Natural Bioactive Molecules: : Impacts and Prospects [1 ed.] 9781783320752, 9781842657805

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Editor Goutam Brahmachari

a Alpha Science International Ltd. Oxford, U.K.

Natural Bioactive Molecules Impacts and Prospects 516 pgs. | 61 figs. | 18 tbls.

Editor Goutam Brahmachari Department of Chemistry Visva-Bharati University Santiniketan Copyright © 2014 ALPHA SCIENCE INTERNATIONAL LTD. 7200 The Quorum, Oxford Business Park North Garsington Road, Oxford OX4 2JZ, U.K.

www.alphasci.com All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the publisher. ISBN 978-1-84265-780-5 E-ISBN 978-1-78332-075-2 Printed in India

Dedication All those who are working globally with bioactive natural products for the cause of human welfare

Foreword For almost 100 years, microbes and plants have contributed to industrial production of important chemicals and to the health of the world’s population by their ability to make valuable natural products. These compounds have shown superiority to those made completely by chemical synthesis. However, in many cases, valuable derivatives of natural products are made via organic synthesis. This book, edited and organized by Prof. Goutam Brahmachari, is an excellent review of natural molecules produced by living organisms. The compounds described in this volume have shown great activity and promise against human diseases, such as Alzheimer’s disease, cardiac disease, cancer, inflammatory diseases, diabetes, oxidative stress, infectious diseases, and many others. Agents slowing down aging or acting as natural food pigments, low-calorie sweeteners, and neuroprotective agents are also covered. The book will be of interest to all scientists, including chemists, geneticists, biochemists, microbiologists and others interested in the use of natural organisms that produce beneficial compounds. This includes academic scientists as well as those working in the biotechnology industry. With the decrease of interest in natural products by the large pharmaceutical companies, it is clear that universities and the biotechnology companies will be the sources of new natural products and their applications. This book will help the research personnel in those institutions to become familiar with the amazing history and prospects of naturally occurring bioactive compounds. For scientists in general, this volume will be an excellent reference book to place on their library shelf. Arnold L. Demain Research Institute for Scientists Emeriti (RISE) Drew University, Madison, New Jersey, USA

Preface This single volume entitled Natural Bioactive Molecules: Impacts & Prospects is an endeavor to focus on the recent cutting-edge research advances in the field of bioactive natural products and their significant contributions in the domain of discovery and development of new medicinal agents; the present book brings together a total of thirteen articles contributed by eminent researchers, well-reputed in their own fields of research from several countries in response to my personal invitation. I am most grateful to the contributors for their generous and timely response in spite of their busy and tight schedules with academics, research, and other responsibilities. The role of natural products in the treatment of diseases has inspired pharmaceutical scientists in their search for new avenues in drug discovery. Bioactive natural products are a rich source of novel therapeutics, and are of great interest and promise in the present day research directed towards drug design and discovery. Many naturally occurring bioactive compounds and/or their derivatives have become drugs of central importance, and represent a high percentage of the drugs used today. Antibiotics, hormones, and statins are the well-known examples. Natural products present in the plant and animal kingdom offer a huge diversity of chemical structures which are the result of biosynthetic processes that have been modulated over the millennia through genetic effects, and hence search for bioactive molecules from nature continues to play an important role in fashioning new medicinal entities. At present, plants, microorganisms, and marine invertebrates represent major sources of natural products for discovering new and novel drugs. Current research trends in the field suggest an optimistic future for natural products in drug discovery; however, novel strategies and innovative approaches in addition to the introduction of more sophisticated technical requirements are still needed today for the development of natural products into new drugs. Medicinal chemistry of such bioactive compounds encompasses a vast area that includes their isolation and characterization from natural sources, structure modification for optimization of their activity and other physical properties, and also total and semi-synthesis for a thorough scrutiny of structure-activity relationships. It has been well documented that natural products played crucial roles in modern drug development, especially for antibacterial and antitumor agents; however, their uses in the treatment of other epidemics such as AIDS, cardiovascular, cancerous, neurodegradative, infective and metabolic diseases have also been extensively explored. The need for leads to solve such health problems threatening the world population makes all natural sources important for the search of novel molecules, diversified and unique structural architectures of which inspired scientists to pursue new chemical entities with completely different structures from

x

Preface

known drugs. Researchers round the globe are deeply engaged with such potent and efficacious naturally occurring bioactive compounds in exploring their detailed chemistry and pharmacology. This book comprising of a variety of thirteen chapters written by active researchers and leading experts working in the field of biologically active natural products, brings together an overview of current discoveries and trends in this remarkable field. The introductory chapter (Chapter-1) presents an overview of the book, and summarizes the contents and subject matter of each technical chapter (Chapter-2 to Chapter-13) so as to offer certain glimpses of the coverage of discussion to the readers before they go for detailed study. These twelve technical chapters are keenly devoted to explore on-going chemical and pharmacological advances on naturally occurring bioactive compounds of potential interest highlighting their chemical transformations and semisynthetic studies, biosynthesis, structure-activity relationships, safety evaluation, clinical studies, metabolism, molecular biology and mode of action. This timely volume encourages interdisciplinary work among chemists, pharmacologists, biologists, botanists, and agronomists with an interest in bioactive natural products. The broad interdisciplinary approach dealt in this book would surely make the work much interesting to the scientists deeply engaged in the research and/or use of bioactive natural products. It will serve not only as a valuable resource for researchers in their own fields to predict promising leads for developing pharmaceuticals to treat various ailments and disease manifestations, but also motivates young scientists to the dynamic field of bioactive natural products research. Representation of facts and their discussions in each chapter are exhaustive, authoritative, and deeply informative; hence the book would serve as a key reference for recent developments in the frontier research on bioactive natural products, and also would find much utility to the scientists working in this area. I would like to express my sincere thanks once again to all the contributors in this volume for the excellent reviews on the chemistry and pharmacology of bioactive natural products that they have produced. It is their participation that makes my effort to organize such a book possible. Their masterly accounts will surely provide its readers with a strong awareness of current cutting-edge research approaches being followed in some of the promising fields of biologically active natural products. I would like to express my sincere thanks and deep sense of gratitude to Professor Arnold L. Demain, Research Institute for Scientists Emeriti (RISE), Drew University, Madison, New Jersey, USA for his keen interest in the manuscript and for writing a foreword to the book. Lastly, but not the least I would like to express my deep sense of appreciation to all of the editorial and publishing staff-members associated with the Publishing House(s) for their keen interest in publishing the work and also for their all-round help so as to ensure that the highest standards of publication are maintained in bringing out the book. Goutam Brahmachari

Contents Foreword Preface List of Contributors 1. Natural Bioactive Molecules: Impacts and Prospects— An Overview

vii ix xiii 1.1-1.6

Goutam Brahmachari 2. Role of Natural Products as a Source of Alzheimer’s Drug Leads: An Update

2.1-2.112

Goutam Brahmachari 3. Natural Product Activators of Sirtuins against Aging

3.1-3.34

Michael D. Scott, Manas K. Haldar, Rinku Dutta and Sanku Mallik 4. Structure-Bioactivity Relations of Acylated Anthocyanins and Their Related Polyphenols

4.1-4.25

Ju Qiu, Norihiko Terahara and Toshiro Matsui 5. Marine-Derived Bioactive Polysaccharides from Microorganisms

5.1-5.21

Sylvia Colliec-Jouault and Christine Delbarre-Ladrat 6. Stevioside and Related Compounds: Molecules of Pharmaceutical Promise Beyond Zero-Calorie Sweeteners

6.1-6.39

Goutam Brahmachari 7. Vitamin B6 Derived Cofactor Pyridoxal-5¢-phosphate: Promising Role in Drug Development Programme

7.1-7.23

Kuheli Chakrabarty and Gourab Kanti Das 8. Allelochemicals from Artemisia vulgaris Linn. (Compositae)

8.1-8.11

R.N. Yadava and Gautam Patil 9. Quercetin — A Ubiquitous Bioflavonoid: Structural Aspects versus Potential Health Benefits

Ajay Kumar Dixit, Shyam Ramakrishnan, and C.C. Lakshmanan

9.1-9.29

xii

Contents

10. Therapeutic Efficacy of Macro and Small Bioactive Molecules in Organ Pathophysiology

10.1-10.30

Jyotirmoy Ghosh and Parames C. Sil 11. Anti-pruritic and Anti-inflammatory Herbal or Natural Products for the Treatment of Skin Disease

11.1-11.13

Katsunori Yamaura and Koichi Ueno 12. Triterpenoid Saponins: Role in Targeted Anti-cancer Therapies

12.1-12.18

Mayank Thakur, Matthias F. Melzig, Hendrik Fuchs and Alexander Weng 13. Natural Bioactive Flavonoids — Recent Developments in Research: A Thorough Update

13.1-13.118

Goutam Brahmachari Index

I.1-I.11

List of Contributors Ajay Kumar Dixit Analytical Center of Excellence (ACE), Bioscience, ITC Life Science & Technology Center, Peenya Industrial Area, Phase 1, Bangalore-560 058, Karnataka, India E-mail: [email protected] Alexander Weng Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité– Universitätsmedizin Berlin, Berlin E-mail: [email protected] C.C. Lakshmanan Analytical Center of Excellence (ACE), Bioscience, ITC Life Science & Technology Center, Peenya Industrial Area, Phase 1, Bangalore-560 058, Karnataka, India Christine Delbarre-Ladrat Ifremer, Laboratory of Biotechnology and Marine Molecules, Ifremer, Rue de l’Ile d’Yeu, BP 21105, Nantes Cedex 03, 44311, France Gautam Patil Natural Products Laboratory, Department of Chemistry, Dr. H. S. Gour Central University, Sagar-470 003 (M.P.) India Gourab Kanti Das Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India E-mail: [email protected] Goutam Brahmachari Laboratory of Natural Products & Organic Synthesis, Department of Chemistry, VisvaBharati University, Santiniketan-731 235, West Bengal, India E-mail: [email protected]; [email protected] Hendrik Fuchs Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité– Universitätsmedizin Berlin, Berlin Ju Qiu Division of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

xiv

List of Contributors

Jyotirmoy Ghosh Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA Katsunori Yamaura Department of Geriatric Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Chiba University 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan E-mail: [email protected] Koichi Ueno Department of Geriatric Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Chiba University 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Kuheli Chakrabarty Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India Manas K. Haldar Department of Pharmaceutical Sciences, North Dakota State University, Sudro Hall, Room: 35A, Department 2665, PO Box 6050, Fargo, North Dakota 58108-6050, USA Matthias F. Melzig Institute of Pharmacy, Free University of Berlin, Berlin, Germany Mayank Thakur Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité– Universitätsmedizin Berlin, Berlin Michael D. Scott Department of Pharmaceutical Sciences, North Dakota State University, Sudro Hall, Room: 35A, Department 2665, PO Box 6050, Fargo, North Dakota 58108-6050, USA Norihiko Terahara Department of Food Science and Technology, Faculty of Health and Nutrition, MinamiKyushu University, 5-1-2 Kirishima, Miyazaki 880-0032, Japan Parames C. Sil Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700 054, India E-mail: [email protected] /[email protected] R.N. Yadava Natural Products Laboratory, Department of Chemistry, Dr. H. S. Gour Central University, Sagar-470 003 (M.P.) India E-mail: [email protected]

List of Contributors

xv

Rinku Dutta Department of Pharmaceutical Sciences, North Dakota State University, Sudro Hall, Room: 35A, Department 2665, PO Box 6050, Fargo, North Dakota 58108-6050, USA Sanku Mallik Department of Pharmaceutical Sciences, North Dakota State University, Sudro Hall, Room: 35A, Department 2665, PO Box 6050, Fargo, North Dakota 58108-6050, USA E-mail: [email protected]; [email protected] Shyam Ramakrishnan Analytical Center of Excellence (ACE), Bioscience, ITC Life Science & Technology Center, Peenya Industrial Area, Phase 1, Bangalore-560 058, Karnataka, India Sylvia Colliec-Jouault Ifremer, Laboratory of Biotechnology and Marine Molecules, Ifremer, Rue de l’Ile d’Yeu, BP 21105, Nantes Cedex 03, 44311, France E-mail: [email protected] Toshiro Matsui Division of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan E-mail: [email protected]

1 Natural Bioactive Molecules: Impacts and Prospects—An Overview Goutam Brahmachari Laboratory of Natural Products & Organic Synthesis, Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India E-mail: [email protected]; [email protected]

ABSTRACT This chapter offers an overview of the present book, and summarizes the contents and subject matter of each chapter with an intention to highlight certain glimpses of the coverage to the readers before they go in-depth.

1.1

INTRODUCTION

This book entitled Natural Bioactive Molecules: Impacts and Prospects is an endeavor to present cutting-edge research in the chemistry of bioactive natural products and helps the reader understand how natural product research continues to make significant contributions in the discovery and development of new medicinal entities. The reference is meant for phytochemists, synthetic chemists, combinatorial chemists as well as other practitioners and advanced students in related fields. The book comprising of twelve technical chapters highlights the impact and prospects of natural bioactive molecules in modern drug discovery processes. It also features chemical advances in naturally occurring organic compounds describing their chemical transformations, and structureactivity relationships. This introductory chapter (Chapter-1) presents an overview of the book, and summarizes the contents and subject matter of each chapter to offer certain glimpses of the coverage of discussion to the readers before they go for detailed study.

1.2

AN OVERVIEW OF THE BOOK

The present book contains twelve technical chapters — Chapter-2 to 13; this section summarizes the contents and subject matter of each of these chapters.

1.2 1.2.1

Natural Bioactive Molecules: Impacts and Prospects

Chapter 2

Chapter-2 by Brahmachari offers a comprehensive update highlighting the role of natural products as a source of Alzheimer’s drug leads, and covers more the 350 such compounds along with 542 references. Alzheimer’s disease (AD) is the most common form of dementia that mainly affects elderly individuals. AD is characterized by progressive and irreversible decline of memory and other cognitive functions. Because of the ageing of populations worldwide, this disorder is reaching epidemic proportions, with a large human, social, and economic burden. It is estimated that 5.4 million people in the United States and 35.6 million people worldwide suffer from AD, and the number is expected to increase to 13.5 million in the United States and 115 million worldwide by 2050. Effective treatments are thus greatly needed. Unfortunately, even after several decades of research efforts, it is still unknown exactly what causes the disease. There are currently only five approved treatments for Alzheimer’s disease, and none of these is a cure. These drugs for AD target cholinergic and glutamatergic transmission thereby improving cognitive symptoms in some patients; however, these agents are not disease modifying, nor do they slow disease progression. Hence, one of the greatest challenges facing medicinal chemists in the 21st century is the discovery and development of compounds for the prevention, treatment, and diagnosis of Alzheimer’s. Several natural products have provided useful chemotypes for the present treatment of Alzheimer’s and other neurodegenerative diseases. Some natural compounds have already reached widespread clinical use as well. The majority of the compounds examined to date with a direct relevance to AD are primarily from plants, with comparatively few molecules derived from marine and microbial sources. The notable successes achieved so far have come from plant-based acetylcholinestrase discovery programs, providing two of the five currently approved drugs for the treatment of AD. Natural molecules can also be subjected to ‘fine-tuning’ by chemical derivatisation and synthesis of analogues for better pharmacokinetics and efficacy. Natural products have always been and continue to be the important medical reservoirs, with considerable number of modern FDAapproved medications from natural sources. In the filed of Alzheimer’s, several conclusions on the possibility of natural product leads are supported from the experimental outcomes; hence, natural products have emerged as promising hope in the drug discovery programs in Alzheimer’s. The main theme of this chapter is to highlight the impact and prospect of bioactive natural products in providing useful leads to the drug discovery and development processes against this devastating disease.

1.2.2

Chapter 3

Chapter-3 by Mallik and his group deals with naturally occurring sirtuin activators and their potential uses against aging. Aging is a continuous and irreversible process that occurs in all living organisms, thereby resulting increased probability of developing diseases, a loss of energy, and ultimately death. Understanding of agents and other devices that affect and slow down aging process are highly demanding historically. Sirtuins (SIRTs), belonging to the NAD-dependent enzyme family, are class III histone deacetyleases (HDACs) which may control the aging process through different mechanisms, and the authors have described on these issues in their review. Several naturally occurring sirtuin activators are reported to extend lifespan. The present chapter

An Overview

1.3

is devoted to sirtuins, natural product sirtuin activators, and the mechanism by which they affect the process of aging. A comprehensive discussion of this important topic would serve the purpose of a source of valuable information to the organic/medicinal chemists working on the same line of the relevant subject.

1.2.3

Chapter 4

In Chapter-4, Matsui and his group have discussed on the structure-bioactivity relations of acylated anthocyanins and their related polyphenols. Anthocyanins have been traditionally used as safe natural food pigments; however, their use is limited due to generally low stability. In recent times, a good number of poly-acylated anthocyanins have been found to be more stable than common ones in neutral aqueous solution. Besides their uses as natural pigments, this group of natural products is also found to exhibit many physiological functions for human health benefits. The authors have nicely reviewed the structure-bioactivity relationship of poly-acylated anthocyanins and their related polyphenols, very particularly focusing on the respective antioxidant and antihyperglycemic effects. Absorption and metabolism of anthocyanin-related compounds are also envisioned in this article. The present article would surely boost the on-going research directed towards the development of high-quality anthocyanin materials for foods, cosmetics, and medicinal products.

1.2.4

Chapter 5

Chapter-5 by Colliec-Jouault and Delbarre-Ladrat focuses on marine-derived bioactive polysaccharides from microorganisms. Polysaccharides and their derivatives find immense applications in human therapeutics. Marine biodiversity is a very interesting source to find not only new carbohydrate molecules with unusual structural patterns, but also enzymes with innovative selectivity or properties that can be used efficiently in processes to produce new drugs and lead molecules. Enzymes are indeed ideal biocatalysts due to their stereo- and regio-selectivity, and their activity in mild conditions. Enzymes capable of generating targeted modifications may categorically be looked upon as glycoside hydrolases or polysaccharide lyases, carbohydrate sulphotransferases, etc. With the increasing number of microbial genomes sequenced, numerous enzyme genes related to polysaccharide biosynthesis and biotransformations are identified and molecular mechanisms of the biosynthesis of the polysaccharides are now better understood. The present chapter offers a thorough inslight in all these scenario.

1.2.5

Chapter 6

Chapter-6 is devoted to stevioside, an ent-kaurene type of diterpenoid glycoside extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni, and related compounds by Brahmachari. Stevioside and related ent-kaurene glycosides have been established as natural zero-calorie/low-calorie sweeteners and many of them in the form of crude plant products are being used commercially in many countries as food additives for sweetening a variety of products; potent sweetness intensities of these glycosides in comparison to sucrose have projected them as cost-effective substitutes of sugar. Beyond their non-caloric sweetness property, these ent-kaurene diterpene glycosides may

1.4

Natural Bioactive Molecules: Impacts and Prospects

also offer a wide range of therapeutic benefits as well. Besides the pharmacological activities and therapeutic applications of stevioside and related compounds, the present article also summarizes current knowledges of the natural distribution of such compounds, their structural features, plausible biosynthetic pathways, pharmacokinetics along with an insight into the structure-sweetness relationship, safety evaluation, and clinical trials of these ent-kaurene glycosides. Although much progress has been made concerning their biological and pharmacological effects of Stevia and steviosides, questions regarding chemical purity and safety remain unsolved. The present article also discusses all these issues that would be helpful to prospective investigators to design future research directions.

1.2.6

Chapter 7

Chakrabarty and Das have contributed on the impacts of vitamin B6 derived cofactor pyridoxal-5¢phosphate in the on-going drug development program in Chapter-7. Pyridoxal-5¢-phosphate (PLP) serves as a versatile enzyme cofactor for various biotransformations. Vitamin B6 derived cofactor PLP itself and the enzymes depending upon PLP are now regarded as promising drugs and drug targets for their catalytic action inside the living cells. Pyridoxal-5¢-phosphate monohydrate (MC-1), a naturally occurring metabolite of vitamin B6 can act as a purinergic (p2) receptor antagonist and prevents cellular calcium overload in preclinical and clinical studies of ischemia reperfusion injury. Various PLP-phosphonate analogues have also been proved to be very good cardio protective agents as well. The authors have discussed critically the role of PLP, PLP conjugates and PLP dependent enzymes as drugs and drug targets considering all the aspects related to medicinal chemistry in their present chapter, which would surely boost the ongoing developments in that direction.

1.2.7

Chapter 8

Chapter-8 by Yadava and Patil deals with the chemistry of allelochemicals isolated from Artemisia vulgaris Linn. (Family: Compositae). The present chapter reports on the isolation and structural elucidation of a new allelochemical characterized as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone7-O-a-L-rhamnopyranosyl-(1Æ3)-O-b-D-arabinopyranosyl-(1Æ4)-O-b-D-xylopyranoside along with two known flavonoids such as 5,4¢-dihydroxy-3,7-dimethoxyflavone and 3,5,7,3¢,4¢pentahydroxyflavone-3-O-a-L-rhamnopyranoside (quercetin-3-O-a-L-rhamnopyranoside) from ethanolic extract of the aerial parts of A. vulgaris Linn. The structures of the isolates were elucidated based on various color reactions, chemical degradations and detailed spectral analyses.

1.2.8

Chapter 9

Dixit and his group have produced a thorough discussion on the promising bioflavonoid, quercetin highlighting its structural aspects versus potential health benefits in Chapter-9. Quercetin and its glycosides are widely distributed in fruits and vegetables as major dietary flavonoids. The bioflavonoid has been found to be responsible for a wide range of biological properties that include antioxidant, anti-inflammatory, anticancer, anti-tumor, anti-atherosclerotic, cardioprotective, antiulcer, anti-allergic, and anti-hyperglycemic activities. Epidemiological evidences suggest that quercetin may play an important role in the prevention of chronic diseases if regularly consumed

An Overview

1.5

in required quantity. Owing to several scientifically validated health benefits and its potential to serve as a preventive tool against several human health problems, there is a re-emergence of keen interest for its use as dietary supplement and functional food ingredients. In this chapter, the authors have overviewed the recent findings on bioactivities, molecular targets, and structural aspects of quercetin and its analogues.

1.2.9

Chapter 10

Chapter-10 provides an insight to the therapeutic efficacy of macro and small bioactive molecules in organ pathophysiology by Ghosh and Sil. Therapeutic efficacy of a variety of plant derived bioactive molecules, both macro and small, as complementary and alternative medicine is promising in the field of organ pathophysiology. Oxidative stress plays an important role in various forms of this pathophysiology. In this chapter, the authors have highlighted the beneficial role of bioactive molecules in various organ pathophysiologies and discussed the underlying mechanism of their protective action. A thorough and critical discussion on the subject has unearthed the multifunctional therapeutic applications of macro and small bioactive molecules of potential therapeutic promise in alternative systems of medicines.

1.2.10

Chapter 11

In Chapter-11, Yamaura and Ueno have presented an overview of anti-pruritic and anti-inflammatory herbal and natural products for the treatment of skin disease. Pruritus being associated with chronic skin diseases such as atopic dermatitis or allergic contact dermatitis is poorly controlled clinically, and has a major effect on the quality of life of patients. There is a growing interest in using herbs and natural products to maintain health or to alleviate chronic conditions. Hence, it becomes mandatory for the healthcare suppliers to provide necessary scientific information to the consumers on such products. The authors, thus, have aimed in their present chapter to focus on the effect of herbal and natural products for alleviating the symptoms of skin disease using a murine model of chronic allergic dermatitis.

1.2.11

Chapter 12

Weng and his group have contributed on the role of triterpenoid saponins in targeted anti-cancer therapies in Chapter-12. Saponins are found to be responsible for many therapeutic potentials; this group of compounds finds such useful therapeutic applications not only for their interesting cell permeability enhancing properties, but also for their promising cytotoxic and cytostatic abilities. In the recent past, there has been an advent of newer technological advances in the utilization of saponins to improve the efficacy of tumor-targeted therapies; saponins are used to increase the cytosolic delivery of targeted anti-tumor drugs or nanoparticles out of intracellular compartments such as endosomes and lysosomes. These approaches take into consideration the ability of triterpenoid saponins to act as transient intracellular lysogens. In this chapter, the authors have focused on the chemical classification of saponins and their usage as intracellular lysogens. In addition, structure–function relationships of saponins are also discussed in this regard. The overall

1.6

Natural Bioactive Molecules: Impacts and Prospects

discussion should invoke a lot of interests to the researchers working with saponins as cytotoxic agents and their uses in tumor therapy.

1.2.12

Chapter 13

In Chapter-13, Brahmachari has offered a thorough update on the recent development in the research of natural bioactive flavonoids covering a variety of over 500 new naturally occurring new bioactive flavonoids reported during the period 2005 to late 2012. Bio-flavonoids comprise a group of phenolic secondary plant metabolites that are widespread in nature. This important group of natural polyphenolics is well known for their multi-directional pharmacological potentials that include antioxidant, anticancer, antitumor, cytotoxic, enzyme inhibitory, anti-inflammatory, antimicrobial, anti-HIV, antidiabetic, anti-platelet aggregation, and neuroprotective activity. Hence, this group of natural polyphenols is now-a-days regarded as promising and significantly attractive natural substances to enrich the current therapy options against a variety of diseases. The present chapter focuses on the natural abundance of new bioactive flavonoids with varying structural skeletons reported during the period, and their significant biological activities including pharmacological efficacies, and absorption and metabolism. Due to promising multi-directional biological activities along with efficient pharmacological/therapeutic applications, bioflavonoids have drawn global attention and have already created a stir in the scientific community at a large. However, more systematic scientific works are demanding and the present-day workers would have to undertake systematic studies in more depth, so that beneficial effects of these important segments of natural components can find safe and effective uses for the betterment of mankind. This overview is anticipated to boost the progress of research on naturally occurring bio-flavonoids in this direction in days coming ahead.

1.3

CONCLUDING REMARKS

This introductory chapter summarizes each technical chapter of the book for which representation of facts and their discussions are exhaustive, authoritative and deeply informative. The readers will find interest in each of the chapters that practically cover wide area of bioactive natural product research. The reference encourages interdisciplinary work among chemists, pharmacologists, biologists, botanists, and agronomists with an interest in bioactive natural products. The present book with a handful of information relating to recent developments in the frontier research on bioactive natural products would surely find much utility to the scientists working in this filed.

2 Role of Natural Products as a Source of Alzheimer’s Drug Leads: An Update§ Goutam Brahmachari Laboratory of Natural Products and Organic Synthesis, Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India.

ABSTRACT Alzheimer’s disease (AD), a devastating neurodegenerative disorder mainly affecting elderly individuals, is the most common form of dementia, and is characterized by progressive and irreversible decline of memory and other cognitive functions. Because of the ageing of populations worldwide, this disorder is reaching epidemic proportions, with a large human, social, and economic burden. It is estimated that 5.4 million people in the United States and 35.6 million people worldwide suffer from AD, and the number is expected to increase to 13.5 million in the United States and 115 million worldwide by 2050. Effective treatments are thus greatly needed. Unfortunately, even after several decades of research efforts, it is still unknown exactly what causes the disease. There are currently only five approved treatments for Alzheimer’s disease, and none of these is a cure. These drugs for AD target cholinergic and glutamatergic transmission thereby improving cognitive symptoms in some patients; however, these agents are not disease modifying, nor do they slow disease progression. While the 1970s heralded a “war on cancer” and the 1980s and 1990s witnessed a monumental effort to vanquish the death sentence of AIDS with novel therapeutics targeting multiple aspects of the viral machinery, one of the greatest challenges facing medicinal chemists in the 21st century is the discovery and development of compounds for the prevention, treatment, and diagnosis of Alzheimer’s. Several natural products have provided useful chemotypes for the present treatment of Alzheimer’s and other neurodegenerative diseases. Some natural compounds have already reached widespread clinical use as well. The majority of the compounds examined to date with a direct relevance to AD are primarily from plants, with comparatively few molecules *Correspondence: [email protected]; [email protected] § This chapter is dedicated to Late Santosh K. Brahmachari — In memory to my beloved father

2.2

Natural Bioactive Molecules: Impacts and Prospects

derived from marine and microbial sources. The notable successes achieved so far have come from plant-based acetylcholinestrase discovery programs, providing two of the five currently approved drugs for the treatment of AD. Natural molecules can also be subjected to ‘fine-tuning’ by chemical derivatisation and synthesis of analogues for better pharmacokinetics and efficacy. Natural products have always been and continue to be the important medical reservoirs, with considerable number of modern FDA-approved medications from natural sources. In the field of Alzheimer’s, several conclusions on the possibility of natural product leads are supported from the experimental outcomes; hence, natural products have emerged as promising hope in the drug discovery programs in Alzheimer’s. More emphasis should be given in finding clinically useful new chemical entities of natural origin in combination with evaluating safety and efficacy of such pure compounds or crude extracts as a whole. The main theme of this chapter is to highlight the impact and prospect of bioactive natural products in providing useful leads to the drug discovery and development processes against this devastating disease. A comprehensive update on the role of natural products against Alzheimer’s is presented herein. An increased collaboration between pharmaceutical companies, basic researchers, and clinical researchers has the potential to bring us closer to developing an optimum treatment for Alzheimer’s disease. We are looking forward for a better medicinal scenario in controlling Alzheimer’s in days coming ahead, and we are hopeful indeed! Keywords: Alzheimer’s disease; Current scenario; Bioactive natural products; Pharmaceutical leads; Drug discovery processes; Clinical trials

2.1 INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia that mainly affects elderly individuals. AD is characterized by progressive and irreversible decline of memory and other cognitive functions including language, judgment, reasoning along with progressive loss of physical functioning and associated neuropsychiatric symptoms, which become severe enough to impede social or occupational functioning (DSM-IV, 1994; Brahmachari, 2011). In 1906 Dr. Alois Alzheimer, a German psychiatrist and neuropathologist, described the first published case of “presenile dementia” based on the observation of amyloid plaques, neurofibrillary tangles and vascular anomalies during the autopsy of his patient, Mrs. Auguste Deter who had died 7 months earlier with severe cognitive defects (Alzheimer, 1907a,b) and the disease became known as Alzheimer’s disease (AD) or simply Alzheimer’s after the name of the inventor (Berchtold and Cotman, 1998). One of the greatest challenges facing medicinal chemists in the 21st century is the discovery and development of compounds for the prevention, treatment, and diagnosis of Alzheimer’s disease (AD). It is estimated that 5.4 million people in the United States (Alzheimer’s Association, 2012) and 35.6 million people worldwide (World Alzheimer’s Report 2010) suffer from AD, and the number is expected to increase to 13.5 million (Alzheimer’s Association, 2012) in the United States and 115 million (World Alzheimer’s Report 2010) worldwide by 2050 because of the aging

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.3

population. It is also a matter of great concern that 1,000 new cases of AD are reported daily throughout the United States (Hebert et al., 2004). Although other major causes of death have been decreasing, deaths attributable to AD have been rising dramatically. Between 2000 and 2008, while deaths from HIV decreased 29%, deaths from stroke decreased 20%, deaths from heart disease decreased 13%, and deaths from several cancers also declined significantly, during the same time period there was a 66% increase in deaths directly related to AD (Alzheimer’s Disease International, 2009; Alzheimer’s Association, 2010; Mangialasche et al., 2010; visit also www.alz. org; www.alzfdn.org; www.phrma.org). In fact, the overall case number in developed countries is estimated to increase by 100% between 2001 and 2040, but by more than 300% in India and China. Recent estimates indicate that nearly 5 million additional new dementia cases are diagnosed per year. Alzheimer’s is predicted to affect 1 in 85 people globally by 2050 (Brookmeyer et al., 2007). The 2003 World Health Report estimates that dementing diseases contribute a greater overall burden of disability than cardiovascular disease, stroke, and cancer. Since Alzheimer’s disease mainly affects elderly individuals, and, because of the ageing of populations worldwide, this disorder is reaching epidemic proportions, with a large human, social, and economic burden (Wimo et al., 2010). In a statistic, the total cost paid for AD care in the United States was approximately $200 billion in 2012 with the majority of that money coming from Medicare and Medicaid. By 2050, that cost is expected to rise to $1 trillion (Alzheimer’s Association, 2012). These statistics highlight the significant opportunity for social and economic impact that may be provided by an effective AD treatment. Effective treatments are thus greatly needed. Unfortunately, even after several decades of research efforts, it is still unknown exactly what causes the disease. There are currently only five approved treatments for Alzheimer’s disease, and none of these is a cure. These drugs for AD target cholinergic and glutamatergic transmission thereby improving cognitive symptoms in some patients; however, these agents are not disease modifying, nor do they slow disease progression. While the 1970s heralded a “war on cancer” and the 1980s and 1990s witnessed a monumental effort to vanquish the death sentence of AIDs with novel therapeutics targeting multiple aspects of the viral machinery, one of the greatest challenges facing medicinal chemists in the 21st century is the discovery and development of compounds for the prevention, treatment, and diagnosis of Alzheimer’s disease (AD). The main theme of this chapter is to highlight the impact and prospect of bioactive natural products in providing useful leads to the drug discovery and development processes against this devastating disease. A comprehensive update on the role of natural products against Alzheimer’s is presented herein.

2.2

NEUROPATHOLOGY OF ALZHEIMER’S DISEASE (AD)

Despite decades of research and many significant advances, the precise neuropathology of AD is still not completely understood; however, amyloid plaques and neurofibrillary tangles are considered as the two primary pathological hallmarks of the disease. Histopathological studies of the AD brain revealed dramatic ultra-structural changes triggered by two classical lesions, the senile plaques, mainly composed of amyloid-b (Ab) peptides, and the neurofibrillary tangles, composed of hyperphosphorylated tau proteins (Davies, 2000; Selkoe, 2001). Amyloid plaques

2.4

Natural Bioactive Molecules: Impacts and Prospects

are insoluble, dense cores of 5-10 nm fibrils containing aggregates of amyloid precursor protein (APP) fragments that are primarily composed 42-amino acid b-amyloid peptide (Ab42) as found in AD patients (Burdick et al., 1992; Roher et al., 1993; Iwatsubo et al., 1994). On the other hand, neurofibrillary tangles contain aggregates of phosphorylated tau, a microtubule-associated protein. Some hypotheses of Alzheimer’s pathology suggest that the aggregation of hyperphosphorylated tau protein causes the degeneration of the microtubule network required for neuronal survival (Mudher and Lovestone, 2002). Ultimately, the insoluble neurofibrillary tangle of tau protein is left as a “tombstone” for dead neurons. Although neurofibrillary tangles can occur independently, and cause neuronal death in frontotemporal dementia (Goedert and Spillantini, 2000), the presence of both lesions in the neocortex is essential to the diagnosis of AD. The pathogenesis of the disease is complex and is driven by both environmental and genetic factors. The molecular identification and characterization of different genes associated with familial AD has provided strong support to the so-called amyloid cascade hypothesis as a causative event in the pathogenesis of AD (Hardy and Selkoe, 2002). This hypothesis states that Ab generated from deregulated proteolysis of the amyloid precursor protein (APP) undergoes accelerated Ab oligomerization, fibril formation, and amyloid deposition in a process that initiates the AD pathology (Hardy and Selkoe, 2002). In the past ten years, the large majority of the pharmacological research on AD has focused on understanding how Ab is generated from APP via b- and g-secretase cleavages, with the goal of designing specific inhibitors that will block Ab production and the associated pathology. Besides, newer approaches aimed at better understanding of various molecular pathways involved in Ab clearance have been gaining considerable attention over the last several years (Tanzi et al., 2004; Vingtdeux et al., 2008). The inflammatory response to the deposition of these amyloid plaques and neurofibrillary tangles is thought to play an important role in producing the “halo” of degenerated neurons, reactive astrocytes and activated microglia around these protein deposits that is observed in microscopic sections (Di Patre et al., 1999). Over time there is gross atrophy of affected regions, including the temporal, parietal, frontal lobes (in particular the ventral forebrain), and the cingulated gyrus. Eventually, neuronal loss leads to global neurotransmitter deficiencies, specifically in norepinephrine and acetylcholine. One of the earliest molecular observations in AD was the finding of a deficiency of overall acetylcholine and decreased activity of enzymes involved in the synthesis and degradation of this neurotransmitter in AD autopsy and biopsy tissue (Collen, 2001; Turner et al., 2004). During the course of the disease, plaques and tangles develop within the structure of the brain. This causes brain cells to die. Current models suggest a prodromal period of amyloid accumulation, followed by a progression of tau pathology, inflammation, and neurodegeneration that tracks cognitive decline. Oxidative damage to proteins, lipids, and DNA in the brains of AD patients likely accompanies the widespread inflammation.

2.3

CURRENT TREATMENTS OF ALZHEIMER’S DISEASE

Early neuropathology of AD is characterized by the loss of basal forebrain cholinergic neurons and loss of cholinergic neurotransmission. As Alzheimer’s progresses, brain cells die and connections

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.5

among cells are lost, causing cognitive symptoms to worsen. While there is no cure for AD or no means to stop the damage Alzheimer’s causes to brain cells, currently there are only five prescription drugs (Figure 1) approved by the U.S. Food and Drug Administration (FDA). These drugs are used for its symptomatic treatments that may help lessen or stabilize cognitive symptoms such as memory loss and confusion, for a limited time by affecting certain chemicals involved in carrying messages among the brain’s nerve cells. Four of these drugs are acetylcholinesterase (AChE) inhibitors, while one modulates N-methyl-D-aspartic acid (NMDA) receptors. Donepezil (Aricept®), galantamine (Razadyne®), rivastigmine (Exelon®) and tacrine (Cognex®) are the four cholinesterase inhibitors prescribed to treat symptoms related to memory, thinking, language, judgment and other thought processes. Memantine (Namenda®), the first Alzheimer drug of the NMDA receptor antagonist-type approved in the United States, is prescribed to improve memory, attention, reason, language and the ability to perform simple tasks; it is used to treat moderate to severe Alzheimer’s. However, these drugs have some adverse side effects that commonly include nausea, vomiting, loss of appetite, headache, constipation and dizziness.

Fig. 1

Five FDA-approved Drugs for the Treatment of Alzheimer’s Disease (AD)

2.6

Natural Bioactive Molecules: Impacts and Prospects

2.4

NATURAL PRODUCTS: PROMISING HOPE AGAINST ALZHEIMER’S DISEASE (AD)

As already stated Alzheimer’s disease is becoming a major challenge worldwide with a tremendous social and economic burden. While the 1970s heralded a “war on cancer” and the 1980s and 1990s witnessed a monumental effort to vanquish the death sentence of AIDs with novel therapeutics targeting multiple aspects of the viral machinery, one of the greatest challenges facing medicinal chemists in the 21st century is the discovery and development of compounds for the prevention, treatment, and diagnosis of the most devastating Alzheimer’s disease (AD). The need is great to identify disease-modifying therapies that slow or stop the neurodegenerative process (Mangialasche et al., 2010; Hubbs et al., 2012). Natural products have been the source of most of the active ingredients of medicines (Vickers and Zollman, 1999). The search for new pharmacologically active agents obtained by screening natural sources such as plants, marines and microbes has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases (Brahmachari, 2010). Natural flora and fauna have always been and continue to be the important medical reservoirs, with considerable number of modern FDA-approved medications from natural sources (Shu, 1998; Newman et al., 2003; Harvey, 2008; Brahmachari, 2011a). In the filed of Alzheimer’s, several conclusions on the possibility of natural product leads have already been supported by the experimental outcomes; the majority of the compounds examined to date with a direct relevance to AD are primarily from plants, with comparatively few molecules derived from marine and microbial sources. Still to date, the greatest successes have been resulted from plant-based AChE discovery programs, which have provided two of the five currently approved drugs for the treatment of AD. Hence, multiple factors are likely the driving force for increased interest in natural supplementation and for use in Alzheimer’s and other neurodegenerative diseases; clearly the lack of a safe, effective, proven therapy is a primary driver for the search for alternatives (Mangialasche et al., 2010; Hubbs et al., 2012). So many raw plant extracts/herbal formulations find immense uses as natural remedies in the treatment of AD and other neurodegenerative diseases (Quid, 1999; Perry et al., 1999; Howes et al., 2003; Howes and Houghton, 2003; Houghton and Howes, 2005; Mukherjee et al., 2007; Brahmachari, 2011). Essentially all traditional natural medical systems including Chinese, Indian, Native American, and medieval European have had various “brain tonics” and memory enhancers (Mehta et al., 1991; Kanowski et al., 1996; Singh and Dhawan, 1997; Schliebs et al., 1997; Jenner et al., 2000; Stough et al., 2001; Perry et al., 2002; Tildesley, 2003; Jamshidi et al., 2003; Ballard et al., 2008; Wang et al., 2008). These include “Ashwagandha” (Withania somnifera; Solanaceae) and Brahmi (Bacopa monnieri L. Pennell; Scophulariaceae) mentioned in Indian Ayurveda as memory enhancers, the common ‘Sage’ plants (Salvia species; Labiatae) described in Roman texts as being “good for the memory”, and Gingko biloba (Ginkgoaceae) discussed in Chinese literature as a possible remedy for memory loss as early as 2800 BC. Indian turmeric (Curcuma longa; Zingiberaceae), which contains an antioxidant and anti-inflammatory compound called curcumin, is found to be very effective in the treatment of AD. Vegetables such as pumpkin, carrot and other foods and spices like zinger, sesame and sunflower seeds that contain various chemical agents

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.7

find very useful for enhancing the function of the brain. Consumption of blueberries/grapes and pomegranate juice has recently been proven to have beneficial effect in AD. Food supplements of vitamin B6 & B12, folic acid, vitamin E, vitamin C and co-enzyme Q10 also have been found to exert beneficial effect in AD patients. Numerous literatures are available on the chemical, pharmacological and clinical studies of natural substances used in the treatment of AD and related diseases, including their medicinal efficacy, safety, and other relevant matters. Good review articles have also been published detailing on the naturally occurring compounds of varying skeletons that showed potential efficacy against AD and other neurodegenerative disorders (Houghton and Howes, 2005; Viegas et al., 2005; Williams et al., 2011a). More than 350 compounds of natural origin are reported so far to exert anti-AD activities; these compounds are tabulated in Table 1 and their respective chemical structures are presented in Figure 2. A few promising natural products that have aroused our hope in controlling Alzheimer’s are presented below:

2.4.1

Physostigmine (1)

The ‘classic’ cholinesterase inhibitor is physostigmine (also called eserine; 1), an alkaloid with a pyrroloindole skeleton first isolated from the Calabar Bean, the seeds of Physostigma venensosum Balf. (Leguminosae) in 1864. The chemical entity was approved by regulatory agencies in Europe and by US Food and Drug Administration (FDA) as an agent to reverse the anticholinergic effects of clinical or toxic dosages of drugs. It is a potent, short-acting and reversible inhibitor of acetylcholinestrase (AChE), and has been shown to improve cognitive functions in vivo and in both normal and AD patients (Julian et al., 1935; Sitaram et al., 1978; McCaleb, 1990; Kamal et al., 2000; Howes and Houghton, 2009). Physostigmine is a carbamate derivative that acts as a competitive substrate for acetylcholine, allowing it to accumulate in the synaptic clefts and to overcome the blockade of muscarinic receptors by the anticholinergic agents. Being a tertiary amine, it is uncharged, lipophilic, and easily crosses the blood-brain barrier (Rulnack, 1973). This action allows physostigmine to reverse toxic CNS effects, whereas other carbamate drugs that are charged quaternary amines (such as neostigmine and pyridostigmine) will only reverse peripheral signs and symptoms (Stilson et al., 2001). Physostigmine’s ability to reverse central effects led to its trade name of Antiliriurn®, since it can reverse the delirium associated with anticholinergic toxicity. Several subsequent clinical trials with small numbers of patients have shown that physostigmine can improve memory, but the results have not been consistent across all the studies. Moreover, a limiting factor has been a high incidence of adverse effects, including nausea, vomiting and diarrhoea. The development of physostigmine has been hindered by its extensive first-pass metabolism and short plasma half-life (approximately 30 minutes), and such short half-life remains a serious disadvantage and requires complex forms of administration (Coelho Filho and Birks, 2008). That is why the drug is almost no longer in use for treating AD; the newer drugs proved to be more effective with a lower side effect profile (Orhan et al., 2009). To improve the pharmacokinetic profile and efficacy, there is a considerable history of the synthesis of analogues of physostigmine — although numerous synthetic derivatives have been developed,

Evodia rutaecarpa T. australis T. australis T. australis Chimarrhis turbinata C. turbinata

Galanthus spp. Narcissus Spp. Lycoris radiata

Voacangine (11)

Voacangine hydroxyindolenine (12)

Rupicoline (13)

Turbinatine (14)

Desoxycordifoline (15)

Galantamine (16)

D. pulchellum D. gangeticum

Indole alkaloid (8)

Tabernaemontana australis

D. pulchellum D. gangeticum

Indole alkaloid (7)

Dehydroevodiamine (9)

D. pulchellum D. gangeticum

Indole alkaloid (6)

Coronaridine (10)

Desmodium pulchellum D. gangeticum

Physosstigma venenosum

Indole alkaloid (5)

Neostigmine (2) Pyridostigmine (3) Rivastigmine (4)

Physostigmine (1)

Alkaloids

Source

Amaryllidaceae

Rubiaceae

Rubiaceae

Apocynaceae

Apocynaceae

Apocynaceae

Apocynaceae

Rutaceae

Fabaceae/Papilionaceae

Fabaceae/Papilionaceae

Fabaceae/Papilionaceae

Fabaceae/Papilionaceae

Fabaceae/Papilionaceae

Family

AChE (1.07)

AChE (1.0)

AChE (0.1)

AChE

AChE

AChE

AChE

AChE (37.8); GSK-3

AChE

AChE

AChE

AChE

AChE (0.25)

Mode of action (IC50 in μM)

Anti-Alzheimer’s compounds, classifications, natural sources and mode of action

Compound (Structure No.)

Table 1

Heinrich and Teoh 2004; Lopez et al., 2002; Elgorashi et al., 2004; Houghton et al., 2006; Howes and Houghton 2009; Geissler et al., 2010; Berkov et al., 2007; Labrana et al., 2002; Rhee et al., 2004

Blackstock et al, 1972; Brown et al., 1978; Cardoso et al., 2003,2004; Elgorashi et al, 2004

Cardoso et al., 2003,2004; Elgorashi et al, 2004

Andrade et al., 2005

Andrade et al., 2005

Andrade et al., 2005

Andrade et al., 2005

Park et al., 1996; Peng et al., 2007

Ghosal et al., 1972; Houghton et al., 2006

Ghosal et al., 1972; Houghton et al., 2006

Ghosal et al., 1972; Houghton et al., 2006

Mohammed et al., 2000; Julian and Pikl, 1935; Kamal et al., 2000; Howes and Houghton, 2009; Houghton et al., 2006

Reference

2.8 Natural Bioactive Molecules: Impacts and Prospects

Crinum spp. Crinum spp. Huperzia serrata

Semi-synthetic derivative of huperzine A H. serrata

Lycopodium sieboldii Berberis spp.

Crinamine (25)

Haemanthamine (26)

Huperzine A (27)

ZT-1 (27a)

Huperzine B (28)

Sieboldine A (29)

Berberine (30)

Chelidonium majus

Crinum spp.

Hamayne (24)

Chelidonine (33)

Narcissus spp.

Assoanine (22)

Ungiminorine (23)

Corydalis speciosa

Narcissus spp.

1-O-acetyllycorine (21)

Argemone mexicana

Narcissus spp. Narcissus spp.

Lycorine (20)

Sanguinarine (32)

Amaryllidaceae

Narcissus spp.

Palmatine (31)

Amaryllidaceae

Narcissus spp.

11-Hydroxygalantamine (18)

Epinorgalantamine (19)

Papaveraceae

Papaveraceae

Papaveraceae

Berberidaceae

Lycopodiaceae

Huperziaceae

―

Huperziaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Amaryllidaceae

Narcissus spp.

Sanguinine (17)

―

Family

Synthetic derivative of galantamine

Source

9-Dehydrogalantaminium bromide (16a)

Compound (Structure No.)

AChE

AChE (0.23)

AChE

AChE (0.23)

AChE (2.0)

AChE

AChE

AChE (0.023)

AChE

AChE

AChE (250)

AChE (86 )

AChE (3.87)

AChE (0.96)

AChE

AChE (9.01)

AChE (1.61)

AChE (0.1)

AChE

Mode of action (IC50 in μM)

Kuznetsova et al., 2002

Santos and Adkilen, 1932; Kuznetsova et al., 2002

Kim et al., 2004

Kuznetsova et al., 2002; Peng et al., 1997

Hirasawa et al., 2003; Canham et al., 2010

Skolnick 1997; Wang et al., 1986; Bai et al., 2000; Kozikowski et al., 1996; Howes and Houghton 2009; He et al., 2007

Ma and Gang, 2004; Ha et al., 2011

Skolnick 1997; Wang et al., 1986; Bai et al., 2000; Kozikowski et al., 1996; Howes and Houghton 2009; Tang et al., 2005; Wang et al., 2006; Ma et al., 2007; Wang et al., 2009

Park et al., 1996; Andrade et al., 2005

Park et al., 1996; Andrade et al., 2005

Park et al., 1996; Andrade et al., 2005

Ingkaninan et al., 2000

Lopez et al., 2002; Elgorashi et al., 2004

Lopez et al., 2002; Elgorashi et al., 2004

Lopez et al., 2002; Elgorashi et al., 2004

Lopez et al., 2002; Elgorashi et al., 2004

Lopez et al., 2002; Elgorashi et al., 2004

Lopez et al., 2002; Elgorashi et al., 2004; Houghton et al., 2004

Mary et al., 1998; Lamirault et al., 2003

Reference

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.9

Buxaceae Buxaceae

Cocculus pendulus C. pendulus C. pendulus C. pendulus C. pendulus Chondodendron tomentosum Solanum sp. Solanum sp. Buxus nyrcana B. nyrcana B. nyrcana B. nyrcana Sarcococca coriacea S. coriacea S. coriacea S. coriacea S. coriacea S. saligna S. saligna S. saligna S. saligna

Alkaloid (36)

Alkaloid (37)

Alkaloid (38)

Alkaloid (39)

Alkaloid (40)

Tubocurarine (41)

α-Solanine (42)

α-Chaconine (43)

Buxakashmiramine (44)

N-Acyl analogue of buxahyrcaninine (45)

N-Acyl analogue of buxahyrcaninine (46)

N-Acyl analogue of buxahyrcaninine (47)

Umafrine C (48)

N-Methylfuntumine (49)

Saracocine (50)

Sarcodine (51)

Isosarcodine (52)

Salignenamide-A (53)

Salignenamide-C (54)

Salignenamide-D (55)

Salignenamide-E (56)

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Buxaceae

Solanaceae

Solanaceae

Menispermaceae

Menispermaceae

Menispermaceae

Menispermaceae

Menispermaceae

Menispermaceae

Papaveraceae

Corydalis spp.

Papaveraceae

Corydalis spp.

Family

Protopine (35)

Source

Corynoline (34)

Compound (Structure No.)

AChE, BChE

AChE, BChE

AChE, BChE

AChE

AChE (10.31), BChE

AChE (49.77), BChE

AChE (20), BChE

AChE (97.6)

AChE (45.8)

AChE (4000)

AChE (630)

AChE (670)

AChE

AChE (146±5.8)

AChE

AChE

GSK (10)

GSK (10)

Anti Ags & clearance

Anti Ags & clearance

Mode of action (IC50 in μM)

Miyazawa et al., 2005

Miyazawa et al., 2005

Miyazawa et al., 2005

Grundy and Still 1985

Miyazawa et al., 1997

Ryan and Byrne 1988; Gracza 1985

Perry et al., 2000

Perry et al., 2000

Perry et al., 2000; Kivrak et al., 2009

Perry et al., 2000; Kivrak et al., 2009

Nukoolkarn et al., 2008

Song et al., 2008

Briggs et al., 1997; Mayer et al., 1997, 1998, 2010; Kem et al., 2000, 2004

Kem et al., 1971,1997; Wheeler et al., 1981

Cimino et al., 1982; Meijer et al., 2000

Sakai et al., 1986; 1987; Hamann et al., 2007

Pappolla et al., 1998

Ono et al., 2002; Nordberg et al., 2002; Oddo et al., 2005

Reference

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.15

S. mittiorrhiza Otostegia limbata Otostegia limbata Otostegia limbata Gutierrezia microcephala Withania somnifera Withania somnifera Cynanchum atratum Origantum majorana Vaccinium oldhami Parthenium argentatum Parthenium argentatum

Furano derivative (165)

Diterpenoid (166)

Diterpenoid (167)

Diterpenoid (168)

Bacchabolivic acid (169)

Withaferin A (170)

Terpenoid compound (171)

Cynatroside B (172)

Ursolic acid (173)

Taraxerol (174)

Argentatin A (175)

Argentatin B (176)

Peltophorum dasyrachis

S. mittiorrhiza

Furano derivative (164)

(+)-(S)-ar-tumerone (179)

S. mittiorrhiza

Cryptotanshinone (163)

Parthenium argentatum

Salvia mittiorrhiza

Dihydrotanshinone (162)

Lycopodium clavatum

Plants

(−)-Fenchone (161)

α-Onocerin (178)

Plants

Toosendanin (177)

Plants

(+)-Fenchone (160)

Source

(+)-Fenchol (159)

Compound (Structure No.)

Caesalpiniaceae

Lycopodiaceae

Compositae/Asteraceae

Compositae/Asteraceae

Compositae/Asteraceae

Vacciniaceae

Labiatae

Asclepiadaceae

Solanaceae

Solanaceae

Asteraceae

Labiatae

Labiatae

Labiatae

Labiatae/Lamiaceae

Labiatae/Lamiaceae

Labiatae/Lamiaceae

Labiatae/Lamiaceae

―

―

―

Family

AChE (191)

AChE (5.2)

AChE

AChE

AChE

AChE (79)

AChE (75), Reduces Aβ levels

AChE

AChE, BChE (62.5)

AChE (8.4), BChE (125)

AChE

AChE (103.7)

AChE (47.2)

AChE (38.5)

AChE

AChE

AChE (7.0), Antioxidant, Reduces Aβ levels

AChE (1.0)

AChE (>1000)

AChE (>1000)

AChE (>1000)

Mode of action (IC50 in μM)

Fujiwara et al., 2010

Orhan et al., 2003

Cespedes et al., 2001

Cespedes et al., 2001

Cespedes et al., 2001

Lee et al., 2004; Kumar et al., 2007; Lee et al., 2004

Chung et al., 2001; Chung et al., 2001; Heo et al., 2002; Wilkinson et al., 2011

Lee et al., 2005

Bhattacharya et al., 1995

Bhattacharya et al., 1995

Calderon et al., 2001

Ahmad et al., 2005

Ahmad et al., 2005

Ahmad et al., 2005

Houghton et al., 2006

Houghton et al., 2006

Ren et al., 2004; Ren et al., 2004; Kim et al., 2007; Wong et al., 2010; Yu et al., 2007; Kim et al., 2002; Ng et al., 2000; Park et al., 2007; Mei et al., 2009; Mei et al., 2010; Mei et al., 2012

Ren et al., 2004

Miyazawa et al., 2005

Miyazawa et al., 2005

Miyazawa et al., 2005

Reference

2.16 Natural Bioactive Molecules: Impacts and Prospects

Umbelliferae ―

Citrus essential oil Leontopodium alpinum Detarium microcarpum Isodon wightii Acacia nilotica Anemarrhena asphodeloides Aralia cordata Aralia cordata Aralia cordata Withania somnifera Centella asiatica One of the animal forms of vitamin A One of the forms of vitamin A A metabolite of vitamin A Mainly in carrots (Daucus carota subsp. sativus) Foods and dietary supplements Ircina variabilis Panax ginseng

(S)-Perillyl alcohol (182)

Silphiperfolene acetate (183)

(5α,8α)-2-Oxokolavenic acid (184)

Melissoidesin (185)

Niloticane (186)

Timosapoprin AIII (187)

7-Oxo-ent-Pimarane-8(14),15diene-19-oic acid (188)

16-Hydroxy-17-isovaleroyloxy-entKauran-19-oic acid (189)

17-Hydroxy-ent-Kaur-15-en-19-oic acid (190)

Withanolide A (191)

Asiatic acid (192)

Retinol (193)

Retinal (194)

Retinoic acid (195)

β-Carotene (196)

Co-enzyme Q10 (197)

Palinurin (198)

Ginsenoside Rg1 (199)

Araliaceae

Irciniidae

―

Apiaceae

―

―

Solanaceae

Araliaceae

Araliaceae

Araliaceae

Asparagaceae

Leguminosae

Lamiaceae

Fabaceae/ Caesalpinioideae

Asteraceae

Rutaceae

Rutaceae

Citrus essential oil

Caesalpiniaceae

Peltophorum dasyrachis

Family

(R)-Limonene (181)

Source

(+)-(S)-Dihydro-ar-tumerone (180)

Compound (Structure No.)

Reduces Aβ levels

GSK (4.5)

Anti Agg & Clearance

Anti Agg & Clearance

Anti Agg & Clearance

Anti Agg & Clearance

Anti Agg & Clearance

Secretase inhibitor

beta-Secretase inhibitor (BACE1)

beta-Secretase inhibitor (23.4)

Secretase inhibitor (18.6)

Secretase inhibitor (24.1)

AChE

AChE

AChE (215)

AChE

AChE

AChE

AChE

AChE (82)

Mode of action (IC50 in μM)

Fang et al., 2012; Shi et al., 2010; Liang et al., 2010; Chen et al., 2012

Alfano et al., 1979; Gordillo et al., 2004

Ono et al., 2005

Ono et al., 2004

Ono et al., 2004

Ono et al., 2004

Ono et al., 2004

Patil et al., 2010

Patil et al., 2010

Jung et al., 2009a

Jung et al., 2009a

Jung et al., 2009a

Lee et al., 2009

Eldeen et al., 2010

Thirugnanasampandan et al., 2008

Cavin et al., 2006

Hornick et al., 2008

Zhou et al., 2009

Zhou et al., 2009

Fujiwara et al., 2010

Reference

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.17

P. ginseng P. ginseng P. ginseng Ginkgo biloba G. biloba

G. biloba G. biloba Cannabis sativa C. sativa

C. sativa C. sativa C. sativa Cornus officinalis C. officinalis

Aralia cordata Polygala tenuifolia

Ginsenoside Re (201)

Ginsenoside Rb1 (202)

Ginkgolide A (203)

Ginkgolide B (204)

Ginkgolide J (205)

Bilobalide (206)

∆9-Tetrahydrocannabinol [THC] (207)

Cannabidiol (208)

HU-210 (209)

WIN-55,212-2 (210)

JWH-133 (211)

Morroniside (212)

Loganin (213)

Oleanolic acid (214)

Tenuifolin (215)

Source

Ginsenoside Rg3 (200)

Compound (Structure No.)

Polygalaceae

Araliaceae

Cannaceae

Cannaceae

Cannaceae

Cannaceae

Cannaceae

Cannaceae

Cannaceae

Ginkgoaceae

Ginkgoaceae

Ginkgoaceae

Ginkgoaceae

Araliaceae

Araliaceae

Araliaceae

Family

Reduces Aβ levels, AChE

Neuroprotective activity

Neuroprotective activity, AChE inhibitory

Neuroprotective activity

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Antioxidant, Reduces Aβ levels

AChE, Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Reduces Aβ levels

Mode of action (IC50 in μM)

Lv et al., 2009; Zhang et al., 2008

Cho et al., 2009; Cho et al., 2009

Li et al., 2005; Zhao et al., 2010; Xu et al., 2009; Oh et al., 2009; Lee et al., 2009

Li et al., 2005; Zhao et al., 2010

Moreno et al., 2011; Ramirez et al., 2005; Moreno et al., 2012

Moreno et al., 2011; Ramirez et al., 2005; Moreno et al., 2012

Moreno et al., 2011; Ramirez et al., 2005; Moreno et al., 2012

Hampson et al., 1998; Iuvone et al., 2004; Esposito et al., 2006a,b; Esposito et al., 2007; Moreno et al., 2011;

Carlini 2004; Eubanks et al., 2006; Walther et al., 2006

Tchantchoyu et al., 2009; Ahlemeyer et al., 1999; Defeudis 2002

Vitolo et al., 2009

Bate et al., 2008; Bate et al., 2004; Xiao et al., 2010; Hu et al., 2011; Lee et al., 2004

Bate et al., 2008; Bate et al., 2004; Wu et al., 2006

Wang et al., 2011; Jung et al., 2001

Liang et al., 2010

Chen et al., 2006; Yang et al., 2009

Reference

2.18 Natural Bioactive Molecules: Impacts and Prospects

A. terreus Penicillium sp. FO-4259-ll A. terreus Penicillium sp. FO-4259-ll

Territrem B (226)

Territrem C (227)

N. galligena

A. terreus Penicillium sp. FO-4259-ll

Territrem A (225)

Ilicicolin F (232)

A. terreus Penicillium sp. FO-4259-ll

Arisugacin D (224)

N. galligena

A. terreus Penicillium sp. FO-4259-ll

Arisugacin C (223)

N. galligena

A. terreus

Arisugacin B (222)

Ilicicolin E (231)

A. terreus

Arisugacin A (221)

Ilicicolin C (230)

A. terreus

Terreulactone D (220)

Nectria galligena

A. terreus

Terreulactone C (219)

N. galligena

A. terreus

Terreulactone B (218)

Colletochlorin B (228)

A. terreus

Terreulactone A (217)

Colletorin B (229)

Aspergillus terreus

Source

Isoterreulactone A (216)

Meroterpenoids

Compound (Structure No.)

Nectriaceae

Nectriaceae

Nectriaceae

Nectriaceae

Nectriaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Trichocomaceae

Family

AChE

AChE

AChE

AChE

AChE

AChE (6.8), BChE (>26)

AChE (7.6), BChE (>20)

AChE (7.6), BChE (>20)

AChE (3.5)

AChE (2.5)

AChE (25.8), BChE (>51600)

AChE (1.0) , BChE (>21000)

AChE (0.42) , BChE (>200)

AChE (0.06) , BChE (>200)

AChE (0.09) , BChE (>200)

AChE (0.23) , BChE (>200)

AChE(2.5), BChE(>500)

Mode of action (IC50 in μM)

Gutierrez et al., 2005

Gutierrez et al., 2005

Gutierrez et al., 2005

Gutierrez et al., 2005

Gutierrez et al., 2005

Otoguro et al., 1997,2000; Omura et al., 1995; Cho et al., 2003; Kuno et al., 1996

Otoguro et al., 1997,2000; Omura et al., 1995; Cho et al., 2003; Kuno et al., 1996

Kuno et al., 1996;Otoguro et al., 2000

Otoguro et al., 1997,2000; Omura et al., 1995; Cho et al., 2003

Otoguro et al., 1997,2000; Omura et al., 1995; Cho et al., 2003

Kuno et al., 1996; Otoguro et al., 1997; Omura et al., 1995; Cho et al., 2003

Kuno et al., 1996; Otoguro et al., 1997; Omura et al., 1995; Cho et al., 2003

Kim et al., 2003; Yoo et al., 2005

Kim et al., 2003; Yoo et al., 2005

Kim et al., 2003; Yoo et al., 2005

Kim et al., 2003; Yoo et al., 2005

Kim et al., 2003; Yoo et al., 2005

Reference

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.19

―

Homalomena occulta H. occulta

H. occulta H. occulta Xestospongia spp. Xestospongia spp. Amycolatopsis rifamycinica Streptomyces spp. A semisynthetic tetracycline derivative A semisynthetic tetracycline derivative

2-[(Z)-Heptadec-11-enyl]-6-hydroxybenzoic acid (236a)

2-[(6Z,9Z,12Z)-Heptadeca-6,9,12trienyl]-6-hydroxybenzoic acid (236b)

2-[(9Z,12Z)-Heptadeca-9,12-dienyl]6-hydroxybenzoic acid (236c)

2-Hydroxy-6-(12-phenyldodecyl) benzoic acid (236d)

Xestosaprol F (237)

Xestosaprol H (238)

Rifampicin (239)

Tetracycline (240)

Doxycycline (241)

Minocycline (242)

Numerous plant sources Buddleja davidii Buddleja davidii

5,7,4¢-Trihydroxy-3,3¢dimethoxyflavone (243)

Linarin (244)

Tilianin (245)

Flavonoids

―

―

Hispidin derivative (235)

Buddlejaceae

Buddlejaceae

―

Streptomycetaceae

Pseudonocardiaceae

Petrosiidae

Petrosiidae

Araceae

Araceae

Araceae

Araceae

―

―

―

Hispidin derivative (234)

Family Hymenochaetaceae

Phellinus pomaceus

Source

Hispidin (233)

Compound (Structure No.)

AChE

AChE

AChE

Anti Agg & Clearance

Anti Agg & Clearance

Anti Agg & Clearance

Anti Agg & Clearance

Secretase inhibitor

Secretase inhibitor

Secretase inhibitor (7.65±0.62)

Secretase inhibitor (7.93±0.38)

Secretase inhibitor (6.28+/−0.63)

Secretase inhibitor (6.23±0.94)

Secretase inhibitor (72)

Secretase inhibitor (40)

Secretase inhibitor (4.9)

Mode of action (IC50 in μM)

Fan et al., 2008

Oinonen et al., 2006

Brühlmann et al., 2004

Familian et al., 2006; Choi et al., 2007; Noble et al., 2009

Loeb et al., 2004

Forloni et al., 2001

Tomiyama et al., 1997a,b; Meng et al., 2008; Loeb et al., 2004

Millan-Aguinaga et al., 2010; Dai et al., 2010a

Millan-Aguinaga et al., 2010; Dai et al., 2010a

Tian et al., 2010

Tian et al., 2010

Tian et al., 2010

Tian et al., 2010

Park et al., 2004

Park et al., 2004

Lee and Yun., 2007; 2008; Park et al., 2004; Klaar and Wolfgang 1997

Reference

2.20 Natural Bioactive Molecules: Impacts and Prospects

Agrimonia pilosa Agrimonia pilosa Agrimonia pilosa Numerous plant sources

Epimedium spp. Numerous plant sources

Numerous plant sources

Numerous plant sources

Numerous plant sources

Numerous plant sources

Numerous plant sources

Psoralea coryfola

Quercitrin (247)

3-Methoxyquercetin (248)

Luteolin (249)

Icariin (250)

Myricetin (251)

Kaempferol (252)

Morin (253)

Apigenin (254)

Fistein (255)

Glabridin (256)

Neocoylin (257)

Source

Quercetin (246)

Compound (Structure No.)

Fabaceae/ Papilionaceae

―

―

―

―

―

―

Berberidaceae

Rosaceae

Rosaceae

Rosaceae

Family

He et ai. 2010; Li et al., 2010a,b

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2013; Choi et al., 2008; Conforti et al., 2010; Liu et al., 2009; Akaishi et al., 2008; Choi et al., 2008, Jang et al., 2009; Pavlica and Gebhardt 2010

Khan et al., 2009; Jung and Park 2007

Khan et al., 2009; Jung and Park 2007

Khan et al., 2009; Jung and Park 2007; Williams et al., 2011; Akaishi et al., 2008

Reference

Secretase inhibitor (0.7 )

AChE

Antioxidant, Anti Agg & Clearance

Secretase inhibitor

Secretase inhibitor

Choi et al., 2008a

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Cui et al., 2008

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Akaishi et al., 2008; Maher 2009

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Shimmyo wt al. 2008

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Shimmyo wt al. 2008

Secretase inhibitors, Brahmachari and Gorai, 2006; BrahAnti Agg & Clearance machari, 2008; Brahmachari and Jash, 2012; Shimmyo wt al. 2008; Akaishi et al., 2008

Antioxidant, Brahmachari and Gorai, 2006; BrahSecretase inhibitors, machari, 2008; Brahmachari and Jash, Anti Agg & Clearance 2012; Williams et al., 2011; Shimmyo wt al. 2008; Akaishi et al., 2008

AChE, MIC

AChE (0.1), Secretase inhibitors (0.5), Antioxidant, Anti Agg & clearance

AChE

AChE

AChE, Antioxidant, Anti Agg & clearance

Mode of action (IC50 in μM)

Brahmachari: Natural Products as Alzheimer’s Drug Leads

2.21

Onosma hispida Numerous plant sources

Sophora flavescens Sophora flavescens Plant sources Camellia sinensis C. sinensis C. sinensis C. sinensis

Numerous plant sources

Numerous plant sources

Agrimonia pilosa

Hispidone (259)

Naringin (260)

Flavanone (261)

Flavanone (262)

Flavanone (263)

(+)-Catechin (264)

(−)-Epicatechin (265)

(−)-Gallocatechin 3-gallate (266)

(−)-Epigallocatechin-3-gallate (EGCG) (267)

Flavan derivative (268)

Flavan derivative (269)

Tiliroside (270) Scopolia carniolica S. carniolica

Scopoletin (271)

Scopolin (272)

Coumarins

Citrus junos

Source

Naringenin (258)

Compound (Structure No.)

Solanaceae

Solanaceae

Rosaceae

―

―

Theaceae

Theaceae

Theaceae

Theaceae

―

Fabaceae/ Papilionaceae

Fabaceae/ Papilionaceae

―

Boraginaceae

Rutaceae

Family

AChE (79)

AChE (79)

AChE

Secretase inhibitor (5.3), Antioxidant

Secretase inhibitor (0.27)

Neuroprotective activity

Secretase inhibitor (0.17)

Anti Agg & Clearance

Anti Agg & Clearance

Secretase inhibitor

Secretase inhibitor

Secretase inhibitor

AChE

AChE (11.6)

AChE, Antioxidant

Mode of action (IC50 in μM)

Lee et al., 2004; rollinger et al., 2004

Lee et al., 2004; rollinger et al., 2004; Fallarero et al., 2008

Khan et al., 2009; Jung and Park 2007

Brahmachari and Gorai, 2006; Brahmachari and Jash, 2012; Williams et al., 2011; Mecocci et al., 2008; Mandel et al., 2008

Brahmachari and Gorai, 2006; Brahmachari and Jash, 2012; Williams et al., 2011

Chen et al., 2000; Hou et al., 2008; Rezai-Zadeh et al., 2005; Okello et al., 2004; Li et al., 2004; Salah et al., 1995; Li et al., 2006; Yang et al., 2007; Mandel et al., 2008

Jeon et al., 2003

Ono et al., 2006; Fruson et al., 2012

Ono et al., 2006

Brahmachari, 2008; Hwang et al., 2008

Brahmachari, 2008; Hwang et al., 2008

Brahmachari, 2008; Hwang et al., 2008

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Kumar et al., 2010

Ahmad et al., 2003

Brahmachari and Gorai, 2006; Brahmachari, 2008; Brahmachari and Jash, 2012; Heo et al., 2004

Reference

2.22 Natural Bioactive Molecules: Impacts and Prospects

A. dahurica A. dahurica A. dahurica A. gigas A. gigas Ammi majus Plant sources Murraya paniculata Celtis chinensis Found in bergamot essential oil, in other citrus essential oils, and in grapefruit juice. Semisynthetic Feroniella lucida Apium graveolens Psoralea spp. Psoralea spp. Coumarin derivative

Imperatorin (277)

Isoimperatorin (278)

Coumarins (279-280)

Decursinol (281)

Decursin (282)

Marmesin (283)

Marmesin glucoside (284)

Murranganone (285)

N-p-Coumaroyl tyramine (286)

Bergapten (287)

4-Methylumbelliferone (288)

Feronielloside (289)

Columbianetin (290)

Psoralen (291)

Isopsoralen (292)

Ensaculin (293)

Six xanthones (294-299)

Plant sources

Umbelliferae

A. acutilobe

Isopimpinellin (276)

Xanthones

Rutaceae

A. acutilobe

Xanthotoxin (275)

―

―

Fabaceae/ Papilionaceae

Fabaceae/ Papilionaceae

―

―

Ulmaceae

Rutaceae

―

Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

Umbelliferae/Apiaceae

A. acutiloba

Umbelliferae/Apiaceae

Angelica acutiloba

Family

(Z)-Ligustilide (274)

Source

(Z)-Butylidenephthalide (273)

Compound (Structure No.)

AChE

AChE

AChE

AChE

AChE

AChE (24.7)

AChE

AChE

AChE (122)

AChE (79.1)

AChE (68)

AChE (67)

AChE (390)

AChE (28)

AChE ( CCS (65%) > (+)-Cat (54%) > FCS (47%) > BCS (41%) > CS (38%) ª BHT (38%) > Caf (35%) > Sin (30%) > Trolox (24%) > a-Toc (22%) > Fer (16%) > Pco (4%) ª Phb (3%). In each acid, as Caf only has a catechol (1,2dihydroxybenzene) structure, it would enhance the antioxidation by stabilizing the generated radicals with many resonance structures after oxidation (Fig. 3a) (Bors et al., 2004). Moreover, for the same reason, cinnamates (Caf, Sin, Fer, Pco) are stronger than benzoate (Phb). The AOCs of non-acylated ANs (some are also de-acylated derivatives such as Cy3S5G or Pn3S5G from YGMs, Da-T from ternatins, and Cy 3-gentiobioside (Cy3Gnt) from alatanin C) were examined. The AOC order was delphinidin 3-glucoside (Dp3G (64%)) > Cy3G (61%) ª Cy3Gnt (60%) > Cy3S5G (32%) ª Da-T (31%) > Pn3S5G (23%) > Pg3S5G (13%) as shown in Fig. 2. The reason why Cy3G, Cy3Gnt and Cy3S5G as well as Dp3G are the strongest of all is considered that since only the Cy(Dp)-based glycosides have a catechol structure in the aglycon B-ring, the Cy(Dp)-based ANs are considered to stabilize more generated radicals than other types (Da-T, Pn3S5G and Pg3S5G) that have only one phenolic hydroxyl group at B-ring 4¢ position (Fig. 3b). Similarly, 3-monosubstituted Cy3G and Cy3Gnt with more resonance structures of the generated radicals enhance their AOCs more than 3,5-disubstituted Cy3S5G. As demonstrated in Fig. 2, AOC intensities of all ANs employed fell between Pg3S5G (Fer equivalent) and YGM-1b (85%) (EGCG equivalent). AN-AOCs were not proportional to degree of acylation (namely Mws of ANs). For instance, AOCs of tetra-p-coumaroylated T-A1 (38%) (Mw 2108) and T-A2 (38%) (Mw1800) were near that of Caf (Mw 180), and di-p-coumaroylated T-A3

Qiu et al.: Acylated Anthocyanins and Related Polyphenols

4.7

Fig. 2 DPPH radical scavenging activity (%) of anthocyanins and related antioxidants. Symbol (•) shows ANs with catechol part(s) in the molecules, ( ) other ANs, and (*) authentic antioxidants. Abbreviations are donated in the text

(30%) (Mw 1492) was almost the same as its deacylated Da-T (Mw 789). In 22 high-AOC ANs (RS > 50%), there were five non- and mono-AR-ANs. Therefore, efficiently AOC-enhanced factor would be mainly eventuated that ANs had catechol structures in the B-ring and/or the acyl moieties (13 ANs given by black circles in Fig. 2).

4.3.3

Structure-Antioxidant Capacity Relations of YGMs

AOC results of ten YGMs, non-acylated YGM-0a (=Cy3S5G) and -0b (=Pn3S5G); mono-acylated YGM-2 and -5b; di-acylated ANs (Cy-based: YGM-1a, -1b, -3; Pn-based YGM-4b, -5a, -6); and related ARs (Caf and Fer), are demonstrated in Fig. 2. The AOC are order of YGM-1b > YGM-3 (80%) ª YGM-4b (79%) > YGM-1a (70%) ª YGM-6 (69%) ª YGM-2 (67%) > YGM-5a (59%) > YGM-5b (54%) > (Caf) > YGM-0a > YGM-0b > (Fer > Pco ª Phb). The results indicate that, (1) acylation with ARs enhances the AOCs of ANs, and the AOC is proportional to the degree of acylation, (2) antioxidative intensities of YGMs are nearly equal to addition of those

4.8

Natural Bioactive Molecules: Impacts and Prospects

Fig. 3

Antioxidant capacity of aromatic acids and non-acylated anthocyanins (G: D-Glucose)

Qiu et al.: Acylated Anthocyanins and Related Polyphenols

4.9

of corresponding de-acylated ANs and acylating acids, e.g. AOC intensity of YGM-3 (80%) ª Cy3S5G (32%) + Caf (35%) + Fer (16%), except for di-caffeoylated YGM-1b and -4b, and (3) Cy-based YGM-1b, -3, -1a and -2 with a catechol moiety are stronger than Pn-based YGM-4b, -6, -5a and -5b, as shown in the case of DAs (Fig. 3b). Thus Cy-based YGMs with both catechol type B-ring and Caf group enhance the AOC additively, resulting in the strongest antioxidant power of YGM-1b (Cy 3-Caf-Caf-S-5-G) of all tested YGMs. HBA (61%), regarded as more caffeoylated analogue of YGM-4b (Fig. 1b), shows less intensity of AOC than YGM-4b. This is accountable that HBA has no catechol moieties in the molecular structure instead bearing three Caf residues.

4.3.4

Structure-Antioxidant Capacity Relation of Ternatins

As shown in Fig. 2, the AOC results of twelve ternatins and related substances gave the order of (Dp3G) > T-D1 (59%), T-D2 (59%) > T-B3 (57%), T-B2 (56%) ª T-B1 (54%) > T-C1 (39%) ª T-C3 (38%) ª T-A1 (38%), T-A2 (38%) > T-C4 (33%) ª T-C5 (32%) ª Da-T ª T-A3 (30%) > (Pco). Since all ternatins have no catechol part in B-ring due to 3¢,5¢-di-glycosylated, they are less intense than Dp3G having catechol structure in B-ring. Ternatin D (T-D1, -D2) series have the highest activity, followed by B-series (T-B1, -B2, -B3), A-series (T-A1, -A2, -A3) and Da-T. T-C series are located between B-series and Da-T. The results show that, 1) only the terminal Pco(s) in 3¢, 5¢-side chains enhance AOCs, and the intensive degree is dependent on the terminal acid numbers (T-D series (di-Pcos) > T-B series (mono-Pco) > T-C series (mono- or non-Pco) > T-A series (nonPco)), 2) AOC intensities are nearly equal to additive intensities of Da-T and terminal Pco(s). Tri- and tetra-Pco ternatins (T-A1, -B1, -D1, -A2, -B2, -D2 etc.) are much more stable than di- and mono-Pco ternatins and other HSAs, suggesting “double-stacking” mechanism that terminal (outer) Pco in the di-acylated side chains attach to inner Pcos which already has stacked with aglycon (Fig. 4). For example, T-D2 binds tightly in folding form through intramolecular doublestacking of 3¢-side chain and normal (single)-stacking of 5¢-side chain in aqueous solutions, and the form efficiently protects itself from water molecule attack leading to loss of color as shown in Fig. 4 (Kondo et al., 1987). This mechanism is supported by some reports, which proved the folding conformation in solution by means of the detailed analysis on NMR data of T-D2 (Yoda et al., 1989; Yui et al., 2000) and the molecular modeling calculation (Igarashi et al., 1989). Therefore, in the case of tri- and tetra-Pco ternatins, the terminal Pco(s) only might enhance the AOC despite of multiple Pcos inner side of the folding molecule. This “burying effect” apparently decreases the AOC expected from AR number, and might be effective to other PAANs (Clifford, 2000; Goto et al., 1984; Jackman et al., 1987; Kondo et al., 1987). The AOC of Tri-Caf HBA seems to be reduced for the same reason. T-C series are ternatins with only one 3¢ (5¢) side-chain. Only T-C1 and -C3 have terminal Pco and their AOC are slightly stronger than T-C4 and -C5 without terminal Pco. In conclusions, the AOCs of AANs were summarized as follows. 1. Intensity of AOCs was nearly equal to the total of those of corresponding DAs and acylating acids, namely additive effect but not synergistic. 2. AOC-enhanced factors relating the structure were as follows:

4.10

Natural Bioactive Molecules: Impacts and Prospects

i AR acylation enhanced the activity more than corresponding de- or non-acylated ANs ii Catechol structures in aglycon B-rings like cyanidin (also Dp and Pt), and/or acylating AR like 5.Caf, powerfully enhanced the activities. 3. In the case of PAANs with three or more ARs, only terminal acids in side chains enhanced the AOC (Fig. 4). 4. Glycosylation patterns affected the AOCs (3-glycosides > 3,5-di-glycosides).

Fig. 4

Conformation of ternatin D2 in neutral aqueous solution (G: D-glucose; P: p-coumaric acid; Dp: delphinidin)

4.4. STRUCTURE-ANTI-HYPERGLYCIMA RELATIONSHIPS 4.4.1

Diabetes and its Prevention

Among the lifestyle-related diseases, diabetes is a serious condition for the individual on the global scale as its rapidly increasing prevalence (WHO Expert Committee on Diabetes Mellitus, 1980).

Qiu et al.: Acylated Anthocyanins and Related Polyphenols

4.11

Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both (Fig. 5) (American Diabetes Association and National Institute of Diabetes and Digestive and Kidney Diseases, 2003). The long-term manifestation of diabetes can result in the development of some serious complications such as neuropathy (nerve damage), nephropathy (renal disease) and vision disorders (retinopathy, glaucoma, cataract and corneal diseases) (Mayfield, 1998). There are two types of diabetes, Type 1 or insulin-dependent diabetes mellitus (IDDM) is characterized by b-cell destruction caused by an autoimmune process, usually leading to absolute insulin deficiency, and diagnosed in childhood. Type 2 or non-insulin dependent diabetes mellitus (NIDDM) is characterized by insulin resistance in peripheral tissue and insulin secretive defect of the b-cells. Persons with fasting blood glucose levels ranging from 110 to 126 mg/dL (6.1 to 7.0 mmol/L) are said to have impaired fasting glucose, while those with a 2 h-postprandial blood glucose level between 140 mg/dL and 200 mg/dL (7.75 mmol/L to 11.1 mmol/L) are said to have impaired glucose tolerance (Zimmet, 1999).

Fig. 5

Strategy for anti-diabetic treatment

It has been recognized that a postprandial hyperglycemia plays role in the development of NIDDM (Baron, 1998; Gavin, 2001). Most anti-diabetic agents that are currently available reduce fasting blood glucose levels, but have little impact on postprandial glycemic excursions and thus do not normalize postprandial hyperglycemia. Therefore, agents that reduce postprandial hyperglycemia have a key role in the treatment of Type 2 diabetes and pre-diabetic states (Baron, 1998). New therapeutic agents, a-glucosidase inhibitors that control postprandial hyperglycemia have been developed (Bischoff, 1994; Toeller, 1994) a-Glucosidase (EC 3.2.1.20, exo-type a-D-glucopyranoside O-linkage hydrolase), a membrane bound enzyme located at the epithelium of the small intestine, catalyzes the cleavage of glucose from disaccharides (Toeller, 1994). Hence, the inhibition is effective in the prevention or treatment of diabetes mellitus and therefore therapeutic inhibitors, such as acarbose (Chiasson et al., 2002) and voglibose (Saito et al., 1998), have received

4.12

Natural Bioactive Molecules: Impacts and Prospects

considerable attention in the past two decades. By contrast, it has been demonstrated that natural agents that slow carbohydrate absorption may mimic the therapeutic drugs (McCarty, 2005). Also, preventing progression to diabetes by taking foods with specific physiological functionality would be a preferable option for people and likely to be practical instead of taking synthetic drugs. This section is, thus, focused on the functionalities of potential a-glucosidase inhibitors from natural medicinal resources, in particular colored phytochemicals.

4.4.2

-GLUCOSIDASE INHIBITION ASSAY

In order to evaluate and survey anti-hyperglycemic natural resources or compounds through a retardation of a-glucosidase activity, an adequate and reliable assay system is essential. The convenient method is based on a spectrophotometric technique using a pseudo-substrate, p-nitrophenyl-a-D-glucopyranoside, and free enzyme from baker’s yeast (Nishioka et al., 1997; Watanabe et al., 1997). However, as Chiba (Chiba, 1997) has pointed out, a catalytic property of a-glucosidase greatly differs from origins: types I (baker’s yeast) and II (mammals). As summarized in Table 1, all the inhibitors gave a variety of a-glucosidase inhibition behavior according to its origin (baker’s yeast, rat, rabbit, and porcine small intestines) (Oki et al., 1999). This finding of poor inhibitory effect against baker’s yeast a-glucosidase as compared to mammalian a-glucosidases agreed with the results of aminocyclitols from Streptomyces hygroscopicus fermentation broth, which showed more potent porcine a-glucosidase inhibitory activity than baker’s yeast (Kameda et al., 1984). These findings, followed by the fact that the oral administration of acarbose in NIDDM subjects was allowed to moderate the postprandial blood glucose level (Chiasson et al., 2002), strongly suggested that the conventional a-glucosidase inhibition study against baker’s yeast may not give us any practical information concerning suppression of glucose production from carbohydrates in the gut. Table 1 Effect of a-glucosidase origin on inhibition profile of natural or synthetic inhibitors when p-nitrophenyl-a-D-glucopyranoside was used as a substrate IC50 (mM) Origin Baker’s yeast Rat (free assay system) (immobilized assay system)

voglibose 26

acarbose

(+)-catechin

no inhibition

130

0.073

63

NI

(0.013)

(0.114)

Rabbit

0.14

62

NI

Pig

0.0017

87

NI

NI: no inhibition

As illustrated in Fig. 6, a-glucosidase in mammalian intestine is anchored in the membrane by the polypeptide chain spanning the bilayer only once in an N (in)/C (out) orientation (Hauri et al., 1982) This indicates that a-glucosidase inhibition studies should be performed with membrane bound or immobilized a-glucosidase assay system but not with free a-glucosidase. Hence, an immobilized a-glucosidase assay system to mimic the membrane-bound condition was newly

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4.13

established (Oki et al., 2000). The assay system using an a-glucosidase immobilized microplate was also proposed for simultaneous inhibition assay (Matsui et al., 2009). The immobilized a-glucosidase assay revealed therapeutic powers of voglibose and acarbose with IC50 values of 13 and 114 nmol/L, respectively, compatible to in vivo potential of each drug (ED50: voglibose; 0.37 mmol/kg, acarbose; 5.42 mmol/kg) (Matsui et al., 2004).

Fig. 6

4.4.3

Schematic representation of membrane-bound a-glucosidase at the small intestine

-Glucosidase Inhibition of Anthocyanins and its Relations with Structure

In a recent report on the STOP-NIDDM trial study (Chiasson et al., 2002), long-term acarbose treatment was effective in borderline subjects, indicating that an appropriate postprandial BGL control by a-glucosidase inhibitors seems to be of benefit for preventing the development of hyperglycemia. Figure 7 represents a-glucosidase inhibition profiles induced by acylated ANs. As shown in Fig. 7, colored plants are good resources as an a-glucosidase inhibitor and have a potential for retarding the action of intestinal maltase activity, among which di-acylated ANs were found to be an excellent natural a-glucosidase (maltase) inhibitor: SOA-4 (IC50; 60 µM), Pg 3-O-(2- O-6O-(E-3-O-(b-D-glucopyranosyl)Caf)-b-D-glucopyranosyl)-6-O-E-Caf-b-D-glucopyranoside)-5O-b-D-glucopyranoside) isolated from a morning glory (Phabilis nil cv. Scarlet O’Hara) red flower, and YGM-6 (IC50; 200 µM) (Matsui et al., 2001a, b). To date, it was found that some other di-acylated ANs occurred in purple-flashed sweetpotato also had a potential to inhibit intestinal maltase action (IC50; YGM-4b: 130 µmol/L, YGM-5b: 276 µmol/L (Terahara et al., 2009)). Interestingly, AN-induced a-glucosidase inhibition was restrictive to maltase inhibition, but not to sucrase inhibition. The preferential inhibition of ANs against intestinal maltase enzyme from Sucrase-Isomaltase (SI) complex of a-glucosidase (Hauri et al., 1982) still remains to be fully understood. However, the anti-hyperglycemic action of di-acylated ANs in Sprague-Dawley rats supported the intestinal maltase inhibition rather than sucrase and/or SGLT1 transport inhibition in the gut (Fig. 8). As a whole, the preferable maltase inhibition by ANs would be of benefit, because the suppression of glucose production from dietary carbohydrates must be effective to improve excess postprandial BGL rise for NIDDM subjects.

4.14

Natural Bioactive Molecules: Impacts and Prospects

Fig. 7

a-Glucosidase inhibitory activity of di-acylated anthocyanins

To clarify structural factors responsible for powerful immobilized a-glucosidase inhibition of the di-acylated ANs, maltase inhibitory activity of their de-acylated ANs were examined. As illustrated in Fig. 9, the de-acylated part, i.e., Pg (or Cy, or Pn) 3S5G, was no longer a potent a-glucosidase inhibitor. This indicated that, in any case, acylation of ANs with Caf or Fer must be important for the expression of maltase inhibition. Among the three de-acylated ANs, Pg-based AN (Pg3S5G) showed the most potent maltase inhibition with IC50 value of 4.6 mmol/L, and the descending order of potency was Pg3S5G (IC50; 4.6 mmol/L) > Cy3S5G (IC50; 14.1 mmol/L) > Pn3S5G (IC50; 18.2 mmol/L). Interestingly, the order was almost same as the case of acylated ANs (SOA-4 (Pg type) (IC50; 60 µmol/L) > YGM-3 (Cy type) (IC50; 193 µmol/L) = YGM-6 (Pn type) (IC50; 200 µmol/L)) (Matsui et al., 2001b). These findings strongly suggested that no replacement at 3¢(5¢)-position of the B-ring would be essential for inhibiting a-glucosidase or maltase action.

Qiu et al.: Acylated Anthocyanins and Related Polyphenols

Fig. 8

4.15

Physiological functions of di-acylated anthocyanins at the small intestinal membrane

Further experiments were done to clarify the structural factors attribute to the powerful maltase inhibition of di-acylated ANs. As described above, a potent maltase inhibition capability was elevated by acylated ANs with phenolic acids, but not by their aglycons. As shown in Fig. 9, acylated moieties of di-acylated ANs were found to show a powerful maltase inhibitory activity (Terahara et al., 2009; Matsui et al., 2004), as the mothers did inhibit maltase. Namely, maltase inhibitory powers of di-acylated moieties of CCS and FCS were compatible with their mother acylated ANs of YGM-4b for CCS (IC50; 214 mM) and YGM-3 or YGM-6 for FCS (IC50; 289 mmol/L), indicating that the ANs-induced maltase inhibition would be caused by an acylated moiety. Figure 9 also clearly demonstrated that the mono-acylated moiety (CS: IC50; 699 mmol/L) or phenolic acids themselves diminish the powers of mother ANs or their di-acylated moieties (Matsui et al., 2001b; Terahara et al., 2009). Ferulic acid and sophorose were far from a-glucosidase inhibitor (Matsui et al., 2004). Similar a-glucosidase inhibitory expression by acylation has also been reported for esterified catechins from tea polyphenols (Honda and Hara, 1993) and for theaflavins (Matsui et al., 2007). These results indicate that the replacement with hydroxyl group at aromatic rings, along with sugars, would be required to elicit the activity. A comparable result that the maltase inhibitory activity of CCS or FCS was much higher than that of CS strongly reveals that the Caf group seems favorable for eliciting maltase inhibition. The importance of the number of esterified Caf moiety to sugars has been also demonstrated in Caf-quinic acids, in which maltase inhibition was markedly enhanced with an increasing number of Caf to quinic acid (QA): IC50 value of 3-CQA; 18,900 mmol/L, 3,4-di-CQA; 1,910 mmol/L, 3,5-di-CQA; 1,890 mmol/L, 3,4,5tri-CQA; 24 mmol/L (Matsui et al., 2004).

4.5

ABSOPTION OF CAFFEOYLSOPHOROSE FROM ACYLATED ANTHOCYANINS INTO RAT BLOOD SYSTEM

Physiological functions of dietary ANs have been paying much attention as discussed above. However, the physiological events of such bioactive compounds must be closely associated with their absorption, metabolism or tissue distribution, and current issues seem to move to the

4.16

Natural Bioactive Molecules: Impacts and Prospects

Fig. 9

Maltase inhibition of di-acylated anthocyanin moieties. Number indicates IC50 value against maltase activity

clarification of bioavailability, along with the evaluation of physiological functionalities (Qiu et al., 2011). This section focuses on the absorption and potential metabolism of AN-related compounds in rats.

4.5.1

Bioavailability Assay

The definition of bioavailability comes from the pharmacology community. The time-dependent change in the blood concentration of an oral dose of the compound is compared to the changes observed when the same dose is administered systemically. This definition takes into account how a compound is distributed to tissues when it is directly introduced into the blood stream. The areas under the concentration-time curves (AUC) for each route of administration is often used for estimating oral bioavailability of acylated ANs (Prasain and Bames, 2007; Miyazawa et al., 1999; Suda et al., 2002; Harada et al., 2004; Ichiyanagi et al., 2006).

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4.5.2 Absorption of Anthocyanins and their Metabolites in Sprague-Dawley Rats There have been several quantitative studies on the absorption of ANs, such as Cy3G (Miyazawa et al., 1999), cyanidin 3-O-b-rutinoside (Cy3R) and delphinidin 3-O-b-rutinoside (Dp3R), and their intact absorption into blood and excretion into urine in rats and humans have been demonstrated (Miyazawa et al., 1999; Matsumoto et al., 2001). Other researches also revealed an intact absorption of acylated ANs such as cyanindin 3-caffeoylsophoroside-5-glucoside (Cy3CafS5G) and peonidin 3-caffeoylsophoroside-5-glucoside (Pn3CafS5G) into rat and human blood systems (Suda et al., 2002; Harada et al., 2004). Intact absorption of acylated ANs into human blood system strongly supported their potential in our body. The pharmacokinetic analyses in some relevant studies on ANs were summarized in Table 2. The Cmax and AUC of CS that is an acylated moiety of ANs (Qiu et al., 2011) were much higher than those of non-acylated ANs, Cy3G, Dp3G, Cy3R or Dp3R (He et al., 2006; Miyazawa et al., 1999), and acylated ANs, Pn3-Cs-G (Suda et al., 2002) or nasunins (Ichiyanagi et al., 2006). Figure 10 shows the absorption behavior of CS in Sprague-Dalwey rats after its single oral administration dosed at 400 mg/kg. CS was rapidly absorbed to blood system and kept the high plasma level up to 3 h. This profile may be because CS entered into enterohepatic circulation (Zhang et al., 2007), like chlorogenic acid and caffeic acid (Lafay et al., 2006). According to the AUC0-6h of CS (108.6 ± 8.1 nmol·h/mL) and its conjugate (50.7 ± 5.7 nmol·h/mL), a half of absorbed CS was converted to conjugates. The lower percentage of CS conjugation (ca. 50%) compared with other compounds (Cy3G > 90% conjugates (Miyazawa et al., 1999); EGCG, 50-90% conjugates (Lambert et al., 2003)), also support the preferable intact absorption and high digestive stability of CS. Table 2 Pharmacokinetic parameters of CS and other reported anthocyanins in rats after their oral administration Compounds

Pharmacokinetic Parameters

Dose (mg/kg bw)

References

Cmax (nmol/mL)

tmax (h)

CS

27.5

0.25

4.68

400

Qiu et al., (2011)

Pn3-Cs-G

0.05

0.5

0.047 (x = 2 h)

—

146

Suda et al., (2002)

cis-Nasunin

0.04

0.25

0.063 (x = 8 h)

—

13

Ichiyanagi et al., (2006)

trans-Nasunin

0.29

0.25

0.44 (x = 8 h)

—

87

Dp3G

0.29

0.25

0.92 (x = 8 h)

—

100

Cy3G

3.49

0.25

3.62 (x = 4 h)

—

320

Miyazawa et al., (1999) Matsumoto et al., (2001)

AUC 0-x h (nmol·h/mL) 108.6 (x = 6 h)

t1/2 (h)

Cy3G

0.84

0.5

1.51 (x = 4 h)

2.08

359

Cy3R

0.85

0.5

2.54 (x = 4 h)

1.36

476

Dp3R

0.58

2.0

1.33 (x = 4 h)

0.79

489

tmax, time to reach maximum concentration; Cmax, maximum concentration; t1/2, half-life; AUC, area under the curve. CS, 6-O-(E)-Caffeoyl(2-O- β-D-glucopyranosyl)-D-glucopyranose; Pn3-Cs-G, Peonidin 3-caffeoylsophoroside-5-glucoside; cis-Nasunin, delphinidin 3-[4-(cis-p-coumaroyl)-L-rhamnosyl(1-6)glucopyranoside]-5-glucopyranoside; trans-Nasunin, delphinidin 3-[4-(transp-coumaroyl)-L-rhamnosyl(1-6)glucopyranoside]-5-glucopyranoside; Cy3G, Cyanidin 3-O-b-glucoside; Cy3R, Cyanidin 3-O-b-rutinoside; Dp3G, Delphinidin 3-O-b-glucoside; Dp3R, Delphinidin 3-O-b-rutinoside

4.18

Natural Bioactive Molecules: Impacts and Prospects

Fig. 10 Time course of plasma concentrations of intact CS and conjugated CS after a single oral administration of CS to rats

An extensive study to clarify the metabolites of absorbed CS was conducted with TOF-MS analysis (Fig. 11). CS derived from di-acylated AN was conjugated by the enzyme to become methylated and glucuronide conjugates, or converted to caffeic, ferulic and isoferulic acids, just like the metabolism of caffeoylquinic acid which was hydrolyzed with esterase (Stalmach et al., 2009) to caffeic acid. The glucuronidation of CS was happened mainly in small intestine but methylation was in liver (Qiu et al., 2011). As the metabolites of CS, caffeic, ferulic and isoferulic acids were further converted to their glucuronides, sulfates, and dihydro derivatives. The caffeic and ferulic acid-related derivative reactions would occur in intestinal epithelium or liver during the metabolic process (Qiu et al., 2011). The dihydro derivatives of caffeic acid and ferulic acid and their subsequent conjugates with glucuronides and sulfates would involve the metabolism in colon or liver depending on the food source and delivery (Adam et al., 2002; Konishi and Kobayashi, 2004; Poquet et al., 2008a). Early researches proposed that caffeic acid is easily absorbed intact in rat and human (Azuma et al., 2000; Olthof et al., 2001; Scalbert et al., 2002). Azuma et al., assumed that ingested caffeic acid, absorbed from the alimentary tract, may be metabolized through the same pathway as that proposed for flavonoids. Flavonoids are present in the common blood circulation in the form of glucuronide, sulfate, and methylated conjugates and are excreted via urine or bile. The glucuronidation of dietary flavonoid occurs in the intestinal mucosa, and then after entering blood circulation, it is sulfated in the liver and methylated in the liver and kidney (Azuma et al., 2000). Intestinal absorption of caffeic acid is influenced by esterification with quinic acid. Caffeic acid is much better absorbed than chlorogenic acid, its ester with quinic acid, common in many fruits and vegetables and particularly abundant in coffee. The intestinal absorption reached 95% and only 33% for caffeic acid and chlorogenic acid, respectively, in human subjects. Alternatively caffeic acid itself may be absorbed, after cleavage of the quinic acid from chlorogenic acids, and subsequently be O-methylated in the liver (Olthof et al., 2001; Scalbert et al., 2002). Ferulic acid as methylated caffeic acid is metabolized mainly as the conjugates form with glucuronides or sulfates. This supports that most ferulic acid passes rapidly across the small intestine as free acid and later is conjugated in the liver for further metabolism (Adam et al., 2002; Poquet et al., 2008b).

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4.19

Fig. 11 CS and its metabolites during absorption COMT, catechol-O-methyltransferase; RA, reductase; GT, UDP-glucuronyltransferase; ST, sulfuryl-O-transferase; EST, esterase. Bold arrows indicate major routes, and dotted arrows indicate minor ones

Transporting mechanism of CS across intestinal membrane is one of our attractive interests in absorption study. In previous reports, chlorogenic acid, caffeic acid (Konishi and Kobayashi, 2004) and hesperidin (Kobayashi et al., 2008) were absorbed mainly via paracellular diffusion through tight-junction. The transport of phenolic acids such as caffeic acid and ferulic acid has also been reported to be involved in intestinal monocarboxylic acid transporter (Konishi and Kobayashi, 2004; Poquet et al., 2008b). However, the transporting pathways of CS and absorbable ANs still remain unclear. Further absorption studies are thus needed to illustrate the underlying mechanisms.

4.6

FUTURE PERSPECTIVE AND CONCLUSIONS

In conclusion, high-stable poly-acylated ANs are found to attain high functionalities depending on the chemical properties of the phenolic acids and the conformational structures of acyl-glycosyl sidechains. The ANs, in particular di-acylated ANs or acylated moieties are regarded to be responsible for preventing Type 2 diabetes and cardiovascular diseases through their antioxidative action. We are hopeful that everyone will be able to share in the health benefits that these phytochemicals may eventually provide.

Abbreviations AAN, acylated anthocyanin; ANs, anthocyanins; AOC, antioxidant capacity; AUC, areas under the concentration-time curves; AR, aromatic acids; BHT, butylated hydroxytoluene; Caf,

4.20

Natural Bioactive Molecules: Impacts and Prospects

caffeic; (+)-Cat, (+)-catechin; CS, 6-O-(E)-caffeoyl-2-O-b-D-glucopyranosyl-D-glucopyranose; DPPH, 1,1-diphenyl-2-picrylhydrazyl; IDDM, insulin-dependent diabetes mellitus; NIDDM, non-insulin dependent diabetes mellitus; SI, Sucrase-Isomaltase; Trolox, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid

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Matsumoto H, Inaba H, Kishi M, Tominaga S, Hirayama M and Tsuda T (2001). Orally administered delphinidin 3-rutinoside and cyanidin 3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms. J Agric Food Chem 49, 1546–1551. Mayfield J (1998). Diagnosis and classification of Diabetes mellitus: New criteria. Am Fam Physician 15, 1355–1362. Mazza G and Miniati E (1993). Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, FL. 92–97. McCarty MF (2005). Magnesium may mediate the favorable impact of whole grains on insulin sensitivity by acting as a mild calcium antagonist. Med Hypotheses 64, 619–627. Miyazawa T, Nakagawa K, Kudo M, Muraishi K and Someya K (1999). Direct intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3,5-diglucoside, into rats and humans. J Agric Food Chem 47, 1083–1091. Nishioka T, Watanabe J, Kawabata J and Niki R (1997). Isolation and activity of N-p-coumaroyltyramine, an alpha-glucosidase inhibitor in Welsh onion (Allium fistulosum). Biosci Biotechnol Biochem 61, 1138–1141. Noda Y, Kneyuki T, Igarashi K, Mori A and Packer L (2000). Antioxidant activity of nasunin, an anthocyanin in eggplant peels. Toxicology 148, 119–123. Odake K, Terahara N, Saito N, Toki K and Honda T (1992). Chemical structures of two anthocyanins from purple sweet potato, Ipomoea batatas. Phytochemistry 31, 2127–2130. Oki T, Masuda M, Furuta S, Nishiba N, Terahara N and Suda I (2002). Involvement of anthocyanins and other phenolic compounds in radical scavenging activity of purple-fleshed sweet potato cultivars. J Food Sci 67, 1752–1756. Oki T, Matsui T and Matsumoto K (2000). Evaluation of alpha-glucosidase inhibition by using an immobilized assay system. Biol Pharm Bull 23, 1084–1087. Oki T, Matsui T and Osajima Y (1999). Inhibitory effect of a-glucosidase, inhibitors varies according to its origin. J Agric Food Chem 47, 550–503. Olthof MR, Hollman PCH and Katan MB (2001). Chlorogenic acid and caffeic acid are absorbed in humans. J Nutr 131, 66–71. Philpott M, Gould KS, Lim C and Ferguson LR (2004). In situ and in vitro antioxidant activity of sweetpotato anthocyanins. J Agric Food Chem 52, 1511–1513. Poquet L, Clifford MN and Williamson G (2008a). Investigation of the metabolic fate of dihydrocaffeic acid. Biochem Pharmacol 75, 1218–1229. Poquet L, Clifford MN and Williamson G (2008b). Transport and metabolism of ferulic acid through the colonic epithelium. Drug Metab Dispos 36, 190–197. Prasain JK and Barnes S (2007). Metabolism and bioavailability of flavonoids in chemoprevention: current analytical strategies and future prospectus. Mol. Pharmaceut 6, 846–864. Qiu J, Saito N, Noguchi M, Fukui K, Yoshiyama K, Matsugano K, Terahara N and Matsui T (2011). Absorption of 6-O-caffeoylsophorose and its metabolites in Sprague-Dawley rats detected by electrochemical detectorhigh-performance liquid chromatography and electrospray ionization- time-of-flight - mass spectrometry methods. J Agric Food Chem 59, 6299–6304. Rossi A, Serraino I, Dugo P, Di-Paola R, Mondello L, Genovese T, Morabito D, Dugo G, Sautebin L, Caputi AP and Cuzzocrea S (2003). Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radical Res 37, 891–900.

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5 Marine-Derived Bioactive Polysaccharides from Microorganisms Sylvia Colliec-Jouault* and Christine Delbarre-Ladrat Ifremer, Laboratory of Biotechnology and Marine Molecules, Ifremer, Rue de l’Ile d’Yeu, BP 21105, Nantes Cedex 03, 44311, France

ABSTRACT In the search of novel fine chemicals and biopharmaceuticals, bacterial polysaccharides offer a source of safe, biocompatible, biodegradable and renewable products with specific biological functions. The bacteria that produce polysaccharides are also a source of key enzymes for the production of tailor-made polysaccharides in the high-value medical field. In the biotechnological challenge for the discovery of original biomolecules and biocatalysts, the bacteria from marine extreme ecosystems contribute to increase chances of success. Since 1989, Ifremer has been involved in the discovery and the description of biotechnologically important microorganisms from hydrothermal deep-sea origin and other marine extreme ecosystems. Microorganisms have been recovered from samples collected all over the world. Screenings performed on these samples led to the discovery of several polysaccharide-producing bacteria. Up to now, more than 15 bacterial strains, belonging to four marine genera, have been described to produce exopolysaccharides (EPS) with both unusual structural features and innovative properties. Each EPS presents an original structure that can be modified to design compounds and improve their specificity. Different processes have been developed to perform chemical depolymerisation and substitution (sulphation) of EPS with the purpose to obtain bioactive derivatives possessing glycosaminoglycanlike (GAG-like) biological properties. The low-molecular-weight oversulphated derivatives can act synergistically with specific growth factors to induce the cell differentiation and present a real potential in cell therapy and tissue engineering. The actions of biologically active polysaccharides are largely dependent on their molecular structure, in particular the composition of the repeating unit, molecular size and sulphation degree.

*Corresponding author: [email protected]

5.2

Natural Bioactive Molecules: Impacts and Prospects

The current processes to modify the EPS, resulting in GAG-like molecules, are performed by chemical methods. But the need for environmentally friendly processes, in particular those derived from the white biotechnology, is important. Enzymes are indeed ideal biocatalysts due to their stereo- and regio-selectivity, and their activity in mild conditions. Enzymes capable of generating targeted modifications are thus looked such as glycoside hydrolases or polysaccharide lyases, and carbohydrate sulphotransferases. In this context, both identification and characterisation of enzymes for the bioprocessing of the bacterial polysaccharides may be carried out by a large screening work of diverse samples. However, these techniques of functional screening are not suitable for certain enzymes such as sulphotransferases because of the high cost of the sulphate donor. With the increasing number of microbial genomes sequenced, numerous enzyme genes related to polysaccharide biosynthesis and biotransformations are identified and molecular mechanisms of the biosynthesis of the polysaccharides are better understood. Future applications will involve traditional and molecular approaches to achieve integrated bioengineering approach for the production of tailor-made polysaccharides suitable for industrial and medical applications. Keywords: Bacterial exopolysaccharide, marine biodiversity, glycosaminoglycan, heparinlike, chemical modification, tissue engineering, hydrogel, drug discovery, biomaterials, wound healing, molecular weight, sulphation, growth factors, cross-linking, glycomics, biosynthesis, metabolic engineering, glycosyltransferase, polysaccharide lyase, glycoside hydrolase, carbohydrate sulphotransferase.

5.1

INTRODUCTION

Polysaccharides are complex macromolecules that are polydisperse, polyfunctional and often poorly characterised. Often located, in plant and animals, at the surface of cells, in the extracellular matrix, or attached to soluble signalling molecules, they participate in many crucial biological processes through the regulation of various key proteins such as chemokines, cytokines, growth factors, morphogens, enzymes and adhesion molecules. With the emergence of the glycobiology and glycomics, and also more detailed understanding in structure-function relationships, the therapeutic potential of complex polysaccharides is now well recognised (Shriver et al., 2004; Turnbull and Field, 2007). Animal-derived molecules for biomedical applications are coming under increasingly stringent regulatory control for fear of contamination by conventional and non-conventional viral agents. Thus the production of polysaccharides by bacteria is relevant for the development of biotechnological molecules for biomedical applications. Microbial production is gradually replacing extraction process as the preferred source of biopolymers and polysaccharides. Several bacterial polysaccharides are already produced commercially through medium to large-scale fermentation such as xanthan, pullulan, dextran and more recently hyaluronic acid. Recent advances in the identification of genes and gene clusters involved in the biosynthesis of these bacterial

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exopolysaccharides has provided potential tools to engineer bacteria for the efficient production of polymers for specific high-value applications. But detailed mechanisms are limited to only few model polysaccharides (Rehm, 2010). Marine environment offers a tremendous biodiversity. This biodiversity due to an exceptional adaptability is matched by a proportional richness in molecular diversity (Arrieta et al., 2010). In the discovery of innovative microbial polysaccharides, the exopolysaccharides produced by marine bacteria provide a valid alternative to traditional polysaccharides from plant, animals or seaweeds. The majority of microbial polysaccharides from marine source is still to be discovered. Marine bacterial polysaccharides present a real potential for natural product discovery and the production of new therapeutics with improved benefit, without risk for both patient and environment. In this present overview, we will present first the main bacterial polysaccharides and exopolysaccharides produced for either industrial applications or research purposes with a focus on described marine exopolysaccharides. Then we will show how the potential for drug discovery can be enhanced by developing biomimetic strategies in order to design bioactive derivatives from bacterial polysaccharides.

5.2 5.2.1

MARINE BACTERIAL EXOPOLYSACCHARIDES (EPS) Bacterial Polysaccharides and Exopolysaccharides

Bacteria synthesize few intracellular polysaccharides but they produce a vast range of extracellular polysaccharides. Extracellular polysaccharides produced by bacteria are mainly components of their cell-wall and also found as extracellular macromolecules. Among the extracellular polysaccharides some are secreted but remain attached to the cell (capsular polysaccharides or CPS) and others can be excreted and released in the bacterial environment (exopolysaccharides or EPS). The EPS released in the bacterial environment can be newly or freshly synthesised or lost from the cell surface as the cell ages or is subjected to high shear forces. Both CPS and EPS often exhibit several physiological functions; they are involved in adhesion and penetration into host and endowed with specific binding properties (Rehm, 2010; Sutherland, 1996). EPS present many great advantages for biotechnological purposes: (i) they are easily produced by fermentation; (ii) they can be easily isolated from bacterial broth at large-scale fermentation and at a competitive cost compared with traditional plant cell wall polysaccharides such as celluloses, pectins, xylanes, and many others; (iii) bacterial EPS are produced in totally controlled environment that is in agreement with the Good Manufacturing Practices, and (iv) EPS are biomolecules and by definition biodegradable and hence they are attractive to avoid accumulation in the environment, and also biocompatible and safe. Most media used for polysaccharide production by fermentation are based on high carbon substrate. Glucose at concentration of 2-5% (w/v) is usually the preferred carbon substrate as it is utilised by a very wide range of microbial species and is also widely available. Several bacterial EPS are already produced commercially at large-scale fermentation with annual world production volumes of around 103 tonnes and 106 tonnes for dextran and xanthan, respectively. Dextran are familiar to laboratory workers as the basis for cross-linked dextran beads used in gel filtration chromatography. Dextran 40 and 70 are widely used as a plasma substitute

5.4

Natural Bioactive Molecules: Impacts and Prospects

for the treatment of shock or impending shock due to hemorraghe, burns, surgery or trauma. Biotechnological production of different EPS, traditionally extracted from animal, terrestrial plant or seaweeds, presents a great interest for researchers and industrialists in view of their potential exploitation as food or pharmaceutical additive or fine chemicals as scaffolds or matrices in tissue engineering, wound dressing, drug delivery and in some particular cases as active component (Tombs and Harding, 1998). Bacterial EPS are industrially and medically very attractive. Some EPS can form viscous solutions and others are soluble in water, they are polydisperse. EPS present a great structural diversity higher than their protein and nucleic acid counterparts. So each EPS is structurally different and presents specific functional properties giving a therapeutic potential that has not been well exploited. Deeper knowledge in the structure-function relationships of these complex macromolecules will improve their development in the biotechnological field. Alginate traditionally extracted from brown seaweeds and the most important polysaccharides from this type of seaweeds is now also produced by soil bacteria Azotobacter vinelandii, Azotobacter chroococcum and Pseudomonas species. Bacterial alginates are a family of both CPS and EPS which, despite their similar composition, vary considerably in their structure. The bacterial alginates are all composed of the same two uronic acids (mannuronic and guluronic acids) as algal alginates, but in addition many of them are highly acetylated and some block structures can be missing such as G blocks in Pseudomonas species. G-rich alginates and M-rich alginates have different physico-chemical and biological properties, G-rich alginate can be used for encapsulation of cells and enzymes whereas alginate rich in M blocks stimulates cytokine production and has higher antitumor activity than G-rich alginate (Sabra et al., 2001; Svanem et al., 1999). Hyaluronic acid (HA), commercially extracted from rooster combs, is now industrially produced by Streptococcus zooepidemicus and Streptococcus equii (Ellwood et al., 1996; Rinaudo, 2008). Recently, recombinant strains have been described to produce HA such as Bacillus subtilis (Widner et al., 2005) and Lactococcus lactis (Chien and Lee, 2007), recognized as safe compared with several Streptococci. Both HA produced by extraction or fermentation are chemically identical, but the fermentation process is more attractive for large-scale production. HA is a linear high molecular weight polysaccharide that belongs to a group of substances known as glycosaminoglycans (GAG). HA is composed of disaccharide repeating units of b-(1-4)-D-glucuronic acid (GlcA) and b-(1-3)-N-acetyl-D-glucosamine (GlcNAc). The HA displays different physical properties (viscoprotection, viscosupplementation, viscoseparation, etc.) and finds number of applications in many medical sectors: ophthalmology, orthopedic surgery, rheumatology, dermatology, plastic surgery and wound healing (Kogan et al., 2007).

5.2.2

Exopolysaccharides from Marine Bacteria

As mentioned above, the bacterial EPS from terrestrial origin present a great structural diversity but this diversity is tremendously increased in the marine environment due to the enormity of the marine biosphere. In fact, marine environment is an extraordinary untapped source of unusual bacteria that remain to be discovered and offer a rich potential for both biotechnology and drug discovery. Aquatic bacterio- and phyto-plankton are known to produce EPS for cryoprotection,

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5.5

halotolerance, attachment to substrate, nutrient uptake and formation of chains, colonies and biofilms (Hoagland et al., 1993). Cyanobacteria can be found in fresh or salt water in different environments. Many cyanobacteria are described for the production of EPS, they are complex anionic heteropolysaccharides and are promising as thickening or suspending agents, emulsifying compounds or chelating agents for the removal of positively charged heavy metal ions (De Philippis et al., 2011). The most described marine cyanobacterial EPS is the spirulan that is a sulphated polysaccharide produced by Arthrospira platensis (formely Spirulina platensis) and consisting of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methyl-rhamnose (acofriose) with sulphate groups. It also contains trace amounts of xylose, glucuronic acid and galacturonic acid. Biological activities of this spirulan have been described : anticoagulant activity as described for heparin, the major drug used to prevent and treat thromboembolic disease (Kaji et al., 2004) and also antiviral activity (Hayashi et al., 1996). Other marine cyanobacterial EPS have been isolated from Polynesian microbial mats, they are both capsular and released EPS consisted of 7 to 10 different monosaccharides with mainly neutral sugars and bearing, for some of them, sulphate groups. The microbial mats may be considered as a great source of yet unexplored cyanobacterial EPS producers for biotechnological applications (Richert et al., 2005). As mentioned above for cyanobacteria, in the classification of bacteria, a clear distinction between marine and non-marine origin is hardly applicable. It is now established that the genus Pseudoalteromonas contains species that exclusively derive from marine waters and are often found in association with marine eukaryotes. They produce biologically active extracellular agents and EPS. EPS producing bacterial strains are common within the genera Pseudoalteromonas and Alteromonas. The bacterial EPS are described to be anti-bacterial components, they can control bacterial attachment, act as protective barriers against toxic molecules (Holmstrom and Kjelleberg, 1999). An EPS-producing bacterium has been isolated from a sponge sample in red sea (Egypt). The characterisations showed that this bacterium is Pseudoalteromonas sp. AM, the produced EPS is a homopolysaccharide constituted mainly of glucose units with a marked antiviral activity against herpes simplex (HSV-I) (Al-Nahas et al., 2011). Two other EPS-producing bacteria have been isolated from coastal regions in India, both are Vibrio sp. The first was a Vibrio furnissii strain VB0S3, the produced EPS is a heteropolysaccharide composed of neutral sugars (glucose and galactose) and also uronic acids; the presence of carboxyl, hydroxyl and amide groups was described. This typical heteropolysaccharide possesses good emulsification activity (Bramhachari et al., 2007). The second was a Vibrio parahaemolyticus isolated from a natural biofilm, the physico-chemical characterisation of the EPS produced by this bacterium showed an average molecular size of 12.278 µm and a composition characteristic of polysaccharides with the presence of neutral sugars (arabinose, galactose, glucose and mannose), uronic acids and amide groups (Kavita et al., 2011). The EPS produced by Vibrio alginolyticus (isolated from a biofilm in the surface coastal waters of bay of Bengal) was also characterised; it is a high molecular weight EPS (>6 × 106 g/mol) composed of glucose, aminoarabinose, aminoribose and xylose and it is unstable at high temperature and high pH (Muralidharan and Jayachandran, 2003). A Vibrio sp. QY101 is a alginate lyase-producing marine bacterium isolated

5.6

Natural Bioactive Molecules: Impacts and Prospects

from a decaying thallus of Laminaria (marine brown seaweed) and the EPS purified from its culture supernatant is a high molecular weight (>500,000 g/mol) heteropolysaccharide primarily constituted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. This EPS can inhibit biofilm formation of many bacteria and also disrupt established biofilm of some strains, these data show the potential of this EPS as active compound in the design of new therapeutic strategies for bacterial biofilm associated infections (Jiang et al., 2011). The microbial biodiversity of extreme marine environments such as sea ice (Southern Ocean), microbial mats in French Polynesia, shallow marine hydrothermal vents and also deep-sea hydrothermal vents have been investigated to find new bacteria and archaea. The Antarctic marine environment is perennially cold and in some cases permanently ice-covered; therefore temperature, and salinity are extreme. Antarctic marine bacterial isolates and the production of EPS have been studied. Two Antarctic marine bacteria have been identified, one from southern ocean particulate material and the other from melted sea ice; they belong to the genus Pseudoalteromonas and produce EPS; they are psychrophiles with an optimal production between –2°C and 10°C. These EPS have high levels of uronic acids such as galacturonic acids and have also sulphate and acetyl groups but some differences were observed in the presence or not of some neutral sugars and amine sugars. They are both sticky and contain trace of metal cations (Hassler et al., 2011; Nichols Mancuso et al., 2004). Microbial mats in French Polynesia can be considered as extreme environment, they are characterised by varying physical and chemical parameters, with pH values ranging from 6 to 10.5, salinity levels ranging from 5 to 42 g/L and temperatures from 20°C during the night to 42°C around mid-day. Microbial mats are laminated communities composed of both phototrophic and chemotrophic prokaryotes and bacteria coexist with cyanobacteria. EPS producing bacteria have been isolated from this particular environmemt rich in sulphurous and non-sulphurous photosynthetic bacteria; these producing EPS bacteria are mesophilic heterotrophic aerobic bacteria belonging to Pseudomonas, Alteromonas, Paracoccus and Vibrio. The produced EPS are heteropolysaccharides composed of neutral sugars (from 11 to 50%) and uronic acids (from 8 to 28%) with the presence of sulphate groups for some of them (from 5 to 29%). One EPS produced under laboratory conditions by Paracoccus zeaxanthificiens subsp. Payriae exhibited a very high binding for both copper and iron (II) salts. It could find applications in heavy metal and radionuclide waste clean-up (Guézennec et al., 2011). Some EPS can also be produced by extremophiles such as thermophiles isolated from shallow marine hydrothermal vents of Vulcano Island (Italy). The thermophilic facultatively aerobic Geobacillus thermodenitrificans bacterium can produce 2 EPS based on mannose and glucose sugars with different ratios. The EPS-2 presents immunomodulatory and antiviral effects. Other thermophiles have been isolated from this environment; they belong to genera Bacillus and Thermus (Nicolaus et al., 2010; Poli et al., 2010). In the discovery of new bacteria, the exploration of the very attractive deep-sea microbial world highlighted an unexpected biodiversity with a remarkable variety of habitat such as cold waters, sediments, animal guts, hydrothermal vent environments characterised by high pressures, high temperature gradients (from 2 to 350°C) and high levels of toxic elements such as sulphides and heavy metals. New thermophiles and hyperthermophiles (maximal growth greater than 90°C)

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and also new mesophiles have been discovered (Deming, 1998). A psychrotolerant bacterium Pseudoalteromonas sp. SM9913 was isolated from deep-sea sediment under optimal growth conditions (15°C, 52 H), the yield of the secreted EPS reached 5.25 g/l of culture medium. The EPS is a linear arrangement of a–(1-6)-Glc with a high degree of acylation (Qin et al., 2007). The screening performed on isolates, collected by the French-manned submersile Nautile during different oceanographic cruises (Starmer in 1989, Hero in 1991, Guaynaut in 1991 and Microsmoke in 1995), led to the discovery of new species of EPS-producing bacteria belonging to known genera such as Pseudoalteromonas, Alteromonas and Vibrio (Guezennec, 2002; Nichols Mancuso et al., 2005). During the Starmer cruise a heterotrophic mesophilic aerobe was isolated and named Alteromonas macleodii subsp fijiensis; this bacterium was collected close to a hydrothermal fluid near an active hydrothermal vent at a depth of 2,000 m in a rift system of the North Fiji Basin. Its ability to produce EPS was demonstrated in synthetic conditions (cultivated in fermentor) (Raguenes et al., 1996). This EPS is an heteropolysaccharide and its repeating unit is a branched hexasaccharide: 1 glucose, 2 galactose, 1 mannose bearing 2 pyruvate groups in C4 and C6 positions and 2 glucuronic acids (Rougeaux et al., 1998). This EPS showed a strong capacity to bind metallic ions such as lead, zinc and cadmium and could be used as a biosorbent to remove toxic metal pollutants from industrial wastewaters (Loaec et al., 1997). A new bacterium was also isolated during the Guaynaut cruise from a sample of fluid collected among a dense population of Riftia pachyptila in the vicinity of an active hydrothermal vent of the Southern depression of the Guaymas basin (Gulf of California): it is a deep-sea aerobic mesophilic heterotrophic bacterium described as a new species of the genus Alteromonas and the proposed name is Alteromonas infernus (Raguenes et al., 1997b). This bacterium A. infernus can produce a water-soluble EPS at laboratory conditions in fermentor; the EPS is a branched heteropolysaccharide and its repeating unit is a nonasaccharide (Fig. 1): 4 glucose residues, 2 galactose, 2 glucuronic acids and 1 galacturonic acid bearing a sulphate group at C2 position (Roger et al., 2004). The biological activities of this sulphated EPS and its oversulphated derivatives have been studied in different medical fields and are presented below in Section 5.3. A very interesting new EPS-producing Vibrio has been discovered from deep-sea environment during the Hero cruise from the dorsal integument of the polychaete annelid Alvinella pompejana living in a rift system of the East pacific Rise. It is a deep-sea facultatively anaerobic heterotrophic mesophilic new organism identified as a new species of the genus Vibrio and the proposed name is Vibrio diabolicus (Raguenes et al., 1997a). It is the first species of Vibrio to be isolated from a vent sample and it can produce a very innovative EPS presenting a chemical resemblance to hyaluronic acid and heparan sulphate after chemical modification. The structure of this EPS consists of a linear tetrasaccharide repeating unit: 2 glucuronic acid, 1 N-acetylated glucosamine and 1 N-acetylated galactosamine residues (Fig. 2). It is very rare to find hexosamine sugars in bacterial EPS (Rougeaux et al., 1999). The biological activities of this unusual GAG-like bacterial EPS have been studied and are described below in Section 5.3.

5.3

EPS DERIVATIVES OBTAINED BY CHEMICAL MODIFICATIONS

As mentioned above the marine EPS present naturally interesting physico-chemical properties and do not need post-fermentation processes to be attractive in a number of industrial applications

5.8

Natural Bioactive Molecules: Impacts and Prospects

Fig. 1

Repeating unit of the marine bacterial EPS produced by Alteromonas infernus

Fig. 2

Repeating unit of the marine bacterial EPS produced by Vibrio diabolicus

such as biosurfactants/bioemulsifiers and also biosorbent to remove toxic metal pollutants (Jain et al., 2012; Satpute et al., 2010). But for some specific applications especially in pharmaceutical industries, it is interesting to modify the EPS in order to improve their physico-chemical and biological properties.

5.3.1

Exopolysaccharides as Biomaterials

Owing to their structural diversity, EPS constitute a large source of very attractive compounds to build biomaterials for tissue engineering (scaffolds, hydrogels, wound dressings and many more), drug vehicles, viscosupplementation, controlled release of drugs. In fact, EPS are renewable, biodegradable, biocompatible, non-toxic as well as some of them are bioactive (Rinaudo, 2008). For example, hyaluronic acid derivatives are often obtained by chemical modifications of their hydroxyl groups (-OH), carboxyl goups (-COOH) and also amino groups (-NHAc). By esterification of the carboxyl groups, the hydrophobic character of hyaluronic acid is increased and can give a 3D scaffold (non-woven pad) for cartilage engineering (Borzacchiello et al., 2007).

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5.9

Crosslinking agents are also used to produce hydrogels with many polysaccharides. Hydrogels are physically or chemically crosslinked polymer networks that are able to absorb large amount of water. The hydrogels based on natural polymers or polysaccharides are close to living tissues and can temporarily replace them during the healing process. Polysaccharides from the GAG family such as chondroitin sulphate and hyaluronic acid have been extensively used to prepare polysaccharides-based products. So the discovery of new original marine bacterial EPS extends the scope of production of tailor-made biomaterials (Van Vlierberghe et al., 2011). The technique of blending consists to mix different bioactive molecules to tailor the final properties of interest of biomaterials; calcium or collagen-alginate wound dressing (or gel) are very useful to limit wound secretions and minimize bacterial contaminations (Laurienzo, 2010). A recent study showed that the incorporation of two-GAG like marine EPS produced by Vibrio diabolicus and Alteromonas infernus into injectable hydrogel can improve the final properties of these biomaterials for cartilage tissue engineering, increasing cell viability, making cell proliferation possible and improving mechanical properties (Rederstorff et al., 2011). Polysaccharide coating can be used for modifying the surface of biomaterials (implants) and adding specific properties such as biocompatibility or antibacterial properties notably in the orthopedic field. The effects of the titanium implant coating with polysaccharides conjugated with vascular endothelial growth factor (VEGF) on the proliferation, differentiation and mineralization of osteoblasts as well as the antibacterial property were investigated. The results showed that this particular coated titanium implant can promote osteoblast functions and concurrently reduce bacterial adhesion (Hu et al., 2010).

5.3.2

Exopolysaccharides as GAG-like derivatives

Glycosaminoglycans (GAG) are present in all animal cell surfaces and in the extracellular matrix (ECM); they are complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. In fact, the large structural diversity of GAG allows specific interactions to bind and regulate a number of key proteins (such as chemokines, cytokines, growth factors, enzymes, adhesion molecules, etc.) involved in cell signalling, cell development and cell integrity. The structural variations in chain length and substitution patterns (sulphation, acetylation…) in polysaccharides are crucial and can modify their affinity for a specific protein and consequently their efficacy. The understanding of the molecular basis of such interactions opens a large potential in the drug discovery to modulate disease processes observed in cancer, inflammatory diseases, pathogen infections, thromboembolic disease, etc. (Bhaskar et al., 2012; DeAngelis, 2012; Gandhi and Mancera, 2008; Mulloy, 2005; Perez and Mulloy, 2005) Heparin belongs to the GAG group (as hyaluronic acid described above) and is still extracted from animals (mainly porcine intestinal mucosa). Heparin is still in widespread clinical use as an intravenous anticoagulant with more than 100,000 kilograms produced annually worldwide. The anticoagulant activity of heparin is due to its affinity to a serine-protease inhibitor, the antithrombin. Heparin inhibits serine-proteases such as thrombin or factor Xa through the catalysis of antithrombin according to its molecular weight. Thus, the low molecular weight

5.10

Natural Bioactive Molecules: Impacts and Prospects

heparin (LMWH with a characteristic average MW of 6.6 mM) in a dose- and Ca2+-dependent manner. Latter on, the same group of investigators elaborated its mechanism of action using whole-cell patch clamp technique to evaluate the effect on KATP from b-cells isolated from mouse islets (Abudula et al., 2008). It was found that in the presence of 16.7 mM glucose, addition of rebaudioside A at the maximally effective concentration of 10−9 M increases the ATP/ADP ratio significantly, while it does not change the intracellular cAMP level. The investigators also showed that rebaudioside A (3) and stevioside (2) at respective doses of 10−9 and 10−6 M reduced the ATP-sensitive potassium channel (KATP) conductance in a glucose-dependent manner. Moreover, rebaudioside A also stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner; the insulinotropic effect of the test compound (3) was supposed to be mediated via inhibition of ATP-sensitive K(+)-channels, which requires the presence of high glucose level (Abudula et al., 2008). The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio; the experimental findings, thus, indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia. Recently, Wang et al., (2012) demonstrated that stevioside may be a promising antidiabetic drug that can act through the modulation of adipose tissue inflammation and systemic insulin resistance. The investigators showed that long-term stevioside treatment ameliorates high-fat diet (HFD)induced insulin resistance in mice; oral administration of the drug for 1 month had no effect on body weight, but it significantly improved fasting glucose, basal insulin levels, glucose tolerance and whole body insulin sensitivity. It was also found that these changes are accompanied with decreased expression levels of several inflammatory cytokines in adipose tissue, including TNF-a, IL6, IL10, IL1b, KC, MIP-1a, CD11b and CD14. Moreover, macrophage infiltration in adipose tissue was remarkably reduced by stevioside. Finally, stevioside significantly suppressed the nuclear factorkappa b (NF-kB) signaling pathway in adipose tissue. Hence, such insulin-sensitizing effect may partly be associated with an attenuated inflammatory state and downregulated NF-kB signaling in adipose tissue (Wand et al., 2012). On the basis of the afore-discussed experimental observations using in vitro cell, whole animal, and human models, it appears that stevioside and related compounds (steviol and rebaudioside A) affect plasma glucose via modulation of insulin secretion and sensitivity, which enhance glucose removal from the plasma. These compounds also inhibit intestinal glucose absorption and glucose generation from the liver by altering the activities of a number of key enzymes involved in glucose synthesis, thereby reducing plasma glucose input. It is note-worthy that the effect of stevioside is

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dependent largely on the plasma glucose level, being observed only when plasma glucose level is elevated (Chatsudthipong and Muanprasat, 2009).

6.7.4

Anti-diarrheal Activity

Shiozaki et al., (2006) demonstrated that stevioside possesses an inhibitory effect on intestinal smooth muscle contraction, stimulation of which results in hypermotility-associated diarrhea. At a concentration of 1 mM, the drug was found to exert inhibitory effect against CaCl2 (10 mM)induced contraction of isolated guinea pig ileum by 40%, and the mechanism was thought to be related to its inhibitory effect on Ca2+ influx into muscle cells. Thus, stevioside may be useful in the treatment of diarrhea resulting from intestinal hypermotility, such as irritable bowel syndrome and inflammatory bowel disease (Chatsudthipong and Muanprasat, 2009). Stevioside and also its major metabolite, steviol (1), were reported to affect ion transport in many types of tissues, such as the kidney, pancreas, and intestine (Pariwat et al., 2008); such effect of stevioside (2), steviol (1), and its analogs on intestinal Cl−-secretion was investigated in detail using human T84 epithelial cell line by Pariwat et al., (2008). Short-circuit current measurements showed that steviol and its analogs isosteviol, dihydroisosteviol and isosteviol 16-oxime inhibit forskolin-induced Cl−-secretion in a dose-dependent manner with IC50 values of 101, 100, 9.6, and 50 mM, respectively, whereas the parent compound stevioside had no such effect. Apical current measurement indicated that dihydroisosteviol targeted the cystic fibrosis transmembrane regulator (CFTR); the inhibitory action of this compound was found reversible and was not associated with changes in the intracellular Camp level. In addition, it did not affect calcium-activated chloride secretion and T84 cell viability. In vivo studies using a mouse closed-loop model of cholera toxininduced intestinal fluid secretion showed that intraluminal injection of 50 mM dihydroisosteviol reduced intestinal fluid secretion by 88.2% without altering fluid absorption, thereby indicating that dihydroisosteviol and similar compounds could be a new class of CFTR inhibitors that may be useful for further development as anti-diarrheal agents.

6.7.6

Enzyme Inhibitory Activity

Stevioside, at concentration level of ~1.5 mM, was found to have no effect on activity of glutamate dehydrogenase of rat or bovine liver (Bracht et al., 1985); however, the drug exhibited inhibitory effects or various enzymatic activities like ATP dependent swelling, NADH oxidase activity, DNP-stimulated ATPase succinate dehydrogenase and succinate oxidase activity at quite high concentration as compared to other ent-kaurane analogs of Stevia (Bracht et al., 1985).

6.8

MUTAGENICITY OF ENT-KAURENE GLYCOSIDES

Pezzuto et al., (1985) reported that stevioside (2) is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system; while the steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of supernatant fraction (S-9) derived from the livers of aroclor 1254-pretreated rats. The investigators (Pezzuto et al., 1985) also indicated that unmetabolized steviol and structurally related species, isosteviol was not active regardless of metabolic activation.

6.24

Natural Bioactive Molecules: Impacts and Prospects

Similarly, chemical reduction of the unsaturated bond linking the carbon atoms 16 and 17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B, that were not mutagenic. In addition, ent-kaurenoic acid (Fig. 1b) was also found to be inactive. The study revealed that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxyl group at position 13 and an unsaturated bond joining the carbon atoms at position 16 and 17 (Pezzuto et al., 1985). The aglycone, steviol (1), was also found to produce dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL (Matsui et al., 1996). Metabolic activation systems containing supernatant fraction (S-9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5, 6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis (Matsui et al., 1996). Steviol was weakly positive in the umu test using S. typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S-9 activation system, was negative in other assays, i.e. the reverse mutation assays using S. typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKMlOl and the rec-assay using Bacillus subtilis. Thus, steviol was found negative in the mouse micronucleus test but the mutagenic in a forward mutation assay, and caused chromosome aberrations and gene mutations in mammalian cells (Matsui et al., 1996) and plasmid mutagenesis (Matsui et al., 1989). Stevioside and steviol were not mutagenic toward S. typhimurium TA97, TA98, TA100, TA102 and TA104 either with or without S-9 mix at doses up to 5 mg per plate. They were not toxic to S. typhimurium even at the highest dose. Neither stevioside nor steviol was mutagenic in S.typhimurium TA1535, TA1537 and E. coli WP2 wvM/pKM101 in the presence of S-9 mix. These results suggested that neither stevioside nor steviol is mutagenic in S. typhimurium TA strains and E. coli WP2 wvM/pKMlOl either with or without metabolic activation. Stevioside (2) was found to induce no significant increase of the mutation frequency of S. typhimurium TM677, even at the highest dose of 10 mg/ml, either with or without S-9 mix. However, steviol induced a significant dose-related increase in the mutation frequency when S-9 mix was present. Steviol increased not only the mutation frequency but also the raw number of 8-AZ resistant colonies (mutants) per plate, ruling out the possibility that the mutagenicity of steviol was an artefact due to the analysis of the data (Procinska et al., 1991). In the absence of S-9 mix, steviol did not give rise to an increase in the mutation frequency. To determine the genetic requirements for the mutagenicity of steviol, the authors compared the sensitivities of three isogenic tester strains in the presence of S9 mix. Of the three strains examined, S. typhimurium TM677 (uvrB, rfa, pKMlOl) exhibited much higher sensitivity toward steviol than did S. typhimurium TM35 (uvrB, rfa) or KH75 (rfa, pKMlOl). These results suggest that steviol is mutagenic to S. typhimurium TM677 in the presence of S-9 mix and also that rfa mutation, deficiency of excision repair and presence of plasmid pKMlOl are all required for the maximum mutagenesis. Suttajit et al., (1993) reported positive results for reverse mutations in the S. typhimurium strain TA98 with and without S-9 extract at a 50 mg/plate for 99% pure stevioside with and without S9 extract. However in another study, Klongpanichpak et al., (1997) did not find stevioside to be mutagenic in TA98 at similar

Brahmachari: Stevioside and Related Compounds

6.25

concentration. However, they used S9 extract from rats, mice, hamsters and guinea-pigs, while Suttajit et al., (1993) showed the strongest result without S-9 extract. The ability of stevioside and rebaudioside A to cause reverse mutations as indicated by TA98 needs to be further investigated, because such mutations suggest the possibility of carcinogenesis. Besides, stevioside was also found to induce no significant increase in the specific p-galactosidase activity of S. typhimurium TA1535/pSK1002 either with or without S-9 mix as observed upon treatment with the compound at various doses ranging from 1250-5000 mg/ml (incubation period, 2 days) (Matsui et al., 1996). However, steviol was reported to induce an increase (~2-fold) in the specific activity of b−galactosidase at concentrations of 313-1250 mg/ml (specific enzymetic activity of 53.6 U/A600 at 1250 mg/ml) in the absence of S-9 mix and 625-2500 mg/ml in the presence of S-9 mix (specific enzymetic activity = 99.9 U/A600 at 2500 mg/ml; incubation period, 2 days). Under the same conditions, the positive controls furylfuramide (specific enzymetic activity = 1759 U/A600 at 0.03 mg/ml without S-9 mix) and 2-aminoanthracene (specific enzymetic activity = 1848 U/A600 at 3.3 mg/ml with S-9 mix) substantially increased the specific activity of b-galactosidase of S.typhimurium TA1535/pSK1002; these results suggested that steviol is weakly positive in the umu test either with or without metabolic activation. It is also interesting to note that mutation frequency of steviol ( 0.60 × 104 at the maximal dose of 10.0 mg/ml) in the absence of S-9 mix is ten times lower than that ( 66.0 × 104 at the same dose) in the presence of S-9 mix (Matsui et al., 1996). Stevioside was also found to cause DNA breakage in blood, spleen, liver, and brain cells in rats (Nunes et al., 2007). Thus it was concluded that metabolically-activated steviol was found to cause dose-related positive responses in several mutagenicity tests, thereby, indicating that a steviol derivative is likely responsible for its mutagenic activity, but the metabolite has not been identified (Brusick, 2008). Although steviol (1) was mutagenic and clastogenic in bacteria and cultured mammalian cells, it did not exhibit any positive response in the mouse micronucleus test. This in vivo test result does not necessarily mean that neither mutagenic nor clastogenic metabolites are generated from steviol in vivo. It could be possible that steviol produced adverse metabolites in vivo but they did not reach the bone marrow, the target organ for the micronucleus test. In fact, dimethylnitrosamine and diethylnitrosamine, potent hepatocarcinogens, do not give rise to a substantial increase in the number of icronucleated cells in mouse micronucleus test, probably because the short-lived active metabolites generated in the liver cannot reach the bone marrow (Proudlock and Allen, 1986; Cliet et al., 1993). It might also be possible that the genotoxic metabolites of steviol could reach bone marrow but that they predominantly induced point mutations, such as base change or frameshifts, rather than chromosome aberrations, so that no micronucleated blood cells were found in the steviol-treated mice. Thus, further work is necessary to predict the genotoxic risk of steviol to human beings. Since steviol requires S-9 activation for mutagenesis and clastogenesis in vitro, the genotoxic damage in the liver of rats or mice should be examined and for this purpose the liver unscheduled DNA synthesis (UDS) assay or a transgenic mutagenicity assay were suggested to be appropriate for the further assessment of the genotoxic potential of steviol in vivo (Matsui et al., 1996).

6.26 6.9

Natural Bioactive Molecules: Impacts and Prospects

SAFETY EVALUATIONS

The toxicology and safety of stevioside used as a sweetener were studied by different investigators (Ishima and Katayama, 1976; Soejarto et al., 1982; Gianfagna et al., 1983; Genus et al., 2007; Nunes et al., 2007). Stevioside (2) does not appear to be carcinogenic (Bracht et al., 1985). Recent studies have demonstrated that a portion of stevioside is absorbed and degraded to steviol, which appear to undergo further metabolism (Genus et al., 2007). Other studies indicate that none of the digestive enzymes from gastro-intestinal tract of different animals and man are able to degrade stevioside into steviol (Wingard et al., 1980; Hutapaeet al., 1997; Koyama et al., 2003a,b). Nevertheless, in feeding experiments with rats and hamsters stevioside was metabolized to steviol by the bacterial flora of the caecum (Geuns, 2003). Although animal studies did not show any adverse effects or toxicity associated with stevioside consumption (Toskulkao et al., 1997; Wasuntarawat et al., 1998), the limited data is available on its metabolism and safety in humans to approve its use as a non nutritive sweetener; only the herbal form of the Stevia plants is allowed for use in foods as a flavor enhancer and also as a tea (Gougeon et al., 2004). Stevioside was found to bear a very low acute oral toxicity (LD50 between 8.2 and 17g/kg) in the mouse, rat and hamster (Mitsuhashi, 1976; Medon et al., 1982). The safety of oral stevioside in relation to carcinogenic activity is evidenced by the works of different groups with rats (Yamada et al., 1985; Xili et al., 1992; Toyoda et al., 1997; Nunes et al., 2007). Stevioside was evaluated for safety by the 51st meeting of the JECFA (Joint FAO/WHO Expert Committee on Food Additives) in 1998 (WHO TRS no. 891). The JECFA considered the toxicity data of steviol glycosides in 1999 but was unable to recommend on ADI (Acceptable Daily Intake) due to insufficient data, including a lack of human metabolism studies, a lack of information on the purities of the product, and lack of adequate in vivo mutagenicity studies; later on, by 2004, JECFA set a temporary ADI of 2 mg/kg bw/day for Stevia at that time and requested extensive additional information to be submitted by 2007 on the effects of steviol glycosides in humans, including special populations such as people with diabeties or hypertension (JECFA, 2004). Melis et al., (2009) evaluated the renal excretion of steviol, and also clarified the actual participation of this compound on the renal excretion of glucose in rats, which has been previously suggested as the preferential action of steviol on the Na+-glucose renal tubular transport system; on the basis of their detailed experimental observations the investigators concluded that steviol is secreted by renal tubular epithelium, causing diuresis, natriuresis, kaliuresis and a fall in renal tubular reabsorption of glucose (Melis et al., 2009). Recently, Maki et al., (2008) demonstrated that consumption of as much as 1000 mg/day of rebaudioside A produced no clinically important changes in blood pressure in healthy adults with normal and low-normal blood pressure. Yamada et al., (1985) demonstrated that no significant effect was observed on spermatogenesis, nor on the interstitial cell proliferation as well as tumor formation in the testes of F344 rats when fed a ration containing up to 1% stevioside (95.2% purity) for 22 months. This observation was supported by various studies on fertility or reproduction in mice, rats or hamsters (Akashi and Yokoyama, 1975; Xili et al., 1992; Mori et al., 1981; Sinchomi and Marcorities, 1989; Yodyingyuad and Bunyawong, 1991). However, steviol, the metabolite of stevioside, was found to be toxic to pregnant hamsters and their foetuses when administered on day 6 through 10 of gestation at doses

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6.27

of 0.5-1.0 g/kg body weight/day (Wood et al., 1955). It was observed that the drug produces decreased maternal weight gain and high maternal mortality; the number of live births per litter and mean fetal weight decreased. Moreover, the maternal kidneys showed a dose-dependant increase in severity of convoluted tubules in the kidneys (Wood et al., 1955). In 2006, the World Health Organization (WHO) performed a thorough evaluation of recent experimental studies of Stevia extracts conducted on animals and humans, and concluded that “stevioside and rebaudioside A are not genotoxic in vitro or in vivo and that the genotoxicity of steviol and some of its oxidative derivatives in vitro is not expressed in vivo.” They also found no evidence of carcinogenic activity of Stevia extracts and suggested the possibility of its health benefits; but at the same time the organization recommended for a further study to determine its proper dosage (Benford et al., 2006). Extensive scientific research by Scientific Committee on Food (SCF) has reported the safety of the common sweeteners like acesulfame K, aspartame, cyclamate, saccharin, sucralose, and also stevioside by several in vitro and in vivo animal studies, tests in human and in some cases of epidemiological studies and also recommended their ADI (Acceptable Daily Intake) — the SCF observed that the consumption of sweeteners in the quantities within the ADI does not constitute a health hazard to consumers (Kroger et al., 2006). Among these synthetic or semi-synthetic sweeteners, stevioside is a naturally occurring molecule of interest because of its low toxicity level.

6.10

CLINICAL TRIALS

Several clinical studies (Melis et al., 1986; Chan et al., 1998; Chan et al., 2000; Jeppesen et al., 2000; Liu et al., 2003; Wong et al., 2004; Chen et al., 2005) reveal that no significant effects on blood pressure and blood sugar occur with high doses of steviol glycoside applied for several months (Sharma et al., 2009). However, two clinical trials on hypertensive patients reported reduction of blood pressure after long-term treatment with stevioside (2) (Sharma et al., 2009). Another clinical trial using stevioside proved a beneficial effect on post-prandial glucose homeostasis in type 2 diabetic patients. Clinical studies were also conducted to examine the effects of rebaudioside A (3) on the blood pressure of healthy subjects and on glucose homeostatis in type 2 diabetics, and it was concluded that 1000 mg/day of rebaudioside A had no clinically significant effects on blood pressure or on glucose homeostats or blood lipids in type 2 diabetic patients. However, no adverse effects were observed in these studies (Toyoda et al., 1997; Carakostas et al., 2008).

6.11

CURRENT MARKET STATUSES

Since the approval of Stevia-derived sweetener ‘Reb A’ as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) in December 2008 for its use in foods and beverages, Stevia-derived sweeteners have received world-wide attention and popularity. GLG Life Tech agreed with Indian-based Global AgriSystem Private Limited to pursue a joint venture in commercializing Stevia-based sweetener in India (Puri et al., 2011). Currently, such zero-calorie sweetener finds United States as its largest market. China occupies the first position in producing and supplying Stevia-based sweetener in the world; the production output is increasing significantly

6.28

Natural Bioactive Molecules: Impacts and Prospects

— it has been estimated that the production of Stevia-based sweetener in 2009 increased to 3096 ton from 2073 ton in 2007, and 80% of the production in China was exported. It is also estimated that China’s Stevia sweetener capacity has been expanding to 11,789 ton/year in 2009 from around 5000 ton/year in 2007 (Pasricha, 2009). China is also an emerging consumption country for Stevia sweetener, with total consumption volume of around 620 ton in 2009 (Puri et al., 2011). India, with its population of 1.14 billion and growing rates of obesity, is anticipated to offer “an untapped market” which creates a significant opportunity for Stevia suppliers as well (Scott-Thomas, 2010). Stevioside has a 20% market share of low-calorie sweeteners in Japan (Kikuchi, 1985). The market potential of these sweeteners ranges between 4 and 8% in Japan, whereas in Eastern countries, the market is valued at US $1200 million (Puri et al., 2011). Pure Circle, a leading supplier of Stevia sweetener, currently has a total of about 15,000 ha contracted in Kenya, Paraguay, Columbia, Indonesia, Vietnam, Thailand and China, for growing Stevia plantations. The companies are also intending for constructing joint extraction facilities in India as the demand rises up. Being natural and low-caloric, Stevia sweetener is progressively achieving world’s attention and is very likely to be approved by many other countries as well in the near future for its consumption as ingredient in food and beverages, and this which would create a global market for the commercialization of Stevia sweetener.

6.12

CONCLUDING REMARKS

Stevioside and related ent-kaurene glycosides have been established as natural zero-calorie/lowcalorie sweeteners, and many of them in the form of crude plant products are being used commercially in many countries as food additives for sweetening a variety of products; potent sweetness intensities of these glycosides in comparison to sucrose have projected them as cost-effective substitutes of sugar. Beyond their non-caloric sweetness property, these ent-kaurene diterpene glycosides may also offer therapeutic benefits, as they have anti-hypertensive, anti-glycaemic, anti-inflammatory, anti-diarrheal, enzyme inhibitory, diuretic, anti-tumor, anticancer, and immunomodulatory actions. It is very interesting to note that their effects on plasma glucose level and blood pressure are only observed when these parameters are higher than normal. Besides the pharmacological activities and therapeutic applications of stevioside and related compounds, the present article also summarizes current knowledge of the natural distribution of such compounds, their structural features, plausible biosynthetic pathways, pharmacokinetics along with an insight into the structure-sweetness relationship, safety evaluation, and clinical trials of these ent-kaurene glycosides. However, these ent-kaurene gycosides have still not been approved as food ingredient in the United States or the European Union; but Stevia in the leaf or extracted form is permitted to be sold in the US only as dietary supplement. It has been demonstrated that steviol, a metabolite of stevioside, produced in the human intestinal microflora is genotoxic and induces developmental toxicity. Remaining uncertainties about the safety of stevioside must be resolved, with particular attention to the reproductive effects that have given rise to FDA concern (FDA, 2007). Lacking this critical data, it will not be possible to proceed with development of stevioside and its derivatives as pharmaceutics or as non-caloric sweeteners for human foods. Another important issue is that most of the available studies of stevioside and related compounds (steviol and rebaudioside A) have been

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performed in cell cultures, isolated tissues, or experimental animals. Only few studies have been carried out in humans; comprehensive clinical studies in humans are needed so as to proceed for any rational design of pharmaceutics related to stevioside. Hence, rigorous researches not only on their prospective uses as pharmaceutical agents including zero-calorie sweeteners, but also on their toxicological evaluation are demanding to resolve issues pertaining to safety concerns. The present overview is devoted to address all such issues that would be helpful to prospective investigators to design future research directions.

Acknowledgements This comprehensive review is based upon the published article entitled “Stevioside and Related Compounds – Molecules of Pharmaceutical Promise: A Critical Overview” by Goutam Brahmachari et al., (2011), Arch Pharm Chem Life Sci 1: 5-19 (Wiley-VCH Verlag GmbH & Co. KGaA). Materials used in this article are reproduced with permission from the Publisher.

Abbreviations ADI: ADME: CHL: CPP: DMBA: DXP: DXR: DXS: ESTs: GA: GAP: GGPP: HMG-coA: ID50: IPP: IPP: IVGT: JECFA: LD50: MEP: MEP: MVA: NIDDM:

Aceptable Daily Intake Absorption, distribution, metabolism, and excretion Chinese hamster lung fibroblast cell line ent-Copalyl pyrophosphate 7,12-dimethylbenz-[a]-anthracene 1-Deoxy-D-xylulose 5-phosphate 1-Deoxy-D-xylulose 5-phosphate reductoisomerase 1-Deoxy-D-xylulose 5-phosphate synthase Expressed sequence tags Gibberellin Glyceraldehyde 3-phosphate Geranylgeranyl pyrophosphate 3-Hydroxy-3-methylglutaryl co-enzyme A 50% Inhibitory Dose Isopentenyldiphosphate Isoprenyl diphosphate Intravenous glucose tolerance test Joint FAO/WHO Expert Committee on Food Additives 50% Lethal Dose 2-C-Methyl-D-erythritol-4-phosphate 2-C-methyl-D-erythritol-4-phosphate Mevanolic acid Non-insulin dependant diabetes mellitus

6.30

Natural Bioactive Molecules: Impacts and Prospects

PEPCK: PKU: SCF: TPA: UDS: US:

Protein levels of phosphoenyl pyruvate carboxy kinase Phenylketonuria Scientific Committee on Food 12-O-tetradecanoyl-phorbol-13-acetate Unscheduled DNA synthesis United States

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Samson SM, Dotzlaf JE, Slisz ML, Becker GW, Van Frank RM, Veal LE, Yeh W-K, Millar JR, Queener SW and Ingolia TD (1987). Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Nature Biotechnology 5, 1207–1214. Sehar I, Kaul A, Bani S, Pal HC and Saxena AK (2008). Immune up regulatory response of a non-caloric natural sweetener, stevioside. Chem Biol Interact 173, 115–121. Sharma M, Thakral NK, Thakral S (2009). Chemistry and in vivo profile of ent-kaurene glycosides of Stevia rebaudiana Bertoni – An overview. Nat. Prod. Rad. 8, 181–189. Shiozaki K, Fujii A, Nakano T, Yamaguchi T and Sato M (2006). Inhibitory effects of hot water extract of the Stevia stem on the contractile response of the smooth muscle of the guinea pig ileum. Biosci Biotechnol Biochem 70, 489–494. Sinchomi D and Marcorities P (1989). Etude de l’activité anti-androgénique d’un extrait de Stevia rebaudiana Bertoni. Plantes médicinales et phtythérapie 23, 282–287. Soejarto DD, Kinghorn AD and Farnsworth NR (1982). Potential sweetening agents of plant origin. III. Organoleptic evaluation of stevia leaf herbarium samples for sweetness. J Nat Prod 45, 590–599. Sponsel VM (2001). The Deoxyxylulose Phosphate Pathway for the Biosynthesis of Plastidic Isoprenoids: Early Days in Our Understanding of the Early Stages of Gibberellin Biosynthesis. J Plant Growth Regul 20, 332–345. Srimaroeng C, Chatsudthipong V, Aslamkhan AG and Pritchard JB (2005a). Transport of the natural sweetener stevioside and its aglycone steviol by human organic anion transporter (hOAT1; SLC22A6) and hOAT3 (SLC22A8). J Pharmacol Exp Ther 313, 621–628. Starratt AN, Kirby CW, Pocs R and Brandle JE (2002). Rebaudioside F, a diterpene glycoside from Stevia rebaudiana. Phytochemistry 59, 367–370. Susuki H, Kasai T and Sumihara M (1977). Effects of oral administration of stevioside on level of blood glucose and liver glycogen of intact rats. Nippon Nogei Kagaku kaishi 51, 171–173. Suttajit M, Vinitketkaumnuen U, Meevatee U and Buddhasukh D (1993). Mutagenicity and human chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni. Environ Health Perspect (Suppl.) 101, 53–56. Takasaki M, Konoshima T, Kozuka M, Tokuda H, Takayasu J, Nishino H, Miyakoshi M, Mizutani K and Lee K-H (2009). Cancer preventive agents. Part 8: chemopreventive effects of stevioside and related compounds. Bioorg Med Chem 17, 600–605. Toskulkao C, Chaturat L, Tenchocharoen P and Glinsukon T (1997). Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species. Drug Chem Toxicol 20, 31–44. Toskulkao C, Sutheerawatananon M, Wanichanon C, Saitongdee P and Suttajit M (1995b). Effects of stevioside and steviol on intestinal glucose absorption in hamsters. J Nutr Sci Vitaminol 41, 105–113. Toskulkao C, Sutheerawattananon M and Piyachaturawat P (1995a). Inhibitory effect of steviol, a metabolite of stevioside, on glucose absorption in everted hamster intestine in vitro. Toxicol Lett 80, 153–159. Totté N, Charon L, Rohmer M, Compernolle F, Baboeuf I and Geuns JMC (2000). Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Lett 41, 6407–6410. Totté N, Van den Ende W, Van Damme EJM, Compernolle F, Baboeuf I and Geuns JMC (2003). Cloning and heterologous expression of early genes in gibberellin and steviol biosynthesis via the methylerythritol phosphate pathway in Stevia rebaudiana Bertoni. Can J Bot 81, 517–522.

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7 Vitamin B6 Derived Cofactor Pyridoxal5¢-phosphate: Promising Role in Drug Development Programme Kuheli Chakrabarty and Gourab Kanti Das* Department of Chemistry, Visva-Bharati University, Santiniketan-731 235 West Bengal, India

ABSTRACT Pyridoxal-5¢-phosphate (PLP) is derived from vitamin B6 and serves as a versatile enzyme cofactor that facilitates many chemical transformations. Particularly, it is a crucial cofactor for various bio transformations of amino acids including racemization, decarboxylation, deamination and transaminations. Many of the PLP-dependent enzymes are essential and therein offer the possibility to target diseases. There are several B6 enzyme inhibitors, which are currently in use as drugs. Some recent developments on drug research reveal that there is immense possibility to use PLP and its novel adducts particularly with amino acids to serve as effective drugs against various diseases caused by parasites. MC-1, an investigational drug acts as purinergic receptor antagonist and is found suitable in the treatment of cardiovascular diseases. PLP phosphonate analogues are also effective as cardioprotective agents. In this chapter, the role of PLP, PLP conjugates and PLP dependent enzymes as drugs and drug targets are discussed critically considering certain aspects related to medicinal chemistry. Keywords: Vitamin B6, pyridoxal-5¢-phosphate, biochemistry, mode of action, drug efficacy, drug development.

7.1

INTRODUCTION

The coenzyme pyridoxal-5¢-phosphate (PLP) (1) is at the heart of chemistry conducted by a number of enzymes (Percudani & Peracchi, 2003). *Corresponding author: [email protected]

7.2

Natural Bioactive Molecules: Impacts and Prospects

PLP can be synthesized de novo from pre-existing precursor in bacteria, fungi, and plants from erythrose 4-phosphate, pyruvate and D-glyceraldehyde 3-phosphate. On the contrary, in human PLP cannot be synthesized de novo and must be supplemented from diet as vitamin B6 that primarily consists of three interrelated vitamers including pyridoxal (2), pyridoxine (3) and pyridoxamine (4).

In the cells, the vitamers are converted into PLP through phosphorylation by pyridoxal/ pyridoxine/pyridoxamine kinase and if necessary, through subsequent oxidation by pyridoxine / pyridoxime-5-phosphate oxidase (Bahn et al., 2002). Structurally, aldehyde group of PLP is bound covalently as internal aldimine to the e-amino group of lysine residues close to N-terminus in a PLP-dependent enzyme (Scheme 1).

Scheme 1

Internal and external aldimine linkage of PLP with enzyme and substrate respectively

Practically, most of the PLP-dependent enzymes are involved in transformation of amino acids like racemization, decarboxylation, deamination and transamination reactions (Percudani & Peracchi, 2003). During catalysis where PLP acts as a cofactor results in the reduction of energy for conversion of amino acid substrates to a zwitterionic species and this enables the apoenzyme to cleave the substrate target bond yielding the product (Richard & Amyes, 2004). The catalytic process starts from the native internal aldimine of PLP-enzyme to the formation of external aldimine linkage of amino group of substrate and PLP forming coenzyme-substrate Schiff base replacing the e-amino group of lysine-enzyme. This zwitterionic coenzyme-substrate Schiff base is the common intermediate for all biological processes involving PLP-dependent enzymes (Scheme 2).

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

Scheme 2

7.3

PLP dependent biological transformations of amino acids

In addition to the PLP enzymes with indispensible roles in the primary metabolism of cells, many other PLP-dependent enzymes act in secondary metabolism as well. These include synthesis of important signalling molecules, polyamines, small-molecule poly-cations that are essential for the growth of eukaryotic cells (Casero and Marton, 2007). Particularly, PLP-dependent enzymes acting in secondary metabolism together with few PLP enzymes of primary metabolism have become targets of medicinal interest (Amadasi et al., 2007). Some recent developments on drug research also reveal that there is immense possibility to use PLP and its novel adducts, particularly amino acid PLP adducts to serve as effective drugs against various diseases caused by parasites, and in cardiovascular diseases as well (Kandzari et al., 2005). There are reports describing the potentiality of PLP-dependent enzymes as antitumor and anticardiovascular agents (El-Sayed, 2010).

7.2 7.2.1

PLP MOLECULE AS A DRUG PLP as Purinergic (P2) Receptor Antagonist

In 1949, it was found that monkeys given a vitamin B6 deficient diet developed atherosclerosis (Rineheart & Greenberg, 1949). Various other studies have confirmed that adenosine tri phosphate (ATP) exerts a positive inotropic action on the myocardium through purinergic receptors but there is very little information regarding the modification of the action of ATP on the heart. Recent studies using isolated perfused rat hearts have revealed that PLP, an active form of vitamin B6, shows antagonism towards ATP-induced positive ionotropic effect (Wang et al., 1999). It was concluded that PLP may antagonizes the action of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. It was also suggested that PLP might serve as a valuable tool for monitoring the role of purinergic receptors in cellular function. Because of these reports, huge research work is going on to find out the possibility of treating heart disease in humans using vitamin B6 as therapeutic agents. It is now established that patients who have suffered a myocardial infarction display lower level of PLP (Serfontein et al., 1985). Research papers published so far have supported the notion that a high dose of supplemental pyridoxine may reduce or even prevent cardiovascular events (Ellis & McCully, 1995). It has been postulated that dietary intake of vitamin B6 increases the level of PLP which is the active coenzyme required for the catabolism of homocysteine. Both markedly and mildly elevated levels of homocysteine, an atherogenic amino acid is now considered to be a risk factor for cardiovascular diseases (Brattstrom & Wilcken, 2000). The clinical relevance of the inverse relationship between homocysteine and

7.4

Natural Bioactive Molecules: Impacts and Prospects

vitamin B levels is still under investigation. It is also reported that pyridoxine supplementation can significantly improve endothelial function and potential beneficial effects in terms of the reduction of transplant coronary artery disease have been noted (Miner et al., 2001). MC-1 (Medicure International Inc, Winnipeg, Manitoba, Canada) is an investigational drug which contains pyridoxal-5¢-phosphate monohydrate. It is a naturally occurring metabolite of pyridoxine and is produced in the mammalian cells by phosphorylation and oxidation reaction. As a purinergic (P2) receptor antagonist it blocks intracellular influx of Ca2+, which in turn reduces cell damage during experimental episodes of ischemia and reperfusion (Kandzari et al., 2005). Coronary Artery Bypass Graft (CABG) surgery is one of the most important therapeutic options for the treatment of multi-vessel coronary artery disease and has become the most common surgical treatment for heart disease in all over the world (Eagle et al., 2004). Although operative mortality has declined over time, there are reports of post operative complications, which include myocardial infarction (MI), recurrent angina, serious arrhythmias, ventricular failure, renal insufficiency, stroke and death. Many of these may attribute to the process of ischemia-reperfusion injury (Yellon and Hausenloy, 2007). Efficacy and safety of pyridoxal-5¢-phosphate (MC-1) in high risk patients undergoing CABG surgery have been examined by MEND-CABG II randomized clinical trial (Alexander, 2008; Tardif et al., 2007). These investigations demonstrate that among intermediate to high-risk patients undergoing CABG surgery MC-1 given immediately before and after following surgery did not give good results. A well-powered trial is necessary for further evaluation the cardio-protective effects of MC-1. On the basis of the reports that the natural cofactor pyridoxal-5¢-phosphate may be useful as cardio-protective drugs, a series of novel analogues of this cofactor have been synthesized and tested for their anti-ischemic properties (Pham et al., 2003).

7.2.2

Design and Synthesis of Phosphonate Analogues of PLP

The phosphate moiety is a common structural feature in various bio-molecules. Particularly, this group serves to target well-defined phosphate binding pockets of a variety of enzymes. So the phosphate mimetic of several natural compounds can be used as drugs to target the phosphatebinding site of a particular enzyme to inhibit certain biological processes. To render stabilization of these synthetic compounds against phosphatase enzymes the replacement of the oxygen atom in P-O-C phosphate bond could give better results. Additionally, the phosphonate substitution pattern PCR1R2C allows for a wide range of structural modifications including variations in both substituents R1 and R2 at the tetravalent carbon centre. The ethyl phosphonate (5) was studied as a structural probe in the detailed investigation into the ionization state of PLP in glycogen phosphorylase (Stirtan & Withers 1996). The 4-formyl derivative of phosphonate derivative (6) served as a precursor for the development of pyridoxal-6-arylazo-5¢-phosphonate based P2 receptor antagonists (Kim et al., 1998).

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

7.5

A series of pyridoxine-5¢-phosphonates (10, 12, 16, 18, 19) was synthesized as agents that potentially mimic the efficacy of the natural cofactor in reducing scar size in rats that have been subjected to ischemia reperfusion injury. The synthesis of pyridoxine-5¢-phosphonates (10) and (12) are described in Scheme 3.

a

Scheme 3 Synthesis of pyridoxine-5¢-phosphonates 10 and 12a Reagents and conditions: (a) MnO2, C7H8, 40°C (b) (t-BuO)2 PO–Na+, THF, 0°C Æ rt (c) HOAc:H2O 4:1, 75°C (d) DAST, CH2Cl2, −78°C Æ rt

The synthesis of a, a-difluoroketophosphonate (16), difluorophosphonate (18) and phosphonate (19) are depicted in the Scheme 4, Scheme 5 and Scheme 6 respectively.

Scheme 4 Synthesis of difluoroketophosphonate 16a Reagents and conditions: (a) LDA/n-BuLi/CHF2PO(OCH2CH3)2, THF, −78°C Æ rt (b) MnO2, C7H8, 60°C, 18h (c) HOAc : H2O 4:1, 80°C, 5h (d) (CH3CH2)3SiBr/CH3CN

a

7.6

Natural Bioactive Molecules: Impacts and Prospects

Scheme 5 Synthesis of difluorophosphonate 18a Reagents and conditions: (a) LDA/THF, (C6H5SO2)2NF, −78°C (b) HOAc:H2O 4:1, 75°C, 18h

a

Scheme 6 Synthesis of phosphonate 19a a Reagents and conditions: (a) [(CH3)2CH]2NCH2CH3/CH2Cl2, SO3-Py/DMSO, −8°C, 1.5h (b) HOAc:H2O 4:1, 75°C, 18h

7.2.3

PLP Phosphonate Analogues as Cardioprotective Agents

In contrast to PLP, the phosphonate analogues described in the earlier section are found to be stable for longer time in aqueous solutions kept at ambient temperature unshielded from sunlight. The phosphonate derivatives (12) and (16) have been shown to reduce infarct size in preliminary experiments in the rat model of ischemia reperfusion injury. It has also come out from the thorough investigation that these phosphonates are also able to alter the pathways of cardiac metabolism (Pham et al., 2003). Glycogen phosphorylase is a PLP dependent enzyme, which plays a crucial role in glucose metabolism. Under normal physiological conditions, the heart primarily utilizes carbohydrates and fatty acids as energy sources in order to maintain its normal functions. Under certain pathological conditions when plasma fatty acid levels become elevated, such as diabetes or myocardial infarction alterations in energy metabolism occur. In that case, there will be an increase in fatty acid metabolism and reduced glucose oxidation in the myocardium (Kantor et al., 1999). Recent studies have revealed that increased fatty acid metabolism is linked to increased ischemic heart injury due to the significant uncoupling between glycolysis and glucose oxidation during reperfusion. This metabolic imbalance finally leads to an increased lactate release along with increased proton generation from glycolytically derived ATP, which in turn leads to greater Na+ and Ca2+ influx. Considering all these a novel therapeutic approach has been proposed to prevent ischemic heart disease based on the concept of modulating myocardial metabolism pathways (Lopaschuk, 1999). By applying specific drugs it is now possible to induce a shift from free fatty

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

7.7

acid towards predominantly glucose utilization by the myocardium to increase ATP generation per unit oxygen consumption (Chagas et al., 2008; Horowitz et al., 2010). The phosphonate analogues described in the text namely (12) and (16) have been tested to investigate their effect on the cardiac metabolism. It has been found that in the rat model these compounds are able to induce a desirable shift away from fatty acid metabolism toward glucose metabolism. Hemodynamic parameters including heart rate, mean arterial pressure and pressure rate index all have remained same in the test animals and that of the control group. Though (12) and (16) are able to induce a shift towards glucose oxidation further work is necessary to specify their actual mode of action.

7.3

PLP DEPENDENT ENZYMES AS POTENTIAL DRUG TARGETS

PLP dependent enzymes play crucial roles in the amino acid metabolism. Polyamines and biogenic amines like histamine and other neurotransmitters are also synthesized by PLP-enzyme catalyzed biotransformation reactions. Particularly, the small signalling molecules, polyamines and smallmolecule polycations are essential for the growth of eukaryotic cells. The B6 enzymes those are involved in primary metabolism have become targets of various therapeutic agents (Casero and Marton, 2007; Seiler, 2003). There are several inhibitors of B6 enzymes, which are currently used as medicine against certain types of neurological disorders, viral diseases like malaria and also against cancers (Amadasi et al., 2007; Kappes et al., 2011; Silverman, 2008). Some of the inhibitors listed in the Table 1 are substrate analogues of the PLP dependent enzymes and they follow distinct mechanisms for their inhibitory action. As for example, vigabatrin a first line drug of epilepsy interacts with the enzyme as normal amino acid substrates do, first with the enzyme bound cofactor PLP. However, after enzymatic activation, covalently modify active-site residues of the target enzyme. DFMO (difluoro-methyl ornithine) was turned out to be a potent therapeutic agent against Trypanosoma brucei, an infectious agent causing African sleeping sickness. This is because DFMO effectively inhibits the trypanosomal ornithine decarboxylase. DFMO is still in use as an effective first-line drug to treat trypanosomiasis and it’s mode of action is very similar to that of vigabatrin (Heby et al., 2007). It is also found that DFMO could suppress the progression of MYC-driven neuroblastoma (Hogarty et al., 2008; Koomoa et al., 2008; Rounbehler et al., 2009). Further clinical studies have shown that DFMO is well tolerated though not as effective as initially thought for treating cancer (Vlastos et al., 2005). Better results in treating cancer are reported when DFMO is used in combination with other drugs (Meyskens et al., 2008; Raul et al., 2007; Sporn and Hong, 2008) but numerous attempts to develop more effective derivatives of DFMO have failed. The mechanism of action of D-cycloserine which is the second line drug for the treatment of tuberculosis is reported to include an aldimine-ketimine transamination step, which is a side reaction catalyzed by mycobacterial alanine racemase, followed by a tautomerization to a stable aromatic adduct (Fenn et al., 2003). On the contrary, carbidopa, a human dopa decarboxylase (hDDC) inhibitor is used to treat Parkinson’s disease irreversibly binds to the cofactor PLP, and thus blocks the active site of the enzyme. Pregabalin, another first line drug for epilepsy, inhibits GABA aminotransferase due to its interaction with a calcium channel (Silverman, 2008).

7.8

Natural Bioactive Molecules: Impacts and Prospects

Table 1

Inhibitors of PLP dependent enzymes used as drugs

B6 Enzyme

Substrate

Drug

GABA aminotransferase (Human)

Usage

Ref.

Epilepsy

Indication

First-line drug

Amadasi and Bertoldi, 2007

Epilepsy

First-line drug

Silverman, 2008

Parkinson’s disease

First-line drug

Amadasi and Bertoldi, 2007

Trypanosomiasis (African sleeping sickness) Hirsutism

First-line drug

Seiler, 2003

Tuberculosis

Second-line drug

Fenn et al., 2003

Vigabatrin GABA aminotransferase (Human) Pregabalin Dopa decarboxylase (Human)

Carbidopa Ornithine decarboxylase (Trypanosoma brucei and human) Eflornithine (difluoromethyl ornithine, DFMO) Alanine rcemase (Mycobacterium tuberculosis) D-Cycloserine

7.3.1 Targeting Human Ornithine Decarboxylase (hODC) Polyamines are ubiquitous super-cations characterized by a charge distribution along the entire length of the carbon backbone. They are essential for a variety of cellular processes ranging from nucleic acid packaging and stabilization, DNA replication, transcription, translation, protein stabilization, cell differentiation and signalling (Wallace et al., 2003). Polyamines accumulate in cancerous tissues and their concentration is elevated in body fluids of cancer patients. hODC, one of amino acid decarboxylases, catalyzes the first committed and rate-limiting step in polyamine biosynthesis by converting the amino acid ornithine to putrescine (1,4-diamino butane) (Wu et al., 2011). Transcription, translation and degradation of the short-lived ODCs are regulated by multiple mechanisms (Pegg, 2006; Origanti and Shantz, 2007). Mammalian ODC is a downstream mediator of MYC-controlled bioprocesses (Wagner et al., 1993) and is up regulated in proliferating cells and implicated as an oncogene in multiple types of tumors (Casero et al., 2005). It was also found that even moderate reduction of intracellular ODC activity retards tumor development, which makes ODC a promising anticancer target.

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

7.3.2

7.9

Coenzyme-substrate Inhibtors of hODC

PLP-amino acid-conjugate inhibitors have to compete with intracellular PLP for occupancy of the same binding site in the target B6 enzyme. Therefore, the ideal candidates are newly synthesized B6 apoenzymes, i.e., freshly synthesized proteins that have not yet bound the cofactor PLP. Short-lived enzymes like ODC and human histidine decarboxylase (HDC) are good targets for PLP-amino acid conjugate inhibitors (Zhao et al., 2003). The coenzyme-substrate conjugate phosphopyridoxyl-ornithine had already been found to be a potent inhibitor of hODC without affecting human S-adenosyl methionine decarboxylase (Coward and Pegg, 1987). However, phosphopyridoxyl-ornithine and similar compounds proved not to inhibit the proliferation of tumor cells due to it’s impermeability of the cell membranes (Coward and Pegg, 1987). The lower cell permeability of these compounds is due to the negative charges of the phosphate and carboxylate group of phosphopyridoxyl-ornithine. To eliminate these negative charges, a pyridoxyl-ornithine methyl ester conjugate has recently been synthesized which serves as a prodrug (Rautio et al., 2008). On cellular uptake, it is hydrolyzed by intracellular esterases and phosphorylated by pyridoxal/pyridoxine kinase to phosphopyridoxyl-ornithine (Fitzpatrick et al., 2010). It has been found that the pro-drug is indeed membrane permeant, but the affinity of this new inhibitor for the target enzyme is significantly low to inhibit the proliferation of the tumor cell lines (Wu et al., 2011). Structure-based drug design has also been used to obtain improved inhibitors with higher affinity for the target enzyme. Guided by fragment-based inhibitor design a number of inhibitors for hODC with additional hydrophobic groups have been synthesized (Wu et al., 2007; Wu and Gehring, 2009). These synthetic compounds having a tert-butoxycarbonyl (BOC) or indole group and a methylated carboxyl group (Scheme 7) have no net negative charge and proved to be cell

Scheme 7

Structures of effective inhibitors of hODC in their pro-drug form

7.10

Natural Bioactive Molecules: Impacts and Prospects

permeant. These new compounds are pro-drugs, which inhibit the hODC on its up taking into the cells. Inside the living cell, pro-drugs are converted to the active ODC inhibitors by several pyridoxal/pyridoxine kinases and by esterases that hydrolyze the ester group of the substrate. The activated coenzyme-substrate conjugates then bind the newly synthesized apoenzyme with high affinity, preferentially affecting only proliferating cells.

7.3.3

Inhibition of Intracellular hODC and of Cell proliferation

Among various synthetic coenzyme-substrate conjugates three compounds namely tertbutoxycarbonyl (BOC)-modified pyridoxyl-L-ornithine methyl ester (POB, 20), pyridoxyl-L-tryptophan methyl ester (PTME, 21), phospho pyridoxyl-L-tryptophan methyl ester (pPTME, 22) have been reported to suppress the proliferation of glioblastoma cells with IC50 values of 50, 50 and 25 mM respectively (Wu et al., 2007; Wu and Gehring, 2009). All these compounds inhibit intracellular ODC activity of cultured human glioblastoma cells. The structural feature which is common to all these inhibitors is the ornithine like side chain Scheme 8 with a –NH-(CHx)4-NH- motif and an adjacent bulky hydrophobic group (Scheme 8). It is revealed from various studies that POB (20), PTME (21) and pPTME (22) all inhibit the activity of intracellular hODC, though the unphosphorylated compounds PTME and POB not significantly inhibit hODC in cell extracts indicating that nonphosphorylated pyridoxyl-amino acid conjugates are less prone to bind to the enzyme. pPTME and nonesterified phosphopyridoxyltryptophan the assumed cellular active form of pPTME effectively inhibit the activity of hODC in cell extracts (IC50 ~ 50 mM). These data indicate that the presence of phosphate group is the necessary condition to show the inhibitory effect of the PLP-amino acid conjugates (Wu and Gehring, 2009). All these hODC inhibitors are found to be effective to inhibit the cell growth in a time-dependent manner among which the pPTME has displayed better pharmacokinetics without a lag phase at low concentrations (Wu et al., 2007; Wu and Gehring, 2009). These compounds are much more efficient in inhibiting proliferation of LN229 glioblastoma cells than DFMO, the well known inhibitor of ODC and more specific towards tumor cells rather than less proliferative normal cells (Wu et al., 2007; Wu and Gehring, 2009). A wide variety of tumor cells are inhibited by POB including myeloma, Jurkat, COS7, SW2 small-cell lung cancer cells, glioblastoma LN18 cells, gliblastoma multiforme cells, and metastasizing lung cancer cells but proliferation of human smooth muscle cells remain unaffected (Wu et al., 2007). The new hODC inhibitor pPTME is as effective as the anticancer drugs Gleevec (Shingu et al., 2009; Ziegler et al., 2008), tamoxifen with an IC50 of ~ 25 mM in inhibiting the proliferation of cultured LN229 glioblastoma cells (Hui et al., 2004). The new hODC inhibitors namely, POB, PTME and pPTME which are capable to inhibit proliferation of glioblastoma cells should now be subjected to proofof-concept studies in animal models.

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

7.3.4

7.11

Inhibitors of Plasmodium ODC

Malaria, one of the most threatening diseases in the world causes billions of fatalities per year particularly, in the third world countries. The causative agent of the most severe form of tropical malaria is Plasmodium falciparum, a protozoan parasite that sequesters in the red blood cells of its human host. There are no vaccines available and recently more cases of drug-resistant malaria have been reported because the conventional antimalarials are losing their efficiency due to rapid spreading of drug-resistant parasites. Therefore, there is an urgency to have inhibitors with a new mode of action beyond the current antimalarial chemotherapies such as chloroquine, artemisinin and the antifolates. The human malaria parasite P. falciparum is able to synthesize PLP de novo as a crucial cofactor during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/ pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. Based on this fact, a new strategy is being developed where synthetic pyridoxal-amino acid adducts are channelled into the parasite (Muller et al., 2009). Trapped upon phosphorylation by the plasmodial PdxK, these compounds block PLP-dependent enzymes and thus impair the growth of P. falciparum. A novel strategy to target the polyamine metabolism has been proposed and tested to inhibit plasmodia, the parasite causing human malaria (Becker et al., 2010; Muller et al., 2000; Muller et al., 2008; Van Brummelen et al., 2009). The key bifunctional enzyme in the plasmodial polyamine biosynthesis plasmodium ODC/AdoMetDC (S-adenosyl methionine decarboxylase) contains two distinct active sites in two domains of a single polypeptide chain (Muller et al., 2000; Muller et al., 2008). Plasmodium ODC/AdoMetDC is proved to be a novel antimalarial target as it is closely related to the proliferation of the parasite cells (Muller et al., 2008). The well-known ODC inhibitor DFMO was shown to inhibit the proliferation of P. falciparum in vitro and also the development of P. berghei in an animal model. However, the survival of the erythrocytic stages of P. falciparum is only moderately effected by DFMO, which is also ineffective against clinical cases of malaria caused by P. falciparum (Wrenger et al., 2001 and Muller et al., 2001). The ineffectiveness of DFMO is mainly due to its poor absorption into the infected cells (Muller et al., 2001). Due to immense difficulties in crystallizing plasmodium proteins the crystal structure of plasmodium ODC/AdoMetDC is still unknown (Birkholtz et al., 2003). The primary structure of plasmodium ODC domain is homologous to that of mammalian ODC and thus belongs to the alanine racemase family of B6 enzymes. Thus targeting the plasmodium ODC to cure malaria without affecting the hODC is the main challenge for antimalarial agents (Birkholtz et al., 2003). Of late, it has been found that a coenzyme-substrate conjugate denominated as PT3 (23) inhibits pfODC (Muller et al., 2009). The structure of PT3 is very similar to that of PTME but contains an additional 6-membered ring between the indole and the coenzyme pyridine ring (Scheme 7) which renders more rigidity to this compound. PT3 was designed in such a way so that it could act as prodrug in which after cellular uptake, the coenzyme moiety become phosphorylated and the methyl ester hydrolyzed. Indeed, PT3, like other pyridoxyl-substrate conjugates could be phosphorylated by purified P. falciparum pyridoxal/pyridoxine kinase in vitro and phosphorylation of the prodrug is essential for its activation (Muller et al., 2009). The proliferation of cultured plasmodium parasites is also inhibited selectively by racemic PT3 (IC50 ~ 14 µM) rather than the proliferation

7.12

Natural Bioactive Molecules: Impacts and Prospects

of various human cancer cell lines (IC50 >100 µM). Further research in this area should be focused on the synthesis of enantiomerically pure PT3 and on the identification of the most effective stereoisomer with an IC50 value possibly even lower than the value showed by racemic PT3.

7.3.5 Targeting Human Histidine Decarboxylase (HDC) Histamine, a biogenic amine is produced from amino acid histidine by the action of histidine decarboxylase (Medina et al., 2003; Moya-Garcia et al., 2009). The human histidine decarboxylase (HDC), a dimeric PLP-dependent enzyme is a very short-lived species with a half-life of 1-2 h. It is now proved that histamine plays important physiologically and pathologically important roles in gastric acid secretion, neurotransmission, inflammation and immune modulation, particularly in allergic reactions by interacting with its specific membrane receptors H1, H2, H3, and H4 (MoyaGarcia et al., 2009). In certain pathological conditions, production of histamine could be controlled by targeting the HDC (Medina et al., 2003; Moya-Garcia et al., 2009; Moya-Garcia et al., 2005; Wu et al., 2008). Different types of inhibitors for HDC were known from long ago i.e, histidine methyl ester (HME, 24) a-fluoromethylhistidine (a-FMH, 25) and His-Phe (26) and more recently, epigallocatechin-3gallate (27) (Scheme 9).

Scheme 9

Structures of known inhibitors of mammalian HDC

a-FMH, a mechanism-based irreversible inhibitor of mammalian as well as pyruvoyl-dependent bacterial HDC, covalently attaches to active-site residues after being activated by HDC according to a inhibitory mechanism similar to that of DFMO (De Graw et al., 1977; Kelley et al., 1977). Most of the inhibitors of HDC are competitive substrate analogs. There was a drive to improve the

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binding affinity of the substrate analogue to the enzyme but was not successful. Histidine methyl ester (HME) was found to inhibit bacterial pyruvoyl-dependent HDC, however, it was proved to be inactive in cells because it was hydrolyzed to histidine, the substrate of HDC, after being taken up by cells (Kelley et al., 1977). Extraction of green tea leaves contains epigallocatechin-3-gallate which is able to inhibit rat HDC in vitro and in rat RBL-2H3 cells, however, the compound also inhibit other B6 enzymes, such as ODC and dopa/aromatic amino acid decarboxylase (Liang et al., 2002; Moya-Garcia et al., 2009). Various experiments indicate that the binding site for the imidazole moiety in hHDC is quite selective and unable to accommodate larger ligands. Out of the nine tested pyridoxyl-amino acid conjugates, only pyridoxyl-histidine methyl ester effectively inhibited the activity of 12-Otetradecanoylphorbol-13-acetate (TPA)-induced hHDC (Maeda et al., 1998). In summary, continuous research is going on to design more potent and target specific inhibitors for mammalian HDC.

7.3.6 7.3.6.1

Other PLP-Dependent Enzymes as Potential Drug Targets Cystathionine-b-synthase and cystathionine-g-lyase

Hydrogen sulfide is one of the gaseous messengers, which acts as mediator in the regulation of vasodilatation, diverse functions of the cardiovascular and nervous system, and inflammation (Mancardi et al., 2009; D’Emmanuele et al., 2009). Cystathionine g-lyase and cystathionine b-synthase two PLP-dependent enzymes are mainly responsible for in vivo production of hydrogen sulfide in mammals. Genetic ablation of cystathionine g-lyase in mice resulted in an 80% decrease of H2S production, increased level of plasma homocysteine and pronounced hypertension (Yang et al., 2008). The known inhibitors of cystathionine-g-lyase and cystathionine−b-synthase are D,Lpropargylglycine and b-cyanoalanine respectively. These inhibitors suffer from low selectivity towards the targets and limited cell-membrane permeability. Consequently, there is a scope to synthesize target specific and membrane permeable inhibitors for cystathionine b-synthase and cystathionine g-lyase.

7.3.6.2

Serine and aspartate racemase

In the brain, D-serine and D-aspartate are synthesized from the respective L-enantiomers. These transformations are catalyzed by two PLP-dependent enzymes, serine and aspartate racemases respectively. D-Serine and D-aspartate are endogenous agonists for N-methyl-D-aspartate (NMDA) receptors in the mammalian brain (Kim et al., 2010; Yoshimura and Goto, 2008). NMDA receptors play an important role in excitatory transmission and synaptic plasticity. Their over excitation has been resulted in various neuro-pathological conditions, including neurodegenerative diseases. Developing inhibitors of D-serine and D-aspartate racemases could possibly offer a novel approach for specific damping of the activity of NMDA receptors. Recently, a new class of hydroxamic acid based inhibitors have been developed for mouse serine racemase (Hoffman et al., 2009).

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Natural Bioactive Molecules: Impacts and Prospects

7.3.6.3

Alanine racemase

Alanine racemase is a homodimeric PLP-dependent enzyme. It catalyzes the conversion of L-alanine to D-alanine, which is a key building block of bacterial cell walls. Alanine racemases are only present in the prokaryotic cells but can’t be found in the eukaryotes and this feature makes the enzyme an attractive target for the development of antimicrobials (Le Magueres et al., 2005; Milligan et al., 2007). D-Cycloserine, a substrate analogue of D-serine is an effective inhibitor of alanine racemase and was marketed originally as a drug against tuberculosis (Fenn & Stamper, 2003). Presently it has become a second line drug due to severe side effects such as central nervous system toxicity. To develop target specific and more effective inhibitors of alanine racemase PLPalanine conjugates may be a worthwhile endeavour.

7.3.6.4

Serine hydroxymethyltransferase

Serine hydroxymethyltransferase is a B6 enzyme and belongs to the aspartate aminotransferase family. It catalyzes the retro-aldol cleavage of serine, which is an essential biotransformation pathway for the synthesis of numerous essential cell components (Metzler, 2003). The products of serine retroaldol cleavage are involved in some metabolic processes which offers many successful anti-cancer targets e.g., dihydrofolate reductase and thymidylate synthase. It is now possible to synthesize novel inhibitors of serine hydroxymethyl transferase as the crystal structures are available including the human enzyme (Renwick et al., 1998).

7.4 7.4.1

PLP-DEPENDENT ENZYME AS A DRUG PLP-dependent Enzymes as Potent Antitumor and Anticardiovascular Agents

Sulfur containing amino acids are pivotal compounds for almost all metabolic cellular processes. The prominent sulfur containing amino acids are cysteine and methionine. Biochemically methionine is activated by methionine adenosyltransferase forming S-adenosylmethionine as a key intermediate following various metabolic pathways. Physiologically, tumors are genetically abnormal cells with uncontrolled and rapid proliferation process. Most of the tumors were reported to be auxotrophs for L-methionine, glutamine, asparagine and arginine due to the absence of intrinsic enzymatic systems synthesizing these amino acids (El-Sayed, 2010). Tumor cells are, thus, dependent for their growth and proliferation on the exogenous supply of these amino acids, usually from diets. Consequently, L-methioninase, L-glutaminase, L-asparaginase and arginine deiminase were frequently used as common anticancer agents by sequestering their corresponding amino acids from the blood plasma (Cheng et al., 2005). On the contrary, normal cells have an active methionine synthase with the ability to synthesize methionine from homocysteine. The inability of tumor cells to synthesize various amino acids mentioned earlier is the biochemical target for many therapeutic strategies (Cellarier et al., 2003). Several approaches were designed for triggering the methionine dependency of tumor cells; for example starvation of the tumor cells from methionine using methionine free diets displays a reliable efficacy against a variety of tumor cells (Goseki et al., 1992). However, this technique is not a viable strategy for many technical,

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therapeutical and economical considerations. Application of L-methioninase, a PLP dependent enzyme necessary for the metabolism of methionine has been found to be useful for the removal of plasma L-methionine which is a potent justifiable strategy towards various methionine dependent tumor cells. L-methioninase was extensively tested as a potent anti-proliferative enzyme towards Lewis lung, human colon, glioblastoma and neuroblastoma (Sato and Nozaki, 2009). L-Methioninase was purified and characterized from different bacterial isolates, particularly Pseudomonas putida enzyme studied extensively on its structure via X-ray crystallographic methods (Kudou et al., 2007; Nikulin et al., 2008). However, there are severe problems in using bacterial enzyme as therapeutic agents due to high immunogenic reactions, rapid plasma clearance and proteolysis especially with multiple dosing, making the patient more vulnerable towards secondary immunogenic disorders by opportunistic pathogens (Sun et al., 2003). It has also been found that extra amount of ammonia is released during the course of tumor therapy due to the deaminating activity of the enzyme causing additional hazards to the kidney and liver as observed for many of anticancer deaminating enzymes (Balcao et al., 2001). Therefore, it is a challenge for many biotechnologists to find out a novel enzyme with less immunogenic activity and high therapeutic potentiality or modification of the currently used enzymes accordingly.

7.4.2

Combination of L-Methioninase and Chemotherapeutic Agents

A new strategy against tumors has been developed based on the synergism between L-methioninase and various chemotherapeutic agents (Yoshioka et al., 1998). Biochemically, starvation of the tumor cells to L-methionine, by the action of L-methioninase or L-methionine depleted diets usually make the tumor cells more vulnerable to any biochemical modulator. The overall concept for all therapeutic strategies is methionine starvation and simultaneous application of phase-specific chemotherapeutic agents. As for example, 5-fluorouracil, a common biochemical modulator which is a thymine analogue competitively binds to thymidylate synthetase causing prompted suppression of DNA synthesis in the tumor cells (Poirson-Bichat et al., 1997). The sensitivity of Lewis lung carcinoma to L-methioninase was reported to increase by about 4.5 fold by addition of 5-fluorouracil (Tan et al., 1998). Similarly doxorubicin, an intercalating agent greatly improves the activity of L-methioninase against human lung carcinoma H460 (Gupta et al., 2003). However, continuous starvation of human serum to methionine may cause hazardous implications to liver at the same time (Kokkinakis, 2006).

7.4.3

L-Methionine Gene Therapy and Selenomethionine as Pro-drugs

Introduction of the L-methioninase encoding genes to tumor cells with regulating their expression is one of the recent challenges for treating of tumor cells. Transduction of bacterial L-methioninase gene via developed adenoviral vector with exogenous L-methioninase display a powerful activity towards human ovarian cancer cells (Miki et al., 2000). Deprivation of the tumor cells from the intrinsic L-methioninase is the potentiality of gene therapy for methionine dependent tumors. Consequently, this technique significantly intensified by combination with external L-methioninase

7.16

Natural Bioactive Molecules: Impacts and Prospects

to remove the serum L-methionine. There is a report of plausible anti-proliferative activity towards human lung cancer by transduction of P. putida L-methioninase gene via retroviral vectors in combination with methioninase treatment (Miki and Xu, 2000). Of late, a novel strategy for reduction of the clinical hazards and augmentation of the pharmacokinetic impact of this enzyme via combination of gene therapy and selenomethionine as pro-drugs has been developed (Yamamoto et al., 2003). Introduction of selenomethionine as non-toxic pro-drug in addition to intracellular and extracellular depletion of L-methionine maximizes the therapeutic potentiality of this approach against tumor cells. Recently, directing of the enzyme via antibodies to the target tumor cells for removal of extracellular methionine in addition to transduced enzyme gene and selenomethionine as pro-drug is the promising strategy against various types of tumors (Zhao et al., 2006). This novel approach is referred to as Antibody Directed Enzyme Pro-drug Therapy (ADEPT).

7.5

CONCLUDING REMARKS

Vitamin B6 derived cofactor PLP itself and the enzymes depending upon PLP are now regarded as promising drugs and drug targets for their catalytic action inside the living cells. Pyridoxal5¢-phosphate monohydrate (MC-1), a naturally occurring metabolite of vitamin B6 can act as a purinergic (p2) receptor antagonist and prevents cellular calcium overload in preclinical and clinical studies of ischemia reperfusion injury. Various PLP-phosphonate analogues have also been proved to be very good cardio protective agents. PLP-dependent enzymes play crucial role in the metabolism of amino acids and also in the synthesis of polyamines essential for cell growth, and hence they are regarded as promising targets for possible pharmacotherapeutic intervention. Various drugs are already available in the market, which are inhibitors of several PLP-dependent enzymes. Reduction of the specific coenzymesubstrate aldimine, the common intermediate formed in the first step of the catalytic process, to a stable secondary amine provides a core template for PLP-substrate conjugate inhibitors of PLP-dependent enzymes. Several critical aspects would have to be considered for the successful implementation of this approach. Regarding the therapeutic potentiality of the PLP-dependent enzymes, the immunogenicity and relative instability are the common limitations from pharmacokinetic point of view. Most of these enzymes which have received considerable attention as therapeutic agents are from bacterial sources. However, it has been established from biochemical and crystallographic studies that there are reliable distinction in the conformation of the surface amino acids and immunogenic sites from prokaryotic to eukaryotic enzymes. Consequently, further biochemical and crystallographic studies to elucidate the catalytic identity and tertiary structure of eukaryotic PLP-enzymes for the maximum therapeutic exploitation of these enzymes need to be resolved. The present overview would surely boost the ongoing developments in that direction.

Abbreviations ATP AdoMetDC ADEPT

Adenosine tri phosphate S-adenosyl methionine decarboxylase Antibody Directed Enzyme Pro-drug Therapy

Chakrabarty and Das: Pyridoxal-5¢-phosphate in Drug Discovery

n-BuLi BOC CABG COS7 DAST DMSO DMFO DNA a-FMH GABA HOAc hDDC hODC HDC HME IC LDA LN229 LN18 MC-1 MI MYC NMDA ODC PLP P2 POB PTME p-PTME PdxK PT3 RBL-2H3 SW2 THF TPA

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n-Butyl Lithium tert-Butoxycarbonyl Coronary Artery Bypass Graft A fibroblast like cell line derived from monkey kidney tissue Diethyl amino sulphur tri fluoride Dimethylsulfoxide Difluoro methyl ornithine Deoxyribonucleic acid a-fluoromethyl histidine Gamma-amino-butyric acid Acetic acid Human dopa decarboxylase Human ornithine decarboxylase Histidine decarboxylase Histidine methyl ester Inhibition concentration Lithium diisopropyl amide Cell line taken from a patient with right frontal parieto-occipital glioblastoma Cell line taken from a patient with a right temporal lobe glioma Medicine International Inc, Winnipeg, Manitoba, Canada Miocardial Infarction It is a regulator gene that codes for a transcription factor N-methyl-D-aspartate Ornithine decarboxylase Pyridoxal-5´-phosphate Purinergic receptor tert-butoxycarbonyl-modified pyridoxal-L-ornithine methylester Pyridoxal-L-tryptophan methyl ester Phospho pyridoxal-L-tryptophan methyl ester Pyridoxine/pyridoxal kinase Cyclized form of pyridoxal-L-tryptophan methyl ester Cell line obtained from the organism Rattus norvegicus Human small cell carcinoma cell line Tetrahydrofuran 12-O-tetradecanoyl-phorbol-13-acetate

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Natural Bioactive Molecules: Impacts and Prospects

Acknowledgements The authors are thankful to the Department of Chemistry, Visva-Bharati, Santiniketan-731 235 for providing basic facilities during the preparation of the manuscript. Financial assistance of CSIR through Research Associate-ship programme (09/202/(0027)/2K10-EMR-I) is also gratefully acknowledged by KC.

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8 Allelochemicals from Artemisia vulgaris Linn. (Compositae) R.N. Yadava* and Gautam Patil Natural Products Laboratory, Department of Chemistry, Dr. H. S. Gour Central University, Sagar-470 003 (M.P.) India

ABSTRACT A new allelochemical A, characterized as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone-7-Oa-L-rhamnopyranosyl-(1Æ3)-O-b-D-arabinopyranosyl-(1Æ4)-O-b-D-xylopyranoside along with two known compounds such as 5,4¢-dihydroxy-3,7-dimethoxyflavone (B) and 3,5,7,3¢,4¢pentahydroxyflavone-3-O-a-L-rhamnopyranoside (quercetin-3-O-a-L-rhamnopyranoside; C) were isolated from ethanolic extract of the aerial parts of Artemisia vulgaris Linn. (Family: Compositae). The structures of the isolates were elucidated on the basis of various color reactions, chemical degradations and detailed spectral analyses. Keywords: Flavonoid glycosides, allelochemicals, Artemisia vulgaris, Compositae, chemical degradations, spectral studies.

8.1

INTRODUCTION

Plant products, especially secondary metabolites obtained from medicinal plants play a very dominant role in synthesizing and designing the analogous of the chemical components leading to the development of effective chemical entities. These chemicals include flavonoids, tannins, alkaloids, aromatic acids etc. They play important role in protecting plants against insects and pathogens. Flavonoids are a group of polyphenols present in substantial amounts (0.5-1.5%) in virtually all land-based plants (Jovanovic et al., 1994; Thompson et al., 1972); they occur in the free states as well as glycosides. In the glycosides, each aglycone may also occur in association with a number of different sugars. Besides their important biological roles in plant pigmentation, flavonoids possess a wide range of biological activities including antiviral (Yamashita et al., 1990; *Corresponding author: [email protected]

8.2

Natural Bioactive Molecules: Impacts and Prospects

Solimani, 1997), anti-inflammatory (Brahmachari, 2010; Pan et al., 2010; González-Gallego et al., 2007), antioxidant (Brahmachari, 2010; Rice-Evans, 2001; Pietta, 2000; Jovanovic et al., 1994), antimicrobial (Brahmachari et al., 2011; Brahmachari and Gorai, 2004; Brit et al., 1986; Mendoza, 1997) and anticancer properties (Chahar et al., 2011; Brahmachari, 2010; Brahmachari, 2008; Wall et al., 1988). Because of their great number and the diversity of their biological activities, these molecules have attracted the wide attention of many researchers. A large number of new potential allelochemicals from plants have been reported by Yadava and his group (Yadava and Reddy, 1998, 2002, 2003a,b; Yadava and Verma, 2003, 2005; Yadava and Jain, 2004, 2005; Yadava and Tiwari, 2005, 2007; Yadava and Singh, 2007; Yadava and Belwanshi, 2009; Yadava and Bhargava, 2010; Yadava and Raj, 2011a,b; Yadava and Satnami, 2011). This chapter incorporates the isolation and structure characterization of a new allelochemical A along with two known compounds B and C from the aerial parts of Artemisia vulgaris Linn. by various color reactions, chemical degradations and detailed spectral analyses. Artemisia vulgaris Linn. (Chopra and Nayar, 1996; Kirtikar and Basu, 1999; Khare, 2004) belongs to the family Compositae, which is commonly known as “Nagadouna or Dona” in Hindi. It is found throughout hilly districts of India and W. Ghats from Konkan southwards to Ceylon. The aerial parts and seeds of this plant has been traditionally used as folk remedy for the treatment of many diseases. The plant is considered to be a valuable stomachic, deobstruent and antispasmodic and also used in the treatment of hysteria. It is also externally used in fomentation given in skin diseases and foul ulcers as an alternative. The expressed juice is used in diseases of children. Earlier workers reported the presence of various chemical constituents from this plant (Regerson et al., 1972; Tang et al., 2000; Thao et al., 2004; Juteau et al., 2005; Zhao et al., 2005; Bhatt et al., 2006).

8.2 8.2.1

EXPERIMENTAL General Procedure

Melting points are uncorrected. The IR spectra were recorded on Shimadzu 8201 PC spectrometer in KBr pellets. 1H and 13C-NMR spectra were recorded on Bruker DRX-300 spectrometer at 300 and 75 MHz , respectively, using CDCl3 as solvent and TMS as internal standard. Mass spectra on Jeol-SX-102 (FAB) mass spectrometer.

8.2.2

Plant Materials

The aerial parts of the plant were collected from Satpuda woody region of Chhindwara District of M.P. (India) and was taxonomically authenticated by the Department of Botany, Dr. H. S. Gour Central University, Sagar (M.P.) India.

8.2.3

Extraction and Isolation

Dried powdered aerial parts (4 kg) of the plant were extracted with 95% ethanol in Soxhlet apparatus for 72 hours. The ethanolic extract of the aerial parts of this plant was concentrated under reduced

Yadava and Patil: Allelochemicals from Artemisia Vulgaris

8.3

pressure to obtain a brown viscous mass which was successively partitioned with petroleum-ether (40-60°C), chloroform, ethyl acetate, acetone and methanol. The methanol soluble fraction of this plant was concentrated under reduced pressure to yield brown viscous substrate (1.75 gm) which was subjected to TLC examination; the solvent system CHCl3:MeOH:H2O (4:6:2) gave three spots indicating it to be mixture of three compounds A, B and C. These compounds were separated and purified by column chromatography over silica gel and eluated with CHCl3 and MeOH in various proportions (2:10, 4:8, 6:6) and studied separately. The eluents obtained from CHCl3:MeOH (4:8) after removal of the solvent and recrystallization from methanol yielded compound B. It gave all the characteristic color reactions of flavonoids. It was identified as 5,4¢-dihydroxy-3,7-dimethoxyflavone by comparison of its spectral data with reported literature values (Echeverri et al., 1991; Martinez et al., 1997; Murillo et al., 2003; Kim et al., 2006). The eluents obtained from CHCl3:MeOH (6:6) after removal of the solvent and recrystalization from methanol gave compound C. It gave all the characteristic color reactions of flavonoids. It was identified as 3,5,7,3¢,4¢-pentahydroxyflavone-3-O-a-L-rhamnopyranoside (quercetin-3-O-a-Lrhamnopyranoside) by comparison of its spectral data with reported literature values (Markham et al., 1978; Chang et al., 2000; Mämmelä et al., 2000; Fabjan et al., 2003; Park et al., 1991, 2011; Manguro et al., 2005). The eluents collected from CHCl3:MeOH (2:10) after removal of the solvent and recrystalization from ethanol gave new compound A, which was identified as given below.

8.2.3.1 8.2.3.1.1

Study of compound A Physical and spectral properties

It was crystallized from acetone to yield 1.10 gm. It gave Molisch test for glycoside and all the specific color reactions of isoflavonoids. It was analyzed for m.f. C34H43O19, m.p. 243-244°C and [M]+ m/z 755 (FABMS), Found (%) C, 54.11; H, 5.78; Calcd. for m.f. C34H43O19 (%) C, 54.03, H, 5.69; UV: (MeOH) lmax (nm) 260, 315, (+AlCl3), 262, 335; (+ AlCl3/HCl), 252, 360; (+NaOAc) 260, 337, (+NaOAc-H3BO3) 265, 378. IR (KBr) n cm−1: 3430, 2955, 2880, 1645, 1585, 1260, 1126, 1065, 875, 840. 1H-NMR: (300 MHz, CDCl3) and 13C-NMR: (75 MHz, CDCl3) (see Table 1 and Table 2 respectively); [M]+ 755 (FABMS) at m/z 608, 477, 344, 343, 329, 326, 316, 313, 182, 154, 183, 162, 165, 137.

Compound A

8.4

Natural Bioactive Molecules: Impacts and Prospects 1

Table 1

H-NMR (300 MHz, CDCl3) Spectrum of the Compound A

S.No.

δ Value

1

Pattern

3.84

Coupling constant (J; Hz)

No.of Protons

s

3

Assignments -OMe-6

2

3.95

s

3

-OMe-3¢

3

3.93

s

3

-OMe-4¢

4

6.58

s

1

H-8

5

7.58

s

8.4

1

H-2¢

6

7.16

d

8.4

1

H-5¢

7

7.65

d

8.3

1

H-6¢

8

9.51

s

1

OH-5

9

10.80

s

10

5.72

d

8.5

1

OH-7

1

H-1≤ (xylose)

11

5.12

d

7.5

1

H-1¢≤ (arabinose)

12

4.98

d

7.3

1

H-1≤≤(rhamnose)

13

1.27

d

6.4

3

Me-6≤≤

14

4.12-4.53

m

21

remaining protons of sugar

13

Table 2

C-NMR (75 MHz, CDCl3) Spectrum of the Compound A

S.No.

Atom

d Value

S.No.

Atom

δ Value

1

C-2

155.2

19

C-4≤

71.1

2

C-3

137.2

20

C-5≤

72.8

3

C-4

175.2

21

C-6≤

62.5

4

C-5

157.6

22

C-1≤¢

102.4

5

C-6

132.2

23

C-2≤¢

73.1

6

C-7

159.6

24

C-3≤¢

72.8

7

C-8

96.8

25

C-4≤¢

70.4

8

C-9

153.4

26

C-5≤¢

68.2

9

C-10

106.2

27

C-1≤¢

100.4

10

C-1¢

118.4

28

C-2≤≤

76.6

11

C-2¢

155.8

29

C-3≤≤

78.8

12

C-3¢

120.4

30

C-4≤≤

76.4

13

C-4¢

133.2

31

C-5≤≤

68.7

14

C-5¢

119.2

32

C-6≤¢

20.4

15

C-6¢

129.1

33

OCH3-3¢

55.94

16

C-1≤

102.6

34

OCH3-4¢

56.52

17

C-2≤

71.5

35

OCH3-6

56.36

18

C-3≤

70.4

Yadava and Patil: Allelochemicals from Artemisia Vulgaris

8.2.3.1.2

8.5

Acid hydrolysis of compound A

Compound A (650 mg) was dissolved in ethanol (25 ml) and refluxed with 25 ml of 10% H2SO4 on water bath for 7-8 hrs. The contents were concentrated under reduced pressure and allowed to cool and residue was extracted with diethyl ether. The ether layer was washed with water and the residue was chromatographed over silica gel using CHCl3: MeOH (4:6) to give compound A-1, which was identified as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone by its UV, IR, NMR and mass spectrum analysis. The aqueous hydrolysate was neutralized with BaCO3 and the BaSO4 filtered off. The filtrate was concentrated and subjected to paper chromatography examination using n-BAW (4:1:5) and aniline hydrogen phthalate as detecting agent, revealed the presence of L-rhamnose (Rf 0.36), D-arabinose (Rf 0.20) and D-xylose (Rf 0.29) (Co-PC). 8.2.3.1.2.1

Study of compound A-1

It was crystallized from ether as light brown amorphous powder (365 mg). It responded to all the characteristic color reactions of isoflavonoids. It has m.f. C18H16O7, m.p. 321-322°C and [M]+ 344 (EIMS); Found (%); C 64.10, H 4.56; Calcd. for m.f. C18H16O7; (%); C 64.28, Compound A-1 H 4.67; UV: (MeOH) lmax (nm) 262, 320, (+AlCl3) 265, 340; (+AlCl3-HCl) 254, 358; (+NaOMe) 284, 344; (+NaOAc) 265, 342; (+NaOAc-H3BO3) 268, 372. IR (KBr) n cm−1: 3434, 2945, 2887, 1642, 1590, 1263, 1126, 860, 830.1H-NMR: (300 MHz, CDCl3) d (ppm); 10.75 (s, OH-5 ), 9.61(s, OH-7 ), 3.92 (3H, s, 6-OMe), 3.73 (3H, s, 3¢-OMe), 3.78 (3H, s, 4¢-OMe), 6.50 (1H, s, H-8), 7.16 (1H, d, J 8.6 Hz, H-2¢), 7.40 (1H, d, J 8.4 Hz, H-5¢), 7.12 (1H, d, J 8.5 Hz, H-6¢). 13C-NMR: (75 MHz, CDCl3) d (ppm); 155.4 (C-2), 137.8 (C-3), 174.5 (C-4), 157.1 (C-5), 132.6 (C-6), 57.6 (OCH3-C-6), 159.0 (C-7), 96.1 (C-8), 153.3 (C-9), 106.8 (C-10), 118.6 (C-1¢), 155.1 (C-2¢), 120.2 (C-3¢), 56.5 (OCH3-3¢), 56.58 (OCH3-4¢), 119.9 (C-5¢), 129.6 (C-6¢). Thus it was identified as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone. 8.2.3.1.3

Permethylation of compound A

50 mg of the compound A was refluxed with MeI (5 ml) and Ag2O (15 ml) in DMF (25 mg) for two days and then filtered. The filtrate was hydrolysed with 10% ethanolic H2SO4 for 5-6 hrs to yield methylated aglycone, identified as 7-hydroxy-5,6,3¢,4¢-tetramethoxyisoflavone and methylated sugars which were identified as 2,3,4-tri-O-methyl-L-rhamnose (RG 1.02), 2,4-di-O-methyl-Darabinose (RG 0.65) and 2,3-di-O-methyl-D-xylose (RG 0.74). 8.2.3.1.4

Enzymatic hydrolysis of compound A

25 mg of the compound A was dissolved in 25 ml of MeOH and hydrolysed with equal volume of takadiastase. The reaction mixture was allowed to stay at room temperature for 48 hours and filtered. The proaglycone and hydrolysate were studied separately. The hydrolysate was concentrated and subjected to paper chromatography examination using n-BAW (4 : 1 : 5) as solvent showed the presence of L-rhamnose (Rf 0.36). The proaglycone was dissolved in MeOH (20 ml) and hydrolysed with equal volume of almond emulsin at room temperature as usual procedure gave aglycone, which was identified as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone and sugars were identified as D-arabinose (Rf 0.20) and D-xylose (Rf 0.29) (Co-PC).

8.6

Natural Bioactive Molecules: Impacts and Prospects

8.2.3.2

Study of compound B

It had m.f. C17H14O6, m.p. 218-219°C; [M]+ m/z 314; (EIMS); Found (%); C 64.91, H 4.51; Calcd. for m.f. C17H14O6; (%); C 64.97, H 4.46; UV: (MeOH) lmax (nm) 255, 370, (+AlCl3), 254, 358; (+AlCl3-HCl), 250, 363; (+NaOAc) 250, 345. IR (KBr) n cm−1: 3398, 2990, 1662, 1615, 1495, 1300, 1260. 1H-NMR: (300 MHz, CDCl3) d (ppm); 12.80 (s, OH-5 ), 6.50 (1H, d, J 2.0 Hz, H-6), 6.51 Compound B (1H, s, H-8), 6.98 (2H, d, J 9.0 H-3¢ and H-5¢), 5.0 (1H, s, OH-4¢), 6.75 (2H, d, J 7.8 Hz, H-2¢ and H-6¢). 13C-NMR: (75 MHz, CDCl3) d (ppm); 157.5 (C-2), 160.2 (C-3), 182.9 (C-4), 164.4 (C-5), 168.2 (C-6),152.8 (C-7), 96.5 (C-8), 128.0 (C-1¢), 118.7 (C-2¢), 115.2 (C-3¢), 146.0 (C-4¢), 117.0 (C-5¢), 119.8(C-6¢). Thus it was identified as 5,4¢dihydroxy-3,7-dimethoxyflavone by comparison its spectral data with reported literature values (Echeverri et al., 1991; Martinez et al., 1997; Murillo et al., 2003; Kim et al., 2006).

8.2.3.3

Study of compound C

It had m.f. C21H20O11, m.p. 249-250°C; [M]+ m/z 448; (EIMS); Found (%); C 56.18, H 4.66; Calcd. for m.f. C21H20O11; (%);C 56.25, H 4.46; UV: (MeOH) lmax (nm) 251, 374, (+AlCl3), 251, 354; (+AlCl3-HCl), 250, 363; (+NaOAc) 250, 345. IR (KBr) n cm−1: 3448, 2904, 1663, 1615, 1492, 1303, 1263. 1H-NMR: (75 MHz, CDCl3) d (ppm); 12.48 (s, OH-5 ), 6.18 (1H, d, 1.9 Hz, H-6), 6.41 (1H, d, J 2.0 Hz, H-8), 7.56 (2H, dd, J 2.2 7.5 Hz, H-2¢ and H-6¢), 6.87 (1H, d, J 8.5 Hz H-5¢), 4.91 (1H, d, 3.7 Hz, H-1≤), 3.98-3.06 (m, the remaining protons of the rhamnose). 13C-NMR: (75 MHz, CDCl3) d (ppm); 156.4 (C-2), 135.9 (C-3), 176.05 (C-4), 160.35 (C-5), 156.04 (C-6),164.14 (C-7), 97.0 (C-8), 125.8 (C-1¢), 116.2 (C-2¢), 147.03 (C-3¢), 147.9 (C-4¢), 115.1 (C-5¢), 119.5 (C-6¢), 98.48 (C-1≤), 75.0 (C-2≤), 49.0 (C-3≤), 69.97 (C-4≤), 66.3 (C-5≤), 100.1 (C-6≤). Thus it was identified as 3,5,7,3¢,4¢pentahydroxyflavone-3-O-a-L-rhamnopyranoside (quercetin-3-O-a-L-rhamnopyranoside) by comparison of its spectral data with reported literature values (Markham et al., 1978; Chang et al., 2000; Mämmelä et al., 2000; Fabjan et al., 2003; Park et al., 1991, 2011; Manguro et al., Compound C 2005).

8.3

RESULTS AND DISCUSSION

The methanol soluble fraction of the plant yielded a compound A with molecular formula of C34H43O19, m.p. 243-244°C, [M]+ 755, (FABMS). It gave Molisch and Shinoda tests (Shinoda, 1928), showing its isoflavonoide glycosidic nature. Its IR spectra showed absorption bands at 3430 (-OH), 2955 (-CH saturated), 2880 (-OCH3), 1645 (-C=O) and aromatic ring- 1590, 1260, 1126, 1065, 875 and 840 cm–1.

Yadava and Patil: Allelochemicals from Artemisia Vulgaris

8.7

In UV spectrum at 260 nm and 315 nm showed its isoflavonoide skeleton. The bathochromic shift of 20 nm with AlCl3 in band I revealed the presence of –OH group at C-5 position in compound A. In 1H-NMR spectrum, a singlet at d 6.58 was assigned to H-8 proton. The anomeric proton signals at d 5.72 (1H, d, J 8.5 Hz), 5.12 (1H, d, J 7.5 Hz) and 4.98 (1H, d, J 7.3 Hz) were assigned to H-1≤ of D-xylose, H-1≤¢ of D-arabinose and H-1≤≤ of L-rhamnose respectively. Characteristic ion peaks appeared at m/z 755 [M]+, 592 [M]+ [L-rhamnose], 460 [M]+ [L-rhamnose-D-arabinose], 344 [M]+ [L-rhamnose-D-arabinose-D-xylose] were obtained by subsequent losses from the molecular ion of each molecule of L-rhamnose, D-arabinose and D-xylose. This confirmed that L-rhamnose was terminal sugar whereas D-arabinose as a middle sugar and D-xylose was attached at C-7-OH position of the aglycone. Acid hydrolysis of compound A with ethanolic 10% H2SO4 gave aglycone A-1, m.p. 321322°C, m.f. C18H16O7, [M]+ 344 (EIMS). It was identified as 5,7-dihydroxy-6,3¢,4¢-trimethoxy isoflavone (known as Junipegenin B or dalspinosin) (Narayanan et al., 2007; Reynaud et al., 2005; Saxena and Chourasia, 2000; Lakshmi et al., 1996; Eu et al., 1991;) by its spectral analysis. The aqueous hydrolysate after the removal of aglycone was neutralized with BaCO3 and the BaSO4 filtered off. The filtrate was concentrated and subjected to PC and sugars were identified as D-xylose (Rf 0.29), D-arabinse (Rf 0.20) and L-rhamnose (Rf 0.36) (Co-PC) (Harborne et al., 1973; Lederer and Lederer, 1957). Periodate oxidation of compound A confirmed that all the sugars were present in the pyranose form (Hakomori, 1964). The position of sugar moieties in compound A were determined by permethylation (Hirst and Jones 1949) followed by acid hydrolysis which yielded methylated aglycone identified as 7-hydroxy-5,6,3¢,4¢-tetramethoxy isoflavone, showed that glycosylation was involved at C-7-OH position of the aglycone and sugars were identified as 2,3,4-tri-O-methyl-L-rhamnose (RG 1.02), 2,4-di-O-methyl-D-arabinose (RG 0.65) and 2,3-di-O-methyl-D-xylose (RG 0.75) (Co-PC) (Lederer and Lederer, 1957), which showed that C-1≤≤ of L-rhamnose was attached to C-3≤¢ of D-arabinose whereas C-1≤¢ of D-arabinose was attached with C-4≤ of D-xylose and C-1≤ of D-xylose was linked with C-7-OH position of aglycone. Enzymatic hydrolysis of compound A with takadiastase enzyme liberated L-rhamnose (Rf 0.36) and 5,7-dihydroxy-6,3¢,4¢-trimethoxy isoflavone-7-O-b-D-arabinopyranosyl-(1Æ4)-O-b-Dxylopyranoside as proaglycone, confirming the presence of a linkage between L-rhamnose and proaglycone. The proaglycone on further hydrolysis with almond emulsin liberated D-arabinose (Rf 0.20) and D-xylose (Rf 0.29) and aglycone confirming the presence of b linkage between D-arabinose and D-xylose as well as between D-xylose and aglycone. Therefore it was concluded that C-1≤≤-OH of L-rhamnose was attached with C-3≤¢-OH of D-arabinose and C-1≤¢-OH of D-arabinose was linked with C-4≤-OH of D-xylose and C-1≤-OH of D-xylose was attached with C-7-OH of the aglycone through b linkage. On the basis of above evidences, the structure of a new compound A was characterized as 5,7-dihydroxy-6,3¢,4¢-trimethoxyisoflavone-7-O-a-L-rhamnopyranosyl-(1Æ3)-O-b-Darabinopyranosyl-(1Æ4)-O-b-D-xylopyranoside.

8.8

Natural Bioactive Molecules: Impacts and Prospects

Study of Compound B: It was analysed for m.f. C17H14O6, m.p. 218-219°C and [M]+ m/z 314; (EIMS). It was characterized as 5,4¢-dihydroxy-3,7-dimethoxyflavone by comparison its spectral data with reported literature values (Echeverri et al., 1991; Martinez et al., 1997; Murillo et al., 2003; Kim et al., 2006). Study of Compound C: It was analysed for m.f. C21H20O11, m.p. 249-250°C and [M]+ m/z 448 (EIMS). It was characterized as 3,5,7,3¢,4¢-pentahydroxyflavone-3-O-a-L-rhamnopyranoside (quercetin-3-O-a-L-rhamnopyranoside) by comparison of its spectral data with reported literature values (Markham et al., 1978; Chang et al., 2000; Mämmelä et al., 2000; Fabjan et al., 2003; Park et al., 1991, 2011; Manguro et al., 2005).

Acknowledgements Authors are thankful to Director, CDRI Lucknow (U.P.) for recording various spectral data and Head, Department of Chemistry, Dr. H. S. Gour Central University Sagar M.P. India, for providing necessary laboratory facilities.

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9 Quercetin — A Ubiquitous Bioflavonoid: Structural Aspects versus Potential Health Benefits Ajay Kumar Dixit*, Shyam Ramakrishnan, and C.C. Lakshmanan Analytical Center of Excellence (ACE), Bioscience, ITC Life Science & Technology Center, Peenya Industrial Area, Phase 1, Bangalore-560 058, Karnataka, India

ABSTRACT Bioflavonoids, a subclass of polyphenols, are low molecular weight compounds ubiquitously present in plant kingdom. Until now, more than 9000 structurally distinct flavonoids have been identified. The diversity in their chemical structures confers them a wide range of biological activities; dietary presence of bioflavonoids indicate their lower toxicities as well. They have been found as common chemical constituents of many traditional medicinal plants world over and are well documented for their general health-promoting properties. Quercetin and its glycosides belong to flavanol, a sub-group of flavonoids. It is one of the widely distributed major dietary flavonoids due to its abundance in fruits and vegetables. In recent years, it has received considerable global attention by the scientific fraternity due to its potent and multi-functional health promoting efficacy. Many of the biological activities attributed to flavonoids are also reported for quercetin, which include antioxidant, antiinflammatory, anticancer, anti-tumoral, anti-atherosclerotic, cardioprotective, anti-ulcer, antiallergic, and anti-hyperglycemic etc. Epidemiological evidences suggest that quercetin may play an important role in the prevention of chronic diseases if regularly consumed in required quantity. Owing to several scientifically validated health benefits and its potential to serve as a preventive tool against several human health problems, there is a re-emergence of keen interest for its use as dietary supplement and functional food ingredients. Overwhelming amount of scientific literature is available on various aspects of large number of flavonoids; therefore, we will confine our discussion only on quercetin, a pharmaceutically *Corresponding author: [email protected]

9.2

Natural Bioactive Molecules: Impacts and Prospects

potent flavonol, widely distributed in nature. In this chapter, we will review the recent findings on bioactivities, molecular targets, and structural aspects of quercetin and its analogues. Keywords: Quercetin, glycosides, bioavailability, metabolites, antioxidant, anticancer, cardioprotective, anti-inflammatory, synthetic analogues, enzyme inhibition

9.1

INTRODUCTION

Plant secondary metabolites form a very large and diversified group of naturally occurring chemicals of plant-origin (Tapas et al., 2008). They represent the non-energetic component of plant metabolism. Many of these secondary metabolites are responsible for various unique characteristics of plants, such as their color and organoleptic properties. Phenolic compounds form one of the main classes of secondary metabolites. Several decades of scientific investigations have revealed health enhancing potential of these molecules. Presently, several of them are also being explored as drugs, dietary supplements and nutraceuticals for the prevention and maintenance of various chronic and degenerative diseases. Flavonoids make a large group of plant-phenolics and are a very important class of plant secondary metabolites (Harborne, 1988). They are low molecular weight bioactive polyphenols, widely distributed in plant kingdom including fruits and vegetables, and are consumed in the daily human diet. Until now, more than 9000 structurally distinct flavonoids have been identified (Buer et al., 2010). The diversity in their chemical structures confers them a wide range of biological activities and their presence in our daily diet indicate their acceptable toxicity profile. Flavonoids promote physiological survival of plant by protecting them from fungal infections and UV radiations. Additionally, flavonoids actively participate in various vital processes of plants, including photosensitization, energy transfer, respiration, photosynthesis control, morphogenesis and sex-determination (Buer et al., 2010). They have also been found to be common chemical constituents of many medicinal plants, responsible for their therapeutic effects. They are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against reactive oxygen species (ROS) (Mariani et al., 2008). Flavonoids are characterized by flavan nucleus with two aromatic rings (A and B rings) interconnected by a three carbon atom heterocylclic ring (ring C), therefore, making a chromane-type C6-C3-C6 carbon skeleton, also called as 2-phenyl-benzo-g-pyrane nucleus (Harborne, 1999) as shown Fig. 1 Basic structure of flavonoids in Fig. 1. Flavonoids differ in their arrangements of hydroxy, methoxy, and glycosidic side groups including variation in A and B rings (Harborne, 1999). The most widespread flavonoids contain a double bond between C-2 and C-3 and a keto function at C-4 of ring C, which is attached to ring B at C-2 (flavone) or at C-3 (isoflavone). As a result of a number of further modifications on all three rings, particularly on ring C, flavonoids represent one of the largest and the most diverse class of plant secondary metabolites. Based on molecular structures they can be divided in eight subclasses, including chalcones, the direct biogenetic precursors of flavonoids (Fig. 2).

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

Fig. 2

9.3

Chemical structures of different sub-classes of flavonoids

These various classes of flavonoids can further get diversified by attachment of one or more sugars to form their glycosides, the form they mostly exist in the nature.

9.2

QUERCETIN AND DERIVATIVES

Quercetin (3,5,7,3¢,4¢-pentahydroxyflavone) (1) is one of the most abundant and ubiquitously distributed dietary polyphenolic bioflavonoid belonging to the flavonol class. Chemically, it is a molecule containing fifteen carbons with three rings and five hydroxyl groups; two each on ring-A and ring-B, and one on ring-C. All the three rings are planar and this molecule is relatively polarized. Quercetin possesses three intermolecular hydrogen bonds; one between the hydroxyl groups in ring B and the other two with the carbonyl group (Maria and Daniel, 2004). In plants, quercetin is mostly present in its glycosidic form i.e. one or more sugars are bound to phenolic hydroxyls through glycosidic linkage. The water-solubility of quercetin increases with increasing number of sugars attached to it. Quercetin was found to be stable in its aglycone as well as its glycosidic form at a wide range of temperature (25 to −80°C) and in acidic pH. Quercetin is not stable under high pH (basic) conditions resulting in opening of the central C-ring (Moon et al., 2008). Quercetin is also implicated in transportation, reactivity, bioavailability and toxicity of metal ions in plants. It possesses three possible chelating sites, chelating with metal ions competitively. These chelating sites binding affinity to metals increases in the following way: catechol > a-hydroxycarbonyl > b-hydroxycarbonyl (Cornard et al., 2005). Structurally, an orthodihydroxy or catechol group in ring B, and a 2,3-double bond, and the 3- and 5-hydroxyl groups with the 4-oxo group offer quercetin its strong anti-oxidant property (Silva et al., 2002; Bros et al., 1990). The anti-proliferation action of quercetin may be attributed to hydroxyl substitutions at carbon 3 of ring B and carbon 5 of ring A. On the contrary, glycosidic forms with sugar substitutions were found to lower its potency (Lao et al., 2009).

9.4

Natural Bioactive Molecules: Impacts and Prospects

Quercetin, being penta-hydroxyl flavonol possesses the possibilities to form a variety of glycosides, and ethers as well as less frequently occurring sulfates and prenyl substituents (Williams and Grayer, 2004). The chemical structures of some of quercetin derivatives are shown in Fig. 3.

No

Chemical name (Trivial name)

Substitutions R1

R2

R3

R4

R5

R6

R7

1

3,5,7,3¢,4¢-pentahydroxyflavone (Quercetin)

OH

OH

H

OH

OH

OH

H

2

Quercetin-3-O-glucoside (Isoquercetin)

OGlc

OH

H

OH

OH

OH

H

3

Quercetin-3-O-rhamnoside (Quercitrin)

ORha

OH

H

OH

OH

OH

H

4

Quercetin-3-O-rhamnosyl-(1Æ6)-glucoside (Rutin)

OP

OH

H

OH

OH

OH

H

5

Quercetin-7-O-glucoside

OH

OH

H

OGlc

OH

OH

H

6

Quercetin-3-O-rhamnoside-3-O-glucoside

ORha

OH

H

OGlc

OH

OH

H

7

Quercetin-6-O-glucoside

OH

OH

Glc

OH

OH

OH

H

8

Quercetin-3-O-(2≤-acetylgalactoside)

OQ

OH

H

OH

OH

OH

H

9

Quercetin-3-sulfate-7-O-arabinoside

OSO3Na

OH

H

OAra

OH

OH

H

10

Quercetin-3-O-glucoside-3¢-sulfate

OGlc

OH

H

OH

OH

OH

H

11

Quercetin-5-methyl ether (Azaleatin)

OH

OMe

H

OH

OH

OH

H

12

Quercetin-7-methyl ether (Rhamnetin)

OH

OH

H

OMe

OH

OH

H

13

Quercetin-3¢-methyl ether (Isorhamnetin)

OH

OH

H

OH

OH

OH

H

14

Quercetin-4¢-methyl ether (Tommarixetin)

OH

OH

H

OH

OH

OMe

H

15

Quercetin-7-methoxy-3-O-glucoside

OGlc

OH

H

OMe

OH

OH

H

16

Quercetin-3¢-methoxy-3-O-galactoside

OGalc

OH

H

OH

OH

OH

H

17

6,5¢-Di-C-prenylquercetin

OH

OH

prenyl

OH

OH

OH

prenyl

Glc: glucopyranosyl; Galc: galactopyroanosyl; Rha: rhamnopyranosyl; Ara: arabinopyranosyl; P: rhamnosylglucoside; Q: 2-acetylglactosyl

Fig. 3

Quercetin and some of its naturally occurring derivatives (Materska et al., 2008)

O-Glycosides of quercetin are most widely distributed derivatives of quercetin in plants. Almost every plant contains compounds of this group, and some like onions contains large quantities of these substances. Glycosides of quercetin are more hydrophilic due to the polar sugar moieties, therefore, can easily be transported to various parts of the plant and stored in the cytosolic vacuoles

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

9.5

of plant cells. The hydroxyl group at C-3 carbon is the most common site for the glycosylation of quercetin. Quercetin 3-O-glycosides occur as monosaccharides with glucose, galactose, rhamnose or xylose. These compounds are found in various fruits and vegetables, e.g., quercetin-3-Oglucoside (2) in mango fruit (Berardini et al., 2005), quercetin-3-O-rhamnoside (3) in spinach (Kuti and Konuru, 2004). Quercetin bonded to disaccharides is also frequently detected in plants, such as, rutin, a 3-O-rhamnosylglucoside of quercetin (4), which is found in significant quantities in tea, spinach etc. (Kuti and Konuru, 2004). In addition to monosaccharides and disaccharides, sugar chains with three-, four- and five saccharide moieties have also been identified in quercetin 3-O-glycoside derivatives (Harborne, 1994; Williams and Grayer, 2004). Another glycosylation site in quercetin is hydroxyl group at C-7 carbon as in case of quercetin-7-O-glucoside (5) which is found in beans (Chang and Wong, 2004). Moreover, glycosylation at C-7 is more frequently accompanied by sugar substitution at C-3 hydroxyl group e.g., quercetin-3-O-rhamnoside-7-Oglucoside (6) found in peppers (Materska et al., 2003). C-Glycosylation forms another type but lesser occurring quercetin derivatives in nature. The most common site for C-glycosylation is the C-6 carbon of quercetin, e.g., in 3,4,7,3¢,4¢-pentahydroxy6-C-glucosyl flavone (7) which was first identified in Ageratina calophylla (Harborne, 1994). The sugar moiety can also be acylated and sulfated, thus forming more possibilities of structural variation among quercetin derivatives (Williams and Grayer, 2004). Aliphatic acids, such as acetic, malonic and 2-hydroxypropionic acid, or aromatic acids, such as benzoic, gallic, caffeic and ferulic acid form ester bond with the hydroxyl groups of quercetin to make acyl derivatives (Harborne, 1994) e.g. quercetin-3-(2¢-acetylgalactoside) (8), is reported from St. John’s wort (Jurgenliemk and Nahrstedt, 2002). Sulfate derivatives of quercetin are relatively rare in nature e.g., quercetin3-sulfate-7-O-arabinoside (9), found in saltbush (Williams and Grayer, 2004) and quercetin-3-Oglucoside-3¢-sulfate (10), found in the cornflower (Flamini et al., 2001). Monoethers of quercetin are also widely distributed (Harborne, 1994). Even, glycosidic ethers of quercetin have also been reported in nature as identified in sage plant viz., quercetin-7-methoxy-3-glucoside (15) and quercetin-3¢-methoxy-3-galactoside (16) (Lu and Foo, 2002). Moreover, the lipophilic derivatives of quercetin are also identified, though less frequently, such as 6,5¢-di-C-prenyl quercetin (17) found in paper mulberry (Son et al., 2001). Quercetin has been reported to have several functional properties such as antioxidant (Mariani et al., 2008), anti-inflammatory (Nair et al., 2006), antibacterial (Metwally et al., 2010), immunomodulatory (Kempuraj et al., 2006), antidiabetic (Li et al., 2009), anti-carcinogenic (Seufi et al., 2009), and cardioprotective (Annapurna et al., 2009; Egert et al., 2009). Epidemiological data suggests that high dietary quercetin intake is associated with decreased rate of cancers such as, colorectal (Kyle et al., 2010), kidney (Wilson et al., 2009), pancreatic (Bobe et al., 2008), prostate (McCann et al., 2005), and lung cancer (Lam et al., 2010; Cui et al., 2008). As a result of these bioactivities, quercetin is considered to be one of potential flavonoid, capable of interacting with and modulating activity of a variety of enzymes, including but not limited to, cyclooxygenase, lipooxygenase, phosphodiesterase, and tyrosine kinase.

9.6

Natural Bioactive Molecules: Impacts and Prospects

9.3

DIETARY SOURCES

Regularly eaten fruits and vegetables, particularly onions, tea, apples, citrus fruits, parsley, red wine, etc. are the primary dietary sources of quercetin. Studies were conducted on the flavonoids (myricetin, quercetin, kaempferol, luteolin, and apigenin) contents of 62 edible tropical plants. Miean and Mohamed (2001) reported the highest total flavonoids content in onion leaves with 1497.5 mg/kg quercetin followed by semambu leaves, bird chillies, black tea, papaya shoots and guava. The major flavonoid in these plant extracts was quercetin, followed by myricetin and kaempferol. As per ‘USDA Database for the Flavonoid Content of Selected Foods’, the quercetin content of some of the selected food items is shown in Table 1 (Nutrient Data Laboratory, 2003). Table 1

Quercetin content in selected food as per USDA database (Nutrient Data Laboratory, 2003).

Food Item (edible portion)

Quercetin mg/100g

Apple, raw Broccoli, Raw Capers, raw

4.42

Food Item (edible portion) Kale, Raw

Quercetin mg/100g 7.71

3.11

Lingonberries, Raw

12.16

180.77

Lovage leaves, raw

170.12

Carrots, Raw

0.07

Onions, red, raw

19.93

Cauliflower, raw

0.03

Peppers, hot chili, green, raw

16.80

3.52

Spinach, raw

Celery, Raw Cocoa powder, Unsweetened

20.13

5.86

Tea leaves, black, dry

204.66 255.55

Cranberries, Raw

14.02

Tea leaves, green, dry

Fennel leaves, raw

48.80

Tomatoes, red, ripe, Raw

0.57

Red and yellow onions (Allium cepa L) are found rich in quercetin content in a survey of 28 vegetables and 9 fruits. Amount of quercetin in onions vary with variety and bulb color type. Regardless of color of the onion bulb and variety, quercetin concentration was found to be highest in their outer rings (Lombard, 2005). Hakkinen et al. (1999) reported the content of quercetin in 25 edible berries collected from Finland and confirmed its presence in all the berries. However, Harnly et al. (2006) reported that the variation in flavonoid content is very large with a relative standard deviation (RSD) of 168%, in an extensive study for the fruits and vegetables encompassing their collection during different seasons from various parts of United States.

9.4

BIOAVAILABILITY

Initially, Kuhnau (1976) has reported the daily human intake of flavonoids to be around one gram, but more recent studies estimate the average dietary intake of flavonoids of around 20–25 mg/day (Hertog, 1993). However, large variations usually occur even within countries depending on the food habits of various groups in that population, with flavonols plus flavones ranging from 6 to 94 mg (Hertog et al., 1995). Quercetin, present in foods as quercetin glycosides, represents 60–75% of the total dietary flavonoid-intake, which has appeared as the basis for its commercialization as a dietary supplement.

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Despite being a multi-functional bioactive molecule, low bioavailability of quercetin poses the major challenge for its use as a therapeutic molecule (Passamonti, 2009). Quercetin exists in nature mostly in the form of glycosides. The glycosides being polar molecules are not able to penetrate the intestinal membranes, therefore, are not easily absorbed in the body. Microfloral enzymes hydrolyze and release the aglycone to make it absorbable (Fig. 4). Although quercetin aglycone is permeable to some extent, the bioavailability is not high due to poor solubility and permeation across membrane and its metabolism (Walgren, 2000).

Fig. 4 Quercetin glycosides and their proposed course of digestion, absorption, metabolism, and excretion in human body (Vargas and Burd, 2010). Reproduced here with permission from the author.

Probably, sugar-free flavonoids (aglycones) can pass through the gut wall, but only lower quantities are found in their free form in plants. It has been proposed by Vargas and Burd (2010) that orally ingested quercetin glycosides get partially hydrolyzed in the oral cavity. Majority of it then gets digested and absorbed at multiple sites along the GI tract (Fig. 4). Quercetin modification takes place during absorption or shortly thereafter and then it enters the circulatory system and then metabolized in the liver by O-methylation, glucuronidation, and/or sulfation to yield various metabolites in varying concentrations viz., 3-O-methylquercetin (18), quercetin-3¢-O-sulphate (19), quercetin-3glucuronide (20), 3¢-O-methylquercetin-7-glucuronide (21) and isorhamnetin-3-glucuronide (22) etc. (Fig. 5). The microorganisms in the colon hydrolyze glycosidic bond to release aglycone,

9.8

Natural Bioactive Molecules: Impacts and Prospects

however, this hydrolysis also degrade quercetin (Murota and Terao 2003; Hollman et al., 1997). Here it is noteworthy that the conversion of quercetin to its metabolites may be helpful in avoiding its harmful effects, as higher concentrations of quercetin aglycone is known to act as a pro-oxidant (Murota and Terao 2003).

Fig. 5 Quercetin metabolites

There has been contradiction among the various studies carried out on the bioavailability of quercetin. In one of the study it was suggested that the glucoside is better absorbed than the aglycone because the glucose transporter (SGLT-1) facilitate its absorption (Hollman et al., 1995). However, Murota and Terao (2003) showed that because of more lipophilic character, quercetin-4¢-O-bglucoside was absorbed more effectively than quercetin-3-O-b-glucoside or quercetin-3,4¢-di-Ob-glucoside. On the contrary, Wittig et al., (2001) showed that complete hydrolysis of quercetin glycosides to their aglycone took place before intestinal absorption, supporting the more widely accepted idea. Furthermore, Day et al., (2001) showed that 1.5 hrs after consumption of onions, only quercetin metabolites viz., quercetin-3¢-sulfate (19) and quercetin-3-glucuronide (20), and isorhamnetin-3-glucuronide (22) were found in human plasma, instead of any glycosidic and aglycone form of quercetin. Scalbert et al., (2002) has proposed that quercetins can exert a positive effect regardless of their poor absorption. In this study, when the rats were fed quercetin in the form of teas and wines (good sources of quercetin), high concentrations of flavonoids were found on the gut lumen of the rats, which resulted in the reduction of oxidative damage to DNA in ceacal mucosal cells thereby exerting the beneficial effect. Furthermore, Azuma et al., (2003) demonstrated that blood plasma concentration of quercetin metabolites increased when quercetin was ingested along

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

9.9

with lipids (e.g., lecithin, fish oil, and beef tallow) and emulsifiers (e.g., sodium caseinate and sucrose fatty acid ester). Therefore, lipids and emulsifiers may facilitate its absorption and increase bioavailability of quercetin.

9.5 9.5.1

HEALTH BENEFITS OF QUERCETIN Quercetin as Antioxidant

Free radicals such as superoxide, hydrogen peroxide, and hydroxyl are reactive molecular species with unpaired electrons that generate other free radicals by oxidizing other molecules, thus initiating a domino effect of free radical stabilization and formation in the cells and its environment (Machlin and Bendich, 1987). At cellular level, oxidative metabolism generates and propagates free radicals, especially in the presence of transition metal ions (e.g., iron and copper) which aid in electron transfers (Uddin and Ahmad, 1995). Free radicals can oxidize macromolecules, such as DNA, proteins, carbohydrates, and lipids (Uddin and Ahmad 1995). They denature proteins and peroxidise unsaturated bonds in membrane lipids; consequently, lipids lose their fluidity (Machlin and Bendich, 1987). As a whole, these oxidative damages by free radicals are collectively referred to as oxidative stress and are associated with several degenerative diseases including cardiovascular, inflammatory, cancer, aging, and stroke related diseases (Machlin and Bendich, 1987). Quercetin has been shown to scavenge free radicals and bind with transition metal ions. Most of the reported biological activities of quercetin can be ascribed to its ability to act as an antioxidant. It is one of the most potent scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Boots et al., 2008). When quercetin reacts with a free radical, it donates a proton and becomes a radical itself — the unpaired electron gets delocalized by resonance, thereby, converting quercetin to a less reactive radical than the parent free radical (Mariani et al., 2008) (Fig. 6). Moreover, the ortho-dihydroxyl moiety in B ring, the 4-oxo group in conjugation with the 2,3-alkene, and the 3- and 5-hydroxyl group, aid in stability of quercetin providing it antioxidant properties by countering free radicals (Hollman and Katan, 1997). Presence of these functional groups increase the number of resonance forms the molecule can exist allowing it to make unpaired electron less available (Mariani et al., 2008).

Fig. 6

DPPH free radical scavenging pathway for quercetin (Goupy et al., 2003)

Quercetin is able to chelate ROS producing metal ions, which in turn protects cells from oxidative DNA damage, prevent the oxidation of low-density lipoproteins (LDL) by scavenging free radicals (Hollman and Katan, 1997; Murota and Terao, 2003). The presence of sugar in

9.10

Natural Bioactive Molecules: Impacts and Prospects

quercetin glycosides can obstruct its antioxidant activities because of engagement of the hydroxyl groups. Therefore, the aglycosylated form usually has higher antioxidant potency than the glycoside form (Murota and Terao, 2003). However, detailed mechanism of anti-oxidant activity of quercetin is not completely understood. Quercetin inhibits nitric oxide synthase activity, which has been known to generate the damaging peroxynitrite from nitric superoxide anions, resulting in a reduction in experimental ischemia–reperfusion injury (Shutenko et al., 1999; Huk et al., 1998). Quercetin also decreases oxidative injury occurring after ischemia or other related conditions by inhibiting the xanthine dehydrogenase/xanthine oxidase system, a well-known generator of reactive oxygen species (Bindoli et al., 1985; Zhu et al., 2004). Recently, Soundararajan et al., (2008) have proposed a novel mechanism for quercetin-induced cyto-protection involving the sterol regulatory element-binding protein-2 (SREBP-2)-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress. Begum and Terao (2002) demonstrated that damaged erythrocytes due to smoking could be protected by quercetin and its conjugate metabolites quercetin-3-O-b-glucuronide (22) and quercetin-3-O-b-glucoside (2). They used basic flavone (quercetin without hydroxyl groups) as control, which showed no effect on the erythrocytes. This study therefore indicated that, structurally, the hydroxyl groups could play a very important role in the antioxidant property of quercetin. Quercetin has also been shown to work synergistically with other antioxidant systems in the body. When quercetin exerts its antioxidant power, it can advance to the highly oxidized states such as semiquinone or even the ortho-quinone state, which can be potentially damaging to the cell (Boots et al., 2003; Kim and Jang, 2009). Quercetin has also shown to activate intrinsic antioxidation pathways involving glutathione (GSH) (Kim and Jang, 2009). Studying the relationship between oxidized quercetin and GSH in a human hepatoma cell line (HepG2), Kim and Jang (2009), found that doses of quercetin up to 100 mM led to antioxidant affects, however, extended exposure of 100 mM quercetin for longer than 30 min led to pro-oxidant/pro-apoptotic effects. Moreover, quercetin was able to chelate ROS producing reactive metal ions, reduce ROS by reacting with hydrogen peroxide, and activate GSH, which in turn quenched ROS to their reduced states (Kim and Jang, 2009). This stimulation of GSH by quercetin is likely to be one of mechanisms by which it can protect the cell from mutagenesis. On the contrary, quercetin may be able to cause cellular damage when it is administered in high dose for longer duration. Interestingly, quercetin also has a unique ability to act as a pro-oxidant depending on its concentration. Quercetin is able to act as a prooxidant at concentrations greater than 40 mM (Watjen et al., 2005). Min and Ebeler, (2009) showed in colorectal adenocarcinoma (Caco-2) cells that treatment at lower concentrations of quercetin (1 mM) resulted in the decrease of doublestranded DNA breakage, whereas higher concentrations of quercetin (100 mM) favored such breakage. Consequently, lower concentrations prevented oxidative DNA damage and increase DNA repair, whereas, higher concentrations led to increased DNA damage, which in turn, led to increased apoptosis, more preferably, in cancerous cells. Moreover, several studies carried out on antioxidant property of quercetin in vivo has further reinforced the fact that antioxidant and prooxidant capacity of quercetin is dependent on its concentration as well as the form of quercetin (glycoside or aglycone or metabolite) in the blood and, presumably, target tissues (Santos et al., 2008; Murota et al., 2007; Justino et al., 2004).

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

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9.11

Anticancer Potential

Quercetin has also been known to be associated with reduced risk of certain types of cancers. Quercetin is able to act as prooxidant at concentrations greater than 40 mM, which may be beneficial in cancer prevention and therapy by countering the transformation and growth processes of cancerous cells (Watjen et al., 2005). Some mutations lead to uncontrolled cell growth, which in turn leads to malignant tumors. At higher dosage, quercetin shows beneficial anti-tumor effects by exerting prooxidant and apoptotic effects. Quercetin at greater than 40 mM concentration is able to increase oxidative stress and cytotoxicity in tumor cells by becoming a ROS itself, damaging the DNA, which in turn induces apoptotic pathways (Metodiewa et al., 1999). Quercetin induces apoptosis in multiple cancer cell lines when administered in doses of more than 40 mM concentrations via the induction of P5, a tumor-suppressor protein which can activate Bax, thereby initiating cell death (Tan et al., 2009; Zhang et al., 2008; Zhang et al., 2009). Quercetin in larger doses with longer exposure time is the pre-requisite for decreased cancer cell viability. Zhang et al., (2008), has proposed that mechanistically quercetin may also induce apoptosis via mitochondrial mediated pathway (Zhang et al., 2008). Recently, Tan et al., (2009) showed in human hepatocellular carcinoma cell line that the expression of P53 increases on treatment with 40–120 mM quercetin, while the level of anti-apoptotic survivin and Bcl-2 proteins decreases. Similarly, quercetin induced apoptosis in human breast cancer cells (MDA-MB-231) via P53 and mitochondria mediated cell-death mechanism. Chien et al., (2009), observed an increase in P53, caspase-9 activation, caspase-3, cytochrome c, and apoptosis in MDA-MB-231 cells treated with 200–250 mM quercetin in vitro. Additionally, they also showed quercetin decreased mitochondrial membrane potential. Zhang et al., (2008) showed in vitro the dose-dependent cytotoxicity of quercetin in p53 mutated human esophageal squamous carcinoma cell line (KYSE-510). The probable mechanism for apoptosis may be via increased cleavage of procaspase-9 and caspase-3 after the treatment of KYSE-510 cells with 80 mM quercetin (Zhang et al., 2008). Quercetin, therefore, shows the ability to initiate apoptosis via the mitochondrial pathway by activating caspase-3, and down regulating caspase-9 (Zhang et al., 2008). A graphical representation (Vargas and Burd, 2010) depicting the involvement of these pathways is given in Fig. 7. Quercetin has even been shown to be capable of differentiating between normal and malignant cells to some extent (Siegelin et al., 2009). Chien et al., (2009) found evidence that quercetin also induced separate pathway, independent of p53 apoptotic pathway known as the death receptor or death-domain pathway. Synergy of quercetin with other death-domain stimulators such as tumor necrosis factor (TNF) a-related apoptosis-inducing ligand (TRAIL) may bring about apoptosis (Siegelin et al., 2009) (Figure 8). Siegelin et al., 2009, reported that quercetin (100 or 200 mM) led to apoptosis of glioma cells via TRAIL sensitization, which selectively kills only cancerous cells. Glioma cells are resistant to TRAIL-induced apoptosis, and neither quercetin nor TRAIL alone caused apoptosis in their cell lines at doses below 300 mM (Siegelin et al., 2009). Galluzzo et al., (2009), have shown that the quercetin in some cervical cancer cell lines induce cytotoxicity via stimulation of the ER-a-P38/mitogen activated protein kinase apoptotic pathway. Quercetin preferentially favors binding to estrogen receptor b (ER-b) over ER-a (Sotoca et al., 2008). Sotoca

9.12

Fig. 7

Natural Bioactive Molecules: Impacts and Prospects

Schematic representation of pro-apoptotic pathways involved in anticancer activity of quercetin (Vargas and Burd, 2010). Reproduced here with permission from the author

et al., 2008, showed ascorbate-stabilized quercetin (> 50 mM) increased quercetin-ER-b binding and apoptosis in breast cancer (T47D-ER-a) and osteosarcoma (U2OSER-a and -ER-b) cell lines (Fig. 8).

Fig. 8

Schematic representation of pro-apoptotic pathways involved in anticancer activity of quercetin (Vargas and Burd, 2010). Reproduced here with permission from the author

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Heat shock protein (HSP) chaperones are responsible for the correct folding and maintenance of proteins in the body. When protein chaperones are unable to perform their duties, cell functionality is decreased and cell death happens. HSP chaperones, specifically, are unregulated in some type of cancerous cells (Wang et al., 2009). Quercetin is able to inactivate these HSP chaperones, seemingly by its ability to inhibit the kinases that aid in HSP induction (Fig. 7) (Wang et al., 2009). Quercetin has also been shown to decrease HSP90 expression in prostate cancer cells. HSP90 is a chaperone protein that aids in the maintenance of oncoproteins, such as insulin-like growth factor binding protein-2 (IGFBP-2) and human epidermal growth factor 2 (HER2) (Aalinkeel et al., 2008). Quercetin is a mutagenic flavonoid, but this mutagenic effect has shown to have no carcinogenic effects (Hertog et al., 1995). It has been found that quercetin reduces the mutant p53 expression in cancer cells and causes G2/M and G1 cell cycle arrest. Quercetin increases the binding site expression of the estrogen receptor II (ER II) in breast cancer cells and inhibits the growth by binding to ER II (Cantero et al., 2006). Cruz-Correa et al., (2006), in a small intervention trial, have demonstrated reduction in polyp number by 60% and reduction in polyp size by 50% from baseline in five patient volunteers with familial adenomatous polyposis, when given curcumin (480 mg) and quercetin (20 mg) orally three times a day for 6 months. Quercetin inhibits tyrosine kinase activation, is the first flavonoid studied for Phase 1 clinical trial in humans (Brit et al., 2001). Lower bioavailability of quercetin poses a bigger challenge for its application for the treatment of cancers, as we know the concentration requirements are much higher than its anti-oxidant effect. Based on molecular dynamics simulations, Boyer et al., (2011) synthesized quercetin analogs viz., 4¢-chloro flavanol (23) and 4¢-methoxy flavanol (24), not having phenolic hydroxyl groups of ring A and ring B (Fig. 9). On testing anticancer activity against breast cancer (MCF7), hepatic cancer (HepG2), prostate cancer (PC3) and colon cancer (HCT15), it was found that 4¢-chloro flavanol (23) showed anticancer activity equivalent to quercetin in HepG2 cells, though the activity was much less for other cell lines. Moreover, this study showed significant reduction in antioxidant and anti-inflammatory activity for (23) and (24), thereby, suggesting that the presence of catechol at ring-B and meta-dihydroxy at ring-A is a must in addition to the presence of C2-C3 double bond along with C-3 hydroxyl in order to have its antioxidant activity (Boyer et al., 2011). Anti-cancer potential of quercetin seems to have not been realized in many of the in vivo studies, owing to its lesser water solubility. Therefore, Ulug et al. (2011) prepared a soluble complex of carboxylated quercetin with propylethylimine to form carboxylated quercetin-polyethylenimine complex (25) and was able to demonstrate its increased anticarcinogenic effect on HeLa cancer cells (Fig. 9).

9.5.3

Cardioprotective Activities

Epidemiological studies have shown an inverse relationship between flavonoid intake and risk of cardiovascular diseases. Quercetin is implicated for inhibition of vasoconstriction and antithrombotic effects. Quercetin has been reported to cause endothelium-dependent relaxation involving nitric oxide (Ajay et al., 2003). Quercetin may act through various anti-inflammatory mechanisms, e.g., regulation of the expression of cellular adhesion molecules and regulation of the secretion of pro-inflammatory cytokines and chemokines (Tribolo et al., 2008; Nair et al., 2006). Quercetin reduced blood pressure both in human and animal studies (Edwards et al., 2007;

9.14

Natural Bioactive Molecules: Impacts and Prospects

Fig. 9

Synthetic analogues and soluble complex of quercetin

Rivera et al., 2008; Egert et al., 2010) and inhibited the platelet activation pathway in human (Hubbard et al., 2006). It improved endothelial function in rat models of hypertension (Sanchez et al., 2006). Quercetin was also shown to reduce oxidation of LDL by inhibiting neutrophil myeloperoxidase, thereby potentially reducing the risk of heart disease and atherosclerosis (Loke et al., 2008a). Interventional/observational studies have shown that both short- and long-term black tea consumption improved endothelial function in patients with existing coronary artery disease (Duffy et al., 2001). In a double-blind crossover study of 49 healthy male subjects with APOE genotype, quercetin showed significant reduction in postprandial systolic blood pressure, postprandial triacylglycerol concentrations, and enhanced HDL-cholesterol concentrations in comparison to placebo. However, the study also showed an increase in TNF-a, potent inflammatory cytokine though values were biologically not significant (Pfeuffer et al., 2013). In several studies, quercetin is reported to inhibit the oxidation of low-density lipoproteins (LDL) which causes the formation of atherosclerotic plaques, thus leads to cardiovascular disease (Hollman and Katan, 1997). Epidemiological study by Graf et al., (2005) showed a reduction of 21% in cardiovascular disease mortality when the intake of quercetin was greater than 4 mg/d. Chopra et al., (2000) gave quercetin (30mg/d) to one group of males and 1g of flavonoid rich red wine powdered extract (containing 3.5 mg/g quercetin) for another group for two weeks. Vitamins C and E plasma concentrations were also measured along with flavonoids. They reported that LDLcholesterol is lowered by quercetin in only hyperlipidemic patients (Chopra et al., 2000). Quercetin and its metabolite quercetin-3¢-sulphate (19) inhibited receptor-mediated contractions of the procine isolated coronary arteries by selectively enhancing the cyclic-GMP-dependent vasodilator glyceryl trinitrate, therefore, showing its potential for patients with angina pectoris (Suri et al., 2010). Rutin (4), a quercetin glycoside, inhibits platelet aggregation via activation of phospholipase C, followed by inhibition of PKC activity and thromboxane A2 formation, thereby leading to the inhibition of the phosphorylation of P47 and intracellular calcium mobilization (Sheu et al., 2004). Quercetin is also capable of regulating platelet function by inhibiting thrombin-induced and collagen-induced platelet activation (Hubbard et al., 2003). Gryglewski et al., (1987), showed that quercetin is about six times more effective than rutin (4) in preventing platelet aggregation in rabbits.

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

9.5.4

9.15

Anti-diabetes and Anti-obesity Potential

Oxidative stress caused by intracellular ROS, plays a crucial role in insulin resistance and in pancreatic b-cell death during the progression of glucose intolerance and development of type 2 diabetes (Piotout et al., 2008). Quercetin is known to have antihyperglycemic activity as it lowers blood glucose, normalizes glucose tolerance tests and protects pancreatic b-cell integrity in diabetic rats (Shetty et al., 2004). However the mechanism of its antihyperglycemic action is not well understood. Recently, Youl et al., (2010) have reported that quercetin potentiates glucose and glibenclamide-induced insulin secretion and protects b-cells against oxidative damage. This study also showed that the ERK1/2 signaling pathway played a very important role in the potentiation of glucose-induced insulin secretion by quercetin (Youl et al., 2010). In another study, Kannappan and Anuradha (2009) have shown in vivo Wistar rat model that quercetin enhances insulin signaling and sensitivity equal to metformin (Kannappan and Anuradha, 2009). One of the therapeutic approaches to address the hyperglycemia is to inhibit the a-amylase and a-glucosidases which are responsible for the hydrolysis of disaccharides (maltose and sucrose) and starches into free sugars which get absorbed in the small intestine. The a-glucosidases include intestinal maltase, sucrase, glucoamylase and isomaltase (Hirsh et al., 1997; Deshpande et al., 2009). Jo et al., (2009) have reported that quercetin and its derivatives showed a significant inhibition of the a-glucosidase but did not show inhibition of porcine pancreatic a-amylase. This quercetin based therapy may lead to lower abdominal side effects arising from excessive inhibition of pancreatic a-amylase. Mono- and diglycoside derivatives of quercetin such as rutin (4) and isoquercetin (2) have shown higher level of inhibition on maltase, glucoamylase, and isomaltase. Therefore, the a-glucosidase inhibitory activity along with its antioxidant activity would be helpful in managing glucose absorption, thereby reducing the spike of glucose in diabetic people (Jo et al., 2009; Li et al., 2009). Topical effects of querectin on diabetes-associated neuropathy have also been reported (Valensi et al., 2005). In a randomized, placebo-controlled, double-blind trial including 34 men and women with type 1 or 2 diabetes and diabetic neuropathy showed that QR-333, a topical preparation containing quercetin, safely reduced the severity of diabetic neuropathy including numbness, jolting pain, and irritation from baseline values, and was well tolerated, and also improved qualityof-life measures (Valensi et al., 2005). On administration of quercetin to obese Zucker rats and the rats fed with a high-sucrose/high-fat diet, significant reduction in blood pressure was observed (Rivera et al., 2008).

9.5.5

Antihypertensive Activity

The blood pressure lowering effect of a fruit and vegetable-rich diet is a necessary dietary lifestyle measure for the management of arterial hypertension (Margetts et al., 1986). Quercetin has been shown to induce a progressive, dose-dependent and sustained reduction in blood pressure when given chronically in several rat models of hypertension, including spontaneously hypertensive rats, L-NAME-treated rats, DOCA-salt hypertensive rats, two-kidney one-clip Goldblatt rats, rats with aortic constriction and Dahl salt-sensitive hypertensive rats (Garcia-Saura et al., 2005; Duarte et al.,

9.16

Natural Bioactive Molecules: Impacts and Prospects

2002). Quercetin exerts antihypertensive effects and improves endothelial function by inhibiting effects of endothelin-1 including increased protein kinase C (PKC) activity (Rivera et al., 2008). Quercetin also prevented morphological and functional changes in the heart, vessels and kidney in elevated ROS associated hypertension. A high dose of quercetin also reduced blood pressure in stage 1 hypertensive patients in a randomized, double-blind, placebo-controlled, crossover study (Edwards et al., 2007). It is proposed that the blood pressure-lowering effect of quercetin could be an important mechanism contributing to the reduced risk of myocardial infarction and stroke observed with flavonoid rich fruit and vegetables diets (Garcia-Saura et al., 2005).

9.5.6

Anti-inflammatory Activity

Quercetin is known to have effects on a variety of inflammatory processes and immune functions (Busse et al., 1984; Camuesco et al., 2004; Comalada et al., 2005; Kawada et al., 1998; Middleton et al., 1998; Nair et al., 2004; Wang and Mazza 2002). Ferandiz et al., (1991), reported that quercetin possesses the capability to modulate arachidonic acid metabolism via inhibition of the enzyme lipoxygenase. Another study by Lee et al., (1982) indicated that quercetin inhibited phospholipase A2 of stimulated neutrophils through inhibition of superoxide radicals by 33% and reduction in release of the proteolytic enzyme b-glucuronidase by 52% in vitro (Ferandiz et al., 1991). Tumor Necrosis Factor (TNF)-a is a major pro-inflammatory cytokine that regulates the growth, proliferation, differentiation and viability of activated leukocytes. Sato et al., (1997) have shown that quercetin suppresses TNF-a induced IL-8 and MCP-1 expression in cultured human synovial cells. Similarly, several studies have demonstrated the inhibitory effects of quercetin on the expression of inflammatory cytokines by cultured cells (Cho et al., 1997; Wadsworth and Dennis, 1999). Nair et al., (2006) have also demonstrated that quercetin inhibits TNF-a production in normal peripheral blood mononuclear cells (PBMCs) via down regulation of NFkB and IkB (Nair et al., 2006). Additionally, they have also shown that quercetin significantly down regulates p24 antigen production, ltr gene expression and viral infectivity in a dose dependent manner (5-50 mM) as compared to HIV infected untreated control PBMCs. Furthermore, quercetin significantly down regulated the expression of the pro-inflammatory cytokine, TNF-a with concomitant up regulation of anti-inflammatory cytokine IL-13 (the latter was determined by measurement of gene expression and protein production). A higher level of IL-13 is known to inhibit TNF-a production (Nair et al., 2009). Quercetin can also reduce inflammation by scavenging free radicals that activate transcription factors generating pro-inflammatory cytokines, which in turn are often found to be elevated in patients suffering from chronic inflammatory diseases (Boots et al., 2008). Alexander et al., (1998) showed that men with the chronic prostatitis had higher levels of both pro-inflammatory cytokines TNF-a and IL-1b in their seminal plasma. In one interventional study, Shoskes et al., (1999) showed that when quercetin (500mg) given twice a day for one month to the workers with chronic prostatitis showed 25% improvement in symptoms in 67% of volunteers. In another study, Shoskes et al., (1999) showed that 82% of men with the chronic prostatitis had at least a 25% improvement on administration with quercetin and Prosta-Q (bromelain and papain, which increases absorption of quercetin). Kumazawa et al., (2006), have shown in collagen-induced

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mouse model of rheumatoid arthritis, oral administration of quercetin led to improvement in symptomatic relief after the onset of arthritis via signal transduction and activation of transcription 1 and NF-kB activations, mitogen-activated protein kinase family phosphorylation, as well as accumulation of lipid rafts (Lee et al., 2008). Therefore, it can be inferred that quercetin has the potential to reduce acute, chronic, and subclinical inflammatory processes.

9.5.7

Antiallergic Activity

It has been proposed that quercetin could have a powerful antihistamine activity and thus, can help in preventing allergic and asthma attacks (Fanning et al., 1983). Middleton et al., (1998) have confirmed that the quercetin also inhibits histamine release. Later studies showed that quercetin inhibited histamine release by affecting intracellular calcium levels and PKC activation (Pearce et al., 1984; Kimata et al., 2000). Moreover, it was shown to cause a decrease in the release of tryptase, monocyte chemotactic protein-1 and IL-6, and down-regulated histidine decarboxylase mRNA in human mast cell line (Kempuraj et al., 2006; Shaik et al., 2006). Quercetin is also a potent inhibitor of leukotriene B4 formation in leukocytes (IC50:2mmol). The major quercetin metabolite, quercetin-3-O-sulfate (19), retained considerable lipoxygenase inhibitory activity, whereas quercetin-3-O-glucuronide (20) maintained antioxidant activity but had no lipoxygenase inhibitory activity at physiological concentrations (Loke et al., 2008b).

9.5.8

Immunomodulator Activity

Quercetin is reported to be the main effective component of some antiabortive herbs. However, there are very few report on how it affects the survival of the fetus. In one of the recent studies, Wang et al., (2011) have demonstrated that when quercetin in combination with bornyl acetate is given to lipopolysaccharide (LPS)-induced pregnant mice, the ratio of CD4+/CD8+ T lymphocytes and IFN-g and IL-4 contents in uterus were lowered than that of the LPS injected mice control. Uterine CD4+ and CD8+ T cells were detected immunohisto-chemically, and IFN-g and IL-4 contents were measured by Enzyme-linked immunosorbent assay (ELISA) with an aim to elucidate the antiabortive effects and the mechanisms of antiabortive action of quercetin. The results showed that quercetin and bornyl acetate had an antiabortive effect through modulation of immunological balance at maternal-fetal interface (Wang et al., 2011).

9.5.9

Memory Enhancing Activity

The lipid peroxidation of brain lipid membranes is thought to lead to neurodegenerative disease, such as Alzheimer’s and Parkinson’s disease (Balazs and Leon 1994). Balazs and Leon (1994) showed that free radical based oxidative stress in the brain membrane lipids lead to extracellular accumulation of amyloid beta-peptide, which precedes neural losses in Alzheimer’s patients. However, antioxidants may reduce the b-amyloid plaques formation (Harman et al., 1976). In this situation, quercetin, which has been shown to cross blood-brain barrier, stops the propagation of lipid peroxidation and also increases glutathione (GSH) levels (Ansari et al., 2008). GSH plays important role in protecting neurons from oxidative damage. Superoxide dismutase (SOD), another important antioxidant enzyme, can convert superoxide radical to less damaging hydrogen peroxide

9.18

Natural Bioactive Molecules: Impacts and Prospects

radical; furthermore, GSH can decompose hydrogen peroxide to oxygen and water, preventing the formation of free radicals (Balazs and Leon 1994).

9.5.10

Hepatoprotective Activity

Quercetin has also shown to have hepatoprotective potential by inhibiting the liver fibrosis. Quercetin is reported to be present in many medicinal plants that have been shown to be hepatoprotective. Gulati et al., (1995) demonstrated significant hepatoprotective effect of quercetin (15 mg/100g body weight) given orally to albino rats and mice which was isolated from Phyllanthus emblica. Janbaz et al., (2004) showed pretreatment of quercetin (10 mg/kg) significantly lowered the paracetamol as well as CCl4 induced serum levels of aminotransferases, aspartate transaminase (AST) and alanine transaminase (ALT)) in rats. Quercetin also prevented the CCl4-induced prolongation in pentobarbital sleeping time confirming their hepatoprotective action through multiple mechanisms.

9.5.11

Antimicrobial Activity

Quercetin appears to exert antibacterial activity against almost all the strains of bacteria known to cause respiratory, gastrointestinal, skin and urinary disorders. Quercetin and its glycosides have been shown to have antibacterial (Akroum et al., 2009; Martini et al., 2004), antiviral and antifungal activity. Quercetin-3-O-glycoside from Mentha longifolia inhibited the growth of causal agents of human urinary, intestinal and respiratory infections i.e. S. aureus, B. cereus, E. coli, P. aeruginosa, B. subtilis at lowest MIC among the compounds evaluated. Recently, Metwally et al., (2010) isolated quercetin and four quercetin glycosides from Psidium guajava L. leaves, all the isolated quercetins showed good antimicrobial activity. Kim et al., (2010) showed that intraperitoneal administration of isoquercetin (2) and quercetin (1) in human influenza virus infected mice significantly decreases the virus titers and pathological changes in the lung. Isoquercetin showed better antiviral effects than that of quercetin. It was also proposed that isoquercetin can be used in the treatment of influenza virus infection in combination therapy with existing drugs (Kim et al., 2010). Thapa et al., (2011) studied antiviral activities against influenza virus of seven synthetic quercetin derivatives (26-32) (Fig. 10) by modifying quercetin (1) at C-3, C-3¢, and C-5 with phenolic ester, alkoxy, and aminoalkoxy. On comparison among all the seven derivatives, the quercetin-3-gallate (26) showed very strong antiviral activity against influenza virus which was comparable to that of epigallocatechin gallate (EGCG) with improved in vitro therapeutic index (Thapa et al., 2011).

9.5.12

Endurance Enhancing Activity

Sport persons involved in high endurance sports often develop diverse inflammatory and oxidative stresses that compromise their immunity. Sport researchers have established that quercetin has the potential to enhance physical and mental performance (Teixeira, 2002; Nieman, 2007 and 2008). Although the underlying mechanisms are not really clear, they are mostly related to reduction of exercise-induced oxidative damage and inflammation by quercetin (McAnulty et al., 2008).

Dixit et al.: Quercetin – Structural Aspects vs. Health Benefits

Fig. 10

9.19

Antiviral synthetic analogues of quercetin

Quercetin supplementation has been demonstrated to reduce incidents of viral infections after extensive exercise (Davis et al., 2008). Nieman et al., (2010) demonstrated that in both animal and human, quercetin stimulates mitochondrial biogenesis, thereby enhancing ATP production, which in turn may work as ergogenic.

9.6

QUERCETIN — HOW FAR IT IS TOXIC?

Ubiquitous presence of quercetin in various common fruits, vegetables and drinks lead to its consumption in a significant quantity in human daily diet. Therefore, it is supposed to be reasonably safe and well tolerated in humans. However, there are few contradictory reports on toxicity of quercetin. Dunnick and Hailey (1992a) reported that high doses of quercetin over several years resulted in the formation of tumors in mice. However, in other long term studies, no carcinogenicity was found. Quercetin has also been shown to cause chromosomal mutations in certain bacteria in the test tube study; however, the significance of these findings for humans is not very relevant. Some doctors showed concern about the possibility of birth defects in the offspring of people supplemented with quercetin at the time of conception or during pregnancy.

9.7

CONCLUSIONS

Quercetin bears the potential for prevention and treatment of many human health concerns and has already attracted enormous attention of scientific community in the last three decades. We might gauge the colossal research interest and attention given to this molecule from the fact that the word “quercetin” returns more than 38700 hits from the ‘SciFinder’!

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Natural Bioactive Molecules: Impacts and Prospects

Since higher concentrations of quercetin are recommended to achieve its expected health benefits, the usual diet is unlikely to provide the required dosage; hence, there remains an enormous scope to commercialize this bioflavonoid in the form of dietary supplement and/or functional foods. It is now well established that upon absorption in the small intestine, quercetin immediately gets metabolized by enzymes in the epithelial cells and further get metabolized in liver. Therefore, bioavailability and stability also poses a big challenge to its application for health benefits. The most important areas for using quercetin as a drug and/or dietary supplement are to develop deeper scientific understanding of bioactivities of its metabolites, their concentrations in various tissues, their effect on various molecular targets, pathways, and mechanism based animal and clinical studies. There is also further scope to carry out elaborate structure-activity relationship (SAR) studies against various targets to enhance the potency and efficacy, thereby developing potential drugs. Despite considerable amount of research on this molecule, perhaps further in vitro, ex vivo, in vivo studies, human clinical trials, and its synergy with other bioactive molecules will help in harnessing its complete health benefits. Disclaimer: All views expressed herein are authors’ views and in no way, expressed or implied, are that of or necessarily represent the positions of ITC Limited, my current employer.

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10 Therapeutic Efficacy of Macro and Small Bioactive Molecules in Organ Pathophysiology Jyotirmoy Ghosha, b and Parames C. Silb* a

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA b

Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700 054, India

ABSTRACT Numerous studies in literature suggest the effective therapeutic strategies of plants used in traditional medicine. Research in the field of natural products (particularly isolation and biological characterization) is in progress so as to explore their remarkable contribution in the domain of new products necessary for clinical science. Therapeutic efficacy of these plant derived bioactive molecules as complementary and alternative medicine is also promising in the field of organ pathophysiology. Oxidative stress plays an important role in various forms of this pathophysiology. In this chapter, we would like to highlight the beneficial role of bioactive molecules in various organ pathophysiologies and also plan to discuss the underlying mechanism of their protective action. These bioactive molecules can be classified into two categories, macro and small. Among the macromolecules, the proteins isolated from the herbal plants play a beneficial role in toxin and drug induced organ pathophysiology. Research at the cellular and molecular level suggests the therapeutic efficacy of two plant proteins: a 43kD protein molecule from Cajanus indicus L and a 35 kD protein molecule purified from the plant Phyllanthus niruri. In addition to macromolecules, the multifunctional therapeutic applications of small bioactive molecules are also well established. Arjunolic acid, a naturally occurring nanometer long chiral triterpenoid saponin isolated from the bark of Terminalia arjuna, is well reputed for multitude of biological activities, including antioxidant, antidiabetic, antifungal, *Corresponding author: [email protected] /[email protected]

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Natural Bioactive Molecules: Impacts and Prospects

antibacterial, anticholinesterase, antitumor, antiasthmatic, wound healing and insect growth inhibitor activity. Mangiferin, a xanthone glucoside isolated from the barks of Mangifera indica also possesses various biological functions and has attracted considerable interest due to its therapeutic potential. D-saccharic acid 1,4-lactone (DSL), a beta-glucuronidase inhibitor (purified from kombucha tea and dietary plants), is the most pharmacologically active lactone and it possesses a number of biological activities. Taurine, a conditionally essential amino acid also has clinical implications for the prevention of diabetes and various drug and toxin induced organ dysfunction. These discussions untie the multifunctional therapeutic application of macro and small bioactive molecules and represent a therapeutic promise of alternative medicine. Keywords: Bioactive molecules; Antioxidant; Natural products; Organ pathophysiology; Oxidative stress; Therapeutic efficacy.

10.1

INTRODUCTION

Naturally occurring new molecules of promise are being extracted, examined and considered to be potential therapeutic targets recently as these molecules are, in general, derived from human consumables and are most likely to be safe and non-toxic. Such chemical substances derived from natural sources (plants, animals, microflora) have been reported to be beneficial for the treatment of human diseases since the dawn of civilization throughout the world. A number of recent reviews and reports described the importance of natural products and the therapeutic properties of extracts from plants in modern as well as complementary and alternative medicine. Use of such extracts has lately increased tremendously and could also serve as one of the valuable sources in pharmaceutical industrial research. Western pharmaceutical industries also directed their investigation on natural products with more intensified manner so as to explore these compounds as an important source of therapeutics, and that research peaked in 1970-1980 resulted in a pharmaceutical landscape heavily influenced by only the natural non-synthetic molecules. In fact, roughly half (49%) of the 877 small molecules introduced between 1981 and 2002 by New Chemical Entities (NCEs), were natural products, semi-synthetic natural product analogues or synthetic compounds based on natural-product PHARMACOPHORES (Koehn and Carter, 2005). It is also reported that approximately one-third of the top-selling drugs in the entire world are either natural products or their derivatives probably because of their wide recognition in the pharmaceutical industry, broad structural diversity and wide range of pharmacological activities. However, because of the competition from other drug discovery methods, natural products related pharmaceutical research has experienced a slow decline beginning in early 1990’s, but has recently gained renewed interests. This book chapter is aimed to present a brief overview of the therapeutic efficacy of macro and small bioactive molecules in organ pathophysiology to motivate the researchers in the field to bring natural product based drug discovery programs back to the forefront of drug discovery. Here we discussed the therapeutic efficacy of two plant proteins: a 43kD protein molecule from Cajanus indicus L and a 35 kD protein molecule purified from the plant Phyllanthus niruri. In addition

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to these macromolecules, we discussed the prophylactic role of some small bioactive molecules available from natural sources.

10.2 10.2.1

ROLE OF MACROMOLECULES IN ORGAN PATHOPHYSIOLOGY Brief History of Cajanus Indicus L

Since ancient times medicinal plants are used against ailments of different pathophysiological states (Kirtikar and Basu, 1935; Ghosh and Biswas, 1973). Among them is Cajanus indicus L is one of the most significant one and is a popular herbal plant in developing tropical countries mainly in Asia and Africa. This is considered the second important pulse crop in India. The two main varieties of Cajanus indicus grown in India are; (i) Cajanus cajan variety bicolor D.C. (Arhar) and (ii) Cajanus cajan variety flavus D.C. (Tur.) (Pandey, 1978). It is reported that protein fractions from Cajanus cajan have been found to possess hypolipidaemic activity in rats fed with high fat-cholesterol diet (Prema and Kurup, 1973). The seeds are also considered to be effective in snakebite (Chopra et al., 1956). This plant has an important medicinal impact and the leaves are known for their hepatoprotective property against different hepatic disorders and pathophysiology (Rastogi and Mehrotra, 1993). In rural India, the aqueous extract of the leaves of Cajanus indicus L is extensively used for the treatment of jaundice and hepatomegaly for a long time.

10.2.2

Active Constituent of Cajanus Indicus Leaves

In 1999, Dutta et al., first reported the hepatoprotective property of the leaves of Cajanus indicus. The investigators showed that a protein isolated from the leaves of the plant species possesses some beneficial role against carbon tetrachloride and beta-galactosamine induced liver injury (Dutta et al., 1999). It could also influence both cell mediated immune and humoral response although its purification procedure, structure and other related properties were not provided by this group (Dutta et al., 1999; Dutta and Bhattacharyya, 2001). Later, Sarkar et al., (2006) purified a protein from the same source, and with the help of a number of in vivo experiments; the beneficial role of the protein was observed in the light of prevention or cure of various organ pathophysiology. The protein is made up of a single polypeptide chain and its isoelectric point was found to be 4.8. Partial sequencing of this molecule showed some amino acid match in the primary structure of plastocyanin, a single electron carrier protein (Sarkar et al., 2006; Sarkar and Sil, 2011), and this similarity provides an idea about its free radical scavenging ability in organ pathophysiology.

10.2.3

Role of Cajanus Indicus Derived Protein in Drug Induced Organ Pathophysiology

Extensive studies on the beneficial effects of the C. indicus derived protein against the most popular analgesic and antipyretic drug acetaminophen has been carried out recently (Sarkar and Sil, 2007; Ghosh and Sil, 2007, 2009). Acetaminophen (APAP) is safe within its therapeutic level but its overdose is a primary cause of liver failure throughout the world. Acetaminophen overdose produces reactive oxygen species (ROS) and reactive nitrogen species (RNS) which gradually lead to fatal hepatic and renal pathophysiology in humans and in experimental animals

10.4

Natural Bioactive Molecules: Impacts and Prospects

(Wrights and Prescott, 1973; Dixon et al., 1973; Ameer and Greenblatt, 1977; Mudge et al., 1978; Srivastava et al., 2010). The main objective was to investigate the mechanism by which this protein provides protection in acetaminophen induced hepatotoxicity. To check this, mitochondrial cell death pathway along with the levels and/or activities of few key proteins involved in cell survival pathways have been investigated. It was found that the intraperitoneal treatment of the protein (2mg/kg body wt) once daily for 4 days prior to oral administration of acetaminophen efficiently prevented the liver injury (Sarkar and Sil, 2011; Ghosh and Sil, 2007). It was also found that the protein administration at the same dose post to acetaminophen administration cured the damages as compared to the control. Acetaminophen-induced liver damage was assessed by measuring the cell death, generation of reactive oxygen and nitrogen species (ROS & RNS), measuring the intracellular antioxidant (glutathione) status in hepatocytes treated with acetaminophen alone or acetaminophen in combination with the protein. Moreover, the involvement of proinflammatory cytokine TNF-a (Ghosh and Sil, 2009, Le and Vilcek, 1987; Decker, 1990) was also examined in acetaminophen and protein treated hepatocytes. Pretreatment of mice with the protein prevented the increase in serum TNF-a following acetaminophen administration. To investigate the involvement of mitochondrial cell death pathway, mitochondrial membrane potential, the adenosine tri phosphate (ATP) content in it and finally the level of intracellular calcium level was measured. Pre treatment of the protein counteracted acetaminophen-induced loss in mitochondrial membrane potential, loss in adenosine tri phosphate and rise in intracellular calcium. Investigating the involved cell signaling pathways, it was found that the protein exerts its protective action via the activation of NF-kB and Akt and deactivation of STAT1. Surprisingly, no role of ERK ½ or STAT 3 was found in the protein-mediated protection of hepatocytes during acetaminophen exposure. Finally, to investigate the nature of cell death in acetaminophen induced hepatotoxicity, DNA fragmentataion by gel electrophoresis, flow cytometry and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was performed (Ghosh and Sil, 2009). Result showed that acetaminophen exposure introduces necrosis as the primary phenomena of cell death and protein treatment decreased the necrotic process as evident from the DNA fragmentation and flow-cytometry studies. Results also showed that some nuclei of these TUNELpositive cells were condensed and contracted, suggesting that acetaminophen also induced apoptosis in hepatocytes compared to the fewer numbers of TUNEL-positive hepatocytes when treated with the protein prior to acetaminophen treatment (Sarkar and Sil, 2011; Ghosh and Sil, 2009).

10.2.4

Role of Cajanus Indicus Derived Protein in Doxorubicin Induced Nephro- and Neurotoxicity

Doxorubicin (Dox), an effective anthracycline antitumor drug, is used for the treatment of different types of cancer. However, its clinical efficacy has now been made limited because of its several acute and chronic side effects. Pal et al., (2012) investigated the nephroprotecive role of this 43 kD protein against doxorubicin induced oxidative impairment and kidney tissue damage. The renal toxicity caused by doxorubicin (20 mg/kg body weight, once) and the protective role of CI protein (3 mg/kg body weight for 4 days, once daily) were evaluated by measuring the activities

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10.5

of kidney specific biomarkers such as blood urea nitrogen (BUN), creatinine, uric acid etc., intracellular antioxidant enzymes activities, reduced and oxidized glutathione levels, intracellular ROS production, lipid peroxidation and protein carbonylation. Administration of doxorubicin also significantly enhanced the levels of TNFa, urinary g-glutamyl transpeptidase (g-GT) activity, total urinary protein and urinary glucose level. In order to investigate the nature of doxorubicin-induced cell death, DNA fragmentation analysis and TUNEL assay were performed. Results clearly showed that doxorubicin exposure caused DNA ladder formation and increased the number of TUNEL positive nucleus as well, suggesting that cell death occurs mainly via apoptosis in this pathophysiology. Investigating the cell signaling mechanism, it appeared that doxorubicin administration markedly decreased mitochondrial membrane potential, disturbed Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased levels of Apaf1, caspase-9/3/8, cleaved PARP protein and ultimately led to apoptotic cell death. Doxorubicin exposure also distinctly stimulated the expression of phosphoJNK, phospho-p38 and phospho-ERK1/2. However, significant changes in the expression of total JNK, p38 and ERK1/2 were not observed between the normal and doxorubicin-exposed groups. Post treatment with CI protein (3 mg/kg body weight, once daily for 4 days), however, reduced all these doxorubicin-induced apoptotic events. In addition to mitochondria-dependent cell death pathway, involvement of mitochondria independent pathway was also investigated in doxorubicin -induced pathophysiology. It is observed that doxorubicin caused up-regulation of Bid, FAS and caspase-8. These results clearly indicate the involvement of extrinsic pathway in this pathophysiology as well. Post treatment of CI protein, however, significantly reduced all these doxorubicin-induced up regulation of pro-apoptotic events and maintained their normalcy. Histological studies also further confirm the beneficial role of the CI protein in this organ pathophysiology (Pal and Sil, 2012). In addition to renal dysfunction, doxorubicin exposure also causes neurotoxicity. The neurotoxic effects caused by doxorubicin (25 mg/kg body weight) and the protective role of CI protein were evaluated by measuring the activities of brain specific enzymes, intracellular ROS production and the other prooxidant-antioxidant indices. Investigating the underlying signaling mechanism, the researchers found that doxorubicin exposure led to apoptotic cell death via mitochondria dependent pathway. In addition, doxorubicin increased NF-k nuclear translocation in association with IKKa/b phosphorylation and IkBa degradation. Post treatment with CI protein (3 mg/kg body weight, once daily for next 4 days), however, reduced doxorubicin-evoked oxidative stress, attenuated translocation of NF-kB and protects the brain tissue from apoptotic death (Pal et al., 2012).

10.2.5

Role of Cajanus Indicus Derived Protein Against Environmental Toxin

Hepatoprotective nature of this protein against a number of chemical agents (like carbon tetrachloride, chloroform, mercuric chloride, galactosamine, sodium fluoride, thioacetamide, cadmium chloride etc) has been reported (Sarkar et al., 2005, 2006; Ghosh et al., 2006; Ghosh and Sil, 2008; Manna et al., 2007a, 2007b; Sinha et al., 2007a, 2007b, 2007c). Activities of the antioxidant enzymes (like SOD, catalase, GPx and GST, etc.) were found to increase in hepatic tissue of CCl4 exposed animals whereas all these factors were back again to the normal level with the protein treatment. Besides, increased peroxidation of lipid membrane and decreased GSH

10.6

Natural Bioactive Molecules: Impacts and Prospects

content was found to toxin-exposed liver whereas the alterations are practically blocked when the toxin was administered along with the protein. Results were confirmed with the histological data where less centrilobular necrosis was found in the protein treated hepatic tissue in toxin-exposed liver compared to that with the toxin the toxin exposure alone (Sarkar and Sil, 2011). Similar results were found for thioacetamide-induced hepatotoxicity. The protein was found to lower the cellular damage caused by thioacetamide in isolated hepatocytes as well as in in vivo system when applied along with the toxin (Sarkar et al., 2005, 2006). Mercury-induced organ toxicity is basically caused by oxidative stress and is associated with increased levels of different cytokines like TNF-a, IL-6 and IL-1B. TNF-a in the blood of HgCl2 induced animals. HgCl2 administration reduced renal GSH content and that ultimately lead to renal failure (Sarkar and Sil, 2011). Protein administration both prior and post to HgCl2 exposure reduced toxin induced ROS generation and TNF-a formation and increased overall anti-oxidant status. Liver and kidney tissue architecture also improved with the protein treatment both prior and post to toxin administration (Ghosh and Sil, 2008). When compared the antioxidant capacity with N-acetyl cysteine (another potent antioxidant molecule), the protein showed similar degree of protection against HgCl2 induced toxicity. In addition, the protein also played beneficial role (both preventive and curative role) in sodium fluoride (NaF) and galactosamine induced oxidative stress (Manna et al., 2007a, 2007b; Sinha et al., 2007a, 2007b, 2007c) and a number of organ pathophysiology (Sarkar and Sil, 2011).

10.2.6

Possible Mechanism

Most of these toxicants, as discussed above, cause organ toxicity via the production of different free radical intermediates which are responsible for damaging the cellular lipid membranes and other macromolecules (Tsyrlov and Lyachovich, 1972) resulting an increased organ specific marker enzyme levels in blood. The protein treatment both prior and post to toxin exposure, on the other hand, inhibits the alterations of those markers suggesting its possible role in protecting tissues from the damage by the toxins. ROS generation and activities of antioxidant enzymes measurements provides support of the beneficial role of this protein. In all the studies, ROS generation peaks high and antioxidant enzymes activities go down after the toxin administration while these alterations are practically reversed on treatment with the protein. These results clearly support scavenging power of the protein in toxin-generated free radicals inside the cell either directly or indirectly by activating some other bioactive molecules. Result of the scavenging of 2,2 diphenyl-1-picrylhydrazyl (DPPH) (Sarkar and Sil, 2011; Ghosh and Sil, 2006) provides a direct proof of the free-radical scavenging property of this protein.

10.2.7

Conclusion

In conclusion, we would like to say that CI protein is potentially useful for the prevention and cure of a number of drug and toxin induced organ pathophysiology and cell death. These remarkable benefit and absence of any noticeable toxicity with CI protein may provide clues in its potential use as a novel promising therapeutic strategy of alternative medicine.

Ghosh and Sil: Bioactive Molecules in Organ Pathophysiology

10.2.7.1

10.7

Role of Phyllanthus niruri

The herb, Phyllanthus niruri (PN) is a natural source of antioxidants and has been shown to play beneficial roles in various organ pathophysiology (Sarkar and Sil, 2010; Tona et al., 1999; Odetola et al., 2000). From a number of clinical studies in humans, this herb has been shown to have practically no side effect (Thyarajan et al., 1990, 1998). Phyllanthus niruri (P. niruri) is also an important medicinal plant (Venkateswaran and Millman, 1987; Barros et al., 2006; Bhattacharjee and Sil, 2007). Earlier, phyllanthin (Harish and Shivanandappa, 2006) and corilagin (Cheng et al., 1995) are well characterized as bioactive compounds isolated from the organic extracts of this herb. Latter, it has been reported that the aqueous extract and the protein isolate of this herb also possess antioxidant activity (Chatterjee and Sil, 2006; Chatterjee et al., 2007; Bhattacharjee and Sil, 2006; Sarkar and Sil, 2007), and a 35 kD unique protein molecule (PNP) with potent antioxidant activity was purified to homogeneity from the aqueous extract having no match with the other proteins present in the database (Sarkar et al., 2009).

10.2.7.2

Phyllanthus niruri protein (PNP) against tertiary butyl hydroperoxide (TBHP)-induced apoptosis

Very recently, Sarkar and Sil (2010) reported the mechanism of the protective action of this antioxidant protein molecule in TBHP-induced oxidative stress and cell death. TBHP exposure caused loss in cell viability and enhanced LDH leakage in a dose-dependent manner. Incubation of hepatocytes with PNP prevented the loss in cell viability and LDH leakage. TBHP-induced reduction in GSH/GSSG ratio and antioxidant enzymes activities have also been found to be prevented by this protein. Moreover, TBHP exposure reciprocally regulated the Bcl-2 family proteins balance, caused mitochondrial membrane permeabilization, disrupted mitochondrial membrane potential, and facilitated cytochrome c release from the mitochondria to cytosol. In addition, DAPI staining, flow cytometric analyses and studies on the activation of caspases confirm that TBHP induced cell death is apoptotic in nature. PNP treatment, on the other hand, counteracted all these adverse changes and maintains normalcy in hepatocytes. Combining all, the results suggest that TBHP increased the Bax expression, while this protein partly suppressed the expression of Bax, indicating that there is a great possibility that it mediated antiapoptotic effect on hepatocytes via the regulation of Bcl-2 and Bax activation (Sarkar and Sil, 2010).

10.3 ROLE OF SMALL BIOACTIVE MOLECULES IN ORGAN PATHOPHYSIOLOGY 10.3.1

Arjunolic Acid

In addition to macromolecules the multifunctional therapeutic applications of small bioactive molecules are also well established. For a long period, a number of medicinal plants with antioxidant activities have been widely used in ayurvedic and other alternative medicine for the treatment of cardiac disorder and many other problems. Among them, different parts of Terminalia arjuna tree ((Roxb.) Wight & Arn (TA) belonging to the family Combretaceae), particularly its fruit and bark, have been used as a human consumable component in water, milk and other drinks for maintaining sound health (Kiritiker and Basu, 1987). The entire plant has been reported to be full of many

10.8

Natural Bioactive Molecules: Impacts and Prospects

bioactive constituents, like flavonoids, ellagic acid, gallic acid, tannins, triterpenoid saponins, oligomeric proanthocyanidins (OPCs), phytosterols etc. (Kiritiker and Basu, 1935; Ghosh et al., 2010a) although major constituents, are usually classified into two general categories, namely, polyphenols (60%-70% of bark) and tannins (20-40%). Triterpene glycosides present in this plant have only been identified but not quantified so far. The other constituents present in it include triterpene saponins (arjunic acid, arjunolic acid, arjungenin, flavonoids (arjunone, arjunolone and luteolin), sterols and phytosterols (e.g. b-sitosterol), proanthocyanidins and minerals (e.g. Ca, Mg, Zn and Cu) (Ghosh et al., 2010a; Facundo et al., 2005). The pentacyclic triterpenes are a large group of natural products, widespread in various plants and constitute an important part of medicinal resources with diverse pharmacological activities (Xiao-An et al., 2010). Among many of these secondary metabolites, the triterpenoids are an important class derived from C30 precursors (Prasad et al., 2004; Ebizuka et al., 2003) although little is known about their beneficial role in the general area of oxidative stress and related organ pathophysiology (Bag and Dinda, 2007). Arjunolic acid (AA: 2,3,23-trihydroxyolean-12-en-28-oic acid), a naturally occurring chiral triterpenoid saponin, has been isolated from the bark of Terminalia arjuna and is well known for various biological functions, including antidiabetic, anti-fungal, anti-bacterial, anticlolinesterase, antitumor (Wille et al., 2001), antiasthmatic (Kalola and Rajani, 2006), wound healing (Chaudhari and Mengi, 2006) and so on (Manna et al., 2010; Masoko et al., 2008; Djoukeng et al., 2005; Kim et al., 2005; Wille et al., 2001; Kalola and Rajani, 2006; Chaudhari and Mengi, 2006; Bhakuni et al., 2002; Hemalata et al., 2010).

10.3.1.1

Extraction of arjunolic acid (AA)

A number of investigators reported the purification of arjunolic acid and checked the homogeneity of preparation by various techniques (Manna et al., 2007c, Ghosh et al., 2010b). A couple of recent reports on its extraction and homogeneous preparation using NMR (1H, 13C), IR, Mass spectroscopy (MS) and optical rotation studies was described by Manna et al and Ghosh et al., (Manna et al., 2007c; Ghosh et al., 2010b).

Ghosh and Sil: Bioactive Molecules in Organ Pathophysiology

10.3.1.2

10.9

Beneficial role of arjunolic acid in drug-induced organ pathophysiology

10.3.1.2.1 Therapeutic potential of arjunolic acid against acetaminophen-induced liver and renal toxicity

Acetaminophen has been considered a widely used analgesic and antipyretic drug and it is safe at its therapeutic dose. However, its overdose causes severe hepatotoxicity, which finally leads to fatal liver injury. Acetaminophen exposure alters a number of biomarkers regarding hepatic oxidative stress, increases ROS production, reduces cellular ATP level and induces necrotic cell death both in vivo and in vitro model (Ghosh et al., 2010b). Arjunolic acid supplementation (80 mg/kg, orally, once), on the other hand, affords significant protection in liver injury by preventing acetaminophen-induced hepatic glutathione depletion and acetaminophen-metabolites formation via metabolic inhibition of the specific forms of cytochrome P450 that bioactivates acetaminophen to NAPQI, the reactive intermediate of this pathway. In addition, when arjunolic acid was applied 4h after acetaminophen exposure it reduces acetaminophen-induced JNK activation, and downstream Bcl-2 and Bcl-xL phosphorylation, thus protects mitochondrial permeabilization (MPT), loss in mitochondrial membrane potential and the release of cytochrome c (Ghosh et al., 2010b). Besides, this molecule afforded complete protection from severe hepatic necrosis and attenuated the acetaminophen-induced liver injury. By performing a series of time dependent experiments on both acetaminophen and arjunolic acid exposure, the investigators (Ghosh et al., 2010b) clearly showed that arjunolic acid remains effective even in relatively late situation (8h after acetaminophen exposure). In this regard, this molecule acts as a better protective agent than N-acetylcysteine (NAC), the most prescribed drug for acetaminophen toxicity. Literature suggests that NAC should be administered relatively early, before a significant increase in ALT occurs because of hepatic damage (Latchoumycandane et al., 2007). This group of workers (Ghosh et al., 2010b) carried out extensive studies on the mechanism of protective action of arjunolic acid on acetaminopheninduced hepatic disorder and showed that acetaminophen induced hepatotoxicity is basically the outcome of two successive events, JNK-independent and JNK-dependent although both of them are required for necrosis to occur. NAPQI, the intermediate metabolite, causes a significant depletion of GSH first and disturbs the covalent binding to macromolecules like proteins, alters hydrogen peroxide level, increases ROS production, activates JNK which in turn, inactivates the antiapoptotic Bcl-2 family proteins by phosphorylation and consequently, the balance of the proapoptotic and antiapoptotic members of this family is altered in favour of the proapoptotic ones. These sequential events leads to mitochondrial permeabilization and helps triggering cytochrome c release to the cytosol which causes a dramatic decrease in the cellular ATP level leading to hepatic necrosis in turn (Ghosh et al., 2010b). In this pathophysiology, arjunolic acid also affords two-way hepatic protection by inhibiting necrosis: at first, by inhibiting cytochrome P450mediated acetaminophen bioactivation and then by inhibiting the phosphorylations of JNK/Bcl-2 family proteins and preventing the alterations of subsequent mitochondrial functions. Besides, arjunolic acid could provide the protection of the organ by scavenging excess ROS produced by acetaminophen exposure (Ghosh et al., 2010b). Acetaminophen exposure not only causes hepatic dysfunction but also causes acute and chronic renal failure. Acetaminophen overdose increases TNF-a production in renal tissue suggesting the

10.10

Natural Bioactive Molecules: Impacts and Prospects

involvement of inflammation in this pathophysiology (Abdel-Zaher et al., 2008). These reports are well correlated with a number of previous studies which showed that the drugs causing nephrotoxicity generally induces renal inflammation via production of TNF-a (Kuhad et al., 2007; Zager, 2007; Ghosh et al., 2010c). In addition to the TNF-a production, acetaminophen exposure could also cause overproduction of NO in the kidney tissue and this increased NO level correlates with the inducible nitric oxide synthase (iNOS) protein expression (Gardner et al., 2002). This is possible in a situation only when TNF-a up-regulates the iNOS expression followed by NO production (Morris and Billiar, 1994). NO, therefore, plays an important role in the pathogenesis of acetaminophen-induced renal toxicity. The mechanism of down-regulation of iNOS by arjunolic acid is not very clear yet; it could, however, be the result of decreased TNF-a formation, as TNF-a could up-regulate iNOS, thereby enhancing the NO production (Ghosh et al., 2010c; Morris and Billiar, 1994). Besides, the carboxyl group of arjunolic acid could directly scavenge NO as was evidenced from its superoxide, hydroxyl and nitric oxide radical quenching ability in a cell free system and thus could reduce the NO level (Ghosh et al., 2010c). Reports by Ghosh et al., (2010c) clearly indicate that exposure of rats with a nephro-toxic dose of acetaminophen altered a number of biomarkers related to renal oxidative stress (like blood urea nitrogen and serum creatinine levels, etc.), decreased intracellular antioxidant enzymes activity of the system, elevated renal tumor necrosis factor-a and nitric oxide levels as well as induced renal death through a caspase mediated mechanism that involves activation of caspase-9 and caspase-3 in the absence of cytochrome c release. Dose dependent studies suggest that arjunolic acid treatment (50 mg/kg body weight for 2 days) both pre and post, on the other hand, mitigated all the alteration of these biomarkers and helps the organ maintaining in its normal physiological state. Investigating the inherent molecular signalling of this pathophysiology, it reveals that maintenance of cellular antioxidant defense mechanism, reduced TNF-a and NO overproduction, and inhibition of caspase-mediated pathways are the key factors contributing to the renal protective effect of arjunolic acid (Ghosh et al., 2010c). Taken together, these findings outline a mechanistic understanding of therapeutic potential of arjunolic acid in acetaminophen-induced hepatic and renal pathophysiology. 10.3.1.2.2

Beneficial role of arjunolic acid against doxorubicin induced cardiac damage

Chemotherapy for cancer treatment causes injury to non-targeted tissues by the therapeutic dosages of most of the anticancer drugs and that impairs the quality of life of patients during and after treatment. Chemotherapeutics usually imply direct or indirect oxidative stress and that might cause toxicity in non-cancerous tissues like heart, testes, etc. Evidence suggests that doxorubicin is the most effective anticancer drug because of its broad-spectrum effects and unmatched efficacy in clinical use although its application has to be limited for the acute and chronic dose-related irreversible cardiotoxicities. A comprehensive understanding of the mechanisms of oxidative injury to heart is, therefore, necessary for the development of strategies to attenuate the cardio toxicity of chemotherapeutic drugs without affecting their efficacy. The findings by Ghosh et al., (2011), suggest that doxorubicin induced cardiac damage is supposed to be the result of mainly two events. Firstly, doxorubicin exposure produces reactive oxygen species (H2O2) in myocytes. This produced H2O2 now activates p53 and p38-JNK mediated signaling pathways in cardiomyocytes.

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10.11

Secondly, this activated p38-JNK promotes translocation of Bax to mitochondria and disrupts the balance between proapoptotic and antiapoptotic proteins. These imbalances in cellular level trigger mitochondrial permeabilization and thereby help in release of cytochrome c, and ultimately lead to activation of caspases and cell death. In this context, treatment with arjunolic acid (25 mg/kg body weight every 2 days for thrice) provided protection in two ways: firstly, it offers cardiac protection via detoxification of reactive oxygen species produced during doxorubicin exposure. Secondly, this molecule plays the same role by inhibiting the phosphorylations of p38, JNK MAPKs, MAPKmediated Bax translocation to mitochondria and subsequent mitochondrial permeabilization (Ghosh et al., 2011). The afore-mentioned discussion outlines a mechanistic understanding of how this bioactive molecule protects heart from doxorubicin-induced toxicity. A critical point needs to be mentioned here about the anticancer effect and cardiotoxicity of doxorubicin. These two distinct effects do not follow identical mechanism (Wang et al., 2004; Kluza et al., 2004). Besides, arjunolic acid itself could inhibit tumor growth (Diallo et al., 1989). In future, if arjunolic acid is considered as an antitumor agent, it is likely that it would not, any way, affect the efficacy of doxorubicin’s usual anticancer effect. 10.3.1.2.3 Cardioprotective effects of arjunolic acid against isoproterenol exposure

Sumitra et al., (2001) reported the cardioprotective effects of arjunolic acid by demonstrating its effect on platelet aggregation, coagulation and myocardial necrosis. Isoproterenol exposure induced myocardial necrosis and also the serum enzyme levels, such as creatine kinase, lactate dehydrogenase and altered the electrocardiographic parameters but arjunolic acid supplementation restored the altered levels and maintained it close to the normal. Histopathological studies of heart suggest an engorgement of coronary vessels due to isoproterenol administration. Arjunolic acid supplementation both pre and post maintained the normal architecture of heart (Hemalata et al., 2010; Sumitra et al., 2001). This study also suggests the antiplatelet and anticoagulant activity of arjunolic acid.

10.3.1.3

Role of arjunolic acid in metal and chemical-induced organ pathophysiology

Arsenic is a well-documented and one of the most widespread environmental toxins, which affects nearly all organ systems. In environment, it exists both in organic and inorganic forms. Absorbed arsenic, irrespective of its state, is widely distributed throughout the body. Although unfortunate, but drinking of As-contaminated ground water is an unavoidable situation for a major percentage of the total populations of the world resulting a suffering from As-related organ dysfunctions (Ghosh et al., 2009, Roy et al., 2009; Das et al., 2009a, 2009b, 2010a; Manna et al., 2008a, 2008b, Sinha et al., 2007d; 2008a, 2008b). The underlying mechanism of As-mediated organ injury and cell death has been studied worldwide for a long period of time although there is no specific antidote to combat against its toxicity so far. 10.3.1.3.1

Arjunolic acid against arsenic-induced toxicity

A group of researchers (Manna et al., 2007c, 2007d) explored the protective role of arjunolic acid against arsenic induced oxidative damages in murine liver. Arsenic exposure in the form of sodium arsenite at a dose of 10 mg/kg body weight for two days significantly reduced the antioxidant

10.12

Natural Bioactive Molecules: Impacts and Prospects

enzymes activities as well as depleted the cellular reduced glutathione and total thiols level. In addition, the same exposure enhanced the leakage of serum marker enzymes (alanine transaminase and alkaline phosphatase) indicating liver damage. DNA fragmentation analysis suggests that As-induced cell death is necrotic in nature. Arjunolic acid pretreatment at a dose of 20 mg/kg body weight for 4 days mitigated all the alterations and maintained their values close to those of normal. Histological findings suggest that arsenic exposure disrupted the liver architecture associated with extensive centrilobular necrosis around the central lobule and bile ducts proliferation. Arjunolic acid could, however, prevent such changes and maintains the normal liver architecture (Manna et al., 2007c, 2007d). In addition to liver, this important molecule has also been reported to protect kidney, brain, testes and heart tissue from arsenic poisoning. (Manna et al., 2008a, 2008b; Sinha et al., 2008a, 2008b). 10.3.1.3.2

Role of arjunolic acid against cadmium induced pathophysiology

Cadmium is one of the most notorious heavy metals and environmental pollutants and it is mainly released from the smelting, burning of fossil fuels and municipal wastes, refining of metals, and cigarette smoking, resulting in the pollution of water, air, and soil. It has been documented that after entering the body, Cd is transported by the blood, especially by red blood cells and albumin, the high molecular weight protein (Bauman and Liu, 1993; Ghosh et al., 2010a) and affects the entire organ systems of the body. Although it is widely distributed in our body most of it accumulates in the liver (Sinha et al., 2009; Pope et al., 1995; ) and causes severe liver dysfunction. Pal et al., (2011) reported the protective role of arjunolic acid in Cd-induced oxidative damage and cell death in isolated primary murine hepatocytes. Cd intoxication significantly enhanced the ALT, ALP and LDH leakage, intracellular reactive oxygen species (ROS) production, reduced hepatocytes viability and decreased antioxidant enzymes activities (Pal et al., 2011). The same exposure also increased the lipid peroxidation and protein carbonylation, which are considered to be the two important parameters for oxidative injury. Flow cytometric analysis suggests that Cd-induced cell death is apoptotic in nature. Investigating the inherent signaling mechanism, Pal et al., (2011) found that Cd markedly increased the levels of caspase-9, 3, disrupted mitochondrial membrane potential, enhanced the release of cytochrome c from mitochondria to cytosol, reciprocally regulated the Bcl-2 family protein balance, cleaved PARP protein and ultimately led to apoptotic cell death. These findings suggest the involvement of mitochondrion dependent pathway in this pathophysiology. The previously mentioned changes due to Cd exposure were found to be associated with increased NF-kB nuclear translocation, IKKa/b phosphorylation and IkBa degradation. During investigation on the involvement of extrinsic pathway in Cd-intoxication, they found that Cd-exposure causes up-regulation of Bid, FAS and caspase 8. Combining results suggest that Cd could trigger both intrinsic and extrinsic apoptotic pathways (Pal et al., 2011). On the other hand, arjunolic acid supplementation (200 mM), however, compensated the deficits in the antioxidant defense mechanisms, attenuated the nuclear translocation of NF-kb, mitigated all the apoptoptic events and protects from Cd-induced hepatocytes death. From the above discussion, it can be hypothesised that the protective effect of arjunolic acid in this pathophysiology is attributed to its antioxidant and free radical scavenging properties. Cd-induced hepatocytes death is due to the ROS formation and mediated via the activation of NF-kB, FAS and Caspase 8. Arjunolic acid supplementation,

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10.13

on the other hand, reduced Cd-induced oxidative stress, attenuated the activation of NF-kB and mitochondrion-dependent and Fas mediated extrinsic signaling pathways (Pal et al., 2011). 10.3.1.3.3

Arjunolic acid against fluoride induced hepatocytes death

Fluorine is never found in the free state in nature and known to be one the most active element. It is usually present as the negatively charged ion, fluoride (F−). The main source of its accumulation is through drinking water and is found in association with the other constituents in the body. Its excess intake in the body usually affect the skeletal systems, teeth and also the structure and function of brain (Shashi et al., 1994), skeletal muscle (Kaul and Susheela, 1974), and spinal cord (Franke, 1976) and causes fluorosis, a slow progressive degenerative organ disorder. Fluoride can attack anyone mercilessly although the developing children and the elderly people suffer the most. Although the exact mechanism of fluoride toxicity is still unclear, some researchers reported that oxidative stress plays a major role in this organ pathophysiology. Ghosh et al., (Ghosh et al., 2008) investigated the beneficial role of arjunolic acid in sodium fluoride (NaF)-induced cytotoxicity and necrotic cell death in murine hepatocytes. NaF exposure significantly decreased the cell viability dose-dependently, increased reactive oxygen species production. NaF-intoxication also increased the activities of the membrane leakage enzymes (ALT, ALP) suggesting the loss of membrane integrity as well as the structure of the cell membrane. The same exposure also increased lipid peroxidation products, protein carbonyl content, depressed antioxidant enzyme activities and glutathione (GSH) and total thiol contents in murine hepatocytes (Ghosh et al., 2008). To justify the impact of fluoride and arjunolic acid on hepatocytes viability all the experimental sets were assessed by flow cytometric analysis. Flow cytometric and DNA fragmentation analyses clearly suggest that fluoride induced hepatocytes death occurs mainly via the necrotic pathway. Arjunolic acid treatment restored membrane leakage enzymes (ALT, ALP) levels that were elevated by fluoride. It also compensated deficits in the antioxidant defense mechanisms, suppressed lipid peroxidation and protected the cells from necrotic death (Ghosh et al., 2008). The findings, as a whole, suggest that arjunolic acid plays a beneficial role against fluoride-induced cellular damage and prevents hepatocytes from necrotic death. 10.3.1.3.4 Mechanism of action of arjunolic acid

Based on the updated studies on the action of arjunolic acid, it can be speculated that in metal and chemical-induced organ pathophysiology, its beneficial effect may be due to: (1) Scavenging of ROS produced during chemical/metal exposure as arjunolic acid itself possesses radical scavenging activity (Ghosh et al., 2010b). Besides, the presence of one carboxylic group also helps in explaining its radical scavenging property. (2) Formation of five-membered complex with the metal atom/ions by two equatorial hydroxyl groups of arjunolic acid. Arjunolic acid, because of the possession of polyhydroxyl groups and two of which are vicinal to each other, could form a five-membered chelate complex with metal atom/ions which probably helps in removing the metal from the system and thereby it inhibits any further oxidative related damage to the specific organ. In conclusion we would like to say that arjunolic acid plays a protective role in drug and environmental toxin-induced oxidative stress although the exact mechanism of its protective action

10.14

Natural Bioactive Molecules: Impacts and Prospects

is not fully understood yet. Future studies are necessary for the identification of functional group(s) and the responsible part of the compound that plays the central role in its protective action.

10.3.1.4

Arjunolic acid in type 1 diabetes

Diabetic mellitus is considered a chronic metabolic disorder and has been emerged as a major cause of morbidity and mortality worldwide due to its complex pathogenesis. This complex pathophysiology is characterized by an imbalance of the regulation of carbohydrate and lipid metabolism by insulin. The etiology of this deadly disease is not clear yet, but several factors like viral infection, autoimmune disease, etc. could play a major role in its pathogenesis and as a result of its persistence (Maritim et al., 2003; Lasker et al., 1993) several organs and tissue damage as well as diseases like cardiovascular diseases, retinopathy, neuropathy, nephropathy, etc could occur either directly or indirectly among the affected population (Kikkawa, 2000). Exogenous insulin treatment and other medications could control the diabetes and its related pathophysiology to some extent; complicacy however, persists and remains highly costly in terms of the longevity and quality of life of the patients (Manna and Sil, 2012a). Arjunolic acid has been reported to possess preventive and therapeutic role in drug and chemical induced organ pathophysiology which led the researchers to investigate beneficial effect in the context of diabetes. The study began with the determination of the prophylactic role of arjunolic acid against streptozotocin (STZ)-induced diabetes in different organs of Swiss albino rats (Manna et al., 2009a, 2009b, 2009c, 2010a, 2010b, 2010c; Manna and Sil, 2012b). STZ administration caused increased production of both ROS and RNS in the spleen and pancreatic tissues of the experimental animals. Investigating the signaling pathways, the authors found that STZ administration caused the activation of phospho-ERK1/2, phospho-p38, NF-kB and destruction of mitochondrial membrane potential, release of cytochrome c as well as activation of caspase-3 keeping the levels of total ERK1/2 and p38 unchanged. Treatment of animals with arjunolic acid both prior and post to the STZ administration effectively reduced these adverse effects by inhibiting the excessive ROS and RNS formation as well as by down-regulating the activation of phospho-ERK1/2, phospho-p38, NF-kB and mitochondrial dependent signal transduction pathways leading to apoptotic cell death (Manna et al., 2009a, 2010a). Diabetic nephropathy (DN), another diabetes associated pathophysiology, whose incidence is up to 47.66% in diabetic population, is reported to be the most common and complicated diabetic microvascular disease due to its complex pathogenesis. In kidney, hyperglycemia activates polyol pathway by increasing aldose reductase (AR) with a concomitant reduction in Na+-K+ATPase activity. Investigating the oxidative stress responsive signaling cascades, they found the activation of PKCd, PKCe, MAPKs and NF-kB (p65) in the renal tissue of the diabetic animals. Arjunolic acid supplementation ameliorated renal dysfunction in diabetic rats by controlling blood glucose level, reducing oxidative stress and inhibiting the activation of polyol pathway (Manna et al., 2009b). In liver, hyperglycemia caused loss in body weight and reduction in serum insulin level; increased formation of HbA1C and advanced glycation end products (AGEs). Activations of iNOS, IkBa/NF-kB and MAPKs pathways and signals from mitochondria have been found to be involved in initiating the apoptotic cell death. Over expression of PARP, reduction in intracellular NAD as well as ATP level, increased DNA fragmentation and apoptotic cell death in the liver tissue of the diabetic animals were also observed in this pathophysiology. Arjunolic acid prevented as

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10.15

well as ameliorated the diabetic liver complication and apoptotic cell death. The effectiveness of arjunolic acid in preventing the formation of ROS, RNS, HbA1C, AGEs, oxidative stress signalling cascades and protecting the PARP mediated DNA fragmentation can speak about its potential uses for diabetic patients (Manna and Sil, 2010b). Another bad effect of hyperglycemia is the vascular inflammation and cardiac dysfunction which causes majority of mortality and morbidity among the diabetic patients. At an early stage, type 1 diabetic mellitus (T1DM) is associated with increased cardiovascular complications, causes alterations in plasma lipid profile, releases membrane bound enzymes, LDH (lactate dehydrogenase) and CK (creatine kinase) and establishes the association of hyperlipidemia and cell membrane disintegration with hyperglycemia and all these events ultimately alter the levels of oxidative stress related biomarkers and decreases the intracellular NAD and ATP concentrations. Besides, hyperglycemia-induced enhanced levels of VEGF, ICAM-1, MCP-1 and IL-6 in plasma indicate vascular inflammation in T1DM and causes cell death mostly via the apoptotic pathway. In molecular level, NF-kB and MAPKs (p38 and ERK1/2) are activated followed by mitochondrial membrane depolarization, cytochrome C release, caspase 3 activation and PARP cleavage. Arjunolic acid, on the other hand, could reduce hyperglycemia by preventing membrane disintegration, vascular inflammation and the activation of oxidative stress induced signaling cascades leading to apoptotic cell death (Manna and Sil, 2012a, 2012b).

10.3.2

Mangiferin

Another important bioactive naturally occurring small molecule is mangiferin (2-C-b-Dglucopyranosyl-1,3,6,7-tetrahydroxyxanthone). It is a polyphenol and is widely distributed in the leaves, barks and roots of the plants belonging to Anacardiaceae and Gentianaceae families (e.g. Mangifera indica, mango). This molecule has attracted considerable interest as it is a potent antioxidant (Moreira et al., 2001) and possesses antitumor, antiviral antidiabetic and immunomodulatory activities (Guha et al., 1996; Yoshimi et al., 2001; Ichiki et al., 1998; Miura et al., 2001; Leiro et al., 2004; Nunez-Selles et al., 2002). Mangiferin is also used for melancholia and nervous debility in traditional Indian medicine (Bhattacharya et al., 1972). As an immunomodulatory agent, this molecule has been reported to inhibit NF-kB activation and a series of pro-inflammatory cytokines (Leiro et al., 2004). Besides, it could increase cellular GSH content, inhibit lipid peroxidation and scavenge ROS because of inherent antioxidant activity (Das et al., 2012a). These properties clearly suggest that in addition to have ROS scavenging activity, this xanthone also possesses the ability to modulate a number of genes expression which play critical roles in regulating inflammation and apoptosis.

10.3.2.1

Mangiferin extraction

Extraction of mangiferin from the bark of Mangifera indica was described in detail by Singh et al., (Singh et al., 2009; Das et al., 2012a). Its purity was confirmed by HPLC, mass and NMR (1H, 13C) spectroscopy, and Reverse-phase HPLC.

10.16 10.3.2.2

Natural Bioactive Molecules: Impacts and Prospects

Mangiferin against D-galactosamine (GAL) induced acute toxicity

D-galactosamine exposure causes elevation in serum ALP, ALT, triglycerides, total cholesterol, lipid-peroxidation, ROS and NO production and causes reduction in the levels of total proteins, albumin, cellular GSH and ultimately induces apoptosis and necrosis in hepatocytes via increased nuclear translocation of NFkB and elevated iNOS protein expression. It is also associated with elevated TNF-a, IFN-g, IL-1b, IL-6, IL-12, IL-18 and decreased IL-10 mRNA and Nrf2, NADPH:quinine oxidoreductase-1, heme oxygenase-1 and GSTa protein expressions. Mangiferin could, however, significantly altered all these GAL-induced adverse effects (Das et al., 2012a). Signal transduction studies on GAL-induced nephro-pathophysiology showed an increased protein expression of Bax, cytochrome c, caspase 3/9 and inducible nitric oxide synthase (iNOS) in the cytosol and NF-kB in nuclear fraction, and a reduced protein expression of Bcl-2 in the cytosol. Mangiferin could, however, successfully reduce D-Gal-induced pathophysiologic alterations and protects kidney tissue from oxidative/nitrosative stress and acute nephrotoxicity via its antioxidant activities (Ghosh et al., 2012a). It can, therefore, be said that mangiferin is worthy of further research as a potential hepatoprotective/nephroprotective agent as this small bioactive molecule has multiple advantageous properties like antioxidantive, antihyperlipidemic as well as anti-inflammatory and absence of any reported noticeable toxicity.

10.3.3

Role of Kombucha Tea (KT) in Cellular Oxidative Stress

Kombucha tea (KT), a sugared black tea, fermented by a consortium of yeast and acetic acid bacteria, is claimed to have many beneficial effects to human health as it is claimed to be a prophylactic agent and beneficial to human health because of its antioxidant and anti-microbial activities (Jayabalan et al., 2007, 2008), although only very few scientific evidences are available in the literature describing this fact. The US Food and Drug Administration evaluated several commercial producers of the starter (kombucha mushroom or tea fungus) and practically found no pathogenic organisms or other hygienic violations in it (FDA, 1995). This tea is traditionally prepared by fermenting sweetened (sucrose) black tea (Camelia sinensis L.) with cellulose pellicle popularly known as a “tea fungus” obtained from previous cultivation, and 10% of old soup and incubated statically under aerobic conditions. The fermentation is allowed to continue for about 14 days. Recent studies suggest that this fermented tea could prevent paracetamol (Pauline et al., 2001) and chromate (VI)-induced oxidative stress in experimental animals (Sai Ram et al., 2000). The active ingredients responsible for beneficial effects of KT have been identified as tea polyphenols, gluconic acid, glucuronic acid and various other compounds produced during fermentation (Jayabalan et al., 2007; Vijayaraghavan et al., 2000; Bhattacharya et al., 2011a). Bhattacharya et al., (Bhattacharya et al., 2011b) very recently investigated whether KT can be used as a protective measure in oxidative damage and cell death. The molecular mechanism underlying the protective action of KT showed that hepatocytes treated with KT in combination with the ROS inducer, TBHP displayed a reduction in ROS generation, prevented the alterations in the antioxidant machineries and blocked the activation of mitochondria dependent apoptotic signaling pathways (Bhattacharya et al., 2011c). The authors found a beta-glucuronidase inhibitor,

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D-saccharic acid-1,4-lactone (DSL), as the most healthful, crucial and pharmacologically active lactone component in KT that possesses a number of biological activities like detoxifying and anticarcinogenic properties (Hanausek et al., 2003). Literature suggests that D-glucarates have also been shown to reduce total serum cholesterol, low-density lipoprotein (LDL)-cholesterol and serum estrogen levels (Walaszek et al., 1996). Recently, Saluk-Juszcak et al., (Saluk-Juszcak et al., 2008) reported the antioxidative properties of DSL and they showed that it decreases the oxidative damage to cellular biomolecules (lipids and proteins) in human blood platelets treated with peroxynitrite or hydroperoxide. They also demonstrated that DSL could reduce the activation of blood platelets under the similar conditions (Saluk-Juszcak et al., 2008). Next, research has been carried out to evaluate whether DSL itself can ameliorate the oxidative stress in hepatocytes exposed to TBHP and observed that it possesses potent antioxidant activity, prevents the alterations in TBHP induced antioxidant machineries and blocks the disruption of mitochondrial membrane potential, release of cytochrome c in the cytosol from mitochondria as well as the activation of mitochondria dependent apoptotic signaling pathways and helps the cells to maintain their normal physiology (Bhattacharya et al., 2011a). Although the antioxidative nature of DSL has been investigated by many researchers, but its beneficial role on oxidative stress related diabetic pathophysiology is poorly described in the existing literature. Studies have, therefore, been carried out to evaluate the beneficial role of DSL in alloxan (ALX)-induced diabetes using rat pancreas tissue as the working model. Alloxan exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intracellular antioxidant machineries whereas DSL supplementation prevented all these changes and restored these alterations close to normal. Investigating the underlying mechanism of the protective action of DSL, it has been observed that it prevented the pancreatic b-cell apoptosis mainly via mitochondria-dependent pathway. Results showed alloxan induced hyperglycema decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocally regulated of Bcl-2 family proteins in the diabetic animals. All these events were also found to be associated with increased level of Apaf-1 and caspases that ultimately led to pancreatic b-cell apoptosis. On the other hand, DSL treatment counteracts all these adverse changes (Bhattacharya et al., 2011d). In conclusion, DSL has been found to be capable of attenuating oxidative stress and diabetes related organ pathophysiology by inhibiting apoptosis via mitochondria-dependent pathway and could be considered as a novel therapeutic strategy to prevent diabetes mellitus in future.

10.3.4

Beneficial Role of Taurine

Taurine (2-aminoethanesulfonic acid), a derivative of the sulphur-containing amino acid cysteine, is the most abundant conditionally essential amino acid in the tissues of many animals and is particularly abundant in aquatic foods. This amino acid is not incorporated into proteins, but it does play major roles in the body. In the liver it acts as a substrate in the conjugation of bile acids (Das et al., 2012b). Taurine possesses multitude of biological function e.g. it exerts membrane stabilizing, osmoregulatory and cytoprotective effects, possesses

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antioxidative properties, regulates intracellular Ca2+ concentration, modulates ion movement and neurotransmitters, reduces the levels of pro-inflammatory cytokines in various organs and controls blood pressure (Das et al., 2012b; Sinha et al., 2007d, 2008c, 2008d, 2009; Manna et al., 2008c, 2008d, 2009c). Usually it does not directly scavange free radicals but as a direct antioxidant, it can only scavenge HOCl; and as an indirect antioxidant it can prevent changes in permeability of membrane due to oxidative impairment (Timbrell et al., 1995; Wright et al., 1986). It protects many organs of our body in pathophysiologic conditions, reduces oxidative stress by enhancing antioxidant enzymes activities and intracellular GSH level, reduces nitrosative stress, prevents alterations of the activities of Na+-K+-ATPase, Ca2+, Mg2+-ATPase during various toxin and drug-induced organ dysfunction (Das et al., 2012b). A biosynthetic route from cysteine and/or the specific uptake from the extracellular space is basically accounted for the intracellular levels of this amino acid. Taurine is synthesised by oxidation followed by decarboxylation of the amino acid, cysteine and this phenomena mainly happens in the liver. It is reported that the biosynthetic capacity of taurine is very low in human, while rodents have high synthetic capacity (Dixon, B. Dixon, 1973). The source of extracellular taurine is dietary intake. Dietary taurine, ingested from meat and especially from sea foods is another alternative source. It is also abundant in human breast milk and is considered to be important in infant brain and retinal development (Das et al., 2012b). With this beneficial background of taurine, we started working on its protective role in environmental toxin and drug induced organ pathophysiology. Results suggest that taurine could protect a number of body organs against chemically exposed pathophysiology (Das et al., 2012b; Sinha et al., 2007d, 2008c, 2008d, 2009; Manna et al., 2008c, 2008d, 2009c). Investigating cell signaling pathways for the As induced hepatic pathophysiology, it has been observed that observed that chronic administration of As produces ROS which subsequently activates PKCd, JNK and helps the progression of apoptosis in liver. Taurine, on the other hand, attenuates As-induced oxidative stress in the liver and thus prevents hepatic apoptosis (Das et al., 2010a). We also observed that acute exposure of As caused oxidative stress and induced apoptosis in rat testes via MAPKinase and NF-kB activation. However, taurine protects this organ by inhibiting oxidative stress and apoptosis via mitochondrial dependent and independent pathways (Das et al., 2009b). In cardiac pathophysiology, As reduced cardiomyocytes viability, increased ROS production and intracellular calcium overload, induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-kB (p65) phosphorylation and MAPKs activation. Taurine suppressed these apoptotic actions, suggesting its protective role in As-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways (Ghosh et al., 2009). Returning to drug-induced organ pathophysiology, it has been observed that acute exposure of acetaminophen caused hepatic tissue injury via the upregulation of CYP2E1 and JNK and in renal tissue via the upregulation of only CYP2E1. Moreover, in both cases, acetaminophen significantly increased the plasma levels of TNFa and NO production, caused DNA fragmentation that ultimately

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leads to cellular necrosis. However, taurine was effective in counteracting acetaminophen-induced hepatic and renal damages, oxidative and nitrosative stress and cellular necrosis (Das et al., 2010b; Das et al., 2010c). It has also been observed that doxorubicin administration impairs survival of cardiac cells via increased ROS generation and intracellular Ca2+ level, activation of p53 and JNK-p38, which ultimately led to NF-kB activation via IKK pathway and mitochondrion-dependent cell apoptosis. Results also suggest that doxorubicin can trigger intrinsic, extrinsic and endoplasmic reticulumassociated apoptotic pathways in testicular pathophysiology. Doxorubicin also triggered activation of JNK, p38MAP kinases and p53. However, taurine could effectively prevent nearly all of these doxorubicin-induced testicular abnormalities, thereby proving to be an effective cytoprotectant (Das et al., 2011; Das et al., 2012c). In addition, the underlying mechanism of the beneficial role of taurine in diabetic pathophysiology has also been highlighted. Taurine significantly reduces oxidative stress and the levels of proinflammatory cytokines in diabetic hearts and improves the insulin-signaling pathway by restoring GLUT4 translocation to the plasma membranes (Das et al., 2012a). Just after the development of cardiomyopathy, taurine could inhibit the increase in heart weight, improved the impaired -dp/dt max (but not +dp/dt max) and also protected cardiac tissue from DNA breakdown and apoptosis in hyperglycemic conditions (Das et al., 2012a, 2012b). The protective effect of taurine on diabetic kidney is mediated via suppression of AGEs, TGFa and ROS (Winiarska et al., 2009; Nandhini and Anuradha, 2003; Higo et al., 2008) in which it reduces the plasma AGEs level probably by functioning as a glycation scavenger and reducing the blood glucose levels (Das et al., 2012b). In addition, taurine inhibits the alteration in the level of Na+-K+-ATPase, decreases the activity of xanthine oxidase, expressions of p47phox and CYP2E1, attenuates the levels of the proinflammatory cytokines and hydroxyproline levels, inhibits the decrease in NO and eNOS and ameliorates ROS-induced up-regulation of PKC, MAPkinases and ultimate renal apoptosis (Das and Sil, 2012b). The beneficial role of taurine in diabetic retinopathy has been reported by several researchers (Das et al., 2012b). After diabetic onset, its supplementation decreases retinal carbonyl dienes, inhibits elevation of retinal glutamate content, normalizes retinal vascular function, improves ultra structure and attenuates induction of glial fibrillary acid protein and thereby protects retinal glial cells from apoptosis (Das et al., 2012b). These impressive benefit and absence of toxicity with taurine supplementation may provide clues in understanding the cardiac, hepatic and renal protective mechanism of taurine. We, therefore, propose that increased dietary taurine intake represents an important nutritional modification that may provide a useful intervention to reduce the worldwide burden from all these toxin, drug and diabetes induced organ dysfunction and its associated complications.

10.3.4.1

Mechanism of the beneficial effect of taurine

Taurine usually forms conjugates with bile acids (mainly cholic acid) to produce taurocholate (Hadi and Suwaidi, 2007; Murakami et al., 2000), controls cholesterol metabolism and prevents atherosclerosis by lowering the levels of oxidized LDL. Taurine possesses membrane stabilizing effect and a possible mechanism could be either its interactions with the cellular polyunsaturated

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fatty acids (Yorek et al., 1984) or its interaction with sites in the membrane related to anion transport and water influx (Pasantes-Morales et al., 1984). It acts as an important organic osmolyte and can regulate the cell volume and could modulate intracellular Ca2+ movements via an osmoticlinked mechanism: firstly by the activation of several Ca2+ transporters and channels (Wright et al., 1984) and secondly via the activation of phospholipase C, which leads to the activation of IP3 and release of Ca2+ from intracellular stores (Hoffmann and Dunham, 1995; Suleiman et al., 1992). Functioning as an inhibitory neurotransmitter and neuromodulator for prevention of excitoxicity, it can inhibit GABA transaminase and allows GABA to stay in the synaptic cleft longer for the binding to the postsynaptic receptor (Freund and Buzsaki, 1992). Besides, it can bind to the GABAA receptor, mimicking the effects of GABA and could exhibit its diuretic action probably by enhancing sodium excretion and preserving potassium and magnesium (Riesenhuber et al., 2006). It inhibits the blood pressure-increasing effect of rennin-angiotensin system (Abe et al., 1988). Renin is activated and angiotensin is formed as a result of the decreased level of taurine in blood which leads the blood vessels to vasoconstrict, salt and water are retained and blood pressure increases. Under these circumstances, taurine suppresses the activation of rennin, destroys the renin-angiotensin feedback loop and helps inhibiting blood pressure increase (Das et al., 2012b). Another important property of this unique amino acid is its anti-inflammatory actions mediated via its reaction with HOCl in presence of myeloperoxidase to form taurine chloramine (Tau-Cl) in activated neutrophils. Tau-Cl is reported to be less toxic and possesses anti-inflammatory properties (Schuller-Levis and Park, 2003). The transcription factor, NF-kB helps controlling numerous gene expressions that is activated during inflammation (Kuhad and Chopra, 2009). One important point to be noted here: taurine can only detoxify the effect of HOCl as a direct antioxidant. Mitochondria are rich in taurine content and that could successfully inhibit mitochondrial ROS production (Schaffer et al., 2009). Besides, taurine-containing modified uridine in mitochondrial tRNA play a crucial role in the electron transport proteins’ translation (Kirino et al., 2004); suggesting taurine depletion could cause a decrease in taurine-modified tRNA and impairs electron transport capacity of the system (Das et al., 2012b).

10.4

CONCLUSIONS

In this book chapter, we discuss about the various bioactivities, multifunctional therapeutic application, antioxidant and signaling properties of macro and small bioactive molecules. From this discussion, it is clear that these molecules play a beneficial role in oxidative stress induced organ pathophysiology. This discussion also unravels the potential use of macro and small bioactive molecules as a novel promising therapeutic strategy.

Abbreviations AA, Arjunolic acid; ALT, alanine transaminase; ALP, alkaline phosphatase; ALX, Alloxan monohydrate; APAP, Acetaminophen; AR, Aldose reductase; ATP, adenosine tri phosphate; CAT, catalase; CI protein, Cajanus indicus protein; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; DSL, D-saccharic acid-1,4-lactone; ERK, extracellular signal-regulated kinases; GAL,

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D-galactosamine; GSH, glutathione; GST, glutathione S-transferase; HO-1,heme oxygenase-1, HDL, high density lipoprotein; JNK, c-Jun-NH2-terminal protein kinase; KT, kombucha tea; LDH, Lactate dehydrogenase; LDL, low density lipoprotein; MAPKs, mitogen-activated protein kinases; MPT, mitochondrial permeabilization; NAPQI, N-acetyl-p-benzoquinone imine; NF-kB, nuclear factor kappa B; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; Nrf2, nuclear erythroid 2-related factor 2; PARP, Poly (ADP-ribose) polymerase; PI3K, Phosphatidylinositol 3-kinases; PKC, protein kinase c; PNP, Phyllanthus niruri protein; ROS, reactive oxygen species; STZ, streptozotocin; TBHP, tertiary butyl hydroperoxide; TNF a, tumor necrosis factor alpha.

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Manna P, Sil PC (2012a). Arjunolic acid: beneficial role in type 1 diabetes and its associated organ pathophysiology. Free Radic Res 46, 815–830. Manna P, Sil PC (2012b). Impaired redox signaling and mitochondrial uncoupling contributes vascular inflammation and cardiac dysfunction in type 1 diabetes: Protective role of arjunolic acid. Biochimie 94, 786–797. Manna P, Das J, Ghosh J, Sil PC (2010a). Streptozotocin induced activation of oxidative stress responsive splenic cell signaling pathways: Protective role of arjunolic acid. Toxicol Appl Pharmacol 244, 114–129. Manna P, Das J, Ghosh J, Sil PC (2010b). Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS, PARP, IkBa/NF-kB, MAPKs, and mitochondria-dependent Prophylactic role of arjunolic acid. Free Radic Biol Med 48, 1465–1484. Manna P, Sinha M, Sil PC (2009a). Protective role of arjunolic acid in response to streptozotocin-induced type-I diabetes via the mitochondrial dependent and independent pathways. Toxicology 257, 53–63. Manna P, Sinha M, Sil PC (2009b). Prophylactic role of arjunolic acid in response to streptozotocin mediated diabetic renal injury: Activation of polyol pathway and oxidative stress responsive signaling cascades. Chem Biol Interact 181, 297–308. Manna P, Sinha M, Sil PC (2009c). Taurine plays a beneficial role against cadmium-induced oxidative renal dysfunction. Amino Acids 36, 417–428. Manna P, Sinha M, Sil PC (2008a). Protection of arsenic-induced testicular oxidative stress by arjunolic acid. Redox Rep 13, 67–77. Manna P, Sinha M, Sil PC (2008b). Arsenic-induced oxidative myocardial injury: protective role of arjunolic acid. Arch Toxicol 82, 137–149. Manna P, Sinha M, Sil PC (2008c). Amelioration of cadmium-induced cardiac impairment by taurine. Chem Biol Interact 174, 88–97. Manna P, Sinha M, Sil PC (2008d). Taurine triggers a chemoprevention against cadmium induced testicular oxidative injury. Reprod Toxicol 26, 282–291. Manna P, Sinha M, Sil PC (2007a). A 43 kD protein isolated from the herb Cajanus indicus L attenuates sodium fluoride-induced hepatic and renal disorders in vivo. J Biochem Mol Biol 40, 382–395. Manna P, Sinha M, Sil PC (2007b). Galactosamine-induced hepatotoxic effect and hepatoprotective role of a protein isolated from the herb Cajanus indicus L in vivo. J Biochem Mol Toxicol 21, 13–23. Manna P, Sinha M, Pal P, Sil PC (2007c). Arjunolic acid, a triterpenoid saponin, ameliorates arsenic-induced cyto-toxicity in hepatocytes. Chem Biol Interact 170, 187–200. Manna P, Sinha M, Sil PC (2007d). Protection of arsenic-induced hepatic disorder by arjunolic Acid. Basic Clin Pharmacol Toxicol 101, 333–338. Maritim AC, Sanders RA (2003). 3rd.Watkins, Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol 17, 24–38. Martı´n R, Tavares JC, Herna´ndez M, Arne´s M, Ruiz-Gutie´rrez V, Nieto ML (2010). Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: A potential therapeutic role. Biochem Pharmacol 79, 198–208. Masoko P, Mdee LK, Mampuru LJ, Eloff JN (2008). Biological activity of two related triterpenes isolated from Combretum nelsonii (Combretaceae) leaves. Nat Prod Res 22, 1074–1084.

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Miura T, Ichiki H, Hashimoto I, Iwamoto N, Kato M, et al., (2001).Antidiabetic activity of a xanthone compound, mangiferin. Phytomedicine 8, 85–87. Moreira RR, Carlos IZ, Vilega W (2001). Release of intermediate reactive hydrogen peroxide by macrophage cells activated by natural products. Biol Pharm Bull 24, 201–204. Mudge GH, Gemborys MW, Duggin GG (1978). Covalent binding of metabolites of acetaminophen to kidney protein and depletion of renal glutathione. J Pharmacol Exp Ther 206, 218–226. Murakami S, Kondo Y, Nagate T (2000). Effects of long-term treatment with taurine in mice fed a high-fat diet: improvement in cholesterol metabolism and vascular lipid accumulation by taurine. Adv Exp Med Biol 483, 177–186. Nandhini TA, and Anuradha CV (2003). Inhibition of lipid peroxidation, protein glycation and elevation of membrane ion pump activity by taurine in RBC exposed to high glucose. Clin Chim Acta 336, 129–135. Newman DJ, Cragg GM, Snader KM (2003). Natural products as sources of new drugs over the period 19812002. J Nat Prod 66, 1022–1037. Newman DJ, Cragg G.M., Snader K.M.. (2000). The influence of natural products upon drug discovery. Nat Prod Rep 17, 215–234. Newman DJ, Cragg GM (2007). Natural products as sources of new drugs over the last 25 years. J Nat Prod 70, 461–477. Nunez-Selles AJ, Velez Castro HT, Aguero-Aguero J, Gonzalez-Gonzalez J, Naddeo F, Simone F De, et al., (2002). Isolation and quantitative analysis of phenolic antioxidants, free sugars, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement. J Agric Food Chem 50, 762–766. Odetola AA, Akojenu SM (2000). Anti-diarrhoeal and gastro-intestinal potentials of the aqueous extract of Phyllanthus amarus (Euphorbiaceae). Afr J Med Sci 29, 119–122. Pal S, Ahir M, Sil PC (2012). Doxorubicin induced neurotoxicity is attenuated by a 43 kD protein from the leaves of Cajanus indicus L via NF-kB and mitochondria dependent pathways. Free Radic Res 46, 785–98. Pal S, Pal PB, Das J, Sil PC (2011). Involvement of both intrinsic and extrinsic pathways in hepatoprotection of Arjunolic acid against cadmium induced acute damage in vitro. Toxicology 283, 129–139. Pal S, Sil PC (2012). A 43 kD protein from the leaves of the herb Cajanus indicus L. modulates doxorubicin induced nephrotoxicity via MAPKs and both mitochondria dependent and independent pathways. Biochimie 94, 1356–1367. Pandey BP (1978). Economic Botany 1st edn., S. Chand and Co. Ltd., New Delhi, 25–26. Pasantes-Morales H, Wright CE, Gaull GE (1984). Protective effect of taurine, zinc and tocopherol on retinol-induced damage in human lymphoblastoid cells. J Nutr 114, 2256–2261. Paterson I, Anderson EA (2005). The renaissance of natural products as drug candidates. Science 310, 451–453 . Pauline TP, Dipti B, Anju S, Kavimani SK, Sharma AK (2001). Studies on toxicity, anti-stress and hepatoprotective properties of Kombucha tea. Biomed Environ Sci 14, 207–213. Pope A, Rall DP (1995). Environmental Medicine Integrating a Missing Element into Medical Education. Washington, DC: National Acad Press. 230–231. Prasad MVV, Anbalagan N, Patra A, Veluchamy G, Balakrishna K (2004). Antiallergic and Anti–asthmatic activities of the Alcoholic Extract of Terminalia Arjuna and Arjunolic acid. Nat Prod Sci 10, 240–243.

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11 Anti-pruritic and Anti-inflammatory Herbal or Natural Products for the Treatment of Skin Disease Katsunori Yamaura* and Koichi Ueno Department of Geriatric Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Chiba University 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan

ABSTRACT There is a growing interest in using herbs and natural products to maintain health or to alleviate chronic conditions. Hence, scientific information on such products should have to be explored in more detailed manner to the consumers and healthcare providers. We herein aim to focus on the effect of herbal and natural products for alleviating the symptoms of skin disease using a murine model of chronic allergic dermatitis. Natsumikan (Citrus natsudaidai) is a citrus fruit that contains several antioxidative nutrients found in higher concentration in the peel than in the pulp. We have shown that extract from immature natsumikan peel attenuates skin inflammation in chronic allergic dermatitis in mice. Bilberry (Vaccinium myrtillus L.) is one of the richest sources of anthocyanins, which are known to have anticancer, wound healing, and anti-allergic effects. We have previously demonstrated that bilberry extract alleviates both hapten-induced pruritus and skin inflammation in a mouse model of chronic allergic contact dermatitis. We have also provided evidence of the inhibitory effect of topical royal jelly on chronic pruritus. These results suggest that some types of herbs and natural products may be beneficial for alleviating symptoms of skin inflammation and pruritus in patients with chronic skin diseases such as allergic contact dermatitis or atopic dermatitis. Keywords: herbs, pruritus, inflammation, allergic contact dermatitis, complementary and alternative medicine, Citrus natsudaidai, bilberry anthocyanins, royal jelly

*Corresponding author: [email protected]

11.2

Natural Bioactive Molecules: Impacts and Prospects

11.1

INTRODUCTION

Pruritus (itch) is often associated with numerous dermatological disorders in populations. It is an uncomfortable sensation that provokes one to scratch it, and this scratching in turn intensifies itching causing further damage to the skin. This vicious cycle is termed as the “itch–scratch cycle” (Pfenninger and Zainea, 2001; Mahtani et al., 2005). Chronic pruritus is associated with many diseases, the most common being chronic renal insufficiency, cholestatic liver diseases, and atopic dermatitis (Talwalkar et al., 2003; Bernhard, 2005). The mechanisms underlying pruritus are complex and poorly understood despite of a century-long research (Graham et al., 1951). The lack of understanding of the neurophysiology and pathophysiology of pruritus has been due to the lack of available reliable animal models for this condition, which has also hampered the development of adequate therapies. In 1995, Kuraishi and co-workers reported a novel method for evaluating pruritus using the scratching behavior of mice as a measure of the degree of pruritus (Kuraishi et al., 1995). Since then, the number of experimental studies has greatly increased, reflecting the increased therapeutic needs of patients with pruritic diseases. Currently used anti-pruritic drugs lack sufficient clinical efficacy. Therefore, effective drugs are urgently needed to improve quality of life (QOL) of affected individuals. Since a long time is required to develop new drugs, it is worthy, in the meantime, to take up investigation on effective herbs and natural products as complementary and alternative medicine (CAM) to alleviate pruritus and also to enhance QOL. CAMs are used by more than 80% of the world’s population and are becoming an increasing component of the US healthcare system, with more than 70% of the population using CAMs at least once and with annual spending reaching as much as $34 billion (Mainardi et al., 2009). Herbs and natural products are very popular for self-medication. The 2002 National Health Information Survey, which was limited with regard to CAMs, showed that 36% of adults used CAM (62% if prayer was included as an alternative therapy), with follow-up surveys demonstrating continued increases, particularly in the use of herbal preparations (Barnes et al., 2004). The National Health Statistics Reports of 2007 describe the most commonly used non-vitamin, nonmineral therapy as natural products (e.g., herbals at 17.7%) (Barnes et al., 2008). However, little evidence-based information is available on the usefulness of herbs and natural products for the alleviation of itching in conditions such as allergic contact dermatitis or atopic dermatitis.

11.2

CITRUS NATSUDAIDAI ALLEVIATES ALLERGIC CONTACT DERMATITIS

Topical exposure to low molecular-weight chemical compounds can cause allergic contact dermatitis (Karlberg et al., 2008). These chemical compounds are termed haptens. To date, the growing list of haptens comprises more than 44,350 substances (Simonsson et al., 2011). Affecting 15–20% of the population in the Western world, allergic contact dermatitis constitutes the most prevalent form of human immunotoxicity (Thyssen et al., 2007). Allergic contact dermatitis occurs in two distinct phases: the sensitization phase, which starts upon first contact with the hapten, and

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11.3

the elicitation phase, which is characterized by the development of the eczematous symptoms induced by subsequent contact with the same hapten. Once an individual is sensitized, there is no cure; the offending compound and any cross-reacting compounds must be avoided. The detailed mechanisms and regulation of allergic contact dermatitis have been widely reviewed (Kimber et al., 2002; Krasteva et al., 1999). At present, allergic contact dermatitis is easily simulated in experimental animal models by repeated application of haptens such as 2, 4-dinitrofluorobenzene (Lee et al., 2007), ovalbumin (Dahten et al., 2008) and 2, 4, 6-trinitro-1-chlorobenzene (TNCB) (Harada et al., 2005; Seike et al., 2010). Natsumikan (Citrus natsudaidai) is a citrus fruit that is recognized for its medicinal properties in many countries. The dried immature fruit is used as an ingredient for kijitsu (Aurantii Fructus Immaturus), which is a crude drug commonly used in some Kampo formulas. Natsumikan has many bioactive components including hesperidin, neohesperidin, naringin, nobiletin, tangeretin, and auraptene (Fig. 1); the anti-inflammatory (Tanabe et al., 2007; Lee et al., 2004), anticarcinogenic (Tanaka et al., 1997), antioxidant (Sugiura et al., 2006), and other pharmacological properties of these components have also been investigated. It has been reported that citrus peel contains several active components at far higher concentrations than in the pulp. We examined the alleviating effect of extract of immature natsumikan peel on skin inflammation in a mouse model of allergic contact dermatitis (Nakayama et al., 2011). The peel contained the following active components per 100 g: hesperidin 11 mg, neohesperidin 370 mg, naringin 780 mg, nobiletin 1.6 mg, tangeretin 3.2 mg, and aurapten 26 mg. Chronic allergic contact dermatitis was induced by repeated application of TNCB in mice, and natsumikan extract was administrated orally for 30 days. Natsumikan showed significant attenuation of the increase in ear swelling induced by TNCB in this model, and improved dermatitis scores (Fig. 2) (Nakayama et al., 2011). Therefore, the extract of immature natsumikan peel might be an effective natural product for treating chronic allergic contact dermatitis. We found that serum derivatives of reactive oxygen metabolites (d-ROM), an indicator of oxidative stress, was increased by repeated application of TNCB. Thus, oxidative stress may be associated with the pathology of dermatitis in this model. In our study, elevations in serum d-ROM in mice with dermatitis were also attenuated by treatment with natsumikan. As described above, there is a high concentration of neohesperidin in the natsumikan peel fraction and it was reported that neohesperidin and its aglycone, hesperetin, are the major secondary metabolites in citrus species having antioxidative properties (Sawa et al., 1999; Kim et al., 2004). Therefore, the inhibitory effect of natsumikan on allergic contact dermatitis in our mouse model may be mediated, at least in part, by these antioxidative components. However, further studies are required to confirm this possibility. Additionally, the beneficial effect of other citrus species was evaluated in similar animal models of contact dermatitis. It was reported that extract of immature Citrus unshiu inhibits ear swelling during both the sensitization and elicitation phases of allergic contact dermatitis in mice (Fujita et al., 2008). It was also demonstrated that extract of immature Citrus hassaku and its flavanone glycosides, naringin and neohesperidin, inhibit contact dermatitis in mice (Itoh et al., 2009). These results suggest that hesperidin, naringin, and neohesperidin might be the active components for treatment of allergic contact dermatitis.

11.4

Natural Bioactive Molecules: Impacts and Prospects

Fig. 1 Structures of some bioactive natural compounds

11.3 ANTHOCYANINS FROM BILBERRY ALLEVIATE CHRONIC PRURITUS Bilberry (Vaccinium myrtillus L.) is one of the best sources of anthocyanins and contains 15 anthocyanin analogues, each composed of one of five anthocyanidins and one of three glucosides (Ichiyanagi et al., 2004). Many pharmacological studies have confirmed the efficacy of bilberry and other anthocyanin-containing extracts with regard to inhibition of cancer cell growth (Zhao et al., 2004), improvement of eyesight (Matsumoto et al., 2003), and a-glucosidase inhibition

Yamaura and Ueno: Anti-pruritic Natural Products

Fig. 2

11.5

Effect of natsumikan extract and dexamethasone administration on ear swelling in mice repeatedly exposed to TNCB. (Nakayama et al., 2011)

Natsumikan (100, 300 and 1000 mg/kg) was administered orally every day from day 1 to 30. Dexamethasone (3 mg/ kg) was administered orally every day from day 1 to day20, and then every other day from day 21 to 30. Each reagent was administered 30 min before the TNCB challenge. Ear thickness was measured 24 h after each TNCB challenge. Each symbol represents the mean ± SEM of n=7–8 mice. *, **: Significantly different from the vehicle group at P 476 (IC50 18.4 vs 31.3 µM). Such structure-activity relationship for the A-type oligomeric proanthocyanidins will be useful as a reference for the development of a-glucosidase inhibitors for blood glucose control in diabetic patients (Lin and Lee, 2010). Kosar and his group (2009) isolated three new homoisoflavone glucosides, Purunusides A-C (496-498), from n-butanol soluble fractions of cold ethanol extract of Prunus domestica, it was also found that the compounds 496-498 showed potent inhibitory activity against the enzyme a-glucosidase with IC50 values of 216.6 ± 0.027, 268.4 ± 0.047, and 203.6 ± 1.7 mM, respectively, comparison with positive control deoxynojirimycin (IC50 281.3 ± 2.8 mM) (Kosar et al., 2009). The enhanced activity is obviously due to the presence of glucose moiety at C-4¢ of the homoisoflavone skeleton. Although the activities were comparable but the compound 498 was the most potent and it might be attributed to 4-hydroxy phenyl ester moiety, which probably acts as a pharmacophoric group (Kosar et al., 2009). Three new prenylated flavanones (377-379) isolated from the n-hexane extract of Dalea boliviana roots were evaluated in vitro in relation to their inhibitory effect on the tyrosinase activity by using a spectrophotometric method; compounds 377 (%inhibition 82.1) and 378 (%inhibition 87.8) appeared to be better inhibitors at a concentration of 100 mM than 379 (%inhibition 6.4) under the same conditions. Hence, a phloroglucinol A-ring with two free

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hydroxy groups is supposed to be essential for inhibitory activity against the tyrosinase enzyme (Peralta et al., 2011). Phytochemical investigation of the figs of Ficus mucuso led to the isolation of three new isoflavone dimer derivatives, mucusisoflavones A-C (503-505). The isolates were evaluated in vitro for their inhibitory properties towards a-glucuronidase enzyme, and found to be highly efficient — the respective IC50 values for the compounds were determined as 0.68 ± 0.01, 13.96 ± 1.23 and 3.08 ± 0.05 mM (Bankeu et al., 2011).

13.3.5

Antiinflammatory Activity

Itoside-N (14), a rarely reported naturally occurring flavone glycoside truxinate ester, isolated from Itoa orientalis, showed significant anti-inflammatory activity against cyclooxygenase-2 (COX-2) with inhibitory rate of 67.3 % at 10 mM concentration (Chai et al., 2008). Two novel chemical entities 19 and 20, isolated from whole plant of Pogonatherum crinitum, also inhibited NO production in activated RAW 264.7 cells to various degrees without affecting the cellular viability; Both of these compounds suppressed LPS-induced NO production, with Emax values of 99.51 ± 0.23% and 92.41 ± 3.22%, respectively (Wang et al., 2008). Three flavonoid glycosidic constituents (22-24) of Dracocephalum peregrinum exhibited anti-inflammatory activity against nityric oxide (NO) and nuclear factor (NF)-kB activity on RAW 264.7 and pNF-kB-luc-293 cells (Fu et al., 2009). The flavonoid 22 showed good inhibitory activity on nitric oxide (NO) production induced by LPS at doses of 100 mg/mL, and 50 mg/mL (% inhibition 56 and 18, respectively); whereas the compounds 23 and 24 were found to possess weak such efficacy against NF-kB at a dose of 100 mg/mL (% inhibition 27 and 38, respectively). Aquisiflavoside (71), a new flavonoid glycoside isolated from the leaves of Aquilaria sinensis (Lour.) Gilg was evaluated to show potent inhibitory activity against nitric oxide (NO) production induced by lipopolysaccharide in macrophage cell line RAW 264.7 in a dose-dependent manner with an IC50 value of 34.95 mM (Yang et al., 2012). Polygonflavanol A (230), a novel flavonostilbene glycoside from the roots of Polygonum multiflorum was also found to inhibit nitric oxide (NO) secretion of RAW264.7 cells in respond to lipopolysaccharide (LPS) (Chen et al., 2012a). The geranyl flavonoid, arcommunol D (454a) isolated from the leaves of Artocarpus communis suppressed the LPS-induced production of nitric oxide (NO) in RAW 264.7 cells with IC50 values of 18.45 ± 2.15 mM (Hsu et al., 2012). Zhang et al. (2012a) isolated two pairs of new diastereomeric flavonolignans, hovenin A–D (226–229) from the seeds of Hovenia acerba. All of these four compounds exhibited significant inhibition on the production of NO and IL-6 in LPSstimulated RAW264.7 cells with IC50 values ranging from 45.11 to 63.47 mM, thereby suggesting that hovenin A–D might be new potential candidates. The new homoisoflavonoid, ophiopogonanone H (305) isolated from the tuberous roots of Ophiopogon japonicus was evaluated for inhibitory effect against NO production induced by lipopolysaccharide in the murine microglial cell line BV-2; the test compound showed potent inhibitory effect on NO production with IC50 values of 20.1 mM (Li et al., 2012a) Dongmo et al. (2007) evaluated that the flavonoids 25 and 145 of the leaves of Acacia pennata possess anti-inflammatory activity as assessed against cyclooxygenase enzymes (COX-1/2); the flavonoid 145 with a 3¢,4¢-subtitution in ring B with a glycosylation at the C-3 position was found

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Natural Bioactive Molecules: Impacts and Prospects

to be the most potent inhibitor of COX-1 (IC50 11.6 mg/mL), whereas flavonoid 25 was inactive. But both the flavonoids 25 and 145 showed weak activity against COX-2 (Dongmo et al., 2007). Hence, 145 may serve as useful lead in anti-inflammatory drug discovery processes. The flavone, yunanensol A (29), isolated from stem bark of Morus yunanensis, showed potent anti-inflammatory activity via inhibiting the release of b-glucuronidase from rat PMNs induced by PAF by 93.6% at concentration of 10–5 mol/L (Cui et al., 2008). The new flavan-3-ol, (+)-afzelechin 5-O-b-Dglucopyranoside (111) isolated from the fruit peels of Wisteria floribunda, exhibited week in vitro anti-inflammatory activity on tumor necrosis factor alpha (TNF-a)-induced nuclear factor kappa-B activation in HepG2 cells with IC50 value >100 mM (Taia et al., 2011). The anti-inflammatory activity of 5,7-dihydroxy-3,6,4¢-trimethoxy-3¢-(4-hydroxy-3-methyl-but-2-enyl)flavone (85), a flavonoid constituent of Dodonaea viscosa was also documented with a flow cytometry TNF-a secretion assay on human Thp-1 cell line (Wabo et al., 2012). The flavonoid glycoside 200 was found to demonstarte moderate anti-inflammatory activity (Tantry et al., 2012). An acylated C-glycosylflavone 34 from Trollius ledebouri was reported to exhibit significant anti-inflammatory effects on TPA-induced ear edema with inhibitory rate of 58.6% (Wu et al., 2006a). Chung et al. (2009) also reported a number of flavonol glycosides 120-121 and 317318 that showed significant anti-inflammatory activity as assessed in proteolytic enzyme matrix metalloproteinases (MMP-9) assay (Chung et al., 2009). The isolated flavonol glycoside 132 from leaves of Alchornea floribunda was also evaluated for anti-inflammatory activity against egg albumen-induced rat paw oedema. The compound exhibited a significant and dose dependent inhibition of rat paw oedema with inhibitory rate of 51.4% at 50 mg/kg concentration, which is higher than that of the standard anti-inflammatory drug, aspirin (% inhibition 45.9 at 100 mg/ kg concentration) (Okoye and Osadebe, 2010). A rare flavonol glycoside 142, isolated from Tephrosia spinosa, was found to exhibit significant anti-inflammatory activity against carrageenin induced paw edema when compared to the standard drug indomethacin (Chakradhar et al., 2005). Yen et al. (2009) reported that the flavonol, morin-3-O-a-rhamnopyranoside (158), isolated from of Muehlenbeckia platyclada, was found to exhibit anti-inflammatory activity by inhibiting neutrophil elastase release with IC50 value of 3.82 ± 0.80 mg/mL and was 15-fold more potent than phenylmethylsulfonyl fluoride (PMSF), the positive control used (Yen et al., 2009). Kaempferol glycoside palmatosides A (163) from the roots of the fern Neocheiropteris palmatopedata exhibited anti-inflammatory activity against COX-1 with inhibitory rate of 52% at 10 µg/mL concentrations (Yang et al., 2010). Conferols A (288) and B (289), two 4-hydroxyisoflavones were isolated from Caragana conferta, and both the compounds showed significant anti-inflammatory activity in the respiratory burst assay with percentage inhibition 78.147 and 89.256 respectively as reported by Khan et al. (2009). Three new 4-hydroxyisoflavans lyratin A (310), lyratin B (311) and lyratin C (312) isolated from the whole plant of Solanum lyratum showed in vitro anti-inflammatory activities by inhibiting the release of b-glucuronidase from polymorphonuclear leukocytes of rats in the range of 30.3−38.6% at 10mM (Zhang et al., 2010). Yadava and Singh (2006) reported that the flavanone glycoside 323 of Echinops echinatus also showed anti-inflammatory activity. A new flavanone glycoside, (−)(2S)-5,6,7,3¢,5¢-pentahydroxyflavanone-7-O-b-D-glucopyranoside (373) isolated from the stems of Lippia graveolens, was found to exhibit weak anti-inflammatory activity

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against TPA-induced oedema model as assessed by Gonza´lez-Gue¨reca et al. (2010). Kuo and his group (2010) isolated three new flavonone glycosides, visartisides A-C (374-376) from Viscum articulatum; all these compounds were evaluated for their anti-inflammatory activities using RAW264.7 cells supplemented with lipopolysaccharide (LPS) to induce cell inflammation and cause nitrite accumulation in the medium. The investigators reported that all the tested flavonone compounds 374, 375, and 376 markedly inhibited NO production in macrophages with IC50 values of 15.6 mM, 25.1 mM, and 14.9 mM, respectively compared with quercetin (IC50 32.1 mM) as positive control (Kuo et al., 2010). Cinnamtannin B-1(506) and cinnamtannin D-1 (507), the two trimeric proanthocyanidins isolated from the bark of Cinnamomum cassia, were found to possess significant in vitro inhibitory activity against cyclooxygenase-2 (COX-2) at micromolar concentrations. Both the compounds were tested in triplicate at 3-log dilutions of 10, 100, and 1000 µg/mL concentrations, with significant inhibition at all levels: trimers 506 (% inhibition: 19, 27, 86) and 507 (% inhibition: 2, 44, 95) (Killday et al., 2011).

13.3.6

Antimicrobial Activity

Flavonoids exhibit a variety of antimicrobial activities. Three flavonoids 126, 127 and 470 from leaf of Piliostigma reticulatum were found to show antimicrobial activity against four bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Proteus vulgaris) and two fungal strains (Aspergillus niger and Candida albicans) (Babajide et al., 2008). Among them piliostigmol (470) exhibited the highest activity against E. coli with minimum inhibitory concentration (MIC) value of 2.57 µg/mL, 3-fold more potent than amoxicillin used as standard (Babajide et al., 2008). The acylated flavonol glycoside 167 isolated from Waltheria indica was also reported to have anti-bacterial potential (Maheswara et al., 2006). Flavonoids 231, 232 and 324 of the aerial parts of Eriophorum scheuchzeri were reported to exhibit antifungal activity against Candida cucumerinum and C. albicans strains (Maver et al., 2005). Compounds 231 and 324 showed activity against C. albicans at both 5 and 10 µg concentrations, while 232 had no activity against C. albicans but showed the highest efficacy among the tested compounds against C. cucumerinum only at 1 mg dose (Maver et al., 2005). Yao-Kouassi et al. (2008) reported that the isoflavonoid glycosides 234 and 235 possess moderate activity against the bacterial strains S. aureus, Enterococcus faecalis, E. coli, and Pseudomonas aeruginosa; however, the compound 236 showed somewhat mild activity (Yao-Kouassi et al., 2008). Antimicrobial activities of the isoflavones 237238 and of the flavonone 332 were tested against a number of microbes, namely E. coli, S. aurones, B. subtilis and C. mycoderma (Chacha et al., 2005). All the tested compounds were found to show less activity against Gram-negative bacteria than those against both the Gram-positive bacteria and fungi (Chacha et al., 2005). Semwal et al. (2009) reported three new flavonoid glycosides such as isoflavone 3¢,4¢,5,6-tetrahydroxy-7-O-{b-D-glucopyranosyl-(1Æ3)-a-L-rhamnopyranoside} (260), isoflavone-3¢,4¢,5,6-tetra-hydroxy-7-O-{b-D-glucopyranosyl-(1Æ6)-b-D-glucopyranosyl-(1Æ6)-b-D-gluco-pyranosyl(1Æ3)-a-L-rhamnopyranoside} (261) and chalcone-6¢-hydroxy2¢,3,4-trimethoxy-4¢-O-b-D-glucopyrano-side (450) from the leaves of Boehmeria rugulosa. All these compounds 260, 261 and 450 showed significant antimicrobial activity against two bacterial strains (S. aureus and Streptococcus mutans) and three fungal pathogens (Microsporum gypseum,

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Natural Bioactive Molecules: Impacts and Prospects

M. canis, and Trichophyton rubrum) in comparison with standard antimicrobial drugs novobiocin and erythromycin (Table 6) (Semwal et al., 2009). The microorganisms, E. coli, S. epidermidis, K. pneumoniae, Aspergillus niger, A. fumigates, and Penicillium citrinum, remained unaffected by the tested compounds 260, 261 and 450 (Semwal et al., 2009). Table 6 Minimum inhibitory concentrations (MICs) of the tested compounds 260, 261 and 450 against the microorganisms comparing with reference standard Test micro-organisms

260 ± SD

261 ± SD

450 ± SD

Novobiocin ± SD

Erythromycin ± SD

S. aureus

18 ± 2

20 ± 2

21 ± 2

21 ± 2

NT

S. mutans

13 ± 4

17 ± 4

25 ± 4

22 ± 2

NT

M. gypseum

11 ± 3

11 ± 2

06 ± 1

NT

15 ± 2

M. canis

15 ± 4

18 ± 3

21 ± 4

NT

18 ± 3

T. rubrum

17 ± 2

17 ± 2

23 ± 3

NT

20 ± 3

Notes: SD, standard deviation; NT, not tested.

The isoflavanone 284, isolated from stems of Erythrina costaricensis, a small tree with brilliant red flowers, exhibited a moderate anti-bacterial activity against methicillin-resistant S. aureus (MRSA) with MIC50 value of 25 mg/mL, while the 283 from the same plant, failed to inhibit the growth of MRSA strains up to the concentration of 50 mg/mL (Tanaka et al., 2008). Dhooghe et al. (2010) isolated six new flavonoid compounds (290, 491-495) from Ormocarpum kirkii and tested them for their biological activities in vitro in an integrated antimicrobial screening panel including Trypanosoma cruzi, Leishmania infantum, T. brucei, chloroquine-resistant Plasmodium falciparum K1, S. aureus, C. albicans, Trichophyton rubrum, and Aspergillus fumigatus. The cytotoxicity was evaluated using MRC-5 cells. The isoflavanone 290 showed a non-selective activity against S. aureus (IC50 6.4 ± 3.1 mM) and T. rubrum (IC50 6.0 ± 2.1 mM), while the compound 491 exhibited moderate but non-selective activity and also found to be cytotoxic. Compound 492 was selectively active against T. rubrum (IC50 7.0 ± 6.4 mM) with only low cytotoxicity (CC50 50.2 ± 16.3 mM). For compound 493, an IC50 of 19.5 ± 13.9 mM was observed only against T. rubrum. 5,5≤-Di-O-methyldiphysin (494) was not selective and showed moderate activity against almost all of their bacterial strains tested with a high cytotoxicity (CC50 11.7 ± 6.2 mM). In contrast, no activity was observed for the mono-glucosylated diphysin derivative 495 as reported by Dhooghe’s groups (2010). Two isoflavanones, 5,7-dihydroxy-2¢-methoxy-3¢,4¢-methylenedioxyisoflavanone (291) and 4¢,5-dihydroxy-2¢,3¢-dimethoxy-7-(5-hydroxyoxychromen-7yl)-isoflavanone (292) were isolated from the roots of Uraria picta (Rahman et al., 2007); the investigators carried out antimicrobial screening of these isolates along with six known compounds including isofavones, terpenes and steroids against two Gram-positive bacteria (S. aureus and B. subtilis), two Gramnegative bacteria (E. coli and Proteus vulgaris) and two fungi (A. niger and C. albicans) — the new isoflavanones 291 and 292 were found to be the most active against S. aureus. In molar concentration, the order of activity against S. aureus was 292>291>known compounds (Rahman et al., 2007). In case of E. coli, the sequence of relative potencies of the compounds was known compounds>292>291 and but against human pathogen C. albicans, the order of activity of the

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13.85

compound was shown known isoflavonones>292>known terpenes>291. The sequence was almost the same for A. niger (Rahman et al., 2007). The isoflavan hildegardiol (313) isolated from the root extract of Hildegardia barteri was assessed to have moderate anti-fungal activity against a variety of Candida species (Meragelman et al., 2005). Smejkal et al. (2008) reported four new antibacterial C-geranylflavonoids (362-365) from Paulownia tomentosa Fruits. The investigators determined antibacterial activity of these four compounds against six bacterial strains such as B. cereus, B. subtilis, Enterococcus faecalis, Listeria monocytogenes, S. aureus and S. epidermidis. The investigators found that compound 362 showed moderate activity, inhibiting only four (Viz. B. cereus, Listeria monocytogenes, S. aureus and S. epidermidis) out of six Gram-positive bacteria with MIC values in the range 16–32 µg/mL. While other three compounds 363-365 were active against all Gram positive bacteria, found with MIC ranging from 2 to 4 µg/mL. Smejkal et al. (2008) also suggested that the presence of 3¢-methoxy-4¢,5¢-dihydroxy and 3¢,5¢-methoxy-4-hydroxy substitution at ring B of flavonoids increases antibacterial activity on the basis of their study. They also suggested that this is due to the increase in planarity of the flavonoid molecules (Šmejkal et al., 2008). Acetylated flavanone glycosides 366-371, from the rhizomes of Cyclosorus acuminatus, showed weak inhibitory activity against S. aureus and E. coli and moderate activity against S. pneumoniae and Haemophilus influenzae with MIC values in the range of 31-128 mg/mL as reported by Fang et al. (2006). Wirasathien et al. (2006) reported that the chalcone 400 isolated from Ellipeiopsis cherrevensis, exhibited anti-microbacterial activity against Mycobacterium tuberculosis with a MIC of 25 mg/ mL. Helichrysone A (401), the bioactive flavonoid isolates of Helichrysum forskahlii, was assessed for anti-bacterial efficacy by Al-Rehaily et al. (2008). The chalcone 401 exhibited weak activity against B. subtilis and S. aureus, respectively with MIC values of 100 mg/mL (Al-Rehaily et al., 2008). Lall et al. (2006) reported that two chalcone derivative 402 and 403, isolated from Helichrysum melanacme, possess anti-tuberculous activity with MIC value of 0.05 mg/mL for both of them. Chokchaisiri et al. (2009) reported that the compound, dihydromonospermoside (414) isolated from the flowers of Butea monosperma, possesess antimycobacterial activity with MIC value 50 mg/mL. The chalcones 417 and 418 are found to be antifungal; the later compound 418 is fungicidal rather than fungistatic in nature (Svetaz et al., 2007). The sesquiterpene coumarin (456), isolated from Ferula sinaica, showed strong anti-bacterial activity against Gram-positive strain, particularly B. cereus (El Bassuony, 2007). A new coumarin compound, pavietin 465, has been isolated from the ethanol extract of Aesculus pavia, which was found to be as lead compound for fungal protection of its source. The investigoter suggested that the isolated compound 465 was found to be selective antifungal agent against Guignardia aesculi (Curir et al., 2007). The coumaroyl glycoside, asphodelin A 4¢-O-b-D-glucoside (466), isolated from Asphodelus microcarpus exhibited moderate antibacterial activity against S. aureus, E. coli and Pseudomonas aeruginosa and low antifungal activity against C. albicans and Botrytis cinerea (El-Seedi, 2007). Das et al. (2009) isolated homoisoflavonoid 469 from aerial parts of the plant Caesalpinia pulcherrima; the compound 469 showed moderate activity with inhibition zone measured in 5-10 mm against the Gram-positive organisms, B. subtilis, B. sphaericus and S. aureus. The antifungal activity of 469 was also found to be moderate against the organisms, A. niger and C. albicans, but it was inactive

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Natural Bioactive Molecules: Impacts and Prospects

against Rhyzopus oryzae (Das et al., 2009). Chemical investigation of the methanolic extract of the leaflets of Cycas circinalis led to the isolation of one new biflavonoid 475, which showed moderate antibacterial activity against S. aureus (IC50 value of 3.9 mM) and methicillin-resistant S. aureus (MRSA; IC50 value of 5.9 mM) as assessed by Moawad et al. (2010). Kaempferol-7-O-(2≤-E-p-coumaroyl)-a-L-arabinofuranoside (172) isoalted from Picea neoveitchii, showed strong anti-fungal activity against Fusarium oxysporum (Song et al., 2011). Cambodianins D (112) and E (509), the two new flavane constituents of dragon’s blood of Dracaena cambodiana showed antibacterial activity against Staphylococcus aureus (SA) and methicillin-resistant S. aureus (MRSA) (Chen et al., 2012). Two unusual substituted acylated flavonol glycosides, elatoside A (201) and elatoside B (202) have been isolated from the ethanolic extract of Epimedium elatum and were evaluated to exhibit modest antimicrobial and PPAR-g ligand binding activity (Tantry et al., 2012a). The new isopropenyl-dihydrofuranoisoflavones, lachnoisoflavones A (263) and B (264) isolated from Crotalaria lachnophora, also showed moderate inhibitory activities against Escherichia coli and Klebsiella pneumoniae (Awouafack et al., 2011). New prenylated flavonoid (219–223) constituents of the roots of Desmodium caudatum were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activities by a disc diffusion method; the test compounds displayed potent inhibitory activities against MRSA with MIC values ranging from 15.6 to 31.3 mg/mL. Comparison of their antibacterial activities suggested that the presence of the 2,2-dimethyl-2H-pyran ring as well as the hydroxy group at C-20 plays important role(s) for the antibacterial activity (Sasaki et al., 2012). A new flavanone 5-methoxy mundulin (396), isolated from the leaves, stem bark and twigs of Mundulea sericea (Willd.) A. Chev., showed a broad spectrum of antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory quantity (MIQ) of 10 µg (Mazimba et al., 2012). Cambodianin A (117) and cambodianin B (118), the two new flavonoid constituents of dragon’s blood of Dracaena cambodiana were evaluated for their moderate antibacterial activities against Staphylococcus aureus and methicillinresistant Staphylococcus aureus (MRSA) (N-68). The another constituent, 4,4¢-dihydroxy2,3¢-dimethoxydihydrochalcone (454) also showed moderate antibacterial activity against S. aureus (Luo et al., 2011). Platyisoflavanone A (303) exhibited moderate in vitro anti-TB activity against Mycobacterium tuberculosis in the microplate alamar blue assay (MABA) with MIC value of 23.7 mM (Gumula et al., 2012). (2S)-5,7,2¢-Trihydroxy-8-methylflavanone (Pisonivanone; 384) isolate of the methanol extract of the combined stem and root of Pisonia aculeata exhibited in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv with MIC value of 12.5 µg/mL (Wu et al., 2011).

13.3.7

Anti-viral Activity

Cao et al. (2010) isolated three flavonoid compounds 38-40 from Selaginella moellendorffii. Among them 39 and 40 displayed in vitro inhibitory activity on hepatitis B virus (HBV) surface antigen (HBsAg) secretion of the HepG 2.2.15 cell line with IC50 values of 0.17 mg/mL and 0.46 mg/mL, and on HBVe antigen (HBeAg) secretion with IC50 values of 0.42 mg/mL and 0.42 mg/mL, respectively. Two flavan derivatives 94 and 95 of Pithecellobium clypearia also were

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13.87

reported to show antiviral activity — cytopathic effect (CPE) reduction assay revealed that both of the compounds 94 and 95 possess antiviral activity against a number of viral strains such as respiratory syncytial virus (RSV), influenza A (H1N1) virus, Coxsackie B3 (Cox B3) virus and herpes simplex virus type 1 (HSV-1) with respective IC50 values of 5, 15.7, 12.5 and 30 µg/mL for compound 94 and 10, 30, 25 and 20 mg/mL for compound 95 (Li et al., 2006). Two chalcones 402 and 403 constituents of Helichrysum melanacme were evaluated to have inhibitory activity against the replication of influenza A virus with an equal IC50 value of 0.1 mg/mL for both of the compounds (Lall et al., 2006). The new isoflavone, 6-hydroxy-7,3¢,4¢,5¢-tetramethoxy-isoflavone (272) isoalted from the roots and stems of Nicotiana tabacum, exhibited moderate anti-TMV activity (22% inhibition of viral replication) at the concentration of 20 mM tested by the halfleaf method (Chen et al., 2012b). The RNA dependent RNA polymerase (RdRp) domain of the nonstructural protein 5 (NS5) of the dengue virus (DENV) appears as a promising target for new drugs since polymerase activity is essential for viral replication and human host cells are devoid of such RdRp activity (Malet et al., 2008). A series of new mono- and dialkylated flavanones, chartaceones A−F (389−394) were isolated from Cryptocarya chartacea and were evaluated for their antiviral activity against the dengue virus (DENV). The dialkylated flavanones chartaceones C−F (391−394) exhibited the most significant NS5 RdRp inhibiting activity, with IC50 values of 4.2 ± 0.1, 1.8 ± 1.2, 2.9 ± 0.3, 2.4 ± 0.3 mM, respectively (Allard et al., 2011). A new biflavonoid, 4¢-methoxydaphnodorin E (502) isolated from the antiviral fraction of Wikstroemia indica against respiratory syncytial virus (RSV) was tested for its in vitro anti-RSV activity with cytopathic effect (CPE) reduction assay. The test compound displayed potent effect with 50% inhibitory concentration (IC50) value of 2.8 mM and selective index (SI) value of 5.4 (Huang et al., 2012). Pedunculosumosides A (57) and C (58) were found to exhibit modest activity of blocking HBsAg secretion with IC50 values of 238.0 and 70.5 mM, respectively (Wan et al., 2011).

13.3.8

Anti-HIV Activity

Feng and his group (2010a) isolated three new prenylated flavonoids 35-37 from the stem bark of Poncirus trifoliate and evaluated for their anti-human immunodeficiency virus-1 (HIV-1) activity; compound 36 showed significant anti-HIV-1 activity (EC50, 0.35 mg/mL; CC50, 50.28 mg/mL) with high therapeutic index (TI) of 143.65. Reutrakul et al. (2007) isolated four bioactive flavonoids 153-154 and 212-213 from Ochna integerrima, which showed strong HIV-1 activities in the syncytium assay using the MC99 virus and the 1A2 cell line system with EC50 values ranging from 14.0−102.4 mg/mL; the investigators also suggested that the flavonoids 153-154 and 212213 with an isoprenyl and sugar groups on ring A showed significant anti-HIV activity (Reutrakul et al., 2007). Two flavan-chalcone dimers 471 and 472, isolated from whole plants of Sarcandra hainanensis, were found to exhibit weak HIV-1 integrase inhibitory activity with IC50 at 18.05 and 25.27 mM, respectively (Cao et al., 2009). Ophioglonin (490), a homoflavonoid isolated from the plant Ophioglossum petiolatum, showed slight anti-hepatitis B virus (HBV) surface antigen secretion at 25 mM using the MS-G2 hepatoma cell line as reported by Lin et al., (2005a). Two new epicatechin derivatives, malaferin B (113) and malaferin C (114) isolated from Malania oleifera were also evaluated for anti-HIV activities (Wu et al., 2012).

13.88 13.3.9

Natural Bioactive Molecules: Impacts and Prospects

Anti-leishmaniasis Activity

Salem and Werbovetz (2006) reported that the isoflavone 239 isolated from Psorothamnus arborescens displayed leishmanicidal activity with IC50 value of 13.0 mM against Leishmania donovani axenic amastigotes. The chalcone 416 also exhibited leishmanicidal (IC50 7.5 mg/mL) and trypanocidal (IC50 6.8 mg/mL) properties (Salem and Werboveta 2005); furthermore, it was found to reduce the number of infected macrophages by at least 96% with no toxicity to the host cell treated with Leishmania mexicana-preinfected macrophages. Mbwambo et al. (2006) observed that the biflavanoid 473 isolated from Garcinia livingstonei possesses mild inhibitory activity against P. falciparum with IC50 value 6.0 mM.

13.3.10

Antiplasmodial Activity

Two new prenylated flavonoids, styracifolins A (27) and B (28), isolated from the stem bark of Artocarpus styracifolius (Moraceae), exhibited antiplasmodial activity against chloroquine-resistant strain of Plasmodium falciparum; the compound 28 showed relatively stronger activity with IC50 value 1.12 ± 0.08 mM than compound 27 (IC50 5.7 ± 0.8 mM) (Bourjot et al., 2010). Dhooghe and his group evaluated in vitro antiplasmodial activity of six new flavonoid constituents (290, 491495) of Ormocarpum kirkii in an integrated antiparasitic screening panel including Trypanosoma cruzi, Leishmania infantum, Trypanosoma brucei, chloroquine-resistant Plasmodium falciparum K1, Staphylococcus aureus, Candida albicans, Trichophyton rubrum, and Aspergillus fumigatus (Dhooghe et al., 2010). The isoflavanone 290 showed a non-selective activity against S. aureus (IC50 6.4 ± 3.1 mM) and T. rubrum (IC50 6.0 ± 2.1 mM), while compound 491 showed moderate but non-selective activity and it was also cytotoxic to MRC-5 cells. Compound 492 was found to be selectively against T. rubrum (IC50 7.0 ± 6.4 mM) with only low cytotoxicity (CC50 50.2 ± 16.3 mM). Compound 493 was found to be active only against T. rubrum with an IC50 of 19.5 ± 13.9 mM. However, 5,5≤-Di-O-methyldiphysin (494) showed moderate activity against almost all parasites non-selectively with a high cytotoxicity (CC50 11.7 ± 6.2 mM); in contrast, no activity was observed for the mono-glucosylated diphysin 495 (Dhooghe et al., 2010). A chalcone 400, isolated from Ellipeiopsis cherrevensis, was found to show antimalarial activity against P. falciparum with an IC50 value of 7.1 mg/mL (Wirasathien et al., 2006). Four new dihydrochalcones 435438 isolated from the leaves of Piper hostmannianu were assessed against both chloroquineresistant (FcB1) and chloroquine-sensitive (F32) strains of P. falciparum by Portet and his group (Portet et al., 2007). The compounds 435-438 exhibited antiplasmodial activity with IC50 values in the range of 60.67−125.23 mM for FcB1 strains and 55.44−101.27 mM for F32 strains of the parasite. IC50 values of the compounds (435-438) for cytotoxicity against MCF7 human cells were found to show in the range 207.12−242.64 mM (Portet et al., 2007). Mucusisoflavone C (505), a new isoflavone dimer from the figs of Ficus mucuso, exhibited a weak inhibitory activity against in vitro Plasmodium falciparum enoyl-ACP reductase (PfENR) enzyme with an IC50 value of 7.69 mM (89.1% inhibition at a concentration of 0.025 mM) (Bankeu et al., 2011). Terpurinflavone (77), a new prenylated flavone from the stem extract of Tephrosia purpurea, also showed promising in vitro antiplasmodial activity against the D6 (chloroquinesensitive) and W2 (chloroquine-resistant) strains of Plasmodium falciparum with IC50 values of 3.12 ± 0.28 and 6.26 ± 2.66 mM (Juma et al., 2011).

Brahmachari: Natural Bioactive Flavonoids

13.3.11

13.89

Proliferative Activity

The flavonol constituent 133 from Euphorbia lunulata showed in vitro proliferative activity against insulin-dependent cell lines (BAF/InsR and BAF/IL10R), and the promising results indicated that the test compound may be the lead compound for the development of a nonpeptidyl insulin substitutional medicine (Nishimura et al., 2005). Wang et al. (2007) reported that two flavonoids 162 and 356, isolated from the rhizomes of Drynaria fortune, also possess the proliferative effects on UMR106 cells; the flavonoid 162 potently stimulated the proliferation of UMR106 cells by 35.9% and 42.6% at concentrations of 10–8 M and 10–6 M, respectively, and the efficacy was found to be greater than 356 (Wang et al., 2007).

13.3.12

Antidiabetic Activity

A flavone xylopyranoside, 4¢,5-Dihyroxy-6,7-dimethoxyflavone-3-O-b-D-xylopyranoside (8), isolated from the roots of Euphorbia leucophylla by Satyanarayana et al. (2006), was found to have significant blood glucose lowering efficacy thereby enhancing the serum insulin levels in normal and diabetic rats. The flavone luteolin 6-C-(6≤-O-trans-caffeoylglucoside) (18) isolated from Phyllostachys nigra showed advanced glycation end products (AGEs) inhibitory effects. As a result, this compound could be offered as a lead for further study in the field of diabetic complications (Jung et al., 2007). Jang et al. (2009) reported that two flavan-3-ols 89 and 90 from Actinidia arguta possess in vitro inhibitory activity on the formation of advanced glycation end products (AGEs) with respective IC50 values of 13.5 and 17.9 mg/mL, respectively (Jang et al., 2009). Two dihydroflavonol glycosides 207 and 208, isolated from the leaves of Stelechocarpus cauliflorus, were also found to inhibit the formation of AGEs and displayed therapeutic potential in the prevention and treatment of diabetic complications (Wirasathien et al., 2007). The isoflavone C-glucoside constituents 252 and 253 of Pueraria iobata were also evaluated to have potent in vitro inhibitory activity against AGEs formation with IC50 values 8.7 and 24.9 mg/mL, respectively (Kim et al., 2006). The investigators suggested that the compound 252 might be worthy of consideration as a therapeutic agent for diabetic complications or related diseases (Kim et al., 2006). Park et al. (2010) reported that the two new dihydrofuranoisoflavanones 286 and 287, were isolated from an n-BuOH soluble fraction from the leaves of Lespedeza maximowiczi. All of the isolates 286 and 287 were evaluated in vitro for their inhibitory activity on the formation of AGEs. Among these, compounds 286 and 287 exhibited such inhibitory activity against AGEs formation with IC50 values of 20.6, and 18.4 mM, respectively (Park et al., 2010). Five homoisoflavonoids 294298, isolated from the EtOAc-soluble fraction of a 90% MeOH extract of the fibrous roots of Polygonatum odoratum, showed effects of sensitizing adipocytes for insulin in a cell-based glucose uptake assay using 3T3-L1 adipocytes compared with the treatment of insulin of 100 nM (Zhang et al., 2010a). Zhang et al. (2010a) suggested that especially, compound 294 and its enantiomer 295 exhibited the most potent glucose uptake estimulatory activity at the concentration of 1 mM. Preliminary structure-activity relationship analysis indicated that compounds with an additional hydroxy group at C-2¢ or C-3¢ (compounds 294, 295, and 296) showed much stronger activities than compounds with only one hydroxy or methoxy group at C-4¢. The results indicate that homoisoflavonoids may be potential insulin sensitizers for the treatment of diabetes (Zhang et al.,

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2010a). Cui and his groups investigated that the insulin and leptin signaling pathway can be regulated by protein tyrosine phosphatase 1B (PTP1B), it has been suggested that compounds that reduce PTP1B activity or expression levels could be used for treating type 2 diabetes and obesity (Cui et al., 2010). In screening efforts by the groups on new PTP1B inhibitors, four new bioactive flavanones 337-340 with dihydrofuran moiety were isolated from the stem bark of Erythrina abyssinica. All the compounds 337-340 inhibited PTP1B activity in an in vitro assay with IC50 values ranging from 15.2 ± 1.2 to 19.6 ± 2.3 mM, whereas RK-682 (positive control) displayed an IC50 value of 4.7 ± 0.5 mM (Cui et al., 2010). One flavonone derivative (351), isolated from the organic extracts of Viscum album, was found to exhibit anti-glycation activity with IC50 value of 264.5 ± 0.9 mM (i.e. % inhibition 74.5) (Choudhary et al., 2010). Yoo et al. (2008) isolated the 2,3-dioxygenated flavanone erigeroflavanone 468 from the flowers of Erigeron annuus, which was found to exhibit potent inhibitory activity against AGEs formation with IC50 value 22.7 mM. Dong et al. (2010) isolated three homoisoflavanones, 3-(4¢-hydroxybenzyl)-5,7-dihydroxy-6-methyl-8methoxychroman-4-one (486), 3-(4¢-hydroxybenzyl)-5,7-dihydroxy-6,8-dimethylchroman-4-one (487), and 3-(4¢-methoxybenzyl)-5,7-dihydroxy-6-methyl-8-methoxychroman-4-one (488) from the active CHCl3-soluble fraction of the EtOH extract of rhizomes of Polygonatum odoratum, and evaluated their in vitro inhibitory activities against AGE formation. All the isolates 486488 exhibited much stronger inhibition of AGE formation with respective IC50 value of 56.30, 46.05 and 107.10 mM and the activity was found to be greater than that of aminoguanidine (IC50 123.48 mM), a well-known glycation inhibitor (Dong et al., 2010). 5,7-Dihydroxy-6,8-dimethyl-4¢methoxyflavone (86) and 8-(2-hydroxypropan-2-yl)-5-hydroxy-7-methoxy-6-methyl-4¢-methoxyflavone (87), isolated from the aerial parts of Callistemon lanceolatus, were found to exhibit blood glucose lowering effect in streptozotocin induced diabetic rats (Nazreen et al., 2010). Caryatin-3¢ methyl ether-7-O-b-D-glucoside (192), isolated from the bark of Pecan tree, Carya illinoinensis (Wangenh) K. Koch, was evaluated to show significant hypoglycaemic effect in streptozocin diabetic rats, possibly through induction of pancreatic insulin secretion from b-cell of islets of Langerhans, its antioxidant effect and aldose reductase (AR) inhibitory effect (Abdallah et al., 2011). Hence, the flavonoid glycoside 192 isolated from pecan bark could be a good source for medical foodstuffs and lead compound as alternatives for ARIs currently used.

13.3.13

Adipogenesis Activity

Adipocytes are regarded as a potential target for obesity and type 2 diabetes; adipose tissue, being composed of adipocytes that store energy in the form of triglycerides, is important for the regulation of energy balance (Nawrocki and Scherer, 2005). Adipogenesis is a process from fibroblast-like preadipocytes to mature adipocytes. Adipocyte differentiation follows a defined program, and in the final stage, the differentiated cells show markers of mature adipocytes, such as gene expression of fatty acid-binding protein (aP2) and glucose transporter 4 (GLUT4) and the massive accumulation of triglycerides inside the cells (Kong and Li, 2006). The increased expression of GLUT4 may promote insulin-stimulated glucose uptake in the adipose tissue and skeletal muscles and reduce the peripheral glucose level (Leney and Tavare, 2009). Nigrasin H (62) and I (63) isolated from the twigs of Morus nigra were evaluated to possess significant adipogenesis activity, characterized by

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increased lipid droplet and triglyceride content in 3T3L1 cells, and induced up-regulation of the expression of adipocyte-specific genes, aP2 and GLUT4 — treatment with 62 and 63 of 10 µM induced a significant up-regulation of both the gene expressions documented by RT-PCR analysis (Hu et al., 2011). Triglyceride contents in 3T3L1 cells were measured as 203.39 ± 9.57% and 250.63 ± 29.59%, respectively for 62 and 63 when treated at a dose of 50 mM (rosiglitazone used as reference standard: 450.32 ± 54.32% at 1 mM) (Hu et al., 2011). The isoprenylated flavonoids, dodoviscins A-I (184-190) isolated from the aerial parts of Dodonaea viscose, were also found to exhibit promising adipogenesis activity (Zhang et al., 2012).

13.3.14

Anti-platelet Aggregation Activity

Chang et al. (2010) reported that five bioactive flavonoid constituents (2, 3, 319, 320 and 322) of the ethyl acetate extract of Calamus quiquesetinervius exhibited inhibitory activity at 50 µM on arachidonic acid (AA)- and platelet activity factor (PAF)-induced platelet aggregation of rabbit platelets (table-7). Notably, compounds 2 and 3 are more potent than that of aspirin (standard). It is indicated that the erythro-configuration in both 2 and 319 (P < 0.05) results in more inhibitory effects than the threo-configuration in 3 and 320. Moreover, the substituted chromanone moiety in 322 replaced by a phenylpropanoid moiety as in 2, 3, 319, 320 and 322 enhances the efficiency to some extent (Chang et al., 2010). Table 7 PAF.

Inhibition of isolates at 50 µM on aggregation of washed rabbit platelets induced by AA or Compounds

Inhibition (%) at 50 μM Arachidonic acid [AA] (100 μM)

Platelet activity factor [PAF] (5 nM)

2

69.1 ± 0.8*

14.5 ± 2.5

3

55.6 ± 1.0

4.7 ± 1.9

319

68.1 ± 0.7*

28.0 ± 0.9

320

53.5 ± 1.3

10.0 ± 2.1

322

43.9 ± 2.3

23.3 ± 1.3

*Indicated P < 0.05, which compared between 2 and 3, 319 and 320, respectively

Dihydrochalcone (444), isolated from leaves of Muntingia calabura, was also found to show significant anti-platelet aggregation activity in vitro as assessed using turbidimetric method in washed rabbit platelets induced by thrombin, arachidonic acid, collagen, and platelet-activating factor with % inhibitions 127, 98.2, 94.1 and 88.7 U/mL, respectively, in 100 µg/mL concentration (Chen et al., 2007).

13.3.15

Melanin Synthesis Inhibitory Activity

Skin pigmentations, such as melasma, freckles, and solar lentigo, can be serious aesthetic problems. They result from increased production and accumulation of melanin. Consequently, inhibition of melanin synthesis regarded as a treatment of a variety skin pigmentations. Melanin synthesis inhibitors have been of interest as target molecules of natural product chemistry. Arung and his

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group isolated 3-prenyl luteolin (26) on activity-guided fraction of Artocarpus heterophyllus extract that possesses anti-melanogenesis activity; the compound 26 exhibited inhibitory activity against mushroom tyrosinase with IC50 value of 76.3 mM, while kojic acid used as positive control exhibited inhibitory activity with IC50 value of 14.1 mM. The compound 26 also showed melanin formation inhibitory activity on B16 melanoma cells with IC50 value of 56.7 mM (Arung et al., 2009). Mori-Hongo et al. (2009) reported that nine flavonoids 209, 210, 285, 346, 426, 480, 481, 482 and 483 from aerial parts of Lespedeza buergeri showed strong inhibition of melanin synthesis in normal human epidermal melanocytes (NHEM); the IC50 of each of these compounds was lower than 2 mM, while that of hydroquinone, a positive control, was 2.2 mM. Thus, these compounds were more potent than hydroquinone, a compound widely used as a skin-lightening drug (MoriHongo et al., 2009).

13.3.16

Estrogenic and Anti-estrogenic Activity

Three prenylated isoflavonoids 249-251, isolated from leaves of Millettia pachycarpa, showed potent antiestrogenic activity in dose-dependent manner as evaluated based on the inhibition of b-galactosidase activity induced by 17b-estradiol (E2) in the yeast two-hybrid assay (Ito et al., 2006); IC50 values of the three isoflavonoids 249-251were determined as 29, 18, and 13 mM, respectively. These values were slightly higher than that of 4-hydroxytamoxifen (positive control; IC50 4.4 mM). The flavanonol rhamnoside acetates 214-218 isolated from the Thai medicinal plant, Smilax corbularia were evaluated for their estrogenic and anti-estrogenic activities using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. The test compounds showed estrogenic activity in both MCF-7 and T47D cells at a concentration of 100 mM, and they enhanced the effects of co-treated E2 on T47D cell proliferation at concentrations of more than 0.1 mM (Wungsintaweekul et al., 2011).

13.3.17

Neutrophils Respiratory Burst Inhibitory Activity

Polymorphonuclear neutrophils (PMNs) are important cells involved in the bactericidal host defense system through the respiratory burst. PMNs respiratory burst plays a critical role in the immune-inflammatory processes. Inhibition of neutrophils respiratory burst has been one of the well-documented methods for the evaluation of anti-inflammatory activity for various synthetic compounds and natural products. The flavone glycoside 45 isolated from the leaves of Aquilaria sinensis was found to show significant inhibitory activity against neutrophils respiratory burst stimulated by phorbol 12-myristate 13-acetate (PMA) with IC50 value 61.25 ± 0.21 µmol/L (Qi et al., 2009).

13.3.18

Neuroprotective Activity

An et al. (2008) isolated (2S)-6,7,4¢-trihydroxyflavan (96) and 4,2¢,5¢-trihydroxy-4¢methoxychalcone (419) along with fourteen known flavonoids and two other known arylbenzofurans from the heartwood of Dalbergia odorifera; among them the new compounds 96 and 419 were found to have protective effect on glutamateinduced oxidative injury in HT22 cells with EC50 values of 17.83, and 7.47 mM, respectively (An et al., 2008). Furthermore, the new chalcone

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derivative 419 was more potent than the positive control trolox (EC50 15.8 mM). From these results, the investigators suggested that the compounds 96 and 419 might be lead molecules having neuroprotective activity against oxidative cellular injuries (An et al., 2008). The flavonol derivative, kaempferol 3-O-(2≤-O-galloylrutinoside) (152), exhibited promising neuroprotective effects on ischemic injury model of cultured rat cortical neurons treated with sodium dithionite in glucose-free medium as reported by Liu et al. (2007).

13.3.19

Osteoclast Differentiation Inhibitory Activity

The aerial parts of Cephalotaxus koreana afforded one bioactive flavonoid glycoside 7 which showed strong inhibitory activities against osteoclast differentiation at concentrations of 0.1 (percentage inhibition 63 ± 4.6) and 1.0 mg/mL (% inhibition 86 ± 6.3) as reported by Yoon et al. (2007).

13.3.20

Anthelmintic Activity

Parasitic diseases caused by helminthes lead to significant health hazards to animals resulting in enormous economic impact. The flavone 48, isolated from Struthiola argentea, was found to show the most potent inhibitory anthelmintic activity in vitro with 90% inhibition of larval motility at 3.1 mg/mL (Ayers et al., 2008).

13.3.21

Nematicidal Activity

Two prenylated flavanones 328 and 329, isolated from Phyllanthus niruri plant, showed dosedependent nematicidal activity against Meloidogyne incognita and Rotylenchulus reniformis nematodes. The LC50 calculated after 72 h showed that compound 329 exhibited nematicidal activity much stronger (LC50 of 14.5 ± 0.96 and 3.3 ± 1.13 ppm, respectively) than the standard bionematicide, Aspergillus niger which has LC50 of 48 ppm after 72 h. This was then compared with another standard, carbofuran having LC50 3.1 ppm (Shakil et al., 2008).

13.3.22

Miscellaneous Activities

Bourjot and his group (2010) isolated two new cytotoxic prenylated flavonoids, styracifolins A (27) and B (28) from the stem bark of Artocarpus styracifolius having antitrypanocidal activity; compound 28 showed relatively strong activity against Trypanosoma brucei bruce with IC50 value of 6.9 ± 0.4 mM than compound 27 (IC50 13.3 ± 4.4 mM) (Bourjot et al., 2010). The flavone glycosides scutellarein-7-O-b-D-apiofuranoside (49) and apigenin-7-O-m-D-apiofuranosyl(1Æ2)-b-D-apiofuranoside (50), and the flavone celtidifoline (5,6,4¢,5¢-tetrahydroxy-7,3¢dimethoxyflavone, 51) isolated from leaves of the ethyl acetate extract of Lantana trifolia exhibited sedative effect in mice and affinity for the benzodiazepine receptor with IC50 values of 187, 670, and 440 mM, respectively (de Santana Juliáo et al., 2010). Lu et al. (2010) reported that the new acetylated flavonoid, kaempferide-7-O-(4≤-O-acetyl)-a-L-rhamnoside (119) from Actinidia kolomikta possesses protective effect on human erythrocytes against AAPH-induced hemolysis. A flavonoid glycoside mutabiloside (144) from Hibiscus mutabilis exhibited significant

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allergy-preventive activity (Iwaoka et al., 2009); mutabiloside (144, 20 mg/kg, p.o.) was found to improve also significantly (p