Food & Function. Vol 01, No 01, October 2010

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Food & Function Downloaded on 21 October 2010 Published on 06 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90001J

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

Volume 1  |  Number 1  |  October 2010  |  Pages 1–132

ISSN 2042-6496

REVIEW Min-Hsiung Pan, Ching-Shu Lai and Chi-Tang Ho Anti-inflammatory activity of natural dietary flavonoids

PAPER Hau Yin Chung, Zhen Yu Chen et al. Hypocholesterolemic activity of onion is mediated by enhancing excretion of fecal sterols in hamsters

2042-6496(2010)1:1;1-D

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Food & Function Downloaded on 21 October 2010 Published on 06 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90002H

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

ISSN 2042-6496

Volume 1  |  Number 1  |  October 2010  |  Pages 1–132

PAPER Gina Borges, William Mullen and Alan Crozier Comparison of the polyphenolic composition and antioxidant activity of European commercial fruit juices

REVIEW David Julian McClements and Yan Li Review of in vitro digestion models for rapid screening of emulsion-based systems

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Food & Function Linking the chemistry and physics of food with health and nutrition 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 Downloaded on 21 October 2010 Published on 06 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90003F

ISSN 2042-6496 CODEN FFOUAI 1(1) 1–132 (2010) Cover See Min-Hsiung Pan, Ching-Shu Lai and Chi-Tang Ho, pp. 15–31. Flavonoids are widely present in the average diet in such foods as fruits and vegetables. Numerous studies have indicated that flavonoids act through a variety of mechanisms to prevent and attenuate inflammatory responses and serve as potential antiinflammatory agents. Image reproduced by permission of Min-Hsiung Pan and Chi-Tang Ho from Food Funct., 2010, 1, 15.

Inside cover See Gina Borges, William Mullen and Alan Crozier, pp. 73–83. Pomegranates contain a myriad of bioactive ellagitannins and anthocyanins. Research shows that in pomegranate juices and drinks sold in Europe, there are large variations in these phytochemicals. Image of ‘‘Wonderful’’ variety pomegranates courtesy of POM Wonderful and reproduced by permission of Alan Crozier from Food Funct., 2010, 1, 73.

EDITORIALS 11 Welcome to the first issue of Food & Function Gary Williamson, Editor-in-chief, and Sarah Ruthven, Managing editor, introduce the inaugural issue of Food & Function.

12 Meet the Food & Function Editorial Board The Food & Function Editorial Board members introduce themselves.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 3–10 | 3

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EDITORIAL STAFF Editor Sarah Ruthven Deputy editor Kathleen Too

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

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

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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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REVIEWS 15 Anti-inflammatory activity of natural dietary flavonoids Min-Hsiung Pan,* Ching-Shu Lai and Chi-Tang Ho*

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Flavonoids may have beneficial properties for various chronic inflammations.

32 Review of in vitro digestion models for rapid screening of emulsion-based systems David Julian McClements* and Yan Li In vitro digestion methods can be used to rapidly screen the ability of different factors to alter the rate and extent of lipid digestion in emulsion-based foods and delivery systems.

60 Combining nutrition, food science and engineering in developing solutions to Inflammatory bowel diseases – omega-3 polyunsaturated fatty acids as an example Lynnette R. Ferguson,* Bronwen G. Smith and Bryony J. James Nutrition, food science and engineering link in developing the evidence base for long chain omega-3 polyunsaturated fatty acids in Inflammatory bowel diseases.

PAPERS 73 Comparison of the polyphenolic composition and antioxidant activity of European commercial fruit juices Gina Borges, William Mullen and Alan Crozier* This paper reports on the use of antioxidant assays and HPLCPDA-MS and fluorescence detection to assess the anthocyanin, ellagitannin and ellagic acid content of pomegranate juices and pomegranate juice blends sold in Europe.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 3–10 | 5

Garlic and Other Alliums The Lore and the Science

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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Eric Block

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

www.rsc.org/books Registered Charity Number 207890

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PAPERS 84 Hypocholesterolemic activity of onion is mediated by enhancing excretion of fecal sterols in hamsters Lei Guan, Hau Yin Chung,* Yalun Su, Rui Jiao, Cheng Peng and Zhen Yu Chen*

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Dietary onion enhances the conversion of cholesterol into bile acid in liver.

90 Suppression of breast xenograft growth and progression in nude mice: implications for the use of orally administered sphingolipids as chemopreventive agents against breast cancer Kirk W. Simon, Larry Tait, Fred Miller, Chun Cao, Kevin P. Davy, Tanya LeRoith and Eva M. Schmelz* Dietary sphingomyelin suppressed progression of breast cancer xenografts in nude mice by reducing proliferation, angiogenesis and reversing aberrant protein expression.

99 Consumption of polyphenolic-rich beverages (mostly pomegranate and black currant juices) by healthy subjects for a short term increased serum antioxidant status, and the serum’s ability to attenuate macrophage cholesterol accumulation Mira Rosenblat, Nina Volkova, Judith Attias, Riad Mahamid and Michael Aviram* Polyphenolic-rich juices with impressive in vitro antioxidant properties, also demonstrate antioxidant effects in vivo when analyzed for short term consumption.

110 Mediterranean diet improves dyslipidemia and biomarkers in chronic renal failure patients Khedidja Mekki,* Nassima Bouzidi-bekada, Abbou Kaddous and Malika Bouchenak Nutritional advice based on a Mediterranean diet reduces dyslipidemia, and protects against lipid peroxidation and inflammation in chronic renal failure patients.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 3–10 | 7

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PAPERS 116 Effects of dietary consumption of cranberry powder on metabolic parameters in growing rats fed high fructose diets Ramesh C. Khanal, Theodore J. Rogers, Samuel E. Wilkes, Luke R. Howard and Ronald L. Prior*

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Cranberry powder attenuates markers of metabolic syndrome in a high fructose fed rat model.

124 Dealcoholized red wine reverse vascular remodeling in an experimental model of metabolic syndrome: role of NAD(P)H oxidase and eNOS activity Marcela Alejandra Vazquez-Prieto, Nicol as Federico Renna, Carina Lembo, Emiliano Ra ul Diez and Roberto Miguel Miatello* Red wine reduced oxidative stress, improved vascular function and remodeling in a model of metabolic syndrome. The antioxidant properties of polyphenols could be responsible for these beneficial effects.

8 | Food Funct., 2010, 1, 3–10

This journal is ª The Royal Society of Chemistry 2010

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First in its class “I attribute this success to the quality of the authors that the editor and editorial board have been able to recruit…” Professor Jonathan Sessler, The University of Texas, USA

Chemical Society Reviews (Chem Soc Rev) publishes the largest number of chemical review articles, making it first in its class for chemical scientists*. With an impact factor of 20.086* and the leading immediacy index for a chemical reviews journal at 5.314, this impressive result underlines the continuing success of the journal. Chem Soc Rev supplies high quality and highly cited articles, covering topical areas of interest across the chemical sciences. Published monthly, it includes themed issues reviewing new research areas, and edited by a specialist guest editor.

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

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EDITORIAL

www.rsc.org/foodfunction | Food & Function

Welcome to the first issue of Food & Function

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DOI: 10.1039/c005532h We are delighted to introduce the inaugural issue of Food & Function. This new timely journal from RSC Publishing will provide a much needed platform devoted to bringing together research which links the chemistry and physics of food with health and nutrition. Monthly issues of Food and Function will publish communications and full papers reporting primary research as well as in-depth reviews focusing on the interaction of food components with the human body, including the influence of the physical properties and structure of food, the chemistry of food components, the biochemical and physiological actions and the nutrition and health aspects of food.

Submissions to Food & Function are handled fairly, quickly and efficiently by our Associate editors, Steven Feng Chen from the University of Hong Kong, and Cesar Fraga from the University of Buenos Aires and UC Davis. The journal is supported by international Editorial and Advisory Boards, and we would like to thank these board members for their support of Food & Function. The members of the Editorial Board are Aedin Cassidy, Kevin Croft, Eric Decker, Alejandro Marangoni, Reinhard Miller, Paul Moughan, Johan Ubbink and Fons

Voragen, and more details about our Editorial Board members can be found in the ‘Meet the Food & Function Editorial Board’ article is this first issue. The journal is backed by a dedicated team of RSC Publishing editors. RSC Publishing has a strong heritage and reputation for publishing science of the very best quality and has a great track record for new journal launches. We are confident that Food & Function will be a high quality publication. You will see that this inaugural issue of Food & Function contains papers that represent the full scope of the journal. Especial thanks must be given to the authors and referees for their support of Food & Function at this early stage. Finally, it is important to note that papers published in issues this year and in 2011 will be free to access, and will therefore have maximum visibility and be widely promoted. We encourage you to sign up to the Food & Function newsletter – Food for Thought. This quarterly newsletter will keep you up to date with all the latest news about the journal. For details

Gary Williamson; Editor-in-chief

Sarah Ruthven; Managing editor

of how to sign up to the newsletter, and register for free access to Food & Function, please visit the journal platform at: www.rsc.org/foodfunction. We hope you enjoy reading the papers in this first issue of Food & Function and will consider submitting some of your research to the journal. Details of how to submit an article to Food & Function are available on our platform. We welcome any comments you have about the journal and invite you to contact us at [email protected]. Gary Williamson, Editor-in-chief Sarah Ruthven, Managing editor

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 11–11 | 11

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EDITORIAL

www.rsc.org/foodfunction | Food & Function

Meet the Food & Function Editorial Board

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DOI: 10.1039/c005531j

vice-president of the Oxygen Club of California. His original research interests in free radical and antioxidants in biological systems were the foundation for his current activities centered on the health effects of plant constituents. Professor Fraga has earned numerous prizes and awards and is one of the most cited scientists in phytonutrients and human health.

Professor Gary Williamson; Editor-in-Chief

Professor Steven Feng Chen; Associate Editor

Professor Gary Williamson is Professor of Functional Food at the University of Leeds, UK, in the School of Food Science and Nutrition. His research interests are in nutritional and food biochemistry. He has over 270 refereed scientific publications, and is one of the most highly cited authors in agricultural sciences according to ISI. Currently, he is also a Visiting Professor of Food Safety at University of Surrey, UK, and Honorary Chair at Harbin Medical University, China. Prior to joining the University of Leeds in 2007, Gary was the Head of Nutrient Bioavailability at Nestle Research Centre, Lausanne, Switzerland, and before that Head of Phytochemicals at the Institute of Food Research, Norwich, UK. In addition to his full-time professorship at the Univeristy of Leeds, Gary is also a Scientific Advisor to the nutrient bioavailability group at Nestle Ltd, specialising in polyphenols.

Supervisor Award, HKU Outstanding Young Researcher Award, and CIFST Outstanding Researcher in Food Science and Technology Award. He is a highly cited scientist (top 1%) in agricultural sciences, and is an elected Fellow of American Institute for Medical and Biological Engineering.

Professor Steven Feng Chen is Professor of Food and Nutritional Science at the University of Hong Kong in the School of Biological Sciences. His research interests are primarily in the areas of functional foods and food biotechnology. He is particularly interested in the development of functional foods and nutraceuticals from plants and algae. He has edited 4 books, filed 6 patents, and published over 200 papers in refereed journals. He has received several prestigious awards such as HKU Outstanding Research Student

Professor Cesar Fraga received his doctoral degree in Biological Chemistry from the University of Buenos Aires in 1985. In 1990 he was appointed Professor of Physical Chemistry at the School of Pharmacy and Biochemistry, University of Buenos Aires, and Researcher at the National Council for Scientific Research Argentina (CONICET), positions that he still holds today. From 2004 Professor Fraga has also had a position of Research Professor at the Department of Nutrition, University of California, Davis. He is

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Professor Cesar Fraga; Associate Editor

Professor Aedin Cassidy, Editorial Board member

Professor Aedin Cassidy is Professor of Diet and Health at the Medical School, University of East Anglia, Norwich, UK. Her research interests are in nutritional biochemistry with a specific interest in food bioactives and cardiovascular disease prevention. She received her PhD from Cambridge University in 1991 where she conducted some of the first studies to elucidate the biological effects of phytochemicals in humans. Prior to joining UEA, Aedin was Head of Molecular Nutrition and Women’s Health at Unilever Research and previously was Reader in Nutritional Biochemistry at the University of Surrey, UK. She was awarded the Nutrition Society Medal in 1999 and in 2008 an award for ‘Outstanding Contribution to Research’ in Tokyo, Japan, for her contribution to the isoflavone research field. Professor Kevin Croft is a Professorial Fellow of the School of Medicine and Pharmacology at the University of Western Australia. He also holds the

This journal is ª The Royal Society of Chemistry 2010

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Professor Kevin Croft, Editorial Board member

following positions: Chair, the Animal Ethics Committee, Royal Perth Hospital; Member of the Board of Postgraduate Studies, University of Western Australia; Director of Research, School of Medicine and Pharmacology, UWA; and was President of the Australasian Society for Free Radical Research, 2006–2007. Kevin’s major research interests are bioactive polyphenols in the diet, the absorption and metabolism of dietary polyphenols, impact on cardiovascular risk factors, human intervention trials, animal models of atherosclerosis, mechanism of vascular protection by polyphenols, lipid metabolism and the role of arachidonic acid metabolism by CYP450 and the role in cardiovascular disease. He has published over 200 peer reviewed papers.

the Food Science Section of the Department of Animal Sciences at the University of Kentucky from 1988 to 1993 after which he joined the Department of Food Science at the University of Massachusetts, Amherst as an Associate Professor in Food Chemistry. He was named the Fergus Clydesdale Endowed Chair from 2002–2007. Professor Decker has been actively conducting research to characterize mechanisms by which lipids oxidize in heterogeneous food systems. His lab has worked extensively in the development of antioxidant technologies for food systems. As part of this research program, he has developed patented technologies to maximize the concentrations and stability of bioactive lipids such as omega-3 fatty acids allowing them to be incorporated into foods at nutritionally significant levels. In addition, Professor Decker has actively collaborated with other scientists to investigate the role of antioxidants, lipids and lipid oxidation products in the molecular basis of disease. Professor Decker has published over 250 peer reviewed journal articles, reviews, and book chapters. He has also organized symposia and workshops on antioxidants, bioactive lipids, lipid oxidation and functional foods.

a Canada Research Chair (2001, renewed in 2006), two Distinguished Researcher Awards from the Ontario Innovation Trust (2002), a Career Award from the Canadian Foundation for Innovation (2002), an E.W.R. Steacie Memorial Fellowship (2002), given to the top 6 Canadian scientists from all disciplines, and the T.L. Mounts Award from AOCS in 2004. Professor Marangoni is a past chair of the Natural Sciences and Engineering Research Council of Canada’s Plant Biology and Food Science Grant Selection Committee and a member of NSERC’s E.W.R. Steacie Memorial Fellowship selection committee. Professor Marangoni has co-founded two high-technology food companies and is the co-recipient of the 2008 Guelph Partners of Innovation ‘‘Innovator of the year’’ award for his discovery of a zero trans, low saturate shortening alternative which could revolutionize the baking industry.

Dr Reinhard Miller; Editorial Board member

Professor Alejandro Marangoni, Editorial Board member

Professor Eric Decker; Editorial Board member

Professor Eric Decker received his B.S. degree in biology from Penn State University in 1982, his M.S. from the Department of Food Science and Human Nutrition, Washington State University, in 1985 and his Ph.D. from the Department of Food Science at the University of Massachusetts, Amherst in 1988 where he was a USDA National Needs Fellow. Professor Decker was an Assistant Professor in

Professor Alejandro Marangoni is a professor and Canada Research Chair in Food and Soft Materials Science at the University of Guelph. His work concentrates on the physical properties of foods, particularly fat crystallization and structure. He has published over 200 refereed research article and four books. He is the recipient of many awards including a 1999 Premier’s Research Excellence Award, the first Young Scientist Award form the American Oil Chemists’ Society (2000),

This journal is ª The Royal Society of Chemistry 2010

Dr Reinhard Miller is group leader at the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany. He studied Mathematics in Rostock and Colloid Chemistry in Dresden. His scientific interests are focused on dynamics of interfacial layers containing proteins, surfactants and other food relevant compounds. Dr Miller edited the book series ‘‘Studies in Interface Science’’ (24 volumes) and is now editor of the ‘‘Progress in Colloid Interface Science’’ series. He has published about 400 papers in refereed journals. At present he is President Elect of the European Colloid and Interface Society. Professor Paul J. Moughan holds the position of Distinguished Professor at Food Funct., 2010, 1, 12–14 | 13

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Professor Paul J. Moughan, Editorial Board member

Massey University, New Zealand, and is Director of the Riddet Institute. The Riddet Institute, a New Zealand government funded Centre of Research Excellence (CoRE), is a partnership between the University of Otago, Auckland University and Massey University and two New Zealand Crown Research Institutes, Plant and Food Research and AgResearch, and is dedicated to research and postgraduate education in the area of food science and human nutrition. He was formerly foundation head of the Institute of Food, Nutrition and Human Health at Massey University, Director of the university’s Mongastric Research Centre and Foundation Scientific Director of the Fonterra-funded Milk and Health Research Centre. Professor Moughan was appointed to the foundation chair in monogastric biology at Massey University in 1993 and his research has encompassed the fields of

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human and animal nutrition, food chemistry, functional foods, mammalian growth biology and digestive physiology. He has published in excess of 300 scientific works. In 1995 he was awarded Doctor of Science and in 1997 was awarded a Personal Chair at Massey University and was elected a Fellow of the Royal Society of New Zealand. He has received several prestigious international awards for his work and is an adviser to the international food and feedstuffs industries. He is a non-executive Director of the Gardiner Foundation, Melbourne, Australia.

Netherlands). After stays as visiting scientist at the University of Bristol (UK) and Moscow State University (Russia), he joined industry. Initially as Senior Scientist at corporate research at Givaudan (Switzerland), in 1999 he switched to the Nestle Research Center in Lausanne (Switzerland) where he was active as R&D specialist, coordinator for Delivery Systems and Scientific Expert Materials Science. His current research interests include food sustainability, the physics of glassy carbohydrates, the statistical mechanics of biopolymer complexes, and the biophysics of stabilization and delivery of fragile bioactive compounds.

Dr Johan Ubbink; Editorial Board member

Dr Johan Ubbink is Senior Consultant at Food Concept & Physical Design, a strategy and technology company focusing on the development of sustainable foods. Trained as an experimental physical chemist at the University of Leiden (The Netherlands), he obtained his Ph.D. in the field of DNA biophysics at Delft University of Technology (The

Professor Fons Voragen, Editorial Board member

Professor Fons Voragen is a Professor in the Laboratory of Food Chemistry of Wageningen University. His research expertise is in the areas of cereal products, enzymes, polysaccharides and proteins. He has published over 300 papers in refereed journals.

This journal is ª The Royal Society of Chemistry 2010

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REVIEW

www.rsc.org/foodfunction | Food & Function

Anti-inflammatory activity of natural dietary flavonoids Min-Hsiung Pan,*a Ching-Shu Laia and Chi-Tang Ho*b

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Received 30th July 2010, Accepted 12th August 2010 DOI: 10.1039/c0fo00103a Over the past few decades, inflammation has been recognized as a major risk factor for various human diseases. Acute inflammation is short-term, self-limiting and it’s easy for host defenses to return the body to homeostasis. Chronic inflammatory responses are predispose to a pathological progression of chronic illnesses characterized by infiltration of inflammatory cells, excessive production of cytokines, dysregulation of cellular signaling and loss of barrier function. Targeting reduction of chronic inflammation is a beneficial strategy to combat several human diseases. Flavonoids are widely present in the average diet in such foods as fruits and vegetables, and have been demonstrated to exhibit a broad spectrum of biological activities for human health including an anti-inflammatory property. Numerous studies have proposed that flavonoids act through a variety mechanisms to prevent and attenuate inflammatory responses and serve as possible cardioprotective, neuroprotective and chemopreventive agents. In this review, we summarize current knowledge and underlying mechanisms on anti-inflammatory activities of flavonoids and their implicated effects in the development of various chronic inflammatory diseases.

Introduction Inflammation is a normal biological process in response to tissue injury, microbial pathogen infection and chemical irritation. This biological process also involves the innate and adaptive immune systems. At a damaged site, inflammation is initiated by migration of immune cells from blood vessels and release of mediators, followed by recruitment of inflammatory cells and release of reactive oxygen species (ROS), reactive nitrogen species (RNS) and proinflammatory cytokines to eliminate foreign pathogens, resolving infection and repairing injured tissues.1,2 Thus, the main function of inflammation is beneficial for a host’s defense. In general, normal inflammation is rapid and self-limiting, but aberrant resolution and prolonged inflammation causes various chronic disorders.3 Chronic inflammation can inflict more serious damage to a host tissue than bacterial infection. Diverse ROS and RNS such as O2 (superoxide anion), OH (hydroxyl radical), H2O2 (hydrogen peroxide), nitric oxide (NO), and 1O2 (singlet oxygen) generated by inflammatory cells injure cellular biomolecules including nucleic acids, proteins and lipids, causing cellular and tissue damage, which in turn augments the state of inflammation.4 These also trigger a series of signaling molecules, inflammatory gene expression and activation of enzymes involved in chronic inflammation. Inflammatory chemicals produced by inflamed and immune cells also attack normal tissues surrounding the infected tissue, causing oxidative damage and extensive tissue inflammation.1,4

a 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 b Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey, 08901-8520, USA. E-mail: [email protected]. edu; Fax: +1-732-932-6776

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Studies show that chronic inflammation is linked to a wide range of progressive diseases, including cancer, neurological disease, metabolic disorder and cardiovascular disease.3,4 Numbers of studies suggest elimination of chronic inflammation as a major way to prevent various chronic diseases.1,3 Epidemiological studies provide convincing evidence that natural dietary compounds that humans consume as food possess many biological activities. Among these natural bioactive compounds, flavonoids are widely recognized for their biological and pharmacological effects, including antiviral, anti-carcinogenic, antioxidant, antimicrobial, anti-inflammatory, anti-angiogenic and anti-thrombogenic properties.1,5 Epidemiologic studies indicate that the incidence of chronic disease and cancer is inversely correlated with the consumption of fruits and vegetables rich in flavonoids,5,6 and this is attributed to their possible anti-inflammatory activities. This review forcuses on the molecular basis of the anti-inflammatory potential of flavonoids, with special emphasis on their effect on molecular mechanisms and signaling pathways involved in inflammation, as agents in reducing or eliminating different chronic inflammation-associated human diseases.

The role of inflammation in human disease Inflammation is a complicated process, driven by preexisting conditions (infection or injury) or genetic changes, that results in triggering signaling cascades, activation of transcription factors, gene expression, increased of levels of inflammatory enzymes, and release of various oxidants and proinflammatory molecules in immune or inflammatory cells.2 In this condition, excessive oxidants and inflammatory mediators have a harmful effect on normal tissue, including toxicity, loss of barrier function, abnormal cell proliferation, inhibiting normal function of tissues and organs, and finally leading to systemic disorders.1,2 Over the past few decades, many studies reveal that chronic inflammation is a critical component in many human diseases and conditions, Food Funct., 2010, 1, 15–31 | 15

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Fig. 1 Chronic inflammation is linked to human diseases.

including obesity, cardiovascular diseases, neurodegenerative diseases, diabetes, aging, and cancers2,4 (Fig. 1). Cardiovascular diseases Cardiovascular disease (CVD) is becoming the leading cause of death in the world. Chronic inflammation, such as atherosclerosis, coronary diseases, cerebrovascular disorder, heart failure and cardiomyopathy is common in CVD.7 In the past, researchers suggested a number of traditional risk factors implicated in the pathogenesis of CVD including age, hypertension, dyslipidemia, hypercholesterolemia, glucose tolerance and metabolic symptoms. However, recent studies focus on the relationship between endothelial dysfunction and inflammatory condition.7,8 Vascular endothelium is very important in regulation of vascular homeostasis, and inhibition of leukocyte adhesion and platelet aggregation by release of mediators such as nitric oxide (NO).8 Increase of NO production, damage of endothelial cells, increase of oxidative stress and an enhanced proinflammatory state lead to the alteration of vascular integrity, the reduction of vasodilator capacity and the appearance of endothelial dysfunction. Atherosclerosis is a chronic inflammatory disease and a major cause of CVD. Recent studies demonstrate that vascular inflammation is the earliest event in the development of atherosclerosis.7,9 The process involves stimulation of cholesterol, oxidized low-density-lipoprotein (ox-LDL) and oxidative free radicals, which initiate activation of vascular endothelial cells and enhance their adhesive property with monocytes by expression of adhesion molecules selectins, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1).10 Once monocytes firmly attach on the surface of endothelium, they transmigrate into the arterial intima and differentiate to macrophages. This transmigration is triggered by chemoattractant molecules 16 | Food Funct., 2010, 1, 15–31

such as monocyte chemoattractant protein-1 (MCP-1), proinflammatory cytokines (TNF-a and ILs) as well as growth factors (PDGF and TGF-b) produced by activated T cells and macrophage.11 Among these, studies indicate that MCP-1 is important for recruitment of monocytes into intima. Differentiated macrophages that expresse scavenger receptors become foam cells via uptake of ox-LDL generated in the intima resulting in formation of fatty streaks.12 The molecules secreted by monocytes, macrophages and arterial cells maintain an inflammatory response within the artery and promote proliferation and migration of vascular smooth muscle cells.9,11 Proliferative smooth muscle cells release fibrogenic mediators and build a dense extracellular matrix around foam cells and monocytes, finally causingfatty streaks to progress into fibrous plaque.13 In pathogenesis and progression of atherosclerosis, chronic inflammation is involved in every stage that is characterized by infiltration of monocytes/macrophages and production of proinflammatory cytokines9,11 (Fig. 2). Hence, the modulation or regulation of the interaction between endothelial and inflammatory cells and the transformation of macrophages to foam cells could be the basis for the beneficial effects that prevent or slow down the progression of this disease. This diversity of cytokine expression and function might also lead to the identification of selective therapeutic targets for the prevention and treatment of atherosclerosis.10,11 Moreover, clinical research shows that elevated levels of systemic inflammatory molecules including IL-6, ICAM-1, P-selectin, E-selectin and C-reactive protein (CRP), are classic acute-phase markers occurring in patients with coronary disease, and, therefore that might be a predictor of cardiovascular risk.14,15 Different treatments of atherosclerosis are associated with reduction of these inflammatory markers, providing a new target for blockage or therapy of atherosclerosis by inhibition of inflammation.9,16 This journal is ª The Royal Society of Chemistry 2010

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Neurological disorders

Fig. 2 Induction of inflammatory responses by ox-LDL in development of atherosclerosis. Stimulated ox-LDL increases chemoattractant concentration and induces monocyte transmigrating into intima then transforming to macrophage. Differentiated macrophage that specific to uptake ox-LDL becomes foam cell and leads to the formation of fatty streak in intima.

Many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis and other nervous system pathologies are associated with inflammatory injury (Fig. 3). This complex inflammatory process involves different cellular components in the central nervous system, such as microglial cells, astrocytes, ependymal cells, macrophages and mast cells.17 Microglial cells are macrophages in the brain and the central nervous system that are the first response to a neuronal injury.18 In the case of Alzheimer’s disease, a cleavage product of amyloid precursor protein (APP), an integral membrane protein, and the aggregation of amyloid b peptides (Ab1-42 and Ab1-40) trigger activation of microglials and astrocytes, following activation of transcription factors such as nuclear factor-kappa B (NF-kB) and activator protein (AP)-1 that induce production of ROS and various proinflammatory mediators including NO, PGE2, ILs and TNF-a.17,18 These proinflammatory cytokines and ROS produced by activated microglial and astrocytes may cause neuronal damage or neurotoxicity resulting in apoptosis and necrosis.17 Moreover, proinflammatory mediators released from microglials and astrocytes can activate each other to amplify inflammatory signals to neurons. Parkinson’s disease is a neurodegenerative disorder in the central nervous system with a pathology similar to Alzheimer’s disease. Aggregation and accumulation of a-synuclein protein of Lewy bodies and loss of dopaminergic neurons are major neuropathological alterations in Parkinson’s disease.19 Lewy bodies and

Fig. 3 Neuroinflammation in Alzheimer’s disease. During the development of Alzheimer’s disease, b amyloid peptide is produced by cleavage of amyloid precursor protein (APP), aggregates and accumulates as b amyloid plaques. Both aggregates and plaques cause neurotoxicity or activation of microglia through up-regulating NF-kB and AP-1 transcription factors, which in turn release ROS and pro-inflammatory cytokines such as NO, PGE2, IL-1, IL-6, and TNF-a that damage cholinergic neuron. These pro-inflammatory cytokines also directly activate astrocytes that also produce cytokines to amplify inflammatory signals and result in neuroinflammation and neurodegeneration. Flavonoids act through avoiding b amyloid induced-neuron injury and death, modulation of pro-inflammatory cytokines and ROS production as well as inhibiting the activation of microglia and astrocyte as neuroprotective mechanisms.

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intracellular and/or extracellular a-synuclein protein aggregates released from neurons lead to the activation of microglial cells,20,21 resulting in the activation of transcription factor NF-kB, gene expression of iNOS, COX-2 and NAPDH oxidase, and production of inflammatory mediators.22 Proinflammatory cytokines such as TNF-a and ILs derived from activated microglial cells also trigger activation of astrocytes. Studies show that released factors from activated microglials and astrocytes have a combinational effect in promoting neurotoxicity.17,22 In neurodegenerative diseases, the activation of immune cells such as microglials and astrocytes is a critical step in neuropathology. Oxidative stress is also important in neurodegenerative diseases, both in Alzheimer’s disease and Parkinson’s disease.17,18,22 Several therapeutic approaches that target inflammatory responses demonstrate the ability to interfere with activation of transcription factor and inhibit function of inflammatory enzymes and production of ROS.23,24 Obesity Obesity, resulting from excessive fat sorted in adipose tissue is a highly prevalent condition that is related to many metabolic disorders throughout the world. Numerous studies indicate the high risk of obesity in the development of cardiovascular disease, type 2 diabetes, hypertension, fatty liver disease and cancer.25 It is now clear that the function of adipose tissue is not only as fat storage, but also as a major endocrine and secretary organ that produces adipokines such as leptin and adiponectin.26 Leptin is an important hormone in the regulation of energy expenditure and caloric intake for maintaining energy balance.27 Clinical and animal studies show that leptin deficiency results in body weight increase and a high risk for the development of type 2 diabetes.27,28

In recent years, many studies document that obesity is significantly associated with a chronic low-grade inflammation29 (Fig. 4). The first connection between obesity and inflammation is evidenced by the release of TNF-a from adipocytes. As the lipid content increases in adipose tissue, adipocytes synthesize TNF-a and several cytokines (IL-1b and IL-6) that change the number and size of cells, influencing lipoprotein lipase and increasing the inflammatory state.30 TNF-a also induces insulin resistance by downregulation of insulin receptor phosphorylation, decrease of glucose uptake and expression of GLUT4 transporter.29,31 Another important inflammatory feature in adipose tissue is recruitment of immune and inflammatory cells such as neutrophils, eosinophils and macrophages.32 Studies in both mice and humans show that while in an obese state, macrophage infiltration is increased in adipose tissue.32 Large adipocytes secret chemotactic signals, such as monocyte chemoattractant protein-1 (MCP-1), to trigger infiltration of macrophages, that then leads to the creation of a chronic, lowgrade inflammation in obesity.32,33 Increased levels of acute phase protein CRP are found in many obese individuals,34 and circulating CRP concentrations are related to the development of cardiovascular disease,14 indicating the association of obesity and cardiovascular disease. Some lines of evidence also suggest that obesity is linked to fat storage in the liver that can lead to the development of fatty liver diseases.25 It is suggested that IL-6 derived from adipocytes may drive the production of CRP in the liver.35 Overexpression of MCP-1 in adipose tissue leads to increase of hepatic triglyceride content.33 In addition, elevated levels of circulating TNF-a in an obese state is often associated with an increase in insulin resistance.31,36 These observations emphasize the correlation among obesity, inflammation and metabolic disorders.

Fig. 4 Obesity in the induction of inflammation. Adipose tissue of visceral obesity induced chronic low-grade inflammation through macrophage infiltration by MCP-1 and secretion of pro-inflammatory factors. However, obesity may cause the high risk in development of several diseases.

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Metabolic disorders Extensive research reveals that inflammation is a characteristic feature of metabolic disorders, including fatty liver disease, type 2 diabetes, chronic kidney disease and heart disease (Fig. 5). Inflammatory responses are considered to be a critical stage of metabolic dysfunction characterized by abnormal proinflammatory cytokine production, increased acute phase protein and activation of inflammatory signaling pathways.37 In addition, obesity correlates with an increase in inflammatory conditions and metabolic syndromes.29 Type 2 diabetes is the most prevalent and serious metabolic disease caused by insulin resistance derived from pancreatic beta cell dysfunction. Both experimental and clinical studies demonstrate that several inflammatory cytokines are closely related to pathogenesis of insulin resistance.38 Increased levels of IL-1b in plasma are shown to have detrimental effects on the function of IL-1 receptor antagonist proteins (IL-1ra) that promote beta cell destruction and alter insulin sensitivity.39 Moreover, IL-6 acts on activation of tyrosine phosphatase and interferes with the interaction between insulin receptor and suppressor of cytokine signaling (SOCS) proteins that result in insulin resistance.40 In patients with chronic kidney disease, elevated levels of serum acute phase proteins such as C reactive protein (CRP), TNF-a and ILs are associated with an increase in chronic inflammatory states and insulin resistance.41 In addition to insulin resistance and Type 2 diabetes, an elevated concentration of CRP and

various cytokines also occur in chronic heart failure with fluid overload and cardiovascular disease.34,41 Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder associated with obesity and insulin resistance that results from abnormal adipokine and cytokine production.42 In liver tissue, these mediators, such as leptin, TNF-a and ILs decrease insulin signaling to hepatocytes by the activation of several signaling molecules and kinases. This results in hepatic insulin resistance, hyperglycemia and fatty liver development caused by increased fatty acid uptake and VLDL production.37,42 Hepatic insulin resistance also stimulates the production of CRP and cytokines that promote atherosclerosis by the inhibition of NO production, an increase in the adhesion property in endothelial cells, and increasing macrophage uptake ox-LDL.40 This information indicates that hepatic insulin resistance is related to the induction of metabolic syndromes and the acceleration of cardiovascular disease progression.

Bone, muscular and skeletal diseases Rheumatoid arthritis is an autoimmune disease that causes joint destruction and functional disability, often characterized by chronic inflammatory responses primarily affecting the synovium of diarthrodial joints (Fig. 6). Besides infections and genetic factors, rheumatoid arthritis is indicated in the interaction between immune cells, such as T cells, B cells, macrophages,

Fig. 5 Proinflammatory cytokines in insulin resistance and metabolic disorders. Insulin synthesized and secreted from b cells in pancreas acts as normal function in different organs and tissues, includes reducing glucose production and output in liver, facilitating glucose uptake in skeletal muscle, and decreasing lipolysis in adipose tissue. Excessive pro-inflammatory cytokines (CRP, IL-1, IL-6, and TNF-a) cause dysfunction of b cell or recruit of inflammatory cells (monocytes and macrophages) that affect both insulin secretion and insulin action, promote pathogenesis of insulin resistance and subsequently reducing insulin-dependent signalling. This local insulin resistance also contributes to its target tissues such as increase concentration of glucose and fatty acids in skeletal muscle, liver and adipose tissue that lead to various metabolic disorders. Flavonoids act through interfering with proinflammatory cytokines-induced b cells dysfunction and cell death, decreasing cytokines production, up-regulation of insulin-dependent signaling and improving glucose uptake in different cell types.

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Fig. 6 Mechanisms of inflammation-associated pathogenesis in rheumatoid arthritis and osteoporosis. In inflamed rheumatoid synovium and bone tissue, pro-inflammatory cytokines produced by recruited inflammatory cells (macrophages, T cells and B cells), endothelial cells and synovial fibroblasts are central to the inflammatory process in rheumatoid arthritis and osteoporosis. This pathological process also involves in innate and adaptive immunity responses. These pro-inflammatory cytokines result in activation of synovial fibroblasts and produce proteases that lead to tissue destruction. In addition, cytokines-trigger activation and differentiation of osteoclasts are important in bone loss. Flavonoids act through reducing recruitment of inflammatory cells, cytokines production, MMPs expression, and activation or differentiation of osteoclasts.

dendritic cells and fibroblasts and as such plays an important role in the pathogenesis of this disease.43 In patients with rheumatoid arthritis, large numbers of activated T cells are present in inflamed joints. Recruited T cells develop a lymphoid structure in synovium with B cells, macrophages and fibroblast-like synoviocytes that create a complex network between cells through secreting various cytokines, such as TNF-a, IL-1 and IL-6.44 Among these cell types in synovium, fibroblast-like synoviocytes is known to produce prostaglandins and proteases that destroy bone and cartilage.45 Moreover, activated B cells and macrophage continuously secrete IL-1 and TNF-a that maintain the synovial fibroblast in an inflamed state.44,45 Enrichment of these immune cells and derived-proinflammatory cytokines in synovium causes varying degrees of joint destruction and also extraarticular organ involvement. Osteoporosis is also a chronic inflammation condition that is characterized by the loss of bone density (Fig. 6). Studies identify proinflammatory cytokines TNF-a, IL-1 and IL-6 as important mediators of bone resorption through increased expression of 20 | Food Funct., 2010, 1, 15–31

receptor activator of NF-kB (RANK), activation and differentiation of osteoclast, and decreased osteoblast survival.46 Despite several in vitro and in vivo studies indicating that proinflammatory cytokines contribute to osteoporosis, and increased levels of IL-1b, IL-6 and TNF-a in whole blood culture from patients with osteoporosis, the mechanism involved in bone loss is still unclear. In addition, recent studies also reveal that elevated systemic CRP is associated with poor bone health.46,47 Chronic inflammatory diseases A continued chronic inflammatory state in different organs and tissues leads to a various chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD), psoriasis, rheumatoid arthritis, chronic pancreatitis and inflammatory bowel disease (IBD) all of which are frequently associated with infiltration of immune and inflammatory cells. For example, inflammatory bowel disease leads to ulcerative colitis (UC) and Crohn’s disease (CD), based on clinical features and This journal is ª The Royal Society of Chemistry 2010

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Fig. 7 Underlying mechanisms in inflammatory bowel disease. As bacteria infection or environmental factors that cause colonic endothelium damage result in recruitment of inflammatory and immune cells from bloodstream. Accumulated inflammatory cells produce pro-inflammatory mediators that trigger proliferation and activation of T cells, lead to differentiate to Th1 and Th2 cells that result in amplification of inflammatory cascade and cause tissue injury. Flavonoids act through decreasing inflammatory cytokines production, reducing recruitment of inflammatory cells and modulation of differentiation and proliferation of T cells.

histopathology, which result from abnormal regulation of the immune system in the intestine.48 In IBD, damage of epithelium in the colon increases recruitment of immune cells such as activated T lymphocytes and leucocytes that are mediated by the expression of adhesion molecules.49 This is thought to be a key step of inflammatory process involved in IBD (Fig. 7). A number of studies indicate that an increased level of proinflammatory cytokines is important for pathogenesis of IBD.48 In general, epithelial cells, paneth cells, enterocytes and immune cells of colon form a complex barrier by the expression of cytokines, chemokines and metabolite from microbes or hosts in order to fight pathogens and to maintain intestinal homeostasis. Also, an alteration of the immune system toward luminal antigens is thought to play a crucial role in pathogenesis in IBD.48 In an immune response, T helper (Th) cells recognize selfantigens (from food consumption or intestinal bacteria) that present from lymphocytes and phagocytes, and then start to produce cytokines. As the specific-antigen occurs, it enhances production of various cytokines in colonic epithelium that promote CD4 and CD8 cells which then differentiate to Th1 and Th2 cells. These two Th cells produce a dramatic amount of proinflammatory cytokines that affect colon mucosa and intestinal cells.50 Studies show that Crohn’s disease is mediated by Th1 cells that are characterized by the production of IL-1, IL-6, interferon-g (IFN-g) and TNF-a, whereas ulcerative colitis is This journal is ª The Royal Society of Chemistry 2010

thought to be a Th2 cell mediated response by the secretion of IL-4, IL-5 and IL-10.48,50 Overexpression of these proinflammatory cytokines is found in the intestinal mucosa from IBD patients and is related to the alteration of intestinal homeostasis and results in an abnormal inflammatory response in the intestinal mucosa. Indeed, increased proinflammatory cytokines in colon mucosa are also linked to the enhanced expression of antiapoptotic molecules, leading to apoptotic resistance and promotion of accumulation of T cells.48,50

Cancers Several lines of evidence indicate that cancer development in humans is a multistep and long-term process which requires six properties, including limitless replication potential, evasion of apoptosis, self-sufficiency in growth signals, insensitivity to antigrowth signals, sustained angiogenesis, and tissue invasion and metastasis.51 Since Virchow observed in 18th century, that cancers frequently occur at sites of chronic irritation, much research confirms the concept that chronic inflammation is a critical component of tumor promotion and progression, including colorectal, gastric, pancreatic, pulmonary, cystic, hepatocellular, ovarian, skin and esophageal cancers.4,52 In view of inflammation involved in different cancers, increasing Food Funct., 2010, 1, 15–31 | 21

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evidence suggests that inflammation should be the seventh hallmarker in cancer development.53 The pathological mechanism of inflammation involved in tumorigenesis is very complicated.54 In tissue injury or inflammatory stimulation, inflammatory cells are recruited and production of pro-inflammatory cytokines and diverse ROS and RNS that induce genetic change which enhances malignant transformation and proliferation of initiated cells. Subsequently, as tumor tissue forms, inflammation continues to promote development of cancer by creating an inflammatory microenvironment, which consists of stromal cells, inflammatory cells and the extracellular matrix from surrounding tissues. The inflammatory and immuosuppressive cytokines and chemokines secreted from these cells not only promotes proliferation, angiogenesis, invasion and metastasis but also suppresses the host’s immune system and facilitates tumor growth and development54,55 (Fig. 8). There are many key molecules that link inflammation and cancer, including transcription factors, signal transducers and activators of transcription 3 (STAT3), nuclear factor-kB (NFkB), nuclear factor of activated T-cells (NFAT), activator protein-1 (AP-1), CCAAT enhancer binding protein (C/EBP), cAMP response element binding protein/p300 (CBP/p300),

activator transcription factor (ATF), downstream genes iNOS, COX-2, interleukin-6 (IL-6), IL-1b, tumor necrosis factor-R (TNF-R), 5-lipoxygenase (5-LOX), hypoxia inducible factor-1a (HIF-1a), and vascular endothelial growth factor (VEGF), resulting in inflammation and tumorigenesis.54,56 Besides the above, inflammatory signaling is regulated by upstream kinases, such as NFkB-inducing kinase (NIK), IkB kinase (PKC), mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase (PI3K)/Akt and protein kinase C (PKC), also important in inflammation linked tumorigenesis. These critical molecules can be considered important in the modulation of an inflammatory response, and thus could block or prevent inflammationassociated carcinogenesis.54,56

Chemoprevention: inflammation as potential target The term ‘‘chemoprevention’’ was coined by Sporn in the mid1970s and is defined as the use of a chemical substance of either natural or synthetic origin to prevent, hamper, arrest, or reverse a disease.57 It is suggested that inflammation is a multifaceted and complicated process implicated in infiltration and activation of various immune and inflammatory cells, cytokine production, signal transduction and molecular mechanism that results in

Fig. 8 Role of inflammation in cancer development. Chronic inflammation is a critical component of tumor promotion and progression, including colorectal, gastric, pancreas, lung, bladder, hepatocellular, ovary, skin and esophageal cancers. In colonic tumorigenesis, inflammatory stimulation, inflammatory cells are recruited and production of pro-inflammatory cytokines and diverse ROS and RNS that induction of genetic change, enhanced malignant transformation and proliferation of initiated cells. Subsequently, as tumor tissue formation, inflammation also promotes development of cancer by creating an inflammatory microenvironment. The inflammatory and immuosuppressive cytokines and chemokines secreted from these cells not only promote proliferation, angiogenesis, invasion and metastasis but also suppress the host’s immune system and facilitates tumor growth and development.

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a widespread physiological state in organ and tissue. There are many clinical drugs and dietary natural compounds that are demonstrated to be able to exert protective action by different mechanisms, on a number of pathological aspects through targeting interference with chronic inflammatory response. For example, anti-inflammatory drugs, including nonsteroidal antiinflammatory drugs (NSAID), are found to have some neuroprotective effects.58 Resveratrol and curcumin are found to prevent cell damage and death through their antioxidative activities that reveal potential neuroprotective activities.59,60 Anti-a4 integrin monoclonal antibody acts by disrupting adhesion and migration of immune cells that attenuate cytokine release in Crohn’s disease individuals.61 Using monoclonal antibodies against B cells function, TNF-a and IL-6 signaling are beneficial for autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease.62 In patients with type 2 diabetes, treatment with an IL-1 receptor antagonist may restore the function of pancreatic b cells and improve insulinresistance.39 Kinase Inhibitors such as SB203580, a p38 MAPK inhibitor, are found to decrease IL-1b production and reduce rheumatoid arthritis in mice.63 Oral intake of tyrosine kinase inhibitor showed a reduction of inflammatory markers and improved quality in rheumatoid arthritis and psoriasis patients.62 In an in vivo study, PPARg agonist reduced the expression of proinflammatory cytokines that then decreased immune complex deposition and renal inflammation, and lowered atherosclerotic lesions.64 PPARg agonist also lowered plasma glucose and serum CRP and TNF-a, and increased the production of adiponectin in adipose tissue, thus improving insulin resistance in humans with type 2 diabetes.62 The relationship between inflammation and cancer is established.54,55 Numerous evidence demonstrates that inflammatory pathways are critical targets in cancer treatment and prevention.65 Many natural bioactive compounds are reported to interfere with the initiation, promotion/progression, and invasion/metastasis of cancer through the control of intracellular signaling cascades as the process of inflammation progresses. These bioactive compounds include flavonoids, flavonolignans, isothiocyanates, proanthocyanidins, terpenoids, and other polyphenolic compounds.1,56 These bioactive compounds act by avoiding the causes of tissue damage, inhibiting signaling pathways and the activation of transcription factors, inhibiting oxidant-generating enzymes and mediators of inflammation, scavenging reactive oxygen and nitrogen species generated by inflammatory cells, and modulating angiogenesis and metastasis.

Flavonoids: new approach in chronic inflammatory diseases Flavonoids are plant secondary metabolites that are ubiquitous in fruits, vegetables, nuts, seeds and plants. These polyphenolic compounds are a subgroup of chemically related polyphenols that possess a basic 15-carbon skeleton and can be represented as C6-C3-C6, consisting of two benzene rings (C6) joined by a linear three carbon chain (C3).1 Based on the differences in the structure of the aglycones C ring, flavonoids can be classified into seven groups: flavones, flavanones, flavonols, flavanonols, isoflavones, flavanols (catechins) and anthocyanidins (Table 1). The structural variation of flavonoids may come from various This journal is ª The Royal Society of Chemistry 2010

patterns of substitution through enzymatic reactions including hydroxylation, methoxylation, sulfonation, acylation, prenylation, or glycosylation. Flavonoids are most frequently present as conjugates in glycosides and polymers that are water soluble and degraded to variable extents in the digestive system.66 There are also a wide variety of types of naturally occurring flavonoids—at least 2000. Some of them exhibit a broad spectrum of pharmacological properties such as antioxidant, free radical-scavenging, anti-inflammatory, anti-carcinogenic, antiviral, anti-bacterial, anti-thrombogenic and anti-atherogenic activities. It is reported that human intake of all flavonoids is a few hundred milligrams to 650 mg per day in our diet.66 Significant scientific evidence shows that flavonoids have many beneficial health effects for human beings. Many studies show that flavonoid intake improves health and fights off chronic diseases.67 Among the biological properties of flavonoids, antiinflammatory activity is attracting growing interest in managing chronic inflammatory diseases. The biochemical and molecular mechanisms, as well as the signaling pathways, of flavonoids implicated in chronic inflammatory diseases are described below. Flavones Apigenin, a flavonoid present in parsley and celery (Fig. 9), is found to inhibit HIF-1a and VEGF expression by blocking PI3K/Akt signaling or LPS-induced pro-inflammatory cytokines expression by inactivating NF-kB through the suppression of p65 phosphorylation.1 Lupus is autoimmune disease, characterized by production of autoantibodies to attack nuclear antigens and immune complex deposition in organs.68 In an in vivo study, apigenin decreased response of Th1 and Th17 cells to major lupus autoantigen, and subsequently suppressed the ability of lupus B cells to produce pathogenic autoantibodies that limit the inflammatory state in SFN-1 mice. Apigenin also downregulated the expression of COX-2 and cellular FLICE-like inhibitory protein (c-FLIP) in immune cells as well as causing activated immune cells to undergo apoptosis, thus suppressing inflammation in lupus.69 In the pathogenesis of rheumatoid arthritis, it is reported that inflammatory cytokines produced by fibroblastlike synoviocytes are involved in joint destruction. Apigenin is known to induce ROS production and cause apoptosis through oxidative stress-activated ERK1/2 pathway in fibroblast-like synoviocytes.70 Moreover, intake of apigenin also showed immunomodulating effects triggered by TNF-a in a mouse model of rheumatoid arthritis.71 Luteolin is prevalent in thyme and also exists in beets, brussels sprouts, cabbage and cauliflower, and is shown to possess great antioxidative activity. It is known that the activation of microglia and cytokine production is important in neurodegenerative diseases. Treatment with luteolin strongly suppressed IFNginduced CD40 expression and the production of TNF-a and IL-6 through downregulated phosphorylation of STAT-1 in microglia.72 Also, luteolin significantly inhibited LPS-induced activation of microglia by inhibiting JNK phosphorylation and activation of AP-1, increasing dopamine uptake and decreasing excessive TNF-a, NO and superoxide production in microglia,73,74 suggesting the neuroprotective effect of luteolin against brain injury. In addition, luteolin was found to regulate MAPK and NF-kB signaling that inhibits TNF-a induced IL-8 Food Funct., 2010, 1, 15–31 | 23

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Table 1 Backbone structures of the different classes of flavonoids

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Groups

Structure

Examples

Flavones

Apigenin, luteolin, tangeretin, nobiletin, 5-hydroxy3,6,7,8,30 ,40 hexamethoxyflavone

Flavonols

Kaempferol, myricetin, quercetin, isorhamnetin

Flavanols

Catechin, gallocatechin, epicatechin, epigallocatechin-3gallate

Flavanones

Naringenin, hesperetin, eriodictyol

Isoflavones

Daidzein, genistein, glycitein

Anthocyanidins

Cyanidin, delphinidin, pelargonidin

production, which is an important inflammatory cytokine involved in maintaining the inflammatory state in inflammatory bowel diseases.75 Luteolin also decreases TNF-a, IL-1 and MCP1 gene expression and increases adiponectin and leptin levels through the enhancement of transcriptional activity of PPARg in 3T3-L1 adipocytes that might improve obesity-driven insulin resistance.76 Citrus peel is a rich source of polymethoxyflavones, such as tangeretin and nobiletin, and exhibits a broad spectrum of biological activities, including modulation of inflammatory-derived cancer development.77 It is shown that tangeretin suppresses IL1b-induced COX-2 expression through inhibiting activation of MAPK and Akt.1 In an in vivo study, nobiletin was found to lower levels of eotaxin, a potent eosinophil chemoattractant cytokine 24 | Food Funct., 2010, 1, 15–31

that relieves the infiltration of eosinophils and airway inflammation in asthmatic rats.78 It is suggested that the inhibition of foam cells forming macrophage and ox-LDL uptake is one of the targets for atherosclerosis. Nobiletin inhibited macrophage foam-cell formation through reducing metabolism of b-VLDL, is primarily taken up by macrophages via the LDL receptor in cultured murine J774A.1 macrophages.79 Several studies demonstrate that nobiletin can improve arthritic diseases as evidenced by decreasing proinflammatory cytokine production in human synovial cells and downregulating gene expression of MMPs in human synovial fibroblasts,80 as well as in collagen-induced arthritic (CIA) mice.81 Nobiletin can also inhibit leukocyte infiltration, protein expression of iNOS and COX-2 as well as tumorigenesis in mouse skin.1 In our previous studies, we reported that a metabolite of nobiletin This journal is ª The Royal Society of Chemistry 2010

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Fig. 9 Representative natural flavonoids and their dietary sources. (A) flavones, (B) flavonols, (C) flavanols, (D) flavanones, (F) isoflavones, (G) anthocyanidins.

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(30 ,40 -didemethylnobiletin) and 5-hydroxy-3,6,7,8,30 ,40 -hexamethoxyflavone, a hydroxylated PMF in citrus peel, inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced skin inflammation and tumor promotion by suppressing MAPK and PI3K/Akt signaling pathways.82,83 Recently, two new flavones isolated from Cirsium japonicum DC, pectolinarin and 5,7-dihydroxy-6,40 -dimethoxyflavone were found to reduce high-carbohydrate/high-fat diet-induced diabetes in rat through decreasing plasma glucose and increasing adiponectin levels that may improve glucose and lipid homeostasis.84 Flavonols Quercetin, an ubiquitous plant secondary metabolite, is found abundant in onions, broccoli, apples, grapes, wine, tea, and leafy green vegetables, is well known as a potent antioxidant and antiinflammatory agent. Recently, it was shown to possess good antiatherosclerotic activity. In human umbilical vein endothelial cells (HUVECs), quercetin treatment strongly attenuated the inflammation-induced upregulated expression of VCAM-1, ICAM-1 and monocyte chemoattractant protein-1 (MCP-1), which may contribute to its interference with the interaction between monocytes and vascular endothelial cells during the early stages of atherosclerosis.85 Oral feeding of quercetin (64-mg/kg body weight daily) significantly inhibited atherosclerotic lesion size in the aortic sinus and thoracic aorta through reducing superoxide production, improving endothelial NO synthase (eNOS) function and decreasing plasma-sP-selectin levels in the apolipoprotein E (ApoE)(-/-) gene-knockout mouse.86 Quercetin also decreased circulating inflammatory markers, including IFNg, IL-1a and IL-4 in high fat diet animal models and therefore may improve inflammation or obesity-associated disorder.87 In addition, consumption of quercetin is found to decrease systolic blood pressure and plasma oxidised LDL in obese subjects (aged 25–65 years) without affecting liver and kidney functions.88 When rats were fed a diet of rutin, a quercetin glycoside, there was markedly attenuated dextran sulfate sodium (DSS) induced gene expression of IL-1b and IL-6 in colonic mucosa and decreased intestinal colitis.89 Quercetin was found to inhibit 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis of rats possibly through downregulation of TNF-a-induced NF-kB activation.90 Kaempferol, another flavonol-type flavonoid present in broccoli, tea and various vegetables, is considered to improve osteoporosis. For example, treatment with kaempferol stimulated differentiation and mineralization of murine pre-osteoblastic cell lines that may contribute to the prevention of bone loss.91 In the pathology of osteoporosis, proinflammatory cytokines TNF-a is important for bone disruption and osteoclastogenic cytokine production. Kaempferol was reported to antagonize TNF-ainduced p65 translocation and production of IL-6 and MCP-1as well as RANKL-triggered osteoclast precursor cell differentiation. These data indicate that kaempferol exerted a profound anti-osteoclastogenic effect.92 Advanced glycation end products (AGE) are oxidative products formed from nonenzymatic reaction of reducing sugars with free amino groups of proteins. AGE is reported to be involved in diabetic complications and various age/inflammation-related chronic diseases through generation of 26 | Food Funct., 2010, 1, 15–31

ROS and activation of inflammatory signaling cascades.93 Studies show that supplementation of kaempferol in mice reduced AGE-induced activation of NADPH oxidase and proinflammatory gene expression through modulating the NFkB signaling cascade.94 When aged Sprague-Dawley rats were fed with a diet containing kaempferol, the activation of T cell was inhibited and COX-2, iNOS and MCP-1 gene expressions of kidney through modulation of NIK/IKK and MAPK signalings were reduced possibly reducing kidney disease.95 In addition, both kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes by acting as agonists of PPARg that may exert a beneficial effect on hyperglycemia and insulin resistance.96 Flavanols Tea is a popular beverage worldwide. It is produced from the leaves of Camellia sinensis. There are more than 300 different kinds of tea made by different manufacturing processes. Among these, green tea has attracted attention for its health benefits contributed by catechin compounds including epigallocatechin3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC). Numerous studies demonstrate the potential of green tea in iron-chelating, radical-scavenging, antiinflammatory and brain-permeable activities thus preventing cardiovascular, chronic and neurodegenerative diseases.97,98 In vivo study shows that pretreatment with ()-catechin protected dopaminergic neurons in the substantia nigra against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity through modulation of the phosphorylation of c-Jun Nterminal kinase (JNK) and GSK-3b.99 Pretreatment with epicatechin also attenuated ox-LDL induced neurotoxicity in mouse-derived striatal neurons.100 EGCG is the most abundant polyphenolic compound in green tea. EGCG is found to inhibit LPS-induced microglial activation, NO and TNF-a production as well as subsequent neuronal injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic culture, suggesting the neuroprotective activity of EGCG may be inhibition of microglial activation.101 Neuronal damage and death caused by excessive NO is one of the pathological mechanisms in neurodegeneration. EGCG was found to inhibit NO-induced PC12 cell death by scavenging ROS.102 Obesity is a low-grade inflammatory state and is predisposed to an increased incidence of diabetes and CVD. A number line of evidence shows that EGCG possesses excellent activity against obesity-associated pathogenesis and metabolic disorders. In high fat diet-induced obesity animal models, supplementation with dietary EGCG reduced body weight gain and body fat, plasma cholesterol and MCP-1 levels, and decreased lipid accumulation in hepatocytes as well as attenuated insulin resistance.103 In addition, feeding EGCG improved high fat diet-induced nonalcoholic steatohepatitis in mice by decreasing triglyceride and cholesterol levels, lipid peroxidation, and expression of a-smooth muscle actin (a-SMA) in liver tissue. The liver protective effect of EGCG in mice on obesitywas further evidenced by expressions of insulin receptor substrate-1 (IRS-1) and phosphorylated IRS-1 (pIRS-1), and decreasing TNF-a and NF-kB signaling in liver tissues thus improving nonalcoholic steatohepatitis and insulin This journal is ª The Royal Society of Chemistry 2010

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resistance.104 Treatment with EGCG on regulatory T cells from obese individuals significantly enhanced the function of regulatory T cells by the production of anti-inflammatory cytokine IL10.105 Moreover, EGCG is reported to inhibit TNF-a stimulated activation of activator protein-1 (AP-1) and secretion of MCP-1 porcine aortic endothelial cells that work against vascular endothelial inflammation and atherosclerosis.106 EGCG also protects against bone, muscular and skeletal diseases. In synoviocytes and chondrocytes, upregulation of MAPK is critical for proinflammatory cytokine-induced signaling that causes production of several mediators of cartilage damage in an arthritic joint. EGCG was reported to modulate IL-1b-induced activation of MAPKs and DNA binding activity of AP-1 in osteoarthritis chondrocytes.107 Synovial fibroblasts produce metalloproteinases (MMPs) induced by proinflammatory cytokine TNF-a, which are involved in destroying cartilage and bone in Rheumatoid arthritis. Treatment with EGCG suppresses TNF-a-induced production of MMP-1 and MMP-3 through downregulating phosphorylation of MAPKs, such as ERK1/2, p38, JNK and the activation of AP-1 transcription factor in RA synovial fibroblasts.108 Furthermore, EGCG suppresses osteoclast differentiation through downregulating expression of nuclear factor activated T cells c1 (NFATc1) and reduces histologic scores in experimental arthritis in mice that may improve osteoporosis and rheumatoid arthritis.109 Flavanones Much of the activity of flavanones from citrus peels appears to have an impact on vascular endothelial cells that reveal protective effect against atherosclerosis and cardiovascular diseases. In high cholesterol-fed New Zealand white rabbits, diet supplemented with naringenin were shown to reduce gene expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and MCP-1 in endothelial cells. Plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein (HDL) were also decreased by naringenin feeding.110,111 These findings suggest that naringenin can prevent hypercholesterolemia caused fatty liver and acts on interfering immune cells and vascular endothelial cells as well as macrophage infiltration against atherosclerosis. Naringin is shown to inhibit TNF-a-induced expression of MMP-9 in vascular smooth muscle cells (VSMC) through modulation of the PI3K/AKT/mTOR/p70S6K pathway and suppression of the transcriptional activity of AP-1 and NF-kB.112 Emerging studies demonstrate that obesity is a major risk factor for atherosclerosis and cardiovascular disease. Adiponectin secreted from adipocytes was found to suppress atherogenesis by inhibiting monocyte adhesion, reducing macrophage uptake ox-LDL and suppressing the accumulation of ox-LDL in vascular wall.113 Studies show that naringin and hesperetin, other flavanones found in large quantity in citrus peel, enhanced adiponectin transcription in differentiated 3T3-L1 adipocytes through activation of PPARg.114 Naringin is also reported to inhibit high fat diet-induced atherosclerosis and normalize hyperinsulinemia and obesity in low-density lipoprotein receptor-null mice.115 Cytokines promote proliferation and migration of vascular smooth muscle cells and play an important role in atherosclerosis. Hesperetin is found to inhibit platelet-derived growth This journal is ª The Royal Society of Chemistry 2010

factor (PDGF)-BB induced proliferation of primary cultured rat aortic vascular smooth muscle cells through regulation of Akt and MAPKs signaling that results in cell cycle arrest.116 Moreover, hesperetin also increased NO releases from human umbilical vein endothelial cells through up-regulation eNOS expression that may improve function of vascular endothelial cells.117 In addition to the anti-atherosclerosis effect, studies also demonstrate the protective activity of naringin and hesperetin against neuroinflammatory injury. Both naringin and hesperetin attenuated LPS/IFNg-induced TNF-a production through glial cells, while naringin showed a greater effect as evidenced by inhibition of iNOS expression by modulation of p38 MAPK and STAT-1 signaling.118 Isoflavonoids The health benefits of isoflavonoids from soybeans was recognized in recent years, especially about its anti-atherosclerotic functions. Consumption of soy-based diets is associated with a lower risk of cardiovascular disease in humans and reduced atherosclerosis in several experimental animals.119 These effects are related to their antioxidant, anti-inflammatory and antithrombogenic properties through inhibition of endothelial and inflammatory cell activation and reduction in recruitment of leukocytes, as well as foam cell formation. Genistein, the major isoflavone abundantly present in soybean, is known for its role in the regulation of vascular function and protection against atherosclerosis.120 Treatment with genistein markedly inhibited TNF-a-induced cell adhesion molecule CD62E and CD106 expression and subsequent monocyte adhesion in HUVECs and human brain microvascular endothelial cells.121 Genistein also decreased the interaction between monocyte and endothelial cells through the activation of PPARg that inhibits of monocyte adhesion in culture cells and animals.122,123 In an in vitro study, genistein inhibited LPS-induced MCP-1 secretion from macrophages that contribute to reduced monocyte migration.124 In vivo administration of genistein inhibits LPS-induced expression of iNOS and nitrotyrosine protein in vascular tissue that prevents hypotension and vascular alterations.125 Genistein is also known to have a potential effect on rheumatoid arthritis, diabetes, metabolic disorders, neurodegenerative diseases and chronic colitis by modulation of inflammatory response. For instance, genistein inhibits production of proinflammatory molecules NO, IL-1b, and HC gp-39, known as markers of cartilage catabolism in LPS-stimulated human chondrocytes.126 In a collagen-induced rheumatoid arthritis rat, genistein modulated Th1-predominant immune response by suppressing the secretion of IFNg and increasing IL-4 production that balances the inflammatory state.127 NAFLD is an obesity-related fatty liver disease caused by proinflammatory cytokine TNF-a and ILs and leads to the dysfunction of hepatocytes and increase fatty acid uptake. Genistein is reported to reduce high fat diet-induced steatohepatitis through decreasing plasma TNF-a levels and improved liver function in rat.128 Supplementation with genistein and daidzein also decreases mRNA levels of TNF-a, IL-1b and MCP-1 in plasma and liver tissue in obese Zucker rats, suggesting a preventive effect of dietary genistein on steatohepatitis through its anti-inflammatory activity.129 Furthermore, intake of high-dose isoflavones Food Funct., 2010, 1, 15–31 | 27

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(genistein, daidzein, and glycitein) significantly reduced serum TNF-a and increased adiponectin levels in women, indicating that isoflavonids could regulate inflammatory conditions and improve metabolic parameters.130 In addition, genistein attenuated LPS-induced loss of dopamine uptake in rat mesencephalic neuron-glia cultures through reducing microglia activation and production of NO and TNFa131 may protect dopaminergic neuron injury-caused pathogenesis of Parkinson’s disease. In Alzheimer’s disease, accumulation of astrocytes at sites of Ab deposition is the earliest neuropathological changes that initiate the inflammatory response. Treatment with genistein reduced Ab-induced production of inflammatory mediator iNOS, COX-2, TNF-a and IL-1b in astrocytes, possibly through activation of PPARs.132 Also, oral treatment with genistein reduced TNBS-induced chronic colitis by inhibiting expression of COX-2 mRNA and protein as well as the myeloperoxidase (MPO) activity in rat colon that exerts beneficial anti-inflammatory effects against inflammatory bowel disease.133 Genistein is a well-known tyrosine kinase inhibitor. In an in vitro study, genistein was found to prevent IL-1b/IFNginduced expression of COX-2 and iNOS as well as produce PGE2 and NO in human islets that may improve insulin resistance and prevent pathogenesis of diabetes.134 Anthocyanidins Anthocyanidins are common plant pigments that give the red and blue colors in some fruits and vegetables such as blueberries and grapes. Epidemiological investigations and animal experiments indicate that anthocyanins may contribute to chemopreventive activities of various chronic inflammatory diseases.135,136 Anthocyanins-rich berries were demonstrated to possess a broad spectrum of biological properties, including antioxidant, cadioprotective, neuroprotective, anti-inflammatory and anticancer.137 In an animal study, cyanidin was reported to reduce PGE2 levels in paw tissues and TNF-a levels in serum in adjuvant-induced arthritis.138 Damage and apoptosis of vascular endothelial cells is frequently observed in atheromatous plaques and contributes to pathology of atherosclerosis. It has been shown that cyanidin inhibited TNF-a-induced endothelial cell apoptosis, elevated expression of eNOS and thioredoxin may improve vascular endothelial cell function and vasculopathy.139 VEGF is known as a major pro-angiogenic and pro-atherosclerotic factor. Both cyanidin and delphinidin, other major anthocyanidins present in pigmented fruits and vegetables, inhibit PDGF-induced VEGF expression through down-regulation of p38 MAPK and JNK signalings in vascular smooth muscle cells.140 Delphinidin also shows protective effects against cardiovascular disease. It is suggested that proliferation of vascular endothelial cells is important in the pathogenesis of atherosclerosis.141 Delphinidin treatment inhibits serum and VEGFinduced bovine aortic endothelial cell proliferation through modulation of ERK and also results in cell cycle arrest.142 Also, delphinidin increased eNOS expression by mediating the MAP kinase pathway, thus preventing bovine aortic endothelial cell apoptosis.143 In addition, delphinidin was found to fight against ox-LDL-induced damage in HUVECs and regulate apoptotic molecule expression.144 These studies suggest delphinidin may be 28 | Food Funct., 2010, 1, 15–31

important in preventing both plaque development and atherosclerosis. Proanthocyanidines and theaflavins Proanthocyanidins (PAs), also called condensed tannins, are ubiquitous and present as the second most abundant group of natural phenolics after lignin. Oligomers and polymers of proanthocyanidins are widely found in the plant kingdom in fruits, cereals, berries, beans, nuts, cocoa and wine. The abundance of proanthocyanidins in plants makes them an important part of the human diet and are reported to exhibit a wide range of biological effects.145 A recent study demonstrated that dietary grape seed proanthocyanidins (GSPs) is effective against ultraviolet (UV) radiation-induced skin tumor in mice. Dietary GSPs inhibited UVB-induced infiltration of proinflammatory leukocytes and the levels of myeloperoxidase, cyclooxygenase-2 (COX-2), prostaglandin (PG) E(2), cyclin D1 and proliferating cell nuclear antigen (PCNA) in the skin and skin tumors.146 PAs are shown to mediate several anti-inflammatory mechanisms involved in the development of cardiovascular disease by modulation of monocytes adhesion in the inflammatory process of atherosclerosis.145 PAs also exhibit in vivo hepatoprotective and anti-fibrogenic effects against dimethylnitrosamine-induced liver injury.147 Theaflavins, a mixture of theaflavin (TF-1), theaflavin-3gallate (TF-2a), theaflavin-30 -gallate (TF-2b), and theaflavin3,30 -digallate (TF-3) are the major components of black tea. We previously reported that TF-3 exerts its anti-inflammatory and cancer chemopreventive actions by suppressing the activation of NFkB through inhibition of IKK activity.148 We also found that epicatechins in green tea and theaflavins in black tea are able to reduce the concentration of methylglyoxal in physiological phosphate buffer conditions.149 Among these black tea components, TF-3 is generally considered to be the more effective component for a protective effect against inflammatory processes.150

Conclusion Chronic inflammation is linked to numerous human diseases. Increasingly epidemiological and experimental studies demonstrate that modulation of inflammatory response by natural phytochemicals plays an important role in the prevention, mitigation, and treatment of many chronic inflammatory diseases. Flavonoids are a group of natural compounds widely present in vegetables, fruits and edible plants that possess potent biological activities. Dietary intake of flavonoids is suggested to prevent and lower the risk of chronic diseases. In this review, we discussed the possible mechanisms by which flavonoids play a role in the regulation of the inflammatory processes associated with atherosclerosis (cardiovascular disease), neurodegenerative diseases, obesity, metabolic disorders, bone, muscular and skeletal diseases, and chronic inflammatory diseases, as well as cancers. The anti-inflammatory activity of flavonoids is seen through several mechanisms involving the modulation of inflammatory signaling, reduction of inflammatory molecule production, diminishing recruitment and activation of inflammatory cells, regulation of cellular function and their This journal is ª The Royal Society of Chemistry 2010

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antioxidative property. Regarding the safety, ability and the antiinflammatory effects of flavonoids, they are likely to have a potential role in preventive and therapeutic roles in chronic inflammatory conditions. However, additional, extensive research of flavonoids in strengthening the network of inflammatory response needs to be studied in the future.

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Abbreviations Ab AGE AP-1 ApoE APP a-SMA ATF CBP/p300 CD C/EBP c-FLIP CIA COPD COX-2 CRP CVD DSS EC ECG EGC EGCG ERK1/2 eNOS GLUT4 GSK-3b HC gp-39 HDL HIF-1a H2O2 HUVEC ICAM-1 IL IL-1ra IBD IFN-g IKK iNOS IRS-1 JNK 5-LOX LPS MAPK MCP-1 MMP MPO MPTP mTOR NADPH oxidase

amyloid b peptide advanced glycation end products activator protein-1 apolipoprotein E amyloid precursor protein a-smooth muscle actin activator transcription factor cAMP response element binding protein/p300 Crohn’s disease CCAAT enhancer binding protein cellular FLICE-like inhibitory protein collagen-induced arthritic chronic obstructive pulmonary disease cyclooxygenase-2 c-active protein cardiovascular disease dextran sulfate sodium epicatechin epicatechin-3-gallate epigallocatechin epigallocatechin-3-gallate external signal regulated kinase 1/2 endothelial NO synthase glucose transporter type 4 glycogen synthase kinase 3b human cartilage glycoprotein-39 high-density lipoprotein hypoxia inducible factor-1a hydrogen peroxide human umbilical vein endothelial cell intercellular adhesion molecule-1 Interleukin IL-1 receptor antagonist protein inflammatory bowel disease interferon-g IkB kinase inducible nitric oxide synthase insulin receptor substrate-1 c-Jun N-terminal kinase 5-lipoxygenase lipopolysaccharides mitogen-activated protein kinase monocyte chemoattractant protein-1 matrix metalloproteinase myeloperoxidase 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mammalian target of rapamycin nicotinamide adenine dinucleotide phosphateoxidase

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NAFLD NF-ATc1 NF-kB NFAT NIK NO NSAID 1O2 O2 OH ox-LDL p70S6K PDGF PGE2 PI3K pIRS-1 PKC PPARg RA RANKL ROS SOCS STAT TGF-b Th cell TNBS TNF-a TPA UC VCAM-1 VEGF VLDL VSMC

non-alcoholic fatty liver disease nuclear factor of activated T cells c1 nuclear factor-kappa B nuclear factor of activated T-cells NF-kB-inducing kinase nitric oxide nonsteroidal anti-inflammatory drugs singlet oxygen superoxide anion hydroxyl radical oxidized low-density-lipoprotein p70 ribosomal S6 kinase platelet-derived growth factor prostaglandin E2 phosphoinositide-3 kinase phosphorylated IRS-1 protein kinase C peroxisome proliferator-activated receptor g rheumatoid arthritis receptor activator for nuclear factor kB ligand reactive oxygen species suppressor of cytokine signaling signal transducers and activators of transcription transforming growth factor-b T helper cell 2,4,6-trinitrobenzene sulfonic acid tumor necrosis factor-a 12-O-tetradecanoyl-phorbol-13-acetate ulcerative colitis vascular cell adhension molecule-1 vascular endothelial growth factor very-low-density lipoprotein vascular smooth muscle cells

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REVIEW

www.rsc.org/foodfunction | Food & Function

Review of in vitro digestion models for rapid screening of emulsion-based systems David Julian McClements* and Yan Li

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Received 6th August 2010, Accepted 7th September 2010 DOI: 10.1039/c0fo00111b There is increasing interest in understanding and controlling the digestion of emulsified lipids within the food and pharmaceutical industries. Emulsion-based delivery systems are being developed to encapsulate, protect, and release non-polar lipids, vitamins, nutraceuticals, and drugs. These delivery systems are also being used to control the stability and digestion of lipids within the human gastrointestinal tract so as to create foods that enhance satiety and reduce hunger. In vitro digestion models are therefore needed to test the efficacy of different approaches of controlling lipid digestion under conditions that simulate the human gastrointestinal tract. This article reviews the current status of in vitro digestion models for simulating lipid digestion, with special emphasis on the pH stat method. The pH stat method is particularly useful for the rapid screening of food emulsions and emulsion-based delivery systems with different compositions and structures. Successful candidates can then be tested with more rigorous in vitro digestion models, or using animal or human feeding studies.

1. Introduction There is growing interest in understanding and controlling the digestibility of lipids within the human gastrointestinal (GI) tract.1–5 The pharmaceutical industry is using this knowledge to design lipid-based delivery systems that either increase the bioavailability of highly lipophilic drugs or that deliver these drugs to specific locations within the GI tract.6,7 The food industry is using a similar approach to design food-grade delivery systems to encapsulate, protect, and release bioactive lipid components, with the aim of either improving their bioavailability or controlling their delivery.4,5,8,9 There are a number of bioactive lipid components that may benefit from encapsulation within this type of delivery system, including u-3 fatty acids, conjugated linoleic acid, butyrate, phytosterols, carotenoids, antioxidants, coenzyme Q, and vitamins A and D.4,10–14 The availability of effective delivery systems for lipophilic bioactive components could lead to the creation of functional foods specifically designed to maintain or improve human health. Functional foods could be designed to increase the digestibility of lipids in individuals with health conditions that impair the normal digestive process.1,15 Functional foods could be designed to control human satiety, satiation, and hunger by controlling the rate and extent of lipid digestion in different regions of the GI tract.16–20 For example, recent studies show that emulsions that remain stable to gravitational separation in the stomach and/or which have a delayed digestion in the small intestine can stimulate the release of gut hormones that induce satiety and reduce food intake.21,22 Functional foods could be designed to deliver bioactive components to specific locations within the GI tract where they can exhibit their functional attributes, e.g., anticancer components could be released in the colon.23 Analytical tools are needed to screen the efficacy of the various emulsion-based delivery systems that have been designed and Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA, 01003, USA

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developed to control lipid digestion and release. Ultimately, the efficacy of newly designed delivery systems should be tested in animal or human feeding studies, but there are ethical, economic, and practical reasons that make this unrealistic. Some prototype delivery systems may be unsafe or unsuitable for human consumption. Animal feeding studies often involve subjecting animals to uncomfortable conditions and/or sacrifice. Feeding studies are usually expensive, time-consuming, and prone to appreciable subject-to-subject variations. Consequently, there is a need for in vitro analytical tools that can be used to screen delivery systems before more extensive animal or human studies are carried out.4,5 The purpose of this article is to provide an overview of some of the most commonly used in vitro testing methods for studying the digestion of emulsified lipids. We begin by providing a brief overview of the physicochemical and physiological processes that occur when emulsified lipids pass through the human gastrointestinal tract. There have been major advances in this area over the past decade or so, which have greatly facilitated the design of in vitro test methods. Next, we define the concept of lipid bioavailability in the context of the digestion and release of lipophilic components. Finally, we describe a number of in vitro methods that have been developed to test the digestion of emulsified lipids, discussing their relative advantages and disadvantages. In particular, we focus on the pH stat method, which is finding increasing utilization as a convenient tool for rapidly screening different emulsion-based systems.

2. Overview of in vivo lipid digestion An understanding of the basic physicochemical and physiological processes that occur as an emulsified lipid passes through the human gastrointestinal (GI) tract is required to develop effective in vitro models that accurately simulate lipid digestion.4,5,8,14,24 After ingestion, emulsified lipids experience a complex series of physical and chemical changes as they pass through the mouth, stomach, small intestine, and large intestine, which affect their ability to be digested and/or absorbed (Fig. 1 and 2). In this This journal is ª The Royal Society of Chemistry 2010

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Fig. 1 Schematic diagram of the physicochemical conditions in the different regions of the the human GI tract. Picture of human body was obtained from http://en.wikipedia.org/wiki/Digestive_tract (Copyright free).

section, we provide an overview of the major physicochemical and physiological events that occur in each region of the GI tract. A more detailed description has been given in several recent review articles.4,5,25,26

2.1.

Passage through the gastrointestinal tract

We begin by providing an overview of the composition and properties of the various regions within the GI tract.

2.1.1. Pre-ingestion. The lipid droplets in foods and beverages have a variety of different compositions, physical states, and structural properties.27 The lipid phase may be either nondigestible (e.g., mineral oils) or digestible (e.g., triacylglycerol oils). It may vary in its physical state and polymorphic form, e.g., being liquid, solid, or partially solid at body temperature. Lipid droplets are usually surrounded by a coating consisting of emulsifier molecules and other adsorbed matter. The most common emulsifiers used in foods are proteins, polysaccharides, surfactants and phospholipids.28–30 Additional materials (such as minerals, solid particles, or biopolymers) may adsorb on top of these emulsifier layers. Consequently, lipid droplet coatings may vary in their electrical charge, thickness, permeability, environmental responsiveness, resistance to displacement, and susceptibility to enzymatic digestion.27 The lipid droplets themselves may vary in their physical dimensions (e.g., particle size distribution and shape) and in their aggregation state (e.g., isolated, flocculated or partially coalesced). There may also be considerable variations in the physicochemical and structural properties of the matrix that surrounds the lipid droplets. The droplets may be simply dispersed within a low viscosity aqueous liquid (as in soft drinks, nutritional beverages, or milk), they may be embedded within larger particles (as in filled hydrogel particles), or they may be distributed within macroscopic gel-like or solid matrices (as in jellies, meat products, cheese, ice cream, or hard candies). Many of these factors may impact the behavior, digestibility, and ultimate fate of lipid droplets within the human GI tract. Indeed, there have been concerted efforts recently to rationally design emulsion-based delivery systems to control lipid digestibility based on knowledge of these and other factors.4,5,14,21,22,31 2.1.2. Mouth. The main function of the mouth is to ingest the foods, and to convert them into a form suitable for swallowing.

Fig. 2 Schematic diagram of the complex physicochemical and physiological processes that may occur during lipid digestion and absorption of emulsified lipids in the human GI tract.

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The composition, structure, and properties of lipid droplets change appreciably during mastication due to the complex physiochemical and physiological processes that occur within the human mouth.32–35 An ingested food or beverage undergoes a number of processes: it is mixed with saliva; it may change its pH, ionic strength, and temperature; it may be acted upon by various digestive enzymes (e.g., lingual lipase, amylase, protease); it may interact with biopolymers in the saliva (e.g., mucin); it may interact with the surfaces of the tongue and mouth; it experiences a complex flow profile; and, it may be physically broken down into smaller pieces by chewing.36–43 Ideally, all of these factors should be simulated in an in vitro digestion model, although in practice many of them are often ignored because they are assumed not to have a major impact on lipid digestion. One of the most important factors that influence the behavior of emulsified lipids in the mouth is their interaction with saliva. Human saliva is usually around pH 5.5 to 6.1 during fasting and around pH 7 to 8 after food ingestion.44 Saliva is typically secreted at a rate of about 0.2 to 4 ml per minute,35 with a total saliva output of 500 to 1500 mL per day.36 Saliva contains water (99%), minerals (<1%), and proteins (0.1–0.2%). The protein fraction is compositionally complex with many different kinds of molecules, including enzymes, immunoglobulins, antibacterial proteins, proline-rich proteins and glycosylated proteins (mucins).45–47 The mucins are capable of inducing coalescence and flocculation of ingested lipid droplets, which has been attributed to depletion and bridging mechanisms.36,41 In practice, it is difficult to accurately simulate the behavior of mucin using in vitro digestion models because there are large variations in the amount, composition, and properties of saliva for a given individual at different times, as well as between individuals.36 A food or beverage usually spends a relatively short time in the mouth before being swallowed,34 with the time depending on the nature of the ingested material, with liquids spending much less time in the mouth than solids that need masticating to break them into smaller pieces.35 Typically, swallowing only takes a few seconds to complete, and involves the integrated movements of parts of the tongue, pharynx, esophagus and stomach.48 The structural organization of the lipids within the mouth depends on their initial structural organization within the food, the duration and intensity of mastication, and the physiological characteristics of the individual consuming the food. A limited amount of lipid digestion may occur during mastication due to the presence of lingual lipases secreted by glands within the mouth. These lingual lipases are usually more important in human infants than in adults, and in rodents than in primates.49 Recently, it has been found that the mouth contains receptors capable of detecting fat and releasing signals that stimulate the body’s ability to digest and absorb lipids.26 Consequently, it may be more important to monitor and control the behavior and digestion of the lipid phase within the mouth than previously believed. The material that is swallowed after mastication of a food is referred to as the ‘‘bolus’’.50 2.1.3. Stomach. The stomach can be considered to be a baglike structure where the food is processed and stored prior to being transferred to the small intestine.51 The rate at which the stomach is emptied can influence the subsequent rate and extent 34 | Food Funct., 2010, 1, 32–59

of nutrient digestion and absorption in the small intestine, as well as influencing the feeling of satiety.52,53 The stomach consists of three main regions with different physiological functions: the cardia (upper section), fundus (middle section) and antrum (lower section). The main function of the fundus is to secrete gastric juice containing acids and digestive enzymes, while the main function of the antrum is to generate mechanical forces that mix, disrupt and transport the stomach contents.52,53 After the bolus is swallowed it rapidly passes through the esophagus and into the stomach (Fig. 1), where it is mixed with the acidic digestive juices containing gastric enzymes, minerals, surface active materials, and various other biological components, and is also subjected to mechanical agitation due to stomach motility.54,55 The pH of the human stomach has been reported to be between 1 and 3 under fasting conditions,44,56,57 but to change considerably after the bolus enters the stomach.44 Usually, there is an appreciable increase in the pH of the stomach contents after food ingestion, followed by a gradual decrease over the next hour or so until a value around pH 2 is reached. The pH-time profile depends on the initial pH, buffering capacity, composition, and quantity of food ingested.44 The high acidity of the stomach plays a number of important physiological roles, including activating enzymes, hydrolysis of food components, and inactivation of microorganisms. In the fasted state, the ionic strength of the stomach contents are around 100 mM, with the major ionic species being: Na+ (70  30 mM); K+ (13  3 mM), Ca2+ (0.6  0.2 mM) and Cl (100  30 mM).57 There is usually an appreciable increase in the ionic strength of the stomach contents after food ingestion due to the additional ions arising from the food.44 The stomach goes through a variety of contractive motions that mix the bolus with the digestive juices, breakdown any large fragments within the food, and transport the resulting material into the small intestine at controlled rate.54,55,58–60 These contractions vary in amplitude, frequency and duration depending on whether the stomach contains food or not. An ingested food component may remain in the stomach for a period ranging from a few minutes to a few hours depending on its quantity, physical state, dimensions, structure, and location. Typically, the amount of food remaining within the stomach after ingestion decreases by about 50% in 30 to 90 min, with fluid components being emptied more rapidly than solid components.54,55 The minimum size of any particles that can pass from the stomach into the small intestine via the pylorus is about 1 to 2 mm.51 Particles with larger dimensions will remain in the stomach until they have been broken down further. These particles may be fragments of a solid or gel-like food that was ingested, or they may be formed within the stomach itself e.g., due to the formation of a biopolymer gel under acidic conditions.61 Thus, the fracture properties of the food material consumed and the degree of mastication within the mouth may have a large impact on the subsequent digestion any encapsulated lipid droplets in the GI tract. Appreciable digestion of emulsified lipids usually begins in the stomach due to the presence of gastric lipase. Human gastric lipase is a highly glycosylated globular protein with a molecular weight of about 50 kDa, that is stable in gastric acid juices over a wide range of pH values (2 to 7).62–64 Human gastric lipase has been reported to have an isoelectric point of 6.6 to 7.9 depending This journal is ª The Royal Society of Chemistry 2010

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on the iso-form,65 consequently it should be positively charged in the highly acidic conditions in the human stomach. The concentration of gastric lipase in the stomach is typically between about 0.5 and 1 mM. Gastric lipase binds to the surface of the lipid droplets, where it converts the encapsulated tricylglycerols (TAG) into diacylglycerols (DAG), monoacylglycerols (MAG), and free fatty acids (FFA).64,66,67 Gastric lipase has a preference for hydrolyzing fatty acids at the sn-3 position of the glycerol backbone of the TAG molecules.26 Lipid hydrolysis usually stops when the droplet surface becomes covered with free fatty acids, which occurs when about 4–40% of the fatty acids have been released from the TAGs, with the amount depending on droplet size.15,26 The extent of digestion is usually greater for smaller droplets (with a larger surface area), than for larger droplets (with a lower surface area), since the former have more surface area. The physicochemical mechanism for the inhibition of gastric lipase by fatty acids is believed to be the trapping of the lipase within a colloidal structure (200 nm) that is comprised of FFA, DAG, MAG and phospholipids.1 The trapped lipase is prevented from coming into close contact with the TAG at the oil–water interface and so its activity is decreased. The FFA released in the stomach may play an important role in the subsequent digestion and absorption of foods: it may promote lipid digestion by enhancing droplet disruption; it may increase solubilization of digestion products in mixed micelles; it may stimulate hormone release, which stimulates the secretion of bile and pancreatic juices; it may increase the binding of colipase; it may increase the activity of pancreatic lipase in the small intestine.49,63 In particular, recent studies indicate that there are fat receptors in the stomach that may trigger the release of biological signals (such as CKK) that stimulate the release of pancreatic lipase and that slow down the emptying of the stomach.26 The partially digested and disrupted food that leaves the stomach and enters the small intestine is usually referred to as ‘‘chyme’’ (Fig. 1). 2.1.4. Small intestine. The small intestine is the region in the GI tract where most of the lipid digestion and absorption processes normally occur. It can be considered to be a tube-like structure (about 2.5 to 3 cm in diameter) consisting of three major regions: duodenum (about 26 cm long); jejunum (about 2500 cm long); and, ileum (about 3500 cm long).51 The actual surface area of the small intestine is much greater than that calculated for a simple smooth tube because of the complex topology of the inner lining, e.g., villi and crypt structures.51 After entering the duodenum, the chyme is mixed with sodium bicarbonate, bile salts, phospholipids, and enzymes secreted by the liver, pancreas and gall bladder.50 The sodium bicarbonate secreted into the small intestine causes the pH to increase from highly acidic (pH 1 to 3) in the stomach to around neutral (pH 5.8–6.5) in the duodenum, where the pancreatic enzymes work most efficiently.63 Nevertheless, studies with human subjects have shown that there may be large variations in both stomach and duodenum pH.44 The bile salts and phospholipids originating from the liver (via the gall bladder) are surface-active and can facilitate emulsification of the lipids by adsorbing to the droplet surfaces.50 Lipid hydrolysis continues within the duodenum This journal is ª The Royal Society of Chemistry 2010

through the actions of lipases originating from the pancreas.62,64 Lipids and lipid digestion products (e.g., FFA, sn-2 MAG, cholesterol, phospholipids, and fat-soluble vitamins) are solubilized within mixed micelles and vesicles consisting of bile salts and phospholipids at the surface of the lipid droplets, and are then transported to the epithelium cells for absorption. The mixed micelles and vesicles must pass through the mucous layer that coats the epithelium walls before it can reach them. In the fasted state, the ionic strength of the small intestine has been reported to be about 140 mM.57 After ingestion of food, there is usually an appreciable increase in ionic strength due to the additional ions arising from the food. The ionic strength is particularly important because it influences the magnitude and range of any electrostatic interactions in the system. The type of ions may also be important since it is known that multivalent cations (such as Ca2+ and Mg2+), may promote precipitation of bile salts and long chain saturated fatty acids in the small intestine, which has a major impact on lipid digestibility.68–70 In addition, multivalent ions may interact with certain kinds of biopolymers (such as alginate) to form gels that can inhibit lipid digestion. Bile salts play an extremely important role in the lipid digestion and absorption processes. Bile salts are surface-active molecules that can adsorb to oil–water interfaces and can form micelles and other association colloids in water. They are therefore capable of facilitating lipid droplet deformation and break up during mechanical agitation, of stabilizing lipid droplets against aggregation, and forming (mixed) micelles that solubilize and transport hydrophobic molecules. In vivo they usually perform these roles in conjunction with other surface active substances, such as phospholipids, MAGs and FFAs. Bile salts are synthesized from cholesterol in the liver. They have structures that differ appreciably from other surfactants, because they do not consist of a hydrocarbon tail and a hydrophilic head group. Instead, they are fairly large rigid ‘‘plate-like’’ molecules that have a hydrophobic side and a hydrophilic side. The hydrophobic side can interact with substances that have a nonpolar character (e.g., other bile salts or lipid droplet surfaces), whereas the hydrophilic side can interact with substances that have a polar character (e.g., water). The ‘‘backbone’’ of bile salt molecules is cholic acid, which may be conjugated with the amino acids taurine or glycine in the liver, thereby increasing its watersolubility.63 In the fasted state, the level of bile salts in the duodenum is around 4.3 to 6.4 mM, while after ingestion of a meal it increases to around 5 to 15 mM.44 Bile salts may adsorb to freshly formed oil–water interfaces, or they may displace other surface active substances already at oil–water interfaces. As mentioned earlier, pancreatic lipase plays a critical role in the lipid digestion process because it is the digestive enzyme responsible for converting TAGs and DAGs into FFAs and MAGs (sn-2 position). Pancreatic lipase preferentially hydrolyses fatty acids in the sn-1 and sn-3 positions on the glycerol backbone, and has little specificity for fatty acid chain length.49 To catalyze this reaction the pancreatic lipase must adsorb to the oil–water interface so that it is in close proximity to the TAG and DAG molecules.71 Lipase usually does this as part of a complex with co-lipase and possibly bile salts.63 The extent of binding of pancreatic lipase to the droplet surfaces depends on the initial interfacial composition and properties, as well as the presence of Food Funct., 2010, 1, 32–59 | 35

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any surface active substances in the continuous phase.63 Competitive adsorption processes occur at the oil droplet surfaces between the enzyme complex, bile salts, phospholipids, digestion products, and other surface-active substances, which could interfere with lipase adsorption to the droplet surfaces.71 Droplet surfaces may be coated with indigestible layers (such as dietary fibers72 or silica particles73) that inhibit the direct access of the lipase to the encapsulated lipids. The electrical charge on the interface may affect any electrostatic interactions between the enzyme complex and the droplet surfaces. The pH optimum of pancreatic lipase is around pH 8.5, but it also operates well around neutral pH.63 A co-enzyme known as ‘‘co-lipase’’ is required for the optimum activity of lipase. It has been proposed that this co-enzyme forms a stoichemetric complex with lipase that adsorbs to the oil–water interface and brings the lipase into close contact with the lipid substrate. Co-lipase is a polypeptide with a molecular weight of about 10 kDa, which has a hydrophobic side that is believed to bind to the oil–water interface, and a hydrophilic side that is believed to bind the lipase. Co-lipase is secreted from the pancreas as pro-co-lipase, and then is converted into its active form when trypsin cleaves off a peptide called enterostatin. This peptide is believed to be a hormone that regulates satiety, reduces fat intake, and inhibits pancreatic secretion.63 The presence of bile salts may either promote or inhibit the activity of pancreatic lipase depending on their concentration.63,74 At relatively low concentrations, they tend to promote lipase activity, which can mainly be attributed to their ability to solubilize lipid digestion products and remove them from the oil– water interface, e.g., sn-2 MAGs and FFAs. If these products are not removed from the droplet surfaces, then the fatty acids would accumulate at the oil–water interface thereby preventing the lipase from adsorbing and getting access to the TAGs and DAGs within the droplets. In addition, if the lipid digestion products were not removed from the site of enzyme activity, then the conversion of TAGs to MAGs and FFAs would be inhibited by the high local concentration of reaction products. On the other hand, relatively high bile salt concentrations may inhibit the ability of lipase to digest emulsified lipids. This effect can be attributed to the ability of the bile salts to compete for the oil– water interface with the lipase, thereby preventing it from coming into close proximity to the lipid substrate.75 One of the key roles of the bile salts is to form micelles that solubilize and transport the lipid digestion products from the lipid droplet surfaces to the intestinal membrane where they are absorbed. It is therefore important that the bile salt concentration exceeds the critical micelle concentration, which is usually around 1 to 2 mM.63 In practice, the bile salt concentrations in the small intestine are above the CMC: 6–15 mM in the duodenum (being higher after consumption of a meal); 10 mM in the jejunum; <4 mM in the ileum (where the bile salts are reabsorbed by the body). Once the lipid digestion products have been transported across the mucous layer in micelles and vesicles they are absorbed by the intestinal enterocyte cells. The mucous layer has been reported to be a stagnant layer (i.e., transport is mainly diffusion controlled) with a thickness between 30 to 100 mm.76 The subsequent fate of the digestion products (FFA and MAG) depends largely on the molecular weight of their hydrocarbon chains. Long chain fatty acids (LCFA) tend to be reassembled into TAGS within the 36 | Food Funct., 2010, 1, 32–59

epithelium cell, packaged into colloidal particles (lipoproteins), and then transported to other tissues via the lymphatic system.15,63,77 On the other hand, short chain fatty acids (SCFA) and medium chain fatty acids (MCFA) tend to be absorbed directly into the portal vein and pass through the liver before entering the systemic circulation.3 2.1.5. Colon. The material that is not absorbed within the small intestine passes from the ileum to the large intestine, which is a tube-like structure with a length of about 1500 mm.78 The large intestine can be divided into four major regions that differ in their physiological functions: caecum, colon, rectum, and anal canal. The colon itself can be divided into four regions, the ascending, transverse, descending, and sigmoid regions. The main physiological functions of the colon are the absorption of water and electrolytes, the fermentation of polysaccharides and proteins, the re-absorption of bile salts, and the formation, storage and elimination of fecal matter.51,78 The pH of the colon varies from region to region,78 with lower pH values occurring in regions where dietary fibers are fermented and short chain fatty acids (SCFA) are released.79 It has been reported that pH values around 5.5, 6.2 and 6.8 represent the proximal, transverse, and distal regions of the colon.80 Any material that is undigested in the upper GI tract will reach the colon. Normally, lipids would be fully digested in the stomach and small intestine, but some undigested lipid may pass through the upper GI tract, particularly in specially designed emulsion-based delivery systems. The droplets in oil-in-water emulsions prepared using a non-digestible lipid phase (such as a mineral oil or alkane) can reach the colon. If lipid droplets are surrounded by an indigestible coating or embedded within an indigestible matrix (e.g., dietary fiber), then they may not be fully digested in the small intestine. Under these circumstances it may be important to consider the processes that occur in the colon. The colon contains a large number of different kinds of anaerobic bacteria species, which are capable of fermenting food components that were not digested in the upper GI tract, e.g., dietary fibers, proteins and peptides.80 Thus, any lipids encapsulated within dietary fiber matrices may only be released after they reach the colon due to bacterial fermentation. It has been suggested that some undigested food ingredients may interfere with the normal metabolic activity of the colonic microbiota, which could alter the health status of the lower GI tract and even the whole body.81,82 Consequently it is important to consider this factor when designing any delivery system that might alter the normal digestibility of food components. 2.2.

Key features of gastrointestinal fluids

A number of the most important processes that occur when emulsified lipids pass through the various regions of the human GI tract were highlighted in Section 2.1. Based on this information, we highlight some of the key parameters that need to be taken into account when developing in vitro models to simulate lipid digestion. 2.2.1. pH. There are considerable variations in the pH of the aqueous medium surrounding the lipid droplets as they pass through the GI tract: mouth (pH z 7); stomach (pH z 1–3); This journal is ª The Royal Society of Chemistry 2010

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small intestine (pH z 6–7); large intestine (pH z 5.5–7). These pH variations may cause considerably changes in the rate and extent of lipid digestion. Solution pH determines the physicochemical properties of many of the components within the GI tract, e.g., charge, solubility, aggregation state, physical interactions, and chemical reaction rates. The water-solubility of many minerals is highly dependent on pH, so that they may go from soluble to insoluble in different regions of the GI tract. The electrical charge of many emulsion droplets is pH-dependent, particularly those stabilized by amphiphilic proteins, polysaccharides, and ionic surfactants. The stability of many emulsions to droplet flocculation and coalescence depends on the magnitude and sign of their electrical charge.27 Protein-coated lipid droplets change from negative to positive when the pH is reduced from above to below their isoelectric point (pI). A positively charged droplet may interact with negatively charged components or surfaces within the GI tract, and vice versa. A protein-coated lipid droplet may aggregate near its isoelectric point since the electrostatic repulsion is no longer sufficient to prevent droplets from coming into close proximity. Some anionic polysaccharides, such as alginate and pectin, have pKa values around 3.5, and so they may have little or no negative charge in the highly acidic conditions within the stomach but have a high negative charge in the neutral conditions in the small intestine. The cationic polysaccharide chitosan, which is commonly used in fabricating emulsion-based delivery systems, has a pKa value around 6.5, and so it has a high positive charge at acidic pH but little or no positive charge at neutral pH. Consequently, any biopolymer matrices held together by electrostatic interactions may change their properties in different GI fluids (e.g., swell, shrink or disintegrate), which could impact the ability of lipase to interact with the lipid droplet surfaces. 2.2.2. Ionic composition. There may be considerable variations in the type and concentration of ions surrounding the lipids droplets, which may impact the electrostatic interactions in the system through electrostatic screening or binding effects. For example, long chain fatty acids may precipitate in the presence of calcium ions, thereby removing them from the lipid droplet surface (which facilitates further digestion), but which may also reduce their subsequent absorption due to calcium soap formation.83 Sufficiently high concentrations of monovalent and multivalent counter-ions can promote extensive flocculation in emulsions containing electrically charged droplets, which may restrict the access of lipase to the oil–water interface and slow down digestion.84 Certain types of mineral ions are capable of promoting the gelation of biopolymers within the GI tract, which would affect the ability of digestive enzymes to reach any entrapped lipid droplets. For example, alginate or pectin form strong gels if there are sufficiently high levels of free calcium ions present in solution.85 If any calcium binding agents are present within a food product, such as EDTA or alginate, they may reduce the amount of free calcium present, which will then alter the ability of calcium ions to interact with other food components.84 2.2.3. Enzyme activity. There are various kinds of enzymes in the mouth, stomach, small intestine and colon that can digest food components, such as lipids (lipases), proteins (proteases), This journal is ª The Royal Society of Chemistry 2010

starch (amylases) and dietary fibers (glycosidases).50 The ability of these enzymes to interact with their specific substrates within a food may impact lipid digestibility and the absorption of encapsulated lipophilic components. Enzyme accessibility to a substrate may be influenced by physical barriers between the encapsulated substrate and the surrounding aqueous phase where the digestive enzymes are normally located. Lipid digestion may not be initiated until the lipid droplets are released from their original emulsifier coatings or from any matrices that they are encapsulated in. The rate of lipid digestion may be decreased by coating lipid droplets with a dietary fiber layer86 or by embedding them within dietary fiber particles.87,88 Similarly, it may be necessary for protein or starch coatings or particles to be digested by proteases or amylases before the lipase can act on the lipids. Enzyme activity may also be influenced by any food components that can bind to them, either specifically or nonspecifically. For example, some foods contain natural enzyme inhibitors, such as peptides from soybeans that inhibit proteases89 and polyphenols from fruits and vegetables that inhibit lipases.90 2.2.4. Surface active components. There are a variety of endogeneous (e.g. proteins, peptides, phospholipids, and bile salts), exogeneous (e.g., surfactants, proteins), and internally generated (e.g., lipid and protein digestion products) surface active substances present within the aqueous phase surrounding the lipid droplets. These substances compete with the surfaceactive substances already present at the oil–water interface, potentially leading to changes in interfacial composition and properties.71,91 Some of these surface active components also play a crucial role in solubilizing lipid digestion products (MAG and FFA) and lipophilic components and carrying them to the epithelium cells for absorption. The mixed micelles and vesicles present responsible for solubilizing and transporting highly lipophilic components within the aqueous phase consist of bile salts and phospholipids secreted by the body, as well as MAG and FFA from the digested lipids. 2.2.5. Flow profiles and mechanical forces. The encapsulated lipids may be exposed to various kinds of forces and flow profiles during their passage through the human body.51 These processes mix the various components together, breakdown structures (lipid droplets, protein particles, hydrogel matrices etc), and transport materials from one location to another. It is therefore important to simulate or model these flow profiles in in vitro digestion models.

3. Key physicochemical events occurring during lipid digestion A number of the key physicochemical events that occur during the lipid digestion process are highlighted in this section (Fig. 2). 3.1.

Matrix disruption

The lipid droplets in a food are surrounded by a matrix that may be liquid-like, gel-like, or solid-like. The disruption of this matrix within the GI tract may have an important impact on lipid digestion, since it determines how easily digestive enzymes and Food Funct., 2010, 1, 32–59 | 37

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other components can access the emulsified lipids, as well as how easily any digestion products can leave the droplet surfaces. If the lipid droplets are dispersed within a simple low viscosity fluid (as in milk, nutritional beverages, or soft drinks), then they will be rapidly dispersed within the digestive juices. The surface of the lipid droplets will then be readily accessible to any surface active components and enzymes present in the surrounding aqueous media. On the other hand, if the lipid droplets are trapped within a gel-like or solid-like matrix, then it may be necessary for this matrix to be disrupted before the digestive components can access the lipid droplet surfaces. Alternatively, the digestive enzymes and other components may have to diffuse through this matrix before they can reach the lipid droplets. The rate at which these molecules diffuse depends on the pore size of the matrix, as well as any specific attractive interactions between the molecules and the matrix material. The initial size of the matrix particles, as well as their response to changes in environmental conditions as they pass through the GI tract, may therefore influence the rate and extent of lipid digestion. The matrix material surrounding a lipid droplet may respond to changing environmental conditions in a number of ways: (i) remain intact; (ii) swell or shrink; (iii) physically, chemically or enzymatically degrade; or (iv) physically fragment. The behavior of a particular matrix material will depend on the type and interactions of the molecules it contains. For example, some biopolymers are enzymatically digested in the stomach or small intestine (e.g., starches and proteins), whereas others are indigestible (e.g., dietary fibers and resistant starch). Electrostatic complexes formed between anionic polysaccharides and proteins at low pH values, may dissociate when the pH is raised above the isoelectric point of the protein because this weakens the electrostatic forces holding them together.92 This kind of information can be used to design matrices that control the digestion and release of lipids within the GI tract.14 3.2.

Alterations in interfacial properties

The lipid droplets in foods are usually coated by an interfacial layer that consists of emulsifiers and any other substances that adsorb to the droplet surfaces, e.g., mineral ions, biopolymers or solid particles.27 After a food is consumed there may be appreciable changes in the properties of the interfacial coatings as the droplets pass through the GI tract. The original emulsifier molecules may be digested by enzymes, e.g., phospholipids by phospolipases, proteins by proteases, or non-ionic surfactants with ester bonds by esterases.93 The ability of the emulsifier molecules to stabilize the lipid droplets against aggregation may be changed considerably after they have been fully or partially hydrolyzed. The original emulsifier molecules (or their digestion products) may be displaced from the droplet surfaces by other surface-active components present in the system.94,95 These surface-active substances may come from the food itself (e.g., proteins, surfactants, phospholipids), or they may be generated as a result of the digestion process (e.g., FFA or MAG), or they may be secreted by the GI tract (e.g., bile salts, phospholipids, or proteins).63 Alternatively, components within the digestive juices may adsorb on top of the original layer of emulsifier molecules, e.g., anionic mucin molecules can form a coating around cationic protein-coated lipid droplets.96,97 All of these changes in 38 | Food Funct., 2010, 1, 32–59

interfacial composition may alter the subsequent susceptibility of the lipids to digestion. 3.3.

Droplet fragmentation, aggregation, and dissolution

There may be considerable changes in the size and aggregation state of the lipid droplets in a food sample as it passes through the GI tract. Bulk fats or large fat droplets may be broken down into smaller droplets by the mechanical forces generated within various regions of the GI tract (such as mastication and swallowing in the mouth, churning in the stomach, passage through the pylorus, and peristaltic movements within the intestines). These processes may be facilitated by the presence of surface active components such as digestion products, phospholipids, bile salts, and proteins. If the interfacial layer surrounding the droplets is not strong enough, then the droplets may coalesce with each other when they collide within the GI tract, which leads to an increase in mean particle size.4,97 If the repulsive interactions between the lipid droplets are insufficiently strong, then the droplets may self-associate and form flocs.84 As the lipid phase within droplets is digested by lipase, there may be a decrease in droplet size due to movement of the digestion products from the droplet interior into the surrounding aqueous phase. Consequently, there may be appreciable changes in the particle size distribution and aggregation state of the lipid phase as it passes through the GI tract,15,63 which may influence the ability of enzymes to adsorb to the oil–water interface. Microscopy studies have provided important insights into the structural changes that occur during lipid digestion.98,99 These studies have shown that digestion of emulsified lipids by pancreatic lipase in the small intestine takes place by a sequence of steps involving the formation of different phases. A liquid crystalline or crystalline phase is observed around the lipid droplet surfaces within the first few minutes of lipolysis, which gets thicker over time. Eventually, any undigested oil within the interior of the droplet may be expelled as a smaller oil droplet, leaving the liquid crystalline or crystalline phase behind. These latter phases are presumably formed due to the high local concentration of surface active lipids (FFA and MAG) at the droplet surfaces. The liquid crystalline phase tends to form at low calcium concentrations, whereas the crystalline phase forms at high calcium concentrations (due to fatty acid calcium soap formation). Nevertheless, in the presence of sufficiently high concentrations of bile salt micelles the lipid reaction products are solubilized and therefore the liquid crystalline phases may not be observed. The amount of bile salt micelles required to solubilize the reaction products depends on their solubilization capacity – once the available micelles have been saturated with digestion products, then liquid crystalline phases may be observed. A variety of different liquid crystalline phases may be formed depending on the nature of the original lipid, solution composition, and digestion time.98,99 3.5.

Solubilization and mass transport processes

The efficiency of the digestion process depends on the mass transport of various reactants, catalysts, and products from one location to another. Digestive enzymes must come into close proximity to their substrates before they can carry out their This journal is ª The Royal Society of Chemistry 2010

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biological actions. Thus, lipase must adsorb to the surfaces of lipid droplets before it can convert the encapsulated TAG molecules into MAG and FFA. These digestion products must then be incorporated within mixed micelles and vesicles, which must be transported across the mucous layer before they can be absorbed by the epithelium cells. Similarly, proteases have to adsorb to droplet surfaces if they are going to hydrolyze the adsorbed protein coatings. Consequently, it is important to be aware of the various kinds of mass transport processes operating in the different regions of the GI tract, and to elucidate the major factors that impact them. Mass transport may occur predominantly by convective or by diffusive processes, depending on the nature of the food matrix and the specific region of the GI tract involved. The various mechanical forces generated by the GI tract tend to mix components together and move them from one location to another, e.g., the peristaltic actions of the intestine. Nevertheless, there are regions within the GI tract where mass transport is mainly diffusion-limited. The movement of digestion products solubilized within micelles and vesicles through the mucous layer is normally considered to be diffusion-limited. If lipid droplets are trapped within viscous, gel-like, or solid matrices, then any digestive enzymes may have to diffuse through these matrices before they can reach their substrates. In this case, the trapped droplets would be digested at a slower rate than free droplets because of the longer time taken for lipase to reach the droplet surfaces.

3.6.

Binding interactions

Foods are highly complex systems that often contain a wide variety of different components, including sugars, salts, proteins, polysaccharides, lipids, vitamins, etc. Many of these components are capable of interacting with each other and forming complexes that could potentially alter the rate and extent of lipid digestion. In this section, we provide a few examples of some binding interactions that could occur, while acknowledging that there are many others possible. Indigestible polysaccharides (dietary fibers) may interact with other molecular species in the GI tract through electrostatic or hydrophobic interactions. For example, cationic chitosan can bind anionic bile salts and free fatty acids under simulated GI conditions, which may have a major impact on lipid digestion and absorption.100 Ionic dietary fibers may bind to the surfaces of oppositely charged lipid droplets where they form a protective coating that inhibits lipase adsorption and activity. Multivalent mineral ions can form electrostatic complexes with oppositely charged species, thereby altering their solubility, aggregation state, and physicochemical properties. For example, calcium ions can form electrostatic complexes with long chain fatty acids, which can promote lipid digestion by removing the FFAs from the lipid droplet surfaces, but which can also reduce the subsequent absorption of the FFAs by forming insoluble soaps. Some food components may be able to bind directly to digestive enzymes and thereby alter their activity or ability to bind to lipid droplet surfaces, e.g., polyphenols. At present there is a fairly poor understanding of how different components within complex foods alter the behavior and digestibility of lipids within the human GI tract. Clearly further This journal is ª The Royal Society of Chemistry 2010

research is required in this area using well characterized complex food products.

4. Lipid bioavailability The term bioavailability has been defined as the fraction of an ingested component (or its products) that eventually ends up in the systemic circulation.101,102 For lipophilic components, the bioavailability (F) can be defined as:101 F ¼ FB  FT  FM

(1)

Here, FB is defined as the bioaccessibility coefficient or fraction of the lipophilic components that is released from the food matrix into the juices of the gastrointestinal tract, FT is defined as the transport coefficient or the fraction of the released lipophilic components that are transported across the intestinal epithelium; and FM is the fraction of the lipophilic components that reaches the systemic circulation without being metabolized. The value of FM depends on the pathway that the lipophilic components follow to reach the systemic circulation, e.g., short chain fatty acids (SCFA) and medium chain fatty acids (MCFA) pass through the portal vein and liver (where they may be metabolized), while long chain fatty acids (LCFA) pass through the lymph system (thereby avoiding the first pass through the liver).3,50 LCFA are reassembled into TAGs in epithelium cells, packaged into colloidal structures, and then leave the epithelium cells, and enter the lymph system. Highly lipophilic bioactive components also tend to be transported via the lymph system, e.g., carotenoids. After the lipophilic components reach the systemic circulation they may be distributed between different tissues, where they may be stored, metabolized, or excreted.102,103 The relative rates of these various processes determine the timedependence of the concentrations of the lipophilic component and its metabolites at specific locations within the body. The concentration-time profile of a specific lipid component at a particular site-of-action will determine its beneficial or adverse affects on human health and wellness. Consequently, it is usually important to measure the concentration of a lipid component at a particular location in order to establish its potential efficacy.103 When determining the bioavailability of bioactive lipophilic components it is important to account for the fact that they may be chemically modified during passage through the gastrointestinal tract prior to or after absorption, e.g., triglycerides are converted to monoglycerides and free fatty acids, whereas some lipophilic bioactives (such as polyphenolics) may be chemically derivitized.102,104

5. Overview of in vitro lipid digestion tests 5.1.

Introduction

A number of in vitro approaches commonly used to study lipid digestion are highlighted in this section. Some of these approaches focus on one particular region of the gastrointestinal tract, whereas others utilize a number of sequential steps to more accurately mimic the entire digestion process.105 In vitro digestion models can therefore be conveniently characterized as:  Single Step Models: One particular region of the GI tract is simulated, e.g., the mouth, stomach, small intestine, or colon. Food Funct., 2010, 1, 32–59 | 39

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The pH-stat method is an example of this type of model, which only simulates digestion in the small intestine (Section 6).  Multiple Step Models: Two or more regions of the GI tract are simulated, e.g., mouth, stomach, small intestine, and colon. A multiple step in vitro digestion model designed to simulate the entire human GI tract is highlighted schematically in Fig. 3. Using either approach, a food sample is prepared and then subjected to one or more treatments designed to simulate specific regions of the human digestive tract, e.g., mouth, stomach, small intestine, and colon. These treatments usually involve mixing the food sample with a simulated digestive fluid of specific composition (e.g., pH, mineral composition, enzyme activity, etc) using controlled mixing conditions. All samples and solutions are normally maintained at 37  C to mimic human body temperature. The level of sophistication of the simulated digestive fluids and mixing conditions used varies widely between different models. The composition, structure, and/or physicochemical properties of the sample being tested can be measured at specific points throughout this process (Section 5.3). Sophisticated analytical instruments specifically designed to simulate the full digestion process are now commercially available, e.g., the TIM lipid absorption system from TNO Quality of Life, The Netherlands.106,107 In this review, we mainly focus on those in vitro methods that use equipment available in most research laboratories, such as glassware, stirrers, and pH meters.

5.2.

Simulation of different regions in GI tract

To accurately model the complex physiological and physicochemical events that occur within the human GI tract it is necessary to simulate the composition, structure, and dynamics of the various intestinal fluids that the lipid droplets encounter.107 In reality, this is often impractical because the in vitro digestion model would become too complicated, time consuming, and expensive to set up and operate. Consequently, researchers often only utilize the key components that are believed to impact the particular system that they are working with, and ignore the other minor components. For example, proteases and amylases may be omitted if the system studied does not contain proteins or starches. Similarly, the wide variety of different monovalent and multivalent ions present in the GI tract may be simulated by simply using NaCl and CaCl2, respectively. One limitation of this approach is that we currently do not have the detailed knowledge

Fig. 3 Schematic diagram of a multiple-step in vitro digestion model to simulate the whole of the GI tract.

40 | Food Funct., 2010, 1, 32–59

of how many of these components influence the behavior of lipid droplets in the GI tract. On the other hand, the simplicity of these models allows one to rapidly screen many samples, and provide some mechanistic understanding of the processes involved. Promising candidate formulations can then be tested using more sophisticated in vitro models, animal feeding studies, or human trials. In the following sections, we highlight some of the most important factors that need to be simulated in each region of the GI tract. 5.2.1. Mouth. The major factors to consider when designing an in vitro digestion step that simulates the human mouth are the potential interactions of the lipid droplets with saliva, tongue, and oral cavity (Section 2.1.2). Most researchers ignore droplet interactions with the tongue and oral cavity because of the difficulty in accurately mimicking these events in the laboratory. Instead, the emulsion to be tested is mixed with a simulated saliva fluid (SSF) under specific conditions (shearing, time, temperature). The compositional complexity of the SSF used in mouth studies varies widely depending on the objectives of the researchers. Some researchers use a simple buffer solution (e.g., pH 7) without any additional components to simulate oral conditions. Other researchers use SSF that contains many of the components found in human saliva, such as acids, buffers, minerals, biopolymers, and enzymes.37,108 Recipes for preparing simulated saliva solutions have been published.43,108 Some researchers simply ignore the oral step altogether (Table 1), assuming that it does not have an impact on lipid digestion. This is likely to depend on the nature of the sample tested, and particularly on emulsifier type. For example, some emulsions undergo extensive flocculation and coalescence in the mouth due to their interactions with the mucin in saliva.36,43 These changes in droplet characteristics (size and interfacial properties) could alter the subsequent digestion of the lipids in the stomach and small intestine. Recently, a number of researchers have developed analytical methods to better understand the interactions of lipid droplets with the tongue and oral cavity.109–111 A number of excellent reviews of the various in vitro/ex vivo/in vivo analytical methods developed have been published.96,97,112 These include methods such as imaging (X-ray, sonography, NMR, and endoscopy), microscopy (optical and confocal fluorescence microscopy), and rheology (shear rheology, large strain deformation, and tribology). These techniques can either be used in isolation or in combination with each other. A particularly innovative approach has been the use of combined tribology and microscopy methods to examine the flocculation, coalescence, and spreading of lipid droplets under simulated oral conditions by using a pig’s tongue as one of the interacting surfaces.96,111 The test sample that results from mixing the food sample with the simulated saliva fluid can be referred to as the ‘‘bolus sample’’. 5.2.2. Stomach. The major factors to take into account when designing an in vitro digestion step to simulate the behavior of lipid droplets within the stomach are the high acidity, specific enzyme activity, mineral composition, and mechanical/flow profile (Section 2.1.3). Previous researchers have used simulated gastric fluids (SGF) with different compositional complexities in their in vitro digestion models (Table 1). The simplest digestion This journal is ª The Royal Society of Chemistry 2010

Parameters Measured Optical microscopy z-potential Particle size Appearance FFA, Glucosamine Optical microscopy z-potential Particle size Protein Adsorbed Optical microscopy z-potential Particle size FFA Microscopical images, droplet size Optical microscopy z-potential Particle diameters Free fatty acid release Confocal microscopy z-potential Particle size Creaming stability Microscopy Creaming stability z-potential Particle size z-potential Particle size, DSC FFA release z-potential Particle size, DSC FFA release z-potential Particle size FFA release Microscopy z-potential Creaming Microscopy Particle size FFA release

Experimental Variables [Bile] [Lipase] Polysaccharides

[Bile] Emulsifier type Emulsifier type

Formulation

Emulsifier type BLG cross-linking

Emulsifier type

Dietary fiber type

Lipid physical state

Emulsifier type Multilayer

Oil type Particle size Lipid content Emulsifier type Polysaccharide type Multilayer formation

Samples Tested

O/W Emulsions O ¼ Tuna E ¼ Lec, Lec/Chit

O/W Emulsions O ¼ soy oil E ¼ LF or BLG

O/W Emulsions O ¼ corn oil E ¼ Cas, WPI, Lec, T20

SEDS O ¼ MCT E ¼ Solutol

O/W Emulsions O ¼ corn oil E ¼ BLG or Lec

This journal is ª The Royal Society of Chemistry 2010

O/W Emulsions O ¼ soybean oil E ¼ Cas, WPI, Lec, T20

O/W Emulsions O ¼ Corn oil E ¼ T20

O/W Emulsions O ¼ Tripalmitin E ¼ SDS

O/W Emulsions O ¼ Corn oil E ¼ Cas, LF or Cas/LF

O/W Emulsions O ¼ Corn oil, MCT E ¼ BLG, T20, Lec

O/W Emulsions O ¼ Fish oil E ¼ Citrem, Chit, Alg

Digestion rate increased with [Bile] and dependent on PS

Protein charge effects displacement by bile salts Digestion rate and extent depends on emulsifier type: Cas, WPI > Lec > T20 Digestion induces a change in lipid composition which affects the solubilization capacity of the lipid phase Protein interfacial cross-linking did not have a big impact on lipid digestion Emulsifier type effects aggregation behavior of lipid droplets throughout in vitro digestion model Dietary fiber type effects aggregation behavior of lipid droplets throughout in vitro digestion, through bridging and/ or depletion mechanisms. Solid fat particles are digested more slowly than lipid fat droplets.

 M: pH 7 (1 h)  S: pH 2 (1 h)  SI: pH 5.3, pancreatic lipase, bile (2 h)  SI: pH 7.5 (2 h)  SI: pH 7.5, CaCl2 (2 h)

 SI: (Pancreatic lipase, bile salts)

 SI: (Pancreatic lipase, bile salts)

 M: (a-amylase, Mucin, BSA)  S: (Pepsin, Mucin)  SI: (Pancreatin, Lipase, Bile salt)

Digestion rate increases with decreasing droplet size, decreasing oil molecular weight, but does not depend strongly on emulsifier type The rate and extent of lipid digestion was decreased when chitosan and chitosan/alginate coatings were present around droplets

 SI: (Pancreatic lipase, bile salts, pH 7)

 SI: (Pancreatic lipase, bile salts, pH 7)

Lipid droplets with different initial protein compositions are all digested

 SI: (Pancreatic lipase, bile salts, pH 7)

 M: pH 7 (1 h)  S: pH 2 (1 h)  SI: pH 5.3, pancreatic lipase, bile (2 h)  SI: pH 7.5 (2 h)  SI: (Pancreatic lipase, bile salts, pH 7)

 SI: (Pancreatic lipase, bile salts, pH 7)

Comments

Digestion Steps Modeled

209

172

208

155

195

192

207

206

131

132

202

References

Table 1 Summary of previous studies using in vitro digestion models used to investigate the digestion and absorption of emulsified food lipids. Samples Tested Key: SEDS ¼ Self-emulsifying delivery system; O ¼ Oil; E ¼ Emulsifier. Digestion Steps Key: M ¼ mouth, S ¼ stomach; SI ¼ small intestine; C ¼ colon. All were carried out at 37  C unless stated. Emulsifier type: LF ¼ lactoferrin; BLG ¼ blactoglobulin; WPI ¼ whey protein isolate; Cas ¼ caseinate; T20 ¼ Tween 20; Chit ¼ Chitosan; Alg ¼ Alginate; MG ¼ monoglyceride; CHO ¼ carbohydrate

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Food Funct., 2010, 1, 32–59 | 41

42 | Food Funct., 2010, 1, 32–59 Optical microscopy z-potential Particle size Appearance FFA, Glucosamine z-potential Particle size FFA release Optical microscopy z-potential Particle size FFA z-potential Particle size FFA Lipolysis products

Lipolysis products

SDS-PAGE Particle size z-potential Optical microscopy Interfacial tension Bioaccessibility FFA released

Particle size z-potential FFA released Interfacial tension Particle size FFA released Protein analysis Particle size z-potential Microstructure FFA released

Polysaccharides

Polysaccharide type TPP concentration Interfacial composition

Bile type [Bile] [Calcium] Triglycerides type

Triglycerides type Emulsifiers Protein type Protein adsorption

Oil amount Emulsifier type NaCl & CaCl2 Emulsifier type

Emulsifier type Microstructure

O/W Emulsions O ¼ Tuna oil E ¼ Lec, Lec/Chit

O/W Emulsions O ¼ Corn oil E ¼lyso-Lec, lyso-Lec/Chit

O/W Emulsions O ¼ Corn oil E ¼ Lec, Lec/Chit, Lec/Chit/Pec

O/W Emulsions O ¼ olive oil E ¼ phosphatidylcholine

Oil O ¼ MCT, soybean oil

O/W Emulsions O ¼ triacylglycerols E ¼ PEG

O/W Emulsions O ¼ Olive oil E ¼ b-Cas/BLG

Oil suspension

SEDDS O ¼ Soybean oil E ¼ Tweens, Spans

O/W Emulsion O ¼ Olive oil E ¼ Galactolipids

Oil bodies & O/W emulsions O ¼ Sunflower seed oil E ¼ Natural, WPI or T20 O/W Emulsion O ¼ Fish oil E ¼ Cas, Cas-CHO complex Emulsifier type

Parameters Measured

Experimental Variables

Samples Tested

Table 1 (Contd. )

Increasing amounts of chitosan reduced the amount of FFA produced. Chitosan was degraded by lipase Coating droplets with non-cross linked or cross-linked chitosan decreased the digestion rate. Coating droplets with chitosan decreased lipid digestion, but having an additional pectin coating increased digestion again. FFA released increased with increasing calcium concentration by amount depending on bile type The rate and extent of lipid digestion was faster for medium chain triglycerides than long chain triglycerides Formulation effects solubilization of encapsulated drugs in mixed micelles Protein hydrolysis by proteases is altered when adsorbed to lipid droplet surfaces

 M: pH 7 (1 h)  S: pH 2 (1 h)  SI: pH 5.3, pancreatic lipase, bile (2 h)  SI: pH 7.5 (2 h)  SI: (Pancreatic lipase, bile salts, pH 7)  SI: (Pancreatic lipase, bile salts, pH 7)

 S: pH 5.5 (0.5 h)  SI: pH 6.25, pancreatic lipase, bile (1 h)  S: pH 2.5 (1 h)  SI: pH 6.5, pancreatic lipase, bile (0.5 h)

Increasing the oil content increases the bioaccessibility of lipophilic components Digestion rate increases with calcium addition, and depends on surfactant type Digestion rate depends on surfactant type

Digestion of oil bodies is slower than emulsion droplets Interfacial covalent (Maillard) caseinate-carbohydrate complexes protect droplets against coalescence

 S: pH 2.0 (1 h)  SI: pH 7.5, pancreatic lipase, bile (0.5 h)  SI: pH 6.5, pancreatic lipase, bile (0.5 h)  SI: pH 7.0, pancreatic lipase, bile (0.5 h)

 SI: pH 7.0, pancreatic lipase, bile  S: pH 1.5, pepsin (1 h)  SI: pH 6.8, pancreatin, bile (2 h)

 SI: (Pancreatic lipase/colipase, bile salts/, pH 7.5)

 SI: (Pancreatic lipase, bile salts, pH 7.5)

Comments

Digestion Steps Modeled

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216

215

84

175

214

178

213

124, 212

211

86

72

210

References

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This journal is ª The Royal Society of Chemistry 2010

This journal is ª The Royal Society of Chemistry 2010 Dietary fibers can inhibit lipid digestion by an amount depending on their structure Gastric lipase has greater activity on the fine emulsion. Free fatty acid concentration plays a key role in the progressive inhibition of lipolysis MCT is hydrolyzed faster than LCT. Lipolysis rate is increased with decreasing particle size. Calcium is a key factor during digestion

 S: human gastric juice (0.5 h)

 S: pH 5.4, gastric juice (0–0.5 h)  SI: pH 7.5, pancreatin, bile (1 h)

Particle size Lipase activity

Particle size Pancreatic lipase activity

Droplet size Triglycerides type Hydrolysis of gastric lipase Droplet size Triacylglycerol composition Calcium pH

O/W Emulsion O ¼ LCT & MCT E ¼ Lec, Sugar esters

O/W Emulsion O ¼ LCT & MCT E ¼ Lec, Sugar esters

Extract Effects

O/W Emulsion O ¼ SCT, LCT E ¼ Lec O/W Emulsion O ¼ Triolein E ¼ Lec

 SI: pH 7.5, pancreatin, bile (0.5 h)

 SI: pH 7.5, pancreatin, bile (0.5/1 h)

Particle size Viscosity FFA released

Oil type

Oil suspension O ¼ MCT, LCT

Polysaccharide type (guar gum, gum arabic, pectin)

Drug solubilization

Oil type

 SI: pH 6.5, pancreatin (10 min)

 S: pH 5.4, gastric juice (0.5 h)  SI: pH 7.5, pancreatin, bile (1 h)

FFA released

Oil type [Lipase]

Oil O ¼ SCT, MCT, LCT E ¼ MG SMEDDS O ¼ SCT, MCT, LCT E ¼ Surfactants

Interfacial covalent (Maillard) caseinate-carbohydrate complexes reduced lipid digestion. Initial digestion rate increased with [Lipase] and depends on oil type

Particle size FFA released

Particle size Viscosity Microstructure FFA released Microstructure FFA released

Emulsifier type

O/W Emulsion O ¼ Fish oil E ¼ Cas, Cas-CHO complex

The digestion rate depends on initial emulsifier type, with MG inhibiting digestion

 TIM Model - Dynamic  S: Acid pH, pepsin, lipase, minerals  SI: z Neutral pH, pancreatin, bile  SI: pH 6.8, pancreatin, bile (0.5 h)

Lipid composition can influence drug solubilization behavior and formulation affects the lipid digestion rate TG-drug suspension has relatively high solubilizing capacity of colloidal phases produced on TG digestion A green tea extract inhibits lipid digestion

FFA released Interfacial tension

Emulsifier type

O/W Emulsion O ¼ Fish oil E ¼ MG, BLG, Lyso-Lec

Comments

Digestion Steps Modeled

 SI: pH 7.5, pancreatin, bile (0.5 h)

Parameters Measured

Experimental Variables

Samples Tested

Table 1 (Contd. )

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125

198

222

221

220

218, 219

163

217

200

References

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models involve adjusting the sample to a highly acidic pH (e.g., pH 1.2) for a fixed period (e.g., 2 h) with some form of mechanical agitation (e.g., stirring). More sophisticated digestion models include a range of different components in the SGF, including acids, buffers, salts, organic molecules, biopolymers, phospholipids, and digestive enzymes.44,57,108 In particular, it is important to note that the stomach contains gastric lipase, which may promote some initial lipid digestion within the stomach. Recipes for preparing simulated gastric juices of varying complexity have been published.2,108,113 A mechanical gut model has been developed to simulate the complex flow profiles, dynamic secretions, and mechanical forces that occur in the human stomach by researchers at the Institute of Food Research (Norwich, UK).114 Most researchers ignore droplet interactions with the surfaces of the stomach in their in vitro digestion models due to the inherent complexity of simulating the stomach’s surface in the laboratory. Nevertheless, these interactions may be important in applications where the lipid droplets are designed to adhere to the stomach wall lining. The test sample that results from mixing the food sample with the simulated gastric fluid can be referred to as the ‘‘chyme sample’’. 5.2.3. Small intestine. The major factors that need to be considered when designing an in vitro digestion step to simulate the behavior of lipid droplets within the small intestine are pH changes (from acid to neutral), enzyme activities (particularly lipase), biological surfactants (particularly bile and phospholipids), and mineral content (particularly calcium). Simulated small intestinal fluids (SSIF) of varying compositional complexity have previously been used within in vitro digestion models (Table 1). The simplest SSIF usually contain a mixture of lipase (or pancreatin) and bile salts (or bile extract) at a pH around neutral. More sophisticated models utilize SSIF that contain buffers, salts, small organic molecules, proteins, enzymes, co-enzymes, bile salts and phospholipids.2,44,108 SSIF composition has been shown to play a major role in determining the rate and extent of lipid digestion determined using in vitro models (see below). It is therefore important to establish an appropriate SSIF composition for a particular sample being tested, e.g., the type and concentration of the components within the SSIF that accurately reflect what happens in vivo. As in the stomach, most researchers ignore the interactions of the lipid droplets with the surfaces of the small intestine in their in vitro digestion models. Nevertheless, a number of researchers have recently examined the impact of the mucous layer on the behavior of lipid droplets and other particles under simulated small intestinal conditions.115 The lipid droplets may become trapped within the mucous layer depending on their size and charge, which is likely to impact how quickly they are digested and absorbed. The test sample that results from mixing the food sample with the simulated small intestinal fluid can be referred to as the ‘‘digest sample’’. 5.2.4. Colon. The colon is one of the most difficult regions to simulate in the laboratory. In vitro testing methods designed to simulate the nutritional and environmental conditions in the human large intestine range from simple static batch microbial 44 | Food Funct., 2010, 1, 32–59

cultures to multiple stage continuous cultures.116–118 A food sample is typically incubated in one or more simulated colonic fluids (SCF) that contain populations of bacteria representative of those normally found in the human large intestine. These bacteria may be cultivated from animal caecal contents or human feces. One difficulty in accurately simulating the human colon is the considerable variations in bacterial populations that exist between individuals. Rather than using bacteria, it is possible to formulate SCF that contain a mixture of enzymes typically produced by colonic bacteria e.g., glycosidases to degrade dietary fibers and proteases to degrade proteins.80,119 Due to the difficulties in setting up and maintaining in vitro colonic models many researchers prefer to go directly to animal models.117 Alternatively, if there is strong evidence that the sample is fully digested and absorbed within the small intestine, then this step can be ignored. 5.3.

In vitro versus in vivo correlations

In vitro studies offer several advantages over in vivo studies, because they are usually faster, less expensive, more versatile, and provide more details about physicochemical mechanisms.2,105,120 Nevertheless, it is extremely difficult to accurately mimic the complex physicochemical and physiological processes that occur in the human digestive tract. For this reason, it is usually advisable to combine in vitro studies with in vivo studies using animals and humans (where possible). In addition, it is important to establish in vitro–in vivo correlations to ensure that any in vitro method being used to test a particular sample is reliable.2,121,122 In this case, the rate and/or extent of lipid digestion may be measured for similar test samples using an in vitro method (e.g., pH stat) and an in vivo method (e.g., human feeding study), and then the results correlated to one another. Eventually, one would like to obtain mathematical models that can predict the real-life performance of a sample from results obtained using an in vitro test model. 5.4.

Physicochemical parameters measured in digestion studies

A variety of analytical techniques can be used to characterize the changes in the properties of emulsified lipids as they pass through simulated GI conditions. A number of the most important and commonly used are highlighted in this section: 5.4.1. Enzyme activity: Formation of digestion products. A variety of digestive enzymes are active in different locations within the human GI tract, including lipases, phospholipases, proteases, amylases, and glycosidases.51,63,80 An accurate in vitro digestion model should therefore contain appropriate types and levels of digestive enzymes, which will depend on the nature of the sample being tested. Lipases. One of the most important parameters to measure in an in vitro digestion model is the rate and extent of lipid digestion due to the activity of gastric and/or pancreatic lipase, i.e., conversion of triacylglycerols (TAG) and diacylglycerols (DAG) into monoacylglycerols (MAG) and free fatty acids (FFA). The most widely used method of measuring lipid digestion is to determine the amount of free fatty acids produced by titration This journal is ª The Royal Society of Chemistry 2010

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with an alkali solution.120 The automated ‘‘pH-stat method’’ based on this principle is discussed in a latter section (Section 6). Nevertheless, other methods can also be used. For example, the amounts of triacylglycerols, diacylglycerols monoacylglycerols, free fatty acids and phospholipids present in the system can be determined at specific digestion times using chromatography methods, such as thin layer chromatography or TLC.123,124 The concentration of specific fatty acids in the lipid and/or aqueous phases can be measured over time using gas chromatography.123 This information can be used to determine which chain lengths, degrees of saturation, and sn-positions of fatty acids on the triacylglycerols molecules are most susceptible to digestion. The human GI tract also contains phospholipases capable of removing free fatty acids from phospholipids and esterases capable of cleaving some non-ionic surfactants, and so it may be important to include these enzymes when testing systems containing these components.125 Proteases. If a lipid droplet is initially coated with a proteinbased emulsifier or if it is initially embedded within a proteinbased matrix, then it may be important to monitor the rate and extent of protein digestion, since this may indirectly influence lipid digestion.93,126 For example, the lipase may be unable to access the lipid droplet surfaces until the protein has been removed by hydrolysis into amino acids. Hence, the lipid digestion rate could depend on the protein digestion rate. Protein digestion can also be measured using the pH-stat method,127 although most previous studies have utilized chromatography (particularly HPLC) or electrophoresis (particularly SDSPAGE) to monitor the conversion of proteins into peptides and amino acids.93,113,128 The concentration and type of specific proteins present within a sample can be determined by combining electrophoresis and mass spectrometry methods.126 Glycosidases and amylases. If a lipid droplet is trapped within a dietary fiber or starch matrix, then it may be important to monitor the digestion of these components by glycosidases or amylases, respectively.80,118 The rate and extent of polysaccharide digestibility is typically monitored by determining the amount of monosaccharides released over time using chemical, enzymatic, chromatography, electrophoresis or spectroscopic methods. As with proteins, the lipid digestion rate may depend on the polysaccharide digestion rate if the lipid droplets are encapsulated within impenetrable polysaccharide matrices. 5.4.2. Particle size distribution and microstructure. The specific surface area of an emulsion is inversely related to the size of the droplets that it contains.129 Lipid digestion is an interfacial phenomenon that requires the lipase molecules to adsorb to oil droplet surfaces before hydrolysis can occur.15,63 Consequently, the rate of lipid digestion often depends on the size of the oil droplets in an emulsion.125,130 In addition, the size of the droplets may change as they pass through the different regions of the GI tract due to fragmentation, coalescence, flocculation, or digestion processes. Consequently, it is often important to have analytical tools to measure the particle size distribution of emulsions as they pass through simulated GI conditions. Optical microscopy techniques are suitable for studying emulsions that contain lipid droplets greater than about 1 mm in radius.98,99 This journal is ª The Royal Society of Chemistry 2010

Specific stains or dyes can be used to highlight particular components within an emulsion and determine its location, e.g., oil soluble dyes,113,131,132 protein stains,133 or polysaccharide stains.100 Thus, one can determine whether a component is present at the oil–water interface or dispersed within the surrounding aqueous phase, and whether its location changes during the digestion process. Electron microscopy (SEM or TEM) can also be used to measure the structural features and organization of the lipid droplets and other colloidal particles present in the digestive fluids. A number of instrumental methods are also available for measuring the particle size distribution (PSD) of emulsions, including static light scattering (SLS), dynamic light scattering (DLS), particle counting, and ultrasonic spectrometry.27,134 Each of these methods has a range of particle diameters that it can reliably detect. For example, SLS instruments can typically detect particles from about 0.1 to 1000 mm, whereas DLS instruments can detect particles in the range 1 nm to 5 mm. Instrumental particle size analyzers are able to provide rapid analysis of the full PSD of emulsion samples in a few minutes, and are therefore extremely convenient for monitoring changes in particle dimensions during the lipid digestion process. They can be used to monitor the breakup, coalescence, flocculation, or degradation of lipid droplets, or to determine the dimensions of other colloidal species present within the digestion fluids such as micelles or vesicles. Nevertheless, these instrumental methods often have a number of limitations when applied to studying lipid digestion. There are often many components within the sample itself or from the simulated GI fluids that can scatter light and therefore contribute to the measured particle size distribution. For example, there may be insoluble matter in the bile extract or pancreatin used to prepare SSIF that obscure the light scattering signal from the lipid droplets. It may therefore be advisable to filter these SSIF solutions prior to utilization in the in vitro digestion model. 5.4.3. Interfacial composition and properties. The composition and properties of the interfacial layer coating the lipid droplets usually changes as an emulsion passes through the GI tract. Consequently, it is useful to have analytical tools that can measure interfacial compositions or at least detect alterations in interfacial compositions. A number of direct and indirect methods have been used to obtain information about interfacial composition. Fluorescent probes can be used to tag surfaceactive species (such as proteins, phospholipids or polysaccharides) to determine changes in their location during the digestion process. The fluorescently-tagged molecules can either be observed directly under a fluorescent microscope or they can be analyzed by fluorescent spectroscopy. Front face fluorescence reflectance spectroscopy measurements can be carried out on emulsion samples without any sample preparation,135,136 whereas fluorescent transmission measurements can be carried out on transparent aqueous solutions after a suitable isolation step e.g., centrifugation, dialysis and/or filtration. Measurements of droplet charge (z-potential) can be used to provide information about changes in interfacial composition, e.g., due to the displacement of one surface-active substance by another.129,131 Knowledge of the droplet z-potential may also be important in its own right, since the charge on the droplets will determine how Food Funct., 2010, 1, 32–59 | 45

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they interact with other components and surfaces within the GI tract. Cationic particles may become trapped within the anionic mucous layer (mucoadhesion), which will alter their ability to be digested and absorbed.137,138 Interfacial tension and rheology measurements are commonly used to detect changes in interfacial composition at planar air–water or oil–water interfaces, providing information that can be related to the behavior of lipid droplet surfaces under similar conditions.94,139 Chemical analysis methods can be used to measure changes in the aqueous phase composition during digestion, which can be used to infer changes in interfacial composition if the total amount of the component in the system is known: CAds ¼ CAq  CTotal. A number of different microscopy methods have been used to examine the structural organization of adsorbed components at the oil–water interface, such as atomic force microscopy and confocal fluorescent microscopy.93,94 5.4.4. Formation of colloidal structures. A number of analytical tools have been used to characterize the various kinds of colloidal structures generated during the lipid digestion process, such as liquid crystals, mixed micelles and vesicles. These methods include light scattering,140,141 X-ray scattering,142,143 neutron scattering,144 electron microscopy,145,146 electron spin resonance and NMR.147 In addition, some techniques can provide information about the distribution of lipid digestion products between the oil, water and colloidal phases, e.g., electron paramagnetic resonance.148 The solubilization of digestion products and lipophilic components within the colloidal structures can usually be determined using standard analytical methods, e.g., chemical, spectroscopic, chromatographic or electrophoresis methods.

shear rheometers, uniaxial compression methods, and flow profiling techniques.27,149–151 The flow profiles in a reaction chamber can be monitored by imaging methods, such as those based on X-ray, NMR or acoustics. Alternatively, they can be simulated using appropriate mathematical models that take into account the geometry of the vessel and the nature of the applied mechanical forces.152 5.4.7. Physical state. A number of studies show that the physical state (solid versus liquid) of lipid droplets influences the rate and extent of their digestion.153–155 Consequently, it may be useful to determine the physical state of lipid droplets during passage through a simulated GI tract if a high melting point fat phase is used. The physical state of lipid droplets can be determined using a number of analytical techniques, including DSC and NMR.27 Information about the packing of the crystals within the lipid droplets can be obtained by X-ray diffraction studies.156,157 5.4.8. Release of lipophilic components. Researchers are often interested in monitoring the release of bioactive lipophilic components encapsulated within lipid droplets during the digestion process. One of the most commonly used methods is to centrifuge the digested sample (Fig. 4a), which typically separates

5.4.5. Binding interactions and aggregation. A number of different kinds of binding interactions may occur in the GI tract that could alter lipid digestibility: biopolymers may bind mineral ions, enzymes, lipid droplets, phospholipids, and surfactants; chelating agents may bind calcium; fatty acids and bile salts may bind calcium (Section 2.1.5). For example, cationic dietary fibers such as chitosan are known to strongly bind anionic bile salts, fatty acids, phospholipids, and lipid droplets, which may interfere with the normal digestion process.100 The method used to study the binding interactions will depend on the nature of the species involved. Some commonly used analytical methods include equilibrium dialysis, isothermal titration calorimetry, centrifugation, specific chemical reactions, chromatography, electrophoresis, and spectroscopy. 5.4.6. Flow profiles and rheology. The rheological characteristics of the digestive fluids in the GI tract may play an important role in determining the rate and extent of lipid digestion.61,113 For instance, their rheology may influence the flow profile and mechanical forces generated within the GI tract, or the mass transport of components through them. The rheology of digestive juices may range from low viscosity liquids, to visco-elastic fluids, to gel-like materials depending on their composition and environmental conditions. Consequently, it is often important to be able to characterize the rheological characteristics of the system. The rheology may be measured using a number of different analytical instruments, such as viscometers, dynamic 46 | Food Funct., 2010, 1, 32–59

Fig. 4 (a) Schematic of an in vitro digestion model used to determine the digestion and release of lipids encapsulated within nano-laminated lipid droplets. The picture of the pH-stat titrator is kindly donated by Metrohm USA, Inc. A triacylglycerol (TAG) is converted into two free fatty acids (FFA) and one monoacylglycerol (MAG) by lipase. (b) Schematic diagram of the three phases typically formed after centrifugation of the material remaining after digestion in simulated small intestine conditions.

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into three different layers (Fig. 4b): a pellet at the bottom, an aqueous phase in the middle, and an oil phase at the top.158 The pellet typically contains dense insoluble matter, such as aggregates containing undigested proteins, dietary fibers, bile salts, free fatty acids, and minerals. For example, insoluble calcium soaps of free fatty acids are usually present in this layer. The aqueous phase typically contains mixed micelles and vesicles that contain bile salts, phospholipids, and lipid digestion products (MAG and FFA). In addition, any lipophilic agents that were originally encapsulated in the lipid droplets may be solubilized within the hydrophobic interiors of these micelles and vesicles, e.g., carotenoids. The cream layer contains any undigested lipid in the form of oil droplets or bulk oil. After centrifugation the different layers present can be collected separately and then analyzed to determine their composition, e.g., using chemical, spectroscopic, electrophoresis, or chromatographic methods. This approach can be used to determine the amount of lipophilic components released from the lipid droplets after digestion, which is important when determining the bioavailability of encapsulated substances.120,159 5.4.9. Absorption of lipophilic components. The absorption of lipophilic components (digestion products or encapsulated agents) by epithelium cells can be simulated in vitro using various physical, biological, or cell-culture models: Physical methods. For some types of lipid it is assumed that the amount of digestion products released from the sample is representative of the amount of digestion products absorbed by the epithelium cells, i.e., the rate limiting step is lipid digestion, rather than absorption. The amount of lipid digestion products released can be determined by measuring their concentration in the aqueous phase, which can be measured directly or after they have been separated from the rest of the material by centrifugation, filtration, or dialysis.160,161 For example, the free fatty acids released from emulsified lipid droplets through the action of pancreatic lipase can simply be measured by acid titration or enzymatic techniques.86,162,163 The concentration of specific bioactive lipids solubilized by mixed micelles/vesicles can be determined using a suitable analytical method, usually after the middle (micelle) phase has been separated from the cream layer and pellet by centrifugation.164–166 Ex vivo permeation methods. A section of the GI tract is cut from an animal after it has been sacrificed, and is then washed to remove any residual components. Part of the intestine is then clamped between two chambers: one of the chambers contains the sample to be analyzed (donor chamber), while the other chamber contains only buffer solution (receiver chamber).167 The transport of the samples across the chamber is then measured over time using suitable analytical methods. Alternatively, the sample to be tested can be placed inside an intact section of GI, which is then placed in an appropriate buffered solution. The amount of material that moves across the intestinal walls and into the surrounding buffer solution can then measured using an appropriate analytical method. Cell culture methods. Caco-2 cells are cell cultures that mimic the human intestinal epithelium.168–170 They can be used in two This journal is ª The Royal Society of Chemistry 2010

different ways to assess the absorption of lipid digestion products: (i) after the digested food material has been left in contact with a membrane coated with Caco-2 cells, the amount of lipid digestion products that pass through the coated membrane can be measured; (ii) the amount of lipid digestion products that are absorbed by the cells themselves can be determined. A suitable analytical technique can then be used to measure the location or quantify the amount of material absorbed, e.g., microscopy, chromatography, spectrometry or chemical methods.

6. pH-stat method 6.1.

Principle of the pH-stat method

The pH-stat method is an analytical tool widely used in pharmaceutical and food research for the in vitro characterization of lipid digestion under simulated small intestinal conditions.120,122,125,171 It is based on measurements of the amount of free fatty acids released from lipids, usually triacylglcyerols, after lipase addition at pH values close to neutral. The sample to be analyzed is placed within a temperature-controlled reaction chamber that contains simulated small intestinal fluid (SSIF) (Fig. 4a). The SSIF should contain appropriate levels of the major digestive components known to influence lipid digestion, such as lipase, co-lipase, bile salts, phospholipids, and mineral ions (Table 2). It is usually assumed that the lipase in the SSIF catalyzes lipid digestion leading to the generation of two FFAs and one MAG per TAG molecule, although further digestion can occur in some situations. The concentration of alkali (NaOH) that must then be added to the digestion cell to neutralize the FFAs produced by lipid digestion, and thereby maintain the pH at the initial pre-set value (e.g., pH 7.0), is recorded versus time (Fig. 4a). The pH-stat method is relatively simple and rapid to carry out and enables comparison of different systems under similar experimental conditions. This technique can therefore be used to rapidly screen the impact of different physicochemical factors expected to affect lipid digestion. Recently, a simple mathematical model was developed to describe the FFA versus time profiles obtained by the pH stat method.172 The percentage of total free fatty acids released (F) as a function of time (t) measured by the pH-stat method is characterized by the following equation:  2 ! 3kMt F ¼ fmax 1  1 þ (1) 2d0 r0 Here, fmax provides a measure of the total extent of digestion (i.e., the maximum percentage of the total FFA present that is released at the end of the reaction), k provides a measure of the rate of digestion (i.e., mmols of FFA released per unit droplet surface area per unit time), d0 is the initial droplet diameter, r0 is the oil droplet density, and M is the molar mass of the oil. A pHstat profile can then be characterized in terms of just two parameters: fmax and k, which can be determined by finding the values which give the best fit between the experimental data and the mathematical model. It should be noted that the pH stat method only simulates the small intestine, and it is usually necessary to simulate the other parts of the digestive tract to get more realistic results. For Food Funct., 2010, 1, 32–59 | 47

48 | Food Funct., 2010, 1, 32–59

O/W Emulsions

Oil SNEDDS O/W Emulsions

O/W Emulsions O/W Emulsions O/W Emulsions Oil suspension

O/W Emulsions

Oil suspension O/W Emulsions

Bile Ca2+ Lipase activity Interfacial composition Influence of dietary fiber Pancreatic lipase activity pH Bile Pancreatic lipase Lipid type Lipolysis time Lipase Bile Lipolysis time Lipid composition

Emulsifier type Ions pH Surfactant/Glycerides Triglycerides Emulsifier type

1000 TBU/mL 800 TBU/mL 0–10 mg/mL 200 tributyrin units/mL

10.5 mg/mL

2 nM (10 nM colipase) 0.4 mg/mL 1 mg/mL (Lipase:colipase ¼ 1 : 5) 0–4.8 mg/mL

0.2% 0.2% 0.2% 0.039 mg/mL 25 mg/mL 0.3% 10%

270–1340 units/mL

1000 IU/mL 2.9 mg/mL

50 mg/mL 0.4% 30.6 mM

2.5 mg/mL

1.6 mg/mL

Lipase

0.5%

0.4%

[CaCl2] [EDTA] [Bile]

O/W Emulsions O ¼ corn oil E ¼ BLG, lecithin, or caseinate

SEDDS

Lipid

Experimental Variables

Samples Tested

5 mM (1.25 mM phosphatidylcholine)

5/20 mM 5 mM (1 mM phosphatidylcholine) 0–25 mg/mL

9.7 mM 2.4 mg/mL 0–8 mM (0.74 mM phospholipid) 0–20 mg/mL

4–16 mM

5 mM 4.2 mg/mL

5 mM (1.25 mM lecithin)

0–5 mg/mL

Bile

5 mM

5 mM 0.045 mM/min 30 mM

0–30 mM

10 mM

4–20 mM

5 mM

5–20 mM

0–20 mM

Calcium

35 min

30 min 30 min 2h

0–2 h 2h 0–3 h 2h

2–24 h 5 min 2h 2h 40 min

20 min

20 min

Digestion times

7.5

7.5 6.5 7.5

7.0 7.5 7.5 6.5

6.5

6.8 6.5–7.0

6.5

7.0

pH

225

124,212 141 224

223 195,210 211 166

83

167 192

175

84

References

Table 2 Summary of the simulated small intestinal fluid compositions used in previous pH-stat studies of the digestion of emulsified lipids. All experiments were carried out at 37  C unless stated

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example, a sample could be passed through a simulated mouth and stomach model, and then analyzed using the pH stat method.

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6.2.

Influence of SSIF composition on the pH-stat method

One of the most important factors affecting the digestion rate determined using the pH-stat method is the composition of the simulated small intestinal fluid (SSIF) used.2 Previous researchers have used various SSIF compositions when carrying out in vitro lipid digestion studies using the pH stat method (Table 2). In addition, a number of workers have examined the influence of specific SSIF components on the rate and extent of lipid digestion. Ideally, the conditions used should closely simulate those found in the human GI tract (Section 2), and the digestion results obtained should correlate closely with those obtained using in vivo studies. The role of the major components within SSIF is highlighted below: 6.2.1. Lipase and other enzymes. Pancreatic lipase is the key component in any in vitro model designed to simulate lipid digestion within the small intestine. Consequently, it is important to use an appropriate type and concentration of pancreatic lipase in the pH-stat method. Previous researchers have used various types and forms of lipase in SSIF, including pancreatin, pancreatic lipases, and non-pancreatic lipases. Pancreatin is a complex mixture of digestive enzymes (lipase, protease, amylase, etc.) and other biological components produced by the exocrine cells of the pancreas, which is typically obtained from animal sources, such as pigs or cows. The chemical composition and functional performance of pancreatin (and consequently its enzyme activity) varies depending on its biological origin, isolation, and purification procedures, and hence there are often considerable batch-to-batch and supplier-to-supplier variations.173 Purified pancreatic lipases are also available commercially that have been isolated from various animal and human sources, although these are more expensive that using pancreatin. In addition, it is important to use an appropriate amount of colipase with pancreatic lipase to ensure its optimum performance. The advantage of using pancreatic lipases is that they are chemically more well-defined, and there is less batch-to-batch variation. Some researchers have used non-pancreatic lipase sources for their in vitro lipid digestion studies, e.g., Candida rugosa, Candida cylindracea, Rhizopus niveus, and Mucor meihei.174 These lipases have the advantage of been highly pure and reproducible, and less expensive than pancreatic lipase. However, they may not accurately mimic the behavior of pancreatic lipase in the lipid digestion process. The catalytic activity of lipase depends on its origin and history. For example, the catalytic activity of an enzyme ingredient may decrease if it is exposed to excessively high temperatures or if it is stored too long. Hence, researchers should be careful not to subject lipase ingredients to temperature abuse, and should prepare fresh lipase preparations for each experiment. The catalytic activity of lipase also depends on solution and environmental conditions, such as temperature, pH, ionic strength, and the presence of chemical denaturants.71,175–177 These conditions should therefore be standardized in any in vitro digestion test. It is therefore recommended that each batch of This journal is ª The Royal Society of Chemistry 2010

lipase should be standardized prior to utilization.120 A commonly used method for determining lipase activity is to measure the amount of free fatty acids released from a fixed amount of a standardized emulsified lipid (e.g., triolein or tributyrin) after a specific digestion time under standardized conditions (pH, ionic strength, temperature).120,167 Nevertheless, the rate of lipid digestion has previously been shown to depend on emulsifier type and droplet size distribution,130,131 and so these parameters should also be standardized. At present there is no consensus on the best values to use for these parameters. We propose using lyso-lecithin as a standard emulsifier since it can form stable emulsions and is readily available from chemical suppliers, and using lipid droplets with a standard mean diameter (d32) around 2 mm since these can be produced using simple high shear mixers and are fairly stable over the experimental time scales involved. The concentration of pancreatic lipase in the human small intestine depends on many factors, including the individual, health status, age, time of day, and type and amount of food consumed.15,63 It is therefore difficult to recommend one definitive lipase level to use in an in vitro test method. Ideally, one should examine a range of lipase concentrations that encompasses those typically found in the human GI tract. However, it is usually assumed that humans have a great excess of lipase in the GI tract, and so it is advisable to use a relatively high level of lipase in in vitro studies, e.g., equivalent to 2.4 mg/mL pancreatin. It should also be noted that there may be various other enzymes present in the small intestine that may directly or indirectly alter the rate of lipid digestion. If lipid droplets are coated by digestible emulsifiers (such as proteins, phospholipids, and some surfactants) or if they are embedded within gel-like or solid particles (such as proteins, or polysaccharides), then the digestion of these components may be important. For example, if lipid droplets are coated by a layer of adsorbed protein molecules, then this layer may have to be digested by proteases before the lipase can reach the triacylglycerols.93 Similarly, if lipid droplets are embedded within a protein hydrogel particle, then it may be necessary for the protein matrix to be fully or partially digested by proteases before the lipase can reach the triacylglycerols.13 Some of these other digestive enzymes are naturally present in pancreatin, e.g., proteases and amylases. On the other hand, it may be necessary to add some or all of these enzymes if pure pancreatic lipase or non-pancreatic lipase is used. For samples containing lipids encapsulated within other digestible components it would be informative to run experiments with and without non-lipase digestive enzymes to determine their influence on the overall lipid digestion rate. An example of the influence of lipase concentration on the rate and extent of lipid digestion of b-lactoglobulin stabilized corn oil-in-water emulsions measured using the pH stat method is shown in Fig. 5a. The rate and extent of lipid digestion increased as the lipase concentration in the reaction vessel increased. At low lipase levels (#0.2 mg/mL), FFAs were released slowly and most of the lipids within the droplets remained undigested after 30 min (<35% FFA released). At intermediate lipase levels (e.g., 0.4 and 0.8 mg/mL), there was an initial period from 0 to 10 min when FFAs were released slowly, followed by another period when the rate of FFA release increased appreciably. At high lipase levels ($2.4 mg/mL), the amount of FFA released increased rapidly with time almost immediately after digestion Food Funct., 2010, 1, 32–59 | 49

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started, and then leveled off at longer times because all the lipids within the droplets had been fully digested. One would expect the rate of lipid digestion to increase as the lipase concentration increased because there would be more total enzyme present in the system to catalyze the conversion of triacylglycerols to free fatty acids.71 In addition, the amount of lipase present at the oil– water interface, where the lipolysis reaction occurs, will increase as the lipase concentration increases.71 Lipase is a surface-active protein that can compete for the oil–water interface with other surface-active components,139 such as the b-lactoglobulin initially coating the lipid droplets or the bile salts added to the reaction vessel. At low lipase concentrations, there may be insufficient lipase present to displace b-lactoglobulin and/or bile from the oil–water interface, and so the enzyme cannot come into close contact with the lipid substrate within the droplets. The observation of an initial slow rate of FFA release at intermediate lipase concentrations may be explained by the finite time required for lipase to adsorb to the lipid droplet surfaces and displace the b-Lg and/or bile coating so as to get access to the triacylglyercols within the droplet core.178 Presumably at higher lipase concentrations the adsorption and displacement processes occur rapidly so that digestion could begin almost immediately after the digestive enzyme was added to the reaction vessel.

Fig. 5 (a) Influence of lipase concentration in the pH-stat reaction vessel on the rate and extent of lipid digestion determined by monitoring the free fatty acids (FFA) released over time (adapted from Li et al. 2010). (b) Influence of bile salt concentration in the pH-stat reaction vessel on the rate and extent of lipid digestion determined by monitoring the free fatty acids (FFA) released over time (adapted from Li et al. 2010). (c) Influence of calcium concentration in the pH-stat reaction vessel on the rate and extent of lipid digestion determined by monitoring the free fatty acids (FFA) released over time (adapted from Li et al. 2010).

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6.2.2. Bile. Bile is another key component in any SSIF used to simulate lipid digestion in the small intestine.2 Typically, researchers use either bile extract or one or more individual bile acids in their simulated small intestinal fluids. Bile extract is a complex mixture of various kinds of molecules typically found in the GI tract (such as bile acids, phospholipids, and minerals), and may therefore more accurately reflect in vivo conditions than individual bile acids, however it tends to be more variable and inconsistent in composition and performance. In addition, bile extract often contains insoluble matter, which can interfere with the analysis of lipid droplets and other colloidal structures in digestion media, and therefore it should be filtered before use. Individual bile acids can be purchased in pure form, which often facilitates the design and interpretation of experimental digestion measurements. On the other hand, using purified bile acids may be less representative of the complex composition of actual small intestinal fluids. For this reason, researchers often utilize a mixture of bile acids and other biological components (such as phospholipids) to reflect the bile composition in the human GI tract. Typically, bile contains four major types of bile acids (cholic, deoxycholic, chenodeoxycholic, and lithocholic acids), as well as their glycine and taurine derivatives.51 Some researchers have tried to simplify the complexity of bile composition by only using the major bile acid components. For example, in a recent study a mixture of sodium taurocholate (NaTC, 52.7%) and sodium glycodeoxycholate (NaGDC, 47.3%) was used to mimic human bile.95,179 An example of the influence of bile concentration on lipid digestion in b-lactoglobulin stabilized corn oil-in-water emulsions measured using the pH stat method is shown in Fig. 5b.180 The rate and extent of FFA release decreased as the concentration of bile extract in the reaction vessel increased from 0 to 20 mg/mL. The suppression of lipid digestion by bile salts is well established in the literature, where it has been attributed to the ability of surface-active bile salts to displace lipase from the This journal is ª The Royal Society of Chemistry 2010

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oil–water interface thereby preventing it from coming into close contact with the lipid substrate.63,71,74,181 Addition of co-lipase has been shown to reverse this effect and increase lipase activity.63 The data shown in Fig. 5b was obtained using an isolated porcine lipase, rather than a crude pancreatin extract containing both lipase and co-lipase, which accounts for the observed decrease in lipase activity with increasing bile salt concentration. In the same study, the ability of bile salts to displace proteins from droplet surfaces was demonstrated using z-potential measurements. 6.2.3. Calcium and other minerals. A number of in vitro digestion studies have highlighted the important role that calcium plays in determining the rate and extent of lipid digestion using the pH-stat method.83,84,182 Calcium ions may impact the lipid digestion process due to a number of different physicochemical mechanisms.  Enzyme activity: Calcium is believed to be a necessary cofactor for the proper functioning of pancreatic lipase62,183–185  Removal of FFA from droplet surfaces: Lipase digestion of emulsified lipids can be inhibited by accumulation of long-chain fatty acids (LCFA) at the droplet surfaces, since this restricts the access of the lipase to the triacylglycerols.15 Calcium is known to precipitate these LCFA, thereby removing them from the lipid droplet surface and allowing the lipase to access the emulsified lipids.98,99 Consequently, calcium ions are able to increase the rate and extent of lipolysis by this mechanism.83,125,182,186  Reduced digestibility of flocculated droplets: Calcium ions are highly effective at causing droplet flocculation in emulsions containing lipid droplets coated with anionic emulsifiers. It is more difficult for the lipase molecules to reach the surfaces of the lipid droplets trapped in the center of a floc, which may slow down the lipid digestion rate in flocculated emulsions.84  Reduced digestibility due to gel formation: Calcium ions may promote the gelation of certain types of biopolymers (such as alginate and pectin), which can lead to the formation of hydrogel matrices that trap lipid droplets and inhibit the diffusion of lipase to the droplet surfaces.  Reduced bioavailability of precipitated FFA: Studies have shown that insoluble soaps are formed between calcium ions and LCFA, which can reduce the bioavailability of these fatty acid digestion products.70,187,188 It is therefore critical to use an appropriate level of calcium in the SSIF used in pH-stat studies. A certain level of calcium is naturally present in human digestive juices and additional amounts may also arise from ingested foods, particularly those having have levels of this mineral.83 Typically, the calcium levels in the human small intestine have been reported to be in the range 5 to 30 mM as reflected in the levels used in most pH-stat digestion models (Table 2). The importance of calcium levels on lipid digestion in corn oilin-water emulsions stabilized by b-lactoglobulin was recently studied in our laboratory.180 The lipid digestion process measured by the pH-stat clearly depended on the level of calcium ions present in the SSIF. The rate and extent of FFA production increased when the calcium level increased from 0 to 10 mM, but decreased when 20 mM calcium was added (Fig. 5c). Two different digestion regimes could be distinguished at 20 mM calcium: Regime I - an initial period from 0 to 30 min when This journal is ª The Royal Society of Chemistry 2010

digestion was relatively slow; Regime II – a later period when the digestion rate increased appreciably. The increase in digestion rate with increasing calcium in Regime I can be attributed to the ability of Ca2+ ions to precipitate LCFA digestion products that tend to accumulate at the oil–water interface and inhibit lipase activity.83,125 The dramatic suppression of FFA production observed in Regime II suggests that the ability of lipase to access the lipid droplet surfaces was restricted, presumably because the high calcium levels promoted extensive droplet flocculation. The concentration of free calcium ions in the small intestine will depend on the presence of any other components that can bind calcium. These components may be naturally present within the human body such as mucins or specific proteins, or they may be present within ingested foods such as chelating agents (EDTA, phosphates) and biopolymers (proteins, peptides and polysaccharides).85,189–191 Recent studies have shown that food components that bind calcium strongly, such as EDTA and alginate, are able to greatly reduce the lipase activity in oil-inwater emulsions.84 This may be important when designing and interpreting pH-stat measurements of lipid digestion in complex food systems. Other mineral ions, such as sodium, potassium, sulfates, phosphates and bicarbonates, may also be present in the digestive fluids in a human’s small intestine.51 These minerals may play an important role in the digestion process since they can affect the magnitude and range of any electrostatic interactions in the system, which may alter the physicochemical properties, solubility, and aggregation state of various components within the system. It is therefore important to use mineral levels that reflect those found in vivo. Typically, a single monovalent salt (such as NaCl or KCl) is used to create an ionic strength that mimics physiological levels (z 150 mM),120 although some studies have used more complex mixtures.192 6.2.4. pH. The pH of the small intestine depends on a number of factors and typically varies from one location to another.193 In the stomach, the droplets are surrounded by a highly acidic environment (pH 1 to 3), but when they enter the duodenum the pH is increased to around neutral (pH 5.8–6.5) due to secretion of sodium bicarbonate.63 Nevertheless, studies with human subjects have shown that there may be large variations in duodenum pH depending on the individual involved, and the type and amount of food consumed.44 In particular, the pH of the stomach contents may increase appreciably after ingestion of a food, before gradually returning to the fasting pH level. As the food passes along the small intestine the pH tends to increase to around pH 7 to 7.5. The pH used in an in vitro digestion model may influence the results for a number of different reasons:  Enzyme activity: Pancreatic lipase has an optimum pH where it exhibits its maximum rate of lipid digestion.175 It has been reported that pancreatic lipase has a maximum activity around pH 8.5,63 but that it also has good activity at pH values around neutral.175  FFA ionization: The ionization of free fatty acids depends on solution pH relative to their pKa value.174,194 In aqueous solution, the ‘‘true’’ pKa of FFAs has been reported to be around 4.7 to 4.9 depending on their chain length, and so they should be predominantly in their ionized anionic form around neutral pH.174 However, in the presence of lipid droplets the ‘‘apparent’’ Food Funct., 2010, 1, 32–59 | 51

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pKa of free fatty acids may be considerably higher than their ‘‘true’’ values due to partitioning effects. The ionized form of FFA tends to partition between the oil and water phases, but the non-ionized form tends to accumulate predominantly in the lipid phase. Consequently, the tendency for any FFA formed at the droplet surfaces to move into the surrounding aqueous phase will depend on the pH relative to the pKa value. The apparent pKa of free fatty acids has been reported to increase with increasing hydrocarbon chain length and with increasing fat content, with values of pKa ¼ 11.2 being reported for lauric acid in a 50% fat system.174 This effect may therefore be important in systems where long chain triacylglycerols are used as the lipid phase and the lipid content is relatively high.  Ingredient properties: The electrical charge, interactions, aggregation, and solubility of many other ionic species may also depend on solution pH. For example, the stability of emulsified lipids to droplet aggregation often depends on pH. Consequently, the rate and extent of lipid digestion will depend on the pH in the reaction vessel, which should be taken into account when comparing measurements on different systems. 6.2.5. Other factors. Various other factors should also be considered when designing an appropriate simulated small intestinal fluid for pH-stat studies. Lipids are normally consumed by humans in a diet that contains a range of other components, including dietary fibers, proteins, carbohydrates, minerals. Many of these components may interfere with the digestion process, either decreasing or increasing the rate and extent of digestion. For example, dietary fibers may interact with the lipid digestion process through a variety of physicochemical mechanisms depending on their molecular characteristics.195 They may bind to components within the SSIF, such as calcium, bile salts or lipase, thereby altering their activity. They may form protective coatings around lipid droplets, or they may promote droplet flocculation, which alters the ability of the lipase molecules to access the lipids. Some food components (e.g., polyphenols) may be able to bind to digestive enzymes and reduce their activity.196 6.3. Proposed standardized pH-stat method for testing emulsified lipids It would be useful to have standardized test conditions for in vitro lipid digestion models so that the results from one study can Table 3 Proposed standardized pH-stat method for testing emulsified lipids using the in vitro digestion model under fed state conditions Experimental Parameter

Proposed Value

pH Reaction Cell Volume Temperature Stirring speed [NaOH] in titration unit Lipid content in reaction cell NaCl in reaction cell CaCl2 in reaction cell Bile Extract in reaction cell Pancreatin (100–400 units/mg protein) in reaction cell

7 37.5 mL 37  C 4 s1 0.1 mM 300 mg 150 mM 10 mM 20 mg/mL 2.4 mg/mL

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easily be compared to those of another within and between laboratories. Ideally, this model should give a measure of lipid digestibility that correlates well with that found for similar systems passing through the human digestive tract. In addition, it would be useful for the test procedure to be relatively inexpensive and easy to implement so that it can be adopted by many different laboratories. The pH-stat method is relatively straightforward to set up and carry out, only requiring an automatic titration unit capable of controlling pH.120 Based on the results of ours and others work we propose the experimental conditions outlined in Table 3 as a basis for a standardized pHstat digestion model. This model is certainly too simplistic to accurately reflect the complex physiological and physicochemical processes that occur in the human gastrointestinal tract, but it does contain the major factors expected to impact lipid digestion and it is relatively simple to implement. Ideally, the rate of FFA release should fall within a reasonably rapid timeframe (e.g., 30 min) so that multiple samples can be conveniently screened. The above conditions should lead to a release rate that falls within this timeframe. Nevertheless, there are often variations in the activity of the lipase within pancreatin from batch-to-batch or during storage, which means that it may be necessary to adjust its concentration so as to obtain a FFA release profile in the appropriate timeframe. The mean particle diameter and emulsifier used in an experiment should always be reported, since the rate of FFA release in the pH Stat method depends on both of these parameters. The effect of particle size may be taken into account by using the mathematical model described above to calculate the FFA release per unit time per unit surface area. 6.4.

Application of pH-stat method

In this section, we demonstrate the usefulness of the pH-stat method using some recent data obtained from our laboratory. 6.4.1. Influence of lipid droplet composition. The composition of the lipid phase present within food and beverage emulsions varies depending on the nature of the product.27 Studies in the pharmaceutical area indicate that lipid type can have a major impact on lipid digestion and absorption.3,121,197 Consequently, it is useful to study the impact of lipid type on the digestion of oilin-water emulsions used in the food industry. We recently used the pH-stat method to study the influence of lipid type on the rate and extent of lipid digestion (Fig. 6a). Oil-in-water emulsions stabilized by b-Lg were prepared with similar droplet sizes and concentrations using either corn oil (long chain triglyceride, LCT) or medium chain triglyceride (MCT) as the lipid phase.172 The rate and extent of lipid digestion was clearly higher when MCT was used than when LCT was used. This effect can be attributed to the fact that the medium chain FFA digestion products arising from MCT have a higher dispersibility in aqueous media than the long chain FFA digestion products arising from LCT.3,121 The medium chain FFAs are able to migrate rapidly away from the droplet surfaces and into the surrounding aqueous phase, and so they do not inhibit the interfacial lipase reaction. On the other hand, the long chain FFAs tend to accumulate at the oil–water interface and inhibit lipase activity until they are removed by being solubilized in This journal is ª The Royal Society of Chemistry 2010

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micelles or precipitated by calcium ions. Similar results have been reported by a number of other researchers using the pH stat method.160,167

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6.4.2. Influence of lipid droplet size. The initial size of the droplets in food and beverage emulsions can be controlled by controlling system composition and homogenization conditions.27 As the droplets pass through the GI tract their size may change due to fragmentation, coalescence, flocculation, or digestion processes (Section 3). The size of the oil droplets within the small intestine should impact their digestion rate since the surface area of lipid exposed to the surrounding aqueous environment is inversely related to the mean droplet diameter.4,27,63,198 Recently, it has been shown that lipid droplet size influences stomach motility and the release of gut hormones, which has important consequences for designing foods to combat obesity.22 It is therefore important to establish the influence of droplet size on the rate and extent of lipid digestion. We recently used the pH stat method to monitor lipid digestion of MCT oil-in-water emulsions stabilized by b-Lg with different mean droplet diameters: d ¼ 195 nm or 14,700 nm (Fig. 6b).172 The rate of FFA released per unit time (FFA% s1) was appreciably higher for the emulsion with the smaller droplets, which should be expected because it has a bigger specific surface area for the lipase molecules to bind.27 This result is consistent with earlier in vitro digestion studies that also found the rate of lipid hydrolysis (expressed per unit time) increased with decreasing droplet size.125,198 On the other hand, when the digestion rate was normalized to the droplet surface area it actually increased with increasing droplet size: k ¼ 0.65 and 3.93 mmol s1 m2 for the small and large droplets, respectively. The overall concentration of lipase within all of the emulsions was the same and so the increase in k with increasing d32 may have occurred because the amount of lipase available per unit droplet surface area increased as the droplet size decreased. Thus, there may have been more lipase molecules adsorbed per unit surface area in the emulsions containing the large oil droplets than those containing the smaller ones.71

Fig. 6 (a) Influence of lipid type (corn oil versus medium chain triglycerides) on the rate and extent of lipid digestion determined by monitoring the free fatty acids (FFA) released over time using the pH stat method (adapted from Li and McClements 2010). (b) Influence of initial mean droplet diameter on the rate and extent of lipid digestion determined by monitoring the free fatty acids (FFA) released over time using the pH stat method (Li et al. 2010). (c) Influence of initial mean droplet diameter on the rate and extent of lipid digestion determined by monitoring the free

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6.4.4. Influence of lipid droplet encapsulation. Encapsulating lipid droplets within hydrogel particles has been suggested as a means of controlling their digestibility.4,129 In these systems the rate of lipid digestion depends on how quickly the lipase in the aqueous phase can access the surfaces of the encapsulated lipids. The rate of lipid digestion can be controlled in a number of ways using this approach, e.g., controlling the permeability of the hydrogel matrix, controlling the dimensions of the hydrogel particles, or controlling the fragmentation or dissociation of the hydrogel particles in the digestive fluids. The influence of encapsulating lipid droplets within hydrogel particles (calcium alginate beads) on the rate and extent of lipid digestion is shown in Fig. 6c. Three samples containing the sample total amount of lipid (MCT) but with different microstructures were prepared: (i) non-encapsulated lipid droplets; (ii) non-encapsulated lipid droplets mixed with unfilled calcium fatty acids (FFA) released over time using the pH stat method (Li et al. 2010).

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alginate beads; and, (iii) lipid droplets encapsulated inside calcium alginate beads. The non-encapsulated (free) lipid droplets were completely digested within the first 25 min of hydrolysis, with the amount of FFA released increasing steeply during the first 5 min and then leveling off at longer digestion times (Fig. 6c). On the other hand, the lipid droplets encapsulated within the calcium alginate beads were digested at a much slower rate, with <8% FFA being released within the first 25 min of digestion. The ability of the calcium alginate beads to retard digestion was attributed to a number of physicochemical phenomena: (i) lipase/co-lipase molecules have to diffuse through the hydrogel matrix before they can adsorb to the lipid droplet surfaces; (ii) FFA and MAG have to diffuse out of the hydrogel particles otherwise they will retard the hydrolysis reaction.15,63 Interestingly, when non-encapsulated (free) oil droplets were mixed with the same type and concentration of unfilled calcium alginate beads, there was no inhibition of lipolysis (Fig. 6c). This demonstrated that the beads themselves were not responsible for retarding the lipid digestion process (e.g., by trapping or binding the lipase or bile). Instead, the delayed digestion rate appears to be due to the fact that the droplets are trapped within the gel network. 6.4.4. Other factors. A number of other factors have been studied recently using the pH stat method, which are summarized below: Emulsifier type. Some studies have shown that emulsifier type has a major impact on the rate of lipid digestion, whereas others have found little effect. Wickham and co-workers found that lipid droplets initially coated by phospholipids were digested more rapidly than those coated by proteins.199 Chu and coworkers found that the rate of lipolysis of lipid droplets could be inhibited by coating them with galactolipids with large hydrophilic head-groups that retard adsorption of bile salts and lipase through steric hindrance.179 Reis and co-workers found that the rate of lipid digestion was lower for droplets initially coated by monoglycerides than those coated by proteins or phospholipids.200 Our laboratory previously found that the resistance of lipid droplets stabilized by different emulsifiers to lipid digestion decreased in the following order: non-ionic surfactant (Tween 20) > phospholipids (lecithin) > protein (caseinate or WPI).131 Nevertheless, in a recent study we found that the rate of lipid digestion was fairly similar for MCT oil-in-water emulsions stabilized by four different types of emulsifier: b-Lg, Tween 20, lecithin and lyso-lecithin.172 Dietary fiber coatings. It has been proposed that the rate of lipid digestion can be controlled by coating lipid droplets with one or more layers of dietary fiber.201 The rationale behind this proposal is the fact that dietary fibers should not be digested in the stomach or small intestine, and therefore they may prevent lipase from adsorbing to lipid droplet surfaces (provided the dietary fibers are not displaced from the droplet surfaces and that the layers formed are impermeable to lipase diffusion). The pH stat method has recently been used to study the impact of dietary fiber type, number of layers, sequence of layers, and layer crosslinking on the rate and extent of lipid digestion.72,86,201,202 These studies found that the lipid digestion rate could be decreased by 54 | Food Funct., 2010, 1, 32–59

depositing one or more dietary fiber layers around the lipid droplets, but that the droplets were still digested eventually. Dietary fiber coatings may therefore be a useful means of retarding lipid digestion in the human GI tract, which could be utilized for the development of functional foods to promote satiety. Droplet physical state. A number of in vitro studies have shown that the physical state of lipid droplets can influence their digestibility by pancreatic lipase.153,154,203,204 These studies found that solid lipid particles were digested by lipase, but at a slower rate than liquid lipid droplets. Recently, we carried out a study using two emulsions with the same lipid type (tripalmitin), but with one containing lipid droplets that were completely liquid and the other containing lipid particles that were completely solid.155 We found that the rate and extent of lipid digestion were greater in the emulsions containing liquid droplets, but that lipid digestion still occurred in the systems containing solid particles. Other researchers have shown that fat crystallization within lipid droplets may also indirectly influence the rate of lipid digestion.52 In this case, emulsions were designed so that they contained partly crystalline droplets that underwent partial coalescence in the small intestine.27,205 The partially coalesced emulsions exhibited slower digestion than stable emulsions, which was attributed to the fact that it was more difficult for the lipase molecules to reach the lipid droplet surfaces within the large clumps of fat droplets in the unstable system. Ingredient interactions. Foods and beverages generally have much more complicated compositions than the simple model systems used in in vitro digestion studies. A number of the components typically found in foods may impact lipid digestion. For example, we recently examined the influence of calcium binding agents (EDTA and alginate) on the rate and extent of lipid digestion.84 We found that both EDTA and alginate could greatly suppress the digestion of triacylglycerols containing long chain fatty acids, which was attributed to their ability to bind free calcium ions. Consequently, there would have been an accumulation of LCFA at the lipid droplet surfaces, which would have inhibited lipase activity.

7. Conclusions There has been growing interest by food scientists in understanding and controlling the digestion of lipids within the human gastrointestinal tract. The main driving forces for this interest are the development of emulsion-based delivery systems to encapsulate, protect and release lipophilic bioactive components within the GI tract, and the possibility of modulating human hunger and appetite by controlling the location and rate of lipid digestion within the GI tract. This article provides an overview of the major physicochemical events that occur during lipid digestion, and reviews a number of in vitro testing methods that have been developed to monitor lipid digestion. In particular, we focused on the pH stat method for simulating lipid digestion in the small intestine, since this method is useful as a rapid screening tool for studying the influence of product composition and structure on lipid digestibility. Considerable advances have been made in this area throughout the past decade, which is providing This journal is ª The Royal Society of Chemistry 2010

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the fundamental knowledge required to rationally develop new food-based strategies to tackle food related diseases, such as heart disease, diabetes, cancer, and hypertension.

Acknowledgements This material is partly based upon work supported by United States Department of Agriculture, CREES, NRI Grants.

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Combining nutrition, food science and engineering in developing solutions to Inflammatory bowel diseases – omega-3 polyunsaturated fatty acids as an example Lynnette R. Ferguson,*ad Bronwen G. Smithbd and Bryony J. Jamesc

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Received 2nd July 2010, Accepted 13th August 2010 DOI: 10.1039/c0fo00057d The Inflammatory bowel diseases, Crohn’s disease and ulcerative colitis, are debilitating conditions, characterised by lifelong sensitivity to certain foods, and often a need for surgery and life-long medication. The anti-inflammatory effects of long chain omega-3 polyunsaturated acids justify their inclusion in enteral nutrition formulas that have been associated with disease remission. However, there have been variable data in clinical trials to test supplementary omega-3 polyunsaturated fatty acids in inducing or maintaining remission in these diseases. Although variability in trial design has been suggested as a major factor, we suggest that variability in processing and presentation of the products may be equally or more important. The nature of the source, and rapidity of getting the fish or other food source to processing or to market, will affect the percentage of the various fatty acids, possible presence of heavy metal contaminants and oxidation status of the various fatty acids. For dietary supplements or fortified foods, whether the product is encapsulated or not, whether storage is under nitrogen or not, and length of time between harvest, processing and marketing will again profoundly affect the properties of the final product. Clinical trials to test efficacy of these products in IBD to date have utilised the relevant skills of pharmacology and gastroenterology. We suggest that knowledge from food science, nutrition and engineering will be essential to establish the true role of this important group of compounds in these diseases.

Introduction Inflammatory bowel diseases (IBD) appear in two forms, Crohn’s disease (CD) and ulcerative colitis (UC). Both are debilitating diseases, for which pharmaceutical treatments are moderately successful, albeit having significant side-effects. We have previously suggested that nutritional (or nutrigenomic) solutions may be appropriate,1,2 and there have been a number of clinical trials in this area. However, if nutritional therapies are to be successfully developed, it is essential that we move beyond pharmaceutical thinking, and put the full power of an integrated set of knowledge on effects of food science and engineering behind new developments. The potential of omega3 polyunsaturated acids (n-3 PUFA) in IBD is reviewed in this light. Fish oil is a common source of n-3 PUFA, and there is reason to believe that increasing fish oils or other sources of n-3 PUFA may have health benefits in IBD.3,4 However, their role is controversial, with variable data among different studies and between individual responses. In all of these reviews and in specific studies identified therein and below, the wide variation in dosages and formulations used, and often lack of information on a Discipline of Nutrition, FM&HS, The University of Auckland, Auckland, New Zealand b Food Science Programmes, FoS, The University of Auckland, Auckland, New Zealand c Department of Chemical and Materials Engineering, FoE, The University of Auckland, Auckland, New Zealand d Nutrigenomics New Zealand; Web: http://www.nutrigenomics.org.nz

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storage conditions and administration methods, makes it difficult to reach an informed opinion on potential benefits, or to recommend dosages for specific treatment goals. The method of presentation of the n-3 PUFA could be expected to have a direct impact on the bioavailability and efficacy of the nutrient. It has been shown that with foods high in naturally occurring n-3 PUFA the cooking method has a direct impact on the amount of EPA and DHA retained in the product.5–7 The options for including PUFA into fortified foods include direct addition of a high n-3 PUFA source such as fish oil, or incorporation of stabilised emulsions or microcapsules. The impact of subsequent food processing steps, such as cooking, is not well reported in the literature. Although many commercial products such as breads and spreads incorporate these ingredients, much of the knowledge of their preparation is proprietary. Where the impact of heating is reported,8,9 the effect on EPA and DHA levels appears to be minimal. By far the greater concern is the oxidative stability of the PUFA during storage, and antioxidants are widely incorporated into PUFA ingredients. Alternatively the n-3 PUFA is incorporated in the form of microencapsulated particles, which have improved oxidation resistance and the potential to protect the PUFA from interaction with the food matrix.10 There are a number of currently available systematic and Cochrane reviews, to effects of enteral nutrition formulas containing n-3 PUFA or of high dose supplementation with either oils or capsules of various sorts on the induction or maintenance of remission in IBD.11–15 However, sample numbers are often small and results are variable. Bassaganya-Riera and This journal is ª The Royal Society of Chemistry 2010

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Hontecillas16 concluded ‘‘there is an urgent need for placebocontrolled, large-scale, multicenter clinical trials’’. We would contend that an equally urgent need is to integrate current knowledge of food science and engineering to ensure strict definitions of sample composition, storage and delivery to optimise the likely effects of these materials, before even planning such important trials.

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Polyunsaturated fatty acids and common dietary sources The chemistry of fatty acids has been well described elsewhere17 but a few characteristics are outlined here. Fatty acids are linear hydrocarbon molecules which vary in the length of their acyl chain and bond type, and this gives rise to their nomenclature. Chains which consist entirely of single bonds are referred to as being saturated, while those with one double bond are monounsaturated, and those with two or more are described as polyunsaturated fatty acids (PUFA).18 Each chain is further characterized by the presence of a methyl group and a carboxyl group at either end of the molecule. The designation omega, u- or n- to PUFA refers to the position of the first double bond from the terminal methyl carbon Hence, n-3 PUFA have the first double bond in the third position, whereas for n-6 PUFA it is in the sixth position. Common names also exist for several. The n-3 PUFA group includes octadecatrienoic usually termed a-linolenic acid (18:3n-3), eicosopentaenoic acid (EPA) (20:5n-3) and docosahexaenoic acid (DHA) (22:6n-3). When inflammatory conditions are being considered, two n-6 PUFA should also be noted and these are linoleic acid (LA) (18:2n-6) and arachidonic acid (AA) (20:4n-6). Structures of some key compounds are illustrated on Fig. 1. Linoleic and a-linolenic acids participate in biochemical pathways. LA can undergo elongation and saturation to AA, and a-linolenic acid can be metabolized to EPA and DHA,4 although this process is less efficient in men than in women.19 AA has been associated with inflammation as this is the precursor for eicosanoids, whereas EPA and DHA have been shown to have ameliorating effects for inflammatory conditions.3 Thus, the

Fig. 1 n-3 Fatty acids. A ¼ a-linolenic acid, 18:3n-3; B ¼ eicosapentaenoic, 20:5n-3; C ¼ docosahexaenoic acid, 22:6n-3.

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relative amounts of LA and a-linolenic acid in a food have some biological relevance, but not all cell types may respond the same way. For example the infiltration of monocytes or macrophage cells in the terminal ileum of SAMP1/Yit mice was impaired by chows containing either fish oil or perilla oil, with the perilla oil being more effective. Unfortunately the complete fatty acid profile of the oils was not reported except to say that the fish oil contained 25–30% EPA and DHA, and the perilla oil was 55– 60% a-linolenic acid with overall concentration of 8% w/w n-3 PUFA.20 Oily fish and fish oil usually contain more EPA and DHA than other foods, but not all extracted oils or oily foods are created equal. Moreover, compositional data can be confusing and unintentionally misleading, if not all the information is considered.21 Fish are biological entities and their tissue composition will reflect their genetics, diet, musculature, sea temperature and global location, season and spawning cycles. Fish store oil in their liver and tissues, primarily as a reserve for gonad development. Wild fish have more freedom to roam or migrate than farmed and as a consequence may have less fatty tissue. Thus, their lipid content and profile may be very different from those of farmed fish. However, the advantage of the farmed fish is that their diet can be controlled and may include supplementation with n-3 PUFA. Nevertheless, one cannot assume that dietary intake will always result in an equivalent change in tissue composition, and the responses may not always be as expected.22 Because of concerns of depletion of fish resources, attention is shifting towards more sustainable production of long chain n-3 PUFA. An oil rich in these products can be produced from microalgae such as Micromonas pusilla,23 and a diet enriched in such an oil shows similar protective properties to one enriched in fish oil, at least in experimental models.24 Other authors (e.g.25) are seeking a sustainable, land-based production system for long chain n-3 PUFA, including metabolic engineering of an artificial pathway that produces such compounds in plants.

Evidence for n-3 PUFA playing a role in human IBD Where dietary supplements or fortified foods are advocated for use, this is usually to address a nutritional deficiency in a population group. However, there is no reason to believe that IBD patients are deficient in these essential fatty acids. For example, no significant differences were found between the long-chain n-3 PUFA status of IBD patients compared with controls, and data did not support the concept of EPA or DHA deficiency in patients with IBD.26 These observations would agree with other studies. However, there seems some reason to believe that IBD patients may show an excessive n-3:n-6 PUFA ratio. The fatty acid composition of plasma phospholipids, anthropometric characteristics, and dietary intake data were measured on 29 UC patients, 20 CD patients, and 31 healthy controls.27 The authors reported a significantly lower lipid intake in IBD patients as compared with controls, but proportionally higher levels of n-6 fatty acids, thereby implicating n-3:n-6 PUFA ratios rather than n-3 PUFA levels per se in the pathophysiology of the disease. The significance of n-3:n-6 PUFA ratios to human health have been highlighted by several authors, including Simopoulos28 and Calder;3,4. A high n-6/n-3 ratio, is found in current Western diets, although it was not a characteristic of traditional diets. There is Food Funct., 2010, 1, 60–72 | 61

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some reason to believe that this high ratio promotes the pathogenesis of many chronic diseases, in addition to IBD. A high dietary LA intake not only leads to LDL oxidation, but also interferes with the incorporation of n-3 PUFA into cell membrane phopholipids. Both n-3 and n-6 PUFA influence gene expression, but whereas n-3 PUFA have anti-inflammatory effects, reducing the expression of TNF-a, IL-1b, IL-6 and IL-8, high levels of n-6 PUFA increase the expression of pro-inflammatory genes. If n-3 PUFA do indeed have a beneficial effect in IBD, this might be expected to most effectively manifest itself in highly controlled studies such as those considering enteral nutrition. Under these circumstances, the entire diet is strictly controlled, and the formulae are unlikely to contain substances that may trigger disease symptoms. A Cochrane Database systematic review compared different enteral nutrition formulas in inducing or maintaining remission in active CD.12 While it was concluded that there was evidence for the nature and amount of fat affecting both outcomes, none of the trials included had comparable formulas with and without n-3 PUFA, or varied the ratios of n-3:n-6 PUFA. The closest was the report that compared an n-6 PUFA-containing formula with one containing monounsaturated fatty acids, and showed the latter to be superior.29 Two enteral supplements enriched with n-3 fatty acids and/or n-6 fatty acids were compared, with results suggestive of superior results for the n-3 enriched formula.30 However, remission was not an endpoint of that study, and both formulas improved clinical and biochemical markers during the course of the experiment. Even in the absence of a deficiency, n-3 PUFA dietary supplements may be beneficial because of their known antiinflammatory effects. Fig. 2 provides an illustration of some of the points at which n-3 PUFA might act to reduce chronic inflammation in IBD. A systematic review studied both published and unpublished trials on the effects of n-3 fatty acids on IBD between 1966 and 2003.4,11 The authors identified 13 controlled trials that assessed the effects on pathologically confirmed rates of induced remission or relapse, or requirements for steroids and other immunosuppressive agents in IBD. However, there was an enormous variability in considered end points across the various studies. Three studies (but only one reaching adequate statistical significance) suggested that n-3 PUFA reduced corticosteroid requirements, but there were no other consistent effects across studies. The therapeutic potential of n-3 PUFA in IBD was again reviewed.13 The authors reported that the most commonly reported adverse effects of fish oil supplements are a fishy aftertaste and gastrointestinal upsets. When recommending n-3 PUFA, the authors suggested that clinicians should be aware of any possible adverse effects. Neither review put a high emphasis on the form of the supplement and/or storage/administration conditions. Two sets of Cochrane Database systematic reviews are available on effects of n-3 PUFA supplementation on maintenance of remission in IBD. A focus on CD14 included data on patients of any age group, who were in remission at the time of recruitment, and were followed for at least six months. The intervention must have been fish oil or n-3 PUFA, given in a pre-defined dosage. The primary outcome was the rate of relapse, while secondary 62 | Food Funct., 2010, 1, 60–72

Fig. 2 Schematic representation of some of the points at which n-3 PUFA may affect the development of chronic inflammation in IBD. Long chain n-3 fatty acids (EPA and DHA) are incorporated into cell membranes where they influence the production of eicosanoids, resolvins, and cytokines. A number of n-3 PUFA act as substrates for the synthesis of eicosanoids, which in turn may directly down-regulate inflammation. This biosynthetic process may also competitively reduce the formation of eicosanoids from n-6 PUFA, which again may reduce inflammation. Long chain n-3 PUFA can also down-regulate the activation of the expression of pro-inflammatory genes, including TNF-a, Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) or various interleukins (IL), possibly through their effects on transcription factors or through their conversion to resolvins. They also have effects on T cell reactivity and antigen presentation (not illustrated).

outcomes included changes in disease activity scores, time to first relapse and adverse events. Six studies met the criteria for eligibility, and showed a marginally significant benefit of n-3 PUFA therapy for maintaining remission. However, the studies were both clinically and statistically heterogeneous, and two large studies showed negative results. There was also evidence for publication bias. Although no serious adverse events were seen, a pooled analysis showed a significantly higher rate of diarrhoea and symptoms of the upper gastrointestinal tract in the n-3 PUFA treatment group. The authors concluded that existing data do not support routine maintenance treatment of CD with n-3 PUFA.15 A further analysis focused on the use of fish oil for induction of remission in UC. Six studies were included, 3 of cross-over design and 3 of parallel design. No data were pooled for analysis due to differences in outcomes and methodology among the included studies. One small study showed a positive benefit for induction of remission, while some of the other included studies showed some positive benefits for secondary This journal is ª The Royal Society of Chemistry 2010

Trial design

n-3 PUFA form and dose Method of administration

Study endpoint

Result

Reference

The initial-onset Japanese Non blinded, Patients were prohibited About 3400 mg/day (2 units) Fatty acid composition In a subset of 20 initial-onset 31Uchiyama et al. (2010) patients were composed non crossover from consuming the main of ALA were ingested of the erythrocyte patients, the mean n-3/nof 12 UC patients (3 sources of dietary n-6 from 7 mL/day of perilla membranes and 6 ratio significantly males, 9 females, mean PUFA: i.e., vegetable oil; (Egoma) oil in addition to disease activity after increased after age 32.9 years and 8 CD seasonings such as a daily intake of 1700 mg 12–18 months intervention. In the patients (5 males, 3 margarine, dressings, and from fish oil (EPA, intervention follow-up group the ratio females, mean age 29.0 mayonnaise; foods DHA), taken as oil (no in the remission group (n years) who had not cooked in vegetable oil; storage recommendation, ¼ 145) was significantly undergone any diet and snacks. The target n-3 PUFA food exchange higher than that in the therapy before dietary for n-3 PUFA ingestion table relapse group (n ¼ 85). intervention. The followwas a total of 5100 mg. The ratio decreased up group was divided into The patients were able to significantly in those who 2 subgroups: the confirm the n-3 and n-6 suffered a relapse after remission and relapse PUFA contents of each the beginning of groups, and fatty acid food using an ‘‘n-3 PUFA treatment. composition was food exchange table’’ compared between the 2 subgroups. Among the UC patients, those in whom the severity was evaluated as mild were assigned to the remission group. The others were assigned to the relapse group. Among the CD patients, those in whom neither endoscopy nor contrast-enhanced radiography revealed an active lesion were assigned to the remission group. The others were assigned to the relapse group. 32 Bjørkkjær et al., 2009 18 out-patients at Randomized, controlled, Seal Oil (purchased from Ten mL were self-adminis- Plasma arachidonic acid Significant and positive tered through a nasoHaukeland University to EPA ratio and double blind pilot trial changes from baseline to JFM Sunile AS, Os, pharyngeal feeding tube Hospital (Bergen, prostaglandin E2 comparing whale oil with Norway) or Whale Oil study end were observed for 10 days, three times Norway) between 18 and seal oil, but with no levels, decreased IBD- in both groups for all (donated by Myklebust daily before meals. 75 years old with IBD, as placebo group. related joint pain and endpoints. There were no Trading AS, Myklebost, assessed by IBD-disease activity, significant differences Norway). The oils were a gastroenterologist, in and improved quality seen between seal oil or protected with nitrogen combination with of life. whale oil. on top in bottles, and presence of joint pain. stored in a refrigerator during the study, otherwise in a 20  C freezer. EPIC1: 363 patients total. Patients were eligible if Epanova capsules consisting 1-g of n-3 PUFA Clinical relapse, or No significant difference in 33Feagan et al. (2008) 188 patients were they had experienced of 50% to 60% EPA and encapsulated in initiation of treatment the relapse rate was found assigned to receive n-3 a disease exacerbation 15% to 25% DHA as a delayed-release soft for active CD. between the patients PUFA and 186 patients within the past year and a free fatty acid, The gelatin capsule (Epanova; treated with n-3 or to receive placebo. were in remission for at placebo capsule consisted Tillotts Pharma AG, placebo. least 3 months but not Ziefen, Switzerland)

Population and numbers

Table 1 Examples of human intervention studies to test effects of n-3 PUFA on IBD

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Trial design

n-3 PUFA form and dose Method of administration

Study endpoint

Result

Reference

longer than 12 months. of 1 g of medium-chain Patients were randomly triglyceride oil. assigned to receive either 4  1g/d of n-3 PUFA or placebo for 52 weeks. No other treatments for CD were permitted. EPIC2: 375 patients. 189 Patients with active disease As for EPIC1. As for EPIC1 As for EPIC1 The relapse rate was 84/187 33Feagan et al. (2008) patients were randomised were treated with (44.9%) and 94/188 (50%) to n-3 PUFA and 190 a standardized 16-week in the n-3 and placebo patients to placebo. tapering course of either groups, respectively prednisone or budesonide. Eight weeks after the initiation of corticosteroid treatment, a disease was assessed. If this was less than an agreed cut off, the patient was eligible for randomization. Patients were randomly assigned to receive either 4 g/d of n-3 PUFA or placebo for 58 weeks. No other treatments for CD were permitted. A total of 38 children (5 to A double blind, placeboTRIOLIP-SOFAR, Italy; Time dependent 5-ASA (50 Primary outcome was A very high relapse rate was 34Romano et al., 2005 16 years of age, 53% controlled trial of one 1.2 g/d of EPA and 0.6 g/ mg/kg/d) + n-3 PUFA in relapse rate within found in the placebo male), 18 in the n-3 day of DHA, as year duration. one year. Time to first group of this study. The GI-resistant capsules PUFA group and 20 in triglycerides, versus relapse was also authors concluded that the controls. They were identical placebo of time recorded, but not enteric coated n-3 PUFA recruited from pediatric dependent 5-ASA (50 mg/ systematically in addition to 5-ASA are gastroenterology centers kg/d) + olive oil presented. effective for maintenance in Italy. At baseline, of remission in pediatric participants were in CD. In this study, remission. compliance was optimal, no patients were lost to follow-up and all patients were analyzed. The median time to first relapse was eight months in the intervention group compared to one month in the placebo group. A total of 78 adults (18 to 67 A double blind, placebo1.8 g/day of EPA and 0.9 g/ Enteric coated fish oil hard Relapse rates over one Significantly fewer patients 35Belluzzi et al., 1996 years of age, 50% men), controlled trial of one day of DHA, as free fatty gelatine capsule with 60 year of follow-up. from the intervention 39 in each arm from year duration. Patients acids, versus identical min delay timed release. Adverse events and group relapsed compared outpatient clinics in Italy. treated with 2.7 g/d of n-3 placebo of 500 mg Oral route of time to first relapse to placebo and it was At baseline, participants PUFA compared with Miglyol 812 (a mixedadministration were also monitored. concluded that enteric were in remission and placebo. acid triglyceride of coated, timed release n-3 high risk for relapse, as fractionated short chain PUFA is highly effective

Population and numbers

Table 1 (Contd. )

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Trial design

n-3 PUFA form and dose Method of administration

Study endpoint

Result

Reference

judged by an increase in fatty acids made up of in maintaining remission at least one of three caprylic and capric acid). in CD. serum inflammatory markers. A multicenter study in A double blind, placebo5g/d of highly concentrated Gelatin n-3 PUFA capsules. Primary outcome was No difference in the relapse 36Lorenz-Meyer et al., 1996 Germany. A total of 135 controlled trial of one n-3 PUFA (3.3 g/day of Oral route of the relapse- free time rate of the intervention adults (17 to 65 years of year duration. Steroids EPA and 1.8 g/day of administration period after group. The estimated 1age, 31% men) with CD, were still administered DHA, as ethyl Esther) randomization. year relapse rate was 69% 70 in the n-3 PUFA arm during the first two versus identical placebo Relapse rate and in both groups. The and 65 in the control arm. months of the study, but of corn oil. adverse events were authors concluded that At baseline, participants no other co-interventions also monitored. gelatin capsules of ethyl were in remission, were allowed. Patients ester fish oil capsules are induced by a course of were instructed to not effective for corticosteroids. consume a diet low in maintenance of remission arachidonic acid and rich in CD. in fiber and were followed every three months until one year. 37 Almallah et al., 1998 18 out-patients (9 male, 9 A double-blind, placebo- Active group: 15 ml of fish Patients received either 15 Sigmoidoscopic score, After 3 months of female) with UC. All had controlled randomised oil extract that provided ml of fish oil extract or 15 histological score, supplementation, all the distal procto-colitis. study, and a parallel a total of 3.2g of EPA and ml of sunflower oil as NK cytotoxicity and patients in the EFA group, for a 6 month 2.4g of DHA. Placebo placebo, and were flow cytometry. group went into treatment period group: 15 ml of sunflower instructed to take 5 ml, 3 Assessments were remission. After 6 months oil. All oil supplements times a day. Oil done at monthly supplementation with included 3% vitamin E. preparations were intervals. EPA/DHA there was supplied by Callanish Ltd a significant (Isle of Lewis, Scotland). improvement in disease There were no special activity as shown by the instructions for storage, reduction of the clinical although the material score, compared with contained a small amount baseline In the placebo of an antioxidant. Oral group, there was no route of administration, significant change in the oil taken as a liquid. clinical score, as compared with baseline. 38 Aslan et al., 1992 17 male patients with mild A double-blind, placebo- Active group: Max-EPA (15 Oral route of administration Disease activity, clinical Significant reduction in to moderate UC. Oral controlled, crossover capsules provided a total and laboratory disease activity index. steroids (<20 mg per day) study. An 8 month of 2.7g of EPA, 1.8g of evaluation, histology The changes were and sulfasalazine were treatment period (3 DHA, and 135 kcal). and mucosal LTB4 determined from baseline allowed if the patient had months per treatment Placebo group: Corn oil levels. Assessments data. taken them for more than arm followed by a two (provided a total of 10.3g were done at monthly 4 weeks. month washout period). of oleic acid, 2.1g of intervals. palmitic acid, 1.8g of linoleic acid, and 135 kcal). 39 Dichi et al., 2000 10 patients (5 male, 5 Randomised, crossover Active group 1: 5.4 g/d fish Fish oil capsule (R.P. Laboratory blood Regression not assessed. female) with mild to study. A 2 month oil (18 capsules, each of Scherer do Brasil parameters, CRP, ERS, and platelet moderate UC. The intervention period, 180 mg of EPA and 120 Encapsulac, Sao Paulo, sigmoidoscopy score, count increased comparison was fish oil followed by 2 month mg of DHA. Active Brazil). Oral route of histologic activity and significantly during versus sulfasalazine. washout period and then group 2: 2 g/ administration. No protein metabolism treatment with fish oil. d sulfasalazine. details of nature of evaluation. The changes were

Population and numbers

Table 1 (Contd. )

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2 month cross-over intervention period.

capsule, purity, or storage.

Assessments were done at monthly intervals.

determined from baseline data. The sigmoidoscopy Score after n-3 PUFA intake was significantly lower than at study entry. 24 out-patients (10 male, 8 Multicenter, randomised, Active group: Max-EPA (18 Fish oil (Max-CPA, R. P. Sigmoidoscopy scores, No significant changes in 40Stenson et al., 1992 female), with active UC. double-blind, placebocapsules provided a total Scherer, Clearwater, global assessment endoscopic score for both controlled, crossover of 3.24g of EPA, 2.16g of Florida) or placebo score, histology and groups. trial. The initial 4-month DHA, and 162 kcal.). (vegetable oil capsules, rectal dialysis. treatment period was Placebo group: vegetable provided by R. P. Assessments were followed by a 1-month oil (18 capsules provided Scherer). Patients done at monthly washout period during 12.36g of oleic acid, 2.52g received either 18 fish oil intervals. which all patients of palmitic acid, 2.16g of capsules or 18 placebo received placebo. Patients linoleic acid, and 162 capsules per day and were then crossed over and kcal). instructed to take 6 began a second 4-month capsules 3 times a day. period during which the The Max-EPA and original treatment placebo capsules were assignments were identical in appearance. reversed. Oral route of administration.

Population and numbers

Table 1 (Contd. )

Trial design

n-3 PUFA form and dose

Method of administration

Study endpoint

Result

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Reference

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outcomes. However, the authors cautioned that results were inconclusive due to small study size and poor study quality. More conclusive evidence can be provided by considering individual clinical trials, examples of which are summarised in Table 1.31–40 An n-3 PUFA diet therapy was considered for IBD patients.31 Dietary guidance on increasing n-3 PUFA and decreasing n-6 PUFA led to a significant increase in the n-3:n-6 ratio in the erythrocyte membrane of IBD patients, and this ratio was significantly higher in the remission group, suggesting an influence on the fatty acid composition of the cell membrane and clinical activity in IBD patients. The Epanova Program in Crohn’s Study (EPIC) was a pair of randomized controlled trials, that were based over 98 centers in Canada, Europe, Israel, and the United States.33 Details of the trial design and data are described in Table 1. The commonly cited conclusion of these trials has been described as ‘‘treatment with n-3 PUFA was not effective for the prevention of relapse in CD’’. However, consideration of the details in the table show differences in a number of events, including CD worsening (or relapse) where the n-3 PUFA group showed fewer subjects, but these individuals also showed an increase in abdominal pain, diarrhea, arthralgia, nasopharyngitis, nausea and fatigue. There were differences between the trials, not only in length of time they went, but also in the entry criteria. What is unclear is how this dose level was set, since it appears higher than commonly found in a dietary supplement. De Ley15 published a Cochrane review on n-3 PUFA and UC. Of the 6 published trials that met their stringent trial design criteria, only 437–40 were full manuscripts, for which details are summarised in Table 1.31–40 Although most of these studies were small and underpowered, the results are generally positive. Other marine sources may prove important for IBD. Liprinol, a preparation from New Zealand green-lipped mussels, is thought to be worth pursuing for anti-inflammatory effects after results indicating that there were benefits in C57BL/6 mice fed a preparation of this in olive oil.41 However, the authors conceded that it was unlikely that the n-3 PUFA were responsible for the ameliorating effects as the dosage was very low, < 1mg per day. Despite optimism in some of the studies, it must be concluded that hard data, directly relevant to human IBD, is limited. The reasons for an apparent lack of activity in many of the studies may include pharmacokinetic limitations and intestinal degradation of the compounds, in particular insufficient sub-mucosal levels of the effective compounds, as well as the known general limitations of studies with free living humans. The studies are typically high dose, and the importance of dose response has not been generally stressed. Furthermore, with very few exceptions,32 there is no indication of storage conditions or measures taken to prevent oxidation of the products.

Possible reasons for failure of some of the n-3 PUFA supplementation trials in IBD There are a considerable number of potential artifacts, if a clear understanding of genetics, nutrition, food science and engineering is not applied to trials of these fatty acids. These fall under several headings, as follows: This journal is ª The Royal Society of Chemistry 2010

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Differences between individuals in response to n-3 PUFA

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42

Simopoulos draws attention to the range of genes that affect the uptake, transport and metabolism of n-3 PUFA. The level and composition of n-3 PUFA in the human body is not only a function of the dietary intake, but also determined by endogenous metabolism, as fatty acid precursors are endogenously elongated and desaturated to physiologically active long chain compounds. D5 and D6 desaturases are the key enzymes involved in this conversion, and these enzymes are highly active in the liver, brain, heart and lung. The fatty acid desaturase 1 gene (FADS1), encodes saturase D5, while fatty acid desaturase 2 (FADS2), encodes saturase D6, both genes being located on chromosome 11 (11q12– 13.1). Genetic variants in FADS1 and FADS2 lead to a highly significant decrease in the endogenous production of arachidonic acid (AA) and its precursors.43,44 Given male/female differences in response to n-3 PUFA, it is of some interest that variants in the FADS1 and FADS2 genes are associated with altered levels of both n-6 and n-3 PUFA in plasma and erythrocyte phospholipids in women during pregnancy, and in breast milk during lactation.45 High density lipoprotein (HDL) particles transport certain lipids through the properties of the apolipoprotein (apo) components, apoA-I and apoE. ApoA-I and apoE act to solubilize phospholipids and stabilize HDL particles, enabling these proteins to be partners with products of the multidrug transporter, ABCA1, in mediating the efflux of cellular phospholipids and cholesterol.46 Variants in the genes encoding these enzymes modulate the transport, and therefore effective functioning of n3 PUFA. For example, there was evidence that the plasma n-3 fatty acid response to an n-3 PUFA acid supplement was modulated by the apoE epsilon4 variant.47 The authors considered the plasma fatty acid response to a dietary supplement of EPA + DHA in carriers of the E4 allele in a group of human volunteers. When the group was separated based on the presence of E4, the baseline EPA and DHA in plasma were 67 and 60% higher, respectively, in E4 carriers. Thus, highly significant gene x diet interactions were found, whereby only non-carriers of the E4 allele had increased levels of EPA and DHA in plasma in response to an n-3 PUFA dietary supplement.47 The effects of n-3 PUFA intake were considered in relation to effects on various genetic polymorphisms, as an example of nutrigenetics in CD.48 The study estimated seven SNPs in interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-a), lymphotoxin alpha (LT-alpha), and IL-6 genes in 116 controls and 99 patients with CD, in relation to the nature and levels of fat intake. A high intake of total, saturated, and monounsaturated fats, and a higher ratio of n-3:n-6 PUFA, was associated with a more active phenotype. Additionally, low intakes of n-3 PUFA and high n-3:n-6 PUFA ratios in patients with the TNF alpha 857 polymorphism were associated with significantly higher disease activity.

Different disease characteristics of selected patient groups Different stages of disease or patient characteristics might explain some of the data. For example, Belluzzi and coworkers35 only included patients at a high risk of relapse, as defined by the presence of an elevated serum concentration of inflammatory This journal is ª The Royal Society of Chemistry 2010

biomarkers. In contrast, patents recruited into the EPIC studies were at substantially lower risk and it is possible that the trials lacked sufficient statistical power to detect the required 15% benefit of treatment. However, the authors noted this could not have been true for EPIC-2, in which 51.2% of patients relapsed33 Poor subject compliance Although no beneficial effect was observed in prevention of relapse in the EPIC trials, the authors noted a statistically significant decrease in the serum concentration of triglycerides in the patients who received the n-3 PUFA supplement. Additionally, an analysis of capsule counts provided confidence that the patients consumed adequate amounts of the dietary supplements, suggesting that poor compliance could not account for the negative results. Different formulations used in the different studies The formulation evaluated in the study by Belluzzi et al/35 was a hard gelatin capsule, whereas the EPIC trials incorporated a soft gelatin capsule.33 Although the authors claimed that a similar concentration of 3 PUFA is incorporated into cell membranes following administration of either preparation, there are some very substantial differences in the properties of these two capsule types. First, there is likely to be difference in the dissolution rate of the hard vs. soft gelatin once it is ingested.49 Secondly, there is a substantial difference in oxidation protection from hard vs. soft gelatin. This is reported for microcapsules,50 and would be equally true of full size capsules, although the surface area to volume ratio means the degradation rate would be faster for the microcapsules. Oxidation of trial products As n-3 PUFA are highly unsaturated, they are very susceptible to oxidation.51,52 Literature reports on studies of oxidative stability of PUFA are contradictory and somewhat controversial, making the reported impact of antioxidants difficult to consistently quantify.53 This might be due to the range of test methods employed, as suggested by Frankel, but equally might reflect the impact of the food matrix itself on the action of antioxidants.54 What is clear is that as n-3 PUFA oxidise, the detection of ‘‘off flavours’’ increases and, with PUFA from marine sources, these manifest as a fishy flavour or odour.55–57 Oxidation also reduces the nutritional benefit of the PUFA and can lead to products that have an adverse effect of health.58,59 To avoid or delay oxidation, PUFA are stabilised by antioxidants. These can be synthetic (for example butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)60 or disodiumethylenediaminetetraacetic acid EDTA,61 or natural extracts such as ascorbic acid, rosemary, oregano or green tea extract.54,62–64 However, once incorporated into a food matrix the efficacy of these antioxidants may be compromised by factors such as pH, in some cases even acting to accelerate oxidation, as occurred in a study of omega 3 enriched mayonnaise54 where ascorbic acid was used as the antioxidant. In that study the proposed mechanism for oxidation acceleration was the based on the role of iron; in the presence of pH below 6.0 the iron ions become accessible and are reduced, by the ascorbic acid, from Food Funct., 2010, 1, 60–72 | 67

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Fe3+ to Fe2+ which is a more efficient oxidation catalyst. Similarly, in a study of baked cereal, the antioxidant EDTA was added to an incorporated fish oil emulsion but actually resulted in increased oxidation.9 In this instance the proposed mechanism was again the reduction of Fe3+ to Fe2+ by EDTA chelates. Without information on the storage time, temperature and humidity of the capsules prior to ingestion the impact of oxidation cannot be quantified, although the hard versus soft gelatin would have had an impact if all else had been equal.

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Bioavailability of n-3 PUFA in different formulations Numerous studies from the food engineering perspective report levels of EPA and DHA potentially available, depending on method of incorporation. Studies from the nutritional perspective report on the bioavailability of these compounds in various food products or dietary supplements, with few details of the method of presentation of the PUFA. Very few studies report the links between bioavailability and method of presentation, though equivalence has been reported between traditional fish oil capsules and microencapsulated PUFA incorporated into food matrices.65–67 The influence of oral breakdown of a food matrix containing PUFA additions, the impact of enzymes on the matrix and PUFA-food matrix interaction during transport through the GI tract have not yet been reported. However, this aspect of the nutritional fortification of foods by PUFA will need to receive attention in the near future and the tools and techniques are available or are being developed. For example the role of oral breakdown and interactions with the food matrix is now being studied in the context of bioavailability of nutrients68 and glycaemic index.69 Potential interactions with other components in the capsules or food matrix n-3 PUFA are not usually found in isolation or in equal amounts among species, or their extracted oils, and this may give rise to equivocal results and make it difficult to attribute a clinical outcome. This was true in the studies by Brox70 and Brunborg71 who compared the effects of seal and cod liver oils. The seal oil had similar amounts of EPA, almost three times as much docosapentaenoic acid (DPA, 22:5n-3), and a third less DHA, than cod liver oil. When administered nasoduodenally for 10 days there was improvement in joint pain and overall disease activity in the seal oil group. However when administered orally for two weeks, despite improvements, there was no significant difference between the CD and UC groups taking either seal oil or cod liver oil.71 The seal oil used in that study had approximately 25% less thiobarbituric acid reactive substances, which are an indication of secondary oxidation products, and 80% less a-tocopherol, but the impact of the quantities of these components of the oils in the study is not known. The important point here is that n-3 PUFA do not occur in isolation, and that the contribution of other fatty acids or components in the oil or food should not be overlooked.

Increasing the dietary intake of n-3 PUFA If we assume that there is sufficient positive data to imply a significant benefit to either healthy humans or IBD patients, it 68 | Food Funct., 2010, 1, 60–72

is important to consider the best source of these. n-3 PUFA occur at naturally high levels in oily fish such as mackerel, salmon, tuna, herring etc.72 Fish is often regarded as being expensive, but the perceived high cost in terms of their n-3 PUFA may not always be valid because of the richness of the source.21 Although fresh farmed salmon, for example, may be of higher cost than other species, the concentration of n-3 PUFA (%/ g) should not be ignored. Nevertheless, in any local market, the full range of available species should be analysed to identify the species which may provide good sources of n-3 PUFA as well as a nutritious meal for a family. The low consumption of fish in a Western diet73 means that alternative means of delivery are being continually explored. Increasing the levels of PUFA in other meat based products by incorporating it into animal feed is one approach74–76 but dietary supplements (capsules) and fortification of foods are the two main routes to increasing intake of n-3 PUFA. Many commercial products are now available from speciality ingredient and nutritional supplement suppliers, and these are often described as stabilised and odourless, both necessary factors for inclusion in foods. Despite the advances in aquaculture, marine-derived oils and fish stocks in general are under threat from diminishing resources. Hence the focus turns back to terrestrial sources and modifications of existing high producing oil crops for sustained supply of n-3 PUFAs. However, native plant oils and plant foods in general are not usually regarded as being such excellent sources of n-3 PUFAs as fish oils because of the lower concentrations of n-3 PUFAs in these. Nevertheless, one cannot discount the native forms entirely. For example, the leaves of purslane (Portulaca oleracea), which for a long time has featured in the diets of many communities, contain 4 mg/g wet weight a-linolenic acid.77,78 Cultivation and plant breeding techniques to enhance production of oil crops are well-established. However, Murphy points out that means of encouraging plants to produce desirable amounts of fatty acids in the organ which is most easy to harvest and process is often challenging.79 Nevertheless, advances in analytical techniques coupled with micro-dissection, have permitted rapid screening of high producing lines and the technical advances likely to arise from biofuel initiatives may well provide more.

Optimising the presentation of dietary omega-3 PUFA Naturally present or direct addition Adding n-3 PUFA, especially those sourced from marine oils, directly to foods has numerous implications in terms of sensory attributes (taste, odour and texture), shelf stability, nutritional quality and safety. One of the main challenges facing food producers is the consumer acceptability of long chain n-3 PUFA fortified products. Direct incorporation of commercial PUFA products into a range of foods, including fat based spreads, yoghurts and fruit juices, has been shown to decrease palatability in direct proportion to an increase in detectable fishy odour.59,80 Even when incorporated into fat based spreads a stabilised commercial fish oil described as ‘‘odourless’’ was detectable, and undesirable, at levels around 0.05% weight.81 Storage often This journal is ª The Royal Society of Chemistry 2010

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emphasises the ‘‘off flavours’’ associated with rancidity, reported for both baked goods82 and fruit juice.80 This is a direct result of the oxidative instability of PUFA.

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Microencapsulation Alternative methods of stabilising PUFA prior to addition to foods include emulsification83 and microencapsulation (an extension of emulsification where a stable emulsion is subsequently dried, often by spray drying.60 Microencapsulation is a very promising technology for encapsulating and protecting PUFA for incorporation into food matrices and for targeted delivery of nutrients.10 Microencapsulation has been used for protecting functional ingredients, such as flavours, for over 40 years84 The active ingredient is enclosed in a capsule that may be made of a variety of materials including polysaccharides, gums, proteins, lipids or synthetic polymers. The particles range between 5 and 1000 microns, and are produced by a number of techniques including, amongst others, spray drying, coacervation, extrusion and self-assembly of liposomes. There are many reviews of the various microencapsulation technologies including details of the capsule material, the particle size and the particle production step.60,84–86 Interestingly, despite widespread application in pharmaceuticals and cosmetics, microencapsulation in food engineering is still a relatively niche technology. This is driven, in part, by the lower margins typically associated with food ingredients; the influence of this aspect of food manufacturing is reviewed, in the context of microencapsulation.85 The advantages of the microencapsulation technique, for fortifying foods with PUFA and other bioactives, include protecting functional ingredients from degradation and concealing undesirable flavours, whilst maintaining acceptable product texture.10 One of the primary advantages for microencapsulation of PUFA is the protection against oxidation (and subsequent detectable ‘‘off’’ flavours) during food storage. Microencapsulation can substantially delay, but not completely eliminate, oxidation.59,81,87 In fact the microencapsulation process can accelerate oxidation of the PUFA during the emulsification stage due to greatly increased surface area.88,89 Notwithstanding the possible oxidation during production of the microcapsules, these particles have been repeatedly shown to increase resistance to oxidation of PUFA when incorporated into foods. When encapsulated with gelatine, n-3 PUFA showed a 3-fold decrease in oxidation products after 4 weeks storage (storage conditions not reported).50 Oxidation resistance reported in this study was best when an additional hardening step (ethanol extraction of chemical cross-linking) was included for the capsule. However, a hardened microcapsule would possibly be discernible, as the capsules in this study were around 40mm diameter, and depending on hardness and shape, particles as small as 15mm can be detected as ‘‘grittiness’’ in foods.90,91 When incorporated into baked cereal snack bars as direct oil addition, oil emulsion and as microencapsulated particles the encapsulated particles showed no significant oxidation products after 11 weeks of storage.9 A further advantage of microencapsulation of n-3 PUFA is the possibility to reduce detectable flavours, such as the fishy flavour associated particularly with marine based fatty acids. Studies have shown the advantage of microencapsulation over direct This journal is ª The Royal Society of Chemistry 2010

addition of fish oil in terms of taste80,82 and, again, the influence of food matrix selection has been recognised. For example when adding PUFA to breads (considered an ideal delivery system as the CO2 generated during proofing and baking protect against oxidation, and bread products have an intrinsically short shelflife60) lower additions are possible to white bread than brown due to the stronger flavours of brown bread.72 Selecting a microencapsulation system (capsule material, active ingredient formulation and encapsulation system) is dictated by the food matrix that is to be fortified.60 An optimum solution for minimising impact on flavour and texture, whilst maintaining the nutritional value of the n-3 PUFA, might not be the most technologically accessible. For example in a study examining addition to bread correct selection of the capsule material (soybean protein extract) ensured minimal impact on flavour of the baked bread or rheology of the dough. However, the authors recommended an alternative encapsulant (methylcellulose) for ease of production, despite a greater impact on taste and texture.92 Many reports of dietary studies list large numbers of commercially available foods that have been eaten by subjects with little or no adverse impact on the favour or texture profile of the product.72,93–95 Bioavailability affected by method of presentation Linking the method of presentation of n-3 PUFA (naturally occurring, direct oil addition, emulsion or microencapsulated) with its bioavailability is a vexed question. Studies from the food engineering perspective tend to concentrate on the PUFA prior to consumption. These studies imply nutritional availability by reporting the amount of EPA and DHA provided in a serving of food.81,96–99 However, studies from the nutritional analysis point of view often do not include details of the method of incorporating PUFA into the food matrix73,93,94 nor controls comparing, for example, direct oil addition with microencapsulation. The impact of the method of presentation of PUFA in a fortified food matrix might be inferred from complementary studies, for example where n-3 PUFA has been incorporated into bread as direct oil addition100 and as microcapsules.101 In both cases, plasma levels of fatty acids indicated the n-3 PUFA were bioavailable. However, in the case of direct oil addition, detail is not provided as to how and when the addition was made. In the case of the microcapsules, whether the bread was served toasted or not and the impact of the toasting step is not reported. Incorporating n-3 PUFA microcapsules as part of a number of commercially available fortified food products has demonstrated good bioavailability (measured by plasma fatty acid profile).102 Moreover, the subjects reported good acceptability of the food products (milk, yoghurt and breads) with no gastrointestinal distress. Some studies have compared the bioavailability of PUFA incorporated into food by direct oil addition and in the microencapsulated form. A study in rats103 found the same distribution of fatty acids in the livers of the animals, whether the PUFA was incorporated directly or in microcapsules. This study might be indicative for incorporation of PUFA into human foods that require no cooking step (such as fat based spreads). However, it is not informative about the potential impact of heat or long term storage, since the samples were stored for up to 14 days at 80  C, substantially colder than commercial cold-store. Food Funct., 2010, 1, 60–72 | 69

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Three studies by separate groups have reported a comparison of bioavailability of n-3 PUFA from traditional gel encased fish oil capsules and microencapsulated fish oil incorporated into a food matrix. The food matrices studied were bread, biscuit and soup65 or milkshake.66,67 In the case of the milkshakes, the microencapsulated fish oil powder was stirred into the drink prior to consumption. In the case of the bread, biscuit and soup, specific details of how and when the microencapsulated fish oil was incorporated are not given. In all cases, the studies reported equivalent bioavailability of n-3 PUFA, as measured by plasma levels of fatty acids and cholesterol, whether administered as capsules or food incorporated microcapsules. These are exemplary studies for comparing delivery vehicles (capsules versus microcapsules) but do not tackle the question of the impact of the food matrix, heating effects or storage time on bioavailability of PUFA. A recent review104 of the influence of emulsion structure on lipid digestion includes an excellent overview of some of the structural analysis techniques that will be required to fully understand how the method of presentation of n-3 PUFA influences their bioavailability. One of the most promising areas of development in microencapsulation is the potential for site-specific delivery of nutrients. Site specific drug delivery has received more attention than site specific nutrient delivery. For example, microencapsulation has been used for delivery of drugs directly to the colon.105–107 Similar approaches are now being taken to increase the intestinal delivery of probiotics,108–110 where encapsulation in alginate capsules has been show to protect the contents against degradation in the upper gastrointestinal (GI) tract. Very few studies are reported for site specific delivery of lipids, though animal studies have shown the feasibility of protecting dietary fats using lecithin-chitosan encapsulation.111 A very recent study has reported site specific delivery of n-3 PUFA to the GI tract in rats.112 The choice of encapsulating material dictated whether fish oil was delivered predominantly to the small intestine or to the large bowel.

Conclusions There is reason to believe that increasing n-3 PUFA and decreasing n-6 PUFA in the diet of IBD patients could be beneficial. However, we suggest that there is an urgent need for consistency in design among clinical trials, and attention paid to method of presenting these to humans, whether as foods, fortified foods or dietary supplements. In particular, we believe that consideration needs to be paid to the source and form of n-3 PUFA. The best available evidence pertains to dietary sources rather than high dose supplements, and the approach using a combination of food exchange tables and lower dose supplements is of significant interest.31 We noted the evidence that feeding Atlantic salmon diets rich in n-3 PUFAs did not always have the expected outcome. Can something be learned from this for other animal or human intervention trials? The duration of the supplementation, stage of life and pre-existing medical conditions are all likely to affect the outcome of supplementation. If dietary supplements are to be recommended to IBD patients, it is essential to ensure that advice is based upon adequate trial design, that is well powered with different patient 70 | Food Funct., 2010, 1, 60–72

groups showing comparable disease activity. Stratifying according to genotype would be beneficial. The dietary supplement or n-3 PUFA-containing foods must be stored or manufactured so as to prevent oxidation, and administered in such as way as to optimise uptake and bioavailability, in order to be quite sure about the benefits that a micro-ingredient might provide to healthy individuals, or those compromised in some way such as the IBD group. Careful characterisation of the exact fatty acid composition of any food or oil supplement, together with knowledge of the target tissue and cell types, should be mandatory to assign with confidence the precise factors giving rise to a clinical outcome.

Abbreviations AA Apo CD COX DHA EPA FADS1 FADS2 HDL IBD IL iNOS LA LDL LT LT-alpha NF-kB n-3 PG PUFA TNF UC

arachidonic acid apolipoprotein Crohn’s disease cyclooxygenase docosahexaenoic acid eicosapentaenoic acid fatty acid desaturase 1 gene fatty acid desaturase 2 gene high density lipoprotein Inflammatory bowel disease interleukin inducible nitric oxide synthase linoleic acid low-density lipoprotein leukotriene lymphotoxin alpha nuclear factor kappa B omega-3 prostaglandin polyunsaturated fatty acid tumor necrosis factor ulcerative colitis

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Comparison of the polyphenolic composition and antioxidant activity of European commercial fruit juices Gina Borges,a William Mullenb and Alan Crozier*a

Downloaded on 21 October 2010 Published on 13 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00008F

Received 14th May 2010, Accepted 30th June 2010 DOI: 10.1039/c0fo00008f Thirty six commercial European fruit juices were tested to ascertain their antioxidant capacity and polyphenolic composition. Six of the products were labelled 100% pomegranate juice, the others included 20 brands of diluted pomegranate juice or pomegranate blended with other fruit juices and 10 different non-pomegranate fruit juices. The antioxidant capacity of all the juices was determined while anthocyanin, ellagitannin and ellagic acid profiles of the 26 pomegranate juices and pomegranate juice blends were obtained using HPLC-PDA-MS2. Additional analysis was conducted on seven of the juices using HPLC with an on-line antioxidant detection system. Three of the ‘‘pure’’ pomegranate juices had the highest ellagitannin content and the highest antioxidant capacity. Only one of these three juices was rich in anthocyanins. The other ‘‘pure juices’’ had differences in their HPLC ‘‘pomegranate’’ fingerprint and also had a lower antioxidant capacity, in some cases lower than that of some of the blended juices. Vitamin C rather than phenolic compounds was the major contributor to the antioxidant capacity for some of the juices. Statistical analysis of both the antioxidant assay and the HPLC on-line antioxidant data demonstrated that the ellagitannins were the major antioxidants in the pomegranate juices. The complexity of the polyphenolic profile of pomegranates necessitates the use of HPLC-PDA-MS2 for a thorough evaluation of juice composition and authenticity.

1 Introduction The evidence that diets rich in fruits and vegetables provide a reduced risk of chronic diseases is compelling.1 Flavonoids and related phenolic compounds that occur in plant-derived foods have been associated with these protective effects. As a consequence of the substantial research in this area, fruit juices are being used increasingly by people who are looking for healthy options as part of the WHO 5-a-day dietary recommendations. This is reflected in a steady global rise in fruit juice consumption. Western Europe is the second largest regional market.2 Of the ten countries with the highest per capita consumption, six are found within this region with a consumption of more that 28 liters/ person/year. Interest in pomegranate (Punica granatum L.) juice and its products has also increased markedly in recent years with a growing number of reports on their potential health benefits. These include pomegranate juice consumption being associated with inhibition of prostrate cancer in men,3 a reduction in serum oxidative stress in plasma of type-2 diabetes mellitus patients,4 reduced atherosclerosis in diabetic patients,5 and potential protection against colon cancer.6 There is enormous variability in antioxidant (AOX) activity and phenolic compounds present in different commercial fruit juices.7,8 Some products were of questionable authenticity with the actual ingredients not matching what was claimed on the label. Pomegranates are characterized by the presence of a Plant Products and Human Nutrition Group, Division of Developmental Medicine, Faculty of Medicine, University of Glasgow, Graham Kerr Building, Glasgow, G12 8QQ, United Kingdom. E-mail: a.crozier@bio. gla.ac.uk; Tel: +44 141 330 4613 b Division of Ecology and Evolutionary Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Graham Kerr Building, Glasgow, G12 8QQ, United Kingdom

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ellagitannins and anthocyanins. However, the levels vary in juices prepared from different pomegranate cultivars,9 maturity stage,9,10 and they are even absent in some commercial products.7,11 Zhang et al.12 used a combination of analytical procedures to develop an ‘‘International Multidimensional Authenticity Specification’’ (IMAS) algorithm to detect a diversity of adulterants of pomegranate juices and drinks. This paper compares 36 European commercial juices derived from pomegranates, and in some instances other fruits, by measuring their total AOX capacity. HPLC-PDA-MS2 was used to obtain fingerprints of pure pomegranate juices and blended pomegranate products to ascertain their composition. In addition, HPLC with on-line AOX detection was used to assess the relationship between the ellagitannin, ellagic acid and anthocyanin content of pomegranate juices and their AOX capacity.

2

Results and discussion

2.1 AOX capacity and vitamin C levels Thirty six juices (Table 1) were investigated initially using the Folin-Ciocalteu assay for total phenols (TP)13 and three different AOX assays. The FRAP14 and TEAC15 AOX assays are simple colorimetric methods based on a single electron transfer reaction and it is assumed that the antioxidant activity is equal to the reducing capacity. The ORAC assay quantifies the peroxyl radical scavenging capacity.16 In terms of overall ranking of the AOX capacity and TP content of the individual juices the four assays yielded very similar results (Table 2). However, for comparative purposes an AOX index was calculated using procedures described by Seeram et al.8 (AOX index ¼ [(sample score/best score)  100]) which gives a composite score taking into account the results obtained with the different methods. The Food Funct., 2010, 1, 73–83 | 73

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Table 1 List of commercial juices analyzed with the ingredients shown in the labelsa Code

Name

Ingredients (as per label)

PG01+ PG02+ PG03+ PG04+ PG05+ PG06+ PG07* PG08* PG09* PG10* PG11* PG12# PG13* PG14* PG15*

BIONA Organic Pomegranate POM Wonderful Rabenshorst Granatapfel Pomegreat Pure Marks & Spencer Pure Pomegranate Juice gn & r, Pur Jus de Grenade Sainsbury’s Pomegranate & Blueberry Pomegreat Ruby Pomegreat de Originale Pomegreat Sapphire Chiquita Welch’s Purple Grape Breaking Wave Pomegranate Juice (Aldi) Rubicon Pomegranate Pomegreat

PG16* PG17* PG18*

Healthy People Pomegreat Pomegreat Granatapfel and Orange

PG19# PG20* PG21* PG22# PG23# PG24* PG25# PG26* PG27* PG28# PG29* PG30*

Becker’s Bester Roter Traubensaft Sainsbury’s Pomegranate Juice Amecke Healthy People Innocent Smoothie Innocent Smoothie Eckes Roter Traubensaft Ocean Spray Cranberry and Pomegranate Rauch Happy Day Innocent Smoothie Fruity King Applesientje Super Fruit

PG31* PG32# PG33* PG34# PG35# PG36#

Coolbest Pomegranate Coolbest SeaBuckthorn Ribena (Really Light) Raspberry & Pomegranate Healthy People Guanabana and appel VIFIT yogurt

Pomegranate (100%) Pomegranate (100%) Pomegranate (100%) Pomegranate (100%) Pomegranate (100%) Pomegranate (100%) Pomegranate (25%), blueberry (5%) Pomegranate (32%), aronia (5%) Pomegranate (30%), grapes (2%), fruit extract, vitamins C & E Pomegranate (28%), blueberry (4%), aronia (4%) Pomegranate (7%), raspberry (18.5%), banana, orange, lemon, grapes Purple grape Pomegranate, grape juice, aonia, berry juice Pomegranate (29%), aronia (7%) Pomegranate (21%), white grapes (3%), elderberry (3%), acai (1.9%), grapefruit (0.5%), lime, vitamins C & E Pomegranate (30%), aronia (7%), vitamins C and E Pomegranate (30%), red grape (7%), vitamins A, C and E, folic acid Pomegranate (20%), mandarin (5%), orange juice (2%), elderberry (3.6%), red grape (0.5%), vitamins C & E Red grape Pomegranate (37%), vitamin C Pomegranate, red and white grape, apple, red currant, cranberry, lemon Apple, acai, raspberries, red grapes, lemon Cranberry, yumberry, blackcurrant, orange Pomegranate (15%), blueberry (4%), acai (3%), banana, orange, grapes, lemon Red grape Pomegranate (14%), cranberry (10.5%), apple (6.5%), vitamin C Pomegranate (22%), aronia, apple, elderberry, vitamin C Guava, mango, goji, orange, apple Pomegranate (5%), grapes (55%) Pomegranate (9%), raspberries (3%), black currant (1.7%) cranberry (1%), strawberry (0.7%), apple, white grapes, vitamins C & E Pomegranate, raspberry, apple, lemon, blackcurrant Kiwi, goji, orange Pomegranate/raspberry (8%), vitamin C Goji, passionfruit, white grapes, pineapple Soursop, apple, soy Passionfruit, goji

a +

100% Pomegranate juices, * reconstituted or blended pomegranate juices, # non-pomegranate fruit juices.

AOX index was very high (>95) for three of the ‘pure’ juices, PG01, PG02, PG03 while values of <54 were obtained for the remainder of the juices including the other three ‘pure’ pomegranate (PG04, PG05, and PG06) (Table 2). The AOX capacity showed great variability not only among the ‘‘pure’’ pomegranate juices but also the ‘‘blended’’ group of samples. Two of the blended juices, PG07 and PG08, containing 25% and 32% of pomegranate, respectively, scored slightly higher than three of the 100% pomegranate juices (PG04, PG05, and PG06). Also of interest was PG20 which contained 37% pomegranate and had an AOX index of 27 which was lower than that of several juices including PG09 and PG10 which contained less pomegranate (Table 2). This may reflect dilution, adulteration and/or reconstitution factors associated with manufacture. It is, however, more difficult to explain in the context of juices from the same label company like PG04 (100% pomegranate; AOX index 49) vs. PG08 (32% pomegranate, 5% aronia; AOX index 51%) both from Pomegreat. The same applies for PG07 (25% pomegranate and 5% blueberry; AOX index 54) and PG20 (37% pomegranate; AOX index 27) from Sainsbury’s. The levels of vitamin C in the blended products ranged from zero to 58 mg/100 74 | Food Funct., 2010, 1, 73–83

ml in PG33. Vitamin C can influence the AOX activity as observed in the FRAP assay where removal of vitamin C with ascorbate oxidase resulted in a marked reduction in the AOX capacity of some of the blended products (Table 2). Most notable were PG33 where there was a 63% decline following treatment with ascorbate oxidase and PG14 where vitamin C made a 21.5% contribution to the FRAP AOX capacity (Table 2). It would appear that vitamin C is added to several of the juices during processing after pasteurisation and it is this supplementation, rather than the polyphenolic constituents of the fruit that boost the AOX capacity of the juice. Very similar AOX profiles were detected with all four assays and when the data were analyzed statistically highly significant correlation values were obtained with the TP, FRAP, TEAC and ORAC assays (Table 3). The analysis of the AOX capacity of juices for comparative purposes using simple colorimetric assays such as FRAP, TP and TEAC, as well as the more complex ORAC method, is of value as the data are well correlated (Table 3). However, more detailed HPLC-PDA-MS2 analysis is required to investigate quality issues and the great variability shown between supposedly similar juices. This journal is ª The Royal Society of Chemistry 2010

This journal is ª The Royal Society of Chemistry 2010

n.d. n.d. n.d. n.d. 2.0  0.0 n.d. 2.0  0.0 n.d. 6.1  0.0 n.d. 1.4  0.0 n.d. 1.1  0.1 38.0  0.1 4.9  0.1 11.9  0.1 1.5  0.0 1.6  0.0 n.d. 7.1  0.1 n.d. n.d. 26.4  0.3 n.d. n.d. 48.2  0.6 24.6  0.2 31.2  0.1 n.d 38.5  0.2 21.4  0.1 21.6  0.1 58.0  0.2 14.7  0.2 16.9  0.1 12.8  0.0

PG01+ PG02+ PG03+ PG04+ PG05+ PG06+ PG07* PG08* PG09* PG10* PG11* PG12# PG13* PG14* PG15* PG16* PG17* PG18* PG19# PG20* PG21* PG22# PG23# PG24* PG25# PG26* PG27* PG28# PG29* PG30* PG31* PG32# PG33* PG34# PG35# PG36#

20.1  0.1 20.7  0.1 19.9  0.6 10.8  0.4 10.7  0.1 10.3  0.2 13.3  0.2 11.9  0.0 9.7  0.1 9.1  0.2 8.0  0.2 8.8  0.0 6.7  0.1 9.3  0.1 7.7  0.0 7.5  0.0 5.6  0.1 7.5  0.2 6.3  0.0 6.7  0.0 7.7  0.0 6.3  0.0 7.6  0.1 6.8  0.1 5.9  0.0 6.2  0.0 6.0  0.1 5.7  0.2 5.6  0.0 5.7  0.0 5.9  0.0 3.3  0.1 3.8  0.0 2.4  0.0 1.8  0.0 1.2  0.0

TP (mmol/L) 55.3  0.2 52.4  0.6 51.8  0.3 25.4  0.3 25.7  0.3 24.1  1.1 34.4  0.5 27.8  0.7 24.7  0.2 21.9  0.3 18.3  0.1 14.1  0.4 16.5  0.1 19.7  0.2 18.5  0.3 15.8  0.4 13.4  0.4 18.7  0.2 9.9  0.2 17.4  0.1 12.8  0.2 11.6  0.1 12.7  0.2 10.7  0.4 9.5  0.1 14.8  0.1 14.7  0.1 10.3  0.1 8.7  0.1 12.5  0.1 13.3  0.3 4.8  0.0 10.5  0.0 3.4  0.0 2.8  0.0 1.8  0.0

FRAP (mmol/L) n.a. n.a. n.a. n.a. 24.5  0.1 n.a. 30.7  0.0 n.a. 23.4  0.1 n.a. 16.6  0.4 14.1  0.4 16.0  0.1 15.5  0.1 17.3  0.1 14.2  0.2 12.7  0.0 18.0  0.1 9.9  0.2 16.1  0.6 n.a. 11.6  0.1 9.9  0.1 n.a. 9.5  0.1 10.9  0.0 11.6  0.2 7.0  0.0 n.a. 7.9  0.0 11.1  0.2 2.8  0.1 3.9  0.0 1.7  0.0 1.0  0.1 0.5  0.0

FRAP-VitC (mmol/L) n.a. n.a. n.a. n.a. 4.3  2.1 n.a. 10.6  0.1 n.a. 5.4  2.3 n.a. 9.3  0.0 0 3.0  0.3 21.5  1.3 6.7  1.6 10.0  0.7 5.7  2.3 3.7  0.0 0 7.9  0.6 n.a. 0 22.4  1.5 n.a. 0 26.7  0.8 21.3  0.7 32.1  1.4 n.a. 36.7  1.4 16.6  2.8 41.4  0.2 63.0  1.0 42.3  1.0 65.5  0.4 67.7  0.0

Vit C (%) Contribution 83.7  0.5 85.8  1.3 82.7  0.4 40.7  0.5 34.5  4.4 35.2  1.6 30.6  1.2 34.2  1.7 18.2  2.2 28.2  0.7 46.5  4.3 33.2  4.9 37.8  6.4 17.6  0.4 16.2  3.6 22.5  2.4 41.9  1.0 15.5  0.4 26.5  1.2 17.8  1.1 20.6  1.6 20.1  0.6 16.0  3.4 22.5  8.0 19.1  0.2 16.9  0.4 16.7  0.9 20.0  0.5 21.8  0.8 16.9  2.0 10.3  1.6 16.0  0.4 7.4  0.8 14.2  0.2 6.0  0.5 4.4  0.2

ORAC (mmol/L) 40.5  1.7 39.7  1.8 41.3  2.7 19.5  0.4 21.4  2.3 17.9  2.1 25.9  1.8 23.6  1.6 20.5  1.7 18.5  2.0 13.2  2.0 10.6  1.0 10.9  1.4 14.1  1.3 13.5  1.6 12.8  1.7 8.5  0.4 13.4  1.5 7.0  0.4 11.5  1.5 9.4  0.8 9.0  0.5 8.6  1.1 7.9  0.8 7.6  0.7 8.4  0.4 7.4  0.3 6.3  0.1 7.2  0.4 7.2  0.5 8.1  0.5 3.0  0.2 5.3  0.1 2.5  0.2 1.6  0.1 0.9  0.2

TEAC (mmol/L) 98 98 96 49 47 44 54 51 40 40 39 38 33 32 30 30 30 30 28 27 27 27 26 24 24 22 22 22 21 20 20 14 12 11 6 4

AOX index

100% 100% 100% 100% 100% 100% 25% 32% 30% 28% 7% 0% n.s 29% 21% 30% 30% 20% 0% 37% n.s. 0% 0% 15% 0% 14% 22% 0% 5% 9% n.s. 0% 8% 0% 0% 0%

Labelled pomegranate content

The AOX index was calculated according to Seeram et al.8 TP in gallic acid equivalents, FRAP in Fe+2 eq., ORAC and TEAC in trolox equivalents. n.d., - not detected; n.a. - not analysed; n.s. - not stated. + 100% Pomegranate juices, * reconstituted or blended pomegranate juices, # non-pomegranate fruit juices.

a

Vit C (mg/100ml)

Code

Table 2 Results for the AOX assays and vitamin C content in the 36 commercial European juicesa

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Table 3 Pearson’s correlation factor for the AOX activity of different assaysa

TP FRAP ORAC TEAC

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a

TP

FRAP

ORAC

TEAC

1 0.986(***) 0.882(***) 0.988(***)

1 0.876(***) 0.989(***)

1 0.863(***)

1

*** Correlation is significant at p < 0.001.

2.2 Qualitative HPLC-PDA-MS2-on-line AOX analysis of pomegranate juices Polyphenolic compounds in the 100% pomegranate juices as well as the other pomegranate juices were analysed by HPLC-PDAMS2 and the identifications used to compile a ‘‘pomegranate fingerprint’’ to compare to those obtained from the blended drinks. At the same time, the AOX capacity contribution of the peaks were measured by an on-line ABTS system.17 Fig. 1–7 show the 520, 280 and 720 nm traces for PG01- PG04, PG06, PG14 and PG33. The anthocyanin profile can be seen at 520 nm and the ellagitannins/ellagic acids at 280 nm while the 720 nm trace depicts the AOX activity associated with each peak. A total of 17 compounds were identified in all 100% juices. The identities of the peaks numbered in the traces (Fig. 1–7) are summarised in Table 4, and their contribution to the ABTS AOX content is evaluated in Table 5. Peak 1 (retention time [Rt] - 6.6 min) had a [M  H] at m/z 1101 and, like peaks 6 and 7, produced MS2 ions at m/z 781, 721, 601 and 301. On the basis of this fragmentation pattern, peak 1 is identified as a punicalagin-like compound. This type of compound has not been described before in pomegranate. This peak was the second major contributor to the ABTS AOX of the ‘pure’ juices ranging between 10.8% and 24.7% in PG03 and PG06 respectively. Peaks 2 and 3 (Rts - 7.2 and 7.5 min) had a negatively charged molecular ion ([M  H]) at m/z 781 which fragmented, yielding a base peak at m/z 721 and other ions at m/z 601 and m/z 301, which are from gallagic acid and ellagic acid moieties. Based on the report of Tanaka et al.,18 the fragmentation pattern and elution order identified these compounds as punicalins A and B. This is one of the typical ellagitannins in pomegranate and one of the major contributors to the AOX capacity with values between 11.7% to 20.9% (Table 5). Peak 4 (Rt - 9.9 min, lmax - 520 nm) was characterized by a positively charged molecular ion ([M  H]+) at m/z 627 which produced two MS2 fragment ions at m/z 465 and 303. This fragmentation pattern and the absorbance spectrum identified this compound as delphinidin-3,5-O-diglucoside, a known pomegranate component.19 Peak 5 (Rt - 10.5 min) had a [M  H] at m/z 933 which yielded daughter ions at m/z 781, 721, 601 and 301. This fragmentation pattern in keeping with published data19 identified this compound as 2-O-galloylpunicalagin. Its contribution to the AOX capacity is minor (2.5% and 3.8%) (Table 5). Peaks 6 and 7 (Rts - 11.1 and 12.1 min) both had a [M  H] at m/z 1083 which produced identical MS2 fragments at m/z 781, 721, and 601. This fragmentation pattern identifies these compounds as punicalagins.19 76 | Food Funct., 2010, 1, 73–83

Fig. 1 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG01 [BIONA Organic Pomegranate] (see Table 1) with detection at 520 nm (anthocyanins), 280 nm (ellagitannins and ellagic acid derivatives) and 720 nm (AOX activity). Peak 1 - punicalagin-like, peak 2 - punicalin A, peak 3 - punicalin B, peak 4 - delphinidin-3,5-O-diglucoside, peak 5 - 2O-galloylpunicalagin, peak 6 - punicalagin A, peak 7 - punicalagin B, peak 8 - cyanidin-3,5-O-diglucoside, peak 9 - granatin A, peak 10 pelargonidin-3,5-O-diglucoside, peak 11 - granatin B, peak 12 - pelargonidin-3,5-O-diglucoside, peak 13 - punicalagin isomer, peak 14 - cyanidin-3-O-glucoside, peak 15 - pelargonidin-3-O-glucoside, peak 16 ellagic acid-O-hexoside and peak 17 - ellagic acid. For identification of peaks see Table 4.

Fig. 2 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG02 [POM Wonderful] (see Table 1). For peak identification see legend to Fig. 1 and Table 4.

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Fig. 3 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG03 [Rabenshorst Granatapfel] (see Table 1). For details and peak identification see legend to Fig. 1 and Table 4.

Fig. 4 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG04 [100% Pomegreat] (see Table 1). For details and peak identification see legend to Fig. 1 and Table 4.

Peak 8 (Rt - 12.2 min, lmax - 520 nm) produced a [M  H]+ at m/z 611 and daughter ions at m/z 449 and 287. This fragmentation pattern and the absorbance spectrum identified this compound as cyanidin-3,5-O-diglucoside, another known pomegranate anthocyanin.19 Peaks 9 and 11 (Rts - 13.2 and 13.7 min) had a [M  H] at m/z 783 and m/z 951 respectively. No fragmentation information was This journal is ª The Royal Society of Chemistry 2010

Fig. 5 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG06 [gn & r 100% Pur, Jus de Grenade (see Table 1). For details and peak identification see legend to Fig. 1 and Table 4.

Fig. 6 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG14 [Rubicon Pomegranate] (see Table 1). For details and peak identification see legend to Fig. 1 and Table 4.

obtained. This is in keeping with the presence of granatin A and B, known constituents of pomegranate.20 Peak 10 (Rt - 13.4 min, lmax - 520 nm) produced a [M  H]+ at m/z 465 and a single MS2 fragment ion at m/z 303. This fragmentation pattern, absorbance spectrum and co-chromatography identified this compound as delphinidin-3-O-glucoside, a known constituent of pomegranates.19 Food Funct., 2010, 1, 73–83 | 77

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Fig. 7 Gradient reversed phase HPLC-PDA-AOX analysis of juice PG33 [Ribena Raspberry and Pomegranate] (see Table 1). For details and peak identification see legend to Fig. 1 and Table 4.

Peak 12 (Rt - 14.2 min, lmax - 520 nm) had a [M  H]+ at m/z 595 and MS2 ions at m/z 433 and 271. This fragmentation pattern and the absorbance spectrum identified this compound as pelargonidin-3,5-O-diglucoside, a minor pomegranate anthocyanin.19 Peaks 13 (Rt - 14.7 min) had a [M  H] at m/z 1083 and, like peaks 6 and 7, produced MS2 ions at m/z 781, 721, 601 and 301. On the basis of this fragmentation pattern, peak 13 is identified as a punicalagin-like compound. There are two known punicalagins, A and B, however, additional isomers occur in pomegranate.18 Peak 14 (Rt - 15.2 min, lmax - 520 nm) yielded a [M  H]+ at m/z 449 and a single MS2 fragment at m/z 287. This fragmentation

pattern, absorbance spectrum and co-chromatography identified this compound as cyanidin-3-O-glucoside.19 Peak 15 (Rt - 17.0 min, lmax - 520 nm) was characterised by a [M  H]+ at m/z 433 which yields a daughter ion at m/z 271. This fragmentation pattern, absorbance spectrum and cochromatography identified this compound as pelargonidin-3-Oglucoside.19 None of the anthocyanins seems to have any effect on the on-line AOX activity, as they are not reflected in the 720 nm ABTS profile. Peak 16 (Rt - 24.8 min) had a [M  H] at m/z 463 and a single MS2 fragment at m/z 301. This is in keeping with the presence of an ellagic acid-O-hexoside conjugate which has previously been reported to occur in pomegranates.19 Peak 17 (Rt - 29.9 min) had a [M  H] at m/z 301 that yielded no MS2 fragment ions. Co-chromatography and the identical fragmentation of a reference compound identified this component as ellagic acid. Both ellagic acid and conjugate were minor contributors to the AOX capacity (Table 5). In addition to these 17 peaks, the 280 nm trace of the PG01 juice (Fig. 1) had a peak with a retention time of 7.9 min that is also present in PG06 (Fig. 5) and appeared in smaller amounts in PG03 (Fig. 3) and PG14 (Fig. 6). This peak did not ionise, so no MS data were obtained to assist identification, nor did it exhibit on-line AOX activity. Overall these results show that the two main groups of polyphenolic compounds in pomegranate juice are anthocyanins and ellagitannins. The spectrum of anthocyanins comprising principally of cyanidin-3,5-O-diglucoside, cyanidin-3-O-glucoside, delphinidin-3,5-O-diglucoside and delphinidin-3-O-glucoside together with smaller amounts of pelargonidin-3,5-O-diglucoside and pelargonidin-3-O-glucoside is in agreement with earlier reports.10,21 This can be used as a convenient fingerprint of pomegranate authenticity. The other potential diagnostic components are the ellagitannins in the form of punicalagins and punicalagin-like (peaks 1, 2, 3 and 13), 2-O-galloylpunicalagin (peak 5), punicalin A and B (peaks 6 and 7) and granatin A and B (peaks 9 and 11) which are the main contributors to the AOX capacity. Ellagic acid and an ellagic acid-hexose conjugate also

Table 4 HPLC-MS2-based identifications of flavonoids and phenolic compounds in pure pomegranate juicesa Peak No.

Rt (min)

[M  H] (m/z)*

MS2 daughter ions

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

6.6 7.2 7.5 9.9 10.5 11.1 12.1 12.2 13.2 13.4 13.7 14.2 14.7 15.2 17.0 24.8 29.9

1101 781 781 627+ 933 1083 1083 611+ 783 465+ 951 595+ 1083 449+ 433+ 463 301

781, 601, 301 601, 301 601, 301 465, 303 781, 721, 601, 301 781, 721, 601, 301 781, 721, 601, 301 449, 287

Punicalagin-like Punicalin A Punicalin B Delphinidin-3,5-O-diglucoside 2-O-Galloylpunicalagin Punicalagin A Punicalagin B Cyanidin-3,5-O-diglucoside Granatin A Delphinidin-3-O-glucoside Granatin B Pelargonidin-3,5-O-diglucoside Punicalagin isomer Cyanidin-3-O-glucoside Pelargonidin-3-O-glucoside Ellagic acid-O-hexoside Ellagic acid

a

303 433, 271 781, 721, 601, 301 287 271 301

[M  H] negatively charged molecular ion; + indicates positively charged molecular ion.

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Table 5 Percentage of contribution of the phenolic pomegranate markers on the HPLC-ABTS on-line AOX activitya

Punicalagin-like Punicalins A–B 2-O-Galloylpunicalagin Punicalagin A + B + isomer Granatin A Granatin B Total ellagitannins Ellagic acid-O-hexoside Ellagic acid Total ellagic acids Anthocyanins Vitamin C Caftaric acid Unidentified compounds a +

PG01+

PG02+

PG03+

PG04+

PG06+

PG14*

PG33*

12.8 19.8 3.2 12 5.5 4.9 58.2 1.6 2.1 3.7 0 0 0 38.2

18.1 11.7 2.6 19.4 7.6 4.9 64.3 1.3 4.8 6.1 0 0 0 29.7

10.8 13.5 3.8 20.4 5.1 4.7 58.3 1.6 3.7 5.3 0 0 0 36.5

15 20.9 2.9 13.2 9.8 6.8 68.6 1.2 2.9 4.1 0 0 0 27.5

24.7 17.6 2.5 14.3 3.9 3.4 66.4 2.1 2.4 4.5 0 0 5.2 23.9

5.6 11.5 0 7.4 1 1.9 27.4 0.5 0.8 1.3 0 62.3 0 9.2

2.8 3.1 0 3.2 0 0 9.1 0 0 0 0 81 0 9.9

100% Pomegranate juices, * reconstituted or blended pomegranate juices.

occur but their presence is not specific to pomegranate as they can be derived from raspberries and other sources.22–24 The 720 nm traces for all the juices tested in the on-line AOX detector is almost identical to the 280 nm fingerprint of ellagitannins/ellagic acids (Fig. 1–7). Thus, as outlined in Table 5, the ellagitannins are the main antioxidants in the five pure pomegranate juices, PGO1–PGO4 and PGO6. The major contributors were the punicalins, punicalagins, and galloylpunicalagin which accounted for 58% to 69% of the total AOX of the juices. Around 30% of the AOX activity was due to an increased background probably due to unresolved oligomeric ellagitannins or proanthocyanidins25 with AOX activity. None of the anthocyanin peaks were associated with the 720 nm AOX peaks. In Fig. 6 and 7, the profiles for juices PG14 and PG33, it can be seen that the predominant AOX is vitamin C, which is responsible for 62.3 and 81%, respectively, of the total AOX activity, with negligible contributions from pomegranate constituents. This confirms the observation made when the juices were analysed in the FRAP assay before and after treatment with ascorbate oxidase (Table 2). 2.3 Quantification of the phenolic pomegranate markers in the 26 juices analyzed The results for the quantification of ellagitannins and anthocyanins and the overall HPLC total phenolics for the 26 pomegranate juices and pomegranate juice blends are presented in Table 6. For the anthocyanin quantification all the peaks appearing in 520 nm traces were quantified in cyanidin-3-Oglucoside equivalents. As expected, the levels of the total HPLC phenolics quantified for the three top samples (PG01, PG02, PG03) were of the order of 2 mmol/L, much higher than the other 23 samples (Table 6), in agreement with the AOX results in Table 2. 2.3.1 Anthocyanins. The levels of anthocyanins did not reflect the AOX indices of the juices. PG01–PG03 all had a high AOX content (Table 2) but PG01 and PG03 contained low amounts of total anthocyanins (68 and 11 mmol/L respectively) relative to PG02 (344 mmol/L). This was visually apparent when comparing This journal is ª The Royal Society of Chemistry 2010

the intense dark red colour of PG02 juice with the dark brownish colour of PG01 and PG03. Anthocyanins are located in the flesh of the arils of the pomegranate fruit and are positively correlated with the juice colour.9 In the case of the blended pomegranate juices, anthocyanins were derived from other fruit in addition to pomegranate. Several, most notably PG11 which comprised 18.5% raspberry and had a 288 mmol/L anthocyanin content, contained substantial amounts of anthocyanins (Table 6) but did not possess high AOX activity (Table 2). Although the total quantities of anthocyanins in the 100% pomegranate juices varied substantially, as discussed above, the 520 nm anthocyanin HPLC profiles were similar with only slight differences in the relative amounts of cyanidin-3,5-O-diglucoside (peak 8) and cyandin-3-O-glucoside (peak 14) (Fig. 1–3). Likewise, a similar profile was obtained with PG04 (Fig. 4) and also PG06 (Fig. 5). In both these juices, however, peak 12, pelargonidin-3,5-O-diglucoside, was much more prominent. PG04 also contained an anthocyanin peak (marked *) which was not detected in the other 100% pomegranate juices. This peak had a [M  H]+ at m/z 949 which produced MS2 fragments at m/z 611, 449 and 287 indicating a cyanidin-based compound. The unusual mass spectrum and the relatively late elution of this component suggest that it might be a cyanidin-O-feruloyl-triglucoside. Among the blended pomegranate products, PG14, a 29% pomegranate, 7% aronia mixture, had an anthocyanin HPLC profile dominated by aronia anthocyanins26 principally in the form of cyanidin-3-O-galatoside and cyanidin-3-O-arabinoside, rather than pomegranate anthocyanins (Fig. 6). PG14 did, however, contain ellagitannins, suggesting that the pomegranate components might be derived from rind rather than arils which, as noted earlier, are the principal source of pomegranate anthocyanins.9 PG33, which is a 8% raspberry/pomegranate blend had a raspberry rather than a pomegranate anthocyanin fingerprint with cyanidin-3-O-sophoroside being the main component24 (Fig. 7). 2.3.2 Ellagitannins. This group of hydrolysable tannins, comprising punicalins, punicalagins, and granatins, occurs mainly in the peel, piths and arils of pomegranate.19,27 PG02, which was high in anthocyanins, contained less punicalins than Food Funct., 2010, 1, 73–83 | 79

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178 261 158 62 58 123 81 78 53 48 0 41 32 25 14 45 29 62 7 16 22 26 25 13 11 6

972 345 512 236 167 230 543 331 375 287 8 248 172 250 63 237 263 199 5 32 121 137 18 37 39 36

22 14 26 5 3 8 12 9 12 7 0 5 2 5 3 2 4 5 0 0 3 3 0 1 2 0

30 18 34 7 7 9 24 16 14 13 0 9 5 10 5 4 9 9 0 0 4 4 0 2 3 0

58 63 125 14 18 21 58 33 23 20 0 5 5 8 20 3 10 18 0 0 6 5 0 2 7 1

181 196 382 48 66 61 169 97 70 58 2 16 33 26 55 11 28 56 0 0 15 13 1 8 19 3

123 136 116 68 78 34 44 37 33 22 0 13 15 18 28 15 16 27 0 0 13 17 13 6 13 2

47 35 48 20 18 11 32 21 21 13 1 17 9 14 10 7 12 9 0 4 7 7 8 0 7 1

1611 1068 1401 460 415 497 963 622 601 468 11 354 273 356 198 324 371 385 12 52 191 212 65 69 101 49

68 85 116 19 23 33 36 37 28 29 3 7 9 16 17 5 17 15 10 4 13 14 4 5 8 3

214 408 515 104 160 96 98 199 65 102 21 32 39 122 34 16 42 52 10 7 26 50 20 25 28 14

282 493 631 123 183 129 134 236 93 131 24 39 48 138 51 21 59 68 20 11 39 64 24 30 36 18

68 344 11 115 155 9 18 30 62 26 288 23 34 52 159 1 46 35 222 101 3 74 151 77 112 14

1961 1905 2043 698 753 635 1115 888 756 625 323 416 355 546 408 346 476 488 254 164 233 350 240 176 249 81

Data expressed as mean values in mmol/L. The standard error (n ¼ 3) values (not shown), were less than 10% of the mean values. + 100% pomegranate juices, * reconstituted or blended pomegranate juices.

a

PG01+ PG02+ PG03+ PG04+ PG05+ PG06+ PG07* PG08* PG09* PG10* PG11* PG13* PG14* PG15* PG16* PG17* PG18* PG20* PG21* PG24* PG26* PG27* PG29* PG30* PG31* PG33*

Punicalins 2-O-Galloyl Punicalagin Total Ellagic acid Total ellagic Total Punicalagin-like A and B punicalagin Punicalagin A Punicalagin B isomer Granatin A Granatin B ellagitannins hexose Ellagic acid Acid anthocyanins Total

Table 6 Quantification of phenolic and polyphenolic compounds in 26 commercial pomegranate juices.a

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PG01 and PG03 and, as a consequence, had lower total ellagitannin content (Table 6). This implies that with PG01 and PG03, proportionally more rind was extracted than with PG02. However, this does not explain the similar total AOX capacity of the three juices (Table 2). Further investigation is required, but this could be a consequence of polymeric ellagitannins and or other high molecular weight compounds with AOX activity in P02 being retained on the HPLC column and therefore not contributing to the on-line AOX measurements. 2.3.3 Ellagic acid. Ellagic acid and an ellagic acid hexose conjugate were detected in substantial amounts in the pure pomegranate juices and typically in smaller amounts in the blended pomegranate drinks. The concentration of ellagic acid and its hexose conjugate ranged from 11 mmol/L (PG24) to 631 mmol/L (PG03) (Table 6). Ellagic acid, which is a product of the hydrolysis of ellagitannins, has been used as a marker for assuring commercial pomegranate extracts are made from genuine pomegranate fruit.11,12 However, this is not necessarily an accurate measure of authenticity as it does not distinguish between pomegranate ellagic acid and ellagic acid derived from other sources of ellagitannins including berries, such as blackberries,22 raspberries,24 and cheaper material such as chestnut bark.23 The Pearson’s correlation coefficients (Table 7) confirmed the significant relationship between the total ellagitannin and ellagic acid contents and the in vitro AOX capacity of the juices measured by TP, FRAP, ORAC and TEAC of the juices. In contrast in vitro AOX capacity was not associated with anthocyanin levels. This in agreement with earlier observations9,19 that anthocyanins make, at best, a very minor contribution to the AOX capacity of pomegranates.

Table 7 Pearson’s correlations coefficient.a

Assay

Total ellagitannins

Total ellagic acid

Total anthocyanins

TP FRAP ORAC TEAC

0.879(***) 0.918(***) 0.730(***) 0.919(***)

0.899(***) 0.895(***) 0.815(***) 0.906(***)

NS NS NS NS

a

*** Correlation is significant at p < 0.001. NS, not significant.

3.3 Extraction of juices A 500 mL aliquot of juice was added to 500 mL of methanol and shaken for 3 min. The mixture was then centrifuged at 13000 g at 4  C for 5 min and the supernatant stored at 80  C prior to analysis. 3.4 Analysis of vitamin C The vitamin C (ascorbic acid) content of the juices was assessed using HPLC-PDA as described by Ross28 with a Surveyor HPLC system (Thermo-Fisher, Scientific, Waltham, MA). Separation was carried out using a 5 mm 250  4.6 mm i.d. Nucleosil C18 column (Phenomenex, Macclesfield, UK) fitted with a C18 guard cartridge. The column was eluted isocratically with a mobile phase comprising 0.05 mM sodium hydroxide, 25 mM myristyltrimethylammonium bromide, 0.06 M acetic acid, 7.5% acetonitrile mobile phase containing 100 mg/L homocysteine and 200 mg/L EDTA. The system was operated at 40  C with a flowrate of 0.6 mL/min and absorbance detection at 265 nm. The amount of ascorbic acid was calculated by reference to 0–200 mM vitamin C calibration curve.

3 Experimental

3.5 Total phenol content

3.1 Chemicals

The TP content of the juices was determined in triplicate, in diluted samples, using the Folin-Ciocalteu assay.13 The data were recorded in gallic acid equivalents (GAE).

5-O-Caffeoylquinic acid, potassium persulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ascorbate oxidase (EC 1.10.3.3) and 20 ,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) were purchased from Sigma-Aldrich (Poole, UK). Ellagic acid was obtained from AASC Ltd (Southampton, UK). Cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside were purchased from Extrasynthese (Genay, France), and methanol was obtained from Rathburn Chemicals (Walkerburn, Scotland). Formic acid and acetic acid were supplied by Fisher Scientific (Loughborough, UK). Punicalagin was purchased from LGC Standards (Teddington, Middlesex, UK).

3.6 Ferric-reducing antioxidant power assay The FRAP assay was used to estimate the AOX capacity of the juices. It measures the ability of a solution to reduce a ferrictripyridyl-triazine complex (Fe3+-TPTZ) to the ferrous form, Fe2+, producing a blue color with absorption at 593 nm. One and a half mL of freshly prepared FRAP reagent (containing the Fe3+-TPTZ in excess at pH 3.6), was added to 50 mL of juice and 150 mL water. The absorbance at 593 nm, measured after a 4 min reaction period, was compared to a 0 to 1 mM Fe2+ standard curve.14

3.2 Juices Thirty six European juices were procured commercially. The brands and ingredients described on the label are shown in Table 1. The first six juices were labelled as ‘‘100% pomegranate juices’’, and the remainder included 20 brands of diluted pomegranate juice or pomegranate blended with other fruit juices and 10 different non-pomegranate fruit juices. This journal is ª The Royal Society of Chemistry 2010

3.7 Contribution of vitamin C to FRAP antioxidant activity Vitamin C reacts almost instantaneously in the FRAP assay. To determine the contribution of vitamin C to the antioxidant activity of the juices, it was selectively destroyed by the addition of ascorbate oxidase. Twenty mL of a 4 U/mL enzyme solution was added to one of a pair of juice aliquots before the FRAP Food Funct., 2010, 1, 73–83 | 81

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reaction. Standard solutions of ascorbic acid (0–0.5 mM), in the presence and absence of the enzyme, were tested.

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3.8 Oxygen radical absorbance capacity assay The principle of the ORAC assay is to monitor the capability of a test antioxidant to quench the fluorescent signal obtained when fluoroscein is exposed to an oxygen radical generator (2,20 -azobis-2-methyl-propionamide). The standard means of ‘‘normalising’’ the data requires comparison of the inhibitory effect of the test agent with that of Trolox, a water-soluble analogue of a-tocopherol. Comparing the area under the curve for 60 min incubations is the conventional means of analysing data obtained with this method.16

3.9 Trolox equivalent antioxidant capacity assay After 12 h in darkness, a stock solution of 7 mM ABTS and 2.45 mM potassium persulfate was diluted with ethanol to an absorbance of 0.70 at 734 nm. Diluted juice samples were mixed with 1 mL of the ABTS solution and after 5 min, absorbance determined at 734 nm. TEAC values were calculated by reference to a Trolox standard curve.15 The same basic procedure was applied with the HPLC on-line antioxidant detection system described below. 3.10 HPLC with PDA, MS2 and AOX detection Analysis was carried out on a Surveyor HPLC system comprising of an autosampler with sampler cooler maintained at 4  C, a PDA detector (Thermo Fisher Scientific), scanning from 200– 600 nm. Samples were analysed on a 250  4.6 mm Gemini C6 phenyl column (Phenomenex, Macclesfield, UK), maintained at 40  C using a 40 min mobile phase gradient of 5 to 60% methanol in 0.1% aqueous formic acid at a flow rate of 1 ml/min. After passing through the flow cell of the PDA detector, the eluate was split and 200 mL directed to a LCQ Advantage ion trap mass spectrometer fitted with an electrospray interface (Thermo Fisher Scientific). Capillary temperature was 300  C, sheath gas and auxiliary gas were 60 and 20 units respectively, the source voltage was 4 kV. Samples were analysed using full scan in both positive and negative ionisation modes, the scan range was from 150–2000 m/z for negative ion and 190–1000 m/z for positive ion. Identifications are based on co-chromatography with authentic standards, where available. Absorbance spectra and mass spectra, using MS2, were used to identify compounds reported previously in the literature. For the detection of components with AOX activity, the remaining 800 mL/min of the HPLC eluate was mixed with an ABTS solution flowing at 0.5 mL/min and the resultant mixture passed through a holding coil before being directed to a P2000 absorbance detector (Nemphlar Bioscience, Lanark, UK) operating at 720 nm.17

3.11 Statistical analysis The data were analyzed by SPSS software ver.14.0 to calculate Pearson correlation coefficients. 82 | Food Funct., 2010, 1, 73–83

4

Conclusions

Although consumption of dietary flavonoids and polyphenolics has been increasingly implicated in health benefits it is remains unclear as to whether or not they function in vivo by directly modulating the body’s AOX network.29 There is growing evidence that in the body, they function in more subtle ways, at low concentrations, by regulating processes such as signal transduction pathways.30 None-the-less, monitoring the AOX and/or total phenolic content of plant-derived foods, including fruit juices, provides a useful initial guide to their potential protective effects as polyphenol-rich products, such as PG01– PG03, are more likely to have a beneficial effects on health effects than the more dilute blended juices (Table 2). It is also necessary to identify at this stage, juices such PG14 and PG33, that contain relatively low levels of fruit but have their AOX capacity boosted by the presence of substantial amounts of vitamin C (Table 2, Fig. 1F and 1G). The results of this study have provided an insight into the differences in both AOX activity and the concentrations of the main phenolic compounds in pure or blended pomegranate juices sold in Europe. While the ellagitannin profile can be used as a fingerprint for confirmation of the origin of the juice, it cannot on its own be used to judge purity or quality. It was, for instance, evident that the PG01 and PG03 juices, both of which had a very high AOX index, were authentic pomegranate juices from their ellagitannin profiles. However, the anthocyanin content of these juices was low compared to PG02 suggesting that juice from the arils had been diluted by more extensive extraction of the rind of the pomegranates. This may have resulted in PG01 and PG03 having an astringent taste which, arguably, may be masked by the addition of sweetener. The dark brown colour of these juices is in keeping with their low anthocyanin content. The anthocyanins, although not associated with AOX activity, readily provide an additional specific fingerprint of pomegranate juice authenticity. The concentration of anthocyanins, along with the ellagitannin profile, can be used as indicators of both authenticity and quality of pomegranate juices. The HPLCPDA-MS methodology utilised in this study provides a means of assessing, not just potential adulteration of pomegranate juices and drinks, but also that of a diversity of other fruit-based beverages.

5

Acknowledgements

This study was funded by a donation from POM Wonderful LLC, Los Angeles, CA, USA.

6

References

1 M. G. L. Hertog, E. J. M. Feskens, P. C. H. Hollman, M. B. Katan and D. Kromhout, Lancet, 1993, 342, 1007–1011; B. Margetts, in Plants: Diet and Health, ed. G. Goldberg, Blackwell Publishing, Oxford, 2003, pp. 49–64. 2 www.FoodandDrinkEurope.com. 3 A. J. Pantuck, J. T. Leppert, N. Zomorodian, W. Aronson, J. Hong, R. J. Barnard, N. Seeram, H. Liker, H. Wang, R. Elashoff, D. Heber, M. Aviram, L. Ignarro and A. Belldegrun, Clin. Cancer Res., 2006, 12, 4018–4026. 4 M. Rosenblat, T. Hayek and M. Aviram, Atherosclerosis, 2006, 187, 363–371.

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5 W. Rock, M. Rosenblat, R. Miller-Lotan, A. P. Levy, M. Elias and M. Aviram, J. Agric. Food Chem., 2008, 56, 8704–8713. 6 S. G. Kasimsetty, D. Bialonska, M. K. Reddy, G. Ma, S. I. Khan and D. Ferreira, J. Agric. Food Chem., 2010, 58, 2180–2187. 7 W. Mullen, S. Marks and A. Crozier, J. Agric. Food Chem., 2007, 55, 3148–3157. 8 N. P. Seeram, M. Aviram, Y. Zhang, S. M. Henning, L. Feng, M. Dreher and D. Huber, J. Agric. Food Chem., 2008, 56, 1415–1422. 9 R. Tzulker, I. Glazer, I. Bar-Ilan, D. Holland, M. Aviram and R. Amir, J. Agric. Food Chem., 2007, 55, 9559–9570. 10 M. I. Gil, J. Cherif, N. Ayed, F. Artes and F. A. Tomas-Barberan, Z. Lebens. Unter. Forschung, 1995, 201, 361–364; E. Shwartz, I. Glazer, I. Bar-Ya’akov, I. Matityahu, I. Bar-Ilan, D. Holland and R. Amir, Food Chem., 2009, 115, 965–973. 11 Y. J. Zhang, D. Wang, R. P. Lee, S. M. Henning and D. Heber, J. Agric. Food Chem., 2009, 57, 7395–7400. 12 Y. J. Zhang, D. Krueger, R. Durst, R. Lee, D. Wang, N. Seeram and D. Heber, J. Agric. Food Chem., 2009, 57, 3961–3961. 13 V. L. Singleton and J. A. Rossi, Am. J. Enol. Vit., 1965, 16, 144–158. 14 I. F. F. Benzie and J. J. Strain, Anal. Biochem., 1996, 239, 70–76. 15 R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, Free Radical Biol. Med., 1999, 26, 1231–1237. 16 D. J. Huang, B. X. Ou, M. Hampsch-Woodill, J. A. Flanagan and R. L. Prior, J. Agric. Food Chem., 2002, 50, 4437–4444. 17 A. J. Stewart, W. Mullen and A. Crozier, Mol. Nutr. Food Res., 2005, 49, 52–60.

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18 T. Tanaka, G. Nonaka and I. Nishioka, Chem. Pharm. Bull. (Tokyo), 1986, 34, 650–655. 19 M. I. Gil, F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft and A. A. Kader, J. Agric. Food Chem., 2000, 48, 4581–4589. 20 T. Tanaka, G. I. Nonaka and I. Nishioka, Chem. Pharm. Bull. (Tokyo), 1990, 38, 2424–2428. 21 F. Hernandez, P. Melgarejo, F. A. Tomas-Barberan and F. Artes, Eur. Food Res. Technol., 1999, 210, 39–42; G. A. Miguel, C. Fontes, D. Antunes, A. Neves and D. Martins, J. Biomed. Biotechnol., 2004, 338–342. 22 T. L. Hager, L. R. Howard, R. Liyanage, J. O. Lay and R. L. Prior, J. Agric. Food Chem., 2008, 56, 661–669. 23 S. A. Vekiari, H. M. Gordon, P. Garcia-Macias and H. Labrinea, Food Chem., 2008, 110, 1007–1011. 24 G. Borges, A. Degeneve, W. Mullen and A. Crozier, J. Agric. Food Chem., 2010, 58, 3901–3909. 25 K. R. Martin, C. G. Krueger, G. Rodriquez, M. Dreher and J. D. Reed, J. Sci. Food Agric., 2009, 89, 157–162. 26 J. Oszmianski and J. C. Sapis, J. Food Sci., 1988, 53, 1241–1242. 27 A. P. Kulkarni, S. M. Aradhya and S. Divakar, Food Chem., 2004, 87, 551–557. 28 M. A. Ross, J. Chromatogr., B: Biomed. Sci. Appl., 1994, 657, 197– 200. 29 M. Serafini, J. Sci. Food Agric., 2006, 86, 1989–1991. 30 A. Crozier, I. B. Jaganath and M. N. Clifford, Nat. Prod. Rep., 2009, 26, 1001–1043.

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PAPER

www.rsc.org/foodfunction | Food & Function

Hypocholesterolemic activity of onion is mediated by enhancing excretion of fecal sterols in hamsters Lei Guan,a Hau Yin Chung,*ab Yalun Su,c Rui Jiao,b Cheng Pengb and Zhen Yu Chen*b

Downloaded on 21 October 2010 Published on 13 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00036A

Received 8th June 2010, Accepted 13th July 2010 DOI: 10.1039/c0fo00036a Onion has been shown to favorably modify the lipoprotein profile. However, research on its underlying mechanism is lacking. The present study investigated the interaction of dietary onion powder with the protein expression of key receptors and enzymes involved in cholesterol metabolism. Thirty-six male hamsters were randomly divided into three groups and fed a high-cholesterol control diet or the two experimental diets supplemented with 1% onion powder (OP-1) or 5% onion powder (OP-5), for a period of 8 weeks. It was found that onion dose-dependently decreased plasma total cholesterol (TC) level. The change in plasma lipoprotein profile was accompanied by a greater excretion of both fecal neutral and acidic sterols. Western blot analysis revealed that onion up-regulated sterol regulatory element binding protein 2 (SREBP-2), liver X receptor alpha (LXRa) and cholesterol-7a-hydroxylase (CYP7A1) with no effect on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDL-R). It was concluded that the hypocholesterolemic activity of onion powder was mediated by enhancement of fecal sterol excretion and up-regulation of LXRa and CYP7A1.

Introduction Onion has been used as an ingredient in food by many cultures. It has been shown that onion possesses various biological activities including being antibiotic, antidiabetic, antioxidant, antiatherogenic, and anticancer.1 Traditionally, onion is also used to treat fever, dropsy, chronic bronchitis, colic, and scurvy. The chemistry of onion has been a subject of the extensive investigations. The active ingredients responsible for these activities in onion are claimed to be quercetin, methiin, propiin, isoalliin, alkyl thiosulfinate, disulfides and polysulfides.2,3 Plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) correlate directly with risk of coronary heart disease (CHD), whereas the high-density lipoprotein cholesterol (HDL) correlates inversely with the risk. Epidemiological studies have shown a correlation between diets rich in onion and a reduced risk of mortality from coronary heart disease CHD.4,5 In healthy subjects receiving 100 g butter fat, onion juice demonstrated a significant protective action against fat-induced increases in serum TC and plasma fibrinogen with a concomitant decrease in coagulation time and fibrinolytic activity.6 Results from animal trials support this notion. Onion powder decreased blood glucose, serum TC and reduced the oxidative stress in STZ-induced diabetic rats.7 In SD rats fed a high-fat highsucrose diet, onion was effective in lowering both plasma and hepatic TC and triacylglycerols (TG).8 When raw onion was added into diet of healthy pigs, a moderate reduction in plasma lipids was also observed.9

a Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China b Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. E-mail: zhenyuchen@ cuhk.edu.hk; [email protected]; Fax: (+852) 26035745 c Institute of Materia Medica, Chinese Academy of Medica Sciences and Peking Union Medical College, Beijing, China

84 | Food Funct., 2010, 1, 84–89

Plasma TC is mainly maintained by sterol regulatory elementbinding protein 2 (SREBP-2) and liver X receptor-alpha (LXRa) in a coordinated manner.10 SREBP-2 governs the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDL-R). HMGR is a rate-limiting enzyme in cholesterogenesis, while LDLR is responsible for the removal of LDL from the circulation. LXRa activates transcription of a gene encoding cholesterol-7a-hydroxylase (CYP7A1), which is a rate-limiting enzyme in conversion of cholesterol to bile acids and responsible for elimination of excessive cholesterol in the liver.10 Despite extensive research on onion, little is known of how consumption of onion interacts with these genes and proteins involved in cholesterol metabolism in vivo. The present study was therefore undertaken to characterize the interaction of dietary onion with SREBP-2, LXRa, HMGR, LDL-R, and CYP7A1 in attempt to explore the underlying cholesterollowering mechanism.

Experimental Diet Red onion (Allium cepa Linn.) was obtained from a local market in Xinjiang, China. Onion was soaked in three volumes of boiling water. The extract was then filtered and spray-dried, leading to production of the white onion powder (OP). Three diets were prepared in the present study with the control diet being mixed with the following ingredients in proportion (g kg1 diet): cornstarch, 508; casein, 242; lard, 50, sucrose, 119; mineral mix, 40; vitamin mix, 20; DL-methionine, 1; cholesterol, 1. The two experimental diets were prepared by adding 1% onion powder (OP-1) and 5% onion powder (OP-5) by weight into the control diet, respectively. The powdered diets were mixed with a gelatin solution (20 g L1) in a ratio of 200 g diet per litre of solution (Table 1). Once the gelatin had set, the diets were cut into pieces of approximately 10 g cubes and stored frozen at 20  C. This journal is ª The Royal Society of Chemistry 2010

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Table 1 Composition (g) of the control and two experimental diets supplemented with 1% onion powder (OP-1) and 5% onion powder (OP-5) Main nutrients

Control

OP-1

OP-5

Corn starch Casein Lard Sucrose Mineral mixture Vitamin mixture DL-Methionine Cholesterol Onion powder Gelatin

508 242 50 119 40 20 1 1 0 20

508 242 50 119 40 20 1 1 10 20

508 242 50 119 40 20 1 1 50 20

Hamsters Golden Syrian male hamsters (n ¼ 36, 100–120 g) were housed in an animal room at 25  C with a 12 : 12 h light-dark cycle. The entire experiment was approved and conducted in accordance with the guidelines set by the Animal Experimental Ethical Committee, The Chinese University of Hong Kong. Hamsters were allowed free access to a standard cereal-based chow diet (PicoLab Rodent Diet20-Lab Diet, Australia) and water for a 2 week acclimation period. Afterwards, all hamsters were allowed free access to the control diet for additional two weeks. They were then randomly divided into three groups (n ¼ 12 epr group), weighed, ear-punched, and fed the control, OP-1 or OP-5 diets for a period of 8 weeks. The body weight was recorded once a week, food consumption was recorded every 2 days, and total fecal output was collected weekly. Blood was collected from the retro-orbital sinus into a heparinized capillary tube under light anesthetization, using a mixture of ketamine, xylazine and saline (v/v/v, 4 : 1 : 5) after 16 h food deprivation at weeks 0, 4, 8 and 12. At the end of 12 weeks, all the hamsters were killed after overnight fasting. Blood was collected via the abdominal aorta. The liver, heart, kidney, testis, perirenal fat and epididymal fat were also removed, rinsed with ice-cold saline, weighed, flash frozen in liquid nitrogen and stored at 80  C until analysis. Plasma lipid and lipoprotein determinations Plasma TC and TG levels were determined using enzymatic kits obtained from Infinity (Waltham, MA, USA) and Stanbio Laboratories (Boerne, TX, USA), respectively. The concentration of HDL was measured after precipitation of LDL and very low-density lipoprotein (VLDL) with phosphotungstic acid and magnesium chloride, using a commercial kit (Stanbio Laboratories). Non-HDL was calculated from the difference between TC and HDL. Determination of organ cholesterol Cholesterol in organs was determined using a method described previously.11 The liver (100 mg) and heart (300 mg) were used to determine the cholesterol level. In brief, the samples with addition of 1 mg stigmastanol, as an internal standard, were homogenized in 15 ml of chloroform–methanol (2 : 1, v/v) and 3 ml saline. The chloroform–methanol phase was removed and dried under a gentle stream of nitrogen gas. After the lipid extract This journal is ª The Royal Society of Chemistry 2010

was mildly saponified in 5 ml of 1 N NaOH in 90% ethanol at 90  C for one hour, 6 ml of cyclohexane were added to extract the total cholesterol. The cyclohexane phase was evaporated to dryness under nitrogen gas, and the cholesterol was converted to its trimethylsilyl (TMS)-ether derivative. The TMS-ether derivative was dissolved in hexane for GC analysis. Determination of fecal neutral and acidic sterols Neutral and acidic sterols in the feces were determined as we described previously with slight modifications.11 Dried fecal samples (300 mg) were mildly hydrolyzed with 1 N NaOH in 90% ethanol at 90  C for 1 h. Then, total neutral sterols were extracted with cyclohexane, and converted into their TMS-ether derivatives. The acidic sterol-containing lower aqueous phase was similarly saponified and converted into their TMS-ether derivatives. The two TMS-ether derivatives were subjected to the GC analysis. Western blotting analysis of liver SREBP-2, LDL-R, HMGR, LXRa and CYP7A1 Liver protein was extracted according to the method described previously by Vaziri and Liang with some modification.12 In brief, the liver sample was homogenized in a homogenizing buffer containing 20 mM Tris-HCL (pH 7.5), 2 mM MgCl2, 0.2 M sucrose and Complete protease inhibitor cocktail (Roche, Mannheim, Germany). The extract was centrifuged at 13 000 g for 15 min at 4  C and the supernatant was collected (total protein). The total protein was centrifuged at 126 000 g for 60 min at 4  C. The pellet was re-suspended in the same homogenizing buffer. The pellet protein was separated by electrophoresis on a 7% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using a semi-dry transfer system. Membranes were blocked in 5% nonfat milk Tris-buffered saline with Tween-20 for 1 h and overnight at 4  C in the same solution containing 1 : 600 anti-LDL-R antibody (Santa Cruz Biotechnology, Inc., California, USA), 1 : 500 anti-HMGR (Upstate USA Inc., Lake Placid, NY, USA), 1 : 200 anti-CYP7A1 (Santa Cruz Biotechnology, Inc., California, USA), 1 : 400 anti-LXRa antibody, or anti-SREBP-2 antibody (Santa Cruz Biotechnology).13 The membrane was then incubated for one hour at 4  C in diluted (1 : 3000) horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz Biotechnology, Inc. California, USA), donkey antirabbit IgG (Santa Cruz Biotechnology, Inc. California, USA) or goat anti-mouse IgG (Calbiochem, EMD Chemicals, Inc., San Diego, CA, USA). Then, membranes were developed with ECL enhanced chemiluminescence agent (Santa Cruz Biotechnology, Inc., California, USA) and subjected to autoradiography on SuperRX medical X-ray film (Fuji, Tokyo, Japan). Densitometry was quantified using the BioRad Quantity one software (BioRad Laboratories, Hercules, USA). Data on abundance of LDLR, HMGR, CYP7A1, LXR and SREBP-2 were normalized with b-actin (Santa Cruz Biotechnology, Inc., California, USA). Statistics Data are expressed as mean  standard deviation (SD). Treatment effects were statistically analyzed among the three groups Food Funct., 2010, 1, 84–89 | 85

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using one-way analysis of variance (ANOVA) and post hoc LSD test on SigmaStat Advisory Statistical Software (SigmaStat version 16.0, SPSS Inc., Chicago, IL, USA). P-values less than 0.05 are regarded as statistically significant.

Results

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Body weight, food intake, food efficiency, and relative organ weight Onion consumption had no effect on body weight among the control, OP-1 and OP-5 groups (Table 2). Food intake was expressed as g per hamster per day, whereas the food efficiency ratio was defined as the body weight gained by each hamster that consumed every 100 g of diet. It was found that food intake and food efficiency did not differ among the three groups. No difference in relative organ weight was seen among the three groups, although hamsters fed the onion powder at both doses had smaller epididymal and peripheral pads than the control group.

Plasma lipoproteins and organ cholesterol level No difference in plasma lipoprotein profiles was seen among the three groups at week 0 (Table 3). At week 4 and 8, OP-5 group had plasma TC decreased by 11.2% and 20.3%, respectively, compared with the control group. Non-HDL concentration in OP-5 groups was also significantly decreased by 20.3% and 23.4%, respectively, compared with that in the control group. Similarly, the ratio of non-HDL to HDL was reduced significantly in OP-5 group compared with that in the control hamsters (Table 3). Although plasma TC and non-HDL in OP-1 group was lower, they were not statistically different from those in the control hamsters. Initially, no difference in plasma TG was seen among the three groups. At week 4, plasma TG showed a decreasing trend in the two experimental group. However, the difference in plasma TG between OP-5 and the control group became statistically significant at week 8 (Table 3). No significant Table 2 Body weight, food intake, food efficiency, and relative organ weight in hamsters fed the control, and two experimental diets supplemented with OP-1 and OP-5

Body weight (g) Initial Final Food Intake (g per hamster per day) Food Efficiency (g per 100 g diet)

Control

OP-1

OP-5

127.1  6.6 138.0  11.8

124.2  10.2 131.0  8.4

124.6  12.1 132.2  10.3

12.19  0.79

11.56  0.56

12.11  0.97

7.32  1.40

7.72  1.27

8.07  1.70

Relative organ weight (g per 100 g body weight) Liver 4.06  0.15 4.06  Heart 0.35  0.03 0.35  Kidney 0.80  0.06 0.80  Epididymal fat 1.52  0.27 1.45  Peripheral fat 0.93  0.15 0.88  Data expressed as mean  SD; n ¼ 12 each group.

86 | Food Funct., 2010, 1, 84–89

0.29 0.02 0.02 0.22 0.14

4.04  0.16 0.36  0.03 0.78  0.09 1.46  0.33 0.87  0.16

Table 3 Changes in plasma TC, HDL, non-HDL, HDL/TC, non-HDL/ HDL in hamsters fed the control diet, and two experimental diets supplemented with 1% onion powder (OP-1) and 5% onion powder (OP-5) for 8 weeks Control

OP-1

OP-5

TC (mg/dl) Initial 4th week 8th week

207.4  36.4 216.4  24.9a 215.1  39.6a

207.2  48.9 199.4  38.5ab 204.7  46.8ab

207.3  48.0 192.2  20.6b 171.4  25.0b

HDL (mg/dl) Initial 4th week 8th week

80.6  14.3 102.8  13.9 96.48  8.0a

84.7  10.2 112.4  20.0 102.83  10.1a

83.9  12.1 102.5  17.1 84.31  7.4b

non-HDL (mg/dl) Initial 126.8  29.9 4th week 118.0  7.7 8th week 107.4  27.8a

122.5  41.0 97.7  41.1 99.5  26.4ab

123.5  42.1 94.1  15.0 82.3  13.4b

HDL/TC Initial 4th week 8th week

0.39  0.06 0.46  0.04 0.45  0.05b

0.42  0.07 0.50  0.11 0.49  0.09ab

0.42  0.07 0.51  0.06 0.52  0.03a

non-HDL/HDL Initial 1.60  0.35 4th week 1.17  0.32 8th week 1.19  0.25a

1.43  0.38 0.99  0.54 0.97  0.23ab

1.48  0.47 0.97  0.21 0.95  0.11b

TG (mg/dl) Initial 4th week 8th week

157.6  45.3 172.9  50.5 166.3  73.6b

147.3  73.2 167.1  43.9 145.4  36.0b

88.5  11.3 3.7  0.3

77.6  13.3 3.6  0.2

155.4  39.7 211.9  61.7 218.3  92.0a

Organ cholesterol (mg/g) Liver 80.2  10.5 Heart 3.7  0.4

Non-HDL¼ [TC]-[HDL]. Data expressed as mean  SD; n ¼ 12. Means at the same raw with different superscripts (a, b, c) differ significantly at p < 0.05.

differences were found in hepatic and heart cholesterol content among the three groups (Table 3). Fecal sterols The total neutral sterols are the sum of cholesterol, coprostanol, coprostanone, dihydrocholesterol, campesterol, b-sistosterol and stigmastanol. Data on fecal analysis were similar in week 4 and 8. To simplify the presentation, only data for week 8 is shown. Supplementation of onion powder into diets caused greater fecal excretion of neutral sterols compared with the control group (Table 4). The total acid sterols were the sum of lithocholic, deoxycholic, chenodeoxycholic, cholic and ursodeoxycholic acids. Similarly, onion demonstrated a dose-dependent increasing trend in fecal excretion of acidic sterols (Table 4). Cholesterol balance Data on the apparent cholesterol absorption was calculated as we previously described.13 Total intake of cholesterol was compared with its excretion in neutral and acidic sterols (Table 5). Net cholesterol equivalent retained was calculated by This journal is ª The Royal Society of Chemistry 2010

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Table 4 Changes in fecal output of individual neutral and acidic sterols in hamsters fed the control, and two experimental diets supplemented with 1% onion powder (OP-1) and 5% onion powder (OP-5) in week 8

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Neutral sterols (mg/day) Coprostanol Coprostanone Cholesterol Dihydrocholesterol Campesterol Stigmasterol b-sitosterol Total Acidic sterols (mg/day) Lithocholic acid Deoxycholic acid Chenodeoxycholic + Cholic acids Ursodecholic acid Total

Control

OP-1

OP-5

0.59  0.14 0.03  0.01 0.79  0.19 0.35  0.06 ND ND ND 1.76  0.38b

0.70  0.01 0.05  0.01 0.81  0.14 0.38  0.02 0.06  0.01 0.11  0.01 0.03  0.01 2.14  0.15ab

0.86  0.05  0.84  0.40  0.10  0.15  0.05  2.45 

1.95  1.25 0.52  0.11 1.14  0.89

3.14  0.74 0.38  0.05 0.68  0.22

3.65  0.76 0.45  0.31 0.57  0.95

0.26  0.05 3.88  0.19b

0.28  0.06 4.49  0.97ab

0.59  0.08 5.25  0.89a

0.43 0.01 0.15 0.03 0.04 0.01 0.02 0.34a

a Data expressed as mean  SD; n ¼ 12. Means at the same raw with different superscripts (a, b, c) differ significantly at p < 0.05.

Table 5 Cholesterol balance in hamsters fed the control diet, and two experimental diets supplemented with 1% onion powder (OP-1) and 5% onion powder (OP-5) for 8 weeks

Food intake (g/d/hamster) Neutral Sterol (mg/d/hamster)# Acidic Sterol (mg/d/hamster) Total Sterol output (mg/d/hamster) Cholesterol Retained (mg/d/hamster) Apparent cholesterol absorption (% intake)

Control

OP-1

OP-5

12.19  0.79

12.04  0.51

12.28  0.67

1.76  0.38

1.94  0.16

2.15  0.31

3.88  0.19

4.49  0.97

5.25  1.12

5.64  0.56

6.53  0.61

6.84  0.69

6.53  1.46

5.03  0.63 a

54.73  6.84

5.17  0.98 ab

46.61  6.75

39.74  3.41b

Data expressed as mean  SD; n ¼ 12. Means at the same raw with different superscripts (a, b, c) differ significantly at p < 0.05. # Excluding the exogenous sterols namely campesterol, stigmasterol and b-sitosterol.

Fig. 1 The relative immunoreactive mass of hepatic SREBP-2, HMGCoA-R, and LDL-R in hamsters fed the control diet, and two experimental diets supplemented containing 1% onion powder (OP-1) and 5% onion powder (OP-5) for 8 weeks. Data were normalized with b-actin and values were expressed as mean  SD, n ¼ 12.

Discussion difference between the intake and the excretion of both neutral and acidic sterols. It was found that the net cholesterol retention was the highest in the control group, followed by OP-1 and OP-5 in decreasing order. The apparent cholesterol absorption was calculated by the equation [(cholesterol intake  excretion of neural and acidic sterols)/cholesterol intake]  100. It was shown that onion powder diet could decrease the apparent cholesterol absorption in a dose-dependent manner. Hepatic SREBP-2, HMGR, LDL-R, LXRa, and CYP7A1 No differences in protein mass of HMGR, LDLR and LXRb were seen among the three groups. However, the immunoreactive mass of liver SREBP-2, LXRa and CYP7A1 was dosedependently increased with the increasing onion powder in diets (Fig. 1 & 2). This journal is ª The Royal Society of Chemistry 2010

The present study demonstrated that supplementation of onion powder in diet could favorably modify the plasma lipoprotein profile by decreasing plasma TC, non-HDL, TG and ratio of non-HDL/HDL. The beneficial effect associated with onion power appeared to be dose-dependent as the supplementation at 5% level had a greater hypocholesterolemic activity than that at 1% supplementation. In general, the present results were in agreement with those previously reported in humans and animal studies, supporting the claim that the regular consumption of onions reduces the risk of CHD.6–9 There is no report to date that has investigated the underlying mechanism by which onion in diet reduces plasma cholesterol level. The present study was the first to demonstrate that onion powder in diets promoted the excretion of total fecal neutral sterols, suggesting it inhibited the cholesterol absorption and Food Funct., 2010, 1, 84–89 | 87

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that of Kumari and Augusti,2 who studied the effect of S-methyl cysteine sulfoxide, an active ingredient in onion, on the excretion of bile acids and neutral sterols, finding it increased the excretion of acidic and neutral sterols by 25 and 37%, respectively. The increase in the excretion of bile acids is likely an additional mechanism by which onion exhibited a cholesterol-lowering activity. No study to date has examined how dietary onion interacts with the protein expressions of SREBP-2, HMGR and LDLR. Our data showed that the addition of onion into diet increased the protein mass of SREBP-2, with LDLR and HMGR being unaffected. SREBP-2 governs the expression of LDLR and HMGR. Theoretically, up-regulation of SREBP-2 should be accompanied by up-regulation of LDLR and HMGR. To explore the underlying mechanism by which dietary onion had no effect on the protein levels of LDLR and HMGR with SREBP-2 being up-regulated, the following explanation is offered. The abundance in HMGR and LDLR was already very low because hamsters in the present study were sacrificed with an empty stomach, so that cholesterol catabolism rate was nil and no effect of dietary onion on these proteins could be seen after the overnight fasting.

Conclusion The present study was the first of its kind to investigate the interaction of dietary onion with the protein expression of hepatic SREBP-2, LDLR, HMGR, LXRa and CYP7A1. Results demonstrated that onion decreased plasma TC in a dosedependent manner. Dietary onion-induced reduction in plasma TC was accompanied by enhanced excretion of both fecal neutral and acidic sterols, most likely by up-regulation of LXRa and CYP7A1.

Abbreviations Fig. 2 The relative immunoreactive mass of hepatic liver X receptor (LXR) and cholesterol-7a-hydroxylase (CYP7A1) in hamsters fed the control diet, and two experimental diets supplemented with 1% onion powder (OP-1) and 5% onion powder (OP-5) for 8 weeks. Data were normalized with b-actin and values were expressed as mean  SD, n ¼ 12.

thus led to reduction in plasma TC. The fecal sterol analysis showed that the three major phytosterols, namely b-sitosterol, campesterol and stigmasterol, were present in the feces of the two onion-fed groups but they were absent in the control group. In addition, the amount of the three phytosterols in feces was in the order OP-5 > OP-1 > control, indicating that onion powder contained phytosterols. This could partially explain the hypocholesterolemic activity of onion because phytosterols compete with cholesterol for absorption. In fact, onion powder demonstrated a dose-dependent increase in the excretion of neutral sterols (Table 4). Cholesterol is mainly eliminated via its conversion to bile acids. The present study clearly demonstrated that dietary onion was able to increase the excretion of bile acids by 16–35% (Table 4). This was partially mediated by up-regulation of LXRa and CYP7A1 proteins (Fig. 2). The observation is in agreement with 88 | Food Funct., 2010, 1, 84–89

CHD CYP7A1 HDL HMGR LDL LDLR LXR SREBP-2 TC

Coronary heart disease Cholesterol 7a-hydroxylase High density lipoprotein cholesterol 3-Hydroxy-3-methylglutaryl-CoA reductase Low-density lipoprotein cholesterol LDL receptor Liver X receptor Sterol regulatory element binding protein 2 Total cholesterol.

References 1 K. T. Augusti, Indian J. Exp. Biol., 1996, 34, 634–640. 2 K. Kumari and K. T. Augusti, J. Ethnopharmacol., 2007, 109, 367– 371. 3 E. Block, Garlic and Other Alliums: The Lore and the Science, Royal Society of Chemistry, Cambridge, UK, 2010. 4 R. K. Sogani and K. Katoch, J. Assoc. Physicians India, 1981, 29, 443–446. 5 M. G. Hertog, E. J. Feskens, P. C. Hollman, M. B. Katan and D. Kromhout, Lancet, 1993, 342, 1007–1011. 6 A. Bordia, H. C. Bansal, S. K. Arora and S. V. Singh, Atherosclerosis, 1975, 21(1), 15–19.

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10 Z. Y. Chen, R. Jiao and K. Y. Ma, J. Agric. Food Chem., 2008, 56, 8761–8773. 11 P. T. Chan, W. P. Fong, Y. L. Cheung, Y. Huang, W. K. K. Ho and Z. Y. Chen, J. Nutr., 1999, 129, 1094–1101. 12 N. D. Vaziri and K. H. Liang, Kidney Int., 1996, 50, 887–893. 13 C. K. Lam, Z. S. Zhang, H. Yu, S. Y. Tsang, Y. Huang and Z. Y. Chen, Mol. Nutr. Food Res., 2008, 52, 950–958.

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7 M. A. Bang, H. A. Kim and Y. J. Cho, Nutr. Res. Pract., 2009, 3(3), 242–246. 8 Y. Yamamoto and A. Yasuoka, Biosci., Biotechnol., Biochem., 2010, 74(2), 402–404. 9 E. Ostrowska, N. K. Gabler, S. J. Sterling, B. G. Tatham, R. B. Jones, D. R. Eagling, M. Jois and F. R. Sunshea, Br. J. Nutr., 2004, 91(2), 211–218.

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PAPER

www.rsc.org/foodfunction | Food & Function

Suppression of breast xenograft growth and progression in nude mice: implications for the use of orally administered sphingolipids as chemopreventive agents against breast cancer Kirk W. Simon,a Larry Tait,b Fred Miller,b Chun Cao,c Kevin P. Davy,c Tanya LeRoithd and Eva M. Schmelz*ce

Downloaded on 21 October 2010 Published on 23 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00108B

Received 2nd August 2010, Accepted 28th August 2010 DOI: 10.1039/c0fo00108b Sphingolipids are lipid messengers involved in the regulation of many different cellular processes. Sphingolipid enzymes and bioactive metabolites have been targets of in vitro and in vivo efforts to suppress cancer growth, progression and metastasis of various cancer types. Dietary sphingomyelin effectively suppressed colon cancer in several rodent models without causing toxic side effects. In the present study, we determined if the effect of sphingolipid metabolites derived from the hydrolysis of dietary sphingomyelin is restricted to the intestinal tract or if their systemic concentrations are sufficient to suppress cancers of distant sites. For these studies, we used MCF10AT1 cells, a model for progressive breast cancer, injected into the mammary fatpad of nude mice as a single cell suspension. The mice were fed 0.1% sphingomyelin supplements in a semi-purified AIN76A control diet when the lesions were palpable. The study was terminated when the first lesions had grown to 5 mm. In the sphingomyelin-fed group, there was a trend to smaller lesion size and, importantly, a delayed progression to more malignant stages without apparent side effects. This may be the result of significantly reduced rates of proliferation and angiogenesis, while no increase of apoptosis was detected. Changes in aberrantly expressed proteins in the sphingomyelin-fed group, such as E-cadherin, VEGF and sphingosine kinase-1, may be associated with the suppression of tumor growth. These results demonstrate that diet-derived sphingolipids can efficiently suppress the growth and progression of MCF10AT1 xenografts, suggesting that dietary sphingomyelin may also be effective against cancers of other sites.

Introduction Breast cancer incidence and mortality has remained high, despite improved diagnostic and treatment efforts to eradicate the disease. An estimated 192,370 women were newly diagnosed in 2009 and 40,170 will die from breast cancer in the United States of America.1 Early detection improves survival,2 but preventive efforts may be more effective at maintaining health and increasing survival. Many in vitro and in vivo studies have demonstrated the preventive potential of dietary compounds; these often lack toxic side effects, and together with an easy route of administration could increase the patients’ compliance with and well-being during the prevention regimen.

a Department of Nutrition and Food Science, Wayne State University, Detroit, MI, USA b Department of Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA c Department of Human Nutrition, Foods and Exercise (HNFE), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA d Department of Anatomic Pathology and Biomedical Sciences & Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA e Department of Human Nutrition, Foods and Exercise (HNFE), Corporate Research Center Building 23 (0913), Room 1011, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. E-mail: [email protected]; Fax: +1 540-231-2947; Tel: +1 540-231-3649

90 | Food Funct., 2010, 1, 90–98

Sphingolipids are membrane-bound lipids with both structural and signaling properties. Sphingolipid metabolites are generated by growth factors, cytokines, cellular stresses and other factors, and as lipid second messengers are involved in the generation of an appropriate cellular response. These responses include the regulation of cell growth, differentiation, cell death, motility, angiogenesis, inflammation and many more processes in a concentration, structure, time and cell type-specific manner.3,4 Several enzymes in sphingolipid metabolism have been identified as targets for drug treatments to generate a sphingolipid pattern that exerts anticancer activities, such as the suppression of cell growth, motility, angiogenesis, etc. Among these enzymes are sphingomyelinases,5,6 glucosylceramide synthase,7 sphingosine kinase 1,8,9 sphingosine-1-phosphate lyase,10 sphingosine-1-phosphate phosphatase11,12 and others. However, the inhibitors are sometimes not sufficiently specific and can cause adverse side effects. Furthermore, cancer cells can exhibit an aberrant expression or activity of these enzymes, rendering the cells resistant to the regulatory stimuli. Thus, the delivery of bioactive metabolites directly to the cancer cells is an attractive alternative. We have shown in previous studies that administration of natural or synthetic complex sphingolipids via the diet suppressed chemically-induced colon cancer13–18 or cancer caused by gene mutations in mice19,20 by up to 80%. There were no side effects of this route of administration in any of our studies, and even very high doses used in other studies were tolerated well.21 This journal is ª The Royal Society of Chemistry 2010

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Diet-derived sphingolipid metabolites are taken up in the gastrointestinal tract, and a small portion is transported to the liver via the lymphatic system, mostly as sphingosine and fatty acids.22–24 Free sphingoid bases can also be re-utilized for the synthesis of more complex sphingolipids such as sphingomyelin (SM), and can be incorporated into chylomicrons and secreted from the intestinal tract; the impact of dietary SM on the content of SM in plasma lipoproteins, however, remains unclear.25 To determine if the diet provides sufficient amounts of bioactive metabolites to prevent cancer of distant sites in the body, in the present study, we investigated if orally administered SM can suppress breast cancer growth and progression. The amounts used in this study, 0.1% by weight, were highly effective against colon cancer (see above) without causing serious side effects, and are about 5–10 times higher than the daily intake in humans, which has been estimated at 0.01 to 0.02% of the diet.26 A recent study demonstrated that in humans, a dose of 250 mg SM is sufficient to increase SM and SM metabolites in the colon.24 The focus of this study is to determine if SM supplements are sufficient to affect breast xenograft growth and progression using, in contrast to a strictly chemopreventive approach, in which the diet is supplemented before cell injection, a study design in which SM is added to the diet when the lesions are palpable. As a model for proliferative breast disease, we used MCF10AT1 cells, pre-malignant human breast epithelial cells derived from the spontaneously immortalized, normal appearing MCF10A cells by transfection with a constitutively active Ha-ras.27 Although ras mutations are rare events in humans and only found in approximately 5% of patients, the aberrant activation of Ras and down-stream targets of Ras are seen in human breast cancer overexpressing ErbB-228 or Notch,29 and it is thought that activated Ras can contribute to transformation, unlimited proliferation,29,30 motility and invasion.31 Ha-ras alone, however, is not sufficient to induce transformation.32 Lesions formed in this system include mild to moderate hyperplasia, atypical hyperplasia, carcinoma in situ, moderately differentiated carcinoma, and undifferentiated carcinoma, as well as histologically normal ducts.32 Thus, the MCF10AT1 cell line represents a model for the slow and heterogeneous progression of preneoplastic breast cells and provides a transplantable xenograft model of human proliferative breast disease with proven neoplastic potential32,33 that is ideal to test the capacity of natural compounds to suppress breast cancer development and progression in vivo.

cells while on the semi-purified AIN76A diet; once the lesions were palpable, the SM group received 0.1% SM in the diet, and the development of the lesions was compared to the controls who did not receive any supplements. SM supplements did not affect the health of the mice as reflected by their general appearance and weight. The mean weight of the control group (27.8  2.3 g) did not differ significantly from the SM-fed group (25.8  0.5 g) (p ¼ 0.663). This confirms earlier studies from our and other’s laboratories that neither a sphingolipid-free diet nor SM supplements to the diet (up to 1% by weight21) cause adverse side effects, and SM is therefore safe to use as a dietary supplement in our studies. Since cells were injected at two conlateral sites per mouse, the development of 30 lesions (2 per mouse) was possible in each group. In the control group, 22 lesions were detected (1.47 per mouse), and 25 in the SM-fed group (1.67 per mouse); this difference is not statistically significant (p ¼ 0.417). There were 2 mice without lesions in each group (13.3%). This lesion development lies in the expected range for this cell line when injected into nude mice in a Matrigel solution.27 The volume of the lesions was lower in the SM-fed group, albeit not statistically significant (99.33  23.99 mm3 in the control and 63.72  7.89 mm3 in the SM-fed group; p ¼ 0.190). This is especially interesting, since we added the SM supplements only after the lesions were already palpable and not as a strictly preventive regimen before cell injection. MCF10AT1 cells were organized as ducts rather than as cell clusters; this confirms previous findings demonstrating that these cells form structures that resemble normal human breast ducts when injected into nude mice in a Matrigel solution.27 The lesions were solid and without a necrotic core. It is important to note the difference in the amount of stromal tissue in individual xenografts. Some lesions consisted mostly of epithelial cells, and there was very little stromal tissue visible (Fig. 1A), while it was more abundant in others, and the epithelial cells appeared as small islands of ducts (Fig. 1B, arrows). Stromal tissue has been shown to generate a microenvironment that regulates breast cell growth, migration, morphology, differentiation, migration and survival.34 In cancer, the cancer cells signal to alter protein secretion of the stroma but stroma activated by inflammatory signals is involved in tumor development and progression (see

Results and discussion Sphingomyelin suppressed tumor development and progression. Sphingolipids are natural components of the diet that are hydrolyzed in the intestinal tract to the bioactive metabolites ceramide and sphingosine, which are taken up by the intestinal cells and either degraded or incorporated into complex sphingolipids.22,23 The exposure to these bioactive metabolites is sufficient to suppress colon cancer in mice.13–20 Only a small fraction of the absorbed sphingolipid metabolites reaches systemic distribution;22,23 therefore, the highest dose effective in colon cancer prevention was used for the present study. Female NCR nude mice (8 weeks old) were injected with MCF10AT1 This journal is ª The Royal Society of Chemistry 2010

Fig. 1 MCF10AT1 xenograft architecture. MCF10AT1 cells are organized either as tightly packed breast-like ducts with little (A) or high (B, arrows) amounts of stromal tissue with no apparent difference between the control group fed the AIN76A diet alone and the SM supplemented group (0.1%, by weight).

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Fig. 3 Suppression of progression. Mice (n ¼ 15 per group) were injected with MCF10AT1 cells while being fed the AIN76A diet. After the lesions were palpable (after 4 weeks), the diet of the SM group was supplemented with 0.1% of SM. Using the criteria in the Experimental, the area of the highest disease stage found in one lesion determined the overall category of the lesion. (different at p < 0.05).

immunohistochemical methods but changes in gene expression levels in the stroma as a result of SM treatment will be considered in future studies using real-time PCR. We next determined the progression of the MCF10AT1 cells using the criteria outlined in the Experimental section to define the different disease stages, as shown in Fig. 2. Most of the xenografts in the SM-fed group progressed to stage 2 (22 or 88%), characterized by moderate hyperplasia, bridging and papillary hyperplasia. Only two areas of stage 3 (8%) and one of stage 5 (4%) were detected in this group. In contrast, half of the xenografts in control animals progressed further and exhibited areas of stage 3 (6 or 27.3%), 4 (2 or 9.1%) and 5 (3 or 13.6%) (different from the SM-fed group at p ¼ 0.0212) (Fig. 3). This lies closely in the range of the progression rate reported for this cell line.27 These results indicate that SM supplements suppressed or delayed the progression of these lesions, confirming our hypothesis that diet-derived SM metabolites are sufficient to exhibit a systemic effect and suppress breast cancer progression without causing side effects. Thus, dietary sphingolipids are not only effective against colon (our previous studies) and liver cancer35 but can successfully target cancer of distant sites in the body. Whether dietary sphingolipids are as effective against more aggressive breast cancer lesions is currently being investigated in our laboratory. Fig. 2 MCF10AT1 xenograft lesions. The lesions were graded using the criteria outlined in the Experimental to approximate the scoring system in place for human surgical specimen: A, stage 0, normal ducts; B, stage 1, mild hyperplasia with multiple layers (arrows); C, stage 2, moderate hyperplasia with bridging (arrow); D, stage 3, atypical hyperplasia; E, stage 4, carcinoma in situ; F, stage 5, invasive carcinoma (arrow); G, fibrocystic structure (arrow) and hyperplastic duct.

recent review34). Thus, the interactions of tumor stroma and epithelial cells can have a profound effect on the progression of the epithelial cells, and the tumor size per se may not truly reflect the preventive potential of the tested compound; the epithelial and stroma geno- and phenotype may be more critical. In this study, the amount of stroma was not the same in sections of different areas of a single xenograft or among the lesions found in one mouse; thus, an association of SM supplements and the epithelial cell to stroma ratio could not be made by 92 | Food Funct., 2010, 1, 90–98

Sphingomyelin reduced cell proliferation but did not induce apoptosis Sphingolipid metabolites regulate cell growth both in vitro and in vivo. Since unlimited proliferation is considered a driving force for progression, the effect of the SM supplements on the rate of proliferation in the breast xenografts was determined using Ki-67 expression as a marker. As shown in Fig. 4, SM supplements significantly suppressed the proliferation of MCF10AT1 xenografts (22.9  2.1% Ki-67 positive nuclei in the control group vs. 11.6  1.7% in the SM-fed group; p < 0.001). One objective of many anticancer treatments is the induction of apoptosis in cancer cells to eradicate tumors. Sphingolipid metabolites induce apoptosis in many cancer cell lines in vitro; therefore, we also evaluated the rate of apoptosis in the MCF10AT1 xenografts. There were very few epithelial cells in This journal is ª The Royal Society of Chemistry 2010

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by the general appearance of the mice, a comparable weight gain and blood parameters we have reported previously (normal blood urea nitrogen, creatinine, albumin, serum glutamate pyruvate transaminase, alkaline phosphatase, etc.14). This may be the result of a limited digestion of complex sphingolipids, which prevents the generation of toxic levels of bioactive sphingolipid metabolites in the intestinal tract.42 Even the feeding of SM over two generations did not affect the health of rats,21 suggesting that the supplementation of the diet with SM is a safe route of administration. Sphingomyelin reduced VEGF expression and the number of blood vessels in the xenografts The growth and progression of tumors are dependent on a sufficient supply of nutrients and oxygen, and thus on the establishment of blood vessels. In addition to the vascular endothelial growth factor (VEGF) secretion by endothelial and stromal cells, breast cancer cells also secrete VEGF to stimulate angiogenesis by enhancing endothelial migration, proliferation and survival,43 but also to attract mesenchymal stem cells that differentiate into stroma.44 To evaluate if the suppression of growth and progression of MCF10AT1 xenografts by dietary SM is associated with a reduced angiogenesis, the expression of VEGF in the xenografts was determined by immunohistochemistry. VEGF was expressed in endothelial cells without apparent differences among the treatment groups, but there was also significant immunostaining of the epithelial cells. The control

Fig. 4 Inhibition of proliferation by orally administered sphingomyelin. Sections from the control (Ctrl) or sphingomyelin-fed (SM) group (n ¼ 15 per group, 5 images per section) were immunostained for Ki-67 as a marker for proliferation and expressed as percent Ki-67 positive cells. *p < 0.001.

either group that were stained positive for activated caspase-3, with most sections being completely negative (not shown). The lack of apoptosis was verified by evaluating the shape of the nuclei in H&E stained sections—condensed or fragmented nuclei of epithelial cells were rarely detected, indicating that orally administered SM did not induce apoptosis in the xenografts. This low rate of apoptosis in MCF10AT1 xenografts is not specific for our study but has been reported before in MCF10AT1 cells grown in three-dimensional cultures36 or as xenografts37,38 These results confirm our previous observations and the reports of other groups that dietary sphingolipids do not induce apoptosis above normal levels in the colon,16,17 liver35,39,40 or spleen.40 Instead, we have previously observed a suppression of aberrant proliferation in the colonic mucosa.16,17 Thus, dietary sphingolipids may exert their antitumor effect against colon and breast cancer in part by reducing the rate of proliferation. This is in contrast to many other natural compounds that clearly show an association of tumor suppression and the induction of apoptosis by many different pathways41 but may be associated with the lack of toxic side effects of dietary sphingolipids, which was apparent This journal is ª The Royal Society of Chemistry 2010

Fig. 5 Regulation of angiogenesis by dietary sphingomyelin. Sections of tumors from the control group (Ctrl) and SM-fed group (SM) were immunostained with VEGF (A, B) or Pecam-1 (C). A moderate to high expression of VEGF was detected in the lower grade ducts of the control group (A) with high levels in higher grades (unspecific luminal staining). Low to moderate levels were seen in the SM-fed group (B). PECAM-1 staining was used to visualize endothelial cells (C), and blood vessels were counted in 10 high-powered viewing fields (HPVF) per section (D). The number of blood vessels per HPVF was significantly higher in the control (Ctrl) group than in the SM-fed group (p < 0.001).

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group expressed moderate to high levels of VEGF in lower grade ducts (Fig. 5A) but higher levels in more advanced stages. Undetectable/very low to moderate levels of VEGF were observed in the SM-fed group (Fig. 5B) (luminal staining is unspecific). Higher grade lesions (stage 5) in either group showed substantial VEGF staining. These results confirm previous findings, demonstrating a reduction of VEGF mRNA expression levels in the colonic mucosa of Min mice after feeding sphingolipids for four weeks.20 Immunostaining for Pecam-1 allowed for the visualization of blood vessels (Fig. 5C) in the xenografts. In the control group, there were 5.41  0.26 blood vessels per high-powered viewing field (Fig. 5D); SM significantly reduced the number of blood vessels to 3.39  0.18 (p < 0.001). There was no consistent difference in the size of these blood vessels between the groups, but it appeared that there were generally more and larger vessels associated with the stromal tissue than with areas of tightly packed epithelial tissue. These results demonstrate the suppression of angiogenesis by SM, possibly via suppression of VEGF expression. It has been shown that stromal cells directly adjacent of tumor cells promote angiogenesis via the activation of gene expression;45 the secretion of VEGF by stromal cells has been

suggested to be a contributing factor.46 Thus, the phenotype of the stroma appears to be critical for tumor growth and progression, and the effect of dietary SM on tumor-associated stroma and its secretion of growth factors and other tumorsupporting proteins needs to be evaluated in more detail. Expression levels of gene products associated with progression of breast cancer The promotion and progression of tumor cells is associated with changes in the expression levels of specific proteins, usually identified as tumor suppressors or promoters. In heterogenic diseases such as breast cancer, this protein expression pattern can be employed to predict the progression of the tumor cells and their response to treatment, and therefore these proteins are potential biomarkers for in vivo efficacy determinations. To confirm the suppression of progression by orally administered SM and to identify targets that are altered by the treatment, we used immunohistochemistry to evaluate changes in gene products that have been associated with breast cancer progression and may be a potential biomarker for SM efficacy against breast cancer in vivo.

Fig. 6 Expression levels and the localization of proteins associated with breast cancer progression are altered with dietary SM. E-cadherin expression in the control group (A low, B high magnification) and the SM-group (C low, D high magnification); H4K16ac in control (E) and SM-group (F); (G) cytokeratin expression confirms the epithelial origin of the ducts; (H) Vimentin expression in the control group; (I, K) Vimentin-positive myoepithelial cells; (L) SK1 expression; (M) ceramide kinase expression (the luminal staining of the duct-like structure is unspecific).

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The loss of E-cadherin is a frequent early event in cancers of many organs such as colon, the ovaries and also breast.47 This has been associated with changes in cellular architecture that may permit the epithelial–mesenchymal transition (EMT), cancer progression and invasion.48,49 In our study, there was weak to no expression of E-cadherin in the control group, even in lower grade lesions (Fig. 6A). Higher magnification showed an expression of E-cadherin in some ducts, predominantly located in the cytoplasm (Fig. 6B). The staining in these cells was sometimes diffuse but also appeared granulated, sometimes localized in the perinuclear area. In contrast, most of the ducts in the SM-fed group exhibited E-cadherin positive staining in at least some cells of each duct (Fig. 6C). Here, E-cadherin was often localized at the membranes (Fig. 6D), but diffuse or sometimes granular cytoplasmic expression was often seen in ducts lacking membranous E-cadherin. The appearance of cytosolic E-cadherin in the early stages has been described in premalignant breast lesions as a result of increased endocytosis, leading to a loss of membrane anchoring that weakens adhesion, and altered cell dissociation, motility and invasive potential.49,50 Later stages of breast cancer, even ductal carcinoma in situ, often demonstrate a complete loss of E-cadherin protein expression.51 Overexpression of E-cadherin in MDA-MB-231 cells partially restored the epithelial morphology with increased cell–cell contacts and adhesion, and reduced migration properties.52 It is not known if there is a different function of the cytosolic and membrane-bound E-cadherin since the cells increased the expression levels in both cellular fractions,52 but the expression and recruitment of tumor suppressors such as PTEN are dependent on the E-cadherin/b-catenin complex at the adherens junctions.53 Natural compounds in the diet can restore the expression of E-cadherin and suppress invasion.54 Thus, the regulation of E-cadherin expression and localization may affect several signaling intermediates restoring cellular differentiation and suppressing proliferation. Since E-cadherin expression is often down-regulated via epigenetic silencing—also reported in breast cancer55—we investigated the global methylation of histones as a marker for transcriptional regulation; the loss of histone 4 lysine 16 acetylation (H4K16ac) has been suggested to be a hallmark of human cancer cells56 and has been found to be associated with larger breast tumors.57 H4K16 hypoacetylation also has been observed in the silencing of E-cadherin.58 As shown in Fig. 6E and 6F, SM feeding leads to an increase in H4K16 acetylation, which followed the stage distribution of the tissue in most cases; in SM-fed mice, positive nuclei were also seen in stage 3 (Fig. 6F). The cytosolic staining seen with this antibody is unspecific since it was still visible in the sections treated with blocking peptides. While these results need to be confirmed in a larger study, and further corroborated by chromatin immunoprecipitation and quantitative PCR analysis for specific epigenetically regulated genes, they suggest that dietary SM indeed affects histone marks involved in epigenetic silencing and thereby potentially influences the expression of genes such as E-cadherin. The mechanisms for gene expression regulation are currently a research focus in our laboratory. Next, we investigated the expression of vimentin, expressed after EMT, and thus indicative of progression. All ducts expressed cytokeratin, confirming their epithelial nature This journal is ª The Royal Society of Chemistry 2010

(Fig. 6G). There was no vimentin expression in the lower grade lesions of either group, but a diffuse staining in higher grade lesions in both groups (Fig. 6H). Since there were less higher grade lesions in the sphingomyelin-fed group, there was also less vimentin staining. Myoepithelial cells also stained strongly positive for vimentin, as has been reported previously.59 These cells closely surround the ducts formed by the MCF10AT1 cells (Fig. 6I). There was no apparent difference in the number of myoepithelial cells between the groups. However, myoepithelial cells have been shown to regulate breast epithelial polarity, morphogenesis, differentiation, etc. via secretion of regulatory factors such as laminin-1, fibronectin, matrix metalloproteinases, ephrin receptor B4, pleiotrophin and others, and are thought to suppress breast cancer progression.60 While there is a reported loss of myoepithelial cells during breast cancer progression,60 the most drastic and consistent changes during this progression occur in myoepithelial cells, specifically in genes encoding secretory and cell surface proteins.61 Since this can alter their function and interaction with breast epithelial cells and affect their role in cancer progression, the changes in their gene expression levels—and functions—in response to sphingolipid metabolites is therefore an important question. We have investigated the expression of other proteins often associated with breast cancer progression but did not find either a consistent or reproducible change in their expression (CXCL4, SDF-1, Her-2, pHer-2 EGFR, pEGFR), or their expression levels were low or not changed by dietary SM (Cyclin D1, N-cadherin, F4/80). However, as reported above, several molecular markers for breast cancer progression were aberrantly expressed in advanced stages and were affected by the dietary SM, suggesting that these proteins may be involved in the suppression of breast cancer progression by dietary SM. Expression of sphingosine kinase-1 and ceramide kinase Changes in the expression of enzymes regulating the sphingolipid metabolism that result in changes in the expression pattern of sphingolipid metabolites have been reported in several cancers, including breast cancer. Sphingosine kinase-1 (SK1) generates sphingosine-1-phosphate, which, in contrast to sphingosine or ceramide, mostly promotes cell growth, motility, inflammation, tumorigenesis, angiogenesis and invasion, and therefore has been implicated in the uncontrolled growth of cancer cells.8,62 SK1 exhibits higher expression levels in estrogen receptor-negative breast tumors and has been associated with a higher rate of metastasis and a worse outcome.63 SK1 expression was detected in the cytosol of approximately half of the fibrocystic structures (depicted in Fig.2G) in the control lesions (Fig. 6L) and only occasionally in the duct-like structures. There was no SK1 expression, even in the higher-grade ductal structures in the SM-fed group, and only an infrequent staining of the fibrocystic structures. Nuclear SK1 was only detected in small areas of one lesion per group, and therefore appears not to be important in the progression of MCF10AT1 lesions. Ceramide kinase is a less well-described member of the sphingolipid metabolic enzyme family that generates ceramide1-phosphate (C1P). In lung cancer cells, C1P increased survival and stimulated proliferation.64 We did not detect drastic differences between the controls and the sphingomyelin-fed group in Food Funct., 2010, 1, 90–98 | 95

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both groups: ceramide kinase was weakly to strongly expressed in the cytosol of few epithelial cells, and often also in the nucleus (Fig. 6M). A high ceramide kinase expression, however, has been shown in estrogen receptor negative breast cancers and was associated with a worse prognosis.65 It has been reported that C1P in macrophages and other immune cells plays a role in inflammatory processes (see the recent review66). Thus, the cancer cells may not be the only critical cell population expressing ceramide kinase; this needs to be investigated in an immune competent model.

Experimental Cell culture MCF10AT1 cells were obtained from the Cell Core at the Karmanos Cancer Institute at Wayne State University, Detroit, MI. Cells were routinely cultured at 37  C in DMEM/Ham’s F12 (1 : 1) (Gibco, Invitrogen), supplemented with 5% horse serum (Gibco), 10 mg ml1 insulin (Sigma, St. Louis, MO), 0.02 mg ml1 EGF (Calbiochem, San Diego, CA), 0.5 mg ml1 hydrocortisone (Sigma) and 0.1 mg ml1 cholera toxin (Calbiochem) in 5% CO2 in a humidified atmosphere.

criteria of Dawson et al. (1996). The lesions were classified according to the highest grade found in the xenograft. The categories were defined as follows: 0 simple small tubules with 1–2 cell layers, no nuclear enlargement. 1 mild hyperplasia, simple small tubules with >2 cell layers but no bridging or architectural complexity, variable nuclear contours. 2 moderate hyperplasia, mildly distended ducts, 4 or more layers of epithelial cells, bridging, irregularly shaped lumen, papillary hyperplasia. 3 atypical hyperplasia, grossly distended ducts, marked cellular proliferation often forming luminal mass, some loss of polarity, cells become monotonous, tendency to clear cytoplasm with distinct borders, enlarged, non-round nuclei, small nucleoli, occasional mitoses. 4 carcinoma in situ, distended ducts filled with uniform cells, rigid intraluminal bridges forming round spaces, occasional central necrosis, distinct cell boundaries, uniform round, hyperchromatic, enlarged nuclei, frequent mitoses. 5 invasive carcinoma, glandular, squamous or undifferentiated.

Animals and diets

Immunohistochemical analyses

Female NCR nude mice (seven weeks old) were purchased from Taconic (Germantown, NY). Upon arrival, they were randomly divided into two experimental groups (n ¼ 15 per group) and weighed to ensure an equal weight. After acclimatization for one week, they were placed on the semi-purified AIN 76A diet (Dyets, Bethlehem, PA). This diet is essentially sphingolipid-free,67 in contrast to the newer formulations that contain soy oil and may contain significant amounts of glucosylceramide. Since sphingolipids are not essential nutrients, placing animals on sphingolipidfree diets is not harmful and has neither caused adverse effects in any of our studies, nor those published by others.

Tissue sections were immunostained using our established procedures.19 Briefly, sections were deparaffinized in xylene, re-hydrated through graded alcohol and steamed in citrate buffer (Target Retrieval Solution, Dako, Carpinteria, CA) for antigen retrieval if suggested for the specific antibody by the manufacturer. Endogenous peroxidase activity was blocked with Peroxoblock (Zymed, Carlsbad, CA). Blocking of the sections for 1 h was followed by overnight incubation at 4  C with the indicated antibodies, without primary antibody or with antibody plus blocking peptide as negative or antibody specificity controls. The sections were rinsed, incubated with the appropriate secondary antibodies for 1 h at room temperature, and treated with the ABC staining systems (Vector, Burlingame, CA) according to the manufacturer’s instructions. The immunocomplex was visualized with diaminobenzidine (Dako). The sections were briefly counterstained with hematoxylin (Zymed) and permanently mounted with Histomount (Zymed). All images were digitally captured on a Nikon Eclipse 80i epifluorescence microscope, equipped with DIC, digital cameras, and acquisition and analysis software (NIS Elements). Images were processed with Adobe Photoshop. Antibodies used in this study were generated against Ki-67 (Dako), activated Caspase-3 (Cell Signaling, Danvers, MA), ceramide kinase (Abgent, San Diego, CA), CRCX4 (R&D), cytokeratin (Sigma), SDF-1 (R&D), E-cadherin (R&D Systems), H4K14ac (abcam), N-cadherin (abcam), EGFR (Upstate Biolabs, Chicago, IL), F4/80 (abcam), Her-2 and phospho-Her-2 (Cell Signaling), VEGF (Santa Cruz), SK1 (Imgenex, San Diego, CA), Vimentin (Dako) and Pecam1 (Santa Cruz). The quantitation of proliferation was achieved by immunostaining for Ki-67. A red filter was used to obscure staining and pictures were taken from 5 randomly selected areas per slide in a blinded manner. The nuclei stained positively for Ki67, all unstained epithelial nuclei were counted, and the rate of proliferation was

Xenografts and treatment Cells were grown to approximately 70% confluence and harvested by gentle trypsination. 1  107 cells were mixed into 100 ml of cold Matrigel (BD, Franklin Lakes, NJ) and subcutaneously injected into the area of two contralateral mammary fatpads per mouse. When the xenografts were palpable (approximately 1–2 mm in diameter, 4 weeks later), one group of mice was placed on the AIN 76A diet containing 0.1% sphingomyelin (by weight) (Avanti, Alabaster, AL), while the control group did not receive any dietary supplements. After the xenografts reached a size of about 5 mm (16 weeks after injection), the mice were killed, the xenografts excised, measured, fixed overnight in 10% neutral buffered formalin, embedded in paraffin and sectioned for immunohistochemical analyses at 4–5mm. The tumor volume was calculated by the formula: V ¼ {W2  L} O 2, where V is the tumor volume, W is the width and L is the length. Determination of progression Hematoxylin and eosin-stained sections of each animal were graded in a blinded manner for progression according to the 96 | Food Funct., 2010, 1, 90–98

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expressed Ki67 positive as a percentage of all nuclei. Data are expressed as mean  SEM.

funds from the Prevention Program, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA.

Determination of angiogenesis

References

Sections were immunostained (as described above) for Pecam1 to visualize blood vessels in the xenografts. 10 high-powered viewing fields per section were randomly selected by a person unaware of the treatment at 40 magnification, and all the visible blood vessels that stained positive for Pecam-1 and/or showed blood cells were counted. Other vessels that might have been lymphatic vessels and microvessels without Pecam1 staining were not counted in any section. Furthermore, the diameter of the blood vessels were not measured since the random angle of sectioning the vessels did not allow for the quantitation of this parameter. 20 tumors per group were analyzed by this method. Data are expressed as the mean number of blood vessels per highpowered viewing field  SEM.

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Statistical analyses Experimental groups were compared using the unpaired t-test after ANOVA for groups that had values sampled from Gaussian distributions. An unpaired t-test followed by Welch’s correction was used when the standard deviations differed significantly among the groups, and the non-parametric Mann– Whitney test was used in groups that did not follow a Gaussian distribution. These statistical analyses were performed with Instat 3.0a (GraphPad Software, Inc.). The association of tumor progression and dietary supplementation was calculated using the one-sided Mantel–Haenszel chi square test.

Conclusion The present study demonstrates for the first time that dietary SM in amounts that are approximately 2.5 to 10 times higher than the estimated human intake of complex sphingolipids26 suppressed the growth and progression of MCF10AT1 xenografts without causing adverse side effects. This was associated with a significantly reduced rate of angiogenesis. Proteins that are aberrantly expressed in breast cancer progression, i.e., E-cadherin, VEGF and enzymes of the sphingolipid metabolism that could generate a pro-tumorigenic microenvironment, i.e., SK1, were targeted by the diet-derived sphingolipid metabolites and exhibited more normal expression levels or protein localization in the SM-fed group, suggesting the possibility that their functions may be restored. Whether these gene products can be used as molecular markers for sphingolipid efficacy in vivo remains to be determined. However, our results demonstrate systemic, in addition to the previously described local, effects of diet-derived sphingolipid metabolites. Whether the amounts of SM that were effective in this pre-malignant breast cancer model can also prevent tumor growth and progression in more advanced models is currently under investigation in our laboratory.

Acknowledgements This study was supported by NCI grant R03 CA101125, The Susan G. Komen Race for the Cure grant BCT0503453 and This journal is ª The Royal Society of Chemistry 2010

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Consumption of polyphenolic-rich beverages (mostly pomegranate and black currant juices) by healthy subjects for a short term increased serum antioxidant status, and the serum’s ability to attenuate macrophage cholesterol accumulation Mira Rosenblat, Nina Volkova, Judith Attias, Riad Mahamid and Michael Aviram*

Downloaded on 21 October 2010 Published on 15 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00011F

Received 17th May 2010, Accepted 16th July 2010 DOI: 10.1039/c0fo00011f The present study analyzed the antioxidative effects of various beverages, in vitro, and also the effect of short term consumption of beverages richest in polyphenols by healthy subjects on serum antiatherogenic properties. Healthy subjects consumed 250 mL of the selected beverages for 2 h, or daily, for up to 1 week. We hypothesized that differences in the anti-atherogenic properties of the studied beverages could be related, not only to the quantity of polyphenols, but also to their quality. Furthermore, we hypothesized that consumption of these juices by healthy subjects for just a short-term, will increase their serum anti-atherogenic properties, as was demonstrated previously in long-term consumption studies. Of 35 beverages studied, both 100% Wonderful-variety pomegranate and 100% black currant juices were the most potent antioxidants in vitro, as they inhibited copper ion-induced LDL oxidation by up to 94% and AAPH-induced serum lipid peroxidation by up to 38%. Furthermore, they increased in vitro serum paraoxonase 1 (PON1) lactonase activity by up to 51%. Consumption of five selected polyphenol rich beverages by healthy subjects increased serum sulfhydryl group (SH) levels and serum PON1 activities after 2 h, and more so after 1 week of drinking these beverages. These effects were most pronounced after the consumption of 100% Wonderful-variety pomegranate and 100% black currant juices. In conclusion, polyphenolic-rich juices with impressive in vitro antioxidant properties, also demonstrate antioxidant effects in vivo when analyzed for short term consumption. In this respect, 100% Wonderful-variety pomegranate and 100% black currant juices were most the potent.

Introduction Macrophage cholesterol accumulation and foam cell formation are hallmarks of early atherogenesis.1 Oxidative stress contributes to the development and progression of atherosclerosis.2 Cholesterol accumulation in macrophages can result from increased uptake of oxidized LDL and/or a decreased rate of HDL-mediated cholesterol efflux from the cells.1,2 Dietary polyphenols, such as those present in some beverages, were shown to be potent antioxidants3 and cardioprotective agents.4–7 Grape juice consumption by hypertensive individuals for 8 weeks increased serum antioxidant potential,8 and exerts hypolipidemic and anti-inflammatory effects in both hemodialysis patients and in healthy subjects.9 Red wine consumption by healthy subjects inhibited LDL oxidation10 and decreased monocyte migration.11 A more potent cardioprotector than grape juice or red wine was found to be 100% Wonderful-variety pomegranate juice (WPJ), as it protected atherosclerotic patients from further atherosclerosis development.12,13 Consumption of WPJ by healthy subjects for at least 2 weeks, significantly The Lipid Research Laboratory, Technion-Israel Institute of Technology Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Rambam Medical Center, Haifa, 31096, Israel. E-mail: [email protected]; Fax: +972-4-8542130; Tel: +972-48542970

This journal is ª The Royal Society of Chemistry 2010

reduced the oxidation of both LDL and HDL and increased HDL-associated paraoxonase 1 (PON1) activity.14 Studies in patients with carotid artery stenosis (CAS) that consumed WPJ for 3 years, clearly demonstrated reduced serum oxidative stress, increased serum PON1 activity, and most importantly - a reduction in atherosclerotic lesion size.12 Furthermore, in diabetic patients, WPJ consumption decreased serum and macrophage oxidative stress, decreased oxidized LDL uptake by their cells,13 and increased PON1 association with HDL.15 Similarly, WPJ in vitro was shown to increase PON1 binding to HDL,16 and also to up-regulate PON1 expression in hepatocytes.17 In all the above studies, the effects of beverages were noted after relatively long periods of consumption (weeks, months, and years). There are a limited number of studies which analyzed the acute effects of beverage consumption on serum oxidative stress and atherogenicity. Plasma antioxidant power was increased postprandially after red wine consumption by healthy subjects.18 Black currant juice consumption for 1 week decreased serum lipid peroxidation and increased urinary excretion of quercetin.19 Similarly, twelve hours after consumption of acai juice by healthy subjects, the plasma antioxidant capacity was significantly increased.20 Finally, consumption of a mixture of berry juices, which include acai and palm fruit as the predominant ingredients, increased the serum antioxidant level, and inhibited lipid peroxidation, two hours post-consumption.21 Food Funct., 2010, 1, 99–109 | 99

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The aim of the present study was to compare the antioxidative effects of polyphenolic-rich beverages (and especially, those of 100% Wonderful-variety pomegranate juice, 100% black currant juice, 100% Concord grape juice, acai juice blend and red wine) in vitro, and also to study the effects of their short term consumption by healthy subjects (either 2 h, or daily intake for 1 week) on serum anti-atherogenic properties.

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In vitro studies Table 1 demonstrates the list of the 35 beverages used in the study. Polyphenols. Total polyphenols were determined spectrophotometrically by the method of Singleton, modified for small volumes.22 Gallic acid served as a standard. Gallic acid stock solution was prepared in water at a concentration of 2 mmol/L. Volumes of 10, 20, 40 and 60 microlitres diluted in 1 mL solution (equivalent to 20, 40, 80 and 120 nmol respectively) were used for the standard curve. Free radical scavenging capacity. The free radical-scavenging capacity of the various beverages was analyzed by the DPPH assay.23 DPPH (1,1-diphenyl-2-picrylhydrazyl) is a radical

generating substance that is widely used to monitor the free radical scavenging abilities (the ability of a compound to donate an electron) of various antioxidants. The DPPH radical has a deep violet color due to its impaired electron, and radical scavenging can be followed spectrophotometrically by the loss of absorbance at 517 nm, as the pale yellow non-radical form is produced. The beverages were diluted with water 1 : 10, v : v. Aliquots of 20 mL from the diluted beverages were mixed with 1 mL of 0.1 mmol DPPH/L in ethanol, and the change in optical density at 517 nm was monitored after 5 min. LDL preparation. LDL was isolated from plasma taken from healthy normolipidemic volunteers, by discontinuous density gradient ultracentrifugation.24 The LDL was washed at d ¼ 1.063 g/mL, dialyzed against 150 mmol/L NaCl, 1 mmol/L Na2EDTA (pH 7.4) at 4  C. The LDL was then sterilized by filtration (0.45 mM), kept under nitrogen in the dark at 4  C and used within 2 weeks. The LDL protein concentration was determined with the Folin Phenol reagent.25 Prior to oxidation, LDL was dialyzed against EDTA-free, phosphate buffered saline (PBS) solution, pH 7.4, and at 4  C. Copper ion-induced oxidation. The beverages were diluted with water 1 : 100, v:v. LDL (100 mg of protein/mL) was then preincubated for 1 h at room temperature with 25 mL/mL of the samples of diluted beverages. Then, 5 mmol/L of CuSO4 was

Table 1 List of beverages tested Category

Brand

Product name

100% Pomegranate juice

POM Wonderful Knudsen Langers Odwalla Naked Welch’s Knudsen Lakewood Knudsen Lakewood Knudsen Currant C Knudsen Wyman’s Frutzzo Bossa Nova Naked Sambazon Naked

100% Pomegranate Juice (Wonderful-variety) Just Pomegranate (variety unknown) 100% Pomegranate Juice (variety unknown) Pomegranate Juice (variety unknown) Pomegranate (variety unknown) 100% Concord Grape Juice Concord Grape Pure Concord Grape Just Black Cherry Pure Black Cherry Just Black Cherry Black Currant Nectar Just Blueberry 100% Wild Blueberry 100% Yumberry Acai Juice- The Original Acai Machine Original Blend Red Machine Green Machine Red Rhapsody Mangosteen/antioxidant fruit Acai Mangosteen Acai Blueberry Mangosteen Dragonfruit Goji Cherry Goji Acai Goji Mangosteen Pomegranate Blueberry Pomegranate Blueberry Green Tea with natural flavor Green Tea with citrus BV Napa Cabernet Sauvignon Robert Mondavi Napa Merlot Zinfandel (California)

100% Concord grape juice 100% Black cherry juice 100% Black currant juice and blends 100% Blueberry juice 100% Yumberry Acai juice blends ‘‘Superfruit’’ blends

Odwalla Lakewood Born Dia Bossa Nova Goji Lania

Green tea Red wine

100 | Food Funct., 2010, 1, 99–109

Minute Maid Tropicana Nestea Lipton Beaulieu Mondavi Cline

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added and the tubes were incubated for 2 h at 37  C. At the end of the incubation, the degree of LDL oxidation was determined by measuring the generated amount of thiobarbituric acid reactive substances (TBARS). The TBARS assay was performed at 532 nm, using malondialdehyde (MDA) for the standard curve.26 AAPH-induced serum lipid peroxidation. Serum samples from healthy subjects were diluted 4 with phosphate buffered saline (PBS) and were pre-incubated with no addition (control), or with five polyphenol rich beverages (2 mL/mL of concentrated beverages) for 1 h at room temperature. Then 100 mmol/L of 2,20 azobis, 2-amidinopropane hydrochloride (AAPH, Wako, Japan) was added to all samples and they were further incubated for 2 h at 37  C.27 The extent of lipid peroxidation was measured by the TBARS assay.26 In vivo studies Subjects. Six healthy male subjects (aged 25–30 years) participated in the study. The subjects drank 250 mL of each beverage after an overnight fast. Blood was collected from the subjects 2 h after consumption. They continued daily beverage consumption for 1 week with the evening meal. After 1 week of beverage consumption, blood samples were collected. After a 1 week ‘‘washout period’’, this protocol was repeated with another beverage until each subject had consumed all 5 of the tested beverages. The order of beverages consumed was: first – acai juice blend (Naked), second – 100% Concord grape juice (Lakewood), third – 100% black currant juice (Knudsen), fourth – 100% Wonderful-variety pomegranate juice (POM Wonderful), and last – red wine (Merlot/Mondavi). The subjects served as their own control, as we compared all post-consumption data to the baseline values. All subjects were non-smokers. The study protocol was approved by the Helsinki Committee of the Rambam Medical Center, Israel Ministry of Health, application no. 3073. Serum biochemical parameters. Serum sodium, potassium, glucose and lipids (total cholesterol, HDL cholesterol and triacylglycerol) concentrations were measured using automated enzymatic tests (Tayco Diagnostics- Agis Commercial Agencies, Israel). Serum oxidative stress parameters Basal serum oxidation status. The extent of lipid peroxidation was measured by the TBARS assay.26 Total thiols (SH groups) in serum. The assay procedure determines the amount of protein bound SH groups, as well as of glutathione.28 An aliquot of 10 mL of the above serum samples was mixed with 200 mL of Tris-EDTA buffer, and the absorbance at 412 nm was measured. To these samples 8 mL of 10 mmol/L DTNB was added, and after 15 min of incubation at room temperature, the absorbance was measured again together with a DTNB blank. The total amounts of SH groups were then calculated.

above serum samples were diluted 1 : 10 with ‘‘activity buffer’’ (1 mmol/L CaCl2 in 50 mmol/L Tris HCl, pH 8.0) and then 5 mL were taken for a total reaction volume of 200 mL. Initial rates of hydrolysis were determined spectrophotometrically at 270 nm for 3 min (every 15 s). The assay mixture included 1.0 mmol/L phenyl acetate in ‘‘activity buffer’’. One unit of arylesterase activity equals 1 mmol of phenyl acetate hydrolyzed/min/mL.29 Serum PON1 lactonase activity towards DHC. The assay was performed in 96 well UV plates, in a total volume of 200 mL per well. The above serum samples were diluted 1 : 10 with ‘‘activity buffer’’ (1 mM CaCl2 in 50 mmol/L Tris HCl, pH 8.0), and 3 mL were then taken for the assay. Lactonase activity was measured using dihydrocoumarin (DHC) as the substrate. Initial rates of hydrolysis were determined spectrophotometrically at 270 nm, for 10 min (every 15 s). The assay mixture included 1 mmol/L DHC in ‘‘activity buffer’’. Non-enzymatic hydrolysis of DHC was subtracted from the total rate of hydrolysis. One unit of lactonase activity equals 1 mmol of DHC hydrolyzed/min/mL.29 Macrophage cholesterol metabolism J774A.1 macrophage cell line. The J774A.1 murine macrophage cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD). The cells were grown in DMEM containing 5% FCS. Serum-mediated changes in net cholesterol mass in macrophages. J774A.1 macrophages were incubated with 20 mL/mL of the above serum samples for 20 h at 37  C. Then the cells were washed and their lipids extracted with hexane: isopropanol (3 : 2; v : v). The hexane phase was collected and dried under N2. The amount of cellular cholesterol was determined using a kit (CHOL, Roche Diagnostics GMbH, Mannheim, Germany). After extraction of cellular lipids, the cells were dissolved in 0.1 mol/L NaOH for measurement of cellular protein by the Lowry assay.25 Serum-mediated cholesterol efflux from macrophages. J774A.1 macrophage cell line was incubated with [3H]-labeled cholesterol (2 mCi/mL) for 1 h at 37  C followed by cell wash in ice-cold PBS (3) and further incubation in the absence or presence of 20 mL/mL of the above serum samples for 3 h at 37  C. Cellular and medium [3H]-labels were quantified30 and serum-mediated cholesterol efflux was calculated as the ratio of [3H]-label in the medium/([3H]-label in the medium + [3H]-label in cells). Statistics. For comparison of the mean differences between paired groups we used the Wilcoxon rank test and a p-value <0.05 was considered significant, and p-values <0.01 were considered highly significant. Results are given as mean  standard error of the mean (SEM).

Results In vitro studies

Serum PON1 activities Serum PON1 arylesterase activity. The assay was performed in a 96 well UV plate, in a total volume of 200 mL per well. The This journal is ª The Royal Society of Chemistry 2010

a. Antioxidative properties of all beverages in a serum-free system. The highest content of total polyphenols was observed in 100% pomegranate and 100% black currant juices (Table 2). All Food Funct., 2010, 1, 99–109 | 101

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100% pomegranate juices studied are rich in polyphenols and, POM Wonderful is the richest in total polyphenol concentration. Unlike 100% black currant (Knudsen) juice however, Currant C brand was relatively low in polyphenols (Table 2). The polyphenol contents of one acai blend (Naked) and that of the red wines were also relatively high (Table 2), whereas very low polyphenol concentrations were observed in the various other juice blends, as well as in green tea, with the lowest polyphenol content observed in green tea (Nestea brand) (Table 2). 100% Pomegranate juice showed very potent free radical scavenging capacity, decreasing the optical density of the DPPH solution by as much as 62%–76% (Table 2). 100% Black currant juice decreased the optical density by 48%, while the red wines and 100% Concord grape juice decreased it by 23%– 37% (Table 2). The Tropicana blend, 100% blueberry (Knudsen), 100% Concord grape (Welch’s), 100% Yumberry (Frutzo) and green tea (Lipton) exhibited very similar, limited free radical scavenging capacity, as demonstrated by only 20%–26% reduction in the DPPH optical density (Table 2). Most beverages from the ‘‘Super fruit’’ blends category, and green tea (Nestea) were very limited in their free radical scavenging abilities (Table 2). All beverages, when used at a concentration of 0.25 mL/mL, inhibited copper ion-induced LDL oxidation, but the extent of inhibition was very different. 100% Pomegranate and 100% black currant juices were the most potent antioxidants (Table 2). Red wines and 100% Concord grape juices were somewhat less potent than the 100% pomegranate and 100% black currant juices (Table 2). Red wine (Mondavi) was the best one among all the tested wine brands, with a reduction in LDL oxidation of as high as 37%. Acai juice blends and some of the superfruit blends were very weak inhibitors of LDL oxidation (reductions of only 3– 11%) (Table 2). Green tea (Nestea) was the weakest inhibitor of LDL oxidation, as it decreased TBARS formation by only 2% (Table 2). Many of the superfruit blends reduced TBARS by only 1%. These results indicate that all pomegranate beverages except Langers and 100% black currant juice have superior total polyphenol concentration, free radical scavenging capacity and are the most potent inhibitors of LDL oxidation. b. Antioxidative properties of the beverages richest in polyphenols in a serum In vitro system. According to the results obtained in the in vitro studies (Table 2), we chose the five beverages containing the highest polyphenol concentrations i.e., 100% Wonderfulvariety pomegranate juice (POM Wonderful), 100% black currant juice (Knudsen), red wine (Mondavi), acai juice blend (Naked) and 100% Concord grape juice (Knudsen) for further in vitro study. All five beverages decreased the serum susceptibility to AAPH-induced lipid peroxidation (as compared to control serum, with no beverage addition), as measured by the TBARS assay (Fig. 1A). 100% Wonderful-variety pomegranate juice, 100% black currant juice and red wine were the most potent antioxidants in this respect, with reduction rates of serum lipid peroxidation of 38%, 38% or 31%, respectively (Fig. 1A). 100% Concord grape juice decreased serum oxidation by 23%, and acai juice blend was the least potent antioxidant juice in this respect, with only 8% inhibition of AAPH-induced serum lipid peroxidation (Fig. 1A). 102 | Food Funct., 2010, 1, 99–109

Paraoxonase 1 (PON1) is associated in serum with HDL, and possesses anti-atherogenic properties.31,32 Antioxidants have been shown to preserve PON1 activity.33 Thus, we next analyzed the effects of these selected beverages on serum PON1 catalytic activities. PON1 lactonase activity (DHC hydrolysis) was significantly increased by 48%, 51% or by 41% on serum incubation with 100% Wonderful-variety pomegranate juice, 100% black currant juice or red wine, respectively, as compared to activity in non-treated (control) serum (Fig. 1B). Grape juice or acai juice were less potent, as they increased serum PON1 lactonase activity by 35% or 29%, respectively (Fig. 1B). A similar pattern was observed upon measuring serum PON1 arylesterase activity (phenyl acetate hydrolysis, data not shown).

In vivo studies All the beverages used contain high concentrations of naturally occurring sugar (which can possibly increase serum glucose and triacylglycerol concentrations, as well as enhance serum oxidative stress). Nevertheless, they also possess potent in vitro antioxidative properties. Thus, we questioned the in vivo effects of the beverages on healthy subjects. In the present study, we analyzed the effects of the five selected polyphenol-rich beverages when consumed for a short period of time (2 h after consumption, or a daily intake for 1 week), on serum glucose and lipid profiles, on serum oxidative stress, on serum PON1 activities and on serum ability to affect cholesterol accumulation in macrophages. a. The effects of consumption by healthy subjects of the polyphenol-rich beverages on serum biochemical parameters. None of the beverages tested (acai juice blend, 100% Concord grape juice, 100% black currant juice, 100% Wonderful-variety pomegranate juice or red wine – consumed for 2 h or for 1 week) significantly affected serum lipids (total cholesterol, HDLcholesterol or triacylglycerol) concentration, compared to baseline (before beverage consumption, Table 3). Similarly, these beverages had no significant effects on serum glucose, sodium or potassium concentrations (Table 3).

b. The effects of beverage consumption by healthy subjects on serum oxidative stress Measurement of TBARS levels in the serum samples (basal oxidative status) before and after 2 h or 1 week of beverage consumption, revealed that all of the five beverages used, did not significantly impact serum basal oxidative stress, which is already low in healthy subjects (Table 4). However, consumption of the five selected beverages increased serum antioxidant status, as measured by the serum concentrations of sulfhydryl (-SH) groups (Fig. 2). Two hours after consumption of 100% Wonderful-variety pomegranate juice, the concentration of serum SH groups significantly increased by 6% (Fig. 2A). After 1 week of consumption, 100% black currant juice or 100% Wonderful-variety pomegranate juice, significantly increased the concentration of serum SH groups by 11% or 8%, respectively (Fig. 2B). In contrast, consumption of all other beverages for 2 h or for one week had no statistically significant effect on the concentration of serum SH groups (Fig. 2B). This journal is ª The Royal Society of Chemistry 2010

This journal is ª The Royal Society of Chemistry 2010 Black Currant Nectar Just Blueberry 100% Blueberry 100% Yumberry Acai Juice- The Original Acai Machine Original Blend Red Machine Green machine Red Rhapsody Mangosteen/antioxidant fruit Acai Mangosteen Acai Blueberry Mangosteen Dragonfruit Goji Cherry Goji Acai Goji Mangosteen Pomegranate Blueberry Pomegranate Blueberry Green Tea with natural flavor Green Tea with citrus BV Cabernet Sauvignon Robert Mondavi Napa Merlot Zinfandel (California)

Currant C Knudsen Wyman’s Frutzzo Bossa Nova Naked Sambazon Naked

a

Minute Maid Tropicana Nestea Lipton Beaulieu Mondavi Cline

Bossa Nova Goji Lania

Odwalla Lakewood Born Dia

Naked Welch’s Knudsen Lakewood Knudsen Lakewood Knudsen

Control represents samples with no beverage added.

Red wine

Green tea

‘‘Superfruit’’ blends

100% Yumberry Acai juice blends

100% Blueberry juice

100% Black currant juice and blends

100% Black cherry juice

100% Concord grape juice

Odwalla

Langers

Knudsen

100% Pomegranate juice (Wonderful-variety) Just Pomegranate (variety unknown) 100% Pomegranate Juice (variety unknown) Pomegranate juice (variety unknown) Pomegranate (variety unknown) 100% Concord Grape Concord Grape Pure Concord Grape Just Black Cherry Pure Black Cherry Just Black Currant

POM Wonderful

100% Pomegranate juice

Product name

Product brand

Category

Table 2 Total polyphenol concentration and antioxidative properties of various beverages tested

3.40 + 0.10 2.80 + 0.20 2.60 + 0.20 1.90 + 0.10 1.60 + 0.18 4.40 + 0.40 1.60 + 0.14 2.00 + 0.10 1.20 + 0.14 1.90 + 0.10 1.10 + 0.10 1.80 + 0.10 1.70 + 0.10 1.00 + 0.06 1.20 + 0.16 0.80 + 0.03 0.60 + 0.06 1.25 + 0.01 1.50 + 0.06 0.20 + 0.02 0.90 + 0.02 3.70 + 0.30 4.70 + 0.40 3.30 + 0.20

4.30 + 0.20 2.70  0.20 3.60 + 0.20 3.30 + 0.20 2.40 + 0.10 1.90 + 0.10 6.80 + 0.10

4.20 + 0.20

3.70 + 0.20

4.70 + 0.10

4.80 + 0.30

Total polyphenols (mg GAE/mL)

CuSO4-induced LDL oxidation (TBARS, % reduction of controla) 93  9 93  9 57  6 84  7 92  8 26  3 11  2 15  2 14  1 51 94  5 11  2 82 51 15  2 51 51 31 10  2 41 51 31 11 21 11 11 31 11 82 11  2 21 82 21  3 37  4 16  2

Free radical scavenging capacity (% O.D. reduction of controla) 76  4 75  2 62  3 71  4 70  4 23  2 32  3 32  3 18  2 15  1 48  4 31  3 23  2 15  1 23  2 11  1 18  2 71 11  2 41 11  1 10  1 10  1 10  1 51 81 51 82 82 26  3 21 20  2 29  3 37  4 27  3

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Fig. 1 The effect of polyphenol-rich beverages on in vitro AAPH-induced serum lipid peroxidation, and on serum PON1 lactonase activity. The serum from healthy subjects was pre-incubated for 1 h with no addition (control), or with 2 mL of the concentrated beverages [POM Wonderful, black currant (Knudsen), red wine (Mondavi), grape (Lakewood), acai (Naked)]. (A) The extent of AAPH-induced serum lipid peroxidation was determined by the TBARS assay. (B) Serum PON1 lactonase activity. Results are expressed as mean  SEM of three different experiments. *p <0.01 vs. control.

c. The effects of consumption of beverages by healthy subjects on serum paraoxonase 1 (PON1) activities PON1 has been shown to be inactivated under oxidative stress.33 Since nutritional antioxidants increase its expression and activities,17,33 we next analyzed the effects of the selected beverages (2 h or 1 week consumption) on serum PON1 catalytic activities (Fig. 3). Two hours after consumption of the selected beverages, serum PON1 lactonase activity was not significantly affected (Fig. 3A). After 1 week of consumption however, 100% black currant juice significantly increased serum PON1 lactonase activities by 20%, and 100% Wonderful-variety pomegranate juice increased it by 5% (Fig. 3B). The other beverages had no significant impact on PON1 lactonase activity after one week of consumption (Fig. 3B). Similar results were also found for serum PON1 arylesterase activity (data not shown).

d. The effects of beverage consumption by healthy subjects on serum-induced changes in J774A.1 macrophage cholesterol content Serum lipoproteins can be taken up by macrophages, leading to cellular accumulation of cholesterol.2,34 Under oxidative stress, lipoproteins undergo oxidation and the oxidized lipoproteins can be taken up by macrophages at an enhanced rate, further contributing to cellular cholesterol accumulation.2 As 100% Wonderful-variety pomegranate and 100% black currant juices were the most potent antioxidants among all the beverages studied, we further analyzed them for their effects on seruminduced macrophage cholesterol accumulation. Serum samples collected 2 h after juice consumption did not significantly affect cellular cholesterol content, but consumption of these juices for 104 | Food Funct., 2010, 1, 99–109

1 week resulted in a significant decrement (8%) in macrophage cholesterol content compared to baseline (Fig. 4A). Finally, we questioned whether the above serum-induced decrement in macrophage cholesterol content could be associated with increased serum-mediated cholesterol efflux from cell cultured macrophages. As shown in Fig. 4B, consumption of 100% Wonderful-variety pomegranate and 100% black currant juices for 2 h or for 1 week, had no significant effect on the extent of the human serum-induced cholesterol efflux rate from the cells, compared to serum obtained at 0 time (before juice consumption).

Discussion In the present study we have demonstrated, for the first time, that 100% Wonderful-variety pomegranate juice or 100% black currant juice, and to a lesser extent 100% Concord grape juice, or red wine, but not acai juice blend, consumed by healthy subjects in the short term (2 h, or daily consumption for 1 week) improved serum anti-atherogenic properties, i.e. increment in serum antioxidative status (SH groups, PON1 activity), and decrement in serum-induced macrophage cholesterol accumulation. Among the 35 beverages analyzed in vitro, 100% Wonderfulvariety pomegranate juice and 100% black currant juice were the most potent antioxidants (free radical scavenging capacity, and inhibition of copper ion-induced LDL oxidation). 100% Black currant juice, which contains the highest polyphenol concentration among all the beverages studied, inhibited LDL oxidation to the same degree as 100% pomegranate juice, but its free radical scavenging capacity was lower than that of 100% pomegranate juice, suggesting that pomegranate polyphenols are more potent antioxidants than black currant juice polyphenols. However, we This journal is ª The Royal Society of Chemistry 2010

159  4 159  5 164  3 86  18 89  14 84  17 51  4 52  3 55  4 85  3 77  2 84  3 144.0  0.7 144.0  0.7 144.0  1.4 4.50  0.11 4.60  0.14 4.40  0.04 154  7 157  9 154  3 92  17 78  15 84  16 49  4 51  4 50  3 86  4 75  4 85  2 146.0  0.6 145.0  0.8 145.0  0.5 4.40  0.10 4.60  0.08 4.40  0.07 h w

h w

h w

h w

Potassium/nmol/L

Sodium/nmol/L

Glucose (mg/dL)

HDL-C (mg/dL)

TG (mg/dL)

0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1

h w

h w

159  7 157  8 157  7 89  17 81  12 84  10 51  5 52  4 51  4 78  4 76  3 81  3 145.0  1.0 144.0  1.6 144.0  0.9 4.80  0.24 4.60  0.13 4.50  0.12 152  7 157  8 157  7 71  14 67  13 71  15 53  4 53  4 52  4 80  2 77  2 81  2 145.0  0.5 144.0  0.4 145.0  1.2 4.50  0.10 4.60  0.10 4.50  0.07 Cho (mg/dL)

158  6 162  7 159  7 80  11 74  11 76  11 54  3 55  3 53  4 87  4 78  2 83  4 144.0  1.2 144.0  1.1 145.0  1.0 4.30  0.09 4.60  0.02 4.60  0.12

100% Wonderful-variety pomegranate juice (POM Wonderful) 100% Black currant juice (Knudsen) 100% Concord grape juice (Lakewood) Acai juice blend (Naked) Serum biochemical parameter

Table 3 Effect of beverages consumption by healthy subjects on serum biochemical parameters. Cho – total cholesterol, TG – triacylglycerol, HDL-C – HDL cholesterol

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Red wine (Merlot/ Mondavi)

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cannot exclude the effects of other potent antioxidants in these beverages. Red wine and 100% Concord grape juice were both less potent than 100% pomegranate juice and 100% black currant juice, while acai juice blend was the weakest antioxidant among those studied in vivo. A previous in vitro comparison of the antioxidant potency of commonly consumed polyphenol-rich beverages in the United States, demonstrated the following order of antioxidant potency: 100% Wonderful-variety pomegranate juice > red wine > 100% Concord grape juice > 100% blueberry juice > 100% black cherry juice, acai juice blend, 100% cranberry juice > 100% orange juice, iced tea beverages, and 100% apple juice.3 This pattern is similar to that shown in the present study, where additional beverages and brands were tested, including 100% black currant juice. In comparison to Knudsen Black Currant juice, Currant C (another black currant juice blend) contains only one-half of the total polyphenol concentration, and demonstrated much lower antioxidative properties than the Knudsen 100% black currant juice. We chose for the in vivo study, the five juices which contained the highest polyphenol concentration: 100% black currant (Knudsen), 100% Wonderful-variety pomegranate (POM Wonderful), red wine (Merlot/Mondavi), acai juice blend (Naked) and 100% Concord grape (Knudsen). The present study is the first to compare the acute effects of the above beverages on serum antiatherogenic properties. We chose 1 week of beverage consumption since we have previously observed a significant reduction in serum oxidative stress after two weeks of 100% Wonderful-variety pomegranate juice or red wine consumption by healthy subjects.14,10 Furthermore, in the current study we evaluated the acute effects 2 h after beverage consumption. Polyphenols in the above beverages were probably absorbed to some extent, as the levels of urinary quercetin,19 or anthocyanins,20,35 or those of plasma catechin,18 were all shown to be significantly increased dose- and time-dependently after acai juice blend, 100% black currant juice or red wine consumption. The bioavailability of the pomegranate active components and metabolites has been demonstrated previously, with ellagic acid detected in the blood and dimethylellagic acid glucuronide (DMEAG), as well as urolithin A and B, found in the urine of most subjects after consumption of 100% pomegranate juice.36–39 An important issue in the current study is that consumption of the beverages (which contain sugars) by healthy subjects for 2 h or for 1 week, did not increase serum glucose levels and did not affect serum lipids or electrolytes (potassium and sodium). Serum PON1 activities (arylesterase and lactonase) were analyzed as an additional marker for oxidative stress. In vitro all the five beverages used significantly increased serum PON1 activities. The mechanism responsible for the PON1 activity increase could be related to the binding of the beverages’ polyphenols to PON1. Such binding may change the enzyme conformation, thus affecting PON1 active site interactions with its substrates. Furthermore, certain polyphenols increase PON1 binding to the HDL, thus stabilizing PON1, as was shown previously upon incubation of serum in vitro with pomegranate juice or its most potent unique polyphenol punicalagin.15,16 Furthermore, 100% Wonderful-variety pomegranate juice consumption over longer periods of time (weeks, months, or years) has been shown to increase PON1 binding to the HDL particles, and as a result, stabilize PON1 and enhance its catalytic Food Funct., 2010, 1, 99–109 | 105

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Table 4 Effect of polyphenol-rich beverages’ consumption by healthy subjects on basal serum oxidation status

Beverage

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Acai juice blend (Naked) 100% Concord grape juice (Lakewood) 100% Black currant juice (Knudsen) 100% Wonderful-variety pomegranate juice (POM Wonderful) Red wine (Merlot/Mondavi)

TBARS/nmol/mL before beverage consumption

TBARS/nmol/mL 2 h after beverage consumption

TBARS/nmol/mL after 1 week of beverage consumption

1.50  0.16 1.60  0.07

1.60  0.18 1.70  0.11

1.60  0.07 1.50  0.09

1.60  0.09

1.70  0.11

1.60  0.12

1.50  0.18

1.50  0.12

1.50  0.08

1.60  0.07

1.60  0.08

1.60  0.11

and biological activities.12,13,15,16 We evaluated also serum sulfhydryl (SH) group concentrations, as they have been shown to be inversely related to oxidative stress, and positively correlated with PON1 activity.40 Although all five selected polyphenol-rich juices inhibited AAPH-induced serum lipid peroxidation in vitro, in vivo their consumption for a short period of time (2 h or 1 week) did not significantly affect AAPH-induced serum lipid peroxidation. Serum SH group concentration and PON1 activity however, were modestly, but significantly increased following beverage consumption, with 100% Wonderful-variety pomegranate and 100% black currant juices being the most potent beverages after 1 week of consumption. Nevertheless, this protection by SH groups and PON1 may be insufficient to protect the serum from free radical-induced oxidative stress. PON1 was shown to protect serum from oxidative stress, by its ability to hydrolyze specific oxidized lipids in lipoproteins (such as specific oxidized phospholipids, lipid peroxides, cholesteryl

linoleate hydroperoxides).41–43 Longer periods of beverage consumption (up to months) may be needed to achieve more impressive effects on serum oxidative stress in healthy subjects. Among the in vivo studied beverages, acai juice blend was the least potent, even though it has a similar concentration of polyphenols. This suggests that acai juice blend polyphenols are weak antioxidants, in comparison to the polyphenols in 100% pomegranate juice, 100% black currant juice or 100% Concord grape juice (and also red wine). Similar to the present study, plasma antioxidant capacity increased twelve hours after consumption of acai juice blend,20 or 2 h post-consumption of a mixture of berries, by healthy subjects.21 In a recent study, 100% black currant juice consumption for 1 week, substantially decreased serum lipid peroxidation,19 but this impressive effect is likely related to the high volume of juice consumed (1500 mL). As 100% Wonderful-variety pomegranate juice and 100% black currant juice were the most potent antioxidants in the

Fig. 2 The effect of polyphenol rich beverages consumed by healthy subjects on the concentration of their serum SH groups. Six healthy subjects consumed POM Wonderful, black currant (Knudsen), red wine (Mondavi), grape (Lakewood), and acai (Naked). Blood samples were collected before and after 2 h or 1 week of beverage consumption. Serum SH group concentrations were determined as described under the methods section. (A) 2 h after consumption of beverages. (B) 1 week after of consumption of beverages. Results are expressed as mean  SEM. *p <0.05 vs. 0 time, **p <0.01 vs. 0 time.

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Fig. 3 The effects of consumption of polyphenol-rich beverages by healthy subjects on their serum paraoxonase 1 (PON1) catalytic activity. Six healthy subjects consumed POM Wonderful, black currant (Knudsen), red wine (Mondavi), grape (Lakewood), and acai (Naked). Blood samples were collected before and after 2 h or 1 week of beverage consumption. Serum PON1 lactonase activity was determined. (A) 2 h after beverage consumption. (B) 1 week after beverage consumption. Results are expressed as mean  SEM. *p <0.05 vs. 0 time, **p <0.01 vs. 0 time.

Fig. 4 The effect of pomegranate juice and black currant juice consumption by healthy subjects on serum-induced cholesterol accumulation in J774A.1 macrophages. J774A.1 macrophages were incubated with 20 mL/mL of the above serum samples for 20 h at 37  C. (A) The cellular total cholesterol content was then determined in the cells’ lipid extract. (B) The extent of serum-mediated cholesterol efflux from the cells was determined as described in the Methods section. Results are expressed as mean  SEM. *p <0.05 vs. 0 time.

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in vitro and in the in vivo studies, we chose them to assess an important feature of atherogenesis, i.e., macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherogenesis.1,2 Consumption of 100% Wonderful-variety pomegranate juice or 100% black currant juice for 1 week, modestly, but significantly, decreased serum-induced cholesterol accumulation in macrophages. The reduction in macrophage cholesterol mass, however, was not the result of increased cholesterol efflux from the cells. In fact, it is probably the result of inhibition of cholesterol-rich lipoprotein uptake by the cells, mediated by serum associated polyphenols and/or polyphenol metabolites.44,45 In conclusion, we showed in the present study that 100% Wonderful-variety pomegranate juice and 100% black currant juice have potent antioxidant properties, both in vitro and in vivo. These findings were observed even after short periods of daily juice consumption (two hours, or for one week), though the antioxidant and anti-atherogenic effects were modest, in comparison to previous studies conducted over longer periods of juice consumption.12–15

Acknowledgements

13 14

15

16

17

18

19

The financial support for this research was obtained from ‘‘POM Wonderful’’ Ltd., Los Angeles, California, USA. All authors declare that they have no conflict of interest. 20

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39 D. Bialonska, S. G. Kasimsetty, S. I. Khan and D. Ferreira, Urolithins, intestinal microbial metabolites of Pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay, J. Agric. Food Chem., 2009, 57, 10181–10186. 40 U. C ¸ akatay, R. Kayali and H. Uzun, Relation of plasma protein oxidation parameters and paraoxonase activity in the ageing population, Clin. Exp. Med., 2008, 8, 51–57. 41 Z. Ahmed, A. Ravandi, G. F. Maguire, A. Emili, D. Draganov, B. N. La Du, A. Kuksis and P. W. Connelly, Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with a peroxynitrite donor, J. Biol. Chem., 2001, 276, 24473–24481. 42 M. I. Mackness, S. Arrol and P. N. Durrington, Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein, FEBS Lett., 1991, 286, 152–154. 43 M. Aviram, M. Rosenblat, C. L. Bisgaier, R. S. Newton, S. L. PrimoParmo and B. N. La Du, Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase, J. Clin. Invest., 1998, 101, 1581– 1590. 44 B. Fuhrman, N. Volkova, R. Coleman and M. Aviram, Grape powder polyphenols attenuate atherosclerosis development in apolipoprotein E deficient (E0) mice and reduce macrophage atherogenicity, J. Nutr., 2005, 135, 722–728. 45 B. Fuhrman, N. Volkova and M. Aviram, Pomegranate juice inhibits oxidized LDL uptake and cholesterol biosynthesis in macrophages, J. Nutr. Biochem., 2005, 16, 570–576.

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PAPER

www.rsc.org/foodfunction | Food & Function

Mediterranean diet improves dyslipidemia and biomarkers in chronic renal failure patients Khedidja Mekki,*a Nassima Bouzidi-bekada,a Abbou Kaddousb and Malika Bouchenaka

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Received 3rd June 2010, Accepted 31st August 2010 DOI: 10.1039/c0fo00032a Dyslipidemia, oxidative stress (OS) and inflammation increase the risk of cardiovascular disease in chronic renal failure (CRF) patients. The aim of this study was to evaluate the effect of nutritional advice on dyslipidemia and biomarkers in CRF patients. 40 CRF patients with dyslipidemia, hypertriglyceridemia and/or hypercholesterolemia were randomly assigned to either the control or the intervention group. The intervention group received nutritional advice adapted to a Mediterranean diet (MD). Patients were assessed at baseline (T0) and after 30 (T1), 60 (T2) and 90 (T3) days for dietary intake and biomarkers. In the intervention group compared to the control group, TG concentrations were decreased by 26% at T3 (p < 0.05), TC concentrations were diminished by 14% at T2 and by 35% at T3 (p < 0.05). A decrease in LDL-C was noted at T2 and T3 (p < 0.05). The TC/HDL-C ratio was diminished at T1, T2 and T3 (p < 0.05). The apo A-I/apo B ratio was elevated at T3 (p < 0.05). HDL-C, apo A-I, apo B concentrations and the TC/LDL-C ratio were similar in the both groups at T1, T2 and T3. Creatinine, urea, glomerular filtration rate (GFR), urate, iron and bilirubin values remained unchanged in both groups. Haemoglobin concentrations were elevated at T1 (p < 0.05). Increased albumin values were observed at T2 (p < 0.05). CRP concentrations were decreased by 29% at T1 (p < 0.05) and 40% (p < 0.01) at T3. Fibrinogen (p < 0.01) concentrations were decreased at T3. In the intervention group compared to control group (p < 0.01), TBARS values were decreased by 16% at T2 and 21% at T3 (p < 0.05). In this study, we demonstrate that the nutritional management of CRF patients before dialysis based on the MD improves food consumption, reduces dyslipidemia and protects against lipid peroxidation and inflammation, allowing patients to enter dialysis with an acceptable nutritional and cardiovascular state.

Introduction Recent advances in the pathophysiology of chronic renal failure (CRF) have highlighted the importance of dietary management of this disease. Indeed, despite continuous progress in the delivery of renal replacement therapy (RRT), new symptoms have appeared. Among them, undernutrition and cardiovascular diseases (CVD) hold a prominent place. The increased prevalence of both CVD morbidity and mortality is evident at all ages among patients with CRF. Both traditional risk factors, including diabetes, dyslipidemia and hypertension, and non-traditional risk factors associated with CRF, including inflammation, oxidant stress and malnutrition, may further increase CVD risk.1–3 Dyslipidemia enhances lipid peroxidation and activates free radical reactions.2 Hypertriglyceridemia, hypercholesterolemia and elevated levels of low-density lipoprotein-cholesterol (LDLC) are identified as key factors for CVD risk in CRF patients. Observational and epidemiological data have suggested the potential predictive role of C-reactive protein (CRP) in coronary heart disease.4–6 a Laboratoire de Nutrition Clinique et M etabolique, D epartement de Biologie, Facult e des Sciences, Universit e d’Oran, 31100, Algeria. E-mail: [email protected]; Fax: +213 41 58 19 44; Tel: +213 41 58 19 44 b Service de N ephrologie, Etablissement Hospitalo-Universitaire Oran, Algeria

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Beneficial outcomes can be strengthened through dietary interventions. Nutritional intervention plays a major role in preserving the overall well-being of CRF patients. In preparation for RRT, dietary monitoring aims to reduce cardiovascular risk factors, prevent malnutrition and slow the progression of renal disease,1–3 all of which can contribute to positive outcomes for patients. There are several potential advantages to prescribing a carefully designed low-protein diet (0.75 g kg1 BW d1) for the treatment of CRF patients.7 Low-protein diets reduce the generation of nitrogenous wastes and inorganic ions, which cause many of the clinical and metabolic characteristics of uremia. Moreover, low-protein diets can diminish the effects of hyperphosphatemia, metabolic acidosis, hyperkalemia and other electrolyte disorders that are the consequence of renal function loss.7 During the past decade, a large body of evidence has related the adherence to a Mediterranean diet (MD) to a decrease in all the causes of mortality, as well as the incidence of coronary heart diseases.8 An MD has long been recommended for its antiatherosclerotic properties.9 Among the effects of an MD diet, the suppression of lipoprotein peroxidation10 and the normalization of endothelial function are observed.11,12 Such diets do not need to be restricted in total lipid intake as long as there is no excess of energy intake over expenditure and vegetable oils are emphasized as the main source of lipids, which are low in saturated fats and partially hydrogenated oils.13 The traditional MD includes the high consumption of olive oil, legumes, unrefined cereals and This journal is ª The Royal Society of Chemistry 2010

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cereal products (whole grain bread, pasta and brown rice), fruits and vegetables, the moderate to high consumption of fish and dairy products (cheese and yogurt), the low consumption of meat and meat products, and moderate wine consumption.14,15 The ratio of monounsaturated (MUFA) to saturated fats is much higher in the MD than in other places of the world, including northern Europe and North America.14,15 The aim of this study is to evaluate the effect of appropriate nutritional advice based on the MD, on feeding, dyslipidemia and biomarkers in dyslipidemic CRF patients. We hypothesize that nutritional management based on a healthy and varied diet will enhance food intake and reduce cardiovascular risk through an adjustment of dyslipidemia and biomarker abnormalities, suggesting that an MD diet may have a preventive role against CVD.

Subjects and methods Subjects A prospective randomized trial study was carried out from January to April 2008 in the hospital of Oran (west of Algeria). Undialyzed patients were included on the basis that they had a moderate CRF with a glomerular filtration rate (GFR) of 60– 89 mL min1 and dyslipidemia (triacylglycerols > 1.7 mmol L1) and/or total cholesterol > 5 mmol L1). Subjects were excluded on the basis that they had diabetic nephropathy, thyroid disease, and the use of anti-inflammatory drugs or antioxidants and vitamins. Creatinine clearance was estimated from serum creatinine using the Cockroft and Gault formula [GFR ¼ (140-age)  BW  1.23/creatinine]. In women, this value was multiplied by 0.85.16 From the 80 patients screened, 40 patients (M/F, 22/18) aged 61  14 years were recruited for the study. CRF in patients was caused by chronic glomerulonephritis (n ¼ 22), vascular nephropathy (n ¼ 8), cystic kidney disease (n ¼ 3) and unknown (n ¼ 7). The demographic and medical characteristics of the population studied are presented in Table 1. Patients consented to participate in the study and were randomized into two groups: the intervention group, which underwent nutritional intervention (n ¼ 20), and the control group (n ¼ 20), which did not receive modified nutritional advice. Table 1 Clinical characteristics of the patientsa

Patients (n) Age/years Weight/kg BMI Sex ratio (M/F) Smokers (%) Employed (%) SBP/mm Hg DBP/mmHg Glucose/g L1 Triacylglycerols/mmol L1 Total cholesterol/mmol L1

CRF patients

Control group

Intervention group

40 61  14 74  15 26.2  5.6 22/18 32 50 125  10 84  1 0.90  0.06 3.8  0.1 6.1  0.7

20 59  12 73  11 25.1  4.2 10/10 29 45 135  8 80  2 0.86  0.01 3.2  0.3 6.5  0.4

20 60  10 76  14 26.9  3.9 11/9 30 38 128  11 83  2 0.95  0.02 3.8  0.1 6.1  0.7

a Data are expressed as mean  standard error. BMI: body mass index (weight kg/height m2); SBP: systolic blood pressure; DBP: diastolic blood pressure.

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Nutritional intervention All patients received nutritional advice based on the NKF K/DOQI (National Kidney Foundation—Kidney Disease Outcomes Quality Initiative) guidelines7 (energy intake of 0.12 MJ kg1 BW d1, protein intake 0.75 g kg1 BW d1, lipid intake 35% and carbohydrates 55% of total energy intake). In the intervention group, dietary recommendations were modified and adapted to a MD, with increased intake of MUFA, PUFA and fibers. To achieve this objective, the subjects were asked to consume olive oil and nuts for seasonings, whole grains (50 g of bread at each meal, 250 g of cereal or starch once a day), fruits (once a day), vegetables (200 g twice daily) and fish (twice a week). A list of foods rich in salt, potassium and phosphorus was provided. In addition, patients received advice about the cooking methods best suited for adherence to a MD. To control the recommendation monitoring, nutritional surveys were carried out at baseline and at 30 (T1), 60 (T2) and 90 (T3) days after the beginning of nutritional intervention. All patients received intervention instructions at the Nephrology ward of the University Hospital of Oran. The purpose of this study was explained to the subjects, and the investigation was carried out with their consent. The experimental protocol was approved by the Committee for Research on Human Subjects of Oran. Dietary survey methods The food consumption survey used the method of ‘‘recall and record’’, repeated every 4 days. Patients were interviewed by trained interviewers using an adapted and structured questionnaire. Each subject was asked to recall everything they had eaten or drunk during the 24 h preceding the interview. The day was chronologically organized into breakfast, lunch and dinner. The meals were structured by entry, principal dish, accompaniments, bread and drinks. The interview was organized with specific questions about the ingredients and methods of preparation, and the additions usually served with a meal, such as butter, milk in coffee and mayonnaise. Subjects were also asked to list the names and quantity of consumed food that was not spontaneously reported. Serving sizes were estimated by the use of the food portion model handbook. Dimensions of dishes, utensils and foods were measured, and the portion sizes were estimated accurately. The consumed foods were converted into various nutrients using the software GENI.17 Assays In all patients’, blood samples were drawn after a 12 h overnight fast by antecubital venipuncture at the beginning (T0), 30 (T1), 60 (T2) and 90 (T3) days after initiating nutritional intervention. Samples were collected and subjected to low speed centrifugation at 3000  g at 5  C for 15 min. They were then preserved with 0.1% Na2 EDTA and 0.02% sodium azide. Triacylglycerols (TG) and total cholesterol (TC) were determined by colorimetric methods (BioMerieux Kits, France). Urea and creatinine were analyzed by colorimetric methods (Kits Biocon). Serum high-density lipoprotein-cholesterol (HDL-C) was determined enzymatically using a CHOD-PAP kit after precipitation of the chylomicrons, very low-density lipoprotein Food Funct., 2010, 1, 110–115 | 111

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cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) with phosphotungstic acid and Mg2+ (BioMerieux Kits, SA-France). Serum LDL-C was determined enzymatically using a CHOD-PAP kit after precipitation of LDL. Serum apolipoproteins (apo) A-I and B were measured by the immunoturbidimetric method (Human kit, Allemagne). Serum lipid peroxidation was estimated by measuring the concentrations of thiobarbituric acid reactive substances (TBARS) according to the method of Quintanilha et al.,18 using tetramethoxypropane (Prolabo) as a precursor of malondialdehyde. C-Reactive Protein (CRP) was measured by the immunoturbidimetric method (Fumouze, France). Colorimetric methods were used for the determination of albumin, urate (Kits Boehringer, Mannheim, Germany), iron and bilirubin (Biolabo kits, France). Fibrinogen levels were measured using automatic nephelometry. Statistical analysis Values are presented as mean and standard error. Data normality and distribution of the variables were tested by the KolmogorovSmirnov test. The difference between the means from the different groups was checked by ANOVA adjusted for multiple comparisons. Differences between groups at the same time point were analyzed using Mann Whitney’s test, whereas differences within groups at different time points were analyzed using Wilcoxson’s test. Levels of p < 0.05 were considered significant. Linear regression analysis was used to determine correlation coefficients between food intake, dyslipidemia and biomarker values. The calculations were performed using STATISTICA 6.0 (for Windows, StatSoft Inc. software, Tulsa, OK, USA).

Results Food intake composition A significant increase in total energy intake (TEI) (Table 2) was noted in the intervention group compared to the control group at T2 and T3 (p < 0.05). These intakes were also increased compared to T0 (p < 0.05). An unbalanced energy distribution was noted at T0. Expressed as a percentage of TEI, protein, carbohydrate and lipid intakes represented, respectively, 8, 65 and 27% at T0. Similar protein intakes were noted at T1, T2 and T3 in the intervention group compared to the control group. Carbohydrate intake was increased at T3 in the intervention group compared to

control group and to T0 (p < 0.05). Lipid intake was increased at T2 and T3 in the intervention group compared to control group and to the baseline value T0 (p < 0.05). An improvement in animal protein intake (Table 3) was noted only at T3 in the intervention group compared to the controls and to T0 (p < 0.05). In parallel, the consumption of vegetable protein decreased at T1 and T3 compared to the controls (p < 0.05). An increase in sugar intake was noted at T2 and T3 (p < 0.05) in the intervention group compared to the control group. Starch consumption decreased at T3 in the intervention group compared to the control group and to T0 (p < 0.05). Fiber and cholesterol intakes were in accordance with nutritional requirements. PUFA and SFA intakes were, respectively, lowered and increased at T1, T2 and T3 in the intervention group compared to the control group (p < 0.05). An increase in MUFA intake was noted at T2 and T3 (p < 0.05). In the intervention group, a decrease was noted in PUFA intake at T1, T2 and T3 compared to T0 (p < 0.05). At T3, the qualitative distribution of nutrients had a tendency to be closer to the recommended diet. In the intervention group compared to the control group (Table 4), a high consumption of cooked vegetables, fruit, bread, cereals, rice, pasta, milk and dairy products was noted at T3 in the intervention group compared to the control group (p < 0.05). However, a significant decrease was noted in fat intake at T3 (p < 0.01) in the intervention group compared to the control group and to T0 (p < 0.05).

Lipids and apolipoproteins A decrease by 26% in TG concentration was noted at T3 (Table 5) in the intervention group compared to the control group, and by 9% compared to T0 (p < 0.05). The TC concentration was diminished by 14% at T2 and by 35% at T3 (p < 0.05) in the intervention group compared to the control group. At T3, values of TC were lower than at T0 (p < 0.05). A decrease in LDL-C was noted at T2 and T3 (p < 0.05) in the intervention group compared to the control group. These values were lower at T3 compared to T0 (p < 0.01). The TC/HDL-C ratio was diminished at T1, T2 and T3 in the intervention group compared to the control group (p < 0.05) and to T0. The apo A-I/apo B ratio was elevated at T3 in the intervention group compared to the control group and to T0 (p < 0.05). HDL-C, apo A-I and apo B concentrations, and the TC/LDL-C ratio, were similar in both groups at T1, T2 and T3.

Table 2 Food intake compositiona Control group

TEI/MJ Proteins/MJ % of TEI Carbohydrates/MJ % of TEI Lipids/MJ % of TEI

Intervention group

Baseline T0

T1

T2

T3

T1

6.8  0.3 0.6  0.02 8 4.1  0.1 65 1.8  0.1 27

6.0  1.5 0.60  0.1 9 4.2  2.1 70 1.3  0.6 21

6.1  0.3c 0.6  0.01 9 3.9  0.2 63 1.9  0.2c 28

6.1  0.2c 0.6  0.04 9 3.3  0.1c 64 1.9  0.2 27

7.5 0.6 9 4.5 60 2.4 32

 0.6  0.07  0.2  0.2

T2

T3

DR

7.9  0.4d 0.7  0.09 9 4.3  0.1 56b 2.7  0.2d 35d

7.6  0.1d 0.7  0.07 10 4.4  0.1d 55b 2.6  0.3d 35d

8 0.8 10 4.4 55 2.8 35

a T0: the beginning of nutritional intervention; T1, T2 and T3: 30, 60 and 90 days after initiating nutritional intervention. DR: dietary recommendations. Data are presented as mean  standard error. b Significant difference between groups at the same time point (Mann Whitney’s test). c Significant difference in relation to the baseline (Wilcoxon’s test). d p < 0.05.

112 | Food Funct., 2010, 1, 110–115

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Table 3 Qualitative food intakea

Downloaded on 21 October 2010 Published on 22 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00032A

Baseline T0 Animal (%) Vegetable (%) Sugar (%) Starch (%) Fiber/g PUFA (%) MUFA (%) SFA (%) Cholesterol/mg

38 62 18 82 36 30 37 33 180

Control group

Intervention group

T1

T1

41 59 20 80 35 35 36 29 190

T2 55 45 19 81 30 32 35 23 175

T3 69 31 21 79 29 30 33 37 225

c

58 42b 25 75 35 25d 38 37b 230

T2 52 48c 30b 70c 34 26d 44b 30b 225

T3

DR

d

57 43d 29b 61d 33 23d 49d 28b 228

60 40 40 60 30 25 50 25 <300

a

T0: the beginning of nutritional intervention; T1, T2 and T3: 30, 60 and 90 days after initiating nutritional intervention. DR: dietary recommendations. Data are presented as mean  standard error. b Significant difference between groups at the same time point (Mann Whitney’s test). c Significant difference in relation to the baseline (Wilcoxon’s test). d p < 0.05.

Renal function and biomarkers Creatinine, urea, GFR, urate, iron and bilirubin values remained unchanged in both groups (Table 6). Haemoglobin concentrations were elevated at T1 in the intervention group compared to the control group and to T0 (p < 0.05). Increased albumin values were observed at T2 (p < 0.05) in the intervention group compared to T0. CRP concentrations decreased by 29% at T1

(p < 0.05) and by 40% at T3 (p < 0.01) in the intervention group compared to the control group. A decrease in the CRP level was noted in the intervention group at T3 compared to T0 (p < 0.05). Fibrinogen concentrations decreased at T3 (p < 0.01) in the intervention group compared to the control group (p < 0.01) and to T0 (p < 0.05). TBARS values decreased by 16% at T2 and by 21% at T3 in the intervention group compared to the control group. These values were lower compared to T0 (p < 0.05). Correlation analyses In the intervention group, inverse relationships were noted between MUFA intake and TC concentration (r ¼ 0.59, p < 0.01) and LDL-C (r ¼ 0.55, p < 0.01). TBARS and CRP concentrations were negatively correlated with cooked, vegetables and fruit intakes (r ¼ 0.60, p < 0.01).

Discussion This study was undertaken in order to evaluate the effect of nutritional advice based on the principles of an MD on dyslipidemia and serum biomarkers in undialyzed CRF patients. During follow-up, food intake and serum biomarkers were assessed at baseline, 30, 60 and 90 days after initiating intervention. A favorable effect on renal function, represented by a stable GFR, was observed. Recent findings have shown that greater adherence to an MD is independently associated with reduced urea and creatinine, and increased creatinine clearance rates, among

Table 4 The intake of food groupsa Control group

Intervention group

Food group

Baseline T0

T1

T2

T3

T1

T2

T3

DR

Cooked vegetables and fruit Bread, cereals, rice and pasta Milk and dairy products Meat, poultry and fish Raw vegetables and fruits Fat Sweet products

255  66 278  55 85  9 35  12 125  26 80  12 95  18

230  15 270  95 95  14c 32  4.0 130  75 85  12 87  13

238  72 267  56 99  28c 39  23 139  13 79  32 79  8.6

238  65 298  15 95  12 45  10 167  62 95  15 89  8

262  85 302  26 121  14c 40  0.64b 138  15 77  25 78  22

393  108 326  89 137  14c 48  14 198  10d 45  33 69  8.6

462  99d 411  85b 160  35d 70  25 255  92 55  0.75c 59  16

500 400 180 50 50 60 60

a T0: the beginning of nutritional intervention; T1, T2 and T3: 30, 60 and 90 days after initiating nutritional intervention. DR: dietary recommendations. Data are presented as mean  standard error. b Significant difference between groups at the same time point (Mann Whitney’s test). c Significant difference in relation to the baseline (Wilcoxon’s test). d p < 0.05.

Table 5 Baseline levels and the effect of 3 months of nutritional intervention on serum lipidsa Control group

TG/mmol L1 TC/mmol L1 HDL-C/mmol L1 LDL-C/mmol L1 Apo AI/g L1 Apo B/g L1 TC/HDL-C TC/LDL-C Apo AI/Apo B

Intervention group

Baseline T0

T1

T2

T3

T1

T2

T3

3.2  0.3 6.5  0.4 2.1  0.5 3.5  1.0 0.9  0.2 0.9  0.1 2.9  0.1 2.1  0.2 0.8  0.1

2.8  0. 6 5.3  1.0 2.7  0.2 3.3  0.2 1.2  0.6 1.0  0.2 2.6  0.1 2.6  0.8 1.5  0.5

3.0  0.1 6.3  1.0 2.5  0.2 3.6  0.2 1.3  0.6 1.0  0.3 2.8  0.2 2.3  0.1 0.9  0.1

3.9  0.1 5.4  0.4 3.0  0.2 3.0  0.2 1.3  0.5 1.0  0.1 2.7  0.4 2.3  0.4 1.1  0.2

3.4  0.4 6.1  0.02 2.5  0.2 3.6  0.2 1.3  0.1 1.1  0.1 2.1  0.2d 2.8  0.9 1.6  0.4

3.1  0.8 5.4  0.9b 2.5  0.4 2.8  0.1b 1.1  0.1 1.1  0.3 1.8  0.1d 3.2  0.1d 0.8  0.4

2.9 4.1 2.8 2.0 1.2 1.0 1.7 2.1 1.8

 0.1d  0.5d  0.6  0.02b,e  0.1  0.1  0.2b,e  0.5  0.1d

a T0: the beginning of nutritional intervention; T1, T2 and T3: 30, 60 and 90 days after initiating nutritional intervention. Data are presented as mean  standard error. b Significant difference between groups at the same time point (Mann Whitney’s test). c Significant difference in relation to the baseline (Wilcoxon’s test). d p < 0.05. e p < 0.01.

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Table 6 Baseline levels and the effect of 3 months of nutritional intervention on renal function and serum biomarkersa Control group

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1

Creatinine/mmol mL Urea/mmol L1 GRF/mL L1 Urate/mmol L1 Iron/mmol L1 Bilirubin/mmol L1 Haemoglobin/g dL1 Albumin/g L1 CRP/mg L1 Fibrinogen/g L1 TBARS/mmol L1

Intervention group

Baseline T0

T1

T2

T3

T1

T2

T3

189.0  70.0 14.2  4.8 75.0  15.0 0.4  0.1 33.0  17.7 6.2  2.6 12.1  0.1 32.2  5.0 6.5  0.9 3.5  0.7 8.4  0.5

150.0  49.0 12.0  5.0 69.0  9.0 0.4  0.1 38.1  15.0 5.6  2.1 10.0  0.1c 30.4  6.0 7.0  0.2 3.0  0.7 9.5  0.2

169.0  49.0 12.0  2.1 72.0  6.0 0.4  0.1 35.0  0.1 7.0  0.1 12.9  0.1 35.0  2.1 7.8  0.8 3.2  1.9 8.0  0.7

110.0  33.0 11.8  4.4 75.0  8.0 0.6  0.2 37.2  0.8 8.1  0. 7 14.1  2.2 38.0  10.0 7.0  0.1 3.4  0.9 7.8  0.3

151.0  57.0 11.0  4.8 70.0  10.0 0.6  0.1 36.0  0.1 7.3  0.1 13.5  0.1d 38.0  6.3 6.4  0.1b 2.9  0.5 7.9  1.5

170.0  56.0 11.5  3.0 72.0  6.0 0.5  0.1 34.6  0.1 6.8  1.3 13.9  1.1 42.2  5.0c 5.8  1.0 2.9  1.5 6.7  0.3d

109.0  47.0 12.1  3.4 77.0  0.9 0.6  0.1 35.1  0.3 7.6  0.1 13.9  5.0 44.1  5.2 4.2  0.2e 2.0  0.1e 6.3  0.01d

a T0: the beginning of nutritional intervention; T1, T2 and T3: 30, 60 and 90 days after initiating nutritional intervention. Data are presented as mean  standard error. b Significant difference between groups at the same time point (Mann Whitney’s test). c Significant difference in relation to the baseline (Wilcoxon’s test). d p < 0.05. e p < 0.01.

healthy men and women.19 Similarly, we showed in a recent study that renal function was not altered by three-month balanced diet intervention.20 This investigation showed that in CRF patients with moderate dyslipidemia, the monitoring of nutritional intake lead to a decrease in TG, TC and LDL-C after 90 days of intervention. Some recent studies have highlighted the beneficial role of an MD on lowering hypertriglyceridemia.19,21,22 In the ATTICA study, the authors established that biochemical and clinical markers were affected favorably by such a diet.19,21,22 Moreover, it has been shown that whole-grain intake is inversely correlated with triacylglycerol concentration.23 In our previous work carried out on haemodialysis patients, hypertriglyceridemia was found to be moderate as compared to its prevalence in developed countries. This could be due to the diet consumed by our population, which was characterized by a high intake of vegetable proteins, complex carbohydrates, fiber and MUFAs.24–26 A negative correlation was noted between MUFA intake and TC in the intervention group. Olive oil, as the major source of MUFAs, exerts an antioxidant action and suppresses the release of arachidonic acid from the lipid constituents of cell membranes.9 The results of our study confirmed that significant changes in serum lipids could be induced by modest dietary modifications. In this study, we used serum TBARS as a marker of OS, and CRP as an inflammation marker. TBARS levels were diminished after 3 months of initiating dietary intervention. Serum lipids were protected against OS by dietary components such as polyphenols and MUFA. The finding that the TBARS concentration was inversely related to cooked vegetable and fruit intake suggests that the key role in protecting lipids against peroxidation is played by some factor(s) present in the diet other than oleic acid. Such an effect may be achieved by polyphenols contained, for example, in cold-pressed olive oil, fruit and vegetable. It has been shown that there is an association between elevated CRP levels and the risk of cardiovascular events and peripheral vascular disease that is independent of traditional risk factors.4–6 In our study, the CRP level was greater than 5 mg L1 at the beginning of the study and then was modified by the diet. Elevated serum CRP levels are correlated with increased risk of death due to stroke.4 With levels of CRP above 5.5 mg L1, the risk is 1.67-times higher than that at levels below 2.1 mg L1; 114 | Food Funct., 2010, 1, 110–115

moreover, this is independent of other risk factors.4 In our patients, CRP concentrations were inversely associated with cooked vegetables, fruit and fish; fish being the best source of omega-3 fatty acids. Indeed, in our patients, sardines were the most frequently consumed fish (twice a week). A strong inverse relationship was found between fish consumption and levels of inflammatory markers related to CV.22 Similarly, it has been demonstrated that a greater adherence to a traditional MD is independently associated with a reduction in CRP and fibrinogen levels.21 The association between elevated plasma fibrinogen and coronary risk may also partly reflect an ongoing inflammatory process.27 However, the exact association between these proinflammatory markers and diet was not clear. Therefore, because fish consumption has been associated with decreased concentrations of these proinflammatory markers, it can be suggested that regular fish intake may suppress inflammation and have beneficial effects on human health. Recently, we also showed that omega-3 fatty acid supplementation (2.1 g day1) was inversely associated with hypertriglyceridemia and CRP in CRF patients eating a balanced diet.20 Zampelas et al. (2005)22 suggested that the benefit is more pronounced if omega-3 fatty acids are consumed in the form of fish rather than in the form of a supplement. In conclusion, the nutritional management of CRF patients before dialysis based on an MD improves food consumption, reduces dyslipidemia and protects against lipid peroxidation and inflammation, allowing patients to enter dialysis with an acceptable nutritional and cardiovascular state.

Acknowledgements This work was supported by the National Agency of Health Research (ANDRS no 02/15/01/01/084).

References 1 V. Chauhan and M. Vaid, Dyslipidemia in chronic kidney disease: managing a high-risk combination, Postgrad. Med., 2009, 121(6), 54–61. 2 V. Filiopoulos, D. Hadjiyannakos, L. Takouli, P. Metaxaki, V. Sideris and D. Vlassopoulos, Inflammation and oxidative stress in dialysis patients: the impact of renal replacement treatment, Int. J. Artif. Organs, 2009, 32(12), 872–882.

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www.rsc.org/foodfunction | Food & Function

Effects of dietary consumption of cranberry powder on metabolic parameters in growing rats fed high fructose diets Ramesh C. Khanal,ab Theodore J. Rogers,a Samuel E. Wilkes,a Luke R. Howardb and Ronald L. Prior*ac

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Received 20th July 2010, Accepted 9th August 2010 DOI: 10.1039/c0fo00089b The effect of dietary consumption of a cranberry powder (CP) containing increased amounts of procyanidins and other phytochemicals on metabolic parameters associated with metabolic syndrome was investigated in growing rats fed a high fructose diet. Dietary treatments were control (starch based), high fructose (HF), and HF containing either 3.3, 6.6, or 33 g CP/kg diet. Fasting plasma glucose and triglycerides tended to be higher with HF feeding and were reduced by feeding CP. The area under curve following an oral glucose tolerance test was 35–50% higher in animals fed HF diet vs. control and was decreased to control levels by the low or medium but not high CP diet. Feeding CP tended to lower fasting plasma insulin. Homeostatic models of insulin resistance (HOMA-IR) and b-cell function (HOMA-BCF) were lowest in animals fed low or medium CP diets (p < 0.05). Rats fed the control starch diet had slightly higher food intake, final body weight, and abdominal fat compared to animals fed other diets. Kidney weight was higher in HF group and feeding CP decreased kidney weight to normal levels. In the fed state, plasma triglyceride was increased with HF diet, whereas insulin was lower in animals fed HF diet. Overall, inclusion of CP in the diet was effective in modulating some aspects of metabolic parameters associated with metabolic syndrome and the medium level of CP in the diet produced a better response than the lower and higher CP levels.

Abbreviations HF LC MC HC HOMA-IR HOMA-BCF QUICKI OGTT

High fructose Low Cranberry powder Medium Cranberry powder High Cranberry powder Homeostatic model of insulin resistance Homeostatic model of b-cell function quantitative insulin-sensitivity check index Oral glucose tolerance test

Introduction Resistance to insulin is a state in which a given concentration of insulin produces subnormal glucose response. Such resistance is characteristic of type II diabetes and of metabolic syndrome. Therefore, some patients with type II diabetes require larger doses of insulin to achieve and control hyperglycemia. In a recent study, cinnamon extract appeared to have moderate effects in reducing fasting plasma glucose concentrations in diabetic patients with poor glycemic control.1 The effectiveness of cinnamon supplementation in patients with type II diabetes has received considerable attention after this study was published in 2003, and a reasonable amount of literature is developing a USDA, Arkansas Children’s Nutrition Center, 15 Children’s Way, Little Rock, AR, 72202, USA. E-mail: [email protected]; Tel: +1-501-743-8949 b Department of Food Science, University of Arkansas, Fayetteville, AR, 72704, USA c USDA, ARS, Little Rock, AR, 72202, USA

116 | Food Funct., 2010, 1, 116–123

relating to the effects of cinnamon on insulin sensitivity and glycemic control in diabetes. The insulinomimetic activity of cinnamon has been shown to be related to its content of A-type procyanidins.2,3 Phenolic phytochemicals are implicated to have potential for prevention and management of many chronic oxidation-linked diseases such as diabetes and cardiovascular disease.4 A-type procyanidins have been found in cranberry extracts and are suggested to be the active component inhibiting the adherence of uropathogenic Escherichia coli to the uroepithelial-cell surfaces.5–7 A-type procyanidins have been found in only a limited number of other foods, such as peanuts and cinnamon.8,9 Other foods are also identified to have A-type linkages,9 but at lower concentrations than in cinnamon or cranberry. Cranberry is a rich source of phenolic phytochemicals that have been shown to have high antioxidant activity10,11 and its procyanidin profile contains A-type linkages in the monomer, dimer, trimer and higher oligomers.9 While the bioavailability of trimers and higher oligomeric procyanidins is questionable, procyanidin monomers and dimers have been shown to be absorbed from the small intestine.12 Cranberry powder (CP) used in the current study is a proprietary product prepared by Decas Botanical Synergies (Carver, MA) and contains increased levels of procyanidins compared to whole cranberry. The objective of this study was to determine if phytochemicals in cranberry were effective in normalizing selected metabolic parameters associated with metabolic syndrome in high fructose fed growing male Sprague-Dawley rats. Along with the current one, a second manuscript comparing the effects of freeze dried whole fruit powders of black berry, blueberry, and cranberry on selected metabolic parameters associated with high fructose feeding has also been submitted. This journal is ª The Royal Society of Chemistry 2010

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Results All animals were fed isocaloric and isonitrogenous diets with all the other major nutrients held constant (Table 1). Control and HF diets had no known source of polyphenols. The CP contained 1.51 mg/g total anthocyanins, 56.2 mg/g procyanidins, 85 mg/g epicatechin, and 137 mg/g catechin. Based upon information provided by the supplier, the CP contained 94.2 mg total phenolics (gallic acid equivalents)/g, 214 mg glucose/g and 70.6 mg fructose/g. The HPLC chromatograms of procyanidins and anthocyanins are given in Fig. 1. The relative distribution of procyanidins is presented in Table 2, which was based upon area under the curve obtained from the HPLC chromatogram; dimers, trimers, tetramers, and polymers were the major procyanidins present in CP contributing approximately 88% of the reported procyanidins, while the monomer was present only in small amounts at slightly more than 3.5% of the total. Among the anthocyanins, peonidin 3-galactoside was present in highest amounts, followed by peonidin 3-arabinoside, cyanidin 3-arabinoside, and cyanidin 3-galactoside, respectively (Table 2). Details related to the intake and urinary excretion pattern of 19 phenolic acids are presented elsewhere.13 Average daily intakes of these different polyphenols by rats in the CP fed groups is presented in Table 3. Since the diets were purified, it was assumed that rats in control or HF group did not receive any of the polyphenols in appreciable amounts that would otherwise confound the results. Animals were randomized to treatments at the beginning of the study such that initial weights were not different (P > 0.95) between treatments (Table 4). Initial body weight was used as a blocking factor in the experimental design, which was incorporated during statistical analysis, to minimize its effect on the eventual results. Rats fed the control starch diet consumed the highest amount of food throughout the experiment (Fig. 2). When analyzed with a repeated measures design, all treatments

Fig. 1 Top panel - Fluorescence trace of HPLC chromatogram of procyanidins extracted from cranberry powder (CP) included in the rat diet. Numbers directly above or by the peaks indicate the degree of polymerization, including the polymer (P). Bottom panel – Diode array traces of HPLC chromatogram of anthocyanins obtained from CP. Peak 1 ¼ Cyanidin 3-galactoside, 2 ¼ cyanidin 3-glucoside, 3 ¼ pelargonidin 3-galactoside, 4 ¼ petunidin 3-galactoside, 5 ¼ cyanidin 3-arabinoside, 6 ¼ peonidin 3-galactoside, 7 ¼ pelargonidin 3-arabinoside, 8 ¼ peonidin 3-glucoside, 9 ¼ malvidin 3-galactoside, 10 ¼ peonidin 3-arabinoside.

Table 1 Composition (g/kg dry weight) of experimental diets.a Ingredients

Control

HF

HF + LC

HF + MC

HF + HC

A. Ingredient composition Casein, 80 mesh L-Lysine Corn starch Maltodextrin Sucrose Fructose Cellulose Soybean oil t-BHQ Vitamin mixture Mineral mixture Choline bitartrate Cranberry powder Total

200 3.0 397.5 132 100 0 50 70 0.014 10 35 2.5 0 1000.014

200 3.0 0 99.5 0 530 50 70 0.014 10 35 2.5 0 1000.014

200 3.0 0 96.2 0 530 50 70 0.014 10 35 2.5 3.3 1000.014

200 3.0 0 92.9 0 530 50 70 0.014 10 35 2.5 6.6 1000.014

200 3.0 0 67.5 0 530 49 70 0.014 10 35 2.5 33.0 1000.014

B. Nutrient composition Crude protein, % Fat, % Crude fiber, % Energy, kcal/kg

20.3 7.0 5.0 3998

20.3 7.0 5.0 4000

20.3 7.0 5.0 3997

20.3 7.0 5.0 3995

20.5 7.1 5.1 3978

a

HF ¼ High fructose, HF + LC ¼ High fructose + low (3.3 g/kg diet) cranberry powder, HF + MC ¼ HF + medium (6.6 g/kg diet) cranberry powder, HF + HC ¼ High fructose + high (33 g/kg diet) cranberry powder.

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Table 2 Relative distribution of procyanidins and anthocyanin profile of cranberry powder Procyanidins, DPa

Distribution, %

Monomer Dimer Trimer Oligomers

1 2 3 4 5 6 7 8 >10

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Polymer

3.65 24.47 30.05 15.06 3.84 2.33 1.48 0.86 18.24

Anthocyaninsb

(mg/g dry weight)

Cyanidin 3-galactoside Cyanidin 3-glucoside Pelargonidin 3-galactoside Petunidin 3-galactoside Cyanidin 3-arabinoside Peonidin 3-galactoside Pelargonidin 3-arabinoside Peonidin 3-glucoside Malvidin 3-galactoside Peonidin 3-arabinoside Total

0.1492 0.0124 0.0062 0.0093 0.1846 0.6359 0.0011 0.1002 0.0090 0.3980 1.5081

a DP ¼ Degree of polymerization. b Two other anthocyanins, peonidin 3,5-digalactoside and delphinidin 3-arabinoside were also detected in small amounts.

Table 4 Least square means  SEM of body weight (g), tissue weight (g and % of body weight) in rats fed a low fructose control diet, a high fructose diet, and high fructose diets with different levels of cranberry powdera

Items

Control HF

HF + LC

HF + MC

HF + HC

SEM P

A. Body weight, g Initial 175.1 Final 518.2 At sac 493.6

175.8 462.2 434.0

174.8 480.9 464.0

175.4 474.1 455.9

175.2 475.5 459.8

2.8 15.8 14.4

0.95 0.07 0.06

B. Tissue weight, g Heart 1.79 Kidney 3.26 Liver 12.2 Abdominal fat 24.6*

1.62 3.23 11.9 16.2x

1.63 3.30 12.8 19.1*x

1.58 3.32 13.5 17.6x

1.62 3.12 12.1 16.6x

0.07 0.15 0.75 2.2

>0.10 >0.10 >0.10 0.003

C. Tissue weight, % BW Heart 0.362 Liver 2.48 Kidney 0.662x Fat 4.94*

0.377 2.75 0.748* 3.75x

0.351 2.73 0.711*x 4.03*x

0.348 2.96 0.726*x 3.80x

0.352 2.62 0.680*x 3.56x

0.012 0.11 0.021 0.24

>0.10 0.06 0.04 0.002

a

HF ¼ High fructose, HF + LC ¼ High fructose + low (3.3 g/kg diet) cranberry powder, HF + MC ¼ HF + medium (6.6 g/kg diet) cranberry powder, HF + HC ¼ High fructose + high (33 g/kg diet) cranberry powder. *x Means without a common superscript are significantly different at the P value indicated in the last column.

Table 3 Calculated average daily consumption of phenolic compounds in rats fed different levels of cranberry powderc

Treatments

PCNa, mg/d

Total phenolics, mg/d

Total ACYb, mg/d

Catechin, mg/d

Epicatechin, mg/d

HF + LC HF + MC HF + HC

3.98 7.75 39.81

6.68 13.0 66.8

106.97 207.92 1069.72

9.72 18.9 97.2

6.03 11.7 60.3

a PCN ¼ procyanidins. b ACY ¼ anthocyanin. c HF ¼ High fructose, HF + LC ¼ High fructose + low (3.3 g/kg diet) cranberry powder, HF + MC ¼ HF + medium (6.6 g/kg diet) cranberry powder, HF + HC ¼ High fructose + high (33 g/kg diet) cranberry powder.

had a significant effect (P < 0.05) on food consumption and cumulative weight gain vs. control (Fig. 2). As a result, animals on control diet were also the heaviest. However, when final and fasted body weight (at the time of sacrifice) and total food intake was analyzed using a randomized complete block design (Table 4), only a tendency (P ¼ 0.06 and 0.07) for the same was observed. Increased body weight, however, did not necessarily result in the higher organ weights (Table 4), except abdominal fat, which was highest in animals fed the control diet. Feeding the high fructose diet decreased abdominal fat accretion, but dietary CP did not significantly (P > 0.1) alter this response. When organ weights were expressed as percent of body weight, high fructose fed animals had the largest kidney weight relative to their body weight compared to animals on control diet, but was not significantly different from other animals on any of the CP diets. A similar trend was observed with liver weights. Cardiac 118 | Food Funct., 2010, 1, 116–123

Fig. 2 Weekly dietary intake (top panel) and cumulative body weight (bottom panel) of rats fed a control low fructose diet, a high fructose diet (HF) and high fructose diets with different levels of cranberry powder (Low, LC; Medium, MC; High, HC).

hypertrophy was not observed in this study as indicated by nonsignificant changes in heart weight as a % of body weight. Both fasting glucose and triglyceride levels were highest in high fructose fed animals compared with the animals on control or CP diets (Table 5). Addition of CP to the diet tended to have positive effects (P ¼ 0.10) in lowering the blood glucose and triglyceride levels. Changes in serum glucose level after the oral glucose load was consistently and significantly (P < 0.05) higher with high This journal is ª The Royal Society of Chemistry 2010

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Table 5 Least square means  SEM of fasting glucose, cholesterol, triglycerides, and insulin levels in rats fed a control low fructose diet, a high fructose diet, and high fructose diets with different levels of cranberry powdera

Items

Control HF

HF + HF + HF + LC MC HC SEM P

Glucose, mg/dL Cholesterol, mg/dL Triglycerides, mg/dL b OGTT Insulin, mU/L c HOMA-IR d HOMA-BCF e QUICKI

182.9 83.5 82.4 10457 49.4 22.4*x 148.2* 0.74

196.1 81.3 82.2 9472 31.0 15.2x 84.1x 0.85

213.1 87.3 125.6 14031 54.2 28.5* 130.2*x 0.69

185.3 74.4 81.7 10466 24.8 11.3x 73.1x 0.95

191.0 79.0 90.1 13788 36.3 17.2*x 102.2*x 0.81

11.8 8.2 12.2 1307 6.7 3.1 17.9 0.09

0.10 0.86 0.10 0.15 0.08 0.04 0.05 0.24

a HF ¼ High fructose, HF + LC ¼ High fructose + low (3.3 g/kg diet) cranberry powder, HF + MC ¼ HF + medium (6.6 g/kg diet) cranberry powder, HF + HC ¼ High fructose + high (33 g/kg diet) cranberry powder. b Oral glucose tolerance test area under curve expressed as mg/dL  minutes1. c Homeostasis model assessment – Insulin resistance (HOMA-IR) ¼ [Fasting glucose (mg/dL)  fasting insulin (mU/L)/405. d Homeostasis model assessment – Beta cell function (HOMA-BCF) ¼ [20  serum insulin (mU/L)]/[plasma glucose (mmol/L)- 3.5]. e Quantitative insulin sensitivity check index (QUICKI) ¼ 1/log HOMA-IR. *x Means without a common superscript are significantly different at the P value indicated in the last column.

Fig. 4 Changes in postprandial serum glucose and insulin levels in rats fed a control low fructose diet, a high fructose diet, and high fructose diets with different levels of cranberry powder (Low, LC; Medium, MC; High, HC).

Fig. 3 Oral glucose tolerance test responses in rats fed a control low fructose diet, a high fructose diet and high fructose diets with different levels of cranberry powder (Low, LC; Medium, MC; High, HC). See Table 3 for AUC.

fructose and high fructose + high CP diets compared to animals in other diets after 60 min of oral glucose load (Fig. 3). The total area under curve after the OGTT, was highest in animals fed the high fructose diet. Addition of CP in the diet, primarily at low and medium levels, lowered the OGTT area under the curve down to control levels (Table 5 and Fig. 3). Serum insulin was low in animals fed diets that contained CP and was highest in animals fed high fructose diet (P ¼ 0.08). No effect of the diet was observed on fasting cholesterol. When the three models of homeostatic assessment scores were calculated in animals fasted overnight, both HOMA-IR and HOMA-BCF were altered by diet, but the QUICKI model of insulin sensitivity was not statistically significant (P > 0.1) among treatments. While insulin resistance was lowest in animals fed medium level of CP in the diet, it was highest in animals fed high This journal is ª The Royal Society of Chemistry 2010

Fig. 5 Changes in postprandial serum cholesterol and triglyceride levels in rats fed a control low fructose diet, a high fructose diet, and high fructose diets with different levels of cranberry powder (Low, LC; Medium, MC; High, HC).

fructose diet. Insulin resistance was reduced by the addition of CP in the diet at all levels (P < 0.04). Similarly, b-cell function was lowest in animals fed medium level of CP in the diet (P 0.05) and highest in animals fed control or high fructose diets. Numerically, QUICKI was lowest in rats fed a high fructose diet and highest in animals fed medium level of CP in the diet. Weekly postprandial blood glucose, cholesterol, and triglycerides were also tested in animals from wk 2 through 7, whereas insulin was tested in animals from wk 5–7. While there were no Food Funct., 2010, 1, 116–123 | 119

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significant effects of diet (P > 0.1) on blood glucose concentration (Fig. 4A), there was a significant treatment  week interaction, a situation in which blood glucose level varied from week to week depending on the dietary treatment. Serum insulin (Fig. 4B), on the other hand, was consistently and significantly higher (P < 0.05) in animals fed the control diet compared to animals on high fructose diet; animals on the three CP diets were in between. The concentration of serum triglycerides was consistently higher (P < 0.05) in animals fed high fructose diet (Fig. 5A) even though they had the least amount of abdominal fat as mentioned previously. Inclusion of CP in the diet of rats reduced plasma triglyceride concentrations. Plasma cholesterol concentration increased as the experiment progressed with the increasing age of the animals (Fig. 5B). Although there was a significant treatment  week interaction (P < 0.05) on plasma cholesterol concentrations, diet had no significant effect (P > 0.1) on its weekly values.

Discussion Polyphenols are one of the major natural dietary compounds believed to be responsible for promotion of health and protection against many chronic diseases, including metabolic syndrome. Originally known as Syndrome X,14 metabolic syndrome refers to the clustering of cardiometabolic risk factors, including abdominal obesity, hyperglycemia, dyslipidemia, elevated blood pressure, increased body mass index/waist circumference, and a decreased high density lipoprotein cholesterol, that are thought to be linked to insulin resistance.15,16 Although it was believed initially to be associated with increased risk of cardiovascular disease, metabolic syndrome has a stronger association with type 2 diabetes than that previously demonstrated for coronary heart disease.17 In the current study, we investigated whether different levels of CP, a concentrated cranberry product, as a source of polyphenols in the diet were effective in modulating some of the metabolic parameters associated with metabolic syndrome. Dietary CP had positive effects in preventing some of the metabolic profiles associated with metabolic syndrome. Response on two of the parameters, fasting blood glucose and triglycerides were borderline and showed a tendency (P ¼ 0.1) to be affected by CP in the diet. Similarly, OGTT responses were slightly improved and similar to animals on control diet when low or medium, but not when the high level of CP was added to the high fructose diet. However, the dietary effect was more pronounced in HOMA scores with insulin resistance being the least in animals when CP was included in the diet. A lower insulin resistance combined with a tendency for reduced blood glucose suggested that animals fed CP were able to utilize the available glucose even at a reduced concentration of circulating insulin, which was reflected in the reduced HOMA-BCF scores when CP was included in the diet. Whereas a higher concentration of fasting insulin was needed to clear out the available glucose in control and HF animals. Lower concentrations of post-prandial glucose and insulin were in line with some previous reports when high fructose diets were fed.18–20 This may be because fructose produces a smaller postprandial rise in plasma glucose than other common carbohydrates21,22 and reduces circulating insulin.20 In the purified diet used in this study, substituting fructose for the starch in the diet removed 75% of the dietary sources of glucose. 120 | Food Funct., 2010, 1, 116–123

Maltodextrin, which is a short chain of molecularly linked dextrose (glucose) molecules (fewer than 20) manufactured by regulating the hydrolysis of starch, was included in the diet to aid in the pelleting process, which limited the possibility of removing all glucose sources from the high fructose diets. Fructose metabolism is not regulated by insulin and it has a lower glycemic index than foods rich in starch.23,24 However, fructose increases post-prandial serum triglyceride levels19,20 as was observed in this study. Only 0.1% of fructose was converted to fatty acids at 240 min and lower insulin excursion after fructose resulted in less activation of adipose tissue lipoprotein lipase and impaired triglyceride clearance.19 It may be one of the reasons why there was lower amount of abdominal fat accretion in HF group than those in control group. Moreover, it has been shown previously that increased abdominal fat may not always be associated with metabolic syndrome.25 While sustained elevation of plasma triglycerides with high fructose feeding suggested its possible contribution to atherogenesis and cardiovascular disease, its reduction by the concentrated cranberry product may be helpful in minimizing the effect. There is a concern that dietary fructose may stimulate energy intake and promote weight gain and obesity.20 As a result, a definitive link through clinical trials may need to be established between the two.21 We actually observed a tendency for lower body weight in HF fed animals compared to animals fed a control diet with equivalent amounts of starch. This was accompanied by a reduced food intake in animals fed HF diet compared to the control diet. We have observed similar results in other experiments with slightly higher or lower level of fructose in the diet (unpublished data). Given the fact that diets were purified and had all the major nutrients at iso-level in the current study (Table 1), results suggested that high fructose feeding may possibly augment some of the factors associated with metabolic syndrome without stimulating energy intake and/or promoting weight gain and obesity. This may partially explain the higher incidence of diabetes and metabolic syndrome among South Asians at lower body mass index and waist circumference.25 The response to high fructose feeding in growing rats in producing elevated fasting plasma triglycerides, enhanced abdominal fat accretion, insulin resistance, and cardiac hypertrophy were not as marked as predicted from the literature using this model.26,27 High fructose feeding in rats is commonly used to induce a strong response on metabolic parameters associated with the metabolic syndrome, albeit more commonly with adult than with growing rats. However, the authors’ experience with feeding high fructose diet in developing a strong response of metabolic syndrome in growing rats is rather mixed. We have conducted several experiments in this regard, all with purified diet and nutrient concentrations held constant (unpublished data). One of the observations that we have consistently made is a slightly reduced food intake, lower body weight at termination of the experiment, and the complete lack of abdominal obesity even after having elevated levels of postprandial plasma triglycerides. A number of previous studies have used a natural ‘‘chow’’ based diet and added fructose to it without properly balancing all the nutrients in the process. As a result, animals may become marginally deficient or deficient in some other nutrients resulting in confounding of the effects on metabolic parameters and symptomatic responses, including those associated with This journal is ª The Royal Society of Chemistry 2010

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metabolic syndrome. To avoid this, a purified diet was used in the current study with all the other major nutrients held constant across treatment. It is possible that when other nutrients are equally balanced and starch is the only item that is completely (or close to) replaced with fructose, a few of the typical signs of metabolic syndrome may not develop at all, while others, such as postprandial plasma triglycerides or insulin resistance may appear. It was observed that balancing all the nutrients across the treatments in purified diets does not generate appreciable oxidative stress in high fructose fed growing animals over those on control diets (unpublished results) that would otherwise be highly conducive for clear signs of metabolic syndrome. A previous study with Sprague-Dawley rats also showed no difference in fasting plasma glucose, cholesterol, triglycerides, and insulin concentration as well as glucose tolerance curves and weight gain between animals fed control (AIN based diet with 53% corn starch), a high fructose (53%) or high fat (25% soybean oil) diet for 3 months.28 The Wistar may be a better rat model in that it is more sensitive to fructose in the diet. Although it is not possible to determine which of the polyphenols present in CP were responsible for any or all of the observed effects, previous data have suggested that procyanidins may be more responsible than anthocyanins in modulating factors associated with metabolic syndrome.29 Similarly, the effect of total or individual phenolic acids on metabolic syndrome is not clear. Whether and how much of the increased excretion of 4-hydroxycinnamic acid and 3-hydroxyphenylacetic acid and decreased excretion of hippuric acid and 4-hydroxyphenylacetic acid13 have contributed to the observed results is not known. This is the first study detailing the effects of different levels of a commercially available concentrated cranberry product on certain parameters associated with metabolic syndrome in growing animals. The doses provided were calculated based upon previous work using procyanidins on insulin sensitivity and metabolic syndrome.29–31 The high dose of CP was based upon previous data,30 which we predicted would be on the high end of a practical dose. Depending upon the method of extrapolation from the rat to the human, the high dose of CP in the rat (33 g/kg diet) would equate to approximately 54 g of CP for a 70 kg human based upon metabolic weight extrapolation between the rat and human which would provide 3 g of procyanidins. Extrapolating based upon caloric intake, a 70 kg human would consume 21 g of the CP or 1.2 g procyanidins per day. The two lower doses were estimated to be practical levels for the rat and when translated to a human would also be reasonable providing 200–600 mg procyanidins per day. Overall, inclusion of CP had positive effects in some of the parameters investigated. The high (33 g/kg diet) dose did not provide any additional benefit and may not been as strong based upon the HOMA-IR estimate. Consumption of high level of procyanidins may affect other physiological processes, such as binding or interacting with protein or lipids, which procyanidins (also called tannins) from grape, tea, or other sources are associated with.32–34 It should be noted, however, that medium level included in the current study is, by no means, the best or optimal dose since there is a wide gap between medium and high dose used in the current study. Further experiments are needed to confirm the findings as well as determine the optimum level of inclusion in the diet. This journal is ª The Royal Society of Chemistry 2010

In summary, the cranberry powder was effective in improving some, but not all, of the metabolic parameters associated with metabolic syndrome investigated in the current study. Of the three levels of CP included in the diet, the medium level at 6.6 g/ kg diet was the most effective in improving factors associated with metabolic syndrome in the high fructose fed growing rats used in the current study. This highlights the importance of performing dose response studies and that more is not always better. Given the mixed polyphenol content of the cranberry product used, it may be unreasonable to pinpoint the effects observed to one or more specific polyphenols or their constituent compounds.

Experimental Chemicals All chemicals used in the study were HPLC grade or higher and were obtained either from Fisher Scientific (Hampton, NH, USA), SigmaAldrich (St. Louis, MO, USA), or SynerMed (Monterey Park, CA, USA). Animals and diet The protocol was approved by Animal Care and Use Committee of the University of Arkansas for Medical Sciences, Little Rock, AR. Metabolic parameters associated with metabolic syndrome in growing rats were investigated by feeding an American Institute of Nutrition (AIN) based purified diet containing 53% by weight of fructose (0% kcal from starch). Isocaloric and isonitrogenous purified diets were formulated according to Table 1. Purified diets were prepared by Research Diets Inc. (New Brunswick, NJ, USA). Male Sprague-Dawley rats (Charles River Laboratories Intl. Inc., Wilmington, MA; 44 d old, 175.0  8.4 g) were used in a randomized complete block design with initial body weight used as the blocking factor. Animals within block were assigned at random to one of the five treatments, 1) Control (starch based diet); 2) Fructose-rich diet (AIN based diet containing 53% by weight of fructose (HF), 0 kcal from starch); 3) Fructose-rich diet with low, 3.3 g CP per kg diet (HF + LC); 4) Fructose-rich diet with medium, 6.6 g CP per kg diet (HF + MC); or 5) Fructose-rich diet with high, 33 g CP per kg diet (HF + HC). Animals were housed two per cage and provided ad libitum access to food and water. Food consumption and body weight changes were monitored weekly. Cages were changed weekly. Analysis of polyphenols Total procyanidins in CP were determined by the aldehyde condensation of 4-dimethylaminocinnamaldehyde (DMAC) method as described previously.35 The CP was also analyzed for monomeric, oligomeric and polymeric procyanidins using a high performance liquid chromatograph (HPLC) on a diol column, details of which have been described previously.36 This was done to determine the distribution of individual procyanidins based on the proportionate area under the curve in the HPLC chromatogram. Procedures for determining total phenolics have been described recently,13 whereas anthocyanins were determined using diode array detector in an Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with Food Funct., 2010, 1, 116–123 | 121

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a binary pump. Details about the HPLC conditions, column description, mobile phase, gradient, etc. have been described previously.37

IR using a formula, QUICKI ¼ 1/log HOMA-IR. Insulin values were expressed as SI units (1 mIU/mL ¼ 6.945 pmol/L). Statistical analysis

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Sample collection and analysis Weekly post-prandial blood samples were collected from the tail vein using a Microvette capillary tube (Sarstedt AG & Co., N€ umbrecht Germany), while fasting trunk blood was collected at the time of sacrifice. Animals were sacrificed on d 57 and 58 of the experiment after euthanizing in a CO2 chamber and decapitating the head. Organ (kidney, heart, liver, total fat) weights were recorded after the sacrifice. Animals were randomized before blood collection and sacrificed such that treatments were evenly distributed across the time frame or when sacrificed in successive days. Plasma was separated from the blood by centrifugation at 3480  g for 15 min at 4  C. Plasma samples were analyzed using commercially available kits for glucose, cholesterol, and triglycerides (Synermed Intl. Inc., Westfield, IN, USA) in a 96-well plate format using a dual pump FluoStar Galaxy (Durham, NC, USA) microplate reader. Plasma insulin was determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Linco Research Inc., St. Charles, MO, USA) in a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). All samples were stored at 80  C prior to analyses. Samples were randomized during each analysis such that treatments were evenly distributed across the plates to minimize the variation caused by running multiple plates and generating multiple standard curves. However, it was not possible to include it in the statistical model given the smaller sample size.

Statistical analyses of fasting glucose, cholesterol, triglycerides, insulin, HOMA-IR, HOMA-BCF, QUICKI, final body weight, organ weights, and organ weights as percent of body weight were carried out in SigmaPlot (Systat Software Inc., San Jose, CA, USA). Treatment, block, and their interactions were included in the model as the fixed factors in a randomized complete block design. Statistical analysis of weekly food intake, weight gain, post-prandial glucose, cholesterol, triglycerides, and insulin were carried out in SAS (SAS, Cary, NC, USA) using PROC MIXED. Treatment, week, and their interactions were included in the model with week as the repeated measures on rats. Changes in serum glucose concentration after the oral glucose load was analyzed in PROC MIXED of SAS using treatment, time and their interaction as the fixed factors with time as the repeated measure on the rats. Compound symmetry was used as covariance structure.

Acknowledgements Financial support was provided in part by Decas Botanicals Inc., the Arkansas Biosciences Institute and the U.S. Department of Agriculture, Agriculture Research Service. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.

Oral glucose tolerance test (OGTT)

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At the end of week 6, an OGTT was performed in six rats/ treatment over a period of three consecutive days. Rats were food deprived for 12 h before the administration of an oral glucose load of 2 g/kg body weight from a 200 g/L glucose solution. Blood samples (150 mL) were collected via tail vein at time 0 (before administration), 30, 60, 90, and 120 min and analyzed for plasma glucose. Animals were randomized before OGTT such that same number of animals per treatment was included in each day.

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Insulin sensitivity indices The Homeostasis model assessment (HOMA) is a computergenerated model consisting of a number of non-linear empirical equations solved numerically to predict glucose and insulin concentrations in the fasting state for any combination of pancreatic b-cell function (HOMA-BCF) and insulin sensitivity (HOMA-IR). The relative-value of the homeostasis model was calculated as an index of insulin resistance (HOMA-IR)17 using the formula: HOMA-IR ¼ Fasting glucose (mmol/L)  fasting insulin (mIU/mL)/22.5. Beta cell function was assessed by the beta cell homeostasis assessment (HOMA-BCF) score: HOMABCF ¼ [20  serum insulin (mU/L)]/[plasma glucose (mmol/L)3.5]. Insulin values were expressed in International Units (1 IU ¼ 0.04167 mg).29,38 Similarly, quantitative insulin-sensitivity check index (QUICKI) was calculated from log-transformed HOMA122 | Food Funct., 2010, 1, 116–123

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17 E. S. Ford, C. Li and N. Sattar, Diabetes Care, 2008, 31, 1898–1904. 18 E. J. Schaefer, J. A. Gleason and M. L. Dansinger, J. Nutr., 2009, 139, 1257S–1262S. 19 M. F. Chong, B. A. Fielding and K. N Frayn, Amer, J. Clin. Nutr., 2007, 85, 1511–1520. 20 K. L. Teff, S. S. Elliott, M. Tsch€ op, T. J. Kieffer, D. Rader, M. Heiman, R. R. Townsend, N. L. Keim, D. D’Alessio and P. J. Havel, J. Clin. Endocrinol. Metab., 2004, 89, 2963–2972. 21 J. P. Bantle, J. Nutr., 2009, 139, 1263S–1268S. 22 J. P. Bantle, D. C. Laine, G. W. Castle, J. W. Thomas, B. J. Hoogwerf and F. C. Goetz, N. Engl. J. Med., 1983, 309, 7–12. 23 R. R. Henry, P. A. Crapo and A. W. Thorburn, Annu. Rev. Nutr., 1991, 11, 21–39. 24 D. J. Jenkins, T. M. Wolever, R. H. Taylor, H. Barker, H. Fielden, J. M. Baldwin, A. C. Bowling, H. C. Newman, A. L. Jenkins and D. V. Goff, Am. J. Clin. Nutr., 1981, 34, 362–366. 25 E. A. Enas, V. Mohan, M. Deepa, S. Farooq, S. Pazhoor and H. Chennikara, J. Cardiometab. Syndrome., 2007, 2, 267–275. 26 S. Delbosc, E. Paizanis, R. Magous, C. Araiz, T. Dimo, J. P. Cristol, G. Cros and J. Azay, Atherosclerosis, 2005, 179, 43–49. 27 M. J. Pagliassotti, P. A. Prach, T. A. Koppenhafer and D. A. Pan, Am. J. Physiol., 1996, 271, R1319–R1326.

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28 A. H. Stark, B. Timar and Z. Madar, Eur. J. Nutr., 2000, 39, 229–234. 29 N. A. Al-Awwadi, C. Araiz, A. Bornet, S. Delbosc, J. P. Cristol, N. Linck, J. Azay, P. L. Teissedre and G. Cros, J. Agric. Food Chem., 2005, 53, 151–157. 30 M. Pinent, M. Blay, M. C. Blade, M. J. Salvad o, L. Arola and A. Ardevol, Endocrinology, 2004, 145, 4985–4990. 31 S. H. Kim, S. H. Hyun and S. Y. Choung, J. Ethnopharmacol., 2006, 104, 119–123. 32 R. A. Frazier, E. R. Deaville, R. J. Green, E. Stringano, I. Willoughby, J. Plant and I. Mueller-Harvey, J. Pharm. Biomed. Anal., 2010, 51, 490–495. 33 P. Sarni-Manchado, V. Cheynier and M. Moutounet, J. Agric. Food Chem., 1999, 47, 42–47. 34 Y. S. Tarahovsky, Plant Signal. Behav., 2008, 3, 609–611. 35 R. L. Prior, E. Fan, H. Ji, A. Howell, C. Nio, M. J. Payne and J. Reed, J. Sci. Food Agric., 2010, 90, 1473–1478. 36 R. C. Khanal, L. R. Howard, C. Brownmiller and R. L. Prior, J. Food Sci., 2009, 74, H52–H58. 37 X. Wu, L. Gu, R. L. Prior and S. McKay, J. Agric. Food Chem., 2004, 52, 7846–7856. 38 D. R. Matthews, J. P. Hosker, A. S. Rudenski and B. A. Naylor, Diabetologia, 1985, 28, 412–419.

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PAPER

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Dealcoholized red wine reverse vascular remodeling in an experimental model of metabolic syndrome: role of NAD(P)H oxidase and eNOS activity Marcela Alejandra Vazquez-Prieto,ab Nicol as Federico Renna,ab Carina Lembo,ab Emiliano Raul Diezab and Roberto Miguel Miatello*ab

Downloaded on 21 October 2010 Published on 22 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00077A

Received 15th July 2010, Accepted 13th August 2010 DOI: 10.1039/c0fo00077a The present study examines the effect of chronic administration of dealcoholized red wine Malbec (DRW) on vascular remodeling and NAD(P)H oxidase and endothelial nitric oxide synthase activity (eNOS) in an experimental model of metabolic syndrome induced by fructose administration. Thirtyday old male Wistar rats were fed a normal rat diet (control) or the same diet plus 10% fructose in drinking water (FFR). During the last 4 weeks of a 10-week period of the corresponding diet, a subgroup of control and FFR (n ¼ 8 each) received DRW in their drinking water. Systolic blood pressure (SBP), a homeostasis model assessment of insulin resistance (HOMA-IR), aortic NAD(P)H oxidase and eNOS activity in the heart and vascular tissue were evaluated. Vascular remodeling was evaluated in the left carotid artery (CA) and interlobar, arcuate and interlobular renal arteries (RA) through lumen to media (L/M) ratio determination. At the end of the study FFR increased the SBP (p < 0.001), HOMA-IR (p < 0.001), and aortic NAD(P)H oxidase activity (p < 0,05) but reduced cardiac and vascular eNOS activity (p < 0.01), L/M ratio in CA (p < 0.001) and RA (p < 0.01) compared with the C group. DRW reduced SBP (p < 0.05), aortic NAD(P)H oxidase (p < 0.05), and recovered eNOS activity (p < 0.001) and L/M in CA (p < 0.001) and RA (p < 0.001) compared with FFR. This study provides new data about the beneficial effect of DRW on oxidative stress and vascular remodeling in the experimental model of metabolic syndrome. Data suggest the participation of mechanisms involving oxidative stress in FFR alterations and the usefulness of natural antioxidant substances present in red wine in the reversion of these changes.

Introduction Metabolic syndrome (MS), characterized by insulin resistance, dyslipidemia and hypertension, is an important risk factor for cardiovascular diseases.1 Rats chronically receiving fructose (FFR) provide a useful experimental model for the study of the interaction between factors clustered in MS.2 Endothelial dysfunction is associated with this experimental model.3 We previously reported a decrease of the endothelial isoform of nitric oxide synthase activity (eNOS) at cardiovascular level and an increase in vascular smooth muscle cell proliferation in primary culture, showing also evidence involving the renin angiotensin system (RAS) in the pathophysiology of these injuries.4,5 Furthermore, it has been demonstrated that angiotensin, acting on AT1 receptors, could induce oxidative stress, through activation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, the most important source of intracellular reactive oxygen species (ROS) in vascular cells.6 ROS play a physiological role in the vessel wall and participate as second messengers in endothelium dependent function, in smooth muscle cell and endothelial cell growth and survival, and in remodeling of the vessel wall.7,8 The major vascular ROS is superoxide anion ($O2), which inactivates nitric oxide (NOc), a Institute of Experimental Medicine and Biology of Cuyo (IMBECU), National Council of Research (CONICET), Mendoza, Argentina b Department of Pathology, School of Medicine, National University of Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina. E-mail: rmiatell@ fcm.uncu.edu.ar; Fax: +54 261 4135242; Tel: +54 261 41305000 ext 2697

124 | Food Funct., 2010, 1, 124–129

the main vascular relaxing factor.9 The relationship between oxidative stress and vascular remodeling had been previously reported in human and animal experimental studies,7 including fructose-fed rats.10,11 The study of the beneficial effect on human health of consumption of natural antioxidants, present in vegetables, fruits and beverages such as red wine, has recently increased in significance. Epidemiological studies suggest that moderate red wine consumption could decrease the risk of cardiovascular mortality,12 mainly attributable to the polyphenol content but also attributable to the alcohol content.13 Polyphenols could favor endothelium-dependent vasodilatation in aorta and human coronary arteries, inhibit vascular smooth muscle cell proliferation.14–16 We previously reported that resveratrol was able to increase the eNOS activity and reduce the systolic blood pressure (SBP) in this model of MS.17 In order to establish the beneficial effects of non-alcoholic constituents of red wine on vascular remodeling, the aim of this study was to determine the effect of chronic administration of dealcoholized red wine (DRW) in fructose-fed rats upon the possible participation of changes in ROS and NOc generation in the development of structural and functional alterations at cardiovascular and metabolic levels. Specifically, ROS production by the NAD(P)H oxidase system, and NOc generation by eNOS were examined in order to establish whether these systems are involved as pathogenic mechanisms in metabolic and structural cardiovascular changes associated with this experimental model. This journal is ª The Royal Society of Chemistry 2010

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Results No differences were observed in food and drink intake between groups throughout the experimental period. Table 1 shows body weight, metabolic variables and SBP. The body weight did not vary among groups. At the end of the study fructose-fed rats developed insulin resistance, increased significantly the triglyceride levels and reduced the HDL cholesterol compared with control groups. Chronic administration of DRW significantly reduced the insulin resistance state and increased the HDL cholesterol. Systolic blood pressure was gradually increasing throughout the experimental period in FFR and reached a significant difference compared to controls. DRW administration to FFR during the last four weeks was able to reduce SBP in a slight but significantly way, without effect on control rats. The NAD(P)H oxidase activity in aortic tissue was higher in FFR, compared with the C group. Administration of DRW significantly reduced the NAD(P)H oxidase activity (Fig. 1). Fig. 2 shows eNOS activity levels, measured in a mesenteric vascular bed (panel A) and heart tissue from left ventricle homogenates (panel B). The eNOS activity was significantly diminished in the FFR group, compared to control rats. DRW chronic administration to FFR was able to return NOc production to control levels in both mesenteric vascular and heart tissue, while DRW to control rats increased significantly the eNOS activity in mesenteric vascular tissue. Arterial wall modifications were detected by structural analysis performed by histological methods, which allow us to observe changes in arteries from different localizations and calibers. Fig. 3 shows lumen : media (L/M) ratios and representative microphotographs observed in arteries from different localizations: left carotid (A), renal interlobar (B), renal arcuate (C), and renal interlobular (D) arteries in each group. The carotid lumen to media ratio in the FFR group was significantly

Table 1 Body weight, SBP, and metabolic parameters from C, C + DRW, FFR, and F + DRW rats. Groups

Body weight/g Plasma glycemia/ mmol L1 Plasma insulin (pmol/L) HOMA-IR Plasma triglycerides/ mmol L1 Plasma HDL/ mmol L1 Plasma total cholesterol/ mmol L1 SBP (mmHg)1 Baseline Week 6 Week 10

C

C + DRW

FFR

FFR + DRW

340  7 4.0  0.2b

324  9 4.0  0.2b

349  7 6.3  0.4a

325  7 5.6  0.2a

72  6c

75  8c

152  7a

118  6b

1.8  0.4c 1.9  0.5c 6.1  0.8a 4.2  0.6b 0.81  0.02b 0.72  0.07b 1.23  0.08a 1.08  0.08a 0.92  0.04a 0.95  0.04a 0.80  0.02b 0.92  0.01a 1.48  0.04 1.40  0.06 1.46  0.08 1.41  0.08

100  1 106  1b 115  1c

102  1 107  1b 116  1c

103  1 130  1a 136  1a

100  1 129  1a 125  1b

Values are expressed as mean  SEM, n ¼ 8; means without a common letter differ, P < 0.05.

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Fig. 1 NAD(P)H oxidase aorta activity from C, C + DRW, FFR, and F + DRW rats. Values are mean  SEM (n ¼ 8). Bars without a common letter differ, P < 0.05.

Fig. 2 eNOS activity in mesenteric vascular bed homogenates (A) and heart tissue homogenates from the left ventricle (B), from C, C + DRW, FFR, and F + DRW rats. Values are mean  SEM (n ¼ 4). Bars without a common letter differ, P < 0.05.

reduced, compared to control rats. Chronic administration of DRW to FFR increased the L/M ratio to control levels. A similar structural pattern was found in interlobar renal arteries (caliber between 120 to 180 mm), in smaller caliber arteries (50 a 120 mm) such as arcuate renal arteries and in very small arteries (10 a 50 mm) such as interlobular renal arteries.

Discussion In the present study we demonstrate that DRW was able to reverse vascular remodeling in fructose-fed rats, an experimental model of MS, associated with an increased eNOS activity and a reduced aortic NAD(P)H oxidase activity. These results suggest that non-alcoholic constituents of red wine reverse the structural and functional changes by mechanisms related to oxidative stress enhanced in this model. We have previously demonstrated the development of endothelial dysfunction in this experimental model, supported by a diminished NOc generation capability and changes in vascular smooth muscle cell proliferative behavior in primary culture.4 These changes could be attributed to a significant increase in ROS production, evaluated through an increased activity of NAD(P)H oxidase, the most quantitatively important source of Food Funct., 2010, 1, 124–129 | 125

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Fig. 3 Lumen to media ratio observed in arteries from different localizations: left carotid (A), renal interlobar (B), renal arcuate (C), renal interlobular (D) arteries described by representative microphotographs and analyzed in the bar graph from C, C + DRW, FFR, and F + DRW rats. Values are mean  SEM (n ¼ 8). Bars without a common letter differ, P < 0.05. Arrows indicate the location of the arteries.

superoxide at vascular level.10,11,18 ROS produced by vascular wall cells can directly inactivate other biologically active free radicals, thereby disturbing vascular homeostasis. One of the main targets of ROS, particularly superoxide anion is NOc, decreasing its bioavailability and favoring the formation of peroxynitrite, a potent vasoconstrictor.19 In this study, we found a decreased eNOS activity in both heart and mesenteric vascular tissue in FFR. DRW administered to FFR was able to restore the activity of this enzyme, suggesting that red wine polyphenols could be responsible for these beneficial effects. The effects of some polyphenols in these variables had been achieved. Quercetin increased the activity of eNOS and downregulates the activity and expression of NAD(P)H oxidase in an experimental model of hypertension.20 Others studies have shown that metabolites of flavonoids inhibit the activity of NAD(P)H oxidase.21,22 In this study, DRW administered to FFR induced a slight but significant decrease in systolic blood pressure, reduced the index of insulin resistance and increased HDL cholesterol. Furthermore, chronic administration of DRW was able to revert the vascular remodeling in FFR in both distribution and resistance arteries. The structural changes in FFR could be associated to vascular smooth muscle cell proliferative behavior previously observed in vitro in this model.4 Red wine administration could protect the NOc inactivation process by ROS and also increased the NOc generating system activity. The final result could be the inhibition of vascular remodeling associated with this experimental model. The beneficial effects of moderate red wine consumption have been demonstrated in several studies. Some mechanisms involved in those effects have pointed to the action of antioxidant properties of different polyphenols present in red wine.23 These substances could induce endothelium-dependent vasodilatation in human aorta and coronary arteries, inhibit vascular smooth muscle cell proliferation and protect ischemic 126 | Food Funct., 2010, 1, 124–129

myocardium.15,16,24,25 In vivo, flavonoids such as quercetin prevent endothelial dysfunction and reduce blood pressure, oxidative stress and end-organ damage in hypertensive animals.26 Quercetin and theaflavin significantly attenuated the atherosclerotic lesion size in aorta arteries in ApoE deficient mice by alleviating inflammation, improving NOc bioavailability, and inducing heme oxygenase-1.27 The prevention of angiotensin II-induced hypertension and endothelial dysfunction by red wine polyphenol extract administration, with a normalization of vascular superoxide anion production and NAD(P)H oxidase expression, has also been described.28 It is important to note that the administration of DRW to the control rats had no positive effects on almost of all variables studied, suggesting that under normal conditions DRW adds no further benefit. Our results are in agreement with epidemiological and experimental evidence demonstrating the beneficial effects of moderated red wine consumption on cardiovascular pathology and contribute to support the hypothesis that the non-alcoholic fraction of wine, represented mainly by phenolic compounds with antioxidant properties, may be the primary factor responsible for this protective effect. Further studies are needed to clarify the molecular mechanism of DRW on vascular alterations.

Experimental Animals and experimental design All procedures were performed according to institutional guidelines for animal experimentation and were approved by the Technical and Science Secretary from the School of Medicine of National University of Cuyo, Mendoza, Argentina. Thirty-dayold male Wistar rats, weighting 90–130 g were housed during the experimental period of 10 weeks in a room under conditions of controlled temperature (21  2  C), humidity and a 12 h This journal is ª The Royal Society of Chemistry 2010

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light/dark cycle. At the beginning of the study, 32 rats were randomly distributed into two groups: one control group (C) (n ¼ 16) and one experimental group (FFR) (n ¼ 16). After six weeks of treatment, the half of C and experimental groups were assigned to receive 10 mL/Kg daily of DRW for four more weeks. The names of each group were assigned as follows: Control (C); C + DRW; FFR: 10% (w/v) fructose solution administration in the drinking water during all the experimental protocol; and F + DRW. All groups were fed the same standard rat diet (GepsaFeeds, Buenos Aires, Argentina) and tap water ad libitum. Administration of 10% fructose (Saporiti Labs., Buenos Aires, Argentina) solution in drinking water was used to achieve the pathological model. The red wine (Malbec grape variety) was provided by the School of Agricultural Sciences, National University of Cuyo. The phenolic characterization of RW Malbec was evaluated by high performance liquid chromatography as previously described.29 One litre of red wine contained 2.9 g of total phenols expressed as gallic acid. The main phenolic content was (expressed as mg L1): non-flavonoids: 18.2 gallic acid; 2 caffeic acid; 4.2 cis-caftaric acid, trans-resveratrol: 1.1, flavonoids: 24.1 catechin; 14.2 epicatechin; procyanidin (11.3 B1; 3.1 B3), flavonols: 4.9 quercetin, and anthocyanins (344 malvidin-3-glucoside; 16.2 peonidin-3-glucoside; 60.3 delphinidin-3-glucoside). Red wine was dealcoholized by rotary evaporation at low pressure and temperature, and then the volume of alcohol evaporated was reconstituted with water, in order to conserve phenolic composition. DRW (10 mL/Kg) was administered in drinking water. The weight of each animal was measured weekly and the energy intake was recorded twice per week during the experimental period in all groups. At the end of the experimental period, and after an overnight fast, the rats were weighed, anesthetized with ketamine (50 mg kg1) and acepromazine (1 mg kg1). Blood was collected from the abdominal aorta into heparinized tubes. Plasma obtained after centrifugation was frozen at 70  C until assayed. Arteries and organs were excised aseptically for the measurement of various parameters described below.

mesenteric resistance arteries and left ventricular cardiac tissue by the conversion of L-[3H]arginine to L-[3H]citrulline, as previously described.4 Mesenteric vessels were homogenized on ice for four 15 s intervals with a Politron homogenizer and then sonicated in a buffer (pH 7.4, 37  C) containing 50 mmol L1 Tris, 20 mmol l1 HEPES, 250 mmol L1 sucrose, 1 mmol L1 dithiothreitol, 10 mg mL1 leupeptin, 10 mg mL1 soybean trypsin inhibitor, 5 mg mL1 aprotinin and 0.1 mmol L1 phenyl methyl sulfonyl fluoride. Heart tissue from left ventricle myocardium was also homogenized on ice for four 15 s intervals with a polytron homogenizer and then sonicated in the same buffer. After centrifugation of the homogenates (100 g, 5 min, 4  C) and determination of the protein content (Bradford method), aliquots of 50 mL were added to 100 mL of a cocktail reaction buffer containing 50 mmol L1 Tris, 20 mmol L1 HEPES, 1 mmol L1 dithiothreitol, 1 mmol L1 NADPH, 0.1 mmol L1 tetrahydrobiopterin, 50 mmol L1 FAD, 50 mmol L1 FMN, and 10 mCi/ml L-[2,3-3H]-arginine (New England Nuclear, Boston MA), and incubated for 30 min at 37  C in a shaking bath in the presence of 10 mg ml1 calmodulin and 3 mmol l1 CaCl2 or with 3 mmol L1 EGTA in the absence of Ca2+/calmodulin. The reaction was stopped by adding 1 mL cold distilled water and the mixture applied to an anion-exchange chromatography column containing Dowex AG 50W-X8 (200–400 Mesh) resin previously saturated with 50 mL of 100 mmol L1 L-citrullin and 2 mL of 50 mmol L1 Tris, 20 mmol L1 HEPES buffer (pH 7.4) and eluted with 2 mL of distilled water. Specifically eluted L-[3H]citrulline concentration was determined by liquid scintillation counting. The calcium-dependent NOS activity was calculated as the difference between activity in the presence and absence of Ca2+/calmodulin. Values were corrected to the amount of protein present in the homogenates and the incubation time (dpm/mg protein/min). Each rat mesenteric vascular bed and heart tissue was processed and eNOS activity measured independently.

Plasma glucose, triglycerides, HDL-cholesterol and total cholesterol concentrations were determined using commercial kits by enzymatic colorimetric methods (Wiener Lab, Rosario, Argentina). Insulin was measured by RIA (Coat-A-Count, Siemens, CA, USA), and insulin resistance was assessed using the homeostasis model assessment (HOMA-IR) described by Mathew et al.30 HOMA-IR was calculated using the following formula: HOMA-IR (mmol L1  mU/mL) ¼ fasting glucose (mmol L1)  fasting insulin (mU/mL)/22.5.

Measurement of vascular NAD(P)H oxidase activity. The lucigenin-derived chemiluminescence assay was used to determine NAD(P)H oxidase activity in the aorta as previously described.18 A 2 cm length segment of thoracic aorta was cut, cleaned, washed, transferred to a tube with 2 mL of Jude’s Krebs buffer (JKB) containing (in mmol L1) 2 HEPES, 11.9 NaCl, 0.46 KCl, 0.1 MgSO4$7H2O, 0.015 Na2HPO4, 0.04 KH2PO4, 0.5 NaHCO3, 1.2 CaCl2, 5.5 glucose; pH 7.40; and equilibrated at 37  C during 30 min. Then the aortic segment was transferred to a tube containing 1 mL JKB and 5 mmol L1 lucigenin and left in darkness at room temperature for 10 min. This concentration of lucigenin does not appear to be involved in redox cycling and specifically detects superoxide anion. To assess NAD(P)H oxidase activity, 500 mmol L1 bNAD(P)H was added and chemiluminescence was immediately measured in a liquid scintillation counter (LKB Wallac Model 1219 Rack-Beta Scintillation Counter, Finland) set in the out-of coincidence mode. Time-adjusted and normalized to tissue weight scintillation counts were used for calculations. Measurements were repeated in the absence and presence of diphenylene iodinium (DPI) (106 mol L1), which inhibits flavincontaining enzymes, including NAD(P)H oxidase.

Measurements of eNOS activity. Ca2+/calmodulin-dependent nitric oxide synthase activity was measured in homogenates from

Tissue preservation. Tissue samples for histopathology were processed as previously reported.10 Samples from all rats were

Systolic blood pressure (SBP). Systolic blood pressure was monitored indirectly in conscious, pre-warmed (32  C) slightly restrained rats by the tail-cuff method and recorded on a Grass Model 7 polygraph (Grass Instruments Co., Quincy, MA, USA). Biochemical determinations

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used in these observations. The kidneys were in vivo perfused with PBS (pH 7.40, 4  C) through the renal artery over 5 min. For histological studies, left kidneys were then perfused with 4% paraformaldehyde solution for 10 min, then additionally fixed by immersion in the same solution for 48 h, introduced to a 30% sucrose solution and kept at 70  C. Five mm thick tissue slices were transversely cut through the entire kidney on a cryostat (Microm HM 505E, Germany) at 26  C and processed for histological studies. Common left carotid arteries were fixed and processed as described above for kidneys. Histopathology and morphometry. Lumen to media ratio in kidney arteries transversal slices from common left carotid artery and left kidney were placed on microscope slides and stained with Masson’s trichrome solution and examined under a light microscope (Nikon Optiphot-2, Kanagawa, Japan). Images were digitalized with a digital camera (GP-KR222 color CCD, Panasonic, Osaka, Japan) and processed with an analysis system Scion Image 4.01 (Scion, Bethesda, MD, USA). To evaluate the renal arterial wall thickening, images from three different artery types were studied in each kidney: interlobar, arcuate and interlobular arteries. The lumen-to-wall media ratio (the internal diameter to the medial thickness) was then calculated. Forty slices from each kidney were processed and 5 to 10 arteries of each type in each slice were analyzed, in order to obtain an average value for each rat. The average values were then used for final analysis. Common left carotid arteries were sectioned transversely. L/M was then calculated in 10 slices from each artery, in order to obtain an average value for each rat and then used for final analysis. Reagents Unless otherwise noted, reagents were purchased from Sigma Chemical Co, MO USA. All other chemicals were of molecular biology or reagent grade. Statistical and data analysis Results were expressed as mean and their deviation errors. The statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls post-test using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California USA. Differences were considered significant at p < 0.05. In the figures and tables, data shown without a common letter differ at a p < 0.05 significance level.

Conclusions The non-alcoholic constituents of red wine increased the eNOS activity, reduced the activity of the enzyme NAD(P)H oxidase and reversed vascular remodeling. The antioxidant properties of polyphenols could be responsible for the beneficial effects of DRW.

Acknowledgements We thank Susana Gonzalez and Cristina Lama for their technical assistance. This work was supported by grants from Program 06/ P01 SECTyP Universidad Nacional de Cuyo, and PIP-5192 CONICET. 128 | Food Funct., 2010, 1, 124–129

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