Evolutionary Diversity as a Source for Anticancer Molecules 0128217103, 9780128217108

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Evolutionary Diversity as a Source for Anticancer Molecules

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Evolutionary Diversity as a Source for Anticancer Molecules Edited by Akhileshwar Kumar Srivastava PCBT Department, CSIR-CFTRI, Mysuru, Karnataka, India

Vinod Kumar Kannaujiya Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Rajesh Kumar Singh Department of Dravya Guna, Faculty of Ayurved, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Divya Singh Central Sericultural Research and Training Institute, Mysuru, Karnataka, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-821710-8 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Senior Acquisitions Editor: Rafael E. Teixeira Editorial Project Manager: Mona Zahir Production Project Manager: Niranjan Bhaskaran Senior Cover Designer: Greg Harris Typeset by SPi Global, India

Dedication This book is dedicated to the late Prof. Sureshwar Prasad Singh, Department of Botany, Banaras Hindu University, Varanasi, India.

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Contents Dedication

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Contributors

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Preface

xix

1. Evolutionary mechanism for biosynthesis of diverse molecules Akhileshwar Kumar Srivastava, Arpana Yadava, and Divya Singh 1.1 Introduction 1.2 Models for evolutionary study 1.3 Evolution of secondary metabolite pathways 1.4 Cell fitness coupling for natural metabolite production 1.5 Chemical diversity of natural products 1.6 Occurrence of flavonoids in the plant kingdom 1.7 Biomolecular activity of secondary metabolites 1.8 Evolution of anticancer drug discovery 1.9 Factors influence the production of secondary metabolites

1 1 4 6 11 12 12 14 15 18

1.10 Future prospective

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References

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2. Impact of ploidy changes on secondary metabolites productions in plants Sanjay Kumar

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2.1 An introduction to ploids (or polyploids)

29

2.2 Morphological effects, meiotic and breeding behavior

31

2.3 Role of ploids (auto, allo and induced) in secondary metabolites production

31

2.4 Perspectives

39

References

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3. Effect of climate change on plant secondary metabolism: An ecological perspective Akhileshwar Kumar Srivastava, Pragyan Mishra, and Amit Kumar Mishra 3.1 Introduction 3.2 Evolutionary theory based on secondary metabolites 3.3 Effect of climate change on secondary metabolites 3.4 Impact of climate change on secondary metabolites of medicinal plants 3.5 The expression of secondary compounds in plants 3.6 Early stage of plant evolution 3.7 Environmental factors triggering the secondary metabolism 3.8 The regulation of plant secondary metabolism by interactions of heat shock and elevated CO2 3.9 Ecological roles of secondary metabolites

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47 51 53 54 55 55 56 63 64

3.10 The ecosystem feedback of plant secondary metabolites for the climate change

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3.11 Secondary metabolites as worthy asset for the biological system: Further support

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3.12 Conclusions and future prospective

69

References

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4. Isolation and characterization of bioactive compounds from natural resources: Metabolomics and molecular approaches Diksha Sharma, V.P. Singh, Rajesh Kumar Singh, C.S. Joshi, and Vinamra Sharma

77

4.1 Introduction

77

4.2 Metabolomics approach

79

4.3 Metabolomics technologies

81

4.4 Molecular approach

86

4.5 Conclusion and future perspectives

96

References

97

Contents

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5. Single-celled bacteria as tool for cancer therapy 103 Ankita Shrivastava 5.1 Introduction 103 5.2 The anti-tumor effect through the release of bacterial substances 106 5.3 The anti-tumor effect through enhancement of human immunity 107 5.4 The anti-tumor effect through the production of biofilms 108 5.5 The anti-tumor effect through the use of viruses along with bacteria 109 5.6 The anti-tumor effect through bacteria-mediated antiangiogenesis therapy 109 5.7 The anti-tumor effect through live tumor-targeting bacteria 110 5.8 The anti-tumor effect through the use of live bacteria as a tumor suppressor 111 5.9 The anti-tumor activity through the use of engineered bacteria 111 5.10 The anti-tumor activity of bacteria in combination with radiotherapy

114

5.11 The anti-tumor activity of bacteria through tumor-specific antigens and antibodies

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5.12 The anti-tumor activity of bacteria through gene transfer

115

5.13 The anti-tumor activity of bacteria through gene silencing

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5.14 The anti-tumor activity of bacteria through gene triggering strategies

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5.15 Future prospective

117

References

118

6. Metabolic pathways for production of anticancer compounds in cyanobacteria Nasreen Amin and Vinod K. Kannaujiya

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6.1 Introduction

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6.2 Diversity and evolutionary significance of cyanobacteria

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6.3 Exploration of secondary metabolites

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6.4 Structural and functional diversity of anticancerous metabolites

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6.5 Biosynthetic pathway

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6.6 Future perspectives

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6.7 Conclusion

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Acknowledgment

142

References

142

7. Prophyletic origin of algae as potential repository of anticancer compounds Ruchita Tripathi, Rachana Shalini, and Rajesh Kumar Singh

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7.1 Introduction

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7.2 Metabolites or bioactive substances present in marine algae having anticancer properties

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7.3 Anticancer therapy via apoptosis

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7.4 Death receptor mediated pathway or extrinsic pathway

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7.5 Other: A typical forms of cell death

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7.6 Anticancer compound isolated from marine algae

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7.7 Anticancer properties of reported marine algal family

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7.8 Conclusions

178

References

178

Further reading

188

8. Metabolic versatility of fungi as a source for anticancer compounds Amit Ranjan, Rajesh Kumar Singh, and Monika Singh 8.1 Introduction 8.2 Plant-fungal interactions and its metabolic diversity 8.3 Genetic aspects of plant-fungal interactions 8.4 Biochemical aspects of plant-fungal interactions 8.5 Signal transduction pathway in plant-fungal interactions

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8.6 The potent anticancer compounds produced by terrestrial endophytic fungi 8.7 The potent anticancer compounds produced by deep-sea sediment fungi 8.8 The potent anticancer compounds produced by algaeassociated fungi 8.9 The potent anticancer compounds produced by mangrove endophytic fungi

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198 199 200 200

8.10 The potent anticancer compounds produced by sponge associated fungi

200

8.11 Conclusion

201

References

202

9. Structural information of natural product metabolites in bryophytes S.J. Aditya Rao

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9.1 Introduction

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9.2 Exploration of bryophytes for medicinal usage

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9.3 Bryophytes as a source of biologically active molecules

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9.4 Different types of secondary metabolites found in bryophytes

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9.5 Bioactive molecules from bryophytes reported with different pharmacological activities

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9.6 Bryophytes as a potential biopharming agents

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9.7 Chemical syntheses of bryophyte components

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9.8 Biotechnological applications for effective utilization of bryophytes for therapy

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9.9 Challenges and future prospects

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9.10 Conclusion

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Acknowledgments

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References

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10. Landscape of natural product diversity in land-plants as source for anticancer molecules Akanksha Srivastava and Richa Raghuwanshi 10.1 Introduction 10.2 Plant diversity and their anticancer potential 10.3 Microbial antitumor products 10.4 Anticancer property of fungi 10.5 Responses of cancer cells to the lichen compounds 10.6 Therapeutic potential of bryophytes against cancer 10.7 Ferns a treasury of anticancer agents 10.8 Anticancer property of gymnosperm 10.9 Anticancer potential of angiosperms

233 233 234 234 236 237 238 239 241 242

10.10 Conclusion

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References

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11. Anticancer natural product from marine invertebrates Rajesh Kumar Singh, Amit Ranjan, Monika Singh, and Akhileshwar Kumar Srivastava

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11.1 Introduction

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11.2 Sponges

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11.3 Cnidaria

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11.4 Bryozoa

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11.5 Molluscs

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11.6 Echinoderms

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11.7 Conclusions

262

References

262

Further reading

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Contents

12. Melatonin: A journey from bovine pineal gland to a promising oncostatic agent Tarun Minocha, Megha Das, Nitesh Kumar Mishra, Soumya Ranjan Mohanty, and Sanjeev Kumar Yadav

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12.1 Introduction

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12.2 Evolutionary history of melatonin

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12.3 Synthesis of melatonin in animals

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12.4 Synthesis of melatonin in plants

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12.5 Role of melatonin in integrity of genome and DNA repair

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12.6 Melatonin and telomerase activity

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12.7 Conclusion

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12.8 Challenge and future perspective

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References

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13. Spice up your food for cancer prevention: Cancer chemoprevention by natural compounds from common dietary spices Jie Gao, Kenza Mamouni, Lei Zhang, and Bal L. Lokeshwar

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13.1 Introduction

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13.2 Role of diet in cancer origin and progression

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13.3 Anticancer activities of select spices used in daily diet

284

13.4 Concluding summary

293

Acknowledgments

294

References

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14. Significance of nutraceuticals in cancer therapy Haritha H. Nair, Vijai V. Alex, and Ruby John Anto

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14.1 History of nutraceuticals

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14.2 Drawbacks in conventional cancer treatments

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14.3 Importance of nutraceuticals in cancer therapy

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14.4 Various nutraceuticals and their application in cancer therapy

313

14.5 Conclusion and future prospective

318

References

319

15. Common techniques and methods for screening of natural products for developing of anticancer drugs Monika Singh, Sukanya Patra, and Rajesh Kumar Singh 15.1 Introduction 15.2 Extraction of compounds 15.3 Fractionation 15.4 Purification 15.5 Crystallization 15.6 Chromatography 15.7 Physical methods for basic structure elucidation 15.8 Antioxidant assay 15.9 Single electron transfer

323 323 324 327 329 329 330 333 335 338

15.10 Hydrogen atom and single electron transfer

339

15.11 Chelation power of antioxidant

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15.12 Lipid oxidation

340

15.13 Anticancer assay

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15.14 Methods to detect ROS

347

15.15 Conclusion

349

References

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Index

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Contributors S.J. Aditya Rao Department of Plant Cell Biotechnology, CSIR—Central Food Technological Research Institute, Mysuru, Karnataka, India Vijai V. Alex Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Nasreen Amin Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India Ruby John Anto Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Megha Das Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Jie Gao Georgia Cancer Center at Augusta University, Augusta, GA, United States C.S. Joshi Patanjali Research Institute, Haridwar, Uttarakhand, India Vinod K. Kannaujiya Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India Sanjay Kumar Department of Botany, Banaras Hindu University, Varanasi, India Bal L. Lokeshwar Georgia Cancer Center at Augusta University; Research Service, Charlie Norwood Veterans Administration Medical Center, Downtown Hospital, Augusta, GA, United States Kenza Mamouni Georgia Cancer Center at Augusta University, Augusta, GA, United States Tarun Minocha Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Amit Kumar Mishra Texas A&M AgriLife Research and Extension Center, Texas A&M University, Uvalde, TX, United States Nitesh Kumar Mishra Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Pragyan Mishra Department of Biotechnology, Microtek College of Management and Technology, Varanasi, Uttar Pradesh, India

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Soumya Ranjan Mohanty Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Haritha H. Nair Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Sukanya Patra School of Biomedical Engineering, Indian Institute of Technology (BHU), Varanasi, India Richa Raghuwanshi Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India Amit Ranjan Centre of Experimental Medicine and Surgery; Department of Kayachikitsa, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Rachana Shalini Department of Botany, J.N. College, Madhubani, Lalit Narayan Mithila University, Darbhanga, Bihar, India Diksha Sharma Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Vinamra Sharma Amity Institute of Indian System of Medicine, Amity University Uttar Pradesh, Noida, India Ankita Shrivastava Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India Divya Singh Central Sericultural Research and Training Institute, Mysuru, Karnataka, India Monika Singh School of Biomedical Engineering, Indian Institute of Technology (BHU), Varanasi, India Rajesh Kumar Singh Department of Dravyaguna, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India V.P. Singh Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Akanksha Srivastava Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Akhileshwar Kumar Srivastava PCBT Department, CSIR-CFTRI, Mysuru, Karnataka, India Ruchita Tripathi Department of Dravyaguna, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Contributors

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Sanjeev Kumar Yadav Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Arpana Yadava School of Biomedical Engineering, IIT, BHU, Varanasi, Uttar Pradesh, India Lei Zhang Bayer U.S. LLC, Pittsburgh, PA, United States

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Preface Evolutionary principles of natural diversity (from prokaryotes to eukaryotes) lead to the emergence, distribution, diversification, and selection of genes involving biosynthesis of secondary metabolites. For the past 40 years, small organic molecules derived naturally from microbes and plants have been used in cancer treatments. Numerous studies on naturally occurring leading compounds have been ongoing in recent years, with the components of marine fauna and flora as well as those of terrestrial microorganisms and plants being investigated for cancer therapy. The different evolutionary mechanisms produce diverse chemical structures of secondary metabolites. Alterations in the chemical structure of secondary metabolites have been some of the best sources of drugs for treatment of severe diseases; however, the identification of anticancer properties at the evolutionary level of natural diversity could provide a clue to the biosynthesis pathway of potential metabolite production that could overcome the limitations of cell toxicity and side effects during chemotherapy. The development of natural products as conventional drugs has been established on the basis of traditional medicine and at present natural products continue to be an important source of pharmaceutical agents. For some time, natural products have been a pillar of cancer treatments. Combinatorial chemistry explores the properties of new and synthetic drugs over a wide range, and natural products provide leading compounds for the development of active components with potential biological properties. The traditional methods used in the discovery of new active compounds have several limitations, such as their complexity and inherent lengthy timescales; hence, research needs faster separation and isolation steps for development of an active compound from the crude extract. Currently, the screening of natural products has become an effective method for rapid selection of metabolites with efficient biological properties. The molecular mechanism of cancer cell development from the differentiated normal cells has been attributed to two major factors: oncogenes and tumor suppressor genes. The activation and inactivation of oncogenes and tumor suppressor genes by natural mutations in either cancer or differentiated normal cells can induce uncontrolled growth and proliferation ending through transformation of cells acquiring carcinogenesis properties. The inactivation of tumor suppressor genes also promotes uncontrolled cell growth. The molecular mechanisms in cancer progression have led to the development of a large number of anticancer regimes; however, the use of chemically synthesized anticancer drugs has produced harmful effects in patients, especially in those with immune system suppression. Hence, the discovery and development of new drugs associated with natural products have been essential in overcoming the harmful effects produced from xix

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chemically synthetic drugs. Alkaloids, flavonoids, terpenoids, polysaccharides, saponins, and others have been recognized as natural bioactive products with potent anticancer activity. Currently, over 60% of anticancer drugs based on natural products are used as conventional drugs derived from plants, marine organisms, and microorganisms. The anticancer activity of natural products often plays a vital role in regulating immune function, inducing apoptosis or autophagy, or inhibiting cell proliferation. This book explains the production of diverse anticancer molecules from different metabolic pathways at the evolutionary level, that is, bacteria to higher plants. It covers the different classes of natural products such as alkaloids, flavonoids, and terpenoids that have opened a wide scope for understanding the preventive and curative role of diverse molecules against oncogenic factors leading to cancer development. Chapters deal with biosynthesis of secondary metabolites through evolutionary mechanisms, alteration in secondary metabolites’ production with climatic changes, the various aspects of natural products derived from bacteria, cyanobacteria, algae, fungi, higher plants, etc., such as isolation, characterization, and assessment of their anticancer properties. This reference will be a welcome addition to the literature, as a single book covering all these essential aspects of natural products from evolutionary diversity has been sorely lacking until now.

1 Evolutionary mechanism for biosynthesis of diverse molecules Akhileshwar Kumar Srivastavaa, Arpana Yadavab, and Divya Singhc a

PC BT DE PART ME NT, CSI R-CFTR I, MYSURU, KARNATAKA, INDIA b S C H OO L O F B I OME DI C A L E N G INE E R I NG, I I T , B HU, V ARA N A S I , U T T A R PR AD E S H , I N DI A c CE NT RAL SE RI CULTURAL RESEARCH AND TRAINING INSTITUTE, MYSURU, K ARNATAKA, INDIA

1.1 Introduction Evolutionary diversity of nature as a best chemist produces diverse chemical products which are completely different from conventional organic synthesis. By own way, the nature optimizes her metabolic pathway efficiently. According to Darwin, evolution is a continuous event of mutation and adaptation through which diversification and natural selection gives an opportunity for the survival of the fittest (Tan et al., 2019). Darwinian evolution also provides an explanation to knowledge-free metabolic pathway alterations through nature (Tan et al., 2019). However, the natural evolvability is very low to preserve of important genetic information for a longer periods (Drake, 1999). Further, screening was not possible for the unexplored phenotype, then further reduced the exploration rate of microbial strains with specific characteristic (Zaccolo et al., 1996). Direct evolution is an in vitro process that develops to imitate the natural evolution at an elevated rate toward a destined aim. The positive explanations of in vitro Darwinian evolution in 1967 (Mills et al., 1967), has fascinated scientific community to understand evolutionary mechanism with potential methods to explore the natural products with novel properties. Gene duplication is a one of the important mechanism having the fresh genetic substances for specific gene evolution and various explanations elucidated about the maintenance of such gene copies from the slipping resulted during silence mutations (Weng, 2014). The genomes in plant have a plenty number of duplicated genes connecting with enlargement of specialized/lineage specific biochemical pathways in plants known as secondary metabolism. Secondary metabolites of plant have different types of important role in chemoecology with modification in precursors of primary metabolism (Kroymann, 2011). The shikimate pathway is most important primary metabolic pathway of plant through which route the aromatic amino acids are formed (Herrmann and Weaver, 1999). The shikimate pathway requires for protein synthesis and chemical or genetic alterations in plants, bacteria and fungi that is lethal due to activeness of glyphosate as an herbicide or by introduction of shikimate pathway biosynthetic genes in the directory of

Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00001-1 © 2021 Elsevier Inc. All rights reserved.

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Evolutionary Diversity as a Source for Anticancer Molecules

embryo lethal genes in Arabidopsis (Pagnussat et al., 2005; Carrington et al., 2018). Therefore shikimate pathway is rigorously managed under vigorous selection. The studies of plant secondary metabolites showed great connection between evolutionary and ecological due to early identification, which many of these components exhibited different ecological function and also showed phylogenetic relations. The complete description about the dissemination of chemical structures through all taxa cannot be described without knowing evolutionary relationships. Immediately, it was clarified that the acquired information on phylogenetic relationships in all plants had not able to assess the biological activity of secondary compounds, e.g., chemically similar secondary metabolites like terpenes have multiple activities like herbivore deterrence, fungal-toxicity, and pollinator attraction; and also act as solvents for higher molecular mass compounds to reduce risk for coagulate and clog transport systems in plants (Theis and Lerdau, 2003). Ecological and evolutionary models emphasized on the development and managements of chemical diversity of secondary metabolites by regulating the chemical contents within plants. Till date, it has not been given much effort on precise models of the evolution of secondary metabolite function and how their biological activity has been changing through time periods, but some researchers investigated such questions on the extent of specific plant clades (Scott Armbruster et al., 1997). Recently, terpenes are used as model at the genetic, physiological, and ecological level for understanding the evolution of chemical function and their ecological impacts on evolutionary processes. Plants produce different patterns of lineage-specific specialized (secondary) metabolites synthesizing from primary metabolites. Plant secondary metabolites play a vital role in plant adaptation and in human nutrition including as in medicine. Despite of the diversification of plant secondary metabolic enzymes, the primary metabolism also contributes in cellular homeostasis during strong selection pressure that is often conserved in all over plant kingdom. However, only some manipulations in primary metabolic pathways are explored in plant system. The biosynthetic pathways of some specific amino acids and lipids have been changed in particular plant descendants. Two other pathways existing in plants for producing primary precursors to generate the two main kinds of plant secondary metabolites: terpenoids and phenylpropanoids. Divergence of these primary metabolic pathways lead to major evolutionary alterations in plant metabolism and chemical diversity through enabling or related properties for the evolution of secondary metabolite pathways (Maeda, 2019). The synthesis of secondary metabolites (SMs) in plants are essential to develop defense mechanism in plants to be fitness against herbivores and microbes and also as signal factors to entice pollinators and fruit dispersers that seems in all higher plants creating wide structural diversity. The evolution of SMs depends on variation in the enzymatic action of primary precursors. It has been explained that some of the genes encoding enzymes of biosynthesis are transferred to plants through ancient horizontal gene transfer (HGT), e.g., from protobacteria or cyanobacteria that converted to mitochondria and plastids later on. Other important factor of SMs are ectomycorrhizal and endophytic fungi which directly develop defense compounds in plants or have been relocated their genes into the genome of host plants through genomic pathways (Wink, 2016).

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

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The diverse secondary metabolites are also produced from the final products and translational stages of the shikimate pathway (Carrington et al., 2018). Phenylalanine generates to several phenylpropanoids namely: lignin, flavonoids, tannins, and hydroxycinnamic acid conjugates which are occurred every parts of plant system (Vogt, 2010). Out of them, mainly hydroxycinnamic ester chlorogenic acid is widely disseminated and highly found in coffee as well as some members of the Solanaceae and Salicaceae families (Niggeweg et al., 2004), although it is not present in some species of model plant Arabidopsis thaliana (Guo et al., 2014). The hydroxycinnamic ester chlorogenic in plants have different biological functions ranging from an insect feeding deterrent (Carrington et al., 2018) to protection against UV radiation (Carrington et al., 2018). Quinate, a precursor of hydroxycinnamic ester chlorogenic and other secondary metabolites is formed in a sidebranch of the shikimate pathway through the reversible reduction of 3-dehydroquinate by involving of quinatedehydrogenase (QDH) enzyme. The QDH of Populus trichocarpa has highly similar sequence with the bifunctional enzyme dehydroquinatedehydratase and shikimate dehydrogenase (DQD/SDH) of the primary shikimate pathway, and these enzymes catalyze involve in same kinds of activities (Guo et al., 2014) and shows a common ancestor link between SDH (primary metabolism) and QDH (secondary metabolism). The shikimate is described as a major intermediate pathway for protein biosynthesis, hence it would be speculated that a pre-duplication progenitor acted on shikimate (Carrington et al., 2018). The scientific community has always been enthusiastic toward the maximum structural diversity in SMs, although researchers have challenging assignment to unravel the main purpose of plants for synthesizing, transporting and accumulating of all different kinds of SMs. The details of maximum identified SMs in the regarding of physiology, biochemistry and ecology are explained by many biologists. Earlier, the SMs had been generally considered as functionless waste products (Wiegrebe, 1986), but later on many reports show its importance for the plants in an ecological aspects (Wink and Mohamed, 2003; Wink, 2015). Now ecologists have explored the implication of SMs on existing producers through its interaction with the biotic and abiotic environment. As envisaged that SMs have developed different structure while evolution of higher plants which is implicated in plant taxonomy and its ecological role as defense against herbivores and pathogens including pollination and seed dispersal mechanisms. All plants groups (from lower to higher) have ability to synthesis secondary metabolites, but they are widely found in angiosperms. The major classes of these metabolites develop from five precursor pathways: acetyl coenzyme A, active isoprene, shikimic acid, glycolysis and citric acid cycle. These pathways with individual or in combination generate various structural diversity and till date about 200,000 secondary metabolites have been characterized (Table 1.1). Further, this structural diversity is enhanced broadly by occurring glycosylation and esterification and sometime by highly involvement of other primary metabolites, e.g., few nonaromatic amino acids and polysaccharides. Generally plants have varieties of the blended SMs and the constituents of these blended SMs vary in plant organs and its developmental events, which belong to different groups of SMs like terpenoids are often accompanied by phenolics. Usually, it has been explored a limited

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Table 1.1 Name of important secondary metabolites (SM) identified from plants (Wink, 2016). Types of secondary metabolites Nitrogen-containing SMs SMs without nitrogen Alkaloids Nonprotein amino acids Amines Cyanogenic glucosides Glucosinolates Alkylamides Lectins, peptides, polypeptides

Monoterpenes (C10) Sesquiterpenes (C15) Diterpenes (C20) Triterpenes, steroids, saponins (C30, C27) Tetraterpenes (C40) Flavonoids, anthocyanins, catechins, tannins Phenylpropanoids, lignin, coumarins, lignans Polyacetylenes, fatty acids, waxes Polyketides Carbohydrates, simple acids

number of SMs and several minor components that are often biosynthetically related to the main constituents (Wink, 2016). The present chapter emphasizes on the evolution of secondary metabolites in plants from different biological pathways.

1.2 Models for evolutionary study The continuous study on secondary metabolites made several attempts to endeavors their occurrence and richness on which basis it was confirmed that these were not just plant waste products. There were two challenges immediately held in development of models for the study of secondary metabolites (i) understanding the origin and management of diversity of chemicals in plant kingdom, and (ii) discovering the ways elemental variation in amounts which are present within and among plants of the same and different species. The interactive role of plants with pests, pathogens, and predators lead to origin of chemical diversity within plants. It has been considered that the origin of secondary metabolites in plant and the progressive evolutionary events were responsible for interaction with phytophagous insects which resulted to the radiation and diversity in such groups (Ehrlich and Raven, 1964). During stepwise methods of escape and radiate has appeared under close scrutiny, the complexity in such defensive plants with insects is well known for generation of the diversity in secondary phytochemicals which play a significant role in prevention of herbivores attack. The advance model of Jones and Firn leads to understand the chemical activity by considering the all aspects of chemical diversity and explains that plants have highly diversified compounds than those are implicated for defense purpose at any single time ( Jones and Firn, 1991). The models of Jones and Firn (1991) and Ehrlich and Raven (1964) have described about the unique diversity in chemical structure (Ehrlich and Raven, 1964). Therefore, these ecological and physiochemical models have investigated within a phylogenetic skeleton if approaches are made to employ them to empirical data (Monson, 1996).

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

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To predict the amount of secondary metabolites occurred in plants by developing a model is more challenging issue. Herbert Simon extended his work toward modeling and linear programming tools by considering economics and other areas (Simon, 1966) who observed a susceptible reservoir of ecology and evolutionary biology. The first attempts were employed by using optimality theory in plants for developing reproductive models (Cohen, 1966). The influential articles followed immediately and thereafter addressed questions related with distribution of secondary metabolites from an optimality perspective (McKey, 1974). These articles have represented a hypothetical outcome that any sources involving in defense mechanism cannot be employed for growth or reproduction. Hence, in evolutionary terms, it has definitely produced for the defense, and this production could be predicted by estimating the foregone reproduction happening from allocation to defense. The advantage of allocation to defense mechanism increases in reproduction in which plants don’t lose biomass to pests or pathogens. Coincidence happened with this clear statement on optimality approach, when Feeny (1976) generated a model of allocation for secondary metabolites which was based on concept of the risk induced by plants (Feeny, 1976). The apparency model is possibly specialized by herbivores (apparent plants) and the “unapparent” plants, ephemeral in their distribution, are more likely to avoid detection by specialists and could bear defensive action that are active at even low concentrations and are mobilized for reapplication. The concept of apparency appears in certain application of risk for calculation of optimal levels of defense in plants. In beginning of 1980s attention started to emphasize on the function of resource availability as a criterion influencing growth rate and the relative costs and profits in synthesizing secondary metabolites for defense. This optimality attempt has been observed in the evolutionary model of Coley et al. (1985) (Coley et al., 1985) and Bloom et al. (1985) (Bloom et al., 1985). In 1992, Zangerl and Bazzaz have considered simultaneously risk and resource availability into an optimality model which has been confirmed intensively effective at predicting allocation to defense (Zangerl and Bazzaz, 1992). The most significant model of John Bryant et al. (1983) not based on optimality principles was kept forth in the early 1980s (Bryant et al., 1983). They crafted that distribution of several defensive components was regulated by basic stoichiometric and hence if availability of resources are much excess than a plant’s capability to utilize it for growth or reproduction; then resources are transferred to secondary metabolites. This model recognized as the carbon-nutrient balance hypothesis, has energized to produce excess heat and a less light, although its intellectual properties has high affinity to the sort of null model which need for further validation for all good optimality simultaneously (Beatty, 1980) rather facing to a general challenge to optimality thinking (Lerdau and Coley, 2002). The challenges dealing by ecologists and evolutionists is less valuable than specific optimality model is highly relevant to particular plants and its secondary metabolites, hence it brings together an advancement at the genetic and physiological bases of secondary metabolite activity with ecological and phylogenetic patterns of compound distribution.

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Evolutionary Diversity as a Source for Anticancer Molecules

1.3 Evolution of secondary metabolite pathways Plant secondary metabolites (PSMs) are synthesized from primary metabolites with different physiological activities. Secondary metabolites have significant function in plants to develop a strong interaction with the environment for their survival and fitness that enables importance of these metabolites like primary metabolites (Kliebenstein, 2013). It is usually speculated the pathways of secondary metabolism develops from precursors associated with primary metabolic network. Single secondary pathways might evolved due to random duplication and consequent mutation in one of the gene copies (Zhang, 2003). The plant kingdom synthesizes more than 100,000 secondary metabolites that are found in specific groups of taxonomy. On the basis of biosynthetic pathway, PSMs are comprised into three main groups like terpenes or isoprenoids, phenolic compounds and nitrogen containing compounds (Verma and Shukla, 2015) (Fig. 1.1). The shikimate pathway generates aromatic amino acids require for protein synthesis in plants. Shikimate dehydrogenase (SDH) a key enzyme of the primary metabolic pathway synthesizes shikimate molecule. The structurally identical quinate; a secondary metabolite is produced in the presence of quinate dehydrogenase (QDH). The SDH and QDH

t

Ligh

Photosyenthesis

CO2

Primary carbon metabolism Erythrose-4-phosphate

3-phosphoglycerate Phosphoenolpyruvate

Shikmic acid pathways

Tricarboxylic acid pathways

Aromatic amino acid

Pyruvate

Acetyl CoA

MEP pathways

Aliphatic amino acid Malonic acid pathways

Nitogen containing secondary metabolites i.e. Alkaloids, glucosinolates, cyanogenic glycosides

Mevalonic acid pathways

Terpenoids compounds i- eisoprene, saponin, mono-, di-, tri-terpenes, carotenoids, phytohormones

Phenolic compounds i.e. Flavanoids, coumarins, lignin, tannins

FIG. 1.1 A schematic diagram of biosynthetic pathway for synthesis of secondary metabolites in plants.

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

7

belonging to the same gene family have deviated into two phylogenetic clades after describing gene duplication just before the division of angiosperm/gymnosperm. Nonseed plants are deviated prior to this duplication harbor only a single gene of this family. The recent members from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens), and lycophytes (Selaginella moellendorfii) have encoded SDH activity in vitro. A regenerated ancestral sequence has represented the node just before to the gene duplication also encoding for SDH activity. QDH activity had been achieved only in seed bearing plants through successive gene duplication. Pinus taeda represented the QDH enzymes of gymnosperms might be translational factor of an evolutionary event encoding for activities of SDH and QDH equally. The second copy in P. taeda retained specificity for shikimate was similar to the activity happening in the angiosperm SDH sister clade. The codon for a tyrosine residue at active site revealed an impression of positive selection at the node of the QDH clade, whereby it has been converted into a glycine. The exchange of tyrosine to a glycine in shikimate-specific angiosperm SDH had enough to have some QDH activity. Hence, few mutations was required to simplify the evolution of QDH genes (Carrington et al., 2018).

1.3.1 Gene clusters for evolution of secondary metabolites Microbes and plants synthesize a large amount of secondary metabolites which involve in major ecological role. Since a longer period, these compounds have been exploited for medicinal values such as antibiotics, anticancer and anti-infective agents and also for other purposes. Gene clusters for secondary metabolic pathways are found mainly in bacteria and filamentous fungi, but recently it has also been observed in plants (Osbourn, 2010). The increasing interest toward secondary metabolites has fascinated a several genome sequencing projects to understand their biosynthetic pathways. Outcomes of these attempts showing the extensive numbers of secondary metabolite gene clusters (SMGCs) have been identified during metabolites production; however, the main challenging of present methods is to dereplicate and categorize the quantity of gene clusters on a wide range. Although, it has been presented an automated system for the genetic dereplication and detection of secondary metabolism genes in fungi. Emphasizing on genus Aspergillus has a rich resource for the secondary metabolite production that has been categorized SMGCs across genomes into SMGC families by implying network assessment. This process explains the diversity and dynamics of secondary metabolism in section Nigri, suggesting that SMGC diversity in this section has the equal magnitude as within the genus. The genome assessment has predicted the gene cluster involving in biosynthesis of malformin, a potent mediator of anti-cancer regimes, in 18 strains. Further, the genetic engineering approach in Aspergillus brasiliensis has been done for validation of the earlier predicted results and also subsequently confirmed the genes encode for the biosynthesis of malformin (Theobald et al., 2018).

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Evolutionary Diversity as a Source for Anticancer Molecules

Exploration of secondary metabolic gene clusters at early stage of growing plants is compared to clusters of microbes. Currently, it had been envisaged that the cluster of genes were not occurred for plant metabolic pathways and it has been definitely observed true in several cases. However, five examples of plant secondary metabolic gene clusters are described in this section (Osbourn and Field, 2009). These are the cyclic hydroxamic acid (DIBOA) cluster in maize (Gierl and Frey, 2001; Osbourn, 2010), triterpene biosynthetic gene clusters in oat and Arabidopsis (Field and Osbourn, 2008; Mugford et al., 2009), and two clusters for producing of diterpenes in rice (Shimura et al., 2007; Field and Osbourn, 2008). The all four clusters of cereals are involved in the synthesis of preformed or stress-induced protective components and are used in plant defense. However, the role of the Arabidopsis thalianol cluster has not been known, although it plays a vital role in ecological interactions (Field and Osbourn, 2008). Out of the five, three plant clusters including the avenacin cluster are found at sub-telomeric site (Qi et al., 2004; Osbourn, 2010). So, the ability of plant gene clusters to be localized toward the ends of chromosomes is similar to actinomycetes and filamentous fungi, although this description is not a definite rule. The mediators for these clustered plant pathways are still unexplored. Nonetheless, the present explanation on Lr34, a gene responsible for disease resistance against various pathogenic fungi in wheat, encodes a ABC transporter employed in the transfer of defense-related metabolites which mediate the neighbors of this gene to assess the role of secondary metabolism (e.g., sugar transferase genes) (Krattinger et al., 2009). Glycosylation of secondary metabolites is also a general process of self-defense in plants, and it has been noticed that the incomplete accumulation of glycosylated avenacins in mutants of oat have growth defects attributable to accumulate of these toxic intermediates (Mylona et al., 2008). Glycosylation has also provided a safeguard against self-poisoning in Actinomycetes (Quiro´s et al., 1994). The first functional cluster of non-homologous genes was recognized as thalianol gene cluster in Arabidopsis model species (Field and Osbourn, 2008) (Fig. 1.2). The avenacin cluster in oat also has been identified as a triterpene cluster (Qi et al., 2004, 2006). However these two great gene clusters have slightly similarities in that both have an oxidosqualenecyclase target gene along with genes for cytochrome P450s and another alternative enzymes, the quantity of gene and its organization in clusters has a bit differences which phylogenetic assessment suggests that their evolution have occurred independent way (Field and Osbourn, 2008). The two diterpene gene clusters in rice also evolved independently, however it has an ancestral cluster containing common precursors for some of the new gene members. In spite of that, subsequent assembly was essential to generate the momilactone and phytocassane clusters (Fig. 1.2) (Swaminathan et al., 2009). The signature genes of all five plant secondary metabolic gene clusters are definitely screened directly or indirectly from genes responsible for plant primary metabolism (Swaminathan et al., 2009; Osbourn, 2010), and these events have compelled to suggest

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

9

i. Thalianol gene cluster (35 kb)

ii. Momilactone gene cluster (170 kb)

Phytocassane gene cluster (245 kb)

Signature enzymes: Triterpene synthase

Tailoring enzymes: CYP85

BAHD acyltransferase

CYP71

Dehydrogenase

Class I diterpene synthase CYP76 Class II diterpene synthase

CYP99

FIG. 1.2 The metabolic gene clusters for evolutional of secondary metabolism in plants (i) the thalianol cluster in Arabidopsis consists of four coexpressed genes encoding a triterpene synthase, two different kinds of cytochrome P450 (CYP) and a BAHD acyltransferase. The first three genes involve the synthesis and consequent changes in thalianol. The BAHD acyltransferase is detected as the fourth component of the cluster, but an acylated downstream product has not still been determined. These clusters seems to be colinear (ii) the momilactone and phytocassane clusters in rice present on chromosomes 2 and 4, respectively, are essential for diterpene production but have originated independently. The main enzymes for these clusters are class I and class II diterpene synthases. The tailoring enzymes are combined with cytochrome P450s and, in the case of the momilactone cluster, a dehydrogenase (Osbourn and Field, 2009).

that such clusters were evolved by gene duplication, neofunctionalisation and genome reorganization instead of horizontal gene transfer (HGT) from microbes (Osbourn and Field, 2009). Presently, the first transcription factor (chitin oligosaccharide elicitor-inducible basic leucine-zipper transcription factor) for production of secondary metabolic gene cluster in plant has been explored (Okada et al., 2009). This transcription factor alters the expression of both momilactone and phytocassane synthesis by upregulating the biosynthetic genes and the upstream pathway for the generation of common precursors. The gene for this transcription factor remains outside of both the clusters which it regulates. Still there is no any confirmation if this transcription factor also controls other secondary metabolic pathways in rice. DNA fluorescence in-situ hybridization (FISH) methods provide a proof for regulation of gene-cluster expression in plant at the chromatin level in which molecular markers for two genes in the oat avenacin cluster had been employed to investigate the relationship between chromatin condensation/decondensation and gene expression. Under

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Evolutionary Diversity as a Source for Anticancer Molecules

constraint developmental regulation, the avenacin genes are expressed particularly in the region of root-tip epidermal cells (Haralampidis et al., 2001; Qi et al., 2006). Highresolution DNA FISH has exhibited that cluster expression is related with tissue specific chromatin decondensation (Wegel et al., 2009).

1.3.2 The evolutionary origin of clusters HGT is used in the transferring of secondary metabolite genes and clusters between bacterial species (Ridley et al., 2008). Transferring the gene clusters from prokaryotes to eukaryotes appreciate in understanding the evolution of functional gene clusters specially for secondary metabolic pathways in filamentous fungi and plants. It is a great proof to explain that genes involving in polyketide synthesis, could be migrated from bacteria into filamentous fungi (Schmitt and Lumbsch, 2009; Marcet-Houben and Gabaldo´n, 2010), and that metabolic gene clusters could be further mobilized between different fungal groups and other genera (Khaldi et al., 2008; Slot and Rokas, 2010). Hence HGT is one of novel way for gene clusters and definitely it has been designed as a primary source for such clusters in the “selfish cluster” model (Walton, 2000). Nonetheless, it has been explored that the genomes of microbes and plants shows a great plasticity and this evidence suggests that clusters could make de novo from existing genetic factors. Such event is probably regulated through strong selection for the evolution of new adaptive biological functions. In case of bacteria, it has been evident that operons could be made through rearrangements which bring closer of distant genes or by deletion of intervening genes (Price et al., 2006), and this process referred as genome defragmentation. The comparative study of genomes in yeast (Saccharomyces cerevisiae) has showed that gene clusters of two catabolic pathways: the GAL and DAL clusters, were necessary for the utilization of galactose and allantoin, respectively-have most likely originated by adaptive gene relocation (Slot and Rokas, 2010). In same way, the existing secondary metabolic clusters in filamentous fungi could be amplified by selecting the new genes from any of the genome (Nomura et al., 2003). The fragmented secondary metabolic clusters are also functional in some cases (Nomura et al., 2003; Bradshaw et al., 2013), explaining that physical clustering is not necessary for biological function. Repetitive DNA sequences and transposable elements immediately adjoin the secondary metabolic clusters in filamentous fungi, and play role in cluster formation and probably also in regulation (Perrin et al., 2007; Shaaban et al., 2010). Although it is a great enigma to be observed if this is also in the case of actinomycetes and plants. An interesting question arises here whether; the generated configurations of all intermediate factors during course of evolution are functional enough to explain their cost of existence. In this aspect, two opposed views have been given on metabolic diversification that all compounds must have a biological function otherwise the gene involved for their synthesis would have been knockout by negative selection (the “functionalist view”), or in other way, it has capability to biosynthesis of structural diversity per se that provides a selective advantage, and hence it is the processes of secondary metabolism rather than the products which have significant role (the “processualist view”) (Slot and Rokas,

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

11

2010). Assessment of sterigmatocystin mutants of Aspergillus nidulans showed the enhanced asexual sporulation is integrated with progressive pathway, consistent with the functionalist view (Wilkinson et al., 2004).

1.4 Cell fitness coupling for natural metabolite production Many cases, the organisms are capable to develop some properties to save itselves from harmful effect caused by selection pressure. The selection pressure leads to produce more metabolites in microbial cells that are capable to reproduce and have a higher growth rate in comparison to low producing cells; hence it appears overwhelming competitions in the low-producing cells in the culture media after subsequent dilution of cultures. This phenomenon could be seen during the regulation of gene expression in microbial cells under selection pressure by employing advance tools for genetic analysis. If E. coli is cultured in toxic level of ethanol, the expression of all genes in tricarboxylic acid (TCA) cycle, and genes associated with glycine, glycine betaine, peptidoglycan, colanic acid and enterobactin synthesis are upregulated to increase ethanol tolerance (Goodarzi et al., 2010). E. coli without glutamate-cysteine ligase (gshA) gene, a main enzyme for the γ-glutamyl cysteine production involving in glutathione (GSH) synthesis, developed another GSH producing pathway from proline synthesis pathway, to save the microbial cell from stressful conditions (Veeravalli et al., 2011). Such results indicate that selection pressure plays a significant role in continuous evolution of metabolic pathway in vivo, since the cell fitness is integrated to desired products. Wild type yeasts synthesis isopentenyl diphosphate that is the natural precursor to carotenoid. The secretion of this antioxidant prevents the cells from being oxidized under oxidative stress. Evolution of β-carotene synthesis pathway in S. cerevisiae with natural metabolite production/cell fitness coupling has completely been explained with an increase in β-carotene production by 3-fold, to 18 mg g 1 [dcw] using periodic hydrogen peroxide shocking strategy (Reyes et al., 2014). Apart from the chemical stress, physical stress could also be applied to enhance the synthesis of chemical products. Shinorine is a compound in mycosporine-like amino acids (MAAs) family, synthesized by marine microorganisms. The absorbance properties of this compound at 333 is identified as an important ingredient for synthesizing of some sun screen products (Balskus and Walsh, 2010). Biosynthesis of shinorine in microbial cell reservoir has also been explained (Yang et al., 2018). However this study has not emphasized on the evolutionary aspects, the higher growth rate of shinorine synthesizing cyanobacteria Synechocystis indicated that the development of this pathway is accomplished in exposure to ultraviolet ray. Deficient of non-ribosomal peptide synthase (NRPS)/polyketide synthase (PKS) gene cluster in cyanobacteria (Weber et al., 2015) initiates to the null effect in synthesis of shinorine in cyanobacteria exposed in ultraviolet ray. Other type of microbial cell as host may result in the physical evolvability of shinorine production. Nevertheless, metabolite production/ cell fitness coupling for chemical synthesis is not always found in nature. This method is only restricted to the pairs of different damaging source that could develop adverse effect on the microbial cell.

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Evolutionary Diversity as a Source for Anticancer Molecules

1.5 Chemical diversity of natural products The present introspection about the generation of drug lead exemplify the notion of having greater molecular diversity within the confined DRUG-LIKE properties (Sadowski and Kubinyi, 1998). It has been identified that the structure of natural-product reveal the nature of high chemical diversity, biochemical specificity and other molecular characteristics which prepare them favorable as lead structures for exploration of drug and that facilitate to segregate it from libraries of synthetic and combinatorial compounds. Several bioinformatician have worked to assess the desirable chemical properties by using computational chemistry for distinguishing the natural compounds from alternative sources of lead drugs. It has been investigated compound libraries associated with molecular diversity the representative combinatorial, synthetic and natural product (Feher and Schmidt, 2003) and “drug-likeness” properties like molecular mass, number of chiral centers, molecular flexibility as analyzed by number of rotatable bonds and ring topology, presence of heavy atoms, as well as LIPINSKI-type descriptors (Lipinski et al., 1997). Other researchers have segregated the natural compounds, conventional drugs or other synthetic molecular libraries depending on the scaffold architecture and pharmacophoric properties (Lee and Schneider, 2001), or other molecular descriptors (Stahura et al., 2000). These studies show that natural compounds contain a higher number of chiral centers and enhanced steric complexity than conventional drugs and combinatorial libraries (Henkel et al., 1999; Feher and Schmidt, 2003). However drug and combinatorial molecules contain a large number of nitrogen-, sulfur- and halogen groups whereas natural products have a greater number of oxygen atoms (Stahura et al., 2000; Feher and Schmidt, 2003). Multivariate statistical assessment of molecular descriptors elucidate that natural compounds form conventional medicines and combinatorial libraries in the ratio of aromatic ring atoms to total heavy atoms (lower in natural compounds), number of solvated hydrogen-bond donors and acceptors (higher in natural products) and by greater molecular rigidity. Natural-product libraries also have a wide distribution of molecular properties like molecular mass, octanol-water partition coefficient and diversity of ring systems in comparison to synthetic and combinatorial counterparts (Stahura et al., 2000; Feher and Schmidt, 2003). Absolutely, less than one-fifth of the ring systems occurred in natural products is expressed conventional drugs. Probably unexpected outcome is that of Schneider and Lee showed that the fraction of natural product structures with two or more “rule-of-five” violations is slight low (approximately 10%) and equal to that of conventional drugs (Stahura et al., 2000).

1.6 Occurrence of flavonoids in the plant kingdom It has been reported that about 9000 flavonoids were identified in the plant kingdom (Williams and Grayer, 2004). Flavonoids has been detected across the plant kingdom, including pteridophytes, gymnosperms, and angiosperms (Tohge et al., 2013). The enormous data on flavonoids from various species suggests to address the distribution of

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

13

flavonoid sub-classes (e.g., chalcones, flavones, flavonols, anthocyanins, and proanthocyanidins) in subgroup of plants (Fig. 1.3). Flavone and flavanone are present in all plant systems except hornworts but no flavonoids have been determined in hornworts yet. Evolution and diversification of plant groups have produced the flavonoid sub-classes in each group, e.g., the flavonoid aglycones are highly diversified in the angiosperms. Despite of flavanones and flavones, other compounds like chalcones, flavonols, and proanthocyanidins are present in various groups. Intriguely, the prenylflavonoids are occurred in liverworts and angiosperms: whereas more than 1000 prenylflavonoids were identified in legumes (Yazaki et al., 2009), prenyldihydrochalcone was explored in the liverworts Radula variabilis and Radula spp. (Asakawa et al., 1982). These data indicate that the two groups of plants achieved the potential to synthesis prenylflavonoids independently, or that many groups have lost the capability to generate prenylflavonoids while evolution. Flavonoid molecules give concise evidence for the presence of the respective flavonoid biosynthetic genes across the plants kingdom. Analytical approaches for exploring the flavonoidsis are essential to acquire the knowledge on evolution of flavonoid metabolism in the plant kingdom.

FIG. 1.3 Diagram shows the evolutionary relationship of flavonoid subclasses in the plant kingdom and red line assigns for the phylogenetic relationships in the bryophyte lineages (Bowman et al., 2017).

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Evolutionary Diversity as a Source for Anticancer Molecules

1.7 Biomolecular activity of secondary metabolites Tool for screening approaches give enormous evidence for any type of biological target, since maximum chemicals (synthetic or natural) remain inactive unless tested at high concentrations. Total 400,000 microbial cultures had been assessed since 10 years and only three biological active antibiotics were explored (Firn and Jones, 2000). Nevertheless, the significance of this evidence to unravel the evolutionary mechanism for production of secondary metabolism has been challenged and that explained the low frequency of activity during screening trials was just outcome from inappropriate screening methodologies. They discussed that, if the “correct” target had been employed, then a very high frequency of biological activity could be obtained. The conclusion of discussion created the conflict on such a fundamental issue associated with the definition of the term “biological activity.” The advance debate on such issue will be possible if definition of biological activity is related in terms of the evolution of secondary metabolism. The study on “biological activity” at a molecular level in vitro could have a different interpretation to “biological activity” studied at an in vivo model. At the molecular level, it has several evidences on certain biological activity against a defined molecular target has a unique feature for a molecule to exhibit, so high-throughput screening protocols have been developed for the determining the biological activity of 100,000 chemicals everyday and that’s why chemical libraries of 1 million compounds are commercially available for drug screening. The experience of several years on longer screening programmes has provided a secure concept for understanding the biological target. The studies on ligand-binding exhibit that high-affinity, reversible, non-covalent interactions of ligand with protein only happen if the ligand has the correct molecular configuration to bind with the three dimensional structure of the protein (Harvey et al., 2000). It has been suggested that this kind of biological activity should be referred as “biomolecular activity,” also defined as the interactive capability of a compound with a biologically active molecule altering the biological function. The immense experimental result shows that at low concentrations (1025 M) for any chemical has less efficient for revealing “biomolecular activity” against any one target protein (Firn and Jones, 1999). Nevertheless, it has been predicted that the frequency of compounds having “biological activity” could be higher when activity is determined by targeting an organism instead of a protein. An organism with several potential protein targets; hence, if one was subjected for screening for a non-specific effect on an unadapted organism and predicted a higher frequency of activity could be obtained in organismbased target in comparison to a screen based on “biomolecular activity.” Furthermore integrative study on organism based screening is subjected for the investigation of chemical potential against several different species. Since the concentration of all chemicals being investigated against an organism is alleviated, according to the laws of mass action, hence the frequency of developing any effect will enhance further. Hence, the low probability of occurring efficient “biomolecular activity” against a certain molecular target at a low concentration (Firn and Jones, 1999) is completely remained same, and so a greater frequency of less specific activity may be determined since a very wide range of unadapted

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

15

organisms is screened by taking a high concentration of every chemical Although, where in such continuity between the extreme definitions of biological activity (efficient biomolecular activity against a particular target versus low-potency “toxicity” against any organism) is selection operating in reference of the chemical interactions with organisms? In terms of evolution, it has been envisaged that the only target organisms had an opportunity to interact with the producer organism. Though, an effect originated in any other organism could not act as a factor for selection. This restricted condition extensively declines the number of possible combining chemical-target of organism (Firn and Jones, 1999). In same way, in evolutionary aspects, it depend on only concentration the which a target organism will have under normal condition physiological effects exhibited and at concentrations above than those achievable in the natural environment could not be of selective significance. So, it has been suggested that the most common evolutionary methods for selection generating on certain parts of the secondary metabolism will have less involvement rather than several target organisms. Moreover, it has been considered that selection favors the organisms for production of effective chemicals at low cost, and that support to the selection of organisms capable of producing highly efficient compounds. High potency results from a strong ligand-protein interaction suggested that it is necessary to specific ligand structure fitting a definite target site of the protein, hence generating the very specific biomolecular effect (Harvey et al., 2000). These notions suggested the constraints which imply to the evolution of “biomolecular activity” would have been more significant in the evolution of secondary metabolism.

1.8 Evolution of anticancer drug discovery Presently, the attempt to explore the new anticancer regimens has been developed by implying the cell-based screening for investigating the antiproliferative effects that targets the certain molecular residues responsible for the development and maintenance of the malignant phenotype in different forms of cancer. The main aim of the formation of molecularly targeted drugs is to enhance the efficacy and selectivity of cancer treatment by comparative study between cancer cells and normal cells. The success of newly developed molecularly targeted regimes like tretinoin for acute promyelocytic leukemia (Avvisati and Tallman, 2003) and imatinib for chronic myelogenous leukemia (Druker et al., 2001) and gastrointestinal stromal tumors (Van Oosterom et al., 2001), gives early clinical proof for the molecularly targeted approach to drug discovery. Mostly the common cytotoxic anticancer medicine were explored by using random high throughput screening of synthetic and natural compounds in cell-based cytotoxicity assays. Instead the number and chemical diversity of these compounds, the mechanistic action of these compounds are limited (Table 1.2), and maximum compounds act as DNAdamaging agents with a low therapeutic index. It has been analyzed from this screening approach that the mechanism of action is not a primary criterion for selection of compounds for further development, so with this reason, no any recent drugs directly targets

16

Evolutionary Diversity as a Source for Anticancer Molecules

Table 1.2 Example of some diverse secondary metabolites having limited molecular target in cancer treatment. Name of secondary metabolites

Molecular targets

Vinca alkaloids Taxanes Camptothecins Dactinomycin Epipodophyllotoxins Anthracyclines

Tubulin-binding agents Tubulin-binding agents Topoisomerase I inhibitors Topoisomerase II inhibitors Topoisomerase II inhibitors Topoisomerase II inhibitors

the molecular residues playing role in malignant transformation. Initially, National Cancer Institute (NCI) high-throughput screen employed the highly chemosensitive P388 leukemia cell line, although this approach of screening was failed to explore those drugs which were functional against the adult solid tumors. In the mid-1980s, the NCI developed a new in vitro disease-oriented screen having 60 human tumor cell lines with nine common forms of cancer (Monks et al., 1991). It has to be determined if selective activity of drugs in vitro against cell lines showing a certain histological form of cancer will have antitumor activity in vivo model (Monks et al., 1991). The molecular levels of studies in different forms of cancer have explored the variety of new potential therapeutic targets. The accumulation of mutations in diverse genes lead for tumorigenesis in various forms of cancer (Kaelin, 1999). For example, the rasactivating mutations and other mutations inactivate p53 and stimulate the cancer cells to circulate intrinsic and extrinsic controls which rigorously regulate the cell cycle, cell division and apoptosis in normal cells. After knowing the genetic changes in cancer cells, the specific transforming mutations are recognized from genetic alterations associated with the inherent genetic instability in cancer cells, although they do not involve directly in development of tumorigenesis (Kaelin, 1999). Such targets in cancer cells are validated in preclinical cancer models. The identified a right molecular targets has led to reasonable target-based drug discovery at the protein level, as previously explained by the formation of imatinib, that was explored by screening of compounds from libraries to inhibit the protein kinase activity in vitro (Druker and Lydon, 2000). Most of the proteins involving in cell cycle, signal transduction, and the regulation of apoptosis are enzymes or receptors potentially responsible for the inhibition of cancer activity through small molecules (Gibbs, 2000a). Another one is target-based treatments employing for clinical evaluation have farnesyl tranferase inhibitors that arrest post translational prenylation of ras, cyclindependent kinase inhibitors, protein kinase C inhibitors, and epidermal growth factor receptor kinase inhibitors (Gibbs, 2000b). Although, target-based screening for exploring the new molecularly targeted cancer treatments has not much influence to develop new drugs further. Unlike the Bcr-Abl fusion protein in CML and ras oncogenes, it has been approached toward the mutations leading anticancer activity, most mutations in cancer cells cause for a loss or inactivation

Chapter 1 • Evolutionary mechanism for biosynthesis of diverse molecules

17

of a protein (e.g., p53 mutations), and screening of several drugs by using of the protein product from mutated tumor suppressor genes is unlikely to explore a small molecule that restore protein function. Moreover, the active components in a target-based screening assessment might not be peculiar for the protein used in the screen, and, as a result, the therapeutic effect of the components at a cellular or individual level might be highly associated with its effect on other unrelated and or higher affinity of targets. Eventually, various molecular targets for new anticancer regimens bind with other proteins within pathways or networks in the cell, and the therapeutic effect appears through inhibition of a specific target might be influenced by the expression or relative levels of such interacting proteins. So, the target-based screening assessment might not be playing role in prediction of drug effect within the context of the whole cell. It has been elucidated that cell-based screening assays would always play a potent role in drug discovery in molecular targeting era (Sausville and Johnson, 2000). The three-stage cell-based method implying in yeast cells could be genetically altered is adaptable to high throughput screening to recognize the regimens, which have a specific effect against mutated cells through gene deletions. Unlike target-based assays, the mechanistic screening is not done in the cell-based assay, although to understand the mechanism of action of selective toxic agents from this screen could explore the new molecular targets like knockdown in pathway by the deletion of a tumor suppressor gene, and then could be proceed for subsequent target-based screening. The pharmacologic aspect shows that this cellbased screen assesses the collateral sensitivity. The genetic mutation increases the sensitive of cell toward the drugs affecting in the related pathways of the cell. This assessment has also a direct implications of the concept based on synthetic lethality that has been explained in yeast earlier (Hartwell et al., 1997). If mutation persisting in either one of two genes exhibit synthetically lethal, although mutations in both genes are always lethal. From this cell-based screening, it has been observed that a drug disrupts the function of the gene’s product, is only lethal in cells, which have developed a mutation in a second related gene. Cell-based assessments have also been subjected to confirm the activity of molecules discovered in target-based screening methods and to determine the pharmacologic effects of compounds at the cellular level. Unanticipated effects in cellular systems indicate toward other targets for the compounds or interactions of the primary molecular target with other particular proteins expressing in the cell. The NCI’s panel of 60 human tumor cell lines also give information on the mechanism of action or molecular target of new compounds, which are undergone for test based on the activity profile of drug obtained during screening assays (Greshock et al., 2010). As new targets are recognized and their expression in each of the 60 cell lines could be explored and correlated with the activity profile of the 70,000 compounds screened earlier without retesting of each agent. The discovery of the topoisomerases as targets for the compounds which were active in yeast cells lacking DNA double-strand break-repair proteins was slightly based on their activity profile in the human tumor cell line.

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Evolutionary Diversity as a Source for Anticancer Molecules

1.9 Factors influence the production of secondary metabolites Such a noteworthy is very important for plants and humans, which production varies in subsequent generations due to climatic and other factors. The content of different PSMs fluctuates among different species and also in the plants belonging to the same species (Barton and Koricheva, 2010). The PSMs produced from biosynthetic pathways is transported to be accumulated at the final destination, are affected by biochemical and cellular factors. The developmental factors also play a vital role in initiation and differentiation of particular cellular structures for the production and accumulation of PSMs (Broun et al., 2006). Their contents are also influenced by many other abiotic factors, e.g., temperature, drought, salinity, altitude, light, UV radiation, wounding and nutrient deficiencies etc. (Gouvea et al., 2012). In general way, it has been observed that secondary metabolite does not achieve stability like qualitative and quantitative traits. Therefore, the influencing factors responsible for variation in PSMs could be comprised into four main categories, (i) genetic, (ii) ontogenic (iii) morphogenetic and (iv) environmental factors: these factors are essential for production and accumulation of PSMs.

1.9.1 Genetic factors So far, it has been acquired very less knowledge on the biosynthetic pathways of PSMs by researchers and is required to garner more information about them by consequent researches; although, some genetic studies explained that array of genes have a regulatory role in the production of secondary metabolites in plants. Nearly thousand genes have been identified in plant genomes in which 15–25% genes is involved in pathways of the secondary metabolism, which leads to the production of PSMs. These genes are controlled by various transcription factors that affect the metabolic flux by influencing the gene expression involving in metabolic pathways (Broun et al., 2006). It is well known that a certain plant species encodes only a specific enzymes having a significant role in production of all the secondary metabolites in the plant kingdom (Pichersky and Gang, 2000). Several products are synthesized from various substrates in secondary metabolism by the specific set of enzymes, indicating a single enzyme catalyze the multiple products from different substrates. Nonetheless, in course of evolution, enzymatic reactions depending on availability of substrate concentration were perverted and that generated the altered enzymes. The altered enzymes lead to the changes in few amino acids that alter the expression of related genes of a particular plant species. Although, enzymes are mainly substrate specific which form a single product from a substrate that is responsible for synthesis of specific secondary metabolites in plant. An enzyme could alter itself by exchanging of one or fewer amino acids after exposure of new substrate which is similar to the old substrate. If altered enzyme forms useful product for the plant then such genetic changes are accepted which support to enhance its synthesis resulting to changes in the expression of responsible gene (Pichersky and Gang,

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2000). Many studies reported that some particular PSMs are only found in one plant species indicating the only specific species have capability to produce these PSMs. This happened in the process of changing which gave rise new enzymes for synthesizing a specific secondary metabolite in a species. The expression of five different genes involve in terpenoid biosynthetic pathway like CcHMGR (mevalonate pathway), CcDXS and CcDXR (methylerythritol-4-phosphate pathway) and the two geranyl-geranyl diphosphate synthases CcGGDPS1 and CcGGDPS2 that code for the enzymes played a significant role in both biosynthetic pathways in distinct tissues of Cistus creticus under abiotic stress and defense signals. The expression profiles of all genes vary in different tissues. The alleviated level of these genes attribute to the increasing of accumulation of isoprenoids in C. creticus (Pateraki and Kanellis, 2010). The study clearly indicates that genes and enzymes both involve intensively in the biosynthetic pathways of PSMs. The transcription factor NaMYC2 perform a vital role in the controlling of biosynthetic pathways in defense response of Nicotiana attenuate (Woldemariam et al., 2013). In addition, various transcription factors are also participated in regulation of defense mechanism in plants and categorized into different families (ERF, bZIP, MYB, bHLH and WRKY), in which only MYC2 transcription factor belongs to bHLH (basic Helix-Loop-Helix) family. MYC2 involves in the regulation of jasmonic acid dependent pathway of defense mechanism and also in other processes, e.g., drought tolerance, light signaling, root growth and circadian clock etc. in plants. In response to herbivore attack, plants generate a regulatory mechanism by inducing several transcription factors that are involved in plant defense; likewise MYC2 transcription factor is also induced. These evidences confirmed that the biosynthesis of PSMs are completely affected by various regulatory genes, enzymes, transcription factors and stresses caused from pathogens leads to unsatbility in accumulation or synthesis of distinct PSMs. The content of these PSMs varies according to their necessity for the plants as defense factors to survive them in adverse conditions.

1.9.2 Ontogenic factors Ontogeny is the entire arrays of process playing significant role in the generation of an organism. It commences from seeds and goes through several developmental events such as seedling, vegetative juvenile to maturing stage which lasts to the senescence stage. It has some particular properties related with every stage like seedling stage relies on preserved seed and seed reserves exhausts at juvenile stage and maturation stage (flowering and fruiting) come under reproduction. Distinguished ontogenic stages might influence the concentration of several primary and secondary plant products, e.g., alkaloids (Elger et al., 2009), phenolics (Donaldson et al., 2006), terpenoids (Langenheim et al., 1986), cyanogenic glycosides (Goodger et al., 2006) and defensive proteins and enzymes in response to defense. Different content level of morphine (alkaloid) have been observed at each developmental stages of Papaver somniferum (Shukla and Singh, 2001). In same way, the PSMs content like; essential oil, phenols, and saponins and their antioxidant activity have variable concentrations at every stages of ontogenic events appeared in Astragalus

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compactus. The composition of these components is changed at flowering stage. Phytol is the major compound at flowering stage; however docosanol, hydrocarbons and sterols are also present in fewer amounts. The large concentration of several hydrocarbons and sterols are found at fructification stage whereas phytol and alcohols are not observed at this stage, but total phenolic content is observed maximum at the fructification stage. The antioxidant activity with maximum IC50 value has been determined at fructification stage and minimum in flowering and vegetative stages. TPC and antioxidant activity has established a positive correlation at fructification stage (Naghiloo et al., 2012). The growth and development show a positive effect on the quality and quantity of essential oil synthesis in Myrtus communis (Rowshan and Najafian, 2012). Apart from this essential oil, phenolic content also changes at various ontogenetic events of Hypericum triquetrifolium (C ¸ irak et al., 2013). These reports on different ontogenic events in terms of the production or accumulation of PSMs have suggested that the various developmental stages of the plant life cycle influence the concentration of secondary plant metabolites. Secondary plant metabolites do not remain a same concentration throughout of the complete plant life, rather they are changed in different environmental conditions according to their need.

1.9.3 Morphogenetic factors Some tissues of plants have specific role like secretion, storage, mechanical support etc. The maximum natural compounds are originated from the secretory tissues of vascular plants. The occurrence of secretory tissues differs in structure, location and in the material secreted. These tissues are catagorised on the basis of the material synthesized by them such as: laticifers, nectaries, ducts, salt glands, hydathodes, mucilage secreting cells, enzyme secreting cells, oil cells, oil secreting trichomes, flavonoid secreting tissues etc. (FAHN, 1988). These different tissues also create a differences in their metabolic pathways (Dey et al., 1988). Array of genes communicates with each other to perform various activity, e.g., cell division, differentiation etc. and the expression of such genes are differentiated into the cell type, site and organ specific (Goldberg et al., 1989). The biosynthetic gene clusters in some plants are essential for the production of the secondary metabolites € tzmann and Osbourn, 2014). PSMs biosynthesis like alkaloids, terpenes and glycosides (Nu is generally limited to a particular tissue (Pichersky and Gang, 2000), hence the synthesis and accumulation of opium poppy latex in the laticifers is a one of the best example of such view (Roberts et al., 1983; Bird et al., 2003). The alkaloids morphine, codeine, thebaine and narcotine are highly accumulated in completely organized capsules and present as maximum amount in all sizes of capsules and least quantity in roots while lancing stage suggest that the capsules having maximum laticiferous cells on their wall are capable to accumulate higher amount of alkaloids in comparison to remaining parts of plant. The maximum accumulation of alkaloids is accomplished in reproductive organs in comparison to vegetative parts of the plant. However, all plant cells have the genes for morphine generation in opium poppy but expressions of these enzymes are regulated by only some cells (Kutchan et al., 1983).

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1.9.4 Environmental factors The interaction of plants with the environment for their existence is affected by various environmental factors including both biotic and abiotic stimuli, which control the synthesis of PSMs in plants (Zhi-Lin et al., 2007). Plants belonging to the same species growing in different environmental conditions have differences in the content of a certain secondary metabolite (Radusˇiene_ et al., 2013). Abiotic and biotic factors develop abiotic and biotic stresses respectively during adverse conditions and in response to such stresses, plants synthesis the particular secondary metabolites to tackle these stresses. Hence, the environmental factors are the one of the major factors playing a significant role in the biosynthesis of PSMs. Biotic stress in plants develops from damaging site caused by other living organisms like bacteria, virus, fungi, parasites, etc. and abiotic stresses are accomplished by light intensity, water availability, temperature variation, type and composition of soil etc. that affect the quality and productivity of plants (Radusˇiene_ et al., 2013). Stresses developed as a result of radiation (light, UV) and chemicals like minerals, gaseous toxins, e.g., oxygen and ozone, pollutants, pesticides, metals (Ni, Cd, Co, Cr, Fe, Zn, Mn, etc.), growth regulators (2,4-D, IAA and NAA) and salts also cause to abiotic stresses in plants (Ramakrishna and Ravishankar, 2011).

1.10 Future prospective Evolution of diverse molecules in plants is resulted from various events like mutations, gene duplication, selection pressure, ecological factors, defensive mechanism etc. The chemical diversity of natural products is results of various metabolic changes during continuous evolution which is novel relevance in drug discovery. The understanding of secondary metabolites developments during evolutionary process has emphasized on their significant roles as mediators of protein-protein interactions in cellular activities and shows a progressive function in synthetic chemistry have revolutionized the methods of material supply and the alteration in biological activity through structural modifications. Despite of frequent metabolism, the accumulating evidence also suggests that pathways and enzymes of primary metabolism of plants are diversified while evolutionary process. These critical changes in primary metabolism attributed to major evolutionary changes in the plant system like evolution of downstream specialized metabolic pathways resulting to chemical diversity. However, it is a matter of study how prevalent phenomenon is beyond the pathways and plant lineages which have been investigated and what influences of such primary metabolic diversification was occurred on metabolism, physiology, and environmental adaptation of different plant species. One of the other interesting questions is how seemingly maladaptive changes in highly conserved and constrained primary metabolism are managed in specific plant lineages; especially the origination of a new downstream pathway that eventually provide adaptive advantage. What are the environmental, anatomical, and genetic factors influencing to primary metabolic pathways for its diversification toward generation of new molecules? Understanding the properties

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of secondary metabolites on the basis of evolutionary origin could anticipate for the any new development or diversification of metabolic pathways. Therefore, the acquired knowledge of metabolic diversification during continuous evolutionary changes on the basis of genetic and biochemical will assist to researchers in redesigning of plant metabolism in a comprehensive manner from primary to secondary metabolism for discovery of anticancer molecules.

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2 Impact of ploidy changes on secondary metabolites productions in plants Sanjay Kumar DEPARTMENT OF BOTANY, BANARAS HINDU UNIVERSITY, VARANASI, INDIA

2.1 An introduction to ploids (or polyploids) The phenomenon of change in chromosome number (either increase or decrease in number) could be defined as the heteroploidy (hetero: different; ploidy: set of chromosomes). It is obvious from the term “heteroploidy” that different types of chromosomes are involved to form a set of chromosomes, and hence ploids. But still there is confusion between the “set of chromosome” and “chromosome complement” for most of us to understand the ploidy level. Therefore, let us clarify these two terms before moving into the ploidy level. A “set” is a well-defined collection of distinct objects, here collection of chromosomes. The most basic properties are that a set (collection of chromosomes) can have elements (here genes on chromosomes), and two sets are equal (means, one and same), if and only if every element (genes on the chromosomes) of each set is an element of other. This property is called the extensionality or extensional equality of sets. Therefore, “set of chromosome” could be correlated with the haploid number (n) of a diploid species or basic chromosome number (x) of polyploid species. Basic chromosome number also refers to the haploid set of chromosomes of a ploid species. So it indicates the haploid number of chromosomes possessed by any species. For instance, haploid contains one set of chromosome, diploid two sets of chromosomes, triploid three sets of chromosomes, hexaploid six sets of chromosomes and so on. On the other hand, “chromosome complement” refers to the “whole set of chromosome” or “complete set of chromosomes” for a species which could be related with the total number of chromosomes possessed by any species. It is hoped that differences are cleared between both the terms and can move into the ploidy level to understand better. The change in chromosome number of an organism could be classified in to euploids (change in set of a chromosome) and aneuploids (change in one or two chromosomes of a set). The change in chromosome set or euploids further classified into monoploids (single set of chromosomes) or haploids (haploid set of chromosomes of a diploid species),

Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00002-3 © 2021 Elsevier Inc. All rights reserved.

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diploids (two complete set of chromosomes) and ploids (more than two sets of chromosomes). Ploids may be of different kinds depending on the number of sets of chromosomes possessed by an organism (e.g., triploid, tetraploid, hexaploid, etc.). The formation of ploids might be deduced from the disjunction (pulling apart) of a complete set of chromosome during gamete formation or duplication of a set within the same species or in different species through hybridization. The formation of ploidy within the same species is called autoploids or autopolyploids, while the formation of ploidy between two different species through hybridization is known as alloploids or allopolyploids. Autopolyploids are homozygous at each locus in the genome; on the other hand, allopolyploids may have varying degree of heterozygosity depending on the divergence of the parental genomes. According to the Winge (1917), a new species (hybrid) may be originated after interspecific hybridization between two unrelated species (F1 hybrid, likely to be sterile because of two unrelated genomes) and doubling the chromosome number of F1 hybrid through a specific chemical (e.g., colchicine) lead to fertile F1 hybrid. Some other ploids are autoalloploids or autoallopolyploids (e.g., decaploid triticale, AABBDDRRRR; ABD ¼ allo; RR ¼ auto) and segmental alloploids or segmental allopolyploids (some chromosomes of one genome have partial homology with chromosomes of other genome). Bivalents and univalents are frequent in segmental alloploids because of partial homology and the pairing of such types of chromosomes is called heterogenetic pairing or allosyndetic pairing. Disomic and tetrasomic inheritance of a ploid organism indicates towards the segmental alloploid. In disomic inheritance, one or two chromosomes become doubled or become twice (two copies of one chromosome or same chromosome), without doubling the set of chromosome and therefore, difficult to compare chromosomes. In tetrasomic inheritance, four homologous chromosomes are found instead of two, i.e., four copies of one chromosome or same chromosome, e.g., autotetraploids (2n ¼ 4  ¼ 48). Autoallopolyploids (Kostoff, 1939) are the allopolyploid organism which shows characteristics of autopolyploid for one or more genomes in that organism. For example, two genomes A and B may be involved in the formation of autoalloploids as AAAABB (A ¼ auto; AB ¼ allo), AABBBB (AB ¼ allo; B ¼ auto), AAAABBBB (A ¼ auto; B ¼ auto; AB ¼ allo). The level of autoalloploids may occur from the hexaploidy and above. Moreover, the level of euploid organisms (either 6 , 7 , 8 , 9 , or 10 ) may be determined by the tolerance power of the nucleus, mitotic and meiotic behavior of chromosomes, gene expression and gene interaction. On the other hand, change in one or two chromosomes from a chromosome set lead to the formation of aneuploids which may be hypoploids (one or two chromosomes less from the chromosome set) or hyperploids (one or two chromosomes extra than the chromosome set). Hypoploids, further classified into nullisomics (lack of two chromosomes from the chromosome set) and monosomics (lack of one chromosome from the chromosome set), while hyperploids into trisomics (addition of one extra chromosome to the chromosome set), tetrasomics (addition of two extra chromosomes to the chromosome set) and pentasomics (addition of three extra chromosomes to the chromosome set).

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2.2 Morphological effects, meiotic and breeding behavior Autopolyploids are homozygous at each locus in the genome (chromosome complement) and have important role in the evolution of vascular plants through autopolyploidization. Approximately, one third of almost all vascular plants and 70% of almost all grasses are polyploids (Ramsey and Schemske, 1998; Wood et al., 2009). Moreover, the first polyploid recognized in the nature was Oenothera lamarkiana (2n ¼ 4  ¼ 28), a tetraploid, which was initially screened as “gigas mutant” by Hugo de Vries (1901). The most universal effect of the autopolyploid is increase in nuclear size and cell size. This is also a fact that the size of the organs and the organism depends on the cell elongation and the number of the cells, but the increase in original cell size may not always lead to the increase in the size of the organ and the organism. This may be because of the reason that the number of cell division is reduced in the autopolyploid (Doyle and Sherman-Broyles, 2017). The most observed and the very common effects of the autopolyploid are the thicker texture of leaves and the petals, shorter and broader leaves, reduced branching, slow growth rate, late flowering, reduction in pollen fertility and seed set (Barker et al., 2016). Meiotic behavior of autopolyploid leads to the formation of the multivalent, bivalent, anaphase dysjunct, unequal distribution of the chromosomes, production of laggards at anaphase I and II, production of micronuclei at diad and tetrad, which results in pollen sterility and reduced seed set (Iqbal et al., 2018). The breeding behavior of autopolyploids leads to the formation of aneuploids (e.g., trisomics, most common, and monosomics or nullisomics) in the progenies of autopolyploids (Sander et al., 2019). Induced autopolyploids are those polyploids which could be induced artificially after the application of suitable chemicals (e.g., colchine) on seed, seedlings, shoot and leaf apex, buds, callus, cambium or meristematic regions of a plant. The effect of colchicine was introduced on mitosis of mice first time, but later on its successful application in inducing polyploidy in flowering plants was reported by Blakeslee (1937).

2.3 Role of ploids (auto, allo and induced) in secondary metabolites production It is well established that nucleus contains two complete sets of chromosomes, but there are some exceptions also, where some organisms contains more than two sets of chromosomes and generally known as polyploid organisms. Triploid (3 sets of chromosomes), tetraploid (4 sets of chromosomes), pentaploid (five sets of chromosomes) are few examples of the polyploidy. A very important role of polyploidy has been deduced in the speciation (formation of a new species), biodiversity dynamics (species diversity formation and effects of human action on it) and adaptability (adjustment of the species in new environment and climatic conditions) of several angiospermic plants such as alfalfa, potato, cassava, banana, apple, and sugar beet (Ainouche et al., 2009; Soltis et al., 2009).

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Polyploids, most probably, associated with the new phenotypes which were not present in their diploid ancestors from where they differentiated into a new polyploidy species and adapted to a new habitat for their normal development (Ainouche and Jenczewski, 2010; Fawcett et al., 2009; Ramsey and Schemske, 2002). Polyploids may differ from the diploids phenotypically through an increase or decrease in leaf, flower (late or longer flowering), fruit, biomass, organ, cell size, gene activity, enzyme variations and high tolerance towards environmental stresses (Stanys et al., 2006; Sugiyama et al., 2005; Glowacka, 2011; Lavania, 2005; Kondorosi et al., 2000; Otto and Whitton, 2000; Saleh et al., 2008; Brochmann et al., 2004). It has been reported that polyploidy may also affect the secondary metabolites in terms of quantity and diversity of the metabolites as well as their chemical structure. One of the possible reasons for the metabolic shift or conformations in the metabolic activity of a polyploid to produce variations in the secondary metabolites might be raised because of the adjustment of the different types of the genomes into the new climatic conditions for their survival and normal function as genome of the polyploid is a combination or mixture of different progenitors, which undergone different structural and functional modifications during the polyploidization, involved in the formation of that polyploid species during course of evolution. It is possible that polyploidization phenomenon may cause deletion, addition, inversion or translocation of genome or part of genome and may be assumed that structural or functional modifications in the genome may induce appearance or disappearance of new functions and phenotypes in the polyploid species. Polyploidization is fairly common in various flowering plant species (e.g., alfalfa, potato, wheat, cassava, banana, apple, sugar beet) with increased level of genetic diversity in the flora and fauna (Comai, 2005; Flagel and Wendel, 2009; Aversano et al., 2012). Usually, the phenotypic characters of polyploid differ from the diploid such as having larger flowers or leaves (Dermen, 1940; Rego et al., 2011; Trojak-Goluch and Skomra, 2013; Sattler et al., 2016; Talebi et al., 2017). It is known that polyploids have more than two sets of homologous chromosomes. The increase in the chromosome number or chromosome set of the species might be related to the phenomenon of genome duplication. The phenomenon of genome duplication is of two type autopolyploid (multiplication of a diploid genome or chromosome complement of intra species) and allopolyploid (consequence of hybridization followed by doubling of the two haploid genomes or chromosome complement of inter species) (Comai, 2005). Although, polyploidy status of the species could be obtained through slightly different phenomenon, but both type of polyploids get benefitted from the genome buffering effect of doubling their genetic information. The buffering effect, epigenetic changes, gain or loss of DNA sequences have negligible detrimental effect on the species viability but, on the other hand, offer for increase in genetic variation and cause the evolution of the new genome. Therefore, polyploidy may be a mechanism of adaptation and played an important role in the plant speciation (Soltis and Soltis, 2000; Ramsey and Schemske, 2002; Bennett, 2004; Jackson and Chen, 2010; Chen, 2010). Previously, there

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were some reports on the study of mechanism of formation of polyploids and their consequences for short as well as long term (Leitch and Bennett, 1997; Ramsey and Schemske, 1998). The high amount of metabolites accumulation in autopolyploids might be the result of the long term or longer period consequences after polyploidization of natural selection (Tsukaya, 2013). Also, the production of secondary metabolites, flavonoids or terpenoids, per gram of tissue was recorded higher (which may be the result of short term consequences after polyploidization in natural selection) in polyploids of Chamomilla recutita, Petunia mitchell, Salvia miltiorrhiza, Artemisia annua, and Panax ginseng, than their diploid progenitor (Gao et al., 1996; Griesbach and Kamo, 1996; Svehlikova and Repcak, 2000; Jesus-Gonzalea and Weathers, 2003; Kim et al., 2004). It is supposed that polyploidy could be involved in producing new features or traits in the appearance or phenotype of an organism which are not present in diploid progenitors, sometimes contributing traits surpassed the phenotype in polyploid organism (Ainouche et al., 2009; Fawcett et al., 2009; Ramsey and Schemske, 2002). Polyploidization may prove a valuable tool in the genetic improvement of crop. Autopolyploids may arise from the single species with high possibility of homologous chromosomes and consequently, autopolyploids are morphologically similar to their diploid progenitors and also, difficult to identify any visible or phenotypic alteration (Soltis et al., 2004, 2007; Parisod et al., 2010; Havananda et al., 2011). It has been established that Autopolyploids have the capacity and being utilized to improve crops, fruits, vegetables and medicinal plants (Levan, 1939; Smith, 1939; Pan et al., 2008). One of the most important feature of autopolyploids for the purpose are autopolyploidization, which have universal phenotypic effect and is believed to be associated with larger stature, thicker texture, prolonged growth, and higher concentration of the metabolites. The higher concentration of metabolites might provide more tolerance to autopolyploids and protects them from biotic and abiotic stresses (Muntzing, 1936; Dhawan and Lavania, 1996; Madlung, 2013). The universal phenotypic effects of autopolyploids had been thoroughly examined and verified in many plant species including animals and fungi and also, found to be connected with autopolyploidy, which contributed to the larger cell size in the various plant species, animals, fungi and bacteria (Fankhauser, 1952; Araki, 2001; Storchova´ et al., 2006; Tsukaya, 2013). In fact, the effects of polyploids could be studied correctly only in autopolyploids, which may be obtained from the chromosomes doubling and differ from donor (diploid) plant only in the genome dosages than in hybridization progenies. It is also reported that chromosome duplications cause metabolic alterations and it has been studied in very few species targeting the specific secondary metabolites such as alkaloids and flavonoids, but lacking in the complete metabolic changes in the species which is associated with the autopolyploidization. Autopolyploidization has significant role in the plant evolution and agriculture along with the cytological, morphological and metabolic changes. It may also have positive correlation between DNA content, morphological traits (such as stomata, pollen, fruit size) and secondary metabolites.

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It is well known that plants produced a variety of natural compounds and large number of methods has been suggested to enhance their production. The various methods for the increase in secondary metabolic compounds may be expensive and not easily carried out in the experiments, but the chromosome doubling is a method which in very effective and inexpensive and also easy to carry out. For instance, Cannabis sativa L. (2n ¼ 2  ¼ 20) cultivated from the ancient times for the fiber, food, and secondary metabolites with therapeutic and recreational properties (Van Bakel et al., 2011). The doubling of chromosome through autopolyploidization could be helpful in the improvement of the cannabinoid compound (a secondary metabolite), but many studies suggested mixed response (Clarke, 1981; Van Bakel et al., 2011; Bagheri and Mansouri, 2015; Mansouri and Bagheri, 2017 Dermen, 1940; Petersen et al., 2003; Talebi et al., 2017; Lata et al., 2009; Lata et al., 2016). Moreover, the mechanism of duplication of a genome may acquire new genes or additional genes in a species which may contribute for the genetic and novel evolutionary traits in the organisms (Ohno, 1970; Grant, 1971). Allopolyploids are the combinations of different genomes. It causes the little modifications in the different genomes during genome organization and produced an allopolyploid. The modifications in the genomes during the genome organization may affect the gene expression of the parental genomes as a result could assumed to be an additive heterosis effects. It has been observed that the increase in chromosome number, additional genomic interactions, and genetic alterations often results into a superior properties in ploid plants as compared to the diploid species, but the mechanism behind this how it occurs yet not fully understood. But whatsoever, polyploidization is a convincing approach for crop improvement. Recently, natural as well as newly formed or hybridized allopolyploids (Arabidopsis, Brassica, Glycine, Gossypium, Tragopogon, and Triticum) provided an important relationship and genomic or genetic consequences of allopolyploidization which contains two different types of genomes and possibility of homeologous chromosomes are high during hybridization and polyploidization and therefore consequent into morphological, physiological and genomic modifications (Doyle et al., 2008, 2004; Soltis and Soltis, 1993; Adams and Wendel, 2004; Chen et al., 2004; Levy and Feldman, 2004; Lukens et al., 2004; Tate et al., 2006; Chapman and Burke, 2007; Dong and Adams, 2011). It has been accounted that autopolyploids have larger cell and organ size which was long established, similarly, tetraploids also have larger cells and organs size than diploids suggested by various studies (Tsukaya, 2013). But it is also reported that polyploids at a higher level than hexaploids results in smaller stature in organs with larger cells due to “high-ploidy syndrome” or less number of cell division with abnormalities (Tsukaya, 2008). The effect of polyploidization has been observed on the leaf metabolic profile and induced tetraploids (2n ¼ 4  ¼ 48) from wild diploid potato species, produced lesser quantity of secondary metabolites such as phenylpropanoids, tryptophan, tyrosine and α-chaconine as compared to the wild diploid potato species, Solanum bulbocastanum (2n ¼ 2  ¼ 24). The production of fewer amounts of secondary metabolites could be

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related to the time required for adjustment, adaptation and evolution of the new combined genomes as a ploidy (tetraploid) after the genomic shock induced by polyploidization. On the other hand, the tetraploids of Scutellaria baicalensis, Artemisia annua, Salvia miltiorrhiza, Papaver somniferum, Datura stramonium, Thymus persicus, Echinacea purpurea, and Tanacetum parthenium, produced higher quantity of secondary metabolites (alkaloids) per gram of tissue than their diploid parents (Gao et al., 2002, 1996; Banyai et al., 2010; Wallaart et al., 2001; Mishra et al., 2010; Berkov and Philipov, 2002; Tavan et al., 2015; Abdoli et al., 2013; Majdi et al., 2010). Also, it could be a fact that an optimum ploidy level for each species may be vital (Caruso et al., 2013). Ploidy may be induced artificially to increase the quantity, production and to improve the quality of the important medicinal compounds used in various pharmaceuticals and aroma industries (Dhawan and Lavania, 1996). But sometimes artificial induction of polyploidy may lead to alterations in the profile of the secondary metabolites and results in less quantity and concentration of the metabolites. Artificially induced autopolyploidization could be obtained in both in vivo and in vitro by the application of the anti-mitotic inhibition chemicals (Dhooghe et al., 2011). The chemicals applied may mimic the natural method to bring the synthetic autopolyploids with improved traits such as larger fruit and flower size, self-compatibility, improved stress tolerance, increased biomass, large quantity of some secondary metabolites and others (Dhooghe et al., 2011). The production and response of artificial polyploidization significantly affects the morphology and physiology of newly formed autopolyploids (Ceccarelli et al., 1992; Stanys et al., 2006; Gaikwad et al., 2009). Artificially induced autotetraploids showed no difference from its diploid donor morphologically, anatomically and modifications through methylation pathways, but instead a random distribution was reported (Aversano et al., 2013). The induced polyploids of Artemisia annua, Chamomilla recutita, Salvia miltiorrhiza, and Scutellaria baicalensis produced high quantity of flavonoids or terpenoids per gram of tissue than their diploid parteners ( Jesus-Gonzalea and Weathers, 2003; Svehlikova and Repcak, 2000; Gao et al., 1996, 2002). Artificial induction of the polyploidy in the ginseng demonstrated the higher amount of ginsenoside (secondary metabolite) in the roots of the plant (Kim et al., 2004). Similarly, the induction of polyploidy in Papaver somniferum proved to be high amount of metabolite production, i.e., morphine (Mishra et al., 2010). But sometimes artificial induction of polyploidy may lead to alterations in the profile of the secondary metabolites and may not always provide high amount of secondary metabolites production, it may give low amount of production, e.g., essential oils from tetraploids of Mentha spicata accounted less amount and concentration per unit dry weight than in the diploid genotypes (Dhawan and Lavania, 1996; Lavania, 2005). Tetraploids (2n ¼ 4  ¼ 48) of wild diploid Solanum commersonii (2n ¼ 2  ¼ 24) produced increased amount of alkaloids phenylpropanoid (PP) and glycoalkaloid (GA) as compared to its diploid progenitor (Birchler and Veitia, 2010). As it is known that polyploidy causes the modifications in the genome of tetraploids which lead to a stronger defense system through increased amount of alkaloids (Caruso et al., 2011).

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Artificially induced tetraploids (2n ¼ 4 ¼ 44) of Hylocereus monacanthus (2n ¼ 2 ¼ 22) and hexaploid (2n ¼ 6 ¼ 66) of Rose (S-75) (2n ¼ 3 ¼ 33) were highly tolerant to extreme drought and may be utilized as a fruit crop in dry land agriculture (Mizrahi and Nerd, 1999; Tel-Zur et al., 2011). But tetraploids and hexaploids of Hylocereus monacanthus and Rose (S-75) indicated multi-level negative alterations such as alterations in meiotic behavior, morphological traits of fruits, seeds and embryos, which may be the result of modifications in metabolic pathways and production of increased amount of alkaloids and closely interrelated (Cohen et al., 2013). The change in chromosome number is a method which can be used to increase the potential of a plant to produce large amount of secondary metabolites through doubling or multiplying the base chromosome number. The method could be obtained through conventional plant breeding among the plants for polyploidy production. It has been successfully implemented in plant breeding program to increase the yield and biomass of several crop species such as potato, red clover, sugar beet and watermelon (Bamakhramah et al., 1984; Cai et al., 2007; Renny-Byfield and Wendel, 2014; Sattler et al., 2016). Moreover, a direct correlation has been reported for the production of secondary metabolites between change in chromosome number and production of large quantities of secondary metabolites (Lavania et al., 2005). There were many reports suggested that increased somatic ploidy level may influence the biomass composition in such a way that could be beneficial for the production of bioenergy and materials. Polyploidy has shown its importance to increase in plant biomass for feed and food production and now interest has been increasing towards the use of these polyploid plants as source of energy and chemical building blocks or production of chemical compounds in large quantities. Therefore, the yield as well as biomass composition of the crops are most important. Plant biomass could be compared with the composition of the cell walls, which mostly contains the high amounts of the polysaccharides and could be depolymerized enzymatically into the monosaccharides (Chen and Dixon, 2007; Vanholme et al., 2012; Van Acker et al., 2013). Polyploids of Salix species (triploids and tetraploids) reduced their lignin content as compared to diploid parents (Morrison, 1980; Serapiglia et al., 2015). Polyploidy has been reported to increase in organ size and improvement of biomass production along with the cell wall composition in several plant species. The combined effect of an altered cell wall composition with an improved biomass production makes induction of polyploidy a promising tool to improve plants for the bio-based economy (Tsukaya, 2008; del Pozo and Ramirez-Parra, 2015; Tavan et al., 2015; Vergara et al., 2016). The desirable traits of the polyploidy for plant breeding may include the buffering of deleterious mutations, increased heterozygosity, and hybrid vigor (Sattler et al., 2016). Secondary metabolites produced in the plants through a complex mechanism for their defense and protection against herbivores and microorganisms, resistance to various abiotic stresses (such as drought, salinity, UV beams), adaptation, survival, to attract animals and other carriers for pollination and seed distribution and continuation of the next

Chapter 2 • Impact of ploidy changes

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generations. The economic value of the secondary metabolites includes as pharmaceutical ingredients, food supplements and in perfumes and agricultural control products. Plants are used as pharmaceutical active ingredients because of the various types of bioactive compounds for most of the world population. Approximately 80% of the developing countries populations obtain their health requirement through the medicinal products and approximately 64% of the world population use medicinal plants for the therapeutic purposes. Also, in developed countries 25% of the prescribed drugs are plant based formulations. Moreover the exploitation and use of medicinal plants for ailments by local healers or folk remedy played an important role for the discoveries of the plant based drugs (Babaoglu et al., 2000). The cultivated potato (Solanum tuberosum) well known for its high nutritional value, and antioxidant activity such as carotenoids, vitamin C, and phenolics and most probably, the wild potato represent an underestimated source of phytocompounds and also used in breeding program and could be utilized to transfer relevant genes to the cultivated potato gene pool and need to be explored (Hale et al., 2012; Camire et al., 2009; Caruso et al., 2013). At present, bioactive compounds have tremendous potential to use along with some challenges and disadvantages in extraction method of plants under natural conditions as amount and quality of bioactive compounds are affected by the climatic conditions. Moreover, higher concentration of metabolites might provide more tolerance to ploids under stress condition and also, protects them from biotic and abiotic stresses (Muntzing, 1936; Dhawan and Lavania, 1996; Madlung, 2013). A molecular analysis between newly created tetraploid (tetraploidization of A. thaliana), naturally occurring tetraploid (Pyrus communis var. sativa) and the diploid parent (A. thaliana) suggested very small differences in the amount metabolites between the tetraploids and the diploids which again suggest that metabolite content or concentrations are not the universal and also not in the direct influence of the ploidy-dependent changes. Molecular analysis of ploidy dependent cell size enlargement failed to detect the significant differences in the mRNA profiles between diploids and polyploids, with few exceptions (Stupar et al., 2007). The phylogenetic analysis of the flowering plants indicated that most of the flowering plants have one or more round of ancient polyploidy organisms ( Jiao et al., 2011; Van de Peer et al., 2009). The various kinds of secondary metabolites may be extracted which directly or indirectly dependent on the different kinds of environmental stresses or other factors. Normally, the secondary metabolites produced in the plants are very less in amount, but sufficient to support normal function of a plant. The secondary metabolites of plants could be induced at higher concentration and amount, if there is any cell damage, reaction to external factors and also function as phytopharmaceuticals. There are lists of different bioactive compounds or secondary metabolites which may be used directly in cosmetics, aroma, perfumery industries (as paint and pigment), food additives, insecticides or as an important component in various formulations. A huge application of herbal extracts or secondary metabolites in modern medicine, it has drawn

38

Evolutionary Diversity as a Source for Anticancer Molecules

the attention of various research workers from all round the world to work in line of medicinal plants. The search for herbal based therapeutic molecules intensified in pharmaceutical industries across the world. The herbal therapeutic molecules may be extracted manually or naturally. The molecule could be extracted from the medicinal plant of interest and synthesized chemically. The manual extractions of herbal therapeutic molecules are conventional, nonrenewable, non-environmental friendly, uneconomical and time-consuming. So, this may be one of the reasons to produce herbal therapeutic molecules by the application of biotechnology or by using different techniques for most of the scientific community (Dubey et al., 2017). The main objectives of tissue culture are to develop a new variety, create genetic diversity in the existing varieties, to save endangered species and production of species that are difficult to propagate (Babaoglu et al., 2000). Similarly, secondary metabolites could be produced through the tissue culture method. It may be used to obtain new tissues, cells or organs (e.g., apical meristem, meristematic cells, cell concentration or callus cells or root), whole plant or plant products (e.g., secondary metabolites) under aseptic conditions (in an environment free from any kind of microorganisms) in an in vitro medium € lgec¸en, 2013). (Atar and C ¸o There are various ways through which bioactive compounds could be produced: Root culture method may be applied to produce the secondary metabolites in the root system of medicinal plant of interest. The part of induced metabolites in the root system could be excised and cultured into the appropriate culture medium for the production of the metabolites (Erkoyuncu and Yorgancılar, 2016). Callus culture is a mass of differentiated cells and formed morphological irregularities on surface of injured tissue. The mass of differentiated and irregular morphological tissues still intact with their mitotic capabilities and played an important role in the production of secondary metabolites (Babaoglu et al., 2000; Erkoyuncu and Yorgancılar, 2016). Micro-propagation is a tissue culture technique used to propagate high number of genetically similar plants quickly from the different plant parts (embryo, seed, body, shoot, root, callus, one cell or pollen grain)and have the potential to form a whole plant € under aseptic condition (Ozkaynak and Samancı, 2005). The technique can be used to produce vegetative parts or vegetative propagation of various medicinal and aromatic plants quickly and in large quantities (Erkoyuncu and Yorgancılar, 2016). Cell concentration is a non-differentiated and non-organized culture method for metabolite production. The difference in the culture medium of cell concentration from that of callus culture is that a thickening agent is added to the callus culture medium whereas it is not added in the cell concentration medium and therefore, cell concentration is a liquid medium (Atar € lgec¸en, 2013). and C ¸o Hairy root culture method may be used for secondary metabolite production as compared to other culture systems as it has the characteristics high growth rate and genetic as well as biochemical stability. Hairy roots may also be used as a source for transgenic plant € lgec¸en, 2013). regeneration (Atar and C ¸o

Chapter 2 • Impact of ploidy changes

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Elicits are chemical compounds (secondary metabolites) produced by the plants with a capability to stimulate any kind of plant defense (Angelova et al., 2006; Namdeo, 2007). Elicitors may be induced from the cell and tissue culture method and could be utilized for the enhancement of the secondary metabolites, e.g., ajmalicine. The secondary metabolite (ajmalicine) content of Catharanthus roseus has been increased through elicitors produced by fungi, Aspergillus and Fusarium (Namdeo et al., 2002). Biotransformation could be defined as the conversion of a small part of chemical molecule into large quantities by application of a biological system. It is an important implementation area for the production of secondary metabolites (e.g., digitoxin, digoxin, essential oils, aroma etc.) using plant tissue and cell cultures (Dave et al., 2014; Baydar and Telci, 2015). Explants are the parts of a plant produced through the culture method and could be utilized for the production of the several secondary metabolic compounds with the advantage of predictability and purity of the compounds (Karuppusamy, 2009). Metabolic engineering is another method which can enhance metabolite productions through over expression or gene silencing pathways (Diretto et al., 2006; Dhawan and Lavania, 1996).

2.4 Perspectives The relationship between the higher concentration of metabolites and the ploidy level of plants may be attributed to the effects of autopolyploidization on the metabolic activity of the plants and the amount of metabolites. Moreover, plants are surviving under the natural stress for longer period after polyploidization caused a good relationship between high secondary metabolites and polyploidization. It may be true for the artificially selected horticultural cultivars for better traits. The amount of primary or secondary metabolites of plants in the form of increased or decreased quantity in polyploids is still not clear as compared to the diploids. A few reports suggested that high concentration or high quantity of the secondary metabolites in the plants most probably could be related to the ploidy of that plant or any other organisms (Banyai et al., 2010; Lavania et al., 2012). The characterization of newly generated polyploids is important and essential for the resourceful selection of the plant materials (Carputo and Barone, 2005). Artificially induced polyploids may be helpful to understand the effect of gene dosage, contribution and improvement on secondary metabolite pathways, to overcome sexual barriers, and possibly, to explain the apparent contradictions of mechanism and metabolite pathways explained in the production of the secondary metabolites. Moreover, there is a need to understand the mechanism behind the alterations in the profile of secondary metabolites, which is the one poorly understood in artificially induced polyploids. Generally, the plants which produced wide variety of metabolites, possibility of chemical characterization, utilization for Human health as well as utilization in plant breeding are received good attention by the scientific community. At present, most of the natural

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Evolutionary Diversity as a Source for Anticancer Molecules

compounds are used as a drugs to diagnose infectious and severe diseases such as cancer, stroke and heart related problems. Some plants can adapt very fast in the environment during their course of evolution, these plants may be identified and utilized to develop genetically improved crops for resistance or tolerant to pest and pathogen attacks. A search for new secondary metabolites or natural compounds should be given first priority for the sustainable conservation and appropriate use of plant diversity (Mazid et al., 2011). A piled information on the secondary metabolites induced from the natural sources recorded in low quantity. Therefore, now it is time to stimulate the research towards the possibility to increase the content of natural compounds in various plant species of interest through polyploidization or by using plant bio-techniques.

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Soltis, P.M., Soltis, D.E., 2000. The role of genetic and genomic attributes in the success of polyploids. Proc. Natl. Acad. Sci. 57, 7051–7057. Soltis, D.E., Soltis, P.S., Pires, J.C., Kovarik, A., Tate, J.A., Mavrodiev, E., 2004. Recent and recurrent polyploidy in Tragopogon (Asteraceae). Cytogenetic, genomic, and genetic comparisons. Biol. J. Linn. Soc. 82, 485–501. Soltis, D.E., Soltis, P.S., Schemske, D.W., Hancock, J.F., Thompson, J.N., Husband, B.C., Judd, W.S., 2007. Autopolyploidy in angiosperms: have we grossly underestimated the number of species. Taxon 56, 13–30. Soltis, D.E., Albert, V.A., Leebens-Mack, J., Bell, C.D., Paterson, A.H., Zheng, C., Sankoff, D., Depamphilis, C.W., Wall, P.K., Soltis, P.S., 2009. Polyploidy and angiosperm diversification. Am. J. Bot. 96 (1), 336–348. Stanys, V., Weckman, A., Staniene, G., Duchovskis, P., 2006. In vitro induction of polyploidy in Japanese quince (Chaenomeles japonica). Plant Cell Tiss. Org. Cult. 84 (3), 263–268. Storchova´, Z., Breneman, A., Cande, J., Dunn, J., Burbank, K., O’tooli, E., Pllman, D., 2006. Genome-wide genetic analysis of polyploidy in yeast. Nature 443, 541–547. Stupar, R.M., Bhaskar, P.B., Yandell, B.S., Rensink, W.A., Hart, A.L., Ouyang, S., Veilleux, R.E., Busse, J.S., Erhardt, R.J., Buell, C.R., Jiang, J., 2007. Phenotypic and transcriptomic changes associated with potato autopolyploidization. Genetics 176, 2055–2067. Sugiyama, T., Cam, H., Verdel, A., Moazed, D., Grewal, G.I., 2005. RNA dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to Si RNA production. Proc. Natl. Acad. Sci. 102 (1), 152–157. Svehlikova, V., Repcak, M., 2000. Variation of Apigenin quantity in diploid and tetraploid Chamomilla recutita (L.) Rauschert. Plant Biol. 2 (4), 403–407. Talebi, S.F., Saharkhiz, M.J., Sharafi, Y., Fard, R.F., 2017. Effect of different antimitotic agents on polyploid induction of anise hyssop (Agastache foeniculum L.). Caryologia 70, 184–193. Tate, J.A., Ni, Z., Scheen, A.-C., Koh, J., Gilbert, C.A., Lefkowitz, D., Chen, Z.J., Soltis, P.S., Soltis, D.E., 2006. Evolution and expression of Homeologous loci in Tragopogon miscellus (Asteraceae), a recent and reciprocally formed allopolyploid. Genetics 173 (3), 1599–1611. Tavan, M., Mirjalili, M.H., Karimzadeh, G., 2015. In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae). Plant Cell Tiss. Org. Cult. 22, 573–583. Tel-Zur, N., Mizrahi, Y., Cisneros, A., Mouyal, J., Schneider, B., Doyl, J.J., 2011. Phenotypic and genomic characterization of vine cactus collection (Cactaceae). Genet. Resour. Crop. Evol. 58, 1075–1085. Trojak-Goluch, A., Skomra, U., 2013. Artificially induced polyploidization in Humulus lupulus L. and its effect on morphological and chemical traits. Breed. Sci. 63, 393–399. Tsukaya, H., 2008. Controlling size in multicellular organs: focus on the leaf. PLoS Biol. 6 (7) e174. Tsukaya, H., 2013. Does ploidy level directly control cell size? Counterevidence from Arabidopsis genetics. PLoS One. 8, e83729. Van Acker, R., Vanholme, R., Storme, V., Mortimer, J.C., Dupree, P., Boerjan, W., 2013. Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana. Biotechnol. Biofuels 6, 46. Van Bakel, H., Stout, J.M., Cote, A.G., Tallon, C.M., Sharpe, A.G., Hughes, T.R., Page, J.E., 2011. The draft genome and transcriptome of Cannabis sativa. Genome Biol. 12 (R), 102. Van de Peer, Y., Maere, S., Meyer, A., 2009. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 10 (10), 725–732.

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3 Effect of climate change on plant secondary metabolism: An ecological perspective Akhileshwar Kumar Srivastavaa, Pragyan Mishrab, and Amit Kumar Mishrac a PCB T DE PARTME NT, CSI R-CFTR I, MYSURU, KARNATAKA, INDIA b DEPARTMENT OF BIOT ECHNOLOGY, MICROTEK COLLEGE OF MANAGEMENT AND TECHNOLOGY, V AR ANASI, UTTAR PRADESH, INDIA c TE XAS A&M AGRILI FE RESE ARCH AND EX TE NSI O N CENTE R, TE XAS A&M UNIVERSITY, UVALDE, T X, UNITED STATES

3.1 Introduction Nowadays, climate change has been a highly remarkable concern for humankind as well as other organisms of the earth. Medicinal plants are very important to human life. WHO has accounted that approximately 60% of the world population and 80% of the population of developing countries depend on traditional medicine, especially plant drugs, for their primary treatment (Kosalge and Fursule, 2009). There are several factors responsible for climate change, however, anthropogenic activities pose a major effect on species and ecological communities at broad-spectrum over the world including medicinal plants. In 2007, the Intergovernmental Panel on climate change (IPCC) explained about climate change is “Unequivocal” (IPCC, 2007). It is expected that the temperatures from 1.4 °C to 5.8 °C would be further escalating by the year 2100, which would be very extreme by 2033 and causing uncertainty in weather conditions such as: (a) more rapidly and excessive monsoon rainfall, (b) highly intermittent and hotter summer days, (c) less frequent and lower dry season rainfall, and (d) stronger and highly frequent storms with high winds. Today it has been realized that the increasing levels of greenhouse gases in the atmosphere (especially increased carbon dioxide followed by methane and ozone) is one of the main reason for climate change. Such changes could influence the secondary metabolites and other components of plants which have mainly therapeutic values for various hazardous diseases. This climatic change is then compelling to biodiversity and ecosystem to rectify the shifting habitat, changing life cycle, and generating the new physical characters. Endemic plant species of certain geographical regions are vulnerable to climate change and might face high challenges in the future (Thomas et al., 2001). The simultaneous interactions of all components of plants have different activities, hence their

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implications could complement or disturb other mechanisms associated with secondary metabolites. As therapeutic regimens, the plant components have shown more potent to cure complex cases including lifestyle diseases. Also, this plays a vital role in reducing the side effect of synthetic chemical remedies. Climate change is significantly affecting both cultivated as well as wild medicinal plants. Therefore, attention toward research considering health significance is required to focus particularly on the accumulation of secondary metabolites in plant systems. Still, very few researches on the medicinal plants with respect to climate change have been accomplished by comparing with other commercial crops. The present time has high demand to work on these group of plants having potential sources of biomolecules and nutraceuticals (Salick et al., 2009). The name of “secondary metabolites” was given by A. Kossel in 1891, who explained that these organic compounds have been accidentally evolved and does not have a primary role in plant life. A vast group of such compounds do not have a direct role in growth, development, and reproduction of plants hence referred to as secondary metabolites. These compounds are distributed in limited taxonomic groups, hence are used as taxonomic markers. Whereas, primary metabolites like carbohydrates, acyl lipids, phytosterols, and organic acids, occurs in all tissues of plants and have an essential metabolic function in the growth and development of plants. Hence, there are no certain parameters to separate these two categories of compounds by using only their chemical structure, precursor molecules and biosynthetic origin, for example, amino acid proline is a primary metabolite whereas its C6 analogous compound, pipecolic acid is an alkaloid. The classes of diterpenes and triterpenes have both primary as well as secondary metabolites (Ahmed et al., 2017). The secondary metabolites attribute to particular odor, color, and taste of plant species. Earlier, these organic compounds were not given more importance by plant biologists because they were believed to be biologically insignificant. Although, in the 1850s its chemical structures and biological activities have been explored profusely by organic chemists. This evidence provided valuable knowledge and changed the thought on previous believes of plant biologists about the importance of secondary metabolites have biologically active and plays a major role in potential defense mechanisms, mainly in the chemical warfare between plants and their pathogens (Grassmann et al., 2002). Some of these secondary metabolites have shown their activities against herbivores and to entice pollinators, allelopathic agents, and protection against toxicity, UV-light shielding, and signal transduction. The current opinions about the secondary metabolite are not sufficient because they have been projected as unimportant compounds instead of having multiple functions in plant life (Vasconsuelo and Boland, 2007). Since the last few decades, scientists have focused on the secondary metabolites with their various potential activities like in human nutrition, cosmetics, drugs, and their fundamental role in plant protection. So, this drastic attention and interest toward the secondary metabolites are not only academic but also because of commercial purposes. Now, climate change has been one of the major issues in the world and it is understood that any variation in climate for a longer time, whether due to natural changes or as a

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result of anthropogenic activity will affect whole vegetation. Such alterations in climate includes global warming, precipitation fluctuations, rising sea levels, and other extreme climatic alterations. It is not well understood how such changes could have an impact on the natural ecosystems, especially on the interactive activity of living organisms. Phytochemical evolution theory explains that the plants develop selective forces which lead to the evolution of defensive mechanisms in plant system in the form of plant secondary metabolites. Such metabolites do not play a significant role in primary plant metabolic processes, e.g., respiration or growth but provide defensive weapons to the plant against herbivore or pathogen attack (Singh et al., 2019). Plant secondary metabolites have a wide range of chemical compounds, which are usually synthesized by the plant species to endure against biotic and abiotic stresses. Historically, the term “secondary metabolites” has been referred to those compounds which are not “primary metabolites,” and are not involved in the growth, development, and reproduction of an organism (Hartmann, 2007). Nonetheless, such a complex classification underrate the significance of secondary metabolites for plants and their interactive role with other organisms, and the larger effects of secondary metabolites could have an impact on ecosystem functioning (Chomel et al., 2016). One of the big challenges for the plant is to develop the resisting capacity against environmental stresses. Plants develop a paramount mechanism for producing their secondary metabolism which allows them to cope with an environmental stress. Likewise, this can be in form of antifeedant components to decrease herbivory (Hopkins et al., 2017), and the production of suitable solutes that allow plants to balance the homeostatic water € m et al., 2000). These environmental issues relations under drought conditions (Holmstro are now being provoked by climate change, and hence, it is required to know the biological effect on secondary metabolism with respect to climate change. It is very difficult to have predictions about major responses of global vegetation to future climatic conditions. Plant secondary compounds also contain high chemical and structural diversity of non-volatile or volatile compounds. Secondary compounds might have been generated with certain physiological and ecological roles for developing the adaptative capability of plants to grow in their environment. Secondary metabolites are synthesized by various metabolic pathways and various secondary compounds appear in particular plant genera or families. In forest ecosystems, completely grown trees contain higher plant biomass and hence these plants have the potential capacity to produce adequate amounts of secondary compounds. The main climate change factors like carbon dioxide (CO2) and warming have paradoxical effects on certain classes of secondary metabolites. The elevated CO2 level stimulates the phenolic components in foliage but overcomes the terpenoids in foliage and emissions. Although, warming conditions decline the phenolic compounds in foliage but enhances the terpenoids in foliage and emissions. Other abiotic stresses produce highly fluctuating effects. Plant secondary compounds aid plants to adapt to climate conditions and to pressure from the presented and invasive pests and pathogens. The indirect acclimatization occurs through the effects of secondary compounds on soil chemistry, and

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nutrient cycling, for example, the generation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into secondary compounds in the wood of living and dead forest trees (Holopainen et al., 2018). Since the immemorial period, plants have been recognized as a rich source of affordable natural compounds, notably the secondary metabolites contain enough structural complexity and have a wide range of biological roles against several diseases including antitumor activity (Nwodo et al., 2015; Seca and Pinto, 2018). Their classification is based on their different synthetic pathways (Delgoda and Murray, 2017). However, a simple classification has mainly three classes (i) terpenoids (polymeric isoprene derivatives and synthesized from acetate through the mevalonic acid pathway), (ii) phenolics (synthesized from shikimate pathways, having one or more hydroxylated aromatic rings), and (iii) the highly diverse group alkaloids (non-protein nitrogen-containing compounds, synthesized from amino acids like tyrosine, with a long back history in medication) (Delgoda and Murray, 2017). Various new cytotoxic secondary metabolites have been characterized from plants every year and generate a source for developing the new regimens to combat cancer. Even, some secondary metabolites with their unique anticancer effects cannot be used in clinical practice due to their physicochemical properties like lower bioavailability and/or their toxic effect. However, the secondary metabolites of the plant often could be great leads for drug development. Hence, the medication in the chemical structure of such potent secondary metabolites is one of the good methods to enhance their anticancer activity and selectivity, increase their absorption, metabolism and excretion properties and lower their toxicity and side effects (Guo, 2017; Yao et al., 2017). A substantial part of the human daily diet is synthesized from the plants or their components, in which nutritional properties are widely studied. Such nutritional components like carbohydrates, lipids, and amino acids are synthesized biosynthetically by photosynthetic green plants that act as primary sources of all food chains on earth that are referred to as primary metabolites. Despite the essential primary metabolites, plants also synthesize approximately million other compounds which are closely associated with plant families and species expressing combinations of similar compounds (Verpoorte, 1998). The selective expression of such compounds has obviously helped plant biologists in the classification of plants into various chemotaxonomic groups. Earlier, the secondary metabolites had been regarded as waste materials, as the progressive evidence has emerged playing a potential role in contributing a certain evolutionary significance to the plants which express them through either directly or indirectly associations with others. Hence the given definition of secondary metabolite has been changed somewhat over the years. The highly accepted definition of secondary metabolites is the naturally produced compounds that do not play a potential role in the internal economy of the plants that synthesize it (Delgoda and Murray, 2017) which makes the direct contradiction to primary metabolites maintaining the fundamental cellular life events. Such secondary metabolites are functionally known to play

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an essential role in the survival of the organisms which synthesizes them through critical interactions with their environment. Plants are continuously influenced by interacting with hasty changing and extensive damage to external environmental factors. Being immobile organisms, plants have developed widely potential defense mechanisms by generating alternative ways that involve a large number of different chemical metabolites as tools to reduce the stress conditions. Hence secondary metabolites participate in the process of plant adaptation to the changing environment. Plants have a greater capacity to generate such metabolites during adverse conditions. Due to biotic and abiotic stresses like temperature, light intensity, herbivory, and microbial attack, plants develop these defense mechanisms and stimulate several complex biochemical pathways (Holopainen and Gershenzon, 2010). Changes in plant systems have been overserved at genetic as well as protein levels which is caused by stress conditions and are reflected as a profound alteration in the metabolite pool of the affected plants (Loreto and Schnitzler, 2010; Ncube et al., 2012). Nonetheless, the biosynthesis of secondary metabolites is strictly controlled that’s why it is usually either confined to particular plant tissues or developmental stages or induced in response to stimulation factors (Ncube et al., 2012). The approach for survival and for the generation of diversity among organisms, plants have the capability to synthesize a specific group of secondary metabolites that are also associated with the selected plant species. To date, the studies of the impact of climate change especially on secondary metabolites of the medicinal plants are severely lacking. Therefore, this chapter provides an overview of climate change effects that can direct synthesizing secondary metabolites in plants with the development of various metabolic pathways.

3.2 Evolutionary theory based on secondary metabolites The primary metabolites are the main precursors for synthesizing secondary metabolites that suggests the first key point of evidence for evolution of secondary metabolites. Few main building blocks of primary metabolites lay the foundation for producing the several known secondary metabolites. These building blocks of primary metabolites are the acetate C2 unit (for the synthesis of polyketides and fatty acids), the phenylalanine/tyrosine derived C9 unit (for the synthesis of phenylpropanoids), the C5 isomeric unit isopentenyl diphosphate and dimethylallyl diphosphate, and some amino acids. The terpenoid and phenylpropanoid mechanisms are more dominant for synthesizing of secondary metabolites in higher plants, whereas the mainly polyketide pathways in microorganisms has highly emerged. Above pathways in plants and microorganisms not only participate in the synthesis of phenylalanine and tryptophan but also involved in secondary metabolism of the shikimate pathway and directly contributes to some secondary metabolite biosynthetic pathways through its end product chorismite (Delgoda and Murray, 2017). Though the presence of limited numbers of building blocks could be seen almost infinite after the combination of these metabolites for the synthesis of novel secondary

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conformations. A large number of distinct secondary metabolites as a breadth of nature’s chemodiversity have already been explored, however, it is required to continue exploration for understanding the chemodiversity of nature. Simply, secondary metabolites could be widely grouped into three fundamental molecular families (i) the phenolics (having products from C2, C5, C91C2 pathways), (ii) terpenes (C10, C15, C20, C30, C40), and (iii) alkaloids (Delgoda and Murray, 2017). The steady evolutionary development of plant-specific metabolic routes is responsible for the synthesis of particular groups of secondary metabolites, e.g., tissue types, organs, and lifestyles, with different taxonomic distributions. Some main metabolic pathways and characters are conserved with the continuous development of new pathways from existing pathways and this explains the second point of evidence for evolution. Such as synthesis of lignin seems to be conserved almost in all vascular plants, is capable to transport of water and nutrients efficiently, along with other different adaptative mechanisms that have developed in gymnosperms and angiosperms after millions of years. The enzymes playing role in the biosynthesis of secondary metabolites have also emerged from the primary enzymes adopting gradually. The group of existing metabolic enzymes could be traced by ancestral proteins which give a shred of evidence that no new enzymes were synthesized but rather the alterations in existing structures by natural selection. Many examples of convergent, parallel, and divergent evolution could be observed in plant metabolic events (Pichersky and Gang, 2000) and these examples have main enzymes like cytochrome P450 (CYP) enzymes that play a major role in the producing of diverse compounds including lignins linked with ancestral primary metabolic enzyme sterol 14-demethylase. Chalcone synthase, an enzyme that participated in the biosynthesis of flavonoid in plants, is another good example that contributes the same protein folds as the fatty acid synthesis enzyme β-ketoacyl ACP synthase III (Weng, 2014). The above findings clearly reflect that the plant enzymes have the capability to accommodate several substrates driving for the increasing of evolutionary diversity and develop the new pathways by facing environmental challenges. Although, gene duplication is the fundamental mechanism for the evolutionary process (Kennedy and Wightman, 2011) which leads to the formation of new copies of genes from the existing genes through multiple processes and cause variation and experimentation with the newly formed genes. About 60 different cannabinoids synthesized in Cannabis sativa, but biological activities have not observed in most of them which support the view that multiple variations of genes can have a slight modification in chemicals with a few numbers of these needed to have a positive outcome, which appears to be nature’s synthetic experimentation method. All genome duplication could also be observed in an angiosperm, Arabidopsis thaliana. It has been understood by studying the evolutionary behaviors and triggers that stimulate the synthesis of secondary metabolites and explains the beneficial purposes in the production of such natural products at a wide scale. Nowadays, the demand for high production of secondary metabolites has been increased due to their multiple uses in

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different industries like pharmaceutical, nutraceutical, cosmetics, and fine chemicals. In spite of field cultivation of plants having diverse chemical nature, it is also processed for producing the metabolites in wide range through plant cell tissue and organ culture, and plant metabolic engineering.

3.3 Effect of climate change on secondary metabolites Climate change is the big cause for threatening to biodiversity and also disturbing the human health and well-being over in coming future (Ramakrishna and Ravishankar, 2011). It has been expected that the productivity of cold weather crops, e.g., rye, oats, wheat, and apples can be reduced by approx. 15% in the next 50 years and strawberries would fall down as much as 32% with continuous changes in climate (Pimm, 2009). Plants are very sensitive to these types of changes and do not able to adapt immediately according to weather changes. It has been observed that the phenolic levels in conifers are increased in ozone exposure, but low ozone exposure did not produce any effect in a concentration of monoterpene and resin acid (Kainulainen et al., 1998). The studies on a limited number of crops have been accomplished to investigate any changes in crop quality under ozone exposure. For example, the productivity of wheat was declined under ozone exposure, but the concentration of grain protein was raised (Pleijel et al., 1999). In addition, ozone had also a positive impact on the quality of potato tubers by decreasing sugars and enhancing the vitamin C concentration (Piikki et al., 2003). Whereas, in rape seeds, the oil, protein and carbohydrate contents were declined under ozone exposure (Ollerenshaw and Lyons, 1999). In addition, ozone fumigation enhanced the level of terpenes and decreased the content of phenolic compounds in leaves of Ginkgo biloba (He et al., 2009). The high CO2 concentration alters the chemical composition in secondary metabolites of plants (Idso and Idso, 1989). The increasing level of CO2 reduces the nitrogen (N) content in seeds, grains, as well as in vegetative parts of the plants leading to the lowering of the protein concentration (Idso and Idso, 1989). Earlier it has been explained that the increased CO2 level raises the phenolics contents and condensed tannins in the leaves. The increased CO2 altered on a decrease/increase in concentrations of some particular monoterpenes and enhanced the total phenolics contents in conifers (Idso and Idso, 1989). It has been observed that concentrations of the monoterpene a-pinene were enhanced under high accumulation CO2 contents (Idso and Idso, 1989). However, Williams et al. (1994) have demonstrated declined level of b-pinene in needles under increased CO2 levels. Many studies have explained about the effect of temperature on the synthesis of secondary metabolites in plants. The higher temperature stimulates the production of secondary metabolites. Although, it has been suggested that the elevated temperature reduces the monoterpene contents in Douglas fir (Pseudotsuga menziesii) (Ramakrishna and Ravishankar, 2011).

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3.4 Impact of climate change on secondary metabolites of medicinal plants Phenological changes The life cycle of plants depend on seasonal changes and the global climate change is influencing species and ecosystems. Though a smaller number of medicinal plant species experience adverse effects from such phenological changes. The main phenological events for medicinal plants are adaptation with climate change and include (i) eruption of bud and unwinding of leaf, (ii) flowering and fruit setting, (iii) falling of leaf during Autumn or dry season, and (iv) related events of winter hardening and breaking as increasing global warming. This would affect the arrival of spring and the length of the growing season (Bidart-Bouzat and Imeh-Nathaniel, 2008).

Shifting ranges The climate change is also responsible for plants to migrate toward the new ranges. The ranges of plants have been started to move toward the poles or to higher elevations in an effort to “reclaim” the right growing zones. A large number of endemic species are under to be extinct over the world due to habitat loss and migratory challenges associated with climate change (Keutgen et al., 1997).

Effect of increased CO2 on medicinal plants The experiments conducted under normal environmental conditions have revealed positive effects of increased CO2 on the productivity and quality of several products and components of medicinal plants. The enhanced CO2 levels (3000 μL CO2/L of air) increased the fresh weight and leaf and number of roots in cultures of lemon basil (Ocimum basilicum L.), oregano (Origanum vulgare L.), spearmint (Mentha spicata L.) and thyme (Thymus vulgaris L.) in comparison to shoots grown in a culture of the same media under ambient air (Tisserat and Vaughn, 2001).

Effect of elevated ozone Changes in the O3 level could affect the production of secondary metabolites of plants. Plant physiological stress enforced by increased O3 levels can trigger for the induction of metabolic pathways like salicylic acid and jasmonic acid pathways, participated in the synthesis of secondary metabolites (Mishra et al., 2016).

Impact of ultraviolet radiation The ultraviolet radiation may cause molecular and cellular damage, such as damage to proteins, DNA, and other biomolecules. However, such kind of radiation could alter plant growth and development and lead to changes in vegetative or reproductive biomass, height, leaf characteristics, and flowering periods (Mishra et al., 2016).

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Global warming and secondary metabolite production The responses of the secondary compounds to elevated temperatures are not well explored, however, the enhanced concentration of volatile organic compounds have been generally determined (Loreto et al., 2006). So, above study suggests that temperature could alter the production of secondary metabolites of plants.

Adaptation with climate change and global warming The vulnerabilities of medicinal plants to climate change near to future could be overcome by preserving the endangered flora and fauna. The cultivation of medicinal plants would be a comprehensive way to protect the venerable species. The conservation of genetic diversity in the natural ecosystem could play a major role in the adaptation of medicinal plants. Traditional local knowledge that could overcome the effect of climate change must be investigated and documented, during promoting the traditional art and craft associated with livelihood by involving local people and income generation through eco-tourism (Mishra et al., 2016).

3.5 The expression of secondary compounds in plants It has been observed that the expression level of secondary metabolites vary with different combinations in various parts of the plant (leaves, roots, shoots, bark), at distinguished stages of growth (seedling, seed, plantlet, mature tree), under distinct environmental pressures (invasive microbes, herbivores), and also various combinations in several classes of plants. However, it is very difficult to reach to general conclusions about secondary metabolite combinations with universal implications, therefore, it has been classified into three categories (Verpoorte, 1998) depending on the way of expression (Table 3.1).

3.6 Early stage of plant evolution Earlier plant forms like green filamentous algae growing in aquatic habitat in the early Ordovician age (B500 million years ago), had been well equipped to conduct the fundamental metabolic processes of photosynthesis, glycolysis, and Krebs cycle to the synthesis of primary metabolites. The special enzymes were observed in the green algae, Chlamydomonas reinhardtii (Weng, 2014), which was more closer to living member of the Charophytes, could enhance the biosynthesis of sterols, glucose, lipids, and nucleic acids and might have played a vital role to balance the homeostasis condition within the aquatic pools of the early environment. The evolution of first land plants was appeared in form liverworts around 450 million years ago, in which shreds of evidence was found from their spores. The transition stage of these early land plants from the aquatic condition to land life had three major challenges like large and rapid temperature changes, desiccation, and direct harsh ultraviolet light.

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Table 3.1 Distinct ways of expression for secondary metabolites in plants species (Delgoda and Murray, 2017). Modes of expression

Definition

Examples

Constitutive

Many secondary metabolites are expressed in plant tissue individually or in many combinations of same compounds to play a particular role.

Constitutively expression requires activation

Some metabolites do not show biological activity in themselves, but following activation like prodrugs do provide some value for the host plant. These metabolites are classified as phytoanticipins.

Induced expression

Some secondary metabolites are expressed due to an environmental pressure which induces for their production. These secondary metabolites are comprised as phytoalexins.

A direct relationship has been observed with the expression of high level of glucosinolates in young oilseed rape cotyledons and the deterred feeding of a slug species, Deroceras reticulatum Allicin in garlic (Allium sativum L.) is an antibacterial compound synthesized from its inactive form alliin. When cellular damage has appeared, then alliin lyase enzyme is capable to mix with the alliin substrate and form allicin as a line of defense to inhibit the pathogens attack. This reaction develops characteristic like aroma and flavor of garlic if it is crushed The antimicrobial agents, anthraquinones, are not observed in healthy Cinchona plants, but are expressed in tissue cultured plants which are infected by fungi.

Other challenges were the need for anchorage on soil and rock, the adaptation of new forms of microorganic occupancies in the new soil environment, and finally competition with other plants for space, resources as well as grazing predators. The development of adaptative mechanisms enable them to overcome such challenges might have been a necessity to thrive in the new environment and it appears that the synthesis of secondary metabolites has played a unique and major role to combat with these biotic and abiotic challenges (Fig. 3.1) (Delgoda and Murray, 2017).

3.7 Environmental factors triggering the secondary metabolism The main factors for the survival of plants with changing environment are to understand the forces behind the evolution and diversity within natural communities. The internal mechanism of plants is able to detect such forces which play an important role in balancing and propagating their biodiversity. Then there could be a hope from a general evolutionary perspective that such a critical factor contributes to any organism can be intrinsically managed in the genome of the organism if survival and stability of species needs to be ensured. The diversification of the biosynthetic pathway led to the synthesis of three main groups of secondary metabolites (Fig. 3.2) like terpenes, phenolic compounds (phenylpropanoids and flavonoids) and nitrogen compounds (alkaloids, glycosides, glucosinolates, and cyanogenic glycosides). These biosynthetic pathways probably provide a force to make sustainable survival of organisms including the plant

Chapter 3 • Effect of climate change on plant secondary metabolism

s

te hy

Algae

p r yo

Pteridophytes

Gymnosperms

57

Angiosperms

B

Cambrian

Ordovician

Devonian

Paleozoic

Mesozoic

Cenozoic

Development of flowers and fruits: Synthesis of volatile compounds were occurred for the pollination.

Development of seeds: Tannins were expressed in seeds and also many other part of plants as antifeedants. Development of roots for holding and absorbing water from the soil: Root synthesised the specific isoflavonoids as defense against invaders. Development of true leaves with trichomes, thorns, hairs and crystals:

The biosynthesis of terpenes, phenolics, alkaloids were occurred to repel the feeders. Vascular development for nutrients supply:

Lignin synthesis were started for providing the strength and rigidity to plants. Transformation to terrestrial life:

Metabolites like: phenylpropanoids, flavonoids, abscisic acid and sporopollenin were synthesised to save from UV damage.

FIG. 3.1 Expression of secondary metabolites in lineage of the green plant phylogeny.

species through generation. The interaction of a plant with the environment is a very common and natural phenomenon. External factors significantly influence the metabolic pathways of plants that alter plant development, growth rates, and segregation of assimilates into major metabolites. These factors could also stimulate random activation of qualitative changes in the synthesis of secondary metabolites (Lommen et al., 2008; Ncube et al., 2012). This can be possibly one of the reasons for marinating plant metabolic diversity. Climatic factors like biotic stresses alter the biosynthetic levels as well as the quality of secondary metabolites in plants (Coley, 1987). The plants could not escape from the extreme environmental condition like light, temperature, and drought, nor migrate to nutritional rich areas, hence they have developed highly complex mechanisms to make the coordination between physiology and metabolism in order to acclimate with the conditions in which they are grown. Secondary metabolites form an integral component of such

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Insects Biotic stress Herbivores Microorganisms etc.

Abiotic stress

Climatic factors (water availability, temperature and solar radiation), drought, freezing etc.

Alterations in primary metabolism

Increaing enzyme activites (PAL, CHS, PPO etc.)

Increasing synthesis of secondary metabolites

Terpenes

Phenolic compounds

Nitrogen compounds

FIG. 3.2 Alterations in the primary metabolism due to biotic and abiotic stress leads to the increased enzymatic activity and synthesis of secondary metabolites and enzymes like phenylalanine ammonia lyase (PAL), chalcona synthase (CHS), polyphenoloxidase (PPO), and peroxidase (POD) are important components in this plant response.

adaptive mechanisms that have developed multiple mechanisms to sense their environment and modify their physiology such as growth and development as per requirement. Constitutive defense mechanisms respond to the different environmental indications although most important, upon exposure to a set of distinct environmental factors, stimulated to plant response takes effect (Table 3.2). Induced defense responses are identified by alterations in a set of traits that reduce the negative effect of the stress factors on plant fitness. Over the period of growth and development, plants experience several biotic and abiotic factors to which they respond by activating their defense system.

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Table 3.2 Effect of environmental factors on secondary metabolite production in medicinal plant species. Environmental factor Light

Moisture

Temperature Ozone

Plant species

Secondary metabolites

Pinus taeda, Salix myrsinifolia, Arabidopsis thaliana, Secale cereal, Artemisia annua, Cornus sanguinea, Frangula alnus, Corylus avellana, Pteridium arachnoideum, Solanum tuberosum, Marchantia polymorpha Artemisia annua, Pachypodium saundersii, Achnatherum inebrians

Terpenoids, alkaloids, flavonoids, flavonol glycosides, hydroxycinnamic acids, tannins, artemisinin, phenolic acids, phytosterols; glycoalkaloids, luteolin, apigenin

Hypericum perforatum, Achnatherum inebrians, Quercus spp., Pachypodium saundersii Pinus taeda, Ginkgo biloba, Betula pendula, Petroselium crispum

Phenolic components, lipophilic resins, artemisinin, tannins, isoprene, anthocyanins, alkaloids Phenolic compounds, isoprene anthocyanins, alkaloids, flavonoids, tannins Tannins, phenolic acids, phenolic compounds

3.7.1 Abiotic Light/solar radiation Solar radiation energy is a fundamental environmental factor essential for plant growth and development. The developed plasticity of plants in many environmental conditions is measured by continuous monitoring of the quality (wavelength), periods and quantity of solar quanta. The survival capacity of plants depends on their capability to take out photosynthetic carbon fixation and biomass productions, and hence it has generated the high sensitiveness and accurate capacities to sense distinguished light spectra and ultraviolet (UV) light of the solar radiation (Kazan and Manners, 2011). For ensuring the maximum advantages from light incidences, the plants have developed biochemical defensive pathways to protect from the damaging higher doses of UV radiation and extreme light radiations. Moreover, some other potential mechanisms have been evolved in plants to absorb the especially beneficial light spectra. As an environmental stress UV-B (280–315 nm) is absorbed by plants or and induces to adaptive mechanisms in plants toward the UV acclimation and survival in sunlight radiation (Rozema et al., 1997). UV-B radiation produces major harmful effects like damaging to plants, alteration in gene transcription and translation, as well as photosynthesis. Plants have the signaling network which converts the input of light into output after sensing the light and eventually helps in plant growth and developments, UV-B radiation widely effects on secondary metabolites such as phenolic compounds, terpenoids, and alkaloids as part of outputs or intermediates of this complex biochemical interaction (Rozema et al., 1997; Ncube et al., 2012). Phenylpropanoid derivatives are one of the main classes of secondary metabolites, which trap certain parts of the UV-B spectral without decreasing the infiltration of photosynthetic quanta into the leaf. It has been speculated that sunscreens phenylpropanoid

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derivatives play a vital role in the absorption of UV-B radiations. The results of many studies on phenylpropanoid mutants and different UV-B radiation have supported the above statements (Laakso et al., 2000; Winkel-Shirley, 2002), however, some reports also suggested that in some species, the content of these metabolites had been decreased (Tegelberg and Julkunen-Tiitto, 2001). These studies have some contrasting results on the range of phenolics which act as potent UV-B protectants. Some of these metabolites are hydroxycinnamic acids, flavonoids, and complex polymeric lignin or tannin-like compounds. The flavonoids are a dominant group among these compounds, though hydroxycinnamic acids and their esters have also been used widely as UV-B protectants in a large range of plant species (Burchard et al., 2000). The structural and chemical diversity of flavonoids depends on their different properties and functions in various plant species. These compounds, instead of providing protection from ultraviolet radiation, also have a major role in the protection of plants against pathogenic and herbivorous attacks (Harborne and Williams, 2000). It has been reported that the higher level of both anthocyanin and flavonol glycosides in the upper epidermal parts of leaf peels of Pisum sativum cv. argenteum mutant had directly been exposed toward visible and UV light irradiation and was confirmed a UV inducible protection role of these metabolites (Hrazdina, 1992). Hence this study suggested that solar radiation has a major contributing role in the level of flavonoids and phenolic acids in plant species. Now, many researchers are attracted toward flavonoids research to understand their pharmacological and pharmaceutical role and explored the different types of plant flavonoids with distinct therapeutic properties such as antibacterial, antiviral, antioxidant, antitumor, anti-hepatotoxic, anti-lipolytic, vasodilator, and antiallergic regimens (Gurib-Fakim, 2006; Ncube et al., 2012). Tannins have been known as antibiotic, antifeedant, and biostatic effects on different organisms (Harborne, 1992), their chemical properties are implicated for the discovery of medicinal compounds with multiple functions against different ailments. Hence tannins have several medicinal properties like antibacterial, antiviral, antidiarrhoeal, free radical scavenging, anti-inflammatory, antitumor and antidote activities (Gurib-Fakim, 2006). Moreover, phenolic compounds especially tannins and flavonoids make potential contributions in investigating the organoleptic properties of fresh fruits, fruit juices, and wine. However, the potential contribution of phenolic compounds for human health would highly depend on the environmental factors, which manage their accumulation levels and quality in plants. The relation of plant adaptation under solar radiation stress and human health is not dependent on only phenolic compounds like antioxidants, however, many other large numbers of metabolites have been explored possessing pharmacological properties and others have harmful effect to humans. It has been reported that upon mechanical or light radiation stress, the glycoalkaloids like α-solanine and α-chaconine is accumulated in potato tubers (Arnqvist et al., 2003; Jansen et al., 2008), and these compounds play an important medicinal role in gastro-intestinal or neurological disorders in humans. Although, the accumulation of such compounds is directly related to the accumulation of phytosterols (Arnqvist et al., 2003) and that have beneficial effects on human health.

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Phytosterols reduce the absorption of cholesterol from fat matrices into the intestinal tract that leads to declining cholesterol contents in human and eventually reduces the risk of cardiovascular diseases. So, it seems a clear relation between light-induced levels of secondary metabolites and the pharmacological benefits. To understand the factors regulating the metabolites contents in different plant’s part will be the primary condition to assure the quality, safety and to enhance the phytopharmaceuticals activities on human health.

Moisture stress Drought conditions of definite ranges of magnitudes and periods have been usually experienced in several environments and could desperately produce adverse effect on plant survival and their resistivity against stress. The reduction in the growth of plants is experienced under drought conditions due to lacking water declines the rate of photosynthesis. The declined water level and elevated temperatures trigger for high production of phenolic compounds in plants (Glynn et al., 2004; Alonso-Amelot et al., 2007). In the deficit of water, plants close their stomata and reduce the photosynthesis activity and hence it can be expected a negative relationship between water deficit and the production of secondary metabolites. A certain function of phenolic components in plant water-relations has been identified that is to accumulate for lipophilic resins in Diplacus and Larrea species (Rhoades, 1977), where an integrative role of antidesiccant and UV screen defense has been speculated. Further, it has been explained that the relationship between xylem pressure and tannin production depends on the degree of water stress affecting on plant system could either be positive or negative (Horner, 1990). The phenolic and saponin contents, as well as their bioactivity, are varied seasonally in medicinal bulbs (Ncube et al., 2011). In some studies, high phenolic compounds have been noticed in several plant species while the winter season, where moisture stress was a predominant factor. Therefore, it has been envisaged that moisture stress plays a major role in contributing to produce such a high level of phenolic compounds during a specific season.

Temperature Temperature stress usually triggers or increases the active oxygen species-scavenging enzymes in plants such as superoxide dismutase, catalase, peroxidase, and several other antioxidants. Temperature stress can alter the physiological, biochemical, and molecular events of plant metabolism like protein denaturation or disturbance of membrane integrity. These several changes could vary the secondary metabolite contents in the plant systems which are generally implicated as an indicator of a stress injury in plants (Zobayed et al., 2005). The higher temperature (35 °C) increases the total peroxidase activity in leaf along with an increase in hypericin, pseudohypericin and hyperforin contents in the shoot system of St. John’s Wort (Zobayed et al., 2005). Even, an exponential increase in the array of volatile organic compounds with increasing temperature linearly has been explained in a range of plant species (Sharkey and Loreto, 1993; Sharkey and Yeh, 2001). It has been suggested that the cold stress stimulated the synthesis of phenolics which subsequently

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accumulated in the cell wall (Christie et al., 1994). After exposure to maize seedlings and oilseed rape plants to temperatures, the higher levels of PAL have been observed that led to a corresponding increase in their phenolic concentration (Christie et al., 1994; Solecka and Kacperska, 1995). In cold stress, the levels of anthocyanins are increased and assumed to protect the plants against this adverse effect (Pennycooke et al., 2005). Ncube et al. (2011) have contributed the increasing levels of total phenolic components derived while the winter season in their research work as being consistent with this fact and supports similar findings from earlier works (Pennycooke et al., 2005; Ncube et al., 2011).

3.7.2 Biotic factors The plants have evolved an extensive of defense mechanisms in their growing places against insects, herbivores, and various microorganisms like bacteria, filamentous fungi, and protozoa that established array of several interactive relations with the host plant. Such consistent interactions of parasitism or mutualism induce the synthesizing of certain secondary metabolites. These natural products synthesizing from such interactions have been categorized into three main groups (a) phytoalexins, (b) phytoanticipins, and (c) signaling molecules, for example, the salicylic acid. The phytoalexins have a low molecular weight of molecules, which is produced and accumulated after exposing the plants to the microorganisms. However, the phytoanticipins are produced first in the plants before the invasion of pathogens (Cheynier et al., 2013).

3.7.3 Multiple stress effect Under natural circumstances, plants are not only affected by a single abiotic factor rather they are likely exposed to different stresses at the same time. The periodic climatic changes escort different stress factors in combination and hence such complex changes could not be always predicated (Holopainen and Gershenzon, 2010). To understand effects of multiple stress on plants, usually, these factors are investigated separately. It has been emphasized that if two or more factors occur together, sometimes their effects can be additive, whereas in other cases the effect of one factor has priority (Gouinguene and Turlings, 2002). In maize, the higher metabolites were observed after a combination of high temperature and induced lepidopteran herbivory than the single applied stress either  and Turlings, 2002). Niinemets (2010) explained that the fungal infection in (Gouinguene maize decreased the emission of stimulated defense metabolites through lepidopteran herbivory alone by about 50% (Niinemets, 2010). The escalated alkaloid levels were observed in Achnatherum inebrians plants after cultivating under salt and drought stress, with the higher levels of ergonovine than ergine (Zhang et al., 2011). The contents of both alkaloids enhanced the long lifespan of the plant growth period. The effect of different stress factors in plants is usually in surrounding environments that are often interactive and suggest that the integrated effect of different stresses is highly diverse. Now, many researchers have explained that the understanding of such diversity is very important in the response of plants to multiple stress combinations which could not often be

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extrapolated from responses to single stress factors. The acquiring knowledge on how multiple stresses affect secondary metabolite accumulation in plants could provide more information to investigate the biological roles of such metabolites in reducing the stress and will provide a clue to explain their optimum productivity and quality, which ensures the quality of phytomedicine.

3.8 The regulation of plant secondary metabolism by interactions of heat shock and elevated CO2 Climate change in the coming future indicate its serious effect on the physiological activities of vegetation, globally. The elevated temperature and atmospheric CO2 levels would alter plant growth, net primary productivity, photosynthetic capability, and other biochemical activities, which play an important role in normal metabolic function. Environmental factors such as, increased temperature (heat shock, Hs) and elevated atmospheric CO2 levels are examples of abiotic stresses, which are integrated by climate change (Raupach et al., 2007; Hansen et al., 2010). The temperature and atmospheric CO2 are identified as main factors to stimulate for the dramatic changes in plant secondary metabolites (Ramakrishna and Ravishankar, 2011; Austen et al., 2019). The synthesis of certain biogenic volatile organic compounds (BVOCs) are increased at high temperatures (Loreto and Schnitzler, 2010; Ramakrishna and Ravishankar, 2011), although the synthesis of phenolic components in plants is normally increased under elevated CO2 (eCO2) (Lambers, 1993), however, such response is usually species-specific (Hartley et al., 2000). Nonetheless, the upregulation of secondary metabolism could provide protection shield to plant against short-term environmental stresses (Ramakrishna and Ravishankar, 2011). The increased emissions of the BVOC isoprene is produced by secondary metabolism through the non-mevalonate pathway (MEP) and is normally related to short-term heat shock in plants (Calfapietra et al., 2013). However, the mechanism of action of isoprene is not completely explored, it seems to inhibit the negative effects on the physiological process under high heat conditions (Singsaas, 2000), possibly through its antioxidative properties protecting plant membranes (Siwko et al., 2007). The isoprene emitting plants are often almost fast-growing, woody species like Salix, which are not subjected to a long time of abiotic stresses like extreme temperature, however, this plays a vital role in reducing the short-term heat shocks through secondary metabolic mechanisms (Sharkey et al., 2008; Loreto and Fineschi, 2015). The isoprene is not only essential for hydrocarbon in order to plant safety against abiotic stress and also gives a larger effect on atmospheric chemistry. The reaction of isoprene with the hydroxyl radical (
OH) could affect the formation of tropospheric ozone (O3) however the oxidation products of isoprene could also produce secondary organic aerosols (Scott et al., 2014), then participated in cloud formation and their consequent albedo impact. While decreasing the capability of the adverse effects of the elevated temperatures, isoprene is energetically very costly, with continuous

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emission of 50% of the carbon fixed during photosynthesis in leaves (Velikova and Loreto, 2005). This emission could even enhance the net content of carbon fixed in the plant leaves, decreasing the stored carbon reserves and the decoupling of isoprene emission from the prevailing rate of photosynthesis (Loreto and Fineschi, 2015). As an outcome, isoprene emissions are highly controlled by plants (Velikova and Loreto, 2005; Austen et al., 2019) with upregulation in the biosynthesis likely to restructure plant carbon partitioning, impacting secondary metabolic processes which are not related with BVOC synthesis as carbon becomes highly limited. It has been described that both factors (Hs and eCO2) affect independently and their interactive effect on woody plant (Salix spp.) secondary metabolism, especially, isoprene biosynthesis has been noticed. In the study, results have shown that during abiotic stress, the plant secondary metabolism is highly changed, with the substrate being deviated to energetically expensive secondary metabolic pathways. Due to anthropogenic activities, the global temperature and atmospheric CO2 levels have increased, so it is important to know the interactions between atmospheric processes and global vegetation, mainly considering the global isoprene emissions have the potential to reduce the atmospheric warming (Austen et al., 2019).

3.9 Ecological roles of secondary metabolites Secondary metabolites are one of the potential compounds of plants, which play a defensive role against herbivores, microbes, viruses or competing plants and signal factors for attracting of pollinating or seed-dispersing animals as shown in Table 3.3. Hence, these metabolites are essential for the plant’s survival and reproductive fitness. Table 3.3

Ecological significance of three main groups of secondary metabolites.

Group

Classification

Biological source

Ecological roles

Alkaloids

Highly diverse and complex, usually contain N

Plants (300 families), bacteria, fungi

Plant defense against herbivores due to toxic properties for the animals or feeding deterrence (bitter taste), antimicrobial activity could decrease the decomposition rate

Phenolic compounds

One or more hydroxylated aromatic rings Plants, bacteria, fungi

Allelopathy; decomposition rate is ceased due to antibacterial activity Pigments and scents, antioxidants, UV protectants, antifungal and antibacterial Antiherbivore and protection against pathogens Complex with proteins and inhibition of enzyme activities Slows decomposition rate Scents and chemical is responsible for signaling between plants and animals, provide resistivity to biotic (insecticidal) and abiotic stress

Phenolic acids Flavonoids

Plants, bacteria, fungi

Tannins

Polymers

Terpenes

Isoprene units

It is highly found in woody species; sometimes in grasses Plants

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3.9.1 Alkaloids After a long research of 200 years on alkaloids, isolation as well as characterization of about 10,000 alkaloid compounds have been done using chemical techniques. Although the distribution of alkaloids in each kingdom is limited, these have been detected in bacteria, fungi, plants, and animals (Chomel et al., 2016). Alkaloids are found in approximately 300 plant families, with certain metabolites usually confined to families like hyoscyamine in Solanaceae (Evans, 2009). Alkaloids are identified with at least one nitrogen atom, and mostly these atoms are well-defined crystalline substances, which exist in a free state as N-oxides or combined with acids to produce salts, both promptly soluble in water (Evans, 2009). Whereas, alkaloid free bases are slightly soluble in water but frequently soluble in organic solvents (Evans, 2009). It has been observed the potential physiological action of alkaloids on animals, such as many of them act as an analgesic or toxic property like cocaine or morphine (Chomel et al., 2016). With such properties, alkaloids have been usually studied in pharmacology and have been implicated since immemorial history in traditional medicine as well as present in modern medical practice. However, it has been not well explored in the context of litter decomposition, soils, and the decomposer subsystem. Alkaloids are toxic components with several functions in plants, as feeding deterrents, allelochemicals, and protection against several infections (Chomel et al., 2016). The present research has continuously explained not only that alkaloids play role in plant metabolism for long period, but also that daily intermittent in alkaloid content: qualitative and quantitative is highly prevalent in some species (Evans, 2009). In various plant families like Poaceae, alkaloids are not produced by the plant, but fungal endophytes play a vital role in the production and accumulation of various types of alkaloids (Schardl et al., 2013). Such endophytic production of alkaloids in plants is capable of the gradual disintegration of litter by influencing the activity of microorganisms and detritivores (Purahong and Hyde, 2011; Iqbal et al., 2012). In contrast, if decaying of fresh alkaloid-laden leaves was compared with alkaloid-free litter, alkaloids were immediately lost from the fresh leaves and differences in decomposition were only meager (Siegrist et al., 2010). Although, to date, there have not been too many studies to generalize the effect of alkaloids on decomposition events or establish their fate in soils.

3.9.2 Phenolic compounds About 10,000 highly diverse groups of phenolic compounds have been isolated with ranging from simple phenolic acids to complex polymers like tannins and lignins (Chomel et al., 2016). Phenolic compounds are synthesized by plants, bacteria, and fungi. In plants, their quantities could be varied from 1% to 25% or more than that of total green leaf dry €ttenschwiler and Vitousek, 2000). Phenolic compounds have been identified mass (Ha with having at least one hydroxylated aromatic ring. In this class of secondary metabolites, different terms are imploded to identify the compounds, which creates confusion due to several criteria implicated in their classification. The term polyphenol is described as the compounds having various hydroxyl groups on one or several aromatic rings, is

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commonly implicated with respect to all family of tannin compounds or even all phenolic components. Later, it could also be qualified as the “total phenols” fraction. Although, a more accurate classification of phenolic compounds related to their structure could be implicated to characterize the simple phenols (C6 skeleton), phenolic acids (C6-C1 or C6-C3), flavonoids (C6-C3-C6) and tannins ((C6-C3-C6)n). In some cases, they could be again categorized on the basis of their degree of polymerization: (i) low molecular weight phenolic compounds present in a maximum number of plants in glycosylated and soluble form and are employed as carbon sources by maximum detritus organisms, but some of them also have certain biological functions, e.g., cinnamic acids, (ii) high molecular weight of phenolic compounds such as tannins that are highly polymerized water-soluble compounds belonging to two subfamilies of several properties. Hydrolyzable tannins are usually found in dicot angiosperms. The condensed tannins are polymers of flavan-3-ols (flavonoids), and sometimes it is known as proanthocyanidins (Chomel et al., 2016). Tannins are highly abundant polyphenolics compounds present in woody €ttenschwiler and species, although these are not found in herbaceous plants (Ha Vitousek, 2000). Phenolic acids are compounds having a phenolic ring and an organic carboxylic acid function. The low molecular weight phenolic acids are readily decomposed by microorganisms and others like caffeic acid, p-coumaric acid, and vanillic acid have been identified with allelopathic activities (Fernandez et al., 2013). Such as, caffeic acid is one of the highly ubiquitous cinnamic acids derived from different types of crops, weed remains and other plants. Caffeic acid checks the growth of fungi (Harrison et al., 2003), bacteria (Bowles and Miller, 1994) and plants (Chomel et al., 2016), as well as early root growth (Batish et al., 2008). Flavonoids contain a highly variable group of molecules containing C6-C3-C6 skeleton and play several biological roles. Their biological activities are capable to contribute to providing color, aroma, and taste to fruits, flowers, and seeds (Mierziak et al., 2014). They are also rich in antioxidants and possess several physical and chemical properties. These could also play as direct oviposition and feeding attractants or deterrents, and they could develop defensive mechanisms in plants against insect pests by altering in their behavior, growth, and development (Mierziak et al., 2014). Flavonoids also communicate information to symbiotic bacteria, e.g., they are well recognized as signaling mediators between rhizobia and the roots of legumes to begin the nodulation (Mierziak et al., 2014). These components also alter in the plant-plant interactions like allelopathic inhibition of germination in target species (Weston et al., 2015). Tannins are extensively identified for their €ttenschwiler and Vitousek, defensive mechanism against herbivores and pathogens (Ha 2000), however, they have acknowledged with special issues during studies of ecosystem functioning for their several biological and chemical characteristics. It has been largely explained that tannins could make insoluble complexes with proteins and other biological polymers or with metal ions at the time of litter decomposition. Such complexes provide the organic matter inaccessible to further decomposition, therefore tannins have been

Chapter 3 • Effect of climate change on plant secondary metabolism

67

€ttenschwiler and Vitousek, increasingly identified for their below-ground effects (Ha 2000). These could also produce toxic effects on microorganisms and decrease enzyme activities, hence it can alter nitrogen and carbon transformations in soils with multiple €ttenschwiler and Vitousek, 2000; Kraus et al., 2003). Most of the studies have aspects (Ha described that nitrogen and carbon mineralization rates have been declined by the addition of tannins. Their production is usually addressed with constitutive activities in some species; however, their production could also be enhanced as a response to environmental stresses such as herbivory. At the genetic level, it has been obtained the remarkable differences among cottonwood hybrids (Populus fremontii L. and P. angustifolia James) in terms of foliar levels of condensed tannins that were ranging from 1% to 10% of leaf dry matter (Schweitzer et al., 2013). The composition and activity of tannins could also alter while tissue senescence (Nierop et al., 2006).

3.9.3 Terpenes Among more than 30,000 identified metabolites, terpenes or terpenoids are a highly diverse group of plant secondary metabolites (Hartmann, 2007). These metabolites are mainly synthesized by plants, and their concentrations generally range 1–2% of dry matter weight, however, higher concentrations could be observed in leaves or in certain parts of plant-like trichomes (Langenheim, 1994). These lipophilic compounds consist of a simple five-carbon building block (isoprene unit). Monoterpenes are made of two isoprene units (C10); they are mainly alpha-pinene that is widely distributed in plants. Sesquiterpenes have three isoprene units (C15; thunbergol), whereas diterpenes contain four (C20). Some terpenes like mono- and sesquiterpenes, are volatile in nature (Langenheim, 1994) and are usually known as volatile organic compounds (VOCs). The terpene family consists of hormones, carotenoid pigments, latex, and most essential oils. Firstly, terpenes were identified in their volatile forms and provide chemical communication among organisms. In addition, terpenes involve in the development of resistance mechanisms in organisms against both biotic and abiotic stresses. Terpenes have multiple functions like to attract the pollinators, protecting the plants from herbivores (Langenheim, 1994), or producing toxic insecticides and insect repellents. The effect of monoterpenes on soil microorganisms is very complex, as they could decrease the activity and growth of microorganisms whereas enhancing these activities in others (Amaral and Knowles, 1998). Several reports suggest that the monoterpenes suppress the N mineralization and net nitrification in the soil (White, 1986, 1994). Exact reasons of this inhibition are not yet well explored, however, the inhibition of the N mineralization and net nitrification can be expected by understanding the direct action of monoterpenes on an enzyme participated in the ammonium oxidation pathway (White, 1988), or suppression of growth in Nitrosomonas europaea, a bacterium oxidizes ammonium to nitrite (Ward et al., 1997). Therefore, it has been demonstrated that other terpenes like betapinene have inducing effects on Nitrosomonas europaea growth.

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3.10 The ecosystem feedback of plant secondary metabolites for the climate change Globally, forests are the main sink for atmospheric CO2 (Pan et al., 2011; Pukkala, 2018). The sequestration of carbon generally occurs in soil carbon (44%), living biomass (42%), deadwood (8%), and litter (5%) (Pan et al., 2011). Declining forest harvesting and providing deadwood as large trees is the most rapid method to enhance CO2 sequestration in boreal forests (Pukkala, 2018). Reducing the decay rate of deadwood decreases the emission of CO2 back to the atmosphere. The contents of both resin acids and stilbenes (Ioannidis et al., 2017) can increase 10% of dry weight in the heartwood of Pinus sp. and significantly decrease the decay rate of deadwood by fungal rot (Karppanen et al., 2007) and by wood borers (Nerg et al., 2004). Plant secondary metabolites from the needle litter are more quickly released into the soil (Kainulainen et al., 2003) and atmosphere (Aaltonen et al., 2011) in comparison to deadwood. In conifer forests, monoterpenes attribute in releasing of around 90% of litter in the beginning of summer and while the needle falls in autumn (Aaltonen et al., 2011). During 19-months of decaying processes, pine needles have lost 96%, 79%, and 86% of initial concentrations of monoterpenes, resin acids and total phenolics, respectively (Kainulainen et al., 2003). The hot conditions in the boreal conifer forests enhance the release of reactive monoterpenes to the forest atmosphere and enhance the production of secondary organic aerosols (SOAs), especially in the evenings (Rose et al., 2018) and highly induce cloudiness ( Joutsensaari et al., 2015; Zhao et al., 2017), there decreasing the penetration of solar radiation to vegetation and reducing warming and drought stress (Niinemets, 2018). Volatile plant secondary metabolites have various biological roles in the species interactions influencing herbivores and their natural invaders and triggering defenses in healthy plants (Blande et al., 2010). Climate change factors also alter the herbivoreinduced volatiles, although knowledge on forest trees and woody plants are not well explored (Holopainen et al., 2017). Non-volatile plant secondary metabolites are secreted from the plant leaf and needle litter, which is sequestered in soil and affected C or net N mineralization in forest soils (Smolander et al., 2012). The growing three seasons in increasing levels of CO2 and O3 did not alter the content of total phenolics and monoterpenes in Pinus sylvestris needles, however, resin acids were enhanced at elevated O3. Although, while decomposition differences in needle litter were lost (Kainulainen et al., 2003). In leaf litter of Betula pendula grown at enhanced CO2 and O3 level, many phenolic components have been increased, whereas the growth of juvenile earthworms declined (Kasurinen et al., 2007) elucidating that climate change reduced the quality of food for soil organisms. The UV-A and UV-B exclusion altered the level of phenolic groups variably in Alnus incana litter, but in birch litter, no significant differences in phenolic components were noticed (Kotilainen et al., 2009).

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3.11 Secondary metabolites as worthy asset for the biological system: Further support The metabolic paths of the secondary metabolites are very complicated and have intricate systems of biosynthesis possessing different pathways, enzymes, and genes that explain support of metabolites function which must be activated in the adaptive process to provide the functional significances for the plants. The hypothesis based on secondary metabolites describes that plants have developed the ability to synthesis these secondary metabolites due to a selective advantage is associated with the following assumptions (Delgoda and Murray, 2017). •

•

•

•

Usually, plants (and other organisms which synthesis secondary metabolites) do not contain an immune system and rely on such compounds for the defense. Therefore, metabolites like phytoalexins and phytoanticipins provide security from pathogens with their antibiotic, antifungal, and antiviral properties, simultaneously with an arsenal of defense processes from enzyme activities and potential resident endophytes. Definitely, it is an adaptive significance to those which synthesis it, for example, to avoid competition; anti-germinative or toxic for other plants (allelopathic); better adapted to adverse environments; and are capable to fight feeding animals like insects or cattle (antifeedant). Some examples are the activation of estrogenic characters through clover or alfalfa, and the secretion of phenolic chemical juglone from the walnut tree which develops bare patches of soil around it. The synthesis of secondary metabolites needs complicated pathways in organism, which are energetically expensive and are unlikely to have developed until the products are beneficial. The genes which synthesize such compounds reside right beside each other, explaining natural selection for it. The resistance genes are also well arranged in plant systems.

3.12 Conclusions and future prospective Climate change induces the production of secondary metabolites in plant systems. It has been explored over 170,000 secondary metabolites in limited plant species so far and still, many secondary metabolites in an evolutionarily diverse group of plant species/lineage are not explored. Several environmental factors, e.g., light, temperature, moisture, etc. play a necessary role to produce diverse groups of secondary metabolites and these diverse group metabolites provide strength and stability to organisms for surviving in inhospitable conditions. However, plant growth and their development might be affected due to alteration in primary metabolites for the synthesis of secondary metabolites.

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The structural diversities in secondary metabolites occur during the evolution of plants in different environmental conditions and they defend plants from various stresses and harmful bacteria, insects, herbivores, etc. Moreover, the diverse structure of several secondary metabolites like alkaloids, flavonoids, terpenes has been investigated against different diseases including cancer. Understandings of the evolutionary behaviors and different factors that stimulate the formation of secondary metabolites would provide worthful ideas to synthesize anticancer molecules on a large scale.

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4 Isolation and characterization of bioactive compounds from natural resources: Metabolomics and molecular approaches Diksha Sharmaa, V.P. Singha, Rajesh Kumar Singhb, C.S. Joshic, and Vinamra Sharmad a DEPARTMENT OF MEDICINAL CHE M IST RY , I NS TI TU TE OF MEDICAL SCIENCES, B ANARAS HINDU UN IVERSITY, V AR ANASI, UTTAR P RADE SH, INDIA b DEPARTMENT OF DRAVYAGUNA, FACULTY O F A YURVEDA, INSTITUT E O F MEDICAL SCIENC ES, BANARAS HINDU UNIVERSITY, VARANASI, UTTAR PRADESH, INDIA c PATANJALI RESEARCH INS T I T U T E , H A R I DW AR , UTTARAKHAND, INDI A d AMITY INSTITUTE OF INDIAN SYSTEM OF MEDIC INE, AMITY UNIVERSITY UTTAR PRADESH, NOIDA, INDIA

4.1 Introduction Nature is the Supreme reservoir of medicinal and food products. Since ancient times mankind has used natural sources in search of food for survival and bioactive compounds for the treatment of various diseases as practiced worldwide in various traditional systems of medicine. Apart of various animal species and natural minerals, the most important source of bioactive compounds, nutraceuticals and dietary supplements has been medicinal plants, also various microorganisms such as fungi, bacteria, etc. Nutraceutical compounds were also considered to be part of medicinal plants which are helpful for treatment and cure of different chronic diseases (Gonza´lez-Sarrı´as et al., 2013). Nutraceutical mainly derived from ‘Nutrition’ and ‘Pharmaceutics,’ which are isolated from herbal products, processed foods, also nutrients. Nutraceuticals are sold in medicinal form, present in food form, used to improve health, prevention of chronic diseases, increase life expectancy (Kalra, 2003). In early times, eating a variety of healthy foods was considered to be best way to get proper nutrients. However, proper nutrients don’t work only by daily dietary schedule, for that purpose dietary supplements were taken so as to balance the nutrients intake. Dietary supplements, mainly lowers risk of health issues such as osteoporosis or rheumatoid arthritis as they comes in the form of capsules, liquids, gel tabs, pills and extracts. Dietary supplements, which may contain minerals, vitamins, amino acids, herbs, plant extracts, Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00004-7 © 2021 Elsevier Inc. All rights reserved.

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or enzymes, supplements the total daily intake, metabolite, or combination of various ingredients (Zhao, 2007). Ancient scholars very well distinguished the benefits of medicinal herbs over the inactive or toxic herbs (Kunle et al., 2012) and more than 50,000 plant species are reported which having medicinal properties (Barboza et al., 2009). Natural source, i.e., medicinal plants were used as potential source of novel lead molecule, leading to drug discovery and development. Aspirin, morphine, digitoxin, quinine and many more were synthesized through scientific validation of herbal medicine. The isolation, characterization and application of bioactive natural products play a highly significant role in drug discovery and development process. The revolution of herbal drug discovery mainly started since starting of 19th century (Wachtel-Galor and Benzie, 2011; Pal and Shukla, 2003), when many drugs were isolated from plants as well as fungi such as Penicillin (antibiotic) from the fungus Penicillium notatum (Fleming, 1929) Paclitaxel from bark of pacific yew tree Taxus brevifolia (Pulici et al., 1997), morphine from Papaver somniferum (Brook et al., 2017), colchicine from Gloriosa superba ( Joshi et al., 2010; Balkrishna et al., 2019), ephedrine (Ephedra species), quinine from Cinchona cordifolia, atropine from Atropa belladonna, caffeine from Coffea Arabica and many more, for the treatment of various diseases (Bapu, 2018). Isolation of these drugs from various plant sources created a vast scope for researcher for discovery and development of bioactive molecule naturally as well as synthetically. In the modern system of medicine, bioactive compounds considered as lead molecule for further therapeutic potential plays appreciable roles due to disease inhibiting capabilities and less toxicity parameters. Traditional Ayurvedic or herbal preparations mostly obtained from plant sources in the form of crude drugs likely to be in extracts or mixture of ingredients form, also as dried herbal powdered form. Ayurvedic products play vital role for treatment various chronic diseases as well as some respiratory disorders or viral infections. These preparations possess their therapeutic effect by acting synergistically or in combination therapy with specific drugs (Parasuraman et al., 2014). However, ethnopharmacological approach of Ayurvedic medicines is very unique and well defines (Sharma and Chaudhary, 2015). In the recent times, Ayurvedic approach is being followed mostly in Asian countries because of their more health benefits and lower side effects, as most of allopathic drugs have specific side effects with its disease treatment properties. Natural products will always play a key role in drug discovery and development. Various strategies for the development of lead compounds from the natural plant follows few methodologies starting from selection of natural product, primary screening, isolation and characterization of compounds or structural elucidation, secondary screening, preclinical development and tertiary screening (Beutler, 2009). On today’s market, majority of drugs have isolated from natural resources by following serendipitous observations, and more efforts have been going on for implementation of rational approach for drug discovery and development process. In late 90s researchers were following a hypothetical approach in the manner by synthesizing new compounds and testing it against various disease on different organs of animals. This approach was

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limited to speed of biological tests. After that time, computational approach was followed using computer aided molecular modeling, where gene technology was the matter of interest and also elucidation of 3D structure of protein. Molecular modeling further boosted in vitro models for drug receptor interaction by inhibiting specific enzymes (Yuan and Xu, 2018). The modern methods used in process of drug discovery and development have generated enormous data, such as bioinformatics, combinatorial chemistry, proteomics, metabolomics, genomics, and ultrahigh throughput screening which further requires powerful tools for mining purpose (Katz and Baltz, 2016). Many approaches have been followed for isolation, structural elucidation, characterization and biological evaluation of novel compounds. In this chapter we have discussed mainly two types of approach used for isolation and characterization of bioactive compounds obtained from natural products sources, i.e., metabolomics approach and molecular approach.

4.2 Metabolomics approach Nowadays, development of plants derived compounds have maintained its traditional value as medicine and still continue as essential source of pharmaceutical drugs. Natural products mainly help in generation of lead molecule, which in turn, further synthesized for its better biological properties, high compatibility, great ADMET properties and lower side effects. But many bioactive compounds face problems while isolation, identification and characterization from various extracts of medicinal plants (Atanasov et al., 2015). Traditional approach followed in drug discovery step comprises of many drawbacks regarding separation and isolation of molecule, its complexity, its feasibility and lot more. For the management of time and proper protocol, Metabolomics approach is better method to be followed for drug discovery and development which works in both pertinent and convenient way. Metabolomics is the systematic study of metabolites (small molecule) within cells, tissue or organisms, bio fluids on the large scale. The interactions of small molecule within the biological system comprises of the term, ‘metabolome.’ Metabolomics includes substrates, intermediates and products of metabolism which are regulated by both environmental and genetic factors. In recent years, increased interest in food metabolomics have been witnessed due to its direct relation with nutrition and human health ( Johnson et al., 2016). Food metabolomics generally evaluates the quality of fruits, beer, meat, vegetables, etc., whereas metabolomics fingerprints profiling comprises of quantification of composition, characterization and authentication of foods (Cubero-Leon et al., 2013). Metabolomics study has been involved in transplant monitoring, clinical chemistry, newborn screening, toxicology and pharmacology, also applied in molecular and personalized medicine. In comparison to other ‘omics,’ metabolomics approach represents and

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Unknown Features

Manual identification

Untargeted Analysis

Metabolite extraction, chromatography & data acquisition

SemiTargeted Analysis

Targeted Analysis

Metabolite assignments from databases or characteristic spectral fragment

Quantitative analysis of set of unknown metabolites

Confirmation using synthetic standard

Novel metabolite discovery

Biomarker discovery

Statistical analysis, pathway enrichment, or biological conclusion

FIG. 4.1 General scheme of different strategies of metabolomics approach.


d
erich et al., 2016). The relamodulates to molecular phenotype of health and disease (Fre tion of genotype-genomics and phenotype-metabolomics demonstrated changes in resultant metabolite and specific gene variations which further provides knowledge about genetic epigenetic phenotypic changes. An extensive variety of metabolites derived from medicinal plants such as carbohydrates, organic acids, steroids, amino acids and lipids have not yet been evaluated and reported completely due to metabolome complexity, because of this complete number of metabolites present in human are still unexplored (Bourgaud et al., 2001). There is a specific database available having details about number of small molecule metabolites present in human body, i.e., The Human Metabolome Database (HMDB), containing biological data, clinical data and chemical data. HMBD reported 114, 166 metabolite entries (December 2019) along with 5702 protein sequences linked to these metabolites’ entries, including both lipid soluble and water-soluble metabolites (Wishart et al., 2018). Metabolomics consists of various strategies, primarily two analytical study is being followed, i.e., ‘global untargeted metabolomics-discovery’ and target-validation-tandem (Fig. 4.1).

4.2.1 Global untargeted metabolomics (discovery) Untargeted discovery global also known as, Hypothesis generating metabolomics, is specifically based on collection and analysis of all detectable metabolite in a sample. As a part of discovery approach, it is based on identification of novel compounds whose chemical potency as a drug and their mechanism of action for the treatment of various diseases is still unexplored and unknown. In order to systematically identify and characterize metabolites, untargeted discovery undergoes comprehensive analysis which could be correlated

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using different databases/libraries search, further qualitative identification and relative quantification can be achieved. These analytical procedures were considered for both endometabolome and exometabolome (Wang et al., 2010). Further three main strategies were followed for untargeted approach including, nontargeting, finger printing and foot printing (Hyotylainen and Wiedmer, 2013). i. Non-targeting profiling: This approach can detect molecular bio signatures and has been successfully applied to environmental and genetic contributions to diseases. Non targeting profiling provide the most appropriate route to detect unexpected changes in metabolites concentration. ii. Metabolomic finger printing: This approach is mainly related to analysis of intracellular metabolites profiling, referred for microbial cultures. This analytical technique can classify different phenotypes of several mutants but still high throughput screening of many mutants is difficult to scale up and requires extracellular space for quenching and extracting of metabolites. This approach can be used as diagnostic tools to evaluate the disease state or to assay success of particular prognosis or treatment. iii. Metabolomic foot printing: This approach is related to analysis of extracellular metabolites profiling, a powerful method to understand the effects of genetic/ environmental signals on biological systems. The main purpose of foot printing is to characterize the low molecular weight metabolites or small molecules secreted or excreted by or consumed by the system. Foot printing mainly helps in elimination of fast metabolism quenching process and time-consuming extraction procedures by following same analytical techniques.

4.2.2 Targeted metabolomics This approach is also known as targeted-validation-tandem, refers to quantitative analysis and identification of species set of metabolites present in biological system. The massive array of metabolites is predestined by the library generated by software generally used for data analysis. Statistical analysis was performed afterwards to attribute the known and unknown biochemical pathways and resultant data is stored in statistical analysis tool to know group separation between phenotype of interest and control by targeted metabolome (Mussap et al., 2018).

4.3 Metabolomics technologies Metabolomics mainly comprises of global metabolic profiling framework which utilizes high resolution analytics together with chemo metric statistical tools such as principal component analysis (PCA) and partial least squares (PLS), to derive integral picture of xenobiotic and endogenous metabolism (Worley and Powers, 2013). Various metabolomes such as amino acids, organic acids, peptides, vitamins, proteins, alkaloids,

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polyphenols and inorganic acids act as small molecule biomarkers that represent the functional phenotype in cell, tissue and organism. Analytical techniques developed for metabolomics are usually referred to comprehensive metabolic profiling, where set of small molecules (known or unknown) were analyzed and detected by various analytical techniques. Separation and identification of these small molecules is made possible by innovational technologies or powerful high resolution detectors namely, UPLC-MS, HPLC, CE/Microfluidics, LC-MS, FT-MS, QOQ-MS, NMR spectroscopy, X-ray crystallography, GC-MS, LIF detection, etc. (Zhang et al., 2012).

4.3.1 Mass spectrometry (MS) MS is intrinsically a highly sensitive method for detection, quantitation and structural elucidation of upwards of several hundred metabolites in a single measurement. The factors that contribute in maintenance of sensitivity and accuracy while detecting the compounds by MS includes method of small molecule extraction, separation, ionization methods, and further detection approaches. Modern MS analysis has very high significance especially for the information related to chemical structure, elemental formula determination (by isotope distribution pattern) and authentication of compound by € hrkop et al., matching reference compound data with specific small molecule (Du 2014). MS allows conversion of picomole to fentomole levels of metabolites (primary and secondary). Modern MS follows different operational principles, instruments and performance. MS technologies include mass analyzer technology, ionization techniques, mass accuracy and resolution power (Bedair and Sumner, 2008). In metabolomics approach various ionization techniques that have studied till date includes, electron spray ionization (ESI), electron ionization, chemical ionization, atmospheric pressure chemical ionization (APCI), extractive ESI and desorption ESI method. Mass analyzers used in metabolomics study having different resolving powers comprises of, high resolution MS and ultra-high-resolution MS such as Orbitrap MS, Fourier transform ion cyclotron resonance MS (FT-ICR-MS), Multipass-TOF MS and also lower resolution instruments such as single quadrupoles, linear and 3D quadrupoles (Dettmer et al., 2007). Mainly in MS analytical technologies two strategies have been followed for metabolomics study in case of chemically complex compounds, i.e., direct mass spectrometer analysis and Mass spectrometer coupled with chromatography.

Direct MS analysis This analytical technique helps to sample crude mixtures without using chromatographic separation technique. In metabolomics, advanced instrumentation is there by using FT-ICR-MS and orbitrap MS of high-resolution power, for accurate mass measurement and tadem mass. FT-ICR-MS is a very powerful tool due to its mass accuracy (1,000,000) which is useful in calculation of empirical formula and identification of compounds (Marshall et al., 1998). FT-ICR-MS technique have a

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disadvantage due to its high cost because of which it is unavailable in many metabolomics laboratories for routine usage. This technique also provides complimentary fragmentation methodology for characterization of post translational modification (phosphorylation and glycosylation) (Parker et al., 2010). In contrast of FT-ICR-MS technique, there is one new technique namely Orbitrap MS analysis that works using electrostatic fields for ions entrapment. Orbitrap MS have excellent mass accuracy (1–5 ppm) and resolving power (typically 150,000), which is basically used in MS analysis through direct method of yeast sphingolipids, bovine lipids and different plant metabolites. Further the use of multipass-TOF-MS has been recently developed for direct MS analysis in metabolomics study having mass accuracy (50 μmol (Gao et al., 2017). A polyketide named (1R,2E,4S,5R)-1-[(2R)-5oxotetrahydrofuran-2-yl]-4,5-dihydroxy-hex-2-en-1-yl(2E)-2-methylbut-2-enoate had produced from the endophytic fungal strain Diaporthe sp. SXZ-19 of Camptotheca acuminate show very mild activity at the concentration of 10 μM (Liu et al., 2013). The α-pinene was potent anticancer compound inducing cytotoxicity via apoptosis in murine melanoma (B16F10-Nex2) cells (Matsuo et al., 2011). Out of nine, one compound altertoxin IV was produced from endophytic fungus Alternaria species G7 from the Broussonetia papyrifera exhibits significant cytotoxic activities against MG-63 and SMMC-7721cell lines with IC50 values of 0.53 and 2.92 μg/mL, respectively (Zhang et al., 2016). A novel fungal endophyte, Trametes hirsute secreted podophyllotoxin and other related aryl tetralin lignans with potent anticancer activity (Stierle et al., 1995).

8.7 The potent anticancer compounds produced by deep-sea sediment fungi Marine fungi are another resource of bioactive compound useful for the drug discovery purposes. Although marine fungi are less explored than terrestrial counterparts however a large number of useful antibacterial, antiviral and anticancerous compounds have been obtained that worked in animal systems (Molinski et al., 2008). Simplicilliumtides A, E, G, and H a linear peptide, were obtained from broth culture of Simplicillium obclavatum EIODSF 020e, a deep-sea-derived fungal strain. Simplicilliumtides A and G exhibited weak cytotoxicity on human leukemia HL-60 cell line (IC50 values was 64.7 and 100 μM) and simplicilliumtides E and H exhibited weak cytotoxicity on K562 cell line (IC50 values was 39.4 and 73.5 μM) (Liang et al., 2016). Five new 20-nor-isopimarane diterpenoids, Asperethers A-E were isolated from the culture of the deep-sea fungus Aspergillus wentii SD-310 exhibited cytotoxic activities against A549 cell line (IC50 values was 20, 16, 19, 17, and 20μM, respectively) (Liang et al., 2016). Circumdatin G, discovered from Aspergillus westerdijkiae SCSIO 05233 a deep-sea sediment sample showed weak antiproliferation activities on K562 and promyelocytic HL-6 cell lines (IC50 values ranging between 25.8 and 44.9 μM) (Fredimoses et al., 2015). A new chromone engyodontiumones H and a known polyketide were isolated from a deep-sea fungus Engyodontium album DFFSCS021cytotoxicity against human histiocytic lymphoma U937 cell line (IC50 values were 4.9 and 8.8 μM, respectively) (Yao et al., 2014). The compounds 5-chlorosclerotiamide and 10-epi-sclerotiamide were the secondary metabolites of the Another deep-sea fungus Aspergillus westerdijkiae

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DFFSCS013 secreted 5-chlorosclerotiamide and 10-epi-sclerotiamide, which showed cytotoxicity against K562 cell line (IC50 was 44 and 53 μM, respectively) (Peng et al., 2013).

8.8 The potent anticancer compounds produced by algaeassociated fungi A indole derivatives varioloid A and varioloid B compounds were isolated from marine algae-derived endophytic fungus Paecilomyces variotii EN-291 exhibited cytotoxic against A549, HepG2, and HCT116 cell lines with IC50 values between 2.6 and 8.2 μg/mL (Zhang et al., 2016). Aspergillus ochraceus Jcma1F17, a marine algae-derived fungus secreted cinnamolide derivative and a known compound insulicolide A, showed cytotoxicity against H1975, K562, U937, 823, Molt-4, 7, HeLa, A549, Huh-7, and HL60 human cancer cell lines and their IC50values range between 1.95 and 6.35 μM (Fang et al., 2014). The epiphytic fungus Microsporum sp. (MFS-YL) isolated from Lomentaria catenata, a marine red alga synthesized physcion which induced apoptosis in HeLa cells and its effect on the expressions of p53, p21, Bcl-2, Bax, caspase-9 and 3 proteins were investigated. It was found that physcion induces cell apoptosis through down-regulation of up-regulation of Bax expression, Bcl-2 expression, and activation the caspase-3 pathway. It also induced the formation of reactive oxygen species (ROS) in HeLa cells (Wijesekara et al., 2014).

8.9 The potent anticancer compounds produced by mangrove endophytic fungi Two compounds spirobrocazines C and brocazine G were isolated from mangrovederived fungus Penicillium brocae MA-231 exhibited moderate activity against A2780 cells with IC50 59μM while brocazine G showed strong activity against A2780 and A2780 CisR cell with the IC50 values of 664 and 661 nM, respectively and it was much better than cisplatin (a positive control), with IC50 values 1.67 and 12.63 μM, respectively (Meng et al., 2016). A mangrove endophytic fungus, Lasiodiplodia sp. 318 isolated from Excoecaria agallocha produced 2,4-dihydroxy-6-nonylbenzoate showed cytotoxicity against MMQ and GH3 cell lines with the IC50 values of 5.2 and 13.0 μM, respectively (Huang et al., 2017). Another endophytic fungus Campylocarpon sp. HDN13-307 isolated from the root of mangrove plant Sonneratia caseolaris, produced Campyridones D and ilicicolin H exhibited cytotoxic against HeLa cell with the IC50 values of 8.8 and 4.7 μM, respectively (Zhu et al., 2016).

8.10 The potent anticancer compounds produced by sponge associated fungi Two compounds cytochalasin K and A unknown compound produced by Fungus Arthrinium arundinis ZSDS1-F3 that was collected from sponge Phakellia fusca showed

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cytotoxicity against K562, A549, Huh-7, H1975, HL60, HeLa, and MOLT-4 cell lines with the IC50 values ranging between 1.1 and 47.4 μM. The cytochalasin K exhibited cytotoxicity against K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa, and MOLT-4 cell lines, with IC50 values of 10.5, 13.7, 10.9, 19.1, 11.1, 47.4, and 11.8 μM, respectively while unknown compound was cytotoxic against K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, HeLa MOLT-4 cell lines with IC50 values of 6.2, 1.1, >50, 14.2, 18.5, 3.4, 18.8, 6.2, 3.2, and 4.1 μM, respectively (Wang et al., 2015). Four compounds aszonapyrone A, 13-oxofumitremorgin B, sartorypyrone A and sartorypyrone B were isolated from coralderived fungus Neosartorya laciniosa (KUFC 7896) inhibit the growth of MCF-7, NCIH460, and A375-C5 cell lines. The MTT assay showed that, among the meroditerpenes tested, aszonapyrone A was the most effective compound having strong growth inhibitory activity with GI50 ¼ 13.6, 11.6 and 10.2 μM while Sartorypyrone B was also potent in growth inhibition, however, it was less active than aszonapyrone A showing the GI50 values 17.8, 20.5, and 25.0 μM for MCF-7, NCI-H460 and A375-C5 cell lines, respectively. Another compound 13-oxofumitremorgin B showed only weak inhibitory activity with GI50 ¼ 115.0, 123.3, and 68.6 μM against MCF-7, NCI-H460 and A375-C5 cell lines, respectively (Eamvijarn et al., 2013). A Red Sea sponge, Xestospongia sp. was collected from deep waters of Sharm Obhur, Jeddah, Saudi Arabia secreated a new polyacetylene, xestospongiamide exhibited antitumor effect against both Ehrlich ascites carcinoma and lymphocytic leukemia with LD50 value of 5.0 μM each (Ayyad et al., 2015). The novel phthalimidine derivatives marilines A1 and A2 was obtained from Stachylidium sp., a marine-derived fungus isolated from the sponge Callyspongia cf. C. flammea. inhibited human leukocyte elastase (HLE) with IC50 value 0.86 μM (Almeida et al., 2012).

8.11 Conclusion Every plant is a pool of numerous fungi which seems to be in a close interface with fungi. The production of bioactive compounds by fungi similar to their host plays a significant role in drug development. It may be alternative source for bioactive compound for pharma industries without any environmental issues. The fungi associated with medicinal plants are chemical synthesizers which have ability to produce secondary metabolites that can be exploited for curing many diseases. In recent year various bioactive metabolites from fungi were isolated and identified. Fermentation of endophytic fungi on large scale for secondary metabolites production has several advantages, like reproducible and dependable productivity. Endophytes can be grown in fermenters to give unlimited supply of secondary metabolites and thus can be exploited commercially. For optimizing various biosynthetic pathways by changes in the culture conditions can be explored as a technique which leads to the production of derivatives and analogs of novel bioactive compounds. The fungal bioactive compounds act as alternative sources without destroying the plant diversity. Several attempts have been made toward fungi to produce natural products. Further research with advanced techniques at molecular level using may offer better visions into fungal biodiversity, genetic constituents and their biochemical

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production. Hence, rigorous work for optimization of bioactive compounds production by fungi is needed to fulfill the requirement of pharma industries, leading to an emerging area of research globally. Recent studies on plant-fungal interactions found that it showed the co-evolutionary relationships between fungi and plants, and the development of new methods for their exploration. This symbiotic association is more complex than previously thought. In recent decades researcher too much progress in understanding the roles of fungi as major interactors with plants, but a lot of remains to be explored, especially regarding associations of non-culturable fungi and/or biotrophic fungal pathogens. Due to advance knowledge of genomics, proteomics, transcriptomics, metagenomics and advanced microscopy tools help us to understand the plant-microbe interactions. there are many types of interactions of fungi with plants such as asmycorrhizal, parasitic or endophytic relation that playing significant roles in plant development and health. It is not clear how a endophyte alters its life style to assimilate with a plant host, in particular the adaptation of parasites into endophytes and vice versa. A detailed understanding of biodiversity and the influence of fungi on plant biology will not only develop our knowledge on ecosystem function and exploiting the potential of endophytic fungi to make sure global food availability.

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9 Structural information of natural product metabolites in bryophytes S.J. Aditya Rao DEPARTMENT OF PLANT CELL BIOTECHNOLOGY, CSIR—CENTRAL F OO D TECHNOLOGICAL RESEARCH INSTITUTE, MYSURU, K ARNATAKA, INDIA

9.1 Introduction The use of bryophytes for healthcare and other therapeutic needs has been understudied due to their minimal use by ethic people, making an assumption that they don’t play a direct role in human health (Alam et al., 2014; Chandra et al., 2017). Although, a fewer report of bryophytes being used for healthcare over vascular (higher) plants in ethnobotanical practices suggests that some possibilities of their ethno-botanical importance in different cultures around the globe. Till date, approximately 136 species of bryophytes have been reported for various ethno-botanical usages (Harris, 2008) but for their medical use, majority are reported from traditional Chinese, Indian Ayurveda and some American practices (Flowers, 1957). The minimal use of bryophytes for healthcare purposes in earlier literature may be due to the reason that they were often got confused with lichens, club mosses or some other vascular plants (Drobnik and Stebel, 2014). Further, unlike vascular plants, the biomass produced by bryophytes is very less and to protect them they might not be used in the traditional medicinal system. Their small and undistinguishable body architecture might have been added for their minimal use. However, the ethnobotanical use of bryophytes by local people of Borneo-polar and tropical regions is may be due to the high biomass of bryophytes grown in these regions, whereas, in dry areas where the biomass is less the records reporting their ethno-botanical use are less. Apart from ethnobotanical reports, the Linnaean bryology also does not mention the use of bryophytes in “Materia medica” of the 18th century influencing greatly the medicinal practitioners of that period to not to use bryophytes for therapeutic purposes (Drobnik and Stebel, 2014). The traditional practices majorly used the similarities of any organ in the human or animal body or in appearances to any plant with known usage to explore the medicinal usage. And hence some liverworts (e.g., Marchantia polymorpha L.) were believed to have effects over liver ailments. Some Philonotis species, Bryum, Mnium, and some other hypnaceous plants were used directly by making a thick bed by Gasuite Indians to alleviate

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burn pain as they produce chilling effects (Sabovljevi
c et al., 2011) while Cheyenne Indians used Polytrichum juniperinum Hedw to produce a similar effect. It was reported that around 40 bryophyte species are being used as crude drugs in traditional Chinese medicine (TCM). Preparations from Conocephalum conicum (L.) Dumort and Marchantia polymorpha with vegetable oils are used to treat boils, cuts, bites, and burns. To treat eye diseases Peat moss Sphagnum teres is used and Haplocladium microphyllum (Hedw.) broth is used to treat tonsillitis, bronchitis, cystitis, and tympanitis. Polytrichum commune Hedw is known for its antipyretic, diuretic and hemostatic properties (Ding, 1982). Like vascular plants, bryophytes also found to possess innumerable health benefits. Being a rich reservoir for complex secondary metabolites their applications in healthcare may not be astonishing. Reports on the presence of secondary metabolites with pharmacological effects already indicated the presence of some novel molecules which are not synthesized in higher plants (Mishra et al., 2014). About 3.2% of mosses and 8.8% of liverworts have already been screened for their unique bioactive constituents. More than 30 different bryophyte plants can be found in the Chinese market and plants like Sphagnum, Marchantia, Riccia, Barbula, Bryum, Octeblepharum, and Fontinalis are found to be effective against cardiovascular diseases, inflammation, fever, lung diseases, infections, wounds and skin diseases (Banerji, 2001; Glime, 2007). Plants produce secondary metabolites as a part of their defense, to adapt to biotic and abiotic stresses, herbivores and also to conserve their resources (Cornelissen et al., 2007; Paramesha et al., 2018; Aditya Rao et al., 2019). These metabolites most of the times come with added advantages of therapeutic benefits. Similar to vascular plants many bioactive secondary metabolites have also been isolated from bryophytes in recent years. Though the knowledge of their ecological relevance is not clear, they can be exploited for therapeutic benefits (Asakawa et al., 2013b). Understanding the role of such bioactive molecules and their mechanism of action in ascertaining the relationship with different ailments often leads to the discovery of novel and potentially active drug candidates (Asakawa et al., 2013a).

9.2 Exploration of bryophytes for medicinal usage With an increase in new and new disease outbreaks throughout the world it has become an inevitable need to search for new and effective drug molecules. In this avenue, many bioactive molecules from bacteria, fungi, lichens, and vascular plants are constantly being explored by medicinal chemists, extensively for medicinal usage. As per plants are concerned, due to the high impact of traditional medical practices the greater importance is always been given to vascular plants and lower plans like bryophytes are constantly being neglected due to their lack in therapeutic utilization. But the exploration of bryophytic plants in recent times has effectively demonstrated the therapeutic effects of many molecules which are found particularly in bryophytes and not in other categories of plants.

Chapter 9 • Natural product metabolites in bryophytes

211

9.3 Bryophytes as a source of biologically active molecules The interest of medicinal chemists and biologists in the chemical analysis of natural products has increased in the last few decades. Among the plant species screened, bryophytes failed to attract attention due to their lack of traditional therapeutic evidence. The methods used earlier for the chemical analysis added-up to the misery, as isolation and characterization procedures were laborious and required highly skilled laborers. Bryophytes being rare in the majority of the plant populations also attributed to their less exploration. But in the last two decades a rapid and unexpected rise in chemical analysis, especially the secondary metabolites have taken place. With advancements in analytical techniques, the identification of bioactive metabolites has resulted in generating a profile of common as well as rare molecules in bryophytes (Table 9.1).

9.4 Different types of secondary metabolites found in bryophytes 9.4.1 Saccharides and lipids Natural products form bryophytes with therapeutic effects entered the mainstream research by medicinal chemists just after the second half of the 20th century and ever since many novel compounds have been described regularly (Asakawa, 2004). Some unique oligo-and polysaccharides (Klavina, 2014) along with rare trisaccharides have been reported from bryophytes which are not found in higher plants (Pejin et al., 2012a,b). Some simple lipids like triglycerides and waxes and complex lipids like glycolipids and phospholipids can be commonly seen in bryophytes. Along with these, many fatty acids with unsaturated band numbers, e.g., eicosa-pentaenoic acid (1), arachidonic acid (2) are also seen in bryophytes, which is specifically a characteristic feature of mosses (Pejin et al., 2011a,b,c, 2012c,d).

OH O

OH CH3

(1)

CH3

O

(2)

9.4.2 Terpenoids All classes of terpenoids are isolated from bryophytes, i.e., mono-, sesqui- and diterpenoids. The terpenoids isolated from bryophytes are closely related to those of vascular

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Evolutionary Diversity as a Source for Anticancer Molecules

Table 9.1 An overview of different secondary metabolites found in different bryophyte classes. Secondary metabolite Terpenes Monoterpenes Sesquiterpenes Diterpenes Triterpenes Tetraterpenes Steroids Prenylquinones Phenols Benzoic acid derivatives Cinnamic acid derivatives Bibenzyl derivatives Bis(bibenzyl) derivatives Phenol ethers Alkylphenols Flavonoids Acylflavonoids Isoflavonoids Biflavonoids Flavones and isoflavone dimmers Chalcone derivatives Aurones Sphagnorubins Lignanes Alkaloids Cyanoglycosides Tetrapyrroles S-Acrylates Azulenes Naphthalene derivatives Phenanthrene derivatives Cyclophanes Vitamins

Liverworts

Mosses

✓ ✓ ✓ ✓ ✓ ✓ ✓

✓

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓

✓ ✓

✓ ✓ ✓ ✓ ✓

Hornworts

✓ ✓ ✓ ✓

✓

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓ ✓ ✓ ✓ ✓ ✓

✓ ✓

✓

✓ ✓

plants. Liverwort species are better-studied as they have a higher accumulation of oil bodies, which is an indication of the existence of a diverse range of terpenoids (He et al., 2013). Hornworts and mosses mainly produce di- and triterpenoids (Zhan et al., 2015). Some triterpenoids (e.g., hopanoids) are characteristic components of mosses. Only four monoterpenoids have been found in peat-mosses (Sphagnum). In total, 18 mono terpenoids, 5 trinorses-quiterpenoids, 72 sesquiterpenoids, 10 diterpenoids,

Chapter 9 • Natural product metabolites in bryophytes

213

and 9 triterpenoids have been isolated from or detected in the genuine mosses— Bryophyta (Asakawa et al., 2013a). Regular plant sterols (e.g., sitosterol, stigmasterol, brassi-casterol and campesterol) are also recorded in bryophytes. Moreover, some leafy liverworts from the genera Scapania, Plagiochila, or Chiloscyphus, possess cholesterol. Less is known, but polyacetylenes and mineral compounds are also isolated from bryophytes (Sabovljevic et al., 2001).

9.4.3 Plant hormones (plant growth regulators) The presence of lunularic acid, a dormancy factor and growth regulator, has been confirmed in liverworts, but not in mosses (Pryce, 1971). Some hornworts lack lunularic acid and are shown to have different pathway for the degradation of D-methionine, compared with liverworts. Species of Sphagnum (i.e., peat-mosses) differ from other mosses, liverworts and tracheophytes in flavonoid composition and complete acetylation of D-methionine. The universal signal molecule-ABA (abscisic acid) is confirmed in mosses 
c et al., 2016). The but not in liverworts, although they react to exogenous ABA (VujiCi influence of the diterpenoids in spore formation is also investigated (Vesty et al., 2016). Auxins and cytokinins have been studied extensively in mosses compared with other plant growth regulators, but still, elucidation in synthesis, co-action and metabolism remain to be investigated (Sabovljevi
c et al., 2014b). Gibberellins are hardly reported to have any effect (Sabovljevi
c et al., 2010), but this can be a consequence of understudy of gibberellins in bryophytes. The production of ethylene is documented in both liverworts and mosses, but the physiological role in bryophytes is not clear (von Schwartzenberg, 2009). The other growth regulators salicylic acid, jasmonates, brassinosteroids or strigolactones are rarely directly or indirectly (through the genes-orthologs present in the bryophyte genome) documented in a few species of bryophytes (Sabovljevi
c et al., 2014b).

9.4.4 Phenylpropanoids Phenylpropanoid metabolism remains at the level of the acids and is directed to the biosynthesis of flavonoids, inferring that early steps on the evolutionary path towards lignification were present in bryophyte ancestors (Weng and Chapple, 2010). The model moss Physcomitrella patens have orthologs gene for the biosynthesis of p-coumaryl alcohol and coniferyl alcohol. This suggests that the biosynthesis of phenylpropanoids was established in the earliest land plants to protect from UV radiation. Due to the lack of lignin, special attention has been given toward polyphenols (phenolic acids, lignans, and complex flavonoids). Some neolignans are unique chemical markers for hornworts. A number of flavonoid glycosides have been detected both in the liverworts and the mosses. Flavonoids such as quercetin, are commonly found in mosses and have been detected in more than two-thirds of entire moss species studied. In contrast, the distribution of flavonoid

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Evolutionary Diversity as a Source for Anticancer Molecules

glycosides has only been detected in about a quarter of all mosses studied so far. This is most likely due to the analytical methods used in the research. Flavonoid glycosides are commonly found in liverworts.

9.4.5 Phenolic components Flavonoids This class of compound is represented by a high number of flavones. They are often present as C-glycosides, e.g., tricetin-6,8-di-C-glucopyranoside(S) (3), tricetin-6-arabino8-glucopyranoside (4), apometzgerin-6,s-di-C-arabino-pyranoside (5), and tricin-6,8-di-Carabinopyranoside (6). Mention should be made also of some O- and O,C-glycosides such as apometzgerin-7-o-glucuronide (7), iso-furcatain-7-O-β-D-ghcopyranoside (8), isoorientin-30 -O-sophoroside and the flavonoid-O-triglycoside (9). Acylated flavonoids are rare in bryophytes but some of the reported ones are ferulyl-isoschaftoside, diosmetin-7-Oglucoside-60 -malonyl ester, and orobol-7-O-glucoside-6-malonyl ester. Bryoflavone (10), heterobryoflavone (11), philonotisflavone (12), and dicranolomin (13) are some bioflavonoids found widespread but only in mosses. Flavonols and particularly anthocyanidins seem to be rarer. So far, the only representatives of the latter which have been isolated are the two 3-deoxyanthocyans of the luteolinidin type, from Bryum species (Musci), although red to violet colored bryophyte organs are encountered. 1

R

2

R R HO

O

3

R R` OH

(3) (4) (5) (6)

O

R1-R3 ¼ OH; R, R0 ¼ β-D-glucopyranosyl R1-R3 ¼ OH; R ¼ β-D-glucopyranosyl; R0 ¼ α-L-arabinopyranosyl R1-R2 ¼ OCH3; R3 ¼ OH; R, R0 ¼ α-L-arabinopyranosyl R1-R3 ¼ OCH3; R2 ¼ OH; R, R0 ¼ α-L-arabinopyranosyl

R

1

R RO

O R

R` OH

O

2

3

Chapter 9 • Natural product metabolites in bryophytes

215

(7) R1-R2 ¼ OCH3; R3 ¼ OH; R ¼ β-D-glucuronyl; R0 ¼ H (8) R1-R3 ¼ C; R2 ¼ OH; R ¼ β-D-glucopyranosyl; R0 ¼ α-L-rhamnopyranosyl (9) R1 ¼ O-sophorosyl; R2 ¼ OH; R3, R, R0 ¼ H

OH OH

HO O

HO

O

HO

O

HO

OH

OH

O

O

OH OH

O

HO

OH

OH

O OH

HO

HO

O

(10)

(11) HO HO

O

HO

O

O

OH

O

O

OH HO

HO HO

O

O

OH

HO HO

(12)

OH

OH

O HO

OH HO

(13)

Other phenolic compounds The structural variants of bibenzyl (14) and bis(bibenzy1) (15) in liverworts are especially remarkable in this connection. Examples of the former are lunularic acid (16), brittonin A (17), prenylbibenzyl and its geranyl analogues, radulanins A and F, perrottetins E-G, marchantins A, C and I, riccardins B and C, plagiochin D (18), 6,7-dihydroxy-4-(3,4-dihydroxyphenyl) naphthalene-2-carboxylic acid, scapaniapyrone (19) and megacerosic and anthocerosic acids.

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Evolutionary Diversity as a Source for Anticancer Molecules

O

H3C

CH3

O

H3C

O CH3

O

OH

CH3 H3C

CH3

O

H3C

H3C

HO

O

O

(14)

(15)

H3C

OH

(16)

OH

HO

CH3

O

O

(17)

(18)

HO

HO

OH

O

O O O OH

(19)

Isoprenoids Monoterpenes Bornyl acetate (20) and β-sabinene (21), bornyl ferulate (22) and limonene (23).

H3C

CH3 CH3

CH3

O CH3

H2C CH3

O

(20)

CH2

O

H3C H3C

H3C

O

CH3

(21)

HO

H3C

O

(22)

CH3

(23)

Sesquiterpenes Barbatene (gymnomitranes) (24), pinguisone (25), aromadendrene (26), myltaylanes and vitranes.

Chapter 9 • Natural product metabolites in bryophytes

CH3

H3C CH3 CH3

CH3

CH3

CH3

O

CH3

H3C

217

CH3

(24)

CH3

O

CH2

(25)

(26)

Diterpenes Isophytol (27) and geranylgeraniol (28), 3-oxolabda-8,14-dien-13-ol and its isomer, 3-oxolabda-8(17),14-dien-13-ol (29), trans-(5R,7S,8S,9S,10S)-labda-12,14diene-7,8-diol, Plagiospirolides A and B.

CHO CHO

CHO CHO

H H

CHO H CHO

HO

H H

H3C

H

H

H

H

H

H

H H

H

(27)

(28)

(29)

Triterpenes and phytosterols A-amyrane, fernane, friedelane (30), D-friedooleanane, hopane (31), lupine (32), neohopane, and serratane 1 (33).

CH3 CH3 H3C

CH3 H3C

CH3 H C H3C3

CH3 CH3

CH3

H3C

(31)

CH3 CH3

H3C

H3C

H3C

H3C

H3C

CH3

(30)

CH3

H3C CH3

H3C H3C

H3C

CH3

(32)

H3C

CH3

H3C CH3

(33)

9.5 Bioactive molecules from bryophytes reported with different pharmacological activities Though bryophytes were kept away from the limelight as far as their medicinal usage is concerned, some studies are suggesting the biological effects of bryophytic extracts. These

218

Evolutionary Diversity as a Source for Anticancer Molecules

biological effects cannot be attributed to a specific molecule as the extract contains a pool of several molecules. The effect can also be contributed by the action of multiple molecules resulted from a synergistic mode of action. Such effects are seen in the extracts of species belonging to genus Rhodobryum where the extract produced antihypertensive effect while its separated fractions unable to produce the same effect in hypertensive rats (Pejin et al., 2011a). However, the fractions of peat moss (Sphagnum) showed antibiotic effects similar to their extract (Sabovljevi
c et al., 2011). Most of the bryophytes have also been studies chemically. There are more than 400 biologically active molecules have been identified from bryophytes in the last two decades (Asakawa, 2007). Therefore many of the bryophytes can be promising natural sources of novel drugs and chemicals for other purposes (Dey et al., 2015). One should note that in many articles, however, the identity and origin of species are insufficiently described. The biological characteristics of the terpenoids and aromatic compounds isolated from the liverworts in our laboratory are: characteristic scents, pungency and bitterness, allergenic contact dermatitis, cytotoxic, anti-HIV and DNA polymerase β inhibition, antimicrobial and antifungal activity, insect antifeedant activity, mortality and nematocidal activity, superoxide anion radical release inhibitory activity, 5-lipoxygenase, calmodulin, hyaluronidase, cyclooxygenase inhibitory activity and NO production inhibitory activity, piscicidal and plant growth inhibitory activity, neurotrophic activity, muscle relaxing activity, cathepsins B and L inhibitory activity, cardiotonic and vasopressin antagonist activity, and anti-obesity activity (Asakawa, 1982, 2007, 2011, 1995a,b; Toyota et al., 1996). Even though bryophytes have not yet formally become important as medicinal or toxic plants preparations from them have indeed been used, notably in the Chinese and North American Indian folk medicine.

9.5.1 Cytotoxicity Several sesquiterpene lactones, such as eudesmanolides, germacranolides, and guaianolides isolated from liverworts exhibit cytotoxic activity against KB nasopharyngeal and P-388 lymphocytic leukemia cells (Asakawa, 1995a,b). The crude ether extracts of the liverworts Bazzania pompeana, Kurzia makinoana, Lophocolea heterophylla, Makinoa crispata, Marsupella emarginata, Pellia endiviifolia, Plagiochila fruticosa, Plagiochila ovalifolia, Porella caespitans, Porella japonica, Porella perrottetiana, Porella vernicosa, and Radula perrottetii showed cytotoxicity against P-388 cells. A series of new, naturally occurring compounds from bryophytes show appreciable cytotoxic and cytostatic properties. Examples are 4-epiarbusculin (40), oxyfrullanolide (41), 8,α-acetoxyzaluzanin C (42), diplophyllin (43), epoxyfrullanolide (44), marchaintin A (39), riccardin B (45), and perrottetin E (46). Further, maytansinoid (34) and 15-methoxyansamitocin P-3 (35), isolated from two moss species, showed a powerful antitumor activity (Sakai et al., 1988). The sesquiterpene aldehyde iso-bicyclogermacrenal (36), from Lepidozea virrea (Hepaticae), inhibits the growth of roots and leaves of rice seedlings. In contrast, the

Chapter 9 • Natural product metabolites in bryophytes

219

diterpene acetate gymnocolin (37), from Gymnocolea inflata (Hepaticae), promotes the germination of wheat seeds. Lunularic acid (16), a benzyl derivative was found to play an important role in growth regulation and development in most of the bryophytes. Sacculatal (38) possesses a potent piscicidal activity, while bis(bibenzyl) (15), marchantin A (39), isolated from various Marchantia species shows distinct antibacterial activity. O

HS

H3C

O O H3C

N

H3C

NH OH

O

Cl

H3C

O

O

O

Cl

CH3

CH3

O

CH3

O

CH3

O N

O

CH3

H3C

N H3C

O H3C

CH3

O

O

CH3

H3C

CH3 O

H3C

O

O

HN HO

O

O

H3C

CH3

O

O

O O

H3C

O

CH3

H3C

CH3

(34)

(35)

O

(36)

(37)

CH3 H3C

CH3

O O

O

CH3

H2C

OH

HO

O

CH3

O

O HO

O

(38)

(39)

(40)

(41) OH

O

O

OH

O

CH3 CH2

O

O HO H2C

CH3

HO

CH3

H2C

HO

CH3

CH2 O CH3

H2C

O

O

H2C

(42)

H3C

OH

O

O

O

H3C

(43)

CH2

O O

(44)

HO

(45)

OH

(46)

9.5.2 Antimicrobial effects of bryophytes Organic extracts of various medicinal plants containing flavonoids have been reported to € tz et al., 1995; show antimicrobial activity (Waage and Hedin, 1985; Rocha et al., 1995; Schu Vaughn, 1995). But most of these studies were carried out using higher plants like angiospers and bryophytes are least explored for their biological effects. However, few of the recent study on bryophytes has shown some of the antibacterial activity against grampositive and gram-negative bacteria (Basile et al., 1999; Mekuria et al., 2005; Zhu et al., 2006).

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Evolutionary Diversity as a Source for Anticancer Molecules

The essential oil of Marchesinia mackaii showed antibacterial activity against Bacillus subtilis, Escherichia coli, Salmonella pullorum, Staphylococcus aureus, and Yersinia enterocolitica (Figueiredo et al., 2002). Sacculatal (38) from Pellia endiviifolia showed potent antibacterial activity against Streptococcus mutans (a causative organism for dental caries). Polygodial was inactive in this same bioassay (Asakawa et al., 2009). Lunularin (47) from Dumortiera hirsuta also showed antimicrobial activity against Pseudomonas aeruginosa (Lu et al., 2006). Lepidozenolide (48) obtained from Lepidozia fauriana showed a positive response to methicillin-resistant S. aureus and the fungi Candida albicans and Trichomonas feetus (Shu et al., 1994). Riccardiphenol C (49) from Riccardia crassa showed antibacterial activity against B. subtilis but not against fungi Candida albicans or Trichophyton mentagrophytes (Perry and Foster, 1995). Glaucescenolide (50) isolated from Schistochila glaucescens, exhibited antifungal activity against Trichophyton mentagrophytes (Scher et al., 2002). Ent-1b-hydroxykauran-12-one (51), a constituent of Paraschistochila pinnatifolia, demonstrated weak antifungal activity against C. albicans (Lorimer et al., 1997). OH

O

H3C

H3C

O

CH3

H3C

CH3

CH3

CH3 OH

(47)

O

O HO

H3C

CH3

O

O

CH3

(48)

(49)

H H3C CH3

OH

(50)

HO

(51)

9.5.3 The antioxidant property of bryophytes Few of the bryophyte species have been studied in context to antioxidant activity. The study suggests that some of the liverworts and moss possess strong antioxidative machinery which helps them to survive in the extreme climate and stress condition. The study conducted on antioxidant activity of the Antarctic mosses Sanioniauncinata (Hedw.) Loeske and Polytrichastrum alpinum (Hedw.) G.L. Sm. var. alpinum has indicated their potential to be used as antioxidants for medicinal and cosmetic purposes (Paudel et al., 2008; Bhattarai et al., 2009). Also, the antioxidant activity of some of the species of bryophyte like Atrichumundulatum (Hedw.) P. Beauv., Polytrichum formosum (Hedw.), Pleurozium schreberi (Brid.) Mitt. and Thudiumtam ariscinium (Hedw.) Schimp. has been screened, and all tested species have shown antioxidant effects lower than the positive control, caffeic acid (Chobot et al., 2008). Bioactivity-guided fractionation of the diethyl ether and methanol extracts of Mastigophora diclados using the DPPH radical scavenging assay resulted in the isolation of diplophyllolide D (52), α-herbertenol (53), herbertene-1,12-diol (54), mastigophorene C (55), mastigophorene D (56), diplophyllin (43), and mastigophorene A (57), among which herbertene-1,12-diol, mastigophorene C, and mastigophorene D showed strong

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221

antioxidant activity. The activities of herbertene-1,12-diol and mastigophorene D were more potent than vitamin C and similar to quercetin (Komala et al., 2010). Marchantin A (58) also showed free-radical scavenging activity (Huang et al., 2010). Marchantin H (59) showed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates (Hsiao et al., 1996). This effect was more potent than those of desferrioxamine or other classical antioxidants. Marchantin H suppressed NADPH-dependent microsomal lipid peroxidation without affecting microsomal electron transport of NADPH-cytochrome P450 reductase. It also inhibited copper-catalyzed oxidation of human low-density lipoprotein. Hsiao et al. (1996) concluded that marchantin H is a potentially effective and versatile antioxidant, and can be used as a chaperone protecting the biomacrocyclic molecule against peroxidative damage. H3C

CH3

H3C H3C OH OH

CH3 CH3

H2C

OH

CH3 H3C

CH3

HO

OH

CH3

HO CH3

O O CH3

(52)

(53)

(54)

H3C H3C

HO

CH3

H3C

H3C H3C

(55)

CH3

H3C

CH3

H3C

H3C

HO

OH

OH

HO OH

HO

OH CH3 CH3

O

CH3

H3C H3C

O

OH HO

O

O

HO OH

CH3

CH3

(56)

HO

HO

HO

(57)

(58)

(59)

9.5.4 Insect antifeedant, mortality, and nematocidal activity Plagiochiline A (60), found in several Plagiochila species, is a strong antifeedant against the African armyworm (Spodoptera exempta) (Asakawa, 1982). Plagiochiline A showed nematocidal activity against Caenorphabdiitis elegans (111 lg/mL). The pungent sacculatal (28) kills tick species Panonychus citri. Plagiochiline A and eudesmanolides from Chiloscyphus polyanthos, and gymnocolin (61) from Gymnocolea inflata also have antifeedant activity against larvae of Japanese Pieris species (Asakawa, 1995a,b). However, their activity is less than plagiochiline A. A series of natural drimanes and related synthetic

222

Evolutionary Diversity as a Source for Anticancer Molecules

compounds were tested for antifeedant activity against aphids. Polygodial (62) from the Porella vernicosa complex and warburganal (63) from the African tree Warburgia ugandensis were the most active substances. Natural polygodial and the synthetic enantiomer showed similar levels of activity as aphid antifeedants. Polygodial (62) killed mosquito larvae and had mosquito repellent activity which is stronger than the commercially available DEET. Plagiochilide isolated from Plagiochila species killed Nilaparvata lugens (Delphacidae) (Asakawa, 1995a,b). O

H3C H3C

H3C CH3

O

CH3

H3C

CH3

CH3

O O

O

O O H3C

(60)

O

O

O

OH

H3C

O

CH3

H3C

O

O

H3C

O

(61)

O

O

(62)

(63)

9.5.5 Plant growth inhibitory activity Almost all crude extracts from liverworts which contain pungent or bitter substances show phytotoxic activity. Polygodial (62) inhibits the germination and root elongation of rice in husk. At a concentration of less than 25 ppm, it dramatically promotes the root elongation of rice (Asakawa et al., 1979; Asakawa, 1982). Yoshikawa et al. (2002) reported that lunularic acid (27) inhibited germination and growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) and gibberellic acid-induced α-amylase induction in embryo-less barley seeds, which has been recognized as specific activity of abscisic acid (ABA). Both lunularic acid and ABA equally inhibited the growth of the liverwort Lunularia cruciatastra in callus. Superimposition between the stable conformers of lunularic acid and those of ABA obtained by computational analysis (MM calculations) has been used to explain why lunularic acid has an ABA-like activity in higher plants. This indicates that both lunularic acid and ABA bind to the same receptor. On the basis of the distribution pattern of lunularic acid and ABA, a hypothesis has been proposed that higher plants have altered their endogenous growth regulator from lunularic acid to abscisic acid in their evolutionary process (Yoshikawa et al., 2002).

9.6 Bryophytes as a potential biopharming agents Bryophytes have indeed penetrated the forefront of modern medicines. Although a vast variety of biopharmaceuticals has been produced in microbial or mammalian cells, plant-based production system possesses several advantages over the mammalian and microbial system, thus, making them interesting alternatives. Bryophytes offer the researchers and the company a high production system that can be grown without antibiotics, hence avoiding the danger of contamination of the final product. Apart

Chapter 9 • Natural product metabolites in bryophytes

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from these advantages, mosses are the only plants known to show a high frequency of homologous recombination. They allow the stable integration of inserted genes into the host cell. A highly complex moss system, compared to bacteria and fungi, permits a much wider array of expression than is possible in other systems. Given the above advantages of mosses over another production system, today, many complex biopharmaceuticals are being produced by moss bioreactors. The moss (Physcomitrella patens) has been successfully grown in a bioreactor that requires only water and minerals to nourish the moss, in the presence of light and CO2 (Greenovation). Consequently, many complex proteins can be produced in moss bioreactor. Other products are human growth factor that is required by the researcher for tissue culture. This plant has successfully been able to produce human proteins (Hohe and Reski, 2002; Decker and Reski, 2008), and is the only plant being used to produce the blood-clotting factor IX for pharmaceutical use.

9.7 Chemical syntheses of bryophyte components Chemical synthesis is very important for acquiring the natural products on a larger scale and for the independent elucidation of structures. Despite the large number and variety of the bryophyte components are isolated and structurally classified, comparatively little is known about syntheses of these naturally occurring compound classes. The investigations undertaken on representative examples in the domains of the phenolic and the nonphenolic components have centered on new types of structure and stereochemical aspects and, above all, on important biological activities. Most compounds found in liverworts are composed of lipophilic mono-, sesqui-, and diterpenoids and aromatic compounds, such as bibenzyls (14) and bis(bibenzyls) (15). The presence of nitrogen or sulfur containing compounds in bryophytes is very rare. However, several nitrogen-containing compounds have been isolated from the Mediterranean liverwort Corsinia coriandrina belonging to the Corsiniaceae (Marchantiales) (Kovtonyuk et al., 2005). The most characteristic chemical phenomenon of liverworts is that most sesqui- and diterpenoids are enantiomers of those found in higher plants although there are a few exceptions such as drimane, germacrane, and guaianes. It is noteworthy that the different species of the same genera, like Frullania tamarisci and F. dilatata (Frullaniaceae), each produces different sesquiterpene enatiomers (Asakawa, 1995a,b). Some liverworts, such as Lepidozia species (Lepidoziaceae), biosynthesize both enantiomers (Toyota et al., 1996). Flavonoids are ubiquitous components in bryophytes and have been isolated from or detected both in the Marchantiophyta and the Bryophyta. The presence of hydrophobic terpenoids is very rare in the Marchantiophyta. A few bitter kaurene glycosides have been found in the Jungermannia species. However, a number of flavonoid glycosides have been detected both in Marchantiophyta and Bryophyta.

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9.8 Biotechnological applications for effective utilization of bryophytes for therapy 9.8.1 In vitro culturing of bryophytes In addition to the difficulties of acquiring large amounts of bryophytes, there is the problem that many of them are spread over wide areas and occur only in small populations. Field cultivation of bryophytes similar to that of useful and medicinal plants is not practicable; even if the required ecological conditions were created, mixed populations would result. Bryophyte cultures offer effectively the same advantages as in vitro cultures of higher plants (Teuscher, 1973). Plants from all climatic zones can be grown in cultures. Comparatively constant production of material can be achieved through controlled culture conditions. Bryophyte cultures have additional advantages: The plants, often only a few millimeters large, can be cultivated easily, even in differentiated form, in fermenters. Since the bryophyte plant itself is haploid, it is possible to select mutants relatively rapid (Knight et al., 1988).

9.8.2 Genetic engineering Transgenic moss The transgenic plants have a low-cost and safe pathogen-free advantage of producing complex recombinant pharmaceutical proteins. However, the high plant-specific protein N-glycosylation was reported to be immunogenic in biopharmaceutical production (Faye et al., 2005). The moss, with the strong regeneration and feasibility of targeted gene replacements, has non-immunogenic humanized glycan patterns for recombinant biopharmaceuticals (Decker and Reski, 2008). Plant protoplasts which had its cell walls removed offer a versatile cell-based experimental system (Yoo et al., 2007). The cell wall is degraded by digestion with different mixture of hydrolytic enzymes example cellulase, hemicellulase. Protoplasts are used to create hybrid cells via protoplast fusion. In some cases, they are totipotent and are thus capable of regenerating into the plant on an appropriate culture medium. Such protoplasts can be used in plant transformation. Mosses, unlike other plant protoplasts, do not need phytohormones for regeneration, nor do they form a callus during regeneration. Instead, they regenerate directly into the filamentous protonemal thus mimicking a germinating moss spore. In general, the transformation frequencies of mosses have been reported to be higher than other plants, nevertheless, they have not reached the levels achieved in eukaryotic microbes such as yeasts. Although the frequency of transformants achieved is on the ascendancy, and the reliability of the transformation procedure is improving too, efforts should be made to continue to find perfect the conditions that will improve both frequency and reliability of the methods used to achieve transgenic moss (Cove, 2000).

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Now the research platform with mosses has not been completed. However, a genetic linkage map has been yielded by crossing a French ecotype to a British ecotype (Kamisugi et al., 2008). We need more time to establish the cDNA library and genome DNA, which could be identified by using next-generation sequencing technologies (Prigge and Bezanilla, 2010). The mechanism of homologous recombination rate with P. patens has not yet been researched enough. The P. patens genome can only represent the portion of the terrestrial plant, which has not been studied by gene knockout. Thus, the mechanism of homologous recombination rate should be investigated. Mosses, especially P. patens, are the most attractive to study due to their simplified molecular biology characteristics, compared to higher plants. The development of transgenic studies in moss could make them model plants for genetic studies and also play a catalytic role in molecular biology in plants similar to that of Arabdopsis. The completely sequenced genome of the model moss Physcomitrella patens contains orthologs of all the eight-core lignin biosynthetic enzymes required for the biosynthesis of p-coumaryl alcohol and coniferyl alcohol, whereas in the green algae they were not recorded (Silber et al., 2008; Beike et al., 2014) reported high contents of polyunsaturated fatty acid in a few species studied. They also reported tissue-specific differences in fatty acid contents and fatty acid desaturase encoding gene expression. The best-studied and completely sequenced model-moss Physcomitrella patens bring insights into developing molecular farming by mosses, e.g., implications of plant glycans in the development of innovative vaccines (Rosales-Mendoza et al., 2016). Bryophyte haploid genomes, high rate of homologous recombinations and facile foreign genes incorporation enable easy knockin and knock-out line productions, following by the green productions of biobetters or biopharmaceuticals (Reski et al., 2015). These examples not only encourage us to search for the new chemical compounds and remedies in bryophytes but also allow us to develop, through bioreactors and molecular farming, green factories for target molecules.

9.9 Challenges and future prospects Discovering patterns in the metabolite profiles can reveal new ecological and biogeochemical relationships as the biochemistry of bryophytes is related to the environment, climate, and biotic interactions (Sardans et al., 2011). Further, bryophytes are important environmental indicators and have been used as predictors of past climate change to validate climate models and potential indicators of global warming (Gignac, 2001). They play a chief role in ecosystem functions, such as soil development (Zhao et al., 2009), nutrient biogeochemical cycling (Uchida et al., 2002; Turetsky, 2003), water retention (Uchida et al., 2002), plant colonization seed germination, seedling growth, and forest renovation (Uchida et al., 2002; Frego, 2007; Zhao et al., 2009).

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9.10 Conclusion The bryophytes hold great potential for novel chemicals, though links between ethnopharmacology and chemistry have not been revealed among hornworts, but only in liverworts and mosses. Bryophytes are already used to produce chemicals and proteins (Simonsen et al., 2009; Ikramt et al., 2015). Along with the studies on novel transformation technology (King et al., 2016) and bioreactors developments, few companies have started to realize the potential of bryophytes. Here we also show that not only the moss Physcomitrella patens should be used, but a whole range of bryophytes are of real interest to produce known and novel natural products. In vitro establishment of single species and propagation of clean material can be a useful biotechnological tool for acquiring enough plant material (Rowntree et al., 2011; Sabovljevi
c et al., 2014a), and also optimizing tool for elicitation of certain secondary metabolite overproduction (Sabovljevi
c et al., 2017). Only a few percent of liverworts and mosses are chemically studied. Further chemical and pharmacological study on the secondary metabolites of bryophytes will give us a number of different new compounds that could be useful for cosmetic, pharmaceutical, medicinal or agricultural fields.

Acknowledgments The author thanks The Director, CSIR-CFTRI and Dr. Nandini P. Shetty, Principal Scientist, CSIR-CFTRI, Mysuru, Karnataka, India for their support.

Conflict of interest None.

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Zhan, X., et al., 2015. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS). Plant Physiol. Biochem. 96, 110–114. https://doi.org/10.1016/ j.plaphy.2015.07.011. Zhao, J., et al., 2009. Progress in the study of algae and mosses in biological soil crusts. Front. Biol. China 4 (2), 143–150. https://doi.org/10.1007/s11515-008-0104-0. Zhu, R.L., et al., 2006. Antibacterial activity in extracts of some bryophytes from China and Mongolia. J. Hattori Bot. Lab. (100), 603–615. https://doi.org/10.18968/jhbl.100.0_603.

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10 Landscape of natural product diversity in land-plants as source for anticancer molecules Akanksha Srivastavaa and Richa Raghuwanshib a

DEPARTMENT OF BOTANY, I NSTITUTE OF SCIE NCE, BANARAS HINDU UNIVERSITY, VARANASI, UTTAR P RADE SH, INDIA b DEPARTMENT OF BOTANY, MAHILA MA HA V I D Y AL AY A , B AN A R A S HINDU UNIVERSITY, V ARANASI, UTTAR P RADESH, INDIA

10.1 Introduction According to world cancer report (2018), cancer is the second leading cause of death and about 9.6 million worldwide are estimated to die from cancer in 2018. More than 36 million (63% global death) die each year from non-communicable diseases, including 14 million people who die too young before the age of 70. Increasing multidrug resistance in pathogenic bacteria and fungi, with the raised number of cancer patients, is causing concern worldwide, especially in the third world countries. WHO launched the Global Action Plan for the Prevention and Control of non-communicable diseases (2013–20) that aimed to reduce by 25% premature mortality from cancer, cardiovascular diseases, diabetes and chronic respiratory diseases by 2025. Cancer is a disease which severely affects the mortality and morbidity in human population. Being a life threatening disease, it has fetched the attention toward preventive and treatment therapies. The treatment of cancer still lacks fully effective and safer drugs for anticancer therapy. Increased morbidity after several therapies of cancer treatment, prolonged toxicity of even well established chemically derived drugs pushed the scientist to explore biological and therapeutic properties of medicinal plants. Natural medicines extracted from plants, have proved to be effective, less toxic and safe and thereby provide a wide scope and opportunity. Decade has witnessed the paradigm shift of treatment modalities toward the natural medicines. According to World Health Organisation (2007), land plant-derived drugs trade was worth US$100 billion and expected to reach US$5 trillion by 2050 (Rajeswara Rao et al., 2007). Researchers attempt to learn the significance of phytochemicals and derive innovative ways for their use in treatment of cancer disease is summarized.

Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00010-2 © 2021 Elsevier Inc. All rights reserved.

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10.2 Plant diversity and their anticancer potential Promising potential of medicinal plants in various treatment modalities of cancer has been evidenced since ages. Sumerians and Akkadians mentioned the use of plants as medicine in their work, around 2600 BC. Egyptian pharmaceutical records, The “Ebers Papyrus,” around 1500 BC, documented over 700 drugs (Borchardt, 2002). The Chinese Materia Medica, about 1100 BC (Huang, 1998), Ayurvedic system of Susruta and Charaka dates from about 1000 BC and Greek physician (AD 100) also documented the use of medicinal plants. The evolution of plants with increasing levels of complexity starting from the earliest algal forms, to the bryophytes, pteridophytes and to the complex gymnosperms and angiosperms enriched them with phytochemicals to survive. Plants in all of these groups continue to thrive, especially in the environments in which they have evolved. The plant kingdom is thus a cistern of bioactive molecules which may give favorable results toward anticancer activities. A glimpse of the plant kingdom diversity exhibiting anticancer potential is depicted in Fig. 10.1. Phytochemicals offer protection against a variety of chronic ailments including cardiovascular diseases, obesity, diabetes, and cancer (Kapoor, 1990; Dev, 1999). Over 10,000 plants are known from over 300 plant families, among which 10–25% are higher plants. Phytochemicals are specific in their functions and selectively target tumor cells less affecting the normal cells. Since carcinogenesis is a multifaceted phenomenon that involves many signaling cascades, phytochemicals are considered suitable candidates for anticancer drug development as they have pleiotropic actions on targeted cells in multiple manners. It has been estimated that only around 10% of the plant kingdom species have been studied for treatment of different diseases (Iqbal et al., 2017). Phytochemicals are derived from different parts of the plant which perform several pharmacological functions. Several plant products such as flavonoids, alkaloids, saponins, lignans, terpenes, taxanes, minerals, vitamins, glycosides, gums, oils, biomolecules, and other primary and secondary metabolites play significant anticancer roles by either inhibiting cancer cell activating proteins, enzymes and signaling pathways, by activating DNA repair mechanism, inducing antioxidant action and so on. Secondary metabolites of plants inhibit the proliferation of cancer cells and induce apoptosis by increasing sub-G1 phase population of cells with lower DNA content and condensation of chromatin. Effort has been made in many comprehensive reviews that highlight the recent progress and milestones achieved in cancer therapies using phytomolecules with their mechanism of action on nuclear and cellular factors (Wang et al., 2012; Iqbal et al., 2017).

10.3 Microbial antitumor products Natural resources have always been used by humans for their welfare. Although land resources have always been an easy source to explore, a great number of antitumor compounds are produced by microorganisms. Keeping in mind its evolutionary significance, phytochemical and pharmacological studies of this group may lead to the discovery of certain novel metabolites having unique therapeutic potential. Cyanobacteria, are arguably a

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FIG. 10.1 Diverse plant kingdom exhibiting anticancer potential.

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group of the photosynthetic oxygen-evolving prokaryotes that originated
2.8 billion years ago (Schopf and Packer, 1987; Schwartzman et al., 2008), could have faced many environmental stresses during evolution, have been perceived as the novel source of antibiotic, antifungal, anticancer, antihelminthic, enzyme inhibitors, photosynthesis inhibitors, hemagglutinins, antiviral, anticoagulant, antiinflammatory, antimalarial, antiprotozoal, antituberculosis, and immunosuppressive activities that show prodigious bioactive potential for drug targeting (Burja et al., 2001; Tan, 2007, 2010, 2013; Nunnery et al., 2010; Mayer et al., 2011; Gerwick and Moore, 2012; Namikoshi and Rinehart, 1996; Jaiswal et al., 2008, €rner, 2008; Prasanna et al., 2010; Vestola et al., 2014; Singh et al., 2005; Volk Sivonen and Bo and Furkert, 2006; Bhatnagar and Kim, 2010; Pearson et al., 2010; Jones et al., 2011). Curacin A, a mixed polyketide, nonribosomal peptide possessing antimitotic and antiproliferative activity, was isolated from L. majuscula (Geders et al., 2007). The most common causes of cancer deaths are cancers of lung (1.59 million), liver (745,000) stomach (723,000), colorectal (694,000), breast (521,000) and esophageal cancer (400,000). Green carbon nanotags (G-Tags) from the harmful cyanobacteria, have been synthesized for use in cancer therapy because of their high solubility, excellent photostability, low cytotoxicity, and ability to induce death in multiple cancer cell lines, including human hepatocellular liver carcinoma (Hep-G2) and human breast adenocarcinoma (MCF-7) (Lee et al., 2014). Lipopetide derived from L. majuscula [somocystinamide A (ScA)] induces apoptosis in tumor and angiogenic endothelial cells (Wrasidlo et al., 2008). Cylindrospermopsin (CYN), synthesized by many fresh water cyanobacteria, has impacts on the cell cycle arrest and double strand breaks in HepG2 cells (Sˇtraser et al., 2013). A number of chemotherapeutic agents (60% of the approved drugs) for cancer are sourced from natural compounds (Cragg et al., 1997). Some earlier studies have shown that filamentous marine cyanobacteria produce a wide range of compounds with pro-apoptotic properties, were the major sources of biomolecules for preventing cancer proliferation, neurodegenerative disorders and infectious diseases (Tan, 2013; Costa et al., 2012). Cell shrinkage, membrane budding, and apoptosis were observed inhuman promyelocytic leukemia (HL-60) cells exposed to the aqueous extracts of unicellular marine strains of Synechocystis and Synechococcus (Martins et al., 2008). Freshwater cyanobacteria Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Arthrospira sp. CCC732 showed anticancer potential using human colon adenocarcinoma (HT29) and human kidney adenocarcinoma (A498) cancer cell lines, along with the normal rat-kidney cells (NRK52E) using Bio-Plex Pro human cancer biomarker panel, cell cycle analysis, and the calcein-based cell viability assay for the purpose. Cell cycle analysis and multiplex assays using cancer biomarkers confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as the anticancer drug resources (Srivastava et al., 2015, 2016).

10.4 Anticancer property of fungi Basidiomycetes have been reported for antitumor activity due to presence of polysaccharides which are different in their chemical composition, configuration and physical properties. There are many fungal species, studied for possible antitumor activity, such as

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Sclerotina sclerotiorum, Schizophyllum commune (Fujimoto et al., 1991), and Grifola frondosa (Ishibashi et al., 2001). Insoluble glucans have been isolated for the first time from the mushroom Lentinus edodes (Chihara et al., 1969). Borchers et al. (2004) reviewed on mushrooms as anticancer immune modulators, the mushroom Agaricus blazei contains compounds [an antitumor glucan with a (1 ! 6)-β-backbone, an (1 ! 6)-α- and (1 ! 4)-αD-glucan complex and a glucomannan with a main chain of (1 ! 2) β-linked D-mannopyranosyl residues] that were found to inhibit tumorigenesis (Itoh et al., 1994; Mizuno et al., 1998, 1999).

10.5 Responses of cancer cells to the lichen compounds Lichens are fascinating symbiotic organisms, biosynthesizing a broad spectrum of secondary metabolites (also called lichen substances) and polysaccharides. They considerably exhibit anticancer effects and potential therapeutic application in medicines, despite only few have been tested for their biological significance. Four typical secondary metabolites of lichens (parietin, atranorin, usnic acid, and gyrophoric acid) have been reported for their antiproliferative/cytotoxic effects for various human cancer cell lines (Backorova et al., 2011). Secondary metabolites lobaric acid and lobarstin derived from lichen Stereocaulon alpnum, from the antarctic region demonstrated several biological activities such as antitumor, antiproliferation, antiinflammation, and antioxidant activities. Lichen-derived usnic acid and zinc sulfate are useful for adjuvant treatment after radiation therapy as it accelerates reepithelization and reduces recurrence in cervical cancer caused by human papillomavirus (HPV). Recent study suggests usnic acid as a non-toxic anticancer drug which reduces the proliferation of human breast cancer cells and lung cancer cells without DNA damage. Atranorin (ATR) is a secondary metabolite, found in a variety of lichen species (Kristmundsdottir et al., 2005). Atranorin, extracted from antarctic Stereocaulon caespitosum exhibits tumor suppressive roles in hepatocellular carcinoma (HCC) through regulation of the cell cycle, cell death, and metastatic potential ( Jeon et al., 2019). Isolated ATR displayed their role in exhibiting antinociceptive effects (Melo et al., 2008) and inhibits leukotriene B4 synthesis in leukocytes, which affects inflammatory processes (Kumar and Muller, 1999). Physciosporin, a chlorinated depsidone, isolated from lichen, Pseudocyphellaria coriacea, Pseudocyphellaria physciospora is well known for its inhibitory activity against lung cancer cell motility. It was first isolated in 1977 from the lichen (Yang et al., 2015). Secondary metabolites formed via the polyketide pathway are unique (Blanco et al., 2005) and are reported to exhibit antibiotic, antimycobacterial, antiviral, antiinflammatory, analgesic, antipyretic, antiproliferative, or cytotoxic activities (Oksanen, 2006; Stocker-Worgotter, 2008). Lichen extracts are therapeutically beneficial probably due to the wide range of biological activity of their endogenous secondary metabolites. The adaptation of lichens to high solar radiation exposure enables them properly to absorb strong ultraviolet radiation, a property which has gained recognition in various cosmetic formulations and sunscreens for skin (Bernard et al., 2003; Muller, 2001).

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10.6 Therapeutic potential of bryophytes against cancer Bryophytes being considered as the first land plants are placed between the algae and pteridophytes and have been divided into three classes as liverworts, hornworts, and mosses. Bryophytes gained importance in recent years for its significant therapeutic values. Bryophytes are a reservoir of naturally occurring bioactive molecules (Asakawa, 1981, 2007; Zinsmeister et al., 1991), that have antifungal (Cheng et al., 2009; Wu et al., 2008), antibacterial, antiviral (van Hoof et al., 1981; Scher et al., 2010; Singh et al., 2006), antiinflammatory (Ivanova et al., 2007), antioxidative (De and De, 2012; Cioffi et al., 2011) and anticancer activity. Bryophytes such as liverworts and mosses has been reported for its cytotoxic activity, related therapeutic potential (Spjut et al., 1986; Spjut and Kingston, 1992) and anticancerous efficacy on various cancer cell lines using multiplex analysis, biochemical markers of apoptosis and necrosis induction such as DNA fragmentation, nuclear condensation, caspases activation etc. (Abhijit and Anuradha, 2015). Modern techniques have enabled isolation and structural elucidation of bioactive molecules from bryophytes.

10.6.1 Bryophytes derived cytotoxic compounds 1. Liverworts: Liverworts contain a number of bioactive molecules. Bryophytes possess a number of terpenoid compounds such as mono, sesqui, di, and triterpenoids, flavonoids, sterols, and characteristic phenolic bibenzyls (Abhijit and Anuradha, 2015). About 80% of the sesqui- and diterpenoids found in higher plants are the enantiomers of those found in hepatics (Asakawa, 2007). (a) Monoterpenes: Cytotoxic activity has been recorded from isoprenyl phenyl ethers of Trichocolea mollissima—methyl 4-[(5-oxogeranyl)oxy]-3-methoxybenzoate as the major cytotoxic agent. Hemi- and monoterpene moieties recorded from the liverwort Conocephalum conicum and Jungermannia vulcanicola, showed cytotoxic activity against human HepG2 cells (Valterova et al., 1992; Adam and Croteau, 1998; Yokouchi et al., 1984; Lu et al., 2006). (b) Sesquiterpenes: Several sesquiterpenoids compounds from bryophytes exhibits cytotoxicity. Diplophyl-line isolated from Diplophyllum albicans and Diplophyllum taxifolium showed significant activity against human epidermoid carcinoma (Ohta et al., 1977). Sesquiterpenoids costunolide and tulipinolide, isolated from the liverworts Conocephalum supra-decompositum, Frullania monocera, Frullania tamarisci, are known tumor growth-inhibitors, among the DNA-damaging natural products (Gunatilakaa and Kingston, 1997). (c) Diterpenoids: Muscicolone, a novel entlabdane type diterpenoid, isolated from the liverwort Frullania muscicola inhibits some human tumor cells by producing cytotoxic effects (Lou et al., 2002). Diterpenoids with their anticarcinogenic potential opens the scope of their use in therapy against several human cancers. Examples of diterpenoids from other liverworts are—cis-clerodane extract from liverwort Gottschelia schizopleura which exhibits cytotoxic activity against liver

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hepato-blastoma (HEP-G2), lung carcinoma (A549), breast ductal carcinoma (MDA-MB-435) (Liu et al., 2009). (d) Triterpenoids: Liverwort reported for triterpenes are Fossombronia alaskana, Fossombronia pusilla (Grammes et al., 1994), Conocephalum japonicum (Toyota and Asakawa, 1993), Nardia scalaris (Benes et al., 1981), Blepharidophyllum densifolium, etc. (Flegel and Becker, 1999). These triterpenoids have shown activity against various cancer cell lines such as the liverwort Ptilidium pulcherrimum showed anticancer activity against the PC3, MDA-MB-231, and Hela cells lines of which ursane triterpenoids has shown moderate cytotoxicity against PC3 cells (Guo et al., 2009). (e) Bibenzyls and bisbenzyls: Various types of secondary metabolites such as Riccardin, from the liverwort Riccardia multifida (Asakawa et al., 1983; Kodama et al., 1988) Marchantin from Marchantia polymorpha and M. tosana (Friederich et al., 1999), Neomarchantins from Schistochila glaucescens (Scher et al., 2002), Plagiochin from M. polymorpha, isoplagiochin from Plagiochila fruticosa (Morita et al., 2009), Perrottetin from Radula perrottetii, dihydroptychantol A (DHA) from Asterella angusta (Sun et al., 2009; Li et al., 2011), Lunularin from Dumortiera hirsute (Lu et al., 2006) have been reported for their significant cytotoxic roles. 2. Hornworts: There is no report on cytotoxic compound from hornwort. Presence xyloglucans in the cell wall of hornworts separate it from liverworts and mosses. Phytochemical analyses were performed in some hornworts such as Anthoceros agrestis, Anthoceros caucasicus, Megaceros flagellaris (Trennheuser et al., 1994), etc. 3. Mosses: Cytotoxicity and antitumor activity has been reported in Polytrichum juniperinum, Claopodium crispifolium, Anomodon attenuates, Isothecium subdiversiforme, and Thamnobrium sandei (Spjut et al., 1988; Sakai et al., 1988; Suwanborirux et al., 1990).

10.7 Ferns a treasury of anticancer agents India is among the 17 M-biodiverse countries in the world with its major part lying within the Indo-Malaya and Palearctic ecozone. Having only 2.5% of the earth’s land area, India accounts for 7–8% of the recorded species of the world including 45,000 species of plants and more than 1300 pteridophytes (Chandra et al., 2008; Ministry of Environment and Forests, 2009). Pteridophytes are widely distributed in the mountainous regions of Himalayas, Western Ghats and Eastern Ghats, grow in moist tropical and temperate forests and eco-geographically threatened regions from sea level to the highest mountains (Dixit, 2000). The Indian Western Ghats harbors about 320 species of ferns, which is used as medicines to cure diseases. In Tamil Nadu, a state from India, 30 pteridophytes were reported to cure various ailments viz., wound healing, kidney problem, chronic disorders, several aches, stomach problems, ulcer, leprosy, ophthalmic, typhoid, urinary bladder, and rheumatism (Sarker et al., 2011) and were also reported as alternative of drug resistance to

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improve cancer chemotherapy and cancers (Gottesman, 2002). Selaginella willdenowii contains isocryptomerin, derivatives of amentoflavone and robustaflavone which is significantly cytotoxic against various cancer cells (Silva et al., 1995). Selaginella tamariscina act as urokinase plasminogen activator, inhibits Lewis lung carcinoma (Yang et al., 2007); inhibited Leukemia cancer cell of HL-60 cell (Lee et al., 1999); degraded leukemia cancer cell of U937 (Lee et al., 1996; Yang et al., 2007); reduced proliferation of nucleus antigen cell from stomach epithelium (Lee et al., 1999); chemopreventive for gastric cancer (Lee et al., 1999); induced apoptosis of cancer cell through DNA fragmentation and nucleus clotting (Ahn et al., 2006); and induced breast cancer apoptosis through blockage of fatty acid synthesis (Lee et al., 2009). Selaginella tamariscina increases p53 gene expression and induce G1 arrest (Lee et al., 1999). Various species of Ophioglossum are used as the antibiotic and anticancer chemotherapeutic agent (Khandelwal, 1989). Swamy et al. (2006) reported that structurally unique biflavonoids isolated from two Indian species of Selaginella viz., S. chrysocaulos and S. bryopteris., Selaginellin A and B have been first isolated and described by Cheng et al. (2008) from Selaginella tamariscina. Li et al. (2007) reported flavonoids from Selaginella delicatula and S. willdenowii, used as anticancer agent. Antioxidant based drug formulations are used for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer (Devasagayam et al., 2004). Peres et al. (2009) evaluated the antioxidant activity of Microgramma vacciniifolia using DPPH assay. Daonian et al. (2010) studied the antioxidant and hepatoprotective activity of Arachniodes exilis. Ferns with strong antioxidant properties include Cyathea latebrosa, Cibotium barometez, Drynaria quercifolia, Blechnum orientale, and Dicranopteris linearis. Paulsamy et al. (2013) reported the antioxidant activities of Actiniopteris radiata and Equisetum ramosissimum methanolic extracts. Many pteridophytes have been reported for their antioxidant potential like Asplenium aethiopicum, Diplazium esculentum, Marsilea crenata, and Stenochlaena palustris (Johnson et al., 2014; Amoroso et al., 2014). Breast cancer is one of the most common and serious malignancies worldwide. Despite intensive cancer control efforts, it remains the second-leading cause of cancer death among women (Harris et al., 2000). George et al. (2018) reported that Pyrrosia heterophylla, a herb used in ethnomedicinal practices to be an effective antiproliferative agent which could induce apoptosis in the human breast cancer cell line MCF7. Amentoflavone extracted from Selaginella tamariscina were effective in inhibiting the proliferation of five types of cancer cells, including HeLa (human cervical carcinoma cells), BEL-7402 (human hepatoma carcinoma cells), MCF-7 (human breast cancer cells), PANC-1 (human pancreatic cancer cells) and showed reliable activity against HL-60 (human leukemia cells) (Jing et al., 2010). Sarker et al. (2011) investigated the antitumor properties of Selaginella ciliaris (80%), Marsilea minuta (82.32%) and Thelypteris prolifera (75.68%) at 1000 ppm. Sri Handayani et al. (2013) evaluated the cytotoxic effect and apoptosis induction by Selaginella plana fractions in carinogenic MCF-7 cell lines. Li et al. (2014) studied the antiproliferation effects of four fractions of Selaginella doederleinii against five human cancer cells. Li et al. (2014) has demonstrated the relatively stronger cytotoxic activity of

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S. labordei, S. tamariscina, S. uncinata, and S. moellendorfii against Bel-7402 and HeLa cells. Kaewsuwan et al. (2015) isolated three coumarin derivatives, interruptins A, B, and C from Cyclosorus terminans that inhibited the growth of MCF-7 human breast and HT-29 human colon cancer cells. Chai et al. (2015) evaluated the glucosidase inhibitory and cytotoxic activities of Blechnum orientale, Davallia denticulata, Diplazium esculentum, Nephrolepis biserrata, and Pteris vittata.

10.8 Anticancer property of gymnosperm Gymnosperms with bioactive compounds having anticancer properties have played a vital role in disclosing the role of phytochemicals in cancer chemotherapy through drugs development (Wani et al., 1971; Cragg and Newman, 2009). The alkaloids and their counterpart target DNA replication or protein synthesis of tumor cells, resulting in apoptosis of the neoplastic cells. Lead alkaloids obtained from gymnosperm derived anticancer drugs are harringtonine, cephalotoxin, and paclitaxel (taxol). The alkaloids and their analog, target DNA replication or protein synthesis in tumor cells leading to apoptosis of the neoplastic cells (Parness and Horwitz, 1981; Faddeeva and Beliaeva, 1997). Ginkgetin, ginkgolide A & B obtained from Ginkgo biloba leaf extract exerts growth inhibitory and apoptotic effects against hepatocarcinoma, ovary, prostate, colon, and liver cancer (Xiong et al., 2016)

10.8.1 Harringtonine Harringtonine and cephalotaxine isolated from Cephalotaxus harringtonia has antineoplastic activity against acute myelogenous leukemia (AML), myelodysplastic syndrome, acute promyelocytic leukemia, and intrathecal and is used in therapy of central nervous system leukemia (Huang and Xue, 1984). The chemical structure differ in structural side chain methylene group having esters series of harringtonine (harringtonine, homoharringtonine, isoharringtonine, deoxyharringtonine) are most active of the series while members lacking the methylene group are inactive (Eckelbargerb et al., 2008). Mechanism of action in homoharringtonine involves inhibition of protein synthesis targeting the ribosomes of cancer cells resulting inhibition of polypeptides synthesis initiation, blockage in the progression of cell cycle from G1 to S phase, G2 into M phase leading to apoptosis (Huang, 1975; Zhou et al., 1995). Homoharringtonine show activity against several kinds of leukemias including resistant ones to standard treatment and produce complete hematological remission in patients in late chronic-phase (O’Brien et al., 1995).

10.8.2 Taxol Cytotoxic activity of taxane dipertene paclitaxel (Taxol), discovered in the 1960s from the bark and leaf of Taxus baccata, Taxus brevifolia Nutt. (Western yew) and T. canadensis were identified as part of the National Cancer Institute (NCI) screening program at Research

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Triangle Institute (RTI) (Wani et al., 1971). Taxol is also isolated from needles, seeds, twig, and bark of T. brevifolia, with other Taxus species of gymnosperms (Vidensek et al., 1990). The yield quantity of taxol varies with genetic, epigenetic and environmental factors (Wheeler et al., 1992). The drug taxol was used against Kaposis sarcomas, breast, colon cancers and solid tumor types besides lung, bladder, prostate melanoma, esophageal, and neck cancers (Saville et al., 1995; Wall and Wani, 1996). The mechanism of action, Taxanes such as docetaxel and paclitaxel represent promising anticancer agents that act by binding to microtubules, apoptotic cell death and mitotic arrest (Parness and Horwitz, 1981). Paclitaxel together with b-tubulin leads to decrease in microtubule dynamics and cell cycle arrest at M phase while docetaxel, a semi synthetic derivative from T. baccata is used in breast, pancreas, prostate, and lung cancers therapies (Schutz et al., 2011; Xie and Zhou, 2017). Paclitaxel derivatives, such as larotaxel, milataxel, ortataxel, and tesetaxel are used either single or together with other therapies for urethral bladder, pancreatic, lung, and breast cancer (Ojima et al., 2016). Studies have shown that of 2069 cancer drugs used in treatment, 248 were taxane-derived drugs, containing 134 with paclitaxel, 105 with docetaxel, and 10 with miscellaneous taxanes, which are used either single or in combination with other anticancer drugs (Cragg and Newman, 2005). Drug-resistance is a common problem in Taxol treated patient thus a combined treatment of Taxol with Bcl-2 (B-cell lymphoma 2), a cell death regulator, inhibits angiogenesis tumor growth, and maintains Taxol sensitivity in cancer cells (George et al., 2009; Yang et al., 2010; Zhou et al., 2010; Morales-Cano et al., 2013).

10.9 Anticancer potential of angiosperms Developed countries in Europe and developing countries such as India and China have started cultivating medicinal plants on a large scale to keep up with increasing demands for alternative natural drugs. Foods with medicinal properties have gained attention on cruciferous vegetables, fruit berries etc. Grapes (Vitris vinifera) and “grape seed extract” has been recognized globally for its human health benefits. Grape stem extracts have demonstrated to have antioxidant properties, prevent DNA damage from reactive oxygen species and shown anticarcinogenic potential against an array of cancer cell lines from cervical cancer, thyroid cancer and many more. Red berries (grapes, blackberry, cranberry, raspberry, or apples and plums, red cabbage and red onion) extracts, cyanidin having antioxidant and radical-scavenging effects, which may act as anticancer agent (Kim et al., 2008). Phenethyl isothiocyanate (PEITC) and sulforaphane isolated from cruciferous plants (watercress, broccoli, cabbage), induce apoptosis in cell lines, have shown potency against melanoma, thus used as chemopreventive agent against breast cancer cells (Hahm and Singh, 2012; Moon et al., 2011), non-small cell lung cancer (Yan et al., 2011), cervical cancer (Huong et al., 2011; Wang et al., 2011), prostate cancer (Xiao et al., 2010; Hwang and Lee, 2010; Powolny and Singh, 2010), and myeloma cell lines ( Jakubikova et al., 2011).

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Lead alkaloids isolated from angiosperms that show anticancer properties is camptothecin. Other important alkaloids are isolated from these life forms include rohitukine, acronycine, thalicarpine, usambarensine, ellipticine, and matrines. Among the alkaloids benzophenanthridines, chelerythrine, and chelidonine isolated from Chelidonium majus, Zanthoxylum clava-herculis, Macleaya cordata, and Toddalia asiatica show anticancer activity. The fagoronine found in Zanthoxylum zanthoxyloides and other species in the genus Zanthoxylum is of immense therapeutic value. Chelerythrine chloride, nitidine, and nitidine chloride were isolated from roots of Zanthoxylum nitidum. Girinimbine and carbazole alkaloid were isolated from Murraya koenigii, and β-carboline alkaloids were isolated from Geissospermum vellosii. Extracts from angiosperm exhibit wide range of therapeutic benefits. Several plant compounds, mainly polyphenols, flavonoids and brassinosteroids (BRs) from angiosperm have shown anticancer properties. A large numbers of plants have been reported for their anticancer potential viz. Annona muricata (Annonaceae) (Chen et al., 2011, 2012a,b), Aloe vera Burm (Asphodelaceae), the bitter melon Momordica charantia (Cucurbitaceae), the neem tree Azadirachta indica (Meliaceae), Moringa oleifera (Moringaceae), Solanum nigrum (Solanaceae), noni Morinda citrifolia (Rubiaceae), the tubulin-interfering vincristine from the periwinkle plant Catharanthus roseus (Apocynaceae), paclitaxel from the Pacific yew Taxus brevifolia (Taxaceae), the topoisomerase I and II inhibitors irinotecan and etoposide, from Podophyllum (Berberidaceae) and the Chinese happy tree Camptotheca acuminate (Nyssaceae). Solowey et al. (2014) revealed the effects of extracts from Urtica membranaceae, Artemesia monosperma, and Origanum dayi over several cancer cell lines from lung, breast, colon and prostate cancers. Extract in combinations demonstrated killing activity specifically over cancer cells without much affecting the normal human cells by drug combinations of Vinca alkaloids, Taxus diterpenes, Podiphyllum lignans, and Camptotheca alkaloids enhances their anticancer effects and better therapeutic agents. Plant secondary metabolite used for anticancer activity can be categorized as the bisindole (vinca) alkaloids, camptothecins, epipodophyllotoxins, and taxanes. Derivatives of vinca alkaloids, vincristine, vinblastine, vinorelbine, vindesine, and vinflunine bind to β-tubulin and inhibit the dynamics of microtubules. Vincristine is used clinically for chemotherapy in adult, and significantly against acute lymphoblastic leukemia in pediatrics. It is also used to treat rhabdomyosarcoma, neuroblastoma, lymphomas, and nephroblastoma (Moore and Pinkerton, 2009; Almagro et al., 2015). However, the antitumor activity gets diminished by prolonged exposure to tumor cells to vincristine. It exhibits a bi-exponential elimination pattern in human blood suggesting diffuse distribution and tissue binding (Douer, 2016). It may also cause temporary or permanent peripheral neuropathy (Velde et al., 2017). However these factors can be overcome by encapsulation of vincristine into liposomes, thereby optimizing its delivery to target tissues with reduced toxicity. Taxanes such as paclitaxel are also microtubule disruptors which inhibit cell cycle phase transitions from metaphase to anaphase causing cell cycle arrest and apoptosis. Vincristine, vinblastine and paclitaxel are few isolated drugs with huge impact on cancer treatment.

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Compounds obtained from angiospermic plants with anticancer potential include polyphenols, flavonoids, and brassinosteroids. Polyphenolic compounds are antioxidant containing flavonoids, tannins, curcumin, resveratrol, and gallacatechins, are considered as potent anticancer compound. Flavonoids of polyphenolic compounds constitute anthocyanins, flavones, flavonols, chalcones.

10.9.1 Polyphenols Polyphenolic compounds such as flavonoids, tannins, curcumin, resveratrol, and gallacatechins have anticancer properties (Azmi et al., 2006). Peanuts, grapes, and red wine contain resveratrol. Green tea contains gallacatechins. Polyphenols being natural antioxidants reduces the risk of cancer and hence are suggested for their inclusion in person’s diet (Azmi et al., 2006; Apostolou et al., 2013). Polyphenols initiates apoptosis by regulating the mobilization of copper ions which are bound to chromatin inducing DNA fragmentation. Resveratrol in the presence of Cu (II), is capable of DNA degradation (Azmi et al., 2006). Cancer agents may be altered through the polyphenol regulating acetylation, methylation or phosphorylation by direct bonding. Curcumin treated cancer cells in various cells lines have shown suppression of the tumor necrosis Factor (TNF) expression through interaction with various stimuli (Gupta et al., 2014).

10.9.2 Flavonoids Flavonoids are physiologically active agents in plants and are well known for their health benefits (Huntely, 2009; Agati et al., 2012). High content of flavonoids (anthocyanins, flavones, flavonols, chalcones) may be found in a single structure of plant such as its seed (Wen et al., 2014). Researchers have found flavonoids to demonstrate cytotoxicity on cancer cells and to have high free radical scavenging activity (Cao et al., 2013). Purified flavonoids exhibit anticancer activities against variety of human cancers such as hepatoma (Hep-G2), cervical carcinoma (Hela), and breast cancer (MCF-7) (Wen et al., 2014). Even low concentrations of flavonoid extracts have demonstrated high percentage of anticancer activity. Kumar et al. (2013) reported that, flavonoids extracted from Erythrina suberosa stem bark (40 -methoxy licoflavanone) and Alpinumi soflavone (AIF) have cytotoxic effects in HL-60 cells (human leukemia).

10.9.3 Brassinosteroids Brassinosteroids (BRs) are naturally occurring compounds found in plants and have demonstrated significant anticancer properties over human tissues (Malı´kova´ et al., 2008; Steigerova´ et al., 2010, 2012). BRs induce apoptosis by interacting with the cell cycle (Malı´kova´ et al., 2008). BRs use in anticancer treatment gains an edge as the cytotoxicity of the agent confines to cancerous cell unaffecting the normal cells. BRs have demonstrated their successful use in investigations to treat a range of cancer cell lines which include; T-lymphoblastic leukemia CEM, multiple myeloma RPMI 8226, cervical carcinoma HeLa, lung carcinoma A-549 and osteosarcoma HOS cell lines, cell lines in breast cancer and prostate cancer (Malı´kova´ et al., 2008; Steigerova´ et al., 2010, 2012).

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10.10 Conclusion Plant derived anticancer agents are effective inhibitors of cancer cell lines. Being helpful in overcoming the side effects of chemotherapy further intensify the search for novel chemotherapeutic agents of plant origin. Drugs derived from vinca alkaloids along with pacitaxel being on late stages of clinical trials have proven the success stories of phytochemicals. At the same time development of nanotechnologies in drug administration has opened new hope in combating the side effects of chemotherapy. Although phytochemicals are emerging as a complementary medicine in cancer treatment but their increasing demand is putting pressure on plant biodiversity which demands effective conservation strategies at the same time. Although the exact mechanism of action of some of these bioactive compounds still remains to be investigated they still continue to serve as an attractive candidate of therapeutic value. Unrevealing the immense pharmacological information through isolation, structural elucidation and determining the mode of action of these active principles could open an exciting aspect of future drug development programs.

Conflict of interest We declare that we have no conflict of interest.

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Lu, Z.Q., Fan, P.H., Ji, M., Lou, H.X., 2006. Terpenoids and bisbibenzyls from Chinese liverworts Conocephalum conicum and Dumortiera hirsuta. J. Asian Nat. Prod. Res. 8 (1–2), 187–192. Malı´kova´, J., Swaczynova´, J., Kola´r, Z., Strnad, M., 2008. Anticancer and antiproliferative activity of natural brasinosteroids. Phtyochemistry 69, 418–426. Martins, R.F., Ramos, M.F., Herfindal, L., Sousa, J.A., Skaerven, K., Vasconcelos, V.M., 2008. Antimicrobial and cytotoxic assessment of marine cyanobacteria—Synechocystis and Synechococcus. Mar. Drugs 6, 1–11. Mayer, A.M.S., Rodriguez, A.D., Berlinck, R.G.S., Fusetani, N., 2011. Marine pharmacology in 2007-8: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 153, 191–222. Melo, M.G.D., Araujo, A.A.S., Rocha, C.P.L., Almeida, E.M.S.A., Siqueira, R.D., Bonjardim, L.R., 2008. Purificationi, physicochemical properties, thermal analysis and antinociceptive effect of atranorin extracted from Cladina kalbii. Biol. Pharm. Bull. 31, 1977–1980. Ministry of Environment and Forests, 2009. Asia-Pacific Forestry Sector Outlook Study II: India Country Report. Working Paper No. APFSOS II/WP/2009/06. FAO, Bangkok, 7 pp. Mizuno, M., Morimoto, M., Minato, K., Tsuchida, H., 1998. Polysaccharides from Agaricus blazei stimulate lymphocyte T-cell subsets in mice. Biosci. Biotechnol. Biochem. 62, 434–437. Mizuno, M., Minato, K., Ito, H., Kawade, M., Terai, H., Tsuchida, H., 1999. Antitumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill. Biochem. Mol. Biol. Int. 47, 707–714. Moon, Y.J., Brazeau, D.A., Morris, M.E., 2011. Dietary phenethyl isothiocyanate alters gene expression in human breast cancer cells. Evid. Based Complement. Alternat. Med. 2011, 462525 Moore, A., Pinkerton, R., 2009. Vincristine: can its therapeutic index be enhanced? Pediatr. Blood Cancer 53, 1180–1187. Morales-Cano, D., Calvino, E., Rubio, V., Herra´ez, A., Sancho, P., Tejedor, M.C., et al., 2013. Apoptos is induced by paclitaxel via Bcl-2, Bax and caspases 3 and 9 activation in NB4 humanl eukaemia cells is not modulated by ERK inhibition. Exp. Toxicol. Pathol. 65, 1101–1108. Morita, H., Tomizawa, Y., Tsuchiya, T., Hirasawa, Y., Hashimoto, T., Asakawa, Y., 2009. Antimitotic activity of two macrocyclic bis(bibenzyls), isoplagiochins A and B from the liverwort Plagiochila fruticosa. Bioorg. Med. Chem. Lett. 19 (2), 493–496. Muller, K., 2001. Pharmaceutically relevant metabolites from lichens. Appl. Microbiol. Biotechnol. 56, 9–16. Namikoshi, M., Rinehart, K.L., 1996. Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. Biotechnol. 17, 373–384. Nunnery, J.K., Mevers, E., Gerwick, W.H., 2010. Biologically active secondary metabolites from marine cyanobacteria. Curr. Opin. Biotechnol. 21, 787–793. O’Brien, S., Kantarjian, H., Keating, M., Beran, M., Koller, C., Robertson, L.E., et al., 1995. Homoharringtonine therapy induces responses in patients with chronic myelogenous leukemia in late chronic phase. Blood 86, 3322–3326. Ohta, Y., Andersen, N.H., Liu, C.B., 1977. Sesquiterpene constituents of two liverworts of genus Diplophyllum: novel eudesmanolides and cytotoxicity studies for enantiomeric methylene lactones. Tetrahedron 33 (6), 617–628. Ojima, I., Lichtenthal, B., Lee, S., Wang, C., Wang, X., 2016. Taxane anticancer agents: a patent perspective. Expert Opin. Ther. Pat. 26, 1–20. Oksanen, I., 2006. Ecological and biotechnological aspects of lichens. Appl. Microbiol. Biotechnol. 73, 723–734.

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11 Anticancer natural product from marine invertebrates Rajesh Kumar Singha, Amit Ranjanb, Monika Singhc, and Akhileshwar Kumar Srivastavad a

DEPARTMENT OF DR AV YAGUNA, FACULTY OF AYURVEDA, INSTITUTE O F MEDICAL SCIENCES, BANARAS HIND U UNIVERSITY, VA RANASI, UTTAR PRADESH, I NDIA b DEPARTMENT OF KAY AC HI KI TSA, I NSTI TUTE O F ME DICAL SCI EN CES, BANARAS HINDU UNIVERSITY, VARANASI, I ND I A c SCHOOL OF BIOMEDICAL ENGINEERING, INDIAN INSTITUTE OF TECHNOLOGY ( BHU), VARANASI, INDIA d P CBT DEP AR TMENT , C SIR-CFT RI, MYSURU, K ARNATAKA, INDIA

11.1 Introduction Cancer remains the second most leading cause of death in the world after cardiovascular diseases. Its treatment includes surgical removal of the malignant tumor tissue, chemotherapy and radiotherapy conventionally. These treatment procedures cause adverse effects on psychological, physiological, and biochemical status of the patients. The chemotherapy and radiotherapy destroy the adjacent normal tissues, leading to rising of some additional adverse symptoms in the patients (Singh et al., 2019). However, chemotherapy remains the most common approach for its treatment which uses synthetic inorganic as well as organic compounds with cytotoxic activity. These synthetic compounds have many adverse effects on body physiology of the cancer patients. The natural products are discovered as alternative to the synthetic compounds and for improving the efficacy of existing therapy against cancer with reduced side effects. The natural compounds are isolated from traditionally used medicinal plants which are compatible to the human physiology. Although, some types of malignancies developed resistance to conventional drugs (Patwardhan et al., 2004). About more than 60% of the anticancer drugs are isolated from natural products or their derivatives. They act as raw drugs for development of lead molecules for the drug development with unique pharmacophores (Cragg et al., 2009; Sharma and Gupta, 2015). These natural products have the potency to induce cytotoxicity through apoptosis in malignant cells. It has been reported that several natural products from different organisms showed anticancer activities. The evolution of organism induces various characteristics in variant population, leading to diversity in chemical constituents of different organism. The biochemical changes and mutations in genetic materials of organisms enhance the fitness in the environment where they use to live. These diversities among the organisms enable them for production of varieties of natural products in different population which increase the survival of organism and Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00011-4 © 2021 Elsevier Inc. All rights reserved.

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enable them to protect from competitors or enemies. Such evolution for survival or protection from other organisms leads to synthesis of bioactive natural products. It is still unclear to explain the vast chemical diversity synthesized by organisms even an absent of environmental factors, their maintenance of chemical diversity for too long time and synthesis of some compounds in absence of their direct role in that organism (Firn et al., 2003). However, human population has been depending on natural products for their survival since its origin. The civilization of human in different zones of the earth, used the local natural resources as food and drugs, leading to the development of different traditional medicine systems in the world. They harvested easy available natural resources mainly terrestrial plants, animals, sea animals, etc. for the purpose till the date but after exploitation of terrestrial natural products and their detailed studies, marine natural products have become the most interesting research area for chemical and biological researches. They have been driven attention of researchers since last four decades when some studies had tried to evaluate the bio-chemical , 1996). Since that time, there has been a lot of importance of few marine isolates (Carte researches has been carried out, especially marine natural products for treatment of neurodegenerative diseases, cancer, inflammation, viral infections, etc. (Khalifa et al., 2019). The development of molecular techniques in pharmacology and chemical biology has facilitated the drug discovery using marine natural products and increase the interest of pharmaceutical industries, leading to excitement in scientific community for research in marine natural , 1996). The phytoplankton, bacteria, fungi, dinoflagellates, products at present time (Carte algae, invertebrates and mangroves are used as source for the marine natural products, in which invertebrates are animals that are lacking vertebral column, belong to Animalia kingdom of classification (Carroll et al., 2019). These animals live in marine habitats and adopted for adverse conditions with several modifications which enable them for synthesis of biological active compounds. The invertebrate marine animals include sponges, Cnidarians, Bryozoan, Molluscs, Ascidians, and Echinoderms.

11.2 Sponges Sponges are primitive multicellular animals which are commonly known as pore-bearer animals. They are composed of different types of cells but there is absence of tissue layer of organ in the body. Their constituent cells form a specialized body form to perform a unique function, filtering of water for their food and oxygen. The sponge body is made up of numerous pore, ostia, canals, and chamber (Spongocoel). The flow of water takes place through dermal ostia to incurrent canal, radial canal, spongocoel, and finally osculum. During water filteration, the food particles and dissolved oxygen are consumed by the sponge for its survival and other physical activities. It is widely occurred in sea water and ranges from millimeter to 6 feet in height. It has a capacity to biosynthesize several compounds of medicinal values. It has contributed about 30% of all natural products reported till the date. The compound isolated or derived from sponges have vast biological activities such as cytotoxicity, induction of cell cycle arrest, apoptosis inducing activity, membrane receptor interaction, enzyme modulation, etc. Some important sponges are listed with their bioactive compounds (Table 11.1).

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Table 11.1 Name of the isolated compounds from different sponges and their anticancer activities. S. no.

Species

Collection site

Compounds

Activities

1

Aaptos sp.

Vietnam

Aaptamine

2

Amphibleptula sp. Aplysina aerophoba Australian spongia Axinella sinoxea

Florida

Microsclerodermin A

Induce apoptotic cell death (Dyshlovoy et al., 2014) Induce apoptotic cell death (Guzman et al., 2015) Induce apoptotic cell death (Florean et al., 2016) Induce apoptotic cell death (Guzman et al., 2013) Anticancer activity (Heidary Jamebozorgi et al., 2019) Microtubule-stabilizing agents (Mooberry et al., 1999) Microtubule-stabilizing (Mooberry et al., 1999) Induce apoptotic cell death (De Stefano et al., 2012) Antiproliferative and induce apoptotic cell death (Umeyama et al., 2010) Cytotoxic and induce apoptotic cell death (Trisciuoglio et al., 2008) Antitumor and cytotoxic activities (Roel et al., 2016) Cytotoxic and antitumor Tumor inhibitor and antileukemic

3 4

Isofistularin-3 Florida

Spongiatriol

Persian Gulf

Saturated fatty acids

Cacospongia mycofijiensis Cacospongia mycofijiensis Cacospongia scalaris Callyspongia sp.

Republic of the Marshall Islands Republic of the Marshall Islands Croatia

Laulimalide Isolaulimalide

Japan

Callyspongidiol

Candidaspongia sp. Crambe crambe

New Guinea

Candidaspongiolide

Mediterranean Sea Indian Ocean Caribbean Sea

Crambescidin Isoquinolinequinones Spongouridine

Indonesia

Smenospongine

Bahamas

Dercitin

Korea

Pectenotoxin

Korea New Guinea

Pectenotoxin Calyculin

Israel

Salarin

Induce apoptosis (Ben-Califa et al., 2012)

Croatia

Cacospongionolide

21

Dinophysis acuminata Dinophysis fortii Discodermia calyx Fascaplysinopsis sp. Fasciospongia cavernosa Forcepia sp.

Cell cycle arrest and induce apoptosis (Kong et al., 2008) Prolong life of tumor-bearing mice and anticancer activity (Carte, 1996) Cytotoxic and induce apoptosis (Kim et al., 2008) Apoptosis induction (Shin et al., 2008) Potent phosphatase inhibitor (Carte, 1996)

22

Geodia japonica

Japan

Geoditin

23

Geodia japonica

Japan

Stellettin

24

H. okadai

Japan

Halichondrin B

Induce apoptotic cell death (De Stefano et al., 2012) Reversible inducer of chromosome condensation (Zhang et al., 2012) Induces oxidative stress and apoptosis (Cheung et al., 2010) Induces oxidative stress and apoptosis (Liu et al., 2006) Antitumor and antileukemic (Carroll et al., 2019)

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Cribochalina sp. Cryptotethia crypta Dactylospongia elegans Dercituss sp.

Scalaradial

Lasonolide

Continued

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Table 11.1 Name of the isolated compounds from different sponges and their anticancer activities—cont’d S. no.

Species

Collection site

Compounds

Activities

25

Korea

Ilimaquinone

26

Hippospongia metachromia Hyrtios sp.

Taiwan

Heteronemin

27

Jaspis sp.

Jaspolide

28 29 30

Jaspis sp. Lanthella sp. Latrunculia biformis Latrunculia magnifica Leiodermatium sp. Leucetta chagosensis Monanchora pulchra Monanchora pulchra Monanchora pulchra Monanchora pulchra Monanchora pulchra Monanchora pulchra Mycale sp.

South China Sea France Vietnam Antarctic Sea

Jaspine Petrosterol Discorhabdin

Red Sea

Latrunculins

Florida

Leiodermatolide

North Pacific Ocean Sea of Okhotsk

Naamidine

Induces death receptor signal and chemosensitizer (Do et al., 2014) c-Met, STAT3 inhibitor and induces apoptosis (Wu et al., 2016) Induces cell cycle arrest and apoptosis (Wei et al., 2008) Induces apoptosis (Salma et al., 2009) Anticancer activity (Nguyen et al., 2009) Inhibits topoisomerase I, II, and indoleamine 2,3-dioxygenase (Li et al., 2019) Altered actin-based cytoskeleton and cytotoxic (Carte, 1996) Cytotoxic and antimitotic activity (Guzman et al., 2016) Induces apoptosis (LaBarbera et al., 2009)

Monanchocidin

Cytotoxic activity (Dyshlovoy et al., 2016)

Sea of Okhotsk

Ptilomycalin

Sea of Okhotsk

Monanchomycalin

Sea of Okhotsk

Normonanchocidin

Sea of Okhotsk

Urupocidin

Sea of Okhotsk

Pulchranin

New Zealand

Mycalamide A

New Zealand New Zealand

Pateamine Mycalamide Latrunculin

Cytotoxic activity and induces apoptosis (Dyshlovoy et al., 2016) Cytotoxic activity and induces apoptosis (Dyshlovoy et al., 2016) Cytotoxic activity and induces apoptosis (Dyshlovoy et al., 2016) Cytotoxic activity and induces apoptosis (Dyshlovoy et al., 2016) Cytotoxic activity and induces apoptosis (Dyshlovoy et al., 2016) Cytotoxic, antileukemic, and antitumor (Hood et al., 2001) Induces apoptosis (Hood et al., 2001) Induces apoptosis (Hood et al., 2001) Anticancer activity (Carte, 1996)

Thailand

Kuanoniamines

Anticancer activity (Kijjoa et al., 2007)

Thailand

Kuanoniamines

Anticancer activity (Kijjoa et al., 2007)

Korea

Dideoxypetrosynol

Korea

Gukulenin A

Anticancer and induces apoptosis (Choi et al., 2004) Anticancer activity (Ahn et al., 2019)

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Mycale sp. Mycale sp. Negombata magnifica Oceanapia sagittaria Oceanapia sagittaria Petrosia sp. Phorbas gukhulensis Psammaplysilla purpurea

Purealin

Myosin phosphorylation inhibitor (Carte, 1996)

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Table 11.1 Name of the isolated compounds from different sponges and their anticancer activities—cont’d S. no.

Species

Collection site

Compounds

Activities

49

Psammaplysilla sp. Psammaplysilla sp. Rhizochalina incrustata Sarcotragus sp.

Korea

Psammaplin

Korea

Psammaplysene

Korea

Rhizocalinin

Induces cell cycle arrest and apoptosis (Ahn et al., 2008) Induces cell cycle arrest and apoptosis (Berry et al., 2009) Induces apoptosis ( Jin et al., 2009)

Korea

Ircinin

Red Sea Coast

Sipholenol

50 51 52 53

Caribbean Sea

Smenamides

55

Siphonochalina sp. Smenospongia aurea Spongia sp.

Indian Ocean

Spongistatins

56 57

Theonella sp. Theonella sp.

Japan Okinawa

Theopederins Onnamide A

58

Theonella swinhoei Xestospongia carbonaria Zyzzya sp.

New Guinea

Motuporin

Palau

Neoamphimedine

Fiji

Pyrroloiminoquinones

54

59 60

Induces cell cycle arrest and apoptosis (Choi et al., 2005) Induces cell cycle arrest and apoptosis (Sobahi et al., 2017) Antitumor activity (Teta et al., 2013) Chemo-sensitizer and antitumor (Carte, 1996) Antileukemic and antitumor (Carte, 1996) Cytotoxic, antileukemic, and antitumor (Carte, 1996) Phosphatase inhibitor (Carte, 1996) DNA topoisomerase II inhibitor and chemosensitizer (Carte, 1996) Cytotoxic, antitumor, DNA topoisomerase II inhibitor, and chemo-sensitizer (Carte, 1996)

These bioactive compounds are very promising and few are under clinical trials. Many of them are potent antileukemic and several research laboratories are working for development of new drugs from these sponge-isolated compounds (Fig. 11.1).

11.3 Cnidaria Cnidarians are invertebrate animals belongs to phylum Cnidaria or coelenterate. It is commonly marine animals with large variation in shape, size and feeding habit. Its body shows a distinct pattern such as radial or approaching bilateral, two-three layers of cells, tentacles bearing nematocysts in mouth, pouch like gastro-vascular cavity, sessile or free swimming, solitary or colonial. Cnidarians are generally carnivorous, but symbiotic cnidarians are also reported. It includes animals like jelly fish, sea-anemones, hydroids, corals, etc. the animals belong to sub-phylum medusozoa such as jellyfish and hydroids use stinging cells to protect themselves from predators. Other protection from mechanical disturbance is achieved by hard reef of corals. The ancient medicine systems have used these animals for treatment of different clinical ailments either in single drug or in compound drug formulation. Scientists re-started investigation for bioactive natural products from cnidarians

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Evolutionary Diversity as a Source for Anticancer Molecules

Antimutagenic activity

Antiinflammatory activity

Marine Anticancer Natural Products (Invertebrates)

Cytotoxic activity

Antiproliferative activity Proapoptotic activity

FIG. 11.1 Showing anticancer activities of natural product isolated from marine invertebrates.

late 1960s, and prostaglandins was isolated from gorgonians of Indo-Pacific sea. This study , 1996). Several catalyzed the discovery of several natural product from cnidarians (Carte natural products have been isolated from cnidarians such as, Palytoxin is a toxic compound which is isolated from Palythoa sp. that attracted researchers for searching of natural products from cnidarians (Moore and Scheuer, 1971). Another natural compound isolated from Lophogorgia sp. was Lophotoxin which is a paralytic toxin (Fenical et al., 1981). An antiinflammatory compound, Pseudopterosins isolated from Pseudopterogorgia sp. which is also showing potent analgesic and antileukemic activity (Look et al., 1986). A bicyclic diterpene glycosides (fuscoside) is isolated from Eunicea fusca which is a nonsteroidal antiinflammatory compound ( Jacobson and Jacobs, 1992). Monanchora pulchra, collected from Urup Island (Sea of Okhotsk), has presented alkaloids: monanchocidins A and B, monanchomycalins B and C, ptilomycalin A, normonanchocidin D, urupocidin A, and pulchranin A (Katanaev et al., 2019). Similarly, Melonanchora kobjakovae, collected from Urup Island (Sea of Okhotsk), has showed the presence of fatty acids such as melonosides A and B, melonosins A and B (Katanaev et al., 2019).

11.4 Bryozoa Bryozoans are a group of moss-like filter-feeding animals with complete digestive system, belong to phylum Ectoprocta. The shape of skeletal chambers is of various types such as tubular, oval, vaselike and rectangular. Its size is usually smaller to 2 mm. It is difficult to collect and isolate compounds from these animals because of its small size and harvesting (Tian et al., 2018). A series of natural products (convolutamides) have been isolated from bryozoans Amathia convoiuta was collected from Florida which shows in vitro cytotoxicity against cancer cells (Zhang et al., 1994). The extracts (B-carboline) of bryozoan Cribricellina cribraria, collected from New Zealand shows potent cytotoxicity (Tian et al., 2018). The cytotoxic isoquinolinequinones (perfragilin) have been isolated from the bryozoan

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Membranipora perfragilis (Choi et al., 1993). The bryozoan Flustra foliacea, collected from Canada has used to isolate indole alkaloids (Holst et al., 1994). The most potent natural product from bryozoan is bryostatins isolated from Bugula neritina (Figuerola and Avila, 2019) which have exceptionally anticancer efficacy against different cancers such as leukemia, renal cancer, melanoma and nonsmall cell lung cancer (Philip et al., 1993; Prendiville et al., 1993).

11.5 Molluscs Molluscs is a group of filter-feeding animals with soft body lacking a true skeleton. The word molluscs were derived from a Latin word “Mollusca” means soft body. These animals belong to the phylum Molluscs and its member animals have an external hard protecting covering, known as shell which is made up of calcium carbonate. The soluble form of calcium carbonate is secreted by a specialized cell layer beneath the shell. Its shell and other products have been reported for medicinal uses in various traditional systems of medicine for preparation of drugs, especially for bone healing. However, several bioactive compounds are synthesized in these organisms for protection from predators and survival. One of them, a neurotoxin is synthesized in their body to protect themselves from predators (Myers et al., 1993). Another important compound isolated is heterocyclic glycoside neosurugatoxin from Japanese mollusk Babylonia japonica which is a reversible antago, 1996). The dolastatins are isolated from Dolabella nist of acetylcholine receptors (Carte auricularia which was collected from the Indian Ocean, shows cytotoxic activity in different cancer cells and antitumor activity in vivo model even at low doses (Schmitz et al., 1991). A potent cytotoxic compound ulapualide isolated from Hexabranchus sanguineus is also showing antifungal activity which is potential to amphotericin B (Khalifa et al., 2019). Chromodorolide is a potent cytotoxic and antimicrobial compound, isolated from Chromodoris cavae collected from Indian Ocean has reported to have tumor growth suppressive activity in vivo mice model (Morris et al., 1991).

11.6 Echinoderms Echinoderms are exclusively marine invertebrates, characterized by pentamerous and radial body symmetry having an endoskeleton of calcareous plates with spines on the body surface. Several echinoderms have reported for anticancer compounds production, and some compounds are potent anticancerous. Lethasterias fusca collected from Posyet Bay (Japan) have steroid glycosides including lethasteriosides A and B, thornasteroside A, anasteroside A, and luidiaquinoside which are bioactive compounds (Ivanchina et al., 2012). Leptasterias ochotensis was collected from Sea of Okhotsk had reported to have steroid glycosides such as leptasteriosides A-F, and leptaochotensosides A-C (Malyarenko et al., 2014, 2015). Hippasteria phrygiana Kuril, collected from Islands, Sea of Okhotsk

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have steroid glycosides hippasteriosides A-D (Kicha et al., 2011). Cucumaria fallax was collected from Sea of Okhotsk has showed triterpene glycoside, fallaxoside D1 (Katanaev et al., 2019). Choriaster granulatus has reported to have anticancer molecules namely, Granulatosides D and E, Echinasterosides C, E and F, Linckoside L4, Echinasteroside B, desulfated Echinasteroside A, desulfated Echinasteroside B, 22,23-Dihydroechinasteroside A, Linckoside E, Laeviuscoloside D, Linckoside B, Linckoside F, Steroid Heptaol and Granulatoside A. Similarly, Culcita novaeguineae has showed anticancer compounds such as Novaeguinosides I and II, Asterosaponin 1–3, Culcinosides A-D, Culcitoside C5, Halityloside D, Linckoside B, Regularoside B, Novaeguinosides A-D, Novaeguinoside E, Echinasteroside C, Linckoside L, Halityloside A, Halityloside B, Linckoside F, and Halityloside E (Lazzara et al., 2019). Thus, echinoderms contribute significantly as a source of marine bioactive compounds which further helps in anticancer drug discovery and development.

11.7 Conclusions The diversities in marine ecosystem have a large number of new scaffolds with potential anticancer effects. The environmental conditions of marine and coastal habitats with aquatic ecosystems are more tough as well as diverse than terrestrial due to the strong selective pressure, leading in a much wide range of phyla and classes of organisms. For example, the ecosystem for coral reef are well-known by their biological competition for habitats and energy resources. The secondary metabolites are produced due to competitive pressure which help them for surviving in such marine ecosystem. Several scientific reports have explained the significant role of such secondary metabolites for surviving of organisms in marine ecosystems. Approximately, to date more than 33 identified phyla, 97% are found in and 45% are exclusive to the marine ecosystem. Among marine organisms, invertebrates are the main source for bioactive molecules in the marine ecosystem which represent 60% of all marine animals. Marine invertebrates have members of phyla like Porifera, Coelenterata, Mollusca, Tunicata, Annelida, and Bryozoa, which are potential source for anticancer compounds. Two-third part of the earth is covered with marine environment which has vast diversity of animals and natural products. Many compounds have reported to rare on the earth but abounded in marine system. The compounds isolated from this resource have showed novel pharmacophore and activities in cancer research. Therefore, marine invertebrates may serve as prime source of anticancer natural products.

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Further reading El-Hawary, S.S., et al., 2019. Bioactive brominated oxindole alkaloids from the Red Sea Sponge Callyspongia siphonella. Mar. Drugs 17 (8), 465. https://doi.org/10.3390/md17080465.

12 Melatonin: A journey from bovine pineal gland to a promising oncostatic agent Tarun Minocha, Megha Das, Nitesh Kumar Mishra, Soumya Ranjan Mohanty, and Sanjeev Kumar Yadav DEPARTMENT OF ZOOLOGY, INSTITUTE OF SCIENCE, BANARAS HINDU UNIVERSITY, VARANASI, INDIA

12.1 Introduction The enigmatic pineal gland that is profoundly found in the center of the brain was considered as one of the dormant organs of vertebrate brain till first half of the 20th century. This dormant vestige has ignited the attention of scholars, philosophers and spirituals from gen erations and has been dubbed the “third eye” and “the principal seat of the soul” by Rene Descartes. Learner and his co-workers at the Yale University School of Medicine in 1959 isolated, purified and characterized methoxy derivative of Serotonin and were the first to define chemical structure of this compound as N-acetyl-5-methoxytrypamine and evoked melatonin. Discovery of a new biological substance from the bovine pineal gland was a major breakthrough in pineal research in the middle of the last century. After melatonin’s discovery in pineal tissue, the entity of the gland quickly changed from that of a futile vestige to one of an active organ of internal secretion. Indeed, the name they selected for their newly discovered molecule, i.e., melatonin, is in part based on its effect on skin pigmentation (“mela” from melanin and “tonin” from serotonin). After melatonin’s discovery in bovine pineal tissue, its isolation and identification are not restricted to the bovine species, in subsequent years melatonin was identified in a various species of flora and fauna (Dubbels et al., 1995; Shi et al., 2016; Tan et al., 2016). One of the prominent attributes of this indolamine is that its synthesis and secretion is stimulated by darkness and decreased by light during 24 h light-dark cycle, irrespective of the habit of the concerned animals. Therefore, it is often regarded as “hormone of darkness” or chronobiotic molecule. The oncostatic behavior of this indolamine came into limelight by the end of 2000 inhibiting growth and development of wide array of tumors by its immunomodulatory, anti-inflammatory, pro-apoptotic, antioxidant, vasoregulatory, and oncostatic properties (Cabrera et al., 2010).

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12.2 Evolutionary history of melatonin Melatonin is one of the most primitive and ancient biological agents revealed on earth and ubiquitously found in almost all the living creatures (Claustrat et al., 2005). Melatonin presumably evolved in primitive bacteria (Alphaproteobacteria and cyanobacteria) and has been retained throughout the evolution of life (Manchester et al., 2015). During the course of evolution these primitive bacteria were engulfed by primitive eukaryotes and eventually developed an endosymbiotic association with their eukaryotic hosts. The ingested α-proteobacteria and cyanobacteria were evolved into mitochondria and chloroplasts respectively and retained the capacity to synthesize melatonin that acts as novel antioxidant (Reiter et al., 2017). The antioxidant behavior of melatonin nullifies the effect of free radicals generated during the process of respiration and photosynthesis (Reiter et al., 2017). Over billions of years of evolution and diversification of different taxa melatonin adopted the pleiotropic nature and performs wide spectrum of remarkable biological right from regulator of circadian rhythms to oncostatic agent. Despite its very long evolutionary history and its multiple functions, the chemical structure of melatonin has remained unchanged for billions of years (Reiter et al., 2017). During the process of melatonin synthesis, the taxon specific variations occurs in the sequence of steps and intermediate molecules (Back et al., 2016).

12.3 Synthesis of melatonin in animals In all vertebrate’s the synthesis of melatonin takes place in the pineal gland. Besides pineal gland, the melatonin is also produced from the extra-pineal organs and functions in autocrine or paracrine manner and involved in local physiological processes (Carrillo-Vico et al., 2004). The synthesis of melatonin involves multiple steps but the initial precursor of melatonin synthesis is an aromatic amino acid, L-tryptophan. Melatonin biosynthesis commences as the pinealocytes convert tryptophan to serotonin through hydroxylation and decarboxylation process respectively. Hydroxylation process involves the conversion of L-tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase (TPH) enzyme and decarboxylation step involves the conversion of 5-hydroxytryptophan to serotonin (5-hydroxytryptamine) by the aromatic amino acid decarboxylase (AAD) enzyme. Arylalkylamine N-acetyltransferase (AANAT) a rate-limiting enzyme that promotes the conversion of serotonin to N-acetyl-serotonin and eventually, hydroxyindole-Omethyltransferase (HIOMT) enzyme converts the N-acetyl-serotonin to melatonin. Once formed, melatonin is released from the pineal gland into the bloodstream. The synthesis of melatonin occurs in a circadian manner with low levels during the day and reaches the peak during the night. This circadian manner is followed by most of the animals despite their diurnal or nocturnal nature (Claustrat et al., 2005).

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12.4 Synthesis of melatonin in plants In the animal system, the synthesis of melatonin takes place in the pineal gland and extrapineal organs also have the tendency to synthesize melatonin. However, there is no such organ in plants, and this difference implies that the melatonin biosynthesis pathway in plants is slightly different from that in animals. Contrary to the animal system, melatonin is also synthesized in various organs of the plants such as the roots, stems and leaves, fruits and seeds. In a wide array of plant species, tryptophan is the initial precursor of melatonin biosynthesis. Tryptophan decarboxylase (TDC) is the enzyme that catalyzes tryptophan to tryptamine, and then tryptamine 5-hydroxylase (T5H) catalyzes tryptamine to serotonin. In the next two steps, serotonin is converted to N-acetyl-serotonin by serotonin N-acetyltransferase (SNAT)/arylalkylamine N-acetyltransferase (AANAT), and then N-acetyl-serotonin methyltransferase (ASMT)/hydroxyindole-O-methyltransferase (HIOMT) catalyzes N-acetyl-serotonin into melatonin. Furthermore, tryptophan is not only the resource of melatonin, but also the precursor of indole-3-acetic acid (IAA), maybe implying the multifunctional role of melatonin in plants. In the plant kingdom, melatonin performs wide array of functions such as plant growth and development, seed protection and germination, root development, fruit ripening, and senescence (Arnao and Herna´ndez-Ruiz, 2015; Hu et al., 2017; Liang et al., 2017; Zhang et al., 2017). In addition, melatonin protects the plants against biotic and abiotic stresses, such as salt, drought, cold, heat and heavy metal stresses.

12.5 Role of melatonin in integrity of genome and DNA repair The integrity of the genome is monitored and maintained by various cell cycle checkpoints. These checkpoints act as surveillance mechanisms for survival and progression of normal cells and also ensures the fidelity of cell division, but when this process gets altered the fidelity and integrity of DNA get vanished which is a prelude for initiation and progression of cancer (Tian et al., 2015). The generation of free radicals is a menace for the various cellular components including DNA, proteins and lipids and any kind of damage to these physical cellular components contributes to the development and progression of cancer. Melatonin acts as a potent free radical scavenger molecule and protects against oxidative damage from ionizing radiation and carcinogenic substances (Galano et al., 2011). The stability and fidelity of the genome is protected and maintained by DNA damage prevention by stimulating DNA repair system, targeting of impaired congregation of centrosome, and suppression of telomerase activity (Block et al., 2015). Melatonin being a natural antioxidant molecule not only protects against DNA damage by scavenging free radicals but also stimulating the DNA repair system. Melatonin is a unique class of antioxidant because not only melatonin but also its metabolites such as AFMK and AMK act as free radical scavengers (Galano et al., 2013). One study has reported that the hydrogen peroxide (H2O2) exposure damages the Integrity of DNA and application of melatonin and its metabolites reduces the activity of hydrogen peroxide (H2O2) and prevents

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from its damaging effects (Sliwinski et al., 2007). In another study, it has been revealed that melatonin protects against UV radiation and X-ray irradiation induced DNA damage by enhancing the expression of various antioxidative enzymes (Fischer et al., 2013) and DNA repair gene such as 8-oxoguanine DNA glycosylase (Ogg1), apurinic/apyrimidinic endonuclease (Apex1), and X-ray repair cross-complementing group 1 (Xrcc1) (Rezapoor et al., 2017). Further analysis showed that melatonin pre-treatment of irradiated rats ameliorates the DNA damage induced by ionizing radiation (Ghobadi et al., 2017) and 45 mg/day of melatonin for 21 days suppresses the radiation-induced oral mucositis in rats (Ortiz et al., 2015). Mitochondrial damage generates free radicals that impair with mitochondrial function and encourage the cell toward demise (Escames et al., 2007). Melatonin administration reduces the mitochondrial oxidative load, increases the activity of ETS, revives the mitochondria and restores the mitochondrial function (Acuna Castroviejo et al., 2011). In addition, melatonin and its catabolite 6-hydroxymelatonin (6-OHM) have the potential to upregulate the activity of complex III (cytochrome bc1), induce cell death in breast MCF-7 and leukemic HL-60 cells by increasing the production of reactive oxygen species (ROS) (Proietti et al., 2017). Melatonin treatment not only diminishes the ATP production but also decrease in viability of breast cancer MCF-7 cells was observed (Scott et al., 2001). Despite, free radical quenching activity, melatonin also increases the expression of various antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase (Antolı´n et al., 1996) and mitigates the expression of pro-oxidative enzymes such as iNOS (Poliandri et al., 2006). In addition, the combined effect of melatonin with rapamycin alters mitochondrial functions that leads to increased ROS production, apoptosis and mitophagy (Shen et al., 2018). Hence, Melatonin not only alone but also in combination proved to be an effective and promising agent to protect DNA from damage.

12.6 Melatonin and telomerase activity Telomere is a network of DNA sequences and enzymatic proteins that covers the end of linear eukaryotic chromosomes. Telomere plays a vital role for maintaining genomic stability and their progressive shortening during successive cell division transforms somatic cell to tumor cell. Telomerase is an enzyme which promotes the elongation of telomeres during successive cell divisions. Both somatic as well as tumor cells possesses the activity of telomerase enzyme. The length of telomeres and activity of telomerase is vital for cancer initiation and survival of tumors as these cells have the capacity for sustained cell division and DNA replication (Akincilar et al., 2016). Melatonin has an immense role in the regulation of telomerase activity. The effect of melatonin on telomerase activity was studied in both in vivo and in vitro experimental conditions and it has been found that melatonin fosters the telomerase activity in normal cells (Akbulut et al., 2009) and suppresses the telomerase activity in cancer cells (Leon-Blanco et al., 2003). An in vitro study on MCF-7 breast cancer cells revealed that the increasing concentration of melatonin was

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found to suppress the activity of telomerase in dose dependent manner. In addition, an in vivo study on nude mice transplanted with MCF-7 xenograft revealed the significant dosedependent inhibition of telomerase activity (Leon-Blanco et al., 2003). Hence, melatonin may be one of the fruitful aspirants to captive the growth of tumor cells by reducing the telomerase activity.

12.7 Conclusion Despite developing several therapeutic agents, cancer still second leading progressive chronic disorder worldwide. Various preventive measures have been taken into consideration Despite the potency of various medicaments such as surgery, chemotherapy, radiotherapy, hormone therapy and immunotherapy. Though these new approaches have enormous therapeutic potential for reducing morbidity, mortality, and improving quality of life but overall survival rates are still unsatisfactory. The scientific journey of melatonin has started five decades ago and extensive research in this molecule has brought clear evidence of its pleiotropic nature into the limelight. These chemotherapeutic medicaments induce ROS and imposes severe side effects however melatonin being a strong antioxidant molecule is very low or no toxic at higher doses. In a nutshell, the discovery of this indolamine has ignited a ray of hope to cancer patients on its benevolent effects in modulation of cancer progression.

12.8 Challenge and future perspective Since the discovery of melatonin various epidemiologic, pre-clinical and clinical studies have been conducted to identify the versatility of melatonin. Melatonin has reflected itself to be a pervasive and functionally manifold molecule with broad spectrum of remarkable functions. Being a pleiotropic nature melatonin not only acts as a therapeutic molecule in cancer but also in other chronic diseases. In addition, more clinical trials are of utmost importance to consider melatonin as a standard therapeutic option to treat myriad of cancer types.

References Acuna Castroviejo, D., Lopez, L.C., Escames, G., Lo´pez, A., Garcia, J.A., Reiter, R.J., 2011. Melatoninmitochondria interplay in health and disease. Curr. Top. Med. Chem. 11 (2), 221–240. Akbulut, K.G., Gonul, B., Akbulut, H., 2009. The role of melatonin on gastric mucosal cell proliferation and telomerase activity in ageing. J. Pineal Res. 47 (4), 308–312. Akincilar, S.C., Unal, B., Tergaonkar, V., 2016. Reactivation of telomerase in cancer. Cell. Mol. Life Sci. 73 (8), 1659–1670. Antolı´n, I., Rodrı´guez, C., Saı´nz, R.M., Mayo, J.C., Urı´a, H., Kotler, M., Rodriguez-Colunga, M., Tolivia, D., Menendez-Pelaez, A., 1996. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. FASEB J. 10 (8), 882–890.

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Arnao, M.B., Herna´ndez-Ruiz, J., 2015. Functions of melatonin in plants: a review. J. Pineal Res. 59, 133–150. https://doi.org/10.1111/jpi.12253. Back, K., Tan, D.X., Reiter, R.J., 2016. Melatonin biosynthesis in plants: multiple pathways catalyze tryptophan to melatonin in the cytoplasm or chloroplasts. J. Pineal Res. 61, 426e37. Block, K.I., Gyllenhaal, C., Lowe, L., Amedei, A., Amin, A.R., Aquilano, K., Arbiser, J., Arreola, A., Arzumanyan, A., Ashraf, S.S., et al., 2015. Designing a broad-spectrum integrative approach for cancer prevention and treatment. Semin. Cancer Biol. 35, S276–S304. vez, F., Loro, J., Reiter, R.J., Quintana, J., 2010. Melatonin decreases cell prolifCabrera, J., Negrı´n, G., Este eration and induces melanogenesis in human melanoma SK-MEL-1 cells. J. Pineal Res. 49 (1), 45–54. ˜ o, S., Reiter, R.J., Guerrero, J.M., 2004. Carrillo-Vico, A., Calvo, J.R., Abreu, P., Lardone, P.J., Garcı´a-Maurin Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance. FASEB J. 18 (3), 537–539. Claustrat, B., Brun, J., Chazot, G., 2005. The basic physiology and pathophysiology of melatonin. Sleep Med. Rev. 9 (1), 11–24. Dubbels, R., Reiter, R., Klenke, E., Goebel, A., Schnakenberg, E., Ehlers, C., et al., 1995. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J. Pineal Res. 18, 28–31. https://doi.org/10.1111/j.1600-079X.1995.tb00136.x. ˜ a-Castroviejo, D., 2007. Attenuation Escames, G., Lo´pez, L.C., Ortiz, F., Lo´pez, A., Garcı´a, J.A., Ros, E., Acun of cardiac mitochondrial dysfunction by melatonin in septic mice. FEBS J. 274 (8), 2135–2147.  ski, K., Hardkop, L.H., Kruse, N., Zillikens, D., 2013. Melatonin enhances antioxiFischer, T.W., Kleszczyn dative enzyme gene expression (CAT, GPx, SOD), prevents their UVR-induced depletion, and protects against the formation of DNA damage (8-hydroxy-20-deoxyguanosine) in ex vivo human skin. J. Pineal Res. 54, 303–312. Galano, A., Tan, D.X., Reiter, R.J., 2011. Melatonin as a natural ally against oxidative stress: a physicochemical examination. J. Pineal Res. 51 (1), 1–16. Galano, A., Tan, D.X., Reiter, R.J., 2013. On the free radical scavenging activities of melatonin' s metabolites, AFMK and AMK. J. Pineal Res. 54 (3), 245–257. Ghobadi, A., Shirazi, A., Najafi, M., Kahkesh, M.H., Rezapoor, S., 2017. Melatonin ameliorates radiationinduced oxidative stress at targeted and nontargeted lung tissue. J. Med. Phys. 42, 241–244. Hu, W., Yang, H., Tie, W., Yan, Y., Ding, Z., Liu, Y., et al., 2017. Natural variation in banana varieties highlights the role of melatonin in postharvest ripening and quality. J. Agric. Food Chem. 65, 9987–9994. https:// doi.org/10.1021/acs.jafc.7b03354. Leon-Blanco, M.M., Guerrero, J.M., Reiter, R.J., Calvo, J.R., Pozo, D., 2003. Melatonin inhibits telomerase activity in the MCF-7 tumor cell line both in vivo and in vitro. J. Pineal Res. 35 (3), 204–211. Liang, C., Li, A., Yu, H., Li, W., Liang, C., Guo, S., et al., 2017. Melatonin regulates root architecture by modulating auxin response in rice. Front. Plant Sci. 8, 134. https://doi.org/10.3389/fpls.2017.00134. Manchester, L.C., Coto-Montes, A., Boga, J.A., Andersen, L.P.H., Zhou, Z., Galano, A., et al., 2015. Melatonin: an ancient molecule that makes oxygen metabolically tolerable. J. Pineal Res. 59, 403–419. https://doi. org/10.1111/jpi.12267. ˜ a-Castroviejo, D., Doerrier, C., Dayoub, J.C., Lo´pez, L.C., Venegas, C., Garcı´a, J.A., Lo´pez, A., Ortiz, F., Acun Volt, H., Luna-Sa´nchez, M., et al., 2015. Melatonin blunts the mitochondrial/NLRP3 connection and protects against radiation-induced oral mucositis. J. Pineal Res. 58, 34–49. Poliandri, A.H., Esquifino, A.I., Cano, P., Jim enez, V., Lafuente, A., Cardinali, D.P., Duvilanski, B.H., 2006. In vivo protective effect of melatonin on cadmium-induced changes in redox balance and gene expression in rat hypothalamus and anterior pituitary. J. Pineal Res. 41 (3), 238–246. Proietti, S., Cucina, A., Minini, M., Bizzarri, M., 2017. Melatonin, mitochondria, and the cancer cell. Cell. Mol. Life Sci. 74, 4015–4025.

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Reiter, R.J., Rosales-Corral, S., Tan, D.X., Jou, M.J., Galano, A., Xu, B., 2017. Melatonin as a mitochondriatargeted antioxidant: one of evolution’s best ideas. Cell. Mol. Life Sci. 74, 3863–3881. https://doi.org/ 10.1007/s00018-017-2609-7. Rezapoor, S., Shirazi, A., Abbasi, S., Bazzaz, J.T., Izadi, P., Rezaeejam, H., Valizadeh, M., SoleimaniMohammadi, F., Najafi, M., 2017. Modulation of radiation-induced base excision repair pathway gene expression by melatonin. J. Med. Phys. 42, 245–250. Scott, A.E., Cosma, G.N., Frank, A.A., Wells, R.L., Gardner Jr., H.S., 2001. Disruption of mitochondrial respiration by melatonin in MCF-7 cells. Toxicol. Appl. Pharmacol. 171, 149–156. Shen, Y.Q., Guerra-Librero, A., Fernandez-Gil, B.I., Florido, J., Garcı´a-Lo´pez, S., Martinez-Ruiz, L., ˜ a-Castroviejo, D., Ortega-Arellano, H., et al., 2018. CombiMendivil-Perez, M., Soto-Mercado, V., Acun nation of melatonin and rapamycin for head and neck cancer therapy: suppression of AKT/mTOR pathway activation, and activation of mitophagy and apoptosis via mitochondrial function regulation. J. Pineal Res. 64(3) Shi, H., Chen, K., Wei, Y., He, C., 2016. Fundamental issues of melatonin-mediated stress signaling in plants. Front. Plant Sci. 7, 1124. https://doi.org/10.3389/fpls.2016.01124. Sliwinski, T., Rozej, W., Morawiec-Bajda, A., Morawiec, Z., Reiter, R., Blasiak, J., 2007. Protective action of melatonin against oxidative DNA damage—chemical inactivation versus base-excision repair. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 634, 220–227. Tan, D.X., Hardeland, R., Back, K., Manchester, L.C., Alatorre-Jimenez, M.A., Reiter, R.J., 2016. On the significance of an alternate pathway of melatonin synthesis via 5-methoxytryptamine: comparisons across species. J. Pineal Res. 61, 27–40. https://doi.org/10.1111/jpi.12336. Tian, H., Gao, Z., Li, H., et al., 2015. DNA damage response—a double-edged sword in cancer prevention and cancer therapy. Cancer Lett. 358, 8–16. Zhang, N., Zhang, H.J., Sun, Q.Q., Cao, Y.Y., Li, X., Zhao, B., et al., 2017. Proteomic analysis reveals a role of melatonin in promoting cucumber seed germination under high salinity by regulating energy production. Sci. Rep. 7, 503. https://doi.org/10.1038/s41598-017-00566-1.

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13 Spice up your food for cancer prevention: Cancer chemoprevention by natural compounds from common dietary spices Jie Gaoa,∗,†, Kenza Mamounia,∗, Lei Zhangb, and Bal L. Lokeshwara,c a

GEORGIA CANCER CENTER AT AUGUSTA UNIVERSITY, AUGUSTA, GA, UNITED STATES b BAYE R U .S . L L C, P IT TS B UR GH, PA, UNITED STATES c RESE ARCH SE RVI CE, CHARLI E NORWOOD VETERANS ADMINISTRATION MEDICAL CENTER, DOWNTOWN HOSPITAL, AUGUSTA, GA, UNI TE D STAT ES

13.1 Introduction The often-quoted Greek physician, Hippocrates’s assertion that “let food be thy medicine, medicine be thy food,” holds even today. Food, like medicine, should be taken quite seriously when needed, and most of the food that we eat could potentially be medicine to address some ailment. Dietary spices play an important role in food preparation. Spices for long considered as merely flavoring agents with minimal nutritional values by regulatory agencies such as the United States Food and Drug Administration. However, in recent times, dietary spices are not only appreciated for their culinary properties but also extensively studied for their potential effects on the body such as cardiovascular, gastrointestinal, nervous systems, and other systems (Kochhar, 2008). Herbs and spices, as defined by the Food and Drug Administration of the United States Government, are “Aromatic vegetable substance in the whole, broken or ground form, the significant function of which in food is seasoning rather than nutrition.” This definition is an understatement for their health-promoting values, in the light of recent findings and have significant utility in cancer prevention and treatment.

∗

Both authors contributed equally.

†

Current address: Department of Clinical Pharmacology, University of Alabama, Birmingham, AL, United States. Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00013-8 © 2021 Elsevier Inc. All rights reserved.

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13.2 Role of diet in cancer origin and progression Cancer is a global epidemic. An estimated 18.1 million people newly developed cancer in the year 2018, according to the International Agency for Research on Cancer, an arm of The World Health Organization. Further, an estimated 9.6 million people died of cancer (Bray et al., 2018; Siegel et al., 2020; Society, 2012). In the United States, by far the most advanced country in medical care, treatment, and related technology, an estimated 606,520 deaths are expected in 2020, ranked second in overall causes of deaths (Siegel et al., 2020; Society, 2018). This large figure is despite all interventions, surveillance, and follow-up systems. It has been widely accepted that a major solution to cancer prevention and perhaps cancer-related deaths lies in prevention rather than attempts to cure (Franceschi and Wild, 2013). Over the past two decades, tremendous insights into the cause and potential preventive strategies have been developed. Some cancer burden has been prevented by existing cancer control strategies, such as prevention of tobacco consumption in various forms (Centers for Disease and Prevention, 2011), changes in dietary intake of harmful foods, increased physical activities, vaccination, etc. (Abbott et al., 2016, 2018; Ma et al., 2016; Neilson et al., 2017; Wang et al., 2017). It should be noted that improper diet or overconsumption of fatty-foods combined with a sedentary lifestyle, and abusive habits constitute over one-third of all causative factors for both cancer incidence and cancer-related death (Organization, 2018). The pro-cancer causing factors can be countered by a better composition of the diet or incorporating food ingredients that detoxify or counter the pro-cancer milieu. Dietary spices used as seasonings and preservatives in food preparation have contributed to eating behaviors. A growing body of epidemiological and case-control clinical trials have established the cancer prevention qualities of herbs and spices (Lopez-Carrillo et al., 1994, 2012, 2016; Lv et al., 2015; Serra et al., 2002). The focus of this article is on the dietary spices containing multiple bioactive compounds some of which, at least in experimental models, have shown significant potential as anticancer and cancer-preventive substances. Table 13.1 lists a select list of common spices used globally and the prominent bioactive compounds identified and tested in the laboratories and some in the clinic for their efficacy and use in human diseases, including cancer (Table 13.1). Although an extensive list of herbs and spices with known bioactive compounds are listed in Table 13.1, the following is a brief survey of the most common spices used globally and studied extensively. While garlic, onion, capsicum, ginger, and turmeric are the most common spices used in main culinary preparations, other spices such as cinnamon, clove, allspice, and cardamom are sparingly used in other preparations such as in breakfast cereals and desserts. The critical overview of various dietary spices and their bioactive compounds presented in this chapter are by no means exhaustive but essential vignette.

Chapter 13 • Edible spices for cancer prevention

Table 13.1

277

Spices with cancer chemopreventive and therapeutic potentials.

S/N

Type of spice

1

Ginger (Zingiber officinale)

Active principles

Activity or property

OH

O

Gingerol

HO OCH3 O

Anti-oxidation (Yanishlieva et al., 2006), anti-cancer (Campbell et al., 2007; Rahmani et al., 2014a,b), antiinflammation (Lee et al., 2009; Pan et al., 2008; Tripathi et al., 2007).

Shogaol

HO O O

Paradol HO OCH3

O Zingerone

HO O HO

OH O

O CH3

O

O

CH3

Curcumin O O

CH3

OCH3 H3C

10 -Acetoxychavicol acetate

CH2

O O CH3

H3C

Zerumbone

H3C CH3

2

Garlic (Allium sativum)

γ-Glutamyl-S-allylcysteine γ-Glutamyl-S-trans-1 propenylcysteine O OH

H

Alliin

(R)

S(s) O

H2C

O– S

S

NH2

S+

Allicin

S

Diallyldisulfide

S

Diallyl sulfide

Anti-oxidation (Dirsch et al., 1998; Rose et al., 2005), anticancer (Berges et al., 2004; Zhang et al., 2014), inhibition of phase I enzymes (Taubert et al., 2006; Yang et al., 2001), induction of phase II enzymes (Singh et al., 1998), anti-inflammation (Ide and Lau, 2001; Keiss et al., 2003), antimetastasis (Liu et al., 2015; Xiao et al., 2014). Continued

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Evolutionary Diversity as a Source for Anticancer Molecules

Table 13.1

Spices with cancer chemopreventive and therapeutic potentials—cont’d

S/N

Type of spice

Active principles

Activity or property

3

Onion (Allium cepa)

5-Hydroxy-3-methyl-4-propylsulfanyl-5H-furan-2-one HO

Anti-oxidation (Fossen et al., 1998; Lee et al., 2012; Prakash et al., 2007; Roldan-Marin et al., 2009), anti-glycolysis (Xintaropoulou et al., 2015), apoptotic activity (Hamdan et al., 2013), anti-cancer (Choi and Park, 2015; Gibellini et al., 2011).

O O

O

5-(Hydroxymethyl) furfural

CH3

Acetovanillone O

CH3

OH O O

Methyl 4-hydroxyl cinnamate

HO O O

O

Ferulic acid methyl ester

HO

O

OH

Gallic acid HO

OH OH O

H3CO

OH Ferulic acid

HO O

OH

Protocatechuic acid OH OH OH OH O

HO

Quercetin OH

OH

O OH O

HO

Kaempferol OH OH

O

Chapter 13 • Edible spices for cancer prevention Table 13.1

279

Spices with cancer chemopreventive and therapeutic potentials—cont’d

S/N

Type of spice

Active principles

4

Curry leaf (Murraya Koenigii L.)

HO

Activity or property OH

O CH3

O

O CH3

O

Curcumin

OH HO

O

OH

Catechin

OH

Anti-oxidation (Nor et al., 2009), apoptotic effects (Ito et al., 2006; Jayakumar et al., 2014), anti-cancer (Bharti et al., 2014; So et al., 1996), antiinflammation (Gupta et al., 2011).

OH OH HO

O

Epicatechin OH OH OH O

HO

OH OH HO

OH

O

O

O

O

OH

H3C HO

Rutin

O HO

OH

OH OH O

HO HO

O

O O

H3C HO

OH

Naringin

O OH OH

O

OH OH OH

O

HO

Quercetin OH

OH

O OH OH

HO

O

OH

Myricetin

OH O

OH

5

Thyme (Thymus vulgaris L.)

OH O

HO

Apigenin OH OH

O

CH3

OH H3C

Anti-oxidation (Bai et al., 2014; Harrison et al., 2014), apoptotic effects (Seo et al., 2014; Shin et al., 2009), anti-inflammatory (Seo et al., 2012), antimutagenic (Mezzoug et al., 2007; Noel et al., 2006).

Thymol

CH3

Continued

Table 13.1

Spices with cancer chemopreventive and therapeutic potentials—cont’d

S/N

Type of spice

6

Peppers (chili, red, and green)

Active principles

Activity or property

N

Piperine

O

O

O

HO H N

O

Capsaicin

Anti-oxidation (Chatterjee et al., 2007; Oyagbemi et al., 2010), apoptotic effects (Chou et al., 2009; Thoennissen et al., 2010), anti-metastasis (Lai et al., 2012), anti-cancer (Kakarala et al., 2010).

O

7

Allspice (Pimenta dioica) berries and leaves

H H O

7″

1″ C

2″

O

O

O 5

HO 2′

3′

HO

2

6

HO

HO 3″

H H

1 6′

1′ OH

OH

4

Ericifolin

3 OME

HO

O

Eugenol HO O

OH

Gallic acid HO

OH OH HO HO O

HO HO

OH

O

HO

O

O

Pedunculagin

O

HO

O O

O

O

HO

OH

HO HO OH HO HOH2C HO

O

O

O C

O O

C

OH HO

HO OH

OH

Nilocitin OH

OH OH HO

OH

OH

OH

O O O O O

HO HO HO HO

HO

O O

OH

OO O HO OH

HO

OH

HO O OH OH

OH

Grandinin

Anti-oxidation (Marzouk et al., 2007; Zhang et al., 2015), anticancer (Zhang and Lokeshwar, 2012; Zhang et al., 2015), antiinflammation (Marzouk et al., 2007).

Chapter 13 • Edible spices for cancer prevention

Table 13.1

281

Spices with cancer chemopreventive and therapeutic potentials—cont’d

S/N

Type of spice

8

Rosemary (Rosmarinus officinalis L.)

Active principles

Activity or property Anti-oxidation (Cheung and Tai, 2007; Marrelli et al., 2015), anti-cancer (Gonzalez-Vallinas et al., 2014, 2015; Johnson, 2011; Yesil-Celiktas et al., 2010), anti-tumorigenesis (Singletary et al., 1996), antiinflammation (Cheung and Tai, 2007; Johnson, 2011; Scheckel et al., 2008).

OH HO HOOC

Carnosic acid

H OH

CH3

HO CH3

Carnosol

O O H H3C CH3

H

Ursolic acid

COOH

H HO OH O

O

OH

OH

Rosmarinic acid

O HO OH

9

Anti-oxidation (Yanishlieva et al., 2006), anti-cancer (de Oliveira et al., 2016; Loizzo et al., 2007).

Sage (Salvia officinalis L.) Manool

OH

CH3 HO H3C

O

CH3

H3C

H OH

H

CH3

HO H3C

H H3C

Pomolic acid Continued

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Table 13.1 S/N

Spices with cancer chemopreventive and therapeutic potentials—cont’d

Type of spice

Active principles

Activity or property

H

COOH

Ursolic

H HO

acid OH HO HOOC

Carnosic acid

H O

Eucalyptol

O

O O O

Carnosol o-quinone

O

10

Oregano (Oreganum vulgare L.)

4-Terpineol OH O HO

OH

Caffeic acid

HO OH

O

Protocatechuic acid OH OH

Carvacrol

Thymol

Anti-oxidation (Marrelli et al., 2015; Nakatani and Kikuzaki, 1987, 1989), anti-cancer (AlKalaldeh et al., 2010; Arunasree, 2010; Begnini et al., 2014; Kubatka et al., 2017; Marrelli et al., 2015).

Chapter 13 • Edible spices for cancer prevention

Table 13.1

283

Spices with cancer chemopreventive and therapeutic potentials—cont’d

S/N

Type of spice

11

Savory (Satureja hortensis L.)

Active principles

Activity or property OH

O

O

OH

OH

Rosmarinic acid

O

Anti-oxidation (Bertelsen et al., 1995; Trojakova et al., 2001; Yanishlieva et al., 2006).

HO OH

Carnosol

Carnosic acid

OH

Carvacrol 12

Cinnamon (Cinnamomum zeylanicum and Cinnamon cassia)

O

Eugenol HO

H O Cinnamaldehyde

O

Anti-inflammatory (Lee et al., 2005), anti-oxidant (Lee et al., 2003; Singh et al., 2007), antimicrobial (Matan et al., 2006), anti-diabetic (Kim et al., 2006), anti-cancer (Kwon et al., 2009; Sadeghi et al., 2019; Schoene et al., 2005).

OH Cinnamic acid

Coumarin

O 13

Clove (Syzygium aromaticum)

O

O

Eugenol HO

β-Caryophyllene

Anti-oxidant (Nagababu et al., 2010), anti-fungal (Singh et al., 2016), anti-microbial (Singh et al., 2016), anti-histaminic (Kim et al., 1998), antiinflammatory (Han and Parker, 2017), anti-cancer (CortesRojas et al., 2014; Liu et al., 2014). Continued

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Table 13.1 S/N

Spices with cancer chemopreventive and therapeutic potentials—cont’d

Type of spice

Active principles CH3

O

H3CO

Activity or property

Eugenin

H3C OH

O

OH

H H HO

14

Saffron (Crocus sativus L.)

Oleanolic acid

O

H

Anti-oxidant (Ochiai et al., 2004), anti-depressant (Shafiee et al., 2018), memory enhancer (Ghadrdoost et al., 2011), cardio protective (Zhang et al., 2017), anti-cancer (Naeimi et al., 2019).

CH3 O

H3C

H Safranal CH3 H3C

CH3

OH

O

O

HO HO

O

Picrocrocin

CH3

OH

OH HO O

HO

OH O OH

HO O

HO O O

OH HO

OH

O

O

HO OH

O O

O

OH

HO

Crocin

13.3 Anticancer activities of select spices used in daily diet 13.3.1 Garlic and onion These two members of the Allium genus (family Amaryllidaceae) that comprises over 500 species are the major “spices” used in the world. A general agreement has been established based on several clinical and epidemiological studies around the world that regular consumption of onion, garlic, and other allium plants has a significant reduction in cancers of esophagus (Chen et al., 2009; Galeone et al., 2006), stomach (Zhou et al., 2011), and prostate (Brasky et al., 2011; Hsing et al., 2002).

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There are extensive studies on various organosulfur compounds isolated from fresh garlic and onions. Alliin (S-allyl cysteine sulfoxide) and isoalliin (trans-(+)-S-(propen-1yl)-L-cysteine sulfoxide) are the major organosulfur components in garlic and onion, respectively (Nicastro et al., 2015). However, the bioactivities are attributed to the thiosulfinate compounds, the metabolic byproducts catalyzed by the enzyme alliinase (Lanzotti, 2006). These thiosulfinate metabolites (e.g., diallyl sulfide, diallyl disulfide, diallyl trisulfide) are responsible for the characteristic flavors and odors, but also exhibit anticancer or chemoprevention properties. These compounds show multiple mechanisms, including the formation, reduction, and detoxification of nitrosamines and heterocyclic amines (HCA), suppression of proliferation of cancer cells, anti-oxidant, and antiinflammation properties (Antony and Singh, 2011). Besides, several clinical trials have been conducted for the anti-cancer effects of garlic. Clinically, all of these compounds or aged garlic extracts exhibit antioxidant and chemo-sensitization properties in colorectal adenomas (Galeone et al., 2006; Lv et al., 2015; Zhu et al., 2014), and gastric cancers (Zhou et al., 2011). Different preparations of garlic and onions are available in a wide variety of dishes, which can influence the activity of alliinase and the bioactive compounds. For example, steaming or cooking can reduce garlic’s bioactivities in addition to the denaturation of alliinase (Song and Milner, 2001). Besides, the levels of thiosulfonates in onion are destroyed by greater than fivefold when cooked whole. In contrast, crushed or chopped onions appear to retain total thiosulfonates even after convection or microwave oven cooking (Cavagnaro and Galmarini, 2012). However, investigators also found storage of garlic over time will lead to an increase in the organosulfur components (Milner, 2006). These results suggested that alliinase are susceptible to high temperatures, and the bioactive thiosulfonates are stable enough while cooking. Consumption of raw allium spices or pre-production of thiosulfonates by crush or chop of garlic and onion is necessary to maintain their different forms of garlic preparations (raw, oil, and aged extract) have been studied in humans and showed controversial results. Although garlic intake is used to reduce the risk of gastric cancer in some studies (Gail and You, 2006; Li et al., 2004), two large prospective US cohorts (up to 30 years of follow-up with repeated measurement of diet and lifestyle factors) found no evidence to support this hypothesis (Kim et al., 2018). Other studies found that garlic consumption was inversely associated with lung cancer (Myneni et al., 2016), hematological malignancies (Walter et al., 2011), and colorectal cancer (Ngo et al., 2007). Diallyl trisulfide (DATS), a bioactive organosulfur compound found in garlic, has been investigated as adjuvant therapy. In xenograft animal models with gastric cancer cells, the combination of DATS and either docetaxel (Pan et al., 2016) or cisplatin ( Jiang et al., 2017) resulted in greater tumor growth inhibition when compared to the groups that received either drug alone. Although these studies provide encouraging results for cancer patients, bioactive garlic compounds specifically associated with anticancer effects need to be isolated, identified and evaluated. Future clinical trials on the effect of garlic should include information on the dosage of active ingredients of standardized garlic preparations for a better comparison of trials.

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13.3.2 Chili pepper/capsicum (Capsicum annum L.) These members of the genus Capsicum (family Solanaceae) are among the most heavily and frequently consumed spices throughout the world and ages. Capsicum fruits are a rich source of health-related compounds such as capsaicinoids, carotenoids, flavonoids, ascorbic acid (vitamin C) and tocopherols (vitamin E) (Wahyuni et al., 2011). Capsaicinoids, which are responsible for the strong and hot taste of Capsicum fruits, consist of several compounds, where capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the main one with 69% typical relative amount (Howard, 2007). Capsaicin activates the transient receptor potential channel, subfamily V member 1 (TRPV1), which leads to the suppression of hyperactive nociceptors and contributes to its pain relief effect (Alawi and Keeble, 2010; Anand and Bley, 2011; Geppetti et al., 2008). Besides, the growth of various human cancer cells has been inhibited by capsaicin through apoptosis or downregulation of the NF-kB signaling components and STAT3-regulated gene products (Bhutani et al., 2007; Hail and Lotan, 2002; Macho et al., 1999; Oyagbemi et al., 2010; Surh, 2002). More importantly, the in vivo anti-tumor activity of capsaicin has been observed in different mouse xenograft models with capsaicin administrated orally, subcutaneously, intraperitoneally, and directly into tumors (Bley et al., 2012).

Potential genotoxicity of capsaicin Potential genotoxicity of capsaicin has been examined both in vitro and in vivo using experimental rat models. The putative reactive metabolite and the genotoxic mechanism of capsaicin were found to be weak (Bley et al., 2012). Although the use of large dose, potentially 20  more than a commonly consumed dose of chili pepper or its extract in the diet was found to have a tumor-promoting potential in liver tumors in rats and mice (Surh and Lee, 1995), However, dose escalation of capsaicin in the diet in mice failed to provide direct proof for carcinogenicity in vivo (Akagi et al., 1998; Chanda et al., 2007). Several epidemiological studies also point out the carcinogenic potential of capsaicin and high chili pepper consumption is associated with an increase in the incidence of gastric cancer and gall bladder cancer (Lopez-Carrillo et al., 1994, 2003; Serra et al., 2002). However, pharmacokinetic analyses of capsaicin after peppers ingestion or topical administration have shown that capsaicin is rapidly absorbed, metabolized with a short plasma half-life, and cleared (Babbar et al., 2009; Chaiyasit et al., 2009; Chanda et al., 2008). Therefore, it is very likely that circulating levels of free capsaicin are quite low in the human body. Thus, the association between the higher rate of certain cancers with chili pepper consumption should be considered inconclusive, especially when the potential contamination of the consumed products with pesticides, insecticides, fertilizers, herbicides, microbicides, etc., are taken into consideration (Bley et al., 2012). Such unambiguous case-control studies have not been reported.

Application of capsaicin for use in the clinic In one report ( Jankovic et al., 2010), capsaicin could slow prostate-specific antigen doubling time (PSAdt) in a patient with PCa, who experienced biochemical failure after

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radiation therapy. When the patient ceased taking capsaicin, a PSA rise or a decrease in its doubling time was consistently observed, suggesting that capsaicin has the potential to be an adjuvant treatment for PCa. Moreover, multiple preclinical studies emphasized the promise of capsaicin as an adjunctive treatment option. When combined with certain chemotherapeutic drugs such as sorafenib (Zhang et al., 2018b), cisplatin (Huh et al., 2011), and camptothecin (Friedman et al., 2017), capsaicin can enhance the anti-tumor activity of these drugs. However, due to its hydrophobicity, low affinity, and short half-life, the use of capsaicin in clinical practice is remarkably limited ( Jiang et al., 2015; Zhang et al., 2019). Recently, various capsaicin-loading nanoparticles have been developed and tested to prolong its retention in the blood circulation and allow active targeting of cancer cells for improved, accurate delivery and target specificity ( Jiang et al., 2015; Lan et al., 2019; Lv et al., 2017). For instance, the efficacy of the capsaicin-loaded nanoparticles against the human glioblastoma U251 cells was significantly superior to capsaicin alone, suggesting that it can provide a promising delivery approach for chemotherapy of gliomas ( Jiang et al., 2015). Given the above findings, the combination of capsaicin and chemotherapeutic drugs or radiation therapy may provide opportunities to increase the sensitivity of chemotherapy or radiation therapy, minimize toxic side effects, and overcome chemotherapy resistance. Therefore, more well-controlled studies are required to assess the safety and efficacy of capsaicin, and further preclinical and clinical trials are needed to clarify the anti-tumor effect of capsaicin when combined with other chemotherapy drugs or radiation therapy.

13.3.3 Ginger Ginger (Zingiber officinale) belongs to the Zingiberaceae family, and its use originated in South Asia. It is used globally not only as a popular spice in cuisine preparation (Park and Pezzuto, 2002) but also as herbal medicine to treat headaches, nausea, colds, arthritis, rheumatological conditions, and muscular discomfort in multiple countries (Grant et al., 2000; Park and Pezzuto, 2002). Most of its medical uses are mainly due to its antioxidant and anti-inflammatory activities of the phenolic components present in it including gingerols, shagaols, paradol (Awang, 2009; Grzanna et al., 2005; Mashhadi et al., 2013), where 6-gingerol (1-[40 -hydroxy-30 -methoxyphenyl]-5-hydroxy-30 -decanone), and 6-shagaol (1-[40 -Hydroxy-30 -methoxyphenyl]-40 -decen-30 -one) which are the major pungent principles and biologically active phenolic compounds (Kundu et al., 2009). Studies have suggested the anti-cancer properties of ginger and its constituents are associated with cell growth inhibition, apoptosis induction, antioxidant, and anti-inflammatory potentials of its active components. In the in vitro studies, both 6-gingerol and 6-shogaol were found to reduce the viability of gastric cancer cells by ligand-induced apoptosis or mitotic arrest (Ishiguro et al., 2007). The anti-proliferation and anti-invasion activities of both components were also well documented against other cancer cells such as cancers of the liver, colon, and rectum (Prasad and Tyagi, 2015), breast (Ling et al., 2010), glioma (Rahman et al., 2014), and pancreatic cancer (Kim and Kim, 2013; Park et al., 2006).

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As for the anti-inflammatory activity, 6-gingerol was found to modulate NF-κB signaling, tumor necrosis factor (TNF), and interleukins (IL-1β and IL-6) in mouse macrophages (Lee et al., 2009; Tripathi et al., 2007) and inhibit TRAIL-induced NF-κB activation (Zhou et al., 2014). Moreover, 6-shogaol can also down-regulate the expression of iNOS and cyclo-oxygenase-2 (COX-2) in LPS-induced murine macrophages and might contribute to the inhibition of carcinogenesis (Pan et al., 2008).

Pre-clinical studies of ginger and its constituents The health benefit in treating and preventing gastrointestinal (GI) cancer of ginger and its components are extensively studied in the context of a healthy diet against cancer. In vivo studies using the rhizome of ginger and its principal active constituent showed the skin carcinogenesis suppression potential (Katiyar et al., 1996) and liver carcinogenesis suppression by scavenging the free radical formation and reducing lipid peroxidation (Yusof et al., 2008). 6-Shogaol inhibits the tumor growth in a pancreatic xenograft model by inducing apoptosis and decreasing cell proliferation (Zhou et al., 2014). More importantly, pharmacokinetics studies have shown that 6-gingerol is rapidly absorbed into plasma, and 6-gingerol glucuronide is the main metabolite ( Jiang et al., 2008; Wang et al., 2009). The plasma concentration of 6-gingerol glucuronide is substantially higher than 6-gingerol regardless of administration manner or dosage ( Jiang et al., 2008), which suggested the glucuronidation of 6-gingerol should be taken into consideration especially with mechanisms studies.

Clinical studies for using ginger Some studies in human volunteers using ginger extract or ginger powder suggested promising beneficial effects in alleviating nausea and vomiting involved with post-operation, chemotherapy, and pregnancy (Arslan and Ozdemir, 2015; Dabaghzadeh et al., 2014a,b; Ryan et al., 2012; Walstab et al., 2013). The mechanism involved in this bioactivity was explained by the 5-HT3 receptor antagonism of 6-gingerol and 6-shogaol and zingerone (Lete and Allue, 2016; Walstab et al., 2013). Although there is no clinical data available for the direct anti-cancer activity of gingers, the current data still indicated the potential of ginger as ancillary therapy for alleviating treatment-related adverse reactions in cancer patients.

13.3.4 Turmeric (Curcuma longa) Turmeric is a member of the Zingiberaceae (ginger) family. Dried turmeric powder is the main ingredient in various Indian, Thai, and Spanish cuisines. Similar to gingers, turmeric is widely used as a spice and natural food coloring in South Asian and Middle Eastern cooking, and as herbal medicine in India and China (Lal, 2012). Turmeric powder contains numerous curcuminoids, in which the polyphenolic compound curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, natural yellow) is the major effective component and is used to treat various respiratory disorders in a traditional Indian Ayurvedic medicine (Ammon and Wahl, 1991). Extensive research

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on curcumin provide evidence for its medicinal and health benefits with antiinflammatory (Brouet and Ohshima, 1995), anti-oxidant (Sharma, 1976), wound healing (Sidhu et al., 1998), hypoglycemic (Sharma et al., 2006), and antimicrobial activities (Negi et al., 1999).

Antitumor activities of curcumin Several studies have shown curcumin can efficiently suppress the activation of various pathways that are elevated in cancers. These pathways are the NF-κB mediated inflammation pathway (Shishodia et al., 2003, 2005; Singh and Aggarwal, 1995), Wnt/β-catenin signaling (Shanmugam et al., 2015), Nrf-2 signaling pathway (Balogun et al., 2003) and those associated with the signal transducer and activator of transcription 3 (STAT3) signaling (Alexandrow et al., 2012; Glienke et al., 2010). These are the mechanisms that regulate cell motility, tumor microenvironment, cell proliferation, angiogenesis, and metastasis. Inhibition of one or more of these pathways may explain the anti-carcinogenic and chemopreventive potential of curcumin observed in various types of cancers (Gupta et al., 2013; Rahmani et al., 2014a,b). The pre-clinical in vitro and in vivo studies have indicated the potential of curcumin against various types of cancer and minimal toxicity even at high doses (Dhillon et al., 2008; Soni and Kuttan, 1992). Many pilots, as well as phase I and II clinical trials of curcumin, have been conducted to investigate its potential in cancer treatment and prevention. Current data indicate that curcumin can potentiate not only the therapeutic effects of established cancer treatment with good tolerability at high dose but also has excellent potential against cancerous lesions (Cheng et al., 2001; Golombick et al., 2012).

Hurdles to clinical application of curcumin The bioavailability of orally administered curcumin to patients was found to be negligible because of the low aqueous solubility, poor gastrointestinal absorption, and rapid elimination (Garcea et al., 2005; Sharma et al., 2001). To overcome this problem, several curcumin formulations, including nanoparticles (Anand et al., 2010; Khalil et al., 2013; Sasaki et al., 2011; Zhongfa et al., 2012), liposomes (Prasad et al., 2014), micelles (Schiborr et al., 2014; Zhang et al., 2018a), and phospholipid complexes (Andrea Giori, 2009; Gupta and Dixit, 2011), have been studied and showed promising results with increased bioavailability, longer circulation, and potent biological activities. Besides, some natural compounds as adjuvants have also been used to increase the bioavailability of curcumin. A study showed that piperine, the main bioactive component of black pepper, when concomitantly administered with curcumin, can enhance the serum concentration, absorption, and bioavailability of curcumin in humans by 154% for 1–2 h post-application (Shoba et al., 1998). Intestinal absorption of curcumin was also found to be higher and stay significantly longer in tissues when administered concomitantly with piperine (Suresh and Srinivasan, 2010). In addition to these research findings, the clinical studies of curcumin are limited by its poor bioavailability. Further, clinical trials in a larger cohort with formulations of curcumin designed with improved bioavailability are needed to validate its anti-cancer potential.

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Clinical trials of curcumin for clinical use: Application in patients Multiple clinical trials have been conducted to evaluate the beneficial effects of curcumin in cancer patients. A phase I clinical study demonstrated that oral curcumin is not toxic to humans up to 8000 mg/day for 3 months (Cheng et al., 2001). Curcumin showed symptomatic relief, as well as improve marker-based tumor detection, and other parameters of various cancer conditions, including skin lesions (Chainani-Wu et al., 2007; Cheng et al., 2001), multiple myeloma (Golombick et al., 2009), head, neck (Kim et al., 2011), and orbital tumors (Lal et al., 2000), lung (Lal et al., 2000), breast (Bayet-Robert et al., 2010), colorectal (Carroll et al., 2011; Cruz-Correa et al., 2006; Garcea et al., 2005; Sharma et al., 2001), and prostate cancers (Ide et al., 2010). A randomized, double-blind, controlled study has been performed with patients who underwent prostate biopsies because of elevated PSA levels but who had negative findings for prostate cancer. They were randomly assigned to take a supplement containing isoflavones and curcumin (0.1 g/day for 6 months) or a placebo. The study showed that PSA level decreased in the treatment group with PSA 10 ng/mL (P ¼ 0.01) (Ide et al., 2010). Another study aimed to determine if oral curcumin could inhibit cancer progression in patients with prostate cancer receiving intermittent androgen deprivation (IAD) (Choi et al., 2019). They reported that a 6-month intake of oral curcumin (1440 mg/day) did not significantly affect the overall off-treatment duration of IAD. However, curcumin treatment resulted in a significantly lower PSA progression in comparison to the control group (P ¼ 0.0259). Regardless, curcumin use suffers from issues related to poor bioavailability due to poor absorption, high metabolism rate, rapid clearance, and elimination from the body, which all limit its therapeutic efficacy (Anand et al., 2007). Piperine, the active component of black pepper, has been described to increase curcumin bioavailability in human volunteers by 2000%, which is an increase of 20-fold, by creating a curcumin complex, ultimately inhibiting hepatic and intestinal glucuronidation (Shoba et al., 1998). However, no clinical testing of this combination or independent verification of these exciting results is available.

13.3.5 Cinnamon Cinnamon is a bark spice obtained from the inner bark of several tree species from the genus Cinnamomum (Lu et al., 2011). The cinnamon extract contains several active ingredients, including eugenol, cinnamaldehyde, cinnamic acid, and coumarin (Liang et al., 2019). Multiple studies have revealed that these active components possess diverse pharmacological functions, including anti-inflammatory, anti-oxidant, anti-microbial, antidiabetic, and anti-tumor effects (Yun et al., 2018). Cinnamon extract inhibits melanoma progression in vivo through apoptosis induction and blockade of NF-κB and AP1 (Kwon et al., 2010). Cinnamon extract decreases the expression of pro-angiogenic factors (EGF, VEGF, and TGF-β) and regulators of tumor progression (Cox-2 and HIF-1α) in melanoma cell lines and a melanoma mouse model (Kwon et al., 2009). Interestingly, the authors showed that the administration of cinnamon extract could significantly inhibit tumor progression by inhibiting angiogenesis while increasing the cytolytic activity of CD8 + T cells

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(Kwon et al., 2009). Eugenol is a critical compound in cinnamon that is found in many plants, including cinnamon, basil, lemon balm, and nutmeg (Kawatra and Rajagopalan, 2015). Eugenol, combined with gemcitabine, showed a strong chemo-sensitizing activity (Hussain et al., 2011). The chemo-sensitizing activity of eugenol was also reported in prostate cancer cells, in which the combination of 2-methoxy-estradiol and eugenol synergistically increased the cytotoxic effects against a castration-resistant, androgen receptor-negative prostate cancer cell line PC-3 (Ghosh et al., 2009). No clinical studies have been conducted to date, using cinnamon or its principle compound have been tested for potential to treat cancer patients has been conducted to date.

13.3.6 Cloves Cloves (Syzygium aromaticum) contain a wide range of bioactive compounds, including eugenol, β-caryophyllene, eugenol, eugenin, and oleanolic acid (Cortes-Rojas et al., 2014). Cloves are known to have active compounds with anti-microbial, anti-fungal, antibacterial, anti-septic, anti-histaminic, anti-inflammatory anti-oxidant, and anticarcinogenic properties (Kello et al., 2020). The in vivo efficacy of ethyl acetate extract of cloves (EAEC) has been investigated using the HT-29 tumor xenograft model (Liu et al., 2014). The authors identified oleanolic acid (OA) as one of the components of EAEC responsible for its anti-tumor activity. Both EAEC and OA display cytotoxicity against several human cancer cell lines. Interestingly, EAEC was superior to OA and the chemotherapeutic agent 5-fluorouracil at suppressing the growth of colon tumor xenografts. In another study, the aqueous extract of clove (AEC) inhibits tumor growth through AMPK/ULK pathway mediated autophagy (Li et al., 2019). Khan et al. (2018) reported that treatment of clove extract in combination with fluorescent magnetic submicronic polymer nanoparticles (FMSP-nanoparticles) enhanced FMSP-nanoparticles mediated cell death in MCF-7 cancer cell line. Despite extensive in vitro studies and testing on animal models, no clinical studies have been reported for antitumor activities of cloves or their extracts.

13.3.7 Saffron Crocus sativus, also known as saffron, is a naturally derived product from the dried stigma of the Crocus sativus flower (Cortes-Rojas et al., 2014). Recent studies have revealed that saffron can act as an anti-oxidant, anti-depressant, memory enhancer, cardioprotective, and anti-inflammatory agent (Naeimi et al., 2019). Saffron and its pharmacologically active components, including crocin, crocetin, and safranal exert their anticancer effects through different mechanisms, including induction of apoptosis, influence on the cell cycle, and regulation of host immune response and anti-inflammatory activities (Srivastava et al., 2010). Crocin is a diester formed from the disaccharide gentiobiose and the dicarboxylic acid crocetin (Mohajeri et al., 2010; Umigai et al., 2012). This compound is the principal constituent and the primary dark red pigment of saffron. Crocin can inhibit the proliferation and induce the apoptosis of

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various tumor cells, such as lung cancer (Chen et al., 2015), liver cancer (Noureini and Wink, 2012), gastric adenocarcinoma (Hoshyar et al., 2013), bladder cancer (Zhao et al., 2008), and leukemia (Rezaee et al., 2013; Sun et al., 2015). Crocetin, a natural apocaratenoid dicarboxylic acid, is the central core of crocin (Giaccio, 2004). It also showed cytotoxic effects in various cellular models and on different kinds of tumors, including leukemia, ovarian and breast carcinoma, colon adenocarcinoma, liver, pancreas, and lung cancer (Gutheil et al., 2012). A study compared the cytotoxicity effects of crocin and crocetin and found that crocetin has 5–18 times more toxicity than crocin due to its structural difference (Kim et al., 2014). For example, an in vivo study indicated that saffron enhanced cell cycle arrest in diethylnitrosamine (DEN)-induced liver cancer in rats (Amin et al., 2011). Similarly, crocetin induced cell cycle arrest in colon cancer cells through a p-53 independent mechanism (Li et al., 2012). Crocetin significantly stimulated apoptosis in pancreatic cells and athymic nude mice, as indicated by the Bax/Bcl-2 ratio (Dhar et al., 2009). Kawata et al. found that crocin significantly decreased the expression of some proinflammatory enzymes (COX-2 and iNOS) and cytokines (IL-6, IL-1β, TNF-α, and interferon γ) in the colorectal mucosa (Kawabata et al., 2012). Crocetin extracted from saffron inhibits the growth of U251 and U87 cells that were subcutaneously injected into animal models (Colapietro et al., 2020). Several studies have found that crocin in combination with various chemotherapy medications, such as cisplatin, doxorubicin, and paclitaxel, as well as crocetin with cisplatin, exerted enhanced anti-cancer effects on multiple cancer cells (Chen et al., 2015; Li et al., 2017; Mollaei et al., 2017; Vali et al., 2015). It has been shown that paclitaxel or radiotherapy combined with crocin has a synergistic effect in inducing the apoptosis of human MCF7 breast cancer cells, not only by inhibiting cell cycle progression through downregulation of cyclin D1 and p21 but also by inducing apoptosis (Ashrafi et al., 2015; Vali et al., 2015). Interestingly, anticancer activities of crocin increased after liposomal encapsulation (Mousavi et al., 2011; Rastgoo et al., 2013), and administration of crocin-coated magnetite nanoparticles (MNP)s was found to be accompanied by regression of precancerous lesions as well as significant upregulation of apoptotic cells, making crocin-coated MNPs more effective than free crocin (El-Kharrag et al., 2017). Due to the promising findings obtained from pre-clinical experiments, the anticancer and chemopreventive activities of saffron need to be investigated in clinical trials.

13.3.8 Jamaican pepper (Pimenta dioica) Almost all parts of Jamaican pepper are used as flavoring agents. However, Allspice is the dried unripe berries of the Jamaican pepper that is most common outside the Caribbean islands and Central America. Whole or powdered Allspice or its essential oil is used in many cuisines and folk medicine (Zhang and Lokeshwar, 2012). The medicinal properties of Allspice are due to its anti-oxidant, pro-apoptotic, and anti-proliferative activities in vitro and experimental animal models. Allspice is highly abundant (> 36% of dry weight; Zhang et al., 2015), which might contribute to its

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anti-cancer property. Specifically, the ethyl acetate extract of Allspice, together with some compounds isolated from leaves and berries of Jamaican pepper plant, shows antioxidant activities (Miyajima et al., 2004; Kikuzaki et al., 2000). The aqueous extract of Allspcie (AAE) exerts a significant anti-proliferative effect in prostate cancer both in vitro and in vivo through the inhibition of androgen receptors (Shamaladevi et al., 2013). AAE inhibits the proliferation of breast cancer cells and tumor growth in mouse xenografts. The main mechanism of antiproliferative and anti-tumor activities of AAE is attributed to cytotoxic autophagy via a down-regulation of mTOR-Akt signaling activity (Zhang et al., 2015). Besides, AAE was also found to decrease blood pressure level when given intravenously to Sprague-Dawley rats (Suarez et al., 1997), but cause depression of the central nervous system (CNS) in a dose-dependent manner in spontaneously hypertensive rats, without significantly affecting blood pressure (Suarez et al., 2000). Among many compounds isolated from Allspice, eugenol is the most abundant compound in Allspice, which composes 60–90% of the essential oil extracted from Pimenta berries. Eugenol is phenyl propene, that was shown to have anti-fungal (Ahmad et al., 2010), anti-inflammatory (Kim et al., 2003), and anti-oxidant effect (Ito et al., 2005). Also, Eugenol was found to have an anti-proliferative effect in various cancer cell lines by apoptosis induction, which is associated with E2F1 down-regulation in breast cancer and melanoma models (Al-Sharif et al., 2013; Ghosh et al., 2005). Quercetin is quite pluripotent with an antiviral, anti-inflammatory (BoeschSaadatmandi et al., 2012; Comalada et al., 2005; Ganesan et al., 2012; Min et al., 2007; Vrijsen et al., 1988). Quercetin to have anti-pulmonary hypertension in rats (Morales-Cano et al., 2014). The anti-cancer effects of quercetin are mainly through the inhibition of cell proliferation in breast cancer cells (Tao et al., 2015), human glioma cells (Pan et al., 2015), leukemia cell (Lee et al., 2015) via different mechanisms including apoptosis and signaling pathway regulations. Also, Quercetin can enhance the chemosensitivity of breast and prostate cancer cells to established chemotherapeutic drugs (Li et al., 2015; Wang et al., 2015). The preclinical studies on allspice are very limited currently, and more research on its chemistry and biology is required to understand its activities on cancer prevention.

13.4 Concluding summary Mounting pre-clinical data have shown have great potential of dietary spices to prevent cancer occurrence through multiple pathways, including anti-proliferation, apoptosis, anti-inflammation, angiogenesis, and immunocompetence, etc. Their low toxicities make the dietary spices more favorable as chemopreventive agents. However, we note that currently available data are still intriguing, and some controversial clinical data are reported from different clinical trials. This inconsistency might be due to the non-standardized experimental design, i.e., sample size, different formulations, dose, etc. Therefore, a standardized protocol for clinical studies of dietary spices is demanded to provide more solid and convincing results. Dose, formulation, duration, cancer type, and cancer stage should be carefully considered in future clinical trials.

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What’s more, considerably more information is also in need to determine if different spices intake together will provide better efficacies and if there are any interactions between the spices and the diet or the medications that cancer patients may consume regularly. Rigorous studies are still required to identify the pharmacokinetics of bioactive components, improve their bioavailabilities and standardize their clinical tests. All of this information will be helpful to define appropriate intervention strategies for maximum benefits from the dietary spices. It should also be pointed out that as natural products, the dietary spices are complicated mixtures of bioactive compounds that can affect each other’s pharmacokinetics, solubility and potentially, pharmacodynamics. Future studies should take this complexity into account and use “fingerprint” approaches to dissect their pleiotropic pharmacological properties.

Acknowledgments The research of the contributing authors was supported by PHS (NIH) Grant No. R01CA156776 and Veterans Health Administration Merit Grant #I01 BX003862-01A2.

Grant support This work was supported in parts by the Veterans Health Administration Merit Award: BX 001517-01 (BLL); the United States’ Public Health Services awards: NIH 5R01CA156776 (BLL). The authors declare no conflict with any commercial entities.

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14 Significance of nutraceuticals in cancer therapy Haritha H. Nair, Vijai V. Alex, and Ruby John Anto D I V I S I O N OF C AN C E R R E S EAR CH, RAJIV GANDHI CENTRE FOR BIOTECHNOLOGY, THIRUV ANANTHA PURAM, KERALA, INDIA

14.1 History of nutraceuticals The term “nutraceutical” was coined by Stephen DeFelice way back in 1989. It is the combination of two words: “nutrient” (a healthful diet component) and “pharmaceutical” (a therapeutic drug). Use of nutraceuticals is based on the saying by Hippocrates, the father of medicine, who said “let food be your medicine”. Nutraceuticals can be described as any non-toxic dietary compounds with extra health benefits in addition to the basic nutritional value found in foods. They are non-specific biological therapies, which can promote general well-being, prevent and control malignant processes. Nutraceuticals can be either plant or animal origin. Majority of the well-known nutraceuticals are phytochemicals. Although the therapeutic use of nutraceuticals has long ancient history, scientific evidences for their medicinal value came to lime light in recent times only. Extensive research has been done on the medicinal value of nutraceuticals owing to their pharmacological safety. Several studies have shown promising results for these compounds in various pathological complications such as diabetes (Baradaran et al., 2013; Nasri, 2013), athrosclerosis (Madihi et al., 2013; Setorki et al., 2013), cardiovascular diseases (CVDs) (Khosravi-Boroujeni et al., 2012), cancer (Anto et al., 2002; Bava et al., 2005; Sreekanth et al., 2011; Vinod et al., 2013a, b; Vinod et al., 2015; Thulasidasan et al., 2017) and neurological disorders (Akhlaghi et al., 2011; Roohafza et al., 2013). In spite of the therapeutic effects against broad spectrum of ailments, this chapter discusses the relevance of various nutraceuticals in cancer treatment together with their usefulness as chemopreventives and chemosensitizers.

14.2 Drawbacks in conventional cancer treatments By definition, cancer is a simple term which describes a group of diseases in which abnormal cells divide without control and invade nearby tissues. But the reality is far more extensive and distressing. Each of the cancer cells are independent units which can grow and form tumor mass. There are several steps defined as multi-step tumorigenesis through which a single cancer cell grows, multiply, form tumor mass, invade neighboring Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00014-X © 2021 Elsevier Inc. All rights reserved.

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tissue and spread to different parts of the body via blood and lymph to form secondary growth. The major treatment strategies for cancer include surgery, radiation therapy, chemotherapy, hormone therapy, adjuvant therapy, immunotherapy, etc.

14.2.1 Chemotherapy In oncology, chemotherapy is defined as the use of cytotoxic chemical substance to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. Even though the mainstay for cancer treatment, currently available chemotherapy regimens fail to accomplish their maximum therapeutic efficacy due to the emergence of acquired and intrinsic chemoresistance. A chemotherapeutic drug is termed as ideal if it has the ability to kill or impair cancer cells with little or no damage to normal cells. This could be mainly achieved by induction of apoptosis in cancer cells while the normal cells are spared. Since most of the currently available chemotherapeutic drugs fail to achieve this goal, they cause massive side effects. These side effects include massive cell death in hematopoietic cells, intestinal epithelial cells and hair matrix keratinocytes, which in turn result in impaired immunity, loss of digestive tract lining and hair loss (Botchkarev et al., 2000). Side effects can be short-term or long term according to the chemotherapeutic which is being used. The short-term side effects will get resolved within few months after the completion of the therapy. Common short-term side effects include fatigue, hair loss, easy bruising and bleeding, frequent infections, anemia (low red blood cell counts) nausea, vomiting, appetite changes, constipation, stomatitis, myelosuppression, alopecia, thromboembolism, etc. while long-term side effects are infertility, weight gain, cardiac dysfunction, chemobrain, early menopause, reduced lung capacity, kidney disease, bone diseases, and secondary leukemia (Partridge et al., 2001).

14.2.2 Chemoresistance Even with arrival of more advanced treatments based on increased understanding of tumor biology, chemotherapy remains as the main stay for cancer treatment. Despite of their invariable talents to tackle cancer, 90% of chemotherapeutics fail to conquer the terrain of cancer due to the presence of a strong barricade called chemoresistance (Longley and Johnston, 2005). Understanding the mechanisms by which chemoresistance arise is of primary importance in developing novel therapeutic approaches to treat cancer. Tumors can be intrinsically resistant to a particular drug or they can gain resistance during treatment. Acquired resistance needs more attention, as the tumors will become resistant not only to the drugs originally used to treat them, but also to other drugs with different mechanisms of action and this cross-resistance makes the scene worse. The mechanism of cellular uptake of many chemotherapeutic drugs is still unknown. Mutations or alterations in expression levels of drug target can have a major impact on drug resistance. Chemoresistance can be an outcome of defective apoptotic

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pathways also. Being the major machinery that accomplishes the cytotoxic effect of the drugs, any defect in the programmed cell death will lead to deterioration of the therapeutic efficacy and eventually contribute to resistance. In most cases, the emergence of chemoresistance is due to the drug-induced activation of survival signals and multidrug resistance genes. On the whole, molecular drug resistance is more complex and multifactorial than they appear and hence remain as intractable problem. The mechanisms that limit the bioavailability of drug and its interaction with tumor microenvironment are also major factors that affect drug sensitivity. Hence, any strategy that can overcome the drug resistance could make a huge impact on cancer cure and thereby increase survival rates.

14.3 Importance of nutraceuticals in cancer therapy Upon prolonged exposure to the chemotherapeutics, the cancer cells try to escape from the ill effects of the drug by inducing up-regulation of pivotal survival signals. Increasing the dosage of the drug could be a possible way to overcome this condition, but that path will be perfectly blocked by the huge side effects ahead (Nair and Anto, 2015). Here comes the significance of search for novel lead molecules from nature which can improve the therapeutic efficacy of the current drugs while being completely non-toxic to the normal cells. Bioprospecting and molecular pharmacology studies have shown that a large number of nutraceuticals, especially, phytochemicals can modulate the survival pathways induced by cancer cells, carcinogens and chemotherapeutics. From ancient days onwards, Indian and Chinese medicine had made use of the anti-cancer properties of various plant and animal derived products. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include epigallocatechin gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin, and tannic acid. They are found to exert significant effect in a broad spectrum of tumors by overcoming chemoresistance to diverse chemotherapeutic drugs. In vitro efficacy of most of the compounds is proved, but the in vivo validation is yet under scrutiny. Since chemoresistance is a tightly regulated process under the control of multiple survival pathways, the inhibition of any single molecule may not be sufficient to circumvent the phenomenon. Hence, compounds that can simultaneously modulate multiple survival signaling pathways might provide a better therapeutic outcome than that of individual inhibitors (Table 14.1).

14.3.1 Chemoprevention and chemosensitization Most of the currently available chemotherapeutics fail to accomplish their maximum outcome not because of their therapeutic inefficacy, but owing to the emergence of acquired and inherited chemoresistance. Cancer chemoprevention refers to the use of any natural or synthetic agent for the inhibition, delay or reversal of carcinogenesis

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Table 14.1

Key target molecules of common phytochemicals (Vinod et al., 2013a, b).

Phytochemical

Common source

Curcumin

Turmeric

Resveratrol

Grapes and berries Soybeans

Genistein Emodin EGCG Quercetin

Polygonum cuspidatum Green tea Fruits and vegetables

Target molecules NF-κB, Bcl-2, Bcl-XL, Bax, Bak, Akt, MAPKs, COX-2, PUMA, Bim, Noxa, p21, p27, VEGF, DR5, survivin, XIAP, MDR, MMPs, EGFR, P-gp, IAPs Cyclin D1, COX-2, ICAM-1, MMP-9, Cyp1b1, NF-κB, MDR1, Bcl-2, Bcl-XL, ERK1/2, Survivin, MRP1, STAT3 DR5, EGF-R, p38, c-FLIP, NF-κB, RANKL, MMP-9, MAPK, PI3K, PKC, AMPK, COX-2, HER-2, Akt, Survivin, EGFR, ERα HER-2/neu, P-gp, XIAP, survivin, MDR1, HIF-1α, MRP1, ROS, NF-κB, AP-1, RhoA, Survivin, β-catenin, Rad51, ERK1/2 PEA15, Bcl2, VEGF, MMPs, DR4, Fas, NF-κB, PCNA, Telomerase, mTOR, EGFR, GRP78, Bcl-2, JNK HIF-1α, Her-2/neu, DR5, c-FLIP, survivin, DRs, Akt, ERK, PKC-α, p53, Hsp72, MRP

before invasion. Several nutraceuticals, specifically those with anti-oxidant properties have attracted the attention of the scientific community as potential cancer-preventive agents (Benetou et al., 2015). Many factors have to be considered before the selection of a compound as a chemopreventive. Two of the most prominent factors are: efficacy and resultant clinical benefit, and toxicity and resultant clinical risk (Rabadi and Bergan, 2017). On prolonged exposure to the chemotherapeutics, the cancer cells try to escape from the ill effects of the drug by inducing up-regulation of pivotal survival signals. One can think of increasing the dosage of the drug to evade the condition, but that path will be perfectly blocked by the huge side effects ahead. Thus, chemosensitization can be a choice that absolutely matches the need. In simple words, chemosensitization can be defined as a process by which a non-toxic compound of either natural or synthetic origin sensitizes the cancer cells for the arrival of a second one, usually a cytotoxic agent without affecting the efficacy of the same, while being completely non-toxic to normal cells. This has an added advantage of minimizing the amount of the chemotherapeutic agent and thereby decreasing the side effects. On the commercial point, it will be of more economic value while thinking about the marketing cost of currently available chemotherapeutic drugs, at least in the case of developing countries where rate of cancer incidence is relatively high (Wcislo, 2014; Nair and Anto, 2015). Several reports have showcased the chemopreventive as well as chemosensitizing efficacy of different nutraceuticals with mechanism based evidences ( Jayaprakasha et al., 2016; Shankar et al., 2007, 2019; Puliyappadamba et al., 2015; Lee et al., 2018; Wang et al., 2007; Kim et al., 2014; Poltronieri et al., 2014; Zhang et al., 2017; Bava et al., 2011; Radhakrishnan et al., 2014; Sreekanth et al., 2011; Vinod et al., 2013a, b, 2015).

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14.4 Various nutraceuticals and their application in cancer therapy 14.4.1 Curcumin Curcumin is a polyphenolic compound isolated from the rhizome of Curcuma longa or turmeric, a member of the ginger family, Zingiberaceae. Turmeric is commonly used in the Indian subcontinent for health benefits, preservation of food and as a dye in textile industry. Curcumin is responsible for the golden yellow color of turmeric and was first isolated in 1815. In 1910, its structure was determined as diferuloylmethane by Lampe and Milobedeska. Turmeric is widely used as a traditional medicine in India to cure various types of ailments like, cough, cold, diabetic wounds, biliary disfunctions, anorexia, sinusitis, etc. Extensive clinical investigations have been done on the various medicinal aspects of curcumin and it has been reported that curcumin has the ability to reduce blood cholesterol, prevent LDL oxidation, inhibit platelet aggregation, suppress thrombosis and myocardial infarction, inhibit rheumatoid arthritis etc. (Aggarwal et al., 2007). The story of turmeric as a traditional remedy in “Ayurvedic medicine” and ancient Indian healing system dates back to almost 5000 years. It has been used through the ages as “herbal aspirin” and “herbal cortisone” to relieve discomfort and inflammation associated with a spectrum of infections (Chakraborty et al., 2014). Curcumin occupies unique position as both chemopreventive and chemosensitizer among the list of natural compounds due to its better success rate in cancer chemotherapy. Numerous in vitro and in vivo studies have established the anti-proliferative, anti-angiogenic, anti-metastatic, and pro-apoptotic properties of curcumin (Aggarwal et al., 2007; Anto et al., 2002; Kunnumakkara et al., 2008; Li et al., 2005; Bava et al., 2005; Sreekanth et al., 2011; Vinod et al., 2013a, b; Puliyappadamba et al., 2015). Curcumin is reported to potentiate the anti-tumor effects of gemcitabine and 5-FU via inhibition of gemcitabine-induced NF-κB and its downstream targets in an orthotopic model of pancreatic cancer (Kunnumakkara et al., 2007). Studies have documented the ability of curcumin to potentiate cytotoxic effect of through the down-regulation of COX-2 and phosphoERK1/2, modulation of cyclin D1, COX-2, matrix metallopeptidase 9 (MMP-9), VEGF, and C-X-C chemokine receptor type 4 (CXCR4) etc. in pancreatic and colorectal cancer (Lev-Ari et al., 2007; Kunnumakkara et al., 2015). Curcumin have been shown to sensitize prostate cancer cells to 5-FU chemotherapy in a p53-independent cell-cycle arrest and inhibition of constitutive NF-κB activation. The cytotoxic effect of 5-FU was found to be enhanced by curcumin in colon cancer cells through down-regulation of COX-2 (Du et al., 2006). Various in vitro and in vivo studies have shown that curcumin is capable of enhancing the chemotherapeutic efficacy of paclitaxel in cervical cancer cells through the down-regulation of paclitaxel-induced activation of NF-κB, Akt, and Bcl-2 (Bava et al., 2005; Sreekanth et al., 2011). Being the most promising chemosensitizer which can be combined with almost all of the common chemotherapeutics, curcumin still has certain drawbacks which shadow its beneficial nature. Poor absorption and low systemic bioavailability are the limiting factors which minimize the reach of adequate concentrations for pharmacological effects in certain tissues, active levels in the gastrointestinal tract have been found in animal and human pharmacokinetic studies (Shehzad et al., 2010) (Figs. 14.1 and 14.2).

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Guggul

Golden seal

Horse chestnut

White ginger

Turmeric

Tea

Cashew

Capsicum

Three-wing-nut

Japanese knotweed

Evodia

Kokum

Black Cumin

Ashwagandha

Beans

Houpu magnolia

Grape

Milk thistle

Figs

Talispatra

Garlic

Cabbage

Coffee

Carrots

Strawberry

Ginger

Tomato

Sesame

Palm

Vanilla

Soybean

Sage

Broccoli

Apple

Mulberry

Fenugreek

Pomegranate

Asian ginger

Kale

Black pepper

FIG. 14.1 Common sources of nutraceuticals (Gupta et al., 2010).

Parsley

Barberry

Chapter 14 • Significance of nutraceuticals in cancer therapy

O

O

315

OH HO

HO

OH OCH3

OCH3

Curcumin

OH

OH

OH O

HO

Resveratrol

OH

OH

O

OH O

OH

H3C

OH

O

OH O

OH

EGCG

OH

Emodin

OH

OH

O

N O

HO

O

Genistein

O

O

Piperine

OH OH O

OH

O

HO

CH3 HO OCH3

OH

[6]-Gingerol

H3C

OH

CH3

CH3

CH3

H3C

CH3

CH3

Lycopene FIG. 14.2 Chemical structures of various nutraceuticals.

CH3 H3C

CH3

O

Quercetin

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14.4.2 Resveratrol Resveratrol is a plant-derived polyphenol which is produced by the enzyme stilbene synthase. It is a phytoalexin produced in response to environmental stress such as harsh climatic changes, exposure to ozone, presence of higher amount of heavy metals, infections by pathogenic microorganisms, etc. Resveratrol is mainly found in skin of red grapes and thus the major component of red wine. It is major component of different types of berries and pines like raspberries, blueberries, mulberries, Scots pine, Eastern white pine, peanuts, knotweed, etc. The presence of resveratrol is considered as the main reason for health benefits offered by these fruits and nuts. Resveratrol has been reported to exhibit a wide range of health-promoting effects for the coronary, neurological, hepatic, and cardiovascular systems (Athar et al., 2009). It has the ability to counter inflammation, viral infection, oxidative stress, and platelet aggregation and the growth of a variety of cancer cells. Resveratrol was first isolated in 1940 as an ingredient of the roots of white hellebore (Veratrum album) which is highly poisonous and later, in 1963, from the roots of Japanese knotweed. It has been shown to sensitize cancer cells to conventional chemotherapeutic drugs by modulating different molecular players of chemoresistance. Resveratrolmediated chemosensitization is mainly due to the direct interaction of resveratrol with different molecular targets including cyclin dependent kinases (Cdks), Bcl-2 family members, p-53, c-Myc, PTEN, mTOR, NF-κB, AP-1, Cox-2, Sirtuins, Nrf2, Akt, IAPs, MMPs, VEGEF, etc. Thus, resveratrol has got unique capability to inhibit multistep tumorigenesis. Extensive studies have been done to verify the chemopreventive as well as chemosensitizing efficacy of resveratrol in breast cancer (Sinha et al., 2016). Most of the studies were concentrated on the efficacy of resveratrol as a phytoestrogen.

14.4.3 Genistein Genistein is an isoflavone with a heterocyclic diphenolic structure found in soybeans. It has been shown to inhibit the growth of various cancer cells in vitro and in vivo without toxicity to normal cells. Most of the research have found that genistein works as a powerful chemosensitizer with anti-metabolites like 5-FU and gemcitabine. Combination of erlotinib and gemcitabine is an FDA approved regimen for treatment of advanced pancreatic cancer. Down-regulation of major survival molecules like NF-κB, Akt, AMPK, TRAIL, MAPKs, etc. is established as the mechanism of action of genistein-mediated chemosensitization and chemoprevention (Vinod et al., 2013a, b).

14.4.4 Emodin Emodin is 6-methyl-1,3,8-trihydroxyanthraquinone isolated mainly from Rheum palmatum, an edible rhubarb. It is also found in many species of fungi, including members of the genera Aspergillus, Pyrenochaeta, and Pestalotiopsis. Emodin is used in combination with paclitaxel against breast cancer and ovarian cancer where it acts as a tyrosine kinase

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inhibitor and suppress growth and transformation of cancer cells (Vinod et al., 2013a, b). Emodin is also a powerful anti-oxidant which is reported to modulate ROS pathways (Yi et al., 2004). NF-κB, AP-1, P-gp, HIF1-α, XIAP, ERK1/2, HER-2, Bcl-2, TRAIL, etc. are the major molecules modulated by emodin-mediated chemoprevention and chemosensitization (Vinod et al., 2013a, b).

14.4.5 EGCG Epigallocatechin gallate (EGCG) is a powerful polyphenolic, chemopreventive compound isolated from green tea. It is well-known for its anti-oxidant properties. Polyphenon E, a well standardized decaffeinated green tea catechin mixture that contains 65% of EGCG along with epicatechin is currently being used in clinical trials (Zhang et al., 2004). Down-regulation of TRAIL-mediated chemoresistance is the major mechanism of action of EGCG in a variety of cancer cells. NF-κB, Akt, IFN-α, EGFR, COX-2, HER-2, m-TOR, etc. are some of the major signaling pathways targeted by EGCG (Vinod et al., 2013a, b).

14.4.6 Quercetin Quercetin is a flavonoid found in fruits and vegetables which possess anti-cancer properties. It is chemically 3,30 ,40 ,5,7-pentahydroxyflavone. Several studies have showcased the ability of quercetin to enhance the efficacy of anti-cancer drugs and by sensitizing cancer cells to chemotherapy. Quercetin is commonly used in combination with doxorubicin, dacarbazine and cisplatin. Down-regulation of HIF1-α, HER2/neu, or by induction of persistent T-cell tumor-specific responses is revealed as the mechanism behind chemosensitizing efficacy of quercetin to doxorubicin chemotherapy. Quercetin also possesses a property to induce immune responses against tumors. The synergistic effect of quercetin and cisplatin could also be mediated through the down-regulation of Bcl-2 and Bcl-xL with the concomitant up-regulation of Bax and the induction of mitochondrial membrane permeabilization (Vinod et al., 2013a, b).

14.4.7 Lycopene Lycopene is the red-pigmented, prominent β-carotenoid present in tomatoes and human plasma. It is an acyclic isomer of beta-carotene also present in fruits like watermelons, pink grapefruits, apricots, pink guavas and papaya. Lycopene is also found in microorganisms but absent in animals. Anti-cancer activities of lycopene are mainly through two different mechanisms: non-oxidative and oxidative. Lycopene can quench singlet oxygen and keep the redox homeostasis in cells. In this way, lycopene prevents oxidation of lipids, proteins and DNA, there by opposing multistage carcinogenesis. Lycopene also suppress carcinogen-induced phosphorylation of p53 and Rb and stop cell division at the G0-G1  ska, 2014). Several studies have shown that lycocell cycle phase (Gajowik and Dobrzyn pene possess radio protective abilities also (Andic et al., 2009).

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14.4.8 Piperine Piperine is the predominant dietary alkaloid found in the fruits and roots of Piper nigrum L. (black pepper) and Piper longum L. (long pepper) species of Piperaceae family. Being responsible for the characteristic pungency and biting taste of pepper, piperine is chemically 1-Piperoylpiperidine. It exhibits anti-oxidant, anti-inflammatory, immunomodulatory, anti-asthmatic, anti-convulsant, anti-mutagenic, anti-mycobacterial, anti-amoebic and anti-cancer activities. Numerous studies have reported the chemoprevetive efficacy of piperine against cancer cells of various origins by activation of apoptotic signaling and inhibition of cell cycle progression (Tanaka, 2013). Piperine is also known for its influence on the redox homeostasis, cancer stem cell (CSC) self-renewal, modulation of ER stress, autophagy, invasion, metastasis, and angiogenesis of cancer cells. It is a potent inhibitor of P-gp and hence reverse multidrug resistance (MDR) in cancer cells. Piperine has a significant effect on the drug metabolizing enzyme (DME) system and acts as bioavailability enhancer for many chemotherapeutic agents (Rather and Bhagat, 2018).

14.4.9 Gingerol [6]-Gingerol constitutes the most pharmacologically active component of ginger. [6]Gingerol exerts its anti-cancer activity through important mediators and pathways of cell signaling including Bax/Bcl2, p38/MAPK, Nrf2, p65/NF-κB, TNF-α, ERK1/2, SAPK/JNK, ROS/NF-κB/COX-2, caspases-3, -9, and p53 (de Lima et al., 2018). Gingerol has the ability to modulate several signaling molecules like NF-κB, STAT3, MAPK, PI3K, ERK1/2, Akt, TNF-α, COX-2, cyclin D1, cdk, MMP-9, survivin, cIAP-1, XIAP, Bcl-2, caspases, and other cell growth regulatory proteins. Various studies have shown that [6]-gingerol exhibits excellent anti-cancer potential specifically against gastrointestinal tract cancers likegastric cancer, pancreatic cancer, liver cancer, colorectal cancer, and cholangiocarcinoma (Prasad and Tyagi, 2015).

14.5 Conclusion and future prospective Even though each of the above-mentioned nutraceuticals exhibit exceptional chemopreventive and chemosensitizing efficacies, the major drawbacks of the clinical usage of these phytochemicals are their poor bioavailability and retention time inside body. Low serum levels, poor tissue absorption and distribution, short half life and rapid metabolism and elimination also contribute greatly for their failure in clinical scenario. Intense research is carried out to increase the bioavailability of these compounds by incorporating chemical modifications, like nano-encapsulation, glucuronidation, polymerization etc. in order to enhance the therapeutic efficacy. The effective use of non-toxic nutraceuticals can enhance the efficacy of the chemotherapeutic drugs, by bringing down the optimal dose of the drug thereby minimizing the cost and side effects of chemotherapy.

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Kunnumakkara, A.B., Guha, S., Krishnan, S., Diagaradjane, P., Gelovani, J., Aggarwal, B.B., 2007. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB-regulated gene products. Cancer Res. 67, 3853–3861. Kunnumakkara, A.B., Anand, P., Aggarwal, B.B., 2008. Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Lett. 269, 199–225. Kunnumakkara, A.B., Diagaradjane, P., Anand, P., Harikumar, K.B., Deorukhkar, A., Gelovani, J., et al., 2015. Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model. Int. J. Cancer 125, 2187–2197. Lee, P.S., Chiou, Y.S., Ho, C.T., Pan, M.H., 2018. Chemoprevention by resveratrol and terostilbene: targeting on epigenetic regulation. Biofactors 44 (1), 26–35. Lev-Ari, S., Vexler, A., Starr, A., Ashkenazy-Voghera, M., Greif, J., Aderka, D., et al., 2007. Curcumin augments gemcitabine cytotoxic effect on pancreatic adenocarcinoma cell lines. Cancer Investig. 25, 411–418. Li, L., Braiteh, F.S., Kurzrock, R., 2005. Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis, signaling, and angiogenesis. Cancer 104, 1322–1331. Longley, D.B., Johnston, P.G., 2005. Molecular mechanisms of drug resistance. J. Pathol. 205 (2), 275–292. Madihi, Y., Merrikhi, A., Baradaran, A., Rafieian-kopaei, M., Shahinfard, N., Ansari, R., et al., 2013. Impact of sumac on postprandial high-fat oxidative stress. Pak. J. Med. Sci. 29, 340–345. Nair, H.H., Anto, R.J., 2015. Triple negative breast cancer: the therapeutic windows yet to be opened? Sci. Lett. J. 4, 175. Nasri, H., 2013. Impact of diabetes mellitus on parathyroid hormone in hemodialysis patients. J. Parathyr. Dis. 1, 9–11. Partridge, A.H., Burstein, H.J., Winer, E.P., 2001. Side effects of chemotherapy and combined chemohormonal therapy in women with early-stage breast cancer. J. Natl. Cancer Inst. Monogr. 2001 (30), 135–142 (J. Natl. Cancer Inst. 2002;94(11):866;J. Natl. Cancer Inst. Monogr. 2003;(31):131 [erratum]). Poltronieri, J., Becceneri, A.B., Fuzer, A.M., Filho, J.C., Martin, A.C., Vieira, P.C., Pouliot, N., Cominetti, M.R., 2014. [6]-gingerol as a cancer chemopreventive agent: a review of its activity on different steps of the metastatic process. Mini Rev. Med. Chem. 14 (4), 313–321. Prasad, S., Tyagi, A.K., 2015. Ginger and its constituents: role in prevention and treatment of gastrointestinal cancer. Gastroenterol. Res. Pract. 2015, 142979. Puliyappadamba, V.T., Thulasidasan, A.K., Vijayakurup, V., Antony, J., Bava, S.V., Anwar, S., Sundaram, S., Anto, R.J., 2015. Curcumin inhibits B[a]PDE-induced procarcinogenic signals in lung cancer cells, and curbs B[a]P-induced mutagenesis and lung carcinogenesis. Biofactors 41 (6), 431–442. Rabadi, L.A., Bergan, R., 2017. A way forward for cancer chemoprevention: think local. Cancer Prev. Res. (Phila.) 10 (1), 14–35. Radhakrishnan, E.K., Bava, S.V., Narayanan, S.S., Nath, L.R., Thulasidasan, A.K., Soniya, E.V., Anto, R.J., 2014. ‘[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling. PLoS One 9 (8), e104401. Rather, R., Bhagat, M., 2018. Cancer chemoprevention and piperine: molecular mechanisms and therapeutic opportunities. Front. Cell Dev. Biol. 15, 6–10. Roohafza, H., Sarrafzadegan, N., Sadeghi, M., Rafieian-Kopaei, M., Sajjadi, F., Khosravi-Boroujeni, H., 2013. The association between stress levels and food consumption among Iranian population. Arch. Iran Med. 16, 145–148.

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15 Common techniques and methods for screening of natural products for developing of anticancer drugs Monika Singha, Sukanya Patraa, and Rajesh Kumar Singhb a SCHOOL OF BIOMEDICAL ENGINEERING, INDIAN INSTITUTE OF TECHNOLOGY ( BHU), V AR AN A SI , I N DI A b DE PARTMENT OF DRAVYAGUNA, FACULTY OF AYURVEDA, INSTITUT E O F MEDICAL S CIENCES, BANARAS HINDU UNIVERSITY, V ARANASI, UTTAR P RADESH, INDIA

15.1 Introduction Cancer is an uncontrolled growth of cells and foremost communal health problem globally (Siegel et al., 2018) and worldwide new incidence is about 6 million cases per year (Srivastava et al., 2005). Some cancer-causing agent such as chemical substances or toxic compound, ionizing radiation, some pathogens and mutation are responsible. Surgery, ionizing radiation and chemotherapy is a treatment for the cancer which use huge number of drugs. The conventional procedure for cancer treatments includes surgical removal of the malignant tissue, chemotherapy and ionizing radiation etc., which causes severe side effect as well as destroying the adjacent normal cells (Singh et al., 2019a). Next alternative drug needs to be discovered, have efficacy against cancer with minimum side effects of chemotherapy and recognized potential in drug discovery and development (Cragg et al., 2009). Only natural occurring anticancer drug have the ability to enhance the immunity against metastatic cells (Cragg et al., 2009). Most of the chemotherapeutic drugs are isolated from the plant such paclitaxel from Taxol etc. (Sharma and Gupta, 2015). The phytochemicals or natural anticancer drugs having the strength to encourage apoptosis in cancer cells are promising to play significant role for treating the cancer (Singh et al., 2019b). The natural products have potential anticancer agents years back, at least, to the Ebers Papyrus in 1550 BCE, but the scientific period of this search is much more recent, beginning with the investigations by Hartwell and co-workers in late 1960s on the application of podophyllotoxin and its derivatives as anticancer agents. A large number of plants, marine, and microbial sources have been tested as leads, and many compounds have survived the potential leads (Srivastava et al., 2005; Shu, 1998). Numerous herbal drugs have been used in cancer treatment such as Podophyllotoxin, Combretastatins, Taxol, Vincristine, Vinblastine, Teniposide and Camptothecin, etc. (Srivastava et al., 2005). Evolutionary Diversity as a Source for Anticancer Molecules. https://doi.org/10.1016/B978-0-12-821710-8.00015-1 © 2021 Elsevier Inc. All rights reserved.

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The aim of plant derived molecule to isolate biologically active molecule or compounds produce patentable entities of higher activity and lower toxicity. Further, which can be used in cancer related treatment, identify, screen and their examination via in vitro and in vivo are necessary. So, plant derived anticancer drug need to identify more and more. Patwardhan et al. (2005) described new approaches to advance and fast track the joint drug discovery and development process are expected to take place mainly from the innovation in drug target elucidation and lead structure discovery. Before clinically trial of drug, it should be necessary that well elucidation of their structure with extraction, identification, cytotoxicity and other parameter of anticancer drug.

15.2 Extraction of compounds Extraction is a process used to purify material/compound without disturbing the properties of the same with maximum yield. It is the very first tool to separate the preferred natural products from the crude materials. Different types of extraction methods are solvent extraction process, distillation, and sublimation. Among the different techniques, solvent extraction technique is the broadly used method. This extraction technique takes place through the following multiple steps: (i) penetration of solvent into the solid matrix; (ii) dissolution of solid in the solvents; (iii) diffusing out the solute from a solid matrix; and (iv) collection of extracted solid from crude material (Zhang et al., 2018a,b). The selection of the solvent is the key attention for this process. Selectivity, solubility, cost, and safety need to be considered in the choice of solvents. According to the law of intermiscibility (like dissolves like), solvents with a polarity value near to the solute polarity performs better and vice versa. Generally, methanol and ethanol are considered as universal solvents for extraction of phytochemical materials. Depending on the fineness of the particle, yield of the extraction process is more. Due to the greater penetration of solvents and diffusion of solutes of the small particle, extraction efficiency can be increased. With an increase in time range also, extraction efficiency increases. Types of different traditional extraction methods are maceration, percolation and reflux extraction. Usually these methods required longer time with large amount of solvent. So some greener extraction methods listed as supercritical fluid extraction (SFC), pressurized liquid extraction (PLE), and microwave-assisted extraction (MAE). Nowadays, these methods are used for the extraction of natural products. The use of lower organic solvent, shorter extraction time, higher selectivity and high yield are the major advantages of these extraction technique over conventional methods (Zhang et al., 2018a,b).

15.2.1 Different types of extraction methods Pressurized liquid extraction (PLE) Pressurized liquid extraction (PLE) is a preset technique with elevated temperature and pressure to get extracted material from solid crude matrices (Fig. 15.1). The major

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Chemical properties FIG. 15.1 An extraction assembles for PLE.

advantages of this technique are lower consumption of solvent enhancing the sample throughput. Therefore, this technique can be considered as an environment-friendly technique as generating small volumes of waste along with reducing costs and time of the complete process. In this process, at first, the solid sample is placed in a stainless-steel vessel and brought to operating pressure (>100 bar) and simultaneously pumping selected solvent into the vessel. The vessel is heated to a selected temperature (50–200 °C) and maintain that value. The remaining extract is flushed from the vessel by a fresh solvent followed by a nitrogen gas purge. The time period of the extraction time is important. A longer extraction time favors solvent absorption by the matrix and increased penetration of solvent into sample defects or interstices. Repeating the extraction with fresh solvent in a cyclic fashion under full automation and provide an option for difficult to extract samples. Some advantages are: solvent consumption is low (15 mL for a 10 g sample); extraction time is short (typically 31 °C and critical pressure may be considered maintained as 74 bar. Selectivity, low temperature, and speed are some advantages of this method. Supercritical fluid can be improved by varying the pressure and temperature, allowing selective extraction (Tanaka et al., 2004). Diffusivities are much faster in supercritical fluids than in liquids, and therefore extraction can occur faster. An extraction with an organic liquid may take several hours, whereas with supercritical fluid, extraction can be completed in 10–60 min (Aizpurua-Olaizola et al., 2015). Mainly SFE process is extensively used for the extraction of polymers, essential oils, pesticides, food materials etc. Due to the high capital investment cost, commercial use is less. Supercritical fluid extraction (SFE) is a well-established process for the recovery of different organics, mainly for nonpolar substances. It allows for selective extraction of different chemicals without additional clean-up steps with little quantity of sample (Pabby and Sastre, 2019). Supercritical fluid extraction (SFE) can be coupled to SFC using a series of switching valves and either a loop or an accumulator trap interface (Poole, 2003).

Microwave-assisted extraction (MAE) The development of the MAE technique was reported by Ganzler and co-workers. Microwaves generate heat and weakening the hydrogen bond. It is interacting with polar compounds such as water and some organic components present in the plant matrix and cause rotation of dipole. The transfers of heat and mass are in the same direction in MAE, which generates a synergistic effect to accelerate extraction and extraction yield. There is a wide range of polarity of solvents used in this process (i.e., heptane to water). This MAE provides many advantages, such as increasing the extract yield, decreasing the thermal degradation and selective heating of vegetal material. MAE is also regarded as a green technology because it reduces the usage of organic solvent. There are two types of MAE methods: solvent-free extraction (usually for volatile compounds in a closed vessel with controlled) and solvent extraction (usually for nonvolatile compounds in the open atmosphere) (Zhang et al., 2018a,b; Vinatoru et al., 2017; Chemat and Cravotto, 2012; Poole, 2020).

Pulsed electric field extraction (PEF) Pulsed electric field extraction considerably increases the extraction yield and decrease the extraction time because it can increase mass transfer during by degrading membrane structures. The effectiveness of PEF treatment depends on several parameters like field strength, specific energy input, pulse number, and treatment temperature. PEF extraction is a nonthermal method and minimizes the degradation of the thermo-labile compounds. Hou et al. obtained the highest yield of the ginsenosides (12.69 mg/g) by PEF using some specific parameters. The yield of the ginsenosides of the PEF extraction method is higher as compared to MAE, heat reflux extraction, UAE and PLE. The entire PEF extraction process took less than 1 s and much less than the other tested methods as described

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earlier (Hou et al., 2010). In a study of antioxidants extracted from Norway spruce bark, Bouras found that much higher phenolic content (eight times) and antioxidant activity (30 times) were achieved very fast and easily after the PEF treatment compared to untreated samples (Bouras et al., 2016).

15.3 Fractionation The natural product is a complicated mixture of several compounds with a number of chemical and physical properties. So, the elementary strategy for separating the components is based on their physical and chemical properties. The process that divides the unpolished mixture of compounds into different chemical classes is defined as fractionation. So many different techniques are used for the fractionation of natural products. It is a separation process in which a certain quantity of a crude mixture (gas, solid, liquid, and enzymes) is divided during a phase transition, into a number of smaller quantities in which the composition varies according to the selected gradient. Fractions are collected based on individual’s specific property. In a single run more than two components can be separated through fractionation.

15.3.1 Fractionating techniques Solvent-solvent partitioning methods This can be used for the separation (fractionating) of different classes of natural products from the initial extract. The separation technique using solvent partitioning involves the use of two immiscible solvents in a separating funnel, and the compounds are distributed between two solvents according to their different partition coefficients (Fig. 15.2). This method is quite easy to handle and highly effective as the first step for the separation of compounds from crude natural product extracts. Solvents with different polarities can be applied in order to separate different polar compounds from the initial plant extract. These techniques also include plant tissue homogenization, maceration, and Soxhlet extraction.

Fractionation based on acid-base nature of solvent A typical fractionation procedure on acid-base nature described here. A plant extract in an organic solvent (e.g., ethyl acetate) is shaken with an inorganic base such as aqueous sodium hydrogen carbonate to separate the carboxylic acids as their water-soluble salts. The more weakly acidic phenolic compounds may only be extracted with a strong base solution such as a sodium hydroxide solution. Extraction of the original solution with an acid such as dilute hydrochloric acid will remove the bases such as the alkaloids, amines as their salts. The neutral compounds remain behind in the organic phase. The acids and phenols may be recovered from the aqueous solution of their sodium salts by treatment with dil. HCl and re-extraction takes place with an organic solvent, and the bases may be recovered by treatment of their salts with NH3 and re-extraction with an organic solvent.

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FIG. 15.2 Separation of the extract into acidic, basic, phenolic and neutral nature.

Fractionating techniques are important to prepare tannin free plant extracts and to remove unwanted matters such as pigments, protein, etc. from the plant extracts which make it easy to proceed with isolating of desired compounds. Presence of plant pigments such as chlorophyll, carotenoids, etc. disturb the isolation of target compounds from the extracts and also may interfere with chemical or biological assays. Therefore, it is good to remove pigments at the beginning of extraction. A scheme is given below for the extraction of plant material from acid, base, and alcoholic medium.

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15.4 Purification Natural plant product extraction is a highly complex and comprise mixture of neutral, acidic, basic, lipophilic, hydrophilic, or amphiphilic compounds. For the testing of biological activity especially in drug discovery and for the structure elucidation of natural potential compounds, a very small quantity of the substance in the pure form is needed. Therefore, the isolation of compounds from their extracts in the pure stage is an important phenomenon in natural product-related research.

15.4.1 Different purifications techniques Distillation Distillation is a procedure that separates a mixture of liquids according to their boiling points. It is an appropriate technique mainly used in chemistry labs, to purify the compounds. There are so many types of distillation processes. Example: simple distillation, fractional distillation, vacuum distillation, hydro distillation, double distillation, etc. For purification of plant products mainly the steam distillation process is used. Steam distillation is a method for distilling compounds that are heat-sensitive. The temperature of the steam is easier to control than the surface of a heating element and allows a high rate of heat transfer without heating at a very high temperature. This process involves bubbling of steam through a heated mixture of the raw material. By Raoult’s law, some of the target compounds will vaporize (according to partial pressure). The vapor mixture is cooled and condensed and usually give a layer of oil and water.

Hydro distillation and steam distillation (HD and SD) Hydro distillation (HD) and steam distillation (SD) are commonly used methods for the extraction of volatile oils. Some natural compounds give decomposition in HD and SD also. The chemical composition and antibacterial activity of the primary essential oil and secondary essential oil from Menthacitrata were significantly affected by distillation methods. Both primary essential oil and secondary essential oil yields by HD were higher than that by SD technique. Yahya and Yunus found that the extraction time affect the quality of the essential patchouli oil extracted. When the extraction time increased, the contents of some components may decreased or increased (Strati et al., 2015; Yahya and Yunus, 2013; Zhang and Su, 2002).

15.5 Crystallization Crystallization is the (natural or artificial) process by which solid forms, where the atoms or molecules are highly arranged into a predefined order called crystal. Some of the ways by which crystals form are precipitating from a solution, or deposition takes place directly from a gas. Features of the resulting crystal depend on some factors like temperature, air

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pressure and in liquid crystals, time of fluid evaporation. Generally, nucleation is a twostep process as nucleation and growth. For crystallization of any chemical compound, you can choose either of the two methods:

15.5.1 Single solvent Dissolve the compound (mix of two compounds) in a solvent in which it is sparingly soluble by heating or any other mechanical way. Then leave the solution in a cool environment. If the compound is of crystalline nature crystals will form.

15.5.2 Mix solvent Dissolve the compound (mix of two compounds) in a solvent in which it is easily soluble. Then add drop by drop a solvent in which it is insoluble. When turbidity appears on the first drop leave it in a cool environment for crystallization.

15.6 Chromatography Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet €del, 1981). Generally chromatographic system is shown in use of column (or a in 1900 (Ro tube) to contain the stationary phase and support, while also allowing the mobile phase and sample to pass through the system. This approach was first described in 1903 by Mikhail Tsvet, who used this method to separate plant pigments into colored bands by using a column that contained calcium carbonate to give support to stationary phase. It is a Greek word, a combination of Chroma (means color) and grapheine (to write) (Hage, 2018). Chromatography is an essential physical method used for the separations of different components present in the mixture according to some physical and chemical properties. The fraction can be separated depending upon their interaction with the mobile phase and stationary phase. The chemical entities used in these two phases are different in their polarities. As plant extract contains compounds with wide polarity range, their affinities are different from mobile and stationary phase, hence the components can be separated. Classification of chromatographic techniques: I. Thin layer chromatography II. Column chromatography III. High-performance liquid chromatography All the above chromatographic methods follow the same principle. In addition to the above chromatographic methods, some other methods are also available. Example: gel permeation chromatography (GPC), size-exclusion chromatography (SEC), and ion exchange chromatography. But these methods are not used in natural products.

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15.6.1 Thin layer chromatography This chromatography is the most commonly used method for the isolation of natural products. This is the cheapest and easiest technique to separate the different components of the natural compound as compared to column chromatography (Wilson et al., 2014). It used less amount of sample for analysis. Commonly alumina or silica (more polar) is used as a stationary phase and different polarities of organic solvents (less polar) are used as mobile phases. This is called the normal phase chromatography. But in reverse phase, silica or alumina are alkyl bonded (less polar) and more polar organic solvents are used as mobile phases. TLC is again of two types. One is Preparative TLC and another one is Analytical TLC. For natural products mainly analytical TLC is used. As a stationary phase, a special finely ground matrix (silica gel, alumina, or similar material) is coated on a glass plate, a metal or a plastic film as a thin layer (
0.25 mm) and a binder like gypsum is mixed into the stationary phase to make it stick better to the slide. In most of the cases, a fluorescent powder is mixed into the stationary phase also (e.g., bright green light when expose it to 254 nm UV light). The major factor in analytical TLC is Rf (distance traveled by analyte/distance travel by solvent) value. Rf stands for retarding factor and this factor mainly depends on the solvent type, grain material present in TLC plate (silica or alumina size), adsorbent and amount of material spotted. To choose the solvent for the mobile phase, the order of polarity of solvent need to know. Order of the polarity is described below: Hexane > Toluene > Diethyl ether > Acetone > THF > Ethyl acetate > Acetonitrile > Isopropanol > Ethanol > Methanol > Water.

15.6.2 Column chromatography Column chromatography is the most effective technique used in the separation of plant extracts into its components in pure form. This is a preparative chromatographic method and the stationary phase with silica gel is packed in a column and the mobile phase (called eluent) is passed through the column after loading the sample (extract) on the top of the stationary phase (i.e., along with silica gel). The mobile phase carries the compounds present in the mixture at a different rate based on their affinities to the stationary and mobile phases. Finally, the components of compounds can be collected along with the mobile phase. Column chromatography is of two types: Normal column chromatography and flash column chromatography. Flash chromatography is also called a medium pressure column chromatography (MPCC). Here solvent is with a high rate flow with high pressure over the stationary phase. This is the best method for separating the components of plant material as plant materials are the complex mixture of multiple unknown components. There are two types of solvent systems are used in column chromatography: (1) isocratic and (2) gradient. In an isocratic system, only one solvent is passed through the

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stationary phase but in the gradient system. Two solvents with predefined ratios have been passed through the stationary phase to separate the components. As plant extracts contain a mixture of complex compounds and they differ in polarities. So natural product chemists generally use gradient system for isolation of compounds. A series of steps need to follow for the performance of column chromatography. The important thing needs to pay attention here is the loading of the column. The loading may be wet loading and dry loading. In Wet loading, the liquid sample directly placed onto the top of the column using a pipet, and allow it to percolate through the top of the sorbent bed. If the sample/plant extract is a solid material it needs to be dissolved in a less polar solvent (If it is not soluble in low polar solvent better to use a small portion of high polar solvent). The procedure for dry loading is; the plant extract is dissolved in a small amount of suitable solvent (polar solvent) which can be easily removed. A portion of silica gel is also added. The proportion used here is 1:1 or 1:3. The resultant mixture is placed in a fume hood to evaporate the solvent. Then with the spatula, the resultant mixture is loaded to the stationary phase. Generally, the researcher is using the dry loading process for natural product extraction (Euerby and Petersson, 2003).

15.6.3 High-performance liquid chromatography (HPLC) High-performance liquid chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with the help of stationary phase. The sample is carried by a carrier gas like helium or nitrogen. This chromatography technique has the ability to separate and identify compounds that are present in sample that can be dissolved in a liquid with small concentrations as low as parts per trillion. Because of this versatility, it is used in a variety of industrial and scientific applications, such as pharmaceutical, environmental, forensics, and chemical laboratory. HPLC is of two types (i.e., normal phase HPLC and reverse phase HPLC). Sample retention time is dependent on the interaction between the stationary phase, molecules being analyzed, and the solvents used. At first, the sample passes through the column and interacts between the two phases at a different rate. Analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will elude the column faster sand later respectively. The pressure makes the technique much faster as compared to column chromatography. This allows using much smaller particles for the column packing materials. The smaller particle has a greater surface area for interaction with the stationary phase and the molecules flowing through it. This results in a much better separation of the components for the plant mixture. The components of the mixture are separated according to the different degrees of interaction with the absorbent particles. The pressurized liquid is typically a mixture of solvents like water, ethanol, methanol, acetone, and acetonitrile and called a mobile phase.

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It is one of the best ways to separate the different components from the plant mixture. But because of its high capital cost, handling of the column, the requirement of skilled professionals, scientists generally avoid this method. Compared to column chromatography, HPLC is highly automated and sensitive (Euerby and Petersson, 2003; Mazzei and Antonio D’avila, 2003; Gerber et al., 2004; Siddiqui et al., 2017).

15.7 Physical methods for basic structure elucidation Structure elucidation is a chemical process helps to predict the partial structure of the synthesized molecule. The essential and first characterization technique to elucidate the structure is FTIR (Fourier transform electron spectroscopy). Then with the help of 1H NMR, 13C NMR, mass spectroscopy, and X-ray crystallography, we can confirm the basic structure of the compound.

15.7.1 FTIR (Fourier transform infrared spectroscopy) The principle behind this technique is, when EMR (electromagnetic radiation) falls on the molecule/sample, the molecule starts to vibrate. Due to vibration, the molecule may stretch or bend. Because of the change in the dipole-moment, the molecule becomes IR active. If there will be no change in dipole-moment than the molecule is IR inactive (Fig. 15.3). This technique is used as a preliminary tool for the partial structure elucidation of an unknown compound. With this tool different functional groups (like dCOOH, dOH, dC]O, NH2, dCHO, O, F, Cl, Br, I, etc.) can be predicted. As all functional group has different stretching or bending vibrations, each vibration will represent a specific absorption/transmission peak in mid IR range (i.e., 4000 cm1 to 400 cm1). IR spectra are the plot of transmittance % (T) vs. wavenumber (cm1). In some cases, the plot may be absorbance vs. wavenumber also. The table given below is the summary of the stretching and bending frequency of some functional groups (Table 15.1).

15.7.2 Nuclear magnetic resonance (NMR) Nuclear Magnetic Resonance (NMR) spectroscopy is a key technique in the structural and functional characterization of molecules (Fig. 15.4) (Louro, 2013). Nuclear magnetic resonance is an analytical chemistry technique used in different areas to predict the structure

EMR Molecule

Due to absorption of energy, bond start to vibrate

FIG. 15.3 Schematic representation of the principle of FTIR.

Due to vibration there will be change in dipole moment

Due to change in dipole moment molecule will IR active

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Table 15.1 groups.

A summary of vibrations for different functional

Functional group

Frequency in wavenumber

Carboxylic acid (dCOOH) Aldehyde (dCHO) Ketone (dC]O) Amine (dNH2) Alcohol (dOH) Ethers (dOd) Nitro (dNO2) Halogens (F, Cl, Br, I)

1725–1700 1740–1720 1725–1705 3500–3100 3600–2400 1300–1000 1550–1350 1400–540

ΔE = hυ

ΔE = hυ

B0 B0 FIG. 15.4 Representing the rotation of electron spin due to application of magnetic field.

of the molecule. This method generally required less amount of sample (in mg) and it is a nondestructive method. Mainly it is useful in detecting the number of protons attached to the molecule. There are so many types of NMR Spectroscopy like 1H NMR, 13C NMR, COSY NMR, 19F NMR, 31P NMR, etc. The principle behind the NMR spectroscopy is that many nuclei have a spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer will takes place from the lower to higher energy levels. The energy transfer takes place at a wavelength that corresponds to a radio frequency. When the spin returns to its lower level and energy is emitted at the same frequencies, the signal that matches this transfer is measured in many ways and processed in a detector to get the final spectrum of the compound. Mainly 1H NMR and 13C NMR are used for the structure elucidation of plant products. Protein NMR, COSY, NOESY, DEPT, FT NMR techniques are used for the secondary and tertiary structure of proteins and enzymes present in the sample. That’s why these techniques are also called as correlation spectroscopy.

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All nuclei carry a charge. So they will possess spin angular momentum. Only those nuclei which have a finite value of the spin quantum number (I > 0) will press along the axis of rotation. This value of I is associated with the mass number and an atomic number of the nuclei. I

Atomic mass

Atomic number

Example

Half Integer Half Integer Integer Zero

Odd Odd Even Even

Odd Even Odd Even

1

H(1/2) C(1/2) 2 H(1) 12 C(0)

NMR ACTIVE

13

INACTIVE

If all protons were equal, then NMR would be useless, but they are not all equal. That is, the frequency needed to achieve resonance varies depending on the proton’s environment. By performing different types of NMR, it is easy to detect what type of proton and how many no of protons, types of splitting pattern are there in the compound. The NMR spectra is a plot of the intensity of NMR signal vs. magnetic field strength. Generally, tetramethylsilane is used as the reference sample here. The solvents used for preparing NMR samples are D2O, DMSO-d6, deuterated benzene and etc. (Nikolic et al., 2019). To avoid the interaction of other hydrogen atoms presents insolvent, the deuterated form of solvents are used. So by combining the result of FTIR and NMR, we can partially elucidate the structure.

15.8 Antioxidant assay All the living cell has the capability to continuously metabolism and generates some ROS or reactive oxygen species over the biochemical reaction as by product which is known as free radicles. Basically, free radicals can be an atom or group of atoms or molecules and has at least one unpaired valence electron. These unpaired electrons are unstable, highly reactive. When oxygen react with certain molecules and further start a chain reaction. Generally, ROS species have a tendency to capture the electron in order to become stable resultant destroy the nearby tissue which ongoing the destructive progression (Abu et al., 2017). Foremost features of an antioxidant have the capacity to capture free radicals. Several antioxidant substances like polyphenols, phenolic acids, and flavonoids scavenge peroxide, hydroperoxide or lipid peroxyl free radicals and therefore hinder the oxidative mechanism that cause the various degenerative diseases. Subsequently, they can reason for oxidative damage to lipids, enzymes, proteins and DNA. Ultimately, responsible for many chronic (cancer, diabetes, aging etc.) degenerative diseases in humans (Aiyegoro and Okoh, 2010). Plants are full of free radical scavenging molecules, for example vitamins, tannins, flavonoids, alkaloids, amines other related metabolites. They are rich in antioxidant properties (Aiyegoro and Okoh, 2010). Herbal or medicinal plant always considered as good antioxidant since prehistoric time.

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15.8.1 Antioxidant measurements valuation technique for the plant extract Several authors reported numerous methods for measurement of antioxidant content in the plant extract. Quite a lot of study has been proposed to investigate the performed analytical methods directing to evaluate the antioxidant ability in plants and continuously remains a persistent area and a sequence of classification has been projected. As the chemical principle fundamental the antioxidant oxidant activity assay and lipid oxidation status evaluation are given below. 1. 2. 3. 4. 5.

Hydrogen atom transfer Single electron transfer Hydrogen atom and single electron transfer Chelation power of antioxidant Lipid oxidation

Hydrogen atom transfer It analyzes the amount of an antioxidant to capture free radicals through hydrogen donation. It is very simple proton-coupled electron transfer reaction processes. These reactions involve transfer of one electron and one proton from one reagent to another. An antioxidant or phenolic compound directly interacts with a free radicle and produce a phenolic radical species along with neutral species. Al-Amiery et al. (2013) described this mechanism is involved with the little bond-dissociation enthalpy through the 5-chlorocurcumin.

ArOH ¼ phenolic compound, X∗ ¼ free radicle, ArO∗ ¼ antioxidant molecule, XH ¼ neutral species.

15.8.2 Total radicle trapping antioxidant parameter or TRAP assay In this technique, a protection providing by an antioxidant on the fluorescence decay of R-phycoerythrin (R-PE) with controlled peroxidation reaction. The fluorescence of R-Phycoerythrin is quenched by ABAP (2,20-azo–bis (2-amidino- propane) hydrochloride) as a radical generator and antioxidant measured this quenching reaction. The antioxidative potential is evaluated by measuring the decay in decoloration. According to Ghiselli et al. (1995) isolated the total plasma antioxidant and calculated length of the lag-phase due to the sample related with standard control (Ghiselli et al., 1995).

Oxygen radicle absorbance capacity or ORAC assay This is innovative investigation which examine the “antioxidant power” of chemical ingredients found in a food or plant material. In this test either bphycoerythrin (b-PE) or fluorescein used as target molecule (Alam et al., 2013).

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Crocin bleaching or beta carotene method This is one of the widely used techniques to monitor antioxidants. The main principle is that linoleic acid (i.e., unsaturated fatty acid) gets oxidized by “reactive oxygen species” (ROS) produced by oxygenated water. The products formed will initiate the b-carotene oxidation, which will lead to discoloration. Antioxidants decrease the extent of discoloration, and that can be measured at 434 nm. The method as described by Kabouche et al. (2007): b-carotene (0.5 mg) in 1 mL of chloroform is added to 25 L of linoleic acid and 200 mg of tween-80 emulsified mixture. Chloroform is evaporated at 40 °C, 100 mL of distilled water saturated with oxygen is slowly added to the residue and the solution is vigorously agitated to form a stable emulsion. 4 mL of this mixture is added into the test tubes containing 200 L of sample prepared in methanol at final concentrations (25, 50, 100, 200 and 400 lg/mL). As soon as the emulsified solution is added to the tubes, zero-time absorbance is measured at 470 nm. The tubes are incubated for 2 h at 50 °C. Vitamin C can be used as standard or reference material. Antioxidant activity is calculated as percentage of inhibition (I%) relative to the control using the following equation: I% ¼ ½1  ðAs  As 120Þ=Ac  Ac 120Þ

where As is initial absorbance, As120 is the absorbance of the sample at 120 min, Ac is initial absorbance of negative control, and Ac120 is the absorbance of the negative control at 120 min.

Lipoprotein peroxidation assay LPO is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging. Malondialdehyde (MDA) is one of the end products in the lipid peroxidation process. Malondialdehyde (MDA) is formed during oxidative degeneration as a product of free oxygen radicals, which is accepted as an indicator of lipid peroxidation. According to Ohkawa et al. (1979) is as follows: the tissues are homogenized in 0.1 M buffer pH 7.4 with a Teflon glass homogenizer. Homogenate LPO is determined by measuring the amounts of MDA produced primarily. Tissue homogenate (0.2 mL), 0.2 mL of 8.1% sodium-dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid and1.5 mL of 8% TBA are added. The volume of the mixture is made up to 4 mL with distilled water and then heated at95 °C on a water bath for 60 min using condenser. After incubation the tubes are cooled to room temperature and final volume is made to 5 mL in each tube. Five mL of butanol: pyridine (15:1) mixture is added and the contents are vortexed thoroughly for 2 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer is taken and its OD is taken at 532 nm against an appropriate blank without the sample. The levels of lipid peroxides can be expressed as n moles of thiobarbituric acid reactive substances (TBARS)/mg protein using an extinction coefficient of 1.56  105 mL cm1.

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15.9 Single electron transfer 15.9.1 N,N-dimethyl-p-phenylenediamine or DMPD assay DMPD radical cation decolorization method has been developed for the measurement of antioxidant activity mainly in food and biological samples. This assay is based on the reduction of buffered solution of colored DMPD in acetate buffer and ferric chloride. The procedure involves measurement of decrease in absorbance of DMPD in the presence of scavengers at its absorption maximum of 505 nm. The activity is expressed as percentage reduction of DMPD. Fogliano et al. (1999) is obtained the radical by mixing 1 mL of DMPD solution (200 mM), 0.4 mL of ferric chloride (III) (0.05 M), and 100 mL of sodium acetate buffer solution at 0.1 M, modifying the pH to 5.25. The reactive mixture has to be kept in darkness, under refrigeration, and at a low temperature (4–5 °C). The reaction takes place when 50 L of the sample (a dilution of 1:10 in water) is added to 950 L of the DMPDÆ + solution. Absorbance is measured after 10 min of continuous stirring, which is the time taken to reach constant decolorization values. The results are quantified in mM Trolox on the relevant calibration curve.

15.9.2 Ferric reducing antioxidant power or FRAP assay Ferric reducing ability of plasma (FRAP, also Ferric ion reducing antioxidant power) is an antioxidant capacity assay that uses Trolox as a standard. This assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements containing polyphenols. The FRAP assay is performed as previously described by Benzie and Strain (12), and is also carried out on a COBAS FARA II spectrofluorometric centrifugal analyzer (Roche). The experiment is conducted at 37 °C under pH 3.6 condition with a blank sample in parallel. In the FRAP assay, reductants (“antioxidants”) in the sample reduce Fe (III)/ tripyridyltriazine complex, present in stoichiometric excess, to the blue ferrous form, with an increase in absorbance at 593 nm. ¢A is proportional to the combined (total) ferric reducing/antioxidant power (FRAP value) of the antioxidants in the sample. The final results were expressed as micromole Trolox equivalents (TE) per gram on dried basis (mol TE/g, db).

15.9.3 Cupric reducing antioxidant capacity or CUPRAC assay The chromogenic oxidizing reagent of the developed CUPRAC method, that is, bis (neocuproine) copper (II) chloride [Cu(II)-Nc], reacts with polyphenols [Ar(OH)n], where the liberated protons may be buffered with the relatively concentrated ammonium acetate buffer solution. In this reaction, the reactive Ar-OH groups of polyphenols are oxidized to the corresponding quinones and Cu (II)-Nc is reduced to the highly colored Cu (I)Nc chelate showing maximum absorption at 450 nm. According to Apak et al. (2008), 1 mL of 102 M of CuCl2, 1 mL of 7.5  103 M neocuproine and 1 M NH4CH3COO solution are added into the glass test tube. Then, 400 μL

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of freshly prepared standard solution is added and diluted to the final volume of 4.1 mL with deionized water. This procedure is repeated for 400 μL, 300 μL, 200 μL, 100 μL and 50 μL additions of freshly prepared solutions of the sample. The prepared solutions are mixed and incubated at room temperature for 30 min. The absorbance at 450 nm is determined against a reagent blank by spectrometer. The calculation of antioxidant capacity of compounds as Trolox equivalents (TEAC values) by the CUPRAC method has been reported.

15.9.4 Potassium ferricyanide reducing power or PFRAP assay The principle of this method is with increase in the absorbance of the reaction mixtures increase there will be increase in the antioxidant activity. In this method, antioxidant compound forms a colored complex with potassium ferricyanide, trichloro acetic acid and ferric chloride, which is measured at 700 nm. The increase in absorbance of the reaction mixture indicates the reducing power of the samples (Jayaprakash et al., 2001). In this method, as described by Oyaizu (1986) 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of K3Fe (CN)6 (1% w/v) are added to 1.0 mL of sample dissolved in distilled water. The resulting mixture is incubated at 50 °C for 20 min, followed by the addition of 2.5 mL of Trichloro acetic acid (10% w/v). The mixture is centrifuged at 3000 rpm for 10 min to collect the upper layer of the solution (2.5 mL), then mixed with distilled water (2.5 mL) and 0.5 mL of FeCl3 (0.1%, w/v). The absorbance is measured at 700 nm against blank sample.

15.10 Hydrogen atom and single electron transfer 1,1-diphenyl-2-picrylhydrazyl (DPPH)-2,2-diphenyl-1-picrylhydrazyl or DPPH is a free radical which have hydrogen acceptor capability to antioxidants. Henceforth, this compound usually used in DPPH assay for the determination of antioxidant activity of medicinal plants, fruits or any other biological substrates. Zheng Chen et al. have used the DPPH radical scavenging assay according to the method reported by Brand-Williams et al. (1995), with some modifications. A control standard solution like DMSO has been prepared and added to a DPPH methanolic solution and the mixtures has shaken energetically and incubate in the dark for half an hour at room temperature. Discoloration was measured at 517 nm at least in triplicate and radical scavenging capacity can be expressed as percentage effect (E%) and calculated using the following equation: Percentage effect ¼

  AbsðControlÞ  Abs ðSampleÞ ∗100 Abs ðControlÞ

2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/(TEAC)-Antioxidant capacity in fruits differs from that of biological samples due to their low pH and very low lipophilic antioxidant capacity. According to Mustafozgen et al., Troloxaliquot is liquified and used to develop a 10–100 mol/L standard curve. All data are then expressed as

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trolox equivalents (TE mol). Assays are conducted by combining antioxidant reactants with 20 L of individual standards or fruit juices. Reactions were allowed to progress at 28 (2° cover extended periods and monitored at specific intervals. All assays of standards and fruit samples were performed in triplicate. (Modified 2,2-Azino-bis-3ethylbenzothiazoline-6-sulfonic Acid (ABTS) Method to Measure Antioxidant Capacity of Selected Small Fruits and Comparison to Ferric Reducing Antioxidant Power (FRAP) and 2,2¢-Diphenyl-1-picrylhydrazyl (DPPH) Methods).

15.11 Chelation power of antioxidant 15.11.1 Ferrozine assay Ferrozine can form a complex with a red color by forming chelates with Fe2+. This reaction is restricted in the presence of other chelating agents and results in a decrease of the red color of the ferrozine-Fe2+ complexes. Measurement of the color reduction determines the chelating activity to compete with ferrozine for the ferrous ions (Soler-Rivas et al., 2000). The chelation of ferrous ions is estimated using the method of Dinis et al. (1994),. 0.1 mL of the extract is added to a solution of 0.5 mL ferrous chloride (0.2 mM). The reaction is started by the addition of 0.2 mL of ferrozine (5 mM) and incubated at room temperature for 10 min and then the absorbance is measured at 562 nm. EDTA or citric acid (Dinis et al., 1994) can be used as a positive control.

15.12 Lipid oxidation 15.12.1 Peroxide value assessment Peroxynitrite (ONOO) is a cytotoxicant with strong oxidizing properties toward various cellular constituents, including sulfhydryls, lipids, amino acids and nucleotides and can cause cell death, lipid peroxidation, carcinogenesis and aging. It is generated in vivo by endothelial cells, Kupffer cells, neutrophils and macrophages. Peroxynitrite radical is a relatively stable species compared with other free radicals but once protonated gives highly reactive peroxynitrous acid (ONOOH), decomposing with a very short half-life (1.9 s) at 37 °C to form various cytotoxicants and that can induce the oxidation of thiol (_SH) groups on proteins, nitration of tyrosine, lipid peroxidation and also nitration reactions, affecting cell metabolism and signal transduction. It can ultimately contribute to cellular and tissue injury with DNA strand breakage and apoptotic cell death, e.g., in thymocytes, cortical cells and HL-60 leukemia cells. Its excessive formation may also be involved in several human diseases such as Alzheimer’s disease, rheumatoid arthritis, cancer and atherosclerosis. Due to the lack of endogenous enzymes responsible for ONOO inactivation, developing specific ONOO scavengers is of considerable importance. The method described by Kooy et al. (1994) involves the use of a stock solution of dihydroxyrhodamine 123 (DHR 123, 5 mM) in dimethylformamide that is purged with nitrogen

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and stored at 80 °C. Working solution with DHR 123 (final concentration 5 L M) is diluted from the stock solution and is placed on ice in the dark immediately prior to the experiment. Buffer solution, 50 mM sodium phosphate (pH 7.4), containing 90 mM sodium chloride and 5 mM potassium chloride with 100 L M diethylene triamine penta acetic acid (DTPA) are purged with nitrogen and placed on ice before use. Scavenging activity of ONOO by the oxidation of DHR 123 is measured on a microplate fluorescence spectrophotometer with excitation and emission wavelengths of 485 nm and 530 nm at room temperature, respectively. The background and final fluorescent intensities are measured 5 min after treatment without 3-morpholino-sydnonimine (SIN-1) or authentic (ONOO). Oxidation of DHR 123 by decomposition of SIN-1 gradually increased whereas authentic ONOO rapidly oxidized DHR123 with its final fluorescent intensity being stable over time.

15.12.2 Thiobarbituric acid reactive substances TBA method, described by Ottolenghi (1959) is as follows: The final sample concentration of 0.02% w/v was used in this method. Two mL of 20% trichloroacetic acid and 2 mL of 0.67% of thiobarbituric acid were added to 1 mL of sample solution. The mixture was placed in a boiling water bath for 10 min and then centrifuged after cooling at 3000 rpm for 20 min. The absorbance activity of the supernatant was measured at 552 nm and recorded after it has reached its maximum.

15.13 Anticancer assay Cancer is an uncontrolled growth of cells which have the capability to invade any part of body. There are a several type of cancer and one of the major issues globally. Some cancercausing agent such as chemical substances or toxic compound, ionizing radiation, some pathogens and mutation are responsible. Surgery, ionizing radiation and chemotherapy is a treatment for the cancer which use huge number of drugs. These chemical drugs have several draw backs like because they damage the cancer to normal cell on the basis rapidly growing. So, in this regard, plant derived drug or natural compound always a new search in cancer research.

15.13.1 Anticancer evaluation method Cell viability assays This is the most used category and specially based on cellular enzymes and proteins. This assay indicates the viability of cells and determine the ability of test molecule whether it has anticancer activity or not. Principally, a substrate is transformed to a colored product through the presence of intracellular enzyme in viable or nonviable cells. Color developed only in viable cells indicate the viability against the test sample. This is the most used assay and can be employed synthetic and natural molecule (Ediriweera et al., 2019). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is one of most commonly used assays for cancer drug screening (Berridge and Tan, 1993; Gerlier and

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Thomasset, 1986). This is a basic and easy assay for the cytotoxicity and one of favorite cytotoxicity assay in the research laboratory. NAD (P)H-dependent oxidoreductase or dehydrogenases in metabolic active cells which can reduce tetrazolium salt (MTT) into purple formazan product. Further, can then be solubilized for spectrophotometric analysis (Berridge and Tan, 1993; Gerlier and Thomasset, 1986). A research supported by (Alsayari et al., 2018) used the MTT assay to assess the Cucumis prophetarum var. prophetarum different fraction for treating liver disorders on different human cancer cell lines (MCF7,MDA-MB-231, HCT-116, A2780, A2780CP and HepG2). Singh et al. (2019a) described the anticancer activity of Amoora rohituka extracts in different solvent with breast cancer cell line (Fig. 15.5). Additionally, other closely associated tetrazolium dye chemical substance such as MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide) in addition WSTs, are used in combination with 1-methoxy phenazine methosulfate (PMS) as the transitional electron acceptor. They can also produce formazan after reduction by oxidoreductase or dehydrogenase generates a water-soluble formazan product, therefore shortening the evaluate test. McCauley et al. (2013) describe the MTT/MTS cell viability and kinase enzyme inhibition method in detailed (McCauley et al., 2013). Resazurin is a nonfluorescent element of alamar Blue reagents enter into viable cells and transformed to resorufin by NADH, NADPH or FADH2-dependent cellular oxidoreductases (Candeias et al., 1998; Anoopkumar-Dukie et al., 2005). Calcein AM, Calcein red-orange, Oregon green 488 or 5-CFDA-AM are alternative nonfluorescent cell membrane penetrable dye family, ability converts to green-fluorescent Calcein by viable intracellular esterases enzyme (Bratosin et al., 2005; Smith et al., 2013). Apart from the above, sulforhodamine B (SRB) assay, the dye SRB have a tendency to bind with proteins or enzymes in viable cells and solubilized under slight acidic conditions (Skehan et al., 1990). New recent bioluminescence assays can be detect apoptosis or necrosis through examine cellular ATP using the luciferase enzyme (Strehler and McElroy, 1957) described earlier and It is more accurate and reproducible method comparative to the conventional method which is based on quantified by colorimetric, fluorometric or luminometric way (Hanahan and Weinberg, 2011). Pikman et al. (2017) and Cusimano et al. (2017) separately used this method to evaluate the outcome of various conventional anticancer drug with CDK4/6 inhibitor in T-cell acute lymphoblastic leukemia cells and oleocanthal in liver and colon cancer cell against the cell viability (Pikman et al., 2017; Cusimano et al., 2017). Similarly, Lactate dehydrogenase leakage assay or LDH assay as name described, along with the tested material, they break the cell membrane and allow the LDH to come out into the medium which can be quantified by calorimetrically and fluorometrically. A study carried out for the GF-AFC and bis-AAF-R110 peptide substrate to examined the viability effect (Niles et al., 2007). Another case defined about the Verteporfin which showed the cell viability effect in HEC-1-A and HEC-1-B endometrial cancer cells (Dasari et al., 2017).

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Electric cell-substrate impedance sensing or ECSI It is a real-time, a noninvasive approach toward quantify the living cells in the presence of electric current. This is an only method which do not use any dye to examine the cellular activity. This technique detects the morphology variations, cell migration, movement, proliferation and other related performances which are govern by the cell cytoskeleton in the presence of test drug. Drs. Ivar Giaever and Charles R. Keese invented this technology in 1973. In this method, cells are allowed to grow on small, planer gold electrode deposited on the bottom of petridish or culture flask. The AC current is impeded according to the number of cells cover the electrode, cell type and the nature of the cell attachment. When cells are stimulated to change their function, the accompanying changes in cell morphology alter the impedance. The data generated is impedance versus time (Wegener et al., 2000; Xiao et al., 2002; Xiao and Luong, 2003; Giaever and Keese, 1993).

DNA synthesis-based assay This is a simple and direct route to encounter during new DNA synthesis. This approach includes the incorporation of a new labeled nucleoside into genetic material and the labeled nucleoside can be detected with a reporter. 3H-thymidine is a thymidine analog, can be used to be incorporated into DNA strand, replacing thymidine through DNA replication and labeling the newly synthesized DNA strand in mitosis (Sidman et al., 1959). Similarly, numerous kinds of methods also established for the quantification of cell proliferation. Several analogous nucleotides like BrdU, EdU and IdU also have been used and quantify them through flow cytometry, immunohistochemistry, and immunocytochemistry (ICC) etc. Specifically, antibodies have been used for the identification of labeled nucleotides such as avidin-biotin, immunoperoxidase and imunoflourescence (Duque and Rakic, 2011; Sidman et al., 1959).

Dye exclusion assays It is determining the number of viable and nonviable cells determination in the cell suspension. Basically, live cells have complete cell membranes that reject certain dyes, such as trypan blue, Eosin, or propidium to come inside, whereas dead cells do not. These dyes do not penetrate the live cell membrane although dead cells do (Altman et al., 1993; Nakamura et al., 2001). Dyeexclusion assays always a key challenge, so it is important to use a suitable dilution of dyes, correct counting and prevention of cell clusters formation during staining are as well essential to progress the precision of assays (Cadena-Herrera et al., 2015). Altman et al. (1993) used the tryptophan blue dye for the comparison along with fluorometric assays (Altman et al., 1993) Whereas, propidium iodide (PI) is used to differentiate viable and nonviable cells and monitored by flow cytometer or flouroscence microscopy (Sasaki et al., 1987). Ethidium homodimer assay is another cell viability assay, which can be used to detect dead cells produce red color upon binding with DNA of dead cells (Haugland et al., 1994).

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Clonogenic assay Another termed is colony formation method constructed on the capability of a single cell to make at least 50 cells colony against specific agent or tested drug. This is very useful technique in oncology area to know the effect of drug and ionization radiation combinedly or separately on a reproductive cell. Which is main feature of cancer cell (Franken et al., 2006) Nicolaas A P Franken et al., find the result of ionizing radiation after treatment, formed colonies firstly secure with glutaraldehyde and then stained with crystal violet further counted using a stereomicroscope (Franken et al., 2006).

Cell migration assays Under the metastasis condition, cancer cells move into neighboring tissue or body part through extracellular matrix from blood circulation. In the development of new drug discovery or herbal plant extraction, this is very important assay:

Wound curative assay In this method, attached cells remain visible to anticancer drug and incubate. Further, a scratch or wound is made by hand with a sterile pipette tip or needle. The size of wound is noted at different intermissions time (Liang et al., 2007). The properties of a drug on cells invasion can too observed with this method (Liang et al., 2007). Rajesh et al. examined the cell migration behavior with this assay A. rohituka leaves extract at different intervals and cells migration visible against MCF-7 cells (Fig. 15.6).

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Boyden chamber assay A chamber made up of two compartments filled by medium and parted through a microporous sheath or membrane. Generally, cells are kept in upper section and are permitted to wander by the holes of the microporous sheath into the lower compartment site, where chemotactic agents are existing. Subsequently, incubation time, microporous membrane fixed and stained. Further, cells that have traveled to the determined lower side of the membrane evaluated through microscopy. So, chamber method also known as filter membrane migration assay, trans-well migration assay, or chemotaxis assay (Chen, 2005).

Capillary chamber cell migration assay This is more similarly Boyden chamber assay; difference exhibit on lower chamber which is connected by small capillary. Relocation and invasion of cancer cells, morphological response based on the time-lapse microscopy (Kramer et al., 2013; Chaubey et al., 2011).

ROS assay There are a lot of indications, suggesting the mediating role of reactive oxygen species (ROS) in cell cycle, stress and death, and it is known as ROS. At different concentrations it would play distinctly different roles and eventually leading to the different rate of cell deaths. So it is highly needed to perform cell death assay with the help of ROS Technique. There are a lot of technique to detect ROS (Fig. 15.7) (Zhang et al., 2018a,b). The reactive oxygen species can be generated from superoxide (O2•), hydroxyl (•OH), peroxyl (ROO•), and alkoxyl (RO•) and certain nonradicals that are either oxidizing agents or easily radical-convertible species, such as hypochlorous acid (HOCl), ozone (O3), singlet oxygen (1O2), and hydrogen peroxide (H2O2).

Electron spin resonance

Chemiluminescent probes

Chromatographic methods ROS detection Fluorescent proteins

Electrochemical biosensor

Spectrophotometric methods FIG. 15.7 Schematic representation of ROS detection.

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Table 15.2

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List of oxygen producing radicals and nonradicals.

Radicals

Nonradicals

Superoxide anionic radical (O •) • Perhydroxyl radical (HO2) • Hydroxyl radical (HO ) 2

Singlet oxygen (1O2) Ozone (O3) Hydrogen peroxide (H2O2)

Chloroplast, mitochondrium and peroxisome, are a major source of ROS production in plants. Along with these peroxidases and amine oxidases are present in cell walls and NADPH oxidase located in the plasma membrane also helps to produce ROS, often in response to stress signals. Oxygen is continuously produced inside the chloroplast due to photosynthetic electron transport and simultaneously removed by reduction and € assimilation (Tripathy and OelmUller, 2012). ROS can be classified into two sub category: (1) radicals (free ROS, i.e., combination of oxygen and hydrogen) and (2) nonradicals as shown in Table 15.2. ROS have important physiological roles in plant development. According to Passardi (in 2004) and Novo Uzal (in 2013) (Passardi et al., 2006; Novo-Uzal et al., 2013), some ROS can be used as antioxidant, cell wall signaling and cell wall loosening agent. In 2006 according to Cona et al., different enzymes show importance of ROS production for development and defense. ROS producing enzyme in plants include NADPH oxidases, amine oxidases, quinone reductases, lipoxyginases, oxalate oxygenase etc. ROS have long been recognized for their roles in mediating the response to abiotic and biotic stress conditions. However, in recent years, a number of studies have uncovered key roles for them during plant growth and development. How ROS can affect basic cellular processes, such as the cell cycle and division need to understand. In plants, exposure to stress is often accompanied by decreased growth and cell cycle arrest, although the mechanisms underlying this response remain largely unexplored. In particular, the molecular factors of the cell cycle that are influenced by ROS are poorly studied in plants. According to Menon and Goswami, it is known that redox cycles are conserved within the cell cycle and that reductive and oxidative signals are required for transitions within the cell cycle. These phase-to-phase progressions and transitions are mainly governed by a complex machinery of interacting cyclins (CYCs) and cyclin-dependent kinases (CDKs) (Menon and Goswami, 2007; Diaz-Vivancos et al., 2015).

15.14 Methods to detect ROS 15.14.1 Fluorescence-dependent methods Fluorescent probes are often oxidant sensitive, and they are nonfluorescent before being oxidized by oxygen species. The most widely used members are dihydroethidium, dichlorodihydrofluorescein and Amplex Red (although cell impermeable). The cell permeable

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ones are helpful to reflect the oxidative state of cellular compartments, providing information on radical production under stimulus.

15.14.2 Dihydroethidium (DHE) staining Dihydroethidium is capable of getting into cells and oxidized by intracellular O•2 to form the specific red fluorescent 2-hydroxyethidium or could be oxidized by other oxidants to generate red fluorescent (Fink et al., 2004; Garvin and Hong, 2008).

15.14.3 Dichlorodihydrofluorescein diacetate (DCFH-DA) It is easy to assume that the deacetylation of DCFH-DA to DCFH and its accumulation in cell traps are essential factors determining the availability and utility of DCFH-DA in reflecting the ROS level. However, different types of cells show different permeabilities to this probe, resulting in different final equilibrium concentrations inside and outside the cells, and equally important, the activity of esterase may vary a lot among cells, consequently, the behavior of DCFH-DA/DCFH in specific cells should be studied before applying it in experiments (Wang and Joseph, 1999).

15.14.4 Amplex red Amplex Red is used for the detection of hydrogen peroxide. H2O2 reacts with the probe in a one-electron pathway, catalyzed by HRP, to form fluorescent resorufin (with Ex of 563 nm and Em 587 nm), so the concentration of H2O2 is reflected by the intensity of resorufin.

15.14.5 Chromatographic method Chromatography methods are rapid, efficient and sensitive in the detection of hydroxyl radicals, while the reaction process and products are complicated, and the treatment of predetection is complex, making it less widely used than other methods. Considering the nonspecificity of fluorescent probes and luminescent probes toward hydroxyl radicals, the chromatography assay could be used as a means of verification of the existence of •OH.

15.14.6 Electrochemical biosensors An electrochemical biosensor formed by alternating layers of cytochrome c and poly (aniline (sulfonic acid)) on a gold wire electrode is applied for the sensitive, selective, and stable quantification of superoxide. This biosensor works based on the following mechanism: superoxide could reduce certain proteins, while these proteins are re-oxidized by the electrode at a suitable potential; during this process an electric current signal is generated and then captured by the sensor, and considering the proportional relationship between the strength of the electric signal and superoxide, the concentration could be determined. The electrochemical biosensor could be used not only in the monitoring of real-time production of superoxide, but also in the detection of its reaction with a chosen antioxidant

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in vitro. The electrode biosensor is helpful in the quantification of superoxide with a high efficiency both in vivo and in vitro, and the combination of the electrode and cytochrome c (Wegerich et al., 2009; Ganesana et al., 2012).

15.15 Conclusion The conventional anticancer drugs have distinct mechanisms of action that vary in their effects on different normal and cancer cells. Screening methods are employed consistently and extensively to reduce cost and time of drug exploration. The anticancer drug screening methods commonly include animal experiments, cell-based screening assays and bioinformatics approaches. This chapter discusses the traditional and advance screening methods for discovery of anticancerous drugs.

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Index Note: Page numbers followed by f indicate figures, and t indicate tables. A Abiotic factors, secondary metabolism and, 59–62 Abiotic stress, 21, 36–37, 63–64, 129–130 Abscisic acid (ABA), 213, 222 Acetovanillone, 277–284t 10 -Acetoxychavicol acetate, 277–284t Achnatherum inebrians, 62–63 Aerosols, 63–64 Alkaloids, 20, 35, 50 biosynthetic pathway, 140 ecological significance, 64t, 65 lead, 241, 243 Allicin, 277–284t Alliin, 277–284t, 285 Alliinase, 285 Allopolyploids, 30, 34 Allosyndetic pairing, 30 Allspice chemopreventive and therapeutic potentials, 277–284t eugenol, 293 medicinal properties, 292–293 Amentoflavone, 239–241 Amoora rohituka extracts, anticancer activity of, 341–342, 343f Amphidinolide, 157–158 Amplex Red, 348 Angiosperms anticancer potential of, 242–244 brassinosteroids (BRs), 244 flavonoids, 12–13, 244 polyphenols, 244 genome duplication, 52 Anoikis, 164 Anthracyclines, 16t Anti-angiogenesis therapy

bacteria, 109–110 inhibitors and mechanisms for, 109–110, 110t Antibacterial activity abietane diterpenes, 141 essential oil of Marchesinia mackaii, 220 of Gymnema sylvestre, 191–192 marchantin, 218–219 Antibiotic resistance gene, 87 Anticancer activity of Amoora rohituka extracts, 341–342, 343f brown marine algae, water extracts of, 158–159 of bryophytes, 238 elatol, 168 fucoidan, 175–176 fucoxanthin, 167 Gracilaria corticata, 159, 160t Gracilaria edulii, 159, 160t sargachromanol, 168 sponges, isolated compounds from, 257–259t Anticancer assay, 341–347 Boyden chamber assay, 346 capillary chamber cell migration assay, 346 cell migration assays, 345 cell viability assays, 341–343 clonogenic assay, 345 DNA synthesis-based assay, 344 dye exclusion assays, 344 electric cell-substrate impedance sensing (ECSI), 344 reactive oxygen species (ROS) assay, 346–347, 346f wound curative assay, 345, 345f Anticancer drug development, natural products screening for anticancer assay, 341–347

355

356

Index

Anticancer drug development, natural products screening for (Continued) antioxidant assay, 335–337 basic structure elucidation, 333–335 chelation power of antioxidant, 340 chromatography, 330–333 crystallization, 329–330 extraction, 324–327 fractionation, 327–328 hydrogen atom transfer, 339–340 lipid oxidation, 340–341 purification, 329 ROS detection, 347–349 single electron transfer, 338–339 Anticancer drug discovery, evolution of, 15–17 Antidiabetic activity cinnamon, 290–291 cyanobacteria, 169–171 of Fusarium equiseti, 191–192 Antigen-presenting cells (APCs), 110–111 Antimicrobial effects of bryophytes, 219–220 Dunaliella salina extracts, 171–172 marine algae, 155–157 naphtho-γ-pyrones, 198–199 Antioxidant(s) chelation power of, 340 enzymatic, 130 nonenzymatic, 130 Antioxidant activity cultivated potato, 37 bryophytes, 220–221 flavonoids, 66–67 grape stem extracts, 243 marine algae, 155–157, 160–161 melatonin, 268 Microgramma vacciniifolia, 240 phycocyanin (PC), 127–128, 165–166 polyphenols, 244 Antioxidant assay, 335–337 plant extract, antioxidant measurement in hydrogen atom transfer, 336 total radicle trapping antioxidant parameter (TRAP) assay

crocin bleaching or beta carotene method, 337 lipoprotein peroxidation (LPO) assay, 337 oxygen radicle absorbance capacity (ORAC) assay, 336 Apigenin, 277–284t Apoplastic effectors, 195–196 Apoptosis, 132–133, 161–163, 310 anticancer therapy via, 161–163, 162t Apoptosis-inducing factor (AIF), 163–164 Apratoxins, 134, 169–171 Aqueous extract of Allspcie (AAE), 292–293 Aqueous extract of clove (AEC), 291 AraC, 116–117 Arachidonic acid, 211 Aromatic amino acid decarboxylase (AAD), 268 Arrays, 95–96 Arylalkylamine N-acetyltransferase (AANAT), 268–269 Ascorbate, 130 Ascorbate peroxidase (APX), 130 Asterosaponin, 261–262 Aszonapyrone A, 200–201 Atranorin (ATR), 237 Autoallopolyploids, 30 Autopolyploidization, 31, 33 Autopolyploids, 30, 33 induced, 31 meiotic and breeding behavior, 31 morphological effects, 31 Avenacin gene cluster, 8–9 Avicins, 140 Ayurvedic medicines, 78, 313 B Bacillus Calmette-Guerin (BCG) vaccine, 106 Bacterial-mediated cancer therapy (BMCT), 106 Bacterial strains role in cancer, 103–105, 105f of Salmonella typhimurium, 107–108 Bacteriocins, 107 “Balanced antagonism” hypothesis, 193 Basidiomycetes, 236–237

Index 357

B-carboline, 260–261 BCL-2 proteins, 163–164, 169–172 β-carotene, 11, 166–167, 337 Betapinene, 67 Bibenzyls, 214, 239 Biflavonoids, 212t, 214, 240 Biliprotein, 165–166, 169–171 Bioactive compounds, 37–38, 77 from bryophytes, 217–222 endophytes, 191–193 gymnosperms with, 241 in marine algae, 157–161, 161t in marine fungi, 199–200 phaeosphoramides, 197 sponges, 256, 257–259t Biofilms bacterial, 108 cancer treatment, 108–109 Biogenic volatile organic compounds (BVOCs), 63–64 Bioluminescence assays, 342 Biomolecular activity, of secondary metabolites, 14–15 Biopharming agents, bryophytes as, 222–223 Biosynthetic gene clusters, 20 Biotic factors, secondary metabolism and, 62 Biotic stress, 21, 56–58 Biotransformation, 39 Bis(bibenzyl), 214, 239 Biselyngbyaside, 132–133 Bis (2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE), 177–178 Blue-green algae, 157 Borophycin, 157, 161t Boyden chamber assay, 346 Brassinosteroids (BRs), 243–244 Brocazine G, 200 Bromophycolide, 173–174 Brown marine algae, 158–159 Bryophytes antimicrobial effects of, 219–220 antioxidant property of, 220–221 bioactive molecules from, 217–222 biotechnological applications in vitro culturing, 224

transgenic moss, 224–225 components, chemical syntheses of, 223 cytotoxic compounds hornworts, 239 liverworts, 238 mosses, 239 cytotoxicity, 218–219 insect antifeedant, 221–222 for medicinal usage, 210 mortality, 221–222 nematocidal activity, 221–222 phenolic components flavonoids, 214–215 isoprenoids, 216–217 plant growth inhibitory activity, 222 plant hormones, 213 as potential biopharming agents, 222–223 secondary metabolites in, 211, 212t lipids, 211 phenylpropanoids, 213–214 saccharides, 211 terpenoids, 211–213 as source of biologically active molecules, 211, 212t therapeutic potential of, 238–239 use of, 209 Bryostatins, 260–261 Bryozoa, anticancer activitiy of, 260–261 C Caffeic acid, 66, 277–284t Caffeine, 78 Calcein AM, 342 Calcein red-orange, 342 Calcium carbonate, 261, 330 Calcium spirulina (Ca-SP), 165 Callus culture, 38 Calothrixin, 132–134, 169–171 Camptothecin (CPT), 16t, 198–199 Cancer, 103, 127, 233, 255, 323 bacterial strains role in, 103–105, 105f breast, 240–241 death, causes of, 236 diet in, 276–283 definition, 309–310

358

Index

Cancer (Continued) diet and, 276–283 spontaneous regression of, 107 Cannabinoids, 52, 168 Capillary chamber cell migration assay, 346 Capillary electrophoresis (CE), 83–84 Capsaicin, 277–284t, 286 anti-tumor activity, 286 clinical applications, 286–287 genotoxicity of, 286 Carbazole, 243 Carbohydrate active enzymes (CAZymes), 195–196 Carbon dioxide (CO2), 325–326 Carcinogenesis, 108, 171–172, 234 Carnosic acid, 277–284t Carnosol, 277–284t Carnosol o-quinone, 277–284t Carotene, 141 Carotenoids, 127–128, 166 Carrageenans, 173–174 Carvacrol, 277–284t β-Caryophyllene, 277–284t, 291 Caspase-activated DNase (CAD), 163–164 Caspases, 161–163 Caulerpenyne, 158 Cell death, 112f, 164, 196 Cell fitness coupling, 11 Cell migration assays, 345 Cell viability assays, 341–343 Cephalotaxine, 241 Chalcona synthase (CHS), 52, 58f Chelerythrine chloride, 243 Chemical diversity, of natural products, 11 Chemical syntheses, of bryophyte components, 223 Chemoprevention, 311–312, 312t Chemoresistance, 310–311 Chemosensitization, 311–312, 312t Chemotherapeutic drugs, 169–171, 286–287, 310, 323 Chemotherapy, 127, 255, 310 Chili pepper/capsicum, 286–287 Chitinase, 196

Chlamydomonas reinhardtii, 6–7, 55 Chlorella, 171–172 Chloroform, 88, 159, 337 Chlorophyceae, 171–173 Chlorophyll, from algae, 166 Chlorophyll a (Chl. a), 127–128 5-Chlorosclerotiamide, 199–200 Chromatography, 330–333 column, 331–332 high-performance liquid chromatography (HPLC), 332–333 mobile phase, 330 stationary phase, 330 thin layer, 331 Chromodorolide, 261 Chromosomes, 29 change in, 29–30, 36 doubling, 34 duplications, 33 homologous, 32–33 Cinnamic acids, 65–66, 212t Cinnamon, anti-tumor activity, 277–284t, 290–291 Circumdatin G, 199–200 Cistus creticus, isoprenoids in, 18–19 Clerosterol, 162t, 172–173 Climate change, 47 effects on secondary metabolites, 53 factors responsible for, 47 heat shock and elevated CO2, 63–64 medicinal plants, impact on CO2 levels, 54 elevated ozone, 54 global warming, 55 phenological changes, 54 shifting ranges, 54 ultraviolet radiation, 54 plant evolution and, 55–56 plant secondary metabolites, 68 Clonogenic assay, 345 Clove, chemopreventive and therapeutic potentials, 277–284t Cloves, antitumor activity, 277–284t, 291 Cnidaria, anticancer activity of, 259–260 Cold stress, 61–62

Index 359

Coley’s toxin, 107 Column chromatography, 331–332 dry loading, 332 gradient system, 331–332 isocratic system, 331–332 wet loading, 332 Combination bacteriolytic therapy (COBALT), 114, 114f Competence stimulating peptide (CSP), 103–105 Complementary DNA (cDNA), 89 Condensed tannins, 65–67 Condriamide-A, 159, 161t Constitutive expression, for secondary metabolites, 56t Correlation spectroscopy. See Nuclear magnetic resonance (NMR) spectroscopy C-phycocyanins (C-PC), 165–166 Crocetin, 291–292 Crocin, 277–284t, 291–292 Crocin bleaching, 337 Crocin-coated magnetite nanoparticles, 292 Cryptophycin, 134, 162t, 169–171 Crystallization, 329–330 mix solvent, 330 single solvent, 330 Culcinosides, 261–262 Culcitoside, 261–262 Cupric reducing antioxidant capacity (CUPRAC) assay, 338–339 Curacin A, 134, 135–136t, 157, 161t, 234–236 Curcumin, 244 antitumor activity, 289 in cancer therapy, 312t, 313–315, 315f clinical application, hurdles in, 289 clinical trials of, 290 structure, 277–284t Curry leaf, chemopreventive and therapeutic potentials, 277–284t Cyanobacteria, 157, 169–171, 234–236 abiotic stress tolerance of, 129–130, 131t anticancerous metabolites, 134, 135–136t, 137f biosynthetic pathway, 134–141, 139f

alkaloids, 140 isoprenoids, 140–141 nonribosomal peptides (NRPs), 139–140 ribosomal peptides, 140 diversity and evolution, 128–133, 129f natural drugs from, 141, 141t pigments, 127–128 secondary metabolites, 128, 133–134, 133f Cyanotoxins, 157 Cylindrospermopsin (CYN), 236 Cytochalasin K, 200–201 Cytochrome c, 163–165, 175–176 Cytochrome P450 (CYP) enzymes, 8–9, 9f, 52 D Dactinomycin, 16t Darwinian evolution, 1 Death-inducing signaling complex (DISC), 163 Deep-sea fungus, 199–200 Dehydroquinatedehydratase (DQD), 3 Dehydrothyrsiferol, 173–174 Desulfated echinasteroside, 261–262 Diallyldisulfide, 277–284t, 285 Diallyl sulfide, 277–284t, 285 Diallyl trisulfide (DATS), 285 Dicer enzyme, 116 Dichlorodihydrofluorescein diacetate (DCFHDA), 348 Diet, in cancer, 276–283 Dietary spices, 275 Dietary supplements, 77–78 Dihydroethidium (DHE) staining, 348 2,4-Dihydroxy-6-nonylbenzoate, 200 N,N-Dimethyl-p-phenylenediamine (DMPD) assay, 338 Dinoflagellates, 157–158 Diphenyl-2-picrylhydrazyl (DPPH) assay, 339 Diphlorethohydroxycarmalol, 162t, 177–178 Direct evolution, 1 Disomic inheritance, 30 Distillation, 329 Diterpenes, 48, 67, 214 Diterpenoids, 140, 223, 238 DNA extraction, 87–88, 88f

360

Index

DNA (Continued) ligase, 87 microarray technology, 95 mutation, 94–95 frameshift, 89–90 point, 88–89 repair, melatonin in, 269–270 synthesis and sequencing, 88–89 DNA binding proteins (DPs), 132 DNA synthesis-based assay, 344 Dolastatins, 132–134, 169–171, 261 Drought stress, 61 Drug discovery, evolution of, 15–17 E Eastern blotting, 94 Echinasterosides, 261–262 Echinoderms, anticancer activity of, 261–262 E. coli, 11, 87, 108 Eicosa-pentaenoic acid, 211 Elatol, 159, 168, 173–174 Electric cell-substrate impedance sensing (ECSI), 344 Electrochemical biosensors, 348–349 Electromagnetic radiation (EMR), 333 Elevated ozone, 54 Elicitors, 39 Elicits, 39 Emodin, in cancer therapy, 312t, 315f, 316–317 Endoglucanase, 195–196 Endophyte-endophyte symbiosis, 193–194 Endophytic fungi, 191–192 anticancer compounds and, 198–199 fermentation of, 201–202 in medicinal plants, 193 symbiotic association of, 191–192 virulence factors, 193–194 Engineered bacteria, 111–113 Ephedrine, 78 Epicatechin, 277–284t, 317 Epigallocatechin gallate (EGCG), 312t, 315f, 317 Epipodophyllotoxins, 16t, 243 10-Epi-sclerotiamide, 199–200 Ericifolin, 277–284t Ethidiumhomodimer assay, 344

Ethyl acetate, 327 Ethyl acetate extract of cloves (EAEC), 291 Eucalyptol, 277–284t Eucheuma serra agglutinin (ESA), 174–175 Eudesmanolides, 218, 221–222 Eugenin, 277–284t, 291 Eugenol, 277–284t, 290–291, 293 Eupenicinicol D, 199 Evolutionary theory, 51–53 Extracellular death factors (EDF), 103–105 Extracellular matrix (ECM), 164 Extraction of compounds, 324–327 microwave-assisted extraction (MAE), 326 pressurized liquid extraction (PLE), 324–325, 325f pulsed electric field extraction (PEF), 326–327 supercritical fluid extraction (SFE), 325–326 types, 324–327 Extrinsic pathway, 163–164 F Fagoronine, 243 Fas-associated death domain (FADD), 163 Ferns, 239–241 Ferric reducing antioxidant power (FRAP) assay, 338 Ferrozine assay, 340 Ferulic acid, 277–284t Ferulic acid methyl ester, 277–284t Flash chromatography, 331 Flavonoids, 244 bryophytes, 214–215 ecological significance, 64t, 66–67 in plant kingdom, 12–13 sub-classes, 12–13, 13f as UV-B protectants, 59–60 Fluorescence-dependent methods, 347–348 Fluorescence in-situ hybridization (FISH), 9–10 5-Fluorouracil (5-FU), 177 Fourier transform infrared spectroscopy (FTIR), 333, 333f, 335t

Index 361

Fourier transform ion cyclotron resonance MS (FT-ICR-MS), 82–83 Fractionation, 327–328 acid-base nature of solvent, 327–328 solvent-solvent partitioning methods, 327, 328f techniques, 327–328 Frameshift mutations, 95 Free radicals, 269–270, 335–336 FTIR. See Fourier transform infrared spectroscopy (FTIR) Fucoidans, 158–159, 165, 175–176 Fucoxanthin, 167, 176–177 Fucoxanthinol, 176–177 Fumarate and nitrate reduction (FNR) regulators, 117 Fungi. See also Plant-fungal interactions algae-associated, 200 anticancer property of, 236–237 mangrove-derived, 200 marine, 199–200 secondary metabolites of, 191–192 sponge associated, 200–201 two-component systems, 197 Fusarium equiseti, antidiabetic activity, 191–192 Fuscoside, 259–260 G Gallic acid, 277–284t γ-irradiation, 117 Garlic, 277–284t, 284–285 Gas chromatography (GC), 83 Gel electrophoresis, 91, 91f Gemcitabine, 313 Gene clusters evolutionary origin, 10–11 for secondary metabolic pathways, 7–10 Gene duplication (GD), 1–2, 52, 194–195 Gene silencing, 116 Gene transfer, 115–116 Gene triggering, 116–117 Genistein, in cancer therapy, 312t, 315f, 316 Genome defragmentation, 10 Genome integrity, melatonin in, 269–270 Gibberellins, 213

Ginger anti-cancer property, 277–284t, 287–288 clinical application, 288 pre-clinical studies, 288 Gingerol, 277–284t, 287, 315f, 318 Ginsenocin, 198–199 Girinimbine, 243 Glaucescenolide, 218 Global warming, 48–49, 55 Glutathione, 130 Glycoalkaloids (GA), 35, 60–61 Glycoprotein, 168 Glycoside hydrolases, 195–196 Glycosylation, 8 Gracilamides, 159, 161t Gracilarioside, 159, 161t Grandinin, 277–284t Granulatosides, 261–262 Green carbon nanotags (G-Tags), 236 Greenhouse gases, 47–48 Gymnema sylvestre, antibacterial activity, 191–192 Gymnosperm, anticancer property of, 241–242 harringtonine, 241 taxol, 241–242 H Hairy root culture method, 38 Halityloside, 261–262 Halomon, 160, 161t Harringtonine, 241 Headspace solid-phase microextraction (HS-SPME), 83 Heat shock, 63–64 Helicobacter pylori, 103–105 Hepatocellular carcinoma (HCC), atranorin (ATR) in, 237 Herbal drugs, 78, 323 Herbal therapeutic molecules, extractions of, 38 Heterogenetic pairing, 30 Heteroploidy, 29 Hexaploids, 36 High-performance liquid chromatography (HPLC), 332–333 Hippasteriosides, 261–262

362

Index

Horizontal gene transfer (HGT), 8–10, 194–195 Hornworts, 211–213, 239 Human Metabolome Database (HMDB), 80 Hydro distillation (HD), 329 Hydrogen atom transfer, 336, 339–340 Hydrogen peroxide (H2O2), 269–270 Hydrolyzable tannins, 65–66 Hydroxyindole-O-methyltransferase (HIOMT), 268–269 6-Hydroxymelatonin (6-OHM), 270 5-(Hydroxymethyl) furfural, 277–284t 5-Hydroxytryptophan, 268 Hyperforin, 61–62 Hypericin, 61–62 Hyperploids, 30 Hypoploids, 30 I Ilicicolin H, 200 Imatinib, 15–16 Indirect acclimatization, 49–50 Indole-3-acetic acid (IAA), 269 Induced expression, for secondary metabolites, 56t Insulicolide A, 200 Intergovernmental Panel on climate change (IPCC), 47 Intermittent androgen deprivation (IAD), 290 Intrinsic pathway, 163–164 In vitro culture, of bryophytes, 224 Isocryptomerin, 239–240 Isoprene, 63–64, 140 Isoprenoids, 140–141, 216–217 J Jamaican pepper (Pimenta dioica), 292–293 Jasmonic acid (JA) pathway, 196–197 K Kaempferol, 277–284t Kanamienamide, 162t, 169–171 14-Keto-stypodiol diacetate, 167

L Lactate dehydrogenase (LDH) leakage assay, 342 Laurinterol, 162t, 173–174 Lead alkaloids, 243 Lepidozenolide, 220 Leptaochotensosides, 261–262 Leptasteriosides, 261–262 Lethasteriosides, 261–262 Lichens anticancer effects, 237 secondary metabolites of, 237 Light-harvesting complexes (LHCs), 171–172 Light/solar radiation, 59–61 Linalool, 169 Lipid oxidation peroxide value assessment, 340–341 thiobarbituric acid reactive substances, 341 Lipids, 211 Lipopeptides, 133 Lipopetide, 236 Lipoprotein peroxidation (LPO) assay, 337 Liquid chromatography (LC), 83 Listeria spp., as tumor-targeting vectors, 112–113 Liverworts, 211–213, 238 Lobaric acid, 235f, 237 Lobarstin, 237 Lophotoxin, 259–260 L-tryptophan, 268 Luidiaquinoside, 261–262 Lung cancer Mycobacterium tuberculosis, 2, 9f phycocyanin, 169–171 physciosporin in, 237 selenium polysaccharide, 172–173 Lunularic acid, 213, 217–218, 222 Lunularin, 218, 220 Lycopene, in cancer therapy, 315f, 317 M Magnetite nanoparticles, crocin-coated, 292 Malformin, 7 Malondialdehyde (MDA), 337 Mangrove-derived fungi, 200

Index 363

Manool, 277–284t Marchantin, 215–216, 218, 220–221, 239 Marine algae, 155–157, 160–161 algal extracts, 160–161, 160t anticancer compound isolated from β-carotene, 166–167 cannabinoids, 168 carotenoids, 166 chlorophyll, 166 elatol, 168 fucoidans, 165 fucoxanthin, 167 glycoprotein, 168 monoterpenes, 169 pheophytin, 166–167 phycocyanin (PC), 165–166 polysaccharides, 164–165 sargachromanol E (SE), 168 siphonaxanthin, 167 stypodiol diacetate, 167 yessotoxins (YTXs), 168 anticancer therapy via apoptosis, 161–163, 162t bioactive compounds in, 157–161, 161t extrinsic pathway, 163–164 family chlorophyceae, 171–173 cyanobacteria, 169–171 phaeophyta, 175–178 rhodophyta, 173–175 intrinsic pathway, 163–164 Marine fungi, 199–200 Marine invertebrates, natural products isolated from, 255–262, 257–259t, 260f Massively parallel signature sequencing (MPPS), 89 Mass spectrometry (MS), 82–84 CE-MS, 84 coupled with chromatographic techniques, 83–84 direct analysis, 82–83 GC-MS, 83 LC-MS, 83–84 UPLC-MS, 84

Maxam-Gilbert method, 88–89 Medicinal plants adaptation of, 55 climate change effects CO2 levels, 54 elevated ozone, 54 global warming, 55 phenological changes, 54 shifting ranges, 54 ultraviolet radiation, 54 endophytic fungi in, 193 environmental factors, effect of, 59t Medium pressure column chromatography (MPCC), 331 Melatonin antioxidant behavior, 268 decarboxylation, 268 in DNA repair, 269–270 evolutionary history of, 268 in genome integrity, 269–270 hydroxylation, 268 synthesis in animals, 268 in plants, 269 and telomerase activity, 270–271 Melonanchora kobjakovae, 259–260 Mertensene, 162t, 173–174 Metabolic effectors, 195–196 Metabolic engineering, 39 Metabolomic finger printing, 81 Metabolomic foot printing, 81 Metabolomics, 79–81, 80f arrays, 95–96 DNA mutation, 94–95 DNA synthesis and sequencing, 88–89 finger printing, 81 food, 79 foot printing, 81 gel electrophoresis, 91, 91f mass spectrometry (MS), 82–84 molecular approach, 86–96 molecular (DNA) cloning, 87–88, 88f molecular hybridization, 92–94 non-targeting profiling, 81

364

Index

Metabolomics (Continued) nuclear magnetic resonance spectroscopy (NMR), 84–86 polymerase chain reaction (PCR), 89–90, 90f targeted, 81 technologies, 81–86 untargeted discovery global, 80–81 Methyl 4-hydroxyl cinnamate, 277–284t Methylerythritol-phosphate (MEP) pathway, 140 Microbial antitumor products, 234–236 Microcolin-A, 157, 161t Microgramma vacciniifolia, antioxidant activity of, 240 Micro-propagation, 38 Microwave-assisted extraction (MAE), 326 Missense mutations, 94 Mitochondrial pathway, 163–164 Moisture stress, 61 Molecular approach arrays, 95–96 DNA mutation, 94–95 DNA synthesis and sequencing, 88–89 gel electrophoresis, 91, 91f molecular (DNA) cloning, 87–88 molecular hybridization, 92–94 polymerase chain reaction (PCR), 89–90 Molecular (DNA) cloning, 87–88 Molecular hybridization, 92–94 Eastern blotting, 94 Northern blotting, 93 Southern blotting, 92, 92f Western blotting, 93 Molecular photocopying. See Polymerase chain reaction (PCR) Molluscs, anticancer activity of, 261 Momilactone gene cluster, 8–9, 9f Monanchocidins, 259–260 Monanchomycalins, 259–260 Monanchora pulchra, 257–259t, 259–260 Monocytes and dendritic cells (MDSCs), 110–111 Monosomics, 30 Monoterpenes, 67, 169, 214, 238 Morphine, 19–20, 78 Mosaic effect theory, 193–194

Mosses, 239 Multiple stress effect, 62–63 Mycobacterium tuberculosis, 103–105, 105f Mycosporine like amino acids (MAAs), 11, 131–132 Mycosterol, 191–192 Myricetin, 277–284t Myrtus communis, 19–20 N Naphtho-γ-pyrones, 198–199 Naringin, 277–284t Natural oxadiazinenocuolin A (NoA), 169–171 Natural products, screening of anticancer assay, 341–347 antioxidant, chelation power of, 340 antioxidant assay, 335–337 basic structure elucidation, 333–335 chemical diversity of, 11 chromatography, 330–333 crystallization, 329–330 extraction, 324–327 fractionation, 327–328 hydrogen atom and single electron transfer, 339–340 isolated from marine invertebrates, 255–262, 257–259t, 260f lipid oxidation, 340–341 purification, 329 ROS detection, 347–349 single electron transfer, 338–339 Neosurugatoxin, 261 Nigriccanoides, 158 Nilocitin, 277–284t Nisin, 107 Nitidine, 243 Nitidine chloride, 243 Nitrogen-containing secondary metabolites (SM), 3–4, 4t Nonribosomal peptides (NRPs), 138f, 139–140 Nonribosomal peptide synthetases (NRPSs), 139, 169–171 Nonsense mutation, 94 Non-volatile plant secondary metabolites, 68

Index 365

Normonanchocidin D, 257–259t, 259–260 Northern blotting, 93 Noscomin, 141 Novaeguinosides, 261–262 Nuclear magnetic resonance (NMR) spectroscopy, 84–86, 333–335, 334f Nullisomics, 30 Nutraceuticals, 77 in cancer therapy, 311–312 chemoprevention, 311–312, 312t chemosensitization, 311–312, 312t curcumin, 312t, 313–315, 315f emodin, 312t, 315f, 316–317 epigallocatechin gallate (EGCG), 312t, 315f, 317 genistein, 312t, 315f, 316 gingerol, 315f, 318 lycopene, 315f, 317 piperine, 315f, 318 quercetin, 312t, 315f, 317 resveratrol, 312t, 315f, 316 chemical structures, 315f drawbacks in cancer treatments, 309–311 history of, 309 sources of, 314f O Okadaic acid, 157–158 Oleanolic acid, 277–284t, 291 Oncology, therapeutic bacteria in, 103, 104t Oncolytic viruses, for cancer treatment, 109 Onion, anticancer activitiy, 277–284t, 284–285 Ontogeny, 19–20 Ophioglossum, 240 Oregano, chemopreventive and therapeutic potentials, 277–284t Osmotin, 196 Oxidative burst, 196 13-Oxofumitremorgin B, 200–201 Oxygen radicle absorbance capacity (ORAC) assay, 336 Ozone, 59t Ozone fumigation, 53

P Paclitaxel, 78, 140, 198, 241–242 Palytoxin, 259–260 Papaver somniferum, 19–20 Paradol, 277–284t, 287 Parthenolide, 140 Pathogenesis-related (PR) proteins, 196 Pediocin, 107 Pedunculagin, 277–284t Pentaploid, 31–32 Pentasomics, 30 Peppers, chemopreventive and therapeutic potentials, 277–284t Peptidyl arginine deaminase (PAD), 103–105 Perfragilin, 260–261 Peroxynitrite (ONOO), 340–341 Peroxynitrous acid (ONOOH), 340–341 Phaeophyta, 175–178 Phaeosphaeri avenari, 197 Phaeosphoramides, 197 Phenethyl isothiocyanate (PEITC), 242 Phenolic acids, 64t, 65–66 Phenolics, 50, 64t, 65 Phenylalanine ammonia lyase (PAL), 58f Phenylpropanoid (PP), 2–3, 35, 59–62, 213–214 Pheophytin, 158, 166–167 Photolyase (PL), 132 Phycobiliproteins (PBPs), 127–128 Phycocyanin (PC), 165–166, 169–171 Physciosporin, 237 Physcomitrella patens, 223–224 Phytoalexins, 6–7, 62 Phytoanticipins, 62, 69 Phytocassane gene cluster, 8–9, 9f Phytochemicals, 234, 312t Phytol, 19–20 Phytosterols, 60–61, 214, 217 Picrocrocin, 277–284t Pineal gland, 267 Pinus taeda, 6–7 Pipecolic acid, 48 Piperine, 195, 277–284t, 289–290, 315f, 318 Plagiochilide, 221–222 Plagiochiline, 221–222

366

Index

Plant(s) anticancer potential, 234, 235f biomass, 36 diversity, 234 evolution, early stage, 55–56 expression of secondary compounds, 55, 56t, 57f growth inhibitory activity, bryophytes, 222 protoplasts, 222–223 secondary metabolites, 234, 243 Plant extract, antioxidant measurement in chelation power of antioxidant, 340 hydrogen atom transfer, 336, 339–340 lipid oxidation, 340–341 single electron transfer, 338–340 Plant-fungal interactions biochemical aspects, 196–197 genetic aspects, 195–196 and metabolic diversity, 192–195 signal transduction pathway, 197 Plant secondary metabolites (PSMs). See Secondary metabolites (SMs) Ploids. See Polyploids Point mutations, 94 Polygodial, 218–220 Polyketides (PKS), 4t, 10, 51, 139–140, 199 Polymerase chain reaction (PCR), 89–90 denaturation, 90 elongation, 90 hybridization, 90 stages, 90f thermal cycling, 90 Polyphenoloxidase (PPO), 58f Polyphenols, 65–66, 244, 311 Polyploidization, 32, 34–35, 39 Polyploids, 29–30 artificial induction, 35 autopolyploidization, 33 biomass production, 36 chromosome doubling, 34 chromosome duplications, 33 chromosome number, change in, 36 genome buffering effect, 32–33 homologous chromosomes, 32–33 phenotypes, 31–32

in plant speciation, 32–33 polyploidization, 32, 34–35 in secondary metabolites production, 31–39 Polysaccharides, isolated from marine algae, 164–165 Porphyran, 159, 161t Post-ribosomal peptide synthesis (PRPs), 140 Post translational modification (PTM), 94 Potassium ferricyanide reducing power (PFRAP) assay, 339 Pratoxins, 169–171 Prenyldihydrochalcone, in Radula spp., 12–13 Prenylflavonoids, in angiosperms, 12–13 Pressurized liquid extraction (PLE), 324–325, 325f Primary metabolites, 50 Proanthocyanidins, 12–13, 65–66 Programmed cell death, 164, 310–311 Proline, 48 Propidium iodide (PI), 344 Prostate-specific antigen doubling time (PSAdt), 288 Prostate-specific antigens (PSA), 115 Protein microarray technology, 96 Protocatechuic acid, 277–284t Pseudohypericin, 61–62 Pseudopterosins, 259–260 Pteridophytes, 239–240 Ptilomycalin A, 257–259t, 259–260 Pulsed electric field extraction (PEF), 326–327 Purification, 329 distillation, 329 hydro distillation (HD), 329 steam distillation (SD), 329 techniques, 329 Pyroptosis, 164 Pyrosequencing, 89 Pyrrosia heterophylla, 240–241 Q Quercetin, 213–214, 277–284t, 293, 312t, 315f, 317 Quinate, 3

Index 367

Quinatedehydrogenase (QDH), 3 Quinine, 78 Quorum-sensing bacteria, 106 R Radiotherapy, 114 Raoult’s law, 329 Reactive oxygen species (ROS), 337 detection methods Amplex Red, 348 chromatographic method, 348 dichlorodihydrofluorescein diacetate (DCFH-DA), 348 dihydroethidium (DHE) staining, 348 electrochemical biosensors, 348–349 fluorescence-dependent methods, 347–348 nonradicals, 347, 347t physiological roles in plant development, 347 radicals, 347, 347t Reactive oxygen species (ROS) assay, 346–347, 346f Recombinant DNA (rDNA), 87 Regularoside B, 261–262 Resazurin, 342 Resveratrol, 244, 312t, 315f, 316 Reverse phase high performance liquid chromatography (RP-HPLC-MS), 83–84 Rhodophyta, 162t, 173–175 Ribosomal peptides, 140 Riccardiphenol C, 218 Root culture method, 38 Rosemary, chemopreventive and therapeutic potentials, 277–284t Rosmarinic acid, 277–284t Rubrofusarin B, 198–199 Rutin, 277–284t S Saccharides, 211 Saffron (Crocus sativus), 291 chemopreventive and therapeutic potentials, 277–284t

pharmacologically active components, 291–292 Safranal, 277–284t, 291–292 Sage, chemopreventive and therapeutic potentials, 277–284t Salicylic acid (SA), 62, 196–197 Salmonella A1-R strain, 113 Salmonella typhi, 103–105 Sargachromanol E (SE), 168 Sartorypyrones, 200–201 Savory, chemopreventive and therapeutic potentials, 277–284t Saxitoxin, 140 Scytonemin, 131–132, 134, 157, 161t Secondary metabolism, environmental factors triggering, 56–63 Secondary metabolite gene clusters (SMGCs), 7 Secondary metabolites (SMs), 1–2, 48 advantages, 69 biomolecular activity, 14–15 biosynthetic pathway, 6, 6f in bryophytes, 211–217, 212t climate change effects, 53 cyanobacteria, 128, 133–134, 133f definition of, 50–51 ecological significance, 64–67, 64t ecosystem feedback of, 68 of endophytes, 191–192 evolutionary theory based on, 51–53 expression in plants, 55, 56t, 57f factors influencing production, 18 environmental interaction, 21 genetic factors, 18–19 morphogenetic factors, 20 ontogenic factors, 19–20 gene clusters, 7–10, 9f history of, 49 of lichens, 237 molecular targets in cancer treatment, 15–16, 16t of plants, 234, 243 types, 3–4, 4t Secondary organic aerosols (SOAs), 68 Selaginella tamariscina

368

Index

Selaginella tamariscina (Continued) amentoflavone, 240–241 p53 gene expression, 240 selaginellin, 240 as urokinase plasminogen activator, 239–240 Selaginella willdenowii, 239–240 Selaginellin, 235f, 240 Selenium-containing phycocyanin (Se-PC), 165–166 “Selfish cluster” model, 10 Serotonin, 267–269 Sesquiterpenes, 4t, 67, 214, 238 Shikimate dehydrogenase (SDH), 3 Shikimate pathway, 1–3, 6–7, 50 Shinorine, 11 Shogaol, 277–284t, 287 Silent mutation, 94 Simplicilliumtides, 199–200 Single-celled bacteria, anti-tumor effect anti-angiogenesis therapy, 109–110, 110t bacterial substances, 106–107 biofilms, production of, 108–109 engineered bacteria, 111–113 gene silencing, 116 gene transfer, 115–116 gene triggering strategies, 116–117 human immunity, enhancement of, 107–108 live bacteria as tumor suppressor, 111 live tumor-targeting bacteria, 110–111, 112f with radiotherapy, 114, 114f tumor-specific antigens and antibodies, 115 viruses, 109 Single electron transfer cupric reducing antioxidant capacity (CUPRAC) assay, 338–339 N,N-dimethyl-p-phenylenediamine (DMPD) assay, 338 ferric reducing antioxidant power (FRAP) assay, 338 hydrogen atom and, 339–340 potassium ferricyanide reducing power (PFRAP) assay, 339 Siphonaxanthin, 162t, 167

SMGCs. See Secondary metabolite gene clusters (SMGCs) Solar radiation energy, 59 Solvent extraction technique, 324 Solvent-solvent partitioning methods, 327, 328f Somocystinamide A, 132–133 Southern blotting, 92, 92f Spices, 275–276, 277–284t Spirobrocazines C, 200 Spirulina, 166–167, 169–171 Sponges anticancer activity of, 256–259, 257–259t associated fungi, 200–201 Spongocoel, 256 Squalene, 141 Steam distillation (SD), 329 Steroid heptaol, 261–262 Structure elucidation, 333–335 Fourier transform infrared spectroscopy (FTIR), 333, 333f, 335t nuclear magnetic resonance (NMR), 333–335, 334f Stypodiol diacetate, 167 Stypoldione, 158 Sulforaphane, 242 Sulforhodamine B (SRB) assay, 342 Sulphated polysaccharide (SPS-CF), 158–159, 161t, 173–174 Supercritical fluid extraction (SFE), 325–326 Superoxide dismutase (SOD), 130, 270 Syctonemin, 131–132 Symbiotic endophytes, 192 Symplostatin, 162t, 169–171 T Tannins, 59–60 condensed, 65–66 ecological significance, 64t hydrolyzable, 65–66 Targeted metabolomics, 81. See also Metabolomics Taxanes, 16t, 241–243 Taxol, 198, 241–242 Telomerase activity, melatonin and, 270–271

Index 369

Telomere, 270–271 Temperature stress, 61–62 Terpenes, 2, 50, 64t, 67 Terpenoids, 2, 211–213 biosynthetic pathway, 18–19 ecological significance, 64t, 67 4-Terpineol, 277–284t Tetraploids, 31–32, 35 Tetrasomic inheritance, 30 Tetrasomics, 30 Thalianol gene cluster, 8–9, 9f Thin layer chromatography (TLC), 331 analytical, 331 preparative, 331 Thiosulfinate metabolites, 285 Thornasteroside A, 261–262 Thyme, chemopreventive and therapeutic potentials, 277–284t Thymol, 277–284t Tissue culture, 38 TNF receptor-associated death domain (TRADD), 163 Topoisomerases, 17 Total radicle trapping antioxidant parameter (TRAP) assay, 336–337 crocin bleaching or beta carotene method, 337 lipoprotein peroxidation (LPO) assay, 337 oxygen radicle absorbance capacity (ORAC) assay, 336 Traditional Chinese medicine (TCM), 210 Transgenic moss, bryophytes, 224–225 Tretinoin, 15 2,3,6-Tribromo-4,5-dihydroxybenzyl methyl ether (TDB), 174–175 Trichoderma harzianum, 194–195 Triploid, 31–32 Trisomics, 30

Triterpene gene cluster, 8–9 Triterpenes, 48, 214 Triterpenoids, 239 Troloxaliquot, 339–340 Trolox equivalents (TE), 338 Tryptophan decarboxylase (TDC), 269 Tuber borchii, 197 Tumor-specific antigens, 115 Turmeric (Curcuma longa), 288–290. See also Curcumin U Ulapualide, 261 Ultra-performance liquid chromatography (UPLC), 84 Ultraviolet radiation (UVR), 54 Urupocidin A, 257–259t, 259–260 UV-B radiations, 59–60 V Varioloids, 200 Vinca alkaloids, 16t, 243 Vincristine, 235f, 243 Volatile organic compounds (VOCs), 55, 67 W Western blotting, 93 Wound curative assay, 345, 345f X Xenohormesis hypothesis, 193–194 Xestospongia sp., 200–201, 257–259t Y Yessotoxins (YTXs), 168 Z Zerumbone, 277–284t Zingerone, 277–284t, 288

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