Food & Function. Vol 01, No 03, December 2010

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Linking the chemistry and physics of food with health and nutrition Volume 1 | Number 1 | 2010 | Pages 1–100

Food & Function Linking the chemistry and physics of food with health and nutrition www.rsc.org/foodfunction

Volume 1 | Number 3 | December 2010 | Pages 225–324

Food & Function

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Volume 1 | Number 3 | 2010

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Food & Function

ISSN 2042-6496

Food & Function Linking the chemistry and physics of food with health and nutrition Food science and nutrition is a highly multidisciplinary area. We know it can be difficult to keep abreast of each other’s work, especially when there is not enough time in the day and the pile of work keeps growing. Wouldn’t it be great if there was a journal which pulled together high impact chemical and physical research linking to human health and nutrition? Just one platform to find what you need in the field, and reach exactly the right audience when you publish your work. Food & Function provides a dedicated venue for physicists, chemists, biochemists, nutritionists and other health scientists focusing on work related to the interaction of food components with the human body.

Go to the website now to submit your research and register for free access!

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Pages 225–324

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ISSN 2042-6496

COVER ARTICLE Harry J. Wichers et al. Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds

2042-6496(2010)1:3;1-B

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Food & Function Linking the chemistry and physics of food with health and nutrition

International Symposia on Advancing the Chemical Sciences

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A new generation of global conferences www.rsc.org/foodfunction

Volume 1 | Number 3 | December 2010 | Pages 225–324

Call for Abstracts Present your latest research at the International Symposia on Advancing the Chemical Sciences (ISACS) - a significant new global symposia series organised by the RSC. Poster and oral abstracts are invited to supplement the following exceptional plenary speaker line-ups:

Challenges in Renewable Energy (ISACS4) 5-8 July 2011 z Boston, USA z www.rsc.org/isacs4 Speakers: Hector Abruña, Fraser Armstrong, James Barber, Allen J Bard, Peter Bruce, Kazunari Domen, Gérard Férey, Leif Hammarström, Cliff Kubiak, Akihiko Kudo, James McCusker, Thomas Moore, Debra Rolison, Michael Strano, Peng Wang, Michael Wasielewski, Andreas Züttel. Poster deadline 6th May 2011, oral deadline 21st January 2011

Challenges in Chemical Biology (ISACS5) 26-29 July 2011 z Manchester, UK z www.rsc.org/isacs5 Speakers: Venki Ramakrishnan, Thomas Steitz, Donna Blackmond, Andrew Ellington, Hermann Gaub, Reza Ghadiri, Andrew Griffiths, Bartosz A Grzybowski, Philip Holliger, Clyde Hutchison, Stefan Knapp, Stephen Mann, David Rees, Hugh Rosen, William Shih, Hiroaki Suga, Erik Winfree. Poster deadline 27th May 2011, oral deadline 21st January 2011

Challenges in Organic Materials & Supramolecular Chemistry (ISACS6) 2-5 September 2011 z Beijing, China z www.rsc.org/isacs6 Speakers: Takuzo Aida, Harry Anderson, Matthew Francis, Philip Gale, Wenping Hu, Myongsoo Lee, David Leigh, Stefan Matile, Colin Nuckolls, Jian Pei, Julius Rebek Jr, Hanadi Sleiman, Samuel Stupp, Benzhong Tang, Xi Zhang. Poster deadline 8th July 2011, oral deadline 18th March 2011

Join in – submit your abstract today! ISSN 2042-6496

www.rsc.org/isacs COVER ARTICLE Subrata Chattopadhyay et al. Comparative healing proerty of kombucha tea and black tea against indomethacin-induced gastric ulceration in mice: possible mechanism of action

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Food & Function www.rsc.org/foodfunction RSC Publishing is a not-for-profit publisher and a division of the Royal Society of Chemistry. Any surplus made is used to support charitable activities aimed at advancing the chemical sciences. Full details are available from www.rsc.org

IN THIS ISSUE

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ISSN 2042-6496 CODEN FFOUAI 1(3) 225–324 (2010) Cover See Harry J. Wichers et al., pp. 254–261. Image reproduced by permission of Robert A. M. Vreeburg from Food Function, 2010, 1, 254.

Inside cover See Subrata Chattopadhyay et al., pp. 284–293. Image reproduced by permission of Subrata Chattopadhyay from Food Function, 2010, 1, 284.

REVIEW 233 Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites Maria Monagas,* Mireia Urpi-Sarda, Fernando Sanchez-Pat an, Rafael Llorach, Ignacio Garrido, Carmen G omez-Cordoves, Cristina Andres-Lacueva and Bego~ na Bartolome Colonic metabolism by the intestinal microbiota plays an important role in the bioavailability and potential bioactivity of flavan-3-ols.

PAPERS 254 Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds Wasaporn Chanput, Jurriaan Mes, Robert A. M. Vreeburg, Huub F. J. Savelkoul and Harry J. Wichers* Gene expression of simultaneous exposing THP-1 cells to LPS and food compounds is a promising screening tool for inflammation modulating studies.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 227–232 | 227

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EDITORIAL STAFF Editor Sarah Ruthven Deputy editor Kathleen Too

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Senior publishing editor Elinor Richards

Food & Function provides a dedicated venue for research relating to the chemical and physical properties of food components and their nutritional and health benefits in humans.

Development editor Anna Simpson Publishing editors Mary Badcock, David Barden, Emma Eley, David Parker, Charles Quigg, Michael Townsend

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Food & Function

Publishing assistants Anna Anderson, Jackie Cockrill Publisher Emma Wilson For queries about submitted articles please contact Elinor Richards, Senior publishing editor, in the first instance. E-mail [email protected]

EDITORIAL BOARD Editor-in-Chief Professor Gary Williamson, University of Leeds, UK Associate Editors Cesar Fraga, University of Buenos Aires, Argentina & University of California, Davis, USA Steven Feng Chen, The University of Hong Kong, China

For pre-submission queries please contact Sarah Ruthven, Editor. E-mail [email protected] Food & Function (print: ISSN 2042-6496; electronic: ISSN 2042-650X) is published 12 times a year by the Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge, UK CB4 0WF. All orders, with cheques made payable to the Royal Society of Chemistry, should be sent to RSC Distribution Services, c/o Portland Customer Services, Commerce Way, Colchester, Essex, UK CO2 8HP. Tel +44 (0)1206 226050; E-mail [email protected] 2010 Annual (print + electronic) subscription price: £1200; US$2232. 2010 Annual (electronic) subscription price: £1080; US$2008. Customers in Canada will be subject to a surcharge to cover GST. Customers in the EU subscribing to the electronic version only will be charged VAT. If you take an institutional subscription to any RSC journal you are entitled to free, site-wide web access to that journal. You can arrange access via Internet Protocol (IP) address at www.rsc.org/ip. Customers should make payments by cheque in sterling payable on a UK clearing bank or in US dollars payable on a US clearing bank. Periodicals postage paid at Rahway, NJ, USA and at additional mailing offices. Airfreight and mailing in the USA by Mercury Airfreight International Ltd., 365 Blair Road, Avenel, NJ 07001, USA. US Postmaster: send address changes to Food & Function, c/o Mercury Airfreight International Ltd., 365 Blair Road, Avenel, NJ 07001. All despatches outside the UK by Consolidated Airfreight. Advertisement sales: Tel +44 (0) 1223 432246; Fax +44 (0) 1223 426017; E-mail [email protected] For marketing opportunities relating to this journal, contact [email protected]

Members Aedin Cassidy, University of East Anglia, UK Kevin Croft, University of Western Australia, Australia Eric Decker, University of Massachusetts, USA Alejandro Marangoni, University of Guelph, Canada

Reinhard Miller, Max Planck Institute of Colloids & Interfaces, Germany Paul Moughan, Riddet Institute, Massey University, New Zealand Johan Ubbink, Food Concept & Physical Design, Switzerland Fons Voragen, Wageningen, The Netherlands

ADVISORY BOARD Hitoshi Ashida, Kobe University, Japan Junshi Chen, Chinese Centre of Disease Control & Prevention, China E. Allen Foegeding, North Carolina State University, USA Vincenzo Fogliano, University of Napoli Federico II, Italy Mike Gidley, University of Queensland, Australia Chi-Tang Ho, Rutgers University, USA Richard Hurrell, ETH Zurich, Switzerland Peter Lillford, University of York, UK Rui Hai Liu, Cornell University, USA

Julian McClements, University of Massachusetts, USA John A. Milner, National Cancer Institute, National Institutes of Health, USA Brent Murray, University of Leeds, UK Patricia Oteiza, University of California at Davis, USA Augustin Scalbert, INRA, France Helmut Sies, University of Dusseldorf, Germany Leif Skibsted, University of Copenhagen, Denmark

David Stuart, The Hershey Company, USA Arthur Tatham, University of Wales Institute, Cardiff, UK Junji Terao, University of Tokushima, Japan George van Aken, NIZO Food Research, The Netherlands Erik van der Linden, TI Food & Nutrition, The Netherlands Jose Vina, University of Valencia, Spain Peter Wood, Agriculture and Agri-Food Canada, Canada

INFORMATION FOR AUTHORS Full details on how to submit material for publication in Food & Function are given in the Instructions for Authors (available from http://www.rsc.org/authors). Submissions should be made via the journal’s homepage: http://www.rsc.org/foodfunction. Authors may reproduce/republish portions of their published contribution without seeking permission from the RSC, provided that any such republication is accompanied by an acknowledgement in the form: (Original Citation)–Reproduced by permission of The Royal Society of Chemistry. This journal is © The Royal Society of Chemistry 2010. Apart from fair dealing for the purposes of research or private study for non-commercial purposes, or criticism or review, as permitted under the Copyright, Designs and

Patents Act 1988 and the Copyright and Related Rights Regulation 2003, this publication may only be reproduced, stored or transmitted, in any form or by any means, with the prior permission in writing of the Publishers or in the case of reprographic reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK. US copyright law is applicable to users in the USA. The Royal Society of Chemistry takes reasonable care in the preparation of this publication but does not accept liability for the consequences of any errors or omissions. ∞ The paper used in this publication meets the  requirements of ANSI/NISO Z39.48–1992 (Permanence of Paper). Royal Society of Chemistry: Registered Charity No. 207890.

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PAPERS 262 Caffeoylquinic acid derived free radicals identified during antioxidant reactions of bitter tea (Ilex latifolia and Ilex kudincha)

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Katharina Franziska Pirker and Bernard Albert Goodman* Free radical oxidation products derived from caffeoylquinic acids have appreciable stability in the complex matrix of the kudingcha extract, and may contribute to the biological properties of the bitter tea beverage, which has a long-standing role in traditional Chinese medicine.

269 Application of response surface methodology to optimize pressurized liquid extraction of antioxidant compounds from sage (Salvia officinalis L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.) M. B. Hossain,* N. P. Brunton, A. B. Martin-Diana and C. Barry-Ryan The present study optimized methanol concentration and temperature for extraction of antioxidant compounds from three Lamiaceae spices using pressurized liquid extraction.

278 Protective role of air potato (Dioscorea bulbifera) of yam family in myocardial ischemic reperfusion injury Hannah Rachel Vasanthi, Subhendu Mukherjee, Diptarka Ray, Karuppiah Shanmugasundara Pandian Jayachandran, Istvan Lekli and Dipak Kumar Das* Cardioprotective activity of tubers of Dioscorea bulbifera is mediated by inhibition of cardiomyocyte necrosis and apoptosis as evidenced by the ventricular function and the pro and antiapoptotic protein expression.

284 Comparative healing property of kombucha tea and black tea against indomethacin-induced gastric ulceration in mice: possible mechanism of action Debashish Banerjee, Sham A. Hassarajani, Biswanath Maity, Geetha Narayan, Sandip K. Bandyopadhyay and Subrata Chattopadhyay* Black tea (BT) and kombucha tea (KT) provides effective healing against indomethacin-induced stomach ulceration in mice.

This journal is ª The Royal Society of Chemistry 2010

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Titles from RSC Publishing

The RSC Publishing books programme contains a superb range of monographs, textbooks, professional references and popular science titles – all related to food science and technology. Our exclusive food science titles also incorporate many of the subdisciplines such as food safety, food preservation and food microbiology and are of central importance to students, teachers, lecturers and professionals working at all levels in the chemical sciences!

The Maillard Reaction

Interface between Aging, Nutrition and Metabolism This book is the proceedings of a meeting held in Australia which brought together the world experts in the Maillard or ‘browning’ reaction. It was held in response to a growing recognition of the role reactive carbonyl compounds play in food technology, nutrition and tissue ageing in biology and medicine. The book highlights efforts to counteract the damage caused by reactive carbonyls which is gaining acceptance as a basis for novel approaches for ageing and preventive medicine. Aimed at both basic and applied researchers, scientists and industries worldwide this book will appeal to those with an interest in the Maillard reaction. Hardback | 252 pages | ISBN 9781849730792 | 2010 | £110.00

Food (5th Edition) The Chemistry of its Components Foreword by Heston Blumenthal 5th Edition

Food The Chemistry of its Components

FOOD The Chemistry of its Components Tom Coultate

Coultate Foreword by Heston Blumenthal 11/08/2008 14:20:15

As a source of detailed information on the chemistry of food, this book is without equal. It investigates components which are present in large amounts (carbohydrates, fats, proteins, minerals and water) and also those that occur in smaller quantities (pigments, flavours, vitamins and preservatives). Food borne toxins, allergens, pesticide residues and other undesirables are also given detailed consideration. Attention is drawn to nutritional and health significance of food components. This classic text has been extensively rewritten for its fifth edition to bring it right up-to-date and many new topics have been introduced. Its accessible style also ensures that anyone with an interest in food issues will find it invaluable!

“very detailed and readable… the author is to be congratulated” The British Heart Foundation “a superb book to have by your side when you read your daily newspaper” New Scientist

Paperback | 500 pages | ISBN 9780854041114 | 2008 | £24.99

The Science of Chocolate (2nd Edition) The second edition of this international best seller has been fully revised and updated describing the complete chocolate making process, from the growing of the beans to the sale in the shops. The reader will discover how confectionery is made and how basic science plays a vital role. There is discussion of the monitoring and controlling of products, and the importance of the packaging. A series of experiments, which can be easily adapted to suit students, are included to demonstrate the physical, chemical or mathematical principles involved. This book is ideal for those studying food sciences working in the confectionery industry or just with a general interest in chocolate.

“…is an excellent read and is strongly recommended for anyone with an interest in chocolate” Chemistry and Industry

Hardback | 250 pages | ISBN 9780854049707 | £24.95

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PAPERS 294 Non-digestible fraction of cooked bean (Phaseolus vulgaris L.) cultivar Bayo Madero suppresses colonic aberrant crypt foci in azoxymethane-induced rats

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H. A. Vergara-Casta~ neda, R. G. Guevara-Gonz alez, M. Ramos-G omez, R. Reynoso-Camacho, H. Guzman-Maldonado, A. A. Feregrino-Perez, B. D. Oomah and G. Loarca-Pi~ na* Non-digestible fraction of cooked bean (Phaseolus vulgaris L.) cultivar Bayo Madero suppresses colonic aberrant crypt foci in azoxymethane-induced rats.

301 Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS-dependent apoptosis An-Chin Cheng,* Mei-Ling Tsai, Chi-Mei Liu, Ming-Fen Lee, Kalyanam Nagabhushanam, Chi-Tang Ho and Min-Hsiung Pan* Garcinol induced apoptosis by mitochondrial pathway in human hepatoma Hep3B cells.

308 Antioxidative and antibacterial effects of seeds and fruit rind of nutraceutical plants belonging to the Fabaceae family Sumitra Chanda,* Sandeep Dudhatra and Mital Kaneria The antioxidant and antibacterial efficacy of six nutraceutical plants of the Fabaceae family.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 227–232 | 231

Garlic and Other Alliums Eric Block This unique book, with a foreword by Nobel Laureate E. J. Corey, outlines the extensive history and the fascinating past and present uses of these plants. The author has carefully sorted out fact from fiction based upon detailed scrutiny of historic documents as well as numerous laboratories studies.

Garlic and Other Alliums

The Lore and the Science

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The Lore and the Science

Readers will be entertained and educated as they learn about early cultivation of garlic and other alliums while being introduced to their remarkable chemistry and biochemistry, much of which prominently features the element sulfur. They will learn how alliums have been portrayed and used in literature, poetry and the arts and how alliums are featured in the world’s oldest cookbook.

Garlic and Other Alliums

The Lore and the Science

Written by Eric Block, Carla Rizzo Delray Distinguished Professor of Chemistry at the University at Albany, State University of New York, well known for his discoveries elucidating the natural product chemistry of the Allium species, Garlic and Other Alliums will make fascinating reading for both scientists and non-scientists alike.

Eric Block

Foreword by E. J. Corey Block

“This is a fascinating book written by an authority on the chemistry of the edible alliums, which include garlic, onions, leeks and chives.” Jim Hanson, Chemistry World, February 2010.

Garlic and Other Alliums_dust jacket.indd 4

03/07/2009 10:54:31

Title: Garlic and Other Alliums Subtitle: The Lore and the Science Author: Eric Block ISBN: 9781849731805 Publication Date: Nov 2009 Format: Paperback Price: £24.99/U.S. $42.00

“This book by Eric Block is a synthesis of his four decades of distinguished work with alliums. His account of this everincreasing knowledge is accessible and will even entertain readers without a deep knowledge of chemistry.” Meriel Jones, Chemistry & Industry, February 2010 “Dr. Block’s book may be the definitive word on the alliums for the moment, but as it and he make clear, there are new flavors to look forward to.” Harold McGee, The New York Times, June 2010

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REVIEW

www.rsc.org/foodfunction | Food & Function

Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites Maria Monagas,*a Mireia Urpi-Sarda,b Fernando Sanchez-Patan,a Rafael Llorach,b Ignacio Garrido,a Carmen G omez-Cordoves,a Cristina Andres-Lacuevab and Bego~na Bartolomea

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Received 5th September 2010, Accepted 25th October 2010 DOI: 10.1039/c0fo00132e Flavan-3-ols, occurring in monomeric, as well as in oligomeric and polymeric forms (also known as condensed tannins or proanthocyanidins), are among the most abundant and bioactive dietary polyphenols, but their in vivo health effects in humans may be limited because of their recognition as xenobiotics. Bioavailability of flavan-3-ols is largely influenced by their degree of polymerization; while monomers are readily absorbed in the small intestine, oligomers and polymers need to be biotransformed by the colonic microbiota before absorption. Therefore, phenolic metabolites, rather than the original high molecular weight compounds found in foods, may be responsible for the health effects derived from flavan-3-ol consumption. Flavan-3-ol phenolic metabolites differ in structure, amount and excretion site. Phase II or tissular metabolites derived from the small intestine and hepatic metabolism are presented as conjugated derivatives (glucuronic acid or sulfate esters, methyl ether, or their combined forms) of monomeric flavan-3-ols and are preferentially eliminated in the bile, whereas microbial metabolites are rather simple conjugated lactones and phenolic acids that are largely excreted in urine. Although the colon is seen as an important organ for the metabolism of flavan-3-ols, the microbial catabolic pathways of these compounds are still under consideration, partly due to the lack of identification of bacteria with such capacity. Studies performed with synthesized or isolated phase II conjugated metabolites have revealed that they could have an effect beyond their antioxidant properties, by interacting with signalling pathways implicated in important processes involved in the development of diseases, among other bioactivities. However, the biological properties of microbederived metabolites in their actual conjugated forms remain largely unknown. Currently, there is an increasing interest in their effects on intestinal infections, inflammatory intestinal diseases and overall gut health. The present review will give an insight into the metabolism and microbial biotransformation of flavan-3-ols, including tentative catabolic pathways and aspects related to the identification of bacteria with the ability to catabolize these kinds of polyphenols. Also, the in vitro bioactivities of phase II and microbial phenolic metabolites will be covered in detail.

I.

Introduction

Proanthocyanidins or condensed tannins are polymers of flavan3-ols and are among the most abundant polyphenols in our diet. Proanthocyanidins exhibit a wide range of biological activities, including antioxidant, anti-carcinogenic, cardioprotective, antimicrobial and neuro-protective activities, as has been demonstrated in many in vitro and ex vivo studies.1 In the last decade, a large body of epidemiological data has been accumulated supporting the assumption that the consumption of flavan-3-olrich food such as cocoa, red wine or tea may reduce the risk of coronary heart disease (CHD).2–4 Proanthocyanidins exhibit a high structural diversity and a wide range of degree of polymerization (DP), and their content varies considerably between the different plant sources. Procyanidins, consisting of (epi)catechin units, are the most abundant type of proanthocyanidins in nature. Propelargonidins and prodelphinidins a Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. E-mail: [email protected] b Department of Nutrition and Food Science, Xarta, INSA, Pharmacy School, University of Barcelona, Av/Joan XXIII s/n., 08028 Barcelona, Spain

This journal is ª The Royal Society of Chemistry 2010

contain (epi)afzelechin and (epi)gallocatechin units, respectively, and are usually mixed with procyanidins. With regard to the interflavanic bond nature, B-type procyanidins [C-4 (upper unit) /C-6 or C-8 (lower unit)] are more abundant than A-type procyanidins, which contain an additional ethertype bond [C-2 (upper unit)–O–C-7 (lower unit)]. Fruits (grapes, apples and pears), legumes, cocoa and beverages such as wine, cider and beer are among the most important sources of B-type proanthocyanidins.5 Polymeric proanthocyanidins with DP >10 represent the largest amount in 21 kinds of food.5 The daily intake of flavan-3-ols in the United States has been estimated to be around 60 mg/day for proanthocyanidins with a DP <2.5 In the Spanish population it has been estimated to be 18–31 mg/day when considering proanthocyanidins with a DP up to 3,6 and 450 mg/day when considering highly polymerized proanthocyanidins.7 Polyphenols are recognized as xenobiotics (i.e. foreign or artificial substances, usually of synthetic origin) by the human organism, and therefore bioavailability is a factor that limits the health benefits derived from proanthocyanidin consumption. Bioavailability of proanthocyanidins is largely influenced by their DP. While monomeric flavan-3-ols are readily absorbed in Food Funct., 2010, 1, 233–253 | 233

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the small intestine, oligomeric and polymeric forms pass intact through the gastrointestinal tract, reaching the colon where they are transformed by the intestinal microbiota before absorption. The scientific evidence accumulated during the last decade indicates that the beneficial effect of these phytochemicals could be attributed to the conjugated metabolites (formed during the phase II metabolism of monomeric flavan-3-ols), and mainly to metabolites derived from the microbial catabolism of proanthocyanidins, rather than to the original forms found in food which have been widely used in most bioactivity studies.8–10 Recent studies have estimated that the amount of non-absorbable polyphenols reaching the colon is very high and that microbe-derived phenolic metabolites excreted in urine represent the largest proportion of polyphenol intake. This recognition is leading to a reformulation of estimated bioavailability values and the potential bioactivity of polyphenols.11 Currently, there is an increasing interest in the determination of the possible health implications derived from the interaction between phenolic compounds and human microbiota, in particular concerning the effect on microbiota composition and gut health. However, the biological properties of microbial-derived metabolites are still largely unknown. The aim of the present review is to provide updated information on metabolites formed from dietary flavan-3-ols, as well as their bioactivity and potential health effects. After giving a general overview about the bioavailability of dietary flavan-3ols in humans, structures of main phase II or tissular metabolites derived from small intestine and liver metabolism are presented.

A special section is dedicated to the microbial catabolism of monomeric flavan-3-ols and proanthocyanidins, describing possible catabolic pathways, microbial reactions, and characteristic metabolites derived from the biotransformation process. Intrisic characteristics of candidate catabolic bacteria and structural flavan-3-ol features limiting bacteria degradation are also discussed. Finally, the main biological activities reported for both phase II or tissular and microbial metabolites derived from flavan-3-ols are reviewed, taking into consideration the results of studies performed with conjugated metabolites at in vivo concentrations.

II. Bioavailability of monomeric flavan-3-ols and proanthocyanidins Bioavailability is a key issue linking polyphenols and health effects. In the case of flavan-3-ols, the degree of polymerization (DP) and galloylation are factors affecting their bioavailability (Fig. 1). Monomeric flavan-3-ols are absorbed in the small intestine and extensively metabolized into glucuronide conjugates by phase II enzymes.12,13 These metabolites can reach the systemic circulation or be eliminated in the bile. Further metabolism into sulfate conjugates and methyl derivatives occurs in the liver. However, oligomers with DP >3 and polymers are not absorbed in the small intestine and reach the colon, where they are subjected to microbial catabolism. Microbial metabolites are further absorbed and metabolized by phase II enzymes, to finally enter the circulation or be eliminated in urine.

Fig. 1 Schematic diagram of organs, reactions and agents involved in the bioavailability of flavan-3-ols.

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II.1

Absorption and metabolism of monomeric flavan-3-ols

With regard to small intestine and liver metabolism, the small intestine is the main site for glucuronidation, which occurs in the luminal part of the endoplasmic reticulum via the superfamily of uridine 50 -diphosphate glucuronosyltransferases (UGTs). In particular, UGT1 is considered to be responsible for the glucuronidation of flavonoids.14 Sulfation and methylation mainly occur in the liver through cytosol sulfotransferases (SULT) and catechol-O-methyltransferase (COMT). Specifically, SULTA1 and SULTA3 are considered to be responsible for the sulfation of ()-epicatechin.15 The preferred positions for conjugation are the hydroxyl groups at C-30 and C-40 (B ring), and C-5 and C-7 (A ring) (Fig. 2). Generally, conjugated metabolites of ()-epicatechin are presented in the form of monoglucuronides (5-, 7- and -30 -O-glucuronides), sulfates (7-O-sulfate), methyl ethers (30 - and 40 -O-methyl) or as combined derivatives (30 -O-methyl-7-O-glucuronide, 40 -O-methyl 5- or -7O-glucuronide).16–19 In the case of ()-epigallocatechin (EGC), the 30 - and 7-O-glucuronides and the 40 -O-methyl and its derivatives (40 -O-methyl-30 -O-glucuronide, 40 -O-methyl-7-Oglucuronide, and 40 -O-methyl-30 -sulfate) have been identified following consumption of green tea.20–24 In general, O-sulfated metabolites of ()-epicatechin are the predominant metabolites in urine samples after the intake of a single nutritional dose of cocoa powder in humans.25 However, O-methyl-O-sulfate derivatives of ()-epicatechin have been found as major urinary metabolites, followed by glucuronide, sulfate and methyl-glucuronide conjugates, after the intake by humans of a single dose of flavonol-rich cocoa powder.26 Major

urinary amounts of (epi)catechin-O-methyl-O-sulfates, followed by sulfates and glucuronide conjugates, have also been reported after the intake of a single dose of tea extracts in humans.27–29 Among O-glucuronides, the 30 -O-glucuronide is the main glucuronide derivative of both ()-epicatechin and EGC in humans.17,22,24,30 EGC-40 -O-methyl is the major methylated metabolite of EGC after tea intake in humans.23 Among the different pairs of diastereomers, ()-epicatechin presents higher absorption than (+)-catechin,16 but the latter is more bioavailable than ()-catechin.31With regard to galloylated monomers, ()-epicatechin-3-O-gallate (ECG) seems to be better absorbed than ()-epigallocatechin-3-O-gallate (EGCG),27–29,32 but is considerably less bioavailable than the non-galloylated monomers. No conjugated metabolites of the 3-O-galloylated flavan-3-ols (ECG and EGCG) have been detected in biological fluids. The possible hydrolysis of ECG after absorption was suggested at first, but no esterases have been described in plasma or liver, being found only at the level of the oral cavity.33 It has been suggested that the low Cmax of EGCG is probably due to not all possible conjugated forms being identified, particularly those conjugated in the gallic acid ring such as EGCG-400 -Omethyl, EGCG-40 ,400 -di-O-methyl, and EGCG-400 -O-glucuronide24,34 (Fig. 2). Recently, a new 7-O-glucopyranosyl-EGCG400 -O-glucopyranoside has also been identified.21 Other studies have confirmed that both ECG and EGCG, but in particular the latter, also appear in unmetabolized form in plasma.28,35–37 Conjugated forms of monomeric flavan-3-ols usually reach a Tmax at 1.5 h after ingestion, which is characteristic of absorption in the small intestine.38

Fig. 2 Chemical structure of conjugated metabolites of ()-epicatechin, ()-epigallocatechin (EGC) and ()-epigallocatechin-3-O-gallate (EGCG).

This journal is ª The Royal Society of Chemistry 2010

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II.2

Absorption and metabolism of dimeric proanthocyanidins

In the last decade, the absorption and metabolism of dimeric proanthocyanidins have been a subject of speculation. It was first thought that procyanidins could be depolymerized into bioavailable monomers under the acidic conditions of the stomach,39 but later studies failed to demonstrate this occurrence in vivo.40–42 In contrast to monomers, glucuronidated or sulfated metabolites of dimeric procyanidins have not been detected in biological fluids,43 although some methylated forms have been reported.44 Procyanidins B1 [epicatechin-(4b/8)-catechin] and B2 [epicatechin-(4b/8)-epicatechin] have been detected in their intact form at very low levels in human plasma (nM range) after consumption of cocoa45 or grape seeds,46 and present the lowest Cmax in plasma among flavonoid compounds.13 Besides dimer B2, procyanidin B5 (epicatechin-(4b/6)-epicatechin) has also been detected in the plasma of rats fed cocoa extracts,47,48 but it was not detected in human plasma after cocoa consumption.45 However, dimer B3 [catechin-(4a/8)-catechin] and trimer C2 [catechin-(4a/8)-catechin-(4a/8)-catechin] were not detected in the plasma of rats fed the corresponding purified compounds.49 Recently, oligomers with DP 2–5 have been

detected in rat plasma after the administration of apple procyanidin fractions (1 g/kg weight) with the same DP.44

III. Microbial catabolism of monomeric flavan-3-ols and proanthocyanidins It has been estimated that 90–95% of dietary polyphenols are not absorbed in the small intestine and therefore accumulate in the colon.50 In the case of flavan-3-ols, in studies performed with ileostomy patients (i.e. patients whose colon has been removed surgically), it was calculated that approximately 70% of the ingested monomeric flavan-3-ols from green tea could pass from the small to the large intestine, with 33% corresponding to the intact parent compounds.29 Recently, it has been reported that after oral administration of [14C]procyanidin B2, 63% of the total radioactivity was excreted via urine, indicating that a large quantity of the parent compound is degraded by the gut microflora.51 The recognition that the colon is a very active organ for the metabolism of flavan-3-ols, particularly proanthocyanidins, has led to a resurgence in the study of the biotransformation of these compounds and other polyphenols by the intestinal

Fig. 3 Metabolic pathway tentatively proposed for the catabolism of monomeric flavan-3-ols and dimeric procyanidins by the intestinal microbiota.

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microbiota8,10 and their implication in the overall bioavailability and bioactivity of polyphenols.

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III.1 First steps of the catabolism of flavan-3-ols: formation of hydroxyphenylvalerolactones and valeric acids The complex catabolism of B-type proanthocyanidins involves C-ring opening, followed by lactonization, decarboxylation, dehydroxylation, and oxidation reactions, among others.10 Although numerous in vitro fermentation and in vivo studies have been carried out in recent years, the accumulated knowledge has only led to partial elucidation of the catabolic route of monomeric and B-type dimeric structures49,52–55 (Fig. 3). In the case of galloylated monomeric flavan-3-ols (ECG and EGCG), the microbial catabolism usually starts with the rapid cleavage of the gallic acid ester moiety by microbial esterases, giving rise to gallic acid which is further decarboxylated into pyrogallol.56–58 The C-ring is subsequently opened, giving rise to diphenylpropan-2ol, which is later converted into 5-(30 ,40 -dihydroxyphenyl)-gvalerolactone (in the case of (epi)catechins) or 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone (in the case of (epi)gallocatechins).56,58,59 The valerolactone ring later breaks, giving rise to 5-(30 ,40 -dihydroxyphenyl)valeric acid and/or 4-hydroxy-5-(30 ,40 -dihydroxyphenyl)valeric acid. The identification of this latter compound was firstly proposed by Khori et al.57 and recently confirmed by Llorach et al.60 in urine samples collected after cocoa consumption in humans, as well as by Stoupi et al.61 after in vitro fermentations carried out with human faeces in the presence of ()-epicatechin and procyanidin B2. Although it was first proposed that 4-hydroxy-5-(hydroxyphenyl)valeric acids could arise from the degradation of diphenylpropan-2-ols, concurrently with hydroxyphenyl-gvalerolactones57 (Fig. 3), it has recently been suggested that they are formed instead from hydroxyphenyl-g-valerolactones, and that an interconversion between both forms [4-hydroxy-5(hydroxyphenyl)valeric acids and 5-(hydroxyphenyl)-g-valerolactones] may exist, but is largely displaced towards the formation of the formers.61 Subsequent biotransformations of these valeric acids give rise to hydroxyphenylpropionic and hydroxybenzoic acids by successive loss of carbon atoms from the side chain through b-oxidation.56 III.2 Metabolites arising from the catabolism of dimeric procyanidins The possible formation of 3,4-dihydroxyphenylacetic acid via aoxidation of 3,4-dihydroxyphenylpropionic acid (as described for tyrosine49,62) in the microbial catabolism pathway of monomeric flavan-3-ols, has been widely debated. Firstly, it was thought that 3,4-dihydroxyphenylacetic acid was only characteristic of the catabolism of dimeric procyanidins;63 however, other authors have recently proposed a-oxidation as a possible pathway for the formation of this compound in the case of both monomers and dimers,61 without discarding other possible pathways, as proposed by Appeldoorn et al.54 in the case of dimers. According to these latter authors, 3,4-dihydroxyphenylacetic acid results from the cleavage of the upper unit of dimeric procyanidins, whereas the lower unit gives rise to 5-(30 ,40 dihydroxyphenyl)-g-valerolactone and to the triggering of the This journal is ª The Royal Society of Chemistry 2010

rest of the previously described route (Fig. 3). The possible depolymerization of dimeric structures into monomeric units, firstly proposed by Groenewoud et al.,64 has been recently confirmed to occur but to a lesser extent,54,61 representing less than 10% in the case of procyanidin B2.61 Other microbial metabolites arising exclusively from the catabolism of dimeric procyanidins have recently been identified, such as 5-(20 ,40 dihydroxyphenyl)-2-ene-valeric acid, as well as other compounds which have been tentatively identified as derivatives from the A-ring of the upper unit, including the interflavanic bond.61 III.3

Last steps of the catabolism of flavan-3-ols

Finally, the last steps of the microbial catabolism of (epi)catechin involve dehydroxylation of 3,4-dihydroxylated phenolic acids at C-40 (preferentially), and C-30 , resulting in 3- and 4-monohydroxylated phenolic acids, respectively.53,61 In the case of (epi)gallocatechins, dehydroxylation preferentially occurs at C-5, resulting in 3,4-dihydroxylated phenolic acids which undergo further dehydroxylation at C-4 and C-3, as mentioned above. However, in the case of hydroxyphenylvalerolactones, the 3,5-dihydroxylated derivative arising from the dehydroxylation of 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone has also been identified, indicating that dehydroxylation at C-40 occurs.20 Once absorbed, the microbial metabolites from flavan-3-ols are mainly metabolized in the liver by phase II enzymes as conjugated derivatives that are subsequently eliminated in urine. At the same time, a portion of microbial metabolites (non-conjugated microbial metabolites) is eliminated in the faeces. Several microbe-derived metabolites that have been detected in urine in their actual conjugated form by targeted analysis including: monoglucuronide and monosulfate of 5-(30 ,40 - and 30 ,50 -dihydroxyphenyl)-g-valerolactone, in addition to the methyl-sulfate derivatives of 5-(30 ,40 ,50 -trihydroxyphenyl)-gvalerolactone.20,22,30,65 In the case of phenolic acids, monoglucuronide and monosulfate conjugates of mono- and di-hydroxyphenylpropionic and p-coumaric acids have been reported.66 Other reactions occurring in the liver and kidney include: glycine conjugation, dehydrogenation, hydroxylation and methylation.53 The excretion of microbial metabolites varies markedly between subjects, and for some individuals it may also vary with the substrate, reaching a very high proportion (up to 50%) of the intake of polyphenols.8 III.4

Main microbial phenolic metabolites found in urine

Several feeding studies have revealed significant changes in the urinary excretion of microbe-derived phenolic acids after the intake of rich sources of flavan-3-ols. Among phenolic acids, mono- and di-hydroxylated phenylpropionic and phenylacetic acids, together with hydroxyhippuric acids, have been found as main urinary microbial phenolic acids derived from flavan-3-ol intake. With regard to cocoa and cocoa-derived products, Gonthier et al.49 reported an increase in 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids in urine after the administration of procyanidin B3 to rats. Similarly, Rios et al.67 reported a significant increase in the urinary excretion of these compounds, as well as in 3-hydroxyphenylpropionic and 3-hydroxybenzoic acids in Food Funct., 2010, 1, 233–253 | 237

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healthy humans after acute consumption of flavanol-rich chocolate. Recently, Urpi-Sarda et al.68 also found increased urinary levels of 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids in humans after chronic consumption of cocoa powder with milk. Other studies have reported an increased urinary excretion of 3-hydroxypropionic and 3-hydroxyphenylacetic acids after human consumption of grape seed polyphenols.69 In the case of green tea, 3-hydroxyphenylpropionic and 4-hydroxyphenylacetic and 4-hydroxyphenylacetic acids significantly increased in human urine.58 Finally, 3-hydroxyphenylpropionic and 3-hydroxybenzoic acids were also reported to increase in the urine of rats fed wine polyphenols.53 Besides these phenolic acids, which are also common to the microbial catabolism of other flavonoids,10 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone and 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone are considered important microbial metabolites and potential biomarkers of flavan-3-ol consumption in humans, as has been confirmed after the intake of green tea,20,22,23,58 cocoa products30 and almond skins.65,70

IV. Intestinal bacteria with ability to catabolize flavan-3-ols It is important to mention that the above difference of opinions concerning the possible catabolic route of monomeric and dimeric flavan-3-ols could be partly attributed to differences in the microbiota composition of faecal samples used in the different studies, suggesting that different pathways could coexist or one predominate over the others, depending on the catabolic capacity of the microbiota. An important limitation in this area is that bacteria belonging to human microbiota with the capacity to catabolize flavan-3-ols have still not been identified. To date, only bacteria with the capacity to catabolize other types of flavonoid compounds, mainly flavonols and flavones, have been described. These bacteria, in general, belong to the Clostridium and Eubacterium groups.10 Among the factors that may limit the identification of flavan3-ol catabolic bacteria, it is important to highlight the wellknown growth inhibitory effects of proanthocyanidins. Another factor that deserves consideration is the structural features of flavan-3-ols as complex non-planar molecules.

IV.1 Inhibitory effects of proanthocyanidins and ‘‘tanninresistant’’ bacteria The growth-inhibitory effects of proanthocyanidins on bacteria have been reviewed by Smith et al.71 Tannins are capable of complexing with polymers and minerals, making nutrients unavailable. In addition, they could have a direct effect by interacting with membranes, cell walls, and/or extracellular proteins. ‘‘Tannin-resistant’’ bacteria have been defined as those bacteria that are able to withstand the inhibitory effect of tannins. ‘‘Resistance’’ implies that some action is required on the part of the organism to withstand the inhibitory effect of tannins, including inducible adaptation or even gene transfer.71 Tanninresistance may also depend on the tannin concentration, structural composition and DP. It is important to highlight the fact that bacteria which are predominant in tannin-rich mediums may 238 | Food Funct., 2010, 1, 233–253

not be resistant per se, but are less affected by nutrient limitations or are better able to access limiting nutrients. ‘‘Tannin-resistant’’ Gram-negative species (Enterobacteriaceae and Bacteriodes) have been isolated from rat faecal samples after prolonged administration of condensed tannins from Acacia angustissima, a forage legume.72 ‘‘Tannin-resistant’’ Gram-positive bacteria have also been identified. Brooker et al.73 isolated a Streptococcus strain (named S. caprinus and close to S. bovis) from the rumen of goats which was able to grow at 2.5% of condensed tannins. A Streptococcus strain (close to S. bovis and S. gallolyticus) has also been isolated from the rumen of sheep, goats and deer.74 Later, Molina et al.75 has also isolated a Eubacterium strain (close to E. cellulosolvens) from the rumen of moose, able to tolerate 0.5 g/L of condensed tannins. Some mechanisms by which bacteria can overcome inhibition by tannins include: modification/degradation of the substrate, dissociation of tannin–substrate complexes, cell membrane modification/repair and metal ion sequestration. It has been reported that Bifidobacterium infantis and Lactobacillus acidophilus are not inhibited by tannins because lactic acid bacteria do not require iron as they do not depend on metal-chelating enzymes, in particular heme enzymes.76 Moreover, in vivo studies have revealed that consumption of grape seed extract, containing 40% of condensed tannins, produced an increase in the bifidobacteria population in healthy individuals.77 Although tannin resistance is the first step in order for bacteria to metabolize condensed tannins, resistance does not guarantee metabolic activity, and the biodegradation pathway of ‘‘tannin-resistant’’ bacteria has not yet been described. IV.2 Structural features of flavan-3-ols limiting bacterial catabolism There is some evidence that the structural characteristics and stereochemistry of flavan-3-ols could be limiting factors for intestinal bacteria to be able to degrade these types of compounds. It has been reported that Eubacterium ramulus was unable to degrade (+)-catechin because of the absence of a functional group at C-4 in this flavonoid structure.78 Similarly, the human bacterium Eubacterium sp. (SDG-2) was able to open the ring of the 3R [()-catechin and ()-epicatechin] and the 3S [(+)-catechin and (+)-epicatechin] forms of monomeric flavan-3ols into 1,3-diphenylpropan-2-ols (Fig. 3), but was incapable of producing the same fission in their galloylated esters.79 However, in no instance was this bacteria able to continue the catabolism up to the formation of 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone. Another characteristic of this bacterium was the ability to dehydroxylate the OH groups in the B ring of 1,3-diphenylpropan-2-ols, but only of the R forms.79 This fact, together with the inability to catabolize the gallate esters, suggests that the spatial configuration of both the original flavan-3-ol molecule and intermediate metabolites may limit the microbial degradation of flavan-3-ols. In fact, in a recent in vitro fermentation study with human faeces it was found that (+)-catechin (2R,3S) was firstly converted into (+)-epicatechin (2S,3S) by intestinal microbiota in order for the biotransformation process to proceed.80 Taken together, these findings suggest that it may be difficult to identify a single bacterium capable of exhibiting the This journal is ª The Royal Society of Chemistry 2010

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whole catabolic pathway proposed in Fig. 3, but rather the catabolism may be carried out by different bacteria with specific catabolic activities that work in sequential form on the appearance of the different intermediate metabolites. Among the different phases of the catabolic pathway, formation of 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone seems to be a limiting step.

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V. Bioactivity of flavan-3-ol metabolites As a consequence of their extensive metabolism in the human organism, the original flavan-3-ol structures present in food are not present in plasma and urine (with the exception of small amounts of gallate ester of monomeric flavan-3-ols and dimeric procyanidins that appear unmetabolized, as mentioned above) but rather appear as a complex series of phase II or tissular metabolites and, particularly, of microbe-derived phenolic metabolites. Therefore, both types of circulating metabolites should be responsible for the health benefits associated with the consumption of dietary sources rich in flavan-3-ols. V.1 Bioactivity of phase II or tissular metabolites derived from small-intestine and liver metabolism One of the limitations of many in vitro and ex vivo studies which have tried to unravel the health effects of flavan-3-ols has been the use of unconjugated structures, as well as the use of test concentrations (mM) at a much higher range than that found in biological fluids (mM range). Taking this into consideration, this section will only try to cover the results of studies performed with conjugated metabolites in the micromolar or submillimolar range (0.5–30 mM) found in plasma (Table 1). In general, the conjugation process (glucuronidation, sulfation and methylation) affects the physico-chemical properties of flavan-3-ols and, in turn, their residence in plasma, their excretion rate, and finally the bioactive properties of the parent compound.13 In particular, sulfation and glucuronidation involve a considerable attenuation of biological activity. The case of methylation seems to be more complex because the incorporation of methyl groups reduces the number of available OH groups, but at the same time increases the lipophilic nature of the compound, which can be advantageous for cellular uptake by passive diffusion.13 Antioxidant activity. The antioxidant activity of flavonoid metabolites has been widely studied, considering the fact that oxidative stress is implicated in the initiation and progression of chronic diseases. In the case of flavonoid compounds (i.e. quercetin), it has been observed that glucuronidation at C-30 and C-40 of the B ring (catechol-type structure) produces a greater loss of antioxidant capacity than when it occurs at C3 of the C ring.81–83 In contrast, glucuronidation at C-7 (A ring) seems to produce a slight increase in antioxidant activity.83 In the case of flavan-3-ol metabolites, ()-epicatechin and its 7-Oglucuronide presented a similar delay of Cu2+-induced LDL oxidation, whereas the activities of the 30 -O-glucuronide and the 40 -O-methyl-30 -O-glucuronide were significantly lower.84 However, in the case of galloylated (epi)gallocatechins, the position of glucuronidation affected the anti-radical capacity This journal is ª The Royal Society of Chemistry 2010

against DPPH differently to the other flavonoids, since EGCG7-O-glucuronide and EGCG-400 -O-glucuronide (galloylation in the gallic acid ring) were less active than the aglycone, whereas the 30 - and 300 -O-glucuronides showed the same activity as the aglycone.24 For non-galloylated (epi)gallocatechins, EGC-7-Oglucuronide and -30 -O-glucuronide were more active than the aglycone.24 In the case of O-methylation, Cren-Olive et al.85 also reported that the catechol B-ring was also the active moiety of (+)-catechin, since the 30 - and 40 -O-methyl ethers and 30 ,40 -di-O-methyl ether showed a much lower inhibition of Cu2+-induced LDL oxidation than the aglycone, but the activity was recovered when these positions were free, as in the 5,7-di-O-methyl analogue. The C-30 and C-40 -O-methyl ethers of ()-epicatechin also showed a lower inhibition of peroxynitrite-induced tyrosine nitration than the parent compound.86 Similarly, O-methylation at position C-30 in ()-epicatechin, ()-epigallocatechin and ()-epicatechin-3-O-gallate elicited a potential inhibition of lipid oxidation of canola oil in comparison to the aglycone.87 In a recent study, C-30 and C-40 -O-methyl ethers of (+)-catechin and ()-epicatechin showed a lower antioxidant capacity than the parent compound, as measured by the ferric-reducing power (FRAP) and by the ability to scavenge the ABTS+ radical cation.88 Moreover, the antioxidant activity of these metabolites was found to be pH dependent, but significant radical scavenging activity was found to be retained at pH 7.4, suggesting that they could act as potential antioxidants under physiological conditions.88 Vascular effects. Epicatechin and its metabolite, epicatechin-7O-glucuronide, have been identified as independent predictors of the vascular effects observed after flavanol-rich cocoa intake.19 Anti-inflammatory effects. In the case of EGCG metabolites, glucuronidation at C-7 affected the ability to inhibit the production of NO or the arachidonic acid metabolism in HT29 cells compared to the aglycone, but it was not affected in the case of glucuronidation at C-30 , C-300 , C-400 .24 Conversely, in the case of ECG, glucuronidation at C-30 decreased such capacity by 20% compared to the aglycone, but it was not affected in the case of the 7-O-glucuronide.24 Inhibition of cellular growth. The effectiveness of ()-epicatechin metabolites on the inhibition of cellular growth has been studied in various types of cell lines. In the case of neuronal cells, it has been reported that 30 -O-methyl-epicatechin was as effective as ()-epicatechin in the inhibition of apoptosis induced by oxidized LDL.89 Similarly, it has been reported that 30 -O-methylepicatechin was as efficient as ()-epicatechin in protecting human fibroblasts against cell death induced by oxidative stress.90 In the case of galloylated flavan-3-ol metabolites, methylation at C-40 and C-400 in ()-epigallocatechin-3-O-gallate (EGCG) produced a 50% decrease in the growth-inhibitory and pro-apoptotic activities of murine osteoclasts, compared to EGCG.91 In another study, methylated derivatives of EGCG at positions C-400 and C-40 -400 (dimethyl derivative) presented less inhibitory capacity than EGCG of the enzyme 20S proteasome, which catalyzes the degradation of intracellular proteins and is associated with cancer.92 Food Funct., 2010, 1, 233–253 | 239

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Schroeter et al.19

FMD after flavanol-rich cocoa ingestion

ABTS/peroxide assay; ABTS/ persulfate assay; Ferricreducing power (FRAP) assay

Due~ nas et al.88

Vascular effects

TEAC assay

Inhibition of peroxynitriteinduced tyrosine nitration

Inhibition of lipid oxidation of canola oil

Pollard et al.86

Pollard et al.86

Su et al.87

Natsume et al.

Inhibition of Cu2+-induced LDL oxidation

EC-7-O-glucuronide

EC-4 -O-methyl

0

C-30 -O-methyl C-40 -O-methyl EC-30 -O-methyl

Cmax  200 nM (2 h)

n.a.

0.1–500 mM

IC50 ¼ 19.0 mM IC50 ¼ 30.7 mM

EC-O-methyl (1 : 1 mix)d EC-5-O-glucuronide EC-O-methyl (1 : 1 mix)d EC-5-O-glucuronide

0.5 mM

0.5 mg/mL

EC-30 -O-methyl EGC-30 -O-methyl EGCG-30 -O-methyl

EC-7-O-glucuronide EC-30 -O-glucuronide EC-40 -O-methyl-30 -O-glucuronide

 EC and EC-7-O-glucuronide are predictors of the increase of FMD.

 C and EC > 30 - and 40 -O-methyl metabolites of C or EC.  Methylated metabolites still retain significant radical scavenging activity at pH 7.4.  Relatively high antioxidant activity was found in the case of C-30 -O-methyl catechin at pH 7.4 compared to C.

 The order of activity was: EC > EC-5-Oglucuronide > EC-O-methyl EC.

 The order of activity was: EC > EC-Omethyl mix > EC-5-O-glucuronide.

 The three metabolites were less effective than EC.

 The order of activity was: EC y EC-7-Oglucuronide (3% lower) [ EC-40 -Omethyl-30 -glucuronide y EC-30 -Oglucuronide.

DPPH radical scavenging activity

Lu et al.24

84

 The order of activity was: EGCG-300 -Oglucuronide ¼ EGCG-30 -O-glucuronide ¼ EGCG > ECG > EGC > EGCG-7-Oglucuronide ¼ EGCG-400 -O-glucuronide > EGC-7-O-glucuronide ¼ EC > EGC-30 -Oglucuronide.

EC50 ¼ 0.084c EC50 ¼ 0.081 EC50 ¼ 0.035 EC50 ¼ 0.037 EC50 ¼ 0.19 EC50 ¼ 0.11

EGCG-400 -O-glucuronide EGCG-7-O-glucuronide EGCG-300 -O-glucuronide EGCG-30 -O-glucuronide EGC-30 -O-glucuronide EGC-7-O-glucuronide

Inhibition of Cu2+-induced LDL oxidation

Result

Cren-Olive et al.85

Concentration

 The order of activity was: C-5,7-di-Omethyl > C > 4-methylcatechol > C-30 ,40 di-O-methyl > C-40 -O-methyl  C-30 -Omethyl > C-30 ,40 ,5,7-tetra-O-methyl.

Metabolite

EC50 ¼ 15.2  1.0 mM EC50 ¼ 11.7  0.9 mM EC50 ¼ 0.63  0.02 mM EC50 ¼ 3.80  0.08 mM EC50 > 100 mM EC50 ¼ 1.00  0.05 mM

Test

C-30 -O-methyl C-40 -O-methyl C-5,7-di-O-methyl C-30 ,40 -di-O-methyl C-30 ,40 ,5,7-tetra-O-methyl 4-Methylcatechol

Antioxidant effects

Reference

Table 1 Biological activity of phase II or tissular metabolites of flavan-3-ols.a,b,e

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This journal is ª The Royal Society of Chemistry 2010

Inhibition of purified 20S proteasome assessed by using a chymotrypsin-like specific fluorogenic substrate

Fe3+ reduction activity ECG-300 -O-methyl ECG-400 -O-methyl ECG-300 ,400 -di-O-methyl EGCG-300 -O-methyl EGCG-400 -O-methyl EGCG-300 ,400 -di-O-methyl EGCG-40 -O-methyl EGCG-40 ,400 -di-O-methyl EGCG-40 -300 ,400 -tri-O-methyl

IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50

¼ 3.43  1.3 mM ¼ 19.12  1.88 mM ¼ 48.25  0.64 mM ¼ 5.63  0.03 mM ¼ 6.91  0.40 mM ¼ 9.81  0.15 mM ¼ 2.45  0.30 mM ¼ 8.23  0.07 mM ¼ 43.03  1.98 mM

EC50 ¼ 87  2.2 mM EC50 > 100 mM EC50 ¼ 70  11 mM EC50 > 100 mM

30 mM

1 mM

30 mM

Cmax  210 nM (2 h)

Cmax  1450 nM (2 h)

2 and 10 mM

Concentration

 The proteasome inhibitory activity for the ECG series was: ECG > ECG-300 -methyl > ECG-400 -methyl > ECG-300 ,400 -dimethyl.  The proteasome inhibitory activity for the ECG series was: EGCG > EGCG-300 -Omethyl > EGCG-400 -O-methyl > EGCG300 ,400 -di-O-methyl.  And: EGCG > EGCG-40 -methyl > EGCG-40 ,400 -dimethyl > EGCG-40 -300 ,400 tri-O-methyl.

 Methylated metabolites at position 40 (B ring) or at position 400 (D-ring) showed markedly cytotoxicity to osteoclasts.  EGCG-40 -O-Me also showed the lowest Fe3+-reducing activity among EGCGs.

 No effects of 30 -O-methyl-EC at 1 mM.  Significant protection against cell death in live-cell population, similar to epicatechin at 30 mM.

 EC-30 -O-methyl protects against oxLDLinduced neuronal injury. MTT reductions: EC (90%) and EC-30 -O-methyl-(93%).  Inhibition of the oxLDL-mediated activation of ERK1/2 and JNK

 At 2 and 10 mM, the order of activity was: EGC-30 -O-glucuronide < EGC-7-Oglucuronide¼ EGC.  At 2 mM, the order of activity was: EGC-7O-glucuronide < EGCG ¼ three glucuronide derivatives of EGCG (30 , 300 and 400 ).

Result

a C: (+)-catechin; EC: ()-epicatechin; ECG: ()-epicatechin-3-O-gallate; EGC: ()-epigallocatechin; EGCG: ()-epigallocatechin-3-O-gallate. b DPPH: 2,2,-diphenylpicrylhydrazyl; ERK1/2: extracellular signal-regulated kinases 1/2; JNK: c-Jun N-terminal kinase; FMD: flow-mediated vasodilation. c EC50 expressed as molar ratio compound/DPPH. d 30 -O-methyl and 40 -O-methylepicatechin (1 : 1 mixture). e n.a.: not available.

Landis-Piwowar et al.92

Nakagawa et al.91

EGCG-30 -O-methyl EGCG-40 -O-methyl EGCG-300 -O-methyl EGCG-400 -O-methyl

EC-30 -O-methyl

Modulation of UVA-induced cell death (FEK4 cells)

Basu-Modak et al.90

Cytotoxicity to murine osteoclasts

EC-30 -O-methyl

EGCG-7-O-glucuronide, EGCG30 -O-glucuronide, EGCG-300 -Oglucuronide, EGCG-400 -Oglucuronide, EGC-30 -Oglucuronide, EGC-7-Oglucuronide, EC-5-O-glucuronide EC-40 -O-methyl-O-b-Dglucuronides (7, 5 and 30 ) EC-7-O-glucuronide

Metabolite

Schroeter et al.89

Inhibition of release of arachidonic acid from HT-29 human colon cancer cells

Test

3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay Activation of signalling pathways ERK1/2 and JNK

Inhibition of cellular growth

Lu et al.

24

Anti-inflammatory effects

Reference

Table 1 (Contd. )

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Food Funct., 2010, 1, 233–253 | 241

Test

242 | Food Funct., 2010, 1, 233–253

Radical scavenging test against superoxide radicals

Ferric-reducing antioxidant potential (FRAP)

Lambert et al.100

Grimm et al.99

Anti-inflammatory effect

Lambert et al.100

5-(30 ,40 -Dihydroxyphenyl)-gvalerolactone; 5-(30 -methoxy-4hydroxyphenyl)-g-valerolactone 5-(30 ,40 ,50 -Trihydroxyphenyl)-gvalerolactone 5-(30 ,40 ,50 -Trimethoxyphenyl)-gvalerolactone

Inhibition of the release of arachidonic acid and production of nitric oxide (NO) by LPS-stimulated murine macrophages (RAW264.7)

5-(30 -Methoxy-40 -hydroxyphenyl)-gvalerolactone

5-(30 ,40 -Dihydroxyphenyl)-gvalerolactone

()-5-(30 ,40 -dihydroxyphenyl)-gvalerolactone; (3-hydroxy-40 methoxyphenyl)-g-valerolactone; 5(30 ,40 ,-dimethoxyphenyl)-gvalerolactone; 5-(30 ,40 ,50 Trimethoxyphenyl)-g-valerolactone

()-5-(30 ,40 ,50 -Trihydroxyphenyl)-gvalerolactone

5-(30 ,40 -Dihydroxyphenyl)-gvalerolactone 5-(30 -Methoxy-40 -hydroxyphenyl)-gvalerolactone

5-(30 ,40 -Dihydroxyphenyl)-gvalerolactone 5-(30 -Methoxy-40 -hydroxyphenyl)-gvalerolactone

Metabolite

Inhibition of MMP-9 secretion

Inhibition of the enzymatic activity of MMP-1, MMP-2 and MMP-9 (matrix metalloproteinases)

3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyletarazolium bromide (MTT) assay on: esophageal squamous carcinoma cells (KYSE150); human colon adenocarcinoma cells (HT-29 and HCT-116); immortalized human intestinal cells epithelial cells (INT407); immortalized rat intestinal epithelial cell line (IEC-6)

Anti-proliferative activity

Grimm et al.

99

Grimm et al.99

Antioxidant effect

Hydroxyphenyl-g-valerolactones

Reference

No effect

IC50 ¼ 20 mM

IC50 ¼ 10–23 mg/mL (MMP-1); IC50  13–23 mg/ mL (MMP-2); IC50  4–19 mg/mL (MMP-9) IC50 ¼ 10–23 mg/mL (MMP-1); IC50 20–22 mg/ mL (MMP-2); IC50  9–10 mg/mL (MMP-9) IC50 ¼ 0.5 mg/mL

>50 mM

IC50 ¼ 15–73 mM

No effect

EC50  25mM

EC50 ¼ 19.65  0.75 mM

EC50 ¼ 10.64  0.42 mM

Concentration

Table 2 Biological activity of phenolic metabolites derived from the catabolism of flavan-3-ols by the intestinal microbiota.a

 Neither compound inhibited the release of arachidonic acid. Only 5-(30 ,40 ,50 trihydroxyphenyl)-g-valerolactone inhibited NO production by 50% at 20 mM.

 Highly potent prevention of MMP-9 release by both metabolites.

 The metabolites were more effective than their metabolic precursor (+)-catechin in MMP inhibition.

 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone was the most active metabolite tested in carcinoma and intestinal cells, whereas 5(30 ,40 ,50 -trimethoxyphenyl)-g-valerolactone only inhibited cell growth by less than 20% at concentrations up to 50 mM.  5-(30 ,40 -dihydroxyphenyl)-g-valerolactone, and its mono- and di-methoxylated derivates were significantly less potent than 5-(30 ,40 ,50 trihydroxyphenyl)-g-valerolactone and inhibited growth of KYSE150 cells by 20– 40% at 50mM, but had no effect on HT-29 cells.

 The order of activity was: 5-(30 ,40 dihydroxyphenyl)-g-valerolactone > (+)-catechin y ascorbic acid > taxifolin > vitamin E.

 The order of activity was: 5-(30 ,40 dihydroxyphenyl)-g-valerolactone > (+)-catechin > ascorbic acid > 5-(30 methoxy-40 -hydroxyphenyl)-gvalerolactone.

Result

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This journal is ª The Royal Society of Chemistry 2010

Test

This journal is ª The Royal Society of Chemistry 2010

Cueva et al.103

Alakomi et al.102

Lee et al.

101

2.5 mM

62.5–1000 mg/mL

3,4-Dihydroxyphenylacetic acid; 3hydroxyphenylacetic acid; 3,4dihydroxyphenylpropionic acid; 4hydroxyphenylpropionic acid; phenylpropionic acid; 3hydroxyphenylpropionic acid

Benzoic acid; 3-hydroxybenzoic acid; 4hydroxybenzoic acid; 3,4dihydroxybenzoic acid; 4-hydroxy-3methoxybenzoic acid; phenylacetic acid; 3-hydroxyphenylacetic acid; 4hydroxyphenylacetic acid; 3,4dihydroxyphenylacetic acid; phenylpropionic acid; 3hydroxyphenylpropionic acid; 4hydroxyphenylpropionic acid; 3,4dihydroxyphenylpropionic acid

Permeability assay through 1-Nphenylnaphthylamine uptake assay

Growth inhibition of pathogenic and non-pathogenic human intestinal bacteria

1.0 mg/mL

Concentration

3-O-Methyl gallic acid; gallic acid; caffeic acid; 4-hydroxyphenylpropionic acid; phenylpropionic acid; 4hydroxyphenylacetic acid

Metabolite

Growth inhibition of pathogenic and non-pathogenic human intestinal bacteria

Effect of intestinal microbiota

Phenolic acids

Reference

Table 2 (Contd. )

 For E.coli strains, the order of activity was: – Benzoic and phenylacetic acids: nonsubstituted [ 4-hydroxy-3-methoxy- > 3hydroxy- > 4-hydroxy- > 3,4-dihydroxysubstituted. – Phenylpropionic acids: non-substituted [ 4-hydroxy- > 3-hydroxy- > 3,4-dihydroxysubstituted.  For Lactobacilli and Staphylocccus aureus strains, the order of activity was: – Benzoic acids: 4-hydroxy- > 3-hydroxy- > non-substituted > 4-hydroxy-3-methoxy- > 3,4-dihydroxy-substituted. – Phenylacetic acids: non-substituted > 3hydroxy- > 4-hydroxy- > 3,4-dihydroxysubstituted. – Phenylpropionic acids: non-substituted > 4-hydroxy- > 3-hydroxy > 3,4-dihydroxysubstituted. – Phenolic acids failed to inhibit the growth of the Gram-negative bacterium P. aeruginosa PAO1 at any concentration tested.

 3,4-Dihydroxyphenylacetic acid, 3hydroxyphenylacetic acid, 3,4dihydroxyphenylpropionic acid, 4hydroxyphenylpropionic acid, phenylpropionic acid, and 3hydroxyphenylpropionic acid efficiently destabilized the outer membrane (OM) of Salmonella enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Infantis. Their activity is based on removal of OM-stabilizing divalent cations.

 Significant inhibition of the growth of: – Clostridium perfringens by 3-O-methyl gallic acid and gallic acid; – Staphylococcus spp. by caffeic acid, 4hydroxyphenylpropionic acid, phenylpropionic acid, 4hydroxyphenylacetic acid; – E. coli and Salmonella spp. by 4hydroxyphenylpropionic acid, phenylpropionic acid, 4hydroxyphenylacetic acid.  Lower inhibition of commensal bacteria and probiotics: Clostridium spp; Bidobacterium spp.; Lactobacillus spp.

Result

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Food Funct., 2010, 1, 233–253 | 243

244 | Food Funct., 2010, 1, 233–253

Rechner et al.105

Anti-thrombotic activity

Gl€aber et al.104

Antioxidant effect

Reference

Table 2 (Contd. )

Effects on epinephrine-stressed platelets

TRAP-induced platelet activation under oxidative stress

TRAP-induced platelet activation

P-selectin expression

Platelet aggregation induced by TRAP, ADP and collagen

Lipid peroxidation in cultured rat hepatocytes challenged with tert-Butyl hydroperoxide (MDA assay)

DPHH radical scavenger assay in cultured rat hepatocytes

Test

10 mM

No effect EC50 > 100 mM EC50 ¼ 30 mM No effect EC50 > 150 mM No effect

3-Hydroxyphenylacetic acid Homovanillic acid 3,4-Dihydroxytoluene Hippuric acid 3,4-Dihydroxyphenylacetic acid 4-Hydroxyphenylacetic acid

Polyphenol mix 1b; 3hydroxyphenylpropionic acid; 4hydroxyphenylpropionic acid; 3,4dihydroxyphenylpropionic acid; dihydroferulic acid; homovanillic acid; 3-hydroxyphenylacetic acid; homovanillyl alcohol; 3hydroxybenzoic acid; phloroglucinol; hippuric acid

EC50 ¼ 4.6 mM EC50 > 500 mM EC50 > 500 mM EC50 ¼ 17 mM EC50 ¼ 7.3 mM EC50 >500 mM

Concentration

3,4-Dihydroxyphenylacetic acid 4-Hydroxyphenylacetic acid 3-Hydroxyphenylacetic acid Homovanillic acid 3,4-Dihydroxytoluene Hippuric acid

Metabolite

 For the agonist TRAP the threshold concentration to induce aggregation increased following the incubation of platelet-rich plasma with dihydroferulic acid (+1.0 mM), homovanillyl alcohol (+0.84 mM) and Polyphenol mix 1 (+1.93 mM). Other compounds tested showed no significant effect on the platelets’ sensitivity towards the agonist TRAP.  None of the tested polyphenol metabolites affected ADP- and collagen-induced platelet aggregation at concentrations up to 100 mM.  Reduction of P-selectin expression on resting platelets was observed following incubation with dihydroferulic acid (29  14%), dihydrocaffeic acid (20  18%), 3hydroxyphenylpropionic acid (19  11%), and Polyphenol mix 1 (16  9%).  Activation of platelet with TRAP increased the P-selectin expression (from 0.5 to 15%). The activation was reduced with dihydroferulic acid (20  17%), 3hydrophenylpropionic acid (21  17%), and Polyphenol mix 1 (12  6).  H2O2 increased response to TRAP. This response was partly reversed with dihydroferulic acid (12  19%), 3hydrophenylpropionic acid (16  10%), and Polyphenol mix 1(13  10).  Only 3-hydroxyphenylpropionic acid (11  12%) significantly reversed epinephrineinduced increase in P-selectin expression.  Epinephrine-induced glycoprotein CD63

 None of the studied metabolites was effective up to 70 mM, with the exception of 3,4dihydroxytoluene.

 The radical scavenging activity decreased in the order: 3,4-dihydroxyphenylacetic acid > 3,4-dihydroxytoluene > homovanillic acid [ hippuric acid ¼ 4-hydroxyphenylacetic acid ¼ 3-hydroxyphenylacetic acid.

Result

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This journal is ª The Royal Society of Chemistry 2010

Monagas et al.108

Inhibition of prostanoid biogenesis

Russell et al.107

Production of pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) in LPS-stimulated peripheral blood mononuclear cells (PBMC) pretreated with the phenolic metabolites

Inhibition of COX-2 protein levels in TNF-a- induced HT-29 cells

Test

Karlsson et al.106

Anti-inflammatory effect

Reference

Table 2 (Contd. )

1 mM

0.1 mmol/L

2-Hydroxybenzoic acid; 4hydroxybenzoic acid; 2,3dihydroxybenzoic acid; 2,4dihydroxybenzoic acid; 2,5dihydroxybenzoic acid; 2,6dihydroxybenzoic acid; protocatechuic acid; 3,5-dihydroxybenzoic acid; gallic acid; vanillic acid; acetovanillone; vanillin; vanillyl alcohol; homovanillic acid; eugenol; cinnamic acid; ocoumaric acid; m-coumaric acid; pcoumaric acid; caffeic acid; 3-(3,4,5trihydroxyphenyl)-acrylic acid; ferulic acid; sinapic acid; ethyl ferulate; coniferyl alcohol; curcumin

3,4-Dihydroxyphenylpropionic acid; 3hydroxyphenylpropionic acid; 3,4dihydroxyphenylacetic acid; 3hydroxyphenylacetic acid; 4hydroxybenzoic acid; 4hydroxyhippuric acid

250–500 mmol/L

Concentration

Phenylpropionic acid; 3hydroxyphenylacetic acid; 4hydroxyphenylpropionic acid

Metabolite

 With the exception of 4-hydroxyhippuric acid for TNF-a secretion, only the dihydroxylated compounds, 3,4dihydroxyphenylpropionic acid and 3,4dihydroxyphenylacetic acid significantly inhibited the secretion of these proinflammatory cytokines in LPS-stimulated PBMC (84.9 and 86.4%, respectively).  The concentrations of IL-6 were reduced by 88.8 and 92.3% with 3,4dihydroxyphenylpropionic acid and 3,4dihydroxyphenylacetic acid, respectively.  Inhibition for IL-1b was 93.1% for 3,4-

 Compounds inhibiting prostanoid production presented the following structure: – 4-hydroxy and 3-methoxyl aromatic substitution pattern (vanillic acid, acetovanillone, vanillin, vanillyl alcohol, homovanillinc acid, ferulic acid, sinapic acid, ethyl ferulate), which significantly inhibited prostanoid biogenesis by up to 81% (vanillin). – 3-carbon side chain (eugenol, cinnamic acid, o-coumaric acid, m-coumaric acid, pcoumaric acid,3-(3,4,5trihydroxyphenyl)acrylic acid, coniferyl alcohol), which significantly inhibited prostanoid biogenesis by up to 75% (coniferyl alcohol).

 % COX-2 inhibition at 250 and 500 mmol/L, respectively was: – phenylpropionic acid: 29.5  14% and 35  7%. – 3-hydroxyphenylacetic acid: 14.7  15% and 39.9  5%. – 4-hydroxyphenylpropionic acid: 61.9  8% and 67  6%.

expression decreased following incubation with 3-hydroxyphenylpropionic acid (15  9%), 3-hydroxyphenylacetic acid (12  7%), 3-hydroxybenzoic acid (17  12%), phloroglucinol (20  7), and Polyphenol mix 1 (13  11%).

Result

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Food Funct., 2010, 1, 233–253 | 245

246 | Food Funct., 2010, 1, 233–253 30 mg/kg

3,4-Dihydroxyphenylpropionic acid; hydroferulic acid; 3,4dihydroxyphenylacetic acid

Measure of prostaglandin E2 (PGE2) production by CCD-18 colon fibroblast cells stimulated with IL-1b

Writhing and paw pressure test in rats

Larrosa et al.109

Larrosa et al.109

Krajka-K uzniak et al.112

Tanaka et al.

111

Protocatechuic acid

Protocatechuic acid

Tumor incidence and multiplicity induced by azoxymethane

Effect on murine hepatic and renal cytochrome P450 and phase II enzymes

80–800 mg/kg

500–1000 ppm

100 mM

3-Hydroxybenzoic acid; 4hydroxybenozoic acid; protocatechuic acid; vanillic acid; phenylacetic acid; 3hydroxyphenylacetic acid; 4hydroxyphenylacetic acid; homoprotocatechuic acid; homovanillic acid; hippuric acid; 3-hydroxyhippuric acid; 3,4-dihydroxyphenylpropionic acid; 3-phenylpropionic acid; 3hydroxyphenylpropionic acid; 4hydroxyphenylpropionic acid; hydroferulic acid; ferulic acid; valeric acid

Anti-proliferative activity and cytotoxicity

50 mg/kg

3,4-Dihydroxyphenylpropionic acid

DSS-induced colitis model

Concentration

Larrosa et al.109

Metabolite

Test

Reference

Table 2 (Contd. )

 Decrease in hepatic and renal EROD and MROD by 20–30% at high concentration (400 and 800 mg/kg).  Decrease in renal PNPH by 28%.  Similar results on the expression of hepatic CYP1A1/1A2 and CYP2E1 proteins.

 Protocatechuic acid administration at 500 ppm and 1000 ppm during the initiation and post-initiation stage significantly inhibited intestinal carcinogenesis.

 The writhing number was reduced by 27, 35 and 40% by hydroferulic acid, 3,4dihydroxyphenylpropionic acid and 3,4dihydroxyphenyl acetic acid.  The three compounds increased the tolerance to the applied pressure of the inflamed paw, but 3,4-dihydroxyphenylpropionic acid was the most potent.

 3,4-dihydroxyphenylpropionic, hydroferulic and 3,4-dihydroxyphenylacetic acids inhibited PGE2 production more than 50%.

 3,4-dihydroxyphenylpropionic acid reduced the expression of the cytokines IL-1b, IL-8, and TNF-a, malonyldialdehyde levels and oxidative DNA damage (measured as 8-oxo20 -deoxyguanosine levels) in distal colon mucosa.

dihydroxyphenylpropionic acid and 97.9% for 3,4-dihydroxyphenylacetic acid.

Result

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This journal is ª The Royal Society of Chemistry 2010

Activation of signalling pathways involved in cancer (MAPK cascades)

Yip et al.114

Effects on rotenone-induced apoptosis in PC12 cells

Protocatechuic acid

Cytotoxicity on HepG2 hepatocellular cells

Yip et al.114

Liu et al.116

Protocatechuic acid

Incubation with LNCaP prostate cell line, HCT116 colonic cell line, and IEC6; normal intestinal epithelial cell line

Gao et al.110

Protocatechuic acid

Hippuric acid

2,4,6-Trihydroxybenzoic acid 3-Hydroxyphenylacetic acid

3-Hydroxyphenylpropionic acid

0.1–1.0 mM

3–300 mmol/L

100 mmol/L (IC50 ¼ 60 mmol/L)

LNCaP: IC50 > 200 mM; HCT116: IC50 > 200 mM LNCaP: IC50 > 200 mM LNCaP: IC50 ¼ 135 mM; HCT116: IC50 ¼ 90 mM LNCaP: IC50 > 200 mM; HCT116: IC50 > 200 mM LNCaP: IC50 > 200 mM LNCaP: IC50 > 200 mM LNCaP: IC50 > 200 mM; HCT116: IC50 > 200 mM

3-Methoxy-4-hydroxyphenylacetic acid 4-Hydroxyphenylacetic acid 3,4-Dihydroxyphenylacetic acid

Concentration

Metabolite

Test

Reference

Table 2 (Contd. )

 Increase in cell viability by 71.15% at 1.0 mM.  Reduction by 12% in the total number of early apoptosis and late apoptosis/necrosis cells at 1.0 mM.  Significant supression of mitochondrial ROS, total glutathione, transmembrane potential, caspase-3-activity at 0.5 and 1.0 mM.

 Detectable activation of the JNK and p38 subgroups of MAPK in HepG2 hepatocellular carcinoma cells at 30 mmol/L; maximum activation was observed at 100– 300 mmol/L

 Cell viability was reduced by 70% at 100 mmol/L.  Dose-dependent cytotoxicity resulted in a IC50 ¼ 60 mmol/L.

 3,4-Dihydroxyphenylacetic acid exhibited anti-proliferative activity in prostate and colon cancer cells.  3,4-Dihydroxyphenylacetic acid was significantly more inhibitory in colon cancer cells (HCT116) compared with an immortalized normal intestinal epithelial cell line (IEC6).

 The activity of phase II enzyme GST was increased at 80 mg/kg.  Inhibition of hepatic NQO1 by 70% at 800 mg/kg  No effects on renal NQO1.

Result

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Food Funct., 2010, 1, 233–253 | 247

a ADP: adenosine 50 -diphosphate; DDS: dextran sodium sulfate; DPPH: 2,2,-diphenylpicrylhydrazyl; EROD: ethoxyresorufin-O-deethylase; JNK: c-Jun N-terminal kinase; GST: glutathione Stransferase; LPS: lipopolysaccharide; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinases; MROD: methoxyresorufin O-demethylase; NQO1: NAD(P)H:quinone oxidoreductase; ROS: reactive oxygen species; TRAP: thrombin-receptor-activating peptide. b Polyphenol mix 1: 1 mM dihydroferulic acid, 1 mM 3-(3-hydroxyphenyl)propionic acid, 1 mM homovanillic acid, 1 mM 3-hydroxyphenylacetic acid, 0.1 mM delphinidin-3-rutinoside, 0.1 mM cyanidin-3-rutinoside, 0.1 mM cyanidin-3-glucoside, 0.1 mM malvidin-3-glucoside.

 3,4-Dihydroxytoluene mimicked the effect of quercetin in primary rat hepatocytes, but much less so in HepG2 cells. EC50 ¼ 50 mM 3,4-Dihydroxytoluene

248 | Food Funct., 2010, 1, 233–253

Gl€aber et al.104

Modulation of lipid metabolism

Effects on cholesterol biosynthesis in cultured heptocytes and HepG2 cells by the incorporation of radiolabeled acetate into the fraction of nonsaponifiable neutral lipids

 Up-regulation of Bcl-2 gene family protein levels from 0.2–1.0 mM.

Concentration Metabolite Test Reference

Table 2 (Contd. )

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Result

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Interaction with cellular signalling pathways. In recent years, it has been suggested that polyphenols may exert their health effects via a mechanism of action beyond their antioxidant activity, and which is more related to its ability to generate an adaptive response at the cellular level that involves interaction with certain key proteins in triggering cell signalling pathways of oxidative stress and exposure to environmental toxins.9 In the case of flavan-3-ols, most studies have been performed mainly with the non-conjugated forms. It has been reported that EGCG induces apoptosis and causes cell-cycle arrest in tumor cells – but not in non-transformed normal cells – through the modulation of nuclear factor kappa-B (NF-kB). NF-kB is a redox-sensitive transcription factor which regulates the expression of proinflammatory cytokines, iNOS, COX-2, growth factors and inhibitors of apoptosis, and is related to inflammatory diseases (atherosclerosis, ulcerative colitis and rheumatoid arthritis), as well as neurodegenerative diseases and cancer.93,94 In another study, EGCG was also found to down-regulate NF-kB-inducing kinase (NIK), death-associated protein kinase (DPAK 1), and rho B and tyrosine protein kinase in PC-9 human lung cancer cells.95 A down-regulation of genes involved in a wide range of physiological functions was found in the mucosa of rats with induced colon carcinogenesis that had been fed wine polyphenols for 16 weeks, being the major pathways down-regulated those involved in the inflammatory response and steroid metabolism.96 With regards to genes involved in relevant process of atherosclerosis, red wine polyphenols were also found to significantly inhibit the proliferation of human vascular smooth muscle cells – but not of human vascular endothelial cells – by reducing the promoter activity and expression of the cyclin A gene.97 Green tea polyphenols have been shown to modulate the regulation of the transcriptional expression of proatherogenic molecules, including the sterol-response element binding protein (SREBP), PPAR-g, IL-8, and apoprotein-E.98 V.2

Bioactivity of microbe-derived phenolic metabolites

The biological activities of microbial metabolites derived from the catabolism of flavan-3-ols are still largely unknown, but in recent years those of hydroxyphenyl-g-valerolactones, and especially of phenolic acids (di- and mono- hydroxylated phenylproponic, phenylacetic, benzoic acids and derivatives) formed from the subsequent catabolism of the former, have started to be unravelled. In contrast to phase II or tissular metabolites derived from small-intestine and liver metabolism as described above, to date, in vitro studies performed with microbe-derived phenolic metabolites have been carried out with unconjugated metabolites (with the exception of hippuric acids) (Table 2). V.2.1 Hydroxyphenyl-g-valerolactones Antioxidant activity. The antioxidant activity of 5-(30 ,40 dihydroxyphenyl)-g-valerolactone and its methyl derivative 5-(30 -methoxy-40 -hydroxyphenyl)-g-valerolactone has been tested against superoxide radicals, as well as by the ferricreducing antioxidant potential (FRAP) test.99 In the radical scavenging test, 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone was more effective than (+)-catechin, ascorbic acid and trolox, whereas 5-(30 -methoxy-40 -hydroxyphenyl)-g-valerolactone did not exhibit antioxidant activity. In the reducing test, the order of This journal is ª The Royal Society of Chemistry 2010

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values was: 5-(3,4-dihydroxyphenyl)-g-valerolactone > (+)-catechin > ascorbic acid > 5-(3-methoxy-4-hydroxyphenyl)g-valerolactone.99

appeared more susceptible to the action of a series of microbial phenolic acids than Gram-negative bacteria, such as E. coli and P. aeruginosa.103

Anti-proliferative activity. 5-(30 ,40 ,50 -Trihydroxyphenyl)-gvalerolactone was more effective in the inhibition of the growth of a series of immortalized and malignant human cell lines than its trimethoxylated derivative, with the exception of HCT-116 colon cancer cells, and immobilized human (INT407) and rat (IEC-6) intestinal cells, which were not sensitive to the growth-inhibitory effects of this compound.100 5-(30 ,40 ,50 -Trihydroxyphenyl)-g-valerolactone was also more effective in the inhibition of the growth of colon (HT-29) and oesophagus (KYSE150) cancer cells than 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone and its mono- and di-methoxylated derivatives.100 However, the growth-inhibitory effects of this metabolite were lower than that of the aglycone, EGCG. Treatment of KYSE150 with 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone at 50 mM resulted in a 40% cell-growth inhibition after 48 h, whereas EGCG resulted in a 50% inhibition at 20 mM.100

Antioxidant activity. Among a series of microbe-derived phenolic acids, 3,4-dihydroxyphenylacetic and 3,4-dihydroxytoluene exhibited the highest radical scavenging activity against DPPH in cultured rat hepatocytes.104 However, only the latter metabolite was found to be effective against the lipid peroxidation of rat hepatocytes challenged with tert-butyl-hydroperoxide.104

Anti-inflammatory effects. The inhibition of NO production in murine macrophage cells (RAW264.7) by 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone and its trimethoxylated derivative has also been described.100 Whereas the former metabolite had IC50 ¼ 20 mM, the latter metabolite did not present any activity. However, none of the metabolites had inhibitory activity towards arachidonic acid metabolism in the same cell model.100 On the other hand, 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone and its methyl derivative 5-(30 -methoxy-40 -hydroxyphenyl)-gvalerolactone had similar inhibitory activity of the enzymatic activity of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9).99 Both metabolites also had similar efficacy in the inhibition of the secretion of MMP-9 from LPS-stimulated human monocytes.99 V.2.2 Phenolic acids Effects on intestinal microbiota. Some phenolic acids, including 3-O-methyl gallic, gallic, caffeic, 4-hydroxyphenylpropionic, phenylpropionic, and 4-hydroxyphenylacetic acids derived from the microbial degradation of tea catechins, were able to inhibit the growth of several pathogenic and nonbeneficial intestinal bacteria without significantly affecting the growth of beneficial bacteria (Lactobacillus spp. and Bifidobacterium spp.).101 Other studies have revealed that dihydroxylated forms (i.e. 3,4-dihydroxyphenylacetic and 3,4-dihydroxyphenylpropionic acids) efficiently destabilize the outer membrane of Salmonella.102 Recently, Cueva et al.103 found that the number and position of substitutions in the benzene ring of phenolic acids and the saturated side chain length influenced the antimicrobial potential of phenolic acids against different microorganisms (Escherichia coli, Lactobacillus spp., Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans), although it was strain-dependent. In general, non-hydroxylated and monohydroxylated phenolic acids were more potent than dihydroxylated or disubstituted phenolic acids. With regard to the saturated side chain, the order of potency, for the same benzene ring-substitution, was benzoic > phenylacetic > phenylpropionic acid. Moreover, Lactobacillus spp. and S. aureus (Gram-positive) This journal is ª The Royal Society of Chemistry 2010

Anti-thrombotic activity. Rechner et al.105 studied the effect of several microbe-derived phenolic acids and their mixture on platelet function through several tests, including: platelet aggregation, P-selectin expression on resting platelets, effect on TRAP-induced platelet activation and epinephrine-stressed platelets. Dihydrocaffeic acid (3,4-dihydroxyphenylpropionic acid), dihydroferulic acid (4-hydroxy-3-methoxyphenylpropionic acid) and 3-hydroxyphenylpropionic acid, as well as the polyphenol mixture, were among the metabolites with the best activity in all tests performed.105 Anti-inflammatory activity. Studies carried out by Karlsson et al.106 showed that faecal samples containing microbial phenolic acids affected cyclooxygenase-2 (COX-2) protein levels in colon cancer cells (HT-29) stimulated with TNF-a. Recently, Russell et al.107 reported that phenolic acids presenting 4-hydroxy-3-methoxy substitution and a one-carbon side chain such as vanillic acid and its derivatives (vanillin, vanillyl alcohol and acetovanillone), as well as a three-carbon side chain (cinnamic, o-, m- and p-coumaric acid, and caffeic acid), inhibited cytokine-induced prostanoid biogenesis in human colonic fibroblasts. A structure–activity relationship has been observed between phenolic acids and their anti-inflammatory effects, since only dihydroxylated phenolic acids (i.e. 3,4-dihydroxyphenylpropionic and 3,4-dihydroxyphenylacetic acids) significantly inhibited the production of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 in peripheral blood mononuclear cells (PBMC) stimulated with LPS, whereas no significant effect was found for the monohydroxylated ones.108 Similarly, Larrosa et al.109 recently found that these dihydroxylated phenolic acids provided the best inhibition of prostaglandin E2 production in cancer cells of fibroblast (CCD-18) stimulated with IL-1b. In vivo experiments with rats have also shown that 3,4-dihydroxypropionic acid was the most potent metabolite in writhing and paw pressure tests in rodents and reduced the expression of cytokines TNF-a, IL-1b, IL-8, as well as the levels of malonaldehyde and oxidative damage to DNA in the distal mucosa of rats with dextran sodium sulfate (DSS)-induced colitis.109 Anti-proliferative activity and cytotoxicity. Among a series of microbial phenolic metabolites, 3,4-dihydroxyphenylacetic acid presented anti-proliferative activity in prostate (LNCaP) and, in particular, in colon cancer (HCT116) cells.110 In vivo studies have also revealed that protocatechuic acid reduces the incidence and multiplicity of cancerous tumors in the colon of rats.111 The modulation of cytochrome P450 and enzymes involved in xenobiotic activation and/or detoxification pathways (phase II Food Funct., 2010, 1, 233–253 | 249

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enzymes) by protocatechuic acid in mouse liver and kidney has also been reported.112 Moreover, protocatechuic acid affected the level of rat hepatic and renal glutathione S-transferase (GST) isoenzymes.113 Cytotoxicity assays have also shown that protocatechuic acid effectively kill the HepG2 hepatocellular carcinoma cells by stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family.114 A similar signalling pathway has been reported to be involved in the apoptosis of human gastric adenocarcinoma cells by protocatechuic acid.115 In a recent study, protocatechuic acid has also shown significant neuroprotective effects on retenoneinduced apoptosis in PC12 cells by ameliorating the mitochondrial dysfunction.116 Modulation of lipid metabolism. It has been reported that 3,4dihydroxytoluene acid inhibits the synthesis of heptocellular cholesterol by inhibiting the incorporation of acetate into HepG2 liver cells.104

VI. Concluding remarks Over the last decade, a large number of epidemiological and interventional studies have demonstrated that there may be an association between flavonoid consumption and human health. Mechanistic studies trying to determine flavan-3-ol health effects have revealed that these polyphenols exhibit a wide range of biological effects. Despite the enormous effort devoted to this area, some results may be misleading, since polyphenol metabolism as xenobiotics has not been considered in a large number of studies which employed structural forms and concentration ranges not found in vivo. Therefore, polyphenol bioavailability is a key issue in the link between polyphenol and human health. In comparison to other micronutrients, knowledge about polyphenol bioavailability is advancing with the progress of analytical instrumentation which allows the identification of new metabolites in vivo. The recognition that some polyphenols, in particular proanthocyanidins, are extensively metabolized by the intestinal microbiota into low molecular weight compounds, and that these metabolites represent a very large percentage of the amount ingested, is bringing into consideration the inclusion of microbial metabolism as part of the bioavailability concept currently adopted for polyphenols. On the basis of these facts, interest is now focused on the study of the bioactivity of microbe-derived metabolites, in addition to phase II or tissular metabolites, as compounds responsible for the health effects of flavan-3-ols. Although advances are being made in the determination of the bioactivity of microbe-derived metabolites, most studies carried out until now have failed, again by not testing the conjugated forms found in vivo. With regards to the bioactivity of actual conjugated forms derived from flavan-3-ol in vivo metabolism, research carried out in the last decade has revealed that flavan-3-ols are multifunctional compounds that may display effects by mechanism(s) of action beyond their antioxidant activity. The health effects derived from the interaction between flavan3-ols and the intestinal microbiota should be a subject of increasing interest. Although some authors have pointed out that polyphenols may be beneficial to gut health by increasing the population of potentially beneficial bacteria or exerting prebiotic 250 | Food Funct., 2010, 1, 233–253

actions, the effects that the interaction between flavan-3-ols and intestinal microbiota may have on the functionality of the metabolic activity of the microbiota and overall gastrointestinal health still remains largely unknown. In fact, for flavan-3-ols to function as a prebiotic, intestinal bacteria with such metabolic capacity should exist in the colon, but they are difficult to identify due to direct or indirect factors inherent in flavan-3-ols. The identification of flavan-3-ol-metabolizing bacteria and their possible use as a probiotic could be a good strategy for increasing the bioavailability and potential bioactivity of proanthocyanidins.

Acknowledgements This work has been funded by the Spanish Ministry for Science and Innovation (AGL2009-13361-C02-01 and CSD2007-00063 Consolider Ingenio 2010 FUN-C-FOOD Projects), and the Comunidad de Madrid (ALIBIRD P2009/AGR-1469 Project). F.S.-P. is the recipient of a contract from the JAE-Doc Program (CSIC). I.G. was the recipient of a fellowship from the I3P Program funding by the European Social Fund. M.U-S. thanks the FPI fellowship from MICINN. R.L. thanks the ‘‘Fondo de Investigaci on Sanitaria’’ post-doctoral program (F.I.S. CD06/ 00161) from MICINN.

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PAPER

www.rsc.org/foodfunction | Food & Function

Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds Wasaporn Chanput,abd Jurriaan Mes,c Robert A. M. Vreeburg,c Huub F. J. Savelkoula and Harry J. Wichers*abc

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Received 9th August 2010, Accepted 11th October 2010 DOI: 10.1039/c0fo00113a An assay was developed to study inflammation-related immune responses of food compounds on monocytes and macrophages derived from THP-1 cell line. First strategy focused on the effects after stimulation with either lipopolysaccharide (LPS) or Concanavalin A (ConA). Gene expression kinetics of inflammation-related cytokines (IL-1b, IL-6, IL-8, IL-10 and TNF-a), inflammation-related enzymes (iNOS and COX-2), and transcription factors (NF-kB, AP-1 and SP-1) were analyzed using RT-PCR. Time dependent cytokine secretion was investigated to study the inflammation-related responses at protein level. LPS stimulation induced inflammation-related cytokine, COX-2 and NF-kB genes of THP-1 monocytes and THP-1 macrophages with the maximum up-regulation at 3 and 6 h, respectively. These time points, were subsequently selected to investigate inflammation modulating activity of three well known immuno-modulating food-derived compounds; quercetin, citrus pectin and barley glucan. Co-stimulation of LPS with either quercetin, citrus pectin, or barley glucan in THP-1 monocytes and macrophages showed different immuno-modulatory activity of these compounds. Therefore, we propose that simultaneously exposing THP-1 cells to LPS and food compounds, combined with gene expression response analysis are a promising in vitro screening tool to select, in a limited time frame, food compounds for inflammation modulating effects.

Introduction Monocytes and macrophages are an important part of the innate arm of the immune system. These cells are involved in inflammatory processes, with a profound capacity to synthesize and secrete pro- and anti-inflammatory cytokines.1,2 THP-1 cells, a human leukemia monocytic cell line, have widely been used as a model to study the immune response capacity of monocytes and monocyte-derived macrophages, because of similarities in their responses when compared to the monocyte fraction present in peripheral blood mononuclear cells (PBMCs).3,4 The innate immune system relies on pathogen recognition receptors (PRRs) such as toll-like receptors (TLRs), to recognize conserved molecular structures of invading pathogens called pathogen associated molecular patterns (PAMPs). PAMPs, like lipopolysacharide (LPS), play a pivotal role in the initiation of a variety of host responses caused by infection with Gramnegative bacteria. Such action leads to systemic inflammatory response, for instance up-regulation of pro-and anti-inflammatory cytokine genes, resulting in secretion of cytokine proteins a Cell Biology and Immunology Group, Wageningen University and Research Centre, Marijkeweg 40, P.O. Box 338, 6700 AH Wageningen, The Netherlands. E-mail: [email protected] b Laboratory of Food Chemistry, Wageningen University and Research Centre, Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen, The Netherlands c Food & Biobased Research, Wageningen University and Research Centre, Bornse Weilanden 9, P.O. Box 17, 6700 AA Wageningen, The Netherlands d Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, 50 Phahonyothin Road, Chatuchak, Bangkok, 10900, Thailand

254 | Food Funct., 2010, 1, 254–261

into the blood stream.5,6 Some transcription factors have been shown to be directly or indirectly related to the receptor-mediated expression of inflammation related-cytokine and inflammation-related enzyme genes. The transcription factor Nuclear Factor (NF)-kB is involved in the transcriptional regulation of the IL-1b, IL-6, IL-8, TNF-a, iNOS and COX-2 genes.7,8 The transcription factor AP-1 is associated with the regulation of IL-8 and TNF-a genes.7,9 Activation of the AP-1 transcription factor occurs by an increased the production of c-Jun and c-Fos proteins which need to form a dimeric complex binding the promoter region of AP-1.10 Expression of the IL-10 gene is regulated by SP-1 transcription factor.11,12 Immune responses are commonly determined by measuring the presence of cytokines in culture medium after challenging cells. However, these assays are often performed with relatively long exposure time (generally hours) in order to obtain cytokine levels above the threshold which can lead to the initiation of further signalling pathways. Furthermore, cytokine protein secretion is only partly related to the expression of cytokinerelated genes and their upstream transcription factors, because of extensive regulation of the transcription and translation processes.13,14 We, therefore, investigated the gene expression kinetics of inflammation-related cytokines, inflammationrelated enzymes and relevant transcription factors, together with time-dependent cytokine protein secretion. Genetically identical THP-1 monocytes and macrophages, which were stimulated with LPS and ConA, were used as a model. Furthermore, the application of this assay for screening immuno-modulatory effects of food-derived components was tested and discussed. This journal is ª The Royal Society of Chemistry 2010

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Results

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Gene expression kinetics of THP-1 monocytes and macrophages stimulated with LPS and ConA Expression of genes for inflammation-related cytokines. Proand anti-inflammatory cytokine gene expression was analyzed in undifferentiated and differentiated THP-1 cells, designated as THP-1 monocytes and THP-1 macrophages, respectively. In this analysis, mRNA expression level of the pro-inflammatory cytokine genes IL-1b, IL-6, IL-8 and TNF-a, and of the antiinflammatory cytokine gene IL-10 were determined by qPCR. In both non-stimulated THP-1 monocytes and macrophages at time zero, IL-8 was the most abundant gene relative to GAPDH, followed by IL-1b and TNF-a genes, respectively (Fig. 1). IL-6 and IL-10 exhibited low abundance in both cell types. All cytokine genes were higher expressed in non-stimulated THP-1 macrophages than in monocytes (Fig. 1). THP-1 cells, like primary monocytes and macrophages, expressed a variety of inflammation-related cytokine genes in response to LPS (Fig. 2A, C). The IL-6 gene showed, among the analyzed cytokine genes, the highest fold change of expression in both THP-1 monocytes and macrophages. Exposure of THP-1 monocytes to LPS strongly induced IL-1b, IL-6, IL-8, IL-10 and TNF-a gene expression, with maximal expression after 3 h of stimulation, except for IL-6 expression which gradually increased throughout the incubation time (Fig. 2A). All inflammation-related cytokine genes of THP-1 macrophages were also highly up-regulated by LPS-stimulation but less than those of monocytes, except IL-6, with a maximal expression after 6 h of stimulation (Fig. 2C). The onset of up-regulation of all analyzed inflammation-related cytokine genes appeared to be earlier in THP-1 monocytes (within 1 h) compared to that of macrophages (within 2 h) (Fig. 2A, C). In all experiments, controls were performed using non-stimulated cells (medium). ConA was chosen as a negative or weak stimulus of the innate immunity. Non-stimulated THP-1 monocytes and macrophages and those-stimulated with ConA showed no or very low effects on gene expression of the inflammation-related cytokine genes (Fig. 3A, C).

Fig. 1 Inflammation-related cytokine gene expression relative to GAPDH (DCt) of THP-1 monocytes and macrophages before stimulation. Data shown are the means + standard deviation (SD bars) from two independent biological replications.

This journal is ª The Royal Society of Chemistry 2010

Expression of genes for inflammation-related enzymes. Apart from inflammation-related cytokine genes, two candidate inflammation-related enzyme genes, COX-2 and iNOS, were also investigated in THP-1 monocytes and macrophages stimulated with LPS and ConA. Incubation of THP-1 monocytes with LPS led to an induction of COX-2 gene expression within 0.5 h and reached the highest expression after 3 h of stimulation (Fig. 4A). For THP-1 macrophages, the first up-regulation of the COX-2 gene was observed after 2 h and the highest expression was after 6 h of stimulation (Fig. 4B). The overall COX-2 gene expression in LPS-stimulated THP-1 macrophages was approximately 10 times higher than that of monocytes. ConA only weakly induced COX-2 gene expression in THP-1 monocytes (data not shown). In contrast to the inflammation-related cytokine genes, the expression level of the COX-2 gene relative to GAPDH (DCt) in LPS-stimulated THP-1 monocytes and macrophages was similar (data not shown). The expression of the iNOS gene could not be detected in both LPS-and ConA-stimulated THP-1 monocytes and macrophages throughout the incubation time, although two different iNOS primer sets in Table 1 have been used (data not shown). Expression of genes for inflammation-related transcription factors. LPS caused considerable up-regulation of NF-kB gene expression in both THP-1 monocytes and macrophages up to 30 h with the highest expression after 3 h and 6 h of stimulation, respectively (Fig. 4). c-Jun and c-Fos gene expression in LPSstimulated THP-1 macrophages was up-regulated for a rather short period 0.5–3 h after stimulation. A bi-phasic pattern was found in c-Fos gene expression of THP-1 macrophages treated with LPS (Fig. 4B). SP-1 gene expression was not affected by LPS stimulation in both THP-1 monocytes and macrophages (Fig. 4). No up-regulation from any of the studied transcription factor genes was observed in ConA-stimulated and non-stimulated THP-1 monocytes and macrophages (data not shown). Time-dependent cytokine secretion of THP-1 monocytes and macrophages stimulated with LPS and ConA Stimulating THP-1 monocytes and macrophages with LPS resulted in a dramatic increase in the secretion of IL-1b, IL-6, IL8, IL-10 and TNF-a (Fig. 2B, 2D). At time zero of THP-1 monocytes and macrophages, all cytokines were present at a basal level of 20 to 30 pg ml1. Inflammatory cytokine production by THP-1 macrophages was higher than in monocytes, likely as a consequence of higher abundance of inflammation-related cytokine genes relative to GAPDH in nonstimulated THP-1 macrophages than in monocytes, as described above (Fig. 1). IL-8 was the most predominant cytokine in the supernatant of both THP-1 monocytes and macrophages (Fig. 2B, D). IL-8 concentration in the supernatant increased over time and only in THP-1 macrophages reached a plateau within the tested time frame after 18 h of stimulation. The relative order in abundance of cytokines secreted from THP-1 monocytes and macrophages was similar to the order of their responsive genes relative to GAPDH (Fig. 1), except for IL-1b from THP-1 monocytes, of which the expression was relatively close to that of the IL-8 gene, but not at protein level. All analyzed cytokines, except for Food Funct., 2010, 1, 254–261 | 255

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Fig. 2 Inflammation-related cytokine gene expression kinetics and cytokine secretion kinetics of THP-1 monocytes (A-B) and THP-1 macrophages (C-D) stimulated with 1 mg ml1 LPS. Gene expression was expressed as relative gene expression towards GAPDH-expression and non-stimulated cells at time zero (DDCt). Data shown from RT-PCR are the means + standard deviation (SD bars) from two independent biological replications.

Fig. 3 Inflammation-related cytokine gene expression kinetics and cytokine secretion kinetics of THP-1 monocytes (A-B) and THP-1 macrophages (C-D) stimulated with 5 mg ml1 ConA and non-stimulated (medium). Gene expression was expressed as relative gene expression towards GAPDHexpression and non-stimulated cells at time zero (DDCt). Data shown are the means from two technical measurements.

TNF-a from monocytes and IL-10 from macrophages, continued to accumulate over the incubation time (Fig. 2B, D). Cytokines secreted from ConA-stimulated THP-1 monocytes 256 | Food Funct., 2010, 1, 254–261

and macrophages were detected in relatively low amounts (Fig. 3B, D), except IL-8 from ConA-stimulated and non-stimulated THP-1 macrophages (Fig. 3D). This journal is ª The Royal Society of Chemistry 2010

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Fig. 4 Inflammation-related enzyme and transcription factor gene expression kinetics of THP-1 monocytes (A) and THP-1 macrophages (B) stimulated with 1 mg ml1 LPS. Gene expression was expressed as relative gene expression towards GAPDH-expression and non-stimulated cells at time zero (DDCt). Data shown are the means + standard deviation (SD bars) from two independent biological replications.

These results suggest that the RNA expression and protein secretion are correlated to a large extent. The lag-phase in upregulation of the mRNA level was approximately 1 h before cytokine proteins were secreted at a detectable concentration. Cytokine production demonstrated more variable kinetics than the expression of corresponding genes, which results in less uniform time points in quantification of effects.

Modulating effects of quercetin, citrus pectin and barley glucan on the expression of inflammation-related genes in LPSstimulated THP-1 monocytes and macrophages The effect of co-stimulation of LPS and purified food compounds was determined after stimulation at 3 h and 6 h of THP-1 monocytes and macrophages respectively, since the maximal gene expression response was observed at these time points (Fig. 2A, C). Quercetin, citrus pectin and barley glucan, were chosen as food-derived compounds. Exposure of the cells to quercetin, citrus pectin and barley glucan or the solvent control (DMSO) and PBS did not show any altered expression of the measured genes (data not shown), indicating that these compounds do not posses direct inflammation-enhancing properties. Data represented in Fig. 5 show the effect of quercetin, citrus pectin and barley glucan on modulation of LPS-induced responses. Quercetin, citrus pectin and barley glucan lowered the LPS-induced expression of most inflammation-related genes expressed by THP-1 monocytes, except TNF-a and COX-2 (Fig. 5A). Different effects were observed from THP-1 macrophages as compared to monocytes for specific food components. After 6 h of stimulation, quercetin reduced expression of all inflammation-related genes of LPS-stimulated THP-1 macrophages, except COX-2 (Fig. 5B). Less inflammatory reducing effects were found for citrus pectin compared to quercetin in THP-1 macrophages. Barley glucan appeared to enhance expression of inflammation-related genes of LPS-stimulated THP-1 macrophages (Fig. 5B) whereas in monocytes it had a reducing effect. Our results indicate that gene expression after simultaneous exposure of LPS with food components to THP-1 monocytes for 3 h and THP-1 macrophages for 6 h is an adequate model to examine inflammation-modulating activity of food compounds. This journal is ª The Royal Society of Chemistry 2010

Discussion In this study, a new test method to investigate immuno-modulating effects of food-derived compounds was developed based on the THP-1 cell line. Two stimuli were chosen with different actions on the cellular responses. LPS is widely used as a potent and prototypical inducer of cytokine production in innate immunity which begins with the orchestration of monocytes.7,15 ConA, a lectin from jack-bean (Canavalia ensiformis), was reported to function as a T-lymphocyte mitogen in adaptive immunity,16 and to control some non-immune responses of THP1 monocytes such as growth, proliferation, metabolism and survival processes.17 Differences in RNA stability, protein translation kinetics, post-translational modification factors and proteolytic processing events, make the production of individual cytokines unique.18 However, a general relation between mRNA and protein level in both LPS-stimulated THP-1 monocytes and macrophages was found in our studies. The higher mRNA expression level relative to GAPDH of inflammation-related cytokine genes and higher cytokine secretion level of LPS-stimulated THP-1 macrophages, as compared to monocytes, could be caused by higher expression of TLR4-mRNA (a PRR for LPS).19 LPS strongly up-regulated inflammation-related cytokine, COX-2 and NF-kB genes in THP-1 monocytes and macrophages, while no expression of the iNOS gene after LPS-stimulation was found. This last finding was in concordance with studies making use of PBMCs, indicating that human peripheral monocytes and their derived macrophages are not able to express the iNOS gene after LPS induction.20–23 However, some PBMCbased studies indicated the ability of LPS to up-regulate the iNOS gene.24,25 No unequivocal explanation for this controversy has been presented yet. Based on literature, it seems that species differences, genetic background, and perhaps details of experimental procedure play a role.7,20–25 The relatively low and short expression of c-Jun and c-Fos in LPS-stimulated THP-1 monocytes and macrophages can be explained by the fact that these genes are typical early response genes with a very short mRNA half life of only 35–45 min.26,27 A bi-phasic pattern of c-Fos gene expression in LPS-stimulated THP-1 macrophages was also reported.26,27 The binding activity of nuclear proteins to SP-1 target genes was constitutive and unchanged by LPS stimulation in THP-1 monocytes and murine macrophages11,28 but it can be Food Funct., 2010, 1, 254–261 | 257

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Table 1 Sequence of Real Time-PCR primers

Gene

Accession number

Primer working concentration/ mM

IL-1b

NM_000576.2

0.1

IL-6

NM_000600.3

0.1

IL-8

NM_000584.2

0.1

IL-10

NM_000572.2

0.4

TNF-a

NM_000594.2

0.1

iNOS (1)

NM_000625.3

0.1

iNOS (2)

NM_000625.4

0.1

COX-2

NM_000963.2

0.1

NF-kB

NM_003998.2

0.4

SP-1

NM_138473.2

0.4

c-Jun

NM_002228.3

0.4

c-Fos

NM_005252.3

0.4

GAPDH

NM_002046.3

0.1

Sequence (50 /30 ) F- GTGGCAATGA GGATGACTTGTTC R- TAGTGGTGGT CGGAGATTCGTA F- AGCCACTCAC CTCTTCAGAAC R- GCCTCTTTGC TGCTTTCACAC F- CTGATTTCTGC AGCTCTGTG R- GGGTGGAAAG GTTTGGAGTATG F- GTGATGCCC CAAGCTGAGA R- CACGGCCTT GCTCTTGTTTT F- CTGCTGCACT TTGGAGTGAT R- AGATGATCTG ACTGCCTGGG F- CATCCTCTTT GCGACAGAGAC R- GCAGCTCAGC CTGTACTTATC F- GGCTGGAA GCCCAAGTACG R- CTCAGGGTC ACGGCCATTG F- CAGCACTTC ACGCATCAGTT R- CGCAGTTTAC GCTGTCTAGC F- TGAGTCCTG CTCCTTCCA R- GCTTCGGTG TAGCCCATT F- GGTGCCTTT TCACAGGCTC R- CATTGGGTGAC TCAATTCTGCT F- TGGAAACGA CCTTCTATGACGA R- GTTGCTGGAC TGGATTATCAGG F- GGATAGCCTC TCTTACTACCAC R- TCCTGTCATG GTCTTCACAACG F- TGCACCACCA ACTGCTTAGC R- GGCATGGACT GTGGTCATGAG

up-regulated during the PMA or vitamin D3 induced differentiation process of THP-1 monocytes to regulate the expression of CD14.29,30 Therefore, it could be argued that SP-1 might not be an appropriate gene to serve as an indicator in LPS exposure of THP-1 monocytes and macrophages. Accumulation of TNF-a in supernatants of both THP-1 monocytes and macrophages declined after 6 and 24 h of stimulation, respectively. The decline of TNF-a-accumulation in our studies was consistent with the findings in several studies which demonstrated that IL-10 can suppress TNF-a production in 258 | Food Funct., 2010, 1, 254–261

human monocytes and macrophages, or even cause diminished levels of TNF-a, IL-1b and IL-8 mRNA upon prolonged stimulation.31–33 However, to drive such mechanisms, IL-10 needs to bind to IL-10R-1 and IL-10R-2 which should cause a decrease in the measurable (unbound) amount of IL-10 in culture supernatants.34,35 Similar to our results, IL-10 cytokine accumulation by LPS-stimulated THP-1 monocytes slightly dropped at 6 h and increased again at 18 h after stimulation, while it dramatically decreased in THP-1 macrophages at 18–30 h after stimulation. The beneficial health effects of quercetin and citrus pectin have been attributed to their anti-inflammatory activity,36–39 while b-glucans show their immunological effect by enhancing innate immunity through induction of cytokine production and phagocytosis.40 Incubating LPS-stimulated THP-1 monocytes for 3 h and macrophages for 6 h with the food compounds revealed different inflammation-modulating effects at mRNA level with similarity as described in the mentioned literature. Studying effects of PAMPs and other (food based) immunomodulating compounds using monocytes and macrophages isolated from PBMCs might be a more realistic model for human immune functioning. However, large variation between blood samples, time and cost efficiency make it difficult to apply this in a high throughput fashion. It has been indicated that the THP-1 cell line has shown to be an accurate model for native and monocytes-derived macrophages for studying LPS responses.3,4,41,42 Our findings suggested that LPS-stimulated THP-1 monocytes and macrophages are a sensitive in vitro system to analyze potential immunomodulatory activity of food components by using a detailed insight into the kinetics of mRNA expression. Therefore, THP-1 monocytes and macrophages could thus be a suitable and reliable model for screening a variety of components prior to a more detailed analysis with human derived cells.

Materials and methods Chemicals and cell culture The human monocytic leukemia cell line THP-1 (American Type Culture Collection, Rockville, Md.) was grown in RPMI 1640 culture medium (Lonza, Switzerland) supplemented with fetal bovine serum (FBS; Invitrogen, UK.) and penicillin/streptomycin (P/S) (Invitrogen) to 10% and 1% respectively, at 37  C in 5% CO2 in a humidified incubator. Cells were sub-cultured twice per week. THP-1 cells change their culture properties after prolonged periods in culture, cells were therefore discarded and replaced by frozen stocks after 25 passages. LPS (E.coli 0111:B4), Concanavalin A (ConA), quercetin, citrus pectin and barley glucan were purchased from Sigma (St. Louis, MO, USA), the latter three of the highest possible purity grade to respectively 99%, $74% galacturonic acid and >95%. Macrophage differentiation and stimulation The mature macrophage-like state was induced by treating THP1 monocytes (106 cells ml1) for 48 h with 100 ng ml1 phorbol 12myristate 13-acetate (PMA; Sigma Chemical) in 12-wells cell culture plates (Greiner, Germany) with 1 ml cell suspension in each well. It has been demonstrated that this differentiation method of THP-1 cells resulted in the expression of macrophage This journal is ª The Royal Society of Chemistry 2010

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Fig. 5 Inflammation-related cytokine gene expression of 700 ng ml1 LPS-stimulated THP-1 monocytes (A) and macrophages (B) with 50 mM quercetin (LPS + Q), 0.75 mg ml1 citrus pectin (LPS + CP) and 100 g ml1 barley glucan (LPS + BG). THP-1 monocytes were collected at 3 h and macrophages at 6 h after stimulation. Gene expression was expressed as relative gene expression towards GAPDH-expression and non-stimulated cells at time zero (DDCt). Data shown are the means + standard deviation (SD bars) from independent biological replications.

specific surface markers CD11b and CD36 and also phagocytic activity.43 Differentiated, plastic-adherent cells were washed once with sterile phosphate-buffered saline (PBS; Sigma Chemical, USA) and RPMI 1640 medium without PMA but containing This journal is ª The Royal Society of Chemistry 2010

10% FBS and 1% P/S. THP-1 monocytes (undifferentiated cells) and THP-1 macrophages (differentiated cells) were treated with ether 1 mg ml1 LPS or 5 mg ml1 ConA, which concentrations were chosen according to our preliminary optimization studies. Food Funct., 2010, 1, 254–261 | 259

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RPMI 1640 medium containing 10% FBS and 1% P/S was used as a control. Both types of cells were harvested at different time points ranging from 0–30 h to investigate gene expression kinetics, while cell-free culture supernatants were collected and stored at 80  C to measure time-dependent cytokine secretion. The experiments were performed by two independent biological replications, started from a new batch of cells.

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Gene expression kinetics by Real-Time PCR Total RNA was isolated by using RNeasy mini kit (Qiagen, USA) with a RNase-free DNase (Qiagen) treatment for 15 min according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from isolated RNA with iScript cDNA synthesis kit (Bio-Rad, USA). Of the synthesized cDNA 200 ng was mixed with 10 ml of IQ SYBR Green Supermix (Bio-rad) and primer pairs in a 20 ml reaction volume and preheated at 95  C for 90 s, followed by PCR for 40 cycles, denaturing temperature of 95  C for 10 s, annealing temperature of 58  C for 10 s, and elongation temperature of 72  C for 15 s, and finally elongation temperature of 72  C for 2 min. Primer sets (see Table 1) were tested by dilution series of cDNA from LPSstimulated THP-1 monocytes to analyze PCR efficiency. Amplified PCR products were also analyzed by ethidium bromide stained agarose gel to check for amplification of a single product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen for normalisation because this gene was stably expressed in both THP-1 monocytes and macrophages, both in challenged and unchallenged cells (data not shown). The PCR products of all samples were subjected to a melting curve analysis to verify the single amplification product. The relative messenger RNA (mRNA) expression were exhibited in two ways; firstly, the expression relative to GAPDH, calculated as DCt [DCt ¼ 2^(CtGAPDH-Ctsample)].44 Secondly, the values expressed as fold change relative to the value at time point zero, calculated as DDCt [DDCt ¼ 2^(DCt GAPDH- DCt sample)].44 All experiments were performed with the same amount of cells (106 cells per ml) and the same quantity of RNA input. qPCR was performed twice on each sample.

Food-derived compounds on LPS-stimulated cells THP-1 cells were stimulated with either 50 mM quercetin, 0.75 mg ml1 citrus pectin, or 100 mg ml1 barley glucan, with and without the presence 700 ng ml1 LPS. The LPS concentration was reduced from 1 mg ml1 to 700 ng ml1 to achieve a lower upregulation of inflammation-related genes. Expression kinetics of inflammation-related genes with 700 ng ml1 LPS were similar to 1 mg ml1 LPS, but with a reduced amplitude (data not shown). The concentrations of food compounds were chosen according to preliminary optimization studies and literature search.39,45,46 After stimulation, THP-1 monocytes and macrophages were harvested at 3 h and 6 h, respectively. Expression of inflammation-related genes was determined. The experiments were performed by two independent biological replications, started from a new batch of cells.

Conclusion A rather short incubation time is required for gene expression analysis, this approach facilitates the use of less sterile samples and allows a more reliable read-out for the early triggering events in which responses by various effector molecules such as cytokines and post-translational events have not yet occurred. This makes it possible to differentiate between primary food-cell signals and secondary cell-cell signals. RT-PCR is a method to analyze gene expression, which is very straight forward, can be performed in almost every molecular lab, is cost-effective compared to microarray analysis, and can be very accurate and informative when using a key selection of functional indicator genes. Analysing cytokine secretion profiling has some major drawbacks compared to gene expression analysis as longer incubation is needed to be detectable resulting in more uncertainties due to occurrence of forward and backward responses from various secreted cytokines. Also, every run needs many extra samples for calibration curves. These remarks together with the findings described in this paper show that gene expression measurement can give reproducible results and even on a wider spectrum of responses than cytokine measurements.

Time-dependent cytokine secretion by cytometric bead array

Acknowledgements

Cytokine secretion (IL-1b, IL-6, IL-8, IL-10 and TNF-a) in cellfree culture supernatants of THP-1 monocytes and macrophages was measured by cytometric bead array (CBA) analysis (BD Biosciences, USA) according to the manufacturer’s guidelines. Briefly, a mixture of 5 capture bead populations (50 ml) with distinct fluorescence intensities coated with capture antibody proteins for the above mentioned cytokines was first mixed with each sample or standard (50 ml) and incubated in the dark for 1 h on an orbital shaker. Additionally, PE-conjugated detection antibodies (50 ml) were added to form sandwich complexes and then incubated in dark for 2 h on an orbital shaker. Subsequently, the samples were resuspended in 200 ml of wash buffer before acquisition on a FACScan cytometer (BD Bioscience). The sample results were analyzed using FCAP Array analysis software. The measurement was performed twice from one of two biological replicates.

This work was supported by the Thai Commission on Higher Education, Ministry of Education, Thailand.

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PAPER

www.rsc.org/foodfunction | Food & Function

Caffeoylquinic acid derived free radicals identified during antioxidant reactions of bitter tea (Ilex latifolia and Ilex kudincha) Katharina Franziska Pirkera and Bernard Albert Goodman*ab

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Received 19th July 2010, Accepted 5th October 2010 DOI: 10.1039/c0fo00085j In order to provide some insight into the chemical basis for the antioxidant behaviour of bitter tea, the Chinese medicinal beverage derived from leaves of Ilex kudincha or Ilex latifolia, free radicals generated during the oxidation of aqueous extracts of dried leaves have been investigated by electron paramagnetic resonance (EPR) spectroscopy. With both beverages, the major components in the EPR spectra after accelerated autoxidation under alkaline conditions or oxidation with the superoxide anion radical were comparable to those derived from reactions of caffeoylquinic acids. Thus these reaction products have sufficient stability for biological activity, and the present results suggest that such molecules contribute appreciably to the antioxidant chemistry of these beverages.

1. Introduction ‘‘Kudingcha’’ or ‘‘bitter tea’’ is a traditional Chinese beverage. It is derived from leaves of the tree Ilex kudincha and has been consumed for centuries in southern China as an alternative to green tea. Similar products may be produced from other members of the Ilex family, e.g. bitter tea from I. latifolia, in southern China and Vietnam, and Yerba Mate from leaves and twigs of I. paraguariensis in several South American countries. In traditional Chinese medicine, several beneficial effects have been associated with kudingcha. These have been summarised by Dharmananda1 and include treatments for the common cold, rhinitis, itching eyes, red eyes, bronchitis, and headache, as well as alleviation of thirst associated with fever or severe diahorrea. Other beneficial effects include preventing the deterioration of the heart and brain functions, and improved digestion. Ahai2 reports uses of kudingcha for relief of hypertension and weight loss, as a diuretic, and as a remedy for sore throats. Triterpene glycosides (saponins) are considered to be the main active components of extracts from Ilex species, and there have been considerable efforts to isolate and identify the various triterpenes and saponins from leaves of I. kudincha,3–6 I. latifolia,7,8 and I. paraguariensis.9,10 However, in view of the wide range of medicinal properties associated with beverages derived from the Ilex species, it is unlikely that their beneficial effects are derived solely from one group of components, such as the saponins. Leaves of the Ilex species contain polyphenols,11,12 which might also be expected to contribute to their biological properties, analogous to the association of such molecules with the beneficial effects of green tea consumption.13 With green tea, it is generally assumed that these beneficial effects of the beverage are

a Health and Environment Department, Environmental Resources & Technologies, Austrian Institute of Technology, A-2444 Seibersdorf, Austria. E-mail: [email protected]; katharina.pirker@ait. ac.at; Fax: +(43) 50550 3520; Tel: +(86) 771 327 2475 b Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning, 520004, Guangxi, China; Fax: +(43) 50550 3434; Tel: +(86) 771 323 7873

262 | Food Funct., 2010, 1, 262–268

associated with the antioxidant (free radical scavenging) properties of its constituent polyphenols,14–17 and recent papers concerning polyphenols in kudingcha have focused on measurements of their overall antioxidant activity and the use of chemical assays to identify individual polyphenol molecules.11,12 However, although such measurements are of undoubted value, they represent just one step towards understanding the chemical behaviour of the beverages, which in turn is just one step towards understanding their biological properties. Although antioxidant assays provide an overall measure of a particular chemical property, they give little information as to the chemical nature of the individual molecules responsible for the activity, and no information on the behaviour of oxidation products which are formed during antioxidant reactions. Identification of components that might feature in antioxidant reactions is provided by chemical analyses, but such measurements give no information on the chemical behaviour of such molecules in a complex mixture, i.e. a beverage, where reactions between individual components could have a major impact on the overall chemistry. Indeed it is one of the principles of traditional medicines that the activity is often dependent on the presence of many molecules, and not on a single molecular species as is the case with pharmaceutical products. The biological properties of traditional medicinal products, however, are likely to be determined to a major extent by molecules with sufficient stability to be transported within the organism, but also with sufficient reactivity to react selectively with specific molecules, cells or tissue types. Thus the next logical step in developing an understanding of the chemical basis for the biological properties of beverages is to identify components or products derived from beverage components that possess such intermediate reactivity. Semiquinone radicals produced by the oxidation of polyphenols are candidates for such behaviour, and measurements made with green tea18 indicate that they have appreciable stability in the complex medium of the beverage. Indeed there have been several recent suggestions that epigallocatechin gallate, the major polyphenol in green tea, could be used as an individual medicine or nutritional supplement for treating a wide range of diseases or disorders, such as cancer,19 This journal is ª The Royal Society of Chemistry 2010

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atherosclerosis,20 blood sugar control,21 and neurodegenerative diseases.22 The principal objective of the present paper was to gain further insight into the oxidative chemistry of components in bitter teas in order to develop further our knowledge of the chemical basis for the biological effects of the beverages. Therefore, we investigated the extent to which free radical generation analogous to that observed during oxidation of green tea might occur in aqueous extracts of I. kudincha and I. latifolia. These measurements were based on the use of electron paramagnetic resonance (EPR) spectroscopy, a physical technique that can specifically characterise paramagnetic molecules such as free radicals, and followed the experimental approach described by Ferreira Severino et al.18 to identify free radical products from leaves of I. kudincha and I. latifolia under accelerated (alkaline) autoxidation and oxidation by the superoxide anion radical (O2c ). The reason for using alkaline conditions for investigation of autoxidation reactions is that autoxidation of polyphenols with O2 at neutral pH is a slow process, but proceeds much more rapidly at high pH. However, although there are many reports of the use of such conditions for accelerating the oxidation reactions of polyphenols, it is recognised that they are not relevant either to the chemistry of teas as beverages, or to the reactions of the phenols after the beverage has been consumed. Additional oxidation measurements using O2c radicals as the oxidising agent were, therefore, performed in order to establish to what extent results obtained from alkaline autoxidation can be extrapolated to other chemical conditions. Also, since the major antioxidant components in bitter tea are di- and monocaffeoylquinic acids,12 additional measurements were made using chlorogenic acid (Fig. 1a) and cynarin (Fig. 1b) as representatives of these two groups of polyphenols.

2. Experimental Bitter tea samples used were a commercial Kudingcha purchased from a Chinese supermarket and tenderised leaves of I. latifolia from Cao Bang, Vietnam (kindly supplied by Prof. Pham Van Ho of the National University of Vietnam, Ho Chi Minh City). Dimethyl sulfoxide (DMSO, >99.9% purity) was bought from

Fig. 1 Molecular structure of (a) chlorogenic acid, (b) cynarin. Proposed structures for radicals derived from chlorogenic acid: (c) initial radical after alkaline autoxidation, (d) initial radical after oxidation by O2c radicals, (e) hydroxylised radical of chlorogenic acid after prolonged alkaline autoxidation.

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Sigma-Aldrich Handels GmbH (Vienna, Austria), chlorogenic acid (5-O-caffeoylquinic acid) (>97% purity) were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and cynarin (1,3-di-O-caffeoylquinic acid) (96% purity) was bought from LGC Standards GmbH (Wesel, Germany). For autoxidation investigations of bitter tea solutions, 200 mg of dried leaves were initially extracted into 10 ml distilled water at 100  C for 10 min; the mixture was then filtered and allowed to cool to room temperature before use. Alternative measurements were made using similar tea extracts that were freeze dried and stored at room temperature in a vacuum desiccator. Stock solutions containing 20 mg ml 1 were prepared in DMSO, and used for oxidation experiments using O2c radicals, for which solutions of KO2 (50 mM) were prepared in DMSO under N2 atmosphere in the presence of the crown ether 18C623 in order to stabilise the O2c radical. Alkaline autoxidation was carried out in two ways. In the first method, a pump-flow system was used and the aqueous tea extract (described above) was mixed with 0.1 M NaOH shortly before the entrance to a flow cell, which was already located inside the resonator of the spectrometer. The flow cell was basically a conventional quartz EPR flat cell, with two arms at the bottom into which reaction solutions could be pumped and mixed immediately below the flat cell. The flow of the two reaction solutions (tea extract and NaOH; stored in separate 50ml beakers) to the two arms of a flow cell was controlled by a peristaltic pump, and the flow rate was adjusted so as to obtain the maximum signal intensity (2ml min 1). In the second method, the aqueous tea extract was mixed with an equal volume of 0.1 M NaOH in a tube outside the spectrometer and the resulting solution then transferred rapidly into a flat cell, which was then inserted into the spectrometer. Chlorogenic acid (10 mM) was also autoxidised under similar conditions to the tea samples described above. With cynarin (10 mM), the mixed solution containing equal volumes of the polyphenol and 0.1 M NaOH in a ratio of 1 : 1, was transferred into a 50ml capillary, sealed on both ends using Cristaseal, and this capillary was then placed inside a 3 mm i.d. quartz tube for the EPR measurements. Oxidation by O2c was carried out by gently mixing the stock solution of the freeze-dried tea in DMSO (20 mg ml 1) with the KO2/DMSO solution in a ratio of 1 : 1 outside the spectrometer, and then transferring the resulting solution rapidly to a flat cell for measurement of their EPR spectra. Similar measurements were carried out with the polyphenols chlorogenic acid or cynarin in DMSO (10 mM). In the case of cynarin, the final reaction solutions were transferred into a 50 ml capillary for recording their EPR spectra. EPR spectra were acquired at room temperature in 1024 points as first derivatives of the microwave absorption using a Bruker EMX CW spectrometer, operating at X-band frequencies (9 GHz), and equipped with a high sensitivity cavity. Microwaves were generated by a Gunn diode and the microwave frequency was recorded continuously with an in-line frequency counter. The measurements of cynarin were carried out on a Bruker EMX plus CW spectrometer using a super high Q cavity (1000 or 4000 points dependent on the modulation amplitude). All spectra were acquired using 2 mW microwave power, 2–20 mT modulation amplitude, and 10, 20 or 100 kHz modulation frequency, the Food Funct., 2010, 1, 262–268 | 263

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actual choice being dependent on the signal intensity and spectrum of the sample. With the exception of the measurements on the tea samples using the pump flow system which produced a steady state concentration of the untable free radicals, the first spectrum in each experiment was recorded immediately after tuning the spectrometer (i.e. about 2–3 min after starting the reaction). Justification of the spectral interpretations and refinement of the parameters for the hyperfine coupling (hfc) constants were obtained by simulations using the Bruker WinSimfonia software.

3. Results 3.1 Alkaline autoxidation The initial EPR spectra produced as a result of accelerated oxidation under alkaline conditions from aqueous extracts of leaves of I. kudincha and I. latifolia are shown in Fig. 2a and 2c along with the initial spectra from chlorogenic acid and cynarin obtained under similar conditions. These signals were unstable and the spectra of the tea samples and chlorogenic acid were acquired using a pump flow system to obtain more acceptable signal-to-noise ratios. The spectra from the two types of bitter tea are different in respect of both the numbers and magnitudes of their 1H hfc constants (see Table 1). The major component in the spectrum from the I. kudincha sample (Fig. 2a) contains six inequivalent 1H hfc constants and is similar to that obtained from autoxidised chlorogenic acid (Fig. 2e). The five largest values are similar to those reported previously for oxidation of chlorogenic acid,24,25 and the radical is assigned to a semiquinone with the unpaired electron located on the caffeic acid moiety (Fig. 1c). The initial spectrum observed with autoxidised cynarin (Fig. 2f) was very unstable and has not been identified. It could not be obtained with sufficient intensity for a reliable simulation, but seems to be different from that of chlorogenic acid. The spectrum obtained with I. latifolia (Fig. 1c) has not yet been identified, but it is also present as a minor component in the spectrum from the I. kudincha sample (Fig. 2a). Its hfc pattern indicates interaction of the unpaired electron with five 1H atoms, two of which are equivalent. Parameters for each of these radicals, refined by computer simulation (Fig. 2b and d), are presented in Table I along with relevant data from the literature. As was mentioned in the previous paragraph, the initial free radicals observed with the bitter tea and polyphenol samples were not sufficiently stable to record their EPR spectra under conventional conditions. When samples were prepared on the bench and then investigated as rapidly as possible, the spectra shown in Fig. 3 were obtained. The major component in the spectra from both bitter tea samples (I. kudincha and I. latifolia; Fig. 3a and b) is similar to the spectra that developed with time with both chlorogenic acid and cynarin (Fig. 3c and d). Its simulation using the parameters reported in Table 1 is presented in Fig. 3e; their similarity to those of the radical formed by autoxidation of 2,4,5-trihydroxy cinnamic acid24,26 suggests radical formation on the hydroxylised aromatic ring of caffeoylquinic acids (Fig. 1e). A number of additional spectra (Fig. 4) corresponding to intermediate radicals were observed in the pump flow measurements after the pump was switched off. The first intermediate 264 | Food Funct., 2010, 1, 262–268

Fig. 2 Initial EPR spectra from solutions of (a) Ilex kudincha, (c) Ilex latifolia, (e) chlorogenic acid, and (f) cynarin during alkaline autoxidation using a pump-flow system for (a), (c), and (e). Simulations (using the parameters in Table 1) of the main spectra from the bitter tea samples are shown in (b) for I. kudincha, and (d) for I. latifolia. 0.1 M NaOH was used and mixed in a ratio of 1 : 1 with either the tea extract (20 mg ml 1) or 10 mM of the polyphenol. All spectra were recorded using 2 mW microwave power. 100 kHz modulation frequency was used for (c) and (f), 20 kHz for (a) and 10 kHz for (e). Modulation amplitudes were 10 mT for (a) and (c), 2 mT for (e), and 20 mT for (f).

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Table 1 Hyperfine coupling constants (in mT) for radicals detected in the bitter tea samples compared with literature data Figure/compound

a(1H)

a(1H)

a(1H)

a(1H)

a(1H)

a(1H)

2b Chlorogenic acid24 Chlorogenic acid25 2d 3e 2,4,5-Tri-OH-cinnamic acid24 2,4,5-Tri-OH-cinnamic acid26 4d 5d 5f 6b

0.263 0.261 0.260 0.380 0.286 0.292 0.283 0.498 0.238 0.2528 0.4202

0.241 0.230 0.232 0.355 0.183 0.180 0.175 0.090 0.220 0.2233 0.0712

0.125 0.120 0.117 0.355 0.133 0.137 0.132 0.034 0.195 0.146 0.0563

0.115 0.120 0.117 0.088 0.041 0.040 0.044 0.034 0.149 0.146 0.0349

0.046 0.078 0.065 0.023

0.009

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0.086 0.0997

Fig. 3 EPR spectra of solutions of (a) Ilex kudincha, (b) Ilex latifolia, (d) cynarin after alkaline autoxidation outside the spectrometer, and (c) alkaline autoxidised chlorogenic acid using a pump-flow system (after the pump was stopped); (e) shows a simulation using the parameters from Table 1. 0.1 M NaOH was used and mixed in a ratio of 1 : 1 with either the tea extract (20 mg ml 1) or 10 mM of the polyphenol. All spectra were recorded using 2 mW microwave power. 100 kHz modulation frequency was used for (a), (b) and (d), and 10 kHz for (c). Modulation amplitudes were 2 mT for (a) 5 mT for (b), 10 mT for (c), and 20 mT for (d).

signal (Fig. 4a and b) from both tea samples has 1H hfc constants similar to those from chlorogenic acid autoxidised outside the spectrometer (Fig. 4c, Table 1). Although it was not possible to assign this signal to a specific radical structure, it is almost This journal is ª The Royal Society of Chemistry 2010

Fig. 4 Intermediate EPR spectra of alkaline autoxidised solutions of (a) Ilex kudincha, (b) Ilex latifolia, after the pump was stopped, and (c) chlorogenic acid after alkaline autoxidation outside the spectrometer. (d) is a simulation of these spectra using the parameters in Table 1. With increasing time spectra (a) and (b) were replaced by (e) Ilex kudincha and (f) Ilex latifolia. 0.1 M NaOH was used and mixed in a ratio of 1 : 1 with either the tea extract (20 mg ml 1) or 10 mM of the polyphenol. All spectra were recorded using 2 mW microwave power and 100 kHz modulation frequency. Modulation amplitudes were 10 mT for (a), (b) and (f), 2 mT for (c), and 3 mT for (e).

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certainly derived from one or more caffeoylquinic acids. This intermediate radical was unstable and its signal was quickly replaced by a spectrum (Fig. 4e and f), which was identical to the radical of hydroxylised caffeoylquinic acids (Fig. 1e), the main signal observed with the tea samples autoxidised outside the spectrometer (Fig. 3). 3.2 Oxidation by superoxide anion radicals

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Oxidation of lyophilised tea extracts of both I. kudincha and I. latifolia by O2c in DMSO solutions produced identical EPR

Fig. 6 (a) 2nd and (c) 3rd EPR spectra observed from DMSO solutions of cynarin (5 mM final concentration) after oxidation by O2c radicals (25 mM final concentration) acquired approx. 4 min (a) and approx. 14 min after the start of the reaction. (b) is a simulation of spectrum (a) using the parameters in Table 1. Spectra were recorded using 2 mW microwave power, 100 kHz modulation frequency and 5 mT modulation amplitude.

Fig. 5 Initial EPR spectra from DMSO solutions of (a) Ilex kudincha (20mg ml 1), (b) Ilex latifolia (20mg ml 1), (c) chlorogenic acid (10 mM), and (e) cynarin (10 mM) after oxidation by O2c radicals (50 mM), mixed in a volume ratio of 1 : 1. Simulations using the parameters in Table 1 are shown for the radicals from (d) chlorogenic acid, and (f) cynarin. All spectra were recorded using 2 mW microwave power. 100 kHz modulation frequency was used for (e), 20 kHz for (a) and (b), and 10 kHz for (c). Modulation amplitudes were 2 mT for (a) and (b), 10 mT for (c), and 5 mT for (e).

266 | Food Funct., 2010, 1, 262–268

spectra (Fig. 5a and b). These spectra could be simulated using five inequivalent 1H hfc constants (Table 1), and were similar to initial spectra from both chlorogenic acid and cynarin oxidised under similar conditions (Fig. 5c and e). This result strongly suggests the formation of a similar radical species with each tea sample, and it is assigned to oxidised caffeoylquinic acid (Fig. 1d) that was proposed for the initial spectrum from autoxidation of the bitter tea samples and chlorogenic acid. The differences in the magnitudes of these hfc constants (Fig. 4d and f, Table 1) and those from the radical obtained from alkaline autoxidation of kudingcha and chlorogenic acid (Fig. 2b) are probably the consequence of the use of different solvent systems (DMSO and water). With cynarin, the initial radical was very unstable and changed rapidly to produce other spectra (Fig. 6a and c). Although these radicals have not yet been identified, the 1st intermediate spectrum (Fig. 6a) is similar to the 1st intermediate spectrum obtained in bitter tea samples under alkaline autoxidation (Fig. 4a and b). The different values for the hfc constants are again most likely to the consequence of using different solvent systems. The 2nd intermediate spectrum showed at least two signals (Fig. 6c) which were not observed with either of the bitter tea samples, a result which suggests that if such radicals are formed in the bitter teas, they have much lower stability than in pure solutions of the polyphenol.

4. Discussion Recent analytical investigations on the composition of bitter teas have identified di- and mono-caffeoylquinic acids as the major polyphenolic components.11,12 In I. kudingcha, Zhu et al.12 found that 67.4% of the total caffeoylquinic acids were accounted by This journal is ª The Royal Society of Chemistry 2010

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two di-caffeoylquinic acids (3,5-di-O-caffeoylquinic acid and 4,5di-O-caffeoylquinic acid) and 26.4% by three mono-caffeoylquinic acids, which included chlorogenic acid. The free radical species formed by oxidation of extracts from I. kudincha and I. latifolia samples that were identified by EPR in the present investigations are also consistent with originating largely from caffeoylquinic acids. The fact that these radicals were able to be identified indicates that the reactions of the semiquinone oxidation products with other molecules in the beverages are sufficiently slow for them to be observed in conventional EPR measurements. In the early stages of the autoxidation reaction of the bitter tea samples, these radicals were accompanied by another, as yet unidentified, free radical whose relative contribution differed greatly between the two samples. There may, therefore, be significant differences in the activity of the two beverages, but this would likely be dependent on whether or not this unstable radical contributes to the biological effects. At the present time, however, it has not been identified. In addition to these initial free radicals, spectra were observed from other unstable intermediates, which are probably also radical reaction products derived from caffeoylquinic acids, because of a resemblance between their spectra and intermediates in the oxidation of chlorogenic acid. Furthermore, after prolonged alkaline autoxidation, a radical compound was detected with similar hfc constants to those from 2,4,5-trihydroxycinnamic acid, indicating oxygenation of the aromatic ring of the caffeic acid moiety. However, both chlorogenic acid and cynarin produced similar spectra, so the EPR method was not able to distinguish between the relative importance of the mono- and dicaffeoylquinic acids in the formation of the relatively stable final radicals due to similar chemical environments of the unpaired electron in the various radical structures. Nevertheless, these results further strengthen the importance of the caffeoylquinic acids in the antioxidant chemistry of the two bitter tea samples. Strong antioxidant activity has been demonstrated previously for aqueous extracts of I. paraguariensis,27,28 and I. kudincha,11,12 and it has been correlated with the total polyphenol contents.11,29 Anesini et al.29 also showed that caffeic and chlorogenic acids were two of the major polyphenols present in I. paraguariensis, and Liu et al.11 and Zhu et al.12 both reported that aqueous extracts of I. kudincha contain large quantities of caffeoylquinic acids, which account for much of the antioxidant activity of the beverages. However, as mentioned in the introduction of this paper, antioxidant assays simply provide an overall measure of a particular chemical property, and give no information on reactions involving oxidation products which are formed during their antioxidant reactions. Also, although chemical analyses provide identification of components that might feature in antioxidant reactions, they give no information on how they might behave in a complex mixture, such as a beverage, where reactions between individual components could have a major impact on the overall chemistry. Furthermore, biological properties are likely to be determined to a major extent by molecules with sufficient stability to be transported within an organism, but with sufficient reactivity to react selectively with specific cells or tissue type. On the basis of the present measurements, the caffeoylquinic acids would appear to be precursors for such molecules in kudingcha and related beverages, and it seems that these molecules show little reactivity with other components in the This journal is ª The Royal Society of Chemistry 2010

beverage, at least on a timescale of several minutes. This conclusion is also consistent with recent work with green tea,18 where it was concluded that polyphenol-derived free radicals have appreciable stability in the complex medium of the beverage. Caffeic and chlorogenic acids have also been shown to have antioxidant activity in other tests,30,31 and the chlorogenic acids are reported to be effective scavengers of the superoxide anion radical.31 In their work with I. paraguariensis, Anesini et al.29 concluded that the contents of caffeic and chlorogenic acids were insufficient to account for all of the peroxidase activity in the leaf extracts, and proposed contributions from other polyphenols, such as quercetin and rutin that are also present in such extracts.32 However, in the present measurements with extracts of I. kudincha and I. latifolia, no evidence was found for the generation of free radicals that correspond to either oxidised quercetin or rutin, although some of the EPR spectral intensity has not yet been assigned, and we suspect that an appreciable fraction of the unidentified activity in the work of Anesini et al.29 is derived from other caffeoylquinic acids. It should also be pointed out that EPR spectroscopy is most sensitive to the detection of free radicals that are stable or have moderate stability, and one of its greatest strengths is its ability to distinguish between individual radicals from closely related structures. However, very unstable free radicals are difficult to detect with conventional EPR techniques, and their formation might not be seen in experiments such as those reported here. Such radicals can be detected using spin trapping techniques, and Goodman et al.33 have shown results from such investigations on ‘‘bitter tea’’ samples, although these radicals could not be identified beyond the fact that there were O- and C-centred radical species. However, it is free radicals with moderate stability, such as those identified in the present work, that are able to react specifically in biological systems and their parent molecules would thus be expected to be major candidates for medicinal properties. Thus the identification of free radicals derived from caffeoylquinic acids during the oxidation of extracts of I. kudincha and I. latifolia strongly suggests that these molecules may play important roles in at least some of the biological properties of these beverages, especially where the cOH or O2c free radicals are involved.

5. Conclusions The EPR spectra from samples of I. kudincha and I. latifolia demonstrate the importance of free radicals derived from caffeoylquinic acids in the oxidation chemistry of these bitter tea beverages; no spectra were observed that could be assigned specifically to saponins or other groups of polyphenol, although some components have not yet been assigned. It is also possible that some active components are not detected, because they do not form radical products with sufficient stability. Nevertheless, caffeoylquinic acids make important contributions to the antioxidant chemistry of various teas from extracts from Ilex species, and products from such reactions are now detected in the present paper. Thus it is important that contributions from these polyphenols are recognised in any attempts to identify quality issues associated with kudingcha and related beverages. Food Funct., 2010, 1, 262–268 | 267

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Acknowledgements This work was supported by the Austrian Ministry of Traffic, Innovation and Technology (BMVIT) and the Austrian Science Fund (FWF). We would like to thank Dr Chris Kay (University College London, UK) for providing use of the EMX plus spectrometer for measurements of the cynarin sample.

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References 1 S. Dharmananda, http://www.itmonline.org/arts/kudingcha.htm. 2 R. K. Ahai, Iconographia Cormophytorum Sinicoru, Beijing Science, 1985, 641. 3 K. Nishimura, T. Miyase and H. Noguchi, J. Nat. Prod., 1999, 62, 1128–1133. 4 M. A. Ouyang, C. R. Yang, Z. L. Chen and H. Q. Wang, Phytochemistry, 1996, 41, 871–877. 5 M. A. Ouyang, H. Q. Wang, Z. L. Chen and C. R. Yang, Phytochemistry, 1996, 43, 443–445. 6 M. A. Ouyang, C. R. Yang and Z. J. Wu, J. Asian Nat. Prod. Res., 2001, 3, 31–42. 7 M. A. Ouyang, Y. Q. Liu, H. Q. Wang and C. R. Yang, Phytochemistry, 1998, 49, 2483–2486. 8 J. Huang, X. Wang, Y. Ogihara, N. Shimizu, T. Akiyama and T. Takeda, Chem. Pharm. Bull., 2001, 49, 765–767. 9 A. T. C. Taketa, E. Breitmaier and E. P. Schenkel, J. Braz. Chem. Soc., 2004, 15, 205–211. 10 S. C. B. Gnoatto, E. P. Schenkel and V. L. Bassani, J. Braz. Chem. Soc., 2005, 16, 723–726. 11 L. Liu, Y. Sun, L. Tanguy, X. Liang, H. Ye and X. Zeng, Food Chem., 2009, 112, 35–41. 12 F. Zhu, Y.-Z. Cai, M. Sun, J. Ke, D. Lu and H. Corke, J. Agric. Food Chem., 2009, 57, 6082–6089. 13 Q. Guo, B. Zhao, M. Li, S. Shen and W. Xin, Biochim. Biophys. Acta, Lipids Lipid Metab., 1996, 1304, 210–222. 14 P. T. Gardner, D. B. McPhail and G. Duthie, J. Sci. Food Agric., 1998, 76, 257–262.

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15 M. A. Morsy and M. M. Khaled, Spectrochim. Acta, Part A, 2002, 58, 1271–1277. 16 S. Valcic, A. Muders, N. E. Jacobsen, D. C. Liebler and B. N. Timmermann, Chem. Res. Toxicol., 1999, 12, 382–386. 17 F. Nanjo, K. Goto, R. Seto, M. Suzuki, M. Sakai and Y. Hara, Free Radical Biol. Med., 1996, 21, 895–902. 18 J. Ferreira Severino, B. A. Goodman, C. W. M. Kay, K. Stolze, D. Tunega, T. G. Reichenauer and K. F. Pirker, Free Radical Biol. Med., 2009, 46, 1076–1088. 19 S. Shankar, S. Ganapathy, S. R. Hingorani and R. K. Srivastava, Front. Biosci., 2008, 13, 440–452. 20 R. Sheng, Z. L. Gu, M. L. Xie, W. X. Zhou and C. Y. Guo, Planta Med., 2009, 75, 113–120. 21 S. Anton, L. Melville and G. Rena, Cell. Signalling, 2007, 19, 378– 383. 22 S. H. Koh, S. M. Lee, H. Y. Kim, K. Y. Lee, Y. J. Lee, H. T. Kim, J. Kim, M. H. Kim, M. S. Hwang, C. Song, K. W. Yang, K. W. Lee, S. H. Kim and O. H. Kim, Neurosci. Lett., 2006, 395, 103–107. 23 J. S. Valentine, A. R. Miksztal and D. T. Sawyer, Methods Enzymol., 1984, 105, 71–81. 24 J. A. Pedersen and B. Ollgaard, Biochem. Syst. Ecol., 1982, 10, 3–9. 25 J. A. Pedersen, Biochem. Syst. Ecol., 2000, 28, 229–253. 26 P. Ashworth, J. Org. Chem., 1976, 41, 2920–2927. 27 R. Filip, S. B. Lotito, G. E. Ferraro and C. G. Fraga, Nutr. Res., 2000, 20, 1437–1446. 28 N. Bracesco, M. Dell, A. Rocha, S. Behtash, T. Menini, A. Gigliucci and E. Nunes, J. Altern. Complement. Med., 2003, 9, 379–387. 29 C. Anesini, G. Ferraro and R. Filip, Food Chem., 2006, 97, 459–464. 30 J. M. Lekse, L. Xia, J. Stark, J. D. Morrow and J. M. May, Mol. Cell. Biochem., 2001, 226, 89–95. 31 N. Nakatani, S.-I. Kayano, H. Kikuzaki, K. Sumino, K. Katagiri and T. Mitani, J. Agric. Food Chem., 2000, 48, 5512–5516. 32 R. Filip, P. L opez, G. Giberti, J. Coussio and G. E. Ferraro, Fitoterapia, 2001, 72, 774–778. 33 B. A. Goodman, K. F. Pirker, T. G. Reichenauer, P. T. Ho and T. D. Ho Huynh, in Natural Antioxidants and Micronutrients, ed. B. Zhao, G. Liu and L. Packer, Medimond, Bologna, 2005, pp. 139–143.

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PAPER

www.rsc.org/foodfunction | Food & Function

Application of response surface methodology to optimize pressurized liquid extraction of antioxidant compounds from sage (Salvia officinalis L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.) M. B. Hossain,*ab N. P. Brunton,b A. B. Martin-Dianaa and C. Barry-Ryana

Downloaded on 02 December 2010 Published on 02 November 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00021C

Received 24th May 2010, Accepted 26th July 2010 DOI: 10.1039/c0fo00021c The present study optimized pressurized liquid extraction (PLE) conditions using Dionex ASE 200, USA to maximize the antioxidant activity [Ferric ion Reducing Antioxidant Power (FRAP)] and total polyphenol content (TP) of the extracts from three spices of Lamiaceae family (sage, basil and thyme). Optimal conditions with regard to extraction temperature (66–129  C) and solvent concentration (32–88% methanol) were identified using response surface methodology (RSM). For all three spices, results showed that 129  C was the optimum temperature with regard to antioxidant activity. Optimal methanol concentrations with respect to the antioxidant activity of sage and basil extracts were 58% and 60% respectively. Thyme showed a different trend with regard to methanol concentration and was optimally extracted at 33%. Antioxidant activity yields of the optimal PLE were significantly (p < 0.05) higher than solid/liquid extracts. Predicted models were highly significant (p < 0.05) for both total phenol (TP) and FRAP values in all the spices with high regression coefficients (R2) ranging from 0.651 to 0.999.

1. Introduction Numerous studies have demonstrated that spices and their extracts have many useful technological and biological properties, for example, it is well known that spice extracts have potent antioxidant properties1,2 and more recent studies have shown that they may possess other useful biological properties such as anti-carcinogenic, anti-diabetic and anti-inflammatory properties.3 This has led to the use of extracts from spices in many food applications.4 The discovery of these beneficial properties has lead to intensive research some of which has indicated that these properties are derived from high levels of antioxidative polyphenols. For example, sage is particularly rich in polyphenols5,6 and several studies have shown that sage (Salvia officinalis L.) essential oil can improve the memory and has shown promise in the treatment of Alzheimer’s disease.4 Basil (Ocimum basilicum L.) extracts have exhibited a spectrum of properties including bactericidal, anti-inflammatory, antioxidative, antiulcer, antidiarrheal, chemopreventive, blood-sugar lowering, a nervous system stimulatory and radiation protection.7–9 Beric et al.10 investigated protective effect of essential oil of basil against oxidative DNA damage and mutagenesis. Thyme (Thymus vulgaris L.) also possesses various beneficial effects, e.g., antiseptic, carminative, antimicrobial, and antioxidative properties.11 Thyme and its extracts are used orally to treat dyspepsia and other gastrointestinal disturbances, cough due to cold, whooping cough, bronchitis, laryngitis and tonsillitis. The literature is indicating that the compounds responsible for these displayed biological activities are polyphenolic secondary metabolites, such as rosmarinic acid, carvacrol, caffeic a School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Ireland. E-mail: [email protected]; Fax: +00353(0)18059550; Tel: +0035318059500 ext 301 b Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland

This journal is ª The Royal Society of Chemistry 2010

acid, ferulic acid, thymol.12 Spice phenolics are an interesting target for optimization of extraction conditions, the extraction parameters can influence the composition of the extract. Moreover, spice extracts also named as oleoresins and essential oils, are traditionally extracted out using conventional solid/liquid extraction at atmospheric pressures.13 Pressurized liquid extraction is relatively a new automated technique, which uses high pressure and this allows the user to carry out extractions at temperatures above the boiling temperature of the solvent.14 The use of high pressures and temperatures increases the target compound’s solubility, solvent diffusion rate and mass transfer, while decreasing the solvent viscosity and surface tension.15 PLE offers many advantages over traditional solid/liquid extraction techniques. Disadvantages of solid/liquid extraction methods include: the use of large quantities of toxic organic solvents, the involvement of elaborate labour pools, the long extraction times, low selectivity and/or low extraction yields. In contrast, PLE uses less solvent, has shorter extraction times, is automated and retains samples in an oxygen and light-free environment.16 Therefore, PLE could be considered as an efficient green extraction technique. A considerable number of studies have focussed on the use of PLE from an environmental perspective.17,18 Several authors have reported the use of PLE to extract specified target compounds from spices.19–22 The technique was recently shown to be particularly useful for maximizing the antioxidative properties of extracts from microalgae,23 anthocyanins from dried red grape skin,24 vitamin E rich oil from grape seeds.25 However, none have concentrated on optimizing the PLE conditions for maximizing the antioxidant activity of spice extracts using response surface methodology (RSM). To maximize antioxidant capacity of the extracts, PLE parameters such as temperature and solvent concentration, must be optimized. RSM is a statistical technique which allows the user to identify optimal conditions for selected responses while minimizing the number of experiments required. Central composite design Food Funct., 2010, 1, 269–277 | 269

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(CCD) is the most popular form of RSM as it has been utilized by a number of researchers to optimize various food processing methods such as milling,26 and extraction.27 The objective of the work was to optimize the analytical scale extraction conditions (temperature and solvent concentration) of PLE using response surface methodologies. PLE has been recognized as a high throughput extraction system for analyzing target analytes but optimized conditions for extraction of antioxidant compounds from spices have not been given previously. Therefore the extraction conditions of PLE were optimized to get significantly (p < 0.05) higher efficiency in extraction than conventional solid/ liquid extraction method. Previous work has indicated that methanol is a highly efficient solvent for analytical scale extraction of polyphenols. Preliminary work on extraction of phenolic antioxidants from spices showed that methanol had higher efficiency as a solvent than ethanol and water. Therefore, this efficient solvent was used to test the PLE process; its optimization and modeling can therefore be used for other solvents.

using conditions specified using RSM guided experimental design. The range of solvent combinations and extraction temperatures used in PLE system are presented in Table 1. The automated PLE cycle was as follows: the cell containing the sample was pre-filled with the extraction solvent, pressurized (103.42 bar), and then heated for 5 min followed by a static period of 5 min. The sample was extracted with methanol concentration and temperature as specified in the design during this 5 min. Then, the cell was rinsed with fresh extraction solvent (60% of the extraction cell volume) and purged with a flow of nitrogen (10.34 bar during 90 s). Extracts (34 mL) were collected into 60 mL glass vials. The solvent used was previously degassed with nitrogen to avoid the oxidation of the analytes under the operating conditions. The extracts were stored at 20  C in darkness until analysis. The experiment was performed in two batches which included three replications in each sample. Prior to HPLC and in vitro antioxidant activity analysis the extract was filtered through a 0.45 mm PTFE filters (Millipore, USA).

2. Materials and methods

2.3.

2.1.

Solid/liquid extractions were carried out according to the method of Shan et al.2 with slight modifications. Briefly, dried and ground samples (0.5 g) were homogenized for 1 min at 24,000 rpm using an Ultra-Turrax T-25 Tissue homogenizer (Janke & Kunkel, IKA-Labortechnik, Saufen, Germany) in 25 mL of 80% methanol at room temperature (23  C). The homogenized sample suspension was shaken overnight with a V400 Multitude Vortexer (Alpha laboratories, North York, Canada) at 1,500 rpm at room temperature (y 25  C). The sample suspension was then centrifuged for 15 min at 2,000 g (MSE Mistral 3000i, Sanyo Gallenkamp, Leicestershire, UK) and immediately filtered through 0.22 mm polytetrafluoroethylene (PTFE) filters. The extracts were kept at 20  C until subsequent analysis. The experiment was performed in two batches which included three replications in each sample.

Samples and reagents

Dried and powdered sage, basil and thyme were sourced from AllinAll Ingredients Ltd., Dublin, Ireland. Folin-Ciocalteu Reagent, sodium carbonate, sodium acetate anhydrous, acetic acid, ferric chloride hexahydrate, 2,4,6-tri(2-pyridyl)-s-triazine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, gallic acid, caffeic acid and rosmarinic acid were purchased from Sigma-Aldrich. 2.2.

Pressurized liquid extraction (PLE)

PLE was performed on a Dionex ASE 200 (Dionex Corp., Sunnyvale, CA) system. Dried and powdered spices (0.5 g) were placed in between two layers of diatomaceous earth in a 22 mL Dionex (ASE 200) stainless-steel cell. The cells were equipped with a stainless steel frit and a cellulose filter (Dionex Corp.) at the bottom to avoid the collection of suspended particles in the collection vial. A dispersing agent (diatomaceous earth) was used to reduce the solvent volume used for the extraction. The extraction cell was arranged in the cell tray and was extracted

2.4.

Conventional solid/liquid extraction

Determination of total phenolic content

The total phenolic content was determined using Folin-Ciocalteu Reagent (FCR) as described by Singelton et al.28 The experiment

Table 1 Combinations of methanol concentration and temperature with their coded terms obtained from RSM and their respective TP and FRAP values

Run

Methanol % (coded term)

Temperature ( C) (coded term)

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

60 (0) 40(1) 60(0) 60(0) 80(+1) 80(+1) 32(a) 60(0) 60(0) 40(1) 60(0) 60(0) 88(+a)

97(0) 75(1) 97(0) 97(0) 120(+1) 75(1) 97(0) 97(0) 66(a) 120(+1) 129(+a) 97(0) 97(0)

270 | Food Funct., 2010, 1, 269–277

TP (g GAE/100 g DW)

FRAP (g Trolox/100 g DW)

Sage

Basil

Thyme

Sage

5.81  0.006 5.37  0.007 5.81  0.012 5.74  0.008 5.40  0.006 4.31  0.004 5.57  0.009 6.11  0.017 5.59  0.014 6.06  0.009 6.16  0.016 5.80  0.004 4.14  0.003

3.05  0.005 2.47  0.003 3.07  0.005 3.08  0.002 3.15  0.006 1.57  0.001 2.70  0.002 3.06  0.004 2.05  0.002 3.36  0.005 3.77  0.004 3.07  0.007 1.97  0.006

2.16  0.002 1.95  0.004 1.98  0.001 1.87  0.005 2.62  0.010 1.75  0.005 2.35  0.002 2.15  0.001 1.44  0.003 2.85  0.006 3.54  0.011 2.17  0.006 2.39  0.003

15.24  14.36  15.17  15.30  15.33  12.77  14.77  15.86  15.30  15.61  16.20  15.22  13.86 

Basil 0.014 0.009 0.018 0.052 0.075 0.037 0.023 0.021 0.065 0.053 0.086 0.056 0.036

7.65  6.91  7.39  7.76  7.10  3.67  6.96  7.84  4.81  9.07  9.42  7.17  4.98 

Thyme 0.026 0.016 0.023 0.043 0.056 0.012 0.033 0.013 0.036 0.056 0.045 0.026 0.015

5.92  5.00  5.54  5.67  6.90  4.79  6.34  6.18  4.08  7.54  9.07  5.82  6.29 

0.017 0.023 0.025 0.014 0.022 0.031 0.035 0.042 0.028 0.017 0.051 0.026 0.036

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was performed in two batches which included three replications in each for both samples and standard. Methanolic gallic acid solutions (10–400 mg/L) were used as standards. In each replicate, 100 mL of the appropriately diluted sample extract, 100 mL methanol, 100 mL FCR and finally 700 mL 20% Na2CO3 were added together and vortexed. The mixture was incubated for 20 min in the dark at room temperature. After incubation the mixture was centrifuged at 13,000 rpm for 3 min. The absorbance of the supernatant was measured at 735 nm by spectrophotometer. The total phenolic content was expressed as gallic acid equivalent [g GAE/100 g dry weight (DW)] of the sample. 2.5.

Ferric ion reducing antioxidant power (FRAP) assay

The FRAP assay was carried out as described by Stratil et al.29 with slight modifications. The FRAP reagent was: 38 mM sodium acetate anhydrous in distilled water at pH 3.6, 20 mM FeCl3$6H2O in distilled water and 10 mM 2,4,6-tri(2-pyridyl)-striazine (TPTZ) in 40 mM HCl in a ratio of 10 : 1 : 1. This reagent was prepared before each experiment. To each sample, 100 mL of appropriately diluted sample extract and 900 mL of FRAP reagent was added, mixed and was incubated at 37  C for 40 min in the dark. In the case of the blank, 100 mL of methanol was added to 900 mL of FRAP reagent. The absorbance of the resulting solution was measured at 593 nm by spectrophotometer. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which is a synthetic antioxidant at concentrations ranging from 0.1 mM to 0.4 mM, was used as a reference antioxidant standard. FRAP values were expressed as g Trolox/100 g DW of the sample. 2.6.

Determination of melanoidins

To estimate the degree of formation of browning compounds such as melanoidins the absorbance at 420 nm was measured; this wavelength has previously been used to detect browning defects in fruit juices.30 The extracts were diluted four times before measuring the absorbance as some of the extracts had very high absorbance (>2.50). 2.7.

HPLC analysis of the extracts

High performance liquid chromatography (HPLC) of the filtered sample extracts were carried out according to the method of Tsao and Yang.31 The chromatographic system (Shimadzu-Model no SPD-M10A VP, Mason Technology, Dublin 8, Ireland) consisted of a pump, a vacuum degasser, a Diode-Array Detector and was controlled through EZ Start 7.3 software (Shimadzu) at 37  C. An Agilent C18 column (15 cm  4.6 cm, 5 mm, Agilent Technologies., USA) was utilized with a binary mobile phase of 6% acetic acid in 2 mM sodium acetate (final pH 2.55, v/v, solvent A) and acetonitrile (solvent B). Solvent A was prepared first by making 2 mM sodium acetate water solution, which was then mixed with acetic acid at a ratio of 94 : 6 by volume. All solvents were filtered through a 0.45 mm membrane filter prior to analysis. The flow rate was kept constant at 1.0 mL/min for a total run time of 80 min. The following gradient program was carried out: 0–15% B in 45 min, 15–30% B in 15 min, 30–50% B in 5 min, 50–100% B in 5 min and 100  5% B in 10 min. The sample injection volume was 10 mL. The standards of This journal is ª The Royal Society of Chemistry 2010

rosmarinic acid and caffeic acid were dissolved in methanol for quantification purpose (100 mg/mL). The detection wavelength of 280 nm was used for the detection of gallic acid. Rosmarinic acid and caffeic acid were detected at 320 nm. Identification of the compounds was achieved by comparing their retention times and UV-Vis spectra with those of authenticated rosmarinic acid standard by using the inline DAD with a 3D feature. Results are expressed as mean values of three assays for each replicated experiment. 2.8.

Statistical analysis

Optimal PLE conditions were determined by RSM which was performed using the Design Expert Version 7.1.3 software (Stat-Ease, Inc., Minneapolis, MN). A CCD was used to investigate the effects of two independent variables, solvent concentration and extraction temperature, on the dependent variables, TP and FRAP. The data obtained from the CCD design was fitted with a second order polynomial equation. The equation was as follows: Y ¼ b0 +

P2

biXi +

i¼1

P2

i¼1

biiX2i +

PP i

j¼i + 1bijXiXj

(1)

where Y is the predicted response; b0 is a constant; bi is the linear coefficient; bii is the quadratic coefficient, bij is the interaction coefficient; and Xi and Xj are independent variables. The adequacy of the model was determined by evaluating the lack of fit, coefficient of regression (R2) and the Fisher test value (F-value) obtained from the analysis of variance (ANOVA). Statistical significance of the model and model variables was determined at the 5% probability level (p < 0.05). The software uses the quadratic model equation shown above to build response surfaces. Three-dimensional response surface plots were generated by maintaining one response variable at its optimal level and plotting that against two factors (independent variables). The input range of the selected variables was determined by preliminary RSM experiments. This was set as 40% (coded as 1) to 80% (coded as +1) for methanol concentration and 75  C (coded as 1) to 120  C (coded as +1). The two independent variables were coded at five levels (a, 1, 0, 1, a), which resulted in an experimental design of 13 experimental points, including five central points. The actual values of the factors with their coded terms for the experimental designs are given in Table 1. Statistical analyses were carried out on the basis of actual values. 2.9.

Model validation

The predictive performance of the developed models describing the combined effect methanol concentration (X1) and temperature (X2) on independent variables (FRAP, TP) of sage, basil and thyme were validated in a separate set of selected conditions. These assessments were carried out by calculating the model performance indices: accuracy factor (AF, eqn (2a)) and bias factor (BF, eqn (2b)).32

AF ¼ 10

S logjVp =VE j ne

 Slog Vp =VE Þ ne BF ¼ 10

(2a)

(2b)

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The criterion used to characterize the fitting efficiency of the data to the model was the multiple correlation coefficients (R2) and their average mean deviation (E, eqn (2a)). Eð%Þ ¼

n 1X VE  Vp  100 ne i¼1 VE

k

k

(2c)

where, ne is the number of experimental data, VE is the experimental value and VP is the predicted value.

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3. Results and discussion 3.1. Optimization of PLE conditions from sage, basil and thyme with respect to total phenols and FRAP values Fig. 1 illustrates response surface plots showing the effect of methanol concentration and temperature on TP and FRAP values of sage, basil and thyme. Using PLE, the optimal temperature for obtaining extracts with high antioxidant activity was 129  C in all the spices tested. In fact, temperature was found to be the dominant factor in maximising the phenolic content and antioxidant activity as measured by FRAP values of the extracts. This result was in agreement with the findings of Zgorka33 who reported that concentrations of isoflavone obtained from Clover (Trifolium L.) using PLE increased with increasing the temperature from 75  C to 125  C with no evidence of thermal degradation of the target analytes. Several other studies using PLE also reported the similar effect of temperature on extraction yield of phenolics from plant materials.34,35 The results were expected since an increase in temperature enhances the solubility of many compounds. High temperatures might also have increased the diffusion rate of the compounds resulting in antioxidant compounds being extracted at much higher rates. Interestingly PLE offers a unique possibility of using high temperature at very high pressure (1500 psi) while preventing degradation of the extracted compounds.33 This is because high pressure generally increases the stability of covalent bonds within bio-molecules. In the present study, evidence of thermal degradation could only be detected at temperatures of 150  C or above. In terms of polyphenol content the spices were ranked in the following descending order sage > basil > thyme. A methanol concentration of 50–60% showed better performance with regard to antioxidant activity and total phenolic content of extracts from sage. Thus RSM assisted optimization demonstrated that optimum PLE conditions for sage were 58% methanol coupled with a temperature of 129  C (Fig. 1a,b and Table 2). Similar results were reported by Akowuah et al.36 using conventional solid/liquid extraction. They found 50% aqueous methanol extract of Orthosiphon stamineus had higher rosmarinic acid content and antioxidant capacity as measured using DPPH scavenging assay than other methanol concentrations (0% and 100%) tested. In the present study both factors, temperature and methanol concentration in the PLE had a significant (p < 0.05) linear effect on the total phenolic content of sage extracts (Fig. 2a,b). Moreover, methanol concentration showed a significant (p < 0.05) quadratic effect on total phenols of sage PLE extracts. The second order polynomial equations presented in Table 2 show the relationship between the factors and response variables. Only the significant model terms were included in the equations. In the case of FRAP values of sage PLE extracts, only 272 | Food Funct., 2010, 1, 269–277

Fig. 1 Contour plots of PLE extracts of sage (a,b), basil (c,d) and thyme (e,f) showing the effect of methanol concentration and temperature on TP (g GAE/100 g DW) and FRAP (g Trolox/100 g DW) values.

temperature had a significant (p < 0.05) effect at a linear level, while methanol concentration showed significant (p < 0.05) quadratic effect. Optimal sage PLE extracts also had a significantly (p < 0.05) higher TP (14%) and FRAP values (20%) as compared to the conventional solid/liquid extracts (Table 3). ANOVA analysis of the designs showed that the models of TP and FRAP values of sage were significant (p < 0.05). The regression coefficient, as obtained from ANOVA, was higher for TP values (R2 ¼ 0.94) than that of FRAP values (R2 ¼ 0.65). It is stipulated that these models could be used for prediction of the data. In fact, the predicted TP and FRAP values of sage correlated strongly with actual values (Pearson’s correlation coefficient ¼ 0.971 for TP and 0.882 for FRAP). Extracts from basil were optimally extracted at 60% methanol and 129  C using PLE (Fig. 1c,d and Table 2). Temperature had a greater effect on the TP and FRAP values of the extracts than methanol concentration (Fig. 2c, d). At optimal methanol concentration (60%), when the temperature was increased from 66  C, lowest temperature used, to 129  C, highest temperature used, the total phenolic content was increased from 2.05 g GAE/ 100 g DW to 3.77 g GAE/100 g DW which corresponds to an 83.9% increase. Over the same temperature range, FRAP values This journal is ª The Royal Society of Chemistry 2010

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Table 2 Second order polynomial equations and regression coefficients of the response variables of pressurized liquid extractions at a temperature range of 66 to 129  C with X1 ¼ Methanol concentration (%), X2 ¼ Temperature ( C), Y ¼ Response variables (TP or FRAP)

Spice name

Response variables

Second order polynomial equation

R2

Optimal PLE (% methanol/ C)

Sage

TP (g GAE/100 g DW)

Y ¼ 1.17639 + 0.132731X1 + 0.0142696X2  0.00130094X12 Y ¼ 6.71513 + 0.197932X1 + 0.0282219X2  0.00164943X12 Y ¼ 1.19361 + 0.0577599X1 + 0.0328361X2 + 0.000383333X1X2  0.000904373X12  0.000146668X22 Y ¼ 2.73633 + 0.170073X1 + 0.0673262X2  0.00183409X12 Y ¼ 0.316668 + 0.0263058 X2 Y ¼ 0.2506 + 0.065017 X2

0.943

58/129

FRAP (g Trolox /100 g DW)

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Basil

TP (g GAE/100 g DW)

FRAP (g Trolox /100 g DW) Thyme

TP (g GAE/100 g DW) FRAP (g Trolox /100 g DW)

were increased by 96% from 4.81 g Trolox/100 g DW to 9.42 g Trolox/100 g DW. Pressurized liquid extracts of basil had almost twice TP and FRAP values than solid/liquid extracts (Table 3). The models for TP and FRAP values were significant (0<0.001) with high regression coefficients of 0.999 (TP) and 0.942 (FRAP). The models also showed very high F-values (3694.20 for TP and 49.29 for FRAP) and a non-significant (p > 0.05) lack of fit. The predicted values for TP and FRAP showed high correlation (Pearson’s correlation coefficient, r ¼ 0.999 for TP and 0.970 for FRAP) when compared with actual experimental data. Total phenolic content and FRAP values of thyme pressurized liquid extracts exhibited a sharp increase with the increase of temperature (Fig. 1e,f). For example TP and FRAP values increased by 46% and 51%, respectively, when the temperature was increased from 75  C to 120  C at 40% methanol concentration. Optimal PLE conditions for thyme were 33% methanol and a temperature of 129  C. Similar to the other tested Lamiaceae spices, thyme showed high regression coefficients (Table 2). Of the three spices, thyme was optimally extracted with the lowest ratio of methanol to water (2 : 1), indicating that thyme may contain more hydrophilic antioxidant compounds than the other two spices examined. For thyme, methanol concentration had no significant (p < 0.05) effect on the level of total phenolic content or FRAP activity. Temperature was the only factor which had significant effect on TP and FRAP of the thyme extracts at a linear level (Fig. 2e, f). As for sage and basil, FRAP and TP values of optimally extracted thyme PLE were significantly (p < 0.05) higher than solid/liquid extracts (Table 3). The models for TP and FRAP in thyme were also very significant (p < 0.0001) and highly predictive. The regression coefficients for TP and FRAP values were 0.819 and 0.890, respectively (Table 2), and the lack of fit was non-significant (p > 0.05). The experimental TP and FRAP values showed a high degree of similarity with the predicted values from the models with Pearson’s correlation coefficients (r) of 0.951 and 0.945 for TP and FRAP respectively. This study also validated the developed model, using a distinct set of data obtained from additional test runs, which is recommended by Carrasco et al.37 The experimental data were plotted against the predicted values from eqn (3) for polynomial models. Predicted values of FRAP and TP obtained using model equations were in good agreement with the This journal is ª The Royal Society of Chemistry 2010

0.651 0.999

60/129

0.942 0.819 0.890

33/129

experimental values. The experimental and predicted values were closely correlated, as demonstrated by regression coefficient (R2). The predicted values were within the 95% confidence interval. Yi ¼ b0 + b1X1 + b2X2 + b11X21 + b22X22 + b12 (X1X2)

(3)

where Yi is predicted response for FRAP and TP content. The applicability of the models was also quantitatively evaluated by comparing the bias and accuracy factors for each of the parameter (eqn (2a) and (2b)). In most cases, as shown in (Table 4), the accuracy factor (AF) values for the predicted model were close to 1.00. The bias factor (BF) values for the predicted models were also close to 1.00, ranging from (0.96– 1.02) for all the parameters studied. These results demonstrate that there was a good agreement between predicted and observed values. Ross et al.38 reported that predictive models should ideally have an AF ¼ 1.00, indicating a perfect model fit where the predicted and actual response values are equal. However, Ross et al.38 and Carrasco et al.37 reported that the AF of a fitted model increases by 0.10–0.15 units for each predictive variable in the model. Since the current model forecasted a response from two predictive variables (temperature, solvent concentration), it was expected to have AF values ranging from 1.20 to 1.3038 or an equivalent percentage error range of 20–30%. Despite some variations, results obtained from the validated predicted model and actual experimental values showed that the established models reliably predicted FRAP and TP content. The predicted values were in close agreement with experimental values (Table 4) and were found to be not significantly different at p > 0.05 using a paired t-test. In addition variations between the predicted and experimental values obtained for total antioxidant activity by FRAP assay and TP content were within acceptable error range as depicted by average mean deviation (E%, Table 4); therefore, the predictive performance of the established model may be considered acceptable. 3.2. Effect of elevated temperatures (>129  C) on rosmarinic acid and other phenolic acids in relation to antioxidant activity of pressurized liquid extracts of Lamiaceae spices Pressurized liquid extracts obtained using optimized parameters had significantly (p < 0.05) higher amounts of rosmarinic acid Food Funct., 2010, 1, 269–277 | 273

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Fig. 2 Standardised pareto chart of sage (a,b), basil (c,d) and thyme (e,f) showing the effect of different factor terms [A: Methanol concentration, B: Temperature, AA: (Methanol concentration)2, BB: (Temperature)2 and AB: Methanol concentration X Temperature] on TP and FRAP values. Bars exceeding the vertical line on the graph indicate that the corresponding factor terms are significant (p < 0.05).

than the conventional solid/liquid extracts (Table 5) in the samples tested. Since all three spices used showed the best performance at 129  C, the highest temperature used in the design, extraction temperatures of 150  C, 175  C and 200  C were tested with the respective optimal methanol concentrations. For all the increased temperatures, a sharp decrease (range: 38– 72%) in the levels of rosmarinic acid in the extracts were observed as shown in Fig. 3 and Table 5. However, total phenolic content and FRAP values increased with higher temperatures as shown in Table 3. One possible explanation for this behaviour is the 274 | Food Funct., 2010, 1, 269–277

degradation of rosmarinic acid into compound/s which had higher antioxidant activity than the parent compound. Other authors have also reported the loss of rosmarinic acid occurred at relatively low temperatures in conventional solid/liquid extraction (105  C).39 In the present study, decreases in rosmarinic acid levels at temperatures of 150  C or higher, were accompanied by increases in the caffeic acid content of the extracts with the highest level being reached at 200  C (Fig. 3 and Table 5). Since rosmarinic acid is an ester of caffeic acid and 3, 4-dihydroxyphenyl lactic acid, caffeic acid may be one of the degradation This journal is ª The Royal Society of Chemistry 2010

Optimal 150  C 175  C 200  C Solid/liquid Optimal 150  C 175  C 200  C Solid/liquid Optimal 150  C 175  C 200  C Solid/liquid

Sage

This journal is ª The Royal Society of Chemistry 2010 0.243  0.002 0.552  0.002 0.583  0.002 0.708  0.004 0.316  0.001 0.428  0.002 0.656  0.003 0.750  0.001 0.822  0.003 0.680  0.001 0.521  0.002 0.682  0.002 0.713  0.003 0.813  0.002 0.375  0.001

1.10  1.63  2.52  2.65  1.07  0.76  1.14  1.41  1.57  0.61  0.64  1.13  1.37  1.41  0.52  0.002 0.006 0.005 0.010 0.002 0.001 0.003 0.002 0.011 0.001 0.002 0.002 0.004 0.006 0.004

Melanoidins (Absorbance at 420 nm)

Gallic acid (mg/g DW) 6.92  0.005 7.37  0.009 7.68  0.016 7.77  0.037 6.08  0.015 4.12  0.011 4.62  0.008 4.83  0.005 5.41  0.014 2.18  0.004 3.32  0.012 3.74  0.007 4.18  0.006 4.79  0.009 2.46  0.003

58/129 80/100 40/70 60/129 80/129 40/70 33/129 80/100 40/70

Sage

Thyme

Basil

Solvent Conc/temp

Spice 0.96 1.01 0.99 0.97 0.98 1.03 1.02 0.97 1.01

BF

TP

1.04 1.01 1.01 1.03 1.02 1.03 1.02 1.03 1.01

AF 0.98 0.99 1.00 0.99 0.98 1.02 1.00 0.99 0.99

BF

FRAP

1.02 1.01 1.00 1.01 1.02 1.06 1.00 1.01 1.03

AF

6.18 4.89 5.40 3.77 2.52 2.32 3.54 2.32 1.52

Predicted

TP

6.92 4.75 5.51 4.12 2.67 2.10 3.32 2.55 1.48

Observed

3.56 1.02 0.64 2.83 1.83 3.53 2.20 3.08 1.00

E%

16.35 14.81 13.96 9.42 5.86 5.84 9.08 6.25 4.30

Predicted

FRAP

TP (g GAE/100 g DW)

Table 4 Bias factor (BF), accuracy factor (AF), actual, predicted and average mean deviation (E%) for the response (TP and FRAP) studied

Thyme

Basil

Treatment

Name of the spice

17.42 15.20 14.15 9.76 6.15 5.52 9.05 6.46 4.45

Observed

17.42  0.045 18.35  0.063 19.12  0.087 20.34  0.104 14.54  0.063 9.76  0.042 10.51  0.062 11.72  0.035 13.15  0.043 5.84  0.009 9.05  0.013 9.94  0.046 11.74  0.074 12.25  0.037 8.20  0.024

2.04 0.84 0.42 1.16 1.55 1.96 0.11 1.07 1.11

E%

FRAP (g Trolox/100 g DW)

Table 3 Content of gallic acid, melanoidin formation indicator, total phenol and antioxidant activity of the extracts obtained at temperatures of 150  C and above in comparison to solid/liquid and optimal extracts

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Food Funct., 2010, 1, 269–277 | 275

A B C D A A A B C A A B C D A Not detected 0.766 1.065 1.352 Not detected 0.044 0.051 1.041 1.214 0.041 0.071 0.181 0.376 0.565 0.032 Same letters in each sample indicate no significant (p $ 0.05) difference.

Thyme

Basil

276 | Food Funct., 2010, 1, 269–277

a

0.429 0.550 0.575 0.631 0.126 0.601 0.897 1.174 1.459 0.068 0.207 0.253 0.288 0.302 0.098 15.881 12.941 10.361 10.440 14.975 4.255 3.755 2.464 2.517 3.410 4.401 3.224 1.462 1.233 3.36 Optimal 150  C 175  C 200  C Solid/liquid Optimal 150  C 175  C 200  C Solid/liquid Optimal 150  C 175  C 200  C Solid/liquid Sage

D B A A C D C B A B C B A A B

B C C D A B C D E A B C D D A

Significancea 3,4-Dihydroxyphenyl lactic acid (mg/g DW) Significancea Caffeic acid (mg/g DW) Significancea Rosmarinic acid (mg/g DW) Treatment Name of the spice

Table 5 Quantity of rosmarinic acid, caffeic acid and 3,4-dihydroxyphenyl lactic acid in optimal PLE extracts, conventional solid/liquid extracts and the PLE extracts obtained at 150  C, 175  C and 200  C with optimal methanol concentrations

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Fig. 3 HPLC chromatograms of PLE extracts obtained at 75  C (I), 129  C (II), 150  C (III), 175  C (IV) and 200  C (V) using respective optimal solvent concentrations in sage (a), basil (b) and thyme (c) showing the changes in peaks of different polyphenols (1 ¼ gallic acid, 2 ¼ caffeic acid, 3 ¼ luteolin-7-O-glucoside, 4 ¼ 3,4-dihydroxyphenyl lactic acid, 5 ¼ apigenin-7-O-glucoside, 6 ¼ rosmarinic acid, 7 ¼ carnosic acid and 8 ¼ ferulic acid).

products. The antioxidant activity as measured by FRAP assay of pure caffeic acid (423.72 g Trolox/100 g DW) was higher than that of rosmarinic acid (406.30 g Trolox/100 g DW). The other possible degradation product 3,4-dihydroxyphenyl lactic acid was also reported to have high antioxidant activity.40 Moreover, the quantity of gallic acid which is a powerful antioxidant (346.21 g Trolox/100g DW as measured by FRAP assay) in the extracts obtained at 150  C or above was significantly higher than that of the extracts obtained at 129  C (Table 3). In fact, the extracts of sage obtained at 200  C contained 2.4 times the gallic This journal is ª The Royal Society of Chemistry 2010

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acid concentration than the extracts obtained at 129  C. Higher concentration of gallic acid, caffeic acid and 3,4-dihydroxyphenyl lactic acid might explain the increase of antioxidant capacity at temperatures of 150  C or above. Quantity of other phenolics such as luteolin-7-O-glucoside, apigenin-7-O-glucoside, carnosic acid of PLE extracts of sage, basil and thyme obtained at 150  C or above did not show significant (p > 0.05) change in comparison to optimal PLE extracts. In addition, the present study observed the formation of Maillard reaction products (melanoidins) at temperatures of 150  C or above. The absorbance at 420 nm of the PLE extracts obtained at 150  C or above was significantly higher than that of optimal and solid/ liquid extracts (Table 3). The increase in antioxidant activity of the extracts obtained at 150  C or above may be related to the production of melanoidins, reported to have antioxidant capacity.41 However, since the Maillard reaction has been reported to produce potentially harmful compounds42 and changed the natural phenolic profile of the extracts, extractions carried out at temperatures in the range of 150–200  C are not recommended. Therefore, PLE at 129  C with respective methanol concentration was determined to be the optimum for extracting antioxidant compounds from the studied spices.

4. Conclusion The present study demonstrated that total phenolic content and FRAP values of the PLE extracts of the spices tested increased significantly with the increase of temperature. PLE temperature of 129  C performed the best. Methanol concentration showed significant effect on the parameters in sage and basil. The optimal methanol concentration for sage and basil was 58% and 60%, respectively. Results of the model validation showed that the developed model had acceptable predictive performance, as assessed by mathematical and graphical model performance indices. The coefficient of determinations (R2) for predicted FRAP and TP content showed good correlation with the experimental data at the 95% confidence level. Thermal degradation of rosmarinic acid was only detected at temperatures higher than 150  C. Optimal PLE extracts of all the spices tested showed significantly (p < 0.05) higher total phenol content and FRAP values than the conventional solid/liquid extracts. Rosmarinic acid content in optimal PLE extracts was also higher than the solid/liquid extracts. Therefore optimised PLE could be used as a high throughput extraction system for spice phenolics.

Acknowledgements This work was supported by the ABBEST scholarship programme of Dublin Institute of Technology, Dublin, Ireland. The authors are thankful to Teagasc, Ashtown Food Research Centre, Ashtown, Dublin for providing laboratory facilities. We would like to thank AllinAll Ingredients Ltd, Dublin 12 for providing spice samples and Hilde Henny Wijngaard for her advice in RSM.

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Protective role of air potato (Dioscorea bulbifera) of yam family in myocardial ischemic reperfusion injury Hannah Rachel Vasanthi,a Subhendu Mukherjee,b Diptarka Ray,b Karuppiah Shanmugasundara Pandian Jayachandran,c Istvan Leklib and Dipak Kumar Das*b

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Received 21st June 2010, Accepted 19th October 2010 DOI: 10.1039/c0fo00048e Hydroalcoholic extract of Dioscorea bulbifera (DB), a yam variety called air potato, was tested for its protective effect on myocardial ischemic/reperfusion (I/R) injury in rats due to apoptosis and necrosis. Myocardial I/R injury was induced by 30 min ischemia followed by 2 h reperfusion by perfusing isolated rat hearts with Krebs Henseilet bicarbonate (KHB) buffer in a Langendorff set up. Pretreatment of DB (150 mg kg1 body weight) for 30 days significantly reduced myocardial infarct size and improved the ventricular function (aortic flow and coronary flow, LVDP, LVmax dp/dt). Role of DB on apoptosis was also evaluated by determining caspase 3 as well as by examining pro-apoptotic and anti-apoptotic proteins Bax and Bcl2 by Western blot analysis followed by TUNEL assay. DB also prevented I/R-mediated down regulation of survival protein Akt and HO-1. Our results indicated that Dioscorea bulbifera could ameliorate myocardial ischemia and reperfusion injury by improving ventricular function and inhibition of cardiomyocyte necrosis and apoptosis.

Introduction

Materials and methods

Ischemia and reperfusion (I/R) causes myocardial infarction potentiated by both necrosis and apoptosis.1 Moreover, accumulating evidence indicates that, apart from necrosis, apoptosis contributes significantly to post ischemic cardiomyocyte death, suggesting that therapeutic intervention that inhibits apoptotic cell death may attenuate I/R-induced cardiomyocyte injury.2,3 Recently, there have been many scientific claims that botanicals provide cardioprotection against (I/R) injury in a variety of experimental models via multiple mechanisms including inhibition of apoptotic cell death.4–6 Dioscorea bulbifera (DB), commonly known as aerial yam or air potato, belonging to the Dioscoreacae family, is widely distributed in India, Ceylon, the Malay Peninsula, Australia, East Africa and Brazil.7 It is one of the important medicinal plants used in indigenous systems of medicine in Asia.8 Dioscorea species are most noted for the abundance of diosgenin, a steroidal saponin used as a precursor for the synthesis of corticosteroids, estrogen, contraceptives, and spiranolactones.9 Recently, diosgenin has been found to ameliorate myocardial infarction by its anti-lipoperoxidative activity.10 Hence, this study aimed to investigate whether the extract of Dioscorea bulbifera could reduce myocardial I/R injury-induced apoptosis in the rat heart.

Chemicals and antibodies

a Department of Biotechnology, School of Life Sciences, Pondicherry University, Puducherry, Tamilnadu, India b Cardiovascular Research Center, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT, 06030-1110, USA. E-mail: [email protected]; Fax: +1 (860) 679-4606; Tel: +1 (860) 679-3687 c CARISM, SASTRA University, Thanjavur, Tamilnadu, India

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The solvents used for plant extraction were of analytical grade and obtained from Sisco Research Laboratory (SRL), India. All other chemicals used were of analytical grade and were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO), unless otherwise specified. Primary antibodies such as Akt, pAkt, Bax, Bcl2, HO-1, pro and cleaved caspase - 3 and glyceraldehyde-6phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Preparation of plant extract Fresh Dioscorea bulbifera (DB) tubers were purchased from Ayurmed Biotech Pvt. Ltd, Mumbai, India, and it was taxonomically identified and authenticated by a taxonomist and a voucher specimen has been preserved for further reference. The tubers were cleaned, dried under shade at room temperature and coarsely pulverized using a mechanical grinder. The dried powder (1 kg) was extracted using a Soxhlet-extractor in 70% ethanol. Hydroalcoholic extracts were evaporated (free of solvents) using a rotary evaporator under reduced pressure at 40  C. A brown concentrated hydroalcoholic extract was obtained (yield 8.52% w/w with respect to the dried starting material). The final product was then stored at room temperature in a dessicator for further use. Animals All animals used in this study received humane care in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving This journal is ª The Royal Society of Chemistry 2010

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animals; the care adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC (Publication Number NIH 85-23, revised 1996). Sprague–Dawley male rats weighing between 250 and 300 g were used for the experiment. The rats were randomly assigned to two groups: control and hydroalcoholic extract of DB treated. The rats were fed ad libitum regular rat chow with free access to water. The experimental rats were gavaged with 1 ml of DB extract dissolved in water (150 mg kg1 of body weight) for 30 days, whereas the control group of rats were gavaged 1 ml of the vehicle for the same period of time. Isolated working heart preparation At the end of 30 days, the rats were anesthetized with sodium pentobarbital (80 mg kg1 of BW, ip) (Abbott Laboratories, North Chicago, IL) and anticoagulated with heparin sodium (500 IU/kg of BW, I.V) (Elkins-Sinn Inc., Cherry Hill, NJ) injection. After euthanising the rats, a thoracotomy was performed, and the heart was perfused in the retrograde Langendorff mode at 37  C at a constant perfusion pressure of 100 cm of water (10 kPa) for a 5 min washout period.11 The perfusion buffer used in this study consisted of a modified Krebs–Henseleit bicarbonate buffer (KHB): sodium chloride, 118 mM; potassium chloride, 4.7 mM; calcium chloride, 1.7 mM; sodium bicarbonate, 25 mM; potassium biphosphate, 0.36 mM; magnesium sulfate, 1.2 mM; and glucose, 10 mM. The Langendorff set up was switched to the working mode following the washout period. At the end of 10 min, after the attainment of steady state cardiac function, baseline functional parameters were recorded. The circuit was then switched back to the retrograde mode, and the hearts were perfused for 15 min with KHB for stabilization.12 The hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. The first 10 min of reperfusion was in the retrograde mode to allow for post-ischemic stabilization and thereafter, in the antegrade working mode to allow for assessment of functional parameters, which were recorded at 30, 60, and 120 min of reperfusion. Assessment of ventricular function Aortic pressure was measured using a Gould P23XL pressure transducer (Gould Instrument Systems Inc., Valley View, OH) connected to a side arm of the aortic cannula; the signal was amplified using a Gould 6600 series signal conditioner and monitored on a CORDAT II real-time data acquisition and analysis system (Triton Technologies, San Diego, CA).12 Heart rate (HR), left ventricular developed pressure (LVDP) (defined as the difference of the maximum systolic and diastolic aortic pressures), and the first derivative of developed pressure (dp/dt) were all derived or calculated from the continuously obtained pressure signal. Aortic flow (AF) was measured using a calibrated flow meter (Gilmont Instrument Inc., Barrington, IL), and coronary flow (CF) was measured by timed collection of the coronary effluent dripping from the heart. Determination of infarct size At the end of reperfusion, a 1% (w/v) solution of triphenyl tetrazolium (TTC) in phosphate buffer was infused into the This journal is ª The Royal Society of Chemistry 2010

aortic cannula for 20 min at 37  C.13 The hearts were excised, and the sections (1 mm) of the heart were fixed in 2% paraformaldehyde, placed between two coverslips, and digitally imaged using a Microtek ScanMaker 600z. To quantify the areas of infarct in pixels, a NIH image 5.1 (a public domain software package) was used. The infarct size was quantified and expressed in pixels. TUNEL assay for assessment of apoptotic cell death Immunohistochemical detection of apoptotic cells was carried out using the terminal dUTP nick-end labeling (TUNEL) method (Promega, Madison, WI).14 The heart tissues were immediately put in 10% formalin and fixed in an automatic tissue-fixing machine. The tissues were carefully embedded in molten paraffin in metallic blocks, covered with flexible plastic molds and kept under freezing plates to allow the paraffin to solidify. The metallic containers were removed, and tissues became embedded in paraffin on the plastic molds. Prior to analyzing tissues for apoptosis, tissue sections were deparaffinized with xylene and washed in succession with different concentrations of ethanol (absolute, 95%, 85%, 70%, 50%). Then the TUNEL staining was performed according to the manufacturer’s instructions. The fluorescence staining was viewed with a fluorescence microscope (AXIOPLAN2 IMAGING) (Carl Zeiss Microimaging, Inc., NY) at 520 nm for green fluorescence of fluorescein and at 620 nm for red fluorescence of propidium iodide. The number of apoptotic cells was counted throughout the slides and expressed as a percent of total myocyte population. Preparation of subcellular fractions Cardiac tissues were homogenized in 1 mL of buffer A (25 mM Tris-HCl, pH 8, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 nM okadaic acid, 0.5 mM EDTA, 1 mM PMSF, and 1 protease inhibitor cocktail) in a Polytron homogenizer. Homogenates were centrifuged at 3000 rpm at 4  C for 10 min, and the nuclear pellet was resuspended in 500 mL of buffer A with 0.1% Triton X-100. Supernatant from the above centrifugation was further centrifuged at 10, 000 rpm at 4  C for 20 min, and the resultant supernatant was used as cytosolic extract. The mitochondrial pellet was resuspended in 200–300 mL of buffer A with 0.1% Triton X-100. The nuclei pellet and mitochondrial pellet were lysed by incubation for 1 h on ice with intermittent tapping. Homogenates were then centrifuged at 14 000 rpm at 4  C for 10 min, and the supernatant was used as nuclear lysate and mitochondrial lysate, respectively. Cytosolic and nuclear extracts were aliquoted, snap frozen, and stored at 80  C until use. Total protein concentrations in cytosolic, and mitochondrial extracts were determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). Western blot analysis Western blot was performed to measure apoptosis-related proteins such as Bax, Bcl2, p-Akt, pro and cleaved caspase - 3 and HO-1. Proteins were separated in SDS-PAGE after estimating the total protein content in each sample. Following electrophoresis, proteins were transferred to nitrocellulose filters. Food Funct., 2010, 1, 278–283 | 279

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Filters were blocked with TBS-T (20 mM Tris HCl pH 7.5, Tween 20) containing 5% (w/v) nonfat dry milk and probed with the respective primary antibodies overnight.14 All primary antibodies were used at the dilution of 1 : 1000. Protein bands were identified with horseradish peroxidase conjugated secondary antibody (1 : 2000 dilutions) and Western blotting luminol reagent (Santa Cruz Biotechnology). GAPDH was used as loading control. The resulting blots were digitized, subjected to densitometric scanning using a standard NIH image program, and normalized against loading control. Statistical analysis The values of myocardial functional parameters, total and infarct volumes and infarct sizes are all expressed as the mean  standard error of the mean (SEM). Analysis of variance test followed by Bonferroni’s correction was first carried out to test for any differences between the mean values of all groups. If differences between groups were established, the values of the treated groups were compared with those of the control group by a modified t-test. The results were considered to be significant if p < 0.05.

Results Effects of DB extract on left ventricular function We first determined if the hearts of DB-treated animals displayed improved ventricular performance compared to those of control

animals. As shown in Fig. 1, aortic flow, LVDP, and LVmax dp/dt of DB treated hearts consistently displayed improved performance compared to the control group during post ischemic reperfusion. The heart rate did not vary significantly between the two groups. Coronary flow of the DB group was higher compared to the control group only at the end of 60 min and 2 h of reperfusion.

Effects of DB on myocardial infarct size Myocardial infarct size at the end of 30 min of ischemia and 2 h of reperfusion as determined by TTC staining method was about 35  1.17% normalized to area of risk (Fig. 2). There was no infarction when the hearts were perfused with the KHB buffer without subjection to ischemia and reperfusion (control) (data not shown). DB significantly reduced myocardial infarct size to about 20  2.64% as compared to the control group.

Effects of DB on cardiomyocyte apoptosis Cardiomyocyte apoptosis was determined by TUNEL assay. As shown in Fig. 3, it was about 39.42  3.5% at the end of reperfusion in case of control group. There were reduced number of apoptotic cells (4.96  0.75%) in the non-treated control hearts perfused with the Krebs–Henseleit bicarbonate buffer without subjection to ischemia and reperfusion. DB significantly reduced the number of apoptotic cardiomyocytes to 16.89  1.7% revealing that DB exhibits anti-apoptotic activity.

Fig. 1 Effect of ischemia/reperfusion on heart rate, aortic flow, LVDP, Maximum first derivative of developed pressure, coronary flow of control rat heart and DB treated rat heart. Results are shown as mean  SEM. * p < 0.05 vs. control.

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expression of pro-apoptotic protein Bax compared to the control value. DB extract significantly increased Bcl2 expression and decreased Bax expression. Taken together, DB treatment reduced the ratio of Bax/Bcl2 as compared to the control hearts. I/R reduced the phosphorylation of Akt, which was prevented with DB (Fig. 5). Similarly there was an upregulation of procaspase - 3 in the DB treated group whereas a significant downregulation was observed in the Cleaved caspase - 3 when compared to that of the ischemic/reperfusion group. To determine the effect of the DB extract on phase II enzyme HO-1 with proven cardioprotective ability, we studied the expression of the HO-1 protein. As shown in Fig. 5, HO-1 was significantly reduced in the ischemic reperfused myocardium, and DB extract prevented the loss of HO-1.

Discussion Fig. 2 Effect of DB on myocardial infarct size. Results are shown as mean  SEM. * p < 0.05 vs. normal, ‡ p < 0.05 vs. ischemic control, n ¼ 3 in each group.

Fig. 3 Effects of DB on cardiomyocyte apoptosis. Panel A is total number of apoptotic cells (green channels). Panel B is total number of cells (red channel). Values are Mean  SEM. * p < 0.05 vs. ischemic control. Representative photomicrographs are shown below the bar graphs. Results are shown as mean  SEM. * p < 0.05 vs. ischemic control.

Modulation of pro- and anti-apoptotic proteins by DB We examined the modulation of the pro- and anti-apoptotic protein signals triggered by DB extract. Fig. 4 shows the results. I/R, regardless of reperfusion time, significantly reduced the expression of the anti-apoptotic protein Bcl2, but increased the This journal is ª The Royal Society of Chemistry 2010

In the present study, we showed that administration of DB extract in vivo improved cardiac function as well as reduced the myocardial infarct size and cardiomyocyte apoptosis following myocardial I/R in rats. We further found that the cardioprotective effects of the yam extract could be explained in part by its modulation of apoptosis-related protein signals. Although there are some reports on the protective effect of wild yam in postmenopausal women for a healthy heart, there are no studies to explore the relationship between the putative anti-apoptotic effects of DB on the functional recovery of the ischemic reperfused myocardium. The present study demonstrated that DB treatment preserved the left ventricular function as reflected by a significant increase in the indices of contractility and relaxation. It is well established that cell death during I/R occurs via necrotic and apoptotic pathways.15,16 The mitochondrial death pathway appears to play an important role in the execution of apoptosis in cardiac myocytes.17 We found that DB reduced I/R injury with an associated reduction in apoptotic cell death as evidenced by the modulation of genes related to apoptosis especially Bcl2 and Bax. It is well known that anti-apoptotic protein Bcl2 has potent cardioprotective effects.18 It exerts cardioprotection by multiple mechanisms such as antioxidant effect, inhibition of pro-apoptotic proteins, mitochondrial membrane stabilization and inhibition of release of cytochrome C. Proapoptotic protein Bax has been reported to be activated in cardiac cells in response to oxidative stress and during ischemia.19 In the present study, Bax activity was down-regulated in the DB treated rat hearts as compared to the control rat hearts resulting in considerable reduction in the Bax/Bcl2 ratio. The altered Bcl  2/Bax ratio attenuates cleavage of Caspase 3 which is well shown in our results. There was a significant expression of Procaspase 3 in the DB treated group and a down regulated expression of Cleaved Caspase 3. Polyphenols and flavonoids from plant products have a direct action on the up regulation of phase II enzyme like HO-1 in a dose dependent manner.21 One of the possible mechanisms by which DB modulates apoptosis may be due to it antioxidant action. The results of the present study suggest such a possibility as DB extract resulted in the overexpression of HO-1. Cardioselective over expression of HO-1 protein improves vascular function by increasing superoxide dismutase and catalase Food Funct., 2010, 1, 278–283 | 281

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Fig. 4 Western blot analysis of Bax, Bcl2 protein from the cytosolic fraction of control, ischemic and DB treated heart samples. GAPDH was used as the loading control. Figures are representative images of three different groups, and each experiment was repeated at least thrice. Bar diagram for Bax/ Bcl2 ratio. Results are shown as mean  SEM. * p < 0.05 vs. ischemic control.

progesterone. A recent study revealed that diosgenin in DB exhibits cardioprotection by reducing lipid peroxidation and membrane liability to lysosomal damage in isoproterenol induced myocardial infarction.10 In conclusion, the results of the present study suggest that Dioscorea bulbifera extract ameliorates rat myocardial ischemia and reperfusion injury with an associated reduction in apoptotic cell death. It is an important endangered medicinal plant that is commercially exploited for the extraction of diosgenin, a precursor of steroid used to produce birth control pills. The results that Dioscorea bulbifera, a yam variety called air potato can also protect the heart from I/R injury demonstrates its effectiveness as molecular target for ischemic heart as it might be useful for potential drug development for this purpose.

Acknowledgements Fig. 5 Western blot analysis of p-Akt, heme oxygenase protein HO-1, procaspase 3 and cleaved caspase 3 from the cytosolic fraction of control, ischemic and DB treated heart samples. GAPDH was used as the loading control. Figures are representative images of three different groups, and each experiment was repeated at least thrice.

activity.22 In addition, up regulation of HO-1 exerts a cardioprotective effect after myocardial I/R in mice, and this effect is probably mediated via the anti-apoptotic action of HO-1.23 The results of the present study indicating significant reduction in the expression of HO-1 after I/R in the control heart is consistent with the results of a previous study.20 Finally, DB prevented I/R-induced down regulation of the survival protein Akt phosphorylation. Dioscorea bulbifera contains a large number of polyphenols and organic acids, some of which may function as antioxidants. It contains significant amount of polyphenols and possesses potent antioxidant action.24 The polyphenolic components of yam range from 13 to 166 mg/100 gm comprising catechins, chlorogenic acids, proanthocyanidins and anthocyanins.25 Moreover it also contains flavonoids such as kampferol3,5-dimethyl ether, myricetin, quercitin 3-O-galactopyranoside, diosbulbin and myricetin 3-O-galactopyranroside.26 Most importantly, Dioscorea bulbifera yam contains the steroid diosgenin, the principal material to manufacture birth control pills as it can overcome the negative effects of DHEA and 282 | Food Funct., 2010, 1, 278–283

The first author is grateful to the Department of Science and Technology, Govt of India for providing the BOYSCAST Fellowship (2007) to work in the laboratory of Dr Dipak K. Das funded by the NIH at UCONN Health Centre, Farmington, CT, USA. The research was supported in part by NIH HL 34360, HL 22559 and HL 33889.

References 1 A. Hamacher-Brady, N. R. Brady and R. A. Gottlieb, Cardiovasc. Drugs Ther., 2006, 20, 445–462. 2 R. A. Gottlieb and R. L. Engler, Ann. N. Y. Acad. Sci., 1999, 874, 412–426. 3 Y. C. Jin and Y. M. Kim, Vasc. Pharmacol., 2009, 50, 71–77. 4 Y. C. Jin, K. J. Kim, Y. M. Kim, Y. M. Ha, H. J. Kim, E. J. Yun, K. H. Bae, Y. S. Kim and S. S. Kang, Exp. Biol. Med., 2008, 233, 280–1288. 5 I. T. Nizamutdinova, Y. C. Jin, J. S. Kim, M. H. Yean, S. S. Kang, Y. S. Kim, J. H. Lee, H. G. Seo and H. J. Kim, Planta Med., 2008, 74, 14–18. 6 S. Mukherjee, H. Gangopadhyay and D. K. Das, J. Agric. Food Chem., 2008, 56, 609–617. 7 E. S. Ayensu and D. G. Coursey, Econ. Bot., 1972, 26, 301–318. 8 C. Djerassi, Steroids, 1992, 57, 631–641. 9 V. P. Kamboj, Herbal Med. Curr. Sci., 2000, 78, 35–39. 10 K. S. Jayachandran, H. R. Vasanthi and G. V. Rajamanickam, Mol. Cell. Biochem., 2009, 327, 203–210. 11 D. T. Engelman, M. Watanabe, R. M. Engelman, J. A. Rousou, E. Kisin, V. E. Kagan, N. Maulik and D. K. Das, Cardiovasc. Res., 1995, 29, 133–140. 12 I. Lekli and S. Das, J. Agric. Food Chem., 2008, 56, 5331–5337.

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13 I. Lekli, G. Szabo, B. Juhasz, S. Das, M. Das, E. Varga, L. Szendrei, R. Geszteyli and J. Varadi, Am. J. Physiol.: Heart Circ. Physiol., 2007, 294, H859–866. 14 S. Mukherjee and I. Lekli, Biochim. Biophys. Acta.: Mol. Basis Dis., 2008, 1782, 498–583. 15 N. Maulik and D. K. Das, Heart Failure Rev., 1999, 4, 165–175. 16 E. Murphy and C. Steenbergen, Physiol. Rev., 2008, 88, 581–609. 17 D. R. Green and J. C. Reed, Science, 1998, 281, 1309–1312. 18 H. R. Vasanthi, S. Mukherjee, I. Lekli, D. Ray, V. Gayathri and D. K. Das, J. Cardiovasc. Pharmacol., 2009, 53, 499–506. 19 A. B. Gustafasson and R. A. Gottlieb, J. Clin. Immunol., 2003, 23, 447–459. 20 C. K. Andreadi, L. M. Howells, P. A. Atherfold and M. M. Manson, Mol. Pharmacol., 2006, 69, 1033–1040.

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PAPER

www.rsc.org/foodfunction | Food & Function

Comparative healing property of kombucha tea and black tea against indomethacin-induced gastric ulceration in mice: possible mechanism of action Debashish Banerjee,a Sham A. Hassarajani,b Biswanath Maity,a Geetha Narayan,b Sandip K. Bandyopadhyaya and Subrata Chattopadhyay*b

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Received 29th May 2010, Accepted 7th October 2010 DOI: 10.1039/c0fo00025f The healing activity of black tea (BT) and BT fermented with Candida parapsilosis and kombucha culture, designated as CT and KT respectively against the indomethacin-induced stomach ulceration has been studied in a mouse model. The KT sample (KT4) produced by fermenting BT for four days, showed the best DPPH radical scavenging capacity and phenolics contents. Hence the ulcer-healing activity of KT4 was compared with those of CT4 and BT. All the tea extracts (15 mg kg1) could effectively heal the gastric ulceration as revealed from the histopathological and biochemical studies, with relative efficacy as KT4 > CT4  BT. The healing capacities of the tea extracts could be attributed to their antioxidant activity as well as the ability to protect the mucin content of the gastric tissues. In addition, the ability of KT4 to reduce gastric acid secretion might also contribute to its ulcer-healing activity. The tea preparation KT4 (15 mg kg1) was as effective as the positive control, omeprazole (3 mg kg1) in ulcer healing.

Introduction The gastro-toxicity of non-steroidal anti-inflammatory drugs (NSAIDs), often leading to gastric ulceration and delayed healing, remains a crucial problem, despite recent pharmaceutical advances.1,2 The currently available synthetic anti-ulcer drugs are expensive, show side effects, and cannot prevent ulcer recurrence.2 Exploration of plants/herbs, especially the edible varieties might provide suitable alternative anti-ulcer formulations. Many taxa of medicinal plants have been assessed worldwide for their antiulcerogenic effects.3 For decades, doctors have recommended dietary adjustments aimed at preventing or treating symptoms of gastritis and ulceration, as diet may moderate the risk for gastritis or peptic ulcer. Camellia sinenesis is widely grown in the tropical humid climate of South East Asia, and decoction of its leaves (tea) is the most popular non-alcoholic beverage worldwide. Different tea preparations such as green tea, black tea, oolang tea etc. are most commonly used. The cytoprotective action of the green teacatechins against ethanol- or restraint plus water-immersion stress-induced acute gastric mucosal injury, and acetic acidinduced chronic gastric ulcers in rats has been reported.4 In addition, epigallocatechin gallate, a constituent of green tea has been suggested to control H. pylori-related chronic inflammations or regress cancer precursor lesions, while a pectin-type acidic polysaccharide from green tea is reported to posses antiadhesive effects against H. pylori.5 It is believed that polyphenols or polyphenol derivatives from green tea may be useful either in prevention or treatment of H. pylori-associated gastric diseases. Although black tea (BT) accounts for 80% of the total tea a IPGME&R, 244B, Acharya Jagadish Chandra Bose Road, Kolkata, 700 020, India b Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India. E-mail: [email protected]; Fax: +91-22-25505151; Tel: +91-22-25593703

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consumption, studies on the pharmacological properties of BT are scarce. The prophylactic action of the tea seed-derived triterpene saponins against ethanol-induced gastric mucosal lesions,6,7 and of BT extract against various ulcerogens8,9 have been reported in rat models. Kombucha or Kargassok tea (KT) is a fermented black tea preparation that is widely consumed in parts of the erstwhile Soviet Union and Central Asia, and has become popular even in Europe and the USA. KT is made by steeping a flat, pancake-like culture, referred to as the Kombucha mushroom, which is actually a symbiotic culture of various yeasts and bacterial species including: Saccharomycodes ludwigii, Schizosaccharomyces pombe, Candida parapsilosis, Acetobacter ketogenum, Bacterium and Torula spp, etc. in brewed black tea and sugar or sucrose for about a week. It is purported to improve general health, aid longevity, boost the immune system, possess anti-oxidant, anti-ageing and chemopreventive properties, and provide relief/cure for chronic conditions such as rheumatism/arthritis, hypertension/arteriosclerosis, stomach/ intestinal/liver disorders and even cancer.10 More recently, its anti-stress and hepato-protective,11 antioxidant and immunopotentiating12 as well as anti-diabetic13 properties have been reported. However, the wide-ranging claims about the health benefits of KT are primarily based on personal observations and testimonials rather than supportive scientific evidence. The primary aim of the present study was to evaluate the healing property of KT and BT against indomethacin-induced acute gastric ulceration of mice and compare the activity with that of the drug, omeprazole (Omez). Factors such as oxidative stress and acid secretion contribute to stomach ulceration. Hence the anti-oxidative property and gastric acid inhibitory capacity of the test samples were also evaluated to rationalize their ulcer healing action. To the best of our knowledge, this is the first report on the stomach ulcer healing property of KT and BT. This journal is ª The Royal Society of Chemistry 2010

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Results

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Standardization of KT preparation and analysis of its composition In this investigation, we prepared two types of fermented teas by fermenting BT separately with kombucha culture, and C. parapsilosis for 2, 4 and 7 days. These are designated as KT and CT, followed by a number, signifying the days of incubation for their preparation. Parameters such as pH, absorbance, viscosity and radical scavenging property of the decoctions were measured. On increasing the fermentation time, a continuous drop of pH from 6.8 to 2.6 was observed with KT. The acid-producing microorganisms present in the kombucha culture are known to produce several organic acids including acetic acid, accounting for the reduction in the pH of KT.14 Besides acetic acid, the presence of glucuronic acid and lactic acid in KT4 was confirmed by TLC analysis. The pH change in the CT samples was much less (6.8 to 5.0). A drop in absorbance during fermentation was observed with KT, which was attributed to the lower availability of free theorubigin anions at the lower pHs. The theorubigin are also known to be degraded by the kombucha culture. The absorbance change amongst the CT samples was not significant. The viscosity of KT also decreased gradually with the increase in fermentation time, possibly due to the increased consumption of the added sugar. It is worth mentioning that the multiplication of the microorganisms in the kombucha culture was confined to the biofim. Hence, this did not increase the viscosity of KT. Amongst the KT samples, KT4 showed maximum total phenolics content, and accordingly best antioxidant capacity, as revealed from the DPPH scavenging assay. Hence, the ulcer healing activity was studied using KT4 only, and the results compared with those of BT and CT4 samples. It was also felt that KT7 would not be ideal for this study as its pH was too low (2.6) due to the generation of more acetic acid, which itself is a strong stomach ulcerogen. The results are summarized in Table 1. Preliminary TLC analyses of BT, CT4 and KT4 revealed the presence of additional compounds in KT4. The three major components of KT4 were isolated and characterized as theophylline, caffeine, and theobromine. Quantification of these compounds by a HPTLC densitogram revealed that fermentation of BT with the kombucha culture led to an increase in the theobromine content from 2.7% to 3.3% along with a substantial reduction in the caffeine level from 8% to 4%. The concentration of theophylline remained nearly the same (77.3%). Optimization of doses of the tea samples for gastric ulcer healing Ulceration in the mice was induced with a single dose of indomethacin (18 mg kg1) and the doses of the tea extracts

were optimized by carrying out the treatment with BT, CT4, and KT4 (10, 15 and 20 mg kg1) for seven days. Omez (3 mg kg1) was used as the positive control. The doses of indomethacin and Omez were decided based on the results of our previous studies.15 To evaluate the immediate effect of the test samples, similar experiments were also carried out under a one-day treatment regime. The extent of ulcer healing was assessed from the macroscopic and histological observations of the glandular portion of the gastric mucosa of the mice. The mice receiving vehicle only showed no mucosal lesions. Indomethacin administration to mice produced acute lesions in the gastric mucosa, measured in terms of macroscopic damage scores (MDS). Treatment with the test samples accelerated the healing of gastric lesions dose-dependently. Table 2 summarizes the dose-dependent ulcer-healing effect of the tea samples on continuing the treatment for 7 days. Optimal ulcer healing was obtained with 15 mg kg1 of all the tea samples, which did not improve significantly at a higher dose (20 mg kg1). Hence, we used this dose (15 mg kg1) of the tea extracts for assaying all other healing and biochemical parameters, which are presented in the following. The comparative MDS reductions due to natural healing, and treatment with BT, CT4, KT4 and Omez on the 1st and 7th days of ulceration are provided in Fig. 1a and Fig. 1b respectively. Treatment with CT4, KT4 and Omez for one day reduced the MDS values by 39.2%, 59.3% and 48.1% respectively, compared to the ulcerated control group. The effect of BT was insignificant. KT4 showed a better effect than Omez. Compared to the 1st dayuntreated mice (group II), the MDS of the 7th day-untreated mice (group III) was less by 34%, due to natural healing. However, the effect was more pronounced in the treated groups. Treatment with BT, CT4, KT4 and Omez for 7 days reduced the MDS by 56.1%, 61.7%, 71.5% and 64.4% respectively, compared to the group III mice. During the production of BT from green tea, a significant part of the catechins is converted to the theaflavins (TF). Our colorimetric assay14 revealed insignificant changes in the TF concentrations in BT and KT4. Hence in a separate experiment, we also studied the ulcer healing activity of TF. Treatment with TF (1 mg kg1) for 7 days was found to provide 81.4% ulcer healing. Histological assessment of ulcer healing The histopathological photographs of mice stomachs belonging to various groups are shown in Fig. 2a–g. Presence of crypts in the mucosal glandular layer with intact submucosa and muscular layer, and vaso congestion in serosa were noticeable in the

Table 1 Physico-chemical and radical scavenging properties of the tea samples Parameters

BT

CT2

CT4

CT7

KT2

KT4

KT7

pH Optical densitya Relative viscosityb Total phenolics (mg GAE g1 extract)c DPPH radical scavenging activity (%)c

6.8 0.44 24.81  2.26

6.4 0.41 1.08 29.16  2.12

5.48 0.39 1.20 33.68  2.28

5.01 0.36 1.16 34.24  2.71

5.45 0.38 0.98 38.52  0.68

4.23 0.15 0.94 44.89  2.10

2.58 0.08 0.89 40.5  1.56

39.77  2.14

43.62  2.44

49.20  3.06

49.70  2.73

59.64  7.81

73.47  4.15

61.06  7.61

a

Measured at 530 nm. b Ratio of the viscosities of the fermented tea samples and BT. c The values are mean  SEM (n ¼ 5).

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Table 2 Comparative healing capacity of BT, CT4, and KT4a

Group

Drug dose/mg kg1

Macroscopic damage scores (MDS)b

MDS reduction (%)c

Ulcerated BT-treated BT-treated BT-treated CT4-treated CT4-treated CT4-treated KT4-treated KT4-treated KT4-treated

– 10 15 20 10 15 20 10 15 20

1.17  0.03 0.62  0.02 0.57  0.03* 0.55  0.03 0.53  0.04 0.45  0.03* 0.43  0.02 0.52  0.02 0.36  0.01* 0.35  0.02

0 47.55 51.07 52.69 54.99 61.18 63.28 56.01 68.98 69.94

a Stomach ulceration in mice was induced by oral administration of indomethacin (18 mg kg1). Different doses of the tea samples were used for these experiments. b The MDS were measured on day seven after indomethacin administration. The values are mean  SEM, from three independent experiments, each with 5 mice per group. *p < 0.01, compared to the untreated control. c Considering a MDS value of 100 for the ulcerated, untreated mice.

normal mucosal epithelium (Fig. 2a). The stomach of the group II mice showed ruptures in different portions of the mucosal epithelial layer. Inflammatory infiltrates containing neutrophils were observed in the lamina propria, muscle coat and serosal layer with altered nucleus/cytoplasmic ratio. The submucosal and muscle layers were also not intact and inflammatory granules were seen in submucosa (Fig. 2b). The acute nature of ulceration was evident from the partial natural healing observed in the untreated control mice. Even on the 7th day of ulceration, multiple areas of ulceration, and hyperemia in the submucosal layer along with inflammatory exudates were noticed (Fig. 2c). All the tea samples showed a potent healing effect, because regenerative changes along the ulcerated margin were noticed in the 7-day treatment groups (Fig. 2d–f). Cryptic proliferation (crypt hyperchromasia) and lack of frank denudation were the prominent features of the healing. However, the effects of different test samples were different. The stomachs of the BTtreated mice showed an intact epithelial layer and mucosal glandular layer. However, the muscle layer was not intact and inflammatory exudates along with hyperemic submucosa were observed in their stomach tissues. The effect of CT4-treatment was also similar. But the presence of crypts as well as vasocongestion in muscle and serosal layers was noticed in the stomachs of the CT4-treated mice. KT4 treatment led to complete regeneration of the intact epithelial and muscle layers. The mucin containing cells were also prominent in the mucosal glandular layer, although hyperemic submucosa was still present. Omez treatment led to normal submucosa and an intact muscle layer. But the epithelial layer was not intact in this group of mice. Amongst the test samples, KT4 and Omez reduced the inflammation significantly and restored the mucosal architecture to near normalcy (Fig. 2f and Fig. 2g). The effects of BT and CT4 were also impressive, not significantly different from each other, but less than that of KT4. For better appreciation of the above results, the histopathological slides were also quantified in terms of damage scores (DS) and inflammatory score (IS). Compared to the ulcerated mice, those treated with BT, and CT4 for one day showed a reduction 286 | Food Funct., 2010, 1, 284–293

Fig. 1 (a) Comparative healing capacities of the tea samples on the 1st day of ulceration, as revealed from the MDS, DS and IS. 1: untreated group, 2: BT group, 3: CT4 group, 4: KT4 group, 5: Omez group. Ulceration in the mice was induced by oral administration of indomethacin (18 mg kg1). Different tea samples (15 mg kg1) and Omez (3 mg kg1) were used as the drugs. The assays were carried out 10 h after indomethacin administration and the values are mean  SEM, from three independent experiments, each with 5 mice per group. *p < 0.05, ** p < 0.01, compared to the untreated control (group II); †p < 0.05, compared to Omez-treatment. (b) Comparative healing capacities of the tea samples under the optimized treatment regime, as revealed from the MDS, DS and IS. 1: Untreated 1st day group, 2: untreated 7th day group, 3: BT group, 4: CT4 group, 5: KT4 group, 6: Omez group. Ulceration in the mice was induced by oral administration of indomethacin (18 mg kg1). Different tea samples (15 mg kg1) and Omez (3 mg kg1) were used as the drugs. The assays were carried out 7 days after indomethacin administration and the values are mean  SEM, from three independent experiments, each with 5 mice per group. *p < 0.05, ** p < 0.01, #p < 0.001, compared to the untreated control (group III).

of DS by 50.0% and 56.3% respectively, while KT4 and Omez reduced the DS by 75.2% and 67.5%. KT4 was more potent than Omez. Likewise, BT, CT4, KT4, and Omez also reduced the IS by 29.5%, 35.5%, 49.0%, and 45.4% respectively, compared to the corresponding untreated group. Compared to the group II mice, natural healing during seven days decreased the DS and IS values of the group III mice marginally (15.3% and 11.4% respectively). Treatment with BT and CT4 for seven days reduced the DS by 70.3% and 71.5%, compared to the group III mice. KT4 and Omez showed This journal is ª The Royal Society of Chemistry 2010

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to the normal value. BT and CT4 reduced it by 13.5% and 18.6% respectively, compared to that of the group II mice. KT4 and Omez reduced the TBARS level almost equally (31%). Even after seven days of ulceration, the TBARS content in the untreated group remained significantly higher (49.5%) than the normal value. Treatment with BT, CT4, KT4 and Omez for seven days reduced it by 27.2%, 23.2%, 33.1%, and 37.1% respectively, compared to the group III mice. The effect of KT4 and Omez was similar, restoring the TBARS level to the normal value. Likewise, the protein carbonyl contents of the ulcerated mice were significantly elevated by 79% and 67.7% respectively on the 1st and 7th days of ulceration, compared to the normal values. One-day treatment with BT reduced it by 16.7%, while CT4, KT4 and Omez reduced it by 26–28%. Treatment with BT, CT4 and KT4 for seven days reduced it by 22.5%, 23.1% and 28.6%, compared to the corresponding untreated mice (group III). Omez showed a significantly better effect than the tea samples, reducing the parameter by 47.0%, compared to that of the untreated group. The results are presented in Tables 3a and 3b. The non-protein thiol (NP-TSH) level in the gastric tissues of the group II mice was similar to that of the normal control. However, the NP-TSH level decreased by 14.2% on the 7th day of ulceration, compared to the normal value. Treatment with BT and KT4 restored it to normalcy. Effect of BT, CT4, and KT4 on gastric mucin Our results (Tables 3a and 3b) showed that indomethacin administration decreased the gastric mucin in mice significantly (36.3%) on the 1st day, compared to that in normal mice. Treatment with BT, CT4, KT4 and Omez for one day augmented the mucin content by 16.5%, 20.9%, 14.6%, and 19.9% respectively, compared to the group II mice. Even after seven days, the mucin content in the untreated mice was only 74.3% of the normal value. Except BT, the other test samples improved the mucin content significantly. The order of efficacy of the test samples was CT4  KT4 > Omez > BT. Anti-secretory effect of BT, CT4, KT4 and Omez

Fig. 2 Histological assessment of acute gastric mucosal injury induced by indomethacin (18 mg kg1) in mice and its prevention by BT, CT4, KT4 (15 mg kg1) and Omez (3 mg kg1). Section of mice stomachs obtained from a: normal control mice; b: untreated control mice 10 h after indomethacin administration; c: untreated control mice seven days after indomethacin administration; d–g: mice treated with BT, CT4, KT4 and Omez for seven days after indomethacin administration. Black, yellow and black dotted arrows indicate areas of mucosal damage, inflammatory cells and areas of cryptic proliferation respectively.

comparable efficacy reducing the DS by 85.3% and 86.8% respectively. The reductions in IS by BT and CT4 (58–59%), KT4 (76%) and Omez (68%) also showed a similar trend. The results are summarized in Fig. 1a and Fig. 1b respectively. Effect of BT, CT4 and KT4 on oxidative stress Indomethacin administration markedly stimulated the TBARS formation in the gastric tissues by 34.5% on the 1st day, compared This journal is ª The Royal Society of Chemistry 2010

Pylorus ligation for 6 h resulted in the accumulation of gastric secretion and an increase in the total acid output of the gastric juice in the untreated mice. The tea extracts and Omez reduced the volume of gastric juice and total acid output (Table 4). CT4 and BT reduced the acid output by 24% while KT4 and Omez reduced the acid concentration by 28.6% and 30.8% respectively, compared to the untreated mice. Likewise, compared to the untreated mice, KT4 and Omez reduced the volume of secreted gastric juice by 25.3% and 19.0% respectively, while the effect of CT4 and BT was much less (10–14%).

Discussion Oxygen free radicals are known to play a role in the induction and pathogenesis of gastrointestinal injury, mediated by various agents including indomethacin.16,17 Extensive research has proved that antioxidants might be effective not only in protecting gastric mucosal injury, but also inhibiting progression of gastric ulcer. Release of preformed mucus plays an important role in promoting epithelial recovery after acute injury.18 Besides Food Funct., 2010, 1, 284–293 | 287

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Table 3a The effects of BT, CT4, KT4 and Omez on the levels of TBARS, protein carbonyls, and mucin in the ulcerated gastric tissue of micea on day one of ulceration

Parameters

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TBARS (nmoles mg1 protein) Protein carbonyls (nmoles mg1 protein) Mucin (mg g1 tissue)

Group I normal control

Group II ulcerated control

Group IV BT-treated

Group V CT4-treated

Group VI KT4-treated

Group VI I Omez-treated

1.16  0.15

1.56  0.15*

1.35  0.10

1.27  0.14

1.08  0.073†

1.08  0.04†

1.24  0.14

2.22  0.30**

1.85  0.11

1.64  0.20†

1.65  0.10†

1.59  0.07†

362.00  17.84

230.70  15.77*

268.67  30.43

279.00  16.37†

264.33  7.62

276.71  8.82†

a Stomach ulceration in mice was induced by oral administration of indomethacin (18 mg kg1). Tea samples (15 mg kg1) and Omez (3 mg kg1) were used as the drugs. The assays were carried out 10 h after indomethacin administration and the values are mean  SEM (n ¼ 15). *p < 0.05, **p < 0.01, compared to normal mice; †p < 0.05, compared to untreated control (group II).

Table 3b The effects of BT, CT4, KT4 and Omez on the levels of TBARS, protein carbonyls, and mucin in the ulcerated gastric tissue of micea on day seven of ulceration

Parameters TBARS (nmoles mg1 protein) Protein carbonyls (nmoles mg1 protein) Mucin (mg g1 tissue)

Group I normal control

Group III ulcerated control

Group VIII BT-treated

Group IX CT4-treated

Group X KT4-treated

Group XI Omez-treated

1.01  0.10

1.51  0.12*

1.10  0.12†

1.16  0.18†

1.01  0.12†

0.95  0.05†

1.27  0.09

2.13  0.17**

1.65  0.13†

1.62  0.10†

1.52  0.10†

1.13  0.04††,#

334.00  11.40

248.00  10.21*

280.00  15.30

314.33  14.77†

311.67  6.23†

301.71  4.41†

a Stomach ulceration in mice was induced by oral administration of indomethacin (20 mg kg1). Tea samples (15 mg kg1) and Omez (3 mg kg1) were used as the drugs. The assays were carried out on day seven after indomethacin administration and the values are mean  SEM (n ¼ 15). *p < 0.05, ** p < 0.01, compared to normal mice; †p < 0.05; ††p < 0.01, compared to untreated control (group III).

Table 4 Effects of BT, CT4, KT4 and Omez administered intraduodenally (i.d.) on the biochemical parameters of gastric juice obtained from pylorus ligature of micea

Treatments

Gastric juice (ml/100 g body wt.)

Total gastric acid (mEq/100g body wt.)

Vehicle BT CT4 KT4 Omez

0.95  0.09 0.85  0.08 0.82  0.05 0.71  0.08* 0.77  0.09*

4.55  0.04 3.45  0.05* 3.46  0.05* 3.25  0.02* 3.15  0.04*

a Immediately after pylorus ligature, the test samples (15 mg kg1) and Omez (3 mg kg1) were injected intraduodenally. The mice were killed 6 h after pylorus ligation, the volume of the gastric juice was measured and the total acid content was determined by titrating with 0.1 N NaOH. The values are mean  SEM (n ¼ 15). *p < 0.05 compared to the untreated mice.

providing significant buffering capacity for the neutralization of luminal acid, the mucoid cap can offer protection against the endogenous aggressors like acid, pepsin, and oxidants produced in the gastric lumen, as well as exogenous damaging agents, such as NSAIDs. The NSAID-produced mucosal hemorrhagic ulcer may be due to a decrease of gastric mucus production.18 Thus, drugs that arrest ulcer progression by antioxidant action, and also increase the synthesis and secretion of gastric mucus would accelerate gastric ulcer healing. The powerful antioxidant property of BT encouraged us to investigate its possible protective effect against indomethacininduced gastric lesions in mice. In addition, we also included KT 288 | Food Funct., 2010, 1, 284–293

for the present investigation because of its proclaimed health benefit against various diseases. In recent years there has been a mounting interest in exploring the possibility of using BT as a supplement among patients. The Food and Agricultural Organisation (FAO) of the United Nations has stressed the need for research on the health benefits of BT in its totality, and not on certain isolated fractions/constituents. Hence, we also used BT as a whole for the studies. The chemical constituents of KT depend on the exact microbiological composition, used for the fermentation as well as the fermentation time. However, these factors are often ignored, leading to substantial confusion regarding its physiological effect. Hence, we followed a scientific approach for the preparation of KT. For this, we identified the microorganisms in the kombucha culture and used it to prepare KT.19 The KT preparation was characterized in terms of several physical and chemical parameters, and evaluated for its ulcer-healing property. Consistent with the findings of a recent report, all the KT samples were found to be acidic.14 During the fermentation process, the yeast invertase hydrolyses sucrose into glucose and fructose, and produces ethanol via glycolysis with preference for fructose as the substrate. Subsequently, the acetic acid bacteria convert glucose and ethanol to gluconic and acetic acids respectively. Other organic acids including lactic acid are also produced by the acetic acid bacteria.20 These acids possess various health promoting attributes, and also make KT acidic. Because KT4 showed the best antioxidant capacity amongst the KT samples, it was chosen for studying the ulcer-healing property and its efficacy was compared with those of CT4 and BT. This journal is ª The Royal Society of Chemistry 2010

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Our macroscopic and histopathological results revealed that indomethacin administration induced marked, but acute damage to the gastric mucosa of mice. All the tea samples reduced the ulcerative damage and inflammation, the efficacy of KT4 being similar to that of Omez. The effects of BT and CT4 were also impressive, but not significantly different from each other, and less than that of KT4. The accelerated healing by the test samples was also evident within 4 h of their administration. However, the effect was more pronounced on continuing the treatment for seven days. Tissue damage is always associated with excess generation of free radicals, leading to excessive lipid peroxidation (LPO) and loss or impairment of protein synthesis.21 These might aggravate tissue damage during stomach ulceration. Hence we assessed LPO (in terms of thiobarbituric acid reactive species [TBARS]) and protein oxidation (in terms of protein carbonyl formation) in the normal, ulcerated and treated groups of mice on day one and seven of the studies. Our results (Tables 3a and 3b) revealed that ulceration in mice was accompanied by a severe oxidative stress, resulting in the oxidation of lipids and proteins of the gastric tissues. These results are consistent with the earlier reports on the indomethacin-induced gastropathy.22,23 Due to their excellent radical scavenging capacity, the tea samples, especially KT4 provided a marked suppression of the oxidative damages and brought most of these parameters to near normalcy. This might decrease the ulcer progression and promote healing of gastric lesions induced by acute intake of indomethacin. Depletion of the gastric mucin level also contributes to the NSAID-mediated gastropathy. Maintenance of mucus production may provide partial, but significant protection against reactive oxygen metabolites. In this study, the decreased mucin secretion in the indomethacin-administered mice indicated reduced ability of the mucosal membrane to protect the mucosa from physical damage and back diffusion of hydrogen ions. Treatment with BT, CT4, KT4, and Omez arrested the gastric mucin depletion significantly. Restoration of the gastric mucin to near normalcy would protect the ulcer crater against irritant stomach secretions (HCl and pepsine) and accelerate ulcer healing. Mucin depletion may result because of oxidative rupture of the disulfide bridges that join the mucus subunits and maintain the structural integrity of the mucus.24 The sulfhydryl compounds (NP-TSH) prevent the mucosal rupture by antioxidant action. The decrease in endogenous thiol (glutathione) in ethanol induced gastric injury and its role in mucosal protection has been demonstrated earlier.23 Hence, we assayed the gastric NP-TSH levels of ulcerated as well as BT and KT4-treated mice. Our results of reduction of gastric NP-TSH in the 7th day-ulcerated mice and its restoration to normal level by BT and KT4 clearly demonstrated that the antioxidative property of BT and KT4 contributes to protection of the gastric mucosa. The ulcerationinduced lipid peroxidation might increase glutathione consumption, reducing the NP-TSH level. The regeneration of the sulfhydryl compounds by the tea samples would help in recycling endogenous antioxidant vitamins, and prevent lipid peroxidation. Suppressors of acid secretion such as proton pump inhibitors (like Omez) and histamine second receptor antagonists have been the mainstay for promotion of ulcer healing.2 In view of this, we This journal is ª The Royal Society of Chemistry 2010

also assessed the healing potential of the tea samples using the pylorus ligation ulcer model. In this study, the test samples, KT4, and Omez could significantly reduce the volume of gastric juice and total acid output. However, KT4 was administered to the duodenum, and the pH of the test solutions was 4.5. Therefore, its effects were secondary resulting from increased secretin generation, neural pathways and/or other mechanisms. Following pyloric ligation, migration of neutrophils into the mucosa has been observed in experimental animals.25 This suggests that besides gastric hypersecretion, oxidative stress is involved in this model of gastric ulceration also. It is therefore possible that the beneficial effect of the tea samples with the pyloric ligated mice may be partly due to their antioxidative property. The suppression of gastric acid by the tea samples also followed the trend of their antioxidant activity. The tea decoction is a complex mixture of products comprising of a group of biopolymers, theaflavins and the water-soluble thearubigins with undefined chemical structures.26 Hence, we did not attempt to analyze the tea decoctions completely. Our HPTLC analysis revealed a gradual reduction in the caffeine level with simultaneous increase in theobromine concentration during the fermentation of BT with the kombucha culture. This might be attributed to N-demethylation of caffeine in the process. Similar oxidative chemical transformation of caffeine was reported earlier.27 Overall, TF was found to account for most of the healing activity of BT in this study. TF also might be the major contributor in the healing action of KT4 because its concentrations in KT4 and BT were practically the same. Presently the exact reason of the better efficacy of KT4 over BT remains elusive and multiple factors including gastric acid suppression may be responsible for this. Due to its higher phenolics content, KT4 is a better free radical scavenger that might account for its superior ulcer healing property compared to BT. Beyond a certain concentration, the three major components of the tea samples (theophylline, caffeine, and theobromine) are known to have adverse effects on gastrointestinal tracks.28 Hence these are unlikely to contribute to the healing activity of the tea samples. In contrast, the significantly reduced caffeine concentration in KT4 may be beneficial for ulcer healing, because caffeine is suggested to aggravate an existing ulcer by stimulating acid secretion. Many organic acid proton donors are known to decrease intestinal secretion.29 Some of these such as lactic acid helps digestive action and reduces acid secretion, while butyric acid strengthens the gut walls.30 Thus, some of the organic acids, produced by the fermentation of sugar by the kombucha culture may also contribute to the ulcer healing. In addition, the low pH of KT4 may be beneficial in controlling bacterial infection that will also reduce inflammation.

Conclusions Overall, our results clearly revealed the healing ability of BT against indomethacin-induced stomach ulceration. The activity is augmented by its fermentation by the kombucha culture, but not significantly by C. parapsilosis alone. The healing action of the BT, CT4 and KT4 was due to their antioxidant action that helped in preventing rupture of the protecting gastric mucin. The increased phenolics content and some of the constituent small Food Funct., 2010, 1, 284–293 | 289

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organic acids in KT4 might account for its superior ulcer healing effect than that of BT. BT is conventionally regarded as harmful for the stomach, while the effect of KT on the stomach has been questioned earlier.31 From this perspective, the present finding is very interesting and dispels doubt about the gastronomical harmful effect of BT and KT. Apparently, the gastrotoxicity (if any) of KT might be due to the unhygienic mode of its preparation, leading to contamination by Apergillus, Bacillus, or even by leaching of lead from the ceramic container used for its preparation. These results, taken together established the potential of KT4 as a potential anti-ulcerogenic formulation.

20  C. The fermented black tea decoctions were designated as CT2, CT4, CT7 (for C. parapsilosis) or KT2, KT4, KT7 (for kombucha culture) respectively, depending on the culture and time used for the fermentation. Determination of phenolic contents Following a known method,33 the amounts of total phenolics in the tea extracts were determined. Gallic acid monohydrate was used as the standard, and the total polyphenolic content is expressed as mg gallic acid equivalent (GAE) g1 of the extract.

Experimental section

Analysis of theaflavin (TF) and thearubigins (TR)

Chemicals and reagents

The theaflavin and thearubigins contents in BT and KT4 were estimated by a known method, with a brief modification.14 The tea extracts (25 ml) was extracted with isobutyl methyl ketone (IBMK, 25 ml), the IBMK extract (1 ml) was mixed with aqueous 45% ethanol (9 ml) and absorbance (A) measured at 380 nm. Ten ml of IBMK phase was extracted with 10 ml of 2.5% disodium hydrogen phosphate. After extraction and phase separation, 1 ml of IBMK phase was mixed with 9 ml of 45% ethanol and absorbance was measured at 380 nm (B). 10 ml of aqueous phase from the first step was extracted with 10 ml of n-butanol. After phase separation, 1 ml of n-butanol layer was mixed with 9 ml of 45% ethanol and absorbance was measured at 380 nm (C). Concentration of theaflavin and thearubigins was calculated from the absorbance values as given below.

Leaves of C. sinenesis (Brooke Bond, Red label) were procured from the local market. Alcian blue, indomethacin, bovine serum albumin (BSA), haematoxylene, alum, eosin, butylated hydroxytoluene (BHT), guanidine hydrochloride, trifluoroacetic acid (TFA), and sucrose were procured from Sigma, St. Louis, MO. Other reagents used were 2-thiobarbituric acid (TBA), ethanol, butanol and ethyl acetate (all from E. Merck, Mumbai, India), trichloroacetic acid (TCA, Thomas Baker, Mumbai, India), hydrogen peroxide (35%, Lancaster, Morecambe, U.K.), 2,4-dinitrophenyl hydrazine (DNPH), disodium hydrogen phosphate and sodium dihydrogen phosphate (BDH, Mumbai, India). Starter cultures The kombucha culture was procured from a home brewer and maintained in the laboratory. It was grown at 28  2  C in a sterilized medium containing tea, sucrose and distilled water. The baby culture was used for further propagation, or for fresh batches of fermentation. The isolated bacteria from the culture were identified as Acetobacter aceti and A. pasteurianus, from their biochemical properties, and by comparison with different Acetobacter strains. The yeasts were identified as Saccharomyces cerevisiae, Zygosaccharomyces bailii and Brettanomyces bruxellensis according to conventional phenotype characterization and the Yeasts Identification Programme.19 This was also confirmed by scanning electron micrographs (data not shown). The Candida parapsilosis culture was maintained on Sabouraud’s dextrose agar. The culture was identified by colonial morphology, formation of pseudohyphae and periodical biochemical tests.32 Preparation of fermented teas Tea leaves (0.5% w/v) were added to distilled water boiled for 15 min, and allowed to steep for 20 min. The decoction was passed through a nylon sieve and autoclaved. Concentrated sucrose solution was added to it, to achieve a final sucrose concentration of 10%. The tea sample was either left unfermented (designated as BT) or allowed to ferment in the presence of C. parapsilosis culture or kombucha culture for 2, 4 and 7 days. At the end of the designated periods, the tea samples were collected, and their volumes and pHs were measured. Each of the extracts were lyophilized, sterilized by millipore filtration and stored at 290 | Food Funct., 2010, 1, 284–293

TF (%) ¼ 4.313  B TR (%) ¼ 13.643  (A + C  B) Characterization of the chemical constituent of KT4 The KT4 extract (50 ml) was successively extracted with chloroform, ethyl acetate and butanol (each 50 ml  3 times). The individual extracts were concentrated in vacuo to obtain 0.514 g, 0.302 g and 0.221 g of the residues respectively. Normal phase preparative TLC (silica gel G, ethyl acetate: methanol: water ¼ 10 : 1.1 : 1 as the solvent), followed by a preparative TLC (RP-18 silica gel plate, same solvent) of the chloroform extract afforded caffeine, which was characterized from its physical and spectral characteristics. The caffeine concentrations in tea samples were also quantified by HPTLC densitograms. Caffeine: mp: 237  C; UV (MeOH) lmax: 275 nm (log 3 3.99); IR (KBr): 1700 cm1; 1H NMR (200 MHz, CDCl3): d 3.15 (s, 3H, 3-CH3), 3.33 (s, 3H, 7-CH3), 3.86 (s, 3H, 1-CH3), 7.1 (s, 1H). Preliminary TLC (silica gel G, ethyl acetate: methanol: water ¼ 10 : 1.1 : 1) investigation of the ethyl acetate and 1-butanol extracts showed a common fluorescent spot at Rf 0.9. The compound was isolated by reverse phase preparative TLC under the above mentioned conditions from the ethyl acetate extract where its concentration was substantial. It was characterized as theobromine. Theobromine: mp: 355  C; UV (MeOH) lmax: 272 nm (log 3 4.01); IR (KBr): 1694 cm1; 1H NMR (200 MHz, CDCl3): d 1.55 (s, 6H, 2  CH3), 3.33 (s, 1H, NH), 3.97 (s, 1H). Following a similar protocol, theophylline was also isolated from the 1-butanol extract, and characterized as above. This journal is ª The Royal Society of Chemistry 2010

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Theophylline: mp: 270  C; UV (MeOH) lmax: 272 nm (log 3 4.00); IR (KBr): 1680 cm1; 1H NMR (200 MHz, CDCl3): d 3.49 (s, 3H, 3-CH3), 3.66 (s, 3H, 1-CH3), 7.25 (s, 1H, 7-H), 12.20 (s, 1H).

0.5 – hyperemia, 1 – one or two lesions, 2 – severe lesions, 3 – very severe lesions, 4 – mucosa full of lesions (lesions – hemorrhagic erosions, hyperemia – vascular congestions). Studies on the histopathological and biochemical parameters

Preparation of the test samples

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The test samples were prepared from BT, CT4, KT4 and Omez as aqueous suspensions in 2% gum acacia as the vehicle, and administered to the mice orally. Any possibility of microbial contamination in the samples was excluded prior to their administration to the mice. Protocol for ulceration and healing studies Male Swiss albino mice, bred at the Dr B. C. Roy Post Graduate Institute of Basic Medical Sciences, Kolkata, India were procured after obtaining clearance from the Animal Ethics Committee of the centre. All the experiments were conducted with strict adherence to the ethical guidelines laid down by European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. In addition, the ethical guidelines, laid by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), constituted by the Animal Welfare Division, Government of India on the use of animals in scientific research was followed. The mice (6–8 weeks old, 25–30 g) were reared on a balanced laboratory diet as per National Institute of Nutrition, Hyderabad, India and given tap water ad libitum. They were kept at 20  2  C, 65–70% humidity, and 12 h day/12 h night cycles. The experiments were performed by two investigators blinded to the group and treatment of animals, which were identified by typical notches in the ear and limbs [performed at a pre-weaning stage to minimize the pain to the animals], and then randomized. Ulceration in the mice was induced by administering indomethacin (18 mg kg1, p. o., single dose) dissolved in distilled water and suspended in the vehicle, gum acacia (2%). The animals were deprived of food, but had free access to tap water, 24 h before ulcer induction. For the standardization of doses, the respective test samples (10, 15 and 20 mg kg1, p. o.) were given to the mice once daily for 7 days, starting the first dose 6 h after the indomethacin administration. In the subsequent six days, the test samples were given at 9 AM on each day. Five mice were taken in each group and each experiment was repeated three times. The mice were sacrificed on the 1st and 7th day, 4 h after administering the last dose of the test samples. The extent of healing was assessed from the macroscopic damage scores (MDS) of the untreated and treated ulcerated mice. Assessment of ulcer healing from MDS The mice were sacrificed after an overdose with thiopental. The stomach from the normal and treated groups were removed rapidly, opened along the greater curvature, and thoroughly rinsed with normal saline. The ulcerated gastric mucosal areas were visualized using a transparent sheet and a dissecting microscope. The MDS was assessed34 by grading the gastric injury on a 0–4 scale, based on the severity of hyperemia and hemorrhagic erosions: 0 – almost normal mucosa, This journal is ª The Royal Society of Chemistry 2010

Based on our MDS results, we assessed the histopathological, and biochemical parameters under the optimized doses of the individual test samples [BT, CT4, KT4 (each 15 mg kg1, p. o.) and Omez (3 mg kg1, p. o.)]. The mice were equally divided in eleven groups as follows: Group I – normal mice; Group II – ulcerated mice and sacrificed after 10 h; Group III – ulcerated mice, and sacrificed after 7 days; Group IV–VII – ulcerated mice, treated with BT, CT4, KT4 and Omez respectively, and sacrificed 4 h after administration of test samples on the 1st day; Group VIII–XI – ulcerated mice, treated with BT, CT4, KT4 and Omez respectively, and sacrificed 4 h after administration of test samples on the 7th day. Group I–III control groups of mice were given the vehicle (0.2 ml) during the period of study. Histological studies The ulcerated portions of the stomach were sectioned after fixing in 10% formol saline solution. After 24 h of fixation followed by embedding in a paraffin block, it was cut into sections of 5 micron onto a glass slide, stained with haematoxylene-eosin and the histology examined under a light microscope. One centimetre length of each histological section was divided into three fields. The damage score (DS) was assessed by scoring each field on a 0– 4 scale as described previously:34 0 – normal mucosa, 1 – epithelial cell damage, 2 – glandular disruption, vasocongestion or edema in the upper mucosa, 3 – mucosal disruption, vasocongestion or edema in the mid-lower mucosa, and 4 – extensive mucosal disruption involving the full thickness of the mucosa. The overall mean value of the damage scores (DS) for each of the fields was taken as the histological ulcer index for that section. Likewise, the inflammatory scores (IS)35 were assigned after reviewing all slides to assess the range of inflammation as follows: 0 – normal mucosa, 1 – minimal inflammatory cells, 2 – moderate number of inflammatory cells, and 3 – large number of inflammatory cells. Histological sections were coded to eliminate an observer bias. Data for the histological analyses are presented as the mean  SEM from the review of a minimum of three sections per animal and five animals per group. Quantification of lipid and protein damages The glandular stomach tissues were pooled from five animals and their wet weights were noted. The tissues were pooled from five animals, rinsed with appropriate buffer, homogenized with a glass-teflon homogenizing tube in a 50 mM phosphate buffer, pH 7.4. The supernatant, obtained after centrifugation at 1200  g was used for the biochemical studies. The lipid peroxidation products were estimated36 with minor modifications. Briefly, 1 ml of each of the tissue homogenates and ice-cold 20% TCA solution containing 0.01% BHT was incubated for 15 min. The samples were centrifuged at 1200  g for 15 min, and the supernatant centrifuged again at 1200  g for Food Funct., 2010, 1, 284–293 | 291

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30 min to obtain the mitochondrial pellets. These were washed with a buffer (150 mM KCl and 20 mM phosphate buffer) and finally suspended in a phosphate buffer (50 mM, pH 7.4). The mitochondrial membrane fraction (1 ml) was treated with TCA/ TBA/HCl (2 ml, 15% TCA, 0.375% TBA, 0.25N HCl) containing 0.01% BHT, heated on a boiling water bath for 15 min, cooled and centrifuged at 3000  g for 5 min, the red chromophore in the supernatant was extracted with 1-butanol (2 ml). The amount of TBARS was calculated from its absorbance at 535 nm (3 ¼ 1.56  105 M1cm1). The protein carbonyl contents were measured following a reported method37 with minor modifications. Briefly, the tissue homogenate was incubated for 60 min with 10 mM DNPH in 2M HCl in a ratio of 1 : 4 with intermittent shaking. After incubating the mixture with ice-cold 20% TCA solution for 15 min, followed by centrifugation at 1200  g for 10 min, the pellet obtained was washed three times with ethanol:ethyl-acetate (1 : 1, 1 ml). The washed pellet was redissolved in 1 ml of guanidine reagent (6M guanidine in 20 mM potassium phosphate buffer, pH 2.3), centrifuged and the carbonyl content of the supernatant was assayed from the absorbance at 362 nm (3 ¼ 22,000 M1 cm1).

pylorus ligation by cervical dislocation under ether anaesthesia. For collection of the gastric juice, the abdomen was opened and another ligature placed around the oesophagus close to the diaphragm. The stomach was removed, inspected internally, and its content drained into a graduated centrifuge tube to determine the total amount of gastric-juice acid (ml/100 g). Following washing the mucosal side of the stomach with distilled water (2 ml), centrifugation at 3000  g for 15 min was carried out. The total acid content (mEq) in the supernatant volume was determined by titration with 0.1 N NaOH.

Non-protein thiol (NP-TSH) assay

References

Following a reported method,38 the gastric mucosal NP-TSH was measured. Briefly, the glandular stomach homogenates were prepared in 0.2 M Tris-HCl buffer, pH 8.2 containing 20 mM EDTA and centrifuged at 1200  g for 15 min. An aliquot of the homogenate (1 ml) was treated with ice-cold 20% TCA (1 ml), centrifuged at 3000  g for 5 min, and the supernatant (1 ml) was added to Tris-HCl buffer (2 ml, 0.8 M, pH 9) containing 20 mM EDTA, and mixed with DTNB (0.1 ml, 10 mM). The NP-TSH content was calculated from the absorbance of the chromogen at 412 nm (3 ¼ 13.6  104 M1 cm1).

1 F. Halter, A. Schamassman, B. M. Peskar and A. S. Tarnawski, Cyclooxygenase-2 implications on maintenance of gastric mucosal integrity and ulcer healing: controversies and perspectives, Gut, 2001, 49, 443–453. 2 J. L. Wallace, Recent advances in gastric ulcer therapeutics, Curr. Opin. Pharmacol., 2005, 5, 573–577. 3 E. Yesilada, and I. A. Gurbuz, Compilation of the studies on the antiulcerogenic effects of medicinal plants. (2003) In: S. Singh, V. K. Singh, J. N. Govil (ed.), Recent Progress in Medicinal Plants, vol. II: Phytochemistry and Pharmacology. SCI Tech Publishing LLC, Houston, USA, pp. 111–174. 4 K. Hamaishi, R. Kojima and M. Ito, Anti-ulcer effect of tea catechin in rats, Biol. Pharm. Bull., 2006, 29, 2206–2213. 5 S. Y. Lee, Y. W. Shin and K. B. Hahm, Phytoceuticals: mighty but ignored weapons against Helicobacter pylori infection, J. Dig. Dis., 2008, 9, 129–139. 6 M. Yoshikawa, T. Morikawa, N. Li, A. Nagatomo, X. Li and H. Matsuda, Bioactive saponins and glycosides. XXIII. Triterpene saponins with gastroprotective effect from the seeds of Camellia sinensis – theasaponins E3, E4, E5, E6, and E7, Chem. Pharm. Bull. (Tokyo), 2005, 53, 1559–1564. 7 T. Morikawa, N. Li, A. Nagatomo, H. Matsuda, X. Li and M. Yoshikawa, Triterpene saponins with gastroprotective effects from tea seed (the seeds of Camellia sinensis), J. Nat. Prod., 2006, 69, 185–190. 8 S. Maity, J. R. Vedasiromoni, L. Chaudhuri and D. K. Ganguly, Role of reduced glutathione and nitric oxide in the black tea extractmediated protection against ulcerogen-induced changes in motility and gastric emptying in rats, Jpn. J. Pharmacol., 2001, 85, 358–364. 9 S. Maity, J. R. Vedasiromoni and D. K. Ganguly, Anti-ulcer effect of the hot water extract of black tea (Camellia sinensis), J. Ethnopharmacol., 1995, 46, 167–174. 10 H. Tietze, Kombucha the Miracle Fungus, Harald Tietze Publ., India, 2000. 11 T. Pauline, P. Dipti, B. Anju, S. Kavimani, S. K. Sharma, A. K. Kain, S. K. Sarada, M. Sairam, G. Ilavazhagan, K. Devendra and W. Selvamurthy, Studies on toxicity, anti-stress and hepatoprotective properties of Kombucha tea, Biomed Environ. Sci., 2001, 14, 207–213. 12 M. Sairam, B. Anju, T. Pauline, D. Prasad, A. K. Jain, S. S. Mongia, S. K. Sharma, B. Singh, R. Singh, G. Ilavazhagan, D. Kumar and W. Selvamurthy, Effect of kombucha tea on chromate(VI)-induced oxidative stress in albino rats, J. Ethnopharmacol., 2000, 71, 235–240. 13 U. S. Hiremath, M. P. Vaidehi and B. J. Mushtari, Effect of fermented tea on the sugar levels of NIDDM subjects, The Indian Practitioner, 2002, 55, 423–425.

Mucin assay Following a reported method,39 the free mucin content in the gastric tissues was estimated by measuring the amount of alcian blue bound to mucus. Briefly, the glandular stomach tissues were incubated with a 1% buffered sucrose solution of alcian blue in (0.1%) sodium acetate at 37  C for 60 min. After incubation, the tissues were washed with sucrose and centrifuged. The supernatant was extracted with MgCl2, and the amount of alcian blue was estimated spectrophotometrically at 610 nm. The quantity (mg) of alcian blue g1 wet glandular tissue was calculated. Determination of gastric juice secretion Mice were randomly divided into predesignated groups of five animals each, which were fasted for 24 h with free access to water. Briefly, a midline incision was made under ether anaethesia following which a portion of the abdomen was opened below the xiphoid process. The pylorus portion of the stomach was lifted and ligated. During this process, care was taken to avoid the traction to the pylorus or damage to its blood supply. The stomach was closed by interrupted sutures.40 Immediately after pylorus ligature, the tea samples (each 15 mg kg1) or the positive control, Omez (3 mg kg1) were injected intraduodenally. The animals were killed 6 h after 292 | Food Funct., 2010, 1, 284–293

Statistical analysis The data are presented as mean  SEM. Parametric data which includes all the biochemical parameters were analyzed using a paired ‘t’ test for the paired data or one way analysis of variance (ANOVA) followed by a Dunnet multiple comparisons post test. Nonparametric data (histology scoring) were analyzed using Kruskal-Wallis test (nonparametric ANOVA) followed by a Dunn’s multiple comparisons post test. A probability value of p < 0.05 was considered significant.

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14 R. Jayabalan, S. Marimuthu and K. Swaminathan, Changes in content of organic acids and tea polyphenols during kombucha tea fermentation, Food Chem., 2007, 102, 392–398. 15 D. Banerjee, B. Maity, A. K. Bauri, S. K. Bandyopadhyay and S. Chattopadhyay, Gastroprotective property of Myristica malabarica against indomethacin-induced stomach ulceration: A mechanistic exploration, J. Pharm. Pharmacol., 2007, 59, 1555–1565. 16 K. Biswas, U. Bandyopadhyay, I. Chattopadhyay, A. Varadaraj, E. Ali and R. K. Banerjee, A novel antioxidant and antiapoptotic role of Omez to block gastric ulcer through scavenging of hydroxyl radical, J. Biol. Chem., 2003, 278, 10993–11001. 17 H. Utsumi, K. Yasukawa, T. Soeda, K.-i. Yamada, R. Shigemi, T. Yao and M. Tsuneyoshi, Noninvasive mapping of reactive oxygen species by in vivo electron spin resonance spectroscopy in indomethacin-induced gastric ulcers in rats, J. Pharmacol. Exp. Ther., 2006, 317, 228–235. 18 K. D. Rainsford, The effects of aspirin and other nonsteroid antiinflammatory analgesic drugs on gastrointestinal mucus glycoprotein biosynthesis in vivo: relationship to ulcerogenic actions, Biochem. Pharmacol., 1978, 27, 877–885. 19 C. H. Liu, W. H. Hsu, F. L. Lee and C. C. Liao, The isolation and identification of microbes from a fermented tea beverage, haipao and their interactions during haipao fermentation, Food Microbiol., 1996, 13, 407–415. 20 C. Dufresne and E. Farnworth, Tea, Kombucha, and health: a review, Food Res. Intnl., 2000, 33, 409–421. 21 S. Szabo and D. Hollander, Pathway of gastrointestinal protection and repair: mechanism of action of sucralfate, Am. J. Med., 1989, 86, 23–31. 22 J. R. Avila, C. A. de la Lastra, M. J. Martin, V. Motilva, I. Luque, D. Delgado, J. Esteban and J. Herrerias, Role of endogenous sulfhydryls and neutrophil infiltration in the pathogenesis of gastric mucosal injury induced by piroxicam in rats, Inflamm. Res., 1996, 45, 83–88. 23 V. Bertrand, F. Guessous, A. L. Le Roy, B. Viossat, H. Fessi, A. El Abbouyi, J. P. Giroud and M. Roch-Arveiller, Copperindomethacinate associated with zwitterionic phospholipids prevents enteropathy in rats: effect on inducible NO synthase, Dig. Dis. Sci., 1999, 44, 991–999. 24 J. J. Bernier and C. Florent, Les defences de l’estomac, Recherche, 1986, 117, 614–621. 25 L. Rastogi, G. K. Patnaik and M. Dikshit, Free radicals and antioxidant status following pylorus ligation induced gastric mucosal injury in rats, Pharmacol. Res., 1998, 38, 125–132.

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26 D. A. Balentine, S. A. Wiseman and L. C. M. Bouwens, The chemistry of tea flavonoids, Crit. Rev. Food Sci. Nutr., 1997, 37, 693–704. 27 R. H. Stadler, J. Richoz, R. J. Turesky, D. H. Welti and L. B. Fay, Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations, Free Radic. Res., 1996, 24, 225–10. 28 J. O. Ibu, Ac. Iyama, C. T. Ijije, D. Ishmael, M. Ibeshim and S. Nwokediuko, The effect of Cola acuminata and Cola nitida on gastric acid secretion, Scand. J. Gastroenterol., 1986, 21(Supp. 124), 39–45. 29 G. S. Forsyth, R. A. Kapitany and D. L. Hamilton, Organic acid proton donors decrease intestinal secretion caused by enterotoxins, Am. J. Physiol, 1981 Sep, 241(3), G227–34. 30 G€ unther Frank; Kombucha: Healthy Beverage and Natural Remedy from the Far East, Ennsthaler, 1991. 31 R. Srinivasan, S. Smolinske and D. Greenbaum, Probable gastrointestinal toxicity of kombucha tea, J. Gen. Int. Med., 1997, 12, 643–645. 32 J. A. Barnett, R. W. Payne and D. Yarrow, Yeasts: Characteristics and Identification, third ed. Cambridge University Press, Cambridge, 1983, pp. 159–160. 33 V. L. Singleton and J. A. Rossi Jr., Colorimetry of total phenolics with phosophomolybdic-phosphotungstic acid reagents, Am. J. Enol. Viticul., 1965, 16, 144–158. 34 D. Dokmeci, M. Akpolat, N. Aydogu, L. Doganay and F. N. Turan, L-Carntine inhibits ethanol-induced gastric mucosal injury in rats, Pharmacol Rep., 2005, 57, 481–488. 35 W. L. Beck and R. Xavier, Mechanism of NSAID induced gastrointestinal injury defined using mutant mice, Gastroenterol., 2000, 119, 699–705. 36 H. Esterbauer, J. Gebicki, H. Puhl and G. Jurgens, The role of lipid peroxidation and antioxidants in oxidative modification of LDL, Free Radic. Biol. Med., 1992, 13, 341–390. 37 P. J. Shah, M. S. Gandhi, M. B. Shah, S. S. Goswami and D. Santani, Study of Mimusops elengi bark in experimental gastric ulcers, J. Ethnopharmacol., 2003, 89, 305–311. 38 J. Sedlak and R. H. Lindsay, Estimation of total protein-bound, and non-protein sulfhydryl groups in tissue with Ellman’s reagent, Anal. Biochem., 1968, 25, 192–205. 39 M. Tariq and A. L. Moutaery, Menadione protects gastric mucosa against ethanol-induced ulcers, Exp. Toxicol. Pathol., 2005, 56, 393– 399. 40 D. A. Brodie, The mechanism of gastric hyperacidity produced by pyloric ligation in the rat, Am. J. Dig. Dis. Sci., 1966, 111, 231–241.

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PAPER

www.rsc.org/foodfunction | Food & Function

Non-digestible fraction of cooked bean (Phaseolus vulgaris L.) cultivar Bayo Madero suppresses colonic aberrant crypt foci in azoxymethane-induced rats Hayde Azeneth Vergara-Casta~ neda,a Ram on Gerardo Guevara-Gonzalez,b Minerva Ramos-Gomez,a Rosalıa Reynoso-Camacho,a Horacio Guzm an-Maldonado,c Ana Angelica Feregrino-Perez,a B. Dave Oomahd a and Guadalupe Loarca-Pi~ na*

Downloaded on 02 December 2010 Published on 08 November 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00130A

Received 31st August 2010, Accepted 25th October 2010 DOI: 10.1039/c0fo00130a The non-digestible fraction (NDF) of common bean (Phaseolus vulgaris L.) cultivar Bayo Madero was evaluated for its chemopreventive effect on azoxymethane (AOM) induced aberrant crypt foci (ACF) in rats. Diets containing cooked beans (CB) or its non-digestible fraction (NDF) were fed to 72 male rats after 2 azoxymethane injections (15 mg kg1 of body weight once a week for 2 weeks). ACF number, short chain fatty acids (SCFA) and b-glucuronidase activity were measured in colon sections from rats sacrificed 7 weeks after the last AOM injection. Food intake and weight gain of rats were unaffected by CB and NDF. CB and NDF suppressed the AOM-induced formation of ACF (0.8 and 1.5 ACF/distal zone, respectively vs. 6.6 ACF/distal zone based on methylene blue stain) and lowered b-glucuronidase activity in cecal, colonic and fecal content compared to AOM group. SCFA production was not significantly different among fecal, cecal and colonic content. These results indicate that CB and NDF from Bayo Madero provide direct chemoprotection against early stage of azoxymethane (AOM)induced colon cancer in rats.

Introduction In 2006, malignant tumors were the third highest cause of death in Mexico with colon cancer contributing 5 and 4.6% for men and women, respectively to this mortality.1 Several studies associate the importance of diet, particularly containing high fiber foods, with prevention and/or development of cancer.2,3 The common bean (Phaseolus vulgaris L.), an important food in the Mexican diet, is rich in several phytochemicals such as polyphenols, carbohydrates, fiber and polysaccharides with potential health benefits.4 Recently, the consumption of common beans has been associated with reduced mortality from colon cancer.5 For example, the phenolic extracts, residue and whole seed of navy beans significantly reduced development of chemically induced colon lesions in obese rats.6 The non-digestible carbohydrates such as fiber (soluble and insoluble), resistant starch and oligosaccharides can be fermented in the large intestine by colonic bacteria to produce short chain fatty acids (SCFA).7 Butyric acid, a major SCFA, plays an important role in suppressing growth in human colon cancer cells.8 In vitro and in vivo studies have demonstrated that butyrate inhibits cell proliferation, induces differentiation and apoptosis, and modulates gene

expression involved in colorectal carcinogenesis, with subsequent reduction in tumor development.9-11 Our previous studies12,13 showed that common bean, cultivars Negro 8025 and Bayo Madero, contained fermentable substrates with a non-digestible fraction that prevented the development of early and late stages of colon cancer by modulating genes and proteins involved in apoptosis, proliferation, cell cycle arrest and inflammation. Cultivar Negro 8025 prevented the development of early stage colon cancer in AOM-induced rats,12 whereas cultivar Bayo Madero elicited chemopreventive effect through several molecular mechanisms in human colon adenocarcinoma HT-29 cells.13 Furthermore, cooked beans of cultivar Bayo Madero showed the highest yield of polysaccharides (55%) and resistant starch (37%) than other bean types (Azufrado, Negro and Pinto) commonly consumed in Mexico based on regional preference.4 This investigation extends our previous study with cultivar Bayo Madero13 and describes the chemopreventive effect of its non-digestible fraction (NDF) on early stage of AOM-induced colon cancer in rats.

Experimental Materials

a

Programa de Posgrado en Alimentos del Centro de la Rep ublica (PROPAC), Research and Graduate Studies in Food Science, School of Chemistry, Universidad Aut onoma de Quer etaro, Quer etaro, Qro, 76010, M exico. E-mail: [email protected]; Fax: +52 (442) 192-1307; Tel: +52 442 192 1307 b School of Engineering, Universidad Aut onoma de Quer etaro, Quer etaro, Qro, 76010, M exico c Biotechnology and Legume Laboratories, Experimental Station El Bajıo, National Research Institute for Forestry, Agriculture and Livestock (INIFAP), Celaya, Mexico d National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, British Columbia, Canada V0H 1Z0

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Seeds of cultivar Bayo Madero were harvested in 2007 at the Bajio Experimental Station of the National Research Institute for Forestry, Agriculture and Livestock (INIFAP), Celaya, Guanajuato, Mexico. This cultivar was selected for its agronomic quality, such as yield and adaptation to the growing conditions in semi-arid regions. Male Sprague Dawley rats (28 days old) were obtained from Harlan, Inc. (Mexico City, Mexico). The care and use of these animals were in compliance with policies and regulations of the Institutional Animal Care and Use Committee of the University of Queretaro, Mexico. This journal is ª The Royal Society of Chemistry 2010

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Azoxymethane (AOM), (+)-catechin, a-amylase, protease, amyloglucosidase, tannic acid, D-(+)-raffinose, D-(+)-stachyose and D-(+)-verbascose standards were purchased from Sigma Chemical Co. (St. Louis, MO). Butyric acid was purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Acetic, propionic, and formic acids and other chemicals were purchased from J. T. Baker (Mexico City, Mexico).

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Thermal treatment Seeds were cooked using a ‘‘traditional’’ cooking process according to the method of Aparicio-Fernandez et al.14 Briefly, beans (50 g) were placed in a beaker with 250 mL of HPLC-grade water and boiled (90  C) covered until they were suitable for consumption (approximately 2.5 h). Cooked seeds and broth were ground in a domestic grinder (Osterizer, Estado de Mexico, Mexico), lyophilized and stored in polyethylene bags at 4  C until use.

Proximate composition Nitrogen, lipid, and ash contents of ground raw and cooked bean samples were determined by procedures of the Association of Official Analytical Chemists (AOAC),15 (methods 920.87, 920.39, and 923.03, respectively). Protein content was calculated as nitrogen  5.85. Non-digestible fraction (NDF) The extraction of non-digestible fraction was performed following the method of Kurtzman and Halbrook.16 Water (1.5 L) was added to cooked bean samples (300 g) and the mixture was shaken for 1 min and centrifuged (Hermle Z323K, Hermle Labortechnik GMBH, Wehingen, Germany) at 9000 g for 10 min. The pellet from the first centrifugation was dissolved in 100 mL of 10% tannic acid, adjusted to pH 4 and centrifuged again (9000 g, 10 min), and the pellet was washed three times with 100 mL acetone and centrifuged for 10 min after each washing to obtain the non-digestible fraction (NDF). NDF samples were lyophilized and stored in amber flasks at 4  C until further analyses.

Soluble and insoluble fiber extraction and quantification Soluble and insoluble fiber fractions were determined following the AOAC method 991.43.15 Sample (1 g of raw, cooked bean or NDF) was incubated with 0.1 mL of a-amylase in 50 mL of 0.08 M sodium phosphate buffer, pH 6.0 at 95  C for 30 min in a water bath at 100  C with manual stirring every 5 min. After cooling, the samples were adjusted to pH 7.5 and treated with 0.1 mL of protease (5 mg mL1, in a water bath at 60  C for 30 min), and finally with 0.3 mL of amyloglucosidase at pH 4.3 with stirring in a water bath at 60  C for 30 min. The suspensions were cooled, vacuum filtered, and washed 3 times with 10 mL of distilled water. The residues corresponding to the insoluble fraction were dried and weighed. The supernatants were precipitated with 80% (v/v) ethanol at a 1 : 4 sample/ethanol ratio for 24 h at room temperature. The precipitates corresponding to the soluble fraction were filtered and the residues were washed 3 times with 10 mL of distilled water, dried and weighed. This journal is ª The Royal Society of Chemistry 2010

Oligosaccharide quantification Oligosaccharides were extracted according to Dıaz-Batalla et al.,17 with some modifications. Raw, cooked bean or NDF samples (500 mg) and 10 mL of HPLC-grade water were placed in a water bath at 80  C for 1 h, with stirring every 5 min. After cooling, the samples were filtered (45 mm, Millipore) and subjected to HPLC for quantification of oligosaccharides as described by Muzquiz et al.18 The sample (25 mL) was injected into an Agilent HPLC system model HP-1100 (Agilent Technologies, Inc., Santa Clara, CA) with a refractive index detector (IDR, 61362A) and fitted with a mBondapack/carbohydrate (3.9 mm  30 cm) column, with acetonitrile-HPLC-grade water (80 : 20, v/v) as mobile phase at 1 mL min1. Column and detector temperatures were maintained at 25  C. Standard curves were determined using raffinose, stachyose and verbascose standards. Quantification of phenolics Total phenolic compounds and condensed tannin extractions were performed in raw and cooked beans, and NDF as described previously by Cardador-Martınez et al.19 with some modifications. Dry sample (1 g) was mixed with 10 mL methanol and the mixture was shaken for 24 h at 25  C protected from light. After incubation, the samples were centrifuged (Hermle Z323K, 8000 g, 10 min) and the supernatants were analyzed for phenolic compounds and condensed tannins. Total phenolic compounds, expressed as mg equivalents of gallic acid per g sample (mg g1), were determined according to the method of Nurmi et al.20 Methanolic extract (40 mL) was mixed with 460 mL of distilled water and 250 mL of 1 N Folin-Ciocalteu reagent. The mixture was vortexed for 5 min and 1250 mL of 20% sodium carbonate was added and shaken during 2 h in the dark. The total phenolic compounds were quantified by spectrophotometry (PerkinElmer Lambda 2S, Waltham, MA) at 760 nm, using gallic acid (up to 16 mg mL1) as a reference standard. Condensed tannins, expressed as mg of (+)-catechin equivalents per g sample (mg g1), were analyzed according to the procedure of Deshpande and Cheryan21 modified for use in a microplate. Briefly, 200 mL of vanillin reagent (0.5% vanillin, 4% HCl in methanol; 1 : 1) were added to 50 ml of methanolic extract and placed in triplicate in a 96-well plate. Condensed tannins were quantified at 495 and 540 nm in a microplate reader (Thermo Electron Corporation, Multiskan Ascent) using (+)-catechin (up to 0.8 mg mL1) as a reference standard. Quantification of resistant starch The resistant starch was measured according to the gravimetric method of Saura-Calixto et al.22 in the NDF sample. The sample (0.1 g) was homogenized with 6 mL of 2 M KOH and placed in a shaker (Maxi Mix II, Thermolyne type 37600 mixer, San Francisco, CA) for 30 min at 25  C. Acetate buffer (3 mL, 0.4 M, pH 4.75) was added and the pH adjusted to 4.75 using 2 N HCL or 0.275 M NaOH. Subsequently, 60 ml of amyloglucosidase were added and the tube placed in a shaking bath at 60  C for 30 min. Samples were centrifuged (Hermle Z323K, 15 min, 3000 g) after incubation. The pellet was resuspended in 10 mL distilled water and centrifuged twice, freeze dried, and weighed. The fraction obtained corresponds to resistant starch. Food Funct., 2010, 1, 294–300 | 295

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In vitro protein digestibility

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The protein digestibility was determined according to the method of Hsu et al.23 in the NDF sample. Sample (354.4 mg) containing exactly 63.8 mg of protein was dissolved in 10 ml of distilled water and adjusted to pH 8. The enzymatic mix (1.58 mg of trypsin, 3.65 mg of chymotrypsin, 0.45 mg of peptidase dissolved in 1 mL of distilled water) was added and the sample was incubated for 10 min at 37  C. Bacterial protease was added and stirred for 10 min at 55  C and change in pH was determined at room temperature. Animal studies Male Sprague Dawley rats (28 days old) were obtained from Harlan, Inc. The rats were maintained at 22–24  C and 12 h lightdark cycle with ad libitum access to commercially prepared basal diet (2018S Harlan Tekland) and water throught the experimental period. After one week of acclimatization, the rats (n ¼ 12) were randomly placed into six groups: (1) Control, subcutaneous injection and intragastric intubation of saline solution and basal diet; (2) cooked beans (CB), CB from cultivar Bayo Madero (5.7 g kg1 of body weight) daily and basal diet; (3) nondigestible fraction (NDF) from cultivar Bayo Madero (2.5 g kg1 of body weight) and basal diet; (4) azoxymethane (AOM), subcutaneous injection of AOM (15 mg kg1 of body weight, dissolved in 1 mL of physiological saline solution) once a week for 2 weeks starting on 3rd week of supplementation and basal diet; (5) CB + AOM and basal diet; (6) NDF + AOM and basal. Both CB and NDF, dissolved in distilled water, were administered intragastrically once a day during the experimental period and the dose was selected according to the rural per capita intake of beans in the Lagunera Region of Mexico.24 All animals were sacrificed 7 weeks after the last injection (Fig. 1) and the colons were removed from caecum to the anus. The distal colon was fixed in phosphate-buffered formalin prior to histological analyses. Cecal and colonic contents (midcolon and distal colon) and feces samples were collected when the animals were sacrificed, immediately frozen and stored at 70  C until analysis. Additionally, feces samples were collected during the experimental period and stored at 70  C for SCFA and pH analyses and b-glucuronidase activity. The consumption of food and water was registered during the experimental period and weight gain was registered once per week. SCFA analysis Short chain fatty acids were determined by the method of Campos-Vega et al.4 Cecal, colonic or fecal samples (1 g) were suspended in 10 mL of HPLC-grade water, homogenized for about 3 min and centrifuged (Hermle Z323K, Hermle Labortechnik GMBH, Wehingen, Germany) at 8000 g for 10 min at 4  C. An aliquot (1.5 mL) of the supernatant was adjusted to pH 2.9–3.1 by adding 1M HCl; 750 mL of the sample were transferred to a flask and 120 mL of formic acid was added. An aliquot (1.5 mL) of the acidified supernatant was loaded into the injection port of an Agilent 6890 Plus coupled to an FID detector (Agilent Technologies Wilmington, DE) and separated on a Carbowax 20 M column (25 m  200 mm  0.10 mm nominal). Helium was 296 | Food Funct., 2010, 1, 294–300

Fig. 1 Experimental protocol for animal treatment. The animals were sacrificed 7 weeks after the last injection with AOM. (1) Control, basal diet; (2) CB, basal diet plus CB (5.7 g kg1 of body weight) daily; (3) nondigestible fraction (NDF), basal diet plus NDF (2.5 g kg1 of body weight); (4) azoxymethane (AOM), basal diet plus subcutaneous injection of AOM; (5) CB + AOM, basal diet plus cooked bean and AOM; (6) NDF + AOM, basal diet plus non-digestible fraction and AOM.

supplied as the carrier gas at 0.5 mL min1. The initial oven temperature was 95  C, maintained for 2 min and ramped at 20  C min1 to 180  C. The temperature of the flame ionization detector (FID) and the injection port was 240 and 200  C, respectively. The flow rates of hydrogen, air, and nitrogen as makeup gas were 30, 300 and 20 mL min1, respectively. The concentration of acetic, propionic, and butyric acids were calculated by using standard curves of acetic, propionic, and butyric acid standards, respectively. Aberrant crypt foci (ACF) analysis Distal colon was removed, washed with physiological saline solution, opened longitudinally, cut in two parts (approximately 1 cm each) and fixed in 10% buffered formalin for at least 24 h. After fixation, one part of the colon was stained with 0.2% methylene blue and examined under a stereomicroscope. ACF were distinguished from adjacent normal crypts by their larger sizes, greater staining intensity, elongated pericryptal area, thickened layer of epithelial cells according to the method of Bird.25 The other part of the distal colon was dissected, embedded in paraffin, cut in parallel to the mucosal surface (5mm) and Hematoxylin and Eosin (HE) stained for histology examination. ACF was reported as the mean of distal zone. b-Glucuronidase activity b-Glucuronidase activity was assayed as described by Fishman et al.26 Cecal, colonic or fecal samples (5 g) kept on ice were placed in a conical tube and 10 mL 0.02M phosphate buffered saline (PBS) were added, homogenized in a polytron, then sonicated for 6 min and centrifuged (Hermle Z323K) at 500 g for 15 min at 4  C. The supernatant (100 mL) was incubated at 37  C for 1 h in the presence of an equal volume of b-D-glucuronide/ phenolphthalein acid conjugate reagent in 800 mL of 0.02M PBS. This journal is ª The Royal Society of Chemistry 2010

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The reaction was stopped by addition of 2.5 mL alkaline glycine, 1 mL of 5% trichloroacetic acid and 1.5 mL of HPLC-grade water and the mixture was allowed to rest for 10 min. Absorbance of liberated phenolphthalein was measured with a spectrophotometer at 540 nm and interpolated to phenolphthalein standard curve (up to 13 mg mL1).

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Statistical analysis Results are expressed as mean  standard error. Statistical significance was determined using Tukey’s procedure (a ¼ 0.05) for partial characterization of common bean (Phaseolus vulgaris L.) and Dunnett test (a ¼ 0.05) for in vivo tests.

Results and discussion

availability for quantification. The total, soluble and insoluble fiber values were similar to those of four bean varieties reported by Bednar et al.31 The yield and the total, soluble and insoluble fiber contents in NDF of Bayo Madero (Table 1) were higher than those reported by Feregrino-Perez et al.12 for cultivar Negro 8025. This difference could be attributed to the variability in bean cultivars, but most importantly to strong environmental effects on bean composition,32 so it was interesting to evaluate the chemopreventive effect of cv Bayo Madero on ACF development. Our results for resistant starch were lower than those reported by Campos-Vega et al.4 for the same Bayo Madero cultivar grown at the same location but different harvest year, suggesting the strong environmental effect on the variability of this component. The resistant starch and the different types of fiber are susceptible fermentation substrates for colonic bacteria, hence the importance of quantification.32

Composition of raw, cooked and NDF beans Table 1 shows the protein, lipid, ash, fiber in raw and cooked beans (Phaseolus vulgaris L.), yield of non-digestible fraction from cooked beans, resistant starch and protein digestibility in NDF of cv. Bayo Madero. Protein content improved from 18.7 to 19.7% with the cooking process. Similar protein contents were reported previously (20 to 29%) for different bean varieties grown in Mexico.27 The protein content in the NDF sample of Bayo Madero (Table 1) was lower (17.1%) than the raw (18.7%) and cooked bean (19.7%) and with high digestibility (81.4%) suggesting that most of the protein is digested and therefore highly bioavailable in the upper gastrointestinal tract and only a small amount reaches the colon, indicating its relatively low contribution to the colonic fermentation. Lipid content was 1.92 and 1.13% in raw and cooked Bayo Madero beans, respectively (Table 1), similar to a previous report28 for Mesoamerican and Andean bean varieties. Ash content, an indicator of iron, potassium and other mineral contents, in raw and cooked beans was higher compared to NDF (Table 1) and within the range reported for four beans cultivars.29 Common beans are an important source of dietary fiber,4 with the soluble fiber increasing significantly with the cooking process (Table 1) similar to those observed for black, navy and red bean cultivars.30 This increase may be due to the thermal dissociation of complex fiber structures that releases the soluble fiber, thereby increasing their Table 1 Chemical composition of raw, cooked and NDF from cultivar Bayo Madero common beans (Phaseolus vulgaris L.)a

Protein Lipid Ash Fiber total Fiber insoluble Fiber soluble Yield of non-digestible fraction Resistant starch Protein digestibility

Oligosaccharide, total phenolic and condensed tannin content Oligosaccharide can inhibit colon carcinogenesis by different mechanisms,7 its quantification is therefore critical. Table 2 shows oligosaccharide content in raw, cooked beans and NDF samples. Total oligosaccharide content decreased with cooking (59.6 to 55.8 mg g1) (Table 2) and similar trends were observed for the individual oligosaccharide, raffinose, stachyose, and verbascose. The results are in agreement with those of DıazBatalla et al.17 for both raw and cooked beans of 10 different Mexican varieties. Total oligosaccharide content of NDF was significantly lower than those of raw and cooked samples, constituting only 28.3% of the total oligosaccharide content in cooked samples. The decrease in total oligosaccharide content in the NDF was due primarily to the extraction method, since substantial amount of oligosaccharide was lost in the supernatants after homogenization with water, tannic acid and acetone washes (data not shown). Total phenolics and condensed tannins (Table 2) were higher in cooked than raw beans. The increase in cooked bean could be attributed to thermal hydrolysis of phenolic complexes formed with other components such as protein and resistant starch in raw bean. Similar phenolic contents have been reported previously for black and pinto cultivars,33 different Mexican bean varieties,34 and for raw and cooked beans.12 NDF, as expected, had significantly higher amount of total phenolics and condensed tannins than raw and cooked samples, because these compounds are concentrated in the extract.

Raw bean

Cooked bean NDF

Food and water consumption and weight gain

18.7  0.1 b 1.9  0.4 ab 4.4  0.1 a 38.5  0.6 b 37.9  0.6 b 0.6  0.1 b

19.7  0.1 a 1.1  0.1 b 4.2  0.1 b 41.2  1.0 b 40.6  1.0 b 0.7  0.1 a 43.8  0.6

17.1  0.1 c 2.2  0.1 a 2.3  0.1 c 51.7  1.3 a 51.1  1.3 a 0.6  0.1 b

Difference in weight gain of rats among groups was not significant (data not shown), suggesting that the induction of colon cancer with AOM had no influence on weight gain or food and water intake of the rats since evaluation was at the early cancer stage.

25.5  0.2 81.4  1.3

ACF analysis

a

NDF: non-digestible fraction. Results, expressed as percentage per gram of dry sample, are the average of two independents experiments with two repetitions  standard error. Different letters by row express significant difference (a ¼ 0.05) by Tukey test.

This journal is ª The Royal Society of Chemistry 2010

Fig. 2 and 3 show the ACF in distal zone stained with methylene blue and hematoxylin and eosin, respectively. The dose of CB or NDF from cultivar Bayo Madero selected according to the per capita rural intake24 of a 70 kg body weight person in the Food Funct., 2010, 1, 294–300 | 297

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Table 2 Oligosaccharide, total phenolic and condensed tannin contents of raw, cooked and NDF from common bean (Phaseolus vulgaris L.) Bayo Madero cultivara

Sample

Raffinoseb (mg g1 dry sample)

Stachyoseb (mg g1 dry sample)

Verbascoseb (mg g1 dry sample)

Total oligosaccharidesb (mg g1 dry sample)

Total phenolicsc (mg eq gallic acid/g dry sample)

Condensed tanninsc (mg eq (+)-catechin/g dry sample)

Raw Cooked NDF

12.3  0.1 a 10.4  0.2 b 1.5  0.1 c

42.9  0.1 b 43.3  0.1 a 13.8  0.1 c

4.6  0.1 a 2.1  0.1 b 0.5  0.1 c

59.6  0.2 a 55.8  0.2 b 15.8  0.1 c

0.7  0.1 b 1.0  0.1 b 13.8  0.7 a

7.4  0.1 c 8.9  0.1 b 14.0  0.1 a

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a NDF: non-digestible fraction. Different letters in each column express significant difference (a ¼ 0.05) by Tukey test. b Results are the average of two independent experiments with duplicates  standard error. c Results are the average of three independent experiments with duplicates  standard error.

Lagunera Region of Mexico did not induce ACF and showed no significant difference compared to the control group by both staining procedures. The results suggest that CB or NDF consumption do not contribute to generation of early lesions in the colon, which is a plus to promote the increase in bean consumption. The NDF + AOM treatment significantly decreased the ACF number in the distal colon section compared with AOM group (1.5  0.72 vs. 6.6  0.40 ACF/distal zone) by methylene blue stain. Hematoxylin and eosin stain showed the same tendency with a significant decrease in the NDF + AOM group compared to AOM group (4.6  0.68 vs. 10.5  1.89 ACF/ distal zone) (Fig. 4). In turn, CB + AOM also decreased the ACF number in the distal colon section compared with AOM group by both stain (0.8  0.20 and 4.4  1.81 ACF/distal zone by methylene blue and hematoxylin and eosin stains, respectively). These data suggest that both CB and NDF can function as preventive treatment at an early or initiation stage of colon cancer. The results are consistent with our previous study,12 where similar chemoprotective effect of polysaccharide extract (PE) from Negro 8025 bean was observed in AOM-induced rats, with significant decrease in ACF in the PE + AOM group compared to AOM group (24.5  8.2 vs. 35.0  18.9 ACF/distal zone) using methylene blue stain. ACF reduction in the present study was 77.3 and 56.2% in the NDF + AOM treatment with methylene blue and HE, respectively, similar to the 70% observed with PE from Negro 8025 bean.12

Fig. 2 Distal colon tissue stained with methylene blue. (a) Control; (b) CB; (c) NDF; (d) AOM; (e) CB + AOM; (f) NDF + AOM. Magnification 40. The ACF (indicated by an arrow), consisting of four large and elliptical crypts and a thicker epithelial lining compared to adjacent crypts were darkly stained. CB: cooked bean; NDF: non-digestible fraction; AOM: azoxymethane.

SCFA production in cecal, colonic, and fecal contents SCFA production was not significantly different among groups in cecal, colonic and fecal contents (Fig. 5). However, the fecal content of the NDF + AOM group showed a slight increase in propionic (3.71  0.03 mM g1 of sample) and butyric acid concentrations (3.34  0.02 mM g1 of sample) compared with those of AOM group (3.48  0.01 and 3.21  0.01 mM g1 of sample of propionic and butyric acids, respectively). The highest SCFA concentration was present in fecal content. Our results differ from those reported by Feregrino-Perez et al.,12 where SCFA production was higher in cecal content relative to colonic and fecal contents in AOM-induced rats and those treated with Negro 8025 bean or its polysaccharide extract. These differences can be due to the bean used, Negro 8025, and the composition of its NDF or its fermentation capacity, thereby suggesting that Bayo Madero and its NDF could produce more SCFA in fecal content. In the present study, the butyric acid concentration in NDF + AOM group was 2.86 mM, 2.63 and 3.34 mM in cecal, colonic and fecal content, respectively, 298 | Food Funct., 2010, 1, 294–300

Fig. 3 Distal colon tissue stained with hematoxylin and eosin. (a) Control; (b) CB; (c) NDF; (d) AOM; (e) CB + AOM; (f) NDF + AOM. Magnification 20. The ACF (indicated by arrows) increased staining intensity of epithelial cytoplasm and presented irregular elongation of the ducts. Some ACF presented conical shape of the focus. CB: cooked bean; NDF: non-digestible fraction; AOM: azoxymethane.

concentration sufficient to produce a protective effect in the colon according to the literature. For example, Hofmanov a et al.35 demonstrated that 3 mM of butyric acid decreased proliferation and induced cell cycle arrest, apoptosis and differentiation on different cell lines of human colon cancer. Furthermore, the presence and activity of other NDF components such as phenolic compounds and galactoligosaccharides, quantifed in this study, and others as bioactive peptides, may influence those preventing ACF development. These compounds have been shown to participate in modulation of genes and This journal is ª The Royal Society of Chemistry 2010

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Fig. 4 Analysis of aberrant crypt foci (ACF) in distal zone stained with (a) methylene blue; (b) hematoxylin and eosin. CB: cooked bean; NDF: non-digestible fraction; AOM: azoxymethane. Each value is the mean of ACF by distal zone  standard error. * Mean statistically significant compared to the AOM treatment (Dunnett a ¼ 0.05). + Mean statistically significant compared to the negative control (Dunnett a ¼ 0.05).

proteins involved in cell cycle arrest, apoptosis, and other cellular processes.36,37,38 Non-digestible galactooligosaccharides increase beneficial bacterial species and have been implicated in interactions between epithelial and bacterial cells, implying that these compounds also have the capacity to inhibit the binding of pathogens to cell surfaces by acting as competitive receptors, resulting in reduced activity of enzymes capable of reactivating the colon carcinogens.39 Both treatments, CB and NDF contain considerable amount of galactooligosaccharide, as described above for the protection mechanism against ACF development. CB and NDF also contain phenolic compounds, that may effectively induce programmed cell death, differentiation, and growth inhibition in transformed colonocytes by acting at the mRNA levels of genes involved in these processes; probably, the SCFA derived from the polyphenols during gut flora-mediated fermentation could also affect these processes.37,40 Finally, bioactive peptides with potential anticancer activity probably present in NDF may pass intact from the gastrointestinal tract and are released into the colon. Bioactive peptides from different legumes have high anti-cancer potential by inducing apoptosis through caspase 3 activation, topoisomerase 2 inhibition or reduction of NF-kappa-B and interleukin-6 activation.36

Fig. 5 Concentration of SCFA in (a) cecal, (b) colonic and (c) fecal contents. CB: cooked bean; NDF: non-digestible fraction; AOM: azoxymethane. Results expressed as mM per g sample, are the average of 3 independent experiments with 3 repetitions  standard error. * Mean statistical significant in comparison to the AOM treatment (Dunnett a ¼ 0.05).

against precancerous lesions induced by the chemical carcinogen (AOM), suggesting that reduction of ACF development may be associated with a decrease in cecal, colonic and fecal activity of this enzyme, because insufficient amount of the enzyme prevents carcinogen release in the colon and thus cannot induce the damage produced in the AOM group. The inhibition of b-glucuronidase activity agrees with those observed by Nakanishi et al.,42 in colonic and cecal contents of AOM-induced rats with a diet high in resistant starch and probiotics C. butyricum. Similarly, Manoj et al.43 found a decrease of b-glucuronidase activity in colonic and fecal contents of DMHinduced rats on diets complemented with black gram and coconut kernel. The results suggest the reduction on b-glucuronidase activity and the presence of other bioactive compounds such as

Effect of CB and NDF on b-glucuronidase activity The b-glucuronidase enzymatic activity is relevant in colon carcinogenesis because it has the ability to hydrolyze many glucuronide conjugates and thus may release active carcinogenic metabolites in the intestinal lumen.41 Diets supplemented with different types of fiber have been suggested to contribute to colon cancer inhibition by increasing non-pathogenic bacteria, fiber fermentation and reduction of bacterial b-glucuronidase activity. CB and NDF consumption in AOM-induced groups significantly decreased the b-glucuronidase activity with respect to AOM (Fig. 6). This decrease was accompanied by protection This journal is ª The Royal Society of Chemistry 2010

Fig. 6 Effect of CB and NDF on b-glucuronidase activity in cecal, colonic and fecal contents. CB: cooked bean; NDF: non-digestible fraction; AOM: azoxymethane. Results are the average of 3 independent experiments with 3 repetitions  standard error. Results are expressed as nmol phenolphthalein liberated/min g1 sample. * Mean statistical difference in comparison to the AOM-treated group (Dunnett a ¼ 0.05).

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galactooligosaccharides, phenolic compounds and bioactive peptides in the CB and NDF mitigate ACF development.

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Conclusions Our results suggest that seeds of the cooked common bean cultivar Bayo Madero are excellent sources of protein, soluble and insoluble fiber and polysaccharide. Furthermore, cooked common bean Bayo Madero (CB) and its non-digestible fraction (NDF) have significant amount of oligosaccharide, resistant starch, total phenolics and condensed tannins, compounds that are all related to a lower risk of developing colon cancer. Bayo Madero and its NDF did not induce the ACF formation and demonstrated a chemoprotective effect against AOM treatment, suggesting that consumption of common beans prevent early stage colon cancer development in vivo. CB and NDF treatments significantly reduced b-glucuronidase activity thereby indicating a strong probiotic effect exerted by these compounds. These findings suggest that the suppression of aberrant crypt foci (ACF) formation is strongly influenced by the decrease in the b-glucuronidase activity in the colon, probably by altering the metabolic activities of the intestinal microflora that prevents AOM release in the colon. On the other hand, this investigation together with our previous similar study12 with Negro 8025 bean indicate that early stage of colon carcinogenesis, evaluated by ACF, in vivo is modulated by different mechanisms depending on the cultivar and composition of the common bean and its non-digestible fraction.

Notes and references 1 Instituto Nacional de Estadısticas, Geografıa e Informatica, Informaci on sobre Tumores Malignos, INEGI, Mexico, 2008, http:// www.inegi.org.mx. 2 A. E. Millen, A. F. Subar, B. I. Graubard, U. Peteres, R. B. Hayes, J. L. Weissfeld, L. A. Yokochi and R. G. Ziegler, Am. J. Clin. Nutr., 2007, 86, 1754–1764. 3 S. A. Bingham, T. Norat, A. Moskal, P. Ferrari, N. Slimani, F. Clavel-Chapelon, E. Kesse, A. Nieters, H. Boeing, A. Tjønneland, K. Overvad, C. Martinez, M. Dorronsoro, C. A. Gonz alez, E. Ardanaz, C. Navarro, J. R. Quir os, T. J. Key, N. E. Day, A. Trichopoulou, A. Naska, V. Krogh, R. Tumino, D. Palli, S. Panico, P. Vineis, H. B. Bueno-de-Mesquita, M. C. Ocke, P. H. M. Peeters, G. Berglund, G. Hallmans, E. Lund, G. Skeie, R. Kaaks and E. Riboli, Cancer Epidemiol., Biomarkers Prev., 2005, 14, 1552–1556. 4 R. Campos-Vega, R. Reynoso-Camacho, G. Pedraza-Aboytes, J. A. Acosta-Gallegos, S. H. Guzman-Maldonado, O. ParedesLopez, B. D. Oomah and G. Loarca-Pi~ na, J. Food Sci., 2009, 74, 59–65. 5 E. Lanza, T. J. Hartman, P. S. Albert, R. Shields, M. Slattery, B. Caan, E. Paskett, F. Iber, J. W. Kikendall, P. Lance, C. Daston and A. Schatzkin, J. Nutr., 2006, 136, 1896–1903. 6 G. Bobe, K. G. Barrett, R. A. Mentor-Marcel, U. Saffiotti, M. R. Young, N. H. Colburn, P. S. Albert, M. R. Bennink and E. Lanza, Nutr. Cancer, 2008, 60, 373–381. 7 K. Swennen, C. M. Courtin and J. A. Delcour, Crit. Rev. Food Sci. Nutr., 2006, 46, 459–471. 8 T. Kautenburger, G. Beyer-Sehlmeyer, G. Festag, N. Haag, S. K€ uhler, A. K€ uchler, A. Weise, B. Marian, W. H. M. Peters, T. Liehr, U. Claussen and B. L. Pool-Zobel, J. Cancer Res. Clin. Oncol., 2005, 131, 692–700. 9 L. Wang, H. S. Luo and H. Xia, J. Int. Med. Res., 2009, 37, 803–811. 10 M. Comalada, E. Bail on, O. de Haro, F. Lara-Villoslada, J. Xaus, A. Zarzuelo and J. Galvez, J. Cancer Res. Clin. Oncol., 2006, 132, 487–497.

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11 J. W. Finley, J. B. Burrell and P. G. Reeves, J. Nutr., 2007, 137, 2391– 2398. 12 A. A. Feregrino-Perez, L. C. Berumen, G. Garcıa-Alcocer, R. G. Guevara-Gonzalez, M. Ramos-G omez, R. ReynosoCamacho, J. A. Acosta-Gallegos and G. Loarca-Pi~ na, J. Agric. Food Chem., 2008, 56, 8737–8744. 13 R. Campos-Vega, R. G. Guevara-Gonzalez, B. L. Guevara-Olvera, B. D. Oomah and M. G. F. Loarca-Pi~ na, Food Res. Int., 2010, 43, 1057–1064. 14 X. O. Aparicio-Fernandez, L. Manzo-Bonilla and G. F. Loarca-Pi~ na, J. Food Sci., 2005, 70, 73–78. 15 Association of Official Analytical Chemists, Official Methods of Analysis, 17th edn, AOAC, Arlington, VA, 2002, Methods 920.87, 920.39, 923.03, 991.43. 16 R. H. Kurtzman and W. U. Halbrook, Appl. Microbiol., 1970, 20, 715–719. 17 L. Dıaz-Batalla, J. M. Widholm, G. C. Fahey, E. Casta~ no-Tostado and O. Paredes-L opez, J. Agric. Food Chem., 2006, 54, 2045–2052. 18 M. Muzquiz, C. Burbano, G. Ayet, M. M. Pedrosa and C. Cuadrado, Biotechnol. Agron. Soc., Environ., 1999, 3, 210–216. 19 A. Cardador-Martınez, E. Casta~ no-Tostado and G. Loarca-Pi~ na, Food Addit. Contam., Part A, 2002, 19, 62–69. 20 K. Nurmi, V. Ossipov, E. Huakioja and K. Pihlaja, J. Chem. Ecol., 1996, 22, 2023–2040. 21 S. S. Deshpande and M. Cheryan, J. Food Sci., 1985, 50, 905–910. 22 F. Saura-Calixto, I. Go~ ni, L. Bravo and E. Ma~ nas, J. Food Sci., 1993, 58, 642–643. 23 H. W. Hsu, D. L. Vavak, L. D. Satterlee and G. A. Miller, J. Food Sci., 1977, 42, 1269–1273. 24 L. M. Del Razo, G. G. Garcia-Vargas, J. Garcia-Salcedo, M. F. Sanmiguel, M. Rivera, M. C. Hernandez and M. E. Cebrian, Food Chem. Toxicol., 2002, 40, 1423–1431. 25 R. P. Bird, Cancer Lett., 1987, 37, 147–151. 26 W. H. Fishman, B. Springer and R. Brunetti, J. Biol. Chem., 1948, 173, 449. 27 C. Reyes-Moreno and O. Paredes-L opez, Crit. Rev. Food Sci. Nutr., 1993, 33, 227–286. 28 F. L. Da Silva, V. Monteze Guimara˜es, E. Gonc¸alves de Barros, M. Alves Moreira, L. A. Dos Santos Dias, M. G. de Almeida Oliveira, I. Chamel Jose and S. Tavares de Rezende, Plant Foods Hum. Nutr., 2006, 61, 87–89. 29 A. Vargas-Torres, P. Osorio-Dıaz, J. Tovar, O. Paredes-L opez, J. Ruales and L. A. Bello-Perez, Starch/Staerke, 2004, 56, 74–78. 30 M. Herrera, E. P. Gonzalez and J. G. Romero, ALAN., 1998, 48, 179– 182. 31 G. E. Bednar, A. R. Patil, S. M. Murray, C. M. Grieshop, N. R. Merchen and G. C. Fahey, J. Nutr., 2001, 131, 276–286. 32 A. Florez, M. Pujola, J. Valero, E. Centelles, A. Almirall and F. Casa~ nas, Food Chem., 2009, 113, 950–956. 33 L. G. Espinosa-Alonso, A. Lygin, J. M. Wildholm, M. E. Valverde and O. Paredes-Lopez, J. Agric. Food Chem., 2006, 54, 4436–4444. 34 E. G. De Mejıa, S. H. Guzman-Maldonado, J. A. Acosta-Gallegos, R. Reynoso-Camacho, E. Ramırez-Rodrıguez, J. L. PonsHernandez, M. M. Gonzalez-Chavira, J. Z. Castellanos and J. D. Nelly, J. Agric. Food Chem., 2003, 51, 5962–5966. 35 J. Hofmanova, A. Vaculova, Z. Koubkova, M. Hyzd’alova and A. Kozubık, Mol. Nutr. Food Res., 2009, 53, S102–S113. 36 E. G. De Mejıa and V. I. Prisecaru, Crit. Rev. Food Sci. Nutr., 2005, 45, 425–445. 37 S. Veeriah, T. Hofmann, M. Glei, H. Dietrich, F. Will, P. Schreier, B. Knaup and B. L. Pool-Zobel, J. Agric. Food Chem., 2007, 55, 2892–900. 38 S. Kuntz, C. Kunz and S. Rudloff, Br. J. Nutr., 2009, 101, 1306–1315. 39 G. Tzortzis, A. K. Goulas, J. M. Gee and G. R. Gibson, J. Nutr., 2005, 135, 1726–1731. 40 U. Wenzel, S. Kuntz, M. D. Brendel and H. Daniel, Cancer Res., 2000, 60, 3823–3831. 41 J. Rafter, Br. J. Nutr., 2002, 88, S89–S94. 42 S. Nakanishi, K. Kataoka, T. Kuwahara and Y. Ohnishi, Microbiol. Immunol., 2003, 47, 951–958. 43 G. Manoj, B. S. H. Thampi, S. Leelamma and P. V. G. Menon, Plant Foods Hum. Nutr., 2001, 56, 13–21.

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PAPER

www.rsc.org/foodfunction | Food & Function

Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS-dependent apoptosis An-Chin Cheng,*a Mei-Ling Tsai,b Chi-Mei Liu,b Ming-Fen Lee,a Kalyanam Nagabhushanam,c Chi-Tang Hod and Min-Hsiung Pan*b

Downloaded on 02 December 2010 Published on 27 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00134A

Received 10th September 2010, Accepted 11th October 2010 DOI: 10.1039/c0fo00134a Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage-inducible gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53-independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl-2 ratio resulted in enhanced apoptosis. Caspase-8 and tBid (truncated Bid) expression also increased in a time-dependent manner. The enzymatic activities of caspase-3 and caspase-9 increased approximately 13-fold and 7.8-fold, respectively. In addition, the proteolytic cleavage of poly-(ADP-ribose)-polymerase (PARP) and DNA fragmentation factor-45 (DFF-45) increased in dose- and time-dependent manners. Our data suggest a promising therapeutic application of garcinol in p53-independent apoptosis in cancers.

1. Introduction Garcinol (Fig. 1A) is a polyisoprenylated benzophenone derived from Garcinia indica and other related species.1 The dried rind of G. indica (cv. Kokum) is used as a garnish in cooking and a constituent in folk medicine in India. Garcinol, similar to curcumin in structure, contains both a phenolic hydroxyl unit and a b-diketone moiety. Many studies have indicated that garcinol is a pleiotropic agent2 whose functions might include antioxidant activity,3 scavenging of free radicals,4 antibacterial activity,5 and inhibition of inflammation.6 Animal studies have shown that garcinol may inhibit colon tumorigenesis7 and 4-nitroquinoline 1-oxide-induced tongue carcinogenesis.8 In addition, our previous studies demonstrated that garcinol could inhibit cell growth in human leukemia HL-60 cells9 and attenuate cell proliferation in nicotine-induced human breast cancer.10 Others have found that garcinol is a potent inducer of apoptosis involved in the regulation of caspases,11 death receptors, and anti-apoptotic proteins.12 Furthermore, garcinol may modulate cell invasion,13 histone acetyltransferases, and global gene expression in cancer.14 Taken together, garcinol can modulate

a Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, 71101, Taiwan. E-mail: [email protected]. tw; Tel: (+886)-6-278-5926 Ext 3307 b Department of Seafood Science, National Kaohsiung Marine University, No.142, Haijhuan Rd., Nanzih District, Kaohsiung, 81143, Taiwan. E-mail: [email protected]; Fax: (+886)-7-361-1261; Tel: (+886)-7-361-7141 Ext 3623 c Sabinsa Corporation, East Windsor, NJ, 08520, USA d Department of Food Science, Rutgers University, New Brunswick, NJ, 08901, USA

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several cell signalling pathways involved in apoptosis and cancer development. Hepatocellular cancer (HCC) is one of the most common cancers worldwide.15 Hepatocellular carcinoma typically occurs in patients with chronic liver diseases due to hepatitis B or C infection, alcohol abuse, or impaired differentiation function resulting in, for example, reduced albumin expression.15b,16 At present, some HCC patients are not eligible for surgical resection procedures due to the metastatic nature of the disease.17 In this study, we investigated the molecular mechanisms of apoptosis by treating Hep 3B cells with garcinol. The mechanism by which garcinol inhibits p53-negative cell proliferation is not clearly understood. The present study provides novel evidence that garcinol may have potent chemopreventive and antitumor effects in human hepatocellular carcinoma cells.

2. Materials and methods 2.1 Cell culture and chemicals The human epidermoid carcinoma cell line A431 (American Type Culture Collection [ATCC]) was cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. The Hep 3B and Hep G2 cell lines were derived from human HCC (ATCC HB-8064 and HB-8065). The COLO 205 cell line (CCL-222; American Type Culture Collection) was developed from a poorly differentiated human colon adenocarcinoma. The A431, Hep 3B, Hep G2, and MCF-7 cell lines were grown in Dulbecco’s modified Eagle’s medium, and the COLO 205 cells were grown in RPMI. Both media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Food Funct., 2010, 1, 301–307 | 301

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Fig. 1 Garcinol induces apoptosis in human cancer cells. (A) Chemical structure of garcinol. (B, C) Garcinol inhibits cell viability and induces apoptosis in a dose-dependent manner. Hep3B, HepG2, A431, MCF-7, and COLO205 cells (2  105 cells per mL) were treated with various concentrations of garcinol for 24 h. Cell viability was determined by MTT assay as described in Materials and Methods. The quantification of sub-G1 cells and apoptotic ratios (%) of the cells were detected by flow cytometry. Each experiment was independently performed three times, and the results are expressed as the mean  SE. (D, E) Induction of DNA fragmentation by garcinol. Hep3B cells were treated with either various doses of garcinol for 24 h or 50 mM garcinol for the indicated durations. Internucleosomal DNA fragmentation was analyzed by agarose electrophoresis. These experiments were performed at least in triplicate; a representative result is presented.

Grand Island, NY), 100 units per mL of penicillin, 100 mg mL1 of streptomycin, and 2 mM l-glutamine (Gibco BRL). Cell lines were maintained at 37  C in 5% carbon dioxide. Garcinol was isolated from the dried rind of G. indica9 to 98% purity. It was dissolved in dimethyl sulfoxide (DMSO). Propidium iodide was obtained from Sigma Chemical Co. (St. Louis, MO). 2.2.

overnight growth, cells were treated with a series of garcinol concentrations for 24 h. The final DMSO concentration in the culture medium was <0.05%. At the end of treatment, 30 mL of MTT was added, and the cells were incubated for 4 h. Cell viability was determined by scanning with an enzyme-linked immunosorbent assay reader at 570 nm (Dynatech MR-7000; Dynatech Laboratories, Chantilly VA).9

Cell viability assay

Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). Briefly, cells were plated into 24 well plates at a density of 1  105 cells per well. After 302 | Food Funct., 2010, 1, 301–307

2.3.

Flow cytometry

The cells were harvested, washed with PBS, resuspended in 200 mL of PBS, and fixed in 800 mL of 100% ethanol at 20  C. This journal is ª The Royal Society of Chemistry 2010

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After being left to stand overnight, the pellets were collected by centrifugation, resuspended in 1 mL of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 mg mL1 RNase), and incubated at 37  C for 30 min. Next, 1 mL of propidium iodide solution (50 mg mL1) was added, and the mixture was allowed to stand in ice water for 30 min. Fluorescence emitted from the propidium iodide-DNA complex was quantitated by FACScan cytometry (Becton Dickinson, San Jose, CA). Quantitation of the number of cells in each stage of the cell cycle was performed with ModFit LT for Mac 3.0 software (Becton Dickinson, San Jose, CA).

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2.4.

Acridine orange staining assay

Cells (5  105) were seeded into 60-mm Petri dishes in RPMI1640 medium and incubated at 37  C for 24 h. After harvesting, 5 mL of suspended cells were mixed on a glass coverslip with an equal volume of acridine orange solution (10 mg mL1 in PBS). Apoptotic cells, showing characteristic chromatic condensation, were easily distinguished from viable cells, showing homogeneously bright staining in the nucleus.

2.8. Subcellular fractionation and determination of cytochrome c release Mitochondrial and cytosolic (S100) fractions were prepared as described previously.9 Briefly, after treatment with 50 mM of garcinol, cells were washed twice in ice cold PBS and then resuspended in homogenizing buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiolthione, 17 mg mL1 phenylmethylsulfonyl fluoride, 8 mg mL1 aprotinin, and 2 mg mL1 leupeptin [pH 7.4]). After a 30 min incubation on ice, cells were passed through a needle 10 times. Unbroken cells and nuclei were pelleted by centrifugation at 750g for 10 min.18 Cytochrome c release was assessed by Western blotting as follows. 2.9.

Western blotting

Hep 3B cells (5  105 cells per mL) were harvested, washed with PBS, and then lysed with digestion buffer (0.5% sarkosyl, 0.5% mg mL1 proteinase K, 50 mM tris (hydroxy methyl) aminomethane (pH 8.0), 10 mM EDTA) at 56  C for 3 h. Lysates were then treated with RNase A (0.5 mg mL1) for another 2 h at 56  C. The DNA was extracted by phenol/chloroform/isoamyl (25/24/1) and analyzed by 1.8% agarose gel electrophoresis. Approximately 20 mg of DNA was loaded into each well of an agarose gel and separated by electrophoresis at 50 V for 120 min in TBE (Tris-borate/EDTA electrophoresis buffer). Gels were visualized under UV light and photographed.

Equal amounts of total cellular proteins (50 mg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA) with transfer buffer composed of 25 mM Tris-HCl (pH 8.9), 192 mM glycine and 20% methanol. Membranes were then probed with primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase. The immunocomplexes were visualized with an enhanced chemiluminescence kit and used film to develop it (Amersham, UK). The intensity of the band was scanned and quantified by densitometry. The primary antibodies used include anti-caspase-3 and -9 (PharMingen, San Diego, CA), anti-Bcl-2, anti-Bid, anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA), anti- PARP (UBI Inc., Lake Placid, NY), antib-actin and GADD 153 (Transduction Laboratory, Lexington, KY), and anti-DFF45/inhibitor of caspase activated DNase (ICAD) (MBL, Naka-Ku, Nagoya, Japan) antibodies.

2.6.

2.10.

2.5.

DNA extraction and electrophoretic analysis

Determination of ROS production

ROS production was monitored by flow cytometry using dichlorodihydrofluorescein diacetate (DCFH-DA). This dye is a stable, nonpolar compound that readily diffuses into cells. It is hydrolyzed by intracellular esterase to yield DCFH, which remains trapped within the cells. Hep3B cells were treated with garcinol (50 mM) for 10 min. DCFH-DA (20 mM) and dihydroethidium (DHE) (20 mM) were added to the medium for a further 30 min at 37  C. Histograms were analyzed using Cell Quest software and were compared with histograms of untreated control cells.18 2.7.

Analysis of mitochondrial transmembrane potential

The change in the mitochondrial transmembrane potential was monitored by flow cytometry. Briefly, Hep3B cells were treated with garcinol (50 mM) for 15 min. The mitochondrial transmembrane potential was measured directly using 40 nM 3,30 dihexyloxacarbocyanine [DiOC6(3)] (Molecular Probes, Eugene, OR). Fluorescence was measured after staining the cells for 30 min at 37  C. Histograms were analyzed using Cell Quest software and were compared with histograms of untreated control cells. This journal is ª The Royal Society of Chemistry 2010

Caspase activity

After garcinol treatment, cells were collected and washed with PBS and suspended in 25 mM HEPES (pH 7.5), 5 mM MgCl2, 5 mM EDTA, 5 mM dithiothione, 2 mM phenylmethanesulfonyl fluoride, 10 mg mL1 pepstatin A, and 10 mg mL1 leupeptin. Cell lysates were clarified by centrifugation at 12 000g for 20 min at 4  C. Caspase activity in the supernatant was determined as described previously.9 2.11.

Isolation of RNA and RT-PCR

Total cellular RNA was isolated with a TRIzol Reagent kit (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Changes in the steady-state concentration of GADD153 mRNA were determined as described.18 2.12.

Statistical analysis

Data are presented as the mean  SE Student’s t-test was used to determine statistical significance of each experimental test condition compared to respective controls. A P-value of <0.05 was considered statistically significant. Food Funct., 2010, 1, 301–307 | 303

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3. Results

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3.1. Cytotoxic and apoptotic effects of garcinol in Hep3B, HepG2, A431, MCF-7, and COLO205 cells To investigate the effect of garcinol on cell growth, we applied various concentrations of garcinol for 24 h to Hep3B, HepG2, A431, MCF-7, and COLO205 cells. Cell viability was determined by the MTT assay. Sub-G1 cells and apoptotic ratios were quantified by flow cytometry. As shown in Fig. 1B and 1C, garcinol exhibited cytotoxic and apoptotic effects on selected human cancer cells in a concentration-dependent manner. Moreover, when treated with 50 mM of garcinol, Hep3B cells exhibited the highest apoptotic ratio among the cell lines studied (Fig. 1C). Therefore, we used Hep3B cells for the subsequent studies to investigate the effects of garcinol on caspases, Bcl-2 family members, and the mechanism of apoptotic cell death.

treated with 50 mM of garcinol in time-course experiments, DNA ladders became visible after 3 h of treatment, and the fragmentation pattern continued up to 24 h (Fig. 1E). In addition, garcinol-induced morphological changes in Hep3B cells were observed by light microscopy. As shown in Fig. 2A–E, Hep3B cells exhibited apoptotic bodies after treatment with garcinol in a dose-dependent pattern. To further characterize garcinol-

3.2. Induction of DNA fragmentation in Hep3B cells by garcinol Apoptosis induces morphological changes including membrane blebbing, cell shrinkage, chromatin condensation, and DNA fragmentation.19 We studied the effect of garcinol on DNA fragmentation by treating Hep3B cells with various concentrations of garcinol for 24 h. DNA ladders emerged at 25 mM of garcinol treatment. The DNA fragmentation response followed a dose-dependent pattern (Fig. 1D). When Hep3B cells were

Fig. 2 Induction of chromatin condensation by garcinol in Hep 3B cells. (A–E) Induction of apoptotic bodies by garcinol. Hep3B cells were treated with 0 mM, 5 mM, 10 mM, 25 mM, 50 mM of garcinol for 24 h. The morphological changes of Hep3B cells were observed by light microscopy. 0 mM (vehicle). (F, G) Induction of chromatin condensation by garcinol. Hep3B cells were treated with 0.05% DMSO as vehicle control or 50 mM garcinol for 24 h, followed by acridine orange treatment. The nuclear staining was examined by fluorescence microscopy (200). Arrowheads indicate nuclear condensation. These experiments were performed at least in triplicate; a representative result is presented.

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Fig. 3 Effect of garcinol on Bcl-2 protein family and DFF-45 degradation in garcinol-treated Hep3B cells. (A)The expression of Bax, Bcl-2, and tBid were analyzed by Western blotting as described in the Materials and Methods. These experiments were performed at least in triplicate, and a representative experiment is presented. (B) Induction of DFF-45 degradation by garcinol. Hep3B cells were treated with various concentrations of garcinol for 24 h or 50 mM of garcinol at various time points. DFF-45 protein was detected by Western blot analysis using specific antibodies against DFF-45. These experiments were performed at least in triplicate, and a representative experiment is presented.

Fig. 4 Induction of reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cytochrome c release by garcinol-induced apoptosis. (A–C) Hep3B cells were treated with 50 mM garcinol for 15 min, incubated with (A) HE, (B) DCFH-DA, or (C) DiOC6 for 30 min, then analyzed by flow cytometry. Data are presented as log fluorescence intensity and expressed as the mean  SE. ‘‘MC’’ indicates the control group; ‘‘MG’’ represents the garcinol group. (D) Hep3B cells were treated with 50 mM garcinol for the indicated durations. Subcellular fractions were prepared and lysed as described, and cytosolic cytochrome c was detected by Western blots. These experiments were performed at least in triplicate, and a representative experiment is presented.

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induced cell death, we examined the nuclear morphology of dying cells with acridine orange, a fluorescent DNA-binding agent. Hep3B cells exhibited significant chromosomal condensation, indicative of apoptotic cell death, after 50 mM of garcinol treatment for 24 h (Fig. 2G).

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3.3. Induction of reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cytochrome c release in garcinolinduced apoptosis To verify whether garcinol has the ability to generate ROS, Hep3B cells were treated with 50 mM garcinol for 15 min followed by HE, DCFH-DA, or DiOC6 for an additional 30 min. The generation of ROS, including superoxide anion (O2) and hydrogen peroxide (H2O2), and the mitochondrial transmembrane potential (DJm) were analyzed by measuring log fluorescence intensity with flow cytometry. Fig. 4 shows that garcinol induced ROS in Hep3B cells. In addition, the mitochondrial membrane potential was reduced in the garcinoltreated cells compared to the control cells (MG : 40.87 vs. MC : 133.19) (Fig. 4C). Mitochondria are specialized organelles

containing an outer membrane and an inner membrane separated by an inter-membrane space that contains many proapoptotic proteins, such as cytochrome c. Fig. 4D illustrates that garcinol treatment gradually increased the release of cytochrome c into the cytosol of Hep3B cells. These observations suggest that garcinol induces apoptosis in Hep3B cells via the mitochondrial pathway. The expression of Bax, Bcl-2 and tBid was evaluated at different time points after garcinol treatment of cells. As shown in Fig. 3A, an apparent increase in Bax, a reduction in Bcl-2 protein expression, and the cleavage of Bid were detected after 1 h treatment with garcinol. The increase in the Bax/Bcl-2 ratio resulted in an enhancement of apoptosis. These data suggest that the cleavage of Bid by active caspase-8 may be one of the mechanisms that contribute to the activation of the mitochondrial pathway during garcinol-induced apoptosis. 3.4. Induction of caspase activity, PARP cleavage, and DFF-45 degradation in garcinol-induced apoptosis To observe the effect of garcinol on the caspases activation, the activities of caspases-1,-3,-8, and -9 were measured in Hep3B

Fig. 5 Induction of caspase activity, PARP cleavage, and DFF-45 degradation by garcinol. (A) Enzymatic activities of caspase-1, -3, -8, and -9 were determined by incubating 50 mg of total protein with corresponding fluorogenic, caspase-specific substrates for 1 h at 30  C. The release of 7-amino-4methylcoumarin (AMC) was monitored by fluorescence spectrophotometry (excitation ¼ 360 nm; emission ¼ 460 nm). These experiments were performed at least in triplicate, and a representative experiment is presented. Asterisk denotes a statistically significant increase compared with values of control, *P < 0.01, **P < 0.001. (B) Hep3B cell were treated with garcinol for the indicated durations, and the expression of caspases-9 and -3 were analyzed by Western blot. Hep3B cells were treated with either various concentrations of garcinol for 24 h or 50 mM of garcinol for various durations. PARP expression (C) was detected by Western blot. These experiments were performed at least in triplicate, and a representative result is presented.

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cells after various durations of treatment with 50 mM of garcinol. As illustrated in Fig. 5A, both caspase-3 and -9 were activated by garcinol in a time-dependent manner, whereas a very low level of caspase-1 activity was detected after the garcinol treatment. Garcinol induced a rapid increase in caspase-3 and caspase-9 activity with approximately 13-fold and 7.8-fold maximal induction, respectively (Fig. 5A). Protein cleavage of pro-caspase-9 and pro-caspase-3 occurred after 0.5 h exposure to garcinol (Fig. 5B). To detect PARP and DFF-45 cleavage as a result of caspase-3 activation, Hep3B cells were treated either with various concentrations of garcinol for 24 h or with 50 mM for varying durations. As shown in Fig. 5C, garcinol induced dose- and timedependent proteolytic cleavage of PARP, resulting in a reduction of the 116 kDa protein and accumulation of the 85 kDa fragment. Garcinol also induced cleavage of DFF-45 in a dosedependent manner. However, cleaved DFF-45 was only detected after 3 h of 50 mM garcinol treatment. (Fig. 3B). 3.5. Induction of GADD153 mRNA and protein expression by garcinol in Hep3B cells Recently, several studies have suggested that GADD153 triggers critical early events in the initiation of apoptosis.20 Thus, we investigated the effect of garcinol on GADD153 mRNA and protein expression in Hep3B cells. As shown in Fig. 6A, garcinol up-regulated GADD153 mRNA in concentration- (upper

Fig. 6 Garcinol induces up-regulation of GADD153 mRNA and protein expression in a dose-dependent manner. Hep3B cells were incubated with either 5 to 50 mM garcinol for 1 h or 50 mM garcinol for 0 to 2 h. (A) Total RNA was then isolated for multiplex relative RT-PCR analysis using gene-specific primers for the target gene, GADD153, and the internal control gene, b-actin. (B) Whole cell lysates were prepared for Western blot analysis of GADD153 and b-actin. This experiment was repeated three times with similar results.

306 | Food Funct., 2010, 1, 301–307

panels) and time-dependent manners (lower panels), with peak induction at 1 h following treatment. Similarly, garcinol treatment increased GADD153 protein expression, with the maximal expression also reached after 1 h (Fig. 6B, upper panels). Furthermore, the level of GADD153 protein expression was increased in a dose-dependent manner (Fig. 6B, lower panels). These results indicate that garcinol induced the expression of both GADD153 mRNA and protein, which may play a role in apoptosis.

4. Discussion In this study, we have shown that garcinol treatment decreases cell viability and strongly induces apoptosis via the ROSdependent pathway in Hep3B cells. Recent studies have suggested numerous apoptosis-inducing chemotherapeutic agents for the treatment of cancer, mutations in p53 block the antitumor effects of chemicals in many cases. Interestingly, garcinol, which has been shown to be pharmacologically quite safe and is used in traditional medicine, was found to induce apoptosis in p53-deficient Hep3B cells. Almost half of hepatocellular cancer cases lack functional p53,15b the absence of which blocks the apoptosis-inducing effects of chemotherapeutic agents.21 However, our initial findings indicate a potential clinical application of garcinol for the treatment of hepatocellular cancers. Garcinol, which contains a phenolic, has a b-diketone and an a,b-unsaturated ketone moiety. Previous studies suggest that a,b-unsaturated carbonyls are susceptible to nucleophilic addition reactions with thiols such as glutathione, the most abundant nonprotein thiol in vivo.22 It is clear that the a,b-unsaturated ketone moiety is essential for exerting cytotoxic activity via depletion of intracellular glutathione.23 Recent studies indicate that intracellular glutathione depletion could result in mitochondrial dysfunction.24 Moreover, oxidative damage to the mitochondrial membrane due to increased generation of ROS has been shown to play an important role in apoptosis. Herein, we demonstrated that garcinol could disrupt mitochondrial function in the early stages of apoptosis and subsequently induce caspase-9, but not caspase-1, activation through the release of cytochrome c. Therefore, we speculate that intracellular generation of ROS could be an important factor in garcinol-induced apoptosis. Recent studies have demonstrated that garcinol can potentiate TRAIL-induced apoptosis through the up-regulation of death receptors and down-regulation of antiapoptotic proteins.25 We also found that garcinol treatment increased caspase-8 activity (approximately 8.3-fold) and levels of tBid in Hep3B cells. These results indicate that garcinol may also induce apoptosis via the death receptor-mediated pathway in Hep3B cells. Taken together, these data show that garcinol induced the generation of ROS (O2 and H2O2) and the expression of GADD153 in Hep3B cells. The increased ROS and GADD153 further resulted in downstream apoptotic events, including an increase in the Bax/Bcl-2 ratio, a reduction in the mitochondrial membrane potential, the release of cytochrome c into the cytosol, the processing of procaspase-9 and procaspase-3, and the cleavage of PARP and DFF-45. Our findings suggest that the apoptosis-inducing effect of garcinol may provide a means for chemotherapy in hepatocellular cancers. This journal is ª The Royal Society of Chemistry 2010

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Acknowledgements This study was supported by the National Science Council NSC 98-2313-B-022-002-MY3 and NSC 98-2321-B-022-001-

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Antioxidative and antibacterial effects of seeds and fruit rind of nutraceutical plants belonging to the Fabaceae family Sumitra Chanda,* Sandeep Dudhatra and Mital Kaneria

Downloaded on 02 December 2010 Published on 25 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00028K

Received 2nd June 2010, Accepted 22nd July 2010 DOI: 10.1039/c0fo00028k In the present study, the seeds and fruit rind of six plants of the Fabaceae family were selected to evaluate their potential as antioxidant and antibacterial agents. The dried powders were individually extracted with various organic solvents by the cold percolation method, were evaluated for antibacterial activity and methanol extracts used for antioxidant activities. Total phenol, protein and sugar contents were also measured. Antioxidant activities were measured by DPPH free radical scavenging activity, superoxide anion radical scavenging activity and reducing capacity assessment. Antibacterial activity was measured by the agar well diffusion method against four Gram positive and four Gram negative bacteria. The methanol extract of the fruit rind of C. indica showed the maximum DPPH free radical scavenging activity, superoxide anion radical scavenging activity, a high reducing capacity assessment and also had the highest total phenol content. There was a direct correlation between the phenol content and the antioxidant activity. The antibacterial activity of all the extracts was more pronounced on Gram positive bacteria than on Gram negative bacteria. Thus, the fruit rind of C. indica showed the best antioxidant and antibacterial activities.

Introduction Reactive oxygen species (ROS) are a class of highly reactive molecules derived from oxygen, and generated by metabolic processes in human beings and by external factors such as pollution, radiation or some dietary habits. ROS are detrimental to cells, as they destroy macromolecules such as DNA, protein and lipids when overproduced during conditions such as excessive exercise, hypoxia and/or in antioxidant system failure. ROS have been implicated in more than 100 diseases from malaria to hemorrhagic shock and AIDS.2 Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and citric acid are commonly used as antioxidants in foods to prevent or retard lipid oxidation. But, according to the toxicologists and nutritionists, synthetic antioxidants can show carcinogenic effects in living organisms,7 enlarge liver size and increase microsomal enzyme activity.3,12 Thus, evaluation of the antioxidative activity of naturally-occurring substances has become a focus of interest in recent years.14 The use of plants and herbs as antioxidants in processed foods is becoming of increasing importance in the food industry as an alternative to synthetic antioxidants.20 The frequency of life threatening infections caused by pathogenic microorganisms has increased worldwide, and is becoming an important cause of morbidity and mortality in immunocompromised patients in developing countries.1 The steadily increasing bacterial resistance to existing drugs is a serious problem, and therefore there is a dire need to search for new classes of antibacterial substances, especially from natural sources. Unlike synthetic drugs, antimicrobials of plant origin Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University, Rajkot, 360 005, Gujarat, India. E-mail: [email protected]

308 | Food Funct., 2010, 1, 308–315

are not associated with side effects and have a great therapeutic potential to heal many infectious diseases.13,29 Any part of a plant can be therapeutically used to treat infectious diseases. There are numerous reports on the antimicrobial activity of crude plant extracts of different parts of plants, for example Punica granatum leaf,25 Nelumbo nucifera rhizome,36 Trapa natans L. fruit rind,30 Satureja cuneifolia,28 Diospyros ebenum Roxb. leaf,8 Pistachia vera green hull33 and Ficus carica latex,5 etc. The Fabaceae family is the second biggest family among the dicotyledons, and out of 12,000 species, 951 species are grown in India. The aim of this work was to analyze the antioxidant and antibacterial activities of seeds and fruit rind extracts of six nutraceutical plant species belonging to the Fabaceae family.

Materials and methods Plant material Fresh pods of six different plants belonging to the Fabaceae family were collected in 2008 from the local market of Rajkot, Gujarat. The ethnobotanical information4 of the studied plants is given in Table 1. The pods were thoroughly washed with tap water, the seeds and fruit rind separated, shade dried, crushed in a homogenizer to a fine powder and stored in air tight bottles. Crude extraction The dry powder of seeds and fruit rind (10 g) was first de-fatted with hexane (100 ml), and then individually extracted with ethyl acetate, methanol and water by the cold percolation method.30 The extraction flasks were kept on a rotary shaker for 24 h at 120 rpm. Thereafter, it was filtered through 8 layers of muslin cloth and centrifuged at 5000 rpm for 15 min. The supernatant was concentrated to dryness under reduced pressure. The This journal is ª The Royal Society of Chemistry 2010

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Table 1 The botanical names, vernacular names and therapeutic use of the screened plants

No.

Botanical Name

Vernacular Name

1

Cajanus indica Spr.

Tuver

2

Pisum sativum L.

Vatana

3

Vicia faba L.

Vaal

4

Vigna mungo L.

Udad

5

Vigna radiate (L.) Wilczek

Mag

6

Vigna unguiculata (Linn.) Walp.

Chori

Therapeutic uses Bechic, diuretic, astringent, strongly antidysenteric, laxative, vulnerant, restoring lost taste, leprosy, cough, mouth ulcers, tumors, bronchitis, heart diseases, piles, biliousness, diarrhoea, weakness and also used in increasing efficiency of liver Contraceptive, fungistatic and spermicidal Hemolytic anemia, Parkinson’s disease, malaria and help in controlling hypertension Nervine disorders, debility, paralysis, facial paralysis, osteo-arthritis, liver disorders and constipation for relief from such ailments Dyspepsia, pyrexia, diarrhoea, skin diseases, leprosy, inflammations, seminal weakness, burning sensation, colic, flatulence, haemorrhages, cough, haemoptysis, agalactia, emaciation, consumption, facial paralysis, hemiplegia, vitiated condition of vata and pitta, fever and general debility Astringent, appetiser, laxative, anthelmintic, anaphrodisiac, diuretic, galactogogue, liver tonic, anorexia, constipation, helmnathiasis, strangury, agalactia, jaundice and general debility

extraction procedure was repeated at least three times, and the dry extract was pooled, weighed and stored at 4  C in air tight bottles. Determination of protein and sugar content The protein content of all the samples was measured by Lowry’s method.19 The reducing, non-reducing and total sugar content of all the samples was estimated using Miller’s reagent.24 Determination of total phenol content The total phenol content in ethyl acetate, methanol and water extracts of the different plants was determined by the Folin– Ciocalteu reagent method.22 0.5 ml of sample (1 mg ml1) and 0.1 ml (0.5 N) of Folin–Ciocalteu reagent were mixed and the mixture incubated at room temperature for 15 min. Then, 2.5 ml of saturated sodium carbonate was added and the resulting This journal is ª The Royal Society of Chemistry 2010

mixture was again incubated at room temperature for 30 min. The absorbance of the mixture was measured at 760 nm using a spectrophotometer. The total phenol values are expressed in terms of gallic acid equivalents per gram of extracted compound. The assay was carried out in triplicate, with mean values being presented. Antioxidant testing assays DPPH free radical scavenging activity. The DPPH (2,2-diphenyl-1-picryl-hydrazyl) free radical scavenging activity of methanol extracts of all the plants was measured according to the method described by McCune and Johns,23 with some modifications. Various concentrations of extracts (100–1000 mg) in methanol were added to a DPPH solution in methanol (0.3 mM). The mixture was incubated for 10 min at room temperature in the dark, after which the absorbance was measured at 517 nm. Ascorbic acid was used as a standard. Superoxide anion radical scavenging activity. The superoxide anion radical scavenging activity of methanol extracts was measured according to the method of Robak and Gryglewski,34 with some modifications. Superoxide anion radicals were generated in phenazine methosulfate (PMS)-nicotinamide adenine dinucleotide-reduced (NADH) systems by NADH oxidation and assayed by nitroblue tetrazolium (NBT) reduction. In this experiment, the superoxide radical were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 0.5 ml of an NBT (300 mM) solution, 0.5 ml of an NADH (936 mM) solution and 0.5 ml of different concentrations of methanolic extract. The reaction was started by adding 0.5 ml of the PMS solution (120 mM) to the mixtures. The reaction mixture was incubated at 25  C for 5 min and the absorbance measured at 560 nm against a blank sample. Gallic acid was used as a standard. The assay was carried out in triplicate and the percentage inhibition calculated. IC50 values were calculated by linear regression analysis. Reducing capacity assessment. The reducing capacity assessment of methanol extracts was determined according to the method of Athukorala et al.,6 with some modifications. Methanol extracts were used at different concentrations (100–500 mg). 1 ml extracts were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [K3Fe(CN)6] (30 mM). The mixture was incubated at 50  C for 20 min. Then, 2.5 ml TCA (0.6 M) was added to the mixture, which was then centrifuged for 10 min at 3000 rpm. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 6 mM), and the absorbance was measured at 700 nm by a spectrophotometer. Ascorbic acid was used as a standard. The assay was carried out in triplicate. The increased absorbance of the reaction mixture is indicated. Antibacterial assay Microorganisms tested. The bacterial strains used to assess the antibacterial properties of the crude extracts included four Gram positive bacteria (Bacillus cereus ATCC11778, Bacillus megaterium ATCC9885, Bacillus subtilis ATCC6633 and Food Funct., 2010, 1, 308–315 | 309

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Corynebacterium rubrum ATCC14898) and four Gram negative bacteria (Pseudomonas aeruginosa ATCC27853, Pseudomonas stutzeri ATCC17588, Pseudomonas pictoruim NCIB9152 and Klebsiella aerogenes NCTC418). The investigated bacterial strains were obtained from the National Chemical Laboratory (NCL), Pune, India. The organisms were maintained on nutrient agar (Hi Media, India) slop at 4  C and sub-cultured before use. The bacteria studied are clinically important examples, causing several infections, and it is essential to overcome them through active therapeutic agents. Determination of antibacterial assay. The in vitro antibacterial activity of the crude extracts was studied against eight bacterial strains by the agar well diffusion method.32,15 Mueller Hinton agar no. 2 (Hi Media, India) was used as the bacteriological medium. The extracts were diluted in 100% dimethylsulfoxide (DMSO) at the concentration of 25 mg ml1. The antibacterial activity was evaluated at a concentration of 2.5 mg well1. The Mueller Hinton agar was melted and cooled to 48–50  C, and a standardized inoculum (1.5  108 CFU ml1, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a solid plate. Wells were prepared in the seeded agar plates. The test compound (100 ml) was then introduced in the well (8.5 mm) and the plates incubated overnight at 37  C. The antimicrobial spectrum of the extract was determined for the bacterial species in terms of zone sizes around each well. The diameter of the zone of inhibition produced by the agent was compared with those produced by the commercial control antibiotics Carbenicillin (100 mg), Imipenen (10 mg), Ceftazidine (30 mg), Ciprofloxacin (5 mg), Cefaclor (30 mg), Amikacin (30 mg), Tetracycline (30 mg), Piperacillin (100 mg), Methicillin (5 mg), Chloramphenicol (30 mg) and Azithromycin (15 mg). These are commonly used antibiotics to treat infections caused by several Gram positive and Gram negative bacteria, and were therefore selected as control antibiotics. DMSO was used as a negative control. The control zones were subtracted from the test zones, and the resulting zone diameter is shown in Table 4 and Table 5. The experiment was performed three times to minimize error, and mean values SEM are presented.

Results and discussion Extractive yield The successful determination of biologically-active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Several parameters such as extraction temperature, solvent type and solvent concentration can influence the yield during the extraction process.17 Traditional healers primarily use water, but plant extracts from organic solvents showed better antimicrobial properties. Therefore, a number of organic solvents, such as chloroform, methanol, ethanol, acetone, dichloromethane, etc., have been used to evaluate the antimicrobial property of different plants.31,35 In the present work, plant extraction was performed in aqueous as well as organic solvents. The extractive yield differed in different solvents, as shown in Table 2. The extractive yield was greater in water (aqueous) than in organic solvents in both the seeds and fruit rind of all the six 310 | Food Funct., 2010, 1, 308–315

plants. The maximum yield was obtained in the aqueous and methanol extracts of the fruit rind of P. sativum. It appears that in the screened plants, polar compounds were greater in concentration than those of non-polar compounds. There are many reports in the literature where the extractive yield varies with different solvents.10,15

Total protein and sugar content Some chemical substances in plants like proteins, carbohydrates, vitamins and fiber also contribute to their antioxidant capacity. The protein content of seeds was more than that of fruit rind in all six plants (Table 2). The reducing sugars were less compared to non-reducing sugars in both the seeds and fruit rind of all six of these plants (Table 2). Amongst the twelve parts, the highest protein content was observed in the seed of V. faba and the highest sugar content was in the fruit rind of C. indica.

Total phenol content Phenolics are antioxidants with redox properties that allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. Phenolic compounds are commonly found in both edible and non-edible plants,18 like fruits and vegetables, their products, leguminous plants, herbs and spices.9 However, there are very few reports on fruit rind, especially plants belonging to the Fabaceae family. In all six plants, the fruit rind had a considerably greater total phenol content than the seeds (Table 3). Among the four solvents, the methanol extracts had considerably more total phenol content following aqueous extraction. Among the twelve parts, the methanol extract of the fruit rind of C. indica had the maximum total phenol content.

Antioxidant testing assays Antioxidants are known to exhibit their biochemical effects through numerous mechanisms, including the prevention of chain initiation, reductive capacity and radical scavenging mechanisms. Several methods have been used to measure the antioxidant activity of biological materials. However, the most commonly used ones are those involving chromogen compounds of radical nature, which stimulate the reductive oxygen species. These methods are popular due to their ease, speed and sensitivity.

DPPH free radical scavenging activity The IC50 values of the DPPH free radical scavenging activity of methanol extracts of seeds and fruit rinds of the six plants is shown in Table 3. The methanolic extracts of the fruit rinds showed varied levels of DPPH free radical scavenging activity, while the methanolic extracts of the seeds showed IC50 values greater than 1000 mg ml1 (Table 3). The fruit rinds of P. sativum and V. faba showed the higher IC50 values (>1000 mg ml1), while in the other four extracts, the value of the IC50 ranged between 39–560 mg ml1. The lowest IC50 value was shown by C. indica fruit rind. This journal is ª The Royal Society of Chemistry 2010

This journal is ª The Royal Society of Chemistry 2010

C. indica

P. sativum

V. faba

V. mungo

V. radiata

V. unguiculata

1

2

3

4

5

6

Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind

Part 0.77 1.4 1.48 0.47 0.58 0.77 0.60 0.50 0.49 0.51 0.72 0.66

Hexane 0.24 0.49 0.40 0.46 0.21 0.52 0.30 0.43 0.20 0.38 0.28 0.46

Ethyl acetate 3.23 4.98 5.39 15.71 3.68 5.84 2.66 2.37 3.79 2.22 5.05 3.08

Methanol 4.44 5.56 11.03 18.67 9.17 13.61 5.51 8.51 6.48 7.69 9.38 8.24

Aqueous

Reducing sugar 1.61  0.12 34.5  0.03 0.92  0.01 3.56  0.01 0.81  0.01 7.95  0.03 0.99  0.01 1.53  0.02 2.08  0.02 3.15  0.03 1.41  0.01 3.04  0.02

Total protein content (mg g1) 62.40  0.02 15.51  0.04 70.20  0.06 13.18  0.03 86.31  0.08 6.89  0.02 77.21  0.12 10.95  0.01 67.50  0.11 16.69  0.06 46.50  0.09 14.25  0.02

22.62  0.06 64.40  0.14 17.14  0.16 1.06  0.01 12.57  0.03 6.52  0.02 14.81  0.07 0.70  0.02 35.56  0.16 4.95  0.03 11.97  0.08 1.14  0.05

Non-reducing sugar

Sugar content (mg g1)

24.23  0.09 98.90  0.13 18.06  0.09 4.62  0.02 13.38  0.06 14.47  0.08 15.80  0.06 2.23  0.02 37.64  0.15 8.10  0.05 13.38  0.06 4.18  0.02

Total sugar

Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind Seed Fruit rind

C. indica

V. unguiculata

V. radiata

V. mungo

V. faba

P. sativum

Part

Plant name

>1000 39 >1000 >1000 >1000 >1000 >1000 560 >1000 220 >1000 480

6.22  0.09 158.98  2.14 8.45  0.12 11.62  0.14 4.35  0.06 14.36  0.07 22.7  0.09 43.95  0.23 16.29  0.09 55.77  0.24 8.33  0.16 32.88  0.27

4.18  0.21 13.62  0.15 6.84  0.08 7.76  0.05 4.57  0.03 11.35  0.08 9.68  0.03 16.5  0.15 7.95  0.05 13.78  0.12 7.56  0.08 8.67  0.11

15.62  0.08 43.13  1.43 11.12  0.08 11.26  0.07 10.9  0.05 42.96  0.14 6.08  0.08 51.99  0.14 20.59  0.12 74.86  0.32 13.78  0.11 47.16  0.17

DPPH

Methanol

Ethyl acetate

Aqueous

IC50 value (mg ml1)

Total phenol content (mg g1)

>1000 420 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000

SO

Table 3 Total phenol content, DPPH free radical scavenging activity (DPPH) and superoxide anion radical scavenging activity (SO) of methanolic extracts of the seeds and fruit rinds of the screened plants

Plant name

No.

% Yield (g g1 dry powder of the plant)

Table 2 The extractive yield, and the total protein and sugar content of the seed and fruit rind of the screened plants

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Fig. 1 Reducing capacity assessments of methanol extracts of the seed and fruit rind of the screened plants.

Table 4 The antibacterial activities of different solvent extracts of seeds of the screened plantsa Zone of inhibition (mm) Gram positive bacteria

Gram negative bacteria

Plant name

Extracts

BC

BS

BM

CR

PA

PS

PP

KA

C. indica

HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ

10.0  0.16 9.5  0.16 8.7  0.05 9.3  0.05 10.5  0.00 12.8  0.33 9.7  0.17 11.7  0.17 10.3  0.47 10.3  0.15 9.5  0.17 9.5  0.17 8.7  0.17 10.3  0.60 8.0  0.29 8.1  0.23 8.5  0.00 9.3  0.17 8.0  0.29 9.8  0.17 9.5  0.00 11.0  0.29 9.5  0.00 9.7  0.17

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

9.3  0.33 9.7  0.33 — — 9.0  0.00 9.0  0.00 — — 9.3  0.33 9.7  0.33 — — 10.0  0.00 10.0  0.00 — — 9.0  0.00 9.0  0.00 — — 9.0  0.00 9.0  0.00 — —

P. sativum

V. faba

V. mungo

V. radiata

V. unguiculata

a HE ¼ hexane; EA ¼ ethyl acetate; ME ¼ methanol; AQ ¼ aqueous; BC ¼ Bacillus cereus; BS ¼ Bacillus subtilis; BM ¼ Bacillus megaterium; CR ¼ Corynebacterium rubrum; PA ¼ Pseudomonas aeruginosa; PS ¼ Pseudomonas stutzeri; PP ¼ Pseudomonas pictoruim; KA ¼ Klebsiella aerogenes;  ¼ SEM value; — ¼ no activity.

312 | Food Funct., 2010, 1, 308–315

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Superoxide anion radical scavenging activity

Reducing capacity assessment

The IC50 values of superoxide anion radical scavenging activity are shown in Table 3. Out of all the methanolic extracts of the seeds and fruit rinds, only the fruit rind of C. indica showed a superoxide anion radical scavenging activity (IC50 ¼ 420 mg ml1), while other methanolic extracts of seeds and fruit rinds showed IC50 values of more than 1000 mg ml1 (Table 3). Here, gallic acid was used as a standard (IC50 ¼ 185 mg ml1).

The reducing capacity assessments of all the methanolic extracts of the seeds and fruit rinds are shown in Fig. 1. The fruit rind of C. indica showed a high reducing capacity assessment compared to all the other extracts, while seeds of V. unguiculata showed a moderate reducing capacity assessment. In the seeds, the reducing capacity assessment of the plants was in the order: C. indica > V. radiata > V. faba > V. mungo > P. sativum >

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Table 5 The antibacterial activities of different solvent extracts of fruit rinds of the screened plantsa Zone of inhibition (mm) Gram positive bacteria

Gram negative bacteria

Plant name

Solvents

BC

BS

BM

CR

PA

PS

PP

KA

C. indica

HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ HE EA ME AQ

14.3  0.17 14.7  0.44 13.3  0.17 8.5  0.00 13.0  0.29 13.0  0.29 9.0  0.29 9.5  0.00 9.5  0.29 12.7  0.17 11.7  0.60 10.0  0.29 9.3  0.44 10.3  0.44 11.0  0.29 11.7  0.17 10.0  0.29 11.7  0.44 10.3  0.60 10.0  0.29 10.7  0.17 10.7  0.17 9.7  0.44 9.5  0.00

— 9.0  0.00 9.0  0.00 — 11.0  0.00 11.0  0.00 — — — 10.0  0.00 — — — — — — — — — — — — — —

— 12.0  0.0 10.3  0.33 — — — — — — 12.0  0.00 — — — — — — — — — — — — — —

— 9.3  0.33 10.7  0.33 — — — — — — 11.7  0.33 — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — —

— 11.7  0.33 12.0  0.00 — — — — — — 10.7  0.33 — — — — — — — — — — — — — —

9.0  0.00 9.7  0.33 9.3  0.33 10.0  0.00 9.7  0.33 9.0  0.00 — — 9.0  0.00 9.7  0.33 9.3  0.33 10.0  0.00 9.0  0.00 9.0  0.00 9.0  0.00 9.0  0.00 9.0  0.00 9.3  0.33 9.0  0.00 9.3  0.33 9.0  0.00 9.0  0.00 9.0  0.00 9.0  0.00

P. sativum

V. faba

V. mungo

V. radiata

V. unguiculata

a HE ¼ hexane; EA ¼ ethyl acetate; ME ¼ methanol; AQ ¼ aqueous; BC ¼ Bacillus cereus; BS ¼ Bacillus subtilis; BM ¼ Bacillus megaterium; CR ¼ Corynebacterium rubrum; PA ¼ Pseudomonas aeruginosa; PS ¼ Pseudomonas stutzeri; PP ¼ Pseudomonas pictoruim; KA ¼ Klebsiella aerogenes;  ¼ SEM value; — ¼ no activity.

Table 6 The antibacterial activities of standard antibiotics against selected Gram positive and Gram negative bacteriaa Zone of inhibition (mm) Gram positive

Gram negative

Antibiotic

BC

BS

BM

CR

PA

PS

PP

KA

Carbennicillin Imipenem Ceftazidimine Ciprofloxacin Cefaclor Amikacin Tetracycline Piperacillin Methicillin Chloramphenicol Azithromycin

10 36 18 19 20 11 17 16 — 10 11

20 37 25 30 35 15 21 17 16 16 14

— 41 — 27 14 23 19 — 14 14 12

22 24 19 16 27 16 20 19 15 15 14

12 27 17 28 — 22 15 — — 34 12

28 38 30 30 — 28 25 22 — 22 32

20 27 12 23 23 20 25 15 14 25 18

— 30 — 10 16 — 10 11 — 13 —

a — ¼ No activity; BC ¼ Bacillus cereus; BS ¼ Bacillus subtilis; BM ¼ Bacillus megaterium; CR ¼ Corynebacterium rubrum; PA ¼ Pseudomonas aeruginosa; PS ¼ Pseudomonas stutzeri; PP ¼ Pseudomonas pictoruim; KA ¼ Klebsiella aerogenes.

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V. unguiculata. In the fruit rinds, the reducing capacity assessment of the plants was in the order: C. indica > V. radiata > V. mungo > V. unguiculata > P. sativum > V. faba. Phenolic compounds are considered to be the most important antioxidative plant components. They also have an ability to scavenge free radicals and active oxygen species, such as singlet oxygen, free radicals and hydroxyl radicals.11 A highly positive correlation between total phenols and the antioxidant activities of many plants has been reported.21,8,15 Natural antioxidants strengthen the endogenous defence mechanism and restore the optimal balance by neutralizing reactive oxygen species. Therefore, screening plants for total phenol content may give a clue as to their antioxidant properties. Thus, the search for crude drugs of plant origin with antioxidant properties has become a central focus of study in recent years. In this study, the methanolic extracts of the fruit rind of C. indica showed a high antioxidant activity as compared to the others that might be due to its chemical composition, which is specifically rich in phenolic compounds.

and economic sources of antioxidants that can be used in the prevention of diseases caused by free radicals. However, they need to be explored as a viable, alternative source to commercially available synthetic and antibiotic drugs. This is the best use of such waste material.

Conclusion From the above study, it can be concluded that the total phenol content, DPPH free radical scavenging activity, superoxide anion radical scavenging activity and also the reducing capacity assessment are at their maximum in the methanolic extract of the fruit rind of C. indica. The best antibacterial activity was shown by the fruit rind of C. indica. It appears that the fruit rind of C. indica could be a potential source of antibacterial and antioxidant agents. However, further studies are needed to isolate and characterize the active compounds that are responsible for the antioxidant and antibacterial activities.

Acknowledgements Antibacterial activity The antibacterial activity of the various solvent extracts exhibited different levels of antibacterial activity against the selected bacterial strains (Table 4 and Table 5). All the extracts showed activity against B. cereus. The hexane and ethyl acetate extracts of the seeds and fruit rinds of all six plants, and the methanolic and aqueous extracts of the fruit rinds of all six plants, except P. sativum, showed activity against K. aerogenes. Ethyl acetate and methanol extracts of the fruit rind of C. indica showed activity against B. subtilis, B. megaterium, C. rubrum and P. pictoruim. Hexane and ethyl acetate extracts of the seeds of P. sativum showed activity against B. subtilis. The ethyl acetate extract of the fruit rind of V. faba showed activity against B. subtilis, B. megaterium, C. rubrum and P. pictoruim. The antibacterial activity of the fruit rind of C. indica against B. cereus was comparable with standard antibiotics like Amikacin, Chloroamphenical and Azithromycin (Table 6). The antibacterial activities of C. indica and V. faba were more pronounced on Gram positive bacteria than Gram negative bacteria. The reason for this difference in sensitivity might be ascribed to differences in the morphological constitution of these microorganisms; Gram negative bacteria have an outer phospholipidic membrane with a lipopolysaccharide component that makes the cell wall impermeable to plant extracts. Gram positive bacteria, on the other hand, are more susceptible, having only an outer peptidoglycan layer that is not as effective a permeability barrier. There are many reports in the literature that Gram positive bacteria are more susceptible to herbal extracts26,16 and Gram negative bacteria are resistant.27,15 The fruit rind of C. indica showed the best antibacterial activity, and therefore it can be further explored as a novel source of natural antibacterial drugs. The present investigation suggests that fruit rind, usually a waste product that is discarded into the environment, possesses potential antibacterial and antioxidant properties. However, further isolation and preparation of bioactive compounds of the fruit rind of C. indica are required to establish in vivo antioxidant activity using different animal models. These are novel, natural 314 | Food Funct., 2010, 1, 308–315

The authors thank Prof. S. P. Singh, Head, Department of Biosciences, Saurashtra University, for providing excellent research facilities. M. K. is thankful to the University Grants Commission, New Delhi, India for providing financial support.

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