Handbook of Green Tea and Health Research (Food and Beverage Consumption and Health)

In: Food and Beverage Consumption and Health Series

HANDBOOK OF GREEN TEA AND HEALTH RESEARCH No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES

Handbook of Green Tea and Health Research Helen McKinley and Mark Jamieson 2009. ISBN 978-1-60741-045-4

In: Food and Beverage Consumption and Health Series

HANDBOOK OF GREEN TEA AND HEALTH RESEARCH

HELEN MCKINLEY AND

MARK JAMIESON EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2009 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Handbook of green tea and health research / [edited by] Helen McKinley and Mark Jamieson. p. ; cm. -- (Food and beverage consumption and health) Includes bibliographical references and index. ISBN 978-1-60876-202-6 (E-Book) 1. Green tea--Health aspects. I. McKinley, Helen. II. Jamieson, Mark. III. Series: Food and beverage consumption and health. [DNLM: 1. Tea. 2. Camellia sinensis. 3. Catechin--analogs & derivatives. 4. Phytotherapy--methods. WB 438 H236 2009] RM251.H36 2009 615'.321--dc22 2009000178

Published by Nova Science Publishers, Inc. Ô New York

CONTENTS Preface

ix

Chapter 1

Central Functions of Green Tea Components M. Furuse,, N. Adachi, S. Tomonaga, H. Yamane and D. M. Denbow

Chapter 2

Green Tea Catechins: A Class of Molecules with Antimicrobial Activity P. Buzzini, P. Vignolini, M. Goretti, B. Turchetti, E. Branda, E. Marchegiani, P. Pinelli and A. Romani

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

1

23

Lipid-soluble Green Tea Polyphenols: Stabilized for Effective Formulation Ping Chen, Douglas Dickinson and Stephen Hsu

45

Assessment of the Antioxidant Capacity of Green Teas: A Critical Review Camilo López-Alarcón and Eduardo Lissi

63

Design and Assessment of the in Vitro Anti-oxidant Capacity of a Beverage Composed of Green Tea (Camellia Sinensis L.) and Lemongrass (Cymbopogon Citratus Stap) D. Fernando Ramos Escudero, Luis Alberto Condezo Hoyos, Mónica Ramos Escudero and Jaime A. Yáñez

81

Teas Are not All the Same: In Vitro and in Vivo Antioxidant Activity and Appetite Modulation in Rats of Green Teas with High and Low Levels of Organic Selenium Abdul L. Molan, Zhuojian Liu and Wenhua Wei Anti-obesity Effects of (-)-Epigallocathchin-3-gallate and its Molecular Mechanism Cheol-Heui Yun, Gi Rak Kim, Min Ji Seo, Hyun-Seuk Moon and Chong-Su Cho

103

125

vi Chapter 8

Chapter 9

Contents Green Tea: Protective Action against Pesticides and other Xenobiotics Present in Human Diet Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi

157

New Method to Improve the Function and Industrial Applicability of Green Tea and Its Byproducts Using Irradiation Technology Cheorun Jo and Myung Woo Byun

177

Chapter 10

Green Tea Catechin as Angiogenesis Inhibitor Kiminori Matsubara and Yoshiyuki Mizushina

197

Chapter 11

Neuroprotective Effect of Theanine on Cerebral Ischemia Nobuaki Egashira,, Kenichi Mishima, Katsunori Iwasaki, Ryozo Oishi and Michihiro Fujiwara

207

Chapter 12

Characterization of the Neuroprotective Activity of the Polyphenol (-)-Epigallocatechin-3-gallate in the Brain Orly Weinreb, Tamar Amit, Moussa B. H. Youdim and Silvia Mandel

219

Chapter 13

Cardiovascular and Metabolic Effects of Green Tea Kamilla Kelemen

Chapter 14

Molecular Basis for the Anti-cancer Activity of EGCG in Vivo: Molecular-Targeting Prevention of Cancer by Green Tea Catechin Yoshinori Fujimura and Hirofumi Tachibana

257

Utility of Epigallocatechin Gallate in the Treatment and Prevention of Breast Cancer: Molecular Mechanisms for Tumor Suppression R. J. Rosengren

301

Chapter 15

Chapter 16

Green Tea Catechins in Colorectal Cancer Seung Joon Baek and Mugdha Sukhthankar

Chapter 17

Inhibitory Effect of Catechin Derivatives from Green Tea on DNA Polymerase Activity, Human Cancer Cell Growth, and TPA (12-O-tetradecanoylphorbol-13-acetate) -induced Inflammation Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina

Chapter 18

Telomerase Regulation in Response to Green Tea Huaping Chen and Trygve O. Tollefsbol

Chapter 19

Green Tea and Chronic Obstructive Pulmonary Disease: A Casecontrol Study in Japan Fumi Hirayama and Andy H. Lee

243

325

347

363

383

Chapter 20

Green Tea and Diabetes Dongfeng Wang, Linge Wang and Li Zhang

393

Chapter 21

Green Tea and Type 2 Diabetes Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song

411

Contents Chapter 22

Chapter 23

Biocatalytic Conversion of Green Tea Catechins to Epitheaflagallin, Epitheaflagallin, 3-O-gallate, and Theaflavins: Production of Promising Functional Foods Nobuya Itoh and Yuji Katsube Preventive Effects of Green Tea Catechins on Dementia Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi and Yukihiko Hara

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419 429

Short Commentary Green Tea and Potential Human Health Effects James E. Trosko Index

451 463

PREFACE After water, tea signifies the second most frequently consumed beverage worldwide. Teas are not all the same; among the many areas of research that are included in this book are the effects of selenium-containing green tea on food consumption and body weight gain. Research shows that tea consumption may have its strongest effect among patients with cardiovascular disease. A specific chapter investigates whether green tea intake can reduce the risk of chronic obstructive pulmonary disease. Research is presented to show that green tea and its major constituent epigallocatechin gallate (EGCG) have a potential chemopreventative and/or treatment for a variety of diseases including breast cancer. Other research sheds new light on the molecular basis for the cancerpreventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer. A further study presents an analysis assessing the progress of research on the mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells. Further chapters look at the relationship of tea to diabetes and a description of the beneficial effects of green tea catechins on neuronal functions and neuronal diseases such as dementia. To improve biological functions and industrial applicability of green tea and its byproducts, research is presented showing irradiation as a useful method. Chapter 1 - Tea (Camellia sinensis) is widely consumed throughout the world and has a number of biologically active substances such as caffeine, catechins, and L-theanine (γglutamylethylamide). Tea consumption is generally known to induce a feeling of relaxation which may be mediated by either catechins, L-theanine, or both, since caffeine stimulates locomotor activities. The catechin (-)-epigallocatechin gallate (EGCG) occurs abundantly in tea. Moreover, frequent consumption of green tea results in high levels of EGCG in the blood and brain. Catechins, which are flavonoids, affect the central nervous system (CNS). The therapeutic effects of flavonoids may involve their binding to γ-aminobutyric acid (GABA)A receptors, which is a major inhibitory neurotransmission system. Recently, EGCG was shown to bind to GABAA receptors in vitro and to induce a sedative effect through GABAA, but not GABAB, receptors in the brain. L-Theanine, a derivative of glutamate, is a unique amino acid occurring only in green tea and a few other plants. After administration L-theanine concentrations were increased in serum, liver and brain, suggesting that L-theanine can cross the blood-brain barrier. Intravenous administration of L-theanine was shown to affect the cortex, hippocampus and amygdala, and increase the alpha-band component of electroencephalograms in rats. More

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recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron excitation in human subjects or influencing the secretion and function of neurotransmitters in the CNS. We discuss the central functions of green tea components such as EGCG and Ltheanine in the CNS. Chapter 2 - A significant part of scientific interest of academy or industry is focused on discovering novel natural antimicrobial drugs. This attention is essentially justified by the expectation that a few of them could play a role in supporting (or even in substituting) some antibiotics of current use. It has been estimated that, although about some tens of novel antimicrobial drugs (either of biological or synthetic origin) are currently launched each year, due to the increasing development of resistant microbial genotypes, their downturn is becoming very rapid. Taking into account these considerations, the enormous scientific and commercial interest in discovering and developing novel classes of molecules exhibiting more or less pronounced antimicrobial properties has oriented the work of a growing part of the scientific community toward large-scale screening programs aimed at discovering novel classes of bioactive molecules. The occurrence in some plants of secondary metabolites exhibiting a more or less pronounced antimicrobial activity is a well-known phenomenon. Among them, green tea polyphenols represent a reservoir of molecules characterized by antioxidant, antiradical and antimicrobial activity. In particular, catechins have proven to be effective towards both prokaryotic and eukaryotic microorganisms. Despite the large number of studies published so far, their actual potentialities and limitations as antimicrobial (mainly antibacterial and antimycotic) drugs have not been critically evaluated. The present chapter represents an overview of the recent literature on the antiviral and antimicrobial properties exhibited by polyphenols, particularly catechins, occurring in green tea composition. Chapter 3 - Green tea polyphenols (GTPs), also referred to as green tea catechins, possess properties that can provide unique health benefits to humans. As indicated in other chapters of this book, studies using molecular, cellular, and animal models, and in human subjects, have demonstrated that these phytochemicals from non-oxidized tea leaves have anti-cancer, antioxidant, anti-microbial, and anti-inflammatory properties. Recently, investigations in our and other laboratories indicated that topical application of GTPs could protect the epidermis against autoimmune disorders, such as psoriasis, prevent or repair UV-induced damage, and suppress scar tissue overgrowth. In addition, specific gene regulation by GTPs, especially epigallocatechin-3-gallate (EGCG), promotes skin cell differentiation, which could lead to improved homeostasis of the skin. Based on these facts, the topical use of products containing GTPs has become more popular, and manufacturers of cosmetic, health care, and household products are adding GTPs or EGCG to their formulations. However, it is important to note that studies described in this book always use “freshly prepared” GTPs or green tea, instead of “pre-prepared” materials. This is because GTPs are potent antioxidants that react rapidly with reactive oxygen species (ROS). As a result, GTPs in most commercially available products have been oxidized and/or epimerized; the biological effects of the resulting compounds are largely unknown. In addition, due to the highly water-soluble nature of these compounds, GTPs in their original form are not lipid-soluble, and therefore not permeable to the skin, a water-proof barrier. Another problem with formulation of GTPs for topical application is the coloration change and precipitation caused by oxidation. Thus, GTPs for topical application (e.g., on skin and

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mucous membranes) must be prepared and used immediately prior to oxidation, coloration and precipitation. These properties of GTPs make it difficult to formulate products containing them that have a reasonable shelf life and maintain their activity and effectiveness. In other words, most of the commercially available “green tea”–containing products are without the full benefits of green tea or GTPs. Therefore, strategies to stabilize and increase the bioavailability of GTPs are needed to provide the full benefits of GTPs to consumers or patients. Recently, it has been shown that lipid esters of GTPs can be formed either enzymatically or chemically. These green tea polyphenol-lipid esters, also referred to as lipid-soluble tea polyphenols (LTPs), could significantly improve formulations of consumer or health care products. We hypothesized that fatty acyl esterification of green tea polyphenol would protect the hydroxyl groups from oxidation and improve skin permeability. In the current study, we compared the activities of LTPs to GTPs for their anti-cancer and gene regulation properties. We examined whether LTPs can be converted into a free GTP (EGCG) in human skin keratinocyte cultures. In addition, the effects of LTPs in a mouse model for psoriasis were evaluated. The results indicate that LTPs effectively cause cancer cell death, induce caspase 14 gene expression both in vitro and in vivo, and improve the skin condition in an animal model for psoriasis. Consistent with these observations, HPLC analysis demonstrated that EGCG in its original form was released from LTPs in situ by human epidermal keratinocytes. These results suggest that LTPs, under appropriate conditions, function similarly to GTPs. More importantly, since the most reactive hydroxyl group(s) is/are protected, and the lipid solubility is dramatically increased by the fatty acyl groups, the biological activity of these compounds can be stabilized, and their bioavailability increased significantly. In conclusion, LTPs are a novel and more effective form of green tea polyphenols for topical applications and other purposes, especially in formulations that require a reasonable shelf life. In addition, LTPs can be a natural additive to consumable products such as salad oil, fish oil, and cooking oil as antioxidants. Chapter 4 - In the last decades, the beneficial influence of green tea on human health has been related to the antioxidant capacity (AC) of its phenolic constituents. The latter has originated systematic studies of the AC of green tea and/or its pure antioxidants. Different methodologies have been used with this purpose. The methods are based on: (1) Estimation of the consumption by additives of stable free radicals (DPPH, and ABTS radical cation); (2) Evaluation of the protection given by antioxidants to a target being oxidized by free radicals (ORAC, TOSC, LDL oxidation assay); (3) Estimation of the steady state of free radicals before and after addition of additives (TAR); (4)Estimation of the reducing power capacity of the additives (FRAP, CUPRAC). The assays differ in the experimental conditions and their chemistry. Therefore, different conclusions could be obtained depending on the methodology used. For example, green tea presents a lower AC than peumus boldus by ORAC (oxygen radical absorbance capacity) method when fluorescein is used as target molecule. However, if pyrogallol red is used as probe, green tea appears with an ORAC index six times higher than peumus boldus. In the present review, we discuss the advantages, and disadvantages of the different methodologies employed to evaluate the AC of green tea. Chapter 5 - Tea is one of the most popular and widely consumed beverages in the world and it is derived from the infusion of tea leaves (Camellia sinensis L.). Different commercial types of tea are available, including black tea, oolong tea (semi-fermented) and green tea,

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which differ on their processing and chemical composition. All these types of tea have been reported to prevent multiple diseases such as cancer, heart conditions, among others. On the other hand, lemongrass (Cymbopogon citratus Stap) is a rich source of essential oils, widely employed in infusions, soaps, and perfumes, and it has been reported to possess gastrointestinal and analgesic properties. In the present study, green tea (Camellia sinensis L.) and lemongrass (Cymbopogon citratus Stap) leaves were collected from Río Azul and Porvenir de Marona, Perú. The anti-oxidant capacity of green tea and lemongrass extracts was evaluated using the DPPH method and it was observed that the IC50 values for green tea was 32.4 ± 0.39 μg/mL and 1350 ± 47 μg/mL for lemongrass. These two plants (green tea and lemongrass) were employed to design multiple infused beverages and it was determined that an infused beverage containing 10 mg/mL total extract (50% green tea and 50% lemongrass) reported a total catechin content of 24.4 ± 0.65 mg/100mL, a DPPH inhibition percentage of 88.6%, and exhibited the greatest acceptance for sensory attributes such as flavor, color, and aroma (values of 6.8, 9.0, and 8.0 respectively) based on Friedman Multiple Comparisons test. The taste panel results also indicated that the optimized acidity and sweetness were to be set at pH 3.1 and 11°Brix, while the optimum infusion time based on the total catechin content was 7 minutes. The pasteurization profile at 90°C for 5 minutes achieved mesophilic microorganisms counts of <10 cfu/mL. The maximum shelf-life of the beverage was achieved at 15ºC based on total catechin content and absence of browning (catechin degradation). Furthermore, the formulated beverage was well accepted by the test panel and presented similar anti-oxidant capacity than commercial green tea based beverages. Chapter 6 - Green tea is a good source of various polyphenolic compounds and minerals which are powerful antioxidants. The effects of selenium-containing green tea (Se-GTE; 1.4 mg selenium/kg) and China green tea (CH-GTE; 0.13 mg selenium/kg) on food consumption and body weight gain were investigated using a rat model. In addition, the total phenolic contents (TPC), antioxidant/antiradical activities of these teas were determined in vitro. Both teas had a satiating influence on experimental rats, as evidenced by their ability to decrease food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE), although a statistically significant decrease over the control rats was achieved only for Se-GTE treatment. In addition, the final body weight of rats gavaged with Se-GTE was significantly lower (P = 0.0063) than that of the water-gavaged control rats and this corresponds to 8.5% reduction in body weight relative to the control group. In contrast, rats gavaged with CH-GTE showed only 1.8% reduction in the final body weight relative to the control group. The reduction in food intake over a short period compared to a control rats preloaded with the same volume of water suggests that the decrease in food intake was mainly a consequence of a satiating effect, rather than a stomach distension effect. The underlying mechanism responsible for this satiating effect was not identified as part of this study. It is also important to mention that water intake for the groups given the tested teas was similar to that of rats given water only and no significant differences were observed. Se-GTE had significantly higher TPC (P < 0.0001), higher ferric reducing antioxidant power (FRAP) (P < 0.01), higher diphenyl-picrylhydrazyl (DPPH) free radical scavenging activity (P < 0.05), higher ferrous-ion chelating activity (P < 0.05-0.01), and higher selenium contents (P < 0.0001) than CH-GTE. A strong positive correlation was found between the TPC, and the FRAP, DPPH, and the ferrous-ion chelating activities in both teas, indicating

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that the polyphenolic compounds are the major contributors of the antioxidant/antiradical activities. When rats were gavaged with water-soluble tea extract (10 ml/kg/day) of Se-GTE for four consecutive days, serum FRAP increased significantly (P = 0.0002) as compared to water-gavaged controls. The level of serum FRAP in rats gavaged with CH-GTE increased slightly when compared with the control rats but not to a significant extent. These results indicate that green tea may have the ability to elevate circulating antioxidant potentials in vivo and this ability is dependent on the type of tea used. The observed results suggest that the reduction in food intake and decrease in body weight in experimental animals may be a consequence of antioxidant mechanisms played by the polyphenolic compounds and the minerals found in green teas. Green tea, especially the one with a high level of organic selenium may provide a good satiety inducer and weight management modulator. Chapter 7 - Development of obesity appears to be influenced by a complex array of genetic, metabolic, and neural frameworks, together with one’s behavior, eating habit, and physical activity. The incidence of obesity is significantly increasing in virtually all societies of the world and causes important pathological consequences such as cardiovascular diseases and type 2 diabetes mellitus. Furthermore, rates of pediatric obesity have increased dramatically over the past decade resulting cardiovascular, metabolic, and hepatic complications. Since ancient times, green tea has been considered as a traditional medicine as a healthful beverage. Major components of green tea including epigallocathchin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) have been received significant scientific attention and public awareness for its beneficial effects on prevention and therapeutic treatment of cardiovascular diseases and cancer. Anti-obesity effects of EGCG have been demonstrated in various in vitro and in vivo models showing that EGCG treatment could reduce food intake, glucose uptake, blood glucose level, and differentiation and growth activity of fat cells together with modulation of lipolytic and lipogenic activities. In these actions, large number of molecules including laminin receptor, fatty acid synthase, glucose-6-phosphate dehydrogenase, cyclin-dependent kinase 2, CCAAT/enhancer binding proteins, mitogen activated protein kinases, AMPactivated protein kinase, phosphatidylinositol-3-kinase, peroxisome proliferator activated receptor, and number of transcription factors including NF-kB were suggested to be involved. This review will focus on the effects of ECGC at molecular level in terms of (1) receptor recognition, (2) modulation of signaling pathways, (3) lipid metabolism and altering the lipogenic and lipolytic activities leading to metabolic changes and apoptosis, and (4) final outcome in vitro and in vivo. Chapter 8 - The indiscriminate usage of synthetic chemicals and pesticides has lead to a widespread contamination of land, water and air with harmful xenobiotics. The exposure to these toxicants results in severe health effects on organisms. Even some natural foods contain harmless chemical species (nitrate) which however become toxic upon certain conditions. Hence it is pertinent to focus attention on commonly consumed plant food materials that can potentially neutralize the toxicity damage caused by environmental agents. One of the most important sources of antioxidants is green tea. This review focuses on the mechanisms of oxidative damage caused by different xenobiotics and the defensive action of green tea in mitigating the damage. It is concluded that tea polyphenols, catechins and flavonoids scavenge reactive oxygen species (ROS) and render the hepato-protective effect. However it

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is important to note that the protective effects of green tea extract are rendered irrespective of the xenobiotic involved thereby suggesting the involvement of a common biochemical pathway. Chapter 9 - Green tea is a well-known biomaterial with various high biological functions. Irradiation was introduced to develop a new processing method to improve color of the extract, resulting in a higher applicability without any adverse change to the beneficial functions such as inhibitory effects of oxidation, melanin hyperpigmentation on the skin, and others. To investigate the application of irradiated green tea leaf extract for a real cosmetic composition, the physiological activities of irradiated green tea leaf extract powder dissolved in butylenes glycol and ethanol were compared to a commercial green tea extract product. Furthermore, a cream lotion was manufactured using the powder and the physiological activities were compared. Results showed that the irradiation of the green tea leaf extract and the freeze-dried powder from the extract had the same physiological activities as the commercial product in a cosmetic composition. Addition of irradiated green tea leaf in a patty can retard lipid oxidation and add the biological functions green tea possessed, however, the intensity of off-odor produced from green tea is a concern. Using irradiated green tea powder, the off-odor problem can be solved. Some research utilizing green tea byproducts were investigated. Irradiation of green tea byproducts extract showed higher biological functions than that of non-irradiated counterparts. Therefore, irradiation technology can be a useful method to improve biological functions and industrial applicability of green tea and its byproducts. Chapter 10 - Many studies suggest that beneficial effect of green tea for human health is mainly attributed to catechins, polyphenol compounds, having anti-oxidative activity. In the last decade, new aspect of green tea catechin has emerged as angiogenesis inhibitor. Angiogenesis is forming new blood vessels from a pre-existing blood vessel and involved in various diseases including tumor growth and metastasis, diabetic retinopathy and atherosclerosis. In addition, it has been demonstrated that obesity is prevented by angiogenesis inhibitor and suggested that angiogenesis is closely associated with Alzheimer’s disease. Thus, angiogenesis inhibitors in food would be expected to prevent these diseases and give beneficial effect on our health. Interestingly, inhibitory effect of green tea catechin on angiogenesis has been demonstrated in various models suggesting green tea catechin could suppress cancer. Furthermore, it has been revealed that a higher consumption of green tea catechin reduces human body fat and is associated with a lower prevalence of cognitive impairment. These evidences suggest that anti-angiogenic activity of green tea catechin might play important roles in human health. Chapter 11 - The present article introduces our study related to the neuroprotective effect of γ-glutamylethylamide (theanine), a component Japanese green tea (Camellia sinensis), on cerebral ischemia. Theanine (1 mg/kg) significantly decreased the size of the cerebral infarcts in a 4 h middle cerebral artery (MCA) occlusion model in mice. However, theanine did not affect the cerebral blood flow, brain temperature and physiological variables (pH, pCO2, pO2 and hematocrit) in this model. Theanine also reduced the alterations of NeuN (neuron), GFAP (astrocyte) and Iba 1 (microglia) expression levels at 24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by bicuculline, γ-aminobutyric acidA (GABAA) receptor antagonist, but not 3-mercaptopropionic acid, glutamate decarboxylase inhibitor. Furthermore, theanine (0.3 and 1 mg/kg) significantly prevented the impairment of spatial memory in rats subjected to twice-repeated cerebral ischemia, 7 days after the second

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reperfusion. In addition, theanine (1 mg/kg) significantly inhibited the decrease in the number of surviving cells in the hippocampal CA1 field in the same rats. These results suggest that theanine directly provides neuroprotection against cerebral ischemia and its neuroprotective effect is mediated, at least in part, by GABAA receptors, and that it may be clinically useful for preventing cerebrovascular disease. Chapter 12 - Standing after water, tea signifies the second most frequently consumed beverage worldwide, which varies its status from a simple ancient drink and a cultural tradition to a nutrient endowed with possible neurobiological-pharmacological actions beneficial to human health. Accumulating evidence suggests that oxidative stress resulting in reactive oxygen species generation plays a pivotal role in neurodegenerative diseases, supporting the implementation of radical scavengers and metal chelating agents, such as natural tea polyphenols for therapy. Vast epidemiology data indicate a correlation between occurrence of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases and green tea consumption. In particular, literature on the putative novel neuroprotective mechanism of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG) strengthens the notion that diverse molecular signaling pathways, participating in the neuroprotective activity of EGCG, thus rendering this natural compound as potential agent to reduce the risk of various neurodegenerative diseases. In the present article, we review the studies concerning the mechanisms of action implicated in EGCG-induced neuroprotection in the brain and discuss the vision to translate these findings into a lifestyle arena. Chapter 13 - Green tea (Camelia sinensis), widespread in the whole world, possesses many health protective properties. Its polyphenolic compounds, mostly flavonols, better known as catechins (e.g. epigallocatechin-3-gallate [EGCG]), are considered to be responsible for the health protective effects. Tea consumption may have its strongest effect among patients with cardiovascular disease. Recently, studies suggested that high flavonoid intake may reduce coronary heart disease by lowering blood lipid levels and inhibiting the oxidation of low-density lipoproteins. Trials showed that short- and long-term tea consumption could reverse endothelial dysfunction in subjects with documented coronary heart disease, providing one possible mechanism for an effect of tea in patients with cardiovascular disease. Interestingly, EGCG has also been shown to have electrophysiological effects by blocking HERG potassium channels, the most important repolarizing potassium channel in the human ventricle that forms the α-subunit of the rapid delayed rectifier current IKr. Inhibition of HERG channels may have profound effects on cardiac repolarization. Furthermore, tea flavonoids have been reported to exhibit metabolic effects in terms of antidiabetic properties. This review summarizes the latest studies on cardiovascular effects of green tea and discusses the possible cardiac health benefits of green tea consumption. Chapter 14 - For the past two decades, many researchers have been investigated the potential cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-Ogallate (EGCG) has been shown to be the most active and major polyphenolic compound from green tea. The mechanisms of action of EGCG have been extensively investigated, but the mechanisms for the cancer-preventive activity of EGCG are not completely characterized and many features remain to be elucidated. Recently we have identified 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. This article reviews some of the reported mechanisms and possible targets for the action of EGCG. Especially, we focus the current understanding of signaling pathway for physiologically relevant EGCG through the 67LR for

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cancer prevention. This information shed new light on the molecular basis for the cancerpreventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer. Chapter 15 - Green tea and its major constituent epigallocatechin gallate (EGCG) have been extensively studied as a potential chemopreventative and/or treatment for a variety of diseases including breast cancer. Experimental evidence is supported by epidemiological studies that have shown an inverse relationship between green tea consumption and the incidence of breast cancer. Numerous studies have demonstrated that EGCG is cytotoxic toward both estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. These studies have highlighted potential mechanisms for the actions of EGCG, such as the induction of apoptosis, the alteration of the expression of cell cycle regulatory proteins critical for cell proliferation, inhibition of the chymotrypsin-like proteasome, inhibition of angiogenesis, as well as inhibition of cell invasion and metastasis. Importantly, these effects occur independently of estrogen receptor expression. This chapter will provide evidence for these events and other molecular mechanisms that significantly contribute to the actions of EGCG in vitro. The utility of green tea extract or EGCG as a breast cancer treatment and chemopreventative has also been extensively investigated using various in vivo models of estrogen receptor-positive and estrogen receptor-negative breast cancer (ie., chemical carcinogenesis and xenograft models). Evidence for EGCG-mediated tumor suppression and the major molecular mechanisms for this effect, such as the induction of apoptosis, the inhibition of angiogenesis and modulation of the expression of cell signaling proteins are also fully examined. These in vivo studies have led to investigations which have focused on ways to improve the actions of EGCG, by either using it as part of a combination therapy or by synthesizing pro-drugs of EGCG. Both of these have been done in order to enhance the bioavailability, stability and efficacy of EGCG. These new compounds and drug combinations have significantly improved the tumor suppression potential of EGCG and provide an exciting future for this multi-faceted phytochemical in the prevention and treatment of breast cancer. Chapter 16 - Colorectal cancer is a global problem that accounts for over 50,000 cancerrelated deaths each year in the United States. Americans have about a one in 20 lifetime risk of developing colorectal cancer. It affects primarily those over 65, but risk starts increasing at age 40. Colorectal cancer develops following disruptions in key cancer-causing genes (oncogenes) like K-ras and β-catenin and tumor suppressor genes like gate keeper APC and p53, and early detection greatly increases the chances of survival. Most cancers are related to a combination of hereditary and environmental factors, and such factors can either contribute to the initiation of cancer or the prevention of tumor development. There is persuasive epidemiological and experimental evidence that a phytochemical-enriched diet may be involved in the prevention of colon cancer. Therefore, the use of dietary compounds for prevention and therapy of colorectal cancer would be of major importance with potentially fewer side effects than therapeutic drugs. Green tea has received much attention as a suitable dietary agent because of its anti-tumorigenic activity. The most active constituents of green tea are catechins, including epigallocatechin 3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC). Many laboratories, including ours, have reported preventive effects with green tea components in cancers of the gastrointestinal tract, lung, skin, prostate, and breast. A mechanistic study indicated that green tea decreased the total levels of early carcinogenesis biomarkers and increased tumor suppressor proteins; in

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addition, reports related to new molecular targets affected by green tea in chemoprevention study have been increased. Since the preponderance of the data strongly indicates significant anti-tumorigenic benefits from green tea polyphenols, this chapter will summarize the current knowledge of molecular targets of green tea research in human colorectal cancer prevention. Chapter 17 - Green tea obtained from the leaves of the plant, Camellia sinensis, is one of the most popular beverages in the world. The major polyphenolic compounds in green tea are catechin derivatives (i.e., flavan-3-ols), and their composition varies depending on the season of harvest and the manufacturing process. The inhibitory activities against DNA polymerases (pols) of catechin derivatives such as (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin gallate (Cg), (-)-epicatechin gallate (ECg), (-)gallocatechin gallate (GCg), and (-)-epigallocatechin gallate (EGCg) were investigated. Among these eight catechins, several inhibited mammalian pols with EGCg being the strongest inhibitor of pols and with IC50 values of 5.1 and 3.8 M, respectively. EGCg did not influence the activities of plant pols, such as pols and , or prokaryotic pols, and had no effect on the activities of DNA metabolic enzymes such as calf primase of pol , T7 RNA polymerase, T4 polynucleotide kinase, or bovine deoxyribonuclease I. Some tea catechins also suppressed human cancer cell growth and/or TPA (12-O-tetradecanoylphorbol13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity for these compounds was the same as that of their anti-inflammatory activity rather than their anticancer activity. Based on these results, the relationship between the structure of tea catechins and their bioactivities is discussed. Chapter 18 - The anti-tumor effect of green tea, especially its major constituent EGCG, has been demonstrated in several animal experiments and its ability to induce apoptosis of most of cancer cell lines has been further documented in cell culture models. A number of mechanisms for how green tea impacts cancer have been proposed. These mechanisms basically include intervention of cell signal transduction pathways or changes of cell epigenetic processes. Telomerase has been recognized as a novel target of green tea. This important enzyme is largely localized to cancer cells, is responsible for the maintenance of telomeres so that cancer cells can escape the replication problem due to their linear chromosomes, and it has been shown to be reactivated in almost all tumor tissues. Telomerase has been found to be inhibited by green tea in telomerase-positive cancer cell lines. This analysis assesses the progress on research of the mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells. A number of mechanisms for how green tea works through this pathway have been proposed. Since telomerase has been identified as a potential molecular target for cancer treatment, and green tea has been shown to inhibit telomerase, clarifying the specific mechanisms for how green tea functions in this pathway should shed new light on the potential to design effective and novel preventive or anti-cancer approaches using green tea. Chapter 19 – Background: Chronic obstructive pulmonary disease (COPD) is a common cause of morbidity and leading cause of death in the world. Cigarette smoking has been established as the principal risk factor for COPD. While 95% of COPD patients are, or have been, cigarette smokers, only 20% of smokers develop COPD. Therefore, other factors may protect against or contribute to the development of the disease. Objective: To investigate whether green tea intake can reduce the risk of COPD. Study design: Case-control study conducted in Aichi, Gifu and Kyoto during 2006. Subjects: A total of 278 eligible patients (244 men and 34 women; mean age 66.5 (SD 6.7) years), with COPD diagnosed by

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Helen McKinley and Mark Jamieson

respiratory physicians as the primary functionally limiting illness within the past four years, were referred from the outpatient departments of six hospitals in central Japan. During the same period, 335 age-matched community-dwelling controls (267 men and 68 women; mean age 65.3 (SD 5.5) years) were recruited from the same catchment areas as the cases. Methods: Interviews were conducted face-to-face to collect information on demographic characteristics and habitual green tea consumption using a structured questionnaire. The reference recall period was set at 5 years before diagnosis for cases or 5 years before interview for controls. Tea drinkers were defined as persons who drunk both Sencha, Bancha, Hojicha and Genmaicha types of green tea once a week or more often. The effect of green tea intake on the COPD risk was assessed by multivariate logistic regression analysis. Results: The prevalence of regular green tea consumption was significantly higher (p < 0.01) among controls (n = 64, 19.3%) than cases (n = 27, 9.7%). Among drinkers, life-long exposure (years of drinking) was similar between the two groups (p = 0.70), and about half of them drank one to three cups of green tea daily. The risk of COPD appeared to decrease with green tea drinking. The adjusted odds ratio was 0.45 (95% confidence interval 0.25-0.81) for tea drinkers relative to non-drinkers after accounting for confounding factors including age, body mass index, gender, marital status, residential location, education level, retirement status, smoking status and alcohol consumption. Conclusion: The preliminary evidence suggests that green tea intake may be protective against COPD for Japanese adults. More research is required to confirm the observed finding and to understand the biological mechanism underlying the disease prevention role of green tea. Chapter 20 - Providing that there has been a dramatic increase in the incidence of diabetes mellitus associated with long-term complications, it is critical to find a natural nutritional material aimed at reducing the prevalence of diabetes which threatens human health over the world . Currently, green tea, only next to water, is the most widely consumed beverage in the world and exerts beneficial bioabilities, such as anti-inflammative, anti-oxidative, antimutagenic, etc. With increasing interest in the health promoting properties of tea and a significant rise in scientific investigation, in vitro and animal studies provide more and more strong evidence that green tea consumption has a great prophylactic and therapeutic effect on diabetes and its complications, which is intensively associated with its mainly bioactive components, such as tea polyphenols and tea polysaccharides. In this article, we will summarize effect of green tea on diabetes, especially anti-diabetic ability of tea polyphenols and polysaccharides, discuss possible mechanisms, and make perspectives and future directions in this area. Chapter 21 - Green tea, which is consumed world wide as a beverage, is known to have many beneficial effects on human health. Green tea contains various biologically-active materials including catechins, flavonols and caffeine. Of them, catechins are the major constituents consisting of 30% of water-extractable materials. Pharmacological implication has been mainly made upon (-)-epigallocatechin-3-gallate (EGCG), as it is the most abundant catechin in green tea extracts. The second biologically important catechin is (-)-epicatechin-3gallate (ECG), which is called one of gallated catechins as EGCG. To date, there is no prominent evidence for green tea consumption to determine whether be beneficial or harmful in metabolic diseases, such as type 2 diabetes and obesity. It may be due to catechins having a variety of function in human body. This chapter will focus on action mechanism of EGCG on ATP-sensitive potassium channels, which manifests as actual phenotypes in cardiac and beta-

Preface

xix

cell function. Additionally, changes by green tea or gallated catechins in insulin resistance will be reviewed. Thereafter, a right method to use EGCG as a supportive regimen for diabetic care and obesity will be introduced. New era should come with modifying natural products fit to human well-being. Chapter 22 - World-wide tea production has reached 2.97 x 106 metric tons/year, and more than 75% of tea products are black tea. In recent years, green tea which contain catechin derivatives such as (-)-epicatechin (EC) (1), (-)-epicatechin gallate (ECg) (2), (-)epigallocatechin (EGC) (3), and (-)-epigallocatechin gallate (EGCg) (4) has been recognized as a useful functional food. However, recent development of enzymatic processes now makes it possible to produce epitheaflagallin (5), epitheaflagallin 3-O-gallate (6) and theaflavin (TF) (7) derivatives from catechins, which are components of black tea. Epitheaflagallin and epitheaflagallin 3-O-gallate are preferentially synthesized from EGC (3) and EGCg (4) in green tea extracts in the presence of laccase and gallic acid. Takemoto and colleagues have developed a Camellia sinensis cell culture system containing peroxidase and hydrolases of ECG (3) and EGCg (4) to produce theaflavin (7) from tea catechins. These biocatalytic processes allow us to preferentially convert catechin derivatives in a crude mixture of green tea into different compounds, and, thus, to improve the composition of the catechins present in green tea. Chapter 23 - Tea is rich in polyphenols which are contained in the leaves and stems of the tea plant. (-)-Epigallocatechin gallate (EGCG), the major and most active component of green tea catechins, acts as an antioxidant in the biological system, and is absorbed and distributed mainly into the mucous membranes of the small intestine and liver; more interestingly it can cross the blood brain barrier. Oxidative stress, a condition of cellular prooxidant-antioxidant disturbance which favors the prooxidant state, induces the production of lipid peroxide (LPO), reactive oxygen species (ROS) and free radicals in membrane lipids. Oxidative stress causes deterioration of a wide variety of cellular enzymes, subsequently exacerbating neurodegenerative process. Aging leads to a decline in memory-related learning ability. Oxidative damage to the brain is associated with age-related cognitive dysfunction but some antioxidants are effective in alleviating this dysfunction for Alzheimer’s disease (AD) model animals. Moreover, a decrease in hippocampal LPO level improves spatial cognition learning in aged rats and an increase in antioxidative activity in the hippocampus prevents or ameliorates the impairment of learning ability in AD model rats produced by the infusion of amyloid-β (1-40) (Aβ1-40) into their cerebral ventricle. Catechins have a protective effect against age-related neurological diseases caused by oxidative damage. Epidemiological studies report that a higher consumption of green tea is associated with lower prevalence of cognitive impairment in elderly people. This chapter describes the beneficial effects of green tea catechins on neuronal functions and neuronal diseases such as dementia. Short Commentary - The scientific understanding of many biological functions involved in maintaining human health (e.g., role of gap junctions in homeostatic control of cell behaviors; multi-state nature of carcinogenesis; role of gap junctions in the tumor promotion step of carcinogenesis; toxicant induced oxidative stress in many human diseases; role of oxidative stress by tumor promoting chemicals; role of green tea components as anti-oxidants; preventive effects of green tea components on toxicant-inhibition of gap junction function and tumor promotion) provides some evidence that green tea could prevent some oxidative stresslinked human diseases. However, as has been demonstrated in experimental examples, it will

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be unlikely that any agent, such as green tea, will be a “silver bullet” to protect against all toxic agents. On the other hand, it does seem it would cause “no harm” and might, in some cases, even be beneficial.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 1

CENTRAL FUNCTIONS OF GREEN TEA COMPONENTS M. Furuse 1,2,*, N. Adachi2, S. Tomonaga2, H. Yamane2 and D. M. Denbow3 Laboratory of Advanced Animal and Marine Bioresources, Faculty of Agriculture and 2Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan 3 Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306, USA 1

ABSTRACT Tea (Camellia sinensis) is widely consumed throughout the world and has a number of biologically active substances such as caffeine, catechins, and L-theanine (γglutamylethylamide). Tea consumption is generally known to induce a feeling of relaxation which may be mediated by either catechins, L-theanine, or both, since caffeine stimulates locomotor activities. The catechin (-)-epigallocatechin gallate (EGCG) occurs abundantly in tea. Moreover, frequent consumption of green tea results in high levels of EGCG in the blood and brain. Catechins, which are flavonoids, affect the central nervous system (CNS). The therapeutic effects of flavonoids may involve their binding to γ-aminobutyric acid (GABA)A receptors, which is a major inhibitory neurotransmission system. Recently, EGCG was shown to bind to GABAA receptors in vitro and to induce a sedative effect through GABAA, but not GABAB, receptors in the brain. L-Theanine, a derivative of glutamate, is a unique amino acid occurring only in green tea and a few other plants. After administration L-theanine concentrations were increased in serum, liver and brain, suggesting that L-theanine can cross the blood-brain barrier. Intravenous administration of L-theanine was shown to affect the cortex, hippocampus and amygdala, and increase the alpha-band component of electroencephalograms in rats. More recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron excitation in human subjects or influencing the

*

Correspondence author. Tel: +81-92-642-2953; Fax: +81-92-642-2953; E-mail address: [email protected] (M. Furuse)

2

M. Furuse, N. Adachi, S. Tomonaga et al. secretion and function of neurotransmitters in the CNS. We discuss the central functions of green tea components such as EGCG and L-theanine in the CNS.

INTRODUCTION Green tea, the most consumed beverage in the world except for fresh water, is a drink made from the steamed and dried leaves of the Camellia sinensis plant, a shrub native to Asia. Green tea is widely consumed in Japan, China, and other Asian nations, and is becoming more popular in Western nations. Black tea is also made from the plant, but unlike green tea, it is made from leaves that have been fermented. Due to differences in the fermentation process, a portion of the active compounds are destroyed in black tea, but remain active in green tea. The active constituents in green tea are a family of polyphenols (catechins) and flavonols which possess potent antioxidant activity. Tannins, large polyphenol molecules, form the bulk of the active compounds in green tea, with catechins comprising nearly 90%. Several forms of catechin are present in the plants (Figure 1). Among them, epigallocatechin-3-gallate (EGCG) is the most abundant flavonoid in tea (Arts et al., 2000) and particularly in green tea (Campbell et al., 2004). Approximately 26% of the solid weight of green tea extract is tea polyphenols, of which 11% are EGCG (Suganuma et al., 1998). Thus, EGCG is primarily responsible for the pharmacological actions of tea. Moreover, frequent consumption of green tea results in high levels of EGCG in the blood and brain (Suganuma et al.., 1998; Kim et al., 2000). Some reports indicate green tea may have the ability to help prevent cancers of the skin, esophagus, stomach, colon, pancreas, lung, bladder, prostate, and breast. Green tea contains chemicals known as polyphenols, which have many beneficial effects such as antioxidant, anticarcinogenic, and antiviral activity (Frei and Higdon, 2003; Tachibana et al., 2004). Compared with the peripheral actions of catechins, the information for the central effect is limited to date. L-Theanine (γ-glutamylethlamide) is a unique amino acid only occurring in green tea and a few other plants, and is a derivative of glutamate (Figure 2). After administration, Ltheanine concentrations increased in the serum, liver and brain (Yokogoshi et al., 1998a, b), suggesting that L-theanine can cross the blood-brain barrier. Intravenous administration of Ltheanine was shown to affect the cortex, hippocampus and amygdala and increase the alphaband component of electroencephalograms (EEG) in rats (Kakuda et al., 2000a). More recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron excitation in human subjects (Kimura et al., 2007) or influencing the secretion and function of neurotransmitters in the central nervous system (CNS) (Terashima et al., 1999). Green tea also contains other active substances such as caffeine. While caffeine stimulates locomotor activities in rats (Mukhopadhyay and Poddar, 1995), tea consumption is generally known to induce a feeling of relaxation (Juneja et al., 1999) which may be mediated by either catechins, L-theanine, or both. We review here the central effects of EGCG and Ltheanine with reference to pharmacology, pharmacokinetics, and toxicology.

Central Functions of Green Tea Components

3

CENTRAL FUNCTION OF EGCG The effects of flavonoids on the CNS have been studied (Zanoli et al., 2000), with particular reference to their role as therapeutics involving γ-aminobutyric acid (GABA)A receptors (Huen et al., 2003). The characteristic structure of flavonoids, which have binding capacity for GABAA receptors, is similar to that of EGCG. Recently, EGCG has been confirmed to bind to GABAA receptors in vitro (Campbell et al., 2004). According to Adachi et al. (2006), intracerebroventricular (i.c.v.) injection of EGCG clearly suppressed the number of vocalizations (Figure 3). The cumulative number of vocalizations increased with time, but those of EGCG-treated groups remained low during the experimental period. Behavioral patterns are shown in Table 1. Central EGCG increased sleep-like behavior, but the reverse was true for active wakefulness.

Figure 1. Structures of tea catechins.

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M. Furuse, N. Adachi, S. Tomonaga et al.

NH2

H N

HO O

O

L-Theanine NH2 OH O H

HO O

O

L-Glutamate Figure 2. Structures of L-theanine and L-glutamate. 1200

Control EGCG 50 μg EGCG 100 μg

Vocalization (count)

EGCG 200 μg 900

600

300

0

1

2

3

4

5

6

7

8

9

10

Time (min) Figure 3. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on cumulative number of vocalizations for 10 min in response to social separation stress in chicks. Data are expressed as means±S.E.M. Significant regression equations were obtained between number of vocalizations and EGCG (μg) as follows: vocalization (count) = 0.001 (SE 2.063 x 10-4) EGCG3 + 0.243 (SE 0.059) EGCG2 – 26.036 (SE 3.895) EGCG + 875.571 (SE 55.016), R2 = 0.872, P<0.0001. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

Central Functions of Green Tea Components

5

Corticosterone concentration (ng/ml)

10

8

6

4

*

*

2

0

EGCG (μg)

0

50

100

200

Figure 4. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on the plasma corticosterone concentration after social separation stress for 10 min in chicks. Data are expressed as means±S.E.M. *, Significantly different from control at P<0.05. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

Table 1. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on various behavioral categories in response to social separation stress for 10 min in chicks EGCG (μg) Active wakefulness Standing/sitting motionless with eyes open Standing motionless with eyes closed Sitting motionless with head drooped (sleep-like behavior) Total

0 543±15

50 172±107*

100 51±51*

200 13±9*

57±15

302±101

164±34

193±67

0±0

0±0

26±26

20±17

0±0

126±87

359±44*

374±66*#

600

600

600

600

Data are expressed as means±S.E.M. in seconds. Significant regression equations were obtained between behavior and EGCG (μg) as follows: active wakefulness (s) = 0.025 (SE 0.006) EGCG2 – 7.604 (SE 1.306) EGCG + 529.67 (SE 49.949), R2 = 0.730, P<0.0001 and sleep-like behavior (s) = 1.868 (SE 0.402) EGCG + 45.776 (SE 48.602), R2 = 0.464, P<0.0001. * and #, Significantly different from control and 50 μg of EGCG respectively at P<0.05. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

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M. Furuse, N. Adachi, S. Tomonaga et al.

Figure 5. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 500 ng of picrotoxin, or EGCG plus picrotoxin on cumulative number of vocalizations in response to social separation stress for 10 min in chicks. Data are expressed as means±S.E.M. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

The i.c.v. injection of EGCG significantly decreased plasma corticosterone release compared with the effect of saline (Figure 4). Figure 5 shows the effect of i.c.v. injection of EGCG with or without the GABAA receptor antagonist picrotoxin on the number of vocalizations during the 10-min social separation stress. The suppressive effect of EGCG on distress-induced vocalizations was attenuated by co-administered picrotoxin. Figure 6 shows the effect of i.c.v. injection of EGCG with or without picrotoxin on plasma corticosterone concentration during social separation stress. Picrotoxin significantly increased the plasma corticosterone concentration compared with the effect of EGCG. Furthermore, picrotoxin attenuated the decrease in plasma corticosterone caused by EGCG. Figure 7 shows the effect of i.c.v. injection of EGCG with or without the GABAB receptor antagonist CGP 54626 (3-N-(1-(3,4-dichlorophenyl)ethylamino)-2-hydroxypropyl cyclohexylmethyl phosphinic acid hydrochloride) on the number of vocalizations during the 10-min social separation stress. Significant effects of EGCG and time were detected, but no significant effect was observed with CGP54626. The effect of EGCG could not be attenuated by CGP54626. These results indicate that EGCG has sedative and hypnotic effects in the brain, acting at least partially through GABAA receptors, which consequently moderates an acute stress response.

Central Functions of Green Tea Components

7

Corticosterone concentration (ng/ml)

30

*

*

24

18

12

6

0

EGCG (μg)

0

0

50

50

Picrotoxin (ng)

0

500

0

500

Figure 6. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 500 ng of picrotoxin, or EGCG plus picrotoxin on plasma corticosterone concentration for 10 min in chicks. Data are expressed as means±S.E.M. *, Significantly different from 50 μg of EGCG alone at P<0.05. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

In addition, an anti-anxiety effect of EGCG was also observed in mice (Vignes et al., 2006). However, green tea consumption does not induce sleep even though green tea contains abundant EGCG. This may be explained by the caffeine in green tea (Kuo et al., 2005). Caffeine acutely stimulates the autonomic nervous system and increases wakefulness (Quinlan et al., 2000). Consequently, it is conceivable that the hypnotic effect of EGCG may be countered by the action of caffeine in green tea. The 4-carbonyl group of flavonoids is important for binding to GABAA receptors (Marder and Paladini, 2002). Apigenin, one of the major flavonoids, binds to GABAA receptors (Dekermendjian et al., 1999). The galloyl group of EGCG contains a carbonyl group which may substitute for the 4-carbonyl group of apigenin (Campbell et al., 2004). Adachi et al. (2007b) investigated whether the galloyl group of EGCG is necessary to act at GABAA receptors. The structure of (-)-epigallocatechin (EGC), one of the catechins, is similar to that of EGCG except for the lack of the galloyl group (Figure 8). The i.c.v. injection of EGC or EGCG clearly suppressed total spontaneous activity and the number of vocalizations (Figure 9). Table 2 shows the effect of EGC or EGCG on various behavioral categories of chicks during 10 min behavior observations. Significant effects of EGC or EGCG were observed on the time for active wakefulness and the time for sleep-like behavior. Compared with the control group, EGC and EGCG increased sleep-like behavior, but the reverse was true for active wakefulness. The suppressive effect of EGC on increasing distress-induced spontaneous activity was attenuated by co-administration of picrotoxin, a GABAA receptor antagonist (Figure 10).

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M. Furuse, N. Adachi, S. Tomonaga et al.

Figure 7. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 10 ng of CGP54626, or EGCG plus CGP54626 on the cumulative number of vocalizations in response to social separation stress for 10 min observation in chicks. Data are expressed as means±S.E.M. Reproduced from Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from Elsevier as the authors’ right.

OH OH O

HO

OH O

OH

OH OH

C O

OH

(-)-Epigallocatechin gallate (EGCG)

OH OH O

HO

OH OH

OH Figure 8. Structural differences in EGCG and EGC. The broken line indicates the galloyl group.

Central Functions of Green Tea Components

9

Figure 9. Effect of i.c.v. injection of EGC (109, 218 nmol) or EGCG (109, 218 nmol) on spontaneous activity (upper panel) and total number of distress vocalizations (lower panel) for 10 min in response to social separation stress in chicks. Data are expressed as means±S.E.M. Groups with different letters are significantly different (P<0.05). Reproduced from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. & Furuse, M. (2007b) Galloyl group is not necessary for a sedative effect of catechin through GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science Publishers Ltd.

In addition, EGC suppressed the number of vocalizations, and co-administration of picrotoxin attenuated this response (Figure 10). A significant effect of EGC was observed on the time for active wakefulness (Table 3). On the other hand, picrotoxin did not decrease the time of active wakefulness. Both EGC and picrotoxin significantly affected the time for sleeplike behavior, respectively. The effects of EGC such as decrease in active wakefulness and increase in sleep-like behavior were attenuated by picrotoxin in the CNS. It is concluded that catechins have sedative effects acting through GABAA receptors under an acute stress condition irrespective of the presence of the galloyl group.

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M. Furuse, N. Adachi, S. Tomonaga et al.

Spontaneous activity (arbitrary unit)

200

a

150 ab 100 b

50

b 0 EGC (nmol) 0 Picrotoxin (pmol) 0

Vocalization (count)

900

0 830

109 0

109 830

a a

a

600

300

b 0 EGC (nmol) Picrotoxin (pmol)

0 0

0 830

109 0

109 830

Figure 10. Effect of i.c.v. injection of EGC (109 nmol), picrotoxin (830 pmol) or both on spontaneous activity (upper panel) and total number of distress vocalizations (lower panel) for 10 min in response to social separation stress in chicks. Values are means±S.E.M. Groups with different letters are significantly different (P<0.05). Reproduced from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. & Furuse, M. (2007b) Galloyl group is not necessary for a sedative effect of catechin through GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science Publishers Ltd.

EGCG possesses two triphenolic groups in its structure. These groups are reported to be important with respect to anticarcinogenic and antioxidant effects. To clarify the antiinflammatory effect of EGCG on Alzheimer's disease (AD), Kim et al. (2007) investigated the effect of EGCG in attenuating the inflammatory response induced by interleukin (IL)1beta+beta-amyloid (25-35) fragment (Abeta) in human astrocytoma, U373MG cells. EGCG significantly inhibited the IL-1beta+Abeta (25-35)-induced IL-6, IL-8, vascular endothelial growth factor and prostaglandin E2 production (Jeong et al., 2007b, Kim et al., 2007). EGCG also attenuated the expression of cyclooxygenase-2 (Jeong et al., 2007b, Kim et al., 2007) and activation of nuclear factor-kappaB induced by IL-1beta+Abeta (25-35) (Kim et al., 2007).

Central Functions of Green Tea Components

11

They demonstrated that EGCG suppressed IL-1beta+Abeta (25-35)-induced phosphorylation of the mitogen-activated protein kinase p38 and the c-Jun N-terminal kinase. In addition, EGCG induced the expression of mitogen-activated protein kinase phosphatase-1. These results provide the possibility for its potential therapeutic application to various neurodegenerative diseases such as AD. Table 2. Effect of i.c.v. injection of EGC or EGCG on various behavioral categories in response to social separation stress for 10 min in chicks

412±49a

EGC (nmol) 109 218 75±50b 0±0b

EGCG (nmol) 109 218 23±23b 42±30b

176±47

232±45

99±54

97±25

265±72

12±12

0±0

36±36

0±0

45±32

0±0b

293±83a

465±49a

480±44a

248±105a

600

600

600

600

600

Saline Active wakefulness Standing/sitting motionless with eyes open Standing motionless with eyes closed Sitting motionless with head drooped (sleep-like behavior) Total

Values are means ± S.E.M. in seconds. Groups with different letters are significantly different (P<0.05). Reproduced with the modification from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. & Furuse, M. (2007b). Galloyl group is not necessary for a sedative effect of catechin through GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science Publishers Ltd.

Table 3. Effect of i.c.v. injection of EGC, picrotoxin or both on various behavioral categories in response to social separation stress for 10 min in chicks Saline

EGC (109 nmol)

P values

Picrotoxin (pmol)

Active wakefulness Standing/sitting motionless with eyes open Standing motionless with eyes closed Sitting motionless with head drooped (sleep-like behavior) Total

EGC x picrotoxin

0

830

0

830

EGC

Picrotoxin

417 ± 51

369 ± 80

64 ± 64

295 ± 70

<0.005

NS

<0.05

183 ± 51

231 ± 80

145 ± 42

305 ± 70

NS

NS

NS

0

0

4±4

0

NS

NS

NS

0

0

387 ± 78

0

<0.0001

<0.0001

<0.0001

600

600

600

600

Values are means ± S.E.M. in seconds.

Reproduced with the modification from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. & Furuse, M. (2007b). Galloyl group is not necessary for a sedative effect of catechin through GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science Publishers Ltd.

12

M. Furuse, N. Adachi, S. Tomonaga et al.

EGCG has neuroprotective effects on oxidative stress-injured neuronal cells, especially motorneurons. Koh et al. (2006) evaluated the effect of EGCG in the amyotrophic lateral sclerosis (ALS) model mice with the human G93A mutated Cu/Zn-superoxide dismutase gene. Treatment with more than 2.9 μg EGCG/g body weight significantly prolonged the symptom onset and life span, preserved more survival signals, and attenuated death signals. They suggested that EGCG could be a potential therapeutic candidate for ALS as a diseasemodifying agent. Parkinson's disease (PD) is known to occur after an 80% loss of dopaminergic nigrostriatal neurons. This loss is enhanced by oxidative stress (Mytilineou et al., 1998). Among the oxidative stresses, nitric oxide (NO) plays a major role (Grunewald and Beak, 1999; Schulz et al., 1995a). In mice (Schulz et al., 1995b) and baboons (Hantraye et al., 1996), inhibition of neuronal NO synthase (nNOS) prevents PD induced by 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). In addition, mutant mice lacking the inducible NOS (iNOS) gene are significantly more resistant to MPTP than their wild-type littermates (Hantraye et al., 1996). Choi et al. (2002) examined whether EGCG attenuates MPTPinduced PD in mice through the inhibition of NOS expression. The oral administration of EGCG prevented the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra and of TH activity in the striatum. These treatments also preserved striatal levels of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Both tea and EGCG decreased expressions of nNOS in the substantia nigra. EGCG plus MPTP treatments decreased expressions of nNOS similar to the EGCG treatment group. Therefore, the preventive effects of tea and EGCG may be explained by the inhibition of nNOS in the substantia nigra. EGCG also has an inhibitory effect on iNOS (Chan et al. 1997; Soliman and Mazzio, 1998). Park et al. (2001) examined the role of iNOS in cocaine-induced locomotor sensitization. Pretreatment with EGCG, 30 min before cocaine administration, totally blocked the development of cocaine-induced locomotor sensitization. Dopamine receptor binding in the nucleus accumbens showed a significant decrease in the density of D2 receptors and the affinity of D1 receptors after cocaine treatment. Pretreatment with EGCG abolished the cocaine-induced changes in these parameters. These results suggest that iNOS may participate in the process of locomotor sensitization through the modulation of dopamine receptors in the nucleus accumbens. Jeong et al. (2007a) investigated the effects of EGCG on the electrical activity of rat substantia nigra dopaminergic neurons using whole-cell patch clamp recordings. The spike frequency was increased, and the resting membrane potential of the cell and the amplitude of after-hyperpolarization were decreased by EGCG. The neuronal activity of dopaminergic neurons is closely linked to dopamine release. When neurons switch from a single-spike firing to bursts of action potentials the release of dopamine increases. The above experimental results suggest that EGCG increases the neuronal activity via inhibition of calcium-dependent potassium currents underlying the after-hyperpolarization, and it could act as a facilitating factor that elicits N-methyl-D-aspartate (NMDA)-dependent bursts of action potentials like apamin or bicuculline methiodide. Lin et al. (2007) reported that the protein binding of EGCG in rat plasma was 92.4±2.5%, and EGCG may potentially penetrate through the blood-brain barrier at a lower rate. The elimination half-life of EGCG was 62±11 and 48±13 min for intravenous (10 mg/kg) and oral (100 mg/kg) administration, respectively. The pharmacokinetic data indicate that the oral bioavailability of EGCG in a conscious and freely moving rat was about 4.95%.

Central Functions of Green Tea Components

13

Ullmann et al. (2003) conducted a randomized, double-blind, placebo-controlled study assessing the safety, tolerability and plasma kinetic behavior of single oral doses of EGCG under fasting conditions. In each group of 10 subjects, eight received oral EGCG in single doses of 50, 100, 200, 400, 800 or 1600 mg, and two received placebo. In each dosage group, the kinetic profile revealed rapid absorption with one plasma peak, followed by a multiphasic decrease consisting of a distribution phase and an elimination phase. Single oral doses of EGCG up to 1600 mg were safe and very well tolerated. Ullmann et al. (2004) further reported the safety, tolerability, and plasma-kinetic behavior of EGCG after ten days' repeated dosing in healthy male volunteers. Orally administered EGCG is rapidly absorbed from the gut. Dose linearity was applied for single-dose application (day 1). After repeated dosing (day 10) dose linearity was applied between the 200 and 400 mg group. Dose escalation to 800 mg was more than dose-proportional in rate and extent, and statistically different from the 200 mg and 400 mg group. An increase in elimination half-life and in the accumulation factor in the 800 mg dosage group indicated dose-dependent saturation of capacity-limited excretion routes or an increase of hepato-duodenal re-circulation. Ten days' repeated administrations of oral dose of EGCG of up to 800 mg per day were found to be safe and very well tolerated.

CENTRAL FUNCTION OF L-THEANINE Time-dependent changes of L-theanine in the brain of rats were investigated during the 24 h after L-theanine administration. When L-theanine was intragastrically administered to rats, the concentrations of L-theanine reached the maximum level in the brain after 5 h (Terashima et al., 1999). L-Theanine was incorporated into brain through the blood-brain barrier via the leucine-preferring transport system (Yokogoshi et al., 1998a). Intravenous administration of L-theanine affected the cortex, hippocampus and amygdala and increased the alpha-band component of EEG in rats (Kakuda et al., 2000a). Evidence from human EEG studies show that L-theanine has a direct effect on the brain (Juneja et al., 1999). L-Theanine significantly increases activity in the alpha frequency band which indicates that it relaxes the mind without inducing drowsiness. However, this effect has only been established at higher doses than that typically found in a cup of black tea (approximately 20 mg). Nobre et al. (2008) reported that L-theanine, at realistic dietary levels, has a significant effect on the general state of mental alertness or arousal. L-Theanine- and theogallin-enriched decaffeinated green tea extract is able to change the physiological pattern of electrical hippocampus activity in a concentration dependent manner. A decrease of population spike amplitude and attenuated long-term potentiation was observed in the presence of L-theanine alone (Dimpfel et al., 2007a). The model Tele-Stereo-EEG (continuous recording of intracerebral field potentials in the freely moving rat to produce an electropharmacogram) has been used and power spectra from Fast Fourier Transformed field potential changes were divided into six frequency bands (delta, theta, alpha1, alpha2, beta1 and beta2). Oral administration of 30 mg/kg L-theanine led to power decreases of nearly all frequencies, being more pronounced during the second and following hours in comparison with the first hour (Dimpfel et al., 2007b).

14

M. Furuse, N. Adachi, S. Tomonaga et al.

Accoding to Dimpfel et al. (2007c), source density analysis of EEG recordings from 12 healthy human volunteers after ingestion of a soft drink containing green tea extract enriched with L-theanine and theogallin revealed a general attenuation of electrical delta power under the condition of eyes open. During a reading test increases of delta and theta power were observed at frontal electrode sites starting with the second hour after administration, significant at the third and fourth hour in comparison to placebo. These changes indicate a higher level of mental performance. Increases of beta 1 power starting with the second hour indicated a higher degree of relaxation. However, no statistical significance was reached. Analysis of visually evoked P300 waves revealed a decrease in latency at the last hour as well as increases of amplitudes at the electrode position Cz (from the first to the third hour, statistically not significant). This type of result in general suggests an improvement of attention. So-called alpha bands (8-14 Hz) can be detected during resting EEG recordings in humans. Independently, alpha band activity has been shown to be a key component in selective attentional processes. L-Theanine ingestion resulted in a substantial overall decrease in background alpha levels relative to placebo while subjects were actively performing a demanding attention task. Despite this decrease in background alpha activity, attention-related alpha effects were significantly greater for L-theanine condition (Gomez-Ramirez et al., 2007). The oral administration of L-theanine to rats via gastric intubation for 2 weeks, caused a 20% lower oxidation level in the cerebral cortex as measured using thiobarbiturate reactive substances as a marker (Nishida et al., 2008). The protein expression levels of phospholipase C-beta 1 and -gamma 1, stress-responsible molecules, were significantly increased in the cerebral cortex after L-theanine administration and the same tendency was observed in the cerebellum, but not the hippocampus. L-Theanine reduced the size of the cerebral infarct and alterations of the number of NeuN (neuron)- and GFAP (astrocyte)-positive cell expression levels at 24 h after middle cerebral artery occlusion in mice. This neuroprotective effect of L-theanine was prevented by bicuculline but not 3-mercaptopropionic acid (glutamate decarboxylase inhibitor) (Egashira et al., 2007). These results suggest that the neuroprotective effect of L-theanine is mediated, at least in part, by GABAA receptors. L-Theanine significantly decreased the size of the cerebral infarcts 1 day after the occlusion. In contrast, L-theanine did not affect the cerebral blood flow, brain temperature or physiological variables in this model (Egashira et al., 2004). These results suggest that L-theanine directly provides neuroprotection against focal cerebral ischemia and may be clinically useful for preventing cerebral infarction. Cho et al. (2008) investigated the protective effects of L-theanine on neurotoxicity induced by PD-related neurotoxicants rotenone and dieldrin in a cultured human dopaminergic cell line, SH-SY5Y, and suggested that L-theanine directly provided neuroprotection against PD-related neurotoxicants and may be clinically useful for preventing PD symptoms. Ischemia-induced neuronal death in the hippocampal CA1 region was significantly prevented in a dose-dependent manner in L-theanine-pretreated groups. These findings indicate that L-theanine might be useful clinically for preventing ischemic neuronal damage (Kakuda et al., 2000b). L-Theanine significantly prevented the impairment of spatial memory in rats subjected to repeated cerebral ischemia, 7 days after the second reperfusion. Moreover, L-theanine significantly inhibited the decrease in the number of surviving cells in the hippocampal CA1 field in the same rats. These results suggest that L-theanine prevents memory impairment induced by repeated cerebral ischemia, in part by protecting against

Central Functions of Green Tea Components

15

neuronal cell death, and that it might be useful for preventing cerebrovascular disease (Egashira et al., 2008). L-Theanine did not increase the concentration of excitatory neurotransmitters in the perfusate when injected into the rat brain striatum, but increased the concentration of glycine in the perfusate (Yamada et al., 2009). Yamada et al. (2009) investigated the glycine and dopamine concentrations in the perfusate, since it has been reported that L-theanine promotes dopamine release in the rat striatum (Yamada et al., 2005; Yokogoshi et al., 1998a). Coinjection of a glycine receptor antagonist, strychnine, reduced L-theanine-induced changes in dopamine. Moreover, an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist inhibited the effects of L-theanine. L-Theanine may induce its inhibitory effect at AMPA receptors. During brain development in infant rats, inhibitory neurotransmission is required for mature brain function. Some neurotransmitters, including dopamine, serotonin, glycine and GABA concentrations, increased in the infant brain, and nerve growth factor mRNA level increased in the cerebral cortex and hippocampus when other rats were fed L-theanine ad libitum after confinement. However, these differences were lost by the end of nerve maturity. From these results Yamada et al. (2007) suggest that Ltheanine enhanced synthesis of nerve growth factor and neurotransmitters during a nerve maturing period and promoted CNS maturation. L-Theanine intake resulted in a reduction in the heart rate (HR) and salivary immunoglobulin A (s-IgA) responses to an acute stress task relative to the placebo control condition. Moreover, analyses of HR variability indicated that the reductions in HR and s-IgA were likely attributable to an attenuation of sympathetic nervous activation (Kimura et al., 2007). These facts suggested that the oral intake of L-theanine could cause anti-stress effects via the inhibition of cortical neuron excitation. The acute effects of L-theanine in comparison with a standard benzodiazepine anxiolytic, alprazolam, and placebo on behavioral measures of anxiety in healthy human subjects using the model of anticipatory anxiety were investigated (Lu et al., 2004). Sixteen healthy volunteers received alprazolam, L-theanine or placebo in a double-blind placebo-controlled repeated measures design. The acute effects of alprazolam and L-theanine were assessed under a relaxed and experimentally induced anxiety condition. The results showed some evidence for relaxing effects of L-theanine during the baseline condition on the tranquiltroubled subscale of the visual analogue mood scale. Alprazolam did not exert any anxiolytic effects in comparison with the placebo on any of the measures during the relaxed state. Neither L-theanine nor alprazalam had any significant anxiolytic effects during the experimentally induced anxiety state. The findings suggest that while L-theanine may have some relaxing effects under resting conditions, neither L-theanine nor alprazolam demonstrate any acute anxiolytic effects under conditions of increased anxiety in the anticipatory anxiety model. Kakuda et al. (2008) suggested that L-theanine would be an inhibitor of different transporters capable of transporting glutamine (Gln) across plasma membranes toward the modulation of the glutamate/Gln cycle required for the neurotransmitter pool of glutamate in neurons. L-Theanine bound to three glutamate receptor subtypes (AMPA, kainate, and NMDA glycine) in rat cortical neurons, but its IC50 of L-theanine was 80- to 30,000-fold less than that of L-glutamate (Kakuda et al., 2002). L-Theanine and a group I metabotropic glutamate receptor (mGluRs) agonist, DHPG, inhibited the delayed death of neurons caused by brief exposure to glutamate, and this effect of L-theanine was abolished by group I mGluR

16

M. Furuse, N. Adachi, S. Tomonaga et al.

antagonists (Nagasawa et al., 2004). Treatment with L-theanine or DHPG alone resulted in increased expression of phospholipase C (PLC)-beta1 and -gamma1, and the action of Ltheanine was completely abolished by group I mGluR antagonists. These findings indicate that group I mGluRs might be involved in the neuroprotective effect of L-theanine by increasing the expression levels of PLC-beta1 and -gamma1. Bryan (2008) reviewed that L-theanine may interact with caffeine to enhance performance in terms of attention switching and the ability to ignore distraction. This is likely to be reflective of higher-level cognitive activity and may be sensitive to the detrimental effects of overstimulation. Kakuda et al. (2000a) investigated the inhibitory action of Ltheanine on the excitation by caffeine at the concentration regularly associated with drinking tea using EEG in rats. The stimulatory effects of caffeine were inhibited by an i.v. administration of L-theanine and the results suggested that L-theanine has an antagonistic effect on caffeine's stimulatory action at an almost equivalent molar concentration. On the other hand, excitatory effects were shown in the rat i.v. administered L-theanine alone. These results suggested two effects of L-theanine, depending on its concentration.

DIRECT COMPARISON OF EGCG AND L-THEANINE FOR A SEDATIVE EFFECT Although tea contains both EGCG and L-theanine that may affect sedation via the CNS, direct comparison of their effect on the stress response is still limited. Adachi et al. (2007a) compared the effects of L-theanine and EGCG at the same level on stress in neonatal chicks using a social stress model. Chicks were injected i.c.v. with either saline as a control, 109 nmol of EGCG, or the same level of L-theanine. The effects of EGCG and L-theanine on total spontaneous activity and distress vocalizations are shown in Figures 11 and 12. EGCG, but not L-theanine, reduced total spontaneous activity and the number of vocalizations. Table 4 shows the effects of EGCG and L-theanine on four behavioral categories in chicks. Compared with the control, EGCG significantly increased sleep-like behavior while decreasing the time of active wakefulness. No significant effects were observed for L-theanine. The failure to observe any effects of L-theanine at a concentration of 109 nmol on the parameters tested should be interpreted with caution. First, it is possible that too low a concentration of Ltheanine was used to observe any stress modifying effect in chicks. When L-glutamate (up to 800 nmol) was i.c.v. administered, 400 and 800 nmol, but not 200 nmol, decreased distress vocalization under separation-stress in chicks (Yamane et al., 2009). Second, it is possible that the L-theanine-related relaxation found in mammals (Kimura et al., 2007) may be mediated primarily through peripheral targets rather than via a direct action on the brain. In this regard, no systemic administration of L-theanine was made. Finally, experimental differences between this and other studies (Kimura et al., 2007) such as species (avian vs. mammalian) and age (neonatal vs. adult animals) may have contributed to the failure of Ltheanine to attenuate stress.

Spontaneous activity (arbitrary unit)

Central Functions of Green Tea Components

a

80

17

a

60

40

20

b

0 Control

EGCG

L-Theanine

Figure 11. Effect of i.c.v. injection of EGCG (109 nmol) or L-theanine (109 nmol) on total number of spontaneous activity during a 10 min period in response to social separation stress in chicks. Data are expressed as means±S.E.M. Groups with different letters are significantly different (P<0.05). Reproduced from Adachi, N., Choi, Y.-H., Suenaga, R., Tomonaga, S., Denbow, D.M. & Furuse, M. (2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission from New Century Health Publishers, LLC.

Vocalization (count)

900

a

a

600

300

b 0 Control

EGCG

L-Theanine

Figure 12. Effect of i.c.v. injection of EGCG (109 nmol) or L-theanine (109 nmol) on total number of distress vocalizations during a 10 min period in response to social separation stress in chicks. Data are expressed as means±S.E.M. Groups with different letters are significantly different (P<0.05). Reproduced from Adachi, N., Choi, Y.-H., Suenaga, R., Tomonaga, S., Denbow, D.M. & Furuse, M. (2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission from New Century Health Publishers, LLC.

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Table 4. Effect of i.c.v. injection of 109 nmol EGCG or L-theanine on various behaviors in response to social separation stress for 10 min in chicks

Active wakefulness Standing/sitting motionless with eyes open Standing motionless with eyes closed Sitting motionless with head drooped (sleep-like behavior) Total

Saline

EGCG

L-Theanine

480±41a

36±22b

425±70a

100±29

128±37

76±23

0±0

48±33

22±22

20±20b

388±72a

77±69b

600

600

600

Values are means ± S.E.M. in seconds. Groups with different letters are significantly different (P<0.05). Reproduced from Adachi, N., Choi, Y.-H., Suenaga, R., Tomonaga, S., Denbow, D.M. & Furuse, M. (2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission from New Century Health Publishers, LLC.

CONCLUSION Tea leaves include several ingredients, some of which are believed to act as medicinal compounds. A feeling of relaxation can be induced by daily tea consumption. A part of this feeling may be mediated by either EGCG, L-theanine, or both. Tea components may be beneficial in a stressful society, and may have medicinal benefits for several mental diseases.

ACKNOWLEDGEMENTS A part of studies described here was supported by grant-in-aid for scientific research from Japan Society for the Promotion of Science (Nos. 16380191, 17208023 and 18208023) to MF. This work was also supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists to ST (No. 18·9592) and HY (No. 20·03662).

REFERENCES Adachi, N., Choi, Y.-H., Suenaga, R., Tomonaga, S., Denbow, D.M. & Furuse, M. (2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110. Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. & Furuse, M. (2007b). Galloyl group is not necessary for a sedative effect of catechin through GABAergic system. Lett. Drug Design Discov., 4, 163-167.

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Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175. Arts, I.C., van De Putte, B. & Hollman, P.C. (2000). Catechin contents of foods commonly consumed in the Netherlands. 2. Tea, wine, fruit juices, and chocolate milk. J. Agric. Food Chem., 48, 1752-1757. Bryan, J. (2008). Psychological effects of dietary components of tea: caffeine and L-theanine. Nutr. Rev., 66, 82-90. Campbell, E.L., Chebib, M. & Johnston, G.A. (2004). The dietary flavonoids apigenin and ()-epigallocatechin gallate enhance the positive modulation by diazepam of the activation by GABA of recombinant GABA(A) receptors. Biochem. Pharmacol., 68, 1631-1638. Chan, M.M., Fong, D., Ho, C.T. & Huang, H.I. (1997). Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea. Biochem. Pharmacol., 54, 1281–1286. Cho, H.S., Kim, S., Lee, S.Y., Park, J.A., Kim, S.J. & Chun, H.S. (2008). Protective effect of the green tea component, L-theanine on environmental toxins-induced neuronal cell death. Neurotoxicology, 29, 656-662. Choi, J.Y., Park, C.S., Kim, D.J., Cho, M.H., Jin, B.K., Pie, J.E. & Chung, W.G. (2002). Prevention of nitric oxide-mediated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineinduced Parkinson's disease in mice by tea phenolic epigallocatechin 3-gallate. Neurotoxicology, 23, 367-374. Dekermendjian, K., Kahnberg, P., Witt, M.R., Sterner, O., Nielsen, M. & Liljefors, T. (1999). Structure-activity relationships and molecular modeling analysis of flavonoids binding to the benzodiazepine site of the rat brain GABA(A) receptor complex. J. Med. Chem., 42, 4343-4350. Dimpfel, W., Kler, A., Kriesl, E. & Lehnfeld, R. (2007a). Theogallin and L-theanine as active ingredients in decaffeinated green tea extract: I. Electrophysiological characterization in the rat hippocampus in-vitro. J. Pharm. Pharmacol., 59, 1131-1136. Dimpfel, W., Kler, A., Kriesl, E. & Lehnfeld, R. (2007b). Theogallin and L-theanine as active ingredients in decaffeinated green tea extract: II. Characterization in the freely moving rat by means of quantitative field potential analysis. J. Pharm. Pharmacol., 59, 1397-1403. Dimpfel, W., Kler, A., Kriesl, E., Lehnfeld, R. & Keplinger-Dimpfel, I.K. (2007c). Source density analysis of the human EEG after ingestion of a drink containing decaffeinated extract of green tea enriched with L-theanine and theogallin. Nutr. Neurosci., 10, 169180. Egashira, N., Hayakawa, K., Mishima, K., Kimura, H., Iwasaki, K. & Fujiwara, M. (2004). Neuroprotective effect of gamma-glutamylethylamide (theanine) on cerebral infarction in mice. Neurosci. Lett., 363, 58-61. Egashira, N., Hayakawa, K., Osajima, M., Mishima, K., Iwasaki, K., Oishi, R. & Fujiwara, M. (2007). Involvement of GABA(A) receptors in the neuroprotective effect of theanine on focal cerebral ischemia in mice. J. Pharmacol. Sci., 105, 211-214. Egashira, N., Ishigami, N., Pu, F., Mishima, K., Iwasaki, K., Orito, K., Oishi, R. & Fujiwara, M. (2008). Theanine prevents memory impairment induced by repeated cerebral ischemia in rats. Phytother. Res., 22, 65-68. Frei, B. & Higdon, J.V. (2003). Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J. Nutr., 133, 3275S-3284S.

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Kuo, K.L., Weng, M.S., Chiang, C.T., Tsai, Y.J., Lin-Shiau, S.Y. & Lin, J.K. (2005). Comparative studies on the hypolipidemic and growth suppressive effects of oolong, black, pu-erh, and green tea leaves in rats. J. Agric. Food Chem., 53, 480-489. Lin, L.C., Wang, M.N., Tseng, T.Y., Sung, J.S. & Tsai, T.H. (2007). Pharmacokinetics of (-)epigallocatechin-3-gallate in conscious and freely moving rats and its brain regional distribution. J. Agric. Food Chem., 55, 1517-1524. Lu, K., Gray, M.A., Oliver, C., Liley, D.T., Harrison, B.J., Bartholomeusz, C.F., Phan, K.L. & Nathan, P.J. (2004). The acute effects of L-theanine in comparison with alprazolam on anticipatory anxiety in humans. Hum. Psychopharmacol., 19, 457-465. Marder, M. & Paladini, A.C. (2002). GABA(A)-receptor ligands of flavonoid structure. Curr. Top Med. Chem., 2, 853-867. Mukhopadhyay, S. & Poddar, M.K. (1995). Caffeine-induced locomotor activity: possible involvement of GABAergic-dopaminergic-adenosinergic interaction. Neurochem. Res., 20, 39-44. Mytilineou, C., Leonardi, E.K., Radcliffe, P., Heinonen, E.H., Han, S.K., Werner, P., Cohen, G. & Olanow, C.W. (1998). Deprenyl and desmethylselegiline protect mesencephalic neurons from toxicity induced by glutathione depletion. J. Pharmacol. Exp. Ther., 284, 700–706. Nagasawa, K., Aoki, H., Yasuda, E., Nagai, K., Shimohama, S. & Fujimoto, S. (2004). Possible involvement of group I mGluRs in neuroprotective effect of theanine. Biochem. Biophys. Res. Commun., 320, 116-122. Nishida, K., Yasuda, E., Nagasawa, K. & Fujimoto, S. (2008). Altered levels of oxidation and phospholipase C isozyme expression in the brains of theanine-administered rats. Biol. Pharm. Bull., 31, 857-860. Nobre, A.C., Rao, A. & Owen, G.N. (2008). L-Theanine, a natural constituent in tea, and its effect on mental state. Asia Pac. J. Clin. Nutr., 17 Suppl 1, 167-168. Park, K., Vora, U., Darling, S.F., Kolta, M.G. & Soliman, K.F. (2001). The role of inducible nitric oxide synthase in cocaine-induced locomotor sensitization. Physiol Behav., 74, 441-447. Quinlan, P.T., Lane, J., Moore, K.L., Aspen, J., Rycroft, J.A. & O'Brien, D.C. (2000). The acute physiological and mood effects of tea and coffee: the role of caffeine level. Pharmacol. Biochem. Behav., 66, 19-28. Schulz, J.B., Matthews, R.T. & Beal, M.F. (1995a). Role of nitric oxide in neurodegenerative diseases. Curr. Opin. Neurol., 8, 480–486. Schulz, J.B., Matthews, R.T., Muquit, M.M., Browne, S.E. & Beal, M.F. (1995b). Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice. J. Neurochem., 64, 936–939. Soliman, K.F.A. & Mazzio, E. (1998). In vitro attenuation of nitric oxide production in C6 astrocyte cell culture by various dietary compounds. Proc. Exp. Biol. Med., 218, 391– 397. Suganuma, M., Okabe, S., Oniyama, M., Tada, Y., Ito, H. & Fujiki, H. (1998). Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue. Carcinogenesis, 19, 1771-1776. Tachibana, H., Koga, K., Fujimura, Y. & Yamada, K. (2004). A receptor for green tea polyphenol EGCG. Nat. Struct. Mol. Biol., 11, 380-381.

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Terashima, T., Takido, J. &Yokogoshi, H. (1999). Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine. Biosci. Biotechnol. Biochem., 63, 615-618. Ullmann, U., Haller, J., Decourt, J.P., Girault, N., Girault, J., Richard-Caudron, A.S., Pineau, B. & Weber, P. (2003). A single ascending dose study of epigallocatechin gallate in healthy volunteers. J. Int. Med. Res., 31, 88-101. Ullmann, U., Haller, J., Decourt, J.D., Girault, J., Spitzer, V. & Weber, P. (2004). Plasmakinetic characteristics of purified and isolated green tea catechin epigallocatechin gallate (EGCG) after 10 days repeated dosing in healthy volunteers. Int. J. Vitam. Nutr. Res., 74, 269-278. Vignes, M., Maurice, T., Lante, F., Nedjar, M., Thethi, K., Guiramand, J. & Recasens, M. (2006). Anxiolytic properties of green tea polyphenol (-)-epigallocatechin gallate (EGCG). Brain Res., 1110, 102-115. Yamada, T., Terashima, T., Kawano, S., Furuno, R., Okubo, T., Juneja, L.R. & Yokogoshi, H. (2009). Theanine, gamma-glutamylethylamide, a unique amino acid in tea leaves, modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats. Amino Acids, 36, 21-27. Yamada, T., Terashima, T., Okubo, T., Juneja, L.R. & Yokogoshi, H. (2005). Effects of theanine, γ-glutamylethylamide, on neurotransmitter release and its relationship with glutamic acid neurotransmission. Nutr. Neurosci., 8, 219-26. Yamada, T., Terashima, T., Wada, K., Ueda, S., Ito, M., Okubo, T., Juneja, L.R. & Yokogoshi, H. (2007). Theanine, γ-glutamylethylamide, increases neurotransmission concentrations and neurotrophin mRNA levels in the brain during lactation. Life. Sci., 81, 1247-1255. Yamane, H., Tsuneyoshi, Y., Denbow, D.M. & Furuse, M. (2009). N-Methyl-D-aspartate and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks. Amino Acids, in press. Yokogoshi, H., Kobayashi, M., Mochizuki, M. & Terashima, T. (1998a). Effect of theanine, γ-glutamylethylamide, on brain monoamines and striatal dopamine release in conscious rats. Neurochem. Res., 23, 667-673. Yokogoshi, H., Mochizuki, M. & Saitoh, K. (1998b). Theanine-induced reduction of brain serotonin concentration in rats. Biosci. Biotechnol. Biochem., 62, 816-817. Zanoli, P., Avallone, R. & Baraldi, M. (2000). Behavioral characterisation of the flavonoids apigenin and chrysin. Fitoterapia, 71 Suppl. 1, S117-S123.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 2

GREEN TEA CATECHINS: A CLASS OF MOLECULES WITH ANTIMICROBIAL ACTIVITY P. Buzzini1,*, P. Vignolini2, M. Goretti1, B. Turchetti1, E. Branda1, E. Marchegiani1, P. Pinelli2 and A. Romani2 1

Department of Applied Biology - Microbiology, University of Perugia, Italy 2 Department of Pharmaceutical Sciences, University of Florence, Italy

ABSTRACT A significant part of scientific interest of academy or industry is focused on discovering novel natural antimicrobial drugs. This attention is essentially justified by the expectation that a few of them could play a role in supporting (or even in substituting) some antibiotics of current use. It has been estimated that, although about some tens of novel antimicrobial drugs (either of biological or synthetic origin) are currently launched each year, due to the increasing development of resistant microbial genotypes, their downturn is becoming very rapid. Taking into account these considerations, the enormous scientific and commercial interest in discovering and developing novel classes of molecules exhibiting more or less pronounced antimicrobial properties has oriented the work of a growing part of the scientific community toward large-scale screening programs aimed at discovering novel classes of bioactive molecules. The occurrence in some plants of secondary metabolites exhibiting a more or less pronounced antimicrobial activity is a well-known phenomenon. Among them, green tea polyphenols represent a reservoir of molecules characterized by antioxidant, antiradical and antimicrobial activity. In particular, catechins have proven to be effective towards both prokaryotic and eukaryotic microorganisms. Despite the large number of studies published so far, their actual potentialities and limitations as antimicrobial (mainly antibacterial and antimycotic) drugs have not been critically evaluated. The present chapter represents an overview of the recent literature on the antiviral and antimicrobial *

Address for correspondence. E-mail: [email protected]

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P. Buzzini, P. Vignolini, M. Goretti et al. properties exhibited by polyphenols, particularly catechins, occurring in green tea composition.

INTRODUCTION Tea is one of the most widely consumed beverages in the world today, second only to water (Stagg, 1980), and its medicinal properties have been widely explored. Three billion kilograms of tea are produced each year worldwide (Kris-Etherton and Keen, 2002). Because of the high rates of tea consumption in the global population, it has been estimated that even small effects in humans could have large implications for public health (Rimm and Diet, 2004). Tea is generally consumed in the forms of green, oolong, and black tea, all of which originate from the leaves of the plant Camellia sinensis. Differences of composition between green tea (popular in the Far East) and other teas (oolong and black teas, usually used in Western countries) are mainly due to the oxidation steps occurring during the fermentation process (Friedman, 2007). In particular, green tea is produced from steaming fresh leaves at high temperatures, thereby inactivating the oxidizing enzymes and leaving the polyphenol content intact.

Figure 1. Structures of most represented flavan-3-ols found in green tea.

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The main polyphenols found in green tea are more commonly hydrolysable and condensed tannins and include 30-40% of the extractable solids of dried green tea leaves. Among them, catechins [e.g. epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate (ECG), gallocatechin (GC), epigallocatechin (EGC), and epicatechin (EC)] are the most represented flavan-3-ols found in green tea (Figure 1). Enzymatic oxidation of EGC (and of its gallate derivative) yields B-ring orto-quinones, which are dimerized spontaneously to give deydrotheasinensis and related quinone dimers (Haslam, 2003). Theaflavins are produced by condensation between cathecol-type and pyrogallol-type catechins. To clarify the oxidation mechanism of green tea catechins occurring during tea fermentation, Tanaka et al. (2002) oxidized pure catechins with a catechin-free homogenate obtained from tea leaves. Oxidation of a mixture of (2)-EC and (2)-EGC yielded a new metabolite, named dehydrotheaflavin, produced by the oxidation of a benzotropolone moiety of the black tea pigment theaflavin (Figure 2). Similar oxidation of a mixture of (2)-EC and (2)-EGCG afforded a new dimer of (2)-EGCG, which was generated by the oxidation and cycloaddition of two pyrogallol rings (Tanaka et al., 2002). OH OR2 OH

HO

O OH

O O

HO

OH OR1 OH

R1

R2

Theaflavin Theaflavin-3-monogallate

H

H

gallate

H

Theaflavin-3’-monogallate

H

gallate

gallate

gallate

Theaflavin-3,3’-digallate Figure 2. Chemical structure of theaflavins.

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Concerning non-antimicrobial activities, the antioxidant properties of green tea polyphenols are well known (Hamilton-Miller, 1995). These molecules are potent free radical scavengers due to the hydroxyl groups in their chemical structure, which can complex (and thus neutralize) free radicals, thus preventing the progression of the disease process due to oxidative stress and subsequent generation of free radicals (Xu et al., 1992; Ichihashi et al., 2000). In particular, in vivo and in vitro biological activities of tea flavan-3-ols include inhibition of carcinogenesis, protection from cardiovascular disease, enhanced loss of body fat, increase of bone density, protection from neurodegenerative diseases, and improvement in type 2 diabetes (Graham, 1992; Hirose et al., 1993; Picard, 1996; Katiyar and Mukhtar, 1997; Lee et al., 1997; Alschuler, 1998; Khafif et al., 1998; Otsuka et al., 1998; Sugiyama and Sadzuka, 1998; Sato et al., 1999; Frei and Higdon, 2003; Cooper et al., 2005; Ramassamy, 2006; Zaveri, 2006; Friedman, 2007). However, the design and interpretation of epidemiological and human intervention studies is limited by so far inadequate information on bioavailability and metabolism/biotransformation of tea flavan-3-ols. Only a small percentage of the flavan-3-ols are absorbed from the small intestine into the intestinal epithelial cells, where they undergo conjugation to sulfated, glucuronidated, or methylated conjugates by phase II enzymes (e.g. sulfotranferase, glucuronosyltransferase, and catecholO-methyltransferase). This process has been critically reviewed in detail by Lambert et al. (2007). Other miscellaneous non-microbiological properties possessed by green tea components have been investigated. Some of the more interesting of these include activation of leukocytes (Sakagami et al., 1992), antimutagenic activity (Hayatsu et al., 1992), lowering of plasma cholesterol levels (Ikeda et al., 1992), protection from the effects of radiation (Uchida et al., 1992), slowing the catabolism of catecholamines and strengthening of capillaries (‘‘vitamin P effect’’) (Stagg and Millin, 1975; Min and Peigen, 1991). More recently, a few studies have postulated a synergistic interaction between caffeine and green tea catechins. This synergy apparently acts by extending sympathetic stimulation of thermogenesis. Green tea catechins have also been shown to markedly inhibit digestive lipases in vitro, resulting in a decreased lipolysis of triglycerides, which may provokes the reduction of fat digestion in humans (Dulloo et al., 2000; Juhel et al., 2000). As above reported, catechins are the major components of green tea, differently from black tea where theaflavins (the oxidized form of catechins) are the major component. It is not clear if catechins and theaflavins have equivalent antioxidant capacity (Lee et al., 2002; Leung et al., 2001). Green tea commercial extracts (GTEs), at different level and content in polyphenol compounds, were currently used as food ingredients or in cosmetics preparations. A GTE, obtained by the hot water-soluble fraction of unfermented leaves, exhibited a superior scavenging activity toward reactive oxygen species (ROS) compared to vitamins C and E (Zhao et al., 1989). More recently, Schroeter et al. (2001) found that GTE has the same protective effects as 10-fold greater concentrations of vitamin C. Likewise, Jarrod et al. (2008) tested in vivo a commercial caffeine-free GTE (Sunphenon 90DCF, Taiyo International, Inc., USA) characterized by the following polyphenol composition: polyphenols > 80%, catechins >80%, EGCG > 45%, caffeine < 1%). The authors observed that physiological performances and endurance ability in mice were greatly enhanced by GTE dietary administration. Independent of running, GTE apparently decreased serum creatine kinase, heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate

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synthase activity of rats. Based on these data, the authors suggested that the antioxidant activity of GTE might be beneficial as therapeutic strategies to improve muscle function in mice. The antimicrobial activity of green tea polyphenols (particularly catechins) is well known. Both industry and academy have been increasingly concerned with the growing number of illness outbreaks caused by microbial pathogens. It has been estimated that, although about some tens of novel antimicrobial drugs (either of biological or synthetic origin) are currently launched each year, their downturn is becoming very rapid. Increasing antibiotic resistance of some pathogens associated with over-consumption of some existing antimicrobial drugs exacerbates this trend. Therefore, there has been an increasing interest in studying and developing novel types of effective plant-derived antimicrobials, including those present in green tea leaves (Friedman, 2007; Buzzini et al., 2007). The main objective of this chapter is to unify and interpret widely scattered information of literature on inhibitory activities of catechins occurring in green tea leaves against viruses, bacteria, yeasts and filamentous fungi.

FUNDAMENTALS OF BIOSYNTHESIS OF GREEN TEA CATECHINS Phenylpropanoid units derived from the shikimate pathway are common structural elements of all flavonoid compounds and of other classes of phenylpropanoids, such as lignin, stilbenes and cinnamate esters. The enzymes catalysing the individual steps of the “phenylpropanoid metabolism” are: phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate CoA ligase. In particular, flavan-3-ols originated from a branch pathway of anthocyanin biosynthesis. Early studies covering enzymatic aspects of proanthocyanidins biosynthesis have been carried out on Ginko biloba and Pseudotsuga menziesii. Such studies reported that dihydroflavonols [(+)-dihydromyricetin (DHM) or (+)-dihydroquercetin (DHQ)] are converted to the corresponding flavan-3,4-diols and catechin derivatives [(+)-GC, or (+)catechin, respectively] (Stafford and Lester, 1984, Stafford and Lester, 1985). The existence of a relationship between the formation of 2,3-trans, catechin-derived series of flavan-3-ols and the consecutive action of a dihydroflavonol reductase (DFR, which produces a leucoanthocyanidin) and a leucoanthocyanidin reductase (LAR) has been postulated. Leucocyanidin (produced by DFR via reaction with dihydroquercetin) was not observed with tea enzyme preparations. Instead, it is immediately further converted to catechin by the LAR present in the enzyme preparation (Punyasiria et al., 2004). The anthocyanidin reductase (ANR) enzyme, recently isolated from Arabidopsis spp. and Medicago spp., was shown to be present in tea leaves with very high activity. It produces EC and EGC from their respective anthocyanidins, thus explaining the very high contents of such compounds. The high ANR activity seems to be essential with respect to the dominance of EC and EGC (as well as their galloyl esters) as the major flavonoid components in tea leaves. EC and EGC, together with catechins, are also the building blocks of proanthocyanidins reported from tea (Kiehne et al., 1997). Thus, ANR together with LAR, may be of great importance for the biosynthesis of proanthocyanidins. The DFR/LAR two-step reaction converted the dihydroflavonols, dihydroquercetin and dihydromyricetin to catechin and GC, respectively, with a high activity. The pathways for the formation of many flavan 3-ol

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derivatives are not known. The occurrence of a galloyl transferase has been recently reported (Garcia et al., 2002). Catechins and EC, however, may also get hydroxylated by F3050H. In general, in green tea is demonstrated a high activity of flavonoid 3’5’-hydroxylase (F3’5’H), since there are more EGC and EGCG than EC and ECG in tea leaves. But also F3’H activity must be present as B-ring dihydroxylated flavonoids like catechin, epicatechin and derivatives are also quite prominent. Future studies will focus on the membrane-bound microsomal enzymes F3’H and F3’5’H that are important for B-ring hydroxylation of tea flavonoids.

ANTIVIRAL ACTIVITY OF GREEN TEA CATECHINS Among the possible modes of antiviral action of green tea polyphenols their ability to prevent viral binding and penetration into cells, and to trigger the host cell self-defence mechanisms are the mostly hypothesized (Friedman, 2007). Weber et al. (2003) studied the effect of four green tea catechins on adenovirus (also labelled as adenoidal-pharyngeal-conjuctival virus, APC) responsible for the infection of mucous membranes of the respiratory and urinary tracts. EGCG was the most effective (IC50 = 109 μM) when added to the cells during the transition from the early to the late phase of viral infection. The same compound exhibited a high selectivity index (SI). On the basis of these evidences, the authors postulated that EGCG could inhibit one or more later steps of viral infection and proposed its use for treating adenovirus infections in humans (Weber et al., 2003). A study by Savi et al. (2006) investigated the structure-activities relationships (SAR) between green tea catechins and Herpes simplex virus (HSV). HSV, also known as cold sore, causes blisters on the mouth and lips (oral and facial infections) and on genitals. The authors observed that catechins exhibit an in vitro anti-HSV activity. The number of hydroxyl groups on the B ring of the catechin molecules as well as the presence (or absence) of galloyl side chains affected the SI (ranging from 1.3 to 13). Prodelphinidin B-2 3’-O-gallate, isolated from green tea leaves, also exhibited an in vitro anti-HSV-2 activity (IC50 = 5.0 μM) (Cheng et al., 2002). In close analogy with studies on anti-APC activity, the proposed mechanism of antiHSV properties of green tea catechins involves the inhibition of viral attachment and penetration into cells. The disturbance of the late stage of viral infection has also been postulated as an auxiliary mechanism (Friedman, 2007). Likewise, EGCG (50 μM) inhibited the expression of lytic proteins of Epstein-Barr virus (EBV, also known as human HSV-4) causing infectious mononucleosis. The inhibition of transcription of early genes governing the initiation of the EBV lytic cascade has been proposed as the anti-EBV mechanisms of EGCG (Chang et al., 2003). The antiviral effects of green tea catechins can be targeted towards HIV (the retrovirus causing the widespread human acquired immunodeficiency syndrome, AIDS) infection. Early studies reported that ECG and EGCG, but not EC or EGC, are powerful antagonists of HIV reverse transcriptase (IC50 from 10 to 20 ng/mL) (Nakane and Ono, 1990). Further studies reported that EGCG is responsible for the observed anti-HIV-1 activity of green tea (Nakane and Ono, 1989; Fassina et al., 2002). Several mechanisms explaining the anti-HIV properties of EGCG have been proposed. Early hypothesis involves the inhibition of the biochemical activity of HIV-1 reverse transcriptase, which causes the blocking of HIV-1 replication in

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human cells (Nakane and Ono, 1989). More recent studies suggested a possible interference of EGCG with HIV-1 viral infection by virion destruction and HIV-1 reverse transcriptase inhibition (Fassina et al., 2002), whereas Yamaguchi et al. (2002) hypothesized that EGCG could induce in vitro virion destruction by deforming phospholipids through the formation of specific bounds with the surface of the viral envelope. Likewise, Kawai et al. (2003) reported that EGCG apparently prevents the attachment of the HIV-1 virion (gp120), to CD4 molecules on T-helper cells. The authors found that EGCG (in concentrations from 25 to 250 μM/L) down-regulated the cell surface expression of CD4 by binding to the CD4 molecule, presumably at a binding site recognized by gp120. Other viruses causing different diseases have been studied as target for the antiviral activity of green tea catechins. Preventive and curative effects of green tea extracts on influenza virus have been claimed in a patent (Shimamura and Hara., 1991). ECGC apparently prevents infection caused by the influenza virus by binding to the viral hemagglutinin (HA) (Nakayama et al., 1993). So, the viral particles (bound by EGCG) cannot attach to the target receptor cells. In vitro studies postulated that changes of viral membrane properties could contribute to the antiviral effect of green tea catechins against the influenza virus. EGCG and ECG were found to be 10–15 times more active against the influenza virus than EGC. The role of 3-galloyl side chain as a factor enhancing antiviral activity of the parent catechin molecule has been hypothesized (Song et al., 2005). As a part of a SAR study, Song et al. (2007) screened the in vitro and in vivo antiviral activity of synthetic EGC and (+)-catechin (C) derivatives characterized by the presence of different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group. Pronounced activity was observed for derivatives carrying moderate chain length (7–9 carbons) as compared to those with aromatic rings. On the contrary, the 5’-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The active derivatives exerted inhibitory effects for all influenza subtypes tested, including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The observed antiviral activity appears to be mediated by the interaction of catechin derivatives with HA and viral membrane (Song et al., 2007). Hepatitis B virus (HBV) infection causes public health problems worldwide and is endemic in some geographical regions (e.g. Asia). Xu et al. (2008) studied the in vitro antiHBV efficacy of green tea catechins and EGCG: IC50 from 5.02 to 10.76 μg/mL were observed, whereas the 50% cytotoxic concentration (CC50) was as higher as 170 μg/mL. Additional studies involving plant and animal viruses have been carried out. EGCG and ECG bound to and inactivated tobacco and cucumber mosaic viruses that cause lesions in leaves (Okada et al., 1971; Okada et al., 1978). Other studies reported that green tea catechins prevent rotavirus and enterovirus from infecting monkey kidney cells in tissue cultures (Mukoyama et al., 1991). This evidence was attributed to the possible interference of green tea catechins with viral adsorption rather than a direct antiviral effect. Additionally, green tea catechins were also proven to be active against the bovine coronavirus and rotavirus (causing diarrhea and gastroenteritis in calves and cattle and resulting in significant losses to agriculture) (Clark et al., 1998).

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ANTIBACTERIAL ACTIVITY OF GREEN TEA CATECHINS Green tea catechins have been tested as antimicrobial drugs against human pathogenic bacteria (e.g. against ocular and cariogenic bacterial microflora causing minor infections). ECGC inhibited gelatinase activity produced by a few ocular bacterial pathogens (IC50 = 200 μM) (Blanco et al., 2003). More recently, Friedman (2007) underlined that the inhibition can delay the invasive spread of the bacteria in the eyes thriving on a gelatin substrate. Moreover, some studies described anticariogenic effects of green tea compounds. Dental caries are caused by a group of acid-producing species of the genus Streptococcus, in particular Streptococcus mutans and Streptococcus sobrinus, which are reported to be the major infective agents of human dental plaque. A key role in such process is played by salivary amylase, which hydrolyses food starch to oligo- and monosaccharides (e.g. maltose, glucose). The fermentation of such carbohydrates by bacterial enzymes occurring in oral cavities provokes the formation of organic acids responsible to dental caries. Green tea components have been seen to inhibit salivary amylase and, consequently, intra-oral hydrolysis of starch (Zhang and Kashket, 1998). Another study showed that EGCG prevented lowering of pH induced by cariogenic bacteria (Hirasawa et al., 2006). Likewise, an extract of green tea was used for inhibiting growth of S. mutans. The analytical characterization of such extract revealed that the main antibacterial components were GC, EGC and EGCG: among them, GC was the most active component (MIC = 250 μg/mL) (Sakanaka et al., 1989). Among pathogenic bacteria, Mycobacterium tuberculosis is a species that causes tuberculosis in humans. Anand et al. (2006) observed that EGCG inhibited the expression of tryptophan-aspartate containing coat protein (TACO) gene in a dose-dependent manner. This effect was coupled with the inhibition of the survival of M. tuberculosis within host macrophages. The species Helicobacter pylori is a urease-producing gastric pathogen that may contribute to the formation of ulcers and to low-grade gastric lymphoma in humans. Mabe et al. (1999) found that EGCG displayed a strong activity against H. pylori (MIC50 = 8 μg/ml). This compound exhibited bactericidal activity at pH 7 but not at pH < 5.0. In vivo studies carried out on infected Mongolian gerbils, reported that H. pylori was eradicated in 10 to 36% of the catechin-treated animals, with significant decreases in mucosal hemorrhage and erosion (Mabe et al., 1999). Likewise, a screening carried out on green tea catechins revealed their anti-H. pylori activity (Shin et al., 2005). Further studies reported that ECGC apparently protects epithelial cells of gastric mucosa against H. pylori-induced apoptosis and DNA damage (Lee et al., 2004). The authors postulated the block of activation of cellular signaling pathways as the mechanism causing protecting activity of epithelial cells. Legionella pneumophila causes Legionnaire disease, an infection of the lungs and other organs. A few authors found that ECGC enhanced the in vitro resistance of alveolar macrophages to infection caused by L. pneumophila (Yamamoto et al., 2004). Similarly, Matsunaga et al. (2001) showed that concentrations as low as 0.5 μg/mL of EGCG inhibited the growth of L. pneumophila in macrophages without any direct antibacterial effect. EGCG selectively up-regulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) and down-regulated the L. pneumophila-induced production of interleukin-10 (IL-10) by macrophages. The authors sauggested that EGCG selectively leads to an enhanced anti-L. pneumophila activity of macrophages.

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Green tea catechins have been also tested as antimicrobial drugs against food-borne pathogen and food-spoilage bacteria. In particular, food-spoilage bacteria can cause spoilage and undesirable changes in a wide range of foods, particularly in processed, preserved, and refrigerated food. Chou et al. (1999) used selected strains of food-borne pathogen and foodspoilage bacteria to test the antibacterial activity of extracts from various tea products. In general, antimicrobial activity decreased when the extents of tea fermentation increased: tea flush and green tea extracts exerted the strongest antibacterial activity. The activity of green tea catechins against the bacterial genera belonging to the Bacillaceae family (Bacillus and Clostridium) was extensively studied. Bacillus cereus is a widely distributed food-borne pathogen causing vomiting and diarrhea in mammals (including humans). Friedman et al. (2006) demonstrated that GCG, EGCG, CG and ECG exhibit antimicrobial activities at nano-Molar levels, whereas catechins without gallate side chains and gallic acid were inactive. Interestingly, most compounds were more active than commercially available antibiotics (e.g. tetracycline or vancomycin) at comparable concentrations. In the same way, studies carried out by Hara et al. (1989; 1995) and by Ahn et al. (1991) showed that green tea catechins strongly inhibited growth of Bacillus stearothermophilus and Clostridium thermoaceticum in vitro. These compounds also reduced the heat-resistance of both species growing in vending machines, which causes sour spoilage in milk and other drinks (Sakanaka et al., 2000). Likewise, Sakanaka et al. (2000) studied the inhibitory action of green tea catechins towards the germination of Bacillus spp. and Clostridium spp. spores. The heat resistance of B. stearothermophilus and C. thermoaceticum spores was reduced by the addition of EGCG. More recently, Juneja et al. (2007) found that green tea extracts characterized by different catechin concentrations (from 141 to 697 mg of total catechins/g of extract) determined the inhibition of Clostridium perfringens spore germination in thawed beef, chicken, and pork. The authors underlined the catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling. Staphylococcus aureus is a highly pathogenic, toxin-producing, food-borne organism. An early study reported that ECGC inhibits the growth of methicillin-resistant S. aureus (MRSA) strains (Toda et al., 1991). Likewise, Ikigai et al. (1993) showed that EC was much less active than EGCG against S. aureus strains. The MICs of EGCG and EC were 73 and 573 μg/mL, respectively, and the bactericidal effect of EGCG was attributed to membrane perturbation (Ikigai et al., 1993; Hamilton-Miller, 1995). The role of catechins (in particular EGC, EGCG ECG) on the anti-MRSA strains was also confirmed by Yam et al. (1997). More recently, Yoda et al. (2004) observed MICs from 50 to 100 μg/mL of EGCG against some species belonging to the genus Staphylococcus (S. aureus, Staphylococcus epidermis, Staphylococcus hominis and Staphylococcus haemolyticus). The authors postulated that the different structure of the cell wall as well as the variable affinities of ECGC to some cell wall components (e.g. peptidoglycans) might determine a differential activity of EGCG against such bacterial species. Furthermore, Si et al. (2006) found that ECG and EGCG were the most active compounds against S. aureus: EGCG exhibited the highest activity (MIC90 = 58 and 37 μg/L for MSSA and MRSA, respectively). Scanning electron microscopy (SEM) studies showed that both ECG and EGCG altered bacterial cell morphology, which might have resulted from disturbed cell division. Analogously, Kim et al. (2004) found that green tea catechins

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exhibited an approximately 5.0 log CFU/mL suppression of S. aureus strains (compared with the control). Physical factors can enhance the anti-S. aureus activity of green tea catechins. An et al. (2004) found that irradiated (at 40 kGy) catechins extracted from green tea leaves increased their antibacterial activities against S. aureus and S. mutans, whereas the major antioxidative activities (e.g. electron donating, inhibition of xanthine-oxydase, metal ion chelating, and inhibition of lipid oxidation) were unchanged after irradiation. The strain O157:H7 of the species Escherichia coli is a food-borne, toxin-producing enteropathogen responsible for a hemorrhagic form of colitis, bloody diarrhea, and hemolytic uremic syndrome. Isogai et al. (1998) reported that green tea catechins protect mice against neurologic and systemic symptoms caused by infection with E. coli O157:H7. Similar studies found that green tea extracts exhibit a wide spectrum of activity against a series of pathogenic bacteria, including strains of E. coli (Yam et al., 1997; Yam et al., 1998). A more recent study (Si et al., 2006) found that green tea extracts strongly inhibit the growth of E. coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, S. aureus, and B. cereus. On the contrary, Kim et al. (2004) found that green tea extracts suppressed the growth of only L. monocytogenes (by approximately 3.0 log CFU/mL), whereas no influence on E. coli O157:H7 and Salmonella enteritidis growth has been noted. Yam et al. (1997) reported that a green tea extract inhibited the growth of several species of food-spoilage bacteria (Proteus vulgaris, Pseudomonas aeruginosa and Serratia marcescens) known to adversely affect the quality of some foods. More recently, Yoda et al. (2004) found that higher levels of EGCG (MIC = 800 μg/mL) were needed to inhibit the growth of some Gram-negative food-borne pathogenic and food-spoilage bacteria (E. coli, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, P. aeruginosa, and S. marcescens). In close analogy with above reported data, ECG and ECGC were also found to inhibit the growth of some phytopathogenic bacteria belonging to the genera Agrobacterium, Clavibacter, Pseudomonas, Erwinia, and Xanthomonas (which contaminates eggplants, grapes, cabbage, lettuce, onions, potatoes, tomatoes, etc.). MIC of about 100 μg/mL were observed (Fukai et al., 1991). Table 1. Some additional examples of antibacterial activity of green tea catechins Species Bacillus anthracis

Active References compound(s) EGCG Dell'Aica et al., 2004

Bacillis cereus

catechins

Hamilton-Miller, 1995; Friedman et al., 2006

Escherichia coli

EGCG

Taguri et al., 2004

Helicobacter pylori

ECG, EGCG Setiawan et al., 2001; Yee et al., 2002; Yahiro et al., 2005

Legionella pneumophila Staphylococcus aureus Vibrio cholerae

EGCG

Matsunaga et al., 2002; Rogers et al., 2005

EGCG

Stapleton et al., 2004; Taguri et al., 2004

EGC, EGCG Ikigai et al., 1990; Toda et al., 1992; Taguri et al., 2004; Bandyopadhyay et al., 2005

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In relation to the activity of green tea catechins on bacteria of technological importance, Goto et al. (1998) reported that these compounds are able to positively affect intestinal dysbiosis in nursing home patients by raising levels of Lactobacillus spp. and Bifidobacterium spp. while lowering levels of Enterobacteriaceae and Bacteroidaceae. Some additional examples of antibacterial activity of green tea catechins are reported in Table 1. Some hypotheses have been recently proposed for explaining the mechanism of antibacterial action of green tea catechins. Accordingly, detailed physicochemical studies suggested that the bactericidal activities of galloylated green tea catechins at the cell membrane level might be due to their specific perturbations carried out over the ordered structure of phosphatidylcholine and phosphatidylethanolamine bilayers constituting bacterial cell wall membranes (Nakayama et al., 2000; Caturla et al., 2003). ECGC was found to be the most effective catechin in causing leakage on E. coli-isolated membranes. Moreover, another study suggested that EGCG could increase antibiotic susceptibility in S. aureus through the inhibition of penicillinase produced by such bacterium (Zhao et al., 2002). In more recent studies additional hypothesis supporting the antibacterial action of green tea catechins have been proposed. Arakawa et al. (2004) and Hayakawa et al. (2004) suggested that the bactericidal action of EGCG may also depend on hydrogen peroxide derived from the reaction of EGCG with oxygen (pro-oxidative activity). Based on the above mentioned hypotheses, Friedman (2007) concluded that the observed antimicrobial effects arise from the interactions of catechins with oxygen, genes, cell membranes, and enzymes.

ANTI-YEAST AND ANTIFUNGAL ACTIVITY OF GREEN TEA CATECHINS Only a few studies have been carried out so far on the antimycotic activity of green tea tannins. Okubo et al. (1991) early examined EGCG for their antifungal and fungicidal activity against pathogenic filamentous fungi belonging to the species Trichophyton mentagrophytes and Trichophyton rubrum. This compound showed fungistatic activity against both Trichophyton species. More recently, Hirasawa and Takada (2004) have studied the susceptibility of the opportunistic pathogenic yeast Candida albicans to various green tea catechin under varying pH conditions. Analogously, Park et al. (2006) reported that EGCG exhibits antimycotic activity against 21 Candida spp. isolates. Among them, the strains belonging to the species Candida glabrata were the most susceptible. Both studies reported that the anti-yeast activity of catechins was pH dependent. The two studies reported contradictory results. Hirasawa and Takada (2004) observed that the MIC90 of EGCG was 2000 μg/mL at pH 6.0, 500–1000 μg/mL at pH 6.5 and 15.6–250 μg/mL at pH 7.0, whereas Park et al. (2006) found that the MIC of EGCG increased several folds as the pH was reduced from 7.0 to 6.0. As a result of a large-scale screening survey on the antimycotic activity of some plant extracts, Turchetti et al. (2005) observed that green tea extracts inhibited growth of yeasts belonging to the species C. glabrata Cryptococcus laurentii, Clavispora lusitaniae, Issatchenkia orientalis, Filobasidiella neoformans and Saccharomyces cerevisiae, as well as

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that of yeast-like microorganisms Prototheca wickerhamii. The authors concluded that the compounds responsible of the observed anti-yeast activity are ECG and EGCG. The mode of action of catechins on eukaryotic microorganisms has been little studied. In early investigations, Toyoshima et al. (1993) suggested that catechins are able to attack the cell membrane of Trichosporon mentagrophytes causing lysis of the conidia and hyphae.

SYNERGISTIC INTERACTIONS BETWEEN CATECHINS AND ANTIBIOTICS Current literature reports studies describing that the combined use of antibiotics and green tea catechins can increase the antimicrobial activity of the formers through specific synergistic interactions. Combinations of EGCG + β-lactames antibiotics exhibited synergistic activities against MRSA strains (Hu et al., 2002). Zhao et al. (2001) suggested that EGCG synergistically increases the activity of β-lactam antibiotics against S. aureus by binding to the peptidoglycan component of the bacterial cell wall. Likewise, Sudano Roccaro et al. (2004), postulated that EGCG acts in synergy with β-lactam antibiotics by reversing tetracycline resistance in Staphylococcus spp. isolates and by inhibiting the specific efflux pump Tet(K). The authors observed an increased accumulation of tetracycline inside bacterial cells as a visible effect of synergy with EGCG. On the contrary, Yanagawa et al. (2003) noted only additive interactions between the β-lactam antibiotic amoxicillin and EGCG against nonresistant and antibiotic-resistant isolates of H. pylori. More recently, Friedman (2007) reported that non-galloylated catechins also potentiated the activity of oxacillin against S. aureus. The combined use of green tea extracts with butylated hydroxyanisole (BHA) was more effective against bacteria and fungi than green tea alone (Simonetti et al., 2004). Glycolic extract from green tea showed a certain activity against E. coli, but only limited activity against S. mutans and no activity against C. albicans. Sub-inhibitory concentrations of BHA increased the microbicidal activity of green tea extracts against S. mutans, non-susceptible E. coli and C. albicans. In addition, green tea extracts in combination with BHA reduced the hydrophobicity of S. mutans and greatly inhibited the formation of pseudomycelium in C. albicans. The authors postulated that the increased antimicrobial activity of green tea extracts (due to synergy with BHA) is related to an impairment of the barrier function in microorganisms and a depletion of thiol groups. Other groups of antibiotics have been studied for their synergistic interactions with green tea catechins. Lee et al. (2005) observed that a combination of catechins and the fluoroquinolone antibiotic ciprofloxacin acts synergistically to alleviate chronic bacterial prostatitis in rats. Likewise, a further study showed that a green tea extract exhibited in vivo synergy with the antibiotic levofloxacin against infection of mice caused by E. coli O157:H7 (Isogai et al., 2001). Hirasawa and Takada (2004) reported that the addition of EGCG to sub-inhibitory concentrations of amphotericin B (AMB, belonging to the group of polyenes) increased the anti-yeast activity of AMB against AMB-susceptible or -resistant C. albicans. Combined treatment with EGCG + AMB (3.12–12.5 and 0.5 μg/mL, respectively) markedly decreased the growth of AMB-resistant C. albicans strains. Since sub-inhibitory concentrations of AMB

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stimulate yeast membrane permeability, the authors suggested that the combined use of AMB and EGCG might stimulate catechin uptake into the cell, and, consequently, the increased intracellular catechin concentration could act as a anti-yeast agent. The same authors also observed that when azole-susceptible C. albicans strains were treated with EGCG + fluconazole (25–50 and 0.125–0.25 μg/mL, respectively), its growth was inhibited by 93.0– 99.4% compared with the growth observed in the presence of fluconazole alone. The combined use of EGCG + fluconazole (12.5 and 10–50 μg/mL, respectively) also inhibited the growth of fluconazole-resistant C. albicans by 98.5–99.7% (Hirasawa and Takada, 2004).

CONCLUDING REMARKS Current literature herein highlight the fact that green tea polyphenols include a series of compounds (e.g. catechins) extensively studied as potential antiviral and antimicrobial drugs. Yet, although some promising results have been reported, most studies remains so far confined to the laboratory scale. In other words, in relation to the commercial exploitation of green tea catechins as antimicrobial drugs a few questions remain still open. First of all, the lack of a rigorous analytical determination of the chemical nature of active components of some green tea extracts is a common feature in most of the literature cited in this chapter. Studies using purified (or partially purified) molecules (e.g. catechins) are still very few in number. The improvement of analytical techniques will indisputably lead to a better knowledge of the antiviral and antimicrobial properties of individual compounds occurring in green tea composition. Hence, the high cost of commercially available pure standards (e.g. EGCG) represents a supplementary problem, whereas synthetic strategies, so far used only for SAR studies carried out at the laboratory-scale, are too costly and timeconsuming procedures. Secondly, the assay protocols employed for the in vitro evaluation of antimicrobial activity of green tea components is an additional problem. Most of the studies reporting MICs did not apply standard CLSI guidelines (CLSI, 2002a; CLSI, 2002b) essential for monitoring the accuracy of the method. As a result, some contradictory results could be the consequence of their low reproducibility level. The well-documented strain-related susceptibility of microbial genotypes is an additional problem. In this sense, the use of undetermined (or even unidentified) strains should be discouraged. Finally, as recently underlined by Friedman (2007), another critical aspect is to determine whether the antimicrobial activities of green tea compounds observed in vitro can be duplicated in vivo. It is important to remember that most of the literature cited in this chapter reported results of in vitro investigations. Even though useful as a preliminary survey, in vivo activity is clearly more significant. As pointed out by some authors (Kawai et al., 2003; Nance and Shearer, 2003), the most crucial aspect of translating the observed in vitro effects of green tea compounds to pharmacologically relevant strategies, is the requirement to achieve physiologically relevant concentrations. Due to the well-known poor bioavailability of some green tea compounds, most of the ingested EGCG does not get into the blood, and a significant fraction is eliminated presystemically (Chow et al., 2001; Pisters et al., 2001; Lee et al., 2002). So, even though the use of capsular administration of green tea or EGCG has been proposed, some authors doubted that, in this form, the blood EGCG concentration could

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remain at a low level than that used in the current in vitro studies (Kawai et al., 2003; Nance and Shearer, 2003). Additional studies should be consequently carried out to clarify these crucial aspects for the possible exploitation of green tea preparations for therapeutic purposes.

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Zhao, WH; Hu, ZQ; Okubo, S; Hara, Y; Shimamura, T. Mechanism of synergy between epigallocatechin gallate and β-lactams against methicillin-resistant Staphylococcus aureus, Antimicrob. Agents Chemother. 2001, 45, 1737 – 1742. Zhao,WH; Hu, ZQ; Hara, Y; Shimamura, T. Inhibition of penicillinase by epigallocatechin gallate resulting in restoration of antibacterial activity of penicillin against penicillinaseproducing Staphylococcus aureus, Antimicrob. Agents Chemother. 2002, 46, 2266–2268. Zhao, BL; Li, XJ; He, RG; Cheng, SJ; Xin, WJ. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell. Biophys. 1989, 14: 175-185.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 3

LIPID-SOLUBLE GREEN TEA POLYPHENOLS: STABILIZED FOR EFFECTIVE FORMULATION Ping Chen2, Douglas Dickinson1 and Stephen Hsu1 1

Medical College of Georgia, USA 2 Zhejiang University, China

ABSTRACT Green tea polyphenols (GTPs), also referred to as green tea catechins, possess properties that can provide unique health benefits to humans. As indicated in other chapters of this book, studies using molecular, cellular, and animal models, and in human subjects, have demonstrated that these phytochemicals from non-oxidized tea leaves have anti-cancer, anti-oxidant, anti-microbial, and anti-inflammatory properties. Recently, investigations in our and other laboratories indicated that topical application of GTPs could protect the epidermis against autoimmune disorders, such as psoriasis, prevent or repair UV-induced damage, and suppress scar tissue overgrowth. In addition, specific gene regulation by GTPs, especially epigallocatechin-3-gallate (EGCG), promotes skin cell differentiation, which could lead to improved homeostasis of the skin. Based on these facts, the topical use of products containing GTPs has become more popular, and manufacturers of cosmetic, health care, and household products are adding GTPs or EGCG to their formulations. However, it is important to note that studies described in this book always use “freshly prepared” GTPs or green tea, instead of “preprepared” materials. This is because GTPs are potent antioxidants that react rapidly with reactive oxygen species (ROS). As a result, GTPs in most commercially available products have been oxidized and/or epimerized; the biological effects of the resulting compounds are largely unknown. In addition, due to the highly water-soluble nature of these compounds, GTPs in their original form are not lipid-soluble, and therefore not permeable to the skin, a water-proof barrier. Another problem with formulation of GTPs for topical application is the coloration change and precipitation caused by oxidation. Thus, GTPs for topical application (e.g., on skin and mucous membranes) must be prepared and used immediately prior to oxidation, coloration and precipitation. These properties of GTPs make it difficult to formulate products containing them that have a reasonable shelf life and maintain their activity and effectiveness. In other words, most of the commercially available “green tea”–containing products are without the full benefits

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Ping Chen, Douglas Dickinson and Stephen Hsu of green tea or GTPs. Therefore, strategies to stabilize and increase the bioavailability of GTPs are needed to provide the full benefits of GTPs to consumers or patients. Recently, it has been shown that lipid esters of GTPs can be formed either enzymatically or chemically. These green tea polyphenol-lipid esters, also referred to as lipid-soluble tea polyphenols (LTPs), could significantly improve formulations of consumer or health care products. We hypothesized that fatty acyl esterification of green tea polyphenol would protect the hydroxyl groups from oxidation and improve skin permeability. In the current study, we compared the activities of LTPs to GTPs for their anti-cancer and gene regulation properties. We examined whether LTPs can be converted into a free GTP (EGCG) in human skin keratinocyte cultures. In addition, the effects of LTPs in a mouse model for psoriasis were evaluated. The results indicate that LTPs effectively cause cancer cell death, induce caspase 14 gene expression both in vitro and in vivo, and improve the skin condition in an animal model for psoriasis. Consistent with these observations, HPLC analysis demonstrated that EGCG in its original form was released from LTPs in situ by human epidermal keratinocytes. These results suggest that LTPs, under appropriate conditions, function similarly to GTPs. More importantly, since the most reactive hydroxyl group(s) is/are protected, and the lipid solubility is dramatically increased by the fatty acyl groups, the biological activity of these compounds can be stabilized, and their bioavailability increased significantly. In conclusion, LTPs are a novel and more effective form of green tea polyphenols for topical applications and other purposes, especially in formulations that require a reasonable shelf life. In addition, LTPs can be a natural additive to consumable products such as salad oil, fish oil, and cooking oil as antioxidants.

INTRODUCTION Green tea polyphenols (GTPs) are under extensive study for their unique properties that can provide protection against a number of human diseases, including neoplastic, cardiovascular, autoimmune, and infectious diseases. During the past decade, anti-cancer, anti-inflammatory, anti-aging and anti-photo-damaging properties of GTPs, especially epigallocatechin-3-gallate (EGCG), have been identified [1-11]. These findings have prompted the addition of GTPs to cosmetic and health care products for topical application. The ingredient labels of many cosmetic and household products list various descriptions of GTPs, such as “green tea extract”, “extract of camellia sinensis”, “green tea polyphenols”, “green tea catechins”, “green tea leaf extracts”, and “EGCG”. In addition, due to their strong anti-oxidant activity, this type of naturally-occurring compound can potentially be used as natural antioxidant food additives in various products, including dietary oils. Unfortunately, certain properties of GTPs create problems for their commercial application. In traditional green tea-consuming countries such as China and Japan, green tea is consumed immediately after it is brewed, i.e., “freshly prepared”. In an effort to bring green tea’s benefits to modern populations, bottled green tea was invented, and it has become popular even in traditionally “green tea brewing” countries. However, an aqueous environment favors oxidation of GTPs, leading to epimerization and polymerization of the catechin monomers. When the monomers are oxidized, their anti-oxidant and biological activities are reduced significantly. A study of bottled tea beverages confirmed that the majority of these drinks no longer contain significant amount of GTPs, but rather the epimer gallocatechin gallate (GCG) and oxidized compounds [12]. In this study, dry leaves of green

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tea had an average EGCG content of 6.87% (see Table 1 in reference 12). In most brandnamed tea beverages tested by this study, the EGCG content was at “trace” levels (< 0.00001%) to 0.00082%. One exception had a higher level (0.058%) [12]. In other words, due to the required step of sterilization by autoclaving, the anti-oxidant and other beneficial effects of most tea beverages have been destroyed prior to their consumption. A second problem with GTPs is that they are highly hydrophilic, resulting in very low solubility in dietary oils, and they form dark precipitates in these oils [13]. Formulation experts have tried to emulsify GTPs with lipid material in order to increase their solubility in dietary oils, but the results have not been satisfactory due to precipitation and coloration [13]. A third problem for the use of GTPs is their relatively very low bioavailability in humans after oral administration as measured by serum concentration analysis [14]. In fact, the majority of GTPs is excreted from the digestive and urinary system, and less than 10 µM of GTPs concentration can be achieved in the circulation [14, 15]. This poor serum bioavailability decreases the potential anti-cancer and anti-inflammatory effectiveness of GTPs, which have been described in many studies that have used much higher concentrations of GTPs. In addition, since the epidermis is not vascularized, the benefit of orally consumed GTPs to the human epidermis is highly questionable [16]. Therefore, for the skin, topical application with high concentrations of GTPs/EGCG has been considered as an optional route of delivery (11, 10% EGCG). However, topical application of a high concentration of GTPs could cause significant skin coloration, although this effect could be used for skin tanning (US Patent 6399046). Other methods, such as “electrical assisted” methods, have been developed to increase the skin permeability to GTPs, but they may not be a suitable strategy to improve skin condition [17]. Taken together, the hydrophilic nature of the water-soluble and unstable polyphenolic GTP molecules prevents them from penetrating lipid barriers such as the stratum corneum. This compounds the fact that GTPs in these products are undergoing oxidation, epimerization, and/or polymerization prior to reaching the customers. Thus, the instability of GTPs in their original monomer form in an aqueous environment at physiological pH, and their impermeability to lipid barriers and insolubility in a hydrophobic environment, prevent their delivery at levels that provide effective benefits to human health. Stability and bioavailability are therefore two major challenges in the development of any effective product with a reasonable shelf life. Green tea researchers and formulation specialists identified these limitations, and attempts to preserve the chemical integrity and activity of GTPs began in the 1990s [13]. Esterification has been proven effective for other bio-molecules such as vitamins C (ascorbyl palmitate) and A (retinyl palmitate), and bioactive peptides, which are widely used in today’s consumer and cosmetic products [18, 19, and international patent WO 1992014830]. Researchers proposed that modification of GTPs with fatty acyl esterification would increase their stability and skin permeability without significant reduction of the antioxidant property. One group of Chinese researchers led by Professor Shuxiong Wang invented a method using an acylation reaction (patent CN1197786A) that chemically modifies GTPs to make GTPesters. This method renders the final product (lipid-soluble tea polyphenols [LTPs] in a dark brown gel-like form) soluble in oil and many hydrophobic solvents. Another method was later developed in China by Jian Hua Zhong and Ping Chen which produces GTP-esters in a solid powder form (CN1231277A). In Japan, similar GTP derivatives were made using the reverse reaction of lipases from Streptomyces rochei (Japanese patent JP6279430). Recently, Kunihiro Kaihatsu’s group in Osaka University synthesized a series of EGCG-esters by an

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enzymatic method using lipase PL from Alcaligenes sp [20]. In 2002, one of us (P Chen) first synthesized and identified the chemical structure of EGCG-4’-hexadecanate [21]. Subsequently, in 2003, the first systematic synthesis, purification, and analysis of EGCGacyl-derivatives in the laboratory was reported, and the purified LTP compound epigallocatechin-3-O-4’-O-hexadecanate was obtained (referred to as EGCG-palmitate hereafter) [22]. The antioxidant activity and stability of EGCG-palmitate was tested in dietary oils. It was found that the antioxidant activity of EGCG-palmitate in soybean salad oil and canola oil is greater than that of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), and similar to that of tertiary butylhydroquinone (TBHQ) [23]. In addition, the potential toxicity of EGCG-palmitate was tested by the Zhejiang Center for Disease Control & Prevention of China. The acute toxicology test result in mice was LD50>5.0 g/kg, and the Ames Test result for mutagenicity was negative [23]. The anti-cancer property of EGCG-palmitate was compared with GTPs and LTPs by determining the inhibition of growth of the human ovarian cancer cell line HO-8910 [24]. The 50% inhibitory concentration (IC50) for EGCG-palmitate was 78.2 µg/ml, while for GTPs it was 72.2 µg/ml, and 88.9 µg/ml for LTP [24]. In a separate study, EGCG-myristate (epigallocatechin-3-O-4’O-myristate) was synthesized [25]. This is an EGCG ester with a 14 carbon fatty acyl chain. The physical characteristics of EGCG-myristate are similar to EGCG-palmitate. It has a whitish powder appearance and is readily soluble in lipid, but not in water. The antioxidant activity of EGCG-myristate is almost identical to EGCG-palmitate when compared with TBHQ, BHA and BHT (in soy bean salad oil) [25]. That is, the food industry, especially dietary oil manufacturers, could benefit from the use of LTPs as an antioxidant. However, it is not known if LTP enters human cells, or if it can be converted to GTPs to induce similar biological effects. It is also not clear whether a stable LTP formulated topical application can improve the skin condition in a mouse model for psoriasiform lesions (the flaky skin mouse), as shown for EGCG. The current study used a powdered form of 18 carbon fatty acyl (stearoyl) and a gel-like 18 carbon monounsaturated fatty acyl (oleic acyl)ester of green tea polyphenols (referred to as LTPStearate and LTPOleate hereafter) to tests their biological effects in vitro and in vivo.

MATERIAL AND METHODS Chemicals and Antibodies EGCG was purchased from Sigma-Aldrich (St. Louis, MO). The solid-form LTPStearate (>90% stearoyl tea polyphenol) was purchased from Yuyao Huidelong Biological BioProducts, Co., Ltd, Zhejiang, China. The gel-form LTPOleate (>90% oleic acyl tea polyphenols) was purchased from Zhejiang Cereals Oils & Foodstuffs Import & Export Company, Ltd, Zhejiang, China. A topical formulation of LTPStearate was made of 0.1% LTPStearate in glycerin and stored at 4o C for daily topical use (the preparation of this formulation is not disclosed due to pending patent). The anti-caspase 14 and anti-human actin (I-19) antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.

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Cell Lines and Cell Culture Pooled normal human primary epidermal keratinocyte (NHEK) cells were purchased from Cambrex (East Rutherford, NJ) and sub-cultured in the specific growth media (KGM-2) provided by the manufacturer. The OSC2 cell line was isolated from a metastatic lymph node of a patient with oral squamous cell carcinoma [26]. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 50/50 MIX medium (Cellgro, Kansas City, MO) supplemented with 10 % (v/v) fetal bovine serum, 100 I.U/ml penicillin, 100 µg/ml streptomycin and 5 µg/ml hydrocortisone.

Morphological Analysis of Cells Treated with LTPStearate OSC2 or NHEK cells were seeded in 24-well tissue culture plates at 2X105 cells/well and incubated overnight. LTPStearate dissolved in vehicle (100% ethanol) at different concentrations was added to each well. Control wells were incubated with vehicle only. At the end of incubation, wells were washed with PBS and photographed at 100X magnification.

Western Blotting To determine changes in the protein level of caspase 14 upon activation by EGCG or LTPStearate, NHEK were exposed to 50 µM EGCG or LTPStearate for various times. Cell lysates were prepared, and samples containing 30 µg protein were separated on 10 % SDS polyacrylamide gels. Western blot analysis was performed using anti-human caspase 14 and actin antibodies (for normalization). The method for Western analysis was previously described [27]. The resulting bands were visualized by enhanced chemiluminescent staining using ECL Western blotting detection reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Animal Treatment and Immunohistochemistry Flaky skin mice were treated daily for consecutive ten days with an LTPStearate topical preparation (0.1% w/v in glycerin) in an area on the back of the animal. On the opposite side of the animal, glycerin was applied topically as control. The skin samples were collected by a biopsy puncture under anesthesia, and the animals were euthanized thereafter. Processing and immunostaining of skin samples have been described previously [28].

HPLC Analysis of EGCG from Cell Lysates Cultures of NHEK cells were divided into two and treated with 0.1% w/v of either LTPOleate or LTPStearate. At 0.5 h, 1.0 h, 2.0 h, and 6.0 h, cells were washed with PBS and lysed

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with 0.5 ml RIPA buffer (pH adjusted to 3.35). An equal amount of cell lysate was also collected from NHEK treated with 100 μM EGCG for 0.5 h as a control for HPLC detection. HPLC detection of EGCG in cell lysates was performed according to a method previously described [29]. The Discovery C-18 reverse-phase column was purchased from Supelco, Sigma-Aldrich (St. Louis, MO). Briefly, the column was eluted with Solution A (30 mM NaH2PO4 in 1.75% CH3CN and 0.125% tetrahydrofuran (pH 3.35) and Solution B (15 mM NaH2PO4 in 58.5% CH3CN and 6.25% tetrahydrofuran (pH 3.45), as described previously [29].

RESULTS LTPStearate Induces Tumor Cell Death as Efficiently as GTPs Our previous study demonstrated that GTPs at 1 mg/ml are a powerful inducer of cell death in OSC2 oral carcinoma cells [30, 31]. To test the effectiveness of LTPStearate, OSC2 cells were incubated with 1 mg/ml LTPStearate (0.1% w/v) for 24 hr. As shown in Figure 1, almost all tumor cells were eliminated by LTPStearate treatment, while control OSC2 cells treated with vehicle only remained viable. This result indicates that LTPStearate possesses the ability of a cell death-inducer for OSC2 cells. The anti-tumor cell effect of LTPStearate was also significant at a lower concentration (0.01% w/v), although less pronounced that at 0.1% w/v (Figure 2), consistent with dose-dependency.

Figure 1. 0.1% LTPStearate induced massive cell death in OSC2 cells. OSC2 oral carcinoma cells were incubated with vehicle or 0.1% LTPStearate (pre-dissolved in 100 ethanol) in cell culture medium for 24 h. The culture dishes were washed by PBS and photographed under light microscope (100 X).

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Figure 2. 0.01% LTPStearate induced significant cell death in OSC2 cells. OSC2 oral carcinoma cells were cultured until reaching confluency, and incubated with vehicle or 0.01% LTPStearate (pre-dissolved in 100 ethanol) in cell culture medium for 24 h. The culture dishes were washed by PBS and photographed under light microscope (100 X).

Figure 3. 0.1% LTPStearate failed to induce cell death in normal human epidermal keratinocytes (NHEK). NHEK cells were incubated with either vehicle or 0.1% LTPStearate for 24 h. The culture dishes were washed with PBS and photographed under light microscope (100 X).

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LTPStearate does not Induce Cell Death in Normal Epidermal Keratinocytes LTPStearate at 1 mg/ml (0.1% w/v) was incubated with primary NHEK (Figure 3). In contrast to the results with the tumor cell line OSC2, no sign of cell death or detachment was observed, indicating that LTPStearate did not induce cytotoxicity in NHEK.

Dose-dependent Induction of Caspase 14, a Marker for Epidermal Terminal Differentiation and Barrier Formation, by LTPStearate The expression of caspase 14 was compared between 50 µM EGCG-treated and 0.01% w/v LTPStearate-treated NHEK. Since 0.01% w/v of LTPStearate contains approximately 0.0025% w/v green tea polyphenols, the polyphenol content was comparable between the two treatments. Figure 4 demonstrated that both materials induced the expression of caspase 14 at the 20 h time point, and continued expression of caspase 14 was observed at 24 h. Western blotting also showed that LTPStearate at 0.001% w/v to 0.01% w/v (10-100 ppm) induced caspase 14 expression in NHEK cells in a dose-dependent manner (Figure 5).

LTPStearate Induces Caspase 14 Expression in Vivo in a Mouse Model for Psoriasiform Lesions Immunostaining of mouse skin treated with LTPStearate in glycerin demonstrated increased caspase 14 expression in the supra-basal layers of the epidermis (Figure 6). This result is consistent with our previous observation that GTPs induce and activate caspase 14 in vivo [28].

Figure 4. Western blot results demonstrate both EGCG and LTPStearate induced caspase 14 expression in NHEK. NHEK cells were treated with either EGCG (50 µM) or LTPStearate for 0, 2, 6, and 24 h. The cell lysates were collected for Western blot using rabbit anti-human caspase 14 and goat anti-human actin antibodies.

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Figure 5. LTPStearate dose-dependently induced caspase 14 protein expression in NHEK cells. Various concentrations of were prepared with DMSO, diluted with cell culture medium, and incubated with NHEK for 24 h. Cell lysates were collected for Western blot with antibodies against human caspase 14, and human actin as an internal control.

LTPOleate and LTPStearate are Converted to Intracellular GTPs in NHEK HPLC analysis of cell lysates of NHEK demonstrated that intracellular EGCG appeared in both LTPOleate and LTPStearate - treated NHEK (Table 1). However, the release time was different between the two (Figure 7). The majority of EGCG released from LTPOleate appeared in the cell lysate collected at 1 h after the addition of LTPOleate, while the majority of EGCG released from LTPStearate appeared at 6 h after treatment (Figure 7). Table 1. Retention time comparison (min) between the two types of LTP and EGCG

*Retention time of EGCG: 28.92 min. Time of EGCG peak was recorded by HPLC using samples from cell lysates collected at different time points after the LTPs was introduced into the cell culture medium.. ND: not determined.

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Figure 6. Immunostaining of skin samples from vehicle-treated and LTPStearate -treated (flaky skin mice) using caspase 14 antibody. Nuclear caspase 14 staining appeared in the junction of the granular layer and the cornified layer in the LTPStearate-treated sample, while the vehicle-treated sample did not exhibit nuclear staining of caspase 14. The supra-basal layers of the LTPStearate-treated sample showed consistent immunostaining of caspase 14, but those of the vehicle-treated sample only showed sporadic caspase 14 expression. The epidermal portion of the samples was photographed at a 400X magnification.

Figure 7. EGCG release from NHEK demonstrates time differences between LTPStearate and LTPOleate. Cell lysates were collected at indicated time points after LTPs was introduced into the cell culture medium. HPLC analysis of the samples showed different release patterns between the two types of LTPs. LTPS: LTPStearate. LTPO: LTPOleate.

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DISCUSSION The earliest Chinese literature indicates that the Chinese grew and used tea plants 3,000 years ago. According to legend, in 2737 B.C., the Chinese Emperor Shen-Nung placed camellia blossom tips into a cup of boiled water and pronounced the beverage healing and refreshing. Tea consumption became popular in China after it was said to cure the ailing troops of General Zhu Ge Liang (181-234 A.D.). Tea was described as able to prevent pandemics and treat patients with eye diseases. During the Tang Dynasty (618-907 A.D.), tea became an essential component of Chinese culture. Chinese author Lu Yu (733-804 A.D.) published the book, Cha Jing (literally, The Book of Tea), systematically chronicling the planting, processing, preparation and consumption of tea. From then, green tea (as well as many other types of teas derived from green tea), became the most popular beverage (second to water) in the world. Today, the medicinal use of green tea and its compounds is being re-visited by scientists and clinicians, and the benefits are being unveiled almost daily. In 2006, the FDA approved an ointment, VeregenTM (MediGene AG, Polyphenon® E Ointment), for treatment of genital warts, an epidermal sexually transmitted disease cause by human papilloma virus (HPV). The active ingredient(s) in this topical ointment is 10-15% Polyphenon E, a highly purified and defined GTPs mixture developed by Dr. Yukihiko Hara and colleagues at Mistui Norin Co. Ltd of Japan [32]. It is important to note that this ointment contains 10-15% of GTPs, far higher than any GTP-containing beverages or topical products. Therefore, this topical ointment treatment for this skin lesion, with an interrupted stratum corneum, still requires a physician’s prescription due to potential side effects. However, it represents the beginning of an era in which green tea components can be used for therapeutic purposes. It also suggests that the anti-microbial, especially the anti-viral, effects of GTPs need to be explored further. Recently, results from various laboratories confirmed that GTPs or EGCG, the most abundant green tea polyphenol, possesses potent inhibitory effects on influenza A and B viruses [33, 34], hepatitis B virus [35], herpes simplex virus (HSV) [36], Epstein-Barr virus [37], adenovirus [38], and human immunodeficiency virus (HIV) [39, 40]. In 2008, Dr. Kaihatsu at Osaka University and colleagues reported that EGCG-palmitate is 24 times more effective than EGCG when compared for their anti-influenza activities [20]. Their findings suggest that EGCG-palmitate (or other types of esters) are suitable candidates for anti-viral formulations. Due to the stability and skin permeability of LTPs in topical formulations, it would only require less than 1% w/v to deliver similar effects in comparison to 10-15% w/v of Polyphenon E. We reported previously that, as measured by the MTT cell viability assay, EGCGpalmitate possesses anti-cancer activity similar to GTPs in the human ovarian cancer cell line HO-8910 [24]. Here we show that LTPStearate at 0.1% w/v induced massive cell death in an oral cancer cell line OSC2 (Figure 1). When the concentration of LTPStearate was decreased to 0.01% w/v, cell death was still clearly evident (Figure 2). In contrast, no cell death was observed in NHEK under identical conditions (Figure 3). This result is consistent with our previous reports that GTPs or EGCG selectively induce apoptosis in cancer cells but not in normal cells [41-47]. The significance of this finding is that the absorption of LTPs, a lipophilic complex, is different than that of GTPs, and it could therefore target internal cancers, such as liver cancer, with higher bioavailability by oral administration. Although the

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delivery route of LTPs into the human body is still not clear, it is postulated to be via the chylomicron pathway. If so, LTPs would be associated with lipoprotein particles only, which significantly reduces potential binding with serum proteins, but increases the level in lipoproteins such as LDL prior to internalization by hepatocytes. That is, in comparison to GTPs the anti-cancer and cardiovascular benefits of LTPs could be better. LTPStearate, even at just 0.01% w/v, significantly induced the expression of caspase 14, a gene product specifically associated with cell differentiation and barrier formation and that causes cell death in cancer cells [48]. The effect of LTPStearate on caspase 14 expression is comparable to that of EGCG (Figure 4), and the induction of caspase 14 by LTPStearate is dosedependent (Figure 5). These results indicate that the effects of LTPStearate on the expression of caspase 14 in epidermal keratinocytes are consistent with the results generated from EGCG [49-51]. However, due to its water-soluble nature EGCG is not bioavailable to human skin by oral consumption [14, 15] or topical application at 50 µM (0.0023% w/v). The concentrations to be delivered in formulations for effectiveness are documented at 10% w/v for EGCG [11] and 10-15% w/v for GTPs [32], which could be very costly (EGCG costs $4000 to $9000 per Kg) and very difficult to formulate. In contrast, bioavailability can be better achieved by LTPs through topical application due to their lipid-soluble nature (<1% w/v). This notion was tested in vivo using a glycerin-based formula (formulation not disclosed due to pending patents) on a mouse model for psoriasiform lesions previously described [28]. The results demonstrated that LTPStearate effectively induced caspase 14 and improved the skin condition in this model (Figure 6), consistent with our previous observation using 0.5% w/v GTPs (freshly prepared immediately before use) [28, 52]. Importantly, based on coloration tests, LTPStearate is stable in various formulas for a minimum of two years (confidential data not shown). Therefore, formulations with LTP for topical use provide a suitable delivery vehicle due to the stability and permeability of LTP. The LTP-containing preparations could be used for inflammatory diseases, wound-healing, scar-treatment [53], photo-protection, and UVinduced damage. In addition, cosmetic products could take advantage of the properties of EGCG-palmitate, which is not only stable but also without significant coloration or precipitation in such formulations. A significant finding was that when LTPs (LTPOleate and LTPStearate) entered NHEK cells, EGCG was released into the cytoplasm. The retention time of EGCG and two types of LTP was compared and showed little difference by HPLC (Table 1). The rate of EGCG release is apparently dependent on the type of acyl group. Figure 7 shows that LTPOleate released most of the EGCG at the 1 h time point, while LTPStearate released the largest amount of EGCG at 6 h. This result suggests that, when the carbon number is identical, an unsaturated fatty acyl group may promote a faster transit through the cell membrane and/or more rapid hydrolysis by lipase/esterase, while a saturated fatty acyl group may require a longer transit time prior to enzymatic intracellular release of EGCG. This observation could help in future formulations aimed at different release times. In conclusion, in addition to the food and oil industries, the physical characteristics and biological effects of LTPs enable both health care and cosmetic industries to produce stable formulations with the full benefits of green tea polyphenols. Potential clinical uses of LTPs (especially EGCG-esters such as EGCG-palmitate) in stable formulations could be treatment of skin disorders associated with: neoplasm, psoriasis, cutaneous lupus, rosacea, actinic keratosis, seborrhreic dermatitis; skin damage caused by trauma, infection, insect bites, and scar formation; and oral lesions due to various causes. In the cosmetic field, the anti-oxidant

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and repair effects of stable formulations of LTPs could be used to protect the skin from environmentally-caused damage and aging, such as results from exposure to ultraviolet (UV) and other types of radiation, pollution, or toxic agents. They could also be used to promote the homeostasis of the skin to improve the appearance. LTP-containing products could also be used for anti-microbial purposes such as anti-viral agents to prevent and inhibit the infection/replication of influenza viruses, herpes simplex viruses, hepatitis viruses, human immunodeficiency virus. Clearly, investigation by scientists and clinicians is warranted to further understand the working mechanisms of LTPs and their metabolism in humans, and to explore their potential for oral administration to combat many diseases such as cancer, diabetes, cardiovascular diseases, and obesity.

ACKNOWLEDGEMENT The authors thank Professor Xian Qiang Yang (Tea Research Institute, Zhejiang University, China), Professor Shuxiong Wang (retired, Zhejiang University, China), and Dr. Yukihiko Hara (Japan) for their friendship, valuable advice, and pioneer work on tea polyphenol chemistry; Dr. Hasan Mukhtar for his pioneer work on tea polyphenol biological effects; Professor. Richard Eckert for his interesting work on the epidermal signaling by green tea; Dr. Santosh Katiyar and Dr. Nihal Ahmad for their work on the cutaneous photoprotection of green tea; Dr. Douglas Walsh (Walter Reed Army Medical Center, USA), and Joseph Wood (US Army Eisenhower Medical Center, Fort Gordon, USA) for collaborating on the flaky skin mouse study; Professor. Chung S. Yang and Dr. Mao-Jung Lee (Rutgers University, USA) for their pioneer physiology and pharmacology work, and technical advice on HPLC analysis; Professor Lihong Xu (Zhejiang University, China) for her contribution to green tea’s protective effects against environmental toxins; Professor Tokio Osaki (Former Vice President, Kochi University, Japan) and Professor Tetsuya Yamamoto (Kochi University, Japan) for their friendship and collaboration on the effects of green tea against oral cancer; Dr. Kunihiro Kaihatsu (Osaka University, Japan) for his informative advice; and Dr. Jian Hua Zhong (Zhejiang University, China), Ms. Daniel Britt, Ms. Haiyan Qin for their technical assistance. The authors thank people at CCA Industries, Inc., USA for their vision and pioneer formulation of effective green tea topical products; The authors also thank the Institute of Catalysts, the Tea Research Institute, and School of Medicine of Zhejiang University, China; the United States Army; the US Department of Veteran Affairs; and School of Dentistry, the Institute of Molecular Medicine & Genetics of Medical College of Georgia, USA for their support.

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[21] Chen P, Wang H, Du Q and Ito Y. Purification of long-chain fatty acid ester of epigallocatechin – 3 - O – gallate by high-speed counter chromatography. Journal of Chromatography A. 2002. 982:163-165. [22] Chen P., Sun D. and Zheng X. Preparation, structure and antioxidant activity of EGCGpalmitate. Journal of Zhejiang University (SCIENCES EDITION). 2003. 30:422-425. [23] Chen P. and Sun D. A novel catechin analog antioxidant for edible vegetable oils. Journal of the Chinese Cereals and Oils Association. 2003. 18:77-79. [24] Chen P., Zhong J., and Sun D. The main active component of lipophilic tea polyphenols and in vitro inhibition activity on ovarian cancer cells HO-8910. Journal of Tea Science. 2003. 23:115-118. [25] Sun D. and Chen P. Preparation, structure and antioxidant activity of EGCG myristate. Journal of Wenzhou Medical College. 2006. 36:225-227. [26] Osaki T, Tatemoto Y, Yoneda K, and Yamamoto T: Tumorigenicity of cell lines established from oral squamous cell carcinoma and its metastatic lymph nodes. Eur J Cancer B Oral Oncol. 1994. 30B:296–301, [27] Yamamoto T, Lewis J, Wataha J, Dickinson D, Singh B, Bollag WB, Ueta E, Osaki T, Athar M, Schuster G, Hsu S: Roles of catalase and hydrogen peroxide in green tea polyphenol-induced chemopreventive effects. J Pharmacol Exp Ther. 2004. 308:317323. [28] Hsu S, Dickinson D, Borke J, Qin H, Winger J, Henna Pearl, Walsh D, Bollag WB, Wood J, and Schuster G. Green tea polyphenols reduced the psoriasiform lesions and regulate caspase 14 by the mitogen-activated protein kinase pathways. Experimental Dermatology. 2007. 16:678. [29] Yang CS; Lee, MJ.; Chen, L. Cancer, Epidemiology, Biomarkers, and Prevention. 1999. 8:83. [30] Hsu S., Singh B., Lewis JE., Borke JL., Dickinson DP, Caughman GB., Drake L., Aiken AC., Huynh CT., and Schuster GS. Chemoprevention of Oral Cancer by Green Tea. General Dentistry. 2002. 50:140-146. [31] Hsu S, Yu F, Huang Q, Lewis J, Singh B, Dickinson D, Borke J, Sharawy M, Wataha J, Yamamoto T, Osaki T and Schuster G. A Mechanism-Based In Vitro Anticancer Drug Screening Approach for Phenolic Phytochemicals. ASSAY & Drug Development Technologies. 2003. 1:611-8. [32] Tatti S, Swinehart JM, Thielert C, Tawfik H, Mescheder A, Beutner KR. Sinecatechins, a defined green tea extract, in the treatment of external anogenital warts: a randomized controlled trial. Obstet Gynecol. 2008. 111:1371-9. [33] Song, J. M., Lee, K. H., and Seong, B. L. Antiviral effect of catechins in green tea on influenza virus. Antiviral Research. 2005. 68(:66-74. [34] Nobuko Imanishi, Yumiko Tuji, Yuko Katada, Miyuki Maruhashi, Satoko Konosu, Naoki Mantani, Katutoshi Terasawa and Hiroshi Ochiai. Additional Inhibitory Effect of Tea Extract on the Growth of Influenza A and B Viruses in MDCK Cells. Microbiology and Immunology. 2002. 46:491-494. [35] Xu J, Wang J, Deng F, Hu Z, Wang H. Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro. Antiviral Res. 2008. 78:242-9. [36] Isaacs CE, Wen GY, Xu W, Jia JH, Rohan L, Corbo C, Di Maggio V, Jenkins EC Jr, Hillier S. Epigallocatechin gallate inactivates clinical isolates of herpes simplex virus. Antimicrob Agents Chemother. 2008. 52:962-70.

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[37] Chang LK, Wei TT, Chiu YF, Tung CP, Chuang JY, Hung SK, Li C, Liu ST. Inhibition of Epstein-Barr virus lytic cycle by (-)-epigallocatechin gallate. Biochem Biophys Res Commun. 2003. 301:1062-8. [38] Weber JM, Ruzindana-Umunyana A, Imbeault L, Sircar S. Inhibition of adenovirus infection and adenain by green tea catechins. Antiviral Res. 2003. 58:167-73. [39] Fassina G, Buffa A, Benelli R, Varnier OE, Noonan DM, Albini A. Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent. AIDS. 2002. 16:939-41. [40] Yamaguchi K, Honda M, Ikigai H, Hara Y, Shimamura T. Inhibitory effects of (-)epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1). Antiviral Res. 2002. 53:19-34. [41] Hsu, S. Green Tea & Oral Cancer. The Journal of Georgia Dental Association. 2001. 31: 32-33. [42] Hsu S., Lewis JE., Borke JL., Singh B., Dickinson DP, Caughman GB., Athar M., Drake L., Aiken AC., Huynh CT., Das B., Osaki T., and Schuster GS. Chemopreventive Effects of Green Tea Polyphenols Correlate with Reversible Induction of p57 Expression. Anticancer Research. 2001. 21:3743-3748. [43] Hsu S., Singh B., Lewis JE., Borke JL., Dickinson DP, Caughman GB., Drake L., Aiken AC., Huynh CT., and Schuster GS. Chemoprevention of Oral Cancer by Green Tea. General Dentistry. 2002. 50:140-146. [44] Hsu S., Yu FS., Lewis J., Singh B., Borke J., Osaki T., Athar M and Schuster G. Induction of p57 Is Required for Cell Survival When Exposed to Green Tea Polyphenols. Anticancer Research. 2002. 22: 4115-4120. [45] Hsu S., Lewis J., Singh B., Schoenlein P., Osaki T., Athar M., Porter AG and Schuster G. Green Tea Polyphenol Targets the Mitochondria in Tumor Cells Inducing Caspase 3-Dependent Apoptosis. Anticancer Research. 2002. 23:1533-1540 [46] Yamamoto T, Hsu S, Lewis J, Wataha J, Ueta E, Osaki T, Luckwood P, Singh B, Dickinson D, and Schuster G. Green Tea Polyphenol Causes Differential Oxidative Environments in Tumor versus Normal Cells. J Pharmacol Exp Ther. 2003. 307:230-6. [47] Hsu S., Singh B., Schuster GS. Inducing Apoptosis in Oral Cancer Cells. Oral Oncology. 2004. 40:461-473. [48] Hsu S, Qin H, Dickinson D, Xie D, Bollag WB, Stöppler H, Pearl H, Vu A, Watkins M, Koehler M and Schuster G. Expression of Caspase 14 Reduces Tumorigenicity of Skin Cancer Cells. In Vivo. 2007. 21:279-83. [49] Hsu S, Bollag W., Lewis J., Huang Q., Singh B., Sharawy M. and Schuster GS. (2003) Tea Polyphenols Induce Differentiation and Proliferation in Epidermal Keratinocytes. Journal of Pharmacology and Experimental Therapeutics. 2003. 306:29-34. [50] Hsu S. Green Tea and the Skin. Journal of the American Academy of Dermatology. 2005. 52:1049-59. [51] 51. Hsu S, Yamamoto T, Borke J, Walsh DS, Singh B, Rao S, Takaaki K, Lapp C, Lapp D, Foster E, Bollag WB, Lewis J, Wataha J, Osaki T and Schuster G. Green tea polyphenol-induced epithelial cell terminal differentiation is associated with coordinated expression of p57/KIP2 and caspase 14. J Pharmacol Exp Ther. 2005. 312:884-90. [52] Walsh D, Borke J, Singh B, Do N, Balagon MV, Abalos RM and Hsu S. Psoriasis is characterized by altered epidermal expression of caspase 14, a novel regulator of

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keratinocyte terminal differentiation and barrier formation. Journal of Dermatological Science. 2005. 37:61-3. [53] Zhang Q, Kelly AP, Wang L, French SW, Tang X, Duong HS, Messadi DV, Le AD. Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/AkT signaling pathways. J Invest Dermatol. 2006. 126:2607-13.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 4

ASSESSMENT OF THE ANTIOXIDANT CAPACITY OF GREEN TEAS: A CRITICAL REVIEW Camilo López-Alarcón1,* and Eduardo Lissi2 1

Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago, Chile 2 Facultad de Química y Biología, Universidad de Santiago de Chile

ABSTRACT In the last decades, the beneficial influence of green tea on human health has been related to the antioxidant capacity (AC) of its phenolic constituents. The latter has originated systematic studies of the AC of green tea and/or its pure antioxidants. Different methodologies have been used with this purpose. The methods are based on: i)

Estimation of the consumption by additives of stable free radicals (DPPH, and ABTS radical cation). ii) Evaluation of the protection given by antioxidants to a target being oxidized by free radicals (ORAC, TOSC, LDL oxidation assay). iii) Estimation of the steady state of free radicals before and after addition of additives (TAR). iv) Estimation of the reducing power capacity of the additives (FRAP, CUPRAC).

The assays differ in the experimental conditions and their chemistry. Therefore, different conclusions could be obtained depending on the methodology used. For example, green tea presents a lower AC than peumus boldus by ORAC (oxygen radical absorbance capacity) method when fluorescein is used as target molecule. However, if pyrogallol red is used as probe, green tea appears with an ORAC index six times higher than peumus boldus. In the present review, we discuss the advantages, and disadvantages of the different methodologies employed to evaluate the AC of green tea. *

Correspondence to C. López-Alarcón; e-mail: [email protected]

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1. INTRODUCTION Green tea, a product made -by a non fermentative process- from leaves of Camellia sinensis, has been considered in traditional Chinese medicine, as a healthful beverage. In the last decades, different beneficial effects on the human health have been attributed to green tea, including prevention of cancer, heart disease, diabetes, and neurodegenerative diseases [1-8]. These associations can be explained, at least partially, in terms of the presence in green tea of free radical scavengers, such as polyphenols [9-13]. Polyphenols, particularly flavonoids, constitute the most interesting group of green tea leaf components. The main flavonoids present in green tea include catechins (flavan-3-ols) and their gallate esters. In addition, green tea contains gallic acid and other phenolic acids, such as chlorogenic acid and caffeic acid, and flavonols such as kaempferol, myricetin, and quercetin [12]. The high polyphenolic concentration present in green tea gives to this beverage their well known antioxidant properties. This antioxidant capacity has been established in many studies using several in vitro methodologies. In the present work, we present and discuss data of the antioxidant ability of green tea obtained by different assays. We present the bases of each methodology, as well as their advantages and shortcomings. In addition, the influence of the experimental procedures employed in green tea extraction is discussed.

2. ANTIOXIDANT CAPACITY ASSAYS AND GREEN TEA The antioxidant capacity of green tea has been established by several in vitro methodologies. The methods involve both different concepts and experimental conditions. The most employed methodologies are based on the evaluation of: the scavenging of stable free radicals by additives, the protection given by antioxidants to a target being oxidized by free radicals, and the estimation of the reducing power capacity of the additives.

Scavenging of Stable Free Radicals by Additives These methods are based on the bleaching induced by additives of stable free radicals such as 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTSy+) and 2,2diphenyl-1-picrylhydrazyl radical (DPPH).

Bleaching of ABTSy+ by Additives This assay is based on the generation of a long-lived ABTSy+ cromophore from the reaction of ABTS with an oxidant. ABTSy+ is a water soluble free radical, characterized by visible absorption maxima at 414, 645, 734, and 815 nm. The addition of an antioxidant or a complex mixture to solutions containing ABTSy+, generates a decay of the original visible bands of ABTSy+, allowing an evaluation of the antioxidant capacity of the sample (commonly is used the decay at 734 nm). The consumption of ABTSy+ given by an additive is compared to that of Trolox (a hydrosoluble analogue of vitamin E), being the assay named TEAC (Trolox equivalent antioxidant capacity). TEAC values are estimated using equations (1) and (2) for pure compounds and beverages, respectively.

Assessment of the Antioxidant Capacity of Green Teas

TEAC =

TEAC =

[ΔUAadditive ] [ΔUATrolox ]

[ΔUA

beverage

]

[ΔUATrolox ]

65

[Trolox ] [ Additive ]

(1)

x f x [Trolox ]

(2)

x

where: ΔUAadditive =

Difference between initial absorbance (UA0 minutes) and after 6 minutes of additive addition (UA6 minutes). ΔUATrolox = Difference between initial absorbance (UA0 minutes) and after 6 minutes of Trolox addition (UA6 minutes). [Trolox] = Trolox micromolar concentration. [Additive] = Additive micromolar concentration. ΔUAbeverage = Difference between initial absorbance (UA0 minutes) and after 6 minutes of beverage addition (UA6 minutes). f= dilution factor

TEAC assay, reported first by Miller et al.[14], was based on the formation of ABTSy+ by the reaction of ABTS with metmyoglobin/ hydrogen peroxide. Some shortcomings have been reported for this methodology [15], being at present ABTS oxidized through manganese dioxide, peroxodisulfate or azocompounds (AAPH) [16,17]. The consumption of ABTSy+ by green tea has been studied by several research groups [18-22]. Pellegrini and coworkers [18] reported that the TEAC value of green tea is 1.7 times higher than black tea. The TEAC values of green and black tea were 6.0 and 3.6 mmoles of Trolox equivalent per liter of solution, respectively. The higher antioxidant activity of green tea was explained in terms of a partial oxidation along the manufacturing of black tea. In this process, oxidative polymerization by polyphenols oxidase, convert catechins or other phenols of green tea in oxidized products [18]. This relation is according to the well known antioxidant capacity of catechins, epicatechins and their gallate esters when they are added as pure compounds to a ABTSy+ solution [19]. The TEAC values reported by Pellegrini et al.(6.0 mM Trolox equivalents/L)[18] are close to the TEAC values reported by Rusak et al (4.59 mM Trolox equivalents/L) [20]. However, the report published by Rusak et al.[20] showed that experimental procedures of green tea extraction are very important, and opposite conclusions can obtained depending of the solvent used, and the time of extraction. For example, using water as solvent extraction, green tea appears with a lower antioxidant capacity (TEAC) than white tea after 5 minutes of extraction. Nevertheless, after 15 or 30 minutes of extraction, the TEAC value of green tea is nearly two times higher than that of white tea. In addition, different TEAC values have been obtained employing others extraction procedures such as soxhlet extractions using methanol or acetone as solvents [21].

Scavenging of DPPH by Additives DPPH is a lipophilic free radical that has been widely used to test the capacity of compounds, plant extracts and foods as free-radical scavengers [23]. DPPH exhibit a maxima absorption band at 517 nm in alcoholic solutions. This band has been employed to follow the

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bleaching of DPPH absorption induced by additives. The antioxidant capacity of the sample generally is expressed as the inhibitory concentration (IC) necessary to reduce the absorbance of DPPH by 50% (IC50), or as the percentage of the DPPH consumed at an arbitrary time. Many studies have shown the consumption of DPPH radical induced by green tea [21,24-27]. Green tea has shown a higher ability to reduce DPPH radical than black tea [24,26,28]. Furthermore, using DPPH assay, green tea has been ranked among the most potent of 52 edible plant products [25]. Bastos et al.[27] reported that water and ethanolic extracts of green tea have a comparable DPPH scavenging activity that the extracts of both green and roasted yerba mate (llex paraguariensis). Nevertheless, the antioxidant activity of ether extracts of green tea showed a higher ability than yerba mate (nearly three times). These results were explained by the authors in terms of the compounds identified by electrospray insertion mass spectrometry (EI-MS) in the ether extract of green tea, such as caffeolylglucose, feruloylquinic acid, methylepicatechin gallate, and 3methyl epigallocatechingallate [27]. The interaction of green tea polyphenols with DPPH included in the bilayer of liposomes has been studied by Chen et al.[29]. The consumption of DPPH was evaluated directly by electron spin resonance (ESR) spectroscopy. The results evidenced that green tea polyphenols react with DPPH in the bilayer of both saturated and unsaturated phospholipids. Therefore, green tea polyphenols not only have an antioxidant activity in aqueous media, but also could react with free radicals involved in lipidperoxidation process.

Evaluation of the Protection Given by Antioxidants to a Target being Oxidized by Free Radicals Among the methodologies most used in this category are ORAC (oxygen radical absorbance capacity) and TRAP (total radical antioxidant potential) assays. Also, the ability of green tea to delay the oxidation of low density lipoproteins (LDL) has been frequently employed. ORAC Methodology ORAC (oxygen radical absorbance capacity) index has been frequently employed in the evaluation of the antioxidant capacity of beverages [30-32]. This methodology measures the protection afforded by an antioxidant to a target molecule that is being oxidized by peroxyl radicals. Trolox and gallic acid have been used as antioxidant standards, and 2,2’-Azo-bis(2amidinopropane) dihydrochloride (AAPH) [33] has commonly been employed as peroxyl radical source. Assuming a simple competitive oxidation of a target molecule (probe) and a given antioxidant (XH) by a peroxyl radical (ROOy), a kinetic model must consider reactions (3) to (6):

AAPH + O2 ⎯⎯→ 2ROO• + N2

(3)

ROO• + probe ⎯⎯→ bleaching

(4)

ROO• + XH ⎯⎯→ X• + ROOH

(5)

Assessment of the Antioxidant Capacity of Green Teas

2ROO• ⎯⎯→ non radicals products

67 (6)

and all the self-reactions and cross-reactions of the radicals produced in steps (4) and (5). In this scheme, and for simplicity, we are not considering the formation of alcoxyl radicals in reaction (6). The protection afforded by antioxidants or beverages is estimated from the changes of area under curve of the kinetics profiles after total or 80% target molecule consumption. ORAC values are estimated according to equation (7) and (8) for pure compounds (additives) and beverages, respectively.

ORAC =

( AUCad − AUC0 ) ( AUCs tan dard − AUC0 )

[s tan dard] [additive]

( AUC − AUC0 ) ORAC = f [s tan dard] (AUCs tandard − AUC0 )

(7)

(8)

where: AUC = Area under curve in presence of the tested beverage. Area under curve in presence of the additives. AUCad = AUCº = Area under curve for the control (target molecule plus AAPH solution). AUCstandard =Area under curve for Trolox or gallic acid. f= Dilution factor, equal to the ratio between the total volume of the working solution (target molecule plus AAPH plus the sample aliquot) and the added sample volume. [standard] = Trolox or gallic acid concentration. [additive] = Additive concentration. ORAC assay was proposed originally by Cao, Alessio and Cutler (1993) using βphycoerythrin (ORAC-PE) as target molecule [34]. Throughout ORAC-PE assay, the antioxidant capacity of many vegetables, fruits, and beverages was studied. Cao et al.[30] found that both, green and black tea, have higher antioxidant activity than vegetables such as garlic, kale, spinach and Brussels sprouts. Moreover, the ORAC-PE value of green tea correlates with catechin derivatives content [35,36], and has been reported to be lower than that of yerba mate (llex paraguariensis) [37]. Ou et al.[38], reported that the use of β-phycoerythrin as target molecule confer to ORAC-PE method some limitations. β-phycoerythrin shows inconsistency from lot to lot, is not photostable, and interacts with polyphenols [38]. Thus, Ou and coworkers [38], proposed an ORAC methodology employing fluorescein (FL) as target molecule (ORAC-FL). At present, FL is the target molecule most employed, and have allowed to estimate the ORACFL index of a large number of phenols, polyphenols, and beverages. However, ORAC-FL for single antioxidants and/or complex mixtures including very reactive compounds is estimated

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generally from kinetics profiles showing a neat induction time (lag time). Furthermore, these lag times have been related to a repair of FL from the secondary radical [39], according to:

FL• + POH →

FLH + PO •

(9) 1.0

F / F0

1.0 0.5

A / A0

0.0 0

400

800

1200

Time / s

0.5

0.0 0

600

1200

1800

2400

3000

Time / s Figure 1. Consumption of pyrogallol red and fluorescein (insert) induced by peroxyl radicals in presence of green tea infusion. Pyrogallol red (5µM) was incubated with AAPH (10 mM) at 37ºC in phosphate buffer 75 mM (pH 7.4) in prescence of green tea infusion: 1.5 µL/mL (…); 2.5 µL/ mL (‘); 5 µL/mL (U). control experiment (in absence of green tea, {). Insert: Kinetic profiles of fluorescein (70 nM) consumption AAPH (10 mM) induced in absence (z), and presence of green tea infusion (0.1 µL/mL, T).

Independently of the origin of the induction times, their generation by additives leads to ORAC-FL indexes more related with stoichiometric factors than the reactivity of antioxidants towards AAPH derived peroxyl radicals [40]. Recently, we have proposed a modified ORAClike methodology that employs pyrogallol red (PGR) as target molecule (ORAC-PGR) [40]. PGR does not produce induction times, even with very reactive polyphenols, giving ORACPGR values more related to the reactivity of the antioxidants towards peroxyl radicals [4042]. The protective effect of green tea extract on FL and PGR bleaching induced by peroxyl radicals is shown in Figure 1. As can be seen in this figure, green tea inhibited the

Assessment of the Antioxidant Capacity of Green Teas

69

consumption of FL and PGR with different protective kinetic profiles. Green tea inhibited the consumption of FL throughout a clear induction time, while it protected PGR without an observable lag time. Therefore, the origin of ORAC values obtained from area under curves employing FL or PGR is different. Also, even relative ORAC values measured for different infusions depend upon the employed methodology [43]. For example, ORAC-FL value of Aloysia citriodora is larger than that of green tea, while if PGR is employed as target molecule, green tea appears as nearly nine times more efficient. Therefore, extreme care must be given to conclusions obtained from ORAC values estimated employing a single target molecule. ORAC assay has been applied to determine the antioxidant capacity of a variety of foods [44,45]. In addition, results obtained in humans indicate that the daily consumption of fruits and vegetables correlated with ORAC values of plasma [46]. These studies allowed to develop a rank for foods and beverages in terms of their ORAC values [45]. Also, it has been proposed that green tea could make a significant contribution to the required total daily antioxidant capacity intake [31]. In addition, in base of ORAC values, a minimum consumption per day of antioxidants has been recommended (5000 ORAC units). The consumption of tea significantly increases the antioxidant capacity of the blood, reducing the risk of oxidative damage to important macromolecules. However, many questions are present related to the assays used to evaluate the antioxidant capacity and the possible effect on the human health [11]. Furthermore, is necessary to develop oxidative damage biomarkers that represent correctly the impact of the consumption of green tea in the human health [47].

TRAP Methodology TRAP is one of the most employed procedures to evaluate the antioxidant status of a biological fluids or food samples. The method, proposed originally by Wayner et al. [48], is based on measurements of induction times in the oxidation of a lipid dispersion exposed to a free radical source with a constant and known rate of free radical production under aerobic conditions. Usually, AAPH is used as peroxyl radical source, and the decrease in oxygen concentration is used to measure the oxidation rate. In addition, a based luminol TRAP method has been proposed as a good and simple alternative to estimate the index [49]. The TRAP index of beverages is estimated according to equation (10):

TRAP =

t green tea tTrolox

f [Trolox]

(10)

Where: t = Induction time in presence of green tea (t green tea) or Trolox (t Trolox). f = Dilution factor. [Trolox] = Trolox concentration. The high antioxidant activity of green tea through a TRAP type assay has been reported in several works [50-53]. Bunkova et al. [50] reported a TRAP value of Chinese Gunpowder and Japanese Sencha green tea of 39 and 38,4 mM Trolox equivalents / L, respectively. In

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addition, using a TRAP index, an increase of the human plasma has been observed after intake of green and black tea infusions [51-53].

LDL Oxidation The peroxidation of low density lipoproteins (LDL) is considered to be a major initiating event in atherogenesis [54]. Therefore, much effort has been devoted to the development of standardized in vitro models for assessment of oxidation resistance of LDL. The LDL oxidation -isolated from human blood samples- can, at least in vitro, be inhibited or delayed by antioxidants, such as phenols or polyphenols [55]. Thus, in vitro oxidation of LDL has been developed also as a model to assess the antioxidant capacity with a more physiologically relevant system [56]. Commonly the oxidation of LDL is initiated by the addition of copper sulfate or AAPH [57,58]. The oxidation of LDL can be easily followed by UV spectroscopy following the increase in absorbance at 234 nm (ε = 29.500 M-1cm-1) due to the formation of conjugated dienes [57]. The kinetic profiles of LDL oxidation are characterized by a lag time during which protective endogenous anti-oxidants are consumed by initiating free radical species. After the consumption of all endogenous anti-oxidants, a lipid radical-propagated peroxidation chain reaction begins in which the polyunsaturated fatty acids contained in the LDL are rapidly oxidized to lipid hydroperoxides. The addition of antioxidants or their complex mixtures to a system containing LDL plus copper sulfate or AAPH, delays the lipoperoxidation process of LDL. Therefore, the estimation of the lag time induced by the antioxidants added is a measure of the protective effect of the additives. The lag time generated by the antioxidants or beverages has been employed to evaluate the antioxidant capacity. In addition, there are several parameters, additional to the lag time, which can be obtained from dienes vs time profiles. For example, it is possible to evaluate the time required to reach half maximum dienes (t ½), and the maximum velocity (v) of the lipid peroxidation, given by the peak of the first derivative [57]. Besides, the maximum diene concentration (diene max) can be estimated from the maximum increase of the absorbance at 234 nm [57]. Green and black teas efficiently delay the in vitro LDL oxidation process [25,59]. Nevertheless, the lag times observed in the oxidation of LDL in presence of both teas depend of the LDL sample, the experimental procedure, and the brand of tea. In some experiments green tea appears with higher antioxidant ability than black tea, but in others the antioxidant capacity of green tea is similar or lower than black tea. For example, Richelle et al. [59] reported that the induction time of LDL oxidation induced by copper or AAPH is in average 248 and 163 minutes in presence of green tea, and black tea (1µL/mL), respectively. Furthermore, a great variability of the results was observed depending of the commercial brand used in the experiment. Green tea values of different brands fluctuated between 186338 minutes, while black tea values laid between 67-277 minutes [59]. Copper sulfate is a initiator of LDL oxidation commonly used in LDL based assays [25,59]. Therefore, the protective effect of green tea or their components could be related to the capacity of the beverage to chelate copper ions. Miller et al. [19] reported that theaflavins are able to chelate copper ions and to inhibit copper-induced LDL oxidation. Nevertheless, the protective effect of green tea was observed at much lower concentrations than the copper sulfate concentration used (5 µM), implying that the inhibition of LDL oxidation by green tea is not only due to its chelating capacity [19]. These works show that LDL susceptibility to oxidative modification is readily inhibited, at least in vitro, by green tea extracts. However, ex vivo studies in healthy volunteers have shown a little or no inhibition of LDL oxidation [11].

Assessment of the Antioxidant Capacity of Green Teas

71

Hodgson et al. [60] reported a greater lag time of LDL oxidation for both black and green tea compared to water. However, these changes within a healthy cohort of 20 men were either borderline (p= 0.05 for black tea) or not significant (p = 0.17 for green tea). Miura et al. [61] detected an increase in lag time (p = 0.05) among 22 healthy young men after they consumed green tea extracts equivalent to seven to eight cups a day for seven days. Interestingly, also plasma β-carotene was higher (p < 0.01) in the tea group after the intervention. Recently, Tinahones et al. [62] reported that the consumption of green tea extract by women for five weeks produced modifications in vascular function and an important decrease in serum oxidizability. Nevertheless, the consumption of green and black tea extracts equivalents to six cups per day does not affect the susceptibility of LDL to oxidation ex vivo in smokers patients [63]. The discrepancy between the effect of tea in vitro and ex vivo on the susceptibility of LDL to oxidation may be due to the inability to achieve concentrations in vivo as great as those obtained with the former methods [11]. Anyway, some reports have been published showing an inverse correlation between catechin intake or green tea intake and coronary heart disease mortality [64,65]. Furthermore, Peters et al. [66] have provided a metanalysis that suggested a decrease in the risk of cardiovascular diseases with increasing green tea consumption.

Estimation of the Reducing Power Capacity of the Additives The methods based on the evaluation of the capacity of antioxidants or their complex mixtures to reduce metals are named FRAP (ferric reducing antioxidant powder) and CUPRAC (cupric reducing antioxidant powder).

FRAP Method FRAP assay was originally developed by Benzie et al.[67] to measure the reducing power of plasma. However, FRAP assay has been used widely to study the antioxidant capacity of pure compounds, foods and beverages [68,69]. The reaction measures the reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) complex to a colored product induced by polyphenols at pH 3.6. The antioxidant capacity is expressed as generated Fe2+ at a fixed time (usually 6 minutes). The FRAP value of green and black tea has been reported by several investigators [20,70,71]. Benzie et al. [70] reported that the FRAP index of green tea is higher than that of black tea. However, as was shown by Benzie et al. [70], high variations of the results between different brands were observed. Interestingly, based on FRAP results it has been proposed that a cup of green tea have a similar antioxidant capacity than 100-200 mg of ascorbic acid. Then, a consumption of several cups of green tea per day would offer the same antioxidant potential (FRAP) as almost 1 gram of vitamin C. CUPRAC Assay CUPRAC assay, proposed originally by Apak et al. [72], is based on the reduction of the complex copper(II)-neocuproine to copper(I)-neocuproine by the combined action of all antioxidants (reducing agents) present in the sample. The complex copper(I)-neocuproine has a characteristic visible band at 450 nm, being the absorption band measured to estimate the

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antioxidant capacity of the sample. Apak et al. [73] reported that the CUPRAC value of green tea was among the higher values of several herbal infusions. Furthermore, they reported that ascorbic acid increase the antioxidant capacity of green tea, being the CUPRAC value in presence of lemon 1.7 times higher than that of green tea alone. In this context, similar results of ascorbic acid contribution to TEAC values have been found by Majchrzak et al. [74].

Limitations The beneficial effects of green tea on the human health have been established in several studies [10,11]. Nevertheless, the published data of the in vitro scavenging activity of green tea involve both different methodologies and experimental procedures. In addition, different values have been reported depending on the commercial brand of the green tea used [70]. Therefore, a comprehensive understanding of the in vitro antioxidant capacity of green tea is difficult and requires an exhaustive analysis of the methodology used and the experimental condition employed in each work. These aspects are stressed in the data of Table 1. As can be seen in Table 1, the antioxidant activity of green tea depends on the methodology used and the procedure of the extraction. Furthermore, within a single assay, the values are dependent of the green tea brand, i.e. in Table 1 using ORAC-PE, LDL, and FRAP assays high fluctuations of the antioxidant capacity values have been published for different green tea brands. In addition, the results obtained using a particular method not always are expressed in the same units, making difficult the comparison of results. For example, employing DPPH test, the results have been reported in terms of IC50, DPPH consumed in percentage or in base of epigallo-catechin-gallate (EGCG) equivalents. In addition, in the analysis of the state of the art, it is necessary to taken into account that the methodologies provide different information. In particular, it must be considered that a given index can be an indication of the total amount of antioxidants (without a discrimination regarding their reactivity, TEAC, DPPH, TRAP, ORAC-FL, FRAP), or be an indication of the quantity and reactivity of the antioxidants present in green tea sample (ORAC-PGR). Moreover, it should be considered that each antioxidant capability assay has their own advantages and limitations. The simplicity to carry out procedures based on the bleaching of ABTSy+ and DPPH, is one of the most important advantage of these assays. However, the free radicals involved (ABTSy+ and DPPH) are very far from those free radicals relevant in oxidative stress situations. The analysis of the kinetics obtained by ABTSy+ assay is complex [75-77] and can be computed as antioxidant compounds that are pro-oxidant in biological systems, such as hydrogen peroxide or organic hydroperoxides [78]. Furthermore, when the consumption of ABTSy+ is measured at a single (long) reaction time, the result obtained is related to stoichiometric factors and gives no information regarding the reactivity of the tested compounds. The interpretation of data obtained by employing DPPH as the stable free radical is less straightforward. When applied to a fixed time at a pure compound, the method provides stoichiometric factors for highly reactive compounds and/or mixtures, and provides reactivity and stoichiometric factors for low reactivity compounds [79,80].

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73

Table 1. Antioxidant capacity values of green tea evaluated by different methodologies Method TEAC

DPPH

ORAC-PE

ORAC-FL ORAC-PGR TRAP LDL FRAP CUPRAC a

Value 6.0 4.59 6.85 8.0 1.8 ≈ 25 ≈ 70 4.14 194.6 59.4 88.4 92.2 80 814 1239-1686 1345.9 12.3 19.1 38.35 191.4 186-338 272-1144 0.94

Units mM Trolox eq /L mM Trolox eq /L mM Trolox eq /L mM Trolox eq /g mM Trolox eq /g DPPH consumed (%)a DPPH consumed (%)a µg/mL (IC50)b µmol EGCG/g (IC50) DPPH consumed (%)c DPPH consumed (%)d DPPH consumed (%)d DPPH consumed (%)e µmol Trolox eq /g µmol Trolox eq /g µmol Trolox eq /g mM Trolox eq /L mM Trolox eq /L mmol Trolox eq /L µmol EGCG/g Minutes (lag time)f µmol /g mmol Trolox/g

Experimental conditions Boiling water (5 min) Boiling water (5 min) Ethanol 70% (5min) Methanol (soxhlet) Boiling water (5 min) Acetone (70%) Methanol Boiling water (1 hour) Ethanol 70% (v/v) Boiling water (5 min) Water (soxhlet) Ethanol (soxhlet) Boiling water (1 h, reflux) Boiling water (30 min) Boiling water (3 min) Boiling water (10 min) Boiling water (5 min) Boiling water (5 min) Boiling water (10 min) Ethanol 70% (v/v) Boiling water (5 min) Boiling water Boiling water (5 min)

Reference [18] [20] [20] [21] [22] [21] [21] [24] [25] [26] [27] [27] [28] [30] [35] [37] [43] [43] [51] [25] [60] [71] [74]

Estimated using a DPPH concentration of 0.1mM, and a concentration of dry green tea extract of 25 µg. b DPPH concentration = 60 µM. c DPPH concentration = 0.2 mM, and a concentration of dry green tea extract = 0.4 mg/mL. d DPPH concentration = 20 mg/mL, and a concentration of green tea extract = 250 µL/mL. c DPPH concentration = 0.25 mM, and a concentration of dry green tea extract = 50 µg/mL.f Lag times obtained with a LDL concentration of 80 µg of cholesterol/mL, and a green tea infusion concentration of 1 µL/mL.

On the other hand, procedures based on the evaluation of the protection given by antioxidants to a target molecule being oxidized by free radicals provide information related to stoichiometric factors and/or reactivity. The TRAP assay is based on the estimation of lag times. Therefore, the index derived from this procedure is only determined by stoichiometric factors. In fact, it only provides an indication of the number of radicals trapped per each antioxidant molecule introduced into the system [81]. The ORAC methodology is a procedure based on the estimation of the area under curve of the protective kinetic profiles. Therefore, the index is related with the origin of the area under curve. Thus, if the target molecule is protected totally by the antioxidants (ORAC-FL), the area under the curve and, in consequence, the ORAC-index is mostly governed by the stoichiometry of the reaction. However, if the area under curve is related only with a decrease in the initial rate of the target molecule consumption (ORAC-PGR), the ORAC index would reflex the reactivity of the additives. Similarly, protection of LDL is a complex system, and is necessary to consider the initiator of the LDL oxidation for the analysis of the results. If copper is used, it must be

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considered the possible chelating effect of the additives. If AAPH is employed as initiator, the delay of the LDL peroxidation chain could be determined by the interaction of the antioxidants with the primary radicals (peroxyl radicals) or with a chain-breaking antioxidant capacity. On the other hand, a disadvantage of LDL assay is that the LDL particles are very different depending of the subject, or even of the diet. Then, the reproducibility, and the comparison of results is not always warranted. Ferric and cupric reducing power assays (FRAP and CUPRAC) have the limitation that they are only titrating all the antioxidants and those molecules with reducing power towards ferric or cupric complexes. In addition, a shortcoming of FRAP methodology is that the pH used is very different to the physiological value. Regarding the validity of these indexes as indicators of the quality of a beverage and/or as useful indicators of pathological situations, there are two questions: • •

Is possible to estimate the antioxidant capacity of green tea employing only one assay? and Is there any relationship between the measured index in a food or beverage and the impact it has on the antioxidant capacity status of the organism?

As expected, there is not a clear-cut yes-or-not answer to these broad questions and it strongly depends on the system considered. For example, using a DPPH-based method [24] it was found that green tea was 6.5 times more powerful as an antioxidant capacity than black tea. However, in preventing the lipid peroxidation of renal homogenates induced by hydrogen peroxide and Fe(II), green tea was only 1.5 time more efficient. Evidently, other factors, such as the distribution and metal chelating characteristics, preclude a quantitative relationship between the amount of antioxidants present in a food or beverage and their biological effect. In in vivo evaluations, the relationship between ingest and observed levels is still more indirect, since factors such as absorption at the gut level and metabolization can be completely different for the different antioxidants present in the samples. Considering the aspects above discussed should be recommendable that for the evaluation of the in vitro antioxidant capacity of foods or beverages should be used similar experimental conditions and as many assays as possible. Recently, Seeram et al. [82] have studied the antioxidant capacity of beverages commonly consumed in United States. Interestingly, the study was developed considering five in vitro antioxidant capacity measuring methodologies (DPPH, ORAC-FL, FRAP, TEAC, and LDL) and different green tea brands. This allows obtaining a more complete profile of the antioxidant capacity, and to correlate the results of different samples. Depending of the assay and the brand, iced green tea showed higher or similar antioxidant ability than iced black and white teas.

3. CONCLUSION Green tea antioxidant potential has been measured by a large number of methodologies. All studies point to a high charge of antioxidants, mostly phenolic compounds. Comparison with other infusions, particularly black tea, is difficult since the reported values are strongly influenced by the employed methodology. The data discussed in the present review

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emphasized two points: i) ranking of infusions or beverages can be proposed only when the same methodology is employed, both the same extraction procedure and the assay employed to evaluate the antioxidant capacity, and ii) to assess the antioxidant capacity of a infusion, several methodologies should be considered, taking into account the factors that condition the measured index (either the amount of antioxidants and/or their reactivity in the free radical scavenging processes).

ACKNOWLEDGMENTS This work was supported by FONDECYT n°11060323 and 1070285.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 5

DESIGN AND ASSESSMENT OF THE IN VITRO ANTIOXIDANT CAPACITY OF A BEVERAGE COMPOSED OF GREEN TEA (CAMELLIA SINENSIS L.) AND LEMONGRASS (CYMBOPOGON CITRATUS STAP) D. Fernando Ramos Escudero1, Luis Alberto Condezo Hoyos2, Mónica Ramos Escudero3 and Jaime A. Yáñez4,* 1

Centro de Investigación de Bioquímica y Nutrición, Facultad de Medicina Humana, Universidad de San Martín de Porres, Lima, Perú 2 Postdoctoral Research, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain 3 Escuela de Postgrado en Agricultura Sostenible, Universidad Nacional Agraria de la Selva, Tingo María, Perú 4 College of Pharmacy, Department of Pharmaceutical Sciences, and Pharmacology and Toxicology Graduate Program, Washington State University, Pullman, WA, USA

ABSTRACT Tea is one of the most popular and widely consumed beverages in the world and it is derived from the infusion of tea leaves (Camellia sinensis L.). Different commercial types of tea are available, including black tea, oolong tea (semi-fermented) and green tea, which differ on their processing and chemical composition. All these types of tea have been reported to prevent multiple diseases such as cancer, heart conditions, among others. On the other hand, lemongrass (Cymbopogon citratus Stap) is a rich source of essential oils, widely employed in infusions, soaps, and perfumes, and it has been reported to possess gastrointestinal and analgesic properties. In the present study, green tea (Camellia sinensis L.) and lemongrass (Cymbopogon citratus Stap) leaves were collected *

Correspondence to: Dr. Jaime A. Yáñez, Ph.D. Schering-Plough Research Institute, Pharmaceutical Sciences and Drug Metabolism, 2015 Galloping Road, Mail Stop K15-3 3700, Kenilworth, NJ 07033, USA. Email: [email protected]

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D. F. Ramos Escudero, L. A. Condezo Hoyos, M. Ramos Escudero et al. from Río Azul and Porvenir de Marona, Perú. The anti-oxidant capacity of green tea and lemongrass extracts was evaluated using the DPPH method and it was observed that the IC50 values for green tea was 32.4 ± 0.39 μg/mL and 1350 ± 47 μg/mL for lemongrass. These two plants (green tea and lemongrass) were employed to design multiple infused beverages and it was determined that an infused beverage containing 10 mg/mL total extract (50% green tea and 50% lemongrass) reported a total catechin content of 24.4 ± 0.65 mg/100mL, a DPPH inhibition percentage of 88.6%, and exhibited the greatest acceptance for sensory attributes such as flavor, color, and aroma (values of 6.8, 9.0, and 8.0 respectively) based on Friedman Multiple Comparisons test. The taste panel results also indicated that the optimized acidity and sweetness were to be set at pH 3.1 and 11°Brix, while the optimum infusion time based on the total catechin content was 7 minutes. The pasteurization profile at 90°C for 5 minutes achieved mesophilic microorganisms counts of <10 cfu/mL. The maximum shelf-life of the beverage was achieved at 15ºC based on total catechin content and absence of browning (catechin degradation). Furthermore, the formulated beverage was well accepted by the test panel and presented similar anti-oxidant capacity than commercial green tea based beverages.

Keywords: Green tea, lemongrass, beverage, catechin, anti-oxidant capacity, 1,1-diphenyl-2picrylhydrazyl (DPPH).

INTRODUCTION Tea is one of the most popular drinks in the world and it is recognized for its high polyphenol content. It is derived from the infusion of tea leaves with or without a fermentation process [1]. The fresh tea leaves are rich in catechins (a polyphenol sub-class), which can total up to 30% of the leaves dry weight [2]. Other polyphenols include flavanols and their glycosides, chlorogenic acid, coumarylquinic acid, theogallin (3-galloylquinic acid). Caffeine is present on an average of 3% along very small quantities of the methylxanthines: theobromine and theophylline and the aminoacid: theanine (N-ethyl L-glutamine) [2]. Green tea differs from black tea in the susceptibility of the enzymes that participate in the fermentation process (i.e. polyphenol oxidase). These enzymes are generally deactivated during the steaming process, and independently of the steaming and/or fermentation process green tea usually contains higher vitamin content than its counterpart (black tea) [3]. It is well recognized that the tea leaves are catechin-rich and that present a polyphenol content between 20 to 35% based on dry matter [4]. The catechin content in green tea leaves vary based on its origin, aging, and treatment [5]. Fresh tea leaves contain a high polyphenol content, especially in the following catechins: (+)-catechin (C), (+)-gallocatechin (GC), (-)epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)epigallocatechin gallate (EGCG) (Figure 1) [1]. While, a green tea cup (2.5 g green tea in 200 mL water), usually contains about 90 mg of (-)-epigallocatechin gallate (EGCG), about 65 mg of (-)-epigallocatechin (EGC), and approximately 20 mg of both (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC) [6].

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Figure 1. Structure of the main polyphenols in green tea.

The consumption of tea has been related with anti-pyretic and diuretic activities, and multiple reports indicate that consuming significant amounts of green tea reduce the risk of developing multiple forms of cancer [7]. Currently, multiple epidemiological and pharmacological studies report that the components of green tea have anti-oxidant capacity, hypocholesterolemic effect, anti-mutagenic, anti-carcinogenic, and anti-tumoral activity [3, 8, 9]. For instance, the anti-mutagenic activity of the green tea catechins has been well correlated with their capacity to scavenge free radicals such as HO· y ROO· [9]. Furthermore, green tea (Camellia sinensis L.) catechins have also been reported by in vitro and in vivo studies to have anti-oxidant capacity, free radical scavenging activity [10], anti-neoplastic properties [11], capacity to inhibit platelet aggregation [12], and anti-bacterial properties [13]. The consumption of commercial beverages based on green tea has a great demand around the world, and most of the time these beverages remain for long time in the commercial shelves before reaching to the final customer. However, it has been reported that the total catechin content in tea beverages gets significantly reduced during the storage due to progressive catechin degradation or epimerization, a phenomenom called browning [14]. For instance, the total catechin content in fresh green tea infusions has been reported to range between 0.9 to 75.0 mg catechin/100 mL, while the total catechin content in commercially available green tea derived beverages range between 0.3 to 35.0 mg catechin/100 mL [14]. However, these differences in total catechin content cannot only be attributed to the progressive catechin degradation (browning) but to the different processing treatments and/or to the multiple other ingredients (such as ginseng, honey, and jasmine) added to the commercial green tea derived-beverages [14]. Lemongrass (Cymbopogon citratus Stap) is a perennial grass that presents multiple species such as: Cymbopogon citratus, Cymbopogon nardus, Cymbopogon flexuosus and Cymbopogon martini [15], that contain characteristic aromas especially in the leaves. Furthermore, lemongrass is a rich source of high-quality essential oils that are used in multiple commercial beverage preparation [16]. Additionally, multiple compounds such as monoterpenes: citronelol, geraniol, and linaldol, triterpenes, sesquiterpenes, and β-sitosterol have been identified in lemongrass extracts [17]. Nevertheless, lemongrass is employed as a therapeutic agent used in different countries; for instance, in the Peruvian jungle lemongrass infusions are utilized to treat different digestive problems and as an anti-neuralgic agent [17]. Because of the attractive phytochemical content and well documented pharmacological attributes of green tean and lemongrass, a functional beverage was designed using these two

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plant species. The technological criteria to design, formulate, and assess the functional beverage in this study include: (a) to determine the anti-oxidant capacity [inhibition of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical] of both plant species (green tea and lemongrass), (b) to design a beverage derived from green tea and lemongrass, considering the ratio between green tea:lemongrass, infusion time, total catechin content, pasteurization, browning (catechin degradation products) and anti-oxidant capacity, and (c) to assess the stability (total catechin content, microbiological activity, and anti-oxidant capacity) of the final beverage formulation.

MATERIALS AND METHODS Plants The fresh tea leaves were collected from the plantations of the Empresa Jardines del Té S.A. (Río Azul, Pucallpa, Perú), while the lemongrass leaves were collected from the Porvenir de Marona area (Pumahuasi, Tingo María, Perú). The tea leaves were processed according to the previously described methodology by Varnam and Sutherland [1]; briefly, the tea leaves were blanched at 100°C for 60 seconds to inactivate degrading enzymes followed by air-drying at room temperature for 2 hours. Then, the leaves were hot-air dried at 100°C for 50 min and ground to 1 mm diameter particles. Finally, the ground leaves were hot-air dried at 60°C for 40 min to achieve a final moisture between 6 to 9% [1]. The fresh lemongrass leaves were quickly washed with water, hot-air dried at 100°C for 40 min, ground to 1 mm diameter particles, and finally the ground leaves were hot-air dried at 50°C for 6 hours [18]. The final ground leaves of green tea and lemongrass were stored in polyethylene bags and placed in a desiccator at room temperature (25 ± 2°C) to maintain the desired moisture (6-9%).

Experimental Design The experimental design for the functional beverage derived from green tea and lemongrass is represented in Figure 2. The beverage design involved different stages: a) formulation based on the following proportions of green tea: 100, 75 and 50% and the difference of lemongrass, and a final extract concentration (green tea:lemongrass) of 5.0, 7.5 and 10.0 mg/mL. b) The two proposed acids to be used in the beverage were citric acid or ascorbic acid. The adequate acidity and sweetness of the beverage was determined by assessing the pH and °Brix, respectively. The assessed sweetness levels of the beverage were 7, 9 and 11°Brix, while the acidity levels studied were pH 3.1, 3.3 and 3.5. The adequate flavor, color and aroma of the beverage were assessed by sensory evaluation. The adequate infusion time was determined by total catechin content analysis and sensory evaluation of color. The assessed infusion times were 3, 5 and 7 minutes. c) The adequate pasteurization conditions (temperature/time) were assessed by microbiological analysis, anti-oxidant capacity, and total catechin content analysis. The pasteurization conditions studied were 85, 90 and 95°C, and 5, 10 and 15 minutes. d) The effect of the light on inducing browning in the

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beverage was assessed after storage for 16 days at room temperature. The first group of bottles was stored for 16 days in light-protected bottles placed in darkness, while the second group of bottles was stored in clear bottles exposed to regular light. e) The stability of the beverage at different temperatures and time periods was assessed by total catechin content analysis, browning, anti-oxidant activity, and microbiological analysis. The assessed storage temperatures were 15, 25 and 35°C, and the time periods were 0, 4, 8, 12 and 16 days. The adequate stability was assessed based on the degree of browning (catechin degradation), microbiological analysis, and anti-oxidant capacity during the storage temperature/time combination.

Commercial Green Tea Based Beverages Commercial green tea based beverages were purchased from a local grocery and were employed to compare their anti-oxidant capacity with our proposed beverage based in green tea and lemongrass. The commercial beverages contain other ingredients; for instance Arizona Green Tea contains green tea, filtered water, high fructose corn syrup, honey, citric acid, natural flavors, ginseng extract and vitamin C. Beberash Iced Tea has as ingredients: purified water, black tea extracts, green tea extracts, sugar, citric acid and vitamin C. Snapple Iced Tea contains water, high fructose corn syrup, natural lime flavor with other natural flavors, citric acid, green tea and ascorbic acid.

Figure 2. Stages of the design and evaluation of the beverage derived from green tea and lemongrass.

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DPPH Radical Scavenging Capacity The previously described method by Brand-Williams et al. [19] was employed. Briefly, the green tea and lemongrass samples were extracted with 95% ethanol for 10 minutes under constant agitation achieving a final stock solution of 100 mg/mL. The reaction was performed using 50 µL of sample and 950 µL of DPPH (100 µM), and the absorbance reading at 515 nm was performed after 1 minute [19]. The assessed extract concentrations were 3, 10, 30, and 100 μg/mL for green tea and 10, 100, 300, and 3000 μg/mL for lemongrass. Based on the results, the necessary concentration of extract (green tea or lemongrass) needed to inhibit the DPPH radical by 50% (IC50) was determined by model fitting using the pharmacokinetic software WinNonlin (Ver. 5.1) (Pharsight Corporation Mountain View, CA). Catechin and quercetin were used as standards. The anti-oxidant capacity of the beverage formulations was determined by allowing the reaction between 50 µL of standard or sample and 950 µL of DPPH (100 µM) for 1 minute [19]. The results are presented in three forms: the anti-oxidant capacity (AC) represents the DPPH radical inhibition percentage; the anti-oxidant value (AOX) represents the final absorbance measured after the 1 minute reaction [20]; and the vitamin C equivalent antioxidant capacity (VCEAC) was calculated based on a calibration curve of DPPH and vitamin C [21].

Total Catechin Content Analysis The content of total catechin was determined by the spectrophotometric method described by Singh et al. [22] using the synthesized diazotized sulfanilamide. The total catechin content analysis was determined by allowing the reaction between 1000 μL of standard or sample and 200 μL diazotized sulfanilamide (1% w/v in acetone) and 200 μL hydrochloric acid (30% v/v) for 1 hour at room temperature. Then, 3.6 mL of water was added and the absorbance values were recorded at 425 nm.

Browning Reaction The methodology to assess the browning reaction was assessed by measuring the total catechin degradation/epimerization products by absorbance at 440 nm following the methodology described by Bradshaw et al. [23].

Microbiological Analysis The microbiological analysis was performed as described previously [24]. Briefly, a sample (10 mL) was homogenized with 90 mL of sterile peptone water (0.1%) by agitation. The samples were diluted (10-1, 10-2, 10-3) by diluting 1 mL sample with 9 mL of peptone water, then an aliquot (1 mL) was plated in duplicate by using deep half plate count agar, and the plates were incubated at 35 to 37°C for 48 h. Then, the colony forming units (cfu) were

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counted using a colony counter and expressed as cfu/mL after correcting for the dilution factor.

Sensory Evaluation Representative samples of the different formulations were evaluated by the Friedman Multiple Comparisons test described previously [25]. The sensory attributes of flavor, color, aroma, acidity, and sweetness were assessed by 9 panelists previously trained. The panelists were students of the quality control course of the Departamento de Ingeniería Alimentaria of the Universidad Nacional Agraria de la Selva, Tingo María, Perú. The samples (approximately 15 mL) were placed in disposable cups and coded with the symbols: π, θ, μ, λ, β, α, φ, ω, η. The panelists were asked to rank order from lowest to highest based on preference.

Statistical Analysis Compiled data were presented as mean and standard error of the mean (mean ± SEM). General Linear Model (GLM) Analysis of Variance (ANOVA) with Duncan’s multiple range test with a p-value < 0.05 been statistically significant (Statistical Analysis System – SAS, Institute Cary, NC).

RESULTS AND DISCUSSION Anti-Oxidant Capacity of Green Tea and Lemongrass The free radical scavenging capacity of green tea and lemongrass was assessed by the stable DPPH radical assay. This assay reported that the green tea extracts (Figure 3A) exhibited higher anti-oxidant capacity than the lemongrass extracts (Figure 3B) at all the concentrations tested, exhibiting a concentration-dependent inhibitory capacity as previously described with other compounds and extracts [19, 26]. Table 1 reports the DDPH radical IC50 values of green tea and lemongrass extracts compared to the standards catechin and quercetin. It can be observed that the green tea extract (IC50 = 32.4 μg/mL) is approximately 40 times more potent than the lemongrass extract (IC50 = 1350 μg/mL), while the controls (quercetin and catechin) report a more potent anti-oxidant capacity (IC50 values of 3.35 and 4.83 μg/mL, respectively) than the green tea extract. Previous reports indicated that the IC50 values for freeze-dried green tea, oolong tea, and black tea samples were 4.14, 27.02 and 47.12 μg/mL, respectively [27]. The variability in IC50 values between our study and previous reports can be attributed to differences in antioxidant capacity methodology, geographical location of the green tea samples, harvesting time, and processing/treatment of the tea as previously reported [14, 22].

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Figure 3. DPPH radical scavenging capacity of the green tea (A) and lemongrass (B) extracts at different concentrations. Data expressed as mean ± SEM, n = 3.

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Table 1. DPPH radical IC50 values of green tea and lemongrass extracts compared to the controls catechin and quercetin Sample

IC50 (µg/mL)

Green tea extract Lemongrass extract Quercetin Catechin

32.4 ± 0.38a 1350 ± 47b 3.35 ± 0.09c 4.83 ± 0.13d

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test. The DPPH radical scavenging capacity was measured after 1 minute reaction.

It can be observed that green tea had a higher anti-oxidant capacity than lemongrass, which might be due to the higher content of anti-oxidant compounds such as flavonoids [28] and the flavonoid sub-class: flavanols also known as green tea catechins [29]. The predominant green tea catechins are: (-)-epicatechin, (-)-epicatechin gallate, (-)epigallocatechin and (-)-epigallocatechin gallate [30].

Formulation of the Beverage Based in Green Tea and Lemongrass The formulation of the beverage included the following stages: to determine the adequate ratio green tea:lemongrass based on the total catechin content, DPPH radical scavenging capacity, and sensory analysis based on flavor, color and aroma. Table 2 reports the total catechin content of the beverages with different green tea:lemongrass proportions and total extract concentration. It can be observed in Table 2 that the formulations SL3P1, SL3P2 and SL3P3 exhibit a lower total catechin content as the green tea proportion decreases, which correlates with previous reports that indicate that the total catechin content increases proportionally with the amount of black tea infused in 200 mL water [31]. Table 2. Total catechin content of the different beverage formulations Formulation

SL3P1 SL2P1 SL3P2 SL2P2 SL1P1 SL3P3 SL1P2 SL2P3 SL1P3

Total extract concentration (mg/mL) 10.0 7.50 10.0 7.50 5.0 10.0 5.0 7.50 5.0

Proportion Green tea (%) Lemongrass (%)

Total catechin content (mg/100mL)

100 100 75 75 100 50 75 50 50

67.5 ± 1.54a 50.2 ± 0.96b 47.2 ± 1.87c 38.0 ± 1.30d 34.0 ± 0.77e 24.4 ± 0.65f 23.5 ± 0.97f 18.2 ± 1.08g 12.4 ± 2.08h

0 0 25 25 0 50 25 50 50

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

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Formulation

SL3P1 SL2P1 SL3P2 SL3P3 SL2P2 SL1P1 SL2P3 SL1P2 SL1P3 Arizona Gree tea Beberash Iced Tea Snapple Iced Tea

Total extract concentration (mg/mL) 10.0 7.50 10.0 10.0 7.50 5.0 7.50 5.0 5.0 NA

Proportion Green tea Lemongrass (%) (%) 100 0 100 0 75 25 50 50 75 25 100 0 50 50 75 25 50 50 NA NA

AC1 (%)

AOX2 (A*min-1)

VCEAC3 (µmol/L)

91.97a 91.76a 90.49a 88.59b 82.25c 82.14c 79.08d 72.00e 64.50f 60.55g

0.076a 0.078a 0.090a 0.108b 0.168c 0.169c 0.198d 0.265e 0.336f 0.386g

37.19a 37.12a 36.63a 35.86b 33.28c 32.25c 32.03d 29.17e 26.14f 24.57g

NA

NA

NA

91.88b

0.101b

36.30b

NA

NA

NA

89.72a

0.080a

37.17a

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test. 1 AC: anti-oxidant capacity, which represents the DPPH radical inhibition percentage. 2 AOX: anti-oxidant value, which represents the final absorbance measured after the 1 minute reaction. 3 VCEAC: vitamin C equivalent anti-oxidant capacity, which was calculated based on a calibration curve of DPPH and vitamin C.

Table 4. Sensory analysis based on flavor, color and aroma evaluated by the Friedman Multiple Comparisons test Formulation

SL3P3 SL3P2 SL2P3 SL1P3 SL1P2 SL3P1 SL2P1 SL2P2 SL1P1

Total extract concentration (mg/mL) 10.0 10.0 7.5 5.0 5.0 10.0 7.50 7.50 5.0

Proportion Green tea Lemongrass (%) (%) 50 50 75 25 50 50 50 50 75 25 100 0 100 0 75 25 100 0

Evaluation parameter Flavor Color Aroma 6.78a 5.78ª,b 5.44b,c 5.33b,c 5.22b,c 4.67b,c 4.47b,c 4.33c 2.78d

9.00a 7.44b 7.11b 6.22c 4.56d 4.00e 2.78f 2.33g 1.56h

8.00a 6.78b 5.89b,c 4.33e 2.11f 5.33c,d 5.33d,e 4.78e 2.44f

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05).

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It can be observed in Table 3 that our green tea based formulations present similar antioxidant capacity than the commercially available beverages. Table 4 reports the sensory analysis based on flavor, color and aroma evaluated by the Friedman Multiple Comparisons test, and it can be observed that the formulations SL3P3 and SL3P2 were statistically more accepted by the panelists and numerically the SL3P3 formulation was superior than the SL3P2 formulation. Based on these results, it was determined that the formulation SL3P3 (10 mg/mL; 50% green tea and 50% lemongrass) exhibited the greatest acceptance by the sensory analysis panel (Table 4). Thus, this was the formulation selected for the next stage. The total extract concentration (10 mg/mL) of green tea:lemongrass correlates with the report by Arts et al. [31], that determined that a concentration of 10 mg/mL of tea presents an adequate total catechin content.

Beverage Acid Selection The selection of the acid was based on its potential in preventing browing as shown in Figure 4. It can be observed that even though ascorbic acid prevents browning better than citric acid up to day 3, citric acid has a higher anti-browning effect throughout one week. Our results indicate that citric acid is more effective in preventing browning and that ascorbic acid acts initially as an anti-oxidant, but after day 4 it appears to act as a pro-oxidant. This was also observed previously by Podmore et al. [32] and Herbert et al. [33] that reported that the presence of ascorbic acid as an ingredient tends to act as an anti-oxidant initially and as a prooxidant in the long-term. Furthermore, Chen et al. [34] reported that ascorbic acid has a protective effect in the short-term, and that citric acid is the ingredient of choice for long-term storage of beverages. Catechins in general are degraded or epimerized in neutral or slightly basic environments; thus, it is critical to use an acid to stabilize catechins and prevent their degradation, so that they can be absorbed from the gastrointestinal tract in their bioactive form [34]. Based on our results, we decided to utilize citric acid to stabilize catechins for a prolonged period of time in our formulated beverage.

Determination of the Adequate Acidity and Sweetness of the Beverage Table 5 reports the sensory evaluation based on the acidity (pH) and sweetness (°Brix) attributes evaluated by the Friedman Multiple Comparisons test. Based on these results, it can be observed that the beverage adjusted to 11°Brix and pH 3.1 reported the highest acceptance than the other formulations. Varnam and Sutherland [1] reported that beverages acidified with citric acid are palatable at pH between 3.08 and 5.4. In the terms of sweetness, Ortega [35] and Carmona [36] reported that an adequate sweetness in Carica candamaecensis Hook (a papaya variety) and cocona (Solanum topiro) beverages range between 12 and 14º Brix, respectively. Thus, the sweetness level of 11°Brix and acidity level of pH 3.1 was selected as adequate for our green tea and lemongrass based beverage.

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Figure 4. Effect of the citric acid and ascorbic acid on browning (catechin degradation) of the beverage. Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

Table 5. Sensory evaluation based on the acidity and sweetness parameters Formulation SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3

º Brix 11.0 11.0 11.0 9.0 9.0 9.0 7.0 7.0 7.0

Parameter pH 3.3 3.1 3.5 3.5 3.3 3.1 3.5 3.3 3.1

Evaluation parameter Sweetness Acidity 8.33a 4.33d a,b 7.89 5.33c b 7.22 2.11e 5.11c 2.44d,e c 4.89 5.33c d 4.11 6.78b e 3.00 4.78d 2.44e,f 5.89b f 2.00 8.00a

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05).

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Infusion Time Table 6 reports the total catechin content after different infusion times. Based on these results it can be observed that longer the infusion time higher the total catechin content, this correlates to previous reports in which black tea reaches the maximum total catechin content after a 5 minute infusion, and that longer infusion times induce catechin degradation [31]. It has to be noted that in Table 2 the total catechin content of the SL3P3 formulation was 24.4 ± 0.65 mg catechin/100 mL, but after adjusting the pH to 3.1 and 11°Brix the catechin content becomes 39.7 ± 0.91 mg catechin/100 mL (Table 6). This phenomena can be explained due to the higher stability of catechin at acidic pH [29, 34, 37] but not at almost neutral environment (pH 6.8) [38]. Table 6. Total catechin content after different infusion times Formulation SL3P3 SL3P3 SL3P3

Infusion time (minutes)

Total catechin content (mg/100mL)

7.0 5.0 3.0

pH 3.1 and 11°Brix 39.7 ± 0.91a 36.2 ± 0.71b 33.2 ± 1.03c

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

Pasteurization To determine the adequate pasteurization conditions, the total catechin content, microbiological analysis, and anti-oxidant capacity of the formulation were assessed. Table 7 reports the total catechin content at different pasteurization conditions. It can be observed that at higher temperature and time of the thermal treatment lower the total catechin content, which could be related to catechin degradation during pasteurization due to longer heat exposure. For instance, Chen et al. [14] reported that pH plays a critical role in catechin stability of green tea beverages, in which 80% of the total catechin content gets degraded at pH 5 to 6 but not at pH 3 to 4 after thermal treatment (120ºC/20 min). Our results indicate that the pasteurization conditions: 85ºC/5 min, 90ºC/5 min and 85ºC/10 min reported the highest catechin retention with only a 9 to 18% catechin content reduction based on an initial catechin content of 39.74 mg catechin/100 mL after infusion. Thus, our results indicate that milder thermal treatments allow for a higher catechin retention, which correlates to the report of Chen et al. [14] that demonstrated that a thermal treatment of 98ºC for 7 hours exhibited a 20% catechin loss in green tea beverages compared to a milder thermal treatment (37ºC for 7 hours).

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Formulation SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3 SL3P3

Pasteurization parameters Temperature (ºC) Time (minutes) 85 5 90 5 85 10 90 10 85 15 95 5 90 15 95 10 95 15

Total catechin content (mg/100mL) 35.7 ± 2.15a 32.8 ± 0.84b 32.4 ± 0.51b,c 30.8 ± 1.24c,d 29.7 ± 1.01e,d 29.0 ± 0.57e,d,f 28.6 ± 0.84e,f 27.2 ± 0.96f,g 26.4 ± 0.79g

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

Table 8. Microbiological analysis after different pasteurization conditions Pasteurization parameters Temperature (ºC) Time (minutes) 85 5 85 10 85 15 90 5 90 10 90 15 Data expressed as mean, n = 3.

10-1 <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL

Dilution 10-2 <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL

10-3 <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL <10 cfu/mL

Furthermore, Table 8 reports the microbiological analysis after different pasteurization conditions. These results indicate that all the thermal treatment combinations were sufficient to maintain the viable mesophile counts below 10 colony forming units (cfu)/mL. The low microbiological counts might also be attributed to the anti-microbial effects of some of the green tea bioactive compounds such as tannins [39], flavanols and theaflavins [40] that exhibit anti-microbial activity against certain microorganisms such as Bacillus subtilis, Bacillus stearothermophilus, Clostridium botulinum and Desulfotomaculum nigrificans [40]. Based on the low microbiological counts and high catechin content the pasteurization conditions: 85ºC/5 min, 90ºC/5 min and 85ºC/10 min were selected and their anti-oxidant capacity was assessed as shown in Table 9. It can be observed that there is no statistically significant differences in anti-oxidant capacity after the three temperature/time pasteurization combinations. Therefore, based on the no stastically significant differences in anti-oxidant capacity and microbiological analysis between the pasteurization combinations, the formulation pasteurized at 90°C for 5 minutes was selected due to its high total catechin content. This formulation was selected to perform the stability tests.

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Table 9. Anti-oxidant capacity after different pasteurization conditions Formulation SL3P3 SL3P3 SL3P3

Pasteurization parameters Temperature (ºC) Time (minutes) 85 5 90 5 85 10

Anti-oxidant capacity AC1 (%) VCEAC2 (µmol/L) 94.0 ± 0.18 38.0 ± 0.07 93.4 ± 0.15 37.8 ± 0.06 93.4 ± 0.10 37.8 ± 0.04

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test. 1 AC: anti-oxidant capacity, which represents the DPPH radical inhibition percentage. 2 VCEAC: vitamin C equivalent anti-oxidant capacity, which was calculated based on a calibration curve of DPPH and vitamin C

Light Stability Figure 5 reports the effect of light over browning (catechin degradation) in beverage formulation bottles protected or not from the light. It can be observed that independently of the light protection browning occurs in similar trends indicating that light exposure is not a critical factor in this green tea and lemongrass based beverage. However, it has to be noted that other factos have been reported to play a critical role in browning and catechin degradation, some of these factors include the pH of the beverage [14] and oxygen content in the bottle [23].

Figure 5. Effect of light on browning (catechin degradation) in presence and absence of light. Data expressed as mean ± SEM, n = 3.

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Temperature Stability The beverage formulation with all the described parameters was bottled and stored at 15, 25 and 35°C and samples were collected at day 0, 4, 8, 12 and 16 to assess total catechin content, browning, anti-oxidant capacity and microbiological analysis. Table 10 reports the total catechin content after storage at different temperatures, and it can be observed that the catechin content losess were of 1, 21 and 45% after 16-day storage at 15, 25 and 35°C, respectively. The initial total catechin content at day 0 was 32.79 ± 0.84 mg catechin/100mL. The effect of temperature over browning (catechin degradation) is shown in Figure 6 and it can be observed that at 35°C catechins are more instable producing more browning, while at temperatures 15 and 25°C there was significantly less browning. However, it also can be observed that up to day 8 there is comparable browning between all the temperatures, but after day 8 there is a temperature-dependant increase in browning. These results correlate with previous reports that demonstrated that browning is significantly produced after 2 days at 45°C in a wine model with ascorbic acid and high catechin content [23]. Furthermore, it has been reported that the stability of catechins is temperature-dependent, with increasing stability at lower temperatures [14]. The anti-oxidant capacity of the beverage stored at different temperatures is shown in Figure 7. It can be observed that after a 16 day-storage at 15°C presents a slightly higher antioxidant capacity (higher DPPH radical percentage inhibited) than the beverage samples stored at higher temperatures. The DPPH radical inhibition was 90.5; 88.6 and 86.4% after 16-day storage at 15, 25 and 35°C, repectively. However, these differences in anti-oxidant capacity between the storage temperatures are relatively small compared to the changes in total catechin content (Table 10) and browning (catechin degradation) appearance (Figure 6), which indicate that the degradation or epimeration sub-products of catechins also possess certain anti-oxidant capacity (DPPH radical scavenging capacity). It has been reported that theaflavins, which are epimerization sub-products of catechins, present similar LDLcholesterol protective effects in humans [41]. Furthermore, it has also been reported that the conversion of catechins to theaflavins induced by the regular processing of black tea do not affect the free radical scavenging capacity of black tea [41]. However, green tea also possesses significant amounts of other more temperature-resistant compounds such as the polyphenols that also significantly contribute to the anti-oxidant capacity of green tea [42]. One of these polyphenols is epigallocatechin gallate (EGCG) that maintains a close relationship between its content and the anti-oxidant capacity of green tea [43]. The microbiological analysis of the beverages stored at different temperatures for 16 days is reported in Table 11. No microbiological (mesophiles) growth was observed during the storage time independently of the storage temperature. This might be attributed to the green tea components that possess bacteriostatic and bacteriodical activities to inhibit bacterial, mold and yeast growth [44]. Even though, there was catechin degradation after storage at different temperatures no microbial growth was observed (<10cfu/mL), which could also indicate that the epimerization products of catechins also have anti-microbial activity. For instance, some of the catechin epimerization products that present anti-microbial activity include gallocatechin gallate (GCG) [44], epigallocatechin (EGC), epicatechin (EC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) [13].

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Table 10. Total catechin content after storage at different temperatures Day 4 8 12 16

15°C 32.0 ± 0.75ª 31.9 ± 0.57ª 31.9 ± 0.22ª 31.7 ± 0.43ª

25°C 30.9 ± 0.38ª 29.8 ± 0.95a,b 28.5 ± 0.57b 25.8 ± 1.21c

35°C 30.2 ± 0.43ª 29.6 ± 0.57ª 25.5 ± 1.42b 19.6 ± 1.30c

Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

Figure 6. Appearance of browning (catechin degradation) after storage at different temperatures. Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

Table 11. Microbiological activity after storage at different temperatures Day

0

8

16

Dilution 10-1 10-2 10-3 10-1 10-2 10-3 10-1 10-2 10-3

Data expressed as mean, n = 3.

15° C cfu/mL <10 <10 <10 <10 <10 <10 <10 <10 <10

25° C cfu/mL <10 <10 <10 <10 <10 <10 <10 <10 <10

35° C cfu/mL <10 <10 <10 <10 <10 <10 <10 <10 <10

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Figure 7. Anti-oxidant capacity of the beverage stored at different temperatures. Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the Duncan’s multiple range test.

CONCLUSION The IC50 values for DPPH radical inhibition for green tea and lemongrass are 32.4 and 1350 μg/mL respectively. The optimized technological parameters for the beverage include a 50:50 ratio of green tea with a total extract concentration of 10 mg/mL, infustion time of 7 minutes, acidity levels of pH 3.1 using citric acid, sweetness levels of 11°Brix, pasteurization for 5 minutes at 90°C. The beverage was stable (not significant browning and no microbiological growth) at 15°C up to 16 days and the final beverage formulation was reported to inhibit the DPPH radical by 93.45%. Furthermore, the formulated beverage was well accepted by the test panel and presented similar anti-oxidant capacity than commercial green tea based beverages.

ACKNOWLEDGMENTS The authors would like to thank the Empresa Jardines del Te (La Divisoria, Tingo María, Perú) for suplying the fresh green tea leaves and to Dr. Manuel Sandoval Chacón, Ph.D. for his scientific support.

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[18] PROMPEX, (Promoción de Exportaciones) - Marco orientador para promover la inversión privada en cultivos de exportación en zonas de desarrollo alternativo. Lima, Perú 1997, 215. [19] Brand-Williams, W.; Cuvelier, M.; Berset, C., Use of free radical method to evaluate antioxidant capacity. Lebensmittel Wissenschaft und Technologie 1995, 28, (1), 25-30. [20] Velioglu, Y. S.; Mazza, G.; Gao, L.; Oomah, B. D., Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural Food Chemistry 1998, 46, (10), 4113-4117. [21] Kim, D. O.; Lee, K. W.; Lee, H. J.; Lee, C. Y., Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J Agric Food Chem 2002, 50, (13), 3713-7. [22] Singh, H. P.; Ravindranath, S. D.; Singh, C., Analysis of tea shoot catechins: spectrophotometric quantitation and selective visualization on two-dimensional paper chromatograms using diazotized sulfanilamide. J Agric Food Chem 1999, 47, (3), 10415. [23] Bradshaw, M. P.; Prenzler, P. D.; Scollary, G. R., Ascorbic acid-induced browning of (+)-catechin in a model wine system. J Agric Food Chem 2001, 49, (2), 934-9. [24] ICMSF, International Commissions on Microbiological Specifications for Foods Microorganismos de los alimentos I: Técnicas de análisis microbiológico. Editorial Acribia: Zaragoza, Spain, 1983; p 431. [25] Ureña, P.; D´Arrigo, M.; Girón, O., Evaluación sensorial de los alimentos : aplicación didáctica. Editorial Agraria: Lima, Perú, 1999; p 197. [26] Okawa, M.; Kinjo, J.; Nohara, T.; Ono, M., DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants. Biol Pharm Bull 2001, 24, (10), 1202-5. [27] Yokozawa, T.; Chen, C. P.; Dong, E.; Tanaka, T.; Nonaka, G. I.; Nishioka, I., Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2 picrylhydrazyl radical. Biochem Pharmacol 1998, 56, (2), 213-22. [28] Cook, N. C.; Samman, S., Flavonoids-Chemistry, metabolism, cardioprotective effects, and dietary sources. J. Nutr. Biochem. 1996, 6, 66-76. [29] Zhu, Q. Y.; Zhang, A.; Tsang, D.; Huang, Y.; Chen, Z. Y., Stability of green tea catechins. Journal of Agricultural Food Chemistry 1997, 45, (12), 4624-4628. [30] Katiyar, S. K.; Ahmad, N.; Mukhtar, H., Green tea and skin. Arch Dermatol 2000, 136, (8), 989-94. [31] Arts, I. C.; van De Putte, B.; Hollman, P. C., Catechin contents of foods commonly consumed in The Netherlands. 2. Tea, wine, fruit juices, and chocolate milk. J Agric Food Chem 2000, 48, (5), 1752-7. [32] Podmore, I. D.; Griffiths, H. R.; Herbert, K. E.; Mistry, N.; Mistry, P.; Lunec, J., Vitamin C exhibits pro-oxidant properties. Nature 1998, 392, (6676), 559. [33] Herbert, V.; Shaw, S.; Jayatilleke, E., Vitamin C-driven free radical generation from iron. J Nutr 1996, 126, (4 Suppl), 1213S-20S. [34] Chen, Z.; Zhu, Q. Y.; Wong, Y. F.; Zhang, Z.; Chung, H. Y., Stabilizing Effect of Ascorbic Acid on Green Tea Catechins. J. Agric. Food Chem. 1998, 46, (7), 2512 2516.

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[35] Ortega, R. A. Obtención del néctar de papayita de monte (Carica candinamaecensis Hook) y su preservación por los métodos de enlatado y embotellado. Universidad Nacional Agraria de la Selva, Tingo María, Perú, 1988. [36] Carmona, R. A. Evaluación de las propiedades reológicas de la pulpa y néctar de dos tipos de cocona (Solanum topiro). Universidad Nacional Agraria de la Selva, Tingo María, Perú, 1990. [37] Zhu, Q. Y.; Holt, R. R.; Lazarus, S. A.; Ensunsa, J. L.; Hammerstone, J. F.; Schmitz, H. H.; Keen, C. L., Stability of the flavan-3-ols epicatechin and catechin and related dimeric procyanidins derived from cocoa. J Agric Food Chem 2002, 50, (6), 1700-5. [38] Wang, L. F.; Kim, D. M.; Lee, C. Y., Effects of heat processing and storage on flavanols and sensory qualities of green tea beverage. J Agric Food Chem 2000, 48, (9), 4227-32. [39] Scalbert, A., Antimicrobial properties of tannins. Phytochemistry 1991, 30, (12), 38753883. [40] Hara, Y.; Watanabe, M., Antibacterial activity of tea polyphenols against Clostridiun botulinum. J. Jpn. Soc. Food Sci. Technol. 1989, 36, (951-955). [41] Leung, L. K.; Su, Y.; Chen, R.; Zhang, Z.; Huang, Y.; Chen, Z. Y., Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J Nutr 2001, 131, (9), 2248-51. [42] Yen, G. C.; Chen, H. Y., Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural Food Chemistry 1995, 43, (1), 27-32. [43] Lunder, L. K. In Catechins of green tea: Antioxidants activity, American Chemical Society, Washington D.C., USA, 1992; Washington D.C., USA, 1992; pp 114-120. [44] Hamilton-Miller, J. M., Antimicrobial properties of tea (Camellia sinensis L.). Antimicrob Agents Chemother 1995, 39, (11), 2375-7.

Reviewed by: Dr. Neal M. Davies, Ph.D. College of Pharmacy, Washington State University, Pullman, WA 99163. [email protected] Dr. Esteban I. Mejía-Meza, Ph.D. SunOpta Fruit Group, Inc. – Healthy Fruit Snacks, Omak, WA 98841. [email protected] Ms. Connie M. Remsberg, Pharm.D. Candidate. College of Pharmacy, Washington State University, Pullman, WA 99163. [email protected]

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 6

TEAS ARE NOT ALL THE SAME: IN VITRO AND IN VIVO ANTIOXIDANT ACTIVITY AND APPETITE MODULATION IN RATS OF GREEN TEAS WITH HIGH AND LOW LEVELS OF ORGANIC SELENIUM Abdul L. Molan1, Zhuojian Liu1 and Wenhua Wei2 1

Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand. 2 Current address: MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK

ABSTRACT Green tea is a good source of various polyphenolic compounds and minerals which are powerful antioxidants. The effects of selenium-containing green tea (Se-GTE; 1.4 mg selenium/kg) and China green tea (CH-GTE; 0.13 mg selenium/kg) on food consumption and body weight gain were investigated using a rat model. In addition, the total phenolic contents (TPC), antioxidant/antiradical activities of these teas were determined in vitro. Both teas had a satiating influence on experimental rats, as evidenced by their ability to decrease food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE), although a statistically significant decrease over the control rats was achieved only for Se-GTE treatment. In addition, the final body weight of rats gavaged with Se-GTE was significantly lower (P = 0.0063) than that of the water-gavaged control rats and this corresponds to 8.5% reduction in body weight relative to the control group. In contrast, rats gavaged with CH-GTE showed only 1.8% reduction in the final body weight relative to the control group. The reduction in food intake over a short period compared to a control rats preloaded with the same volume of water suggests that the decrease in food intake was mainly a consequence of a satiating effect, rather than a stomach distension effect. The underlying mechanism responsible for this satiating effect was not identified as part of this study. It is also important to mention that water intake for the groups given the tested teas was similar to that of rats given water only and no significant differences were observed. Se-GTE had significantly higher TPC (P < 0.0001), higher ferric reducing antioxidant power (FRAP) (P < 0.01), higher diphenyl-picrylhydrazyl (DPPH) free radical scavenging activity (P < 0.05), higher ferrous-ion chelating activity (P < 0.05-

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Abdul Lateef Molan, Zhuojian Liu and Wenhua Wei 0.01), and higher selenium contents (P < 0.0001) than CH-GTE. A strong positive correlation was found between the TPC, and the FRAP, DPPH, and the ferrous-ion chelating activities in both teas, indicating that the polyphenolic compounds are the major contributors of the antioxidant/antiradical activities. When rats were gavaged with water-soluble tea extract (10 ml/kg/day) of Se-GTE for four consecutive days, serum FRAP increased significantly (P = 0.0002) as compared to water-gavaged controls. The level of serum FRAP in rats gavaged with CH-GTE increased slightly when compared with the control rats but not to a significant extent. These results indicate that green tea may have the ability to elevate circulating antioxidant potentials in vivo and this ability is dependent on the type of tea used. The observed results suggest that the reduction in food intake and decrease in body weight in experimental animals may be a consequence of antioxidant mechanisms played by the polyphenolic compounds and the minerals found in green teas. Green tea, especially the one with a high level of organic selenium may provide a good satiety inducer and weight management modulator.

Keywords: Green tea; selenium; Satiety; Rats; Antioxidant; antiradical activity; FRAP

BACKGROUND Obesity is a complex multifactorial syndrome caused by an increase in food availability, high-fat diet, and sedentary lifestyle and is becoming a pandemic which has been rapidly developing for three decades (Hill et al., 2003). This syndrome seems to affect all ages as some studies have shown that in many developed countries child obesity levels have doubled in the last two decades (Lobestein et al., 2004) and are set to double again, probably over a shorter period. Lobstein (2006) mentioned in his review that the impending disease burden has been described by many researchers as ‘a public health disaster waits to happen’, ‘a massive tsunami’, and ‘a health time-bomb’, and politicians are aware that the amount of time they have left to make their decisions is rapidly declining. Obesity is a risk factor for several chronic disorders such as the cardiovascular diseases, hypertension, sleeping apnea, osteoarthritis of weight-bearing joints, reduced fertility, asthma, and some cancers (Rippe, 1998; Shore and Johanson, 2005). In contrast, weight loss is known to reduce blood pressure, lipid levels, and the incidence of type 2 diabetes mellitus (Sheard, 2003). Unlike cardiovascular diseases and cancer, there is no functional food available for obesity (St-Onge, 2005) and hence efficient, effective and satisfying treatments are required. Nutritional research strategies that target aspects of the physiology of obesity are critically important if a workable solution to this health challenge is to be found. One potential area is to discover foods/food ingredients that can induce greater satiety through physiological modulation, thus reduce overall food intake. Selenium (Se) has been considered as an essential micronutrient for animals and the human body (Navarro-Alarcon and Lopez-Martiez, 2000). Selenium deficiency is a serious nutritional and health problem in many countries (Ge and Yang, 1993). Tinggi (2003) reported in his review that diseases associated with Se deficiency are still a cause of concern in many countries, and in particular in countries of low Se status such as Finland and New Zealand. In both countries, policies to increase Se status in the human population by adding

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Se fertilizers to agricultural crops or by importation of Se-rich foods have been introduced (Aro et al., 1995; Thompson and Robinson, 1996). Plant foods, meats and sea foods are the major dietary sources of Se but the content of the soil where the plants are cultivated or where the animals are raised (Toyran et al., 2008). Selenium present in most vegetables is in highly available form, which is around 85-100% while meat products have a Se bioavailability of approximately 15% (Navarro-Alarcon et al., 1998). Selenium deficiency has been linked to the development of many health problems such as heart diseases and cancer (Finley and Penland, 1998). On the other hand, some studies have shown that Se has many biological activities such as protection against some cancers, enhancement of neuropsycological function, and maintenance of a healthy immune system (Clark et al., 1996; Finley and Penland, 1998; Toyran et al., 2008).

SATIETY AND SATIATION Humans usually eat during meals until they are comfortably full (satiation), after which they do not eat for a certain time (satiety) (Blundell et al., 1996). It is well known that internal factors and external environmental factors determine the extent of satiety and satiation (Birch et al., 1989). Appitite is one of the most important internal factors, which can be measured in either with the help of objective ratings (hunger, desire to eat, prospective consumption, and fullness) (Graaf et al., 2004), or by measuring actual food intake (the amount of food eaten within a certain context). The expression of appetite is reflected in the relationship between the level of psychological events and behavior, the peripheral physiology, and the central nervous system (Bundell et al., 1996; Geliebter et al., 1996; Graaf et al., 2004). The physiological measures that relate to subjectively rated appetite, actual food intake, or both are defined as biomarkers of satiety and satiation (Graaf et al., 2004). According to Graaf et al. (2004), determination of the biomarkers of satiation and satiety could be used as a tool or index with which to measure the satiating efficiency of foods. These tools may serve as evidence for the ability of certain food or food ingredients to enhance satiety, reduces appetite, or both (Diplock et al., 1999). In addition, these biomarkers help to understand the physiological mechanisms behind regulation of food intake and energy balance in humans. Graaf et al. (2004) in their review, discussed in detail the internal biomarkers and divided them into the following types: brain biomarkers or measures which relate to pleasantness of food and sensory-specific satiety, physical biomarkers which relate to stomach distension and volume of food consumed; hormonal signals (including cholecystokin (CCK), Glucagon Like Peptide-1 (GLP-1), bombesin or gastrin-releasing peptide, ghrelin, enterostatin, glucosedependent insulinotropic polypeptide (GIP), pancreatic polypeptide, and somatostatin); physiological biomarkers or measures such as body temperature and diet-induced thermogenesis; and biochemical measures (glucose uptake, insulin and leptin levels). They concluded that a number of physiologic measures are available that can serve as biomarkers of satiation, satiety, or both. With respect to satiation (meal termination), physical and chemical measures of stomach distension and blood plasma concentrations of CCK and GLP1 are useful. For satiety and meal initiation, glucose dynamics within a short time frame (less than 5 min), leptin concentrations during longer-term negative energy balance (more than 2–4

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days), and ghrelin concentrations at both the short-term and long-term intervals are physiological markers (Graaf et al., 2004).

CHEMICAL COMPOSITION OF TEA Fresh tea leaf is unusually rich in the flavanol group of polyphenols known as catechins. The catechins may constitute up to 30% of the dry leaf weight (Graham, 1992). The other polyphenols in tea include flavonols (quercetin, kaempferol, myricetin), and their glycosides, and depsides such as chlorogenic acid. Caffeine is present at an average level of 3% along with very small amounts of methylxanthines, theobromine and theophylline. Anthocyanidins are also found in the leaf. The amino acid theanine is unique to tea. In addition to phenolic compounds, the tea leaf contains vitamins and several minerals. Vitamin C is lost during the processing of the fresh leaf, but carotenoids and vitamin K are present in brewed tea. Tea also contains aluminium, potassium, fluoride and manganese (Balentine et al., 1997).

R3 OH HO

O

OH R2 R1

OH R1

R2

R3

Monomers

H

OH

H

Catechin (C)

OH

H

H

Epicatechin(EC)

H

OH

OH

Gallocatechin(GC)

OH

H

OH

Epigallocatechin (EGC)

Figure 1. Basic Structure of green tea catechins.

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The polyphenols in tea are oxidised to theaflavins and thearubigin in the presence of polyphenoloxidase and oxygen. The oxidised constituents are unique to black tea. Approximately 10% of the flavonoids in black tea are catechins, 10% are theaflavins, and 70% are thearubigins (Balentine et al., 1997; Higdon and Frei, 2003). The common tea polyphenols belong to three main groups called flavan-3-ol (flavonoids), flavonols and flavones. The chemical structure of major tea catechins (-) epigallocatechin 3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)epicatechin 3-gallate (ECG) and (+)-catechin (C), are illustrated in Figure 1.

HEALTH BENEFITS OF GREEN TEA It is well known that fruits and teas are good sources of antioxidants and have profound effects on human health. Green tea is derived from the leaves and fine stems of the leguminous shrub, Camellia sinensis and has been consumed as a beverage for thousands of years (Graham, 1992). Tea is one of the principle sources of polyphenols in the human diet because of its high level of consumption combined with its relatively high polyphenol content compared to other polyphenol sources (Hollman and Arts, 2000). The health promoting effects of green tea are mainly attributed to its polyphenol content (Wolfram et al., 2006). Recent research has demonstrated the potential of polyphenols in the prevention of chronic diseases. In vitro and animal studies have demonstrated that flavonoids have antioxidant and antimutagenic activities (Peterson and Dwyer, 1998), anti bacterial (Sakanaka et al., 2000) and antiparasitic activities (Paveto et al., 2004; Molan et al., 2003, 2004). More recent research has also shown the influence of flavonoids on metabolic reactions in vitro (Goto et al., 1999; Anderson et al., 2002). The antioxidant effect of tea has been suggested to have beneficial effects on Alzheimer’s disease and Parkinson’s disease (Behl et al., 1998; Pillai et al., 1999; Checkoway et al., 2002; Tan, et al., 2003). However, the effect of tea on such pathologies has not been fully established. Several experimental studies indicate a strong chemo-preventive action of tea against gastrointestinal tract and colorectal cancers (Mei et al., 2005). A case control study of 882 Japanese subjects by Ohno et al (1985) indicated a protective effect of green tea on bladder cancer. Other studies have shown an inverse relationship between tea intake and prostate cancer (Jain et al., 1998), breast cancer (Tavani et al., 1998), skin cancer (Zhao et al., 1999) and mucosal Leukamia (Lee et al., 2004). Animal studies have shown a beneficial effect of long-term feeding of tea catechins on the development of obesity ( Murase et al., 2002; Choo, 2003; Klaus et al., 2005). It has been concluded that the stimulation of hepatic lipid metabolism might be a factor responsible for the anti-obesity effects of tea catechins and that long-term consumption of tea catechins is beneficial for the suppression of diet-induced obesity. In some of these studies, It was concluded that this suppressive effect was resulted mainly from increase in brown adipose tissue thermogenesis through beta-adrenoceptor activation while in other studies, the antiobesity action was attributed to the ability of green tea to modulates the glucose uptake system in adipose tissue and skeletal muscle and to suppress the expression and/or activation of adipogenesis–related transcription factors.

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Wolfram et al. (2006) reported in their review that six studies have investigated the antiobesity effects of green tea and green tea catechins in humans and most of these studies reported decreased body weight and fat mass. Some of these studies have suggested that the body fat-lowering effects of green tea extracts/catechins may be associated with an increase in the thermogenesis and fat oxidation. In one study, the antiobesity effect of an 80% ethanolic dry extract from green tea (AR25) was evaluated in moderately obese patients (Chantre and Lairon, 2002). After three months, body weight was decreased by 4.6% and waist circumference by 4.48%. In vitro, AR25 exerted a direct inhibition of gastric and pancreatic lipases and a stimulation of thermogenesis. The authors suggested that the green tea extract to be a natural product for the treatment of obesity by inhibition of lipasess and stimulation of thermogenesis. Tea consumption has been identified as an independent factor protecting against the risk of hip fractures in mainly women over the age of 50 (Johnell et al., 1995; Kanis et al., 1999; Hegarty et al., 2000). Hegarty et al. (2000) found that those who drank tea had greater bone mineral density compared to that of those who did not consume tea. Higher bone mineral density of the lumber spine, greater trochanter and Ward’s triangle were independent of smoking status, hormone replacement therapy and coffee drinking. The aim of this study was to investigate the appetite suppression capacity of two types of teas that differ in their selenium contents and also to evaluate possible correlations between antioxidant activity and satiety effect. The total phenolic contents, the ferric reducing antioxidant power, free radical scavenging activity and ferrous-ion chelating activity of both CH-GTE and Se-GTE were also investigated in vitro.

MATERIAL AND METHODS Chemicals and Standards 2, 4, 6-Tripyridyl-s-triazine (TPTZ), sodium acetate, ferric chloride and gallic acid, FolinCiocalteu’s phenol reagent and ferrous sulfate were purchased from Sigma (Australia).

Preparation of Tea Extracts China green tea (CH-GTE) was purchased from retail shops while selenium-containing green tea (Se-GTE) was obtained from China. Se-GTE is not presently available on the market. The aqueous extracts were made by adding 100 ml water (100 oC) to 1 g (1% extract) tea leaves and allowed to brew for 10 minutes with stirring. Suspension was filtered through Whatman No. 1 filter paper to retain the clear solution.

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Mineral Analysis of Dry Tea Material Dry leaves of both Se-GTE and CH-GTE were ground into fine powders and sent to the New Zealand Laboratory Services, Ruakura Research Centre, Hamilton, New Zealand for mineral analysis.

Total Polyphenols Contents (TPC) Determination The amount of total phenolic content (TPC) in tea extracts was determined according to the Folin-Ciocalteu procedure as used by Molan et al. (2008a) with some modifications. Briefly, an aliquot of 12.5 µl of water-soluble extract was mixed with 250 µl of 2% sodium carbonate solution in 96-well microplates and allowed to react for 5 minutes at room temperature. Then 12.5 µl of Folin-Ciocalteu phenol reagent (50 %) was added and allowed to stand for 30 minutes at room temperature before the absorbance of the reaction mixture was read at 650 nm using a plate reader. Calibration was achieved with an aqueous gallic acid solution (100-1000 µg/ml). The TPC of the extract was expressed as mg gallic acid equivalent (GAE) per gram of tea leaves on dry basis.

Ferric Reducing Antioxidant Power Assay The capacity to reduce ferric ions was determined using Ferric reducing antioxidant power (FRAP) assay as described by Benzie and Strain (1996). Briefly, an aliquot of 8.5 µl of tea (1% or 2% water extracts) was added to 275 µl of diluted FRAP reagent using micoplate and the plates were incubated at 37 oC for 30 minutes before measuring the absorbance at 395 nm using a plate reader. The working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L TPTZ (2,4,6-tripyridyl-striazine) in 40 mmol/L hydrochloric acid and with 1 volume of 20 mmol/L ferric chloride. Standard curve was prepared using different concentrations (200-2000 μmol/L) of FeSO4.7H2O. The antioxidant capacity based on the ability to reduce ferric ions of the extract was expressed as micromole FeSO4 equivalents per litre of aqueous extracts. . All solutions were used on the day of preparation and all determinations were performed in triplicate.

Scavenging of Diphenyl-picrylhydrazyl (DPPH) Radicals This assay detects scavenging of free radicals by the tested compound through the scavenging activity of the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical. This assay was performed using a previously described method (van Amsterdam et al., 1992) with some minor modifications. Briefly, 25 μL of tea extracts was allowed to react with 250 μL of 0.2 mM DPPH in 95% ethanol in a 96-well microplate. The plate was then incubated at 37 °C for 30 minutes after which the absorbance was measured at 550 nm using a microplate reader. Scavenging capacity of the sample was compared to that of ascorbic acid as positive control (0.1- 1.0 mM ascorbic acid).

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The antiradical activity was calculated as a percentage of DPPH decolouration relative to a negative control using the following equation: Antiradical activity (%) = (absorbance of control incubation – absorbance of the tea extract/ absorbance of control incubation) x 100.

Ferrous-ion Chelating Assay The ferrous-ion chelating (FCA) assay used by Singh and Rajini (2004) was followed with slight modifications. Solutions of 2 mM FeSO4 and 5 mM ferrozine were prepared. Diluted FeSO4 (100 µl) was mixed with 100 µl of sample, followed by 100 µl of diluted ferrozine. Assay mixtures were allowed to equilibrate for 10 minutes before measuring the absorbance at 560 nm using a plate reader. Tea extracts were assayed in duplicate. The ability of the tea extracts to chelate ferrous ions was calculated relative to a negative control using the equation: Chelating activity % = (absorbance of control incubation – absorbance of the tea extract/ absorbance of control incubation) x 100.

Animals and Housing Thirty male Sprague-Dawley rats aged eight weeks that had been weaned onto a balanced semi-synthetic diet were housed individually in hanging wire-mesh stainless steel cages in a room with a temperature of 22 ± 1 0C and a 12-h light: dark cycle (light on at midnight) and they had free access to water throughout the study. The rats were obtained from the Animal Unit, Massey University, Palmerston North, New Zealand. For the first seven days, the rats were habituated to a 20-h deprivation schedule whereby a feeder containing more than one day’s expected intake of food was made available to each rat for a total of four hours (1000 - 1400h; 2 hours at the end of the light phase, and 2 hours at the beginning of the dark phase). This is to accommodate for the rat’s natural behaviour of nocturnal eating’s. This regimen is in line with recent procedures reported in the literature (Froetschel et al., 2001).

Pre-meal Administration This study involved giving the rats a 10 ml fluid pre-meal/kg body weight, containing either water or tea extracts. This pre-meal dosing was by pharyngeal gavage with a soft silicon rubber tube attached to a 3-ml syringe. The rats were habituated to this procedure, during the acclimation period. Thus, for the last three days of the acclimation period premeals of 2 ml sterile water at room-temperature were gavaged daily 30 minutes before the feeding period. The same experimental diet was offered to all treatment groups during the feeding period. It was formulated to meet the nutrient requirements of growing rats as per the formulation AIN-93G (Reeves et al., 1993).

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The amount of preload given to each rat was individually calculated based on body weight. The dose was calculated based on the assumption that a 75 kg person would take 3 cups (250 ml) of 1% extract of CH-GTE or Se-GTE, which is equivalent to 10 ml per kg body weight. In the present study, the rats were weighed at the beginning of the experiment and the body weight (g) was multiplied by 0.01 to determine each individual pre-meal load. For the remainder of the study (from day 8 to day 12) the rats were divided randomly into 3 equal groups (n = 10) and matched for body weight. The rats in the first group were fed the basal diet (AIN-93G; Reeves et al, 1993) and gavaged daily with 10 ml of deionized water/kg body weight to serve as a control group while the rats in the second and the third groups were gavaged daily with the same volume of water soluble extracts from CH-GTE and Se-GTE, respectively. The pre-meal was given to each rat 30 minutes before the normal meal time, and impact of the pre-meal was determined by monitoring changes in each or rat’s total food intake for that day. This was repeated for a period of four days. The rats were weighed at the beginning and at the end of the experiment. The food was presented to the rats in metal cups and food intake measured daily, as described previously (Molan et al., 2008b). Water intake was also measured daily. At the end of the study, the rats were euthanized by inhaling CO2 and the blood from the hearts was collected immediately after death.

Serum Antioxidant Capacity Serum antioxidant capacity was measured using the FRAP assay. Blood was collected from each rat in the control group and those gavaged with CH-GTE or Se-GTE. Samples were allowed to clot at room temperature for 25 minutes. Samples were then immediately centrifuged (1000 g) for 15 minutes at 4 0C to recover serum. Serum was extracted and then stored at – 80 0C for further use.

Statistical Analysis The data were recorded as (ANOVA) and Tukey multiple differences between the means. Office Excel 2003. Differences significant.

means ± standard errors. One way analysis of variance comparisons were carried out to test for any significant Linear regression analysis was carried out by Microsoft between means at 5% (P < 0.05) level were considered

RESULTS Mineral Contents The Se-GTE and CH-GTE powdered leaves were analyzed for mineral content (Table 1). Elemental analysis indicates that the Se-GTE leaves have a ten-fold higher mean total selenium concentration (1.4 versus 0.13 mg of Se/kg; P < 0.0001). Moreover, Se-GTE contains higher concentrations of phosphorus, potassium, iron, zinc and copper than CH-GTE (Table1), but CH-GTE contains more calcium and manganese.

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Table 1. The mineral contents of selenium green tea (Se-GTE) and China green tea (CH-GTE) Mineral Phosphorus Potassium Calcium Sodium Iron Manganese Zinc Copper Selenium

Unit g/100g g/100g g/100g g/100g mg/kg mg/kg mg/kg mg/kg mg/kg

Value Se-GTE 0.46 ± 0.083 1.78 ± 0.055 0.33 ± 0.077 < 0.02 407 ± 13.0 1159 ± 140.5 49.1 ± 11.1 25.5 ± 2.1 1.4 ± 0.075

CH-GTE 0.25 ± 0.005 1.5 ± 0.085 0.49 ± 0.015 < 0.02 305.0 ± 64 1619 ± 428.0 43.5 ± 11.5 16.0 ± 3.8 0.13 ± 0.002

Total Polyphenol Contents and Antioxidant Activities Se-GTE had significantly higher (P < 0.0001) TPC than CH-GTE at both 1% and 2% concentrations (Table 2). Aqueous extracts from Se-GTE also showed significantly higher reducing activity as measured by FRAP (P < 0.01), higher free radical scavenging activity as measured by DPPH assay (P < 0.05) and higher ferrous-ion chelating activity (P< 0.05- 0.01) than CH-GTE. The results of this study showed that the TPC values were strongly correlated with the FRAP (R2 = 0.70 and 0.90 for CH-GTE and Se-GTE, respectively), DPPH (R2 = 0.92 and 0.76, respectively) and FCA (R2 = 0.69 and 0.71, respectively) values for both CH-GTE and Se-GTE. Table 2. Antioxidant activity [ferric reducing antioxidant power (FRAP; µmol/L) values; DPPH scavenging activity and ferrous-ion chelating activity (FCA)], total phenolic contents (TPC; mg GAE/g dry leaves) and correlation (R2) between antioxidant values and TPC of China green tea (CH-GTE) and selenium-containing tea (Se-GTE) FCA (%) 34.1 ± 0.57 39.3 ± 1.3* 0.69 (CH-GTE) 2 and 0.71 (SeR GTE) versus TPC The mean of duplicate incubations of two separate experiments and standard errors are presented. Statistical analysis based on ANOVA, * P < 0.05; ** P< 0.01 and *** P< 0.0001 compared to values in the same column. CH-GTE Se-GTE

TPC (mg/g DL) 34.1 ± 4.2 60.5 ± 2.9***

FRAP (µmol/L) 9002.8 ± 60.6 9947.6 ± 37** 0.70 (CH-GTE) and 0.90 (Se-GTE) versus TPC

DPPH (%) 69.4 ± 0.76 71.5 ± 0.6* 0.92 (CH-GTE) and 0.76 (SeGTE) versus TPC

Teas Are not All the Same Control

(A)

113 CH-GTE

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*

*

10 8 6 4 2 0 1

2

3

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Days after oral adminstration

% reduction in total food intake relative to control group

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Figure 2. Effects of oral administration of water extracts from two teas on food consumption (A) and % reduction in food intake (B) in rats fasted for 20 h. Rats were divided into 3 groups of ten rats each. The experimental rats were gavaged with ~2 ml of tea extracts daily for 4 consecutive days. The control group was gavaged with ~2 ml of distilled water. Data are expressed as means ± SEM. * P < 0.05 versus control group and $$ P < 0.01 versus CH-GTE.

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(A)

Final body weight (g)

250

**

200 150 100 50 0 Control

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% reduction in body weight relative to control group

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8 6 4 2 0 CH-GTE

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Figure 3. Effects of oral administration of tea extracts on body weight. A: final body weight and B: % reduction in body weight in rats. Data are expressed as means ± SEM. Significantly different from the value in the control group: ** P < 0.01 versus control group and $ P < 0.05 versus CH-GTE.

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Effect of Tea Extracts on Food Intake, Water Intake and Body Weight Gain in Rats The animal protocol was approved by the Massey University Animal Ethics Committee. All rats consumed their diets as expected and demonstrated acceptable weight gains. Before gavaging, there was no difference between rat groups in food intake. The results of this experiment are shown in Figures 2-4. There was no difference between the rats gavaged with water and those gavaged with CH-GTE in the daily food intake during the whole experimental period. During the first day, there was no significant in food intake difference between the rats gavaged with water and those gavaged with Se-GTE. However, the food intake in rats gavaged with Se-GTE extract for 2 (13.9 ± 0.8 g/day in control vs. 11.37 ± 0.98 g/day in rats gavaged with Se-GTE; P = 0.0194), 3 (12.9 ± 0.6 g/day in control vs. 10.38 ± 0.34 g/day in rats gavaged with Se-GTE; P = 0.0204) and 4 (13.23 ± 0.22 g in control vs. 11.48 ± 0.39 g in rats gavaged with Se-GTE; P = 0.0300) days was significantly lower than their counterparts in the control group (Figure 2A). In general, rats gavaged with CH-GTE and Se-GTE consumed 4.9% and 13.8% less food than their counterparts in the control group, respectively (Figure 2B). The final body weight of rats gavaged with Se-GTE extract was significantly lower (225.2 ± 3.7 g in controls vs. 206.0 ± 5.9 g in rats gavaged with tea; P = 0.0003) than those in the control group (Figure 2A). This is corresponding to 8.53 % reduction in body weight (Figure 3B). In contrast, no significant difference in final body weight was detected between control rats and those gavaged with CH-GTE [225.2 ± 3.7 g in controls vs. 221.1 ± 1.25 g in rats gavaged with tea; P= 0.0063; Figure 3A]. Again, Se-GTE was more effective (P = 0.0378) than CH-GTE at reducing the body weight gain (Figure 3B). Overall, water intake in rats preloaded with CH-GTE or Se-GTE was not significantly different when compared with the rats given water only (Data not shown).

In Vivo Antioxidant Status Figure 4 shows the differences in serum FRAP between rats gavaged with CH-GTE, SeGTE and rats gavaged with water only. The FRAP value, an indicator of total antioxidant defence, was significantly affected by gavaging with teas, especially Se-GTE [750.3 ± 65 µmol FeII/L in controls vs. 834.3 ± 29.8 in rats gavaged with CH-GTE (P > 0.05) and 1237.6 ± 84.9 µmol FeII/L in rats gavaged with Se-GTE; P = 0.0002]. In addition, the FRAP value of the serum in rats gavaged with Se-GTE was significantly higher (P = 0.0008) than that in rats gavaged with CH-GTE.

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$

Serum FRAP level (micromol/L)

1400

* *

1200 1000 800 600 400 200 0 Control

CH-GTE

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(B) % increase in serum antioxidant level

100

$$

80 60 40 20 0 CH-GTE

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Figure 4. Effect of oral administration of water-soluble extracts from two teas CH-GTE and Se-GTE) on antioxidant status (FRAP values; micromole/L) in sera collected from the rats 4 days after gavaging. A: antioxidant activity and B: % increase in antioxidant activity relative to control group. Data are expressed as means ± SEM. ** P < 0.01 versus control group. $ P < 0.05 and $$ P < 0.01 versus CHGTE.

DISCUSSION Both teas had a satiating influence on experimental rats, as evidenced by their ability to decrease food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE), although a statistically significant decrease over the control rats was achieved only for Se-GTE treatment. The reduction in food intake, observed over four hours in rats given a preload of Se-GTE in

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comparison to rats in a control group given the same preload volume of water, has led to the conclusion that the decrease in food intake was mainly the consequence of a satiating effect of the tea rather than simply a stomach distension effect. It is well known that food intake is regulated by a variety of peripheral factors and by central neuroendocrine systems. Various hormones, including cholecystokinin, glucagon-like polypeptide-1, glucagon, somatostatin, and bombesin, have been reported to affect food intake (Morley, 1987; Kalra et al., 1999). Although the precise mechanisms which underlie the satiating effects of Se-GTE are not fully understood, it may trigger receptors for amino acids which have been detected in the wall of the upper intestine (Mei, 1992). The afferent fibers from these receptors may inform certain brain centres that a source of energy and/or a specific nutrient has been ingested. Alternatively, such receptors may play a role in inducing satiety by triggering the release of hormones such as cholecystokinin (CCK), which might act directly at the central and/or peripheral levels to stimulate pancreatic juices and reduce gastric emptying. Kao et al. (2000) found that epigallocatechin gallate (EGCG), but not related catechins, significantly reduced food intake; body weight; blood levels of testosterone, estradiol, leptin, insulin, insulin-like growth factor I, glucose, cholesterol, and triglyceride in male rats. The authors observed similar effects in lean and obese male Zucker rats and suggested that the effect of EGCG was independent of an intact leptin receptor. The authors concluded that EGCG may interact specifically with a component of a leptin-independent appetite control pathway. Further study is required to determine the mechanism by which SeGTE suppresses the food intake. In addition, the final body weight of rats gavaged with Se-GTE decreased significantly when compared with the water-gavaged control rats and this corresponds to 8.53% reduction in body weight relative to the control group. In contrast, rats gavaged with CH-GTE showed only 1.8% reduction in the final body weight relative to the control group. The reduction in body weight may be due to the reduction in food intake or to other factors. Choo (2003) reported that in Sprague-Dawley rats fed a high diet, consumption of a water extract of green tea for 2 weeks resulted in decreased body fat accumulation. The author concluded that green tea reduces body fat and increases energy expenditure, which is partially mediated via betaadreoreceptor activation. Several animal studies have shown a beneficial effect of long-term feeding of green tea catechins on the development of obesity (Murase et al., 2002; Choo, 2003; Klaus et al., 2005; Kao et al., 2006). In some of these studies, it was concluded that this suppressive effect was resulted mainly from increase in brown adipose tissue thermogenesis through betaadrenoceptor activation while in other studies, the anti-obesity action was attributed to the ability of green tea to modulate the glucose uptake system in adipose tissue and skeletal muscle and to suppress the expression and/or activation of adipogenesis-related transcription factors. The gavaging technique guaranteed that the rats received orally a define amount of load and prevented several technical problems. In the present study, the load was delivered by gavage, which eliminated the role of the orosensory cues in the feeding response. The physiological response may differ if the orosensory factors are also taken into account (Taha and Fields, 2005). The time period in which rats had access to food was restricted to 4h/day in an attempt to provide a more controlled and exaggerated appetite response at feeding (Froetschel et al., 2001). This time interval set for meal-feeding was considered to be the minimal time that

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would still allow the rats to consume enough food and grow comparably to those with 24-h access to food (Froetschel et al., 2001). The ability of Se-GTE to reduce the food intake coupled with the decrease in body weight gain compared with their counterparts given water (control group), suggests that this tea may be a good satiety inducer and weight management modulator. Recently St-Onge (2005) reported that the inclusion of foods or the replacement of habitual foods with others that may enhance energy expenditure (EE) or improve satiety may be a practical way to maintain a stable body weight or to assist in achieving weight loss; such foods may act as functional foods in body weight control. Se-GTE had significantly higher TPC, higher FRAP, higher DPPH free radical scavenging activity, higher ferrous-ion chelating activity, and higher selenium contents than CH-GTE. A strong positive correlation was found between the TPC, and the FRAP, DPPH, and the ferrous-ion chelating activities in both teas, indicating that the polyphenolic compounds are the major contributors of the antioxidant/antiradical activities. This is consistent with previous studies which have shown that polyphenols are major contributors of the antioxidant activity (Khokhar and Magnusdottir, 2002; Schotsmans et al., 2007; Molan et al., 2008b). The results of this study also demonstrated that consumption of both teas increased the antioxidant capacity of serum in rats and Se-GTE was significantly more effective than CHGTE in this respect. Significant increase in FRAP was observed in rats gavaged with Se-GTE compared to the control rats gavaged with the same volume of water. The increased antioxidant capacity in serum (FRAP) following the oral gavage of Se-GTE indicated a direct absorption and/or an enhanced production of antioxidants. The antioxidants responsible for the increased antioxidant capacity following the consumption of Se-GTE are likely phenolic compounds and selenium. Green tea leaves contain 10–30% (dry weight, DW) of polyphenols, including catechins, flavonols, flavanones, phenolic acids, glycosides and the aglycones of plant pigments (Pan et al., 2003). In green tea, the major flavonoids present include catechin (flavan-3-ol) such as epicatechin, epicatechin-3-gallate, epigallocatechin and epigallocatechin-3-gallate (EGCG; Balentine et al. 1997). It has been reported that tea leaves are the only food products containing EGCG (Graham, 1992; Chu and Juneja, 1997). In addition to phenolic compounds, tea leaves contain vitamins, several minerals, caffeine and anthocyanidins, aluminum, potassium, fluoride and manganese (Chao et al., 1995). Other studies have shown that polyphenolic compounds in green tea extracts have been found to be potent antioxidants and free-radical scavengers due to the ability to act as hydrogen or electron donors (Higdon and Frei, 2003; Molan et al., 2008a). There is a possibility that the presence of selenium in the green tea sample produced a synergistic effect with the green tea resulting in the significantly increased in the antioxidant activity and other biological activities observed in the present study. Indeed other studies regarding selenium and green tea have also reported on a synergistic effect between selenium and green tea. Hu et al., (2000) studied the physiological function of selenium (Se)-enriched tea fertilized with sodium selenite and naturally high-Se tea in rats and found that the selenium biological utilization rates were 65.41, 68.05 and 70.49% for sodium selenite, Se-enriched tea and naturally high-Se tea, respectively. In another study, the effect of foliar application of Se on increasing the antioxidant activity of tea was investigated (Xu et al., 2003) and the results showed that the radical scavenging ability of the tea extracts were in the following order: Seenriched tea obtained by fertilization with selenate, Se-enriched tea obtained by fertilization

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with selenite and then regular tea. The higher antioxidant activity shown by Se-GTE over that of regular CH-GTE may be attributed to the synergistic effect of tea polyphenols and selenium. Amantana et al. (2002) compared the antimutagenic activities of regular green tea and selenium-enriched green tea obtained by foliar application of selenite toward the hetrocyclic amine 2-amino-3-methylimidazol [4,5-f] quinoline (IQ) in the Salmonella assay. The authors found that selenium-enriched green tea exhibited concentration-dependent inhibition of IQ-induced mutagenesis and was significantly more effective than regular green tea tested under the same conditions. They suggested an enhancing (coantimutagenic) effect of selenium in combination (synergism) with green tea in vitro. More recently, Li et al. (2008) found that enrichement of selenium endowed green tea with higher antioxidant and antitumor activity. Certainly, the presence of selenium in the diet is of major importance because selenium deficiency was found to be associated with many defects, such as heart disease, depression, hypothyroidism and a weakened immune system (Zimmerman and Kohrle 2002; Beck et al., 2003). Recent studies have confirmed the fact that supplementation of selenium from natural food materials in the form of organic selenium is safe and suitable as compared with consumption of inorganic selenium (Hu et al., 2002). Decreased serum antioxidant status has been suggested as a risk factor in cardiovascular disease (Kaplan and Aviram, 1999), and cancer (Ames et al., 1995). On the other hand, increasing the serum antioxidant status has been suggested as a possible method of reducing the risk of many chronic degenerative disorders (Kaplan and Aviram, 1999; Georgopoulos, 1999; Vendemiale et al., 1999).

CONCLUSIONS The current study has shown a reducing effect of Se-GTE pre-meals on subsequent meal intake. The underlying mechanism responsible for this response was not identified as part of this study. Se-GTE showed statistically significant satiety activity as evidenced by its ability to reduce food intake (up to 13 %) when orally gavaged into rats compared with the food intake of control rats given a gavage of water only. The final body weight of the rats given Se-GTE was significantly lower than that observed in the control groups. In addition, administration of Se-GTE was associated with a significant increase in serum antioxidant status above the control group for the FRAP assay. The ability of Se-GTE to reduce the food intake coupled with the decrease in body weight gain compared with their counterparts given water (control group), suggests that this tea may be a good satiety inducer and weight management modulator.

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Pan, X., Niu, G. and Liu, H. (2003). Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chem. Eng. Process, 42:129–133. Paveto, C., Guida, M.C., Esteva, M.I., Martino, V., Coussio, J., Flawia, M.M. and Torres, H.N. (2004). Anti-trypanosoma cruzi activity of green tea (Camellia sinensis) catechins. Antimicro. Agents Chemother., 48: 69-74. Peterson, J. and Dwyer, J. (1998). Flavonoids: dietary occurrence and biochemical activity. Nutr. Res., 18: 1995-2018. Pillai, S.P., Mitscher, L.A., Menon, S.R., Pillai. C.A., Shankel, D.M. (1999). Antimutagenic/antioxidant activity of green tea components and related compounds. J. Enviro. Pathol.. Toxicol. Oncol., 18:147-58. Reeves, P. G., Nielsen, F. H. and Fahey, G. C. (1993). AIN93 purified diets for laboratory rodents: final report to the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr., 123: 1939-1951. Rippe, J. (1998). Obesity as a chronic disease: modern medical and lifestyle management. J. Am. Diet. Assoc., 98: S9-S15. Sakanaka, S., Junelia, LR, and Taniguchi, M. (2000). Antimicrobial effects of green tea polyphenols on thermophilic spore-forming bacteria. J. Biosci. Bioeng., 90: 81-85. Schotsmans, W., Molan, A. L. and MacKay, B. (2007). Controlled atmosphere storage of rabbiteye blueberries enhances postharvest quality aspects. Posthar. Biol. Tec., 44: 277285. Sheard, N.F. (2003). Moderate changes in weight and physical activity can prevent or delay the development of type 2 diabetes mellitus in susceptible individuals. Nutr. Rev., 61: 7679. Shore, S.A. and Johanson, R.A. (2005). Obesity and asthma. Pharmacology and Therapeutics (online article). Singh, N. and Rajini, P.S. (2004). Free radical scavenging activity of an aqueous extract of potato peel. Food Chem., 85, 611–616. St-Onge, M. P. (2005). Dietary fats, teas, dairy, and nuts: potential functional foods for weight control? Am. J. Clin. Nutr., 81:7-15. Taha, S.A. and Fields, H.L. (2005). Encoding of palatability and appetitive behaviors by distinct neuronal populations in the nucleus accumbens. J. Neurosci., 25: 1193-1202. Tan, EK, Tan, C, Fook-Chong, S, Lum SY,Chai, A, Chung, H, Shen, H, Zhao, Y, Teoh, ML, Yih, Y, Pavani, R, Chandran, VR, Wong, MC. (2003). Dose-dependent protective effect of coffee, tea and smoking in Parkinnson’s disease: a study in ethnic Chinese. J. Neurol. Sci., 216:163-167. Tavani, A., Pregnolato, A., La Vecchia, C., Favero, A. and Franceschi, S. (1998). Coffee consumption and the risk of breast cancer. Eur. J. Cancer Prev., 7:77-82. Thomson, C.D. and Robinson, M.F. (1996). The changing selenium status of New Zealand residents. Eur. J. Clin. Nutr., 50: 107-114. Tinggi, U. (2003). Essentiality and toxicity of selenium and its status in Australia: a review. Toxicol. Lett., 137: 103-110. Toyran, N., Severcan, F., Severcan, M. and Turan, B. (2008). Effects of selenium supplementation on rat heart apex and right ventricle mycardia by using FTIR spectroscopy: A cluster analysis and neural network approach. Food Chem., 110: 590597.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 7

ANTI-OBESITY EFFECTS OF (-)-EPIGALLOCATHCHIN3-GALLATE AND ITS MOLECULAR MECHANISM Cheol-Heui Yun1, Gi Rak Kim1, Min Ji Seo1, Hyun-Seuk Moon2 and Chong-Su Cho1,* 1

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea; 2 Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-9410, USA

ABSTRACT Development of obesity appears to be influenced by a complex array of genetic, metabolic, and neural frameworks, together with one’s behavior, eating habit, and physical activity. The incidence of obesity is significantly increasing in virtually all societies of the world and causes important pathological consequences such as cardiovascular diseases and type 2 diabetes mellitus. Furthermore, rates of pediatric obesity have increased dramatically over the past decade resulting cardiovascular, metabolic, and hepatic complications. Since ancient times, green tea has been considered as a traditional medicine as a healthful beverage. Major components of green tea including epigallocathchin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) have been received significant scientific attention and public awareness for its beneficial effects on prevention and therapeutic treatment of cardiovascular diseases and cancer. Anti-obesity effects of EGCG have been demonstrated in various in vitro and in vivo models showing that EGCG treatment could reduce food intake, glucose uptake, blood glucose level, and differentiation and growth activity of fat cells together with modulation of lipolytic and lipogenic activities. In these actions, large number of molecules including laminin receptor, fatty acid synthase, glucose-6-phosphate dehydrogenase, cyclin-dependent kinase 2, CCAAT/enhancer binding proteins, mitogen activated protein kinases, AMP-activated protein kinase, phosphatidylinositol-3-kinase, *

To whom correspondence should be addressed. Tel: +82-2-880-4636; Fax: +82-2-875-2494; E-mail: [email protected]

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1. INTRODUCTION Green tea is made differently during the manufacture processing compared with oolong tea, black tea, and pu-erh tea, which allows the fresh tea leaves to wither for decrease of their moisture content, sequentially fermenting polyphenols. This fermentation makes polymerization of monomeric catechins, which leads to the major formation of flavins, namely theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3’-gallate (TF-2b), and theaflavin-3,3’-digallate (TF-3), and thearubigins, consequently decreasing catechin content [1, 2]. On the contrary, green tea leaves are processed under steam followed by partial withering to prevent fermentation. The other important aspect of process is drying and rolling, which allows yielding a dry and stable product [3]. Therefore green tea generally posses higher content of catechins. The catechins are the main compounds in green tea, and are polyphenolic flavonoids as phenol derivates. The four major catechins are (-)epigallocatechin-3-gallate (EGCG), which represents approximately 59% of the total contents; (-)-epigallocatechin (EGC) (approximately 19%); (-)-epicatechin-3-gallate (ECG) (approximately 13.6%); and (-)-epicatechin (EC) (approximately 6.4%) [4]. EGCG, the richest catechin in green tea, has a large number of positive health effects in the prevention of lifestyle-related diseases including cardiovascular disease, carcinogenesis and obesity. Obesity, characterized by increased number and size of fat cells due to the processes of so-called mitogenesis and differentiation, has increased at an alarming rate in recent years and it is obvious that excess body weight is now a critical health concern worldwide. Indeed obesity became a common disease associated with risks of cancer, type 2 diabetes, hypertension, and cardiovascular disease [5]. The prevalence of obesity is reaching epidemic proportions in many Western countries. That is the reason why development of drugs to treat obesity or implementation of a dietary regimen to prevent obesity becomes a public awareness and health goal. Obesity arises from the imbalance between energy intake and its expenditure. These processes are regulated by genetic, endocrine, metabolic, neurological, pharmacological, environmental, and nutritional factors [5, 6]. According to the requirements for treating obesity, EGCG has been focused on its physiological effects. Simply, EGCG can reduce body weight and body fat. This contention was further supported by experimental data where the EGCG or EGCG-containing green tea extract not only reduces food uptake, lipid absorption, blood triglyceride, cholesterol, and leptin levels but also stimulates energy expenditure, fat oxidation and regulates various enzymes related to lipid anabolism and catabolism [7-10]. Moreover, EGCG appears to modulate the mitogenic, endocrine, and metabolic functions of fat cells [11]. Accordingly, a thorough examination of the signal element through which EGCG executes modulation of preadipocyte mitogenesis will certainly help in the prevention and control of the development of obesity. The mechanisms of action of EGCG seem to involve several but, certain pathways including (1) the decrease in the energy intake, (2) the increase in energy expenditure, and (3)

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the alterations in the activities of fat, liver, muscle, and intestinal cells. In this review, we focus on the signal transduction and mechanisms which induce anti-obese effects caused by EGCG.

2. EGCG RECEPTORS The first step for the ligand to initiate cellular function in many cases, if not all, is to interact with appropriate receptor(s). Recently the receptor for EGCG has been identified. It is known as laminin receptor (LR), approximately 67 kDa accessory integrin protein, and was first isolated from cancer cells [12]. The Kd value for the binding affinity of EGCG to LR appears to be about 40 nM [13]. As expected, EGCG competes with laminin to bind LR [13]. It has been suggested that the LR is in association with a lipid raft of the plasma membrane for increasing suppressive effect of EGCG on IgE receptor of B-cells [14]. Through this, the possible interaction relationship between the EGCG receptor and other types of receptor in particular cells has been studied. It is to note that the LR is not only found on cancer cells, but normal cells, such as muscle cells, macrophages, neutrophils, endothelial cells, epithelial cells, hepatocytes, interstitial cells, and neuronal cells [15]. On these normal cells, the LR may also serve as an EGCG receptor to regulate the effects of EGCG. Basically, LR is a heterodimeric structure, a member of the integrin family of cell adhesion receptors, and occurs in many types on the membrane of numbers of different normal cells [13, 16, 17], such as integrin α5 and α6 in murine adipocytes [18]. It has been clearly shown that certain types of LRs and laminin are present in preadipocytes and adipocytes, which relate to their activity. Activities of fat cells are regulated by their receptors on the membrane or in the cells. Thus, EGCG or green tea may have an effect on fat cells or other types of cells through the changes in the activation and expression of these receptors. EGCG can alter the activities or expressions of other membrane and nuclear receptors. For example, EGCG can displace the bound estrogen receptors α and β, each encoded by a separate gene, ESR1 and ESR2, respectively, to 17b-estradiol [19]. To note that the estrogen receptor plays a major role as a DNA binding transcription factor and regulates gene expression. It has been reported that tyrosine phosphorylation of the insulin receptor in rat hepatoma cells and of IGF-I receptor in HepG2 cells was increased when treated with EGCG [20]. Such effect of EGCG on insulin receptor and IGF-I receptor appears to be cell type-dependent. EGCG down-regulates the expression of the androgen receptor in LNCaP prostate cancer cells [21]. It is important to note that MCF-7 and LNCaP cancer cells are fat-synthesizing cell lines. In addition, EGCG can directly and indirectly inhibit the activation or gene expression of almost all receptors for growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) receptors, and human epidermal growth factor receptor 2 (HER2/neu, also known as ErbB-2). It has been suggested that EGCG can stimulate the apoptotic receptor CD95 (FAS/APO-1) in cancer cells [22, 23], suggesting that EGCG potentially induce apoptosis through activating CD95 expressed on certain cancer cells. Interestingly, some of these receptors are known to be expressed in or on adipocytes and other normal cell types that are involved in the regulation of adipose tissues [24].

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3. SIGNALING PATHWAY Cells are equipped with a membrane that separates between inner and outer environment. However, they are able to respond to extracellular stimuli such as mitogens and hormones, and convert their signals into cellular processes. This conversion is usually mediated by the binding of designated extracellular ligands to specific transmembrane receptors, which are consequently activated to further transmit the signals of the different ligands through intracellular signaling pathways. These pathways, operating within complex networks of interacting or counteracting proteins, transmit the signals to various intracellular targets, thereby regulating inducible cellular processes such as transcription, translation, proliferation, differentiation and apoptosis. Cells receive constantly clues from their environment via activation of surface receptors and extracellular matrix.

3.1. ERK PATHWAY The MAPK family is an essential part of the signal transduction machinery in signal transmissions by external stimuli, and contains three major MAPK subfamilies: Extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) [25, 26]. ERKs are composed of two similar (85% sequence identity) protein kinases, originally called ERK1 and ERK2. ERK1/2 signaling cascade is one of the key signaling pathways, which are involved in functions including the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells [27]. A large variety of extracellular ligands can stimulate this ERK signaling cascade and, as a consequence, the ERK1/2 signaling cascade regulates many distinct and even opposing cellular processes. As the ERK1/2 pathway is responsive primarily to mitogens and growth factors, and plays key roles in cell proliferation, survival, and differentiation, this section will focus mainly on the action mechanism of ERK1/2 pathway in relation to (pre)adipocytes. Obesity arises from an imbalance in energy intake and energy expenditure that leads to the pathological growth of adipocytes. Obesity is known to be induced by the hypertrophy of adipocytes and the generation of new adipocytes from precursor cells [28]. Both two processes are dependent on the regulation of adipocyte differentiation. The adipocyte differentiation process at the beginning requires a precise proliferative step called mitotic clonal expansion (MCE), which is initiated by adipogenic stimuli, such as insulin and known to activate the ERK pathway. During MCE, a complete inhibition of ERK pathway are required to induce complete differentiation [29], suggesting a positive role for ERK in adipocyte differentiation in relation to the expression of the crucial adipogenic regulators, C/EBP α, β, γ and , and PPARγ. It has been proposed that EGCG could serve as a signal element to regulate cell growth [30-33] and modulate the mitogenic and adipogenic signaling in preadipocytes [34-36]. EGCG induced a decrease in phosphorylated ERK1/2 in 3T3-L1 preadipocytes but did not alter the total levels of MEK1, ERK-1, ERK-2, p38, phospho-p38, JNK, or phospho-JNK [37], suggesting that EGCG acts specifically on the ERK. This contention is also partially supported by the fact that exposure to EGCG induced a down-regulation in the phosphorylated ERK1/2 of preadipocytes without altering total levels of MEK1 and ERK1/2

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proteins [38]. In addition, transient amplification of phospho-ERK1/2 content by transfecting wild type of MEK1 cDNA or its constitutively active mutant cDNA to 3T3-L1 preadipocytes prevented EGCG-induced decreases in their cell numbers. Taken all together, these findings demonstrate that a suppressive effect of EGCG on the proliferation of preadipocytes is likely mediated via ERK, but not p38 and JNK, MAPK-dependent pathway and is an important factor in preadipocyte proliferation [38]. The ERK-dependent effect of EGCG observed in 3T3-L1 preadipocytes was further confirmed that two specific inhibitors of ERK MAPK, PD98059 and U0126, alone can inhibit cell growth and MEK1 activity [38]. It is intriguing to note that EGCG appears to work differently from PD98059 and U0126 in reducing levels of phosphorylated ERK1/2 proteins. Since PD98059 is known to prevent MEK1 activation by Raf while U0126 directly protects ERK from being phosphorylated by MEK1, one might assume that the target molecule of EGCG could be upstream of Erk pathway. It is possible that EGCG induces a decrease in phosphorylated ERK1/2 from preadipocytes via reducing the phosphorylation of MEK1 in association with Raf as reported for H-Ras-transformed cells [39]. In cultured cells, ERK1/2 are phosphorylated under the regulation of a variety of factors, including growth factors, G protein-coupled receptors, tyrosine kinase receptors, and Raf and MEK1 kinases [27, 40]. It has been shown that EGCG significantly prevented the increase of phosphorylated ERK1/2 by either IGF-I or IGF-II, or concomitantly reduced IGF-I receptor activity [38, 41]. The study further proved that EGCG caused a decrease in the phosphotyrosine-IGF-I receptor and an association of the IGF-II receptor with Giα-2 protein [42-44]. Although the Erk-mediated EGCG effects are obvious, more detailed and thorough studies are needed to confirm the precise target and its associated mechanisms, such as PI3KAkt-Bad/phosphorylated Bad pathway.

3.2. CDK2 PATHWAY Cyclin-dependent kinases (Cdks) are known to play a role in the regulation of the eukaryotic cell cycle and therefore, it has been implicated in the control of gene transcription, responsible for cell division [45, 46]. The Cdks are proteins of 34~40kDa possessing Ser/Thrspecific protein kinase activity. Cdk catalytic subunits do not act alone and the way they trigger cell cycle events is completely dependent on associated cyclin subunits [47]. In mammals, there are at least 10 different Cdks, Cdk1 to Cdk10. Progress of the cell cycle appears to be controlled mainly by Cdk1 and Cdks2, 4 and 6. The role of Cdk2 in mediating the cell-cycle inhibitory and tumor-suppressing activities of p21Cip1 and p27Kip1 appears to be dispensable [47-49], which could be explained by compensatory activities of other kinases, most likely Cdk1 activity through its association with Cyclin A, a partner of Cdk2. It has been suggested that EGCG modulates cell mitogenesis and growth arrest of most cancer cells via Cdk pathway [38, 50, 51] and can serve as the main controller of mitogenesis and mitotic clonal expansion of preadipocytes [52]. In fact, EGCG appears to down-regulate adipocyte differentiation through a specific type of Cdks, for instance Cdk2 signaling pathway in preadipocytes [38]. This was further confirmed by transfection study that the decrease of Cdk2 activity together with the increase of G1 arrest induced by EGCG in preadipocyte were inhibited by the transfection of Cdk2 [34, 38]. Furthermore the transfection

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of dnCdk2 to preadipocytes slowed down the growth of preadipocytes, as expected. These results demonstrated that the effect of EGCG-induced preadipocyte anti-mitogenesis and growth arrest is indeed dependent on a Cdk2 pathway and requires inactivation of the Cdk2 protein. The other hand, cyclin D1 is a G1 cyclin associated with Cdk4 and Cdk6 proteins, which are required for cell cycle G1/S transition, and therefore, cyclin D1 protein expression by EGCG [53, 54], suggesting the possibility of Cdk4- and Cdk6-related effects of EGCG on preadipocyte growth arrest. However, the effect of EGCG on the reduction of cyclin D1 via Cdk4 and/or Cdk6 has yet to be further confirmed. Decrease in the expression of cell division cycle 2 (Cdc2) protein by EGCG [50, 51] suggests the possibility of the action of EGCG on the G2/M phase of preadipocytes since Cdc2 serves for the predominant Cdk in the early G2/M transition of the cell cycle [33]. Regulation of Cdk2 activity is regulated at multiple levels including the synthesis of subunits and the association of inhibitory proteins p21 and p27 [38, 51]. Therefore all these observations suggest that EGCG is responsible for the increased association of Cdk2 with p21 and p27, thereby leading to low Cdk2 activity and a subsequent in the percentage of G0/G1 arrest. It is also possible that EGCG acts on the CDK inhibitor (CKI) family to reduce Cdk2 activity in the particular type of preadipocyte. It would be of interest if any of CKIs, for instance p53 [54], is involved in the action of EGCG-mediated growth arrest in preadipocytes. Decreases in cyclin D1 protein expression of preadipocytes by EGCG could be also involved in the association of Cdk2 with p21 and p27, since the sequestration of p21 and p27 is mediated through the increase of synthesis rates of the induction of cyclin D1 and cyclin D2 protein [51]. Further studies to determine whether EGCG affects the association of cyclin D1 together with CKIs would help clarify this notion. In smooth muscle cells, increased Cdk2 activity by endothelin is mediated via either the activation of Erk and Cdc25A (a phosphatase) or the inactivation of WEE1 (an inhibitory kinase) but is prevented by the inactivation of ERK and the activation of WEE1, whereas Cdk2 activity is restored through dephosphorylation at Tyr15 by Cdc25A [55]. Therefore, activities of WEE1 and Cdc25A are, respectively, inactivated and activated through phosphorylation by the activation of ERK, and vice versa [38]. It is also probable that the reduction in Cdk2 activity by EGCG in 3T3-L1 preadipocytes could be mediated by the inactivation of ERK. Because Cdk2 activity is also regulated by the association of stimulatory proteins, such as cyclin E [56, 57], further investigations are needed to clarify whether EGCG can change the production of cyclin E protein in association with Cdk2. It has been suggested that EGCG may result in altered cyclin-Cdk2 protein complexes in 3T3-L1 preadipocytes [29] as reported in mouse liver cells [55]. These observations together with the results mentioned in the previous sections suggested that the anti-mitogenic effect of EGCG on 3T3-L1 preadipocytes could be dependent on the MAPK and CDK pathways and is likely mediated through decreases in their activities. Overall, the anti-adipogenic effect of EGCG is important not only in reducing adipocyte differentiation but also in inhibiting adipocyte proliferation, suggesting that two cell cycle control kinases, ERK and cyclin-dependent kinase (CDK), are required for these inhibitory effects [58]. Future studies on characterizing its oxidative stress induced by interaction of EGCG and its receptor are also needed to elucidate the mechanisms of how EGCG signals reduce the activities of MEK and CDK proteins.

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3.3. AMP-ACTIVATED PROTEIN KINASE (AMPK) AMP-activated protein kinase (AMPK), a metabolite-sensing protein kinase, is known to play a major role in energy homeostasis by coordinating a number of adaptive responses in ATP-depleting metabolic status such as ischemia/reperfusion, hypoxia, heat shock, oxidative stress, and/or exercise [59-61]. Thus, it is certain that AMPK is sensitively regulated by the allosteric binding of AMP under not only physiological but also pathological conditions when ATPs are depleted [61, 62]. The prolonged and persistent activation of AMPK has a link to p53-dependent cellular senescence, suggesting its role as an intrinsic regulator of the cell cycle in mammalian cells [62, 63]. It has been suggested that AMPK cascades have emerged as novel targets for the treatment of obesity and type 2 diabetes [60, 64, 65]. AMPK is also known to be activated by aminoimidazole-4-carboxamide riboside (AICAR), which is converted to a nucleotide that mimics the effect of AMP. Furthermore, the pro-apoptotic potential of the activated AMPK was observed under the AMPK over-expressed conditions in the various cells [66, 67], which was confirmed by long-term treatment with AICAR has prevented development of diabetes in animal models [68]. EGCG treatment inducing the activation of AMPK caused inhibition of adipocyte differentiation in 3T3-L1 cells [35], suggesting that AMPK activation is necessary for the inhibition of adipocyte differentiation and anti-obesity effects by EGCG. Therefore, the mechanism that affects AMPK regulation with physiological stimuli or anti-obesity agents, for instances activation of ROS and/or using naturally occurring compounds like EGCG treatment, appears as a promising target for the development of strategies for the treatment of obesity [35, 69]. Generally, reactive oxygen species (ROS) have been suggested as upstream molecules of AMPK-activated signals since AMPK cascades respond to the intracellular level of AMP and to the AMP:ATP ratio, which is highly sensitive to oxidative stress [66], and therefore one of the major AMPK activation mechanisms was suspected to be ROS. It is not surprising that various therapeutic effects involving the release of ROS have been reported [70-75]. Overall, the anti-proliferatory and lipolytic effects of EGCG have been attributed to their ability to modulate various signaling pathways, specifically those that control the differentiation and cell proliferation of (pre)adipocytes.

3.4. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS) The peroxisome proliferator-activated receptor (PPAR) subfamily of nuclear receptors controls many different number of target genes involved in both lipid metabolism and glucose homeostasis [62]. By forming heterodimers with the retinoid X receptor, they regulate the transcription of many genes in a ligand-dependent manner [2]. Three isotypes (α, γ, β/δ) of PPAR have been identified. PPARα is predominantly expressed in tissues with a high rate of fat catabolism (e.g. liver and muscle). A study showed that green tea including cathechins increases PPARα expression in skeletal muscle (but not in liver) and increases PPARδ expression in visceral and subcutaneous adipose tissues. Green tea catechins activated PPARα in cell culture [76] and increased PPARα and PPARγ expression in a mammalian liver [35]. The expression of PPARγ is high in adipose tissue, where it triggers adipocyte differentiation and induces the expression of genes critical for adipogenesis [77-79]. Antioxidants (via

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intracellular redox state) could activate the transcription factors, NF-kB and activator protein1 (AP-1) [80, 81], which further regulate PPAR [82]. EGCG appears to not only promote energy expenditure but also stimulate the oxidation of lipids by the known inhibitory effect on catechol-O-methyltransferase, an enzyme that degrades noradrenaline [83] suggesting that EGCG enhances lipid catabolism perhaps through counteract with the PPARδ agonistinduced lipogenesis. PPARγ is expressed at high levels in white and brown adipose tissues and to a lesser extent in macrophages, colon, kidney, and liver [78, 79, 84], where it plays a major role in many different biological processes including adipogenesis, glucose homeostasis, atherogenesis, inflammation, and tumor susceptibility [4]. In general, the enforced expression of PPARγ and C/EBP stimulates adipogenesis, suggesting the essential roles of these transcription factors in regulating adipogenesis [77, 84]. EGCG-induced down-regulation of PPARγ expression appeared to function similarly to the negative effects of EGCG on lipid accumulation and on adipocyte differentiation [35]. The combined expression of PPARγ and C/EBP have synergistic effects in promoting fat cell conversion in myoblasts [85], indicating that these genes must be involved in the fat accumulation. Both transcription factors, PPARγ and C/EBP, coordinate the expression of genes including aP2 involved in creating and maintaining the adipocyte phenotype [86, 87]. The aP2 is a member of the intracellular lipid binding protein family causing direct modification of cholesterol trafficking and inflammatory responses [88, 89]. In fact, it has been demonstrated that fat cells from insulin-resistant individuals display decreased expression of PPARγ target genes, for instance aP2, and increased lipid partitioning together with a subsequent enlargement of the existing adipose cells [90, 91]. PPARγ2 and C/EBP, almost exclusively found in the adipose tissues, are linked to the adipocyte differentiation [86, 92], indicating that they are most likely playing a crucial role both in the expression of adiposespecific genes and in the manifestation of the mature adipose phenotype. It has been demonstrated that the negative impact of EGCG on adipogenesis was accompanied by the reduction of PPARγ2 protein together with the attenuation of C/EBP expression, indicating that EGCG down-regulated the expression of maker genes during adipocyte differentiation [91]. Binding of PPARγ2 to two specific sites in the calreticulin gene was found to stimulate the expression of calreticulin, the major Ca2+-binding protein of the endoplasmic reticulum lumen. In addition, PPARγ2 was found to be down-regulated in cells over-expressing calreticulin [93, 94]. Therefore, there appears to be a negative feedback loop in which PPARγ2 stimulates the expression of calreticulin, which, in turn, inhibits the activity and expression of PPARγ2. It is to note that an increase in the cellular Ca2+ level induced activation of receptors or channels [95] has been shown to inhibit the differentiation of preadipocytes. A recent study [93] demonstrated that this process is repressed by increasing intracellular Ca2+ and is dependent on the expression of calreticulin. Taken all these reports together, although we have gained much insight on the action of EGCG on adipocytes, further studies are needed to ensure certain in-depth understanding on the mechanism of EGCG’s action in the regulation of mitogenesis of preadipocytes especially in relation to activation of PPARs, C/EBP and aP2, and/or regulation of Ca2+ pool.

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3.5. P53 AND NF-ΚB Human prostate cancer cells, LNCaP, are fat-synthesizing cell lines and therefore, it could be worthwhile to look into the effect of EGCG in relation to action mechanism of LNCaP cells. The p53 is the most frequently altered tumor suppressor in human malignancies. Indeed more than 50% of solid tumors having a loss of normal p53 expression due to deletion or point mutation [96, 97]. It is also responsible for cell cycle arrest upon DNA damage [98], which is also a key regulator of apoptosis. The p53-dependent apoptotic response is well-documented as an anticancer mechanism [99]. It has been suggested that EGCG could be potentially important cancer chemopreventive agent because of its ability to selectively induce apoptosis in cancer, but not in normal, cells. Indeed, EGCG treatment caused a significant decrease in the percentage of viable cell along with a concurrent induction of apoptosis while the percentage of apoptotic cells does not coincide with the proportion of viable cells. This is probable that EGCG not only induces apoptosis but also is responsible for G1 arrest [52]. A study showed that EGCG treatment induces an increase in the cellular levels of p53 in LNCaP cells coincided with the phosphorylation of p53 at serine 6, 15, 20, 37 and 392 [100, 101], suggesting that the difference in cell viability and apoptosis caused by cell cycle arrest can be induced by EGCG.. The importance of p53 in EGCG-mediated apoptosis was revealed in a study using PC-3 cells (null for p53), which are not very sensitive to EGCG-mediated apoptosis compared to LNCaP cells containing wild-type p53 [101]. This notion was confirmed that PC-3 cells became very sensitive to EGCG-mediated G1 arrest and apoptosis when wild-type p53 was introduced into PC-3 cells [102]. EGCG-induced stabilization of p53 caused an increase in its transcriptional activity, thereby resulting in an up-regulation of its downstream target p21/WAF1, which further inhibits Cdk2, 4 and 6 causing cell cycle arrest [55, 103]. This was further supported by several independent studies, which show that targeted over expression of p21/WAF1 increases apoptosis [22, 53]. The exact mechanism by which p21 promotes the apoptosis is not currently clear, but could be related to its ability to interact and possibly regulate the components of DNA repair system [104, 105]. It is important to note that EGCG treatment can induce the increase of another transcriptional target of p53, proapoptotic protein Bax (Figure 1). Indeed, a study showed that EGCG treatment resulted in up-regulation of Bax via p53 and a parallel down-regulation of Bcl-2, which might ultimately initiate the activation of the casapase cascade leading to apoptosis [106]. EGCG effectively decreased NF- B transcriptional activity in LNCaP cells, which may be related to decrease in the nuclear levels of the p65 subunit of NF- B [101]. A number of studies suggest that NF- B activates the expression of anti-apoptotic proteins Bcl-2 and BclxL [107, 108]. EGCG-induced decrease in NF- B transcriptional activity is accompanied by a decrease in the levels of the anti-apoptotic protein Bcl-2. It is to note that genetic approaches using dominant-negative NF- B together with Bcl-2 reporter study will help to understand the possible mechanism. Moreover, a direct role of p53-dependent repression of Bcl-2, which might be independent of NF- B, cannot be ruled out. It is also important to note that EGCG reduced the cell viability of preadipocytes, induced the appearance of DNA fragmentation, and increased the activity of the caspase-3 [50], suggesting that the inhibition

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of cell mitogenesis and induction of apoptosis could be attributable to decrease in the number of preadipocytes by EGCG.

3.6. RESISTIN Resistin, one of the adipokines, was initially shown to be induced during adipogenesis and released in large amounts from white adipose tissue in mice [109, 110]. The release of resistin was increased in obese mice and involved in insulin resistance [111, 112], suggesting that adipocyte-derived resistin is related to obesity and diabetes. Thus, it has been proposed as a biomarker for insulin resistance or of adipose tissue mass [113]. In fact, resistin has an inhibitory effect on insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes [111]. Moreover, the increase in adipocyte number may have caused a rise in local resistin production, inhibiting insulin action on glucose uptake in adipose tissue and, thus, preventing further adipocyte differentiation [114, 115].

Figure 1. Signal transduction pathways mediating EGCG-induced apoptosis and cell cycle arrest (Na and Surh, Molecular Nutrition and Food Research 2006, 50: 152 – 159; reprinted with permission).

It has been shown that intracellular resistin protein significantly decreased after EGCG treatment, while no change was found on the extracellular level. It is probable that EGCG could play a role to modulate the distribution of resistin between the intracellular and extracellular compartments [116]. It has also been reported that EGCG suppressed the expression of resistin, both mRNA and protein, in a dose- and time-dependent manner in 3T3–L1 adipocytes [116]. In particular, resistin mRNA expression in adipose tissues of obese

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humans is higher than that in normal subjects [113, 117]. In humans, the expression of resistin in adipocytes is low compared with that in rodents, but resistin mRNA is readily detectable in circulating mononuclear cells, which suggests that human resistin may be regulated by a different mechanism or may have a different role than that in rodents [118]. Since its discovery, resistin has been found to possess numerous physiological and immunological actions. For instance, resistin regulates fasted blood glucose, causes dyslipidemia, suppresses insulin-stimulated glucose uptake in adipocytes and muscle cells, inhibits dopamine and norepinephrine release in the hypothalamus, and promotes endothelial cell activation, smooth muscle proliferation, inflammatory responses and inflammatory bowel diseases [109, 110, 113, 117, 119]. The expression of resistin gene appears to be regulated by nutritional, endocrine, genetic, pharmacological, immunological and developmental factors, and the action mechanism of resistin is emerging. For example, resistin promotes smooth muscle cell proliferation through the activation of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) [120, 121]. Other study showed that over-expression with MEK1 blocked EGCG-inhibited resistin mRNA expression, suggesting that EGCG could down-regulate the expression of resistin via MEK1-dependent pathway [116]. Further investigations are necessary to clarify the action mode of EGCG in relation to its target molecule(s). As discussed previously, EGCG has been reported as a chemopreventative agent that blocks excessive body weight. Whether EGCG exerts its effects through the control of resistin is unknown and minimal attention has given to this subject so far, although one assumes such effect since resistin is known to cause insulin resistance and inhibit adipocyte differentiation.

4. LIPID METABOLISM In epidemiological studies a significant inverse relationship between tea drinking and plasma cholesterol levels has been reported. The effect of a purified form of EGCG on lipid metabolism has been proven through animal studies to get better understanding of the underlying mechanism. These studies have shown that catechins inhibited cholesterol absorption and lowered plasma cholesterol [122-126]. Chinese green tea and Jasmine tea containing high amount of EGCG more effectively reduced cholesterol levels in rats when compared to other teas with lower EGCG [127]. EGCG, administered by intraperitoneal injection, did not inhibit cholesterol synthesis but oral administration of EGCG decreased cholesterol absorption from rat intestine [124, 128]. These reports suggest that green tea or more specifically EGCG can cause to decrease the cholesterol absorption from the gut. Green tea catechins affect lipid metabolism by several different pathways and prevent the appearance of atherosclerotic plaque. Among several lipid metabolic pathways, absorption of cholesterol is considered to be one of the major pathways. The principal steps in the absorption of cholesterol are emulsification in the stomach, hydrolysis of the ester bond by a specific pancreatic esterase, micellar solubilization (often used as a powerful alternative for dissolving hydrophobic drugs in aqueous environments), and absorption in the proximal jejunum, re-esterification within the intestinal cells, and transport to the lymph by chylomicrons [129, 130]. Due to the insolubility of cholesterol in water, solubilization of cholesterol in mixed micelle is essentially required for its efficient absorption [131, 132]. A

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micelle is polymolecular aggregates that act as a carrier and a solubilizer of cholesterol and other lipids. A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic head regions in contact with surrounding solvent resulted in sequestering the hydrophobic tail regions in the center of micelle. Once solubilized cholesterol is formed, and then transferred from the micelle to the cell membrane of the intestinal brush border [129]. Thus, the action of EGCG that influence cholesterol uptake could interfere with the (1) forming micelle, or (2) affinity either of micelles for membranes or (3) of cholesterol for micelles. These alterations in the intestinal lumen affect hepatic cholesterol metabolism and may affect synthesis and catabolism of lipoproteins as well. The action mechanism of EGCG on the cholesterol absorption was studied on the basis of micelle size and cholesterol solubility. The addition of pure EGCG to mixed micelles decreased the intermicellar cholesterol concentration by 65% [124]. These findings were further supported by a study showing that green tea catechins reduced the cholesterol absorption from the intestine by reducing solubility of cholesterol in mixed micelles [133]. In addition, it was shown that pure EGCG induced an increase in the mean particle size of the micelles [134], suggesting that the change in the micelle size could affect the solubility of cholesterol in micelles and/or the affinity of micelles for membrane. Thus, the hypocholesterolemic activity of EGCG appears to be due to the inhibition of intestinal cholesterol absorption by reducing micellar solubilization of cholesterol. Overall, intake of green tea extracts decreased the absorption of triglycerides and cholesterol [8, 135], and, at the same time, increased a fat excretion [8]. Although number of studies reported that green tea catechins decreased total plasma cholesterol and blood triglyceride levels, the efficacies differ among studies [7, 124, 136-139], probably because of the animal models used and a particular component of catechins, for instance EGCG, were different. Green tea ingestion causes decrease in the LDL cholesterol [140, 141], concomitantly with increase in the HDL cholesterol. The addition of EGCG to the diet significantly lowered total plasma cholesterol and non-HDL cholesterol levels [124]. The ratio of non-HDL: HDL-cholesterol, an indicator of atherosclerosis risk, was also positively affected in the animals fed with a diet containing 1% of EGCG, suggesting that green tea polyphenols exert beneficial effects on the suppression of high-fat diet-induced obesity by modulating lipid metabolism and the risk of associated diseases, including diabetes and coronary diseases. It has been postulated that the absence of change in body composition after EGCG treatment, where it could trigger a shift of fat distribution from visceral to subcutaneous adipose depots.

5. MODULATION OF LIPOLYTIC ENZYMES The lipid substrate is either dispersed as an emulsion in aqueous medium or present as fat droplets, and the enzyme acts at the interface between the lipid and aqueous phases. Lipases are water-soluble enzymes that catalyze the hydrolysis of ester bonds in water–insoluble, lipid substrates. Lipases perform essential roles in the digestion, transport and processing of dietary lipids (e.g., triglycerides, fats, oils) in most, if not all, living organisms. Human pancreatic lipase is the major enzyme responsible for lipid breakdown in the digestive systems, which

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convert triglycerides to free fatty acids and monoglycerides [142, 143]. It is a diverse family dependent on their substrates and the positions in substrates of the bonds that they hydrolyze [143]. The activity of pancreatic lipase is inhibited by EGCG due to a catechin-induced lipid emulsification process since the addition of EGCG not only dose-dependently reduces cholesterol solubility in biliary micelles but also alters the size of the mixed lecithin/taurocholate/cholesterol micelles [134], suggesting that the reduced lipid emulsification and digestibility may be responsible for lowering intestinal cholesterol absorption, total fat absorption, and serum triglyceride and cholesterol levels. It is to note that the EGCG, but not (+)-catechin (C), appears to stimulate the activity of hormone-sensitive lipase, which exists within adipocytes and is responsible for lipid mobilization from adipose tissues to other peripheral tissues [134, 144]. Inhibition of adrenaline- and adrenocorticotropic hormone-induced lipolysis by EGCG, but not C nor EC, in the primary fat cells were confirmed by decreased release of fatty acids [35, 145]. No changes in the expression of lipoprotein lipase, a cell surface enzyme that hydrolyzes triglycerides in lipoprotein and is responsible for the transport of lipids from the blood to tissues, were observed in obese rats treated with EGCG [35]. EGCG appears to affect the activity of various types of lipases dependent on the physiological conditions of animals and the presence of the specific type of hormones in the experimental models. Therefore clear-cut evidence together with defined mechanism for the effects of EGCG on a variety of lipases has yet to be determined.

6. INHIBITION OF LIPOGENIC ENZYMES Green tea catechins are known to possess anti-lipogenic activity [146] where they inhibit the activity and/or expression of lipogenic enzymes including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), glyceol-3-phosphate dehydrogenase (G3PDH), and stearoyl-CoA desaturase-1 (SCD1) [22,11,10,55]. ACC is the rate-limiting step in fatty acid synthesis for catalyzing the conversion of acetyl-CoA to malonyl-CoA [68]. The green tea EGCG reveals the inhibitory activity on rat liver ACC in vitro [125] and in vivo [134]. Interestingly neither (+)-catechin, EC, nor EGC has shown such effect. It has been reported that when EGCG was incubated with chicken FAS, the second enzyme to catalyze the conversion of malonyl-CoA to fatty acyl-CoA, it inhibited FAS activity [147]. In general, EGCG-mediated inhibition of FAS activity is composed of reversible fast-binding inhibition and irreversible slow-binding inactivation [147]. The expression of hepatic ME and G6PDH, known to generate NADPH for fatty acid biogenesis, has decreased in obese mice treated with EGCG [8, 139]. The activity and expression of G3PDH, the rate-limiting step in TG biosynthesis, are also decreased by EGCG treatment [148]. Furthermore, gene expression of SCD1, the rate-limiting enzyme in the synthesis of monounsaturated fatty acids in adipose tissue, was suppressed in EGCG-treated obese mice [8], suggesting that EGCG or green tea can reduce fatty acid and TG synthesis by inhibiting lipogenic enzymes. Several cholesterol-related enzymes are also regulated by green tea catechins, supporting its potential hypocholesterolemic effect. For instance, it has been reported that EGCG from green tea is an inhibitor of fatty-acid synthase (FAS). Its inhibition of FAS appears to be

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composed of reversible fast-binding inhibition, through which 52 μM EGCG can inhibit 50% of the activity of FAS, and irreversible slow-binding inactivation. Therefore, the inhibitory activity may be caused by specific binding to the enzyme or by scavenging reactive oxygen species required for the monooxygenase reaction. EGCG also inhibited the activity of two and other cholesterol-biosynthetic enzymes, lanosterol 14α-demethylase oxidosqualene:lanosterol cyclase [149]. Inhibition of EGCG on cholesterol-biosynthetic enzymes could cause the low plasma cholesterol levels, together with the inhibition of micelle formation [124] and other steroid-related enzymes including 11β-hydroxysteroid dehydrogenase [150], 5α-reductase [151], and aromatase [152] and the stimulation of fecal cholesterol excretion [8]. In EGCG-treated rats, decreased activities of 5α-reductase and aromatase could be related to weight loss of androgen-dependent organs, such as prostates and seminal vesicles, and to low blood estrogen levels and the subsequent weight loss of estrogen-dependent organs, such as uterus [55, 152]. These observations may explain the beneficial use of EGCG in the prevention of prostate and breast cancers in patients [153, 154]. There are controversial observations in the green tea’s regulation of energy expenditure and fat oxidation in humans [10, 69, 155-158]. This controversy may be attributable to the protocols employed, the source and purity of green tea extracts, the number of times administered, the period and dose of administration, and the method of administration (capsules vs. tea drinking).

7. GLUCOSE TOLERANCE AND INSULIN SENSITIVITY High level of circulating glucose (or hyperglycemia) can cause a serious problem with diabetes mellitus type 2 (type 2 diabetes), a metabolic disorder that is primarily characterized by insulin resistance [159]. Once chronic hyperglycemia becomes apparent, it damages insulin target tissues and aggravates insulin resistance, forming a vicious circle that is collectively called glucotoxicity [160, 161]. It has been suggested by epidemiological observations and laboratory studies that EGCG has a certain anitdiabetic properties and an effect on glucose tolerance and insulin sensitivity [7, 69]. Furthermore, EGCG is reported to lower glucose production by decrease of the expression of genes that control gluconeogenesis [162]. Dietary supplementation with EGCG markedly ameliorates glucose tolerance in diabetic rodents [163, 164]. It has been shown that rats fed with standard chow with green tea had lower fasting plasma levels of glucose, insulin, triglycerides, and free fatty acid than the control rats fed with standard chow with deionized distilled water [164]. Some reports have also suggested that EGCG involved on the regulation of the glucose level in blood by improved insulin secretion through rehabilitation of damaged beta-cells [165, 166]. However, exact mechanism(s) leading to this result have not been documented in detail yet. Researches on the relationship between green tea and obesity-related insulin resistance syndrome has shown that green tea enhances in vitro insulin activity [167], enhances insulin sensitivity in human subjects [168] and rat [165], and reduces hypertriacylglycerolaemia in mice [137]. These results indicate that EGCG indeed has insulin-potentiating activity. Indeed, EGCG appears to mimic the performance of insulin by increasing the tyrosine phosphorylation of both the insulin receptor and insulin receptor substrate-1 [167], which is

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the first stage of insulin-stimulated glucose uptake. Some reports indicated that the phosphorylation of IRS-1 was induced at high glucose level and subsequently repressed hepatic glucose utilization through suppressing Akt downstream signaling [169]. Interestingly, the supplementation of EGCG alleviated this insulin signaling blockade by improving the function of IRS-1 molecules [35]. In addition, EGCG mimics insulin actions by inducing PI3K-sensitive phosphorylation of transcription factor FOXO1a (Forkhead box O1a) which is sensitive to scavengers of free radicals [170]. Based on the fact that serine/threonine phosphorylation can modulate both positive and negative signaling transmission via IRS, it has been shown that some high IRS-1 serine phosphorylation (i. e., Ser307) leads to an insulin-signaling blockade by inhibiting insulininduced IRS-1 tyrosine phosphorylation [171]. In parallel with this observation, it has been reported that EGCG treatment significantly decreases high glucose-induced IRS-1 Ser307 phosphorylation, suggesting critical role of the serine phosphorylation of IRS-1 in the development of insulin resistance [35]. Furthermore, these EGCG effects seem dependent, at least in part, on the cellular AMPK phosphorylation, suggesting a new molecular mechanism for antidiabetic activities of EGCG in addition to the previously described one (see AMPK section). It has been suggested that EGCG has indeed inhibitory role to Ser307 phosphorylation in IRS-1 and this effect was abolished by inhibition of cellular AMPK [35]. However, more studies are needed to answer the question on whether such effect was direct interaction of EGCG with IRS-1 or AMPK or both. It seems fair to say that EGCG indeed regulates glucose homeostasis and displays some antidiabetic benefits. A number of studies were devoted to investigate how high glucose condition preferentially impairs insulin downstream signaling and what role EGCG plays to alleviate this insulin resistance state. These alterations provide an uptake for excess circulating glucose that would be cleared by maintaining glucose homeostasis and thereby protect individuals from glucotoxicity damages. Further studies to determine the cellular molecule(s) responsible for direct interaction with EGCG will certainly lead to the identification of specific molecular target(s) for the generation of therapeutic agents useful in the management of insulin resistance disease like diabetes.

8. ANTIMITOGENIC AND APOPTOTIC EFFECTS The mitogenic signaling in mammalian cells is carried out mainly by growth factors that interact with receptors localized at the plasma membrane. Most of these receptors have a tyrosine kinase activity domain that is localized at the cytoplasmic region of the molecule and often alter the gene expression patterns and induce mitogenesis [172]. Typically, the binding of an agonistic ligand to its cognate GPCR triggers the activation of multiple signal transduction pathways that act in a synergistic and combinatorial manner to relay the mitogenic signal to the nucleus and promote cell proliferation [172]. A rapid increase in the activity of phospholipases C, D, and A2 leading to the synthesis of lipid-derived second messengers, Ca2+ fluxes and subsequent activation of protein phosphorylation cascades, including PKC/PKD, Raf/MEK/ERK, and Akt/mTOR/p70S6K is an important early response to mitogenic avtivity [173, 174].

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Homeostasis in multicellular organisms is maintained by a well-controlled balance between cell proliferation and cell death. Cell death is a fundamental cellular response that has a crucial role in shaping our bodies during development and in regulating tissue homeostasis by eliminating unwanted cells. Several types of cell death have been described; apoptosis (type I), cell death associated with autophagy (type II) and necrosis or oncosis (type III) and few others (e.g., mitotic catastrophe, anoikis, excitotoxicity, Wallerian degeneration, and cornification) [175]. Among these, apoptosis is a complex and highly regulated process that is involved to eliminate superfluous, aged, injured or infected cells from the body in an ordered and controlled sequence of events [176]. Once the decision to die has been made by an individual cell, the ‘execution’ phase of apoptosis is rapid where some cell types showing unique morphological changes as soon as 15 min after exposure to an apoptotic stimulus [177]. All cells undergoing apoptosis show typical, well-defined morphological changes, including plasma membrane blebbing, chromatin condensation with margination of chromatin to the nuclear membrane, nuclear fragmentation, and finally the formation of apoptotic bodies [177]. Apoptosis has been also characterized by several biochemical criteria, including different kinetics of phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane, changes in mitochondrial membrane permeability, release of intermembrane space mitochondrial proteins, and caspase-dependent activation and nuclear translocation of a caspase-activated DNase resulting in internucleosomal DNA cleavage, which is controlled both positively and negatively by B-cell lymphoma protein-2 (BCL2) family members [177, 178]. Identification of these morphological and biochemical markers makes it possible to distinguish the apoptosis from other forms of cell death. There are two classical pathways in apoptosis [179, 180]. In the extrinsic pathway, ligation causes cell surface receptors such as CD95/Fas to bind and oligomerize the cytoplasmic adaptor molecule FADD. The subsequent binding of the prodomains of procaspases 8 and/or 10 to FADD leads to presumed oligomerization of these procaspases causing a conformational change that result in acquisition of enzymatic activity. In the intrinsic pathway, release of cytochrome c, which is released from damaged mitochondria, results in oligomerization of the cytoplasmic scaffolding molecule Apaf-1 (apoptotic protease activating factor-1) caspase-9 (a member of the CED-3-like Cys protease family). Activated caspase-9 in turn cleaves and activates downstream caspases, including caspase-3, caspase-6 and caspase-7, which carry out the execution phase of apoptosis. There are in vitro and in vivo observations suggesting that green tea EGCG appears to modulate the mitogenic, endocrine, and metabolic functions of fat cells. Green tea EGCG inhibited preadipocyte proliferation [35] in dose-, time-, catechin-, and growth phasedependent manners suggesting anti-mitogenic effect of EGCG and different growth stages of preadipocytes have different sensitivities and/or signals dependent on the microenvironmental milieu such as types and sources of catechins. This contention was supported by the light of fact that the amounts of phospho-Erk-1 and phospho-Erk-2 in log-phase, but not latent or confluent, preadipocytes were significantly reduced by EGCG treatment [38]. In addition to the inhibitory effect on preadipocyte proliferation, apoptosis was also enhanced by incubation with EGCG during differentiation, suggesting that EGCG can reduce adipose tissue mass both by inhibiting maturation and by increasing cell death. The mechanism of EGCG in inducing apoptosis seems to be complicated. It has been shown that treatment of 3T3-L1 preadipocytes with EGCG for 48 hours reduced the level and activity of Cdk2 and decreased protein expression of cyclin D1 in a dose dependent manner

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[50]. Furthermore, EGCG significantly increased protein levels of the cyclin kinase inhibitors p21 and p27 and increased binding of p21 and p27 to Cdk2 [56, 181]. It has also been suggested that EGCG can regulate other various antiapoptotic and apoptotic factors including Bcl2, Bad, Bax, and p53, although further studies are required to determine precise mechanisms [56]. It is worthwhile to mention that Cdk2 and caspase-3 have been found to serve as mitogenic and apoptotic signal transducers in eukaryotic cells [50]. It is probable that decreases in Cdk2 expression and activity of 3T3–L1 preadipocytes by EGCG could link to the activation of caspase-3 via changing the mitochondrial transmembrane potential, cytochrome c release, and the caspase-9 activity (an activator of caspase- 3) as reported for the apoptotic effect of the tea polyphenol, theasinensin A [182]. Unfortunately, the Cdk2 pathway required for the mechanism of apoptotic action of EGCG is still not clear (see Cdk pathway in this chapter). However, it is evident that Cdk2 appears to control G1 checkpoint in the cell cycle via regulating the formation of retinoblastoma (Rb) protein and E2F, a transcriptional factor being able to interact either with p53 or with caspase 3 to mediate apoptosis [183]. Accordingly, decreases in Cdk2 expression and activity of 3T3-L1 preadipocytes by EGCG may cause an increase in cell death through promoting the accumulation of the Rb and E2F complex in a p53- or caspase-3-dependent pathway. This notion was indirectly supported by observation that p53-upregulated and caspase-3-related Cdk inhibitors, p21 and p27, were increased when EGCG was added to 3T3-L1 preadipocytes [183, 184]. Overall, EGCG has been reported to induce growth arrest and apoptosis in 3T3-L1 preadipocytes. EGCG is able to block adipogenesis and increase apoptosis in mature adipocytes. The apoptotic effect of EGCG on preadipocytes appears to be dependent on the Cdk2 and caspase-3 pathways and is likely mediated through alterations in their activities. The mechanistic results seem to offer a possible utility in the treatment of obesity using this compound, EGCG.

9. BODY WEIGHT CONTROL Obesity has increased at an alarming rate in recent years and is now one of the major health problems worldwide. It is well known that obesity is the result of both increased adipocyte size and/or increased adipocyte number. Currently interest in the role of plant ingredients in weight control has focused their effects on interfering with the sympathoadrenal systems [185]. The action mechanisms of green tea and EGCG involve diverse routes including (1) the decrease in the energy intake [186], (2) the increase in energy expenditure [157], and (3) the alterations in the activities of fat, liver, muscle, and intestinal cells [11, 15, 137], resulting reduced body weight. The effects of long-term feeding with tea catechins, especially EGCG, have been intensively studied, and some reports suggest a potential role of green tea in body weight control. In vitro studies with green tea extracts containing 25% of catechins have significantly inhibited the gastric and pancreatic lipases. Thus, the lipolysis of long-chain triglycerides is reduced in a 37% [187]. It has been suggested that green tea extracts interfere in the fat emulsification process before enzymes’ action, which is indispensable step for lipid intestinal absorption [124, 137]. It is important to note that although green tea extracts are advisable for the treatment of overweight patients

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whose body mass index ranges over 25 kg/m2, only when and if they do not present special sensitiveness to xantic bases [188]. EGCG treatment in rats causes reduction of subcutaneous fat by 40–70% and abdominal fat by 20–35%, but not epididymal fat [139]. The loss of fat in different proportion in obese rats by EGCG treatment suggests the potential selective effect of EGCG on adipose tissues. Decreased adipose fats may explain the observed decreases in adipose tissue mass and the sequent hypolipidemia of animals treated with EGCG [139, 189]. It is important to note that although the effective dose of EGCG on reducing body weight is 30–50 mg of EGCG/kg body weight, rats gradually adapt and higher doses of EGCG are needed to reduce and/or prevent body weight increases within 1 week [190]. As expected, the body weight loss is reversible as animals regain the body weight when EGCG administration is stopped.

Figure 2. A proposed mechanism of the action of green tea EGCG on obesity.

Overall, pre-clinical and clinical studies have shown that body weight and fat mass of human subjects and animals given green tea catechins decreased significantly. An elevation of the expression of PPARδ, LPL and GLUT4 in adipose tissue without any effect on plasma non-esterified fatty acids (NEFA) concentration seems to enhance fat depots. Further preclinical and clinical studies will help us to fight against obesity and its associated diseases including cardiovascular diseases and type 2 diabetes mellitus.

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10. CONCLUSION Obesity is closely associated with high blood cholesterol and has a high risk for developing diabetes and cardiovascular diseases. Therefore, the management of body weight and obesity is increasingly becoming important to maintain healthy cholesterol profiles and to reduce cardiovascular risks. Interest in the health benefits of tea has been increased and led to the inclusion of tea extracts in dietary supplements and functional foods. Specifically, EGCG is known to have a beneficial effect on human health that reduced adipocyte differentiation and decreased triglyceride levels. Recent reports on the action mechanism of anti-obesity effects by EGCG have been proposed its use as a chemopreventive against obesity, diabetes, cancer, neurodegenerative disorders, and cardiovascular diseases. Studies showed that proliferation of preadipocyte was inhibited by EGCG in dose-, time-, and growth phasedependent manner. EGCG decreased levels of phosphor-ERK1/2, Cdk2, PPARγ, C/EBP and cyclin D1 proteins, and increased levels of Go/G1 growth arrest. EGCG also causes dosedependent inhibition of lipid accumulation in maturing preadipocytes. It is interesting to note that EGCG significantly stimulated the glucose uptake for the antiobesity action, which was accompanied by a decrease in translocation of glucose transporter 4 (GLUT4) in adipose tissue, while it significantly stimulated the glucose uptake with GLUT4 translocation in skeletal muscle. EGCG appears to effectively reduce the body weight via a decrease food uptake, lipid absorption, and serum lipids, triglyceride, and cholesterol levels, as well as stimulating energy expenditure, fat oxidation, high-density lipoprotein levels, and fecal lipid excretion (Figure 2). Finally, although we have gained in-depth knowledge on the action mechanism of EGCG at cellular and molecular level in vitro and in vivo, further detailed and thorough studies will certainly ensure better understanding of the regulatory mechanism of EGCG and its associated signaling pathways, which will eventually, lead to define therapeutic effect of EGCG against obesity, diabetes, and other related diseases.

ACKNOWLEDGEMENT This work was supported by a grant of Biogreen 21 program (20080101-080-038-0010200), Rural Development Administration and GRCMVP for Technology Development Program of Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea.

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[170] Anton S, Melville L, Rena G. Epigallocatechin gallate (EGCG) mimics insulin action on the transcription factor FOXO1a and elicits cellular responses in the presence and absence of insulin. Cell Signal2007 Feb;19(2):378-83. [171] Waltner-Law ME, Wang XL, Law BK, Hall RK, Nawano M, Granner DK. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem2002 Sep 20;277(38):34933-40. [172] O'Bryan JP, Mohney RP, Oldham CE. Mitogenesis and endocytosis: What's at the INTERSECTIoN? Oncogene2001 Oct 1;20(44):6300-8. [173] Ferrell JE, Jr. MAP kinases in mitogenesis and development. Curr Top Dev Biol1996;33:1-60. [174] Rozengurt E. Signal transduction pathways in mitogenesis. Br Med Bull1989 Apr;45(2):515-28. [175] Tang PS, Mura M, Seth R, Liu M. Acute lung injury and cell death: how many ways can cells die? Am J Physiol Lung Cell Mol Physiol2008 Apr;294(4):L632-41. [176] Yasuhara S, Asai A, Sahani ND, Martyn JA. Mitochondria, endoplasmic reticulum, and alternative pathways of cell death in critical illness. Crit Care Med2007 Sep;35(9 Suppl):S488-95. [177] Krysko DV, Vanden Berghe T, D'Herde K, Vandenabeele P. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods2008 Mar;44(3):205-21. [178] Bouchier-Hayes L, Munoz-Pinedo C, Connell S, Green DR. Measuring apoptosis at the single cell level. Methods2008 Mar;44(3):222-8. [179] Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene2006 Aug 7;25(34):4798-811. [180] Chowdhury I, Tharakan B, Bhat GK. Current concepts in apoptosis: the physiological suicide program revisited. Cell Mol Biol Lett2006;11(4):506-25. [181] Nihal M, Ahmad N, Mukhtar H, Wood GS. Anti-proliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate on human melanoma: possible implications for the chemoprevention of melanoma. Int J Cancer2005 Apr 20;114(4):513-21. [182] Pan MH, Liang YC, Lin-Shiau SY, Zhu NQ, Ho CT, Lin JK. Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells. J Agric Food Chem2000 Dec;48(12):6337-46. [183] Fan G, Ma X, Wong PY, Rodrigues CM, Steer CJ. p53 dephosphorylation and p21(Cip1/Waf1) translocation correlate with caspase-3 activation in TGF-beta1-induced apoptosis of HuH-7 cells. Apoptosis2004 Mar;9(2):211-21. [184] Santoni-Rugiu E, Duro D, Farkas T, Mathiasen IS, Jaattela M, Bartek J, et al. E2F activity is essential for survival of Myc-overexpressing human cancer cells. Oncogene2002 Sep 19;21(42):6498-509. [185] Hsu CH, Tsai TH, Kao YH, Hwang KC, Tseng TY, Chou P. Effect of green tea extract on obese women: a randomized, double-blind, placebo-controlled clinical trial. Clin Nutr2008 Jun;27(3):363-70. [186] Klaus S, Pultz S, Thone-Reineke C, Wolfram S. Epigallocatechin gallate attenuates diet-induced obesity in mice by decreasing energy absorption and increasing fat oxidation. Int J Obes (Lond)2005 Jun;29(6):615-23.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 8

GREEN TEA: PROTECTIVE ACTION AGAINST PESTICIDES AND OTHER XENOBIOTICS PRESENT IN HUMAN DIET Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi Centre for Rural Development and Technology Indian Institute of Technology Delhi, Hauz Khas New Delhi-110016, India

ABSTRACT The indiscriminate usage of synthetic chemicals and pesticides has lead to a widespread contamination of land, water and air with harmful xenobiotics. The exposure to these toxicants results in severe health effects on organisms. Even some natural foods contain harmless chemical species (nitrate) which however become toxic upon certain conditions. Hence it is pertinent to focus attention on commonly consumed plant food materials that can potentially neutralize the toxicity damage caused by environmental agents. One of the most important sources of antioxidants is green tea. This review focuses on the mechanisms of oxidative damage caused by different xenobiotics and the defensive action of green tea in mitigating the damage. It is concluded that tea polyphenols, catechins and flavonoids scavenge reactive oxygen species (ROS) and render the hepatoprotective effect. However it is important to note that the protective effects of green tea extract are rendered irrespective of the xenobiotic involved thereby suggesting the involvement of a common biochemical pathway.

Key words: xenobiotics, antioxidants, reactive oxygen species, polyphenols

1.1. GREEN TEA PRODUCTION AND CONSUMPTION The most popular beverage in the world is tea prepared from the leaves of Camellia sinensis [1, 2]. Over 25 million kilos of tea are produced annually, with the bulk being

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consumed by Asians, North Africans and Middle Easterners. From dried Camellia sinensis leaves are produced the three principal types of tea products: Black Tea, Oolong Tea and Green Tea. Black Tea is produced by the fermentative oxidation of green tea's astringent phytochemicals, the catechin polyphenols, producing related compounds called theaflavins. This process removes an appreciable amount of the bitterness characteristic of green tea, yielding a milder, and more (subjectively) flavored beverage. Oolong tea results from a partial oxidation of green tea polyphenols. Green tea is produced with the goal of preserving the leaf polyphenols. After the leaves have been picked, the polyphenoldegrading enzymes are inactivated by steaming or pan roasting and drying [3]. By tradition the Orient, North Africa and the Middle East favor green tea and the rest of the world drinks black tea. Oolong tea production is confined to China and Taiwan. Black tea accounts for 80% of the world tea crop [1, 2].

1.2 GREEN TEA - ACTIVE COMPONENTS Tea leaves harbor a myriad of plant chemicals, phytochemicals, possessing a broad spectrum of biological actions. These include antioxidant, anticancer, anti -hypertensive, anticholesterolemic and antimicrobial activities. The polyphenols are the active players in green tea, mediating both taste profile and biological actions. From a chemical perspective, green tea polyphenols are catechins, phytochemicals composed of several linked ring-like structures. Attached to each structure are chemical tags called phenol groups, and because there are many phenol groups, these catechins are called polyphenols. In native green tea, approximately 15-30% of the weight of the leaf is composed of polyphenols. The major anti-inflammatory and anti-oxidative polyphenols present in green tea are (−)-epigallocatechin gallate (EGCG),(−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (+)-epicatechin (EC), (−)-gallocatechin gallate (GCG) and (−)-catechin [14, 15, 16]. Over 50% of this polyphenol fraction is comprised of (-) Epigallocatechin Gallate(EGCG), the most biologically active and influential polyphenol in green tea. Other components include the unique amino acid theanine, carotenoids, chiorophyll and caffeine [3]. Anthocyanidins, plant pigments are also found in green tea. Caffeine occurs in green tea leaves at a level of 3%; brewed green tea contains approximately 35-50 mg of caffeine per cup, contrasted to a cup of coffee, which contains between 75-95mg [3].

1.3. MECHANISM OF ACTION OF ACTIVE COMPONENTS Green tea polyphenols, and especially EGCG, have been shown to not only protect against undesirable prooxidant attack but also to detoxify radicals produced from the environmental toxins paraquat. Additionally, EGCG has been shown to be over 200 times more potent than vitamin E in protecting fats in the brain, which are exceptionally susceptible to oxidative stress [3].

Green Tea

Figure 1. Chemical structure of tea catechins [50].

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EGCG is a potent antioxidant, since the multiple available phenolic groups capture prooxidants and free radicals, there by affording protective action against oxidative stress [3]. Hence, the antioxidative properties of green tea are due to its ability to scavenge reactive oxygen species (ROS), to inhibit their generation and to chelate transition metal ions that can participate in free radical transformations and in the processes of lipid peroxidation [1, 4, 5]. In addition tea polyphenols have been found to prevent inflammation, eliminate the excess free radicals and stimulate the regeneration of damaged cells or tissues [12, 13, 16]. These are also associated with the inhibition of redox-sensitive transcription factors, inhibition of prooxidant enzymes and induction of phase II enzymes [22].

1.4. GREEN TEA: PROTECTIVE EFFECT AGAINST XENOBIOTICS The indiscriminate usage of synthetic chemicals and pesticides has lead to a widespread contamination of land, water and air with harmful xenobiotics. The exposure to these toxicants results in severe health effects on organisms. Even some natural foods contain harmless chemical species (nitrate) which however become toxic upon certain conditions. Hence it is pertinent to focus attention on commonly consumed plant food materials which are natural sources of relevant antioxidants such as polyphenols which can neutralize the toxicity damage caused by environmental agents. One of the most important sources of antioxidants is green tea. This review focuses on the mechanisms of oxidative damage caused by different xenobiotics and the defensive action of green tea in mitigating the damage. The xenobiotics selected include alcohol, carbon tetrachloride, pesticides, benezene, nitrosoamines, PAHs, nitroquiunoline oxide and dopamine.

i) Alcohol Ethanol is known to induce rapid lipid peroxidation and activation of NF-κB, which are critical events responsible for mucosal hemorrhages and edema, in vascular smooth muscle cells and endothelial cells [17, 18, 16]. The alcohol oxidation to acetaldehyde and acetate is accompanied by ROS formation [6, 5]. The erythrocytes are especially exposed to the action of ROS as they contain large amount of iron and oxygen which promote free radical processes [7,5]. Both, ethanol and its metabolites, can react with cell components, including biological membrane. Ethanol reduces the cell membrane surface hydration and affects the membrane protein–lipid structure [8, 5]. Acetaldehyde and ROS can react with proteins and thus modify their structure and functions [9–11, 5]. Moreover, ROS is also responsible for lipid peroxidation [6, 5]. Polyunsaturated fatty acids, originating from membrane phospholipids, are particularly vulnerable to peroxidation. The erythrocyte membrane, in particular, is constantly subjected to oxidative stress due to high content of peroxidizable [11, 5]. The influence of green tea on total antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane phospholipids in ethanol intoxicated rats was examined. It was found that ethanol administration caused, in term, decrease in TAS and increase in the level of all phospholipids and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface

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charge density of erythrocyte membrane. The ingestion of green tea partially prevented decrease in erythrocyte antioxidant abilities observed during ethanol intoxication. Moreover, long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed during chronic ethanol intoxication [5]. A study was designed to evaluate the efficacy of green tea polyphenols in protecting against alcohol-induced gastric damage and to elucidate the underlying mechanisms. Intragastric administration of ethanol to male Sprague–Dawley rats caused significant gastric mucosal damage, which was accompanied by elevated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as transient activation of redoxsensitive transcription factors, such as NF-κB and AP-1, and mitogen-activated protein kinases (MAPKs). Oral administration of the green tea polyphenolic extracts (GTE) significantly ameliorated mucosal damages induced by ethanol and also attenuated the ethanol-induced expression of COX-2 and iNOS. Inactivation of MAPKs, especially p38 and ERKl/2, by GTE might be responsible for inhibition of ethanol-induced expression of COX-2 and iNOS [16].

ii) Carbon-tetrachloride Oxidative stress has been thought to be a major cause of CCl4-induced liver injury, in which CCl4 is metabolized by cytochrome P450 in liver cells to yield the trichloromethyl free radical. These radicals cause lipid peroxidation, which produces hepatocellular damage and enhanced production of fibrotic tissue [21] and the depletion of antioxidant status [19, 20, 21]. In this regard, reduction of oxidative stress may be a potential and effective therapeutic strategy for prevention and treatment of hepatic fibrosis. The effects of epigallocatechin-3gallate (EGCG) on hepatic fibrogenesis were studied. The rat model of carbon tetrachloride (CCl4)-induced hepatic fibrosis was used to assess the effect of daily intraperitoneal injections of EGCG on the indexes of fibrosis. Histological and hepatic hydroxyproline examination revealed that EGCG significantly arrested progression of hepatic fibrosis. EGCG caused significant amelioration of liver injury (reduced activities of serum alanine aminotransferase and aspartate aminotransferase). The development of CCl4-induced hepatic fibrosis altered the redox state with a decreased hepatic glutathione and increased the formation of lipid peroxidative products, which were partially normalized by treatment with EGCG, respectively. Moreover, EGCG markedly attenuated HSC activation as well as matrix metalloproteinase (MMP)-2 activity. Therefore, the administration of EGCG may be an optional therapeutic and preventive measure against oxidative stress-induced liver injury and hepatic fibrosis [21].

iii) Pesticides Pesticides have been used in agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors. Occupational exposure to pesticides is becoming a common and increasingly alarming worldwide phenomenon. Approximately 3 million acute poisonings and 220,000 deaths from pesticide exposure have been reported annually [23–25, 32]. The health effects caused by this occupational exposure are enormous.

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Chlorpyriphos- O, O-diethyl-O- (3, 5, 6-trichloro-2-pyridinyl) phosphorothioate, is classified as a moderately hazardous, Class II insecticide by the WHO [26, 32]. It belongs to the phosphorothioate class of insecticides. Its acute toxicity varies according to the species and route of exposure [27, 32]. The chief mechanism of action of organophosphorus pesticides occurs by the inhibition of neuronal cholinesterase activity, a key enzyme that is involved in neurotransmission [28, 32]. Acute and chronic exposure to chlorpyriphos results in considerable liver damage as evidenced by changes in aspartate transaminase (AST),1 and alanine transaminase (ALT) [29, 32]. These toxic effects probably occur through the generation of reactive oxygen species causing damage to the various membranous components of the cell. This is proved by marked changes to the overall histoarchitecture of liver [30, 32]. Hepatic atrophy has also been observed when repeated doses of chlorpyriphos are given [31, 32]. This paper reports the effect of green tea administration following subacute toxicity caused by exposure to organophosphorus pesticide chlorpyriphos in liver of rats. Four groups containing five male Sprague–Dawley rats each were selected. Group I served as control. Group II rats were permitted free access to solubilised crude extract of green tea (1.5%w/v in water) as the sole drinking fluid. Group III rats were given a single daily oral dose of chlorpyriphos (30 mg/kg bodyweight in corn oil). Group IV rats received oral dose of pesticide and green tea extract simultaneously. All rats were sacrificed after 15 days. Significant damage to liver was observed via increased serum levels of transaminases and alkaline phosphatase. Lipid peroxidation showed a 5-fold increase in pesticide exposed rats compared to control. In contrast, levels of antioxidant GSH, glutathione-dependent enzymes like glutathione peroxidase (GPx), glutathione S-transferase (GST) and free radical scavengers like catalase (CAT) and superoxide dismutase (SOD) were significantly lower than those of the control group reinforcing oxidative damage. The use of green tea extract appeared to be beneficial to rats, although not to a great extent in significantly reducing and reversing the damage sustained by pesticide exposure and favors recovery since a decrease in lipid peroxidation and enhancement of antioxidant GSH level is also observed following supplementation with green tea extract. Green tea treatment may either replenish the levels of antioxidant directly or spare the endogenous pool of GSH from being exhausted by the free radicals generated [32]. Fenitrothion- (O,O-dimethyl-O-(3-methyl-4-nitrophenyl) phosphorothioate is an organophosphorus insecticide widely used for controlling a wide range of insects and other pests. Although fenitrothion exhibits low mammalian toxicity, biochemicals, morphological and functional alterations in animal tissues have been reported. The prolonged administration of fenitrothion increased the concentration of corticosterone and glucose in the plasma of male rats. It also increases the weight of the adrenal gland of male rats and altered its functions [33, 44]. Dermal, inhalation and oral exposure to fenitrothion inhibit acetylcholinesterase enzyme (AChE)1 in plasma, erythrocytes and brain of mammals [34,35,44], in addition to a considerable liver and kidney damage evidenced by elevation in serum AST and ALT [36,37,44]. Another studies recorded an increase in serum cholesterol and alternation in cell membrane fluidity and lipid content [38, 44]. Acute and chronic exposure to fenitrothion induced ultra structural changes in liver and kidney cells of rats with complete distortion of the nuclear membrane, total loss of nuclear intactness and abnormally enlarged smooth endoplasmic reticulum [39, 44]. These toxic effects probably occur through the generation of reactive oxygen species (ROS) causing damage to various membranous components of the cell [40, 44]. The biochemical basis of oxidative damage is becoming

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clearer as recent studies point to the production of ROS as a secondary means of toxicity [41, 44]. Cells succumb to oxidative damage when endogenous store of antioxidants is used up by the oxidant exposure. The antioxidant machinery is composed of enzymes like glutathione Stransferase (GST) [42, 44], and non-enzymatic components are primarily composed of thiols and glutathione (GSH) [43, 44]. The ameliorative effect of daily administrated dose of green tea extract (60 mg polyphenols/animal/day) was investigated on albino rats Rattus norvegicus (150–180 gm) intoxicated with 1/30 and 1/60 LD50 fenitrothion organophosphate insecticide for 28 days. Blood samples were taken at 14 and 28 days for further biochemical parameters. Histopathological studies were carried out in the liver and kidney at the end of the experiment. Significant inhibition in plasma cholinesterase (ChE), a biomarker of Ops, was recorded. Damage in the liver and kidney tissues was observed and confirmed with elevation of plasma alanine aminotransferase (ALT), aspartate aminotaransferase (AST), albumin, urea and creatinine, as well as an elevation in the oxidative stress (OS) marker malondialdehyde (MDA). Decrease in total glutathione (GSH) content in erythrocytes and fluctuation in glutathione S-transferase (GST) activity in plasma was also observed. Green tea supplementation (60 mg/animal/day) partially counteracts the toxic effect of fenitrothion on oxidative stress parameters and repairs tissue damage in the liver and kidney, especially when supplemented to 1/60 LD50 intoxicated animals depending on the duration. It seems that enzyme and metabolite markers of these organs need more time to be restored to the control level [44]. Supplementation with green tea extract to fenitrothion intoxicated animals counteracts plasma lipid peroxidation biomarker (MDA) [45, 44]. Green tea treatment may either replenish the levels of antioxidant directly or spare the endogenous pool of GSH from being exhausted by the generated free radical [46, 44]. Meanwhile, green tea polyphenols stimulate the transcription of phase II detoxifying enzymes mediated by an antioxidantresponsive element [47, 44]. This could explain the significant elevation in the glutathione Stransferase enzyme activity recorded in the green tea treated groups in the current study. In spite of the previous ameliorative effect of green tea supplementation on some antioxidant parameters, it needs more time for ameliorating membrane damage in the liver parenchymal cells to prevent over leakage of liver enzyme markers (AIT and AST) in plasma This hypothesis is supported by the histopathological findings in liver and kidney tissues in the groups supplemented with green tea. Less damage was clearly noticed in liver with green tea supplementation especially with the group of intoxicated with low dose fenitrothion and green tea. The liver architecture was preserved withless hemorrhage and less cell degeneration; however, inflammatory cells were noticed [44]. Paraquat - (1, 1-dimethyl-4, 4-bipyridilium; Pq) is a commonly used herbicide. Although the exact mechanism of Pq toxicity is not completely elucidated, numerous arguments suggest that free-radical generation by Pq plays an essential role in the mechanism of its toxicity [48, 49]. EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver microsome system containing 40 mM FeSO4. Forty micromolar EGCg inhibited MDA production significantly. EGCg may inhibit the Pq-induced MDA production by at least two mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess amounts of FeSO4 were added to the reaction mixture, which indicated that EGCg reduced iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the microsome-Pq system. EGCg inhibited iron-driven lipid peroxidation presumably not only by

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chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the reduction of ferric (Fe3+) to ferrous (Fe2+) that catalyzes the Fenton reaction. Chelating and radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq toxicity, which may explain the protective effects of EGCg against Pq toxicity [49]. Rotenone and dieldrin- A common denominator in the pathogenesis by pesticides rotenone and dieldrin is the involvement of oxidative stress-mediated apoptotic process [50, 51, 52, 53]. Gamma-glutamylethylamide (L-theanine), a natural glutamate analog in green tea, has been shown to exert strong anti-ischemic effect. The protective effects of L-theanine on neurotoxicants, rotenone and dieldrin in cultured human dopaminergic cell line, SHSY5Ywere investigated. The experiments revealed that L-theanine (500 mM) attenuated both rotenone- and dieldrin-induced morphological changes of nuclei and HO-1 elevation which is related to overproduction of ROS [53]. Tamoxifen- A recent study has shown that tamoxifen-induced hepatotoxicity in female rats that occurs due to oxidative stress can be reversed by green tea administration [54, 32].

iv) Nitrosoamines Nitrite is an important additive in production of cured meat products in terms of maintaining desirable color, texture, and especially with respect to preventing toxin formation by Clostridium botulinum [55,71]. Recent evidence has suggested that nitrite is bactericidal for pathogenic gastrointestinal, oral and skin bacteria when ingested and mixed with gastric acid [56, 67]. However, significant concerns still exist because the nitrite may react with amines and amino acids to produce N-nitrosamines, which are known to be carcinogenic, mutagenic and teratogenic [57–59, 71]. The formation of N-nitrosamines in cured meat products is dependent on the cooking method, residual and/or added nitrite concentration, ascorbate or a-tocopherol concentration, nitrosamine precursors, moisture content, lean-toadipose tissue ratio, presence of nitrosation catalysts and inhibitors, and possibly the smoking process [60–62, 71]. High nitrate and amine-rich food intake has been shown to increase the risk of endogenous formation of carcinogenic N-nitroso compounds (NOCs) [63, 64, 71]. Although nitrate itself is relatively nontoxic, approximately 5% of ingested nitrate is converted to the more toxic nitrite form in the oral cavity [65, 66, 71]. Since the discovery in 1992 that N-nitroso compounds were carcinogenic to rodents [67, 71], this class of versatile carcinogens has been shown to induce tumors in most bodily organs of 40 animal species [68, 71]. Further, human exposure to endogenously formed NOCs has been related to increased risk of gastric, esophageal, nasopharyngeal and bladder cancers [69, 71]. The endogenous nitrosation occurs to a great extent when the precursors are ingested in foods. Nitrite can react with secondary amines under the acidic conditions of the stomach to form N- nitrosamine. Nitrosamine formation in the acidic environment of the stomach can take place when both nitrite and secondary amines are present. Nitrite is readily protonated to nitrous acid (HNO2), with two molecules of nitrous acid forming nitrogen trioxide (N2O3), which is the actual nitrosating species that reacts with unprotonated amines to form nitrosamines [70,71]. In vitro experiments were performed to test inhibition of nitrite-mediated N-nitrosation by individual catechins, green tea. The extent of inhibition was measured via nitrosamine formation. The results show that N-dimethylamine nitrosation is partially inhibited by green tea with or without enzymatic modification. The inhibition of nitrosation by catechins is a

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result of competition between the secondary amines and catechins for nitrite in the reaction mixture [71]. Carcinogenic N-nitroso compounds, which are easily produced under the acidic conditions in the stomach, might cause stomach cancer [72, 76]. Soy sauce has the highest mutagenicity when various Japanese food- stuffs are treated with nitrite and it is one of the most important foods concerning to the relationship of Japanese food consumption and the high gastric cancer mortality in Japan [73, 76]. Tyramine is a major mutagen precursor in soy sauce treated with nitrite, and 1-methyl-1, 2, 3, 4- tetrahydro-b-carboline-3-carboxylic acid (MTCCA) is a minor one [74, 76]. However, MTCCA becomes the most potent mutagen in soy sauce when it is treated with nitrite in the presence of ethanol, while the muta- genicity induced from nitrite-treated tyramine strongly decreases in the presence of ethanol [75, 76]. It has been shown that the mutagenicity of 1-methyl-1, 2, 3, 4-tetrahydro-b-carboline-3carb-oxylic acid (MTCCA), a major mutagen precursor in soy sauce on treatment with nitrite and ethanol, was strongly decreased by the addition of hot water extracts of green, black and oolong teas in the reaction mixture when it was treated with 50 mM nitrite at pH 3.0, 378C for 60 min in the presence of 7.5% ethanol. The mutagenicity-decreasing activity of the teas was scarcely decreased by washing the teas with chloroform and benzene and was partly decreased by butanol and ethyl acetate. Typical polyphenols such as catechins were shown to have the antimutagenicity dose dependently. The antimutagenicity and the reducing power of tea extracts gave a positive good correlation. The results suggest that the mutagenicity of MTCCA on treatment with nitrite in the presence of ethanol may be decreased by the mixed fractions of lyophilic components such as polyphenols, which have high reducing power such as catechins and the other compounds which have little reducing power including the derivatives of the catechins and so on. Although the antimutagenicity of teas and catechins was also considerably effective when they were added after the nitrosation, while that of black tea and some catechins was less effective [76]. High nitrate and amine-rich food intake has been shown to result in an increased risk of endogenous formation of carcinogenic N-nitroso compounds (NOCs) [77, 78, 79]. Vegetables and vegetable products are an important source of nitrate in an average diet. The formation of carcinogenic nitrosamines under simulated gastric conditions was studied during the incubation of amine rich food and nitrate, and its possible inhibition by adding green tea extracts. The green tea extracts were effective in reducing the formation of Nnitrosodimethylamine (NDMA). During four weeks of test (designated EW1, EW2, EW3 and EW4; experiment week 1, 2, 3 and 4 diets) volunteers consumed a diet of low nitrate and amine (EW1)and consumed a fish meal rich in amines as nitrosatable precursors in combination with intake of nitrate-containing drinking water without (EW2)or with green tea extracts (EW4, respectively). The intake of nitrate-containing drinking water (340 mg nitrate/100 ml) resulted in a significant rise in mean salivary nitrate and nitrite concentrations and in mean urinary nitrate levels. Mean urinary nitrate was increased to 455.0±66.2, 334.6±67.8 and 333.4±50.7 mg/18 h after the nitrate intake of EW2, EW3 and EW4, respectively. Significant increases in urinary dimethylamine and trimethylamine levels were observed in consumption of diets (EW2, EW3, and EW4) rich in amine and nitrate. Green tea extract in EW3 and EW4 enhanced the increase of urinary dimethylamine and trimethylamine levels. Urinary excretion of N-nitrosodimethylamine in consumption of diet rich in nitrate and amine (EW2) increased to 6504.4±2638.7 ng/18 h from 257.0±112.0 ng/18 h of low nitrate and amine diet (EW1). Korean green tea in nitrate and amine rich diet reduced the excretion

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of N-nitrosodimethylamine to 249.7±90.6 respectively, compared with 6504.4±2638.7 ng /18 h after ingestion of TD1 diet [79].

v) PAHs Many of the polycyclic aromatic hydrocarbons (PAHs), some of which are common environmental pollutants, are known to have carcinogenic and mutagenic effects [80, 83]. The PAHs themselves are relatively inert biologically and essentially act as precarcinogens that must first undergo metabolic activation by the cytochrome P-450-dependent and other enzymes to their biologically active ultimate carcinogenic metabolites [81, 83]. The cytochrome P-450 enzyme system is present and inducible in mouse skin [82, 83]. Benzo[a]pyrene (BP) is a prototype of PAHs that is metabolized by sequential reactions of this enzyme system and epoxide hydrolase to the chemically reactive ultimate carcinogenic metabolite BPDE-2 (7f3, 8a-dihydroxy- 9a, 1 Oa-epoxy- 7,8,9,1 O tetrahydrobenzo[a]pyrene) [81,83]. The effect of pretreatment of skin of Sencar mice with topically applied green tea polyphenols (GTP) on the skin tumor initiating activity of ( +- )- 7f3,8a-dihydroxy- 9a, 1 Oaepoxy- 7,8,9,1 O-tetrahydrobenzo[a]pyrene (BPDE-2) had been evaluated. The animals were pretreated with the plant phenols (GTP 24 mg/mouse) for 7 days after which they received a single topical application of 200 nmol of BPDE-2 as the initiating agent. Beginning 7 days following initiation animals received twice weekly applications of 3.24 nmol of l2-0tetradecanoyl phorbol-13-acetate (TPA). GTP afforded significant protection against skin tumor induction. These inhibitory effects were verified both by prolongation of the latency period and subsequent development of tumors. Our results suggest that tannic acid and GTP have substantial potential for protecting against the skin tumorigenic response to BPDE-2 and the mechanism of inhibition may involve inactivation of the reactive carcinogenic moiety [83].

vi) Benzene Benzene is a ubiquitous environmental contaminant as well as an important industrial chemical. It is used in the manufacture of a wide variety of consumer goods and is a constituent of gasoline and tobacco smoke [84, 85,105]. Chronic exposure to benzene is known to induce aplastic anemia, leukemia, and other related blood disorders [86,105]. However, the precise mechanisms by which benzene induces these effects are unknown. Benzene itself is unlikely to be the actual toxicant but rather, is converted to bioactive metabolites which cause myelotoxicity [87,105]. Benzene is metabolized to benzene oxide by P450IIEl monooxygenase enzymes in the liver. Benzene oxide gives rise to phenolic metabolites, such as phenol (PH), catechol (CT), hydroquinone (HQ) and 1,2,4-benzenetriol (BT) and open-ring products, such as t,t muconaldehyde and t,t muconic acid [88,105]. The phenolic metabolites can be further oxidized by peroxidases to the corresponding reactive semiquinones and quinones, e.g. CT is converted to 1,2-benzoquinone (1,2-BQ), HQ to 1 ,4 benzoquinone (1,4- BQ) and BT to 2-hydroxy-1,4 BQ [88,105]. It has been reported that several metabolites of benzene are capable of reacting with both DNA and protein [89,

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90,105], reducing bone marrow cellularity and developing colony forming unit-erythroid in mice [91,105]. These compounds also decrease host resistance to microorganisms and tumors [92,105], and result in cytochrome P450 destruction [93,105]. In addition to these findings, administration of metabolites of benzene also induces sister chromatid exchanges, micronuclei and aneuploidy [94- 97,105], and inhibits the synthesis of DNA and RNA [98, 99,105]. They can also induce oxidative DNA damage [96,100,105] and DNA strand breakage [l01, 102,105] in target cells. One mechanism by which benzene induces these genotoxic effects may be mediated by the generation of one or more active oxygen species (AOS) such as superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet oxygen [88,105]. According to earlier studies, several polyphenolic metabolites of benzene such as HQ, CT, BT and related polyphenol, PG, can produce AOS by autooxidation and they are highly mutagenic [103,104,105]. Hence, especially in the presence of cuprous and iron [96,105], benzene metabolites can increase the level of 8-hydroxy deoxyguanosine (8OHdG), suggesting that metabolites of benzene can induce oxidative DNA base modification in cells [ 96,100,102,105] and may therefore play a role in benzene-induced genotoxicity, myelotoxicity and leukemia. Autooxidation of polyphenolic metabolites of benzene, such as hydroquionone (HQ), catechol (CT), 1, 2, 4-benzenetriol (BT) and pyrogallol (PG), produced several kinds of active oxygen species (AOS). BT and PG induced DNA breaks in the absence of metal ions, especially when producing AOS such as H202, 02-, HO. or ΔgO2. HQ and CT did not result in double-strand DNA breaks, except when ferrous ion was added, indicating the participation of the Fenton reaction. Polyphenolic fractions isolated from green tea (GTP) exerted inhibitory effects on the autooxidation of BT and suppressive effects on H202, or HO. generated from phenolic metabolites of benzene in the presence of S9 or an in vivo system [105].

vii) 4-Nitroquiunoline 1-oxide 4-Nitroquiunoline 1-oxide (4-NQO), a water soluble oral carcinogen, produce papilloma and invasive squamous cell carcinoma, resulting in clinical and histologic changes which are similar to those observed in neoplasms of humans (106,111). 4-NQO is known to induce multistep carcinogenesis (107,111). 4-NQO is also known to induce H-ras mutation in chromosome 7 leading to head and neck squamous cell carcinoma in experimental murine models (108,111). Carcinoma is preceded by a sequence of hyperplasia–papilloma/dysplasia– carcinoma, similar to that of human oral cancer (109,111). 4-NQO is also known to cause intracellular oxidative stress (110,111), which leads to lipid peroxidation. 4-NQO at the concentration of 1.5mMwas found to induce lipid peroxdation in 5% liver homogenate in phosphate buffered saline and extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min was studied by assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was found that addition of 4- NQO caused an increase in OH and TBARS level and a decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10 mg/ ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, it was concluded that green tea polyphenols, were found to nullify 4-NQO induced lipid

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peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to carcinogenesis (111).

viii) 6-hydroxydopamine (6-OHDA) 6-hydroxydopamine (6-OHDA), is known to induce dopaminergic neurodegeneration via oxidative stress(112,113). 6-OHDA (350 and 50 µM) activated the iron dependent inflammatory redox sensitive nuclear factor-κB (NF-κB) in rat pheochromocytoma (PC12) and human neuroblastoma (NB) SH-SY5Y cells, respectively. Immunofluorescence and electromobility shift assays showed increased nuclear translocation and binding activity of NF-κB after exposure to 6-OHDA in NB SH-SY5Y cells, with a concomitant disappearance from the cytoplasm. Introduction of GT extract (0.6, 3 µM total polyphenols) before 6-OHDA inhibited both NF-κB nuclear translocation and binding activity induced by this toxin in NB SH-SY5Y cells. Neuroprotection was attributed to the potent antioxidant and iron chelating actions of the polyphenolic constituents of tea extracts, preventing nuclear translocation and activation of cell death promoting NF-κB. Brain penetrating property of polyphenols may make such compounds an important class of drugs for treatment of neurodegenerative diseases (113).

CONCLUSION There is evidence that synthetic antioxidants cause toxicity [114; 115, 116] through oxidative stress so it is important to focus on plant food materials that are a part of the human diet. Green tea is a popular beverage world over and is known to be a rich source of potent antioxidants. Hence it is important to investigate the role of green tea as a natural antioxidant in order to prevent or retard oxidative stress from environmental agents. It is clear from the above discussion that tea polyphenols, catechins and flavonoids scavenge reactive oxygen species (ROS) [117, 32] and render the hepato-protective effect. However it is important to note that the protective effects of green tea extract are rendered irrespective of the xenobiotic involved thereby suggesting involvement of a common biochemical pathway [118, 32].

FUTURE SCOPE Most of these studies related to oxidative stress as a consequence of pesticide exposure have been performed in experimental animals. And studies on oxidative stress in human subjects exposed to different pesticides have not received much attention as yet. It is therefore important to investigate oxidative stress, and derangement of the antioxidant defense system in stemming from the ingestion of pesticides in human poisoning cases. There is also need to investigate the protective effects of green tea on oxidative stress induced by other xenobiotics and natural agents.

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Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi tetrahydrobenzo[a]pyrene by tannic acid, green tea polyphenols and quercetin in Sencar mice. Cancer Letters, 42 (1988) 7- 12. L.A. Wallace, Major sources of benzene exposure, Environ. Health Perspect.. 82 (1989) 165-169. L.A. Wallace, The exposure ofgeneral population to benzene, Cell Biol. Toxicol.. 5 (1989) 297-3 14. G.F. Kalf, Recent advances in the metabolism and toxicity of benzene. CRC Crit Rev. Toxicol. 18 (1987) 141-159. D. Sammett, E.W. Lee, J.J. Kocsis and R. Snyder, Partial hepatectomy reduced both the metabolism and toxicity of benzene, J. Toxicol. Environ. Health, 5 (1977) 785-792. V.V. Subrahmanyam, D. Ross, D.A. Eastmond and M.T. Smith, Potential role of free radicals in benzene-induced myelotoxicity and leukemia, Free Rad. Biol. Med., I1 (1991) 495-515. T.A. McDonald, S. Waidyanatha and S.M. Rappaport, Production of benzoquinone adducts with hemoglobin and bone-marrow proteins follwoing administration of [13C6] benzene to rats, Carcinogenesis, 14 (1993) 1921-1925. G. Levay and W.J. Bodell, Potentiation of DNA adduct formation in HL-60 cells by combinations of benzene metabolites, Proc. Natl. Acad. Sci. USA, 89 (1992) 71057109. D.J. Neun, A. Penn and C.A. Snyder, Evidence for strain-specific differences in benzene toxicity as a function of host target cell susceptibility, Arch. Toxicol., 66 (1992) 1 l-17. B.W. Manning, D.O. Adams and J.G. Lewis, Effects of benzene metabolites on receptor-mediated phagocytosis and cytoskeletal integrity in mose peritoneal macrophages, Toxicol. Appl. Pharmacol.,126 (1994) 214-223. P. Soucek, B. Filipcova and 1. Gut, Cytochrome P450 destruction and radical scavenging by benzene and its metabolites, evidence for the key role of quinones. Biochem. Pharmacol., 47 (1994) 2233-2242. K. Morimoto, S. Wolff and A. Koizumi, Induction of sister-chromatid exchanges in human lymphocytes by microsomal activation of benzene metabolites, Mutat. Res., 119 (1983) 355-360. L. Zhang, P. Venkatesh, M.L. Robertson Creek and M.T. Smith, Detection of 1,2,4benzentriol induced aneuploidy and microtubule disruption by fluorescence in situ hybridization and immunocytochemistry, Mutat. Res., 320 (1994) 315-327. L. Zhang, M.L. Robertson, P. Kolachana, A.J. Davison and M.T. Smith, Benzene metabolite,1,2,4benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL 60 cells, Environ. Mol. Mutat., 21 (1993) 339-348. D.A. Estmond, Induction of micronuclei and aneuploidy by the quinone-forming agents benzene and O-phenylphenol, Toxicol. Lett., 66 (I 993) 105- 118. P. Pellack-Walker, J.K. Walker, H.H. Evans and J.L. Blumer, Relationship between the oxidation potential of benzene metabolites and their inhibitory effect on DNA synthesis of L5178YS cells, Mol. Pharmacol., 28 (1985) 560-566. G.B. Post, R. Snyder and G.F. Kalf, Inhibition of mRNA synthesis in rabbit bone marrow nuclei in vitro by quinone metabolites of benzone, Chem.-Biol. Interact., 50 (1984) 203-211.

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[100] P. Kolachana, V.V. Subrahmanyam, K.B. Meyer, L. Zhang and M.T. Smith, Benzene and its phenolic metabolites produce oxidative DNA damage in HL60 cells in vitro and in the bone marrow in vivo, Cancer Res., 53 (1993) 1023-1026. [101] S.A.S. Walles, Mechanisms of DNA damage induced in rat hepatocytes by quinones. Cancer Lett. 63 (1992) 47-52. [102] Y. Li and M.A. Trush, DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(lI)/Cu(I) redox cycle and reactive oxygen generation Carcinogenesis, 14 (1993) 1303-1311. [103] J.K. Lin and SF. Lee, Enhancement of the mutagenicity of polyphenols by chlorination and nitrosation in Salmonella typhimurium, Mutat. Res.. 269 (1992) 217-224. [104] S.F. Lee and J.K. Lin, Generation of hydrogen peroxide, superoxide anion and the hydroxyl free radical from polyphenols and active benzene metabolites: their possible role in mutagenesis, J. Biomed. Sci., 1 (1994) 125-130. [105] Shern Fwu Lee, Yu Chih Liang, Jen Kun Lin. Inhibition of 1,2,4-benzenetriolgenerated active oxygen species and induction of phase II enzymes by green tea polyphenols. Chemico-Biological Interactions 98 (1995) 283-301. [106] Tanaka, T., Makita, H., Ohnishi, M., Hirose, Y., Wang, A., Mori, H., Satoh, K., Hara, A., Ogawa, H., 1994. Chemoprevention of 4-Nitroquinoline 1-Oxideinduced oral carcinogenesis by dietary curcumin and hesperidin: comparison with the protective effect of beta-carotene. Cancer Research 54, 4653–4659. [107] Sugimura, Takashi, 1992. Multistep carcinogenesis: a 1992 prospective. Science 258, 603–607. [108] Hendler, F.J., Yuan, B., Oechlsi, M.N., Lentseh, E.J., Menes, J.C., Shum-Siu, A.,Hu, L.H., Hawkins, B., Heniford, B.W., 1996. 4-NQO oral carcinogenesis, a murine model of human head and neck cancer. In: Waerner, J.A., Lippert, B.M., Rudert, H.H. (Eds.), Head and Neck Cancer — Advances in Basic Research. Elsevier science, pp. 79–87. [109] Stenman, G., 1981. Malignancy of 4-NQO induced oral squamous cell carcinomas in rat. Acta Otolaryngology 92, 557–561. [110] Nunoshiba, T., Demple, P., 1993. Potent intracellular oxidative stress exerted by the carcinogen 4-NQO. Cancer Research 53, 3250–3252. [111] Periasamy Srinivasan, Kuruvimalai Ekambaram Sabitha, Chennam Srinivasulu Shyamaladevi.Attenuation of 4-Nitroquinoline 1-oxide induced in vitro lipid peroxidation by green tea polyphenols. Life Sciences 80 (2007) 1080–1086. [112] Cohen G, Heikkila RE. The generation of hydrogen peroxide, superoxideradical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents. Journal of Biological Chemistry 1974;249:2447–52. [113] Yona Levites, Moussa B.H. Youdim, Gila Maor, Silvia Mandel. Attenuation of 6hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-κB) activation and cell death by tea extracts in neuronal cultures. Biochemical Pharmacology 63 (2002) 21–29. [114] Kaur, C., & Kappor, H. C. (2001). Antioxidants in fruits and vegetables – The millennium’s health. International Journal of Food and Science Technology, 36, 703– 725. [115] Prior, R. L., & Cao, G. (2000). Flavonoids: Diet and health relationships. Nutrition in Clinical Care, 3, 279–288.

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[116] Rabia Alghazeer, Suhur Saeed, Nazlin K. Howell. Aldehyde formation in frozen mackerel (Scomber scombrus) in the presence and absence of instant green tea. Food Chemistry 108 (2008) 801–810. [117] N. Kerry, C.A. Rice–Evans, Inhibition of peroxy nitrite-mediated oxidation of dopamine by flavonoid and phenolic antioxidant and their structural relationships, J. Neurochem. 73 (1999) 247–253. [118] A. Augustyniak, E. Waszkiewicz, E. Skrzydlewska, Preventive action of green tea from changes in the liver antioxidant abilities of different aged rats intoxicated with alcohol, Nutrition 21 (2005) 925–932.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 9

NEW METHOD TO IMPROVE THE FUNCTION AND INDUSTRIAL APPLICABILITY OF GREEN TEA AND ITS BYPRODUCTS USING IRRADIATION TECHNOLOGY Cheorun Jo1 and Myung Woo Byun2,* 1

Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, 305-764, Republic of Korea 2 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, 580-185, Republic of Korea

ABSTRACT Green tea is a well-known biomaterial with various high biological functions. Irradiation was introduced to develop a new processing method to improve color of the extract, resulting in a higher applicability without any adverse change to the beneficial functions such as inhibitory effects of oxidation, melanin hyperpigmentation on the skin, and others. To investigate the application of irradiated green tea leaf extract for a real cosmetic composition, the physiological activities of irradiated green tea leaf extract powder dissolved in butylenes glycol and ethanol were compared to a commercial green tea extract product. Furthermore, a cream lotion was manufactured using the powder and the physiological activities were compared. Results showed that the irradiation of the green tea leaf extract and the freeze-dried powder from the extract had the same physiological activities as the commercial product in a cosmetic composition. Addition of irradiated green tea leaf in a patty can retard lipid oxidation and add the biological functions green tea possessed, however, the intensity of off-odor produced from green tea is a concern. Using irradiated green tea powder, the off-odor problem can be solved. Some research utilizing green tea byproducts were investigated. Irradiation of green tea byproducts extract showed higher biological functions than that of non-irradiated counterparts. Therefore, irradiation technology can be a useful method to improve biological functions and industrial applicability of green tea and its byproducts.

*

To whom all correspondence should be addressed : Dr. Myung Woo Byun, Phone) +82-63-570-3400, Fax) +8263-570-3409, Email) [email protected]

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Keywords: green tea, irradiation, biological function, application

INTRODUCTION Separation of bioactive compounds from natural resources, which have been consumed safely for a very long time by human beings, and their application to various fields including the food, pharmaceutical, and cosmetic industry have been actively studied (Choi et al., 1989). Among them, green tea has the longest history in the world and is used in about 160 countries every day as a drinking tea. Green tea is one of the 3 most popular beverages besides coffee and cocoa (Kim, 1996). Green tea is composed of about 30% polyphenols (dry basis) such as flavanol, flavandiol, flavonoid, and phenol acid. These polyphenols are wellknown to have various excellent biological activities, for example, the inhibition of tooth decay (Sakanaka et al., 1989), inhibition of allergies (Yeo et al., 1995), reduction of blood pressure (An, 1998), prevention of gout (An et al., 1996) and the inhibition of oxidation. Oak et al. (2005) reported that the polyphenols from green tea and red wine have the antioxidant properties mostly because of their ability to directly scavenge reactive oxygen species such as a hydroxyl radical and a superoxide anion. Especially, the inhibition effect of green tea polyphenol on lipid oxidation is higher than that of synthetic antioxidant, butylated hydroxytoluene (BHT) (Wanasundara & Shahidi, 1998; Chen et al., 1996). On the other hand, food irradiation is known to be the best method for controlling pathogenic microorganisms and one of the best alternatives to the chemical fumigants or preservatives usually used for a sanitation treatment for international trade (WHO 1999). Irradiation technology has been officially adopted by international organizations (WHO/IAEA/FAO) and experts due to its effectiveness in food, wholesomeness and economic benefits. Besides the sanitary purposes, irradiation has been studied to reduce or eliminate undesirable or toxic materials including food allergens (Lee et al., 2000; Lee et al., 2001), carcinogenic volatile N-nitrosamines in sausage (Ahn et al., 2002; Ahn et al., 2003; Jo et al., 2003e), biogenic amines (Kim et al., 2003, Kim et al., 2004), embryotoxicity of gossypol (Jo et al., 2003c), and enhancement of the antioxidant power of phytic acid (Ahn et al., 2004). In addition, irradiation has been shown to enhance the color of low-nitrite meat products (Byun et al., 2000a) and to develop low-salt fermented traditional foods (Byun et al., 2000b; Lee et al., 2002; Kim et al., 2002; Jo et al, 2004). Byun et al. (2002) observed the breakdown of chlorophyll by irradiation, which can be used in oil processing. Based on this result, an application for color removal of green tea leaf extract (Jo et al., 2003a) was developed. Then, the commercial application of irradiation for the color improvement of plants-derived products without changing their beneficial biological activities was tested in foods or cosmetics (Jo et al., 2003b; Byun et al., 2004a). In this chapter, some of the background research and the selected results of the related studies are introduced and discussed for the application of irradiation technology to improve functions and industrial applicability for the green tea industry.

New Method to Improve the Function and Industrial Applicability of Green Tea … 179

COLOR IMPROVEMENT OF GREEN TEA LEAF EXTRACT BY IRRADIATION Green tea leaf has been used mostly for brewing. This is mainly because of its deep dark color and off-flavor, which makes it very difficult to apply the proper amount for the compositions of cosmetics, medicine or foods. The feasibility of using irradiation to develop a new processing method to obtain a light-colored material while maintaining its biological function has been conducted (Jo et al., 2003a). Before the irradiation was applied for color enhancement of green tea leaf extract, the authors were working for the irradiation effect on soybean oil. The presence of green pigments of chlorophyll in soybean oil is of interest not only for their impact on the color of the finished product but also for their potential role in oxidative stability, especially for photooxidation (Usuki et al., 1984; Fakourelis et al., 1987). Chlorophylls not only cause an undesirable color change in vegetable oils but impair the hydrogenation process (Daun, 1982) and promote oxidation in the presence of light although they may be antioxidants under dark conditions (Abraham & deMan, 1986). A study was conducted to investigate the possibility of chlorophyll breakdown by irradiation to inhibit the photooxidation during storage without accelerating lipid oxidation during the irradiation process (Byun et al., 2002). Results showed that the oil sample containing 3 ppm of chlorophyll showed no detectable chlorophyll after being irradiated at 2 kGy either with or without N2 flush (Table 1). The non-irradiated control sample stored in the dark to avoid a photooxidation showed no change in chlorophyll levels during 6 hr storage. Results on peroxide values (POV) indicate that irradiation increased lipid oxidation but the chlorophyll breakdown in the sample irradiated at 20 kGy did not induce the photooxidation when exposed to light (Table 2). Irradiation of samples without oxygen (treated by continuous N2-flush), did not develop lipid oxidation during the irradiation process or photooxidation during storage under light (Byun et al., 2002). The POV value of 20 kGy-irradiated samples with N2-flushing remained 0 during the entire storage regardless of lighting conditions, indicating that irradiation destroyed virtually all of the chlorophyll, resulting in a complete protection from photooxidation. The results suggested that irradiation of oils conducted in the absence of oxygen can be used to eliminate residual chlorophyll. On the basis of chlorophyll breakdown by irradiation, a new processing method was introduced for brighter-colored natural green tea leaf extract (GTLE) material for cosmetics, medicine or the food industry (Jo et al., 2003a) because natural products with high functions cannot be directly applied to the food or cosmetic composition due to their undesirable dark color. An irradiation of GTLE (70% ethanol) showed higher Hunter color L*-value and lower a*- and b*-values, resulting in a color change of solution to bright yellow from dark brown (Table 3). To apply for the industry properly, this color enhancement process using irradiation should not reduce the biological activity of GTLE, and there was no difference in the radical scavenging (Table 4) and tyrosinase inhibition effect (the in vitro test for skin whitening effect) by irradiation regardless of storage temperature (4 and 25°C). However, one of the problems was the change of brighter color of the extract produced from irradiation to darker (decreasing trend in Hunter color L*-value) during storage. The storage temperature was important in this color change and lower storage temperature was better to minimize color change. Also, the addition of vitamin C in the extract was effective in reducing this color change (Jo et al., 2003a). This technology was transferred to a commercial cosmetic company

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in Korea (SunBiotech Co., Ltd.) and the problem of color change was solved in the formulation of cosmetic compositions. Similar results were obtained from a series of studies using different natural materials with various biological functions such as persimmon leaf (Diospyros kaki L. folium), licorice (Glycyrrhiza Uralensis Fischer) root and its stolon (Jo et al., 2003d), Japaneses honeysuckle (Byun et al., 2004b), and root of Curcuma aromatica (Kim et al., 2006). Table 1. Chlorophyll content (ppm) of 20 kGy-irradiated and photooxidized linoleic acid solution (1% in methanol) containing chlorophyll b (3 ppm) by HPLC Irradiation Unfoiled

Foiled5

0 kGy 20 kGy 20 / N24 0 kGy 20 kGy 20 / N24

0 2.91a2 nd nd 2.88 nd nd

Photooxidation time (hr)1 1 2 4 2.99a 2.34a 1.81b nd nd nd nd nd nd 3.05 3.02 2.85 nd nd nd nd nd nd

6 1.51b nd nd 2.73 nd nd

SEM3 0.144 0.108 -

1

Light intensity was 3,300 lux at 25°C. Different letters (a, b) within a row differ significantly at P<0.05. 3 SEM: Pooled standard errors of the mean (n = 10). 4 Sample was bubbled with ultra pure N2 gas during irradiation. 5 Bottles were covered by aluminum foil to avoid photooxidation. The data was published at Byun et al. (2002), J. Am. Oil Chem. Soc. 79, 145-150. 2

Table 2. Peroxide value of photooxidized linoleic acid solution (1% in methanol) containing chlorophyll b (3 ppm) by gamma irradiation

Unfoiled

Foiledg

1

Irradiation 0 kGy 20 kGy 20 / N25 SEM6 0 kGy 20 kGy 20 / N25 SEM6

0 0cy3,4 1243.7x 0y 7.81 0y 1251.7x 0y 10.41

Photooxidation time (hr)1 1 2 4 24.0 cy 27.5cy 49.7by 1251.6x 1286.3x 1253.4x 0y 0z 0z 12.90 3.82 3.38 0y 0y 0y 1275.4x 1262.1x 1251.8x 0y 0y 0y 6.19 3.88 11.10

6 79.0ay 1268.8x 0z 3.03 0y 1266.5x 0y 14.82

Light intensity was 3,300 lux at 25°C. SEM: Pooled standard errors of the mean. n = 20 3 Different letters (a-c) within a row differ significantly at P<0.05. 4 Different letters (x-z) within foiled and unfoiled columns differ significantly at P<0.05. 5 Sample was bubbled with ultra pure N2 gas during 20 kGy-irradiation. 6 SEM: n = 12. 7 Bottle were covered by aluminum foil to avoid photooxidation. The data was published at Byun et al. (2002), J. Am. Oil Chem. Soc. 79, 145-150. 2

SEM2 4.86 11.44 17.40 -

New Method to Improve the Function and Industrial Applicability of Green Tea … 181 Table 3. Hunter color L*-value of green tea leave extract from 70% ethanol solution after gamma irradiation Storage Temperature 4°C

25°C

Irradiation (kGy) 0 5 10 20 SEM2 0 5 10 20 SEM2

0 75.37dz 90.83ay 93.63ax 96.22aw 0.010 74.82az 87.33ay 89.23ax 89.98aw 0.008

Storage (week) 1 2 75.65bz 76.21az 88.35by 87.50cy 90.14bx 88.70cw 91.19bw 88.19cx 0.010 0.005 73.27bz 72.98cz 81.88bx 80.21cw 82.16bw 79.73cx 80.87by 77.55cy 0.012 0.015

3 75.57cz 86.28dx 87.12dw 86.04dy 0.022 71.99dz 80.21dw 79.23dx 76.51dy 0.015

SEM1 0.005 0.013 0.021 0.008 0.007 0.011 0.016 0.007

a-d

Different letters within the same row differ significantly (P<0.05). Different letters within the same column with the same storage temperature differ significantly (P<0.05). 1 Standard errors of the mean (n = 12). 2(n = 12). The data was published at Jo et al. (2003), Radiat. Phy. Chem. 66, 179-184. w-z

Table 4. Scavenging effect of DPPH radical of green tea leave extract from 70% ethanol solution after gamma irradiation Storage Temperature 4°C

25°C

a,b

Irradiation (kGy) 0 5 10 20 SEM2 0 5 10 20 SEM2

0 48.74az 48.35az 51.71ay 53.33ax 0.363 39.96a 44.85a 45.14a 45.44a 1.087

Storage (week) 1 2 30.57b 27.37b 33.20b 28.82c 26.74b 27.73b 16.73b 29.70b 4.282 1.080 33.36b 23.83c 33.53b 28.58c 34.85b 27.51b 33.00b 28.57b 2.445 0.975

3 31.66by 36.95bx 30.69by 29.98by 0.818 25.58cy 31.08cx 29.98bx 33.33bx 0.982

SEM1 2.232 1.078 1.805 3.301 0.795 0.758 2.450 1.368

Different letters within the same row differ significantly (P<0.05). Different letters within the same column with the same storage temperature differ significantly (P<0.05). 1 Standard errors of the mean (n = 8). 2(n = 8). The data was published at Jo et al. (2003), Radiat. Phy. Chem. 66, 179-184. x-z

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IRRADIATION OF THE POLYPHENOLS SEPARATED FROM GREEN TEA LEAF When the polyphenols of the green tea leaf were separated by Sephadex LH-20 column and thin layer chromatography and irradiated at a higher dose (40 kGy), the major antioxidative activities, including electron donating, inhibition of xanthine oxidase, metal ion chelating, and inhibition of lipid oxidation were still maintained or even better after irradiation treatment (Figures 1 and 2, An et al., 2004). The anti-microbial activities against Staphylococcus aureus and Streptococcus mutans were higher in the irradiated green tea polyphenols, which showed inhibition of microorganisms tested at a lower concentration than those of the non-irradiated counterpart. Ranges of inhibition zone for growth Escherichia coli, S. aureus, S. epidermidis, and S. mutans at 1 mg/disc were 9.3, 10.1, 22.5, and 9.3 mm in non-irradiated control while 10.8, 11.0, 25.0, and 11.7 mm in irradiated counterpart, respectively. Generally, the polyphenol is very stable except for thiolysis, reaction with strong acid (McGraw et al., 1993). The irradiated polyphenol might not change its chemical structure but some change in activity is expected of functional groups such as –OH or –COOH, resulting in higher antimicrobial activity.

Electron donating ability (%)

100 IP NP 80

a

a

a a

a

a

a

a a

a

100

200

60

40

20

b b

0 1

5

10

50

Concentration (ppm) Figure 1. Electron donating ability of irradiated polyphenol isolated from green tea. IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 5 replicates and those with different alphabet letters are significantly different at P<0.01.

New Method to Improve the Function and Industrial Applicability of Green Tea … 183

50 IP NP

Inhibition rate (%)

40

a b

30 c d 20

e f

10

g

g

gh h

0 1

5

10

50

100

Concentration (ppm) Figure 2. Inhibition rate of irradiated polyphenol isolated from green tea on xanthine oxidase. IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 5 replicates and those with different alphabet letters are significantly different at P<0.01.

100 IP NP

Inhibition rate (%)

80

a b

60 c c 40 d

20

0

d e e

e e

1

5

e e

10 50 100 Concentration (ppm)

200

Figure 3. Inhibition rate of the irradiated green tea polyphenol on the collagenase activity. IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3 replicates and those with different alphabet letters are significantly different at P<0.05.

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Collagen synthesis rate (%)

50 IP NP

40

30 a

20

ab bcd

cd

10

ab

bc

cd

d 0 1

5

10

50

Concentration (ppm) Figure 4. Collagen biosynthesis enhancing activity of the irradiated green tea polyphenol on the human fibroblast. IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3 replicates and those with different alphabet letters are significantly different at P<0.05.

Growth inhibition rate (%)

100

a

IP NP

a b

80

c 60

40 d

20

d

de f ef

ef ef

1

5

ef

0 10

50

100

200

Concentration (ppm) Figure 5. Growth inhibition rate of the irradiated green tea polyphenol on the malignant melanoma (SKMEL-2). IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3 replicates and those with different alphabet letters are significantly different at P<0.05.

New Method to Improve the Function and Industrial Applicability of Green Tea … 185 Another study was focused on the changes of physiological activity of irradiated green tea polyphenols on the human skin (An et al., 2005). The important factors for the human skin wrinkles are reactive oxygen species, ultraviolet light, and the reduction of collagen biosynthesis (Grove et al., 1989; Griffiths et al., 1992). The assessment of anti-wrinkle effect on human skin including collagenase inhibition and collagen biosynthesis was measured form the irradiated green tea polyphenols. Results showed that collagenase inhibition effect was higher in the irradiated sample (65.3%) than that of the non-irradiated control (56.8%) at 200 ppm of concentration (Figure 3, An et al., 2004). Collagen biosynthesis rates using human fibroblast were 19.4% and 16.3% in the irradiated and the non-irradiated polyphenols, respectively (Figure 4, An et al., 2004). The tyrosinase inhibition effect, which is related to the skin-whitening effect, showed a 45.2 and 42.9% in the irradiated and non-irradiated polyphenols, respectively, at a 100 ppm level. A higher than 90% growth inhibition on skin cancer cells (SK-MEL-2 and G361) was demonstrated in both the irradiated and nonirradiated polyphenols (Figure 5, An et al., 2004). Thus, the results showed that the irradiation of green tea polyphenol did not change and even increased its anti-wrinkle, skin-whitening, and anticancer effects on the human skin. These results also indicate that irradiation of the polyphenols separated from green tea leaf can be applicable in the food, pharmaceutical, and cosmetic compositions with improved microbiological safety, already proved and commercialized.

APPLICATION OF IRRADIATED GREEN TEA ON MEAT PRODUCTS Irradiation of meat to control pathogens and extend shelf-life accelerates lipid oxidation and produces off-odors under aerobic conditions (Ahn et al., 1999). To overcome these adverse effects of irradiation on meat products irradiated freeze-dried GTLE powder was applied to pork patties (Jo et al., 2003b). The reason of irradiation was to obtain a brightcolored GTLE for better appearance of the additive in pork patties. The development of lipid oxidation of pork patties was lower and radical scavenging effect was greater in the samples with non-irradiated or irradiated green tea extract powder than those of control samples (without additive, Table 5). The pork patties with green tea powder had a higher Hunter color a*-value and less cooking loss than that of control, indicating that the addition of GTLE may improve the quality of the pork patties. Especially, sensory panelists preferred the odor of the raw pork patties and color of the cooked pork patties added with irradiated GTLE than that with non-irradiated GTLE (Table 6). It because the odor of native green tea adversely affected on the sensory quality of pork patty added with GTLE and irradiation of GTLE may reduce the native green tea odor. It has been used mainly two methods to achieve healthier meat and meat products; avoiding undesired substances or reducing them to appropriate limits, and increasing the levels of other substances with beneficial properties (JimmenezColmenero et al., 2001; Arihara, 2006). Currently, exogenous antioxidants including phenolic compounds, tocopherols, plant derivatives and chelating agents are added to meat products. From this study, it can be concluded that irradiated, freeze-dried GTLE powder can be used for producing functionally-improved meat products with additional off-odor reduction from plant-derived natural material in the products.

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Table 5. TBARS values (mg malondialdehyde/kg meat) of raw and cooked pork patties with added nonirradiated or irradiated freeze-dried green tea leaf extract powder (0.1%)

0 Raw pork patties Trt A 0.38cx Trt B 0.28by Trt C 0.30by SEM2 0.021 Cooked pork patties Trt A 1.13bx Trt B 0.48z Trt C 0.58ay SEM2 0.027

5

Storage day 10

15

SEM1

0.56bx 0.42ay 0.36az 0.016

0.56bx 0.43ay 0.35ay 0.025

0.69ax 0.48ay 0.39ay 0.028

0.029 0.024 0.016

1.22abx 0.42y 0.36by 0.048

1.38ax 0.42y 0.44by 0.043

1.21abx 0.44y 0.31by 0.048

0.044 0.042 0.042

Abbreviations: Trt A, only pork patties; Trt B, patties with nonirradiated, freeze-dried green tea leaf extract powder (0.1%); Trt C, patties with irradiated at 10 kGy, freeze-dried green tea leaf extract powder (0.1%). a-c Different letters within the same row differ significantly (P<0.05). x-z Different letters within the same column with raw and cooked pork patties differ (P<0.05). 1 Pooled standard errors of the mean (n = 6). 2(n = 8). The data was published at Jo et al. (2003), Meat Sci. 64, 13-17.

Table 6. Sensory scores of raw and cooked pork patties with nonirradiated or irradiated freeze-dried green tea leaf extract powder (0.1%)

Parameter Trt A Trt B Trt C SEM2

Color 8.6 8.7 7.9 0.47

Raw Odor 6.9b 5.4c 9.0a 0.90

Color 6.78b 8.10a 8.66a 0.47

Odor 6.61 7.41 5.97 0.80

Cooked1 Taste 5.98 8.16 6.52 0.75

Tenderness 6.30 6.67 6.17 0.69

a-c

Different letters within the same column differ (P<0.05). Patties was cooked on a preheated pan for 3.5 min. The cooked patties was sliced into small pieces, served to the panelists individually and evaluated independently in 2 different times at Day 1 and Day 3. A 15 line-scale was provided to the panelists and scored; very undesirable (0) to very desirable (15) for color, strong off-odor (0) to very mild odor (15) for odor, very poor taste (0) to very tasty for taste, and very tough (0) to very tender (15) for tenderness. 2 Pooled standard errors of the mean (n = 60). The data was published at Jo et al. (2003), Meat Sci. 64, 13-17. 1

Another example is shown using the pectin-based edible coating material containing 0.5% irradiated GTLE powder to pork patties (Kang et al., 2007). The edible coatings may serve as carrier for antimicrobial compounds in order to maintain high concentrations of presevatatives on the surface of foods (Gennadios et al., 1997) in addition to the protection of moisture loss. During storage at 10°C for 14 days, lipid oxidation decreased (Figure 6, Kang et al., 2007) and radical scavenging activity increased (Figure 7, Kang et al., 2007) in the pork

New Method to Improve the Function and Industrial Applicability of Green Tea … 187 patties coated by the pectin-based material containing GTLE powder. In addition, the coated patties had higher moisture content throughout the storage than control in both aerobic- and vacuum-packaging. A number of total aerobic bacteria were significantly reduced by the coating treatment as well as by irradiation (Figure 8, Kang et al., 2007). No difference was detected in sensory characteristics due to the coating treatment containing irradiated GTLE powder. The results demonstrate that the GTLE added to pectin-based coating has positive effects on the quality of irradiated pork patty. 20

20 Control CP CGP

TBARS (mg malonaldehyde/kg) value

15

Vacuum (3 kGy)

15

10

10

5

5

20 0

20 0 Aerobic (0 kGy)

Aerobic (3 kGy)

15

15

10

10

5

5

0

0

7

0

14

7

14

TBARS (mg malonaldehyde/kg) value

Vacuum (0 kGy)

0

Storage time (days)

Figure 6. 2-thiobarbituric acid reactive substance value (TBARS; mg malondialdehyde/kg sample) of gamma-irradiated pork patty coated by a pectin-based coating material during a storage at 10°C.

APPLICATION OF IRRADIATED GREEN TEA ON COSMETIC COMPOSITION GTLE powder was prepared using irradiation (20 kGy) to improve its color and antioxidative activity including electron donating and superoxide dismutase-like activities was compared with a commercially available green tea powder produced especially for cosmetic composition (Byun et al., 2004a). The commercial product was removed its dark color by adsorbents through column chromatography, which is time-consuming process with

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high cost. The superoxide dismutase-like activity of 1% irradiated green tea powder dissolved in butylenes glycol showed 48.0% but that of commercial product showed only 25.1% (Table 7). This indicates that the commercial color removal process may reduce the activity of green tea powder originally present. When 1% of the irradiated GTLE powder was diluted 400 and 800 times, the electron donating ability showed 78.0 and 51.7%, respectively, which was not different from the commercial one. No difference in tyrosinase inhibition activity was also found. Results showed that 1% of the irradiated green tea powder dissolved in butylenes glycol for cosmetic composition was higher or at least had the same physiological activity with the commercial product. After 1% of the irradiated GTLE solution dissolved in buytlene glycol was prepared, the solution was mixed (15%) to a cosmetic composition for manufacturing a cream lotion to conduct a real application study. The electron donating activity of the cream lotion prepared from the solution of the irradiated green tea leaf extract powder did not differ from the cream lotion prepared from the solution of a commercial product. Therefore, irradiation of GTLE for cosmetic composition may be a cost-effective method to improve industrial applicability. 40

40

30

Electron donating ability (%)

Vacuum (3 kGy)

Control CP CGP

30

20

20

10

10

40 0

40 0 Aerobic (0 kGy)

Aerobic (3 kGy)

30

30

20

20

10

10

0

0

7

14

0 Storage time (days)

7

14

Electron donating ability (%)

Vacuum (0 kGy)

0

Figure 7. Electron donating ability (%) of gamma-irradiated pork patty coated by a pectin-based coating material during a storage at 10°C.

New Method to Improve the Function and Industrial Applicability of Green Tea … 189

12

12

10

Total plate count (Log CFU/mg)

Vacuum (3 kGy)

Control CP CGP

10

8

8

6

6

4

4

2

2

12 0

12 0 Aerobic (0 kGy)

Aerobic (3 kGy)

10

10

8

8

6

6

4

4

2

2

0 0

7

0

14

7

14

Total plate count (Log CFU/mg)

Vacuum (0 kGy)

0

Storage time (day)

Figure 8. Total plate count of gamma-irradiated pork patty coated by a pectin-based coating material during a storage at 10°C.

Table 7. Physiological activities (%) of irradiated green tea leaf extract powder (1%) dissolved in butylene glycol prepared from laboratory and commercially available one for a cosmetic composition Physiological activity (%) Superoxide dismutase-like DPPH radical scavenging Tyrosinase inhibition 1

Dilution factor 1 400 800 1 5

Irradiated1 48.0±0.8 78.0±0.9 51.7±0.6 81.1±1.5 44.0±0.8

Dried green tea leaf was extracted, irradiated at 20 kGy, and freeze-dried. Commercially available green tea leaf extract powder for cosmetic composition. The data was published at Byun et al. (2004), Raidat. Phy. Chem. 71, 487-489. 2

Commercial2 25.1±0.4 78.0±0.6 53.7±0.9 82.5±0.9 29.0±0.3

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APPLICATION OF GREEN TEA BYPRODUCTS USING IRRADIATION Food processing industries create large quantities of byproducts, which are difficult to dispose of as they have a high biological oxygen demand. Plant material wastes from these industries sometimes contain high levels of phenolic compounds that can have an adverse environmental impact (Kang et al., 2006). On the other hand, agricultural and industrial byproducts are excellent sources of natural antioxidants or functional materials. Several studies have reported that these byproducts have already been used as potential sources of functional and nutritional materials (Lapornik et al., 2005; Esposito et al., 2005; Šližytė et al., 2005). For instance, every year, about 50 tons of green tea byproducts are produced in Boseong area, the biggest green tea production area in Korea, but the economic value of green tea byproducts is much lower (about 1/100) when compared with the green tea leaf (Ki, 2004). Green tea stem extract powder, a major byproduct of the green tea industry, was prepared and investigated for its biological activities after a irradiation treatment for the purpose of a brightened color. The color, total phenolic contents, antioxidative activity, tyrosinase inhibition activity, cancer cell proliferation inhibition effect, and antimutagenicity were measured and compared with those of a GTLE powder (Lee et al., 2006). Hunter L* values of the irradiated GTLE and stem extracts were increased in a comparison with the non-irradiated extracts. The content of total phenolic compounds of the GTLE and stem extract was 424.2 and 11.45 mg/g in non-irradiated sample and 335.5 and 18.65 mg/g in 20 kGy-irradiated sample, respectively. The electron donating ability was not different between GTLE and stem extract in both non-irradiated and irradiated conditions. Ferric reducing antioxidant power (FRAP) of the samples showed similar results to the radical scavenging capacity. The both GTLE and stem extract showed an inhibition of tyrosinase and also a cancer cell proliferation inhibition effect. Especially, the antimutagenicities of the stem extracts showed a strong inhibition effect (96.20 and 57.77%) on 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp) and 2-nitrofluorene (NF), respectively (Table 8). The antioxidative and antigenotoxic activities of GTLE and stem extracts was compared after 20 kGy of irradiation (Lee et al., 2008). In this study, in vitro antioxidative activities including a radical scavenging, a hydrogen peroxide inhibition, the tyrosinase inhibition activities, and the reducing power were tested. GTLE had higher antioxidative activities than the stem extract. Irradiation of 20 kGy to GTLE showed a decreasing tendency in radical and hydrogen peroxide scavenging activities and reducing power, while that to stem extract showed an increasing tendency of the antioxidative activities. Ahn et al. (2004) reported that the FRAP of phytic acid increased significantly by irradiation. Tyrosinase inhibition activity showed no difference by irradiation in both the GTLE and stem extract. Overall, an irradiation had positive influences on the antioxidative activity in the stem extract more than in the GTLE including a color improvement. Antigenotoxic effect of the green tea extracts on an oxidative DNA damage in human leucocytes by a DNA comet assay also indicated a protective effect of GTLE (Figure 9, Jo et al., 2008). The irradiation of the GTLE began to decrease the DNA damage significantly at 10 μg/mL which showed a higher inhibition activity than the non-irradiated GTLE. The non-irradiated and irradiated green tea stem extract showed similar inhibition trends and they were comparable to that of the GTLE. This

New Method to Improve the Function and Industrial Applicability of Green Tea … 191 result also indicates that the green tea bypdroducts can be considered as a cost-effective functional ingredient for industrial applications. Furthermore, irradiation of green tea byproducts may have beneficial effects on its functional activity. Table 8. Antimutagenicity of the green tea leaf and byproduct extract powders on S. Typhimurium TA 981 Mutagen2

Samples

Green tea leaf extract Trp Green tea leaf extract irradiated at 20 kGy Green tea byproduct extract Green tea byproduct extract irradiated at 20 kGy SEM4 Green tea leaf extract NF Green tea leaf extract irradiated at 20 kGy Green tea byproduct extract Green tea byproduct extract irradiated at 20 kGy SEM4

Revertant colonies / plate 51.0±1.41 51.5±3.54 60.0±4.24 57.0±2.83 119.0±11.31 110.5±6.36 111.5±9.19 106.5±6.36

Inhibition rate (%)3 96.77 96.74 96.20 96.39 0.141 54.92 58.14 57.77 59.66 2.295

Abbreviations: Trp, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; NF, 2-nitrofluorene. 1 Concentration of samples was 10 mg per plate. 2 Concentration of Trp and NF were 1 and 3 μL per plate, respectively. 3 Means with the same letter in each column are not significantly different (P<0.05). 4 Standard error of the mean (n = 8). The data was published at Lee et al. (2006), J. Food Sci. 71, C269-C274.

Table 9. The 50% inhibition concentration (IC50, μg/mL) of green tea leaf and stem extract against superoxide radical and hydroxyl radical

0 Superoxide radical Green tea leaf Green tea stem SEM1 Hydroxy radical Green tea leaf Green tea stem SEM1 a,b

Irradiation dose (kGy) 20

2.33by a 2.77ax 0.062

3.25ax 2.09by 0.110

0.066 0.107

1.31a 1.38a 0.014

0.62by 0.95bx 0.020

0.013 0.021

Different letters (a-b) within the same row are significantly different (P < 0.05). Different letters (x-y) within the same column are significantly different (P < 0.05). 1 Standard error of the means (n = 6). The data will be published at Lee et al. (2008), J. Food Biochem. 32, 782-794. x,y

SEMb

Cheorun Jo and Myung Woo Byun

80

80

70

70

Fluorescence in tail (%)

Fluorescence in tail (%)

192

60 50 40 30 20 10 0

60 50 40 30 20 10 0

control

0

0.1

1

10

control

100

0

80

80

70

70

60 50 40 30 20 10 0

0.1

1

10

100

μg /ml of sample + 200μM H2O2

Fluorescence in tail (%)

Fluorescence in tail (%)

μg /ml of sample + 200 μM H2O2

60 50 40 30 20 10 0

control

0

0.1

1

10

100

μg /ml of sample + 200 μM H2O2

control

0

0.1

1

10

100

μg /ml of sample + 200μM H2O2

Figure 9. Antigenotoxic effect of irradiated green tea leaf and stem extracts on oxidative DNA damage in human leucocytes. A, Non-irradiated green tea leaf extract; B, Irradiated green tea leaf extract at 20 kGy; C, Non-irradiated green tea stem extracts; D, Irradiated green tea stem extract at 20 kGy.

Another study was conducted with a similar method and indicated that irradiation decreased total phenolic contents in GTLE from 98.6 to 79.91 mg/g but that of green tea stem extract was increased from 67.83 to 71.94 mg/g after irradiation. Total flavanol and ascorbic acid contents also showed the same trends with the total phenolic contents. The 50% inhibition concentration (IC50, (g/mL) of green tea leaf and stem extract against superoxide radical and hydroxyl radical also demonstrated that green tea stem extract showed higher activity when irradiation was applied (Table 9). However, catechins and caffeine content of the both GTLE and stem extracts decreased by irradiation at 20 kGy (Table 10). From the results, irradiation of green tea extract may have beneficial effects to improve industrial applicability by enhancing the color with shorter time and lower cost than conventional method and biological functions. In addition, the green tea byproducts, the remainder of green tea processing, has still been considered as a potential functional ingredient for industrial applications, in terms of an increased cost-effectiveness, when compared with the green tea leaf. Irradiation of green tea byproducts may also have beneficial effects on its functional activities.

New Method to Improve the Function and Industrial Applicability of Green Tea … 193 Table 10. Effect of irradiation on catechins and caffeine in green tea leaf and stem extracts. All values are on a dry basis (mg/g) Irradiation dose (kGy) Epicatechin Epicatechingallate Epigallocatechin Epigallocatechin gallate Gallocatechin Gallocatechingallate Caffein Gallic acid Total catechin Total epicatechins Total monomeric flavanol

Green tea leaf 0 20 4.19b 4.84a 1.11a 0.70b 10.01a 7.42b 8.14a 6.32b 0.58a 0.58a 0.07a 8.69a 6.96b 0.17a 0.09c 0.65 0.58 23.45 19.28 24.1 19.86

Green tea stem 0 20 3.21c 1.35d -c -c 4.20c 2.90d 4.57c 2.91d 0.30b 0.20c 0.54c 0.64c 0.12b 0.09c 0.30 0.20 11.98 7.16 12.28 7.36

SEM1 0.03 0.24 0.06 0.01 0.12 0.02

a-d

Different letters within the same row are significantly different (P < 0.05). Standard errors of the mean (n = 12). The data will be published at Lee et al. (2008), J. Food Biochem. 32, 782-794. 1

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Arihara, K. (2006). Strategies for designing novel functional meat products. Meat Sci. 74, 219-229. Byun, M. W., Kim, J. H., Lee, J. W., Park, J. W., Hong, C. S. & Kang, I. J. (2000a). Effects of gamma radiation on the conformational and antigenic properties of heat-stable major allergen in brown shrimp. J. Food Prot. 63, 940-944. Byun, M. W., Lee, K. H., Kim, D. H., Kim, J. H., Yook, H. S., & Ahn, H. J. (2000b). Effects of gamma radiation on sensory qualities, microbiological and chemical properties of salted and fermented squid. J. Food Prot. 63, 934-939. Byun, M. W., Jo, C., Lee, K. H. & Kim, K. S. (2002). Chlorophyll breakdown by gamma irradiation in a model system containing linoleic acid. J Am. Oil Chem. Soc. 79, 145-150. Byun, M. W., Jo, C., Lee, J. W., Jo, S. K. & Kim, K. S. (2004a). Application of radiation technology to develop green tea leaf as a natural resource for the cosmetic industry. Raidat. Phy. Chem. 71, 487-489. Byun, M. W., Jo, C., Jeon, T. W. & Hong, C. H. (2004b). Effect of gamma irradiation on color characteristics and biological activities of extracts of Lonicera japonica with methanol and acetone. LWT Food Sci. Technol. 37, 29-33. Chen, Z. Y., Chan, P. T., Ma, H. M., Fung, K. P., & Wang, J. (1996). Antioxidant effect of ethanol tea effects on oxidation of canola oil. J. Am Oil Chem. Soc., 73, 375-380. Choi, U., Shin, D. H., Chang, Y. S., & Shin, J. I. (1989). Sceeening of natural antioxidant from plant and their antioxidative effect. Korean J. Food Sci. Technol., 24, 142-148. Daun, J. K. (1982). The relationship between rapeseed chlorophyll, rapeseed oil chlorophyll and percentage green seeds. J Am. Oil Chem. Soc. 59, 15-18. Esposito, F., Arlotti, G., Bonifati, A. M., Napolitano, A., Vitale, D. & Foglitano, A. (2005). Antioxidant activity and dietary fiber in durum wheat bran byproducts. Food Res. Int. 38, 1167-1173. Fakourelis, N., Lee, E. C. & Min, D. B. (1987). Effects of chlorophyll and β-carotene on the oxidation stability of olive oil, J. Food Sci. 52, 234-235. Gennadios, A., Hanna, M. A. & Kurth, L. B. (1997). Application of edible coating on meats, poultry and seafoods: a review. LWT Food Sci. Technol. 30, 337-350. Griffiths, C. E., Wang, T. S., Hamiton, T. A., Voorhees, J. J. & Ellis, C. N. (1992). A photonumeric scale for the assessment of cutaneous photodamage. Arch. Dermatol. 128, 347-351. Grove, G. I., Grove, M. J. & Leyden, J. J. (1989). Optical profilometry. An objective method for quantification of facial wrinkles. J. Am. Acad. Dermatol. 21, 631-637. Jimenez-Colmenero, F., Cabrallo, J. & Cofrades, S. (2001). Healthier meat and meat products, their role as functional foods. Meat Sci. 59, 5-13. Jo, C., Son, J. H., Lee, H. J. & Byun, M. W. (2003a). Irradiation application for color removal and purification of green tea leaves extract. Radiat. Phy. Chem. 66, 179-184. Jo, C., Son, J. H., Sohn, C. B. & Byun, M. W. (2003b). Functional properties of raw and cooked pork patties with added irradiated, freeze-dried green tea leaf extract powder during storage at 4°C. Meat Sci. 64, 13-17. Jo, C., Yook, H. S., Lee, M. S., Kim, J. H., Song, H. P., Kwon, J. S. & Byun, M. W. (2003c). Irradiation effects on embryotoxicity and oxidative properties of gossypol dissolved in methanol. Food Chem. Toxicol. 41, 1329-1336.

New Method to Improve the Function and Industrial Applicability of Green Tea … 195 Jo, C., Son, J. H., Shin, M. G. & Byun, M. W. (2003d). Irradiation effects on color and functional properties of persimmon (Diospyros kaki L. folium) leaf extract and licorice (Glycyrrhiza Uralensis Fischer) root extract during storage. Lebensm. –Wiss u –Technol. 67, 143-148. Jo, C., Ahn, H. J., Son, J. H., Lee, J. W. & Byun, M. W. (2003e). Packaging and irradiation effect on lipid oxidation, color, residual nitrite content, and nitrosamine formation in irradiated cooked pork sausage. Food Control 14, 7-12. Jo, C., Lee, W. D., Kim, D. H., Kim, J. H., Ahn, H. J. & Byun, M. W. (2004). Quality attributes of low salt Changran Jeotkal (aged and seasoned intestine of Alaska Pollock, Therage chalcogramma) developed using gamma irradiation. Food Control 15, 435-440. Jo, C., Jeong, S. M., Kim, S. Y., Park, E. J., and Lee, S. C. (2008). Effect of irradiation on the antioxidative and antigenotoxic activities of green tea leaf and stem extract. Int. J. Food Sci. Technol. 43, 400-405. Kang, H. J., Chawla, S. P., Jo, C., Kwon, J. H. & Byun, M. W. (2006). The studies on development of functional powder from citrus peel. Bioresource Technol. 97, 614-620. Kang, H. J., Jo, C., Kwon, J. H., Kim, J. H., Chung, H. J. & Byun, M. W. (2007). Effect of pectin-based edible coating containing green tea powder on the quality of irradiated pork patty. Food Control 18, 430-435. Ki, M. S. 2004. A study on the elevation of the brand value of bosung green tea. KFMAS 22:155-178. Kim, D. H., Jo, C., Yook, H. S., Park, B. J. & Byun, M. W. (2002). Enhancement of preservation characteristic of Meju, an intermediate material for Korean legume-based fermented soy sauce, Kanjang, by irradiation. Radiat. Phy. Chem. 64, 317-312. Kim, J. H., Ahn, H. J., Kim, D. H., Jo, C., Yook, H. S., Park, H. J. & Byun, M. W. (2003). Irradiation effects on biogenic amines in Korean fermented soybean paste during fermentation. J. Food Sci. 68, 80-84. Kim, J. H., Ahn, H. J., Jo, C., Park, H. J., Chung, Y. J. & Byun, M. W. (2004). Radiolysis of biogenic amines in model system by gamma irradiation. Food Control 15, 405-408. Kim, J. K., Jo, C., Hwang, H. J., Park, H. J., Kim, Y. J. & Byun, M. W. (2006). Color improvement by irradiation of Curcuma aromatica extract for industrial application. Radiat. Phy. Chem. 75, 449-452. Kim, J. T. (1996). Science and culture of tea. Borimsa Publishing Co., Seoul, Korea Lapornik, B., Prosek, M. & Wondra, A. G. (2005). Comparison of extracts prepared from plant byproducts using different solvents and extraction time. J. Food Engineer. 71, 214222. Lee, J. W., Yook, H. S., Lee, K. H., Kim, J. H. & Byun, M. W. (2000). Conformational changes of myosine by gamma irradiation. Radiat Phy Chem 58, 271-277. Lee, J. W., Kim, J. H., Yook, H. S., Kang, K. O., Lee, W. Y., Hwang, H. J. & Byun, M. W. (2001). Effects of gamma radiation on the allergenicity and antigenicity properties of milk proteins. J. Food Prot. 64, 272-276. Lee, K. H., Ahn, H. J., Jo, C., Yook, H. S. & Byun, M. W. (2002). Production of low salted and fermented shrimp by irradiation process. J. Food Sci. 67, 1772-1777. Lee, N. Y., Jo, C., Sohn, S. H., Kim, J. K. & Byun, M. W. (2006). Effect of gamma irradiation on the biological activity of green tea byproduct extracts and a comparison with green tea leaf extracts. J. Food Sci. 71, C269-C274.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 10

GREEN TEA CATECHIN AS ANGIOGENESIS INHIBITOR Kiminori Matsubara1,2,* and Yoshiyuki Mizushina2,3 1

Department of Human Life Sciences Education, Graduate School of Education, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8524, Japan 2 Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan 3 Department of Nutritional Science, Kobe-Gakuin University, Kobe, Hyogo 651-2180, Japan

ABSTRACT Many studies suggest that beneficial effect of green tea for human health is mainly attributed to catechins, polyphenol compounds, having anti-oxidative activity. In the last decade, new aspect of green tea catechin has emerged as angiogenesis inhibitor. Angiogenesis is forming new blood vessels from a pre-existing blood vessel and involved in various diseases including tumor growth and metastasis, diabetic retinopathy and atherosclerosis. In addition, it has been demonstrated that obesity is prevented by angiogenesis inhibitor and suggested that angiogenesis is closely associated with Alzheimer’s disease. Thus, angiogenesis inhibitors in food would be expected to prevent these diseases and give beneficial effect on our health. Interestingly, inhibitory effect of green tea catechin on angiogenesis has been demonstrated in various models suggesting green tea catechin could suppress cancer. Furthermore, it has been revealed that a higher consumption of green tea catechin reduces human body fat and is associated with a lower prevalence of cognitive impairment. These evidences suggest that anti-angiogenic activity of green tea catechin might play important roles in human health.

*

Corresponding author. Kiminori Matsubara, Ph.D., Department of Human Life Sciences Education, Graduate School of Education, Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-8524, Japan. Tel; +81-82424-6854; Fax; +81-82-4227133; E-mail; [email protected]

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INTRODUCTION Angiogenesis is a phenomenon forming new blood vessels from a pre-existing blood vessel. This physiological process is necessary to supply nutrients and oxygen for development, wound healing and reproduction [1]. On the other hand, pathological blood vessel formation is involved in various diseases including tumor growth and metastasis, rheumatoid and other diseases [2]. Recently, it has been demonstrated that metabolic disorder causes various diseases such as diabetic retinopathy, atherosclerosis, and obesity, which are associated with angiogenesis. Thus, suppression of pathological angiogenesis could prevent these diseases. In fact, therapeutic strategy for inhibiting-angiogenesis has been developed especially in cancer patients. This angiogenesis targeting therapy was proposed by Dr. Judah Folkman more than 30 years ago [3]. Folkman’s group demonstrated that tumor growth depends on angiogenesis to obtain nutrients and oxygen and to remove metabolites [4]. After that, extensive studies have been conducted on angiogenesis inhibitors, and data obtained from a lot of experiments and clinical trials for cancer therapy using angiogenesis inhibitors support the usefulness of this strategy. This simple and elegant concept provides a strong strategy for cancer therapy. In addition, as mentioned above, angiogenesis is involved in various diseases, thus angiogenesis inhibitors could be used as drugs for various diseases. There are many candidates for anti-angiogenesis drugs in clinical trials. One of the most well-known angiogenesis inhibitors in cancer treatment is a humanized anti-VEGF antibody, bevacizumab (Avastin; Genentech), which blocks the role of angiogenic factor, vascular endothelial growth factor (VEGF). This anti-angiogenesis cancer agent validates the usefulness of targeting angiogenesis in metastatic colorectal cancer [5]. Many other VEGF inhibitors are in cancer clinical trials. In addition, angiogenesis inhibitors are potential drugs in non-oncology indications, such as age-related macular degradation, rheumatoid arthritis, psoriasis, endometriosis and cerebral oedema [5]. On the other hand, many diseases associated with angiogenesis are life-style relating diseases. Therefore, to prevent such diseases many people would intake healthy food, from which various biologically active substances have been isolated and characterized such as polyphenols in red wine, green tea, herbs, vegetables. In fact, many of these polyphenols exert pharmacological effects, and their anti-angiogenic activity is one of their attractive effects [6]. As epidemiological studies have shown that red wine and green tea consumption would lower the risk of coronary heart diseases and cancer [7-13], anti-angiogenic polyphenols from red wine and green tea have attractive attention [14]. In this review, we focus on the anti-angiogenic effect of green tea catechin and summarize the mechanisms how green tea catechin exerts the anti-angiogenic activity and beneficial effects for human health.

GREEN TEA CATECHIN AND ANGIOGENESIS The first report of anti-angiogenic effect of dietary polyphenol would be genistein from soybean [15]. After this report, many research have been conducted on anti-angiogenic activity of dietary polyphenols. In 1999, Cao and Cao, a Swedish research group, reported that green tea catechin has anti-angiogeinc activity [16]. They demonstrated anti-angiogenic activity of green tea catechin, especially epigallocatechin gallate (EGCG) being abundant

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green tea catechin, by cornea and chorioallantoic membrane (CAM) assays and endothelial cell model. Furthermore, it is notable that oral administration of green tea catechin suppressed angiogenesis in vivo model, showing the usefulness of consumption of green tea. The mechanisms by which green tea catechin, mainly EGCG, exerts anti-angiogenic activity have been extensively studied. Suppressive effects of green tea catechin on in vitro angiogenesis models have been demonstrated, and the molecular targets of green tea catechin have been revealed; inhibition of VEGF receptor signaling [17, 18] and membrane-type I matrix metalloproteinase [19], suppression of vascular endothelial (VE)-cadherin tyrosine phosphorylation and Akt activation [20] and downregulation of transcription factors, Ets-1, cFos, and c-Jun [21]. As reactive oxygen species (ROS) are known as stimulants for angiogenesis [22, 23], antioxidant activity of green tea catechin may in part account for the anti-angiogeinc activity. As green tea catechin is considered a good model to develop angiogenesis inhibitors, green tea catechin derivatives are expected to exert stronger anti-angiogenic activity. We synthesized green tea catechin derivatives and their activities were compared [24, 25]. The green tea catechin derivatives conjugated with fatty acid enhanced anti-angiogenic activity and also strongly inhibited DNA polymerase activity. Further investigation on green tea catechin derivatives could find more effective angiogenesis inhibitors.

GREEN TEA CATECHIN AND CANCER Cancer is the most serious medical problem in the world. Epidemiological studies have shown that consumption of green tea lowers the risk of cancer, and green tea catechin suppresses tumorigenesis and tumor growth [9, 10, 12]. Green tea catechin inhibits cancer cells proliferation at least in part by inhibiting DNA polymerases [26] and induces apoptosis [27]. Thus, green tea catechin would directly prevent cancer cell growth. However, the concentration of green tea catechin was relatively low than IC50 in cancer cell growth inhibition [27]. This fact puts an idea that green catechin exerts anti-cancer effect through a different mechanism. As the mechanism, inhibition of angiogenesis emerged, and antiangiogenic activity of green tea catechin was ascertained [16]. VEGF-induced angiogenesis in cornea of mice drinking green tea was suppressed. Therefore, anti-cancer effect of green tea catechin would be due to inhibition of cancer cell growth and angiogenesis, which is a critical for tumor growth [2, 3]. Many cancer cells produce VEGF to induce angiogenesis. Green tea catechin also suppresses VEGF production from many cancer cells [28-30]. Fibroblast growth factors are known as other important pro-angiogenic factors. Green tea catechin also suppresses fibroblast growth factor production [31]. Furthermore, green tea catechin suppresses cellular matrix degradation enzymes such as gelatinases and matrix metalloproteinases [32, 33]. These enzymes are involved in not only angiogenesis but also cancer cell metastasis. Thus, green tea catechin may reduce cancer cell metastasis. However, the relationship between green tea consumption and low mortality of cancer seems to be controversial [34-36] due to the complex mechanisms of cancer.

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GREEN TEA CATECHIN AND CARDIOVASCULAR DISEASES Coronary heart disease has been serious medical problem in Western countries. Patients are increasing in Asian and other countries. It is well-known that “French paradox”; red wine consumption lowers coronary heart disease in French people [7]. Interestingly, epidemiological studies have shown an inverse relationship between green tea consumption and coronary heart disease [9-11]. Recent a large population-based epidemiological research in Japan supports that green tea consumption reduces mortality due to cardiovascular disease [34]. The formation and progression of atherosclerosis lesions are considered to be excessive vascular remodeling and chronic inflammation resulting in lipid accumulation and complex lesions in the arterial lumen [37, 38]. Following description of a review in which details of atherosclerosis initiation and progression are explained [38], the revealed mechanisms of preventive effects of green tea catechin are summarized. Initiating events of atherosclerosis are recruitment of macrophages and their subsequent up-take of low-density lipoprotein (LDL)-derived cholesterol. As oxidative modifications in the lipid and apolipoprotein B (apo B) components of LDL initiate formation of atherosclerosis and induce LDL aggregation causing macrophage degradation [39], inhibition of the oxidative component formation would be very important. Green tea catechins prevent LDL oxidation [40] due to their anti-oxidant property and LDL aggregation [41]. However, green tea catechins have no effect on oxidized LDL-induced endothelial cell and monocyte THP-1 cell interaction [42], which is involved in atherosclerosis development. Vascular smooth muscle cells (VSMCs) also play important role in atherosclerosis. VSMCs secrete VEGF in atherosclerotic plaques inducing plaque angiogenesis [43, 44], which is observed in atherosclerotic lesions. Angiogenesis inhibitors reduce intimal new blood vessel formation and plaque growth [45]. As green tea catechin has anti-angiogenic activity and inhibits platelet derived growth factor-induced VEGF expression in VSMCs [46], regular consumption of green tea could reduce atherosclerotic plaque development. In addition, green tea catechin induces proliferating VSMC apoptosis and prevents VSMC invasion [47, 48].

GREEN TEA CATECHIN AND OBESITY Obesity or overweight has become serious medical problem in United States and other developed countries because obesity or overweight substantially increases the risk of various diseases including type 2 diabetes mellitus, hypertension, dyslipidemia, coronary heart disease, and colon cancer [49]. Thus, body weight control by pharmacological and nonpharmacological treatments is very important to reduce the risks of overweight or obesityrelating diseases. As obesity or overweight patients have excess adipose tissue, a strategy to reduce the mass of adipose tissue seems to be a simple one. However, body weight control is regulated by complex systems, and adipocytes have unique characteristics secreting various hormones and differentiated from stem cells [50]. Thus, there are many targets for treatment of overweight patients. In 2002 a new concept for body weight control emerged from U. S. research group; adipose mass can be regulated by the vasculature [51]. Most organs in adult body do not increase the mass however adipose tissue can increase the mass like tumor growth. As all organs including adipose tissue need blood supply, growing adipose tissue

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mass accompanies new blood vessel formation. Rupnick et al. demonstrated that angiogenesis inhibitors could suppress adipose tissue growth [51]. The effectiveness of this strategy to suppress overweight using angiogenesis inhibitors is confirmed by the other group [52]. These basic experiments suggest that food-derived angiogenesis inhibitors can suppress adipose tissue growth. There are many food-derive angiogenesis inhibitors; green tea catechins, curcumin, resveratrol, etc. Interestingly, green tea catechins have only been demonstrated to have anti-obesity effect for humans [53, 54]. The mechanisms by which green tea catechins suppress obesity are demonstrated to disturb the absorption of glucose and lipids [55-57] and to stimulate lipid catabolism in the liver [58]. In addition these mechanisms, at least in part, anti-angiogenic effect of green tea catechins would contribute to suppression of adipose tissue growth.

GREEN TEA CATECHIN AND DEMENTIA Dementia and Alzheimer’s disease (AD) are a public health problem in modern society [59-61]. The number of aged population is growing, and these diseases will become more serious concern in the world. Several risk factors for vascular dementia have been reported [59, 62-65]. In addition, green tea catechin has been shown to have neuroprotective effect and to ameliorate neurodegenerative diseases such as AD and Parkinson disease [66]. Molecular mechanisms of neruoprotective and neurorescue activity of green tea catechin have been revealed; protective effects against β-amyloid (A β)-induced neurotoxicity, modulation of survival and cell cycle genes and promotion of neurite outgrowth activity [67-69]. On the other hand, anti-angiogenic activity of green tea catechin may play an important role in AD prevention because it has been suggested that drugs that reduce the risk of AD have antiangiogenic activity [70]. In consistent with these studies, a recent epidemiological research shows that a higher consumption of green tea is associated with a lower prevalence of cognitive impairment [71]. These scientific data suggest that the intake of green tea would be useful to prevent dementia. In summary, angiogenesis is involved in various major diseases, which are rapidly increasing in the world as mentioned in this chapter. Consumption of green tea catechin lowers the risks of such diseases. The anti-angiogenic activity of green tea catechin would contribute to the lower prevalence of such diseases as observed in Asian countries. Further investigation on green tea catechin would give clinical significance of many diseases, and green tea would be recognized as “Asian Paradox” [72] like “French Paradox”.

ACKNOWLEDGMENTS Our works on anti-angiogenic natural products including green tea cactechin and derivatives are supported by “Academic Frontier” Project for Private Universities (KobeGakuin University): matching fund subsidy from Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT), 2006-2010, (K. M. and Y. M.). K. M. acknowledges a Grant-in-Aid for Young Scientists (B) (No. 18700608) from MEXT.

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[64] Cummings, J. L. Alzheimer's disease. N Engl J Med 2004, 351, 56-67. [65] Haan, M. N.; Wallace, R. Can dementia be prevented? Brain aging in a populationbased context. Annu Rev Public Health 2004, 25, 1-24. [66] Mandel, S.; Youdim, M. B. Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases. Free Radic Biol Med 2004, 37, 304-17. [67] Levites, Y.; Amit, T.; Mandel, S.; Youdim, M. B. Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J 2003, 17, 952-4. [68] Levites, Y.; Amit, T.; Youdim, M. B.; Mandel, S. Involvement of protein kinase C activation and cell survival/ cell cycle genes in green tea polyphenol (-)epigallocatechin 3-gallate neuroprotective action. J Biol Chem 2002, 277, 30574-80. [69] Reznichenko, L.; Amit, T.; Youdim, M. B.; Mandel, S. Green tea polyphenol (-)epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth. J Neurochem 2005, 93, 1157-67. [70] Vagnucci, A. H., Jr.; Li, W. W. Alzheimer's disease and angiogenesis. Lancet 2003, 361, 605-8. [71] Kuriyama, S.; Hozawa, A.; Ohmori, K.; Shimazu, T.; Matsui, T.; Ebihara, S.; Awata, S.; Nagatomi, R.; Arai, H.; Tsuji, I. Green tea consumption and cognitive function: a cross-sectional study from the Tsurugaya Project 1. Am J Clin Nutr 2006, 83, 355-61. [72] Sumpio, B. E.; Cordova, A. C.; Berke-Schlessel, D. W.; Qin, F.; Chen, Q. H. Green tea, the "Asian paradox," and cardiovascular disease. J Am Coll Surg 2006, 202, 813-25.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 11

NEUROPROTECTIVE EFFECT OF THEANINE ON CEREBRAL ISCHEMIA Nobuaki Egashira1,2,*, Kenichi Mishima1, Katsunori Iwasaki1, Ryozo Oishi2 and Michihiro Fujiwara1 1

Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan 2 Department of Pharmacy, Kyushu University Hospital, Fukuoka 812-8582, Japan

ABSTRACT The present article introduces our study related to the neuroprotective effect of γglutamylethylamide (theanine), a component Japanese green tea (Camellia sinensis), on cerebral ischemia. Theanine (1 mg/kg) significantly decreased the size of the cerebral infarcts in a 4 h middle cerebral artery (MCA) occlusion model in mice. However, theanine did not affect the cerebral blood flow, brain temperature and physiological variables (pH, pCO2, pO2 and hematocrit) in this model. Theanine also reduced the alterations of NeuN (neuron), GFAP (astrocyte) and Iba 1 (microglia) expression levels at 24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by bicuculline, γ-aminobutyric acidA (GABAA) receptor antagonist, but not 3mercaptopropionic acid, glutamate decarboxylase inhibitor. Furthermore, theanine (0.3 and 1 mg/kg) significantly prevented the impairment of spatial memory in rats subjected to twice-repeated cerebral ischemia, 7 days after the second reperfusion. In addition, theanine (1 mg/kg) significantly inhibited the decrease in the number of surviving cells in the hippocampal CA1 field in the same rats. These results suggest that theanine directly provides neuroprotection against cerebral ischemia and its neuroprotective effect is mediated, at least in part, by GABAA receptors, and that it may be clinically useful for preventing cerebrovascular disease.

*

Address correspondence to: Nobuaki Egashira, Ph.D. Department of Pharmacy, Kyushu University Hospital, 3-11 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Telephone: 81-92-642-5920; FAX: 81-92-642-5937; Email: [email protected]

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Keywords: γ-Gutamylethylamide; Theanine; Cerebral ischemia; Neuroprotection; Middle cerebral artery; Cerebrovascular disease; GABAA receptor.

ABBREVIATIONS Theanine, MCA, GABA, AMPA, NMDA, EGCG, CBF, BBB, SHR, DPPH, CNS, GAD,

γ-glutamylethylamide; middle cerebral artery; γ-aminobutyric acid; α-amino-3-hydroxy-5-methyl-4-isoxazole; N-methy-D-aspartate; epigallocatechin gallate; cerebral blood flow; blood-brain barrier; spontaneously hypertensive rats; 2,2-Diphenyl-1-picryl-hydrazyl; central nervous system; glutamate decarboxylase

INTRODUCTION In general, Japanese people drink several cups of tea including Japanese green tea (Camellia sinensis) daily to take a break or relieve stress. The physiological and pharmacological actions of various components of Japanese green tea such as catechins, caffeine, γ-aminobutyric acid (GABA) have been studied [1-6]. The administration of these components has affected blood pressure and stress. Moreover, catechins such as epigallocatechin gallate (EGCG) have antioxidative [7] and anti-ischemic effects [8]. γ-Glutamylethylamide (theanine), a component of Japanese green tea, is a natural glutamate analog (Figure 1). While theanine has been known to have relaxing properties, the pharmacology of theanine is relatively unknown. Several studies have shown evidence for multiple pharmacological effects on various neurochemical systems. These pharmacological effects include: (1) inhibition of glutamate reuptake by inhibition of the glutamate transporter [9]; (2) increase in GABA concentrations [10]; (3) increases in dopamine release in the striatum in rats [11]; (4) decreases or no change in brain serotonin levels in rats [12, 13]; (5) inhibition of the binding of α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA), kainate and N-methy-D-aspartate (NMDA) receptors [14]; and inhibition of lipid peroxidation [15]. The present article introduces our study related to the neuroprotective effect of theanine on cerebral ischemia.

Neuroprotective Effect of Theanine on Cerebral Ischemia

COOH

COOH

CHNH 2 CH 2

CHNH2 CH2

CH 2 CONHC2H 5

CH2 COOH

L-theanine

L-glutamic acid

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Figure 1. Chemical structures of theanine and glutamic acid.

EFFECT OF THEANINE ON CEREBRAL INFARCTION IN MICE Kakuda et al. [16] reported that global cerebral ischemia-induced delayed neuronal death in the hippocampal CA1 region was prevented by i.c.v. injection of theanine in gerbils. We previously reported that systemic injection of theanine (1 mg/kg, i.p.) reduced the size of cerebral infarcts following occlusion of the middle cerebral artery (MCA) for four hours in mice [17]. In our study, theanine significantly reduced the infarct volume when it was injected immediately before and 3 h after the occlusion. Theanine also reduced the infarct volume when it was injected 3 h after the occlusion. Moreover, we showed that theanine did not affect cerebral blood flow (CBF), brain temperature or physiological variables such as pH, pCO2, pO2 and the hematocrit of plasma. Kakuda et al [16] also reported that the i.c.v. injection of theanine did not decrease brain temperature in gerbils. In addition, it has been reported that a high dose (2000 mg/kg, i.p.) of theanine did not alter blood pressure in spontaneously hypertensive rats (SHR) [18]. We also found that theanine (1-100 mg/kg, i.p.) had no effect on the blood pressure in SHR (N Egashira et al., unpublished data). These findings suggest that theanine provides neuroprotection directly, against focal cerebral ischemia. Kimura et al. [10] reported that 30 min after the i.p. injection of theanine labeled with 14 C, the amide was incorporated into the mouse brain without any metabolic change. Yokogoshi et al. [11, 19] also reported that theanine administered orally was transported into the brain through the blood-brain barrier (BBB). These findings suggested that theanine passes easily through the BBB. The neuroprotective effect of systemic administration of theanine on the cerebral ischemia provided further evidence for the permeability of the BBB to theanine. Maruyama and Takeda [20] suggested that theanine is a competitive antagonist of glutamate receptors. Kakuda et al. [14] also reported that theanine bound both AMPA and NMDA receptors in rat cortical neurons, and its binding activity for AMPA receptors was 10fold that for NMDA receptors. It has been reported that AMPA receptor antagonists provide protection against ischemia induced by MCA occlusion in rats [21-24] and forebrain ischemia in gerbils and rats [25, 26]. We also reported that a selective competitive AMPA receptor antagonist could provide neuroprotective activity and improve the spatial memory impaired by repeated ischemia in rats [27]. These findings suggested that there were antagonistic effects of theanine on the AMPA receptor providing neuroprotective action. However, this

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was suggested as one of the mechanisms, because the binding activity of theanine to AMPA receptors was lower than that of L-glutamic acid [14]. Furthermore, we found that theanine (0.1-100 μM) did not prevent the cell damage induced by glutamate (100 μM) or kainate (1 mM) in rat cultured hippocampal neurons (N Egashira et al., unpublished data). Theanine has been reported to inhibit lipid peroxidation [15]. We reported that vitamin E isoforms such as α-tocotrienol and γ-tocopherol, which inhibit lipid peroxidation, prevented focal cerebral ischemia induced by MCA occlusion [28]. These isoforms were injected i.v. immediately before and 3 h after the occlusion. These findings suggest that the period of radical formation is a few hours after the occlusion in this model. Theanine also reduced infarct volume when it was injected 3 h after the occlusion [17]. However, we found that vitamin E isoforms markedly exhibit 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radicalscavenging activity, whereas theanine did not exhibit its activity (N Egashira et al., unpublished data). Therefore, it is unlikely that theanine prevented the focal cerebral ischemia through radical-scavenging activity.

INVOLVEMENT OF GABAA RECEPTORS IN NEUROPROTECTIVE EFFECT OF THEANINE ON FOCAL CEREBRAL ISCHEMIA IN MICE Following an i.p. injection of theanine into mice, 14C-labeled theanine was incorporated into the brain, without metabolic change and resulted in elevation of intracerebral levels of GABA within 30 min [10], suggesting that theanine is transported into the brain through the BBB and alter intracerebral GABA levels. GABA is the principal inhibitory neurotransmitter in the mammalian brain. GABAA receptors are mainly located postsynaptically and mediate the majority of inhibitory synaptic transmission in the central nervous system (CNS). GABA synthesis and GABAA receptor expression is reduced following occlusion of the MCA, suggesting that these receptors play a role in cerebral ischemia [29, 30]. It has been shown that tiagabine, a GABA reuptake inhibitor, and muscimol, a GABAA receptor agonist, have a neuroprotective action on cerebral ischemia in rats [31, 32]. Moreover, the anesthetics propofol and isoflurane have protective effects on cerebral ischemia, which can be reversed by bicuculline, a GABAA receptor antagonist [33, 34]. These findings suggest that activation of the GABAA receptor might play a role in inhibiting neuronal death induced by cerebral ischemia. We investigated whether the GABAA receptor was involved in neuroprotection by theanine following ischemic brain damage in the MCA occlusion mouse model [35]. The GABAA receptor antagonist bicuculline (10 mg/kg, i.p.) prevented the inhibition of infarct by theanine although when administered alone, bicuculline had no effect on the size of infarct (Figure 2). Our findings showed that the neuroprotective effect of theanine is mediated, at least in part, by GABAA receptors (Figure 3). In our studies, theanine was effective by injection 1 h before reperfusion in MCA model. Moreover, theanine-treatment 30 min before reperfusion is also effective (N Egashira et al., unpublished data). However, we found that theanine-treatment immediately after reperfusion had no effect on the focal cerebral ischemia in same model (N Egashira et al., unpublished data). An i.p. injection of theanine was reported to increase the intracerebral level of GABA 30 min after injection of theanine [10]. These findings suggest that the neuroprotective effect of theanine is due to act indirectly on

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GABAA receptors during reperfusion. Therefore, theanine could prevent the focal cerebral ischemia by injection 30-60 min before reperfusion. In this study, the number of NeuN (neuron) and GFAP (astrocyte) positive cells was decreased, while the number of Iba1 (microglia) positive cells was increased in the cerebral cortex at 24 h after MCA occlusion, and theanine (1 mg/kg, i.p.) inhibited these alterations [35]. Iba1 immunoreactive cells rapidly appear at 3.5 h after reperfusion [36]. Therefore, the decrease of Iba1 expression levels is a result of neuroprotection by theanine.

Infarct volume (mm3)

100

#

80

* *

60 40 20 0 Vehicle

Theanine 1 mg/kg (i.p.) Bicuculline 10 mg/kg (i.p.)

Distance from frontal pole 2mm 4mm 6mm 8mm Vehicle

Theanine 1 mg/kg (i.p.) Bicuculline 10 mg/kg (i.p.)

Figure 2. Effects of theanine and bicuculline on infarct volume following MCA occlusion in mice. Theanine and bicuculline were injected i.p. 3 h after the occlusion. Slices were immediately stained with 2% 2,3,5-triphenyltetrazolium chloride 24 h after MCA occlusion. **P<0.01 compared with vehicle, #P<0.05 compared with theanine alone. (This figure was reproduced from reference#35 under permit).

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Glutamatergic neuron GABAnergic neuron

Theanine

+

GABA Glutamate pre GABAA receptor

post NMDA receptor AMPA receptor

Fig. 3

Neuroprotective effect

Figure 3. Theanine prevents the cerebral infarcts following occlusion of the MCA. The neuroprotective effect of theanine is mediated, at least in part, by GABAA receptors.

In general, glutamic acid is metabolized into GABA by glutamate decarboxylase (GAD). When theanine was administered intragastrically to rats, the concentration of theanine in the brain was increased, however the concentration of glutamic acid in the brain remained unchanged [19]. These findings suggest that theanine increases the intracerebral level of GABA through a mechanism other than metabolism by GAD. We also found that GAD inhibitor 3-MPA had no effect on the protective effect of theanine on focal cerebral ischemia [35]. Therefore, GAD is not involved in the neuroprotective effect of theanine on focal cerebral ischemia.

EFFECT OF THEANINE ON REPEATED ISCHEMIA-INDUCED IMPAIRMENT OF SPATIAL MEMORY AND NEURONAL CELL DEATH We previously reported that repeated ischemia (10 min x 2 times, 1 h interval) caused cell death and acetylcholine dysfunction in the hippocampus, and the impairment of spatial memory in radial maze test in rats [37]. We also reported that a AMPA receptor antagonist YM-90K but not a NMDA receptor antagonist MK-801 prevented both this impairment of spatial memory and cell death, suggesting that AMPA receptor may play an important role on

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the impairment of the spatial memory in the rats subjected to repeated ischemia [27]. It has been determined that the AMPA receptor has four subunits, designated GluR1 through GluR4. AMPA receptors exist as either Ca2+-impermeable or Ca2+-permeable channels depending on their subunit consititution; that is, the presence of the GluR2 subunit renders heteromeric AMPA receptor assembly Ca2+-impermeable, while an AMPA receptor without GluR2 subunit shows Ca2+-permeability [38, 39]. It has also been determined that a marked reduction of GluR2 expression in vulnerable neurons prior to cell death following cerebral transient ischemia, and overexpression of Ca2+-permeable via AMPA receptor lacking the GluR2 subunit promoted delayed cell death of hippocampal CA1 neurons following the cerebral ischemia [40]. The decrease of GluR2 mRNA is the triggers for the degeneration of the CA1 pyramidal cell [41]. Moreover, we have reported that repeated ischemia reduced ratios of GluR2 subunit variants to those of GluR1 [42]. AMPA receptors play an important role in mediating cerebral ischemia [43]. Cerebral ischemia produces two main changes in the AMPA receptors: up-regulation [44] and conformational changes characterized by decrease of GluR2 mRNA subunit in hippocampal CA1 neurons [45]. These changes, together with overstimulation of AMPA receptors by increased glutamate, induce excessive Ca2+ influx, leading to intracellular Ca2+ overload which triggers mainly an apoptotic type of cell death with a more delayed onset [43]. We found that theanine significantly prevents the impairment of spatial memory and inhibits the decrease in the number of surviving cells in the hippocampal CA1 field in rats subjected to repeated cerebral ischemia [46]. These results suggest that theanine prevents memory impairment induced by repeated cerebral ischemia, at least in part, by protecting against neuronal cell death. Maruyama and Takeda [20] suggested that theanine is a competitive antagonist of glutamate receptors. Kakuda et al. [14] reported that theanine binds both AMPA and NMDA receptors, and that its binding affinity for AMPA receptors is 10fold that for NMDA receptors. It has been reported that AMPA receptor antagonists provide protection against ischemia induced by MCA occlusion in rats [21-23] and forebrain ischemia in gerbils and rats [25, 26]. We also reported that a selective, competitive AMPA receptor antagonist could provide neuroprotective activity and reduce the impairment in spatial memory induced by repeated cerebral ischemia in rats [27]. Theanine might provide a neuroprotective action via antagonistic effects on AMPA receptors. This mechanism was suggested as just one of the multiple mechanisms, because the binding affinity of theanine to AMPA receptors is lower than that of L-glutamic acid [14]. Arias et al. [47] reported that the neuroprotective effect of a combination of AMPA and NMDA receptor antagonists was greater than the sum of the effects of the individual antagonists in an in vitro model of cerebral ischemia. Therefore, the neuroprotective effect of theanine may be due to its antagonistic effects on both AMPA and NMDA receptors. It has also been reported that i.p. injection of theanine increases the intracerebral level of GABA 30 min after injection [10]. GABA reuptake inhibitors and GABAA receptor agonists have a neuroprotective action on cerebral ischemia in rats [31, 32]. Also, the anesthetics propofol and isoflurane have protective effects on neuronal damage induced by cerebral ischemia, which can be reversed by bicuculline, a GABAA receptor antagonist [33, 34]. Moreover, we previously found that the neuroprotective effect of theanine was reversed by bicuculline in MCA occlusion mice [35]. Therefore, theanine might prevent the impairment of spatial memory and neuronal cell death induced by repeated cerebral ischemia not only by blocking the glutamate receptors, but also by acting on the GABAA receptors.

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This study shows that i.p. injection of theanine significantly prevents the impairment of spatial memory and neuronal cell death induced by repeated cerebral ischemia. We previously demonstrated that i.p. injection of theanine reduced the size of cerebral infarcts in MCA occlusion mice [17]. These findings suggest that systemic administration of theanine provides neuroprotection against cerebral ischemia. Kimura and Murata [10] reported that 30 min after i.p. injection of theanine, it was incorporated into the mouse brain without any metabolic change. In addition, Yokogoshi et al. [11, 18] reported that theanine, when administered orally, was transported into the brain through the BBB, suggesting that theanine passes easily through this barrier. Moreover, theanine exerts a neuroprotective action against cerebral ischemia when administered systemically, providing further evidence for the permeability of the BBB to theanine. We previously showed that an effective dose of theanine did not alter CBF, brain temperature or physiological variables such as pH, pCO2, pO2 and the hematocrit of plasma, in MCA occlusion mice [17]. Moreover, Kakuda et al. [16] reported that i.c.v. injection of theanine into gerbils did not decrease brain temperature. In addition, a high dose of theanine (2000 mg/kg, i.p.) did not alter blood pressure in rats [18]. In this study, we found that theanine did not affect body temperature in rats subjected to repeated cerebral ischemia (N. Egashira et al., unpublished data). Thus, theanine provides neuroprotection against cerebral ischemia without changes in CBF, body temperature, brain temperature or physiological responses.

CONCLUSION In conclusion, the findings of our study showed that systemic administration of theanine protected against the focal cerebral ischemia induced by MCA occlusion without affecting CBF and brain temperature. Our findings also show that systemic administration of theanine prevents the impairment of spatial memory and neuronal cell death induced by repeated cerebral ischemia. These results suggest that theanine may be clinically useful for preventing cerebrovascular disease. Moreover, the results of this study imply that GABAA receptors are involved in the neuroprotective mechanism of theanine following cerebral ischemia.

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[34] Elsersy, H., Mixco, J., Sheng, H., Pearlstein, R.D., & Warner, D.S. (2006). Selective gamma-aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. Anesthesiology, 105, 81-90. [35] Egashira, N., Hayakawa, K., Osajima, M., Mishima, K., Iwasaki, K., Oishi, R., & Fujiwara, M. (2007). Involvement of GABAA receptors in the neuroprotective effect of theanine on focal cerebral ischemia in mice. J Pharmacol Sci, 105, 211-214. [36] Ito, D., Tanaka, K., Suzuki, S., Dembo, T., & Fukuuchi, Y. (2001). Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke, 32, 1208-1215. [37] Chung, E.H., Iwasaki, K., Mishima, K., Egashira, N., & Fujiwara, M. (2002). Repeated cerebral ischemia induced hippocampal cell death and impairments of spatial cognition in the rat. Life Sci, 72, 609-619. [38] Ozawa, S., Kamiya, H., & Tsuzuki, K. (1998). Glutamate receptors in the mammalian central nervous system. Pro Neurobio, 54, 581-618. [39] Jia, Z., Agopyan, N., Miu, P., Xiong, Z., Henderson, J., Gerlai, R., Taverna, F.A., Velumian, A., MacDonald, J., Carlen, P., Abramow-Newerly, W., & Roder, J. (1996). Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron, 17, 945-956. [40] Colbourne, F., Grooms, S.Y., Zukin, R.S., Buchan, A.M., & Bennett, M.V. (2003). Hypothermia rescues hippocampal CA1 neurons and attenuates down-regulation of the AMPA receptor GluR2 subunit after forebrain ischemia. Proc Natl Acad Sci U S A, 100, 2906-2910. [41] Tanaka, H., Grooms, S.Y., Bennett, M.V., & Zukin, R.S. (2000). The AMPAR subunit GluR2: still front and center-stage. Brain Res, 886, 190-207. [42] Hatip-Al-Khatib, I., Iwasaki, K., Egashira, N., Ishibashi, D., Mishima, K., & Fujiwara, M. Comparison of single- and repeated-ischemia-induced changes in expression of flip and flop splice variants of AMPA receptor subtypes GluR1 and GluR2 in the rats hippocampus CA1 subregion. J Pharmacol Sci, 103, 83-91. [43] Choi, D.W. (1996). Ischemia-induced neuronal apoptosis. Curr Opin Neurobiol, 6, 667–672. [44] Driscoll, M. (1996). Cell death in C. elegans: molecular insights into mechanisms conserved between nematodes and mammals. Brain Pathol, 6, 411-425. [45] Davies, S.N., Lester, R.A.J., Reymann, K.G., & Collingridge, G.L. (1989). Temporally distinct pre- and post-synaptic mechanism maintain long-term potentiation. Nature, 338, 500-503. [46] Egashira, N., Ishigami, N., Pu, F., Mishima, K., Iwasaki, K., Orito, K., Oishi, R., & Fujiwara, M. (2008). Theanine prevents memory impairment induced by repeated cerebral ischemia in rats. Phytother Res, 22, 65-68. [47] Arias, R.L., Tasse, J.R.P., & Bowlby, M.R. (1999). Neuroprotective interaction effects of NMDA and AMPA receptor antagonists in an an vitro model of cerebral ischemia. Brain Res, 816, 299-308.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 12

CHARACTERIZATION OF THE NEUROPROTECTIVE ACTIVITY OF THE POLYPHENOL (-)EPIGALLOCATECHIN-3-GALLATE IN THE BRAIN Orly Weinreb*, Tamar Amit, Moussa B. H. Youdim and Silvia Mandel Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel

ABSTRACT Standing after water, tea signifies the second most frequently consumed beverage worldwide, which varies its status from a simple ancient drink and a cultural tradition to a nutrient endowed with possible neurobiological-pharmacological actions beneficial to human health. Accumulating evidence suggests that oxidative stress resulting in reactive oxygen species generation plays a pivotal role in neurodegenerative diseases, supporting the implementation of radical scavengers and metal chelating agents, such as natural tea polyphenols for therapy. Vast epidemiology data indicate a correlation between occurrence of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases and green tea consumption. In particular, literature on the putative novel neuroprotective mechanism of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG) strengthens the notion that diverse molecular signaling pathways, participating in the neuroprotective activity of EGCG, thus rendering this natural compound as potential agent to reduce the risk of various neurodegenerative diseases. In the present article, we review the studies concerning the mechanisms of action implicated in EGCG-induced neuroprotection in the brain and discuss the vision to translate these findings into a lifestyle arena.

*

Correspondence should be addressed to: Dr. Orly Weinreb. Department of Pharmacology, Technion- Faculty of Medicine; P.O.B. 9649, Haifa 31096, Israel. Tel: +972-4-8295291; Fax: +972-4-8513145; E-mail: [email protected]

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ABBREVIATIONS AD Aβ ARE COMT BBB DA EC ECG EGCG EGC MAPK ERK1/2 GAP-43 6-OHDA HSP90 HIF-1 3-HK iNOS MEK1 MPP+ MPTP OS PD PKC PC12 cells R-APO ROS GSH  sAPPα TNF-α TfR SNPC VEGF UPS

Alzheimer’s disease; amyloid-beta peptide, antioxidant response elements; catechol-O-methyltransferase; blood-brain barrier; dopamine; (–)-epicatechin; epicatechin-3-gallate; (-)-epigallocatechin-3-gallate; (–)-epigallocatechin; extracellular mitogen-activated protein kinases; extracellular signal-regulated kinases; growth associated protein-43; 6-hydroxydopamine; heat shock protein 90; hypoxia inducible factor-1; 3-Hydroxykynurenine; inducible nitric oxide synthase; mitogen-activated protein kinase 1; 1-methyl-4-phenylpyridinium; N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; oxidation stress; Parkinson’s disease; protein kinase C; rat pheochromocytoma cells; R-apomorphine; reactive oxygen species; reduced glutathione; soluble amyloid precursor protein; tumor necrosis-alpha, transferrin receptor; substantia nigra pars compacta; vascular endothelial growth factor; ubiquitin proteasome system.

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Tri-hydroxyl group (pyrogallol structure)

B A

C

Galloyl group

Figure 1. The chemical structure of eight polyphenolic flavanol-type compounds namely, (+)-catechin (C), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechingallate (CG), (-)-epicatechin gallate (ECG), (+)-gallocatechin gallate (GCG) and (-)-epigallocatechin-3- gallate (EGCG).

INTRODUCTION The natural polyphenols present in beverages obtained from plants, fruits and vegetables such as olive oil, red wine and teas, have been suggested to be beneficial and favorable to human health (see reviews, [1, 2]). Flavonoids are the largest group of polyphenols, mainly divided into anthocyanins, glycosylated derivative of anthocyanidin, present in colorful flowers and fruits, and anthoxantins, colorless compounds which are further divided into several monomeric compound categories including flavones, isoflavones, falvanols flavans, and flavonols [3]. Flavonoids have a diphenypropane (C6C3C6) skeleton consisting of an aromatic ring (Figure 1), which is condensed, to a heterocyclic ring and attached to a second aromatic ring [3]. The abundant phenolic hydroxyl groups on the aromatic rings confer the antioxidant and the iron chelating activities of these compounds [4]. The importance of polyphenolic flavonoids in enhancing cell resistance to oxidative stress (OS) goes beyond the simple scavenging and iron chelating activities and is mostly interesting in those pathologies, where OS and iron are involved. Numerous studies in the last decade have shown that polyphenols have in-vitro and in-vivo activities in preventing and/or reducing the deleterious effects of oxygen-derived free radicals, associated with several chronic and stress related human diseases [5, 6]. Several lines of evidence suggest that OS resulting in reactive oxygen species (ROS) generation, either through an enzyme or metal catalyzed processes, play a pivotal role in clinical disorders, such as atherosclerosis, ischemia-reperfusion injury, cancer, stroke and neurodegenerative disorders [5, 6]. Special interest has been assigned to the therapeutic feature of antioxidants and nutritional approach

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in neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) [7, 8]. Oxidative damage to neuronal molecules and increased accumulation of iron in specific brain areas are considered major pathological aspects of PD and AD [9]. Although the etiology of both disorders and their respective dopaminergic or cholinergic neuronal degeneration remains elusive, the chemical pathology of PD shows many similarities to AD, involving increase in iron concentration in the affected areas, release of mitochondrial cytochrome c, protein aggregation, OS, loss of tissue reduced glutathione (GSH), reduction in mitochondrial complex I activity and increased lipid peroxidation [10, 11]. A large investigation on PD and cognitive impairment found a moderate risk reduction in black (fermented), oolong (semi-fermented) and green (not-fermented) teas consumers compared to non tea drinkers [12-14]. The favorable properties ascribed to tea consumption are believed to rely on its bioactive flavanol class-related catechins and their derivatives, demonstrated to act as radical scavengers and exert indirect antioxidant effects through activation of transcription factors, signaling regulators and antioxidant enzymes (for reviews see [1,15,16]). In line with this evidence, particular attention has been placed to study the neuroprotective action of antioxidants, iron-chelating and anti-inflammatory agents, tea catechins and especially, the major component of green tea, (-)-epigallocatechin-3-gallate (EGCG) [17-19]. The revelation of novel molecular targets, possibly implicated in their neuroprotective action include: calcium homeostasis [20], the extracellular mitogen-activated protein kinases (MAPK) [21] and protein kinase C (PKC) signaling pathways [22, 23] and regulation of antioxidant enzymes [24], antioxidant response element (ARE) [25], cell death and cell survival genes and proteins associated with mitochondrial function, such as Bcl-2 family members [26-28], amyloid precursor protein (APP) pathway [22, 29] and iron regulators and sensors encoding genes and proteins, such as transferrin receptor (TfR) and hypoxia-inducible factor (HIF) prolyl-hydroxylases [29-31] (Figure 2). This review aims to compile the most recent literature regarding the mechanisms of action implicated in EGCG-induced neuroprotection in the brain, which might be a reflective outcome of its brain permeable, antioxidant and iron chelating properties.

GREEN TEA POLYPHENOLS Green tea is the most widely consumed beverage aside from water in Japan, China, and other Asian nations and is becoming more popular in Western countries. Green tea belongs to the Theacease family derived from two plant varieties, Camellia sinesis and assamica [32]. The first scientific recognition of medicinal properties of green tea was in the 16th century, using extracts as therapeutic mean to cure fever, headache, stomach and articulation pain [33]. To date, green tea has attracted attention for its health benefits, particularly with respect to its potential for preventing and treating cancer, cardiovascular diseases, inflammatory diseases, aging and neurodegenerative diseases in human [34-36].

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Figure 2. A schematic model of EGCG neuroprotective mechanism of action indicating potential pathways of activity with respect to its suggested multifunctional effects in neuronal and extra-neuronal tissues. Full explanation is discussed in the text.

Catechins account for 25-35% of the green tea dry extract and consist of eight polyphenolic flavonoid-type compounds, namely, (+)-catechin (C), (-)-epicatechin (EC), (+)gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechingallate (CG), (-)-epicatechin gallate (ECG), (+)-gallocatechin gallate (GCG) and EGCG, (Figure 1). The most abundant polyphenol is EGCG, thought to particularly contribute to the beneficial effects attributed to green tea, such as its neuroprotective and antioxidant properties. It is well established that the catechins contain free radical scavenging properties and act as biological antioxidants [18, 37]. It has been demonstrated that they can scavenge both superoxide and hydroxyl radicals, as well as the 1,1-diphenyl-3-picrylhydrazyl radical, peroxyl radicals, nitric oxide, carboncenter free radicals, singlet oxygen and lipid free radicals, and peroxynitrite by preventing the nitration of tyrosine [37-39]. Additionally, catechins chelate metal ions such as copper (II) and iron (III) to form inactive complexes and prevent the generation of potentially damaging free radicals [40]. Catechins have been found to be more efficient radical scavengers than vitamin E and C [18, 37]. Relative antioxidant activity among tea catechins has been found to be EGCG=ECG >EGC>EC [41, 42]. EGCG accounts for more than 10% of the extract dry weight, 20-35 mg per cup of green tea, followed by EGC >EC ≥ ECG [41, 42]. The metabolism of green tea catechins has been studied in various animals including human [43, 44]. Orally catechin administration to human is absorbed, metabolized and excreted within 24 h [45]. Study with healthy green tea consumers revealed levels of EGCG, EGC and EC in the plasma in a dose-dependent concentration, varying between 0.2 and 2% of the ingested amount, with maximal concentration 1.4-2.4 h after ingestion [46]. In addition, after ingestion of 1.2 g of green tea solids (dissolved in two cups of warm water), the plasma

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samples collected at 1 h from human volunteers contain 46-268 ng/ml [44]. The half-life for EGCG is about 5 h and for EGC and EC it varies between 2.4 and 3.4 h [47, 48]. Several reports indicated that tea polyphenols can be attained in the brain and exert neuroprotective effect simply by drinking. Previously, it was reported that EC metabolites (epicatechin glucuronide and 3’-O-methylated epicatechin glucuronide), formed after oral ingestion of EC by rats, had gained entry to the brain [49]. Furthermore, study with labeled EGCG demonstrated a wide distribution of radioactivity in mouse organs including brain, after oral administration and small amount of [3H] EGCG excretion in the urine after direct administration [50]. Recently, the absorption and pharmacokinetic of EGCG in various brain regions of adult and fetal rats have been demonstrated by oral and intravenous administration, indicating that EGCG is the most abundant catechin in brain tissue [51] and may potentially penetrate through the blood-brain barrier (BBB) [52]. In vitro model of BBB demonstrated that various flavonoids and some metabolites were able to traverse the BBB and that the potential for permeation was consistent with compound lipophilicity [53].

THE BENEFICIAL EFFECTS OF EGCG IN NEURODEGENERATIVE DISEASES Human Epidemiology and Clinical Trials Notwithstanding the uncertainty on the capacity of tea polyphenols to penetrate the brain and lack of well-controlled clinical data, green tea polyphenols were suggested to inversely correlate with the incidence of dementia and brain aging and neurodegeneration, such in PD and AD. Previous epidemiological studies have shown a reduced risk of PD associated with consumption of 2 cups/day or more of black tea [54]. In support, Tan et al [55] found an inverse association between black tea and PD, based on a 12 year prospective study of over 63,000 men and women, that was due to black tea ingredients separate from its caffeine content. In a cross-sectional study aimed at investigating an association between consumption of green tea and cognitive function in elderly Japanese subjects, it was found that higher consumption of green tea is associated with lower prevalence of cognitive impairment [13]. Despite the fact that the prevalence of PD is much lower in tea consumers, the association of green tea drinking and risk of AD and other neurodegenerative diseases is not well established. No case-control study has been accomplished that points to a beneficial effect associated with tea consumption in AD, although treatment with the extract EGb761 derived from Gingko biloba leaves, another natural flavonoid that does not contain catechins, improved cognitive performance of AD patients [56]. These findings emphasize the importance of well-designed controlled studies to assess a risk reduction of PD and AD in consumers of green and black tea. Indeed, a randomized, double-blind and efficacy study in Beijing China is under completion, to determine the safety, tolerability and potential neuroprotective effects of a green tea polyphenol enriched preparation, in de novo PD patients without taking any anti-Parkinsonism drug (sponsored by the Michael J. Fox Foundation for Parkinson's Research).

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Neuroprotection in Vitro and in Animal Models of Neurodegenerative Diseases Neuroprotective in-vivo studies employing the parkinsonism-inducing neurotoxin, Nmethyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have shown that both green tea extract and EGCG possess highly potent activities in preventing mice striatal dopamine (DA) depletion and substantia nigra (SN) dopaminergic neuron loss [24]. One possible mechanism underlying the effectiveness of green tea and EGCG against MPTP neurotoxicity, may involve its catechol-like structure, since it is known that catechol-containing compounds are potent radical anti-oxidants and ferric ion chelators [39]. In agreement, the iron chelators, radical scavengers and catechol derivative compounds R-apomorphine (R-APO), a DA receptor agonist and its S-isomer, induced neuroprotection in animal models of PD [57, 58]. The catechol structural resemblance may account for a recently reported inhibitory effect of green tea polyphenols on [3H] DA uptake by presynaptic transporters. This inhibition was suggested to block the metabolic product of MPTP, the neurotoxin 1-methyl-4phenylpyridinium (MPP+) uptake (because of competition for the vesicular transporter) thereby protecting DA-containing neurons against MPP+-induced injury [59]. In-vitro studies also demonstrated inhibition of MPP+ and 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by EGCG [26]. Furthermore, EGCG, at a low IC50 concentration (0.2 μM), inhibited the activity of the enzyme catechol-O-methyltransferase (COMT) in rat liver cytosol homogenates [60]. DA and related catecholamines are physiological substrates of COMT. The COMT inhibitors entacapone and tolcapone, clinically prescribed to PD-affected individuals, dose-dependently inhibited the formation of the major metabolite of levodopa, 3O-methyldopa, thereby improving its bioavailability in the brain [61]. In addition, the implication of the iron chelation property of EGCG in neuroprotection in PD animal models has been strengthened by the observations that both MPTP and 6-OHDA significantly increased iron in SN pars compacta (SNPC) of mice, rats and monkeys [62, 63]. Indeed, iron accumulation in the brain has been reported in a range of neurodegenerative disorders [64] and demonstrated to accumulate in neurons and microglia in SN of PD patients [65]. Although AD epidemiological studies did not report any established outcome relative to green tea consumptions, in-vitro observations showed that EGCG prevents amyloid beta peptides (Aβ)-induced neurotoxicity, [22, 66] and EC reduces nascent Aβ fibrils, elongation of the fibrils, and destabilization of the formed assemblies [67]. In addition, EGCG was able to regulate the proteolytic processing of APP under in-vivo and in-vitro conditions [22, 29], suggesting that green tea polyphenols might be potentially promising therapeutic agents not only for PD, but also for AD. EGCG promoted the non-amyloidogenic α-secretase pathway of APP in neuronal cell cultures resulting in a consequential augment in soluble APPα (sAPPα) [22]. This increase was inhibited by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, indicating that this effect was mediated via α-secretase processing [22]. Also, long-term treatment of mice with EGCG resulted in decreases in cell-associated, fulllength APP levels, as well as increases in the sAPPα levels in the hippocampus [22]. New supportive data came from a study conducted in an Alzheimer's transgenic mice model ("Swedish" mutant APP overexpressing, APPswTg) mice, showing that EGCG promoted sAPPα generation and decreased Aβ levels and plaques via promotion of the nonamyloidogenic α-secretase proteolytic pathway [68, 69]. Recently, long-term administration

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of EGCG provided prophylactic benefits on rat spatial cognitive learning impairment caused by Aβ cerebral ventricleinfusion [70]. Supplementary preclinical models of other neurodegenerative diseases reported neuroprotective effects of EGCG, such as prolongation of the symptom onset and life span and attenuation of death signals in ALS mice model expressing the human G93A mutated Cu/Zn-superoxide dismutase (SOD1) gene [71, 72] and in a variety of cerebral ischemia animal models [33, 73].

THE MECHANISMS UNDERLYING EGCG-INDUCED NEUROPROTECTION Antioxidant and Iron Chelating Mechanism of Action The protective effect of EGCG against neurological diseases may involve its radical scavenging and iron chelating activity and/or regulation of antioxidant protective enzymes. The inhibition of enzymes, whose activity may promote OS or an increase of antioxidant enzyme activities, might have beneficial significance to neuroprotection. Previous studies reported that EGCG elevated the activity of two major antioxidant enzymes, superoxide dismutase (SOD) and catalase in mice striatum [24] (Figure 2). In addition, tea polyphenols were found to be potent scavengers of free radicals, such as singlet oxygen, superoxide anions, hydroxyl radicals and peroxy radicals, in a number of invitro systems [18, 74, 75]. In the majority of these studies, EGCG was shown to be more efficient as a radical scavenger than its counterparts ECG, EC and EGC, which may be attributed to the presence of the trihydroxyl group on the B ring (Figure 1) and the gallate moiety at the 3’ position in the C ring [18]. EGCG attenuated hydroxykynurenine (3-HK) induced cell death, as well as the increase in ROS concentrations and caspase-3 activity in neuronal culture, presumably via its antioxidant activity [76]. In rat brain tissue, green tea and black tea extracts were shown to inhibit lipid peroxidation promoted by iron-ascorbate in brain mitochondrial membranes [77]. A similar effect was also reported using brain synaptosomes, in which the four major polyphenol catechins of green tea were shown to inhibit iron-induced lipid peroxidation [39]. In this regard, it has been shown that EGCG attenuated paraquat-induced microsomal lipid peroxidation and increased the survival rate of paraquat-poisoned mice [78, 79]. In addition, Higuchi et al. [78] suggested that EGCG inhibited paraquate-induced malondialdehyde production in rat liver microsome system containing FeSO4 by two possible mechanisms: one was by scavenging of superoxide radicals, which were responsible for the reduction of ferric to ferrous, catalyzed by the Fenton reaction. The other was through iron-chelating activity, given that the inhibition disappeared when excessive amount of FeSO4 were added to the reaction, indicating that EGCG inhibited iron driven lipid peroxidation by pulling out available iron. The ability of green tea polyphenols and EGCG in particular to chelate metal ions, such as iron and copper, may contribute to their antioxidant/neuroprotective activity by inhibiting transition metal-catalysed free radical formation. The two points of attachment of transition metal ions to the flavonoids molecule are: the o-diphenolic groups in the 3’,4’-dihydroxy positions in the B ring, and the keto structure 4-keto, 3-hyroxy or 4-keto and 5-hydroxy in the

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C ring of the flavonols [4, 80]. The ability of green tea polyphenols to act as relatively potent metal chelators [39, 81] may be of major significance for treatment of neurodegenerative diseases, where iron accumulation has been shown in brain areas where neurodegeneration occurs. Most importantly, green tea was reported not to affect iron absorption in healthy human subjects [82]. The localization of iron and ferritin in PD patients is restricted to specific brain areas [9, 83, 84] in the SNPC, but not the reticulata [83]. Similarly, AD pathogenesis is associated with iron accumulation and is linked to the characteristic neocortical Aβ deposition, phosphorylation of tau and tangle formation, which may be mediated by abnormal interaction with excess of free-chelatable iron. Ionic iron can, in turn, participate in the Fenton reaction with subsequent generation of ROS, thought to initiate the processes of OS and inflammatory cascade resulting in production of cytotoxic cytokines (tumor necrosis-alpha (TNF-α), interluekin-1 and -6) in the microglia and in the surrounding neurons [85, 86] and activation of transcription factors and nuclear factor-kappa B (NF-κB) [87, 88]. Indeed, a marked increase in NF-κB immunoreactivity was found in the nucleus of melanized dopaminergic neurons of the Parkinsonian SNPC, compared to normal brains [89]. EGCG was found to inhibit the nuclear translocation of NF-κB in in-vitro systems: immunofluorescence and electromobility shift assays showed that introduction of green tea extract before 6-OHDA, inhibited both NF-κB nuclear translocation and binding activity in human neuroblastoma SH-SY5Y cells [77, 90]. Furthermore, the reduced activity of NF-κB by EGCG and the theaflavin-3,3'-digallate polyphenol from black tea, was associated with inhibition of lipopolysaccharide (LPS)-induced TNF- α production [91] and the enzyme inducible nitric oxide synthase (iNOS) [87, 92, 93], which is responsible for the production of the short-live free radical, nitric oxide in activated macrophages. Interestingly, recent studies have identified a novel link between iron and AD, associated with an enhancement of endogenous APP translation and subsequent Aβ formation, via activation of an iron responsive element (IRE-type II) in the 5' untranslated region (UTR) of APP mRNA [94]. This finding opened a new potential therapeutic avenue aimed at reducing amyloidosis with iron-complexing drugs that modulate APP mRNA translation. In support, a recent in vitro study demonstrated that EGCG reduced full-length APP in SH-SY5Y cells without altering APP mRNA levels, while exogenous iron supplementation reversed its effect [29], suggesting a post-transcriptional action, presumably by the mechanism of chelating intracellular iron pools (Figure 2). This is further supported by the observation that EGCG suppressed translation of a luciferase reporter gene driven by the IRE-type II-containing sequences of APP [29]. Furthermore, it was found that EGCG markedly reduced secreted Aβ levels in the conditioned medium of Chinese hamster ovarian cells, overexpressing the "Swedish" mutated APP (CHO/ΔNL) [29] and in primary neuronal cells derived from transgenic mice bearing the APP "Swedish" mutation [69]. More recently, Friedlich et al [95] have described a putative IRE in the 5'-UTR of PDrelated α-synuclein mRNA and predicted that this RNA structure may have the potential to function as a post-transcriptional regulator of its protein synthesis in response to iron and redox events, resembling the pattern seen with APP and the iron-associated protein ferritin. This finding can explain, in part, previous observation demonstrating that the iron chelating compounds R-APO and EGCG, prevented iron-dependent up-regulation of α-synuclein in the SNPC of MPTP-treated mice, resulting in neuroprotection of SN dopaminergic neurons [96]. Thus, the radical scavenging and free-iron complexing activities of green tea polyphenols

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may directly influence aggregation and deposition of either Aβ or α-synuclein in brains of AD and PD patients, respectively.

Regulation of Neuronal Survival PKC/MAPK Pathways Associated with Bcl-2 Family Members Emerging evidence suggests that the antioxidant activity of green tea polyphenols cannot be the exclusive mechanism responsible for their neuroprotective action, but rather, their ability to alter signaling pathways may significantly contribute to the cell survival effects. Modulation of cellular survival and signal transduction pathways has significant biological consequences that are important in understanding the various pharmacological and toxicological responses of antioxidant drugs. A number of intracellular signaling pathways have been described to play central functions in EGCG-promoted neuronal protection against a variety of extracellular insults, such as the MAPK [31, 97, 98], PKC [23, 26, 99, 100] and phosphatidylinositol-3-kinase (PI-3 kinase)-Akt [101-103] pathways, as described in Figure 2. Given the critical role of MAPK pathways in regulating cellular processes that are affected in neurodegenerative diseases, the importance of MAPKs as transducers of extracellular stimuli into a series of intracellular phosphorylation is being increasingly recognized. OS seems to be a major stimulus for MAPK cascade, which might lead to cell survival/cell death. Among the MAPKs, the extracellular signal-regulated kinases (ERK1/2) are mainly activated by mitogens and growth factors [104], while p38 and c-jun-N-terminal kinase (JNK) respond to stress stimuli [105, 106]. Previous in-vitro studies [25] demonstrated the potency of EGCG to induce ARE-mediated defensive genes and MAPKs pathways, including the cell survival signaling regulators, p44/42 ERK 1/2, JNK and p38 MAPK [107]. The role of ERK1/2 signaling seems to be connected to attenuation of neuronal death and cellular injury by OS [108]. EGCG counteracted the decline in ERK1/2 induced by 6-OHDA [26] and induced phosphorylation of ERK1/2 in serum-deprived SH-SY5Y cells [31]. Nonetheless, EGCG at its neuroprotective concentrations (1-10 μM), did not affect the levels of ERK1/2 phosphorylation by itself, in the absence of any exogenous damage, in neuronal cell line [26]. EGCG neuroprotective activity also involves the intracellular signaling mediator PKC [22, 23], thought to have an essential role in the regulation of cell survival and programmed cell death [109, 110]. A rapid loss of neuronal PKC activity is a common consequence of brain neurodegeneration [111, 112]. The induction of PKC activity in neurons by EGCG (110 μM) is thought to be a prerequisite for neuroprotection against several neurotoxins, such as Aβ [22], serum withdrawal [23, 100, 103, 113] and 6-OHDA [26] since inhibition of PKC phosphorylation completely abolished the protection induced by EGCG and by a PKC activator, phorbol 12-myristate 13-acetate (PMA). These in-vitro results were supported by a previous report [22], who showed that EGCG oral administration to mice (2 mg/kg) caused a significant increase of the PKC isozymes α end ε protein levels in the membrane and cytosolic fractions of hippocampus. These isoforms play a crucial role in cell survival and differentiation pathways [114] and may be involved in APP regulatory processing associated with the pathogenesis of AD [115, 116]. Indeed, previous studies in brains of AD patients demonstrated reduction of PKCε activity [117].

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The mechanism by which PKC activation leads to neuroprotection begins to be elucidated. Studies with extra-neuronal tissues support a role for PKCα as a kinase of the antiapoptotic Bcl-2, probably through direct or indirect phosphorylation of this cell survival protein [118]. Neuroprotective experimental studies demonstrated that the protective effect of EGCG involved reduction of the apoptotic markers, cleaved caspase 3, its downstream cleaved substrate poly-ADP-ribose-polymerase (PARP), a nuclear zinc finger DNA-binding protein that detects and binds to DNA strand breaks and Bad, a member of a group of “BH3 domain only” proteins of the Bcl-2 family [23, 28]. This is supported by the observation that EGCG could not overcome neuronal death under PKC pathway blockade, suggesting that this cascade is essential for the neuroprotection and neurorescue effects of EGCG [23]. Recently, we have identified a novel pathway in the neuroprotective mechanism of action of EGCG, which involves a rapid PKC-mediated degradation of Bad protein by the ubiquitin proteasome system (UPS) in SH-SY5Y cells [27]. Bad has been suggested to link survival signals to the mitochondrial cell death machinery. Thus, the newly described role of Bad during the initial response to EGCG-induced cell signaling, may potentially contribute to the illumination of the EGCG mechanism of neuroprotection/neurorescue action. In addition, EGCG was shown to induce a rapid translocation of the isoform PKCα to the membrane compartment in human astroglioma or rat pheochromocytoma PC12 cells [23, 119], as well as upregulation of PKCε mRNA expression and a concentration-dependent activation of PKCε in serum-deprived in SH-SY5Y cells [100]. These findings are supported by animal studies showing that two weeks oral consumption of EGCG prevented the extensive depletion of PKCα and counteracted the robust increase of Bax protein in the striatum and SNPC of mice intoxicated with MPTP [103]. A previous study in human epidermal keratinocytes indicated that EGCG promoted cell survival by increasing the ratio of Bcl-2 to proapoptotic Bax and phosphorylation of Bad through ERK and AKT signaling pathways [120]. Using mitogen-activated protein kinase 1 (MEK1) inhibitor (PD98059), EGCG induced only the phosphorylation of serine (Ser)136 of Bad, and when the authors used PI-3 kinase inhibitor (LY294002), EGCG induced only the phosphorylation of Ser112 of Bad. These results indicated that EGCG affects both the ERK pathway, which is involved in phosphorylation of Bad at Ser112 and the PI-3 Kinase/AKT pathway, involved in phosphorylation of Bad at Ser136 (Figure 2). Nonetheless, a study with high concentrations with EGCG reported cell proliferation arrest of tumor cells and inhibition of ERK1/2 and AKT phosphorylation, which was associated with reduced phosphorylation of Bad [121]. This biphasic mode of biological activity of EGCG relies on its concentrationdependent window of pharmacological action: EGCG exhibits pro-oxidant and pro-apoptotic activity at high concentrations, which are responsible for its anti-cancer-cell death effect, while lower doses exert neuroprotection against a wide spectrum of neurotoxic compounds [16, 122]. A biphasic mode of action has been described for most of the typical radical scavengers and antioxidants, such as ascorbic acid (vitamin C) [123] and iron chelators, such as R-APO [124]. When SH-SY5Y cells were challenged with 6-OHDA or reduced content of serum, a low concentration of EGCG (<10 μM) abolished the induction of proapoptoticrelated mRNAs and the decrease in Bcl-2, Bcl-w and Bcl-xL [28, 77]. The neuroprotective effect of EGCG is thought to be mediated through down-regulation of pro-apoptotic genes, as shown for mdm2, caspase-1, cyclin dependent kinase inhibitor p21 and TNF-related apoptosis-inducing ligand, TRAIL rather than up-regulation of anti-apoptotic genes [28].

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Theses findings support the assumption that complementary mechanisms, in addition to antioxidant activity, are involved in the neuronal survival effect of EGCG.

Promoting of Neuronal Outgrowth Our recent proteomic studies [31] have demonstrated that EGCG increases the level of the binding protein, 14-3-3 gamma. By their interaction with more than 100 binding partners, 14-3-3 protein family members modulate the action of proteins that are involved in cell cycle and transcriptional control, signal transduction, intracellular trafficking, regulation of ion channels and expression of cytoskeletal components [125]. In this regards the neurorescue/neuroprotective activity of EGCG may be associated with the induction of 14-33 gamma, interacting with kinases of the PKC pathway and Bad and consequently preventing neuronal death (Figure 2). Indeed, EGCG has been shown to be neuroprotective via activation of PKC signaling, inhibition of activation of caspase-3 and its substrate, poly ADP-ribose polymerase cleavage and induction of Bad degradation [23, 27, 31, 126]. A previous study has demonstrated that overexpression of 14-3-3 gamma contributed to the regulation of the dynamics of glial fibrillary acidic protein (GFAP) filaments, which may facilitate the stability of the cytoskeleton, and thus play a specific neuroprotective role in the brain of patients with AD [127]. In fact, recent proteomic analysis showed that EGCG dose-dependently increased the expression levels of the stabilizer proteins of chromatin organization and DNA, histone H1 member 4 and cytoskeletal proteins, such as the actin binding protein tropomyosin 3 and beta-tubulin IV [31]. Since cytoskeletal proteins play a crucial role in promoting neurite outgrowth [128], these results suggest that induction of the structural proteins by EGCG are associated with its differentiation features including neurite extension, cell body elongation and up-regulation of the growth associated protein-43 (GAP-43)[23, 100] in rat pheochromocytoma PC12 cells [23] and human neuroblastoma SH-SY5Y cells [100].

Regulation of Hypoxia Inducible Factor-1 (HIF-1) Pathway An emerging target for neuroprotection associated with iron chelation implicates the activation of a hypoxia signal transduction pathway that culminates in the stabilization of the transcriptional activator HIF-1 and increased transcription of genes mediating compensatory survival processes in response to OS [129, 130]. The presence of HIF-1 within the cells is under the strict control of a class of iron-dependent and oxygen-sensor enzymes named the HIF prolyl-4-hydroxylases [129]. This family of enzymes hydroxylates critical proline and asparagine residues in HIF upon high oxygen levels and iron overload, targeting it for degradation by the UPS. This may explain the decrease in HIF-dependent cell survival genes described in neurodegenerative diseases, such as phosphofructokinase and the angiogenic vascular endothelial growth factor (VEGF) [131]. In this scheme, iron chelators would stabilize HIF-1α, which in turn would heterodimerize with its partner, HIF-1β in the nucleus, bind to an hypoxia responsive element in regulatory genes and transactivate the expression of established protective genes, including VEGF, erythropoietin, p21waf1/cip1, glucose transporter1 and the glycolytic enzymes aldolase and enolase-1 [132, 133]. Indeed, EGCG and ECG

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were shown to induce HIF-1α protein and HIF-1 activity and increase mRNA expression levels of GLUT-1, VEGF, and p21waf1/cip1, whereas this effect was blocked by iron and ascorbate, indicating that these catechins may activate HIF-1 through the chelation of iron [134, 135]. Applying a neurorescue paradigm in neuronal culture, we have recently found that EGCG decreased mRNA transcript and protein levels of the beta-subunit of prolyl-4hydroxylase and the protein levels of two molecular chaperones, which are associated with HIF-regulation/degradation, the immunoglobulin-heavy-chain binding protein, BiP and the heat shock protein 90 (HSP90) [31] [100](Figure 2). In support, previous finding demonstrated that EGCG directly binds and inhibits HSP90 in mouse hepatoma cells [136]. Inhibition of HSP90 is considered a requirement for the rapid hypoxic stabilization of HIF1α, which otherwise might be degraded by unspecific pathway [137]. Thus, it is possible that the protective effect of EGCG under OS/hypoxic conditions may combine the suppression of hydroxyl radical formation via Fenton chemistry, as well as inhibition of iron-dependent prolyl hydroxylase. Another link between hypoxia and iron is reflected by the hypoxic-mediated positive regulation of the iron regulatory proteins, IRP1 and IRP2 and consequential transactivation of their target mRNAs, ferritin and transferrin receptor (TfR). Interestingly, the free ironinduced proteasomal-mediated degradation of IRP2 involves also activation of a prolyl hydroxylase and is inhibited by iron chelators [138-140]. Thus, it is possible that IRP2 is a substrate for this enzyme, in a similar way as HIF, signaling it for protein degradation. Thus, the reduction in the chelatable iron pool by EGCG may result in the inhibition of prolyl hydroxylases and consequently, in the concerted activation of both HIF and IRP2. As HIF-1 and IRPs coordinate the expression of a wide array of genes, involved in cellular iron homeostasis, survival and proliferation [141], their activation could be of major importance in neurodegenerative diseases. In support, recent findings suggest the application of low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases as novel neurological therapy for neurodegenerative diseases [130].

CONCLUSIONS Two main aspects are significantly contributing to the raising concept viewing green tea consumption of relevance to brain health: the factors and events that influence the incidence and progression of PD and AD are becoming better defined and understood; in parallel, the experimental evidence documenting the neuroprotective properties of green tea catechins, both in cell culture and animal studies is persistently increasing. It becomes evident that syndromes, such as AD and PD will require multiple drug therapy to address the varied pathological aspects of the disease. Therefore, the poly-pharmacological activities of green tea catechins may be of significance for neuroprotection. Earlier viewed as a mere antiimflammatory and antioxidant EGCG is at the present time considered a multimodal acting molecule, invoking various cellular neuroprotective/neurorescue mechanisms involving ironchelation, scavenging of oxygen and nitrogen radical species and activation of PKC signaling pathway and pro-survival genes (Figure 2). Their non-toxic, lipophilic (and thus brain permeable) nature is advocated for "ironing out iron" from those brain areas, where it preferentially accumulates in neurodegenerative diseases [19]. The chelation of the reactive

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free-iron pool by EGCG and consequent reduction in full-length APP translation, would contribute to the decreased Aβ generation/fibrillization, which together with the promotion of the non-amyloidogenic pathway and induction of neurite outgrowth, may converge in a slowdown in the process of nerve neruonal loss in AD [113]. Future efforts in the understanding of the protective mechanism of action of EGCG must concentrate on deciphering the cell targets, affected by this compound and other green tea catechins. Further preclinical studies are needed to clarify if EGCG and its metabolites, at sufficient concentrations, can reach the brain and may alter cell-signaling pathway and whether these effects can be successfully translated into prospect human studies to affect the progression of neurodegenerative disorders.

ACKNOWLEDGEMENT We thank the support of Rappaport Family Research, Technion- Israel Institute of Technology.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 13

CARDIOVASCULAR AND METABOLIC EFFECTS OF GREEN TEA Kamilla Kelemen* Department of Cardiology, Angiology and Pneumology University of Heidelberg, INF 410, 69120 Heidelberg, Germany

ABSTRACT Green tea (Camelia sinensis), widespread in the whole world, possesses many health protective properties. Its polyphenolic compounds, mostly flavonols, better known as catechins (e.g. epigallocatechin-3-gallate [EGCG]), are considered to be responsible for the health protective effects. Tea consumption may have its strongest effect among patients with cardiovascular disease. Recently, studies suggested that high flavonoid intake may reduce coronary heart disease by lowering blood lipid levels and inhibiting the oxidation of low-density lipoproteins. Trials showed that short- and long-term tea consumption could reverse endothelial dysfunction in subjects with documented coronary heart disease, providing one possible mechanism for an effect of tea in patients with cardiovascular disease. Interestingly, EGCG has also been shown to have electrophysiological effects by blocking HERG potassium channels, the most important repolarizing potassium channel in the human ventricle that forms the α-subunit of the rapid delayed rectifier current IKr. Inhibition of HERG channels may have profound effects on cardiac repolarization. Furthermore, tea flavonoids have been reported to exhibit metabolic effects in terms of anti-diabetic properties. This review summarizes the latest studies on cardiovascular effects of green tea and discusses the possible cardiac health benefits of green tea consumption.

INTRODUCTION Green tea (Camelia sinensis), one of the most popular beverages in the world, especially recommended in chinese medicine, gained increased public awareness because of many *

Correspondence to [email protected] or [email protected]

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health protective characteristics like anti-inflammatory, antioxidant, antihypertensive, antidiabetic, antiobesity and antimutagenic properties. Epidemiological evidence linking tea or flavonoid consumption with coronary heart disease or total mortality among healthy adults is conflicting [1, 2], but tea consumption may have its strongest effect among patients with cardiovascular disease [3, 4]. Recently, studies suggested that high flavonoid intake may reduce coronary heart disease by lowering blood lipid levels, inhibiting the oxidation of low-density lipoproteins and inhibiting inflammatory reaction (4-6]. A randomized crossover trial showed that short- and long-term tea consumption could reverse endothelial dysfunction in patients with documented coronary heart disease [3], providing a possible mechanism for an effect of tea in patients with cardiovascular disease. In our own study, we demonstrated for the first time that green tea flavonoid EGCG blocks the human ether a-go-go related gene (HERG), which encodes the cardiac rapid delayed rectifier potassium current IKr [7]. The cardiac refractory period can be prolonged by blocking HERG. Thus, the heart might be less susceptible for cardiac arrhythmias by blocking IKr. Apart from its direct effects on cardiovascular disease (CVD), green tea has also been found to affect CVD risk factors. Several animal studies and investigations in human subjects showed a reduced risk of type 2 diabetes mellitus due to an improved insulin sensitivity and glucose metabolism [8, 9]. Furthermore, green tea possesses hypotensive properties, which can be partly attributed to vasodilating effects [10]. It also reduces other CVD risk factors like obesity through higher fat oxidation and higher energy expenditure [11-13]. Green tea contains polyphenols, flavonols, also known as catechins, which are considered to be responsible for the health protective effect. The primary catechins in green tea are epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin3-gallate (EGCG), which account up to 30-40% of the dry weight of green tea [14]. One cup of green tea contains about 90 mg EGCG [14]. Pronounced cardiovascular health benefits can be achieved by regular consumption of 5-6 or more cups of green tea per day [1]. Additionally, green tea contains also proteins, amino acids, lipids, fiber, pigments, minerals and carbohydrates. The content of catechins in green tea depends on the processing of the leaves. Several studies were performed to investigate the bioavailability and biotransformation of green tea catechins [15-17] with discrepant results for plasma EGCG levels [18, 19]. A study by Lee et al. [16] found a mean peak plasma EGCG level of 0.17 µM in humans after drinking the equivalent of ~ 2 cups of green tea, which was consistent with two other studies [15, 17]. However, another group found that oral intake of 525 mg EGCG could reach plasma levels of 4.4µM [18, 19]. Concentrations of EGCG up to 20 µM are achievable in the oral cavity after drinking green tea, perhaps in the stomach and intestines, where a direct contact between EGCG and the epithelial cells exists. The methods of green tea preparation and differences in tea strength might affect bioavailability, plasma cetechin levels and the functional properties.

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Figure 1. Chemical formula of green tea flavonoids EGCG, EGC, ECG and EC.

1.1. EFFECTS OF GREEN TEA ON CARDIOVASCULAR DISEASE / CORONARY HEART DISEASE Myocardial infarction is a major cause of death in industrialized countries. The impact of cardiovascular disease on the economic budget in industrialized countries is enormous and increasing. For example, in Germany the costs for treatment of acute myocardial infarction in the 90s were already in the range of 1.5 billion Euro, for coronary artery disease in general 4.45 billion Euro [20]. In the United States, expenses for cardiovascular disease were $ 393 billion for the year 2005 [21]. Reducing cardiovascular disease would have a great impact on the economy of industrialized countries. Several studies that are discussed in this chapter showed positive effects of green tea consumption on coronary heart disease. A couple of Japanese and Chinese studies could prove that the positive influence of green tea on cardiovascular health is directly correlated with increasing consumption of green tea. For example in the Ohsaki study [22], a prospective cohort study, including 40,530 Japanese who were followed for 11 years, the consumption of green tea resulted in an inverse association with mortality due to all causes and due to cardiovascular disease. This observation was especially true for women, although altogether both sexes had a benefit from green tea intake. However, the benefit was more pronounced in women. A study by Kono et al., including 1306 Japanese men, could show an inverse correlation between the consumption of nine cups or more of green tea daily and the serum cholesterol level [23]. Studies by Imai et al. confirmed the latter result and expanded this observation to an inverse

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relationship between green tea consumption of more than 3 cups daily and not only serum cholesterol levels but also triglycerides and low-density lipoproteins [24]. However, these studies showed no effect on high density lipoprotein levels. An animal study by Yang et al. investigated the effects of green tea in a rat model of hyperlipidemia that was induced by administration of a high-sucrose diet [25]. Green tea consumption decreased total plasma triglycerides and cholesterol. High density lipoprotein (HDL), which has cardioprotective characteristics, was not changed. In liver and heart, green tea prevented the lipotrophic effects of the diet. Green tea didn’t modify protein absorption or bile acid concentration, but markedly reduced fat absorption. Regarding the risk of mortality due to heart disease, another prospective study by Hertog et al. including 12,763 and 25 follow-up years showed an inverse relationship between mortality due to heart disease and flavonoid intake in seven countries [26]. Concerning coronary artery disease (CAD) Hirano et al. and Ohmori et al. examined 203 Japanese patients and found that green tea consumption of approximately 6 cups of green tea per day was inversely related to the incidence of coronary artery disease during coronary angiography [27, 28]. Another finding of this study was the observation that patients who drank green tea on a regular basis also frequently consumed fish that is rich in omega-3-fatty acids and vegetables and soybeans that also additionally might have contributed to the lower incidence of CAD and myocardial infarction in these patients. It is known that CAD has several risk factors. Among others, free radicals have been shown to play an important role in heart disease. The free radicals of special interest in CAD are oxygen free radicals. Oxidized free radicals are believed to cause tissue damage at the cellular level, causing damage to the DNA, mitochondria and cell membrane. They have often been attributed to cause aging, cancer and heart disease. Green tea is thought to antagonize effects of excessive alcohol intake, smoking, and various chemical exposures that increase the amount of free radicals in the body [29, 30]. To prevent free radical damage, the body has a defense system of antioxidants. Antioxidants are a defense mechanism of the human body to protect cells from free radical damage. They interact with free radicals and neutralize their effects. Another study investigated moderate green tea consumption for 42 days (2 cups per day) in 24 healthy volunteers and found an increased antioxidant capacity and decreased plasma peroxides and reduced oxidative damage and glutathione peroxidase activity in lymphocytes [31]. Several studies tested the effect of green tea on other cardiovascular risk factors beside cholesterol and LDL levels like hypertension and diabetes and surprisingly found also positive outcomes for these risk factors, which will be discussed separately below. Thus, a reduction of cardiovascular disease needs to be seen in the context of altogether CAD risk factors lowering characteristics of green tea.

1.2. EFFECTS OF GREEN TEA ON HYPERTENSION The major risk factor for stroke and cardiovascular disease is hypertension, affecting millions of people worldwide. Chinese medicine postulated hypotensive effects of green tea for decades. However, there are only few studies examining the long-term effects of green tea on blood pressure and the available data were conflicting until a Chinese study tested the

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effect of tea (green tea and oolong tea) in detail for the past decades on the risk of newly diagnosed hypertension in 1507 subjects. The study population was divided in habitual tea drinkers of 120 ml/d or more for at least 1 year and nonhabitual tea drinkers. This study could show that the daily consumption of 120-600 ml green tea per day for at least one year reduced the risk of developing hypertension by 46%. A consumption of more than 600 ml green tea per day showed a reduction of hypertension by 65% [32]. These results were adjusted to socioeconomic and dietary factors and physical activity and altogether lifestyle factors. Another study, a Norwegian epidemiological study, examined the effects of tea consumption on blood pressure and found that the mean systolic blood pressure decreased with increasing tea consumption [33]. Concerning these positive effects of tea on hypertension the question arises through which possible mechanism tea might exert its hypotensic effect. There are several animal studies showing different possible mechanisms. One of these animal studies examined the effect of green tea extracts on angiotensin IIinduced hypertension in rats [30]. Rats were treated with drinking water with or without green tea extracts and angiotensin II for 13 days. In the angiotensin II without green tea extracts group blood pressure, left ventricular mass index, media- to lumen ratio and hydroperoxide radicals, as a measure for oxidative stress, were increased. In the Green tea group hypertension and target organ damage induced by high angiotensin II dose could be prevented. Oxidative stress markers like plasma hydroperodxides and nitrotyrosine were decreased by green tea. Altogether factors elevated with endothelial dysfunction like heme oxygenase 1 (HO-1), NADPH oxidase endothelial p22phox subunit and the superoxide dismutase SOD-1 were measured and found to be increased by angiotensin II and decreased below baseline levels by green tea extracts. Endothelial dysfunction plays a key role in the triggering and progression of cardiovascular disease and predicts mortality [34, 35]. Several studies showed that green tea flavonoids have antioxidant and endothelium-relaxing and thus vasodilatatory effects [36, 37]. Another study showing endothelial dysfunction improving effects of the green tea flavonoid EGCG observed in vascular endothelial cells that EGCG activates endothelial nitric oxide synthase (eNOS) and increases production of nitric oxide via a phosphaditylinositol (PI) 3-kinase/Akt-dependent pathway [38]. The antioxidative effects of green tea were investigated by Hakim et al. in a randomized, controlled study in 133 smokers [39]. Consumption of 4 cups of green tea compared to water for 4 months decreased urinary 8hydroxydeoxyguanosin, a sensitive marker of oxidative DNA damage. Another theory concerning hypotensive characteristics of green tea concerns theanine (γglutamylethylamide), which is one of the major components of amino acids in green tea and belongs to the neurotransmitter family in the brain. Theanine administration could reduce blood pressure through a not yet understood pathway in sponatenously hypertensive rats [40, 41].

1.3. EFFECTS OF GREEN TEA ON METABOLISM Cardiovascular and metabolic diseases are the major causes of death in industrialized countries. The metabolic syndrome describes a disease including several cardiovascular risk factors like diabetes mellitus type II, obesity, hypertension, hyperlipidemia, which is

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predicted to continue to grow in the coming years in western countries. All statistics reflect an increase in the prevalence of obesity and diabetes mellitus type II and mortality because of successive diseases like cardiovascular disease, blindness, neuronal damage, renal failure and diabetic foot disease. Type II diabetes mellitus is a disease that is a strong risk factor of coronary artery disease and that was associated with 2.9 million deaths worldwide in the year 2000. Diabetic patients have a lower life-expectancy than non-diabetics [42]. The rapid spread of diabetes mellitus II is considered to be due to increasing obesity, the major risk factor of diabetes mellitus II. In a retrospective cohort study including 17,413 Japanese subjects, consumption of 6 or more cups of green tea per day was found to reduce the development of diabetes by 33% compared to consumption of less than 1 cup per week [8]. A study with healthy young volunteers investigated the effects of green tea on glucose tolerance. The consumption of 1.5 g green tea extract was found to significantly reduce plasma glucose levels during the glucose tolerance test [43]. Another cross-over study investigating oolong tea (1.5 liter with 390 mg EGCG) intake in 20 patients with diabetes mellitus II resulted in significantly decreased plasma glucose levels [44]. A study by Shimada K. et al. [45] showed a decreased hemoglobin A1c, increased serum LDL and adiponectin in 22 subjects with coronary artery disease that consumed approximately 1 liter of oolong tea daily (45 mg EGCG). Furthermore, an animal study in obese but otherwise healthy dogs observed that consumption of green tea (80 mg/kg EGCG per day) reduced glucose and insulin levels, suggesting that green tea improves insulin sensitivity [46]. Several animal models for diabetes exist like streptozotocin – and alloxan-induced diabetes. In a number of studies, Babu et al .[47] examined the effects of green tea in rats with streptozotocin-induced diabetes. They administered green tea extracts (300 mg/kg for 4 weeks) orally in rats and thus reduced lipid peroxides and activity of antioxidant enzymes as well as increased gluthatione, an antioxidant that protects cells from toxins such as free radicals, in the heart. In another study they observed reduced blood glucose levels, decreased lipid peroxides, triglycerides and protein glycation in the heart of diabetic rats [48]. Lipid peroxidation refers to the oxidative degradation of lipids resulting in cell damage. Glycation is the non-enzymatic addition or insertion of sugar molecules into proteins that causes damage to proteins or other molecules and is considered to be a significant contributor to many diseases of aging. Yamabe et al. [49] tested the effects of EGCG (25, 50, 100 mg/kg for 50 days) in rats with streptozotocin-induced diabetes and subtotal nephrectomy. The authors observed that EGCG reduced hyperglycemia, proteinuria, and lipid peroxidation. EGCG also reduced the accumulation of renal advanced glycation end-products in the kidney. In alloxaninduced diabetic rats administration of green tea extracts (100 mg/kg for 15 days) increased glutathione and superoxide dismutase, which catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide and is an important antioxidant defense in nearly all cells exposed to oxygen. Furthermore, lipid peroxidation was decreased as well as blood glucose levels. Liver and kidney function was improved [50]. Wu et al. investigated the effects of green tea on insulin sensitivity in rats that received green tea in their drinking water (370 mg/kg for 12 weeks). They found decreased fasting plasma levels of glucose, insulin, free fatty acids and triglycerides [51]. Adipocytes from rats receiving green tea showed a better capacity for glucose uptake and increased specific insulin binding. Another major cardiovascular and metabolic risk factor includes overweight and obesity. A multi-center study by Chantre et al. investigated the consumption of green tea extracts (279

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mg EGCG) for 12 weeks in 70 moderately overweight subjects [52]. The investigators found a 4.6% decrease in body weight and a 4.5% reduction in waist-to hip ratio. Kovacs et al. conducted a study that looked for the effects of green tea on the regain of body weight following consumption of a low-energy diet for 4 weeks in 104 overweight subjects (323 mg EGCG for 12 weeks). The authors found a continuation of loosing body fat in the group with only low caffeine intake [53]. All other groups continued to gain weight again. An animal model for obesity, where rats were fed a high-fat diet and then administered green tea extracts in their drinking water for two weeks showed a decrease of body fat [54]. These effects resulted from increased energy expenditure and a moderate reduction of food digestability. Furthermore, the thermogenic capacity was increased. The reduction of body fat and the increase of energy expenditure was partially mediated by β-adrenoreceptor activation. In Zucker rats, consumption of green tea resulted in attenuated body weight gain and decreased adipose tissue weight as well as reduced plasma cholesterol levels, whereas food intake was not affected [55]. In another animal study with mice, consumption of EGCG, in addition to endurance training, increased exercise capacity and β-oxidation, the process by which fatty acids are broken down in mitochondria and/or in peroxisomes to generate AcetylCoA, an important molecule in metabolism, used in many biochemical reactions . The expression of fatty acid translocase/CD 36 mRNA was increased in skeletal muscle. This study showed that endurance exercise along with green tea consumption can improve exercise capacity and stimulates lipid metabolism in mice.

1.4. POTENTIAL EFFECTS OF GREEN TEA ON CARDIAC AMYLOIDOSIS Amyloidosis is a rare disease which affects also the heart among other organs. There are several subtypes of cardiac amyloidosis, which is characterized by infiltration of the heart from insoluble protein deposits resulting in restrictive cardiomyopathy with heart failure and conduction abnormalities leading to potentially life-threatening arrhythmias. The therapeutic strategy depends on the subtype of amyloidosis and includes chemotherapy, stem cell therapy and often requires liver transplantation. Table 1. The different types of amyloidosis, the involved proteins and the affected organs Amyloidosis type Primary Amyloidosis (ALAmyloidosis) Reactive Amyloidosis (AAAmyloidosis) Hereditary Amyloidosis (ATTR)

Protein Light chain

Atrial Amyloidosis (AANF) Senile systemic Amyloidosis Dialysis-related Amyloidosis (β2microglobulin)

Atrial natriuretic factor Transthyretin β2-microglobulin

Amyloid A Mutant transthyretin

Affected organs Heart, kidney, liver, nerval system Heart, kidney, liver Heart, nerval system, kidney, eyes Limited to the heart Diffuse organ mnifestation Bones, joints

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Patients with cardiac amyloidosis predominantly die because of electromechanical dissociation or other diagnoses that don’t benefit from an implantation of an intracardiac defibrillator (ICD). A study by Bauer et al. tested nineteen patients with histologically proven cardiac amyloidosis and a history of syncope and/or ventricular extra beats (Lown grade IVa or higher) that received an ICD [56]. As a result of the study the investigators noted that only two patients had a benefit of the implanted ICD to treat sustained ventricular tachycardia. Most of the patients died due to electromechanical dissociation. The study investigators concluded that only a small part of cardiac amyloidosis patients benefit from an ICD. So far, not a single study exists that may prove therapeutic effects of green tea in cardiac amyloidosis. However, there is one case report published from the University of Heidelberg [57], where one patient with cardiac amyloidosis (AL-Amyloidosis) underwent a selftest with green tea after receiving several chemotherapies that could stabilize the disease, but were accompanied by side effects. The patient, an emeritus professor of internal medicine and haematology, was announced to have AL-Amyloidosis at the age of 72. He received two different chemotherapies, the first according to the “Boston scheme” (4 mg melphalan daily for 21 days) without success, the second according to the “Palladini scheme” (0,22 mg/kg melphalan and 40 mg dexamethasone for 4 days every 28 days) that could stabilize the cardiac amyloidosis. The patient then attended a lecture by Prof. E.E. Wanker on effects of EGCG, the main phenol in green tea, on light chains and amyloid fibrils in Huntington`s disease [58]. Since that lecture he consumed 1.5 to 2.0 liters of green tea daily without any other treatment. Before the consumption of green tea the patient’s interventricular septum was 16.5 mm and constant for 20 months due to the success of the second chemotherapy (cardiac amyloidosis is associated with cardiac hypertrophy). However, since the consumption of 1.5 to 2.0 liters of green tea daily, the thickness of the interventricular septum decreased month by month to 13.2 mm. Since then the light chains didn’t increase any more and his renal insufficiency stabilized. Also the patient’s “quality of life improved dramatically”, he says. Now, he is almost 80 years old and the cardiac amyloidosis is greatly stabilized without any other treatment than green tea. Randomized trials are urgently needed to further examine the effects of green tea and especially EGCG in cardiac amyloidosis. Possibly, treatment with EGCG might soon become an alternative therapeutic option for cardiac amyloidosis.

1.5. EFFECTS OF TEA ON ARRHYTHMIA The Determinants of Myocardial Infarction Onset Study (The Onset Study) was a prospective multicenter study from 1989 to 1996, where 3882 patients with acute myocardial infarction were enrolled in the United State [59]. Patients were asked according to a standardized questionnaire to report about their tea and coffee drinking habits. They were divided in non tea drinkers, moderate tea drinkers with < 14 cups per day and heavy tea drinkers with > 14 cups per day and coffee drinkers. However, this study didn’t differentiate between the different kinds of teas (black tea, green tea, white tea…). The first phase of the study found that tea consumption was associated with lower mortality among patients with acute myocardial infarction whereas coffee intake showed no effect on mortality outcome.

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Figure 2. EGCG blocks HERG potassium channels, that is the molecular correlate of the cardiac rapid delayed rectifier potassium current IKr. By blocking IKr ,which results in smaller currents, repolarization will be prolonged.

Unexpectedly the investigators also found that tea consuming patients had a lower prevalence of ventricular arrhythmia after myocardial infarction. However, this question was not the focus of the study, so that no adjusted analysis was conducted and a second phase of the Onset Study was started, where the occurrence of ventricular arrhythmia was assessed in detail and correlated with the different tea drinker groups and the coffee drinker group with adjustment for potentially confounding factors. The investigators found that of the 3882 patients 445 patients developed ventricular arrhythmia during hospitalization, in detail 370 patients developed ventricular tachycardia and 87 ventricular fibrillation. The results of this second phase of the study were that moderate tea intake was associated with a lower prevalence of ventricular arrhythmia and coffee drinkers had a higher incidence of ventricular arrhythmia. Heavy tea drinkers had an intermediate prevalence of ventricular arrhythmia. Possible explanations for a lower incidence of ventricular arrhythmia among tea drinkers might be an improved gap junctional intercellular communication due to the flavonoid content in tea. An experimental study with rat liver epithelial cells [60] showed that with increasing levels of epicatechin, which is found in green tea, in cell culture the number of communicating gap junctional cells increased so that gap junctional communication was improved. Since intercellular communication in the heart is also similar to the liver, with connexins playing the most important role, it might be assumed that cardiac intercellular gap junctional communication might also be positively affected by flavonoids. Another

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experimental study in rat hearts [61] describes the positive minimizing effect of catechins on ischemia-reperfusion injury and thus leading to less ischemia-triggered arrhythmia. In our own in vitro study in xenopus laevis oocytes [7] we demonstrated for the first time, that green tea flavonoid EGCG blocks the human ether a-go-go related gene (HERG), which is the molecular correlate of the cardiac rapid delayed rectifier potassium current IKr. Prolonging repolarization of the cardiac action potential is the mechanism of many antiarrhythmic agents, e.g. dofetilide [62], a class III-antiarrhythmic agent. Characteristically, class III-antiarrhythmic drugs prolong the cardiac refractory period and make the heart less susceptible for cardiac arrhythmias by blocking IKr. According to a study by Prineas et al. [63] coffee intake however is associated with a greater prevalence of premature ventricular contractions.

CONCLUSION Tea consumption had its origin in China almost 5,000 years ago. Green tea, a worldwide consumed beverage, has been used as traditional medicine in areas such as China, Japan, India and Thailand. Green tea has gained scientific attention due to its antioxidant, antinflammatory, antihypertensive, antidiabetic and antimutagenic properties. Due to its high content of polyphenolic flavonoids, mainly EGCG, green tea has especially shown exciting cardiovascular health benefits. A number of animal as well as randomized human studies have proven the benefits of green tea for cardiovascular and metabolic diseases concluding that 200-300 mg of EGCG or 5-6 cups of green tea per day protects cardiovascular and metabolic health. In times of growing cardiovascular and metabolic disease, a beverage that has scientific evidence for its health protective properties and that is easily available for everybody might have a tremendous impact on the health of the world population and an increased impact on the economic budget. A balanced diet combined with regular green tea consumption and physical activity as well as life style changes may offer primary prevention against cardiovascular disease.

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[37] Fitzpatrick DF, Hirschfield SL, Ricci T, Jantzen P, Coffey RG. Endothelium-dependent vasorelaxation caused by various plant extracts. J Cardiovasc Pharmacol 1995 Jul;26(1):90-95. [38] Lorenz M, Wessler S, Follmann E, Michaelis W, Dusterhoft T, Baumann G, et al. A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem 2004 Feb 13;279(7):6190-6195. [39] Hakim IA, Harris RB, Brown S, Chow HH, Wiseman S, Agarwal S, et al. Effect of increased tea consumption on oxidative DNA damage among smokers: a randomized controlled study. J Nutr 2003 Oct;133(10):3303S-3309S. [40] Yokogoshi H, Kato Y, Sagesaka YM, Takihara-Matsuura T, Kakuda T, Takeuchi N. Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci Biotechnol Biochem 1995 Apr;59(4):615-618. [41] Yokogoshi H, Kobayashi M. Hypotensive effect of gamma-glutamylmethylamide in spontaneously hypertensive rats. Life Sci 1998;62(12):1065-1068. [42] Gu K, Cowie CC, Harris MI. Mortality in adults with and without diabetes in a national cohort of the U.S. population, 1971-1993. Diabetes Care 1998 Jul;21(7):1138-1145. [43] Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004 Aug 26;4:18. [44] Hosoda K, Wang MF, Liao ML, Chuang CK, Iha M, Clevidence B, et al. Antihyperglycemic effect of oolong tea in type 2 diabetes. Diabetes Care 2003 Jun;26(6):1714-1718. [45] Shimada K, Kawarabayashi T, Tanaka A, Fukuda D, Nakamura Y, Yoshiyama M, et al. Oolong tea increases plasma adiponectin levels and low-density lipoprotein particle size in patients with coronary artery disease. Diabetes Res Clin Pract 2004 Sep;65(3):227234. [46] Serisier S, Leray V, Poudroux W, Magot T, Ouguerram K, Nguyen P. Effects of green tea on insulin sensitivity, lipid profile and expression of PPARalpha and PPARgamma and their target genes in obese dogs. Br J Nutr 2008 Jun;99(6):1208-1216. [47] Babu PV, Sabitha KE, Shyamaladevi CS. Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats. Chem Biol Interact 2006 Aug 25;162(2):114-120. [48] Babu PV, Sabitha KE, Shyamaladevi CS. Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca2+ -ATPase and Na+/K+ -ATPase activity in the heart of streptozotocin-diabetic rats. Chem Biol Interact 2006 Aug 25;162(2):157-164. [49] Yamabe N, Yokozawa T, Oya T, Kim M. Therapeutic potential of (-)-epigallocatechin 3-O-gallate on renal damage in diabetic nephropathy model rats. J Pharmacol Exp Ther 2006 Oct;319(1):228-236. [50] Sabu MC, Smitha K, Kuttan R. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol 2002 Nov;83(1-2):109-116.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 14

MOLECULAR BASIS FOR THE ANTI-CANCER ACTIVITY OF EGCG IN VIVO: MOLECULAR-TARGETING PREVENTION OF CANCER BY GREEN TEA CATECHIN Yoshinori Fujimura1 and Hirofumi Tachibana1,2,3,* 1

Innovation Center for Medical Redox Navigation, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 2 Bio-Architecture Center, 3Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

ABSTRACT For the past two decades, many researchers have been investigated the potential cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-O-gallate (EGCG) has been shown to be the most active and major polyphenolic compound from green tea. The mechanisms of action of EGCG have been extensively investigated, but the mechanisms for the cancer-preventive activity of EGCG are not completely characterized and many features remain to be elucidated. Recently we have identified 67kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. This article reviews some of the reported mechanisms and possible targets for the action of EGCG. Especially, we focus the current understanding of signaling pathway for physiologically relevant EGCG through the 67LR for cancer prevention. This information shed new light on the molecular basis for the cancer-preventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer.

*

Corresponding author: Hirofumi Tachibana, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail address: [email protected]; Tel/Fax: +81-92-642-3008

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1. INTRODUCTION Tea is one of the most widely consumed beverages in the world. Green tea, black tea, and oolong tea are all derived from the leaves of Camellia sinensis plant and contain an assortment of compounds, the most significant components of which are polyphenols. Among all teas consumed in the world, green tea is best studied for its health benefits. It has been demonstrated that tea constituents exhibit various biological and pharmacological properties such anti-carcinogenic, anti-oxidative, anti-allergic, anti-virus, anti-hypertensive, antiatherosclerosis, and anti-hypercholesterolemic activities [1-8]. Major principles for these activities were shown to be a group of polyphenols, catechin. A typical green tea beverage, prepared in a proportion of 1 g leaf to 100 ml water in a 3-min brew, usually contains 250350 mg tea solids, and catechins account for 30-42% of the dry weight of the solids [9]. Catechins contain a benzopyran skeleton with a phenyl group substituted at the 2-position and a hydroxyl (or ester) function at the 3-position. Variations to catechin structure include the stereochemistry of the 2,3-substituents and the number of hydroxyl groups in the B- and Dring. Belonging to the flavan-3-ol class of flavonoids, major catechins found in tea leaves are (–)-epigallocatechin-3-O-gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin-3-Ogallate (ECG), (–)-epicatechin (EC) and their structures are shown in Figure 1. Among the green tea catechins, EGCG is the most abundant, representing ~16.5 wt% of the water extractable fraction of green tea leaves, and most active catechin in various kinds of physiological activities. Because EGCG is not found to a plant except tea, EGCG is regarded as a constituent characterizing green tea. Recently, double-blind, placebo-controlled study on oral administration of green tea catechins (EGCG, 50%) in volunteers with high-grade prostate intraepithelial neoplasia demonstrated that green tea catechins have potent in vivo chemoprevention activity for human prostate cancer [10,11]. This impressive evidence has fueled interest in the role of EGCG as chemoprevention of cancer. This chapter will discuss the effects of green tea polyphenol EGCG on signal transduction pathways that are related to cancer chemoprevention based on the biological importance of the target, and especially we focused the current understanding of EGCG signaling pathway through the 67-kDa laminin receptor (67LR) as the target molecule mediating anti-cancer effect of the physiologically relevant EGCG

2. ANTI-OXIDANT AND PRO-OXIDANT Tea polyphenols such as EGCG are well known for their anti-oxidant activities. They have been reported to inhibit carcinogen-induced DNA damage and tumor promoter-induced oxidative stress [12,13]. These results are consistent with the commonly mentioned idea that tea prevents cancer because tea polyphenols are anti-oxidants. It is unclear, however, whether this is a general mechanism for cancer prevention, especially in human carcinogenesis when strong carcinogenesis and tumor promoters are not known to be involved. When carcinogenactivation and tumor promotion were active areas of research, these events had been proposed as the targets of tea polyphenol action. With the advancement of research on signal transduction pathways targeted by tea polyphenols, many studies have been carried out in cell

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lines. However, whether some of the phenomena observed in cell lines occur in vivo still remains unclear.

Figure 1. Chemical structures of green tea catechins and their analogues.

There two major problems in extrapolating results observed in cell lines to animal models. (i) The concentration used in cell line systems, for example, EGCG at 10-100 μM, or

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higher concentrations, are much higher than those observed in the plasma or tissues in experimental animals or humans after ingestion of tea or related tea preparations [12]. (ii) The oxygen partial pressure in a cell culture system (160 mmHg) is much higher than that in the blood or tissues (<40 mmHg). EGCG is unstable under cell culture conditions, and the halflife, less than 2 h, can be extended several folds by the addition of superoxide dismutase (SOD), suggesting a role for superoxide radical in the oxidation and polymerization of EGCG [14-16]. Similar to other anti-oxidants, EGCG and other tea polyphenols may also act as prooxidants. They can be oxidized to form phenolic radicals, superoxide radical and hydrogen peroxide. These species may trigger a variety of biochemical reactions and biological responses. In studies of EGCG and other polyphenolic compounds in cell culture, the addition of SOD and catalase is recommend to stabilize EGCG and to avoid possible artifacts. Thus, according to this observation, we also added those enzymes to the cell culture systems [1721]. It is not clear whether pro-oxidants produced by EGCG-generated reactions occur in low oxygen partial pressure conditions in vivo in cells, which generally have strong anti-oxidative capacity and low oxygen partial pressure. Recently, EGCG has been shown to form quinone and dimmer quinone during the autoxidation of EGCG in vitro, but none of these oxidation products were observed in plasma samples of mice after treatment with 50 mg/kg EGCG i.p. daily for 3 days [22]. Therefore the roles of EGCG autoxidation in biological activities of this compound in vivo remain to be investigated further. The difference between in vitro and in vivo systems should be considered in studies attempting to elucidate the mechanisms of action of EGCG.

3. BIOAVAILABILITY OF EGCG The bioavailability and biotransformation of tea catechins following tea ingestion has been investigated, and a Tmax (time to reach maximal concentration) in the plasma of 1.5 to 2.5 h after consumption of decaffeinated green tea solids (1.5, 3.0, and 4.5 g) [23]. The catechins levels decreased and were not detectable by 24 h. Whereas EGCG and ECG were not detected in the urine, 90% of the urinary EC and EGC were excreted by 8 h. Most of the ingested EGCG apparently does not get into the blood, and absolute EGCG is preferentially excreted through the bile to the colon [24]. EGC and EC appear to be more bioavailable, but the fractions of these compounds that appeared in the plasma are also low, and only 3.3 and 8.9% of the ingested EGC and EC were excreted in the urine. Glucuronidation, sulfation, methylation, and ring-fission metabolism represent the major metabolic pathways for green tea catechins [25]. Plasma EC and EGC were present mainly in the conjugated form such as glucuronide and sulfate conjugates, whereas 77% of the EGCG was in the free form [26]. EGC but not EC is also methylated (4’-O-methyl-EGC) in humans. EGCG has also been shown to undergo methylation. The maximum plasma concentration of 4’,4”-di-O-methylEGCG is 20% that of EGCG but the cumulative excretion of 4’,4”-di-O-methyl-EGCG is 10fold higher than that of EGCG over 24 h [27]. The EGCG levels in plasma, lung, and liver are much higher than in rats when the same polyphenol preparation is given to mice [28]. Although most of published studies in cell culture systems used 10-100 μM of EGCG, the blood level of EGCG after consuming the equivalent of 2-3 cups of green tea was 0.1-0.6 μM and for an equivalent of 7-9 cups was still lower than 1 μM [23,29]. The rather poor

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bioavailability of tea catechins needs to be considered when we extrapolate results obtained in vitro to situations in vivo. The biological activities of EGCG in vivo depend on its bioavailability at the specific sites of interest. For example, EGCG is more accessible to the oral cavity and intestinal tract due to direct contact after ingestion. The tissue levels of EGCG of internal organs depend on the systemic bioavailability after oral ingestion. The limited systemic bioavailability is due to its multiple phenolic groups, its rapid conversion to methylated, glucuronidated, and sulfated metabolites, and its efflux by multiple drug resistant related proteins [12]. The peak EGCG (including the free and conjugated forms) concentrations observed in the plasma and tissue levels of animals or in human plasma after ingestion of tea or polyphenols are usually lower than 1 μM [12]. When large pharmacological doses of tea polyphenols are given by oral administration, peak plasma concentrations of 7.5 μM have been observed in human [30]. EGCG is excreted mostly through the feces, and urinary levels of EGCG are very low or undetectable [12].

4. POSSIBLE DIRECT TARGETS FOR THE ACTION OF EGCG Currently, there is much interest in the design and development of chemopreventive agents that act on specific molecular and cellular targets. Searching for high-affinity proteins that bind to EGCG is the first step to understanding the molecular and biochemical mechanisms of the anti-cancer effects of tea polyphenols. Several proteins that can directly bind with EGCG have been identified in vitro models and these topics were shown in Table 1 [17-19,31-56]. Some are summarized below.

4.1. Matrix Metalloproteinase The progression of human tumors involves the matrix metalloproteinase (MMP) family. Two particular members of this family, MMP-2 and MMP-9, seem to play an important role in tumor invasion and metastasis. They are involved in the turnover of basement membrane collagen under basal conditions and of other matrix proteins during angiogenesis, tissue remodeling, and repair. EGCG exerted dose-dependent inhibition of both MMP-2 and MMP9. The concentrations giving 50% inhibition (IC50) were 20 and 50 μM, respectively [57]. The invasion of HT1080 fibrosarcoma cells through a Matrigel basement membrane was inhibited with an IC50 less than 0.1 μM EGCG. In addition to inhibitory effect of EGCG on activity of MMPs, EGCG has also been found to inhibit the intracellular metalloproteolytic activity of the anthrax lethal factor (LF; IC50 = 0.1 μM), which has a major role in the development of anthrax [58]. The inhibition of LF-induced cleavage of MAPKK, which is the natural substrate of LF, was involved in the protection of LF-induced death of macrophages by EGCG.

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4.2. Proteasome The proteasome is a massive multicatalytic proteinase complex found in all eukaryotic cells and is responsible for degrading most of the cellular proteins. The ubiquitin/proteasome-

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dependent degradation pathway plays an essential role in up-regulation of cell proliferation, down-regulation of cell death, and development of drug resistance in human tumor cells, suggesting that the proteasome is a novel and promising target for cancer chemotherapy. It has been reported that EGCG potently and specifically inhibited the chymotrypsin-like activity of the proteasome both in cell-free systems (IC50 = 86 nM) and tumor cell lines (1-10 μM) [33]. In addition, in silico docking study suggested that a particular pose of EGCG could lead to potential covalent modification of the N-terminal threonine (Thr 1) of the proteasome β5 subunit in the chymotrypsin-like active site [59]. This could be accomplished via nucleophilic attack of a pair of electrons from the hydroxyl group of Thr 1 to the ester carbon of EGCG. Numerous substrates of the proteasome have been identified including cyclins, cyclin-dependent kinase inhibitors, p53, Bcl-2, and IκB among others. These proteins have various functions like regulation of the cell cycle, protection of apoptosis, and transcriptional regulation [60]. The inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of two natural proteasome substrates, p27Kip1 and IκB-α, followed by growth arrest in the G1 phase of the cell cycle.

4.3. Extracellular Signal-regulated Protein Kinase 1/2 (ERK1/2) and Akt MAPKs have been implicated in many physiologic process, including cell proliferation, differentiation, and death. MAPK has received increasing attention as a target molecule for cancer prevention and therapy. There are three major types of MAPKs in mammalian cells, the extracellular signal-regulated protein kinases (ERK), the p38 MAPKs, and c-Jun NH2terminal kinases (JNK). Activation of the MAPK pathways may cause the induction of phase II detoxifying enzymes, and its inhibition may inhibit AP-1-mediated gene expression. Once activated, MAPKs (ERK, JNK, and p38) can activate a variety of transcription factors, including ELK and c-Jun, a component of AP-1, thus leading to changes in the expression of genes that play critical roles in cell proliferation, migration and apoptosis. It has been shown that treatment of H2O2 resulted in phosphorylation of ERK1/2, JNK, and p38 in human epidermal keratinocytes. H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was found to be significantly inhibited when these cells were pretreated with EGCG. These findings demonstrate that EGCG has the potential to inhibit oxidative stress-mediated phosphorylation of MAPK signaling pathways [61]. EGCG inhibited the phosphorylation of ERK1/2, and p38 MAPK activity in human fibrosarcoma HT1080 cells [4]. EGCG (20 μM) inhibited the phosphorylation of MAP/ERK kinase 1/2 (MEK1/2), ERK1/2, and ELK-1 in 30.7b Ras 12 cells (H-ras transformed JB6 mouse epidermal cell line) [62]. This study suggested that EGCG decreased the association between RAF-1 and MEK1, and EGCG competitively inhibited the phosphorylation of ELK-1 by ERK1/2 possibly by competing for the binding site on ERK1/2. Epidermal growth factor receptor (EGFR) is often overexpressed in neoplastic cells, activating signal transduction pathways that promote cell proliferation and tumor progression [63,64]. Antagonists to EGFR are currently under intensive investigation for cancer therapy [65]. In addition, downstream EGFR targets, including ERK1/2 and Akt, are considered viable drug targets. EGCG inhibited epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of ERK1/2 and Akt at the concentration range of 10-50 μM in the immortalized cervical cell line ECE16-1 [66]. In addition to

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inhibiting EGFR activation, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and Akt kinase activity at the concentration of more than 5 μM.

4.4. DNA Methyltransferase Hypermethylation of DNA is a key epigenetic mechanism for the silencing of many genes, including those for cell cycle regulation, receptors, DNA repair, and apoptosis [67-70]. In this aberrant methylation, the cytosine of the CpG island in or near the promoter region of the newly synthesized DNA strand is methylated by 5-cytosine DNA methyltransferase (DNMT). The methylated CpG island has higher binding affinity to methyl-CpG binding domain proteins that recruit transcriptional corepressors such as histone deacetylases, resulting in chromosome condensation and transcription repression [71,72]. The inhibition of DNMT would block the hypermethylation of the newly synthesized DNA strand, resulting in the reversal of the hypermethylation and the re-expression of the silenced genes [73-75]. The inhibitors of DNMT have been shown to inhibit cancer cell growth, induce cancer cell apoptosis, and reduce tumor volume in mice [76-79]. There is high potential for developing this group of inhibitors for cancer therapy. It has been reported that EGCG can inhibit DNMT activity and reactive methylation-silenced genes in cancer cells [37]. With nuclear extracts as the enzyme source and polydeoxyinosine-deoxycytosine as the substrate, EGCG dosedependently inhibited DNMT activity, showing competitive inhibition with a Ki of 6.89 μM. Studies with structural analogues of EGCG suggest the importance of D and B ring structures in the inhibitory activity. Molecular modeling studies also support this conclusion, and suggest that EGCG can form hydrogen bonds with Pro1223, Glu1265, Cys1225, Ser1229, and Arg1309 in the catalytic pocket of DNMT. Treatment of human esophageal cancer KYSE 510 cells with 5-50 μM EGCG for 12-144 h caused a concentration- and time-dependent reversal of hypermethylation of key tumor suppression gene p16, retinoic acid receptor β, the DNA repair gene hMLH1, and methylguanine methyltransferase. Some of these genes were also reactivated in colon cancer HT29 cells and prostate cancer PC3 cells. The observed effective dose of EGCG, Ki of 6.89 μM, or IC50 of 20 μM, is achievable in the oral cavity (in the saliva) after drinking green tea, and perhaps in the stomach, esophagus, and intestines where there is direct contact between EGCG and the epithelial cells. This effective concentration, however, is higher than those in the internal organs, which depend on the systemic bioavailability of EGCG [12]. There, the extent of DNMT inhibition in vivo would depend on the bioavailability of EGCG in a particular organ site.

4.5. Bcl-2-family Proteins Bcl-2-family proteins are important regulators of apoptosis. Anti-apoptotic members of this family, such as Bcl-2 and Bcl-x, contain on the surface a hydrophobic groove in which they can bind the BH3 domain of the pro-apoptotic counterparts [80]. This binding is crucial for the regulation of apoptosis in vivo, with pro-and anti-survival proteins neutralizing each other’s function through dimerization. The observation that antiapoptotic Bcl-2 family members, such as Bcl-2 and Bcl-xL, are generally overexpressed in many cancer cells [80]

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has stimulated a growing interest in the discovery of small molecules targeting such proteins, as potential anti-cancer therapeutics [81,82]. A study using a combination of nuclear magnetic resonance binding assay, fluorescence polarization assay, and computational docking analysis demonstrated the direct binding of EGCG to the BH3 pocket of the anti-apoptotic Bcl-2 family proteins and the inhibitory effect of EGCG on the binding of BH3 peptide to Bcl-2family proteins, Bcl-2 and Bcl-xL with Ki values of 335 and 490 nM, respectively [36]. On the contrary, C, EC, and EGC did not interact with their proteins, and showed no inhibition of the binding at 100 μM. Although the BH3 domain was recognized as one of the binding site of EGCG in cell-free systems, the functional importance of this binding still requires more investigation. Furthermore, whether EGCG can bind directly the intracellular Bcl-2-family proteins in cell-culture systems or in vivo still remains unclear.

4.6. Vimentin Vimentin, one of the type III intermediate filament (IF) proteins, is a major component of IFs and is expressed during development in a wide range of cells, including mesenchymal cells and in a variety of cultured cell line and tumors [83,84]. IFs are essential for structure and mechanical integration of the cellular space and a variety of cellular functions such as mitosis, locomotion, and organizational cell architecture, and vimentin is readily phosphorylated by numerous protein kinases such as cycline-dependent kinases 2 (Cdc2) and cAMP-dependent protein kinase (PKA), therefore, regulating their functions [85-87]. The proteolytic derivatives indicate that the amino-terminal domain, but not the carboxyl-terminal domain, has a direct effect on filament stability and polymerization [88]. Vimentin was recently discovered as a high-affinity EGCG binding protein with a Kd of 3.3 nM [40]. The protein was isolated from cell lysates of JB6 C141 mouse epidermal cells by EGCGSepharose 4B column, and identified by two-dimensional electrophoresis and MALDI-TOFMS. Functional studies showed that EGCG inhibited the phosphorylation of vimentin at Ser55 and Ser50 by Cdc2 (IC50 = 17 μM) and PKA (IC50 = 2 μM), respectively. Vimentin knockdown (by siRNA) in JB6 C141 cells resulted in a lower proliferation rate and the cells became much less responsive to inhibition by EGCG at the concentraion of more than 15 μM. In this study, the biological consequences of EGCG have been demonstrated, but the effective concentration for the inhibition of cell growth is much higher. The molecular basis for this difference needs to be investigated. Whether this is general mechanism that are responsible for the inhibition of carcinogenesis in animal models or humans or are artifacts of the cell culture system also needs to be determined.

4.7. Urokinase-plasminogen Activator (uPA) uPA is primarily associated with the degradation and regeneration of the basement membrane and extracellular matrix that leads to metastasis. It also aids in anti-thrombolytic activities to remove blood clots and helps stimulate angiogenesis in tumor cells. uPA is a trypsin-like protease that converts the zymogen plasminogen into active plasmin. Plasmin facilitates the release of several proteolytic enzymes, including gelatinase, fibronectin, fibrin,

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laminin, and latent forms of collagenases and stromelysins [89]. Inhibition of uPA can decrease tumor size or even complete remission of cancers in mice [90,91]. It has been reported that EGCG inhibited the activity of uPA [53]. With the use of molecular modeling, it was shown that EGCG binds to urokinase, blocking His57 and Ser195 of the urokinase catalytic triad and extending toward Arg35 from a positive charged loop of urokinase. However, the effective concentration of needed to inhibit urokinase (2-10 mM) was at least 3 or 4 orders of magnitude higher than the expected tissue level (close to 1 μM).

4.8. Dihydrofolate Reductase (DHFR) DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8tetrahydrofolate, which acts as a coenzyme for several one-carbon group transfer reactions that include steps in nucleotide biosynthesis. Consequently, inhibition of DHFR, resulting in the disruption of DNA biosynthesis, is the basis of chemotherapeutic action of a range of DHFR inhibitors, generically known as “antifolate.” Tumor cells that grow rapidly require a higher concentration of dTTP than normal cells, and therefore are more sensitive to antifolates. EGCG was recently reported to an inhibitor of DHFR [39]. It exhibited kinetic characteristics of a slow tight binding inhibitor of 7,8-dihydrofolate reduction with bovine liver DHFR (Ki = 0.11 μM), but acted as classic reversible competitive inhibitor with chicken liver DHFR with a much larger Ki (10.3 μM). EGCG also inhibited lymphoma cell growth (IC50 = 20 μM), G0/G1 phase arrest of the cell cycle, and the induction of apoptosis. Folate depletion increased the sensitivity of these cell lines to the antifolate activities of EGCG. The suggestions of this paper that EGCG may be an antifolate reagent and that tea consumption may be related to folate deficiency are rather speculative, and further examination. If the EGCG blood level could be maintained at 20 μM for a long term, antifolate effects might be produced. However, this is not an achievable blood level of EGCG through oral consumption of even a large quantity of tea. The highest peak plasma level of EGCG after the oral administration of 1200 mg of EGCG (equivalent to ten cups of tea) is 7.5 μM, and this amount is associated with nausea [30]. The very low Ki value observed for bovine liver DHFR might be due to the fact that very low levels of the enzyme were used in the assay, and the inhibition was due to the strong binding activity of EGCG to the enzyme (a slow tight binding inhibitor, as reported), not necessarily by binding to the active site.

5. THE 67-KDA LAMININ RECEPTOR AS A GREEN TEA CATECHIN RECEPTOR It should be noted that most of the effects of EGCG in cell culture systems and cell-free systems have been obtained with relatively high concentrations than observed in the plasma or tissues of animals or in human plasma after administration of green tea or EGCG. The pharmacokinetic studies in humans indicate that the peak plasma concentration after single dose of EGCG is <1.0 μM. Furthermore, the intracellular levels of EGCG are much lower than the concentrations observed in the extracellular levels. Therefore, it is not clear whether

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the activities observed with high EGCG concentrations in vitro can be observed in vivo and the proposed EGCG-binding molecules as mentioned above are responsible for in vivo physiological activities of EGCG. A target molecule for physiological achievable concentration (<1.0 μM) of EGCG still remained unclear.

5.1. The 67-kDa Laminin Receptor (67LR) The 67-kDa laminin receptor (67LR) is a nonintegrin receptor for laminin, fibronectin, and type IV collagen [92,93]. Its full-length cDNA encodes only a 37-kDa polypeptide, which has been reported to be a ribosome-associated cytoplasmic protein, and the exact structure and maturation mechanism by which the 67LR is synthesized from the precursor of 37-kDa polypeptide is unclear. Expression of the 67LR has been shown to be upregulated in neoplastic cells compared with their normal counterparts and directly correlate with an enhanced invasive and metastatic potential in many malignancies [94,95]. The receptor has been implicated in laminin-induced tumor cell attachment and migration, as well as in tumor angiogenesis, invasion, and metastasis [96-98]. Surface expression of the 67LR has also been reported to be a dominant laminin-binding protein expressed in neutrophils, macrophages, and monocytes, which suggests that the receptor may play an important role in the regulation of cell adherence via the basement membrane laminin [99-101]. 67LR appears to has an important role in host defense by being expressed in cells including T lymphocytes and mast cells, but there is relatively little information about allergy-relevant cells such as basophils, eosinophils, and B lymphocyte [102,103]. It has been reported that the expression of 67LR in mast cells is related to the adhesion to laminin and may contribute to the tissue distribution of mast cells and to mast cell accumulation at sites of tissue injury and inflammation [102,104]. In the immune system, 67LR has also been shown to be able to modulate granulocytemacrophage colony-stimulating factor (GM-CSF) signaling by inhibiting GM-CSF-its receptor (GMR) complex formation through its interaction with GMR [105]. Furthermore, it has been suggested that the 67LR expression in human T cells is upregulated by stimulating the neuropeptides GnRH-II and GnRH-I which trigger its homing to specific organs [103]. Recently, it has become clear that acting as a receptor for laminin is not the only function of the 67LR. This protein also act as a receptor for pathogenic prion protein, cytotoxic necrotizing factor 1 from E. coli, Sindbis virus, Venezuelan equine encephalitis virus, Dengue virus, and adeno-associated virus [106-111].

5.2. Plasma Membrane Microdomain “Lipid Rafts” To date, most of the research on physiological activities of EGCG has been connected its action with anti-oxidative activity. On the other hand, it has been suggested that EGCG and its derivatives exhibit biological activities through interactions with the cellular membranes [112]. The study, using the liposome system, on the interaction of tea catechins with the lipid bilayers implicates that the affinity of catechins for the lipid bilayers may be responsible for various kinds of actions [113]. We also found that tea catechins, having potent anti-cancer activity, can bind to the cell surface, while catechins, having lower activity did not [38].

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Therefore, further investigation about the cellular interaction of EGCG may contribute to the elucidation of the mechanism of EGCG actions. Recently, plasma membrane microdomains referred to as ‘lipid rafts’ have received much attention as potential regulators and organizing centers for signal transduction and membrane traffic pathways [114]. The already impressive catalog of raft involvement in diseases, including cancer, Alzheimer disease, Parkinson disease, prion diseases, atherosclerosis, hypertension, diabetes, allergic response, bacterial infections, and viral infections, continues to grow (Figure 2) [115]. These microdomains have been characterized as sphingolipid/cholesterol-rich domains in the plasma membranes. Lipid rafts have also been shown to interact with various kinds of extracellular factors including cytokines and growth factors, neuroproteins, and viruses [116-119]. A large number of raft proteins and raft-associated proteins have been identified [116-118,120-141]. However, little is known about the interaction of these microdomains with low-molecular compounds such as tea catechins. EGCG has been found to inhibit epidermal growth factor or platelet-derived growth factor-mediated tumor cell growth by reducing the autophosphorylation of their receptors [52], and both of these receptors were observed in lipid rafts [116,117]. The formation of senile plaques containing the β amyloid peptide (Aβ) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer’s disease. It has been demonstrated that APP interacts with lipid rafts and which are critically involved in regulating Aβ generation [142]. Aβ has been known to be toxic to neurons in rat primary hippocampal cultures [143], and such neurotoxicity has been shown to be attenuated by treatment with EGCG [144]. These circumstances have fueled interest in the role of lipid rafts as a platform for researching the function of EGCG, but there has been no direct data proving the relationship of EGCG with lipid rafts. To explore a potential role of lipid rafts in binding of EGCG to the plasma membrane, a study of raft integrity was performed using MβCD, a cholesterol-removing agent that disturbs raft function [145]. A surface plasmon resonance (SPR) assay revealed that EGCG was able to bind to the cell surface of the human basophilic KU812 cells, chronic myelogenous leukemia cells, and this binding was inhibited upon treatment with MβCD [146]. In addition, we found that the disruption of lipid rafts caused a significant reduction of EGCG activities such as the inhibition of histamine release and the suppression of the high-affinity-IgE receptor (FcεRI) expression [146,147]. To elucidate the interaction of EGCG with the lipid rafts, the amount of EGCG in the lipid rafts was directly measured by sucrose gradient ultracentrifugion and high-performance liquid chromatography (HPLC) methods. The level of EGCG in the raft fraction was much higher than the non-raft fraction, indicating that EGCG does not homogenously bind to the membrane of the cells, but interacts with the cells in a heterogeneous fashion [146]. This suggests that these lipid rafts may play a possible role in the interaction of EGCG with the cells. In the study on a disruption of lipid rafts, the treatment with MβCD clearly decreased the binding of EGCG, and this treatment also led to a reduced amount of EGCG in the raft fraction isolated from MβCD-treated cells [146]. These results suggest that lipid rafts is responsible for the interaction of EGCG with the cell surface of the human chronic myelogenous leukemia KU812 cells and plays an important role in mediating EGCG signaling. These facts raised a possibility that the primary target of EGCG may be a membrane molecule localized in lipid rafts on the cell surface. Therefore, we attempted to investigate the cell-surface molecule acting as an EGCG receptor, which can mediate anti-cancer activity of EGCG.

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Figure 2. Plasma membrane microdomain “lipid rafts” and its related diseases. Rafts contain proteins attached to the outer leaflet of the bilayer by their GPI anchors, proteins binding to the inner leaflet by acyltails (for example, the Src-family kinase). The lipid bilayer in rafts is asymmetric, with sphingomyelin and glycosphingolipids enriched in the outer leaflet and glycerolipids (for example, phosphatidylserine and phosphatidylethanolamine) in the inner leaflet. Cholesterol is present in both leaflets and fills the space under the head groups of sphingolipids or extends the interdigitating fatty acyl chain in apposing leaflet. Raft-related diseases are reviewed in ref. [115].

5.3. Discovery of 67LR as a Green Tea Catechin Receptor To elucidate the detail molecular basis for the action of EGCG, the same as drugs, it is necessary to identify the molecular target triggering a specific signaling of EGCG. As you know, toll-like receptor teaches us the principal role in the lipopolysaccharide signaling [148] and the necessity of identification of the specific receptor as a signal initiator for generating cellular responses for understanding the specific cellular signaling of foreign or functional substances (Figure 3). However, the molecular target for physiologically relevant EGCG that

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can mediate its anti-cancer effect still remained unknown. Recently we found that all-transretinoic acid (ATRA) enhances the binding of EGCG to the cell surface of cancer cells when the binding was monitored on the basis of the increase in response units in a SPR assay [38]. To identify candidates through which EGCG inhibits cell growth, we used a subtraction cloning strategy involving cDNA libraries constructed from cells treated or untreated with ATRA. We isolated a single target that allows EGCG to bind to the cell surface. An analysis of the DNA sequence identified this unknown cell surface candidate as the 67LR. In fact, the expression of this 67LR was enhanced by ATRA treatment. Human lung cancer A549 cells were used to assess how effectively the 67LR elicits EGCG-mediated growth inhibition. Cells transfected with empty vector and treated with EGCG showed no growth inhibition. However, cells transfected with the gene encoding 67LR and treated with EGCG demonstrated considerable inhibition as compared with the cells treated with H2O. We next tested whether the growth inhibitory activity of EGCG correlates with the binding strength of EGCG to the cell surface. We found increased binding of EGCG to the cell surface of cells transfected with 67LR. EGCG binding to the 67LR-transfected cells was inhibited by treatment with an antibody to 67LR. We then measured the binding affinity of EGCG to 67LR in equilibrium binding experiments using SPR. Kd measurements were made with a purified recombinant 67LR protein. The predicted Kd value for the binding of EGCG to the 67LR protein is 39.9 nM [38].

Figure 3. The importance of a primary target for triggering a specific signaling of EGCG.

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Figure 4. Tumor size in C57BL/6N inoculated with 67LR-, eEF1A, or MYPT1-knockdown B16 cells. C57BL/6N mice were subcutaneously inoculated with B16 cells stably transfected with control shRNA or either the 67LR (A), eEF1A (B), or MYPT1 (C) shRNA expression vector. Peroral administration of 0.1% EGCG was started 1 day before the tumor cell inoculation. After 15 days, tumor size were measured, and these data were represented as the mean±S.E. of 6 or 7 mice (*, P < 0.05 versus untreated control).

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Most of the 67LR protein was found to exist in the raft fraction rather than the non-raft fraction [149], and this distribution pattern correlated well with the plasma membraneassociated EGCG level after treating the cells with EGCG as previously reported [146]. Disruption of these rafts by cholesterol depletion was shown to cause a decrease in the binding of EGCG to the cell surface [146]. A defect in the integrity of rafts induced by MβCD treatment led to a reduced expression of 67LR on the cell surface [149]. These facts suggest that a reduction of lipid raft-associated 67LR may be responsible for the cell-surface binding of EGCG. The concentrations of EGCG shown to have an effect (10-100 μM) in these previous studies are much higher than those observed in the blood or tissues. However, the EGCG concentrations observed in the plasma and tissue levels after ingestion of tea or polyphenols are usually lower than 1 μM [12]. To investigate whether the 67LR can confer a sensitivity to EGCG at physiologically relevant concentrations, we treated the 67LR-transfected A549 cells with two concentrations of EGCG (0.1 and 1.0 μM); these concentrations are similar to the amount of EGCG found in human plasma after drinking more than two or three cups of tea [150]. The growth of the transfected cells was inhibited at both of these concentrations [38]. In addition, this growth-suppressive effect was completely eliminated upon treatment with anti-67LR antibody before the addition of EGCG. Although we have identified 67LR as a cell surface receptor for EGCG that mediates EGCG-induced cell growth inhibition [38], there is no validation of its implication in EGCG-induced cancer prevention in vivo. We investigated the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with 67LR-ablated B16 cells [17]. We confirmed both silencing of 67LR by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 μM EGCG on cell growth in 67LR-ablated B16 cells in vitro. As shown in Figure 4A, tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with 67LRablated B16 cells, suggesting that 67LR functions as an EGCG receptor not only in vitro but also in vivo. Together, these observations demonstrate that the cell-surface 67LR is the receptor for antitumor action of EGCG at the physiologically relevant concentration. Characterization of the mechanisms by which 67LR regulates cell proliferation may provide a new approach to cancer prevention.

5.4. The Inhibition of Cancer Cell Growth by EGCG through the Green Tea Catechin Receptor Phosphorylation of the myosin regulatory light chain (MRLC) at Thr18/Ser19 was shown to increase the actin-activated Mg-ATPase activity of myosin II and the assembly of myosin II filaments, and regulate the association between myosin II and filamentous actin (F-actin) [151,152]. The association of myosin II with F-actin results in the formation of stress fibers in interphase cells and the contractile ring in dividing cells (Figure 5A) [153]. During cytokinesis an actomyosin-based contractile ring is formed at the equator of dividing cells, then gets constricted, and finally disappears at the end of cytokinesis [154]. In higher

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eukaryotes, it is well established that the state of MRLC phosphorylation is cell cycle dependent. To examine the effect of EGCG on cell morphology and actin cytoskeleton, we stained HeLa cells, human cervical adenocarcinoma, for F-actin with Alexa Fluor 488 phalloidin [19]. As shown in Figure 5B, control HeLa cells exhibited an organized network of F-actin containing stress fibers spanning across the cell body and a thin cortical F-actin rim at the margins. This rim was so thin that cell-cell junctions were not distinguishable. When the cells were incubated with the indicated concentrations of EGCG for 20 min, the cells retracted and left intercellular gaps. In addition, disappearance of the stress fibers in the central cell body was observed upon treatment with EGCG, and the peripheral actin rim became thicker, providing an outline of the cell-cell junctions. Because stress fiber formation has been known to require Thr18/Ser19 phosphorylation of MRLC [153], we examined the effect of EGCG on the MRLC phosphorylation by Western blot analysis using phosphorylated MRLC-specific antibodies [19]. EGCG dose-dependently decreased the diphosphorylation of MRLC at The18/Ser19. This result correlates well with EGCG-induced actin cytoskeleton remodeling. EGCG has been reported to inhibit cancer cell proliferation directly by affecting the signaling pathway involved in cell growth [43,155]. However, the concentration range of EGCG shown to have an effect (20-100 μM) in previous studies is much higher than that observed in blood or tissues. To investigate whether the physiologically relevant concentrations of EGCG can reduce the MRLC phosphorylation, we treated HeLa cells for 24 h with three concentrations of EGCG (0.1, 1 or 10 μM). The MRLC phosphorylation was reduced at concentrations between 1 and 10 μM. The phosphorylation of MRLC at Thr18/Ser19 has been shown to be necessary for formation of the contractile ring in dividing cells [153]. To examine the effect of EGCG on contractile ring formation, HeLa cells were treated with 50 μM EGCG for 10 min and stained for F-actin [19]. At low magnification, the contractile ring in dividing cells not treated with EGCG exhibited spot-like aggregated F-actin (Figure 5B). However, in EGCG-treated cells these spot-like F-actin condensations disappeared and the cell peripheral actin rim was able to be visualized. We then double stained the dividing cells for F-actin and the phosphorylated MRLC utilizing Alexa Fluor 488 phalloidin and the phospho-MRLC (Thr18/Ser19) specific antibody, respectively. At high magnification it is clearly observed that EGCG inhibited the assembly of F-actin and the phosphorylation of MRLC in the cleavage furrow of dividing cells. We next examined the effect of EGCG on the growth and cell cycle of HeLa cells [19]. Treatment of the cells with 50 μM EGCG inhibited cell growth and significantly reduced the diphosphorylation of MRLC at The18/Ser19. Given that EGCG inhibited the contractile ring formation by reducing the MRLC phosphorylation and thus inhibited the cell growth, to determine if EGCG treatment could alter the cell cycle, we subjected EGCG-treated cells to FACS analysis using propidium iodide staining to measure the DNA content. EGCG treatment for 48 h significantly increased the percentage of cells in the G2/M phase. These results suggest that the suppressive effect of EGCG on the MRLC phosphorylation continued at least for 48 h and the increase of G2/M phase cells resulted from the effect of EGCG

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Figure 5. EGCG-induced actin cytoskeleton rearrangement and cell morphology. (A) The interaction between acitin and myosin filaments. Phosphorylation of the myosin II regulatory light chain (MRLC) at Thr18/Ser19 was shown to increase the actin-activated Mg-ATPase activity of myosin II and the assembly of myosin II filaments, and regulate the association between myosin II and filamentous actin (F-actin). The association of myosin II with F-actin results in the formation of stress fibers in the interphase cells and the contractile ring in dividing cells. (B) HeLa cells were treated with 50 μM EGCG in DMEM. The cells were fixed and stained with Alexa Fluor 488 phalloidin.

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EGCG has been known to produce H2O2 when it is added to the media of several cultured cell lines, and H2O2 may exert biological effects on cell growth. It has been reported that 30 μM EGCG can induce apoptosis in H661 lung cancer cells, and this effect could be abolished by the addition of catalase (50 U/ml) in the culture medium [156]. We examined whether catalase can change the effect of EGCG on cell growth and MRLC phosphorylation [19]. The addition of catalase could not alter the EGCG-induced reduction of the MRLC phosphorylation level, the inhibition of the cell growth or the accumulation of the cells in G2/M phase. To analyze whether the suppressive effect of EGCG on the MRLC phosphorylation is mediated by the 67LR, RNA interference (RNAi)-mediated gene silencing was utilized to knock down the expression of the 67LR [19]. HeLa cells were transiently transfected with three short hairpin RNA (shRNA) expression vectors for the 67LR. The 67LR silencing was observed when each of the shRNA expression vectors was trasnsfected separately (data not shown), however when we combined all three shRNA expression vectors, the highest silencing was observed. We confirmed the elimination of the EGCG inhibitory effect on the growth of cells transfected with all three shRNA expression vectors. EGCG significantly reduced the phosphorylation of MRLC in the cells transfected with the empty vector, however in the cells transfected with shRNA expression vectors for the 67LR, the same concentration of EGCG only slightly reduced the phosphorylation. These results indicate that 67LR does indeed mediate the suppressive effect of EGCG on MRLC phosphorylation. Taken together, these findings suggest that EGCG inhibits the cancer cell growth by reducing the MRLC phosphorylation and this effect is mediated by the 67LR. Colorectal cancer is one of the most prevalent types of cancer in the Western world [157]. Epidemiological studies have suggested that the consumption of green tea may decrease colon cancer risk [158]. The Wnt-pathway appears to play an important role particularly in colon carcinogenesis [159]. The essential event in Wnt-signaling is the stabilization of β-catenin. The resulting accumulation of β-catenin increases the pool of nuclear β-catenin bound to transcription factor TCF/LEF in complexes that can activate certain genes, including oncogene c-Myc. As the main binding partner of β-catenin at cell.cell junctions, E-cadherin plays a pivotal role in β-catenin stabilization and function. E-cadherin commonly repressed in epithelial carcinogenesis and its binding with β-catenin suppressed Wnt-signaling [160]. Previous studies have reported that EGCG-treatment downregulated the β-catenin protein expression [41] or upregulated E-cadherin protein expression [161], suggesting that EGCG suppressed Wnt-signaling. However, it is still not known how a physiological concentration of EGCG induces cell growth inhibition in colorectal cancer cells. Recently, we found for the first time that a physiologically achievable concentration of EGCG inhibited cell cycle progression of human colon adenocarcinoma Caco-2 cells through 67LR without affecting components of Wnt-signaling. Further, we found that EGCG at a physiological concentration decreased the phosphorylation of MRLC at Thr-18/Ser-19 in Caco-2 cells through 67LR, suggesting that an activation of myosin cytoskeleton is involved in anti-proliferative effect of EGCG at a physiological concentration [18]. Caco-2 cells exhibited higher 67LR protein expression than HeLa cells. EGCG inhibited the growth of Caco-2 cells even at the concentration of 1 μM. On the other hand, compared with Caco-2 cells, HeLa cells were less sensitive to EGCG because its growth was inhibited at only 10 μM EGCG. These results suggest that the expression level of 67LR may be

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elevated in the cells that are sensitive to the effect of EGCG at a physiological concentration. EGCG dose-dependently decreased the G0/G1 and S fractions while increasing the G2/M fraction in Caco-2 cells. The inhibition of cell cycle progression was significantly observed even at 1 μM EGCG. These results indicate that a physiologically achievable concentration of EGCG inhibited cell growth of Caco-2 cells by accumulating the cells in G2/M phase. We investigated the effect of EGCG on the protein expression of E-cadherin, β-catenin, and c-Myc, which are important Wnt-signaling components, in Caco-2 cells by Western blot analysis. E-cadherin protein expression was not affected by EGCG at 1 or 5 μM, though it was slightly reduced by 10 μM EGCG treatment for 72 h. β-Catenin protein expression were slightly affected by EGCG at 1 or 5 μM but dose- and time-dependency were not observed. cMyc protein expression was slightly reduced by 10 μM EGCG treatment for 72 h. Since these results did not correlate with EGCG-induced cell growth inhibition as mentioned above, it is suggested that E-cadherin, β-catenin, and c-Myc are not involved in anti-proliferative effect of EGCG at a physiological concentration on Caco-2 cells. Although it may be proposed that suppression of Wnt-signaling by EGCG is the mechanism for EGCG-induced cell cycle arrest at G0/G1 phase, our results suggest that cell growth inhibition induced by a physiological concentration of EGCG in Caco-2 cells is dependent on other mechanisms than suppression of Wnt-signaling. We further examined the effect of EGCG on MRLC phosphorylation at Thr-18/Ser-19 by Western blot analysis. EGCG treatment for 24 h dose-dependently reduced the level of MRLC phosphorylation. Moreover, decrease in the phosphorylation of MRLC was observed even at 1 μM EGCG and correlated well with EGCG-induced cell growth inhibition. To investigate whether the inhibitory effect of EGCG on cell cycle progression is mediated by 67LR, we knockdown the expression of 67LR in Caco-2 cells using RNAimediated gene silencing. 67LR knockdown significantly attenuated 1 μM EGCG-induced inhibition of cell growth and accumulation of the cells in G2/M phase. Furthermore, 1 μM EGCG-induced reduction of the phosphorylation of both MRLC (The-18/Ser-19) was also attenuated in 67LR-shRNA expressing cells. These results suggest that 67LR mediates the suppressive effect of EGCG at a physiological concentration on cell cycle progression and the phosphorylation of MRLC and MYPT1.

5.5. Induction of Apoptosis by EGCG through the Green Tea Catechin Receptor Recently, EGCG has been shown to be able to induce growth arrest and subsequent apoptotic cell death in multiple myeloma (MM) cells including IL-6-dependent cells and primary patient MM cells in vitro, while having no significant effect on growth normal cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts [162]. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. The expression of 67LR was significantly elevated in myeloma cell lines and patient samples compared to normal PBMCs. RNAi-mediated inhibition of 67LR resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that 67LR plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways

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of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-kappaB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expressions of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific anti-myeloma activity of EGCG and provide the rationale for its clinical evaluation.

5.6. Anti-allergic Action of EGCG through the Green Tea Catechin Receptor 5.6.1. Inhibition of Histamine Release Mast cells and basophils play a central role in immediate allergic reactions mediated by IgE. Binding of multivalent allergens to specific IgE attached to the FcεRI on the surface of mast cells or basophils leads to the release of both preformed and newly generated inflammatory mediators such as histamine, cytokines, chemotactic factors, and arachidonic acid metabolites. These mediators ultimately cause various symptoms including atopic dermatitis, hay fever, bronchial asthma, and food allergy [163,164]. The early phase of cell activation of mast cells and basophils includes the phosphorylation and activation of protein tyrosine kinases and their substrates, generation of the second messengers such as inositol trisphosphate and diacylglycerol, and elevation of intracellular Ca2+ levels [165,166]. The late phase of the activation, which occurs after the influx of Ca2+, includes the fusion of secretory granules with the membrane and dramatic morphological changes due to remodeling of actin cytoskeleton, which undergo extensive membrane ruffling [167-169]. Understanding the molecular events in this degranulation process is thought to hold promise for a therapeutic intervention leading to the attenuation of various allergic diseases. Screening and evaluation of anti-allergic factors based on the inhibition of histamine release has been most frequently performed. Recently, we reported that EGCG inhibited the calcium ionophore A23187-induced histamine release from the human basophilic KU812 cells and could not inhibit the increase of the intracellular Ca2+ level after stimulation with A23187 [147]. This result suggested that the effect of EGCG on histamine release occurs after the elevation of the intracellular Ca2+ concentration. The increase of intracellular Ca2+ activates degranulation-related cytoskeleton, and the rearrangement of cytoskeleton requires the interaction between myosin and actin filaments [167-169]. Phosphorylation of the MRLC at Thr18/Ser19 has been shown to regulate the association between myosin II and F-actin [151,152]. Thr18/Ser19 phosphorylation of MRLC has been reported to be temporally correlated with degranulation in the rat basophilic RBL-2H3 cells, and the inhibition of MRLC phosphorylation has been shown to impair the degranulation [167,170]. Although EGC, having no ability to inhibit histamine release, showed no inhibitory effect on MRLC phosphorylation, EGCG clearly reduced the level of phosphorylated MRLC [147]. After treatment of KU812 cells with the anti-67LR antibody, cells were incubated with EGCG, and further challenged with A23187. The reductive effect of EGCG on the histamine release was almost completely inhibited in cells treated with the anti-67LR antibody. Experiment using such 67LR-downregulated cells revealed a significant abrogation of the inhibitory effect of EGCG on degranulation.

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Furthermore, the lowering effect of EGCG on the phosphorylation of MRLC was also inhibited by either treatment with the anti-67LR antibody or 67LR-knockdown. These findings indicate that the inhibitory effect of EGCG on degranulation was caused by a modification of myosin cytoskeleton through the binding of EGCG to 67LR on the cell surface. Activation of mast cells and basophils results in extensive changes in cell morphology, the formation of F-actin, and extensive redistribution of myosin and actin-filaments within the cells [168,171-174]. To examine the effect of EGCG on the actin cytoskeleton, we stained Factin with Alexa Fluor 488 phalloidin. A wavy pattern of stained F-actin was observed in the cells stimulated with A23187 alone, suggesting that A23187 leads to remodeling of the actin cytoskeleton in KU812 cells. Generally, degranulation-inducing stimuli have been known to induce dramatic change of actin cytoskeleton such as membrane ruffling, wavy form of membrane [169,175]. Thus, the present data implicated that A23187 induced the actin remodeling similar to membrane ruffling in KU812 cells. When the cells were stimulated with A23187 in the presence of EGCG, membrane ruffling was inhibited and a biased F-actin accumulation was observed. Furthermore, this EGCG-induced actin remodeling was abolished in both anti-67LR antibody-treated cells and 67LR-knockdowned cells. Generally, the association between F-actin and myosin II has been shown to be regulated by phosphorylation of the MRLC at Thr18/Ser19 [151,152]. Our findings indicated that EGCGinduced actin remodeling is caused by lowering MRLC phosphorylation mediated through the binding of EGCG to the 67LR. Thus, these cytoskeletal modifications may have an important role in the inhibition of histamine release by EGCG.

5.6.2. Suppression of the High-affinity IgE Receptor Expression FcεRI plays a central role in the induction and maintenance of IgE mediated allergic responses such as atopic dermatitis, bronchial asthma, allergic rhitis, and food allergy. The FcεRI molecule on these cells is a tetrameric structure of one α chain, one β chain, and two disulfide-linked γ chains. In addition, a trimeric form of FcεRI which lacks the β chain is found in human. Among the three subunits forming FcεRI, the α chain is the specific component of FcεRI that mostly extends out to the extracellular region and directly binds to IgE. Analysis of α chain-deficient mice demonstrated that IgE was unable to bind to the cell surface of mast cells, thereby inabling the induction of degranulation through IgE binding [176]. Thus, it is expected that the downregulation of FcεRI expression in mast cells and basophils may lead to the attenuation of the IgE-mediated allergic symptoms. The effect of several major tea catechins, (+)-catechin (C), EC, EGC, ECG, and EGCG, on the cell-surface expression of FcεRI in KU812 cells was studied [177]. Flow cytometric analysis showed that only EGCG was able to decrease the cell-surface expression of FcεRI after a 24-h treatment in a dose-dependent manner, while other catechins did not. Moreover, immunoblot analysis revealed that the total cellular expression of the FcεRI α chain decreased upon treatment with EGCG. KU812 cells treated with EGCG expressed lower levels of FcεRI α and γ mRNA than nontreated cells. These results suggest that EGCG has an ability to down-regulate FcεRI expression, and this suppressive effect may be due to the down-regulation of FcεRI α and γ mRNA levels. We also found that EGCG has an ability to inhibit the phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) [146]. This inhibition was involved in downregulation of FcεRI expression by EGCG. Moreover, the

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inhibitory effect elicited by EGCG on ERK1/2 was prevented by disruption of lipid rafts. Thus, these results suggest that the interaction between EGCG and the lipid rafts is important for EGCG’s ability to downregulate FcεRI expression, and ERK1/2 may be involved in this suppression signal. We also examined the effect of the 67LR knockdown on EGCG’s abilities [149]. SPR analysis showed a reduction of the binding of EGCG to the cells transfected with the 67LRshRNA vector, indicating that the EGCG’s binding to the cells lowered upon the decrease of 67LR expression. The suppressive effect of EGCG was inhibited by the knockdown of 67LR. Furthermore, the ability of EGCG to decrease the phosphorylation of ERK1/2 was reduced in the 67LR-knocked down cells. These results indicate that the effect of EGCG on ERK1/2 phosphorylation correlates with the expression of 67LR, which implies that the 67LR is the molecule responsible for transducing the EGCG’s downregulatory signaling of the FcεRI.

5.6.3. Anti-allergic Effect of O-methylated Catechin through the Green Tea Catechin The O-methylated derivative of EGCG, (–)-epigallpcatechin-3-O- (3-O-methyl)-gallate (EGCG3”Me) and (–)-epigallpcatechin-3-O- (4-O-methyl)-gallate (EGCG4”Me), as shown in Figure 1, which was isolated from tea leaves such as Tong-ting oolong tea or cultivars ‘Benifuuki’ and ‘Benihomare‘ (Camellia sinensis L.), has been shown to inhibit allergic reactions [178,179]. The inhibitory effects of O-methylated EGCG on mouse type I and IV allergies in vivo more potently than EGCG [178,180]. It is suggested that the higher antiallergic activity of O-methylated EGCG may be due to the higher absorption and stability in vivo [181]. These catechins also strongly inhibited mast cell activation through the prevention of tyrosine phosphorylation (Lyn, Syk and Btk) of cellular protein and histamine/ leukotriene release, interleukin-2 secretion after FcεRI cross-linking [182]. Therefore, these facts may encourage allergic patients to drink tea rich in EGCG3”Me. Indeed, a double-blind clinical trial to treat allergic cedar pollinosis patients with ‘Benifuuki’ green tea rich in EGCG3”Me was carried out, and promising results have been obtained by using a protocol of drinking of 1.5 g of tea powder with water twice a day for 13 weeks [181]. We have been found that EGCG3”Me can inhibit histamine release and suppress the FcεRI expression the same as EGCG [179,183]. RNAi-mediated knockdown of 67LR expression resulted in a decreased activity of EGCG3”Me [20]. The suppression of MRLC phosphorylation through the cell-surface binding to the 67 LR contributes to the inhibitory effect of EGCG3”Me on the histamine release. The 67LR also mediated the EGCG3”Meinduced suppression of FcεRI expression by reducing ERK1/2 phosphorylation. These results suggest that anti-allergic effects of EGCG3”Me may be triggered by the inhibition of MRLC or ERK1/2 phosphorylation mediated through the cell-surface 67LR. Recently, we examined the effects of methylated derivatives of EGCG, EGCG4”Me and (–)-4’-O-methyl-epigallocatechin-3-O-(4-O-methyl) gallate (EGCG4’4”diMe) as shown in Figure 1, on FcεRI expression and ERK1/2 phosphorylation, and each of their cell-surface binding activities was measured [184]. EGCG4”Me, which is methylated at the 4’-position, suppressed FcεRI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG4’4”diMe, which is methylated at both the 4’- and 4”positions, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG4”Me could bind to the cell surface even though the binding activity was lower than that of EGCG. EGCG4’4”diMe could not bind. These results suggest that the trihydroxyl

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structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4’-position in the B ring and the 4”-position in the D ring (gallate moiety) are crucial for the cell-surface binding activity of EGCG. EGCG is known to be unstable and is degraded easily in animal bodies. On the other hand, EGCG3”Me and EGCG4”Me are absorbed efficiently and are more stable than EGCG in animal and human plasma, suggesting the reason for the methylated derivatives of EGCG having potent inhibitory activities to allergies in vivo, whereas the biological activities in vitro are lower than those of EGCG. EGCG has been reported to undergo methylation, and EGCG4’4”diMe has been shown to be a major metabolite of EGCG in plasma [27]. Although our studies on methylated EGCGs may contribute to the elucidation of physiological activities of EGCG in vivo, further investigation of the relationship between metabolites of EGCG and 67LR is necessary for a better understanding of the molecular basis of EGCG activities in vivo.

6. IS THE 67LR A SPECIFIC RECEPTOR FOR GALLATE DERIVATIVES ? Tea also contains other biologically active compounds such as caffeine [185]. To compare the ability of 67LR to mediate a response to other tea constituents, we examined caffeine and other tea polyphenols (Figure 6). None of these other compounds affected the growth of 67LR-expressing A549 cells, nor could they bind to the cell surface [38]. EGCG is the only gallate (gallic acid ester) we tested, suggesting that the gallate moiety may be critical for 67 LR binding and subsequent activity.

Figure 6. The interactions between tea constituents and 67LR-transfected cells. (A) Growth inhibitory activities of tea constituents (indicated by bars, 5 μM) on A549 cells transfected with either the gene encoding 67LR (red) or vector only (blue) were examined. (B) The interaction between tea constituents (5 μM) and A549 cells transfected with the 67LR vector (red line) or the empty vector (blue line) was measured using a SPR assay.

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Recently, we investigated the structure-activity relationship of major green tea catechins and their corresponding C-2 epimeric isomers, as shown in Figure 1, on cell-surface binding and inhibitory effect for histamine release [21]. Galloylated catechins; EGCG, (–)gallocatechin-3-O-gallate (GCG), ECG, and (–)-catechin-3-O-gallate (CG) showed the cellsurface binding to the human basophilic KU812 cells by SPR analysis, but their nongalloylated forms (EC, C, EGC, and GC) did not. The overlapped SPR sensorgram of each catechins showed that the order of the binding strength based on the resonance unit was EGCG > GCG > CG ECG. On the other hand, gallic acid (GA) and pyrogallol (PG), as shown in Figure 1, which are the model molecules of galloyl group or B-ring, could not bind to the cell surface the same as nongalloylated catechins (C, EC, GC, and EGC). These observations indicate that only a structural moiety of galloylated catechins (CG, ECG, GCG, and EGCG) such as basic flavan-3-ol structure (C, EC, GC, and EGC), B-ring (PG), or the galloyl moiety (GA and PG) is not sufficient to exert the cell-surface binding. Furthermore, these findings suggest that the combination of basic flavan-3-ol structure and galloyl group has an essential role in cell-surface binding of tea catechins. The binding strengths of pyrogallol-type catechins (3’,4’,5’-trihydroxyl structure of the B-ring; GCG and EGCG) were higher than those of catechol-type catechins (3’,4’-dihydroxyl structure of B-ring; CG and ECG). Unlike catechol-type catechins, in pyrogallol-type catechins, a remarkable difference in binding activities was observed. These results indicate that the galloyl group and the number of hydoxyl group on the B-ring as well as the configuration of the B-ring are responsible for the cell-surface binding of tea catechins. These patterns were also observed in their inhibitory effects on histamine release. RNAi-mediated downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. Among four galloylated catechins, the maximum reduction of the binding was observed in EGCG. The reduction rates of the binding strength of pyrogallol-type catechins (GCG and EGCG) were significantly higher than those of catechol-type catechins (CG and ECG). Although there is a bulky sterical difference between the C-2 epimers by the B-ring confuguration, no significant difference in the binding was found between either pyrogallol- or catechol-type catechins. These results suggest that the 67LR is involved in the cell-surface binding of four galloylated catechins and that the number of the hydoxyl group on the B-ring contributes to the 67LRdependency of galloylated catechins on the cell-surface binding. As to the inhibitiory effect of tea catechins on histamine release, the order of the 67LR-dependent reduction rate was EGCG > GCG > ECG > CG. The reduction rates of inhibitory activities of pyrogallol-type catechins (GCG and EGCG) were higher than those of catechol-type catechins (CG and ECG). This 67LR-dependency was similar to a result of the dependency on their cell-surface bindings. The influence of different configuration of the B-ring on the 67LR-dependency was much smaller than those of different hydroxylation of the B-ring. These results suggest that the 67LR is involved in the inhibitory effect of four galloylated catechins on histamine release, and that the pattern of B-ring hydroxylation is a major structural determinant for the 67LRdependency of galloylated catechins. Taken together, our findings suggest that the combination of galloyl group with basic flavan-3-ol structure may be necessary for the binding of tea polyphenols to the cell-surface 67LR

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7. GREEN TEA CATECHIN RECEPTOR SIGNALING MOLECULES 7.1. Genetic Suppressor Element (GSE) Methodology In an attempt to elucidate the pathways involved in the anticancer action of EGCG, we applied genetic suppressor element (GSE) methodology. GSEs are short cDNA fragments encoding peptides acting as dominant inhibitors of protein function or antisense RNAs inhibiting gene expression [186]. GSEs behave as dominant selectable markers for the phenotype associated with the repression of the gene from which they derived, thus allowing identification of this gene. For example, this strategy previously allowed the demonstration that kinesin heavy chain is involved in the control of cell response to various DNA-damaging agents [187]. For identifying genes mediating cell sensitivity to EGCG, we selected GSEs conferring resistance to EGCG. To search for the mediators of EGCG-induced cell growth inhibition in B16 mouse melanoma cells, we utilized a targeted genetic screen with a GSE complementary DNA library, which was prepared from a mouse embryo. Among genetic elements protecting cells from EGCG-induced cell growth inhibition, we isolated a GSE that encoded the N terminus of eukaryotic translation elongation factor 1A (eEF1A) [17]. eEF1A is an important component of the eukaryotic translation apparatus and is also known as a multifunctional protein that is involved in a large number of cellular processes [188].

7.2. Eukaryotic Translation Elongation Factor 1A (eEF1A) To investigate the role of eEF1A in EGCG-induced cell growth inhibition, we used stable RNAi [189] to silence eEF1A expression in B16 cells. Remarkably, silencing of eEF1A attenuated the inhibitory effect of 1 μM EGCG on cell growth [17]. In contrast, overexpression of eEF1A enhanced the inhibitory effects of 1 μM EGCG on cell growth. This concentration is similar to the amount of EGCG found in human plasma after drinking more than two or three cups of green tea [150]. EGCG is the only known polyphenol present in plasma in large proportion (77-90%) in a free form, although the other catechins are highly conjugated with glucuronic acid and/or sulfate group [190]. Based on these considerations, the activities observed at 1 μM EGCG are relevant to the in vivo situations. Given this, we investigated the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with eEF1A-ablated B16 cells as shown in Figure 4B [17]. Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with eEF1A-ablated B16 cells, indicating that eEF1A is involved in EGCG-induced cancer prevention. EGCG induces growth inhibition in many cell lines; however, the efficacy of inhibition varied, depending on the cell lines used [191]. We hypothesized that the expression level of eEF1A in a cell line correlates to the efficacy of EGCG-induced cell growth inhibition in that cell line. We investigated the expression levels of eEF1A in B16 cells and the following human cancer cell lines: hepatoma HepG2, breast carcinoma MCF-7, cervical carcinoma HeLa, and squamous cell carcinoma A431 [17]. The levels of eEF1A expression in B16 cells, HepG2 cells, and MCF-7 cells were relatively higher than those in HeLa cells and A431 cells.

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EGCG appeared to display different efficacies of growth inhibition in these cell lines, with estimated IC50 values of 9.7 μM for MCF-7 cells, 22.7 μM for HepG2 cells, 51.6 μM for HeLa cells, and 52.8 μM for A431 cells, respectively. The expression level of eEF1A is elevated in cell lines that are more sensitive to the effect of EGCG. These results support our conclusion that eEF1A serves as a mediator for EGCG-induced cancer prevention.

7.3. Myosin Phosphatase-targeting Subunit (MYPT1) and Remodeling of Actin Cytoskeleton MRLC phosphorylation controls the activity of myosin II, a major motor protein in animal cells, which is involved in a wide range of processes, including muscle contraction, cell locomotion, cell division, and receptor capping [192]. The phosphorylation of MRLC is regulated by two classes of enzymes: MLC kinases and myosin phosphatase [193]. Myosin light chain kinase and Rho-kinase seem to be the two major kinases that phosphorylate MRLC in vitro as well as in vivo [193]. Myosin phosphatase is composed with three subunits: a 37-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a 110-130-kDa myosin phosphatase-targeting subunit (MYPT1) [194]. The activity of myosin phosphatase is known to be regulated by phosphorylation of MYPT1, and two major sites, Thr-696 and Thr853, have been extensively investigated and identified as an inhibitory site [194]. Previously, we reported that EGCG-induced cell growth inhibition may result from the reduction of the phosphorylation of myosin regulatory light chain (MRLC) at Thr-18/Ser-19 through 67LR in HeLa cells [19]. The activity of myosin phosphatase is known to be inhibited by phosphorylation of its targeting subunit MYPT1 at Thr-696 and Thr-853 [194]. We tested the effect of EGCG on the phosphorylation of MYPT1 at Thr-696 and Thr-853 [17]. Intriguingly, although the phosphorylation level at Thr-853 was unaffected by EGCG, EGCG induced the dephosphorylation of MYPT1 at Thr-696. Further, this effect correlated with EGCG-induced reduction of the MRLC phosphorylation, suggesting that EGCG activates myosin phosphatase by reducing the MYPT1 phosphorylation level at Thr-696. Next, we investigated whether MYPT1 is involved in anticancer action of EGCG in vivo. In B16 cells, physiological concentrations of EGCG reduced the MYPT1 phosphorylation at Thr-696 and the MRLC phosphorylation. We confirmed both silencing of MYPT1 by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 μM EGCG on cell growth in MYPT1-ablated B16 cells in vitro. We tested the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with MYPT1-ablated B16 cells (Figure 4C). Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with MYPT-1-ablated B16 cells, suggesting that MYPT1 is indispensable for EGCG-induced cancer prevention. In addition to this role of MYPT1 in EGCG-induced inhibition of cancer cell growth, a study on RNAi-mediated knockdown of MYPT1 expression demonstrated the importance of MYPT1 as the signaling component mediating the inhibitory effect of EGCG on histamine release and MRLC phosphorylation on the basis of the defect of both activities (unpublished data).

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Figure 7. Model of possible EGCG signaling pathway through 67LR in vivo.

It has been reported that MYPT1 directs the action of myosin phosphatase to not only MRLC but also other F-actin-binding proteins that influence cell contractility, morphology, and proliferation [195-198]. We found that EGCG induces a dynamic remodeling of actin cytoskeleton. The cells treated with EGCG exhibited a filopodial-like structure, and then after 3 h, the cell body retracted and left intracellular gaps, suggesting that the EGCG-induced filopodial-like projections are simple residual contact sites that have not yet been released from the substrate [17]. Together, it is suggested that EGCG-induced actin cytoskeleton remodeling results from not only the reduction of the MRLC phosphorylation but also the activation of myosin phosphatase.

7.4. A Hierarchy of EGCG Signaling Molecules Further, to establish whether MYPT1 is indeed involved in the suppressive effect of EGCG on MRLC phosphorylation and cell growth, we used stable RNAi to silence MYPT1 expression in HeLa cells [17]. Western blot analysis indicated that stable RNAi for MYPT1 specifically silenced MYPT1 protein expression in HeLa cells with no effect on the expression of 67LR and eEF1A. Silencing of MYPT1 prevented both EGCG-induced reduction of the MRLC phosphorylation and cell growth inhibition, suggesting that EGCGinduced dephosphorylation of MYPT1 at Thr-696 results in the activation of myosin phosphatase and inhibition of cell growth.

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It has been reported that eEF1A binds to the ankyrin repeat of MYPT1 [198]. It is tempting to speculate that both 67LR and eEF1A are upstream signaling components responsible for EGCG-induced dephosphorylation of MYPT1 at Thr-696. To test this hypothesis, we used stable RNAi to silence the expression of 67LR or eEF1A in HeLa cells. We confirmed specific silencing of each target protein by stable RNAi in HeLa cells and attenuation of the inhibitory effect of EGCG on cell growth in these cells [17]. In both 67LRablated HeLa cells and eEF1A-ablated HeLa cells, the inhibitory effect of EGCG on both the phosphorylation of MYPT1 at Thr-696 and the phosphorylation of MRLC was attenuated. In addition, EGCG-induced actin cytoskeleton rearrangement was no longer observed in MYPT1-, eEF1A-, or 67LR-ablated HeLa cells. The involvement of MYPT1 in downstream EGCG-triggered signaling from both 67LR and eEF1A was further documented by confirming abrogation of 1 μM EGCG-induced reduction of the MYPT1 phosphorylation level at Thr-696 and the MRLC phosphorylation in 67LR- or eEF1A-ablated B16 cells. These results suggest that MYPT1 is involved in downstream EGCG signaling from both 67LR and eEF1A (Figure 7). It has been reported that MYPT1 binds to eEF1A [198], and more than half of the total eEF1A (>60%) binds to the actin cytoskeleton [199]. Because other findings indicate that eEF1A is also implicated in microtubule binding, bundling, or severing [200,201], a potential role for the protein in regulating cytoskeleton organization has also been proposed. Characterizing the mechanisms by which EGCG induces reduction of the MYPT1 phosphorylation at Thr-696 and reorganization of actin cytoskeleton through eEF1A should help in more precise understanding of cytoskeleton organization, although further experiments are necessary.

8. CONCLUSION Recent studies have highlighted the importance of genetically determined factors in evaluating the role of green tea intake in the development of breast cancer [202-204]. In a case-control study conducted among Asian-American women, green tea intake appeared to reduce breast cancer risk [203]. Reduction in risk was strongest among persons who had the low-activity catechol-O-methyltransferase (COMT) alleles, but not high-activity COMT alleles, suggesting that these individuals were less efficient in eliminating green tea catechins and may derive the most benefit from these compounds. Yuan et al. reported a low risk of breast cancer among women with higher green tea intake and the low-activity genotype of angiotensin-converting enzyme gene among Singapore Chinese women [202]. A nested casecontrol study suggested protective effect of green tea against breast cancer among women with high-activity genotypes of the methylenetetrahydrofolate reductase and thymidylate synthase genes [204]. This effect was even stronger among those who were low consumers of dietary folate. These observations indicate that further study to elucidate the key molecules determining EGCG responsiveness is indispensable for a better understanding of EGCG activity in vivo. Chemoprevention by edible phytochemicals is now considered to be an inexpensive, readily applicable, acceptable, and accessible approach to cancer control and management [205]; however, little is known about the mechanism of the chemopreventive action of most phytochemicals, including EGCG. Although previous studies have proposed various different

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mechanisms for cancer-preventive action of EGCG [205-207], it remains unclear which EGCG-induced molecular events are relevant in vivo. The activities affected by lower concentrations of EGCG are likely to be more relevant in vivo because of the limited bioavailability of EGCG. The essence is the identification of the primary target and the demonstration of specific mechanisms of action in animal models and human tissues. Here we described that each of 67LR, eEF1A, and MYPT1 is indispensable for EGCG-induced cancer prevention in vivo, and these proteins mediate physiological concentrations of EGCGtriggered unique signaling for cancer prevention. Our findings suggest that these proteins are "master proteins," which determine the efficacy of cancer-preventive activity of EGCG and have important implications for development and use of EGCG as a cancer-chemopreventive agent. Probably, only a tumor with a high expression level of these "master proteins" has sensitivity to physiological concentrations of EGCG, while lower expression of those molecules causes “EGCG-resistance”. Our results not only illuminate the mechanisms for the cancer-preventive activity of EGCG but should help in the design of new strategies to prevent cancer and underscore the importance of tailoring cancer therapy on the basis of tumor genotype. The integrity of lipid rafts has been shown to be important for the pathogenesis of cancer [115,208]. Epigenetic regulation of genes encoding raft components and its roles in cell transformation, angiogenesis, immune escape, and metastasis has been reviewed earlier [115,138,140,208-212]. The role of rafts in prostate cancer is intriguing and discussed recently [213,214]. These contributions would help to best understanding current concerns in raft-associated signaling in regulation of cancer, and will help to select strategies for better management of neoplasm. Therefore, both orienting research toward newly proposed interactions (EGCG-67LR-eEF1A-MYPT1-Cytoskeleton axis), as shown in Figure 7, and investigation focused on lipid rafts as a signaling platform regulating the 67LR-mediated action of EGCG may unravel some of the complex aspect of EGCG-induced anti-cancer signaling. Finally, in addition to cancer-chemopreventive and anti-allergic properties, EGCG has been shown to possess diverse physiological activities, and we are curious to know whether EGCG signaling through the 67LR relates to other beneficial effects of EGCG.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 15

UTILITY OF EPIGALLOCATECHIN GALLATE IN THE TREATMENT AND PREVENTION OF BREAST CANCER: MOLECULAR MECHANISMS FOR TUMOR SUPPRESSION R. J. Rosengren* Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand

ABSTRACT Green tea and its major constituent epigallocatechin gallate (EGCG) have been extensively studied as a potential chemopreventative and/or treatment for a variety of diseases including breast cancer. Experimental evidence is supported by epidemiological studies that have shown an inverse relationship between green tea consumption and the incidence of breast cancer. Numerous studies have demonstrated that EGCG is cytotoxic toward both estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. These studies have highlighted potential mechanisms for the actions of EGCG, such as the induction of apoptosis, the alteration of the expression of cell cycle regulatory proteins critical for cell proliferation, inhibition of the chymotrypsin-like proteasome, inhibition of angiogenesis, as well as inhibition of cell invasion and metastasis. Importantly, these effects occur independently of estrogen receptor expression. This chapter will provide evidence for these events and other molecular mechanisms that significantly contribute to the actions of EGCG in vitro. The utility of green tea extract or EGCG as a breast cancer treatment and chemopreventative has also been extensively investigated using various in vivo models of estrogen receptor-positive and estrogen receptor-negative breast cancer (ie., chemical carcinogenesis and xenograft models). Evidence for EGCG-mediated tumor suppression and the major molecular mechanisms for this effect, such as the induction of apoptosis, the inhibition of angiogenesis and *

Address for correspondence: Rhonda J. Rosengren. Department of Pharmacology & Toxicology. 18 Frederick Street, Adams Building, University of Otago, Dunedin, New Zealand, 9001. E-mail: [email protected]; Tel: +64 3 479 9141; Fax +64 3 479 9140.

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R. J. Rosengren modulation of the expression of cell signaling proteins are also fully examined. These in vivo studies have led to investigations which have focused on ways to improve the actions of EGCG, by either using it as part of a combination therapy or by synthesizing pro-drugs of EGCG. Both of these have been done in order to enhance the bioavailability, stability and efficacy of EGCG. These new compounds and drug combinations have significantly improved the tumor suppression potential of EGCG and provide an exciting future for this multi-faceted phytochemical in the prevention and treatment of breast cancer.

INTRODUCTION Flavanoids are plant-derived polyphenolic compounds found in fruits, vegetables, herbs, tea and wine [1] and are divided into several different classes based on small variations in their structure. One such class is the flavan-3-ols, also termed the catechins, which are differentiated by di- or tri-hydroxyl group substitutions on the B-ring and meta-5,7-dihydroxy substitution on the A ring [2]. Catechins are particularly abundant in green tea (Camellia sinensis), accounting for 30-40% of its dry weight [3-5]. The major catechins contained in green tea are (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)epicatechin gallate (ECG), (-)-epicatechin (EC) and catechin, with EGCG comprising the greatest proportion of these catechins [4, 5]. Since tea is the second most widely consumed beverage next to water, both tea and EGCG are generally considered to be non-toxic. However, the literature on this topic is not consistent. For example, EGCG (500 mg/kg/d as part of the diet, for 13 weeks) did not produce weight loss in male or female Sprague-Dawley rats [6]. In contrast, green tea extract (GTE) (5% of the diet/d, for 90 d) caused a loss of body weight in male but not female rats [7] and EGCG (81 mg/kg/, 8d, ip) decreased body weight in both rat sexes [8]. Even though the LD50 of GTE in mice has been calculated to be 3.09 g/kg for females and 5 g/kg for males [7], weight loss, hepatotoxicity and mortality have been reported following the administration of much lower doses of EGCG. Specifically, a single dose of EGCG (150 mg/kg, ip) produced mortality in femake mice [9] and repeated administration of EGCG (50 mg/kg/d, 7d, ip) elicited hepatotoxicity, weight loss and mortality in male and female mice [10-12]. However, all three parameters were more severe in females compared to males [10]. While female mice were more susceptible to EGCG-mediated hepatotoxicity, this has not been demonstrated in rats. Specifically, female Wistar rats were administered GTE (2.5 g/kg/d, po, 6 weeks or 2 g/kg/d, po, 12 weeks) and no liver injury was seen in any treatment group [13]. The conflicting evidence in rodents makes extrapolation to humans difficult. However, there have been reports of liver injury in humans following consumption of GTE. For example, In Europe, there have been 14 reported cases of severe hepatitis following ingestion of an ethanolic extract of green tea, which caused the product to no longer be marketed [14]. A further 3 cases of liver injury have been reported following the consumption of either green tea (6 cups/day, 4 months) [15] or a micronized powder of Camellia sinensis) [16]. However, clinical trials with high doses of GTE (2.2 g/m2, tid, 6 months) have only shown minor sideeffects most likely related to caffeine in the extract (insomnia, restlessness, gastrointestinal complaints) [17]. Additionally, EGCG (1.6 g, as one bolus oral dose) was given to 8 volunteers without any clinical or biological adverse reactions [18]. Therefore, it is probably

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safe to conclude that green tea / EGCG can be consumed without unwanted side effects in most individuals. This is also supported by the numerous health benefits associated with green tea / EGCG consumption. These have been consistently demonstrated in numerous epidemiological studies which have examine the role of green tea consumption and breast cancer risk [19-21]. In this chapter, evidence will be given for the potential application of EGCG in the prevention and treatment of breast cancer as well as the molecular mechanisms that underpin these actions of EGCG.

EPIDEMIOLOGICAL EVIDENCE - BREAST CANCER RISK ASSOCIATION WITH GREEN TEA CONSUMPTION AND GENOTYPE Epidemiological studies that have analyzed green tea consumption and the risk of developing breast cancer have yielded conflicting results. Some studies have shown no association with green tea drinking [19-21], while others have demonstrated a chemopreventative effect [22] (Table 1). It is important to note that most of the published epidemiological studies were not specifically designed to examine the relationship between tea consumption and cancer risk and therefore the amount and frequency of tea consumption was not always accurately determined [23]. However, the results from a meta-analysis of 4 studies (three cohort studies from Japan and one case control study from the USA) showed that breast cancer risk was significantly reduced following green tea consumption (OR = 0.77, 95% CI = 0.61-0.97) [24]. Additionally, a decreased risk of recurrence of breast cancer has also been shown to correlate with green tea consumption [25, 26] (Table 1). Therefore, the overall evidence suggests a positive correlation between green tea consumption and the development and recurrence of breast cancer. In order to more fully understand the chemopreventative actions of green tea, studies have genotyped breast cancer patients to determine whether genetic polymorphisms could influence the actions of green tea. One such case-control study in Chinese-, Japanese-, and Filipino-American women demonstrated that green tea intake was associated with a reduction in breast cancer risk, but only in women possessing a low-activity allele of catechol-Omethyltransferase (COMT) [22]. COMT is responsible for the rapid methylation of the catechins in green tea and, therefore, differences in the methylation capacity between individuals may alter the chemopreventative activity of green tea. The findings of Wu et al. (2003) [22] suggest that chemoprevention by green tea in women possessing the low-activity COMT allele may result from decreased metabolism and thus an increased bioavailability of catechins. However, women with a low activity COMT would also be at a higher risk for developing breast cancer due to a decreased production of the pro-apoptotoic and antiangiogenic metabolite of estradiol, 2-methoxy-estradiol [28]. Therefore, further work needs to be conducted to determine if the reduction in risk is related to catechin metabolism or the overall higher risk of the patients. Other enzymes were also shown to be important as green tea consumption was also associated with a reduced risk of breast cancer in women who expressed the high-activity, but not the low-activity, angiotensin-converting enzyme [29]. Changes in the activity of this enzyme support the hypothesis that a possible mechanism of chemoprevention by EGCG and other green tea catechins involves their inhibition of reactive oxygen species via the

Table 1. Green Tea Intake and the Risk of the Development or Recurrence of Breast Cancer Type

Population Profile

Risk Ratio (95% CI)

Recurrence

472 Japanese women with Stage I, II or III breast cancer

Green tea consumption: Stage I and II Stage III Green tea consumption: Stage I ≤3 cups/day 3-5 cups/day ≥6 cups/day Stage II ≤3 cups/day 3-5 cups/day ≥6 cups/day Stage III ≤3 cups/day 3-5 cups/day ≥6 cups/day Green tea consumption: ≤ 1/day 2-4/day ≥ 5/day Green tea consumption: ≤ 1/day 2-4/day ≥ 5/day Green tea consumption: 1-2 cups/day 3-4 cups/day ≥ 5 cups/day Green tea consumption: 0 - 85.7 ml/day ≥ 85.7 ml/day

1,160 Japanese women

Development

34,759 women in Hiroshima & Nagasaki, Japan

23,667 women, members of the Life Span Study Cohort

Combined 2 cohort studies conducted in rural Japan -17,353 women cohort 1 -24,769 women cohort 2 Chinese, Japanese and Filipino women residing in the US

*Statistically significant. Table modified from Stuart et al., (2006) [27].

Outcome 0.56 (0.35 - 0.91)* 1.88 (0.79 - 4.54) 0.43 (0.22 - 0.84)* 0.37 (0.17 - 0.80)* 0.59 (0.23 - 1.52) 0.71 (0.35 - 1.44) 0.80 (0.38 - 1.69) 0.51 (0.18 - 1.46) 1.01 (0.50 - 2.05) 1.06 (0.51 - 2.17) 0.87 (0.33 - 2.27)

Reference

Green tea intake was associated with a reduced risk of recurrence of Stage I and II breast cancer Green tea intake was associated with a reduced risk of recurrence in Stage I breast cancer

[25]

No association

[19]

No association

[20]

No association

[21]

Green tea intake is associated with a reduced risk of the development of breast cancer

[22]

[26]

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angiotensin-converting enzyme. A further mechanism of chemoprevention by green tea catechins involves the alteration of circulating steroid hormone levels. Specifically, in a study of 130 postmenopausal Chinese women, it was shown that women who regularly consume green tea had lower plasma levels of estrone, estradiol and androstenedione compared with non- or irregular green tea-drinkers [30]. In this study, the changes in hormone levels were not dependent upon the genotype of COMT. Therefore, these findings suggest that alteration of steroid hormone levels may contribute to the chemopreventative activity of green tea. However, in order to obtain a more thorough picture of the effect of green tea / EGCG, more investigations must be conducted with both GTE and pure EGCG.

IN VITRO EFFECTS AND MOLECULAR MECHANISMS OF EGCG EGCG is cytotoxic toward breast cancer cells regardless of their ER status. Specifically, after EGCG treatment, cell number was significantly decreased from control in both ERpositive (MCF-7, T47-D and BT474) [31-34] and ER-negative (MDA-MB-231, Hs578t, MBA-MB-468 and BT-20) human breast cancer cells lines, [31-41]. Importantly, EGCG displayed a greater cytotoxic potency in ER-negative breast cancer cell lines compared to ERpositive cell lines [33, 34]. Results from in vitro ER binding and reporter gene assays as well as in vivo functional assays demonstrated that EGCG weakly bound both ERα and ERβ, but could not antagonize estradiol-mediated responses in vivo [11]. These results demonstrate that EGCG is not a strong antagonist of the ER and its cytotoxic action in breast cancer cells occurs independently of the ER. Furthermore, EGCG is cytotoxic toward breast cancer cells that grow in response to stimulation via HER2, an isoform of the epidermal growth factor receptor (EGFR), but are also resistant to trastuzumab immunotherapy. Specifically, EGCG (~87-349 µM) dose-dependently decreased the proliferation of both trastuzumab-resistant BT474 human breast cancer cells and JIMT-1 cells (derived from a patient who displayed clinical resistance to trastuzumab immunotherapy) [42]. Therefore, EGCG is cytotoxic toward a variety of human breast cancer cells and this effect is mediated via ER-independent mechanisms that are critical in the regulation of cell proliferation. The major mechanisms by which EGCG conveys its cytotoxicity toward breast cancer cells are outlined in the following sections.

EGCG-MEDIATED INDUCTION OF APOPTOSIS AND MODULATION OF CELL SIGNALING PATHWAYS There is an abundance of literature obtained from research using a variety of human cancer cell lines that has demonstrated that EGCG induces apoptosis [43-47]. It would then be expected that EGCG would induce apoptosis in most, if not all, breast cancer cell lines. However, the role of EGCG in breast cancer has only more recently become a focus of this potential drug and most of this work has been conducted in ER-negative breast cancer cells. Specifically, EGCG or green tea extract induced apoptosis in ER-negative MDA-MB-468 [38] and MDA-MB-231 cells [33, 40, 41, 48, 49], as well as trastuzumab–resistant BT474 and JINT-1 cells, but only following high concentrations in these cells (>170 µM) [42].

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Mechanisms through which EGCG-induced apoptosis was mediated includes; cell cycle arrest [31, 36, 39], changes in intracellular signaling cascades [50-53], inhibition of proteasomal chymotrypsin-like activity [49], upregulation of p53 and changes in the ratio of Bax/Bcl-2 [38]. For example EGCG has caused ER-negative breast cancer cells to arrest in the G1 phase of the cell cycle. Specifically, treatment of MDA-MB-231 cells with EGCG (87 µM) for 12 h produced a 34% increase in the number of cells in the G1 phase of the cell cycle compared to control [39], while a lower concentration of EGCG (25 µM) only increased the number of G1 phase cells by 4% [40, 41]. Therefore, the ability of EGCG to induce cell cycle arrest is clearly dose-dependent. Further studies have demonstrated that changes in the cell cycle were driven by EGCG-mediated alterations in the protein expression of the cyclins, cyclin dependent kinases (cdks) and their inhibitors (CDKIs). Specifically, studies in MDA-MB-231 cells have shown that EGCG (87 µM) decreased the protein expression of Cyclin D and Cylcin E as well as cdk4 and cdk1 by 50% [39], while the CDKIs, p21 and p27 were increased [36]. A similar result has also been reported in MCF-7 cells where 30 µM of EGCG-induced the expression of p21 and p27 and this correlated with an 1.5-fold increase in the number of cells arrested in the G1 phase of the cell cycle [31]. The induction of apoptosis and alterations in the cell cycle are likely to be the result of EGCG-mediated changes in intracellular pathways. It has been documented that EGCG alters the phosphorylative activity of the EGFR and its downstream targets in breast cancer cells (for an overview of EGFR and its downstream targets see Figure 1). Specifically, the treatment of MDA-MB-231 cells with EGCG inhibited both basal and TGF-α-induced autophosphorylation of the EGFR [36]. It was further established that EGCG inhibited constitutive and TGF-α-induced AKT and STAT3 activity in these cells, but not the expression of phosphorylated ERK. Furthermore, EGCG inhibited the activity of the HER2 and this correlated with a decrease in downstream effects of EGFR activation, such as c-fos promoter activity [37]. Another important protein that governs cell survival is the transfactor NF-κB. NF-κB has overlapping roles in many mitogenic signaling pathways as it is capable of promoting and repressing the expression of proteins involved in cell growth, apoptosis, inflammation, the stress response as well as other important physiological processes [54-56]. Therefore, it is vital for tumor growth, and is a key player in ER-negative breast cancer as it is overexpressed in both ER-negative breast cancer cell lines and in tumors from patients [57]. Therefore, it is an important target in ER-negative breast cancer treatment strategies. Studies in cancer cell lines have demonstrated that EGCG inhibits NF-κB [36, 50-53]. For example, MDA-MB-231 cells were used to demonstrate that EGCG inhibited both the basal and inducible activity of the NF-κB complex [36]. This supported previous work showing that EGCG elicited doseand time-dependent inhibition of both the activation and translocation of NF-κB via the suppression and cytoplasmic degradation of IkBa [52, 53]. Further studies demonstrated that EGCG exhibited a concurrent effect on p53 and NF-κB, which caused a change in the ratio of BAX/Bcl-2 and thus favored apoptosis [50]. This effect would significantly impact the growth of many breast cancer cell lines but would not be relevant to MDA-MB-231 cell growth, as these cells lack a functional form of p53 [58].

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Figure 1. Schematic diagram of intracellular cell signaling cascades that are activated following phosphorylation of the EGFR. Dimerization of the EGFR leads to the activation of various signaling pathways such as PI3K/Akt and Ras/Raf/ERK. These regulate key downstream regulators of cell growth such as NFκB and mTOR.

EGCG modulates other important cell survival pathways in MDA-MB-231 cells. One of these is the hepatocyte growth factor (HGF)/Met signaling pathway which is involved in proliferation, survival, motility and invasion [59]. Met is a tyrosine receptor kinase which autophosphorylates upon activation by HGF and its downstream targets include the PI3K/AKT and MAPK pathways [59-62]. Studies with EGCG have shown that concentrations as low as 0.6 µM inhibited HGF-induced Met phosphorylation, and subsequent AKT and ERK activation and this correlated with a 67% decrease in HGFinduced invasion in MDA-MB-231 cells [59]. Importantly, the authors also demonstrated that EGCG (5 µM) inhibited the basal invasion capabilities of MDA-MB-231 cells by 50%, which demonstrates that EGCG decreases both basal cancer cell invasion and that induced by HGF. Apoptosis can also be modulated in cancer cells by the chymotrypsin-like proteasome [63]. Inhibition of this specific proteasome rapidly and selectively induces apoptosis in cancer cells but not normal cells [64, 65]. Importantly, this effect has been shown in human cancer

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cells that are resistant to numerous other anticancer agents. In MDA-MB-231 breast cancer cells EGCG inhibited chymotrypsin-like activity by 24% and this correlated with an increase in ubiquitinated IkBa, p27 and Bax [49]. All three of these proteins work to decrease cell proliferation and increase apoptosis, as IkB prevents the activation of the transfactor NF-kB [66]. Overall these studies show that, EGCG induces apoptosis in breast cancer cells by modulating intracellular signaling pathways that control cell cycle progression, cell motility/invasion and proteasomal degradation. Since these effects are not dependent on the ER, EGCG is able to decrease cell growth and induce a strong apoptotic response in ERnegative breast cancer cells. Thus EGCG has the potential to be used as a drug that will positively impact on the lives of women with ER-negative breast cancer.

EGCG AS AN INHIBITOR OF ANGIOGENESIS Numerous studies have demonstrated that EGCG inhibits a variety of processes involved in angiogenesis. This action elicited by EGCG is extremely relevant to tumor suppression since tumor growth is an angiogenic-dependent process, with new blood vessels supplying nutrients, oxygen and growth factors that enhance tumor proliferation and expansion [67]. In breast cancer, neovascularization has been shown to contribute to the long-term aggressiveness of the tumor and correlates to increased metastasis [68, 69]. EGCG has been documented to inhibit endothelial cell growth in a dose-dependent manner in vitro, which correlated to a significant decrease in new blood vessel formation in an in vivo angiogenesis model [70]. Furthermore, in vitro studies have shown that EGCG inhibits the production of vascular endothelial growth factor (VEGF) and matrix metalloproteinases [71, 72]. All isoforms of VEGF have been established as potent angiogenic agents [73] and the levels of this growth factor are closely correlated to the induction and maintenance of the neovasculature in breast cancer [74, 75]. VEGF initiates angiogenesis via the binding to its receptors (VEGFR-1 and VEGFR-2) [76]. VEGFR-2 activation is responsible for most of the mitogenic and chemotactic effects of VEGF [77] and inhibition of angiogenesis occurs through the inhibition of both VEGF and VEGFR-2 [78, 79]. Importantly, EGCG has been shown to inhibit VEGFR-2 phosphorylation in vitro but this study was not conducted in human breast cancer cells [80] and thus, further work needs to be conducted to confirm this in breast cancer models. EGCG also decreases cancer cell invasion and metastasis [81, 82] by inhibiting cell adhesion function via an inhibition of E-cadherin. Macrophages and other inflammatory cells also promote angiogenesis and EGCG has decreased inflammation by suppressing the overexpression of both cyclooxgenase [83] and nitric oxide synthatase [84]. Another potential mechanism for EGCG is via binding to the metastatsis-associated 67-kDa laminin receptor. While this study was conducted in lung cancer cells, the results showed that growth inhibition occurred only in cells transfected with the 67-kDa laminin receptor and that EGCG bound to this receptor with a Kd of 39.9 nM [85]. Interestingly, none of the other green tea catechins were able to bind to this receptor and thus the effect was specific for EGCG. However, epicatechin gallate was not tested and thus the gallate moiety may prove to be critical for activity. Additionally, these experiments were not performed in breast cancer cells and thus it

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has yet to be determined if this response is relevant to the antiangiogenic effect of EGCG in breast cancer, as there are many additional facets to this process. In breast cancer, another factor involved in angiogenesis, invasiveness and metastasis is the oncogene Wnt. Constitutively active Wnt has been linked to increased proliferation and invasiveness in breast cancer through the stabilization of β-catenin. In breast cancer, high βcatenin levels correlates with poor prognosis [86-88] Additionally, the percentage of breast cancers with high β-catenin expression is estimated to be approximately 50% [89]. Therefore, modulation of Wnt could significantly impact on a large number of breast cancer patients. Importantly, in MDA-MB-231 cells EGCG (25-100 µM) blocked Wnt signaling in a dosedependent manner, via the induction of HBP1 [90]. Induction of HBP1 occurred via an increase in the stabilization of mRNA and not through transcriptional induction. HBP1 plays an important role in invasive breast cancer, as a loss HBP1 is associated with invasive breast cancer and 30% of patients with invasive disease express HBP1 mutants [89]. Importantly, downstream effects of Wnt signaling were also modulated by EGCG, as c-mcy (a relevant Wnt target gene) was also decreased [90]. These results correlated with a decrease in the migration of MDA-MB-231 cells. Specifically, EGCG (50 and 100 µM) decreased the migration of cells toward fibronectin treated medium by 50 and 75%, respectively and also decreased the invasion of cells through Matrigel [90]. This demonstrates that EGCG has the ability to modulate breast cancer invasiveness by increasing the stability of HBP1, which decrease Wnt signaling, c-mcy expression and ultimately cell migration. In breast cancer, invasion and metastasis is more extensive in breast cancer cells that lack the alpha isoform of the ER (ERα). Breast cancer cells that are positive for ERα have a more epithelial architecture while; ERα-negative cells have a more invasive phenotype. Importantly, this effect is not static and the transition from an epithelial to a more mesenchymal phenotype (EMT) has been shown to occur. During EMT, cancer cells lose the expression of E-cadherin and γ-catenin, proteins that promote cell to cell contact and gain markers of a mesenchymal, and thus more migratory and invasive phenotype, such as Snail, vimentin, N-cadherin and fibronetin [91, 92]. Recently it has been shown that EMT can be inhibited and the modulation of ERα signaling by FOXO3a (a forkhead family transcription factor) is a key mechanistic driver of this effect. Evidence for this includes the fact that ERα synthesis can be controled by FOXO3a, [93, 94] and FOXO3a is also greatly reduced in ERαnegative breast cancers that are driven by HER2 [72, 93]. Importantly, EGCG repressed the invasive phenotype of breast cancer cell growth that was driven by HER2 [95]. Specifically, treatment of NF639 breast cancer cells with EGCG (87 µM) increased the protein expression of E-cadherin, γ-catenin, FOXO3a and ERα and decreased the protein expression of Snail [95]. The increase in FOXO3a by EGCG promotes ERα signaling and a less malignant phenotype in breast cancer cells. The phenotype of these breast cancer cells changes to a more epithelial type partly due to the inhibition of Snail, which represses the expression of Ecadherin [92]. Further evidence for the role of FOXO3a in this phenotypic change is the fact that FOXO3a inhibited cell migration, invasiveness in Matrigel as well as TGF-β1 stimulated invasion [95]. Importantly, ERα signaling was required for this change toward a more epithelial phenotype. Therefore, EGCG plays an important role in decreasing the invasiveness of HER2 driven breast cancer cells by upregulating ERα through the stimulation of FOXO3a gene expression. These studies provide further evidence for the clinical usefulness of EGCG,

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as inactive FOXO3a expression was shown to correlate with the poor survival of breast cancer patients [96].

EGCG AS A NEW CLASS OF CHEMICAL CHAPERONES IN VITRO Since EGCG has a wide range of effects in a variety of cancer models, new modes of action have been proposed. One of these is a potential role for EGCG as a new class of chemical chaperones [97]. This theory was put forward due to the fact that both EGCG and protein chaperones interact transiently with numerous proteins. For example, both EGCG and chemical chaperones regulate endoplasmic reticulum stress, as the GADD153 gene is induced by both stress [98] and EGCG [99, 100]. More evidence for this theory is the structural similarity between the chemical chaperone trehalose and EGCG, as both have eight hydroxyl groups. To test this theory, in silico analysis was used to assess the mobility and flexibility of the galloyl group and the results showed that EGCG existed in variable conformations and thus could interact with numerous molecular targets [97]. The presence of the galloyl group also provided more possible conformations compared to the other green tea catechins. These results support previous surface plasmon resonance studies that demonstrated that the binding of EGCG to DNA and RNA was reversed [101, 102] in a similar manner to that of chemical chaperones. While this theory will be strengthened by further tertiary structural analysis of the EGCG-protein complex, the results to date provide evidence to suggest that EGCG acts as a new class of chemical chaperones.

IN VIVO EFFECTS AND MOLECULAR MECHANISMS OF EGCG The majority of studies investigating the effects of green tea constituents in in vivo breast cancer models have focused on green tea mixtures rather than purified individual catechins. The earliest studies focused on chemical-induced mammary carcinogenesis in rats and demonstrated a protective effect of green tea compounds on tumor burden and survival [35, 103-107]. However, it is still unclear whether this protection is greater at the pre- or postinitiation stage. Various studies using either GTE or purified EGCG have also been conducted using breast cancer cell xenografts in mice [39, 108, 109]. For example, MDA-MB-231 breast cancer cell xenografts were used to illustrate that tumor growth, tumor weight and endothelial vessel density decreased following GTE (1.25 – 2.5 g/l) consumption compared to control [108]. Delayed tumor growth onset, rate of tumor growth, tumor volume and metastasis was also shown following the administration of a green tea polyphenol mixture in the drinking water of BALB/c mice inoculated with 4T1 mouse mammary carcinoma cells [110]. These effects were associated with an increase in the Bax/Bcl2 ratio and caspase-3 activation, demonstrating that the induction of apoptosis is a major mechanism for the tumor suppression. There have also been studies that focused on the effects of purified EGCG. Liao et al. (1995) [109] was the first to demonstrate that EGCG (1mg/mouse/day, i.p., 14 days) reduced tumor size in female athymic nude mice inoculated with MCF-7 cells. More recently, Thangapazham et al. (2007) [39] conducted a study using female athymic nude mice

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inoculated with MDA-MB-231 human breast cancer cells. Mice received 3 mg of green tea polyphenols (GTP) in the drinking water or EGCG (1 mg) by oral gavage. The treatments began on the day of cell inoculation and continued for 10 weeks. This treatment protocol more closely represents chemoprevention because the drug treatment began before palpable tumors had formed. Additionally at the conclusion of the study the control tumors were onethird the size (~30 mm3) of tumors from typical xenograft studies that begin when the tumor volume reaches ~100 mm3. In this model, both EGCG and the green tea mixture suppressed tumor growth (45 and 61%, respectively) and decreased incidence (10 and 20%, respectively) and this suggests that EGCG is predominantly responsible for the chemopreventative actions of green tea. Further examination of tumor sections demonstrated that GTP increased the number of apoptotic cells by 3.5-fold and EGCG increased apoptotic cells by 2.6-fold [39]. PCNA staining also revealed that proliferation of tumor cells was decreased by both treatments. These findings have been supported by other more recent studies that followed a more standard treatment protocol, as treatment with EGCG (25-50 mg/kg/d, 5-10 weeks) began once palpable tumors (derived from MDA-MB-231 cell xenografts) had formed [34, 49, 111]. Results in all of these studies showed a modest but statistically significant suppression of ERnegative breast cancer xenografts following EGCG administration. Mechanisms for this suppression were determined and the results from both Western blotting and immunohistochemistry demonstrated that EGCG (25 mg/kg, ip) caused a significant decrease in Akt, bRaf, VEGF and VEGFR-1 [34, 111]. While no other markers for changes in apoptosis or angiogenesis were determined in these studies, Akt is critical for breast cancer cell proliferation [112] and numerous other studies have shown that EGCG inhibits angiogenesis [71, 72, 81, 82]. Apoptosis was also linked with tumor suppression following 50 mg/kg of EGCG for 31 days, as Bax, cleaved PARP and caspase-3 activity were increased in tumors from treated mice compared to tumors from control mice [49]. These authors also demonstrated that chymotrypsin-like activity was decreased and this correlated with a modest increase in IkBa protein expression. Importantly, this study also showed that apoptosis in cancer cells could be induced by in vivo inhibition of the chymotrypsin-like proteasome and these results were further supported by in vitro studies in MDA-MB-231 cells. Overall, recent studies in ER-breast cancer models have shown that EGCG elicits modest tumor suppression through an induction of apoptosis and modulation of cell signaling proteins.

IMPROVING THE EFFICACY AND BIOAVAILABILITY OF EGCG Many of the in vitro effects elicited by EGCG are produced following the use of high concentrations (~80-150 µM). Due to the instability of EGCG and its extensive first pass metabolism [113], these concentrations are not achievable in vivo where plasma concentrations are aproximately 1-10-fold lower [114, 115]. Therefore, clinical trials with EGCG and GTP have relied on extremely high doses. While patients in these clinical studies were relatively free from side effects, the sheer volume of the medication required in these trials was not well tolerated [17, 115] and the maximum tolerated dose of oral, once-daily GTE was determined to be 3 g/m2 per day [116]. Therefore, stability and bioavailability problems, which result in the need for very high doses, are significant hurdles that EGCG

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must overcome in order to make significant clinical progress. One way to circumvent this problem would be to provide EGCG as a pro-drug. The first attempts at this involved the introduction of peracetate groups, in order to protect the reactive hydroxyl groups of EGCG. In cell free conditions this peracetate-EGCG was efficiently converted back to the parent compound [117]. Further studies with this compound in MDA-MB-231 breast cancer cells demonstrated that it was converted to EGCG, which accumulated in 2.4-fold greater amounts compared to standard EGCG following treatment at 50 µM [49]. In all in vitro assays examined, peracetate-EGCG was more potent than standard EGCG (namely, chymotrysinlike activity, PARP cleavage, caspase-3 activity, and protein expression of Bax, p27 and IkBa) [49]. Importantly, these in vitro results correlated with tumor suppression in vivo. Specifically, peracetate-EGCG (50 mg/kg/d, 31d, sc) decreased tumor growth by 54% compared to control, while EGCG decreased tumor growth by 23% [49]. Mechanisms which were elicited in vitro were also shown to be relevant to tumor suppression as peractetateEGCG inhibited tumor proteasome activity in vivo, as IkBa, p27, and Bax proteins were all accumulated in tumors from peracetate treated mice [49]. This study showed that there are numerous advantages to administering EGCG in peracetate form. Specifically, peracetateEGCG was more stable in neutral and slightly alkaline pH than EGCG and more readily absorbed into the tumor cells where it was subsequently hydrolyzed back to EGCG. While these results are promising, peracetate-EGCG was parentally administered at a high dose, namely 50 mg/kg. Studies using a more realistic route of administration have also been performed. Specifically, orally administered O-acyl derivatives of EGCG have also shown anti-tumor activity in a skin cancer model. The results with these O-acyl derivatives are relevant because they have improved bioavailability, stability and also convert back to EGCG in vivo [118]. An important component of this work was that the O-acyl derivatives were synthesized using green tea leaves as the starting material. Four O-acyl derivatives were synthesized with increasing acyl side chains and one derivative contained a branched side chain. These derivatives were administered orally at 50-53 mg/kg for a total of 20 weeks. All of the derivatives reduced incidence, number of skin tumors and percent survival of the mice [119]. Furthermore, there was a significant decrease in the anti-tumor activity with an increase in the size and branching of the acyl side chain. One downfall of this study was that EGCG was not examined alongside the derivatives to demonstrate the increased potency of the derivatives. However, the study provides important evidence for anti-tumor activity of orally administered derivatives of EGCG. While these results are even more promising due to the fact that the derivative was given orally, the compounds need to be examined in breast cancer models in order to demonstrate efficacy toward this type of cancer. Other attempts to improve the effectiveness of EGCG have involved the use of combination therapy. These studies have used both a conventional breast cancer drug (tamoxifen) and a natural polyphenolic compound (curcumin) in combination with EGCG. Specifically, The combination of EGCG and curcumin was synergistically cytotoxic toward ER-negative, but not ER-positive breast cancer cells [34]. These results correlated with a 263±16% increase in the proportion of cells in G2/M phase and a 40±4% decrease in G0/G1 phase cells compared to control following EGCG (20 µM) + curcumin (3 µM) [34]. These in vitro results translated to in vivo efficacy as EGCG (25 mg/kg, ip) + curcumin (200 mg/kg, po) significantly decreased tumor volume compared to all other treatment groups and 49% compared to control in an MDA-MB-231 xenograft model of ER-negative tumorigenesis

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[34]. A decrease in angiogenesis was proposed as the major mechanism for this effect as the protein expression of VEGFR-1 was decreased 78% following treatment with EGCG + curcumin. Other combination studies with tamoxifen showed an even greater tumor suppression, as EGCG (25 mg/kg,ip) + tamoxifen (75 µg/kg, po) decreased tumor volume and tumor weight 71 and 78%, respectively compared to control [111]. The mechanism for this effect is likely to be initiated via modulation of EGFR activity, as protein levels of the EGFR and its active phosphorylated form were both significantly reduced (78% compared to control) by EGCG + tamoxifen treatment. The similar decrease in both the unphosphorylated and phosphorylated forms indicates that the signaling capacity of the pathway as a whole was reduced by approximately 78% by the combination treatment [111]. Combination treatment also caused a similar reduction in the protein expression of mammalian target of rapamycin (mTOR). The inhibition of mTOR is of particular significance, as this is a key regulator of protein synthesis within the cell (Figure 1). Furthermore, studies in other cancer models have shown that a dual suppression of EGFR and mTOR resulted in significant tumor suppression [120]. Importantly, the combination of EGCG and tamoxifen also decreased angiogenesis by decreasing the protein expression of both VEGF and VEGFR-1 in the tumor, as shown by both Western blotting and immunohistochemistry [111]. A final component to the mechanism involved a decrease in the protein expression of cytochrome P5401B1 (CYP1B1), a protein that plays a variety of roles in both the treatment and development of breast cancer. Specifically, inhibition of CYP1B1 is critical to breast tumor formation, as CYP1B1 null mice are protected against DMBA-induced mammary tumors compared to wild-type mice [121]. Additionally, CYP1B1 can inactivate the cancer dug flutamide [122] and is also induced in breast cancer cells following treatment with docetaxel [123]. Therefore, it plays an important role in the development of resistance to chemotherapy. Since there was a 78% decrease in the tumor protein expression of CYP1B1 following treatment with EGCG + tamoxifen, this provides another critical component to the mechanism of action of this drug combination. An important aspect of this combination therapy is the fact that the doses of both drugs were much lower than those typically used in similar studies. For example, combination therapy with a PDK-1/Akt inhibitor has been used to sensitize MDA-MB-231 cells to the effects of tamoxifen. In this study tamoxifen (60 mg/kg) was unable to suppress the growth of MDA-MB-231 xenografts, but the addition of OUS-03012 (100 mg/kg) showed significant tumor suppression of 50% compared to control [124]. This result further demonstrates that tamoxifen can be used in combination therapies for the treatment of ER-negative breast cancer. However, it was much more effective when combined with EGCG, as this combination produced a greater level of tumor suppression (71%) at a much lower dose of tamoxfien (75 µg/kg) [111]. Overall these combination studies have shown tumor suppression following the lowest dose of EGCG used to date and thus provide another role for the use of EGCG in ER-negative breast cancer. The only research using a drug combination that administered green tea orally was conducted with GTE and tamoxifen. In this study, GTE (2.5 g/l) was administered in the drinking water and tamoxifen (20 mg) was inoculated subcutaneously as a slow-release pellet. Treatment continued for 52 days after the establishment of MCF-7 cell xenografts in mice. The results showed that combination treatment elicited tumor suppression that was greater than each individual treatment and was also 81% smaller than tumors from control mice [48].

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These results correlated with a significant decrease (46%) in blood vessel density in the tumors from GTE + tamoxifen treated mice. Additionally, TUNEL analysis revealed a 2.5fold increase in the number of apoptotic tumor cells following combination treatment, compared to control [48]. Importantly, the author also reported a dose-dependent increase in the amount of three major green tea catechins (EC, ECG, EGCG) in the mammary fat pad of mice that drank GTE (0.625 – 2.5 g/l) for 4 days. Interestingly, EGCG (2.5 g/l) accumulated to the highest level of all the green tea catechins (45 ng/g) and this demonstrates that green tea catechins administered orally can reach the mammary tissue. Demonstrating that the drug reaches the target tissue is an important component of breast cancer drug design. However another important aspect will be providing the drug at a dose that is low enough to ensure that patient compliance remains high. This balance between high oral activity and a low oral dose is a significant challenge for future research investigating EGCG as a treatment for breast cancer.

CONCLUSION EGCG induces apoptosis in ER-positive and ER-negative breast cancer cells in vitro and the effect is not influenced by the hormone receptor status of the cell line. Many mechanisms contribute to this effect, which cumulate in the induction of apoptosis and the inhibition of angiogenesis. Importantly, many of the in vitro effects elicited by EGCG have also been documented in ER-negative xenograft models following treatment with either EGCG or GTE. This demonstrates that in vitro studies are useful for dictating / predicting in vivo results with this multifaceted phytochemical. The latest research with EGCG indicates that there are new roles for this compound in treatment-resistant breast cancer, including those cancers resistant to trastuzumab (Herceptin). This new in vitro role for EGCG suggests a promising future for this drug. Especially since in vivo studies have demonstrated that the efficacy of EGCG can be increased by synthesizing pro-drug analogs of EGCG as well as through the use of combination therapy with EGCG. These latest studies provide a basis for the development of highly active, orally available analogs of EGCG that could, one day, significantly impact on the lives of women with treatment-resistant breast cancer

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[111] Scandlyn, MJ; Stuart, EC; Somers-Edgar, TJ; Menzies, AR; Rosengren, RJ. A new role for tamoxifen in estrogen receptor negative breast cancer when it is combined with epigallocatechin gallate. British Journal of Cancer, 2008 99,1056-1063. [112] Ju, X; Katiyar, S; Wang, W; Liu, M; Jiao, X; Li, S; Zhou, J; Turner, J; Lisanti, MP; Russell, RG; Mueller, SC; Ojeifo, J; Chen, WS; Hay, N; Pestell, RG. Akt1 governs breast cancer progression in vivo. Proceedings of the National Academy of Sciences of the United States of America, 2007 104,7438-7443. [113] Lu, H; Meng, X; Yang, CS. Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate. Drug Metabolism and Disposition, 2003 31,572-579. [114] Henning, SM; Niu, Y; Lee, NH; Thames, GD; Minutti, RR; Wang, H; Go, VL; Heber, D. Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement. American Journal of Clinical Nutrition, 2004 80,1558-1564. [115] Chow, HH; Hakim, IA; Vining, DR; Crowell, JA; Ranger-Moore, J; Chew, WM; Celaya, CA; Rodney, SR; Hara, Y; Alberts, DS. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clinical Cancer Research, 2005 1511,4627-4633. [116] Laurie, SA; Miller, VA; Grant, SC; Kris, MG; Ng, KK. Phase I study of green tea extract in patients with advanced lung cancer. Cancer Chemotherapeutics and Pharmacology, 2005 55,33-38. [117] Lam, WH; Kazi, A; Kuhn, DJ; Chow, LMC; Chan, ASC; Dou, QP; Chan, TH. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (−)-epigallocatechin gallate [(−)-EGCG]. Bioorganic and Medicinal Chemistry, 2004 12,5587-5593. [118] Landis-Piwowar, KR; Kuhn, DJ; Wan, SB; Chen, D; Chan, TK; Dou, QP. Evaluation of proteasome-inhibitor and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs. Internation Journal of Molecular Medicine, 2005 15,735-742. [119] Vyas, S; Sharma, M; Sharma, PD; Singh, TV. Design, semisynthesis, and evaluation of O-acyl derivatives of (-)-epigallocatechin-3-gallate as antitumor agents. Journal of Agricultural Food Chemistry, 2007 55,6319-6324. [120] Buck, E; Eyzaguirre, A; Brown. E; Petti, F; McCormack, S; Haley, JD; Iwata, KK; Gibson, NW; Griffin, G. Rapamycin synergizes with the epderimal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreastic, colon, and breast tumors. Molecular Cancer Therapeutics, 2006 5,2676-2684. [121] Buters, JTM; Sakai, S; Richter, T; Pineau, T; Alexander, DL; Savas, U; Doehmer, J; Ward, JM; Jefcoate, CR; Gonzalez, FJ. Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas. Proceedings of the Nattional Academy of Sciences of the United States of America, 1999 96,1977-1982. [122] Rochat, BM; Murray, JM; Figg, GI; McLeod, WD. Human CYP1B1 and anticancer agent metabolism: mechanism for tumor-specific drug inactivation? Journal of Pharmacology and Experimental Therapeutics, 2001 296,537-541. [123] Martinez, VG; O'Connor, R; Clynes, M. CYP1B1 expression is induced by docetaxel: effect on cell viability and drug resistance. British Journal of Cancer, 2008 98,564-570.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson, pp.

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 16

GREEN TEA CATECHINS IN COLORECTAL CANCER Seung Joon Baek and Mugdha Sukhthankar Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA

ABSTRACT Colorectal cancer is a global problem that accounts for over 50,000 cancer-related deaths each year in the United States. Americans have about a one in 20 lifetime risk of developing colorectal cancer. It affects primarily those over 65, but risk starts increasing at age 40. Colorectal cancer develops following disruptions in key cancer-causing genes (oncogenes) like K-ras and β-catenin and tumor suppressor genes like gate keeper APC and p53, and early detection greatly increases the chances of survival. Most cancers are related to a combination of hereditary and environmental factors, and such factors can either contribute to the initiation of cancer or the prevention of tumor development. There is persuasive epidemiological and experimental evidence that a phytochemical-enriched diet may be involved in the prevention of colon cancer. Therefore, the use of dietary compounds for prevention and therapy of colorectal cancer would be of major importance with potentially fewer side effects than therapeutic drugs. Green tea has received much attention as a suitable dietary agent because of its anti-tumorigenic activity. The most active constituents of green tea are catechins, including epigallocatechin 3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC). Many laboratories, including ours, have reported preventive effects with green tea components in cancers of the gastrointestinal tract, lung, skin, prostate, and breast. A mechanistic study indicated that green tea decreased the total levels of early carcinogenesis biomarkers and increased tumor suppressor proteins; in addition, reports related to new molecular targets affected by green tea in chemoprevention study have been increased. Since the preponderance of the data strongly indicates significant antitumorigenic benefits from green tea polyphenols, this chapter will summarize the current knowledge of molecular targets of green tea research in human colorectal cancer prevention.

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1. INTRODUCTION Cancer, or malignant neoplasia, is an array of diseases wherein a group of cells display uncontrolled growth and metastasis. Cancer affects all age groups, even fetuses, but risk tends to increase with age. According to the American Cancer Society, cancer killed 7.6 million people in the world during 2007 [1; 2]. Cancer is caused by both external and internal factors that may work together or in a pattern to initiate or promote carcinogenesis. The lifetime risk (the risk of developing a disease during one’s lifetime or dying of the disease) for developing cancer in men is 1 in 2, and in women, it is 1 in 3. Cancer is second only to heart disease as the leading cause of death in the United States. Specifically, colorectal cancer is one of the most prevalent causes of cancer-related mortality in the western world [3]. The process of cancer development may be divided into at least three stages: initiation, promotion and progression [4]. Most advanced cancers are incurable, hence it is important to prolong or block the process of carcinogenesis through chemoprevention, which turns out to be a feasible strategy for cancer control and management [5]. Thus, further development of therapeutic and preventive means of controlling this disease are clearly needed, particularly as they pertain to gastrointestinal cancer [6; 7]. Epidemiological studies have suggested that nutrition plays an important role in carcinogenesis, and dietary factors have been estimated to account for up to 80% of cancers of the gastrointestinal tract. Approximately 30% of cancer morbidity and mortality might be prevented with proper adjustment of diets [8; 9; 10; 11; 12]. The basic theory of chemoprevention is to reduce the occurrence of cancer either by slowing, blocking, or reversing the development of the disease by the administration of natural or synthetic compounds [13]. In addition, the molecular mechanisms responsible for potential human health benefits derived from dietary components must be studied in vitro and validated in preclinical studies in animal models before strong support can be given for more extensive clinical trials.

2. COLORECTAL CANCER Colorectal cancer is the third most common cancer in the United States [14]. It develops slowly over many years and usually begins as a polyp: a benign growth of tissue that starts in the lining and grows towards the center of the colon or rectum. Early detection and removal of polyps may prevent formation of adenocarcinomas, which account for over 95% of colorectal cancers. Colorectal cancer is caused due to mutations in both tumor suppressors and oncogenes [15]. Broadly, there are two types of colorectal cancers: hereditary and sporadic.

2.1. Hereditary Cancers Familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) are the two best-known hereditary colorectal cancers.

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Familial Adenomatous Polyposis Familial adenomatous polyposis is very rare. It causes less than 1% of all colorectal cancer cases. FAP is caused by mutations in the adenomatous polyposis coli (APC) gene, a tumor suppressor. One of the many roles of APC is regulating cell replication, and hence it earned the name “GATE KEEPER”. Around five hundred APC mutations have been identified so far [16]. The polyps themselves are benign but because of their usual high numbers, some of them might undergo a mutation in the normal copy of the APC gene and trigger the development of cancer. Hereditary Non-polyposis Colorectal Cancer Hereditary non-polyposis colorectal cancer (HNPCC, also known as Lynch syndrome) is caused by a mutation in one of the DNA mismatch repair (MMR) genes. The hMSH2 and hMLH1 are most commonly mutated MMR genes in HNPCC, and the other genes involved are hMSH6, PMS1 and PMS2 [17]. The aberrant operation of DNA mismatch repair systems lead to Microsatellite instability (MSI) and cause naturally occurring, highly repeated short DNA sequences, called microsatellites, to get shorter or longer than expected.

2.2. Sporadic Colorectal Cancer In most people without an inherited mutation predisposing them to colorectal cancer or a family history of it, a series of mutations is needed for colorectal cancer to develop (Fig. 1). It often takes many decades for these mutations to accumulate –hence the majority of colorectal cancer cases occur in the elderly. Mutations in APC are seen in 70 to 80% of sporadic tumors and often occur early in the development of colorectal cancer. During early stages of colorectal tumorigenesis, three ras genes are mutated (K-ras, N-ras, and H-ras) [18]. The p53

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gene is another tumor suppressor gene that is known to be mutated. The p53-encoding gene mutations in colorectal cancer occur in specific conserved regions of the gene and might be present in over 50% of the colorectal cancers [19]. In addition, the DCC (deleted in colorectal cancer) gene is mutated in 70% of colorectal cancers, and MMR genes are inactivated in around 15% of sporadic cases.

3. DIFFERENT SIGNALING PATHWAYS INVOLVED IN COLORECTAL CARCINOGENESIS Many signaling pathways play a pivotal role in colorectal tumorigenesis. Specifically, the cyclooxygenases (COX), p53, Wnt, and TGF-β pathways are important in the development of colorectal tumorigenesis (Fig. 2). COXs, key enzymes responsible for eicosanoid production, can profoundly influence cancer development, progression and therapeutic response. COX-2 is highly inducible by various cytokines, growth factors, and tumor promoters [20]. Elevated COX-2 levels are associated with inflammation, neoplastic transformation and metastasis [21; 22], and increased expression of mRNA and protein is found in the great majority of colorectal cancers [23; 24]. Mutations also commonly occur at the p53 tumor suppressor protein locus in many forms of cancer, including colorectal cancer. Thus, it is logical when studying colorectal cancer to consider the p53 mutation and the status of COX-2 expression.

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The over-activation of the Wingless/Wnt signaling pathway also leads to the expression of genes that favor cell growth and thus, contributes to many different cancers including colorectal cancer. APC and β-catenin are two major genes involved in this pathway [25]. In normal cells, APC interacts with β-catenin and forms a macromolecular complex with axin and glycogen synthase kinase-3 β (GSK-3β), in which β-catenin is subsequently directed towards ubiquitin-proteasomal degradation by phophorylation of GSK-3 β [26; 27; 28]. If this complex is not formed, then β-catenin increases in cytoplasm and translocates to the nucleus where it acts on transcription of many downstream genes. The β-catenin expression has been shown in 90% of the sporadic colorectal cancers and in 65% of HNPCC’s [29; 30]. Transforming growth factor-β (TGF- β) is part of a superfamily of proteins known as the transforming growth factor β superfamily. TGF- β initiates signaling by formation of heteromeric complexes of type I and type II serine/threonine kinase receptors [31; 32; 33]. The role of TGF-β in tumorigenesis is quite complex, but it appears that TGF-β functions both as a tumor suppressor in early tumor onset and as an oncogene during late tumor progression [34; 35]. TGF- β receptor II somatic mutations are found in patients with HNPCCs and in cancers that show microsatellite instability [34; 35]. De-regulation of one of the above pathways lies at the heart of the development and progression of human colorectal malignancies.

4. HEALTH BENEFITS OF GREEN TEA Green tea has been known to have many health benefits. It has been shown to decrease low-density lipoprotein and increase high-density lipoprotein [36; 37]. Subsequently, green tea has been shown to inhibit hypertension [38] and also to prevent cardiac hypertrophy [39]. Green tea has been used as a diet drink in women as it raises metabolic rates, fastens fat oxidation and ameliorates insulin sensitivity and glucose tolerance [40]. Green tea can even lead to the inhibition of HIV virus binding and hence be used as a supportive therapy for HIV patients [41] because oxalates present in tea help with HIV and general infections by cleaning up free iron, which leaves one less task for the immune system.

5. GREEN TEA AND CANCER Abundant experimental and epidemiological evidence accumulated mainly in the past decade from several centers worldwide provides a convincing argument that polyphenolic antioxidants present in green tea can reduce cancer risk in a variety of animal tumor bioassay systems. Tea consumption also assures protection against cancers induced by chemical carcinogens that involve the lung, forestomach, esophagus, duodenum, pancreas, liver, breast, colon, and skin in mice, rats, and hamsters [42]. In addition, many epidemiological studies suggest that green tea consumption shows beneficial effects on many cancers [43].

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6. GREEN TEA CATECHINS Four major polyphenol catechins in green tea include epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and, epicatechin (EC) (Fig. 3). In experimental models, catechins show a wide range of protective effects, including cardioprotective, chemoprotective, and antimicrobial properties. Catechin and epicatechin are epimers, with (-)-epicatechin and (+)-catechin being the most common optical isomers found in nature. Catechin derives its name from the plant catechu from where it was first isolated. Epigallocatechin and gallocatechin contain an additional phenolic hydroxyl group, which makes them stronger anti-oxidant agents compared to epicatechin and catechin, respectively, similar to the difference in pyrogallol compared to pyrocatechol. Catechin gallates are gallic acid esters of the catechins, such as EGCG (epigallocatechin gallate), which is the most abundant catechin in tea. EGCG is about 25-100 times more potent than Vitamin C in its antioxidant property [44].

7. GREEN TEA CATECHINS IN CANCER Green tea catechins have recently gained significant acceptance as cancer preventive agents. We and numerous other scientists have reported the cancer preventive activities of EGCG and green tea extract: inhibition of angiogenesis and prevention of cancer metastasis, induction of apoptosis, inhibition of inflammation, translocation of different genes, cell cycle regulation, growth prevention of tumors in vivo, inhibiting activation of IGF-1 and alteration of the expression of various oncogenes and tumor suppressor genes [45; 46; 47]. The role of green tea catechins in colorectal cancers is more significant as the catechins are absorbed into the gut [48]; however, their role in other cancers is also important. Recent studies point out that dietary cancer chemopreventive agents modulate multiple signaling pathways that interrupt the carcinogenic process and are also capable of extending one or more stages of carcinogenic process [13; 49].

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7.1. Effect on Angiogenesis and Cancer Metastasis Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels in growth and development, as well as in wound healing. It is also an important step in the progression and conversion of tumors from a dormant state to a malignant state. Tumors induce blood vessel growth by secreting various growth factors which can induce capillary growth into the tumor and supply required nutrients allowing for tumor expansion [50]. There could be either mechanical or chemical stimulation of angiogenesis. Chemical stimulation, as the name suggests, is performed by various chemical substances or angiogenic proteins, including several growth factors like VEGF, bFGF, and TGF-β [51]. In addition, the role of various proteases such as urokinases, MMP, and tissue inhibitor of matrix metalloproteinases (TIMP) has also been extensively studied in tumor angiogenesis [52]. These proteins have been linked to be a molecular target of green tea catechins.

VEGF The vascular endothelial growth factor (VEGF) belongs to a sub-family of growth factors; the platelet-derived growth factor family, which are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF has been known to be a key mediator of the abnormal angiogenesis in many hematologic malignancies and has been shown to be secreted by diverse types of malignant cells [53; 54; 55]. It is a small molecule (~45 kDa) with diverse biological activities that include the regulation of embryonic vascular development, extracellular matrix remodeling, generation of inflammatory cytokines, and enhancement of vascular permeability, through a VEGF receptor (VEGF-R) [56; 57]. EGCG inhibits transcriptional regulation of VEGF by decreasing the transcript levels of VEGF [58]. This effect is mediated by the VEGF promoter region, which contains several potential binding sites for the transcription factor activator protein-1 [59], cfos and c-jun [60]. ERK-1 and 2 have been reported to be important signaling cascades that lead to over-expression of VEGF mRNA [61]; however, EGCG inhibits kinases that cause activation of ERK-1 and 2 and hence decreases VEGF [62]. EGCG is also known to chelate strong metal ions [63] and some receptor kinases dependent on divalent cations for their activity, and EGCG can also inhibit the activity of receptor kinases by chelating the divalent ions [64]. Basic Fibroblast Growth Factor The bFGF protein (FGF-2) is a member of the fibroblast growth factor family [65]. In normal tissue, basic fibroblast growth factor is present in basement membranes and in the sub endothelial extracellular matrix of blood vessels. Our recent study in human colorectal cancer cells with EGCG treatment provided insight on a new mechanism by which EGCG downregulates bFGF both in vitro and in vivo [46]. The bFGF protein is degraded in the presence of EGCG through the ubiquitin/proteasome pathway. The proteasome complex includes three activities: chymotrypsin-like, trypsin-like and caspase-like [66] and chymotrypsin-like activity is involved in tumor survival [67]. Interestingly, our results showed that EGCG decreases the chymotrypsin-like activity and increases the trypsin-like activity of the 20S

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proteasome, subsequently degrading bFGF at the post-translational level. EGCG also increases ubiquitination in a dose-dependent manner [46]. In addition, bFGF suppression by EGCG is not only seen in culture but also seen in animal studies. We demonstrated the decrease in tumor volume and number in the APCMin/+ mice fed EGCG in their drinking water, as assessed by ELISA using the small intestinal tumor tissue lysates [46].

MMP and uPA MMP-9 is a collagenase involved in extracellular matrix degradation during tumor metastasis and inflammatory disorders [68]. It has been reported that EGCG inhibits MMP-9 secretion in cancer cells [69; 70]. Similary, uPA is a serine protease also called urokinase plasminogen activator. It is present in several physiological locations, such as the blood stream and the extracellular matrix. Plasminogen is the primary physiological substrate of plasmin. The activation of plasmin leads to activation of a proteolysis cascade leading to either thrombolysis or extracellular matrix degradation [71]. Swiercz et al. reported that EGCG inhibits uPA, thereby blocking metastasis and acting as a tumor inhibitor [72].

7.2. Effect on Apoptosis and Cancer Progression Programmed cell death in multicellular organisms is known as apoptosis. A series of biochemical events is involved in the process of apoptosis that leads to a variety of morphological changes, including blebbing, cell membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis helps to maintain tissue integrity and function and helps to eliminate damaged or unwanted cells [73]. EGCG induces apoptosis via different signaling pathways such as inhibiting NF-κB activation, binding to Fas, activating tumor necrosis factor-α, arresting cell cycle at G0/G1, and binding to and suppressing anti-apoptotic Bcl-2 family proteins [74; 75; 76]. EGCG inhibits growth of transformed cells, but not normal cells, primarily via induction of apoptosis [74; 76; 77] and by altering several protein expressions that are involved in apoptosis pathways.

NAG-1 NAG-1, or non-steroidal anti-inflammatory drug (NSAID)-activated gene is a proapoptotic and anti tumorigenic protein [78]. It is a secreted protein belonging to the TGF-β superfamily. NAG-1 expression causes the inhibition of cell growth of several epithelial cells as well as primitive hematopoietic progenitors [79]. NAG-1 is also known as macrophage inhibitory cytokine-1 (MIC-1) [80], placental transforming growth factor-β [81], prostatederived factor [82], growth differentiation factor 15 [83], and placental bone morphogenetic protein [84]. It has been shown to be highly expressed in mature intestinal epithelial cells; however, its expression is decreased in human colorectal carcinoma and neoplastic intestinal polyps of APCMin/+ mice [85]. It has been shown that EGCG or ECG can increase NAG-1 expression in human colorectal cancer cells and that NAG-1 induction is one of the mechanisms of catechin-induced apoptosis leading to blocking of colorectal tumorigenesis [45]. In addition, EGCG is known to produce oxidative DNA damage at high concentrations [86]. This DNA damage can cause induction of p53 and cause apoptosis.

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p53 The p53 gene encodes the transcription factor p53, which is a very important regulator of the cell cycle, plays a crucial role as a tumor suppressor, and hence is involved in preventing carcinogenesis [87]. The p53 protein can cause repair of damaged DNA by activating DNA repair proteins, and subsequently it can hold the cell cycle at the G1/S phase if DNA damage is detected, whereas it can initiate apoptosis if the DNA damage is irreversible. EGCG induces p53 tumor suppressor protein, followed by the induction of apoptotic related genes [88]. Amin et al. showed that EGCG can modulate the cancer cell growth pattern in a p53dependent manner [89]. In addition, EGCG induces p53 proteins not only in colorectal cancer cells, but also in other cancer cells [79].

ATF3 Activating transcription factor 3 (ATF3) belongs to the mammalian activation transcription factor (ATF)/cAMP responsive element-binding (CREB) protein family of transcription factors. ATF3 is known to be activated by a variety of physiological and pathological stimuli to the cells [90]. Overexpression of ATF3 in human colorectal cancer cells lead to apoptosis and cell cycle arrest [91]. The human colorectal cancer cells, HCT-116 (p53 wild type) and SW480 (p53 mutant), treated with 50 µM of EGCG and ECG, respectively, caused a high induction of ATF3 [92]. This result implied that catechins can induce ATF3 in a p53-independent manner. Furthermore, it has been shown that catechins can act as both antioxidant and pro-oxidant agents [93], and that ECG generates oxidative stress in the media, followed by the induction of ATF3 [92]. EGR-1 Early growth response gene-1 is an inducible zinc finger transcription factor and an immediate-early gene induced by stress or injury, mitogens, growth factors, cytokines, hypoxia, and differentiation factors [94]. It is mainly involved in progress of vascular diseases. EGR-1 is also known to modulate the genes involved in growth control and survival. Expression of EGR-1 can have a dual effect; it can cause either promotion or inhibition of cell growth, which depends on cell type and environment. EGR-1 has been also shown to act as a tumor suppressor gene, and its loss can lead to progression of cancer. Many researchers have shown that EGR-1 induces apoptosis and is an important factor in neuronal apoptosis [95]. It exhibits its pro-apoptotic function by directly binding to p53 [96], NAG1[97] and PTEN promoters [98]. We have previously shown that EGR-1 phosphorylation enhances ATF3 expression in colorectal cancer cells [91], and then we demonstrated that EGR-1 played an important role in ECG-induced ATF3 expression in the human colorectal cancer cell line HCT-116 [92].

7.3. Effect on Cell Adhesion Cell or cellular adhesion is an interaction of a cell to another cell or to a surface. This interaction or adhesion is brought about by special molecules called cell adhesion molecules (CAM), which interact with other molecules on the other cells or on the other surface. The

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CAMs are transmembrane receptors and include molecules like integrins, cadherins and selectins.

Integrins Integrins are CAMs that interact with other cells and extracellular matrix and define cellular shape and mobility while playing an important role in regulating the cell cycle. They are usually attached to the cellular plasma membrane where they help to transfer a signal from one cell to another and influence cell growth and differentiation. All these properties make the integrins an important component in cell migration, invasion, and platelet interaction, hence its role in tumor growth and metastasis [99]. These integrin molecules regulate the interaction between tumor cells and other adhesion molecules such as fibronectin, laminin and collagens [100]. The adhesive interactions between tumor cells and these molecules lead to tumor growth, invasion and metastasis [101; 102]. EGCG has been shown to impair the adhesion between tumor cells and proteins such as fibronectin and laminin by binding to them [103; 104]. Suzuki et al. demonstrated the binding of EGCG to β1 integrin, and this binding led to disruption of β1 integrin and hence impairment of cell adhesion [105]. EGCG and ECG have also been shown to bind matrix proteins and inhibit smooth muscle cell adhesion with integrin receptor β1; EGCG can also inhibit laminin-induced smooth muscle cell migration [106]. Cadherins Cadherins play a crucial role in cell adhesion and do not allow cancer cells to migrate or metastasize. However, the perturbation of cadherin function can lead to temporary or permanent non-attachment of tumor cells and hence promote the invasion and metastasis of such loose cells. E-cadherin is also known to bind β-catenin, which is an intracellular anchoring protein. This adherence is important for epithelial cell homeostasis [107; 108]. In the development of intestinal tumors in APCMin/+ mice, aberrant β-catenin signaling is a key molecular event [109]. E-cadherin is a tumor and invasion suppressor protein that plays a crucial suppressive role in the progression from adenoma to carcinoma in colorectal cancers [110]. EGCG has been shown to increase E-cadherin protein levels in vitro in HT29 cells, and this is due to translocation of β-catenin from the cytoplasm to the nuclei and then to the plasma membrane. EGCG also increases E-cadherin protein levels by attenuating transcriptional repression, slug/snail zinc finger protein family, or by causing posttranscriptional modification of caveolin-1, a protein that plays an important role in the endocytosis of E-cadherin [111; 112; 113; 114].

7.4. Effect on Cell Cycle Regulators Cell cycle is a series of processes taking place in an eukaryotic cell that leads to its replication. There are two major events involved: interphase, divided into 4 phases, G0, G1, S, and G2, in which the cell grows and accumulates nutrients, and mitosis wherein the cells divide into two daughter cells. It is very important for the cell cycle to function properly, and certain check points are needed in order to detect and repair any kind of genetic damage so that the cell cycle does not go unchecked leading to uncontrolled growth. The regulatory

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molecules governing the ordered processes of cell cycle are cyclins and cyclin dependent kinases (CDKs). CDKs once activated by cyclins cause phosphorylation of target proteins, which either activates or inactivates the proteins or causes them to move into the next phase of the cell cycle.

Cyclin D1 Cyclin D1 is produced in response to growth factors. It is an important cell cycle regulator that is involved in G1-S transition and in regulation of proliferation and differentiation [115]. Dysregulated degradation of cyclin D1 is associated with its increased levels in various cancers [116]. Jeong et al. reported decreased expression of cyclin D1 in HT-29 human colorectal cancer cell lines by EGCG [117], and this result was confirmed by other groups, showing that EGCG and ECG decreased the expression of cyclin D1 [118]. In addition, we have reported that ECG can significantly suppress the expression of cyclin D1 in oral cancers [119]. GAK GAK (Cyclin G-associated kinase) is a serine/threonine kinase that is associated with cyclin G [120]. EGCG has been shown to down regulate the expression of GAK and hence arrest cell cycle progression from the G1 phase to the S phase by de-activating cyclin G [121]. p21 This regulator belongs to the CIP/KIP family which are inhibitors of cyclin-dependent kinases [122]. EGCG has been shown to increase the expression of p21 and hence lead to cell cycle arrest [123].

8. EGCG IN OTHER CANCERS Breast Cancer EGCG induces apoptosis and significantly decreases invasion of breast cancer cells, and induction of apoptosis is by increasing the pro-apoptotic agent BAX and decreasing the antiapoptotic agent BCL-2 [124]. EGCG also reduces invasion of cells by 24-28% on matrigel and decreases the expression of MMP-9 [125]. EGCG has also been shown to decrease the estrogen receptor α function, which is a pivotal pathway in breast tumorigenesis [126].

Lung Cancer EGCG has been shown to inhibit lung tumorigenesis in several different animal model systems. This includes lung tumorigenesis in A/J mice induced by 4-(methylnitrosamino)-1(3pyridyl)-1-butanone (NNK), N-nitrosodiethylamine, benzo[a]pyrene, N-nitrosomethylurea, or cisplatin [127]. EGCG mostly accounts for decreased tumor number, size and incidence. All these parameters are associated with the anti-proliferative, pro-apoptotic, and anti-

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angiogenic activities of EGCG and inhibition of protein kinases involved in signal transduction and cell cycle regulation.

Prostate Cancer Prostate cancer is the second leading cause of cancer-related death in men in the United States. Incorporating chemopreventive agents such as EGCG into the diet can delay the early onset of prostate cancer. EGCG exerts its chemopreventive effect in the prostate via regulation of the sex steroid receptor, growth factor-signaling, and inflammatory pathways. Harper et al. showed that EGCG inhibited early but not late stage prostate cancer. EGCG can also reduce cell proliferation and induce apoptosis by decreasing androgen receptor, IGF-1, COX-2 and MAPK signaling [128].

Pancreatic Cancer Pancreatic cancer can be a deadly disease and lead to higher mortality as it is very difficult to diagnose. By the time it is diagnosed, it has reached the late stages. EGCG has been shown to inhibit growth and induce apoptosis in human pancreatic cancer cells. EGCG can effectively inhibit viability, capillary tube formation and migration of HUVEC on its own and these effects are enhanced in presence of an ERK inhibitor in vitro. In the case of in vivo studies, AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume, proliferation, angiogenesis, VEGF-R2 and metastasis (MMP-2, MMP-7, MMP-9 and MMP-12) and induction of apoptosis, caspase-3 activity and growth arrest (p21/WAF1). In addition, there was a reduced ERK activity and enhanced p38 and JNK activities in tumor samples from EGCG-treated mice. Hence all these results show that EGCG inhibits pancreatic cancer growth, invasion, metastasis and angiogenesis and can be used in the management of pancreatic cancer prevention and treatment [129].

Skin Cancer Of all the possible causes of skin cancer, exposure to ultra violet radiation is the main one. Sevin et al. carried out a study in 12-week-old Wistar albino rats that were exposed to UV light and used ointment containing 2% EGCG. They found that topical application of EGCG was effective in preventing the skin cancer before UV exposure or had a protective effect; however, once damage was caused after exposure to UV, EGCG didn’t show any significant recovery [130]. Another study focused on EGCG-induced caspase-14 expression in an A431 human epidermoid cancer cell line. Caspase-14 is a member of the caspase family associated with epithelial cell differentiation, planned cell death, and barrier formation. Induction of caspase-14 by EGCG led to growth inhibition and reduction in tumorigenicity of A431 cells [131]. IL-12 is an interleukin known to induce immune responses and antiangiogenic activities. Based on these studies, EGCG can induce IL-12 and can prevent photocarcinogenesis through an EGCG-induced, IL-12-dependent, DNA repair mechanism [132].

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9. SUMMARY Most of the beneficial effects of green tea are due to the major catechin EGCG, which accounts for at least 50% of the total catechin content of green tea. EGCG has hydroxyl groups that make it a potent anti-oxidant agent, and its activity is 25-100% more potent than vitamin C and E. Other catechins also play a role in blocking colorectal carcinogenesis. ECG increases NAG-1 expression and induces apoptosis to prevent carcinogenesis. Colorectal cancer occurs due to mutations in both tumor suppressors as well as oncogenes. Different signaling pathways govern colorectal tumorigenesis. As shown in Fig. 4, many researchers have shown that EGCG acts on all these pathways in some way. EGCG also acts on other cancers such as lung, pancreas, breast, prostate, and skin and many other cancers. Green tea and its catechins have emerged up to be very effective dietary agents that are easily available, non- expensive, and most importantly have no side effects and can prevent cancer growth and metastasis.

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[119] Lim YC, Lee SH, Song MH, Yamaguchi K, Yoon JH, Choi EC, Baek SJ. Growth inhibition and apoptosis by (-)-epicatechin gallate are mediated by cyclin d1 suppression in head and neck squamous carcinoma cells. Eur J Cancer 2006;42:32603266. [120] Kanaoka Y, Kimura SH, Okazaki I, Ikeda M, Nojima H. Gak: A cyclin g associated kinase contains a tensin/auxilin-like domain. FEBS Letters 1997;402:73-80. [121] Ahn WS, Huh SW, Bae SM, Lee IP, Lee JM, Namkoong SE, Kim CK, Sin JI. A major constituent of green tea, egcg, inhibits the growth of a human cervical cancer cell line, caski cells, through apoptosis, g(1) arrest, and regulation of gene expression. DNA Cell Biol 2003;22:217-224. [122] Kei-ichi Nakayama KN. Cip/kip cyclin-dependent kinase inhibitors: Brakes of the cell cycle engine during development. BioEssays 1998;20:1020-1029. [123] Manson MM. Cancer prevention - the potential for diet to modulate molecular signalling. Trends in Molecular Medicine 2003;9:11-18. [124] Khan N, Afaq F, Mukhtar H. Apoptosis by dietary factors: The suicide solution for delaying cancer growth. Carcinogenesis 2007;28:233-239. [125] Thangapazham RL, Passi N, Maheshwari RK. Green tea polyphenol and epigallocatechin gallate induce apoptosis and inhibit invasion in human breast cancer cells. Cancer Biol Ther 2007;6:1938-1943. [126] Farabegoli F, Barbi C, Lambertini E, Piva R. (-)-epigallocatechin-3-gallate downregulates estrogen receptor alpha function in mcf-7 breast carcinoma cells. Cancer Detection and Prevention 2007;31:499-504. [127] Yang CS, Liao J, Yang GY, Lu G. Inhibition of lung tumorigenesis by tea. Exp Lung Res 2005;31:135-144. [128] Harper CE, Patel BB, Wang J, Eltoum IA, Lamartiniere CA. Epigallocatechin-3-gallate suppresses early stage, but not late stage prostate cancer in tramp mice: Mechanisms of action. Prostate 2007;67:1576-1589. [129] Shankar S, Ganapathy S, Hingorani SR, Srivastava RK. Egcg inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci 2008;13:440-452. [130] Sevin A, Oztas P, Senen D, Han U, Karaman C, Tarimci N, Kartal M, Erdogan B. Effects of polyphenols on skin damage due to ultraviolet a rays: An experimental study on rats. J Eur Acad Dermatol Venereol 2007;21:650-656. [131] Hsu S, Qin H, Dickinson D, Xie D, Bollag WB, Stoppler H, Pearl H, Vu A, Watkins M, Koehler M, Schuster G. Expression of caspase-14 reduces tumorigenicity of skin cancer cells. In Vivo 2007;21:279-283. [132] Meeran SM, Mantena SK, Elmets CA, Katiyar SK. (-)-epigallocatechin-3-gallate prevents photocarcinogenesis in mice through interleukin-12-dependent DNA repair. Cancer Res 2006;66:5512-5520.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 17

INHIBITORY EFFECT OF CATECHIN DERIVATIVES FROM GREEN TEA ON DNA POLYMERASE ACTIVITY, HUMAN CANCER CELL GROWTH, AND TPA (12-OTETRADECANOYLPHORBOL-13-ACETATE) -INDUCED INFLAMMATION Yuko Kumamoto-Yonezawa1, Hiromi Yoshida1,2 and Yoshiyuki Mizushina1,2,* 1

Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, KobeGakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan 2 Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan

ABSTRACT Green tea obtained from the leaves of the plant, Camellia sinensis, is one of the most popular beverages in the world. The major polyphenolic compounds in green tea are catechin derivatives (i.e., flavan-3-ols), and their composition varies depending on the season of harvest and the manufacturing process. The inhibitory activities against DNA polymerases (pols) of catechin derivatives such as (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin gallate (Cg), (-)epicatechin gallate (ECg), (-)-gallocatechin gallate (GCg), and (-)-epigallocatechin gallate (EGCg) were investigated. Among these eight catechins, several inhibited mammalian pols with EGCg being the strongest inhibitor of pols α and λ with IC50 values of 5.1 and 3.8 μM, respectively. EGCg did not influence the activities of plant pols, such as pols α and λ, or prokaryotic pols, and had no effect on the activities of DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 *

Correspondence to: Yoshiyuki Mizushina Ph.D., Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan; Tel: +81-78-974-1551 (ext.3232); Fax: +81-78-974-5689; E-mail: [email protected]

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Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina polynucleotide kinase, or bovine deoxyribonuclease I. Some tea catechins also suppressed human cancer cell growth and/or TPA (12-O-tetradecanoylphorbol-13acetate)-induced inflammation, and the tendency of the pol inhibitory activity for these compounds was the same as that of their anti-inflammatory activity rather than their anticancer activity. Based on these results, the relationship between the structure of tea catechins and their bioactivities is discussed.

Keywords: Green tea; Catechin derivatives; DNA polymerase; enzyme inhibitor; DNA replication; Cancer cell growth inhibition; Anti-cancer activity; Anti-inflammation

ABBREVIATIONS pol, DNA polymerase (E.C.2.7.7.7); C, (+)-catechin; EC, (-)-epicatechin; GC, (-)-gallocatechin; EGC, (-)-epigallocatechin; Cg, (+)-catechin gallate; ECg, (-)-epicatechin gallate; GCg, (-)-gallocatechin gallate; EGCg, (-)-epigallocatechin gallate; TPA, 12-O-tetradecanoylphorbol-13-acetate.

1. INTRODUCTION Green tea, a popular beverage, is consumed worldwide. It contains an infusion of the leaves from Camellia sinensis, which is rich in polyphenolic compounds known as catechins, especially, (-)-epigallocatechin gallate (EGCg), (-)-epicatechin gallate (ECg), (-)epigallocatechin (EGC), and (-)-epicatechin (EC) [1, 2]. The catechin composition of green tea varies depending on the season of harvest and the manufacturing process. Green tea leaves usually contain about 1 % EC, 2-3 % EGC, 1-2 % ECg, and 5-8 % EGCg [3]; therefore, the main constituent of catechin is EGCg. The human genome encodes 14 pols, which conduct cellular DNA synthesis [4]. Eukaryotic cells reportedly contain three replicative types (pols α, δ and ε, mitochondrial pol γ, and at least twelve repair types (pols β, δ, ε, ζ, η, θ, ι, κ, λ, μ, and σ, and REV1) [5]. We have been studying selective inhibitors of each pol from natural materials including foods such as vegetables for more than 10 years [6-13]. It is considered that food materials containing pol inhibitory compounds could be used as bioactive functional foods with anticancer, anti-virus, and immuno-suppression activities etc. Polyphenols, such as curcumin [7, 8] and anthocyanin compounds [12], were found to inhibit the activities of mammalian pols. This review reports the inhibitory effect of catechin derivatives [11], as contained in green tea, on bioactivities including DNA polymerase inhibition, human cancer cell growth inhibition, and anti-inflammation, and the relationship of these bioactivities of catechin derivatives is discussed.

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2. EFFECT OF CATECHIN DERIVATIVES ON THE ACTIVITIES OF MAMMALIAN DNA POLYMERASES The assay method for pol activity was described previously [15, 16]. The substrates of the pols were poly(dA)/oligo(dT)12-18 and 2'-deoxythymidine 5'-triphosphate (dTTP) as the DNA template-primer and nucleotide substrate (i.e., 2'-deoxynucleotide 5'-triphosphate (dNTP)), respectively. As described above, during searches for natural inhibitors of mammalian pols it was found that catechin compounds from the leaves of dried green tea, Camellia sinensis, inhibited these activities. Eight analytical grade catechin derivatives (i.e., flavan-3-ols) were purchased commercially, including (+)-catechin (C, compound 1), (-)-epicatechin (EC, compound 2), (-)-gallocatechin (GC, compound 3), (-)-epigallocatechin (EGC, compound 4), (+)-catechin gallate (Cg, compound 5), (-)-epicatechin gallate (ECg, compound 6), (-)gallocatechin gallate (GCg, compound 7), and (-)-epigallocatechin gallate (EGCg, compound 8), and two part compounds of catechin derivatives as pyrogallol (compound 9) and gallic acid (compound 10). The chemical structures are shown in Figure 1. This study investigated whether compounds 1 - 10 inhibited the activities of mammalian pols α, β, and λ. The relative activity of each pol with a set concentration (100 μM) of the test compounds is shown in Figure 2. Immuno-affinity purified calf pol α was used as a replicative pol, and E. coli expressed recombinant mammalian pols β and λ were prepared and used as repair related pols. Compounds 1 (C) and 2 (EC) did not influence the activities of pols α, β or λ. Compounds 9 (pyrogallol) and 10 (gallic acid), which are the structural parts of flavan-3-ol, also did not inhibit these pol activities. Compounds 3 to 8 inhibited the pol activities, with the effects on pols α and λ being stronger than that on pol β. The compounds that inhibited the relative activity of pol α by more than 50 % were 3 (GC), 4 (EGC), 5 (Cg), 6 (ECg), 7 (GCg), and 8 (EGCg). Compound 8 (EGCg) had the strongest inhibitory effect on the three pols of all the catechin derivatives tested. Therefore, on the properties of EGCg were examined in the subsequent experiments. OH

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OH

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Figure 1. Structure of catechin derivatives. Compound 1, (+)-catechin (C); compound 2, (-)-epicatechin (EC); compound 3, (-)-gallocatechin (GC); compound 4, (-)-epigallocatechin (EGC); compound 5, (+)catechin gallate (Cg); compound 6, (-)-epicatechin gallate (ECg); compound 7, (-)-gallocatechin gallate (GCg); compound 8, (-)-epigallocatechin gallate (EGCg); compound 9, pyrogallol; and compound 10, gallic acid.

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Calf polƒ¿ Rat polƒÀ Human polƒÉ

Compound 1 (C) Compound 2 (EC) Compound 3 (GC) Compound 4 (EGC) Compound 5 (Cg) Compound 6 (ECg) Compound 7 (GCg) Compound 8 (EGCg) Compound 9 (Pyrogallol) Compound 10 (Gallic acid) 0

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DNA polymerase activity (%) Figure 2. Inhibitory effect of catechin derivatives on the activities of mammalian DNA polymerases. Compounds 1 to 10 (100 μM each) were incubated with calf pol α, rat pol β and human pol λ (0.05 units each). Percent relative activity is shown. The enzymatic activities were measured as described previously [15, 16]. Pol activity in the absence of the test compounds was taken as 100%. Data are shown as the means ± SEM of three independent experiments.

3. EFFECTS OF EGCG ON THE ACTIVITIES OF DNA POLYMERASES AND OTHER DNA METABOLIC ENZYMES As shown in Table 1, EGCg inhibited the activities of all the mammalian pols tested, and calf pol α and human pol λ were strongly inhibited with IC50 values of 5.1 and 3.8 μM, respectively. The inhibitory effect on pols β and λ was the weakest and strongest, respectively, of the mammalian pols tested, and the inhibition of pol β was 12.3-fold weaker than that of pol λ, although the amino acid sequence of pol β is similar to pol λ, and these pols are both classified as family-X pols [1, 17]. On the other hand, the activity of plant pols

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such as cauliflower pol α and rice pol λ, prokaryotic pols such as the Klenow fragment of E. coli pol I, Taq pol, and T4 pol, and DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I, were not influenced by EGCg. EGCg should be classified as an inhibitor of mammalian pols.

4. EFFECT OF CATECHIN DERIVATIVES ON CULTURED HUMAN CANCER CELL GROWTH Catechin derivatives are of interest in developing of functional foods for cancer chemotherapy. Replicative pols are regarded as targets of anti-cancer drugs because they play central roles in DNA replication, which is indispensable for the proliferation of cancer cells. Therefore, the growth effects of these compounds on a human cancer cell line (promyelocytic leukemia cell, HL-60) were investigated in culture using MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay [18]. Table 1. IC50 values of compound 8 (EGCg) on the activities of various DNA polymerases and other DNA metabolic enzymes Enzyme Mammalian DNA polymerases Calf DNA polymerase α Rat DNA polymerase β Human DNA polymerase γ Human DNA polymerase δ Human DNA polymerase ε Human DNA polymerase η Human DNA polymerase ι Human DNA polymerase κ Human DNA polymerase λ Plant DNA polymerases Cauliflower DNA polymerase α Rice DNA polymerase λ Prokaryotic DNA polymerases E. coli DNA polymerase I (Klenow fragment) Taq DNA polymerase T4 DNA polymerase Other DNA metabolic enzymes Calf primase of DNA polymerase α T7 RNA polymerase T4 polynucleotide kinase Bovine deoxyribonuclease I

IC50 value (μM) Compound 8 (EGCg) 5.1 ± 0.3 46.8 ± 1.8 11.0 ± 0.5 17.2 ± 0.9 17.6 ± 0.9 11.6 ± 0.6 12.7 ± 0.6 13.5 ± 0.7 3.8 ± 0.2 >100 >100 >100 >100 >100 >100 >100 >100 >100

The compound was incubated with each enzyme (0.05 units). Enzymatic activity in the absence of the compound was taken as 100 %.

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Compound 1 (C) Compound 2 (EC) Compound 3 (GC) Compound 4 (EGC) Compound 5 (Cg) Compound 6 (ECg) Compound 7 (GCg) Compound 8 (EGCg) Compound 9 (Pyrogallol) Compound 10 (Gallic acid) 0

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Figure 3. Effects of catechin derivatives on HL-60 cancer cell growth. The compounds (100 μM each) were incubated with the human cancer cells (promyelocytic leukemia cell line, HL-60). Percent relative activity is shown. Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [18]. The growth rate of the cancer cells in the absence of the test compounds was taken as 100 %. Data are shown as the means ± SEM of five independent experiments.

As shown in Figure 3, 100 μM of compound 4 (EGC) had the strongest growth inhibitory effect on this cancer cell line of the compounds tested, and the compounds that prevented more than 50 % of cancer cell growth, were 3 (GC), 4 (EGC), 7 (GCg), and 9 (pyrogallol). Compounds 8 (EGCg) and 10 (gallic acid) moderately suppressed cell growth, although EGCg was the strongest inhibitor of mammalian pols (Figure 2). On the other hand, compounds 1 (C), 2 (EC), 5 (Cg), and 6 (ECg) had no effect on cancer cell growth.

5. EFFECT OF CATECHIN DERIVATIVES ON ANTI-INFLAMMATORY ACTIVITY Although TPA (12-O-tetradecanoylphorbol-13-acetate) promotes tumor formation [19], it is also known to cause inflammation and is generally used as an artificial inflammation inducer for the screening of anti-inflammatory agents [20]. Tumor promoter-induced inflammation can be distinguished from acute inflammation, which is exudative and accompanied by fibroblast proliferation and granulation. Using mouse inflammatory tests, the anti-inflammatory activities of catechin derivatives (compounds 1 – 10) were examined. The application of TPA (0.5 μg) to a mouse ear induced edema; the weight increase of the ear disk at 7 hr after application was 241 %. As expected, the inhibitory effect of compound 8 (EGCg) on inflammation was the strongest among the compounds tested at an applied dose of at least 250 μg, and the level of inhibition was 65.6 % (Figure 4). Compound 7 (GCg) was the second

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strongest inhibitor of inflammation with an inhibitory effect of 58.0 %. Compounds 3 (GC) and 4 (EGC) and compounds 5 (Cg) and 6 (ECg) moderately suppressed inflammation at levels of 30 to 32 % and 25 to 27 %, respectively. On the other hand, compounds 1 (C) and 2 (EC) and the part compounds of catechin derivatives, compounds 9 (pyrogallol) and 10 (gallic acid), had little effect on inflammation. These results suggested that some catechin derivatives caused a remarkable reduction in TPA-induced inflammation, indicating that EGCg possesses anti-inflammatory activity.

Compound 1 (C) Compound 2 (EC) Compound 3 (GC) Compound 4 (EGC) Compound 5 (Cg) Compound 6 (ECg) Compound 7 (GCg) Compound 8 (EGCg) Compound 9 (Pyrogallol) Compound 10 (Gallic acid) 0

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Anti-inflammatory activity (%) Figure 4. Anti-inflammatory activity of catechin derivatives toward TPA-induced edema on mouse ear. Compounds 1 to 10 (250 μg each) were applied individually to one ear on each mouse, and after 30 min TPA (0.5 μg) was applied to both ears. Edema was evaluated after 7 hr. The inhibitory effect is expressed as the percentage of edema. Data are shown as the means ± SEM of five independent experiments.

6. RELATIONSHIP OF THE THREE BIOACTIVITIES OF CATECHIN DERIVATIVES To confirm if there is a relationship between mammalian pol inhibition, human cancer cell (promyelocytic leukemia cell line, HL-60) growth inhibition, and anti-chronic inflammation, catechin derivatives (compounds 1 - 10) were compared for their inhibitory effects on these three bioactivities (Figure 5). Pol λ inhibition had the largest correlation (correlation coefficient = 0.9979) with the anti-inflammatory activity of all combinations of bioactivities (Figure 5A). On the other hand, neither pol λ inhibitory activity nor antiinflammatory activity was related to the inhibition of human cancer cell growth because the correlation coefficient between these activities and the cell growth inhibitory activity was less

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than 0.2 (i.e., 0.1703 in Figure 5B and 0.1851 in Figure 5C, respectively). These results led to the speculation that TPA-induced inflammation may involve a process requiring pol λ.

R2 = 0.9979

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Figure 5. The relationship between pol λ inhibition, cancer cell growth inhibition, and antiinflammation by catechin derivatives. (A) Pol λ inhibitory activity of 100 μM of each compound versus anti-inflammatory activity of 250 μg of the same compound. (B) Pol λ inhibitory activity of 100 μM of each compound versus cancer cell growth inhibitory activity of 100 μM of the same compound. (C) Cancer cell growth inhibitory activity of 100 μM of each compound versus anti-inflammatory activity of 250 μg of the same compound. Numbers 1 to 10 in the figures indicate compounds 1 to 10, respectively. The values of correlation coefficients are shown in each figure.

Pol λ is a pol X family polymerase [17]. Although the in vivo biochemical function of pol λ is unclear as yet, pol λ appears to work in a similar manner to pol β. Pol β, which is widely known to have roles in the short-patch base excision repair (BER) pathway [21-24], plays an essential role in neural development [25]. Recently, pol λ was found to contain 5'deoxyribose-5-phosphate (dRP) lyase activity, but no apurinic/apyrimidinic (AP) lyase activity [26] and to be able to substitute pol β in in vitro BER, suggesting that pol λ also participates in BER. Northern blot analysis indicated that transcripts of pol β were abundantly expressed in the testis, thymus, and brain in rats [27], but pol λ was efficiently transcribed mostly in the testis [17]. Bertocci et al. reported that mice in which pol λ was knocked down were not only viable and fertile but also displayed a normal hyper-mutation pattern [28]. TPA not only causes inflammation, but also influences cell proliferation and has physiological effects on cells because it is a tumor promoter [29]. Therefore, antiinflammatory agents are expected to suppress both mammalian cell proliferation and DNA replication / repair in nuclei related to the action of TPA. Since pol λ is a repair-related polymerase [30], the result that the molecular target of catechin derivatives was pol λ is in good agreement. If so, the pol λ inhibitor could be an inhibitor of chronic inflammation. A positive relationship between anti-inflammatory and pol λ-inhibitory activities was found,

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which may be useful as a new and convenient in vitro assay to screen for novel anti-chronic inflammatory compounds. On the other hand, the suppression of human cancer cell growth had almost the same tendency as the inhibition of mammalian pol α among the compounds (Figure 2 and Figure 3), suggesting that the cause of the cancer cell influence might be through the activity of replicative pols such as pols α, δ, and ε.

7. STRUCTURE AND ACTIVITY RELATIONSHIP OF CATECHIN DERIVATIVES Eight catechin derivatives (compounds 1 – 8) and two part compounds of them (compounds 9 and 10) were prepared, and compound 8 (EGCg) was the strongest inhibitor of pol λ and had the strongest anti-inflammation activity of the compounds tested. In the structure of EGCg (Figure 6), the essential moieties of the structure for these activities were considered as: <1> the gallic acid moiety, and <2> the hydroxyl group in the pyrogallol moiety. Compounds 7 (GCg) and 8 (EGCg) contain these moieties; therefore, these compounds should have the strongest activities of both pol λ inhibition and antiinflammation. On the other hand, catechin derivatives lacking moiety <2> (i.e., compounds 1 (C), 2 (EC), 5 (Cg), and 6 (ECg)) had no effect on the growth inhibition of human cancer cells (promyelocytic leukemia cell line, HL-60), suggesting that this moiety must be essential for anti-cancer activity.

OH

<2>

OH HO

O

OH OH O C

OH

OH

O

<1>

OH

Figure 6. Chemical structure of compound 8 (EGCg). Essential groups (i.e., <1> and <2>) for pol λ inhibitory activity and anti-inflammatory activity in catechin derivatives are shown.

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8. DISCUSSION Previous studies demonstrated that the existence of an effective daily amount of green tea polyphenols for cancer prevention in humans. Among green tea polyphenols comprising catechin derivatives, compound 8 (EGCg) has cancer preventive activities in vitro, in cell culture, and in vivo [31-36]. EGCg is able to inhibit the migration of bronchial tumor cells and could be an attractive candidate to treat tumor invasion and cell migration [37]. EGCg can also induce apoptosis and suppress the formation and growth of human cancers including colorectal cancers (CRC) [38]. Furthermore, green tea polyphenols were shown to affect several biological pathways. The anti-proliferative action of EGCg on pancreatic carcinoma is mediated through programmed cell death or apoptosis as evident from nuclear condensation, caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage [39]. The biological effects of green tea and tea polyphenols, including anti-oxidative activity toward low-density lipoproteins [40], anti-carcinogenic [41], and anti-bacterial actions [4244], have been examined extensively both in vitro and in vivo. Catechin derivatives have also been receiving attention for their protective effects against cardiovascular disease and cancer [45-49]. The amounts required are assumed to be equivalent to 10 Japanese-size cups of green tea per day, and it contains 2.5 g green tea extract. Catechin derivatives such as EGCg have a wide-range of target organs, such as the digestive tract, lungs, liver, pancreas, breast, bladder, prostate, and skin [1, 2]. The human intake from the above sources would be approximately 0.2 g of EGCg per day, and this concentration in the human body (60 kg) is equivalent to 7.2 μM (3.3 μg/ml, molecular weight of EGCg is 458.37). This dose would be able to significantly inhibit the activities of mammalian pols such as pol λ in vitro, and might give the bioactive effects for human health in vivo. In conclusion, green tea, containing catechin derivative, is a potential functional food for preventing cancer and inflammation, and tea catechin derivatives, especially EGCg, could be considered as possible candidates for anti-cancer and anti-inflammation agents.

ACKNOWLEDGEMENTS We are grateful for the donations of calf pol α, rat pol β, human pol γ, human pols and ε, human pols η and ι, human pol κ, and human pol λ by Dr. M. Takemura of Tokyo University of Science (Tokyo, Japan), Dr. A. Matsukage of Japan Women's University (Tokyo, Japan), Dr. M. Suzuki of Nagoya University (Nagoya, Japan), Dr. K. Sakaguchi of Tokyo University of Science (Chiba, Japan), Dr. F. Hanaoka and Dr. C. Masutani of Osaka University (Osaka, Japan), Dr. H. Ohmori of Kyoto University (Kyoto, Japan), and Dr. O. Koiwai of Tokyo University of Science (Chiba, Japan), respectively. This work was supported in part by a Grant-in-Aid for Kobe-Gakuin University Joint Research (A), and the “Academic Frontier” Project for Private Universities: matching fund subsidy from the Ministry of Education, Science, Sports, and Culture of Japan (MEXT), 2006 - 2010, (H. Y. and Y. M.). Y. M. acknowledges a Grant-in-Aid for Young Scientists (A) (No. 19680031) from MEXT, Grants-in-Aid from the Nakashima Foundation (Japan), Foundation

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of Oil & Fat Industry Kaikan (Japan), and The Salt Science Research Foundation, No. 08S3 (Japan).

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Fujiki, H. & Suganuma, M. (2002). Green tea and cancer prevention. Proc. Japan Acad., 78, 263-270. [2] Fujiki, H. (2005). Green tea: Health benefits as cancer preventive for humans. Chem. Rec., 5, 119-132. [3] Chen, Z., Zhu, Q. Y., Tsang, D. & Huang, Y. (2001). Degradation of green tea catechins in tea drinks. J. Agric. Food Chem., 49, 477-482. [4] Hubscher, U., Maga, G. & Spadari, S. (2002). Eukaryotic DNA polymerases. Annu. Rev. Biochem., 71, 133-163. [5] Kimura, S., Uchiyama, Y., Kasai, N., Namekawa, S., Saotome, A., Ueda, T., Ando, T., Ishibashi, T., Oshige, M., Furukawa, T., Yamamoto, T., Hashimoto, J. & Sakaguchi, K. (2002). A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.). Nucleic Acids Res., 30, 1585-1592. [6] Nishida, M., Hada, T., Kuramochi, K., Yoshida, H., Yonezawa, Y., Kuriyama, I., Sugawara, F., Yoshida, H. & Mizushina, Y. (2008). Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.). Food Chem., 108, 551-560. [7] Mizushina, Y., Takeuchi, T., Kuramochi, K., Kobayashi, S., Sugawara, F., Sakaguchi, K. & Yoshida, H. (2007). Study on the molecular structure and bio-activity (DNA polymerase inhibitory activity, anti-inflammatory activity and anti-oxidant activity) relationship of curcumin derivatives. Curr. Bioactive Compounds, 3, 171-177. [8] Takeuchi, T., Ishidoh, T., Iijima, H., Kuriyama, I., Shimazaki, N., Koiwai, O., Kuramochi, K., Kobayashi, S., Sugawara, F., Sakaguchi, K., Yoshida, H. & Mizushina, Y. (2006). Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase λ. Genes Cells, 11, 223-235. [9] Mizushina, Y., Nakanishi, R., Kuriyama, I., Kamiya, K., Satake, T., Shimazaki, N., Koiwai, O., Uchiyama, Y., Yonezawa, Y., Takemura, M., Sakaguchi, K. & Yoshida, H. (2006). β-sitosterol-3-O-β-D-glucopyranoside: a eukaryotic DNA polymerase λ inhibitor. J. Steroid Biochem. Mol. Biol., 99, 100-107. [10] Mizushina, Y., Yagita, E., Kuramochi, K., Kuriyama, I., Shimazaki, N., Koiwai, O., Uchiyama, Y., Yomezawa, Y., Sugawara, F., Kobayashi, S., Sakaguchi, K. & Yoshida, H. (2006). 5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase. Arch. Biochem. Biophys., 446, 69-76. [11] Mizushina, Y., Saito, A., Tanaka, A., Nakajima, N., Kuriyama, I., Takemura, M., Takeuchi, T., Sugawara, F. & Yoshida, H. (2005). Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors. Biochem. Biophys. Res. Commun., 333, 101-109. [12] Nakai, C. M., Kamiya, K., Satake, T., Kuriyama, I., Iijima, H., Ikawa, H., Yoshida, H. & Mizushina, Y. (2005). Novel topoisomerase I and II inhibitors, parameritannins A-2 and A-3, are selective human cancer cytotoxins. Int. J. Pharmacol., 1, 138-151.

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[29] Nakamura, Y., Murakami, A., Ohto, Y., Torikai, K., Tanaka, T. & Ohigashi, H. (1998). Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate. Cancer Res., 58, 4832-4839. [30] Garcia-Diaz, M., Bebenek, K., Sabariegos, R., Dominguez, O., Rodriguez, J., Kirchhoff, T., Garcia-Palomero, E., Picher, A. J., Juarez, R., Ruiz, J. F., Kunkel, T. A. & Blanco, L. (2002). DNA polymerase λ, a novel DNA repair enzyme in human cells. J. Biol. Chem., 277, 13184-13191. [31] Nakachi, K., Matsuyama, S., Miyake, S., Suganuma, M. & Imai, K. (2000). Preventive effectts of drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple targeting prevention. Bio-Factors, 13, 49-54. [32] Okuda, T. (2005). Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry, 66, 2012-2031. [33] Wang, Z. Y., Hong, J. Y., Huang, M. T., Reuhl, K. R., Conney, A. H. & Yang, C. S. (1992). Inhibition of N-nitrosodiethylamine- and 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone-induced tumorigenesis in A/J mice by green tea and black tea. Cancer Res., 52, 1943-1947. [34] Yang, C. S. & Wang, Z. Y. (1993). Tea and cancer. J. Natl. Cancer Inst., 85, 10381049. [35] Lu, Y. P., Lou, Y. R., Lin, Y., Shih, W. J., Huang, M. T., Yang, C. S. & Conney, A. H. (2001). Inhibitory effects of orally administered green tea, black tea, and caffeine on skin carcinogenesis in mice previously treated with ultraviolet B light (high-risk mice): relationship to decreased tissue fat. Cancer Res., 61, 5002-5009. [36] Gupta, S., Hastak, K., Ahmad, N., Lewin, J. S. & Mukhtar, H. (2001). Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc. Natl. Acad. Sci. USA, 98, 10350-10355. [37] Hazgui, S., Bonnomet, A., Nawrocki, R. B., Milliot, M., Terryn, C., Cutrona, J., Polette, M., Birembaut, P. & Zahm, J. M., (2008). Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells. Respir Res ., 9, 33. [38] Kumar, N., Shibata, D., Helm, J., Coppola, D. & Malafa, M. (2007). Green tea polyphenols in the prevention of colon cancer. Front Biosci., 12, 2309-2315. [39] Qanungo, S., Das, M., Haldar, S. & Basu, A. (2005). Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis, 26, 958-967. [40] Miura, S., Watanabe, J., Tomita, T., Sano, M. & Tomita, I. (1994). The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low density lipoprotein. Biol. Pharm. Bull., 17, 1567-1572. [41] Dreosti, I. E., Wargovich, M. J. & Yang, C. S. (1997). Inhibition of carcinogenesis by tea: the evidence from experimental studies. Crit. Rev. Food Sci. Nutr., 37, 761-770. [42] Amarowicz, R., Pegg, R. B. & Bautista, D. A. (2000). Antibacterial activity of green tea polyphenols against Escherichia coli K 12. Nahrung, 44, 60-62. [43] Sakanaka, S., Kim, M., Taniguchi, M. & Yamamoto, T. (1989). Antibacterial substances in Japanese green tea extract against Streptococcus mutants, a cariogenic bacterium. Agric. Biol. Chem., 53, 2307-2311.

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[44] Mabe, K., Yamada, M., Oguni, I. & Takahashi, T. (1999). In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob. Agents Chemother, 43, 17881791. [45] Ahmad, N. & Mukhtar, H. (1999). Green tea polyphenols and cancer: biologic mechanisms and practical implications. Nutr. Rev., 57, 78-83. [46] Buschman, J. L. (1998). Green tea and cancer in humans: a review of the literature. Nutr. Cancer, 31, 51-57. [47] Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B. & Kromhout, D. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet , 342, 1007-1011. [48] Imai, K. & Nakachi, K. (1985). Cross sectional study of effects of drinking green tea on cardiovascular and liver disease. Biochem. Med. J., 310, 693-696. [49] Yang, C. S. (1999). Tea and health. Nutrition, 15, 946-949.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 18

TELOMERASE REGULATION IN RESPONSE TO GREEN TEA Huaping Chen1 and Trygve O. Tollefsbol1,2,3,4,* 1

Department of Biology, University of Alabama at Birmingham, AL 35294, USA 2 Center for Aging, University of Alabama at Birmingham, AL 35294, USA 3 Comprehensive Cancer Center, University of Alabama at Birmingham, AL 35294, USA 4 Clinical Nutrition Research Center, University of Alabama at Birmingham, AL 35294, USA

ABSTRACT The anti-tumor effect of green tea, especially its major constituent EGCG, has been demonstrated in several animal experiments and its ability to induce apoptosis of most of cancer cell lines has been further documented in cell culture models. A number of mechanisms for how green tea impacts cancer have been proposed. These mechanisms basically include intervention of cell signal transduction pathways or changes of cell epigenetic processes. Telomerase has been recognized as a novel target of green tea. This important enzyme is largely localized to cancer cells, is responsible for the maintenance of telomeres so that cancer cells can escape the replication problem due to their linear chromosomes, and it has been shown to be reactivated in almost all tumor tissues. Telomerase has been found to be inhibited by green tea in telomerase-positive cancer cell lines. This analysis assesses the progress on research of the mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells. A number of mechanisms for how green tea works through this pathway have been proposed. Since telomerase has been identified as a potential molecular target for cancer treatment, and green tea has been shown to inhibit telomerase, clarifying the specific mechanisms for how green tea functions in this pathway should shed new light on the potential to design effective and novel preventive or anti-cancer approaches using green tea.

*

Corresponding Author. Department of Biology, 175 Campbell Hall, 1300 University Boulevard, Birmingham, AL 35294-1170, USA. Tel: +1-205-934-4573; Fax: +1-205-975-6097; Email: [email protected]

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Keywords: Green tea, EGCG, Telomerase, Cancer

INTRODUCTION Cancer still accounts for a high number of deaths worldwide. It is characterized by unlimited cell division, invasion into adjacent tissues, and may lead to metastasis which can spread to other tissues in the body through lymph or blood. The occurrence of cancer is a multistage process which includes initiation, promotion, and progression of carcinogenesis. Cancers can be caused by aberrant genetic material of the transformed cells. They are formed due in part to the interaction between genes of humans and environmental carcinogens, mutation in replication or inherited factors. These abnormalities include mutation of DNA sequences and epigenetic changes of the genome. The expression levels of two sets of genes are often changed in cancer. There are oncogenes which are responsible for the division of cells that have been activated, and tumor suppressor genes which are often inactivated as a result of disruption in the balance that controls proliferation of cells. Surgery, chemotherapy, radiation therapy, immunotherapy, and monoclonal antibody therapy have been used for treating cancer patients depending on the progress of the cancer and the health status of the patients as well as other factors.

EGCG

ECG

EC Figure 1. Molecular structures of the major polyphenol constituents of tea.

EGC

Telomerase Regulation in Response to Green Tea

frying or steaming

Fresh leaves

fermentation

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Prevent oxidation of polyphenols

Green tea

Partial oxidation of polyphenols

Oolong tea

Oxidation of polyphenols

Black tea

Figure 2. Processes by which various types of teas are prepared.

Tea is considered to be the most popular beverage worldwide next to water. Its use originates from China, and it is regularly consumed by a large number of individuals in the Asian countries. Tea is made from the leaves of Camellia sinensis. Several biologically active molecules are found in its leaves and the most important group is polyphenols, although other constituents include caffeine, flavonols such as quercetin and myricetin, and theobromine[1]. The primary polyphenols in tea are: epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin (EC), gallocatechin, and catechin (Figure 1). EGCG is the major and most active constituent of tea. There are currently three major types of tea: green tea, black tea, and oolong tea. They vary from each other in the procedure by which they are prepared and correspondingly the constituents they contain. Green tea is made by frying or steaming the fresh leaves of this plant. The oxidative enzymes in leaves can be heat-inactivated and thus the polyphenols in leaves can be protected in this way. Black tea is made by crushing the leaves and then letting the oxidation of polyphenols occur which is mediated by enzymes contained in the leaves, a process known as fermentation. Oolong tea is made by partial oxidation of the polyphenols in the leaves, and the ratio of the oxidized polyphenols in oolong tea lies between green tea and black tea (Figure 2). Thus, the distinguishing feature of green tea is that it is processed to prevent the oxidation of polyphenols, while the majority of the polyphenols in black tea have been oxidized into dimeric theaflavin molecules and polymeric thearubigins during its production, and theaflavins render black tea’s color and taste. Since traditional chemotherapy often produces unsatisfactory and toxic effects, non-toxic or less cytotoxic drugs for cancer prevention or treatment are therefore warranted. The effects of green tea have been investigated in a wide array of systems varying from animal models to cell lines, and inhibition of tumors and apoptosis of cancer cells have been observed. A variety of mechanisms have been proposed based on these studies. Here, we will briefly evaluate the biological effect of green tea first, and will then focus on the telomerase inhibition effect of green tea which has been regarded as a potential mechanism that imparts its chemopreventive and anticancer effects.

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BENEFICIAL EFFECT OF GREEN TEA Epidemiological studies indicate that consuming green tea is associated with a decrease in the occurrence of cancer in Asian people. In addition, research from animal models shows that green tea can inhibit tumor formation in a number of tissues such as lung, oral cavity, esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas. Cell culture experiments also demonstrate that EGCG can inhibit the proliferation of cancer cells. Here we would like to briefly review those effects.

INHIBITION OF TUMOR GROWTH IN VIVO AND ANGIOGENESIS INHIBITION Tea and its constituents have been shown to inhibit tumor formation in different organs in animal models. The organs affected include but are not limited to lung, oral cavity, esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas [2]. EGCG has been shown to block angiogenesis which is the process involving the formation of blood vessels. This process therefore deprives the tumor cells of the nutrients that they need to grow [3, 4]. Oral administration of GTPs (green tea polyphenols) in the drinking water for mice can inhibit expression of angiogenic factors such as matrix metalloproteinases (MMPs), and their natural inhibitor, TIMP1, which is upregulated in response to GTPs [5].

PROLIFERATION INHIBITION AND INDUCTION OF APOPTOSIS OF CANCER CELLS IN VITRO Green tea and its major constituent, EGCG, have been shown to inhibit proliferation and induce apoptosis of a series of tumor cell lines. For instance, It has been shown that the exposure of human lymphoid leukemia Molt 4B cells to epigallocatechin gallate (EGCG) and epigallocatechin (EGC) led to both growth inhibition and the induction of programmed cell death (apoptosis) in a concentration- and time-dependent manner [6]. Green tea extract and EGCG have also been demonstrated to inhibit growth and induce apoptosis of human stomach cancer KATO III cells [7]. Another study has found that tea polyphenols (EGCG and EGC) can inhibit growth and induce apoptosis in human cancer cell lines such as lung tumor cell lines H661 and H1299, with estimated IC50 values of 22 µM, while the lung cancer cell line, H441, and colon cancer cell line, HT-29, have weak responses to these polyphenols, with IC50 values 2- to 3-fold higher [8]. Further work has indicated that tea polyphenol-induced apoptosis and the growth inhibitory activities may be mediated by hydrogen peroxide generated by polyphenols [8]. EGCG has also been shown to inhibit telomerase in MCF-7 breast cancer cells leading to suppression of cell viability and induction of apoptosis [9, 10].

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OTHER BENEFICIAL EFFECTS Besides the effects mentioned above, a series of other effects of green tea have also been reported, including antidiabetes properties, anti-inflammatory [11], anti-arthritic [12, 13], antibacteria, anti-viral [14, 15] and neuroprotective effects [16]. Due to the limitation of space, these effects will not be discussed in detail here.

POTENTIAL MECHANISMS PROPOSED To address those effects that have been observed in vivo and in vitro, a number of mechanisms pertaining to how EGCG mediates its cancer prevention properties have been proposed. These include the direct causes and the signal pathways that may be involved. The direct causes may include anti-oxidatant properties, telomerase inhibition, DNMT1 inhibition, and inhibition of the ubiquitin-proteasome and topoisomerase I [17]. Signal pathways involved may include mediation of growth factor-mediated pathways such as those initiated or carried out by epidermal growth factor receptor (EGFR) [18], the mitogen-activated protein (MAP) kinase-dependent proteins [19], activator protein 1 (AP-1) [20], nuclear factorB (NF-kappaB) [21], matrix metalloproteinases [5] and other potential targets.

ANTI-OXIDATION AND PRO-OXIDANT ACTIVITY Polyphenols have been shown to have anti-oxidation effects in several in vitro studies. For example, it has been demonstrated that 40 µmol/L of EGCG can inhibit the production of hydrogen peroxide in UVB-treated normal human keratinocytes (NHEK) by 66–80% [22]. This effect was correlated with inhibition of UVB-induced phosphorylation of ERK1/2, jun N-terminal kinase (JNK), and p38. Similar inhibitory activity of EGCG was observed when hydrogen peroxide was directly added to this cell culture system [22]. However, in vivo analyses of anti-oxidative effects of green tea on tumor inhibition are very limited in number. EGCG and theaflavin-3,3’-digallate (TFdiG) have also been shown to inhibit lipid peroxidation in vitro [23]. Meng Q et al [24] reported that treatment of middle-aged male Fischer 344 rats with high doses of EGCG resulted in significant decline in the concentration of hydroxy-2'-deoxyguanosine in the plasma while maintaining a better mitochondrial potential in the peripheral lymphocytes and preventing the deletion of the ND4 region from mitochondrial DNA in the liver compared with low dose treatment. EGCG has been recently shown to modulate the activity of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2), which further induces expression of glutathione S-transferase, glutathione peroxidase, glutamate cysteine ligase, hemeoxygenase-1, and other antioxidant enzymes [25]. On the other hand, polyphenols have been demonstrated to have a pro-oxidant activity. For instance, the polyphenols may generate superoxide radicals and hydrogen peroxide in cell culture systems [26]. Interestingly, this is the cause for the instability of EGCG in cell culture conditions, where its halflife is less than 2 h [27], as the radicals generated due to this activity may cause an artificial impact on cell growth. Hong et al. [27] showed that administration of 50 µmol/L of EGCG to HT29 cells in McCoy’s 5A medium results in the production of up to

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23 µmol/L of hydrogen peroxide. Moreover, adding catalase to the medium before EGCG treatment can partially or completely block apoptosis in H661 lung cancer cells and Rastransformed human bronchial cells [28, 29]. These data indicate that many of the effects observed in cell culture systems may originate from hydrogen peroxide production secondary to polyphenol administration. However, since oxygen partial pressure in vivo (<40 mmHg) is much lower than that in cell culture systems (152 mmHg), and there are many anti-oxidative enzymes present in tissues, such as superoxide dismutase and glutathione peroxidase, the probability that pro-oxidant activity of EGCG plays a role in its anticancer mechanism in vivo is thought to be rather low. To address issues related to the poor bioavailability of EGCG, peracetate protections have been introduced into its hydroxyl groups to generate a more stable form which can be converted into (–)-EGCG under cell-free conditions, and enhanced effects on proteasome inhibition, growth suppression, and apoptosis induction in the breast cancer cell line MDAMB-231 have been observed compared with cells treated with unmodified EGCG [30-32]. However, other modifications of EGCG such as methylation can decrease the occurrence of apoptosis of cancer cells [33].

RECEPTOR FOR EGCG A 67-kDa laminin receptor (67LR) has been identified as a cell surface receptor for EGCG that may allow cancer cells to respond to physiologically relevant concentrations of EGCG, thereby mediating the anticancer effect of EGCG [34]. Eukaryotic translation elongation factor 1A (eEF1A) has been further identified as a component responsible for the anticancer activity of EGCG. By using a direct genetic screen, eEF1A can be up-regulated by EGCG via 67LR, and eEF1A can induce the activation of myosin phosphatase by dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) which is a subunit of myosin phosphatase [34]. It is proposed that the activated myosin phosphatase will then dephosphorylate its substrates such as MRLC, which could induce the rearrangement of actin cytoskeleton and lead to cell growth inhibition. Evidence also shows that silencing of 67LR, eEF1A, or MYPT1 in tumor cells abolish the tumor growth inhibition effect of EGCG in vivo [34-36]. However, due to the limited number of cell lines that has been tested in this manner, whether or not this is a common mechanism that accounts for the anticancer effect of green tea awaits follow up and further investigation. And it remains unresolved whether 67LR can mediate all the effects of EGCG on cancer cells. Moreover, caution should be taken when mechanisms pertaining to anti-cancer effects are analyzed, since the concentration of EGCG administered in most of these in vitro studies are impossible to be attained in humans consuming green tea, and the instability of its major constituent, EGCG, may worsen this situation leading to artificial results. These possible mechanisms do provide potential explanations on how green tea functions in humans, however, they need to be further verified and corroborated.

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THE SIGNIFICANCE OF CANCER PREVENTION TARGETING TELOMERASE Another mechanism that has been proposed for the cancer prevention effect of EGCG is based on telomerase inhibition. Here we would like to first review some background information about telomerase.

TELOMERE ATTRITION EFFECT All of the ends of eukaryotic chromosomes are characterized by ribonucleoprotein structures referred to as telomeres. A telomeric region consists of DNA sequence and many telomere-related proteins. Some of these proteins are found exclusively in telomeres, whereas others can be found in other sites of cells [37]. The DNA sequence in human telomeres is composed of repeats of 5’-TTAGGG-3’, and they can form a structure called G-quadraplex. There is a single-strand G-rich overhang of between 50 and 300 nucleotides located at the 3’ end of telomeric DNA that can fold back onto duplex telomeric DNA to form a “T-loop” structure [38]. Telomeres have been proven to play an important role in maintaining the stability of the whole genome and protecting chromosomes from end fusions [39]. Due to the inability of polymerases to fully replicate the end of a duplex DNA (3’ end replication problem of linear chromosome), a cell would lose 50-70 base pairs of its telomere sequence with each cell division. The more times the cell divides, the shorter the telomeres become and once the telomeres have been attenuated, DNA damage signals are generated and cell senescence and apoptosis ensue [40]. To compensate for the loss of DNA sequence in each cell division, organisms have developed at least two different mechanisms to circumvent this problem, one is telomerasedependent and the other one is telomerase-independent, which is referred to as alternative lengthening of telomeres (ALT). Telomerase is an enzyme that can add telomeric (TTAGGG)n hexamer nucleotide repeats to the 5’ ends of telomeric DNA to maintain the necessary telomere length. This important enzyme has three components: telomerase reverse transcriptase (TERT), the RNA component of telomerase (TERC), and the TERC-binding protein dyskerin [41, 42]. TERT is the catalytic component of this enzyme, it has reverse transcription activity, and its expression is tightly regulated. TERC functions as the template for TERT. Although it has been recently shown that TERC expression also contributes its share to regulate telomerase activity [43, 44], the activity of telomerase is mainly regulated by the expression of TERT due to the tighter regulation of its transcription. Several detailed mechanisms have been proposed pertaining to how telomerase inhibition affects cellular functions. One theory is that once telomeres have been attenuated to a certain length, the structure of telomeres will be changed, and a DNA damage signal will be generated [40]. Cell lines will experience senescence thereafter, and research shows that significant shortening of just one telomere can induce senescence of cells, while another theory is based on telomerase’s extra-telomere effects [45, 46].

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EXTRA-TELOMERE EFFECT Besides the classic role of maintaining the minimal length of telomeres, telomerase has been proposed to have other effects such as promoting the transformation of cells. To verify whether telomerase has an extra-telomere effect, Stewart et al [45, 46] introduced telomerase into the immortal ALT cell line, GM847, which is a telomerase-negative cell line. They found that this imparts a tumorigenic phenotype to this cell line, while expression of oncogenic HRas cannot lead to transformation which further indicates the potential central role of hTERT in tumorigenesis. Additional investigation consisted of expressing a mutated telomerase that cannot exert its telomere-maintaining role in this cell line which also resulted in transformation, thus providing direct evidence of the extra-telomeric effect of telomerase [45, 46]. This effect can also impact many genes involved in proliferation, and the anticancer effects via inhibition of telomerase can in some cases precede telomere shortening [47, 48]. Knocking out the expression of hTERT accompanies growth inhibition of MDA-MB-157 human mammary cancer cells, human ovarian cancer SKOV-3 cells, and human embryonic kidney (HEK 293) cells [49]. p53 and p21 have been shown to involved in this process [49].

DIFFERENT EXPRESSION LEVEL OF TELOMERASE IN SOMATIC AND CANCER CELLS Telomerase is expressed in embryonic cells and adult male germline cells [50], but exhibits barely detectable telomerase activity in normal somatic cells with the exception of proliferating cells in tissues undergoing renewal [50-55]. In normal somatic cells, telomeric DNA sequence will shorten after each cell division until telomere length reaches a critically short threshold, and cell senescence will follow in response to the signal generated in the cascade. In contrast to normal cells, telomerase activity has been detected in the early stages of most common cancers and the higher the activity of telomerase, the poorer the prognosis of cancer [56]. Ablation of telomerase expression leads to telomeric attrition and growth inhibition of neoplastic cells [57, 58]. About 90% of cancer cells have been shown to have elevated telomerase activity and these cells have no net loss of average telomere length [59]. Normal human cells are not able to undergo neoplastic transformation in the absence of telomerase activity [60] indicating that compounds that suppress telomerase have significant potential in cancer prevention. Collectively, these lines of evidence have led many to the opinion that novel approaches targeted to telomerase would be an excellent approach for cancer prevention or therapy. Owing to its relative universality compared with other targets for cancer therapy as well as the dependence of cancer cells on telomerase and its specificity for cancer cells, telomerase has been considered as a promising target for treatment of cancer. This area of research has been exponentially expanding in recent years [61-63]. Therapies targeting telomeres have also been developed in a number of laboratories. However, considering that many population doublings may be required for cancer cells to exhaust their telomeres through telomerase inhibition, the opportunities that drug-resistant cancer variants may be generated are high, while telomerase’s extra-telomeric effect has also shed a new light on strategies that targeted telomerase inhibition in chemoprevention. As a

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pleiotropic molecule, EGCG can also function on multi-targets in cancer cell lines, thus making it a promising drug against cancer.

TELOMERASE INHIBITION BY GREEN TEA Green tea had been shown to have growth inhibition effects on all kinds of tumors and cancer cells and several mechanisms have been proposed to address how green tea works in this manner as discussed above. Here we would like to focus on the telomerase inhibition effect of green tea. It has been shown that 1 µmol EGCG can induce 50% inhibition (IC50) of telomerase in a cell free system, and that the telomerase inhibition effect of EGCG in U937 and HT29 cells can occur by treating these cells with 15 µmol EGCG for 24 h [64]. Prolonged passage of these two cell lines in the presence of nontoxic concentrations could reduce telomere length and induce the cell lines to enter into crisis and senescence [64]. The synthesized compounds MST-312, MST-295, and MST-199, based on the structure of EGCG, have been shown to cause human monoblastoid leukemia U937 cells to undergo progressive telomere shortening and eventual reduction of growth rate accompanied by induction of the senescence-associated β-galactosidase activity with a nontoxic dose. The telomere shortening effect of MST-312 is strongest, being effective at only 1–2 µmol, which was 15- to 20-fold lower than that of EGCG in this system [65]. EGCG and other selected polyphenols have been shown to undergo structural rearrangements at physiologically permissible conditions that result in remarkably increased telomerase inhibition. For instance, briefly incubating EGCG in neutral or slightly alkaline medium or human or mouse plasma (pH 6.8) at 37° can lead to rapid chemical degradation of the parent EGCG compound which is accompanied by a dramatic enhancement in telomerase inhibition ( 20-fold), and the same effect is observed when other major plant polyphenols are administered to incubate with medium [66]. It is interesting that 5 µmol EGCG can induce marked shortening of telomeres in telomerase-positive U937 leukemia cells and eventually lead to cellular senescence while it has no effect on the telomerase-negative Saos-2 osteosarcoma cells or normal human foreskin fibroblasts and two immortal cell lines also with negative- and positive- telomerase activity even at 10 µmol in long-term treatment [66]. Studies from animal models have also shown the pivotal position of telomerase inhibition by EGCG in cancer prevention. Nude mice bearing telomerase-dependent xenograft tumors responded to prolonged oral administration of EGCG, while it has no effect on those bearing telomerase-independent xenograft tumors cloned from a single human cancer progeny [67]. This serves as an encouraging example that EGCG and likely other structurally-related dietary polyphenols may act as prodrug-like molecules that undergo structural changes and further lead to inhibition of telomerase [67]. EGCG has also been shown to inhibit telomerase and induce apoptosis in drug-resistant lung cancer cells [68]

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POTENTIAL MECHANISMS THAT MEDIATE TELOMERASE INHIBITION BY GREEN TEA Telomerase can be regulated at different levels through multiple pathways. This includes regulation at both the transcription level and post-transcription level. Studies show that telomerase activity requires molecular chaperones for appropriate assembly of the holoenzyme [69, 70]. Telomerase activity is also regulated positively by phosphorylation of hTERT by protein kinase C and negatively by dephosphorylation by protein phosphatase 2A [71, 72]. It has also been reported that telomerase can be regulated by its translocation from the cytoplasm to the nucleus [73-75], and alternate splicing also plays a role in the regulation process, where the hTERT α splice variant has been recognized as a dominant negative inhibitor of telomerase activity [76]. Although expression level of hTERC has been indicated to be also tightly regulated, previous research showed that copy number of the TERT and TERC telomerase subunit genes were increased in cancer cells, and the growth advantage of the cells with more copies of TERT and TERC have been indicated [77]. However, the expression level of hTERT is still a major rate-limiting factor that regulates telomerase in comparison with other factors. The hTERT gene is located at human chromosome band 5p15.33. There are several transcriptional factor binding elements in the promoter region of hTERT, such as two canonical E-box (CACGTG). The expression of hTERT has been shown to be tightly regulated by various cellular factors, which can be divided into positive regulators and negative regulators. Positive transcriptional factors include c-Myc [78], negative regulators include the c-Myc antagonist, Mad [79], while some transcriptional factors are still controversial such as E2F1 [80]. Several CpG islands have also been found in its promoter region which suggests the potential epigenetic regulation mechanism involved in this process [81].

POTENTIAL MECHANISM BEING PROPOSED Covalent Modification of Telomerase EGCG has been suggested to decompose to form a galloyl radical in cell-free system at neutral pH, and the galloyl radical can covalently modify telomerase, thereby inhibiting its activity [64], The concentration of EGCG required for this effect varies from high nanomolar to low micromolar levels [64], and this is connected with the pro-oxidative activity of EGCG which has already been discussed above. However, whether this mechanism functions in vivo remains to be investigated.

Epigenetic Regulation of Telomerase Expression The activity of telomerase has been shown to be regulated at the transcription level in cancer cells [9, 10], and epigenetic regulation has been indicated as an important mechanism controling gene expression in the transcriptional level. These mechanisms of epigenetic

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regulation include methylation of CpG islands, histone modification and noncoding RNA. Epigenetic regulation of transcription of hTERT has been documemted. Increasingly, evidence has been put forward indicating that epigenetic mechanisms are also involved in the interaction between green tea treatment and the expression of hTERT [10].

Methylation Mechanism The methylation mechanism involves the methylation of CpG islands such that methylation-sensitive transcription factors (methyl CpG binding proteins, MeCPs) may bind, leading to transcriptional repression. Previous work also revealed that hypermethylation of the hTERT CpG island accompanies its abnormal expression in cancer cells [82]. A DNA demethylating agent, 5-aza-2-deoxycytidine (5-AZC), has been shown to induce demethylation of the hTERT promoter and expression of the hTERT transcript in normal and transformed cells [81, 83], while CTCF has been shown to bind to the demethylated region and decrease the expression of hTERT [84]. Zinn et al [85] reported that using methylationspecific PCR and bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells, all cancer cell lines retain alleles with little or no methylation around the transcription start site despite being densely methylated in a region 600 bp upstream of the transcription start site. Both active chromatin and inactive chromatin marks have been found in the promoter region of hTERT, indicating that active chromatin marks are associated with unmethylated DNA while inactive chromatin marks are associated with methylated DNA in the region around the transcription start site [85].

EGCG

Other epigenetic mechanisms? hypomethylation

CpG island

hTERT

DNMT

E2F1 hypoacetylation at H3 lys9 E2F1 Histon

CpG island hTERT promoter

Figure 3. Epigenetic mechanism by which the expression of hTERT is regulated in MCF-7 cells. EGCG can bind to DNMT1 and disrupt its function, thereby causing hypomethylation of the hTERT promoter. E2F1, which can only bind if its recognition sequence is unmethylated, would then bind to the promoter, and transcription of hTERT would be suppressed. EGCG can also cause hypoacetylation of the hTERT promoter at H3 lys9, which may contribute to silencing of hTERT. Other epigenetic mechanisms remain to be investigated.

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EGCG can bind to DNMT1 [86], which can disturb its function of methylation, thereby decreasing the methylation level of the genome, including the promoter region of hTERT [10] (Figure 3). Investigation from our laboratory indicated that EGCG treatment can lead to hypomethylation of the hTERT promoter region in MCF7 breast cancer cells [10]. An increase of binding of E2F1 to the hTERT promoter has also been identified which may suppress the transcription of the hTERT gene, and hypoacetylation at H3 lys9 has been observed in the hTERT promoter region in response to EGCG which is connected with inactivation of gene expression [10]. While methylation of CpG islands can lead to transcriptional repression directly, it can also regulate chromatin structure by interacting with other proteins [87], and modification of nucleosomal histones can also play a role in the regulation of hTERT expression.

Histone Modification Mechanism Histone modification has been indicated as another epigenetic mechanism that can regulate gene expression. Adjustment of the higher structure of chromatin accompanies activation or inactivation of genes. For example, acetyl-H3K9 and dimethyl-H3K4 accompanies gene expression, while trimethyl-H3K9 and trimethyl-H3K27 accompanies gene inactivation [85]. As mentioned above, hypoacetylation at H3 lys9 occurs in the hTERT promoter when EGCG is administrated to MCF-7 cells [10]. Further epigenetic analysis of cancer cells during treatment of EGCG is currently ongoing in our laboratory. These studies will help to elucidate how dietary green tea imparts its chemopreventive and anticancer effects and may lead to novel approaches to cancer prevention through control of not only of dietary factors, but also through epigenetic processes.

Relationship between Telomerase Inhibition and other Mechanisms Several mechanisms have been proposed to explain the growth inhibition effect and induction of apoptosis on cancer cells by green tea, however, each of these mechanisms has their own system. For those different systems, investigators’ methods may also favor those responses that they were ready for while omitting other possible mechanisms. Which response is the primary response of cancer cells to polyphenols, and which is the subsequent event that follows the first response has not yet been resolved. It is also possible that those mechanisms may work together to cause arrest and apoptosis of cancer cells.

CONCLUSION In comparision to other treatments targeting telomerase, investigations have shown us the merits of green tea, such as doing no harm to normal cells, and its constituents have a variety of beneficial effects which enhance the health of individuals. However, over consumption of

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green tea may cause potential harmful effects in humans [88], such as nervousness, sleep disorders, vomiting, headaches, epigastric pain, tachycardia caused by its caffeine content, accumulation of too much aluminum which may cause neurodegenerative disorders [89] and inhibition of iron bioavailability [90]. Thus, the design of drugs that possess the beneficial effects without any harmful effects is urgent and studies on the functional mechanisms of green tea are showing promise in this regard. A number of mechanisms have been suggested based on previous studies of green tea and its major constituent, EGCG, and those results provide useful insightful into understanding how green tea may function to prevent cancer. However, current knowledge on the detailed mechanisms through which green tea exerts its anticancer effect is still fragmentary. Results from different studies are hard to compare due to different systems that have been used. Some common problems with these studies is that often the concentrations of EGCG that have been used are too high, and another issue is the limited bioavailability of EGCG since it is reported that EGCG degrades quickly in media. It is not fully clear whether the responses observed in cell culture systems would also occur in animals or humans following oral administration [91]. Corresponding detailed metabolism and kinetics analysis of green tea and EGCG in each cell culture systems as well as in vivo studies are therefore warranted, which may help us to understand the mechanisms more clearly, and the difference between cell culture systems and what occurs in vivo. For example, artificial results may be produced since hydrogen peroxide may be generated when EGCG is administrated to cell culture medium; therefore future experiments need to incorporate this information to exclude responses potentially caused by hydrogen peroxide. Another topic worth our attention is that all the mechanisms that have been proposed in cell culture systems should be reproduced in animal models and also eventually in humans. Animal models and analysis of in vivo studies cannot be omitted [92], since ultimately those responses observed in humans after consuming green tea are most relevant to the cancer prevention effect of this beverage. Findings from animal models should be analyzed with caution when incorporated into information from human studies due to the discrepancies that exist in metabolism and signaling networks that can occur between humans and other animals. Due to the huge difference between monolayer cultures of human tumor cell lines or human xenograft tumors and tumors that exist in patients, new experimental models urgently await to be developed [93]. In this regard, complex three-dimensional culture systems have attracted much attention of the scientific community [94-96]. Moreover, for different cell lines that have been tested, their genetic background should be taken into account when we analyze the data in each treatment. Also, different cell lines have their own metabolic patterns. Telomerase inhibition by green tea has been regarded as an important mechanism through which the anticancer effect of green tea works. However, as we had discussed above, the logic linkage between telomerase inhibition and other mechanisms is still controversial, which necessitates further investigation. To clarify the role that telomerase inhibition conferred by green tea plays in chemoprevention, more cell lines need to be tested, and strategies that can differentiate between the telomerase inhibition effect and other mechanisms should be adopted. Considering that telomerase is a complex protein with several components, additional potential mechanisms that target other components of telomerase besides hTERT also warrant further investigation.

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ACKNOWLEDGEMENT This work was supported by a grant from the National Cancer Institute (R01 CA129415).

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[92] Yang C, Lambert J, Hou Z, Ju J, Lu G, Hao X: Molecular targets for the cancer preventive activity of tea polyphenols. Mol Carcinog 2006, 45(6):431-435. [93] Burnworth B, Arendt S, Muffler S, Steinkraus V, Brocker EB, Birek C, Hartschuh W, Jauch A, Boukamp P: The multi-step process of human skin carcinogenesis: a role for p53, cyclin D1, hTERT, p16, and TSP-1. Eur J Cell Biol 2007, 86(11-12):763-780. [94] Stark H, Baur M, Breitkreutz D, Mirancea N, Fusenig N: Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J Invest Dermatol 1999, 112(5):681-691. [95] Margulis A, Andriani F, Fusenig N, Hashimoto K, Hanakawa Y, Garlick J: Abrogation of E-cadherin-mediated adhesion induces tumor cell invasion in human skin-like organotypic culture. J Invest Dermatol 2003, 121(5):1182-1190. [96] Kunz-Schughart L, Freyer J, Hofstaedter F, Ebner R: The use of 3-D cultures for highthroughput screening: the multicellular spheroid model. J Biomol Screen 2004, 9(4):273-285.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 19

GREEN TEA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A CASE-CONTROL STUDY IN JAPAN Fumi Hirayama and Andy H. Lee School of Public Health and National Drug Research Institute, Curtin University of Technology, Perth, WA, Australia

ABSTRACT Background: Chronic obstructive pulmonary disease (COPD) is a common cause of morbidity and leading cause of death in the world. Cigarette smoking has been established as the principal risk factor for COPD. While 95% of COPD patients are, or have been, cigarette smokers, only 20% of smokers develop COPD. Therefore, other factors may protect against or contribute to the development of the disease. Objective: To investigate whether green tea intake can reduce the risk of COPD. Study design: Case-control study conducted in Aichi, Gifu and Kyoto during 2006. Subjects: A total of 278 eligible patients (244 men and 34 women; mean age 66.5 (SD 6.7) years), with COPD diagnosed by respiratory physicians as the primary functionally limiting illness within the past four years, were referred from the outpatient departments of six hospitals in central Japan. During the same period, 335 age-matched community-dwelling controls (267 men and 68 women; mean age 65.3 (SD 5.5) years) were recruited from the same catchment areas as the cases. Methods: Interviews were conducted face-to-face to collect information on demographic characteristics and habitual green tea consumption using a structured questionnaire. The reference recall period was set at 5 years before diagnosis for cases or 5 years before interview for controls. Tea drinkers were defined as persons who drunk both Sencha, Bancha, Hojicha and Genmaicha types of green tea once a week or more often. The effect of green tea intake on the COPD risk was assessed by multivariate logistic regression analysis. Results: The prevalence of regular green tea consumption was significantly higher (p < 0.01) among controls (n = 64, 19.3%) than cases (n = 27, 9.7%). Among drinkers, lifelong exposure (years of drinking) was similar between the two groups (p = 0.70), and about half of them drank one to three cups of green tea daily. The risk of COPD appeared

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Fumi Hirayama and Andy H. Lee to decrease with green tea drinking. The adjusted odds ratio was 0.45 (95% confidence interval 0.25-0.81) for tea drinkers relative to non-drinkers after accounting for confounding factors including age, body mass index, gender, marital status, residential location, education level, retirement status, smoking status and alcohol consumption. Conclusion: The preliminary evidence suggests that green tea intake may be protective against COPD for Japanese adults. More research is required to confirm the observed finding and to understand the biological mechanism underlying the disease prevention role of green tea.

INTRODUCTION Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disease (COPD) is the fifth most common cause of morbidity and sixth leading cause of death in the world (Murray & Lopez 1997). It is likely to be the third most frequent cause of death by 2020, just behind coronary and cerebrovascular diseases (Murray & Lopez 1997). COPD is characterized by airflow limitation that is not fully reversible, with chronic cough, sputum production, and breathlessness on exertion being common symptoms. The disease results from the interaction of host factors and environmental exposures (McKenzie et al. 2003; Celli & MacNee 2004; Pauwels & Rabe 2004). Host factors include genetic predisposition and airway hyper-responsiveness, while environmental exposures include polluted air, inhaled particles and gases. Cigarette smoking has been established as the principal cause of COPD. Although 95% of COPD patients are, or have been, cigarette smokers, only 20% of smokers develop COPD (Madison & Irwin 1998). Therefore, other factors may protect against or contribute to the development of the disease. Because of the high burden and societal cost associated with COPD, new methods of prevention are important. It is possible to reduce the incidence of COPD through an appropriate diet (Sridhar 1995).

Green Tea Tea is the second most common beverage in the world, just behind water (Peters, Poole & Arab 2001). Of all tea production and consumption worldwide, 78% is black tea, 20% is green tea and 2% is oolong tea (Jankun et al. 1997). Japanese consumed 138,000 tonnes of tea per year in 2006, of which 73% are green tea (Iruma City Museum 2008). The common method of preparation is to brew dry tea leaves in a teapot using hot water without milk and sugar (Ministry of Agriculture Forestry and Fisheries 2005). Teas are made from a leaf extract of the plant Camellia sinensis and classified into three main types, depending on the manufacturing process. Green tea is non-fermented, oolong tea is semi-fermented, while black tea is fermented (Regional Agricultural Administration Office 2007). The common kinds of green tea in Japan are Sencha, Bancha, Hojicha, and Genmaicha. Sencha is harvested in early spring and makes up 75% of all tea production in Japan. It is initially steamed for 15 to 45 seconds, knead, and then roasted (Regional Agricultural Administration Office 2007). Bancha has lower quality and more coarse than Sencha. It is harvested from the second flush

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of Sencha leaf between summer and autumn, and is made from the same process as Sencha. Both Hojicha and Genmaicha mix Sencha and Bancha tea leaves and are roasted in high temperature, but Genmaicha also combines with roasted brown rice. Green tea is rich in catechins, such as (-)-epigallocatechin-3-gallate (EGCG) and (-)epicatechin gallate (ECG), a subclass of flavonoids (Cabrera, Gimenez & Lopez 2003). The level of catechins in green tea, which ranges from 51.5 to 84.3 mg/g, is higher than those of black tea (5.6-47.5 mg/g) and fruit tea (8.5-13.9 mg/g) (Khokhar & Magnusdottir 2002). It has been noted that catechins, especially EGCG, may prevent the formation of a mutated cell (Liang et al. 2007).

Green Tea and Disease Prevention Green tea has been shown to reduce the risk of several diseases (Crespy & Williamson 2004) including cognitive impairment (Kuriyama et al. 2006), stroke (Fraser, Mok & Lee 2007), prostate cancer (Jian et al. 2004), ovarian cancer (Zhang et al. 2004), lung cancer (Liang et al. 2007), and can decrease mortality of cardiovascular diseases (Kuriyama et al. 2006). A review of 78 relevant articles on lung cancer concluded that high level of green tea intake over a long period may be protective against tobacco carcinogens (Liang et al. 2007). However, only two studies examined the association between tea consumption and COPD. A Turkish case-control study of 40 male smokers with COPD (mean tea intake 7 glass/day) and 36 male non-smokers without COPD (mean tea intake 16 glass/day) reported that black tea drinking was protective against COPD among smokers, with odds ratio (OR) 0.90, 95% confidence interval (CI) 0.82-0.98 (p < 0.001). The study recommended 6 to 10 cups (960-1600 ml) of tea drinking per day as a useful dietary habit (Celik & Topcu 2006). On the other hand, a cross-sectional study of 13651 adults in the Netherlands showed no association between tea consumption and the prevalence of COPD symptoms (Tabak et al. 2001), though the risk of COPD was not evaluated. More research is needed to ascertain the effect of tea in view of the limited studies in the literature. Therefore, this study aims to investigate whether green tea intake can reduce the risk of COPD for Japanese adults.

METHODS Study Design and Subjects A case-control study was conducted in central Japan in 2006. Three hundred COPD patients diagnosed by respiratory physicians were recruited from the outpatient departments of six hospitals in Aichi, Gifu and Kyoto. Patients were included in this case-control study provided that they (i) were aged between 50 and 75 years; and (ii) had COPD as the primary functionally limiting illness which was diagnosed within the past four years. Subjects were excluded if they had a recent stroke, dementia or other health conditions that prohibited them from answering the questions. Twenty-two eligible patients were subsequently excluded due to missing or incomplete demographic and lifestyle details. The remaining 278 patients (244 men and 34 women) were available for analysis. No statistically significant differences were

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found between the included and excluded cases in terms of clinical and other variables. Permission to recruit patients and access to medical records were granted by the participating hospitals in Japan. During the same period, 400 community-dwelling adults, aged between 50 and 75 years, were recruited from the same catchment areas as the cases. This convenience sample of subjects was interviewed about their demographic and lifestyle habits when they attended community centres or undertook health checks at hospitals. The same exclusion criteria as cases were adopted, resulting in 335 eligible controls (267 men and 68 women). The distribution of controls was: orthopaedic and physiotherapy clinics (n = 136, 40.6%), medical check centres (n = 119, 35.5%), community centres (n = 46, 13.7%), and other places such as universities and shopping malls (n = 34, 10.1%). Approval of the study protocol was obtained from the Human Research Ethics Committee of Curtin University of Technology (approval number HR 90/2005).

Questionnaire and Interview A structured questionnaire was administered face-to-face by the first author to collect information on demographic and lifestyle characteristics. Questions on habitual tea consumption were taken from the Japan Public Health Center-based prospective study on cancer and cardiovascular disease (Ishihara et al. 2003a). Validity and reproducibility of these questions had been established for Japanese adults (Ishihara et al. 2003b). In our study, a green tea drinker was defined as a person who drank Sencha, Bancha, Hojicha and Genmaicha kinds of green tea once per week or more often. Participants were first classified as either ‘ever’ or ‘never’ green tea drinkers. The ever drinkers were then asked about their frequency and duration of drinking, and whether they had changed their habitual tea consumption pattern five years ago. Demographic and lifestyle information, including age, gender, weight (kg), height (m), marital status (single, divorced or separated; married), education level (high school or below; college or university), residential location (urban; rural), retirement status (working; retired), together with cigarette smoking (non-smoker; current smoker) and alcohol consumption (non-drinker; drinker) in daily life, was obtained from each participant. The reference recall period was set at 5 years before diagnosis for cases or 5 years before interview for controls. For the cases, each interview was conducted in the presence of their next-of-kin to minimize recall error, and appointment was made via their respiratory physician. The purpose of the study was explained to each participant before obtaining their formal written consent. Confidentiality of the information provided, and the right to withdraw without prejudice, were ensured and maintained throughout the study. All interviews, averaging 30 minutes in duration, took place in the hospital outpatient departments for cases and their place of recruitment for controls.

Statistical Analysis Descriptive statistics were first applied to summarise participant characteristics. After comparing the tea consumption pattern between cases and controls using chi-square and t tests, multivariate logistic regression analysis was performed to assess the effect of green tea

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intake on the COPD risk. Besides green tea drinking, independent variables considered in the regression model were age, gender, body mass index (BMI = weight/height2), marital status, education level, residential location, retirement status, smoking status and alcohol consumption; definition of the categorical variables were given in the previous section. Data entry, screening and statistical analyses were undertaken using the SPSS for Windows package version 13.

RESULTS Table 1 presents the demographic profile of the participants. The majority of subjects were male with mean age similar between case and control groups. The mean BMI was significantly lower for case than control subjects (p < 0.001). Most of the participants were married, had high school or below education, resided in the rural area and retired. A substantial proportion of cases (22.5%) continued to smoke after their diagnosis of COPD while over half of the participants consumed alcohol on at least a monthly basis. Table 1. Demographic profile of case and control groups Characteristic Mean age (SD) years Mean BMI (SD) kg/m2 Gender Male Female Marital status Single, divorced or separated Married Education level High school or below College or university Residential location Rural Urban Retirement status Retired Working Smoking status Non-smoker Current smoker Alcohol consumption Non-drinker Drinker

Cases (n = 278) 66.5 (6.7) 21.9 (3.6)

Controls (n = 335 ) 65.3 (5.5) 23.4 (2.8)

244 (87.8%) 34 (12.2%)

267 (79.7%) 68 (20.3%)

43 (15.6%) 233 (84.4%)

53 (15.9%) 281 (84.1%)

221 (80.1%) 55 (19.9%)

211 (63.6%) 121 (36.4%))

224 (82.4%) 48 (17.6%)

245 (73.8%) 87 (26.2%)

153 (55.4%) 123 (44.6%)

142 (42.6%) 191 (57.4%)

214 (77.5%) 62 (22.5%)

271 (81.1%) 63 (18.9%)

120 (43.2%) 158 (56.8%)

115 (34.3%) 220 (65.7%)

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The prevalence of regular green tea consumption was significantly higher (p < 0.01) among controls (n = 64, 19.3%) than cases (n = 27, 9.7%). As shown in Table 2, about half of the tea drinkers drank one to three cups of green tea daily five years ago. Overall, the frequency and duration of drinking were similar between the two groups of drinkers; most of whom did not change their consumption pattern when compared with five years ago. Results from the logistic regression analysis are given in Table 3. The risk of COPD appeared to decrease with green tea drinking (p = 0.007). The adjusted OR was 0.45 (95% CI 0.25-0.81) for tea drinkers relative to non-drinkers, after accounting for significant confounding factors such as gender, BMI, residential location, education level, retirement status, smoking status and alcohol consumption. Table 2. Consumption pattern of green tea drinkers

Sencha/Bancha Drink weekly 1-3 cups per day > 3 cups per day Hojicha/Genmaicha Drink weekly 1-3 cups per day > 3 cups per day Years of drinking Compared with drinking pattern 5 years ago2 No change Increasing tea intake Decreasing tea intake 1 2

Cases (n = 27)

Controls (n = 64)

6 (22.2%) 13 (48.1%) 8 (29.6%)

7 (10.9%) 38 (59.4%) 19 (29.7%)

6 (22.2%) 15 (55.6%) 6 (22.2%) 49.3 (SD 16.4)

19 (29.7%) 32 (50.0%) 13 (20.3%) 54.5 (SD 13.9)

17 (68.0%) 6 (24.0%) 2 (8.0%)

49 (79.0%) 9 (14.5%) 4 (6.5%)

p1 0.346

0.776

0.704 0.582

cases versus controls. Missing data present.

DISCUSSION In this case-control study of community-dwelling Japanese adults, green tea intake was associated with a decreased risk of COPD after accounting for significant confounders. An extensive literature search found no published report that investigated the relationship between green tea consumption and the development of COPD. The average duration of tea drinking was about 50 years among drinkers. Therefore, our preliminary finding supported a review of the protective role of green tea against lung cancer, which suggested that a long exposure to green tea is needed in order to reduce the damage caused by tobacco carcinogens (Liang et al. 2007). In addition to green tea drinking, several variables were found to significantly influence the risk of COPD. Firstly, the effect of BMI was consistent with a 10-year longitudinal study of 458 males and 192 females in the USA, which showed that a lower BMI could increase the risk of COPD for men (Harik-Khan, Fleg & Wise 2002). Secondly, women appeared to be more likely to develop COPD than men with adjusted OR 2.72. However, recent reviews

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indicated little difference in COPD prevalence between genders (Global Initiative for Chronic Obstructive Lung Disease 2007; World Health Organization 2007), although two studies had reported that females were more susceptible to severe COPD development than males (Silverman et al. 2000; Xu et al. 1994). Thirdly, living in the rural area could elevate the risk of COPD (OR 1.70). A similar observation was reported by a study conducted in south China involving 3286 adults (Liu et al. 2007). Fourthly, cigarette smoking has been established as the principal cause of COPD. It is therefore not surprising that smokers in this study were subjected to a much higher risk of COPD than non-smokers. In terms of alcohol consumption, however, regular drinkers seemed to have half the likelihood of developing COPD when compared with non-drinkers. Although heavy alcohol consumption is known to have deleterious effects on the lungs, recent and lifetime wine intake may improve pulmonary function (Schünemann et al. 2002). Similarly, a study of 15294 American adults observed altered pulmonary function and a reduced risk of lung restriction at modest levels of alcohol intake (Sisson et al. 2005). Finally, retirement status and education level were found to affect the COPD risk, despite such apparent association had not been reported in the literature. The major strength of the present study was that information on green tea consumption was obtained using a short validated questionnaire from the Japan Public Health Center-based prospective study on cancer and cardiovascular disease (Ishihara et al. 2003a). A reference recall period of five years was adopted to minimise recall error and to avoid possible changes in tea exposure, because the diagnosis of the disease was confirmed for all patients within the past four years. Moreover, data collection was conducted solely by the first author in order to minimise inter-interviewer bias. Several limitations should be considered in conjunction with the findings. The assessment of green tea intake was based on self-report, which might lead to some misclassification of drinking status when estimating the effect of the beverage. Although multivariate logistic regression analysis was used to adjust for the effects of demographic and lifestyle variables, other potential confounding factors such as habitual physical activity, dietary intake and comorbidities were not taken into account. In particular, data were not collected on the consumption of foods and other beverages. The lack of detailed and specific information on quantity of green tea intake posed another limitation on the conclusion drawn with respect to dose-response relationship. Finally, the evidence concerning the protective effect of green tea may be regarded as preliminary, in view of the moderate sample size especially the relatively small number of female participants recruited into the study.

CONCLUSION Although this case-control study has suggested a protective role of green tea against COPD for Japanese adults, more research is required to ascertain the exact effect of green tea intake and whether the observed finding can be generalized to other populations. In addition to large scale case-control studies, long-term prospective cohort studies collecting detailed tea consumption information would provide important epidemiological evidence on both morbidity and mortality. Experimental and animal studies will also complement and provide additional supporting evidence to understand the biological mechanism underlying the

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protective role of green tea. The increasing knowledge would be beneficial towards the prevention of this disease.

ACKNOWLEDGEMENTS The authors are grateful to the Japanese respiratory physicians for recruitment of COPD patients, and to all participants who volunteered their time for the project.

Table 3. Risk of COPD from logistic regression analysis

Green tea drinking: Non-drinker Drinker Age BMI Gender: Male Female Marital status: Single, divorced or separated Married Education level: High school or below College or university Residential location: Urban Rural Retirement status: Working Retired Smoking status: Non-smoker Current smoker Alcohol consumption: Non-drinker Drinker

Adjusted OR

95% CI

p

1.00 0.45 1.01 0.87

0.25 – 0.81 0.97 – 1.05 0.82 – 0.93

0.007 0.663 <0.001

1.00 2.72

1.07 – 6.91

0.035

1.00 0.87

0.47 – 1.62

0.657

1.00 0.59

0.37 – 0.93

0.024

1.00 1.70

1.04 – 2.80

0.036

1.00 0.62

0.40 – 0.97

0.036

1.00 75.31

23.82 – 238.05

<0.001

1.00 0.49

0.32 – 0.76

0.001

REFERENCES Cabrera, C, Gimenez, R & Lopez, MC 2003, 'Determination of tea components with antioxidant activity', J Agric Food Chem, vol. 51, no. 15, pp. 4427-35. Celik, F & Topcu, F 2006, 'Nutritional risk factors for the development of chronic obstructive pulmonary disease (COPD) in male smokers', Clin Nutr, vol. 25, no. 6, pp. 955-61.

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Celli, BR & MacNee, W 2004, 'Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper', Eur Respir J, vol. 23, no. 6, pp. 93246. Crespy, V & Williamson, G 2004, 'A review of the health effects of green tea catechins in in vivo animal models', J Nutr, vol. 134, pp. 3431S-40S. Fraser, ML, Mok, GS & Lee, AH 2007, 'Green tea and stroke prevention: emerging evidence', Complement Ther Med, vol. 15, no. 1, pp. 46-53. Global Initiative for Chronic Obstructive Lung Disease 2007, Global Strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease National heart lung and blood institute and World Health Organization, 2007. Retrieved May 4, from http://www.goldcopd.dk/index_uk.htm Harik-Khan, RI, Fleg, JL & Wise, RA 2002, 'Body mass index and the risk of COPD', Chest, vol. 121, no. 2, pp. 370-6. Iruma City Museum 2008, Tea museum. Retrieved June 20, from http://www.alit.city. iruma.saitama.jp/07tea-museum/index.html Ishihara, J, Sobue, T, Yamamoto, S, Sasaki, S & Tsugane, S 2003a, 'Demographics, lifestyles, health characteristics, and dietary intake among dietary supplement users in Japan', Int J Epidemiol, vol. 32, no. 4, pp. 546-53. Ishihara, J, Soube, T, Yamamoto, S, Yoshimi, I, Sasaki, S, Kobayashi, M, Takahashi, K, Iitoi, Y & Akabane, S 2003b, 'Validity and reproducibility of a self-administered food frequency questionnaire in the JPHC study cohort II: study design, participant profile and results in comparison with cohort I', J Epidemiol., vol. 13, no. 1 Supple, pp. S134-47. Jankun, J, Selman, SH, Swiercz, R & Skrzypczak-Jankun, E 1997, 'Why drinking green tea could prevent cancer', Nature, vol. 387, no. 6633, p. 561. Jian, L, Xie, LP, Lee, AH & Binns, CW 2004, 'Protective effect of green tea against prostate cancer: A case-control study in southeast China', Int J Cancer, vol. 108, pp. 130-5. Khokhar, S & Magnusdottir, SG 2002, 'Total phenol, catechin, and caffeine contents of teas commonly consumed in the United kingdom', J Agric Food Chem, vol. 50, no. 3, pp. 565-70. Kuriyama, S, Shimazu, T, Ohmori, K, Kikuchi, N, Nakaya, N, Nishino, Y, Tsubono, Y & Tsuji, I 2006, 'Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study', Jama, vol. 296, no. 10, pp. 1255-65. Liang, W, Binns, CW, Jian, L & Lee, AH 2007, 'Does the consumption of green tea reduce the risk of lung cancer among smokers?' Evid Based Complement Alternat Med, vol. 4, no. 1, pp. 17-22. Liu, S, Zhou, Y, Wang, X, Wang, D, Lu, J, Zheng, J, Zhong, N & Ran, P 2007, 'Biomass fuels are the probable risk factor for chronic obstructive pulmonary disease in rural South China', Thorax, vol. 62, no. 10, pp. 889-97. Madison, JM & Irwin, RS 1998, 'Chronic obstructive pulmonary disease', Lancet, vol. 352, no. 9126, pp. 467-73. McKenzie, DK, Frith, PA, Burdon, JG & Town, GI 2003, 'The COPDX Plan: Australian and New Zealand Guidelines for the management of Chronic Obstructive Pulmonary Disease 2003', Med J Aust, vol. 178 Suppl, pp. S7-39. Ministry of Agriculture Forestry and Fisheries 2005, Green tea consumption, from http://www.maff.go.jp/soshiki/syokuhin/heya/m_report/h1702report.pdf

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Murray, CJ & Lopez, AD 1997, 'Alternative projections of mortality and disability by cause 1990-2020: Global Burden of Disease Study', Lancet, vol. 349, no. 906, pp. 1498-504. Pauwels, RA & Rabe, KF 2004, 'Burden and clinical features of chronic obstructive pulmonary disease (COPD)', Lancet, vol. 364, no. 9434, pp. 613-20. Peters, U, Poole, C & Arab, L 2001, 'Does tea affect cardiovascular disease? A metaanalysis', Am J Epidemiol, vol. 154, no. 6, pp. 495-503. Regional Agricultural Administration Office 2007, Magic of green tea, Kyoto. Retrieved June 20, from http://www.maff.go.jp/kinki/keikaku/shigen/futaba/futabafiles/futaba14.pdf Schünemann, HJ, Grant, BJ, Freudenheim, JL, Muti, P, McCann, SE, Kudalkar, D, Ram, M, Nochajski, T, Russell, M & Trevisan, M 2002, 'Evidence for a positive association between pulmonary function and wine intake in a population-based study', Sleep Breath, vol. 6, no. 4, pp. 161-73. Silverman, EK, Weiss, ST, Drazen, JM, Chapman, HA, Carey, V, Campbell, EJ, Denish, P, Silverman, RA, Celedon, JC, Reilly, JJ, Ginns, LC & Speizer, FE 2000, 'Gender-related differences in severe, early-onset chronic obstructive pulmonary disease', Am J Respir Crit Care Med, vol. 162, no. 6, pp. 2152-8. Sisson, JH, Stoner, JA, Romberger, DJ, Spurzem, JR, Wyatt, TA, Owens-Ream, J & Mannino, DM 2005, 'Alcohol intake is associated with altered pulmonary function', Alcohol, vol. 36, no. 1, pp. 19-30. Sridhar, MK 1995, 'Nutrition and lung health', BMJ, vol. 310, pp. 75-6. Tabak, C, Arts, ILJAC, Smit, HA, Heederik, D & Kromhout, D 2001, 'Chronic obstructive pulmonary disease and intake of catechins, flavonols, and flavones', Am J Respir Crit Care Med, vol. 164, pp. 61-4. Watson, L, Margetts, B, Howarth, P, Dorward, M, Thompson, R & Little, P 2002, 'The association between diet and chronic obstructive pulmonary disease in subjects selected from general practice', Eur Respir J, vol. 20, no. 2, pp. 313-8. World Health Organization 2007, Chronic obstructive pulmonary disease (COPD), 2007. Retrieved July 25, from http://www.who.int/en/ Xu, X, Weiss, ST, Rijcken, B & Schouten, JP 1994, 'Smoking, changes in smoking habits, and rate of decline in FEV1: new insight into gender differences', Eur Respir J, vol. 7, no. 6, pp. 1056-61. Zhang, M, Lee, AH, Binns, CW & Xie, X 2004, 'Green tea consumption enhances survival of epithelial ovarian cancer', Int J Cancer, vol. 112, pp. 465-9.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 20

GREEN TEA AND DIABETES Dongfeng Wang, Linge Wang and Li Zhang College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

ABSTRACT Providing that there has been a dramatic increase in the incidence of diabetes mellitus associated with long-term complications, it is critical to find a natural nutritional material aimed at reducing the prevalence of diabetes which threatens human health over the world . Currently, green tea, only next to water, is the most widely consumed beverage in the world and exerts beneficial bioabilities, such as anti-inflammative, anti-oxidative, antimutagenic, etc. With increasing interest in the health promoting properties of tea and a significant rise in scientific investigation, in vitro and animal studies provide more and more strong evidence that green tea consumption has a great prophylactic and therapeutic effect on diabetes and its complications, which is intensively associated with its mainly bioactive components, such as tea polyphenols and tea polysaccharides. In this article, we will summarize effect of green tea on diabetes, especially antidiabetic ability of tea polyphenols and polysaccharides, discuss possible mechanisms, and make perspectives and future directions in this area.

Keywords: tea polysaccharides, tea polyphenols, green tea, diabetes, mechanism

1. INTRODUCTION During the last century changes in human behavior and lifestyle have resulted in a dramatic increase in the incidence of diabetes over the world. According to a widely accepted estimation, the number of diabetic patients would reach 366 million by the year 2030 [1]. Diabetes mellitus is a disorder of metabolism caused by the relative or absolute lack of insulin and is associated with long-term complications that affect eye, heart, blood vessel, and kidney function.

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Though oral hypoglycemic agents and insulin is the mainstay of treatment of diabetes, they have prominent side effects and fail to significantly alter the course of diabetic complications. Lifestyle modifications including appropriate diet and exercise programs have been found to be greatly effective in the management of the disease. Diet therapy especially is showing a bright future in the therapy of diabetes mellitus. Green tea is a widely consumed beverage and its origins date back thousands of years in China. The legendary Chinese emperor, Shen Nung, discovered the detoxifying and healthmaintaining properties of green tea around 2700 B.C. In recent years, green tea is being widely studied for its beneficial effect in treatment and prevention of human diseases. It is considered to be anti-inflammative, anti-oxidative, anti-mutagenic, and anti-carcinogenic and can prevent cardiac disorders. What is more, it has been over 30 years since people studied the effect of green tea on diabetes. Domestic and international researches have already reported that green tea could be prophylactic and therapeutic against diabetes. Now we introduce the summary on the relationship between green tea and diabetes as follows.

2. GREEN TEA CONSUMPTION AND DIABETES Green tea, which is prepared by drying fresh tea leaves with no fermentation during processing, retains the original color of the tea leaves and contains various bioactive components, including polyphenols, polysaccharides, caffeine, amino acids, and other trace compounds such as lipidsand vitamins. Since ancient times, green tea consumption has been known to maintain and improve health. Initial research on the benefits of green tea was carried out in China and Japan because of the local customs. Curing diabetes with coarse green tea is a popular folk prescription in both China and Japan. A prospective epidemiological study done in Japan found that men and women reported drinking 6 or more cups of green tea per day had one-third less the incidence of type 2 diabetes mellitus over 5 years [2]. But what is the reason? On one hand, green tea extract was found to have an obvious hypoglycemic effect. Tsuneki H et al. reported that green tea had an effect on blood glucose levels and serum proteomic patterns in diabetic mice and promoted glucose metabolism in healthy human [3]. Many reports show the anti-hyperglycemic effect of green tea in both type 1 and type 2 diabetic animal models. This effect may be associated with the inhibitory effects on aglucosidase activity and glucose transport ability in small intestinal mucosa [4]. Jerzy Ju kiewicz et al. suggested that supplementation of the diet with green tea extract significantly reduced maltase as well as saccharase and lactase (at higher dose) activities, which may be indicative of a reduced level of absorbable glucose and, thus, may be beneficial in the amelioration of the diabetic state [5]. On the other hand, green tea was reported to increase insulin sensitivity. Taiwanese investigators demonstrated recently that green tea increased insulin sensitivity in Sprague– Dawley rats and ameliorated insulin resistance and increased glucose transporter IV content of adipocytes isolated from the epididymal fat pads [6]. Green tea was reported to regenerate pancreatic beta cells in streptozotocin (STZ)-induced diabetic rats to maintain normal insulin levels [7].

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What is more, the reduction in the risk of diabetic complications by green tea drinking has been observed in epidemiological studies. Diabetes leads to modification of collagen such as advanced glycation and cross-linking which play an important role in the pathogenesis of diabetes mellitus. Pon Velayutham et al. have investigated the effect of green tea on modification of collagen in STZ-induced diabetic rats and shown that green tea is effective in reducing the modification of tail tendon collagen in diabetic rats,which demonstrates that green tea may have a therapeutic effect in the treatment of glycation induced complications of diabetes. The same scholars also reported that green tea by ameliorating myocardial collagen characteristics may provide a therapeutic option in the treatment of cardiovascular complications of diabetes [8]. As we all know, Paraoxonase (PON1) is an antioxidant enzyme that protects lipoproteins against oxidative modification [9, 10]. Lipoprotein oxidation is believed to play an essential role in the pathogenesis of atherogenesis [11, 12]. Recently, Sibel Tasa has suggested that green tea could slow the progression of atherogenesis by reducing oxidation of lipoproteins and preserving paraoxonase activity [7]. The anti-hyperglycemic and antioxidant effects of green tea may be ascribed to this activity. In addition, diabetes-induced hyperlipidemia, oxidative stress and protein glycation impair cellular calcium and sodium homeostasis associated with abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To explore the cardioprotective mechanism of green tea in diabetes, Pon Velayutham et al. have done several investigations revealing that green tea extract indeed has a therapeutic effect and suggesting that a possible mechanism may be associated with the attenuation of the rise in [Ca2+] and [Na+] by ameliorating Ca2+-ATPase and Na+/K+-ATPase activities [13]. Above all, it can conclude that green tea consumption has a great prophylactic and therapeutic effect on diabetes and its complacations, which is intensively associated with its bioactive components, such as polyphenols, polysaccharides and other trace compounds such as lipidsand vitamins.

3. TEA POLYSACCHARIDES AND DIABETES It has been established that non-starch polysaccharides are likely to be associated with less risk of diabetes. Polysaccharide is one of the main components in lower-grade green tea which is considered to cure diabetes especially in China and Japan. With the fast development of biological and chemical technologies during the last 20 years, great advances have been made in the studies of tea polysaccharides on both structure and anti-diabetes activity.

3.1. Chemistry of Tea Polysaccharides Tea polysaccharides (TPS) is a kind of macromolecular glycoprotein with a molecular weight of approximately 1.07–1.10×105 Da. It is soluble in hot water (about 76%) of which aqueous solutions behave as slightly acidic Newtonian fluids, but insoluble in the high concentration solutions of such organic solvents as alcohol, ether, acetone, acetic ether etc [14].

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Wang DF et al. investigated the composition and chemical characterisitics of TPS by methods of UV, IR, GC, and found that its polysaccharide part was composed of arabinose, xylose, fructose, glucose and galactose with a proportion of 5.52:2.21:6.08:44.20:41.99. The protein part was composed of 16 normal amino acids, among which Val, Ala, Gly, Glu were the major proportion [15]. Moreover, Nie SP et al. showed that TPS was composed of seven kinds of monosaccharides, namely ribose, rhamnose, arabinose, xylose, mannose, glucose, galactose in molar ratio of 71:5.88:13.70:1.99:1.00:1.84:33.75, and eighteen amino acids via HPGPC, FT-IR, GC–MS technologies [16]. Although there are many discrepancies due to different preparation and purifying methods, the polysaccharide part of TPS is mainly composed of arabinose, xylose, glucose and galactose obviously, and it contains many natural amino acids, a majority of which have one amino and two carboxyls.

3.2. Method with Celerity and Availability for Assaying Content of TPS In order to effectively valuate TPS pharmacological activities and functions, Wang DF et al. found an available method to assay the content of TPS. Standards of TPS is got first and then used to build a standard curve by anthrone sulfuric acid method, then the content of TPS in tea extract is determined quantitatively [17]. 3 ,5 A490nm

OD280nm/OD490nm

3

A280nm

2 ,5 2 1 ,5 1 0 ,5 0

0

5 10 15 20 25 30 35 40 45 50 55 60

n u m b e r o f th e tu b e Figure 1. TPS fractions separated on Sepherose Fast Flow.

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The water extraction was concentrated to 1/15 volume in a rotary evaporator under reduced pressure and subsequently precipitated by adding ethanol (1:4,V:V). The precipitation was collected by centrifugation and desiccated in vacuum. Then the coarse TPS had been gotten. The coarse TPS was firstly loaded onto a Sepharose Fast Flow column and two main fractions were acquired (Figure 1). The first fraction was chosen for further purification because of the more content of polysaccharide. It was directly loaded onto Sephadex G100 column for further purification. And then the main fraction was collected and named TPS-1 (Figure 2). The purified TPS-1was white powder. Figure 3 illustrated the character of the purified TPS-1 on HPLC. It was a single peak, which indicated the purity of TPS-1. The results illuminated the purified TPS could be used as standards.

3.3. Anti-diabetes Activity of Tea Polysaccharides In recent years, along with in-depth study in the functions of tea, TPS, a kind of glycoprotein, has aroused public concern, especially in its hypoglycemic activity. A number of studies have revealed that TPS indeed improve the status of diabetes. 0,25

A490nm

OD280nm/OD490nm

0,2

A280nm

0,15

0,1

0,05

0 0

2

4

6

8

10

number of tube Figure 2. TPS fraction separated on SephadexG100.

12

14

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Figure 3. TPS-1 performed on HPLC.

Table 1. Contents of TPP, Catechins, Caffeine, and TPS in Various Grades of Green Tea (Grams per 100 g)

TPP Catechins Caffeine TPS *

First 22.03± 0.31 14.07 ± 0.97 4.00± 0.21 0.23± 0.10

Second 23.70 ±0.97 14.01 ± 0.78 4.09 ±0.37 0.29 ± 0.12

Grade* Thirst Fourth 21.10 ± 18.55 ± 1.01 0.31 12.56 ± 9.87± 0.71 0.51 3.85 ± 0.11 3.25 ± 0.71 0.31 ± 0.17 0.46± 0.21

Fifth 19.05 ± 0.37 9.63± 0.91

Sixth 17.23 ± 0.85 8.33± 0.79

2.87 ± 0.05 0.49 ± 0.15

3.01± 0.07 0.58± 0.15

The first grade tea consisted of 90% one leaf and a bud and 10% two leaves and a bud; the second grade, 60% one leaf and a bud and40% two leaves and a bud: the third grade, 30% one leaf and a bud, 30% two leaves and a bud, and 40% three leaves and a bud; thefourth grade, 10% one leaf and a bud, 20% two leaves and a bud, 50% three leaves and a bud, and 20% tender banjhi; the fifth grade, 10%two leaves and a bud, 30% three leaves and a bud, 50% tender banjhi, and 10% leaves; the sixth grade, 80% tender banjhi and 20%leaves.

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Table 2. Effect of TPS on BG in Normal Mice and Model Mice (n= 10, X± SD)

Control 9.6 ±0.6 a b

Normal mice 1.2 2.4 mg/mL mg/mL 8.8 ± 0.7 8.1± 0.4a

4.0 mg/mL 8.0± 0.6b

control 17.9 ± 1.3

Model mice 4.0 6.0 mg/mL mg/mL 16.7± 1.4 13.0± 2.1a

8.0 mg/mL 12.1 ± 1.5b

Compared with the control group p < 0.05. Compared with the control group p < 0.01.

Earlier in 1991, Isigaki K reported that the soluble polysaccharide in tea had an obvious hypoglycemic effect. Wang DF et al. has investigated the activity of TPS for over 20 years and gained plenty of valuable findings as follows. Treatment of diabetes with coarse tea in both China and Japan may be related to TPS and the high content of TPS in coarse tea(Table 1), which indicates that TPS is one of the main components related to its hypoglycemic activity. Additionally, the contents of TPP, catechin, and caffeine in the sixth-grade tea were less than those in the first-grade tea by 20, 40, and 25%, respectively, but the content of TPS in the sixth-grade tea was twice as high as that in the first one, which is to say that the content of TPS increases with the age of green tea, which is why that d diabetics drink coarse tea for curing diabetes in folk of China . When mice (7 weeks old) were injected with purified TPS, the levels of blood glucose (BG) in normal mice and model mice with high BG were decreased significantly by averages of 13.54%, and 22.18%, respectively (Table 2). Wang DF et al. also reported that a 100-120 kDa fraction, essentially composed of polysaccharides (90%) with substantial amounts of arabinogalactan proteins, contained the hypoglycemic activity, which indicated that a soluble tea polysaccharide is the major hypoglycemic factor in tea and that this polysaccharide may be developed to a potential natural hypoglycemic functional ingredient [17, 18]. Ni DJ et al. has compared the hypoglycemic effect of TPS from different teas, and found that all TPS have significant effects, especially TPS from green tea has an obvious dose-effect relationship [19]. Juśkiewicz J et al. [5] have done further investigation and found that the effect of green tea extract, added to the diet at 2 levels of 0.01% and 0.2% the extraction for diabetic rats, seems to be dose dependent in the case of the following examined parameters: small intestinal saccharase and lactase activities, urinary albumin excretion , thiobarbituric acid-reactive substances content in kidneys' tissue, superoxide dismutase activity in the serum, and levels of total antioxidant capacity and integral antioxidant capacity of lipophilic substances. Although the higher dose of green tea extract did not completely protect against STZ-induced hyperglycemia and oxidative stress in experimental rats, this study suggests that green tea extract ingested at high amounts may prove to be a useful therapeutic option in the reversal of diabetic dysfunction. Chen HX et al. have demonstrated that the effects of the molecular weight and protein content of the polysaccharide conjugates on the improvement of the bioactivities appeared to be significant, and especially a relatively low molecular weight and a high protein content appeared to be important for the antioxidant activity [20]. Wang LY et al. have found that TPS group showed lower incidence of diabetes, higher serum C-peptide level, decreased lymphocytic inflammation in pancreatic islets, stronger proliferation of CD8 T subsets and lower ratio of CD4/CD8 subgroup in splenocytes as compared with NS group, which indicated that TPS seems to prevent the onset of type 1 diabetes in nonobese diabetes(NOD)

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mice [21]. Chen JG et al. have found that TPS could significantly ease the diabetic mice’ symptoms, reduce the fasting plasma glucose, and there is a dose-response relationship. This result indicated that TPS could decrease blood glucose and alleviate symptoms of diabetes [22]. The other scholars have all suggested that TPS indeed have a remarkable hypoglycemic activity in treating the diabetics. Although TPS could decrease blood glucose and alleviate symptoms of diabetes, the detailed mechanism has not been assured. Many scholars have investigated the mechanism as follows. As we all know, antioxidants are important in diabetes, with low levels of plasma antioxidants implicates as a risk factor for the development of the disease and circulating levels of radical scavengers impaired throughout the progression of diabetes. Many of the complications of diabetes, including retinopathy and atherosclerotic vascular disease, the leading cause of mortality in diabetics, have been linked to oxidative stress [23-29]. Chen HX et al. has found that TPS exerted significant inhibitory effects on hydroxyl, superoxide radicals and lipid peroxidation, and it could improve the activity of superoxide dismutase (SOD) (p < 0.05). These results indicated that TPS was a potent antioxidant and there appeared to be a direct connection between antioxidant activity and hypoglycemic activity [30]. The same scholars have also proved that TPS extracted from low-grade green tea exerted a significant hypoglycemic effect on both normal and experimental mice model of high blood sugar. They proposed that TPS could enhance glucose tolerance, which was beneficial to alleviate the symptoms of diabetic mice, through such mechanisms as weakening the damage to the islet p cell induced by alloxan, improving the antioxidant capacity of liver, or enhancing the activity of hepatic glucokinase [20]. In recent years, an increased number of studies have shown that the chronic, sub-clinical and non-specific inflammation is closely related to the development of T2DM and its complications such as artherosclerosis, diabetic nephropathy and retinopathy. According to that inflammation can result in insulin resistance and the dysfunction of pancreatic β-cells, the control of inflammation will become a new treatment. Wang DF et al. has found that TPS treatment was beneficial not only for the subsequent production of interleukin (IL) 2 in spleen cells of adjuvant arthritis (AA) rats but also because it prohibited the body from producing too much IL-1 in AA rats [17]. These results suggested thatTPS could prevent diabetes by controlling the body proinflammatory cytokine. Moreover, researches have revealed that there may be a possible relationship between TPS and insulin. Li BQ et al. has reported that treatment of TPS to model mice could reduce the blood serum glucose and consequently improve the glycogen activity, which suggested the effect of TPS on the glucose metabolism is similar to insulin [31]. Rui LL has also testified TPS can not only effectively reduce blood sugar levels in type 2 diabetes mice, but also improve glucose and lipid metabolism [32], which implies the mechanism may be related to increased insulin sensitivity. Quan JH et al. have shown that TPS exerts an obvious hypoglycemic effect on diabetic rats by delaying intestinal digestion and absorption, which may be associated with the inhibitory effects on a-glucosidase and amylase activity and glucose transport ability in small intestinal brush border vesicles [33]. According to previous reports, there are other several main mechanisms for polysaccharides to act on the serum glucose level, to decrease the content of liver glycogen, to stimulate the release of insulin, or to influence the activities of metabolizing enzymes [34].

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Above of all, we can conclude that TPS exerts its hypoglycemic activity through a number of mechanisms. Indeed, TPS has more bioactivity that can benefit diabetics besides the above, for example, the effect of protecting the body from radiation, delaying thrombosis time, improving the blood vessel system, and decreasing lipid content.

4. TEA POLYPHENOLS AND DIABETES Polyphenols are plant metabolites occurring widely in plant food and exhibit outstanding antioxidant and free radical scavenging properties. Green tea is produced by inactivating the polyphenol oxidase in the leaves of camellia sinensis which preserves abundant natural polyphenols, such as catechins(once called vitamin P), orgallotannins, flavonols, flavandiols, and phenolic acids. In particular greent tea catechins and their derivatives are known to contribute beneficial health effects ascribed to tea by their antioxidant, anti-mutagenic, and anti-carcinogenic properties [6].The important catechins of green tea are (−)-epicatechin (EC), (−)-epicatechin3-gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin- 3-gallate (EGCG), among which EGCG is the most abundant and its chemical structure is shown as follows [35]. Since the discovery that tea polyphenols have unique chemical structures and are major ingredients of unfermented tea, they have been found to possess widespread biological functions and health benefits. Epidemiologic observation and laboratory studies have indicated that polyphenolic compounds present in the tea may reduce the risk of a variety of illnesses.

Figure 4. The structure of EC(A), EGC(B) ,ECG(C) and EGCG(D).

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In recent years, more and more researches have shown that green tea polyphenols (GTP) has anti-hyperglycemic action on both normal and diabetic animals. At the same time, GTP also has the therapeutic effect on diabetic complications.

4.1. The Hypoglycemic Activity of Tea Polyphenols As we all know, obesity is associated with high blood cholesterol and a high risk for developing diabetes and cardiovascular disease. The obesity-preventive effects of green tea and its main constituent EGCG are widely supported by results from epidemiological, cell culture, animal, and clinical studies in the last decade. In addition, it has been shown that dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus. Researches have shown that GTP especially EGCG injected into rats significantly reduced food intake, body weight, blood levels of insulin, glucose, cholesterol and triglyceride. Sabu MC et al. has found that GTP has an obvious effect on serum glucose level in normal and diabetic mice. Administration of GTP (500 mg/kg.b.wt.) to normal rats increased glucose tolerance significantly (P<0.05) at 60 min. GTP is also found to reduce serum glucose level in alloxan-induced diabetic rats significantly at a dosage of 100 mg/kg.b.wt. Continued daily administration (15 days) of the extract 50, 100 mg/kg.b.wt produce, respectively, 29% and 44% reduction in the elevated serum glucose level produced by alloxan administration [36]. Ding RF et al. have also reported that GTP exerted an inhibitive effect on increased blood sugar, an improvement in glucose tolerance and the level of blood insulin in diabetic rats [37], indicating that there is a tight connection between the hypoglycemic effect and time of GTP administration. Nowadays, people have investigated the hypoglycemic effect of GTP only on alloxan- or streptozocin-induced diabetic rats, and with rare reports on clinical research, and some others do not sustain the hypoglycemic effect of GTP. Thus there is a long way to go for our research work.

4.2. The Therapeutic Effect of Tea Polyphenols on Diabetic Complications It is well known that diabetes is a metabolic disease which effects not only the glucose metabolism but also lipid and protein metabolism, predisposing to markedly increased cardiovascular mortality and nephropathy.

4.2.1. The Effect of Tea Polyphenols on Cardiovascular Complication Atherosclerosis accounts for some 80 % of all diabetic mortality, about three-quarters of the cardiovascular deaths from diabetes result from coronary artery disease. Researches have reported that GTP may slow the progression of atherogenesis by reducing oxidation of lipoproteins and preserving paraoxonase activity by its anti-hyperlipidemic and anti-oxidative effects. Experimental and clinical studies indicate the extensive chemical modification of collagen, an important constituent of most of the tissues, in the etiology of diabetes and diabetic complications. The modification is due to nonenzymatic glycosylation of protein,

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which results in deleterious effects on collagen. It is not the initial glucose adducts, but subsequent oxidation products known as advanced glycation end products (AGE), particularly the cross-linking AGE, which cause the damaging effect [38]. The enhanced myocardial collagen content, collagen glycation and the resulting advanced glycation end products (AGE) which exhibit the characteristics of increased cross-linking are proposed for the stiffness of myocardium in diabetes. Pon Velayutham et al. has suggested green tea flavonoids could cure cardiovascular complications of diabetes via ameliorating myocardial collagen characteristics, mainly with its typical antioxidant and the anti-hyperglycemic activities [8]. In addition, previous studies have reported that supplementation with antioxidants prevents lipid peroxidation, protein glycation and inhibition of Na+/K+-ATPase or Ca2+ATPase activity caused by hyperglycemia, which could lead to cardiac dysfunction in diabetes [13]. The same scholars have measured the changes in the levels of calcium, sodium, potassium and the activities of Na+/K+-ATPase and Ca2+-ATPase in green tea treated diabetic rat hearts to explore the cardioprotective mechanism of green tea in diabetes, and found that green tea exerts a great therapeutic effect on cardiac dysfunction of diabetes, with a possible mechanism associated with the antioxidant activity of green tea catechins, which could reduce the factors responsible for disturbed fluidity and stability of the membranes by preventing the oxidative stress, protein glycation and hyperlipidemia.

4.2.2. The Effect of Tea Polyphenols on Diabetic Nephropathy Diabetic nephropathy (DN) is one of the most serious chronic microvascular diabetic complications. A great number of factors can result in DN. In particular active oxygen free radicals and lipid peroxidation play an important role in the etiology of DN. Renal damage is a well-known consequence of diabetes, and the early identification of micro albuminuria is considered to be clinically relevant. A therapeutic intervention should start early enough to be effective or to delay the development of end-stage renal disease. Otherwise, there will be serious consequences such as renal failure accompanied by uremia [39, 40]. Wang B et al. has studied a protective effect of tea polyphenols on diabetic rats and found that tea polyphenols can inhibit the reduction of SOD in early experimental animal serum and the level of lipid peroxides. Additionally, GTP exerted a reduction in albuminuria level and an inhibitory effect on the increase of TGF-B1 mRNA and protein expression in early DN mice, which indicated that GTP had an obvious preventive effect on the damage of kidney in diabetes [41, 42]. Zhao QL et al. also affirmed the protective effects of tea polyphenols on diabetic rats. They found that administration of GTP can significantly decreased the level of albuminuria, creatinine, cholesterol and triglycerides, and mitigated the renal damage in diabetic mice, compared with model mice without treatment [43]. There are many scholars who believe that GTP exerts its effect on DN by the typical antioxidant activity, but it still requires further study on whether it works through direct effect or indirect effect of regulating oxidative balance.

4.3. The Anti-Diabetic Mechanism of Green Tea Polyphenols In all, GTP indeed has both anti-hyperglycemic action and the therapeutic effect on diabetes and its complications. Many researchers are engaged in the prevention and treatment of diabetes using tea polyphenols and the mechanisms of its actions are emerging based on

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the various laboratory data. These mechanisms may be related to certain pathways, such as through its antioxidant activity, inhibition of intestinal glucose transporter and related enzyme activities, mimic insulin by decreasing the expression of genes that control gluconeogenesis, increasing insulin sensitivity, and enhancing basal- and insulin-stimulated glucose uptake, etc. These effects may be attributed to the anti-diabetic effect of green tea.

4.3.1. The Great Antioxidant Activity of Tea Polyphenols The polyphenolic fraction of green tea, has been reported to have multiple pharmacological actions, among which the antioxidant activities are the most widely recognized [44]. GTP has been shown to possess potent antioxidant activity that is several folds higher than that of Vitamin C and Vitamin E [45]. Different methods of evaluation the antioxidant activity of polyphenols suggest that they exhibit scavenging activity against superoxide radicals, free radicals and hydrogen peroxide. In addition to directly quenching reactive oxygen species, tea flavonoids can chelate iron and copper preventing the metalcatalyzed free radical formation [46-49]. Thus GTP can inhibit body damages induced by free radicals. Increased oxidation stress has been implicated in the pathogenesis of DM. Hyperglycemia induced protein glycation generates superoxide free radicals [50-54], leading to lipid peroxidation and formation of reactive products, which may play an important role in the development of diabetes and its complications such as DN. Administration of GTP can effectively improve the body antioxidant potential and increase the antioxidant enzymes such as the activity of SOD, GSH –PX and POD which are lower in diabetes mellitus [55, 56]. In addition, Li et al. reported that EGCG does not affect glucose-stimulated insulin secretion under high energy conditions, and further showed that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation [57]. Those results strongly support the notion that GT consumption helps maintain and improve health by increasing antioxidant defense and cellular metabolic activities in various tissues of normal rats and provides a useful therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes mellitus. Hence we can conclude that the anti-diabetic activity of GTP is mainly associated with its antioxidant activity. 4.3.2. The Inhibition of Intestinal Glucose Transporter and Related Enzyme Activities Intestinal glucose uptake can be achieved by the sodium-dependent SGLT1. There is a site combined with Na+ on this glucose transporter, and this modification makes transfer of glucose easier. The glucose transportation through cell membrane depended on the effect of SGLT1. Yoko Kobayashi et al. have found that EGCG can inhibit intestinal glucose uptake by sodium dependent glucose transporter, SGLT1, through a competitive mechanism, which can indicate its increase in controlling blood sugar [58]. In addition, researches have shown that GTP exert hypoglycemic effect by inhibiting the related enzyme activities. Japanese scholars have been engaged in the related research since 1990, and found that green tea catechins have the obviously inhibitory effects on aglucosidase and amylase activity, which directly slow down the intestinal glucose taken [59]. This mechanism may be well ascribed for the anti-hyperglycemia activity of GTP.

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4.3.3. Insulin-like Activities and Insulin-enhancing Activity of GTP Several known compounds found in tea were testified to enhance insulin, with the greatest activity due to EGCG, followed by ECG, tannins, and theaflavins. As support for this result, Anderson et al. reported that tea contains in vitro insulin-enhancing activity and the predominant active ingredient is EGCG. The green tea flavonoid has also been shown to have insulin-like glucose-lowering activities [60] as well as insulin-enhancing activity in mammals [61]. However, EGCG, the principal catechin in green tea, differs from insulin in that it affects several insulin-activated kinases with slower kinetics. Furthermore, EGCG regulates protein-tyrosine-phosphorylation by modulating the redox state of the cell [60], and mimics insulin at least in part by repression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase, which is a key ratelimiting enzyme in hepatic gluconeogenesis, a process which is thought to contribute to increased glucose production in diabetes [62].What is more, other scholars have reported that green tea catechins possess a strong hypoglycemic effect by enhancing basal- and insulinstimulated glucose uptake, increases insulin sensitivity and ameliorates insulin resistance in diabetes.

5. OTHER COMPONENTS OF GREEN TEA AND DIABETES The preventive and therapeutic effects of green tea on diabetes result from a combination of many components. There are many other substances, apart from TPS and polyphenols, which have positive effects on therapy of diabetes. For example, tea pigments have a better therapeutic effect on DN by decreasing the quantity of microspheres protein and significantly inhibiting the production of endothelin and the activity of platelet [63, 64], and can cure diabetic cardiovascular disease through the possible mechanism of lowing cholesterol and triglycerides and increasing high-density lipoprotein, Vitamin C could normalize the tenacity and permeability of microvascular, beneficial for the diabetes [65]. In addition, there are other components improving glucose metabolism, such as Vitamin B, pantothenic acid.

6. PERSPECTIVES AND FUTURE DIRECTIONS It is apparent that green tea is a source of a wide range of phytochemicals that are digested, absorbed and metabolized by the body, and that tea constituents exert their antidiabetic effects at the cellular level. Because of several potential benefits of green tea, we propose that this beverage may act as a supportive agent in diabetes mellitus. For the promotion of health, there are various factors that matters like type of green tea and preparation shown important, but so will the frequency and timing of intake as these factors directly affect the pharmacokinetics and ultimate disposition of the polyphenols and polysaccharides within tissues. Future research needs to define the actual magnitude of health benefits, establish the safe range of tea consumption associated with these benefits and elucidate potential mechanisms of action.

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Further progress in the evaluation of the effects of green tea on humans depends on the development of new experimental systems. Exploration at the cellular level allows a better understanding of the underlying mechanisms regulating functions in normal and pathologic states. Development of more specific and sensitive methods with more representative models along with the development of good predictive biomarkers will give a better understanding of how tea interacts with endogenous systems and other exogenous factors. In order to understand the effect of green tea consumption on diabetes, additional research on the pharmacokinetics of tea constituents as well as their mechanisms of action is needed. Definitive conclusions concerning the protective effect of green tea have to come from well-designed observational epidemiological studies and intervention trials. The development of biomarkers for tea consumption, as well as molecular markers for its biological effects, will contribute to better future studies in this area.

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[27] Godin DV, Wohaieb SA, Garnett ME, Goumeniouk AD. Antioxidant enzyme alterations in experimental and clinical diabetes. Mol Cell Biochem 1988, 84(2), 223231. [28] Collier A, Wilson R, Bradley H, Thomson JA, Small M. Free radical activity in type 2 diabetes. Diabet Med 1990, 7(1), 27-30. [29] Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991, 40(4), 405-412. [30] Chen HX, Zhang M, Xie BJ. Components and antioxidant activity of polysaccharide conjugate from green tea. Food Chem 2005, 90(1-2), 17-21. [31] Li BQ, Zhang HL, Shu QL, Zhang BC, Ge SF. Studies on the extraction of polysaccharide from middle and lower middle grade green tea and the effectiveness on blood-glucose depressing. Tea Sci 1996, 16(1), 67-72. [32] Rui LL, Xiao JZ, Cheng YY. Anti diabetic activity of tea polysaccharide on KK-A mice. J China-Japan Friendship Hospital 2005, 19(2), 93-96. [33] Quan JS, Yin XZ, Kazushi O. Study on hypoglycemic mechanism of tea polysaccharide. Chin J Public Health 2007, 23(3), 295-296. [34] Liu C, Liu ZY. Studying progress in herbal polysaccharides on hypoglycemic activities. J Anhui TCM College 1999, 18(5), 83–85. [35] Almajano MP, Carbó R, Jiménez JAL, Gordon MH. Antioxidant and antimicrobial activities of tea infusions. Food Chem 2008, 108, 55-63. [36] Sabu MC, Smitha K, Ramadasan K. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol 2002, 83(1-2), 109-116. [37] Ding RF, He PM, Jie GL. Study on hypoglycemic mechanism of tea polysaccharides and polyphenols. Tea Sci 2005, 25(3), 219-224. [38] Reiser KM. Nonenzymatic glycation of collagen in ageing and diabetes. Proc Soc Biol Med 1991, 196(1), 17-29. [39] Aybar MJ, Sánchez Riera AN, Grau A, Sánchez SS. Hypoglycemic effect of the water extract of Smallanthus sonchifolius (yacon) leaves in normal and diabetic rats. J Ethnopharmacol 2001, 74, 125-132. [40] Mathiesen ER, Rønn B, Storm B, Foght H, Deckert T. The natural course of microalbuminuria in insulin-dependent diabetes: a 10-year prospective study. Diabet Med 1995, 12(6), 482-487. [41] Wang B, Deng H, Su N, Liu DF. An experimental study of the effect of tea polyphenols on the expression of reactive oxygen species in diabetic rats. J southeast Univ (med sci ed) 2002, 21(1), 40-48. [42] Wang B, Li K, Deng H, Su N, Zhao GH, Li C. Effect of tea polyphenols on expression of TGF-β1 in the kidney of streptozocin-induced diabetic nephropathy rats. J ChinaJapan Friendship Hospital 2003, 17(5), 286-289. [43] Li QZ, Liu YL, Yao L, Liu GX, Zhang YS, Shi J, Meng AG, Yin H, Wang HY. The protective effects of TP on diabetic mice. J Environ Occup Med 2003, 20(3), 233-235. [44] McKay DL, Blumberg JB. The role of tea in human health: An update. J Am Coll Nutr 2002, 21(1), 1-13. [45] Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995, 22(4), 375-383.

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[46] Pillai SP, Mitscher LA, Menon SR, Pillai CA, Shankel DM.Antimutagenic, antioxidant activity of green tea components and related compounds. J Environ Pathol Toxicol Oncol 1999, 18(3), 147-158. [47] Salah N, Miller NJ, Paganga G, Tijburg L, Bolwell GP, Rice Evans C. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 1995, 322(2), 339-346. [48] Hong JT, Ryu SR, Kim HJ, Lee JK, Lee SH, Kim DB, Yun YP, Ryu JH, Lee BM, Kim PY. Neuroprotective effect of green tea extract in experimental ischemia-reperfusion brain injury. Brain Res Bull 2000, 53(6), 743–749. [49] Kashima, M. Effects of catechins on superoxide and hydroxyl radical. Chem Pharm Bull (Tokyo) 47, 279–283. [50] Atalay M, Laaksonen DE. Diabetes, oxidative stress and physical exercise. J Sports Sci Med 2002, 1, 1-14. [51] Memisogullari R, Taysi S, Bakan E. Antioxidant status and lipid peroxidation in Type II diabetes mellitus. Cell Biochem Funct 2003, 21(3), 291-296. [52] Raskin P, Jovanovic L, Berger S, Schwartz S, Woo V Ratner R. Repaglinide/troglitazone combination therapy:improved glycemic control in Type 2 diabetes. Diabetes Care 2000, 23(7), 979-983. [53] Cunningham J, Leffell M, Mearkle P, Harmatz P. Elevated plasma ceruloplasmin in insulin-dependent diabetes mellitus: evidence for increased oxidative stress as a variable complication. Metabolism 1995, 44(8), 996-999. [54] Lipinski B. Pathophysiology of oxidative stress in diabetes mellitus. J Diabet Complications 2001, 15(4), 203-210. [55] Sabu MC, Smitha K, Kuttan R. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol 2002, 83(1-2), 109-116. [56] Leena P, Balaraman R. Effect of green tea extract on cisplatin induced oxidative damage on kidney and testes of rats. Ars Pharm 2005, 46(1), 5–18. [57] HS Moon, HG Lee, YJ Choi, TG Kim, CS Cho. Proposed mechanisms of (−)epigallocatechin-3-gallate for anti-obesity. Chem Biol Interact 2007, 167, 85–98. [58] Kobayashi Y, Suzuki M, Satsu H, Arai S, Hara Y, Suzuki K, Miyamoto Y, Shimizu M. Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. J Agric Food Chem 2000, 48(11), 56185623. [59] Matsumoto N, Ishigaki F, Ishigaki A, Iwashina H, Hara Y. Reduction of blood glucose level by tea catechin. Biosci Biotechnol Biochem 1993, 57(4), 525- 527. [60] Waltner-Law ME, Wang XL, Law BK, Hall RK, Nawano M, Granner DK. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem 2002, 277(38), 34933-34940. [61] Anderson RA, Polansky MM. Tea enhances insulin activity. J Agric Food Chem 2002, 50(24), 7182-7186. [62] Anton S, Melville L, Rena G. Epigallocatechin gallate (EGCG) mimics insulin action on the transcription factor FOXO1a and elicits cellular responses in the presence and absence of insulin. Cellular Signalling 2007, 19(2), 378-383.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 21

GREEN TEA AND TYPE 2 DIABETES Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song Department of Physiology & Chronic Disease Research Center, Keimyung University School of Medicine, 194 Dongsan-Dong, Jung-Gu, Daegu, 700-712 Korea

ABSTRACT Green tea, which is consumed world wide as a beverage, is known to have many beneficial effects on human health. Green tea contains various biologically-active materials including catechins, flavonols and caffeine. Of them, catechins are the major constituents consisting of 30% of water-extractable materials. Pharmacological implication has been mainly made upon (-)-epigallocatechin-3-gallate (EGCG), as it is the most abundant catechin in green tea extracts. The second biologically important catechin is (-)-epicatechin-3-gallate (ECG), which is called one of gallated catechins as EGCG. To date, there is no prominent evidence for green tea consumption to determine whether be beneficial or harmful in metabolic diseases, such as type 2 diabetes and obesity. It may be due to catechins having a variety of function in human body. This chapter will focus on action mechanism of EGCG on ATP-sensitive potassium channels, which manifests as actual phenotypes in cardiac and beta-cell function. Additionally, changes by green tea or gallated catechins in insulin resistance will be reviewed. Thereafter, a right method to use EGCG as a supportive regimen for diabetic care and obesity will be introduced. New era should come with modifying natural products fit to human well-being.

INTRODUCTION Type 2 diabetes can be characterized by both fasting and postprandial hyperglycemia. Hyperglycemia causes various tissues in the body to suffer from increased oxygen and nitrogen free radicals, exacerbating various diabetic complications (Hong et al., 2004). Therefore, antioxidative manipulation is a method for diabetic prevention and treatment. Green tea catechins have chargeable moiety and specific lipophilicity according to their

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molecular structure. Therefore, green tea catechins may act as modulators of a variety of function in human body.

OXIDATIVE STRESS AND GREEN TEA Green tea catechins, in particular (-)-epigallocatechin-3-gallate (EGCG) which is the most abundant and biologically active components of green tea polyphenols, are known to possess both antioxidant and prooxidant activity (Mandel et al., 2004; Salah et al., 1995), which probably depends on its molecular structure or its concentration to be used. Ironically, EGCG protects normal cells against oxygen radical insults (Xia et al., 2005), whereas it induces apoptosis of cancer cells by generating intracellular reactive oxygen species (ROS) (Qanungo et al., 2005). This discrepancy may be due to 1) occurrence of a specific signal cascade in cancer cells which is targeted by catechins but not manifested in normal cells; 2) greater susceptibility of cancer cells than normal cells to ROS, which can be more generated in cancer cells having a higher energy need; 3) There is also discrepancy between results in in vitro and in vivo study to evaluate the effect of EGCG on possible free radical toxicity, suggesting that other existing factors present in vivo may modify the EGCG effect. Therefore, it appears difficult to make an exact answer about whether EGCG is antioxidant, prooxidant, or both in the body. Instead, it may depend on targeted tissue-specific environment that affects the actions of EGCG (Albright et al., 2004). In other words, the action mechanism of EGCG associated with ROS would be expressed differently according to conditions in related tissues. In diabetes, many tissue cells, such as pancreatic beta cells and vascular endothelial cells, suffer from increased oxidative stress mainly by prolonged hyperglycemia. This situation is somewhat like those of cancer cells, which are also stressful owing to increased ROS and thereby susceptible to EGCG. In normal rats, a moderate concentration of EGCG injection (5 mg/kg, i.p.) does not induce any significant glucose intolerance during intraperitoneal glucose tolerance test (IPGTT). However, in streptozotocin (STZ)-induced diabetic rats which are expected to have severe oxidative stress, EGCG treatment causes much more blood glucose elevation during IPGTT compared with non-EGCG-treated, STZcontrol rats (Yun et al., 2006; Figure 1). In histological examination, the pancreatic beta cells in the EGCG-treated rats are more damaged than in the control (Yun et al., 2006). This result may suggest that EGCG may act as a prooxidant rather than as an antioxidant at least in the rat beta cells conditioned with STZ. The plasma EGCG concentration achievable by the injection of 5 mg EGCG is nanomolar levels (Chen et al., 1997; Kao et al., 2000), which is readily reachable in human plasma by moderate daily green tea consumption.

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Figure 1. Effect of EGCG on change in blood glucose level in response to high glucose loading (1.5 g/kg i.p.) in control and streptozotocin-treated diabetic rats. EGCG was intraperitoneally injected in each diabetic rat at a dose of 0 to 50 mg/kg/day for 4 consecutive days (Yun et al., 2006; Permission from Elsevier).

ATP-SENSITIVE POTASSIUM (KATP) CHANNELS AND GREEN TEA Ten micromolar concentration of EGCG can be achievable in human plasma by oral intake of 15 cups of green tea in the fasting state (Chow et al., 2001; when supposed 100 mg EGCG per one cup). At this concentration, EGCG reduces ATP sensitivity of beta-cell ATPsensitive potassium (KATP) channels (Jin et al., 2007; Figure 2), which are inhibited by increased intracellular ATP/ADP ratio. It is well known that glucose-induced KATP-channel inhibition in beta cells triggers insulin secretion, through elevation of membrane potential and cytosolic Ca2+ levels. The reduced ATP sensitivity of the channel by EGCG may cause diminished insulin secretion in response to high glucose after a meal. This effect is EGCGspecific, not demonstrated in other catechins, such as (-)-epigallocatechin (EGC), (-)epicatechin (EC) and ECG. The binding sites on the KATP channel for α-, β- and γ-phosphate tails of ATP are known to be R201, K185 and R50 residues of the cytoplasmic termini on the inwardly-rectifying potassium channel Kir6.2 (Anticliff et al., 2005). The positively-charged residues on the channel interact with the negatively-charged phosphate tails of ATP to close the channel. EGCG possesses many negatively-chargeable hydroxyl groups, and then hinders the interaction of the channel with ATP. This inhibitory mechanism of EGCG is also applicable to the binding to the channel of phosphatidylinositol polyphosphates (PIP) (Jin et

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al., 2007; Figure 3), which can increase the channel open probability in contrast to ATP. The positive residues R54 in the N-terminus, R176 and R177 in the C-terminus on Kir6.2 appear to be PIP-binding sites (Ribalet et al., 2006). The inhibition of PIP-binding to the channel by EGCG is more effective than of the ATP-binding because the PIP inhibition of EGCG can occur at EGCG concentration around 1 μM, which is equivalent to 2-3 cups green tea ingestion in humans. Therefore, there is little possibility that regular daily oral ingestion of green tea may cause impaired glucose-stimulated insulin secretion by EGCG-mediated decrease in the channel ATP sensitivity. Reduced PIP sensitivity by 1 μM EGCG is more relevant, and may hinder cardiac KATP channel activation against hypoxia or ischemia. However, it appears that cardiac-type KATP channels (Kir6.2/SUR2A) are more sensitive to EGCG inhibiting the channel ATP sensitivity than beta-cell type KATP channels (Kir6.2/SUR1). Hence, the inhibitory mechanism of EGCG on the channel ATP sensitivity may be helpful for prevention of ischemic heart, by increasing the channel open probability. Which mechanism of EGCG associated with the PIP or ATP binding is dominant in actual cardiac tissues should be further clearly determined.

Figure 2. Effect of 10 μM EGCG on ATP-induced inhibition of KATP currents expressed in plasma membrane of Xenopus frog oocytes (Jin et al., 2007; Permission from Elsevier).

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Figure 3. Effect of 1 μM EGCG on PIP2-induced activation of KATP (Kir6.2/SUR1) currents expressed in plasma membrane of Xenopus frog oocytes (Jin et al., 2007; Permission from Elsevier).

INTESTINAL NUTRIENT ABSORPTION AND GREEN TEA One-hundred micromolar concentration of EGCG is difficult to be reachable in human plasma, because it may cause severe unexpected side effects in the circulation. Nevertheless, EGCG at this concentration inhibit the KATP channel allosterically via interacting with cellular lipid membrane (Baek et al., 2005). ECG as well as EGCG is critical for this action. Therefore, gallated catechins appear to perturb lipid bilayer, and thus modify the lipid function itself as well as protein functions embedded in it. The lipid perturbation effects of gallated catechins are useful for inhibiting micelle formation in the alimentary tract, thereby decreasing cholesterol absorption into the circulation (Raederstorff et al., 2003). This mechanism has been emphasized to reveal the beneficial effect of green tea consumption to prevent obesity. This range of concentration of EGCG can also inhibit Na+-glucose cotransporters in the alimentary tract, thereby diminishing glucose absorption as well (Kobayashi et al., 2000). Taken together, green tea catechins, in particular gallated catechins,

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seem to be effective in reducing absorption of the body energy source, over-intake of which causes detrimental metabolic diseases.

GLUCOSE INTOLERANCE AND GREEN TEA However, problem may arise that gallated catechins also inhibit cellular glucose uptake in the circulation. A recent report demonstrated that ECG or EGCG inhibits glucose movement through plasma membrane of red blood cells, competing the binding to glucose transporter type 1 with glucose (Naftalin et al., 2003). The concentration of gallated catechins to exert this action is around 100 nM for ECG and 1 μM for EGCG, which is readily achievable in human plasma. Although additional observation has not been reported to date as for relation of the gallated catechins with other glucose transporters in the body, it may be associated with insulin resistance: Normal glucose flow into the cells is a requisite to reduce blood glucose levels in postprandial period, which is helped by secreted insulin. In case glucose removal is hindered by EGCG into tissues, greater secretion of insulin is needed to normalize tissue glucose utilization as well as blood glucose level. Beta-cell overload is a well known factor to facilitate diabetic progression. Truly, in-blood EGCG at that concentration elevates blood glucose levels during IPGTT compared to control without EGCG (Jin et al., 2007; Figure 4). Therefore, the luminal effect to be positive and the circulating effect to be negative of gallated catechins against the metabolic syndromes should be put together to selectively exploit their beneficial role. In addition, no matter how green tea is helpful for managing diabetes and obesity, the effect may be useless at concentration of EGCG beyond its endurable blood level in humans.

CONCLUSION Green tea EGCG may act as a prooxidant in diabetic beta cells, which suffer from ROS increased by chronic hyperglycemia. However, by decreasing ATP sensitivity of KATP channels, EGCG may help the cardiac tissues with surviving against ischemic or reperfusion injury. These effects may be relevant because it occurs at EGCG concentration around 1 μM. Since the beta-cell channel ATP sensitivity is decreased at EGCG around 10 μM, it may not be problem during regular normal intake of green tea. Biological meaning of PIP sensitivity change of the KATP channel by EGCG remains to be further determined. The gallated catechins in the alimentary tract may be helpful for controlling diabetes and obesity by blocking glucose and cholesterol absorption, but the circulating gallated catechins may be harmful for diabetes by blocking normal blood glucose utilization of tissues. Selecting beneficial action of green tea catechins is needed for appropriate future usage of them in diabetes and obesity.

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REFERENCES Albright, C.D., Salganik, R.I., Van Dyke, T. (2004). Dietary depletion of vitamin E and vitamin A inhibits mammary tumor growth and metastasis in transgenic mice. J Nutr 134, 1139-1144. Antcliff, J.F., Haider, S., Proks, P., Sansom, M.S., Ashcroft, F.M. (2005). Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit. Embo J 24, 229-239. Baek, W.K., Jang, B.C., Lim, J.H., Kwon, T.K., Lee, H.Y., Cho, C.H., Kim, D.K., Shin, D.H., Park, J.G., Lim, J.G., Bae, J.H., Yoo, S.K., Park, W.K., Song, D.K. (2005). Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)epigallocatechin-3-gallate. Biochem Pharmacol 70, 1560-1567. Chen, L., Lee, M.J., Li, H., Yang, C.S. (1997). Absorption, distribution, elimination of tea polyphenols in rats. Drug Metab Dispos 25, 1045-1050. Chow, H.H., Cai, Y., Alberts, D.S., Hakim, I., Dorr, R., Shahi, F., Crowell, J.A., Yang, C.S., Hara, Y. (2001). Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 10, 53-58. Hong, J.H., Kim, M.J., Park, M.R., Kwag, O.G., Lee, I.S., Byun, B.H., Lee, S.C., Lee, K.B., Rhee, S.J. (2004). Effects of vitamin E on oxidative stress and membrane fluidity in brain of streptozotocin-induced diabetic rats. Clin Chim Acta 340, 107-115. Jin, J.Y., Park, S.H., Bae, J.H., Cho, H.C., Lim, J.G., Park, W.S., Han, J., Lee, J.H., Song, D.K. (2007). Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP. Pharmacol Res 56, 237247. Kao, Y.H., Hiipakka, R.A., Liao, S. (2000). Modulation of endocrine systems and food intake by green tea epigallocatechin gallate. Endocrinology 141, 980-987. Kobayashi, Y., Suzuki, M., Satsu, H., Arai, S., Hara, Y., Suzuki, K., Miyamoto, Y., Shimizu, M. (2000). Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. J Agric Food Chem 48, 56185623. Mandel, S., Weinreb, O., Amit, T., Youdim, M.B. (2004). Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases. J Neurochem 88, 1555-1569. Naftalin, R.J., Afzal, I., Cunningham, P., Halai, M., Ross, C., Salleh, N., Milligan, S.R. (2003). Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose-binding site of the human erythrocyte glucose transporter GLUT1. Br J Pharmacol 140, 487-499. Qanungo, S., Das, M., Haldar, S., Basu, A. (2005). Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis 26, 958-967. Raederstorff, D.G., Schlachter, M.F., Elste, V., Weber, P. (2003). Effect of EGCG on lipid absorption and plasma lipid levels in rats. J Nutr Biochem 14, 326-332.

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Ribalet, B., John, S.A., Xie, L.H., Weiss, J.N. (2006). ATP-sensitive K+ channels: regulation of bursting by the sulphonylurea receptor, PIP2 and regions of Kir6.2. J Physiol 571, 303-317. Salah, N., Miller, N.J., Paganga, G., Tijburg, L., Bolwell, G.P., Rice-Evans, C. (1995). Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 322, 339-346. Xia, J., Song, X., Bi, Z., Chu, W., Wan, Y. (2005). UV-induced NF-kappaB activation and expression of IL-6 is attenuated by (-)-epigallocatechin-3-gallate in cultured human keratinocytes in vitro. Int J Mol Med 16, 943-950. Yun, S.Y., Kim, S.P., Song, D.K. (2006). Effects of (-)-epigallocatechin-3-gallate on pancreatic beta-cell damage in streptozotocin-induced diabetic rats. Eur J Pharmacol 541, 115-121. Acknowledgments: Supported by KOSEF (R13-2002-028-03002-0)

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 22

BIOCATALYTIC CONVERSION OF GREEN TEA CATECHINS TO EPITHEAFLAGALLIN, EPITHEAFLAGALLIN, 3-O-GALLATE, AND THEAFLAVINS: PRODUCTION OF PROMISING FUNCTIONAL FOODS Nobuya Itoh1,* and Yuji Katsube2 1

Department of Biotechnology, Faculty of Engineering (Biotechnology Research Center), Toyama Prefectural University, Kurokawa 5180, Imizu, Toyama 939-0398, Japan 2 Kracie Seiyaku, Ltd., 3-1, Kanebo-machi, Takaoka, Toyama 933-0856, Japan

ABSTRACT World-wide tea production has reached 2.97 x 106 metric tons/year, and more than 75% of tea products are black tea. In recent years, green tea which contain catechin derivatives such as (-)-epicatechin (EC) (1), (-)-epicatechin gallate (ECg) (2), (-)epigallocatechin (EGC) (3), and (-)-epigallocatechin gallate (EGCg) (4) has been recognized as a useful functional food. However, recent development of enzymatic processes now makes it possible to produce epitheaflagallin (5), epitheaflagallin 3-Ogallate (6) and theaflavin (TF) (7) derivatives from catechins, which are components of black tea. Epitheaflagallin and epitheaflagallin 3-O-gallate are preferentially synthesized from EGC (3) and EGCg (4) in green tea extracts in the presence of laccase and gallic acid. Takemoto and colleagues have developed a Camellia sinensis cell culture system containing peroxidase and hydrolases of ECG (3) and EGCg (4) to produce theaflavin (7) from tea catechins. These biocatalytic processes allow us to preferentially convert catechin derivatives in a crude mixture of green tea into different compounds, and, thus, to improve the composition of the catechins present in green tea.

*

Corresponding author. Tel.: +81-766-56-7500 ext. 560; fax: +81-766-56-2498; e-mail: [email protected]

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Nobuya Itoh and Yuji Katsube

Keywords: Laccase/peroxidase-catalyzed oxidation; epitheaflagallin; epitheaflagallin 3-Ogallate; theaflavins; green tea catechins; functional food; inhibition of pancreatic lipase

1. LACCASE-MEDIATED CONVERSION OF GREEN TEA CATECHINS TO EPITHEAFLAGALLIN AND EPITHEAFLAGALLIN 3-O-GALLATE IN THE PRESENCE OF GALLIC ACID [1] We recently found that laccase (EC 1.10.3.2) was able to synthesize two benzotropolone derivatives, epitheaflagallin (5), and epitheaflagallin 3-O-gallate (6) (Figure 1), from crude tea catechins in the presence of oxygen and gallic acid. These compounds have been previously synthesized from EGC (3)/EGCg (4) in the presence of catechol/pyrogallol by Takino and Imagawa. [2] Using purified compounds including (1), (2), (3), (4), we confirmed that (5) and (6) were synthesized from (3) and (4), respectively, through laccase oxidation reaction with gallic acid. The reaction involves the preferential oxidation of gallic acid and the pyrogalloyl group (B ring) in (3) and (4) to the quinone intermediate galloquinone, [3] followed by the Michael addition of the oxidized pyrogalloyl group in (3)/(4) and subsequent carbonyl addition across the ring and decarboxylation to yield benzotropolone skeletone. Recently, Sang et al. reported the enzymatic synthesis of tea theaflavin derivatives from catechin derivatives in the presence of hydrogen peroxide and peroxidase (EC 1.11.1.7). [4,5] In peroxidase oxidation, the catechoyl/pyrogalloyl/galloyl groups in EC (1)/ECg (2)/EGC (3)/EGCg (4) are easily oxidized to form the quinone intermediates that subsequently yield the various theaflavin-related compounds. In our oxidation system, preferential conversion of (3) and (4) is possible, and very little EC (1) and ECG (2) reacted with the galloquinone intermediate in the reaction mixture. Laccase-dependent preferential oxidation of gallic acid and the pyrogalloyl group in (3) and (4) to the galloquinone intermediate makes it possible to convert (3) and (4) in crude tea catechins to (5) and (6). The proposed mechanism for this reaction is supported by the observation that the addition of gallic acid to the reaction mixture accelerates the production of (5) and (6), as determined by HPLC. We hypothesized that the low level of production of (5) and (6) in the absence of exogenously supplied gallic acid is due to free gallic acid in the green tea extract. We also confirmed that a laccase reaction gradually oxidizes authentic (3)/(4) in the absence of gallic acid to produce other, as yet uncharacterized, compounds. However, these reactions were suppressed in crude green tea extracts when gallic acid was added. Matuo et al. reported that (3) was oxidized by a Japanese pear homogenate containing polyphenol oxidase to yield quinone dimers, one of which was converted to theaflavin-related compounds. A minor reaction converted the quinine dimer to (5) and to hydroxytheaflavin. [6] Therefore, laccase oxidation of (3)/(4) in the absence of gallic acid may form these, or similar, compounds.

Biocatalytic Conversion of Green Tea Catechins … OH

O

HO

OH

HO

421

OH

O

O

OH

HO

HO

-O

O OH

O C

O

gallic acid

O O

OH

H

+ OH

O

HO

OH

H -CO2

OH

C O

O

O

O O

HO

enzymatic oxidation

OR2 O

-CO2

OH

HO

OR2

+H2O

OH

OH OH OH

O OH

O

HO

OH

O

HO

R1 OR2 OH

EC 1: R1=H, R2=H ECG 2: R1=H, R2=gallate EGC 3: R1=OH, R2=H EGCG 4: R1=OH, R2=gallate

OR2 OH

5: R2=H 6: R2=gallate

Figure 1. The proposed mechanism of epitheaflagallin (5) and epitheaflagallin 3-O-gallate (6) formation.

The reaction mixture for the production of (5) and (6) consisted of 1.0 g green tea extract (Camellia extract 30S, Taiyo Kagaku Corp., Yokkaichi, Mie, Japan), 0.48 g gallic acid monohydrate, and 10,000 units of laccase from Trametes sp. (DaiwaY120, Amano Enzyme Co., Ltd., Nagoya, Japan). The reaction was allowed to proceed for 2 h at 45°C without adjustment of the pH. Enzymatic activity was spectrophotometrically assayed at 30°C by measuring the formation of quinonimine dye from phenol and 4-aminoantipyrine (pH 4.5) at 505 nm, according to the manufacturer’s protocol. One unit of the enzyme was defined as the amount that increased the absorbance at 505 nm by 0.1 OD/min. The purified products (5) (orange in color) and (6) (orange-red in color) were analyzed by MS and NMR and the structures of them were coincided with the data from the chemically synthesized epitheaflagallins reported by Takino et al. [7 ]and Nonaka et al. [8] The reaction was not optimized for each substrate in the crude tea catechins, and the conversion yields of (5)/(6) were less than 20%, although (3)/(4) in the reaction mixture was entirely consumed. These data suggest that the reaction did not proceed stoichiometrically, but we have not yet determined which products were synthesized in addition to (5) and (6). We hypothesized that highly polymerized compounds of (3)/(4) were formed by oxidation in the presence of gallic acid, since there were no additional peaks corresponding to small molecules in HPLC chromatogram under our experimental conditions.

422

Nobuya Itoh and Yuji Katsube Table 1. Epitheaflagallin (5) and epitheaflagallin-3-O-gallate (6) content of black tea extracts Sample1 2

Content (mg/g extract)

A

B

C

D

Epitheaflagallin (5)

0.35

0.46

N.D.3

0.25

Epitheaflagallin-3-O-gallate (6)

0.57

0.84

N.D.

0.47

1

FD/SD black tea extracts with different lot numbers were purchased from San-Ei Gen F.F.I., Inc., Osaka, Japan. 2 The amounts of (5) and (6) were calculated from the areas under the peaks on an HPLC chromatogram using calibration curves prepared from purified (5) and (6). 3 N.D.: not detected.

To detect the presence of (5) and (6) in natural tea products, four kinds of black tea extract (A-D) were analyzed (Table 1). We confirmed that epitheaflagallin derivatives were minor components of all the black tea extracts tested, with the exception of one sample. The black tea extracts contained a mean of approximately 0.1% (w/w) of (5) and (6) together. The amount of (6) was approximately 1.8 times higher than that of (5) in each of the three samples. These results indicate that epitheaflagallin derivatives are naturally synthesized as minor components during the tea fermentation process. [9] The bio-oxidation process that converts (3) and/or (4) to epitheaflagallin derivatives could be applied to the production of useful functional food components, as described in Section 3.

1.1. Epitheaflagallin (5) Orange solid; 1H-NMR (CD3OD) δΗ (ppm) 2.82, 2.93 (2H, m), 4.27 (1H, m), 4.81 (1H, s), 5.97, 5.98 (each 1H, d, J=2.4 Hz), 6.89 (1H, s), 7.33 (1H, d, J=1.0 Hz), 7.50 (1H, s), 13CNMR (CD3OD) δC (ppm) 29.7, 67.0, 81.5, 95.9, 96.7, 99.8, 111.9, 116.2, 117.1, 133.4, 134.6, 135.6, 135.7, 152.60, 152.65, 155.0, 156.9, 157.8, 158.1, 183.4, ESI-TOF-MS m/z 401.0869 [M+H]+ for C20H16O9, MALDI-TOF-MS m/z 401 [M+H]+, 423 [M+Na] +, E1%1cm 371 (281 nm), 446 (306 nm).

1.2. Epitheaflagallin 3-O-gallate (6) Deep-orange solid; 1H-NMR (CD3OD) δΗ (ppm) 2.91, 3.07 (2H, m), 5.04 (1H, s), 5.67 (1H, m), 5.99 (1H, d, J=2.2 Hz), 6.02 (1H, d, J=2.2 Hz), 6.64 (1H, s), 6.89 (2H, s), 7.33 (1H, d, J=1.2 Hz), 7.46 (1H, s), 13C-NMR (CD3OD) δC (ppm) 27.1, 69.3, 80.4, 95.9, 96.9, 99.2, 110.2, 112.0, 116.1, 116.5, 121.1, 133.5, 134.3, 134.5, 135.8, 139.9, 146.4, 152.5, 152.7,

Biocatalytic Conversion of Green Tea Catechins …

423

155.1, 156.8, 158.0, 158.1, 167.4, 183.4, ESI-TOF-MS m/z 553.0993 [M+H]+ for C27H20O13, MALDI-TOF-MS m/z 553 [M+H] +, 575 [M+Na] +, E1%1cm 494 (280 nm), 469 (302 nm).

2. PEROXIDASE-MEDIATED CONVERSION OF GREEN TEA CATECHINS TO THEAFLAVINS IN CAMELLIA SINENSIS CELL CULTURES [5, 10] Theaflavins are orange or orange-red in color and possess a benzotoropolone skeleton, similar to the epitheaflagallins. Theaflavins are major pigments of black tea, which account for 2-6% of the dry weight of black tea extracts, and consist of four major derivatives: theaflavin (7), theaflavin 3-O-galate (8), theaflavin 3’-O-galate (9) and theaflavin 3,3’-di-Ogalate (10) (Figure 2). In 2003, Sang et al. reported a very simple method to produce theaflavin derivatives using horseradish peroxidase (HRP) and H2O2. [5] HRP is commercially available, inexpensive, and is able to catalyze the oxidation of tea catechins as efficiently as the endogenous polyphenol oxidase and peroxidase in the leaves of C. sinensis. The HRP/H2O2 system is a good mimic of pigment formation in black tea fermentation. As shown in Figure 2, the possible mechanism of formation of the benzotropolone skeleton of theaflavins is quite similar to the formation of epitheaflagallins. In the case of peroxidase oxidation, however, not only pyrogalloyl but also pyrocatechoyl and/or galloyl groups in the catechins are oxidized to quinone type intermediates to form the benzotropolone skeleton from the various tea catechins. For example, theaflavin is synthesized from EC (1) and EGC (3), theaflavin 3-Ogallate from EC (1) and EGCg (4), theaflavin 3’-O-gallate from ECg (2) and EGC (3), and theaflavin 3, 3’-di-O-gallate from ECg (2) and EGCg (4). The HRP/H2O2 system is a very convenient method for obtaining standard theaflavins when purified tea catechins are available. Although purified tea catechins are available, they are too expensive to produce functional food components. We applied the HRP/H2O2 system to the production of epitheaflagallins, but a large amount of purpurogallin was formed as a by-product, which is not suitable for food materials. Peroxidase catalyzed oxidation is apparently stronger than the laccase-mediated reaction and, therefore, is difficult to control in the conversion of tea catechin mixtures. Recently, Takemoto et al. of Shizuoka Prefectural University, Shizuoka, Japan, developed a Camellia sinensis cell culture system, which possesses high peroxidase activity, and can convert EC (1) and EGC (3) in green tea into theaflavin (TF, 7). This callus cell culture readily converts (1) and (3) to TF when H2O2 (at a final concentration of 0.045%) is added to reaction mixture, consisting of 6 mg (1), 6 mg (3), 1 ml acetone, 15 ml of 0.1 M phosphate buffer (pH 6.0), 2.5 ml of C. sinensis callus cell culture broth (peroxidase, 15.5 U/ml), 2.5 ml Gamborg’s (B5) medium (total volume, 20 ml). The reaction was allowed to proceed for 30 min and the yield of (7) was 48%. This process was the same as the method reported by Sang et al. in terms of the peroxidase oxidation of purified catechin derivatives. Takemoto and colleagues focused on increasing the yield of (7) from green tea extract. These extracts usually contains many catechin derivatives, but EC (1), ECg (2), EGC (3), and EGCg (4) make up ~10% (w/w), ~10%, >20%, ~50%, respectively, of the total catechin. Thus, the amount of (1) and (3) in green tea extracts are not sufficient to produce high amount of (7). Thus, Takemoto et al. developed C. sinensis callus cells, which possess not only peroxidase

424

Nobuya Itoh and Yuji Katsube

activity, but also a tannase-like (EC 3.1.1.20) hydrolase activity. The latter enzyme catalyzes the hydrolysis of ECg (2) to EC (1) and gallic acid, and EGCg (4) to EGC (3) and gallic acid (Figure 2). As a result, production of (7) from the green tea extract increased to levels suitable for practical production of (7) from green tea extracts. [11]

3. POTENTIAL ANTI-OBESITY BENEFITS OF EPITHEAFLAGALLIN 3-OGALLATE AND THEAFLAVIN 3-O-GALLATE THAT INHIBIT PANCREATIC LIPASE [12] There has been little physiological information regarding the effects of epitheaflagallins on human health. Epitheaflagallin 3-O-gallate (6) inhibits some inflammation-related matrix metallo-proteases (MMP-2,7, MT1-MMP), resulting in an anti-inflammatory effect, [13] and also possesses growth inhibitory activity toward human colon cancer cells. [5] In contrast, theaflavins, as well as EGCg in green tea, have attracted considerable interest because they possess potential benefits for human health, including antioxidant, [14] and antiviral [15,16] activities, inhibition of α-/β-glucosidase (anti-diabetic effect), [17] suppression of cytochrome P-450 activity (anti-mutagenicity), [18] inhibition of angiotensin converting enzyme (ACE) activity, [19] and cancer-preventing activity. [20] We have hypothesized that epitheaflagallins, especially epitheaflagallin 3-O-gallate (6), possess beneficial effects on human health as well, because of their structural similarity to TFs and (4). Recently, many beneficial effects of green tea have been attributed to its abundant catechin, EGCg (4), [21] and 3 O-gallate forms of catechins. In 2005, we initiated research to elucidate the functions of epitheaflagallins in human health. During the course of our study, we found that (6) and TF 3O-gallate (8) can inhibit pancreatic lipase. Figure 3 displays the dose-dependent inhibition of pancreatic lipase by the laccase-treated green tea catechins, the purified (6) and (8). Xenical, an anti-obesity drug which strongly inhibits pancreatic lipase, was used as a positive control. Although the effects of (6) (observed IC50: 1.2 mg/ml) and (8) (observed IC50: 0.4 mg/ml) were less dramatic than Xenical (observed IC50: 0.04 mg/ml), these compounds may still be suitable functional foods able to suppress the absorption of lipids during digestion. Interestingly, EGCg (4) and epitheaflagallin (5) had no effect on pancreatic lipase inhibition, indicating that the benzotropolone ring and the 3-O-gallate structure are necessary to inhibit pancreatic lipase. Figure 4 shows the changes in triglyceride concentration in the serum of rats after oral administration of lipid emulsions to rats that had been fasted overnight and then given the lipids in the presence or absence of laccase-treated green tea extract or (6). Epitheaflagallin 3-O-gallate (6) exhibited an inhibitory effect on lipid absorption in vivo in rats. In Japan, black oolong tea is commercially available as a food for qualified health use to suppress the absorption of dietary lipids. As epitheaflagallins and TFs have a mild, black tealike taste, a new tea drink rich in (6) and (8) could be popular for sale in Japan in the near future. Our research group has also confirmed the inhibitory effects of epitheaflagallins on glucosidases and on metastasis of human colon cancer. The detailed results concerning the beneficial effects of epitheaflagallins will be reported elsewhere.

Biocatalytic Conversion of Green Tea Catechins … O

OH

OH

O

OH

425

OR2 OH

O

HO

HO OH

H 2O 2 + P eroxidas e

OH OH

O

HO

E C (1)

OH

OH

OR1

OH

OH

O O

O

HO

OH OH

OH

T F (7): R 1 =H, R 2 =H T F  3‐O ‐gallate: R 1 =gallate, R 2=H T F  3’‐gallate: R 1=H, R 2 =gallate T F  3,3’‐di‐O ‐gallate: R 1 , R 2 =gallate

OH

E G C (2) H 2O 2 + P eroxidas e

T annas e/internal hydrolas e E C g (3) E G C g (4)

E C (1) E G C (2)

Figure 2. Schematic diagram of the process to produce TF from green tea extract using C. sinensis cell cultures.

Figure 3. Inhibition of pancreatic lipase by epitheaflagallin and TF derivatives.

Nobuya Itoh and Yuji Katsube

Changes in serum

riglyceride concentration (mg/dl)

426 160 140 120 100 80 60 40 20 0 -20

0

50

100

150

200

250

300

350

-40 Time after administration of lipid emulsion (min) epitheaflagallin 3-O-gallate (20 mg/kg)

control laccase-treated green tea (500 mg/kg)

Figure 4. Inhibitory effects of epitheaflagallins on absorption of triglycerides in rats.

ACKNOWLEDGEMENTS This work was supported by a grant for the “Toyama Medical Bio-Cluster” from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

REFERENCES [1]

[2] [3] [4]

[5]

Itoh, N.; Katsube, Y.; Yamamoto, K.; Nakajima, N.; Yoshida, K. (2007) Laccasecatalysed conversion of green tea catechins in the presence of gallic acid to epitheaflagallin and epitheaflagallin 3-O-gallate. Tetrahedron, 63, 9488-9492. Takino, Y. & Imagawa. H. (1964) Studies on the mechanism of the oxidation of tea leaf catechins. Agric. Biol. Chem., 28, 125-130. Takino, Y.; Imagawa. H.; Aoki, Y.; Ozawa, T. (1963) Studies on the mechanism of the oxidation of tea leaf catechins. Agric. Biol. Chem., 27, 562-568. Sang, S.; Tian, S.; Meng, X.; Stark, R.E.; Rosen, R.T.; Yang, C.S; Ho, C.-T. (2002) Theadibenzotropolone A, a new type pigment from enzymatic oxidation of (−)epicatechin and (−)-epigallocatechin gallate and characterized from black tea using LC/MS/MS. Tetrahedron Lett., 43, 7129-7133. Sang, S.; Lambert, J.D.; Tian, S,; Hong, J.; Hou, Z.; Ryu, J.-H.; Stark, R.E.; Rosen, R.T.; Huang, M.-T.; Yang, C.S; Ho, C.-T. (2004) Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities. Bioorg. Med. Chem., 12, 459-467.

Biocatalytic Conversion of Green Tea Catechins … [6]

[7]

[8]

[9] [10] [11] [12] [13] [14]

[15]

[16]

[17] [18]

[19] [20]

[21]

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Matsuo, Y.; Tanaka, T.; Kouno, I. (2006) A new mechanism for oxidation of epigallocatechin and production of benzotropolone pigments. Tetrahedron, 62, 47744783. Takino, Y.; Ferretti, A.; Flanagan, V.; Gianturco, M.A.; Vogel, M. (1967) Spectral evidence for the structure of three flavanotropolones related to theaflavin, an orangered pigment of black tea. Can. J. Chem., 45, 1949-1956. Nonaka, G.; Hashimoto, F.; Nishioka, I. (1986) Tannins and related compounds XXXVI. Isolation and structures of theaflagallins, new red pigments from black tea. Chem. Pharm. Bull. 34, 61-65. Tanaka, T.; Kouno, I. (2003) Oxidation of tea catechins: chemical structures and reaction mechanism. Food Sci. Technol. Res. 9, 128-133. Takemoto, M. (2007) JPN Patent Application, No. 2007-143461 (Japanese). Takemoto, M. (2007) JPN Patent Application, No. 2007-182217 (Japanese). Itoh, N.; Matsunaga, T.; Ogasawara, M.; Katsube, Y.; Yamamoto, K. (2007) JPN Patent Application, No. 2007-285650 (Japanese). Akizawa, T.; Yahara, M.; Hashimoto, F. (2000) JPN Patent Application, No. 2000226329 (Japanese). Shiraki, M.; Hara, Y.; Osawa, T.; Kumon, H.; Nakayama, T.; Kawakishi, S. (1994) Antioxidative and antimutagenic effects of theaflavins of black tea. Mutation Res., 323, 29-34. Nakayama, M.; Suzuki, K.; Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. (1993) Inhibition of the infectivity of influenza virus by tea polyphenols. Antiviral Res., 21, 289-99. Ono, K.; Nakane, H.; Fukushima, M.; Chermann, J.C.; Barré-Sinoussi, F. (1991) Inhibition of reverse transcriptase activity by a flavonoid compound, 5,6,7trihydroxyflavone. Biochem. Biophys. Res. Commun., 174, 56-62. Honda, M.; Hara, Y. (1993) Inhibition of rat small intestinal sucrose and α-glucosidase activities by tea polyphenols. Biosci. Biotech. Biochem., 57, 123-124. Apostolides, Z.; Balentine, D.A.; Harbowy, M.E.; Hara, Y.; Weisburger, J.H. (1997) Inhibition of PhIP mutagenicity by catechins, and by theaflavins and gallate esters. Mutation Res., 389, 167-72. Hara, Y.; Matsuzaki, T.; Suzuki, T. (1987) Angiotensin I converting enzyme inhibiting activity of tea components. Nippon Nougeikagaku Kaishi (Japanese), 61, 803-808. Matsumoto, N.; Kori, T.; Okushio, K.; Hara, Y. (1996) Inhibitory effects of tea catechins, black tea extract and oolong tea extract on hepatocarcinogenesis in rat. Jpn. J. Cancer Res., 87, 1034-1038. Liao, A; Kao, Y.-H.; Hiipakka, R. A. (2001) Green tea: biochemical and biological basis for health benefits. Vitamins and Hormones, 62, 1-94.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 23

PREVENTIVE EFFECTS OF GREEN TEA CATECHINS ON DEMENTIA Michio Hashimoto1,*, Md Abdul Haque1, Kohinoor Begum Himi1 and Yukihiko Hara2 1

Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo, Shimane 693-8501, Japan 2 Mitsui Norin Co., Ltd., Nishishinbashi, Minato-ku, Tokyo 105-8427, Japan

ABSTRACT Tea is rich in polyphenols which are contained in the leaves and stems of the tea plant. (-)-Epigallocatechin gallate (EGCG), the major and most active component of green tea catechins, acts as an antioxidant in the biological system, and is absorbed and distributed mainly into the mucous membranes of the small intestine and liver; more interestingly it can cross the blood brain barrier. Oxidative stress, a condition of cellular prooxidant-antioxidant disturbance which favors the prooxidant state, induces the production of lipid peroxide (LPO), reactive oxygen species (ROS) and free radicals in membrane lipids. Oxidative stress causes deterioration of a wide variety of cellular enzymes, subsequently exacerbating neurodegenerative process. Aging leads to a decline in memory-related learning ability. Oxidative damage to the brain is associated with age-related cognitive dysfunction but some antioxidants are effective in alleviating this dysfunction for Alzheimer’s disease (AD) model animals. Moreover, a decrease in hippocampal LPO level improves spatial cognition learning in aged rats and an increase in antioxidative activity in the hippocampus prevents or ameliorates the impairment of learning ability in AD model rats produced by the infusion of amyloid-β (1-40) (Aβ1-40) into their cerebral ventricle. Catechins have a protective effect against age-related neurological diseases caused by oxidative damage. Epidemiological studies report that a higher consumption of green tea is associated with lower prevalence of cognitive impairment in elderly people. This chapter describes the *

Corresponding author: Michio Hashimoto. Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo, Shimane 693-8501, Japan; E-mail: [email protected]. Telephone: +81 853 20 2112; Fax: +81 853 20 2110.

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Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi et al. beneficial effects of green tea catechins on neuronal functions and neuronal diseases such as dementia.

INTRODUCTION Tea is the most widely consumed beverage in the world (mean consumption: approximately 120 ml/day/percapita) [McKay and Blumberg, 2002]. It has only recently been studied extensively as a health-promoting beverage to prevent a number of chronic diseases such as cancer [Inoue et al., 1998; Mittal et al., 2004], cardiovascular diseases [Geleijnse et al., 1999; Negishi et al., 2004], diabetes [Anderson and Polansky, 2002; Wu et al., 2004a; 2004b] and neurodegenerative diseases including Parkinson’s disease (PD) [Choi et al., 2002; Pan et al., 2003; Weinreb et al., 2004] and Alzheimer’s disease (AD) [Levites et al., 2003; Mandel et al., 2005; Bastianetto et al., 2006]. Oxidative damage to neuronal biomolecules such as DNA, proteins, and lipids causes the aging process and contributes to the pathogenesis of various neurodegenerative disorders [Halliwell, 1989] including age-related decline in learning and memory [Foster, 2006]. Oxidative stress is clearly evident in AD brain manifested by lipid peroxidation (LPO) and protein oxidation, along with other markers of oxidative damages [Butterfield et al., 2007]. Most individuals with amnestic mild cognitive impairment (MCI), an earliest phase of AD [Petersen et al., 2001; Petersen, 2004], also demonstrate elevated oxidative stress markers in their brain [Keller et al., 2005; Wang et al., 2006; Bufferfield et al., 2006; 2007]. Hippocampus is an indispensable part of the brain in order to form spatial memory and it is one of the first regions to suffer oxidative damage in AD [Mortimer et al., 2004]. Spatial cognition-related memory refers to the ability to recall previously navigated cues oriented in a specific arrangement in an individual’s environment. Any lesion or damage in the hippocampus has been reported to compromise the spatial learning in rats. According to cognitive map hypothesis, an intact hippocampal function is the prerequisite for accumulating, processing and storing spatial information of an individual’s environment [Jeffery and Hayman, 2004]. Infusion of amyloid β peptide (1-40) (Aβ1-40) into rat brain increases the concentrations of LPO and reactive oxygen species (ROS), and correlates with an impairment of reference and working memory; this indicates a deficit in spatial learning ability, a well-known characteristic of AD [Hashimoto et al., 2002; 2005a]. While tea contains a number of bioactive chemicals, it is particularly rich in polyphenolic compounds known as catechins which contribute to the beneficial effects ascribed to tea. The principal catechins in green tea are (-)-epicatechins (EC), (-)-epicatechin gallate (ECG), (-)epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCG). Among them EGCG is the most abundant and active component of green tea. It can pass the blood brain barrier [Suganuma et al., 1998; Nakagawa et al., 1997a; Abd EI Mohsen et al., 2002] and causes neuroprotective activity. Among different biological properties of green tea catechins, the potent antioxidant and radical scavenging properties have led to them a number of diseases associated with oxidative insults. It has been demonstrated that EGCG protects neurons against conditions such as hypoxia [Burckhardt et al., 2008], ischemia [Lee et al., 2000; Lee et al., 2004], AD [Mandel et al., 2005], and PD [Checkoway et al., 2002; Tan et al., 2003]. Epidemiological and nutritional studies demonstrate that a higher consumption of green tea

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(two cups/day or more) lowers the prevalence of cognitive impairment in elderly people [Kuriyama et al., 2006] and reduces risk of PD [Checkoway et al., 2002]. In animal studies, administration of green tea catechins improves spatial cognitive learning ability in normal young rats [Haque et al., 2006] and prevents impairment of learning ability in AD model rats [Haque et al., 2008]. Green tea extract and EGCG also effectively prevent N-methyl-4phenyl-1,2,3,6-tetrahydropyridine-induced mouse striatal dopamine depletion and substantia nigra dopaminergic neuron loss [Levites et al., 2001], and suppress cognitive impairment in aged SAMP10 mice, a model of brain senescence with cerebral atrophy and cognitive dysfunction [Unno et al., 2004]. We addressed the beneficial effects of the green tea catechins on neuronal functions with special emphasis on dementia.

SOURCES, ABSORPTION AND DISTRIBUTION OF GREEN TEA Tea belongs to the Theacease family and comes from two main varieties: Camellia sinensis var. sinensis and Camellia sinensis var. assamicsa. Depending on the manufacturing process, teas are classified into three major types: ‘non-fermented’ green tea (produced by drying and steaming the fresh leaves); ‘semi-fermented’ oolong tea (produced when the fresh leaves are subjected to a partial fermentation stage before drying); and ‘fermented’ black tea (post-harvest fermentation stage before drying and steaming). The mean chemical composition of green tea leaves are as follows (% of dry weight of tea leaves): 15 proteins, 4 aminoacids, 26 fibres, 7 other carbohydrates, 7 lipids, 2 pigments, 5 minerals, 30 phenolic compounds (especially flavonoids). The main flavonoids present in green tea include catechins (flavan-3-ols), and the four major catechins are EGCG (approximately 60% of the total catechins), EGC (approximately 19%); ECG (approximately 13.6%) and EC (approximately 6.4%). In Figure 1, a possible metabolic route of EGCG is shown. When oral administration of EGCG to rats, part of EGCG is absorbed in the intestine and enters into liver via the portal vein [Okushio et al., 1995; 1996]. During this process most of the EGCG undergoes conjugation in the intestinal mucosa and/or in the liver, and a part of the EGCG is further methylated in the liver. Most of the EGCG metabolites are then excreted in the bile [Kida et al., 2000] and a part of the EGCG enters the blood circulation, peaking at 1-2 h after administration [Unno and Takeo, 1995; Nakagawa et al., 1997b, Chen et al., 1997]. On the other hand, most of the unabsorbed EGCG moves into the cecum and large intestine and then undergoes degradation by intestinal bacteria to 5-(3', 5'-dihydroxyphenyl)-γ-valerolactone (M-1) with EGC as an intermediate. A great part of M-1 is absorbed in the body, undergoing glucuronidation in the intestinal mucosa and/or liver, to form 5-(5'-hydroxyphenyl)-γvalerolactone 3'-O-β-glucuronide (M-2), which enters the blood circulation is distributed to various tissues, and finally excreted in the urine. The occurrence of tea catechin EGCG in free form in the various tissue organelles of rats is sown in Table 1.

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portal vein

blood circulation bioavailability < 0.3%

EGCG

tissue < 0.2%

EGCG EGCG-conj EGC+ GA

bile (6%) EGCG EGCG-conj Me-EGCG-conj

M-1

M-2

liver

kidney

M-1 M-2

EGCG, ECG, M-1

M-2 urine (32%)

feces (35%)

Figure 1. Possible metabolic route of epigallocatechin-3-gallate (EGCG) orally administered to rats. GA, gallic acid; M-1, 5-(3',5',-dihydroxyphenyl)-γ-valerolactone; M-2, 5-(5'-hydroxyphenyl)-γvalerolactone 3'-O-β-glucuronide; EGCG-conj, EGCG conjugates; Me-EGCG-conj, Methylated EGCG conjugates. Data from Kohir et al., (2001).

Table 1. EGCG detected in blood and tissue organelles of rats Tissue

60 min after administration a

EGCG detected c (%)

Plasma (nmol/mL)

12.3 ± 6.2

0.024 b

Liver (nmol/g) Brain (nmol/g)

48.4 ± 19.7 0.5 ± 0.3

0.19 b 0.0003 b

Small intestine mucosa (nmol/g)

565 ± 153

0.45 b

Colon mucosa (nmol/g)

68.6 ± 40.4

0.013 b

Data are expressed as the mean ± SD of six rats. EGCG: (-)-epigallocatechin gallate, a Rats received a single oral administration of EGCG (500 mg/Kg body weight) after 24 h of food deprivation. b Percentage against ingested EGCG. c Calculated from the blood mass and from the tissue weights of rats. Data from Nakagawa and Miyazawa, (1997a).

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OXIDATIVE STRESS AND NEURODEGENERATION Oxidative stress is a condition of cellular oxidant-antioxidant imbalance in favor of prooxidant state, i.e a relative increase in the ratio of free radicals to antioxidants [Markesbery 1997; van Rensburg et al., 2006]. Patients with neurodegenerative diseases, such as AD, PD and amyotrophic lateral sclerosis (ALS), clearly exhibit higher oxidative stress (ROS concentrations) in affected brain regions. The brain zones demonstrating the highest levels of oxidative stress are typically the areas most structurally affected by diseases: hippocampus, amygdale, parietal cortex, and other neocortical zones [Markesbery, 1997; Butterfield et al., 2007]. Others tissues of AD patients also manifest oxidative stress. van Rensburg et al. [2006] reported that the blood of AD patients demonstrates increased oxidative stress and abnormally poor antioxidant status compared to healthy controls. According to the free-radical theory of AD, an increased production of LPO and ROS, which are produced with free radicals in membrane lipids, causes deterioration of a wide variety of cellular enzymes, subsequently exacerbating the neurodegenerative process [Yatin et al., 1999]. A lower LPO level in hippocampus is inversely associated with spatial cognition learning memory both in young and aged rats [Gamoh et al., 1999; 2001]. In AD brain, markers for lipid peroxidation such as 4-hydroxynonenal (4-HNE) and malondialdehyde, and marker of protein oxidation such as nitrated proteins are identified in cerebral cortex and hippocampus. 4-HNE can diffuse from the site of its production, potentially modify neuronal organelles and changes their functions [Butterfield et al., 1997]. Approximately 2-5% of the oxygen consumed by a cell is subsequently converted to free radicals [Floyd and Hensley, 2002; Wickens 2001]. The brain is poor in antioxidative enzymes such as catalase, glutathione peroxidase and superoxide dismutase, compared with other organs of the body such as the heart, liver, and kidneys [Mizuno and Ohta, 1986; Cohen, 1988]. On the other hand, the brain alone utilizes 20% of total oxygen demand of the body [Cui et at., 2004]. Therefore, the damaging effects are dependent obviously on the balance between cellular oxidative damage and the antioxidative defenses. AD is histochemically characterized by the deposition of amyloid β peptide (Aβ) in the hippocampus as well as in the cerebral cortex, and also by neuronal degeneration and loss of cognition [Wilcock and Esiri, 1982; Selkoe, 2001]. Genetic mutations and other mechanisms that potentially lead to increase Aβ deposition contribute to neurotoxicity. Though it is not conclusive how exactly Aβ contributes to the disease process, oxidative stress is believed to involve the mechanism of Aβ-induced neurotoxicity [Behl et al., 1992; 1994; Butterfield et al., 1994; Schubert et al., 1995] and the pathogenesis of AD [Markesbery 1997; Yankner 1996]. Aβ causes lipid peroxidation in brain cell membranes [Behl et al., 1994; Harris et al., 1995; Mark et al., 1999], alters the conformation of neuronal membrane proteins [Subramaniam et al., 1997; Pocernich et al., 2001] and induces disorder of cellular functions.

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Odds ratios of cognitive impairment

2.00

< 3 cups/wk (reference)

1.80

4-6 cups/wk or 1 cup/d

1.60

> 2 cups/d

1.40

P for trend = 0.0006

1.20 1.00 0.80

*

0.60 0.40 0.20 0.00 n=170

n=108

n=725

Figure 2. Odds ratios (ORs) for the association between different frequencies of green tea consumption and cognitive impairment. The bars indicate adjusted ORs for the association between green tea consumption frequencies and cognitive impairment; error bar represent the corresponding 95% CIs. Multivariate logistic regression analysis was used to calculate ORs for cognitive impairment relative to the consumption frequencies of green tea, with the lowest frequency category (≤3 cups/wk) treated as the reference group. Cognitive impairment was defined as a Mini-Mental State Examination score < 26. *P< 0.001. 1 cup = 0.1L. Data from Kuriyama et al., (2006).

GREEN TEA CATECHINS AND BRAIN FUNCTION Influence of Green Tea Catechins on Cognitive Function Green tea catechins currently show a profound beneficial effect on cognitive function both in animal and humans. An epidemiological cross-sectional study involving 1003 Japanese subjects 70 years old or older, demonstrated the relationship between the consumption of green tea and cognitive function. The study clearly showed that a higher consumption of green tea is associated with lower prevalence of cognitive impairment in humans [Kuriyama et al., 2006]. Drinking more than 2 cups a day of green tea slashed odds of cognitive impairment in elderly Japanese men and women by 64% (Figure 2). The results might partly explain the relatively lower prevalence of dementia, especially AD, in Japan compared to Europe and North America [Ritchie and Lovestone, 2002].

Preventive Effects of Green Tea Catechins on Dementia A

B

4

Control (n=8)

4

3

2

a b

1

0

Working Memory Errors

Reference Memory Errors

435

Catechins (n=9) 3

2

1

a b

0 0

2

4

6

8

Blocks of Six Trials

10

0

2

4

6

8

10

Blocks of Six Trials

Figure 3. Reference (A) and working (B) memory-related learning ability in the radial maze task of rats administered water alone (control, n = 8), or green tea catechins (0.5% polyphenon E; n = 9) for 26 wk. Values are means ± SEM in each block of six trials. Groups without a common letter differ, P < 0.05. Data from Haque et al., (2006).

In animal experiments with rats, long-term administration of green tea catechins in the form of Polyphenon E (PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%) mixed with water (0.5% w/v) improved spatial cognition learning ability when measuring eight-arm radial maze task (Figure 3). The radial maze estimates two types of memory function, reference and working memory without any harmful stress to the rats. Reference memory involves utilizing information that remains constant over time whereas working memory involves holding information that is pertinent only within a short period of time. The lower numbers of reference memory errors (RMEs; entry into unbaited arm within one trial) and working memory errors (WMEs; repeated entry into arms that had already been visited within same trial) implies a higher acquisition of spatial learning ability in rats. 0.5% PE-administered rats had significantly lower LPO levels both in plasma and hippocampus and higher ferric reducing antioxidant power (FRAP) levels (an indicator of plasma antioxidant status) (Table 2). A significant positive correlation between the hippocampal LPO levels and the number of RMEs, and a negative correlation between plasma FRAP levels and the number of RMEs were observed in block 10 of the radial maze task in controls and in 0.5% PE-administered rats (Figure 4). These results indicate that the lower LPO and higher FRAP levels, combined with higher acquisition of memory performance, are likely to be the effects of PE on scavenging and/or preventing radical formation at the neuronal level. Similarly, it is reported that dietary administration of green tea catechins prevents memory regression and DNA oxidative damage in aged mice [Unno et al., 2007]. Thus, these findings suggest that continued intake of green tea catechins might promote healthy ageing of the brain in older persons.

Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi et al.

A r = - 0.570 p = 0.017

r = 0.520 p = 0.032

3.0 Reference M emory Errors

B 3.0.

2.0

2.0

1.0

1.0 Control (n=8) Catechins (n=9)

0.0 0

0.25

0.50

1

0.75

150

TBARS levels in hippocampus (nmol/mg protein)

Reference M emory Errors

436

0.0 200

250

300

350

FRAP levels in plasma ( mol/L)

Figure 4. Correlations between the numbers of reference memory errors and each of the hippocampal TBARS (Figure 4A) and the plasma FRAP (Figure 4B) levels in controls and green tea catechinsadministered rats. (o), control rats; (●), green tea catechins (0.5% polyphenon E)-administered rats. Data from Haque et al., (2006).

Table 2. Effects of green tea catechins (0.5% PE) administration on oxidative status of plasma, cerebral cortex and hippocampus in rats Plasma

Cerebral cortex

Hippocampus

TBARS

FRAP

TBARS

ROS

TBARS

ROS

Control (n=8)

4.0 ± 0.1

224 ± 10

1.45 ± 0.10

0.21 ± 0.03

0.63 ± 0.09

0.13 ± 0.03

Catechins (n=9)

3.0 ± 0.1*

271 ± 11*

1.27 ± 0.08

0.19 ± 0.04

0.33 ± 0.03*

0.05 ± 0.01*

Values are means ± SEM. Rats were orally administered either water (control rats) or green tea catechins (Polyphenon E, PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%, catechins rats) for 26 weeks. The levels of lipid peroxide were measured as TBARS (thiobarbituric acid reactive substance) indicated in nmol malondialdehyde/mL for plasma and nmol malondialdehyde/mg protein for brain tissues. Reactive oxygen species (ROS) is indicated in pmol/min/mg protein. The antioxidant potential of plasma was mesured as ferric reducing antioxidation power of plasma (FRAP) is indicated in μmol/L. *P<0.05. Data from Haque et al., (2006).

Preventive Effects of Green Tea Catechins on Dementia B

A

Working Memory Errors

Reference Memory Errors

Aβ (n=13)

4.0

3.5

3.0 a 2.5 b 2.0

1.5

437

Catechins + Aβ (n=12)

3.0

2.0 a 1.0

b

0.0 0

1

2

3

4

5

Blocks of Six Trials

6

0

1

2

3

4

5

6

Blocks of Six Trials

Figure 5. Effects of green tea catechins (0.5% polyphenon E) preadministration to Aβ1–40-infused rats on reference and working memory-related learning ability in the eight-arm radial maze task. Each value represents the numbers of reference memory errors (A) and that of working memory errors (B) as the means ± SEM in each block of six trials. Groups without a common letter for the main effects are significantly different at P<0.05. Data from Haque et al., (2008).

Preventive Effect of Green Tea Catechins on Cognitive Impairment We reported previously that preadministration of docosahexaenoic acid protects against the impairment of learning ability in an animal model of AD, rats infused with Aβ1–40 into the cerebral ventricle [Hashimoto et al., 2002]. It has been demonstrated that long-term preadministration of PE prevents cognitive impairment in AD model rats (Figure 5). The preventive effect of green tea is measured by an eight-arm radial maze. Randomized twofactor (block and group) ANOVA demonstrates significant main effects of blocks of trials and groups on the number of RMEs and that of WMEs, indicating that PE preadministration prevents Aβ1–40-infused learning impairment of AD model rats. Chronic administration of EGCG prevents cognitive impairment in Alzheimer’s transgenic mice [Rezai-Zadeh et al., 2008]. As described previously, an epidemiological Japanese study also showed that a higher consumption of green tea is associated with lower prevalence of cognitive impairment in humans [Kuriyama et al., 2006]. Therefore, these findings suggest that green tea catechins might be an effective prophylaxis for cognitive impairment in AD patients.

Antioxidant Effects of Green Tea Affect Brain Function The impact of an antioxidant rich diet and specific food groups on degenerative diseases has been widely recognized. It has been reported that a decrease in LPO level or an increase in antioxidative defense in hippocampus prevents and/or ameliorates the impairment of learning ability in Aβ1-40-infused AD model rats [Hashimoto et al., 2002; 2005a]. The infusion of Aβ1-40 increases the cortico-hippocampal LPO and ROS concentrations in the AD rats, while PE preadministration suppresses these increase of LPO and ROS concentrations

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(Table 3). Regression analysis revealed inverse correlations between plasma EGCG concentrations and hippocamapl ROS levels (Figure 6), suggesting that the antioxidative action of EGCG is involved in reducing the Aβ-induced oxidative stress in the hippocampus. Significantly positive correlations between the numbers of RMEs and the levels of TBARS in plasma and hippocampus and between the numbers of WMEs and the ROS levels in hippocampus were found (Table 4). In addition, PE preadministration increased plasma FRAP levels and the FRAP values were negatively correlated with the numbers of WMEs (Table 4). These results suggest that the antioxidative action of PE is involved in the prevention of cognitive impairment in AD model rats. Catechins 1.25

Catechins + A

Hippocampal ROS (pmol/min/ mg protein)

1.00

0.75

0.50 r = - 0.586 P = 0.007

0.25

0.00 0

10

20

30

40

50

Plasma EGCG (ng/ml)

Figure 6. Relationship between plasma EGCG concentrations and hippocampal ROS levels after longterm administration of green tea catechins (0.5% polyphenon E).

Table 3. Oxidative status of cerebral cortex and hippocampus of Aβ and PE+Aβ rats TBARS (nmol/mg protein)

ROS (pmol/min/mg protein)

Cerebral cortex (n=13)

3.13 ± 0.23

0.223 ± 0.02

PE + A (n=12)

2.74 ± 0.22*

0.130 ± 0.02*

3.54 ± 0.05

0.332 ± 0.03

A

Hippocampus A

(n=13)

PE + A (n=12)

2.10 ±

0.13*

0.162 ± 0.01*

Values are means ±SEM. Rats were orally administered either water or green tea catechins (Polyphenon E, PE). Twenty weeks after the PE administration, rats were infused with amyloid β 1-40 peptide (Aβ140 ) into cerebral ventricle. Aβ, the water-preadmiistered Aβ1-40-infused rats; PE+Aβ, 0.5% PEpreadministered Aβ1-40-infused rats. The levels of lipid peroxide were measured as TBARS (thiobarbituric acid reactive substance). ROS, Reactive oxygen species. *P<0.05. Data from Haque et al., (2008).

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Table 4. Correlation coefficients between learning ability and oxidative stress on plasma and brain of rats Plasma TBARS FRAP

Cerebral cortex TBARS ROS

Hippocampus TBARS ROS

RMEs P-value

+ 0.324 0.023

- 0.272 0.058

NS

NS

+ 0.440 0.016

+ 0.280 0.051

WMEs P-value

NS

- 0.296 0.039

NS

NS

+ 0.245 0.089

+ 0.292 0.041

The numbers of reference memory errors (RMEs) and working memory errors (WMEs) in the final session shown in Figure 5 was used as an indicator of learning ability.The data of TBARS and ROS in cerebral cortex and hippocampus shown in Table 3 were used. Differences with P < 0.05 were considered significant NS, not significant. Data from Haque et al., (2008).

GREEN TEA CATECHINS AND ANTIOXIDATIVE ACTION IN BRAIN Tea catechins are powerful hydrogen-donating antioxidants and free radical scavengers [Salah et al., 1995; Nanjo et al., 1996; Rice-Evans, 1999]. The potent radical scavenging properties of green tea polyphenols have been attributed to the presence of the ortho-3′,4′dihydroxy moiety in the B ring of their molecule which participates in electron delocalization and stabilization of the radicals. The gallocatechins possess a trihydroxyl group in the B ring (3′, 4′, 5′-OH) and the gallates contain a galloyl moiety attached to flavan-3-ol at the 3 position (C ring), adding three more hydroxyl groups, as in the case of ECG and EGCG (Figure 7). Both the galloyl moiety and the three hydroxyls in the B ring confer better antioxidant activity [Nanjo et al., 1996]. In addition to their radical scavenging action, green tea catechins possess well-established metal-chelating properties. This is likely to be mediated through the 3′,4′-dihydroxyl group in the B ring [Hider et al., 2001] as well as via the gallate group [Guo et al., 1996; Kumamoto et al., 2001] which neutralize ferric iron to form redoxinactive iron and protect cells against oxidative damage. Green tea polyphenols have been found to be more effective antioxidants than vitamins E and C on a molar basis, as indicated by their reduction potentials (Rice-Evans et al., 1995). In reducing ferrous ion-induced lipid peroxidation, the IC50 values of several antioxidants are as follows: 3.32 μM for EGCG, 75.65 μM for trolox, 7.63 μM for lipoic acids and 15.48 μM for melatonin (Lee et al., 2003). OH

OH HO 7 6

8

O

A

C

1 2 3 4

5

OH

2 3 4 B 1 6 5

x =

HO 7 6

OR1 OH OH OH

2 3 4 B 1 6 5

OH 8

A 5

OH

O 1 C 4

OH

2 3

OR2

R1= H: (-)- Epicatechin (EC) R2= H: (-)- Epigallocatechin (EGC) R1= x: (-)- Epicatechin gallate (ECG) R2= x: (-)- Epigallocatechin gallate (EGCG)

Figure 7. The basic structural formulas of tea catechins.

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Catechins increase the body's endogenous antioxidants to reduce oxidative damage. In ageing, cells of the nervous system are vulnerable to oxidative damage. The antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) play an important role in preventing oxidative damage. Among various isoforms of SOD, the mitochondrial manganese-dependent superoxide dismutase (MnSOD) contributes a major role to detoxify superoxide radicals and prevents neuronal cell death in the brain [Keller et al., 1998; Gonzalez-Zulueta et al., 1998; Maragos et al., 2000]. SOD neutralizes superoxide radicals to form hydrogen peroxide and molecular oxygen. The efficacy of SOD as an antioxidant depends on cooperation with other enzymes, such as CAT and GPx. CAT detoxifies high concentrations of H2O2 [Baud et al., 2004; Kono and Fridorich, 1982] although its activity is much lower in the brain [Ho et al., 1997]. The activity of GPx and GR is also important in regulating the level of H2O2 [de Haan et al., 1998; Dringen et al., 2005]. GPx limits the production of LPO by directly catalyzing the conversion of peroxidated lipid into lipid alcohol [Christophersen, 1968]. GR indirectly facilitates to inactivate oxygen radicals [Huang and Philbert, 1996]. A decrease in GR activity increases GSSG accumulation and results in a depletion in the intracellular GSH pool. This eventually predisposes the cells towards oxidative stress. We found that six months administration of green tea catechins to rats enhances gene expression and activity of SOD, CAT, GPx and GR in brain (unpublished data). These findings are consistent with the report [Kishido et al., 2007] that consumption of green tea catechin prevents the decline in GPx activity and protein oxidative damage in ageing mouse brain. One month administration of catechin-containing antioxidant preparation also demonstrates an increase in SOD activity in the mitochondrial fraction of the striatum and the midbrain and a decrease in TBARS formation in the cortex and cerebellum of aged rats [Komatsu and Hiramatsu, 2000]. Phenolic antioxidants activate the expression of some stress-response genes [Chen et al., 2000], probably binding to the antioxidant regulatory element (ARE) present in the promoter of their respective genes. The NF-E2-related transcription factor 2 (Nrf2) is known to activate ARE-mediated gene expression of many antioxidants enzymes [Rushmore et al., 1991]. It has been reported that the transcriptional activities of the stress-response genes correlate with increased activity and nuclear binding of the transcription factors Nrf2 to the ARE sequences contained in their promoters via activation of the MAPK pathway [Owuor and Kong, 2002]. Green tea polyphenols demonstrate the ability to affect cell signaling pathways, in particular the MAPK signaling pathways inside brain cells which play a critical role in neurodegenerative diseases. Therefore green tea polyphenols may also protect cells against toxic insults through MAPK's activation of transcription factors, which provide the neuronal cells with acquired antioxidant defense capacity to survive against oxidative stress. The pathological accumulation of Aβ in the brain leads to oxidative damage, neuronal destruction and, finally to the clinical syndrome of AD. Since a reduction of Aβ level in cerebral cortex and hippocampus ameliorates the impairment of memory learning in Aβinfused AD model rats [Hashimoto et al., 2005b], the regulation of Aβ synthesis would be an important target to prevent Aβ-induced cognitive impairment. EGCG decreases Aβ1–40,42 levels and attenuates Aβ plaques across the hippocampal and cortical brain regions in TgAPPsw mice, a mouse model of AD [Rezai-Zadeh et al., 2008].

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Figure 8. Possible targets of EGCG to prevent neurodegeneration. EGCG, a main green tea catechin may increase cell survivability or prevents neuronal cell death either directly preventing oxidative stress or indirectly modulating MAPK cascade or Aβ synthesis pathways. Long-term administration of EGCG may down regulate β-secretase 1 (BACE 1) gene expression, up regulates transthyretin (TTR) gene expression in brain and these combine effects may reduce Aβ levels either inhibiting Aβ production and/or accelerating Aβ clearance and finally reduce Aβ-induced LPO and ROS levels and prevent neuronal cell death. EGCG may also activate PKCα and ε, the two specific isoforms of PKC, increase α-secretase cleavage activity and modulates non amyloidigenic pathway for APP processing. EGCG may inhibit NFκb signaling pathways, a known pathway for cell death, and decreases apoptosis relatedgene expression such as bax and bad. EGCG may also activate PI3 kinase pathway which is associated with antiapoptosis signaling in neurons. For details please see text.

The possible targets of EGCG-mediated prevention of neurodegeneration are diagrammed in Figure 8. EGCG may increase cell survivability or prevent neuronal cell death in neuronal diseases such as AD either by directly attenuating oxidative stress or indirectly modulating MAPK cascade or APP processing pathways. APP is the precursor molecule of Aβ, first cleaved by either α-secretase or β-secretase 1 (BACE1) and the resultant membraneattached fragments are processed by γ-secretase (Presinilin 1, 2; PS1, PS2). Up regulation of BACE1 increases the production of Aβ. Long-term administration of green tea catechins has been shown to decrease BACE1 gene expression in the hippocampus (unpublished data). Alternatively, EGCG activates PKCα and ε, the two specific isoforms of PKC which increase α-secretase cleavage activity and promotes non amyloidigenic pathway for APP processing to produce and secretes a soluble form of APP (sAPPα) [Levites et al., 2003]. Administration of green tea catechins up regulates the mRNA expression of transthyretin, a carrier protein of thyroxin and retinol, in the hippocampus and the cerebral cortex (unpublished data). This protein also acts as an Aβ scavenger, and prevents Aβ aggregation and plaque formation in brain [Schwarzman et al., 1994]. Taken together it is suggested that EGCG administration could reduce Aβ levels either by inhibiting Aβ production and/or accelerating Aβ clearance in

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conjunction with facilitating the non-amyloidogenic α-secretase pathway, and the combination of these effects may lead to reduced Aβ-induced oxidative stress (such as decrease LPO and ROS levels) and prevent related neuronal cell death. Other cell signaling pathways may also be affected by green tea to improve neuronal survivability. It has been reported that EGCG inhibits neuronal translocation of NFκb to the nucleus [Levites et al., 2002; Aktas et al., 2004], a known pathway for cell death. In addition EGCG decreases apoptosis related-gene expression such as bax and bad [Weinreb et al., 2003; Kalfon et al., 2007] and activates PI3/AKt kinase pathway [Koh et al., 2004] which are involved in antiapoptotic signaling in neurons. From these observations, it is suggested that the potency which neuronal cells achieve by the antioxidative action of green tea catechins may prevent oxidative insults, Aβ-induced or otherwise at least facilitating antioxidant defense.

CONCLUSION Green tea catechins manipulate multiple desired targets in the central nervous system. Administration of green tea catechins reduces brain oxidative stress and improves spatial cognitive learning ability in normal rats and prevents cognitive impairment in Aβ-infused AD model rats. Green tea catechins decrease Aβ levels and Aβ plaques in AD model mice. Furthermore, catechins increase the activity and gene expression of antioxidant enzymes in rat brains. All the evidence taken together suggests that green tea catchins strengthen antioxidant defenses in the brain against oxidative stress, Aβ-induced, and/or otherwise, and it seems to be a therapeutic strategy in preventing neurodegenerative diseases where oxidative stress is implicated. However, detailed long-term research is required to clarify the mechanism of how green tea catechins contribute to the prevention of age-related dementia in particular to cognitive impairment.

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In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Short Commentary

GREEN TEA AND POTENTIAL HUMAN HEALTH EFFECTS James E. Trosko* Center for Integrated Toxicology, Food Safety Toxicology Center Dept. Pediatrics/Human Development, College of Human Medicine Michigan State University, East Lansing, Michigan 48824, USA

Keywords: Green tea; gap junctions; adult stem cells; cell-cell communication; intra-cell signaling; anti-oxidants

INTRODUCTION: CAN GREEN TEA ASSIST IN MAINTAINING HEALTH

IN THE COMPLEX HUMAN HOMEOSTATIC –CYBERNETIC,

HIERARCHICAL “MEAT MACHINE?” The aims of any scientific advice, bearing on whether any nutritional social policy , should (a) promote health or avoid harm; (b) have intended beneficiaries of the advice; and (c) balance the strengths of the scientific evidence with the expected consequences of scientific advice[ 1]. The intent of this short “Commentary” is directed to examine the incomplete scientific knowledge bearing on the alleged health benefits of drinking green tea or taking nutritional supplements of green tea chemical components. Before one can begin to assess the growing and, in many cases, conflicting studies on the potential role of green tea’s effect on human health, it is the fundamental assumption of this “Commentary” that one must understand how the components of green tea might interact with both endogenous and exogenous toxicant/toxins’ mechanisms of toxicity and how these mechanisms contribute to the pathogeneses of various chronic diseases. To do this, a brief *

Correspondence to be sent: James E. Trosko, Ph.D. Dept. Pediatrics/ Human Development, College of Human Medicine, Michigan State University. East Lansing, Michigan 48824; Phone: 517-884-2053; Fax: 517-4326340; E-mail: [email protected]

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overview of philosophical concepts of the biology of human health, including how biologicaland cultural- evolutionally factors have influenced dietary practices, will be made prior to some scientific concepts of how green tea might influence various disease- contributing mechanisms. From the moment of conception of the single fertilized egg, through embryonic-, fetal-, neonatal-, adolescent- development, mature adult and geriatric periods of life, cell functions of proliferation, differentiation, apoptosis, adaptive responses of terminally-differentiated cells and senescence of individual cells contribute to the 100 trillion cells that make up the multiple tissues, organs, organ systems, that lead to consciousness, behavior, and human culture. The biological evolutionary advance that allowed the transition from colonies of single cell organisms to the metazoan, exemplified by the human being, included the genes that encouraged group cell adherence for the development of new phenotypes of (a) growth control; (b) cell differentiation; (c) apoptosis; (d) cell senescence; and (e) choice of cell “mortality” for somatic cells and for the individual or of “immortality” for the germ line for the species and for adult or somatic stem cells for tissue growth/ repair. From this grouping of cells that gained specialized physiological functions ( neurons; hepatocytes; retinal cells; etc.), a delicate orchestration of molecular information transfer, triggered by the interaction of the information coded in the individual genomes and the specific historic environmental, dietary, social/cultural factors, had to occur, in order to prevent disruption of development, of early diseases, and of reproductive success. This is beautifully illustrated by Markert [2]: “Cells interact and communicate during embryonic development and through inductive stimuli mutually direct the divergent courses of their differentiation. Very little cell differentiation is truly autonomous in vertebrate organisms. The myriad cell phenotypes present in mammals, for example, must reflect a corresponding complexity in the timing, nature, and amount of inductive interactions. Whatever the nature of inductive stimuli may be, they emerge as a consequence of specific sequential interactions of cells during embryonic development. The first embryonic cells, blastomeres, of mice and other mammals are all totipotent. During cleavage and early morphogenesis these cells come to occupy different positions in the three-dimensional embryo. Some cells are on the outside, some inside. The different environments of these cells cause the cells to express different patterns of metabolism in accordance with their own developing programs of gene function. These patterns of metabolism create new chemical environments for nearby cells and these changed environments induce yet new programs of gene function in responding cells. Thus a progressive series of reciprocal interactions is established between the cellular environment and the genome of each cell. These interactions drive the cell along a specific path of differentiation until a stable equilibrium is reached in the adult. Thereafter little change occurs in the specialized cells and they become remarkably refractory to changes in the environment. They seem stably locked into the terminal patterns of gene function characteristic of adult cells. The genome seems no longer responsible to the signals that were effective earlier in development. Of course, changes can occur in adult cells that lead to renewed cell proliferation and altered differentiation as seen in neoplasms, both benign and malignant, but such changes are very rare indeed when one considers the number of cells potentially available for neoplastic transformation. Possibly, mutations in regulatory DNA of dividing adult cells can occasionally lead to new and highly effective programs gene function that we recognize as

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neoplastic or malignant. However, most genetic changes in adult cells can probably lead to cell death since random changes in patterns of gene activity are not likely to be beneficial.”

Clearly, this assured species survival but it could not prevent the inevitability of aging, the diseases of aging and ultimate death of the individual. From the organization of molecular, biochemical, cellular, tissue, organ, organ system units and the interaction of physical/social environmental factors, the hierarchical emergence of new phenotypes occurred [3]. Many systems had to be developed to facilitate the molecular transfer of critical and specific information to regulate the selective expression of genes in the total genome. This included the information from (a) the cell- matrix interaction; (b) cell-cell adhesion molecules; (c) secreted cell communication ions and molecules; ions and selective small molecules through gap junctional intercellular communication protein channels of coupled cells. Within the developing embryo, the existence of pluripotent stem cells, with unlimited proliferation potential, gave rise to multi-potent stem cells with restricted differentiation potential. From these stem cells, bi-polar stem cells were generated and from these uni-polar stem cells one differentiated cell lineage was born. Progenitor cells, born from these adult stem cells, were committed to proliferate with a finite life span, leading ultimately to terminal differentiation, apoptosis or senescence.

THE MIS-MATCH OF BIOLOGICAL EVOLUTIONARY MEANS FOR DIETARY SURVIVAL WITH THE RADIPLY-DEVELOPING CULTURALLY-EVOLVED MEANS FOR DIETARY SURVIVAL Given that the human body is hierarchical/cybernetic system of different cell types (stem, progenitor, and differentiated) that generate tissues, organs and organ systems [4, 5], it should be clear that biological evolution, utilizing oxygen to generate energy from dietary-supplied, caloric/nutrient-laden foods, shaped multiple systems to deal with the by-products of oxidative metabolism, namely the release of reactive oxygen species (ROS). [6, 7]. The early cultural evolution of the human being shaped how the human species survived a “feastfamine” dietary experience. With the emergence of farming practices, the first new rapidly developing cultural dietary experience collided with the very slow biologically-evolved mechanism, designed for “feast and famine” survival [8] Within the last century, food preservation, food distribution, food processing and new foods entered the continued collision of cultural dietary practices on a relatively stable biological basis for how foods/nutrition helped to maintain health sufficient for the survival of the human species. Clearly within a hundred or so years, the average human median life span was about the mid- 40’s to today’s of over 70’s. As a consequence of the improvement of agriculture, sanitation, and maybe, so medical interventions, we have the “privilege of experiencing the effluence of our affluence”, that is, living longer, due to the reduction of acute infectious lethal diseases, only to experience chronic diseases of both growing older and having newer dietary practices. Recently, in spite of the rapid increase in the population of human beings, maldistribution of resources, and political/economic injustices, the numbers of over-weight and

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obese individuals over-weight individuals are increasing around the world[9,10] than . Clearly, it seems clear that this increase in individuals’ weight could be responsible, in large part, to the emergence of many chronic diseases (cancer, diabetes, hypertension, cardiovascular diseases, etc.), acting together with smoking, lack of exercise, poor nutritional diets [11]. However, what might be missed is the fact that some of the most nutritious foods are becoming the most expensive, and the most poorly-nutritious and highly caloric-laden foods are the least expensive. The coupling of the non-renewal fossil fuel or current energy crisis with the energy-dependent agri-business can be seen as a major contributing factor. What should not be ignored is that cultural evolution has also brought new eating timehabits. With the early biological evolved biochemical systems to deal with “feast-famine” having coincided with the diurnal rhythms, new patterns of living outside these biologicallygoverned diurnal rhythms probably influenced how our dietary/nutrient patterns affect our homeostatic regulation for a healthy life. Then, with the introduction of agriculture, a more or less stable, if not seasonal eating pattern emerged. With cultural evolution introducing new forms of food production, food preservation, storage, transport , processing, and preparation, for many, eating three or more times at day challenged or biologically-evolved mechanisms to maintain homeostatic control of a disease-free life.

EPIDEMIOLOGICAL ASSESSMENTS FOR DIETARY FACTORS AFFECTING HUMAN HEALTH While it can not be denied that the science of epidemiology can provide solid insights to some factors associated with various human diseases, fundamentally, it can not be used to understand underlying mechanisms by which any factor, might or might not affect human health. Moreover, in any epidemiological study of human populations, what might or might affect the individual within that affected population can not be rigorously assessed. With both (a) genetic-, gender-, developmental state- differences that might pre-dispose or offer resistance to diseases and (b) multiple external agents that might synergize or antagonize the agent(s) being studied, it is very understandable why many epidemiological studies do not agree on the alleged affect of any suspected health related agent. In the case of dietary/nutrient epidemiological studies on human beings is the fact that multiple decade studies, with many individuals, together with “matched” controls, and with multiple identical or equally-rigorously-designed and executed studies must be planned. In addition, unless basic mechanistic knowledge of how any dietary/nutrient factor might be toxic (via mutagenic, cytotoxic, or epigenetic mechanisms), is known, unforeseen consequences might result with the pathogenesis of any disease. This will reduce the likelihood that the epidemiological data be used in any meaningful intervention strategy. That was the case of the pre-mature intervention Alpha-Tocopherol Beta-Carotene (ATBC) and Beta-Carotene and Retinol Efficacy Trial (CARET) studies, in which the objective was to reduce the lung cancer risk in heavy cigarette smokers, but which led to the cessation of the human trial occurred because of the higher incidence of lung cancer in the treated individuals versus the placebo group [12]. Lastly, with the unbelievable alteration of the human diet within decades that is occurring now by the new processing of foods, the types of foods, amounts of foods, the interaction of

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what is in and on foods, the manner by which foods are preserved, prepared, transported, stored and the effect of economic stresses, no matter what any epidemiological insights might have been gained, the likelihood of its having rigorous scientific utility is diminished. Moreover, even with valid information gain from these time-shaped results, the question remains, “Will implementation of the results for the future risk reduction of diseases be effective or even harmful?”

GREEN TEA AND HUMAN HEALTH Without doing a rigorous evaluation of all the epidemiological studies reported, one example will be used to illustrate the aforementioned factors. Also, what might seem obvious in trying to assess the potential benefit of green tea, it should be remembered that drinking green tea for centuries occurred in cultures whose caloric intact and specific dietary components (lots of vegetables, some fruits, raw fish, plenty of soy products, and a minimum of red meats, dairy products, sugar, etc.) are not shared by those populations in which green tea is proposed to be used. Human cancer is one health endpoint that has been frequently used to assess the effect of any dietary /nutrient exposes might have on cancer frequencies. Whether caloric restriction or caloric excess is studied or dietary factors, such as fats or red meats, as “carcinogenic”, or fruits, vegetables, fibers, essential metals/vitamins as cancer preventive agents [13], a few of many examples of peer-reviewed reports on green tea that state there is a positive- [[14-17] , negative- [ 18] , both positive and negative-effects in the same population but in different sexes [20],or no significant side effects or adverse effects [ 21] with the agent being studies of the health effect being measured.. In order to understand how human cancers are produced, the current understanding must be reviewed. First, human carcinogenesis is a multi-stage, multi-mechanism process [22-24], consisting of a single cell in the human body being irreversibly altered, in such a fashion that it remains immortal in the first stage to become a cancer. All human beings have such “initiated”, pre-malignant cells in our body. Three fourths of us will reach death without having a detected malignant cancer. One fourth of us will be diagnosed with a malignant cancer before they die. The difference between these two groups is the fact that those that get a malignant cancer had at least one of their “initiated cells” “promoted” to a large mass ( e.g., polyps in the colon; papillomas in the skin, nodules in the breast) by many endogenous (genetic/developmental stage , gender and exogenous factors - cell death, wounding, growth factors, life style agents, such as cigarette smoke chemicals, hormones, infectious biological factors, inflammatory physical agents-asbestos, exogenous drugs, environmental pollutants and dietary factors.)[24]. The three fourths of us ,who are lucky to die without having to deal without treating a cancer, have our initiated cells suppressed by endogenous factors ( genetic, gender, developmental state) or by exogenous factors ( dietary/ medicinal, life-style, such as exercise). The question is how those inevitable initiated cells are either “promoted” (clonally expanded to a large mass of abnormal, immortal cells which can eventually accrue the hallmarks of cancer [25], the additional genetic/epigenetic changes needed to become a

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malignant cell. This also implies that this promotion step, which could take decades in the case of human cancers, can be interrupted or even reversed [26]. Furthermore, the promotion phase would be the most efficacious phase of carcinogenesis to design intervention cancer prevention strategies [27]. The fact that the “initiation” step can be prevented from increasing (one does not have expose one’s skin to too much sun light), one can never reduce the risk to the initiation phase to zero. If this is a correct assumption, then it should be helpful to understand the characteristics of the promotion and anti-promotion processes [26, 28]. Most, if not all tumor promoting agents/conditions need “threshold” levels to cause an initiated cell to escape the mitogenic suppressing effect of surrounding normal cells, caused by either secreted anti-mitogenic factors or direct transfer of ions or small molecules via gap junction channels of contiguous cells [29]. Tumor promoters also have species-, gender-, cell-type-, and organ- specificies. Promoting conditions could occur via indirect actions of cell death or cell removal, for instance would-healing after surgery or tissue repair after cytotoxic exposures to viruses or cytotoxic chemicals, parasites or bacteria, such as asbestos particles, all of which induce inflammatory agents [24]. In addition, if an initiated cell is exposed to a tumor promoter, the exposure must be sustained for a regular and chronic period of time. Most notably, not all tumor promoters work via the same mechanism [30], for example, phorbol esters stimulate protein kinase C and its cascading signal transduction mechanism. However, TCDD, a promoter of skin and liver tumors appear to work through the Aryl hydrocarbon receptormediated signaling [31]. Although most, if not all of these promoting agents and conditions are associated with altering the redox state of the cell, inducing oxidative stress –inducing intra-cellular signaling [32-34], this might be a necessary, but insufficient, causative factor for tumor promotion By implication, any potential anti-tumor promoter would not be expected to be able to ameliorate the promoting mechanisms of all the different kinds of tumor promoting conditions [30]. Not only would one expect that the anti-promoting agent specifically interfere with the specific mechanism of the promoter, in order to be an effective chemopreventive agent, but it must be there at the exact time where it might block the start of the promoting signaling, but also at the right concentration, for the entire chronic exposure period. One also must remember, that both the promoting agent/condition, by being organ specific, and the anti-promoting agent must be residing in the same organ site. For example, if the tumor promoter acts in the lung, but the anti-promoter’s active chemical does not get distributed to the brain in sufficient time/concentration, it might not be effective.

THE “BIOLOGICAL ROSETTA STONE”: GAP JUNCTIONAL INTERCELLULAR COMMUNICATION AND HUMAN HEALTH It must be noted that gap junction intercellular channel, composed of a hexamer of six transmembrane proteins, “connexin”, that form a hemi-channel , “connexon”, which unite with the connexon of the contiguous cell, exists in all organs of the human body [35]. Twenty connexin genes code for these proteins which are expressed in a cell type/organ-specific manner. The function of these gap junctions can be modulated at the transcriptional, translational and posttranslational levels[29] by both exogenous and endogenous factors,

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which then can alter the intra-cellular physiology, intracellular signaling and ultimately, the gene expression of the cells[36].While all metazoans share the common cellular structures, such as endoplastic reticulum, nuclei, mitochondrial, receptors, spindle fibers, the gap junction has to be singled out as absolutely critical for maintaining, directly and indirectly, the phenotypes of growth control, differentiation and apoptosis [37 ]. Without these gap junctions, the ability to segregate cells during development to specialize and function independently of neighboring cells, the complex homeostatic regulation of the hierarchical biological system of the human being could not exist. Interference of GJIC during embryogenesis, fetal development could lead to death or birth defects as indicated by either retinoid or thalidomide modulation of gap junction function in human beings [38, 39]. Interference of GJIC in the mature reproductive, immune or neuronal tissues by chemicals, such as DDT, can lead to various toxicities [40]. While it has not been universally accepted, one cellular mechanism has been proposed, based on the observation that most, if not all, tumor promoters and promoting conditions can reversibly inhibit gap junctional intercellular communication , that inhibition of gap junction function, via multiple biochemical mechanisms, allows initiated cells to escape mitotic suppression of surround normal cells.[41]. To support this hypothesis, known tumor promoters of the skin, liver, mammary gland, lung and bladder can reversibly inhibit gap junction function in a threshold manner, both in vitro and in vivo. [29]. In addition many anti-tumor promoters, such as retinoids, caretinoids, reveratrol. Even chemotherapeutic agents have been shown to restore gap junctional communication in non-communicating cancer cells, such as with lovastatin, SAHA, [42, 43] Given that gap junction genes (connexins) appeared during the evolutionary-transition from the single cell organism to the multi-cellular metazoan [3], when growth control, differentiation and apoptosis appeared, it should not be surprising that the cancer cells, which do not have growth control, cannot terminally-differentiate or apoptose properly, do not have functional GJIC [44]. Having discovered that going from the normal cell to an initiated cell, which can be expanded by the inhibition of cell-cell communication via tumor promoters until the cell becomes malignant or tumor promoter –independent, it demonstrates the evolutionary importance of cell-cell communication in the cybernetic regulation needed to maintain the hierarchical nature of all the interacting units of the human body and how these gap junctions were beautifully designed to respond to environmentally-induced redox stress triggered signaling in order to modulate adaptive genes [45]. From this vantage point, by understanding the fundamental role that gap junctions play in the homeostatic regulation of all the cellular, tissue, organ and organ system, including the ultimate human brain function, consciousness, it should not be surprising to label the gap junction function one viewed the translation of the hieroglyphics as a “biological Rosetta Stone”.

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GREEN TEA, GAP JUNCTIONAL INTERCELLULAR COMMUNICATION, AND POTENTIAL PROTECTION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION’S ROLE IN HOMEOSTATIC REGULATION OF HUMAN HEALTH Given all the many contradicting studies at the molecular, in vitro, experimental animal and epidemiological studies on green tea’s effect on prevention of various biological/health endpoints, is there some evidence that there is or is not any potential health benefit from drinking green tea? The answer seems potentially positive. While green tea is a solution of many chemicals, several of which have anti-oxidant potential, it might not be unreasonable to predict that any acute or chronic disease endpoint, correlated with oxidative stress-induced signaling, might be prevented by the timing, threshold concentrations, chronic exposure and linkage of specific cross-talk/interaction of the components of green tea and the toxin/toxicants. Of course, positive chemo preventive effects at the molecular/ in vitro and whole animal level might or might not have any practical relevance to human beings. The former test systems can be controlled, while the green-tea drinking behavior in human beings can not. The major example to highlight this point in this short Commentary will be the demonstration that green tea could ameliorate the tumor promoting effect of PCP. PCP is known to induce oxidative stress [46]. Moreover, after rats were initiated with pentachlorophenol (PCP), chronic exposures to PCP at a threshold and higher levels, induced liver tumors. PCP, in vitro, is not a genomic DNA damaging agent, but an epigenetic agent [47]. In vitro, at not cytotoxic levels, but at threshold or above levels, could inhibit GJIC. On the other hand, pre-treatment of the in vitro cells with green tea components, prevented the GJIC inhibitory effects of PCP. More striking, treatment of the rats exposed to both green tea and PCP in the drinking water, ameliorated the promoting effect of the PCP on liver tumors. While it can be argued that this example is still “correlative” not causal, it clearly is consistent with the hypothesis that green tea has components capable of protecting the tumor promoting ( and possibly other gap junctional-dependent health effect in other organs) of chemicals that work like PCP. Of the several epidemiological studies of green tea, “cherrypicking” one example , namely that of comparison of American and Japanese smokers might serve to illustrate how green tea could be beneficial to human health [17]( Recognizing the hazards of experimental design; cultural differences in diet/behavior; sampling errors, etc.). However, with the experimental demonstration that green tea polyphenol, {(-)epigalllocatechin-3-gallate –EGCG}, suppresses cigarette smoke condensate-induced redox intracellular signaling in normal human bronchial epithelial cells [48] In this example, there was a correlation between the green tea drinking of the Japanese men who smoked and their risk to lung cancers compared to American men. While it might have been even more interesting to compare a number of American smokers who drank that amount of green tea as the Japanese and compared them to Japanese smokers who did not drink green tea( Unfortunately, the numbers in either category are probably too small to obtain statistically significant results). It should be noted that if there is a demonstrated validated health effect of green tea, there must be an underlying biological mechanism.

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However, even if there might be a demonstrated mechanistic biological effect of green tea (of which there are many), it does not necessarily imply there will be a health effect. In summary, the scientific understanding of many biological functions involved in maintaining human health (e.g., role of gap junctions in homeostatic control of cell behaviors; multi-state nature of carcinogenesis; role of gap junctions in the tumor promotion step of carcinogenesis; toxicant induced oxidative stress in many human diseases; role of oxidative stress by tumor promoting chemicals; role of green tea components as anti-oxidants; preventive effects of green tea components on toxicant-inhibition of gap junction function and tumor promotion) provides some evidence that green tea could prevent some oxidative stresslinked human diseases. However, as has been demonstrated in experimental examples, it will be unlikely that any agent, such as green tea, will be a “silver bullet” to protect against all toxic agents. On the other hand, it does seem it would cause “no harm” and might, in some cases, even be beneficial.

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[31] Weiss C, Faust D, Schreck I, etc. TCDD deregulates contact inhibition in rat liver oval cells via AH receptor, JunD anf Cyclin A. Onvcogene 27:2198-2207, 2008.32. [32] Klaunig J E, Kamendulis L M, The role of oxiudative stress in carcinogenesis. Ann Rev Pharmacol Toxicol 44: 239-267, 2004. [33] Upham B L, Wagner J G.. Toxicant-induced oxidative stress in cancer. Toxicol Sci 64: 1-3, 2001. [34] Cerruti P. Proxidant states and tumor promotion. Science 227: 375-381, 1985. [35] Evans W H, Martin P E M. Gap junctions: Structure and Function. Molec Membr Biol 19: 121-136, 2002. [36] Trosko J E, Chang C C, Upham B L, Wilson M R. Epigenetic toxicology as toxicant – induced changes in intracellular signaling leading to altered gap junctional intercellular communication. Toxicol Letters 102-103: 71-78, 1998. [37] Trosko , J.E., “ Gap junction intercellular communication as a ‘Biological Rosetta Stone’ in understanding , in a Systems manner, stem cell behavior, mechanisms of epigenetic toxicology, chemopreventyion and chemotherapy”. J. Membr. Biol.218: 93100, 2007. [38] Maden M: Vitamin A and pattern formation in the regenerating limb. Nature 295: 672675, 1982. [39] Lenz W: A short history of thalidomide embryopathy. Teratology 38: 203-215, 1988. [40] Trosko JE, Ruch J J. Gap junctional Intercellular communication epigenetic toxicology. In: Encyclopedia of Toxoicology. 2nd Edition, Wexler P, ed. Elsevier Press, Oxford pp. 402-407, 2005. [41] Yotti, L.P., J.E. Trosko and C.C. Chang: "Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter". Science 206:1089-1091, 1979. [42] Ruch, R.J., B.V. Madhukar, J.E. Trosko and J.E. Klaunig: "Reversal of ras- induced inhibition of gap junctional intercellular communication, transformation, and tumorigenesis by lovastatin”. Mol. Carcinog. 7:50-59, 1993. [43] Ogawa, T., Hayashi, T., Tokunou, M., Nakachi, K., Trosko, J.E., Chang, C.C., and Yorioka, N., “Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin43 gene locus”. Cancer Res. 65: 9771-9778, 2005. [44] Trosko JE: The role of stem cells and gap junctional communication in carcinogenesis. J Biochem Molec Biol 36: 43-48, 2003. [45] Upham, B.L. and Trosko, J.E., “Carcinogenic tumor promotion, induced oxidative stress signaling, modulated gap junction function and altered gene expression”. Antioxidants & Redox Signaling, in press. [46] Umemura T, Kai S, Hasegawa R, Sai K, Kurokawa Y, Williams G M. Pentachlorophenol (PCP) produces liver oxidative stress and promoters but promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice. Carcinogenesis 6: 11151120, 1999. [47] Sai K, Kanno J, Hasegawa R, Trosko J E, Inoue T. Prevention of the down regulation of gap junction intercellular communication by green tea in the liver of mice fed pentachlorophenol. Carcinogenes 21: 1671-1676, 2000. [48] Syed D N, Afaq F, Kweon M-H, Hadi N, Bhatta N, Spiegelman V S, Mukhtar H. Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF- κB activation in normal human bronchial epithelial cells. Oncogene 26: 673-682, 2007.

INDEX A Aβ, 152, 438 A1c, 248 aberrant methylation, 264 abnormalities, 249, 364 absorption, 13, 55, 64, 65, 71, 74, 77, 78, 118, 122, 126, 135, 136, 137, 141, 143, 151, 152, 154, 201, 205, 224, 227, 237, 246, 279, 340, 381, 400, 415, 416, 417, 424, 426 abusive, 31 ACC, 137 accessibility, 291 accounting, xviii, 302, 384, 388 accuracy, 35 ACE, 299, 316, 424 acetaldehyde, 160, 169 acetate, vi, xvii, 108, 109, 160, 165, 166, 228, 256, 347, 348, 353, 361 acetone, 65, 86, 194, 395, 423 acetylation, 148, 291, 461 acetylcholine, 212 acetylcholinesterase, 162, 170 acidic, 93, 164, 165, 395 acidity, xii, 82, 84, 87, 91, 92, 98 acquired immunodeficiency syndrome, 28 ACS, 99 actin, 48, 49, 52, 53, 230, 272, 273, 274, 277, 278, 284, 285, 294, 296, 297, 298, 299, 368 actinic keratosis, 56 action potential, 12, 252 activators, 292 active oxygen, 43, 167, 175, 403 active site, 263, 266 acute, 4, 5, 6, 7, 8, 9, 15, 17, 18, 19, 21, 22, 48, 161, 162, 214, 245, 250, 315, 341, 353, 453, 458

acute myeloid leukemia, 341 acute stress, 4, 5, 6, 7, 8, 9, 15, 17, 18, 19, 22, 214 acylation, 47 ad hoc, 123 Adams, 174, 301, 318, 379 adaptation, 459 additives, xi, 46, 63, 64, 66, 67, 68, 70, 73 adducts, 163, 174, 403 adenocarcinoma, 145, 150, 273, 275, 287, 299, 317, 377 adenocarcinomas, 326 adenoma, 334, 344 adenosine, 298 adenovirus, 28, 42, 55, 60, 377 adhesion, 99, 127, 169, 204, 267, 293, 294, 308, 333, 334, 339, 343, 344, 382, 453 adhesive interaction, 334 adipocyte, 128, 129, 130, 131, 132, 134, 135, 141, 143, 144, 145, 147, 148, 149 adipocytes, 127, 128, 131, 132, 134, 135, 137, 141, 146, 150, 200, 394 adipogenic, 128, 130, 148, 149 adiponectin, 150, 248, 255 adipose, 107, 117, 120, 127, 131, 132, 134, 136, 137, 140, 142, 143, 147, 153, 164, 200, 249, 256 adipose tissue, 107, 117, 120, 127, 131, 132, 134, 137, 140, 142, 143, 147, 164, 200, 249, 256 adjustment, 251, 326, 421 adolescents, 144 ADP, 229, 230, 358, 413 adrenal gland, 162 adrenaline, 137 adrenocorticotropic hormone, 137 adriamycin, 41 adsorption, 29 adult, 16, 40, 200, 224, 315, 370, 446, 451, 452, 453 adult stem cells, 451, 453

464

Index

adults, xviii, 233, 244, 253, 255, 296, 384, 385, 386, 388, 389, 406 advanced glycation end products, 403 aerobic, 69, 185, 187 aerobic bacteria, 187 aerodigestive tract, 344 Africa, 158 agar, 86 age, xvi, xvii, xix, 16, 108, 169, 198, 250, 325, 326, 338, 377, 383, 384, 386, 387, 399, 429, 430, 442, 444, 453 ageing, 408, 435, 440, 445 agent, xv, xvi, xx, 12, 35, 37, 60, 83, 133, 135, 166, 198, 219, 252, 268, 286, 317, 322, 325, 335, 337, 373, 377, 378, 405, 454, 455, 456, 458, 459 agents, xiii, xx, 30, 57, 71, 131, 139, 147, 157, 160, 168, 174, 175, 222, 225, 252, 261, 282, 308, 321, 322, 330, 333, 336, 337, 353, 356, 358, 394, 443, 454, 455, 456, 457, 459 aggregates, 136 aggregation, 172, 200, 204, 222, 228, 441 aggressiveness, 308 aging, 46, 57, 82, 206, 222, 224, 234, 246, 248, 378, 430, 444, 453 aging process, 430 agonist, 15, 132, 210, 225 agricultural, 105, 190 agricultural crop, 105 agriculture, 29, 161, 453, 454 AIDS, 28, 37, 60, 377 air, xiii, 84, 157, 160, 319, 384 AKT, 147, 229, 240, 291, 306, 307 Alabama, 363 alanine, 161, 162, 163 alanine aminotransferase, 161, 163 Alaska, 195 albumin, 163, 399 albuminuria, 403 alcohol, xviii, 120, 160, 161, 169, 176, 202, 235, 246, 384, 386, 387, 388, 389, 395, 440, 443 alcohol consumption, xviii, 120, 235, 384, 386, 387, 388, 389, 443 alcohols, 173 aldolase, 230 alertness, 13 alkaline, 162, 312, 371 alkaline phosphatase, 162 alkaloids, 37 alleles, 285, 303, 373 allergens, 178, 277 allergic reaction, 277, 279 allergy, 267, 277, 278, 297 allosteric, 131, 404

alpha, ix, 1, 2, 13, 14, 30, 99, 145, 148, 149, 150, 151, 220, 227, 238, 240, 241, 295, 297, 298, 299, 309, 320, 340, 341, 345, 380, 443 alpha activity, 14 alpha-tocopherol, 340 ALT, 162, 369, 370 alternative, 69, 135, 148, 154, 250, 341, 369 alternatives, 178 alters, 137, 147, 298, 306, 433, 448 aluminium, 106 aluminum, 118, 180, 375 alveolar macrophage, 30, 39, 42 Alzheimer disease, 205, 268 Alzheimer's disease, 10, 107, 197, 201, 205, 206, 219, 220, 222, 234, 236, 237, 240, 241, 268, 295, 443, 444, 446, 448, 449 amelioration, 161, 394, 444 American Cancer Society, 326, 338 American Heart Association, 254 amide, 209 amine, 119, 164, 165, 172, 173, 321 amines, 164, 165, 178, 195 amino, ix, 1, 2, 15, 20, 22, 79, 106, 117, 119, 158, 164, 190, 191, 208, 215, 244, 247, 265, 351, 394, 396 amino acid, ix, 1, 2, 20, 22, 79, 106, 117, 158, 164, 215, 244, 247, 351, 394, 396 amino acids, 22, 79, 117, 164, 215, 244, 247, 394, 396 ammonia, 27 AMPA, 15, 20, 208, 209, 212, 213, 215, 216, 217 amplitude, 12, 13 Amsterdam, 109, 122, 124 amygdala, ix, 1, 2, 13 amylase, 30, 42, 400, 404 amyloid, xix, 10, 201, 220, 222, 225, 234, 236, 238, 240, 250, 268, 295, 429, 430, 433, 438, 442, 443, 444, 446, 447, 448, 449 Amyloid, 233, 249, 294, 443, 447 amyloid beta, 225, 443, 444 amyloid fibrils, 250 amyloid precursor protein, 220, 222, 234, 236, 238, 240, 268, 295 amyloid β, 433, 446 amyloidosis, 227, 249, 250, 256 amyotrophic lateral sclerosis (ALS), 12, 20, 222, 226, 237, 433 anabolic, 148 anabolism, 126 analgesic, xii, 81 analog, 59, 164, 208 analysis of variance, 111 analytical techniques, 35

Index anaphylaxis, 297 androgen, 127, 138, 145, 146, 336 androgens, 417 androstenedione, 305 anesthetics, 210, 213 aneuploidy, 167, 174 angiogenesis, xiv, xvi, 148, 197, 198, 199, 200, 201, 202, 203, 206, 261, 265, 267, 286, 292, 301, 308, 309, 311, 313, 314, 319, 330, 331, 336, 339, 340, 341, 345, 366, 376 angiogenic, 198, 199, 201, 203, 230, 308, 331, 340, 366 angiography, 246 Angiotensin, 427 angiotensin converting enzyme, 193, 424 angiotensin II, 247 angiotensin-converting enzyme, 285, 303, 305 animal models, x, 45, 131, 136, 204, 225, 226, 248, 259, 265, 286, 314, 326, 365, 366, 371, 375, 391, 394 animal studies, xviii, 19, 37, 58, 107, 117, 135, 170, 229, 231, 244, 247, 332, 389, 393, 431 animal tissues, 162 animals, xiii, xix, 16, 30, 49, 104, 136, 137, 142, 163, 166, 168, 223, 260, 261, 266, 375, 402, 429 ANOVA, 87, 111, 112, 437 antagonism, 216, 217 antagonist, xiv, 6, 7, 15, 124, 207, 209, 210, 212, 213, 216, 305, 372 antagonistic, 16, 38, 209, 213 antagonists, 16, 28, 120, 209, 213, 215, 217, 289 anthocyanin, 27, 348 anthracene, 75, 321, 322 anthrax, 37, 261, 290 antiangiogenic, 309 anti-angiogenic, xiv, 197, 198, 199, 200, 201, 303 anti-angiogenic, 336 antiapoptotic, 141, 133, 229, 234, 264, 265, 289, 335, 341, 342, 442 antiarrhythmic, 252 antibacterial, x, 23, 30, 31, 32, 33, 36, 37, 43 anti-bacterial, 83, 358 antibacterial properties, 36 antibiotic, 27, 33, 34 antibiotic resistance, 27 antibiotics, x, 23, 31, 34, 38, 42 antibodies, 48 antibody, 54, 198, 202, 270, 272, 273, 277, 278, 364 anticancer, 37, 133, 146, 154, 158, 185, 282, 283, 290, 308, 317, 322, 365, 368, 370, 374, 375, 378 anti-cancer, x, xi, xvii, 45, 46, 47, 48, 55, 199, 229, 258, 261, 265, 267, 270, 286, 348, 352, 357, 358, 363, 368

465

anticancer activity, 368 antidiabetic, 139, 244, 252 antigen, 297 antigenicity, 195 anti-HIV, 28, 37, 60, 377 anti-inflammatory agents, 222, 353, 356 antiobesity, 107, 108, 143, 244 antioxidative, 143, 196, 343, 427, 439 antioxidative activity, xix, 187, 190, 429 antioxidative potential, 448 antiradical activity, 104, 110 antisense, 282, 319 antisense RNA, 282 antitoxin, 37 antitumor, 41, 119, 122, 272, 322 anti-tumor, xvi, xvii, 50, 83, 312, 319, 325, 363, 456, 457 antitumor agent, 322 antiviral, x, 2, 23, 28, 29, 35, 37, 424 anxiety, 7, 15, 21 anxiolytic, 15 aorta, 253, 255 APC, xvi, 28, 325, 327, 329 aplastic anemia, 166 apnea, 104 APO, 127, 220, 225, 227, 229 apoptosis pathways, 154, 332, 342 apoptotic, 127, 131, 133, 140, 141, 146, 149, 150, 164, 213, 229, 234, 240, 264, 276, 308, 311, 314, 332, 333, 335, 342, 448 apoptotic cells, 133, 311 apoptotic effect, 141, 146 APP, 222, 225, 227, 228, 232, 268, 441 appetite, 105, 108, 117, 120, 121, 122 application, x, xiv, 11, 13, 40, 45, 46, 47, 48, 56, 76, 118, 166, 170, 177, 178, 188, 194, 231, 239, 303, 336, 353, 360 aqueous solution, 136, 395 arabinogalactan, 399 arachidonic acid, 277, 320 argument, 329 Arizona, 85, 90 aromatic rings, 29, 221 arousal, 13 arrest, 129, 130, 133, 134, 141, 143, 146, 147, 149, 150, 229, 263, 266, 276, 289, 306, 316, 317, 321, 333, 335, 336, 342, 345, 374 arrhythmia, 251 arrhythmias, 244, 249, 252 ARS, 78 arteries, 204 artery, xiv, 14, 207, 208, 209, 216, 245, 246, 248, 252, 254, 255, 402

466

Index

arthritis, 58, 376, 400 articulation, 222 aryl hydrocarbon receptor, 241, 289 asbestos, 455, 456 ascorbic, 71, 72, 79, 84, 85, 91, 92, 96, 109, 192, 193, 229, 232 ascorbic acid, 71, 72, 79, 84, 85, 91, 92, 96, 109, 192, 193, 229, 232 Asia, 2, 21, 29, 36, 151 Asian, 2, 200, 201, 206, 222, 285, 299, 316, 365, 366 Asian American, 316 Asian Americans, 316 Asian countries, 201, 365 aspartate, 12, 22, 30, 161, 162, 163, 208 assessment, 70, 76, 77, 170, 185, 194, 215, 389, 443 asthma, 104, 123, 277, 278 astringent, 158 astrocyte, xiv, 14, 21, 207, 211 astrocytes, 241, 445 astrocytoma, 10, 20, 377 asymmetry, 332 ATF, 241, 333 atherogenesis, 70, 132, 204, 395, 402 atherosclerosis, xiv, 78, 136, 149, 151, 197, 198, 200, 202, 204, 221, 252, 258, 268, 444 atherosclerotic plaque, 135, 200 atherosclerotic vascular disease, 400 atmosphere, 123, 193 atopic dermatitis, 277, 278 ATP, xviii, 131, 411, 413, 414, 416, 417, 418 ATPase, 255, 272, 274, 295, 395, 403, 407 atrophy, 162, 431 attachment, 28, 29, 226, 267, 292, 293, 332, 334 attitudes, 338 atypical, 294 Australia, 108, 123, 383 autoimmune, x, 45, 46, 442 autoimmune disorders, x, 45 autonomic nervous system, 7 autooxidation, 167 autophagy, 140 avian influenza, 29 awareness, xiii, 125, 126, 243 Aβ, 433, 438, 440, 441, 442

B B cell, 122, 148 Bacillus, 31, 32, 94 Bacillus subtilis, 94 background information, 369 bacteria, 27, 30, 31, 32, 33, 34, 37, 40, 41, 123, 164, 187, 431, 456

bacterial, 30, 31, 33, 34, 36, 39, 41, 42, 96, 107, 152, 268 bacterial cells, 34, 41 bacterial infection, 42, 268 bacteriostatic, 96 bacterium, 33, 40, 196, 361 barrier, ix, x, xix, 1, 2, 12, 13, 34, 45, 56, 61, 208, 209, 214, 220, 224, 235, 336, 429, 430 barriers, 47 basal layer, 52, 54 base pair, 369 basement membrane, 261, 265, 267, 292, 331, 343 basic fibroblast growth factor, 331, 340 basophils, 267, 277, 278 Bax, 133, 141, 149, 229, 306, 308, 310, 311, 312, 317 BBB, 208, 209, 210, 214, 220, 224 B-cell, 127, 140, 292 B-cell lymphoma, 140 B-cells, 127 bcl-2, 240, 291, 341, 342 Bcl-2, 133, 150, 153, 222, 228, 229, 263, 264, 289, 291, 306, 332 Bcl-xL, 133, 229, 264 beef, 31, 38, 381 behavior, xiii, 3, 5, 7, 9, 11, 13, 16, 18, 105, 125, 361, 393, 452, 458, 459, 461 Beijing, 224 beneficial effect, ix, xiii, xiv, xviii, xix, 2, 47, 64, 72, 107, 117, 125, 136, 143, 191, 192, 197, 198, 223, 224, 234, 286, 329, 337, 374, 394, 411, 415, 424, 430, 434 benefits, x, xi, xv, xvii, 18, 37, 45, 46, 47, 55, 56, 139, 143, 155, 178, 222, 226, 232, 243, 244, 252, 258, 303, 321, 325, 326, 329, 359, 394, 401, 405, 424, 427, 451 benign, 326, 327, 452 benzene, 165, 166, 167, 174, 175 benzodiazepine, 15, 19, 20 benzoquinone, 166, 174 beta cell, 394, 412, 413, 416 beta-carotene, 175, 459 bevacizumab, 198, 202 beverages, xi, xvii, 24, 46, 55, 64, 66, 67, 69, 70, 71, 74, 75, 76, 77, 78, 80, 81, 83, 85, 89, 90, 91, 93, 96, 98, 178, 221, 243, 258, 347, 389 BH3, 229, 264 bias, 389 bile, 151, 246, 260, 431 bindings, 281, 321 bioactive compounds, 94, 178 bioassay, 329

Index bioavailability, xi, xvi, 12, 26, 35, 46, 47, 55, 56, 105, 171, 225, 235, 244, 260, 261, 264, 286, 288, 298, 302, 303, 311, 312, 322, 368, 375, 381 biocatalytic process, xix, 419 biochemistry, 40, 124 biogenesis, 137 biogenic amines, 178, 195 biological activity, xi, 46, 179, 195, 229 biological consequences, 228, 265 biological processes, 132 biological responses, 260 biological systems, 72, 459 biologically active compounds, 280 biomarker, 134, 163 biomarkers, xvi, 69, 105, 296, 325, 406 biomaterial, xiv, 177 biomolecules, 430 biopsy, 49 biosynthesis, 27, 40, 41, 137, 184, 185, 266 biotransformation, 26, 75, 244, 260, 287, 377 birth, 457 black tea, xi, xix, 2, 13, 24, 25, 26, 36, 39, 65, 66, 67, 70, 71, 74, 75, 76, 78, 79, 81, 82, 85, 87, 89, 93, 96, 101, 107, 126, 143, 144, 158, 165, 196, 224, 226, 227, 238, 250, 252, 254, 258, 287, 288, 316, 320, 322, 361, 365, 381, 384, 385, 419, 422, 423, 424, 426, 427, 431, 442 bladder, 2, 40, 107, 122, 153, 164, 173, 358, 457 bladder cancer, 107, 122, 153, 164, 173 bleaching, 64, 66, 68, 72 blindness, 248 blocks, 135, 198, 238, 244, 251, 252, 318, 320, 341, 377, 437 blood clot, 265 blood glucose, xiii, 125, 135, 205, 248, 253, 255, 394, 399, 400, 406, 409, 412, 413, 416 blood plasma, 78, 105 blood pressure, 104, 153, 178, 208, 209, 214, 215, 246, 247, 254, 255, 287, 447 blood stream, 332 blood supply, 200 blood vessels, xiv, 197, 198, 308, 331, 366 blood-brain barrier, ix, 1, 2, 12, 13, 208, 209, 220, 224, 235 blot, 49, 52, 53, 273, 276, 277, 284, 356 blueberry, 77, 122 body composition, 136 body fat, xiv, 26, 108, 117, 120, 126, 197, 205, 249, 256 body mass index (BMI), xviii, 142, 384, 387, 388, 390 body temperature, 105, 214

467

body weight, ix, xii, xiii, 12, 103, 104, 108, 110, 111, 114, 115, 117, 118, 119, 121, 126, 135, 141, 142, 143, 200, 205, 249, 302, 402, 432 Boeing, 233 bolus, 302 bonds, 137 bone density, 26 bone marrow, 40, 149, 167, 174, 175, 293 borderline, 71 boreal forest, 407 Bose, 144, 344 Boston, 250 botulinum, 101 bounds, 29 bovine, xvii, 29, 36, 49, 266, 348, 352, 379 brain, ix, xiv, xv, xix, 1, 2, 4, 5, 6, 7, 8, 13, 14, 15, 16, 19, 20, 21, 22, 105, 117, 158, 162, 170, 171, 207, 208, 209, 210, 212, 214, 215, 216, 217, 219, 222, 224, 225, 226, 227, 228, 230, 231, 232, 233, 234, 235, 237, 238, 239, 240, 241, 242, 247, 255, 295, 356, 409, 417, 429, 430, 433, 435, 436, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 456 brain damage, 210, 237 brain development, 15 brain injury, 170, 409 brain microvascular endothelial cell, 293 branching, 312 breakdown, 136, 178, 179, 193, 194 breast cancer, ix, xvi, 107, 123, 138, 145, 148, 150, 152, 203, 285, 291, 299, 301, 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 335, 341, 345, 366, 368, 374, 376, 380 breast carcinoma, 145, 146, 203, 282, 289, 316, 317, 319, 345, 376, 379, 446 breathlessness, 384 broad spectrum, 158 bronchial asthma, 277, 278 bronchial epithelial cells, 361, 458, 461 Brussels, 67 budding, 294 buffer, 50, 68, 109, 423 building blocks, 27 bundling, 285, 289 by-products, ix, 453

C Ca2+, 132, 139, 149, 213, 240, 255, 277, 298, 395, 403, 407, 413 cabbage, 32 cadherin, 199, 203, 275, 276, 309, 334, 344 cadherins, 334, 344 caffeic acid, 64

468

Index

caffeine, ix, xviii, 1, 2, 7, 16, 19, 20, 21, 26, 37, 118, 120, 121, 123, 153, 158, 192, 193, 208, 224, 234, 235, 249, 253, 280, 302, 342, 361, 365, 375, 391, 394, 399, 406, 411, 443 calcium, 12, 111, 124, 149, 217, 222, 277, 297, 338, 395, 403 calibration, 86, 90, 95, 422 calmodulin, 240, 296, 299 caloric restriction, 455 calreticulin, 132 calvaria, 149 CAM, 199, 333 cAMP, 255, 265, 333 cancer progression, 295, 320, 322, 339 cancer treatment, xvi, xvii, 198, 301, 306, 363 cancerous cells, 317 Candida, 33, 37 candidates, 55, 198, 270, 358 capacity, xi, xii, 3, 13, 26, 38, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 96, 98, 100, 108, 109, 111, 118, 120, 122, 190, 224, 234, 246, 248, 249, 260, 303, 313, 399, 400, 440 capillary, 331, 336 carbohydrates, 30, 244, 431 carbon, 48, 56, 160, 161, 170, 223, 263, 266 carbon tetrachloride, 160, 161, 170 carboxyl, 265 carboxylic, 165, 173 carcinogen, 167, 173, 175, 258, 316, 321 carcinogenesis, xvi, xix, 26, 37, 57, 75, 126, 167, 168, 173, 175, 202, 235, 258, 265, 275, 287, 291, 296, 301, 310, 314, 315, 321, 325, 326, 333, 336, 337, 338, 340, 342, 361, 364, 376, 382, 455, 456, 459, 460, 461 carcinogenic, 83, 164, 165, 166, 172, 178, 258, 330, 358, 394, 401, 455 carcinogens, 37, 164, 172, 329, 364, 385, 388 carcinoma, 49, 50, 51, 59, 145, 146, 147, 167, 203, 234, 282, 287, 289, 290, 292, 296, 310, 316, 317, 319, 332, 334, 341, 344, 345, 358, 376, 377, 379, 381, 446 carcinomas, 175, 339 cardiac arrhythmia, 244, 252 cardiovascular disease, ix, xiii, xv, 26, 57, 71, 79, 104, 119, 125, 126, 142, 143, 200, 202, 204, 206, 222, 234, 243, 244, 245, 246, 247, 248, 252, 253, 254, 358, 361, 385, 386, 389, 391, 392, 402, 405, 407, 430, 454, 460 cardiovascular risk, 143, 246, 247, 254 carotene, 71, 99, 194, 460 carotenoids, 106, 158, 407 carrier, 136, 186, 441

casein, 120 caspase, xi, 40, 46, 48, 49, 52, 53, 54, 56, 59, 60, 133, 140, 141, 150, 154, 226, 229, 230, 277, 310, 311, 312, 317, 331, 336, 345, 358, 361, 417 caspase-dependent, 140, 361, 417 caspases, 140, 233, 443 CAT, 162, 167, 440 catabolic, 148 catabolism, 26, 126, 131, 136, 201, 205 catalase, 59, 162, 167, 226, 260, 275, 368, 433, 440, 442, 445 catechol, 26, 132, 148, 166, 167, 220, 225, 236, 281, 285, 303, 322, 420 catecholamines, 26, 225 category a, 66, 458 cation, xi, 63, 64, 76, 79 cattle, 29 causation, 152, 234 cavities, 30 CD95, 127, 140 CDK, 130, 146 Cdk inhibitor, 141, 146, 289, 316 CDKIs, 306 CDKs, 335 cDNA, 129, 267, 270, 282 cecum, 431 cell adhesion, 127, 294, 308, 333, 334, 343, 344, 453 cell body, 230, 273, 284 cell culture, xvii, xix, 21, 50, 51, 53, 54, 131, 225, 231, 234, 251, 260, 265, 266, 289, 358, 363, 367, 375, 402, 419, 423, 425 cell cycle, xvi, 38, 129, 130, 131, 133, 134, 141, 147, 150, 201, 206, 230, 234, 263, 264, 266, 273, 275, 276, 301, 306, 308, 317, 318, 330, 332, 333, 334, 335, 336, 345 cell death, xi, 15, 19, 46, 50, 51, 52, 55, 56, 140, 141, 154, 168, 172, 175, 212, 213, 214, 217, 222, 226, 228, 229, 237, 239, 263, 276, 332, 336, 358, 366, 376, 440, 441, 446, 453, 455, 456 cell differentiation, x, 45, 56, 239, 452 cell division, 31, 129, 130, 283, 364, 369, 370 cell growth, xvii, 128, 129, 145, 199, 264, 265, 266, 268, 270, 272, 273, 275, 276, 282, 283, 284, 285, 288, 306, 307, 308, 309, 316, 329, 332, 333, 334, 342, 348, 353, 354, 356, 357, 367, 368, 378 cell invasion, xvi, 205, 301, 307, 308, 341, 344, 382 cell line, xvi, xvii, 14, 48, 49, 52, 55, 59, 127, 133, 145, 150, 164, 228, 239, 259, 263, 265, 266, 275, 276, 282, 289, 291, 292, 296, 301, 305, 306, 314, 317, 318, 319, 320, 333, 335, 336, 342, 343, 344, 345, 352, 353, 354, 357, 363, 365, 366, 368, 370, 371, 373, 375, 376, 378, 381, 453

Index cell lines, xvi, xvii, 59, 127, 133, 259, 263, 266, 275, 276, 282, 289, 291, 296, 301, 305, 306, 317, 318, 319, 335, 342, 343, 363, 365, 366, 368, 371, 373, 375, 376, 378, 381 cell membranes, 33, 293, 433 cell signaling, xvi, 229, 299, 302, 307, 311, 440, 442, 451 cell surface, 29, 137, 140, 204, 267, 270, 272, 278, 279, 280, 281, 298, 316, 368 cellular adhesion, 333, 339 cellular response, 445 cellular signaling pathway, 30 central nervous system, ix, 1, 2, 105, 208, 210, 215, 216, 217, 232, 241, 442, 444 cerebellum, 14, 440 cerebral amyloidosis, 237 cerebral blood flow, xiv, 14, 207, 208, 209 cerebral blood flow (CBF), 209 cerebral cortex, 14, 15, 211, 294, 433, 436, 438, 439, 440, 441 cerebral ischemia, xiv, 14, 19, 207, 208, 209, 210, 212, 213, 214, 216, 217, 226, 239, 446 cerebrospinal fluid, 238 cerebrovascular, xv, 15, 207, 214, 254, 384 cerebrovascular disease, xv, 15, 207, 214, 384 cerebrovascular diseases, 384 ceruloplasmin, 409 cervical cancer, 345 cervical carcinoma, 234, 282 c-Fos, 199, 203, 306, 331 channels, xv, xviii, 132, 213, 230, 243, 251, 253, 411, 413, 416, 417, 418, 453, 456 chaperones, 231, 310, 372 charge density, 161 chelating agents, xv, 185, 219 chelators, 225, 229, 230, 231, 233, 241 chemical degradation, 371 chemical properties, 194 chemical structures, 41, 349, 401, 427 chemicals, xiii, xix, 2, 153, 157, 158, 160, 173, 430, 455, 456, 457, 458, 459 chemiluminescence, 78 chemoprevention, xvii, 38, 153, 154, 241, 258, 299, 303, 305, 311, 325, 326, 338, 340, 370, 375, 447, 459, 460 chemopreventive agents, 147, 261, 330, 336 chemoresistance, 291 chemotaxis, 293 chemotherapeutic agent, 457 chemotherapies, 250 chemotherapy, 154, 249, 250, 263, 295, 313, 318, 352, 364, 365, 459, 460, 461 chicken, 31, 38, 137, 178, 266

469

chicks, 4, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 22 children, 120, 122, 144 Chile, 63 China, xii, 2, 45, 46, 47, 48, 55, 57, 103, 108, 112, 120, 158, 222, 224, 252, 365, 389, 393, 394, 395, 399, 408, 460 Chinese medicine, 64, 246 Chinese women, 285, 299, 305, 316 chloride, 108, 109, 211 chlorination, 175 chloroform, 165 chlorogenic acid, 64, 82, 106 chlorophyll, 178, 179, 180, 193, 194 chlorpyriphos, 162, 170, 171 chocolate, 19, 100 cholecystokinin, 117, 120 cholesterol, 26, 38, 73, 96, 117, 126, 132, 135, 136, 137, 143, 149, 151, 152, 162, 200, 205, 245, 246, 249, 254, 268, 272, 295, 299, 340, 402, 403, 405, 415, 416, 444 cholinergic, 222 cholinesterase, 162, 163, 170 chondrocytes, 148, 376 chromatid, 174 chromatin, 140, 230, 291, 332, 344, 373, 374, 381 chromatograms, 100 chromatography, 59, 182, 187 chromosome, 167, 264, 369, 372 chromosomes, xvii, 363, 369 chronic cough, 384 chronic disease, 107, 123, 152, 234, 430, 451, 453, 454, 458 chronic disorders, 104 chronic myelogenous, 268 chronic obstructive pulmonary disease, ix, 390, 391, 392 chymotrypsin, xvi, 263, 301, 306, 307, 311, 331 cigarette smoke, xvii, 39, 383, 384, 454, 455, 458, 461 cigarette smokers, xvii, 383, 384, 454 cigarette smoking, 120, 235, 386, 389, 443 ciprofloxacin, 34, 39 circulation, 13, 47, 415, 416, 431, 448 cisplatin, 335, 409 citrus, 195 c-jun, 228, 331, 342, 378 classes, x, 23, 27, 283, 302, 360 classical, 140 cleavage, 140, 196, 230, 236, 261, 273, 312, 358, 441, 452 clinical disorders, 221 clinical syndrome, 440 clinical trial, 78, 154, 198, 279, 302, 311, 326

470

Index

clinical trials, 78, 198, 302, 311, 326 clinics, 386 cloning, 270 clostridium botulinum, 37, 94, 164 cluster analysis, 123 clusters, 295 c-Myc, 275, 276, 372, 380 CNS, ix, x, 1, 2, 3, 9, 15, 16, 208, 210, 444 Co, 15, 48, 55, 100, 180, 195, 421, 429 CO2, xiv, 111, 207 coatings, 186 cocaine, 12, 21 cocoa, 38, 78, 101, 178 coding, 295 coenzyme, 152, 266 coffee, 21, 108, 123, 158, 178, 250, 251, 252, 445, 448 cognition, xix, 217, 429, 430, 433, 435, 444 cognitive, xiv, xix, 16, 197, 201, 206, 222, 224, 226, 233, 236, 237, 385, 429, 430, 431, 434, 437, 438, 440, 442, 444, 446, 447 cognitive activity, 16 cognitive deficit, 237, 444 cognitive deficits, 237, 444 cognitive dysfunction, xix, 429, 431 cognitive function, 206, 224, 233, 434, 446 cognitive impairment, xiv, xix, 197, 201, 222, 224, 233, 236, 385, 429, 430, 431, 434, 437, 438, 440, 442, 443, 445, 447, 448 cognitive map, 430 cognitive performance, 224 cohort, 71, 245, 248, 255, 296, 303, 304, 389, 391 cold sore, 28 colitis, 32 collaboration, 57 collagen, 58, 61, 185, 261, 267, 292, 344, 376, 395, 402, 406, 408 colon, xvi, 2, 132, 145, 147, 200, 203, 260, 264, 275, 287, 292, 317, 319, 320, 322, 325, 326, 329, 338, 339, 340, 341, 344, 361, 366, 373, 377, 378, 424, 455, 460 colon cancer, xvi, 147, 200, 264, 275, 287, 319, 325, 338, 339, 340, 361, 366, 373, 377, 378, 424, 460 colon carcinogenesis, 275 colony-stimulating factor, 267, 293 colorectal cancer, xvi, 107, 198, 275, 296, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 338, 339, 340, 358 combination therapy, xvi, 302, 312, 313, 314, 409 common symptoms, 384 communication, 99, 251, 256, 451, 453, 457, 459, 460, 461 community, x, xviii, 23, 375, 383, 386, 388

co-morbidities, 389 competition, 165, 225 complementary DNA, 282 complete remission, 266 complex systems, 200 complexity, 452 compliance, 153, 314 complications, xiii, xviii, 125, 150, 393, 394, 395, 400, 402, 403, 404, 408, 411 components, x, xiii, xvi, xviii, xix, 2, 18, 19, 26, 27, 30, 31, 35, 39, 41, 42, 55, 64, 70, 77, 83, 96, 123, 125, 133, 158, 160, 162, 165, 169, 200, 208, 215, 230, 234, 247, 258, 275, 276, 285, 286, 295, 315, 325, 326, 369, 375, 378, 390, 393, 394, 395, 399, 405, 409, 412, 419, 422, 423, 427, 443, 444, 451, 455, 458, 459 composition, x, xii, xiv, xvii, xix, 24, 26, 35, 37, 81, 99, 120, 121, 160, 169, 177, 179, 187, 189, 234, 315, 347, 348, 378, 396, 419, 431 COMT inhibitor, 225 conception, 452 condensation, 25, 140, 264, 332, 358 conduction, 249 confidence, xviii, 384, 385 confidence interval, xviii, 384, 385 configuration, 281 confinement, 15 confounders, 388 conjugated dienes, 70 conjugation, 26, 431 consciousness, 452, 457 consensus, 120, 447 consent, 386 consumer goods, 166 consumers, xi, 46, 222, 223, 224, 285 contaminant, 166 contamination, xiii, 157, 160 content analysis, 84, 86 contractions, 252 control condition, 15 control group, xii, 7, 103, 111, 113, 114, 115, 116, 117, 118, 119, 162, 387, 399 controlled studies, 224 conversion, 41, 96, 128, 132, 137, 145, 149, 261, 331, 420, 421, 423, 426, 440 cooking, xi, 46, 164, 172, 185 cooling, 31, 38 COPD, xvii, 383, 384, 385, 387, 388, 389, 390, 391, 392 copper, 70, 71, 73, 78, 111, 175, 223, 226, 237, 404 corn, 85, 162 cornea, 199, 202 coronary arteries, 204

Index coronary artery disease (CAD), 245, 246, 248, 252, 254, 255, 402 coronary heart disease, xv, 71, 75, 79, 198, 200, 202, 243, 244, 245, 252, 254, 362 coronavirus, 29, 36 correlation, xv, 71, 112, 165, 219, 234, 245, 291, 354, 356, 435, 458 correlation coefficient, 354, 356 correlations, 108, 438 cortex, ix, 1, 2, 13, 14, 294, 440 cortical neurons, 15, 209, 239 corticosterone, 5, 6, 7, 162 cosmetics, 26, 178, 179 Costa Rica, 99 cost-effective, 188, 191, 192 costs, 56, 245 cough, 384 coupling, 454 covalent, 263, 291 covering, 27 COX-2, 20, 161, 328, 336 CpG islands, 372, 373, 374 CPI, 298 CRC, 120, 174, 358 creatine, 26, 449 creatine kinase, 26, 449 creatinine, 163, 403 CREB, 241, 333 crops, 105 cross-linking, 279, 395, 403, 406 cross-sectional, 206, 224, 233, 254, 385, 434, 446 cross-sectional study, 206, 224, 233, 254, 385, 434, 446 cross-talk, 458 Cryptococcus, 33 CSF, 267 C-terminus, 414 cues, 117, 430 cultural differences, 458 cultural factors, 452 culture, 49, 50, 51, 55, 195, 226, 231, 260, 265, 275, 289, 293, 316, 332, 352, 366, 367, 375, 377, 382, 423, 452 culture conditions, 260, 367 culture media, 289, 377 curcumin, 175, 201, 312, 316, 348, 359 curing, 399 customers, 47 cyclin D1, 130, 140, 143, 320, 335, 382 cyclin-dependent kinase inhibitor, 150, 263, 277, 345 cyclin-dependent kinases, 129 cyclins, 263, 306, 335 cyclodextrins, 295

471

cyclooxygenase, 10, 161, 320, 339 cyclooxygenase-2, 10, 161 cyclooxygenases, 328 cysteine, 367 cytochrome, 140, 141, 154, 161, 166, 167, 222, 313, 424 cytokine, 39, 40, 153, 332, 342, 400 cytokine response, 39 cytokines, 227, 268, 277, 328, 331, 333 cytokinesis, 272, 296 cytoplasm, 56, 168, 294, 329, 334, 372 cytoprotective, 445 cytosine, 264 cytoskeleton, 144, 230, 273, 274, 275, 277, 278, 284, 285, 295, 368 cytosol, 152, 225 cytosolic, 228, 413 cytotoxic, xvi, 29, 175, 227, 267, 293, 301, 305, 312, 316, 365, 376, 377, 426, 454, 456, 458 cytotoxic action, 305 cytotoxic agents, 175 cytotoxicity, 39, 52, 99, 148, 305, 317, 360 cytotoxins, 359

D dairy, 123, 455 dairy products, 455 Dallas, 254 data collection, 389 daughter cells, 334 de novo, 224, 331 death, xvii, 12, 14, 15, 20, 50, 55, 111, 140, 209, 210, 212, 213, 215, 216, 217, 226, 228, 229, 230, 234, 245, 247, 256, 261, 263, 277, 295, 326, 336, 338, 383, 384, 441, 442, 443, 453, 455, 457 deaths, xvi, 161, 248, 325, 364, 402 decay, 64, 76, 178 decisions, 104 defects, 119, 338, 457 defense, 124, 168, 246, 248, 267, 404, 437, 440, 442 defenses, 433, 442 defibrillator, 250, 256 deficiency, 104, 105, 119, 120, 266 deficit, 430 deficits, 237, 444 definition, 387 degenerative disease, 437 degradation, xii, 82, 83, 84, 85, 86, 91, 92, 93, 95, 96, 97, 198, 199, 200, 204, 229, 230, 231, 234, 241, 242, 248, 263, 265, 306, 308, 329, 332, 335, 344, 371, 431, 445 degradation pathway, 263 degrading, 84, 262, 332

472

Index

dehydration, 169 dehydrogenase, xiii, 125, 137, 138, 152 delivery, 47, 56 delocalization, 439 dementia, ix, xix, 201, 206, 224, 236, 385, 430, 431, 434, 442, 448 demographic characteristics, xviii, 383 dendritic cell, 20, 40 dengue, 293 denitrification, 173 density, 12, 14, 19, 20, 26, 40, 66, 70, 78, 108, 121, 143, 151, 204, 246, 253, 294, 310, 314, 319, 329, 361, 405, 406, 407 dental caries, 30 dental plaque, 30, 37 deoxynucleotide, 349 deoxyribose, 356, 360 Department of Agriculture, 75, 78, 170 dephosphorylation, 130, 154, 283, 284, 285, 368, 372 depolarization, 361, 417 deposition, 227, 228, 433 deposits, 249 depression, 119 deprivation, 110, 120, 432 derivatives, xvii, xix, 27, 28, 29, 39, 40, 47, 58, 67, 165, 185, 199, 201, 203, 204, 222, 233, 265, 267, 279, 297, 298, 312, 322, 347, 348, 349, 350, 351, 352, 353, 354, 356, 357, 358, 359, 361, 401, 419, 420, 422, 423, 425, 426, 447 dermatitis, 56 destruction, 29, 167, 174, 440 detachment, 52 detection, xvi, 49, 50, 154, 235, 325, 326, 338 detoxification, 444 detoxifying, 163, 263, 377, 394 developed countries, 104, 200 developmental factors, 135 dexamethasone, 250 diabetes, ix, xviii, 26, 57, 64, 121, 126, 131, 134, 136, 138, 139, 143, 144, 150, 151, 246, 247, 248, 253, 255, 268, 393, 394, 395, 397, 399, 400, 402, 403, 404, 405, 406, 407, 408, 409, 411, 412, 416, 430, 454 diabetes mellitus, xviii, 121, 138, 247, 393, 394, 395, 404, 405, 407, 409 diabetic nephropathy, 255, 400, 408 diabetic patients, 393 diabetic retinopathy, xiv, 197, 198 diacylglycerol, 277 diarrhea, 29, 31, 32 dienes, 70

diet, xvi, 74, 104, 105, 107, 110, 111, 117, 119, 120, 122, 123, 136, 151, 153, 154, 165, 168, 172, 173, 202, 205, 246, 249, 252, 254, 256, 302, 325, 329, 336, 345, 384, 392, 394, 399, 437, 454, 458, 459 dietary, xvi, 13, 19, 21, 26, 37, 39, 46, 47, 48, 75, 78, 79, 99, 100, 105, 120, 121, 123, 126, 136, 143, 144, 147, 150, 153, 172, 175, 194, 198, 202, 234, 247, 285, 299, 325, 326, 330, 337, 338, 340, 345, 371, 374, 380, 385, 389, 391, 402, 424, 435, 452, 453, 454, 455 dietary fat, 120, 338 dietary fiber, 194 dietary intake, 389, 391 dietary supplementation, 144, 402 dieting, 120 diets, 78, 115, 123, 165, 326, 454 differentiated cells, 128, 452 differentiation, x, xiii, 45, 56, 60, 61, 125, 126, 128, 129, 130, 131, 132, 134, 135, 140, 143, 144, 145, 147, 148, 149, 228, 230, 239, 263, 318, 332, 333, 334, 335, 336, 340, 342, 379, 382, 452, 453, 457, 460 digestibility, 137 digestion, 26, 136, 400, 424 digestive tract, 358, 445 dihydroquercetin, 27, 41 dimer, 25, 420 dimeric, 101, 365 dimerization, 264 direct action, 16 disability, 392 disaster, 104 discrimination, 72, 154 disease progression, 20, 237 diseases, ix, xii, xiii, xiv, xv, xvi, xix, 29, 57, 71, 81, 104, 105, 120, 125, 126, 135, 142, 197, 198, 200, 201, 219, 221, 226, 231, 247, 248, 252, 269, 301, 326, 385, 429, 430, 441, 444, 453, 454, 455 disorder, 393, 433 dispersion, 69 disposition, 405 dissociation, 250 distilled water, 113, 138 distraction, 16 distress, 6, 7, 9, 10, 16, 17 distribution, 13, 21, 74, 134, 136, 152, 224, 235, 267, 272, 288, 319, 386, 417, 443, 446, 448, 453 disulfide, 278 diuretic, 83 division, 31, 129, 130, 147, 283, 364, 369, 370 DMFA, 172 DNA, vi, xvii, 30, 127, 133, 140, 149, 150, 166, 167, 172, 174, 175, 190, 192, 199, 203, 229, 230, 241,

Index 246, 247, 255, 258, 264, 266, 270, 273, 282, 289, 290, 291, 295, 310, 321, 327, 332, 333, 336, 342, 343, 345, 347, 348, 349, 351, 352, 356, 359, 360, 361, 364, 369, 370, 373, 380, 381, 430, 435, 445, 447, 452, 458 DNA damage, 30, 133, 149, 167, 174, 175, 190, 192, 247, 255, 258, 332, 333, 342, 369, 445 DNA polymerase, xvii, 199, 203, 347, 348, 351, 352, 359, 360, 361 DNA repair, 133, 150, 264, 333, 336, 345, 360, 361 DNA strand breaks, 229 DNase, 140 docetaxel, 313, 322 docosahexaenoic acid, 437, 444 dogs, 248, 255 dominance, 27 donations, 358 donors, 118 dopamine, 12, 15, 22, 135, 160, 176, 208, 215, 220, 225, 236, 240, 431, 448 dopaminergic, 12, 14, 20, 21, 164, 168, 222, 225, 227, 233, 237, 238, 431, 446 dopaminergic neurons, 12, 20, 227, 238 dosage, 13, 402 dose-response relationship, 389, 400 dosing, 13, 22, 110, 288, 322 Down syndrome, 241 down-regulation, 128, 132, 133, 217, 229, 238, 263, 278 drinking, xviii, 16, 58, 108, 120, 121, 135, 138, 161, 162, 165, 178, 199, 202, 224, 244, 247, 248, 249, 250, 254, 264, 272, 279, 282, 286, 287, 290, 303, 310, 311, 313, 316, 319, 332, 361, 362, 366, 383, 385, 386, 387, 388, 389, 390, 391, 394, 395, 451, 455, 458, 460 drinking pattern, 388 drinking water, 165, 247, 248, 249, 310, 311, 313, 332, 366, 458 drowsiness, 13 drug design, 290, 314 drug resistance, 263, 298, 322 drug targets, 263 drug therapy, 231 drug treatment, 311 drug-resistant, 370, 371, 380 drugs, x, xvi, 23, 27, 30, 31, 35, 39, 126, 135, 168, 198, 201, 227, 228, 236, 252, 269, 286, 302, 313, 325, 352, 365, 375, 455 dry matter, 82 drying, 84, 126, 158, 394, 431 duodenum, 329 duration, 163, 386, 388 dyslipidemia, 135, 200, 255, 407

473

dysplasia, 167 dysregulation, 146, 147

E E. coli, 32, 33, 34, 267, 349, 352 eating, xiii, 125, 454 E-cadherin, 275, 276, 296, 308, 309, 334, 382 edema, 160, 353, 354, 446 Education, 197, 387, 390 EEG, 2, 13, 14, 16, 19, 20 egg, 122, 452 eicosanoid, 328 elderly, xix, 75, 121, 224, 253, 327, 429, 431, 434 electric charge, 160 electrochemical detection, 235 electron, 31, 32, 66, 77, 118, 182, 187, 190, 439 electron microscopy, 31 electron spin resonance, 66, 77 electrons, 263 electrophoresis, 265 ELISA, 332 elongation, 225, 230, 282, 298, 299, 368 embryo, 282, 452, 453 embryogenesis, 457 embryonic development, 452 emulsification, 135, 137, 141 emulsions, 424 enantiomers, 240 encapsulated, 153 encephalitis, 267, 293 encephalomyelitis, 442 encoding, 123, 222, 270, 280, 282, 286, 328 endocrine, 121, 126, 135, 140, 152, 315, 417 endocrine system, 121, 152, 315, 417 endocrinological, 152 endocytosis, 154, 334 endometriosis, 198 endometrium, 340 endoplasmic reticulum, 132, 154, 162, 310 endothelial cell, 99, 127, 135, 160, 170, 199, 200, 203, 204, 247, 296, 308, 319, 340, 341, 412 endothelial cells, 99, 127, 160, 170, 203, 204, 247, 293, 340, 341, 412 endothelial dysfunction, xv, 243, 244, 247, 252, 254, 340 Endothelin, 410 endothelium, 247, 295, 319 endotoxemia, 238 end-stage renal disease, 403 endurance, 26, 249 energy, 105, 117, 118, 122, 126, 128, 131, 132, 138, 141, 143, 147, 153, 154, 244, 249, 253, 404, 412, 416, 453, 454

474

Index

enlargement, 132 enolase, 230 enterovirus, 29, 39 environment, 46, 47, 93, 121, 128, 164, 333, 412, 430, 452 environmental factors, xvi, 325 environmental impact, 190 enzymatic, xix, 27, 48, 56, 140, 164, 351, 419, 420, 426 enzymatic activity, 140 enzyme induction, 377 enzyme inhibitors, 143 enzyme secretion, 152 enzymes, xvii, xix, 24, 26, 27, 28, 30, 33, 40, 82, 84, 126, 136, 137, 152, 158, 160, 162, 163, 166, 170, 171, 173, 175, 199, 222, 226, 230, 241, 248, 260, 263, 265, 283, 303, 328, 347, 352, 365, 367, 368, 400, 404, 429, 433, 440, 442, 446 eosinophils, 267 Epi, 36, 39 epidemic, 126 epidemiology, xv, 120, 202, 219, 234, 454 epidermal cells, 265 epidermal growth factor, 127, 147, 240, 263, 268, 287, 291, 295, 296, 305, 317, 367 epidermal growth factor receptor, 127, 147, 240, 287, 291, 296, 305, 317, 367 epidermis, x, 31, 45, 47, 52 epigenetic, xvii, 264, 291, 363, 364, 372, 373, 374, 454, 455, 458, 459, 461 epigenetic mechanism, 264, 373, 374, 454 epigenetics, 291 epithelial cell, 26, 30, 60, 127, 148, 244, 251, 256, 264, 292, 318, 332, 334, 336, 339, 361, 379, 409, 417, 458, 461 epithelial cells, 26, 30, 127, 148, 244, 251, 256, 264, 318, 332, 339, 361, 379, 409, 417, 458, 461 epithelial ovarian cancer, 392 epoxy, 166, 173 Epstein-Barr virus, 28, 36, 55, 60, 380 equilibrium, 270, 452 Erk, 129, 130, 140, 205 ERK1, 128, 129, 135, 143, 147, 220, 228, 229, 240, 263, 278, 279, 291, 367 erosion, 30 erythrocyte, 160, 169, 417 erythrocytes, 152, 160, 162 erythroid, 167, 367 erythropoietin, 230, 241 Escherichia coli, 32, 38, 99, 182, 293, 359, 361 ESI, 76, 422, 423 esophageal cancer, 264, 287 esophagus, 2, 264, 329, 366

ESR, 66, 235 essential oils, xii, 81, 83 ester, 48, 59, 135, 136, 258, 263, 280, 322, 378, 417 ester bonds, 136 esterase, 56, 135, 170 esterification, xi, 46, 47, 135 esters, xi, 27, 46, 47, 55, 56, 64, 65, 76, 143, 240, 256, 330, 427, 442, 456 estimating, 389 estradiol, 117, 127, 150, 303, 305 estrogen, xvi, 127, 138, 145, 150, 239, 301, 315, 316, 317, 320, 322, 323, 335, 345 estrogen receptors, 127 ethanol, xiv, 37, 49, 50, 51, 73, 86, 109, 160, 161, 165, 169, 170, 173, 177, 179, 181, 194, 397 ethics, 459 ethyl acetate, 165 etiology, 120, 173, 222, 402, 403 eukaryotes, 273 eukaryotic cell, 129, 141, 262, 334 Euro, 245 Europe, 120, 121, 302, 434 evolution, 453, 454 excess body weight, 126 excision, 150, 356, 360 excitation, x, 1, 2, 15, 16 excitotoxicity, 140 exclusion, 386 excretion, 13, 136, 138, 143, 165, 224, 260, 399 execution, 140 exercise, 131, 147, 249, 394, 454, 455 exertion, 384 exocytosis, 152 experimental condition, xi, 63, 64, 72, 74, 287, 421 experimental design, 84, 458 exploitation, 35, 36 exposure, xiii, xviii, 15, 57, 93, 95, 128, 140, 157, 160, 161, 162, 166, 168, 170, 171, 174, 235, 336, 366, 383, 388, 389, 456, 458 external environment, 105 extracellular matrix, 128, 265, 292, 331, 332, 334 extraction, 64, 65, 72, 75, 76, 123, 195, 381, 397, 399, 408 extrapolation, 302 eyes, 5, 11, 14, 18, 30, 249

F failure, 16, 153 familial, 338 family, 2, 31, 127, 128, 130, 137, 140, 153, 222, 229, 230, 241, 247, 261, 264, 269, 289, 290, 291, 294, 309, 318, 327, 331, 332, 335, 336, 342, 351, 356, 359, 431

Index family history, 327 family members, 140, 222, 264 famine, 453, 454 FAO, 178 Far East, 24 farming, 453 Fas, 127, 137, 140, 145, 277, 332 fasting, 13, 138, 248, 400, 411, 413 fat, xiii, 26, 104, 108, 117, 120, 122, 125, 126, 127, 131, 132, 133, 136, 137, 138, 140, 141, 142, 143, 144, 147, 151, 153, 154, 172, 244, 246, 249, 253, 256, 314, 329, 340, 361, 394 fatigue, 144 fats, 123, 136, 142, 158, 455 fatty acids, 137, 142, 148, 160, 246, 248, 249, 360, 443 Fatty liver, 144 fax, 419 FDA, 55 feces, 261 feedback, 132, 152, 379 feeding, 107, 110, 117, 141, 287 females, 253, 302, 388 fermentation, 2, 24, 25, 30, 31, 36, 41, 82, 126, 195, 365, 394, 422, 423, 431 ferric ion, 109, 225 ferritin, 227, 231, 232, 236, 237 ferrous ion, 110, 167, 439 fertility, 104 fertilization, 118, 121 fertilizers, 105, 120 fetal, 49, 224, 235, 452, 457 fetuses, 326 fever, 222 FGF-2, 331 fiber, 194, 244, 273 fibers, 117, 272, 273, 274, 287, 296, 378, 455, 457 fibrils, 225 fibrin, 265 fibrinogen, 293 fibroblast, 127, 184, 185, 199, 203, 331, 353 fibroblast growth factor, 127, 199, 203, 331, 340 fibroblast proliferation, 353 fibroblasts, 61, 150, 240, 276, 371, 379, 380 fibrogenesis, 161 fibronectin, 265, 267, 293, 309, 334, 344 fibrosarcoma, 261, 263, 287, 344 fibrosis, 148, 161, 170, 376 filament, 265, 289, 292, 299 Filipino, 303, 304 Filobasidiella neoformans, 33 Finland, 104, 120 fish, xi, 46, 165, 246, 455

475

fish meal, 165 fish oil, xi, 46 fission, 260 flavonoid, xv, 2, 21, 27, 75, 78, 89, 176, 178, 223, 224, 237, 243, 244, 246, 247, 251, 252, 253, 287, 405, 427 flavonoids, ix, xiii, xv, 1, 3, 7, 19, 20, 22, 28, 37, 38, 41, 64, 75, 76, 89, 99, 100, 107, 118, 120, 126, 153, 157, 168, 169, 221, 224, 226, 232, 233, 235, 237, 243, 245, 247, 251, 252, 253, 254, 258, 289, 314, 340, 362, 385, 403, 404, 408, 431, 444, 447 flavor, xii, 82, 84, 85, 87, 89, 90, 91, 179 flavors, 85 flexibility, 310, 341 flow, xiv, 14, 207, 208, 416 fluctuations, 72 fluid, 110, 162, 238 fluorescence, 174, 265 fluorescence in situ hybridization, 174 fluoride, 106, 118 flushing, 179 focal cerebral ischaemia, 216 folate, 266, 285, 294, 338 Folate, 266 food additives, 46 food allergy, 277, 278 food industry, 48, 179 food intake, xii, xiii, 103, 104, 105, 111, 113, 115, 116, 117, 118, 119, 120, 121, 125, 147, 152, 164, 165, 249, 315, 402, 417 food production, 161, 454 food products, 118 Ford, 150, 380 forebrain, 209, 213, 216, 217 Forestry, 143, 384, 391 fossil, 454 fossil fuel, 454 Fourier, 13 Fox, 224 fragmentation, 133, 140, 172, 332 free radical, xi, xii, xix, 26, 63, 64, 65, 66, 69, 70, 72, 73, 75, 76, 77, 83, 87, 96, 100, 103, 108, 109, 112, 118, 124, 139, 160, 161, 162, 163, 174, 175, 221, 223, 226, 232, 246, 248, 401, 403, 404, 411, 412, 429, 433, 439, 443, 446, 448, 449 free radical scavenger, 26, 64, 162, 439 free radicals, xi, xix, 26, 63, 64, 66, 72, 73, 77, 83, 109, 124, 139, 160, 162, 174, 221, 223, 226, 232, 246, 248, 403, 404, 411, 429, 433, 446 free-radical, 65, 118, 163, 433 freeze-dried, xiv, 87, 177, 185, 186, 189, 194 fresh water, 2 friendship, 57

476

Index

frog, 414, 415 fructose, 85, 241, 396, 406, 448 fruit juice, 19, 79, 100 fruit juices, 19, 79, 100 fruits, 67, 69, 99, 100, 107, 175, 221, 302, 455 frying, 365 FTIR, 123 FT-IR, 396 FTIR spectroscopy, 123 fuel, 147, 153, 454 fumigants, 178 fungi, 27, 33, 34 fungicidal, 33, 40 fusion, 277

G G protein, 129, 294, 298 GABA, ix, 1, 3, 15, 19, 21, 208, 210, 212, 213 GABAB, ix, 1, 6 GABAergic, 4, 5, 6, 7, 8, 9, 10, 11, 18, 19, 21, 214 Gadus morhua, 196 galloyl, 7, 8, 9, 27, 28, 29, 143, 281, 310, 372, 420, 423, 439 Gamma, 164 gamma radiation, 194, 195 gamma-aminobutyric acid, 215, 216, 217 gas, 180 gases, 384 gasoline, 166 gastric, 14, 30, 38, 39, 42, 108, 117, 120, 122, 141, 150, 161, 164, 165, 169, 173, 204, 292, 341, 377, 460 gastric mucosa, 30, 161, 169 gastrin, 105 gastritis, 41 gastrocnemius, 26 gastroenteritis, 29 gastrointestinal, xii, xvi, 81, 91, 107, 122, 164, 302, 315, 325, 326, 338, 406 gastrointestinal tract, xvi, 91, 107, 122, 325, 326 GC, xvii, 7, 25, 27, 30, 82, 106, 221, 223, 281, 341, 347, 348, 349, 350, 353, 354, 396 GE, 122, 145, 148, 316, 319, 320, 340, 341, 403 gel, 47, 48 gelatin, 30 gels, 49 gender, xviii, 384, 386, 387, 388, 392, 454, 455, 456 gender differences, 392 gene expression, xi, 19, 46, 127, 137, 139, 147, 148, 149, 150, 151, 234, 263, 276, 282, 293, 296, 309, 317, 342, 344, 345, 372, 374, 379, 440, 441, 442, 457, 461 gene promoter, 381

gene silencing, 275, 276, 291, 381 generation, xv, 26, 64, 68, 100, 128, 139, 148, 160, 162, 163, 167, 175, 219, 221, 223, 225, 227, 232, 268, 277, 289, 331, 361 genes, xvi, 28, 33, 131, 132, 138, 201, 206, 222, 228, 229, 230, 231, 233, 234, 236, 255, 263, 264, 275, 277, 282, 285, 286, 289, 291, 295, 298, 321, 325, 327, 329, 330, 333, 339, 342, 343, 364, 370, 372, 374, 381, 404, 405, 440, 446, 448, 452, 453, 456, 457, 460 Geneva, 196 genistein, 198 Genistein, 147, 202 genital warts, 55 genome, 348, 364, 369, 374, 452, 453 genomes, 452 genomic, 150, 338, 458 genomics, 148 genotoxic, 40, 167 genotype, 285, 286, 299, 305, 344 genotypes, x, 23, 35, 285 Georgia, 45, 57, 60 geriatric, 452 germ line, 452 Germany, 36, 243, 245 germination, 31 GFAP, xiv, 14, 207, 211 ginger, 297 Ginkgo biloba, 236 ginseng, 83, 85 GIP, 105 gland, 321, 457 glass, 385 glial, 230, 241, 292 glial fibrillary acidic protein, 230, 241, 292 glial fibrillary acidic protein (GFAP), 230 glioblastoma, 289 GLP-1, 105 glucagon, 117 gluconeogenesis, 138, 153, 404, 405 glucose, xiii, 30, 105, 107, 117, 125, 131, 132, 134, 135, 137, 138, 139, 143, 145, 147, 150, 152, 153, 154, 162, 201, 205, 230, 244, 248, 253, 255, 329, 340, 394, 396, 399, 400, 402, 403, 404, 405, 406, 408, 409, 412, 413, 415, 416, 417, 448 glucose metabolism, 150, 244, 255, 394, 400, 402, 405, 406 glucose tolerance, 138, 147, 153, 248, 329, 340, 400, 402, 412 glucose tolerance test, 248, 412 glucosidases, 424 GLUT, 231 GLUT4, 142, 143

Index glutamate, ix, xiv, 1, 2, 4, 14, 15, 16, 20, 164, 207, 208, 209, 212, 213, 215, 239, 367 glutamate decarboxylase, xiv, 14, 207, 208, 212 glutamic acid, 22, 209, 210, 212, 213, 215 glutamine, 15, 20, 82, 404 glutathione, 21, 161, 162, 163, 167, 220, 222, 232, 246, 248, 367, 368, 433, 440, 443, 445 glutathione peroxidase, 162, 246, 367, 368, 433, 440, 443, 445 glycation, 248, 255, 395, 403, 404, 406, 407, 408 glycerin, 48, 49, 52, 56 glycerol, 152 glycine, 15 glycogen, 239, 329, 400 glycogen synthase kinase, 239, 329 glycol, xiv, 177, 188, 189 glycolipids, 360 glycoprotein, 395, 397, 407 glycosides, 82, 106, 118 glycosyl, 294 glycosylated, 221, 295 glycosylation, 402 GM-CSF, 267 gossypol, 178, 194 gout, 178 government, iv GPCR, 139 GPI, 269, 294 GPx, 162, 440 grain, 100 Gram-negative, 32, 42 granules, 277, 293 granulocyte, 58, 267, 293 grapes, 32 grass, 83 grouping, 452 groups, xi, xii, xviii, 3, 10, 14, 26, 28, 34, 46, 65, 103, 107, 110, 111, 113, 115, 119, 158, 160, 162, 163, 182, 221, 226, 233, 249, 251, 258, 261, 269, 280, 310, 312, 326, 335, 337, 357, 368, 383, 387, 388, 413, 420, 423, 437, 439, 455 growth factor, 10, 15, 117, 127, 128, 129, 139, 146, 147, 148, 170, 199, 200, 202, 203, 204, 220, 228, 239, 240, 263, 268, 287, 290, 291, 293, 295, 296, 305, 307, 308, 316, 317, 319, 322, 328, 329, 331, 333, 335, 336, 340, 341, 342, 367, 381, 455 growth factors, 127, 128, 129, 139, 170, 199, 203, 228, 268, 308, 328, 331, 333, 335, 455 growth inhibition, 185, 199, 270, 276, 282, 284, 287, 308, 336, 340, 354, 357, 366, 368, 370, 371, 374, 380 growth rate, 353, 371 GSK-3, 329, 445

477

GST, 163 GTE, xii, xiii, 26, 103, 104, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 161, 302, 305, 310, 311, 313, 314 guidelines, 35 gut, 13, 37, 74, 135, 296, 330

H H. pylori, 30, 34 H1, 230 H1N1, 29 H3N2, 29 Haifa, 219 half-life, 12, 13, 224, 260 hanging, 110 harm, xx, 374, 451, 459 harmful effects, 375 harvest, xvii, 347, 348, 431 harvesting, 87 hay fever, 277 hazards, 458 HBV, 29 HDL, 136, 246 head and neck cancer, 175 headache, 222 healing, 55, 56, 456 health care, x, xi, 45, 46, 56 health effects, xiii, 75, 126, 157, 160, 161, 215, 232, 287, 361, 391, 401 health problems, 105, 141 health status, 364 heart, xii, xv, 15, 26, 64, 71, 75, 79, 81, 105, 119, 123, 198, 200, 202, 243, 244, 245, 246, 248, 249, 251, 252, 254, 255, 256, 314, 326, 329, 362, 391, 393, 407, 414, 433 heart disease, xv, 64, 71, 75, 79, 105, 119, 200, 243, 244, 246, 254, 314, 326 heart failure, 249 heart rate, 15 heart rate (HR), 15 heat, 31, 93, 101, 131, 194, 220, 231, 241, 365 heat shock protein, 220, 231, 241 heating, 381 height, 386 Helicobacter pylori, 30, 32, 39, 41, 42, 362 helper cells, 29 hemagglutinin, 29 hematocrit, xiv, 207, 209, 214 hematologic, 331 hematological, 171 hematopoietic, 332, 340 heme, 247 heme oxygenase, 247

478

Index

hemoglobin, 174, 248 hemolytic uremic syndrome, 32 hemorrhage, 30, 163 hemorrhages, 160 hepatic fibrosis, 161, 170 hepatic necrosis, 172 hepatitis, 42, 55, 57, 59, 302, 315 hepatitis a, 315 hepatitis B, 29, 42, 55, 59 hepatitis d, 315 hepatocarcinogenesis, 338, 427, 461 hepatocellular, 146, 161 hepatocellular carcinoma, 146 hepatocyte, 237, 307 hepatocyte growth factor, 307 hepatocytes, 56, 127, 150, 151, 175, 452 hepatoma, 127, 145, 231, 234, 282 hepatotoxicity, 164, 170, 171, 302, 315 HER2, 127, 148, 305, 306, 309, 317 herbal, 72, 77, 79, 408 herbicide, 163 herbs, 198, 302 herceptin, 314 hereditary non-polyposis colorectal cancer, 326 hERG, 253 herpes, 55, 57, 59 herpes simplex, 55, 57, 59 heterocycles, 36 heterogeneous, 268 high blood cholesterol, 143, 402 high density lipoprotein, 246, 295 high fat, 151 high risk, 143, 256, 402 high school, 386, 387 high temperature, 24, 385 high-density lipoprotein, 143, 329, 405, 406, 407 high-fat, 104, 120, 136, 144, 249, 256 high-performance liquid chromatography, 77, 235, 268 high-risk, 361 high-speed, 59 high-throughput screening, 382 hip, 108, 121, 249 hip fracture, 108, 121 hip fractures, 108 hippocampal, xv, xix, 14, 207, 209, 210, 213, 215, 217, 236, 268, 295, 429, 430, 435, 436, 437, 438, 440, 444, 445, 446 hippocampus, ix, xix, 1, 2, 13, 14, 15, 19, 212, 217, 225, 228, 429, 430, 433, 435, 436, 437, 438, 439, 440, 441 Hippocampus, 430 Hiroshima, 197, 304, 315

histamine, 268, 277, 278, 279, 281, 283, 297 histological, 412 histone, 148, 230, 264, 373, 461 HIV, 28, 37, 39, 42, 55, 60, 238, 329, 377 HIV-1, 28, 39, 42, 60, 238 HNE, 433 HO-1, 164, 247 holoenzyme, 372, 378 homeostasis, x, 45, 57, 131, 132, 139, 222, 231, 334, 395, 445 homogenized, 86 homolog, 299 homology, 234 homovanillic acid, 12 Honda, 42, 60, 427 honey, 83, 85 hormone, 108, 137, 305, 314, 342 hormones, 117, 128, 137, 200, 455 hospital, 386 hospitalization, 251 hospitals, xviii, 383, 385, 386 host, 28, 30, 167, 174, 267, 384 hot water, 26, 165, 384, 395 household, x, 45, 46 HPLC, xi, 37, 46, 49, 50, 53, 54, 56, 57, 76, 144, 180, 268, 397, 398, 420, 421, 422 HPV, 55 HR, 15, 77, 147, 340, 386 H-ras, 167, 263, 288, 327 HRP, 423 HSC, 161 HSP90, 220, 231 human behavior, 393 human brain, 293, 457 human exposure, 164 human genome, 348 human immunodeficiency virus, 39, 42, 55, 57, 60, 294 human papilloma virus, 55 human subjects, x, 1, 2, 15, 45, 138, 142, 168, 227, 244, 253, 288 humans, x, 14, 20, 21, 24, 26, 28, 30, 31, 39, 45, 47, 57, 58, 69, 79, 96, 99, 105, 108, 135, 138, 145, 147, 167, 172, 201, 204, 205, 232, 244, 253, 254, 255, 260, 265, 266, 288, 298, 302, 314, 321, 338, 340, 358, 359, 362, 364, 368, 375, 406, 414, 416, 434, 437, 447 Huntington's disease, 256 hydration, 160 hydro, 47, 77, 136, 166, 173 hydrocarbon, 241, 289, 456 hydrochloric acid, 86, 109 hydrocortisone, 49

Index hydrogen, 33, 59, 65, 72, 74, 118, 167, 175, 190, 203, 248, 260, 264, 289, 366, 367, 375, 404, 420, 439, 440, 442, 443 hydrogen bonds, 264 hydrogen peroxide, 33, 59, 65, 72, 74, 167, 175, 190, 203, 248, 260, 289, 366, 367, 375, 404, 420, 440, 442, 443 hydrogenation, 179 hydrolases, xix, 419 hydrolysis, 30, 42, 56, 135, 136, 196, 424 hydrolyzed, 312 hydroperoxides, 70, 72, 80, 407 hydrophilic, 47, 77, 136 hydrophobic, 47, 135, 264 hydrophobicity, 34 hydroquinone, 166, 175 hydroxyl, xi, 20, 26, 28, 29, 46, 167, 175, 178, 191, 192, 221, 223, 226, 231, 258, 263, 280, 302, 310, 312, 330, 337, 357, 368, 400, 409, 413, 439 hydroxyl groups, xi, 26, 28, 46, 221, 258, 280, 310, 312, 337, 368, 413, 439 hydroxylation, 28, 241, 281 hydroxyproline, 161 hydroxypropyl, 6 hypercholesteremia, 286 hyperglycemia, 138, 248, 399, 403, 404, 407, 411, 412, 416 hyperlipidemia, 151, 246, 247, 254, 395, 403 hypermethylation, 264, 291, 373 hyperplasia, 167 hypertension, 104, 126, 145, 200, 215, 246, 247, 254, 268, 329, 340, 454 hypertensive, 158, 208, 209, 215, 216, 247, 255, 258, 447 hypertrophy, 128, 145, 250, 329, 340 hypnotic, 6, 7 hypocholesterolemic, 83, 136, 137 hypomethylation, 373, 374 hypotensive, 244, 246, 247 hypothalamic, 121 hypothalamus, 135, 147 hypothesis, 28, 33, 163, 285, 303, 430, 446, 449, 457, 458 hypothyroidism, 119 hypoxia, 131, 220, 222, 230, 231, 234, 237, 241, 333, 414, 430, 443 hypoxia-inducible factor, 222, 241 hypoxia-ischemia, 237 hypoxic, 231

I IAEA, 178 ICD, 250

479

ice, 12, 76, 311, 313, 361, 400 identification, 41, 139, 172, 235, 269, 282, 286, 293, 403, 443, 447 IgE, 127, 268, 277, 278, 296, 297 IGF, 127, 129, 146, 330, 336 IGF-1, 127, 146, 330, 336 IGF-I, 127, 129 IKr, xv, 243, 244, 251, 252 IL-1, 10, 30, 336, 400 IL-10, 30 IL-6, 10, 276, 418 IL-8, 10 illumination, 229 immortal, 370, 371, 378, 379, 455 immortality, 452 immune response, 336 immune system, 105, 119, 267, 329 immunocytochemistry, 174 immunodeficient, 292 immunofluorescence, 227 immunoglobulin, 15, 231, 297 immunohistochemistry, 311, 313 immunological, 135 immunomodulatory, 39 immunomodulatory agent, 39 immunoreactivity, 227 immunotherapy, 305, 364 impairments, 217 implementation, xv, 126, 219, 455 in situ, xi, 46, 319 in situ hybridization, 174 in utero, 235 inactivation, 130, 137, 138, 166, 203, 287, 322, 374 inactive, 31, 223, 310, 373, 380, 439 incidence, xiii, xvi, xviii, 104, 125, 224, 231, 246, 251, 301, 311, 312, 315, 335, 384, 393, 394, 399, 454 inclusion, 118, 143 incubation, 49, 110, 140, 165 incurable, 326 independent variable, 387 India, 157, 252 Indian, 157 indication, 72, 73 indicators, 74, 122 Indigenous, 407 indirect effect, 403 indole, 190, 191 inducer, xiii, 50, 104, 118, 119, 353 induction, xvi, 22, 56, 68, 69, 70, 130, 133, 134, 146, 153, 160, 166, 175, 203, 228, 229, 230, 232, 238, 263, 266, 278, 289, 296, 301, 306, 308, 309, 310, 311, 314, 316, 317, 319, 320, 330, 332, 333, 335,

480

Index

336, 341, 342, 366, 368, 371, 374, 376, 377, 378, 379, 446 induction time, 68, 69, 70 industrial, ix, xiv, 166, 177, 178, 188, 190, 191, 192, 195 industrial application, 191, 192, 195 industrialized countries, 245, 247 industry, x, 23, 27, 48, 178, 179, 190, 194 inert, 166 infarction, 14, 19, 215, 216, 245, 250 infection, 28, 29, 30, 32, 34, 36, 38, 39, 41, 42, 56, 60, 120, 377 infections, 28, 30, 36, 39, 42, 268, 329 infectious, 28, 46, 453, 455 infectious disease, 46 infectious diseases, 46 infectious mononucleosis, 28 inflammation, xvii, 132, 148, 160, 200, 267, 306, 308, 314, 328, 330, 348, 353, 354, 356, 357, 358, 376, 399, 400, 424, 460 inflammatory, x, xvii, 10, 45, 46, 47, 56, 132, 135, 149, 151, 158, 163, 168, 222, 227, 244, 252, 277, 308, 331, 332, 336, 342, 343, 348, 353, 354, 356, 357, 359, 361, 367, 424, 426, 455, 456 inflammatory bowel disease, 135 inflammatory cells, 163, 308 inflammatory disease, 56, 222 inflammatory mediators, 277 inflammatory response, 10, 132, 135, 361 inflammatory responses, 132, 135, 361 influenza, 29, 39, 41, 55, 57, 58, 59, 427 influenza a, 55 infusions, xii, 69, 70, 72, 74, 77, 78, 81, 83, 235, 315, 408 ingest, 74 ingestion, 14, 19, 39, 58, 78, 136, 147, 161, 166, 168, 223, 235, 253, 260, 261, 272, 288, 302, 315, 340, 414, 442 inhalation, 162, 170 inherited, 327, 364, 443 inhibitor, xiv, xvii, 14, 15, 130, 137, 145, 146, 150, 152, 171, 193, 197, 202, 203, 205, 207, 210, 212, 225, 229, 239, 266, 291, 297, 313, 322, 323, 331, 332, 336, 347, 348, 352, 353, 354, 356, 357, 359, 360, 361, 366, 372, 378, 381 inhibitors, xiv, 36, 37, 129, 141, 143, 152, 164, 169, 197, 198, 199, 200, 201, 203, 204, 213, 215, 225, 231, 241, 264, 266, 282, 290, 291, 306, 318, 335, 342, 343, 348, 349, 359, 380 inhibitory effect, xiv, 12, 15, 29, 39, 41, 55, 100, 130, 132, 134, 140, 144, 145, 166, 167, 174, 177, 197, 204, 225, 261, 265, 272, 275, 276, 277, 279,

281, 282, 283, 285, 317, 321, 348, 349, 351, 353, 354, 361, 394, 400, 403, 404, 424, 458 initiation, xvi, 28, 105, 120, 166, 200, 310, 321, 325, 326, 338, 364, 456 injection, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 17, 18, 135, 209, 210, 213, 214, 412 injections, 161 injury, 42, 154, 161, 171, 172, 221, 225, 228, 236, 252, 256, 267, 302, 333, 416, 444 innervation, 122 inoculation, 271, 311 inorganic, 119 iNOS, 12, 161, 220, 227 inositol, 277, 295 insecticide, 162, 171 insecticides, 162, 170 insects, 161, 162 insertion, 66, 248 insight, 132, 331, 392 insomnia, 302 instability, 47, 311, 327, 329, 367, 368 insulin, xix, 105, 117, 120, 127, 128, 132, 134, 135, 138, 139, 144, 145, 146, 150, 151, 153, 154, 244, 248, 255, 256, 329, 393, 394, 400, 402, 404, 405, 406, 407, 408, 409, 411, 413, 416, 442, 448 insulin resistance, xix, 134, 135, 138, 139, 144, 150, 151, 394, 400, 405, 406, 407, 411, 416, 448 insulin sensitivity, 138, 244, 248, 255, 256, 329, 394, 400, 404, 405, 448 insulin signaling, 139, 145, 151 insulin-like growth factor, 117, 127, 146 insulin-like growth factor I, 117 insults, 228, 412, 430, 440, 442 integration, 265 integrin, 127, 144, 334, 344 integrins, 334, 343 integrity, 47, 174, 268, 272, 286, 332, 377 intensity, xiv, 177, 180 interaction, 21, 26, 29, 66, 74, 77, 127, 130, 139, 171, 200, 204, 217, 227, 230, 267, 274, 277, 279, 280, 295, 299, 333, 334, 344, 364, 373, 384, 413, 452, 453, 454, 458 interaction effect, 217 interaction effects, 217 interactions, 33, 34, 37, 39, 78, 267, 280, 286, 334, 343, 344, 452, 459 interface, 136 interference, 29, 275, 344 interleukin, 10, 30, 40, 240, 279, 336, 345, 376, 400 interleukin-1, 30, 40, 345, 376 interleukin-2, 279 internalization, 56, 293 international trade, 178

Index interphase, 272, 274, 296, 334 interpretation, 26, 72 interstitial, 127 interval, 117, 212 intervention, xvii, 26, 71, 277, 363, 403, 406, 454, 456 interview, xviii, 383, 386 interviews, 386 intestinal tract, 261 intestine, 117, 135, 136, 195, 431 intoxication, 161, 169 intracellular signaling, 128, 228, 306, 308, 457, 458, 461 intracerebral, 13, 210, 212, 213 intragastrically, 13, 212 intraperitoneal, 135, 161, 412 intravenous, 12, 224 intrinsic, 131, 140, 154, 360 invasive, 30, 167, 267, 309, 319, 320 iodine, 124 ion channels, 230 ions, 70, 78, 109, 110, 160, 164, 167, 223, 226, 331, 445, 453, 456 iron, 100, 111, 124, 160, 163, 167, 168, 221, 222, 223, 225, 226, 227, 229, 230, 231, 233, 234, 236, 237, 238, 241, 329, 375, 377, 381, 404, 439, 445, 446 irradiation, ix, xiv, 32, 177, 178, 179, 180, 181, 182, 185, 187, 190, 192, 193, 194, 195, 196, 343 IRS, 139 ischemia, xiv, 14, 19, 131, 207, 208, 209, 210, 212, 213, 214, 215, 216, 217, 221, 226, 234, 237, 239, 252, 256, 409, 414, 430, 446 ischemic, 14, 20, 164, 208, 210, 215, 216, 217, 414, 416, 445 ischemic brain injury, 445 island, 264, 291, 373, 381 isoflavones, 221 isoforms, 210, 216, 228, 308, 315, 440, 441 isolation, 296 isomers, 281, 330 isozyme, 21 isozymes, 228, 239 Israel, 219, 232 Italy, 23, 76

J JAMA, 40, 120, 204, 254 Japan, vi, xviii, 1, 2, 18, 46, 47, 55, 57, 165, 197, 200, 201, 204, 207, 222, 252, 254, 257, 303, 304, 315, 316, 347, 358, 359, 383, 384, 385, 386, 389, 391, 394, 395, 399, 408, 419, 421, 422, 423, 424, 426, 429, 434, 445, 460

481

Japanese, xiv, xviii, 38, 40, 47, 58, 69, 107, 153, 165, 173, 196, 202, 204, 207, 208, 224, 245, 246, 248, 253, 254, 303, 304, 316, 358, 361, 384, 385, 386, 388, 389, 390, 404, 406, 420, 427, 434, 437, 458, 460 Japanese women, 304 jejunum, 135 JNK, 20, 40, 128, 228, 238, 239, 263, 336, 367, 377 joints, 104, 249 Jordan, 287, 448 Jun, 11, 20, 40, 128, 144, 145, 146, 148, 149, 150, 151, 154, 155, 172, 199, 203, 239, 253, 254, 255, 256, 263, 320 Jung, 20, 57, 203, 204, 236, 239, 319, 341, 377, 411, 445, 446

K K+, 255, 395, 403, 407, 418 kappa, 170, 227, 238, 341, 442 kappa B, 170, 227, 238, 442 keratinocyte, xi, 46, 49, 61, 382 keratinocytes, xi, 46, 51, 56, 58, 146, 229, 240, 263, 290, 318, 367, 377, 418 kidney, 29, 132, 162, 171, 248, 249, 287, 370, 393, 403, 408, 409 kidneys, 433 killing, 296 kinase, xiii, xvii, 11, 40, 125, 128, 129, 130, 131, 135, 139, 141, 146, 147, 149, 151, 153, 206, 220, 228, 229, 234, 238, 239, 240, 241, 247, 255, 256, 263, 265, 269, 277, 283, 286, 290, 294, 295, 296, 297, 298, 307, 318, 319, 321, 323, 329, 335, 341, 343, 345, 348, 352, 367, 372, 441, 442, 445, 456 kinase activity, 129, 139, 146, 238, 264, 318, 449 kinases, 128, 129, 130, 146, 147, 154, 169, 220, 228, 230, 238, 239, 240, 263, 265, 277, 283, 289, 291, 294, 297, 298, 306, 316, 331, 335, 336, 405 kinetic model, 66 kinetics, 67, 68, 72, 78, 140, 170, 235, 375, 405 Kirchhoff, 360, 361 Kobe, 58, 197, 201, 347, 358 Korea, 125, 143, 177, 180, 190, 195, 411 Korean, 165, 173, 193, 194, 195, 196

L L1, 128, 129, 130, 131, 134, 140, 141, 145, 146, 149, 150, 152 laboratory studies, 138, 401 laccase, xix, 419, 420, 421, 423, 424 lactams, 43 lactase, 394, 399 lactation, 22

482

Index

Lactobacillus, 33 laminin, xiii, xv, 125, 127, 144, 257, 258, 266, 267, 287, 288, 292, 293, 295, 296, 308, 334, 344, 368, 378 Laminin, 266, 267, 292, 293, 343 land, xiii, 157, 160 large intestine, 431 large-scale, x, 23, 33 larvae, 122 larval, 122 latency, 14, 166 LDL, xi, 56, 63, 66, 70, 72, 73, 74, 79, 96, 136, 200, 204, 246, 248 leakage, 33, 163 learning, xix, 226, 429, 430, 431, 433, 435, 437, 439, 440, 442, 444 lecithin, 137 left ventricular, 247 Legionella, 30, 32, 39, 40 Legionella pneumophila, 30, 32, 39, 40 legume, 195 lemongrass, xii, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 95, 98 leptin, 105, 117, 126 Lesion, 204 lesions, 29, 48, 56, 59, 200, 241 lethal factor, 37, 261, 290 lettuce, 32 leucine, 13 leukaemia, 122 leukemia, 144, 166, 174, 268, 290, 292, 297, 341, 352, 353, 354, 357, 366, 371, 376 leukemia cells, 144, 268, 297, 371, 376 leukemic, 40 leukemic cells, 40 leukocyte, 169 Leukocyte, 40 leukocytes, 26, 293 leukoplakia, 38 levodopa, 225 life cycle, 42, 60 life span, 12, 226, 453 life style, 252, 455 lifespan, 379 lifestyle, xv, 40, 58, 123, 126, 219, 247, 385, 386, 389, 393 lifestyles, 391 life-threatening, 249 lifetime, xvi, 325, 326, 389 ligand, 127, 131, 139, 149, 229, 277, 295 ligands, 20, 21, 128 lignin, 27 likelihood, 389, 454, 455

limitation, 74, 367, 384, 389 limitations, x, 23, 47, 67, 72, 389 linear, xvii, 363, 369 linkage, 375, 458 links, 147 linoleic acid, 180, 194 lipase, 48, 56, 136, 137, 155, 420, 424, 425 lipases, 26, 47, 108, 137, 141 lipid metabolism, xiii, 107, 126, 131, 135, 136, 249, 254, 400 lipid oxidation, xiv, 32, 177, 178, 179, 182, 185, 186, 193, 195 lipid peroxidation, 26, 70, 74, 78, 160, 161, 162, 163, 167, 169, 170, 171, 175, 208, 210, 222, 226, 234, 236, 237, 248, 255, 287, 367, 400, 403, 404, 407, 409, 430, 433, 439, 444, 445, 446, 447, 448 lipid peroxides, 248, 403, 443 lipid profile, 254, 255 lipid rafts, 152, 268, 269, 279, 286, 293, 294, 295, 299 lipids, xix, 132, 136, 137, 143, 151, 152, 201, 202, 244, 248, 424, 429, 430, 431, 433 lipolysis, 26, 38, 137, 141, 152 lipophilic, 55, 58, 59, 65, 77, 151, 231, 399 lipopolysaccharide, 40, 227, 238, 269, 294, 320 lipoprotein, 40, 56, 76, 78, 121, 137, 151, 200, 204, 215, 246, 255, 295, 329, 361, 406, 407 lipoproteins, 56, 66, 70, 78, 136, 169, 253, 395, 402 liposome, 267 liposomes, 66, 77 lipoxygenase, 320 liquid chromatography, 77, 235, 268 Listeria monocytogenes, 32 liver, ix, xix, 1, 2, 22, 55, 58, 127, 130, 131, 132, 137, 141, 144, 147, 150, 151, 152, 161, 162, 163, 166, 167, 170, 171, 172, 176, 201, 205, 215, 225, 226, 246, 249, 251, 254, 256, 260, 266, 287, 293, 302, 315, 329, 358, 362, 366, 367, 400, 429, 431, 433, 456, 457, 458, 461 liver cancer, 55 liver cells, 130, 161, 293 liver damage, 162 liver disease, 58, 144, 150, 254, 362 liver transplant, 249 liver transplantation, 249 localization, 227, 296, 319, 341 location, xviii, 87, 384, 386, 387, 388, 390 locomotion, 265, 283 locomotor activity, 21 locus, 328, 461 London, 299 long period, 385 longevity, 40

Index longitudinal study, 388 long-term potentiation, 13, 217 losses, 29 lovastatin, 457, 461 love, 232 low molecular weight, 231, 399 low risk, 285 low-density, xv, 40, 76, 78, 200, 215, 243, 244, 246, 255, 294, 329, 358, 406 low-density lipoprotein, xv, 76, 78, 200, 215, 243, 244, 246, 255, 329, 358, 406 low-level, 459 LPO, xix, 429, 430, 433, 435, 437, 440, 441, 442 LPS, 227 LSI, 35 LTP, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 217 luciferase, 227 lumen, 132, 136, 200, 247 luminal, 295, 416 lung, xvi, 2, 42, 148, 154, 260, 270, 275, 287, 289, 292, 295, 308, 321, 322, 325, 329, 335, 337, 341, 345, 366, 368, 371, 373, 380, 385, 388, 389, 391, 392, 454, 456, 457, 458, 460 lung cancer, 270, 275, 289, 308, 321, 322, 366, 368, 371, 380, 385, 388, 391, 454, 458, 460 lungs, 30, 358, 389 lupus, 56 lymph, 49, 59, 135, 364 lymph node, 49, 59 lymphatic, 340 lymphocyte, 267, 292 lymphocytes, 174, 246, 367, 380 lymphoid, 366, 376 lymphoma, 30, 140, 266 lymphomas, 322 lysine, 169, 381 lysis, 34

M M1, 320 machinery, 128, 163, 229 machines, 31 mackerel, 176 macromolecules, 69 macrophage, 121, 149, 200, 204, 267, 293, 294, 332, 342 macrophages, 30, 36, 39, 127, 132, 144, 174, 200, 227, 238, 253, 261, 267 Madison, 384, 391 magnetic, 265 maintenance, xvii, 105, 155, 256, 278, 308, 363 males, 302, 388 malic, 137

483

malignancy, 316 malignant, 184, 309, 326, 331, 343, 452, 455, 456, 457 malignant cells, 331, 343, 455 malignant melanoma, 184 malondialdehyde, 163, 186, 187, 226, 433, 436 malondialdehyde (MDA), 163 maltose, 30 Mammalian, 321, 349, 352, 378 mammalian brain, 210 mammalian cell, 131, 139, 263, 293, 298, 314, 356 mammalian cells, 131, 139, 263, 293, 298, 314 mammals, 16, 31, 129, 162, 217, 405, 452 management, xiii, 104, 118, 119, 123, 139, 143, 215, 285, 286, 326, 336, 391, 394 manganese, 65, 106, 111, 112, 118, 144, 171, 440, 445, 446 manganese superoxide, 445, 446 manganese superoxide dismutase, 445, 446 manipulation, 411 manners, 140 manufacturer, 49 manufacturing, xvii, 36, 65, 188, 347, 348, 384, 431 MAPK, 20, 128, 129, 130, 145, 146, 220, 222, 228, 233, 239, 263, 307, 336, 377, 440, 441 MAPKs, 151, 161, 228, 233, 263, 443 mapping, 20, 288 marital status, xviii, 384, 386, 387 market, 108 marrow, 174 Maryland, 125, 443 mass spectrometry, 66, 288 mast cell, 61, 267, 277, 278, 279, 293, 297 mast cells, 267, 277, 278, 293, 297 matrix, 128, 150, 161, 199, 203, 261, 265, 286, 292, 308, 331, 332, 334, 366, 367, 376, 424, 453 matrix metalloproteinase, 150, 161, 199, 203, 261, 287, 308, 331, 366, 367, 376 matrix protein, 261, 334 maturation, 15, 140, 267 Mb, 193 MCA, xiv, 207, 208, 209, 210, 211, 212, 213, 214 MCI, 430 MDA, 163, 305, 306, 307, 308, 309, 310, 311, 312, 313, 316, 317, 368, 370 meals, 105, 110, 119 mean systolic blood pressure, 247 measurement, 78 measures, 15, 66, 71, 105 meat, 105, 164, 172, 178, 185, 186, 194 media, 49, 66, 247, 275, 289, 333, 375, 377 median, 453 mediation, 367

484

Index

mediators, 277, 282 medication, 311, 315 medicinal plants, 100, 361, 407 medicine, xiii, 64, 125, 179, 202, 243, 246, 250, 252, 295, 459 Mediterranean, 121, 253 meiosis, 128, 360 MEK, 130, 139, 149 melanin, xiv, 177 melanoma, 154, 282, 320, 344 melatonin, 240, 439, 448 membrane permeability, 35, 140 membranes, 33, 36, 136, 216, 226, 267, 299, 403, 447 memory, xiv, xix, 14, 19, 207, 209, 212, 213, 214, 216, 217, 429, 430, 433, 435, 436, 437, 439, 440, 444, 448 memory deficits, 444 memory performance, 435 men, xvii, 71, 120, 121, 150, 153, 202, 204, 224, 245, 253, 326, 336, 383, 385, 386, 388, 394, 407, 434, 458, 460 mental state, 21 Merck, 36 messenger RNA, 149 messengers, 139, 238, 277 meta-analysis, 79, 202, 303, 316, 392 metabolic, xiii, xv, xvii, xviii, 107, 125, 126, 131, 135, 138, 140, 144, 147, 148, 150, 151, 166, 198, 209, 210, 214, 225, 241, 243, 247, 248, 252, 253, 260, 329, 347, 352, 375, 402, 404, 411, 416, 431, 432, 461 metabolic disorder, 138, 198 metabolic pathways, 135, 260 metabolic rate, 253, 329 metabolic syndrome, 144, 151, 247, 416 metabolism, xiii, 26, 27, 57, 75, 99, 100, 107, 121, 124, 126, 131, 135, 136, 150, 152, 173, 174, 212, 215, 223, 232, 235, 239, 244, 249, 253, 254, 255, 260, 287, 303, 311, 320, 322, 375, 393, 394, 400, 402, 405, 406, 442, 446, 452, 453 metabolite, 25, 131, 163, 166, 170, 174, 225, 280, 288, 303 metabolites, x, 12, 23, 39, 58, 75, 78, 160, 166, 167, 174, 175, 198, 224, 232, 235, 253, 261, 277, 280, 288, 401, 431, 445 metabolizing, 400 metabotropic glutamate receptor, 15 metal chelators, 76, 227 metal ions, 160, 167, 223, 226, 331, 445 metalloproteinases, 199, 308, 331, 366, 367 metals, 71, 232, 455

metastasis, xiv, xvi, 197, 198, 199, 261, 265, 267, 286, 287, 301, 308, 309, 310, 319, 320, 321, 326, 328, 330, 332, 334, 336, 337, 342, 345, 364, 417, 424 metastasize, 334 metastatic, 49, 59, 198, 267, 299, 320 metazoan, 452, 457 metazoans, 457 Methamphetamine, 446 methanol, 65, 180, 194 methicillin-resistant, 31, 38, 41, 43, 99 methylation, 148, 236, 260, 264, 280, 289, 291, 295, 303, 322, 368, 373, 374, 381 Methylation, 373, 378, 381 methylenetetrahydrofolate reductase, 285 metric, xix, 419 mGluR, 15 mGluRs, 15, 21 micelle formation, 138, 415 micelles, 136, 137 microbial, x, 23, 27, 35, 36, 45, 55, 57, 94, 96, 182, 193 microcirculation, 170, 410 microflora, 30 microglia, xiv, 207, 211, 225, 227 microorganisms, x, xii, 23, 34, 82, 94, 167, 178, 182 microsatellites, 327 microscope, 50, 51 microscopy, 31 microsomes, 171 microspheres, 405 microtubule, 174, 285 microtubules, 299 microvascular, 203, 319, 403, 405 midbrain, 440 Middle East, 158 middle-aged, 153, 367 migration, 263, 267, 289, 292, 309, 334, 336, 344, 358 mild cognitive impairment, 430, 443, 445, 447, 448 mild cognitive impairment (MCI), 430 milk, 19, 31, 100, 195, 384 minerals, xii, xiii, 103, 104, 106, 118, 244, 431 Mini-Mental State Examination, 434 Ministry of Education, 201, 358, 426 mitochondria, 140, 246, 249 mitochondrial, 140, 141, 171, 222, 226, 229, 240, 348, 361, 367, 377, 417, 440, 445, 448, 457 mitochondrial DNA, 367, 448 mitochondrial membrane, 140, 226, 361, 417 mitogen, xiii, 11, 59, 125, 146, 161, 220, 222, 229, 233, 239, 290, 367, 377, 443 mitogen activated protein kinase, xiii, 125, 146

Index mitogen activated protein kinases, xiii, 125 mitogen-activated protein kinase, 11, 59, 161, 220, 222, 229, 233, 239, 290, 377, 443 mitogenesis, 126, 129, 132, 134, 139, 154 mitogenic, 126, 128, 130, 139, 140, 141, 306, 308, 316, 341, 456 mitosis, 128, 147, 265, 334 mitotic, 128, 129, 140, 457 mixing, 109 MK-80, 212 MLC, 283 MMP, 161, 203, 204, 205, 261, 289, 331, 332, 335, 336, 377, 424 MMP-2, 204, 205, 261, 289, 336, 424 MMP-9, 261, 332, 335, 336, 377 MMPs, 261, 366 MnSOD, 440 mobility, 310, 334 model fitting, 86 model system, 99, 194, 195, 335 modeling, 19, 264, 266 models, x, xiii, xiv, xvi, xvii, 45, 70, 125, 131, 136, 137, 167, 170, 197, 199, 204, 225, 226, 248, 259, 261, 265, 286, 301, 308, 310, 311, 312, 313, 314, 326, 330, 338, 363, 365, 366, 371, 375, 394, 406 moderates, 6 modern society, 201 modulation, xiii, xvi, 12, 15, 19, 104, 125, 126, 144, 153, 201, 239, 295, 302, 309, 311, 313, 317, 343, 417, 443, 457 moieties, 357 moisture, 84, 126, 164, 186 moisture content, 126, 164, 187 molar ratio, 396 mold, 96 molecular biology, 338 molecular markers, 406 molecular mechanisms, xvi, 301, 303, 326 molecular medicine, 295 molecular oxygen, 440 molecular structure, 359, 412 molecular weight, 231, 358, 395, 399 molecules, x, xiii, 2, 14, 23, 26, 28, 29, 35, 47, 74, 125, 131, 136, 139, 151, 164, 222, 248, 256, 265, 267, 281, 285, 286, 294, 333, 334, 335, 365, 371, 421, 453, 456 monkeys, 225 monoclonal, 319, 364 monoclonal antibodies, 319 monoclonal antibody, 364 monocyte, 40, 200, 204, 294 monocytes, 267, 293 monolayer, 375

485

monomer, 47 monomeric, 77, 126, 193, 221 monomers, 46 mononuclear cell, 135, 276 mononuclear cells, 135, 276 monosaccharides, 30, 396 monoterpenes, 83 monounsaturated fat, 48, 137 monounsaturated fatty acids, 137 mood, 15, 21 Moon, 145, 150, 152, 380, 409 morbidity, xvii, 326, 383, 384, 389 Morocco, 37 morphogenesis, 203, 382, 452 morphological, 140, 162, 164, 277, 332 morphology, 31, 273, 274, 278, 284 mortality, 71, 165, 170, 199, 200, 204, 244, 245, 246, 247, 248, 250, 254, 296, 302, 326, 336, 385, 389, 391, 392, 400, 402, 452, 460 mosaic, 29 motorneurons, 12 mouse, xi, 21, 46, 48, 52, 56, 57, 130, 166, 209, 210, 214, 224, 231, 235, 239, 263, 265, 279, 282, 287, 293, 294, 297, 310, 315, 321, 342, 344, 353, 354, 360, 361, 371, 431, 440, 445, 448 mouse model, xi, 46, 48, 56, 210, 440 mouth, 28 movement, 416 MPA, 212 MPP, 220, 225, 236 MPTP, 12, 20, 21, 220, 225, 227, 229, 236, 238 mRNA, 15, 22, 134, 135, 174, 213, 227, 229, 231, 249, 278, 291, 309, 328, 331, 403, 441 MRSA, 31, 34 MS, 58, 66, 76, 151, 153, 155, 253, 254, 256, 265, 317, 319, 321, 338, 396, 421, 422, 423, 426, 444, 445, 446 MSI, 327 MTHFR, 299 mucosa, 30, 320, 394, 406, 431 mucous membranes, xi, xix, 28, 45, 429 multicellular organisms, 140, 332, 379 multiple myeloma, 276, 292, 296, 340 multiple sclerosis, 241 multivariate, xviii, 383, 386, 389 murine model, 167, 175, 238 murine models, 167 muscle, 27, 107, 117, 127, 130, 131, 135, 141, 143, 147, 150, 151, 160, 172, 200, 205, 249, 283, 296, 298, 334, 344 muscle cells, 127, 130, 135, 150, 160, 200, 205, 298 muscle contraction, 283 mutagen, 165

486

Index

mutagenesis, 119, 175 mutagenic, xviii, 83, 164, 166, 167, 173, 393, 394, 401, 454 mutant, 12, 129, 225, 333 mutants, 309, 361 mutation, 133, 167, 227, 327, 328, 339, 356, 364, 443 mutations, 39, 326, 327, 329, 337, 339, 433, 452 MYC, 381 Mycobacterium, 30, 36 myeloid, 144, 170, 341 myeloma, 276, 292, 296, 340 myoblasts, 132, 148 myocardial infarction, 79, 245, 246, 250, 251, 252, 254, 256 myocardium, 403 myosin, 272, 274, 275, 277, 278, 283, 284, 287, 296, 297, 298, 368, 378 myricetin, 64, 106, 365

N NA, 90, 149, 264, 359 Na+, 255, 395, 403, 404, 407, 415 NADH, 316 Nash, 319 nasopharyngeal carcinoma, 381 National Academy of Sciences, 173, 318, 320, 322 National Institutes of Health, 125 natural, x, xi, xiii, xv, xviii, xix, 19, 21, 23, 43, 46, 85, 99, 108, 110, 119, 145, 152, 157, 160, 164, 168, 178, 179, 185, 190, 194, 196, 201, 202, 208, 219, 221, 224, 261, 263, 312, 321, 326, 344, 348, 349, 366, 376, 393, 396, 399, 401, 408, 411, 422 natural food, xiii, 119, 157, 160 natural resources, 178 nausea, 266 neck, 167, 175, 203, 296, 317, 345 necrosis, 30, 99, 140, 154, 172, 220, 227, 238, 332, 380 nematodes, 217 neonatal, 16, 22, 452 neonates, 76 neoplasia, 258, 287, 326, 460 neoplasm, 56, 286 neoplasms, 167, 452 neoplastic, 46, 83, 263, 267, 328, 332, 339, 370, 452 neoplastic cells, 263, 267, 370 neovascularization, 202, 204, 308 nephrectomy, 248 nephropathy, 402, 403 nerve, 15, 232, 239, 443 nerve cells, 239, 443 nerve growth factor, 15, 239

nervous system, 239, 240, 440 nervousness, 375 Netherlands, 19, 100, 122, 314, 385 network, 123, 146, 149, 273 NeuN, xiv, 14, 207, 211 neural development, 356 neural network, 123 neurobiological, xv, 219 neuroblastoma, 168, 227, 230, 234, 294, 343 neurodegeneration, 168, 206, 224, 227, 228, 233, 234, 236, 441, 443, 445, 446 neurodegenerative, xv, xix, 11, 21, 26, 40, 64, 143, 168, 201, 206, 219, 221, 222, 224, 225, 226, 227, 228, 230, 231, 232, 236, 240, 375, 417, 429, 430, 433, 440, 442, 444, 447 neurodegenerative disease, xv, 11, 21, 26, 40, 64, 168, 201, 206, 219, 222, 224, 226, 227, 228, 230, 231, 232, 240, 417, 430, 433, 440, 442, 444, 447 neurodegenerative disorders, xv, 143, 219, 221, 225, 232, 375, 430 neurodegenerative processes, 236 neuroendocrine, 117 neuroendocrine system, 117 neurogenesis, 360 neurological disease, xix, 226, 429 neuronal apoptosis, 217, 333 neuronal cells, 12, 127, 227, 233, 238, 440, 442 neuronal death, 14, 20, 209, 210, 215, 216, 228, 229, 230 neuronal degeneration, 222, 433, 448 neuronal loss, 236 neuronal survival, 230 neurons, 12, 15, 20, 21, 40, 210, 213, 217, 225, 227, 228, 236, 238, 239, 268, 294, 295, 430, 441, 442, 444, 452 neuropathology, 446 neuropeptides, 267, 293 neuroprotection, xv, 14, 20, 120, 206, 207, 209, 210, 214, 216, 217, 219, 222, 225, 226, 227, 228, 229, 230, 231, 233, 234, 239, 241, 442, 445, 446 neuroprotective, xiv, xv, 12, 14, 16, 19, 21, 201, 206, 207, 208, 209, 210, 212, 213, 214, 216, 217, 219, 222, 223, 224, 226, 228, 229, 230, 231, 234, 238, 240, 367, 417, 430 neuroprotective drugs, 238 neurotoxic, 170, 229 neurotoxicity, 14, 21, 171, 201, 225, 236, 268, 295, 433, 444 neurotoxins, 228 neurotransmission, ix, 1, 15, 22, 162 neurotransmitter, 15, 22, 210, 247 neurotransmitters, x, 2, 15 neurotrophic, 216

Index neutrophil, 292 neutrophils, 127, 267, 292 New York, 173, 314 New Zealand, 103, 104, 109, 110, 123, 301, 391 Newton, 239, 407 Newtonian, 395 NF-kB, xiii, 126, 132, 150, 308, 318 NF-κB, 133, 160, 161, 168, 175 Ni, 197, 347, 399, 407 Nielsen, 19, 123 nigrostriatal, 12 NIH, 148, 149 nitrate, xiii, 157, 160, 164, 165, 172, 173 nitric oxide, 12, 19, 20, 21, 144, 161, 220, 223, 227, 233, 238, 247, 255, 308, 320, 443, 444 nitric oxide (NO), 12, 193 nitric oxide synthase (NOS), 12, 19, 20, 21, 144, 161, 220, 227, 238, 247, 255, 294, 320 nitrite, 164, 173 nitrogen, 164, 231, 241, 411 nitrosamines, 164, 165, 172, 178, 193 nitroso compounds, 164, 165, 172, 173 nitrosoamines, 160 Nixon, 342 NMDA, 12, 15, 208, 209, 212, 213, 216, 217, 444 NMDA receptors, 209, 213 N-methyl-D-aspartate, 12 NMR, 421, 422 N-N, 172 nodules, 455 non toxic, 233 non-enzymatic, 163, 248 non-insulin dependent diabetes, 407 non-smokers, 385, 389 nontoxic, 164, 371 noradrenaline, 132 norepinephrine, 135, 215 normal, 49, 51, 55, 111, 127, 133, 135, 204, 227, 236, 266, 267, 276, 307, 317, 318, 327, 329, 331, 332, 339, 342, 356, 367, 370, 371, 373, 374, 394, 396, 399, 400, 402, 404, 406, 408, 412, 416, 431, 442, 456, 457, 458, 461 normalization, 49 North Africa, 158 North America, 236, 407, 434 N-ras, 327 Nrf2, 367, 377, 440 N-terminal, 11, 40, 128, 228, 239, 263, 367 nuclear, 10, 54, 127, 131, 133, 140, 162, 168, 169, 175, 227, 229, 237, 238, 264, 265, 275, 296, 318, 320, 332, 342, 358, 367, 380, 440, 446, 448 nuclear factor, 380 nuclear factor-κB, 168

487

nuclear magnetic resonance, 265 nuclear receptors, 127, 131 nuclei, 164, 174, 334, 356, 457 nucleotides, 149, 369 nucleus, 12, 123, 139, 227, 230, 329, 372, 442 nucleus accumbens, 12, 123 nursing, 33 nursing home, 33 nutraceutical, 99, 443 nutrient, xv, 110, 117, 147, 219, 453, 454, 455 nutrients, 198, 308, 331, 334, 366, 459 nutrition, 326, 453, 460 nutritional supplements, 451 nuts, 123

O obese, 108, 117, 120, 127, 134, 137, 142, 144, 153, 154, 248, 253, 255, 454 obese patients, 108 obesity, v, xiii, xiv, xviii, 57, 104, 107, 108, 117, 120, 121, 122, 124, 125, 126, 131, 134, 136, 138, 141, 142, 143, 144, 145, 150, 152, 154, 155, 197, 198, 200, 205, 244, 247, 248, 256, 296, 402, 409, 411, 415, 416, 424, 459 observations, xi, 7, 46, 130, 138, 140, 225, 272, 281, 285, 442 occlusion, xiv, 14, 207, 209, 210, 211, 212, 213, 214, 216 occupational, 161 OCs, 164, 165 odds ratio, xviii, 384, 385 odors, 185 oedema, 198 oil, xi, 46, 47, 56, 162, 172, 178, 179, 180, 193, 194, 221 oils, xii, 46, 47, 48, 76, 81, 83, 136, 179, 196 oligodendrocytes, 442 oligomeric, 122 oligomerization, 140 olive, 194, 221 olive oil, 194, 221 omega-3, 246 oncogene, 275, 290, 291, 295, 309, 318, 329, 344, 380, 381 oncogenes, xvi, 325, 326, 330, 337, 364 oncogenesis, 460 oncology, 60, 198, 298, 315, 317, 318, 338 oncosis, 140 online, 123 oocytes, 252, 414, 415 Opioid, 120 Ops, 163 optical, 330

488

Index

optimization, 196 oral, 12, 13, 14, 15, 28, 30, 38, 40, 47, 49, 50, 51, 55, 56, 57, 59, 113, 114, 116, 118, 135, 152, 153, 162, 164, 167, 170, 172, 175, 199, 224, 228, 229, 235, 244, 258, 261, 264, 266, 272, 282, 283, 287, 288, 302, 311, 314, 315, 322, 335, 361, 366, 371, 375, 394, 413, 424, 431, 432, 442, 445, 460 oral cancers, 335 oral cavity, 164, 244, 261, 264, 366 oral hypoglycemic agents, 394 oral leukoplakia, 38 oral squamous cell carcinoma, 49, 59, 175 orchestration, 452 organ, 37, 247, 249, 264, 321, 452, 453, 456, 457 organelles, 319, 431, 432, 433 organic, xiii, 30, 72, 104, 119, 291, 395 organic solvent, 395 organic solvents, 395 organism, 31, 74, 457 organization, 230, 285, 296, 298, 453 organizations, 178 organophosphorous, 171 orthopaedic, 386 osteoarthritis, 104 osteosarcoma, 371 outpatient, xviii, 383, 385, 386 ovarian cancer, 48, 55, 59, 341, 370, 385 ovariectomized, 152, 340 ovariectomized rat, 152, 340 overload, 213, 230, 340, 416 overproduction, 164 overweight, 141, 144, 153, 200, 248, 253 oxidability, 79 oxidants, xix, 42, 70, 225, 239, 258, 260, 447, 451, 459 oxidation products, 41, 260, 403 oxidation rate, 69 oxidative damage, xiii, xix, 69, 157, 160, 162, 170, 237, 246, 343, 377, 409, 429, 430, 433, 435, 439, 440, 445, 446, 448 oxidative stress, xv, xix, 12, 26, 72, 130, 131, 158, 160, 161, 163, 164, 167, 168, 169, 175, 203, 219, 221, 232, 233, 238, 239, 240, 241, 247, 255, 258, 263, 290, 333, 361, 377, 395, 399, 400, 403, 404, 408, 409, 412, 417, 430, 433, 438, 439, 440, 441, 442, 443, 445, 446, 447, 456, 458, 459, 461 oxide, 19, 20, 21, 144, 160, 161, 166, 167, 175, 220, 223, 227, 233, 238, 247, 255, 308, 320, 443, 444 oxidizability, 71 oxygen, xi, 33, 43, 63, 66, 69, 77, 78, 95, 107, 160, 167, 175, 179, 190, 198, 221, 223, 226, 230, 231, 232, 238, 241, 242, 246, 248, 260, 308, 368, 403, 411, 412, 420, 433, 436, 438, 440, 446, 453, 459

P P300, 14 p38, 11, 20, 128, 161, 228, 238, 239, 263, 336, 367, 377 p53, xvi, 130, 131, 133, 141, 145, 147, 149, 150, 154, 205, 263, 277, 306, 317, 318, 325, 327, 328, 332, 333, 339, 342, 343, 370, 382 pachytene, 360 packaging, 187, 193 PAHs, 160, 166 pain, 222, 375 PAN, 40 pancreas, 2, 329, 337, 358, 366 pancreatic, 105, 108, 117, 135, 136, 137, 141, 147, 148, 152, 153, 155, 336, 345, 358, 361, 394, 399, 400, 412, 417, 418, 420, 424, 425 pancreatic cancer, 147, 336, 345, 361, 417 Pancreatic cancer, 336 pancreatic islet, 399 pandemic, 104 pantothenic acid, 405 paper, 100, 108, 162, 266 paracrine, 341 paradox, 200, 202, 206 parameter, 90, 92 paraoxonase, 121, 395, 402, 406, 407 parasites, 456 parenchymal, 163 parenchymal cell, 163 parietal cortex, 433 Parkinson, 201, 219, 268 Parkinson disease, 201, 268 Parkinson’s, xv, 107, 120, 171, 172, 219, 220, 222, 430, 443 Parkinson’s disease, 107, 120, 171, 172, 220, 222, 430, 443 Parkinsonism, 20, 224, 225 PARP, 229, 311, 312, 358 particles, 29, 56, 74, 84, 122, 384, 456 pasteurization, xii, 82, 84, 93, 94, 95, 98 patents, 56 pathogenesis, 78, 120, 164, 171, 227, 228, 232, 286, 379, 395, 404, 430, 433, 454 pathogenic, 30, 31, 32, 33, 40, 164, 178, 267 pathogens, 27, 30, 38, 185 pathology, 169, 222, 232, 236, 447 pathways, xvii, 27, 59, 121, 126, 128, 130, 134, 135, 139, 140, 141, 145, 146, 151, 154, 203, 223, 228, 239, 256, 258, 260, 263, 268, 276, 282, 299, 306, 307, 308, 317, 318, 328, 329, 332, 336, 337, 342, 358, 363, 367, 372, 377, 379, 404, 440, 441, 442, 447

Index patients, ix, xi, xv, xvii, 33, 40, 46, 55, 71, 120, 122, 138, 141, 150, 198, 200, 224, 225, 227, 228, 230, 238, 240, 241, 243, 244, 246, 248, 250, 251, 252, 254, 255, 256, 279, 303, 306, 309, 310, 311, 315, 316, 322, 329, 339, 364, 375, 383, 384, 385, 389, 390, 391, 393, 433, 437 PC12 cells, 206, 220, 229, 230, 233, 239 PCP, 458, 461 PCR, 373 PDGF, 127, 204 PDK, 313 pectin, 186, 187, 188, 189, 195 pediatric, xiii, 125 peer, 455 penicillin, 43, 49 penumbra, 216 peptide, 105, 220, 231, 234, 265, 268, 399, 430, 433, 438, 443, 444, 446, 449 peptides, 47, 225, 282, 290, 447 performance, 14, 16, 138, 224, 235, 435, 444, 447 peripheral blood, 276 peripheral blood mononuclear cell, 276 peritoneal, 174 permeability, xi, 35, 46, 47, 55, 56, 140, 204, 209, 213, 214, 319, 331, 405 permeation, 224 permit, 211 peroxidation, 70, 74, 160, 162, 163, 167, 169, 210, 226, 248 peroxide, xix, 179, 190, 367, 368, 375, 429, 436, 438, 443, 445 Peroxisome, 131 peroxisomes, 249 peroxynitrite, 223, 234, 445 personal, 338 Perth, 383 perturbation, 31, 334, 415, 445 perturbations, 33 pesticide, 161, 162, 168 pesticides, xiii, 157, 160, 161, 162, 164, 168, 172 pests, 162 81, 408, 444, 447 PGR, 68, 72, 73 pH, xii, xiv, 30, 33, 47, 50, 68, 71, 74, 82, 84, 91, 92, 93, 95, 98, 109, 165, 207, 209, 214, 312, 371, 372, 421, 423, 445 phagocytosis, 154, 174 pharmaceutical, 178, 185 pharmacodynamics, 173 pharmacokinetic, 12, 36, 86, 224, 236, 266, 417 pharmacokinetics, 2, 405, 406 pharmacological, xv, 2, 83, 126, 135, 198, 200, 208, 219, 228, 229, 231, 258, 261, 396, 404

489

pharmacology, 2, 57, 148, 208, 216 phenol, 108, 109, 121, 126, 158, 166, 169, 178, 196, 250, 391, 421 phenolic, xi, xii, 19, 63, 64, 74, 77, 79, 80, 100, 103, 106, 108, 109, 112, 118, 160, 166, 167, 175, 176, 185, 190, 192, 221, 232, 260, 261, 315, 330, 340, 401, 431, 443 phenolic acid, 64, 80, 118, 232, 315, 340, 401 phenolic acids, 64, 80, 118, 232, 315, 340, 401 phenolic compounds, 74, 77, 79, 106, 118, 185, 190, 431 phenotype, 132, 145, 282, 309, 320, 370 phenotypes, xviii, 411, 452, 453, 457 phenotypic, 309 phenylalanine, 27 pheochromocytoma, 168, 220, 229, 230, 240 philosophical, 452 phorbol, 166, 228, 240, 256, 456 phosphate, xiii, 68, 125, 137, 152, 167, 356, 360, 413, 423 phosphatidylcholine, 33 phosphatidylethanolamine, 33, 153, 269 phosphatidylserine, 140, 269 phosphinic acid, 6 phosphoenolpyruvate, 405 phospholipase C, 14, 16, 21 phospholipids, 29, 66, 160 phosphoprotein, 294 phosphor, 143 phosphorus, 111 phosphorylation, 11, 20, 127, 129, 130, 133, 138, 139, 145, 148, 199, 202, 203, 227, 228, 229, 240, 263, 265, 273, 275, 276, 277, 278, 279, 283, 284, 285, 286, 288, 290, 292, 293, 295, 296, 297, 298, 306, 307, 308, 319, 333, 335, 342, 367, 372, 377, 378, 405 Phosphorylation, 272, 274, 277 photochemical, 124 photooxidation, 179, 180 phycoerythrin, 67 physical activity, xiii, 123, 125, 247, 252, 389 physical exercise, 409 physicians, xviii, 383, 385, 390 physicochemical, 33 physiological, xiv, xv, 13, 14, 20, 21, 26, 47, 74, 104, 105, 117, 118, 126, 131, 135, 137, 154, 177, 185, 188, 198, 207, 208, 209, 214, 225, 257, 258, 267, 275, 276, 280, 283, 286, 287, 306, 318, 319, 331, 332, 333, 356, 378, 424, 452 physiology, 57, 104, 105, 444, 457 physiotherapy, 386 phytochemicals, x, 45, 99, 100, 150, 158, 285, 299, 340, 405

490

Index

PI3K, 129, 135, 139, 153, 307, 318 pigments, 118, 158, 179, 244, 405, 423, 427, 431 pilot study, 153 PKC, 220, 228, 229, 230, 231, 233, 441, 446 PKD, 139 placebo, 13, 14, 15, 154, 236, 258, 454 placental, 332, 342 plants, ix, x, xii, 1, 2, 23, 55, 82, 100, 105, 178, 221, 361, 407 plaque, 200, 204, 441 plaques, 200, 225, 440, 442 plasma, 5, 6, 7, 12, 13, 15, 26, 69, 70, 71, 78, 79, 105, 120, 127, 135, 136, 138, 139, 140, 142, 151, 162, 205, 209, 214, 223, 235, 244, 246, 247, 248, 249, 253, 254, 255, 260, 261, 266, 268, 272, 280, 282, 288, 292, 294, 295, 298, 305, 311, 319, 334, 340, 367, 371, 400, 407, 409, 412, 413, 414, 415, 416, 417, 435, 436, 438, 439, 447 plasma levels, 138, 244, 248, 305 plasma membrane, 15, 127, 139, 140, 268, 272, 292, 294, 295, 319, 334, 414, 415, 416 plasminogen, 265, 292, 294, 332, 342 platelet, 83, 127, 148, 200, 268, 293, 331, 334, 341, 405 platelet aggregation, 83 platelet derived growth factor, 200 platelets, 202 play, x, xiv, 23, 95, 117, 129, 131, 134, 167, 197, 200, 201, 210, 212, 221, 228, 230, 246, 261, 263, 267, 268, 275, 277, 328, 334, 337, 352, 369, 374, 395, 403, 404, 440, 457 PLC, 16 PMA, 228 point mutation, 133 poisoning, 168, 170 polarity, 147 polarization, 265 politicians, 104 pollutants, 166, 455 pollution, 57 polyacrylamide, 49 polycyclic aromatic hydrocarbon, 166, 173 polyethylene, 84 polymerase, xvii, 229, 230, 347, 352, 356, 358, 359, 360 polymerization, 46, 47, 65, 126, 260, 265, 296, 299 polymorphism, 299, 316 polymorphisms, 303 polymorphonuclear, 40, 293 polynucleotide, xvii, 348, 352 polynucleotide kinase, xvii, 348, 352 polyp, 326 polypeptide, 105, 117, 267

polyphenolic compounds, xii, xiii, xv, xvii, 40, 103, 104, 118, 243, 260, 302, 347, 348, 401, 430 polyphosphates, 413, 417 polyps, 326, 327, 332, 338, 455 polysaccharide, 396, 397, 399, 407, 408 polysaccharides, xviii, 393, 394, 395, 399, 400, 405, 407, 408 Polysaccharides, 395, 397, 407 polyunsaturated fat, 70 polyunsaturated fatty acids, 70 PON1, 395 pools, 227 poor, 35, 47, 186, 260, 309, 310, 368, 433, 454 population, 13, 24, 58, 104, 174, 200, 201, 206, 247, 252, 255, 340, 370, 392, 453, 454, 455, 460 pores, 37 pork, 31, 38, 185, 186, 187, 188, 189, 194, 195 portal vein, 431, 447 positive correlation, xii, 104, 118, 303, 435, 438 positive influences, 190 positive relation, 356 positive relationship, 356 postmenopausal, 305, 316 post-translational, 241, 332 potassium, xv, xviii, 12, 106, 111, 118, 243, 244, 251, 252, 253, 256, 403, 411, 413, 417 potassium channels, xv, xviii, 243, 251, 253, 411, 417 potato, 123 potatoes, 32 poultry, 151, 194 powder, xiv, 47, 71, 170, 177, 185, 186, 187, 189, 190, 194, 195, 279, 302, 397 powders, 109, 191 power, xi, xii, 13, 14, 63, 64, 71, 74, 79, 103, 108, 109, 112, 165, 178, 190, 435, 436 PPARγ, 131 precipitation, x, 45, 47, 56, 397 preclinical, 226, 232 pre-clinical, 142 pre-clinical, 326 precursor cells, 128 pre-existing, xiv, 197, 198, 331 preference, 87 prejudice, 386 premature ventricular contractions, 252 preservatives, 178 pressure, 104, 148, 153, 178, 208, 209, 214, 215, 246, 247, 254, 255, 260, 287, 340, 368, 397, 447 presynaptic, 225 prevention, xiii, xvi, xviii, 57, 58, 64, 107, 120, 125, 126, 138, 144, 147, 148, 151, 152, 161, 169, 172, 173, 178, 201, 202, 203, 234, 236, 252, 257, 258,

Index 263, 272, 279, 282, 283, 286, 288, 289, 298, 302, 303, 321, 325, 330, 336, 338, 339, 342, 345, 358, 359, 361, 365, 367, 369, 370, 371, 374, 375, 384, 390, 391, 394, 402, 403, 411, 414, 438, 441, 442, 456, 458 preventive, ix, xv, xvi, xvii, xix, 12, 21, 58, 75, 107, 145, 161, 170, 200, 232, 235, 257, 286, 289, 290, 299, 325, 326, 330, 340, 358, 359, 363, 382, 402, 403, 405, 437, 448, 455, 456, 458, 459, 460 prion diseases, 268, 445 probability, 368, 414 probe, xi, 63, 66, 77 processing pathways, 441 procyanidins, 101, 196 prodrugs, 322, 378 production, xix, 10, 21, 30, 37, 40, 42, 69, 118, 130, 134, 138, 154, 158, 161, 163, 164, 167, 170, 171, 190, 193, 199, 203, 226, 227, 232, 238, 247, 303, 308, 317, 328, 342, 365, 367, 378, 384, 400, 405, 409, 419, 420, 421, 422, 423, 424, 427, 429, 433, 440, 441, 445 progenitors, 332 progeny, 371 prognosis, 150, 309, 319, 370 prognostic marker, 320 program, 143, 154 proinflammatory, 121, 400 prokaryotic, x, xvii, 23, 347, 352 proliferation, xvi, 128, 129, 130, 131, 135, 139, 140, 143, 145, 148, 151, 152, 190, 199, 229, 231, 263, 265, 272, 273, 284, 290, 301, 305, 307, 308, 309, 311, 316, 317, 318, 319, 335, 336, 339, 352, 353, 356, 360, 364, 366, 370, 399, 452, 453 proliferation potential, 453 promote, 56, 57, 132, 139, 160, 179, 226, 263, 308, 309, 326, 334, 435, 451 promoter, 256, 258, 264, 288, 291, 306, 331, 341, 344, 353, 356, 361, 372, 373, 374, 377, 381, 440, 456, 457, 461 promoter region, 264, 331, 372, 373, 374, 381 promyelocytic, 352, 353, 354, 357 pro-oxidant, 72, 78, 91, 100, 160, 229, 240, 260, 333, 367 property, 47, 168, 200, 225, 330, 341 prophylactic, xviii, 226, 393, 394, 395 prophylaxis, 437 propionic acid, 15 propofol, 210, 213, 216 prostaglandin, 10, 339 prostate, xvi, 2, 107, 121, 127, 133, 138, 145, 146, 147, 148, 204, 241, 258, 264, 286, 287, 292, 299, 316, 317, 321, 325, 332, 336, 337, 341, 345, 358, 361, 366, 385, 391, 460

491

prostate cancer, 107, 121, 127, 133, 145, 147, 148, 204, 241, 258, 264, 286, 287, 292, 299, 316, 336, 345, 385, 391, 460 prostate carcinoma, 146, 147, 317 prostatitis, 34, 39 proteases, 331, 424 proteasome, 262 protection, xi, 26, 46, 56, 63, 64, 66, 67, 73, 75, 95, 105, 166, 179, 186, 209, 213, 228, 261, 263, 310, 329, 444 protective factors, 205 protective genes, 230 protective mechanisms, 234, 444 protective role, 388, 389, 447 protein aggregation, 222 protein binding, 12 protein family, 132, 230, 333, 334, 342 protein function, 282, 415 protein kinase C, 206, 220, 222, 234, 239, 240, 295, 297, 372, 445, 456 protein kinase C (PKC), 222 protein kinases, xiii, 125, 128, 161, 220, 222, 233, 239, 263, 265, 297, 336, 443 protein oxidation, 169, 430, 433, 449 protein synthesis, 227, 313 proteinase, 262 proteins, xiii, xvi, 28, 56, 125, 128, 129, 130, 133, 140, 143, 146, 153, 160, 169, 172, 174, 195, 222, 229, 230, 231, 240, 241, 244, 248, 249, 261, 262, 264, 265, 268, 269, 277, 284, 286, 289, 291, 292, 294, 295, 298, 301, 306, 308, 309, 310, 311, 312, 325, 329, 331, 332, 333, 334, 335, 339, 341, 342, 367, 369, 373, 374, 399, 430, 431, 433, 443, 448, 456 proteinuria, 248 proteolysis, 332 proteolytic enzyme, 265 proteomics, 234, 239, 295, 443 protocol, 115, 279, 311, 386, 421 protocols, 35, 138 prototype, 145, 166 proximal, 135 Pseudomonas, 32 Pseudomonas aeruginosa, 32 psoriasis, x, xi, 45, 46, 56, 198 Psoriasis, 60 public, xiii, 24, 29, 104, 122, 124, 125, 126, 144, 201, 243, 397 public awareness, xiii, 125, 126, 243 public health, 24, 29, 104, 122, 124, 144, 201 purification, 41, 48, 194, 397, 407 PVA, 406, 407 pyramidal, 213

492

Index

pyrene, 166, 174, 335 pyretic, 83 pyrimidine, 149

Q quality control, 87 quality of life, 250 quercetin, 64, 86, 87, 89, 106, 174, 204, 237, 289, 343, 365 Quercetin, 89 questionnaire, xviii, 233, 250, 383, 386, 389, 391 quinine, 420 quinone, 25, 77, 174, 260, 288, 420, 423 quinones, 25, 166, 174, 175

R RAC, 63, 67, 72, 73 radiation, 26, 57, 194, 195, 336, 364, 376, 401 Radiation, 177 radiation therapy, 364 radical, xi, xv, 63, 64, 66, 68, 69, 70, 72, 76, 77, 78, 79, 80, 83, 84, 86, 87, 88, 89, 90, 95, 96, 98, 100, 109, 118, 123, 160, 163, 167, 169, 171, 174, 175, 178, 179, 181, 185, 186, 189, 190, 191, 192, 196, 210, 219, 222, 223, 225, 226, 227, 229, 231, 233, 234, 239, 246, 260, 372, 400, 404, 408, 409, 412, 430, 435, 439, 443, 445, 447 radical formation, 210, 226, 231, 404, 435 Ramadan, 360 random, 453 range, 31, 83, 87, 89, 90, 91, 92, 93, 94, 95, 97, 98, 162, 225, 245, 263, 265, 266, 273, 283, 310, 330, 358, 405, 415 rapamycin, 313 ras, xvi, 325, 327, 461 rat, xii, 12, 13, 15, 16, 19, 20, 37, 39, 103, 110, 111, 115, 123, 127, 135, 137, 138, 147, 148, 149, 151, 161, 163, 168, 170, 171, 175, 209, 215, 216, 217, 220, 225, 226, 229, 230, 235, 237, 239, 246, 251, 253, 256, 268, 277, 287, 295, 297, 299, 302, 321, 342, 344, 351, 358, 403, 406, 407, 412, 413, 427, 430, 442, 445, 446, 447, 448, 461 ratings, 105 reactants, 167 reaction mechanism, 41, 427 reaction time, 72 reactive nitrogen, 241 reactive oxygen, x, xiii, xv, xix, 26, 45, 131, 138, 148, 150, 157, 160, 162, 168, 175, 178, 185, 199, 219, 220, 221, 303, 404, 408, 412, 429, 430, 453

reactive oxygen species, x, xiii, xv, xix, 26, 45, 131, 138, 148, 150, 157, 160, 162, 168, 178, 185, 199, 219, 220, 221, 303, 404, 408, 412, 429, 430, 453 reactive oxygen species (ROS), x, xiii, xix, 26, 45, 131, 157, 160, 162, 168, 199, 221, 412, 429, 430, 453 reactivity, 68, 72, 73, 75, 78 reading, 14, 86 reagent, 108, 109, 266 reagents, 49 recall, xviii, 383, 386, 389, 430, 446 receptor agonist, 210, 213, 225 receptor-negative, xvi, 301, 317, 320, 323 receptor-positive, xvi, 301, 316 receptors, ix, xv, 1, 3, 6, 7, 9, 12, 14, 15, 19, 20, 22, 117, 127, 128, 129, 131, 132, 139, 140, 207, 208, 209, 210, 212, 213, 214, 215, 216, 217, 264, 268, 290, 292, 293, 294, 296, 308, 318, 329, 334, 340, 341, 457 reciprocal interactions, 452 recognition, xiii, 126, 222, 373 recovery, 162, 336 rectum, 326 recurrence, 303, 304, 316 red blood cell, 237, 416 red blood cells, 237, 416 red meat, 455 red wine, 178, 196, 198, 200, 202, 204, 221, 376 redistribution, 278 redox, 132, 148, 160, 161, 168, 175, 227, 240, 257, 405, 439, 443, 456, 457, 458, 461 reductases, 41, 152 reduction, xii, xiii, 15, 22, 26, 47, 71, 93, 103, 104, 113, 114, 115, 116, 117, 130, 132, 142, 161, 164, 178, 185, 213, 215, 222, 224, 226, 228, 229, 231, 232, 246, 247, 249, 266, 268, 272, 275, 276, 279, 281, 283, 284, 285, 286, 303, 313, 336, 341, 354, 371, 395, 402, 403, 439, 440, 453, 455 refractory, 244, 252, 452 regenerate, 394 regeneration, 160, 265, 406 regional, 21, 235 regression, xviii, 4, 5, 111, 321, 383, 386, 388, 389, 390, 434, 435, 448 regression analysis, xviii, 111, 383, 386, 388, 389, 390, 434 regression equation, 4, 5 regular, xviii, 85, 96, 119, 200, 244, 246, 252, 382, 383, 388, 389, 414, 416, 456 regulation, x, xi, 45, 46, 105, 121, 122, 127, 128, 129, 131, 132, 133, 138, 146, 147, 148, 150, 152, 205, 213, 217, 222, 226, 227, 228, 229, 230, 231, 238, 239, 241, 263, 264, 267, 278, 286, 295, 298,

Index 305, 318, 319, 329, 330, 331, 335, 336, 339, 341, 342, 343, 344, 345, 369, 372, 374, 379, 381, 418, 440, 441, 454, 457, 461 regulators, 128, 147, 222, 228, 264, 268, 277, 307, 372 rehabilitation, 138 relationship, ix, xvi, xvii, 22, 27, 36, 74, 96, 105, 107, 122, 127, 135, 138, 145, 165, 172, 194, 199, 200, 235, 246, 253, 254, 268, 280, 281, 288, 296, 301, 303, 348, 354, 356, 359, 361, 388, 389, 394, 399, 400, 406, 434 relationships, 19, 28, 99, 175, 176, 232, 291, 340 relaxation, ix, 1, 2, 14, 16, 18, 20 relaxation effect, 20 relevance, 124, 146, 171, 172, 231, 447, 458, 459 remission, 266 remodeling, 200, 261, 273, 277, 278, 284, 331 renal, 74, 248, 250, 255, 403 renal disease, 403 renal failure, 248, 403 repair, x, 45, 57, 68, 133, 150, 261, 264, 327, 333, 334, 336, 338, 345, 348, 349, 356, 360, 361, 452, 456 repair system, 133, 327 reperfusion, xv, 14, 131, 207, 210, 221, 252, 256, 409, 416 replication, xvii, 28, 39, 57, 291, 327, 334, 348, 352, 356, 363, 364, 369 repolarization, xv, 243, 251, 252 repression, 133, 264, 282, 334, 373, 374, 405 repressor, 291, 320, 344, 381 reproduction, 198 research, ix, xiv, xvii, xviii, 18, 65, 104, 107, 177, 178, 198, 200, 201, 258, 267, 286, 305, 313, 314, 325, 363, 366, 369, 370, 372, 384, 385, 389, 394, 402, 404, 405, 406, 424, 442 researchers, xv, 47, 104, 257, 333, 337, 403 reservation, 453 reservoir, x, 23 residential, xviii, 384, 386, 387, 388 residues, 169, 230, 360, 413 resistance, xix, 27, 30, 31, 34, 41, 42, 70, 134, 135, 138, 139, 144, 150, 151, 167, 221, 263, 282, 286, 298, 305, 313, 322, 394, 400, 405, 406, 407, 411, 416, 442, 448, 454 resistin, 134, 135, 150, 151 resources, 178, 453 respiratory, xviii, 28, 383, 385, 386, 390 responsiveness, xv, 257, 285, 384 restrictive cardiomyopathy, 249 Resveratrol, 343 retail, 108 retention, 56, 93

493

reticulum, 132, 154, 162, 310, 457 retinoblastoma, 141 retinoic acid, 264, 270 retinoic acid receptor, 264 retinoids, 457 retinol, 441, 459 Retinol, 454 retinopathy, 400 retirement, xviii, 384, 386, 387, 388, 389 retrovirus, 28 reveratrol, 457 reverse transcriptase, 28, 39, 369, 378, 380, 381, 427 Reynolds, 149, 232 rheumatoid arthritis, 198 Rho, 283, 298 Rho-kinase, 283, 298 rhythms, 454 ribonucleic acid, 39 ribose, 229, 230, 358, 396 ribosome, 267 rice, 352, 359, 385 right ventricle, 123 rings, 25 risk, ix, xv, xvi, xvii, 31, 42, 69, 71, 75, 79, 83, 104, 108, 119, 121, 123, 136, 143, 144, 164, 165, 170, 198, 199, 200, 201, 202, 204, 215, 219, 222, 224, 235, 244, 246, 247, 248, 252, 253, 254, 256, 275, 285, 296, 299, 303, 304, 315, 316, 325, 326, 329, 338, 340, 361, 362, 383, 385, 387, 388, 390, 391, 395, 400, 401, 402, 406, 407, 431, 445, 454, 455, 456, 458, 460 risk factors, 144, 201, 244, 246, 247, 254, 340, 390 risks, 41, 120, 126, 143, 200, 201, 235, 338, 443 RNA, xvii, 167, 227, 275, 290, 292, 310, 321, 347, 352, 369, 373, 379, 380 RNAi, 272, 275, 276, 279, 281, 282, 283, 284, 285 rodent, 123, 321 rodents, 123, 135, 138, 151, 164, 302 rods, 42 rolling, 126, 169 room temperature, 84, 85, 86, 109, 111 room-temperature, 110 ROS, x, xiii, xix, 26, 45, 131, 157, 160, 162, 164, 168, 199, 220, 221, 226, 227, 412, 416, 429, 430, 433, 436, 437, 438, 439, 441, 442, 453 rosacea, 56 rosiglitazone, 150 rotavirus, 29, 36, 39 rubber, 110 rural, 304, 386, 387, 389, 391

S S phase, 333, 335

494

Index

Saccharomyces cerevisiae, 33 safety, 13, 185, 224 SAHA, 457 saline, 6, 7, 8, 16, 167 saliva, 172, 264 Salmonella, 32, 119, 175 salt, 178, 195, 215 salts, 151 sample, 54, 64, 66, 67, 70, 71, 72, 86, 109, 110, 118, 179, 185, 187, 190, 386, 389, 422 sampling, 458 sampling error, 458 sanitation, 178, 453 SAR, 28, 29, 35 SAS, 87 saturated fat, 56 saturation, 13 scaffolding, 140 Scanning electron, 31 scar tissue, x, 45 scavenger, 226, 441 school, 386, 387, 390 scientific community, x, 23, 375 scientific knowledge, 451 scientific understanding, xix, 459 scientists, 55, 57, 330 Scomber scombrus, 176 scores, 186 screening programs, x, 23 SDS, 49 search, 282, 388 searches, 349 secrete, 200 secretion, x, 2, 138, 152, 153, 240, 279, 289, 297, 332, 404, 413, 416 sedation, 16 sedative, ix, 1, 6, 9, 10, 11, 17, 18, 22 sedentary, 104 sedentary lifestyle, 104 seeds, 194, 381 selective attention, 14, 20 selectivity, 28 selenium, ix, xii, xiii, 103, 104, 108, 111, 112, 118, 119, 120, 121, 122, 123, 124 self, 296 self-report, 253, 389, 406 SEM, 31, 87, 88, 89, 90, 92, 93, 94, 95, 97, 98, 113, 114, 116, 180, 351, 353, 354, 435, 436, 437, 438 seminal vesicle, 138 senescence, 131, 369, 370, 371, 378, 380, 431, 443, 445, 448, 452, 453 senile, 268 senile plaques, 268

sensing, 131, 242 sensitivity, 138, 244, 248, 255, 256, 266, 272, 282, 286, 298, 329, 394, 400, 404, 405, 413, 416, 445, 448 sensitization, 12, 21 sensors, 222 separation, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18 septum, 250 sequencing, 373 series, 27, 32, 35, 47, 99, 180, 228, 327, 332, 334, 366, 367, 452 serine, 133, 139, 229, 329, 332, 335 serotonin, 15, 22, 208, 215, 297 serum, ix, xiii, 1, 2, 22, 26, 47, 49, 56, 71, 104, 111, 115, 118, 119, 121, 137, 143, 150, 161, 162, 171, 206, 215, 228, 229, 233, 234, 239, 245, 248, 254, 255, 341, 394, 399, 400, 402, 403, 406, 424, 448 severity, 150, 202 sex, 336, 338 sex steroid, 336 sexually transmitted disease, 55 Shanghai, 58, 296, 460 shape, 334 shaping, 140 shock, 131, 220, 231, 241 shoot, 100 short period, xii, 103, 435 short-term, 91, 106, 315 Shp-2, 343 shrimp, 194, 195 side effects, xvi, 55, 250, 303, 311, 325, 337, 394, 415, 455 sign, 52 signal transduction, xvii, 127, 128, 139, 203, 228, 230, 239, 258, 263, 268, 299, 317, 336, 339, 363, 377, 447, 456 signalling, 343, 345 signals, 12, 105, 128, 130, 131, 140, 145, 147, 226, 229, 290, 299, 317, 321, 369, 452 silicon, 110 silver, xx, 459 similarity, 310, 424 Singapore, 235, 285, 299, 316 siRNA, 265 sister chromatid exchange, 167 sites, 14, 132, 150, 261, 267, 283, 284, 299, 331, 369, 413 skeletal muscle, 107, 117, 131, 143, 147, 249 skeleton, 221, 258, 297, 423 skin, x, xi, xiv, xvi, 2, 38, 40, 45, 46, 47, 48, 49, 52, 54, 55, 56, 57, 58, 75, 100, 107, 120, 124, 164, 166, 173, 177, 179, 185, 193, 240, 312, 325, 329,

Index 336, 337, 342, 345, 358, 361, 366, 376, 382, 455, 456, 457 skin cancer, 38, 40, 107, 185, 312, 336, 345, 376 skin disorders, 56 sleep, 3, 5, 7, 9, 11, 16, 18, 375 sleep disorders, 375 small intestine, xix, 26, 366, 429 smoke, 387 smokers, xvii, 71, 79, 247, 255, 383, 384, 385, 389, 391, 454, 458 smoking, xvii, 108, 120, 123, 164, 235, 246, 338, 383, 384, 386, 387, 388, 389, 392, 443, 448, 454 smooth muscle, 130, 135, 150, 151, 160, 200, 205, 296, 298, 334, 344 smooth muscle cells, 130, 150, 160, 200, 205 social environment, 453 social policy, 451 social stress, 16 socioeconomic, 247 SOD, 162, 167, 226, 247, 260, 400, 403, 404, 440 SOD1, 226 sodium, 108, 109, 118, 121, 395, 403, 404, 409, 417 software, 86 soil, 105 solid tumors, 40, 133, 315 solubility, xi, 38, 46, 47, 136, 137, 205 solvent, 65, 76, 136 solvents, 47, 65, 195 somatic cell, 370, 452 somatic cells, 370, 452 somatic mutations, 329 somatic stem cells, 452 somatostatin, 105, 117 soy, 48, 165, 173, 195, 455 soybean, 48, 179, 195, 198, 246 Spain, 81, 99, 100 spatial, xiv, xix, 14, 207, 209, 212, 213, 214, 217, 226, 429, 430, 431, 433, 435, 442, 444 spatial information, 430 spatial learning, 430, 435 spatial memory, xiv, 14, 207, 209, 212, 213, 214, 430 specialized cells, 452 species, x, xiii, xv, xix, 16, 26, 30, 31, 32, 33, 45, 70, 80, 83, 84, 131, 138, 148, 150, 157, 160, 162, 164, 167, 168, 175, 178, 185, 199, 219, 220, 221, 231, 232, 241, 260, 303, 404, 408, 412, 429, 430, 436, 438, 452, 453, 456 specificity, 151, 295, 370 spectrophotometric, 86, 100 spectrophotometric method, 86 spectroscopy, 66, 70 spectrum, 32, 158, 229

495

speculation, 355 speed, 59 spermatocyte, 360 S-phase, 149 sphingolipids, 269 spin, 66, 77, 163 spinach, 67, 360 spindle, 457 spine, 108 spleen, 400 sporadic, 54, 291, 326, 327, 329 spore, 31, 40, 123 SPR, 268, 270, 279, 280, 281 Sprague-Dawley rats, 110, 117, 256, 302, 321, 448 SPSS, 387 sputum, 384 squamous cell, 49, 59, 167, 175, 282, 296, 317 squamous cell carcinoma, 49, 59, 167, 175, 282, 296, 317 St. Louis, 48, 50 stability, xvi, 47, 55, 56, 84, 85, 93, 94, 96, 179, 194, 230, 265, 279, 302, 309, 311, 312, 369, 403 stabilization, 133, 230, 275, 309, 339, 439 stabilize, xi, 46, 91, 230, 250, 260 stages, 84, 89, 140, 326, 327, 330, 336, 370 stainless steel, 110 standard error, 87, 111, 112, 180, 181, 186, 191, 193 standards, 35, 66, 86, 87, 108, 396, 397 staphylococci, 41 Staphylococcus, 31, 32, 34, 38, 41, 42, 43, 99, 182 Staphylococcus aureus, 31, 32, 38, 41, 42, 43, 99, 182 starch, 30, 42, 395 starch polysaccharides, 395 starvation, 149, 341 Statistical Analysis System, 87 statistics, 248, 338, 386 steady state, xi, 63 steel, 110 stellate cells, 148 stem cell therapy, 249 stem cells, 200, 451, 452, 453, 459, 460, 461 sterile, 86, 110 sterilization, 47 steroid, 138, 305, 316, 336, 359 steroid hormone, 305 stiffness, 403 stilbenes, 27 stimulus, 140, 228 stock, 86 stoichiometry, 73 stomach, xii, 2, 41, 103, 105, 117, 120, 135, 164, 165, 222, 244, 264, 366, 376

496

Index

storage, 77, 83, 85, 91, 96, 97, 101, 123, 179, 181, 186, 187, 188, 189, 193, 194, 195, 236, 454 strain, 32, 35, 38, 174, 315 strains, 31, 32, 33, 34, 35, 42 S-transferase (GST), 162, 163 strategies, ix, xi, xvi, 27, 35, 46, 104, 131, 257, 286, 306, 370, 375, 402, 456 strength, 244, 270, 281, 389 Streptomyces, 47 striatum, 12, 15, 22, 208, 226, 229, 440 stroke, 151, 221, 246, 385, 391, 444, 447 stromal, 149 structural changes, 162, 371 structural protein, 230 students, 87 subacute, 162 subcellular, 294 subsidy, 201, 358 substances, ix, 1, 2, 14, 40, 185, 196, 198, 269, 331, 361, 399, 405 substantia nigra, 12, 20, 220, 225, 236, 237, 238, 431 substantia nigra pars compacta, 220 substitution, 302 substrates, 136, 172, 225, 263, 277, 349, 368 subtraction, 270 sucrose, 246, 254, 268, 427 sugar, 85, 248, 384, 400, 402, 404, 455 suicide, 154, 345 sulfate, 70, 108, 260, 282 sulfuric acid, 396 summer, 385 Sun, 59, 150, 170, 236, 253, 288, 289, 299, 316, 381, 407 superoxide, 12, 162, 164, 167, 175, 178, 187, 189, 191, 192, 223, 226, 247, 248, 260, 361, 367, 399, 400, 404, 407, 409, 433, 440, 444, 445 superoxide dismutase, 12, 162, 167, 187, 226, 247, 248, 260, 368, 399, 400, 433, 440, 444, 445 supplements, 37, 78, 143, 234, 338, 460 supply, 198, 331 suppression, xvi, 32, 39, 58, 107, 108, 136, 149, 198, 199, 201, 203, 231, 240, 264, 268, 276, 279, 286, 301, 306, 308, 310, 311, 312, 313, 332, 339, 341, 345, 348, 357, 366, 368, 376, 377, 424, 445, 446, 457 suppressor, xvi, 133, 282, 291, 298, 325, 327, 328, 329, 330, 333, 334, 343, 364 suppressors, 326, 337 surfactant, 136 surgery, 319, 364, 456 surveillance, 338 survivability, 441

survival, xvi, 12, 30, 36, 58, 128, 146, 154, 201, 206, 222, 226, 228, 229, 230, 231, 233, 234, 240, 264, 306, 307, 310, 312, 316, 325, 331, 333, 360, 392, 443, 446, 453 survival rate, 226 survival signals, 12, 229 surviving, xv, 14, 207, 213, 416 susceptibility, 33, 35, 40, 70, 82, 132, 174, 204, 322, 412, 443 switching, 16 symbols, 87 sympathetic, 15, 26, 37 symptom, 12, 226 symptoms, 14, 32, 277, 278, 384, 385, 400, 407 synaptic transmission, 210 syndrome, 104, 138, 327 synergistic, 26, 34, 118, 119, 132, 139, 317 synergistic effect, 118, 119, 132 synthesis, 15, 48, 130, 135, 136, 137, 139, 167, 174, 210, 227, 309, 313, 340, 348, 360, 420, 426, 440, 441 systems, 136, 141, 172, 200, 208, 226, 227, 259, 260, 263, 265, 266, 327, 329, 365, 367, 374, 375, 406, 452, 453, 454, 458, 459 systolic blood pressure, 247

T T cells, 267, 293, 376, 377, 442 T lymphocytes, 267 T2DM, 400 tachycardia, 250, 251, 375 Taiwan, 158 tamoxifen, 164, 172, 289, 312, 313, 316, 317, 322, 323 tangles, 448 tannin, 42 tannins, 25, 33, 94, 100, 101, 196, 361, 405 TAR, xi, 63 target organs, 358 targets, xv, xvii, 16, 20, 40, 75, 128, 131, 147, 148, 169, 199, 200, 222, 232, 257, 258, 261, 263, 290, 294, 306, 307, 310, 321, 325, 338, 340, 343, 352, 367, 370, 371, 382, 441, 442, 460 taste, xii, 82, 158, 186, 365, 424 tau, 227, 236, 447 tau pathology, 236, 447 T-cell, 290 T-cell receptor, 290 technology, xiv, 177, 178, 179, 194 telomerase, ix, xvii, 363, 365, 366, 367, 369, 370, 371, 372, 374, 375, 376, 378, 379, 380, 381, 446 telomere, 369, 370, 371, 378, 379, 380 telomere shortening, 370, 371, 380

Index telomeres, xvii, 363, 369, 370, 371, 378, 380, 381 temperature, xiv, 14, 84, 93, 94, 96, 105, 109, 110, 179, 181, 207, 209, 214, 239, 385 tendon, 395, 406 Tennessee, 325 tension, 296 teratogenic, 164 testes, 409 testis, 356 testosterone, 117 tetracycline, 31, 34, 41 tetrahydrofuran, 50 Texas, 254 TGF, 154, 306, 309, 328, 329, 331, 332, 403, 408 Thailand, 252 thalidomide embryopathy, 461 theaflavin, xix, 25, 126, 146, 227, 238, 290, 365, 367, 419, 420, 423, 426, 427 T-helper cell, 29 theory, 169, 247, 310, 326, 369, 433, 448, 460 therapeutic agents, 139, 225 therapeutics, 3, 236, 265, 379, 380, 444 therapy, xv, xvi, 108, 146, 147, 198, 219, 231, 249, 256, 263, 264, 286, 290, 291, 313, 318, 319, 325, 329, 340, 342, 364, 370, 394, 405, 448 thermal treatment, 93, 94 theta, 13, 14 thiobarbituric acid, 167, 187, 399, 436, 438, 446 Thomson, 123, 408 three-dimensional, 375, 452 threonine, 139, 263, 329, 335 threshold, 370, 456, 457, 458 thrombosis, 401 thrombotic, 216 thymus, 356, 360 thyroid, 124 thyroxin, 441 time frame, 105 time periods, 85 timing, 405, 452, 458 TIMP, 331 tissue, x, 20, 21, 29, 41, 45, 49, 107, 117, 120, 131, 134, 137, 140, 142, 143, 147, 161, 163, 164, 200, 205, 222, 224, 226, 235, 246, 249, 256, 261, 266, 267, 272, 288, 293, 314, 326, 331, 332, 342, 361, 399, 412, 416, 431, 432, 448, 452, 453, 456, 457 tissue homeostasis, 140 TLR, 39 TNF, 30, 148, 150, 220, 227, 229, 238 TNF-alpha, 148, 150, 238 tobacco, 29, 42, 152, 166, 234, 385, 388 tobacco smoke, 166 Tocopherol, 58, 454

497

tocopherols, 185, 407 Tokyo, 153, 205, 286, 358, 409, 429 tolerance, 138, 147, 153, 248, 329, 340, 400, 402, 412 toll-like, 269, 296 tomato, 32 tonic, 36 total plasma, 136, 246 totipotent, 452 toxic, xiii, xx, 57, 157, 160, 162, 164, 178, 231, 268, 302, 365, 440, 447, 454, 459 toxic effect, 162, 365 toxicities, 457 toxicity, xiii, 21, 48, 123, 157, 160, 162, 163, 164, 168, 169, 170, 171, 172, 174, 206, 237, 239, 315, 412, 442, 443, 446, 451 toxicological, 228 toxicology, 2, 48, 173, 459, 461 toxin, 11, 31, 32, 42, 164, 168, 458 toxins, 19, 57, 158, 172, 248 TPA, vi, xvii, 166, 347, 348, 353, 354, 355, 356 trade, 178 tradition, xv, 158, 219 traffic, 268 training, 249 transaminases, 162 transcript, 231, 238, 331, 343, 373 transcriptase, 28, 39, 369, 378, 380, 381, 427 transcription, xiii, 28, 107, 117, 126, 127, 128, 129, 131, 132, 139, 148, 150, 154, 160, 161, 163, 169, 199, 203, 222, 227, 230, 238, 241, 263, 264, 275, 291, 309, 320, 329, 331, 333, 343, 344, 369, 372, 373, 374, 381, 409, 440 transcription factor, xiii, 107, 117, 126, 127, 132, 139, 154, 160, 161, 169, 199, 203, 222, 227, 238, 263, 275, 309, 320, 331, 333, 343, 344, 373, 381, 409, 440 transcription factors, xiii, 107, 117, 126, 132, 160, 161, 169, 199, 222, 227, 263, 333, 373, 381, 440 transcriptional, 133, 141, 147, 227, 230, 263, 264, 309, 320, 331, 334, 344, 372, 373, 374, 381, 440, 456 transcriptomics, 234 transcripts, 356 transduction, 134, 154, 294, 297, 339 transfection, 129, 319 transfer, 266, 334, 404, 452, 453, 456 transferrin, 220, 222, 231 transformation, 146, 286, 288, 290, 295, 328, 370, 452, 461 transformations, 160 transforming growth factor, 329, 332, 342 transforming growth factor-β, 332

498

Index

transgenic, 151, 225, 227, 236, 237, 417, 437, 447 transgenic mice, 151, 225, 227, 236, 237, 417, 437, 447 transgenic mouse, 237 transition, 28, 130, 160, 226, 309, 335, 344, 452, 457 transition metal, 160, 226 transition metal ions, 160, 226 translation, 128, 227, 232, 282, 298, 368, 457 translational, 456 translocation, 140, 143, 154, 168, 227, 229, 238, 239, 306, 330, 334, 372, 380, 442 transmembrane, 128, 141, 334, 456 transmission, 139, 210 transplantation, 249 transport, 13, 20, 135, 136, 137, 394, 400, 406, 454 transportation, 404 trastuzumab, 305, 314 trauma, 56 treatment-resistant, 314 trend, 27, 179 trial, 40, 59, 144, 153, 154, 236, 244, 279, 315, 435, 454, 459 triggers, 131, 139, 213, 413 triglyceride, 117, 121, 126, 136, 137, 143, 151, 402, 424 triglycerides, 26, 38, 136, 137, 138, 141, 246, 248, 403, 405, 426 trimethylamine, 165 trochanter, 108 Trp, 190, 191 trypsin, 265, 331 tryptophan, 30 tuberculosis, 30, 36 tubular, 203 tumor cells, 50, 229, 263, 265, 291, 311, 312, 314, 334, 342, 343, 358, 366, 368 tumor growth, xiv, 197, 198, 199, 200, 272, 282, 283, 306, 308, 310, 311, 312, 319, 334, 368, 417 tumor invasion, 261, 358 tumor metastasis, 332 tumor necrosis factor, 30, 99, 238, 332, 380 tumor progression, 263, 329, 378 tumorigenesis, 42, 199, 296, 312, 321, 327, 328, 329, 332, 335, 337, 338, 339, 344, 345, 361, 370, 376, 379, 461 tumorigenic, xvii, 166, 318, 325, 332, 370 tumors, 40, 133, 164, 166, 167, 261, 265, 276, 306, 311, 312, 313, 315, 321, 322, 327, 330, 331, 334, 336, 339, 342, 365, 371, 375, 376, 456, 458 tumour, 203, 238, 292, 319, 343, 344 tumour growth, 203, 319 turnover, 261 two-dimensional, 100, 265

type 1 diabetes, 399 type 2 diabetes, xiii, xviii, 26, 104, 123, 125, 126, 131, 138, 142, 150, 153, 200, 244, 253, 255, 394, 400, 402, 406, 408, 411 type 2 diabetes mellitus, xiii, 104, 123, 125, 142, 153, 200, 244, 394, 402 tyramine, 165 tyrosine, 12, 127, 129, 138, 139, 148, 199, 223, 234, 277, 279, 294, 307, 319, 343, 405 tyrosine hydroxylase, 12

U U.S. Department of Agriculture (USDA), 75, 78, 170 ubiquitin, 220, 229, 242, 262, 329, 331, 339, 367 ubiquitous, 166 ultrastructure, 42 ultraviolet, 38, 57, 75, 124, 185, 318, 345, 361, 376 ultraviolet B, 318, 361 ultraviolet light, 185 uncertainty, 224 underlying mechanisms, 75, 161, 406, 454 United Kingdom, 121 United States, xvi, 57, 74, 80, 200, 245, 318, 320, 322, 325, 326, 336 universality, 370 universities, 386 urea, 163 urease, 30 urinary, 28, 40, 47, 122, 165, 247, 253, 260, 261, 288, 399 urinary bladder, 40, 122 urinary bladder cancer, 122 urinary tract, 28 urine, 22, 58, 215, 224, 235, 253, 260, 288, 431 urokinase, 266, 292, 294, 332, 342 uterus, 138 UV, x, 45, 56, 57, 70, 76, 150, 336, 396, 418 UV exposure, 336 UV light, 336

V vacuum, 187, 397 validation, 77, 272 validity, 74 values, xii, xvii, 11, 64, 65, 67, 68, 69, 70, 72, 73, 74, 82, 86, 87, 89, 98, 112, 116, 179, 186, 190, 193, 265, 283, 347, 351, 352, 356, 366, 438, 439 vancomycin, 31 variability, 15, 39, 58, 70, 87, 253, 288 variable, 31, 310, 377, 409 variables, xiv, 14, 207, 209, 214, 386, 387, 388, 389 variance, 111

Index variation, 254 vascular dementia, 201 vascular disease, 333, 400 vascular endothelial growth factor (VEGF), 10, 122, 127, 198, 200, 202, 203, 204, 220, 230, 234, 308, 311, 313, 316, 317, 319, 331, 336, 340, 341, 376 vasculature, 200, 205, 331 vasculogenesis, 331 vector, 270, 271, 275, 279, 280 vegetable oil, 59, 179 vegetables, 67, 69, 77, 78, 100, 105, 175, 198, 221, 246, 302, 314, 348, 455 VEGF expression, 200, 204, 234 vein, 341 velocity, 70 ventricle, xv, xix, 226, 243, 429, 437, 438 ventricular arrhythmia, 251, 256 ventricular fibrillation, 251 ventricular tachycardia, 250, 251 vessels, 331 Vibrio cholerae, 32, 42 vimentin, 241, 265, 289, 292, 309 viral envelope, 29 viral infection, 28, 29, 120, 268 virus, 28, 29, 36, 39, 40, 41, 42, 55, 57, 58, 59, 60, 258, 267, 293, 294, 321, 329, 348, 380, 427 viruses, 27, 29, 39, 55, 57, 268, 456 visible, 34, 64, 71 vision, xv, 57, 219 visualization, 100, 380 vitamin A, 417 vitamin C, 26, 71, 85, 86, 90, 95, 179, 229, 337 vitamin C, 100, 106, 240, 330, 404, 405 vitamin E, 64, 158, 210, 223, 407, 417, 449 vitamin K, 106 vitamins, 26, 47, 79, 106, 118, 394, 395, 439, 455 vocalizations, 3, 4, 6, 7, 8, 9, 10, 16, 17 vomiting, 31, 375

W Wallerian degeneration, 140 Ward’s triangle, 108 warts, 59 wastes, 190

499

water, ix, x, xii, xiii, xv, xviii, 24, 37, 38, 45, 47, 48, 55, 56, 64, 65, 66, 71, 73, 77, 82, 84, 85, 86, 89, 103, 104, 108, 109, 110, 111, 113, 115, 116, 117, 118, 119, 135, 136, 138, 157, 160, 162, 165, 167, 219, 222, 223, 247, 248, 249, 258, 279, 302, 310, 311, 313, 332, 365, 366, 384, 393, 397, 408, 411, 435, 436, 438, 458 water-soluble, x, xiii, 38, 45, 47, 56, 104, 109, 116, 136 weight control, 118, 123, 141, 200 weight gain, 115, 119, 153, 381 weight loss, 104, 118, 138, 142, 153, 155, 253, 256, 302 weight management, xiii, 104, 118, 119 Weinberg, 317, 380, 381, 460 well-being, xix, 411 wells, 49 Western countries, 24, 126, 200, 222, 248 wheat, 194 wild type, 129, 333 wine, 19, 96, 100, 198, 302, 389, 392 Wistar rats, 302, 315 withdrawal, 228, 239 Wnt signaling, 309, 320, 329 women, xvii, 71, 108, 121, 153, 154, 202, 224, 245, 253, 285, 299, 303, 304, 305, 308, 314, 326, 329, 383, 385, 386, 388, 394, 434, 460 working memory, 430, 435, 437, 439 World Health Organization (WHO), 162, 170, 178, 196, 389, 391, 392 wound healing, 198, 331

X xenobiotics, xiii, 157, 160, 168 xenografts, xvi, 292, 301, 310, 311, 313, 314, 321,371, 375

Y yeast, 33, 34, 37, 42, 96 yield, 161, 196, 420, 423

Z ZAP-70, 290 Zinc (Zn), 12, 111, 112, 170, 171, 226, 229, 333, 334