containing selenium for reduction of hydroperoxides, D-T diaphorase, thioredoxin reductase, nitric oxide synthase (NOS), heme oxygenase-1 (HO-1), eosinophil peroxidase (EPO), and glutathione-regenerating enzyme systems. The nonenzymatic antioxidants, which are less specific than the enzymatic ones, can also scavenge other radicals, both organic and inorganic, e.g., glutathione, histidine peptides, the iron-binding proteins transferring and ferritin, dihydrolipoic acid, reduced CoQ10, melatonin, urate, and plasma protein thiols. The small molecular weight compounds react with oxidizing chemicals, reducing their capacity for damaging effects. Several essential minerals including selenium, copper, manganese, and zinc are involved in the structure or catalytic activity of these enzymes. There are two tiers of antioxidant defense mechanisms against ROS-mediated lipid peroxidation. Low molecular mass compounds primarily acting against peroxyl radicals provide first line of defense, called “chain-breaking antioxidants (CBAs),” which can terminate the propagation of free radical-mediated reactions and/or interrupt the autocatalytic chain reaction of lipid peroxidation. The main cellular CBAs include tocopherol, ascorbic acid, GSH, uric acid, carotenoids, ubiquinone, polyphenols, etc., (Acworth et al., 1997). The antioxidant enzymes constitute the second line of defenses which provide primary and secondary defenses. Primary antioxidant enzymes like SOD, CAT, and GPxs are mainly preventive and can decompose ROS and prevent the initiation of lipid peroxidation. Secondary defenses typically are involved in the removal of LOOH to terminate the autocatalytic chain reaction by excision or repair of any lesions caused by ROS. In the event of ROS-induced lipid peroxidation, GPxs and GSHs are involved by catalyzing GSH-dependent reduction of LOOH (PL-OOH and FA-OOH) through their peroxidase activity. Among four seleniumdependent GPx isoenzymes, GPx-1, GPx-2, and GPx-3 are primarily involved in the reduction of H2O2 and FA-OOH, whereas GPx-4 displays activity toward PL-OOH and cholesterol hydroperoxides (Thomas et al., 1990).

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Antioxidants

Antioxidants are present in low concentrations relative to the oxidizable substrate and able to significantly delay or reduce the oxidation of this substrate (Halliwell et al., 2000). This is done by the antioxidant itself becoming oxidized—one antioxidant molecule reacts with one free radical. They have the properties of anticancer, antiviral, and anti-inflammatory activities, effects on capillary fragility, and an ability to inhibit human platelet aggregation (Benavente-Garcia et al., 1997). They can also modulate lipid peroxidation involved in atherogenesis, thrombosis, and carcinogenesis, and their known properties include free radical scavenging, strong antioxidant activity, inhibition of hydrolytic and oxidative enzymes (phospholipase A2, cyclooxygenase, lipoxygenase), and anti-inflammatory logical effects of flavonoids are correlated with their antioxidant activities. These antioxidants can be classified as watersoluble or lipid-soluble, depending on lipophilic region of cell membranes. 1. 2. 3. 4.

Hydrophilic antioxidants: Ascorbic acid (vitamin C) and urate. Lipid-soluble antioxidants: Ubiquinols, retinoids, carotenoids, and tocopherols. Endogenous antioxidants: Plasma proteins, glutathione (GSH), and urate. Dietary antioxidants: Ascorbic acid, carotenoids, retinoids, flavonoids, polyphenols, and tocopherols.

The synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been restricted recently for safety concerns and the cause of liver swelling and influence on liver enzyme activities (Martin & Gilbert, 1968). Therefore, the identification and development of safer, natural antioxidants are more beneficial for which plants being a rich source of natural antioxidants and vegetables and fruits like tomato, cabbage, onion, tea, bean, citrus fruits, grapes, and its wine are of great concern (Kaur & Kapoor, 2001). They are the sources of vitamins C and E, carotenoids, flavonoids, thiol (SH) compounds, and iron, copper, selenium, and zinc minerals.

28.3.1 Phytoconstituent as antioxidant Antioxidants of herbal origin are preferred to costly inputs of allopathic medicines and technology due to ease in availability and low price. There are about 45,000 plant species in India, with concentrated hotspots in the region of Eastern Himalayas, Western Ghats, and Andaman and Nicobar Island. The officially documented plants with medicinal potential that are 3000, but traditional practitioners use more than 6000. India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world. The use of plants as a source of diverged medications is the prime objective of ethnobotany where the search for antioxidants remains underneath. Among several phytoconstituents of medicinal herbs, polyphenols are the most potent bioactive compounds to exhibit multiple and diverged functions.

28.3.1.1 Polyphenol Polyphenols with one or more phenolic hydroxyl groups attached to the carbon-based aromatic phenyl ring compounds are easily oxidized to quinones by reactive oxygen species. Dietary polyphenols with over 8000 structural variants are secondary metabolites of fruits, vegetables, wine, and tea; extra virgin olive oil, chocolate, and other cocoa products are derivatives and/or isomers of flavones, isoflavones, flavonols, catechins, and phenolic acids. Phenolic acids account for about one-third of the total intake of polyphenols in our diet, and flavonoids account for the remaining two-thirds. They are subdivided into different groups by the number of phenolic rings and the structural elements that link these rings (Butterfield et al., 2002; Schaffer et al., 2007). Phenolic compounds are a large, diverse group of secondary plant metabolites that are widespread in the plant kingdom and include phenolic acids, flavonoids, and tannins. Polyphenol compounds may accumulate as end products from the shikimate pathway and acetate pathways (Carey, 2003) and have several important physiological functions in plants as these are essential for the regulation of growth and participate in the reproduction by attracting pollinating insects and also protect plants against UV radiation, harmful insects, and infections (Korkina, 2007). These are largely distributed over the vegetable kingdom and are subdivided into classes according to the chemical structure of each substance (Arts & Hollman, 2005). Phenolic compounds have specific health effects, even though they are nonnutritive compounds. A considerable amount of research has been conducted on the antioxidant activity and scavenging activity of phenolic compounds (Escarpa & Gonzalez, 2001). Feeding studies recently conducted with poultry showed that plant extract obtained from oregano prevented lipoperoxidation in muscle tissue and may be complementary to vitamin E (Giannenas et al., 2005). A recent report confirmed in rats the ability of plant-rich polyphenols, including grape extract, to exhibit a significant antioxidative protective effect in plasma and liver (Gladine et al., 2007). Present in high concentrations in the stomach, they might exert local antioxidant effects by scavenging various types of reactive species or chelating transition metal ions (Halliwell, 2007). Plant polyphenols can act as reducing agents by donating hydrogen and quenching singlet oxygen.

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Phenolic acids

It includes hydroxybenzoic acid and hydroxycinnamic acids which constitute the majority of phenolic acids found in plant tissue and which are derived from nonphenolic molecules, benzoic and cinnamic acids, respectively (Harborne & Williams, 2000; Wrolstad et al., 2005). Hydroxycinnamic acid compounds (p-coumaric, caffeic acid, ferulic acid) occur most frequently as simple esters with hydroxy carboxylic acids or glucose, while the hydroxybenzoic acid compounds (p-hydroxybenzoic, gallic acid, ellagic acid) are present mainly in the form of glucosides (Cabrini et al., 2001; Clifford, 2000). The glycosylated derivatives or esters of quinic, shikimic, or tartaric acid of hydroxycinnamic acids are found as bound forms instead of free existence, but phenolic acids of pomegranate, coffee, and blueberries occur as esters or glycosides conjugated with other natural compounds such as flavonoids, alcohols, hydroxy fatty acids, and sterols. Antioxidant properties of phenolic acids play a vital role in the stability of food products, as well as in the antioxidative defense mechanisms of biological systems. Common phenolic acids include caffeic acid, chlorogenic acid, catechin, epicatechin, p-hydrobenzoic acid, and ferulic acid. 28.3.1.1.2

Caffeic acid

Caffeic acid is a hydroxycinnamic acid commonly found in many fruits, vegetables, seasonings, and beverages and acts as an antioxidant in vitro and may contribute to the prevention of cardiovascular disease and appears to contribute to the resistance of roots to microbial infections, fungi, and insects (Harrison et al., 2003; Olthof et al., 2001). 28.3.1.1.3 Chlorogenic acid Chlorogenic acid is an ester of caffeic acid and quinic acid and is a major phenolic compound in coffee and has been isolated from the leaves and fruits of plants. It has antioxidant activity in vitro, which may contribute to reduction of diseases (Gonthie et al., 2003; Olthof et al., 2001). 28.3.1.1.4

Catechin

Catechins are classified as proanthocyanidins. Catechin has been deemed a powerful antioxidant because it is easily oxidized (Nijveldt et al., 2001). 28.3.1.1.5 p-hydroxybenzoic acid p-Hydroxybenzoic acid is a phenolic derivative of benzoic acid. It is primarily known as the basis for the preparation of its esters, which are used as preservatives in cosmetics. One study indicated p-hydroxybenzoic acid acts as an antioxidant against peroxyl radicals, and its sources are red fruits, berries, onions, and cereals (Manach et al., 2004; Mattila et al., 2005, 2006; Yeh & Yen, 2003). 28.3.1.1.6

Ferulic acid

Ferulic acid is a phenolic acid belonging to the hydroxycinnamic group, and it is found in the leaves and seeds of many plants including whole wheat, oats, coffee, apples, peanuts, and pineapples. It is an antioxidant which neutralizes free radicals and seems to reduce the risk of many types of cancer (Mori et al., 1999). 28.3.1.1.7

Flavonoids

Flavonoids, a class of polyphenolic compounds, have been studied with interest due to their essential functions in plant physiology (e.g., resistance to pathogens and predators), their organoleptic properties (e.g., pigmentation, flavor), and their effects on human health (Bravo, 1998; Croft, 1998; Dreosti, 2000; Ross & Kasum, 2002). Currently, almost 6500 flavonoids are known and, being the major phenolic compounds, are present in the human diet (approximately 2/3 of the total phenols), followed by the phenolic acids (approximately 1/3 of the total phenols) (Scalbert & Williamson, 2000; Tapiero et al., 2002). 28.3.1.1.8 Flavonols Quercetin is the main flavonol in the human diet, present in many fruits, vegetables, and beverages such as tea or wine and usually occurs as O-glycosides, with D-glucose as the most frequent sugar residue. More than 170 different quercetin glycosides have been identified (Soleas et al., 1997; Souquet et al., 2000; Yang et al., 2001).

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28.3.1.1.9

Epicatechin

Epicatechin is a flavanol, which has been classified as an antioxidant. High concentrations of epicatechin have been found in cocoa, particularly dark chocolate (Mayer & Stern, 2003). Its concentration in cocoa may contribute to the higher antioxidant activity compared to green tea and wine (Lee et al., 2003). 28.3.1.1.10

Stilbenes

Stilbenes, 1, 2-diphenylethylene nucleus with hydroxyls in aromatic rings, exist in the form of monomers/oligomers. But, resveratrol, a glycosylated form, exists as both cis and trans isomers among which trans-resveratrol possesses a trihydroxy-stilbene skeleton (Delmas et al., 2006). 28.3.1.1.11

Resveratrol

Resveratrol is a flavonoid that is more specifically classified as a hydroxystilbene. It has been isolated and identified in peanut plants, skins of certain red grapes, blueberries, and some and has response to some stress such as fungal invasion. Many health effects, such as anticancer, antiviral, neuroprotective, anti-inflammatory, have been attributed to its pines (Chen et al., 2002; Chung et al., 2003; Liu et al., 2004; Wang et al., 2005). 28.3.1.1.12 Anthocyanin Anthocyanins are considered to contribute to the healthiness of fruits and berries for their antioxidant, anticarcinogenic, anti-inflammatory, and antiangiogenic properties (Kong et al., 2003; Rossi et al., 2003). Anthocyanins can also improve the nutritional value of processed foods by preventing the oxidation of lipids and proteins in the food products (Kahkonen et al., 2001, 2003; Viljanen et al., 2004). 28.3.1.1.13 Tannins Tannins are a group of water-soluble polyphenols (500 to 3000 MW) complexed with alkaloids, polysaccharides, and proteins and can be grouped as condensed and hydrolyzable catagories. Further, hydrolyzable tannins are divided into gallo-tannins and ellagi-tannins on the basis of structural characteristics. Diferuloylmethanes have two aromatic rings substituted with hydroxyls and linked by aliphatic chain containing carbonyl groups.

28.4

Antioxidative effect of phytoconstituents

Many pharmacological activities of polyphenols are linked to their ability to act as antioxidants and free radical scavengers, to chelate metal and interact with enzymes, adenosine receptors, and biomarkers. They also attenuate oxidative stress by acting as effective ROS and RNS scavengers as shown in vitro and although to a lesser degree, in vivo. A growing body of literature points to the importance of natural antioxidants from many plants, which may be used to reduce cellular oxidative damage, not only in foods, but also in the human body. This may provide protection against chronic diseases, including cancer and neurodegenerative diseases, inflammation, and cardiovascular disease. The effect of some of the dietary polyphenols on different afflictions due to oxidative stress is enlisted in Table. 28.1. TABLE 28.1 Effect of dietary polyphenols on different afflictions due to oxidative stress. Dietary polyphenols

Mechanism of effects

Conditions/models

References

Epigallocatechin, EGCG, ECG

Inhibits lipoxygenase and cyclooxygenase

Colon mucosa and tumor tissues

Hong et al. (2001)

EGCGECG

Activates MAPK proteins by REmediated gene expression

Hep G2-ARE-C8 cell

Chen et al. (2000)

Hydroxytyrosol

Increases CAT and SOD

Cholesterol-rich diet-fed rats

Fki et al. (2007)

Inhibits 1,2-lipoxygenase and 5lipoxygenase, reduces leukotrieneB4 production

Rat platelets and PMNL

Kohyama et al. (1997)

(Continued )

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TABLE 28.1 (Continued) Dietary polyphenols

Mechanism of effects

Conditions/models

References

Catechin, proanthocyanidin-B4

Increases CAT, GST, SOD activities, and cellular GSH content

Cardiac H9C2 cells

Du et al. (2007)

Curcumin

Inhibits CYP-1A2, 3A4, 2B6, 2D6, 2C9, and cyto-chrome-P-450 NADPH reductase

Plasmids

Appiah-Opong et al. (2007)

Inhibits mitochondrial proton F0F1-ATPase/ATP synthase

Rat brain

Zheng and Ramirez (2000)

Activates ARE and Nrf2 and increases GSTP1 expression

HepG2 cells

Nishinaka et al. (2007)

Increases CAT, SOD activity, decreases (MDA), (NO22), (NO32), MPO level, and serum transaminases

Rat

Shen S. Q. Zhang Y. Xiang J. J. and Xiong C. L. (2007)

Kaempferol-3-Ogalactoside

Inhibits human recombinant synovial phospholipase A2 (PLA2)

Mice

Gil et al. (1994)

EGCG, quercetin, kaempferol, morin, apigenin, daidzein, ECG

Inhibits mitochondrial proton F0F1-ATPase/ATP synthase

Rat brain

Zheng and Ramirez (2000)

Ellagic acid, gallic acid, corilagin

Inhibits tyrosinase, xanthine oxidase, and superoxide radical

Substrate of L-tyrosine

Rangkadilok et al. (2007)

Dihydrocaffeic acid

Scavenging intracellular ROS

EA.hy926 endothelial cells

Huang et al. (2004)

Caffeic acid, (1)-catechin

Inhibits peroxy-nitrite-mediated oxidation of dopamine

Dopamine

Kerry and Rice-Evans (1999)

Quercetin

Prevents LDH leakage and increases SOD, CAT, GSH, and GPx activity.

Mouse liver

Molina et al. (2003)

Decreases MDA and lipoperoxidation, increases Cu/Zn SOD and GPx mRNA.

Hep-G2 cells

Alı´a et al. (2006)

Increases NADPH: quinone oxidoreductase-1 (NQO1) expression and activity

MCF-7 breast carcinoma cells

Valerio et al. (2001)

Enhances γ-glutamylcysteine synthetase (γ-GCS)

Hep-G2 cells

Scharf et al. (2003)

Enhances ARE binding, Nrf2mediated activity of transcription and reduces Keap1 protein level

Hep-G2 cells

Tanigawa et al. (2007)

Inhibits O-acetyl-transferase, sulfotransferase, increases GSH level and SOD, and decreases MPO and oxidized GR levels

Wister rats with KBr

Cadenas and Barja (1999)

Reduces O-acetyl-transferase and sulfotransferase catalysis

Primary cultures

Dubuisson et al. (2002)

Inhibits expression and activity of CYP 1A1 / 1A2

Microsomes and HepG2 cells

Ciolino and Yeh (1999)

Inhibits mitochondrial proton F0F1-ATPase/ATP synthase

Rat brain and liver F0F1-ATPase

Zheng and Ramirez (2000)

Inhibits 12-lipoxygenase and 15-lipoxygenase

J774A-1 and muscle cells

Schewe et al. (2001)

Resveratrol

(-)-Epicatechin, procyanidin, EGCG, ECG

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28.4.1 Mechanism of action Protective mechanisms include scavenging/detoxification of reactive oxygen species (ROS), blocking ROS production, and sequestration of transition metals along with enzymatic and nonenzymatic antioxidant defenses. Dietary polyphenols have strong antioxidant capacities by which cell functions are regulated (Hartman et al., 2006; Hollman et al., 1997). They might have effects like inhibition/reduction of telomerase, cyclooxygenase (Hussain et al., 2005; O’Leary et al., 2004), lipoxygenase (Sadik et al., 2003), and the interaction with signal transduction pathways and cell receptors (Kong et al., 2000; Wiseman et al., 2001). Moreover, polyphenols can affect caspase-dependent pathways (Monasterio et al., 2004), cell cycle regulation (Way et al., 2005), and platelet functions (Murphy et al., 2003). They offer indirect protection by activating endogenous defense systems and by modulating cellular signaling processes such as nuclear factor kappa B (NF-κB) activation, activator protein-1 (AP-1) DNA binding, glutathione biosynthesis, phosphoinositide 3 (PI3) kinase/protein kinase B (Akt) pathway, mitogen-activated protein kinase (MAPK) proteins, extracellular signalregulated protein kinase (ERK), and c-jun N-terminal kinase (JNK). There are three possible mechanisms by which polyphenols can exert their intracellular molecular effect. In the first mechanism, the intracellular polyphenol glucuronide is the active compound which has to be transported over the plasma membrane and may alter cellular physiology. Second, the transported glucuronide may be deconjugated by intracellular enzymes with glucuronidase activity to release polyphenol aglycone for intracellular effects. Third, the polyphenol glucuronide being deconjugated extracellularly by glucuronidases and quercetin aglycone is subsequently taken up into the cell to alter cellular physiology. Further, polyphenols can interact with extracellular/membrane targets to trigger an intracellular response (Frank, 2004). The antioxidant potential of phenolic compounds has been shown in a number of in vitro studies. They are capable of direct chain-breaking antioxidant action by radical scavenging: in addition to being capable of scavenging several nonphysiological radicals such as DPPH* and ABTS* 1 (Cai et al., 2006; Kosar et al., 2003; Payet et al., 2005). Also, they are capable of hydroperoxide decomposition and scavenging a variety of reactive species such as superoxide, hydroxyl, peroxyl, and hypochlorous acid radicals with significance in vivo (Halliwell et al., 2005). They can also suppress the formation of reactive oxygen species by chelating transition metal ions capable of catalyzing oxidative reactions (Mira et al., 2002). It is mainly by virtue of these properties the phytochemicals exert their protective effects and receive more and more attention as potential therapeutic agents against several chronic degenerative diseases (Kris-Etherton et al., 2002). In addition to radical scavenging, metal chelation, reductive action, and enhancement of hydroperoxide decomposition, polyphenols have been suggested to inhibit the oxidation of LDL by binding certain proteins containing catalytic metal sites, for example, in copper-induced oxidation of human LDL in vitro. Combining phenolic compounds with each other resulted in synergistic protection against LDL lipid and protein oxidation (Cirico & Omaye, 2006; Yeomans et al., 2005). The direct antioxidant capacity of phenolic compounds is essentially due to the ease with which a hydrogen atom from an aromatic hydroxyl group can be donated to a free radical and the stabilization of the phenoxyl radical by delocalization of unpaired electrons around the aromatic ring (Frankel & Meyer, 2000; Rice-Evans et al., 1996). Even though details depend on the radical used, the following general structure-activity rules seem to apply for phenolic acids because radical scavenging action requires (1) a free hydroxyl group, (2) the presence of an aromatic ortho-dihydroxyl moiety, (3) number of free hydroxyl groups, and (4) electron-donating substituent(s) such as methoxy group(s) next to aromatic hydroxyl group(s) enhance the hydrogen donation ability (Cai et al., 2006; Siquet et al., 2006). The antioxidant activity of flavonoids arises from their ability to donate a hydrogen atom from an aromatic hydroxyl group to a free radical and yield a resonance-stabilized phenolic radical (Ross & Kasum, 2002). In the case of flavonoids, the structural criteria for maximal radical scavenging are (1) the ortho-dihydroxy structure in the B ring, (2) the 2, 3-double bond in conjugation with the 4-oxo function in the C ring, and (3) the 3- and 5-hydroxy groups with the 4oxo function in the unsaturated C ring (Cai et al., 2006; Heijnen et al., 2002). Phenolic antioxidants such as flavonoids can be antioxidative and prooxidative, depending on the balance between their reduced forms, which act as antioxidants and their oxidized forms (phenoxyl radicals or quinone/quinone methide intermediates), which have prooxidant activities (Galati & O’Brien, 2004). The balance between antioxidant and prooxidant characteristics of flavonoids has been attributed not only to their structural features, but also to the concentration, suggesting the induction of antioxidant defense metabolism by low concentrations and ROS production at high concentrations (Raza & John, 2005). Extensive conjugation between 3-OH and B-ring catechol groups, together with abundant β, 4-8 linkages, endows a polymer with significant radical scavenging properties by increasing the stability of its radical (Castillo et al., 2000). Polyphenols clearly improve the status of different oxidative stress biomarkers as predictors of disease risk like cardiovascular risk and the appropriateness of the different methods used (Williamson & Manach, 2005). Prominent plant polyphenols have received considerable attention in recent years due to their diverse pharmacological properties

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including antioxidant and hepatoprotective activity (Murugesh et al., 2005) and also may play a role in the prevention of other diseases, such as cancer (Issa et al., 2006; Yang et al., 2001), cardiovascular diseases (Arts & Hollman, 2005; Huxley & Neil, 2003), osteoporosis, diabetes mellitus, atherosclerosis, and neuro dysfunctions (Masella et al., 2005). Those are strong antioxidants, but are also known to interfere with signal transduction pathways (Hou et al., 2004; Mandel et al., 2004; Williams et al., 2004), and inflammation can interact with a number of proteins involved in cell proliferation and may induce apoptosis and prevent tumor growth (Middleton et al., 2000; Nijveldt et al., 2001). In contrast, evidence for protective effects of polyphenols against cancers, neurodegenerative diseases, and brain function deterioration is still largely derived from animal experiments and in vitro studies.

28.4.1.1 Metabolism The metabolism of several polyphenols is now well understood. Generally, the aglycones can be absorbed from the small intestine, but some dietary polyphenols in form of esters, glycosides, or polymers cannot be absorbed in the native. These compounds must be hydrolyzed by intestinal enzymes/colonic microflora. They are conjugated in the intestinal cells and later in the liver by methylation/sulfation/glucuronidation. As a consequence, the forms reaching the blood and tissues are different from those present in food, and it is very difficult to identify all the metabolites and to evaluate their biological activity (Natsume et al., 2003; Setchell et al., 2003; Zhang et al., 2003). Isoflavones and phenolic acids like caffeic acid and gallic acid are the most well-absorbed polyphenols, followed by catechins, flavanones, and quercetin glucosides, but with different kinetics. On the other hand, large molecular weight polyphenols such as proanthocyanidins, galloylated tea catechins, and anthocyanins are least absorbed (Gonthier et al., 2003). Hydroxytyrosol, the major olive oil phenolic compound, is dose-dependent absorbed from olive oil (Visioli et al., 2000). The major metabolites identified in in vitro and in vivo studies were an O-methylated derivative of hydroxytyrosol, glucuronides of hydroxytyrosol, and a novel glutathionyl conjugate of hydroxytyrosol (Corona et al., 2006; Tuck et al., 2002). The polyphenols reaching the colon are extensively metabolized by the microflora into a wide array of low molecular weight phenolic acids (Manach et al., 2005).

28.4.1.2 Absorption Glycosylation of resveratrol is protected from oxidative degradation, and glycosylated resveratrol is more stable and more soluble so is readily absorbed in the gastrointestinal tract (Regev-Shoshani et al., 2003). On the other hand, quercetin glucosylation facilitates its absorption; in fact, the efficiency of quercetin glucosides absorption is higher than that of the aglycone itself (Morand et al., 2000). Glucosides, being transported into enterocytes by sodium-dependent glucose transporter, are hydrolyzed by a cytosolic β-glucosidase (Day et al., 1998). However, the effect of glucosylation on absorption is less clear for isoflavones than for quercetin (Manach et al., 2004). Proanthocyanidins, due to their polymeric nature and high molecular weight absorption, have a limit through the gut barrier whereas oligomers larger than trimers are unlikely to be absorbed in the small intestine in their native forms (Halliwell et al., 2000). Hydroxycinnamic acids, when ingested in the free form, are rapidly absorbed by the small intestine and are conjugated similar to flavonoids (Clifford, 2000; Cremin et al., 2001). However, these compounds are naturally esterified in plant products, and this impairs their absorption because intestinal mucosa, liver, and plasma do not possess esterases capable of hydrolyzing chlorogenic acid to release caffeic acid (Olthof et al., 2001; Rechner et al., 2001) so hydrolysis can be performed only by the colonic microflora (Gonthier et al., 2003; Olthof et al., 2001). The polyphenols that are not absorbed in the small intestine reach the colon, where the microflora hydrolyzes glycosides into aglycones and extensively metabolizes the aglycones into various aromatic acids. Aglycones are split by the opening of the heterocycle at different points depending on chemical structure and thus produce different acids that are further metabolized to derivatives of benzoic acid. Intestinal microflora affects the metabolism of isoflavones glucosides since they are hydrolyzed to aglycones or transformed into active metabolites such as equol from daidzein (Atkinson et al., 2005; Franke et al., 2004).

28.4.1.3 Conjugation and plasma transport The absorbed polyphenols are conjugated by methylation, sulfation, and glucuronidation. It is a common metabolic detoxification process of xenobiotics facilitating their biliary and urinary elimination by increasing their hydrophilicity. Catechol-O-methyl transferase catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to polyphenols such as quercetin, luteolin, caffeic acid, catechins, and cyaniding (Wu et al., 2002). The methylation generally occurs in the C3’ position of the polyphenol in preference to the C4’ position: catechol-O-methyl transferase activity is highest in the liver and the kidneys among other tissues. Sulfotransferases catalyze the transfer of sulfate moiety from 3’-

492

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phosphoadenosine-5’-phosphosulfate to hydroxyl group on polyphenols. UDP-glucuronosyl transferases are membranebound enzymes located in the endoplasmic reticulum in many tissues which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to polyphenols, steroids, bile acids, and many other dietary constituents. Glucuronidation occurs in the intestine and in the liver with the highest rate in the C3 position (Spencer et al., 1999). The relative importance of these three types of conjugation appears to vary according to the nature of the substrate and the dose ingested. The balance between sulfation and glucuronidation of polyphenols also seems to be affected by species and sex the conjugation mechanisms are highly efficient, and free aglycones are generally either absent/present in low concentrations in plasma after consumption of nutritional doses. Polyphenol metabolites circulate in blood protein-bound forms where albumin represents the primary protein responsible for the binding. The binding to albumin has variable affinity according to their chemical structure (Dangles et al., 2001) and may have consequences for the rate of clearance of metabolites and for their delivery to cells and tissues. It is possible that the cellular uptake of metabolites is proportional to their unbound concentration. The biological activity of polyphenols whether is due to its free or albumin-bound form is unclear (Dufour et al., 2007).

28.4.1.4 Plasma concentrations The plasma concentration of polyphenols varies according to their nature and food source. In fact, the maximum concentrations are most often reached 1 hour
2 hour after ingestion except those which require to be degraded prior to absorption (Aziz et al., 1998).

28.4.1.5 Tissue uptake Polyphenols are able to penetrate tissues like intestine and liver where they are metabolized. The plasma concentration of polyphenols is not directly correlated with that in target tissues. Moreover, the distribution between blood and tissues differs between various kinds of polyphenols (Maubach et al., 2003).

28.4.1.6 Excretion Polyphenols and their derivatives are eliminated chiefly in urine and bile. Extensively conjugated metabolites are more likely to be eliminated in the bile whereas small conjugates such as mono-sulfates are preferentially excreted in urine. The total amount of metabolites excreted in urine is roughly correlated with maximum plasma concentrations (Hong et al., 2002).

28.4.1.7 Toxicity Dietary polyphenols at higher doses/under certain conditions may exert toxic prooxidant activities (Rucinska et al., 2007). They are metabolized by peroxidase to form prooxidant phenoxyl radicals which in some cases are reactive to co-oxidize GSH/NADH and are accompanied by extensive oxygen uptake and reactive oxygen species formation. Incubation of hepatocytes with dietary polyphenols with phenol rings was found to partially oxidize hepatocyte GSH to GSSG while polyphenols with a catechol ring were found to deplete GSH through the formation of GSH conjugates. Dietary polyphenols with phenol rings also oxidized human erythrocyte oxy-hemoglobin and caused erythrocyte hemolysis more readily than polyphenols with catechol rings. It is concluded that polyphenols containing a phenol ring are generally more prooxidant than polyphenols containing a catechol ring. Flavonoids can induce oxidative damage and nick DNA via the production of radicals in the presence of Cu and O2, Al, Zn, Ca, Mg, and Cd, and they stimulate phenoxyl radical-induced lipid peroxidation (Sakihama et al., 2002). It leads to the formation of phenoxyl radicals as the primary oxidized products which can also initiate lipid peroxidation. It is concluded that the prooxidant cytotoxicity of dietary polyphenols is due to the formation of ROS, role of phenoxyl radical/phenol redox couple, and stimulation by metals (Fujisawa et al., 2004; Nemeikaite-Ceniene et al., 2005; Sakihama et al., 2002).

28.5

Conclusion

Oxidative stress has direct effect on the pathogenesis of potentially severe conditions. For chronic periods, increasing the level of free radicals can cause structural defects in nuclear and/or mitochondrial DNA owing to alterations in cellular structures and enzymatic activities or aberrations in gene expression. The importance of oxidative metabolism in the context of health and disease cannot be overlooked. For their normal functioning, cells need to maintain equilibrium between prooxidants and antioxidants, failing where they stand to face consequences of oxidative stress. Also, it has

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also been reviewed that in addition to metabolic products, other external agents can have the potentiality to generate ROS and RNS. As a matter of fact, oxidative stress can be reduced by approaching a balanced lifestyle and so as to nutrition which plays a critical role because the best treatment against oxidative stress is antioxidants. Plant-derived bioactive molecules have gained pivotal attention in recent years, given their therapeutic relevance in both disease prevention and treatment, whether using whole plants, plant extracts, or even the isolated constituents with its authenticated phytochemical profiles. Herbal or ethnomedicinal drugs are usually effective against stress owing to the presence of some bioactive compounds. However, there are many ethnomedicinal prescriptions which are considered effective against certain diseases, but the exact nature of their effects is not clearly understood. It might hold promise for add-on treatment for several diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative disorders. That is why larger randomized trials are needed to obtain clear scientific evidence on the benefits or risks of antioxidant supplementation during treatment. The risk of antioxidant supplementation is due to its toxicity which should not be overruled. Toxicity or adverse effects of herbal drugs, associated with many herbal medicines, have been reported in numerous studies. Therefore, considering the growing body of experimental evidence about the medicinal efficacy of herbs, it is necessary to identify their active principles responsible for such effects. This should be followed by studies to explore the mechanisms of action of active principles of interest. It is hoped that the progress in the refinement of our analytical tools will pave the way for the identification of more chemicals of therapeutic value from plant origin. Therefore, hidden wealth of knowledge about naturally occurring medicinal compounds which are yet to be explored in the years to come is expected that phytochemicals-based drugs will be object of growing interest for inflammation and oxidative stress-related diseases.

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Chapter 29

Phytochemicals: an immune booster against the pathogens Kena Premshankar Anshuman Department of Microbiology, Sir P.P. Institute of Science, MK Bhavnagar University, Bhavnagar, Gujarat, India

29.1

Introduction

Phytochemicals are chemical compounds produced by plants. The term derived from Greek “phyto” meaning “plant.” It is produced by the plant by primary and secondary metabolism and imparts color, aroma, and flavor. It also protects the plant from infection and predators (Harborne et al., 1999; Molyneux et al., 2007). It has tremendous antimicrobial and antioxidant capacity. Other beneficial effects on human health are increased immunity by stimulating immune system, preventing damage of DNA, and reducing the growth rate of cells (Kumar, 2019). There are many phytochemicals like polyphenols, flavonoids, isoflavonoids, alkaloids, phytoestrogens, terpenoids, carotenoids, limonoids, phytosterols, fibers, etc. They act as antioxidant against lung, urinal, cancer, and liver diseases related to stomach, heart, bone, etc. (Thakur et al., 2020). Phytochemicals are rich in vegetables, fruits, whole grains, nuts, seeds, etc. Tens of thousands of phytochemicals have been identified, and still many more are yet to be discovered. Such compounds are found in many medicinal plants such as garlic, tomatoes, various leafy green vegetables, herbs, etc. In India, the Western Ghats are rich regions of such plant biodiversity. Out of 7500 plant species, 43,000 are used in various medicines, cosmetics, hygiene fragrances, and food supplements (Singh et al., 2020). Various parts of medicinal plants such as leaves, stems, flowers, seeds, fruits, and roots can be used for many diseases and to form different products from it (Kawaii et al., 2000). The plant produces large number of bioactive compounds. High concentrations of phytochemicals that accumulate in fruits and vegetables protect against free radical damage (Suffredini et al., 2004). Approximately, 20% of known plants have been used in pharmaceutical studies, impacting the healthcare system in positive ways such as treating cancer and harmful diseases (Naczk & Shahidi, 2006). Numerous studies have attempted to screen vegetables and fruits for antioxidant activity by using different oxidation systems. Research suggests that the consumption of vegetables and fruits increases health benefits. Wide varieties of vegetables particularly dark green leafy, cruciferous, and deep yellow orange while in fruits particularly citrus and deep yellow orange one reduce disease risk and provide immunity (Duyn & Pivonka, 2000). It also helps in coronary heart disease, protects against strokes, and prevents cataract formation and role in chronic obstructive pulmonary disease, diverticulosis, and hypertension (Duyn & Pivonka, 2000). Soong and Barlow (2004) investigated the antioxidant activity and phenolic content of various fruit seeds. According to a report, published by the World Health Organization (WHO), 70%
80% of the global population used herbs for medicinal purposes. Since age to till date, about 53,000 species of herbs have been used for medicinal purposes (Qadir & Raja, 2021). Herbs have always been used for flavor and fragrance in the food industry, and some of them have been found to exhibit antimicrobial properties (Baharlouei et al., 2011). Baskar et al. (2007) also found antioxidant activity of 32 herbs belonging to 21 different families that has been screened and found positive correlation between the total antioxidant activity and total phenolic content. Antioxidants are grouped as natural or synthetic antioxidants. Some synthetic antioxidants like BHT, BHA, propyl gallate, and tertbutylhydroquinine are commonly used. Synthetic antioxidants have recently been shown to cause health problems due to their toxicity and carcinogenicity, such as liver damage. Therefore, the development of safer antioxidants from natural sources has increased. Plants have been used as an excellent source of traditional medicines to treat Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00009-8 © 2023 Elsevier Inc. All rights reserved.

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different diseases. Many of these medicinal plants are indeed good sources of phytochemicals. Some typical examples of the most commonly used ingredients in ethnic foods are tamarind, cardamom, lemongrass, and galangal basil. These spices or herbs have been shown to contain antioxidants (Javanmardi et al., 2003). Today, due to some uncontrolled diseases, people in rural and urban become conscious of having natural medicinal plants, vegetables, fruits, and its product in diet. Phytochemical-rich diet reduces many diseases and boosts up immunity.

29.2

Secondary metabolites

Based on chemical structure and biosynthetic chemical compound in plant, they are grouped as primary and secondary metabolites (Wink, 2003). They are unique to species or genera (Harborne, 1993). Phytochemicals have various pharmacological and physiological activities (Ma et al., 2011; Sohail et al., 2021; Yanez et al., 2013). It has very good antioxidant property, which can be used as an effective natural antioxidant source in nutraceuticals (Velu et al., 2018). Generally, it helps to reduce diseases like cardiovascular disease, cancer, type-2 diabetes, neurodegeneration, etc. Some other activities are: (1) immune function, (2) anti-inflammation, (3) antihepatoma, (4) antimicrobial effect, (5) anticancerous, (6) therapeutic, etc. (Sohail et al., 2021; Zhang et al., 2015). Primary modes of action of phytochemicals are: 1. 2. 3. 4. 5. 6.

Entry into target cell. Bind with specific receptor site in target cell. Inhibit replication. Destroy viral polymerases and proteases (which are essential for viral replication). Protect plant against bacterial, fungal, and viral diseases. It direct interacts with receptor cell membrane, nucleic acid.

The most important secondary metabolites are phenolic compounds, flavonoids, alkaloids, terpenoids, carotenoids, and glycosides.

29.2.1 Phenolic compounds Phenolic compounds are excellent antioxidant compounds. Phenolic compounds like phenolic acid, stilbenes, and lignans are found in many plants. Other polyphenols include both flavonoids and nonflavonoids. Generally, it targets spike (S) glycoprotein of SARS-CoV-2 and destroys its structure. Therefore, it is effective for coronavirus infection (Sohail et al., 2021). Some other polyphenols are curcumin, kaempferol, pterostilbene target to S1 domain of protein, while other polyphenolic compounds like quercetin, apigenin, luteolin, fisetin, etc., target S2 domain of SARS-CoV-2 spike protein (Pandey et al., 2020). Curcumin is curcuminoid polyphenols responsible for the bright yellow color of the Indian spice turmeric (Curcuma longa L.) and has been used in diet for centuries and also in the Ayurvedic system of medicines. It cures inflammation (Ammon & Wahl, 1991). Research suggested that there is a strong correlation between total antioxidant activity and phenolic content. According to Jain et al. (2010), Asclepiadaceae and Periplocoideae presented high antioxidant activity with the presence of a strong correlation between antioxidant activity and phenolic content. Moreover, the phenolic content and antioxidant activity of parsley (Petroselinum crispum) and cilantro (Coriandrum sativum) have been tested and found a strong correlation between total antioxidant activity and phenolic content (Singh et al., 2002).

29.2.2 Phytoestrogens Phytoestrogen is a naturally occurring polyphenolic and nonsteroidal compound found in plants and plant-based foods. It works the same as estrogens produced by the body, so it is also known as dietary estrogens. It is very much beneficial special to women to rebalance hormones (Aronson, 2016). Phytoestrogen is a polyphenolic compound. There are mainly four groups of phytoestrogen, namely, flavonoids, isoflavonoids, stilbenes, and lignans (Aronson, 2016). It is generally found in soybeans (Glycine max L.), red clover (Trifolium pretens), and white clover (Trifolium repens L.). Other dietary sources of phytoestrogen are hops, beer, apples, onions, parsley, capsicum pepper, tofu, etc. (Mostrom & Evans, 2011).

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Phytoestrogens have great medicinal effects. It also acts as antioxidant, anti-inflammatories, and antineoplastics. In addition, it reduces atherosclerosis, osteoporosis, angiogenesis, diabetes, etc. (Mostrom & Evans, 2011).

29.2.3 Flavonoids Flavonoids are major class of polyphenols and are mostly found in vegetables and fruits. They are the largest group to study phytochemicals. More than 6000 flavonoids occur in plants (Panche et al., 2016). It is used in many medicinal formulations to cure diseases like atherosclerosis, Alzheimer, cancer, etc. (Leyva-Lopez et al., 2016; Rengasamy et al., 2019). Citrus flavonoids have a wide range of antibacterial, antifungal, antiviral, antidiabetic, and anticancer activities (Burt, 2004; Ortuno et al., 2006). Citrus fruits cure arthritis, rheumatism, prostate and colon cancer, diabetes, heart disease, and high fever (Mohanapriya et al., 2013).

29.2.4 Alkaloids Alkaloids are plant secondary metabolites that contain unique structure (amine-type structure with basic nitrogen atom). Due to its unique structure, many alkaloids are referred to as “DNA intercalators,” for example, berberine, emetine, sanguinarine. These intercalators have many functions as it inhibits transcription, replication, and translation of genetic material, stabilizing structure. So, it has ability to inhibit coronavirus replication and development within the host cell (Velu et al., 2018). It has over 12,000 cyclic nitrogen-containing compound. They are found in over 20% of plant species, for example, caffeine, nicotine, cocaine, etc. (Zulak et al., 2006). Caffeine is the most widely used psychoactive substance in the world, and it is a popular additive to many products. Caffeine (purine alkaloids) increased alertness and improved attention in human (Haskell et al., 2005), while nicotine is a pyridine alkaloid. It is derived from the American plant Nicotiana tabacum. It stimulates effect and improves attention and memory (Heishman et al., 2010). Cocaine (tropane alkaloid) increases motor activity in human. But higher dose leads to restlessness and increases anxiety, insomnia, tachycardia, and death (Reissig et al., 2009). Actually, alkaloids are typical natural products of plant but are also found in bacteria, fungi, and animals. Most alkaloids have pharmacological and toxicological activities. Some interfere with protein and lipid at the cell periphery (neuroreceptors and ion channels) and also target DNA, RNA, cytoskeleton, biomembrane, and related processes (Wink, 2020b). Many alkaloids are lipophilic and so easily enter into cell by diffusion. Steroidal alkaloids can specifically enter with receptors and ion channels and interfere with membrane integrity (Wink, 2020b). Many quinoline and isoquinoline alkaloids, that is, quinine, skimmianine, dictamine, cinchonine, β-carboline, have been effective against virus diseases including SARS-CoV-2 infections (Wink, 2020a). So, we can use alkaloidcontaining plants in diet, and they could be promising candidates in the treatment of COVID-19. Only benzophenanthridine alkaloids (sanguinarine) have antibacterial activity like microbial antibiotic. It is widely used as an antiplaque agent in mouthwash and toothpaste preparations (Martindale, 1993). Alkaloid primarily acts as feeding deterrents and toxins to insect and other herbivores (Harborne, 1993). Some are in common usage as psychotropic medicines, social drugs, or hallucinogens (Kennedy & Wightman, 2011). Some alkaloid-based psychotropics are appropriate for use as nootropics in healthy populations, for example, atropine (tropane alkaloid) impairing memory in primates and humans (Bartus & Dean, 2009). Among fruits, lemon is one of the most important citrus fruits. It has alkaloids having anticancer and antibacterial effects of crude extract of different parts such as leaves, stems, roots, flowers, seeds, etc. (Kawaii et al., 2000).

29.2.5 Terpenes All classes of terpenes are found in legumes (triterpenes). This is a powerful defense compound against microbes and herbivores. Triterpenes and triterpene saponins are widely distributed in the plant kingdom (Wink, 2003). Terpenes are a diverse group of more than 30,000 lipid-solube compounds, for example, Ginkgo biloba, Melissa officinalis (lemon balm), sage, valerian, etc. Terpenes also recognized as “terpenoids/isoprenoids” are modified terpenes. The common plant sources of terpenes are tea, thyme, cannabis, Spanish sage, and citrus fruits (Cox
Georgian et al., 2019). It has a wide range of medicinal value. Sage is worked as cognition enhancer and also treatment for cognitive decline stretches back to ancient Greek (Kennedy & Wightman, 2011). Essential oils from sage extract inhibit human

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AChE (Savelev et al., 2003; Savelev et al., 2004). Cannabis is used in pain and side effects of chemotherapy in cancer patients such as nausea, anxiety, depression (Cathcart et al., 2015). Terpenes form a major constituent of essential oil in plant. The oil extracts were tested for human virus infections like herpes simplex virus-1 (HSV1), Dengue virus type-2, and Junin virus (Cox
Georgian et al., 2019). Another terpene is curcumin acting as antioxidant, anti-inflammatory, anticancer, antiseptic, antiplasmodial, astringent, digestive, etc. (Cox
Georgian et al., 2019). This group of chemicals is also present in a host of spices, flavors, and food. It forms essential components of our diet to provide taste and healthy eating, for example, carotenoids (Kennedy & Wightman, 2011). It has also antimicrobial properties. Monoterpenes act as antigerminative, phytotoxic allelopathy (De Almeida et al., 2010; De Martino et al., 2010). Due to its antimicrobial properties, it is widely used in traditional and modern medicine.

29.2.6 Carotenoids Carotenoid is the basic source of plant pigment such as yellow, orange, and red. It is widely distributed in nature (Sugawara et al., 2009), especially in higher plants leaves, flowers, and fruits (Mattea et al., 2009). It is responsible for the characteristic color and plays important biological role as antioxidant and regulator of membrane fluidity (Umeno et al., 2005). Polyphenols and carotenoids are the major phenolic compounds of apples (Malus domestica L.) including caffeic, quinic, and p-coumaric acids. These polyphenols can act as antioxidants. β-carotene, ascorbic acid, and many phenolics play dynamic roles in delaying aging, reducing inflammation, and preventing certain cancers (Duthie et al., 1996). Zeaxanthin is the dihydroxy form of β-carotene found in corn and in many vegetables, while lycopene is mostly found in tomatoes and tomato products (Nishino et al., 2002). Apricots, carrots, pumpkin, and sweet potatoes are rich sources of α-carotene and β-carotene. Pink grapefruit, tomatoes, and watermelon are source of β-carotene, lycopene, phytofluene, and phytoene. β-carotene is isolated and identified from peel, pulp, and seed fractions of Canarium odontophyllum Miq. (Prasad et al., 2011). The carotenoid of fruits, vegetables, and animal products are usually fat-soluble and associated with lipid fraction, especially the lipid portion of human tissue cell (El-Qudah, 2008). Approximately, 700 carotenoids found in nature and 600 natural carotenoids have been identified (Aizawa & Inakuma, 2007; Rao & Rao, 2007). Only 50 have provitamin A activity (Okada et al., 2008). α-carotene, β-carotene, and B-cryptoxanthin are important precursors of vitamin A (Park et al., 2009; Thane & Reddy, 1997). Carotene has many medical effects as provitamin A, antioxidant/prooxidant, anticancer, anti-obesity, effect on bone components, etc. (Jaswir et al., 2011; Nishino, 1998). Due to its tremendous beneficial effect, carotenoids are used in pharmaceuticals, neutraceuticals, animal feed additives, and as coloring agents in foods (Das et al., 2007; Mortensen, 2009).

29.2.7 Phytosterols Sterols of plant origin are referred to as “phytosterols.” Natural sources of phytosterol are vegetables, fruits, nuts, and oils. Commercial sources of sterols are from seed oils like corn, soybean, and rapeseed oil (Bot, 2019). It reduces cholesterol blood levels, effective in the treatment of patients having hypercholesterolemia, and promotes heart health (Bouic, 2001). It has strong anticancer activity (Sexton & Lomas, 2018; Woyengo et al., 2009). Cardiovascular diseases are also increasing day by day. A new approach to decrease cholesterol by the use of phytosterol as a functional food ingredient is a good option (Rashid et al., 2021). American Heart Association and the European Current Dietary Guidelines recommended phytosterol as a therapeutic option for treating patients with high blood cholesterol (Lichtenstein et al., 2006). Some phytosterols are also shown to have antioxidant activities, but the mechanism of antioxidation is different from traditional phenolic compounds (Wang, 2008).

29.3

Phytotherapy

Phytotherapy is the use of plants or herbs as medication to prevent or cure human and animal diseases. It improves the quality of life. Phytotherapy-based research and phytotherapeutic medicine have potential cures and remedies for today’s viral infections like SARS, MERS, and CoV-2 and also many other bacterial and fungal acute or chronic diseases (Rao & Agarwal,1999; Zhang et al., 2015).

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Essential oil has been used in phytotherapy for preservation, flavor, and safety of foods for ages to till dates, for example, clove oil, garlic oil, bergamot oil, etc. (Avila
Sosa et al., 2016; Goni et al., 2016; Leyva et al., 2016). Hops are used for phytotherapy, and the extract of hop cones is used in sleeping disorders and mood disturbances such as restlessness and anxiety (Heinlein & Buettner, 2014). Many fruits and vegetables show antimicrobial activities due to the presence of organic acids, vitamins, secondary metabolites, and their interaction with each other. Sap of fruits contains 80%
90% sugar and acid, citric acid (Adode, 2002). Many gram-positive/negative bacteria and yeast, for example, Staphylococcus aureus, Enterococcus faecalis, Salmonella tphi, Escherichia coli, Proteus vulgaris, Candida albicans, etc., can be easily killed by such natural remedies (Deshwal & Kaur, 2018).

29.4

Phytomedicine

Plant or plant-based medicines are also known as “phytomedicine.” They are also known as “herbal medicines,” “phytopharmceutical,” and “botanicals.” It is available as concentrated extract, pill/capsule, or as ointments/compresses (Tupas & Gido, 2021). It is derived from traditional Chinese, Japanese, Indian, and European herbal medicine systems and used for the development of novel antibacterial, antifungal, and antiviral diseases (Reichling & Schnitzler, 2011; Sohail et al., 2021). It is involved in preventive, healing, and therapeutic procedure. Various plant and botanical sources are used in drugs, nutraceuticals, proteins, and gum resins (Gul et al., 2021). Chinese traditional medicine is recommended by their government for the treatment of SARS-CoV-12 (Yang et al., 2020). Herbal extracts of Anthemis hyaline, Nigella sativa, and Citrus sinensis reduce coronavirus replication and downregulated TRP genes (Ulasli et al., 2014). Ayurveda is one of the world’s oldest natural medicinal systems, originated about 3000 years ago in India. It is very popular for preventing and curing diseases, especially chronic diseases. Herbal medicines are safe, have better physiological compatibility, and are cost-effective. India is a gold mine of medicinal plants and a rich repository of traditional medicinal knowledge (Bhattacharjee et al., 2019). Traditional medicinal plants, such as Sarpagandha, aloe, guggal, aonla/amla, isubgol, etc., are widely used.

29.5

SARS-CoV-2

At the end of 2019, outbreak of COVID-19 became pandemic. Actually, it emerged as unexplained pneumonia cases in Wuhan, China, in December 2019 and spread worldwide (Chan et al., 2020). It is also declared as Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) in January 2020 (Sohail et al., 2021). There were some other PHEICs like H1N1, Polio, Ebola, Zika, etc., from 2009 to 2019. The disease is named as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. In the middle of 2020, SARS-CoV-2 has been declared a clinical threat to the general public worldwide (Gorbalenya et al., 2020; Lai et al., 2020; Sun et al., 2020; Velavan & Meyer, 2020). Thus, since 2009 the world is exposed to such severe viral infections and could be exposed to some novel ones in the coming years too. In COVID-19 treatment, the use of antibiotics and natural medicinal plant in diet increases immunity tremendously. Recovery rate of the patient also varies fast. Moreover, nosocomial infection is caused by bacteria, fungi, and viruses, and handwashing with lemon juice reduces the spread of such nosocomial infection and provides a cheap alternative to alcohol-based hand washers (Okeke et al., 2015; Van Eldere, 2003). In the future to fight against such severe bacterial, fungal, and viral infections, including natural medicinal plants or its product in routine diet is beneficial. Advanced technologies such as nanodrug delivery systems have been used in the formulation of a drug to improve efficiency in targeting drugs (Pradhan et al., 2021). Thus, traditional medicinal plants play a great role in human health and source of the drug. Plant-based products improve human brain functions and immunity too. Still more research on different medicinal plants and its secondary metabolite is required.

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Chapter 30

Phytochemicals: recent trends and future prospective in COVID-19 Dhwani Upadhyay1, Arti Gaur1, Maru Minaxi2, Vijay Upadhye3 and Prasad Andhare2 1

Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India, 2Biological Sciences, PDPIAS, Charotar University of Science and

Technology, Changa, Gujarat, India, 3Center of Research for Development (CR4D), Department of Microbiology, Parul Institute of Applied Sciences (PIAS), Parul University, Waghodia, Gujarat, India

30.1

Introduction

30.1.1 SARS-CoV-2 and COVID-19 Humans are susceptible to mild to life-threatening respiratory infections caused by coronaviruses, a group of viruses that infect a wide variety of animals. SARS-CoV-2, a newly discovered coronavirus, surfaced around the end of 2019 and spread an uncommon kind of viral pneumonia over the Chinese city of Wuhan. This new coronavirus disease, also known as coronavirus disease 2019 (COVID-19), has quickly spread over the world due to its high infectiousness (Hu et al., 2021). The coronaviruses are a group of spherical, positive-strand RNA viruses that possess an envelope and have spikes protruding from their surfaces. There are two main groups of human coronaviruses: alphacoronaviruses and betacoronaviruses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused by its recent emergence and human-to-human transmission has triggered a worldwide pandemic of coronavirus disease 2019 (COVID19), which is associated with substantial mortality and morbidity (Narang et al., 2020). Novel SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, is the seventh member of the coronavirus family. Other human-infecting members of this coronavirus family include the agent of severe acute respiratory syndrome (SARS-CoV) and the culprit of Middle East respiratory syndrome (MERS-CoV) (MERS-CoV). SARS-CoV and SARS-CoV-2 each have genomes of around 30 kilobases (kb) in length, with 29,727 and 29,811 nucleotides, respectively; the SARS-CoV-2 genome encodes for 29 proteins, whereas the SARS-CoV genome has just 14 open reading frames (Suhail et al., 2020).

30.1.2 Plants’ role in COVID-19 treatment There is currently no licensed therapy for SARS-CoV-2, despite the widespread reports of research studies in recent days on the discovery of viable therapies against this global health disaster. In this overview, the effectiveness of medicines derived from plants has been emphasized, with a particular emphasis on those substances that have been generated in plants (termed “secondary metabolites” or “PSMs”). Numerous pharmaceutical molecules, superior for treating severe disorders, have been isolated from plant metabolites because of their diverse multifunctional chemical structures. Potential alternative medications and lead molecules for drug repurposing and synthesis have been described for some of them in this chapter (Bhuiyan et al., 2020). Patients infected with COVID-19 have been treated with a variety of traditional herbal treatments, either alone or in conjunction with conventional pharmaceuticals, with positive reinforcements since the outbreak. The anti-SARS-CoV-2 effects of various herbal remedies (extracts) and refined compounds might be due to direct prevention of viral replication or entrance. Curiously, certain medications may inhibit SARS-CoV2 infection by blocking the ACE-2 receptor or the serine protease TMPRRS2. There is evidence that natural compounds can block proteins involved in the SARS-CoV-2 life cycle. These proteins include papain-like and chymotrypsin-like proteases (Benarba & Pandiella, 2020). Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00032-3 © 2023 Elsevier Inc. All rights reserved.

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30.1.3 Phytochemicals and their role in COVID-19 More bioactive and less toxic phytochemicals may be the most effective option for most disorders. Phytoconstituents (herbal extracts as crude, or processed extracts, mixtures, and decoctions), micronutrients, and animal products have been used as therapies for a wide range of diseases and other health problems by humans since antiquity. As a result, scientists have begun to recognize the value of plant-derived natural compounds. Flavonoids, terpenoids, alkaloids, phenolic and essential oils, and so on are the most common phytocompounds (Sharanya et al., 2021). Against coronavirus, crude extract or purified chemicals extracted from herbal medicines and/or herbs, such as Artemisia annua, Agastache rugosa, Astragalus membranaceus, Cassia alata, Ecklonia cava, Gymnema sylvestre, Glycyrrhizae uralensis, Houttuynia cordata, Lindera aggregata, Lycoris radiata, Mollugo cerviana, Polygonum multiflorum, Pyrrosia lingua, Saposhnikoviae divaricata, Tinospora cordifolia, etc., have demonstrated potential inhibition effect (Adhikari et al., 2021). Roughly, 80% of the populace presently use herbal treatments as their primary source of health care. Numerous new components with antiviral properties have been found in plants native to Nigeria. As an example, Sida cordifolia has been studied and found to have antihuman immunodeficiency virus (HIV) properties in its native condition. Boerhavia diffusa is another species with powerful antiviral properties. Molecular weight estimates the active component extracted from B. diffusa valued around 16,000 and 20,000 (Oladele et al., 2020). Numerous scientific journals have testified the antiviral actions of plants, such as the study showed by recent data that aqueous purified products of plants species from the Lamiaceae family (Rosmarinus officinalis, Melissa officinalis, Mentha x piperita, Prunella vulgaris, Salvia officinalis, and Thymus vulgaris) unveiled antiviral activity against herpes simplex virus type 1 and type 2 and the most imperative results versus an acyclovir-resistant strain of HCV-1 (Jalal et al., 2021). Phytochemicals like gingerol shogaol, oleanolic acid, resveratrol rhoifolin, kaempferol, almond oil, rosmarinic acid, ursolic acid, lycorine, hederagenin, nigellidine, α-hederin, ethyl cholate, apigenin, nobiletin, chalcone, tangeretin, hesperidin, allicin, epigallocatechin gallate, diallyl trisulfide ajoene, apigenin, artemisinin, aloenin, glucobrassicin, curcumin, piperine, anthraquinone, flavonoids, hydroxychloroquine, and jensenone are testified to demonstrate the antiviral properties. Enhanced bond affinity for SARS-CoV-2 6LU7 and 6Y2E proteases, as well as suppression of SARS-CoV-2 M protease (Mpro) and spike (S) glycoprotein, may represent the mode of action of these bioactive molecules (Tegen et al., 2021). Once tested, in vitro cultures have the potential to function as factories producing bioactive substances and phytochemicals that can be manufactured in large quantities of consistent quality in an effort to combat COVID-19. In a similar vein, manipulating the habitat and the molecules of these in vitro cells might yield tailored therapeutic candidates that can be tested for their effectiveness against COVID-19. The phytochemicals that are produced by in vitro cultivation have the added benefit of being consistent both in terms of their yield and quality. Nevertheless, because the traditional plant-based substances may, under certain circumstances, show themselves to be harmful, the engineered generation of prospective phytochemicals can get around this obstacle (Khan et al., 2021).

30.1.4 List of various targetable sites in SARS-CoV-2 infection with human cell To identify the target sites for the development of effective treatment against the COVID-19 disease, it is essential to have extensive knowledge about the pathogenesis of SARS-CoV infection. Enveloped viruses are capable of entering the host cell via direct plasma membrane fusion or via endocytosis, where membrane fusion would rely on pH-dependent proteases and optimal intra-endosomal conditions. The life cycle of SARSCoV-2 initiates with its invasion into the nasopharynx mucosa. For further reciprocal action, the host transmembrane serine protease 2 (TMPRSS2) must cleave the virus spike (S) glycoprotein in order for it to connect with the host angiotensin-converting enzyme 2 (ACE2) receptor and enable viral entrance into the host cell (Heald-Sargent & Gallagher, 2012). In S protein, there are two subunits, namely, S1 and S2, and after this cleavage, now receptor-binding domain (RBD) of S1 subunit will be exposed and directly will bind with the angiotensin-converting enzyme-2 (ACE2) receptor of the epithelial cells in the nasopharynx. After the severance of S1 subunit by host transmembrane proteases, the spring-loaded S2 subunit of spike protein refolds and becomes conducive for membrane fusion (Madu et al., 2009). In the light of this, the two main mechanisms by which viruses enter the body are receptor binding and proteolytic activation. After then, cascade of biological processes including viral assembly, replication, and endosome development take place. Once the virus enters the host cell, it gets disassembled to release the nucleocapsid and the viral genome (Perlman & Netland, 2009; Zhou et al., 2020). The viral genome consists of long single-stranded RNA having 26,000 to 32,000 bases and is wrapped by the nucleocapsid protein (Fig. 30.1). The genomic RNA of SARS-CoV-2 contains about six open reading frames (ORFs). Using ribosomes of host cells, then open reading frames 1a and 1b are translated into two polyproteins (pp1a and pp1b), which encode 16 nonstructural proteins (nsps), whereas the remaining ORFs

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FIGURE 30.1 The pathogenesis of SARS-CoV infection.

encode structural and auxiliary proteins (Katsnelson, What; Letko et al., 2020; Matheson & Lehner, 2020; Shang et al., 2020). These polyproteins are broken down by two proteases, namely, the primary protease (3CLpro, nsp5) and the papain-like protease (PLpro, nsp3), which results in the production of nsp216 that is a component of the replication
transcription complex (RTC) (V’Kovski et al., 2019). The RNA-dependent RNA polymerase (RdRp, nsp12) (Hoffmann et al., 2020) and the helicase are two of them (nsp13). These nonstructural proteins are then involved in producing subgenomic RNA (sgRNA), which code for four main structural proteins, for instance, the proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N) (Letko et al., 2020). Consequently, these structural and auxiliary proteins are produced as a result of sgRNA transcription. Replicase then rearranges the endoplasmic reticulum (ER) to generate double-membrane vesicles, which are crucial in the control of viral genome replication, transcription, and exit from the host cell. The sgRNAs are then introduced into the ER and subsequently transferred to the intermediate compartment between the ER and Golgi for viral budding. The N protein-encased genome eventually assembles to integrate new virions, which are carried in the vesicle and released outside through exocytosis (McBride et al., 2007). Thereafter, the main infective body sites like nasal, pulmonary, and oral release freshly encapsulated virus which infects additional cells and spreads to internal organs leading to many organ deficits in the disease’s development. Additionally, an uncontrolled “cytokine storm” of hyper-inflammatory cytokines, such as interleukin IL-6, tumor necrosis factor-alpha (TNF-), and IL-1b, is powerfully triggered by the invasive virus and the assaulted cells (Fig. 30.2). The target sites for the drug preparation and management of infection can be broadly divided into two categories: 1. Virus-based targets and 2. Host-based targets.

30.2

Virus-based targets

30.2.1 Structural-based proteins For SARS-CoV-2 to infiltrate host cells, entry is the initial step. Structure-related proteins are crucial to this process. As it was already noted, S, M, E, and N proteins make up the main structural proteins of SARS-CoV2. Key components of these structural proteins are found to be potent targets for therapeutic strategies intended to prevent viral invasion. In COVID-19, a number of phytochemicals that were thought to have anti-entry properties were repurposed.

30.2.1.1 Spike protein The most important structural protein used as target site for COVID-19 treatment is spike (S) protein. It is a type-I transmembrane protein with clove-shaped structure. Glycosylated spike (S) protein is a major inducer of host immune

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FIGURE 30.2 Targetable sites in SARS-CoV-2 infection with human cell.

responses (Du et al., 2009). As a key player in the interaction between the virus and the cell receptor, it is a crucial target for antiviral medicines (Wrapp et al., 2020). According to recent research, the flavonoids fisetin, quercetin, and kaempferol can act as a lead molecule to combat SARS-CoV-2 by binding to the spike protein (Pandey et al., 2020). On the basis of molecular docking studies, many potent inhibitors from selected flavonoid and non-flavonoid compounds (kaempferol, curcumin, pterostilbene, hydroxychloroquine, fisetin, quercetin, isorhamnetin, genistein, luteolin, resveratrol, and apigenin) were identified. The famous antimalarial medication hydroxychloroquine also demonstrated adequacy against SARS-CoV-2 for in silico experiments. The results during simulation investigation for phytochemicals like fisetin, quercetin, and kaempferol showed low binding free energy with hACE2-S protein complex, proving that they can interfere with the complex and inhibit the viral entry and further signal cascades. One more natural-based compound stilbene is also found to be powerful deterrants of spike glycoprotein
ACE2 complex (Wahedi et al., 2020). Furthermore, dithymoquinone phytochemical from Nigella sativa was also found to inhibit the spike glycoprotein
ACE2 interface with 21.4 kcal/mol more binding affinity than chloroquine (Kulkarni et al., 2020). According to one recent investigation, it is proved that glycyrrhizic acid is also a well-known lead chemical to combat COVID-19. By evaluating the binding activity of glycyrrhizic acid at the interface of the spike protein RBD
ACE2 via surface plasmon resonance and nano bit assay, it has been evident that it is a low-toxic, broad-spectrum anti-coronavirus candidate with both cytoplasmic and membrane actions. It also has strong anti-inflammatory and immune-modulating impact (Kulkarni et al., 2020). The antimalarial drug chloroquine (CQ) and its analog hydroxychloroquine (HCQ), which are commonly used to combat autoimmune disorders, may also be used to treat COVID-19 because they raise endosomal pH, which prevents SARS-CoV-2 from fusing with the host cell membranes (Vincent et al., 2005).

30.2.1.2 Envelope, nucleocapsid, and membrane proteins The coronavirus E (envelope) protein is the smallest structured transmembrane protein (Kuo et al., 2007). It has two distinct domains: the hydrophobic domain and the charged cytoplasmic tail. It performs essential biological tasks for the pathogenicity of the host and the structural integrity of coronaviruses (Venkatagopalan et al., 2015). Different coronavirus families’ members share a conserved nucleocapsid (N) protein as the other structural protein (Chang et al., 2016). It consists of two protein domains, N-terminus and C-terminus. Binding of RNA is the principal activity of the N-terminal domain (NTD), whereas dimerization is the primary function of the C-terminal domain (CTD) (Chang et al., 2016; McBride et al., 2014). Conversely, the membrane (M) protein’s primary job is to keep the viral envelope in its proper structure. It accomplishes this function through interactions with other coronavirus proteins, incorporation of the Golgi complex into new virions, and stabilization of the N protein. Consequently, a virus structure cannot exist without these E, N, and M structural proteins; thus, they can be considered prospective targets for the development of anti-COVID-19

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drugs. Numerous antivirals, antibacterial, anti-inflammatory, antitumor, and antiasthmatic medications have been identified to have comparably strong affinity for these targets, preventing the viral replication in the host cells (Schoeman & Fielding, 2019).

30.2.2 Nonstructural proteins 30.2.2.1 Proteases Both the major protease (3CLpro or Mpro, Nsp5) and the papain-like protease (PLpro, Nsp3) are essential to the life cycle of SARS-CoV-2, notably in replication (V’kovski et al., 2021). They broke the polyproteins to release different NSps. Polyphenols from different medicinal plants inhibit the major protease (Mpro) or papain-like protease and signaling pathways, respectively, as part of their method of coronavirus suppression. For instance, papyriflavonol A, kazinol F, kazinol J, 30 -(3-methylbut-2-enyl)-30 ,40 ,7-trihydroxyflavane, broussoflavan A, and broussochalcone A are some of the key drugs demonstrating greater inhibitory effects on the Mpro of SARS-CoV and SARS-CoV-2. The aforementioned compounds had strong binding affinity (between 7.6 and 8.2 kcal/mol) and were appropriately docked at the active site residues (His41 and Cys145) of Mpro (Park et al., 2017). Numerous tea polyphenols, including gallocatechin-3-gallate, epigallocatechin gallate, and epicatechin gallate, have also shown strong binding affinity and were proven to be effective SARS-CoV-2 Mpro inhibitors (Ghosh et al., 2020). They stabilized the complexes by noncovalent, polar, and hydrophobic interactions with residues like His41 and Cys145. Gallocatechin-3-gallate had the lowest binding affinity while epigallocatechin gallate had the highest binding affinity among these three polyphenols. Additionally, many tea plants-derived substances, including oolonghomobisflavan-A, theaflavin-3-O-gallate, and theasinensin D, had strong Mpro inhibiting potentials (Bhardwaj et al., 2020). Further research also proved andrographolide from Andrographis paniculata as an inhibitor of Mpro (Enmozhi et al., 2020). For the experimental research against Mpro, bioactive substances derived from Indian spices were also utilized. The compounds were located in the zinc database, and the findings showed that carnosol had the greatest binding affinity of 8.2 kcal/mol. Similarly, rosmanol (7.99 kcal/mol) and arjunglucoside-I (7.88 kcal/mol) demonstrated a robust and persistent binding affinity with favorable ADME characteristics. Computing techniques have shown luteolin’s effectiveness against SARS-CoV-2, but its comparative analysis with chloroquine molecule showed luteolin has a lower affinity for the active site of Mpro (Yu, Chen, et al., 2020). Quercetin, an antioxidant, also has a comparable impact. Additionally, in silico research revealed that the terpenoids caesalmin B, ursolic acid, bonducellpin D, carvacrol, and oleanolic acid, as well as the flavonoids 5,7-dimethoxyflavanone-40 -O-D-glucopyranoside, had a higher binding affinity for the Mpro of both SARS-CoV-1 and SARS-CoV-2 (Wu et al., 2020). Ocimum sanctum, Withania somnifera, and T. cordifolia are some very popular medicinal plants. Molecular docking and simulation studies of these plants revealed that their phytochemicals like vicenin, isorientin, and 40 -O-glucoside 2v-Op-hydroxybenzoate from O. sanctum, withanoside V and somniferine from W. somnifera (Ashwagandha), and tinocordiside from T. cordifolia also have good inhibitory potential toward Mpro of SARS CoV-2 (Shree et al., 2020).

30.2.2.2 RNA-dependent RNA polymerase (RdRp) For RNA transcription and viral replication, RdRp is a crucial enzyme. It is a conserved nonstructural protein of the virus (NSP 12) that is essential to SARS-CoV’s life cycle. At the C-terminus of the polymerase, the RdRp domain possesses a conserved Ser-Asp-Asp motif (Subissi et al., 2014). Clinical medications and novel compounds are investigated for their impact on RdRp as one of the antiviral drug development techniques since selective inhibition of RdRp by these agents could not result in major side effects and toxicity on host cells (Chu et al., 2006). Natural substances and their derivatives with antiviral, anti-inflammatory, and antitumor properties, such as betulonal from Cassine xylocarpa and gnidicin and gniditrin from Gnidia lamprantha, show strong binding affinity to the Ser-Asp-Asp motif of RdRp (Wu et al., 2020). According to several recent research, many phytochemicals have a high affinity for RdRp and are effective antiRdRp drugs. An analysis of five distinct phytochemicals shows that isoskimmiwallin has seven times the force and around 1.5 times the work of the control medication (remdesivir). Terflavin A, a different phytochemical, shows the second highest force. Additionally, terchebulin and terflavin C exhibit similar resistance to RdRp, while cinnamtannin has the weakest resistance among the five tested ligands, though it exhibits a slightly stronger resistance than the control (remdesivir). Comparing cinnamtannin, terflavin A, and terflavin C to isoskimmiwallin and terchebulin, the latter two show less work. Consequently, it was noted that all five medicines that are powerful against RdRp had strong binding

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forces. The overall result suggests that isoskimmiwallin and terflavin A can be good inhibitor candidates against RdRp (Arpana Parihar et al., 2022).

30.2.2.3 Helicases Nsp 13 belongs to superfamily 1 helicase, which is a class of ubiquitous multifunctional motor proteins called helicases. It takes part in the virus’s double-stranded (ds) DNA and RNA unraveling in the 50 -30 direction, which is fueled by ATP hydrolysis (Kwong et al., 2005). It has a helicase domain (Hel) and a metal-binding domain (MBD) at the end. While the C-terminal creates a helicase domain with a conserved motif, the N-terminal forms a Zn-binding domain. According to research, the replication, transcription, and translation of SARS-COV-2 depend on Nsp13-dependent unwinding (Frick & Lam, 2006). Helicases are therefore a possible therapeutic target for COVID-19. Recently, numerous powerful helicases encoded by SARS-COV-2 inhibitors have been described in diverse scientific publications. Preclinical research is being done on a number of inhibitors, including bananins, ADKs, 5-hydroxychromone derivative, and SSYA10
001, to treat SARS-COV-2 with respect to helicase as a target site.

30.2.2.4 The viral virulence factors Nsp1, Nsp3c, and ORF7a are three virulence proteins found in coronaviruses that interfere with the host’s innate immunity and aid viral immunological escape, according to research (Kamitani et al., 2006). The 40S subunit of ribosome and Nsp1 interact to cause mostly host mRNA degradation and suppress type-I interferon production (Kamitani et al., 2006). The capacity of Nsp3c to connect with the host’s adenosine diphosphate (ADP)-ribose, on the other hand, enables coronaviruses to evade the host’s inherent defenses (Narayanan et al., 2008). By preventing BST-2 glycosylation, ORF7a direct binding to bone marrow matrix antigen 2 (BST-2) reduces the function of BST-2 (Forni et al., 2016). BST-2 is in charge of preventing newly constructed coronaviruses from being released from host cells. These data imply that Nsp1, Nsp3c, and ORF7a may represent promising targets for the production of COVID-19 antiviral drugs (Taylor et al., 2015). Numerous therapeutic medications and herbal remedies with antibacterial and antiinflammatory properties, including piperacillin, cefpiramide, streptomycin, lymecycline, and tetracycline, were shown to have relatively significant binding affinities to these three target proteins, according to conducted studies (Dinarello et al., 1986).

30.3

Host-based targets

30.3.1 Host proteins 30.3.1.1 ACE2 For the development of therapeutics against COVID-19, angiotensin-converting enzyme 2 (ACE2) is regarded as a potent host target. Many phytochemicals are found to be effective against this target site. The essential oil component of garlic showed a potent inhibitory effect against the SARS-CoV-2 receptor ACE2. It consists of organosulfur compounds with tremendous pharmacological activities. GCMS analysis of this essential oil showed 18 such compounds which interact well with ACE2, including allyl disulfide (28.1%), diallyl tetrasulfide (6.5%), allyl methyl trisulfide (6.7%), allyl trisulfide (22.8%), and allyl (E)-1-propenyl disulfide (8.2%) (Kulkarni et al., 2020). Molecular docking and simulation studies revealed strong binding interaction with amino acids Pro565, Trp566, Ala396, Gln102, Gln101, Glu208, Gly205, Gln98, Asn210, Lys94, Lys562, Val209, and Ser563 for all those important organosulfur compounds. Additionally, the phytochemical chloroquine may prevent the cellular ACE2 receptor from being glycosylated, which would prevent SARS-CoV from attaching to the cell membrane (Vincent et al., 2005). For molecular docking studies to identify the binding studies with ACE2 receptor, a protein model for RBD was constructed, and thereafter, many isolated phytochemicals have been tested. The investigation revealed that hesperidin, emodin, and chrysin showed potential activity against the drug target (Kuo et al., 2007). The receptor domain residues interacting with ACE2 were found to be Tyr449, Tyr453, Asn487, Phe486, Tyr489, Gln493, Gly496, Gln498, Thr500, Gly502, and Tyr505. Essential oil compounds from star anise, basil, holy basil, eucalyptus, cinnamon, clove, thyme, geranium, oregano, and ajwain were also analyzed against the spike protein, and out of all, carvacrol, cinnamaldehyde, cinnamyl acetate, geraniol, L-4terpineol, and anethole displayed better binding affinity.

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30.3.1.2 TMPRSS2 The type-II transmembrane serine protease enzyme transmembrane protease serine 2 (TMPRSS2) cleaves the viral spike protein, which is activated by protease and causes virus membrane fusion on the cell surface (Chang et al., 2016; McBride et al., 2014; Schoeman & Fielding, 2019; Venkatagopalan et al., 2015). The initial step in facilitating host cell entry is the viral hemagglutinin protein binding to ACE2. TMPRSS2 enzymatic activity can be inhibited to stop some coronaviruses from infecting host cells. Neoandrographolide, a phytochemical, is thought to be a possible TMPRSS2 inhibitor (Glowacka et al., 2011).

30.3.2 Epigentic mechanism 30.3.2.1 Cytokines toxicity Glycoproteins called cytokines act as chemical messengers in the immune system’s response to pathogens. The body produces a large number of cytokines from our immune cells. The pro-inflammatory cytokine among these, which are generated in reaction to viruses like SARS-COV-2, have a harmful effect. SARS-COV-2 produces mild or severe acute respiratory syndrome and releases pro-inflammatory cytokines including interleukin (IL)-1 and IL-6 after infecting the upper and lower respiratory tract (Dinarello et al., 1986). It frequently triggers the release of pro-IL-1 from the lung’s toll-like receptor (TLR), which is then cleaved by caspase-1. This is followed by the activation of the inflammasome and the creation of active mature IL-1, which is a mediator of fever, lung inflammation, and fibrosis (Conti, Gallenga, et al., 2020). It is anticipated that suppressing IL-6 and the pro-inflammatory IL-1 family will have a therapeutic effect on COVID-19. Non-inflammatory cytokines like IL-37 and IL-38 could do this. Class II histocompatibility complex, IL-1, IL-6, and tumor necrosis factor molecules can all be suppressed by IL-37 through binding to the IL-18Ra receptor (Conti, Lauritano, et al., 2020; Heoharides et al., 2019). In an inflammatory condition brought on by COVID-19, IL-37 inhibits IL-1 to have a unique therapeutic effect. Similar to IL-1, IL-38, the newest member of the IL-1 family, is a suppressor cytokine that inhibits IL-1 and other pro-inflammatory IL family members. Additionally, it suppresses inflammation in viral infections, especially COVID-19-related viral infections, making it a potential target therapeutic cytokine for the treatment (Lauritano et al., 2020).

30.3.3 Pathways The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are a crucial signaling route for several different types of cytokines and are crucial for the immune system’s cellular response to external signals. JAK pathway inhibitors had a crucial impact in preventing and suppressing the production of cytokines (Russell et al., 2020). Baricitinib, fedratinib, and ruxolitinib are found to be powerful and selective JAK-STAT signaling inhibitors that work well against the negative effects of elevated cytokine levels, especially those seen in COVID-19 patients (Kontzias et al., 2012). Additionally, tylophorine-based substances and inositol-required transmembrane kinase/endoribonuclease 1a showed anti-COVID-19 activity in various trials (Kontzias et al., 2012; Russell et al., 2020).

30.3.3.1 Alkaloids The term “alkaloids” refers to a vast category of naturally occurring chemical amalgams that play a significant role within the biggest group of bioactive compounds found in plants. Numerous alkaloids find their way into the modern diet, both in the form of foods and beverages. Some examples of these are the alkaloids found in coffee seeds (caffeine), tea leaves (theophylline and caffeine), cacao seeds (theobromine and caffeine), potatoes (solanine), and tomatoes (tomatine). Throughout the course of the COVID-19 epidemic, which had an impact on the entire globe, quinine and its derivatives showed up rather regularly. The glycosylation of SARS-CoV cellular receptors is inhibited by chloroquine’s presence in the body. Additionally, it raises the endosomal pH, which is necessary for virus/cell fusion, and as a result, it exhibits antiviral action across a wide spectrum (Rashed & Rashed, 2021). There is a large array of actions that can be carried out by quinoline and quinazoline alkaloids, which are N-based heterocyclic alkaloids. Antiviral properties have been found for many of these molecules. Alkaloids with their nitrogen-containing structures are among the most prevalent metabolites in plant families such as Amaryllidaceae, Apocynaceae, Papaveraceae, Asteraceae, and Solanaceae with possible biologically active compounds and therapeutic actions. The majority of alkaloids are synthesized from a small number of amino acid antecedents, including phenylalanine, tyrosine, tryptophan, ornithine, and lysine. These precursors are transformed into adaptable central intermediates, resulting in the generation of diverse alkaloids (Majnooni et al., 2021).

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Since 1951, cepharanthine has been employed in Japanese medicine largely to cure radiation-induced leukopenia, alopecia areata, and alopecia pityrodes, in addition to exudative middle-ear catarrh and snake bite. In addition, CEP was discovered as perhaps the most targeted therapy against SARS-CoV-2-related pangolin coronavirus, a less virulent model for SARS-CoV-2, in a comprehensive screening of 2406 clinically licensed pharmaceuticals. CEP suppresses viral entry and replication at small levels against SARS-CoV-2 and related viruses; it was found lately as the most powerful coronavirus inhibitor among 2406 clinically authorized medication repurposing candidates in a preclinical prototype (Rogosnitzky et al., 2020). Chelerythrine is a quaternary ammonium alkaloid that has a structure 2,3,7,8-tetrasubstituted benzophenanthridine. It has been isolated from the roots of many medicinal plant families, including Fumariaceae, Papaveraceae, and Rutaceae. Chelerythrine possesses broad-spectrum antiviral and anti-inflammatory properties and might be diagnosed and treated by COVID-19 victims. Chelerythrine has the ability to suppress a hyperinflammatory immune reaction by modulating key signaling pathways that are implicated in SARS-CoV-2 transmission. These pathways include alterations in the activity of Nrf2, NF-B, and p38 MAPK. Furthermore, chelerythrine’s potent protein kinase C 2 / 2 inhibition effect makes it useful for preventing cerebral vasospasm and lowering eryptosis, and it also has favorable benefits in reducing pulmonary inflammation and fibrosis (Valipour et al., 2021). Norquinadoline A, deoxytryptoquivaline, and deoxynortryptoquivaline show high affinity to the three targets of SARS-CoV-2 notably to protease, spike glycoprotein, and human angiotensin-converting enzyme. Such alkaloids consequently have the potential for future investigation as potential multitarget medicines in the combat against COVID-19 (Ismail et al., 2021). Had been previously studied by in human lung cells (MRC-5), the cytopathic impact of HCoVOC43 was able to be inhibited by the alkaloids tetrandrine, fangchinoline, and cepharanthine, with corresponding EC50 values of 295.6, 919.2, and 729.7 nM. The alkaloids were shown to be lethal to MRC-5 cells, with CC50 values of 15.51, 12.40, and 10.54 M and SI values of .40, 11, and 13, respectively, (Verma et al., 2020). Studies using molecular docking have shown that a multitude of the phytochemicals found in Solanum surattense has the ability to bind firmly to the C3-like protease found in SARS-CoV-2. This raises the likelihood that these phytochemicals could act as medicinal applications against the virus. There are around 200 different alkaloids that have been isolated from various Solanum species. Each of the following alkaloids shares the same C27 cholestane skeleton and may be categorized according to one of five distinct structural types: solanidine, spirosolanes, solacongestidine, solanocapsine, or jurbidine. Even though a lot of research has been done on the anticancer properties of alkaloids, there are still quite a few of them that should be looked at as potential antiviral medicines. The interaction of the alkaloid carpesterol with the C3-like protease resulted in a binding energy of -8.3 kcal/mol. It had an effect on Arg40, Cys85, Phe134, and Pro184, four of the twenty-four amino acids. It is fascinating to note that the molecule has been recognized as a potential lead drug for the treatment of human hepatitis B viral capsid protein as a result of an in silico approach (Ogunyemi et al., 2020). A rich supply of chemopreventive phytochemicals may be found in the African herbs and medicinal flora that are used in traditional medicine. It has been suggested that some phytochemicals derived from African herbs and medicinal trees might act as antagonists of the SARS-CoV-2 RdRp. Seeing as this enzyme is so important to the machinery that controls viral replication and transcription, it was identified as a prime candidate for the development of novel therapeutics, and the Food and Drug Administration (FDA) has given its blessing to the development of lead modulators like remdesivir. The highest binding propensity and protein interface displayed by alkaloids, such as 100 -hydroxyusambarensine, cryptoquindoline, and cryptospirolepine (the top docked compounds), paint a picture of alkaloids as possible inhibitory RdRp of SARS-CoV-2, SARS-CoV, and HCV. A preserved binding mechanism was exhibited by the alkaloids, particularly the indole alkaloids (10-hydroxyusambarensine and strychnopentamine), from Strychnos usambarensis to the SARS-CoV-2 RdRp and HCV (Ogunyemi et al., 2020).

30.3.3.2 Flavonoids As antivirals, numerous flavonoids have been discovered to impede the targets of SARS and MERS coronaviruses in a wide range of ways. These include restricting the enzyme reactions of viral proteases (3CLpro and PLpro), meddling with spike glycoproteins, and repressing the interaction of ACE2 receptors. ACE2 receptors not just play a significant role in cardiovascular diseases, but they can also be a major element in viral contaminations and pneumonia. Particularly, the hydroxyl group at the seventh position of flavonoids is indeed for attacking the receptor sites toward 3CLpro and PLpro. Quercetin is among the most essential flavonoids and is classified as a flavonol. As confirmed by Alzaabi et al., quercetin was a promising anti-SARS-CoV-2 option. By inhibiting 3CL and PLpro proteases, various flavonols have shown antiviral efficacy toward coronaviruses (such as SARS-Cov and MERS-CoV). Since the spike glycoproteins of SARS-CoV and SARS-CoV-2 have a greater degree of sequence similarity, flavonols could also inhibit the entrance of SARS-CoV-2 into host cells. In addition, it has been shown that the spike protein of the new virus

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interacts with the ACE2 receptor with greater fidelity than SARS-CoV. Consequently, inhibiting ACE2 via a competitive binding seems to be an efficient strategy for preventing SARS-CoV-2 outbreaks (Alzaabi et al., 2021). Another extensive and functionally varied class of secondary metabolites that plants create is called flavonoids. Flavonoids are synthesized by plants. The basic flavonoid skeleton is comprised of 15 carbon atoms, and it is comprised in such a way that the two major aromatic rings (A and B) are linked by three carbon atoms (C6-C3-C6), which may be interconnected to a third ring. In addition, the two key aromatic rings (A and B) are attached by three carbon atoms (C6-C3-C6) (C). Various flavonoids have demonstrated anti-SARS-CoV-2 action in laboratory studies. Numerous research has indicated that the flavonoids baicalin and its aglycone baicalein are able to prevent SARS-CoV-2 transmission in vitro. Additionally, a few of these studies have demonstrated that these flavonoids are able to reduce Mpro activity (Espan˜o et al., 2021). By binding to cellular receptors, flavonoids prevent viruses from entering cells, and they also prevent viral multiplication and translation. COVID-19 may be directed toward the Mpro enzyme because of its significance in viral replication and translation. Many such flavonoids, including azithromycin, mangiferin, procyanidin—2,7dimethoxyflavaN-40 -O-D-glucopyranoside, amentoflavone, hidrosmin, diosmin, gallocatechin gallate, elsamitrucin, pectolinarin, quercetin, and isoquercetin, have been suggested by computational research findings to have strong affinity for recent scenarios. Flavonoids like hesperetin, myricetin, flavonoid, and caflanone have shown strong binding interactions for ACE-2, rendering them relevant sites of investigation over SARS-CoV-2 in addition to the Mpro, another potential therapeutic target (Das et al., 2021). The antiviral capability of naturally produced flavonoid molecules is rather significant. The flavonoid scutellarein, which is derived from the root of Scutellaria baicalensis (Lamiaceae), has been demonstrated to block the nsP13 helicase of SARS-CoV-2 by modifying its ATPase action. In reply to COVID-19, the pharmaceutical study has demonstrated the possible medicinal value of baicalin and baicalein, which are additional particular flavone glycosides of S. baicalensis (Sytar et al., 2021). Apples, berries, grapes, onions, green tea, and Ginkgo biloba are just a few of the crops and items that contain the flavonoid known as quercetin (3,30 ,40 5,7-pentahydroxyflavone). Quercetin is classed as a flavonoid. The therapy of cell cultures with quercetin was effective in inhibiting influenza strains and blocking the entrance of the H5N1 virus. In addition, quercetin has been shown to be useful in the treatment of viral infections because of its possible antiviral effects, which include the inhibition of viral proteases, reverse transcriptase, polymerases, and reverse transcriptase; the adhering of viral capsid proteins; and the suppression of DNA gyrase (Ali et al., 2022). A flavonoid repertoire was utilized in recent research to investigate the possibility that these particular phytochemicals demonstrated an antagonistic effect toward SARS-CoV 3CLpro. The substances rhoifolin, herbacetin, and pectolinarin were shown to have detrimental influence over 3CLpro, with IC50 values of 27.45, 33.17, and 37.78 M, accordingly. Pectolinarin was also reported to have strong inhibitory potential (Verma et al., 2020). The phenolic flavonoid known as naringin is a member of the flavonoid family. It possesses anti-neuroinflammatory and antiviral actions, and it has the ability to be employed in the prevention and therapy of COVID-19. Through its suppression of highly mobile group box 1 (HMGB1) in COVID-19, naringin is indeed able to reduce the levels of expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), interleukin-1 beta, and interleukin-6. Additionally, the expression level of p38MAPK was reduced, which inhibited the synthesis of inflammatory mediators by HMGB1 and the lung damage that was linked with it. In the light of the fact that pro-inflammatory mediators play a crucially detrimental role in the neurological indications caused by COVID-19, naringin appears to be a promising antiinflammatory and antiviral option in the fight against associated neuronal symptoms (Fakhri et al., 2021). Mechanisms of flavonoids-mediated inhibitory activity of SARS-CoV-2: an bioinformatic research claimed that hesperidin has an inhibition against SARS-CoV-2 by binding to SARS-CoV-2 Mpro, the receptor-binding domain of S protein (RBD-S), and the peptidase domain of ACE-2. This recommended that hesperidin has an inhibition effect against SARS-CoV-2. Research performed entirely in computer simulations found that quercetin could be able to limit the spread of SARSCoV-2. The binding attraction of quercetin to Mpro was found to be quite high. Cyanidin and genistein were revealed to have a binding interaction with Mpro and RdRp that is equivalent to that of nelfinavir and lopinavir, according to the findings (Alzaabi et al., 2021).

30.3.3.3 Terpenes and terpenoids Plants and fungi are by far the most prevalent sources of bioactive compounds, which are referred to as terpenes. Terpenes are the biggest and perhaps most varied category of naturally occurring substances. Their compositional identity is denoted by a five-carbon unit, which further serves as the foundation for their categorization. The majority of terpene molecules contain cyclic structures, and these compounds are able to be changed and subdivided into

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monoterpenes, diterpenes, triterpenes, sesterpenes, and sesquiterpenes. Sesquiterpenes are also a kind of terpene. They offer considerable benefits in aromatherapy, particularly monoterpenes and sesquiterpenes of small molecular weight, which are less oxygenated and provide natural ingredients in their organoleptic properties due to their volatility. Carvone, geraniol, citral, and citronellal have demonstrated antiviral efficacy against herpes simplex virus type 1 (HSV-1) equivalent to acyclovir, the usual medicine utilized to treat the illness, as well as several additional cases (Boone et al., 2020). There is a class of terpenoids known as quinone methide triterpenes, and they are exclusively widely distributed in plants belonging to the Celastraceae group. One such species is Tripterygium regelii. These extracts exhibited a reasonable degree of inhibition activity toward 3CLpro, with an IC50 value ranging from around 2.6 to 10.3 M. The availability of a quinone methide moiety has a substantial impact on 3CLpro attenuation, as determined by SAR analyzation. In 2012, an in vitro study was conducted to investigate the anti-HCoV efficacy of triterpenoids that had been extracted from the leaf of Euphorbia neriifolia. After being incubated with HCoV, 3-friedelanol, which has the architecture of a triterpenoid, demonstrated more strong antiviral action and enhanced the survivability of the cells (Boozari & Hosseinzadeh, 2021). Natural oils that have synthetic ingredients and anti-inflammatory actions can also be eligible for testing to see whether or not they have the ability to speed up recuperation from ageusia and anosmia. Cannabinoid receptor 2 (CB2) ligands were discovered in 2008, and one of them was a sesquiterpene called ß-caryophyllene. This sesquiterpene is present in a variety of plants and spices, such as lavender and black pepper. It was discovered that improving reepithelialization of mouse cutaneous wounds by topical administration of ß-caryophyllene was possible. The results of an RNA sequencing study on skin that had been exposed to ß-caryophyllene showed that several epidermal stem cell marker genes were up substantially in the skin that had been implemented with ß-caryophyllene (Gli1, Lgr5, Sox9, Lrig1). All such genes are also engaged in the turnover of the tongue’s epidermis. This points to the idea that ingesting ß-caryophyllene through the mouth might trigger the signaling pathways to the multiplication of epidermal stem cells in the tongue, which would be analogous to what took place in the skin. In addition, metabolic pathways that are associated with inflammation and the immune system were inhibited. According to the findings among these investigations, ß-caryophyllene could be one of the most promising choices, particularly for promoting rejuvenation and, as a result, for promoting the restoration of a person’s taste buds (Koyama et al., 2021). The far more powerful abietane terpenoid, which was characterized by pi-alkyl/alkyl interactions with 3CLpro, had the greatest ligand efficacy. The abietane has demonstrated a broad spectrum of activity in both practical and in silico analyses; nevertheless, the precise link between structure and biological activity is not yet fully known (Swain et al., 2021). Several terpenoids have been utilized in the therapeutic treatment of COVID-19 due to their potent antiviral activities, which have been shown to be effective against viruses such as SARS-CoV-2 and hepatitis C virus. Terpenoids such as (-)—pinene, (-)—pinene, and 1,8-cineole have an unique mechanism to impede infectious bursal disease virus (IBV) and split its replication cycle. This is accomplished by high affinity to the N protein of the virus, which prevents the N protein from interfering with the viral genomic RNA. The simulations of the terpenoids, which corroborated the earlier data by demonstrating that these compounds are able to effectively attach to five amino acid residues at the catalytic site at the N-terminus of the N protein, provided evidence in favor of the hypotheses (TyrA140, TyrA92, As A138, PheA137, and ProA134). According to the findings of a research investigation, these amino groups are significantly maintained across all of the avian coronavirus subtypes. Consequently, terpenoids and isoprenoids, which are organic compounds generated from terpenes, can be considered potentially powerful antiviral medicines versus practically all IBV variants and as a plausible candidate for further research because of their ability to inhibit the replication of IBV (Attia et al., 2020). On Vero E6 cells, a number of anti-cytopathogenic phytochemicals, comprising diterpenoids, triterpenoids, and sesquiterpenoids, were found to be effective against an anti-SARS-CoV-induced cytopathogenic impact. Furthermore addition, anti-SARS-CoV action has been attributed to sesquiterpenes, abietane, labdane diterpenes, and lupane triterpenes. Saikosaponins, also known as triterpene saponins, were identified from Radix Bupleuri, according to studies. Between these, saikosaponins (A, B2, C, and D) strongly suppressed HCoV-229E virus infection with IC50 values of 8.6, 1.7, 19.9, and 13.2 M. This value represents the concentration at which 50% of the infection is controlled. Inhibition of viral attachment and penetration phases, as well as increased activity versus HCoV229E, was seen with saikosaponins B2 (Idrees et al., 2021). Moreover, ursolic acid and pentacyclic triterpenoid both have the ability to strongly block the activity of proteases. The results of a molecular docking simulation work that was centered on an incorporated molecular modulation strategy unveil that it acts as a prospective suppressor of the key protease (Mpro) of SARS-CoV2 and the chymotrypsin-like 3CLpro protein. These proteins are synthesized after the refining polypeptide a/b and encompass the transcription replication machinery of SARS-CoV2. Therefore, the ursolic acid that is found in Hyptis suaveolens has the potential to be utilized as a pharmaceutical agent in the treatment of COVID-19 virus (Mishra et al., 2021).

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30.3.3.4 Polyphenols Phytochemicals with aromatic rings that include one or multiple hydroxyl groups are referred to be phenolic compounds and polyphenols, respectively. The word “phenol” comes from “phenolic,” which refers to a simple aromatic phenyl ring that only has one hydroxyl group. Tannins have a reputation for being extremely powerful protein inactivators, and they also have antiviral properties. This behavior may also be observed when phenols or tannins are cultured with viruses. Polyphenols attach to virus particles in the envelope, which frequently inhibits a virus from docking with its infected cells. As a result, most polyphenols exhibit antiviral properties; however, this only holds true if the virus particles are still whole and have not yet been ingested by the host cell (Wink, 2020). Tannins are polyphenolic compounds that belong to a varied class of chemicals that can fasten to one another to develop either remediable or irreparable complexes. The majority of the components that make up these structures are proteins, polysaccharides like cellulose, hemicellulose, and pectin, alkaloids, nucleic acids, minerals, and so on. It has been reported that the tannin known as epigallocatechin-3-gallate offers antidiabetic effect due to the fact that it combines with glucose molecules and induces receptor cells to make use of carbohydrates (Subissi et al., 2014). Tannins have been demonstrated to exhibit antiviral action in experiments conducted against the human immunodeficiency virus, bovine adeno-associated virus, and noroviruses (Singh et al., 2021). Increasing research revealed that polyphenols and micronutrients boost defense ability, which is defined as resilience to infection, by modifying immune homeostasis; this might have a substantial effect in lowering COVID-19, especially for cytokine storms (-). Epigallocatechin-3-gallate, often known as EGCG, has had an action that prevents neutrophils from migrating across monolayers of endothelial cells, which then in turn might lower vascular permeability. Additionally, it has the capacity to inhibit neutrophil elastase, a proteolytic protein that has been linked to an upsurge in alveolar epithelial permeability. It is possible that the homeostatic imbalance that results from the breakdown of endothelial cell barrier integrity and the formation of reactive oxygen compounds by neutrophils is the cause of acute respiratory distress syndrome (ARDS) in COVID-19 patients. This illness was found in these individuals (Chowdhury & Barooah, 2020). Researchers are looking at the antibacterial and antiviral activities of polyphenolic chemicals that may be present in plants sourced from both the land and the sea. One variety of polyphenolic chemical is called phlorotannin present in a brown alga which is the source of this one-of-a-kind polyphenolic component. Researchers have determined that phlorotannins generated from brown algae are the most potent inhibitor of SARS-CoV-2 Mpro. Dieckol and 6,60 -bieckol, two phlorotannins that were derived from E. cava, a kind of brown algae that may be consumed, were shown to be Mpro inhibitors, ultimately helping to control COVID-19 (Rahman et al., 2022).

30.3.4 Effects of phytochemicals from honey against COVID-19 Honey is a gifted complex natural product produced by honey bees from nectar, fruits effluvium, and aphid’s extractions. It is made up of a number of bioactive substances having potent therapeutic effects. It has been widely used for its antibacterial qualities since ancient times (Meo et al., 2017). The geographic and botanical origin, bee species, and climate of honey all have a significant impact on its chemical composition (Al-Hatamleh et al., 2020). As a result, different types of honey produced by different types of bees are sold in the market. The chemical composition of 100 g of honey is approximately 64.9%
72.6% carbohydrates, 35.6%
41.8% fructose, 25.3%
28.2% glucose, 16.9%
18% water, 1.8%
2.7% maltose, 0.23%
1.21% sucrose, and 0.50%
1% proteins, vitamins, amino acids, and minerals (Cianciosi et al., 2018). The secondary metabolites found in honey are primarily responsible for their therapeutic effects. These secondary metabolites are phenolic compounds that act as antioxidants and have a variety of chemical structures, such as phenolic acids and polyphenols (flavonoids), and their chemical compositions mostly rely on the source of the substance (Cianciosi et al., 2018). These phenolic compounds’ modes of action can be generally split into two categories: 1. Immunity-boosting mechanism and 2. Antiviral mechanism.

30.3.4.1 Immunity-boosting mechanism A well-known immune system booster, honey, increases lymphocyte cell proliferation and controls the release of proinflammatory cytokines including IL-6, LI1beta, and tumor necrosis factor (TNF), among others (Abuharfeil et al., 1999; Tonks et al., 2003). Honey’s mode of action against viral infection and for the expansion of immunity is mostly due to the antioxidant properties of the phenolic components of secondary metabolites that are found in honey (Fig. 30.1) (Miguel et al., 2017). According to previous reports, the severity of the infection in COVID-19 acute

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FIGURE 30.3 Mechanism of honey’s phenolic chemicals against COVID-19 infection.

respiratory syndrome is mostly caused by the significant production of pro-inflammatory cytokines (Conti, Ronconi, et al., 2020). Honey can therefore be a helpful adjunct in the management of infection. Antioxidants may change signal transduction pathways linked to oxidative stress, which are crucial for cellular responses like inflammation, survival, cellular proliferation, and death (Dharmaraja, 2017; Dzialo et al., 2016). Additionally, they shield vital cellular components (lipids, amino acids/proteins, and DNA) from the destructive effects of oxidative stress, which promotes lymphocyte activation and proliferation. Nevertheless, inhibiting the pathways for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and mitogen-activated protein kinases (MAPK) causes complex cellular processes that lower pro-inflammatory genes and so limit the production of pro-inflammatory cytokines (Yahfoufi et al., 2018). These antioxidants also lead to less production of arachidonic acid, which prevents the phospholipids oxidation of membranes and, thus, reduces the production of important inflammatory mediators leukotrienes and prostaglandins (Adhikari et al., 2021; Sohail et al., 2021) (Fig. 30.3).

30.3.4.2 Antiviral mechanism Numerous studies have demonstrated that honey has effective antiviral capabilities against viruses like the H1N1 influenza virus, the DEN virus, the varicella zoster virus, and the Rubella virus. When phenolic compounds from honey were tested with molecular docking studies for their potential effects against SARS-CoV2, it revealed that some of the major components, including the flavonoid hesperidin, rosmarinic acid, caffeic acid, galangin, caffeic acid phenethyl ester (CAPE), and chrysin, can stop the spread of viruses by blocking the 3-chymotrypsin-like cysteine protease (3 CLpro) enzyme in the virus (Cheung et al., 2019; Hashem, 2020; Wu et al., 2020). Other investigations have also shown that methylglyoxal, a carbonyl produced during the conversion of DHA during honey ripening, blocks and creates virion assembly (Behbahani, 2014). Through its impact on the S-RBD binding site and subsequent inhibition of the interaction with ACE2, the flavonoid hesperidin of honey has also been reported to lower viral load. Hence, this evidence is making the first step of future research studies to explore the direct and indirect medicinal values of the phytochemicals from honey against the COVID-19 infection.

30.4

Conclusion and future prospective

Lastly, in order to get the most health benefits from various plant components, you may either take them in their natural, unprocessed state or transform them into extracts and teas. In addition, phytochemicals have the potential to be a great source of healthier and less expensive treatments that are comparable to commercial ingredients. More particularly, plants that have the ability to inhibit the spread of influenza and malaria are possible contenders (Sohail et al., 2021). As emphasized by the WHO-China Joint Mission on COVID-19, the utilization of the possibilities of phytomedicine, which is potentially the earliest and most diverse of all therapeutic systems, is an essential part of the global

TABLE 30.1 Important phytochemicals structures, classification, and their applications. Structure

Phytochemical

Source

Application

References

7-Methoxycryptopleurine

Boehmeria pannosa

In vitro blocking of the S and N proteins, 3CLpro Inhibitor. EC50 5 58 nM

Yang et al. (2010)

Berbamine

Berberis vulgaris L. (Berberidaceae)

In vitro blocking of the E proteins and the calcium transition. EC50 5 14.5 μM, 2.3 mM

Gao et al. SARS, Huang et al. (2021), Huang et al., Berbamine, Liang et al. (2019), Yan et al. (2018)

Cepharanthine Cepharanthine

Stephania cepharantha Hayata (Menispermaceae)

In vitro blocking of the expression of S and N proteins, RdRp inhibitor. EC50 5 0.83 μM

Gad et al. (2020), He et al. (2021), Jan et al. (2021), Kim, Min, Jang, Lee, et al. (2019), Ohashi et al. (2021)

Lycorine

Amaryllidaceae species

In vitro Mpro inhibitor. EC50 5 15 nM, 0.15 μM 0.47 μM

Li et al. (2005), Shen et al. (2019), Zhang et al. (2020)

Alkaloids O O N

O O

O N

O

H O O

OH O

N

H O O H

N

O H O

N

O O

OH HO H

H

O O

N

(Continued )

TABLE 30.1 (Continued) Structure N

Phytochemical

Source

Application

References

Quinine

Cinchona bark (Cinchona officinalis)

In vitro Mpro and S proteins inhibitor (in silico study). EC50 5 10.7 μM

Gendrot et al. (2020), Große et al. (2021), Roza et al. (2021)

Emodin

Roots of the Chinese rhubarb

Inhibition of the S protein and ACE2 interaction

Ho et al. (2007)

Gallocatechin gallate

Green tea

Interaction with catalytic residues of major protease (Mpro)

Ghosh et al. (2021)

6,60 -Bieckol

Brown algae, Ecklonia cava

Inhibits major protease (Mpro)

Gentile et al. (2020)

OH

H O N

Polyphenol OH

O

OH

HO O OH OH HO

O

OH

HO

O OH

O

OH OH OH

OH

O O

OH HO HO

OH

O

O

O

OH HO

O OH

OH O

OH

OH OH

OH

OH

OH

O

OH

HO

O

OH

O

Ecklonia stolonifera

SARS-CoV-2 MPRO inhibition of viral life cycle inside host

Letko et al. (2020)

Tetra-O-galloyl-β-D-glucose (TGG)

Rhodiola rosea and Syzygium aromaticum

Viral replication and transcription. Bind to the SARS- CoV surface protein, hindering virus entry into its host cells

Toto et al. (2020), Yi et al. (2004)

Quercetin

Capers Buckwheat Onions

In vitro SARS-CoV-2 proteases (3CLpro, PLpro), ACE2 receptor, glycoproteinRBD Spike

Chiow et al. (2016), Derosa et al. (2021), Inocencio et al. (2000), Wang et al. (2015)

Hesperidin

Lemon, orange, and various polyherbal formulations

In vitro MERS-CoV S protein

Yamamoto et al. (2016)

Naringenin

Citrus fruits and tomatoes

In vitro SARS-CoV-2 protease (3CLpro), ACE2 receptor, NFkB

Tutunchi et al. (2020), Wilcox et al. (1999), Yu, Liu, et al. (2020), Zhao et al. (2017)

O

O OH

Dieckol

OH

OH O

OH

O OH

OH

OH HO HO O O

HO O

HO

OH

O O

OH

O

HO

OH HO

HO

Flavonoid OH OH HO

O OH OH

OH

HO HO

O

O

OH O

O O HO HO

O

O

OH OH

OH

HO

O

O

O

OH

(Continued )

TABLE 30.1 (Continued) Structure O HO

O

Phytochemical

Source

Application

References

Hesperetin

Citruses

In vitro SARS-CoV-2 protease (3CLpro), glycoprotein-RBD spike, ACE2 receptor

Bellavite and Donzelli (2020), Lin et al. (2005), Wang et al. (2019)

Curcumin

Turmeric

In vitro MERS-CoV S protein

Yamamoto et al. (2016)

Betulinic acid

White birch (Betula pubescens), ber tree (Ziziphus mauritiana), sycamore

In vitro SARS-CoV-2 MPRO inhibition of viral life cycle inside host

Wen et al. (2007)

Salvinorin A

Salvia divinorum

In vitro SARS-CoV-2 MPRO inhibition of viral life cycle inside host

Shaghaghi and Molecular

Bilobalide

Ginkgo biloba

In vitro SARS-CoV MPRO inhibition of viral life cycle inside host

Shaghaghi and Molecular

OH

O

O

OH

R1

R2

HO

OH

R1 = OCH3, R2 = OCH3 (Curcumin) R1 = OCH3, R2 = H (Demethoxycurcumin) R1 = H, R2 = H (bis-Demethoxycurcumin) Terpenoids

H OH H O H HO

H

O O

O

H O

O H

O

O

HO HO

O O

O O

O

O

O

Tanshinone

Salvia miltiorrhiza

In vitro SARS-CoV MPRO, SARS-CoV PLPRO inhibition of viral life cycle inside host

Park et al. (2012)

Celastrol

Tripterygium wilfordii Hook F

In vitro SARS-CoV MPRO inhibition of viral life cycle inside host

Ryu et al. (2010)

O O

O OH O HO

OH

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Recent Frontiers of Phytochemicals

response to the COVID-19 pandemic. Phytomedicine is one of the oldest and most diverse of all therapeutic systems. A combination of 230 different species have been identified as having the propensity to provide a supply of components for the antiviral response to the 2019 new coronavirus. Of these species, 30 encompass affirmed anti-COVID-19 secondary metabolites, 90 are used conventionally to handle at least 3 typical signs of COVID-19, 10 possess immunostimulant activity, 52 have anti-inflammatory function, 14 include antiviral activity, and 78 plants are cataloged as being used to treat malaria (Fongnzossie Fedoung et al., 2021). Additionally, 14 of these life-forms have antiviral activity. The fact that herbal medicines have bioactive constituents that might be included in the formulation of drugs to treat a variety of conditions with little adverse effects has attracted a substantial amount of attention in recent years. Since antiquity, medicinal plants have emerged as one of the most popular therapy options for a variety of viral infections due to the fact that they are healthier, inexpensive, and less hazardous than other treatment methods. The versatile character of phytochemicals and unique chemical variety have captured the interest of researchers looking to create leads originating from natural sources in the battle against COVID-19. The refined preparations are traditionally utilized in Ayurvedic, Siddha, Unani, and Chinese health fields. It is considered that they are acceptable for human ingestion, with few or no negative effects. Indigenous herbal remedies are utilized in foods to boost immunity throughout the initial stages of development (Venkatagopalan et al., 2015), a practice that is further encouraged by the World Health Organization (WHO) and acknowledged in the health objectives. Even though developing potent bioactive merchandise that are effective against a particular disease, such as COVID-19, is a faster process than developing vaccines, it is still a challenging task due to the wide variety of organic metabolites, the synthetic ambiguity of those metabolites, and the extraction of those metabolites. In natural products research, computational methods for bioactive chemicals are a helpful method for decreasing the amount of time spent in phytochemical screening of many natural goods extracts. This is accomplished by simulating the process of performing the screening in a laboratory setting. The term for this method is known as in silico studies by molecular docking (da Silva Antonio et al., 2020). Many times, a molecular repertoire of plant-derived drugs including antiviral characteristics and a range of modes of action is where these plant-based medications get their start as a therapeutic candidate. In traditional medicine, the treatment of viral illnesses often involves the use of natural products from plants or extracts. This practice has been used for centuries. Because the outbreak puts people’s lives and ways of making a living in danger, it makes sense to focus on naturally occurring substances that have the capacity to fight viruses. The business for herbal supplements with specialized nutraceutical qualities is massive. Because phytochemicals have low specificity, low bioavailability, and poor dispersion when examined in vivo, the majority of the work that focuses on the health benefits of phytochemicals is undertaken in vitro. This is because in vivo testing produces inconsistent results. The most optimistic of the instruments and tactics that are employed to augment these functionalities are the incorporation of structural alterations into the molecules, the employment of permeation enhancers, and nanotechnology. These three methods are listed in the order of promise. The next phase of epidemiologic studies should concentrate on conducting clinical trials on COVID-19 individuals. The goal of this study is to demonstrate a reduction in the multiplication of the virus within the body of the patient as well as a lessening of the clinical symptoms. In addition, including phytochemicals in one’s diet has a number of benefits, including the fact that they have a good safety record and do not cause any severe adverse effects. Nevertheless, in order to evaluate bioactive treatments against an extremely infectious virus such as SARS-CoV-2 (3CLpro), one requires costly and properly covered research facilities in which to undertake antiviral tests or utilize molecular modeling techniques (molecular docking) for computer-aided drug design and/or exploration. It is a fallacy to believe that any or all compounds found in molecular docking to entangle specific proteins of the virus will demonstrate their ability to suppress the virus in vivo. Notwithstanding, molecular docking has proven to be a useful tool for screening new compounds against essential proteins of a variety of pathogenic microbes or potential targets in the human body, which allows scientists to continue their quest for a treatment. Computational methods, which are affiliated with search space minimization, financial viability, and high versatility, may be particularly helpful for speedily discovering a powerful and effective inhibitor of the COVID-19 virus. This is because virtual screening reduces the dimensionality required to locate a potential inhibitor, which in turn reduces the cost of doing the investigation (Table 30.1).

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Chapter 31

Phytochemicals—a safe fortification agent in the fermented food industry Renitta Jobby1,2, Sneha P. Nair1, Vaishnavi Murugan1, Simran Khera1 and Kanchanlata Tungare3 1

Amity Institute of Biotechnology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India, 2Amity Centre of Excellence in

Astrobiology, Amity University Maharashtra - Pune Expressway, Mumbai, Maharashtra, India, 3School of Biotechnology and Bioinformatics, D. Y. Patil Deemed to be University, Belapur, Maharashtra, India

31.1

Introduction

“Let food be thy medicine and medicine be thy food” by Hippocrates is of immense relevance nowadays owing to increasing awareness about healthy diet habits and the advent of new diseases (Valero-Cases et al., 2020). Phytochemicals are chemical compounds synthesized by plants and are present in edible foods like seeds, fruits, legumes, vegetables, nuts, grains, and tea (Arte´s-Herna´ndez et al., 2021). These bioactive constituents accumulate in all the parts of the plant and assist to protect itself against environmental hazards like pollution, stress, pathogens, UV exposure, and consumption by insects or other animals (Koche et al., 2016). The amount of phytochemical differs in plants based on its variety and climatic conditions required for growth. It harbors superior health-promoting properties like antimicrobials, antioxidants, anti-inflammatory, and anticancer. It also stimulates the immune system and modulates hormone metabolism and thus can be used to delay or prevent chronic diseases in humans (Koche et al., 2016). However, some phytochemicals can be toxic to humans and might interfere with the nutrient absorption process in humans. In the Ayurvedic literature of India, phytochemicals are used in the preparation of many drugs and formulations. The application of phytochemicals spreads beyond the pharmaceutical sector to cosmetics, health and hygiene, fragrance, and food supplement industries (Njeru et al., 2013). It is found that these phytochemicals only produce negligible side effects as compared to traditional pharmaceutical therapies in disease treatment. Due to such increased benefits of phytochemicals, they are incorporated into fruit- and vegetable-based beverages (Arte´s-Herna´ndez et al., 2021). Prebiotic and probiotic fermented beverages are potential carriers of bioactives. Such beverages with bioactive compounds can be used as an effective supplement for lactose-intolerant patients by using nondairy products instead of dairy products for fermentation. In this chapter, we have discussed phytochemicals and its nutritional benefits when incorporated into fermented food products leading to its global demand.

31.2

Types of phytochemicals

Phytochemicals are categorized according to their function, chemical, and physical structure. On the other hand, phytochemicals can be classified as primary and secondary metabolites based on their role in plant metabolism. Primary metabolites include amino acids, peptides, purines, pyrimidines, and chlorophylls, while secondary metabolites include alkaloids, polyphenols, terpenoids, organosulfur compounds, phytosterols, and carotenoids (Koche et al., 2016).

31.2.1 Alkaloids Nitrogen-containing compounds (amino acids, amines, purines, pyrimidines) are known as alkaloids. Camptothecin, theobromine, caffeine, berberine, vinblastine, and vincristine are the major plant alkaloids. Alkaloids are commonly Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00016-5 © 2023 Elsevier Inc. All rights reserved.

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found in coffee beans, leaves, barks, and roots of plants and are found to have anticancer and antioxidant properties (Njeru et al., 2013; Koche et al., 2016).

31.2.2 Polyphenols Polyphenols are further divided into flavonoids, phenolic acids, tannins, lignins, and stilbenes. Fruits, vegetables, oilseeds, and legumes are the main sources of polyphenols. Polyphenols have mitigating action against free radicals and its mediated cellular signaling, inflammation, aggregation of platelets, allergies, and hepatotoxins. Further, flavonoids are subclassified as flavanols, flavones, isoflavonoids, flavanones, and anthocyanins (Koche et al., 2016).

31.2.3 Terpenoids These are derivatives of isoprene molecules with a carbon skeleton built from one or more of the C15 units. Terpenes like thymoquinone, acetic acid, D-limonene, and parthenolide have antimicrobial, antiviral, antiallergic, chemotherapeutic, and antihyperglycemic properties. They are found in liverworts, mosses, algae, lichens, and mushrooms (Koche et al., 2016).

31.2.4 Organosulfur compounds The main organosulfur compounds are isothiocyanates and sulforaphane. They are sulfur-containing organic compounds, which serve as an essential element. They are reported to possess antiaging, antioxidant, antibacterial, antiinflammatory, and immunomodulatory properties. They have a characteristic pungent odor and therefore are found in garlic and onions. They are also found in Brussels sprouts, cauliflower, and broccoli (Koche et al., 2016).

31.2.5 Phytosterols Plant steroids known as phytosterols may or may not function in the body as weak hormones. Phytosterols like β-sitosterol, campesterol, and stigmasterol reduce cholesterol levels and prevent coronary heart disease. Phytosterols are present in small quantities in whole grains, vegetables, fruits, unrefined plant oils, nuts, seeds, and legumes. They suppress the growth of diverse tumor cell lines (Ostlund et al., 2002).

31.2.6 Carotenoids The major carotenoids include lycopene, β-carotene, ~ -carotene, lutein, and zeaxanthin. Carotenoids are found mainly in carrots, oranges, green leafy vegetables, and tomatoes. They are reported to be anti-cancerous in nature owing to their excellent antioxidant properties (Ramos et al., 2011).

31.2.7 Other phytochemicals Phytoestrogen, tannins, and saponins are other types of phytochemicals. Phytoestrogen is a nonsteroidal phytochemical also called dietary estrogen. Legumes, cereals, red grapes, berries, red wines, whole grains, and peanuts are the principal sources of phytoestrogen. They offer defense against cancer, bone loss, diabetes, and cardiovascular diseases, diabetes (Koche et al., 2016). Another type is tannins, which are a heterogeneous group of high molecular
weight polyphenolic compounds. They are further classified into gallotannins, condensed tannins, ellagitannins, and complex tannins (Koche et al., 2016). Major sources of tannins are grapes, blueberries, tea, legumes, chocolate, corn, and sorghum. Tannins are known to decrease the frequency of chronic diseases (Njeru et al., 2013). Glycosylated steroids, triterpenoids, and steroid alkaloids are the major saponins. They are antimicrobial, shield the plants from pest infestation, and inhibit the growth of mold. Saponins are commonly found in legumes, sugarbeet, onions, garlic, oats, tea, and yams (Njeru et al., 2013).

31.3

Health benefits of phytochemicals

Phytonutrients are those phytochemicals which are produced by plants via several pathways that contribute to both health improvement and disease prevention (Upadhyay & Dixit, 2015). Some of the health benefits that phytochemicals possess are as follows.

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31.3.1 Oxidative stress amelioration In the body, there is a balance maintained between the reactive oxygen species (ROS) production and antioxidant protection. But at times when this balance gets disrupted, it leads to oxidative stress. Large biomolecules like proteins, DNA, and lipids can become oxidatively damaged owing to the excess production of ROS, thus increasing the risk of cancer and cardiovascular disease (Kanchanlata et al., 2018). They delay or neutralize the free radicals that cause damage to vital organs by interacting with these free radicals, leading to chain termination thus having antioxidant properties (Kanchanlata et al., 2012). The higher the phytochemical content, the greater its radical scavenger efficiency. The scavenging activity decides whether the antioxidants belong to the primary (antioxidants donate hydrogen ion) or secondary (chelate prooxidants convert hydroperoxides to nonradical form) group. Synthetic antioxidants like butyl hydroxyanisole (BHA), propyl gallate (PG), butylhydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) have proven to be toxic, demanding natural alternatives. Phytochemicals like tannins, carotenoids, polyphenols, ascorbic acid, green tea, essential oils, and black tea can thus provide a safer alternative solution (Adebo & Gabriela Medina-Meza, 2020). The antioxidant activity is responsible for the risk of human diseases. The greater the antioxidant activity, the lower the risk of diseases. This antioxidant content can be increased by alteration in the food handling process, like fermenting as a microbial culture has the ability to increase the phytochemical content by breaking down the products, increasing secondary metabolite production.

31.3.2 Reducing inflammation One of the significant factors, that is chronic inflammation, may contribute to the pathogenesis of diseases like type 2 diabetes and cancer. Resveratrol, anthocyanins, and curcumin are phytochemicals that harbor the potential to reduce inflammation by decreasing nuclear factor-B activity and prostaglandin production, inhibiting enzymes, and increasing cytokine production (Costa et al., 2013).

31.3.3 Cardiovascular protection In developed nations, one of the leading causes of death is cardiovascular disease (CVD). The pathogenesis and severity of CVD are correlated with thrombosis and coronary artery blockage caused by platelet aggregation and adhesion under pathophysiological conditions. Flavonoids, polyphenols, dehydroglyasperin C, and other phytochemicals that behave as antioxidants have been reported to offer cardiovascular protection by altering molecular processes that prevent platelet aggregation (Costa et al., 2013).

31.3.4 Anti-obesity activity Citrus fruits have higher concentrations of antioxidants, and it has a potential inhibitory effect on glucosidase and pancreatic lipase due to the presence of phytochemical flavanones. In vitro resveratrol has been reported in previous literature to inhibit adipogenesis and exert its anti-obesity effect (Taing et al., 2012). In high fat-induced obese mice model, caffeine and chlorogenic acid both have been reported to significantly act as reducing body weight, visceral fat mass, plasma leptin levels, and insulin levels; however, chlorogenic acid was more effective than caffeine (Cho & Irudayaraj, 2013).

31.3.5 Anti-diabetes activity Diabetes is a multifactorial disorder that is linked to hyperglycemia, hyperlipidemia, and oxidative stress. Resveratrol comes under the category of phytoalexin which is naturally produced by certain kinds of spermatophytes in response to injury. It is known to show antihyperglycemic effects associated with its stimulatory action on intracellular glucose transport (Barbosa et al., 2013). In vitro analysis on mice has found that it may affect the expression of genes for the onset of diabetes and increase the expression of insulin in pancreatic cells (Xie et al., 2013).

31.3.6 Anticancer activity Phytochemicals such as polyphenols that are antioxidant in nature can inhibit cancer cell proliferation and subsequent death. Anticancer activity can be exerted by downregulating the expression of cancer-related genes which can be achieved by phytochemicals such as cyanidin, kaempferol, and genistein (Kumar et al., 2012).

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TABLE 31.1 Phytochemical classes and subclasses with their activity against different microorganisms. Phytochemicals group

Subclass

Examples

Activity

Against microorganisms

Polyphenols

Flavanoids Non-flavanoids

Galangin, Myricetin Tannins, coumeric acid Phenolic acids

Antibacterial Antimicrobial Antibacterial


Escherichia coli, Streptococcus mutans Staphylococcus aureus, E. coli

Carotenoids

Carotene Xanthophyll

Lycopene, α-carotene Lutine, zeaxanthin

Antimicrobial

E. coli, Salmonella paratyphi, Enterobacter spp.

Alkaloids




Sanguinarine, Quinine Tomatidine, Compound 8

Antimicrobial Antibacterial

E. coli, Bacillus subtilis


Allicin, isothiocyanates

Antimicrobial

Gram-positive and gram-negative bacteria, molds

P-cymene, Pinene Thymol, menthol

Antimicrobial (low) Antimicrobial (high)

Range of bacteria and fungi

Sulfur-containing groups Terpenes and terpenoids




31.3.7 Antimicrobial activity Plants have been used in conventional medicine for years against diverse pathogenic invaders due to the therapeutic value of the molecules in them. The secondary metabolites help to defend the plant by evolving in vivo from microorganisms and their toxins. Due to the existing structural diversity among the different groups of phytochemicals, the mode of action differs and, at times, may act through multiple modes of action. The complete mechanisms are not well understood. The cell death responsible mechanism depends upon degrading the cell wall, disrupting the cytoplasmic membrane, biofilm formation, coagulating the cytoplasm, toxins, depleting protons, leaking the intracellular contents, and virulence factor suppression (Ada´mez et al., 2012; Prakash et al., 2020). The different phytochemical groups exhibiting antimicrobial and/or antibacterial activity against distinct species are summarized in Table 31.1. Polyphenols play a major role in destabilizing the membrane of bacteria, leading to an increase in the permeability of the membrane. This group inhibits nucleic acid synthesis and enzymes, interferes with cell metabolism, and further inactivates microbial adhesins. Alkaloids cause an increase in DNA intercalation and membrane permeability. Sulfur-containing groups bind to sulfhydryl groups of external proteins of cell membranes. Terpenes and terpenoids are responsible for increasing fluidity and permeability, causing disturbance to embedded membrane proteins. They inhibit respiration in gram-negative and gram-positive bacteria (Borges et al., 2015; Prakash et al., 2020).

31.4

Fortification in the fermentation industry

Fermentation is a process in which the biological activity of the organism is utilized during their growth, development, reproduction, and even in death. Under controlled microbial growth, the conversion of food components through enzymatic action forms the fermented food products. Some examples are bread, wine, yogurt, kefir, kombucha, sauerkraut, tempeh, etc. For thousands of years, people have used fermentation as a technique to improve the flavor and aroma of final food items as well as the shelf life of perishable foods (Buckenhu¨skes, 1993). For the purpose of preventing the deficiency of one or more nutrients in the food sample, fortification is carried out wherein the addition of one or more essential nutrients takes place (Codex Alimentarius Commission Report, 1991). Nutritionists have explained that fortifying food products using natural resources fruits, cereals, etc., increases the intake of nutrients (Nestle 2013). Fortification is now done to grain products, snack food, artificial sweeteners, bottled water, and even fermented foods. Fortification of fermented food products provides a beneficial effect of probiotic microorganisms with added important amounts of dietary nutrients. Some of the popular food fortification types are mentioned as follows.

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31.4.1 Vitamin fortification Vitamins are essential micronutrients required in small quantities for the proper functioning of the metabolism in the human body. They play the role of cofactors in the body. Yogurt can be thought of as a vitamin source, which is one of the fermented milk products. The goal of supplements is to boost the nutritional value and taste of food by making up for the vitamins and minerals that were lost during processing and manufacturing (Borenstein, 1971). Vitamin D is essential for healthy skeletal development, fundamental for controlling the body’s serum calcium and phosphorus concentrations. In vitro analysis has shown that vitamin D is associated with antiproliferative, differentiative, and apoptotic effects on prostate cancer. Type I diabetes, hypertension, multiple sclerosis, and some other cancers are caused due to the deficiency of vitamin D (Holick, 2004). Dairy products that have been fortified with vitamins A and C are reported to have better nutritional value, which raises consumer acceptance of those products. When there is too much intake of vitamin A, it leads to hypervitaminosis. When the intake of vitamins is more than 100 μg/day, health complications can occur. Different countries have kept different fortification levels to be added to the product like Some cases where the fortification leads to enhanced nutrient: 1. In the United States, multiple death cases of pellagra were reported due to the deficiency of vitamin B complex. After some years, bread was enriched with yeasts, a rich source of vitamins, and this action led to the reduction of cases of pellagra. 2. Infants are supplemented with vitamin D of about 6 and 15 mg, respectively. To prevent rickets, these amounts were sufficient (FAO/WHO report, 1998).

31.4.2 Iron fortification In India, fermented dairy products are generally deficient in iron, and therefore, iron-fortified fermented products like yogurt enhance the relative iron bioavailability (Vande Woestyne et al., 1991). The kind of mineral and the quantity of additive added to the product affect the properties of fortified dairy products. Generally, ferrous bisglycinate, ferrous sulfate microencapsulated, or ferrous lactate is used for fortification. Fortification of yogurt can be a significant and successful technique for managing iron deficiency causing diseases like anemia; although there are still issues with adding iron to yogurt as in some cases where iron absorption does not take place properly it might lead to poor cognitive function in children, iodine utilization getting affected, outcomes of pregnancy will be poor, etc., (Zimmermann & Hurrell, 2007). Some cases where the fortification leads to enhanced nutrient (Miglioranza et al., 2012): 1. In Vietnam, fortification of iron in biscuits leads to reduction of anemia risk and enhanced deworming efficacy in schoolchildren. 2. In Kuwait, fortification in wheat-based biscuits leads to women aged between 18 and 35 years and increased 88% body Fe stores

31.4.3 Calcium fortification Calcium is required for many biological processes, including blood clotting, nerve transmission, muscle contraction, cell adhesion, and skeletal support. Deficiency of calcium leads to diseases such as osteoporosis, affecting both elderly women and men. The use of micronized tricalcium citrate is the top-end choice in yogurt and other dairy products, giving rise to technological property and nutritional value (Deeth & Tamime, 1981). A concentration of more than 1 g/L of tricalcium citrate can be used.

31.4.4 Fortification with phenolics A vast majority of secondary metabolites are produced by plants. There is a decreased risk of cardiovascular diseases and cancer development due to the usage of phenolics which have gained a major prominence due to their ability to reduce the oxidative stress, reducing inflammation and anticlotting property (Fresco et al., 2010). In the food industry and dairy industry, fruit extracts, powders, and juices have the potential to be employed as functional additives

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(Coı¨sson et al., 2005). The amount of phenolics in dairy products is severely limited as a result of high amounts of phenolics in cattle feed. As a result, plant-based additions had been used to raise yogurt’s phenol level (O’Connell & Fox, 2001).

31.5

Effect of fermentation on phytochemicals

The fermentation process helps to disrupt the nutrient and antinutritional factors, allowing the free release of nutrients and phytochemicals, which is then accessible by the digestive system. Phenylpropanoid biosynthesis and shikimate pathways in the plant growth phase produce phytochemicals (Xu et al., 2021; Zhang et al., 2015). Cinnamic acid produced as a product of the catalyzation of L-phenylalanine in presence of phenylalanine ammonia-lyase can synthesize many phenolic components, which can be converted into tannins, flavonoids, and other compounds. Although the fermentation effect on phytochemicals is not specific, the effects were studied in soybeans (Hubert et al., 2008) and other cereals or pseudocereals (Ðorðevi´c et al., 2010; Oghbaei & Prakash, 2016; Wang et al., 2015). Few phytochemicals can interact with either carbohydrates, proteins, or minerals, which makes them inaccessible (El Hag et al., 2002; Taylor & Duodu, 2015), and hence, there is a significant focus on freeing the phytochemicals (Hubert et al., 2008). During fermentation, the strain of microorganisms acts upon the cereal grain matrices by breaking them and releasing phytochemicals that can be used by the microorganisms, reducing their concentration (El Hag et al., 2002; Hubert et al., 2008). A significant increase in total phenolic, as well as flavonoids, was observed while studying the antioxidant profiles of four kinds of cereal during fermentation using Bacillus subtilis and Lactiplantibacillus plantarum (Wang et al., 2015). Lactobacillus rhamnosus was found to be more efficient than Saccharomyces cerevisiae in releasing total phenolics during the fermentation of cereals (Ðorðevi´c et al., 2010). A significant loss of carotenoids was observed when highcarotenoid biofortified maize was fermented, depending upon the duration of the process (Li et al., 2018; Ortiz et al., 2017). Using probiotics (living organisms beneficial for health) in fermentation or beverage fortification may lead to enhancing their phytochemical content, specifically phenolic compounds. The breakdown of macromolecular polyphenol or anthocyanin structures into smaller phenols is the underlying hypothesis for excess phenolic production. Fermentation of blueberry juice with L. plantarum for 24 h at 37 C increased phenolic and anthocyanin levels by 43% while further continuing it for 2 h at 4 C led to an increase of 15%. Other Lactobacillus species also stimulated an increase of phenolic (49% TPC increase after fermentation with Lactobacillus paracasei) (Bontsidis et al., 2021), TAC (74% increase after fermentation with Lactobacillus acidophilus) (Hashemi et al., 2021) as well as other compounds such as riboflavin, β-carotene, and sulforaphane (broccoli juice fermented with Pediococcus pentosaceus) (Xu et al., 2021) (Table 31.2).

TABLE 31.2 Effect of fermentation on phytochemical content. Fermented food

Impact of fermentation on phytochemical content

References

Lotus seed, adlay, chestnut, walnut

Increased flavonoid and phenolic extract

Wang et al. (2015)

High-carotenoid biofortified maize

Modest carotenoid losses after 24 and 72 h but bigger losses after 120 h. Reduced bioavailability

Ortiz et al. (2017)

Cocoa (6 days)

Reduced antioxidant capacity and polyphenols contents

Albertini et al. (2015)

Sorghum (24 h)

Reduced phytic acid, trypsin inhibitors, and tannins

Osman (2004); Taylor & Duodu (2015)

Soybeans (48 h)

Decrease in phytosterols, glycosylated saponins, and tocopherols

Hubert et al. (2008)

Soy milk

Increased antioxidative activity

Wang et al. (2006)

Milk and milk products

Presence of galacto-oligosaccharide, casein phosphopeptides, and conjugated linoliec acid

Korhonen (2009)

Merlot grape pomace

Increased phenolic compounds

Correˆa et al. (2017)

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31.6

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Use of phytochemicals as a safe fortifying agent

Phytochemicals are majorly understood to have advantages due to their distinct characteristics, delivering numerous health benefits. The bioactive compound or phytochemical content in raw plants is considerably high and gets reduced while handling and processing food. Phytochemicals, in their natural state, are bound and less bioavailable than existing in free form for use which can be resolved by fermenting and making them more readily bioavailable than the naturally existing quantities (Jimenez-Garcia et al., 2018).

31.6.1 Cantaloupe (C. melon) incorporated into yogurt Functional foods such as yogurt have numerous advantages, but enriching them with the fruit cantaloupe affects its overall composition, thus making cantaloupe yogurt more beneficial than natural yogurt. The nutritional value of cantaloupe is due to the presence of polyphenols, vitamin C, potassium, fibers, and carotenoids. Using the pulp of this fruit to enhance yogurt is the conventional method as followed in various other fruit additions to yogurt, but since cantaloupe is a seasonal fruit, dried fruit can be used to increase shelf life. Natural yogurt (NY), cantaloupe puree yogurt (CPY), dry cantaloupe yogurt (CDY), and yogurt with dry cantaloupe and its puree (CPDY) were prepared using standard procedures to compare the results among the four. Extraction procedures were performed to determine the quantity of phytochemicals such as phenolics, polyphenols, and carotenoids enhanced in the different samples containing cantaloupe extracts than the normal yogurt. An increase in the total phenolic content of enriched yogurts was observed as compared to NY. Cantaloupe puree consists of 18.44 mg GAE/100 mg, while dry cantaloupe consists of 264.72 mg GAE/100 mg of total phenolic content. Natural yogurt consists 5.68 mg GAE/100 mg while CPY, CDY, and CPDY consisted of 7.48 mg GAE/100 mg, 9.94 mg GAE/100 mg, and 9.53 mg GAE/100 mg of total phenolic content, respectively. Carotenoids, natural pigments, also showed a significant difference in values, adding to the health benefits impacted by beta-carotene (BC). Cantaloupe puree consists of 1460.7 micrograms BC/100 g, while dry cantaloupe consists of 2214.9 μg BC/100 g of carotenoid content. Natural yogurt consists of 5.68 micrograms BC/100 g, while CPY, CDY, and CPDY consisted of 285.7 μg BC/100 g, 500.6 μg BC/100 g, and 397.7 μg BC/100 g of carotenoids, respectively. Yogurt enhanced with cantaloupe fruit increased the antioxidant compounds (phytochemicals) and also increased its nutritional value, ashes, impacted pH, and fat contents (Kermiche et al., 2018; Akın, 2006).

31.6.2 Soy isoflavones used in the fermentation of probiotics and beverages Soybeans are a rich source of phytochemicals, especially isoflavones. Isoflavones exhibit an estrogen-like structure and have antioxidant effects, antiangiogenic effects, etc. Naturally present as glycosides in soybeans, isoflavones are not readily bioavailable in huge quantities, which limits the efficiency of absorption of these components in the intestinal tract. Fermenting foods with isoflavones contributes to reducing the unpleasant soy milk odor while improving the absorption efficiency. Fermenting soy milk using bifidobacteria and lactic acid bacteria reduces components responsible for unpleasant odor like n-pentanal and n-hexanal. It also reduces the astringent taste by reducing group A saponin glycosides. Normally on ingestion, the serum isoflavone maximum concentration of soy milk is 0.95 μmol/L, while that of fermented soy milk is 2.04 μmol/L. Hence, the overall fermented soy milk functionality is improved (Takagi et al., 2015).

31.6.3 Whole-bread preparation using cupuassu (Theobroma grandiflorum) peel Bread, when enhanced with cupuassu peel flour, shows high phytochemical values of compounds like tannins, phenolic contents, and phytic acid with high dietary fiber. The agro-industrial by-products of cupuassu, like pulps and peels, are a source of bioactive compounds which, on fermentation, are advantageous. Cupuassu peel was separated from the pulp and seeds in the fruits. Bread, an important energy source, was made by replacing wheat with cupuassu peel flour in quantities of 0% (control), 3%, 6%, and 9%. The total phenolic compounds (Folin
Ciocalteu colorimetric method), tannins (Price et al. protocol), and phytic acid (extraction and colorimetry) were determined using standard procedures. The phenolic compound in vitro digestibility at 0%, 3%, 6%, and 9% was found to be 3.32, 3.57, 3.83, and 3.98 mg/g. Similarly, digestibility of tannins at 0%, 3%, 6%, and 9% was 0.84, 0.91, 0.94, 1.08 mg/g, respectively. Phytic acid showed digestibility at 0%, 3%, 6%, and 9% of different values—1.19, 1.98, 2.10, 2.62 mg/g, respectively. Cupuassu peel flour enhances the phytochemical contents in the bread, making it a safe fortifying agent in fermentation that yields bread of high nutritional quality (Salgado et al., 2011).

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31.7

Recent Frontiers of Phytochemicals

Limitations

Plant-based foods have been used for ages for consumption and energy. While phytochemicals obtained from plants exert health benefits, their mechanisms are poorly understood. Phytochemicals are a renewable source, although the demand for raw materials will rise exponentially. This, in turn, will give rise to the requirement for land space, energy, labor, and time. Considering the optimal time period for the growth of this raw material would be a crucial parameter (Tiwari et al., 2015). The availability of specific raw materials also depends on the season. With an increase in the requirement for raw materials, other sources would be used extensively. It would no longer be cost-effective. Furthermore, the literature available is limited and subject to the nutritional values of phytochemicals only. Efficient and extensive research needs to be carried out using different model systems. Bioavailability study measures the ingestion of materials that are digested and reach the circulatory tract to their designated tissues for the biological activities that need to be carried out. Since the bioavailability of the phytochemicals required for health benefits is not understood, it cannot be measured (Epriliati & Ginjom, 2012). The transport activity and metabolism of phytochemicals and biomarkers exhibiting advantages remain unknown. Forming a regulatory authority to keep the contents in check according to dosages should also be maintained. Also, high levels of phytochemical dosages result in antinutritional instincts that cause damage to the cellular organ. Improper or uncooked food, resulting in the consumption of excess lectins, might lead to nutritional deficiencies and immune reactions. Thus, a proper and strictly recommended dietary allowance of phytochemicals should be framed and taken into consideration during fortification so as to avoid the excess addition of these bioactive constituents (Rao, 2003).

31.8

Conclusion

Fortification is indeed a very significant step in the food industry as it enhances the nutritional value of food products and reduces micronutrient deficiencies. There are numerous health benefits that phytochemicals possess such as they act as antioxidant, antimicrobial, reducing inflammation, etc. The use of phytochemicals as a fortifying agent offers a wide range of health benefits in disease prevention and treatment. Fermented foods like soy milk which contains soybean serve as a phytochemical source and enhance the overall nutritional value. On the other hand, functional foods like yogurt can be enriched with a natural phytochemical-rich source that not only enhances the nutritional value of traditional yogurt but also increases the antioxidant property, pH, and other parameters. Therefore, phytochemicals can be used as a safe fortifying agent in the fermentation industry. However, further research is being conducted to study its impact on the human body in disease treatment and micronutrient deficiency reduction.

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520. Available from https://doi.org/10.1016/S01406736(07)61235-5.

Chapter 32

Molecular docking study of bioactive phytochemicals against infectious diseases Sanjeev Kumar Sahu1, Thatikayala Mahender1,2, Iqubal Singh1, Pankaj Wadhwa1, Paranjeet Kaur3 and Kuldeep Bansal4 1

School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India, 2Avanthi Institute of Pharmaceutical Sciences,

Hayathnagar, Hyderabad, India, 3Chitkara of College of Pharmacy, Chitkara University, Punjab, India, 4Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Abo Akademi University, Turku, Finland

32.1

Introduction

Many of the natural products used by humans as fragrances, flavors, nutraceuticals, and especially pharmaceuticals have an important role in pharmaceuticals, for example penicillin; the penicillin is the first known antibiotic isolated from the fungus Penicillium notatum (Brown et al., 2014; Clement, 2014; Cragg & Newman, 2014; Frampton, 2013; Saito, 2014). The natural products are the secondary metabolites of plants with different functional groups like flavonoids, alkaloids, tannins, terpenes, and phenolic compounds with many benefits to health (Chapter-9 Computer-Aided Drug Design Studies in Food Chemistry, 2018; Sun et al., 2009; Wang et al., 2011). The natural products have many activities like cardioprotective effects (Chan et al., 2013), anti-inflammatory activity (Karioti et al., 2015), cosmetic activity (Schwab et al., 2015), anticancer (Lu et al., 2003) and are used to treat neglected diseases like Alzheimer’s (Kennedy et al., 2009; Mills et al., 2005; Mishra et al., 2008). The chemical modification of natural products produces new molecules which are effective in drug discovery and curing the diseases by minimizing the side effects (Scotti et al., 2012). The infectious diseases are caused by microorganisms, and infectious diseases are currently lead killers of people including children and young adults worldwide. In fact, infectious diseases killed many people, from 1918 to 1919: the Spanish flu killed around 20 to 100 million people, tuberculosis killed 1 billion people, smallpox killed 300 to 500 million people in the 20th century, AIDS killed approximately 2.1 million people, TB killed 1.7 million people, malaria killed approximately 1 million people every year due to lack of proper treatment, and the COVID-19 killed millions of peoples in the last 3 years due to lack of proper treatment (Selgelid, 2012).

32.1.1 Molecular docking Molecular docking is a computer-assisted technique, one of the most widely used molecular modeling methods. It allows for determining the orientation and conformation of the ligand with the active site of the target of receptor or enzymes and allows to verify the affinity between drug
receptor and drug
enzyme, as well as to observe the residues participating in the interaction. Currently, the molecular docking approach has been used in modern drug design to understand drug
receptor and drug
enzyme interactions. According to the literature, computational techniques can support and help in making drug design as well as knowing the mechanism (Azevedo et al., 2012; Chen et al., 2002; Dave & Panchal, 2012; Devi et al., 2015; Heberle & Azevedo, 2011; Kothandan & Ganapathy, 2014; Molegro, 2008, 2010; Morris & Lim-Wilby, 2008; Naeem et al., 2013; Scotti et al., 2012; Shekhar, 2008; Talele et al., 2010; Vipin et al., 2015). Vincristine and vinblastine obtained from Catharanthus roseus are well-known anticancer agents (Moudi et al., 2013). The gymnemic acid obtained from Gymnema sylvestre is used in the treatment of diabetes and obesity Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00011-6 © 2023 Elsevier Inc. All rights reserved.

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(Pothuraju et al., 2014). The curcumin obtained from the Curcuma longa have many medicinal properties like antiviral, antiseptic, and antimicrobial properties (Fan et al., 2013).

32.2

Molecular docking studies of plant products as anti-coronal agents

A novel coronavirus is identified as the causative agent of severe acute respiratory syndrome (SARS). The viral coronavirus main proteinase (3CLpro) controls the activities of the coronavirus replication (Anand et al., 2003), while infecting host cells, coronaviruses assemble a multi-subunit of RNA synthesis complex of viral nonstructural proteins (NSPs), where RNA-dependent RNA polymerases are responsible for the transcription and replication of the viral genome (Kirchdoerfer & Ward, 2019). The spike protein of SARS coronavirus attaches the virus to its cellular receptor, angiotensin-converting enzyme-2 (ACE2) through S mediates. This interaction is responsible for the residue changes that facilitate efficient cross-species infection and human-to-human transmission (Li et al., 2005). Qazi et al. (2021) reported the molecular docking study of plant constituents and food products like marmin, malic acid, benzylamine, khusinol oxide against the target of coronavirus main proteinase (3CLpro, PDB ID: 1UJ1), RNAdependent RNA polymerase (PDB ID: 6NUR). The marmin (Fig. 32.1, Table 32.1) formed hydrogen bonds with residues of Met17, Asp95, hydrophobic bonds with Trp31, Ala70 of coronavirus main proteinase (3CLpro), and hydrogen bond with residue Asp623, hydrophobic bonds with residue Thr455, Lys621, Asp623 of RNA-dependent RNA polymerase (RdRp). The malic acid (Fig. 32.2, Table 32.1) formed hydrogen bonds with residues Glu14, Met17, Gly71 with coronavirus main proteinase (3CLpro) and Ser15, Gln18, Gln19, Gly413 of RNA-dependent RNA polymerase (RdRp). The benzylamine (Fig. 32.3, Table 32.1) formed hydrogen bonds with residue Met17 of coronavirus main proteinase (3CLpro), hydrophobic bonds with Ala70, Lys97 of RNA-dependent RNA polymerase (RdRp). The khusinol oxide (Fig. 32.4, Table 32.1) formed the hydrogen bonds with residues Arg188, Thr190, Gln192 of coronavirus main proteinase (3CLpro) and Ser15, Gly413 of RNA-dependent RNA polymerase (RdRp) (Qazi et al., 2021). Similarily, the interaction with RNA-dependent RNA polymerase (PDB ID: 6NUR) for these plant constituents and food products reported in Figs. 33.4
33.8. Lestari et al. (2020) reported the molecular docking study of quinine against the target human angiotensinconverting enzyme-2 receptor (PDB ID: 6VW1). The quinine (Fig. 32.9, Table 32.1) interacted with residues of His34, Glu37, and Lys353 of angiotensin-converting enzyme-2 receptor (PDB ID: 6VW1) (Lestari et al., 2020). Shawky et al. (2020) reported the molecular docking study of rutin, racemosin B, and verbascoside against the target of 3-chymotrypsin-like protease (3CLpro PDB ID: 5R7Y). The rutin (Fig. 32.10, Table 32.2) formed hydrogen bond with Leu14, Glu166 and hydrophobic interaction with the residues Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163 of coronavirus main proteinase (3CLpro), the racemosin B (Fig. 32.11, Table 32.2) formed hydrogen bond with residues Leu141 and Glu166 and hydrophobic bonds with the residues Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163 of 3-chymotrypsin-like protease (3CLpro), and the verbascoside (Fig. 32.12, Table 32.2) formed the hydrogen bonds with the Leu141 and Glu166 FIGURE 32.1 (A) Complex of coronavirus main proteinase (3CLpro, PDB ID: 1UJ1) with marmin. (B) Interaction between marmin with amino acids of coronavirus main proteinase.

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TABLE 32.1 Binding interaction of plant products with two SARS-COV-2 targets proteins: 3-chymotrypsin main proteinase (3CLpro PDB ID: 1UJ1, 5R7Y), RNA-dependent RNA polymerase (RdRP PDB ID: 6NUR). S. no 1

2

3

4

Name

Binding energy

Interactions

26.4 kcal/mol (3CLpro)

Hydrogen bonds MET 17, ASP 95 and hydrophobic bonds TRP 31, ALA 703CLpro (1UJ1).

25.6 kcal/mol (RdRp)

Hydrogen bond Thr556 and hydrophobic bonds Thr455, Lys621, Asp623.

24.4 kcal/mol (3CLpro)

Glu 14, Met 17, Gly 71 with 3CLpro (1UJ1).

23.9 kcal/mol (RdRp)

Ser 15, Gln 18, Gln 19, Gly 413 with RdRp (6NUR).

23.8 kcal/mol (3CLpro)

Ala 70, Lys 97

23.5 kcal/mol (RdRp)

Ser15, RdRp (6NUR).

21.7 kcal/mol (3CLpro)

Arg188, Thr190, Gln192, 3CLpro (1UJ1).

21.6 kcal/mol (RdRp)

Ser15, Gly413 with RdRp (6NUR).

FIGURE 32.2 (A) Complex of coronavirus main proteinase (3CLpro, PDB ID: 1UJ1) with malic acid. (B) Interaction between malic acid with amino acids of coronavirus main proteinase.

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FIGURE 32.3 (A) Complex of coronavirus main proteinase (3CLpro, PDB ID: 1UJ1) with benzylamine. (B) Interaction between benzylamine with amino acids of coronavirus main proteinase.

FIGURE 32.4 (A) Complex of coronavirus main proteinase (3CLpro, PDB ID: 1UJ1) with khusinol oxide. (B). Interaction between khusinol oxide with amino acids of coronavirus main proteinase.

FIGURE 32.5 (A) Complex of RNA-dependent RNA polymerase (PDB ID: 6NUR) with marmin. (B) Interaction between marmin with amino acids of RNA-dependent RNA polymerase.

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FIGURE 32.6 (A) Complex of RNA-dependent RNA polymerase (PDB ID: 6NUR) with malic acid. (B) Interaction between malic acid with amino acids of RNAdependent RNA polymerase.

FIGURE 32.7 (A) Complex of RNA-dependent RNA polymerase (PDB ID: 6NUR) with benzylamine. (B) Interaction between benzylamine with amino acids of RNA-dependent RNA polymerase.

FIGURE 32.8 (A) Complex of RNA-dependent RNA polymerase (PDB ID: 6NUR) with khusinol oxide. (B) Interaction between khusinol oxide with amino acids of RNA-dependent RNA polymerase.

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FIGURE 32.9 (A) Complex of angiotensin-converting enzyme-2 receptor (PDB ID: 6VW1) with quinine. (B) Interaction between quinine and 3-chemotrypsin-like main proteinase.

FIGURE 32.10 (A) Complex of coronavirus main proteinase (3CLpro PDB ID: 5R7Y) with rutin. (B) Interaction between rutin with amino acids of coronavirus main proteinase.

and hydrophobic bonds with the residues Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163 of 3-chymotrypsin-like protease (3CLpro) (Shawky et al., 2020).

32.3

Molecular docking studies of plant products as anti-leishmanial agents

Leishmanolysin is a major surface antigen of the promastigote insect form of the parasitic protozoan Leishmania, and it plays a very important role in preventing complement-mediated lysis of Leishmania after infection of the mammalian host by the sand fly vector (Metcalf & Etges, 2006). The reactive oxygen species generated inside the macrophages kills

TABLE 32.2 Binding interaction of plant products with human angiotensin-converting enzyme-2 receptor (PDB ID: 6VW1). S. no

Binding energy

Interactions

1

Name

24.89 kcal/mol

His34, Glu37, and Lys353

2

212.632 kcal/mol

Hydrogen bond: Leu141 and Glu166 and hydrophobic bonds: Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163.

3

211.844 kcal/mol

Hydrogen bond: Leu141 and Glu166 and hydrophobic bonds: Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163.

4

211.721 kcal/mol

Hydrogen bond: Leu141 and Glu166 and hydrophobic bonds: Met49, Leu141, Cys145, Met165, Leu167, Pro168, Thr25, Thr26, His41, Asn142, Ser144, His163.

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FIGURE 32.11 (A) Complex of coronavirus main proteinase (3CLpro PDB ID: 5R7Y) with racemosin B. (B) Interaction between racemosin B with amino acids of coronavirus main proteinase.

FIGURE 32.12 (A) Complex of coronavirus main proteinase (3CLpro PDB ID: 5R7Y) with verbascoside. (B) Interaction between racemosin B with amino acids of coronavirus main proteinase.

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553

the parasites, and the trypanothione reductase enzyme neutralizes the reactive oxygen species by generation hydrogens (Ukhyil, 2019). Leishmanolysin is the important surface protein antigen of promastigote, and it is a membrane-bound zinc proteinase having an important role during the infection (Bouvier, 2004). Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Shah, et al. (2019), Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Syed Shahd, et al. (2019), summarized the molecular docking study of methyl 3,4-dihyroxybenzoate, octadecyl benzoate against the target Leishmania tropica promastigote surface gp63 enzyme (PDB code: 1LML). The methyl 3, 4dihyroxybenzoate (Fig. 32.13, Table 32.3) formed the hydrogen bond interaction with the residues Glu265, Leu344 of L. tropica promastigote surface gp63 enzyme, and the octadecyl benzoate (Fig. 32.14, Table 32.3) formed the hydrogen

FIGURE 32.13 (A) Complex of Leishmania tropica promastigote surface gp63 enzyme with methyl 3,4-dihyroxybenzoate. (B) Interaction between methyl 3, 4-dihyroxybenzoate and L. tropica promastigote surface gp63 enzyme.

TABLE 32.3 Binding interaction of plant products with Leishmania tropica promastigote surface gp63 enzyme (PDB code: 1LML), trypanothione reductase (PDB ID: 4APN), leishmanolysin (PDB ID: 1LML), human calcium/calmodulindependent protein kinase type-IV (PDB ID: 2W4O). S. no 1

Name

Binding energy

Interactions

25.3 kcal/mol

Glu265, Leu 344.

(Continued )

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TABLE 32.3 (Continued) S. no

Name

Binding energy

Interactions

2

25.6 kcal/mol

Ser449

3

261.54 kcal/mol (for TR)

Gly16, Ala159 (for TR).

233.24 kcal/mol (for GP63)

Trp226 (for GP63).

4

26.01 kcal/mol

H-bond-Ala63. Hydrophobic interactions Leu54, Thr62, Met78, Glu79, Ala81, Gln85, Thr131, Ala133, Cys139, Asp140.

5

26.41 kcal/mol

H-bonds Glu79. Hydrophobic interactions Gln19, Thr62, Met78, Ala81.

bonds with the residue H-bond of Ser449 of L. tropica promastigote surface gp63 enzyme (Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Shah, et al., 2019). Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Shah, et al. (2019), Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Syed Shahd, et al. (2019), presented the molecular docking study of β-sitosterol (Figs. 32.15 and 32.16, Table 32.3) against the target of trypanothione reductase (TR, PDB ID: 4APN) and leishmanolysin (GP63, 1LML). The β-sitosterol formed hydrogen bonds with Gly16, Ala159 of trypanothione reductase and Trp226, Asp234 of leishmanolysin (Shah, Ullah, Ayaz, Sadiq, Hussain, Ali Shah, Syed Shahd, et al., 2019).

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FIGURE 32.14 (A) Complex of Leishmania tropica promastigote surface gp63 enzyme with Octadecyl benzoate. (B) Interaction between octadecyl benzoate and L. tropica promastigote surface gp63 enzyme.

FIGURE 32.15 (A) Complex of trypanothione reductase with β-sitosterol. (B) Interaction between β-sitosterol and trypanothione reductase.

Grover et al. (2012) concluded the molecular docking study of withaferin A, withanone against the target human calcium/calmodulin-dependent protein kinase type-IV (2W4O). The binding energy of withaferin A (Fig. 32.17, Table 32.3) formed the hydrogen bond with the residue Ala63 and hydrophobic interactions with the residues Leu54, Thr62, Met78, Glu79, Ala81, Gln85, Thr131, Ala133, Cys139, Asp140 of human calcium/calmodulin-dependent protein kinase type-IV, and withanone (Fig. 32.18, Table 32.3) formed the hydrogen bond with Glu79 and hydrophobic interactions with Gln19, Thr62, Met78, Ala81 of human calcium/calmodulin-dependent protein kinase type-IV (Grover et al., 2012).

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FIGURE 32.16 (A) Complex of leishmanolysin (GP63, 1LML) with β-sitosterol. (B) Interaction between β-sitosterol and leishmanolysin.

FIGURE 32.17 (A) Complex of human calcium/calmodulin-dependent protein kinase type-IV (2W4O) with withaferin A. (B) Interaction between withaferin A and human calcium/calmodulin-dependent protein kinase type-IV.

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FIGURE 32.18 (A) Complex of human calcium/calmodulin-dependent protein kinase type-IV (2W4O) with withanone. (B) Interaction between withanone and human calcium/calmodulin-dependent protein kinase type-IV.

32.4

Molecular docking studies of plant products as antitubercular agents

Baptista et al. (2021) performed the molecular docking study of α-cubebin (Fig. 32.19, Table 32.4) against the target polyketide synthase Pks13 (PDB ID: 5V3X). The α-cubeb formed hydrogen bonds with the residues Asp1644 and Gln1633 and hydrophobic interactions with the residues Tyr1637, Ser1636, Phe1670, Ile1643, Tyr1663, Tyr1674, Ala1667, Asn1640 of polyketide synthase (Baptista et al., 2021). Pawar et al. (2020) reported the molecular docking study of flavonoids naringenin and quercetin against the glutamate racemase (PDB ID: 5HJ7). The naringenin (Fig. 32.20, Table 32.4) formed several interactions with amino acid residues Ser13, Gly14, Val15, Asn41, Gly42, Pro43, Tyr44, Gly45, Ile52, Ser77, Ala121, Cys185, His187 Val199 of glutamate racemase, and the quercetin (Fig. 32.21, Table 32.4) made several interactions with amino acid residues of MTB-MurI at Gly14, Val15, Asp38, Glu153, Arg154, Gly155, Ala246, Phe247, and Lys249 of glutamate racemase (Pawar et al., 2020). Qasaymeh et al. (2019) reported the molecular docking study of flavonoids present in the aerial parts of Pelargonium reniforme, Pelargonium sidoides against protein kinase G enzyme of mycobacterium tuberculosis (PDB ID: 2PZI). The binding energy of isoorientin 200 -O-gallate (Fig. 32.22, Table 32.4) made several interactions with amino acid residues Lys241, Ser239, His159, Ser239, Ile292, Val179, Ala158 of protein kinase G enzyme, and the binding energy of isovitexin 2v-O-gallate (Fig. 32.23, Table 32.4) found to be 12.6 kJ/mol made several interactions with amino acid residues Lys241, Met232, Ala158, Ile292, Val235, Val179 of protein kinase G enzyme (Qasaymeh et al., 2019). Shilpi et al. (2015) performed the molecular docking study of pteleoellagic acid, 3,30 -di-O-methylellagic acid 4-Oα-rhamnopyranoside against the Mycobacterium tuberculosis enoyl-ACP reductase (InhA PDB ID:1BVR), MabA (PDB ID:1UZN), pantothenate kinase from Mycobacterium tuberculosis (MtPanK, PDB ID: 3AF3). The pteleoellagic acid (Figs. 32.24
32.26, Table 32.4) made several interactions with amino acid residues Ile95, Gly96, Lys165, Thr196 of MabA, Gly90, Gly139, Lys157, Gly184, Thr188, Ala100, Val101, Gly102, Lys103, Thr105, Arg108 of pantothenate kinase from Mycobacterium tuberculosis, and the 3,30 -di-O-methylellagic acid 4-O-α-rhamnopyranoside (Figs. 32.27
32.29, Table 32.4) made several interactions with amino acid residues Ser94, Tyr158 of InhA Arg25,

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FIGURE 32.19 Complex of polyketide synthase Pks13 (PDB ID: 5V3X) with α-cubebin.

FIGURE 32.20 (A) Complex of glutamate racemase with naringenin. (B) Interaction between naringenin and glutamate racemase.

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FIGURE 32.21 (A) Complex of glutamate racemase with quercetin. (B) Interaction between naringenin and glutamate quercetin.

FIGURE 32.22 (A) Complex of protein kinase G enzyme of Mycobacterium tuberculosis (PDB ID: 2PZI) with isoorientin 200 -O-gallate. (B) Interaction between isoorientin 200 -O-gallate protein kinase G enzyme.

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FIGURE 32.23 (A) Complex of protein kinase G enzyme of Mycobacterium tuberculosis (PDB ID: 2PZI) with isovitexin 2v-O-gallate. (B) Interaction between isovitexin 2v-O-gallate protein kinase G enzyme.

FIGURE 32.24 (A) Complex of Mycobacterium tuberculosis enoyl-ACP reductase with pteleoellagic acid. (B) Interaction between pteleoellagic acid and Mycobacterium tuberculosis enoyl-ACP reductase.

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FIGURE 32.25 (A) Complex of MabA with pteleoellagic acid. (B) Interaction between pteleoellagic acid and MabA.

FIGURE 32.26 (A) Complex of pantothenate kinase from Mycobacterium tuberculosis with pteleoellagic acid. (B) Interaction between pteleoellagic acid and pantothenate kinase.

Ser92, Asn88, Ser140, Tyr153 of MabA and PanK at Lys103, Ser104, Tyr182, Tyr123, Arg238 of pantothenate kinase from Mycobacterium tuberculosis (Shilpi et al., 2015). Radha et al. (2015) concluded the molecular docking study of quercetin, piperine, xanthone with mycobacterium tuberculosis hypoxic response regulator (PDB ID: 3c3w). The binding energy of quercetin (Fig. 32.30, Table 32.4) formed hydrogen bond interaction with the residue 155Arg, the piperine (Fig. 32.31, Table 32.4) formed hydrogen bond interaction with the residue of 156Thr, 159Gly, and the xanthone (Fig. 32.32, Table 32.4) formed hydrogen bond interaction with the residue 156Thr of mycobacterium tuberculosis hypoxic response regulator (Radha et al., 2015).

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FIGURE 32.27 (A) Complex of Mycobacterium tuberculosis enoyl-ACP reductase with 3,30 -di-O-methyl ellagic acid 4-O-α-rhamnopyranoside. (B) Interaction between 3,30 -di-O-methyl ellagic acid 4-O-α-rhamnopyranoside and Mycobacterium tuberculosis enoyl-ACP reductase.

FIGURE 32.28 (A) Complex of MabA with 3,30 -di-O-methyl ellagic acid 4-O-α-rhamnopyranoside. (B) Interaction between 3,30 -di-O-methylellagic acid 4-O-α-rhamnopyranoside and MabA.

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FIGURE 32.29 (A) Complex of pantothenate kinase from Mycobacterium tuberculosis with 3,30 -di-O-methylellagic acid 4-O-α-rhamnopyranoside. (B) Interaction between 3, 30 -di-O-methylellagic acid 4-O-α-rhamnopyranoside and pantothenate kinase.

FIGURE 32.30 Complex of mycobacterium tuberculosis hypoxic response regulator with quercetin.

FIGURE 32.31 Complex of mycobacterium tuberculosis hypoxic response regulator with piperine.

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TABLE 32.4 Binding interaction of plant products with thioesterase domain in complex with inhibitor TAM1 (PDB ID: 5V3X), Glutamate Racemase Mycobacterium tuberculosis (MurI) (PDB ID: 5HJ7), protein kinase G enzyme of Mycobacterium tuberculosis (PDB ID: 2PZI), Mycobacterium tuberculosis enoyl-ACP reductase, MabA from Mycobacterium tuberculosis, pantothenate kinase from Mycobacterium tuberculosis (PDB ID: 1BVR, 1UZN, 3AF3), Mycobacterium tuberculosis hypoxic response regulator (PDB ID: 3c3w). S. no

Binding energy

Interactions

1

Name

211.0 kcal/mol

H-bonds-Asp1644 and Gln1633, Hydrophobic interactions with the residues Tyr1637, Ser1636, Phe1670, Ile1643, Tyr1663, Tyr1674, Ala1667, Asn1640, and Arg1641.

2

27.15 kJ/mol

Ser13, Gly14, Val15, Asn41, Gly42, Pro43, Tyr44, Gly45, Ile52, Ser77, Ala121, Cys185, His187 Val199.

3

27.15 kJ/mol

Gly14, Val15, Asp38, Glu153, Arg154, Gly155, Ala246, Phe247, and Lys249.

4

213.2 kJ/mol

Lys241, Ser239, His159, Ser239, Ile292, Val179, Ala158

(Continued )

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TABLE 32.4 (Continued) S. no

Binding energy

Interactions

5

2 12.6 kJ/mol

Lys241, Met232, Ala158, Ile292, Val235, Val179.

6

28.4 kcal/mol

Ile95, Gly96, Lys165, Thr196.

29.4 kcal/mol

Gly90, Gly139, Lys157, Gly184, Thr188.

29.7 kcal/mol

Ala100, Val101, Gly102, Lys103, Thr105, Arg108.

27.8 kcal/mol,

Ser94, Tyr158.

210.8 kcal/mol

Arg25, Ser92, Asn88, Ser140, Tyr153.

211.3 kcal/mol

Lys103, Ser104, Tyr182, Tyr123, Arg238.

26.4215 kcal/mol

155Arg.

7

8

Name

(Continued )

TABLE 32.4 (Continued) S. no

Name

Binding energy

Interactions

9

27.54 kcalkcal/mol

156Thr, 159Gly

10

27.18 kcal/mol

156Thr.

FIGURE 32.32 Complex of mycobacterium tuberculosis hypoxic response regulator with xanthone.

32.5

Conclusion

The molecular docking studies help to understand the interaction between functional groups of the ligand and amino acids at the targeted site of the protein. The present study concluded that many secondary metabolites of the plants may cure the infectious diseases, and small modification in the structure produces efficient molecules, showing the good interaction with the active site of the target. The present study may be helpful to scientists in the development of the effective drugs to treat infectious diseases.

References Anand, John, K. A., Ziebuhrparvesh, J., Wadhwani., & Hilgenfeld, R. M. (2003). Coronavirus main proteinase (3CLpro) structure: Basis for design of anti-SARS drugs. Science, 300(5626), 1763
1767. Azevedo, L. S., Moraes, F. P., Xavier, M. M., Pantoja, E. O., Villavicencio, B., Finck, J. A., Proenca, A. M., Rocha, K. B., & de Azevedo, W. F. (2012). Recent progress of molecular docking simulations applied to development of drugs. Current Bioinformatics., 7(4), 352
365. Baptista, R., Bhowmick, S., Shen, J., & Mur, L. A. J. (2021). Molecular docking suggests the targets of anti-mycobacterial natural products. Molecules, 26(475), 1
14. Bouvier, J. (2004). Leishmanolysin. Handbook of Proteolytic Enzymes (pp. 764
769). Academic Press. Brown, D. G., Lister, T., & May-Dracka, T. L. (2014). New natural products as new leads for antibacterial drug discovery. Bioorganic & Medicinal Chemistry Letters., 24(2), 413
418. Chan, J. Y. Y., Yuen, A. C. Y., Chan, R. Y. K., & Chan, S. W. (2013). A review of the cardiovascular benefits and antioxidant properties of allicin. Phytotherapy Research, 27(5), 637
646. Chapter-9 Computer-Aided Drug Design Studies in Food Chemistry. (2018). Published in natural and artificial flavoring agents and food dyes a volume in handbook of food bioengineering (pp. 261
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Chen, Y. Z., Li, Z. R., & Ung, C. Y. (2002). Computational method for drug target search and application in drug discovery. Journal of Computational Biophysics and Chemistry, 01, 213
224. Clement, J. A. (2014). Recent progress in medicinal natural products drug discovery. Current Topics in Medicinal Chemistry, 14(24), 2758-2758. Cragg, G., & Newman, D. (2014). Natural products and drug discovery and development: A history of success and continuing promise for the future. Planta Med, 80(10), 750-750. Dave, K., & Panchal, H. (2012). Review on chemogenomics approach: Interpreting antagonist activity of secreted frizzled-related protein 1 in glaucoma disease with in-silico docking. Current Topics in Medicinal Chemistry, 12, 1834
1842. Devi, R. V., Sathya, S. S., & Selvaraj, M. (2015). Evolutionary algorithms for de novo drug design
A survey. Applied Soft Computing Journal, 27, 543
552. Fan, X., Zhang, C., Liu, D. B., Yan, J., & Liang, H. P. (2013). The clinical applications of curcumin: Current state and the future. Current Pharma Design, 19(11), 2011
2031. Frampton, C. S. (2013). An introduction to the special issue on pharmaceuticals, drug discovery and natural products. Structure Communications, 69, 1205
1206. Grover, A., Katiyar, S. P., Jeyakanthan, J., Dubey, V. K., & Sundar, D. (2012). Blocking Protein kinase C signaling pathway: Mechanistic insights into the anti-leishmanial activity of prospective herbal drugs from Withania somnifera. BMC Genomics, 13(7), 1
11. Heberle, G., & Azevedo, W. F. de (2011). Bio-inspired algorithms applied to molecular docking simulations. Current Medicinal Chemistry, 18(9), 1339
1352. Karioti, A., Milosevic-Ifantis, T., Pachopos, N., Niryiannaki, N., Hadjipavlou-Litina, D., & Skaltsa, H. (2015). Antioxidant, anti-inflammatory potential and chemical constituents of Origanum dubium Boiss., growing wild in Cyprus. Journal of Enzyme Inhibition and Medicinal Chemistry, 30 (1), 38
43. Kennedy, D. A., Hart, J., & Seely, D. (2009). Cost effectiveness of natural health products: A systematic review of randomized clinical trials. Evidence-Based Complementary and Alternative Medicine, 6(3), 297
304. Kirchdoerfer, R. N., & Ward, A. B. (2019). Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nature Communications, 10(1), 2342. Kothandan, G., & Ganapathy, J. (2014). A short review on the application of combining molecular docking and molecular dynamics simulations in field of drug discovery. Journal of the Chosun Natural Science, 7, 75
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1151.

Chapter 33

Phytochemicals in structure-based drug discovery Amit Kumar1, Jaya Baranwal2, Amalia Di Petrillo3, Sonia Floris4, Brajesh Barse5 and Antonella Fais4 1

Department of Electrical and Electronic Engineering, University of Cagliari, Cagliari, Italy, 2DBT-ICGEB Centre for Advanced Bioenergy Research,

International Centre for Genetic Engineering & Biotechnology, New Delhi, India, 3Department of Medical Sciences and Public Health, University of Cagliari, Monserrato, Italy, 4Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy, 5Confederation of Indian Industry (CII), New Delhi, India

33.1

Introduction

Phytochemicals are naturally occurring bioactive plant molecules that provide humans with several health benefits (Hasler & Blumberg, 1999). Phytochemicals are chemical compounds found in plants that defend them from bacteria, viruses, and fungi. A diet rich in vividly colored fruits and vegetables, entire cereals, and legumes containing phytochemicals may reduce the risk of acquiring some malignancies, diabetes, hypertension, and cardiovascular disease. Plants are a highly sought-after ingredient in nutraceuticals due to their bioactive compounds and influence on human health. Phytocompounds are known to play an important role in plant adaptation to their environment and are also a rich source of pharmaceuticals. The use of plants and their extracts in the production of herbal medicines created the foundation for contemporary therapeutic sciences (Firenzuoli & Gori, 2007). Phytochemicals are classified according to many chemical structure categories, including alkaloids, curcumin, flavonoids, terpenes, phenolics, plant steroids, lignans, saponins, and glucosides (Fig. 33.1).

FIGURE 33.1 Classes of phytochemicals (Suresh & Abraham, 2020). Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00015-3 © 2023 Elsevier Inc. All rights reserved.

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The vast majority of plant-produced organic molecules usually referred to as secondary metabolites does not appear to be directly involved in plant growth and development (Teoh, 2016). The roles of the majority of secondary metabolites are still unclear. In contrast, main metabolites, such as nucleotides, phytosterols, amino acids, acyl lipids, and organic acids are present in all plants and perform vital and typically obvious metabolic functions.

33.1.1 Phytochemicals—medicinal properties The use of natural products in ancient times and folk medicine all over the world is what led to the use of therapeutic drugs in modern medicine. Pharmaceutical corporations have relied on natural plant products in their hunt for novel medications. In-depth research is being constantly pursued to identify active compounds with curative qualities in polyphenol-rich herbal medicine plant sources. They may serve as antioxidants, nutritional protectors, or cancerpreventing agents. Scientists have found numerous phytochemicals, but only a tiny proportion of it has been thoroughly investigated. β-carotene and other carotenoids, vitamin C, vitamin E, and folic acid, are examples of phytochemicals that are commonly encountered (Glaser et al., 2015). Phytochemicals can be employed as chemotherapeutic or chemopreventive agents to prevent, reverse, or slow tumor growth. Proton-motive force disruption, active transport, cytoplasmic membrane disruption, electron movement, and cell contents coagulation are all thought to be involved in phytochemical activity (Kotzekidou et al., 2008). Phytoconstituents have various mechanisms of action, including antioxidant, anticancer, anti-inflammatory, anti-ulcer, antidiabetic, and multifunctional targets (Table 33.1).

33.1.1.1 Antimicrobial phytochemicals Synthetic antimicrobial chemicals have become widely employed to prevent and cure microbial infections since the discovery of efficient and powerful antimicrobial molecules. A variety of phytochemicals have the potential to become effective antimicrobial agents that might be used to prevent or treat microbial and viral infections. Although there are some promising in vivo effects in inhibiting pathogenic microbes without harming beneficial bacteria in the gastrointestinal tracts, more research on the safety and efficacy of these phytochemicals is needed to determine whether they could offer therapeutic benefits over current therapies (Patra, 2012).

33.1.1.2 Antiviral phytochemicals Natural materials offer a novel approach to the development of antiviral medicines with impressive pharmacological properties. Approximately, 25% of the medications administered today are derived from plants. Plant products are the source of many anticancer and anti-infective medications. Herbalists have been using traditional herbs to treat a variety of human and animal problems since ancient times, particularly in Asia. In many regions of the world, people still rely on traditional plants and their products for their health, lifestyle, and basic health care. Globally, around 2500 species of medicinal plants have been cataloged for the treatment of a wide variety of ailments and diseases (Kapoor et al., 2017). TABLE 33.1 Phytochemicals with medicinal properties and applications. Phytochemicals

Properties

Uses/treatment

References

Tannins

Antibacterial, antioxidant

Diarrhea, dysentery

Chung et al. (1998)

Alkaloids

Anti-inflammatory, antifungal, antimicrobial

Pain relivers, antidote for barbiturate and morphine intoxication

Zhang et al. (2013)

Phytosterols

Anti-inflammatory, antineoplastic, antipyretic, and immune-modulating

Effective at lowering cholesterol levels in the body

Zyriax et al. (2022)

Saponins

Anti-inflammatory

Cholesterol reduction, hypertension

He et al. (2022)

Phenols

Antioxidant

Sore throat, sore mouth

Palmero & Arena (2001)

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TABLE 33.2 Medicinal plants seed extracts as a source of phytochemicals. Botanical name

Family name

Common name

References

Aframomum melegueta

Zingiberaceae

Alligator pepper, grains of paradise

Ilic et al. (2014)

Abelmoschus esculentus

Malvaceae

Okra, lady’s finger

Borokini et al. (2022)

Butyrospermum paradoxum

Sapotaceae

Shea butter tree

Ojo et al. (2021)

Carica papaya

Caricaceae

Pawpaw

Singh et al. (2021)

Cannabis sativa

Cannabaceae

Indian hemp

Odieka et al. (2022)

Linum usitatissimum

Linaceae

Flax or linseed

Chera et al. (2022)

Lepidium sativum

Brassicaceae

Garden cress

Ramadan & Oraby (2020)

33.1.1.3 Anticancer phytochemicals Plants and their derivatives have been used for therapeutic purposes since antiquity. Folk medicine practitioners utilize a variety of herbal mixtures with various philosophies and cultural backgrounds to treat various ailments. Ayurveda is the science of good health and well-being, according to India’s ancient Vedic literature (Behere et al., 2013). It is an amalgamation of traditional and cultural beliefs for the treatment of ailments. In today’s healthcare system, a modern medication research program based on Ayurvedic notions has received widespread support. Natural compounds generated from plants are harmless to normal cells and are also better tolerated; therefore they are gaining interest in current medicinal development.

33.1.1.4 Plants as the dominant source The plant kingdom is a renewable, low cost, and largely unexplored source of biologically active molecules. According to the World Health Organization report (WHO, 2022), around 6.5 billion people worldwide use traditional medicine for their primary health care, and 80% of people in poor countries and 85% of traditional medicine rely on plant extracts (Wilson & Peter, 1988). Natural products from plants provide unlimited opportunities for new drugs because their chemical diversity could give rise to a range of biological activities. Plant-derived drugs account for 25% of drug prescriptions in the United States (Pezzuto, 1997), and from 1983 to 1994, 39% of newly approved drugs were of natural origin, which also include original natural products, semisynthetically derived products from natural origin, and synthetic products based on natural product models. A review of 87 authorized anticancer medicines revealed that 62% of them were of natural origin or derived from natural product modeling. In recent years, researchers have been attempting to better valorize fruits as a natural source of bioactive products not only because of their nutrient intake, but also because their consumption is strongly associated with reduced risk of developing a wide range of chronic diseases. Moreover, the biological properties of extracts obtained from seeds contribute to increase in the use of this part of the fruit that is usually discarded. Some examples of phytochemical analysis performed on seed extracts of medicinal plants are reported in Table 33.2.

33.2

Phytochemicals screening of plant extracts

The activity of the extracts is determined by the extraction process and solvent, as well as their concentration and structure. Phytochemicals extraction using solvents: Different types of solvents, such as methanol, hexane, and ethyl alcohol, have been used for antioxidant extraction from diverse plant components, such as leaves and seeds. Various solvents with varying polarity must be employed to extract different phytochemicals from plants with a high degree of precision (Wong & Kitts, 2006). Extremely polar solvents, such as methanol, has proven to be particularly potent antioxidants. The polarity of the solute of interest determines which solvents are employed to extract biomolecules from plants. To minimize the number of similar compounds in the required yield, several solvents might be utilized successively. Microwave-assisted extraction (MAE): This method is used for extracting bioactive chemicals from a broad variety of plants and natural wastes. Microwaves emit electromagnetic radiation in wavelength range of 1
100 cm and

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frequency ranging between 300 MHz and 300 GHz (Ballard et al., 2010). Recent technological advances have made it possible to limit bioactive chemical loss without extending the extraction time. Therefore, microwave-assisted extraction has been proven to be an effective technology in a variety of domains, particularly in the area of medicinal plants. Moreover, this approach minimizes the losses of the collected biological components (Santelli et al., 2004). Bioactive molecule purification: Purification and isolation of phytochemical compounds from plants are a technology that has recently experienced significant advancement (Altemimi et al., 2015). This current approach allows for the parallel development and availability of various complex bioassays on the one hand, while also providing accurate isolation, separation, and purification procedures on the other hand. Using paper thin-layer and column chromatography technologies, several bioactive compounds have been identified and purified. Column chromatography and thin-layer chromatography (TLC) are still widely employed due to their ease of use, low cost, and availability in a wide range of stationary phases. Silica, alumina, cellulose, and polyamide have the highest value for phytochemical separation. TLC has long been employed in column chromatography to evaluate chemical fractions. Some analytical instruments have employed column chromatography using silica gel and TLC to separate bioactive compounds. UV
Visible spectroscopy: In both pure and biological mixtures, UV
visible spectroscopy is used for qualitative investigation and the identification of certain classes of substances. Since aromatic compounds are potent chromophores in the UV region, UV
visible spectroscopy is used for a quantitative study. UV
visible spectroscopy is used to identify natural substances (Poli et al., 2021). Tannins, anthocyanins, polymer dyes, and phenols are examples of phenolic chemicals that produce iron complexes that may be identified by ultraviolet/visible (UV
vis) spectroscopy. Furthermore, it was discovered that spectroscopic UV
vis methods are less selective and simply provide information on the composition of the overall polyphenol content. Total phenolic extract (280 nm), flavones (320 nm), phenolic acids (360 nm), and total anthocyanins have been determined using UV
vis spectroscopy (520 nm) techniques. When compared to other approaches, this technique is not time-consuming and economical (Urbano et al., 2006). Infrared spectroscopy: When infrared light passes through a sample of an organic substance, some of the frequencies are absorbed; however, certain frequencies are transmitted through the sample without being absorbed. When a molecule is exposed to infrared light, it undergoes vibrational alterations, which are known as infrared absorption. As a result, infrared spectroscopy may be thought of as vibrational spectroscopy. The vibrational frequencies of different bonds (C
C, C 5 C, C
C, C
O, C 5 O, O
H, and N
H) are different. The presence of these types of bonds in an organic molecule may be detected in the infrared spectrum by looking for the distinctive frequency absorption band (Kemp, 1991). Fourier transform infrared spectroscopy (FTIS) is a high-resolution analytical method generally used to detect chemical ingredients and deduce structural compounds. To fingerprint natural compounds or powders, FTIR provides a quick and nondestructive method. Nuclear magnetic resonance spectroscopy (NMR): The magnetic characteristics of particular atomic nuclei, such as the nucleus of the hydrogen atom, the proton, carbon, and a carbon isotope, are the focus of NMR. Many researchers have been able to examine molecules using NMR spectroscopy, which records the differences between the various magnetic nuclei and so provides a clear image of where these nuclei are in the molecule. Furthermore, it shows which atoms are present in adjacent groups, and is able to determine how many atoms are present in each of these habitats. Several attempts have been undertaken in the past to isolate individual phenols using preparative or liquid chromatography, semi-preparative thin-layer chromatography, and column chromatography, with the structures, confirmed later by NMR offline (Bringmann et al., 1999). Identification using mass spectrometry: In mass spectrometry, organic molecules are bombarded with electrons or lasers and transformed into charged ions, which are very energetic. The relative abundance of a fragmented ion is shown against the mass/charge ratio of these ions in a mass spectrum. The relative molar mass of a molecule may be calculated with great accuracy using mass spectrometry, and an accurate molecular formula can be established using the information of where the molecule has been broken. When a pure standard is unavailable, the combination of HPLC and Ms allows for quick and precise identification of chemical components in medicinal plants (Tsugawa et al., 2021). LC-MS and GC-MS have recently become popular for the study of phytochemicals. Because of its high ionization efficiency, electrospray ionization is a recommended source. Streamlining the drug discovery process: Computer-aided drug design tools are beneficial to screen phytochemicals (and their derivatives) for novel scaffolds capable of efficiently binding to the active site of the crucial receptors. The process of drug development begins with exploration of natural resources for the identification of new leads (Fig. 33.2). The knowledge of plants to treat specific disorders can be then utilized to identify the target protein and the phytocompounds to be screened. The proteins that are known to play a key role in the progression of the disease are identified while knowledge of network pharmacology provides a list of probable multi-target inhibitors and their targets. Molecular docking is a well-established tool in computer-aided drug discovery for rapid screening and prediction of the

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FIGURE 33.2 Flow chart for phytochemicals as lead compound for structure-based drug discovery.

binding characteristics of molecules to a target protein from their three-dimensional structures. Ligand binding is the key step in enzymatic reactions and, thus, for their inhibition. Therefore, a detailed understanding of interactions between small molecules and proteins can form the basis for a rational drug design strategy. The top-ranked hits are then further screened for chemical absorption, distribution, metabolism, excretion, and toxicity properties, which ensures there is a higher probability for the bioactives to pass through the drug discovery pipeline. Molecular docking procedure follows the induced-fit model, and hence, there are high chances of missing out on novel scaffolds. In this scenario, molecular dynamics (MD) simulation can be performed to include conformational changes of the target protein that could have an impact on the accessibility of the binding pocket to the ligand. MD simulation is a technique founded upon the basic principles of classical mechanics that provide a dynamic picture of the individual particles of the system at a microscopic level. The conformation of the protein complexes in the local minima adjacent to the global minima is extracted from the MD simulations and the bioactive re-docked for ensemble docking. This step ensures that the limitations of rigid docking are addressed. The complexes of the top hits can then be simulated (based on their coefficient of similarity) for their binding free energy. Relative alchemical binding free

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energy estimation relies on the transformation of the ligand into its closest neighbor (based on similarity) allowing the computation of binding free energy (ΔG). When the bioactives are not similar, the ΔG estimation could be calculated using molecular mechanics Poisson
Boltzmann surface area (Miller et al., 2012) or by using solvated interaction energy methods (Kumar et al., 2014). The top hits could then be isolated/procured (if commercially available) for in vitro analysis.

33.3

Phytochemicals from Phytolacca dioica L. seeds extracts—case study I

Phytolacca dioica belongs to the Phytolaccaceae family native to Pampa, South America. It is otherwise known as Belhambra (English) or Belhambraboom (Afrikaans) tree and can easily be recognized by its massive trunk, simple and somewhat fleshy leaves borne on pinkish stalks with a pendulous cluster of berries (van Wyk, 2002). The leaves and berries of P. dioica have been reported to be rich sources of triterpenoid saponins, which have been described as displaying important biological activities such as molluscicidal, anti-inflammatory, antifungal, antibacterial effects and are often used for healing skin wounds (Di Maro et al., 2007; Escalante et al., 2002; Quiroga et al., 2001). Moreover, a number of ribosome-inactivating proteins (RIPs), that are potentially useful for the development of immunotoxins for tumor therapy and the production of transgenic plants endowed with specific parasite resistance, have been isolated from the plant (Del Vecchio Blanco et al., 1998). The fatty acid composition of the hexane extract from the P. dioica seeds showed that the major fatty acid identified was oleic acid (18:1 n-9), which is very important for the nutritional value of oil and, in fact, is involved in LDL cholesterol reduction, increasing HDL cholesterol and promoting insulin resistance, and it has been reported as an antiapoptotic and anti-inflammatory agent via downregulation of cyclooxygenase-2 and inducible nitric oxide synthase through the activation of nuclear factor-kappa B (NF-κB) (Orsavova et al., 2015). Recently, several compounds from P. dioica seeds were identified and extracted in order to discover new molecules with new biological activities (Di Petrillo et al., 2019). The main phenolic compounds identified by HPLC-Ms analysis in the ethyl acetate extracts of P. dioica seeds were found mostly consist of neo-lignans, isoamericanol B1 and related isomers (Fig. 33.3), and caffeoyl-threonic acid. Isoamericanol B1 and isomers were further extracted with high-speed counter-current chromatography showing a high antioxidant power, explaining in part the antioxidant activity determined in the P. dioica seeds extract (Di Petrillo et al., 2019). The isolated compounds were tested on xanthine oxidase (XO) and tyrosinase (TYR) enzymes in order to evaluate their inhibitory activity for the development of new drugs which act both on the oxidizing and uric acid production

FIGURE 33.3 2D chemical structure for the three classes of isoamericanols.

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TABLE 33.3 ADME properties of three classes of isoamericanol compounds. Physicochemical properties

Isoamericanol A

Isoamericanol B

Isoamericanol C1

Molecular formula

C18H18O6

C19H20O5

C27H26O9

Molecular weight (g/mol)

330.33

328.36

494.49

Rotatable bonds

4

4

6

H-bond acceptor atoms

6

5

9

H-bond donor atoms

4

2

5

Molar refractivity

87.87

91.18

129.20

Polar surface area (A˚2)

99.38

68.15

138.07

Lipophilicity (consensus)

1.65

2.82

2.24

Water solubility

Soluble

Moderate

Moderate

Pharmacokinetics

Isoamericanol A

Isoamericanol B

Isoamericanol C1

Gastrointestinal absorption

High

High

High

Blood
brain barrier permeation

No

Yes

No

P-glycoprotein substrate

Yes

Yes

No

Cytochrome P450 2D6 inhibitor

No

Yes

No

Cytochrome P450 3A4 inhibitor

No

No

Yes

Druglikeness (Lipinsky rule)

Yes

Yes

Yes

activity of XO and on the melanin production of TYR. The mixture of isoamericanols B1, B2, C1, and C2 showed inhibitory activity on TYR with IC50 of 0.110 6 0.02 mg/mL and inhibitory activity on XO with IC50 of 0.145 6 0.05 mg/mL. Given the potential use in the pharmaceutical industry of isoamericanol, the physicochemical properties of three classes of these compounds were calculated using Swiss-ADME (Daina et al., 2017) web tool (Table 33.3). Due to the critical importance of pharmacokinetics, drug discovery was reported, in addition to simple molecular attributes, the lipophilicity, water solubility, and drug likeliness characteristics for the three classes. Among the three classes of isoamericanol, A and B display similar molecular flexibility reflected by their same number of rotatable bonds, while C1 exhibits the highest flexibility. Further, isoamericanol C1 possesses the highest number of hydrogen (H-bond) acceptor and donor atoms, molecular refractivity, and polar surface area. On the other hand, isoamericanol B showed the highest lipophilic behavior, while the best water solubility characteristics were noted for isoamericanol A. Concerning pharmacokinetics, all three classes displayed high gastrointestinal absorption, while only isoamericanol B showed promising blood
brain barrier permeation characteristics. These findings suggest that P. dioica seeds extract may possess constituents with good medicinal properties that could be exploited to treat the diseases associated with oxidative stress, XO activity, and hyperpigmentation. Furthermore, given the limited literature on these compounds, further studies could identify new biological activities.

33.4 Phytochemicals composition and biological properties of seed extracts from Washingtonia filifera—case study II The palm family includes a range of plant species of particular importance in the field of nutrition, some of which may also have pharmacological interest. Washingtonia palms are a genus that belong to the Arecaceae family and Coryphoideae subfamily that includes two species: Washingtonia filifera and Washingtonia robusta (BenahmedBouhafsoun et al., 2015). They differ in subtle characteristics, and even palm experts have trouble distinguishing them. W. filifera (Linden ex Andre´) H. Wendl. ex de Bary known as California fan palm, desert fan palm, or Washington palm is the only palm native to California and is considered as the largest one in the United States but has been cultivated in Egypt and elsewhere (Hemmati et al., 2015).

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Fruits, including seeds of W. filifera have been analyzed for their nutritional composition, and they were revealed to possess a higher concentration of carbohydrates than proteins (Cornett, 1987). W. filifera fruits and seeds are also relevant sources of dietary oils, and the percentage compositions of its seeds are ash 1.37%, oil 16.30%, protein content 3.46%, total carbohydrate 77.19%, and moisture 3.22%. The major nutrients (mg/100 g) found in the seeds are potassium, magnesium, calcium, and phosphorus (Nehdi, 2011). Phytochemical investigation of this species detected lipids, trisaturated and unsaturated glycerides, proteins, leucoanthocyanins, flavonols, C-glycosylflavones, and flavonoid sulfates. While the latter compounds, the flavonoid sulfates, are not widely distributed in the plant kingdom, they occur in many members of the Palmae, especially in such important palm genera such as Washingtonia (El-Sayed et al., 2006). In particular, authors have studied the antioxidant activities of the aerial part of W. filifera and reported the presence of eight known flavonoids, luteolin 7-O-glucoside, luteolin 7-O-glucoside 200 -sulfate, tricin 7-O-glucoside, tricin 7-Orhamnopyranoside (100 -600 ) glucopyranoside, orientin, isoorientin, vitexin, and isovitexin 7-O-methyl ether, together with two newly described compounds, luteolin 7-O-glucoside 400 -sulfate and 8-hydroxyisoscoparin (i.e., 8hydroxychrysoeriol 6-C-glucoside). C-Glycosylation at different positions of luteolin significantly affects its antioxidant, anti-Alzheimer’s disease (AD), antidiabetic, and anti-inflammatory activities. The differences among these bioactivities of luteolin and its C-glycosylated derivatives are due to the nature as well as the position of the glycosylation (Choi et al., 2014). Natural products from plants provide unlimited opportunities for new drugs because their chemical diversity could give rise to a range of biological activities. The main compounds identified by HPLC-Ms analysis in the alcoholic extracts of W. filifera seeds were found mostly consisting of flavan-3-ols, in particular B-type procyanidin dimers (B1
B4), catechin, protocatechuic acid, and p-hydroxybenzoic acid. Conversely, very low amounts of phenolic compounds were detected in the pulp extracts. Recent interest in these compounds has been stimulated by the potential health benefits arising from their great antioxidant activity. The antioxidant activity of W. filifera seed extracts was evaluated by the ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) spectrophotometric assay and by measuring reactive oxygen species (ROS) levels using human keratinocytes as cell line. W. filifera seeds have been revealed to possess good antioxidant activity. In literature, it is reported that B-type procyanidins exhibit a wide range of biological, pharmacological, and chemoprotective properties against oxygen free radicals. The previous study has shown that the antiradical activity of procyanidins is strong at high concentrations, and it appears that epicatechin and epicatechin polymers are better antioxidants than catechin and catechin polymers and B-type procyanidins are better antioxidants than A-type procyanidins. Moreover, it appears that procyanidins extracts are more effective superoxide radical scavengers than the antioxidant vitamin C and trolox. It is well known that free radicals and ROS play a major role in the development of oxidative stress. An important enzyme that has been reported to proliferate during oxidative stress is XO, which catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid, and in both steps, there is the formation of free radicals. Overproduction or reduced excretion of uric acid leads to abnormal amounts of uric acid in the body, causing hyperuricemia and gout. Therefore, compounds that can inhibit XO may reduce both the circulating levels of uric acid and the production of ROS. W. filifera seed extracts showed good inhibitory activity against XO (Floris et al., 2019). Procyanidin B1 also showed good inhibitory activity against XO, and this means that procyanidin B1 could contribute to the inhibitory activity of the extracts against XO. Molecular docking studies were also carried out to predict the best ligand pose within the XO binding site of ligand procyanidin B1, procyanidin B2, catechin, and epicatechin (Fig. 33.4). For procyanidin, ligands were observed with two most probable binding sites: binding site 1, which is located distant from the protein active site, and binding site 2 that was found to be near the XO active site. Instead, the binding region for the ligands catechin and epicatechin is in the active site of XO. Spectrophotometric experimental data performed on seed extracts indicated a mixed-type inhibition against XO enzyme; thus, considering that the concentration of procyanidin is pronounced in the seed extracts, a plausible explanation for mixed-type inhibition can be established from the spatial location of the predicted binding sites for the procyanidin ligands that are different from the active site (Fig. 33.5). Oxidative stress can lead to many illnesses, including AD. Indeed, oxidative stress is involved in neuronal damage, due to the neurodegeneration promoted by highly reactive compounds. In a healthy brain, acetylcholinesterase (AChE) degrades acetylcholine (Ach) while butyrylcholinesterase (BChE) plays only a supportive role. However, several studies have shown the importance of BChE within the nervous system to be pivotal in the late stages of AD. A welldocumented strategy toward the effective management of AD is developing inhibitors that suppress the ChEs enzymes from breaking down ACh and therefore increasing both the level and the duration of the neurotransmitter action. In this regard, W. filifera seeds showed good cholinesterase inhibition, selective for BChE, which is of interest, because in

FIGURE 33.4 2D chemical structures of molecules identified from extracts. FIGURE 33.5 Molecular docking of the compounds against XO enzyme. The two predicted binding sites (BS1, BS-2) for procyanidin B1 and B2 are encircled in pink.

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patients with AD, BChE activity progressively increases, while AChE activity remains unchanged. The inhibitory activity against BChE of a procyanidin B1 standard was also evaluated, and the results showed that anti-BChE activity observed in seed extracts cannot be mainly attributed to procyanidin B1, even if it is present in high concentration. The docking results obtained for procyanidin B1 against BChE are shown in Fig. 33.6. The production of free radicals and ROS is also believed to be related to increasing hyperglycemia that is associated with type 2 diabetes (T2D), which is mainly characterized by pancreas β-cell dysfunction and postprandial hyperglycemia. One of the causes of β-cell dysfunction is the formation of the toxic islet amyloid polypeptide (IAPP) aggregates that lead to the formation of cytotoxic fibrils that accumulate on β-cells leading to their death. Thus, the inhibition of the formation of cytotoxic aggregates of IAPP could be one of the therapeutic approaches in the treatment of T2D. Another therapeutic approach to treat diabetes is to decrease postprandial hyperglycemia. This can be achieved by the inhibition of carbohydrate hydrolyzing enzymes like α-amylase and α-glucosidase that break down starch and disaccharides to glucose, thereby moderating the postprandial blood glucose elevation. In this respect, W. filifera seed extracts showed significant inhibitory activities against α-amylase and α-glucosidase and a complete inhibition of the formation of the IAPP aggregates (Floris et al., 2021). These results become important in the prevention or progression of diabetes. The main compounds identified in W. filifera seeds were also subjected to docking simulations to determine their binding affinities against IAPP, α-amylase, and α-glucosidase. Among the four compounds (Table 33.4), catechin displayed better binding energy values for all the three protein targets.

FIGURE 33.6 Molecular docking of the procyanidin B1 against BChE enzyme. The interaction picture between BChE and ligand was drawn using Ligplot software (Laskowski & Swindells, 2011). Hydrophobic interactions are represented by red spokes radiating toward the interacting ligand atoms, while hydrogen-bonded interactions in dashed green lines.

TABLE 33.4 Predicted binding energy for the compounds against IAPP, α-amylase, and α-glucosidase. Compounds

IAPP

α-amylase

α-glucosidase

Catechin

26.9 kcal/mol

27.1 kcal/mol

28.1 kcal/mol

Protocatechuic acid

24.9 kcal/mol

25.5 kcal/mol

25.3 kcal/mol

p-hydroxybenzoic acid

24.5 kcal/mol

25.2 kcal/mol

25.2 kcal/mol

Procyanidin B dimer

27.8 kcal/mol

28.5 kcal/mol

24.9 kcal/mol

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Considering the effect of W. filifera seed extracts on target enzymes for AD and the correlation between AD and T2D, W. filifera seeds can emerge as a promising natural source of bioactive compounds for these diseases. Taking into account the implication of oxidative stress in skin aging, W. filifera seed extracts were also evaluated for their effect on enzymes involved in these processes. Skin aging is a physiological process related to UV-induced damage that leads to ROS formation that in turn can initiate complex molecular pathways including the activation of enzymes that degrade extracellular matrix. Among them, elastase causes the degradation of elastin and therefore the appearance of wrinkles as a result of its increased activation, and collagenase degrades collagen and therefore causes changes in the thickness of the skin. Another enzyme implicated in the skin aging process is tyrosinase, which is the key enzyme in melanin synthesis and represents a physiological process which plays a crucial role in preventing UVinduced skin damage by absorbing UV sunlight. However, excess production or abnormal accumulation of melanin causes skin problems such as the typical age spots. Thus, inhibitors of all these enzymes represent increasingly important ingredients in cosmetics and medications to prevent skin aging. W. filifera seeds extracts could act in the prevention of premature aging, acting simultaneously on several fronts: at the beginning of the process via their photoprotective effect and therefore reducing UV rays absorption; then, they showed a good antioxidant effect with methanol extract preventing ROS formation in cellular system without cell toxicity; finally, all the extracts inhibit collagenase, elastase, and tyrosinase, even if the latter with a minor extent (Era et al., 2021). All these studies contribute to valorize W. filifera seeds as a source of bioactive compounds and assume particular importance considering that the seeds are an inedible part of the fruit that is usually discarded. Future prospects include further studies in order to test the inhibitory activity of the main compounds identified in the extracts with possible therapeutic potential.

33.5

Phytochemicals—opportunities and challenges

The global phytochemicals market is predicted to grow as more phytochemicals are used in cosmetics, food and beverages, and pharmaceuticals. Sales of phytochemicals are predicted to grow at a 7% CAGR between 2021 and 2031, driven by increased demand in a variety of applications. The usage of phytochemicals in the cosmetics industry is improving due to rising concerns about wellness and skincare, which will drive market expansion during the forecast period. New skincare products with phytochemicals as an active component have been launched by cosmetic industry behemoths. Furthermore, research into the production of novel food products and dietary supplements generated from phytochemicals is projected to help the industry thrive. The creation of products generated from plant extracts is becoming more important to major corporations. Phytochemicals, for example, are in high demand as the demand for plantbased diets and goods develops. Phytochemicals are also being used to treat malignancies of several organs by blocking angiogenesis. As a result, sales of phytochemicals will increase across pharmaceutical, personal care, and other end-use applications.

33.5.1 Phytochemicals as vegan food ingredients Consumers want multicomponent items that are readily available on the market and can help them cope with their fastpaced lifestyle. As the demand for nutrient-rich health foods grows, so does the desire for healthy foods made with healthier components. Researchers are looking for new food additives to improve the product’s functionality and nutritional value. As a result, to produce a sufficient volume of vegan, nutrient-rich food for human consumption, companies are turning to phytochemicals.

33.5.2 Plant-based ingredients Manufacturers in the food and beverage business are benefiting from the use of materials derived from plants, which has created a rather stable situation. Chemicals derived from plant extracts are becoming more popular among manufacturers since they may be utilized in a variety of food products and are linked to a variety of health advantages. In the food sector, plants including gooseberry, turmeric, ginger, beetroot, and ginseng are regularly used to create consumerfriendly healthy food products. Phytochemicals are found in all plants and play a crucial role in protecting the plant from environmental challenges. Including phytochemicals in one’s diet can help to minimize the risk of a variety of chronic and lifestyle diseases. These compounds have been linked to the consumption of food products associated with diets rich in fruits, vegetables, legumes, and cereals.

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33.5.3 Dietary supplements As the need for nutritional supplements rises, the global demand for phytochemicals is expected to rise. Changing lifestyles, demanding work schedules, and an increase in unhealthy and imbalanced eating habits are all leading to an increase in the risk and occurrence of a variety of health problems. Another aspect driving demand for phytochemicals is the rising popularity of natural supplements and components. Phytochemical demand is increasing in response to increased demand for natural products and increased consumption of dietary supplements. Phytochemicals have recently gained prominence because of their ability to reduce illness risk and offer antiaging properties. LDL cholesterol, or bad cholesterol, can be reduced by phytochemicals, lowering the risk of heart disease, cancer, and diabetes. Demand for phytochemicals is on the rise and is projected to continue to witness an upward trend, as health conditions such as cardiovascular disease, obesity, and others become more common.

33.5.4 Effect of COVID-19 on phytochemicals demand The phytochemicals market has benefited from the COVID-19 crisis. It provided opportunities for the pharmaceutical business to expand into other areas. Consumption of plant-based foods and dietary supplements increased as consumers became more health-conscious. As a result, the demand for phytochemicals will increase. Meanwhile, worldwide regulatory authorities devised guidelines for maintaining food cleanliness and food safety laws to prevent COVID-19 transmission through food. The demand for clean-label phytochemicals will be aided by these improvements. By 2021 to 2031, a global economic recovery would result in higher per capita income and the adoption of healthier lifestyles, strengthening the market (Editor, 2022).

33.5.5 Transfer of phytochemicals into pharmaceuticals—Challenges Plant-derived natural compounds displaying beneficial effects in curing human diseases are expected to increase considering the growing interest in this research field. It can seem as an encouraging alternative route compared to synthetic drugs that have failed to definitively cure diseases. Numerous mechanistic studies have investigated the therapeutic potential of natural bioactives in human diseases. However, the efficacy of bioactive compounds has been rather limited in clinical studies. The first impediment of natural bioactives in clinical use is to infer the right dose required in humans from the biological response reported in vitro or in vivo models. Another main issue is bioavailability, due to the complex nature of bioactive compounds and also the different mechanisms of absorption that could strongly influence and even drive their pharmacological effects. Finally, a considerable limitation that can affect the therapeutic potential of phytochemicals can be related to the design and execution of clinical trials, wherein identification of the exact mechanism of action and, hence, of a therapeutic target is of crucial importance (Piccialli et al., 2022).

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6.

Chapter 34

Modulation of drug resistance in leukemia using phytochemicals: an in-silico, in-vitro, and in-vivo approach Urja Desai1, Medha Pandya2, Hiram Saiyed1 and Rakesh Rawal3 1

Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India,

2

Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar, Gujarat, India, 3Department of Life Sciences, School

of Sciences, Gujarat University, Ahmedabad, Gujarat, India

34.1

Introduction

Leukemia is an uncontrolled proliferation of blood cells. Leukemia means “white blood” (leukos, “white”; haima, “blood”), because a delay in the maturation of the transit-amplifying cells causes a significant increase in immature white blood cells to circulate, turning the blood from red to creamy white. The hallmarks of leukemogenesis encompass recurrent nonrandom chromosomal translocations (Daga et al., 2018). Leukemia is classified clinically or pathologically into acute and chronic forms based on how rapidly the disease develops and the kind of blood cell involved (Vincent et al., 2001). There are mainly four types of leukemia: acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and acute myelogenous leukemia (AML). Many of the outstanding triumphs in cancer treatment have resulted from novel investigations and trials in leukemia. These include combination chemotherapy, stem cell transplantation, “differentiation” therapy, monoclonal antibody therapy, and targeted treatment (Greaves, 2016). Doxorubicin, cytarabine, ibrutinib, idelalisib, rituximab, nilotinib, and imatinib are among the most commonly used chemotherapeutic drugs in the treatment of leukemia. The study shows that doctors are preferring to use chemotherapy or hematopoietic stem cell transplantation (HSCT), and the major issue with this treatment is that leukemia cells can easily relapse after the treatment. The characteristics of leukemia have become more advanced in recent years, and the treatment of this disease must also be more advanced. In addition, organ transplantation can be a good option, and they will have a lifelong risk of immunosuppression. Additionally, the effect of HSCT can be dangerous, and there is proof that “the donor-derived immune system” can easily attack the “recipient’s leukemic cells.” After the relapse of the HSCT, the chemotherapy control procedure is utilized to mitigate the effect. The killer immunoglobulin receptor and KIR ligand interact and allow the donor’s T cells to recognize the antigens related to leukemia (Maacha et al., 2019). However, it happens much too often that leukemia escapes control after HSTC and uses chemotherapy to control the disease although acute leukemia shares several mechanisms of immune evasion that can be found in solid tumors. Unfortunately, hematological malignancies have less research in this area. A deeper understanding of this process is required to create an effective and reasonable immunotherapy for acute leukemia. Resistance to chemotherapy is one of the most difficult challenges in cancer treatment. Ninety percent of cancerrelated mortality is caused by the emergence of drug resistance, which renders chemotherapeutic drugs useless. Drug resistance is the ability of cancer cells to decrease the effectiveness and potency of chemotherapy agents (Nikolaou et al., 2018). Intrinsic resistance, which occurs in cancer when malignant cells develop resistance without having previously been exposed to chemotherapeutic drugs, causes a subpar response to initial therapy (Gottesman, 2002). In some situations, cancer cells initially respond well to chemotherapy but thereafter have a poor response because they have acquired resistance (acquired resistance). Prior studies on cell lines and animal models demonstrated that drug resistance in cancer may be gained by a variety of mechanisms, including drug efflux via the ATP-binding cassette (ABC) transporter, changing the expression of proteins targeted by anticancer medicines, drug detoxification, and evasion of apoptosis (Aris, 2000). Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00008-6 © 2023 Elsevier Inc. All rights reserved.

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Phytochemicals (flavonoids, carotenoids, allicin, polyphenols, hydrolyzable tannins, lignans, and phytosterols) provide flavor and color to vegetables, fruits, and herbs. Phytochemicals have different anti-inflammatory, antioxidant, antiviral, antibacterial, anticarcinogenic, antiproliferative, and cellular repair properties. Phytochemicals are important resources for innovative medications and cancer therapy. Taxol analogs, vinca alkaloids such as vincristine and vinblastine, and podophyllotoxin analogs are common examples (Lichota & Gwozdzinski, 2018). These phytochemicals frequently function by altering molecular pathways involved in cancer development and progression. The precise mechanisms include boosting antioxidant status, neutralizing carcinogens, reducing proliferation, promoting cell cycle arrest and apoptosis, and immune system control (George et al., 2021). The development of efficient and side-effectfree phytochemical-based anticancer therapy begins with the assessment of natural extracts (from dry/wet plant material) for possible anticancer biological activity, which is then followed by the extraction of active phytochemicals based on bioassay-guided fractionation and testing for in vitro and in vivo effects. Increasingly, information is available for the phytochemicals and drug targets in online repositories and biological databases. Scientists have consistently used standard pharmacology tools such as in vivo and in vitro models to explore these theories. However, in the recent decade, there is an increase in the development and application of computational (in silico) methodologies to pharmacology hypothesis creation and testing. Databases, quantitative structure
activity relationships, pharmacophores, machine learning, data mining, network analysis tools, and computer-based drug designing tools are examples of in silico methodologies which are increasingly used for screening of phytochemicals against bacterial, viral, and cancer targets (Juneja et al., 2021; Pandya et al., 2022; Menamadathil et al., 2022). Fig. 34.1 depicts the general path to target various disease mechanisms: phytochemicals from the plant to the computational and molecular laboratory. Drug combinations that work synergistically are anticipated to be enormously beneficial for different cancers in which chemotherapeutic and targeted strategies have failed or frequently exhibit cases of drug resistance (Pezzani et al., 2019; Tinoush et al., 2020). A beneficial approach is multidrug therapy, which combines the direct blocking or killing of harmful substances (such as pathogens or cancer cells) with the activation of the body’s natural defenses or repair processes. It results from a progressive rejection of the mono-drug therapy dogma, which had been widely accepted for decades and constituted the foundation of pharmacological research (Doos et al., 2014). In terms of phytotherapy research, traditional Chinese medicine, Ayurveda, and traditional phytomedicines have just recently begun to be scientifically validated and valued. Furthermore, over the last 20 years, there has been an increase in the use of conventional

FIGURE 34.1 Phytochemicals from the plant to the computational and molecular laboratory to target multiple disease processes. Created in BioRender.com.

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drugs in combination with complementary and alternative medicine, which includes not only homeopathy, naturopathy, chiropractic, and energy medicine, but also ethnopharmacology and phytotherapy. In this chapter, we summarize natural products that have the potential to overcome drug resistance in leukemia and the synergistic effects of phytochemicals with conventional therapeutic drugs. This chapter provides a summary of physiologically active substances that target leukemic cells, as determined by in silico, in vitro, and in vivo tests or clinical investigations, in order to investigate their therapeutic potential in the treatment of leukemia. The diverse set of natural chemicals with antileukemic potential provides a foundation for academics and hematologists to improve scientific and clinical research on the creation of innovative alternative medicines in the fight against leukemia.

34.2

Drug resistance: therapeutic failure in leukemia

Drug resistance is the reduction in a drug’s effectiveness in treating a disease or condition, such as an antibiotic or an antineoplastic (Housman et al., 2014). In other words, a disease develops a resistance to medication. Resistance to treatment with anticancer drugs is caused by a variety of factors, including individual variances in patients and somatic cell genetic changes in cancers, even those from the same tissue of origin. Resistance to chemotherapeutic medicines is a major concern in the treatment of leukemia (Wang et al., 2019). Many mechanisms are implicated in this, such as the cell’s failure to undergo apoptosis in response to chemotherapy or the drug’s failure to reach and/or effect its intracellular target. The main mechanism through which many malignancies acquire resistance to chemotherapeutic drugs and result in treatment failure is multidrug resistance (MDR) (Bukowski et al., 2020). Patients with various blood malignancies and solid tumors are affected. Malignant cell populations commonly coexist in tumors; some are drug-sensitive, while others are drug-resistant (Tyner et al., 2022). Chemotherapy destroys drug-sensitive cells but also leaves behind a greater percentage of drug-resistant cells. Chemotherapy may fail as the tumor regrows because the surviving tumor cells are now resistant. There are categories of mechanisms that can enable or promote direct or indirect drug resistance in human cancer cells which are depicted in Fig. 34.2. Drug activation: Drug activation is a complicated process in which chemicals interact with many proteins. These interactions can change, partially destroy, or combine the drug with other molecules or proteins, which activates it. Anticancer drugs need to metabolize in order to work (Housman et al., 2014). Through lessened drug activation, cancer cells might also acquire resistance to such therapies. For instance, cytarabine (Ara-C: A nucleoside drug) is activated in the therapy of AML following many phosphorylation reactions that change it into Ara-C triphosphate. A reduction in Ara-C activation caused by a mutation in this pathway may result in Ara-C drug resistance (Lamba, 2009). Alteration of drug target: The molecular target of a drug and changes to this target, such as mutations or altered expression levels, have an impact on the treatment’s effectiveness. These kinds of target changes in malignancies may eventually result in drug resistance. Imatinib is a tyrosine kinase inhibitor that selectively targets the BCR-ABL protein and causes remission in CML patients. Point mutations in the ABL gene and gene amplification in the BCR-ABL fusion can both lead to imatinib resistance (Shah et al., 2002). Drug efflux: Cancer drug-resistance mechanisms entail minimizing drug buildup by promoting drug efflux via ABC transporters including P-gp, BCRP, MRP1, and LRP. They are all categorized by the presence of two different domains—a highly conserved nucleotide-binding domain and a more variable transmembrane domain. ATP hydrolysis at the nucleotide-binding site triggers a conformational shift that pushes a specific substrate out of the cell when it binds to the transmembrane domain. This efflux mechanism is critical in preventing toxic overload (Ughachukwu & Unekwe, 2012). DNA damage repair: Anticancer drug resistance is clearly influenced by DNA repair in reaction to chemotherapeutic treatments that either directly or indirectly disrupt DNA. By triggering nucleotide excision repair or other fundamental DNA repair mechanisms, DNA damage response mechanisms can repair drug-induced damage (Li et al., 2021). Cancer cell heterogeneity: This population of heterogeneous cells contains a small number of stem cells, most of which are treatment-resistant. Some of these cancer cells with resistance may be circulating and have the ability to develop tumors in distant tissues (Dagogo-Jack & Shaw, 2018). However, both cancer cells in the bloodstream and those in solid tumors exhibit heterogeneity. Two coexisting dominant clones were discovered in a recent AML research. One was drug-resistant, whereas the other was drug-sensitive. It is conceivable that the proliferation of cancer cells from the drug-resistant clone is what causes this illness to recur in people after effective therapy. Resistance to apoptosis: The extrinsic route or the intrinsic pathway is activated to cause apoptosis (mitochondrialmediated). Activating and inhibiting proteases, reactive oxygen species, and members of the Bcl-2 family are just a few of the numerous mechanisms that control the release of mitochondrial cytochrome c as a result of chemotherapy.

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FIGURE 34.2 Factors responsible for drug resistance in cancer cell. Created in BioRender.com.

Overexpression of the antiapoptotic Bcl-2/BcI-xL proteins corresponds with chemoresistance in human leukemia, and the balance between pro- and antiapoptotic Bcl-2 family members regulates mitochondrial membrane integrity (Deng et al., 2000).

34.2.1 Proteins/genes responsible for drug-resistance leukemia 34.2.1.1 ATP-binding cassette transporters Human physiology, toxicology, pharmacology, and a variety of diseases, such as acute myeloid leukemia, are all known to be significantly influenced by ATP-binding cassette (ABC) transporters (Dean et al., 2005). On the basis of their sequence homology scores, 49 human ABC transporter genes have been sequenced and divided into 8 subfamilies, ABCA through ABCG and ANSA (arsenite and antimonite transporter) (Mahadevan & List, 2004). Since the beginning or middle of the 1970s, it has been recognized that P-glycoprotein (P-gp), which serves as a drug efflux pump and mediates the MDR of mammalian cancer cells. The recruitment of drug processing and metabolizing enzymes, the activity of conjugating enzymes, the modification of DNA repair activity, the reduction of cell sensitivity to apoptosis, and the mutation of particular drug targets are some of the mechanisms causing multidrug resistance. The ABC transporterencoding genes ABCB1, which encodes a P-gp, and ABCC1, also known as MRPL ABCG2 or BCRP, have been investigated the most in stem cells. These are the main genes that have been linked to both in vitro and in vivo multidrug resistance in tumor cells.

Modulation of drug resistance in leukemia using phytochemicals Chapter | 34

34.2.1.1.1

587

P-glycoprotein (ABCB1/MDR1)

P-glycoprotein also known as MDR1 is an ABC protein found in almost all living creatures, from prokaryotes to humans. They are plasma membrane “pumps” that actively extrude various lethal chemicals from cancer cells and are so referred to as “efflux pumps.” P-gps are present in multigene families in a variety of organisms. In mice (MDR1, MDR2, MDR3) (Devault & Gros, 1990), rats (MDR1 and MDR2) (Brown et al., 1993), hamsters (P-gp1, P-gp2, P-gp3) (Endicott et al., 1991), and humans (MDR1. MDR2) (Chin et al., 1989), P-gp isoforms are expressed by a small family of closely similar genes, although only MDR1 is implicated in multidrug resistance. 34.2.1.1.2

ABCCl (MRP1)

MRP1 is made up of 1531 amino acids and is a 190 kDa plasma membrane efflux pump (Cole, 2014). It has two ATPbinding domains as well as three membrane-spanning helices. MRP1 overexpression gives resistance in tumor cells to a wide range of hydrophobic drugs, including doxorubicin, daunorubicin, vinblastine, vincristine, and colchicine (Choi et al., 2005). The successful expulsion of chemotherapy drugs depends on glutathione; P-gp, the primary protein causing the multidrug-resistance phenotype, and MRP1 have a similar method of action. ABCG2 (BCRP): The 72-kDa breast cancer-resistance protein (BCRP) is the second member of the human ATPbinding cassette (ABC) transporter superfamily and is hence also known as ABCG2 (Mao, 2005). Current experimental data show that BCRP may act as a homodimer or homotetramer. Due to its ability to increase drug efflux, overexpression of BCRP is linked to high levels of resistance to several anticancer drugs, such as anthracyclines, mitoxantrone, and camptothecins.

34.2.1.2 Cancer stem cells and drug resistance Cancer stem cells (CSCs), also known as tumor-initiating cells, are thought to be responsible for drug resistance and cancer recurrence due to their capacity to self-renew and differentiate into a variety of cancer cell lineages. Bonnet and Dick published the first clear evidence of CSCs in Bonnet and Dick (1997), when they discovered a subset of leukemia cells expressing the surface marker CD341 but not CD382. After transplantation, the CD341/CD38 subpopulation was capable of beginning tumor development in NOD/SCID recipient mice.

34.2.1.3 Hypoxia-inducible factor-1-mediated resistance The major transcription factor driving hypoxia responses has been identified as hypoxia-inducible factor (HIF), and HIF target genes overlap significantly with those implicated in dysregulated tumor metabolism. HIF-l α stimulates the expression of various genes involved in fermentative glucose metabolism, including glucose transporters, glycolytic enzymes, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1, and the monocarboxylate transporter. Chemoresistance is associated with HIF-l α upregulation (Weidemann & Johnson, 2008).

34.3

Combination index method and synergism

The value of nutraceuticals as potential cancer treatments is becoming more universally recognized. Significant advances in modulating critical signaling pathways/molecules that affect tumorigenicity have been reported, suggesting that they offer a promising future (Ahmad et al., 2015). Natural products can mitigate oral mucositis, various organrelated toxicity, and hematopoietic system damage caused by chemotherapy and radiotherapy. Purified phytochemicals obtained from herbs, bioactive component-enriched fractions, and crude extracts from plants, as well as herbal formulations, can effectively reduce the side effects of chemotherapy and radiotherapy, mainly because of their antioxidative and anti-inflammatory properties. Furthermore, some natural compounds can reduce the oral mucositis caused by chemotherapy and radiation therapy by acting as an anti-infective, as well as reduce the side effects of the treatment by regulating the gut microbiota, the liver, the kidneys, the immune system, the hematopoietic system, the cardiovascular system, the energy metabolism, and the nervous system (Zhang et al., 2018). Natural compound combinations have the potential to be more effective than single-agent therapy. The goal is to enhance efficacy while minimizing potential negative effects, same as with pharmacologic medicines. The study and development of agents derived from natural products are attracting attention in the scientific community, and substantial progress has already been gained in this area (Sauter, 2020). Today, natural products are effective for the treatment of cancer. A significant number of anticancer drugs used in clinics are either natural or derived from natural products from a variety of sources, including plants, animals, and microbes of those of marine origin. Classic examples of drugs

588

Recent Frontiers of Phytochemicals

developed from plants include vincristine, irinotecan, etoposide, and paclitaxel. Drugs derived from microorganisms include actinomycin D, mitomycin C, bleomycin, doxorubicin, and l-asparaginase. The first drug derived from a marine source is cytarabine. The discovery of natural chemicals with anticancer activity has been significantly aided by largescale anticancer drug discovery and screening programs like those funded by the National Cancer Institute (NCI). Combinatorial synthesis, high-throughput screening, and their accompanying techniques have increased naturalproduct-based drug advances in recent years (Nobili et al., 2009). Drug synergy can be achieved by increasing intracellular retention via enhanced drug absorption or reduced drug elimination (Jia et al., 2009). Understanding how compounds interact with one another, whether synergistically, additively, or antagonistically, can have a wide range of practical implications. These include toxicity tests to determine whether or not a mixture of common household chemicals is safe to use, therapeutic uses such as medicine combinations, and measures to reduce selectivity and resistance (Bulusu et al., 2016). The calculation of the combination index (CI) equation directly indicates the degree of synergy between medications. The variables IC50(A) and IC50(B) in the equation reflect the concentrations at which medications A and B, respectively, achieve a 50% cell growth inhibition rate, whereas the variables IC50(A)pair and IC50(B)pair represent the concentrations at which the combined effects of the drugs result in a 50% growth inhibition rate. Values of CI of 1, ,1, and . 1 are indicative of addition, synergistic, and antagonistic effects, respectively (Chou, 2006, 2010; Chou & Talalay, 1984; Zhao et al., 2004). Traditional combinations of free drugs do not usually exhibit good synergy due to variations in drug properties such as solubility, permeation, stability, half-life, distribution in tumors, and metabolism. It is also vital to have a sensible design of combined drug administration that adheres to well-accepted principles of combination chemotherapy, such as separate pharmacological mechanisms of the combined medications (e.g., targeting various cell cycles), no crossresistance, no overlapping adverse effects, and synergistic antitumor benefits (Tardi et al., 2009; Waterhouse et al., 2006). Various diseases and abnormalities can be treated using natural products. Numerous investigations are being carried out to evaluate the translational potential of naturally occurring substances, from the laboratory to the clinical levels (Singla et al., 2022).

34.4

Phytochemicals as chemosensitizer and modulators

34.4.1 Computational approach to target multidrug resistance The computational approach is a rapidly expanding field that uses an integrated method to describe the specific target of a disease and investigate how drugs or other molecules interact with that target. Computer-based drug discovery is a very efficient method in terms of both time and money. There are several online and offline tools and applications that have been utilized in computer-based drug development to simplify and reduce costs (Manjunathan et al., 2022). It is possible to anticipate how molecules would interact with a target’s drug-binding site, such as P-glycoprotein, BCRP, or MRP1, using in silico studies. In order to conduct in silico investigations, the target protein must have a threedimensional structure; if one is not available, a homology modeling technique can be used to estimate the protein’s structure (Dave et al., 2015). Molecular docking is one of the most commonly used computational methods in structurebased drug design due to its capability to calculate the binding confirmation of small molecule ligands to suitable target binding sites. Finding the phytochemicals that have the highest affinity for the drug-binding site and should be employed as lead compounds for additional research can be assisted by molecular docking studies. Various in silico studies on phytochemicals with multidrug-resistance targets are depicted in Table 34.1. Several types of phytochemicals in the group of alkaloids, flavonoids, curcuminoids, terpenes, carotenoids, lignans, and polyketides have been investigated for MDR-reversing activity. For multidrug-resistance proteins like P-gp and BCRP (EGCG, curcumin, quercetin, piperine, and taxifolin), MRP-1 (resveratrol, curcumin, quercetin, EGCG, piperine, emodine) MDR1 in leukemia and other malignancies are summarized in Fig. 34.3. These phytochemicals also have the synergistic ability to reverse MDR in cancer cells (Talib et al., 2022). Mohammed et al. (2022) reported that flavonoids from Suaeda vermiculata demonstrated remarkable inhibitory effect on ABC transporters. Its binding affinity and docking position are identical to those of doxorubicin. Isorhoifolin, luteolin 7-O-glucoside (cynaroside) demonstrated fair binding energy with P-glycoprotein. Other flavonoids kaempferol-3-O-rutinoside and apigenin-7-O-glucoside have inhibitory effect on MRP protein. BCRP demonstrated in silico interaction with kaempferol-3-O-rutinoside, isorhoifolin, and luteolin 7-O-glucoside (cynaroside). In vitro activity followed by in silico analysis also demonstrated that flavonoids from S. vermiculata (5-O-methyl visamminol), (N-transferuloyl tyramine), (atractylenolide-III), and (ginsenoside-Rh2) also identified as the potential ATP-binding cassette

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TABLE 34.1 In silico interactions of phytochemicals with multidrug resistance targets. Substrate

Phytochemical

References

P-glycoprotein

4’-Demethylknipholone 2’-β-D-glucopyranoside

Kushwaha et al. (2021)

P-glycoprotein

Cannabisin M, cannabisin N

Kazemi et al. (2021)

P-glycoprotein

Quercetin

Mohana et al. (2016)

P-glycoprotein

Quinic acid, kaempferol-O-rutinoside, rutin, and isorhamnetin-Orutinoside

Elkady et al. (2020)

P-glycoprotein

Aflavine and quercetin

Mohana et al. (2016)

P-glycoprotein

Piperine and its analog (natural product analog—Pip1 ((2E,4E)-5(benzo[d][1,3]dioxol-5-yl)-1-(6,7-dimethoxy-3,4dihydroisoquinolin-2(1H)-yl)penta-2,4-dien-1-one)
)

Syed et al. (2017)

P-glycoprotein

Tetrandrine

Liao et al. (2019)

P-glycoprotein

Acridones

Gade et al. (2018)

P-glycoprotein

Chrysin, epigallocatechin(EGCG)

Wongrattanakamon et al. (2017)

P-glycoprotein

Apigenin

Saeed et al. (2015)

P-glycoprotein

Naringenin

Ferreira et al. (2018)

ABCB1(P-gp)

Isorhoifolin, luteolin 7-O-glucoside (cynaroside)

Mohammed et al. (2022)

ABCC1(MRP)

Kaempferol-3-O-rutinoside, apigenin-7-O-glucoside

Mohammed et al. (2022)

ABCG2(BCRP)

Kaempferol-3-O-rutinoside, isorhoifolin, luteolin 7-O-glucoside (cynaroside)

Mohammed et al. (2022)

BCRP

Naringenin

Ferreira et al. (2018)

MRP1

Carbohydrazide derivatives

Ferreira et al. (2018)

MRP1

Curcumin

Sreenivasan et al. (2012)

modulators during the study (Mohammed et al., 2022). Flavonoids including quercetin, quinic acid, kaempferol-O-rutinoside, rutin, and isorhamnetin-O-rutinoside, chrysin, and epigallocatechin have also been shown to be effective MDR reversal agents in several investigations (Table 34.1). Tetrandrine, an alkaloid of the benzylisoquinoline class, has been shown to exhibit inhibitory properties. It has a comparable docking location to verapamil and a similar binding affinity. The amino acids Ala729, Ala987, Ileu306, Ileu340, Leu339, Leu65, Leu975, Met69, Met986, Phe303, Phe336, Phe343, Phe728, Phe732, Phe983, Tyr307, and Tyr310 create a lipophilic pocket that holds the main portion of tetrandrine. Additionally, tetrandrine’s positively charged methylamine moiety interacted cationically with Phe343 and negatively with Phe336 through an aryl ether (Liao et al., 2019). Syed et al. (2017) studied piperine, a different category of alkaloids, and found that hydrophobic interactions with P-gp occurred in the following positions: Leu339, Met69, Met986, Phe72, Phe336, Phe728, Phe983, Tyr953, and Val982. In addition, Tyr307 and an H-bond were created. Two piperine analogs that demonstrated improved hydrophobic interaction with the majority of the aforementioned amino acids were developed (Syed et al., 2017). Kazemi and coworkers (2021) investigated the lignanamides from Cannabis sativa were docked against P-gp to identify possible binding affinities of these phytochemicals. Two well-known P-gp inhibitors, tariquidar and zosuquidar, are used as control ligands. When compared to tariquidar and zosuquidar, cannabisin M and cannabisin N were found to have greater binding affinities to the drug-binding pocket of P-gp. These results suggest that cannabisin M and cannabisin N may make promising P-gp inhibitors. Numerous naringenin derivatives, including hydrazones and azines, were designed by Ferreira et al. (2018) to enhance MDR reversal in P-gp and BCRP. The majority of these two groups’ members were very selective. It has been demonstrated that, like fumitremorgin C, a common inhibitor of BCRP, the active substances bind to hydrophobic residues such as transmembrane helix 4 and 5 in the middle of an active pocket of BCRP. The fine interaction between the

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FIGURE 34.3 Multidrug resistance reversal or prevention of drug efflux by nutraceuticals. Created in BioRender.com.

transporter MRP1 and the carbohydrazide derivatives of flavonones was also seen as a result of the consequent flexibility (Ferreira et al., 2018).

34.4.2 In vitro analysis of phytochemicals as multidrug resistance reversal Phytochemicals have been shown to reverse MDR in cancer cells via synergism, in addition to influencing the function or expression of transporters. When evaluating a drug combination, the combinational index, the isobologram, and the correlation coefficient of linear regression, which depends on the medium effect equation, are the best tools to use. Chou in 2006 reported “theoretical foundation experimental design and computerized simulation in combinational pharmacological trials” as efficient methodologies. In recent years, the idea of synergy in natural product mixes has garnered increased attention, and the significance of multitarget combination therapy has risen to the forefront of the medical community. Multiple mechanisms contribute to synergy, such as (1) pharmacological synergism through multitarget effects, (2) pharmacokinetic synergism via modulation of drug transport, permeation, and bioavailability, (3) decimation of adverse effects, and (4) targeting of disease-resistance mechanisms (Caesar & Cech, 2019). One of the primary issues with preclinical screening is the creation of new high-tech techniques for testing anticancer medicines. The major worry is the poor association of in vitro processes. Due to the prompt screening and use of ineffective drugs, the selection of inappropriate processes, sometimes in vitro testing results in a decrease of both money expenses and time. The advancement of new high-tech “preclinical in vitro screening” is crucial due to the rising incidence of oncology. Combinations of drugs have shown effectiveness in treating a wide variety of life-threatening conditions, such as cancer, HIV, AIDS, tuberculosis, malaria, diabetes, hypertension, MRSA, and many others. Researchers looked at the pharmacological effects of alkaloids, flavonoids, curcuminoids, stilbenoids, terpenes, carotenoids, lignans, and polyketides on MDR. In the majority of experiments, it was capable of discriminating between molecules with greater and lower effectiveness (Tinoush et al., 2020). Various in vitro studies related to phytochemicals

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TABLE 34.2 In vitro anticancer effect of phytochemicals in leukemia. Phytochemical

Source

Cell line

References

Luteolin

Vegetables and fruits such as celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins, and chrysanthemum flowers are luteolin rich

MOLM-13 and Kasumi-1 cells

Deng et al. (2017)

EGCG

Green tea

NB4 and HL60 cells

Britschgi et al. (2010)

Quercetin

Citrus fruits, apples, onions, parsley, sage, tea, and red wine. Olive oil, grapes, dark cherries, and dark berries

MV4
11 and HL-60 cells

Shi et al. (2020)

Chrysin

Passionflower, silver linden, and some geranium species; and in honey and bee propolis

MO7e cells

Lee et al. (2007)

Resveratrol

Grapes, wine, grape juice, peanuts, cocoa, and berries of Vaccinium species, including blueberries, bilberries, and cranberries

CD341 CD382 KG1a cells

Peng et al. (2015)

Apigenin

Parsley, chamomile, celery, vine spinach, artichokes, and oregano

K652 and K562/IMA3 cells

Solmaz et al. (2014)

Aromatic (ar)-tumerone

Turmeric oil

U937

Lee (2009)

Rhizochalin

Rhizochalina incrustata

THP-1

Fedorov et al. (2009)

Asterosaponi

Astropecten monacanthus

HL-60

Thao et al. (2014)

as anticancer agent on different leukemia cell lines are summarized in Table 34.2. Natural compounds like EGCG, (2)-epicatechin, quercetin, and green tea, chrysin, resveratrol, apigenin, 40 -methoxy-5-epi-ancistecrorine A1, aromatic (ar)-tumerone, rhizochalin, Asterosaponi, Morus alba demonstrated antileukemic potential. Experimental studies on various cell lines or primary cultures, as well as preclinical and clinical studies, have revealed natural compounds with antileukemic activity; these results may lead to the inclusion of these compounds in future therapeutic protocols for various forms of leukemia, including AML, CML, ALL, and CLL. Even though the results in vitro are promising, most bioactive compounds have not yet been tested in preclinical or clinical studies (Cotoraci et al., 2021). The rapid acceleration of the discovery, examination of drugs, and development leads to growth in the importance of the in vitro system. There are multiple two-dimensional and three-dimensional models present in the pharmaceutical sector. The study discusses various testing modes and their properties and their scientific applications. As a result, multiple physicians are choosing in vitro testing in a variety of domains such as “pharmacology and preclinical research.” In the case of searching the novel compounds of anticancer drugs, the in vitro process is most suitable and highly accepted by scientists. In vitro system allows an intense initial screening of possible anticancer medication without the risk of any damage. Curcumin is a secondary metabolite and bioactive component of the turmeric spice, which is obtained from the ground rhizome of the Curcuma longa plant, a member of the ginger family Zingiberaceae (Ewon & Bhagya, 2019). This complex molecule, having a broad range of pharmacological activities, for instance, anti-inflammatory, antioxidant, antibacterial, and anticancer properties, has been widely applied in Indian traditional medicine to prevent and treat various disorders (Rahmani et al., 2018; Sumathi et al., 2017;), for instance, combining P-gp-reversing substances like ningalin, ardeemin, and limonin with P-gp-substrate medications like vinblastine, paclitaxel, and doxorubicin (Chou, 2006; El-Readi et al., 2010). Multiple studies have shown that administering a larger dose of cytarabine is just as effective as an intermediate dose, but that doing so increases hematologic, neurologic, and organ toxicities without increasing antileukemic effects (Di Francia et al., 2021; Lo¨wenberg, 2013; Wu et al., 2017, Ye et al., 2021) Consequently, a medication or combination that generates a synergistic or additive impact and allows for a lower cytarabine dose is necessary. Putative mechanism of natural compounds in chemotherapeutic synergism was well studied by our group in which we find the synergism of hesperidin, silibinin, and curcumin in combination with standard chemotherapy drugs (cytarabine) in CD341 primary leukemic cells as well as in leukemic cell line THP-1. In our study, the use of silibinin and

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hesperidin showed 50% cell inhibition at 16.2 and 50.12 μM, respectively. On the other hand, the use of fixed doses of hesperidin and silibinin in conjunction with cytarabine at different concentrations reduced the IC50 value of cytarabine by 5.9 and 4.5 folds, respectively. Analyses of drug interactions revealed that silibinin and hesperidin displayed a synergistic effect with cytarabine in ratios ranging from 1:50 to 1:250 (Desai et al., 2015). Similar finding demonstrated that silibinin in combination with doxorubicin was used on colon carcinoma LoVo cell lines (Colombo et al., 2011). Lo¨wenberg et al. (2011) documented that a greater dose of cytarabine was just as effective as an intermediate dose; however, at larger doses, cytotoxicity increased. Therefore, alternative therapy or combination will be required which produces synergistic or additive effects and reduces the dose of cytarabine. Hence, our experimental data suggest that silibinin and hesperidin can be used alone or in combination as chemotherapeutic drugs to minimize cytotoxicity in leukemia patients. Hesperidin also known as hesperetin 7-rutinoside is an anticancer flavonoid (Ahmadi & Shadboorestan, 2016) and in combination with fisetin has been reported to inhibit cellular proliferation via triggering programmed cell death in acute promyelocytic leukemia HL-60 cells and K562 chronic myeloid leukemia cells through activation of caspase-3 and JAK/STAT pathway, and genes of JAK/STAT pathway have also been identified as candidates of CML therapy (Adan & Baran, 2015, 2016). For the first time, we were able to demonstrate the synergistic effects of cytarabine and curcumin on primary AML cells. Our findings show that curcumin treatment at various concentrations significantly reduces cell proliferation and curcumin treatment in combination with cytarabine also has a synergistic effect on primary AML cells. Reductions in MDR1, LRP, and BCRP averaged 35.75%, 31.30%, and 27.97%, respectively. The levels of FLT3-ITD and wild-type FLT3 also went down by 46.60% and 65.86%, respectively. This strongly suggests that curcumin can be used alone or in combination with current chemotherapy regimens to provide a synergistic impact and may be a promising chemosensitizer for modulating MDR in AML (Shah et al., 2016). In addition, it has been observed that dietary curcuminoids not only cause cell death in the human leukemia HL-60 cell line at a low concentration of 3.5 g/mL (Kuo et al., 1996), but also have cytotoxic effects on the leukemic cell lines K562, U937, and HL-60 (Duvoix et al., 2003). Curcumin being a chemosensitizer and reducing cytotoxicity when used in conjunction with Ara-C therapy can inhibit the mRNA expression of the multidrug-resistance genes, including MDR1, LRP, BCRP, and FLT3. We found that curcumin had the ability to downregulate the expression of MDR1, LRP, BCRP, and FLT3 in primary cultured cells. Consequently, in order to improve clinical outcomes associated with leukemia, it is crucial to develop a method to overcome drug resistance. The advantage of utilizing curcuminoids as MDR inhibitors is their comparatively broad therapeutic index when compared to the bulk of other third-generation MDR inhibitors. One way to make therapy for AML work better is to combine Ara-C with natural compounds like hesperidin, silibinin, and curcumin, which may make the cells more sensitive to the cytotoxic effects of the drugs. However, there is currently no published clinical trial data supporting the use of this approach. Curcumin inhibits the production of nitric oxide and has antileukemic effects. Study was conducted by Ghalaut and colleagues on curcumin and imatainib alone and in combination given to 50 patients of CML for 6 weeks. Nitric oxide levels were found to be significantly decreased in patients’ groups. This suggests that curcumin may aid in the treatment of CML by acting as an adjuvant to imatinib in reducing nitric oxide levels (Ghalaut et al., 2012). Amoora rohituka stem bark is one of the components of Canarib used in the Ayurvedic system of medicine in India for the treatment of human cancers. Amooranin is a triterpene acid with a unique structure isolated from the stem bark of A. rohituka that has anticancer effects both in vitro and in vivo (Rabi et al., 1994). In 2003, Ramachandran et al. demonstrated that amooranin has chemosensitizing effect on doxorubicin cytotoxicity and may be a competitive inhibitor of P-gp-mediated drug efflux. In multidrug-resistant leukemia (CEM/VLB) cell lines, amooranin and DOX combination showed modulation of DOX cytotoxicity. Apoptosis resistance obtained from members of the inhibitor of apoptosis proteins (IAPs), such as XIAP, and drug extrusion by P-glycoprotein (P-gp) overexpression are two processes that are connected to MDR. In 2012, deSouza et al. suggested that modulation of P-gp activity by using cyclosporine A did not induce cytotoxic effects in leukemia cells, independently of P-gp expression. However, under the modulation condition, they saw that vincristine significantly increased apoptosis in resistant cells, which was accompanied by a decline in the expression of the apoptosisinhibiting protein XIAP. Preclinical and clinical evidence suggests that phytochemicals can alter many signaling pathways involved in cancer genesis and progression. Combinations of phytochemicals with conventional chemotherapeutic drugs promote cell death, inhibit cell growth and invasion, sensitize malignant cells, and strengthen the immune system, making them compelling cancer therapy options. Combination therapy is more effective than single-agent therapy for preventing or treating cancer, whether the agents used are natural or chemical. Different in-vitro studies related to synergistic effects of phytochemicals with standard chemotherapeutic agents in leukaemia are summarised in Table 34.3.

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TABLE 34.3 In vitro synergistic effects of phytochemicals with conventional chemotherapeutic agents. Phytochemical

Source

Combination of phytochemical and drug

References

Hesperidin

Citrus fruits

Hesperidin 1 cytarabine

Desai et al. (2015)

Silibinin

Milk thistle, Silybum marianum.

Silibinin 1 cytarabine

Desai et al. (2015)

Curcumin

Curcuma longa

Curcumin 1 cytarabine

Shah et al. (2016)

Terpenoid

Amoora rohituka stem bark

Amooranin 1 doxorubicin

Ramachandran et al. (2003)

Alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol, aromadendrene, β-sitosterol-Oglucoside, and β-carotene)

Glaucium flavum, green tea, peppermint and other mint plants, yellow, orange, and green leafy fruits

Secondary metabolites 1 digitonin and doxorubicin

Eid et al. (2012)

Catharanthus roseus

Madagascar periwinkle plant.

Cyclosporine A 1 vincristine

de Souza et al. (2012)

Vinca alkaloids

Periwinkle plant, Catharanthus roseus (basionym Vinca rosea), and other vinca plants

Vinca alkaloids 1 combretastatin

Munayi (2016)

Ardisiacrispin B

Ardisia crispa plant

Ardisiacrispin B 1 cisplatin

More et al. (2021)

34.4.3 In vivo analysis of phytochemicals as multidrug resistance-reversing agents Our present understanding of tumor biology is greatly influenced by animal models of human malignancies. Animal models facilitated the identification and validation of therapeutic targets and biomarkers in preclinical oncology. As a result, cancer patients received better treatment. Technological advances in genetic engineering and single-cell “omics” provide a significant opportunity to enhance the informative value of preclinical models in the quest to understand and treat a wide range of cancer types. Papie˙z et al. (2016) reported that curcumin altered the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-L-cysteine significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In vitro results were verified on a BNML model. Curcumin pretreatment enhanced the antileukemic activity of etoposide drug in BNML rats and more effectively induced apoptosis in BNML cells than etoposide alone. However, it protected non-leukemic B cells from apoptosis (Papie˙z et al., 2012). Curcumin appears to boost etoposide’s proapoptotic activity in BNML cells in vivo. Similarly, in one other experimental study, curcumin reduced the growth of B6p210 and B6T315I cells and triggered apoptosis. Additionally, curcumin raised p53 levels while decreasing NF-kB levels. Curcumin reduced c-Abl levels in cells expressing the wild-type BCRABL oncogene but not the mutant variant. Additionally, curcumin therapy reduced the number of white blood and GFP cells while statistically significantly improving survival in diseased mice (William et al., 2008). Indole-3-carbinol is a natural product with antitumor properties already clinically tested. Indole-3-carbinol causes cytotoxicity in CLL cells but not in healthy lymphocytes. Across every CLL cell tested, such as those with p53 deficiency and/or F-ara-A resistance, indole-3-carbinol effectively synergized with F-ara-A. The cell death pathway included both p53-dependent and p53-independent apoptosis. IGHV mutation stage and patient resistance seemed to have no influence on the combination of indole-3-carbinol 1 F-ara-efficacy A’s in the CLL. Additionally, the combinatory indole-3-carbinol 1 F-ara-A therapy was able to overcome CLL survival and treatment resistance that was brought on by coculturing CLL cells on stroma cells. The combination indole-3-carbinol 1 fludarabine therapy did not cause any side effects in mice (Perez-Chacon). A natural sesquiterpene called parthenolide has been known to stimulate apoptosis in acute myeloid leukemia cells. The effects of parthenolide on leukemia-initiating cell types in pediatric ALL were examined by Diamanti et al. in 2013. Parthenolide was effective against bulk B- and T-ALL cells, according to apoptosis tests, but less so against the CD34(1)/CD19(2), CD34(1)/CD7(2), and CD34(2) subpopulations. Functional investigations showed that parthenolide

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TABLE 34.4 In vivo effects of phytochemicals as multidrug resistance reversals. Phytochemical

Source

Animal mode

References

Indole-3-carbinol

Broccoli, Brussels sprouts, cabbage, collards, cauliflower, kale, mustard greens, turnips, and rutabagas

C57bl/6 mice

Perez-Chacon et al. (2016)

PEITC

Turnips and radish

TCL1-Tg:p53 1 mice

Liu et al. (2016)

Allanxanthone C and macluraxanthone

Stem barks of Allanblackia monticola and Calophyllum inophyllum, Garcinia opaca, and other organisms

Xenograft murine model of human CLL

Loisel et al. (2010)

Parthenolide

Extract from Magnolia grandiflora

NOD/LtSz-scld IL-2Rγ cnull mice

Diamanti et al. (2013)

Curcumin

Curcuma longa

B6 mice

William et al. (2008)

Emodin

Plant resin usually obtained from the rhubarb

BALB/c nude mice

Chun-Guang et al. (2010)

Curcumin

Curcuma longa (Turmeric)

Brown Norway rats with acute myeloid leukemia (BNML)

Papie˙z et al., 2012

(2)-Epicatechin

Cocoa, dark chocolate, red wine, and tea

Brown Norway rats

Papie˙z et al., 2012

Quercetin

Citrus fruits, apples, onions, parsley, sage, tea, and red wine. Olive oil, grapes, dark cherries, and dark berries such as blueberries, blackberries, and bilberries

NOD.CB17-Prkdcscid/J mice

Maso et al. (2014)

Quercetin and green tea

Citrus fruits, apples, onions, parsley, sage, tea, and red wine. Olive oil, grapes, dark cherries, and dark berries

NOD/SCID mice

Calgarotto et al. (2018)

therapy in NOD/LtSz-scid IL-2R(c)-null mice blocked the engraftment of various leukemia-initiating cell populations. The effect of phytochemicals in vivo is depicted in Table 34.4. A natural anthraquinone derivative known as emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) that was extracted from the Rheum palmatum L. has been shown to have anticancer effects on a number of human malignancies, including liver cancer and lung cancer. Emodin showed antitumor action in K562 cells in vitro and in vivo by apoptosis induction, according to research by Chun-Guang and colleagues. The process involved increasing Bax and Bcl-2 gene expression as well as activating the caspase-3, caspase-8, and caspase-9 cascades. Another report showed that emodin alters the AMPK/mTOR signaling pathways and activates autophagy in rat renal tubular cells to reduce cisplatin-induced apoptosis in culture. For the treatment of cisplatin-induced nephrotoxicity, emodin may be useful (Liu et al., 2016). In a study published in 2010, Loisel et al. examined the effects of the two xanthones allanxanthone C and macluraxanthone on a murine xenograft model of human CLL that was created by implanting chronic leukemia B cells with the CD5 gene into SCID mice. In P39 leukemia cells, quercetin induced a significant amount of apoptosis, which was followed by the overexpression of Bax, the upregulation of Bcl-2, the downregulation of Mcl-1, the release of cytochrome c, and the activation of caspases. Additionally, quercetin increased FasL protein expression (Maso et al., 2014). Additionally, quercetin treatment in vivo dramatically decreased tumor volume in P39 xenografts and supported in vitro findings concerning apoptosis, autophagy, and cell cycle arrest. According to the findings of the study, quercetin has anticancer activity both in vitro and in vivo, making it a promising antitumor drug for hematologic malignancies (Maso et al., 2014).

34.5

Conclusions and future prospects

Natural products are becoming more and more popular because of their affordability and superiority over conventional medications’ adverse effects. Bioactive phytochemicals and formulations might be used as a starting point to create safer anticancer drugs. The majority of today’s pharmaceuticals and medicinal plants are developed in India.

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Their potential as an anticancer agent must be investigated. For this objective, many plants and their constituent phytochemicals have been examined, but only a very small number have advanced to the clinical stage. It is necessary to turn them into druggable forms with adequate bioavailability. Therefore, therapeutic intervention based on the combination of anticancer agents may provide strong and beneficial therapeutic effects. Many phytochemicals, including alkaloids, flavonoids, curcuminoids, stilbenoids, terpenes, carotenoids, lignans, and polyketides, have been studied for MDRreversing action. The fact that phytochemicals typically have low cytotoxicity in the human body makes them a possible source of adjuvant chemicals against cancer. Treatment for multidrug-resistant cancer types may be improved by using MDR-reversing phytochemicals as adjuvants in conjunction with anticancer drugs. In the future, natural product analysis using nanotechnology and analytical techniques will be a prominent tool for locating biologically active chemicals with distinctive structures and modes of action. Natural product analysis, which combines nanotechnology and analytical approaches, is a leading strategy for discovering physiologically active compounds with different structures and modes of activity. The multitarget strategy for drug development has created an appealing niche for medical scientists since it lessens the burden of the multidrug strategy for cancer care as well as the side effects associated with it. Natural phytochemicals are still a viable alternative to synthetic scaffolds because they have a wide range of anticancer activity and high structural diversity, both of which may be employed either directly or as building blocks for novel therapies. These natural compounds have tremendous theranostic potential, and there will be a lot of future studies into natural theranostic agents.

Acknowledgment Among all authors, Dr. Medha Pandya thankfully acknowledges Dr. Bharatsinh M Gohil Head, Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, for kind support.

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39. Aris, P. (2000). Cancer multidrug resistance. Nature Biotechnology, 18, IT18-20. Bonnet, D., & Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Medicine, 3(7), 730
737. Britschgi, A., Simon, H. U., Tobler, A., Fey, M. F., & Tschan, M. P. (2010). Epigallocatechin-3-gallate induces cell death in acute myeloid leukaemia cells and supports all-trans retinoic acid-induced neutrophil differentiation via death-associated protein kinase 2. British Journal of Haematology, 149(1), 55
64. Brown, P. C., Thorgeirsson, S. S., & Silverman, J. A. (1993). Cloning and regulation of the rat mdr2 gene. Nucleic Acids Research, 21(16), 3885
3891. Bukowski, K., Kciuk, M., & Kontek, R. (2020). Mechanisms of multidrug resistance in cancer chemotherapy. International Journal of Molecular Sciences, 21(9), 3233. Bulusu, K. C., Guha, R., Mason, D. J., Lewis, R. P., Muratov, E., Motamedi, Y. K., . . . Bender, A. (2016). Modelling of compound combination effects and applications to efficacy and toxicity: State-of-the-art, challenges and perspectives. Drug Discovery Today, 21(2), 225
238. Caesar, L. K., & Cech, N. B. (2019). Synergy and antagonism in natural product extracts: When 1 1 1 does not equal 2. Natural Product Reports, 36 (6), 869
888. Calgarotto, A. K., Maso, V., Junior, G. C. F., Nowill, A. E., Vassallo, J., & Saad, S. T. O. (2018). Antitumor activities of quercetin and green tea in xenografts of human leukemia HL60 cells. Scientific Reports, 8(1), 1
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Chapter 35

Phytochemical and bioactive potentialities of Melastoma malabathricum Mansi Tiwari, Mridula Saikia Barooah and Deepjyoti Bhuyan Department of Food Science and Nutrition, College of Community Science, Assam Agricultural University, Jorhat, Assam, India

35.1

Introduction

Melastoma malabathricum, commonly known as Malabar melastome or Indian rhododendron, is a shrub native to South Asian region. Locally, it is also known by several other names such as Senduduk in Malay community, Phutukola in Assamese dialect, Bit-Bit in Karbi, Thung-khu in Bodo, Phutuki in Bengali, Bol khakhu or Khakhuchi among Garo, Ankarke in Kannada, Athirani in Malayalam, Lakeri in Marathi, Bui-lu-kham in Mizo, and Angeri or Chulesi in Nepali. The term “Melastoma” originated from a Greek word meaning “black mouth.” It was named so because the fruit pulp is often eaten by children which stains mouth and tongue black (Koay, 2008). It is an evergreen plant which reaches an average height of 1.80
2.90 m with a spread of around 2.00 m. The plant has an erected, branched, slender, and scabrous stem which is covered densely with minute, rigid, appressed, and ciliate scales. The leaves are petiolate, narrow with blades narrowly tapered at both ends. The surface of leaf is slightly rough in texture, especially on the adaxial part. They typically have a length of 5
12 cm with 3
5 longitudinal veins. The plant has flower with color ranging from pink to violet or mauve which is imparted by the anthocyanin pigments present in it. It has 5 petals and 10 stamens of different kinds. Of the stamens, five are larger in size with yellow filament and purple colored curved upper part, while the other five are small yellow colored straight filament with yellow anther (Susanti et al., 2007). The fruits of plant are encapsulated berries covered with scaly, bristle-tipped calyx containing many minute non-endospermous seeds having red, sweet, astringent pulp (Adams, 1972; Bodkin, 1991). M. malabathricum is the only species of the genus “Melastoma” that has been classified as a weed and is found extensively growing in the roadside, wasteland areas, and previously cleared land (Valkenberg & Bunyapraphatsara, 2001). The plant also possesses a unique ability to grow in poor nutrient soil such as that with phosphorus deficiency and acidic nature (Figs. 35.1
35.3). Since ages, the plant has been used in many traditional medicines (Rajenderan, 2010). Different plant parts such as leaves, roots, barks, stems, and fruits of the species have been recognized in Malay, Indian, and Indonesian folk medicines owing to its ethno-medicinal properties for the treatment of ailments such as diarrhea, dysentery, cuts and wounds, skin infections, stomach ulcer, scarring of smallpox, and piles (Begum & Nath, 2000; Ong & Norzalina, 1999). There are numerous research findings concerning the presence of many phytochemical constituents in plants mainly polyphenol and tannin. Apart from these, other phytochemical constituents such as saponins, flavonoids, triterpenes, flavan-3ols, anthocyanins, ellagic acid, cyanidin, malvidin-3,5-diglucoside, β-sitosterol, ursolic acid, 2-hydroxyursolic acid, gallic acid, kaempferol, malabathrin B, malabathrin C, malabathrin D, strictinin, (2)-epicatechin, quercetin, quercitrin, and rutin (Das & Kotoky, 1988; Dinda & Saha, 1986, 1988; Lowry, 1968, 1976; Manzoor-I-Khuda et al., 1981; Nazlina et al., 2008; Nuresti et al., 2003; Susanti et al., 2008; Wong et al., 2004; Yoshida, Nakata, Hosotani, Nitta, et al., 1992; Yoshida, Nakata, Hosotani, Okuda, et al., 1992) have also been reported. The plant exhibits several bioactivities such as antioxidant (Marjoni & Zulfisa, 2017; Zakaria et al., 2011), antibacterial (Choudhury et al., 2011; Diris et al., 2016; Omar et al., 2012), antiviral (Nazlina et al., 2008; Zakaria et al., 2006), anti-cancerous (Balamurugan et al., 2013; Kumar et al., 2021; Roslen et al., 2014), anti-inflammatory (Kumar et al., 2016), antipyretic (Zakaria et al., 2006), antiulcerogenic (Halim et al., 2017; Zabidi et al., 2012; Zainulddin et al., 2016), anticoagulant (Caroline et al., 2010; Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00024-4 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 35.1 Melastoma malabathricum leaf. Leaves of M. malabathricum showing its longitudinal vein.

FIGURE 35.2 Melastoma malabathricum flower. Flower of plant M. malabathricum showing its petals and sepals.

Khoo et al., 2014), and hepatoprotective effects (Amin, 2013; Kamisan et al., 2013; Mamat et al., 2013) which are evident from the pharmacological and clinical trials. Therefore, in this chapter an attempt has been made to comprehend the scientific evidence relating to ethno-medicinal and pharmacological potentialities of the plant.

35.2

Ethno-medicinal practices

Traditional medicines have been used in the treatment of human ailments since ages. As per the World Health Organization, a traditional medicine is defined as “The sum total of the knowledge, skill, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness.” In literature, there is evidence suggesting numerous plants being used traditionally for healing purpose, owing to its efficacy against various diseases with little or no side effects. The plant M. malabathricum has been used in many traditional folklore practices by indigenous tribes and communities due to its ethno-medicinal potency. Different parts of the plant, that is leaves, flowers, roots, shoots, and barks, have been used as ethnomedicines (Table 35.1) for the treatment of ailments such

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FIGURE 35.3 Melastoma malabathricum fruit. Unripe fruits of plant M. malabathricum.

TABLE 35.1 Ethno-medicinal uses of various parts of Melastoma malabathricum in different countries/regions. Plant parts

Country/region

Medicinal uses

References

Leaves and roots

China, India

Leaves and roots are used in the treatment of diarrhea, dysentery, wound healing, scarring, homeostasis. Root decoction has been used to cure excessive vaginal discharge and irregular menstruation.

Burkill (1966), Editorial Committee of Flora of China (1984), Kumar et al. (2013)

Leaves

India, Malaysia, Indonesia

Ground leaves in form of paste are effective on cuts and wounds to stop bleeding. Liquid of boiled leaf is effective in diarrheal treatment

Burkill (1966), Indu and Razali (1998), Jaganath and Ng (2000), Latiff and Zakri (2000)

Young leaves

Malaysia

Young leaf consumption on empty stomach twice daily helps in relieving diarrhea and dysentery

Indu and Razali (1998), Lin (2005), Jaganath and Ng (2000), Sajem and Gosai (2006)

Leaves and roots

Indonesia

Leaves and roots are used to treat dysentery, diarrhea, hemorrhoids, leukorrhea, wounds and cut; to treat infection during confinement and to prevent scarring of smallpox and piles.

Kumar et al. (2013), Yoshida, Nakata, Hosotani, Okuda et al. (1992)

Shoots and flower

India

Use of plant shoot as toothbrush relieves toothache. Flower is used for treating stomach ache and cancer

Mishra et al. (2016)

Leaves, shoots, and roots

India

Leaves are chewed and applied in cuts and wounds due to their blood-coagulating property. Dried leaves are used to provide prevention against scarring due to smallpox, piles, gastric ulcer, dysentery, and also as a tonic. Oral intake of shoots is known to treat puerperal infection, high blood pressure, and diabetes. Root of plant finds application in the therapy for rheumatism, arthritis, toothache, and epilepsy.

Begum and Nath (2000), Jaganath and Ng (2000), Kumar et al. (2016)

(Continued )

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TABLE 35.1 (Continued) Plant parts

Country/region

Medicinal uses

References

Leaves and roots

Malaysia

Leaves and roots have been used to treat dysentery, diarrhea, hemorrhoids, leukorrhea, wounds and cuts. They are also used as treatment against infection during confinement.

Kumar et al. (2013), Ong and Norzalina (1999), Begum and Nath (2000)

Flowers

India

Flowers are applied as a treatment for sore mouth in children

Paul et al. (2011)

Whole plant and roots

Malaysia

Used in toning up of uterus, strengthening womb, and accelerating the healing process after childbirth

Indu and Razali (1998), Jaganath and Ng (2000)

Leaves and roots

Bangladesh

Leaf juice is used as a diuretic and in the treatment of various urinary disorders. Root juice or water extract of roots is used as a cure against leukorrhea and jaundice

Rahmatullah et al. (2009)

Bark

Tahiti

Bark decoction is used as a gargle to treat diarrhea, dysentery, and several skin infections.

Umali-Stuart and StiuartSantiago (2010), Jain and Filipps (1991)

Whole plant

Taiwan region

Whole herb is utilized to treat traumatic injury, bacterial dysentery, and cleanse the serum toxins.

Wang et al. (2008)

Flower, seeds, and leaves

Malaysia

A combination of flower, seeds, and leaves are used in the reduction of vaginal discharge and indigestion

Indu and Razali (1998), Jaganath and Ng (2000)

Leaves

India

Leaves in fresh or dried forms are used as antipyretics, wound healer, and gastro-protector.

Ringmichon et al. (2010)

Leaves and flower

Malaysia, India, China

Flower and leaf combination has been effective in the treatment of cholera, diarrhea, prolonged fever, dysentery, leukorrhea, wounds, and skin diseases.

Burkill (1966), Koay (2008), Perry (1980), Sharma et al. (2001)

Flower

Philippine

Used to treat cancer, hemorrhoidal bleeding, and as a nervous sedative.

Burkill (1966), Koay (2008), Perry (1980), Sharma et al. (2001)

Flower

Indonesia

Flower is used as an optional ingredient treatment for anemia associated with gastrointestinal bleeding and epigastric pain.

Elliott and Brimacombe (1987)

Leaves

Bangladesh

The leaf juice functions as a diuretic and against urinary infections

Rahmatullah et al. (2009)

as diarrhea, dysentery, leukorrhea, ulcer, skin infection, cuts and wounds, etc., (Begum & Nath, 2000; Bharadwaj & Gakhar, 2005; Yao & Liu, 2010). The seed of plant is being used widely in the Chinese preparation of “Poh chi” pills for diarrheal treatment (Tan & Yeo, 2009). The roots and tender plant leaves are used in the preparation of decoction which is beneficial for treating diarrhea (Indu & Razali, 1998; Lin, 2005). Traditionally, a handful of fresh leaves consumed raw has been found effective against dysentery (Sajem & Gosai, 2006). The shrub M. malabathricum is used in postpartum toning up of the uterus for wound strengthening and accelerating the healing process. Flower, seeds, and leaves have been used together in traditional medicine to reduce excessive vaginal discharge and indigestion (Indu and Razali, 1998). In the Malaysian region, young premature leaves are taken orally twice a day on an empty stomach to cure dysentery (Sajem & Gosai, 2006). The Bodo tribes of Assam use flower of the plant for application on sore mouth of children (Paul et al., 2011). Consumption of flower has also been proven beneficial in the treatment of stomach ache and cancer (Mishra et al., 2016). Several records of the utility of roots and leaves in the treatment of diarrhea, dysentery, ulcer, wound, scar formation, and postpartum complications (Kumar et al., 2013) are also present in traditional systems of medicine. In parts of Indian states, raw leaves of M. malabathricum are chewed and applied to cuts or wounds to stop bleeding due to their blood-coagulating property (Kumar et al., 2016). In traditional Chinese medication, decoction of fresh root is prepared which is used to treat leukorrhea and excessive bleeding during menstrual cycle (Liu & Fu, 2008). The local dwellers of Dibrugarh district of Assam, India, applies roots of M. malabathricum during rheumatoid therapy (Kumar et al., 2016). In Manipur, the Naga people use the freshly dried leaves as an ailment in cuts and wounds, fever, and stomach disorders (Ringmichon et al., 2010). Dehydrated leaves and root powder are applied by indigenous people to cure scar caused due to pox, quick expedition of healing process, and ease pain caused by hemorrhoid (Begum & Nath, 2000;

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Kumar et al., 2013; Ong & Norzalina, 1999). In Indonesia and Malaysia, leaves of plant have been used as a popular drug called “daun halendong” for treating various ailments such as diarrhea, dysentery, ulcer, leukorrhea, cuts and wounds (Wong et al., 2011; Yoshida, Nakata, Hosotani, Nitta, et al., 1992; Yoshida, Nakata, Hosotani, Okuda, et al., 1992).

35.3

Phytochemical constituents

In present days, the number of people turning to traditional medicines as alternative to conventional therapeutic drugs is increasing rapidly. In view of this, research investigation aiming at identifying the potential phytochemicals and their pharmacological potency is also increasing. The shrub M. malabathricum is chemically diverse in nature and has gained importance in recent years due to its ethno-medicinal properties. A number of compounds including tannins, flavonoids, steroids, organic acids, etc., have been isolated and identified by researchers. Flavanoids, tannins, and organic acids have been recognized as the principal active constituents in Melastoma spp. (Yao & Liu, 2010). Studies have reported the presence of fatty acids and aliphatic constituents (Das & Kotoky, 1988; Dinda & Saha, 1986), phenolic acids (Lowry, 1968), tannins (Alwash et al., 2013; Yoshida, Nakata, Hosotani, Nitta, et al., 1992; Yoshida, Nakata, Hosotani, Okuda, et al., 1992), flavonoids (Dinda & Saha, 1988; Wong et al., 2011), anthocyanin (Lowry, 1976), amino acids (Dinda & Saha, 1985), etc., in different parts of the plant. The phytochemical constituents of leaf crude extract of M. malabathricum were studied using methanol crude extract of leaf. The extract was identified for its phytochemical constituents by gas chromatography
mass spectrophotometry (GC/Ms). A total of ten phytochemical constituents accounting for 59.2% of relative area were identified. The phytochemical compounds 5-hydroxymethyl furfural, pyrogallol, phytol, palmitic acid methyl ester, palmitic acid, 8,11-octadecadienoic acid methyl ester, stearic acid, methyl ester, trans-squalene, and tocopherol were identified in the study from methanolic crude leaf extract (Diris et al., 2016). Alwash et al. (2013) reported the isolation of kaempferol-3-O-(2",6"-di-O-trans-p-coumaroyl)-β-D-glucopyranoside from methanolic leaf extract. The identification of phytochemicals in plant started long back. In 1968, Lowry identified the presence of ellagic acid in the methanolic extract of M. malabathricum bark. The anthocyanin compound malvidin-3,5-diglucoside was also detected by Lowry from the aqueous extract of M. malabathricum flower (Lowry, 1968). Later, another anthocyanin compound (cyanidin-(Cy-) 3-glucoside and Cy-3,5-diglucoside) was also screened in the water extract of M. malabathricum fruit (Lowry, 1976). Several other past studies have demonstrated the presence of 40-methylpeonidin 7-O-D-glucoside (Mohandoss & Ravindran, 1993), kaempferol (Das & Kotoky, 1988; Mohandoss & Ravindran, 1993; Susanti et al., 2007), kaempferol 3-O
D-xyloside (Mohandoss & Ravindran, 1993), kaempferol 3-O-glucoside, kaempferol 3-O(200,600-di-O-p-trans-coumaroyl) glucoside and naringenin (Susanti et al., 2007), p-hydroxybenzoic acid and gallic acid (Das & Kotoky, 1988) in the flowers, b-sitosterol and melastomic acid in the roots (Manzoor-I-Khuda et al., 1981), quercetin 3-O-L-rhamnosyl-(1
2)-D-galactoside (Dinda & Saha, 1988), 1-octyl docosanoate, 11-methyl-1-tritriacontanol (Dinda & Saha, 1986) in the aerial parts, 32-methyl-1-tritriacontanol, sitosterol, ursolic acid (Das & Kotoky, 1988), and several tannins (Yoshida, Nakata, Hosotani, Nitta, et al., 1992; Yoshida, Nakata, Hosotani, Okuda, et al., 1992) in the dried leaves (Table 35.2). The screening of hexane, dichloromethane, ethyl acetate, methanol, and 50% aqueous methanol extract of leaf, flower, and fruit of M. malabathricum was done for estimation of different phytochemical constituents present in the study of Giri & Rajbhandari, 2018. The phytochemical screening showed the presence of phenolics, flavonoids, and tannins in the ethyl acetate, methanol, and 50% aqueous methanol extract only, and glycosides were reported only in the 50% methanol extract of fruit. Mamat et al. (2013) screened the phytochemical constituents using HPLC technique and demonstrated the presence of flavonoid, triterpene, tannin, saponins, and steroid. Several tannins such as the hydrolyzable tannin oligomers (nobotanin B, malabathrins B, malabathrins C, malabathrins D), the hydrolysable tannin monomers (1,4,6-tri-O-galloyl-b-D-glucose, 1,2,4,6-tetra-O-galloyl-b-D-glucose, strictinin, casuarictin, pedunculagin, nobotanin D, pterocarinin) have been reported to be isolated from the dry leaves of M. malabathricum (Yoshida, Nakata, Hosotani, Nitta, et al., 1992; Yoshida, Nakata, Hosotani, Okuda, et al., 1992). Nuresti et al. (2003) reported the presence of β-sitosterol, α-amyrin, uvaol, sitosterol 3-O-b-D-glucopyranoside, quercetin, quercitrin, and rutin in the light pink magenta petals of M. malabathricum. The phytochemical constituents of M. malabathricum flower were also isolated by Susanti et al. (2007) in which the phytochemicals were identified and isolated using ESR spectrophotometry method. The ethyl acetate extract of flower yielded compounds which were identified as naringenin, kaempferol, and kaempferol-3-O-D-glucoside, and the methanol extract yielded kaempferol-3-O-(200,600-di-O-p-trans-coumaroyl) glucoside and kaempferol-3-O-Dglucoside. Of these compounds isolated, naringenin and kaempferol-3-O-(200,600-di-O-p-trans-coumaroyl) glucoside showed biological activity against cell proliferation of MCF7.

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TABLE 35.2 List of phytochemical compounds isolated from different parts of Melastoma malabathricum. Compound name

Plant part

Type of extract

References

5-hydroxymethyl furfural, pyrogallol, phytol, palmitic acid methyl ester, palmitic acid, 8,11-octadecadienoic acid methyl ester, stearic acid methyl ester, transsqualene, tocopherol

Leaves

Methanolic

Diris et al. (2016)

Kaempferol-3-O-(2v,6v-di-O-trans-p-coumaroyl)-β-Dglucopyranoside

Leaves

Methanolic

Alwash et al. (2013)

Phenolics, flavanoids, tannins

Leaves Flower Fruits

Ethyl acetate, methanol, and 50% aqueous methanol

Giri & Rajbhandari (2018)

Flavanoid, triterpenes, tannins, saponin, steroids

Leaves

Methanol extract

Mamat et al. (2013)

Naringenin, kaempferol, kaempferol-3-O-D-glucoside

Flower

Ethyl acetate extract

Susanti et al. (2007)

Kaempferol-3-O-(200,600-di-O-p-trans-coumaroyl) glucoside, kaempferol-3-O-D-glucoside

Flower

Methanol extract

Susanti et al. (2007)

Cyanidin-(Cy-) 3-glucoside, Cy-3,5-diglucoside

Fruit

Water extract

Lowry (1976)

Kaempferol, kaempferol-3-O-β-D-glucopyranoside, kaempferol-3-O-α-L-rhamnopyranoside, kaempferol 3O-β-D-galactopyranoside, quercetin, kaempferol 3-O(2,v6v-di-O-E-p-coumaryl)-β-D-galactopyranoside, ellagic acid

Flower

Ethyl acetate-soluble part of 90% aqueous methanolic extract

Ali et al. (2001), Wong et al. (2004)

β-Sitosterol-3-O-β-D-glucopyranoside, asiatic acid, glycerol, 1,2-dilinolenyl-3-O-β-D-galactopyranoside, glycerol, 1,2-dilinolenyl-3-O-(4,6-O-isopropylidene)β-D-galactopyranoside, 2-hydroxyursolic acid

Leaves

90% aqueous methanolic extract

Ali et al. (2010)

Malabathrins B, C, D, isoquercitrin 6v-0-gallate 1,4,6tri-0-galloyl
D-glucose, 1,2,4,6-tetra-O-galloyl-b-Dglucose, strictinin, pedunculagin, casuarictin, pterocarinin C, nobotanins B, D, G, H, J

Leaves

70% acetone extract

Yoshida, Nakata, Hosotani, Nitta, et al. (1992)

Malabathrins A, E, and F

Leaves

70% acetone extract

Yoshida, Nakata, Hosotani, Okuda, et al. (1992)

β-sitosterol, melastomic acid

Roots

Ethanol extract

Manzoor-I-Khuda et al. (1981)

Quercetin-3-O-α-L rhamnosyl-(1 - 2)-α-D galactoside, 4-methylpeonidin-7-O-β-D-glucoside, 1octyl decanoate A, kaempferol-3-Oβ-D-xyloside

Aerial




Dinda and Saha (1988)

Kaempferol-3-Oβ-D glucoside A, naringenin

Flower

Ethyl acetate extract

Koay (2008)

Kaempferol-3-Oβ-D glucoside A, kaempferol-3-O(2,6- di-O-p-trans-coumaroyl)-β-glucoside

Leaves

Methanol extract

Koay (2008)

α-Amyrin A

Stem

n-Hexane extract

Koay (2008)

Betulinic acid, serrat-14-en-16-one 2-(2-hydroxyvinyl)1-methyl-4-propoxyphthalate

roots

n-Hexane extract

Koay (2008)

Betulinic acid

Fruit

Ethyl acetate extract

Koay (2008)

2,5,6-Trihydroxynaphtoic carbonic acid, methyl-2,5,6trihydroxynaphthalene carbonate, flavonol glycoside derivative

Leaves

n-Hexane, ethyl acetate, and methanol extracts

Koay (2008)

4-O-caffeoylquinic acid, quercimeritin, digiprolactone, 3-O-trans-coumaroylquinic acid, artemisinin, norbergenin

Leaves

Water extract

Lestari et al. (2022)

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The urs-12-ene pentacyclic triterpenoids, ursolic acid, 2α-hydroxyursolic acid, asiatic acid, β-sitosterol 3-O-β-D glucopyranoside, glycerol 1,2-dilinolenyl-3-O-β-D-galactopyranoside, and glycerol 1,2-dilinolenyl-3-O-(4,6-O-iso-propylidene)-β-D-galactopyranoside were isolated in the study of Ali et al. (2010) from the 90% aqueous methanolic extracts of fresh leaves of M. malabathricum. The ethyl acetate-soluble portion of 90.00% aqueous methanolic extract of M. malabathricum flower was subjected to isolation and identification of phytochemicals in which the compounds ellagic acid, quercetin, kaempferol, kaempferol 3-O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranoside, kaempferol 3-O-(2,v6v-di-O-E-p-coumaryl)-β-D-galactopyranoside, and kaempferol 3-O-β-D-galactopyranoside were identified (Ali et al., 2001; Wong et al., 2004). Yoshida, Nakata, Hosotani, Nitta, et al. (1992) cited the isolation of 3-dimeric hydrolyzable tannins, namely, malabathrins B (9), malabathrins C (14), and malabathrins D (17). A flavonoid, glycoside (isoquercitrin 6v-0-gallate), and 11 hydrolyzable tannins were also isolated. Among the 11 hydrolyzable tannins that were isolated, 7 of them were monomeric hydrolyzable tannins, that is 1,4,6-tri-0-galloyl
D-glucose, 1,2,4,6-tetra-Ogalloyl-b-D-glucose, strictinin, casuarictin, pedunculagin, nobotanin D, and pterocarinin C. Apart from these, other four tannins identified were hydrolyzable tannin oligomers, that is nobotanins B, nobotanins G, nobotanins H, and nobotanins J. Further investigation of the leaf extract led to the isolation of another seven C-glucosidic ellagitannins including three new complex tannins, namely, malabathrins A, E, and F (Yoshida, Nakata, Hosotani, Okuda, et al., 1992). Koay (2008) in their research investigation cited the presence of 2,5,6-trihydroxynaphtoic carbonic acid, flavonol glycoside derivative, and methyl-2,5,6-trihydroxynaphtalene carbonate in the n-hexane, ethyl acetate, and methanol extracts of M. malabathricum leaves. Betulinic acid, serrat-14-en-16-one, and 2-(2-hydroxyvinyl)-1-methyl-4-propo-xyphthalate were identified in the n-hexane extract of M. malabathricum roots. There are reports on the yield of compounds kaempferol-3-O-β-D-glucoside, kaempferol, and naringenin from ethyl acetate extract of M. malabathricum flowers and kaempferol-3-O-(2,v6v-di-O-p-transcoumaroyl)-glucoside and kaempferol-3-O-β-D-glucoside from the methanol extract of M. malabathricum flowers. Additionally, betulinic acid has been reported in ethyl acetate extract of M. malabathricum fruits. Apart from the above-reported components, the presence of some essential amino acids (Asp, Leu, Tyr, Phe, His, Lys, Thr, Ser, Glu, Pro, Gly, Ala, Val, Met, Trp, and Arg) were also seen in the study of Dinda and Saha (1988) and Yeoh et al. (1992) in the leaf of M. malabathricum.

35.4

Pharmacological potentialities

In the present times, many modern drugs have been developed and are also proving to be effective against many ailments. They, however, in addition to health ameliorating potential also pose several side effects and health hazards in long run. Therefore, many modern-day researches are aiming to explore a wide range of plants and their healthpromoting role as a substitute for modern pharmacopeia. Many in vitro and in vivo studies have been conducted to study the pharmacological potency of M. malabathricum which have been highlighted under this section.

35.4.1 Antioxidative potential The antioxidative effects of many plant parts are under investigation. The oxidative alteration caused by the reactive oxygen species in the protein, lipid, DNA, and a wide range of cellular components are responsible for a wide range of diseases such as neurodegenerative disease, wrinkled skin, DNA damage, cardiovascular disease, carcinogenesis, etc. (Takashi et al., 2007). Studies have shown the extract of M. malabathricum to be effective against free radical damage, thereby delaying or stopping the onset of many diseased conditions. The antioxidative effect of M. malabathricum leaves was studied by Karupiah and Ismail (2013). The antioxidant potential of leaves was determined by β-carotene lineolate bleaching method using three different solvent extracts (methanolic extract, ethyl acetate extract, and ethanolic extract). During this estimation process, hydroperoxide free radical is produced by linoleic acid which attacks the β-carotene molecules leading to a rapid discoloration. In presence of any antioxidant compound in the system, this β-carotene discoloration can be stopped by its action on the free radicals produced. All three solvent extracts of M. malabathricum showed significant antioxidant activity with the highest activity shown by chloroform extract followed by methanol and ethyl acetate extract in the range of 44.41% to 83.28%. The polyphenolic compounds such as quercetin, quercetrin, rutin, kaempferol, gallic acid which are known for their antioxidative potency might be responsible for the free radical scavenging actions. The activity was comparable with the commercial antioxidant, thus making it a potential natural remedy against oxidative damage. With the aim to determine the chemical constituents and possible potential of M. malabathricum as an antioxidant, a research investigation was carried out by Ismail et al. (2021). The antioxidant potency of crude ethanol extract of M. malabathricum leaf was investigated by its DPPH radical scavenging action taking five different concentrations of

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extracts, that is 100, 200, 300, 400, and 500 ppm. In the study, vitamin C and butylated hydroxy anisole (BHA) were used as standard during estimation. A dose-dependent increase in DPPH scavenging activity was recorded which was comparable to the standard vitamin C and BHA. This potent antioxidant activity was attributed to the flavonoid and tannin content of leaf extract. Water extract from M. malabathricum leaf has shown to exhibit potential antioxidant activity in the research findings of Lestari et al. (2022). In the study, the leaves of M. malabathricum were extracted at different temperatures of 25 C 6 2 C and 90 C 6 2 C, extract concentration of 10 and 5 g leaves/100 mL water, and period of 15, 30, and 45 minutes. The maximum antioxidant activity was recorded by the extract prepared from M. malabathricum leaves at concentration 5 g leaves/100 mL of water at 90 C 6 2 C for 15 minutes. The antioxidant activity recorded in the study was higher than that obtained for vitamin C indicating a strong antioxidative potential of M. malabathricum water extract which was attributed to the presence of phytochemicals gallic acid, 4-O-caffeoylquinic acid, 3-O-transcoumaroylqinic acid, quercetin, quercimeritrin, norberginin, artemisinin I, and digiprolactone.

35.4.2 Antimicrobial potential M. malabathricum has demonstrated positive effects against various microbial strain. Das et al. (2021) in their study evaluated the antimicrobial activity and minimum inhibitory concentration (MIC) of aqueous and methanolic extract of leaf, flower, fruit, and stem against two major bacterial pathogens, Escherichia coli and Staphylococcus aureus, involved in human health by agar well diffusion method and 96-well microtiter plate method. The methanolic leaf extract of M. malabathricum showed antimicrobial activity against E. coli but not against S. aureus while the methanolic extract of flower showed antimicrobial activity against S. aureus and not E. coli. In the study, the greater inhibition was shown by the flower extract of M. malabathricum which was mainly attributed to the presence of active components like kaempferol-3-O-β-D-glucoside, kaempferol, and naringenin (Susanti et al., 2007). The leaf M. malabathricum has been reported to exhibit antibacterial effect against bacteria Salmonella typhi ATCC 14028, E. coli ATCC 25922, and S. aureus ATCC 25923. The testing of antibacterial activity was done using disk diffusion method in which zone of inhibition was measured. The ethanol extract of leaves showed greater activity against S. typhi compared to flower extract which showed maximum activity against E. coli. The difference in the activity was due to different phytoconstituents present in both the extracts. Flavanoid screened the extracts that have the ability to form complex with soluble extracellular protein and bacterial cells (Bilal et al., 2017). Flavanoid functions by inhibiting nucleic acid synthesis, cytoplasmic membrane function, and energy metabolism (Maftuch et al., 2016). Alkaloids present in the extract also exhibit antibacterial property by forming an intercellate with double-helix DNA and uncoupling respiration (Bilal et al., 2017). Tannin has been reported to be associated with the inactivation of microbial adhesion, including bacterial filament toxicity and binding to protein wall for bacterial growth inhibition (Pandey & Kumar, 2013). Omar et al. (2012) studied the antimicrobial potency of methanolic crude extract of M. malabathricum fruit and flower against a total of 32 microbial species. The species employed were a group of gram-positive bacteria, gramnegative bacteria, and fungi which are involved in food spoilage. The testing of antimicrobial activity was performed using the disk diffusion method, and those species showing growth inhibition zone were further subjected to minimum inhibitory concentration determination. In the study, greater effectiveness of the extracts was seen against gram-positive bacteria compared to the gram-negative bacteria, and no activity demonstrated against fungal species tested. The high sensitivity to the gram-positive bacteria may be attributed to their cell wall and membrane structure and composition (Shan et al., 2007). The outer membrane of gram-negative bacteria is composed of lipopolysaccharide molecule which acts as a barrier to certain antibiotic penetration. Gram-negative bacteria also have enzymes in the periplasmic space which is capable of breaking down foreign molecule (Duffy & Power, 2001). Unlike gram-negative bacteria, grampositive bacteria do not have any specialized cell membrane and cell wall structure, thus offering ease in the entry of the antibacterial substances and thereby destroying cell wall and cytoplasmic materials (Gao et al., 1999).

35.4.3 Wound-healing potential There are reports of M. malabathricum being used in traditional medicine in the treatment of wound (Joffry et al., 2012). Nurdiana and Marziana (2013) studied the wound-healing property of aqueous extract of M. malabathricum in Sprague Dawley rats. For the study purpose, 12 rats were selected and were separated into 4 groups with each group having a total of 3 number of rats. The rats were allowed to acclimatize to the laboratory condition for 7 days and then were excised with around 1 cm2 area on the back with a scalpel. The group of rats was then treated to check the

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wound-healing potency of different test compounds. The first group was treated with aqueous extract of M. malabathricum, second with commercially available flavin (positive control group), third with poviderm (positive control group), and the fourth group was subjected to saline administration (negative control group). The treatment to all the groups was given for 2 weeks, and the wound area was examined periodically at an interval of 3 days. The rate of wound contraction showed the highest percentage in M. malabathricum-treated group (93%), followed by the group treated with flavin (88%), poviderm (86%), and saline (77%), respectively. The high percentage of wound concentration of M. malabathricum was due to the presence of flavonoid sand tannins, both of which have anti-inflammatory and antimicrobial properties. These constituents also have the ability to control microbial colonization which might be responsible for wound healing in rats (Sunilson et al., 2008). Rasad et al. (2012) in their study on “Effect of in vitro treatment of M. malabathricum on fibroblast proliferation” determined the wound-healing property by assessing the rate of fibroblast proliferation both prior to and after treating with methanolic and aqueous extract. The extracts used in the study were diluted to the following concentration of 100, 50, 25, and 12.5 μg/mL with distilled water. The fibroblast proliferation activity of the prepared extract was then assessed by using methylene blue assay. A significant proliferation of cell of 39.3% and 20.7% was observed at 25 and 12.5 μg/mL concentration of aqueous extract. The methanolic extract on the other hand showed a decrease in cell proliferation to 13.2% and 16.4% at 48 hours which became almost negligible at 72 hours, thereby indicating a dosedependent inhibitory effect at all concentrations.

35.4.4 Antidiarrheal property The diarrhea-preventing activity of M. malabathricum leaf extract was studied in animal models. The leaves collected from Dandeli, Joida Taluk, Hubli district of North Karnataka were shade dried, powdered, and exposed to Soxhlet extraction using ethanol. The antidiarrheal effect of extract at 100, 200, and 400 mg/kg body weight was evaluated in normal and healthy Wistar Albino rats. The experimental rats were divided in such a way that each group has six numbers of rat. All the rats were subjected to oral administration of castor oil (10 m/kg body weight concentration) to induce diarrhea prior to performing test. The group I animal was subjected to castor oil for 7 days, group II with 100 mg/kg body weight ethanolic extract of M. malabathricum for 7 days, group III with 200 mg/kg body weight ethanolic extract of M. malabathricum for 7 days, group IV with 400 mg/kg body weight ethanolic extract of M. malabathricum, and group V with standard drug, loperamide (2 mg/kg body weight). A significant reduction (93.67%) in diarrheal episode was evident in test group treated with ethanol extract of M. malabathricum in a dose-dependent manner. A comparable reduction in number of fecal episodes in group treated with 400 mg/kg dose of ethanol extract of M. malabathricum was seen with that treated with loperamide (94.84%) indicating antidiarrheal effects (Karuppasamy et al., 2013). Sunilson et al. (2009) evaluated the possible antidiarrheal activity of M. malabathricum leaf extract in Swiss mice. The mice were divided into five groups in which the first group was given only distilled water (control), the second, third, and fourth groups were given M. malabathricum water extract at doses 100, 200, and 500 mg/kg, respectively, and the fifth group was subjected to 0.5 mL of loperamide (5 mg/kg body wt.). The quantity of fecal matter collected at the end of 12 hours was dried and weight recorded followed by measurement of percent reduction in the fecal output. The mice were then kept fasted overnight and later subjected to castor oil administration (0.5 mL per mouse) to induce diarrhea. Protection of mice against castor oil-induced diarrhea was seen in the mouse treated with M. malabathricum leaf water extract in a dose-dependent manner.

35.4.5 Anti-ulcer property M. malabathricum has been used traditionally in the prevention of ulcer owing to its phytochemicals present in it which is responsible for its bioactivity. Several studies have shown the plant to exhibit anti-ulcer property (Balan et al., 2014; Zabidi et al., 2012). Balamurugan et al. (2013) studied the anti-ulcer property of a widely available shrub M. malabathricum using rat model. The rats were given 8 mL/kg of 90% v/v ethanol to all tested animal for inducing gastric ulcer. A control group was formed which was treated with 4% w/v aqueous tween 80 (10 mL/kg body weight) for 7 days, and two test groups were subjected to 250 and 500 mg/kg body weight of ethanolic extract of M. malabathricum leaf extract and a positive control group in which rats were treated with omeprazole (20 mg/kg body wt.). The treatments were given to rats 30 minutes prior to administration of 90% v/v ethanol. A significant activity against gastric ulcer was seen in group treated with 500 mg/kg body weight. This anti-ulcer effect could be due to its phytoconstituents, flavonoids,

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tannins, terpenoids, and saponnins which have been reported in several studies as potent gastroprotective agents (Berenguer et al., 2006; Borrelli & Izzo, 2000). Zabidi et al. (2012) reported the anti-ulcer property of M. malabathricum leaf extract in rat models which is mainly related to the ability of extract to enhance the synthesis of prostaglandin. The prostaglandin has the ability to stimulate mucus and bicarbonate production, thus protecting gastric mucosa from ulcer formation. The anti-ulcer activity can also be due to the antioxidant and antiproliferative action of extract reported (Zakaria et al., 2011). Furthermore, it may be associated with its phytochemicals present which have been reportedly exhibiting anti-ulcer properties (Izzo et al., 1994; Souza et al., 2007; Ye¸silada & Takaishi, 1999). The gastroprotective effect of the chloroform extract of M. malabathricum leaves was investigated by Zakaria et al. (2016). The chloroform extract was used in the study to extract only nonpolar compound and test their gastroprotective action. The gastroprotective effect and gastroprotection mechanism were analyzed by ethanol-induced gastric ulcer assay and pyloric ligation assay. The extract exhibited a significant gastroprotective effect in ethanol-induced gastric ulcer via antimicrobial and antisecretory effects. Treatment with extract reduced the total and free acidity and increased the pH of gastric juice and gastric wall mucus, thereby inducing gastroprotection effect.

35.4.6 Hepatoprotective potential Liver is the key organ for human metabolism. It is subjected to many environmental pollutants and chemicals on an everyday basis (Opoku et al., 2007). These chemicals may result in hepatic injury on prolonged exposure. Numerous plant-based herbal formulation have been made owing to their hepatoprotective actions (Chattopadhyay, 2003). The leaves of M. malabathricum have been used in many traditional medicines and have also been scientifically proven to exhibit pharmacological activities such as anti-inflammatory and antioxidant (Joffry et al., 2012). Kamisan et al. (2013) studied the hepatoprotective effects of the methanolic extract of M. malabathricum leaf in rat models on Sprague Dawley rats. Prior to the assay, the test animals were kept fasted for 48 hours. The test rats were administered with 200 mg/kg silymarin and propane extract orally with dimethyl sulfoxide as the vehicle. Hepatotoxicity was artificially induced in the rats using either paracetamol or carbon tetrachloride. The leaf extract was then administered in doses 50, 250, and 500 mg/kg of methanolic extract of M. malabathricum. The extract exhibited significant hepatoprotective activity against paracetamol or carbon tetrachloride-induced liver model by improving the function of liver which was evident from histopathological studies of liver.

35.4.7 Antidiabetic potential As per International Diabetes Federation, the prevalence of diabetes is estimated to reach around 330 million by the year 2025. To cater to the increased rate of diabetes in the world, a number of insulin and drug therapies are available, many of which may have adverse effects on human health. Numerous herbs and plants have been found to exhibit antidiabetic potential. Antidiabetic effect of ethanol extract of M. malabathricum leaf was evident in alloxan-induced diabetic rat in the study of Balamurugan et al. (2014) which was possibly caused by increasing the insulin secretion by regeneration of damaged b-cells of pancreas in rats (Sezik et al., 2005). A lowering of serum urea and creatinine level was achieved in diabetic rat on treatment with leaf extract. A reduction in HbA1C was also evident in the study in the group treated with leaf extract. These effects were due to the presence of flavonoids, triterpenoids, steroids, and phenolic acids that have potent antidiabetic effects (Alagammal et al., 2012; Anitha et al., 2012). Flavanoids found in the extract are known for its regeneration capacity of damaged β 2 cells and its activity as secretagogues (Alagammal et al., 2012) in alloxan-induced rats. In the study of Kumar et al. (2013), the methanol extract of M. malabathricum was seen to result in a significant reduction in the blood glucose levels of streptozocin-induced diabetic rat both before and after glucose loading. The antidiabetic activity was introduced by increasing plasma insulin level in test rats. It was suggested the mode of action similar to glibenclamide was exerted by the methanolic leaf extract.

35.4.8 Antinociceptive property Zakaria et al. (2006) studied the antinociceptive activity of M. malabathricum leaf aqueous extract in experimental animal using abdominal constriction, hot plate, and formalin test. In the study, the aqueous extract of M. malabathricum was seen to exhibit antinociceptive effect in a dose-dependent manner. The extract has the ability to influence peripheral and central nociceptive mechanism and also to inhibit the pain occurring from chemical and thermal stimuli.

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This ability of the extract to affect the early and late phase of formalin test (Chen et al., 1995) indicates its potential as a centrally acting agent and can be employed in relieving chronic pain (Cowan, 1990), thus showing its effectiveness in reducing pain occurring from direct nociceptor stimulation or with the release of inflammatory mediators (Amalou et al., 2005).

35.4.9 Anti-cancerous property Several studies have displayed the anti-cancerous and antiproliferative action of the plant M. malabathricum. As per a study conducted by Susanti et al. (2007), the ethyl acetate extract of M. malabathricum flower was shown to exhibit structural changes in MCF-7 cancer cells. Two phytochemicals (naringerin and kaempferol-3
0-(2,v6v-di-0-p-transcoumaroyl) glucoside) isolated in the study exerted anti-cancerous activity against MCF-7 cells by inhibiting its growth and multiplication. The mechanism involved in inhibiting cancer-forming cells by M. malabathricum extract includes free radical scavenging action and lipid peroxidation inhibition (Nishanthini et al., 2013). The effect of M. malabathricum leaf extract on diethylnitrosamine and ferric nitrilotriacetate induced renal carcinogenesis, oxidative stress, and renal hyperproliferation in rat models which was studied by Verma et al. (2016). A potent chemoprotection was seen to be exhibited by the extract which was evident from macroscopic and histopathological investigations. The chemoprevention action of methanol extract of M. malabathricum leaves was demonstrated in the research investigation of Kooi et al. (2014). The study was performed using 7,12-dimethylbenz(α)anthracene (DMBA) mouse skin carcinogenesis model. A total of 40 ICR strain mice were divided into six groups with each group having eight mice. On the dorsal side of each mice, a 4 cm2 area was shaved for chemical application. The mice were given a single dose of 100 μL/100 μg DMBA or 100 μL acetone on the shaved area. In the later phase, test solutions were applied for 30 minutes which was followed by tropical application of tumor promoter (100 μL croton oil). Tumor was examined on a weekly basis for a total of 15 weeks. Results of examination revealed a significant reduction in burden, incidence, and volume of tumors in the methanol extract M. malabathricum-treated group, indicating an anticancer potency of the extract. The DMBA used in the study initiates tumor formation through the generation of reactive oxygen species which is responsible for causing damage to the genetic material (Das et al., 2010). Henceforth, the chemoprevention action of the methanol extract of M. malabathricum leaves could be due to its antioxidative potential, imparted by its phytochemical constituents (Surh, 2002; Zakaria et al., 2011).

35.5

Conclusion and future perspective

Today, the utilization of herbal medicine in the treatment of various ailments is being recognized and acknowledged. The global concern for preventive and therapeutic action of natural products has given a new dimension to the traditional system of medicine. M. malabathricum is an indigenous herb native to Southeast Asia which have been used for long in the traditional pharmacopeia. There are claims on the plant being effective in treating many ailments such as diarrhea, dysentery, cuts and wounds, sore throat, ulcer, etc. Certain research investigation has been made to elucidate the phytochemical constituents of the plant and the in vitro and in vivo pharmacological properties of different parts of M. malabathricum. In spite of many claims made in traditional folklore, there are still very less clinical studies concerning the plant. There is a need for in-depth research on the therapeutic efficacy of the plant and their mode of action using animal models. Further, toxic studies of the plant extracts also need to be taken into consideration for future research studies.

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503. Available from https://doi.org/10.1159/000337406. Zainulddin, W. N. W., Zabidi, Z., Kamisan, F. H., Yahya, F., Ismail, N. A., Din, N. S. S., Mamat, S. S., Hassan, H., Mohtarrudin, N., & Zakaria, Z. A. (2016). Anti-ulcer activity of the aqueous extract of Melastoma malabathricum L. leaf in rats. Pakistan Journal of Pharmaceutical Sciences, 29(1), 35
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256. Available from https://doi.org/10.1016/j.jams.2011.09.016.

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Chapter 36

Bioactivity of essential oils and its medicinal applications Abdel Rahman Al Tawaha1, Rose Abukhader2, Ali Qaisi3, Abhijit Dey4, Abdel Razzaq Al-Tawaha5 and Iftikhar Ali6 1

Department of Biological Sciences, Al Hussein Bin Talal University, Maan, Jordan, 2Faculty of Medicine, Jordan University of Science and

Technology, Irbid, Jordan, 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Jordan, Amman, Jordan, 4Department of Life Sciences, Presidency University, Kolkata, West Bengal, India, 5Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, Serdang, Selangor, Malaysia, 6Center for Plant Sciences and Biodiversity, University of Swat, Charbagh, Pakistan

36.1

Introduction

Plants have been used for different purposes since early mankind’s history. The most established proof of the utilization of plants for well-being purposes has been found on a Sumerian dirt chunk from Nagpur, which is roughly 5000 years of age (Jamshidi-Kia et al., 2018). They were first utilized as powders, teas, tinctures, poultices, and many others. Within the early 19th century, medicinal plants have been separated and analyzed. Phytochemical analysis began with the work of Serturner who separated morphine from opium in 1803. Plants have extraordinary significance with their metabolites of around 250,000 plant species existing. These plants are known to share a comparative chemical profile vital for a living cell (essential metabolites). In addition, they create a wide assortment of phytochemicals (auxiliary metabolites) (Suntar, 2020). More than a tenth of the plant species (over 50,000 species) are utilized in pharmaceuticals and cosmetics, for example, as antitumor, anti-inflammatory, antidiabetic, and antioxidants (Jamshidi-Kia et al., 2018). Identifying scientific research of natural-source chemical compounds allows for the evaluation of their activities and the determination of the next research steps in the quest for alternative medical applications based on active compounds found in plants. This is particularly important given the rise in antibiotic resistance among microorganisms (Adamczak et al., 2020). MDR, or multidrug resistance, is a major danger to public health as well as to plants and animals. In health care, MDR is becoming more of a problem. Many international investigations have recently been conducted to determine the presence of medicinal herbs used in microbial diseases (Adamczak et al., 2020; Chandra et al., 2017). Many infectious microorganisms, such as bacteria, viruses, fungi, and protozoa, have evolved over time, and there is an alarmingly high number of antibiotic-resistant species able to withstand the protective effects of antimicrobials, according to metadata collection from various parts of the world (Biharee et al., 2020). MDR is divided into three categories: primary, secondary, and clinical resistance. When an organism has never met the drug of interest in a specific host, it is said to have developed primary resistance. After being exposed to drugs, an organism develops secondary resistance (Tanwar et al., 2014). Clinical resistance refers to the infectious species being suppressed by a dose of an antimicrobial agent that is linked to a high probability of drug resistance or infection recurrence within an organism due to weakened host immune responses. This implies that the microorganism is suppressed by a dose of antimicrobial that is greater than what can be safely accomplished with standard dosages (Biharee et al., 2020; Tanwar et al., 2014). Extracts of medicinal herbs are thought to be an effective substitute for resistance-modifying drugs (Gupta & Birdi, 2017). Plant extracts and other herbal remedies are chemical compounds including a diverse range of primary and secondary metabolites, and their activity may be due to the collaboration of various ingredients (Gupta & Birdi, 2017). Antibacterial effects can be found in a variety of natural substances, including organosulfur compounds, alkaloids, flavonoids, phenolic acids, carotenoids, terpenes, coumarins, tannins, and some metabolites (amino acids, organic acids, and peptides) (Chandra et al., 2017; Gutierrez-Grijalva et al., 2018; Ozcelik et al., 2011).

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Flavonoids are a remarkable class of bioactive compounds with low toxic effects. Compounds in this class, including flavonols, flavones, and flavanones, are widespread secondary metabolites contained in a variety of fruits, vegetables, and wild plants (Gutierrez-Grijalva et al., 2018). Flavonoids are by-products of green plant secondary metabolic processes. The term "flavonoid" comes from the Latin word "flavous," which translates as "clear, pure yellow" (Rana & Gulliya, 2019). Apart from their name, most flavonoids are not white; in particular, they can be purple, red, or blue (Wang et al., 2018a). Flavonoids are a group of polyphenols and consist of aromatic rings. Seventy percent of plants contain flavones and flavonoids found as pigments and have potent antioxidant activity, such as quercetin and kaempferol (Biharee et al., 2020). Flavonoids can be found in the leaves and stems, as well as the fruits and flowers of plant species in the Asteraceae, Apiaceae, Brassicaceae, Betulaceae, Fabaceae, Ericaceae, Lamiaceae, Hypericaceae, Passifloraceae, Liliaceae, Primulaceae, Polygonaceae, Rosaceae, Ranunculaceae, Rubiaceae, and Rutaceae families. Two flavonols, quercetin and kaempferol, and flavones, apigenin and luteolin, are among the most common flavonoids found (Adamczak et al., 2020; Wang et al., 2019). Rutin is a glycoside variant of quercetin that is abundant in buckwheat (Fagopyrum esculentum), rue (Ruta graveolens), Styphnolobium japonicum flower buds, apricots, peaches, and citrus (Chua, 2013; Enogieru et al., 2018). The principal active components in hawthorn leaves and flowers are apigenin derivatives such as vitexin, isovitexin, and vitexin 2v-O-rhamnoside (Adamczak et al., 2020). Orientin and isoorientin, the 8- and 6-C-glucosides of luteolin, have been found in a variety of agricultural crops, including buckwheat, maize silk, acai berries (Euterpe oleracea, E. precatoria), and Moso bamboo stems (Phyllostachys edulis) (Yamaguchi et al., 2015; Yuan et al., 2016). Naringin is a flavanone glycoside extracted from grapes and citrus (Alam et al., 2014).

36.2

Chemical structure of flavonoids

There are thousands of flavonoids known to exist in nature, which are chemically characterized as compounds with a phenylchromanone structure of C6-C3-C6 with differing hydroxyl substituents. A biphenyl propane skeleton, specifically two benzene rings (rings A and B), is connected by a three-carbon chain to form a complete pyran ring in the fundamental flavonoid structure (heterocyclic ring containing oxygen, the C ring). An α-pyrone (flavonols and flavanones) or its dihydro derivative is a six-membered ring compressed with a benzene ring (flavonols and flavanones) (Kumar & Pandey, 2013; Rana & Gulliya, 2019). The molecular structure of a flavonoid is centered on a C15 skeleton with a chroman ring (ring A merged with pyran ring C) connected to the second aromatic ring B in positions 2, 3, or 4 and represents various flavonoid structural groups (Kumar & Pandey, 2013). Flavonoids are chemically categorized into four classes based on the second, third, and fourth positions of the aromatic ring (ring B) to the benzopyrano (chromano) moiety, respectively: 2phenylbenzopyrans, 3-phenylbenzopyrans, and 4-phenylbenzopyrans, with aurones, anthocyanidins, and other minor flavonoids existing in natural environment (Batra & Sharma, 2013). Fig. 36.1 shows the basic flavonoid structure with its classes. Also, Fig. 36.2 illustrates common flavonols as they are the most abundant flavonoids in foods and are among the most important class of flavonoids that show potent antibacterial activities (Farhadi et al., 2019). Until now, a large number of flavonoids have been discovered, and hundreds of derivatives have been synthesized by substituting a different moiety in the basic design of flavonoids (Biharee et al., 2020). Flavonoids have a high structural diversity due to the presence of various hydroxyl groups and unsaturation on ring C. Changes in various functional groups, such as hydroxyl, methoxyl, carbonyl, and olefinic, will increase this variability (Biharee et al., 2020). The fact that flavonoids have such a wide diversity and bioactivity stems from their continuous alterations (Chen et al., 2018). The composition and mechanism of antimicrobial activities of flavonoid glycosides, as well as their prenylated, geranylated, methoxylated, and hydroxylated derivatives, vary widely (Cushnie & Lamb, 2011). Given the large number of the latest flavonoids constantly getting extracted, many systems of naming flavonoids are presently used, which may create some inconsistency. The most appropriate system is to use a simple name that refers to the compound’s category or the plant from which it was first processed (Gorniak et al., 2019). Another option is to use a semi-systematic name, in which the name’s core is derived from the subclass, such as 3,5,7,30 ,40 -pentahydroxyflavone. In the third method, the flavonoids are named using their systematic chemical nomenclature, such as 3,4-dihydro-2-phenyl-2H-1-benzopyran (flavan). While this approach is overly complicated for naming known flavonoids, it is the most exact and therefore better than other nomenclature methods for describing novel components (Gorniak et al., 2019) The International Union of Pure and Applied Chemistry (IUPAC) developed one of the flavonoid nomenclature guidelines. These guidelines set regulations for the generic nomenclature of flavonoids, including examples of appropriate trivial names and names generated from trivial names, as well as semi-systematic and completely systematic names that adhere to IUPAC guidelines (Rauter et al., 2018).

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FIGURE 36.1 Basic flavonoid structure with its classes (Biharee et al., 2020).

36.3

Flavonoids activity against multidrug-resistant microbes

MDR, or multidrug resistance, is a severe hazard to people’s health as well as plants and animals. In health care, MDR is becoming more of a problem. Several worldwide analyses have recently been conducted to investigate the incidence of medicinal herbs used in harmful microbial infections. Extracts of medicinal herbs are thought to provide an alternate source of resistance-modifying chemicals (Adamczak et al., 2020; Farhadi et al., 2019; Jamshidi-Kia et al., 2018). Plant extracts and other herbal preparations are complex combinations comprising a diverse range of primary and secondary metabolites, and their activity could be due to the combination of several chemical compounds. Furthermore, these preparations may exhibit a variety of biological and pharmacological activity processes, such as the ability to adhere to protein domains, immune system response regulation, proliferation, programmed cell death, and metabolic pathways (Adamczak et al., 2020; Biharee et al., 2020; Jamshidi-Kia et al., 2018). It should be emphasized, however, that because plants come in contact with the surroundings and other life forms, their chemical properties and active ingredient levels can vary greatly. Furthermore, the manufacturing process for herbal medical treatments is quite complicated since it involves non-standardized operations such as plant cultivation, collecting vegetable raw material from various regions of the world, extract preparation, and product manufacturing in compliance with local good manufacturing practice regulations (Adamczak et al., 2020; Biharee et al., 2020; Jamshidi-Kia et al., 2018). As a result, it can be stated that adopting pure chemical substances from natural sources as a complimentary option would be a viable alternative. A large number of studies evaluated the inhibitory effects of plant flavonoid-containing extracts against a variety of infectious microorganisms, such as bacteria, viruses, fungi, and protozoa, and several mechanisms have been proposed for the observed antibacterial activities of flavonoids. The following sections review, according to the mechanisms of antimicrobial activity, a collection of recent scientific papers that have reported flavonoid compounds with activity against multidrug-resistant microbes.

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FIGURE 36.2 Structures of flavonols (Gorniak et al., 2019).

36.3.1 Inhibitory activity against cell envelope synthesis Because both phospholipids and lipopolysaccharides are required for gram-negative bacteria’s existence, and fatty acid synthase is involved in their biosynthesis, inhibiting fatty acid synthase restricts bacterial cell envelope construction. Because bacterial type-II fatty acid synthase (FAS-II) varies from mammalian FAS-I in several aspects, we can

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effectively identify FAS-II and produce a potent antimicrobial drug (Biharee et al., 2020; Gorniak et al., 2019; Wallace et al., 2015). The malonyl CoA-acyl carrier protein transacylase fabD (MCATs), which regulates bacterial FAS-II, has been shown to be downregulated by 5,6,7,40 ,50 -pentahydroxyflavone and 5-hydroxy-40 ,7-dimethoxyflavone. As a result, these two flavones are thought to be potential medicines for stopping the growth of bacteria (Elmasri et al., 2017). Attributed to the prevalence of mycolic acid, which is one of the unique survival characteristics of the mycobacterial cell wall, treating mycobacterium is extremely difficult. Both mammalian form FAS-I and bacterial form FAS-II are necessary for mycolic acid production (Bedi et al., 2020; Biharee et al., 2020; Dong et al., 2015; Gorniak et al., 2019). Several flavonols such as quercetin, kaempferol, fisetin, myricetin, and morin, as well as flavones like luteolin, baicalein, taxifolin, and hesperetin, have been shown to inhibit FAS-I (Bedi et al., 2020; Biharee et al., 2020; Dong et al., 2015). Peptidoglycan is an integral part of bacterial cell walls, and inhibiting its formation is a frequent mode of activity for antimicrobials and flavonoids (Gorniak et al., 2019). Galangin, kaempferide, and kaempferide-3-O-glucoside flavonols were found to have not only antimicrobial efficacy against amoxicillin-resistant E. coli, but also the potential to overcome resistance by inhibiting peptidoglycan and ribosome formation (Eumkeb et al., 2012). Catechins have also been discovered to impede bacterial cell wall production by attaching to the peptidoglycan surface. A synergistic impact of EGCG and DL-cycloserine also suppressed cell wall production (an inhibitor unrelated to penicillin-binding protein). Additionally, because both EGCG and lactams (benzylpenicillin, oxacillin, methicillin, ampicillin, and cephalexin) attack peptidoglycan directly or indirectly, EGCG enhances β-lactams action (Gorniak et al., 2019). The D-alanine: D-alanine ligase (Ddl, EC 6.3.2.4) has been identified as a potential antimicrobial therapeutic destination, prompting extensive inhibitor investigation. Quercetin, which has a flavone core made up of two aromatic rings joined by a heterocyclic pyrone ring, has been shown to have antibacterial properties against the H. pylori Ddl (HpDdl) enzyme. An experiment was conducted as a molecular docking investigation of quercetin and its derivatives at the active site of HpDdl in this environment. A few of the substances tested have a higher affinity for and ability to interact with the HpDdl enzyme. Some of them emerge as plausible lead molecules or a novel class of medications with improved pharmacological activities after docking research and absorption, distribution, metabolism, and toxicity studies (Singh et al., 2013). Quercetin and apigenin suppress D-alanine:D-alanine ligase, which is responsible for the development of the terminal dipeptide of the peptidoglycan precursor UDP-MurNAc-pentapeptide, according to kinetic analysis (Gorniak et al., 2019). Those two compounds bind to the D-alanine:D-alanine ligase active center. Quercetin, on the other hand, displayed lower activity than apigenin, which can be related to its extra
OH groups, which increase its attraction to the enzyme. Sakuranetin, a flavonoid identical to apigenin (this has a 7-methoxy group rather than a 7-hydroxy group and no double bonding on the C ring), on the other hand, has no inhibitory action. Moreover, quercetin’s hydrophilicity prevents it from penetrating bacterial cells (Gorniak et al., 2019; Singh et al., 2013).

36.3.2 Inhibitory activity against DNA synthesis Flavonoids have been found to be capable of inhibiting topoisomerases, which adds to their antimicrobial activities. DNA gyrase, for example, is an important enzyme for DNA synthesis that is only found in prokaryotes, making it a promising target for antimicrobial medications (Gorniak et al., 2019). Furthermore, helicases are ubiquitous motor molecules that split and/or reorganize nucleic acid compounds in adenosine triphosphate (ATP)-fueled processes. Their role in the replication of nucleic acid is analogous to that of topoisomerases and gyrases. These proteins have been proposed as flavonoids’ molecular targets. Flavones and flavonols, two types of pharmacophores that can bind to nucleic acids, have been investigated as helicase inhibitors. The flavone luteolin, as well as structurally similar flavonols notably morin and myricetin, has been reported to suppress E. coli replicative helicases like DnaB and RecBCD helicase/ nuclease. Myricetin was also found to suppress gram-negative bacterial contamination and to be a powerful inhibitor of a variety of DNA and RNA polymerases, and also viral reverse transcriptases and telomerases (Gorniak et al., 2019; Shadrick et al., 2013). According to a research paper, quercetin can attack subunit B of DNA gyrase from Mycobacterium smegmatis and Mycobacterium tuberculosis (Suriyanarayanan et al., 2013). Gyrases are DNA topologymodifying enzymes that are only found in prokaryotic cells, making them a desirable target for antimicrobial agents. Quercetin has been shown to decrease DNA gyrase’s supercoiling action, and it adheres to the subunit B of DNA gyrase by interacting with residues in the Toprim domain of the enzyme. This domain has been discovered to be critical for DNA splitting and rejoining. The minimum inhibitory concentration (MIC) of quercetin against M. smegmatis and Mycobacterium TB was determined to be 100 g/mL, indicating that DNA gyrase may be a possible target for quercetin in Mycobacterium (Suriyanarayanan et al., 2013). Furthermore, the relevant flavonoids chrysin and kaempferol strongly suppressed E. coli DNA gyrase (Fang et al., 2016). These studies revealed that flavonoid hydroxyl groups are stronger at interacting with gyrase than methoxy

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groups, though adding an extra 50 -OH to myricetin significantly reduced its gyrase inhibitory activity (Fang et al., 2016). Molecular modeling experiments indicate that flavonoids disrupt DNA supercoiling by competitively interfering with the ATP binding site of the DNA gyrase B subunit, which is the second mechanism of DNA gyrase inhibition (GyrB). The binding of flavonoids to DNA preserves the DNA
gyrase combination, resulting in DNA cleavage activation (Singh et al., 2013). Furthermore, Fang et al. (2016) and Singh et al. (2013) found that 3-hydroxyl, 5-hydroxyl, 7hydroxyl, and 4-carbonyl groups react with essential GyrB sequences to make flavonoids more active. Helicases are widespread motor enzymes that isolate and/or reorganize oligonucleotides in ATP-fueled reactions, and they have even been proposed as flavonoids’ target molecules (Shadrick et al., 2013). Furthermore, many medications, including antibacterial drugs, attack dihydrofolate reductase (DHFR). The DHFR is a key antioxidant in the folic acid biosynthetic pathway, as it produces pyrimidines and purines as a substrate [36]. EGCG was reported to inhibit DHFRs in M. tuberculosis. EGCG also had synergistic interactions with other folic acid pathway blockers such as ethambutol (Raju et al., 2015). Raju et al. (Raju et al., 2015) chose seven polyphenols to investigate M. tuberculosis in their study. Estimated effectiveness of the phytochemicals ranged from 3 to 183 μm. In a high-throughput microtiter analysis, these compounds were evaluated against bacterial and human enzymes. The highest effectiveness and specificity were found with epigallocatechin gallate. Five of the seven polyphenols with minimum inhibitory concentration levels of 15 m were investigated for synergistic efficacy with ethambutol, a first-line medication, and para-aminosalicylic acid, a second-line folate inhibitor. By decreasing the minimal inhibitory concentration of these medications, epigallocatechin gallate, magnolol, and bakuchiol showed a mild synergistic interaction. Following in vivo verification, these basic phytoconstituents could be viewed as candidates for further research or for the synthesis of semisynthetic derivatives to be employed in combination therapy for improved antituberculosis action.

36.3.3 Inhibitory activity against ATP synthesis Membrane potential is critical for mammalian and microbial life because it is the primary supply of energy for all biochemical reactions in the biological system (Murray et al., 2014). Flavonoids from Dorstenia ssp., such as isobavachalcone and 6-prenylapigenin, cause bacterial cell membrane depolarization in S. aureus, while licochalcone from Glycyrrhiza inflata reduces aerobic capacity (Biharee et al., 2020; Dzoyem et al., 2013). Furthermore, flavonoids were discovered to impair E. coli ATPase by connecting to the polyphenol binding pocket of ATP synthase, which is critical for ATP synthesis (Chinnam et al., 2010). As a result, it is possible that this pathway will also disrupt other microbes. Baicalein, morin, epicatechin, and flavanonols like silibinin and silymarin were revealed to be the most capable of inhibiting E. coli ATPase (Chinnam et al., 2010; Silva et al., 2016). On pure F1 or membrane-bound F1Fo E. coli ATP synthase, the suppressive impact of 17 bioflavonoid substances was investigated. The level of inhibition by bioflavonoid substances was found to be varied. Absolute inhibition was achieved with morin, silymarin, baicalein, silibinin, rimantadine, amantidine, or epicatechin. Hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin inhibited the enzyme in a partial way (40%
60%), while galangin, daidzein, or luteolin had no effect. In the efficient inhibition of ATP synthase, the primary skeleton, size, geometry, shape, and distribution of functional groups on inhibitors all played a role. The bioflavonoid molecules utilized in this work inhibited F1-ATPase and ATP production roughly equally in ATPase and growth experiments, implying a relationship between bioflavonoids’ positive impacts and their inhibitory activity on ATP synthase (Chinnam et al., 2010).

36.3.4 Inhibitory activity against bacterial toxins Bacterial hyaluronidases (formed by both gram-positive and gram-negative microbes) come into direct contact with host cells or disguise the bacterial surface from the host’s immune system. The deterioration of hyaluronan by hyaluronidase improves the permeability of connective tissues and lowers the viscosity of bodily secretions during disease pathogenesis (Girish & Kemparaju, 2007). In Streptococcus agalactiae, flavonols such as myricetin and quercetin have been found as hyaluronic acid lyase (Hyal B) blockers. The quantity of hydroxyl groups in the flavonoid composition enhanced the inhibitory impact of the flavonoids. Streptomyces hyalurolyticus (Hyal S) and Streptococcus equisimilis (Hyal C) hyaluronate lyases, on the other hand, were only mildly inhibited (Gorniak et al., 2019). One of the most prominent pathogenic elements developed by S. aureus is α-hemolysin (Hla), a member of the bacterial pore-forming β-barrel toxic substances. Pinocembrin, a honey flavanone, was found to decrease S. aureus α-hemolysin secretion in a dose-dependent way (Soromou et al., 2013). Pinocembrin inhibited hemolysin synthesis and

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reduced hemolysin-mediated cell damage when used at low dosages. Pinocembrin also safeguarded mice from S. aureus pneumonia in vivo, according to the results (Soromou et al., 2013). Pinocembrin’s mode of action on Neisseria gonorrhoeae bacterial surfaces has also been investigated. Despite the fact that pinocembrin-induced cell lysis was reported in the study, the modes of action of this molecule are still unknown (Rasul et al., 2013; Ruddock et al., 2011). Green tea catechins, such as EGCG and gallocatechin gallate (GCG), have also been found to inhibit the production of verotoxin from enterohemorrhagic E. coli cells, and green tea catechins can be applied to avoid food contamination resulting from E. coli (Gorniak et al., 2019).

36.3.5 Inhibitory activity against biofilm formation Infectious diseases caused by bacterial biofilms account for a large portion of all microbial and chronic infectious diseases, as well as food poisoning (Abdullahi et al., 2016; Jamal et al., 2018). One of the most important characteristics of bacteria that form biofilms is that they develop 10 to 1000 times greater resistance to antimicrobials than their planktonic counterparts (Kon & Rai, 2016). Bacterial biofilm construction and development, as well as associated bacterial rigidity, motility, and quorum sensing, have all been shown to be influenced by anti-biofilm phytochemical compounds (Borges et al., 2013). Biofilms are microbial populations that are sessile and encased in a network of extracellular polymeric substances (EPSs), including proteins, nucleic acids, and polysaccharides (Borges et al., 2013; Cos et al., 2010). This microbial pattern is an illustration of physiological adaptation that is more difficult to eradicate, and it is one of the most common causes of persistent infections (Simoes, 2011). Biofilms are linked to a variety of health issues, including periodontitis, endocarditis, osteomyelitis, cystic fibrosis, and infections caused by surgical implantation (Borges et al., 2013; Hancock et al., 2010; Simoes, 2011). Biofilm development is involved in more than 80% of bacterial infections in human populations (Borges et al., 2013; Hancock et al., 2010). Antimicrobials’ limited absorption or deactivation in the extracellular polymeric matrix (1), external membrane construction (2), a modified microbial metabolic condition (3), the existence of persister cells (4), genetic adaptability (5), and resistance caused by the antibacterial agent itself after the application of sublethal dosages (6) are all contributing factors for the increased resistance of bacteria in biofilm (Borges et al., 2013; Hancock et al., 2010). Flavonoids induce bacterial accumulation by causing partial lysis, which causes membrane fusion and decreases active nutrient availability due to a smaller membrane region. As a result, flavonoids cannot be presumed to promote biofilm formation (Kragh et al., 2016). Flavonoids, on the other hand, have been shown to inhibit biofilms by various research groups. Isovitexin, EC, and 5,7,40 -trihydroxyflavanol, for instance, have been shown to have anti-biofilm action against S. aureus (Awolola et al., 2014). Likewise, S. mutans biofilm formation was reduced by 55%
66% when exposed to 2%
15% EC. The goal of the research by El-Adawi (El-Adawi, 2012) was to determine how a grape seed extract (GSE) affected S. mutans proliferation and biofilm formation. The findings showed that GSE and its principal ingredients (gallic acid, catechin, and epicatechin) have the capacity to suppress S. mutans proliferation and biofilm development, with GSE and epicatechin being the most effective (80.98% and 66.25%, respectively). Furthermore, the GSE could capture up to 85% of free radicals and entirely prevent DNA damage. As S. mutans performs a significant role in the development of dental caries in humans, GSE, particularly epicatechin, demonstrated a noteworthy impact on this possibly cariogenic species in the oral cavity and could be employed to reduce this possibly cariogenic species in the oral cavity. In E. coli, Vibrio spp., and Salmonella typhimurium, quorum sensing, specifically autoinducer-2-mediated cell
cell signaling, has been suggested as a major regulatory factor for biofilm formation. Citrus flavonoids like apigenin, kaempferol, quercetin, and naringenin, for example, are potent antagonists of cell
cell signals (Vikram et al., 2010). Additionally, quercetin increases the development of several iron siderophore proteins, reducing the level of Fe31 needed for Pseudomonas aeruginosa biofilm synthesis (Ouyang et al., 2016). While chrysin, phloretin, and naringenin impaired QS synthase/receptor combinations, LasI/R, and RhlI/R, kaempferol, epicatechin gallate, and EGCG were found to trigger the removal of AHL molecules from LuxR-type transcription factors (Paczkowski et al., 2017; Roy et al., 2018). Anti-adhesion properties of cranberry A-type proanthocyanidins have also been discovered against the gram-negative bacterium P. aeruginosa. In an in vivo study of infection employing G. mellonella, cranberry proanthocyanidins inhibited P. aeruginosa swarming movement, dramatically damaged P. aeruginosa biofilm formation, and potentiated the antibacterial action of gentamicin. The analysis also demonstrated that after treatment with proanthocyanidins, considerably different proteins were synthesized (Ulrey et al., 2014). Flavones like 6-aminoflavone, 6-hydroxyflavone, apigenin, and chrysin, as well as isoflavones like daidzein and genistein, and a dihydrochalcone called phloretin, were found to inhibit E. coli biofilm synthesis (Lee et al., 2011).

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Moreover, phloretin (a natural, nontoxic apple flavonoid) reduced enterohemorrhagic E. coli O157:H7 biofilms the most effectively without influencing planktonic cell proliferation. Derriobtusone A, an auronol that hindered biofilm development in E. coli, had a consistent impact though the planktonic development of E. coli was only slightly suppressed. At doses of 250 and 500 g/mL, derriobtusone A decreased the biomass and colony-forming unit (cfu) of S. aureus biofilms, and it demonstrated extremely effective antioxidant activity in scavenging DPPH radicals and preventing carotene oxidation. The chemical could be a powerful antimicrobial against S. aureus and associated biofilm, as well as a nontoxic antioxidant (Vasconcelos et al., 2014). Phloretin inhibited biofilm in a dose-dependent manner and did not damage biofilms of commensal E. coli K-12 and E. coli ATCC 4157 that are not pathogenic (Lee et al., 2011).

36.3.6 Membrane-disrupting activities The bacterial cell membrane is essential for osmoregulation, respiratory, and transport processes, as well as peptidoglycan biosynthesis and cross-linking, and lipid biosynthesis. As a result, membrane disturbance leads to metabolic impairment and microbial destruction (Hartmann et al., 2010). Numerous flavonoids, particularly catechins, have been extensively researched for antimicrobial activities in gram-positive and gram-negative cultures. Flavonoids interfere with phospholipid bilayer through two processes. The first is concerned with the separation of nonpolar substances into the membrane’s hydrophobic core, while the second is concerned with the hydrogen bond formation at the membrane interface between the polar heads of lipids and the more hydrophilic flavonoids (Tsuchiya, 2015). Catechins have been shown to disrupt the bacterial membrane by attaching to the lipid bilayer and blocking or hindering the formation of intracellular and extracellular enzymes (Reygaert, 2014) Furthermore, the latest researches using cell models have revealed that several polyphenols, such as epicatechin, epigallocatechin gallate, and the flavonol quercetin, possess prooxidative inhibitory effect (Bouayed & Bohn, 2010). It was also discovered that catechins destroy bacteria by causing oxidative damage, which results in the formation of reactive oxygen species (ROS) that attack membranes. The creation and treatment of bacterial liposomes validated catechin’s suppressive mechanism of oxidative stress through membrane permeabilization. Membrane degradation with morphological changes can be seen in SEM pictures of treated and control microbes (Fathima & Rao, 2016). Additional flavonoids have been found to have membrane-disrupting properties as well. The flavonols quercetin, rutin (quercetin-3-O-rhamnoglucoside), and tiliroside reduced bilayer density and interrupted the lipid monolayer composition, respectively, (Sanver et al., 2016). Due to microbial cell aggregation which affects cell structure and disrupts biofilms, artificial lipophilic 3-arylideneflavanones were found to be highly effective against S. aureus, S. epidermidis, and Enterococcus faecalis. In the presence of 3-arylideneflavanone 2c in the culture media, bacteria’s initial adherence to an abiotic surface was reduced. Biofilm development was reduced over prolonged culture as a result of this action. The amount of living biofilm cells was reduced by 3-arylideneflavanone 2e. E. faecalis biofilms were more vulnerable to 3-arylideneflavanone 2c activity than S. aureus biofilms. The observation that 3-arylideneflavanones are lipid soluble, induce bacterial aggregation, and alter membrane structure, rendering them susceptible to SYTO 9/propidium iodide stains, suggests that the cytoplasmic membrane may be a target region for their activities (Budzynska et al., 2011). It is also important to notice that catechins and other flavonoids can harm bacterial membranes, preventing bacteria from secreting toxins. Phloretin inhibited toxin genes (hlyE and stx 2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgA and csgB), and tens of prophage genes in E. coli O157:H7 biofilm colonies, according to the research’s global transcriptome analyses (Lee et al., 2011).

36.3.7 Inhibitory activity against efflux pumps Another key cause of resistance is active efflux of drugs from microbial species, since pushing out antibacterial compounds by efflux pumps leads to a reduction or submaximal inhibitory concentration within the microorganism. The efflux pump is inhibited by flavonoids, which have been described to have antibiotic potentiating effect. When silibinin is combined with ciprofloxacin, it has been shown to improve antibiotic efficacy by inhibiting MRSA’s efflux systems (Gorniak et al., 2019). By affecting the efflux via potassium secretion, quercetin, morin, rutin, and luteolin have been shown to have a strong inhibitory activity against MRSA. Antibiotic and flavonoids screening experiments revealed flavanoids’ antibacterial properties. When tested flavonoids were mixed with antibiotics that were resistant, the inhibition zones grew larger. Ampicillin, cephradine, ceftriaxone, imipenem, and methicillin all demonstrated an additive impact with quercetin alone. Morin 1 rutin 1 quercetin had the largest potassium permeability, which improved further when combined with imipenem. Morin and rutin had little effect on test bacteria when used separately, but when combined, they were effective (Amin et al., 2015).

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Rutin has also been shown to disrupt MDR in P. aeruginosa and to behave as effective gentamycin-resistance inhibitors (Deepika et al., 2018). When administered with ciprofloxacin, silybin, a compound extracted from the milk thistle seeds, was found to improve antibiotic effectiveness by inhibiting MRSA efflux pumps. A fluorescent experiment was used to assess the capacity of silybin to block ciprofloxacin outflow from MRSA. Silybin inhibited the generation of the quinolone-resistance protein NorA (norA) and quaternary ammonium-resistance proteins A/B (qacA/B) efflux genes in MRSA, according to polymerase chain reaction research. This indicated that silybin could successfully disrupt MRSA41577 efflux mechanism. MRSA41577 cultured with silybin for 16 hours had a 36% and 49% decrease in norA and qacA/B production, respectively, compared to the control. MRSA41577 susceptibility to antibiotics was recovered after silybin inhibited the expression of these genes, showing that efflux pump inhibitors, which function by reducing MRSA’s efflux mechanism, may break MRSA resistance to antibiotics, making the bacteria susceptible to these treatments (Wang et al., 2018b).

36.3.8 Inhibitory activity against bacterial motility Bacterial adherence, survivability, and motility to populate greater areas and enter host cells are all aided by biofilm since multiple cell components known as appendages, such as a capsule, pili, and other surface proteins known as adhesive proteins which are required for bacterial adherence and colonization, are expressed at the same time in bacteria, with the exception of the flagellum, which is needed for bacterial motility (Harshey, 2003). Since bacterial motility and attachment take place at different moments, flavonoids can swiftly stop bacterial movement by reducing swarming motility. This prevents bacterial adherence and colonization. Nobiletin, sinensetin, morin, luteolin, naringenin, quercetin, and epigallocatechin gallate are some of the flavonoids that have been described in the literature to suppress bacterial swarming and twitching movement (Biharee et al., 2020; Silva et al., 2016). In addition, a research looked at the anti-biofilm and anti-quorum sensing activities of several flavonoids in Pseudomonas aeruginosa and showed quercetin to be the only molecule that effectively reduced both biofilm development and twitching movement (Boris et al., 2015).

36.4

Conclusion

Flavonoids represent a class of polyphenols with aromatic rings that are pigments reported in 70% of plants and have antioxidant, antiproliferative, and antimicrobial properties. The aim of this paper is to review the existing published literature on flavonoids’ potential as complementary and alternative medicine against multidrug-resistant microbes. A literature survey of relevant studies was conducted by searching different scientific search engines, such as Science Direct, PubMed, and Google Scholar using related keywords such as “flavonoids,” “antimicrobial activity,” and “multidrug-resistant microbes.” The reviewed reports included recent peer-reviewed papers, without language restriction. Several subgroups of flavonoids, such as flavonols, flavanones, flavones, and chalcones have been extensively studied for their activities against multidrug-resistant microbes, and they were found extremely potent agents, with strong antimicrobial impact attributed to several mechanisms of action such as inhibition of cell envelope, DNA, ATP, bacterial toxins, and biofilm syntheses, as well as membrane-disrupting activities and inhibitory activity against efflux pumps. This review paper can lead to a conclusion that flavonoids would have promising medicinal use in the treatment of infections induced by multidrug-resistant microbes. It is now undeniable that modern antimicrobial agents, or at the very least compounds that would improve the efficacy of existing drugs, are urgently required. Various flavonoids have strong antimicrobial properties, which are attributed to a range of antimicrobial mechanisms, such as inhibition of cell envelope and nucleic acid syntheses, as well as membrane-disrupting actions, as discussed in our review. Bacterial efflux pumps are inhibited. Because flavonoids are found in so many plant-based foods and drinks, most of them are considered nontoxic. Flavonoid-rich foods and drinks are also linked to a variety of beneficial effects. Flavonoids may also be excellent food preservatives due to their low toxicity and natural occurrence. As a result, flavonoids may be a valid alternative to synthetic chemicals that are increasingly criticized by consumers.

Ethics declarations Ethical approval Not required.

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Consent to participate All authors have their consent to participate.

Consent to publish All authors have their consent to publish their work.

Authors contributions All authors contributed equally to this work.

Funding The author(s) received no specific funding for this work.

Competing interests The authors declare no competing interests.

Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

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134. Wang, M., Firrman, J., Liu, L., & Yam, K. (2019). A review on flavonoid apigenin: Dietary intake, ADME, antimicrobial effects, and interactions with human gut microbiota. BioMed Research International7010467. Wallace, J., Bowlin, N. O., Mills, D. M., Saenkham, P., Kwasny, S. M., Opperman, T. J., Williams, J. D., Rock, C. O., Bowlin, T. L., & Moir, D. T. (2015). Discovery of bacterial fatty acid synthase type II inhibitors using a novel cellular bioluminescent reporter assay. Antimicrobial Agents and Chemotherapy, 59, 5775. Wang, D., Xie, K., Zou, D., Meng, M., & Xie, M. (2018b). Inhibitory effects of silybin on the efflux pump of methicillin-resistant Staphylococcus aureus. Molecular Medicine Reports, 18, 827
833. Yuan, L., Wang, J., Wu, W., Liu, Q., & Liu, X. (2016). Effect of isoorientin on intracellular antioxidant defence mechanisms in hepatoma and liver cell lines. Biomedicine & Pharmacotherapy 5 Biomedecine & Pharmacotherapie, 81, 356
362. Yamaguchi, K. K. D. L., Pereira, L. F. R., Lamarao, C. V., Lima, E. S., & Da Veiga-Junior, V. F. (2015). Amazon acai: Chemistry and biological activities: A review. Food Chemistry, 179, 137
151.

Chapter 37

Essential oils as anticancer agents Vilas Jagatap1, Iqrar Ahmad1, Aakruti Kaikini2 and Harun Patel1 1

Division of Computer-Aided Drug Design, Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and

Research, Dhule, Maharashtra, India, 2Department of Diabetes and Obesity, King’s College London, London, United Kingdom

37.1

Introduction

Cancer is a debilitating disease having the highest global fatality rate. In the year 2021, the United States alone reported about 608,570 cancer-related fatalities and 898,160 new cancer cases (Mardani et al., 2022). Over the past several decades, cancer has emerged to be one of the greatest concerning diseases in the world (Mardani et al., 2022). It is a multifactorial illness that causes abnormal cells to multiply and invade unregulated resulting in the development of tumors. Dietary changes, alcohol and cigarette use, chronic illnesses, exposure to radiation and chemicals, and changes in lifestyle and environmental pollution are a few of the factors responsible for the sharp increase in cancer prevalence (Gautam et al., 2014). According to the WHO and International Agency for Cancer Research, in the last 5 years, there were 18.1 million new instances of cancer disease, 9.6 million cancer deaths, and 43.8 million individuals living with cancer (Kumar & Jaitak, 2019). Men were more likely than women to get the disease and succumb to it. It is predicted that by 2050, there would be 27 million additional cancer diagnoses and 17.1 million fatalities every year (Kumar & Jaitak, 2019). For both sexes, lung cancer was the leading cause of death, followed by colorectal, stomach, liver, and breast cancers (Kumar & Jaitak, 2019). Chemotherapy, radiation, hormone therapy, targeted therapy, and immune therapy are only a few of the therapeutic options available for cancer. The success rate of cancer therapy is lowered by the adverse effects connected with various treatment approaches (Menon et al., 2021). Since the beginning of time, people have employed plants as medicines (Ijaz et al., 2018). Plant-based medicines provide several benefits. Medicinal and aromatic plants are rich sources of various secondary metabolites like flavonoids, terpenoids, glycosides, alkaloids, essential oils, resins, tannins, etc. They are used to treat and prevent some of the most aggressive illnesses, including cancer, hepatitis, AIDS, etc. (Ijaz et al., 2018). Essential oils (EOs) are complex mixtures of aromatic and volatile chemicals synthesized by secondary plant metabolism. Mechanical pressing or hydro- and steam distillation are methods used to isolate essential oils from various plants. The major components of essential oils are monoterpenes and sesquiterpenes, but aromatic and phenolic components are also present. EOs are known to elicit a wide range of biological effects due to their antibacterial, antifungal, cytotoxic, antioxidant, and antimutagenic properties (Jugreet et al., 2020; Patel & Gogna, 2015). EOs have gained considerable attention recently because of their ability to treat cancer (Patel & Gogna, 2015). They have been shown to have a significant brutal impact on malignancies of the brain, oral, lung, breast, liver, pancreatic, gastric, ovary, and prostate (Patel & Gogna, 2015). Considering the huge applications of essential oils in medicine, in this chapter, we have discussed potent essential oils and their main volatile components (Table. 37.1) that are effective against a variety of cancers.

37.2

Anticancer potential of essential oils

Asif and his team investigated the in vitro anti-colon cancer activity of essential oils containing oleo-gum resin extract from Mesua ferrea. MTT assay for cell viability was performed to assess the cytotoxic potential of oils containing oleogum resin extract in a panel of cell lines. The extract demonstrated profound cytotoxicity toward two human colon cancer cell lines, namely, HCT 116 and LIM1215, with IC50 values of 17.38 6 0.92 μg/mL and 18.86 6 0.80 μg/mL, Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00037-2 © 2023 Elsevier Inc. All rights reserved.

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TABLE 37.1 Essential oil-bearing plants and their major components useful as anticancer agents. Sr. No

Essential oilbearing plant

Main component

Cancer cell line

IC50 value

References

1

Mesua ferrea

Isoledene, elemene

Human colon cancer cell lines HCT 116 and LIM1215

17.38 6 0.92 and 18.86 6 0.80 μg/mL

Asif et al. (2019)

2

Illicium verum

Trans-anethole

Human colon cancer cell lines HCT 116

50.34 6 1.19 μg/mL

Asif et al. (2016)

3

Cymbopogon citratus, Cymbopogon giganteus

Citral

Prostate cell lines LNCaP and PC-3, glioblastoma cell lines SF-767 and SF-763

6.36, 32.1, 45.13 and 172.05 μg/mL

Bayala et al. (2018)

4

Thymus fallax

Carvacrol and thymol

Human colorectal adenocarcinoma DLD-1 cell lines

0.347 mg/mL

C¸etinus et al. (2013)

5

Boswellia sacra

Boswellic acid

Human breast cancer cell lines like T47D, MCF7, MDA-MB-231

Cytotoxic to human breast cancer cell lines in different dilutions like 1:900 for TD47, 1:1000 for MCF 7, and 1:950 for MDA-MB-231

Suhail et al. (2011)

6

Amomum tsao-ko

1,8-Cineole

Hepatocellular carcinoma (HepG2) cell line

31.80 6 1.18 μg/mL

Yang et al. (2010)

7

Colombian Lippia alba

Citral

Human cervical carcinoma (HeLa) cell line

CC50 value 5 , 0.1 μg/mL

Mesa-Arango et al. (2009)

8

Frankincense or Boswellia tree

Boswellic acid

Bladder transitional carcinoma J82 cells

Cytotoxic to cancer cells even after dilution 1: 1100 in .24 h.

Frank et al. (2009)

9

Casearia sylvestris

β-caryophyllene, α-humulene

Human cancer cells such HeLa, A-549, and HT-29

ED50 value 5 63.3, 60.7, and 90.6 μg/mL

Silva et al. (2008)

10

Zanthoxylum rhoifolium

Synergistic effect of β-caryophyllene, α-humulene, α-pinene, myrcene, and linalool

HeLa (human cervical carcinoma), A-549 (human lung carcinoma), HT-29 (human colon adenocarcinoma)

ED50 value 5 82.3, 90.7, and 113.6 μg/mL

Silva et al. (2007)

11

Commiphora gileadensis

β-caryophyllene

Human MoFir cancer cell line

2.5 μL/mL

Amiel et al. (2012)

Rosewood Aniba rosaeodora

Linalool

Human epidermoid cancer cell line A431, on immortal HaCaT cells

400 nL/mL

Sœur et al. (2011)

12

Citrus

D-limonene

Human prostate cancer DU-145 cell line

9.4 mM

Rabi and Bishayee (2009)

13

Origanum majorana

Carvacrol

Human tumor cell lines Hep2 and HT29 and Vero

85.63 6 2.38, 13.73 6 1.31, and 70.13 6 1.72 mg/mL

Bouyahya et al. (2021)

14

Perilla frutescens

Perilla ketone

Human gastric cell line (MGC-803) and human lung carcinoma (A549) cell lines

17.82 6 5.12 μg/mL 21.31 6 0.98 μg/mL

Chen et al. (2020)

15

Curcumae rhizoma

Curcumol

LoVo colorectal cancerous cell line

0.11 μM

Chen et al. (2021) (Continued )

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TABLE 37.1 (Continued) Sr. No

Essential oilbearing plant

Main component

Cancer cell line

IC50 value

References

16

Agarwood (Aquilaria crassna)

Synergistic effect of β-caryophyllene, 1phenanthrenecarboxylic acid, azulene, naphthalene, and cyclodecene

Pancreatic cell line MIA PaCa-2

11 6 2.18 μg/mL

Dahham et al. (2016)

17

Thymus numidicus

Thymol

Colonic cancer (HCT116) and breast adenocarcinoma (MCF7)

26.9 and 11.7 μg/mL

Elaissi et al. (2021)

18

Araucaria heterophylla, Araucaria bidwillii

α-Pinene, beyerene

Human liver cancer cell line (Hep-G2), human breast adenocarcinoma (MCF-7), and human colon cancer (Caco-2) cell line

0.70, 3.20, and 1.10 μg/ mL, 1.67, 1.10, and 1.32 μg/ml

Elkady and Ayoub (2018)

19

Moringa oleifera and M. peregrina

Oleic acid

Zebrafish (Danio rerio) embryos

LD50 values of 21.24 6 0.44 and 25.11 6 0.547 μg/mL

Elsayed et al. (2020)

20

Nigella sativa

Thymoquinone

Colon cancer (HT29), lymphoblastic leukemia (CEMSS), and promyelocytic leukemia (HL60) cell lines

8, 5, and 3 μg/mL

Mollazadeh et al. (2017)

21

Mentha spicata




Human ductal breast epithelial tumor T47D, human colon cancer HCT116, and human breast adenocarcinoma MCF-7 cell lines

LD50 value of 324 6 81, 975 6 156, and 279 6 52 μg/mL for T47D, MCF-7, and HCT-116 cell lines

Mahendran et al. (2021)

22

Ginger

6-shogaol

Human cervical carcinoma (HeLa) cell line

14.75 μM

Mahomoodally et al. (2021)

23

Schinus polygamus bark, S. polygamus leaf

dl-Limonene and E-caryophyllene

Human liver cancer cells (HepG2) and human colon cancer cells (Caco-2)

1.56 and 7.55 μg/mL, 3.78 and 24.12 mg/mL

Torky et al. (2021)

24

Cupressus sempervirens

α-Pinene, 3- carene, cedrol, terpinolene, and sabinene

Amelanotic melanoma C32 cell line

104.90 μg/mL

Loizzo et al. (2008)

25

Salvia rubifolia

y´-Muurolene, 1-epicubenol, transpinocarvyl acetate, thujone, α-pinene, and p-cymene

Melanoma cancer cell line (M14)

12.5 μg/mL

Cardile et al. (2009)

26

Morinda citrifolia

Nordamnacanthal and β-morindone

Human lung cancer (A549) cell line

40 μg/mL

Rajivgandhi et al. (2020)

27

Ballota undulata, Ballota saxatilis, Ballota nigra

Germacrene D, linalool

Hepatoma HepG2 and breast cancer MCF-7 cell line

100 μg/mL

Rigano et al. (2017)

28

Citrus pyriformis, Citrus jambhiri

D-limonene

Hepatocellular carcinoma (HepG2) cell line

374.36, 588.06 g/mL

Singh et al. (2021) (Continued )

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TABLE 37.1 (Continued) Sr. No

Essential oilbearing plant

Main component

Cancer cell line

IC50 value

References

29

Virola surinamensis bark and leaves

Aristolene, α-farnesene

Human colon carcinoma HCT116 cells

For EOB:9.41 to 29.52 μg/mL (HCT116 and B16
F10), for. EOL: 7.07 to 26.70 μg/mL (HepG2 and HCT116)

da Anunciac¸ao et al. (2020)

30

Alpinia oxyphylla Miq. (AOEOs), Kaempferia galanga L. (KGEOs), Amomum kravanh Pierre ex Gagnep. (AKEOs), Alpinia galanga (Linn.) Wild (AGEOs)




Prostate cancer cell line (LNCaP), Murine melanoma cell line (B16)

18.42
113.76 μg/mL, 54.11
101.31 μg/mL

Zhang et al. (2020)

31

Platycladus orientalis

Citral, γ-terpinene, and D-limonene

Human normal liver LO2 cell line, breast cancer MCF-7 cell line, human lung carcinoma (A549) cell lines

200 μg/mL

Zhu et al. (2020)

32

Juniperus oxycedrus

α-pinene and β-myrcene

Estrogen receptor-positive breast cancer cells




El-Abid et al. (2019)

33

Nigella sativa

Thymoquinone

Pancreatic cancer cell line




Ahmad et al. (2021)

34

Cardamom

1,8-cineole and limonene




Dose of 100 and 200 mg/kg/day for 26 weeks causes anticancer effect

Ashok Kumar et al. (2020)

35

S. triloba




DMBA/TPA mouse model of skin carcinogenesis

Obstructs tumor multiplicity by 78% and reduces tumor weight by 80%.

Gali-Muhtasib (2006)

36

Coriander

Linalool

Breast cancer cell line




Laribi et al. (2015)

respectively (Fig. 37.1). Isoledene and elemene were identified as the major components in the oil by GC-MS and HPLC analysis (Asif et al., 2019). In another study, Asif and collaborators reported the anti-colon cancer action of Illicium verum fruit essential oil. Among various cell lines tested, the essential oil showed the highest cytotoxicity against the HCT 116 colon cell line with an IC50 value of 50.34 6 1.19 μg/mL (Fig. 37.2). Induction of apoptosis and inhibition of key steps of metastasis were the mechanisms attributed to the antitumor activity of the oil. Trans-anethole was identified as a major chemical in volatile oil and was confirmed by GC-MS analysis (Asif et al., 2016). Bayala and colleagues tested the antimetastatic effects of essential oils derived from Cymbopogon species such as Cymbopogon citratus (DC.) Stapf. and Cymbopogon giganteus Chiov. on prostate cancer and glioblastoma cell lines using MTT assay. The essential oil of C. citratus demonstrated maximum cytotoxicity on prostate cancer cell lines, LNCaP and PC-3, with IC50 values of 6.36 and 32.1 μg/mL, respectively (Fig. 37.3). The volatile oil also showed promising results on glioblastoma cell lines, SF-767 and SF-763, with IC50 values of 45.13 and 172.05 μg/mL, respectively (Fig. 37.3). Citral was identified to be the major component of the essential oil following chemical and functional study whose activity was statistically equal to the activity of essential oil. Consequently, the antiproliferative effect of essential oil may be due to citral (Bayala et al., 2018).

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FIGURE 37.1 Anticancer activity of isoledene and elemene against colon cancer.

FIGURE 37.2 Anticancer activity of trans-anethole against colon cancer.

FIGURE 37.3 Anticancer activity of citral against prostate and glioblastoma cancer.

FIGURE 37.4 Anticancer activity of carvacrol and thymol against colorectal cancer.

FIGURE 37.5 Anticancer activity of boswellic acid against breast cancer.

Eren and group examined the in vitro concentration-dependent cytotoxic effect of Thymus fallax essential oil against human colorectal adenocarcinoma DLD-1 cell lines. Essential oil (carvacrol and thymol) was found to be cytotoxic to DLD-1 cell lines (IC50 5 0.347 mg/mL) (Fig. 37.4). Carvacrol, p-cymene, thymol, and γ-terpinene were found to be the major constituents in oil and were responsible for cytotoxic activity. Antioxidant effect of the oil was attributed as the mechanism for cytotoxic property (C¸etinus et al., 2013). Mahmoud and their team demonstrated that Boswellia sacra essential oil prepared from hydrodistillation had tumor cell-specific cytotoxicity in multiple human breast cancer cell lines such as T47D, MCF7, MDA-MB-231, etc. The plant oil was found to be rich in constituents such as α-pinene, α-thujene, β-pinene, myrcene, and boswellic acid. Boswellic acid was the major component among all other constituents. The volatile oil was found to be cytotoxic to human breast cancer cell lines in different dilutions which were reported as their corresponding IC50 values: 1:900 for TD47, 1:1000 for MCF 7, and 1:950 for MDA-MB-231 cell line (Fig. 37.5) (Suhail et al., 2011).

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The cytotoxic potential of the volatile oil extracted from Amomum tsao-ko was studied by Yang and coworkers. The essential oil demonstrated cytotoxicity toward HepG2, Hela, Bel-7402, SGC-7901, and PC-3 cell lines with HepG2 carcinoma cell line having the lowest IC50 value of 31.80 6 1.18 μg/mL. 1,8-Cineole was the principal constituent found in the essential oil of Amomum tsao-ko (Fig. 37.6) (Yang et al., 2010). Ana Cecilia Mesa-Arango and coauthors evaluated the cytotoxic activity of citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown using tetrazolium dye method. Citral and carvone chemotypes showed dose-dependent cytotoxic effect against HeLa cells. Citral exhibited the highest cytotoxicity against HeLa tumor cell lines with a 50% cytotoxic concentration (CC50) value ,0.1 μg/mL (Fig. 37.7) (Mesa-Arango et al., 2009). Mark Barton Frank and his collaborators prepared frankincense oil from hardened gum resins obtained from Boswellia trees. Boswellic acid was found to be the main component of frankincense oil which is well known for its antitumor properties. Frankincense oil was evaluated for its antitumor activity and action on signaling pathways in bladder cancer cells. They found that frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells. The essential oil was found to be cytotoxic to cancer cells even after dilution at 1: 1100 in .24 hours (Fig. 37.8) (Frank et al., 2009). Saulo Lus da Silva and colleagues inspected the cytotoxic effects of Casearia sylvestris sw essential oil on human cancer cells, namely, HeLa, A-549, and HT-29. Native Americans from Brazil, Peru, and Bolivia utilize the herb C. sylvestris, also known as guac¸atonga, to treat a variety of illnesses, including cancer. The presence of carsearins and a larger percentage of powerful cytotoxic sesquiterpenes, particularly β-caryophyllene and α-humulene, are the major reasons due to which the essential oil possesses antitumor effects. The essential oil displayed median effective dose (ED50) values of 63.3, 60.7, and 90.6 μg/mL against HeLa, A-549, and HT-29 tumor cells (Silva et al., 2008). Saulo Luis da silva and his research team extracted volatile oil from Zanthoxylum rhoifolium Lam. leaves and evaluated its antimetastatic action against HeLa, A-549, HT-29, and Vero cell lines. Essential oil displayed ED50 values of 82.3, 90.7, and 113.6 μg/mL against HeLa, A-549, and HT-29 tumor cells, respectively. They also tested some of the terpenes from essential oil including β-caryophyllene, α-humulene, α-pinene, myrcene, and linalool to investigate their likely contribution toward the cytotoxic action of essential oil. It was confirmed that the antitumor activity of essential oil was due to the synergistic interaction of all the components present in it (Silva et al., 2007).

FIGURE 37.6 Anticancer activity of 1,8-cineole against liver cancer.

FIGURE 37.7 Anticancer activity of citral against cervical cancer.

FIGURE 37.8 Anticancer activity of boswellic acid against bladder cancer.

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Eitan Amiel and his group described the antiproliferative and proapoptotic effect of the essential oil derived from Commiphora gileadensis and its derivative compound β-caryophyllene on human lymphocytes B transformed through Epstein
Barr virus (MoFir) cancer cell line. Phytochemical screening results confirmed that β-caryophyllene is a key constituent in the essential oil of the plant. β-Caryophyllene demonstrated selective activity against the tumor cells (IC50 value: 2.5 μL/mL toward the MoFir cell line) and strongly induced apoptosis along with DNA ladder and caspase-3 catalytic activity (Fig. 37.9) (Amiel et al., 2012). The antitumor effects of essential oil from rosewood Aniba rosaeodora (REO) were studied by Jeremie Soeur and colleagues on human epidermoid cancer cell line A431, immortal HaCaT cells, transformed normal HEK001 keratinocytes, and primary normal NHEK keratinocytes. Essential oil exhibited anticancer activity by inducing selective apoptosis in malignant and precancerous skin cells whereas normal skin cells were not substantially harmed by it. Additionally, researchers discovered that at specific doses, REO specifically killed precancerous HaCaT cells and A431 human epidermoid carcinoma cells. Linalool is one of the main chemical constituents of REO. The authors mainly evaluated the effect of REO on viability of different cancer cells by performing MTT assay. They observed that REO at a concentration of 400 nL/mL induced significant cell death in A431 and HaCaT cells while it was only slightly toxic toward HEK001 and NHEK cells (Sœur et al., 2011). Thangaiyan Rabi and his colleagues used human prostate cancer DU-145 cells to test the in vitro anticancer potential of D-limonene in conjunction with docetaxel. This study depicted that D-limonene boosted the antitumor effect of docetaxel against prostate cancer cells without being toxic to normal prostate epithelial cells. It killed prostate cancer DU-145 cells more effectively with an IC50 value of 9.4 mM (Fig. 37.10) (Rabi & Bishayee, 2009). Abdelhakim Bouyahya and his research group investigated antiproliferative effect of Origanum majorana L. essential oil on three human tumor cell lines, Hep2, HT29, and Vero. The results showed that O. majorana essential oil inhibited the proliferation of Hep2, HT29, and Vero with IC50 values of 85.63 6 2.38, 13.73 6 1.31, and 70.13 6 1.72 mg/mL, respectively (Fig. 37.11). The antitumor potential of the volatile oil may be due to carvacrol which was identified as the main chemical constituent (Bouyahya et al., 2021). Fengli chen and collaborators studied the cytotoxic activity of essential oil isolated from Perilla frutescens (L.) Britt. Plant essential oil was separated by ultrasound pretreatment combined with microwave-assisted hydrodistillation method. It exerted promising cytotoxic activity against MGC-803 and A549 cell lines. The IC50 value of essential oil was found to be 17.82 6 5.12 μg/mL against MGC-803 and 21.31 6 0.98 μg/mL against A549, indicating the potential

FIGURE 37.9 Anticancer activity of β-caryophyllene.

FIGURE 37.10 Anticancer activity of D-limonene against prostate cancer.

FIGURE 37.11 Anticancer activity of carvacrol against liver and colon cancer.

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of this plant’s essential oil in anticancer drug development (Fig. 37.12). Perilla ketone was found to be the main oxygenated monoterpene in oil which might be responsible for activity (Chen et al., 2020). Yi Chen and his research team studied anticancer effects of β-elemene, furanodiene, furanodienone, germacrone, curcumol, and curdione named terpenoids obtained from Curcumae rhizoma. They found that curcumol showed the most potent anticancer activity against LoVo colorectal cancerous cell line with IC50 of 0.11 μM (Fig. 37.13) (Chen et al., 2021). Sad and his team explored the effectiveness of Agarwood (Aquilaria crassna) essential oil in the inhibition of tumor and initiation of programmed cell death in MIA PaCa-2 pancreatic cell line. Hydrodistillation technique was utilized for the separation of volatile oil from A. crassna. FTIR and GC-MS were used to analyze the separated essential oil. The chemical profile of oil depicted the presence of β-caryophyllene, 1-phenanthrenecarboxylic acid, azulene, naphthalene, and cyclodecene. The volatile oil showed potent cytotoxic activity against MIA PaCa-2 cells with an IC50 value of 11 6 2.18 μg/mL. The observed anticancer effect of agarwood was attributed to the combined effect of various biologically active plant constituents present in the essential oils (Dahham et al., 2016). Ameur Elaissi and his collaborators studied the antiproliferative potential of volatile oil separated from Thymus numidicus L. Aerial parts of the plant were subjected to extraction using hydrodistillation process to obtain essential oil, and volatile oil constituents were analyzed by GC-MS analysis. Thymol was identified as the main component of T. numidicus L. essential oil. Further, MTT assay was performed to assess the in vitro antiproliferative potential of essential oil on two human cancer cell lines, namely, the colonic (HCT116) and breast adenocarcinoma (MCF7). The essential oil (thymol) exhibited reasonable antitumor activity against both cancer cell lines, with IC50 values of 26.9 and 11.7 μg/mL, respectively (Fig. 37.14) (Elaissi et al., 2021). Wafaa M. Elkady and coworkers evaluated the antiproliferative effect of essential oil obtained from two Araucaria species mainly Araucaria heterophylla (Salisb.) and Araucaria bidwillii Hook. Both the volatile oils were initially analyzed by GC and GC-MS, and further cytotoxicity was assessed on three cancer cell lines, namely, Hep-G2, MCF-7, and Caco-2, using MTT assay. Both oils demonstrated potent cytotoxicity against all tested cancer cell lines (Hep-G2, MCF-7, and Caco-2) with IC50 values of 0.70, 3.20, and 1.10 μg/ mL for A. heterophylla and 1.67, 1.10, and 1.32 μg/ mL for A. bidwillii, respectively. α-Pinene was identified as the major constituent in A. heterophylla and beyerene for A. bidwillii (Elkady & Ayoub, 2018). E.A. Elsayed and his research group investigated in vivo antiangiogenic effects of essential oils from Moringa oleifera and M. peregrina seeds on zebrafish (Danio rerio) embryos. Both the plant seeds are rich in unsaturated omega fatty acid with oleic acid being the major component. Seed oils from M. oleifera and M. peregrina were lethal to zebrafish embryos at lethal dose (LD50) values of 21.24 6 0.44 and 25.11 6 0.547 μg/mL, respectively (Fig. 37.15) (Elsayed et al., 2020). FIGURE 37.12 Anticancer activity of perilla ketone against gastric and lung cancer.

FIGURE 37.13 Anticancer activity of curcumol against colorectal cancer.

FIGURE 37.14 Anticancer activity of thymol against breast cancer.

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FIGURE 37.15 Anticancer activity of oleic acid on zebrafish embryos.

FIGURE 37.16 Anticancer activity of thymoquinone against colon, lymphoblastic leukemia, and promyelocytic cancer.

FIGURE 37.17 Anticancer activity of 6-shogaol against cervical cancer. FIGURE 37.18 Anticancer activity of dllimonene and E-caryophyllene against liver and colon cancer.

Hala Gali-Muhtasib investigated the chemoprotective capabilities of oil extract or essential oil of S. triloba in the 7,12-dimethylbenzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) mouse model of skin carcinogenesis. They observed that topical application of the essential oil to the skin of Balb-c mice interrupted the tumor appearance by 4 weeks, obstructed the tumor multiplicity by about 78%, and reduced the weight of tumors by 80% (Gali-Muhtasib, 2006). Hamid Mollazadeh and his research group studied the cytotoxicity and the antiproliferative effects of thymoquinone (TQ) obtained from Nigella sativa essential oil toward HeLa cell line. TQ depicted time-dependent cytotoxic and antiproliferative actions toward the HeLa cell line with IC50 values of 2.80 6 0.10 and 5.37 6 0.12 mg/mL, respectively. Further, they also investigated the cytotoxic and antiproliferative effects of TQ on the HT29, CEMSS, and HL60 cell lines. IC50 values of TQ were found to be 8, 5, and 3 μg/mL, respectively (Fig. 37.16) (Mollazadeh et al., 2017). The anticancer properties of coriander seed essential oil were revealed by Bochra Laribi et al. Linalool was identified as the primary component of the oil and was believed to possess anticancer properties (Laribi et al., 2015). Ganesan Mahendran and his collaborators investigated the antiproliferative effect of Mentha spicata oils toward different types of cancerous cell lines, namely, human ductal breast epithelial tumor T47D cell line, human colon cancer HCT-116, and human breast adenocarcinoma MCF-7 cell lines. The oil exhibited LD50 values of 324 6 81 μg/mL, 975 6 156 μg/mL, and 279 6 52 μg/mL for T47D, MCF-7, and HCT-116 cell lines, respectively, demonstrating its therapeutic potential in treating a few types of cancers (Mahendran et al., 2021). Mahomoodally and coworkers reported the activity of 6-shogaol against cervical cancer using HeLa cells. The 6-shogaol showed IC50 value of 14.75 μM against HeLa cells (Fig. 37.17). Additionally, the essential oil of ginger was also evaluated for its anticancer potential against SiHa cell line. The essential oil of ginger showed a potent IC50 value of 38.6 μg/mL, which was found to be more effective than α-zingiberene. Apart from cervical cancer, ginger essential oil and its components were also found to be effective in the inhibition of breast, prostate, liver, blood, and colorectal cancer (Mahomoodally et al., 2021). Heba A. S. El-Nashar and investigators studied the cytotoxic activities of essential oils from Schinus polygamus (Cav.) cabrera leaf and bark. The S. polygamus bark (SPB) and S. polygamus leaf (SBL) oils inhibited human liver cancer cells (HepG2) with IC50 values of 1.56 and 7.55 μg/mL, respectively (Fig. 37.18).

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S. polygamus essential oil also inhibited human colon cancer cells (Caco-2) with IC50 values of 3.78 and 24.12 mg/ mL, respectively. GC-MS analysis gave an idea about the composition of bark and leaf essential oil obtained from S. polygamus. dl-Limonene and E-caryophyllene were found as the main constituents in SPB and SBL essential oil (Torky et al., 2021). M. R. Loizzo and collaborators evaluated anti-melanoma activity of essential oil from Cupressus sempervirens L. on amelanotic melanoma C32 cell line. They reported an IC50 value of 104.90 μg/mL against C32 cell line. α-Pinene, 3-carene, cedrol, terpinolene, and sabinene were identified as the main components from the essential oil (Loizzo et al., 2008). Venera Cardile and the research group tested the anti-melanoma potential of Salvia rubifolia essential oil using the M14 cell line. The chemical constituents reported in the essential oil of the plant were Ƴ-muurolene, 1-epi-cubenol, trans-pinocarvyl acetate, thujone, α-pinene p-cymene, etc. The plant essential oil exhibited an IC50 value of 12.5 μg/mL against M14 melanoma cancer cell line (Cardile et al., 2009). Govindan Rajivgandhi and coauthors studied the anticancer activity of chitosan-loaded Morinda citrifolia essential oil against A549 human lung cancer cells. Chitosan nanoparticles were prepared by using the essential oil of M. citrifolia followed by evaluation of their cytotoxic effect against A549 cancer cell line. Nanoparticles showed 54% inhibition of cancer cell growth. IC50 value was found to be 40 μg/mL. GC-MS analysis revealed the presence of total of 29 compounds of which nordamnacanthal and β-morindone were found as the major constituents followed by L-scopoletin, morindadiol, and α-morenone (Rajivgandhi et al., 2020). Daniela Rigano and his team investigated the cytotoxic effects of essential oils from three different Ballota species, namely Ballota undulata, Ballota saxatilis, and Ballota nigra ssp. Foetida is primarily grown in Europe. The cytotoxicity of all three essential oils was evaluated on hepatoma HepG2 and breast cancer MCF-7 cell line. They found that the essential oil of B. undulata exhibited the maximum antiproliferative activity against HepG2 cells with a percentage inhibition value of 81.36%. Moreover, the essential oil from B. saxatilis demonstrated maximum antiproliferative activity against MCF-7 cells with a percentage inhibition value of 24.18% at a concentration of 100 μg/mL (Fig. 37.19). The sesquiterpene germacrene D was identified as the main component in essential oil of B. undulata responsible for the cytotoxicity against different human cancer cell lines. Linalool was found as a prime constituent in B. saxatilis (Rigano et al., 2017). Sharada H. Sharma and research group reported various terpenoids useful in the treatment of colon cancer. Terpenoids like D-limonene, perillyl alcohol, carvacrol, D-carvone, geraniol, carnosol, pseudolaric acid, triptolide, kahweol, betulinic acid, oleanolic acid, escin, glycyrrhizic acid, lycopene, and β-carotene were reported for activity against colon cancer. Terpenoids are a major part of essential oil obtained from plants. All the above terpenoids listed showed activity against colon cancer via cell cycle arrest and apoptosis induction. Some terpenoids interrupted various stages of the adenoma
carcinoma sequence of colon cancer (Sharma et al., 2017). The cytotoxic effects of Citrus pyriformis and Citrus jambhiri peel essential oils (EOs) on the hepatocellular carcinoma (HepG2) cell line were investigated by Singh and his colleagues. The IC50 values for these two essential oils were 374.36 and 588.06 g/mL, respectively. Further, they explored the cytotoxic effect of essential oil on pancreatic cancer (MIA-PaCa-2) cell line using MTT assay and reported the IC50 values of 213.87 and 512.45 g/mL, respectively. Citrus reticulata peel essential oil lowered cell viability by regulating lipid metabolic pathways, promoting cell cycle arrest, inducing apoptosis, and inhibiting the proliferation of pulmonarycarcinoma A549 cell lines. The presence of a larger concentration of D-limonene may be the cause of the stronger cytotoxic action of C. pyriformis peel essential oil (Fig. 37.20) (Singh et al., 2021). Talita A. da Anunciacao and colleagues investigated the in vitro inhibition of human colon carcinoma HCT116 cells by essential oils obtained from the bark and leaves of Virola surinamensis. Hydrodistillation process was used to separate essential oil of bark and leaves of V. surinamensis. In vitro cytotoxic activity was determined in a panel of cultured

FIGURE 37.19 Anticancer activity of germacrene D and linalool against liver and breast cancer.

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FIGURE 37.20 Anticancer activity of D-limonene liver and pancreatic cancer.

FIGURE 37.21 Anticancer activity of aristolene and α-farnesene against colon and liver cancer.

cancer cells, namely, HCT116, HepG2, HL-60, B16-F10, and MCF-7, and also in a non-cancerous cell line MRC-5 by the Alamar blue assay. Furthermore, analysis of the oil by gas chromatography coupled with mass spectroscopy revealed aristolene and α-farnesene to be the main components in essential oil from bark and leaves, respectively. They found that essential oil from bark showed IC50 values ranging from 9.41 to 29.52 μg/mL for HCT116 and B16
F10, while essential oil from leaves showed IC50 values from 7.07 to 26.70 μg/mL for HepG2 and HCT116. The IC50 value for the non-cancerous MRC-5 cell was 34.7 and 38.93 μg/mL for essential oil from bark and leaves, respectively (Fig. 37.21) (da Anunciac¸ao et al., 2020). Lanyue Zhang and coworkers evaluated bioactivities of essential oils obtained from four genuine Zingiberaceae herbs, namely, Alpinia oxyphylla Miq. (AOEOs), Kaempferia galanga L. (KGEOs), Amomum kravanh Pierre ex Gagnep. (AKEOs), and Alpinia galanga (Linn.) wild (AGEOs). The four essential oils revealed marked cytotoxicity against LNCaP cells (IC50 value of 18.42
113.76 μg/mL) and B16 cells (IC50 value of 54.11
101.31 μg/mL); KGEOs demonstrated maximum cytotoxic activity among the four essential oils (Zhang et al., 2020). Jing-Jing Zhu and investigators studied anticancer activities of essential oil separated from Platycladus orientalis against LO2, MCF-7, A549 cell lines using MTT assay. The maximum safe concentration of essential oil was found to be 200 μg/mL using human normal liver LO2 cells. The essential oils were found to have high concentrations of alkenes, including citral, γ-terpinene, and D-limonene, which are essential for numerous cellular functions, including cell cycle regulation (Zhu et al., 2020). Hassan et al. explored anticancer properties of essential oils obtained from fruits and leaves of Juniperus oxycedrus L. on estrogen receptor-positive breast cancer cells. The main constituents responsible for antitumor activity of the plant were α-pinene and β-myrcene. They also reported that essential oils from fruits had advanced efficacy on MCF-7 cells compared to essential oils of leaves (El-Abid et al., 2019). Ahmad and group described that thymoquinone from N. sativa essential oils exhibits outstanding anticarcinogenic, antimutagenic, and antineoplastic activity against various tumor cells. This compound is useful against pancreatic cancer and acts by inducing apoptosis and inhibiting the proliferation of pancreatic ductal adenocarcinoma cells. Its effectiveness in treating hepatic, renal, and prostate cancer is also proved scientifically by other researchers (Ahmad et al., 2021). In vivo anticancer potential of volatile oil of cardamom capsules was studied by Ashok Kumar and his colleagues. The main active ingredients in the oil are 1,8-cineole and limonene, which have been shown to protect against cancer growth.

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They found lowered blood creatinine and urea levels, as well as LDH activity, in rats subjected to diethyl nitrosamine (DENA)-induced oxidative stress after receiving cardamom essential oil (CEO) at doses of 100 and 200 mg/kg/day for 26 weeks (Ashok Kumar et al., 2020).

37.3

Conclusion and future perspective

Cancer is an extremely perilous disease characterized by uncontrolled and unregulated growth of normal cells. It is one of the leading causes of death globally. Currently, treatment options are scarce and are limited in practice due to the emergence of multidrug resistance. Moreover, chemotherapy and radiation therapies are debilitating, and many patients find it difficult to bear them. Furthermore, early detected cancers can be treated to prevent metastasis of cancer; however, this becomes difficult as cancer progresses. Thus, there is a dire need for the identification and development of new anticancer agents as therapeutics. Medicinal and aromatic plants have always provided remarkable medicines to treat diverse diseases from ancient times. Essential oils separated from aromatic plants have shown great potential in curing various cancers of the blood, stomach, colon, prostate, ovary, mouth, lung, etc. They mainly consist of various terpenoid molecules of monoterpene, sesquiterpene, triterpene, and tetraterpene classes. EOs have applications in various other sectors like cosmetics, nutraceuticals, perfumes, flavors, and medicine. Keeping in mind the potential of EO for these huge applications, in this chapter we have discussed essential oils having medicinal and aromatic plants and their potent chief volatile components which have shown promising results in the prevention and cure of lethal cancer illness. We have additionally provided their IC50 values, cancer cell lines used for the study, and the mechanism by which EO and their components showed anticancer activity. We believe that this will be a piece of valuable information for the upcoming natural chemist for finding new hits against various malignancies.

Abbreviations EOs AIDS HCT 116 LIM1215 GC-MS HPLC IC50 value LNCaP PC-3 SF-767 SF-763 DLD-1 T47D MCF7 MDA-MB-231 HepG2 Hela Bel-7402 SGC-7901 µg/mL CC50 value J82 A-549 HT-29 ED50 value HaCaT MTT nL/mL mM

Essential oils acquired immuno deficiency syndrome human colon cancer cell line human colon cancer cell line gas chromatography-mass spectroscopy high performance liquid chromatography concentration at which a substance exerts half of its maximal inhibitory effect prostate cancer cell line prostate cancer cell line glioblastoma cell line glioblastoma cell line human colorectal adenocarcinoma cell line human breast cancer cell line human breast cancer cell line human breast cancer cell line human liver carcinoma cells cervical cancer cell line hepatocellular carcinoma cell lines human gastric cancer cell line microgram per mililiter 50% cytotoxic concentration bladder transitional carcinoma cell line non-small-cell lung carcinoma human colorectal adenocarcinoma cell line effective dose for 50% of the population keratinocyte cell line from adult human skin microculture tetrazolium assay nanoliter per mililiter millimolar

Essential oils as anticancer agents Chapter | 37

mg/mL MGC-803 FTIR Caco-2 LD50 value µM SiHa HL-60 B16
F10 MCF-7aro CEMSS MoFir

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milligram per milliliter human gastric cell line Fourier-transform infrared spectroscopy human epithelial colorectal adenocarcinoma cell line measurement of a lethal dose of a substance micromole cell line isolated from fragments of a primary uterine tissue sample human leukaemia cell line murine melanoma cell line breast cancer cell line lymphoblastic leukemia Epstein-Barr virus-transformed human B lymphocytes.

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Chapter 38

Molecular docking study of bioactive phytochemicals against cancer Sandhya Jain and Surya Prakash Gupta Department of Pharmaceutical Sciences & Technology, Faculty of Pharmaceutical Sciences & Technology, AKS University Satna, Satna, Madhya Pradesh, India

38.1

Introduction

Naturally happening compounds from plants called phytochemicals serve as important sources for novel drugs and are also sources for the remedy of most cancers. Some ordinary examples encompass taxol analogs, vinca alkaloids such as vincristine, vinblastine, and podophyllotoxin analogs (Avonce et al., 2006). These phytochemicals frequently act via regulating molecular pathways which are implicated in the increase and development of most cancers. Phytochemicals and derivatives (Beena and Joji, 2010) found in vegetation are promising alternatives to enhance treatment efficiency in cancer patients and reduce unfavorable reactions. A number of these phytochemicals are naturally happening biologically, lively compounds with enormous anticancer ability. Most cancers are a complex disease compared to many other human sicknesses and hence could have many such potential molecular objectives for the improvement of therapeutics. Out of about 30,000 human genes, around 6000
8000 websites are expected to have potential pharmacological targets, out of which the best 400 encoded proteins were proved to be powerful within the process of drug development until now. Hopkins has discovered a new drug discovery approach (Chaitra and Jeyanthi, 2011) referred to as network pharmacology which stresses upon the idea of “network target, multicomponent therapeutics.” This has changed the idea of one gene, one goal, and one disorder. The conventional drug discovery methods such as animal experiments and in vitro analyses (Łopie´nska-Biernat et al., 2018) are time-consuming, high priced, and hard in nature; it has been predicted that around 12 years and 2.7 billion USD, on average, are required for a brand-new drug discovery through conventional techniques. Exploring computational prediction equipment can assist screen a bulk quantity of molecules for their anticancer ability in a very short period, without tons of cost and sacrificing animals. This may provide the initial statistics for further in vitro and animal testing, which may enhance the success rate of the study (Lipinski, 2004). So nowadays computer-aided drug discovery strategies had been broadly used to increase the effectiveness of the drug discovery and development pipeline in drug research, subject to the cause and systems of interest. Molecular docking is one of the most applied digital screening methods that is used to predict the interplay between receptors and ligands. This approach could predict both the binding affinity and the structure of protein
ligand complex which is valuable information for lead optimization (Oleg and Arthur, 2010). A new generation of cancer therapy has begun with the identification and characterization of diverse target proteins that govern cell growth, differentiation, motility, and apoptosis. Many researches are underway globally to investigate their scientific consequences of phytochemicals with anticancer properties.

38.2

Molecular docking of bioactive phytochemicals with anticancer properties

Clinical evidences indicate that phytochemicals have significant antitumor potential. Approximately, 50% of approved anticancer pills from 1940 to 2014 originate from natural products or without delay derived therefrom (Newman & Cragg, 2020). Some of the splendid anticancer phytochemicals in this regard are described in the present evaluation. These phytochemicals had been tested for most anticancer efficacy at both in vitro and in vivo levels. They possess complementary Recent Frontiers of Phytochemicals. DOI: https://doi.org/10.1016/B978-0-443-19143-5.00001-3 © 2023 Elsevier Inc. All rights reserved.

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and overlapping mechanisms to slow down the carcinogenic process by using scavenging free radicals (Lee et al., 2013), suppressing survival and proliferation of malignant cells (Yan et al., 2018), as well as diminishing invasiveness and angiogenesis of tumors (Lu et al., 2018a). They exert a wide and complex variety of actions on unique molecular targets and signal transduction pathways such as membrane receptors (Deng et al., 2017), kinases (Dou et al., 2018), downstream tumor-activator or -suppressor proteins (Adams et al., 2010), transcriptional elements (Zhang et al., 2017b), microRNAs (miRNAs) (Cojocneanu et al., 2015), cyclins, and caspases (Yan et al., 2018). All research articles showed promising binding affinity (Harish et al., 2013) toward the two target proteins in the molecular docking research (Garrett et al., 1998). From the findings, the plant may additionally enable us to develop reliable and effective drugs against various diseases. Further investigations to analyze its bioactivity and scientific trials are essential for the discovery of new drugs formulations (Raghunath et al., 2015). There are a few extra study papers. These consist of and show the bioactive phytochemicals docking study of some compounds which are very useful in the prevention and remedy of cancer. Group et al. (2001) worked on proteins such as EGFR, mTOR, p53R2, CTLA-4, and CDK8 and showed that it can serve as potential drug targets and their inhibition can control cancer progression. Ralte et al. (2022) worked on GC
Ms and molecular docking analyses of phytochemicals from the underutilized plant, Parkia timoriana, and proved it is by far a discovered candidate of anti-cancerous and anti-inflammatory agent. Devkarb et al. (2017) worked on 68 phytochemicals; they have been screened using pharmacokinetic parameters, which led to 38 lead molecules. Further, these 38 molecules were docked against PR, EGFR, mTOR, p53R2, CTLA4, and CDK8, resulting in the top 10 potential binders. Furthermore, bioavailability and toxicity evaluation of these top 10 molecules discovered that the phytochemicals (Farina et al., 2014) which include ursolic acid, enterolactone, parthenolide, berberine, and its derivative berberastine were observed to be safe and nontoxic. Therefore, these molecules may be taken into consideration as effective against the target proteins. Mendie and Hemalatha (2022) worked on molecular docking of phytochemicals targeting GFRs as therapeutic sites for cancer: an in silico study, and after completion of their research, they explored the potentials of various phytochemicals to inhibit cancer growth and development through modulation of growth factor receptors. Based totally on our evaluation, seven phytochemicals (Andrographolide, Curdione, Nectandrin B, Nimbolide, Salvicine, Withaferin A, and Hecogenic) possessed drug-likeness and pharmacokinetics activities that are comparable with the standard drugs (Dovitinib and Gefitinib). This exhibits that bioactive compounds have the potential to bind with GFRs causing inhibition of growth factors which in turn hinders cancer cell proliferation. In addition, research is needed to set up the pharmacodynamics and kinetic properties of these phytochemicals; also, the mechanism of action of these phytochemicals as nanoparticle carrier of anticancer drug for effective cancer treatment may be determined. Mahnashi et al. (2021) achieved their research on cytotoxicity, antiangiogenic, antitumor, and molecular docking research on phytochemicals isolated from Polygonum hydropiper L. They were isolated potent compounds which displayed enormous cytotoxicity against MCF-7, HeLA, and NIH/3T3 cells. CAM assay revealed antiangiogenic potentials, and antitumor assay indicates tumor-suppressing effect by our test samples. Molecular docking discovered the mode of action of compounds is mediated through the inhibition of EGFR, HER2, and VERGR receptors. Ghalloo et al. worked on phytochemical profiling, in vitro organic activities, and in silico molecular docking studies of Dracaena reflexa, and this study explored the methanolic extract, n-hexane, chloroform, and n-butanol fractions of D. reflexa regarding phytochemical analysis (Elizabeth et al., 2006) and biological activities. The comparative study data showed that the n-butanol fraction contained the best general phytochemicals in comparison to the other extract/ fractions, which showed its maximum antioxidant and enzyme inhibition (Kapil et al., 2019) (tyrosinase and cholinesterase) activities. The n-hexane fraction, due to the presence of antibacterial compounds, was also evaluated against Gram-positive and Gram-negative bacterial traces, which exhibited moderate to good antibacterial activity (Hossain et al., 2010). The GC
Ms evaluation of the n-hexane fraction provided tentative identification of antibacterial (benzyl benzoate, dodecane, tetradecane, and 5,9-undecadien-2-one, 6,10-dimethyl-, (E)-) and different potential secondary metabolites. The tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibition activities of Dracaena reflexa were in addition justified by using in silico molecular docking research (Huey et al., 2007) of GC-Ms-diagnosed ligands, beta-sitosterol, 9,12-octadecadienoic acid, octadecatrienoic acid, methyl ester, nutrition E, alpha-cadinol, n-hexadecanoic acid, and N-hydroxy-N0 -[2-(trifluoromethyl)phenyl] pyridine-3-carboximidamide, with these enzymes. Further research on fractionation, isolation, and structure elucidation of pure compounds of the extract/fractions of D. reflexa is currently in progress. Lutfiyaa et al. finished and studied on phytochemicals, and in this study, in silico analysis was performed to discover the therapeutic potential of numerous phytocompounds to inhibit angiogenesis through modulation of VEGF receptors. The general evaluation suggested that the phytocompounds have the ability to firmly bind with VEGF and

Molecular docking study of bioactive phytochemicals against cancer Chapter | 38

647

retard its development. These compounds can be similarly investigated using in vitro and in vivo studies for its pharmacodynamics and kinetic properties. Rosdi et al. worked on molecular docking studies of bioactive compounds from Annona muricata Linn. as capacity inhibitors for Bcl-2, Bcl-w, and Mcl-1 antiapoptotic proteins.

38.3

Conclusion

Eventually, we have given the general view of viable therapeutic relevance of selected bioactive phytochemicals. In the future, it will be realized that the inhibition of bioactive phytochemicals is a very valuable device in research, and in silico-based in vitro work will reduce the effort of inhibitor layout. Although pharmaceutical researchers have taken into consideration bioactive phytochemicals as prime applicants for drug improvement, however nonetheless the sector is in its infancy. Given the function of bioactive phytochemicals in innumerable organic processes, it seems nearly certain that bioactive phytochemical inhibitors will be a necessary part of the drugs of the next era.

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124. Beena, J., & Joji, R. (2010). Analysis of the essential oils of the stems, leaves andrhizomes of the medicinal plant costus pictus from southern india. International Journal of Pharmacy and Pharmaceutical Sciences, 2(2), 100
101, issn-0975
1491. Chaitra, P., & Jeyanthi, G. P. (2011). In silico drug designing approaches for latentautoimmune diabetes in adults (LADA). International Journal of Pharma and Bio Sciences, 2, B16
B27. Cojocneanu Petric, R., Braicu, C., Raduly, L., Zanoaga, O., Dragos, N., Monroig, P., Dumitrascu, D., & Berindan-Neagoe, I. (2015). Phytochemicals modulate carcinogenic signaling pathways in breast and hormone-related cancers. OncoTargets and therapy, 8, 2053
2066. Available from https://doi.org/10.2147/OTT.S83597. Deng, Y., Zhang, Y., & Lemos, B. (2017). Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Scientific Reports, 7, 46687. Available from https://doi.org/10.1038/srep46687. Elizabeth, W. P., Uwe, H., Eleftherios, M., Thomas, R., & Dena, T. (2006). Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans. Infection and Immunity, 74(10), 5877
5887. Farina, M., Preeti, B., & Neelam, P. (2014). Phytochemical evaluation, antimicrobial activity, and determination of bioactive components from leaves of Aegle marmelo. BioMed Research International, 497606, 11. Garrett, M. M., David, S. G., Robert, S. H., Ruth, H., & William, E. H. (1998). Automated docking using a lamarkian genetic algorithm and empirical binding freenergy function. Journal of Computational Chemistry, 19, 1639
1662. Harish, B. M., Devaraju, K. S., Gopi, A., Saraswathi, R., Babu, R. L., & Chidananda Sharma, S. (2013). In silico binding affinity study of calcineurin inhibitors to calcineurin and its close associates. Indian Journal of Biotechnology, 12, 213
217. Hossain, M. A., Kabir, M. J., Salehuddin, S. M., Rahman, S. M., Das, A. K., Singha, S. K., Alam, M. K., & Rahman, A. (2010). Antibacterial properties of essential oils and methanol extracts of sweet basil Ocimum basilicum occurring in Bangladesh. Le Pharmacien Biologiste, 48(5), 504
511. Huey, R., Morris, G. M., Olson, A. J., & Good Sell, D. S. (2007). A semi empirical free energy force field with charge based desolvation. Journal of Computational Chemistry, 28, 1145
1152. Kapil, S., Petit, C., Drago, V. N., Ronning, D. R., & Sucheck, S. J. (2019). Synthesis and in vitro characterization of trehalose-based inhibitors of mycobacterial trehalose 6-phosphate phosphatases. Chembiochem: A European Journal of Chemical Biology, 20(2), 260
269. Lipinski, C. A. (2004). Lead- and drug-like compounds: The rule-of-five revolution. Drug Discovery Today: Technologies, 1(4), 337
341. Łopie´nska-Biernat, E., Molcan, T., Paukszto, Ł., Jastrze˛bski, J. P., & Myszczy´nski, K. (2018). Modelling studies determing the mode of action of anthelmintics inhibiting in vitro trehalose-6-phosphate phosphatase (TPP) of Anisakis simplex s.l. Experimental Parasitology, 184, 46
56. Newman, D. J., & Cragg, G. M. (2020). Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. Journal of Natural Products, 83(3), 770
803. Available from https://doi.org/10.1021/acs.jnatprod.9b01285. Oleg, T., & Arthur, J. O. (2010). Auto Dock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31(2), 455
461, 2010. Raghunath, S., Konkimalla, V. B., & Jagnyeswar, R. (2015). In silico phylogenetic analysis and molecular modelling study of 2-haloalkanoic acid dehalogenase enzymes from bacterial and fungal origin. Advances in Bioinformatics, 2016, 1
10. Yan, D., Scott, R. L., Biederman, J. A., Barron-Gafford, G., Yang, X., Moore, D. J. P., Dannenberg, M. P., Wang, X., Knowles, J. F., Yang, J., Smith, W. K. (2018). Quantifying plant physiological response to water stress with high-frequency, near-surface observations of chlorophyll fluorescence and photochemical reflectivity. Abstract B31N-2694 presented at 2018 AGU Fall Meeting, Washington, D.C., 10-14 Dec.

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A ABC. See ATP-binding cassette (ABC) ABCC1, 586
587 ABCG2. See ATP-binding cassette (ABC) Abnormal endocrine function, 144 Abraxane, 264t ABTS. See 2,20 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) Acanthospermolides, 406 Acanthospermum hispidum, 406 Acanthus ilicifolius, 388, 390
391 ACC. See Acetyl CoA carboxylase (ACC) Accelerated solvent extraction (ASE), 20
21, 28
29, 43, 63, 68. See also Microwave-assisted extraction (MAE) advantages and disadvantages of, 43 potential applications of, 43 principles and working mechanisms, 43 ACE2. See Angiotensin-converting enzyme 2 (ACE2) Acetaminophen-induced nephrotoxicity, 115 Acetic acid, 406 Acetone, 34 Acetyl CoA carboxylase (ACC), 375
377 Acetylation, 225 Acetylcholinesterase (AChE), 297
298, 576
578 AChE. See Acetylcholinesterase (AChE) Achillea A. clavennae essential oil, 373t A. fragrantissima EO, 373t Acids, 74 Acorus calamus, 467t Acridine, 267
268 Acrolein, 484
485 ACS. See American Cancer Society (ACS) ACs. See Anthocyanins (ACs) ACSOs. See Alk(en)yl cysteine sulfoxides (ACSOs) Activator protein 1 (AP-1), 311
313, 490 Active component extraction, solvents used for, 33t Active compounds, 67
68 Active transfer channels, 465 Acute lymphocytic leukemia (ALL), 583 Acute myelogenous leukemia (AML), 264
265, 583 Acute respiratory distress syndrome (ARDS), 521

Acyclic monoterpenes, 355, 355f Acyl homoserine lactones (AHLs), 401
405, 436 Acylase, 401 AD. See Alzheimer’s disease (AD) Adenoma bracteosum, 165 Adenosine triphosphate (ATP), 621 hydrolysis, 585 inhibitory activity against ATP synthesis, 622 production, 622 Adiantum philippense, 423 Adipic semialdehyde, 484 Adjuvant chemotherapy, 263
264 Adrenal glands, 143
145 Adsorbents, 70t Adsorption, 69 Adsorption chromatography, 5
6 Aedes aegypti, 196
197 Aerobic metabolism, 98 Aeromonas hydrophila, 405 Aetheroleum, 353 Affinity chromatography, 5
6 AG. See Aloe gel (AG) Agar, 385t Agelas linnaei, 407 Ageratum conyzoides, 374 Aging, 114, 144 Aglycones, 359
360, 491 Agri-food by-products, phytochemicals from, 87
88 two-dimensional representation of bioactive phytochemicals, 87f AHLs. See Acyl homoserine lactones (AHLs) AIDS. See Anti-inflammatory medicines (AIDS) AIF. See Alpinumisoflavone (AIF) AIPs. See Autoinducing peptides (AIPs) Air-drying, 31 AIs. See Aromatase inhibitors (AIs); Autoinducers (AIs) AITC. See Allyl isothiocyanate (AITC) Ajwain essential oil, 373t ALA. See Alpha-linolenic acid (ALA) Alcohols, 74, 163, 353, 372, 629 Aldehyde dehydrogenase (ALDH), 281
282 Aldehydes, 353, 372 ALDH. See Aldehyde dehydrogenase (ALDH) Alginate, 385t Alk(en)yl cysteine sulfoxides (ACSOs), 10
11

Alkaloids, 46, 74, 86
87, 99t, 102
104, 184, 195
196, 203, 250, 261
262, 267
269, 295
296, 320
321, 339, 390
391, 401, 407, 503, 517
518, 535
536, 538, 545, 608 anticancer alkaloids with future perspective, 219 aporphinoid alkaloids, 215 cephalotaxus alkaloids, 217
219 different members of, 268t emetine and related alkaloids, 216
217 indole alkaloids, 205
210 camptothecin, 209 montamine, 210 vinblastine, 205 vincristine, 206 vindesine, 207 vinflunine, 208 isoquinoline alkaloids, 210
214 as microtubulin disrupting agents, 228
231 side effects of vinca alkaloids, 230
231 therapeutic relevance, 230 vinca alkaloids, 228
229 vinca domain, 229
230 pyrrolizidine alkaloids, 219 saponins, 189 taxus alkaloid, 214
215 theoretical relevance, 203
204 targeted pathways in cancer treatment, 204 types of alkaloids, 203
204 ALKP. See Serum alkaline phosphate (ALKP) ALL. See Acute lymphocytic leukemia (ALL) Allium A. cepa, 297 A. sativum, 297, 467t Allium cepa L. See Onions (Allium cepa L.) Allium sativum. See Garlic (Allium sativum) Allyl isothiocyanate (AITC), 423 Aloe barbadensis, 297 Aloe gel (AG), 131 Alpha-linolenic acid (ALA), 18 5-Alpha-reductase inhibitors, 183 α dimmers, 227f α-amyrin, 190, 406 α-amyrin acetate, 190 α-solanine, 268t α-tubulin, 225
226 Alpinia galanga essential oil (AGEOs), 639 Alpinia oxyphylla essential oil (AOEOs), 639 Alpinumisoflavone (AIF), 168t, 173t

649

650

Index

ALS. See Amyotrophic lateral sclerosis (ALS) Alzheimer’s disease (AD), 297, 463, 474t, 576 phytoconstituents-based nanoformulations for, 469
471 Alzheimer’s disorders, 102 Amalgamation, 571 American Cancer Society (ACS), 261
262 Amines, 74, 296 Amino acid transporter protein (ASCT2 protein), 465 Amino acid(s), 74, 104, 111, 172, 226, 557, 605 7A aminonoscapine, 237 AML. See Acute myelogenous leukemia (AML) Amomum A. kravanh, 639 A. tsaoko, 634 Amoora rohituka, 592 aMPV. See Avian metapneumovirus (aMPV) AMR. See Antimicrobial resistance (AMR) Amyotrophic lateral sclerosis (ALS), 463, 474t phytoconstituents-based nanoformulations for, 472 Analgesic, 360 Analogs origin of phytochemicals, 240f toxicity remarks of coumarin and, 239 Analyte, 75 Anaphase-promoting complex (APC), 235
236 Anastrozole, 262
263 Ancient medicine, 369
370 Androgen deprivation therapy, 182 Angiogenesis, 185 Angiotensin-converting enzyme 2 (ACE2), 512, 516, 546 Annona muricata, 646
647 Anopheles A. gambiae, 196
197 A. stephensi, 377 Anoxybacillus flavithermus, 417t, 419 Anthocyanidins, 337, 385t, 390
391 Anthocyanins (ACs), 10, 44, 46
47, 85, 97t, 488, 540, 605 compound, 605 pigments, 601 Anthroquinoids, 74 Anti-cancerous property, 611 Anti-diabetes activity, 537 Anti-inflammatory, 360, 391
392 Anti-inflammatory medicines (AIDS), 545 Anti-obesity activity, 537 Anti-ulcer property, 609
610 Antibiotics, 74, 624 resistance, 617 Anticancer, 101
102, 360 ability, 645 activity, 537 agents, 545 perceptions of phytochemicals as anticancer agents in history, 249 alkaloids with future perspective, 219 drugs, 163, 303, 335, 585 discovery, 336
337

phytochemicals, 313, 571 potential of essential oils, 629
640 of 1,8-cineole against liver cancer, 634f of 6-shogaol against cervical cancer, 637f of aristolene and α-farnesene against colon and liver cancer, 639f of boswellic acid against bladder cancer, 634f of boswellic acid against breast cancer, 633f of carvacrol against liver and colon cancer, 635f of carvacrol and thymol against colorectal cancer, 633f of citral against prostate and glioblastoma cancer, 633f of curcumol against colorectal cancer, 636f of D-limonene against prostate cancer, 635f of D-limonene liver and pancreatic cancer, 639f of dllimonene and E-caryophyllene against liver and colon cancer, 637f of germacrene D and linalool against liver and breast cancer, 638f of isoledene and elemene against colon cancer, 633f of oleic acid on zebrafish embryos, 637f of perilla ketone against gastric and lung cancer, 636f of thymol against breast cancer, 636f of trans-anethole against colon cancer, 633f Anticholesteremic phytochemicals, 100
101 Anti
coronal agents binding interaction of plant products with human angiotensin-converting enzyme-2 receptor, 551t with two SARSCOV-2 targets proteins, 547t molecular docking studies of plant products as, 546
550 Antidiabetic, 101 potential, 610 Antidiarrheal property, 609 Antifungal activity of EOs, 361 Anti
leishmanial agents, molecular docking studies of plant products as, 550
556 Antimicrobial resistance (AMR), 401, 435 Antimicrobial(s), 360, 413, 617 activity, 23, 538 antibacterial activity, 360
361 antifungal activity, 361 of EOs, 360
361 phytochemical classes and subclasses with activity against different microorganisms, 538t agents, 570 phytochemicals, 570 potential, 608 properties of phytochemicals, 86
87 Antinociceptive property, 610
611

Antioxidant(s), 163, 311, 360, 486
488, 501
502, 535, 570, 607 activity, 23 beverages rich in, 134
136 capacity, 24 defense mechanism, oxidative stress and, 310
311 phytoconstituent as antioxidant, 486
488 properties of phytochemicals, 86
87 Antioxidative potential, 607
608 Antiplatelet, 360 Antipyretics, 114 Antitubercular agents binding interaction of plant products with thioesterase domain in complex with inhibitor TAM1, 564t complex of mycobacterium tuberculosis hypoxic response regulator with piperine, 563f with quercetin, 563f with xanthone, 566f complex of polyketide synthase Pks13 with α-cubebin, 558f molecular docking studies of plant products as, 557
565 Antitumor activity of extract oils, 361
362 Antiviral, 102
103 mechanism, 522 phytochemicals, 570 AOEOs. See Alpinia oxyphylla essential oil (AOEOs) Aonla RTS, 131 AP-1. See Activator protein 1 (AP-1) APC. See Anaphase-promoting complex (APC) APG. See Apigenin (APG) Apigenin (APG), 168t, 173t Apocarotenoids, 272
273 Apoptosis, 164, 170, 185, 585
586, 639 Aporphine, 210 Aporphinoid alkaloids, 215 structure of, 215f Apple, 133 Aqueous methanol, 605 Aquilaria crassna, 636 Arachidonic acid, 375 Araucaria heterophylla, 636 Arbutus species, 86 ARDS. See Acute respiratory distress syndrome (ARDS) Aromatase inhibitors (AIs), 262
263, 265
266 Aromatherapy, 353 Aromatic plants, 629 Artemisia A. arborescens, 357 essential oils, 373t Arugampul (Cynodon dactylon), 134 Asclepiadaceae, 502 Ascorbic acid, 123
124, 195
196 ASCT2 protein. See Amino acid transporter protein (ASCT2 protein) ASE. See Accelerated solvent extraction (ASE) Aspergillus A. flavus, 361

Index

A. ochraceopetaliformis SSP13, 437 Aspirin, 183 ATP. See Adenosine triphosphate (ATP) ATP-binding cassette (ABC), 278
281, 586
587 transporter, 583 transporters, 586
587 ABCCl, 587 P-glycoprotein, 587 Auto-sampler, 72 Autoinducers (AIs), 401, 455
456 Autoinducing peptides (AIPs), 401, 436, 456 Autophagy, 164, 170 phytochemicals induce cancer cell apoptosis and, 325
326 Avian metapneumovirus (aMPV), 196 Avicenna officinalis, 388 Avicennia marina, 387
388, 390
391 Avicenniaceae A. alba, 390
391 A. marina, 390
391 A. officinalis, 390
391 Ayurveda, 369, 483, 584
585 mahua flower, 110
111 Ayurvedic system, 592 Azadirachta indica, 297 2,20 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), 135 Azithromycin (AZM), 437 AZM. See Azithromycin (AZM) Azulenes, 357

B B-glucans, 10 Bacillus, 116
117 B. cereus, 372, 417t, 418
419 B. pumilus, 115 B. subtilis, 86
87, 115, 193, 406 Bacopa monnieri, 467t Bacteria, 9, 397
399, 617 Bacterial biofilm, 623 Bacterial hyaluronidases, 622 Bacterial motility, inhibitory activity against, 625 Bacterial toxins, inhibitory activity against, 622
623 Baking process, 136 Ballota B. nigra, 638 B. saxatilis, 638 Basella alba, 165 BBB. See Blood
brain barrier (BBB) BC. See Beta-carotene (BC); Breast cancer (BC) BChE. See Butyrylcholinesterase (BChE) BCRP. See ABCC1 Breast cancer-resistance protein (BCRP) BCS. See Breast-conserving surgery (BCS) Beetroot (Beta vulgaris var. rubra L), 12 classification of main groups of betalains present in beetroot, 13f Benzo[c]phenanthridine, 210 Benzopyrrole, 267
268

Benzyl isothiocyanate (BITC), 102, 423 Benzylamine, 546 Benzylisoquinoline, 210 class, 589 Berberine, 204, 211, 268t, 470
471 structure of, 211f Bersama engleriana, 165 Beta vulgaris var. rubra L. See Beetroot (Beta vulgaris var. rubra L) Beta-carotene (BC), 541 β dimmers, 227f β-carotene, 123
124, 385t β-carotene discoloration, 607 β-carotene lineolate bleaching method, 607 β-caryophyllene, 634 anticancer activity of β-caryophyllene, 635f β-Sitosterol, 274, 406 β-tubulin, 225
226 β
glucan, 126 Betulin, 445 Beverages, 124, 131 classification of, 124, 124t consumer demand for beverages, 138 fermented beverages, 132 fruits-based beverages, 130
132 health safety of drinks, 138 market of nutraceutical or functional beverages, 125 micronutrient-fortified beverage, 133
134 need for, 123
124 nonalcoholic beverages, 126
128 prebiotic beverages, 136
137 probiotics beverages, 128
130 rich in antioxidants and herbs, 134
136 flowchart for preparation of herbalblended lime-based ready-to-serve beverage, 135f soft drinks, 125 sports or energy drinks, 137
138 storage study of beverages, 138 types of, 124
125 whey-based beverages, 132
133 BHA. See Butyl hydroxyanisole (BHA); Butylated hydroxyanisole (BHA) BHT. See Butylated hydroxytoluene (BHT); Butylhydroxytoluene (BHT) Bi-tyrosine, 484 Bicarbonate production, 610 Bicyclic monoterpenes, 355, 356f, 357 Bifidum longum, 130 Binding energy, 557 Bioactive compounds, 9
10, 50, 85, 104, 463
464, 535, 569, 617
618 Bioactive concept, 19 Bioactive food components, 95
96 Bioactive molecule purification, 572 Bioactive phytochemicals, 85
86, 91
92 diverse spectrum of applications of bioactive phytochemicals derived from edible flowers, 86f docking, 646 uses of, 91f Bioactive substances, 383 Bioactivities, 369, 601
602

651

Biochemical tests, 75
76 Biodiesel, 113 Biofilm(s), 397, 413, 455 biofilm-forming pathogens in food industry, 417t biofilms-forming organisms, 416f detachment, 398
399 development and health hazards, 413
418 factors influencing biofilm development, 414 important variables in bacterial cell attachment and biofilm formation, 414t microorganisms associated with biofilms and their health hazards, 416
418 stages in, 415
416 formation, 397
399 inhibitory activity against, 623
624 stages in, 398f future perspective, 408 inactivation mechanism, 399
401, 400f maturation, 415 mode of action of phytochemicals on, 424 occurrence, 418
420 organic acids, 406 phenolics, 401
405 phytochemicals, 407 in biofilm inhibition, 401 phytochemicals in biofilm inhibition, 420
425 sulfur-and nitrogen-containing phytochemicals, 407
408 terpenoids, 405
406 17 bioflavonoid, 622 Biogenic nanoparticles, 195
196 Biological activity, 113
116 of phytochemicals, 98
103 Biological carcinogens, 163 Biological diversity Act (2002), 31 Biological response, 580 Biological system, 622 Biomedicals, 383 applications of marine-based phytochemicals, 390
392 Biopolymers, 398
399 Biphenyl propane skeleton, 618 Bisabolol, 375 Bisphosphonate therapy, 182 BITC. See Benzyl isothiocyanate (BITC) Black rice, 98 Bladder cancer, 217 Bladder cancer cells, 634 anticancer activity of boswellic acid against bladder cancer, 634f Bleekeria vitiensis, 218 Blood cells, 583 Blood glucose levels, 610 Blood pressure (BP), 297 Blood
brain barrier (BBB), 463
465, 575 Bodo tribes, 604
605 Boronia megastigma, 357 Boswellia sacra, 633 Boswellic acid, 633 anticancer activity of boswellic acid against breast cancer, 633f

652

Index

BP. See Blood pressure (BP) Brachytherapy, 265 Brain cancer, 101
102, 163 Breast cancer (BC), 101
102, 145, 163, 179, 228, 261
262 CAM in breast cancer treatment, 266
267 cell, 633 challenges and perspectives, 284
285 definition, subtypes, and conventional therapies, 262
263 subtypes along with receptor expressions and representative cell lines, 262t diet and dietary phytochemicals in chemosensitization, 283
284 perils of conventional BC therapies, 263
266 chemotherapy, 263
265 hormone therapy, 265
266 radiotherapy, 265 phytochemicals, 267
274 and ER(1) breast cancer, 274
275 and HER(2) breast cancer, 276 interventions in healing cancer-associated MDR, 278
283 used for TNBC, 276 role of phytochemicals in modulating noncoding RNA expression in BC cells, 276
278 window in breast cancer therapy, 267
274 alkaloids, 267
269 cardiac glycosides, 274 carotenoids, 271
273 flavonoids, 271 phytosterols and phytostanols, 273
274 terpenoids, 269
271 Breast cancer-resistance protein (BCRP), 587 Breast-conserving surgery (BCS), 265 Broccoli, 102 Bromopyrrole alkaloids, 407 Bruker 700 MHz nuclear magnetic resonance spectrometer, 80f Brusatol, 276 Buttermilk-based fermented drink, 136 Butyl hydroxyanisole (BHA), 537 Butylated hydroxyanisole (BHA), 486, 607
608 Butylated hydroxytoluene (BHT), 486 Butylhydroxytoluene (BHT), 537 Butyrylcholinesterase (BChE), 576
578

C c-di-GMP, 436 c-jun N-terminal kinase (JNK), 212, 490 c-mitosis. See Colchicine mitosis (c-mitosis) 13 C-NMR spectra, 78 C18-resins, 49 CA. See Caffeic acid (CA) Cabazitaxel, 340 Cabbage, 102 Cabinet drying, 117 Caco-2. See Colon cancer cells (Caco-2) CADD. See Computer-aided drug design (CADD)

Caenorhabditis elegans, 406 Caffeic acid (CA), 338, 371
372, 457
459, 487 Caffeine, 204, 250, 503 CAG. See Cytosine-adenine-guanine (CAG) CagA. See Cytotoxin-associated gene A (CagA) CAGR. See Compound Annual Growth Rate (CAGR) Cake, 110 Calcium fortification, 539 Calmodulin-dependent protein kinase type-IV, 555 Calotropis procera, 165 CAM. See Complementary and alternative medicine (CAM) Camellia sinensis, 296 cAMP. See Cyclic adenosine monophosphate (cAMP) Camphor laurel, 357 Camptotheca acuminate, 209, 341 Camptothecin (CPT), 203, 209, 249
250, 255, 282, 303, 341 derivatives, 339 structure of, 209f Campylobacter jejuni, 417t Cananga odorata, 213 Cancer, 96, 103, 114, 163, 179, 181, 203, 247, 255, 267, 309, 335, 345, 361
362, 629 alkaloids and other nitrogen-containing constituents, 296 cells, 179, 208, 225
227, 239, 250, 255, 281
282, 309, 325, 585 factors affecting microtubule dynamics in, 226
227 factors responsible for drug resistance in cancer cell, 586f proliferation, 646 ROS-dependent cellular metabolic pathways in, 311 chemotherapy resistance and contributing factors, 164f drug delivery approach to improve phytochemical drugability, 172 genesis, 170
172 historical perspective of plant-derived drugs used popularly in, 318
325 insights on phytochemicals as dietary recommendation in, 172
175 intracellular stress in, 227 invasion, metastasis, angiogenesis, and stemness-related pathways in, 319f molecular mechanism of phytochemicals in preventing cancer, 169
170 apoptosis and autophagy, 170 genome instability, 169
170 modulation of membrane, 170 targeting cell proliferation, 169 targeting immune surveillance and inflammation, 170 targeting molecular pathway of cancerous cell, 169 targeting oxidative stress and redox signaling, 169

nanodrug delivery of phytochemicals in treating, 89
90 other important phenolic compounds studied on Cancer targets, 322
325 chalcones, 322
324 flavones, flavanones, isoflavones, and flavanols, 325 flavonols, 324
325 phytochemicals currently in use as cancer therapeutics, 339
342 camptothecins, 341 plant-derived anticancer agents, 341
342 podophyllotoxins, 341 taxanes, 340 vinca alkaloids, 339
340 phytochemicals for cancer prevention by targeting cellular signalling transduction pathways, 313
318 phytochemicals in clinical and preclinical stages for preventing cancer, 172 phytochemicals unexplored, 164
168 polyphenols, 295
296 role of phytochemicals in diseases, 296
299 strategies to improve phytochemical drugability, 170
172 targeting microtubules in, 227
228 α and β dimmers, 227f breast cancer, 228 colon cancer, 228 oral squamous cell carcinoma, 228 ovarian cancer, 227
228 terpenes and terpenoids, 295 treatment, 228
229, 247
248, 267, 310, 645 alkaloids, 320
321 flavonoids, 321
322 future prospects of phytochemicals in, 345 important secondary metabolites in, 320
322 plant-derived phytochemicals currently in use for various, 251 targeted pathways in, 204 terpenes, 320 Cancer stem cells (CSCs), 278
281, 309
310, 587 Cancerous cell, targeting molecular pathway of, 169 Candida C. albicans, 115, 378, 405
406 C. glabrata, 405
406 C. parapsilosis, 405
406 C. tropicalis, 378 Cannabidiol, 256t Cannabis sativa, 589 Cantaloupe into yogurt, 541 Cantaloupe puree yogurt (CPY), 541 Canthospermolides, 406 Cape Leadwort, 189 Cape Plumbago, 189 Capecitabine, 264t Capensindin-3-rhamnoside, 190 Capensinidin, 190 Capensisone, 190 Capillary electrophoresis, 72
73, 73f

Index

Capparis spinosa, 155 Capsaicin, 99t, 101
102, 170, 204 CAR. See Carvacrol (CAR) Carbapenemase, 436 Carbohydrates, 74, 295 Carbohydrazide, 589
590 Carbon nuclear magnetic resonance spectroscopy (13C-NMR), 7 Carbon tetrachloride-induced liver model, 610 Carbonated beverages, 137 Carboplatin, 264
265, 264t Carcinogenesis, 169, 250
251, 335, 361
362, 637 role of oxidative stress in, 310
313 Carcinogenicity, 180 Cardamom essential oil (CEO), 639
640 Cardiac glycosides, 274 Cardiovascular disease (CVD), 96, 103, 297, 536
537, 569 Cardiovascular protection, 537 Cardiovascular system, 587 Carotenoid(s), 12, 85, 87, 97t, 104, 184, 261
262, 271
273, 338, 504, 536 anticancer properties of phytochemicals, 339t different members of, 273t Carrageenan, 385t Carvacrol (CAR), 400
401, 406, 422 and thymol against colorectal cancer, anticancer activity of, 633f Casearia sylvestris, 634 Cassipourol, 406 Castanea sp., 296 CAT. See Catalase (CAT) Catalase (CAT), 485 Catechin(s), 99t, 487, 620
621 hydrate, 256t polymers, 576 Catechol, 457
459 Catechol-O-methyl transferase, 491
492 Catharanthus, 228
229 C. roseus, 205, 228
229, 255, 268, 303, 337, 545 Cauliflower, 102 CBAs. See Chain-breaking antioxidants (CBAs) CC. See Cervical cancer (CC) CCC. See Countercurrent chromatography (CCC) CCE. See Countercurrent extraction (CCE) CDC. See Centers for Disease Control and Prevention (CDC) Cdcs. See Cell division cycle proteins (Cdcs) CDKs. See Cyclin-dependent kinases (CDKs) CDY. See Dry cantaloupe yogurt (CDY) Celastrus paniculatus, 467t Cell death, 315
316 division, 180 envelope synthesis, inhibitory activity against, 620
621 injury, 313 metabolism, 251

targets of phytochemicals in cell cycle pathways, 316
317, 318f tissues, 3 Cell division cycle proteins (Cdcs), 313 Cellular attachment, 415 Cellular components, 607 Cellular protein Ki67, 262
263 Cellular system, 579 Cellular toxicity, 170 Centaurea Montana, 210 Centella asiatica, 136, 377, 401, 467t Centers for Disease Control and Prevention (CDC), 185 Central nervous system (CNS), 102, 463
464 CEO. See Cardamom essential oil (CEO) Cephaelis ipecacuanha, 216 Cephalotaxine, 217
218 Cephalotaxus alkaloids biological source, mechanism of action, and applications of, 217
219 cephalotaxine, 217
218 homoharringtonine, 218
219 Cephalotaxus harringtonia, 217 structure of, 217f Cepharanthine, 518 Cereals, 137 cereal grains, 126 cereal-based fermented nonalcoholic beverages, 126
127 process flowchart for manufacturing of Kunun-zaki, 127f Cerebral ischemia, 472
473, 474t Cerebrovascular diseases, 472 Cervical cancer (CC), 255
256, 637 anticancer activity of 6-shogaol against, 637f anticervical cancer phytochemicals and mechanism of action, 256t CF. See Cystic fibrosis (CF) Chain of polymers of isoprene, 354 Chain-breaking antioxidants (CBAs), 485 Chalcones, 322
324 chalcones in cancer, 323f Chamazulene, 375 Chamomile (Matricaria chamomilla), 357 Characterization methods, 75
81 1 H-NMR and 13C-NMR spectra, 78 gas chromatogram, 77
78 GC
Ms spectrum, 79 mass spectrometry, 79 of phytochemicals, 2f two-dimensional NMR spectrum, 80
81 UV and visible spectrum, 78 X-ray spectroscopy, 81 Checkpoint kinase (Chks), 313 Chemical assays, 23 Chemical compounds, 535, 569
571, 617 Chemical ionization mass spectrometry (CIMS), 6
7 Chemoprevention, 267, 283 phytochemicals in, 300
302 Chemosensitization, diet and dietary phytochemicals in, 283
284 Chemosensitizer, 592 phytochemicals as, 588
594

653

Chemotherapeutic agents, phytochemicals as, 303 Chemotherapeutic drugs, 264
265, 345, 583, 585 Chemotherapeutic medicines, 585 Chemotherapy, 163
164, 180, 182, 247
248, 261
265, 300, 303, 315
316, 335, 583, 585, 629 BC patients undergoing neoadjuvant and adjuvant chemotherapy, 265f chemotherapy-resistant cancer cells, 236 commonly used chemotherapeutic drugs for BC treatment, 264t drugs, 591
592 phytochemicals, 300f conjugation with, 303
304 resistance and contributing factors, 164f Chemotoxicity, phytochemical in alleviation of, 303 Chitosan nanoparticles, 638 Chks. See Checkpoint kinase (Chks) Chloroform, 193 Chlorogenic acid, 487 Chlorophylls, 72, 535 Chloroquine (CQ), 514 Chol. See Cholesterol (Chol) Cholesterol (Chol), 206, 385t Choriocarcinoma, 151 Chresta spp, 67
68 Chromatographic techniques, 5
6, 23, 68
74. See also Non-chromatographic techniques applications of, 74
75 non-chromatographic techniques, 75 capillary electrophoresis, 72
73 CCC, 73
74 electrophoresis techniques, 6 GLC, 70 HPLC, 71
72 HPTLC, 72 paper chromatography, 69 TLC, 69
70 Chromatography, 5, 68
69 Chromobacterium violaceum, 401, 422 Chromosomal translocations, 583 Chronic diseases, 144, 535 Chronic kidney disease (CKD), 297 Chronic lymphocytic leukemia (CLL), 583 Chronic myelogenous leukemia (CML), 583 Chronic myelomonocytic leukemia (CMML), 342 Chronic renal disease, 102 Chronic wounds, 418 Chronic-degenerative disorders, 96 3-chymotrypsin-like protease (3CLpro), 522, 546
550 CI. See Combination index (CI) CI-MS. See Chemical ionization mass spectrometry (CI-MS) Cilantro (Coriandrum sativum), 502 Cinchona officinalis, 384
387 Cinnamaldehyde, 371
372, 375 Cinnamic acid, 371
372, 445 Cinnamic aldehyde, 405

654

Index

Cinnamomum C. camphora, 357 C. verum, 457
459 C. zeylanicum, 361 Cinnamon, 376t EOs, 375 oil, 422
423 Cinnamon cassia, 238
239 CIP-36, 341 Ciprofloxacin, 624 Circadian rhythm, 86 Circulatory extraction, 66 Circulatory tract, 542 Cisplatin, 249, 264
265 Citric acid, 195
196, 406 Citrus C. aurantium, 356
357 C. jambhiri, 638 C. medica, 374 C. pyriformis, 638 C. reticulata, 638 flavonoids, 503 fruits, 325 CKD. See Chronic kidney disease (CKD) CLA. See Conjugated linoleic acid (CLA) Classical isolation methods, 68 Clinical trials, phytochemicals in, 325 Clitoria ternatea, 136 Clivorine, 219 CLL. See Chronic lymphocytic leukemia (CLL) Closed vessel systems, 39
40 Clostridium perfringens, 360
361 Clove, 14 essential oils, 373t structure of Eugenol, 16f CML. See Chronic myelogenous leukemia (CML) CMML. See Chronic myelomonocytic leukemia (CMML) CNS. See Central nervous system (CNS) Cocaine, 204 Coffee extraction, 46
47 Colcemid, 205 Colchicine, 225 as microtubule-disrupting agent, 233
235 colchicine binding site and interplay with microtubule, 234 mechanism of action, 234 side effects of, 235 therapeutic relevance, 234
235 Colchicine mitosis (c-mitosis), 234 Colchicum autumnale, 233
234 Cold percolation, 50, 66 Cold pressing, 37 Collection process, 29
31 ecological consideration, 30 area of sustenance and habitat management, 30 equipment for collection, 30 significance of preservation and restoration of species, 30 quality consideration, 29
30

climatic condition and area of collection, 30 collection of healthy plant at unerring phenological phase, 30 at liberty from undesirable stuffs during collection, 30 taxonomical authenticity of species, 29
30 social consideration, 30
31 availability for local use, 30 cultural ethics, 31 health stature of collectors, 31 reasonable pricing and sharing of benefits, 31 Colombian Lippia, 634 Colon cancer, 179, 228 Colon cancer cells (Caco-2), 638 anticancer activity of carvacrol against liver and, 635f dl-limonene and E-caryophyllene against liver and, 637f isoledene and elemene against colon cancer, 633f trans-anethole against colon cancer, 633f Colonization, 625 Colorectal cancer, 101
102 Column chromatography, 68, 77, 572 Combination index (CI), 588 method and synergism, 587
588 Combretastatins, 225 Commiphora gileadensis, 635 Complementary and alternative medicine (CAM), 159, 266
267, 310 CAM-based approaches, 261
262 gut microbiota in gastrointestinal malignancy, 326
329 historical perspective of plant-derived drugs used popularly in cancer, 318
325 mode of action of phytochemicals for cancer prevention by targeting cellular signalling transduction pathways, 313
318 phytochemicals induce cancer cell apoptosis and autophagy, 325
326 plant-derived drugs, 329 role of oxidative stress in carcinogenesis, 310
313 Composite flours, 104 Compound Annual Growth Rate (CAGR), 125 Computational approach to target multidrug resistance, 588
590 Computational prediction equipment, 645 Computational techniques, 545 Computer-aided drug design (CADD), 445 Computer-aided drug discovery, 572
573, 645 Computer-based drug discovery, 588 Confectionery market, 104 Coniine, 204 Conjugated linoleic acid (CLA), 18 Constant refluxing method, 2
3 Consumers, 130 demand for beverages, 138 Conventional chromatographic techniques, 6

Conventional methods. See also Nonconventional methods of extraction, 21
22 hydrodistillation, 22 maceration, 22 Soxhlet extraction, 21 of phytochemical extraction, 63 Copper, 198
199 Cordyceps militaris, 303 Coriander seed essential oil, 637 Coriandrum sativum, 467t Coriandrum sativum. See Cilantro (Coriandrum sativum) Coronavirus disease 2019 (COVID-19), 511, 545, 580 effects of phytochemicals from honey against COVID-19, 521
522 phytochemicals and role in, 512 effect on phytochemicals demand, 580 plants’ role in COVID-19 treatment, 511 Coryphoideae, 575 Cosmetics, 535 Coumarin, 104, 239 therapeutic activities, 238
239 mechanism and binding site against microtubule, 239 origin of phytochemicals, 240f therapeutic relevance, 239 toxicity remarks of coumarin and analogs, 239 Countercurrent chromatography (CCC), 73
74, 574 DCCC, 74 HSCCC, 74 schematic representation of, 74f Countercurrent extraction (CCE), 48, 50, 64, 66
67 advantages of countercurrent extraction process, 48 COVID-19. See Coronavirus disease 2019 (COVID-19) COX. See Cyclooxygenase (COX) CPCR. See Cytoskeletal protein-related coding (CPCR) CPT. See Camptothecin (CPT) CPY. See Cantaloupe puree yogurt (CPY) CQ. See Chloroquine (CQ) Crassulaceae, 185 Cretan propolis, 295 Crocus sativus, 136, 165 Crohn’s disease, 298 Cronobacter sakazakii, 406, 418 Croton C. stellatopilosus, 357 C. sublyratus, 357 Cruciferous vegetables, 102 Crude plant extracts against biofilm formation in ESKAPE pathogens, 438 Cryotherapy, 182 Cryptococcus C. laurentii, 422 C. neoformans, 422 Crystallization method, 5, 68 CSCs. See Cancer stem cells (CSCs)

Index

Culex quinquefasciatus, 196
197 Cumin seeds (Nigella sativa L.), 373t, 376t Cuminum cyminum, 373t EO, 376t Cupressus sempervirens L., 638 CUR. See Curcumin (CUR) Curcuma C. longa, 251
252, 297, 467t, 545 plant, 101 Curcuma longa L. See Turmeric (Curcuma longa L.,) Curcumae rhizome, 636 Curcumin (CUR), 10, 99t, 101
102, 235
236, 251
254, 469
471, 502, 591 binding of curcumin polyphenol with microtubule, 236 plant-derived compounds reported having anticancer potential, 254f side effects of curcumin, 236 therapeutic relevance of curcumin against microtubule, 236 Curcuminoids, 169
170 Curcumol against colorectal cancer, anticancer activity of, 636f CVD. See Cardiovascular disease (CVD) Cyanidin, 343
344 Cyanogenic glycosides, 296 Cyclic adenosine monophosphate (cAMP), 151 Cyclic amino acid, 204 Cyclic sesquiterpenes, 356 Cyclin-dependent kinases (CDKs), 313, 316
317 Cyclooxygenase (COX), 375 COX1
1, 313
314 COX1
2, 211 Cyclophosphamide, 264
265, 264t, 310 Cymbopogon C. citrates, 632 C. giganteus, 632 C. winterianus, 375 Cynodon dactylon. See Arugampul (Cynodon dactylon) Cystic fibrosis (CF), 418 Cyt-c. See Cytochrome c (Cyt-c) Cytarabine, 591 Cytochrome c (Cyt-c), 234
235 Cytokines toxicity, 517 Cytoplasmic membrane, 538, 624 disruption, 570 Cytosine-adenine-guanine (CAG), 473 Cytoskeletal protein-related coding (CPCR), 227 Cytoskeleton filaments, 225 Cytotoxic action, 634 Cytotoxic podophyllotoxins, 203 Cytotoxic treatment, 247
248 Cytotoxin-associated gene A (CagA), 329

D D-limonene, 635 anticancer activity of D-limonene against prostate cancer, 635f

anticancer activity of D-limonene liver and pancreatic cancer, 639f DL-cycloserine, 620
621 DAD. See Diode array detector (DAD) Daidzein, 343 Dairy products, 539 Dalton’s lymphoma ascites cells (DLA), 374 DASH. See Dietary Approaches to Stop Hypertension (DASH) Date palm, 137
138 Daun halendong, 604
605 DCCC. See Droplet countercurrent chromatography (DCCC) Death receptor (DR), 325 Decoction method, 2
3, 37 Dehydrated leaves, 604
605 Delphinidin, 343
344 DENA. See Diethyl nitrosamine (DENA) Deoxynortryptoquivaline, 518 Deoxytryptoquivaline, 518 Derjaguin
Landau
Verwey
Overbeek theory, 398
399 Destructive process, 149 Detachment of biofilm, 415
416 Detection, 163 Detyrosination, 225 DFS. See Disease-free survival (DFS) DHFR. See Dihydrofolate reductase (DHFR) DHT. See Dihydrotestosterone (DHT) Diabetes, 96, 123
124, 149, 155, 296
297, 537 causes of, 149 herbs used in, 155 Diabetes mellitus (DM), 101, 145
149 Diallyl sulfide, 407
408 Dialysis, 5, 68 Diaporthe phaseolorum SSP12, 437 Diarrheal treatment, 602
604 Diatomaceous earth, 49 Dibromoisophakellin, 407 Dibromophakellin, 407 Dibutyl phthalate, 112 Diene, 484
485 Diet, 186 phytochemicals in chemosensitization, 283
284 Dietary antioxidants, 486 Dietary Approaches to Stop Hypertension (DASH), 297 Dietary estrogen. See Nonsteroidal phytochemical Dietary fiber, 88 Dietary phytochemicals, 310 in chemosensitization, 283
284 Dietary polyphenols, 490 Dietary recommendation in cancer, insights on phytochemicals as, 172
175 Dietary supplements, 44
45, 580 and in preventing cancer, 175t Diethyl nitrosamine (DENA), 611, 639
640 Differential leukocyte count (DLC), 115 Differentiation therapy, 583 Diffusible signal factors (DSFs), 436 Digestive system, 540

655

Digital rectal exam (DRE), 181 Digital screening methods, 645 Dihydrofolate reductase (DHFR), 622 Dihydrotestosterone (DHT), 183 Diiodotyrosine (DIT), 151 Dimethyl sulfoxide, 610 Dimethylallyl diphosphate (DMAPP), 354, 371 7,12-dimethylbenz(α)anthracene (DMBA), 611, 637 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, 193 Dimyrcene, 357 Diode array detector (DAD), 71
72 Dipeptidyl peptidase-4 (DPPIV), 216
217 2,2-diphenyl-1-picrylhydrazyl (DPPH), 193
194 1,1-diphenyl-2-picrylhydrazyl (DPPH), 135 Dipteryx odorata, 238
239 Direct steam distillation, 67 Disease-free survival (DFS), 263, 278
281 Disease(s), 133 and conditions, 144 stage, 181 Disinfectants, 399 Disk diffusion method, 115, 608 Distillation, 37
38 hydrodistillation, 37 hydrosteam distillation, 38 steam distillation, 37
38 DIT. See Diiodotyrosine (DIT) Diterpenes (C20), 295, 356
357, 371, 405
406, 519
520 structure, 358f Diterpenoids (C20), 269 DLA. See Dalton’s lymphoma ascites cells (DLA) DLC. See Differential leukocyte count (DLC) DM. See Diabetes mellitus (DM) DMAPP. See Dimethylallyl diphosphate (DMAPP) DMBA. See 7,12-dimethylbenz(α)anthracene (DMBA) DMF. See N,N dimethyl-formamide (DMF) DNA gyrase inhibition (GyrB), 621
622 DNA, 537 damage, 623 repair, 585 DNA-damaging species, 169 intercalators, 503 ladder, 635 synthesis, 621
622 topology, 621 Docetaxel, 249, 264t, 635 13-Docosenamide, 190 Donor-derived immune system, 583 Dopamine, 204 Dosing, 170 Double maceration, 66 Double-helix DNA, 608 DOX. See Doxorubicin (DOX) Doxorubicin (DOX), 249, 264t, 282, 583, 591
592 cytotoxicity, 592

656

Index

DPPH. See 2,2-diphenyl-1-picrylhydrazyl (DPPH); 1,1-diphenyl-2-picrylhydrazyl (DPPH) DPPH scavenging method, 23 DPPIV. See Dipeptidyl peptidase-4 (DPPIV) DR. See Death receptor (DR) Dracaena reflexa, 646 Dragon fruit, 13
14 chemical structure of betacyanin, 16f DRE. See Digital rectal exam (DRE) Drinks, health safety of, 138 Droplet countercurrent chromatography (DCCC), 6, 74 Drug delivery approach to improve phytochemical drug ability, 172 systems, 154
155, 436
437 Drug discovery, 545, 645 opportunities and challenges, 579
580 effect of COVID-19 on phytochemicals demand, 580 dietary supplements, 580 phytochemicals as vegan food ingredients, 579 plant-based ingredients, 579 transfer of phytochemicals into pharmaceuticals, 580 phytochemicals, 570
571 anticancer phytochemicals, 571 antimicrobial phytochemicals, 570 antiviral phytochemicals, 570 composition and biological properties of seed extracts from Washingtonia filifera, 575
579 from Phytolacca dioica L. Seeds extracts, 574
575 plants as dominant source, 571 screening of plant extracts, 571
574 process, 572
573 Drug resistance, 585
587, 617 proteins/genes responsible for drugresistance leukemia, 586
587 ATP-binding cassette transporters, 586
587 cancer stem cells and drug resistance, 587 hypoxia-inducible factor-1-mediated resistance, 587 Drugs, 182, 205, 240 activation, 585 development, 335, 445 drug-loaded carrier system, 172 drug-resistance gene, 164 drug-resistant cancer cells, 247
248 synergy, 588 target, 317
318 targeting systems, 154
155 therapies, 610 Dry cantaloupe yogurt (CDY), 541 Drying, 31
32, 116 air-drying, 31 freeze-drying, 31 microwave-drying, 32 oven-drying, 31

DSFs. See Diffusible signal factors (DSFs) Dynamic instability, 215

E E-14-hexdecenal, 112 E-caryophyllene, 638 anticancer activity of dl-limonene and Ecaryophyllene against liver and colon cancer, 637f EA. See Ellagic acid (EA) EAC. See Ehrlich ascites carcinoma (EAC) EAE. See Enzyme-assisted extraction (EAE) EBRT. See External beam radiation therapy (EBRT) EBV. See Epstein
Barr virus (EBV) Eclipta alba plant, 164
165 ECM. See Extracellular matrix (ECM) EDC. See Environmental endocrine-disrupting chemical (EDC) Edible flowers, 86 eDNA. See Extracellular DNA (eDNA) Efflux pumps (EPs), 424 inhibitory activity against, 624
625 phytochemicals as, 424 EGCG. See Epigallocatechin-3-gallate (EGCG) EGF. See Epidermal growth factor (EGF) EGFR. See Epidermal growth factor receptor (EGFR) EHEC. See Enterohemorrhagic Escherichia coli (EHEC) Ehrlich ascites carcinoma (EAC), 374 EI-MS. See Electron impact mass spectrometry (EI-MS) Eicosanoids, 375 EIEC. See Enteroinvasive Escherichia coli (EIEC) Elaeagnus angustifolia, 88 Electromagnetic radiation, 571
572 Electron impact mass spectrometry (EI-MS), 6
7 Electrophoresis, 5 techniques, 6 Electrospray ionization (ESI), 79, 572 Electrospray ionization mass spectrometry (ESI-MS), 6
7 Elemene, 629
632 against colon cancer, anticancer activity of isoledene and, 633f ELISA. See Enzyme linked immunosorbent assay (ELISA) Ellagic acid (EA), 10, 175t, 405, 605 Ellagitannins, 131, 405 Emblica officinalis. See Gooseberry (Emblica officinalis) Emetine, 210 and related alkaloids, 216
217 structure of, 216f Emodin, 594 EMTs. See Epithelial
mesenchymal transitions (EMTs) Encapsulation technologies, 105 EndMT. See Endothelial-to-mesenchymal transition (EndMT)

Endocrinal targeted approaches, 261
262 Endocrine, 263 disorder, 149 treatment with help of targeted drug delivery, 155 factors affecting, 144
145 aging, 144 diseases and conditions, 144 environmental factors, 145 genetics, 145 stress, 144
145 glands, 143
145, 150 endocrine system and disorders, 143 main hormone-producing glands, 143
144 novel phytomedicinal formulations to target endocrine galnds ans hormone for treatment of various major endocrine disorders, 155
158 hormones, 143
145, 150 major endocrinological disorder and natural products/herbs used in treatment of endocrinological disorder, 155 occurs due to sedentary lifestyle, 149
150 endocrine glands and endocrine hormones, 150 rationale, 149
150 physiology and mechanism behind, 151
154 thyroid gland, 151 thyroxine, 152 Triiodothyronine, 152
154 relation between sedentary lifestyle and, 150 system, 143, 149 and disorders, 143 therapy, 265
266 Endocrine receptors (ER), 263 Endocrinological disorders, 149 endocrinological disorder and natural products/herbs used in treatment of, 155 diabetes, 155 herbs used in diabetes, 155 herbs used in polycystic ovary syndrome, 155 polycystic ovary syndrome, 155 Endogenous antioxidants, 486 Endoplasmic reticulum (ER), 512
513 ER(1) breast cancer, 274
275 different cardiac glycosides, 275t Endorinological disorders, 145
149 causes of diabetes, 149 ovaries in thyroid disorders, 146
147 thyroid in polycystic ovary syndrome, 147
149 Endothelial-to-mesenchymal transition (EndMT), 228 Energy drinks, 137
138 Enfleurage, 49
50 Enhanced solvent extraction system (ESE), 43 Enterococcus faecalis, 624 Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. (ESKAPE), 433
434

Index

impact of bacterial biofilm, 435 biofilm and historical perspectives, 433
434 biofilm formation, 434 biofilm-mediated drug resistance in, 435
436 regulation of specific virulence genes associated with biofilms, 436 current trends in biofilm inhibition, 445
446 future perspectives, 446 mitigation of biofilm architecture, 436
437 microbial secondary metabolites for biofilm inhibition, 437 synthetic and semisynthetic derivatives as biofilm inhibitors, 437 phytochemicals-based mitigation strategies against biofilm formation, 438
444 ultrastructure of biofilm communities, 434
435 Enterohemorrhagic Escherichia coli (EHEC), 419 Enteroinvasive Escherichia coli (EIEC), 419 Enteropathogenic Escherichia coli (EPEC), 419 Enterotoxigenic Escherichia coli (ETEC), 419 Envelope protein, 514
515 Enveloped viruses, 512 Environmental endocrine-disrupting chemical (EDC), 145 Environmental pollutants, 610 Environmental pollution, 629 Enzymatic antioxidants, 485 Enzymatic extraction, 67 Enzymatic reactions, 572
573 Enzyme linked immunosorbent assay (ELISA), 75 Enzyme-assisted extraction (EAE), 22, 32, 47 Enzymes, 111 inhibition, 646 EO-ACs. See Essential oil and active components (EO-ACs) EOs. See Essential oils (EOs) Eosinophil peroxidase (EPO), 485 EPEC. See Enteropathogenic Escherichia coli (EPEC) Ephedrine, 204 Epicatechin, 488 Epidermal growth factor (EGF), 315 Epidermal growth factor receptor (EGFR), 170, 276 EGFR-2, 208 Epidermoid carcinoma cells, 635 Epigallocatechin, 10, 168t, 173t gallate, 101
102, 625 Epigallocatechin-3-gallate (EGCG), 10, 101
102, 256t, 267, 283
284, 470, 521 Epipodophyllotoxin, 339 Epirubicin, 264t Epithelial
mesenchymal transitions (EMTs), 192
193, 227, 261
262, 324
325 Epithilones, 225 EPM. See Extracellular polymeric matrix (EPM) EPO. See Eosinophil peroxidase (EPO) EPs. See Efflux pumps (EPs)

EPS. See Extracellular polymeric substances (EPS) Epstein
Barr virus (EBV), 327
328, 635 ER. See Endocrine receptors (ER); Endoplasmic reticulum (ER); Estrogen receptor (ER) Ergosterol, 385t Eriodictyol, 255 ERK. See Extracellular signal-regulated kinase (ERK) Eruca vesicaria EO, 376t Erythroxylum coca, 384
387 ESBL. See Extended-spectrum β-lactamase (ESBL) Escherichia coli, 86
87, 115, 190, 360
361, 372, 417t, 419, 608 ESE. See Enhanced solvent extraction system (ESE) ESI. See Electrospray ionization (ESI) ESI-MS. See Electrospray ionization mass spectrometry (ESI-MS) ESKAPE. See Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. (ESKAPE) Essential fatty acids, 137 Essential oil and active components (EO-ACs), 422 Essential oils (EOs), 3, 40, 44
47, 353, 369, 420
423, 421t, 629, 638 anticancer potential of essential oils, 629
640 antidiabetic agents, 375
377 antioxidant and anti-inflammatory properties, 374
375 application of EO in pharmaceutical industry, 377
378 bioactive components, 370
372 biological activity of, 360
362, 372
373 anticancer activity, 361
362 antimicrobial activity, 360
361 antimicrobial properties, 372
373, 373t cancer-preventing function, 374 cardiovascular diseases, 375 chemical structure of flavonoids, 618 chemistry of, 353
360 phenylpropanoids, 358
359 terpenes, 354
357 composition, 369 consolidated data on antidiabetic potential of, 376t EO-based microencapsulation, 362 EO-based nano-emulsion, 362 essential oil-bearing plants and major components useful as anticancer agents, 630t extraction of, 68 flavonoids activity against multidrugresistant microbes, 619
625 inhibitory activity against ATP synthesis, 622 inhibitory activity against bacterial motility, 625

657

inhibitory activity against bacterial toxins, 622
623 inhibitory activity against biofilm formation, 623
624 inhibitory activity against cell envelope synthesis, 620
621 inhibitory activity against DNA synthesis, 621
622 inhibitory activity against efflux pumps, 624
625 membrane-disrupting activities, 624 future perspective, 378 important properties, 377 medicinal applications of, 362
363 nitrogen-and sulfur-containing compounds in, 359
360 phenylpropanoids occurrence in, 359 therapeutic potential, 369
370 therapeutic properties, 370f Esters, 37
38, 353 Estimation process, 607 Estrogen receptor (ER), 262
263 ETEC. See Enterotoxigenic Escherichia coli (ETEC) Ethanol, 33, 38
39, 41
42, 46, 64, 193 ethanol-induced gastric ulcer assay, 610 Ethereal oils, 353 Ethers, 353 Ethno-medicinal practices, 602
605 ethno-medicinal uses of parts of Melastoma malabathricum in different countries, 603t Ethnomedicines, 602
604 Ethnopharmacology, 584
585 Ethosomes, 158 Ethyl acetate, 33, 64, 87 Etoposide, 249 Etymology, 9 Eucalyptus E. camaldulensis, 374 essential oil, 376t E. globulus EO, 374 Eudesmol, 357 Eugenia caryophyllata, 361
362, 378 Eugenol, 406, 422, 457
459 Euphorbia E. hierosolymitana, 165 E. neriifolia, 165, 519
520 E. peplus, 341 Euterpe oleracea, 618 Evodiamine, 268t Exclusive reagent precipitation method, 5 Excoecaria agallocha, 387 Exhaustive extraction, 36 Exosomes, 473
474 Extended-spectrum β-lactamase (ESBL), 436 External beam radiation therapy (EBRT), 265 Extracellular DNA (eDNA), 434
435 Extracellular matrix (ECM), 227
228 Extracellular polymeric matrix (EPM), 422 Extracellular polymeric substances (EPS), 397
399, 414, 433, 455, 623 Extracellular signal-regulated kinase (ERK), 311, 490

658

Index

Extraction, 1 accelerated solvent extractor, 68 assisted by microwave, 67 assisted by ultrasound, 67 of bioactive compounds, 63 characterization of phytochemicals, 23
24 antimicrobial activity, 23 antioxidant activity, 23 antioxidant capacity, 24 assessment of MIC, 24 determination of TFC, 23 determination of TPC, 23 classification of extraction method, 35
50 accelerated solvent extraction, 43 countercurrent extraction, 48 decoction, 37 distillation, 37
38 enfleurage, 49
50 enzyme-assisted extraction, 47 expression, 37 infusion, 36 maceration, 35
36 matrix solid-phase dispersion, 49 microwave-assisted extraction, 39
42 negative pressure cavitation extraction, 49 percolation, 36 phytonic process, 48
49 pulsed electric field extraction, 48 reflux extraction, 47
48 solid-phase microextraction, 47 Soxhlet extraction, 38
39 supercritical fluid extraction, 45
47 ultrasound-assisted extraction/sonication, 43
45 with cold fat, 49
50 conditions, 33
50 extraction methods, 34
50 solvent system for extraction, 33
34 current techniques for extraction of phytochemicals, 21
23 conventional methods of extraction, 21
22 nonconventional methods for plant extraction, 21 direct steam distillation, 67 enzymatic extraction, 67 extraction of essential oil, 68 extraction with supercritical gases, 67 factors affecting, 20
21 factors affecting extraction efficiency of bioactive compounds, 21f factors to be considered for selecting method of extraction, 35 methods, 34
50, 63
68 microwave-assisted extraction, 67 modified percolation, 66
67 organic solvent extraction, 64
66 of phytochemicals, 1
4, 2f pressing method, 3 solvent extraction method, 1
3 steam distillation method, 3 sublimation method, 3
4 phytochemicals and therapeutic effect, 9
10

phytochemicals from different food sources, 10
21 beetroot, 12 clove, 14 dragon fruit, 13
14 factors affecting extraction techniques, 20
21 finger millet, 16 fish, 16
17 flaxseed, 18 garlic, 11
12 ginger, 12 kiwi, 12 meat, 18 onion, 10
11 pomegranate, 18
19 tomato, 10 turmeric, 12 whole grain, 14
16 process, 117 selection approach for suitable, 50 comparison of different extraction methods, 51t Soxhlet extraction setup, 65f techniques, 32, 34
35, 64, 105 temperature, 41 time, 46 ultrasonic extraction, 67
68

F FA. See Ferulic acid (FA) FAB-MS. See Fast atom bombardment mass spectrometry (FAB-MS) Fagopyrum esculentum, 618 FAP. See Fibroblast activation protein (FAP) Farnesyl diphosphate (FPP), 354 FAS-II. See Type-II fatty acid synthase (FASII) Fast atom bombardment mass spectrometry (FAB-MS), 6
7 Fat, 111 Fatty acid, 111, 172, 250, 620
621 ester, 190 FD-MS. See Field desorption mass spectrometry (FD-MS) FDA. See US Food and Drug Administration (FDA) Fenugreek, 376t Fermentation, 538 effect on phytochemicals, 540, 540t substrates, 137 Fermented beverages, 132 Fermented dairy products, 104
105, 132 Fermented food industry effect of fermentation on phytochemicals, 540 fortification in the fermentation industry, 538
540 calcium fortification, 539 fortification with phenolics, 539
540 iron fortification, 539 vitamin fortification, 539 health benefits of phytochemicals, 536
538

anti-diabetes activity, 537 anti-obesity activity, 537 anticancer activity, 537 antimicrobial activity, 538 cardiovascular protection, 537 oxidative stress amelioration, 537 reducing inflammation, 537 limitations, 542 phytochemicals as safe fortifying agent, 541 cantaloupe incorporated into yogurt, 541 Soy isoflavones used in the fermentation of probiotics and beverages, 541 whole-bread preparation using cupuassu peel, 541 types of phytochemicals, 535
536 alkaloids, 535
536 carotenoids, 536 organosulfur compounds, 536 phytochemicals, 536 phytosterols, 536 polyphenols, 536 terpenoids, 536 Fermented products, 539 Ferrous sulfate, 539 Ferulic acid (FA), 338, 470
471, 487 FGF. See Fibroblast growth factor (FGF) Fibers, 87, 97t, 137, 385t, 390
391 Fibroblast activation protein (FAP), 216
217 Fibroblast growth factor (FGF), 315 Fibroblast proliferation, 609 Ficus deltoidea. See Fig tree (Ficus deltoidea) Field desorption mass spectrometry (FD-MS), 6
7 Fig tree (Ficus deltoidea), 185 Finger millet, 16 structure of finger millet arabinoxylan, 18f Fiscus carica, 165 Fisetin, 256t, 324 Fish, 16
17 chemical structures of EPA and DHA, 18f Flagellum, 625 Flash chromatography, 77 Flavan-3-ols, 337 Flavanoid functions, 608 Flavanols, 325 Flavanones, 325, 337 Flavones, 325, 337 Flavonoid(s), 10
11, 74, 85, 97t, 99t, 101, 104, 131, 195
196, 250
251, 261
262, 271, 282, 321
322, 335
337, 342, 385t, 390
391, 487, 503, 518
519, 537, 545, 617
618 activity against multidrug-resistant microbes, 619
625 inhibitory activity against ATP synthesis, 622 inhibitory activity against bacterial motility, 625 inhibitory activity against bacterial toxins, 622
623 inhibitory activity against biofilm formation, 623
624 inhibitory activity against cell envelope synthesis, 620
621

Index

inhibitory activity against DNA synthesis, 621
622 inhibitory activity against efflux pumps, 624
625 membrane-disrupting activities, 624 chemical structure of, 618 basic flavonoid structure with classes, 619f compounds for anticancer activity, 344
345 different members of, 272t different subclasses of, 271f flavonols in cancer, 324f mechanism action of, 343
344 naturally occurring flavonoids, 344f naringenin, 557 Flavonols, 337, 487 Flaxseed, 18, 19f Flour-based confectionery products, 104 Flours, 104 Flowers, 110 effect of geographical distribution on flower composition, 112 phytochemical profiling of mahua, 113t phytochemical screening of mahua flower extract, 112t processing, 116
117 collection of flowers, 116 drying, 116 methods of preservation, 117 postharvest spoilage of flowers, 116
117 preprocessing, 116 Fluorouracil, 264t 5-fluorouracil (5-Fu), 249, 283
284 Foeniculum vulgare, 373t Foetida, 638 Folin
Ciocalteu colorimetric method, 541 Follicle-stimulating hormone (FSH), 146 Food biofilm on food contact surfaces, 418
419 constituents, 9 intake, 103 phytochemicals from different food sources, 10
21 products, 579 biofilms in, 419
420 safety laws, 580 Forests, 109 Forkhead box O (FOXO), 297
298 Formic acid, 406 Fortification in fermentation industry, 538
540 calcium fortification, 539 fortification with phenolics, 539
540 iron fortification, 539 vitamin fortification, 539 of mango pulp, 133 with phenolics, 539
540 Fortunella margarita, 373t Fourier transform infrared (FTIR), 10 Fourier transform infrared spectroscopy (FTIS), 23, 75, 77f, 572 FTIR spectroscopic analysis, 196 FOXO. See Forkhead box O (FOXO) FPP. See Farnesyl diphosphate (FPP)

Fractional distillation, 5, 68 Frankincense oil, 634 Free radicals, 87, 114, 193
194, 483
485 Freeze-drying, 31 Freon-22, 45 Friedreich ataxia, 463 Fructose-1,6-bisphosphate, 190 Fruit(s), 98, 110, 123 fruit-based functional beverages, 123 fruits-based beverages, 130
132 juices, 100 processing technology, 67 pulp, 133 FSH. See Follicle-stimulating hormone (FSH) FTIR. See Fourier transform infrared (FTIR) FTIS. See Fourier transform infrared spectroscopy (FTIS) Fucoidans, 385t Fucosterol, 385t Fucoxanthin, 272
273 Fucoxanthinol, 385t Fucoxanthins, 385t Fumitremorgin C, 589
590 Functional bakery and confectionery, 104 Functional beverages, 103
104, 123, 125 beverages rich in antioxidants and herbs, 134
136 flowchart for preparation of herbalblended lime-based ready-to-serve beverage, 135f classification of, 124, 124t consumer demand for beverages, 138 fermented beverages, 132 fruits-based beverages, 130
132 health safety of drinks, 138 market of nutraceutical or functional beverages, 125 micronutrient-fortified beverage, 133
134 need for, 123
124 nonalcoholic beverages, 126
128 prebiotic beverages, 136
137 probiotics beverages, 128
130 product, 103 soft drinks, 125 sports or energy drinks, 137
138 storage study of beverages, 138 types of, 124
125 whey-based beverages, 132
133 Functional components, 9
10 Functional dairy products, 104
105 Functional drinks, 103
104, 123
124 Functional foods, 95, 125 concept, 105 Mahua as, 116
118 processing of flowers, 116
117 value-added food products, 117
118 market, 105 phytochemicals as bioactive ingredients for biological activities of phytochemicals, 98
103 health-promoting ability of phytochemicals, 96
97 phytochemicals-based functional foods, 103
105

659

phytonutrients, 95
96 Functional herbal RTS, 135 Functional nutraceuticals, 125 Functional nutrients, 123
124 Fungal species, 608 Fungi, 384, 617 Fusarium solani, 117

G G6Pase. See Glucose-6-phosphatase (G6Pase) GA. See Gallic acid (GA); Glycyrrhizin (GA) Galanthus nivalis, 467t Galenicals, 32 Gallic acid (GA), 14, 256t Gallocatechin gallate (GCG), 622
623 Garlic (Allium sativum), 11
12 chemical structures of main organosulfur compounds in garlic, 12f Gas chromatogram, 77
78 schematic diagram of basic gas chromatograph, 78f Gas chromatography (GC), 6, 47, 70, 371 Gas chromatography
mass spectrometry (GCMS), 6
7, 79, 190, 605 analysis, 112 spectrometer, 80f spectrum, 79 Gas
liquid chromatography (GLC), 23, 70 schematic representation of standard GLC, 71f Gasoline, 204 Gastric adenocarcinoma (GC), 327
328 Gastric ulcer, 609
610 Gastrointestinal absorption, 575 Gastrointestinal malignancies (GMs), 329 gut microbiota in, 326
329 Gastrointestinal tracts, 570 Gastroprotection mechanism, 610 GC. See Gas chromatography (GC); Gastric adenocarcinoma (GC) GC-MS. See Gas chromatography
mass spectrometry (GC-MS) GCG. See Gallocatechin gallate (GCG) GCK. See Glucokinase (GCK) Geissospermine, 204 Gel filtration chromatography, 5
6 Gene(s), 145, 181 gene-mediated therapy, 163 mutation, 309 Generally recognized as safe (GRAS), 406 Genetic(s), 145 hormones synthesized and secreted by dedicated endocrine glands, 146t material, 611 nomenclature, 618 Genome instability, 169
170 Genomic instability, 374 Geobacillus stearothermophilus, 417t Geranyl diphosphate (GPP), 354 Geranylgeranyl diphosphate (GGPP), 354 GFR. See Growth factor receptor (GFR) GGPP. See Geranylgeranyl diphosphate (GGPP)

660

Index

Gherkin hybrid zefir, 165 Ginger (Zingiber officinale), 12 ginger plant, rhizome, and active components, 13f Gingko, 136 G. biloba, 136, 467t Ginsenosides, 470
471 Glass, 418 GLC. See Gas
liquid chromatography (GLC) Glioblastoma cell, 632 anticancer activity of citral against prostate and, 633f Global phytochemicals, 579 GLS. See Glucosinolates (GLS) Glucokinase (GCK), 375
377 Glucopyranoside, 190 Glucose, 149 Glucose transporter 1 (GLUT1), 465 Glucose-6-phosphatase (G6Pase), 190, 375
377 dehydrogenase, 483 Glucosidase, 537 Glucosides, 491 Glucosinolates (GLS), 296, 359
360, 420, 423 Glucuronidation, 172, 491
492 GLUT-4, 153, 375
377 GLUT1. See Glucose transporter 1 (GLUT1) Glutamate semialdehyde, 484 Glutathione (GSH), 311
313, 483, 485 glutathione-dependent system, 483 reductase, 483 transferase, 483 Glutathione peroxidase (GPx), 483, 485 Glyceryl monooleate (GMO), 471 Glycine max L. See Soybeans (Glycine max L.) Glycone, 359
360 Glycosides, 250, 385t, 390
391, 618 Glycosidic compounds, 46 Glycosylated steroids, 536 Glycosylation of resveratrol, 491 Glycyrrhiza glabra, 467t Glycyrrhizic acid, 638 Glycyrrhizin (GA), 168t, 173t GMO. See Glyceryl monooleate (GMO) GMs. See Gastrointestinal malignancies (GMs) Gold, 198
199 Gooseberry (Emblica officinalis), 131 GPP. See Geranyl diphosphate (GPP) GPx. See Glutathione peroxidase (GPx) Gram-positive bacteria, 456 Grape seed extract (GSE), 623 Graphene, 49 GRAS. See Generally recognized as safe (GRAS) Grinding, 32 Griseofulvin, 384 Ground samples, 32 Growth factor receptor (GFR), 315 Growth factor signalling targets, 315 GSE. See Grape seed extract (GSE) GSH. See Glutathione (GSH) Guaiol, 357 Gut microbiota in gastrointestinal malignancy, 326
329

Gut microorganisms, 328 Gymnema sylvestre, 545

H 1

H-NMR spectra, 78 Haber
Weiss reaction, 484 Habitat management, area of, 30 Harringtonine, 218 HATR. See High-attenuated total reflectance (HATR) Hb. See Hemoglobin (Hb) HCL-116. See Human colorectal carcinoma (HCL-116) HCQ. See Hydroxychloroquine (HCQ) HD. See Huntington’s disease (HD) HDL. See High-density lipoprotein cholesterol (HDL) Head cancer, 101
102 Healing cancer-associated MDR, phytochemical interventions in, 278
283 Healing process, 602
604 Health, 181 benefits of phytochemicals, 536
538 anti-diabetes activity, 537 anti-obesity activity, 537 anticancer activity, 537 antimicrobial activity, 538 cardiovascular protection, 537 oxidative stress amelioration, 537 reducing inflammation, 537 health-promoting ability of phytochemicals, 96
97 major phytonutrients of nutraceutical importance, sources, and health benefits, 97t health-providing ingredients, 124 safety of drinks, 138 Healthcare system, 571 Healthy foods, 579 Heart disease, 114 HeLa cell, 637 Helicases, 516 Helichrysum italicum, 295 Helicobacter pylori, 327
328 Helicobacter pylori Ddl enzyme (HpDdl enzyme), 620
621 Hematopoietic stem cell transplantation (HSCT), 583 Heme oxygenase-1 (HO-1), 485 Hemoglobin (Hb), 115 Hemolytic uremic syndrome (HUS), 419 Hepatitis B virus, 180 Hepatitis
Barr virus, 180 Hepatocellular carcinoma cell (HepG2 cell), 638 Hepatoprotective potential, 610 Hepatotoxicity, 610 HepG2 cell. See Hepatocellular carcinoma cell (HepG2 cell) HER2. See Human epidermal growth factor receptor 2 (HER2) Herb-mixed beverage, 135

Herbal beverage, 135
136 Herbal drug delivery system, 157 Herbal formulations, 587 Herbal medical treatments, 619 Herbal medicines, 154
155, 158
159, 335, 569 factors responsible for increased selfmedication with, 159 Herbal nutraceuticals, 463 Herbal phytomedicines in modern system advantages of, 159 factors responsible for increased selfmedication with herbal medicine, 159 Herbal supplements, 172 Herbal targeted drug delivery concept, 154
155 Herbal-blended lime-based ready-to-serve beverage, flowchart for preparation of, 135f Herbs, 136, 501 beverages rich in, 134
136 used in diabetes, 155 used in polycystic ovary syndrome, 155 Hereditary cancer, 181 Herpes simplex virus type 1 (HSV-1), 519
520 Herpes simplex virus type 2 (HSV-2), 361 Hesperetin 7-rutinoside. See Hesperidin Hesperidin, 101, 103, 591
592 Heterocyclic alkaloids, 204 Hexane, 38
41, 45, 87 Hexylcyclohexane, 112 HHPE. See High hydrostatic pressure extraction (HHPE) HHT. See Homoharringtonine (HHT) Hibiscus sabdariffa, 297 Hibiscus sabdariffa extract (HSE), 134
135 HIF. See Hypoxia-inducible factor (HIF) High blood pressure, 96 High extraction temperature, 39
40 High hydrostatic pressure extraction (HHPE), 20
21 High-attenuated total reflectance (HATR), 75 High-density lipoprotein cholesterol (HDL), 100 High-energy beams, 182 High-intensity focused ultrasound, 182 High-performance capillary electrophoresis (HPCE), 6 High-performance liquid chromatography (HPLC), 6, 23, 68, 77, 605 High-performance liquid drop countercurrent chromatography, 68 High-performance thin-layer chromatography (HPTLC), 72 differences between HPTLC and TLC, 73t High-pressure liquid chromatography (HPLC), 71
72 schematic representation of standard HPLC, 71f High-pressure solvent extraction (HPSE), 43 High-resolution mass spectrometry (HR-MS), 6
7

Index

High-speed countercurrent chromatography (HSCCC), 6, 74 Highly mobile group box 1 (HMGB1), 204, 519 HIV, 180 HLV. See Hybrid liposome vesicle (HLV) HMGB1. See Highly mobile group box 1 (HMGB1) HO-1. See Heme oxygenase-1 (HO-1) Hodgkin’s breast cancer, 205 Hodgkin’s lymphoma, 205 Holarrhena antidysenterica (L. ), 88 Homoharringtonine (HHT), 217
219, 342 structure of, 218f Homology modeling technique, 588 Hormonal therapy, 163 Hormone(s), 143 hormone-producing glands, 143
144 metabolism, 535 therapy, 182, 265
266, 629 Host-based targets, 516
522 epigentic mechanism, 517 host proteins, 516
517 pathways, 517
521 Hot air drying, 117 Hot extraction process, 47
48 HPCE. See High-performance capillary electrophoresis (HPCE) HpDdl enzyme. See Helicobacter pylori Ddl enzyme (HpDdl enzyme) HPLC. See High-performance liquid chromatography (HPLC); High-pressure liquid chromatography (HPLC) HPSE. See High-pressure solvent extraction (HPSE) HPTLC. See High-performance thin-layer chromatography (HPTLC) HPV. See Human papillomavirus (HPV) HR-MS. See High-resolution mass spectrometry (HR-MS) HSCCC. See High-speed countercurrent chromatography (HSCCC) HSCT. See Hematopoietic stem cell transplantation (HSCT) HSE. See Hibiscus sabdariffa extract (HSE) HSV-1. See Herpes simplex virus type 1 (HSV-1) HSV-2. See Herpes simplex virus type 2 (HSV-2) HTN. See Hypertension (HTN) HTT. See Huntingtin (HTT) Human breast cancer cell lines (HCC1937), 168 Human colorectal carcinoma (HCL-116), 164
165 Human epidermal growth factor receptor 2 (HER2), 262
263 HER(2) breast cancer, 276 Human herpes virus 8, 180 Human non-small cell lung cancer cells (NSCLC cells), 217 Human papillomavirus (HPV), 180, 255, 327
328

Human prostate cancer cells, effects of specific plant families extracts on, 184
185 crassulaceae, 185 juglandaceae, 184
185 moraceae, 185 Human T-cell lymphotropic virus, 180 Huntingtin (HTT), 473 Huntington’s disease (HD), 297, 463, 474t HUS. See Hemolytic uremic syndrome (HUS) Hyal S. See Streptomyces hyalurolyticus (Hyal S) Hybrid liposome vesicle (HLV), 472
473 Hybridoma technology, 75 Hydrocarbons, 38
39, 45 Hydrodistillation, 22, 37, 633 Hydrogen bonds, 546 Hydrogen peroxide (H2O2), 374
375 Hydrolysate tannins, 131 Hydrolysis products, 420 Hydrolyzable components, 37
38 Hydrolyzable tannin oligomers, 605, 607 Hydrophilic antioxidants, 486 Hydrophilic phytochemicals, 1
2 Hydrophobic interactions, 398
399, 555 Hydrosteam distillation, 38 Hydroxybenzoic acid, 487 Hydroxychloroquine (HCQ), 514 Hydroxycinnamic acid, 338, 487, 491 6-hydroxydopamine (6-OHDA), 471 Hydroxyl free radicals, 87 radicals, 484 radicals, 374
375 7-hydroxylation pathway, 239 5-hydroxymethyl furfural (5-HMF), 445 4-hydroxynonenal (4-HNE), 484
485 Hypercholesterolemia, 100 Hyperglycemia, 296
297 Hypericum perforatum, 467t Hypertension (HTN), 86, 123
124, 297 Hypothalamus, 143 Hypothesis linking adiposity and raised thyroid stimulating hormone, 148f Hypoxia-inducible factor (HIF), 587 HIF-1-mediated resistance, 587 HIF-1α, 311
313 Hyptis spicigera, 374

I I/R. See Ischemia/reperfusion (I/R) I3C. See Indole-3-carbinol (I3C) IAPP. See Islet amyloid polypeptide (IAPP) IAPs. See Inhibitor of apoptosis proteins (IAPs) IBD. See Inflammatory bowel disease (IBD) ICMR National Cancer Registry Program (NCRP), 150 ICU. See Intensive care unit (ICU) IDF. See International Federation for Dairy (IDF) IE. See Infective endocarditis (IE) Ifosfamide, 310 IGF. See Insulin-like growth factor (IGF)

661

IHME. See Institute for Health Metrics and Evaluation (IHME) IL-1β. See Interleukin-1beta (IL-1β) Illicium verum, 632 IM. See Ingenol mebutate (IM) Imatinib, 585 Imidazole, 267
268 Immersion method, 2 Immune reactions, 542 Immune responses, 617 Immune system, 535, 587 Immune therapy, 629 Immunity regulators, 98
100 Immunity-boosting mechanism, 521
522 Immunoassay, 75, 77 Immunoglobulin receptor, 583 Immunonutrients, 98
100 Immunonutrition, 98 Immunotherapy, 163, 180, 182 Immunotoxins, 574 Impurities, 3 In silico approaches for phytochemicals-based mitigation of biofilm formation, 445 In vitro tests, 91 In vivo application, 91 Indian gooseberry, 155 Indian nutraceutical market, 125 Indian rhododendron. See Melastoma malabathricum Indole-3-carbinol (I3C), 407
408, 593 Indolizidine, 267
268 Induced pluripotent stem (IPS), 309
310 Infection, 413 Infectious diseases, 545 Infectious microorganisms, 617 Infective endocarditis (IE), 418 Inflammation, 96, 114, 170 Inflammatory bowel disease (IBD), 298
299 phytochemicals, 298t Inflammatory disorder, 116 Infrared spectroscopy (IR spectroscopy), 6, 76
77, 572 Infusion, 36 Ingenol mebutate (IM), 341 Inhibition, 400 Inhibitor of apoptosis proteins (IAPs), 592 Innate immune system, 170 Institute for Health Metrics and Evaluation (IHME), 150 Insulin, 153 insulin-dependent diabetes mellitus, 296
297 resistance, 149, 574 secretion, 610 Insulin-like growth factor (IGF), 315 Intensive care unit (ICU), 151 Intensive laboratory technology, 29 Interleukin-1beta (IL-1β), 375 Internal circadian clock, 86 International Agency for Cancer Research, 629 International Federation for Dairy (IDF), 104
105 International Union of Pure and Applied Chemistry (IUPAC), 618

662

Index

Intestinal tract, 541 Intracellular stress in cancer, 227 Inulin, 137 Iodine utilization, 539 Ion exchange chromatography, 5
6 Ipomoea cairica, 377 IPP. See Isopentenyl diphosphate (IPP) IPS. See Induced pluripotent stem (IPS) IR spectroscopy. See Infrared spectroscopy (IR spectroscopy) Irinotecan, 209, 249 Iron, 198
199 deficiency, 539 fortification, 539 Irradiation time, 41
42 Ischemia/reperfusion (I/R), 472 ISL. See Isoliquiritigenin (ISL) Islet amyloid polypeptide (IAPP), 578 Isoamericanol B1, 574 Isobologram, 590 Isoborneol, 372 Isoflavin, 42 Isoflavones, 99t, 137, 256t, 325, 337, 343, 541 Isoflavonoids, 49, 97t, 385t, 390
391 Isolated pure phytochemicals, 1 Isolation of phytochemicals, 1, 2f of phytoconstituents, 4
6 chromatography techniques, 5
6 classical isolation methods, 4
5 modern separation technologies, 5
6 schematic representation of common polarity gradient extraction method, 4f Isoledene, 629
632 and elemene against colon cancer, anticancer activity of, 633f Isoliquiritigenin (ISL), 256t, 324 Isopentenyl diphosphate (IPP), 354, 371 Isoprene, 370
371 chain of polymers of, 354f molecules, 536 Isoprenoids, 251, 295 Isopropanol, 38
39 Isoquinoline, 267
268 biological source, mechanism of action, and applications of isoquinoline alkaloids, 210
214 berberine, 211 liriodenine, 213 noscapine, 212 sanguinarine, 213
214 Isoshinanolone, 190 Isothiocyanates (ITCs), 85, 97t, 102, 370, 421t, 423, 536 ITCs. See Isothiocyanates (ITCs) IUPAC. See International Union of Pure and Applied Chemistry (IUPAC)

J JAK/STAT pathway. See Janus kinase/signal transducers and activators of transcription pathway (JAK/STAT pathway)

JAK2/STAT3. See Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), 228 Janus kinase/signal transducers and activators of transcription pathway (JAK/STAT pathway), 517 JNK. See c-jun N-terminal kinase (JNK) Juglandaceae, 184
185 Juice, 130 Juniperus J. oxycedrus EO, 374, 639 Jurinea bucharica, 165

K Kaempferia galanga L. (KGEOs), 639 Kaempferia parviflora, 136 Kaempferol, 405 Kaposi’s sarcoma, 205 Katharometer, 77
78 Ketones, 353, 372 Kigelia Africana, 168 Kiwi, 12 bioactive compounds of kiwi, 15f Klebsiella pneumoniae, 193, 401
405 Kunun-zaki, 127 Kvass, 127 Kynurenines, 484

L L-di-hydroxy-phenylalanine (L-DOPA), 484 L-DOPA. See L-di-hydroxy-phenylalanine (LDOPA) LA. See Licochalcone A (LA) LA6. See Lactobacillus plantarum A6 (LA6) LAB. See Lactic acid bacteria (LAB) Lactate dehydrogenase (LDH), 472
473 Lactic acid, 406 Lactic acid bacteria (LAB), 127
128 Lactobacillus L. acidophilus, 100
101 L. delbrueckii sp. bulgaricus, 391
392 L. paracasei, 540 L. rhamnosus, 540 GR-1, 128 L. sakei, 127 Lactobacillus plantarum A6 (LA6), 130 Lactobacillus rhamnosus GG (LGG), 130 Lactonase, 401 Laminaran, 385t Latex, 418 Lavandula angustifolia, 377 Lavender essential oils, 371 Lawsonia inermis, 88 LC
MS. See Liquid chromatography
mass spectrometry (LC
MS) LDH. See Lactate dehydrogenase (LDH) LDL-C. See Low-density lipoprotein cholesterol (LDL-C) LDLR. See Low-density lipoprotein receptor (LDLR)

Lead molecules, 620
621 Leea indica, 164
165 Leishmania tropica, 553
554 Leishmanolysin, 550
553 Lemon balm essential oil, 376t Lethal subtype of prostate cancer, 180 Letrozole, 262
263 Letterer
Siwe disease, 205 Leucine hydroxides, 484 Leukemia combination index method and synergism, 587
588 drug resistance, 585
587 proteins/genes responsible for drugresistance leukemia, 586
587 phytochemicals as chemosensitizer and modulators, 588
594 computational approach to target multidrug resistance, 588
590 in vitro analysis of phytochemicals as multidrug resistance reversal, 590
592 in vivo analysis of phytochemicals as multidrug resistance reversing agents, 593
594 phytochemicals from plant to computational and molecular laboratory to target multiple disease processes, 584f Leukemogenesis, 583 Leukorrhea, 604
605 Leukotrienes (LTs), 375 LGG. See Lactobacillus rhamnosus GG (LGG) Licochalcone A (LA), 323
324 Lignans, 74 Ligularia hodgsonii, 219 Limonoids, 97t, 385t, 390
391, 406 Linalool, 361, 635 anticancer activity of germacrene D and Linalool against liver and breast cancer, 638f Linear tubulin heterodimers, 225 Lipid(s), 44
45, 295, 398
399, 484
485, 537 hydroperoxides, 484
485 lipid-soluble antioxidants, 486 peroxidation inhibition, 611 Lipopolysaccharide (LPS), 375, 620
621 molecule, 608 Liposomes, 157 Lipoteichoic acid (LTA), 375 Lipoxygenase (LOX), 375 Lippia multiflora, 374 Liquid chromatography, 371, 572 Liquid chromatography
mass spectrometry (LC
MS), 6
7, 72 Liquor, 110 Liriodenine, 213 structure of, 213f Listeria monocytogenes, 360
361, 397, 417t, 418 Liver cancer anticancer activity of 1,8-cineole against, 634f aristolene and α-farnesene against colon and, 639f

Index

lncRNAs. See Long noncoding RNA (lncRNAs) Lobules, 262 Long noncoding RNA (lncRNAs), 276
278 Lophophora williamsii, 204 Loranthus micranthus, 297 Lou Gehrig’s disease. See Amyotrophic lateral sclerosis (ALS) LoVo colorectal cancerous cellm, 636 Low-density lipoprotein cholesterol (LDL-C), 147
148, 580 Low-density lipoprotein receptor (LDLR), 211 Low-fat probiotic beverage, 128 LOX. See Lipoxygenase (LOX) LPS. See Lipopolysaccharide (LPS) LTA. See Lipoteichoic acid (LTA) LTs. See Leukotrienes (LTs) Lumnitzera racemosa, 388 Lung adenocarcinoma, 165
168 Lung cancer, 217 anticancer activity of perilla ketone against gastric and, 636f Lutein, 97t, 385t, 390
391 Luteolin, 256t Lycopene, 10, 97t, 98, 99t Lycopodium serratum, 467t Lycorine, 268t Lymph nodes of pelvis, 181 Lyophilization, 31 Lysergol, 86
87

M M-loop. See Microtubule loop (M-loop) MABA. See Microplate Alamar blue assay (MABA) MAbs. See Monoclonal antibodies (MAbs) Maceration, 22, 35
36, 50, 65
66 Macrophages, 550
553 Madhuca M. indica, 109 M. longifolia, 114 MAE. See Microwave-assisted extraction (MAE) Magnetic nuclei, 572 Mahua, 109 biofertilizer, 112
113 botanical description, 109
110 flowers, 117 as functional food, 116
118 health benefits of, 118 Mahua-deoiled cake, 110 microscopy of mahua, 110 NTFP, 109 nutritional and phytochemical profiling, 111
112 comparative nutritional profile, 112 effect of geographical distribution on flower composition, 112 nutritional analysis of mahua, 111 oil, 110 pharmaceutical uses and pharmacological importance, 112
116 biodiesel, 113

biological activity, 113
116 industrial uses, 112
113 traditional uses, 110
111 trees, 109
110 uses of different parts of mahua, 110 cake, 110 flowers, 110 fruits, 110 Mahua oil, 110 seeds, 110 Maize (Zea mays (L.), 126 Malabar melastome. See Melastoma malabathricum MALDI-MS. See Matrix-assisted laser desorption mass spectrometry (MALDIMS) Maleic acid, 406 Malic acid, 406 Malignant cells, 325, 585 Malignant neoplasm, 309 Malondialdehyde (MDA), 471, 484
485 Malonyl CoA-acyl carrier protein transacylase fabD (MCATs), 620
621 MALT. See Mucosa-associated lymphoid tissue (MALT) Mammalian cell cycle, 316
317 Mandragora autumnalis, 165
168 Mangifera indica, 297 Mango fruit, 130 Manual injection valve, 72 MAPK. See Mitogen-activated protein kinase (MAPK) MAPs. See Microtubule-associated proteins (MAPs) Marine-based phytochemicals, 384, 393f bioactive potential, 387
389 antibacterial activity, 388 anticancer agents, 389 antifungal activity, 388 antiviral agent, 389 biomedical applications, 390
392 biomedical use and sources, 385t, 388f effective against cancer treatment, 389t metabolic process, 387 phytochemicals from marine resources, 384
387 portrays ROS with assorted biological molecules, 392f Market of nonalcoholic beverages, 128 of nutraceutical or functional beverages, 125 Marqibo, 206 Mass spectra, 6, 76
77 Mass spectrometry (Ms), 79, 190 Mass spectroscopy (Ms), 70, 639 Matricaria chamomilla. See Chamomile (Matricaria chamomilla) Matrix metalloproteinases (MMPs), 317
318 MMP-2, 217 MMP-9, 217 Matrix solid-phase dispersion, 49 Matrix-assisted laser desorption mass spectrometry (MALDI-MS), 6
7 MBL. See Metallo-β-lactamase (MBL)

663

MCATs. See Malonyl CoA-acyl carrier protein transacylase fabD (MCATs) MCF-7. See Michigan Cancer Foundation (MCF-7) Mcl-1. See Myeloid cell leukemia sequence-1 (Mcl-1) MD. See Molecular dynamics (MD); Multidrug (MD) MDA. See Malondialdehyde (MDA) MDAs. See Microtubule-destabilizing agents (MDAs) MDR. See Multidrug resistance (MDR) MDS. See Molecular dynamics simulation (MDS); Myelodysplastic syndromes (MDS) Meat, 18 products, 105 representative meat-based bioactive compounds, 19f Medicinal herbs, 483, 617 Medicinal plants, 27, 102, 189, 198
199, 571, 617 research, 32 seed extracts as source of phytochemicals, 571t Mediterranean diet, 85 Melaleuca alternifolia, 361 Melastoma, 601 Melastoma malabathricum, 601 ethno-medicinal practices, 602
605 flower, 602f fruit, 603f leaf, 602f list of phytochemical compounds isolated from different parts of, 606t pharmacological potentialities, 607
611 anti-cancerous property, 611 anti-ulcer property, 609
610 antidiabetic potential, 610 antidiarrheal property, 609 antimicrobial potential, 608 antinociceptive property, 610
611 antioxidative potential, 607
608 hepatoprotective potential, 610 wound-healing potential, 608
609 phytochemical constituents, 605
607 Melissa officinalis, 467t EO, 372 Meloidogyne incognita, 115 Membrane degradation, 624 membrane-bound zinc proteinase, 550
553 membrane-disrupting activities, 624 modulation of, 170 protein, 514
515 Menstrual cycle, 144 Mental stressors, 144
145 Mentha M. balsamea, 296 M. piperita, 361 M. spicata, 637 M. suaveolens ssp. insularis, 422 Menthol, 401 MERS-CoV, 102

664

Index

Mesua ferrea, 629
632 Metabolic syndromes, 86 Metal chelating, 424 Metal chelators, phytochemicals as, 424 Metallo-β-lactamase (MBL), 436 Metastasis, 247
248, 262, 632 Methanol, 33, 41
42, 46, 64, 87 Methicillin-resistant Staphylococcus aureus (MRSA), 422 Methicillin-sensitive Staphylococcus aureus (MSSA), 422 Methotrexate, 264t Methyl alcohol (CH3OH), 388 Methyl jasmonate, 256t Methylene chloride, 75 Methyl
erythritol phosphate pathway, 354 MIC. See Minimum inhibitory concentration (MIC) Michigan Cancer Foundation (MCF-7), 164
165 Microbial biofilms, 433 Microbial colonization, 608
609 Microbial diseases, 617 Microbial infections, 96 Micrococcus, 116
117 M. luteus, 115 Microcolony development, 398
399 formation, 415 Microelements, 124 Micronutrient malnutrition, 133 micronutrient-fortified beverage, 133
134 micronutrient-fortified foods, 134 Microorganisms, 397
399, 545, 617 associated with biofilms and their health hazards, 416
418 Microplate Alamar blue assay (MABA), 197 MicroRNAs (miRNAs), 276
278, 645
646 Microscopy of mahua, 110 Microtubule loop (M-loop), 234 Microtubule-associated proteins (MAPs), 225 Microtubule-destabilizing agents (MDAs), 239
240 alkaloids as microtubulin disrupting agents, 228
231 colchicine as microtubule-disrupting agent, 233
235 coumarin’s background and therapeutic activities, 238
239 curcumin, phenolic compound, disrupts microtubule function, 235
236 binding of curcumin polyphenol with microtubule, 236 mechanism underlying polyphenols as microtubulin-binding target, 235
236 therapeutic relevance of curcumin against microtubule, 236 factors affecting microtubule dynamics in cancer cells, 226
227 intracellular stress in cancer, 227 molecular basis of microtubule dynamics, 226

noscapine therapeutic agents disrupting microtubule dynamics, 236
238 targeting microtubules in cancer, 227
228 taxol as therapeutic agent disrupting cell polymerization, 231
233 Microtubule-stabilizing agents (MSAs), 239
240 Microtubule-targeting agents (MTAs), 239
240 Microtubules (MTs), 208, 225 colchicine binding site and interplay with, 234 mechanism and binding site against microtubule, 239 site interplay of taxanes with, 232
233 Microtubulin alkaloids as microtubulin disrupting agents, 228
231 vinca alkaloids and their mechanism of action against microtubulin, 229 Microwave-assisted extraction (MAE), 20
23, 28
29, 35, 39
42, 44, 64, 67, 98, 571
572. See also Accelerated solvent extraction (ASE) advantages and disadvantages of, 42 method, 3 potential applications of, 42 practical issues for, 40
42 dielectric constant of some commonly used solvents, 41t principles and mechanisms, 39
40 Microwave(s), 39
40, 635
636 energy density, 42 extraction, 67, 371 microwave-drying, 32 power, 41 Millets (Pennisetumglaucum (L. )), 126 Millettia pinnata, 165 Minerals, 95
96, 111 Minimal residual disease (MRD), 264
265 Minimum inhibitory concentration (MIC), 24, 197, 608, 621 assessment of, 24 Mintel Business Market Research report, 124
125 miRNA regulation in BC, enlists roles of different dietary phytochemicals in, 279t miRNAs. See MicroRNAs (miRNAs) Miscible solvents, 34 Mitochondrial cytochrome c, 585
586 Mitogen-activated protein kinase (MAPK), 217, 311, 490, 522 Mitomycin-C, 249 Mitoxantrone (MTX), 282 MMN. See Multiple-micronutrient (MMN) MMPs. See Matrix metalloproteinases (MMPs) Modern allopathic medicine, 369
370 Modern separation technologies, 5
6 chromatography techniques, 5
6 Modified percolation, 66
67 cold percolation, 66 countercurrent extraction, 66
67 Modifiers, 46

Molecular docking, 545
546, 572
573, 576, 645 of compounds against XO enzyme, 577f of procyanidin B1 against BChE enzyme, 578f study of bioactive phytochemicals with anticancer properties, 645
647 study of bioactive phytochemicals plant products as anti-coronal agents, 546
550 plant products as anti-leishmanial agents, 550
556 plant products as antitubercular agents, 557
565 Molecular dynamics (MD), 573
574 Molecular dynamics simulation (MDS), 445 Molecular mechanism of phytochemicals in preventing cancer, 169
170 apoptosis and autophagy, 170 genome instability, 169
170 modulation of membrane, 170 targeting cell proliferation, 169 targeting immune surveillance and inflammation, 170 targeting molecular pathway of cancerous cell, 169 targeting oxidative stress and redox signaling, 169 Molecular modeling methods, 545 Momordica charantia, 377 Mono-drug therapy, 584
585 Monoclonal antibodies (MAbs), 75, 163 Monoclonal antibody therapy, 583 Monocyclic monoterpenes, 355, 356f Monocyclic sesquiterpenes, 357 Monoterpenes (C10), 295, 355, 371, 405
406, 519
520, 635
636 acyclic, 355f bicyclic, 356f chemical structure of leaf alcohol, 356f monocyclic, 356f tricyclic, 356f Monoterpenoids (C10), 269 Montamine, 210 structure of, 210f Moraceae, 185 Morganella morganii, 193 Morinda citrifolia, 638 Moringa oleifera, 165, 636 Moroccan Labiatae, 361 Morphine, 204, 210 Mosloflavone, 445 Motor neuron disease, 463 MRD. See Minimal residual disease (MRD) MRI, 181 MRPL ABCG2. See ABCC1 MRSA. See Methicillin-resistant Staphylococcus aureus (MRSA) Ms. See Mass spectrometry (Ms); Mass spectroscopy (Ms); Multiple sclerosis (Ms) MS. See Spectrographic analysis (MS) MSAs. See Microtubule-stabilizing agents (MSAs)

Index

MS
MS. See Tandem mass spectrometry (MS
MS) MSSA. See Methicillin-sensitive Staphylococcus aureus (MSSA) MTAs. See Microtubule-targeting agents (MTAs) MTs. See Microtubules (MTs) MTX. See Mitoxantrone (MTX) Mucoid substances, 3 Mucosa-associated lymphoid tissue (MALT), 327
328 Multidrug (MD), 278
281 therapy, 584
585 Multidrug resistance (MDR), 247
248, 261
262, 310, 341, 459, 585, 617 flavonoids activity against multidrugresistant microbes, 619
625 structures of flavonols, 620f in vitro analysis of phytochemicals as multidrug resistance reversal, 590
592 in vivo analysis of phytochemicals as multidrug resistance reversing agents, 593
594 Multiple sclerosis (Ms), 463, 474t Multiple-micronutrient (MMN), 134 Multistage extraction, 66 Murraya koenigii, 297 Mycobacterium M. smegmatis, 190, 621 M. tuberculosis, 190, 557, 621 Mycolic acid, 620
621 Mycosis fungoides, 205 Mycosporine, 385t, 390
391 Myelodysplastic syndromes (MDS), 342 Myeloid cell leukemia sequence-1 (Mcl-1), 217 Myricetin, 325, 621 Myrosinases, 359
360

N N,N dimethyl-formamide (DMF), 34 N protein-encased genome, 513 N-hydroxy-N0 -[2-(trifluoromethyl)phenyl] pyridine-3-carboximidamide, 646 N-terminal constitutes amino acids, 226 NAFLD. See Nonalcoholic fatty liver disease (NAFLD) Nano-based formulation using plant-derived phytochemicals for biofilm inhibition, 445
446 Nano-biotechnology, 195
197 morphological analysis of ZnONPs, 196f Nanocarriers, 247
248 Nanocomposite (NC), 446 Nanodecanol, 190 Nanodrug delivery of phytochemicals in treating cancer, 89
90 anticancer properties of diverse phytochemicals, 89t recent advances in drug delivery with phytochemicals, 89f Nanoencapsulation, 391
392 Nanoformulations in tackling neurodegeneration, 466
475

Nanoliposomes, 470 Nanomaterials, 465 Nanomedicine, 199 Nanoparticles, 157, 195
196, 198
199, 466
469 Nanostructured lipid carriers (NLCs), 469 Nanotechnology, 198
199, 446 in neurodegenerative disorders, 465 Naringenin, 255, 343 Naringin, 256t, 618 National Cancer Institute (NCI), 318, 587
588 National Center for Complementary and Integrative Health (NCCIH), 266 National Centre for Disease Informatics and Research (NCDIR), 150 Natural antioxidants, 486 Natural bioactive compounds, 10 Natural compounds, 85
86, 590
591 Natural food sources, 98 Natural materials, 570 Natural oils, 520 Natural products, 3
4, 10, 63, 68, 72, 203, 545, 645
646 brief summary of various extraction methods for, 28t endocrinological disorder and natural products/herbs used in treatment, 155 Natural yogurt (NY), 541 Natural-source chemical compounds, 617 Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT), 320 NBD. See Nucleotide-binding domain (NBD) NBP. See Nucleotide binding pocket (NBP) NC. See Nanocomposite (NC) NCCIH. See National Center for Complementary and Integrative Health (NCCIH) NCDIR. See National Centre for Disease Informatics and Research (NCDIR) NCDs. See Noncommunicable diseases (NCDs) NCI. See National Cancer Institute (NCI) ncRNA. See Noncoding RNAs (ncRNA) NCRP. See ICMR National Cancer Registry Program (NCRP) NDDS. See Novel drug delivery system (NDDS) Neck cancer, 101
102 Negative pressure cavitation extraction, 49 Nelumbo nucifera, 136 Neoadjuvant chemotherapy, 263
264 Neoplastic cells, 262 Neoplastic disease, 163 Neoxanthin, 272
273 NEPC. See Neuroendocrine prostate tumor (NEPC) Nervous system, 576
578 Neurodegenerative diseases, 96, 463 Neurodegenerative disorders, 102, 297
298, 463
464 etiology, 464f key issues associated with neurodegenerative diseases, 464

665

limitations of nanotechnology-based approaches for management of, 476
477 nanoformulations in tackling neurodegeneration, 466
475 phyto-nanomedicine in management of neurodegenerative disorders, 466 phytochemicals-based nanoformulations in tackling neurodegeneration, 469f phytoconstituents and general mechanism of actions pertaining to neuroprotection, 465
466 phytoconstituents-based nanoformulations for, 473
475, 474t Alzheimer’s disease, 469
471 amyotrophic lateral sclerosis, 472 Parkinson’s disease, 471 stroke, 472
473 significance of nanotechnology in, 465 Neuroendocrine prostate tumor (NEPC), 180 Neuropathy-related disorders, 233 Neuroprotection, 463 Neuroprotective phytochemicals, 102 Neutral alumina, 49 New product development (NPD), 123 NF-κB. See Nuclear factor-kappa B (NF-κB) NHEK keratinocytes, 635 Nicotine, 204 Nigella sativa, 155, 297, 637 Nigella sativa L. See Cumin seeds (Nigella sativa L.) Niosomes, 157 Nitric acid, 70 Nitric oxide (NO), 87, 374
375, 592 Nitric oxide synthase (NOS), 485 Nitrogen, 401 nitrogen-compounds in essential oils, 359
360, 360f nitrogen-containing compounds, 295, 535 nitrogen-containing constituents, 296 nitrogen-containing phytochemicals, 407
408 3-nitropropionic acid (3-NP), 473 Nitrous oxide, 45 Nitroxoline, 424 NLCs. See Nanostructured lipid carriers (NLCs) NMR. See Nuclear magnetic resonance (NMR) NO. See Nitric oxide (NO) Nocardia, 116
117 Nocodazole, 205 Non-chromatographic techniques, 75, 77. See also Chromatographic techniques FTIR, 75 immunoassay, 75 phytochemical screening assay, 75 Non-heterocyclic alkaloids, 204 Non-lymphoma, 205 Nonalcoholic beverages, 126
128 cereal-based fermented nonalcoholic beverages, 126
127 market of, 128 Nonalcoholic fatty liver disease (NAFLD), 86 Noncoding RNAs (ncRNA), 276
278

666

Index

Noncommunicable diseases (NCDs), 150 Nonconventional methods. See also Conventional methods for plant extraction, 21 EAE, 22 MAE, 22
23 PEF, 23 supercritical extraction, 22 UAE, 22 Nonenzymatic antioxidants, 485 Nonnarcotic opium, 236
237 Nonpolar compounds, 45 Nonpolar solvent, 40
41 Nonprotein amino acids, 296 Nonsteroidal anti-inflammatory medicines (NSAIDs), 313
314 Nonsteroidal phytochemical, 536 Nonstructural angiotensin-converting enzyme 2 (ACE2), 102
103 Nonstructural proteins (NSPs), 515
516, 546 helicases, 516 proteases, 515 RdRp, 515
516 viral virulence factors, 516 Nontimber forest products (NTFPs), 109 Norcarotenoids, 272
273 Normal cells, 309 Normal-phase partition chromatography, 5
6 Norquinadoline A, 518 Norspermidine, 401 Norterpenes, 357 structure of damascone, 358f NOS. See Nitric oxide synthase (NOS) Noscapine, 212 structure of, 212f therapeutic agents disrupting microtubule dynamics, 236
238 collective phytochemicals, 237f mechanism of action, 237 noscapine binding site, 237 therapeutic relevance of noscapine against cancer, 238 toxicity remarks of noscapine on subjected patients, 238 Novel drug delivery system (NDDS), 155 Novel extraction techniques, 50 Novel herbal drug delivery systems, types of, 157
158 Novel phytomedicinal formulations in pharmacy to target endocrine glands and hormone for treatment of major endocrine disorders list of, 155
158 types of novel herbal drug delivery systems, 157
158 NPACT. See Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT) NPD. See New product development (NPD) Nrf2. See Nuclear factor erythroid 2-related factor 2 (Nrf2) NSAIDs. See Nonsteroidal anti-inflammatory medicines (NSAIDs)

NSCLC cells. See Human non-small cell lung cancer cells (NSCLC cells) NSPs. See Nonstructural proteins (NSPs) NTFPs. See Nontimber forest products (NTFPs) Nuclear factor erythroid 2-related factor 2 (Nrf2), 169, 297
298, 311
313 Nuclear factor kappa-light-chain-enhancer activated B cells, 522 Nuclear factor-kappa B (NF-κB), 311
313, 490, 574 Nuclear magnetic resonance (NMR), 6
7, 23, 76
77 spectroscopy, 78, 572 Nucleic acids, 398
399 Nucleocapsid (N), 102
103 protein, 514
515 Nucleotide binding pocket (NBP), 226 Nucleotide-binding domain (NBD), 226, 281
282 Nutraceuticals, 124, 163, 391
392 beverages, 123 market of, 125 Nutrition, 337 acquisition, 398
399 Nutritional analysis of mahua, 111 Nutritional profiling, 111
112 NY. See Natural yogurt (NY) Nypa fruticans, 387

O 12-O-tetradecanoylphorbol-13-acetate (TPA), 637 Obesity, 103, 186 Obesumbacterium proteus, 401
405 OBP. See Odorant-binding protein (OBP) Ocimum O. americanum, 374 O. aromaticus, 457
459 O. basilicum, 373t, 374 O. gratissimum, 375 O. sanctum, 297, 457
459, 467t Ocimum sanctum. See Tulsi (Ocimum sanctum) Ocotea quixos, 375 8,11-octadecadienoic acid methyl ester, 605 9,12-octadecadienoic acid methyl ester, 190 8-octadecanone, 112 9-octadecenamide, 190 Octadecenoic pathway, 355 Octadecyl benzoate, 553
554 Octylcyclohexane, 112 Odorant-binding protein (OBP), 196
197 Oleanolic acid, 638 Oleic acid on zebrafish embryos, anticancer activity of, 637f Oleo-gum resin extract, 629
632 Oleuropein, 10 Oligosaccharides, 137 Omacetaxine, 218 Omega-3 fatty acids, 97t Omega-3 polyunsaturated fatty acids (PUFAs), 16 Omeprazole, 609
610

Onco-miRs, 276
278 Oncogenes, 181 Onions (Allium cepa L.), 10
11 Open vessel system, 39
40 ORAC. See Oxygen radical absorbance capacity (ORAC) Oral mucositis, 587 Oral squamous cell carcinoma (OSCC), 228 Orange, 133 Oregano, 376t Organ transplantation, 583 Organic acids, 401, 406, 570, 605 Organic compounds, 74 Organic micronutrients, 87 Organic molecules, 77, 570 Organic solvent extraction, 64
66 extraction methods, 65f maceration, 65
66 percolation, 66 Organosulfur, 12 compounds, 335
336, 338, 536 Origanum, 373t O. compactum, 361 O. majorana L., 635 O. vulgare, 360
361, 376t Origanum vulgare L. oregano (OVEO), 422 Orthotyrosine, 484 OS. See Overall survival (OS) OSCC. See Oral squamous cell carcinoma (OSCC) Osteomyelitis, 418 OTC. See Overthe-counter (OTC) Otitis media, 418 Ovarian cancer, 179, 217, 227
228 Ovaries in thyroid disorders, 146
147 pathophysiology of thyroid disorder, 147f Oven drying, 31, 117 OVEO. See Origanum vulgare L. oregano (OVEO) Overall survival (OS), 262
263 Overthe-counter (OTC), 158, 362
363 Oxazole, 267
268 Oxidation, 484 Oxidative stress, 169, 193
194, 483, 539
540, 576
578, 611 amelioration, 537 antioxidants, 486
488 antioxidative effect of phytoconstituents, 488
492 absorption, 491 conjugation and plasma transport, 491
492 excretion, 492 mechanism of action, 490
492 metabolism, 491 plasma concentrations, 492 tissue uptake, 492 toxicity, 492 defense, 485 effect, 484
485, 488t and free radicals, 483
485 role of oxidative stress in carcinogenesis, 310
313

Index

oxidative stress and antioxidant defense mechanism, 310
311 plant-derived antioxidants for amelioration of, 311
313 ROS-dependent cellular metabolic pathways in cancer cells, 311 Oxides, 353 Oxidized lutein, 256t Oxidoreductase, 401 Oxindole, 407 2-oxo-histidine, 484 Oxygen free radicals, 576 Oxygen radical absorbance capacity (ORAC), 23
24 Oxygenated products, 484
485

P p-coumaric acid (PCA), 338, 371
372, 405 p-cymene, 370
371 P-glycoprotein (P-gp), 281
282, 586
587, 592 P-gp. See P-glycoprotein (P-gp) p-hydroxybenzoic acid, 487, 605 p-nitrophenyl butyrate (PNPB), 194
195 Paclitaxel (PTX), 214
215, 225, 231, 249, 264t, 282 PAHs. See Polycyclic aromatic hydrocarbons (PAHs) Palladium, 198
199 Panax ginseng, 467t Pancreas, 143, 148
149 Pancreatic cancer, 101
102, 179 anticancer activity of D-limonene liver and, 639f cell, 217 Pancreatic ductal adenocarcinoma cells, 639 Papaver somniferum, 212, 236
237, 384
387 Papaverine, 204 Paper chromatography, 69 Paraoxonase, 401 Parasitic protozoan leishmania, 550
553 Parathyroid, 143 Parkia timoriana, 646 Parkinson’s disease (PD), 297, 463, 474t phytoconstituents-based nanoformulations for, 471 Parkinson’s disorders, 102 PARP. See Poly(ADP-ribose) polymerase (PARP) Parsley (Petroselinum crispum), 502 Parthenolide, 593
594 Partition, 69 chromatography, 5
6 Passive transfer channels, 465 Pathogenesis, 398
399, 537 Pathogenic bacteria, 413
414 Pavine, 210 PBE. See Piper betle ethyl acetate extract (PBE) PCA. See p-coumaric acid (PCA) PCOS. See Polycystic ovary syndrome (PCOS)

PD. See Parkinson’s disease (PD); Pharmacodynamic (PD) PDGF. See Platelet-derived growth factor (PDGF) Peach, 133 Pear, 133 Pectinatus spp., 417t Pediococcus pentosaceus, 540 PEF. See Pulsed electric field (PEF) PEG. See Polyethylene glycol (PEG) PEITC. See Phenylethyl isothiocyanate (PEITC) Pelargonium graveolens EO, 376t Penicillium notatum, 545 Pentadecan-8-one, 112 Pentylenetetrazol-induced seizures (PTZ), 115 PEPCK. See Phosphoenolpyruvate carboxykinase (PEPCK) Peppermint oil, 401 Peptidoglycan, 620
621 Percolation, 36, 66 method, 2 reserved percolation, 36 Percolator, 36, 66 Perilla frutescens L., 635 Periodontitis, 418 Periplocoideae, 502 Periwinkle, 205 Peroxidation of PUFAs, 484
485 Peroxynitrate anion, 374
375 Persistent infections, 413 Persister cells, 400 Pertinent equipment, 30 Pertuzumab, 263 Petroleum ether, 193 Petroselinum crispum. See Parsley (Petroselinum crispum) Peumus boldus, 215 Peyote, 204 PFE. See Pressurized fluid extraction (PFE) PG. See Propyl gallate (PG) PGE2. See Prostaglandin E2 (PGE2) PGs. See Prostaglandins (PGs) Phakellin-based alkaloids, 407 Pharmaceutical analysis, 72
73 Pharmaceutical applications, 371 Pharmaceutical(s), 337, 390
391 transfer of phytochemicals into, 580 uses and pharmacological importance, 112
116 Pharmacodynamic (PD), 445 Pharmacokinetic (PK), 445 activities, 646 Pharmacological potentialities, 607
611 anti-cancerous property, 611 anti-ulcer property, 609
610 antidiabetic potential, 610 antidiarrheal property, 609 antimicrobial potential, 608 antinociceptive property, 610
611 antioxidative potential, 607
608 hepatoprotective potential, 610

667

wound-healing potential, 608
609 Pharmacophores, 621 Pharmacy, application of phytomedicine in modern drug development in, 158 PHEIC. See Public Health Emergency of International Concern (PHEIC) Phenanthrene alkaloid, 204 1-phenanthrenecarboxylic acid, 636 Phenolic(s), 46, 184, 250, 295, 337
338, 372, 401
405, 420, 421t, 423, 605 acids, 42, 104, 487, 605 compounds, 9, 12, 88, 172, 183
184, 235
236, 502, 545 contents, 541 fortification with, 539
540 Phenols, 189, 250
251 Phenylalanine, 371
372 ammonia lyase, 371
372 Phenylchromanone, 618 Phenylethyl isothiocyanate (PEITC), 423 Phenylpropanoids, 354, 358
359, 370
372 biosynthesis of, 358
359 nitrogen-and sulfur-containing compounds in essential oils, 359
360 phenylpropanoids occurrence in essential oils, 359 chemical structures of phenylpropenes, 359f Phenylpropenes, chemical structures of, 359f PHFI. See Public Health Foundation of India (PHFI) Phloretin, 457
459, 623
624 Phloroglucinol, 385t Phlorotannins, 385t, 390
391 Phomopsis tersa, 437 Phosphoenolpyruvate carboxykinase (PEPCK), 375
377 Phosphoinositide 3 (PI3), 490 Phosphorus deficiency, 601 Phosphorylation, 225 Photodynamic therapy, 163 Phthalide isoquinoline, 210 PHWE. See Pressurized hot water extraction (PHWE) Phyllanthus P. amarus, 297 P. emblica, 136 Phyllostachys edulis, 618 Physical stressors, 144
145 Phyto-nanomedicine in management of neurodegenerative disorders, 466, 467t Phyto-polyphenols, 483 Phytochemicals, 6, 9, 27
29, 74
75, 95
96, 101
103, 112, 203, 225, 249, 251, 267, 295, 298, 310, 335, 383, 397, 407, 413, 463, 501
502, 536, 569
571, 584 from agri-food by-products, 87
88 dietary fiber, 88 phenolic compounds, 88 alkaloids and other nitrogen-containing constituents, 296 anti-biofilm potential of phytochemicals, 402t

668

Index

Phytochemicals (Continued) antioxidant and antimicrobial properties of, 86
87 applications of chromatography techniques, 74
75 approach for extraction, isolation, and characterization of phytochemicals, 2f BC, 267
274 bioactive, 85
86 in biofilm inhibition, 401, 420
425 for biofilm prevention and control with mechanism of action, 421t biological activities of, 98
103 anticancer, 101
102 anticholesteremic, 100
101 antidiabetic, 101 antioxidant, 98 antiviral, 102
103 immunity booster, 98
100 mechanism of action of phytonutrients in the prevention of disorders, 99t neuroprotective, 102 renoprotective, 102 camptothecin, 255 in cancer, 300
304 in alleviation of chemotoxicity, 303 in chemoprevention, 300
302 as chemotherapeutic agents, 303 in chemotherapy, 300f in conjugation with chemotherapy, 303
304 for cancer prevention by targeting cellular signalling transduction pathways, 313
318 anti-inflammatory targets, 314, 314f apoptosis targets, 315
316 genome stability, 319f growth factor signalling targets, 315 invasion, metastasis, angiogenesis, and stemness-related pathways in cancer, 319f targets in other important pathways, 317
318 targets of phytochemicals in cell cycle pathways, 316
317 cervical cancer and phytochemicals, 255
256 characterization, 23
24, 75
81 antimicrobial activity, 23 antioxidant activity, 23 antioxidant capacity, 24 assessment of MIC, 24 determination of TFC, 23 determination of TPC, 23 as chemosensitizer and modulators, 588
594 computational approach to target multidrug resistance, 588
590 in vitro analysis of phytochemicals as multidrug resistance reversal, 590
592 in vivo analysis of phytochemicals as multidrug resistance reversing agents, 593
594 classes of, 569f

classification of phytochemicals, 250
251 alkaloids, 250 polyphenol, 250
251 terpenoid, 251 thiols, 251 classification of phytochemicals source and effectiveness against cancer, 338
339 alkaloids, 339 carotenoids, 338 organosulfur compounds, 338 phenolics, 338 in clinical and preclinical stages for preventing cancer, 172 dietary supplements and probable role in preventing cancer, 175t phytoconstituents in clinical trial on different types of cancers, 174t phytoconstituents in preclinical trial on different types of cancer, 173t in clinical trials, 325 composition and biological properties of seed extracts from Washingtonia filifera, 575
579 constituents, 605
607 and conventional medical practice, 184 curcumin, 251
254 current limitations and future of phytochemicals, 91
92 uses of bioactive phytochemicals, 91f current scenario and future perspective, 257 as dietary recommendation in cancer, 172
175 from different food sources, 10
21 beetroot, 12 clove, 14 dragon fruit, 13
14 factors affecting extraction techniques, 20
21 finger millet, 16 fish, 16
17 flaxseed, 18 garlic, 11
12 ginger, 12 kiwi, 12 meat, 18 onion, 10
11 pomegranate, 18
19 tomato, 10 turmeric, 12 whole grain, 14
16 drug delivery approach to improve phytochemical drug ability, 172 effective phytochemicals herbs, 155 endocrine disorder treatment with help of targeted drug delivery, 155 extraction methods, 1
4, 63
68 pressing method, 3 solvent extraction method, 1
3 steam distillation method, 3 sublimation method, 3
4 flavonoids, 342 compounds for anticancer activity, 344
345

future prospects of phytochemicals in cancer treatment, 345 health-promoting ability of, 96
97 identification of, 6
7 spectral technologies, 6
7 importance of, 337 induce cancer cell apoptosis and autophagy, 325
326 interventions in healing cancer-associated MDR, 278
283 enlists roles of different dietary phytochemicals in miRNA regulation in BC, 279t secondary metabolites and ABC transporters, 281
283 isolation and purification of phytoconstituents, 4
6 from marine resources, 384
387 mechanism action of flavonoids, 343
344 with medicinal properties and applications, 570t in modulating noncoding RNA expression in BC cells, 276
278 plant metabolites validated chemotherapeutic activities, 277t molecular mechanism of phytochemicals in preventing cancer, 169
170 nanodrug delivery of phytochemicals in treating cancer, 89
90 natural products and phytochemicals against cancer, 336t novel extraction conditions for extraction conditions, 33
50 extraction methods for natural products, 28t pharmaceutical importance of phytoconstituents, 27t pre-extraction conditions, 29
32 selecting pre-extracting sample preparation, 32 selection approach for suitable extraction method, 50 perceptions of phytochemicals as anticancer agents in history, 249 pharmacological aspects of, 88 Elaeagnus angustifolia, 88 Holarrhena antidysenterica (L. ), 88 Lawsonia inermis, 88 phytochemicals currently in use as cancer therapeutics, 339
342 phytochemicals-based functional foods, 103
105 functional bakery and confectionery, 104 functional dairy products, 104
105 functional drinks/beverages, 103
104 meat products, 105 phytochemicals-based mitigation strategies against biofilm formation, 438
444 crude plant extracts against biofilm formation in ESKAPE pathogens, 438 pharmacologically relevant medicinal plants reported for quorum sensing inhibition and mitigation of biofilm mechanics, 439t

Index

phytochemicals involved in inhibition of biofilm formation in ESKAPE pathogens, 438
444 plant-derived phytochemicals reported for attenuation of quorum sensing-regulated virulence and biofilm inhibition, 441t from Phytolacca dioica L. Seeds extracts, 574
575 phytomedicine, 505 phytotherapy, 504
505 from plant to computational and molecular laboratory to target multiple disease processes, 584f plant-derived phytochemicals currently in use for various cancer treatments, 251 polyphenols, 295
296 profiling, 111
112 of mahua, 113t in prostate cancer, 183
184 quercetin, 255 as quorum-sensing inhibitors, 457
459 role of phytochemicals in diseases, 296
299 CVD, 297 diabetes, 296
297 hypertension, 297 IBD, 298
299 neurodegenerative disorders, 297
298 as safe fortifying agent, 541 cantaloupe incorporated into yogurt, 541 Soy isoflavones used in the fermentation of probiotics and beverages, 541 whole-bread preparation using cupuassu peel, 541 saponins, 100
101 SARS-CoV-2, 505 screening assay, 75 preliminary phytochemical screening tests, 76t screening of mahua flower extract, 112t screening of plant extracts, 571
574 flow chart for phytochemicals as lead compound for structure-based drug discovery, 573f secondary metabolites, 502
504 separation techniques, 68
74 serve, 645 strategies for identification of phytochemicals with pharmaceutical potential, 248 anticancer phytochemical synthesis, optimization, characterization, and potential application, 249f strategies to improve phytochemical drug ability, 170
172 factors contribute to limiting bioavailability of phytochemicals, 171f synthetic analogs for plant-derived compounds, 250 target areas, 424
425 change in bacterial static properties, 425 control of cellular motility, 425 preventing microbial adhesion, 424
425 techniques for extraction of, 21
23

conventional methods of extraction, 21
22 nonconventional methods for plant extraction, 21 terpenes and terpenoids, 295 and therapeutic effect, 9
10 types of, 535
536 alkaloids, 535
536 carotenoids, 536 organosulfur compounds, 536 phytochemicals, 536 phytosterols, 536 polyphenols, 536 terpenoids, 536 unexplored, 164
168 different methods of suppression of cancer progression by phytochemicals, 165f medicinal plants and phytochemicals and role in cancer treatment, 166t phytochemicals used in types of cancer, 168t as vegan food ingredients, 579 vinca alkaloids, 255 in vitro analysis of phytochemicals as multidrug resistance reversal, 590
592 in vitro anticancer effect of phytochemicals in leukemia, 591t in vitro synergistic effects of phytochemicals with conventional chemotherapeutic agents, 593t in vivo effects of phytochemicals as multidrug resistance reversals, 594t in vivo analysis of phytochemicals as multidrug resistance reversing agents, 593
594 Phytochemistry, 190
191 phytochemical constituents present in P. auriculata, 191t phytochemical diversity of P. auriculata, 194f Phytocompounds, 459 Phytoconstituents, 156 as antioxidant, 486
488 antioxidative effect of, 488
492 and general mechanism of actions pertaining to neuroprotection, 465
466 isolation and purification of, 4
6 pharmaceutical importance of, 27t phytoconstituents-based nanoformulations for Alzheimer’s disease, 469
471 Parkinson’s disease, 471 stroke, 472
473 Phytoestrogen, 97t, 174
175, 502
503, 536 Phytolacca dioica L. Seeds extracts phytochemicals from, 574
575 2D chemical structure for three classes of isoamericanols, 574f Phytomedicinal herbs effective, 155 to treat endocrine disorder with help of targeted drug delivery, 155 Phytomedicine, 505

669

application of phytomedicine in modern drug development in pharmacy, 158 Phytonic process, 48
49 advantages of, 49 use of, 49 Phytonutrients, 95
96, 384, 536 classification of phytonutrients, 96f Phytosomes, 157 Phytostanols, 273
274 Phytosterol, 97t, 169, 273
274, 504, 536 consumption, 100
101 Phytotherapy, 96, 504
505 PI3. See Phosphoinositide 3 (PI3) Pigmented rice, 98 Pine oils, 355 Pinidine, 204 Pinocembrin, 622 Pinus spp., 354 P. ponderosa, 295 Piper P. longum, 282 P. nigrum, 282 Piper betle ethyl acetate extract (PBE), 423 Piperidine, 267
268 Piperine, 204, 268t, 282, 471, 561 complex of mycobacterium tuberculosis hypoxic response regulator with, 563f Piperlongumine, 268t Piplartine, 268t Pituitary gland, 143 PK. See Pharmacokinetic (PK) 2-plamitoylglycerol, 190 Plant-based Anticancer Compound-ActivityTarget database (NPACT), 313
314, 329 Plant(s), 1, 9, 27
29, 189 alkaloids, 250 contain azalein, 190 defense metabolites, 320 as dominant source, 571 endophytes, 384 essential oil, 635
636 extracts, 63, 103 phytochemicals screening of, 571
574 metabolites, 102 oils, 353 particle size, 46 plant-based beverages, 104 plant-based foods, 95 plant-based ingredients, 579 plant-derived anticancer agents, 341
342 plant-derived antioxidants for amelioration of oxidative stress, 311
313 phytochemicals in clinical trials, 312t ROS-dependent anticancer mechanism, 312t plant-derived chemical substances, 203 plant-derived compounds, 249 plant-derived drugs, 329, 335 common dietary phytochemicals, 325 historical perspective of plant-derived drugs used popularly in cancer, 318
325

670

Index

Plant(s) (Continued) important secondary metabolites in cancer treatment, 320
322 other important phenolic compounds studied on cancer targets, 322
325 phytochemicals in clinical trials, 325 plant-derived medicinal products, 95 plant-derived nutraceuticals, 98
100 plant-derived phytochemicals, 96 in use for various cancer treatments, 251 sources, 95
96 steroids, 536 synthetic analogs for, 250 Plasma membrane, 587 Plasmodium berghei, 190 Plasmodium falciparum, 190 Platelet-derived growth factor (PDGF), 315 Platinum, 198
199 Platostoma rotundifolium, 406 Platycladus orientalis, 639 Plaunotol, 357 PLE. See Pressurized liquid extraction (PLE) Plumbagin, 189
190, 192
193 Plumbaginaceae, 189 Plumbago auriculata, 189 future perspectives, 198
199 medicinal uses, 193
195 anticancer and cytotoxic activity, 193 antimicrobial activity, 193 antiobesity, 194
195 antioxidant activity, 193
194 antiulcer activity, 195 nano-biotechnology, 195
197 other properties, 198 phytochemistry, 190
191 plumbagin, 192
193 traditional uses, 189 PNPB. See p-nitrophenyl butyrate (PNPB) Podophyllotoxins, 341 as anticancer agents, 341f derivative, 341 Podophyllum P. emodi, 303 P. peltatum, 303 Pogostemon cablin, 377
378 Poisson
Boltzmann surface, 573
574 Polar phytochemicals, 1
2 Polar solvents, 33
34, 64 Polar supercritical fluids, 45 Polarity gradient extraction method, 4f Poly(ADP-ribose) polymerase (PARP), 322 inhibitors, 263 Polyacetylenes, 104 Polyamine, 401 Polycarbonate, 418 Polycyclic aromatic hydrocarbons (PAHs), 68 Polycyclic musk, 390
391 Polycystic ovarian syndrome, 145
149 Polycystic ovary syndrome (PCOS), 145
146, 149, 155 herbs and phytoconstituents used in the treatment of endocrine disorders, 156t herbs used in, 155 thyroid in, 147
149

Polyethylene glycol (PEG), 400
401 Polyglutamylation, 225 Polygonum hydropiper L., 646 Polymerase chain reaction, 625 Polyphenol, 250
251, 486
488, 570 anthocyanins, 488 caffeic acid, 487 catechin, 487 chlorogenic acid, 487 epicatechin, 488 ferulic acid, 487 flavonoids, 487 flavonols, 487 p-hydroxybenzoic acid, 487 phenolic acids, 487 quercetin, 303 resveratrol, 488 stilbenes, 488 tannins, 488 Polyphenolics, 156 compounds, 98, 607 Polyphenols, 87, 97t, 99t, 131, 184, 195
196, 295
296, 335
336, 521, 536, 538, 617
618 as microtubulin-binding target, 235
236 Polysaccharide, 111, 385t, 390
391, 398
399 Polyterpenoids, 269 Polyunsaturated fatty acids, 375 Polyvinyl chloride (PVC), 418 Pomegranate, 18
19 chemical constituents of, 20f juice, 131 Pongamia pinnata, 389 Populus P. alba, 405 P. nigra, 405 Porphyrin, 385t Postharvest spoilage of flowers, 116
117 Potassium bromide (KBr), 75 Potassium permanganate, 70 Potent anticancer drugs, 164 Powdered samples, 32 PR. See Progesterone receptor (PR) Prader
Willi syndrome, 145 Pre-extracting sample preparation, selecting, 32 fresh or dried samples, 32 ground or powdered samples, 32 Pre-extraction conditions, 29
32 collection, 29
31 drying, 31
32 grinding, 32 storage, 32 Prebiotics, 100
101, 136 beverages, 136
137 process flowchart for preparation of functional beverage, 136f Precipitation, 5, 68 Premalignant cells, 186 Preprocessing methods, 116 Preservation, methods of, 117 Pressing method, 3 Pressurized fluid extraction (PFE), 43, 68 Pressurized hot water extraction (PHWE), 43

Pressurized liquid extraction (PLE), 22, 43, 64, 68, 98, 371 Primary defenses, 485 Primary hypothyroidism, pathophysiology of polycystic ovaries in patients with, 148f Primary metabolites, 1, 370 Prion disease, 463 Proanthocyanidins, 405, 491 Probiotics, 97t, 103, 128 bacteria, 128 beverages, 128
130 preparation of low-fat probiotic beverage from whey and sorghum, 129f drink, 130 Procarcinogens, 169 Processing methods, 29 Procyanidin B1, 576 Progesterone receptor (PR), 262
263, 266 Prolactin, 146 Promastigote, 550
553 Proniosomes, 157
158 gel system, 157 Propionibacterium acnes, 406 Propyl gallate (PG), 537 Prostaglandin E2 (PGE2), 313
314, 375 Prostaglandins (PGs), 375, 610 Prostate cancer, 101
102, 164
165, 179
181 causes of, 180
181 general causes of, 180 genetic causes of, 180
181 test to identify prostate cancer, 181 effects of specific plant families extracts on human prostate cancer cells, 184
185 phytochemicals, 183
184 and conventional medical practice, 184 prevention of, 183 5-Alpha-reductase inhibitors, 183 aspirin, 183 medicines, 183 mineral, vitamins, and supplement, 183 physical activity, diet, and body weight, 183 risk factors, 185
186 age, 185 diet, 186 environmental exposures, 186 obesity, 186 past of family, 186 race, 185 symptoms of, 181 advanced symptoms, 181 treatments, 181 bisphosphonate therapy, 182 chemotherapy, 182 cryotherapy, 182 high-intensity focused ultrasound, 182 hormone therapy, 182 immunotherapy, 182 prostate cancer vaccine, 182
183 proton beam radiation, 182 radiation, 182 surgery, 181
183 types of, 179
180 neuroendocrine prostate tumor, 180

Index

sarcomas of prostate glands, 180 small-cell carcinoma, 179
180 transitional cell carcinomas of prostate gland, 180 vaccine, 182
183 Prostate-specific antigen test (PSA), 181 Prostatic hyperplasia, 179 Prostatic stromal sarcoma, 180 Proteases, 515 Protein tyrosine kinase (PTK), 166t Protein(s), 74, 111, 137, 189, 398
399 protein kinase G enzyme, 557 protein-based beverages, 137 protein-rich beverage, 133 protein
ligand complex, 645 proteins/genes responsible for drugresistance leukemia, 586
587 ABCCl, 587 ATP-binding cassette transporters, 586
587 cancer stem cells and drug resistance, 587 hypoxia-inducible factor-1-mediated resistance, 587 P-glycoprotein, 587 Proteus vulgaris, 115, 193 Protoalkaloids, 204, 267
268 Protoberberine, 210 Proton beam radiation, 182 Proton nuclear magnetic resonance spectroscopy (1H-NMR), 7 Protopine, 210 Protozoa, 617 PSA. See Prostate-specific antigen test (PSA) Pseudo-alkaloids, 204, 267
268 Pseudo-cereals, 104 Pseudomonads, 419 Pseudomonas spp., 116
117, 417t, 419 P. aeruginosa, 115, 193, 398
399, 401
405, 623, 625 PAO1, 422
423 P. fluorescens, 397 Pseudopterosin, 385t, 390
391 Psidium guajava, 377 Pteleoellagic acid, 557
561 PTK. See Protein tyrosine kinase (PTK) PTX. See Paclitaxel (PTX) PTZ. See Pentylenetetrazol-induced seizures (PTZ) Public Health Emergency of International Concern (PHEIC), 505 Public Health Foundation of India (PHFI), 150 PUFAs. See Omega-3 polyunsaturated fatty acids (PUFAs) Pulsed electric field (PEF), 48 extraction, 22
23, 48 treatment, 23 Punica granatum, 297 Punicalagins, 131 Purification of phytoconstituents, 4
6 Purines, 296 Purple rice, 98 PVC. See Polyvinyl chloride (PVC) Pyloric ligation assay, 610 Pyocyanin, 401

Pyrimidines, 296 Pyrrolidine, 267
268 Pyrrolizidine, 267
268 alkaloids, biological source, mechanism of action, and applications of, 219 clivorine, 219

Q QQ. See Quorum quenching (QQ) QR. See Quercetin (QR) QS. See Quorum sensing (QS) QSIs. See Quorum sensing inhibitors (QSIs) Quantitative X-ray spectrum analysis, 81 Quassinoids, 276 Quercetin (QR), 10, 42, 97t, 169, 255, 256t, 325, 405, 470
471, 487, 557, 594, 620
621 complex of mycobacterium tuberculosis hypoxic response regulator with, 563f quercetin-3-O-rhamnoglucoside, 624 Quinine, 204, 546 Quinine oxidoreductase (NQOI), 169 Quinoline, 268t Quinolizidine, 267
268 Quinolone, 267
268 Quorum quenching (QQ), 401 Quorum sensing (QS), 398
405, 423, 434
436, 455 biofilm formation and, 455
456 economic impact, 456f steps involved in process quorum sensing in bacteria, 456f clinical studies, 459 mechanism of phytochemicals involved in quorum-sensing inhibition, 459 mechanism of quorum sensing in bacteria, 456 molecular mechanism of quorum sensing in gram-positive bacteria, 457f phytochemicals as quorum-sensing inhibitors, 424, 457
459 bioactive phytochemicals in biofilm inhibition with evidence from studies, 458t grouping of phytochemicals as QS inhibitors, 457
459 necessities and low falls in QS inhibition, 459 taxa and habitats intersected and interacted with QS inhibition, 459 potential anti-biofilm agents derived from plants under clinical evaluation, 460t systems in different bacteria, 457t Quorum sensing inhibitors (QSIs), 436
437

R (R)-Bgugaine, 407 Radiation, 629 therapy, 163, 182, 261
263, 265 treatment, 180 Radiolabeled 13C NMR, 78 Radiotherapy, 265, 335

671

Ragi, 16 Rauvolfia serpentina, 136 RB flask. See Round-bottomed flask (RB flask) RB3-SLD. See Vinblasin-RB3 protein stathmin-like domain (RB3-SLD) RBD. See Receptor-binding domain (RBD) RBD-S. See Receptor-binding domain of S protein (RBD-S) RdRp. See RNA-dependent RNA polymerase (RdRp) Reactive nitrogen species (RNS), 374
375, 483 Reactive oxygen radicals, 297 Reactive oxygen species (ROS), 169, 192
194, 227, 265, 283
284, 310
311, 345, 374
375, 483, 490, 537, 550
553, 607, 611, 624 ROS-dependent anticancer mechanism, 312t, 576 ROS-dependent cellular metabolic pathways in cancer cells, 311 Reactive oxygen stress, 251
252 Reactive phenoxy radicals, 374
375 Ready-to-drink (RTD), 124
125 Ready-to-serve (RTS), 130
131 Real time PCR (RT-qPCR), 198 Receptor-binding domain (RBD), 512 Receptor-binding domain of S protein (RBDS), 519 Recycling process, 49 Red clover (Trifolium pretens), 502 Red rice, 98 Red-fleshed dragon fruit, 13 Redox signaling, 169 Reducing inflammation, 537 Reflux extraction, 47
48 Refluxing method, 2
3 Renal cell carcinoma (RCC-45), 164
165 Renoprotective, 102 REO. See Rosewood Aniba rosaeodora (REO) Replication
transcription complex (RTC), 512
513 Reserved percolation, 36 Resveratrol, 10, 97t, 102, 256t, 391
392, 406, 471, 488 Resveratrol (3,5,4-trihydroxystilbene) (RSV), 10 Retention factor(Rf), 69 value, 70 Reverse T3 (rT3), 152 Rh-123. See Rhodamine-123 (Rh-123) Rheum palmatum L., 594 Rheumatic arthritis, 116 Rheumatoid therapy, 604
605 Rhizoma coptidis, 211 Rhizophora R. mangle, 391 R. mucronata, 388 R. racemosa, 387 R. stylosa, 390
391 Rhodamine-123 (Rh-123), 282 Ribosome-inactivating proteins (RIPs), 574 RIPs. See Ribosome-inactivating proteins (RIPs)

672

Index

RNA-dependent RNA polymerase (RdRp), 515
516, 546 RNS. See Reactive nitrogen species (RNS) ROS. See Reactive oxygen species (ROS) Rosa damascene, 357 Rosemary essential oils, 373t Rosewood Aniba rosaeodora (REO), 635 Rosmarinus officinalis, 295, 378, 457
459, 467t Rotenoids, 391 Round-bottomed flask (RB flask), 38 RT-qPCR. See Real time PCR (RT-qPCR) rT3. See Reverse T3 (rT3) RTC. See Replication
transcription complex (RTC) RTD. See Ready-to-drink (RTD) RTS. See Ready-to-serve (RTS) Rubus rosaefolius, 405 Russian olive, 88 Rutin, 283, 618

S S glycoprotein. See Spike glycoprotein (S glycoprotein) Saccharomyces cerevisiae, 540 Safflower essential oil, 376t Saikosaponins, 520 Salacia reticulata, 297 Saline administration, 608
609 Salix sp., 384
387 Salmonella spp., 406, 418 S. enterica, 417t, 419 S. enteritidis, 360
361 S. typhi, 115 S. typhimurium, 377, 406, 623 Salt glands, 189 Salting-out method, 5, 68 Salvia S. officinalis, 467t S. rubifolia, 638 Sample preparation process, 47 Sanguinarine, 86
87, 213
214, 268t structure of, 213f Santalum S. album, 357, 457
459 S. spicatum, 356
357 Santolina insularis, 361 Saponin(s), 67
68, 74, 100
101, 104, 111, 195
196, 385t, 390
391 Sarcomas of prostate glands, 180 Sargassum serratifolium, 87 SARS. See Severe acute respiratory syndrome (SARS) SARS-CoV-2. See Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Sartans, 297 Satureja khuzestanica, 269 SBL. See Schinus polygamus leaf (SBL) Scabrous stem, 601 Scanning electron microscope (SEM), 196 SCC. See Squamous cell carcinoma (SCC) Schinus polygamus, 637 Schinus polygamus bark (SPB), 637

Schinus polygamus leaf (SBL), 637 Schkuhria pinnata, 168 Second cancer, 264
265 Secondary defenses, 485 Secondary metabolites, 1, 9, 295, 370, 413, 457
459, 502
504 and ABC transporters, 281
283 alkaloids, 503 carotenoids, 504 flavonoids, 503 phenolic compounds, 502 phytoestrogens, 502
503 phytosterols, 504 synthesis, 398
399 terpenes, 503
504 Secondary plant metabolites, 353 Sedentary lifestyle endocrine disorder occurs due to, 149
150 relation between endocrine disorder and, 150 Seed extracts from Washingtonia filifera, phytochemicals composition and biological properties of, 575
579 Seeds, 110 Selective estrogen receptor downregulators (SERDs), 265
266 Selective estrogen receptor modulators (SERMs), 262
263, 265
266, 274
275 SEM. See Scanning electron microscope (SEM) Sensor kinases, 456 Separation techniques, 1, 5, 68
74, 77 chromatographic techniques, 68
74 Sephadex, 248 chromatography, 77 SERDs. See Selective estrogen receptor downregulators (SERDs) Seriphium plumosum, 168 SERMs. See Selective estrogen receptor modulators (SERMs) Serum alkaline phosphate (ALKP), 114 Serum glutamic oxaloacetic transaminase (SGOT), 114 Serum glutamic pyruvic transaminase (SGPT), 114 Sesamol, 470
471 Sesquiterpenes (C15), 295, 356
357, 371, 519
520 acyclic sesquiterpenes, 357f Sesquiterpenoids (C15), 269, 371 Sesterpenes, 519
520 Sesterterpenoids (C25), 269 Severe acute respiratory syndrome (SARS), 546 Severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), 102
103, 505, 511 pathogenesis, 513f targetable sites in SARS-CoV-2 infection with human cell, 512
513 SF. See Supercritical fluid (SF) SFE. See Supercritical fluid extraction (SFE) SGOT. See Serum glutamic oxaloacetic transaminase (SGOT)

SGPT. See Serum glutamic pyruvic transaminase (SGPT) sgRNA. See Sub-genomic RNA (sgRNA) Shake flask extraction, 68 SHRs. See Spontaneously hypertensive rats (SHRs) Siderococcus, 116
117 Sidha, 483 Signal transducer and activator of transcription 3 (STAT3), 228 Silica, 248 gel, 49, 70 Silicon, 418 Silver, 198
199 Silver nanoparticles (AgNPs), 196
197, 445
446 antibacterial activity of green synthesized AgNPs, 197f Silver nitrate (AgNO3), 196
197 Singlet oxygen, 87 Skim milk, 128 Skin aging process, 578 Skin cancer, 179 SLNs. See Solid lipid nanoparticles (SLNs) SM. See Sphingomyelin (SM) Small molecule phenolic compounds, 42 Small-cell prostate cancer, 179
180 Smoothies, 130 SMPs. See Soluble microbial products (SMPs) SOCS3. See Suppressor of cytokine signaling 3 (SOCS3) SOD. See Superoxide dismutase (SOD) SOD-1. See Superoxide dismutase-1 (SOD-1) Soft drinks, 125 Solanidine, 204 Solanum lycopersicum. See Tomato (Solanum lycopersicum) Solar drying, 117 Solid lipid nanoparticles (SLNs), 469 Solid-phase extraction (SPE), 47, 64 Solid-phase micro-extraction, 47, 64 Solid-state NMR spectroscopy, 78 Solid
liquid techniques, 34
35 Solubility of analytes, 46 Soluble microbial products (SMPs), 455 Solvent(s), 41
42, 75 extraction, 68, 369 method, 1
4 extracts, 87 precipitation method, 5 solvent-to-sample ratio, 40
41 system, 64 for extraction, 33
34 properties of solvent for extraction, 34t selection of solvents, 34 solvents used for active component extraction, 33t Sonication, 43
45, 68 Sonification, 64 Sorghum (Sorghum bicolour (L.), 126 Sound waves, 43
44, 182 Soxhlet extraction, 21, 38
39, 68, 98, 609 advantages and disadvantages of, 39 method, 50

Index

practical issues for, 38
39 setup, 65f Soy isoflavones used in fermentation of probiotics and beverages, 541 Soybeans (Glycine max L.), 502 SPB. See Schinus polygamus bark (SPB) SPE. See Solid-phase extraction (SPE) Species significance of preservation and restoration of, 30 taxonomical authenticity of, 29
30 Spectral technologies, 6
7 Spectrographic analysis (MS), 23 Spectrophotometric experimental data, 576 Spectroscopic techniques, 77 Spermatophytes, 537 Sphingomyelin (SM), 206 Spike glycoprotein (S glycoprotein), 102
103 Spike protein, 513
514 Spinal muscular atrophy, 463 Spinocerebellar ataxia, 463 Spontaneously hypertensive rats (SHRs), 377 Sports drinks, 103, 137
138 Sports nutrition market, 137 Squalamine, 86
87 Squalene, 371 Squamous cell carcinoma (SCC), 327
328 Stainless steel, 418 Staphylococcus, 115 S. aureus, 86
87, 115, 190, 193, 417t, 422, 608 Starch, 195
196 STAT3. See Signal transducer and activator of transcription 3 (STAT3) Stationary phase, 68
69 Steam distillation, 37
38, 369, 629 method, 3 Stem cells, 309
310 transplantation, 583 Steroidal saponins, 335
336 Steroids, 67
68, 101, 183
184, 250 Sterol, 390
391 Stilbenes, 488 Stirring effect, 42 Straight-chain components not containing any side chain, 355 Streptococcus S. agalactiae, 622 S. pyogenes, 193 Streptomyces hyalurolyticus (Hyal S), 622 Streptomycin, 384 Streptozocin, 610 Stress, 144
145 Stroke, phytoconstituents-based nanoformulations for, 472
473 Structural-based proteins, 513
515 Stylissa massa, 407 Styphnolobium japonicum, 618 Suaeda vermiculata, 588
589 Sub-genomic RNA (sgRNA), 512
513 sub-MICs. See Subminimum inhibitory concentrations (sub-MICs) Subcritical water extraction (SWE), 43, 98 Sublimation, 31

method, 3
4 Subminimum inhibitory concentrations (subMICs), 423 Sugars, 111, 189 alcohols, 104 syrup, 117 Sulfate polysaccharide, 385t Sulforaphane, 10, 536 Sulfotransferases, 491
492 Sulfur-containing compounds, 401 in essential oils, 359
360, 360f Sulfur-containing phytochemicals, 407
408 Supercritical CO2, 45 Supercritical fluid (SF), 3, 22, 46 Supercritical fluid extraction (SFE), 3, 20
22, 28
29, 38, 45
47, 64, 98, 369, 371 advantages and disadvantages of, 46
47 potential applications of, 46 practical issues for, 45
46 principles and mechanisms, 45 Supercritical gases, extraction with, 67 Superdex, 248 Superoxide anions, 87, 374
375 radicals, 374
375 Superoxide dismutase (SOD), 485 Superoxide dismutase-1 (SOD-1), 283
284 Superoxide radicals, 484 Suppressor of cytokine signaling 3 (SOCS3), 228 Surface attachment, in biofilm formation, 398
399 Surfactant-mediated techniques, 64 Surgical interventions, 261
262 Sustenance, area of, 30 SWE. See Subcritical water extraction (SWE) Synthetic antioxidants, 486, 501
502 Syzygium aromaticum, 296 EO, 376t

T T2D. See Type 2 diabetes (T2D) Tandem mass spectrometry (MS
MS), 6
7, 72 Tannins, 131, 189, 195
196, 250, 488, 536, 545, 605 Target multidrug resistance computational approach to, 588
590 multidrug resistance reversal or prevention of drug efflux by nutraceuticals, 590f in silico interactions of phytochemicals with multidrug resistance targets, 589t Targeted drug delivery, 155 Targeted therapies, 163, 247
248, 263, 629 Targeting cell proliferation, 169 demonstrates an example of β-sitosterol mechanism of, 169f Targeting immune surveillance, 170 Targeting molecular pathway of cancerous cell, 169 Targeting oxidative stress, 169 Taxanes, 227, 340 diterpenoids, 339

673

interplay of taxanes with microtubule site, 232
233 phytochemicals, 232f side effects of taxanes on treated patients, 233 Taxines, 214 Taxol, 203, 214
215, 282, 321, 384 mechanism of action of taxol phytochemicals, 232 structure of, 214f as therapeutic agent disrupting cell polymerization, 231
233 interplay of taxanes with microtubule site, 232
233 mechanism of action of taxol phytochemicals, 232 side effects of taxanes on treated patients, 233 taxane-based phytochemicals, 231f therapeutic relevance of taxol concerning microtubulin dynamics, 233 therapeutic relevance of taxol concerning microtubulin dynamics, 233 Taxomyces andreanae, 231
232 Taxonomical authenticity of species, 29
30 Taxus T. baccata, 214 T. brevifolia, 214, 231, 340 T. chinensis, 231 Taxus alkaloid biological source, mechanism of action, and applications of, 214
215 taxol, 214
215 TBA. See Thiobarbituric acid (TBA) TBG. See Thyroxine-binding globulin (TBG) TBHQ. See Tertiary butylhydroquinone (TBHQ) TCCU. See Transitional cell carcinoma of urothelium (TCCU) TDDS. See Transdermal drug delivery system (TDDS) TEM. See Transmission electron microscope (TEM) Terminalia T. arjuna, 297 T. chebula, 136, 467t Terpenes, 183
184, 250, 269, 295, 320, 354
357, 370
372, 406, 503
504, 519
520, 536, 545 biosynthesis of, 354 hemi-, mono-, sesqui-, di-, tri-, and tetraterpene natural products, 355f chain of polymers of isoprene, 354f diterpenes, 357 monoterpenes, 355 norterpenes, 357 sesquiterpenes, 356
357 straight-chain components not containing any side chain, 355 Terpenoids, 97t, 101, 104, 251, 261
262, 269
271, 295, 320, 337, 370
371, 385t, 390
391, 401, 405
406, 519
520, 536, 638 different members of, 270t

674

Index

Terpinen-4-ol, 375 Tertiary butylhydroquinone (TBHQ), 537 Testicular cancer, 205 Testing anticancer medicines, 590 Tetrahydrocurcumin, 283 Tetrandrine, 86
87, 589 Tetraterpenes, 371, 405
406 Tetraterpenoids (C40), 269 Tetratricopeptide repeats 2 (IFIT2), 283 TFC. See Total flavonoid content (TFC) TGFBI. See Transforming growth factor betainduced (TGFBI) Theobroma cacao, 296 Theobroma grandiflorum, 541 Theobromine, 204 Therapeutic drugs, 605 Therapeuticals, 391 Therapeutics, 163, 391 Thermodynamic methods, 398
399 Thermolabile compounds, 44 Thermosensitive compounds, 40, 49 Thin-layer chromatography (TLC), 6, 23, 69
70, 371, 572 different adsorbent used to separate various compounds, 70t setup, 69f Thiobarbituric acid (TBA), 484
485 Thiols, 251 Thiotepa, 310 3D culturing models, 247
248 Thrombosis, 537 Thromboxane A2, 375 Thyme (Thymus vulgaris), 303 Thymidylate synthase (TS), 283
284 Thymol, 406, 636 anticancer activity of thymol against breast cancer, 636f Thymoquinone (TQ), 637, 639 Thymus T. caramanicus, 269 T. fallax, 633 T. glandulosus, 361 T. numidicus L., 636 Thymus vulgaris. See Thyme (Thymus vulgaris) Thyro-globin, 151 Thyroid, 145
149 gland, 143, 151 ovaries in thyroid disorders, 146
147 in polycystic ovary syndrome, 147
149 hypothesis linking adiposity and raised thyroid stimulating hormone, 148f pathophysiology of polycystic ovaries in patients with primary hypothyroidism, 148f Thyroid-stimulating hormone (TSH), 146, 151 Thyrotrophs, 151 Thyrotropin hormone (TRH), 146 Thyroxine (T4), 152 Thyroxine-binding globulin (TBG), 151 Tight junctions, 465 Time-consuming extraction and isolation process, 29 Tinospora cordifolia, 135, 467t Tissue plasminogen activator (tPA), 391
392

TLC. See Thin-layer chromatography (TLC); Total leukocyte count (TLC) TLR4. See Toll-like receptor 4 (TLR4) TMD. See Transmembrane domain (TMD) TMPRSS2. See Transmembrane serine protease enzyme transmembrane protease serine 2 (TMPRSS2) TNBC. See Triple-negative breast cancer (TNBC) Tocopherol, 123
124 Toll-like receptor 4 (TLR4), 470 Toluene, 41 Tomato (Solanum lycopersicum), 10 chemical structures of lycopene, β-carotene, and vitamin A, 11f substance designs of fundamental organosulfur and phenolic intensifies in onion, 11f Toona ciliata, 168 TOP I. See Topoisomerase I (TOP I) Topoisomerase I (TOP I), 209 Topoisomerases, 621 Topotecan, 209, 249 Total flavonoid content (TFC), 23, 77 determination of, 23 Total leukocyte count (TLC), 115 Total phenolic content (TPC), 23 determination of, 23 Total phenolics (TP), 10 Total soluble solids (TSSs), 125, 131 TP. See Total phenolics (TP) TPA. See 12-O-tetradecanoylphorbol-13acetate (TPA) tPA. See Tissue plasminogen activator (tPA) TPC. See Total phenolic content (TPC) TQ. See Thymoquinone (TQ) TR. See Trypanothione reductase (TR) TR-alpha, 152 Traditional chemotherapy, 310 Traditional medicinal plants, 505 Traditional medicine, 27, 602
604 Traditional method for liquid
liquid extraction, 34 TRAIL. See Tumor necrosis factor
related apoptosis-inducing ligand (TRAIL) Trans-cinnamate 4-monooxygenase, 371
372 Trans-pinocarvyl acetate, 638 Transdermal drug delivery, 158 Transdermal drug delivery system (TDDS), 157 Transfer RNA (tRNA), 218 Transferosomes, 158 Transforming growth factor beta-induced (TGFBI), 227
228 Transitional cell carcinoma of urothelium (TCCU), 230, 340 Transitional cell carcinomas of prostate gland, 180 Transmembrane domain (TMD), 281
282 Transmembrane serine protease enzyme transmembrane protease serine 2 (TMPRSS2), 517 Transmission electron microscope (TEM), 196 Transresveratrol, 42

Transthyretin (TTR), 151 Trastuzumab, 263, 276 Tray drying, 117 TRH. See Thyrotropin hormone (TRH) Tricalcium citrate, 539 Trichloromethane (CHCl3), 388 Trichomes, 189 Tricyclic monoterpenes, 355, 356f Tricyclic sesquiterpenes, 357 Tridax procumbens, 377 Trifolium pretens. See Red clover (Trifolium pretens) Trifolium repens L. See White clover (Trifolium repens L.) 5,7,40 -trihydroxyflavanol, 623 Triiodothyronine (T3), 152
154 Triple maceration, 66 Triple-negative breast cancer (TNBC), 263, 276 Tripterygium T. regelii, 519
520 T. wilfordii, 89 Triptolide, 89 Triterpenes (C30), 74, 295, 371, 405
406, 519
520 Triterpenoids (C30), 67
68, 269 promote, 269 tRNA. See Transfer RNA (tRNA) Tropone, 267
268 Trypanothione reductase (TR), 554 enzyme, 550
553 Tryptanthrin, 268t TS. See Thymidylate synthase (TS) TSGs. See Tumor-suppressor genes (TSGs) TSH. See Thyroid-stimulating hormone (TSH) TSSs. See Total soluble solids (TSSs) TTR. See Transthyretin (TTR) Tuberculosis, 545 Tubulin, 215 heterodimers, 225 Tulsi (Ocimum sanctum), 134 Tumor necrosis factor
related apoptosisinducing ligand (TRAIL), 217, 322 Tumor-initiating cells. See Cancer stem cells (CSCs) Tumor-suppressor genes (TSGs), 276
278 Tumor(s), 263, 309 cells, 203, 281, 635 DNA, 247
248 therapy, 574 Turmeric (Curcuma longa L.,), 12 main compounds found in rhizomes, 14f Turner syndrome, 145 Two-dimension (2D) models, 247
248 NMR spectrum, 80
81 Bruker 700 MHz nuclear magnetic resonance spectrometer, 80f representation of bioactive phytochemicals, 87f structures of vinca alkaloids, 229f Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), 7 techniques, 78

Index

Two-phase solvent system, 4 Type 1 5’-deiodinase, 152 Type 2 5’-deiodinase, 152 Type 2 diabetes (T2D), 86, 149, 578 Type 3 5’-deiodinase, 152 Type-1 diabetes mellitus, 296
297 Type-II fatty acid synthase (FAS-II), 620
621 TYR enzymes. See Tyrosinase enzymes (TYR enzymes) Tyrosinase enzymes (TYR enzymes), 574
575, 578 Tyrosine kinase inhibitor, 585

U UAE. See Ultrasound-assisted extraction (UAE) UE. See Ultrasonic extraction (UE) Ulcer, 114 Ultrafiltration, 68 Ultrasonic extraction (UE), 3, 43
44, 67
68 Ultrasonic sonication, 43
44 Ultrasound frequency, 44 Ultrasound waves, 67
68 Ultrasound-assisted extraction (UAE), 20
22, 28
29, 35, 43
45, 371. See also Accelerated solvent extraction; Microwave-assisted extraction advantages and disadvantages of, 44 operating conditions, 44 potential applications of, 44
45 practical issues for, 44 principles and mechanisms, 43
44 Ultraviolet (UV), 76
77 Ultraviolet
visible (UV-vis), 6, 77 spectroscopy, 78, 572 spectrum, 6
7, 78 Ulvan, 385t Unani, 483 United States Department of Agriculture (USDA), 321
322 Uric acid, 576 Urothelial carcinoma of prostate, 180 US Food and Drug Administration (FDA), 206, 233
234, 239, 420
422 US Food and Medication Administration, 342 USDA. See United States Department of Agriculture (USDA) Utathione-S-transferase-omega-1, 283
284 Uterine cancer, 179 UV. See Ultraviolet (UV) UV-vis. See Ultraviolet
visible (UV-vis)

V Valeriana officinalis, 457
459 Valine hydroxides, 484 Value-added food products, 117
118 Van Wyk and Grumbach syndrome, 146 Vanillin, 405 VAs. See Vinca alkaloids (VAs)

Vascular endothelial growth factor (VEGF), 230, 315, 646
647 VBL. See Vinblastine (VBL) VCR. See Vincristine (VCR) VDS. See Vindesine (VDS) Vegan food ingredients, phytochemicals as, 579 Vegetables, 98, 123 Vegetation, 645 VEGF. See Vascular endothelial growth factor (VEGF) Venera Cardile, 638 Vero cytotoxigenic Escherichia coli (VTEC), 419 VFL. See Vinflunine (VFL) Vibrio spp., 405 V. cholerae, 415 Vinblasin-RB3 protein stathmin-like domain (RB3-SLD), 229
230 Vinblastine (VBL), 205, 208, 225
226, 269, 339
340 structure of, 205f Vinblastine, 249 Vinca alkaloids (VAs), 205
206, 225, 228
229, 255, 321, 339
340, 645 2D structures of vinca alkaloids, 229f mechanism of action against microtubulin, 229 side effects of, 230
231 vinca alkaloids used as anticancer agents, 340f Vinca domain, 229
230 Vincristine (VCR), 206, 208, 228
229, 249, 339
340 structure of, 206f therapy, 206 Vincristine-induced peripheral neuropathy (VIPN), 206 Vindesine (VDS), 207, 228
229 structure of, 207f Vinflunine (VFL), 208, 230 structure of, 208f Vinorelbine (VRL), 208, 228
229 Violacein, 401 VIPN. See Vincristine-induced peripheral neuropathy (VIPN) Viral virulence factors, 516 Virgin drinks, 126 Viridiflorol, 405
406 Virola surinamensis, 638
639 Virus-based targets, 513
516 nonstructural proteins, 515
516 structural-based proteins, 513
515 Viruses, 617 Vitamin A, 311
313 Vitamin C, 87, 311
313 Vitamin E, 311
313, 485 Vitamins, 12, 95
96, 111, 335
336, 539 fortification, 539 Vitis coignetiae, 401
405 Vitis vinifera, 296 Volatile oil, 353, 636 VRL. See Vinorelbine (VRL)

675

VTEC. See Vero cytotoxigenic Escherichia coli (VTEC)

W WA. See Withaferin A (WA) Warfarin, 457
459 Washingtonia filifera, 575 phytochemicals composition and biological properties of seed extracts from, 575
579 molecular docking of compounds against XO enzyme, 577f 2D chemical structures of molecules identified from extracts, 577f Washingtonia robusta, 575 Water, 3, 41
42 distillation, 369 extract, 608 water
dichloromethane, 34 water
ether, 34 water
hexane, 34 Water-soluble polysaccharides, 111 Watermelon fruit, 137
138 Wheat flour, 104 structures of bioactive phytochemicals present in, 17f Whey, 128 whey-based beverages, 132
133 Whey protein concentrate (WPC), 131 White clover (Trifolium repens L.), 502 WHO. See World Health Organization (WHO) Whole grain, 14
16, 17f Whole-bread preparation using cupuassu peel, 541 Wholemeal wheat flour, 104 Widdrol, 357 Withaferin A (WA), 168t, 173t Withania somnifera, 155, 297, 467t World Health Organization (WHO), 27, 63, 158, 247, 501, 505, 571, 602
604, 629 Wound-healing potential, 608
609 WPC. See Whey protein concentrate (WPC)

X X-ray diffraction, 196
197 X-ray spectroscopy, 81 X-ray spectrometer, 81f X-ray spectrum analysis, 81 Xanthine oxidase (XO), 574
576 Xanthohumol, 323 Xanthone, 561 complex of mycobacterium tuberculosis hypoxic response regulator with, 566f XO. See Xanthine oxidase (XO) Xylene, 41 Xylocarpus granatum, 388

Y Yeast, 128

676

Index

Z Zanthoxylum rhoifolium, 634 Zeaxanthin, 85, 97t, 272
273, 385t Zerumbone, 276 ZFRs. See Zucker fatty rats (ZFRs) Zinc oxide nanoparticles (ZnO NPs), 196, 445
446

antibacterial activity of green synthesized AgNPs, 197f degradation of Congo red and malachite green, 198f morphological analysis of ZnONPs, 196f Zingiber officinale, 374

Zingiber officinale. See Ginger (Zingiber officinale) Zizyphus jujube, 467t ZnO NPs. See Zinc oxide nanoparticles (ZnO NPs) Zone capillary electrophoresis, 72
73 Zucker fatty rats (ZFRs), 377