Flavonoid Pharmacokinetics E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 547

Veröffentlichungsjahr: 2012

Beschreibung

SETS FORTH A FRAMEWORK FOR THE ANALYSIS AND STUDY OF FLAVONOIDS More and more dietary supplements contain flavonoids. These products are typically viewed as food rather than drug products by regulatory agencies and therefore not subjected to rigorous clinical trials before they are marketed to the general public. As a result, the use of flavonoid-containing supplements presents a potential public health risk. From discovery to therapeutic application, this book is a comprehensive guide to both achiral and chiral flavonoids, enabling researchers to perform essential preclinical and clinical pharmacokinetics studies in order to ensure the efficacy of flavonoids marketed for therapeutic use. Moreover, the book examines the safety and toxicology of flavonoids as well as flavonoid-drug interactions. With contributions from a multidisciplinary team of leading researchers, Flavonoids Pharmacokinetics reviews and synthesizes the most recent research findings and results from preclinical and clinical studies. The book begins with a comprehensive overview of polyphenols and flavonoids. Next, the book covers: * Methods of analysis of achiral flavonoids * Preclinical pharmacokinetic of flavonoids * Toxicology and safety of flavonoids * Methods of analysis for chiral flavonoids * Clinical pharmacokinetics of flavonoids * Flavonoids and drug interactions Throughout the book, the authors provide examples that demonstrate the use of pharmacokinetics concepts during the preclinical and clinical drug development process. Flavonoid Pharmacokinetics is written for pharmaceutical, food, and nutritional scientists and students, offering the tools they need to thoroughly analyze and test flavonoids and flavonoid-containing supplements to ensure their safety and efficacy.

Leseprobe

Table of Contents

COVER

TITLE PAGE

COPYRIGHT PAGE

DEDICATION

FOREWORD

PREFACE

CONTRIBUTORS

1 POLYPHENOLS AND FLAVONOIDS: AN OVERVIEW

1.1 INTRODUCTION

1.2 SYNTHESIS

1.3 SOURCES

1.4 PHARMACOLOGICAL ACTIVITIES OF SELECTED FLAVONOIDS

1.5 CONCLUSIONS

REFERENCES

2 ANALYSIS OF FLAVONOIDS THROUGH CHROMATOGRAPHY

2.1 INTRODUCTION

2.2 POLYPHENOLS

2.3 CHROMATOGRAPHY

2.4 DETECTION

2.5 METHOD DEVELOPMENT

2.6 VALIDATION

2.7 SUMMARY

2.8 FUTURE DIRECTIONS

2.9 CONCLUSIONS

REFERENCES

3 CHIRAL METHODS OF FLAVONOID ANALYSIS

3.1 INTRODUCTION

3.2 FLAVONOIDS AND CHIRALITY

3.3 SEPARATION OF ENANTIOMERS THROUGH CHROMATOGRAPHY

3.4 ENANTIOMERIZATION AND RACEMIZATION

3.5 CURRENT METHODS: PROS AND CONS

3.6 FLAVANONES

3.7 CATECHINS

3.8 CONCLUSIONS

REFERENCES

4 PRECLINICAL PHARMACOKINETICS OF FLAVONOIDS

4.1 INTRODUCTION

4.2 ABSORPTION

4.3 DISTRIBUTION

4.4 METABOLISM

4.5 EXCRETION

4.6 CONCLUSIONS

REFERENCES

5 CLINICAL PHARMACOKINETICS OF FLAVONOIDS

5.1 INTRODUCTION

5.2 METHODS OF ANALYSIS

5.3 FLAVONOID PHARMACOKINETICS

5.4 DRUG INTERACTIONS

5.5 CONCLUSIONS

REFERENCES

6 TOXICOLOGY AND SAFETY OF FLAVONOIDS

6.1 INTRODUCTION

6.2 FLAVONOIDS AND THE GASTROINTESTINAL TRACT

6.3 FLAVONOIDS AND HEPATIC SIDE EFFECTS

6.4 FLAVONOIDS AND THE KIDNEY

6.5 FLAVONOIDS AND BLOOD DISORDERS

6.6 FLAVONOIDS AND CANCER

6.7 FLAVONOIDS AND THE ENDOCRINE SYSTEM

6.8 FLAVONOIDS AND OCULAR SAFETY

6.9 FLAVONOIDS AND THE CENTRAL NERVOUS SYSTEM

6.10 ALLERGY-LIKE RESPONSES AND FLAVONOIDS

6.11 FLAVONOIDS AND PREGNANCY

6.12 OVERVIEW AND CONCLUSIONS

REFERENCES

7 FLAVONOIDS AND DRUG INTERACTIONS

7.1 INTRODUCTION

7.2 FLAVONOID–DRUG INTERACTIONS MEDIATED BY FOOD, BEVERAGE, AND HERBAL INTAKE

7.3 BEVERAGE INTAKE

7.4 FOOD INTAKE

7.5 FLAVONOID–DRUG INTERACTIONS

7.6 FLAVONOID–DRUG INTERACTIONS MEDIATED BY TRANSPORTERS

7.7 EXPERIMENTAL TECHNIQUES TO STUDY FLAVONOID–ABC TRANSPORTER INTERACTIONS

7.8 P-GLYCOPROTEIN (P-GP, ABCB1)

7.9 INTERACTION BETWEEN P-GLYCOPROTEIN AND CYP450

7.10 MULTIDRUG RESISTANCE PROTEIN 2 (MRP2, ABCC2)

7.11 BREAST CANCER RESISTANCE PROTEIN (BCRP, ABCG2)

7.12 FLAVONOID MODULATION OF ABC TRANSPORTER EXPRESSION

7.13 ORGANIC ANION TRANSPORTERS (OATS)

7.14 CYTOCHROME P450

7.15 CONCLUSIONS

REFERENCES

INDEX

Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data is available.

ISBN: 9780470578711

Dedicated to my wife Claudia Davies

and daughters Cassandra, Daniela, and Catalina,

who have encouraged and supported me throughout the journey.

—Neal M. Davies

Dedicated to my mother Lillian Emperatriz Farfán Azpilcueta

and my grandmother Hilda Regina Farfán Aspilcueta,

whose inspirations live on with this book,

and to all my family for their

unconditional love, encouragement,

and understanding.

—Jaime A. Yáñez

FOREWORD

Natural products have been used for hundreds and even thousands of years as food products and for therapeutic benefit. Even today a large part of the world’s population relies on plants for their well-being. What at first may seem surprising is that natural products continue to be popular in developed countries, including the US, Canada, Australia, the UK, and Europe. However, this becomes understandable when one considers the factors that have led to the interest in and the continuing development of natural products in the marketplace. Among these are concerns associated with development of pharmaceuticals, particularly the increasing cost of maintaining a pipeline and bringing the small number of successful molecules to market. Developed societies continue to look toward natural products for many reasons, including a desire to maintain more control of their health care into older age, particularly given the greater awareness of side effects that have become apparent with some recently introduced drugs. Despite the explosion of biotechnology, the pharmaceutical industry continues to utilize natural products for small molecule drug discovery, and over half of small drug molecules available today continue to have their origins in natural products. The science behind natural products thus continues to be important and, indeed, essential if such agents are to continue to be used safely and effectively and as sources of new discoveries and therapies.

Polyphenols are being recognized more and more as important components of plant natural products. There are some striking examples of the importance of such agents in health care, including components of green tea, red wine, and chocolate for cardiovascular disease protection and as adjunctive cancer management. There are thousands of such compounds present in plants, and to date we have only just begun to identify a relatively small percentage of such molecules from a small percentage of plants that have been screened from nature’s bounty. Among the polyphenols are the group of molecules known as flavonoids. While thousands of flavonoids have been identified many more remain unknown and undiscovered. The potential for understanding the scientific basis of traditional medicine and for developing new therapies based on flavonoids remains enormous.

Advances in natural products must be based on the use of a multitude of techniques and practices. Greater application of research methodologies is key to such development. Only through understanding the structure and function relationships of molecules such as the flavonoids can we hope to apply what is commonly described as “reverse pharmacology”—that is, the discovery and improvement of therapies starting from traditional knowledge built over many generations in many cultures and then designing chemical, pharmacological, and clinical studies to validate and extend the value and understanding of such therapies in the context of today’s requirements for evidence-based therapeutic approaches.

This book covers the fundamental techniques that can be applied to natural product research and describes the science and methodology behind these techniques. It then provides extensive examples of the outcomes from applications of these techniques. Before the new chemical entities can be described, a variety of experimental methods of isolation, separation, and identification are needed—a complex process in natural medicines where one expects multi-component and multi-target actions underpinning clinical effectiveness. Furthermore, such understanding of the chemistry of flavonoids is essential if new molecules can be developed to overcome normal limitations of natural products and, hence, to provide wider application in modern therapy. Principles that are common in pharmaceutical research, such as identifying metabolism and describing the role of chirality of natural products, are demonstrated in this scientific evaluation; and flavonoids provide excellent examples of this. Only certain molecules have a primary role in many herbal medicines, while other components support the clinical effectiveness of the herb (such as moderating absorption and reducing toxic effects). The molecules responsible for all these attributes are often unknown. As an example, willow bark is used for arthritis and pain; yet, while it is known that salicyn is a major component and a source of salicylic acid, it has become evident that the levels of salicylate produced in the body are insufficient to explain the clinical results. Hence, one must search for other perhaps minor, but potent, components which account for the efficacy of willow bark. These may well be flavonoids and other components known to be also present in the preparations, given the wide range of pharmacological actions that have been described for such compounds.

A major limitation of herbal natural product medicines for current therapeutic applications is the bioavailability of their various components. Thus the effects obtained with natural medicines are generally slow to develop, often require high concentrations, and generally produce milder effects compared to, say, analgesics like paracetamol or aspirin (as is the case in the willow bark example above). Understanding metabolism and pharmacokinetics of the natural product components is key to understanding and ultimately improving the effectiveness of natural products, while still maintaining the benefits of their lower toxicity. This is an emerging and less-researched area of natural medicines, and the authors of this book are world experts in this specialized pharmaceutical area. They provide an excellent rationale for the experimental methods required in undertaking pharmacokinetic experiments, including the ADME parameters of absorption, distribution, metabolism, and excretion. They follow this up with a comprehensive list of examples of pharmacokinetic studies that have been undertaken preclinically and clinically in the flavonoid area. This is a major and important contribution of this book. Such studies also lead to the appropriate use of natural products, particularly with respect to the potential for interactions, both positive and negative, between herbal natural medicines and pharmaceutical drugs and other foods and supplements. In my own research, we have undertaken development of an herb–drug interaction database on such interactions; and this book provides an excellent source for studies of such interactions.

Overall, natural product state-of-the-art research is continuing to grow; and the need for more sophisticated research is growing. Flavonoids is an important class of molecules in natural medicines and various other complementary medicine products which, while appreciated for a long time, rely on state-of-the-art methodology for their understanding and for new discoveries. Having a source of information on underpinning scientific methodology and extensively documented natural product research outcomes, as provided by this book, will be invaluable for all those interested in this area or wanting to gain a greater appreciation of the potential of this approach.

BASIL D. ROUFOGALIS, PhD, DScProfessor EmeritusUniversity of Sydney

PREFACE

There has been an increase in pharmaceutical and biomedical therapeutic interest in natural products as reflected in the sales of nutraceuticals and functional foods and in the global therapeutic use of traditional medicines over the last decade. The use of traditional medicines is based on knowledge, skills, and practices founded on experiences and theories from different cultures. Traditional medicines are used to prevent and maintain health, which may ultimately improve and/or treat physical and mental illnesses. The present day use of these products encompasses almost every aspect of our daily lives from health and beauty, dietary supplements, performance enhancement supplements, and food and beverage to overall health and well-being products. Over the last 30 years, scientific investigations have illustrated the therapeutic bioactivity of flavonoids in chronic disease studies and have piqued the interests of scientists from the diverse fields of nutrition, food, horticulture, and pharmaceutical sciences. Additionally, increased interest by nutraceutical manufacturers has created an abundance of flavonoid-containing dietary supplements on the market. These products and others like them are consumed by a large percentage of the Western population. Since dietary supplements are viewed by most regulatory agencies as food rather than drugs, many of these products are produced without having passed standards of safety or efficacy. The use of flavonoid-containing nutraceuticals presents a potential public health risk that could be ameliorated by flavonoid-specific research generated from a variety of fields. Hence, the objective of this book is to provide the framework for fundamental concepts and contemporary practice of methods of analysis for achiral and chiral flavonoids, preclinical and clinical pharmacokinetics, as well as toxicology and safety of flavonoids and their possible drug interactions.

It is our belief that this book provides the basic concepts to a novice graduate student and the advanced knowledge to a veteran pharmaceutical, food, or nutrition scientist. Chapter 1 provides a comprehensive overview of polyphenols and flavonoids. The methods of analysis of achiral flavonoids using chromatography are covered in Chapter 2, while methods of analysis for chiral flavonoids are described in Chapter 3. Chapters 4 and 5 present the advanced concepts of preclinical and clinical pharmacokinetics of flavonoids, respectively. The toxicology and safety of flavonoids is presented in Chapter 6, while the reported flavonoid–drug interactions are detailed in Chapter 7. The various topics of this book can be adapted by scientists to their specific research needs.

This book contains diverse topics that required a multidisciplinary effort, which would not have been possible without the great efforts of our contributors. We really appreciate the expertise, willingness, and patience of our contributors during the completion process of this book project. We would like to express our sincere thanks to Mr. Jonathan Rose for his support, patience, and confidence in us. We would also like to express our appreciation to our families and colleagues for their support and encouragement. Finally, we would like to thank Professor Basil Roufogalis, an innovator and world leader in herbal medicine research and education, for writing such an inspiring foreword for this book.

NEAL M. DAVIES, PhDJAIME A. YÁÑEZ, PhD

CONTRIBUTORS

Preston K. Andrews, Department of Horticulture and Landscape Architecture, Washington State University, Pullman, WA, USA

Nagendra V. Chemuturi, Drug Metabolism and Pharmacokinetics, Alcon Research, Ltd., a Novartis Company, Fort Worth, TX, USA

Neal M. Davies, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada

Karen D. Gerde, College of Pharmacy, Department of Pharmaceutical Sciences, Washington State University, Pullman, WA, USA

Stephanie E. Martinez, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada

Connie M. Remsberg, Division of Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, CA, USA

Jonathan K. Reynolds, College of Pharmacy, Department of Pharmacotherapy, Washington State University, Pullman, WA, USA

Casey L. Sayre, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada

Jody K. Takemoto, John A. Burns School of Medicine, Department of Cell and Molecular Biology, University of Hawaii, Honolulu, HI, USA

Karina R. Vega-Villa, College of Pharmacy, The University of Oklahoma, Oklahoma City, OK, USA

Scott W. Womble, Drug Metabolism and Pharmacokinetics, Alcon Research, Ltd., a Novartis Company, Fort Worth, TX, USA

Jaime A. Yáñez, Drug Metabolism and Pharmacokinetics, Alcon Research, Ltd., a Novartis Company, Fort Worth, TX, USA

1

POLYPHENOLS AND FLAVONOIDS: AN OVERVIEW

Jaime A. Yáñez, Connie M. Remsberg, Jody K. Takemoto, Karina R. Vega-Villa, Preston K. Andrews, Casey L. Sayre, Stephanie E. Martinez, and Neal M. Davies

1.1 INTRODUCTION

There has been an increase in pharmaceutical and biomedical therapeutic interest in natural products as reflected in sales of nutraceuticals and in the global therapeutic use of traditional medicines.1–9 Use of traditional medicines is based on knowledge, skills, and practices based on experiences and theories from different cultures that are used to prevent and maintain health, which may ultimately improve, and/or to treat physical and mental illnesses.10 The popularity of these products encompasses almost every aspect of our daily lives from health and beauty, dietary supplements, performance enhancement supplements, food and beverage to overall health and well-being products.1 It is apparent that this growing demand for phytotherapies could be very profitable for nutraceutical and pharmaceutical companies. Nutraceutical as well as pharmaceutical companies are interested in many of these naturally occurring compounds that can be extracted from plants and be further modified, synthesized, formulated, manufactured, marketed, and sold for their reported health benefits. Pharmaceutical companies are also using these natural compounds as lead drug candidates that can be modified and formulated to be potential new drug candidates. From drug discovery and development to marketing, between 15 and 20 years may lapse with billions of dollars spent on drug development and research of pharmaceuticals.11,12 Consumers are looking for beneficial health-related products that have efficacy at a low cost to the consumer, while the nutraceutical industry is struggling to develop therapies at a low cost and to bring them to the market. Through scientific studies, natural products can be scrutinized using pharmaceutical approaches to develop and provide alternative or adjunctive therapies.

The drug discovery process is expensive and time-consuming. It has been estimated to take 10–15 years and $800 million to get a drug to the approval process.13 Part of this cost is due to advances in technology whereby drug manufacturers have adopted a target-based discovery paradigm with high throughput screening of compound libraries. This approach, although expected to have vast potential, has not necessarily proven itself. Reviews of new chemical entities have shown that natural products or derivatives of natural products are still the majority of newly developed drugs. For instance, 63% of the 974 new small molecule chemical entities developed between 1981 and 2006 were directly isolated from nature or semisynthetic derivatives of a natural product.14 This trend continues even into this century where approximately 50% of new small molecule chemical entities approved from 2000 to 2006 have a natural origin.14 It is apparent that natural products are important compounds to be explored in the drug discovery process. More importantly, however, there remains a multitude of bioactive compounds yet to be systematically characterized. It is estimated that of the 250,000–750,000 higher plant species, only 10–15% have been screened for potential therapeutic agents.15 Characterizing bioactive molecules in microbial and marine life is even more limited. Nonetheless, natural products remain a reservoir of potential therapeutic agents.

It has been reported that 5000–10,000 compounds are screened before a single drug makes it to the market, and on average, it takes 10–15 years to develop a single drug.16 Of the successfully developed drugs, 60% have a natural origin, either as modified or unmodified drug entities, or as a model for synthetic drugs—not all of them used for human diseases—and it is estimated that 5–15% of the approximately 250,000–750,000 species of higher plants have been systematically screened for bioactive compounds.15 Structure–activity relationship (SAR) programs are generally employed to improve the chances of phytochemicals being developed as drug entities.17 Further studies to develop more drugs of natural origin have been limited in part due to their structural complexity, which is sometimes incompatible with high throughput formats of drug discovery and high extraction costs.16 The potentially long resupply time and unforeseen political reasons such as warfare in developing nations also limit the development of plant-based drugs.17 As a result, plants remain and represent a virtually untouched reservoir of potential novel compounds. Nevertheless, the number of drugs developed each year based on natural products has remained constant over the last 22 years.17

A class of molecules with well-documented therapeutic potential is the polyphenols. Polyphenols are small molecular weight (MW) compounds (MW 200–400 g/mol) that occur naturally. They are produced as secondary metabolites that serve to protect the plant from bombardment of pathogens and ultraviolet (UV) radiation. Upon environmental threat, the plant host activates one of the synthesis pathways and polyphenol structures are produced and subsequently secreted.18 Which specific polyphenol is produced depends largely on its host, the region of origin, and the environmental stimuli. Many polyphenols are synthesized by the phenylpropanoid pathway. Several classes of polyphenols exist including flavonoids, stilbenes, isoflavonoids, and lignans. Polyphenols of all classes are found in a wide range of plants and plant by-products such as herbal supplements and beauty products.

1.2 SYNTHESIS

An understanding of the biosynthesis of natural compounds will enable researchers to further investigate possible therapeutic uses based on the activity of phytochemicals in plants. Plant chemicals are often given the moniker “phytochemicals” and can be classified either as primary or secondary metabolites.19 Primary metabolites are widely distributed in nature and are needed for physiological development in plants. On the other hand, secondary metabolites are derived from the primary metabolites, are limited in distribution in the plant kingdom, and are restricted to a particular taxonomic group (Fig. 1.1). Secondary metabolites usually play an ecological role; for example, they act as pollinator attractants, are involved in chemical defense, are often end products from chemical adaptations to environmental stresses, or are synthesized in specialized cell types at different developmental stages of plant development or during disease or are induced by sunlight.19

Figure 1.1. Biosynthetic origin of some plant-derived compounds. Major groups of secondary metabolites are indicated by ovals.

Allelochemicals are phytotoxic compounds produced by higher plants that include flavonoids. Like other secondary metabolites, flavonoids have complex structures where multiple chiral centers are common.19 Flavonoids consist of a C15 unit with two benzene rings A and B connected by a three-carbon chain (Fig. 1.2). This chain is closed in most flavonoids, forming the heterocyclic ring C; however, chalcones and dihydrochalcones present as an open ring system.20 Depending on the oxidation state of the C ring and on the connection of the B ring to the C ring,21 flavonoids can be classified into various subclasses. Flavonoids can undergo hydroxylation, methylation, glycosylation, acylation, prenylation, and sulfonation; these basic chemical metabolic substitutions generate the different subclasses: flavanols, flavanones, flavones, isoflavones, flavonols, dihydroflavonols, and anthocyanidins.20,21 Flavonoids in nature are naturally most often found as glycosides and other conjugates; likewise, many flavonoids are polymerized by plants themselves or as a result of food processing.21

Figure 1.2. Basic chemical structure and numbering pattern of flavonoids.

1.2.1 Synthesis of Flavonoids

In plants, primary metabolites such as sugar are associated with basic life functions including, but not limited to, cell division, growth, and reproduction.22 On the other hand secondary metabolites are involved in the adaptive necessity of plants to their environments, such as pigmentation, defense from toxins, and enzyme inhibition;23–25 additionally, these secondary metabolites can have pathogenic or symbiotic effects.26 Secondary metabolites including polyphenols have been associated with having many health benefits.27 The abundance of polyphenols in foodstuffs is apparent, although they often have not been adequately characterized; however, an assortment of polyphenols is prevalent in unprocessed and processed foods and beverages and nutraceuticals.28

Structurally, polyphenols or phenolics have one or more aromatic rings with hydroxyl groups and can occur as simple and complex molecules.29 Polyphenols can be subdivided into two major groups: hydroxybenzoic acids and hydroxycinnamic acids (Fig. 1.3). Examples of hydroxybenzoic acids include gallic and vanillic acids. They are typically found in the bound form as a smaller entity of a ligand or tannin or are linked to a sugar or an organic acid in plant foods.25 Alternatively, hydroxycinnamic acid examples include p-coumaric and caffeic acids. These molecules are found esterified with small molecules, bound to cell walls, and/or proteins.25 A subcategory of p-coumaric acid derivatives is the flavonoids (flavonones, flavanones, flavonols, flavanols [proanthocyanidins, catechins, epicatechins, procyanidins, prodelphinidins], and anthocyanins) as these are the most abundant polyphenols in our diets (Fig. 1.4).30,31 Flavonones and isoflavones can be predominantly found in citrus fruits and soy products, respectively. Proanthocyanidins are complex polymeric flavanols found in conjunction with flavanol catechins from apples, pears, grape, and chocolate products; these flavonoids are primarily responsible for the astringency of foods. Anthocyanins are located in an assortment of fruits (cherries, plums, strawberries, raspberries, blackberries, and currants). In addition to these polyphenol subclasses, in nature, flavonoids are also prevalent as a glycoside (parent compound or aglycone with a sugar moiety attached) as this sugar moiety helps to facilitate water solubility and transportability of the aglycone.26,32,33 Another important factor to consider is that the distribution of polyphenols in plant tissues is heterogenous; thus, the seed, pericarp, flavedo, and albedo contain polyphenols in different proportions.31

Figure 1.3. Chemical structures of polyphenols. Representative chemical structures of hydroxybenzoic acids, hydroxycinnamic acids, flavonoids, ligands, and stilbenes.

Adapted from Manach et al.63

Figure 1.4. General structures for polyphenols.

Flavonoids are synthesized via the phenylpropanoid pathway and are derived from estrogen.34 The phenylalanine structure from phenolic compounds is transformed to cinnamate by the enzyme phenylalanine ammonia-lyase (PAL). The cinnamate 4-hydroxylase (C4H) converts cinnamate to p-coumarate, and then an acetyl-CoA group is added by the CoA ligase enzyme to yield cinnamoyl-CoA. Lastly, this product is transformed by chalcone synthase (CHS) to yield a general chalcone structure. Stilbenoids are synthesized in much the same fashion except for the C4H enzymatic step (Fig. 1.5).

Figure 1.5. Phenylpropanoid pathway and chalcone synthesis.

The chalcone structure is further metabolized by the chalcone isomerase (CHI) to the general chiral flavanone structure. From the general chiral flavanone structure, the other derivatives, namely, dihydroflavonols, flavonols, flavones, flavan-3-ols, flavan-3,4-diols, isoflavonoids, and neoflavonoids, are further metabolized by a well-characterized enzymatically derived process (Fig. 1.6). Anthocyanidins and anthocyanins are derived from flavan-3,4-diols by leuocyanidin oxygenase (LO) and anthocyanidins-3-O-glucosyltransferase, respectively. Chromones are synthesized from isoflavonoids through the chromone synthase (ChS), while lignans and coumarins are derived from neoflavonoids by lignan synthase (LS) and coumarin synthase (CS), respectively (Fig. 1.6).

Figure 1.6. Synthesis pathway of chiral flavanones and other flavonoid derivatives. CHI, chalcone isomerase; K4R, kaempferol-4-reductase; FLS, flavonol synthase; F3D, flavanone-3-dioxygenase; IFS, isoflavonoid synthase; NFS, neoflavonoid synthase; LO, leucocyanidin oxygenase; A3G, anthocyanidin-3-O-glucosyltransferase; ChS, chromone synthase; LS, lignan synthase; CS, coumarin synthase.

In addition to flavanone, other small natural compounds found in a wide variety of food and plant sources exist. These compounds, namely, flavonoids, isoflavonoids, and lignans, have generated much scientific interest in their potential clinical applications in the possible dietary prevention of different diseases. Flavanones, stilbenes, lignans, isoflavonoids, and other flavonoid derivatives are similar in structure and provide host-protective purposes. They share the common parent compound, estrogen, in their synthesis and are differentiated based on key structural differences, specific plant hosts, and the environment (Fig. 1.7).

Figure 1.7. Relationship between stilbenes and dihydrochalcones to other polyphenols.

1.3 SOURCES

In 1936, Professor Szent-Györgyi reported the isolation of a substance that was a strong reducing agent acting as a cofactor in the reaction between peroxidase and ascorbic acid. This substance was initially given the name “vitamin P”; this substance has been subsequently categorized as the flavonoid rutin. Professor Szent-Györgyi’s seminal investigations identified rutin and reported its isolation from both lemons and red pepper.35 Since this time, more than other 4000 flavonoids have being identified and studied. Flavonoids are a group of polyphenolic compounds of low MW36 that present a common benzo-γ-pyrone structure.37 They are categorized into various subclasses including flavones, flavonols, flavanones, isoflavanones, anthocyanidins, and catechins.

Consumption of polyphenols could be close to 1 g/day in our diet, making polyphenols the largest source of antioxidants.38 Dietary sources of polyphenols include fruits, vegetables, cereals, legumes, chocolate, and plant-based beverages such as juices, tea, and wine.38 Extensive biomedical evidence suggests that polyphenolic compounds no matter their class may contribute to the prevention of cardiovascular disease, cancer, osteoporosis, diabetes, and neurodegenerative diseases.39–41 As polyphenols are found in plant sources consumed regularly or that are used in traditional medicine, there is a necessity to study these potentially beneficial compounds. Additionally, potential health benefiting properties such as antiinflammatory, antiproliferative, and colon protection may call for development of these compounds into future therapeutic agents. The average human diet contains a considerable amount of flavonoids, the major dietary sources of which include fruits (i.e., orange, grapefruit, apple, and strawberry), vegetables (i.e., onion, broccoli, green pepper, and tomato), soybeans, and a variety of herbs.42,43 Due to the constant and significant intake of these compounds in our diet, the United States Department of Agriculture (USDA) has created a database that contains the reported average content of these compounds in different foodstuffs.44 Among the classes of flavonoids, flavanones have been defined as citrus flavonoids44–46 due to their almost unique presence in citrus fruits.44,47–57 However, flavanones have been also reported in tomatoes,35,58–60 peanuts,61,62 and some herbs such as mint,63 gaviota tarplant,62,64 yerba santa,62,65 and thyme.62,66 Flavonoids are consumed in the human diet; the calculated flavonoid intake varies among countries since cultural dietary habits, available flora, and weather influence what food is consumed and, therefore, the amount and subclasses of flavonoids ingested.21 However, in the Western diet, the overall amount of flavonoids consumed on a daily basis is likely in the milligram range. It has been determined that the consumption of selected subclasses of flavonoids may be more important in determining health benefits than the total flavonoid intake. The content of flavonoids is also potentially influenced by food processing and storage conditions, which can result in transformation of flavonoids, and loss of flavonoid content.21

Flavonoids in general have been studied for more than 70 years in in vivo and in vitro systems. They have been shown to exert potent antioxi­dant activity 48,59,67–69 in some instances, stronger than α-tocopherol (vitamin E).70 They have been also shown to exhibit beneficial effects on capillary permeability and fragility,23,37,48,68,71–77 to have antiplatelet,23,37,48,67,68,71–76 hypolipidemic,67,78–81 antihypertensive,51,67,82 antimicrobial,67 antiviral,23,37,48,67,68,71–76,83,84 antiallergenic,85 antiulcerogenic,67 cytotoxic,67 antineoplastic,47,50,67,86–90 antiinflammatory,23,37,48,67,68,71–76 antiatherogenic,67,91 and antihepatotoxic67 activities. There are multiple chiral flavanones; however, they have been generally thought of as achiral entities and their chiral nature, in many cases, has not been recognized or denoted. Furthermore, the USDA database reports these compounds as achiral entities and uses the aglycone terminology interchangeably with the glycosides.92

The importance of considering the chiral nature of naturally occurring compounds and xenobiotics has been previously reviewed by Yáñez et al.93 The chirality of flavonoids was initially examined by Krause and Galensa’s studies in the early 1980s.62,94,95 Chirality plays an important role in biological activity; disciplines like agriculture, nutrition, and pharmaceutical sciences have long recognized the existence of natural chiral compounds; however, developed methods of analysis have often failed to stereospecifically separate and discriminate compounds into their respective antipodes. The advantage of chiral separation methods includes a more thorough appreciation of the stereospecific disposition of natural compounds including flavonoids. Moreover, the lack of configurational stability is a common issue with chiral xenobiotics. Some chiral flavonoids have been reported to undergo nonenzymatic interconversion of one stereoisomer into another in isomerization processes such as racemization and enantiomerization.93 Racemization refers to the conversion of an enantioenriched substance into a mixture of enantiomers. Alternatively, enantiomerization refers to a reversible interconversion of enantiomers. The importance of isomerization in stereospecific chromatography as well as in the pharmaceutical manufacturing process has been described.93 Therefore, the development of chiral methodology to analyze this kind of xenobiotics is necessary.

The study of the stereochemistry of flavonoids comprises mainly C-2 and C-3; nevertheless, the majority of natural flavonoids possess only one stereochemical isomer at the C-2 position. C-2 and C-3 act as chiral centers of dihydroxyflavonols and are important in flavonoid metabolism. The nomenclature of flavonoids with two chiral centers remains a topic of debate since the use of symbolism (+/−) or 2,3-cis or -trans seems to be inadequate to describe four possible enantiomers.96 It is also argued that the R, S nomenclature for absolute configuration is confusing for flavonoids because the designation of R or S changes at C-2 depending on the priority of neighboring groups, even though the stereochemistry remains the same.96 An alternative nomenclature system was proposed by Hemingway et al.97 based on that used for carbohydrate chemistry. In this system, the prefix ent- has been used for the mirror images. However, scientific consensus has not been reached on stereochemical lexicon cognates, and, to date, all these systems of nomenclature still remain being used and appearing in the biomedical, biochemical, agricultural, and food science literature.

1.4 PHARMACOLOGICAL ACTIVITIES OF SELECTED FLAVONOIDS

Humans have utilized and/or consumed polyphenols for health benefits. For centuries, alternative medicine has been practiced in different countries as exemplified by the use of plant extracts as traditional medicinal folk agents in the prevention and treatment of an assortment of ailments like menses, coughing, digestive problems, and so on. There are a variety of health benefits that can be attributed to the use/consumption of polyphenols including antioxidant, anticancer, antihyperlipidemic, antiallergenic, antibacterial, antiviral, and antiinflammatory.25 Conversely, there are also toxic effects associated with the use/consumption of polyphenols such as anemia due to the inhibited absorption of nutrients and minerals and inhibitory effects on cytochrome P450 enzymes (P450) resulting in potential drug–drug interactions. Current uses of polyphenols, in addition to their dietary health-related benefits and herbal remedies, are their use as dietary supplements and as pharmaceutical leads; thus, the reported intake of polyphenols is in the tens to hundreds of milligrams per day in human diets.21,31

The World Health Organization (WHO), published a comprehensive study and analysis in September 2008 naming the leading causes of mortality in the world in 2004 to include cardiovascular and pulmonary ailments and cancer accounting for approximately 22.9 million deaths.98 These statistics remain consistent with the data published in 2007 with similar primary causes of mortality as seen in 2002.99 There appears to be evidence that suggests that the leading causes of death are often multifactorial and intertwined, for example, dyspnea, malignant pericardial effusion, malignant pleural effusion, and superior vena cava syndrome, all of which are cardiopulmonary and/or vascular problems.100 Biomedical literature suggests etiologies of cardiovascular and pulmonary ailments and cancer have been linked to diet and nutrition, environment, exercise, genetics, hormones, lifestyles, radiation, sex, and weight; however, direct correlations of the disease, etiologies, and pathogenic mechanisms have not been fully elucidated. Contemporary Western medicine provides a variety of options to prevent and treat cardiovascular and pulmonary ailments and cancer. It is becoming increasingly popular and apparent that there is a need for other effective means to prevent, treat, and develop newer drugs or alternatives to disease treatment for both the consumer and the nutraceutical and pharmaceutical industry at a lower cost.

There are a several assay methodologies to determine the total polyphenolic content of a sample through the use of the Folin–Denis and Folin–Ciocalteu reagents and complexation with aluminum III ion.101–103 The Folin–Denis or Folin–Ciocalteu reducing reagents are able to form phosphomolybdic–phosphotungstic–phenol complexes, which can be monitored at a visible wavelength of 760 nm via reduction–oxidation reaction. These assays may have some inherent falsely elevated values because of interference as there may be other components in the sample that are also reducing reagents. As previously mentioned, the total phenolic content of the sample can be quantified; thus, this method is a nonspecific measurement of polyphenol content. Alternatively, complexation of polyphenols with aluminum III ion can be used to determine the quantity of polyphenols in the sample monitored at a wavelength of 425 nm. This method is dependent upon the aluminum ion complexing with the carbonyl and hydroxyl groups of the polyphenol. Again, these processes are not specific for a particular polyphenol; therefore, it is necessary to develop analytical methods to quantify individual polyphenols in a sample to enable determination of a correlation between the amount of a polyphenol in a sample and a health-related benefit.

1.4.1 Hesperidin and Hesperetin

Hesperidin ((+/−) 3,5,7-trihydroxy-4′-methoxyflavanone 7-rhamnoglucoside) C28H34O15, MW 610.56 g/mol, experimental octanol to water partition coefficient (XLogP) value of −1.1 (Fig. 1.8), is a chiral flavanone-7-O-glycoside consumed in oranges and in other citrus fruits and herbal products.104 The rutinose sugar moiety is rapidly cleaved off the parent compound to leave the aglycone bioflavonoid hesperetin (+/−3,5,7-trihydroxy-4′-methoxyflavanone) C16H14O6, MW 302.28 g/mol, XLogP value of 2.174 (Fig. 1.9), also a chiral flavonoid. There is current interest in the medical use of bioflavonoids, including hesperetin, in the treatment of a variety of cancers and vascular diseases.105

Figure 1.8. Structure of hesperidin. The asterisk (*) denotes a chiral center.

Figure 1.9. Structure of hesperetin. The asterisk (*) denotes a chiral center.

1.4.1.1 Antifungal, Antibacterial, and Antiviral Activity 

Hesperidin extracted from grapefruit (Citrus paradise Macf., Rutaceae) seed and pulp ethanolic extracts has been related to have antibacterial and antifungal activity against 20 bacterial and 10 yeast strains.106 The level of antimicrobial effects was assessed employing an in vitro agar assay and standard broth dilution susceptibility test. It was observed that hesperidin exhibits strong antimicrobial activity against Salmonella enteritidis (minimum inhibitory concentration [MIC] of 2.06% extract concentration—m/V), while its activity against other bacteria and yeasts ranged from 4.13% to 16.5% m/V.106 Furthermore, hesperidin has also been observed to have protective effects in infected mice with encephalomyocarditis (EMC) virus and Staphylococcus aureus that were administered with hesperidin before or coadministered with the lethal viral-bacterial dose.107

In the case of the aglycone hesperetin, it has been shown to have MIC > 20 µg/mL against Helicobacter pylori. However, neither hesperetin nor other flavonoids and phenolic acids inhibited the urease activity of H. pylori.108 Furthermore, hesperetin has shown to be an effective in vitro agent against severe acute respiratory syndrome (SARS) (or similar) coronavirus (CoV) infections.109 Hesperetin inhibits the SARS-CoV replication by interacting with the spike (S) glycoprotein (S1 domain) in the host cell receptor and fusing the S2 domain with the host cell membrane activating the replicase polyproteins by the virus-encoded proteases (3C-like cysteine protease [3CLpro] and papain-like cysteine protease) and other virus-encoded enzymes such as the NTPase/helicase and RNA-dependent RNA polymerase. The blocking of the S1 may play an important role in the immunoprophylaxis of SARS.109 Similar activities have also been observed for hesperetin against the replication of the neurovirulent Sindbis strain (NSV) having 50% inhibitory doses (ID50) of 20.5 µg/mL. However, its glycoside, hesperidin, did not have inhibitory activity, indicating the possibility that the rutinose moiety of flavanones blocks the antiviral effect.110 Nevertheless, hesperetin has also been reported to be effective against the replication of herpes simplex virus type 1 (HSV-1), poliovirus type 1, parainfluenza virus type 3 (Pf-3), and respiratory syncytial virus (RSV) in in vitro cell culture monolayers employing the technique of viral plaque reduction.83

1.4.1.2 Antiinflammatory Activity 

The inflammatory process involves a series of events encompassed by numerous stimuli such as infectious agents, ischemia, antigen–antibody interactions, and chemical, thermal, or mechanical injury. The inflammatory responses have been characterized to occur in three distinct phases, each apparently mediated by different mechanisms: an acute phase characterized by local vasodilatation and increased capillary permeability, a subacute phase characterized by infiltration of leukocyte and phagocyte cells, and a chronic proliferative phase, in which tissue degeneration and fibrosis occur.111 Different animal models have been developed to study the different phases of an inflammatory response. In the case of testing acute inflammatory response, the carrageenan-induced paw edema in mice112 and the xylene-induced ear edema113 are widely employed. Methods to test the proliferative phase (granuloma formation) include the cotton pellet granuloma model.114 Another model that allows the assessment of acute and chronic inflammation is the adjuvant–carrageenan-induced inflammation (ACII) model to induce adjuvant arthritis.115 Hesperidin and hesperetin were tested under these models, and it was observed that only hesperetin had a positive effect in reducing the carrageenan-induced paw edema in mice by 48% and 29% after 3 and 7 hours postinflammatory insult.111 In the case of the xylene-induced ear edema model, both hesperidin and hesperetin had a positive effect by reducing the edema by 45% and 44%, respectively.111 Similar observations were observed in the cotton pellet granuloma, whereas hesperidin and hesperetin inhibited granuloma formation by 30% and 28%, respectively.111 In the case of the ACII model, hesperidin exhibited activity in the acute phase (day 6) by causing a reduction in paw edema of 52% and exhibited a more moderate reduction in the chronic phase (7–21 days) by reducing the paw edema by 36%, 44%, 47%, 38%, and 31% at 7, 8, 10, 12, and 16 days postinflammatory insult, respectively.111 Different mechanisms to elucidate how hesperidin, hesperetin, and other polyphenols might carry their antiinflammatory activity have been proposed. Among these, it has been observed that after carrageenan injection, there is an initial release of histamine and serotonin during the first 1.5 hours with a posterior release of kinin between 1.5 and 2.5 hours, followed with a release of prostaglandins until 5 hours.116–118 Thus, it is believed that hesperidin and hesperetin might be involved with a variety of steps during the development of inflammation.

Other studies have reported that hesperidin downregulates the lipopolysaccharide (LPS)-induced expression of different proinflammatory (tumor necrosis factor-alpha [TNF-α], IL-1 beta, interleukin-6 [IL-6]) and antiinflammatory mediators (IL-12), cytokines as well as cytokines (KC, MCP-1 and MIP-2), while enhancing the production of other antiinflammatory cytokines (IL-4 and IL-10).119 In this study, mice were challenged with intratracheal LPS (100 µg) 30 minutes before treatment with hesperidin (200 mg/kg oral administration) or vehicle. After 4 and 24 hours, bronchoalveolar lavage fluid was collected, observing that hesperidin significantly reduced the total leukocyte counts, nitric oxide production, and inducible nitric oxide synthase (iNOS) expression.119 These results correlate with in vitro studies that have demonstrated that hesperidin suppresses the expression of IL-8 on A549 cells and THP-1 cells, the expression of TNF-α, IL-1 beta, and IL-6 on THP-1 cells, and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (responsible for cell adhesion) on A549 cells. The suppression of these inflammatory mediators is regulated by nuclear factor-kappa B (NF-κB) and AP-1, which are activated by IκB and mitogen-activated protein kinase (MAPK) pathways, indicating that hesperidin might interact within these pathways to exert its antiinflammatory activity.119

1.4.1.3 Antioxidant Activity 

Hesperidin and its aglycone, hesperetin, have been assessed in various in vitro chemical antioxidant models (cell-free bioassay systems). It has been observed that both hesperidin and hesperetin exhibited similar patterns of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities.120 Similar results have been reported elsewhere for hesperidin, an antioxidant that was comparable in efficacy to Trolox® (positive control).121 Furthermore, hesperetin alone has been reported to effectively scavenge peroxynitrite (ONOO−) in a concentration-dependent manner. Peroxynitrite (ONOO−) is a reactive oxidant formed from superoxide (*O2−) and nitric oxide (*NO), which can oxidize several cellular components, including essential protein, nonprotein thiols, DNA, low density lipoproteins (LDLs), and membrane phospholipids.122

Both hesperidin and hesperetin have also been assessed for their antioxidant capacity in vivo. It has been observed that hesperidin (25 mg/kg body weight [BW] p.o.) offers protection against lung damage induced by a subcutaneous injection of nicotine at a dosage of 2.5 mg/kg BW for 5 days a week. Hesperidin treatment resulted in a decreased level of all the marker enzymes, the recovery of the in vivo antioxidant status back to near baseline level,123 and different matrix metalloproteinases (MMPs) were downregulated.124 Hesperidin (60 mg/kg BW/day p.o. for 9 days) has also been shown to increase the free SH-group concentration (SHC), hydrogen-donating ability (HDA), and natural scavenger capacity, and to decrease the hepatic malonaldehyde content and dien conjugate (DC) in male Wistar albino rats with alimentary-induced fatty livers.125 Furthermore, hesperidin in the same animal models has been reported to increase both the total scavenger capacity (TSC) and the activity of superoxide dismutase (SOD) in liver homogenates, and to induce slight changes in the Cu, Zn, Mn, and Fe contents of liver homogenates.126 Similar results were observed for hesperidin (100 and 200 mg/kg p.o. for 1 week) in CCl4-induced oxidative stressed rats, whereas the thiobarbituric acid-reactive substances (TBARSs) decreased and the glutathione (GSH) content, SOD, and catalase (CAT) levels increased in liver and kidney homogenates.127 In the case of hesperetin, it was observed to be a potent antioxidant, inhibiting lipid peroxidation initiated in rat brain homogenates by Fe2+ and L-ascorbic acid. Hesperetin was found to protect primary cultured cortical cells against the oxidative neuronal damage induced by H2O2 or xanthine and xanthine oxidase (XO). In addition, it was shown to attenuate the excitotoxic neuronal damage induced by excess glutamate in the cortical cultures.120

1.4.1.4 Anticancer Activity 

In vitro tests have shown that hesperidin reduces the proliferation of many cancer cells.128 For instance, hesperidin (100 µM) has been shown to reduce the cell viability (65 ± 0.05%) of human colon cancer cells, SNU-C4 based in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.129 It was proposed that hesperidin treatment decreased the expression of B-cell CLL/lymphoma 2 (BCL2) mRNA and increased the expression of BCL2-associated X protein (BAX) and of the apoptotic factor caspase-3 (CASP3) inducing apoptosis.129 Another study, less mechanistic in nature, observed that hesperidin and hesperetin at smaller concentrations (1 µM) inhibit the neoplastic transformation of C3H 10T1/2 murine fibroblasts induced by the carcinogen 3-methylcholanthrene.130

Hesperetin has been reported to affect the proliferation and growth of a human breast carcinoma cell line, MDA-MB-435, with an IC50 of 22.5 µg/mL and to exhibit low cytotoxicity (>500 µg/mL for 50% cell death).88 Furthermore, hesperetin has also been reported to significantly inhibit cell proliferation of MCF-7 cells in a concentration-dependent manner by causing cell cycle arrest in the G1 phase. In the G1 phase, hesperetin downregulates the cyclin-dependent kinases (CDKs) and cyclins while upregulating p21(Cip1) and p27(Kip1) in MCF-7 cells. Hesperetin also decreases CDK2 and CDK4 together with cyclin D. In addition, hesperetin increases the binding of CDK4 with p21(Cip1) but not p27(Kip1) or p57(Kip2), indicating that the regulation of CDK4 and p21(Cip1)1 may participate in the anticancer activity pathway of hesperetin in MCF-7 cells.131

The ApcMin/+ mouse model and the azoxymethane (AOM) rat model are the main animal models used to study the effect of dietary agents on colorectal cancer.132 Different chemopreventive agents in the AOM rat model have been analyzed,132,133 and it was observed that hesperidin and hesperetin-rich foods are able to suppress colon adenocarcinoma and/or consistently inhibit adenoma and aberrant crypt foci (ACF) in several independent rat studies.90,132,134–136 Other animal studies have reported that hesperidin has the capacity to inhibit tumor initiation and promotion in CD-1 mice skin. Subcutaneous application of hesperidin did not inhibit 7,12-dimethylbenz(a)anthracene-induced tumor initiation but did inhibit 12-O-tetradecanoyl-13-phorbol acetate-induced tumor promotion.137 Furthermore, male imprinting control (ICR) mice that were N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) (500 µg/mL) induced for urinary bladder tumors were fed with hesperidin (1 mg/mL), diosmin (1 mg/mL), and combination (4.9 mg/mL diosmin and 0.1 mg/mL hesperidin) for 8 weeks. It was observed that hesperidin and diosmin alone or in combination significantly reduced the frequency of bladder carcinoma and preneoplasia. Also, a significant decrease in the incidence of bladder lesions and cell-proliferation activity estimated by enumeration of silver-stained nucleolar-organizer-region-associated proteins (AgNORs) and by the 5-bromodeoxyuridine (BUdR)-labeling index was observed.90 However, other research groups have observed that hesperidin (100 µg/mL) and diosmin (100 µg/mL) alone or in combination (900 µg/mL diosmin and 100 µg/mL hesperidin) provide no pathological alterations during the initiation and postinitiation phases of esophageal carcinogenesis initiation with N-methyl-N-amylnitrosamine (MNAN) in male Wistar rats.138

1.4.1.5 Cyclooxygenase-1 and -2 Inhibitory Activity 

Hesperidin has been assessed for its inhibitory effect on LPS-induced overexpression of cyclooxygenase-2 (COX-2), iNOS proteins, overproduction of prostaglandin E2 (PGE2) and nitric oxide (NO) using mouse macrophage cells. Treatment with hesperidin suppressed production of PGE2, nitrogen dioxide (NO2), and expression of iNOS protein. In the case of COX-2, hesperidin did not affect the protein levels expressed. Thus, hesperidin has been reported to be a COX-2 and iNOS inhibitor, which may explain its antiinflammatory and antitumorigenic efficacies in vivo.139 Furthermore, hesperetin and hesperidin in the concentration range 250–500 µM have been shown to potently inhibit the LPS-induced expression of the COX-2 gene in RAW 264.7 cells, also demonstrating the antiinflammatory activity of these compounds. The ability of hesperetin and hesperidin to suppress COX-2 gene expression has been suggested to possibly be a consequence of their antioxidant activity.140

1.4.1.6 Antiadipogenic Activity 

Obesity is biologically characterized at the cellular level to be an increase in the number and size of adipocytes differentiated from fibroblastic preadipocytes in adipose tissue. It has been reported that hesperidin inhibits the formation of 3T3-L1 preadipocytes by 11.1%. Apoptosis assays indicate that hesperidin increased apoptotic cells in a time- and concentration-dependent manner. Treatment of cells with hesperidin also decreased the mitochondrial membrane potential in a time- and dose-dependent manner. The cell apoptosis/necrosis assay demonstrated that hesperidin increased the number of apoptotic cells but not necrotic cells. Hesperidin treatment of cells caused a significant time- and concentration-dependent increase in the CASP3 activity. Western blot analysis indicated that treatment of hesperidin also markedly downregulated poly ADP-ribose polymerase (PARP) and Bcl-2 proteins, and activated CASP3, Bax, and Bak proteins. These results indicate that hesperidin efficiently inhibits cell population growth and induction of apoptosis in 3T3-L1 preadipocytes.141 Furthermore, in the same in vitro, model hesperidin has been recently reported to inhibit intracellular triglyceride and glycerol-3-phosphate dehydrogenase (GPDH) activity by 40.2 ± 3.2% and 37.9 ± 4.6%, respectively.142

1.4.1.7 Other Reported Activities 

Hesperidin and its aglycone, hesperetin, have been shown to have a very weak estrogenic effect, and its regular use can alleviate certain symptoms related with menopause and dysmenorrhea.143,144 For instance, in a controlled clinical study, 94 menopausal woman with hot flashes were given a daily formula for 1 month containing 900 mg hesperidin, 300 mg hesperidin methyl chalcone, and 1200 mf vitamin C. After 1 month of treatment, the symptoms of hot flashes were completely relieved in 53% and reduced in 34% of the women.145

1.4.2 Naringin and Naringenin

Naringin ((+/−) 4′,5,7-trihydroxyflavanone 7-rhamnoglucoside) C27H32O14, MW 580.53 g/mol, XLogP value of −1 (Fig. 1.10), is a chiral flavanone-7-O-glycoside present in citrus fruits, tomatoes, cherries, oregano, beans, and cocoa.146–151 After consumption, the neohesperidose sugar moiety is rapidly cleaved off the parent compound in the gastrointestinal tract and liver to leave the aglycone bioflavonoid naringenin ((+/−) 4′,5,7-trihydroxyflavanone) C15H12O5, MW 272.25 g/mol, XLogP value of 2.211 (Fig. 1.11). The ratio between the amount of naringenin and naringin varies among different food products. For instance, citrus fruits contain higher amounts of the glycoside naringin, while tomatoes have higher amounts of the aglycone naringenin.148

Figure 1.10. Structure of naringin. The asterisk (*) denotes a chiral center.

Figure 1.11. Structures of naringenin. The asterisk (*) denotes a chiral center.

1.4.2.1 Antifungal, Antibacterial, and Antiviral Activity 

Naringin present in grapefruit (C. paradise Macf., Rutaceae) seed and pulp ethanolic extracts has been related to have antibacterial and antifungal activity against multiple bacteria, fungi, and yeast strains.106,152 Naringin was assessed employing an in vitro agar assay and standard broth dilution susceptibility test, and it was observed that it exhibited the strongest antimicrobial effect against S. enteritidis (MIC of 2.06% extract concentration—m/V) and an MIC ranging from 4.13% to 16.5% m/V for the other tested bacteria and yeasts.106 Similar results have been reported for naringin present in Argentine Tagetes (Asteraceae)153 and in Drynaria quercifolia.154

Naringenin isolated from ethanol extracts of propolis from four different regions of Turkey and Brazil exhibited to have MIC values ranging from 4 to 512 µg/mL for all the analyzed bacterial strains. Death was observed within 4 hours of incubation for Peptostreptococcus anaerobius, Peptostreptococcus micros, Lactobacillus acidophilus, and Actinomyces naeslundii, while 8 hours for Prevotella oralis and Prevotella melaninogenica and Porphyromonas gingivalis, 12 hours for Fusobacterium nucleatum, and 16 hours for Veillonella parvula.155 Similar results were found for naringenin-rich ethanol extracts of propolis having MIC values of 2 µg/mL for Streptococcus sobrinus and Enterococcus faecalis; 4 µg/mL for Micrococcus luteus, Candida albicans, and Candida krusei; 8 µg/mL for Streptococcus mutans, S. aureus, Staphylococcus epidermidis, and Enterobacter aerogenes; 16 µg/mL for Escherichia coli and Candida tropicalis; and 32 µg/mL for Salmonella typhimurium and Pseudomonas aeruginosa.156 Similar MIC values have been observed for naringenin isolated from the capitula of Helichrysum compactum.157 Naringenin has also been shown to have MIC > 20 µg/mL against H. pylori. However, neither naringenin nor other flavonoids and phenolic acids inhibited the urease activity of H. pylori.108

Naringenin has also been reported to have antiviral activity. For instance, naringenin exhibited an inhibitory effect on the replication of the NSV having a 50% inhibitory dose (ID50) of 14.9 µg/mL. However, its glycoside, naringin, did not have inhibitory activity.110 Similar results were observed for naringin, which was also ineffective on the replication of HSV-1, poliovirus type 1, Pf-3, and RSV in in vitro cell culture monolayers employing the technique of viral plaque reduction.83 Furthermore, naringenin has demonstrated activity against HSV-1 and type 2 (HSV-2) infected Vero cells in a virus-induced cytopathic effect (CPE) inhibitory assay, plaque reduction assay, and yield reduction assay.158 However, both naringin and naringenin are ineffective in inhibiting poliovirus replication.159

1.4.2.2 Antiinflammatory Activity 

Naringenin has been reported to have poor or no effect over different inflammatory mediators in vitro. For instance, naringenin was ineffective in inhibiting endothelial adhesion molecule expression or in attenuating expression of E-selectin and ICAM-1, VCAM-1, and TNF-α-induced adhesion molecule expression in human aortic endothelial cells.160 In another study, naringenin also exhibited virtually no effects on cytokines, metabolic activity, or on the number of cells in the studied cell populations of stimulated human peripheral blood mononuclear cells (PBMCs) by LPS.161 Furthermore, the lack of ability of naringenin to inhibit the activity of NOS-2 has been reported; however, the induction of NOS-2 protein in LPS-treated J774.2 cell was evident by Western blotting techniques.162

However, naringin has been reported to regulate certain inflammatory mediators and to possess antiinflammatory activity. Naringin (10, 30, and 60 mg/kg intraperitoneal [i.p.]) dose dependently suppressed LPS-induced production of TNF-α in mice. To further examine the mechanism by which naringin suppresses LPS-induced endotoxin shock, an in vitro model, RAW 264.7 mouse macrophage cells, was utilized. Naringin (1 mM) suppressed LPS-induced production of NO and the expression of inflammatory gene products such as iNOS, TNF-α, inducible cyclooxygenase (COX-2), and IL-6 as determined by RT-PCR assay. Naringin was also found to have blocked the LPS-induced transcriptional activity of NF-κB in electrophoretic mobility shift assay and reporter assay. These findings suggest that suppression of the LPS-induced mortality and production of NO by naringin is due to inhibition of the activation of NF-κB.163

Similarly, a separate study assessed the effect of naringin in an endotoxin shock model based on Salmonella infection. Intraperitoneal (i.p.) infection with 10 cfu S. typhimurium aroA caused lethal shock in LPS-responder but not in LPS-nonresponder mice. Administration of 1 mg naringin 3 hours before infection resulted in protection from lethal shock, similar to LPS-nonresponder mice. The protective effect of naringin was time- and dose dependent. Treatment with naringin resulted not only in a significant decrease in bacterial numbers in spleens and in livers, but also in a decrease in plasma LPS levels. In addition, naringin markedly suppressed TNF-α and normalized the activated states of blood coagulation factors such as prothrombin time, fibrinogen concentration, and platelet numbers caused by infection.164

1.4.2.3 Antioxidant Activity 

Different in vitro chemical and biological assays have reported that naringin and naringenin have considerable antioxidant properties. For instance, naringin has been reported to scavenge the DPPH, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and nitric oxide (NO) radicals in vitro in a concentration-dependent manner.165 Furthermore, naringin and naringenin have been assessed in the beta-carotene–linoleic acid, DPPH, superoxide, and hamster LDL in vitro models to measure their antioxidant activity. Using the beta-carotene–linoleate model, naringin (10 µM) and naringenin (10 µM) exhibited an 8% and 9% inhibition, respectively, whereas both compounds demonstrated negative free radical scavenging activity using the DPPH method and a 25% and 30% inhibition of superoxide radicals for naringin and naringenin, respectively. Naringin and naringenin increased the lag time of LDL oxidation to 150 minutes (a 32% increase from baseline levels). Thus, indicating that both compounds have significant in vitro antioxidant properties.166 Furthermore, naringin has been reported to have a positive effect in iron-induced oxidative stress and in a variety of cellular processes like respiration and DNA synthesis. For this, HepG2 cells were treated with 0.5, 1.0, 2.5, and 5.0 mM naringin 1 hour before exposure to 0.1, 0.25, 0.5, and 1.0 mM ferric iron. Pretreatment of HepG2 cells with naringin resulted in inhibition of lipid peroxidation, arrested the iron-induced depletion in the GSH concentration, and increased various antioxidant enzymes like glutathione peroxidase (GSHPx), CAT, and SOD.167

Naringin has also demonstrated antioxidant properties in different in vivo animal models. A comparison study between grapefruit juice and naringin reported that the total antioxidant activity of a quantity of red grapefruit juice was higher than that of naringin. Animals received a cholesterol-rich diet and after administration of naringin (0.46–0.92 mg p.o.) or red grapefruit juice (1.2 mL), it was observed that diets supplemented with red grapefruit juice and, to a lesser degree, with naringin improved the plasma lipid levels and increased the plasma antioxidant activity.168

1.4.2.4 Anticancer Activity 

Naringin and naringenin have been reported to have anticancer activities. For instance, naringenin has been reported to induce cytotoxicity in cell lines derived from cancer of the breast (MCF-7, MDA-MB-231), stomach (KATOIII, MKN-7), liver (HepG2, Hep3B, and Huh7), cervix (Hela, Hela-TG), pancreas (PK-1), and colon (Caco-2), as well as leukemia (HL60, NALM-6, Jurkat, and U937). Naringenin-induced cytotoxicity was low in Caco-2 and high in leukemia cells compared to other cell lines. Naringenin dose dependently induced apoptosis, with hypodiploid cells detected in both Caco-2 and HL60 by flow cytometric analysis.169 Furthermore, naringenin at concentrations higher than 0.71 mM has been reported to inhibit cell proliferation of HT29 colon cancer cells,170 while naringin has been reported to induce cytotoxicity via apoptosis in mouse leukemia P388 cells and to slightly increase the activities of the antioxidant enzymes, CAT, and GSHPx in these cells.171

Naringin and naringenin have also been assessed for its effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50) was around 20 µg/mL for naringin and naringenin with low cytotoxicity (>500 µg/mL for 50% cell death).88 Two possible mechanisms that could modulate breast tumor growth have been proposed, one via inhibition of aromatase (CYP19) and the other via interaction with the estrogen receptor (ER). Multiple in vitro studies confirmed that naringin and naringenin act as aromatase inhibitors potentially reducing tumor growth. It is thought that in the in vivo situation, breast epithelial (tumor) cells communicate with surrounding connective tissue by means of cytokines, prostaglandins, and estradiol forming a complex feedback mechanism. It has been reported that naringenin affects MCF-7 proliferation with an EC50 value of 287 nM and acts as an aromatase inhibitor with an IC50 value of 2.2 µM. These results show that naringenin can induce cell proliferation or inhibit aromatase in the same concentration range (1–10 µM).172 The second proposed mechanism is related to the ER, and it has been observed that naringenin exerts an antiproliferative effect only in the presence of ERα or ERβ. Moreover, naringenin stimulation induces the activation of p38/MAPK leading to the proapoptotic CASP3 activation and to the poly(ADP-ribose) polymerase cleavage in selected cancer cell lines. Notably, naringenin shows an antiestrogenic effect only in ERα-containing cells, whereas in ERβ-containing cells, naringenin mimics the 17beta-estradiol effects.173 Nevertheless, naringenin-mediated growth arrest in MCF-7 breast cancer cells has also been observed. Naringenin was found to inhibit the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation, as shown by impaired phosphorylation of the downstream signaling molecule Akt. Naringenin also inhibited the phosphorylation of p44/p42 MAPK. Inhibition of the MAPK pathway with PD98059, a MAPK kinase inhibitor, reduced insulin-stimulated glucose uptake by approximately 60%. The MAPK pathway therefore appears to contribute significantly to insulin-stimulated glucose uptake in breast cancer cells.174

In the case of human prostate cancer cells (PC3) stably transfected with activator protein 1 (AP-1) luciferase reporter gene, the maximum AP-1 luciferase induction is of about threefold over control after treatment with naringenin (20 µM). At higher concentrations, naringenin demonstrated inhibition of AP-1 activity. The MTS assay for cell viability at 24 hours demonstrated that even at a very high concentration (500 µM), cell death was minimal for naringenin. Furthermore, induction of phospho-C-Jun N-terminal kinase (JNK) and phospho-ERK activity was observed after a 2-hour incubation of PC3-AP-1 cells with naringenin. However, no induction of phospho-p38 activity was observed. Furthermore, pretreating the cells with specific inhibitors of JNK reduced the AP-1 luciferase activity that was induced by naringenin, while pretreatment with MAPK (MEK) inhibitor did not affect the AP-1 luciferase activity.175 It was also observed that naringenin induced apoptosis of human promyeloleukemia HL60 cells by markedly promoting the activation of CASP3, and slightly promoting the activation of caspase-9, but with no observed effect on caspase-8.176 The apoptosis-induced mechanism of naringenin has also been linked with the activation of NF-κB and the degradation of IκBα, which has been observed in human promyeloleukemia HL60 cells,176