Food & Function, Vol 01, No 02, November 2010

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Food & Function Downloaded on 10 November 2010 Published on 02 November 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90006K

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

Volume 1  |  Number 2  |  November 2010  |  Pages 133–224

ISSN 2042-6496

COVER ARTICLE H. Ashida et al. Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes

2042-6496(2010)1:2;1-C

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Food & Function Downloaded on 10 November 2010 Published on 02 November 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90007A

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

Volume 1  |  Number 2  |  November 2010  |  Pages 133–224

ISSN 2042-6496

COVER ARTICLE E. Jeffery et al. Glucoraphanin hydrolysis by microbiota in the rat cecum results in sulforaphane absorption

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

IN THIS ISSUE

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ISSN 2042-6496 CODEN FFOUAI 1(2) 133–224 (2010) Cover See M. Ueda, T. Furuyashiki, K. Yamada, Y. Aoki, I. Sakane, I. Fukuda, K.-i. Yoshida and H. Ashida, pp. 168–174. Image reproduced by permission of Hitoshi Ashida from Food Funct., 2010, 1, 168.

Inside cover See R.-H. Lai, M. J. Miller and E. Jeffery, pp. 162–167. Image by Jason Lindsey. Reproduced by permission of Elizabeth Jeffery from Food Funct., 2010, 1, 162.

REVIEWS 141 Interfacial design of protein-stabilized emulsions for optimal delivery of nutrients Amir Malaki Nik, Amanda J. Wright and Milena Corredig* During transit through the gastrointestinal tract, the composition of the oil–water interface is in continuous evolution.

149 Mechanisms underlying the cholesterol-lowering properties of soluble dietary fibre polysaccharides Purnima Gunness and Michael John Gidley* Interactions of bile salt micelles with soluble dietary fibres are reviewed as a potential mechanism for reduction of serum cholesterol.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 135–140 | 135

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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

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

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

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REVIEWS 156

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Effects of eggs on plasma lipoproteins in healthy populations Maria Luz Fernandez* The lack of association between egg consumption and coronary heart disease risk may be due to two major reasons: (1) Eggs are good sources of anti-oxidants that might protect against lipoprotein oxidation and (2) Clinical studies show that LDL and HDL increase in individuals who experience an elevation in plasma cholesterol following egg consumption. Thus the LDL/ HDL ratio, a key biomarker of coronary heart disease risk, is maintained.

PAPERS 161 Glucoraphanin hydrolysis by microbiota in the rat cecum results in sulforaphane absorption Ren-Hau Lai, Michael J. Miller and Elizabeth Jeffery* Glucoraphanin from broccoli is hydrolyzed by gut microbiota to the anticarcinogen sulforaphane in situ.

167 Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes Manabu Ueda, Takashi Furuyashiki, Kayo Yamada, Yukiko Aoki, Iwao Sakane, Itsuko Fukuda, Ken-ichi Yoshida and Hitoshi Ashida* Gallate-type catechins in green tea decrease insulin-induced glucose uptake and GLUT4 translocation in adipose tissue.

174 Modulation of doxorubicin-induced genotoxicity by squalene in Balb/c mice Bhilwade Hari Narayan, Naoto Tatewaki, Vijayasree Vayalanellore Giridharan, Hiroshi Nishida and Tetsuya Konishi* The present study aims to evaluate the protective effect of squalene against the genotoxicity of the chemotherapeutic agent doxorubicin (Dox) using two genotoxicity assays, the micronucleus assay and the comet assay.

This journal is ª The Royal Society of Chemistry 2010

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Titles from RSC Publishing

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

Garlic and Other Alliums The Lore and the Science

Foreword by 1990 Nobel Laureate E.J. Corey

Garlic and Other Alliums

The Lore and the Science

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

Garlic and Other Alliums

The Lore and the Science

Eric Block

Written by Eric Block, 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.

Foreword by E. J. Corey

Block

Hardback | 450 pages | ISBN 9780854041909 | 2009 | £29.95

Paperback | 450 pages | ISBN 9781849731805 | 2009 | £24.99

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

Food The Chemistry of its Components

FOOD The Chemistry of its Components Tom Coultate

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

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

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

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

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

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

Hardback | 250 pages | ISBN 9780854049707 | £24.95

www.rsc.org/books Registered Charity Number 207890

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PAPERS 180

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Oxidative cascade reactions yielding polyhydroxytheaflavins and theacitrins in the formation of black tea thearubigins: Evidence by tandem LC-MS Nikolai Kuhnert,* Michael N. Clifford and Anja M€ uller We have characterised black tea thearubigins using standard and advanced analytical techniques and confirmed the oxidative cascade hypothesis for thearubigins formation showing evidence for theaflavin and theacitrin polyhydroxylation and quinone formation.

200 Antioxidant, anti-inflammatory and anti-browning activities of hot water extracts of oriental herbal teas Li-Chen Wu,* Amily Fang-Ju Jou, Si-Han Chen, Chia-Ying Tien, Chih-Fu Cheng, Nien-Chu Fan and Ja-an Annie Ho* Inulae Flos might be a potential antioxidant source, a noteworthy inhibitor of pro-inflammatory cytokine production, and an anti-browning agent.

209 Antioxidant capacity in cultivated and wild Solanum species: The effect of wound stress Christina B. Wegener* and Gisela Jansen Wounding of tubers enhances the antioxidant potential in cultivated and wild potato species.

This journal is ª The Royal Society of Chemistry 2010

Food Funct., 2010, 1, 135–140 | 139

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

Garlic and Other Alliums

The Lore and the Science

Downloaded on 10 November 2010 Published on 02 November 2010 on http://pubs.rsc.org | doi:10.1039/C0FO90008G

The Lore and the Science

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

Garlic and Other Alliums

The Lore and the Science

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

Eric Block

Foreword by E. J. Corey Block

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

Garlic and Other Alliums_dust jacket.indd 4

03/07/2009 10:54:31

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

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

www.rsc.org/books Registered Charity Number 207890

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REVIEW

www.rsc.org/foodfunction | Food & Function

Interfacial design of protein-stabilized emulsions for optimal delivery of nutrients Amir Malaki Nik,a Amanda J. Wrightb and Milena Corredig*a

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Received 28th July 2010, Accepted 24th August 2010 DOI: 10.1039/c0fo00099j Proteins are often used as ingredients in food emulsions, as their amphiphilic structures provide electrostatic and steric stabilization. Significant attention has recently been directed at understanding how the composition and structure of oil–water interfaces change during digestion and how these can be manipulated to enhance the delivery of nutrients contained within the oil droplets. These efforts have necessitated the development of more sophisticated in vitro digestion models of greater physiological relevance and increased efforts in research to identify the role of the various digestive parameters on interfacial dynamics. The changes occurring at the oil–water interface will affect the adsorption of gastro-intestinal lipases and, ultimately, affect lipid digestion. The composition of a protein-stabilized oil droplet changes continuously during digestion, because of proteolysis and the formation of peptides with different affinities for the interface. In addition, natural bio-surfactants such as phospholipids and bile salts, other surface- active molecules present in foods, and the products of lipolysis (i.e. mono and diglycerides, lysophospholipids), all compete for access to the interface, and contribute to the dynamic changes occurring on the surface of the oil droplets. A better understanding of how to tailor the composition of oil droplet surfaces in food emulsions will aid in optimizing lipid digestion and, as a result, delivery of lipophilic nutrients. This review focuses on the physico-chemical changes occurring in protein-stabilized oil-in-water emulsions during gastric and small intestine digestion, and on how interfacial engineering could lead to differences in fatty acid release and the potential bioavailability of lipophilic molecules.

Introduction Proteins are a major component in foods, not only because of their nutritional properties but also their various processing functionalities (gelling, emulsifying, foaming, etc.). Knowledge of their digestive metabolism has, primarily, been obtained from in vivo studies based on nitrogen or amino acid balances, or from in vitro experiments using relatively simple proteolytic cocktails. More recently, however, there has been increased interest in understanding the breakdown of food proteins during digestion, as clear evidence has emerged that the food matrix plays an important role in the bioavailability of nutrients. This has necessitated the development and use of increasingly sophisticated in vitro digestion models, resulting in a better understanding of the molecular details occurring during transit of food in the gastro-intestinal (GI) tract1–3 and will enable better microstructural engineering of foods for optimal delivery of bioactives and nutrients. Proteins encode in their sequence a number of bioactive peptides with recognized effects on human health. These include, for example, peptides with iron-binding capacity, immunestimulation, antimicrobial, angiotensin I-converting enzyme inhibition, or opioid activities.4 The ability of these peptides to act depends on their release during digestion, which, in turn, a Department of Food Science, University of Guelph, Guelph, Ontario, Canada. E-mail: [email protected]; Fax: (+519) 824-6631; Tel: (+519) 824-4120 ext 56101 b Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada, N1G 2W1

This journal is ª The Royal Society of Chemistry 2010

depends on the changes occurring to the protein structure, as this influences the protein’s susceptibility to specific enzymes. Proteins in foods form structural hierarchies, and these structures affect the proteolytic patterns during digestion. Differences in protein digestion depending on processing history and interaction with other molecules present in foods have relevance in terms of the potential allergenicity of protein-based products.1 For example, it was recently demonstrated, using in vitro gastrointestinal models, that heat5 or high pressure6,7 treatments can improve the digestibility of b-lactoglobulin (b-LG). Furthermore, the kinetics of b-LG hydrolysis are also significantly altered when the protein is adsorbed at an oil–water interface.8,9 Thus, understanding the biophysical mechanisms related to protein digestion is important to modulate peptide bioactivity. For example, it would be desirable to design protein structures that are predictably digested to form specific peptides during digestion. However, this requires more research into the complex interplay between foods and the digestive environment. There are also implications of protein digestion in terms of the release and absorption of molecules contained within proteinbased food matrices. For instance, differences in the proteins present at an emulsion interface lead to changes in the physicochemical characteristics of the emulsion droplets, including size, charge, and aggregation state. All of these characteristics contribute to the stability of an emulsion, and change the accessibility of the interface to important hydrolytic enzymes such as the proteases and lipases. A number of studies have been published on the role played by the surface area and interfacial composition on the ability and rate of lipase to digest lipids in oil Food Funct., 2010, 1, 141–148 | 141

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droplets.10–13 Indeed, it has been demonstrated that the postprandial plasma triglycerol response is affected by the size of emulsion droplets.14 In addition, gastric emptying rates are affected by the physical state of an emulsion (i.e. stable, aggregated, creamed).15 Since lipolysis is a prerequisite for the release and absorption of molecules contained within the oil droplets of an emulsion, research to address the relationships between protein digestion, interfacial changes, lipolysis, and bioavailability is warranted. Most advances in elucidating digestion mechanisms have been made using model systems, including protein-stabilized oil-inwater emulsions. Such research is laying the foundation for the design of more complex matrices and mixed food systems. It is also identifying where opportunities may exist to engineer structures that allow for slowing down digestion or tailoring the delivery of bioactive molecules to particular sections of the human GI tract. However, studies with relatively simple systems have also clearly illustrated the complex and very intricate dynamics occurring during digestion. In this regard, the specifics of protein digestion, particularly under physiological conditions, require more careful consideration and study. Given the complexity of the interactions at and competition for the interface, it is critical that attention be directed specifically at understanding protein digestion, in light of the presence of biosurfactants and other ingredients present in foods. This review aims to provide an overview of recent knowledge on the digestion of protein-stabilized emulsions with an emphasis on how proteinstabilized interfaces can be designed to modulate lipolysis and the release of molecules encapsulated within oil droplets.

Physico-chemical properties of protein-stabilized emulsions During homogenization of oil in an aqueous solution, the size of the oil droplets is dramatically reduced, improving their physical stability. Water-soluble surfactants quickly adsorb onto the interface and decrease the surface tension, contributing to the stability of the droplets to flocculation or coalescence. Due to their amphiphilic nature, proteins are often used in food emulsions. Because of their interfacial adsorption, they unfold, to different extents and expose their hydrophilic portions to the aqueous phase, while their hydrophobic moieties favor the oil phase. During homogenization, flexible proteins spread to cover the maximum possible interfacial area. However, if the concentration is not sufficient to cover the entire surface, the emulsion droplets will coalesce. The difference in density between the oil and the aqueous phase leads to creaming of the oil droplets, and the rate of creaming will depend on the square of the size of the droplets as well as the viscosity of the continuous phase. This emphasizes the importance of an efficient homogenization process and proper formulation. The conformation proteins adopt at an interface depends on their state of minimum conformational energy. Either as individual molecules or as aggregates, proteins create a thick and charged layer at the interface, causing steric and charge repulsion when the droplets come into close contact with one another. Emulsion stability is affected by the strength and type of interactions occurring between droplets and these depend on the structure, concentration, and composition of the adsorbed layer. 142 | Food Funct., 2010, 1, 141–148

Milk proteins are amongst the most employed emulsifiers in foods. Their changes at the interface have been well studied16,17 and highlight the various interfacial behaviours of proteins. Monomeric and globular proteins (such as b-LG, whey protein isolate, or lysozyme) cover interfaces with surface loads of about 2–3 mg m2 and partially unfold and rearrange upon adsorption.17–19 On the other hand, more flexible proteins, such as monomeric caseins, adsorb readily at the interface forming loops (with part of the molecules adsorbed and other parts protruding in the water phase) and trains (where a portion is at the interface while the other protrudes in the aqueous phase) with hydrophilic moieties protruding into the water phase, forming thicker hydrodynamic layers of a few nanometres in size (8–10 nm). These proteins spread at the interface very efficiently, with very low surface coverage (less than 1 to 3 mg m2). In systems containing cysteine residues, disulfide cross-linking can also occur at the interface, creating more elastic and brittle surfaces, as for example, in the case of whey proteins.20 When present in abundance, globular proteins can form multilayers,18 while supramolecular assemblies of proteins such as casein micelles, calcium caseinate or soy proteins instead adsorb as aggregates. In this case, a much higher concentration is needed to form stable emulsions, the proteins form thicker interfaces, and the surface coverage exceeds 10–20 mg m2.21,22 Therefore, protein-stabilized interfaces differ in terms of protein loads, conformations, and supramolecular assemblies. Oil-in-water emulsions are thermodynamically unstable and can destabilize in several ways, including through physical mechanisms related to the droplets’ colloidal properties and by chemical means (such as oxidation or hydrolysis). The type of destabilization depends on the properties of the interfacial layer,16 and the influence of interfacial composition on oil-inwater emulsion stability has been well described.16,17 Upon collision, oil droplets may flocculate. This means they associate with one another, but still retain their identity. The behaviour of the oil droplets now resembles that of one oil droplet of bigger size, corresponding to the size of the floc. Flocculation can be reversible or irreversible.16 When the surfactant (for example a protein aggregate) is shared between two droplets, bridging flocculation occurs. This is often observed when polymers with a tendency to interact with the protein adsorbed at the interface are present in the continuous phase. For example, charged emulsion droplets will interact with oppositely charged polysaccharides, forming bridged flocs. When sufficient interacting biopolymer is present in the aqueous phase, the emulsion droplets may become completely covered by a secondary charged layer that re-stabilizes the system. On the other hand, when a non-adsorbing polymer is present in the continuous phase and it does not interact with the oil droplet surface, an osmotic pressure gradient develops in the proximity of the oil droplets. This drives the oil droplets closer to one another, ultimately causing the formation of flocs. This type of flocculation, called depletion flocculation, is reversible and the flocs will disrupt upon dilution. As the stability of an emulsion is determined greatly by the properties of the adsorbed layer, environmental conditions affecting the protein structure will ultimately affect the emulsion’s physico-chemical properties. Changes in pH and the presence of ions, for example, can have a substantial impact on This journal is ª The Royal Society of Chemistry 2010

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the charge and hydration of the interfacial layer, ultimately affecting the stability of the droplets upon collision. The presence of other polymers or surface-active molecules in the continuous phase is also important in determining the type of interface and stability of oil droplets. For example, polymers that only interact at certain pH and ionic strengths may not adsorb or desorb from the interface, ultimately causing destabilization of an emulsion, as described above. Also, hydrophilic polysaccharides can form a thick secondary layer at the interface, driven by electrostatic interactions and further stabilization of the interface may occur, decreasing the accessibility of proteases and lipase.23,24 In addition, the presence of other surface-active molecules can cause competitive displacement or co-adsorption at the interface, ultimately modifying the properties of the oil droplets. Products of lipolysis, such as the mono- and diglycerides as well as biosurfactants such as bile salts and phospholipids will cause desorption of the original protein at the interface. It has also been shown that phospholipids can interact with proteins in solution as well as on the surface of the oil droplets, thus changing the quality of the interface and affecting the accessibility of proteases to the substrate.25,26 Despite their inherent instability, a solid understanding of the molecular interactions occurring enables formulation of emulsions for optimal stability during processing and storage. While many studies have considered the effect of protein composition on the stability aspects of emulsions, relatively little is understood in terms of the changes in interfacial quality of the protein layer and the associated physicochemical changes occurring during gastrointestinal digestion.

Physico-chemical changes of protein-stabilized emulsions during gastrointestinal transit Structure modifications of emulsion-based foods initiate in the oral cavity. Although the residence time is relatively short (i.e. 5–20 s in the case of beverage emulsions), changes in droplet structure in the mouth can be very significant, particularly as they relate to flavor release and sensory perception. The physical changes which occur during oral processing are non covalent in nature and depend on the droplets’ surface charge.27,28 They are the result of mixing with the salivary components, changes in environmental conditions (such as temperature, pH, and ionic strength), physical contact with the oral surfaces, and the frictional forces present.29 The presence of mucins (i.e. negatively charged glycoproteins) and an ionic environment were recently cited as the most important factors affecting emulsion stability in the mouth. Mucins induce reversible droplet flocculation through a depletion mechanism in the case of weakly negatively charged or neutral oil droplets (b-LG or Tween 20-stabilized emulsions at pH 6.7). In contrast, with positively charged oil droplets (lysozyme or lactoferrin-stabilized emulsions), the mucins form electrostatic complexes, causing bridging flocculation.28–30 The presence of ions at certain concentration promotes emulsion destabilization as well. Indeed parotid saliva causes emulsion aggregation, depending on the initial charge of the emulsion.31 Aggregation in the mouth results in changes in viscosity and different sensory qualities. For example, bridging flocculation between salivary proteins and lysozyme-stabilized oil droplets leads to the perception that an emulsion is astringent.32 This journal is ª The Royal Society of Chemistry 2010

Once an emulsion reaches the stomach, the droplets are diluted and exposed to acidic conditions. Shear forces also result in mixing with other food ingredients and the proteins undergo substantial hydrolysis by pepsin. After the stomach, the emulsion droplets are processed in the duodenum where lipolytic and proteolytic enzymes are present, together with inorganic salts and bio-surfactants. As previously mentioned, the bile salts and phospholipids both affect protein digestibility as they have high interfacial activity and compete for the interface. Phospholipids can also form complexes, both in solution and at the interface. Of great importance are the interfacially active molecules produced during digestion, as they also compete for adsorption at the interface, or form supramolecular structures (i.e. micelles, vesicles, mixed complexes) in solution. The net result of these physico-chemical changes is that the state of an emulsion interface constantly changes during GI transit. Since the composition of the oil–water interface affects the ability of the lipolytic enzymes (i.e. pancreatic lipase and colipase) to adsorb, there are consequences for lipid digestion and the release of bioactives contained within the oil droplets. Fig. 1 represents schematically the physical changes occurring during GI transit, as well as the exchanges occurring at the oil–water interface. Proteolysis occurs at neutral pH in the small intestine through the action of pancreatic proteases, including trypsin, chymotrypsin and membrane peptidases. Proteins have very different susceptibility to proteases, and the profile of peptides liberated during digestion is affected by a number of factors. For example, a-lactalbumin (a-LA) and b-casein (b-Cas) in solution are very susceptible to pepsinolysis.8,9,33 In contrast, native b-LG in solution shows resistance to pepsinolysis at low pH 5,34; However, during in vitro duodenal incubation, in the presence of trypsin and chymotrypsin, b-LG is completely hydrolyzed within 30 min.35 Changes in protein hydrolysis patterns are of particular significance in terms of protein allergenicity and the role played by processing and molecular interactions. As previously mentioned, other components such as salts and phospholipids can strongly affect the structure and accessibility of a protein to proteolytic enzymes. For example, the supramolecular structures formed in the GI tract between proteins and phospholipids could modify protein digestion patterns. Of utmost importance are the changes in protein conformation induced by adsorption on the oil water interface, as they affect protein digestion. Emulsification was recently reported to increase the susceptibility of b-LG and b-Cas to in vitro pepsinolysis.8 Digestibility of b-Cas at the interface was doubled compared to that of the protein in solution, with the formation of one major peptide of about 6 kDa. A 60% increase in pepsinolysis of b-LG was also reported when this protein was adsorbed at the oil–water interface.8 Moreover, there were consequences of the newly formed peptides in terms of interfacial properties. Using drop tensiometry, increases in surface tension were observed during pepsinolysis of b-Cas adsorbed at the interface.8 Fig. 2 illustrates the differences between the gastric digestion of proteins in solution compared with the same proteins adsorbed at an interface. In particular, Fig. 2 shows the changes in the polypeptide patterns of solubilized versus interfacial a-LA (Fig. 2A) and b-LG (Fig. 2B) during exposure to simulated gastric conditions. When a 10% oil-in-water emulsion stabilized with a-LA was subjected to pepsinolysis, the adsorbed protein Food Funct., 2010, 1, 141–148 | 143

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Fig. 1 Schematic diagram of the physico-chemical changes occurring in emulsion droplets during transit through the gastrointestinal tract. Interfacial composition changes occur in the stomach and the small intestine. The proteolytic activity of pepsin results in compositional changes at the interface, and different types of droplet destabilization will occur, depending on the type of interface formed. Phospholipids can form aggregates with the proteins in solution or can bind or displace proteins at the interface. In the small intestine and after a drastic change in pH further hydrolysis of the interfacial layer by pancreatic proteases also alters the interfacial composition. Furthermore, competition for the interface occurs between pancreatic lipase, colipase, bile salts and phospholipids, as well as the products of hydrolysis. The contribution of phospholipase A2, which hydrolyzes phospholipids to lysophospholipids and free fatty acids, is also important. Overall, accessibility of pancreatic lipase to the oil–water interface lead to triglyceride hydrolysis into fatty acids and monoglycerides. The product of lipolysis will be removed from the interface by incorporation into bile salt micelles.

showed a higher level of resistance to pepsinolysis compared with the same protein in solution. The conformational changes occurring in a-LA at the interface resulted in a very limited hydrolysis, with only a few sites accessible to pepsin.9 In contrast, when a 10% oil-in-water emulsion was stabilized with b-LG, the protein was fully hydrolyzed under in vitro gastric conditions, with no residual protein observed after 1 h of digestion (Fig. 2). Protein concentration can also affect the polypeptide patterns resulting from in vitro digestion of emulsions.9 When 10% oil-inwater emulsions prepared with 0.5 and 1.5% whey protein isolate (WPI) are exposed to in vitro gastric digestion, exchanges occur between the interfacially adsorbed and the unadsorbed proteins. These exchanges, in turn, affect the susceptibility of the proteins to hydrolysis. Interestingly, regardless of protein concentration (i.e. if the protein is all at the interface or present partly unadsorbed in solution), subsequent incubation of the whey proteinstabilized emulsions in simulated duodenal fluids lead to rapid hydrolysis of both b-LG and a-LA by trypsin and chymotrypsin.9 The same authors also showed that heat treatment of the emulsions before digestion also lead to an increase in the extent of gastric hydrolysis, especially for the unadsorbed protein fraction. Again, these observations stress the importance of 144 | Food Funct., 2010, 1, 141–148

emulsion formulation and processing history to ensure delivery of nutrients during digestion. The composition of the interface determines the physical properties of the emulsion droplets and their state of aggregation. A number of studies have demonstrated that droplet stability during in vitro digestion is strongly dependent on emulsifier type.8,10,36 For example, the stability of WPI-stabilized emulsions during in vitro digestion is significantly lower than that of emulsions stabilized with Tween 20, a low molecular weight emulsifier. Substantial flocculation and coalescence has been shown in b-Cas-stabilized emulsions subjected to simulated gastric condition containing pepsin.8 Of interest, subsequent incubation of this emulsion with duodenal fluids resulted in reemulsification of the oil droplets and disruption of the flocs. Proteolysis and the newly formed peptides weaken the steric and electrostatic forces involved in emulsion droplet stabilization. In another example, WPI-stabilized emulsions (0.5% protein, 10% oil) showed extensive aggregation during incubation with simulated gastric fluids containing pepsin. Although the oil droplets were stable against the low pH and ionic strengths associated with the gastric environment, pepsinolysis of the interfacial layer caused destabilization.37 The instability was This journal is ª The Royal Society of Chemistry 2010

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Fig. 2 MALDI-TOF mass spectra as reported by Malaki Nik et al. (2010) for a-lactalbumin (A) and b-lactoglobulin (B) solutions and the cream phases of 10% oil-in-water emulsions after pepsin digestion, expressed as m/z (where z ¼ 1). Untreated protein solutions are also shown. Surface adsorption significantly altered the hydrolysis pattern of the proteins. While native a-lactalbumin was fully hydrolyzed by pepsin, adsorbed a-lactalbumin still showed a peak at about 13 kDa after digestion (A). In the case of b-lactoglobulin, while the protein was resistant to hydrolysis in solution, it was fully hydrolyzed when adsorbed at the interface, with a major peak at about 6 kDa (B).

dependent on protein concentration: during exposure to gastric conditions, 1.5% WPI emulsions were relatively more stable than those prepared with 0.5% WPI. The presence of intact proteins, along with a higher concentration of surface-active peptides liberated during pepsinolysis, may have played a role in stabilizing the system. Of critical importance to the interfacial dynamics during digestion are the bile salts (BS). BS are strong anionic detergents, consisting of rigid steroid backbones and short aliphatic side chains.38 They are present as mixed micelles in the aqueous phase, but rapidly adsorb at the oil–water interface, decreasing the interfacial tension. They can either partly or fully displace other surface-active molecules from an interface.10,27,39,40 Phospholipids (PL), mainly phosphatidylcholine (PC) are another major constituent of biliary fluids, also secreted by the gastric and duodenal mucosa. PL, alone or in combination with BS, play a major role in modulating gastro-duodenal proteolysis.3 For example, it was recently reported that the presence of PC protected b-LG against trypsin and chymotrypsin hydrolysis. However, this effect seemed to depend on the ratio of PL to This journal is ª The Royal Society of Chemistry 2010

protein, with greater protection afforded when a higher proportion of PC was present.26,35 BS promote the digestion of dietary lipids by aiding in reemulsification and contributing to pancreatic lipase adsorption at the interface. However, they also promote absorption of the products of lipid digestion by solubilizing them in mixed micelles.38 Moreover, the presence of BS can modulate proteolysis by trypsin and chymotrypsin, as was recently demonstrated for b-LG, bovine serum albumin, and myoglobin in solution.41 This behaviour may be a result of BS increasing the accessibility of the proteolytic enzymes by altering the protein structure. BS can also penetrate and disrupt b-LG films, thereby displacing the proteins from both air–water and oil–water interfaces42 and facilitating pancreatic protease activity.9 The above discussion highlights the complex and dynamic interplay between foods and the digestive environment and underscores the challenges in adequately mimicking digestion using in vitro models. Most reports on the in vitro changes in protein-covered oil droplet stability during digestion are specific to only the mouth, stomach, or small intestine. However, surfaceactive molecules, BS and PL, as well as the products of lipid digestion, play a major role in altering the composition at the interface, by competing for adsorption and by forming mixed micelles in the aqueous phase. In addition, colipase is recognized for its role in overcoming the inhibition of pancreatic lipase by BS, and phospholipase A2 for its role in cleaving the sn-2 position of PL. This co-factor (colipase) and enzyme also contribute to interfacial changes. Despite the roles played by all these constituents during digestion, studies of protein-stabilized emulsions typically do not include the full range of the digestion co-factors or utilize concentrations, which are expected to approximate physiological relevance. Of course, the challenges in doing so are substantial and this is a criticism of in vitro digestion modelling in general. The influence of protein digestion on protein-stabilized oil droplet stability, in the context of exposure to gastric and small intestinal conditions, is clearly illustrated in Fig. 3, where 10% oil in water emulsions prepared with 1.5% soy protein isolate was first incubated with simulated gastric fluids containing pepsin, and then subjected to simulated duodenal fluids containing pancreatic proteolytic enzymes as well as pancreatic lipase, and bio-surfactants (PL and BS) as described elsewhere.37 The average droplet diameter and surface charge (z-potential) of the initial emulsion were 180 nm and 40 mV, respectively. Incubation with gastric fluids caused a significant increase in particle size and a shift in z-potential to a positive value of about +10 mV. Immediately after the addition of intestinal fluids, the emulsion droplet size decreased because of the disruption of bridged flocs. Further proteolysis of the interfacial film by the pancreatic proteases (trypsin and chymotrypsin), interfacial adsorption of BS, and the release of highly surface-active lipolysis products (mainly monoglycerides), all contributed to a further decrease in the mean droplet diameter. The presence of BS along with the change in pH caused a rapid decrease in the charge, reaching a plateau at about 70 mV.

Impact of interfacial composition on lipid hydrolysis The hydrolysis of dietary lipids begins in the stomach with partial hydrolysis of triglycerides (TG) by the pre-duodenal (gastric and Food Funct., 2010, 1, 141–148 | 145

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Fig. 3 Changes in z-potential (C, left hand axis) and average droplet diameter (D3,2) (B, right hand axis), for a 10% oil in water emulsion stabilized by 1.5% soy protein isolate. The emulsion was first incubated with simulated gastric fluids containing pepsin, and then subjected to simulated duodenal and bile fluids containing lipase, phospholipase A2, bile salts and phospholipids as described elsewhere.37 Data are the average of three independent experiments and bars represent standard deviation.

lingual) lipases. In healthy adults and under physiological conditions, only 10–30% of lipid digestion occurs in the gastric phase, but this facilitates the subsequent hydrolysis by pancreatic lipases.43 Lipid hydrolysis continues in the upper small intestine through the action of pancreatic triglyceride lipase (PTL), the main lipid digestion enzyme. PTL catalyzes TG breakdown at the oil–water interface and its activity is critically determined by the quality of the interface. In solution, PTL’s active site is covered by an amino acid lid that makes the protein hydrophilic. However, in the presence of an interface, the hydrophobic environment induces opening of the lid, exposing a small (i.e.  5 nm) hydrophobic region that anchors the enzyme on the surface of the emulsion droplet.13 While BS can inhibit adsorption of PTL at the interface, this effect is overcome through the formation of a PTL-colipase complex (1 : 1 ratio), which ensures displacement of BS and optimal binding of PTL at the interface. Interfacial composition is a key factor in determining the rate of lipid hydrolysis,11,12 and compounds that bind to or interact with the interface have the potential to alter PTL activity. Therefore, the impact of original emulsifier on PTL activity depends on its stability against enzymatic degradation, its ability to control the stability of the emulsion droplets, and its ability to remain at the interface when in competition with other interfacially active molecules present during digestion. The rate of lypolysis is higher for protein-stabilized emulsions compared with emulsions stabilized with phospholipids or low molecular weight surfactants (i.e. Tween 20).10,24,44 However, the type of protein present affects PTL activity. For example, non-covalent inhibition of PTL activity (i.e. when the inhibitor does not bind to the enzyme, but affects its activity) has been observed by certain proteins, including bovine serum albumin (BSA), b-LG, and soy proteins, because PTL access to the surface is hindered.40 Importantly, the inhibition observed is overcome in the presence 146 | Food Funct., 2010, 1, 141–148

of BS, as they penetrate and disrupt the interfacial protein films.42 Inhibition of PTL activity has also been reported in the presence of dietary fiber (pectin and/or chitosan).24,45 For example, in vitro PTL activity decreases when lipid droplets are coated with the cationic biopolymer chitosan.24 However, an in vivo study showed that chitosan did not affect the rate of lipolysis during digestion in rats.46 This may be in part explained by in vitro studies with gum Arabic, where the activity of human PTL depends heavily on the presence and concentration of BS.47,48 Research on the effects of mixed interfaces on lipid digestion is in its infancy; however, it is clear that using physiologically relevant in vitro systems it is possible to derive information on the mechanisms related to competition at interfaces. It is important to note that when polysaccharides are present with proteins at the interface, interactions are electrostatic in nature, and the dynamics of adsorption and digestion will change depending on the pH and the ionic conditions. Since PL are present in the GI tract and contained within many food products, their consideration and inclusion in in vitro models is warranted. The presence of PL, either in the form of vesicles or mixed micelles, also can inhibit PTL interfacial adsorption through steric hindrance by the phospholipids’ head groups, but this can be overcome by the action of pancreatic phospholipase A2 (PLA2).49 The products of phospholipase hydrolysis (free fatty acids and lyso-phospholipids) tend to dissolve in the mixed micelles and leave the interface. Incubation of soybean oil (10%) emulsion stabilized with 0.5% WPI in simulated duodenal fluids containing PLA2 led to a significantly higher percentage of lipid hydrolysis than in the absence of this enzyme.37 Fig. 4 illustrates the impact of interfacial protein type and concentration on digestion of emulsion droplets stabilized with WPI (0.5 and 1.5%) or soy protein isolate (SPI) (1.5%). These emulsions were incubated in a two-step in vitro digestion model

Fig. 4 Amount of free fatty acids liberated from emulsion droplets stabilized with 0.5% whey protein isolate (B), 1.5% whey protein isolate (;), and 1.5% soy protein isolate (C) subjected to two-step in vitro digestion model (gastric and duodenal) containing bile salts, phospholipids, colipase, phospholipase A2 along with digestive enzymes.37 Data are the average of three independent experiments and bars represent standard deviation.

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containing BS, PL, colipase, PTL and PLA2, as described elsewhere.37 Production of free fatty acids (FFA) increased rapidly in all the emulsions within the first 20 min of duodenal incubation, and no lag phase was observed. The final plateau differed between the emulsions with the most extensive lipid hydrolysis observed in the SPI- versus the WPI-stabilized emulsions. In addition, the emulsions prepared with 1.5% WPI had a lower FFA maximum compared to the same emulsion prepared with 0.5% WPI. This clearly shows that both composition and concentration of the proteins initially present in an emulsion affect not only the emulsion stability during GI transit, but also the extent and kinetics of lipid hydrolysis.

Micellar solubilization and the release of lipophilic bioactives The bioavailability of lipophilic micronutrients and bioactives can be highly variable and depends on many dietary and physiological factors. It has long been acknowledged that the solubilization of these molecules in the aqueous, micellar phase of the digestate is an important and perhaps limiting step, in their absorption. Inclusion in the micellar phase allows lipophilic molecules within the intestinal lumen to approach the intestinal epithelium for absorption. Therefore, micellization is now routinely quantified in in vitro studies and is considered to be an estimate of so-called ‘bioaccessibility’.50–52 Bioaccessibility depends on a molecule’s chemical structure, the physicochemical properties of the carrier, the extent of lipolysis, and the actual incorporation in the micelles.53 Studies of bioaccessibility enable a better understanding of bioavailability. For example, carotenoids are known to be more bioavailable when solubilized in oil droplets, compared to when they are present as crystals in vegetable tissues.54 This has been attributed to an easier transfer to the micellar phase in the case of the oilsolubilized molecules. BS, especially, play a fundamental role in formation of the micellar phase during digestion. The presence of micelles is also of great importance in the self-regulation of PTL activity by sn-2 monoglycerides.12 It is important to note that the micellar phase is not homogeneous, but consists of a series of colloidal structures, including multilamellar and unilamellar vesicles, mixed micelles, and micelles.50,55 The type of micellar structures formed is highly dependent on the concentration of the individual species involved (i.e. BS, PL, cholesterol, monoglycerides, fatty acids, etc.). Therefore, lipolysis, which itself depends on the interface, influences the micellization capacity. Fig. 5 shows how in vitro modelling can be used to investigate lipolysis in protein-stabilized emulsions and the relationship with bioaccessibility. The lipophilic bioactive molecule b-carotene (BC) is shown as an example. BC was solubilized in 10% oil, 0.5% WPI emulsion and exposed to simulated gastric and duodenal conditions, as previously described.37 The micellization of BC was markedly influenced by the extent of lipid hydrolysis. A positive correlation (r2 0.95) was observed between the extent of lipolysis and transfer of the carotenoid into the aqueous micellar phase. However, comparing the experimental data with a theoretical relation between lipolysis and micellization, the results suggest that the extent of lipolysis was greater than the BC micellization. For example, at 70% lipid hydrolysis, only 50% of This journal is ª The Royal Society of Chemistry 2010

Fig. 5 Relation between lipid hydrolysis and incorporation of b-carotene (BC), as a model of lipophilic bioactives, in the aqueous micellar phase during digestion of a 10% oil and 0.5% whey protein isolate stabilized emulsion. Broken line represents a 1 : 1 relationship between % lipid hydrolysis and % micellization of BC. Data are the average of three independent experiments and bars represent standard deviation.

BC was incorporated in the aqueous phase, suggesting that BC tended to remain in the oil phase. This agrees with earlier observations that the degree of hydrophobicity of a molecule affects its bioavailability. Xanthophylls, for example, tend to be more bioavailable than carotenes.56 As they are more hydrophilic than carotenes, the xanthophylls are postulated to locate closer to the surface of an oil droplet and to more readily partition into the micelles.54,57

Conclusions Food manufacturers have learnt how to manipulate the interfacial composition of emulsions to achieve stability system during processing and storage. In contrast, our understanding of how to manipulate protein-based emulsions for desired outcomes in the GI tract is still in its infancy. Not only protein type, but also protein concentration of an initial emulsion plays an important role in the release of fatty acids and, as a consequence, bioaccessibility of bioactive components. Furthermore, consideration of how the presence of BS and PL, as well as the products of pepsinolysis and lipolysis, affect the accessibility of the digestive enzymes, is critical to understanding the changes occurring to protein-stabilized emulsions during GI transit, at the molecular level. Recent studies have stressed the need for physiologically relevant in vitro models, especially given the complexity of the biochemical and biophysical changes and the interactions that occur between the emulsion constituents and the GI environment. Different results are observed, in terms of emulsion proteolysis and lipolysis, depending on the absence or presence of the various digestive co-factors. Once a better understanding exists in terms of the fundamental molecular interactions and changes occurring during GI transit, it will be possible to develop strategies for optimal lipid digestion and bioavailability of lipophilic nutrients. Food Funct., 2010, 1, 141–148 | 147

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31 M. H. Vingerhoeds, T. B. J. Blijdenstein, F. D. Zoet and G. A. van Aken, Food Hydrocolloids, 2005, 19, 915–922. 32 M. H. Vingerhoeds, E. Silletti, J. De Groot, R. G. Schipper and G. A. van Aken, Food Hydrocolloids, 2009, 23, 773–785. 33 F. J. Moreno, A. R. Mackie and E. N. C. Mills, J. Agric. Food Chem., 2005, 53, 9810–9816. 34 M. Dalgalarrondo, E. Dufour, J. M. Chobert, C. Bertrandharb and T. Haertle, Int. Dairy J., 1995, 5, 1–14. 35 G. Mandalari, A. M. Mackie, N. M. Rigby, M. S. J. Wickham and E. N. C. Mills, Mol. Nut. Food Res., 2009, 53, S131–S139. 36 A. Sarkar, D. S. Horne and H. Singh, Food Hydrocolloids, 2010, 24, 142–151. 37 A. Malaki Nik, M. Corredig and A. J. Wright, Food Digestion, 2010, in press. 38 S. Mukhopadhyay and U. Maitra, Curr. Sci., 2004, 87, 1666–1683. 39 G. Fave, T. C. Coste and M. Armand, Cell. Mol. Biol., 2004, 50, 815– 831. 40 M. G. Ivanova, I. Panaiotov, A. G. Bois, Y. Gargouri and R. Verger, J. Colloid Interface Sci., 1990, 136, 363–374. 41 J. Gass, H. Vora, A. F. Hofmann, G. M. Gray and C. Khosla, Gastroenterology, 2007, 133, 16–23. 42 J. Maldonado-Valderrama, N. C. Woodward, A. P. Gunning, M. J. Ridout, F. A. Husband, A. R. Mackie, V. J. Morris and P. J. Wilde, Langmuir, 2008, 24, 6759–6767. 43 H. L. Mu and C. E. Hoy, Prog. Lipid Res., 2004, 43, 105–133. 44 D. J. McClements, E. A. Decker, Y. Park and J. Weiss, Food Biophys., 2008, 3, 219–228. 45 M. Beysseriat, E. A. Decker and D. J. McClements, Food Hydrocolloids, 2006, 20, 800–809. 46 G. Y. Park, S. Mun, Y. Park, S. Rhee, E. A. Decker, J. Weiss, D. J. McClements and Y. Park, Food Chem., 2007, 104, 761–767. 47 A. Tiss, F. Carriere, I. Douchet, S. Patkar, A. Svedsen and R. Verger, Colloids Surf., B, 2002, 26, 135–145. 48 A. Tiss, F. Carriere and R. Verger, Anal. Biochem., 2001, 294, 36–43. 49 B. Borgstrom, Gastroent., 1980, 78, 954–962. 50 C. J. H. Porter and W. N. Charman, Adv. Drug Delivery Rev., 2001, 50, S127–S147. 51 A. Rube, S. Klein and K. Mader, Pharm. Res., 2006, 23, 2024–2029. 52 C. H. M. Versantvoort, A. G. Oomen, E. van de Kamp, C. J. M. Rompelberg and A. J. A. M. Sips, Food Chem. Toxicol., 2005, 43, 31–40. 53 T. Sugawara, M. Kushiro, H. Zhang, E. Nara, H. Ono and A. Nagao, J. Nutr., 2001, 131, 2921–2927. 54 G. T. Rich, A. L. Bailey, R. M. Faulks, M. L. Parker, M. S. J. Wickham and A. Fillery-Travis, Lipids, 2003, 38, 933–945. 55 C. J. H. Porter, A. M. Kaukonen, A. Taillardat-Bertschinger, B. J. Boyd, J. M. O’Connor, G. A. Edwards and W. N. Charman, J. Pharm. Sci., 2004, 93, 1110–1121. 56 K. H. het Hof, I. A. Brower, C. E. West, E. Haddeman, R. P. M. Steegers-Theunissen, M. van Dussldorp, J. A. Weststrate, T. K. A. B. Eskes and J. G. A. J. Hautvast, Amer. J. Clin. Nut., 1999, 70, 261–268. 57 D. A. Garrett, M. L. Failla and R. J. Sarama, J. Agric. Food Chem., 1999, 47, 4301–4309.

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REVIEW

www.rsc.org/foodfunction | Food & Function

Mechanisms underlying the cholesterol-lowering properties of soluble dietary fibre polysaccharides Purnima Gunness and Michael John Gidley*

Downloaded on 10 November 2010 Published on 30 September 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00080A

Received 16th July 2010, Accepted 31st August 2010 DOI: 10.1039/c0fo00080a A number of studies have shown a positive relationship between diets rich in soluble dietary fibres (SDF) such as b-glucan, pectin, guar gum and psyllium, and reduced serum cholesterol and thus a decreased risk of cardiovascular disease (CVD). Three major biological mechanisms have been proposed to explain the cholesterol-reducing effects of SDF: prevention of bile salt (BS) re-absorption from the small intestine leading to an excess faecal BS excretion; reduced glycemic response leading to lower insulin stimulation of hepatic cholesterol synthesis; and physiological effects of fermentation products of SDF, mainly propionate. Evidence for the latter mechanism is inconclusive, whereas in vivo, ex vivo and in vitro experiments suggest that BS micelles ‘‘bind’’ to SDF preventing their reabsorption. Whereas, glycemic responses to SDF have been studied extensively, the nature of interactions between bile salt micelles and SDF that lead to incomplete BS re-absorption are poorly defined. Three potential physicochemical mechanisms are proposed together with suggestions for in vitro experiments to test them.

1. Introduction Over the past 50 years there has been a growing interest in the cholesterol-reducing effects of soluble dietary fibres (SDF). The United States Food and Drugs Administration (FDA) has accepted health claims for food containing oat products (oats, oat bran and oat flour), barley and psyllium husks1 for their cholesterol reducing properties. It is well established that cholesterol is a major risk factor for cardiovascular disease (CVD).2 Many studies have shown a positive relationship between diets rich in soluble dietary fibres such as b-glucan (bG) from oats and barley, pectin from fruits, guar gum and psyllium, and reduced serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C). The FDA has therefore recommended incorporation of 3 g/d of bG or 7 g/d of soluble dietary fibre from psyllium seed husks to diets low in cholesterol and saturated fat in order to help reduce TC and LDL-C1,3 and thus lower the risk of CVD. Arabinoxylan (AX), the major SDF in wheat and rye, is believed to have similar cholesterol-reducing health benefits to bG,4,5 but has been studied less extensively. The human body obtains cholesterol from two sources: de novo synthesis mainly in the liver accounting for approximately 700–900 mg/d, and directly from the diet accounting for up to 300–500 mg/d.6 Cholesterol is eliminated from the body by excretion from the gastrointestinal tract (approximately 600 mg/ d), conversion to bile acids/salts (BA/BS) (approximately 400 mg/ d), loss of dead skin cells (approximately 100 mg/d), synthesis of steroid hormones (approximately 50 mg/d), and by integration into cell membranes of actively dividing cells.7 The liver maintains cholesterol homeostasis. Hepatocyte cholesterol is catabolised into bile acids (BA) and steroid hormones, secreted as is

Centre for Nutrition and Food Sciences, The University of Queensland, Hartley Teakle Building, St Lucia, Queensland, 4072, Australia. E-mail: [email protected]; Fax: +61 7 3365 1177; Tel: +61 7 3365 2145

This journal is ª The Royal Society of Chemistry 2010

in bile, used in cell membranes or stored as cholesteryl esters. The major route by which hepatic cholesterol is eliminated is via BA synthesis through a cascade of 14 enzymatic reactions with cholesterol 7a-hydroxylase being the rate limiting enzyme.8 Cholesterol homeostasis is also maintained by modulation of 3hydroxy 3-methylglutaryl co-enzyme A reductase (HMG-Co AR), a rate limiting enzyme in cholesterol biosynthesis and by cholesterol-carrying low density lipoprotein (LDL) receptors.7 In vivo studies have shown that ingestion or administration of some viscous SDF results in a 35 to 65% excess excretion of BA/ BS in the faeces9,10 leading to a substantial reduction in total plasma cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) by diversion to synthesis of BA/BS, but with no effect on high density lipoprotein-cholesterol (HDL-C), the ‘good’ cholesterol.11–13 It is believed that the excess BS arises because SDF hinders BS reabsorption into the enterohepatic circulation. SDF may also lower TC and LDL-C levels through effects on postprandial glycaemia. Viscous SDF reduces the rate of intestinal absorption of glucose with subsequent decrease in insulin production by the pancreas.14 As insulin is an activator of HMGCo AR, it is possible that reduced insulin levels could lead to a decrease in cholesterol synthesis.15 A further potential role for viscosity is interference in efficient emulsion formation, particularly the ability to form the very small mixed micelles (ca 10 nm) associated with efficient absorption from the digestive tract.16 In addition, other studies have shown that SDF causes an increase in short chain fatty acids (SCFA) such as acetate, propionate and butyrate, products of their bacterial colonic fermentation. These SCFA, particularly propionate, may also indirectly cause a decrease in blood cholesterol through inhibition of hepatic cholesterol synthesis.17,18 Therefore, the properties that could contribute to the cholesterol-reducing effect of SDF in animals and humans are the prevention of BS re-absorption (with subsequent excess faecal excretion), and decreased hepatic cholesterol synthesis Food Funct., 2010, 1, 149–155 | 149

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modulated by insulin and/or by fermentation products such as propionate.19

2. Proposed biological mechanisms

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2.1 Excess faecal BS excretion In humans, primary bile acids such as cholic acid (CA, 3a,7a,12a-trihydroxy-5b-cholanic acid) and chenodeoxy cholic acid (CDCA, 3a,7a-dihydroxy-5b-cholanic acid) are synthesised de novo in the liver from cholesterol as their sole precursor and are conjugated by N-acyl linkage with glycine or taurine before being temporarily stored in the gall bladder. As the conjugated BA are stronger acids than the un-conjugated forms, they remain ionised at the pH prevailing in the biliary tract and the small intestine; they are therefore termed ‘‘bile salts’’ (BS).20 In the gall bladder, the concentration of conjugated BS is high (300 mM) compared with their critical micelle concentrations (8.86 mM for glycocholate,21 2.5 mM for glycochenodeoxycholate22 in 0.15 M NaCl) causing aggregation of these amphipathic molecules and the formation of mixed micelles with biliary cholesterol and phosphatidylcholine.23 The liberation of gastric chyme and stimulation by cholecystokinin cause the flow of bile from the gall bladder into the duodenum and proximal jejunum, where BS micelles help in the solubilisation of food components such as cholesterol, fat soluble vitamins, and other lipids.20 Diffusion of micelles then delivers solubilised components as well as both biliary and dietary cholesterol across the unstirred water layer covering the luminal side of the enterocytes, thus facilitating uptake of cholesterol and other lipophilic components by the enterocytes.24 Once this role is fulfilled, the BS micelles transit the remainder of the small and large intestine where they are progressively reabsorbed into the enterohepatic circulation by the hepatic portal vein. In the terminal ileum the conjugated BS are reabsorbed from the intestinal lumen into the enterocytes by apical sodium-dependent bile acid transporters (ASBT). The BS are then passed into the hepatic portal vein via heterodimer protein transporters, organic solute transporter a/organic solute transporter b (OSTa/OSTb) present on the basolateral membrane of the enterocytes.25 Bacteria present in the lumen of the ileum cause the deconjugation of some of the conjugated BS resulting in the passive absorption of free BS starting from the jejunum and throughout the small intestine.26,27 Therefore, in the terminal ileum there is a mixture of conjugated and deconjugated BS.28 The BS which escape intestinal absorption undergo bacterial enzymatic transformation (e.g. dehydroxylation at C-7 and epimerisation) in the large intestine to form secondary BS.29 Thus, the primary BS chenodeoxycholic acid is converted to lithocholic acid (LCA, 3a,12a-dihydroxy-5b-cholan-24-oic acid) and cholic acid to deoxycholic acid (DCA, 3a-hydroxy-5bcholan-24-oic acid). DCA is passively absorbed across the colonic epithelium into the enterohepatic circulation while most of the LCA is excreted in the faeces.30 The enterohepatic circulation is very efficient at reabsorbing and recycling approximately 95% of the BS at each passage through the intestine and back to the liver. Small amounts, 400 to 600 mg/d are lost by faecal excretion but are compensated by hepatic synthesis from cholesterol25 thus maintaining cholesterol homeostasis.31 The pool of BS in bile is therefore composed of both primary (e.g. 150 | Food Funct., 2010, 1, 149–155

CDCA, CA) and secondary BS (e.g. DCA and trace of LCA) and this pool size is maintained at a constant level as the daily BS losses are replenished by biosynthesis.20 The presence of viscous SDF in the small intestine has been shown to prevent at least some BS from being re-absorbed into the enterohepatic circulation, resulting in excess excretion of BS in faeces.32,33 This causes a depletion of BA in the liver and consequently cholesterol is rapidly catabolised in the hepatocyte to replenish the BA pool via activation of cholesterol 7ahydroxylase. Further, cholesteryl esters are also metabolised and production of LDL-C surface membrane receptors are increased to enhance the uptake of LDL-C from the blood stream, thus lowering blood cholesterol concentration.34 2.2 Low glycemic response Insulin is known to have an influence on carbohydrate metabolism, protein synthesis and lipogenesis. In addition, this hormone also activates HMG-Co AR which promotes the synthesis of hepatic cholesterol.35 Viscous SDF such as oat bG,36 guar gum and pectin37 slow down the digestion of macronutrients by delaying gastric emptying, slowing down the transport/mixing of digestive enzymes, and increasing the resistance of the unstirred water layer lining the mucosa to intestinal absorption.38,39 Consequently, glucose and other macronutrient absorption is reduced.36,40 This decreased postprandial glucose level is accompanied by a decline in insulin level,41 potentially resulting in the inhibition of HMG-Co AR and a subsequent reduction in hepatic cholesterol synthesis.14,42 The hormonal action on cholesterol biosynthesis is complex. In an ileostomy study Lundin et al. (2004)42 reported that a diet high in rye taken as isocaloric meals seven times per day as opposed to meals taken three times per day, decreases blood glucose and insulin levels and lowers the excretion of urinary C-peptide leading to a reduction in cholesterol. The authors argued that the bile salt excretion mechanism is not sufficient as in another study rye did not show any cholesterol-lowering effect even though an excess BS was excreted in the ileostomy bags.43 Further, Jones et al.44 also found that cholesterol synthesis was dependent on meal frequency and correlated with insulin levels. Lundin et al.42 explained their results as the combined action of reduced insulin and enhanced BS excretion leading to a decrease in plasma cholesterol. As insulin plays a central role in so many dietinduced responses, it is difficult to design experiments to determine its exact role in controlling cholesterol levels. Furthermore, the same types of rheological effects of SDF in digesta are likely to cause both a reduced insulin response and restriction of bile salt re-absorption, making the two mechanisms difficult to disentangle. 2.3 Fermentation products of SDF As soluble dietary fibres are neither digested nor absorbed in the small intestine, they undergo anaerobic bacterial fermentation in the caecum and colon, to produce short chain fatty acids (SCFA) such as acetate, propionate, and butyrate. Butyrate is utilised by the colonocytes as their primary source of energy45 whereas acetate and propionate are at least partially absorbed into the hepatic portal vein.17,18 It has been proposed that the SCFA, This journal is ª The Royal Society of Chemistry 2010

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particularly propionate, deplete plasma cholesterol by inhibiting hepatic cholesterol metabolism via one or more of several mechanisms including reduction of HMG-Co AR activity46,47 and inhibition of acetyl-Co A reductase which catalyses the synthesis of acetyl-Co A from acetate.18 These proposed mechanisms are in line with the fact that propionate intervenes in glycolysis, gluconeogenosis and ketogenosis in the liver.48 The role of propionate in blood cholesterol reduction has been studied in vitro18,48 and in vivo by supplementing the diet with propionate49,50 and by intracaecal infusion of propionate.51,52 The in vitro studies were conducted on isolated rat liver cells incubated in propionate at different concentration levels from 1.0 to 2.5 mM18 and it was found that the inhibition of cholesterol biosynthesis increased with propionate concentration. These concentrations are lower than previously obtained in similar experiments (18 mM) and are closer to the concentration of propionate in the hepatic portal vein (0.8 mM).49 However in the latter report, no change in hepatic cholesterol synthesis was observed in the whole animal experiment in which rats were fed propionate even though there was a decrease in blood cholesterol. Similarly, with the intracaecal infusion experiments no cholesterol reducing role could be attributed to propionate. Moreover, only a few studies on the effects of SCFA on hepatic cholesterol metabolism in humans have been reported.53,54 Results obtained from these experiments are inconsistent, and further human studies are needed to underpin the role of propionate in blood cholesterol reduction. If the effect of propionate on hepatic cholesterol metabolism is involved in the cholesterol-lowering properties of SDF, it is not likely to be the dominant mechanism. This is because SDF fermentation in the colon occurs irrespective of the molecular size of the polysaccharides, whereas cholesterol-lowering effects are associated with viscosity i.e. only polymeric SDF.55 From this overview of the three potential mechanisms, it is concluded that excess bile salt secretion can play a role in reducing plasma cholesterol and has the benefit of being a specific, measurable, and testable hypothesis. A central role for insulin cannot be ruled out but is very difficult to test directly, whereas a role for propionate and other fermentation products is not currently established. The remainder of this review will discuss potential physicochemical mechanisms leading to bile salt excretion. It is noted that there is a close parallel with mechanisms that attenuate macronutrient digestion and which could therefore contribute to the insulin-based mechanism as well.

3. Physicochemical mechanisms 3.1 In vivo and ex vivo results Ileostomy studies are very useful in understanding the relationships between viscosity, ileal BS content and consequently blood cholesterol, as samples collected contain only BS with little or no SDF microbial fermentation. Studies comparing native viscous bG with less viscous, hydrolysed low molecular weight bG, showed an increase in BS excretion and synthesis in patients fed native bG in their breakfast cereal.33 The increase in BS synthesis as measured by an increase in serum concentration of 7a-hydroxy-4-cholesten-3-one (a marker for BA synthesis56) This journal is ª The Royal Society of Chemistry 2010

suggests a decrease in blood cholesterol in humans57 and in pigs.4 Viscous but non-fermentable substances such as hydroxypropyl methyl cellulose (HPMC) have been used to show that increased intestinal viscosity is positively related to a decrease in plasma cholesterol in hamsters.55 Using the same animal model, Carr et al.58 showed that HPMC (Methocel, K100M: 22% methyl substitution and 8.1% hydroxypropyl substitution) at different viscosity grades (14-1698 cP59) caused an increase in lumen viscosity compared with cellulose which is very slowly fermentable but not viscous. The cholesterol reducing effect of the viscous HPMC suggests that viscosity and not fermentability is primarily responsible for the cholesterol reducing behaviour of SDF as HPMC is poorly fermented by colonic bacteria.60 In a recent human study, Reppas et al.61 have confirmed and extended these findings by showing that HPMC (particularly high viscosity grades) reduces plasma cholesterol. An effect of bG molecular weight (and therefore viscosity) on plasma cholesterol has recently been demonstrated in a human intervention trial.62 This study showed that oat bG provided as a component of breakfast cereal reduced plasma cholesterol to similar extents if it had a high (ca 2 MDa) or intermediate (ca 500 kDa) molecular weight, but that the effect was halved for a lower molecular weight bG of ca 200 kDa.62 Studies on interactions between viscous soluble polymers and BA/BS have been undertaken in vitro,63 in vivo64 and ex vivo using in vivo samples.55 Most of these studies have been interpreted to suggest that the SDF interact with BA in one or both of two different ways, either they are bound to the BA/BS molecules65,66 or the bile salt micelles are entrapped in a viscous or gelatinous network formed by the polymer.9,33,67 However, the nature of this entrapment and the differences between ‘viscous’ networks (which are temporary and caused by polymer entanglement or particle packing) and ‘gelatinous’ networks (which are essentially permanent and based on physical cross-links)68 have not been investigated. This is a difficult area to investigate and the rheology of digesta in vivo is poorly understood due to its complex multi-phase nature68 and limited access to samples. In ileostomy studies,33 the increase in BS ileal content and hepatic BA synthesis were attributed to the entrapment of BA by SDF in the small intestine. The investigators supported the idea that high viscosity oat-bran bG has an effect on the BS-cholesterol mixed micelles in the intestinal lumen. They argued that, as the relative amounts of BS and cholesterol ileal excretion were similar to the ratio of BS to cholesterol (1 mol to 0.08 mol) in mixed micelles found in other ileostomy studies,9,57 oat-bran bG entraps the entire micelles. Other reports have speculated that SDF forms a barrier which prevents the formation of BA micelles and increases the unstirred water layer lining the intestinal mucosal surface.69–71 There are therefore three hypotheses (Fig. 1–3) that have been proposed for explaining the incomplete re-absorption of BS and their subsequent (partial) excretion: Hypothesis 1: SDF increases the barrier properties of the unstirred layer between micelles and intestinal absorptive cells (Fig. 1) Hypothesis 2: SDF and BS are associated/complexed at a molecular level (Fig. 2) Hypothesis 3: SDF forms a local matrix that entraps BS micelles (Fig. 3) Food Funct., 2010, 1, 149–155 | 151

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Fig. 3 Entrapment of mixed bile salt-cholesterol micelles in a SDF network.

Fig. 1 Illustration of the potential barrier property of SDF in the small intestine.

investigated specific interactions between barley bG and glycocholic acid using solid state 13C nuclear magnetic resonance (NMR) spectroscopy. However, no evidence for specific molecular interactions was found. The methods, results and limitations of the reported techniques to analyse interactions between SDF and BS/BA are summarised in Table 1.

4. Future prospects

Fig. 2 Schematic representation of molecular interactions between SDF and mixed bile salt-cholesterol micelles in the intestinal lumen.

3.2 In vitro studies Many investigations of SDF interactions with BA have been undertaken in vitro, mostly through binding type experiments. Although there are a number of different types of approach, a common in vitro binding experiment involves mixing BA with the SDF source to be tested, incubating, centrifuging,63,65,72 filtering using a dialysis membrane73–75 and measuring the unbound BA. A major drawback of most of these studies is the use of bile salt concentrations (typically 1 mM) lower than the critical micellar concentration. The majority of studies allow a certain time for interactions to occur and then determine the unbound fraction. A few studies73,75 have reported the time course of BS micelle passage across membranes in the presence and absence of SDF. This is not only a closer approximation to the re-absorption thought to occur in vivo but has also been carried out at sufficiently high BS concentrations to ensure micelle formation. There has been very little data reported on the nature of any molecular interactions between BS micelles and SDF. One of the few examples is the report of Bowles et al.76 who 152 | Food Funct., 2010, 1, 149–155

Despite the interest in the topic, very few experiments have been performed that could be used to identify the nature of the molecular interactions between SDF and BA/BS molecules and/ or micelles even though numerous reports exist on the in vitro binding of SDF and BA as summarised in Table 1. However, methods and approaches can be proposed to test each of the hypotheses represented in Fig. 1–3. Thus, to study whether the SDF forms a barrier preventing mixed BA-cholesterol micelles from reaching the intestinal absorptive cells (Fig. 1), the simplest model is to use dialysis membranes or other semi-permeable barriers that allow passage of BS molecules but not polymeric SDF. The first reports of this type of experiment have started to appear,73,75 but there is a lot of scope for further studies, potentially extending to monolayers of epithelial cells as a more realistic model. The nature of molecular associations i.e. whether the SDF is associated/complexed on a molecular level with BA/BS, and/or forms a local network that entraps the BS micelles can be studied by a variety of structural techniques. Examples include high resolution NMR spectroscopy, small angle X-ray and neutron scattering, together with microcalorimetry to define the energetics of any interactions and binding stoichiometry as a function of SDF and BS type and concentration. Solution state 13C NMR spectroscopy is a suitable technique for characterising SDF/BS interactions, at least in model systems, as both components are sufficiently mobile in solution to give narrow resonances. First results using this methodology77 show clear evidence for (i) close molecular interactions (Fig. 2) This journal is ª The Royal Society of Chemistry 2010

 Wide range of polymers and particulate suspensions  bG (barley, oat flours & brans)

63, 72 and references cited therein, 74 and references cited therein

This journal is ª The Royal Society of Chemistry 2010 C NMR of hydrated (25% water) mixtures of bG and either glycocholic acid or Congo Red dye. Both rigid (CP/MAS) and mobile (SP/MAS) segments probed.

13

 Processed barley bG

76

Bile salts (1 mM) mixed with 250 mg fibre in 10 mL buffer for 2 h at 37  C. Unbound bile sample (1 mL) separated by ultrafiltration at 3 Bar with MWCO 3000 and analysed by HPLC Bile salts (10 mM) mixed with fibres in pH 7 buffer. Dialysed (MWCO 6–8 kDa for proteins) with agitation. Equilibrated dialysate concentration used to infer binding.

 Barley meal and extrudates

75

 Oat/barley bG products

Taurocholic acid (16 mM) mixed with fibre in pH 7 buffer and dialysed (molecular weight cut off (MWCO) 1200)) against same buffer. Time course of dialysed BA compared with and without fibre

 Soybean fibre  Barley flour  Barley bG

In vitro simulation of gastrointestinal conditions, typically involving treatment of fibre sample with acid and pepsin followed by neutralisation and addition of bile salts. ‘Bound’ bile salts separated by centrifugation Analysis of unbound BA by HPLC after derivatisation and fluorescence detection

Methods used to analyse fibre-BA interactions

73 and references cited therein

 Arabinoxylan (rye, wheat flours & brans)  Pectin (fruit & vegetable cell wall materials)

SDF types studied

Reference

Table 1 In vitro studies of interactions between bile salts and soluble dietary fibres: methods, findings and limitations.

No measurable change in bG chemical shifts and no rigidification of BA in presence of bG. Congo Red immobilised by bG.

Equilibration complete within 5 h. Many phenolic compounds also bind

Extrusion or autoclaving increase bile binding. Dihydroxy bile acid more bound than trihydroxy.

Fibre-containing samples delayed but did not completely prevent transport of taurocholic acid compared with control.

Non-hydrated particulate fibres show low (e.g. resistant starch) or very low (e.g. cellulose or lignin) binding

Many soluble polymers and hydrated particulate fibre preparations ‘bind’ bile salts to significant extent.

Main findings

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Does not rule out dynamic interactions

Limited sensitivity and broad NMR signals

Structural basis of ‘binding’ not clarified - many factors inferred to contribute

Bile salts below critical micelle concentration Small sample of unbound bile recovered after ultrafiltration

Single BA used Transport across dialysis membrane slow even in absence of fibre (e.g. 33% after 18 h73)

‘Binding’ may be reversible under in vivo conditions of reabsorption Bile salt concentrations typically 1 mM or lower, less than both critical micelle and typical in vivo concentrations (5–20 mM in small intestine)

Centrifugation step not representative of in vivo conditions

Limitations

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between BS micelles and a barley b-glucan (systematic chemical shift changes in BS resonances as a function of b-glucan concentration) and (ii) the dynamic nature of the interaction (no measurable change in line widths for either BS or SDF resonances). In contrast, a wheat arabinoxylan was found to cause increased line widths but no systematic chemical shift changes for BS micelles,77 consistent with local aggregates of arabinoxylan causing entrapment and reduced mobility of micelles (Fig. 3). The presence of aggregates within ‘solutions’ of arabinoxylan has also been shown to result in reduced diffusion of a probe molecule of the same size range as BS micelles.78 Small angle scattering (X-ray and/or neutron) gives information on the size, shape and surface characteristics of entities with sizes in the range 1–100 nm. As both micelles and polymers are in this size range, they can be investigated both separately and in combination. If there are specific molecular interactions between micelles and the polymer (Fig. 2), then scattering patterns of mixtures of the two would be predicted to be non-additive, with the expectation79 that association of BS micelles would result in a shrinking of the SDF polymer chain. Small angle neutron scattering has the advantage80 that selective contrast matching should be able to be used to examine the structure of BS micelles or SDF in the presence of the other component. A combination of structural studies with stoichiometric and calorimetric analyses can confidently be predicted to shed further light on the interactions of pure bile salts and SDF. The challenge will then be to extend results from model systems to biological bile and whole foods. This will be difficult given the greater heterogeneity and the presence of many other components, but is necessary in order to connect in vitro data to in vivo data. If successful, this will allow mechanism-based prediction of potential in-body BS binding of SDF from different origins and in different food formulations.

References 1 FDA, Food labelling: Health claims; soluble fibre from certain foods and coronoary heart disease, Report 0020–6563, Federal Register 73(37), USA, 2008. 2 T. A. Pearson, S. N. Blair, S. R. Daniels, R. H. Eckel, J. M. Fair, S. P. Fortmann, B. A. Franklin, L. B. Goldstein, P. Greenland, S. M. Grundy, Y. L. Hong, N. H. Miller, R. M. Lauer, I. S. Ockene, R. L. Sacco, J. F. Sallis, S. C. Smith, N. J. Stone and K. A. Taubert, Circulation, 2002, 106, 388–391. 3 D. J. A. Jenkins, C. W. C. Kendall, V. Vuksan, E. Vidgen, T. Parker, D. Faulkner, C. C. Mehling, M. Garsetti, G. Testolin, S. C. Cunnane, M. A. Ryan and P. N. Corey, Am. J. Clin. Nutr., 2002, 75, 834–839. 4 H. N. Laerke, C. Pedersen, M. A. Mortensen, P. K. Theil, T. Larsen and K. E. B. Knudsen, J. Sci. Food Agric., 2008, 88, 1385–1393. 5 H. W. Lopez, M.-A. Levrat, C. Guy, A. Messager, C. Demigne and C. Remesy, J. Nutr. Biochem., 1999, 10, 500–509. 6 J. M. Dietschy, Klin. Wochenschr., 1984, 62, 338–345. 7 D. W. Russell, Cardiovasc. Drugs Ther., 1992, 6, 103–110. 8 D. W. Russell and K. D. R. Setchell, Biochemistry, 1992, 31, 4737– 4749. 9 A. Lia, G. Hallmans, A. S. Sandberg, B. Sundberg, P. Aman and H. Andersson, Am. J. Clin. Nutr., 1995, 62, 1245–1251. 10 Z. Madar and A. Stark, Agro Food Industry Hi-Tech, 1995, 6, 40–42. 11 B. H. Arjmandi and D. Reevesm Robert, J. Nutr., 1992, 122, 246. 12 C. Moundras, S. R. Behr, C. Remesy and C. Demigne, J. Nutr., 1997, 127, 1068–1076. 13 A. S. Truswell, Eur. J. Clin. Nutr., 2002, 56, 1–14. 14 A. T. Erkkila and A. H. Lichtenstein, Journal of Cardiovascular Nursing, 2006, 21, 3–8.

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15 K. S. Juntunen, D. E. Laaksonen, K. Autio, L. K. Niskanen, J. J. Holst, K. E. Savolainen, K.-H. Liukkonen, K. S. Poutanen and H. M. Mykkanen, Am. J. Clin. Nutr., 2003, 78, 957–964. 16 Z. Haikal, B. Play, J.-F. Landrier, A. Giraud, O. Ghiringhelli, D. Lairon and D. Jourdheuil-Rahmani, Lipids, 2008, 43, 401–408. 17 S. Bridges, J. Anderson, D. Deakins, D. Dillon and C. Wood, Am. J. Clin. Nutr., 1992, 56, 455–459. 18 R. S. Wright, J. W. Anderson and S. R. Bridges, Proc. Soc. Exp. Biol. Med., 1990, 195, 26–29. 19 Z. Y. Chen, R. Jiao and K. Y. Ma, J. Agric. Food Chem., 2008, 56, 8761–8773. 20 R. Z. Vlahcevic, M. D. Heuman and B. P. Hylemon, in Hepatology- A textbook of liver disease, ed. D. Zakim and D. T. Boyer, W.B. Sauders Company, Philadelphia, 1996, vol. 1, ch. 13, pp. 376–417. 21 S. Reis, C. G. Moutinho, C. Matos, B. de Castro, P. Gameiro and J. L. F. C. Lima, Anal. Biochem., 2004, 334, 117–126. 22 K. Matsuoka, M. Suzuki, C. Honda, K. Endo and Y. Moroi, Chem. Phys. Lipids, 2006, 139, 1–10. 23 A. F. Hofmann, News Physiol. Sci., 1999, 14, 24–29. 24 D. Q. H. Wang, Annu. Rev. Physiol., 2007, 69, 221–248. 25 P. A. Dawson, T. Lan and A. Rao, J. Lipid Res., 2009, 50, 2340– 2357. 26 E. Krag and S. F. Phillips, J. Clin. Invest., 1974, 53, 1686–1694. 27 E. R. Schiff, N. C. Small and J. M. Dietschy, J. Clin. Invest., 1972, 51, 1351–1362. 28 A. F. Hofmann, Front. Biosci., 2009, 2584–2598. 29 J. P. Hamilton, G. Xie, J.-P. Raufman, S. Hogan, T. L. Griffin, C. A. Packard, D. A. Chatfield, L. R. Hagey, J. H. Steinbach and A. F. Hofmann, Am. J. Physiol.: Gastrointest. Liver Physiol., 2007, 293, G256–263. 30 J. M. Ridlon, D.-J. Kang and P. B. Hylemon, J. Lipid Res., 2005, 47, 241–259. 31 J. Y. L. Chiang, J. Lipid Res., 2009, 50, 1955–1966. 32 J. A. Marlett and M. H. Fischer, J. Nutr., 2002, 132, 2638–2643. 33 L. Ellegard and H. Andersson, Eur. J. Clin. Nutr., 2007, 61, 938–945. 34 M. S. Brown and J. L. Goldstein, Angew. Chem., Int. Ed. Engl., 1986, 25, 583–602. 35 M. R. Lakshmanan, C. M. Nepokroeff, G. C. Ness, R. E. Dugan and J. W. Porter, Biochem. Biophys. Res. Commun., 1973, 50, 704–710. 36 K. Queenan, M. Stewart, K. Smith, W. Thomas, R. G. Fulcher and J. Slavin, Nutr. J., 2007, 6, 6. 37 D. J. A. Jenkins, A. R. Leeds, T. M. S. Wolever, D. V. Goff, K. Alberti, M. A. Gassull and T. D. R. Hockaday, Lancet, 1976, 308, 172–174. 38 J. Salas-Salvado, M. Bullo, A. Perez-Heras and E. Ros, Br. J. Nutr., 2006, 96, S45–51. 39 D. Lairon, B. Play and D. Jourdheuil-Rahmani, J. Nutr. Biochem., 2007, 18, 217–227. 40 I. Bourdon, W. Yokoyama, P. Davis, C. Hudson, R. Backus, D. Richter, B. Knuckles and B. O. Schneeman, Am. J. Clin. Nutr., 1999, 69, 55–63. 41 J. G. Potter, K. P. Coffman, R. L. Reid, J. M. Krall and M. J. Albrink, Am. J. Clin. Nutr., 1981, 34, 328–334. 42 E. A. Lundin, J. X. Zhang, D. Lairon, P. Tidehag, P. Aman, H. Adlercreutz and G. Hallmans, Eur. J. Clin. Nutr., 2004, 58, 1410–1419. 43 J. X. Zhang, E. Lundin, G. Hallmans, H. Adlercreutz, H. Andersson, I. Bosaeus, P. Aman, R. Stenling and S. Dahlgren, Am. J. Clin. Nutr., 1994, 59, 389–394. 44 P. J. Jones, C. A. Leitch and R. A. Pederson, Am. J. Clin. Nutr., 1993, 57, 868–874. 45 S. A. L. W. Vanhoutvin, F. J. Troost, H. M. Hamer, P. J. Lindsey, G. H. Koek, D. M. A. E. Jonkers, A. Kodde, K. Venema and R. J. M. Brummer, PLoS One, 2009, 4, e6759, Article No.: e6759. 46 W. J. L. Chen, J. W. Anderson and D. Jennings, Proc. Soc. Exp. Biol. Med., 1984, 175, 215–218. 47 M. A. Levrat, M. L. Favier, C. Moundras, C. Remesy, C. Demigne and C. Morand, J. Nutr., 1994, 124, 531–538. 48 P. M. Nishina and R. A. Freedland, J. Nutr., 1990, 120, 668–673. 49 R. J. Illman, D. L. Topping, G. H. McIntosh, R. P. Trimble, G. B. Storer, M. N. Taylor and B. Q. Cheng, Ann. Nutr. Metab., 1988, 32, 97–107. 50 C. S. Venter, H. H. Vorster and D. G. Van der Nest, J. Nutr., 1990, 120, 1046–1053. 51 K. E. Beaulieu and M. I. McBurney, J. Nutr., 1992, 122, 241–245.

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52 T. M. S. Wolever, F. Brighenti, D. Royall, A. L. Jenkins and D. J. A. Jenkins, Am. J. Gastroenterol., 1989, 84, 1027–1033. 53 T. Todesco, A. Rao, O. Bosello and D. Jenkins, Am. J. Clin. Nutr., 1991, 54, 860–865. 54 T. Wolever, P. Spadafora and H. Eshuis, Am. J. Clin. Nutr., 1991, 53, 681–687. 55 D. D. Gallaher, C. A. Hassel, K. J. Lee and C. M. Gallaher, J. Nutr., 1993, 123, 244–252. 56 G. Sauter, F. Berr, U. Beuers, S. Fischer and G. Paumgartner, Hepatology, 1996, 24, 123–126. 57 J. Zhang, G. Hallmans, H. Andersson, I. Bosaeus, P. Aman, P. Tidehag, R. Stenling, E. Lundin and S. Dahlgren, Am. J. Clin. Nutr., 1992, 56, 99–105. 58 T. P. Carr and D. D. Gallaher, J. Nutr., 1996, 126, 1463–1469. 59 D. D. Gallaher, C. A. Hassel and K. J. Lee, J. Nutr., 1993, 123, 1732– 1738. 60 M. J. Ferguson and G. P. Jones, J. Sci. Food Agric., 2000, 80, 166– 170. 61 C. Reppas, S. Z. Swidan, S. W. Tobey, M. Turowski and J. B. Dressman, Eur. J. Clin. Nutr., 2009, 63, 71–77. 62 T. M. Wolever, S. M. Tosh, A. L. Gibbs, J. Brand-Miller, A. M. Duncan, V. Hart, B. Lamarche, B. A. Thomson, R. Duss and P. J. Wood, Am. J. Clin. Nutr., 2010, DOI: 10.3945/ ajcn.2010.2917. 63 T. S. Kahlon, M. M. Chiu and M. H. Chapman, Cereal Chem., 2009, 86, 329–332. 64 G. Dongowski, M. Huth, E. Gebhardt and W. Flamme, J. Nutr., 2002, 132, 3704–3714.

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65 G. Dongowski, Food Chem., 2007, 104, 390–397. 66 S. Sayar, J. L. Jannink and P. J. White, J. Agric. Food Chem., 2006, 54, 5142–5148. 67 S. S. Cho and C. Clark, in Handbook of dietary fiber, ed. S. S. Cho and M. L. Dreher, Marcel Dekker, Inc., New York, 2001, pp. 473–495. 68 M. A. Eastwood and E. R. Morris, Am. J. Clin. Nutr., 1992, 55, 436–442. 69 E. Theuwissen and R. P. Mensink, Physiol. Behav., 2008, 94, 285–292. 70 J. A. Marlett, in Dietary fiber in health and disease, ed. D. Kritchevsky and C. Bonfield, Plenum Press, New York, 1997, vol. 427, pp. 109– 121. 71 C. Edwards, Adv. Exp. Med. Biol., 1990, 270, 95–104. 72 H. J. Kim and P. J. White, J. Agric. Food Chem., 2010, 58, 628–634. 73 S. H. Han, S. W. Lee and C. Rhee, Nutr. Res. Pract., 2009, 3, 149–155. 74 M. Huth, G. Dongowski, E. Gebhardt and W. Flamme, J. Cereal Sci., 2000, 32, 115–128. 75 H. T. Simonsen, M. S. Nielsen, N. J. Christensen, U. Christensen, T. V. l. Cour, M. S. Motawia, B. P. M. Jespersen, S. B. Engelsen and B. L. Mller, J. Agric. Food Chem., 2009, 57, 2056–2064. 76 R. K. Bowles, K. R. Morgan, R. H. Furneaux and G. D. Coles, Carbohydr. Polym., 1996, 29, 7–10. 77 P. Gunness, B. M. Flanagan and M. J. Gidley, J. Cereal Sci., 2010, 10.1016/j.jcs.2010.07.009. 78 K. J. Shelat, F. Vilaplanaa, T. M. Nicholson, H. K. Wong, M. J. Gidley and R. G. Gilbert, Carbohydr. Polym., 2010, 82, 46–53. 79 J. Mata, J. Patel, N. Jain, G. Ghosh and P. Bahadur, J. Colloid Interface Sci., 2006, 297, 797–804. 80 A. Lopez-Rubio and E. P. Gilbert, Trends Food Sci. Technol., 2009, 20, 576–586.

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REVIEW

www.rsc.org/foodfunction | Food & Function

Effects of eggs on plasma lipoproteins in healthy populations† Maria Luz Fernandez*

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Received 19th July 2010, Accepted 6th September 2010 DOI: 10.1039/c0fo00088d Extensive research has not clearly established a link between egg consumption and risk for coronary heart disease. This lack of connection can be explained by two major reasons: First, eggs are a good source of numerous nutrients including lutein and zeaxanthin, potent antioxidants, which may exert a protective effect against lipoprotein oxidation. Second, it has been well established that dietary cholesterol increases the concentrations of both circulating LDL and HDL cholesterol in those individuals who experience a higher increase in plasma cholesterol following egg consumption (hyper-responders). It is also important to note that 75% of the population experiences a mild increase or no alterations in plasma cholesterol concentrations when challenged with high amounts of dietary cholesterol (normal responders and hypo-responders). Egg intake has been shown to promote the formation of large LDL and HDL subclasses in addition to shifting individuals from the LDL pattern B to pattern A, which is less atherogenic. For these reasons, dietary recommendations aimed at restricting egg consumption should be taken with caution and not include all individuals. We need to acknowledge that diverse healthy populations experience no risk in developing coronary heart disease by increasing their intake of cholesterol but in contrast, they may have multiple beneficial effects by the inclusion of eggs in their regular diet.

Introduction Dietary guidelines aimed at reducing the risk for coronary heart disease (CHD) recommend no more than 300 mg of dietary cholesterol per day, a recommendation that bears on egg consumption.1 Although the American Heart Association has modified its previous policy of eating only two eggs per week to allowing the consumption of one yolk per day while restricting the rest of the dietary cholesterol,2 eggs continue to be regarded with caution by the majority of the population. It is important to note that guidelines from other countries such as Canada,3 New Zealand4 and the European countries5 do not support the idea of recommending an upper limit for dietary cholesterol and their policies are restricted to control the intake of saturated fat and trans fat as major determinants of blood cholesterol concentrations. Extensive research does not support a relationship between egg intake and CHD incidence.6,7 A review of multiple casecontrolled studies measuring intake of cholesterol and disease incidence, reported that a relationship could not be clearly established between this dietary component and increase in CHD risk. Furthermore, data gathered from the Lipid Research Clinics Prevalence Follow-up Study,8 which examined both men and women (n ¼ 4546) found no significant relationships between deaths attributable to CHD and dietary cholesterol intake. Analyses of several studies,9–11 including the elderly population,12 have also failed to find an association between the incidence of CHD and egg consumption. More recent studies Department of Nutritional Sciences, The University of Connecticut, 3624 Horsebarn Road Extension, Storrs, CT, 06269. E-mail: maria-luz. [email protected]; Fax: +1 (860) 486-3674; Tel: +1 (860) 486-5547 † There was no financial support for this review; Maria Luz Fernandez has no conflicts of interest in any of the information presented in this manuscript.

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also indicate the lack of correlation between egg intake and risk for coronary heart disease, or stroke.13,14 It is noteworthy to mention that recent studies have also shown that diabetic individuals may not benefit from egg consumption14 and that this may increase their risk for all cause mortality.15 However, the intent of this review is to focus on healthy populations. The lack of association between egg intake and CHD reported in these epidemiological studies4–10,12–14 could partly be explained by the fluctuations in response to dietary cholesterol among all individuals, which varies from no changes, slight increase or higher increases in plasma cholesterol. However, it is important to note that for those individuals (25% of the population) who have a higher response to dietary cholesterol, there is a consistent increase in both plasma LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) concentrations with no alterations in the LDL-C/HDL-C ratio, a major determinant of CHD risk. This inter-individual variation in response to dietary cholesterol can be attributed in part to differences in absorption rates,16 the body’s ability to down regulate cholesterol synthesis17 or to increase biliary excretion.18,19 These variations could have a significant genetic component, mediated in part by candidate genes involved in lipoprotein metabolism. The presence of a variant allele may influence the metabolism of cholesterol in a way that deviates from classical understanding.

Gene polymorphisms and response to dietary cholesterol There are very limited studies addressing the effects of different genotypes on the response to dietary cholesterol. One study reported that individuals identified as homozygous for the variant adenosine binding cassette transporter (ABC)G5 allele (G/G) have greater plasma total cholesterol response to dietary cholesterol intake.20 In contrast to this report, we have found This journal is ª The Royal Society of Chemistry 2010

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that individuals possessing the C/C genotype experienced a greater increase in both LDL cholesterol (P < 0.05) and a trend for lutein (P ¼ 0.08) after consuming 3 eggs for one month compared to those individuals with the C/G (heterozygote) or G/G genotypes (homozygotes).21 These results, although obtained from a small number of subjects, suggest that the ABCG5 polymorphism may play a role in the plasma response to dietary cholesterol and carotenoids.21 The effect of the A278-C promoter polymorphism on the rate limiting enzyme of conversion of cholesterol to bile acids, cholesterol 7a-hydroxylase (CYP7A1) was studied in 496 normolipidemic individuals.22 Subjects were challenged with a mean intake of 742 mg per day of dietary cholesterol for 3–4 weeks. All subjects had a significant increase in HDL-C following the dietary cholesterol challenge. The CYP7A1 polymorphism was found to have a significant effect on the increases in HDL cholesterol.22 However, the APOC3 and APOC4 polymorphisms although associated to plasma lipid parameters, have not been found to be related to the responses to dietary cholesterol.23

Eggs and plasma cholesterol responses in different populations The current dietary recommendations of no more than 300 mg of dietary cholesterol per day pose a controversial issue for those individuals who might derive health benefits by including eggs in their diets while simultaneously, they are not increasing their risk for CHD. An analysis of various cholesterol-feeding studies, conducted over a 50 year period, has produced evidence that a modest increase in total cholesterol of 0.056–0.061 mmol L1 (95% CI 0.051–0.069 mmol L1) can be predicted in response to a 100 mg per day increase in dietary cholesterol.7 If this moderate increase is used as a reference, those who experience elevations in TC higher than 0.061 mmol L1 (95% CI 0.064–0.14 mmol L1) would be classified as hyper-responders to dietary cholesterol. The hypo-responders would be those who experience an increase of <0.036 mmol L1 (95% CI 0.025–0.049 mmol L1).7 Clinical trials conducted in children,24 younger adults25,26 and the elderly27,28 have clearly demonstrated that while hyperresponders, which correspond to 25% of the population exhibit increases in both LDL-C and HDL-C with the result of no changes in the LDL-C/HDL-C ratio. These responses to dietary cholesterol are independent of fat intake.24–28 Recent studies in my laboratory have also shown that during a weight loss intervention, participants consuming 3 eggs per day had no increases in LDL-C while a significant increase in HDL-C was observed.29 In contrast, subjects consuming egg substitutes had no changes in HDL-C following the intervention.29 Further, when lipoprotein particle size and subclasses were analyzed by nuclear magnetic resonance (NMR), significant increases in both LDL and HDL particles were observed as well as an increase in lecithin cholesterol acyl transferase (LCAT) and cholesterol ester transfer protein (CETP) activities.30 These data suggest that dietary cholesterol provided by eggs enhances reverse cholesterol transport. Fig. 1 presents comparisons between subjects consuming 3 whole eggs or the equivalent amount of egg substitutes on different parameters of HDL metabolism.30 This journal is ª The Royal Society of Chemistry 2010

Responses to dietary cholesterol in children Eggs are a central food item in Mexico, which spans all socioeconomic classes. Because the northern part of Mexico is characterized by dyslipidemias conducive to the metabolic syndrome and CHD,31,32 we evaluated the effects of consuming two whole eggs per day compared to egg whites only, on plasma lipids and the atherogenicity of the LDL particle in Mexican children aged 10–12 y.24 We reported that the increases in plasma cholesterol due to dietary cholesterol was present in 1/3 of the children and was associated with increases in both LDL and HDL with no alterations in the LDL-C/HDL-C ratio. However, when we adjusted the body weight to 70 kg, the response to dietary cholesterol was not present in any of the children. In addition, egg consumption resulted in the formation of buoyant LDL particles associated with pattern A. The significance of this finding derives from the high prevalence of pattern B LDL present in this population.33 A predominance of LDL particles in this pattern B sub-class has been shown to be associated with a three-fold increase in CHD risk,34 which may be due to the easy entry of this particle into the arterial wall and its high susceptibility to oxidation. Thus, egg consumption did not alter the LDL-C/HDL-C ratio in these Mexican children and there was a shift of LDL size to a less atherogenic particle. The potential beneficial effects of eggs in children suffering from Smith-Lemli-Opitz syndrome, which is a condition of impaired cholesterol synthesis and birth defects related to mental retardation, was evaluated.35 Dietary cholesterol provided by eggs increased both LDL-C and HDL-C in these subjects suggesting that egg intake may be a potential therapeutic effect for this condition.

Responses to dietary cholesterol in young adults Early reports from the Framingham study show a lack of association between dietary cholesterol and heart disease.36 Other studies conducted in the 1980s demonstrated that there were no differences in plasma cholesterol and triglyceride concentrations in young males consuming either 150 mg per 1000 kcal or 500 mg per 1000 kcal of dietary cholesterol.37 Similar results were reported in young adult males when they ate 400 versus 1400 mg of dietary cholesterol for 4 weeks.38 More recent studies conducted in 40 men aged 20–50 years old25 and in 51 pre-menopausal women, 50% of which were of Hispanic origin26 reported that men and women classified as hypo-responders to dietary cholesterol (70% of the population), had no changes in LDL or HDL-C after consuming 3 eggs per day for 30 days. In contrast, those individuals who were classified as hyper-responders did experience an increase in both LDL-C and HDL-C. In addition, intake of eggs resulted in the production of larger LDL particles with no increased susceptibility to oxidation.39 These results indicate that pre-menopausal women and men with initial plasma cholesterol concentrations that place them at a low risk for CHD do not experience the development of an atherogenic lipoprotein profile following the consumption of additional dietary cholesterol, regardless of their response classification. A study carried out in 18 healthy lacto-vegetarian Indians demonstrated an increase in blood cholesterol in subjects after Food Funct., 2010, 1, 156–160 | 157

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Fig. 1 Comparisons in different parameters of HDL metabolism in subjects who consumed 3 eggs (EGG) or the equivalent of 3 egg substitutes (SUB) at baseline (black bar) and at week 12 (white bar). NS ¼ non significant; * P < 0.05; ** P < 0.001.

4 weeks of consuming one boiled egg per day.40 However, the mean LDL-C and HDL-C were not different after 8 weeks of egg consumption although some hyper-responders still presented elevations in total cholesterol after this period of time.40

Responses to dietary cholesterol in adults By the year 2020, the number of people worldwide over the age of 60 is expected to reach one billion, which suggests that the incidence of age-related disease will continue to increase. The physiologic and economical changes, as well as the increased risk of chronic disease, associated with advancing age places the elderly at an interesting crossroads. Widely accepted risk factors that have been identified for CHD may not be applicable to elderly populations. Although elevated plasma cholesterol concentrations have been shown to predict CHD risk in middleaged individuals, this parameter does not seem to be relevant for the elderly demographic.41 However, low-fat diets are commonly prescribed to many elderly individuals in an attempt to lower elevated cholesterol concentrations. Unfortunately, this can result in an increase in dietary carbohydrates. This change in diet composition can be detrimental, causing increases in triglycerides, which are generally accompanied by low HDL-C levels. Low HDL-C has been identified as the best indicator of CHD risk in elderly individuals.27 Furthermore, the consumption of a diet high in simple sugars can cause changes in lipoprotein metabolism that result in the production of smaller, denser LDL particles.42 In a study conducted in post-menopausal women and men 60 y or older, similar results were observed compared with the younger populations,27,28 no changes in the LDL-C/HDL-C in those individuals classified as hyper-responders and a shift to the larger, more buoyant LDL particle. Furthermore, the increases in HDL were associated with larger HDL particles and the number of LDL particles and apo B concentrations did not change following egg consumption confirming that egg intake resulted in the formation of larger LDL.43 In this study,27 participants were 158 | Food Funct., 2010, 1, 156–160

challenged by eating 3 eggs per day. We decided to investigate the effects of consuming only one egg per day for 12 weeks in a population of subjects aged 40 to 65 y.44 Surprisingly, we observed no increases in LDL cholesterol, while an increase in HDL cholesterol was reported for all subjects43 implying once more the role of dietary cholesterol in promoting reverse cholesterol transport. Similar results were reported in a study conducted in 110 elderly Japanese subjects who were fed additional 750 mg of dietary cholesterol for 4 weeks.28 Subjects experienced a significant increase in HDL-C with no changes in LDL-C at the end of the dietary treatment. However, there were significant increases in the larger LDL particle as well as the less dense HDL (HDL2).28 Different plasma lipid responses (total cholesterol, LDL-C, HDL-C and LDL-C/HDL-C ratio) to egg consumption between hyper and hypo-responders in various studies are summarized in Table 1.

Responses to dietary cholesterol during insulin resistance To evaluate whether insulin resistance with or without obesity influences the response to dietary cholesterol,45 197 healthy subjects participated in a randomized crossover design and were fed 0, 2 and 4 eggs per 4 weeks with one month washout between periods. The subjects were classified as insulin sensitive (IS n ¼ 65), insulin resistant (IR n ¼ 75) and obese insulinresistant (OIR n ¼ 58). IR and IS subjects had a significant increase in LDL-C of 7.8 and 3.3% only after consuming 4 eggs while OIR subjects had no changes in LDL-C when consuming 0, 2 or 4 eggs. In contrast HDL-C was significantly increased for all groups even after the consumption of 2 eggs. These studies suggest that dietary management of OIR individuals should focus more on restricting calories rather than dietary cholesterol.28 This journal is ª The Royal Society of Chemistry 2010

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Table 1 Responses in plasma LDL cholesterol, HDL cholesterol and LDL/HDL cholesterol ratio in diverse populations during the egg period or after 12 weeks of intervention. The data are separated into hyper-responders (Hyper-R) and hypo-responders (Hypo-R)

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LDL-C Population

Intervention

Childrena

2 eggs/d for 4 weeks

Womenb

3 eggs/d for 4 weeks

Menc

3 eggs/d 12 weeks, weight- loss

Men/Womend

1 egg/d for 12 weeks

a

Hyper-R

HDL-C Hypo-R

Hyper-R

LDL-C/HDL-C Ratio Hypo-R

Hyper-R

Hypo-R

Ballesteros et al., 2004 (ref. 24). b Herron et al., 2002 (ref. 26). c Mutungi et al., 2008 (ref. 29). d Ata et al., 2010 (ref. 44).

Eggs and carotenoids Eggs also are major sources of lutein and zeaxanthin, two potent antioxidants, which in addition to their protective effects against macular degeneration and cataract formation,44,46 they may also play a role in decreasing the susceptibility of the LDL particle to oxidation.47 Epidemiological studies indicate an inverse relationship between intake of these carotenoids and both cataract and age-related macular degeneration.47 These carotenoids circulate in plasma mostly carried in the HDL particle. Plasma lutein and zeaxanthin have been shown to increase significantly after egg supplementation in moderately hypercholesterolemic men and women.48 We have demonstrated that in pre-menopausal women and younger men classified as hyperresponders, there was a significant increase in lutein and zeaxanthin compared to those individuals classified as hyporesponders.49 We also found that female hyper-responders had significantly higher plasma lutein concentrations than male hyper-responders following egg consumption despite the finding that the dietary intake of lutein + zeaxanthin was similar between the groups. One explanation for why female hyper-responders had higher plasma lutein levels than men could be found in lipoprotein analysis. Female hyper-responders had significantly higher plasma HDL-C levels (76.0  14.0 mg dL1) than males (48.7  7.8 mg dL1) therefore they experienced the same carotenoid response possibly due to the fact that HDL is the major lipoprotein transporting lutein in plasma. When LDL and HDL subclasses were evaluated in an elderly population, a significant correlation was found between large HDL and lutein and zeaxanthin content following intake of 3 eggs for 4 weeks.43

Conclusion This review of epidemiological data and results from recent clinical trials related to egg consumption present reliable evidence that in multiple populations including children, young adults and the elderly, there are consistent results on both plasma cholesterol distribution in lipoprotein subclasses and on the formation of larger LDL and HDL. The maintenance of the This journal is ª The Royal Society of Chemistry 2010

LDL-C/HDL-C ratio, the presence of more buoyant LDL without increases in the susceptibility of LDL to oxidation, the increased concentration of large HDL and the higher plasma lutein and zeaxanthin seen in these individuals clearly suggest that healthy populations benefit from egg consumption.

Acknowledgements The sole author has the responsibility for all the parts of the manuscript.

References 1 A. H. Lichenstein, L. J. Appel, M. Brands and M. Carnethon, et al. Diet and Lifestyle recommendations Revision 2006, A scientific statement from the American Heart Association Scientific Committee Circulation, 2006, 114, pp. 82–96. 2 American Heart Association, 2004, www.americanheart.org. 3 J. Genest, R. McPherson, J. Frolhlich, T. Anderson and N. Campbell, et al. 2009 Canadian Cardiovascular Society/Canadian guidelines for the diagnosis and treatment of dyslipidemia and prevention of cardiovascular disease in the adult- 2009 recommendations, Can. J. Cardiol., 2009, 25, 567–579. 4 Ministry of Health: Health and Nutrition guidelines for healthy adults, New Zealand: Ministry of Health, 2003,18–21. 5 Task force members: I. Graham, D. Atar and K. Borsch-Johnsen, et al. European guidelines on cardiovascular disease prevention in clinical practice: executive summary. Fourth Joint Task Force of the European Society of Cardiology and Other societies on cardiovascular disease prevention in clinical practice, Eur. Heart J., 2007, 28, 2375–2414. 6 F. B. Hu, M. J. Stampfer, E. B. Rimm and J. E. Manson, et al. A prospective study of egg consumption and risk of cardiovascular disease in men and women, JAMA, J. Am. Med. Assoc., 1999, 281, 1387–1394. 7 W. H. Howell, D. J. McNamara, M. A. Tosca and B. T. Smith, et al. Plasma lipid and lipoprotein responses to dietary fat and cholesterol, Am. J. Clin. Nutr., 1997, 65, 1747–1764. 8 K. L. Esrey, L. Joseph and S. A. Grover, Relationship between dietary intake and coronary heart disease mortality: lipid research clinics prevalence follow-up study, J. Clin. Epidemiol., 1996, 49, 211–216. 9 D. J. McNamara, Dietary cholesterol and atherosclerosis, Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 2000, 1529, 310–320. 10 S. B. Kritchevsky, A review of scientific research and recommendations regarding eggs, J. Am. Coll. Nutr., 23, 596S–600S.

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47 48 49

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Food & Function Downloaded on 10 November 2010 Published on 22 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00110D

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

Volume 1  |  Number 2  |  November 2010  |  Pages 133–224

ISSN 2042-6496

COVER ARTICLE E. Jeffery et al. Glucoraphanin hydrolysis by microbiota in the rat cecum results in sulforaphane absorption

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PAPER

www.rsc.org/foodfunction | Food & Function

Glucoraphanin hydrolysis by microbiota in the rat cecum results in sulforaphane absorption Ren-Hau Lai,a Michael J. Millerab and Elizabeth Jeffery*ab

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Received 6th August 2010, Accepted 21st September 2010 DOI: 10.1039/c0fo00110d In the absence of the plant enzyme myrosinase, such as in cooked broccoli, glucoraphanin is considered to be hydrolyzed by bacteria in the lower gut to produce the bioactive isothiocyanate sulforaphane. Simulated digestion using US Pharmacopeia methods caused no loss of glucoraphanin, confirming that glucoraphanin is not destroyed by digestive enzymes during passage through the digestive tract and is able to reach the rat cecum intact. Introduction of glucoraphanin (150 mmol/kg BW) directly into the cecum resulted in appearance of isothiocyanates in the mesenteric plasma by 120 min. In contrast, introduction of sulforaphane (150 mmol/kg BW) directly into the cecum resulted in the appearance of isothiocyanates in the mesenteric plasma within 15 min. Plasma levels remained constant for over an hour. Anaerobic incubation ex vivo of cecal microbiota from male F344 rats with glucoraphanin resulted in very low levels of the hydrolytic metabolite erucin nitrile, showing that hydrolysis of glucosinolates is carried out by cecal microbiota, but metabolism ex vivo by microbiota did not reflect not reflect metabolism in situ. These data are the first to report direct evidence of hydrolysis of glucoraphanin to sulforaphane in the cecum of rats and to show that sulforaphane is able to cross the cecal enterocyte for systemic absorption.

Introduction In epidemiological studies, vegetable intake has been shown to reduce cancer risk, including cancer of the colon, lung, breast, and prostate.1,2 In particular, cruciferous vegetables may provide greater protective benefit than many other vegetables or fruits.3,4 Broccoli, Brassica oleracea L., is well known as providing a high content of fiber, minerals and vitamins, all of which may aid in reducing carcinogenesis. Broccoli is rich in antioxidants, including ascorbic acid, tocopherols, carotenoids, and flavonoids, which may also play important roles in chemoprotection.5,6 But in addition to these components shared with many other plant foods, crucifers contain glucosinolates, which are considered to play the major role in reduction of cancer risk by crucifers.7 Glucoraphanin (GRP), 4-methylsufinylbutyl glucosinolate, is a major glucosinolate in broccoli and precursor to the bioactive isothiocyanate sulforaphane (SF). The hydrolysis of glucosinolates requires the enzyme myrosinase (EC 3.2.1.147), a thioglucoside glucohydrolase, which is endogenous to the plant and physically separated from glucosinolates.8 However, once raw broccoli undergoes tissue disruption from chewing, the myrosinase gains access to GRP and catalyzes hydrolysis within the gastrointestinal tract. Upon cooking of broccoli, myrosinase is heat inactivated, yet low levels of SF metabolites appear in urine following ingestion of cooked crucifers, suggesting that hydrolysis has occurred.9 Data supporting a role for microbiota include the lack of glucosinolate hydrolysis products in urine of germ-free rats10 and in urine of subjects undergoing bowel cleansing together with antibiotic treatment11 suggesting that rats and humans lack

a thioglucoside glucohyrodrolase and that the gut microbiota are required for GRP conversion to SF. These conclusions are supported by studies of purified GRP metabolism in rats.12 Absorption of SF across the upper intestinal wall (jejunum) has been evaluated in a clinical study where a tube was passed through the stomach to the jejunum and a broccoli soup containing myrosinase was introduced. Sulforaphane and a SFglutathione conjugate (SF-GSH) were later effluxed into the perfusate collected from the jejunum.13 There are no studies directly evaluating GRP or SF metabolism and absorption in the lower gastrointestinal tract (cecum or colon), although this is implied by the appearance of urinary SF metabolites following oral administration of purified GRP to rats.14 Furthermore, there are reports that the rat colon exhibits induced levels of quinone reductase following feeding with purified GRP.15 Our long-term goal is to enhance SF bioavailability, by improving SF formation by gut microbiota. However, it is not yet clear by what mechanism and to what extent GRP is hydrolyzed and/or SF absorbed in the lower gut; both steps are required for systemic bioavailability. It therefore becomes critical to study the relationship between GRP, microbiota and the gastrointestinal tract. The objectives of this study were 1) to evaluate simulated digestion of GRP in the upper gastrointestinal tract; 2) to determine if GRP is hydrolyzed by rat cecal microbiota ex-vivo; 3) to determine if GRP is hydrolyzed in the rat cecum in situ and if the hydrolysis product(s) are absorbed across the cecal wall.

Materials and methods Materials

a

Division of Nutritional Sciences, University of Illinois, 905 S Goodwin Ave, Urbana, IL 61801, USA b Food Science and Human Nutrition, University of Illinois, 905 S Goodwin Ave, Urbana, IL 61801, USA. E-mail: [email protected]

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Semi-purified GRP (30.4% pure) was prepared from Premium Crop broccoli seed (Brassica oleracea L.) as previous described.14 Sulforaphane and SF nitrile standards were purified from Food Funct., 2010, 1, 161–166 | 161

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broccoli seed as previously described.16 Erucin standard was purchased from LKT laboratories (St. Paul, MN). Both MRS and RCM media were purchased from Beckton Dickenson (Franklin Lakes, NJ). Dichloromethane and HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

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Simulated upper gut digestion of glucoraphanin The simulation model of upper gastrointestinal enzymatic digestion was modified from the methods laid out in the US Pharmacopeia.17 Briefly, the major enzymes, salivary a-amylase, gastric pepsin, and intestinal pancreatin, were tested sequentially and in triplicate. First, 0.5 mM GRP was incubated with a-amylase (100 U/mL) at 37  C for 3 min to simulate oral digestion. After withdrawing a 450 mL sample for analysis, pepsin (0.32 : 1 w/v, pepsin:GRP) and concentrated HCl (to bring down the pH to 2.0) were added and incubated at 37  C for 2 h. After 2 h, a 450 mL sample was collected for analysis. The remainder was added to a mixture of 0.8 mg/L pancreatin (8-fold USP specifications) with 25 g/L bile salts mixture (SigmaAldrich), and the pH was titrated to 7.5 with 0.5 N NaHCO3. The incubation time of the simulated intestinal digestion was 2 h. Samples collected from each step of the simulation were hydrolyzed in excess myrosinase to form SF, which was estimated by GC as previously described.16 Animals and treatments Male F344 rats from Harlan Inc. (Indianapolis, IN) were housed individually in hanging wire bottom cages under controlled conditions (12 h light-dark cycle, 22  C, and 60% humidity). They were fed AIN93G diet and tap water ad libitum throughout the study. Food was available 24 h a day and provided fresh daily. Animal use was approved by the Animal Care and Use Committee of the University of Illinois. Animals were acclimated for three days, during the last two days of which all animals received saline daily by gavage. Six male F344 rats, weighing 235  4.1 g (mean  SD) were used for in situ cecal metabolism. Rats were anesthetized using ketamine: xylazine (87 mg/mL: 13 mg/mL; 1.0 mL/kg body weight). Anesthesia was maintained with ketamine, 30% of the starting dose, given hourly as necessary. The surgical procedure was modified from Johnson et al.18 Briefly, the lower gastrointestinal tract was exposed by a midline incision. The gut was closed by surgical thread ties at the cecal-colonic junction to prevent flow from the cecum to the colon. Sulforaphane or GRP (150 mmol/kg body weight) was injected into the cecum through the ileum, which was immediately closed at the ileocecal junction, using surgical thread. A catheter was installed into a mesenteric vein 2–3 cm from the cecum (20G x 200 , Exelint Inc., Los Angeles, CA). Before the installation, catheters and syringes were flushed with heparinized saline (25 U/mL) to prevent clotting. Blood samples (approximate 400 mL) were collected at 15, 30, 45, 60 min from the animals administered SF and at 30, 60, 90, 120, 150 min from the animals administered GRP. During surgery, a heating pad was used to maintain body temperature, and saline-soaked gauze was placed over the 162 | Food Funct., 2010, 1, 161–166

exposed gut to maintain hydration. Blood samples were placed on ice until centrifugation at 1000 x g at 4  C for 12 min to separate the plasma (Eppendorf Centrifuge 5415D; Westbury, NY). The plasma was then immediately collected and frozen at 80  C until analysis. Total isothiocyanates (isothiocyanates and their glutathione metabolites) were measured by a modification of the cyclocondensation assay,19 as previously described.20 For hydrolysis ex vivo, rats were administrated either saline (control group, n ¼ 6) or 150 mmol GRP/kg body weight by gavage daily for 4 days (GRP pretreated group, n ¼ 6), since pretreatment might stimulate metabolism. The rats were fasted for 16 h prior to termination and killed 24 h after the last treatment. The abdominal cavity was opened by a midline incision to expose the gastrointestinal tract and the cecum was ligated and excised proximally and distally to the ileal and colonic ties, respectively, to maintain anaerobic conditions. Ceca were immediately transported in an air-tight bag at 37  C to an anaerobic chamber (Coy, Grass Lake MI; 90% N2, 5% H2 and 5% CO2). Inside the anaerobic chamber, cecal contents were removed and diluted with sterile distilled water, 1 : 10 (w/v). After mixing well, 100 mL samples were diluted into 10 mL medium (MRS, RCM, or 0.5 mM phosphate buffer pH 6.8; see below for choice of media) and divided into 5 aliquots for anaerobic incubation at 37  C for 0, 3, 6, 12, or 24 h. After measuring the pH, incubations were stopped, and microbiota were removed by centrifugation (6000 x g for 10 min) at 4  C followed by filtration, using a 0.2 mm syringe filter, and stored at 80  C until analysis of GRP, isothiocyanate and nitrile.

Choice of media for glucosinolate metabolism by cecal microbiota In order to support growth of rat cecal bacterial strains with the greatest potential for GRP hydrolysis, we performed a search for bacterial genes most similar to myrosinase from white mustard (thioglucoside glucohydrolase, EC3.2.1.147, Sinapis alba). Using the Basic Local Alignment Search Tool (BLAST), we probed the bacterial protein database archived at the National Center for Biotechnology Information, against the known amino acid sequence of myrosinase.21 An integrated framework analysis for identification and characterization of taxonomic and phenotypic variation of enzymes (CHISEL) was then used to identify which bacteria contained the best matches to white mustard myrosinase.

Statistical analysis For the incubation of cecal microbiota ex vivo, significant differences (p # 0.05) in metabolism between microbiota from rats gavaged with saline (control group) and with GRP (pretreated group) were determined by Student’s t-test using SAS software (SAS, Inc., Cary, NC) at each time. For the cecal metabolism of GRP in situ, samples from each time point were compared to zero time, and a p-value # 0.05 was accepted as significantly different. This journal is ª The Royal Society of Chemistry 2010

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Results Simulated gastrointestinal digestion of glucoraphanin No digestive enzymes had any effect on GRP in vitro. The recovery of GRP following simulated oral/ amylase digestion (100.5  2.5%), gastric/HCl-pepsin digestion (100.7  0.7%), and intestinal/pancreatin-bile digestion (101.3  1.2%) were not different from the original dose (Fig. 1).

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Cecal metabolism of glucoraphanin in situ In order to determine if SF can be absorbed from the cecum, we injected purified SF directly into the cecal lumen and measured mesenteric plasma levels of total isothiocyanate (free isothiocyanate plus glutathione metabolites). Total isothiocyanate increased from 0 to 105 mM by 15 min, the first time point (Fig. 2). Blood samples were collected every 15 min for the next 45 min, and total isothiocyanate levels were not statistically different from the level at 15 min. Although only one animal survived surgery past 60 min, this last plasma sample contained a total isothiocyanate level very similar to the concentration at 15 min. Plasma total isothiocyanate levels in rats administered GRP (150 mmol/kg body weight) showed a very different pattern. Mesenteric plasma total isothiocyanate levels did not rise significantly until 120 min (Fig. 3). At 120 min, plasma total isothiocyanate levels rose to 9.1  1.8 mM, less than 10% of the value following SF administration. Only one animal remained alive until 150 min after GRP administration, and plasma levels were 10.9 mM, very similar to the 120 min value.

Fig. 3 Total isothiocyanate equivalents in mesenteric plasma of rats treated with Glucoraphanin. Rats were dosed with 150 mmol GRP/kg BW directly into the cecum. Values were compared to the zero time value using the Student’s t-test. The asterisk indicates significantly different from 0 h (p # 0.05). Mean  SE, n ¼ 3 (n ¼ 1 at 150 min).

Choice of media for support of glucosinolate metabolism by cecal microbiota CHISEL analysis identified 7 taxonomy-specific clusters for thioglucoside glucohydrolase from domains in either Eukarya or Bacteria (Table 1). The two most similar models SF001079-4-BFirmicutes (phylum which includes Lactobacillus, Clostridium and many other genera) and SF001079-5-B-Lactobacillales, (order which includes Lactobacillus species), include bacterial species abundant in the human gastrointestinal tract. The specific proteins identified from these two models are members of the glycoside hydrolase family 1.22 Activities associated with the glycoside hydrolase family 1 include beta-glucosidase, betagalactosidase, 6-phospho-beta-galactosidase, 6-phospho-betaglucosidase, lactase-phlorizin hydrolase, and beta-mannosidase activities. From these data, we chose to perform microbial metabolism ex-vivo using media that support lactobacilli and clostridia growth for GRP metabolism. Glucoraphanin metabolism by cecal microbiota ex-vivo Body weight and food intake of GRP-pretreated animals were not different from the control, saline gavaged rats (data not shown). Cecal microbiota were transferred anaerobically, as

Fig. 1 Effect of digestive enzymes on glucoraphanin recovery. Glucoraphanin (0.5 mM) was incubated with amylase, HCl/pepsin then pancreatin/bile salts and samples taken to determine oral, gastric and intestinal recovery of glucoraphanin, respectively. Mean  SE, n ¼ 3.

Table 1 Bacteria identified as containing proteins similar to thioglucoside glucohydrolase (myrosinase) from brassica Model

Description

Score

E-Value

SF001079_2_E_ Brassicaceae SF001079_3_E_ Magnoliophyta SF001079_4_B_ Firmicutes SF001079_5_B_ Lactobacillales

3.2.1.147 thioglucosidase (Eukaryotic version) 3.2.1.21 beta-glucosidase (Eukaryotic version) 3.2.1.21 beta-glucosidase (Bacterial version) 3.2.1.86 6-phospho-betaglucosidase (Bacterial version) 3.2.1.86 6-phospho-betaglucosidase (Bacterial version) 3.2.1.85 6-phospho-betagalactosidase (Bacterial version) 3.2.1.21 beta-glucosidase (Bacterial version)

1447.8

0

663.8

1.1e198

329.6

4.3e98

127.0

4.3e37

96.3

7.6e28

91.0

2.9e26

23.3

6.9e06

SF001079_5_B_ Gammaproteobacteria Fig. 2 Total isothiocyanate equivalents in mesenteric plasma of rats treated with Sulforaphane. Rats were dosed with 150 mmol SF/kg BW directly into the cecum. Values were compared to the zero time value using the Student’s t-test. The asterisk indicates significantly different from 0 h (p # 0.05). Mean  SE, n ¼ 3 (n ¼ 1 at 60 min).

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SF001079_6_B_ Bacilli SF001079_1_B_ Bacilli

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Table 2 The change in pH and glucoraphanin levels during glucoraphanin metabolism by rat cecal microbiota incubated in MRS, RCM or buffered medium pH

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Control MRS 0h 6.30  0.06 3h 5.39  0.21 6h 4.55  0.25 12h 4.08  0.08 24h 4.01  0.03 RCM 0h 6.27  0.12 3h 5.31  0.18 6h 4.81  0.18 12h 4.64  0.09 24h 4.71  0.07 Buffered 0h 6.89  0.01 3h 6.76  0.02 6h 6.75  0.01 12h 6.74  0.01 24h 6.75  0.01

Glucoraphanin (mM) GRP Pretreated

Control

GRP Pretreated

6.33  0.07 5.46  0.26 4.65  0.27 4.12  0.09 4.03  0.05

0.49  0.42  0.26  0.28  0.30 

0.01 0.05 0.04 0.04 0.05

0.46  0.01 0.37  0.03 0.21  0.02 0.19  0.03a 0.21  0.03a

6.36  0.12 5.31  0.18 4.82  0.18 4.66  0.09 4.72  0.08

0.50  0.38  0.17  0.07  0.00 

0.00 0.10 0.04 0.02 0.00

0.48  0.00 0.23  0.11 0.08  0.05 0.03  0.01a 0.02  0.02

6.88  0.02 6.76  0.01 6.75  0.02 6.75  0.01 6.76  0.01

0.48  0.29  0.02  0.01  0.01 

0.01 0.05 0.00 0.00 0.00

0.44  0.00 0.16  0.08a 0.02  0.00 0.01  0.00 0.01  0.00

a Glucoraphanin content remaining at termination of metabolism was significantly different in cecal samples from glucoraphanin-pretreated rats compared to samples from control rats, (p # 0.05).

described in methods, and incubated in an anaerobic chamber with GRP (0.5 mM) in MRS medium (supports growth of lactobacilli and others) or RCM medium (supports growth of clostridia, bifidobacteria and others). The pH was recorded at the end of the incubation (Table 2). The pH, an indicator of microbial growth, dropped to 4.0 in MRS and to 4.7 in RCM medium by 24 h. In the MRS medium, GRP hydrolysis in microbiota from the GRP-pretreated rats was slightly but significantly greater than from the control group at both 12 and 24 h (p # 0.05; Table 2), suggesting that there was some change in the microbial population toward improved metabolism of GRP. A similar pattern was seen in GRP metabolism in microbiota grown in RCM medium. However, the effect of GRP pre-treating was smaller than that in MRS medium and only significant at 12 h (Table 2). Sulforaphane was found as product in only one cecal incubation in MRS medium, and in no RCM incubations. The SF-positive incubation was from a control animal, and the level of SF in that sample was 0.16 mM at 3 h, decreasing to 0.07 mM by 24 h. In addition, a small peak identified as erucin nitrile was found in all samples, increasing over time in the MRS medium, but remaining very small (data not shown). We have previously identified erucin nitrile by GC/MS, but have not developed quantification and no erucin nitrile standard is commercially available.12 Nitriles are alternative products to isothiocyanates, but they lack bioactivity.16 Because a low pH environment supports nitrile formation during myrosinase-dependent metabolism of glucosinolates, we considered that metabolism in buffer, to maintain neutral pH, might support greater isothiocyanate formation. Using phosphate buffer (pH 6.8) as the medium, the pH was stable throughout the 24 h incubation (Table 2). The microbiota from GRP-pretreated rats were significantly more able (p # 0.05) to support GRP disappearance (metabolism) at 3 h, compared to 164 | Food Funct., 2010, 1, 161–166

GRP metabolism in microbiota from control rats (Table 2). At 24 h, almost 100% of the GRP was metabolized by the microbiota from both the GRP-pretreated and the control rats. However, even with the pH controlled, erucin nitrile was the only detectable hydrolysis product.

Discussion Digestive enzymes from the upper gastrointestinal tract did not destroy GRP. Not all glucosinolates respond similarly, since progoitrin and sinigrin, unsaturated aliphatic glucosinolates, are reported to be lost during digestion. A study feeding pigs rapeseed showed that only 60% of intact glucosinolates (mostly progoitrin) reached the colon, measured by examining the ileal digesta.23 Another study found that sinigrin was either absorbed or destroyed during passage through the rat gastrointestinal tract, so that total AITC in colon and cecum could only account for 55% of the dose administrated.24 However, simulated digestion is far less complex than an animal feeding study and a variety of factors could affect recovery in vivo, such as absorption of intact glucosinolates.12 Reports of effects of digestive enzymes on specific glucosinolates in vitro also suggest that individual glucosinolates are differently affected.25,26 Our data are the first to indicate that GRP is not degraded by upper gastrointestinal digestive enzymes in vitro. The doses of GRP and SF chosen to study cecal metabolism in situ approximated an oral dose of 60 mg GRP/kg body weight, which we have shown is effective (colonic quinone reductase was upregulated) but not toxic to the cecum.14,15 When SF was introduced into the cecum, SF/ isothiocyanates were found in the mesenteric blood within 15 min. Most broccoli feeding and SF dosing studies report peripheral blood levels and cannot be compared to the present study.27,28 In a study that sampled mesenteric blood during constant infusion of SF (10 mM) through a portion of the ileum of Sprague-Dawley rats, comparably early appearance of SF and metabolites in plasma was reported.29 That study estimated SF and glutathione metabolites separately, by LC-MS/MS, and found that most SF was conjugated rather than free, showing that SF metabolism had occurred in the enterocyte during absorption.29 The mechanism of SF absorption at the enterocyte could be passive or mediated by transporters, such as multi-drug resistance associated proteins (MRP) or P-glycoprotein.30 Sulforaphane has been shown to upregulate MRP-1 and -2 in human cells, suggesting that pretreating with broccoli/SF can alter SF uptake.31,32 Active transport was also suggested by Petri and colleagues, who found SF-GSH efflux into the jejunum of humans given a broccoli soup by feeding tube.13 Athough our data are consistent with an active transport system at the cecum, with mesenteric plasma total isothiocyanates constant for an hour or more, there are insufficient data to determine if transport is active or passive (Fig. 2). Although the analysis we used does not allow us to differentiate between SF, other isothiocyanates and glutathione metabolites of isothiocyanates, it was chosen because it captures total SF and SF metabolites and because it is sufficiently sensitive to be used with plasma samples of less than 200 mL.33 The environment inside the cecum is delicate, and the microbiota are sensitive to even slight changes, including nutrients, temperature or oxygen tension. Specialized media have been This journal is ª The Royal Society of Chemistry 2010

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Fig. 4 Scheme of possible routes for glucoraphanin hydrolysis to form erucin nitrile.

developed to support optimal growth of specific bacteria, although there is no reliable method to provide nutrient support of the entire mixed population of microbiota within the cecum. A comparison of bacterial enzymes to the amino acid sequence of white mustard myrosinase suggested that lactobacilli and clostridia might be most likely to hydrolyze glucosinolates (Table 1). MRS medium is widely used to support the growth of lactobacilli,34 whereas RCM is used to support growth of clostridia and bifidobacteria, both of which are abundant gut microorganisms.35,36 A GRP dose (0.5mM) was almost completely metabolized in RCM by 12 h, whereas almost 50% remained in the MRS medium, even at 24 h, indicating a possible key role for clostridia or bifidobacteria in GRP hydrolysis. Unexpectedly, the 0.5 mM phosphate buffer pH 6.8 ‘‘control’’ medium supported the most robust GRP metabolism, depleting the GRP to less than 5% by 6 h, possibly suggesting the importance of pH maintenance for activity of a bacterial thiohydrolase, and thus for metabolism of GRP. Pretreating rats with GRP resulted in only slight, although significant, improvement in GRP metabolism in both MRS and RCM media, suggesting that four days’ treatment with GRP was unable to greatly alter the microbial profile toward one with improved GRP metabolism. In a study where subjects ate 14 g crucifers/kg body weight/day for 14 days, there were measurable changes in the profile of microbiota.37 Cruciferous vegetables typically consist of 40% of dry weight as fiber which could have altered the profile.37 Future studies comparing metabolism of GRP in digesta from rats administered broccoli and GRP may be able to distinguish between effects of GRP and broccoli fiber on GRP metabolism. Hydrolysis of glucoraphanin was supported by microbiota, although the only hydrolysis product present in all incubations was erucin nitrile (Fig. 4). The isothiocyanate erucin, the reduced form of SF, has similar bioactivity to SF.38 Reduction of SF to erucin occurs following i.p. administration of SF.39 Erucin nitrile could be formed by reduction of SF nitrile in the anaerobic environment of the cecum or by hydrolysis of glucoerucin (Fig. 4). When rats were dosed with GRP, the reduced glucosinolate glucoerucin was found in urine.12 Also, incubation of GRP with E. coli resulted in glucoerucin appearance This journal is ª The Royal Society of Chemistry 2010

concomitantly with GRP disappearance; at the completion of 24 h, the major hydrolytic product was erucin nitrile (data not shown). Therefore, it is possible that GRP was reduced to glucoerucin and hydrolyzed to erucin nitrile. One cecal preparation (from a control rat) displayed more rapid metabolism of GRP than other preparations, regardless of the medium used. Whereas erucin nitrile was formed in all media, incubation in MRS medium also resulted in SF formation, accounting for 50% of the GRP disappearance at 3 h. However, much of the SF was lost by 24 h. When SF was incubated in MRS medium for 24 h, even without lactobacilli, there was a loss of almost 50% of the SF during this time (data not shown). A similar lack of stability has previously been reported for allyl isothiocyanate in MRS medium.40 However, when SF was incubated at room temperature in buffer, recovery was 100% at 24 h (data not shown). Since SF was not lost from the buffered incubations, it appears unlikely that the cause for lack of SF appearance in most incubations was SF degradation. Nitrile is less labile than isothiocyanates, decreasing less than 20% during 48 h incubation, providing possible rationale for our finding nitrile in our incubations.41 Overall, results from incubation of GRP with microbiota ex vivo show that microbiota in the gastrointestinal tract have the ability to hydrolyze GRP but, as with other reports of in vitro studies of glucosinolate metabolism by microbiota, these studies did not show abundant isothiocyanate formation. Furthermore, metabolism ex vivo did not reflect metabolism in situ, where SF was formed.

Conclusion This study provides direct evidence that GRP was hydrolyzed by F344 rat cecal microbiota, both in situ and ex vivo. It is known that the unstable intermediate formed during glucosinolate hydrolysis can undergo non-enzymatic rearrangement to a nitrile, particularly in an environment of low pH when ferrous iron is present (Fig. 4). This may explain why nitrile is the main hydrolytic product in MRS and RCM incubations, particularly later in the incubation when bacterial growth and metabolism have lowered the pH. This is the first study to show direct Food Funct., 2010, 1, 161–166 | 165

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evidence of GRP hydrolysis to bioactive SF and SF absorption across the cecal wall of rats.

Acknowledgements Thanks to H. Gaskins and G. Rivera for support in running the database searches. This work was supported by a grant from USDA/ AFRI, NIFA 2009-02961.

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17 United States Pharmacopeial Convention Inc., The United States Pharmacopeia, United States Pharmacopeial Convention Inc., Rockville, MD, 2000. 18 B. M. Johnson, W. Chen, R. T. Borchardt, W. N. Charman and C. J. Porter, J. Pharmacol. Exp. Ther., 2003, 305, 151–158. 19 Y. Zhang, K. L. Wade, T. Prestera and P. Talalay, Anal. Biochem., 1996, 239, 160–167. 20 J. Cramer and E. H. Jeffery, Nutrition and Cancer, submitted. 21 L. Rask, E. Andreasson, B. Ekbom, S. Eriksson, B. Pontoppidan and J. Meijer, Plant Mol. Biol., 2000, 42, 93–113. 22 B. Henrissat, Biochem. J., 1991, 280(Pt 2), 309–316. 23 I. Maskell, University of Newcastle upon Tyne, 1990. 24 L. Elfoul, S. Rabot, N. Khelifa, A. Quinsac, A. Duguay and A. Rimbault, FEMS Microbiol. Lett., 2001, 197, 99–103. 25 I. Maskell and R. Smithard, Br. J. Nutr., 1994, 72, 455–466. 26 H. G. M. Tiedink, C. E. Malingre, L. W. van Broekhoven, W. M. F. Jongen and G. R. Fenwick, J. Agric. Food Chem., 1991, 39, 922–966. 27 L. Ye, A. T. Dinkova-Kostova, K. L. Wade, Y. Zhang, T. A. Shapiro and P. Talalay, Clin. Chim. Acta, 2002, 316, 43–53. 28 A. V. Gasper, A. Al-Janobi, J. A. Smith, J. R. Bacon, P. Fortun, C. Atherton, M. A. Taylor, C. J. Hawkey, D. A. Barrett and R. F. Mithen, Am. J. Clin. Nutr., 2005, 82, 1283–1291. 29 S. Agrawal, B. Winnik, B. Buckley, L. Mi, F. L. Chung and T. J. Cook, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2006, 840, 99–107. 30 B. Holst and G. Williamson, Nat. Prod. Rep., 2004, 21, 425–447. 31 J. R. Bacon, G. W. Plumb, A. F. Howie, G. J. Beckett, W. Wang and Y. Bao, J. Agric. Food Chem., 2007, 55, 1170–1176. 32 K. E. Harris and E. H. Jeffery, J. Nutr. Biochem., 2007, 19, 246–254. 33 Y. Zhang, C. G. Cho, G. H. Posner and P. Talalay, Anal. Biochem., 1992, 205, 100–107. 34 Y. Sawatari, T. Hirano and A. Yokota, J. Gen. Appl. Microbiol., 2006, 52, 349–356. 35 S. Kajiwara, H. Gandhi and Z. Ustunol, J. Food Prot., 2002, 65, 214–218. 36 S. C. Long, P. C. Arango and J. D. Plummer, Can. J. Microbiol., 2005, 51, 413–422. 37 F. Li, M. A. Hullar, Y. Schwarz and J. W. Lampe, J. Nutr., 2009, 139, 1685–1691. 38 R. Munday and C. M. Munday, J. Agric. Food Chem., 2004, 52, 1867–1871. 39 K. Kassahun, M. Davis, P. Hu, B. Martin and T. Baillie, Chem. Res. Toxicol., 1997, 10, 1228–1233. 40 B. Combourieu, L. Elfoul, A. M. Delort and S. Rabot, Drug Metab. Dispos., 2001, 29, 1440–1445. 41 D. L. Cheng, K. Hashimoto and Y. Uda, Food Chem. Toxicol., 2004, 42, 351–357.

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Food & Function Downloaded on 10 November 2010 Published on 21 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00105H

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

Volume 1  |  Number 2  |  November 2010  |  Pages 133–224

ISSN 2042-6496

COVER ARTICLE H. Ashida et al. Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes

2042-6496(2010)1:2;1-C

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PAPER

www.rsc.org/foodfunction | Food & Function

Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes Manabu Ueda,a Takashi Furuyashiki,a Kayo Yamada,a Yukiko Aoki,a Iwao Sakane,b Itsuko Fukuda,c Ken-ichi Yoshidaa and Hitoshi Ashida*a

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Received 31st July 2010, Accepted 13th October 2010 DOI: 10.1039/c0fo00105h In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 mM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. ()-Epicatechin (EC) and ()-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the doseand time-dependent manner, whereas ()-catechin 3-gallate (Cg) and ()-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and timedependent manner. When the cells were treated with 50 mM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCl/z without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 30 -kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCl/z in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.

Introduction Glucose transporters (GLUTs) are membrane proteins and play a role in transporting blood glucose into various tissues.1 GLUT1 is the most ubiquitous form expressed in brain, endothelial cells, erythrocytes, kidney, skeletal muscle and adipose tissue, and its expression level is usually related to the rate of cellular glucose metabolism. GLUT4 locates endoplasmic reticulum in skeletal and cardiac muscle and adipose tissue, and is translocated to the plasma membrane upon stimulation with binding of insulin to insulin receptor (IR) and subsequent activation of various protein kinases.2 In muscle and adipose tissue, GLUT4 translocates rapidly to the plasma membrane following insulin action, though more than 95% of GLUT4 is sequestered to intracellular compartment in the absence of insulin.3 Therefore, GLUT4 plays an important role in the maintenance of the postprandial blood glucose level by the insulin-dependent action. Insulin-induced translocation is initiated by the binding of insulin to IR a-subunit, resulting in phosphorylation of tyrosine residues in the SH2 domain of IR b-subunit (IRb).4 The activated IRb phosphorylates insulin receptor substrate-1 followed by phosphorylation of the p85 regulatory subunit of a Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe, 657-8501, Japan. E-mail: [email protected]; Fax: +81 78 803 5878 b Central Research Institue, Ito En Ltd., Megami, Makinohara, Shizuoka, 421-0515, Japan c Research Center for Food Safety and Security, Graduate School of Agricultural Science, Kobe University, Kobe, 657-8501, Japan

This journal is ª The Royal Society of Chemistry 2010

phosphatidylinositol 30 -kinase (PI3K) that induces phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). In turn, PDK1 phosphorylates Akt (also termed protein kinase B: PKB). Akt has three isoforms (Akt1–3, also known as PKB a, b, and g) in mammals, each encoded by a different gene. Akt2 rather than the Akt1 or Akt3 isoform appears to control GLUT4 trafficking in muscle cells and adipose tissue.5 Substantial evidence indicates that protein kinase C l and z isforms (PKCl/z) act downstream of PDK1 to relay insulin signals for GLUT4 translocation. It has been reported that treatment with a PKC inhibitor or the transfection of kinase-inactive forms of PKC z and l in 3T3-L1 cells decreased the insulin-induced translocation of GLUT4.6 A recent study showed that RNAi-induced knockdown of PKC l impaired the glucose uptake activity in 3T3-L1 cells.7 These results indicate that GLUT4 translocation is regulated by a complex cascade consisting of various protein kinases. Tea is one of the most popular beverages consumed worldwide8,9 and tea leaves originate from the plant Camellia sinensis. Tea contains various polyphenols such as catechins, teaflavins, flavanols, and phenolic acids.10 Especially, green tea contains abundant catechins; the major tea catechins are ()-epigallocatechin gallate (EGCg), ()-gallocatechin 3-gallate (GCg), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECg), ()-epicatechin (EC), ()-catechin 3-gallate (Cg), (+)-gallocatechin (GC), and (+)-catechin (C). Green tea has been regarded as possessing health promoting effects: recent studies demonstrated that green tea and tea catechins have anti-tumor,11 antiinflammatory,12 and anti-oxidative effects.13 Moreover, it has been reported that green tea and tea catechins have an Food Funct., 2010, 1, 167–173 | 167

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anti-obesity effect.14 Our previous report demonstrated that the intake of green tea for 3 weeks increased glucose uptake activity accompanied by the translocation of GLUT4 in skeletal muscle of male Wistar rats, while it decreased glucose uptake activity and the translocation in adipose tissue.15 Moreover, we showed that catechins stimulated glucose uptake activity in L6 myotubes and EGCg promoted the translocation of GLUT4 in skeletal muscle at physiological concentrations through activation of PI3K.16 However, it was unclear how green tea decreased glucose uptake activity and inhibited the translocation of GLUT4 in adipose tissue. Therefore, in this study, we investigated the effects of tea catechins on glucose uptake activity and the mechanism by which catechins modulate the translocation of GLUT4 in 3T3-L1 cells.

Materials and methods Chemicals and antibodies Catechins were obtained from Kurita Kogyo (Tokyo, Japan). For the glucose uptake assay, [3H]-3-O-methyl-D-glucose (3OMG) was obtained from DuPont/NEN Research Products (Boston, MA). For the Western blot analysis, anti-GLUT4 goat IgG, anti-GLUT1 goat IgG, anti-b-actin mouse IgG, anti-IRb rabbit IgG, anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), anti-Akt rabbit IgG, anti-PKCl rabbit IgG, and anti-phospho-PKCl/z rabbit IgG from Cell Signaling Technology, Ins. (Danvers, MA), anti-PY20 mouse IgG from Transduction Laboratories Ltd. (San Diego, CA), and antiphospho-Akt rabbit IgG from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of the highest grade available commercially. Fractionation of green tea extract Dried green tea leaves (Camellia sinensis) (500 g) were extracted with ethanol, and the extracts were separated into six fractions as described.17 Briefly, green tea leaves were percolated with 75% ethanol (F1), and the extract was partitioned between each organic solvent and water after the extract had been dried by evaporation in vacuo. Step-wise partition was carried out by using hexane (F2), chloroform (F3), ethyl acetate (F4), and nbutanol (F5) against the ethanol extract. Cell culture and glucose uptake assay Cultures and the differentiation of 3T3-L1 cells were performed as described.18 The differentiated adipocytes on the 24-well plate were serum-starved for 18 h in DMEM containing 0.2% BSA at 37  C. The cells were incubated with various catechins at 30 mM for 30 min in the presence or absence of 100 nM insulin in KrebsRinger phosphate-HEPES buffer (KRH; 50 mM HEPES, pH 7.4, 137 mM NaCl, 4.8 mM KCl, 1.85 mM CaCl2, and 1.3 mM MgSO4). Then a final concentration of 6.5 mM (0.5 mCi) [3H]-3OMG was added and incubated for 1 min at 37  C. As positive and negative controls, 100 nM insulin and DMSO (final concentration, 0.1%) as a vehicle were given to the cells for 15 min, respectively. The uptake was terminated by immediate washing the adipocytes four times with ice-cold KRH, and the 168 | Food Funct., 2010, 1, 167–173

cells were solubilized with a 0.5% SDS solution. Non-specific uptake was measured in the presence of 20 mM cytochalasin B as an inhibitor of glucose transporters. The radioactivity was measured by a liquid scintillation counter with a scintillation cocktail. Immunoprecipitation and immunoblotting The differentiated adipocytes were incubated with 50 mM EC and EGC in the absence of insulin, and Cg and EGCg in the presence of 100 nM insulin for 30 min. The membrane fraction and cell lysate were prepared as described.19 To detect phosphorylation of IR, the cell lysate (300 mg) was incubated with 20 mg of a 50% protein A/G plus-agarose suspension (Santa Cruz Biotechnology) for 1 h at 4  C and centrifuged to remove the agarose resin to which was bound the non-specific protein. The supernatant was incubated with 5 ml of anti-PY20 overnight at 4  C. A new 20 mg of the protein A/G plus-agarose suspension was added to this mixture, and incubated for another 1 h at 4  C. After the agarose resin was washed four times with an ice-cold RIPA buffer [10 mM Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.5 mM 1 mM phenylmethylsulfonyl floride (PMSF), 10 mg ml1 leupeptin, 1 mM Na3VO4, 10 mM NaF, and 0.5 mM dithiotheritol (DTT)], it was subjected to SDS-PAGE followed by Western blot analysis to detected phosphoryated IR. To detect GLUT4 translocation, the plasma membrane fraction was subjected to Western blot analysis as described.19 To detect the expression and phosphorylation of proteins involved in the insulin signaling pathway, cell lysate was subjected SDS-PAGE followed by Western blotting. Statistical analysis Statistical analyses were performed with Student’s t-test. Significance was defined as p< 0.05.

Results Effects of tea catechins on glucose uptake activity in 3T3-L1 cells We investigated the effects of tea catechins on glucose uptake activity in the cells because green tea, which contains variously polyphenols especially catechins, modulates glucose uptake activity in vivo.15 The cells were incubated with various catechins at 50 mM in the presence or absence of 100 nM insulin. In the absence of insulin, C, EC, GC, EGC and EGCg increased glucose uptake activity significantly, approximately 2.2-, 3.5-, 3.1-, 3.5-, and 2.2-fold, respectively (Fig. 1A) compared with DMSO as a negative control. In the presence of insulin, Cg, ECg, GCg, and EGCg decreased insulin-induced glucose uptake activity significantly, approximately 0.2-, 0.5-. 0.5-, and 0.6-fold, respectively (Fig. 1B) compared with insulin as a positive control. EC and EGC, the two most effective compounds, increased the activity in a dose- and time-dependent manner (Fig. 2A, B), and treatment with no less than 5 mM for 30 min and with 50 mM for no less than 5 min had a significant effect. Cg and EGCg decreased the insulin-induced activity in a dose- and timedependent manner (Fig. 2C, D) and treatment with no less than 5 mM for 30 min and with 50 mM for no less than 1 min had This journal is ª The Royal Society of Chemistry 2010

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Fig. 1 Effect of catechins on glucose uptake activity in the absence or presence of insulin in 3T3-L1 cells. The cells were treated with 50 mM catechins in the absence (A) or precsence (B) of insulin for 30 min. Values are calculated relative to DMSO-treated cells (A) or insulin-treated cells (B) and expressed as the means  SE (n ¼ 4). * Significant difference from the DMSO-treated cells (A) or insulin-treated cells (B), p < 0.05 by Student’s t-test. C, Catechin; EC, epicatechin; GC, gallocatechin; EGC, epigallocatechin; Cg, catechin gallate; ECg, epicatecin; GCg, gallate gallocatechin gallate; and EGCg, epigallocatechin gallate.

a significant effect. These results suggested that nongallate-type catechins increased glucose uptake activity in the absence of insulin whereas gallate-type catechins decreased insulin-induced activity in 3T3-L1 cells. Interestingly, EGCg revealed both effects.

The effects of tea catechins on the translocation of GLUT4 in 3T3-L1 cells We further investigated the effects of EC, EGC, Cg, and EGCg on the translocation of GLUT4 to the plasma membrane in 3T3L1 cells. In the absence of insulin, EC and EGC at 50 mM significantly increased the amount of GLUT4 in the plasma membrane fraction compared with DMSO as the vehicle control (Fig. 3A). On the other hand, in the presence of 100 nM insulin, This journal is ª The Royal Society of Chemistry 2010

Fig. 2 Dose- and time-dependent effect of selected catechins on glucose uptake activity in 3T3-L1 cells. The cells were treated with EC and ECG at the indicated concentration for 30 min (A) or at 50 mM for the indicated period (B) in the absence of insulin. The cells were treated with Cg and EGCg at the indicated concentration for 30 min (C) or at 50 mM for the indicated period (D) in the presence of insulin. Values are calculated relative to the DMSO-treated cells (A, B) or insulin-treated cells (C, D) and expressed as the mean  SE (n ¼ 4). * Significant difference from the DMSO-treated cells (A, B) or insulin-treated cells (C, D), p < 0.05 by Student’s t-test.

Cg and EGCg at 50 mM significantly decreased the amount of GLUT4 in the plasma membrane fraction compared with insulin as the positive control (Fig. 3A). As to the GLUT1, the amount of it on the plasma membrane fraction was remained unchanged. Moreover, these catechins did not affect the expression levels of GLUT4 in the cells (Fig. 3B). Thus, nongallate-type catechins promoted the translocation of GLUT4, whereas gallate-type catechins decreased the insulin-induced translocation.

The effects of tea catechis on the insulin signaling pathway in 3T3-L1 cells Since the insulin-induced translocation of GLUT4 requires the activation of several proteins in the insulin signaling pathway, we investigated the activation of IRb, Akt, and atypical PKC in 3T3L1 cells after treatment with EC, EGC, Cg, and EGCg. Although, EC and EGC did not affect phosphorylation of IRb, and its downstream Akt (Fig. 4), they significantly phosphorylated PKCl/z compared with DMSO-treated cells. In Food Funct., 2010, 1, 167–173 | 169

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Fig. 3 Effect of catechins on GLUT4 translocation in 3T3-L1 cells. The cells were treated with 50 mM catechins for 30 min in the absence or presence of insulin. GLUT4 and GLUT1 proteins on the plasma membrane and expression levels of GLUT4 and b-actin in cell lysates were detected by Western blotting analysis. Values are the mean  S.E. of triplicates. * Significant difference from the DMSO-treated cells, p < 0.05 by Student’s t-test.

insulin-treated cells, Cg and EGCg significantly decreased phosphorylation of PKCl/z in the presence of insulin compared with insulin-treated cells without affecting the insulin-induced phosphorylation of IR, and Akt. Moreover, these catechins did not affect expression levels of these proteins in the cells under our experimental conditions.

The effects of EC and EGC on the glucose uptake activity in the PI3K inhibitor-treated cells To confirm whether EC- and EGC-increased glucose uptake activity is involved in the activation of the insulin signaling pathway, we used wortmannin and LY294002 as the PI3K inhibitors. Both inhibitors decreased either insulin-induced or EC- and EGC-induced glucose uptake activity (Fig. 5). These 170 | Food Funct., 2010, 1, 167–173

Fig. 4 Effect of catechins on insulin signaling pathway in 3T3-L1 cells. The cells were treated with 50 mM catechins for 30 min in the absence or presence of insulin. The phosphorylation and expression levels of IR, Akt, and PKCz/l were detected by Western blotting. Values are the mean  S.E. of triplicates. * Significant difference from the DMSOtreated cells, † significant difference from insulin-treated cells, p < 0.05 by Student’s t-test.

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Fig. 5 Effects of LY294002 and wortmaninn on the EC- and EGC-induced glucose uptake activity in 3T3-L1 cells. Serum-starved cells were incubated with 100 mM LY294002 or 100 nM wortmaninn for 10 min, and treated with 50 mM catechins for another 30 min. DMSO and 100 nM insulin were used for a negative and positive control, respectively. Data are expressed as the mean  SE (n ¼ 4). * Significant difference from control, p < 0.05 by Student’s t-test.

results suggested that EC and EGC promoted the translocation of GLUT4 through the activation of PI3K. Effects of tea fractions on glucose uptake activity in 3T3-L1 cells Finally, we checked the effects of tea fractions on glucose uptake in 3T3-L1 cells using [3H]-3-OMG. The ethanol extract of tea was suspended in water and partitioned with hexane, chloroform, ethyl acetate, n-butanol, and water step-wisely (Fig. 6A). All the fractions expected for the water fraction decreased insulininduced glucose uptake activity in the cells, with the ethyl acetate fraction being the most effective and significantly decreased glucose uptake compared with insulin-treated cells (Fig. 6B). This fraction is known to be in the most catechin-rich fraction in the ethanol extract of tea.17

Discussion The incidence of hypoglycemia is increasing year by year all over the world. Compounds are therefore required which regulate blood glucose levels. Our previous report demonstrated that an intake of green tea decreased glucose uptake activity and the translocation of GLUT4 in adipose tissue of Wistar rats.15 In this study, we examined the effects of eight catechins on glucose uptake in 3T3-L1 cells to obtain the effective compound on glucose transport system. We found interesting results that nongallate-type catechins increased glucose uptake activity in the absence of insulin whereas gallate-type catechins decreased in 3T3-L1 cells (Fig. 1). Moreover, we also found that these effects were accompanied by GLUT4 translocation; i.e., nongallatetype catechins promoted the translocation without insulin whereas gallate-type catechins decreased the insulin-induced This journal is ª The Royal Society of Chemistry 2010

translocation (Fig. 3). This is the first report for the structure– activity relationship of catechins on the glucose transport system, although this relationship is specific in adipose tissue but not in muscle cells.16 Nomura et al. reported that EGCg decreased insulin-induced glucose uptake activity but catechin had no effect in mouse MC3T3-G2/PA6 adipose cells.20 In adipose tissue, GLUT1 and GLUT4 play a role in regulating glucose uptake into the cells. Tea catechins and insulin did not affect glucose uptake activity in the preadipocytes, which had not expressed GLUT4 (data not shown), suggesting that tea catechins did not affect the function of GLUT1. We also checked that gallic acid and its related compounds did not affect glucose uptake activity in the presence or absence of insulin in 3T3-L1 cells (data not shown). It is, therefore, strongly suggested that the presence of galloyl ester at the C3 position plays an important role in the different mechanisms regulating GLUT4 translocation in adipose cells. It was reported that hydrophobic group at C3 position enhanced the function of catechins. Park et al. reported that 3-O-acyl and alkyl-()-epicatechins showed stronger anticancer effect than epicatechin.24 Stapleton et al. demonstrated that 3-O-acyl-()-catechins revealed higher anti-staphylococcal activity than catechin because these compounds have higher affinity to the cytoplasmic membrane than catechin.25 Moreover, Kajiya et al. showed that the galloyl group at C3 position affected the affinity for lipid bilayer membrane by measurement of the incorporated amount of catechins into liposome.26 Therefore, we proposed a hypothesis that gallate-type catechins bind strongly to the lipid bilayer membrane of adipose cells and decrease the function of glucose transport system compared to nongallate-type catechins. As the mechanism by which nongallate-type catechins increase glucose uptake activity in 3T3-L1 cells, our results indicated that Food Funct., 2010, 1, 167–173 | 171

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Fig. 6 Green tea fractions modulate glucose uptake activity in 3T3-L1 cells. (A) Green tea was extracted with ethanol and fractionated with organic solvents, as described in Materials and methods. (B) The cells were treated with each fraction for 15 min and co-incubated with 100 nM insulin for an additional 15 min. Data are expressed as the mean  SE (n ¼ 4). *Significant difference from control, †significant difference from insulin-treated cells, p < 0.05 by Student’s t-test.

the translocation of GLUT4 was promoted through the activation of PI3K without phosphorylation of IRb (Fig. 4 and 5). This mechanism is similar to that of EGCg in skeletal muscle cells,16 and actually EGCg slightly increased glucose uptake activity without insulin in 3T3-L1 cells (Fig. 2). On the other hand, our results suggested that gallate-type catecins decreased the insulininduced translocation of GLUT4 by inhibiting phosphorylation of PKCl/z, downstream of PI3K (Fig. 5). Harmon et al. showed that narigenin, grapefruit flavanone, did not affect phosphorylation of IR and IRS-1 but inhibited PI3K activity, resulting in decreasing the insulin-induced glucose uptake in 3T3-L1 cells.21 We proposed that gallate-type catechins inhibit PI3K activity or the TC10 pathway in 3T3-L1 cells. TC10 is a member of the Rho family of small GTP-binding protein and plays an important role in the control of insulin action in adipocytes.22 Insulin promotes 172 | Food Funct., 2010, 1, 167–173

phosphorylation of Cbl, a substrate for insulin receptor tyrosine kinase, followed by activation of TC10. Recent study showed that TC10 promoted phosphorylation of PKCl/z through a PI3K-independent pathway in 3T3-L1 cells.23 In skeletal muscle cells, EGCg did not inhibit the effect of insulin on the translocation of GLUT4 and glucose uptake activity,16 and TC10 expression was lower than in adipose tissue.22 Therefore, gallatetype catechins might inhibit the Cbl-TC10 pathway in 3T3-L1 cells. Further investigation will be required to examine the effects of catechins on the TC10 pathway. In this study, we also showed that the ethyl acetate fraction of green tea had the strongest inhibitory effect on glucose uptake (Fig. 6). It is known that the ethyl acetate fraction contained large amounts of catechins especially gallate-type catechins such as EGCg than other fractions.17 Other fractions except water fraction also decreased insulin-induced glucose uptake activity in the cells (Fig. 6). The hexane fraction contained mainly lipids and pigments such as lutein and chlorophyll a and b, and the chloroform fraction contained abundant caffeine.17 It was reported that caffeine decreased insulin-induced glucose uptake in rat adipose cells through inhibition of Akt activity.27 As to the butanol fraction, this fraction contained saponin28 and flavonoid glycosides.17 From these results, several active compounds including gallate-type catechins will orchestrate to inhibit the glucose transport system in adipocytes. Indeed, our previous report demonstrated the intake of green tea decreased glucose uptake and GLUT4 translocation in adipose tissue of rats.15 Among the active compounds in tea, EGCg might mainly affect glucose transport in adipose tissue in vivo. It had reported that EGCg has anti-obesity and hypoglycemic effects.29–31 Wolfram et al. showed that the intake of EGCg for 5 months reduced blood glucose levels of C57BL/6J mice fed a high-fat diet.29 Bose et al. demonstrated that long-term EGCg treatment (3.2 g kg1 diet, 16 weeks) reduced body weight gain, fat weight, and liver weight and short-term EGCg treatment (4 weeks) decreased mesenteric fat weight and blood glucose levels in C57BL/6J mice fed a high-fat diet.30 Chen et al. also showed that EGCg decreased body fat mass in Sprague Dawley rats fed a high-fat diet.31 On the other hand, our previous studies showed that catechins increased glucose uptake activity in skeletal muscle, the most important tissue for glucose utilization.15,16 Therefore, we assume that catechins modulate glucose transport in skeletal muscle and adipose tissue, resulting in a reduction in adipose tissue weight and blood glucose levels. In conclusion, green tea extracts decreased the glucose uptake activity in 3T3-L1 cells, and gallate-type catechins contributed to this effect. Moreover, nongallate-type catechins stimulated glucose uptake activity without insulin in 3T3-L1 cells accompanied with GLUT4 translocation. Therefore, tea catechins modulate the glucose transport in adipose tissue, affecting blood glucose levels.

Acknowledgements Part of this study was supported by Creation of Innovation Centers for Advanced Interdisciplinary Research Areas in Special Coordination Funds for Promoting Science and Technology Program by MEXT, Japan and by a grant from the This journal is ª The Royal Society of Chemistry 2010

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Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (M.U.).

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17 I. Fukuda, I. Sakane, Y. Yabushita, R. Kodoi, S. Nishiumi, T. Kakuda, S. Sawamura, K. Kanazawa and H. Ashida, J. Agric. Food Chem., 2004, 52, 2499–2506. 18 T. Furuyashiki, H. Nagayasu, Y. Aoki, H. Bessho, T. Hashimoto, K. Kanazawa and H. Ashida, Biosci., Biotechnol., Biochem., 2004, 68, 2353–2359. 19 S. Nishiumi and H. Ashida, Biosci., Biotechnol., Biochem., 2007, 71, 2343–2346. 20 M. Nomura, T. Takahashi, N. Nagata, K. Tsutsumi, S. Kobayashi, T. Akiba, K. Yokogawa, S. Moritani and K. Miyamoto, Biol. Pharm. Bull., 2008, 31, 1403–1409. 21 A. W. Hormon and Y. M. Patel, Biochem. Biophys. Res. Commun., 2003, 305, 229–234. 22 L. JeBailey, A. Rudich, X. Huang, C. Di Ciano-Oliveira, K. Kapus and A. Klip, Mol. Endocrinol., 2004, 18, 359–372. 23 M. Kanzaki, S. Mora, J. B. Hwang, A. R. Saltiel and J. E. Pessin, J. Cell Biol., 2004, 164, 279–290. 24 K. D. Park, S. G. Lee, S. U. Kim, S. H. Kim, W. S. Sun, S. J. Cho and D. H. Jeong, Bioorg. Med. Chem. Lett., 2004, 14, 5189–5192. 25 P. D. Stapleton, S. Shah, J. M. T. Hamilton-Miller, Y. Hara, Y. Nagaoka, S. Uesato and P. W. Taylor, Int. J. Antimicrob. Agents, 2004, 24, 374–380. 26 K. Kajiya, S. Kumazawa and T. Nakayama, Biosci., Biotechnol., Biochem., 2001, 65, 2638–2642. 27 T. Akiba, K. yaguchi, K. Tsutsumi, T. Nishioka, I. Koyama, M. Nomura, K. Yokogawa, S. Moritani and K. Miyamoto, Biochem. Pharmacol., 2004, 68, 1929–1937. 28 Y. M. Sagesaka, T. Uemura, N. Watanabe, K. Sakata and J. Uzawa, Biosci., Biotechnol., Biochem., 1994, 58, 2036–2040. 29 S. Wolfram, D. Raederstorff, Y. Wang, S. D. Teixeira, V. Elste and P. Weber, Ann. Nutr. Metab., 2005, 49, 54–63. 30 M. Bose, J. D. Lambert, J. Ju, K. R. Reuhl, S. A. Shapses and C. S. Yang, J. Nutr., 2008, 138, 1677–1683. 31 N. Chen, R. Bezzina, E. Hinch, P. A. Lewandowsli, D. CammeronSmith, M. L. Mathai, M. Jois, A. J. Sinclair, D. P. Begg, J. D. Wark, H. S. Weisinger and R. S. Weisinger, Nutr. Res., 2009, 29, 784–793.

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PAPER

www.rsc.org/foodfunction | Food & Function

Modulation of doxorubicin-induced genotoxicity by squalene in Balb/c mice Bhilwade Hari Narayan, Naoto Tatewaki, Vijayasree Vayalanellore Giridharan, Hiroshi Nishida and Tetsuya Konishi*

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Received 29th July 2010, Accepted 23rd September 2010 DOI: 10.1039/c0fo00102c The present study aims to evaluate the protective effect of squalene against the genotoxicity of the chemotherapeutic agent doxorubicin (Dox) using two genotoxicity assays, the micronucleus assay and the comet assay. Different groups of mice were fed squalene at the doses of 1 and 4 mmol g1 body weight (100 or 400 ml as squalene oil) either at 4 h before or 1 h after Dox (20 mg kg1) treatment. 24 h after the Dox treatment, bone marrow erythrocytes were evaluated for the incidence of micronuclei, and the induced DNA strand breaks were examined in heart tissue by the alkaline comet assay. As expected, Dox significantly induced micronuclei in polychromatic (immature) erythrocytes, as well as in total erythrocytes. The frequency of Dox-induced micronucleated erythrocytes was significantly reduced in the mice treated with squalene both before and after Dox administration. Squalene itself obviously did not induce any micronuclei in bone marrow erythrocytes. The comet assay also demonstrated a significant increase in DNA damage, especially DNA single strand breaks in the Doxtreated group of mice as compared to the control. The Dox-induced DNA damage was also effectively reduced by squalene when it was administered either before or after the Dox treatment. Squalene did not induce any significant DNA damage by itself. Compared to the pre-treatment of squalene, post treatment gave rise to more effective prevention against Dox-induced DNA damage. The data suggest that the complimentary use of squalene with Dox will be beneficial to reduce the adverse effect of Dox in cancer chemotherapy, such as the increased incidence of undesirable mutagenic side effects.

1. Introduction Human beings are exposed to a variety of physical, chemical and biological stressors that can induce genetic transformations. This is a substantive factor leading to chronic diseases, such as arteriosclerosis, heart diseases and cancers, which are the main causes of human death.1 All of these disorders more or less associate with genomic disturbances, and thus the search for antimutagenic compounds and the evaluation of their mechanism of action deserves special attention because of their possible significance in the protection of human health.2 Short-term mutagenicity assays have been effectively used for screening mutagens and potential carcinogens in human environments. The same methodologies are applicable for the identification of antimutagens or anticarcinogens. The micronucleus assay, a well known cytogenetic mutagenicity test, is one of these methods and has been proven suitable for such studies.3,4 The frequency of micronuclei is a reliable measure of both chromosome loss and breakage, and thus it is unique compared to other cytogenetic tests. However, this test can only be conducted in rapidly dividing cells, and thus mainly measures chromosomal damage induced in the bone marrow, thereby providing a limited assessment of the genotoxic potential of chemicals. The alkaline comet assay is, on the other hand, an in vivo genotoxicity assay to complement the in vivo micronucleus assay, and is gaining the status of a standard in testing and regulatory agencies, since it

Niigata University of Pharmacy & Applied Life Sciences, Department of Functional and Analytical Food Sciences, Higashijima 738-1, Niigata, 956-8603, Japan. E-mail: [email protected]

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can detect a broad spectrum of DNA damage, including DNA single and double strand breaks, base damage, alkali-labile DNA adducts, and other DNA lesions associated with diverse reactive oxygen species in virtually any tissue.5–7 The comet assay is recommended as a follow-up to a negative or equivocal in vivo micronucleus assay result, and also as a means to measure genotoxicity in target tissues other than bone marrow.8,9 In the present study, we focus our attention on the protective effect of squalene as a food factor against Doxorubicin (Dox) genotoxicity. Dox is an anthracycline anticancer drug that is commonly used for the treatment of many types of human cancers such as solid tumors, leukemia, soft tissue sarcoma and breast cancer.10 However, its clinical usefulness is restricted due to the toxicity frequently observed in cardiac tissue.11 The cumulative administration of Dox leads to abnormal cardiac function such as electrocardiographic changes, congestive heart failure and cardiomyopathy.12,13 The cause of Dox cardiotoxicity is multifactorial, but the major cause of Dox-induced cardiotoxicity can be attributed to the formation of reactive oxygen species (ROS), leading to myocyte apoptosis.14,15 The prevention of Dox-induced cardiotoxicity is the primary request so as to exploit the full therapeutic potential of Dox in cancer chemotherapy. Squalene is a physiological compound known to be a key precursor of steroid biosynthesis in organisms and is a natural product belonging to the terpenoid family. It occurs widely in nature from vegetable oil to fish oil. For example, common human dietary fat and oil contain approximately 0.002– 0.03% on average, but its content in olive oil is rather higher (0.2–0.7%). It is also known that squalene is highly concentrated in the liver This journal is ª The Royal Society of Chemistry 2010

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oil of the blue shark.16,17 The anticarcinogenic potential of squalene was proposed a long time ago,18,19 but only limited studies have been reported to support its anticarcinogenic function. For example, Newmark20 and also Rao et al.17 reported that squalene shows preventive activities against the action of several carcinogens. On the other hand, Senthilkumar et al.21 demonstrated the protective role of squalene in the tissue defence system of rats towards cyclophosphamide-induced toxicity, in that they observed increased levels of antioxidant enzymes, such as SOD, and non-enzyme antioxidants, such as reduced glutathione, in heart tissue. Furthermore it is reported that the prior administration of squalene prevents isoprenaline-induced adverse changes in plasma and heart tissue by inhibiting lipid peroxidation and exerting an antioxidant effect by maintaining the level of non-enzymatic free radical scavengers such as reduced glutathione at near normal.22 It is therefore suggested that squalene is an attractive food factor for targeting integrative or complementary applications in cancer chemotherapy. The aim of the present study is to evaluate the protective potential of squalene against the genotoxicity of chemotherapeutic agent Dox.

2. Materials and methods Dox was obtained from Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan and was dissolved in phosphate buffered saline (PBS). The Dox dose (20 mg kg1) was determined based on its effectiveness at inducing micronuclei.4 Squalene (>99% purity) was a generous gift from Nissei Marin Technology Co., Ltd., Tokyo, Japan. 2.1.

Animals

Male Balb/c mice (8–10 weeks, 22  1.5 g body weight) were adapted in the animal house for a period of 1 week before the experiments under controlled temperature (21  C) and humidity (50  10%) conditions with a 12 h light–dark cycle. Water and diet were given ad libitum. Animal experiments were carried out according to the guidelines of the Niigata University of Pharmacy and Life Science Animal Ethics Committee. 2.2.

2.3.

Micronucleus assay

The bone marrow micronucleus assay was performed according to protocol described by Schmid23 with a slight modification, as reported previously by Bhilwade et al.24 In brief, mice were sacrificed 24 h after Dox treatment by cervical dislocation. Both femur bones were removed clear from the adhering tissues. The marrow was flushed in a 5 ml eppendorff tube containing fetal bovine serum and centrifuged at 1500 rpm for 5 min. The cell pellet was mixed thoroughly, bone marrow smears were made on a glass slide, and they were stained in May–Gruenwald and Giemsa as follows: 5 min in undiluted May–Gruenwald (0.25% in methanol), 3 min in diluted May–Gruenwald solution (1 : 1, May–Gruenwald : distilled water), rinsed in distilled water three times and then stained with diluted Giemsa (1 : 6 of the Giemsa stock : distilled water) for 10 min following thorough washing with distilled water. The slides were dried, cleared for 5 min in xylene and mounted in DPX. Two slides were made from each animal. Coded slides were scored for the incidence of micronucleated polychromatic erythrocytes (Mn-PCEs) and micronucleated normochromatic erythrocytes (Mn-NCEs) at 100 magnification under oil. (Fig. 1). Approximately 2000 PCEs with or without micronuclei and a corresponding number of NCEs were analyzed per animal. 2.4.

Comet assay

The comet assay was carried out by the method described by Chaubey et al.,6 which is based on the original work of Singh et al.25 Briefly, heart tissue was removed immediately after sacrificing the mice and washed with chilled PBS. The tissue was dissociated into cells using a cell dissociation sieve-tissue grinder kit under ice cold conditions and processed for the comet assay. Approximately 1  105 cells were mixed with 1.5 ml of 0.8% low melting agarose solution prepared in 0.9% saline at 38  C, and

Experimental design

Squalene is an oil with a specific density of 0.867, and thus 100 and 400 ml volumes were directly given orally to mice by intubation (1 and 4 mmol g1 body weight, respectively). Mice were randomly divided into 9 groups (n ¼ 4). Group I served as the control and were treated intraperitoneally with 400 ml PBS. Group II received 400 ml of mineral oil as a squalene reference. Groups III and IV were orally administered squalene at doses of 100 and 400 ml, respectively. Group V animals intraperitoneally received Dox dissolved in PBS at a dose of 20 mg kg1. Group VI and VII animals were orally administered 100 and 400 ml of squalene, and then Dox was received 4 h after the squalene administration. Group VIII and IX animals were treated with Dox following squalene administration at doses of 100 and 400 ml, respectively, 1 h after Dox treatment. All animals were sacrificed 24 h after Dox treatment, and bone marrow and heart tissues were removed for the micronucleus test and the comet assay, respectively. This journal is ª The Royal Society of Chemistry 2010

Fig. 1 Photomicrographs showing mouse bone marrow erythrocytes stained with May–Gruenwald–Giemsa (A–C) and comets from heart cells stained with SYBR green-II. (D–F). A: micronucleus in polychromatic (immature) erythrocyte, bluish in colour. B: micronucleus in normochromatic (matured) erythrocytes, golden yellow in colour. C: polychromatic erythrocytes containing two micronuclei. D: comet in a normal cell. E: comet in a doxorubicin treated cell. F: comet in a cell of mice treated with Dox following squalene oral administration of 400 ml 1 h after Dox.

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poured onto fully frosted microscope slides. After solidification, the slides were kept in lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, and 10% DMSO and 0.1% Triton X-100) for 1 h at 4  C. After lyses, the slides were set in a horizontal electrophoresis apparatus and kept in alkaline buffer (30 mM NaOH, 1 mM Na2-EDTA, pH 13.0) at room temperature for 20 min to unwind the DNA strands. Electrophoresis was carried out for 30 min at 25 V, 300 mA using a power supply. After electrophoresis, the slides were washed gently in a neutralizing buffer (0.4 M TrisHCl, pH 7.5) to remove the alkaline buffer and detergent, and stored on wet tissue paper in a closed plastic box at 4  C until observation. The slides were stained with SYBR green II, and at least 50 cells were captured per slide at 20 magnification using a fluorescence microscope (Olympus (BH2-RFCA), Japan) equipped with green light excitation with a 590 nm barrier filter. (Fig. 1). The comet images were analyzed by the digital imaging software ‘‘CASP’’. Two parameters, tail moment (TM) and tail length (TL), were extended from the images directly. TM is the product of the tail length and the percentage DNA in the tail.26 The percentage reduction of micronucleated erythrocytes in the micronucleus assay and the DNA damage in the comet assay were calculated by the formula below to evaluate the anti-genotoxicity potential of squalene according to a method reported elsewhere.27 %Reduction ¼

ðmean score in AÞ-ðmean score in BÞ 100 ðmean score in AÞ-ðmean score in CÞ

Where A is the group of cells from Dox-treated mice (positive control), B is the group of cells from squalene plus Dox-treated mice and C is the control. 2.5.

Statistical analysis

All the data are expressed as mean  SD. Statistical analysis of the data was carried out by one-way ANOVA using the Tukey– Kramer test. The differences were considered significant at the 95% confidence limit.

3. Results 3.1.

Micronucleus assay

The genotoxicity of squalene was first examined using mineral oil as a reference. The induction of micronuclei observed in both groups treated with 100 and 400 ml squalene, respectively, was slightly higher than that observed in the buffer control, although the differences were not statistically significant. Furthermore, the level was the same as that of the mineral oil reference. It was thus concluded that squalene itself, even in a 400 ml dose, does not have any mutagenic effects. The frequencies of Mn-PCEs, Mn-NCEs and total micronucleated erythrocytes (Mn-Es) in the bone marrow of mice treated with squalene alone or in combination with Dox are summarized in Table 1. As expected, the frequency of Mn-PCEs and total Mn-Es increased significantly (p < 0.01) in animals receiving Dox. Both pre-treatment with squalene 4 h before and post treatment 1 h after the Dox treatment, respectively, showed a significant reduction (p < 0.01) in the frequency of Mn-PCEs and Mn-Es when compared to the group treated with only Dox. 176 | Food Funct., 2010, 1, 174–179

It was revealed that the frequency of both Mn-PCEs and Mn-Es were more markedly reduced when squalene was given after the Dox treatment (post-treatment) than before (pre-treatment). The percentage reduction of Mn-Es in the squalene pre-treated group was 44.72 and 33.23% at 100 and 400 ml, respectively, while in the post-treated group it was 56.69 and 59.53%, respectively. Similarly, the percentage reduction of Mn-PCEs was 52.90 and 44.58% at 100 and 400 ml in the squalene pre-treated groups, respectively, and 61.71 and 69.47% in the post-treated groups (Table 2). The PCEs/NCEs ratio, which reflects the proliferation rate of bone marrow, was significantly decreased (p < 0.05) in the Dox-treated group as compared to the control and squalenetreated groups. The PCEs/NCEs ratio in the group treated with a combination of a higher dose of squalene and Dox was observed to be significantly higher (p < 0.05) as compared to the Dox-treated group (Table 1). 3.2.

Comet assay

Studies were carried out to evaluate cellular DNA damage with or without squalene administration in Dox-treated mice. The values of TM and TL are shown in Fig. 2. As is evident from the figure, the values of TM and TL increased significantly in mice treated with Dox at a dose of 20 mg kg1 compared to cells from untreated control mice. Cells from the mice administered with squalene either 4 h before or 1 h after Dox treatment showed significant inhibition (p < 0.01) of DNA damage measured in terms of TM and TL. The results also indicate that the inhibition of Dox-induced DNA damage by squalene was relatively more efficient in the post-treated group compared to the pre-treated group (Table 2). Although squalene treatment alone showed a little DNA damage, it was not a significant difference from that of the control.

4. Discussion In the present study, both the micronucleus assay and the comet assay were cooperatively used to study the modulatory effect of squalene against Dox-induced genotoxicity at a chromosomal level as well as a DNA level. The results clearly show that squalene efficiently prevents Dox-induced DNA damage and protects heart tissue. These two methods cover different aspects of genotoxic events. Essentially, the micronucleus assay detects chromosomal damage that persists for at least one mitotic cycle, while the comet assay identifies repairable DNA damage or alkali-labile sites.28 Some of the DNA lesions, such as single and double strand breaks, and adduct formation, led to permanent fetal damage, and the micronucleus test is an excellent means of evaluating any permanent damage in genetic material. On the other hand, the comet assay is a very sensitive method for measuring DNA strand breaks at a single cell level and thus is sensitive to acute DNA lesions. Moreover, it was recently shown that the comet assay detected nearly 90% of carcinogens that were negative or equivocal in the micronucleus assay. Therefore, a combination of the micronucleus assay and the comet assay is recommended for the broad assessment of in vivo genotoxic potential.29,30 This protocol allows evaluation of two distinct genotoxicity end points in the same animal. The National Toxicological Program (NTP) is presently using this combined This journal is ª The Royal Society of Chemistry 2010

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Table 1 Modulation of Dox-induced micronucleated erythrocytes in mouse bone marrow by squalenea Treatment

&Mn-Es

&Mn-PCEs

&Mn-NCEs

PCEs/NCEs ratio

Control

1.71  0.19 (28/16336) 2.01  0.51 (33/16369) 2.25  0.65 (37/16437) 2.18  0.46 (36/16500) 8.06  1.93b (144/17073) 5.22  1.97c (87/16652) 5.95  1.90 (99/16629) 4.46  0.37d (75/16812) 4.28  0.54d (71/16579)

2.08  0.22 (21/8063) 2.98  0.42 (24/8053) 3.34  0.74 (27/8083) 3.21  0.63 (26/8099) 16.39  4.26b (132/8052) 8.82  2.89d (71/8045) 10.01  3.74d (81/8091) 7.56  0.82d (61/8066) 6.45  0.79d (52/8056)

0.85  0.45 (7/8273) 1.08  0.61 (9/8316) 1.20  0.63 (10/8354) 1.19  0.63 (10/8401) 1.33  0.37 (12/9021) 1.86  1.36 (16/8607) 2.11  0.57 (18/8538) 1.60  0.29 (14/8746) 2.23  0.45 (19/8523)

0.97  0.02

Mineral oil Squalene 100 ml Squalene 400 ml Dox 20 mg kg1

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Squalene 100 ml (4 h) + Dox Squalene 400 ml (4 h) + Dox Dox (1 h) + squalene 100 ml Dox (1 h) + squalene 400 ml a c

0.97  0.01 0.97  0.01 0.96  0.02 0.89  0.04b 0.93  0.03 0.95  0.01c 0.92  0.02 0.95  0.01c

Numbers within parenthesis are actual numbers of micronucleated cells/total erythrocytes scored. b p < 0.01, significantly different from the control. p < 0.05. d p < 0.01, significantly different from the Dox group.

protocol as part of its efforts to evaluate the genotoxicity of substances of public health concern.31 As shown in Table 1 and Table 2, when we evaluated the frequency of micronuclei (as genetic marker) in PCEs, NCEs and the total erythrocytes (PCEs and NCEs) in bone marrow using the micronucleus assay, and the extent of DNA damage in heart tissue by the comet assay, the numbers of micronucleated erythrocytes increased by Dox treatment was markedly reduced in mice administered with squalene, either before or after the Dox treatment. In the same way, the Dox-mediated DNA strand breaks were inhibited in mice pre- and post-administered with squalene. The present study is thus the first in vivo demonstration of the modulation effect of squalene on Dox-induced chromosomal or DNA damage in mice assessed by the two genotoxicity evaluation methods mentioned above. Regarding the genotoxicity of squalene itself, however, Fan et al.32 have reported that no induction of sister chromatid exchanges and micronuclei occurs in squalene-treated Chinese hamster ovary-K1 cells. Table 2 Percentage inhibition of micronucleated erythrocytes in total erythrocytes (Mn-Es) and immature (polychromatic) erythrocytes (MnPCEs), and DNA damage (tail moment and tail length), by squalene in Dox-induced mice Micronucleus assay

Comet assay

Group

Mn-Es

Mn-PCEs

Tail moment

Tail length

Pre-treatment 4 h (squalene 100 ml) Pre-treatment 4 h (squalene 400 ml) Post-treatment 1 h (squalene 100 ml) Post-treatment 1 h (squalene 400 ml)

44.72

52.90

51.08

38.25

33.23

44.58

78.35

58.37

56.69

61.71

82.26

55.46

59.53

69.47

85.41

55.40

This journal is ª The Royal Society of Chemistry 2010

Dox is an antineoplastic agent in the anthracyclin antibiotic family that is used widely in the treatment of human cancer. The drug is metabolically activated to a free radical form and interacts with molecular oxygen to generate superoxide radicals.33 The superoxide radicals can react with hydrogen peroxide to form highly reactive hydroxyl radicals through the iron-catalyzed Haber–Weiss reaction. Secondarily-derived hydroxyl radicals can cause protein and DNA damage and initiate lipid peroxidation.34 Thomas et al.35 showed that DNA damage is an early event in Dox-induced cardiac myocyte death in the H9c2 cardiac cell line derived from embryonic rat heart. It is thus highly likely that the Dox-mediated induction of micronuclei and DNA damage observed here is due to the ROS including free OH radicals produced by Dox. It has been reported that squalene is an antioxidant molecule having a high scavenging activity towards ROS, especially towards singlet oxygen.36,37 Although it is not yet clear how the singlet oxygen is involved in Dox-induced DNA lesion formation, its antioxidant property is likely to be associated with the mechanism underlying the protective effect of squalene against Dox-induced mutagenicity and DNA or chromosomal damage. This idea is supported by our present observation that the squalene administered after Dox treatment gave effective protection against the production of DNA damage in both micronuclei and the comet assay, and the effect was also dependent on the dose of squalene. Since squalene is rapidly metabolized after intake,38 post-administration will be more effective at maintaining the plasma and tissue levels, and thus a higher ROS scavenging activity of squalene is implicated in the case of post-administration than pre-administration. It is also important to note that squalene itself did not increase the frequency of micronucleated erythrocytes in mouse bone marrow, even with 400 ml administration (4 mmol g1 body weight), indicating that squalene has essentially no genotoxicity, because it is well known that certain molecules with a high ROS Food Funct., 2010, 1, 174–179 | 177

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cisplatin and carboplatin-induced cytotoxicity in vivo without protecting tumor growth.46 Therefore, a further study is needed to confirm these mechanisms. In summary, the combination of both assays in the present work proved to be adequate and useful for the evaluation of the genotoxicity of Dox. Both assays evidenced the protective function of squalene against Dox-induced genotoxicity in bone marrow and heart tissue. The extent of DNA damage was measured qualitatively by the comet assay. On the other hand, the micronucleus assay measured chromosomal damage and provided evidence of cytotoxicity. Although the comet assay is observed to be the more sensitive method, the micronucleus assay was also informative, and its usefulness should be considered for the evaluation of genotoxicity. The results obtained by these studies indicate that squalene as a food factor might be an effective antimutagen and might be applicable for the reduction of the adverse effect of Dox in complementary or integrative cancer chemotherapy.

Acknowledgements

Fig. 2 Single cell gel electrophoresis analysis of DNA damage in mouse heart cells treated with squalene and Dox. A: Tail moment (TM), B: Tail length (TL).

scavenging potential in vitro, such as catecholamines, show cytotoxicity rather than oxidative stress prevention.39 Furthermore, our experiments suggest that treatment with squalene is effective for reducing the mutagenic effect of Dox, both in heart tissue and bone marrow, even at 100 ml or less, because the inhibitory action of squalene is almost saturated above 100 ml administration in the comet and micronucleus assay results. On the other hand, in the several experimental models,32,40 squalene was demonstrated to detoxify the adverse effects of diverse chemicals such as hexachlorobiphenyl, hexachlorobenzene, arsenic, theophylline, phenobarbital and strychnine.41 These compounds are not strong antioxidants by themselves, but their cell toxicity is associated with oxidative stress.42–44 Therefore, this also supports the idea that the primary mechanism involved in the antigenotoxic effect of squalene is expected to be an antioxidant effect. However, as De Flora and Ramel45 suggested, multiple mechanisms for antimutagen action and the possible involvement of mechanisms other than the direct scavenging of ROS cannot be excluded, such as through the modulation of antioxidant enzymes, damage repair or the metabolic inactivation of Dox. These effects might be reflected in the present observation that the dose dependency of squalene action was more clearly shown in the pre-treated group, both in the micronucleus assay and the comet assay. Moreover, the present study revealed that in the micronucleus assay, the PCEs/ NCEs ratio, which reflects the cytotoxicity in the erythropoietic system, was reduced in the Dox-treated group as compared to the control and squalene groups. However, the ratio became higher in the group treated with a combination of Dox and squalene. This is consistent with the observation by Das et al. that squalene selectively protects mouse bone marrow progenitors against 178 | Food Funct., 2010, 1, 174–179

We thank Nissei Marin Technology Co., Ltd., Tokyo, Japan for supporting our squalene research project. We are also grateful to Kyowa Hakko Kirin Co., Ltd., Japan for providing doxorubicin.

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www.rsc.org/foodfunction | Food & Function

Oxidative cascade reactions yielding polyhydroxy-theaflavins and theacitrins in the formation of black tea thearubigins: Evidence by tandem LC-MS† Nikolai Kuhnert,*a Michael N. Cliffordb and Anja M€ullera

Downloaded on 10 November 2010 Published on 14 October 2010 on http://pubs.rsc.org | doi:10.1039/C0FO00066C

Received 9th July 2010, Accepted 16th September 2010 DOI: 10.1039/c0fo00066c LC-MSn and direct infusion-MSn have been applied for the first time to the characterisation of crude thearubigins isolated from black tea. The data generated have been used to test two hypotheses of thearubigin structure: (i) that a significant fraction of the thearubigins consist of polyhydroxylated derivatives of the better-known catechin dimers (theaflavins, theaflavin mono- and di-gallates, theacitrins) in redox equilibrium with their associated quinones; and (ii) that a significant fraction of the thearubigins consist of dicarboxylic acids generated by oxidative cleavage of aromatic diols. The data were consistent with the polyhydroxylation hypothesis and did not support the dicarboxylic acid hypothesis. Evidence is presented for the presence in crude thearubigins of at least 29 hydroxylated theaflavins (with between one and six oxygen insertions), at least 12 theaflavin mono-gallates (with between one and six oxygen insertions), at least nine theaflavin di-gallates (with between one and four oxygen insertions), and at least ten theacitrin mono-gallates (with between one and four oxygen insertions). Evidence is also presented for at least ten mono- or di-quinone forms of the parent compounds and hydroxylated derivatives in each of these homologous series. A general method for the analysis of complex mixtures by tandem LC-MS is furthermore introduced and established.

Introduction Black tea is, second only to water, the most consumed beverage globally with an average per capita consumption of around 550 ml a day. Annual production of tea leaves reached a record high in 2008 with a global harvest of 3.75 Mt.1 Production of dried tea comprises 20% green, 2% oolong and the remainder black. In January 2006, tea prices were US$1.56 kg1 and had increased to a record high in June 2008 of US$3.40 kg1, making black tea one of the most economically important agricultural products.1,2 Despite its importance, the majority of black tea’s chemical composition remains unresolved if not mysterious. Black tea is produced from the young green shoots of the tea plant (Camellia sinensis), which are converted to black tea by a manufacturing process, in which the green tea shoots are so-called fermented. Within the fermentation, an enzymatic oxidation process, the major chemical constituents of the green tea leaves, flavan-3-ols or catechins 1–6, mainly epigallocatechin 3 and its gallate ester 1, that account for 10–25% of the dry weight of a fresh green tea leaf, (representative structures shown in Scheme 1) are consumed and chemically transformed. These substrates are oxidised and extensively transformed into novel dimeric, oligomeric and polymeric compounds, few of which have been fully characterised. This material was originally referred to as oxytheotannin.3 Selected well characterised dimeric

a Functional Materials and Nanomolecular Science Research Centre, Jacobs University Bremen, 28759 Bremen, Germany. E-mail: n.kuhnert@ jacobs-university.de; Tel: +0049 421 200 3120 b Centre for Nutrition and Food Safety, Faculty of Health and Medical Sciences, The University of Surrey, Guildford, GU2 7XH, United Kingdom † Electronic supplementary information (ESI) available: Supplementary tables, structures of polyphenols, and chromatograms. See DOI: 10.1039/c0fo00066c

180 | Food Funct., 2010, 1, 180–199

structures such as theacitrins 7, theaflavins 8–11, theasinensins 12 and theanaphthoquinones 13 are shown in Scheme 1. Oxytheotannin was subsequently divided into the reddishorange, ethyl-acetate-soluble theaflavins, and the brownish water-soluble (or ethyl acetate-insoluble) thearubigins. Although first observed in 1959,4 the term thearubigins was not introduced until 1962,5 but even 50 years later these oligomeric and polymeric transformation products remain poorly characterised. The components of black tea can be divided into two classes. Firstly, a series of well characterised small molecules, including alkaloids such as theobromine and caffeine, carbohydrates and amino acids, and a series of glycosylated flavonoids, that together account for 30–40% of the dry mass of a typical black tea infusion. Secondly, the heterogeneous fraction of polyphenolic fermentation products that account for the remaining 60–70%. These fermentation products are again divided in two distinct classes of compounds: Firstly the orange-red theaflavins containing benztropolone ring systems and secondly the heterogeneous fraction of the thearubigins (TRs) constituting between 60 and 70% of the dry mass of an average black tea infusion.6 Substantial progress has been made in the isolation and structure elucidation of the theaflavins over the last 40 years with many compounds of this class being isolated and identified, whereas the structures of the thearubigins, that were discovered in 19594,7 and named as such by Roberts in 19628 still retain their mysterious nature and remain a challenge for scientists. Two recent reviews, one by Harbowy and Balentine and one by Haslam summarise the state of knowledge on the chemical structure of the TRs.9,10 It is commercially important to elucidate the chemical structure of the TRs and hence their function for a variety of reasons. Structural characterisation will improve the understanding of the components contributing to taste, colour and shelf-life of This journal is ª The Royal Society of Chemistry 2010

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Scheme 1 Structures of green tea catechin derivatives 1–6 and structures of formal dimers of catechins 7–13 found in black tea.15

black tea based products. Furthermore structure elucidation of the TR fraction will allow the identification of chemical compounds responsible for any of the various and wide ranging beneficial biological activities arising from the human consumption of black tea and black tea based products.11 We have reported on the characterisation of black tea thearubigins by a series of standard and advanced analytical techniques including ESI-FT-ICR mass spectrometry and MALDI-TOF mass spectrometry. Our observations and conclusions were as follows.12–14 Thearubigins comprise several thousand compounds, an order of magnitude higher than previously expected, with around 10 000 molecular ions resolved in a single direct infusion ESIFTICR MS experiment. Around 1500 molecular formulas have been assigned using these data.12 The unusual Gaussian shaped hump characteristic of thearubigin chromatograms can be explained by the large number of compounds present combined with peak broadening arising from aromatic–aromatic interactions, non-covalent interactions such as hydrogen bonding, and dis-equilibration–re-equilibration during chromatography. Data obtained by ESI-FT-ICR and MALDI-TOF mass spectroscopy, Diffusion NMR, AFM and size exclusion chromatography, demonstrate that components of the thearubigins do not exceed 2100 Da and are therefore unlikely to contain oligomers comprising more than seven catechins units. This journal is ª The Royal Society of Chemistry 2010

Although not identical, thearubigins from 15 different sources are remarkably similar with respect to all their spectroscopic fingerprints. Petrolomics style as well as novel data interpretation strategies were adopted and developed to visualise these enormously complex data. Several homologous series were detected, with mass increments corresponding to oxygen insertion, loss of hydrogen, and addition of gallate, hexose, deoxy-hexose, and, particularly, water increments. This interpretation permitted the formulation of an experimentally based hypothesis to explain the formation and structure of approximately 90% of the thearubigins, whose molecular formula has been assigned, with a mass below 1000 Da, which we have named ‘‘oxidative cascade hypothesis’’.12 The hypothesis assumes three levels of chemical reaction types producing the thousands of compounds observed within the thearubigin fraction. Level 1: The six catechin building blocks oligomerise using the four types of dimerisation mechanisms previously described in the literature. These four mechanisms comprise a type I mechanism (theasinensin type), a type II mechanism (theaflavin type) a type III mechanism (theacitrin type) and a type IV mechanism (theanaphthoquinone type). The rules of connectivities have been discussed by Drynan et al. in detail.15 At this level oligomers comprising two to seven catechin units or two to four gallated catechin units are formed. Food Funct., 2010, 1, 180–199 | 181

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Level 2: Any oligomer reacts via an ortho-quinone intermediate with water as the most abundant nucleophile in the fermenting tea leaf. In this reaction an aromatic hydrogen is replaced by a phenolic OH group, thus formally an oxygen is inserted into an aromatic CH group until finally all aromatic hydrogens are replaced by phenolic OHs. Compounds like polyhydroxytheaflavins or polyhydroxytheacitrins are formed. This level represents a true oxidative cascade reaction, since with the introduction of a new phenolic OH group the aromatic nuclei become more electron rich and hence with each oxygen insertion step easier to oxidise. Regioisomers are possible at each step of oxygen insertion. Level 3: Any of the polyhydroxylated oligomers of catechins are in a redox equilibrium with their corresponding quinone structures (both ortho- and para-quinoid structures are feasible). Again with increasing numbers of phenolic OHs groups present this oxidation step will be favoured. Evidence for quinone structures in the TR fraction were clearly obtained using mass spectrometry and Circular Dichroism spectroscopy. This contribution reports our testing of this hypothesis using, in particular structures produced at levels 2 and 3 by direct infusion MSn and LC–MSn experiments

Materials and methods Chemicals and reagents (–)-Epicatechin, (–)-EGCG, theaflavin, theaflavin-3-gallate, theaflavin-30 -gallate and theaflavin-3,30 -digallate and the 15 world teas were provided by Unilever, Colworth (UK). All other chemicals and reagents were purchased from the Sigma Aldrich Company. Preparation of thearubigins Freshly ground black tea leaves (8 g) were added to 150 ml freshly boiled water and kept for 10 min in a Thermos flask, which was inverted every 30 s. The flask contents were filtered through a Whatman No 4 filter paper to remove the leaves, and Table 1 Numbering of TR samples including their yields, geographic origin and number of well resolved peaks floating on the thearubigin hump in LC analysis

Sample number

Black tea leaf description

Yield [mg, %]

Number of well-resolved peaks in negative mode LC-MS

I II III IV V VI VII VIII IX X XI XII XIII XIV XV

Kenya Darjeeling Lipton Blend Vietnam Dust Turkish Tiger Hill Kenyan BP1 Java Broken Indian BB21 Darjeeling White Leaf Ceylon UVA Ceylon Standard EBOP Ceylon GMD Assam Argentine BOP

560, 7 780, 1 401, 5 489, 5 687 8 783, 1 621, 8 710, 9 410, 5 567, 6 490, 5 601, 7 730, 8 480, 6 510, 6

15 18 22 28 30 29 22 27 25 25 24 27 19 26 30

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the remaining brew allowed to cool to room temperature. Caffeine sufficient to achieve 20 mM was added to the brew, stirred to ensure dissolution, and allowed to stand at 4  C for two hours, and centrifuged at 23,300  g for 20 min. The resulting precipitate was recovered and suspended in boiling water, and partitioned against aliquots of ethyl acetate (40 ml) until no further color was extracted (usually 5). The ethyl acetate-supernatant was removed and evaporated to dryness under nitrogen below 35  C, and the residue (TF fraction) recovered in 10 ml distilled water. The aqueous phase was partitioned at 80  C against two volumes of chloroform, the decaffeinated liquid stored overnight at 80  C, and freeze-dried. The freeze-dried material (TR fraction) was stored at 20  C until required and reconstituted as required for the analysis. The thearubigins were obtained as orange to light brown fluffy powders. Individual yields are stated in Table 1.

LC-MSn The LC equipment (Agilent 1100 series) comprised a binary pump, an auto sampler with a 100 mL loop, and a DAD detector with a light-pipe flow cell (recording at 400 and 254 nm and scanning from 200 to 600 nm). This was interfaced with an iontrap mass spectrometer fitted with an ESI source (Bruker Daltonics HCT Ultra) operating in the negative ion mode Auto MSn mode to obtain fragment ion m/z. Tandem mass spectra were acquired in Auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 Volt, starting at 30% and ending at 200%. MS operating conditions (negative mode) had been optimised using theaflavin-3-gallate 15 with a capillary temperature of 300  C, a dry gas flow rate of 10 L/min, and a nebulizer pressure of 10 psi. As necessary, MS2, MS3 and MS4 fragment-targeted experiments were performed to focus only on compounds producing a parent ion at m/z 563.2, 579.2, 595.2, 611.2, 627.2 for theaflavin derivatives, 715.2, 731.2, 747.2, 763.2, 779.2 for theaflavin gallate derivatives, 867.2, 883.2, 899.2, 915.2, 931.2 for theaflavin digallate derivatives, 759.2, 775.2, 791.2, 807.2, 823.2 and 911.2 for theacitrin derivatives.

High resolution LC-MS High Resolution LC-MS in the negative ion mode was carried out using the same HPLC equipped with a MicrOTOF Focus mass spectrometer (Bruker Daltonics) fitted with an ESI source and internal calibration was achieved with 10 mL of 0.1 M sodium formate solution injected through a six port valve prior to each chromatographic run. Calibration was carried out using the enhanced quadratic calibration mode. It should be noted that in TOF calibration the intensities of the measured peaks have a significant influence on the magnitude of the mass error with high intensity peaks resulting in detector saturation displaying larger mass errors. Where necessary this was avoided by using a more dilute sample. All MS measurements were carried out in the negative ion mode. This journal is ª The Royal Society of Chemistry 2010

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HPLC analysis

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The extracted thearubigins were analysed by HPLC using an Agilent 1200 HPLC pump with a 5 ml loop, coupled to an Agilent 1100 autosampler, an Agilent 1100 DAD-UV-VIS detector. Black tea extracts and thearubigin extracts were reconstituted at 3 g/l in 1 : 1 MeOH/H2O and filtered through a 0.45 mm HPLC filter prior to injection of a volume of 3 ml. HPLC analysis used a POLARIS 5-C18-A column (length 250 mm, diameter 3 mm, particle size 5 mm) with a step gradient elution employing acetonitrile (MeCN) and water containing 0.005% formic acid, as follows: 8% MeCN from 0 to 50 min, then changing to 31% MeCN for 10 min then changing to 25% MeCN for a further 5 min. Direct infusion tandem MS A thearubigin solution (TR IV, TR VI and TR XII) of 5 g/l in water was infused at a flow rate of 180 ml/min into an ion trap mass spectrometer (Bruker HCT ultra) in the negative ion mode using the instrument settings above. MS2 experiments were carried out manually with an isolation width of 1 Da for targeted masses in the mass range between m/z 500 to 1000 with 30– 50 MS2 spectra summed up per 1 Da. Data analysis Data were analysed using Bruker Data Analysis 4.0 software. Micro TOF data were analysed (TICs, EICs) after external enhanced quadratic calibration. Ion trap data were analysed in terms of EICs, TICs and neutral loss chromatograms (NLCs) using the implemented software routines. Reaction of TR with KMnO4 To a solution of TR XII (5 ml, 5 g/l) was added at room temperature 1 ml 0.1 M KMnO4 solution and the solution stirred for 5 min. until discoloration of the KMnO4 took place. The resulting solution was centrifuged at 5000 rpm, filtered and subjected to LC-MS analysis using the conditions above.

would commonly be found when analysing less complex mixtures, and some allowance must be made for these peculiarities when interpreting the spectra.

ESI-LC-TOF MS data In a first set of experiments thearubigins were isolated from fifteen commercial black teas, presenting a selection of geographical and sensory variations (see Table 1). As isolation procedure a method previously used by Roberts was employed comprising caffeine precipitation of TRs.7,12 Crude TR samples from 15 commercial teas were analysed by ESI-LC-TOF MS in the negative ion mode. All fifteen showed the typical Gaussian TR hump and some well-resolved peaks floating thereon. At 400 nm the TR hump was particularly pronounced. Representative chromatograms are shown in Fig. 1. In comparison with the original black tea infusion, we observed a significant reduction in the intensities of the floating wellresolved peaks in the UV-VIS chromatogram (>90%) and in the negative ion mode total ion chromatogram (TIC) (80–90%). In the UV chromatogram typically between 15 and 20 well-resolved peaks were observed, whereas in the TICs between 15 and 30 were observed (See Table 1). In our earlier paper we detected in these crude TR fractions substances that had been previously reported and for which there was unambiguous NMR data in the literature.15 The tandem MS data obtained in this investigation confirm these previous assignments (see Table S1 and S2 in supplementary information†). By reference to a review of black tea composition,15 a listing was prepared of other components that might be present in the crude TR samples, and these were sought by extracting the appropriate ion chromatograms from the ESI-LC-TOF-MS data. When appropriate ions were found these were probed by tandem MS. These high resolution and tandem MS data confirm the presence of previous literature assignments for around forty compounds. The tandem MS results and selected extracted ion chromatograms (EIC) are shown in the supplementary informations (Figures S2–S5†).

Results and discussion The LC–MSn and direct infusion–MSn results are presented and discussed in three main sections. The first deals with confirming the presence in the TR fractions analysed of substances previously reported in black tea. The second tests in four representative homologous series (A–D) our hypothesis of progressive hydroxylation and aromatic diol–quinone equilibria. Finally, an alternative dicarboxylic acid hypothesis is evaluated. Because the thearubigins are much more complex than most other extracts of foods and beverages that are routinely analysed by LC–MS it is important to appreciate that the data generated differ significantly. For example, because the chromatogram is so crowded, even a peak that appears well-resolved in the UV-Vis trace yields several intense MS peaks accompanied by several weaker signals. Similarly, during direct infusion MS, even with a 1 Da window set for the ion trap, numerous regio- and stereoisomers will be trapped and these might be accompanied by multiply-charged ions, each yielding multiple fragment ions. Accordingly, the resultant fragment spectra are less clean than This journal is ª The Royal Society of Chemistry 2010

Constant neutral loss chromatograms In an LC-tandem MS experiment following LC separation molecular ions of analytes present are fragmented. In the fragmentation process the molecular ion produces a fragment ion and a neutral species. By using data analysis algorithms the presence of fragment ions (in so called all MSn searches) and neutral losses (in so called neutral loss chromatograms) with desired masses can be sought and therefore chromatographic peaks corresponding to a particular mass and having particular fragmentation characteristics can be located. In the preceding paper,12 we hypothesised that the crude TR contained novel compounds belonging to several homologous series. These were sought by using constant neutral loss analyses (CNL) of the tandem MS data. NLCs were prepared for the gallate increment (C7H5O4 m/z 152), the hexose increment (C6H10O5 m/z 162), and the deoxy-hexose increment (C6H12O4 m/z 146), and are shown in Fig. 2. Clearly, these postulated structural increments are ubiquitous in the sample. Food Funct., 2010, 1, 180–199 | 183

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Fig. 1 HPLC chromatogram of sample TR IV a) TIC in negative ion mode, b) TIC of all MSn in negative ion mode and c) UV trace monitored at 400 nm showing well-resolved peaks and thearubigin hump.

The possible significance of the hexose and deoxy-hexose increments will be examined in the future. We suggest tentatively that such homologous series might include compounds similar to theaflavonin and desgalloyl-theaflavonin, in which one of the precursors is a flavonol glycoside.12,15 Further NLCs were obtained for smaller increments such as loss of water, loss of CO2 or demethylation again showing that our HSA hypothesis and analysis has considerable value (see supplementary information Figure S3†). Interestingly, neutral losses of CO2 are largely absent from the data and we shall return to this point at a later stage in this paper. Probing by tandem MS the progressive hydroxylation hypothesis for thearubigins structure In the preceding paper, we put forward a mechanistic hypothesis for the formation of TRs combined with a structural hypothesis for around 90% of the 1500 TR components so far identified by molecular formula.12 This hypothesis accommodates a wide range of TR components but is far from complete because 184 | Food Funct., 2010, 1, 180–199

multiply charged ions, components with masses above 1000 Da, and positive ion mode data have so far not been considered, with other structures certainly being present. This hypothesis proposes an initial formation of well-known catechin dimers (7– 13).16,17 These dimers are oxidised to ortho-quinones that in turn add water as a nucleophile to yield polyhydroxylated dimers. The oxidation to quinones and addition of water continues until all aromatic hydrogens are replaced by OH functionalities. In turn, the polyhydroxylated oligomers of catechins are in a redox equilibrium with their quinone counterparts by a two electron oxidation followed by loss of two protons. This hypothesis now requires evaluation using tandem MS experiments. The traditional chemistry approach to identifying a novel compound would be to prepare a crude isolate, purify it, and then obtain NMR, MS, IR and elemental composition data for a single component. If possible, synthesis would be used to confirm the identification. We have shown that the black tea TR fraction consists of at least 5000 components, ignoring isomers. If allowance is made for the presence of isomers then some 30 000 to 50 000 substances would be expected. The best current LC This journal is ª The Royal Society of Chemistry 2010

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Fig. 2 a) TIC in negative ion mode of sample TR VI, b) constant neutral loss chromatogram of neutral loss at m/z 152 (gallate), c) constant neutral loss chromatogram of neutral loss at m/z 162 (hexose), d) constant neutral loss chromatogram of neutral loss at m/z 146 (deoxy-hexose).

methods resolve only some 200 to 300 compounds and the isolation and purification from this of a single TR is far beyond the current capabilities of separation science. Synthesis of compounds (our interpretation would suggest around 30 000 target compounds) selected from those that we believe to be present is feasible but would not greatly assist when the mixture occurring naturally cannot be resolved.18 A full analysis of all 500 homologous series identified would be far beyond the scope of this paper but is in principle possible with the data available and the analysis strategies introduced here for the first time.12 For evaluation of our hypothesis, we chose four homologous series (A–D, see Table 2) to which the addition of oxygen had been identified previously, for which a pure standard of the parent molecule was available, and for which the ESIFTICR MS molecular ion was sufficiently intense to allow fragment spectra to be obtained. To facilitate description of the TR chemistry we introduce a compound nomenclature in a simple and logical manner to describe the hydroxylated derivatives and associated quinone forms. Taking theaflavin 8, for example, the hydroxylated This journal is ª The Royal Society of Chemistry 2010

derivatives will be 8 + O1, 8 + O2, etc. With the exception of the fully hydroxylated derivative (8 + 07), there will be many regioisomers. The designation 8 + Ox where x is any positive integer from one to seven (or as otherwise appropriate for the theoretical maximum) insertions will be used to refer to the totality of the oxygenated derivatives. Putative quinones are designated as 8 + O1H2 or 8 + O2H4 indicating the loss of two or four hydrogens respectively from the parent compound within the homologous series. Our basic experimental strategy was first to generate extracted ion chromatograms at the mass of the parent ion for each member of the four homologous series A–D under investigation, and when the signals were strong enough to obtain MS2 and MS3 spectra. This approach established that signals were obtained at m/z values corresponding to the majority of predicted hydroxylated derivatives in each of the four homologous series. In many cases, predominantly for two or three oxygen insertions, several regioisomers can be detected at distinct retention times. The signals for the associated quinones and the more extensively hydroxylated derivatives Food Funct., 2010, 1, 180–199 | 185

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Table 2 Four representative homologous series A–D of molecular formulas from black tea thearubigin samples TR IV with one oxygen incrementally added (m/z value of [M-H] ion added below molecular formula)

Series

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Parent compound O1 1 O inserted O2 2 O inserted O3 3 O inserted O4 4 O inserted O5 5 O inserted O6 6 O inserted

A: Theaflavins C29H24Ox

B: Theaflavin mono-gallates C36H28Ox

C: Theaflavin di-gallates C43H32Ox

D: Theacitrin mono-gallates C37H28Ox

8 C29H24O12 563.2 8 + O1 C29H24O14 579.2 8 + O2 C29H24O15 595.2 8 + O3 C29H24O16 611.2 8 + O4 C29H24O17 627.2 8 + O5 C29H24O18 643.2 8 + O6 C29H24O19 659.2

9 C36H28O15 715.2 9 + O1 C36H28O16 731.2 9 + O2 C36H28O17 747.2 9 + O3 C36H28O18 763.2 9 + O4 C36H28O19 779.2 9 + O5 C36H28O20 795.2 9 + O6 C36H28O21 811.2

11 C43H32O19 867.3 11 + O1 C43H32O20 883.3 11 + O2 C43H32O21 899.3 11 + O3 C43H32O22 915.3 11 + O4 C43H32O23 931.3

7 C37H28O18 759.2 7 + O1 C37H28O19 775.2 7 + O2 C37H28O20 791.2 7 + O3 C37H28O21 807.2 7 + O4 C37H28O22 823.2 7 + O5 C37H28O23 839.2 7 + O6 C37H28O24 855.2

were on occasions too weak to provide higher order fragmentation spectra. To circumvent this problem, our second approach was to use direct infusion MSn. In this operating mode, a signal can be maintained for much longer (minutes) than in LC–MS (seconds) permitting optimisation of the trapping and fragmentation. However, in general, the procedure must be automated and there are other significant practical limitations. For example, setting an isolation width of 1 Da and investigating the mass range 600 m/z to 1000 m/z generates 400 spectra per sample. Even with a 1 Da isolation width between 5 and 10 molecular ions (see mass table in ref. 13) are isolated from the TR sample in the ion trap and fragmented at the same time, resulting in fragment ions originating from a considerable number of molecular ions. Furthermore, each molecular ion can in theory correspond to up to ten different regio- and stereoisomers, increasing the number of structurally distinct parent ions to over 100. To complicate matters yet further, a large number of direct infusion MS2 fragment spectra show base peaks at m/z values higher than that of the parent ion. This confirms that doubly charged precursor ions of higher molecular mass are present in the sample, as previously indicated by MALDI-TOF MS.12 To our knowledge, this is the first time that such tandem MS experimental strategies have been implemented to characterise a complex polyphenolic mixture of dietary significance. Four hundred MS2 spectra were obtained at intervals of 1 Da with an isolation width of 1 Da each for two randomly selected TR samples, TR XII and TR IV. Our rationale for data interpretation is as follows. The fragmentation pattern and mechanism of fragmentation for the first member in each homologous series (e.g. theaflavin 8, theaflavin 3, 30 -digallate 11, etc.) has been determined experimentally with authentic standards or obtained from literature data.15 186 | Food Funct., 2010, 1, 180–199

For flavonoids, it has been well established that variations in B-ring and A-ring hydroxylation do not alter the mechanism of fragmentation. For example, the B-ring hydroxylation series (epi)afzelchin, (epi)catechin and (epi)gallocatechin (and associated proanthocyanidins) fragment by the same mechanism, with the RDA fragment increasing by 16 Da in parallel with the mass of the parent molecule. Similarly, it has been demonstrated for various classes of flavonoids analysed by negative ion LC–MS that the extent of A-ring hydroxylation is easily determined from the fragments observed.19,20 For ester fragmentation, e.g. degallation in MS2 for series of structurally related compounds identical fragmentation mechanisms have been observed.21,22 Accordingly, we believe that it is reasonable to expect that similar, if not identical, fragmentation mechanisms will apply to all members of any one of the homologous series that we are investigating. Therefore, if an ion corresponding to an expected hydroxylated derivative fragmented in the same manner as the parent molecule, this would be interpreted as consistent with our hypothesis. The presence of other fragment ions, potentially arising from coeluting and/or simultaneously trapped species would be considered not to invalidate this interpretation. Although it was anticipated that in each series most intermediate hydroxylation levels would be observed, the apparent absence of some would not invalidate the interpretation because some regioselectivity in the nucleophilic addition of water to the quinone can reasonably be expected. Similarly, although it was expected to detect some regio-isomers during LC–MS, failure to detect all the theoretical forms might arise because of co-elution, or some simply being below the limit of detection. It was anticipated that generally retention time would decrease relative to the parent molecule as hydroxylation increased unless internal hydrogen bonding significantly increased the hydrophobicity. However, if all the hydroxylated derivatives in any series eluted after the parent molecule, this would cast doubt on the identification. This journal is ª The Royal Society of Chemistry 2010

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Homologous series A: Theaflavins An authentic purified reference standard of theaflavin 8 shows a rather complex MS2 spectrum with a base peak at m/z 462.9, of uncertain structure, and a further characteristic fragment at m/z 425.1. This fragment can be rationalised in terms of a retro Diels– Alder fragmentation (RDA) at one of the two benzopyran moieties with a neutral loss of an enone at m/z 137 (C7H6O3) and an enol fragment at m/z 425 (C22H15O9). The mechanism of fragmentation is shown in Scheme 2 and the fragmentation of theaflavin 8 during tandem MS has been discussed by Mulder.23 The enol fragment has five sites available for hydroxylation, two in the A-ring and three in the fused ring system, whereas the eliminated benzopyran fragment has two sites in the catechin A-ring. Accordingly, the neutral mass loss of either 138, 154 or 170 amu is diagnostic for the extent of hydroxylation in this part

of the molecule. It should be noted, however, that certain flavanol and/or theaflavin tautomers (see Fig. 5) would be susceptible to an additional hydroxylation at C4 (and/or C40 ) increasing the theoretical maximum hydroxylation in theaflavin 8 from seven to nine, and the maximum insertions in the RDAeliminated benzopyran fragment from two to three. Direct infusion ESI-FTICR MS and LC-MS measurements12 detected the starting member theaflavin 8 C29H24O12 (at m/z 563.2) and putative hydroxy theaflafin 8 + O1 C29H24O13 (at m/z 579.2), 8 + O2 C29H24O14 (at m/z 595.2), 8 + O3 C29H24O14 (at m/z 611.2) and 8 + O4 C29H24O15 (at m/z 627.2). As shown in Fig. 3, the EICs prepared from LC-MS data located these same masses (although 8 + O1 was rather weak), with evidence for several regioisomers at 8 + O2, 8 + O3 and 8 + O4 (Scheme 3). In order to evaluate the assignment of these peaks as hydroxytheaflavins EICs were prepared at the masses corresponding to

Scheme 2 Mechanism of fragmentation of theaflavin 8 and retro Diels–Alder fragmentations of selected members of series A compounds 8 + Ox (regioisomers selected randomly).

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Fig. 3 EIC chromatograms for parent ions in homologous series A: a) EIC of ion 8 at m/z 563.2, b) EIC of ions 8 + O1 at m/z 579.2, (arrows indicating location of peaks of low intensity), c) EIC of ions 8 + O2 at m/z 595.2, d) EIC of ions 8 + O3 at m/z 611. e) EIC of ions 8 + O4 at m/z 627.2.

the expected RDA fragment ions at m/z 425.1, 441.1, 457.1, 473.1 and 489.1. Selected data for one TR sample are shown in Table 3 and supplementary information (Fig. 4). A close inspection of the MS2 and MS3 spectra of these peaks revealed the following: The peak at 46.1 min retention time (RT), corresponding to theaflavin 8, produces the known RDA fragment at m/z 425. Five 8 + O1 regio-isomers were detected at m/z 579, all eluting faster than theaflavin 8. The possibility that some of these might be (epi)theaflavic acid gallates, previously characterised black tea components,15 was excluded because it was not possible to detect losses of 44 amu (decarboxylatoin) or 152 amu (degallation). Two 8 + O1 regio-isomers show a transition from m/z 579 to m/z 425 indicating that one oxygen has been inserted in the RDA-eliminated benzopyran moiety. One suffered a loss of 138 amu indicating that the hydroxyl had been inserted in the benzotropolone (see Scheme 2). Two lost 122 amu suggesting that the RDAeliminated benzopyran moiety contained one less hydroxyl than is found in theaflavin 8, and accordingly two hydroxyls had been inserted in the benzotropolone moiety. This is discussed below. Six 8 + O2 regio-isomers were detected at m/z 595, all eluting faster than theaflavin 8. Four 8 + O2 regio-isomers transitioned from m/z 595 to m/z 425 indicating that two hydroxyls had been inserted in the RDA-eliminated benzopyran moiety. Two 8 + O2 regio-isomers transitioned from m/z 595 to m/z 489 suggesting that the RDA-eliminated benzopyran moiety contained two less hydroxyls than is found in theaflavin 8, and that four hydroxyls 188 | Food Funct., 2010, 1, 180–199

had been inserted in the benzotropolone moiety. This is discussed below. Only one 8 + O3 regio-isomer was detected at m/z 611, eluting faster than theaflavin 8. This transitioned from m/z 611 to m/z 457 indicating that one additional hydroxyl had been inserted in the RDA-eliminated benzopyran and the other two were in the benzotropolone moiety. Single 8 + O4, 8 + O5 and 8 + O6 regio-isomers were found but the signals were too weak to yield higher order spectra. The direct infusion tandem MS experiments confirmed the foregoing and yielded additional data, with parent ion–fragment ion transitions as follows: 1. For 8 + O2, additional isomers were detected with two insertions in the benzotropolone (m/z 595 to m/z 457), one insertion in the benzopyran with the second insertion in the benzotropolone (m/z 595 to m/z 441), and with three insertions in the benzotropolone associated with a benzopyran lacking one hydroxyl. 2. For 8 + O3, additional isomers were detected with three insertions in the benzotropolone (m/z 611 to m/z 473), one insertion in the benzotropolone plus two insertions in the benzopyran (m/z 611 to m/z 441), and three insertions in the benzopyran (m/z 611 to m/z 425). 3. For 8 + O4 one isomer with four insertions was detected (m/z 627 to m/z 425, loss of 202, RDA fragment with four oxygens inserted) pointing to potential addition of hydrogen peroxide to a quinone. The presence in a black tea infusion of This journal is ª The Royal Society of Chemistry 2010

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Fig. 4 EIC chromatograms extracted from all MSn data for RDA fragment ions in homologous series A: a) EIC of fragment ion at m/z 425.1, b) EIC of fragment ion at m/z 441.1, c) EIC of fragment ion at m/z 457.1, d) EIC of fragment ion at m/z 473.1, e) EIC of fragment ion at m/z 489.1.

H2O2 at concentrations of around 30–60 mM was previously established by Subramanian.24 So addition of H2O2 a much more powerful nucleophile, due to its a-effect, if compared with water, seems feasible in black tea, to form an aromatic hydroperoxide. Unfortunately the hydroperoxide moiety appears as a neutral loss in the mass spectrum and can therefore not be easily further investigated to further substantiate this possibility. However, we expand our structural hypothesis on TR formation at this point to include as well H2O2 as a potential nucleophile involved in TR formation. It must be noted that when fragmentation data indicate that two (or four) oxygens have been inserted in a particular moiety, one (or two) might be a peroxide. Additional isomers were detected with three insertions in the benzopyran plus one in the benzotropolone (m/z 627 to m/z 441), two insertions in the benzopyran plus two in the benzotropolone (m/z 627 to m/z 457), one insertion in the benzopyran plus three in the benzotropolone (m/z 627 to m/z 473), and four in the benzotropolone (m/z 627 to m/z 489). 4. For 8 + O5 Additional isomers were detected with three insertions in the benzopyran plus two in the benzotropolone (m/z 643 to m/z 457). 5. For 8 + O6 one isomer with four insertions was detected (m/z 659 to m/z 457) pointing to potential addition of hydrogen peroxide to a quinone.24 Additional isomers were detected with three insertions in the benzopyran plus three in the benzotropolone (m/z 659 to m/z 473), and two insertions in the benzopyran plus four in the benzotropolone (m/z 659 to m/z 489). This journal is ª The Royal Society of Chemistry 2010

Several transitions, e.g. m/z 595 to m/z 489 or m/z 579 to m/z 457, corresponding to neutral losses of 122 and 106 respectively, have been observed in these experiments. Assuming these are RDA fragmentations, this suggests the absence of one or two OH functionalities at one A ring of the theaflavin. While such compounds are feasible, to our knowledge neither they nor their putative dehydroxylated precursors have been reported in tea. It is suggested that they might form through a vinylogous dehydroxylation similar to that seen in conversion of EGCG to tricetanidin 13 and possibly involving the tautomer that is susceptible to hydroxylation at C4. All data are summarised in Table 3. Selected MS2 spectra are shown in the supplementary information (Figure S6†). In no case was the theoretical maximum oxygenation exceeded and collectively the data argue strongly for the presence of at least 29 polyhydroxy-theaflavins 8 + Ox in TR samples. Only one compound structurally similar to 8 + O1 has been previously reported in the literature by Matsuo.25 Homologous series B: Theaflavin mono-gallates Authentic purified reference standards of theaflavin 3-gallate 9 and theaflavin 30 -gallate 10 show MS2 spectra with a base peak at m/z 563.2 corresponding to a degallated theaflavin (C29H23O12). The mechanism of fragmentation is shown in Scheme 4 and further details on theaflavin tandem MS have been discussed by Mulder.23 The theaflavin mono-gallates have eleven sites Food Funct., 2010, 1, 180–199 | 189

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Fig. 5 Selected MS2 spectra of quinone series C obtained by direct infusion experiments of sample TR XII in negative ion mode showing degallated fragments: a) MS2 of parent ion 11H2 at m/z 865 showing a fragment at m/z 713.1; b) MS2 of parent ion 11H4 at m/z 863 showing a fragment at m/z 711.1; c) MS2 of parent ion 11 + O1H2 at m/z 881 showing a fragment at m/z 729.1; d) MS2 of parent ion 11 + O1H4 at m/z 879 showing a fragment at m/z 727.1.

available for oxygen insertion, three in each A-ring, two in the gallate moiety and three in the benzotropolone moiety. If oxygens have been inserted in the gallate moiety then ‘degallation’ mass losses of 170 or 186 amu would be expected. The theaflavin mono-gallates 9/10 + Ox homologous series B commences with theaflavin 3-gallate 9 and 30 -gallate 10 C36H28O15 (at m/z 715.2). Direct infusion MS experiments have indicated the presence of putative hydroxy theaflavin monogallates 9/10 + O1 at m/z 731.2 (C36H28O16), 9/10 + O2 at m/z 747.2 (C36H28O17), 9/10 + O3 at m/z 763.2 (C36H28O18), 9/10+O4 ar m/z 779.2 (C36H28O19), 9/10 + O5 at m/z 795.2 (C36H28O20) and 9/10 + O6 at m/z 811.2 (C36H28O21) this last with low intensity, indicating the insertion of up to six oxygens (see Scheme 4). Direct infusion MS demonstrated the loss of gallate from 9/10 + O1 to 9/10 + O6, inclusive. LC-MS data were searched for these masses. Tandem LC-MS detected an MS2 mass loss of 184 in two regio-isomers of 9/10 + O3 consistent with the presence of one gallate residue bearing two additional hydroxyls (Table 4). MS3 indicated that the third hydroxyl was inserted in the RDAeliminated benzopyran moiety. These hydroxylated derivatives were appreciably more hydrophobic than the 9/10 + O2 and the 190 | Food Funct., 2010, 1, 180–199

9/10 + O1 regio-isomers suggesting the presence of internal hydrogen bonds involving the hydroxylated gallate residue in 9/10 + O3, but they eluted in advance of the parent theaflavin mono-gallates. Gallate hydroxylation was not detected in 9/10 + O4. The EICs corresponding to these six oxygenation levels were prepared (see supplementary information Figures S5 and S6†) and demonstrated the presence of regio-isomers as follows: Three 9/10 + O1 regio-isomers with an m/z 731.2 to m/z 579 transition Three 9/10 + O2 regio-isomers with an m/z 747.2 to m/z 595 transition Three 9/10 + O3 regio-isomers with an m/z 763.2 to m/z 611 transition One 9/10 + O4 regio-isomer with an m/z 779.2 to m/z 627 transition The signals for 9/10 + O5 and 9/10 + O6 were too weak to be reliably distinguished from background. Results are summarised in Table 4. Selected MS2 spectra are shown in the supplementary information (Figure S6†). Collectively, the data argue strongly for the presence of at least 12 polyhydroxy theaflavin mono-gallates 9/10 + Ox in TR samples. This journal is ª The Royal Society of Chemistry 2010

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Table 3 Selected tandem MS data for theaflavin series A (C29H24Ox)

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Mass of Retention times Fragment ions observed Mass loss Fragment ions observed Compound Molecular MS1 to MS2 a in direct infusion MS2 [m/z] No formulas C29H24Ox parent ion[m/z] from EICs [min] in LC-MS2 EICs [m/z] 8

C29H24O12

563.2

46.1

461 (100%), 425 (70%)

102, 138

8 + O1

C29H24O13

8 + O2

C29H24O14

8 + O3

C29H24O15

579.2 579.2 579.2 579.2 579.2 595.2 595.2 595.2 595.2 595.2 595.2 611.2

37.1 28.3 17.3 16.1 11.8 23.3 20.8 19.3 17.9 16.3 12.8 13.1

441.1 457.1 425.1 457.2 425.2 489.2 425.1 425.1 489.1 425.1 425.1 457.2

138 122 154 122 154 106 170 170 106 170 170 154

8 + O4

C29H24O16

627.2

8 + O5 8 + O6

C29H24O17 C29H24O18

643.2 659.2

461.2 (100%), 425.1 (70%), 457.1 (10%) 457.1 (20%), 425.1 (30%)

425.1 (100%), 441.1 (12%), 457.1 (3%), 473.1 (3%), 489.1 (3%)

425.1 (2%), 441.1 (2%), 457.1 (5%), 473.1 (5%) 425.1 (5%), 441.1 (5%), 457.1 (80%), 473.1 (20%) 457.2 (35%) 457.1 (3%), 473.1 (12%), 489.1 (25%)

a 138 ¼ normal benzopyran; 154 ¼ one extra hydroxyl in benzopyran; 170 ¼ two extra hydroxyls in benzopyran; 122 ¼ see text – possibly a Benzopyran from which one hydroxyl has been eliminated; 106 ¼ see text – possibly a benzopyran from which two hydroxyls have been eliminated.

Homologous series C: Theaflavin 3, 30 -di-gallates

Scheme 3 Homologous series A of oxygen insertion into theaflavin 8 (regioisomers selected randomly).

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An authentic purified reference standard of theaflavin 3, 30 -digallate 11 shows MS2 spectra with base peak at m/z 715.2 corresponding to a mono-gallated theaflavin (C36H27O15). The mechanism of fragmentation is shown in Schemes 4 and 5. The oxygen insertion homologous series for theaflavin di-gallates (series C) commences with theaflavin 3, 30 -di-gallate 11 at m/z 867.2 (C43H34O18). Direct infusion ESI-FTICR MS (13) detected the parent molecule 11 (C36H27O15, m/z 867), and putative hydroxy theaflavin-digallates 11 + O1 at m/z 883.2 (C43H34O19), 11 + O2 at m/z 899.2 (C43H34O20), 11 + O3 at m/z 915.2 (C43H34O21), 11 + O4 at m/z 931.2 (C43H34O22), 11 + O5 at m/z 947.2 (C43H34O23) and 11 + O6 at m/z 963.2 (C43H34O24). The signals for 11 + O5 and 11 + O6 were of low intensity. At MS2, all showed the loss of gallate. Selected MS3 data confirm the structure of the MS2 fragment ions at m/z 715.2 and 731.2 as theaflavin di-gallates or hydroxy theaflavin di-gallates, respectively. All data are summarised in Table 5. Selected EICs and selected MS2 spectra are shown in the supplementary information (Figure S7†). The EICs corresponding to these six oxygenation levels were prepared and demonstrated the presence of regioisomers as follows: Three 11 + O1 regio-isomers with an m/z 883.2 to m/z 731.2 transition Two 11 + O2 regio-isomers with an m/z 899.2 to m/z 747.2 transition Three 11 + O3 regio-isomers with an m/z 915.2 to m/z 763.2 transition One 11 + O4 regio-isomer with an m/z 931.2 to m/z 779.2 transition It was not possible to obtain satisfactory fragmentation spectra for 11 + O5 and 11 + O6. Collectively, the data argue strongly for the presence of at least nine polyhydroxy theaflavin di-gallates 11 + Ox in TR samples. Food Funct., 2010, 1, 180–199 | 191

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Scheme 4 Mechanism of fragmentation of theaflavin mono gallate 9 and theaflavin digallate 11.

Homologous series D: Theacitrin mono-gallates An authentic purified reference standard of theacitrin 3-gallate 7 shows an MS spectrum with base peak at m/z 759.2 (C37H27O18). The mechanism of fragmentation is shown in Scheme 6 and involves two fragmentation routes, firstly the loss of a gallate moiety to give a base peak at m/z 607.1 (C30H23O14) and secondly loss of water to yield a fragment at m/z 751.2 (see Scheme 6).12 Using tandem LC-MS a second regioisomer of theacitrin gallate 33 possibly theacitrin 30 -gallate) could be observed, but its structure has never been unambiguously confirmed by NMR spectroscopy (15).

Therefore we consider in the discussion of this homologous series only derivatives of 7 with other possible regioisomers feasible as well. The oxygen insertion homologous series (D) shown in Scheme 7 commences with theacitrin 3-gallate 7 at m/z 759.2 (C37H27O18). Direct infusion MS detected putative hydroxy theacitrin mono-gallates 7 + O1 at m/z 775.2 (C37H27O19), 7 + O2 at m/z 791.2 (C37H27O20), 7 + O3 at m/z 807.2 (C37H27O21), 7 + O4 at m/z 823.2 (C37H27O22), 7 + O5 at m/z 839.2 (C37H27O23) and 7 + O6 at m/z 855.2 (C37H27O24), the last two with low intensities (see Scheme 7). At MS2, all showed the loss of gallate

Table 4 Selected tandem MS data for theaflavin-mono-gallate series B C36H28Ox

Compound No

Molecular formula C36H28Ox

9 10 9/10 + O1

C36H28O15

9/10 + O2

C36H28O17

9/10 + O3

C36H28O18

9/10 + O4 9/10 + O5 9/10 + O6

C36H28O19 C36H28O20 C36H28O21

a

C36H28O16

Mass of parent ion [m/z]

Retention times from EICs [min]

Fragment ions observed in LC-MS2 EICs [m/z]

715.2 715.2 731.2 731.2 731.2 747.2 747.2 747.2 763.2 763.2 779.2 795.2 811.2

49.2 50.6 38.8 22.5 23.8 17.7 14.5 13.3 44.9 35.6 43.8

563.2 (100%) 563.2 (100%) 579.2 579.2 579.2 595.2 595.2 595.2 579.2, 611.2 579.2, 611.2 627.2

Mass Loss from MS1 to MS2 152 152 152 152 152 152 152 152 184, 152 184, 152 152 152 152

a

MS3 ions of MS2 [m/z]

Mass Loss from MS2 to MS3 b

425.1 (100%) 425.2 (100%) 425.1

138 138 154

Fragment ions observed in direct infusion MS2 [m/z] 563.2 579.2 595.2

425.2, 457.1

154, 154

611.2 627.2 643.2 659.2

152 – loss of gallate; 184 ¼ loss of gallate containing two extra hydroxyls; 138 ¼ normal benzopyran; 154 ¼ one extra hydroxyl in benzopyran;.

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This journal is ª The Royal Society of Chemistry 2010

779.2 (30%)

763.2 (40%)

747.2 (8%)

715.2 (100%), 563.2 (40%) 731.2 (100%) 563.2 (100%) 579.2

152 152 152 152 152 152 152 152 152 152 715.2 (100%) 731.2 731.2 731.2 747.2 747.2 763.2 763.2 763.2 779.2 51.2 37.5 34.2 33.6 25.5 22.6 21.1 18.6 16.3 (broad) 9.9 C43H32O21

C43H32O22

C43H32O23

11 + O2

11 + O3

11 + O4

867.3 883.3 883.3 883.3 899.3 899.3 915.3 915.3 915.3 931.3 C43H32O19 C43H32O20 11 11 + O1

Retention times from EICs [min] Mass of parent ion [m/z] Molecular formula C43H32Ox

Selected MS3 data confirm the structure of the MS2 fragment ions at m/z 607 and 623 as a theacitrin mono-gallate and a hydroxy theacitrin mono-gallate, respectively. All data are summarised in Table 6. The EICs corresponding to these six oxygenation levels were prepared (see supplementary information Figure S8†) and demonstrated the presence of regioisomers as follows:

Compound No

Scheme 5 Suggested mechanism of formation of oxygenated homologous series of compounds through successive oxidations and nucleophilic additions of water starting from theaflavin mono gallates 9 and 10 and theaflavin digallate 11 including expected fragmentation pathways (regioisomers selected randomly).

Table 5 Selected tandem MS data for theaflavin di-gallate series C C43H32Ox

Fragment ions observed in LC-MS2 EICs [m/z]

Mass Loss from MS1 to MS2

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MS3 ions of MS2 [m/z]

Fragment ions observed in direct infusion MS2 [m/z]

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Scheme 6 Mechanism of fragmentation of theacitrin-3-gallate 7.

Scheme 7 Homologous series D of theacitrin-3-gallate 7 with successive oxygen insertion (regioisomers selected randomly).

Three 7 + O1 regio-isomers with m/z 775 to m/z 623.2 and m/z 757 transitions for loss of gallate and water, respectively were located in the chromatograms. Three 7 + O2 regio-isomers with an m/z 791 to m/z 639 transition were located in the chromatograms. Two 7 + O3 regio-isomers with an m/z 807 to m/z 655 transition for loss of gallate were located in the chromatograms. Two 7 + O4 regio-isomers one of with an m/z 823 to m/z 671 transition for loss of gallate were located in the chromatograms. It was not possible to obtain satisfactory fragmentation spectra for 7 + O5 and 7 + O6. The MS signals for 7 + O3 and 7 + O4 were appreciably stronger than for 7 + O1 and 7 + O2. Somewhat contrary to expectations, only the 7 + O4 regioisomers eluted faster than the parent compound. These data 194 | Food Funct., 2010, 1, 180–199

argue strongly for the presence of at least 10 polyhydroxy theacitrin 3-gallates 7 + Ox in TR samples. Selected data for one TR sample are shown in Table 6. Selected tandem mass spectra are shown in supplementary information (Figure S9†). Aromatic diol–ortho-quinone equilibria As part of the polyhydroxylation hypothesis we suggested that each class of polyhydroxy dimers is in a redox-equilibrium with their quinone counterparts. Scheme 8 illustrates such a redoxequilibrium. An exhaustive investigation of these aromatic diol– quinone equilibria for all suggested and identified homologous series would require several hundred targeted MS, and a lengthy discussion, both of which are outside the scope of this paper. This journal is ª The Royal Society of Chemistry 2010

671.2 (40%), 809.2 (8%)

655.2 (10%), 793.2 (6%)

639.2 (5%)

607.2 (100%), 741.2 (40%) 623.2 (15%) 589 (100%), 427.1, 301.1 605.2

152, 18 152 152 152 152 152 152 152 152 152 607.2 (100%), 741.2 (40%) 623.2 623.2 623.2 639.2 639.2 639.2 655.2 655.2 671.2 759.2 775.2 775.2 775.2 791.2 791.2 791.2 807.2 807.2 823.2 823.2 C37H28O18 C37H28O19

C37H28O20

C37H28O21

C37H28O22

7 7 + O1

7 + O2

7 + O3

7 + O4

15.4 34.6 28.4 18.7 21.3 19.0 17.2 25.8 22.3 14.6 14.4

MS3 ions of MS2 [m/z] Mass Loss from MS1 to MS2 Fragment ions observed in LC-MS2 EICs [m/z] Retention times from EICs [min] Mass of parent ion [m/z] Molecular formula C37H28Ox Compound No

Table 6 Selected tandem MS data for theacitrin mono-gallate series D C37H28Ox

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Fragment ions observed in direct infusion MS2 [m/z]

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Scheme 8 Redox equilibrium between polyhydroxy-theaflavins and their quinone counterparts (regioisomers selected randomly).

LC–MS and direct infusion–MS data are presented in Table 7 for the quinones associated with homologous series A–D and these are discussed below. Representative MS2 spectra from direct infusion experiments are shown in supplementary information (Figure S9†). For all four series, it was possible to detect mono-quinone (i.e. –2H) and di-quinone (i.e. –4H) derivatives of the parent compound and derivatives that had undergone up to four hydroxylations. In the theaflavin series (A) the expected retroDiels–Alder fragment was always observed, and the patterns of hydroxylation (insertions in the RDA-eliminated benzopyran and in the benzotropolone) were consistent with those reported for the non-quinoid forms (Table 7). It should be noted that an alternative formal loss of 2H by a direct aromatic coupling (theasinensin type coupling) is not consistent with our fragmentation data. The mass of the RDA-eliminated benzopyran observed during direct infusion-MS was either m/z 138, m/z 152, m/z 154, m/z 168, m/z 170 or m/z 172 corresponding respectively to an unmodified benzopyran moiety, a mono-hydroxy-monoquinone derivative, a mono-hydroxy derivative, a di-hydroxymono-quinone derivative, a di-hydroxy derivative, and a di-hydroxy derivative in which additional reduction has occurred (Scheme 9). In some instances, the quinone(s) were clearly present in the benzotropolone moiety. During direct infusion-MS2, the quinones associated with the theaflavin mono-gallates, the theaflavin di-gallates and the theacitrin mono-gallates (with the exception of 7 + O3H4) all produced a fragment at m/z 152 that is probably the degallation as observed with the non-quinoid forms, but which might also be an RDA-eliminated mono-hydroxy-mono-quinone benzopyran fragment. Under the conditions employed in this investigation, the non-quinoid forms eliminate gallate in preference to the bemzopyran moiety. Food Funct., 2010, 1, 180–199 | 195

196 | Food Funct., 2010, 1, 180–199

Theacitrin mono-gallate Series D

Theaflavin digallates Series C

Theaflavin mono-gallate series B

Theaflavins series A

Series

8 + O2H4 8 + O3H2 8 + O3H4 8 + O4H2 8 + O4H4 9/10H2 9/10H4 9/10 + O1H2 9/10 + O1H4 9/10 + O2H2 9/10 + O2H4 9/10 + O3H2 9/10 + O3H4 9/10 + O4H2 9/10 + O4H4 11H2 11H4 11 + O1H2 11 + O1H4 11 + O2H2 11 + O2H4 11 + O3H2 11 + O3H4 11 + O4H2 11 + O4H4 7H2 7H4 7 7 7 7 7

7 + O3H4

595.2 C29H24O15 611.2 C29H24O16 627.2 C36H28O15 715.2 C36H28O16 731.2 C36H28O17 747.2 C36H28O18 763.2 C36H28O19 779.2 C43H32O19 867.3 C43H32O20 883.3 C43H32O21 899.3 C43H32O22 915.3 C43H32O23 931.3 C37H28O18 759.2 C37H28O19 775.2 C37H28O20 791.2 C37H28O21 807.2

+ O1H2 + O1H4 + O2H2 + O2H4 + O3H2

8H2 8H4 8 + O1H2 8 + O1H4 8 + O2H2

Compound

C29H24O12 563.2 C29H24O13 579.2 C29H24O14

Parent compound Molecular formula and mass

Table 7 Selected tandem MS data for diol–quinone equilibria

C37H24O21

803.2

773.2 771.2 789.2 787.2 805.2

755.2

C37H24O18 C37H26O19 C37H24O19 C37H26O20 C37H24O20 C37H26O21

929.3 927.3 757.2

911.2

C43H28O22 C43H30O23 C43H28O23 C37H26O18

743.2 761.2 759.2 777.2 775.2 865.2 863.2 881.3 879.3 897.2 895.3 913.2

591.2 609.2 607.2 625.2 623.2 713.2 711.2 729.2 727.2 745.2

C29H20O14 C29H22O15 C29H20O15 C29H22O16 C29H20O16 C36H26O15 C36H24O15 C36H26O16 C36H24O16 C36H26O17 C36H24O17 C36H26O18 C36H24O18 C36H26O19 C36H24O19 C43H30O19 C43H28O19 C43H30O20 C43H28O20 C43H30O21 C43H28O21 C43H30O22

561.2 559.2 577.2 d 575.2 593.2

Quinone parent ion [m/z]

C29H22O12 C29H20O12 C29H22O13 C29H20O13 C29H22O14

Quinone Molecular formula 423.1 (70%) 421.0 (45%) 425.1 100%) 423.1 (30%) 421.1 (5%), 423.1 (5%), 441.1 (12%) a 421.1 (60%), 439.1 (100%) 471.1 (3%), 469.1 (5%), 439.1 (12%) 455.1 (20%), 471.1 (5%) 453.1 (5%), 471.1 (15%), 485.1 (5%) 561.2 (20%) Not observed 577.2 (20%) 575.2 (25%) 593.2 (MS3 at 455.1 and 423.1) 591.2 (80%) 563.2, 609.2 607.2 (10%) 625.2 (45%) 623.2 (50%) 713.2. (100%) 711.2 (90%) 729.2 (100%) 727.2 (100%) 745.2 (15%) 743.2(30%) 761.2 (40%)(MS3 at 609.2 1 and 591.2) 759.2 (100%) (MS3 at 607.2, 589.2 and 463.1) 777.1 (20%) 775.2 (35%) 739.2 (15%), 607.2 (100%), 605.1 (15%) 737.2 (5%), 601.2 (40%), 603.1 (20%), 605.1 (20%) 755.2 (5%), 621.2 (8%) 619.1 (4%) 637.1 (10%) 635.1 (10%) 653.2 (50%), 775.2 (25%), 789.1 (10%) 653.2 (50%)

Fragment ion observed in MS2 of direct infusion tandem MS corresponding to expected quinone fragment

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150

18, 152 152 152 152 152, 30, 18

152 152 18, 150, 152 18, 154, 152, 150

152 (152, 170, 296)

92 198, 152 152 152 152 152 152 152 152 152 152 152 (152, 170)

152 152 152 (138, 170)

170, 152 138 168, 138 170, 154 170, 152, 138 152

138 138 138 138 172, 170, 152

Mass loss at MS2 (MS3)b

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a Also observed in LC-MS. b Normal benzopyran; 152 ¼ gallate or mono-hydroxy-mono-quinone benzopyran; 154 ¼ one extra hydroxyl in benzopyran; 168 ¼ di-hydroxy-mono-quinone benzopyran; 170 ¼ two extra hydroxyls in benzopyran; 172 ¼ two extra. c Hydroxyls in benzopyran in which one double bond has been reduced. d Similar fragmentation would be expected for dihydrotheaflavins reported by Tanaka et al.25

18, 152 18, 152 803.2 (40%), 669.1 (20%) 801.2 (30%), 667.2 (35%) 821.3 819.3 7 + O4H2 7 + O4H4 C37H28O22 823.2

C37H26O22 C37H24O22

Compound Parent compound Molecular formula and mass Series

Table 7 (Contd. )

Quinone Molecular formula

Quinone parent ion [m/z]

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Fragment ion observed in MS2 of direct infusion tandem MS corresponding to expected quinone fragment

Mass loss at MS2 (MS3)b

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At MS3, the theaflavin mono-gallate derivative 9/10 + O2H2 also yielded benzopyran fragments at m/z 138 and m/z 170 corresponding to an unmodified benzopyran and a di-hydroxy benzopyran, respectively. 9/10 + O3H2 uniquely produced a fragment at m/z 198 that is presumably a di-quinoid form of the m/z 202 fragment produced by 8 + O4 and 8 + O6. At MS3, the theaflavin di-gallate derivatives 11 + O3H2 and 11 + O3H4 yielded fragments at m/z 170 and m/z 152 corresponding to an RDA-eliminated di-hydroxy benzopyran moiety and either the second gallate or a mono-hydroxy-mono-quinone benzopyran moiety, respectively. During direct infusion–MS2 of the quinones associated with the theacitrin mono-gallate derivatives the elimination of water was frequently observed as previously reported for the nonquinoid forms. Fragments at m/z 154 (7H4) and m/z 150 (7H2, 7H4 and 7 + O3H4) were also observed. These correspond to an RDA-eliminated mono-hydroxy benzopyran moiety and a quinoid form of the gallate moiety, respectively. Of the 40 masses probed by direct infusion MSn all yielded fragments that could be rationalised by these substances being

Scheme 9 Fragmentation of theaflavin digallate 11 + O1 and its orthoquinone derivatives (regioisomers selected randomly).

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mono- or di-quinone forms of the parent molecules and polyhydroxylated derivatives belonging to homologous series A–D.

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The evaluation of an alternative hypothesis for thearubigins structure After several discussions with colleagues it became necessary to evaluate an alternative hypothesis for TR structure. This alternative hypothesis would explain the increased number of oxygens and reduced number of hydrogens by oxidative cleavage of aromatic polyol moieties, producing dicarboxylic acids. This transformation was first proposed by Roberts.26 It was considered initially as a possible feature of the theacitrins and has been proposed to explain the formation of theaflavates or theaflavic acid,15 and is thus worthy of serious consideration. This hypothesis and our original hypothesis are not necessarily mutually exclusive. We evaluated this dicarboxylic acid hypothesis by probing the data using the appropriate Homologous Series Analysis (HSA), i.e. (–C2H2) or (+O, –CH). These homologous series were not detected in the data. As a further check, we sought neutral losses of 44 a.m.u (–CO2) that would have been expected if the dicarboxylic acids were present in the TR. The neutral loss chromatogram (NLC) obtained clearly indicated that such fragmentation is of very limited occurrence. Metal oxidising agents such as FeCl3, KMnO4 or K2Cr2O7 are well known to cleave aromatic diols oxidatively yielding dicarboxylic acids. As a final precaution, to ensure that the protocol employed could indeed detect these dicarboxylic acids if present, a TR sample was deliberately oxidised by treatment with a small amount of KMnO4 solution, sufficient to achieve decoloration. The sample was subsequently analysed by tandem LC-MS and a neutral loss chromatogram at m/z 44 obtained. The data are shown in the supplementary information (Figure S10†). It can clearly be seen that a chemical oxidising agents induces oxidative aromatic ring cleavage and formation of carboxylic acids, whose presence can be established in a complex mixture using neutral loss analysis. These neutral losses are, however absent in the TR samples, suggesting the absence of ring cleaved derivatives.

Conclusion In conclusion we have provided an innovative general strategy for the mass spectrometric characterisation of complex materials (materials in which too many compounds are present to allow chromatographic separation). Such complex materials are ubiquitous in food chemistry (complex polyphenols in tea and cocoa, matured red wines, non-phenolic components in Maillard reactions, etc.), biological systems and environmental samples such as waste or NOMs. Previous complex mixture analysis has remained at the step of counting and sorting compounds at the high resolution mass spectrometry level and has failed to provide structural hypotheses.27–31 Our general strategy for complex mixture analysis comprises the following steps: 1. Chemical characterisation of the material using traditional spectroscopic methods to identify elemental composition, functional groups and molecular weight distributions. 198 | Food Funct., 2010, 1, 180–199

2. Ultra high resolution mass spectrometry to establish the number of compounds present and to establish molecular formulas. 3. Data analysis (van Krevelen analysis, Kendrick analysis, unsaturation analysis and homologous series analysis) that allow visualisation and interpretation of the tremendously complex data. 4. Formulation of one or several structural and mechanistic hypotheses for the mixture of components under consideration. 5. Critical evaluation of the structural hypothesis by tandem LC-MS and direct infusion tandem MS. It should be noted that, despite the enormous power of tandem MS a structural hypothesis is required prior to a meaningful design of tandem MS experiments and data interpretation. More importantly we have characterised thearubigins isolated from fifteen commercial teas by LC-MS and direct infusion tandem MS. In the process, we have confirmed the structure of around 45 previously assigned major TR components by tandem LC-MS. More importantly, we have tested our newly developed mechanistic and structural hypothesis for thearubigins structure. All the data obtained are consistent with the hypothesis previously formulated—the evidence is strong and in some cases compelling that a significant portion of the thearubigins are indeed poly-hydroxylated dimers of catechins that are in redox equilibrium with their quinone counterparts. We originally proposed that oxygenation arose by nucleophilic addition of water to a quinone and now expand this to include nucleophilic addition of hydrogen peroxide. The alternative dicarboxylic acid hypothesis could not be confirmed. This evidence comes from a careful investigation of tandem MS spectra, in which fragments expected to arise from homologous series of such compounds, are indeed observed. Thus, in this contribution we have suggested and confirmed, within the restriction of the technologically possible, the structures of more than 150 new thearubigin components. The methodology introduced will in the future allow a further confirmation of many of the 1500 molecular formulas assigned in the thearubigins so far. Fifty years after their discovery the structure and mechanism of formation of at least a significant part of the thearubigins has been unravelled. We have shown that black tea must be considered as the ultimate master of creating chemical diversity by turning only a handful of starting materials (the catechins) in the presence of two reagents (water and oxygen) into a myriad of thousands of structurally distinct reaction products. This structural diversity is programmed into the functional groups of simple tea polyphenols helping nature to achieve this enormous chemical diversity surpassing any complexity previously observed in nature.

Notes and references 1 S. Poulter, Daily mail online, dailymail.co.uk, 27th June 2008. 2 Price 2007, FAO newsroom 2007, http://www.fao.org/news/ newsroom-home/en/. 3 A. E. Bradfield and M. Penney, Journal of the Society of Chemistry and Industry, 1944, 63, 306–310. 4 E. A. H. Roberts and M. Myers, J. Sci. Food Agric., 1959, 10, 167–179.

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5 E. A. H. Roberts., Economic importance of flavanoid substances: tea fermentation, in The Chemistry of Flavanoid Compounds, ed. T. A. Geissman, Pergamon Press, Oxford, 1962, pp. 468–512. 6 A. J. Charlton, A. L. Davis, D. P. Jones, J. R. Lewis, A. P. Davies, E. Haslam and M. P. Williamson, J. Chem. Soc., Perkin Trans. 2, 2000, 317–322. 7 R. Jaiswal, T. Sovdat and N. Kuhnert, J. Agric. Food Chem., 2010, 58, 5471–5484. 8 E. A. H. Roberts, J. Sci. Food Agric., 1958, 9, 381–390. 9 M. E. Harbowy and D. A. Balentine, Crit. Rev. Plant Sci., 1997, 16, 569–581. 10 E. Haslam, Phytochemistry, 2003, 64, 61–73. 11 E. J. Gardener, C. H. S. Ruxton and A. R. Leeds, Eur. J. Clin. Nutrition, 2006, 1–16. 12 N. Kuhnert, Arch. Biochem. Biophys., 2010, 501, 37–51. 13 N. Kuhnert, J. W. Drynan, J. Obuchowicz, M. Witt and M. N. Clifford, Rapid Commun. Mass Spectrom., 2010, RCM-100370.R1, manuscript in press. 14 N. Kuhnert, M. N. Clifford and A.-G. Radenac, Tetrahedron Lett., 2001, 42, 9261–9264. 15 J. W. Drynan, J. Obuchowicz, M. N. Clifford and N. Kuhnert, Nat. Prod. Rep., 2010, 27, 417–462. 16 F. Hashimoto, G.-I. Nonaka and I. Nishioka, Chem.Pharm.Bull., 1992, 40, 1383–1389. 17 Y. Takino and H. Imagawa, Agric.Biol.Chem., 1963, 27, 666–667. 18 In future studies we intend to employ enzymic and electrochemical oxidation of pure flavanols and simple mixtures (for example EC and EGC) as previously employed in our laboratories but this

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19

20 21 22 23 24 25 26 27 28 29 30 31

time to monitor by LC-MSn. See e.g. S. C. Opie, M. N. Clifford and A. Robertson, J. Sci. Food Agric., 1995, 67, 501–505. S. de Pascual-Teresa and J. C. Rivas-Gonzalo, Application of LC– MS for the identification of polyphenols, in Methods in Polyphenol Analysis, ed. C. Santos-Buelga and G. Williamson, Royal Society of Chemistry, Cambridge, 2003, pp. 48–62. F. Cuyckens and M. Claeys, J. Mass Spectrom., 2004, 39, 1–15. M. N. Clifford, S. Stoupi and N. Kuhnert, J. Agric. Food Chem., 2007, 55, 2797–2807. M. N. Clifford, Z. Wang and N. Kuhnert, Phytochem. Anal., 2006, 17, 384–393. P. J. Mulder, C. J. van Platerink, W. Schuyl and J. J. M. van Amelsvoort, J. Chromatogr., B: Biomed. Sci. Appl., 2001, 760, 271–279. N. Subramanian, P. Venkatesh, S. Ganguli and V. P. Sinkar, J. Agric. Food Chem., 1999, 47, 2571–2578. Z. Matsuo, T. Tanaka and I. Kouno, Tetrahedron, 2006, 62, 4774–4783. E. A. H. Roberts, J. Sci. Food Agric., 1958, 9, 381–390. C. A. Hughey, R. P. Rodgers and A. G. Marshall, Anal. Chem., 2002, 74, 4145–4149. R. L. Sleighterr and P. Hatcher, J. Mass Spectrom., 2007, 42, 559–574. Z. G. Wu, R. P. Rodgers and A. G. Marshall, Anal. Chem., 2004, 76, 2511–2516. T. Tanaka, C. Mine and I. Kouno, Tetrahedron, 2002, 58, 8851– 8856. M. N. Clifford, J. Kirkpatrick, N. Kuhnert, H. Roozendaal and P. R. Salgado, Food Chem., 2008, 106, 379–385.

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PAPER

www.rsc.org/foodfunction | Food & Function

Antioxidant, anti-inflammatory and anti-browning activities of hot water extracts of oriental herbal teas Li-Chen Wu,*a Amily Fang-Ju Jou,b Si-Han Chen,b Chia-Ying Tien,b Chih-Fu Cheng,a Nien-Chu Fanb and Ja-an Annie Ho*bc

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Received 16th June 2010, Accepted 13th August 2010 DOI: 10.1039/c0fo00047g Traditionally, antioxidants are used to scavenge reactive oxygen species (ROS), which are harmful by-products of aerobic metabolism. Inulae Flos, Horsetail, Chinese Leucas, Broomweed and Indian Wikstroemia are five herbal teas commonly consumed by Asians. Our aim was to investigate the hot water extracts of these five herbal teas for their total phenolics/flavonoid contents and antioxidant capacities. Furthermore, with inflammation and hyper-pigmentation considered as two biological processes associated with elevated cellular oxidative stress, Inulae Flos water extract was chosen for further evaluation of its inhibitory effects on the production of LPS-induced inflammatory mediators (such as, TNF-a, IL-6, IL-1b) in RAW 264.7 cells and its anti-tyrosinase activity. Our findings suggest that Inulae Flos might be an alternative source as a potential antioxidant, and a noteworthy inhibitor of production of pro-inflammatory cytokines in a dose-dependent manner. Moreover, it could also serve as a potential natural food additive to prevent browning.

Introduction Many nutritional supplements on the market are derived from herbal extracts for their health benefits, such as antioxidation and immunomodulation.1,2,3,4 An increasing number of natural dietary supplement products are being developed in response to the increasing focus of consumers in terms of personal health. It is known that excess reactive oxygen species (ROS) tend to attack susceptible biomolecules such as nucleic acid and protein, causing oxidative imbalance of the antioxidant system. The resulting oxidative stress may lead to aging, inflammation, and other chronic diseases.5,6 It has also been reported that a wide array of diseases, ranging from coronary heart disease to cancer, are caused by inflammation.7 Undoubtedly oxidative stress underlies many human diseases, and the exploration of new, rich sources of natural antioxidants for scavenging cellular free radicals has become the top priority in developing functional foods or nutritional supplements. The consumption of herbal tea, particularly green tea, has been proven to scavenge free radicals and thus effectively reduce the risks of various chronic diseases due to its abundance of antioxidants.8–13 Antioxidants can therefore be regarded as a natural anti-inflammatory approach which helps improve health and reduce inflammation without the use of prescription medicine. Therefore, there has been an increasing interest in searching for new antioxidant nutraceuticals which have the potential health benefits to present a substitution for corticosteroids (a common anti-inflammation drug) as an approach to get rid of the root cause that leads to inflammations. a Department of Applied Chemistry, National Chi Nan University, Puli, Nantou, 545, Taiwan. E-mail: [email protected]; Fax: +886-49-2917956 b Department of Chemistry, National Tsing Hua University, Hsin-chu, 300, Taiwan c Department of Biochemical Science and Technology, National Taiwan University, Taipai, 106, Taiwan. E-mail: [email protected]; Fax: +886-23366-2271

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Antioxidants such as vitamin C, vitamin E, carotenoids and polyphenolics have been found for their additional bio-function as inhibitors of tyrosinase. Tyrosinase inhibitors have important applications in the food industry and the field of biomedicine. Tyrosinase inhibitors not only possess anti-browning ability in the processing of meats, vegetables and fruits,14–16 but they also exert anti-melanogenesis in mammalian cells. Tyrosinase, a multifunctional copper-containing enzyme (mono- and diphenolase activities) catalyzes melanin synthesis via the hydroxylation of tyrosine to o-diphenol and the oxidation of o-diphenol to highly reactive o-quinones,17 which then spontaneously polymerize to form compounds of high molecular weight or brown pigments, or undergo nucleophilic attack by amino acids and proteins, polyphenols, or water to form Michael type addition products18 that enhance the production of the brown color. Browning in foods, on the other hand, occurs in the presence of oxygen when the polyphenol oxidase (PPO) converts phenolic compounds into dark colored pigments. Since the fresh-cut produce (i.e., fruits and vegetables) industry is one of the fastest growing food sectors of the market, and chlorine solutions are used extensively to sanitize and extend the shelf-life of such products, concerns have been raised toward the possible formation of carcinogens from such chlorine usage. Though sodium sulfite, citric acid, cysteine, potassium sulfite, sulfur dioxide, sodium meta-bisulfite and oxyresveratrol are existing alternative commonly-used food additives that function to prevent browning in foods (i.e., fruits, vegetables and meats), substantial research efforts in exploring alternative sources of anti-browning agents are still been encouraged.14,19 Inulae Flos, Horsetail, Chinese Leucas, Broomweed, and Indian Wikstroemia, found in Asia, Europe and Pacific islands, are normally used as herbal teas for the treatment of sore throats, bronchitis, and digestive disorders by Asians. However, little information is available on the inhibitory effects of these herbal teas on antioxidation and inflammation activity.20 In this study, we quantified the hot water extracts of five commonly-used This journal is ª The Royal Society of Chemistry 2010

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herbal teas which grew locally in Taiwan for their total phenolic content, flavonoid content, and antioxidant capacity. Moreover, studies were conducted to evaluate their inhibitory effect on the production of LPS-induced inflammatory mediators (such as IL-6, IL-1b, and TNF-a) in RAW 264.7 cells. In addition, the hot water extract of Inulae Flos was chosen to be investigated for its potential anti-tyrosinase activity.

Materials and methods

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Reagents and materials All herbs, Inulae Flos, Equisetum spp. (Horsetail), Leucas chinensis (Chinese Leucas), Scoparia dulcis L. (Broomweed), Wikstroemia indica (L.) C. A. Mey. (Indian Wikstroemia), were obtained from a local market in Kaohsiung, Taiwan. Ascorbic acid, ascorbate oxidase, aluminium chloride anhydrous (AlCl3), 2-20 -azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS), bovine serum albumin (BSA), catechin, DL-3,4-dihydroxyphenylalanine (DL-DOPA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-(4,5dimethylthiazaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), Folin-Ciocalteau phenol reagent, ferric trichloridehexahydrate (FeCl3$6H2O), gallic acid, Griess reagent (modified), 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox), sodium nitrite (NaNO2), sodium acetate (CH3COONa), Tween-20, 2,4,6-tripyridyl-s-triazine (TPTZ) and 3,30 ,5,50 -tetramethyl benzidine (TMB) liquid substrate system for ELISA were purchased from Sigma Chemical (St. Louis, MO, USA). Dulbecco’s modified Eagles’s medium (DMEM), mushroom tyrosinase and penicillin/streptomycin solution were obtained from Hyclone (Logan, Utah, USA). Fetal bovine serum (FBS) was acquired from Biological Industries (Kibbutz BeitHaemek, Israel). Raw 264.7 cell line was obtained from the Culture Collection and Research Center (CCRC, Hsinchu, Taiwan). Anhydrous sodium carbonate (Na2CO3) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Acetic acid (CH3COOH) and potassium persulfate (K2S2O8) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Hydrochloric acid (HCl) was purchased from Riedel-de Ha€en (Seelze, Germany). Sulfuric acid (H2SO4) was purchased from Fluka (Buchs, Switzerland). Mouse IL-1b, and mouse IL-6 were purchased from Invitrogen (Carlsbad, CA, USA). Mouse TNF-a ELISA was purchased from Bender Medsystems (Burlingame, CA, USA).

measured using the microplate reader (Sunrise, Tecan Trading AG, Switzerland). The structural characterization of natural flavonoids presented in the Inulae Flos herbal tea extract was conducted by Fourier Transform Mass Spectrometry (Varian 901-MS, Palo Alto, CA, USA). The HPLC system was controlled by the Agilent Technologies 1200. Separation and retention studies were carried out on a LiChroCART125-4 RP18 (5 mm, 250 mm  2mm i.d.) column, which was purchased from Merck (Darmstadt, Germany). HPLC measurements were performed using the methanol–water (70 : 30 v/v) mobile phase at a flow rate of 0.8 mL min1. An isocratic elution was used for analysis of naringenin and amentoflavone. All injection volumes were 20 mL. The detection of naringenin and amentoflavone was carried out using UV-Vis spectrophotometry set at a wavelength of 283 nm and 370 respectively. Determination of total phenolic content The total phenolic content of the five oriental herbal tea extracts was determined using a modified Folin-Ciocalteu colorimetric method. In short, 0.125 mL of a known dilution of extract was mixed with 0.5 mL of Folin-Ciocalteu reagent, and allowed to react for six min. It was followed by the addition of 1.25 mL, 7% Na2CO3 solution to the mixture, and the total was brought up to 3 mL with D. D. water, and the color development was allowed to occur after 90 min. Finally the absorbance of reaction mixture was read at 760 nm. All tests were in triplicate. The measurement was compared to a calibration curve of prepared gallic acid solutions and expressed as gallic acid equivalent in milligrams.4 Determination of flavonoid content The flavonoid content was determined using a modified colorimetric method, in which 0.25 mL of the known dilution of extract was diluted with 1.25 mL of D. D. water, followed by the addition of a 75 mL 5% NaNO2 solution and allowed to stand for 5 min. Subsequently 150 mL of a 10% AlCl3 solution was added. After 6 min, 0.5 mL of 1 M NaOH solution was put in, and the mixture was diluted with another 0.0275 mL of D. D. water. Immediately after the solution was well-mixed, the absorbance of the solution at 510 nm was measured in comparison with the standard curve of prepared catechin solutions. All tests were in triplicate. The results were expressed as milligrams of catechin equivalents.1,4

Sample preparation - extraction and isolation

DPPHc radical-scavenging activity

The extraction of five herbal teas was initiated by soaking the dried herbs (50 g) with distilled and deionized water (D. D. water, 400 mL; resistivity not less than 18 MU$cm). Heating individual herbal tea under reflux at 100  C for 3 h was then carried out. After filtration, the supernatant was collected, followed by lyophilization to obtain extract powder. All crude extracts were stored in airtight packaging at 20  C until use.

The radical-scavenging activity was determined using the DPPHc method described previously.4 In brief, 0.5 mL of the extract dissolved in 80% ethanol was reacted with ethanolic 0.5 mM DPPHc solution (0.25 mL) and 100 mM acetate buffer (pH 5.5, 0.5mL). An 80% ethanol solution was used as a blank solution; the ethanolic 0.5 mM DPPHc solution, in the absence of sample or standard, served as the control. The decrease in absorbance of DPPH at 517 nm after 30 min of standing was measured. All tests were performed in triplicate. The antioxidant activity of the test sample is expressed as the median effective concentration for radical-scavenging activity (EC50), the amount of tested extract (mg) required for a 50% decrease in absorbance of DPPH radicals, expressed in terms of ascorbic acid equivalents. The

Apparatus Lyophilization was conducted using a Thermo Electron Corporation lyophilizer (Model ModulyoD-115, Waltham, MA, USA). The absorbance (optical density) of each sample was This journal is ª The Royal Society of Chemistry 2010

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inhibition (%) of DPPH absorbance was calculated based on the expression (Acontrol  Atest)  100%/Acontrol, where Acontrol is the absorbance of the control (containing only DPPHc solution) and Atest is the absorbance of the test sample. The absorbance of DPPHc was plotted against the antioxidant concentrations as standard curves to calculate EC50. Results of the assay are expressed in terms of vitamin C equivalents.

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ABTSc+ assay ABTSc+ reagent was prepared by mixing 2.45 mM potassium persulfate (K2S2O8) with 7 mM ABTS salt in 0.01 M phosphatebuffered saline (PBS, pH 7.4) and allowed to react for 16 h at room temperature in the dark. The resultant ABTSc+ radical cation was diluted with 0.01 M PBS (pH 7.4) to give an absorbance of ca. 0.70 at 734 nm. The standards and sample extracts were diluted 100-fold with ABTSc+ solution to a total volume of 1 mL and allowed to react for 3 more min. Control (with no standard or sample) was used as a blank and 0.99 mL of PBS were added to these controls instead. All tests were in triplicate. The inhibition (%) of ABTSc+ absorbance was calculated based on the expression (Acontrol  Atest)  100%/Acontrol. The absorbance of ABTSc+ was plotted against the antioxidant concentrations as standard curves to calculate EC50. Trolox, the water-soluble a-tocopherol (vitamin E) analog, served as a standard, and the results of the assay were expressed relative to trolox in terms of TEAC (trolox equivalent antioxidant capacity).4 FRASC assay Working FRASC reagent was first prepared by mixing 80 mL of 300 mM acetate buffer with 10 mL of 10 mM TPTZ (2,4,6tripyridyl-s-triazine), which was dissolved in 40 mM HCl; and 10 mL of 20 mM FeCl3$6H2O. One pair well set is used to account for the presence of total antioxidant in the sample (socalled experimental well), and another well is used to represent the background (so-called background well). 10 mL of D. D. water was added into the antioxidant wells (experiment wells), while 10 mL of 4 IU mL1 ascorbate oxidase was added into the background wells. Ascorbic acid served as the standard and was prepared to generate 0, 2, 4, 6, 8, 10 nmol of ascorbic acid/well.21 100 mL of test samples or ascorbic acid standards were dispensed into a paired set of wells in a 96-well plate. After the addition of 100 mL of the working FRASC reagent into the wells containing either ascorbic acid standard or test samples, the measurement of absorbance at 593 nm was taken within 2–3 min. All tests were in triplicate. The ascorbic acid concentration in the sample (mM) was calculated based on the expression of (Ae  Ac)  100/slope of the standard curve, where Ae is the absorbance of the experiment well, and Ac is the absorbance of the control well with ascorbate oxidase. Cell culture The mouse macrophage cell line Raw 264.7 were used in all experiments, which were maintained at 37  C and 5% CO2 in Dulbecco’s modified Eagles’ medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were plated at a density of 5  104 cells mL1 in 202 | Food Funct., 2010, 1, 200–208

a 96-well plate, and allowed to attach for 8 h, followed by the 24 h incubation with various herbal tea extract. Cell viability A colorimetric MTT assay was utilized to measure the mitochondrial activity in viable cells based on the conversion of MTT to formazan crystals by mitochondrial enzymes. The cell culturing procedure was as described above. Aliquots (20 mL) of the MTT stock solution were pipetted into each well and the plate was continued to be incubated at 37  C in a humidified 5% CO2 incubator. After 4 h, the medium was removed by aspiration and DMSO (100 mL) was added to each well to dissolve the formazan. Ten min later, the optical density of each well was measured spectrophotometrically at 570 nm. Results are exhibited from three replicate runs. Measurement of nitric oxide in LPS-treated RAW 264.7 cells The cell culturing procedure was as previously described. The cells were then incubated with 1 mg mL1 of lipopolysaccharide (LPS) to induce an inflammatory process. After the harvest of the cells, the supernatants were collected for the measurement of nitric oxide (NO) by Griess Reagent method. The nitrite concentration in the samples was determined by adding 100 mL of Griess reagent (0.4 g Griess reagent powder dissolved in 10 mL D. D. water) to 100 mL sample. The absorbance at 540 nm was then acquired thereafter to incubate for 10 min.22 The inhibitory effect was standardized with respect to the control group treated with LPS but without extracts in terms of A/A0 (%), where A is the value of A540 generated by the Griess reaction at a given concentration of a herbal tea, and A0 is that obtained from the control group treated with LPS but without extracts. Again all tests were in triplicate. Assessment of pro-inflammatory mediators in LPS-treated RAW 264.7 cells by ELISA The cell culturing procedure was again as described above. The cells were then incubated with 1 mg mL1 of lipopolysaccharide (LPS) to induce the elevated amount production of pro-inflammatory cytokines, tumor necrosis factor-a (TNF-a), Interleukin6 (IL-6) and Interleukin-1b (IL-1b), which are three major pro-inflammatory mediators. In order to investigate the potential anti-inflammatory effects of Inulae Flos, its hot water extract was added to the LPS-treated cultured cells (5  104 cells/well) at final concentrations of 50, 100 and 200 mg mL1. After 24 h of co-culturing, 100 mL of medium samples were sampled and subjected to analysis of selected pro-inflammatory cytokines (IL1b, IL-6 and TNF-a). Various cytokine levels in cell supernatant were determined by enzyme-linked immunosorbent assays (ELISA) according to the manufacturers’ instructions (Mouse IL-1b/IL-6 CytoSet, Invitrogen, Carlsbad, CA, USA; Mouse TNF-a module set, Bender MedSystems, Burlingame, CA, USA), in which most of the following reaction occurred at ambient temperature unless described otherwise. 100 mL of corresponding capture antibody (1–100 mg mL1) was added to a 96-well plate and incubated at 4  C overnight. Each well was subsequently washed with washing buffer (0.05% Tween 20 in phosphate buffered saline, PBS) once, after which 300 mL of This journal is ª The Royal Society of Chemistry 2010

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assay buffer (0.05% Tween 20 in PBS containing 0.5% (w/v) BSA) was added and allowed to incubate for 1 h to block nonspecific binding sites. It was followed by the addition of 100 mL of standard samples into designated wells, and thereafter 50 mL of working detection antibody (biotin-antibody conjugate, 0.2 mg mL1) was added into each well and allowed to incubate for 2 h. After the removal of unbound working detection antibody, 100 mL of streptavidin-HRP solution (0.2 mg mL1) was added to each well and incubated for 30 min. Finally 100 mL of TMB substrate solution was dispensed to each well and incubated for 30 min in the dark. At the very last, 100 mL of stop solution (1.8 N H2SO4) was added to terminate the reaction. The absorbance at 450 nm (reference absorbance at 650 nm) was obtained by an ELISA reader. The inhibitory effect was standardized with respect to the control group treated with LPS but without extracts in terms of A/A0 (%), where A is the value of A450 generated by the ELISA assay at a given concentration of an herbal tea, and A0 is that obtained from the control group treated with LPS but without extracts. All tests were in triplicate. Enzyme inhibition assay and enzymatic kinetic analysis This assay was performed based on the modification of procedures reported by Mason and Peterson.23 Mushroom tyrosinase was used for this assay because of its commercial availability. The inhibitory effects of the five herbal tea extracts on tyrosinase were measured by determining the oxidation rate of DL-DOPA as a substrate. The activity of tyrosinase was determined spectrophotometrically by examining dopachrome formation at 475 nm. In short, 0.8 mL of 0.625 mM DL-DOPA solution was mixed with 0.1 mL of Inulae Flos extracts separately as inhibitors (diluted with 0.05 M PBS, pH 6.5) to a final concentration of 2 mg mL1. The tyrosinase solution (100 units, 0.1 mL) was then added to the mixture and immediate measurement of the absorbance at 475 nm was taken and continuously monitored for 6.5 min to determine the initial rate of increase in the dopachrome concentration. The inhibitory effect of Inulae Flos is expressed as the percentage required to provide 50% inhibition (IC50). To evaluate the kinetics of the potential inhibitor candidate Inulae Flos, the tyrosinase activity corresponding to the different concentrations of DL-DOPA was investigated. For various concentrations of inhibitors (0, 4, 6 mg mL1), four replicates of the absorbance reading for varied concentrations of DL-DOPA (0.08, 0.12, 0.16, 0.2 mM) were converted to the reciprocal of the value of absorbance. The reciprocal of velocity was then plotted versus the reciprocal of concentration of the substrate. The apparent Michaelis constant (Km), the maximum velocity (Vmax) and the type of inhibition were analyzed using a Lineweaver– Burk plot.

had similar levels of total phenolic and flavonoid contents, with total phenolic contents of 558.45  53.62 and 517.24  10.38 gallic acid equivalents/100 g dried herb respectively, and the total flavonoid content of 340.59  4.37 and 342.79  19.31 mg catechin equivalents/100 g dried herb, respectively. Statistical analysis revealed that the total phenolic and flavonoid contents of Broomweed and Chinese Leucas showed no significant difference (p > 0.05), but were higher than the contents of the dried herbs of Horsetail and Indian Wikstroemia, whose total phenolic contents were around 170.11  1.72 and 158.196  0.69 gallic acid equivalents/100 g dried herbs respectively, whereas flavonoid contents were 178.16  0.59 and 117.35  1.56 mg catechin equivalents/100 g dried herbs, correspondingly. We thus concluded that Inulae Flos possessed significantly higher phenolic and flavonoid contents than those of other herbal tea hot water extracts (p < 0.05). Compared to the most widely consumed tea beverages such as green tea and black tea, Inulae Flos in relativity possesses an intermediate amount of polyphenolic content and flavonoid among the three, whereas those of green tea are the highest and black tea the lowest.24 The structure identification of effective compounds in the hot water extract of Inulae Flos was conducted by Fourier Transform Mass Spectrometry. Mass spectra gave two distinct ion peaks at m/z 453.2 and 555.1 [M + H+], and it is believed that these two peaks represented glycosylated naringenin and amentoflavone, respectively. To confirm the assumption, HPLC (Agilent Technologies 1200 series) analysis was carried out after an acid treatment of crude extract was performed. Such pre-treatment was to ensure the release of the free forms of polyphenolic and flavonoid from what is commonly present in plants such as glycosides or esters, or those that are bound to cell walls.25 Two major peaks were shown in the chromatograph of Inulae Flos, whose retention times are identical to those of naringenin and amentoflavone standards (data not shown), confirming the presence of naringenin and amentoflavone in the hot water extract of Inulae Flos. Assessment of antioxidant capacity Three assay systems, namely the DPPHc and ABTSc+ radicalscavenging assays and the FRASC assay were selected to evaluate the antioxidant activities of the five herbal tea hot water extracts. While DPPHc and ABTSc+ approaches have been applied widely to measure the antioxidant activities of

Results and discussion Content of phenolics compounds (total phenolics and flavonoids) The total phenolic content and flavonoid content of the five herbal tea extracts were determined. Of the five tested samples, the phenolics and flavonoid contents of Inulae Flos were the highest, with 1452.35  21.33 mg gallic acid equivalents/100 g dried herb and 1220.75  48.35 mg catechin equivalents/100 g dried herb, respectively. The Broomweed and Chinese Leucas This journal is ª The Royal Society of Chemistry 2010

Fig. 1 Assessment of the antioxidant activities of the herbal tea extracts using 3 approaches (DPPHc radical-scavenging assay, ABTSc+ radicalscavenging assay, and FRASC assay).

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Fig. 2 MTT assays performed to measure the survival rate of RAW 264.7 cells after treatment with different concentrations of the five herbal teas’ hot water extracts. The cell growth of the treated group was standardized with respect to the untreated control group in terms of A/A10 (%), where A is the value of A570 generated by the MTT assay at a given concentration of a herbal tea extract, and A0 is that obtained from the untreated control group. The inset table shows the IC50 values for the cytotoxicity of the five dried herbs. (aNot detected.)

Fig. 3 (a) Inhibitory effects of the five herbal teas’ hot water extracts on nitric oxide production. Significant difference from the control value: *p < 0.05. (b) Inhibitory effect of Inulae Flos extract on the production of LPS-induced pro-inflammatory cytokines. Significant difference from the control value: *p < 0.05.

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polyphenolics, FRASC assay is used to determine specifically the ascorbic acid content presented in the samples. The addition of ascorbate oxidase to a parallel sample removed the ascorbic acid, leaving a background value.26 As illustrated in Fig. 1, Inuale Flos showed the greatest antioxidant capacity (p < 0.05) than that of the other four herbal tea extracts as determined by these three assays.

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Cytotoxicity MTT assay was used to investigate the cytotoxicity of the five herbal teas prior to the experiments of their anti-inflammatory effects on LPS-induced RAW 264.7 cells. MTT is reduced by mitochondrial dehydrogenase to form formazan, an insoluble purple compound. We measured the cytotoxicity in terms of the intensity of the purple compound. Dead cells, on the other hand, did not form any purple formazan because the enzyme was degraded and lack of regular function. Among the five herbal teas, the Inulae Flos exhibited the highest cytotoxic effect on RAW 264.7 cells, as evident by its lowest IC50 of 389.8 mg mL1 listed in the inset table of Fig. 2. The macrophage cells were then

treated separately with 0, 100, 200 300, 400, 500 and 600 mg mL1 of the five herbal tea extracts to determine the maximum concentration that RAW 264.7 cells could tolerate and exhibit at least 80% viability. Cytotoxicity for each group was expressed as a percentage of the control group (without co-treatment with herbal tea extracts). As shown in Fig. 2, 200 mg mL1 was chosen as the maximum concentration for all five herbal tea extracts, at where it exhibited 80% cell viability. Moreover it was also found that a greater level of Inulae Flos might exert an antiproliferative effect on RAW 264.7 cells. Evaluation of anti-inflammatory effect Three pro-inflammatory cytokines, namely tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and interleukin-1b (IL1b) were evaluated in activated inflammatory cells. They are responsible for inducing neutrophil proliferation, fever and acute inflammatory phase, increasing the expression of adhesion factors on endothelial cells, and enabling the transmigration of leukocytes. Incidentally, it has been well understood that nitric oxide is not only a signaling molecule but also plays an important

Fig. 4 (a) Dose-dependent inhibitory effect of Inulae Flos on mushroom tyrosinase. (b) Lineweaver–Burk plots for the inhibition of Inulae Flos on mushroom tyrosinase for the catalysis of DL-DOPA.

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role in regulating inflammation through the up-regulation of leukocyte and endothelial adhesion molecules.22,27,28 Consequently, the quantitative assessment of the pro-inflammatory cytokines and nitric oxide can be regarded as the biomarkers for studying inflammatory response. The anti-inflammatory effects of the five herbal tea extracts in this study were evaluated through a Griess reagent method for determining NO production, and ELISA assays for screening in vitro levels of pro-inflammatory cytokine, TNF-a, IL-6, IL-1b. The percentage of inhibition for each was expressed as the ratio of NO production (measured at 540 nm) for each experimental group to that of LPS-induced cell control group. As shown in Fig. 3(a), only the Inulae Flos extract (with various concentrations: 50, 100, and 200 mg mL1) demonstrated a dose-dependent suppression on NO production (p < 0.05) with an EC50 of 139.74 mg mL1. However, no significant change was observed for the other 4 herbal tea extracts, and therefore their EC50 were undeterminable. Furthermore, the percentage of inhibition for each herbal tea extract was expressed as the ratio of pro-inflammatory cytokines (IL-1b, IL-6 and TNF-a) production (measured at 540 nm, reference at 650 nm) of each experimental group to that of the LPS-induced cell control group. Pro-inflammatory cytokines are secreted mostly by activated macrophages, and are involved in the up-regulation of inflammatory reactions. As shown in Fig. 3(b), LPS-induced cell groups treated with 50, 100, and 200 mg mL1 of Inulae Flos extract led to the suppression of the production of IL-6 at 50%, 19%, and 9% of the control group respectively, while TNF-a was lowered to 68%, 60%, and 43% of control respectively, and IL-1b was reduced to 59%, 48%, and 39% of control respectively. The treatment of Inulae Flos extract applied on the LPS-induced cells demonstrated the reduced production of the selected cytokines in a concentration-dependent manner (p < 0.05) without observed cytotoxicity (cell viability > 80%). Inhibitory effects of Inulae Flos hot water extracts on the activity of mushroom tyrosinase An enzyme activity assay was performed to determine the kinetics and mode of the inhibitory effect for Inulae Flos hot water extracts. We observed a dose-dependent inhibitory effect of Inulae Flos hot water extracts on the oxidation of DL-DOPA by mushroom tyrosinase (Fig. 4(a)); the IC50 was 4.35 mg mL1, which was equivalent to 26.8 mg mL1 in terms of dry herb weight. The inhibition kinetics of the Inulae Flos were subsequently analyzed by a Lineweaver–Burk plot. As seen in Fig. 4(b), the three lines, which were obtained from the uninhibited enzyme and two different concentrations of Inulae Flos extracts, intersected on the horizontal axis, indicating that Inulae Flos hot water extract-mediated inhibition of the concentration of DLDOPA to dopachrome is based on a noncompetitive inhibitory effect. The kinetic parameters, such as Km and Vmax, and inhibition rate constants (KI) for the mushroom tyrosinase/Inulae Flos system, were calculated to be 1.48 mM, 3.48 OD (optical density)/min, and 10.56 mg mL1 respectively. Melanogenesis is the process of protecting skin cells from the attack of free radicals resulting from various environmental 206 | Food Funct., 2010, 1, 200–208

Fig. 5 Chemical structures of amentoflavone (a) and naringenin (b).

factors (i.e., UV radiation) or from potential cellular injury caused by aging or cancer. However, hyper-pigmentation may cause negative impacts on agriculture commodities or on health and medical conditions. The search for tyrosinase inhibitor alternatives are therefore of great focus. We found that Inulae Flos water extracts noncompetitively inhibited the activity of tyrosinase, indicating that some constituents present in the extract would bind to both the enzymes (E) and the enzymesubstrates (ES). Noncompetitive inhibitors for tyrosinase, such as 3,4-dihydroycinnamic acid, 4-hydroxy-3-methoxycinnamic acid (isolated from Pulstillla cernua roots), and oxyresveratrol were previously reported.14,29,30 We thereby suggested that amentoflavone and naringenin found in Inulae Flos water extract revealed structural commonness of the presence of a resorcinol moiety as shown in Fig. 5.

Conclusions The hot water extracts of five herbal teas which are commonly used for relieving pain and inflammation in Asian countries were subjected to phytochemical characterization and bioactivity evaluation. Data obtained herein (summarized in Table 1) suggested that water extracts isolated from Inulae Flos possessed the greatest amount of total polyphenolics and flavonoids, and was the most effective antioxidant among the studied tea types for scavenging free radicals. In addition, the hot water extracts of Inulae Flos demonstrated a dose-dependent behavior in suppressing the production of nitric oxide and pro-inflammatory cytokines (IL-6, TNF-a, and IL-1b) in LPS-induced RAW 264.7 This journal is ª The Royal Society of Chemistry 2010

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Table 1 (a) Total phenolic contents, total flavonoid contents, antioxidation activities, ascorbate concentrations of the five herbal tea extracts and their inhibitory effects on nitrate production and anti-inflammatory effects. (b) The kinetic parameters and rate constants for the inhibition of mushroom tyrosinase by Inulae Flos (a) a

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Total phenolic content Total flavonoid contentb DPPHc scavenging abilityc ABTSc+ scavenging abilityd FRASC assaye Inhibitory effect on nitrate contents Anti-inflammatory effects

Inulae Flos

Horsetail

Chinese Leucas

Broomweed

Indian Wikstroemia

1452.35  21.33 1220.75  48.35 117.1  0.8 191.6  1.2 141.7  9.2 +f +

170.11  1.72 178.16  0.59 10.98  0.22 22.83  0.09 66.86  4.11 NDg —h

517.24  10.38 342.79  19.31 32.65  0.38 57.93  0.29 43.97  1.54 ND —

558.45  53.62 340.59  4.37 10.48  0.15 17.23  0.25 39.97  1.03 ND —

158.196  0.69 117.35  1.56 43.27  0.81 59.95  0.72 51.60  3.08 ND —

a mg gallic acid equivalents/100 g dried herb. b mg catechin equivalents/100 g dried herb. c mmol vitamin C equivalents/g in terms of EC50. d mmol trolox equivalents/g in terms of EC50. e mM of ascorbate concentration. f demonstrated inhibitory effect in dose-dependent manner. g not detected. h not tested.

(b) Km Vmax Kl Inhibition type

macrophages. Furthermore, Inuale Flos water extract containing amentoflavone and naringenin might also be a potentially useful alternative as an anti-browning agent or tyrosinase inhibitor that could be applied in fresh-cut produce and meat industries, or as cosmeceutical products for treating hyper-pigmentation or skinwhitening. In summary, Inulae Flos herbal tea extract, with respect to its possession of the most abundant antioxidant, its high effectiveness in suppressing inflammation among the studied herbal teas, and its inhibitory effect on the production of melanin, may serve as a potential dietary nutraceutical supplement to keep human beings healthy. Furthermore, it holds promise for becoming a natural food additive as an antibrowning agent.

Acknowledgements This study was supported by the National Science Council (NSC) in Taiwan, under grants NSC 97-2113-M-260-006-MY2 (Wu), 98-2627-M-260-002 (Wu), and 98-2113-M-007-013-MY3 (Ho). The authors thank Mr. Allan Yeh for his editorial assistance.

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Antioxidant capacity in cultivated and wild Solanum species: The effect of wound stress Christina B. Wegener* and Gisela Jansen

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Received 8th July 2010, Accepted 20th August 2010 DOI: 10.1039/c0fo00063a Wild potatoes are of increasing interest as a gene pool in breeding. In this study, 23 genotypes of two cultivated (S. tuberosum subsp. andigena, S. phureja) and two wild Solanum species (S. chacoense, S. pinnatisectum) were evaluated for contents of soluble phenols and soluble proteins as well as their antioxidant capacity measured as ascorbic acid and trolox equivalent. Amounts of phenols present in tuber tissue ranged from 0.25 to 2.84 mg kg1 fw. On average, S. pinnatisectum (pnt) exhibited 3.9-fold greater quantities of phenols in its tuber tissue than the other Solanum species. In pnt tissue, high phenol content coincided with high levels of soluble proteins and antioxidants. It is concluded that an involvement of individual accessions of pnt in breeding could be profitable for the antioxidant potential and thus for the nutritional value of new potato cultivars. The results also revealed that soluble phenols as well as proteins present in tuber tissue substantially contributed to the total antioxidant capacity of potatoes. Moreover, it was found that quantities of soluble phenols, proteins and antioxidants increased notably upon wounding the tubers, a fact which underlines the role of all these components in wound stress responses of potatoes.

Introduction A high level of tolerance to biotic and abiotic stress as well as enhancing health-related quality traits like antioxidants, vitamins and anticancer compounds are the most important topics for plant breeding in the future.1 Also breeding of potatoes is focused on these traits.2–4 Particularly, plant phenols comprising hydroxycinnamates, flavonoids, tannins, lignin etc. are of great importance in this context. The latter are mainly derived from cinnamic acid synthesised via the phenylpropanoid metabolism of plants.5 Phenolic compounds are not only associated with expression of disease resistance in plants,6 they also act as radical scavengers7 and are thus part of the plant antioxidant system diminishing undesired effects of oxidative stress on metabolism and cells, as caused by various environmental stresses.8 When consumed in the diet, plant phenols are incorporated in an antioxidant network protecting animal and human cells against oxidative damage.9,10 In addition, they are involved in a variety of essential physiological functions associated with acclimation of plants to stressful environments,7 and similar to reactive oxygen species (ROS), plant phenols are inducible by environmental stresses.11 Accordingly, it has been argued that adaptation of plants to environmental stresses coincides with an improvement of the nutritional value.12 In this context, the rich genetic resource comprised by wild potatoes could be an interesting source,1 which should increasingly be exploited in the future in order to improve the stress tolerance and with it the nutritional value of cultivated potatoes. In previous work a multitude of accessions of wild, tuber-bearing Solanum species and Andean cultivated potatoes as well as an international

Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, Institute for Resistance Research and Stress Tolerance, Experimental Station for Potato Research, Rudolf-Schick-Platz 3, OT Groß L€ usewitz, D-18190 Sanitz, Germany. E-mail: [email protected]

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assortment of S. tuberosum subsp. tuberosum has been evaluated with respect to quality traits like concentrations of dry matter, crude protein and starch in tuber tissue including starch quality characteristics.13 In the present study, two cultivated (S. tuberosum subsp. andigena, adg; S. phureja, phu) and two wild, tuber-bearing Solanum species (S. chacoense, chc; S. pinnatisectum, pnt), each represented by several accessions and genotypes (Table 1) were assessed for contents of soluble phenols and proteins in tuber tissue as well as their antioxidant capacity measured as ascorbic acid (ACE) and trolox (TXE) equivalent. Furthermore, the effect of wounding, as one of the major stress factors for plants in nature,11 on the level of soluble phenols, proteins and antioxidants has been examined. Investigating the aftermath of wound stress on the level of antioxidants in tuber tissue is a novel aspect in context with wild potatoes. Apart from the investigation of stress-inducible responses as a prerequisite for the development of stress tolerant crops, whose plants are able to produce high yield under stress conditions,12 the effect of wounding the tubers is interesting with respect to general quality characteristics of potatoes which are offered on the markets or processed in the food industry, especially in the potato processing industry.

Table 1 Series, species, abbreviations and number of genotypes of cultivated and wild Solanum species involved in the tests

Series

Number of Abbreviations genotypes

Species

Tuberosa - cultivated S. tuberosum subsp. andigena Tuberosa - cultivated S. phureja Yungasensa S. chacoense Pinnatisecta S. pinnatisectum Altogether

adg

5

phu chc pnt

6 6 6 23

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Material and methods

Assay of soluble proteins

Plant material

The tissue slices excised from the cylinders prepared from (i) fresh and (ii) wounded tubers as described for the assay of soluble phenols were pooled and 3 g of slices were ground under liquid nitrogen using a mortar and pestle. The homogenate was centrifuged at 15 000  g for 10 min at 4  C. Amounts of soluble proteins were determined in the supernatant (¼ cell sap fraction) by means of a Bradford assay using a RotiR-Quant reagent (Roth, Karlsruhe, Germany) according to the manufacturer recommendations. The absorbance was measured at 595 nm on a UV spectrophotometer (Kontron Instruments). Standards were prepared from bovine serum albumin (Cohn fraction V; Sigma-Aldrich, Taufkirchen, Germany). Amounts of soluble proteins were calculated as milligrams per millilitre of extract. Analyses were carried out in duplicate (SD # 5%).

The experiments were carried out in Groß L€ usewitz, near the Baltic Sea. Seed tubers of two cultivated (adg, phu) and two wild, tuber-bearing Solanum species (chc, pnt), each represented by several accessions and genotypes (Table 1) were from the Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Potato Genebank, Groß L€ usewitz, Germany. The Solanum species used in the test series were chosen according to information about evaluation results on resistance properties to potato diseases available in the literature.14 Ten plants per genotype were grown in pots of 130 mm diameter under a shelter from April to October in 2008. Fertilizer, insecticides, fungicides and all other treatments were conducted according to local agronomic practice. After harvest, tubers were stored in a controlled environment at 5  C. All analyses started in November and finished in December of the test year.

Preparation of potato cylinder samples Twenty tubers were taken as an average sample from each genotype and halved. A cork borer of 5 mm diameter was used to cut two cylinders from the outer region of each half of each tuber. In order to test the effect of wounding on concentrations of total soluble phenols (A), soluble proteins (B) and antioxidants (C), two cylinder samples were taken per genotype and assay: the first one was excised from (i) fresh tuber tissue and a second was prepared (ii) 24 h after wounding the tubers. Before preparing the second sample, the tuber halves of each experimental set were stored for 24 h at 20  C with the wound-surface upward on moist filter papers placed in a plastic box which was covered with a glass plate.

Assay of total soluble phenols Preparation of extracts from (i) fresh and (ii) wounded tuber tissue for assaying total soluble phenols was carried out as detailed.15 A 1 mm thick slice was excised from the upper wound region of each cylinder cut from the tuber half as described above. The tissue slices were pooled, and 1 g of the slices was ground under liquid nitrogen using mortar and pestle. The homogenate was suspended in 4 mL of methanol (Roth, Karlsruhe, Germany). The suspension was stirred slightly and after 1 h centrifuged at 6000  g for 10 min at 4  C. The supernatant was removed and the plant material re-extracted. The total amount of phenols present in the combined extracts was determined using Folin-Ciocalteu reagent (Sigma-Aldrich, Taufkirchen, Germany) according to Cahill & Mc Comb.16 The absorbance was measured at 725 nm on a UV spectrophotometer (Kontron Instruments, Neufahrn, Germany). Standards were prepared from p-coumaric acid (Sigma-Aldrich, Taufkirchen, Germany). Amounts of soluble phenols (¼ coumaric acid equivalent) were expressed in grams per kilogram of fresh weight (fw). Extractions and measurements were performed in duplicate (SD # 5%). 210 | Food Funct., 2010, 1, 209–218

Assay of the antioxidant activity The tissue cylinders prepared from (i) fresh and (ii) wounded tuber tissue as described above were cut into 3 mm slices, after the peel region was removed from each cylinder by means of a scalpel. The slices were pooled, and 3 g of the tissue slices were ground under liquid nitrogen by mortar and pestle. The homogenate was suspended in a solution of 85% (v/v) ice cold ethanol. The suspension was stored on ice, occasionally shaken, and after 1 h centrifuged for 10 min at 8000  g and 4  C. The supernatant was removed and used for measurement of the antioxidant activity on a Photochem instrument, utilizing an ACW-kit for water soluble and ACL-kit for lipid soluble antioxidants, as detailed.15 The Photochem instrument as well as kit reagents were supplied by the AnalytikJena AG (Germany). This automated photochemiluminescent (PCL) method is based on a photochemical generation of free radicals combined with their detection by chemiluminescence as described recently.17 The antioxidant activity was calculated by means of an ascorbic acid calibration curve for hydrophilic antioxidants and a trolox calibration curve for lipid soluble antioxidants, using the Photochem software package. Results were expressed in microgram equivalents in antioxidant activity of the reference compound, i.e. as ascorbic acid (ACE) and trolox equivalents (TXE) per milligram of fresh weight, respectively. Measurements were performed in duplicate with SD # 5%. Statistical methods Conventional statistical methods were used for the analyses of the data. The differences between pnt and the other Solanum species with respect to soluble phenols, proteins and antioxidants comprising ACE and TXE were assessed using unpaired t-test, at the 0.05 level of significance. Differences in all these traits between extracts derived from (i) fresh tuber tissue and those prepared (ii) 24 h after wounding the tubers were valued by means of t-test for paired samples, and P < 0.05 was considered significant. Correlation coefficients (Pearson) were calculated between amounts of the individual substances, i.e. soluble phenols, proteins, water (ACE) and lipid soluble antioxidants (TXE), found in fresh tissue and those detected 24 h after wounding the tubers. Moreover, correlations were assessed between soluble phenols and antioxidant capacity including This journal is ª The Royal Society of Chemistry 2010

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ACE and TXE as well as between soluble proteins and both types of antioxidant activity. In addition, the relationship between phenols and antioxidant capacity as well as between proteins and the latter were examined by regression analyses.

Results

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Soluble phenols The Solanum genotypes involved in the tests differed considerably in their contents of soluble phenols (Table 2A). Within potato samples prepared from (i) fresh tuber tissue the amounts of phenols ranged from 0.25 to 2.84 mg kg1 fw, while comparative values for (ii) wounded tuber tissue ranged from 0.36 to

2.60 mg kg1 fw. Of the 23 genotypes involved in the study, pnt 31598-2 exhibited the highest levels of soluble phenols in its fresh and wounded tuber tissue with values of $2.60 g kg1 fw, followed by pnt 31598-3. All accessions of S. tuberosum subsp. andigena had less soluble phenols in their fresh tissue with values of <0.50 g kg1 fw (Table 2A). On average, the wild Solanum species S. pinnatisectum displayed the highest phenol contents (Table 2A). This was observed in samples from fresh tissue, which had 3.9-times the phenol concentrations of the other Solanum species, and also in samples taken 24 h after wounding (2.5). In tissue samples prepared after 24 h, the amounts of soluble phenols were on average greater (+28%) than in those derived from fresh tuber tissue (Table 2A). The differences in phenols

Table 2 Concentrations of soluble phenols and soluble proteins measured in fresh and wounded tuber tissue of the Solanum species/genotypes. Harvest 2008 (A) Soluble phenols (g kg1 fw)

adg 31881-1 31881-2 34155-1 34155-3 34155-5 Average Range Significancea chc 30161-12 30161-17 30180-3 30180-4 30180-13 30180-16 Average Range Significancea phu 31455-1 31455-2 31455-4 31467-2 31567-4 31567-8 Average Range Significancea pnt 31598-2 31598-3 31598-4 31606-1 31606-2 31606-3 Average Range Significancea All (n ¼ 23) Average Range Significancea Correlationb

(B) Soluble proteins (mg mL1)

Fresh tissue

Wounded tissue

Fresh tissue

0.43 0.39 0.34 0.48 0.34 0.40 0.34–0.48 P < 0.001

0.82 0.90 0.76 0.81 0.70 0.80 0.70–0.90

0.61 0.25 0.56 0.34 0.64 0.66 0.51 0.25–0.66 P < 0.001

0.77 0.36 0.73 0.45 0.79 0.73 0.64 0.36–0.79

0.31 0.55 0.52 0.45 0.67 0.44 0.49 0.31–0.67 P < 0.01

0.48 0.90 1.14 0.97 0.84 0.78 0.85 0.48–1.14

2.84 2.12 1.60 1.58 1.06 1.74 1.82 1.06–2.84 ns

2.60 2.10 1.76 1.53 1.41 1.87 1.88 1.41–2.60

11.40 13.52 13.12 11.96 14.44 12.84 12.88 11.40–14.44 ns

12.16 14.40 12.64 11.48 14.36 12.80 12.97 11.48–14.40

0.82 0.25–2.84 P < 0.0001 0.97, P < 0.01

1.05 0.36–2.60

9.48 2.80–14.44 P < 0.05 0.97, P < 0.01

9.93 3.32–14.40

2.80 7.28 3.44 11.48 4.96 5.99 2.80–11.48 P < 0.05

Wounded tissue

3.32 8.04 3.76 11.92 5.08 6.42 3.32–11.92

12.32 12.00 11.00 7.08 12.68 11.72 11.13 7.08–12.68 ns 10.16 5.56 6.24 4.56 11.48 5.92 7.32 4.56–11.48 ns

11.16 11.68 11.32 9.68 12.76 11.48 11.35 9.68–12.76 11.68 6.00 7.20 4.08 14.36 6.92 8.37 4.08–14.36

a Significance of the difference between fresh and wounded tuber tissue (n ¼ 23). b Correlation between amounts of soluble phenols and proteins detected in fresh tissue and those found 24 h after wounding (23 genotypes); ns, not significant P $ 0.05.

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between fresh and wounded tissue were statistically significant, when all genotypes were regarded, and also within the species adg, chc and phu. But, they were not significant within S. pinnatisectum. Individual genotypes of this wild Solanum species, e.g. pnt 31598-2, had even slightly lower quantities of soluble phenols in their wounded tuber tissue (Table 2A). Interestingly, S. tuberosum subsp. andigena which had only small amounts of phenols in its fresh tissue, revealed the greatest increase in phenols after wounding among the species involved in this study. Of the 23 genotypes tested so far, the S. phureja accession phu 31455-4 displayed the highest raise in phenols after 24 h (Table 2A). There was a clear relationship between amounts of phenols present in fresh tissue and those found 24 h after wounding the tubers (Table 2A). Furthermore, soluble phenols accumulated in fresh and wounded tuber tissue correlated with the antioxidant activity, including ACE and TXE, and also with soluble proteins as discussed below. Soluble proteins The Solanum genotypes also varied notably in their soluble protein concentrations (Table 2B). In potato samples taken from (i) fresh and (ii) wounded tuber tissue the quantities of proteins ranged from 2.80 to 14.44 mg mL1 and from 3.32 to 14.40 mg mL1, respectively. Among the 23 genotypes, pnt 316062 and pnt 31598-3 exhibited the greatest concentrations of soluble proteins in their fresh and wounded tuber tissue (Table 2B). On average, S. pinnatisectum revealed higher protein contents in both tissue types than the other three Solanum species had (Table 2B). Of the four species tested so far, S. tuberosum subsp. andigena had on average the lowest protein values in its fresh and wounded tuber tissue (Table 2B). Amounts of soluble proteins were greater on average in potato samples prepared 24 h after wounding than in those derived from

fresh tuber tissue (Table 2B). This difference was statistically significant when all genotypes were regarded (P < 0.05, +5.0%) and within adg (P < 0.05, +7.2%). Within the other Solanum species the protein values were also increased in their tendency after wounding the tubers, however, these differences were statistically not significant. Among the genotypes tested so far, phu 31567-4 exhibited the greatest raise of proteins in response to wounding (Table 2B), whereas individual accessions of S. pinnatisectum (e.g. 31598-4) showed even smaller amounts of soluble proteins in their wounded tuber tissue. There was a significant correlation between quantities of soluble proteins detected in fresh tissue and those found after wounding the tubers (Table 2B). Moreover, a correlation between soluble proteins and soluble phenols could be detected with respect to fresh tuber tissue (r ¼ 0.52, P < 0.01, n ¼ 23). In the case of wounded tissue, the correlation between these last two components was statistically not significant (r ¼ 0.36, P $ 0.05, n ¼ 23). Soluble proteins were also correlated with the ascorbic acid (ACE) as well as with the trolox equivalent (TXE) as mentioned below. Antioxidant activity The different genotypes of cultivated and wild Solanum species varied notably in their antioxidant capacity measured as ascorbic acid (ACE: Fig. 1) and trolox equivalent (TXE: Fig. 2). In potato samples taken from fresh tissue, the ACE and TXE values ranged from 0.06 to 4.22 mg mg1 fw and from 0.08 to 3.98 mg mg1 fw, respectively. In the case of wounded tuber tissue, the ascorbic acid and trolox equivalent ranged from 0.28 to 4.11 mg mg1 fw and from 0.23 to 4.26 mg mg1 fw, respectively. Among the genotypes tested here, pnt 31598-2 exhibited the highest ACE values in both types of tuber tissue (Fig. 1). The latter also reached the highest TXE in its fresh tissue (Fig. 2), whereas in wounded tuber tissue, pnt 31598-3 displayed the highest trolox

Fig. 1 Antioxidant activity measured as ascorbic acid equivalent (ACE) in extracts prepared from (i) fresh tuber tissue and in those taken (ii) 24 h after wounding the tubers of different Solanum genotypes.

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Fig. 2 Antioxidant activity measured as trolox equivalent (TXE) in extracts prepared from (i) fresh tuber tissue and in those taken (ii) 24 h after wounding the tubers of different Solanum genotypes.

equivalent. Of the four Solanum species analysed so far, S. pinnatisectum had on average the highest antioxidant potential comprising the ascorbic acid (Fig. 1, Table 3A) as well as the trolox equivalent (Fig. 2, Table 3B). Potato samples taken after 24 h had on average higher ACE (Table 3A, +44%) and TXE values (Table 3B, +26%) than those derived from fresh tuber tissue. These differences were statistically significant, when all genotypes were regarded and within

Solanum species adg, chc and phu. Also S. pinnatisectum exhibited on average higher ACE (Table 3A) and TXE values after wounding its tubers (Table 3B), however, in this case the differences were statistically not significant. Individual genotypes of pnt had even slightly lower ACE (e.g. pnt 31598-4) and TXE levels (e.g. pnt 31606-3) in their wounded tissue. Of the 23 genotypes assayed in this study, chc 30161-12 exhibited the greatest raise in the ascorbic acid equivalent after wounding

Table 3 Average antioxidant activity measured as ascorbic acid (ACE) and trolox equivalent (TXE) in fresh and wounded tuber tissue of the different Solanum species (A) ACE (mg mg1 fw) Species adg Average Range Significancea chc Average Range Significancea phu Average Range Significancea pnt Average Range Significancea All Average Range Significancea Correlationb

(B) TXE (mg mg1 fw)

Fresh tissue

Wounded tissue

Fresh tissue

Wounded tissue

0.15 0.08–0.24 P < 0.001

0.59 0.33–0.73

0.27 0.21–0.30 P < 0.01

0.62 0.37–0.76

0.54 0.19–0.85 P < 0.05

0.95 0.31–1.51

0.59 0.32–0.79 P < 0.05

0.74 0.47–0.96

0.12 0.06–0.21 P < 0.05

0.53 0.28–0.86

0.18 0.08–0.26 P < 0.05

0.48 0.23–0.80

2.46 0.89–4.22 n.s.

2.67 1.04–4.11

2.84 1.60–3.98 n.s.

3.11 1.55–4.26

0.84 0.06–4.22 P < 0.0001 0.98, P < 0.01

1.21 0.28–4.11

1.00 0.08–3.98 P < 0.01 0.96, P < 0.01

1.26 0.23–4.26

a Significance of the difference in ACE and TXE between fresh and wounded tuber tissue (n ¼ 23). b Correlation between antioxidant activity detected in fresh tissue and that measured 24 h after wounding (23 genotypes); ns, not significant P $ 0.05.

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(Fig. 1), and pnt 31598-3 was most efficient in the enhancement of its trolox equivalent within 24 h (Fig. 2). Moreover, it was found that S. pinnatisectum differed significantly from the other three Solanum species (adg, chc and phu) with respect to concentrations of soluble phenols (Table 4A), soluble proteins (Table 4B) as well as antioxidants measured as ACE and TXE (Table 4C) - a result noticed in the case of fresh and wounded tuber tissue. There was a clear correlation between the ACE values detected in fresh and those found in wounded tuber tissue (Fig. 1, Table 3A; r ¼ 0.98, P < 0.01), a result which was similarly noticed with respect to TXE values (Fig. 2, Table 3B; r ¼ 0.96, P < 0.01). The ACE was significantly correlated with the TXE in the case of both tissue types (Fresh tissue: r ¼ 0.94, P < 0.01; Wounded tissue: r ¼ 0.95, P < 0.01). Furthermore, ACE and TXE values were significantly correlated with soluble phenols and proteins (Table 5).

Discussion The evaluation of cultivated and wild, tuber-bearing Solanum species may lead to individual accessions bearing valuable traits and genes worth introducing into new potato cultivars by breeding. In this respect, several of the 23 genotypes tested in this study could be interesting candidates. Soluble phenols The quantities of total soluble phenols found in tuber tissue of the wild and cultivated Solanum species involved in this study concurred with results reported elsewhere.18–20As expected, the different genotypes revealed a wide range in phenol contents of their tuber tissue (Table 2A). On the one side, there was the accession chc 30161-17 (S. chacoense) with its small amounts of soluble phenols, and on the other side, the two accessions pnt 31598-2 and 31598-3 (S. pinnatisectum) with their relatively high phenol contents (Table 2A). With it, these last two accessions reached similar high phenol levels as purple-fleshed potato breeding clones studied recently,15 whereas most of the other genotypes had less phenols in their tuber tissue and were thus comparable with actual, white/yellow fleshed potato cultivars.18

The fact that S. pinnatisectum exhibited significantly higher quantities of soluble phenols than the other three Solanum species (i.e. adg, chc and phu) had (Table 4A) concurred with former results, obtained in 2006 and 2007.21 The effect of wound stress on soluble phenols Wound stress is a continuous threat for potatoes in agricultural practice, during storage and transportation as well as in potato commerce because wounds are a major entree for plant pathogenic microorganisms causing tissue decay associated with quality decrease. Hence, a rapid wound healing is needed, and this process requires sufficient amounts of simple phenols in the wound area. It was important therefore, that soluble phenols were increased after wounding the tubers of wild and cultivated Solanum species (Table 2A). Especially, S. tuberosum subsp. andigena which had relatively low phenol contents in its fresh tissue displayed the greatest raise of phenols in response to wounding its tubers. The differences in phenols between fresh and wounded tuber tissue were statistically significant within the three Solanum species adg, chc and phu, but interestingly, not within S. pinnatisectum. For example, pnt 31598-2 had smaller phenol quantities in extract samples taken after 24 h than in those derived from fresh tuber tissue (Table 2A). This tendency might be based on the fact that S. pinnatisectum displayed considerably higher basic levels of phenols in its fresh tissue than the other Solanum species had. A similar differentiated woundinduced alteration of plant phenols was noticed in white/yellow and purple flesh potato cultivars and clones,15,22 and a weaker effect on phenols induced by wounding was also reported for a red-lettuce that was very rich in phenols.23 Plant phenols are not only involved in pathogen defence due to their antimicrobial activities,24–27 they also function as precursors in the biosynthesis of suberin and lignin, both complex polymers associated with wound healing28 and defence responses.29,30 It has been suggested recently, that chlorogenic acid (CGA) as one of the major simple plant phenols31 could act as a reservoir that can rapidly be mobilized7 to form phenylpropanoid products such as lignin, phytoalexins and cell wall cross-linking compounds.32–34 This may explain why Solanum genotypes exhibiting small amounts of phenols in their tissue, like adg, increased their

Table 4 Average concentrations of soluble phenols, proteins and antioxidants (ACE, TXE) in fresh and wounded tuber tissue of S. pinnatisectum and the other three Solanum species (adg, chc, phu), and significance of the difference between these two groups Type of analyses (A) Soluble phenols (g kg1 fw) Fresh tissue Wounded tissue (B) Soluble proteins (mg mL1) Fresh tissue Wounded tissue (C) Antioxidant activity ACE (mg mg1) Fresh tissue Wounded tissue TXE (mg mg1) Fresh tissue Wounded tissue a

Other species (17 genotypes)

Significancea

1.82 1.88

0.47 0.76

<0.01 <0.001

12.88 12.97

8.28 8.85

<0.0001 <0.001

2.46 2.67

0.27 0.70

<0.05 <0.05

2.84 3.11

0.35 0.61

<0.001 <0.01

S. pinnatisectum (6 genotypes)

Significance of the difference between pnt and the other three Solanum species.

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Table 5 Correlation coefficients between the antioxidant activity comprising ACE and TXE values and soluble phenols as well as between antioxidant activity and soluble proteinsa Assay (2008) Antioxidant activity ACE Fresh tissue Wounded tissue TXE Fresh tissue Wounded tissue

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a

Soluble phenols

Soluble proteins

0.93** 0.85*

0.52** 0.45*

0.96** 0.91**

0.57** 0.50*

Significant: **P < 0.01, *P < 0.05.

phenol concentrations much more in response to wounding than those with relatively high basic levels of phenols, such as pnt 31598-2 (Table 2A). Obviously, the latter had no compelling need to refill its reserves, in contrary, in tissue of pnt 31598-2 the phenol quantities were diminished after 24 h. Its tissue may have started previously to integrate portions of simple phenols into more complex polymers such as lignin or intermediates, whereas the other genotypes, like those of adg, aspired to fill up their phenol reserves in the meantime. It can be assumed therefore that in tuber tissue of pnt 31598-2 the wound response is advanced. Altogether these results underline the role of plant phenols within wound stress responses actually directed to healing damaged tissue as soon as possible and to prevent further damage.35 But besides their function within stress responses, plant phenols could contribute to the nutritional value of potatoes as discussed below. Antioxidants Regarding their consumption potatoes are considered as a significant antioxidant source in human nutrition.36 Different potato cultivars had antioxidant activities that excelled onion, carrots and bell pepper.18 Besides ascorbic acid, a-tocopherol and bcarotene, plant phenols such as CGA also play a role as potent antioxidants.18,37 It was to be expected therefore that similar to that observed with phenols, the 23 genotypes involved in this study varied notably in their antioxidant activity measured as ascorbic acid (ACE: Fig. 1) and trolox equivalent (TXE: Fig. 2). On average, S. pinnatisectum displayed the highest antioxidant capacity, and pnt also differed significantly in its antioxidant activity from the other three Solanum species (Table 4C), again a result which coincided with soluble phenols (Table 4A) and proteins (Table 4B). Similar high amounts of antioxidants were found in tuber tissue of pnt during another test performed in 2007.21 The effect of wound stress on antioxidants Investigating the effect of wound stress on the level of antioxidants was a novel aspect with respect to wild Solanum potatoes. It was important to find out that the antioxidant activity, including water (ACE: Fig. 1, Table 3A) and lipid soluble antioxidants (TXE: Fig. 2, Table 3B) raised on average 24 h after wounding the tubers of cultivated and wild Solanum species a result that concurred with phenols (Table 2A) discussed above. The differences in ACE (Table 3A) and TXE (Table 3B) values This journal is ª The Royal Society of Chemistry 2010

between fresh and wounded tuber tissue were statistically significant when all genotypes were regarded and within the species adg, chc and phu. On average, also the wild Solanum species S. pinnatisectum increased its antioxidant potential in response to wounding (Table 3A and 3B). However, in the case of pnt the differences were statistically not significant. Similar to that observed with soluble phenols, there were several genotypes among pnt (e.g. pnt 31598-2) that revealed slightly reduced antioxidant activities after wounding. It was also interesting to note that the cultivated Solanum species adg and phu displayed greater enhancements of ACE (Fig. 1; Table 3A) and TXE (Fig. 2; Table 3B) values 24 h after wounding than the two wild potato species chc and pnt. However the latter, above all S. pinnatisectum exhibited on average multiple higher basic levels of water and lipid soluble antioxidants in the non-wounded tissue than adg and phu had (Table 3A). It seems that in the tuber tissue of pnt the level of antioxidants was sufficiently high and thus no tremendous increase of the latter was noticed. This tendency may also underline the role of antioxidants in wound stress responses of tuber tissue. A similar differentiated wound-induced alteration was found in context with plant phenols as discussed before. Apart from this fact, the enhancement of antioxidant capacity by wounding, especially within those Solanum species which had low concentrations of antioxidants in their fresh tissue (adg, phu) was not surprising, because there is growing evidence that plants respond to stress factors, such as wounding, by enhancing their radical scavenging capacity.7,9,38 A wound-induced increase of soluble phenols and antioxidant capacity in potatoes was also reported by Reyes & Cisneros-Zevallos,22 who in addition found an elevated activity of phenylalanine ammonia-lyase (PAL), an enzyme which is responsible for the synthesis of phenolic metabolites.5 The relationship between soluble phenols and antioxidant activity In addition, results of the present tests reflect a close relationship between antioxidants and soluble phenols. Thus, a significant correlation could be observed between the ascorbic acid equivalent and soluble phenols as well as between the trolox equivalent and the latter (Table 5). In addition, quantities of soluble phenols showed a linear relationship with ACE (fresh: R2 ¼ 0.86; wound: R2 ¼ 0.72) and TXE values (fresh: R2 ¼ 0.93; wound: R2 ¼ 0.82). A similar clear relationship between phenols and antioxidants was reported for purple and white/yellow fleshed potato breeding clones and cultivars15 as well as for Andean potato cultivars studied elsewhere.19 Altogether, these results underline the notion that amounts of phenols in tuber tissue can be an indicator for the antioxidant capacity of potatoes,39 and vice versa. Plant phenols are well known to function as efficient antioxidants7 due to a hydrogen-donating activity of the phenolic hydroxyl groups.9,40 For example, L-tyrosine, a monophenolic amino acid was found to exhibit strong antiradical activities.41,42 Moreover, caffeic and chlorogenic acid which are both among the major free phenolics in potatoes,36 and especially concentrated in the peel, correlated with the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH$) scavenging activity.43 In this context, it should be mentioned that pnt 31598-2 with its high amounts of antioxidants (ACE: Fig.1; TXE: Fig. 2) was also found recently to have high content of chlorogenic acid in its Food Funct., 2010, 1, 209–218 | 215

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tuber tissue (2.68 g kg1 fdm).21 In potato peel extracts caffeicand chlorogenic acid contributed to about 57% of total antioxidant activity measured as TXE,44 and both phenolic acids exceeded the antioxidant activity of a-tocopherol as reported elsewhere.45 Moreover, caffeic acid exhibited a stronger inhibiting activity on lipid peroxidation than trolox, an analogue of a-tocopherol,43 and also methanolic potato extracts had a better antioxidant capacity than the latter.46 Potato peel extracts containing high quantities of gallic-, caffeic acid and CGA inhibited lipid peroxidation and protected human erythrocyte membrane proteins from oxidative damage,47 while a potato peel powder was demonstrated to ameliorate oxidative stress in streptozotocin diabetic rats.48 All this indicates, that high amounts of simple plant phenols are associated with high antioxidant activities. Accordingly, plant phenols seem to have the ability to positively affect the antioxidant system in plant, animal and human cells. In this context it should also be mentioned, that caffeic acid and CGA can be absorbed in the small intestine of humans49 - i.e. part of hydroxycinnamic acids from food will enter into the blood circulation and could hereby induce biological effects in the human body. For example, plant phenols are well known to be anti-carcinogenic, anti-inflammatory, vasodilatory, antibacterial, immune-stimulating, anti-allergic etc.40,50 due to their radical scavenging activities.

Soluble proteins As observed in context with soluble phenols (Table 2A) and antioxidants (Fig. 1 and 2), the 23 Solanum genotypes varied in their contents of total soluble proteins (Table 2B). It was important to note, that also in this respect S. pinnatisectum exceeded on average the other three Solanum species involved in the tests (Table 2B, Table 4B). In particular, its two accessions pnt 31606-2 and pnt 31598-3 had significant soluble protein concentrations (Table 2B). On the other hand, the species S. tuberosum subsp. andigena exhibited on average the lowest amounts of proteins in its fresh tuber tissue - a result which was in agreement with soluble phenols.

The effect of wound stress on soluble proteins Wounding of plant tissue is normally associated with an increase in wound-related proteins within a few hours.51,52 For example, protein kinases, proteinase inhibitors,53 pathogenesis related proteins,51 PAL and peroxidase54 were found to be induced in plant tissue by wounding. It was to be expected therefore, that the protein contents increased on average 24 h after wounding the tubers (Table 2B). However, the increase of soluble proteins was less significant than that observed in the case of phenols (Table 2A) and antioxidants (Fig. 1 and 2; Table 3A and 3B). Only small differences with respect to proteins between wounded and non-wounded potato tubers were also reported by Logemann et al.52 Nevertheless, the enhancement of proteins by wounding, above all in tuber tissue of adg (Table 2B), a species with a relatively low level of soluble proteins in its fresh tissue, may underline their importance in wound stress responses of potatoes. 216 | Food Funct., 2010, 1, 209–218

The relationship between soluble proteins and antioxidant activity It was interesting to note that soluble proteins correlated not only with soluble phenols (fresh tissue: r ¼ 0.52), but also with water and lipid soluble antioxidants (Table 5). It appears therefore, that besides plant phenols, soluble proteins also accumulated in tuber tissue to support the antioxidant capacity of potatoes. Although, their effect seems to be less strong than that of phenols, a notion which is reflected by the results of regression analyses (fresh tissue, ACE: R2 ¼ 0.27 and TXE: R2 ¼ 0.31; wounded tissue, ACE: R2 ¼ 0.21 and TXE: R2 ¼ 0.25). Patatin, used as a trivial name for a family of glycoproteins, accounts for 30–40% of total soluble proteins accumulated in potatoes.55 Besides its function as a major storage protein in tuber tissue, it was reported to exhibit lipid acyl hydrolase and acyltransferase activity with a large number of lipid substrates,56 and moreover, patatin has been demonstrated by a series of in vitro tests to possess efficient radical scavenging activities.57 Especially, the cystein and tryptophan residues in the patatin molecule are assumed to mediate its antioxidant activity.57 It is imaginable therefore, that patatin contributed partly to the antioxidant potential of cultivated and wild Solanum species. Logemann et al.52 reported that wounding leads to suppression of patatin transcription in favour of defence and repair mechanisms, a fact which may explain why soluble proteins (Table 2B) raised less strong upon wounding than soluble phenols (Table 2A) and antioxidants (Fig. 1 and 2; Table 3A and 3B) which are both associated with plant defence responses.6,26 Characteristics of S. pinnatisectum All these results imply that an involvement of S. pinnatisectum, a Mexican wild diploid Solanum species, with its relatively high contents of soluble phenols (Table 2A) and proteins (Table 2B) as well as water (Fig. 1) and lipid soluble antioxidants (Fig. 2) in potato breeding could be useful in order to improve the efficiency of the antioxidant system in tuber tissue of new cultivars. In addition, S. pinnatisectum was found recently to have high quantities of starch in its tuber tissue.13 However, tubers of this Solanum species are relatively small, i.e. about 10 to 15 mm diameter, and several backcrosses with cultivated potatoes will be necessary to get an acceptable tuber size. Moreover, this Solanum species could contain higher levels of glycoalkaloids (GA) including a-solanin and a-chaconine than are found in S. tuberosum.58 Apart from undesirable flavor,59 GA’s are known to be poisoning.60,61 Therefore, the quantities of GA in parental genotypes and progenies derived from such crosses have to be checked in order not to exceed the accepted concentrations of <200 mg kg1 fresh weight.62 Ergo - a transfer of desired properties from pnt to new potato cultivars by conventional breeding is not very easy.

Conclusions The results revealed that soluble phenols and soluble proteins present in tuber tissue substantially contribute to the total antioxidant capacity of potatoes. It was important to note, that (1) soluble phenols and (2) soluble proteins as well as (3) water and (4) lipid soluble antioxidants increased notably upon wounding This journal is ª The Royal Society of Chemistry 2010

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the tubers of cultivated and wild Solanum species, a result which underlines the importance of all these components in wound stress responses of potatoes. Furthermore, it was interesting to find out that S. pinnatisectum exhibited significantly higher concentrations of total soluble phenols and proteins as well as water and lipid soluble antioxidants in its fresh and wounded tuber tissue than the other Solanum species had. Accordingly, an involvement of individual S. pinnatisectum accessions, e.g. pnt 31598-2 and 31598-4, in potato breeding could be profitable for the antioxidant potential and with it the nutritional value of new potato cultivars. This last aspect could be of great importance for the potato processing industry.

Abbreviations fw fdm ACE TXE PCL CGA GA

fresh weight freeze-dried matter ascorbic acid equivalent trolox equivalent photochemiluminescence chlorogenic acid glycoalkaloids

Acknowledgements Ilona Schollenberg is thanked for excellent technical assistance.

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

Food

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Function

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Volume 1  |  Number 1  |  2010

w al Ne rn u Jo

Food & Function

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