FLAVONOIDS Chemistry, Biochemistry and Applications FLAVONOIDS Chemistry, Biochemistry and Applications Edited by Øyvind M. Andersen Kenneth R. Markham Boca Raton London New York A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc. 2021_Discl.fm Page 1 Friday, November 4, 2005 2:14 PM Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-2021-6 (Hardcover) International Standard Book Number-13: 978-0-8493-2021-7 (Hardcover) Library of Congress Card Number 2005048626 This book contains information obtained from authentic and highly regarded sources. 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QP671.F52F53 2005 612’.01528--dc22 2005048626 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group and the CRC Press Web site at is the Academic Division of Informa plc. http://www.crcpress.com AndersenandMarkham/Flavonoids:Chemistry,Biochemistry,andApplications #2021_C000 FinalProof page 3 8.11.2005 9:11pm Editors ØyvindM.Andersen,afullprofessorofchemistrysince1993,hasspecializedinthechemistry of natural compounds. Since 2002, he has been head of the Department of Chemistry, University of Bergen, Norway. He received his Ph.D. degree in 1988 fromtheUniversityofBergen.Heisauthororcoauthorof100journal articles,sevenbookchapters,andfivepatents,andhassupervisedover 40 M.Sc. and Ph.D. students in the fields of anthocyanins and other flavonoids. Theactivities ofDr.Andersen’s researchgroup haveledto the establishment of several flavonoid-based companies. Most of his research projects concentrate on structural elucidation of new com- pounds, while others are methodology based and relate to NMR spec- troscopyandvariouschromatographictechniques.Someoftheprojects focus on anthocyanin properties with the objective of exploring their pharmaceutical poten- tial and their use as colorants in food. Kenneth R. Markham (Ken) has recently retired from his position as group leader and distinguished scientist at Industrial Research Ltd., a New Zealand Crown Research Insti- tute. Following completion of his B.Sc. and M.Sc. (Hons) at Victoria University, Wellington, New Zealand, and his Ph.D. in chemistry (University of Melbourne, Australia, 1963), he returned to Chemistry Division,D.S.I.R.,inLowerHutt,NewZealand,toworkonxanthone chemistry.Hisinterestinflavonoidsbeganduringa2-yearpostdoctoral withProfessorTomMabryattheUniversityofTexasatAustin(1965– 1967). The coauthored book The Systematic Identification of Flavo- noids resulted from this interaction. On his return to New Zealand, Dr. Markham established and led the ‘‘Natural Products’’ section, predominantly devoted to the study of flavonoids with particular em- phasis on the chemotaxonomy of New Zealand bryophytes, ferns, and gymnosperms. This workwascarriedoutinconjunctionwithbothlocalbotanistsandcolleaguesattheUniversity ofSaarbrucken(ProfessorsZinsmeister,Mues,andGeiger).In1979,heworkedasavisiting scientistwithProfessorJeffreyHarborneattheUniversityofReading.Thisagainresultedin the publication of a popular book, Techniques of Flavonoid Identification. Dr. Markham’s flavonoid research continued at Industrial Research Ltd. and has led him into studies as diverse as plant chemotaxonomy, plant evolution, plant UV protection, historical Antarctic ozone levels, propolis and bee pollen bioactives, G.E. modification of flower color, and subcellular chemistry, to name but a few. His work has been reported in some 280 publica- tions, including 18 invited chapters and two books, and has been recognized over the years through awards such as the Chemical Society’s Easterfield Medal, Fellowship of the Royal Society of New Zealand, a Ministerial Award for Excellence in Scientific Research, and the 1999 Pergamon PhytochemicalPrize. AndersenandMarkham/Flavonoids:Chemistry,Biochemistry,andApplications #2021_C000 FinalProof page 4 8.11.2005 9:11pm AndersenandMarkham/Flavonoids:Chemistry,Biochemistry,andApplications #2021_C000 FinalProof page 5 8.11.2005 9:11pm Preface It is with great pleasure that we accepted the offer by CRC Press to assemble and edit this compilation of reviews on flavonoids and their properties and functions for the present volume. We considered the volume timely in that the last book of this general type, The Flavonoids—AdvancesinResearchSince1986(editedbyJeffreyB.Harborne),appearedover a decade ago. Since then, advances in the flavonoid field have been nothing short of spectacular. These advances are particularly evident in the contributed chapters that cover: the discovery of a variety of new flavonoids; the application of advanced analytical tech- niques;geneticmanipulationoftheflavonoidpathway;improvedunderstandingofflavonoid structuresandphysiologicalfunctionsinplantsandanimals;and,perhapsmostimportantly, the significanceof flavonoids tohumanhealth. Whilsttheupdatingaspectofthechaptersisseenastheprimecontributionofthisbook, an effort also has been made to include a summary of previous knowledge in the field to enable the reader to place new advances in this context. Chapters 1 and 2 review the applicationofcontemporaryisolation,quantification,andspectroscopictechniquesinflavo- noid analysis, while Chapter 3 is devoted to molecular biology and biotechnology of flavo- noid biosynthesis. Individual chapters address the flavonoids in food (Chapter 4) and wine (Chapter 5), and the impact of flavonoids and other phenolics on human health (Chapter 6 and,inpart,Chapter16). Chapter 8 reviews newlydiscoveredflavonoidfunctionsin plants, whileChapter9isthefirstreviewofflavonoid–proteininteractions.Chapters10to17discuss the chemistry and distribution of the various flavonoid classes including new structures reported during 1993 to 2004. A complete listing of all known flavonoids within the various flavonoid classes are found in these later chapters and the Appendix, and to date a total of above 8150 different flavonoidshasbeen reported. Itisdifficulttooverstatetheimportanceofrecentadvancesinresearchonflavonoids,and we are sure that the information contained within this book will prove to be invaluable to a wide range of researchers, professionals, and advanced students in both the academic and industrial sectors. We are greatly indebted to our authors, and are delighted that so many of the world’s leadingresearchersinavarietyofflavonoid-relatedfieldshavebeenwilling,sogenerously,to share their knowledgeand experiencewithothers through theircontributionto thisvolume. We are also very grateful to Lindsey Hofmeister, Erika Dery, Jill Jurgensen, and Tanya Gordon at Taylor and Francis, and Balaji Krishnasamy at SPI Publisher Services for their supportand interest throughoutthe preparationof this book. Øyvind M.Andersen andKennethR. Markham HistoricalAdvancesin theFlavonoidField —A Personal Perspective Having been associated with flavonoid research for the past 40 years, and having witnessed thespellbindingchangesthathavetakenplaceinthefieldduringthistime,itistootempting by far not to take this opportunity to document for future researchers a brief personal perspective on developments in the flavonoid field over this period. I emphasize that this is AndersenandMarkham/Flavonoids:Chemistry,Biochemistry,andApplications #2021_C000 FinalProof page 6 8.11.2005 9:11pm but a personal perspective on progress, and as such will surely exhibit some bias and deficiencies. To the aggrieved I offer my apologies. In the early 1960s, flavonoids were widely viewed as metabolic waste products that were stored in the plant vacuole. Whilst there was interest at that time in their function as flower colorants, and in their distribution between plant taxa, the earliest investigations of their biosynthesishadjustbegun.InthisrespectitisinformativetonotethatTomGeissman’s1962 compilation of reviews in The Chemistry of Flavonoid Compounds includes nothing at all on biological function, and details only paper chromatography and absorption spectroscopy as analytical tools. At this time too, information on flavonoid distribution within the plant kingdom was still incomplete. For example, even as late as 1969 Bate-Smith wrote (in Chemical Plant Taxonomy edited by T. Swain) that flavonoids are rarely found in any but vascular plants. But within a few years of this statement, Markham, Porter, and others reported the widespread presence of flavonoids in mosses and liverworts and even their occurrence in an alga, Nitella hookeri. This, incidentally, remains the sole example of the occurrenceofflavonoidsinalgae.Todate,flavonoidshavebeenfoundinallmajorcategories of green plants except forthe Anthocerotae. By 1967 little had changed with regard to the application of physical techniques to flavonoid structure determination, with NMR and GLC yet to make an impact on the field. About this time, however, there was an upsurge in the application of flavonoid distribution to the emerging field of chemotaxonomy. Leading these researches were groups attheUniversityofTexasatAustin(ledbyTomMabry,RalphAlston,andBillyTurner)and attheUniversityofReading(ledbyJeffreyHarborne).AlstonandTurner’spioneeringwork onthetrackingofplanthybridizationthroughflavonoidanalysesisstillquotedtoday,asalso ismuchoftheanthocyaninstructureandflavonoiddistributionalworkdetailedbyHarborne in his1967 book, Comparative BiochemistryoftheFlavonoids. Rapid advances in the application of physical techniques to flavonoid structure analysis beganappearingintheliteratureinthemid-tolate-1960s,andthesewerewelldocumentedin a series of books beginning in 1970 with Mabry, Markham, and Thomas’ The Systematic Identification of Flavonoids, which reviewed, for the first time, the considerable advances made in the application of shift reagents in UV–visible absorption spectroscopy, the use of GLCforsugaranalyses,andtheapplicationof1HNMRspectroscopytoflavonoidstructure analysis.Atthistime,CCl -solubleTMSetherderivativeswerewidelyusedforNMRstudies 4 intheabsenceofreadilyobtainabledeuteratedsolvents.Thesesamederivatives,togetherwith permethylated derivatives, were commonly also used to make flavonoid glycosides suffi- ciently volatile for early applications of electron impact mass spectrometry to flavonoid structure analysis (first summarized in the 1974 ‘‘Advances’’ book, The Flavonoids, Chapter 3). Contemporaneously, and detailed in the above volume, rapid advances were being reported in the structure analysis of C-glycosides (Chopin and Brouillant, Chapter 12) and inthebiosynthesisofflavonoids(HahlbrockandGrisebach,Chapter16).Agrowingaware- nessofthephysiological,metabolic,andevolutionaryvalueofflavonoidswasalsobeginning to emerge. Tony Swain, for example, was in the initial stages of formulating his innovative interpretationsoftheevolutionofflavonoids,andthepartthattheir‘‘chemoecology’’played in theevolution of plants (e.g., seeChapter 20in The Flavonoids,1974). The next major advance in flavonoid structural techniques was the application of 13C NMR spectroscopy. This has arguably had the greatest impact on flavonoid structure analysis since the invention of paper chromatography around 1900. For the first time, complete flavonoid structures, including flavonoid aglycones together with sugar types and linkages, could be determined using a single technique. Admittedly, the development of advanced two-dimensional techniques has further revolutionized structure analysis since the earliest applications of this technique to flavonoids in the mid-1970s, and the appearance of AndersenandMarkham/Flavonoids:Chemistry,Biochemistry,andApplications #2021_C000 FinalProof page 7 8.11.2005 9:11pm the first review article by Markham and Chari in 1982 (The Flavonoids — Advances in Research, Chapter 2). Advances in technology have diminished the sample size required for spectralanalysisbymore than100timesforboth13Cand1HNMRtechniques.Intheearly 1960s, 100-mg samples were required for proton work and in the late 1970s the same sized sampleswererequired for carbon-13 studies. Modern flavonoid researchers will also be aware of the impact that the more recently developedmassspectrometrytechniquessuchasFAB,MALDI-TOF,andelectrosprayhave hadontheabilityofresearcherstoelucidatecomplexflavonoidglycosidicstructuresthrough the ready determination of accurate molecular weights and limited fragmentation patterns. Similarly,thedevelopmentofadvancedmethodsofseparationsuchascapillaryelectrophor- esis, HPLC, and, latterly, HPLC–MS, has recently revolutionized the qualitative and quan- titative analysis of flavonoid mixtures. Chemotaxonomic studies involving comparative distributionaldatahaveaccordinglybeenvastlyfacilitated.Techniquessuchasthosereferred to above have enabled previously intractable flavonoid structural problems to be solved. Particularly good examples of this are to be found amongst the many complex structures currentlybeingreportedfor‘‘blue’’flowerpigmentsbasedonanthocyaninselaboratedinan often intricate manner with large numbers of sugars, acyl groups, other flavonoids, and occasionally including metalions. Returningonceagaintothequestionsoffunctionanduses,theoldconceptofflavonoids beingmerelytheby-productsofcellularmetabolism,whicharesimplycompartmentalizedin solution in the cell vacuole, is well and truly past its use-by date. For a start, studies have revealedthatflavonoidsarealsocommonlyfoundontheoutersurfacesofleavesandflowers, albeit only the aglycone form. Additionally, flavonoids have been shown over the past few yearstobefoundinthecellwall,thecytoplasm,inoilbodies,andassociatedwiththenucleus andcellproteins,aswellasinthevacuole.Eveninthevacuole,flavonoidsarenotnecessarily found free in solution. For example, protein-bound flavonoids have been isolated from lisianthus and other flowers in which a structurally specific binding has been identified (in anthocyanic vacuolar inclusions). It is probable that flavonoid location and specific protein binding properties will ultimately prove to relate directly totheir function in plants. Amongstthemanyfunctionsnowknowntobeperformedbyplantflavonoidsarethoseof UV protection, oxidant or free radical protection, modulation of enzymic activity, allelo- pathy, insect attraction or repulsion, nectar guides, probing stimulants, viral, fungal, and bacterialprotection,nodulationinleguminousplants,pollengermination,etc.,anditislikely thatthisisonlythetipoftheiceberg.Flavonoids,itwouldseem,havebeenvitalcomponents of plants, ever since their (purported) development at the time plant life emerged from the aquaticenvironment,andneededprotectionfromUVlightinanatmospherelackingtoday’s protectiveozonelayer.Thecontinuedwidespreadaccumulationofflavonoidsbyvirtuallyall land-based green plants lends support tothis view. Intriguingly, it is now possible to exert some precise control over plant flavonoid com- position.Manipulationoftheflavonoidbiosyntheticpathwayinplantsviageneticengineer- ing has progressed rapidly in recent years. This has been expedited by the extensive information made available through the earlier studies of flavonoid biosynthesis pioneered inthe1960sand1970s(seeabove).Geneticmanipulationoftheflavonoidpathwayinplants has enormous potential to, for example, produce new flower colors, enhance the nutritional value of crops, and improve crop protection from UV light, microorganisms, insects, and browsing animals. Indeed, much of this work has been underway for some time and shows great promise. Plantflavonoidshavebeenshowninrecentyearstobeofvitalsignificancetomankindas well as to plants. They have been strongly implicated as active contributors to the health benefits of beverages such as tea and wine, foods such as fruit and vegetables, and even,